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Regulation of in vitro immunoglobulin secretion in healthy individuals and multiple sclerosis patients O'Gorman, Maurice R. G. 1988

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REGULATION OF IN VITRO IMMUNOGLOBULIN SECRETION IN HEALTHY INDIVIDUALS AND MULTIPLE SCLEROSIS PATIENTS By Maurice R.G. O'Gorman Hon. B. Sc., University of Western Ontario M . S c , University of British Columbia A THESIS SUBMITTED IN PARTIAL FULLFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF Ph.D. i n The Faculty of Graduate Studies Pathology Department, We accept this thesis as conforming to the required standard University of British Columbia June 1988 © Maurice (Mo.) Raymond Gerard O'Gorman In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for. financial gain shall not be allowed without my written permission. Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE -6G /81) 11 A b s t r a c t R E G U L A T I O N O F IN VITRO IMMUNOGLOBULIN S E C R E T I O N IN H E A L T H Y INDIVIDUALS AND M U L T I P L E S C L E R O S I S PATIENTS. Mitogen driven differentiation of mononuclear cells is a useful model of antibody synthesis and secretion in humans. We have studied Pokeweed mitogen (PWM) induced immunoglobulin secretion in vitro in both healthy individuals and multiple sclerosis patients. Within the healthy population we have identified individuals who consistently secrete low levels of IgG in response to PWM and others who secrete very high levels. The underlying mechanisms involved in low response are not well understood. We have observed that the peripheral blood mononuclear cells (PBMC) obtained from low responders differ from those obtained from high responders in each of the following: Their T-helper cell subset contains a higher ratio of T suppressor-inducer cells over T helper-inducer cells; their PBMC contain a higher level of in vivo radiation-sensitive suppression; their PBMC generate a lower autologous mixed lymphocyte response; and their B lymphocytes secrete lower amounts of IgG when mixed with heterologous high responder T helper cells. These results suggest the response involves the interactions between T helper cell subsets, T suppressor cells and B lymphocytes and that the level of response is the sum of the contribution of each subset. PWM induced immunoglobulin secretion was measured in multiple sclerosis patients during different^ phases of clinical disease activity. Relapsing-remitting multiple sclerosis patients in early relapse secreted less immunoglobulin than patients with prolonged I l l relapse, suggesting that immune function varies with clinical disease activity. Testing the level of PWM induced immunoglobulin secretion in relapsing-remit t ing multiple sc le ros is patients during the clinically stable phase suggested that those patients who secreted high levels of IgG in response to PWM were more likely to suffer a clinical relapse within 6 months than those patients who secreted a low amount. Chronic progressive multiple sclerosis patients secreted higher amounts of immunoglobulin in this assay than healthy control individuals. This group of multiple sclerosis patients also had; (i) reduced Concanavalin A (Con A) suppressor cell activity measured both by the ability to suppress a/ Con A induced proliferation and bl PWM induced IgG secretion in heterologous cell cultures and; (ii) reduced percentages of T cells expressing T suppressor and T suppressor-inducer markers. The treatment of chronic progressive multiple sc leros is patients in vivo with lymphoblastoid interferon resulted in a dramatic reduction in level of PWM induced immunoglobulin secretion without alteration in Concanavalin A induced suppression or in the percentages of T cells expressing subset specific markers. The PWM induced IgG secretion assay is a valuable technique for investigating the regulation of humoral immunity in both health and disease. IV Table of contents Abs t rac t 11 Table of contents IV List of Tables IX List of Figures XII List of Abbreviations XV Acknowledgement XVII Chapter 1: CELL-MEDIATED IMMUNE FUNCTIONS IN MULTIPLE SCLEROSIS. 1 1:1 Introduction: 1 1:2 Pathology: 2 1:3 Immune Cell Subset Enumeration: 7 1:3:i Peripheral Blood 7 1:3:ii Cerebrospinal Fluid 14 1:4 Suppressor Cell Function Abnormalities: 15 1:5 IgG Secretion: 1 7 1:5:i IgG Secretion in vivo 1 7 1:5:ii In vitro IgG Secretion in MS 24 1:6 Stimulation Assays: 2 9 1:6:i Search for the Elusive MS-Specif ic Antigen 29 1:7 Cell Cytotoxicity Assays: 3 6 1:8 MS: Diagnostic, Evolution: 41 1:9 Summary: 43 1:10 References: 45 Chapter 2: REGULATION OF IN VITRO PWM INDUCED IGG SECRETION IN HUMANS 8 9 V 2:1 Introduction 89 2:2 Materials and Methods 9 2 2:2:i Subjects: 9 2 2:2:ii Cell separation techniques: 92 2:2:ii:a Fractionation of Mononuclear Cells 92 2:2:ii:b E Rosette Cell Subset Separation Technique 9 2 2:2:ii:c Removal of Monocytes 9 2 2:2:iii PBMC Cultures: 9 3 2:2:iii:a PWM induced Proliferation 9 3 2:2:iii:b PWM induced IgG secretion assay 9 3 2:2:iii:c Staphylococcus Aureus Cowan Strain 1 (SAC) Induced IgG Secretion 9 5 2:2:iii:d Reconstitution experiments 9 5 2:2:iii:e Autologous Mixed Lymphocyte Reaction (AMLR) 9 6 2:2:iv Lymphocyte Subset Labelling and Analysis: 9 6 2:3 Results 9 8 2:3:i PWM Induced IgG Secretion: ...9 8 2:3:ii Relationship between DNA Synthesis and IgG Secret ion: 9 9 2:3:iii Staphylococcus Aureus Cowan Strain 1 (SAC) induced IgG secretion: 9 9 2:3:iv IgG Secretion in monocyte depleted cultures: 9 9 2:3:v Cell subset mixing experiments: 1 00 2:3:vi Effect of Irradiation of E+ cells in the PBMC of LR individuals: 100 VI 2:3:vii Autologous Mixed Lymphocyte Reaction (AMLR) and PWM induced IgG secretion: 1 01 2:3:viii T-helper Cell Subset Enumeration and PWM induced IgG secretion: 1 01 2:4 Discussion: 103 2:5 References: 1 22 Chapter 3: IMMUNE FUNCTION AND DISEASE ACTIVITY IN MULTIPLE SCLEROSIS 1 28 3:1 Introduction 128 3:2 Materials and Methods 1 33 3:2:i Patients: 133 3:2:i:a MS patient Subgroups 133 3:2:i:b RR-stable MS patients 134 3:2:i:c Chronic Progressive MS 134 3:2:ii Immune function studies: 135 3:2:ii:a PWM induced IgG secretion 135 3:2:ii:b Con A suppressor cell assay: 135 3:2:ii:c ConA induced suppression of PWM induced IgG secretion 1 36 3:2:ii:d T cell subset enumeration: 137 3:2:ii:e T helper cell subset labelling and analys is 1 37 3:3 Results 139 3:3:i:a PWM induced IgG secretion in Relapsing Remitting MS patients 139 3:3:i:b PWM induced IgG secretion in Stable MS as a prognostic indicator 139 VII 3:3:i:c Chronic progressive MS patients 140 3:3:ii Con A induced suppression of Con A induced pro l i ferat ion 1 41 3:3:iii ConA induced Suppression of PWM Induced IgG secretion in CP-MS-A , and controls 141 3:3:iii Surface phenotype of T cells in MS patients and controls 1 42 3:3:iv Surface phenotype of CD4+ cells in MS patients and controls 1 42 3:4 Discussion 143 3:5 References 1 63 Chapter 4 : REDUCTION OF IMMUNOGLOBULIN G SECRETION IN VITRO FOLLOWING LONG TERM LYMPHOBLASTOID INTERFERON 1 73 4:1 Introduction 173 4:2 Materials and Methods 1 76 4:2:i Patients: 176 4:2:ii Interferon: 176 4:2:iii IgG secretion in vitro: 177 4:2:iv Con A suppressor cell assay: 1 77 4:2:v T cell subset enumeration: 178 4:2:vi Lymphocyte subset mixing experiments: 178 4:2:vi:a E- cells + T helper cells 178 4:2:vi:b Monocyte deprived PBMC + monocytes 179 4:2:vii Statistics 1 79 4:3 Results 180 4:3:i MS patients and healthy controls: 180 4:3:ii MS Patients during the interferon trial: 180 VIII 4:3:ii:a Before treatment 180 4:3:ii:b After 1 week of treatment 1 81 4:3:ii:c At 1 month and 6 months of treatment 181 4:3:ii:d Six months after the last injection 182 4:3:iii Subset mixing experiments 182 4:4 Discussion 184 4:5 References 1 93 Chapter 5: SUMMARY AND HYPOTHESIS 1 97 5:1 References 207 IX List of Tables Table 2:1. The effect of four different lots of fetal calf sera on amount of IgG (ng/ml) secreted by 1X106 PWM stimulated P B M C obtained from 2 different individuals 109 Table 2:ll. The effect of different concentrations of Pokeweed Mitogen (PWM) on the level of IgG sec re ted by P B M C into the cul ture supernatant 11 0 Table 2:lll. The rate of PWM induced IgG secretion in PBMC obtained from healthy individuals 111 Table 2:IV. The level of IgG secreted into the culture supernatant by different concentrat ions of PWM stimulated PBMC 11 2 Table 2:V. Level of IgG secreted in response to PWM and to S A C in whole P B M C cultures and in macrophage depleted (Mo-ve) cultures 115 Table 2:VI. The amount of vivo radiation sensitive suppression in the P B M C of 2 low responders and 2 high responders calculated using 2 different methods 11 7 Table 2:VII. In vitro PWM induced IgG secretion and autologous mixed lymphocyte reaction in P B M C cultures derived from the same whole blood X sample of 2 low responder (LR) and 2 high responder (HR) individuals 11 8 Table 2:VIII. The ratio of T-suppressor/inducer cells to T-helper/ inducer cel ls (Tsi/Thi) and the level of PWM induced IgG secretion in the peripheral blood mononuclear cells of healthy male and female subjects 119 Table 3:l. PWM induced IgG secretion in Relapsing Remitting (RR) patients during remission: Patients were separated into 2 groups based on the length of time since since their last relapse 1 53 Table 3:ll. PWM induced IgG secretion in stable MS patients with relapsing remitting d isease . Patients are sub-grouped according to the results of further follow-up as indicated in fig. 3:1 1 54 Table 3:lll. PWM stimulated IgG secretion (10 days) in Controls and 2 groups of MS with chronic progressive (CP) disease, CP-Act ive and C P -Stable 1 56 Table 3:IV. Suppression of PWM induced IgG secretion by Con A preactivated PBMC obtained from healthy controls (HC), other neurological disease controls (OND) and chronic progressive multiple sclerosis (CP-MS) patients 160 XI Table 3:V The percentage of CD4+, DC5+, CD8+ and the CD4:CD8 ratio in chronic-progressive MS patients and healthy controls 161 Table 3:VI. Surface phenotype of CD4+ T helper cells and PWM induced IgG secretion in the P B M C obtained from 2 MS groups and 2 control groups 1 62 Table 4:l. Percentages of CD4+, CD5+, CD8+ cells and the CD4 :CD8 ratios in the MS patients (a) before treatment and the control group, and (b) at var ious time points fol lowing their assignment to the IFN (n=14) or PLA (n=16) groups 1 88 Table 4:ll. Pokeweed mitogen induced IgG secretion (ng/ml) in co-cultures of 10 5 E- cells and 10 5 T helper cells from two IFN-patients and one PLA-pat ien t 191 Table 4:lll. Pokeweed mitogen induced IgG secretion (ng/ml) in co-cultures of 2x10 5 monocytes (plastic adherent cells) and 8x10 5 monocyte depleted PBMC obtained from two PLA and two IFN patients 192 XII List of F igures Fig. 2:1. Results of repeated PWM induced IgG secretion by P B M C obtained from 8 healthy individuals, 4 of these individuals consistently generated a high response (HR) and the other 4 indiv iduals consistent ly generated a low response (LR) 11 3 Fig. 2:2. A- DNA synthesis measured by 18 hr pulses with tritiated thymidine at the end of the indicated days in PWM stimulated cultures of 2 . 5 x 1 0 5 P B M C obtained from 3 different subjects and B- Amount of IgG in the supernatants of these same cultures 114 Fig. 2:3. PWM induced IgG secretion (mean±SEM) in heterologous mixed cultures of .5x10 6 E- cells and .5x10 6 E+ cells 11 6 Fig. 2:4. Two color cytometric analysis of PBMC co-express ing the Leu3a and 2H4 A g , (T-suppressor/inducer, Tsi) or the Leu3a and 4B4 Ag, (T-helper/inducer, Thi) in the blood of one HRand one LR 120-121 F ig .3 :1 . Diagrammatic representat ion of the procedure used to study the prognostic value of measuring PWM induced IgG secretion in the XIII per iphera l b lood mononuc lear ce l l s of Relapsing Remitting (RR) MS patients during the stable phase of their disease 151 Fig. 3:2. The amount of IgG secreted by the PWM st imulated P B M C of R R - M S patients in remission correlates with the length of time since their last attack, r=.739, p<.01 152 Fig 3:3. Frequency distribution of the percentage of heal thy contro l ind iv idua ls or chron ic progressive MS patients whose P B M C secrete IgG at a given level when stimulated with PWM in vitro for 7 days 1 55 Fig. 3:4. Frequency distribution of the percentage of heal thy contro l ind iv idua ls or chron ic progressive MS patients whose P B M C secrete IgG at a given level when stimulated with PWM in vitro for 10 days 1 57 Figure 3.5. Concanavalin A induced suppressor cell activity in the PBMC of 25 chronic progressive MS and 18 Healthy controls 158 Fig. 3:6. A dose response effect on the suppression of IgG secretion in PWM stimulated P B M C cultures is observed by adding increasing numbers of Con A pretreated suppressor cells 159 Fig. 4:1. IgG production (ng/ml) in response to PWM by P B M C obtained from MS patients and controls, (left side, mean±SEM). Right side, XIV ConA induced suppression in MS patients and controls (meantSEM) 187 Fig. 4:2. IgG production (mean±SEM) in response to PWM by PBMC obtained from 30 MS patients at various time points following their inclusion into IFN or PLA-treated groups 189 Fig. 4:3. Con A suppression in MS patients following inclusion into the IFN- or PLA-treated groups before treatment and again after 1 week of daily subcutaneous injections of Wellferon® (lymphoblastoid interferon) 1 90 XV L i s t of A b b r e v i a t i o n s Ab-Ant ibody ADCC-Ant ibody dependent cellular cytotoxicity AET-Amino-ethy l isoth iouronium-bromide Ag-Ant igen Ag-ARFC-Ant igen active rosette forming cell AMLR-Autologous mixed lymphocyte reaction BBB-Blood brain barrier Con A-Concanavalin A CP-A-Chron ic -p rogress ive -a t tack CP-Chron ic -progress ive CP-S-Chron ic -p rog ress ive -s tab le CSF-Cerebrospinal fluid CTL-Cytotoxic T cell DNA-Deoxyribonucleic acid EKDSS-Extended Kurtzke disability status scale E- roset te-Ery thocyte- roset te FACS-Fluorescence activated cell sorter FITC-Fluorescein isothiocyanate G F A P - G l i a fibrillary acid protein HBSS-Hank's balanced salt solution HR-High responder IFN-lnterferon Ig-lmmunoglobulin IgA-lmmunoglobulin of subtype A XVI IgG-lmmunoglobulin of subtype G IgM-lmmunoglobulin of subtype M K cells-Kil ler cells LR-Low responder mAb-Monoclonal antibody MBP-Myel in basic protein MIF-Migration inhibition factor MRI-Magnetic resonance imaging MS-Mult iple sclerosis NAWM-Normal appearing white matter NK-Natural killer cell PBMC-Peripheral blood mononuclear cells PE-Phycoerythr in PGE-Prostaglandin E PWM-Pokeweed mitogen R R - A - R e lapsing-remit t ing-at tack RR-Relaps ing- remi t t ing RR-S-Re laps ing- remi t t i ng -s tab le SAC-Staphylococcus Aureus Cowan strain 1 SRBC-Sheep red blood cells Th-T helper Thi-T helper-inducer Ts-T suppressor Tsi-T suppressor-inducer XVII A c k n o w l e d g e m e n t I would first and foremost like to acknowledge my friend and supervisor, Dr. Joel Oger., for without his confidence, encouragement and guidance I would not have completed this project. Secondly I must thank my wife (and son) for their compassion and understanding of the long hours, the frustrations and disapointments and of course the accomplishments which accompanied my progression through graduate studies. Lastly I would like to thank my parents and family for their support and encouragement. -1 -Chapter 1 C E L L MEDIATED IMMUNITY IN M U L T I P L E S C L E R O S I S 1:1 I n t r o d u c t i o n : Multiple sc lerosis (MS) is an inflammatory demyelinating disorder of the white matter of the central nervous system (CNS). Abnormalit ies of cell-mediated immunity have been implicated as contributing to the pathogenesis. Three observations support the involvement of immune mechanisms in the destruction of the myelin sheath: (1) a discrete inflammatory infiltrate is present in active lesions; (2) lymphocyte subsets and functions seem to be altered in blood and cerebrospinal fluid (CSF) ; (3) pathological similarities reminiscent of MS are found in chronic relapsing experimental allergic encephalomyel i t is . We will review the abnormalities of cellular immunity which have been identified in blood and C S F of MS patients. Cellular immunity can be monitored by assaying the functional activity of regulatory and effector subsets of lymphocytes. Effector lymphocytes include: NK cells (natural killer cell activity), K cells (which mediate antibody-dependent cell cytotoxicity), T cytotoxic cells (mediating T-ce l l -dependen t cytotox ic i ty) , and B ce l l s (which secre te immunoglobulins). Regulatory cells include T helper and T suppressor cells. The former respond to antigen challenge by proliferation and DNA synthesis and are involved in the induction of effector functions - 2 -and T suppressor cell function, while the latter serve to down-regulate the immune response. T suppressor cells may also be controlled by contrasuppressor cel ls. Lymphocyte subsets with different functional capacit ies express unique surface structures, al lowing for their identif ication, essent ial ly using monoclonal antibodies. The major problem with the studies of cell-mediated immunity in MS is that a specific antigen, against which the immune response is mounted has not been recognized. This explains why the majority of immune function studies reported in this disease have not addressed antigen specific stimulation. Abnormal response to a specific antigen may, in fact, not be necessary for MS to develop. 1:2 P a t h o l o g y : The elementary lesion in MS is called 'plaque' after Charcot [1868]. Plaques are circumscribed areas of demyelination with relative preservation of the axons. They vary in size from a few millimeters to 1 or 2 cm. They are widespread in the white matter of the C N S including the spinal cord and show some predilection for the periventricular area. Plaques within the CNS are classified according to their activity and age: (a) acute active (recent), (b) active chronic (moderately recent), or (c) silent chronic (old). Although this terminology is generally accepted it has been suggested that a fourth group of healing or resolving plaques be included in the classification [Traugott and Raine, 1984; Prineas et al., 1984]. 'Shadow plaques' have - 3 -also been descr ibed and are thought to represent incomplete remyel inat ion. Despite many publications referring to the 'early lesion' in MS, very little is known of the initial stage of plaque evolution. The lack of knowledge in this area stems from the chronic nature of the d isease and pathologists often equate early lesions with the abnormalities found at the edge of active plaques. Acute lesions are seen only rarely and are character ized by the presence of inflammatory cel ls, edema and little or no gliosis [Prineas et al. , 1984]. Lymphocytes and macrophages are seen in the parenchyma and within the plaques have a predilection for perivascular areas. Lymphocytes are generally absent from the normal-appearing white matter (NAWM). The silent chronic lesions have well-demarcated edges and exhibi t ex tens ive as t rocy tos is with very few oligodendrocytes and few inflammatory cells. These types of lesions are also referred to as 'burnt-out plaques' and occur more frequently in patients with a long history. The chronic active plaques (which are the most common and hence often referred to as the characteristic MS plaque) have well-demarcated edges and a variable degree of inflammatory cell infiltrates. The pathological changes observed within these lesions vary from mildly active to highly active. The former is cha rac te r i zed by as t rocy t ic g l ios is and mild hypercellularity (mostly foamy cells) at the edges, the latter is characterized by myelin-laden macrophages at the center and dense mononuclear cell infiltrates at the edge. Outside the plaques, in the NAWM, many of the sections reveal abnormalities, the most frequent of them being diffuse gliosis [Allen, 1981]. The phenotypes of the - 4 -immune cells in both plaque and non-plaque areas (usually referred to as NAWM) have been intensely investigated. Raine's group [Traugott et al . , 1982, 1983a, b; Traugott and Raine, 1984] reported that the distribution of T cell subsets and macrophages varied with lesion activity. Ia+ macrophage were most numerous in the center of the chronic active lesion and decreased consistently towards the lesion edge. The distribution of the T cells followed a reverse gradient. TII+ cells (CD2+, pan T cells) were rare in the center of the plaque but the density increased towards the edge where it reached a maximum both within the parenchyma and in perivascular areas. Numerous TII+ cells were observed in the NAWM. T4+ (T helper) cells showed a similar pattern. T4+ cells were the predominant cell type in the NAWM. T8+ ce l ls (suppressor /cytotox ic) showed a predi lect ion for the perivascular areas at the edge of the lesions and in a narrow zone of the NAWM. T4+ cells were large and expressed the la+ antigen which might indicate that they were activated. In many of the chronic active plaques these authors [Traugott et al., 1982] found a close association between la+ macrophages and T lymphocytes (both T4+ and T8+). This close association between lymphocytes and macrophages may possibly give some insight into the pathogenesis of the lesion progression. Within less active chronic plaques, la+ cells were infrequent at the lesion center but accumulated in large numbers at the lesion edge and deep into the NAWM. Booss et al. [1983] also studied the cellular infiltrates in plaque and non-plaque sections of CNS material obtained from MS patients at autopsy. Within the C N S , the predominant T cell subset was the cytotoxic/suppressor ce l l . This group also observed that the - 5 -proportions of T cells carrying specific subset markers was not the same in the perivascular infiltrates as the proportions of T cell subsets within the C N S parenchyma. Within the parenchyma it was observed that the T8+ cel ls outnumbered the Leu 3a+ (T helper/inducer) by a ratio of 3:1. Interestingly, the T8+ cells also outnumbered the T3+ cells (pan T cells). This was the case for both plaque and non-plaque areas in most of their counts. In a similar investigation, Hauser et al. [1986] reported that T8+ cells within the perivascular infiltrates predominated by as much as 50:1. In none of the cases did the T4+ cells outnumber the T8+ cells. Similarly, Nyland et al. [1982] reported that the OKT8 monoclonal antibodies stained many of the cells in the parenchyma of plaques. In summary, three independent groups [Nyland et al., 1982; Booss et al., 1983; Hauser et al., 1986] reported that within the parenchyma of active plaques and in the NAWM, T8+ were the predominant phenotype. In contrast Traugott et al. [1982] and Traugott and Raine [1984] reported that T4+ cells outnumbered the T8+ cells in the parenchyma of both active plaques and non-plaques. Within the perivascular areas, T4 and T8 are reported to be equally represented [Booss et al . , 1983], preponderantly T8+ [Traugott et al . , 1982; Traugott and Raine, 1984; Hauser et al., 1986] and prepon-derantly T4+ [Nyland et al., 1982], or Leu 3a+ [Brinkman et al., 1982b]. In addition to the T cell specific markers, several groups have observed large numbers of la+ (MHC Class II) cells at the plaque edge, within plaques and also in the NAWM [Traugott, 1987; Hofman et al., 1986; Hauser et al., 1986]. The la+ cells show a wide range of morphologies resembling macrophage, lymphocytes and astrocytes. - 6 -Hofman et al. [1986] recently confirmed by a double staining technique that the majority of the la+ cells at the edge of chronic active plaques were GFAP+ astrocytes. This observation is interesting in light of the evidence indicating that cultured rat astrocytes are able to present Ag to T-cells and in the process become la+ [Fontana et al., 1984]. la antigen expression on astrocytes can be induced by gamma interferon [Hirsch et al.,1983], a lymphokine secreted by activated T cells. The presence of IL2 receptor bearing cells within the brain tissue of MS patients suggests that the T cells in MS brains may be activated [Hofman et al., 1986]. Traugott [1987] reported on the espression of HLA Class I and Class II Ag on astrocytes and endothelial cells in brain samples of MS patients and healthy controls. In normal C N S , Class I Ag were expressed on most endothelial cells and the Class II Ag were absent. Astrocytes in these samples were Class II negative and occasionally Class I positive. In chronic MS the number of Class I positive endothelial cells was low but Class II positive endothelial cells were scattered throughout the C N S . MHC Class I and Class II positive astrocytes were more common in active lesions that in silent les ions. The presence of these cellular infiltrated is one of the major tenets of the immunological theory. However the distribution of the T cell subsets and the relationship to the stage of the disease is not resolved. - 7 -1:3 Immune Cell Subset Enumeration: 1:3:i Peripheral Blood; During the last decade we have witnessed the accumulation of a plethora of data concerning the enumeration of various lymphocyte subsets and the associated changes occurring with different clinical manifestations of MS. There have been many conflicting reports and these will be summarized in this section. Interested readers may also refer to recent reviews by Reder and Arnason [1985] and Baumhefner and Tourtellotte [1985, 1986] for more details. B cells were originally recognized by the binding of fluorescein-conjugated antisera to their surface immunoglobulins T cells by their binding to sheep erythrocytes ( S R B C ) . When peripheral blood mononuclear cells (PBMC) and S R B C are mixed, S R B C bind to the surface of T cells, forming what is known as an E-rosette and these can be readily identified by light microscopy. Most investigators have observed that the percentage of B cells in stable MS is normal [Lisak et al., 1975; Traugott et al., 1979; Hauser et al., 1981; Hammann et al., 1984, and many others]. A few investigators have observed a slight increase. Oger et al. [1975] reported that the number of complement receptor bearing PMNC (B cells and monocytes) was increased in a group of MS patients. Reddy and Goh [1976] and Schauf et al. [1977] also observed an increased proportion of B cells in MS patients. Recently, Link et al. [1984] reported a decrease in B cells in MS. The percentage of B cells during acute relapse has similarly been reported to be: normal, increased, or decreased and one report indicates that in chronic progressive MS the percentage of B cells is normal [Nordal and - 8 -Froland, 1978]. It would appear that the number of B lymphocytes in the peripheral blood of MS is for the most part not abnormal. The percentage of T cells in the peripheral circulation of MS patients has been reported as normal in patients with stable disease and also in groups of patients that have not been classified as to disease activity [see Reder and Arnason, 1985]. Most authors have shown a reduced number of SRBC-binding cells during active disease [Oger et al . , 1975; Huddlestone and Oldstone, 1979; Merrill et al . , 1980; Sandberg-Wollheim, 1983, and others] although some groups have reported normal numbers [Goust et al., 1978; Traugott et al., 1979; Kelley et al., 1981]. A subset of T cells shows a high affinity for S R B C and has been termed 'avid' T cells. Oger et al. [1975] and others [Utermohlen et al., 1978; Dore-Duffy and Zurier, 1979] have reported that avid T cells are reduced in MS. It is suspected that the reduced number of avid T cells may be due to an increased level of prostaglandin [Dore-Duffy and Zurier, 1979]. Interestingly, T suppressor cells have more receptors for S R B C [Howard et al., 1981] and the reduced number of avid T cells may have been an early recognition of the alteration of this subset (see below). Another subset of T cells is identified by the rapidity of their binding to S R B C . This subset, termed 'active' T-cells, has been reported to be decreased in stable and active MS [Kately and Bazzell, 1979; Traugott et al., 1979; Manconi et al., 1980; Rukavina et al., 1984]. Other groups have failed to find abnormalities although the number of patients studied was small [Goust et al., 1978; Offner et al. , 1978; Kam-Hansen, 1980]. The relationship between active and avid rosetting cells has not been explored. - 9 -Before the advent of monoclonal antibody technology, T cells were separated into regulatory helper and suppressor subpopulations based on their receptors for IgM and IgG immune complexes, respectively [Moretta et al., 1977]. Approximately 75% of the T cells bind to IgM-coated ox RBC (T mu cells) while approximately 20% bind to IgG-coated ox RBC (T gamma cells). The number of T gamma cells reportedly fluctuates with d isease activity; decreasing prior to relapse and rising above normal levels during early remission [Huddlestone et al., 1979, 1982; Traugott et al . , 1982; Wicher and Holub, 1982]. Several groups have reported increased T gamma levels in chronic progressive disease [Santoli et al., 1978; Goust et al., 1980; Merrill et al . , 1980; Traugott et al., 1982]. Additionally, Merrill et al. [1980] found increased T gamma level during attacks. The availabil ity of mouse monoclonal antibodies directed against unique cell surface molecules on T cells and T cell subsets produced a veritable explosion of immunological research. Not only did these molecules provide the most accurate method for enumerating T cell subsets but they also allowed for the isolation of specif ic regulatory and effector T cell subsets. The monoclonal antibody used most commonly to identify total T cells, i.e. pan T cell markers, are OKT3, Leu4 and Leu1. These monoclonal antibodies bind a 19-kilodalton protein found on almost all peripheral blood T cells. The original observation that OKT3-posit ive cells were reduced in MS regardless of disease activity [Reinhertz et al., 1980] is supported by some groups [Reder et al., 1984; Craig et al., 1985] although others have not observed any difference in the percentage of T cells (i.e. -1 0-Leu4, OKT3 or Leu1+ cells) between stable MS and controls [Oger et al., 1984; Thompson et al., 1985; Hirsch et al., 1985]. In active disease, most groups have observed a slight decrease in percentages of T cells expressing the pan T cell markers [Paty et al., 1981, 1983; Antel et al., 1984b; Tjernlund et al., 1984; Reder et al., 1984; Thompson et al., 1985; Albala et al., 1985] but again other groups have not observed significant differences [Mingioli and McFarlin, 1984; Bach et al., 1985; Hirsch et al., 1985]. The monoclonal antibodies Leu 2a and OKT8 identify both the regulatory T suppressor cells and effector cytotoxic T cells. OKT5 also recognizes both subsets, but appears to stain only a certain proportion of the cells identified by either Leu 2a or OKT8. OKT5 cells were reported to be drastically reduced in active MS [Reinhertz et al., 1980; Thompson et al., 1985] although Rice et al. [1983a, b, 1984a, b] and Paty et al. [1983] failed to confirm this observation. OKT8-posit ive cells are significantly decreased during acute relapse in most hands [Bach et al., 1980, 1985; Huddlestone and Oldstone, 1982; Antel et al., 1984b; Thompson et al., 1985; Oger et al., I985; Craig et al., 1985] although others have reported no change [Rice et al., 1984a, b; Reder et al., 1984; Mingioli and McFarlin, 1984; Hirsch et al., 1985]. The general consensus is that the percentage of OKT8-positive cells is somewhat reduced in the chronic progressive form of MS although a few recent reports have not found this [Zabriskie et al., 1 985; Hirsch et al., 1985]. Similarly, most authors agree that Leu 2a is decreased during relapse as well as in the chronic progressive form of the d isease , although this observation is not unanimous. Explanations for vagaries obtained in enumerating the percentages of - 1 1 -T suppressor cells in MS are numerous: different techniques, i.e. flow cytometry versus f luorescence microscopy, different criteria for selection of parameters in flow cytometry, difficulty in assessing the clinical status of the patients and fluctuating results observed in normal individuals may all be implicated. The majority of the reports on the percentages of helper T cells in stable and active disease suggests that the numbers are normal [Compston, 1983; Brinkman et a l . , 1983; Antel et a l . , 1984b; Kastrukoff and Paty, 1984; Rice et al., 1984a, b; Thompson et al., 1985; Bach et al. , 1985; Hirsch et al. , 1985; Albala et al. , 1985] although conflicting observations have been reported [Reinhertz et al., 1980; Mingioli and McFarlin, 1984; Salk et al., 1982]. The mechanism responsible for the reduced number of suppressor lymphocytes and the relationship of reduced numbers to active clinical disease in MS is not known. The reduction of cells bearing the suppressor/cytotoxic phenotypic markers could be a primary pathological event leading to systemical ly reduced suppressor cell function, thus allowing for an autoimmune response. Results of functional suppressor assays in vitro support this notion (see below) although the mechanisms by which immune reactivity is directed specifically towards the C N S remains largely unexplored. Another explanation for the reduced suppressor/cytotoxic cells in the circulation may be that they home specifically to the CNS (see above) where they play a role in active demyelination [Traugott et al., 1983a, b] or alternatively in the suppression of the demyelinating response [Booss et al., 1983]. - 1 2 -Another possibility for the reduction in suppressor/cytotoxic cells is that they share antigenicity with the target cells which are preferentially destroyed in the C N S . In support of this concept it has been observed that ovine oligodendrocytes express an antigen which cross-reacted with the OKT8 monoclonal antibody [Oger et al., 1982b]. This reaction was independent of Fc receptor binding. Further investigation of shared antigenicity between rat, calf and human ol igodendrocytes and the lymphocyte subsets identified by the monoclonal antibody OKT3, OKT4 and OKT8 showed no cross-reactivity [Hirayama et al., 1983]. Yet another explanation for the reduction of suppressor/cytotoxic cel ls during active d isease relates to the modulation of the cell surface markers. An antibody present in the circulation which binds to the OKT8+ cell subset could conceivably alter the binding of the subset-specific monoclonal antibody. If this were the case the cell would remain in the circulation although it would not be detected by conventional methods [Antel et al., 1982]. Anti-lymphocyte antibodies, which have been detected in the sera of MS patients [Kuwert and Bertrams, 1972; Schocket et al., 1977; Lisak et al., 1979; McMillan et al., 1980] would be candidates to support this mechanism. Further evidence suggesting modulation of surface antigens was reported by Paty et al. [1983]. This group observed a fluctuation in a subpopulation of T suppressor/cytotoxic cells which is Leu 2a+ (OKT8+) and OKT5-. The variations in the enumeration of this subset were thought to reflect the variable expression of the cell surface antigens. The fluctuations in this subpopulation were not related to clinical activity. Prostaglandins have been reported to modulate OKT8 expression. Prostaglandin E (PGE) was shown to hinder - 1 3 -the increased level of OKT8 expression usually observed in culture; aspirin on the other hand was shown to increase the spontaneous increase of OKT8+ cells in culture [Dore-Duffy et al., 1983]. These results are interesting in view of the fact that in vitro prostaglandin production is increased in MS [Dore-Duffy and Zurier, 1981; Merill et al., 1983]. Recently Rose et al. [1985] described the selective loss of a subset of T helper cells in the peripheral blood of patients with active MS. T cells identified by the monoclonal antibody 3AC5 (CD45R) were shown to be selectively reduced in patients with clinically active disease. Morimoto et al. [1987] have confirmed these results using a different monoclonal antibody (2H4) identifying the same antigen. T helper cells bearing this antigen induce suppressor cells [Morimoto et al., 1985]. Recently, this group [Morimoto et al., 1986] has shown that the antigen itself (identified by the antibody 2H4) may be involved in concanava l in -A (Con-A)- induced suppress ion . Con-A- induced suppression has consistently been shown to be reduced in MS (see below) and we wonder if the reduced expression of this subset of helper cells may not be related to reduced Con-A-induced suppression in active MS. Recent reports have indicated that cells expressing the CD45R Ag will upon activation switch irreversibly to the expression of the CDw29 Ag [Abkar et al . , 1988; Serra et al., 1988]. CDw29 positive cells have been shown to be T helper inducer cells in the in vitro PWM IgG secretion system [Morimoto, 1985]. We wonder if activation of T cells in MS might be responsible for the reduced number of 2H4 positive T helper cells observed. - 1 4 -The percentage of natural killer cells, which are large granular lymphocytes, has been reported as normal both in chronic progressive disease and during clinical attacks [Merrill et al., 1982b; Rice et al., 1983a; Oger et al., 1983], although this is not unanimous (see below). 1:3:ii Cerebrospinal Fluid; T lymphocytes predominate in the C S F of both MS patients and controls, the majority being T4+ cells [Brinkman et al., 1983; Hauser et al . , 1983b]. The percentage of cells bearing the T cell surface marker is increased during attacks [Allen et al., 1976; Naess, 1979] with the OKT4 cell subset preferentially increased and the OKT8+ cell subsets decreased [Oger et al., 1982b; Hauser et al., 1983c; Kolar et al., 1984]. B cells are present in decreased amounts relative to the percentage in blood (see section 'IgG Secretion'). This observation is interesting in light of the numerous B cells and plasma cells observed within the C N S parenchyma [Link et al., 1984] and would suggest either that B cells multiply in the C N S or are specifically retained inside the parenchyma. - 1 5 -1:4 Suppressor Cell Function Abnormalities: Not only are the T suppressor cells reduced in number during active clinical disease, their function is also altered. Concanavalin A (Con A) -induced suppressor cell activity has been the main assay used [Shou et al. , 1976]. In this assay, suppression is induced by stimulating peripheral blood mononuclear cel ls (PBMC) with a suboptimal amount of Con A, a potent T cell mitogen. After 3 days cells are washed and their DNA synthesis is blocked by mitomycin C. Irradiation should be avoided as it abrogates suppression [Siegal and Siegal, 1977]. Cells are then added to freshly isolated responder cells which are stimulated with Con A and suppression is measured as a ratio between 1) the amount of Con A-induced DNA synthesis in the cultures containing responder cells and Con A-stimulated suppressor cells versus 2) the amount of DNA synthesized in the cultures containing responder cells and unstimulated cells. Several groups have consistently demonstrated a reduction in suppression during active cl inical d isease [Arnason and Antel, 1978; Antel et al., 1978, 1979, 1986; Neighbor and Bloom, 1979; Sheremata et al., 1982; Haahr et al., 1983; Tjernlund et al., 1984; Gonzalez et al., 1979; Oger et al., 1985; O'Gorman et al . , 1987]. Additionally, Con A-induced suppression appears to fluctuate with changes in clinical disease activity falling prior to an attack and rising to levels above those seen in normal controls during the remitting phase of the disease [Antel et al., 1978, 1979]. Difficulties have arisen in determining the exact mechanism of Con A-induced suppression although a recent report suggested the - 1 6 -functional suppressor defect in MS patients is in the OKT8 cell subset [Antel et al., 1986]. Suppressor factors are involved and candidates include interferon gamma and alpha. Direct cytotoxicity through stimulation of cytotoxic T cells and NK cells could also be operative. In a recent longitudinal study of 7 multiple sclerosis patients (MS) with the relapsing-remitting form of the disease, patients were monitored monthly by cl inical examination, magnetic resonance imaging (MRI) and Con A suppressor cell function. Oger et al [1986] observed a correlation between MRI-defined attacks and reduction of suppression. Five clinical attacks have occurred; only 2 of them were preceded by a reduction in Con-A-suppression. However, out of 4 instances where suppressor cell function was temporarily reduced, 3 corresponded to the maximal size of new lesions on MRI [Oger et al., 1986]. No meaningful variation of suppressor/cytotoxic markers was being recognized [Kastrukoff et al., 1986]. The Con-A-induced suppressor cells and suppressor factors have also been tested for their ability to suppress the response of PBMC to other mitogens. When phytohemagglutinin or Staphylococcus aureus protein A were used to stimulate the target cel ls, no difference between MS patients and controls was observed [Gonzalez et al. , 1979]. Suppressor cells have also been induced by a variety of other techniques including myelin basic protein (MBP) [Wicher et al., 1979]. This group observed that MBP did not induce suppression in lymphocytes obtained from active MS patients, while lymphocytes obtained from stable MS patients did suppress. Other methods of measuring suppression, either induced or spontaneous, have been -1 7-devised [e.g. by the autologous mixed lymphocyte reaction; Hafler et al. , 1986]. Most reports indicate reduced suppression in progressive MS; this defect seems to be more elusive during attacks [Neighbor and Bloom, 1979; Wallen et al., 1981; Huddlestone and Oldstone, 1982]. Suppressor cells are also involved in regulating the level of IgG secretion and the next sect ions will review the abnormalit ies associated with this fuction in vivo and in vitro. 1:5 IgG Secretion: 1:5:i IgG Secretion in vivo; Analysis of the proteins in the cerebrospinal fluid (CSF) of MS patients has revealed quantitatively elevated and qualitatively altered IgG [for reviews, see Ivanainen, 1981; Oger et al., 1983; Walsh et al., 1983]. These two abnormalities aid in the diagnosis of MS and have been recently added to diagnostic criteria used for research purposes [Poser et al., 1983]. Elevated levels of IgG within the central nervous system (CNS) of MS patients was first reported by Kabat et al. [1942]. The presence of discrete bands of IgG observed after electrophoresis of C S F (which are not present in serum), the relative increase in IgG in C S F as compared with serum and the results of isotopic tracer studies of IgG in C S F and serum suggest that IgG is synthesized within the blood-brain barrier (BBB) and not simply the result of transudation. Intra-BBB IgG synthesis is present in over 90% of clinically definite MS patients [Tourtellotte, 1970; Walsh et al., 1983; and many -1 8-others]. The average MS patient synthesizes 20 mg of IgG within the BBB per day [Tourtellotte and Ma, 1978]. Tourtellotte et al. [1983] estimate that 1-3 X 10 9 plasma cells are required to synthesize 20-30 mg of immunoglobulin. Quantitative assessments of the number of lymphocytes observed in the C N S by Prineas and Wright [1978] and Adams [1977] support this estimate. That IgG synthesis within the C N S can be quantitated reliably is not universally accepted [Lefvert and Link, 1984; Hische and Van der Helm, 1987]. Different methods for measuring or detecting intra-BBB IgG synthesis exist. A recent study by Hische and Van der Helm [1987] suggests that the dimensionless IgG index provides better discriminating properties with respect to the diagnosis of MS. The IgG synthesis rate on the other hand provides a physical quantity of IgG produced within the BBB and may some day be applied to the quantitation of antibody directed at the putative MS antigen [Tourtellotte et al . , 1985]. Lefvert and Link [1985] suggest that the demonstration of ol igoclonal IgG bands is the most appropriate identifier of intrathecal IgG synthesis. Although most groups agree that there is intrathecal IgG synthesis in MS, the controversy surrounding the reliability of quantitating synthesis rates, the usefulness of a dimensionless IgG index and the reliance on the presence of oligoclonal bands rages on. Discrete bands of IgG observed after the electrophoresis of MS C S F were first reported by Lowenthal et al. [1960] and Delmotte [1971]. These bands now known to be IgG and referred to as oligoclonal bands are the result of intra-BBB IgG synthesis. Ebers et al. [1983] reported that the presence of p lasma cel ls within MS brains -1 9 -correlated with the detection of oligoclonal bands, suggesting that the IgG in the C S F is secreted by these cells. C S F oligoclonal IgG has been reported to be present in 85-95% of clinically definite MS patients [Johnson et al., 1977; Ebers and Paty, 1980; Hershy and Trotter, 1980]. Tourtellotte et al. [1983] reported that by increasing the sensit ivity of isoelectr ic focusing by immunofixation and silver staining, oligoclonal banding could be detected in greater than 99% of their clinically definite MS patients. Oligoclonal bands of IgG have been identified in the serum of some MS patients [Laurenzi, 1981; Staley et al., 1986]. Staley et al. [1986] observed that paired electrophoretograms of the C S F and serum revealed identical patterns in contrast to Tourtellotte's group (see below). Oligoclonal bands of IgG have also been observed by isoelectric focusing of the tears of MS patients [Coyle et al., 1987]. Although oligoclonal bands of IgG are of diagnostic importance, this phenomenon is not specific for MS and oligoclonal banding has been demonstrated in a variety of neurological conditions associated with chronic antigenic stimuli such as neurosyphil is, progressive rubella encephalitis, subacute sclerosing panencephalitis and chronic fungal meningitis [Johnson, 1980; Miller et a l . , 1983]. A small proportion of pathologically proven cases of MS does not show oligoclonal banding [Farrell et al., 1985]. A hypothesis accounting for the presence of oligoclonal bands in the C S F , brain and occasionally (27%) in serum of MS patients holds that these represent antibodies directed against the etiological agent. This has been shown to be the case in subacute sclerosing panencephalit is where as much as 80% of the total intrathecal^ - 2 0 -produced IgG and most of the oligoclonal bands react with the viral antigen, i.e. measles virus [Vandvik et al., 1973, 1976; Waldmann et al., 1974; Tourtellotte et al., 1981 ]. When IgG is eluted from brain samples at acid pH it is thought to yield IgG bound specifically through the Fab' fragment. Mattson et al. [1981] observed that the electrophoretic pattern of acid-eluted IgG varied from plaque to plaque in a single patient. Oligoclonal bands can also be generated by activation of lymphocytes in vitro: Oger et al. [1981] have generated oligoclonal bands in supernatants of lymphocytes isolated from the peripheral blood following activation by an aspecific mitogen, both MS and control cel ls could generate oligoclonal bands. The latter observation confirms that an aspeci f ic stimulus may elicit an oligoclonal pattern of secreted IgG. Defining the antigen specificity of the IgG in MS serum and C S F has been and continues to be a major goal in MS research [Catz and Warren, 1986]. Adams and Imagawa [1962] were the first to note that MS serum contained higher levels of antibodies to measles virus than control sera. This has been amply confirmed [Salmi et al., 1979, 1982; Detels et al., 1981; Albrecht et al., 1983] and extended to antibodies to other viruses as well [Norrby et al., 1974; Forghani et al . , 1978; Salmi et al., 1979]. Interestingly, in MS C S F , simultaneous increases in the titers of antibodies to more than one virus have been detected [Norrby et al., 1974; Arnadottir et al., 1979; Salmi et al., 1979]. IgG eluted from the plaques of an MS brain [Mattson et al., 1982] varies in specificity from plaque to plaque. These latter observations coupled with the detection of C S F antibodies both free and bound to protein constituents and cells of the CNS such as MBP [Panitch et al., 1980; -21 -Ruutianen et al., 1981; Wajgt and Gorny, 1983; Catz and Warren, 1986; Chou et al., 1983], myelin-associated glycoprotein [Wajgt and Gorney, 1983] and oligodendrocytes [Abramsky et al., 1977; Traugott et al., 1981; Pedersen et al., 1983; Gorney et al., 1983] and the ubiquitously increased levels of IgG in the C S F suggest to us that most of the IgG produced intrathecal^ is nonspecific. The increased immunoglobulin can also be interpreted as reflecting deregulation of B cell functions, i.e. 'nonsense antibodies'. Failure to detect cross-reactive idiotypes between the IgG obtained from different MS patients supports this theory [Ebers, 1982]. Many groups have provided evidence which does not support the 'nonsense antibodies' theory. Greater than 90% of the C S F IgG has no known antigen specificity [Walsh et al . , 1983]. This has led to arguments both for and against the 'nonsense antibody' theory. It has been suggested that factors other than Ag specificity could account for the different electrophoretic profiles seen in different areas of the same MS brain by one-d imensional electrophoresis [Walsh and Tourtellotte, 1986]. Using sensitive two-dimensional e lectrophoresis, the latter group establ ished that patterns generated by CSF- lgA, IgM and IgG were stable over several years. Additionally in contrast to Mattson et al. [1982] 2 dimentional e lectrophoresis revealed that the major spots (clones) were distributed uniformly throughout the brain and C S F of MS patients. However some clones were more prominent in certain areas. These results are consistent with both allotypic and idiotypic analyses [Salier et a l . , 1983; Ebers, 1982] demonstrating the relatively homogeneous distribution of specific IgG allotypes and idiotypes in different regions of MS brains. Walsh and Tourtellotte [1986] also observed that the patterns of light and heavy chains in brain and C S F differed from serum. This group suggests that the bands or spots present only in the C S F (oligoclonal IgG) represent the synthesis of IgG with specificity for a putative MS antigen related directly to the cause of MS. The search for the specificity of the majority of C N S IgG remains an area of active invest igat ion. In addition to the intrathecal production of whole IgG, free kappa and lambda light-chain monomers and dimers without free heavy chains were recently identified in the C S F of all MS patients studied (n = 10) and in none of the neurological disease controls (n = 14) [Rudick et al., 1985]. Other groups have also detected free light chains in the C S F of MS patients [Zetterwall and Link, 1979; Laurenzi et al . , 1980; Link and Laurenzi, 1979; De Carli et al., 1986]. The significance of the free light chains in the C S F of MS patients is not known. Rudick et al. [ 1985] have suggested that the synthesis of free light chains may be associated with: (a) defective B cells, because it has not been shown that the cells secreting light chains also secrete whole IgG, (b) 'pseudoneoplastic B cells' under autonomous control (free light chains are often detected in the blood of patients with monoclonal gammopathy and plasma cell dyscrasias [Dammaco and Waldenstrom, 1968] or (c) the free light chains which appear to be monoclonal or oligoclonal could represent the discrete population of restricted specificity important in the pathogenesis of MS. An elevated IgM index (intrathecal secretion of IgM) has also been reported in the C S F of MS patients [Forsberg et al., 1984; Sindic et al., 1982] although increased C S F IgA has only occasionally been observed . A comprehensive investigation of the number of immunoglobulin-secreting cells and the IgG secretion index in the C S F (IgG, IgM and IgA) by a research group in Sweden [Henriksson et al., 1985] noted that in MS there was a higher percentage of immunoglobul in-secreting cel ls (due predominantly to the high concentration of IgG-secreting cells) in the C S F than in the peripheral blood. Other studies of the number of B cells in the C S F and peripheral blood have observed a higher percentage in the latter [Kam-Hansen et al . , 1978; Brooks et al. , 1983]. These studies, however, were not detecting the immunoglobulin-secreting cells, i.e. the final stage of differentiation of B cells. The number of immunoglobulin-secreting cells (IgG, IgM and IgA) in the C S F of the MS patients was also elevated compared to healthy controls and neurological controls. Although the majority of the MS patients had IgA-secreting cells, only 1 (out of 37) had an elevated C S F IgA index. The majority of the patients had elevated C S F IgM indices. Again the specificity of this class of immunoglobulin remains to be determined. There was no association between the number of immunoglobulin-secreting cells or the C S F immunoglobulin indices in the C S F and the level of clinical activity in this group of MS patients [Henriksson et al., 1985]. - 2 4 -1:5:ii In vitro IgG Secretion in MS; P B M C cultured in vitro with pokeweed mitogen (PWM) are stimulated to divide and differentiate into immunoglobulin-secreting cells. This observation has led to the use of this assay as a model of the in vivo humoral immune response. PWM-induced IgG secretion has proved to be invaluable in determining the mechanisms of B cell activation and differentiation and also in unravelling the complex circuitry of lymphocyte subsets and molecules involved in regulating the antibody response. In active MS, the amount of IgG secreted by PWM-stimulated PBMC is increased over the level observed in control PBMC cultures in the majority of reports [Oger et al., 1982; Goust et al., 1982; Antel et al., 1984b]. Investigations aimed at resolving the immunopathogenetic mechanisms responsible for altered in vitro IgG secretion in MS have involved cell subset reconstitution and mixing experiments. Levitt et al . [1980] were the first to perform this sort of experiment in the study of MS. In allogeneic cultures, T cells isolated from MS patients with severe but stable MS induced 2- to 4-fold increases over normal T cells in the number of plasma cells generated from PWM-stimulated normal B cells. In contrast, B cells isolated from these MS patients differentiated poorly in response to PWM whether the T cells were from normal donors or MS patients. This group concluded that in MS patients, B cell differentiation was diminished and their T cells provided excess help in the differentiation of B lymphocytes. It was not determined if the excess help was the result of increased T helper cell activity or decreased T suppressor cell activity. Henriksson et al. [1986] studied PWM-induced immunoglobulin secretion in MS patients, healthy controls and acute aseptic meningoencephalit is patients. There were no differences in the amount of immunoglobulin secreted in the supernatants of unstimulated or PWM-stimulated PBMC cultures between the MS and the aseptic meningoencephalitis groups. As there were only 11 MS patients studied, classification of clinical disease in the MS group was not possible. We have shown that IgG secretion is increased in C P - M S [Oger et al., 1986a] but decreases during attacks [Oger et al . , 1986b], suggesting that careful clinical grouping is essent ia l . Henr iksson also studied P W M stimulation of C S F lymphocytes and found no response to PWM in the MS group, as opposed to the aseptic meningoencephalitis group, whose C S F lymphocytes secreted significant amounts of immunoglobulin into the supernatant. Henriksson et al. [1986] suggest that the lack of response in the MS C S F lymphocytes is due to the fact that they were activated in vivo. This hypothesis is further supported by their previous observation that the number of immunoglobulin-secreting cells is higher in the C S F than in the peripheral blood in MS (see earlier) [Henriksson et al., 1985]. Hauser et al . [1985] also observed that the amount of immunoglobulin secreted by peripheral blood lymphocytes (PBMC) isolated from MS patients was not higher than that of healthy controls or other neurological d isease controls. Interestingly, this group observed that unstimulated P B M C isolated from MS when cultured for 7 days secreted higher levels of IgG than the two control groups. Fifty eight percent of the active MS group (n= 50) had PBMC which secreted greater than 2,400 ng/ml in the absence of PWM stimulation compared to only 14% in the inactive MS group (n = 21). In a series of subset reconstitution experiments, this group observed that the function of the OKT8+ suppressor cells was not different from the controls and was, therefore, not responsible for the increased spontaneous immunoglobulin secretion [see further, Antel et a l . , 1984b]. The amount of IgG secreted spontaneously by 10 5 PBMC over a period of 7 days appears to be inordinately high in this report. Additionally, PWM stimulation did not augment IgG secretion above the level observed in unstimulated cultures. We wonder if perhaps their medium was somewhat stimulatory. It is interesting that both Henriksson et al . [1986] and Hauser et al. [ 1985] observed that the P B M C of MS patients 'tended to secrete more IgG in PWM-stimulated cultures than controls' although in neither case was this significant. Similarly, Kelley et al. [1981] found no differences in the amount of IgG secreted by PWM-stimulated PBMC isolated from MS patients and normal controls but their sample size was small. This group also measured suppression of PWM-induced IgG secretion in reconstituted autologous and allogeneic cultures of B and T lymphocytes isolated from MS patients and healthy controls. Autologous cocultures of B and T cel ls obtained from MS patients exhibited significantly lower suppression indices than similar cultures from healthy controls. It was observed that MS B cel ls were somewhat insensit ive to suppression by T suppressor cells isolated from healthy individuals, and that T cells isolated from active MS patients were no different from normal control T cells in their ability to suppress normal B cells. A series of allogeneic coculture experiments suggested that the decreased suppressor index in autologous cultures of MS PBMC was not due to a specific defect in either the T cell subset or the B cell subset (i.e. there is not simply a defect in the Ts cell subset alone). Goust et al. [1982] observed that P B M C isolated from patients with active MS secreted more IgG in response to PWM than the PBMC of healthy age-matched controls. Mixing of normal B cells with MS T cells isolated from active patients resulted in marked suppression of PWM-induced IgG secretion. The suppression was greater than that observed in allogeneic cell mixtures of normal B and T cells or when T cells from patients with stable MS were mixed with normal B cells. Oger et al. [1986b] recently measured PWM-induced IgG secretion serially over a period of approximately 6 months in a group of relapsing and remitting patients; this group also had an MRI examination of the head done serially. In 2 patients with large MRI lesions, PWM-induced IgG secretion fell at a time where MRI lesions were maximal [Oger et al., 1986b]. We wonder if this may not correlate with the study of Goust et al . [1982] although their 'active' MS group includes chronic progressive disease which we find have elevated PWM-induced IgG secretion [Oger et al., 1986a]. Goust et al. also measured sodiumperiodate-induced suppression of IgG secretion in whole PBMC cultures of MS patients and healthy controls. Sodium periodate treatment led to significant suppression of PWM-induced IgG secretion in normal individuals and stable MS but not in patients with active d isease. This observation of reduced suppression in active MS is in agreement with the results obtained in the Con-A-induced suppressor cell assay, as reviewed earlier. It is interesting that in allogeneic reconstitution experiments Levitt et al. [1980] found increased plasma cell differentiation in cultures of MS T cells with normal B cells while Goust et al. [1982] found decreased IgG secret ion in similar reconstitution exper iments. These - 2 8 -discrepancies could result from differences among the MS populations studied or a lack of synchronism between the generation of plaque-forming cells and IgG secretion [Ades et al., 1980]. Oger et al . [1982a, b, 1986a] and Antel et al. [1984b] have reported that the MS patient population contains a greater proportion of patients secreting a 'high' level of IgG compared with age-and sex-matched controls. The P B M C of normal adults secrete highly variable amounts of IgG in response to PWM [Keightly et al., 1976] although the amount of IgG secreted by any one individual's P B M C remains relatively constant over time [Antel et al., 1984b]. One group has reported that a low response appears to be governed by the functional state of the OKT8+ (CD8+ suppressor/cytotoxic) cells and not by their absolute number [Antel et al., 1984b]. The OKT8+ cells isolated from high responder MS patients or healthy controls suppressed PWM-induced IgG secretion less well than the OKT8+ cells from low responders. T helper cells (OKT4+) obtained from high-responder MS and high-responder control subjects did not differ in their ability to reconstitute a high response in mixing experiments using allogeneic E-cells (B cells and monocytes) pooled from several donors. In summary we conclude that the quantitative and qualitative immunoglobulin abnormalities reported above are one of the hallmarks of M S . The cause of these abnormalities both in vivo and in vitro remains an enigma. Persistent stimulation and or immune dysfunction have not been ruled out. An interesting hypothesis put forth by Roos [1985] suggests that intrinsic B cell abnormalities such as DNA translocations or abnormal gene rearrangements could lead to B cell prol i ferat ion, increased immunoglobul in secret ion and even immunodysregu la t ion . Recen t s tud ies have shown genet ic abnormalities in the P B M C of MS patients; increased sister-chromatid exchange [Karki et al., 1986; Seshadri et al., 1983] and an increased sensitivity to ionising radiation [Gipps and Kidson, 1981] have been reported. These abnormalities could lead to increased mutations in the B lymphocytes of MS patients. We think that the in vitro and in vivo IgG abnormalities reviewed here are potential clues to unravelling the pathogenesis of MS. 1:6 Stimulation Assays : 1:6:i Search for the Elusive MS-Specific Antigen; One of the ultimate goals in the investigation of cellular immunity in MS is to identify the target of the immune response in the C N S . Alternatively and equally rewarding would be the identification of a generalized immune dysfunction which could potentially lead to autoreactivity and to the various clinical manifestations of MS. The following two sections are intended as an outline of the various techniques employed to achieve these goals. Readers interested in a more detailed summary of individual investigations in this area of research may to the annotated bibliography published by Baumhefner and Tourtellotte [1986] and a recent review by Reder and Arnason [1985]. In vivo cell-mediated hypersensitivity has been measured by a variety of techniques. The most popular technique thought to correlate with in vivo ce l l -media ted immunity is the lymphoblast ic - 3 0 -transformation assay . A proliferative response to an antigen (measured by the incorporation of radiolabeled DNA precursors during replication) above background is thought to represent in vivo sensitization [Hirschhorn, 1968]. Similarly, lymphocytes exposed to an antigen to which they have been previously sensitized release lymphokines: factors known as macrophage migration inhibitory factor and leukocyte migration inhibitory factor [Rocklin, 1974]. The release of these factors in vitro is also thought to be indicative of in vivo sensitization [Rocklin et al., 1971]. Another more recently developed in vitro method used to measure cel l -mediated immunity is the antigen-active rosette-forming cel l (Ag -ARFC) assay [Felsberg and Edelman, 1977]. Lymphocytes exposed to an antigen to which they have been previously sensitized will upon interaction with S R B C form a greater number of active rosettes than lymphocytes which have not been previously exposed to the antigen. Sensitization in this assay has been shown to correlate with delayed-type hypersensitivity reactions [Felsberg and Edelman, 1977; Hashim et al., 1977]. Other assays have been employed to measure specif ic sensit ization. Levy et al. [1976] described a rosetting test where PBMC bind to measles-infected cells. The PBMC of MS patients showed increased adherence to virally infected cells when compared with control P B M C . Others, however, have failed to confirm this observation [Salmi and Fray 1977; Dore-Duffy et al . , 1979]. Another assay was developed which measured specif ic sensit izat ion, a leukocyte adherence inhibition assay [Angers et al., 1979]. If P B M C were exposed to an antigen to which they had been sensitized, their -31 -ability to adhere to glass was decreased. These results were not conf i rmed. The lymphocytes from peripheral blood and C S F of MS patients appear to be spontaneously activated (i.e. spontaneously dividing in the absence of antigen [Hughes et al., 1977; Lisak and Zweiman, 1977; Fraser et al . , 1979; Noronha et al . , 1980; Hauser et al . , 1983a; Brinkman et al., 1984; Hafler et al . , 1985]. Additional studies with monoclonal antibody indicate that early-activation antigens are present on a large proportion of T lymphocytes in the peripheral blood compartment [Golaz et al., 1983; Hafler et al., 1985] and C N S of MS patients [Hofman et al., 1986]. Spontaneous activation of lymphocytes has previously been shown to be increased in patients whose immune system had recently been specifically stimulated [Virolainen, 1971; Lalla et al., 1973; Cook and Dowling, 1980]. Such increased activation in the peripheral blood and C N S of MS patients could represent reactivity to the etiological agent; it is also possible that it is secondary to the leaking of some factor from damaged CNS tissue (e.g. myelin or neuroleukin). Other investigators have not observed increased spontaneous reactivity in the lymphocytes isolated from MS patients [Haahr et al., 1983; Lisak and Zweiman, 1977; Brinkman et al., 1982a, and others]. Studies of lymphoblastic transformation in MS are extremely numerous. Lymphoblastic transformation has been measured in response to nonspecific mitogens, viruses and neural antigens. The results have been variable. For example, measles-virus-induced blastogenesis has been reported to be equal in MS and control lymphocytes [Dau and Peterson, 1970; Knowles and Saunders, 1970; Cunningham-Rundles et al., 1977; Stewart et al., 1977; Symington and MacKay, 1978]. In other studies, the response of MS lymphocytes to measles virus was weaker than that of control lymphocytes [McFarland and McFarlin, 1979; lllonen et al., 1981; Sagar et al., 1981] and further studies have shown that MS lymphocytes responded more strongly than control lymphocytes [Walker and Cook, 1979; Walker et al., 1982]. Reports involving blast transformation to neural antigens also reveal many inconsistencies, however, the majority of reports suggest that MS lymphocytes react to MBP [Colby et al. , 1977; Gosseye-Lissoir et al., 1977; Hughes et al., 1977; Lisak and Zweiman, 1977; Lisak et al., 1981; Wicher et al., 1981; Wicher and Holub, 1982; Frick, 1982] as well as cerebrosides and gangliosides [Offner and Konat, 1980; Frick, 1982]. Although lymphocytes from MS patients are more reactive to these neural antigens than those of healthy control individuals, the magnitude of this response is very limited (stimulation indexes < 3). This is in contrast with a reliably brisker response to M B P of pat ients with acute demyel inat ing encephalomyelitis [Lisak and Zweiman, 1977]. Several authors found a stronger reaction to MBP during the acute exacerbations of the disease than when the disease was stable [Bartfeld et al., 1972; Myers, 1972; Sheremata et al., 1976; Colby et al., 1977; Wicher et al., 1981]. However this was not always the case [Behan et al., 1972; Casparary and Field, 1974]. Groups measuring the amount of MBP released into the C S F [Lisak et al., 1981; Jacques et al., 1982; Massaro et al., 1985; Matias-Guiu et al.,1986; Thompson et al., 1987] found the levels correlated with the severity of clinical signs. MBP cross-reactive material in the C S F of MS patients is detectable for 2-3 weeks after an exacerbation. This material is also present in the C S F of patients with cerebrovascular d isease, patients with inflammatory diseases of the CNS and generally in patients with acute destruction of C N S myelin [Cohen et al., 1976; Whitaker et al., 1980; Lisak et al., 1981]. The presence of such material in C S F does not have diagnostic specificity but can be used as a means to determine recent myelin injury. Lisak et al. [1981] reported elevated basic protein levels and enhanced basic protein reactivity in M S , acute demyelinating encephalomyel i t is and other neurological d iseases ; interestingly, there was no correlation between these two parameters in any of the above diseases. This would suggest that C S F lymphocytes are not simply sensitized by the MBP released into the C S F . An analogous situation occurs in guinea pigs with E A E , where extensive demyelination is uncommon, however, there is a strong in vitro MBP-induced proliferative response [Waksman and Adams, 1962; Lisak and Zweiman, 1974]. The Ag-active rosette-forming cell assay was first performed by Felsberg and Edelman [1977]. It is believed that this system represents in vivo delayed hypersensitivity [Hashim et a l . , 1977; Felsberg and Edelman, 1977]. In 1977, Hashim et al. reported that lymphocytes from MS patients exposed to MBP in vitro formed a greater number of active rosettes than did the lymphocytes of healthy controls. They found, however, that patients with C N S tumors and patients with cerebrovascular disease also had elevated levels of MBP-active rosette-forming cells. Offner et al. [1980] measured the number of bovine-cerebroside and bovine-ganglioside-active rosette-forming cells. They observed an increased frequency of these neural-antigen-active-rosette-forming cells in MS patients when compared to other neurological disease controls and healthy individuals but concluded that the sensitivity of MS patients' lymphocytes to the neural glycolipids was not specific and was simply a response of the immune system to antigens liberated from demyelinating lesions [Offner et al., 1980]. Traugott et al [1981] employed this assay to investigate the reactivity of MS lymphocytes to bovine oligodendrocyte antigens and M B P . They similarly concluded that oligodendrocytes and M B P -reactive P B M C occur more frequently in MS than other neurological d iseases. However, these neural-antigen-reactive clones are not specific for MS and their occurrence does not correlate with disease activity. In a recent study, Hashim and Brewen [1985] followed a group of MS patients serially using the M B P - A R F C assay. This group observed that the level of sensitivity to MBP was influenced by c l in ical status, degree of neurological deficit and particular treatment course. They suggested that increased numbers of MBP-A R F C correlated with the degree of C N S destruction. Production of the leukocyte migration inhibition factor and macrophage migration inhibition factors (MIF) in response to viral and brain antigens as a measure of cell-mediated immunity in MS have also resulted in conflicting reports. Production of macrophage MIF in response to MBP has been reported in MS [Bartfeld et al., 1970; Rocklin et al., 1971; Sheremata et al., 1976]. Behan et al [1972], however, failed to detect significant macrophage MIF production in response to MBP. It appears that in MS patients the production of macrophage MIF correlates with the clinical severity of the disease [Sheremata et al., 1976; Pekarek et al., 1977]. The production of leukocyte MIF, a factor different from macrophage MIF [Rocklin, 1974], has been detected in response to MBP in patients with MS and the production of this factor has been directly related to the time course of MS. Strandgaard and Jorgensen [1972] failed to detect sensitivity to MBP in this assay. Production of these factors by lymphocytes of MS patients in response to viral antigen has also been investigated. Symington and MacKay [1978] and Walker and Cook [1979] observed that the level of macrophage MIF produced in response to measles virus correlated with the activity of the disease. Detels et al. [1981] later confirmed this observation. Walker and Cook [1979] noted that macrophage MIF produced in response to PWM, herpes and parainfluenza virus was reduced in MS patients. Other groups have confirmed these findings [Sever et al., 1976; Kinnman et al., 1978; Visscher et al., 1979]. This decreased response was also observed in other neurological diseases, suggesting that the altered immune response is secondary to C N S lesions rather than specific for the disease [Walker and Cook, 1979]. Our conc lus ions would be that all the myel in- or oligodendrocyte-specific antigens which have been tested to date have revealed a certain degree of sensitization of MS patients' lymphocytes but this has also been true for neurological disease controls. None has been shown to be specific for MS. In 1968, Knowles et al. [1968] observed that MS sera caused a significant decrease in the level of blastogenic response of normal lymphocytes, whereas normal sera had no effect. This 'lymphotoxic' factor increased with the severity of the illness [Van den Noort and - 3 6 -Stjernholm, 1971] and was reduced following steroid therapy. This may be secondary to the elevation of P G E in the serum of MS patients as reported by Dore-Duffy and Zurier [1981]. P G E is secreted by macrophages and inhibits blastogenesis. Along the same line, Ziola and Hader [1985] found that the level of thymidine incorporation (a measure of DNA synthesis) induced by viruses in unfractionated PBMC from MS patients was not different from that induced in control PBMC. In MS P B M C , removal of the glass adhering cells resulted in increased thymidine incorporation in response to viral antigens. This was not seen following the same procedure in the P B M C of healthy controls. This group suggests that DNA synthesis in PBMC should be studied after removal of this glass-adhering subset. 1:7 Cell Cytotoxicity Assays: The effector branch of the immune system includes B cells but also cytotoxic T cells, NK cells and K cells (the latter mediate the antibody-dependent cell cytotoxicity, A D C C ) . Their activity is also regulated by T cells and lymphokines. Cytotoxicity assays have been developed to measure the specific activity of NK and K effector cells. MS patients have depressed numbers and activity of NK cells [Benzcur et al., 1980; Hauser et al., 1981; Merrill et al., 1982a, b]. Oger et al. [1986a] have confirmed a reduced NK cell function in the peripheral blood of a group of chronic progressive MS patients and have shown that in a limited number of RR patients NK cell function was reduced during the development of large new MRI-recognized lesions [Kastrukoff et al., 1986]. In vitro production of interferon is also reduced [Neighbour and Bloom, 1979; Neighbour et al., 1981; Benzcur et al . , 1980; Salonen et al . , 1982; Vervliet et al . , 1983]. Vervliet et al. [1985] also studied the production of interferon by the leukocytes isolated from patients suffering from other neurological diseases and neurological injuries. They concluded that the defective interferon response of the PBL cultures was not a specific defect of MS patients. Tovell et al. [1983] also failed to observe significant differences between MS patients and other neurological disease controls with respect to the production of interferon in culture in response to various viruses. In the C S F of MS patients virtually no NK cells are found [Merrill et al., 1982a, b]. K cell activity, measured as A D C C has been found to be elevated in both peripheral blood and cerebrospinal fluid compartments of MS patients as compared to control subjects [Frick and Stickl, 1980; Mar, 1980; Merrill et al . , 1982b, c]. NK cell activity measured in MS patients remains controversial as other reports have concluded that both cytotoxic activity and interferon production are normal [Santoli et al., 1981; Haahr et al., 1983; Rauch et al., 1985]. Interferon is known to boost both NK and A D C C activity [Herberman et al., 1981; Hirsch and Johnson, 1984], and it has been argued that the defect in MS NK cell number and function is related to a defect in interferon production. Merrill et a l . [1984] recently reported that MS patients have fewer interferon-producing cells in their blood than controls with other neurological disease or normal controls. This group has also suggested that the decreased interferon production may be the indirect result of both increased production of P G E , an abnormality already explored by Dore-Duffy and Zurier [1981], - 3 8 -and an increased sensitivity of NK cells from MS patients to the inhibitory effects of P G E [Merrill et al., 1983]. Measles-virus-induced suppression is reduced in MS [Utermohlen and Zabriskie, 1973; Utermohlen et a l . , 1978]. This virus-induced suppression can be abrogated by anti-interferon serum, suggesting that the suppression is indeed mediated by interferon [Levy et al., 1976]. It is possible that the reduced ability of MS patients to produce interferon in response to virus chal lenge contributes to the persistence of a virus in the C N S although attempts to isolate any specif ic virus from MS brains have been repeatedly negative. Additionally impaired interferon production may lead to altered NK funct ion. This abnormality of the interferon system has been the basis for therapeutical trials in MS. The clinical results of such a trial, underway in our institution, will be published soon [Kastrukoff et al., submitted] but preliminary results of studies of lymphocyte functions have been informative. Following subcutaneous injection of interferon (Wellferon®), we have noticed reduced IgG secretion following PWM stimulation lasting the duration of the treatment period [O'Gorman et al., 1987]. A temporary boost of the depressed NK cell function was found both in interferon- and placebo-treated patients [L. Kastrukoff, personal commun.]. This latter finding is in keeping with the experience of the Scripps Clinic [Rice et al., 1984b]. No effect of interferon on the decreased Con A suppressor cell function was observed [O'Gorman et al., 1987]. A recent clinical trial of gamma interferon in the treatment of MS was interrupted by the overseeing committee when most of the patients in the interferon arm of the trial developed attacks. This occurred despite complete correction of the NK cell function defect [Panitch et al., 1987]. This unfortunate series of events, nevertheless confirms at least partially that immune mechanisms are invoved in the determination of attacks in M S . Gamma interferon activates astrocytes and renders them la positive. We wonder if these glial cells may then act as antigen presenting cells and promote an immune reaction within the C N S . The first cellular cytotoxic immune reaction against a neural antigen was documented in 1964 by Berg and Kallen. They observed that in 17 of 40 cases the PBMC isolated from MS patients reacted against rat neonatal glial cells. Most of the patients not showing a positive response were in an inactive phase of the disease. Halpern et al . [1969] later confirmed these results using embryonic cultures from rat brain tissue. Eight of 9 patients in an active phase of the disease had positive results, whereas patients in remission were negative. The cytotoxic effects appeared to be specific for brain tissue as fibroblasts were unaffected. Lumsden [1971] demonstrated myel in-sensi t ized lymphocytes which caused demyelination and destruction of oligodendrocytes in fully myelinated tissue. The nerve cells and astrocytes were not affected. Hauw et al. [1975] reported similarly that the lymphocytes from 14 acute MS patients reacted against embryonic human brain tissue. These results should, however, be interpreted carefully as Kim et al. [1985] have documented the presence of la antigens on the surface of glial cells in culture. More recently, Frick [1982] measured the cytotoxic activity of MS patients' lymphocytes against autologous target cells coated with - 4 0 -bovine M B P , purified bovine cerebrogangliosides, and a fragment of human basic protein. In patients with stable disease the level of cytotoxicity was greater than that observed in controls but similar to that of patients with other organic neurologic d iseases . The cytotoxicity was highest during active d isease both in chronic progressive and relapsing forms. Cytotoxicity was highest using human myelin protein peptide and Frick suggested that a cytotoxic response against this peptide fragment of 20% or greater was specific for MS since cytotoxicity of this degree was not found in healthy persons or patients with other neural diseases. It is interesting to note that there was an inverse relationship between the specific lymphocyte stimulation and the degree of specific cytotoxicity in the PBMC of these MS patients. These results would suggest that the cells exhibiting higher cytotoxicity were already activated. These studies, however, should be taken with some reservation as the techniques used were mostly qualitative. Recent investigations have involved the cloning of T-cells isolated from the peripheral blood of MS patients. Results from a group at the NIH [Goodman et al., 1986; Jacobson et al., 1985] claim to have demonstrated the only antigen specific abnormality in MS. The majority of the PBMC from their group of MS patients either failed to generate clones of HLA-restricted measles-virus-specif ic CTL cells or had significantly reduced CTL responses compared to either normal individuals or other neurological disease controls. The significance of this finding is not known. Either MS patients have a reduced ability to generate measles-virus-specific CTL or these virus-specific CTL are sequestered in the C N S . Hafler et al. [1986] have generated IL-2--41 -dependent T cell clones directly from the plaques of MS brains. None of the clones proliferated in response to MBP or proteolipid protein. However a common rearrangement of the T cell receptor 8-chain suggests that there may be an expansion of a common T cell clonotype in MS. 1:8 MS: Diagnostic, Evolution: A high degree of discrepancy can be found in reviewing the literature on immune abnormalities in MS. Indeed this is attributable to the variability of the assay techniques used but can also come from difficulty in diagnosis and staging of patients reported in individual studies. MS is an extremely variable disease both in terms of presentation and evolution. This renders the diagnosis difficult. In the present state of knowledge a definite diagnosis is only acquired following pathological examination. This occurs seldom by brain biopsy and most often the diagnosis is proved only following autopsy. Cl in ica l diagnost ic c lassi f icat ions have thus been establ ished. Originally this was done an a purely clinical basis [Schumacher et al., 1965; McAlpine et al., 1972; Rose et al., 1976; McDonald and Halliday, 1977]. Most of these classif ications established three degrees of certainty in the clinical diagnosis: clinically definite, probable and possible. These classifications did not take into consideration the results of C S F findings (oligoclonal bands, intra-BBB IgG synthesis) nor the results of evoked-potential studies. Recently it has been proposed that this could lead to a better definition of the disease for research purposes and the terms 'laboratory-supported MS' has been coined [Poser et al., 1983]. Imprecisions in the diagnostic criteria used in individual studies are such that this may have introduced a large degree of heterogeneity in the groups of patients studied and discrepancies in some studies reported in this review. The staging of MS patients into homogeneous groups also presents some difficulties. MS presents itself either as a relapsing-remitting disease or as a progressive disease. In the relapsing-remitting form of the disease, attacks are followed by remissions where no clinical evolution is apparent. We have, however, presented evidence using MRI, that new lesions may appear even though the patient seems to be clinically stable or improving [Li et al., 1984]. Thus, the disease can in fact be active (as judged by MRI) at a time when patients are clinically stable. Conversely, not all clinical attacks have been recognized by MRI associated changes. This may explain some of the discrepancies seen when results in patients with stable disease are opposed to results obtained during attacks. An unknown percentage of patients who are reported as clinically stable may in fact be active when more objective methods of assessing disease activity are used. 'Progressive disease' clinically describes patients who have increasing disability without frank attacks. There is evidence that this group also covers different clinical situations. Some patients with a clinically progressive evolution have been through a relapsing-remitting phase and this may represent the effect of aging on the immune process or alternatively represent a biological process different from progressive demyelination (absence of remyelination? astrocytosis?). This group with progressive disease developing after a relapsing-remitting course can be opposed to other progressive MS patients who have a progressive evolution from the beginning of the disease. In this last group, the disease is also different from the relapsing-remitting group by starting at a later age, affecting the spinal cord more often than the brain, leading more rapidly to invalidation and showing different HLA phenotypes [Madigand et al., 1982]. The term 'acute' MS can be found in the literature to describe attacks. However, when it is not used to clinically describe patients with rapidly fatal disease, (malignant MS of McAlpine et al. [1972]), it is a misnomer. 1:9 Summary: Ample evidence points towards the presence of abnormalities in the regulation of the immune function in MS patients. Suppression induced by mitogens or autologous mixed lymphocyte response as well as changes in in vitro IgG secretion and NK cell function are the cornerstone of the observed abnormalities. These, however, can only be recognized in groups of patients homogeneous for clinical and MRI parameters. It has not yet been possible to determine if these abnormal findings are primary or secondary events. It is also possible that they represent genetically inherited traits which predispose - 4 4 -certain individuals to generate an autoimmune process following apparently trivial insult to the C N S . 1:10 References: Abkar, AN. , Terry, L , Timms, A., Beverley, P C L , and Janossy, G. Loss of CD45R and gain of UCHL1 reactivity is a feature of primed T cells. J . Immunol. 140; 2171 (1988). Abramsky, O.; Lisak, R.P.; Silberberg, D.H.; Pleasure, D.E.: Antibodies to oligodendroglia in patients with multiple sclerosis. New Engl. J . Med. 297: 1207 (1977). Adams, C.W.M. : Pathology of multiple sclerosis: progression of the lesion. Br. med. Bull. 33: 15-20 (1977). Adams, S . M . ; Imagawa, D.T.: Measles virus antibodies in multiple sclerosis. Proc. Soc. exp. Biol. Med. 111: 562 (1962). Ades, E.W.; Hinson, A.; Munoz, J . : Polyclonal activation of B-cells: lack of correlation between extracellular immunoglobulin and plasma cell differen- tiation. Fed. Proc. 39. 3 (1980) Albala, M.M.; Davignon, D.; Fas, L.D.; Clark, D.D.: Normal T-cell subsets and lymphocyte activity in multiple sclerosis. Cl in. exp. Immunol. 61: 542- 547 (1985). Albrecht, P.; Tourtellotte, W.W.; Hicks, J.T.; Sato, H.; Boone, E .J . ; Potvin, A .R . : Intra-blood-brain barrier measles virus antibody synthesis in multiple sclerosis patients. Neurology 33: 45 (1983). Al len, I.V.: The pathology of multiple sclerosis: fact, fiction and hypothesis. Neuropath. Appl. Neurobiol. 7: 169 (1981). Allen, J . G . ; Sheremata, W.; Cosgrove, J .B.R. ; Osterland, K.; Shea, M.: Cerebral spinal fluid T and B lymphocyte kinetics related to exacerbations of multiple sclerosis. Neurology 26: 579 (1976). Angers, J .W. ; Korik, L ; Franklin, L.S. : The leukocyte adherence inhibition assay as a diagnostic test for multiple sclerosis: study of a substance found in MS blood. J . Lab. Clin. Med. 93: 528 (1979). Antel, J . P . ; Arnason, B.G.W.; Medof, M.E.: Suppressor cell function in multiple sclerosis: correlation with clinical disease activity. Ann. Neurol. 5: 338 (1979). Antel, J .P . ; Bania, M.B.; Reder, A.; Cashman, N.: Activated suppressor cell dysfunction in progressive multiple sclerosis. J . Immunol. 137: 137 (1986). Antel, J . ; Oger, J . J .F . ; Jackevicius, S. ; Kuo, H.H.; Arnason, B.G.W.: Modulation of T-lymphocyte differentiation antigens: potential relevance for multiple sclerosis. Proc. Natl. Acad. Sc i . USA 79: 3330 (1982). Antel, J .P . ; Peeples, D.M.; Reader, A T . ; Arnason, B.G.W.: Analysis of T regulator cell surface markers and functional properties in multiple sclerosis. J . Neuroimmunol. 6: 93 (1984). Antel, J . P . ; Rosenkoetter, M.; Reder, A. ; Oger, J . ; Arnason, B.G.W.: Multiple sclerosis: relation of in vitro IgG secretion to suppressor cell number and function. Neurology 34: 1155 (1984b). Antel, J .P . ; Weinrich, M.; Arnason, B.G.W.: Mitogen responsiveness and suppressor cell function in multiple sclerosis: influence of age and disease activity. Neurology 28: 999 (1978). Arnadottir, T.; Reunanen, M.; Neurman, O.: Measles and rubella virus antibodies in patients with multiple sclerosis: a longitudinal study of serum and C S F specimens by radioimmunoassay. Archs Neurol. 36: 261 (1979). Arnason, B .G.W. ; Antel, J . : Suppressor cell function in multiple sclerosis. Ann. Immunol. 129C: 159 (1978). Bach, M.A.; Martin, C ; Cesaro, P.; Eizenbaum, J .F. ; Degas, J.D.: T-cell subse ts in mult iple sc le ros i s : a longi tudinal study of exacerbating-remitting cases. J . Neuroimmunol. 7: 331 (1985). - 4 8 -Bach, M.A.; Phan-Dinh-Tuy, F.; Tournier, E.; Chatenoud, L.; Bach, J .F. : Deficit of suppressor T-cells in active multiple sclerosis. Lancet ii: 1221 (1980). Bartfeld, H.; Atoynatan, T.: In vitro (delayed) hypersensitivity in MS to central nervous system antigens. Int. Archs Allergy appl. Immunol. 39: 361 (1970). Bartfeld, H.; Atoynatan, T.; Donnenfeld, H.: In vitro cellular immunity to central nervous system antigens in multiple sc lerosis; in Wol fgram, E l l i son , S tevens , Andrews, Mult iple sc le ros is , (Academic Press, London 1972). Baumhefner, R.W.; Tourtellotte, W.W.: Cellular immunology in multiple sclerosis. A review through 1984; in Cruse, Lewis, Concepts Immunopathol., vol. 2, p. 151 (Karger, Basel 1985). Baumhefner, R.W.; Tourtellotte, W.W.: Peripheral, cerebrospinal fluid and C N S immune cell subsets and functional alterations in multiple sclerosis; in Hommes, Mertin, Tourtellotte, Immunotherapies in MS, p. XXXVI (Stuart Phillips Publications, England 1986). Behan, P.O.; Behan, W.M.H.; Feldman, R.G.; Kies, M.W.: Cell-mediated hypersensitivity to neural antigens. Archs Neurol. 27: 145 (1972). Benzcur G; Petranyi G.Y.; Palffy G.Y.; Varga, M.; Talas, M.; Kotsky, B.; Foldes, I.; Hollan, S.V. : Dysfunction of natural killer cells in multiple sc leros is : A possible pathogenic factor. C l in . exp. Immunol. 39: 657 (1980). Berg, O.; Kallen, B.: Effect of mononuclear blood cells from multiple sclerosis patients on neuroglia in tissue culture. J . Neuropath. Exp. Neurol. 23: 550 (1964). B o o s s , J . ; Es i r i , M .M . ; Tourtel lot te, W.W. ; M a s o n , D.Y.: Immunohistological analysis of T-lymphocyte subsets in the central nervous system in chronic progressive multiple sclerosis. J . neurol. Sci . 62: 219 (1983). Brinkman, C .J .J . ; Nillesen, W.M.; Hommes, O.R.: T- cell subpopulations in blood and cerebrospinal fluid of multiple sclerosis patients. Effect of cyclophosphamide. Clin. Immunol. Immunopath. 29: 341 (1983). Br inkman, C . J . J . ; N i l lesen, W . M . ; Hommes, O .R . : Lymphocyte subpopulation in multiple sclerosis: spontaneous and mitogen-induced activity. Clin. Immunol. Immunopath. 31: 364 (1984). Brinkman, C.J .J . ; Nillesen, W.M.; Hommes, O.R.; Larmers, K.J.B.; de Pauw, B.E.: Spontaneous and mitogen-induced activity of lymphocytes of different density in multiple sclerosis. Eur. Neurol. 21: 366 (1982a). - 5 0 -Brinkman, C . J . J . ; Ter Laak, H.J . ; Hommes, O.R. : T-lymphocyte subpopulations in multiple sclerosis lesions (Letter). New Engl. J . Med. 307: 1644 (1982b). Brinkman, C .J .J . ; Nillesen, W.M.; Hommes, O.R.: T- cell subpopulations in blood and cerebrospinal fluid of multiple sclerosis patients. Effect of cyclophosphamide. Clin. Immunol. Immunopath. 29: 341 (1983). Brooks, B.R.; Hirsch, R.L.; Cayle, P.: Cellular and humoral immune responses in human cerebrospinal fluid; in Wood, Neurology of cerebrospinal fluid, p. 263 (Plenum Press, New York 1983). Casparary, E.A.; Field, E .J . : Lymphocyte sensitization to basic protein of brain in multiple sclerosis and other neurological diseases. J . Neurol. Neurosurg. Psychiat. 37: 70 I (1974). Catz, I.; Warren K.: Intrathecal synthesis of auto-antibodies to myelin basic protein in multiple sclerosis. Can. J . neurol. Sc i . 13.- 21 (1986). Charcot, J .M. : Gaz. Hop. Paris. 41: 554 (1868). Chou, C . H . J . ; Tourtellotte, W.W.; Kibler, R.F. : Failure to detect antibodies to myelin basic protein or peptide fragments of myelin basic protein in C S F of patients with MS. Neurology 33: 24 (1983). -51 -Cohen, S.R.; Herndon, R.M.; McKhann, G.M.: Radioimmunoassay for myelin bas ic protein in cerebrospinal f luid. An index of active demyelination. New Engl. J . Med. 295: 1455 (1976). Colby, S . P . ; Sheremata, W.; Ba in , B.; Eylar, E .H . : Cel lu lar hypersensitivity in attacks of multiple sclerosis. Neurology 27: 132 (1977). Compston, A. : Lymphocyte subpopulations in patients with multiple sclerosis. J . Neurol. Neurosurg. Psychiat. 46: 105 (1983). Cook, S.D.; Dowling, P.C.: Multiple sclerosis and viruses and overview. Neurology 30: 80 (1980). Coyle, P.K.; Sibony, P.A.; Johnson, C : Oligoclonal IgG in tears . Neurology 37; 853 (1987). Craig, J .C . ; Hawkins, S.A.; Swallow, M.W.; Lyttle, J.A.; Patterson, V.H. ; Merrett, J .D. ; Haire, M.: Subsets of T lymphocytes in relation to T lymphocyte function in multiple sclerosis. Clin. Exp. Immunol. 61: 548 (1985). Cunningham-Rundles, S.; Dupont, B.; Posner, J .B. ; Hansem, J.A.; Good, R.A.: In vitro lymphocyte transformation of MS patients to paramyxovirus antigens. Acta Neurol. Scand. 55: suppl. 63, p. 145 (1977). Dammaco, F.; Waldenstrom, J . : Serum and urine light chain levels in benign monoclonal gammopath ies, multiple myeloma and Waldenstrom's macroglobulinemia. Clin. exp. Immunol. 3: 911 (1968). Dau, P .C . ; Peterson, R.D.A.: Transformation of lymphocytes from patients with multiple sclerosis :Use of an encephalitogen of human origin with a report of a trial of immunosuppressive therapy in multiple sclerosis. Archs Neurol. 23: 32 (1970). De Carli, C ; Menegus, M.; Rudick, R.: Differences in the intrathecal humoral immune response in patients with multiple sclerosis and CNS infections (Abstract). Neurology 36: suppl. 1, p. 322 (1986). Delmotte, P.: Gel isoelectric focusing of C S F proteins: a potential diagnostic test. Z. klin. Chem. Biochem. 9: 334 (1971). Detels, R.; Myers, L.M.; Ellison, G. ; Visscher, B.R.; Malmgren, R.M.; Madden, D.L.; Sever, J.L. . : Changes in immune response during relapse in MS patients. Neurology 31: 492 (1981). Dore-Duffy, P.; Kuv, H.-H.; Dowling, M.M.: Role of prostaglandin E in modulation of T-cell differentiation antigens. Implications for multiple sclerosis. Neurology, Minneap. 33: suppl. 2, p. 142 (1983). Dore-Duffy, P.; Zurier, R.B.: E-rosette formation in normals and patients with multiple sc lerosis: Effect of prostaglandin and aspirin. Clin. Immunol. Immunopath. 13: 261 (1979). Dore-Duffy, P.; Zurier, R.B. : Lymphocyte adherence in multiple sclerosis: Role of monocytes and increased sensitivity of MS lymphocytes to prostaglandin E. Cl in. Immunol. Immunopath. 19: 303 (1981). Dore-Duffy, P.; Zurier, R.B.; Donaldson, J . ; Nystrom, S .S . ; Viola, M.V.; Rothman, B.; Thompson, H.G.: Lymphocyte adherence in multiple sclerosis. Neurology 29: 232 (1979). Ebers, G .C . : A study of C S F idiotypes in multiple sclerosis. Scand. J . Immunol. 16: I 51 (1982). Ebers, G.C. ; Farell, M.; Kaufmann, J .C. : Oligoclonal bands in MS: clinical pathological correlation. Neurology 33: suppl. 2, p. 141 (1983). Ebers, G.C. ; Paty, D.W.: C S F electrophoresis in one thousand patients. Can. J . neurol. Sci. 7: 275 (1980). Farrell, M.A.; Kaufmann. J .C .E . ; Noseworthy, J .H. : Armstrong, H.A.; Ebers. G . C . : Oligoclonal bands in MS clinical pathologic correlation. Neurology 35: 212 (1985). Felsberg, P .J . ; Edelman, R.: The active E-rosette test :A sensitive in vitro correlate for human delayed-type hypersensit ivi ty. J . Immunol. 118: 62 (1977). Fontana, A.; Fierz, W.; Wekerle, H.: Astrocytes present myelin basic protein to encephalitogenic T cell lines. Nature 307:273 (1984). Forghani, B.; Cremer, N.E. ; Johnson, K .P. : Viral antibodies in cerebrospinal fluid of multiple sclerosis and control patients. Compar i son between rad io immunoassay and convent ional techniques. J . Clin. Microbiol. 7: 63 (1978). Forsberg, P.; Henriksson, A.; Link, H.; Ohman, S.: Reference values for CSF-IgM, CSF-lgM/S-IgM ratio and IgM index, and its application to multiple sclerosis and aseptic meningoencephalitis. Scand. J . Lab. Invest. 44: 7 (1984). Fraser, K.B.; Millar, J.D.H.; Haire, M.; McCrae, J . : Increased tendency to spontaneous in vitro lymphocyte transformation in cl inically active multiple sclerosis. Lancet ii: 715 (1979). Frick, E.: Cell-mediated cytotoxicity by peripheral blood lymphocytes against basic protein of myelin, encephal i togenic peptide, cerebrosides and gangliosides in multiple sclerosis. J . neurol. Sci . 57: 55 (1982). Frick, E.; St ickl , H.: Antibody-dependent lymphocyte cytotoxicity against basic protein of myelin in multiple sclerosis. J . neurol. Sc i . 46: 187 (1980). Gipps, E.; K idson, C : Ionizing radiation sensitivity in multiple sclerosis. Lancet ii: 947 (1981). Golaz, J . ; Steck, A.; Moretta, L.: Activated T lymphocytes in patients with multiple sclerosis. Neurology 33: 1371 (1983). Gonzalez, R.L.; Dau, P.C. ; Spitler, L.E.: Altered regulation of mitogen responsiveness by suppressor cells in multiple sclerosis. Clin. exp. Immunol. 36: 78 (1979). Goodman, A.; Jacobson, S.; Flerlage, M.; McFarland, H.F.: Virus specific cytotoxic T lymphocytes in multiple sclerosis (Abstract). Ann. Neurol. 20.- 161 (1986). Gorny, M.K.; Wroblewska, Z. ; Pleasure, D.; Miller, S.L. ; Waigt, A. ; Koprowski, H.: C S F antibodies to myelin basic protein and ol igodendrocytes in multiple sclerosis and other neurological diseases. Acta neurol. Scand. 67: 338 (1983). Gosseye-Lissoir, F.; Delmotte, P.; Carton, H.: Biochemical findings in multiple sc le ros is . V. Transformation of lymphocytes from patients with multiple sclerosis by human basic protein. J . Neurol. 216: 197 (1977). Goust, J .M. ; Chenais, F.; Carries, J .E. ; Hames, C.G.; Fudenberg, H.H.; Hogan, E.L.: Abnormal T-cell subpopulations and circulating immune complexes in the Guillain-Barre syndrome and multiple sclerosis. Neurology 28: 421 (1978). Goust, J .M . ; Hoffman, P.M.; Pryjma, J . ; Hogan, E.L.; Fudenberg, H.H.: Defective immunoregulation in multiple sclerosis. Ann. Neurol. 8: 526 (1980). Goust, J . M . ; Hogan, E.L.; Arnaud, P.: Abnormal regulation of IgG production in multiple sclerosis. Neurology 32: 228 (1982). Haahr, S. ; Moller-Larsen, A.; Pedersen, E.: Immunological parameters in multiple sclerosis patients with special reference to the herpes virus group. Clin. exp. Immunol. 51: 197 (1983). Hafler, D.A.; Duby, A.D.; Seidman, J .G . ; Weiner, H.L.: Analysis of T-cell receptor beta chain rearrangements in T-cells cloned directly from active plaques (Abstract). Neuroimmunol. Symp. London, Ont., 1986, p. 46. Hafler, D.A.; Fox, D.A.; Manning, M.E.; Schlossman, S.F. ; Reinhertz, E.L.; Weiner, H.L.: In vivo activated T-lymphocytes in the peripheral blood and cerebral spinal fluid of patients with multiple sclerosis. New Engl. J . Med. 312: 1405 (1985). Halpern B.; Bakouche P.; Lasfargues, M.: Destruction des cellules nerveuses, cultivees in vitro par les lymphocytes de malades de sclerose en plaques. Presse med. 77: 2103 (1969). Hammann, K.F.; Steldern, D.V.; Diedrich, M.P.; Hopf, H.C.: Changes in the number of complement-binding leukocytes in the peripheral blood of multiple sclerosis (MS) patients indication to a reduction of E A C 3bi-binding T-cells in the early acute phase of MS. Cl in. Immunol. Immunopath. 32: 12 (1984). Hashim, G.A.; Brewen, M.: Myelin basic protein- responsive blood T-lymphocytes in patients with multiple sclerosis. J . Neurosci. Res. 13: 349 (1985). Hashim, G.A.; Pierce, J .C . ; Ramey, W.G. ; Munther, A .S . ; Burrows, W.B.; Fitzpatrick, H.F.: Myelin basic protein stimulated rosette forming T-cells in multiple sclerosis. Neurochem. Res. 3: 37 (1977). Hauser, S.L. ; Atul, K.B.; Gilles, F.; Kemp, M.; Kerr, C ; Weiner, H.L.: Immunohistochemical analys is of the cel lu lar infiltrate in multiple sclerosis lesions. Ann Neurol. 19:578 (1986). Hauser, S.L.; Ault. K.A.; Johnson, D.; Hoban, C ; Weiner. H.L.: Increased IgG secretion by unstimulated mononuclear cells in active multiple sclerosis and functional assessment of the T8 subset. Cl in. Immunol. Immunopath. 37: 312 (1985). - 5 8 -Hauser, S.L.; Ault, K.A.; Levia, M.J.; Garovoy, M.R.; Weiner, H.L.: Natural killer cell activity in multiple sclerosis. J . Immun. 127: 1114 (1981). Hauser, S.L. ; Hoban. C . J . ; Reinhertz, E.L.; Weiner, H.L. Increased spontaneous IgG production by cultured mononuclear cells in multiple sclerosis. Neurology 33. suppl. 2, p. 180 (1983a). Hauser, S.L.; Reinhertz, E.L.; Hoban, C .J . ; Schlossman, S.F.; Weiner, H.L.: Immunoregulatory T- cel ls and lymphocytotoxic antibodies in active multiple sclerosis: weekly analysis over a six- month period. Ann. Neurol. 13: 418 (1983b). Hauser, S.L.; Reinhertz, E.L.; Hoban, C .J . ; Schlossman, S.F.; Weiner, H.L.: C S F cells in multiple sclerosis -.Monoclonal antibody analysis and relationship to peripheral blood T-cell subsets. Neurology 33: 575 (1983c). Hauw, J . J . ; Berger, B.; Escourol le, R.: Etude de la cytotoxicite lymphocytaire sanguine au cours de la sclerose en plaques; in Schuller, Immunopathologie du systeme nerveux ( INSERM, Paris 1975). Henriksson, A.; Kam-Hansen, S.; Forsberg, P.; Gradien, M.: Cerebrospinal fluid lymphocytes from patients with multiple sclerosis do not increase immunoglobulin or measles antibody production after stimulation with pokeweed mitogen. J . Neuroimmunol. 11: 15 (1986). Henriksson, A.; Kam-Hansen, S. ; Link, H.: IgM, IgA and IgG producing cel ls in cerebrospinal fluid and peripheral blood in multiple sclerosis. Clin. exp. Immunol. 62: 176 (1985). Herberman, R.B.; Ortaldo, J.R.; Rubenstein, M.: Augmentation of natural and antibody-dependent cell-mediated cytotoxicity by pure human leukocyte interferon. J . clin. Immunol. 1: 149 (1981). Hershy, L.A.; Trotter, J.L.: The use and abuse of the cerebrospinal fluid IgG profile in the adult: a practical evaluation. Ann. Neurol. 8: 426 (1980). Hirayama, M.; Lisak, R.P.; Kim, S .U. ; Pleasure, D.E.; Silberberg, D.H.: Absence of expression of OKT8 antigen on cultured human, calf and rat oligodendrocytes. Nature, Lond. 301: 152 (1983). Hische, E.A.H.; van der Helm, H.J.: Rate of synthesis of IgG within the blood-brain barrier and the IgG index compared in the diagnosis of multiple sclerosis. Clin. Chem. 33: 113 (1987). Hirsch, M.R.; Wietzerbin, J . ; Pierres, M.; Goridis, C : Expression of la antigens by cultured astrocytes treated with gamma-interferon. Neurosci. Lett. 41:199 (1983). Hirsch, R.L.; Johnson, K.P. : Interferon enhances natural killer cell activity in vitro in multiple sclerosis. Neurology 34: suppl., p. 111 (1984) . Hirsch, R.L.; Ordonez, J . ; Panitch, H.L.; Johnson, K.P.: T8 antigen density on peripheral blood lymphocytes remains unchanged during exacerbations of multiple sclerosis. J . Neuroimmunol. 9: 391-398 (1985) . Hirschhorn, K.: Discussion of lymphocyte transformation. Fed. Proc. 27:31 (168). Hofman, F.M.; von Hanwehr, R.I.; Dinarello, C.A.; Mizel, S.B.; Hinton, D.; Merri l l , J . E . : Immunoregulatory molecules and IL-2 receptors identified in multiple sclerosis brain. J . Immunol. 136: 3239 (1986) . Howard, F.; Ledbetter, J . ; Wong, J . ; Bieber, C P . ; Stinson, E.B.; Herzenberg, L.A.: A human T lymphocyte differentiation marker defined by monoclonal antibodies that block E rosette formation. J . Immunol. 126: 2117 (1981). Huddlestone, J .R. ; Oldstone, M.B.A.: T-suppressor (Tg) lymphocytes fluctuate in parallel with changes in the cl inical course of patients with multiple sclerosis. J . Immunol. 123: 1615 (1979). -61 -Huddlestone, J.R.; Oldstone, M.B.A.: Suppressor T cells are activated in vivo in patients with multiple sclerosis coinciding with remission from acute attack. J . Immunol. 129: 915 (1982). Hughes, R.A.C.; Gray, I.; Clifford-Jones, R.; Stern, M.: Immune response to myelin basic protein in multiple sclerosis. Proc. R. Soc. Med. 70: 874 (1977). Illonen, J . ; Reunanen, M.; Salmi, A.; Tiilikainen, A.: Lymphocyte blast transformation responses and viral antibodies in relation to HLA antigens in multiple sclerosis. J . neurol. Sci . 49: 117 (1981). Ivanainen, M.V.: The significance of an abnormal immune response in patients with multiple sclerosis. J . Neuroimmunol. 1: 141 (1981). Jacobson, S. ; Flerlage, M.; McFarland, H.F.: Impaired measles virus-specific cytotoxic T cell responses in multiple sclerosis. J . exp. Med. 162: 839 (1985). Jacques, C ; Delassalle, A.; Rancurel, G.; Raoul, M.; Lesourd, B.; Legrand, J .C . : Myelin basic protein in C S F and blood. Archs Neurol. 39: 557 (1982). Johnson, K.P. ; Arrigo, S . C . ; Nelson, B .J . ; Ginsberg, A. : Agarose electrophoresis of cerebrospinal fluid in multiple sc leros is . Neurology 27: 273 (1977). Kabat, E.A.; Moore, D.H.; Landow, H.: An electrophoretic study of the protein components in cerebrospinal fluid and their relationship to the serum proteins. J . clin. Invest. 21: 571 (1942). Kam-Hansen, S . : Distribution and function of lymphocytes from the cerebrospinal fluid and blood in patients with multiple sclerosis. Acta neurol. Scand. 62. suppl. 75, p. 1 (1980). Kam-Hansen, S . ; Fryden, A. ; Link, H.: B and T lymphocytes in cerebrospinal and blood in multiple sclerosis, optic neuritis, and mumps meningitis. Acta neurol. Scand. 58: 95 (1978). Karki, N.T.; l l lonen, J . ; Reunanen, M.: Increased sister-chromatid exchange rate and its regression during prolonged incubation in lymphocyte cultures from patients with multiple sclerosis. Mutat. Res. 160: 215 (1986). Kastrukoff, K.F. ; Oger, J . ; Paty, D.W.: Multiple sclerosis (MS): correlation of peripheral blood lymphocyte (PBL) phenotypes and natural killer (NK) cell activity with disease activity assessed clinically and by MRI (Abstract). Ann. Neurol. 20: 164 (1986). Kastrukoff, L.; Paty, D.W.: A serial study of peripheral blood lymphocytes in relapsing remitting MS. Ann. Neurol. 15: 250 (1984). Kately, J .R . ; Bazze l , S . J . : Immunological dysfunction in multiple sclerosis. Diminution of "active" thymus-derived lymphocytes and presence of immunomodulating serum factors. Clin. Exp. Immunol. 35: 218 (1979). Keightly, R.G.; Cooper, M.D.; Sawton AR. The T cell dependence of B-cell differentiation induced by pokeweed mitogen. J . Immunol. 1 17: 1538 (1976). Kelley, R.E.; Ellison, G.W.; Myers, L.W.; Goymerac, U.; Larrick, S.B.; Kelly, C .C . : Abnormal regulation of in vitro IgG production in multiple sclerosis. Ann. Neurol. 9: 267 (1981). Kim, S .U . ; Moretto, G. ; Shin, D.H.: Expression of la antigens on the surface of human oligodendrocytes and astrocytes in culture. J . Neuroimmunol. 10: 141 (1985). Kinnman, J . ; Link, H.; Moller, E.: Influence of measles virus antigen on leukocyte migration in multiple sclerosis and controls. Acta neurol. Scand. 58: 2612 (1978). Knowles, M.; Hughes, D.; Caspary, E.A.: Lymphocyte transformation in multiple sclerosis: inhibition of unstimulated thymidine uptake by a serum factor. Lancet ii: 1207 (1968). Knowles, M.; Saunders, M.: Lymphocyte stimulation with measles antigen in multiple sclerosis. Neurology 20: 700 (1970). Kolar, O.J.; Rice, P.H.; Bauer, D . C ; Defalque, R.J . ; Danielson, C.F. ; Farlow, M.R.; Wright, J .H . : Clinical implications of studies involving cerebrospinal fluid T-cell subpopulations; in Scarlati, Matthews, Multiple Sclerosis: Present and Future, p. 203 (New York, Plenum Press 1984). Kuwert, E.; Bertrams, J . : Leukocyte iso- and auto-antibodies in multiple sclerosis with special regard to complement-dependent cold-reacting autolymphocytotoxins. Europ. Neurol. 7: 65 (1972). Lalla, M.; Vesikari, T.; Virolainen, M.: Lymphoblast proliferation and humoral antibody response after rubella vaccination. Cl in. exp. Immunol. 15: 193 (1973). Laurenzi, M.A.: Immunochemical characterization of immunoglobulins and viral antibodies synthesized within the central nervous system in patients with multiple sclerosis and controls. Acta neurol. Scand. 63: suppl. 84, p. 61 (1981). Laurenzi, M.A.; Mavra, M.; Kam-Hansen, S.; Link, H.: Oligoclonal IgG and free light chains in multiple sclerosis demonstrated by thin layer polyacrilamide gel isoelectric focusing and immunofixation. Ann. Neurol. 8: 241 (1980). Lefvert, A.K.; Link, H.: IgG production within the C N S : a critical review of proposed formulae. Ann. Neurol. 17: 13-20 (1985). Levitt, D.; Giffin, N.K.; Egan, M.L.: Mitogen-induced plasma cell differentiation in patients with multiple sclerosis. J . Immunol. 124: 2117 (1980). Levy, N.L.; Auerbach, P .S. ; Hayes, E.C. : A blood test for multiple sclerosis based on the adherence of lymphocytes to measles-infected cells. New Engl. J . Med. 294: 1423 (1976). Li, D.; Mayo, J . ; Fach, S.; Robertson, W.; Kastrukoff, L.; Oger, J . ; Paty, D.W.: Lack of correlation between clinical and MRI findings in MS. Neurology 34: 278 (1984). Link, H.; Kam-Hansen, S. ; Henriksson, A. : Studies on B-lymphocyte function in multiple sclerosis; in Scarlato, Matthews, Multiple sclerosis: present and future, p. 71 (Plenum Press, New York 1984). Link, H.; Laurenzi, M.A.: Immunoglobulin class and light chain type of oligoclonal bands in C S F in multiple sclerosis determined by agarose gel electrophoresis and immunofixation. Ann. Neurol. 6: 107 (1979). Lisak, R .P . ; Levinson, A .1 . ; Zweiman, B.; Abdou, N.I.: T and B lymphocytes in multiple sclerosis. Cl in. exp. Immunol. 22: 30 (1975). Lisak, R.P.; Mercado, F.; Zweiman, B.: Cold reactive anti-lymphocyte antibodies in neurologic disease. J . Neurol. Neurosurg. Psychiat. 42: 1054 (1979). Lisak, R.P. ; Zweiman, B.: In vitro and in vivo immune responses to homologous myelin basic protein in experimental al lergic encephalomyelitis. Cell Immunol. 11: 212 (1974). Lisak, R .P . ; Zweiman, B.: In vitro cel l -mediated immunity of cerebrospinal fluid lymphocytes to myelin basic protein in primary demyelinating diseases. New Engl. J . Med. 297: 850 (1977). Lisak, R.P. ; Zweiman, B.; Whitaker, J . N . : Spinal fluid basic protein immunoreactive material and spinal fluid lymphocyte reactivity to basic protein. Neurology 31: 180 (1981). Lowenthal, A.; van Sande, M.; Karcher, D.: The differential diagnosis of neurological diseases by fractionating electrophoretically the C S F G-glob- ulins. J . Neurochem. 6: 51 (1960). Lumsden, C .E . : The immunogenesis of multiple sclerosis plaques. Brain Res. 28: 365 (1971). Madigand, M.; Oger, J . ; Fauchet, R.; Sabouraud, 0.; Genetet, B.: HLA profile in MS. J . neurol. Sci . 53: 519(1982). Manconi, P.E.; Marrosu, M.G.; Cianchetti, C ; Ennas, M.G.; Mangoni, A.; Zaccheo, D.: Lymphocyte subpopulations in cerebrospinal fluid and peripheral blood in multiple sclerosis. Acta neurol. Scand. 62: 165 (1980). Mar, P. : Ant ibody-dependent cel lu lar cytotoxicity in multiple sclerosis. J . Neurol. Sci . 47: 285 (1980). Massaro, A .R. ; Mitchett, F.; Laudisio, A.; Bergonzi, P.: Myelin basic protein and S-100 antigen in cerebrospinal fluid of patients with multiple sclerosis in the acute phase. Ital. J . neurol. Sc i . 6: 53 (1985). Mat ias-Guiu, J . ; Ruiba, A. ; Mart inez-Vazquez, J . M . ; Colomer, R.; Agustin, C : Concentration of myelin basic protein in cerebrospinal fluid in prognosis of multiple sclerosis (Letter). Cl in. Chem. 32: 915 (1986). Mattson, D.; Roos, R.P. ; Arnason, B.G.W.: Comparison of agar gel electrophoresis and isoelectric focussing in MS and S S P E brains. Ann. Neurol. 9: 34 (1981). Mattson, D.H.; Roos, R.P.; Arnason, B.G.W.: Oligoclonal IgG in multiple s c l e r o s i s and subacu te sc le ros ing panencepha l i t i s . J . Neuroimmunol. 2: 261 (1982). - 6 8 -McAlpine, D.; Lumsden, C . E . ; Acheson, E.D.: Multiple sclerosis, a reappraisal; 2nd ed. (Churchill Livingston, London 1972). McDonald, W.I.; Halliday, A .M. : Diagnosis and classification of MS. Br. med. Bull. 33: 4 (1977). McFarland, H.F.; McFarlin, D.E.: Cellular immune response to measles, mumps and vaccinia viruses in multiple sclerosis. Ann. Neurol. 6: 101 (1979). McMillan, S.A.; Haire, M.; Middleton, D.: Antibodies to lymphocytes and smooth muscle in the sera of patients with multiple sclerosis. Clin. Immunol. Immunopath. 16: 374 (1980). Merrill, J . E . ; Biberfeld, G. ; Kolmodin, G. ; Landin, S. ; Norrby, E.: A T-lymphocyte subpopulation in multiple sclerosis patients bearing Fc receptors for both IgG and IgM. J . Immunol. 124: 2758 (1980). Merrill, J .E . ; Ellison, G.W.; Myers, L.W.: Cytotoxic activity of peripheral blood and cerebrospinal spinal fluid lymphocytes from patients with multiple sclerosis and other neurological d iseases: analysis at the single cell level of the relationship of cytotoxic effector cells and interferon producing cells. Cl in. Immunol. Immunopath. 31: 390 (1984). Merrill, J .E . ; Jondal, M.; Seeley, J . ; Ullberg, M.; Siden, A.: Decreased NK killing in patients with multiple sclerosis : An analysis on the level of the single effector cel l in peripheral blood and cerebrospinal fluid in relation to d isease activity. Cl in. exp. Immunol. 47: 419 (1982a). Merrill, J .E . ; Myers, L.W.; Ellison, G.W.: Regulation of natural killer cell cytotoxicity by prostaglandin E in the peripheral blood and cerebrospinal fluid of patients with multiple sclerosis and other neurological diseases. Part 2. J . Neuroimmunol. 4: 239 (1983). Merrill, J . E . ; Scott, A.; Myers, L ; Ell ison, G. : Cytotoxic activity of peripheral blood and cerebrospinal fluid lymphocytes from patients with multiple sclerosis and other neurological d iseases: analysis at the single cell level using morphological and surface marker phenotype criteria. J . Neuroimmunol. 3: 123 (1982b). Merrill, J .E . ; Wahlin, B.; Sinden, A.; Perlmann, P.: Elevated direct and IgG enhanced A D C C activity in MS patients. J . Immunol. 128: 1728 (1982c). Miller, J .R. ; Burke, A.; Bever, C.T.: Occurrence of oligoclonal bands in multiple sclerosis and other C N S diseases. Ann. Neurol. 13: 53 (1983). Mingioli, E.S. ; McFarlin, D.E.: Leukocyte surface antigens in patients with multiple sclerosis. J . Neuroimmunol. 6: 131 (1984). - 7 0 -Moretta, L ; Webb, S.R.; Grossi , C .E . ; Lydyard, P.M.; Cooper, M.D.: Functional analysis of two human T-cell subpopulations: help and suppression of B- cell responses by T-cells bearing receptors for IgM and IgG. J . exp. Med. 146: 184 (1977). Morimoto, C ; Hafler, D.A.; Weiner, H.L.; Letvin, N.L.; Hagan, M.; Daley, J . ; Schlossman, S .F . : Selective loss of the suppressor-inducer T-cell subset in progressive multiple sclerosis. New Engl. J . Med. 316: 67 (1987). Morimoto, C ; Letvin, N.L.; Budd, C . E . ; Hagan, M.; Takeuchi, T.; Schlossman, S.F. : The role of the 2H4 molecule in the generation of suppressor function in Con-A-activated T-cells. J . Immunol. 137: 3247 (1986). Morimoto, C ; Letvin, N.L.; Distaso, J.A.; Aldrich, W.R.; Schlossman, S.F.: The isolation and characterization of the human suppressor inducer T cell subset. J . Immunol. 124: 1503 (1985). Morimoto, C , N. L. Letvin, A. W. Boyd, M. Hagan, H . 'M. Brown, M. M. Kornacki and S. F. Schlossman. The isolation and characterization of the human helper inducer T cell subset. J . Immunol. 134:3762 (1985). Myers, L.: Leukocyte migration inhibition in multiple sclerosis; in Wolfgram, Ell ison, Stevens, Andrews, Multiple sclerosis, pp. 383-402 (Academic Press, New York 1972). -71 -Naess, A. : T-lymphocytes in cerebrospinal fluid from patients with neurological diseases. Eur. Neurol. 18: 183 (1979). Neighbour, P.A.; Bloom, B.R.: Absence of virus-induced lymphocyte suppression and interferon production in multiple sclerosis. Proc. Natl. Acad. Sci . USA 76: 476 (1979). Neighbour, P.A.; Miller, A . E . ; Bloom, B.R.: Interferon responses of leukocytes in multiple sclerosis. Neurology 31: 561 (1981). Nordal, H.J.; Froland, S .S. : Lymphocyte populations and cellular immune actions in vitro in patients with multiple sclerosis. Clin. Immunol. Immuno- path. 9: 87 (1978). Noronha, A .B .C . ; Richman, D.P.; Arnason, B.G.W.: Detection of in vivo stimulated cerebrospinal fluid lymphocytes by flow cytometry in patients with multiple sclerosis. New Engl. J . Med. 303: 713 (1980). Norrby, E.; Link, H.; Olsson, J . E . : Comparison of antibodies against different viruses in cerebrospinal fluid and serum samples from patients with multiple sclerosis. Infect. Immunity 10: 373 (1974). Nyland, H.; Matre, R.; Mork, S.; Bjerke, J .R. ; Naess, A.: T lymphocyte subpopulations in multiple sclerosis lesions (Letter). New Engl. J . Med. 307: 1643 (1982). Offner, H.; Konat, G . : Stimulation of active E-rosette forming lymphocytes from multiple sclerosis patients by gangliosides and cerebrosides. J . Neurol. Sci . 46: 101 (1980). Offner, H.; Konat, G . ; Raun, N.E.; Clausen, J . : E- rosette-forming lymphocytes in multiple sc leros is patients: basic protein stimulation of rosette forming cells. Acta Neurol. Scand. 57: 380 (1978). Oger, J . ; Antel, J .P . ; Kuo, H.H.; Arnason, G. W.: Influence of azathioprine (Imuran) on immune function in multiple sclerosis. Ann. Neurol. 11: 177 (1982). Oger, J . ; Antel, J .P . ; Noronha, A.; Arnason, B.G.W.: Changes in T-cell subpopulations in the cerebrospinal fluid of multiple sclerosis patients. Neurology 32: 148A (1982a). Oger, J . ; Arnason, A.G.W.; Wray, S.M. ; Kistler, J .P . : A study of T and B cells in multiple sclerosis. Neurology, Minneap. 25: 444 (1975). Oger, J . ; Jackevicius, S.; Antel, J .P . ; Arnason, B.G.W.: Reduced OKT8+ and OKT3+ cells in blood of progressive MS patients. Ann. N.Y. Acad. Sci . 436: 506 (1984). Oger J . ; Kastrukoff, L ; O'Gorman, M.; Paty, D.: Progressive multiple sc leros is : abnormal immune functions in vitro and aberrant correlation with enumeration of lymphocyte subpopulations. J . Neuroimmunol. 12: 37-48 (1986a). Oger, J . ; Kastrukoff, L.; Paty, D.W.: Multiple sclerosis: relationship between suppressor cell function, IgG secretion in vitro and the attacks of multiple sclerosis (MS) as studied by serial clinical and MRI examinations. Ann. Neurol. 20: 161 (1986b). Oger, J . ; Mattson, D.; Roos, R.; et al.: Isoelectrofocusing of IgG secreted by lymphocytes in multiple sclerosis and controls. Neurology 21: 144 (1981). Oger, J . ; O'Gorman, M.; Kastrukoff, L ; Paty, D.: In vitro simultaneous test ing of three immune parameters helps differentiate progressive MS from controls. Neurology 35: 313 (1985). Oger, J . ; Roos, R.; Antel, J .P . : Immunology of multiple sclerosis. Neurol. Clin. 1: 655 (1983). Oger, J . ; Szuchet, S.; Antel, J .P . ; Arnason, B.G.W.: A monoclonal antibody against human T-suppressor lymphocytes binds specifically to the surface of cultured ol igodendrocytes. Nature, Lond. 295: 66 (1982b). O'Gorman, M.R.G.; Oger, J . ; Kastrukoff, L : Reduction of immunoglobulin G secret ion following long term lymphoblastoid interferon - 7 4 -(Wellferon) treatment in multiple sclerosis patients. J . clin. exp. Immunol. 67: 66 (1987). Panitch, H.S.; Hirsch, R.L.; Haley, AS. , Johnson, KP. Exacerbations of multiple sclerosis in patients treated with gamma interferon. Lancet 1; 893 (1987). Panitch, H.S.; Hooper, C . J . ; Johnson, K.P.: C S F antibody to myelin basic protein. Measurement in patients with multiple sclerosis and subacute sclerosing panencephalitis. Archs Neurol. 37: 206 (1980). Paty, D.W.; Kastrukoff; L.F.; Hiob, L.: Chronic progressive multiple sclerosis has low suppressor cell levels. Trans. Am. neurol. Ass. 106: 276 (1981). Paty, D.W.; Kastrukoff, L.; Morgan, N.; Hiob, L.: Suppressor T-lymphocytes in multiple sclerosis: analysis of patients with acute relapsing and chronic progressive disease. Ann. Neurol. 14: 445 (1983). Pedersen, J .S . ; Walker, M.; Toh, B.H.; De Aizpurua, H.J.; Lolait, S . J . ; Bernard, C.C.A. : Flow microfluorometry detects IgM autoantibody to oligodendrocytes in multiple sclerosis. J . Neuroimmunol. 5: 251 (1983). Poser, C M . ; Paty, D.W.; Scheinberg, L.; McDonald, W.I.; Davis, F.A.; Ebers, G . C ; Johnson, K.P.; Sibley, W.A.; Silberberg, D.H.; Tourtelotte, W.W.: New diagnostic criteria for multiple sc lerosis: guidelines for research protocols. Ann. Neurol. 13: 227 (1983). Prineas, J .W.; Kwon, E.E. ; Eun-Sook, C ; Sharer, L.R.: Continual breakdown and regeneration of myelin in progressive multiple sclerosis plaques. Ann. N.Y. Acad. Sci . 246: 11 (1984). Prineas, J.W.; Wright, R.G.: Macrophages, lymphocytes and plasma cells in the perivascular compartment in multiple sclerosis. Lab. Invest. 38: 409-421 (1978). Rauch, H.C.; Montgomery, I.N.; Kaplan, J . : Natural killer cell activity in multiple sclerosis and myasthenia gravis. Immunol. Invest. 14: 427 (1985). Reddy, M.M.; Goh, K.O.: Band T-lymphocytes in man III B, T, and "null' lymphocytes in multiple sclerosis. Neurology 26: 997 (1976). Reder, A.T.; Antel, J .P . ; Oger, J .J .F. ; McFarland, T.A.; Rosenkoetter, M.; Arnason, B.G.W.: Low T8 antigen density on lymphocytes in active multiple sclerosis. Ann. Neurol. 16: 242 (1984). Reder, A.T.; Arnason, B.G.W.: Immunology of multiple sclerosis; in Koetsier, Handbook of clinical neurology, vol. 3 (47), chapt. 12, p. 337 (Elsevier, Amsterdam 1985). Reinhertz, E.L.; Weiner, M.L.; Hauser, S .M. ; Cohen, J.A.; Distoso, J.A.; Schlossman, S .F . : Loss of suppressor T cells in active multiple sclerosis. New Engl. J . Med. 303: 125 (1980). Rice, G.P.A. ; Casali, P.; Merigan, T.C.; Oldstone, M.B.A.: NK cell activity in patients with MS given alpha IFN. Ann. Neurol. 14: 333 (1983a). Rice, G.P.A.; Finney, D.; Braheny, S.L.; Knobler, R.L.; Sipe, J .C . ; Oldstone, M.B.A.: Disease activity markers in multiple sclerosis : another look at suppressor cells defined by monoclonal antibodies OKT4, OKT5 and OKT8. J . Neuroimmunol. 6: 75 (1984a). Rice, G.P. ; Sipe, J .C. ; Braheng, S.L.; Knobler, R.L.; Finney, D.A.; Oldstone, M.B.A.: The failure of monoclonal antibody defined lymphocyte subsets to monitor d isease activity in patients with multiple sclerosis. Ann. N.Y. Acad. Sci. 436: 271 (1983b). Rice, G .P . ; Talbot, P.; Waefel, E.M.; Vandirk, B.; Braheny, S. ; Knobler, R.L.; Sipe, J . ; Oldstone,M B A. Immunological observations in patients with multiple sc leros is treated with human alpha interferon (Abstract). Neurology 34: 112 (1984b). Rocklin, R.E. : Products of activated lymphocytes: leukocyte inhibitory factor distinct from migration inhibitory factor (MIF). J . Immunol. 112: 1461 (1974). Rocklin, R.E. ; Sheremata, W.A.; Feldman, R.G. : The Guillain-Barre syndrome and multiple sclerosis: in vitro cellular responses to nervous tissue anti- gens. New Engl. J . Med. 284: 803 (1971). Roos, R.P. : B-cell abnormalities in multiple sclerosis. Archs Neurol. 42: 73 (1985). Rose, A .S . ; Ellison, G.N. ; Myers, L.W.; Tourtelotte, W.W.: Criteria for the clinical diagnosis of MS. Neurology 26: (6: Part. 2): 20 (1976). Rose, L.M.; Ginsberg, A.H. ; Rothstein, T.L.; Ledbetter, J.A.; Clark, E.A.: Selective loss of a subset of T-Helper cells in active multiple sclerosis. Proc. Natl. Acad. Sci . USA 82: 7389 (1985). Rudick, R.A.; Peter, D.R.; Bidlack, J .M. ; Knutson, D.W.: Multiple sclerosis: free light chains in the cerebrospinal fluid. Neurology 35: 1443 (1985). Rukavina, D.; Sepcic, J . ; Doric, M.; Ledic, P.; Zaputovic, L ; Eberhardt, P.: Lymphocyte subpopulations in the blood and cerebrospinal fluid of multiple sclerosis patients in active disease. Acta Neurol. Scand. 69: 182 (1984). Ruutianen, J . ; Arnadottir, T.; Molnar, G. : Myelin basic protein antibodies in the serum and C S F of multiple sclerosis and subacute sclerosing panencephalitis. Acta neurol. Scand. 64: 196 (1981). - 7 8 -Sagar, H.J.; Allonby, I.D.; Hughes, P.: Cell-mediated immunity to viral antigens and tuberculin in multiple sclerosis. Acta neurol. Scand. 63: 81 (1981). Salier, J .P . ; Glynn, P.; Goust, J .M. ; Cuzner, M.L.: Distribution of nominal and latent IgG (Gm) allotypes in plaques of multiple sclerosis brain. Clin. exp. Immunol. 54: 634 (1983). Salk, P.L.; Wegemer, D.E.; Ward, E.M.; McClure, J .E . ; Goldstein, A.L.; Romine, J . S . ; Salk, J . : Decreased circulating T-lymphocytes and increased serum thymosin alpha-1 levels in patients with chronic progressive multiple sclerosis. Fed. Proc. 41: 808A (1982). Salmi, A.; Fray, H.: More about blood tests for MS. New Engl. J . Med. 297: 398 (1977). Salmi, A. ; Panelius, M.; Vainionpaa, R.: Antibodies against different viral antigens in the C S F of patients with multiple sclerosis and other neurological diseases. Acta neurol. Scand. 36: 261 (1979). Salmi, A.; Ziola, B.; Hovi, T.; Reunanen, M.: Antibodies to coronavirus OC 43 and 229E in multiple sclerosis. Neurology 32: 292 (1982). Salonen, R.; Illonen, J . ; Reunanen, M.; Nikoskelainen, J . ; Salmi, A.: PPD-, P W M - and PHA-induced interferon in stable multiple sclerosis. Association with HLA-Dw2 antigen and clinical variables. Ann. Neurol. 11: 279 (1982). Sandberg-Wollheim, M.: Lymphocyte populations in the cerebrospinal fluid and peripheral blood of patients with multiple sclerosis and optic neuritis. Scand. J . Immunol. 17: 575 (1983). Santoli, D.; Hall, W.; Kastrukoff, L.; Lisak, R.P.; Perussia, B.; Trinchieri, G. ; Koprowski, H.: Cytotoxic activity and interferon production by lym- phocytes from patients with multiple sclerosis. J . Immunol. 126: 1274 (1981). Santoli, D.; Moretta, L.; Lisak, R.; Gilden, D.; Kaprowski, H.: Imbalances in T-cel l subpopulat ions in multiple sc leros is patients. J . Immunol. 120: 1369 (1978). Schauf, C.L. ; Schauf, V.; Davis, F.A.; Strelkaukas, A . J . ; Mizen, M.R.: Lymphocyte subpopulations in multiple sc lerosis: comparison with neuroelectric blocking activity. Neurology 27: 822 (1977). Schocket, A.L. ; Weiner, H.L.; Walker, J . ; Mcintosh, K.; Kohler, P.M. : Lympho-cytotoxic antibodies in multiple sclerosis. Clin. Immunol. Immunopath. 7: 15 (1977). Schumacher, G.A.; Beebe, G. ; Kibler, R.E.; Kurland, L.T.; Kurtzke, J .F . ; McDowell, F.; Nadler, B.; Sibley, W.A.; Tourtellotte, W.W.; Willmon, T.L.: Problems of experimental trials of therapy in MS. Ann. N.Y. Acad. Sci . 122: 552 (1965). - 8 0 -Serra, HM., Krowka, J F . , Ledbetter, JA. , and Pilarski, LM. Loss of CD45R (Lp220) represents a post-thymic T cell differentiation event. J . Immunol. 140; 1435 (1988). Seshadri, R.; Sutherland, G.R.; Baker, E.: S C E , X- radiation sensitivity and mutation rate in multiple sclerosis. Mutat. Res. 110: 141 (1983). Sever, J . ; Fuccillo, D.A.; Madden, D.L.: Multiple sclerosis: attempts to demonstrate altered immune responses and viruses. Neurology 26: suppl., p. 72 (1976). Sheremata, W.; Cosgrove, J.B.R. ; Eylar, E.H.: Multiple sclerosis and cell-mediated hypersensitivity to myelin A1 protein. J . neurol. Sci . 27: 413 (1976). Sheremata, W.; Rzepel ia, A . J . ; Berger, J . ; Sazant, A. ; Castro, A.: Inhibition of concanavalin A inducible suppressor cell function in multiple sclerosis by adrenocorticotropic hormone. Ann. Neurol. 12: 103A (1982). Shou, L.; Schwartz, S.A.; & Good, R.A. Suppressor cell activity after concanavalin A treatment of lymphocytes from normal donors. J . exp. Medicine. 143, 1100 (1976). -81 -Siegal, E.P. ; Siegal, M.: Enhancement by irradiated T-cells of human plasma cell production: dissection of helper and suppressor functions in vitro. J . Immun. 118: 642 (1977). Sindic, C.J .M. ; Cambiaso, C.L.; Depre, A.; Laterre, E.C.; Masson, P.L.: The concentrations of IgM in the cerebrospinal fluid of neurological patients. J . neurol. Sci . 55: 339 (1982). Staley, M.J.; Schmierrer, D.M.; Milligen, K.S.; Yeo, P.T.: Oligoclonal bands are found in electrophoretograms of serum of patients with multiple sclerosis (Letter). Clin. Chem. 32: 709 (1986). Stewart, G .J . ; Basten, A.; Guinan, J . ; Bashir, H.V.; Cameron, J . ; McLeod, J . G . : HLA-Dw2, viral immunity and family studies in multiple sclerosis. J . neurol. Sci . 32: 153 (1977). Strandgaard, S. ; Jorgensen, P.N. : Delayed hypersensitivity to myelin antigen in multiple sc leros is investigated with leukocyte migration method. Acta neurol. Scand. 48: 243 (1972). Symington, G.R. ; MacKay, 1.R.: Cell-mediated immunity to measles virus in multiple sclerosis: correlation with disability. Neurology 28: 109 (1978). Thompson, A . J . ; Brazil, J . ; Martin, E.A.; Hutchinson, M.; Whelan, C.A.; Feighery, C : Suppressor T-cell changes in active multiple - 8 2 -sclerosis: analysis with three different monoclonal antibodies. J . Neurol. Neurosurg. Psychiat. 48: 1062 (1985). Thompson, A . J . ; Brazil, J . ; Hutchinson, M.; Feighery, C. Three possible laboratory indexes of d isease activity in multiple sclerosis. Neurology 37; 515 (1987). Tjernlund, U.; Cesaro, P.; Tournier, E.; Degos, J.D.; Bach, J.F. ; Back, M.A.: T-cel l subsets in multiple sc leros is : a comparat ive study between cell surface antigens and function. Cl in. Immunol. Immunopath. 32: 185 (1984). Tourtellotte, W.W.; Ma, B.I.; Brandes, D.B.; Walsh, M.J.; Potvin, A.R.: Quanti-fication of de novo central nervous system IgG measles antibody synthesis in S S P E . Ann. Neurol. 9: 551 (1981). Tourtellotte, W.W.: On C S F IgG quotients: A review and a new formula. J . Neurol. Sci. 10: 279 (1970). Tourtellotte, W.W.; Ma, B.I.: Multiple sclerosis: the blood-brain-barrier and the measurement of central nervous system IgG synthesis. Neurology 28: 76-83 (1978). Tourtellotte, W.W.; Shapshak, P.; Straugaitis, S .M. : Do all MS have intra-blood-brain IgG synthesis. Neurology 33: suppl. 2, p. 194 (1983). - 8 3 -Tourtellotte, W.W.; Straugaitis, S . M . ; Walsh, M.J . ; Shapshak, P.; Baumhefner, R.W.; Potvin, A.R. ; Syndulko, K.: The basis of intra-blood-brain barrier IgG synthesis. Ann. Neurol. 17: 21-27 (1985). Tovell, D.R.; McRobbie, I.A.; Warren, K.G. ; Tyrell, D.L.J.: Interferon production by lymphocytes from multiple sclerosis and non-MS patients. Neurology 33: 640 (1983). Traugott, U.: Multiple sclerosis: Relevance of Class I and Class II MHC-expressing cells to lesion development. J . Neuroimmunol. 16:283 (1987). Traugott, U.; Raine, C .E . : Further lymphocyte characterization in the central nervous system in multiple sclerosis. Ann. N.Y. Acad. Sci . 246: 163 (1984). Traugott, U.; Reinhertz, E.L.; Raine, C .E . : Multiple sclerosis: heterology among early T-jo. cells and T-ycells. Ann. Neurol. 11: 182 (1982). Traugott, U.; Reinhertz, E.L. ; Raine, C . S . : Multiple sc leros is : distribution of T-cel l subsets within active chronic lesions. Science 219: 308 (1983a). Traugott, U.; Reinhertz, E.L.: Raine, C .S. : Distribution of T-cells, T-cell subsets and la positive macrophages in lesions of different ages. J . Neuroimmunol. 4: 201 (1983b). - 8 4 -Traugott, U.; Scheinberg, L .C. ; Raine, C . S . : Multiple sclerosis circulating antigen reactive lymphocytes. Ann. Neurol. 6: 425 (1979). Traugott, U.; Scheinberg, L.C.; Raine, C .S . : Lymphocyte responsiveness to oligodendrocytes in multiple sclerosis. J . Neuroimmunol. 1: 41 (1981). Utermohlen, V. ; Farmer, J . ; Kornbluth, J . ; Kornstein, M.: The relationship between direct migration inhibition with measles antigen and E-rosettes in normals and patients with multiple sclerosis. Clin. Immunol. Immunopath. 9: 63 (1978). Utermohlen, V.; Zabriskie, J .B . : A suppression of cellular immunity in patients with multiple sclerosis. J . exp. Med. 138: 1591 (1973). Van den Noort, S. ; Stjernholm, R.L.: Lymphotoxic activity in multiple sclerosis serum. Neurology 21: 783 (1971). Vandvik, B.; Norrby, E.: Oligoclonal IgG antibody response in the central nervous system to different measles virus antigens in subacute sclerosing panencephalitis. Proc. natl. Acad. Sc i . USA 70: 1060 (1973). Vandvik B.; Norrby E.; Nordal, H.K.; Degre, M.: Oligoclonal measles virus-specif ic IgG antibodies isolated from cerebrospinal fluids, brain extracts and sera from patients with subacute sclerosing - 8 5 -panencephalitis and multiple sclerosis. Scand. J . Immunol. 5: 979 (1976). Vervliet, G . ; Carton, H.; Billiau, A.: Interferon-gamma production by peripheral blood leukocytes from patients with multiple sclerosis and other neurological diseases. J . clin. exp. Immunol. 59: 391 (1985). Vervliet, G . ; Claeys, H.; Van Hauer, H.; Carton, H.; Vermylen, C ; Meulepas, E.; Billiau, A.: Interferon production and natural killer (NK) activity in leukocyte cultures from multiple sc lerosis patients. J . neurol. Sci . 60: 137 (1983). V i ro la inen, M. : Blast transformation in vivo and in vitro in carbamazepine hypersensitivity. Clin. exp. Immunol. 9: 429 (1971). Visscher, B.R.; Myers, L.W.; Ellison, G.W.: HLA haplotypes and immunity in multiple sclerosis. Neurology 29: 1561 (1979). Wajgt, A.; Gorny, M.: Antibodies to myelin basic protein and to myelin assoc ia ted glycoprotein in multiple sc le ros is . Ev idence of intrathecal production of antibodies. Acta neurol. Scand. 68: 337 (1983). Waksman, B.; Adams, R.D.: A histological study of the early lesion in experimental allergic encephalomyelit is in the guinea pig and rabbit. Am. J . Path. 41: 162 (1962). - 8 6 -Waldmann, T.A.; Durm, M.; Broder, S.; Blackman, M.; Blaese, R.M.; Strober, W.: Role of suppressor T-cells in pathogenesis of variable hypogammaglobulinaemia. Lancet ii: 609 (1974). Walker, J . E . ; Cook, J .D. : Lymphoblastic transformation in response to viral antigens in multiple sclerosis. Neurology 29: 1341 (1979). Walker, J .E . ; Cook J.D.; Harrison, P.; Stastny, P.: HLA and the response of lymphocytes to viral antigens in patients with multiple sclerosis. Human Immunol. 4: 71 (1982). Wallen, W . C ; Houff, S.A.; Ivanainen, M.; Calabrese, U.P.; DeVries, G.M.: Suppressor cell activity in multiple sclerosis. Neurology 31: 668 (1981). Walsh , M.J . ; • Tourtellotte, W.: Temporal invariance and clonal uniformity of brain and cerebrospinal IgG, IgA and IgM in multiple sclerosis. J . exp. Med. 163: 41 (1986). Walsh M.J . ; Tourtellotte W.W.; Potvin, A .R . ; Potvin, J . H . : The cerebrospinal fluid in multiple sclerosis; in Hallpike, Adams, Tourtel lotte, Mult iple sc le ros is . Pathology, d iagnos is and management, pp. 275-358 (Chapman & Hall, London 1983). - 8 7 -Weiner, H.L.; Hafler, D.A.; Fallis, R.J. ; Johnson, D.; Ault, K.A.; Hauser, S.L.: T-cell subsets in patients with multiple sclerosis. Ann. N.Y. Acad. Sci . 246: 281 (1984). Whitaker, J .N. ; Lisak, R. P.; Bashir, R.M.; Fitch, O.H.; Seyer, J .M. ; Krance, R.; Lawrence, J .A. ; Chien, L.T.; O'Sull ivan, P.: Immune reactive myelin basic protein in the cerebrospinal fluid in neurological disorders. Ann. Neurol. 7: 58 (1980). Wicher, V. ; Holub, R.: Cellular response to basic protein and T-suppressor cells in multiple sclerosis. Immunol. Commun. 11: 485 (1982). Wicher, V. ; Olszewski , W.; Milgrom, F.: Age-related reactivity of lymphocytes from multiple sclerosis patients to myelin basic protein. Int. Archs Allergy Appl. Immunol. 66: 136 (1981). Wicher, V.; Olszewski, W.; Milgrom, F.: Dual response of lymphocytes from multiple sclerosis patients to myelin basic protein. Clin. exp. Immunol .37: 114 (1979). Zabriskie, J .B. ; Mayer, L.; Fu, S.U. ; Yeadon, C ; Cam, V.; Plank, O : T-cell subsets in multiple sclerosis: lack of correlation between helper and suppressor T-cells and the clinical state. J . clin. Immunol. 5: 7 (1985). - 8 8 -Zetterwall, O.; Link, H.: Electrophoretic distribution of kappa and lambda immunoglobulin light chain determinants in serum and cerebrospinal fluid in multiple sclerosis. Cl in. exp. Immunol. 7: 365 (1979). Ziola, B.; Hader, W.J . : Adherent cells suppress measles and herpes simplex I virus induced blastogenesis in multiple sclerosis lymphocytes. J . Neuroimmunol. 7:315 (1985). - 8 9 -Chapter 2 REGULATION OF IN VITRO PWM INDUCED IgG SECRETION IN HUMANS 2:1 Introduction Within the healthy population there is a large variation in the amount of IgG secreted by peripheral blood mononuclear cells (PBMC) in response to Pokeweed mitogen (PWM). In any given individual the amount of IgG secreted is fairly consistent [Wu et a l . , 1976; Rosenkoetter et al., 1984]. We have measured PWM induced IgG secretion repeatedly in a group of healthy individuals over a period of approximately 2 years. The response varied markedly from individual to individual but was relatively stable in each of them. Within this group certain individuals consistently secreted very low amounts of IgG (low responders, LR) while others secreted very high amounts of IgG (high responders, HR). In order to gain insight into the mechanisms underlying low response in the PWM induced B cell differentiation assay, we compared the phenotype and function of the regulatory cells in P B M C cultures obtained from healthy individuals presenting HR or LR characteristics. PWM induced proliferation and differentiation of B cells in vitro is a popular model of in vivo Ig secretion and has been used extensively to define the regulatory activities of various T cell subsets which have been identif ied by surface molecu les. Differentiation of B cel ls leading to immunoglobulin secretion involves the complex interaction of T cells, B cells, monocytes, T-cell factors and surface molecules [O'Garra et al., 1988; Gordon et al., 1987]. The mechanisms involved in the control of T-T and T-B interactions are not completely understood. CD4+ T helper cells play a central role in regulating PWM induced B cell differentiation: the addition of this subset to B cells and monocytes in the presence of PWM induces IgG synthesis and secretion [Reinhertz et al.,1979]. The further addition of CD8+ cells to the system results in suppression [Thomas et al., 1980]. Suppression of PWM induced IgG secretion is radiation sensitive while help is relatively resistant [Siegal and Siegal, 1977; Fauci et al., 1978; Wasserman et al., 1979]. The CD4+ T cell subset has recently been shown to be heterogeneous in both phenotype and function. Following activation CD4+ cells some CD4 cells become la+. Both CD4la+ and CD4+la-, cell subpopulations are required to induce maximal Ig secretion by B lymphocytes [Reinhertz et a l . , 1981]. In the serum of certain patients with juvenile rheumatoid arthritis (JRA) antibodies have been detected which allow the separation of CD4+ cells into a helper-inducer (T4+JRA-) and a suppressor- inducer population (T4+JRA+) for P W M induced Ig synthesis [Morimoto et al., 1982]. Monoclonal antibodies such as TQ1 [Reinhertz et a l . , 1982], 2H4 [Morimoto et al . , 1985a] and 4B4 [Morimoto et al., 1985b] further subdivide the CD4+ cells into inducers of help (T4+JRA-TQ- and T4+4B4+) and inducers of suppression (T4+2H4+) in the PWM system. The autologous mixed lymphocyte reaction (AMLR) is a measure of the proliferative response of CD4+ T cells to the HLA-Dr Ag present -91 -on irradiated autologous E- cells [Palacios et al., 1981]. T cells isolated from an AMLR exert help (after 3 days) or suppression (after 6 days) in PWM stimulated Ig synthesis [Kotani et al., 1986] and it has been suggested that the AMLR may represent an important mechanism by which immune responses are regulated [Hirsch, 1986]. It has been observed that CD4+2H4+ cells proliferate well upon stimulation in a 5 day AMLR while the CD4+4B4+ cells proliferate poorly [Morimoto et all., 1985b]. We report here that the IgG secretion response of the B cells, the phenotype of the T helper cells, the amount of in vivo radiation sensitive suppression and the AMLR all differ between LR and HR 2:2 Materials and Methods 2:2:i Subjects: In a 2 year period we have studied healthy individuals between the ages of 18 and 50 by using the PWM induced IgG secretion assay. Twenty-seven individuals have been reassayed on 5 or more occasions. 2:2:ii Cell separation techniques: 2:2:ii:a Fractionation of mononuclear cells; P B M C were isolated from heparinized venous blood by Ficoll-hypaque density gradient centrifugation according to the method of Boyiim [1968]. The cells were washed at least 4 times in cold HBSS and resuspended in RPMI 1640 with 10% FCS, 2 mM L-Glutamine, and 2 mg of gentamicin per 100 ml (RPMI complete). 2:2:ii:b E-Rosette cell Subset separation technique; P B M C were separated into sheep erythrocyte binding (E+) and non-binding (E-) subsets using 2-aminoethylisothiouronium-bromide treated S R B C by a method similar to that developed by Saxon et al. [1976]. The E" cells contained <2% Leu 1+ cells and <1% E rosette positive cells and the E + cell subset contained >95% E rosette positive 2:2:ii:c Removal of monocytes; The protocol was derived from Ly and Mishell, [Ly and Mishell, 1974]. Sephadex G-10 (Pharmacia, Piscataway, NJ) was washed 3 times, autoclaved for 10 min, and washed again 3 times in RPMI. Columns were prepared in 10 ml syringes plugged with nylon wool. The settled Sephadex was washed again with 30-50 ml of RPMI with 20% F C S ; after warming, 3x10 7 PBMC were added and the columns were incubated at 37°C for 30 min. Non-adherent cells were then eluted from the column with 25-35 ml of RPMI. The percentage of monocytes remaining in the non-adherent population as assessed by morphology and esterase staining was always less 4%. 2:2:iii PBMC Cultures: 2:2:iii:a PWM induced proliferation; PWM (Gibco, Grand Island, NY) induced tritiated thymidine (TdR, Amersham Canada Ltd., Oakville, Ont.) incorporation was measured in triplicate cultures of 2x10 5 P B M C (200 u.l). Cells were cultured in round bottom microtitre plates with PWM for 72 hr. at 37°C with 5% CO2. During the last 6 hr., cells were pulsed with one microcurie of TdR, after which they were harvested onto glass fiber filters and the radioactivity (cpm) was counted in a Beckman LS9000 liquid 2:2:iii:b PWM induced IgG secretion assay; Pokeweed-mitogen-induced IgG secretion was carried out in vitro as reported previously [Oger et al., 1982]. Briefly, mononuclear ce l l s ( P B M C ) iso lated by F ico l l -hypaque densi ty gradient centrifugation from the peripheral blood were washed at least five times and 1 0 6 P B M C were set up in I ml cultures of RPMI 1640 with 10% fetal calf serum (Flow Lab), 2 mM L-glutamine and 2 mg of gentamicin per 100 ml with PWM (1 /300 final dilution) and without PWM added. After 7 or 10 days at 37°C and 5% CO2 in air, the cultures were centrifuged (400xg) for 10 min and the cell free supernatants IgG content of the supernatants was measured by an ELISA [modified from Voller, Bidwell & Bartlett, 1976]. Microtitre plates (Dynatech, Immulon I) were coated with IgG purified from goat anti-human IgG serum (Cappel) at 10 ug/ml in carbonate-bicarbonate buffer (pH 9.6) by incubation overnight at 4°C. The plates were washed 3 times with P B S and 0.05% (v/v) Tween 20. Serial dilutions of known concentrations of human IgG (from the U.S. National reference preparation for Specific Human Serum Proteins, Centers for Disease Control, Atlanta, GA.) were used as standards in parallel with samples to be tested and added to the wells for 1 h at room temperature. Plates were washed as above and a 1:1000 dilution of alkaline phosphatase conjugated goat IgG anti-human IgG (Tago) was added. After 1 h in the dark at room temperature plates were washed and 100 u,l of p-nitrophenyl phosphate (Sigma 104-105) was added (1 tablet per 10 ml of diethanolamine buffer). After 45 min the absorbance at 405 nm was read on a microELISA spectrophotometer (Dynatech, MR Individuals who consistently (at least 5 assays over a period of 6 months to 2 years) secreted large amounts of IgG ie. >1000 ng were classif ied as high responders (HR), individuals who consistently secreted very low amounts of IgG ie. <1000 ng were classified as low responders (LR) and individuals who secreted variable amounts of IgG were classified as intermediate responders (IR). We studied only the In adjusting the parameters of the IgG secretion assay we observed that the amount of IgG secreted varied with the lot of F C S (Table 2:l) used and that there was a large plateau in the amount of IgG secreted at dilutions of PWM between 1:50 and 1:500 (Table 2:ll). Additionally when the cultures were allowed to incubate for greater than 7 days, the LR individuals' responses remained lower than that of high responders (Table 2:111). We had some cultures from LR and HR which were allowed to grow for up to 12 days. The amount of IgG secreted increased in the order of hundreds of nanograms in the LR cultures and in the order of thousands in the HR cultures (data not shown). Individual responses remained either high or low when the cell concentrations of the cultures remained within 20% of 1X10^ PBMC/ml (Table 2:IV) . 2:2:iii:c Staphylococcus Aureus Cowan strain 1 (SAC) induced IgG secretion; P B M C cultures were stimulated with Staphylococcus Aureus Cowan Strain 1 (SAC), ("Pansorbin"®, Calbiochem, La Jol la, CA, 1:20,000 final dilution) for 10 days, after which time the cell free supernatants were harvested and the amount of IgG was measured by 2:2:iii:d Reconstitution experiments; E+ cells obtained from LR (or HR) individuals were mixed with E-cells obtained from HR (or LR) and the amount of IgG secreted in response to PWM was measured after 7 days in culture. The optimal ratio of E " E+ cells in all PWM stimulated cultures was 1:1, (final In other experiments E- cells were mixed 1:1 with autologous irradiated E+ cells (350 rads and 1350 rads with Cobalt 60 in a Gammacell 220, Atomic Energy of Canada Ltd., Ont). Irradiation inhibits in vivo suppression which can be measured by a Suppressor Index, (SI), [Huddlestone et al., 1982; Kelly et al., 1980] calculated as fo l l ows : - 9 6 -Sl = 1-IgG secreted in cultures containing untreated T cells IgG secreted in cultures containing irradiated T cells. 2:2:iii:e Autologous mixed lymphocyte reaction (AMLR); The AMLR was performed using a modification of the procedure reported by Hafler et al., [1985]. Briefly, E - cells were irradiated (3000 rads) and mixed 1:1 with 1x10 5 autologous E + cells in round bottom microtitre plates. Control cultures consisted of E + cells in media alone. After culture at 37°C for 72 hr in a humidified chamber with 5% CO2, cells were pulsed for 6 hr with 1 u.Ci of TdR and harvested onto glass fiber filters. The results are expressed as the 2:2:iv Lymphocyte subset labelling and analysis: Aliquots of 1x10 6 P B M C were labelled with a combination of either Leu3a-PE (1:25, Becton Dickinson, Mountain View, CA) and 2H4-FITC (1:10, Coulter Immunology, Hialeah, FA) or Leu3a-PE (1:25) and 4B4-FITC (1:20), (Coulter) on ice for 30 min. Controls consisted of unlabeled cel ls. Analysis was done by flow cytometry (FACS IV, Becton Dickinson). Scatter gates were set on the lymphocyte peak and 4 x 1 0 4 cells per sample were counted for the amount of red and/or green fluorescence. Percentage of Leu3a+2H4+ cells, (ie. Tsi, positive for red and for green) was calculated from the 3 dimentional histograms. The percentage of Leu3a+4B4+ cells (ie. Thi, also positive for red and for green) was also determined from the 3 dimentional - 9 7 -histograms generated on the F A C S IV. Results are expressed as a ratio of Leu3a+2H4+/Leu3a+4B4+ (ie. Tsi/Thi). - 9 8 -2:3 Results 2:3:i PWM induced IgG secretion: The amount of IgG secreted by the P B M C obtained from this group of healthy individuals varied from 90 ng to over 10,000 ng. Of the 27 individuals repeatedly tested over a period of 6 months to 2 years, 5 (4 male,1 female) consistently secreted <1000 ng and were classi f ied as low responders (LR), 13/27 (4 male, 9 female) consistently secreted >1000 ng, and were c lassi f ied as high responders (HR). The remaining individuals tested (9/27, 4 male, 5 female) secreted variable amounts of IgG in response to P W M , and were classified as intermediate responders (IR). The ages of the HR group (34+3 years) did not differ significantly from the LR group (36±3 years). In Fig. 2:1 we show the results of repeated measurements of PWM induced IgG secretion in 4 representative LR and 4 representative HR (please note the results are expressed in log). The following ser ies of experiments were des igned to investigate the mechanism of low response at a cellular level. Except for cell subset enumeration assays where individuals were not selected, the subjects used in this investigation had been previously characterized as to the amount of IgG secreted in the PWM IgG secretion assay. - 9 9 -2:3:ii Relationship between DNA synthesis and IgG secretion: As seen in Fig. 2:2b, the 3 individuals tested the IgG secretion response varied markedly both in terms of kinetics and amount. The 2 HR secreted 5600 and 2500 ng by day 9 but the LR secreted only 300 ng. In contrast the level of thymidine incorporation was very similar both in terms of amount and kinetics in all three individuals, peaking at day 6 or 7 and being reduced to background by day 9. 2:3:iii Staphylococcus Aureus Cowan strain 1 (SAC) induced IgG secretion: The PBMC of three of the LR and 3 HR individuals were tested for their ability to secrete IgG in response to S A C . The 3 LR secreted more IgG in response to S A C (870±86 ng) than they did in response to PWM (335±47 ng) but their response to S A C remained significantly less than the response to S A C of the 3 HR (7057+1206 ng, p<.001, by Student t-test) 2:3:iv IgG Secretion in monocyte depleted cultures: Following monocyte depletion, PWM induced IgG secretion was reduced by 71 ±8% (for individual data points see Table 2:V). In contrast when S A C was used to stimulate monocyte depleted cultures, IgG secretion increased by 139±79%. In the individuals who had the highest response to S A C , IgG secretion was marginally reduced by monocyte depletion (-9% and -10%). This is in contrast with the 2 individuals who had the lowest - 1 0 0 -responses in whole PMNC to S A C and exhibited marked increases after monocyte depletion (+366% and +211%). This result suggests that monocytes have an inhibitory role in low response to S A C . 2:3:v Cell subset mixing experiments: In 3 separate experiments, the PBMC of 4 different HR and 4 different LR individuals were separated into E+ and E- subsets, mixed in the various heterologous combinations (eg. HR E+ and LR E" etc.) and stimulated with PWM. Results are presented in Fig. 2.3. Cultures of HR E + and HR E- subsets contained the most IgG whereas cultures of LR E+ and LR E" contained the least IgG. Cultures of HR E+ and LR E- or LR E+ and HR E - contained intermediate amounts of IgG (Fig. 2:3). It is of note that the amount of IgG secreted in autologous PWM stimulated cultures of E+ and E- cells, correlated with the amount of IgG secreted by whole PBMC cultures (r=.84, data not shown). 2:3:vi Effect of irradiation of E+ cells in the PBMC of LR i nd i v i dua l s : We observed a dose response effect when E+ cells irradiated at 2 different doses (350 and 1350 rads) were added to autologous E- cells at a ratio of 1:1. Calculation of the suppressor index revealed that there was more radiation sensitive T suppressor cell activity in the P B M C of 5 LR (0.67+0.12 at 1350 rads and 0.48+0.08 at 350 rads) compared to 5 HR subjects (0.32±0.11 at 1350 rads and 0.31+0.08 at 350 rads, p<.05, Student t-test), but at 1350 rads the increase in the - 1 0 1 -amount of IgG secreted was similar in LR cultures (+1006+234 ng) and HR cultures (+771+ 355 ng). Results of a representative experiment are shown in table 2:VI. After irradiation of the E+ cells, the amount of IgG secreted in the autologous reconstituted LR cultures was significantly lower than the amount of IgG secreted in the autologous reconstituted HR cultures. 2:3:vii Autologous Mixed Lymphocyte Reaction (AMLR) and PWM induced IgG secretion: We have compared the 3 day AMLR in 4 LR and 5 HR individuals. The AMLR was higher (27,643±8441 cpm) in the HR individuals than the A M L R in the LR individuals, (8,103+2124 cpm, p<.05, t-test). Table 2:VII shows a representative experiment where the AMLR response and PWM induced IgG secretion response were measured in para l le l . 2:3:viii T-helper Cell Subset Enumeration and PWM induced IgG secretion: The combina t ion of L e u 3 a and 2H4 ident i f ies T suppressor/ inducer (Tsi) cells which help CD8+ suppressor cells [Morimoto et al. , 1985a], and the combination of Leu3a and 4B4 identifies T helper/inducer (Thi) cells which provide helper signals for PWM induced IgG secretion [Morimoto et al., 1985b]. Following dual staining with Leu3a-PE and 2H4-FITC or Leu3a-PE and 4B4-FITC (see Fig. 2:4) we calculated the Tsi/Thi ratio within the T helper cell subset and correlated this ratio with the amount of IgG secreted in - 1 0 2 -P B M C cultures obtained from several individuals (table 2:VIII). In the 9 male subjects tested, the Tsi/Thi ratio was inversely correlated with the amount of IgG secreted in response to PWM (r= .85, p<.01), no such correlation was found in the 10 females tested. In males the Tsi/Thi ratio was higher in the LR (1.8±.3) than in the HR subjects tested (0.7±.1, p<.02, Student t-test), again no such difference as found in the females tested. - 1 0 3 -2:4 Discuss ion: In a first series of experiments healthy individuals were repeatedly tested in the in vitro PWM-induced IgG secretion assay. We have confirmed and extended earlier findings [Wu et a l . , 1976; Rosenkoetter et al., 1984] that the amount of IgG secreted in response to PWM varies extensively between individuals but less in a given individual. In a group of 27 different healthy subjects we were able to recognize individuals who consistently secreted high or low amounts of IgG. Approximately 50% of the individuals were high responders, 18% of were low responders and the remaining 32% were classified as intermediate responders. Due to the variable responses in the intermediate responders these were not included in the further evaluation of the factors involved in regulation of high and low response. PBMC obtained from HR and LR individuals were used to explore the cellular mechanisms controlling the level of PWM induced IgG secretion. Differences in the PWM induced pro-liferative responses between the P B M C of HR and LR did not account for the large differences in IgG secretion observed. These results corroborate those of Wu et al. [1976] who reported that there was no correlation between the frequency of cells containing cytoplasmic Ig and the incorporation of TdR in PWM stimulated cells. Similarly Haynes and Fauci [Haynes and Fauci, 1979] reported that non-response to S R B C in PWM stimulated PBMC was not a result of a defect in the PWM induced lymphocyte blastogenesis. These results confirm that low IgG - 1 0 4 -secretion response is not due to an inability of the P B M C to pro l i ferate. PBMC of both LR and HR secreted more IgG in response to SAC than they did in response to PWM, however the response to SAC in LR subjects remained less than the response to S A C in the HR subjects. As S A C stimulation of monocytes depleted cultures resulted in large increases in the amount of IgG secreted we conclude that a low response to S A C is essentially under the control of suppressor monocytes. When PWM was used as the polyclonal stimulator, removal of the monocyte subset resulted in reduced IgG secretion both in HR and in LR subjects. The requirement for monocytes in PWM induced IgG secretion is well known [Rosenberg and Lipsky, 1979; Moretta et al . , 1977] but suppressor monocytes in S A C stimulated PBMC cultures have not been reported previously. It is generally accepted that the T cells regulate the amount of IgG secreted in PWM stimulated cultures [Moretta et a l . , 1977; Reinhertz et al., 1980; Thomas et al., 1981]. We have confirmed and extended this observation. Initially we observed that in hetero-logous cell subset mixing experiments cultures containing LR E+ cells contained less IgG than cultures containing HR E + cells. However we also observed that cultures containing HR E+ cells and LR E- cells contained less IgG than cultures of HR E+ and HR E- suggesting that the E- cells are involved in determining the level of response (see further). It has been reported that the amount of IgG secreted in PWM stimulated P B M C cultures is determined by the functional integrity of the regulatory T suppressor cell subset [Rosenkoetter et al., 1984]. - 1 0 5 -Irradiation of the T lymphocyte subset inhibits suppression but does not appear to affect help in PWM induced differentiation of B cells to Ig secreting cells [Fauci et al., 1978; Wasserman et al., 1979]. We calculated the level of in vivo radiation sensitive suppression using 2 different methods. We first calculated the net increase in IgG contained in cultures with irradiated E+ cells over the amount contained in control cultures. This net increase was similar in both HR and LR individuals. We also calculated the "suppressor index" which other investigators have used [Huddlestone et al., 1982; Kelly et al., 1980] to measure in vivo radiation sensitive suppression. This index revealed high radiation sensitive T suppressor cell activity in the P B M C of LR individuals. This observation is in agreement with past results generated by Antel et al. [Rosenkoetter et al . , 1984] where they reported that the level of suppressor activity within the CD8+ subset was inversely correlated with the amount of IgG secreted in whole PBMC cultures. We are not convinced however that the use of this index is representative of in vivo suppression as it incorporates the baseline PWM induced IgG secretion response (see materials and methods) and thus introduces a bias. The AMLR measures the proliferative response of CD4+ lymphocytes [Kotani et al., 1984] to autologous HLA-Dr Ag on B cells and monocytes [Palacios and Moller, 1981]. It has been shown that the T cells isolated after a 3 day AMLR posses potent helper activity in the synthesis of Ig by autologous PWM stimulated B cells [Morimoto et al., 1985b]. We compared the AMLR after 3 days in culture in HR and LR and found that the response was lower in the cultures of the 4 LR than in the 5 HR cultures. If the level of proliferation after a 3 day - 1 0 6 -AMLR correlates with CD4+ T helper cell activity, this would indicate increased T helper cell activity in the P B M C of HR individuals compared to the helper activity in the PBMC of LR individuals. The T helper cell subset is composed of at least 2 functionally distinct subpopulations which can be identified by mutually exclusive surface markers. Cel ls expressing the Leu3a+4B4+ phenotype (T helper-inducer cells, Thi) provide help in PWM induced IgG secretion responses [Morimoto et al . , 1985b] whereas cells expressing the Leu3a+2H4+ markers (T suppressor-inducer cells, Tsi) help the CD8+ suppressor cells suppress PWM induced IgG secretion [Morimoto et al 1985a]. We have found that only in males did the ratio of Tsi/Thi cells within the Th cell subset correlate inversely with the amount of IgG secreted in response to PWM. Morimoto [Morimoto et al., 1987] has reported that the percentage of T helper cells expressing the T suppressor/inducer marker was inversely correlated with the amount IgG secreted by PWM stimulated P B M C in active MS patients, (this group did not comment on the sex of their patients). It appears that in male subjects LR may be due to an increased Tsi/Thi ratio in the Th cell subset. We observed that only one of the 5 consistent LR was female whereas 9 of the 13 HR were female. This difference indicates a trend suggesting that females secrete more IgG in response to PWM than males, however our sample size is too small to make a general statement concern ing the populat ion of healthy indiv iduals. Differences in the immune systems of males and female have been reported previously. Abo et al have reported that the number of -1 0 7 -natural killer cells is higher in the peripheral blood of adult women than men [Abo et al., 1982]. In this series of experiments we have confirmed that the T helper and T suppressor cells are involved in the generation of low response but we also report here that there are properties of the E-subset which may play a role in controlling the amount of IgG secreted. We have 2 lines of evidence implicating the E- subset in the control of the level of response to PWM: first of all the PBMC obtained from LR also secreted less IgG in response to S A C and second, cultures of LR E- cells secreted less IgG than the E- cells obtained from HR when both were reconstituted with heterologous HR E + cells. E- cells are composed of B cells and monocytes. In PWM stimulated P B M C cultures, depletion of the monocytes reduced IgG secretion in both HR and LR subjects. This would indicate the necessity for processing PWM by monocytes. Monocytes appear not to be responsible for a low response to PWM but they could be implicated in low response to other antigens as suggested by their contribution to a low response to S A C . As monocyte function is not involved in low response to PWM, we would suggest that the B lymphocyte subset is. This would confirm the suggestion of other groups that B cells themselves are involved in low response [Haynes and Fauci, 1979; Antel et al., 1983]. In summary we have shown that the Tsi/Thi ratio, the T suppressor index, the AMLR and the B cell response differ in HR and LR individuals and it is possible that these in vitro functions vary together. It would now be interesting to measure the production and/or response of purified lymphocytes to lymphokines which are known to - 1 0 8 -be operative in the induction or suppression of B lymphocyte proliferation and differentiation. - 1 0 9 -Table 2:l. The effect of four different lots of fetal calf sera on amount of IgG (ng/ml) secreted by 1X10 6 PWM stimulated PBMC obtained from 2 different individuals. Lot of Fetal Calf Sera Subject A§ B1f Cf D¥ mean±SEM mean±SEM mean±SEM mean±SEM LR#1 472±22 371±14 352±7 369±4 HR#2 2262+240 3614±203 2455+231 1066+249 § - Fetal calf sera obtained from GIBCO (Grand Island, NY), lot # 19N2655 H - Fetal calf sera obtained from GIBCO, lot # 11N2262 t - Fetal calf sera obtained from Flow (Mclean, VA), lot # 29101121 ¥ - Fetal calf sera obtained from Flow, lot # 29101116 -11 0-Table 2:11. The effect of different concentrat ions of Pokeweed Mitogen (PWM) on the level of IgG secreted by PBMC into the culture supernatant. Subject PWM concentration (final dilution) 0 1/600 1/500 1/400 1/300 1/200 1/100 1/50 #1 330§ NAU NA 3614 NA 2371 1675 1458 #2 160 1178 1128 3172 2878 3886 2850 2850 #3 315 3572 3104 3988 2854 2838 3088 4328 #4 408 359 412 371 399 367 395 406 §- IgG secreted (ng/ml) by 1X106 PBMC %- NA-results not available - 1 1 1 -Table 2:lll. The rate of PWM induced IgG secretion in PBMC obtained from healthy individuals. Subject Day 05 Day 06 Day 07 Day 08 Day 09 #1 889§ 1292 3373 4217 5175 #2 775 1168 1504 2216 1362 #3 30 30 53 176 121 #4 1 20 173 229 282 160 §- IgG, ng/ml secreted by 1 x 10 6 PWM stimulated PBMC -1 1 2 -Table 2:1V. The level of IgG secreted into the culture supernatant by different concentrations of PWM stimulated PBMC Subject Concentration of PBMC/ml 5.0x10 6 2.5x10 6 1.0x106 0.5x10 6 0.25x10 6 0.1x106 #1 275§ 1222 1200 1412 134 50 #2 150 88 56 50 48 54 §-ng of IgG secreted into 1 ml of culture supernatant -1 1 3 -g G ( n g ) 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 4.00 3.50' 3.00 2.50' 2.00 1.50-1.00' 0.50 0.00-4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 HR#1. HR#2 1 2 3 4 5 6 7 8 9 10 11 12 HR#3 1 2 3 4 5 6 7 LR#1 1 2 3 4 5 6 7 8 9 101112131415 LR#3 1 3 5 7 9 11 13 15 17 19 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 1 2 3 4 5 6 7 4.00r 3.50 • 3.00 2.50 2.00 1.50 1.00 0.50 0.00 HR#4 4.00 3.50 • 3.00 2.50 2.00 1.50 1.00 0.50 0.00 1 2 3 4 5 6 7 LR#2 1 3 5 7 9 11 13 15 17 19 21 23 4 .00 T LR#4 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 3 4 5 6 Number of Assays Fig. 2:1. Results of repeated PWM induced IgG secretion by PBMC obtained from 8 healthy individuals, 4 of these individuals consistently generated a high response (HR) and the other 4 individuals consistently generated a low response (LR). The ordinate represents the amount of IgG (expressed in logio) secreted into 1ml of culture supernatant by 1X106 cells over 7 days. The abscissa represents the number of times the subject was tested. The horizontal line at 1000 ng (log 3.0) represents an arbitrary cutoff point between a high and a low response. -11 4 -A. o •) 1 1 1 1 1 1 3 4 5 6 7 8 9 Days in Culture • - HR #1 -o- LR #1 -X- HR #2 B. 6000 ^ 3 4 5 6 7 8 9 Days in Culture • • - HR #1 ° - LR #1 -X- HR #2 Fig. 2:2. A- DNA synthesis measured by 18 hr pulses with tritiated thymidine at the end of the indicated days in PWM stimulated cultures of 2.5xl05 PBMC obtained from 3 different subjects and B- Amount of IgG in the supernatants of these same cultures. -1 1 5-T a b l e 2 : V . L e v e l of IgG s e c r e t e d in r e s p o n s e t o P W M a n d to S A C in w h o l e P B M C c u l t u r e s a n d in m a c r o p h a g e d e p l e t e d (M0-ve) c u l t u r e s Subject Culture % esterase +ve Cells Amount of IgG Secreted in response to: PWM SAC (% Change)* #1 PBMC 33 620 1820 M0 - v e 4 186 (-70)t 8484 (+366) #2 PBMC 21 2130 2646 M0 - v e 3 534 (-75) 8228 (+211) #3 PBMC 13 6900 8960 M 0 -ve 1 3634 (-47) 8150 (-9) #4 PBMC 1 5 13740 16340 M0 - v e 3 924 (-93) 14676 (-10) *% change in macrophage depleted cultures = IgG secreted in M0 - ve cultures 1-IgG secreted in whole PBMC cultures treduction = -, and increase = + x100 -1 1 6-(3 1 2 0 0 -1 1 0 0 -1 0 0 0 -900 800 7 0 0 -6 0 0 -5 0 0 -4 0 0 -3 0 0 -2 0 0 -100 0 HR E-+ HR E+ LR E-+ HR E+ HR E-+ LR E+ LR E-+ LR E+ Heterologous Subset Cultures Fig. 2:3. PWM induced IgG secret ion (mean+SEM) in heterologous mixed cultures of .5x10 6 E~ cells and .5x10 6 E+ cells. The subsets were designated HR (high responder) or LR (low responder) when the autologous reconstitution was greater than or less than 1000 ng respectively. The data represent the cumulat ive results of 3 different experiments involving a total of 4 LR individuals and 4 HR i nd i v i dua l s . -1 1 7-Table 2:VI. The amount of vivo radiation sens i t ive suppression in the PBMC of 2 low responders and 2 high responders calculated using 2 different methods Subject PWM Induced E- + E+(400 rads) E- + E+(1350 rads) IgG Secretion A l g G t Tsi§ A l g G t Tsi§ (ng)* LR#1 261 21 .34 1137 .67 LR#2 335 295 .81 1571 .96 HR#1 1447 732 .48 .1030 .57 HR#2 2970 130 .04 231 .07 * IgG secreted (ng) by 1 x 10 6 PBMC stimulated with PWM and cultured for 7 days t AlgG= (IgG secreted in cultures of 5x10 5 E- cells and 5x10 5 irradiated E+ cells)- (IgG secreted in cultures of 5x10 5 E- cells and 5x10 5 untreated E+ cells). § Tsi = (IgG secreted in cultures of 5x10 5 E- cells and 5x10 5 untreated E+ cells) 1-(IgG secreted in cultures of 5x10 5 E- cells and 5x10 5 irradiated E + cells). -1 1 8-Table 2: V11. In vitro PWM induced IgG secret ion and autologous mixed lymphocyte reaction in PBMC cultures derived from the same whole blood sample of 2 low responder (LR) and 2 high responder (HR) individuals Subject PWM Induced Autologous Mixed IgG Secretion Lymphocyte Reaction (ng/ml)1f (cpm)§ LR#1 169 5665 LR#2 940 4163 HR#1 1102 34437 HR#2 2027 28222 1J-The amount of IgG secreted into 1 ml of supernatant by 1x10 6 PBMC stimulated with PWM. §-The amount of tritiated thymidine incorporated by .5x10 6 E+ cells stimulated with .5x10 6 irradiated autologous E- cells after 3 days in culture. -1 1 9-Table 2:VIII. The ratio of T-suppressor/lnducer cells to T-helper/inducer cells (Tsi/Thi) and the level of PWM induced IgG secretion in the peripheral blood mononuclear cells of healthy male and female subjects. Male Level of PWM induced Tsi/Thi Female Level of PWM induced Tsi/Thi Subjects IgG secretion response Subjects IgG secretion response (no/ml) (ng/ml) LR#1* 136§ 2.2 LR#2 150 2.9 LR#3 300 1.5 LR#1 130 0.8 LR#4 421 1.2 LR#2 172 1.5 LR#5 832 1.3 LR#3 196 0.9 Mean±SEM 367±127 1.8±.3 166±19 1.1+.2 HR#1 1058 1.7 HR#2 1640 1.6 HR#1 1305 0.8 HR#3 2000 0.9 HR#2 2080 1.0 HR#4 3526 1.2 HR#3 3412 0.5 HR#5 3660 1.7 HR#4 5282 0.4 HR#6 6910 1.4 Mean ± SEM 2998±871 0.7±.11f 3132±866 1.4+.1 *LR- low response, ie. this individual secreted less than 1000 ng of IgG in the PWM IgG secretion assay. IfTsi/Thi in LR males different from Tsi/Thi in HR males at p<.025, Student t-test. §amount of IgG secreted correlates inversely with the Tsi/Thi ratio, r =-.85. p<.01. - 1 2 0 -Fig. 2:4. Two color cytometric analysis of PBMC using the Leu3a and 2H4 mAb, (T-suppressor/inducer, Tsi) or the Leu3a and 4B4 mAb, (T-helper/inducer, Thi) in the blood of one HR and one LR. This is a 3 dimentional histogram where cell number is expressed along the vertical axis, the amount of red fluorescence (Leu3a+) is displayed along the Y axis and the amount green fluorescence (either 2H4+ or 4B4+) is displayed along the X axis. Cells expressing both ant igens are shown in the center of the diagrams. Indiv idual d iag rams represent two co lo r f requency histogram of: Ai/ the PBMC obtained from an HR (2148±369 ng), labelled with Leu3a and 2H4 (Leu3a+2H4+=14.3%) and Aii. the PBMC of the same individual labelled with Leu3a and 4B4 (Leu3a+4B4+=32.1%) and Bi. the PBMC obtained from an LR (161±9 ng) labelled with Leu3a and 2H4 (Leu3a+2H4+=44.1%) and Bi i. the PBMC of the same i n d i v i d u a l l a b e l l e d w i th L e u 3 a a n d 4 B 4 , (Leu3a+4B4+=10.9%). The Ts i/Thi ratio in this high responder was 0.4 and the Tsi/Thi ratio in this low responder was 4.0. vertical ax -121--1 2 2 -2:5 References: Abo, T., M. D. Cooper, and C. M. Balch. Postnatal expansion of the natural killer and killer cell population in humans identified by the monoclonal HNK-1 antibody. J . Exp. Med. 155:321 (1982). Antel, J . P., J . J . -F . Oger, L. G . Wrabetz, B. G. W. Arnason and J . E. Hopper . Mechan i sms respons ib le for reduced in vitro immunoglobulin secretion in aged humans. Mech. Age. Develop. 23:11 (1983). Boyum, A. Separation of leukocytes from blood and bone marrow. Scand. J . Clin. Lab. Invest (Suppl. 97) 21; 1. (1968). de Vries, J . E., A. P. Caviles, JR. , W. S. Bont and J . Mendelsohn. The role of monocytes in human lymphocyte activation by mitogens. J . Immunol. 122:1099 (1979). Fauci , A. S., K. R. Pratt and G . Whalen. Activation of human B lymphocytes. VIII. Differential radiosensitivity of subpopulations of lymphoid cel ls involved in the polyclonal ly- induced P F C responses of peripheral blood B lymphocytes. Immunology 35:715 (1978). - 1 2 3 -Gordon, J . , and Guy, G. R. The molecules controlling B lymphocytes. Immunol. Today 8:339 (1987). Hafler, D. A., M. Buchsbaum and H. L. Weiner. Decreased Autologous mixed lymphocyte reaction in multiple sclerosis. J . Neuroimmunol. 9:339 (1985). Haynes, B.F., A. S. Fauci. Activation of human B lymphocytes. X111. Characterization of multiple populations of naturally occurring immunoregulatory cells of polyclonally induced in vitro human B cell function. J . Immunol. 123:1289 (1979). Hirsch, R. L. Defective autologous mixed lymphocyte reactivity in multiple sclerosis. Clin. exp. Immunol. 64:107 (1986). Huddlestone, J . R., and M. B. A Oldstone. Suppressor T cells are activated in vivo in patients with multiple sclerosis coinciding with remission from acute attack. J . Immunol. 129:915 (1982). Kelly, R. E., G. W. Ellison, L. W. Myers, V. Goymerac, S. B. Larrick and C. C. Kelly. Abnormal regulation of in vitro IgG production in multiple sclerosis. Ann. Neurol. 9:267 (1980). Kotani, H., S. Takada, Y. Ueda, Y. Murakawa, N. Suzuki and T. Sakane. Activation of immune regulatory circuits among OKT4+ cells by -1 2 4 -autologous mixed lymphocyte reactions. Clin. exp. Immunol. 56:390 (1984). Ly, I. A., and R. I. Mishell. Separation of mouse spleen cells by passage through columns of Sephadex G-10. J . Immunol. Methods 5:239 (1974). Moretta, L , S. R. Webb, C. E. Grossi, P. M. Lydyard and M. D. Cooper. Functional analysis of two human T-cell subpopulations: help and suppression of B-cell responses by T cells bearing receptors for IgM or IgG. J . exp. Med. 146:184 (1977). Morimoto, C , D. A. Hafler, H. L. Weiner, N. L. Letvin, M. Hagan, J . Daley, and S. F. Schlossman. Selective loss of the suppressor-inducer T-cell subset in progressive multiple sclerosis. Analysis with Anti-2H4 monoclonal antibody. N. Eng. J . Med. 316:67 (1987). Morimoto, C , E. L. Reinhertz, Y. Borel, E. Mantzouranis, A. D. Steinberg and S. F. Schlossman. Autoantibody to an immunoregulatory inducer population in patients with juvenile rheumatoid arthritis. J . Clin. Invest. 67:753 (1982). Morimoto, C , N. L. Letvin, A. W. Boyd, M. Hagan, H. M. Brown, M. M. Kornacki and S. F. Schlossman. The isolation and characterization of the human helper inducer T cell subset. J . Immunol. 134:3762 (1985b). -1 2 5 -Morimoto, C , N. L. Letvin, J . A. Distaso, W. R. Aldrich, and S. F. Schlossman. The isolation and characterization of the human suppressor inducer T cell subset. J . Immunol. 134:1508 (1985a). O'Garra, A., S. Umland, T. DeFrance and J . Christiansen. B-cell factors' are pleiotropic. Immunol. Today 9:45 (1988). Oger, J . , Antel, J P . , Kuo, HH. , and Arnason BGW. Influence of Azathioprine (Imuran) on immune function in multiple sclerosis. Ann. Neurol. 11; 177 (1982). Palacios, R. and G. Moller. HLA-DR antigens render resting T-cells sensitive to interleukin 2 and induce production of the growth factor in the autologous mixed lymphocyte reaction. Cel l . Immunol. 63:143 (1981). Reinhertz, E. L., C. Morimoto, A. C. Penta and S. F. Schlossman. Regulation of B cell immunoglobulin secretion by functional subsets of T lymphocytes in man. Eur. J . Immunol. 10:570 (1980). Reinhertz, E. L., C. Morimoto, A. C. Penta, and S. F. Schlossman. Supopulations of the T4+ inducer T cell subset in man. Evidence for an amplifier population preferentially expressing la antigen upon activation. J . Immunol. 126:67 (1981). Reinhertz, E. L., C. Morimoto, K. A. Fitzgerald, R. E. Hussey, J . A. Daley and S. F. Schlossman. Heterogeneity of human T4+ inducer as -1 2 6 -defined by a monoclonal antibody that delineates two functionally unique subpopulations. J . Immunol. 128:463 (1982). Reinhertz, E. L , P.C. Kung, G. Goldstein and S. F. Schlossman. Further characterization of the human inducer T cell subset defined by monoclonal antibody. J . Immunol. 123:2894 (1979). Rosenberg, S. A., and P. E. Lipsky. Monocyte dependence of Pokeweed mi togen- induced differentiation of immunoglobul in-secret ing cells from human peripheral blood mononuclear cells. J . Immunol. 122:926 (1979). Rosenkoetter, M., A. T. Reder, J . J . -F . Oger, and J . P. Antel. T cell regulation of polyclonally induced immunoglobulin secretion in humans. J . Immunol. 132:1779 (1984). Saxon, A., J . L. Feldhaus and R. A. Robbins. Single step separation of human T and B cells using AET treated S R B C rosettes. J . Immunol. Methods. 12:285 (1976). Siegal, F., and M. Siegal. Enhancement by irradiated T cells of human plasma cell production: Dissection of helper and suppressor functions in vitro. J . Immunol. 118:642 (1977). Thomas, Y., J . Sosman, O. Irigoyen, L. M. Friedman, P. C. Kung, F. Goldstein, and L. Chess. Functional analysis of human T cell subsets defined by monoclonal antibodies. I. Collaborative T-T -1 2 7 -interactions in the immunoregulation of B cell differentiation. J . Immunol. 125:2402 (1980). Thomas, Y., L. Rogozinski, O. H. Irigoyen, S. M. Friedman, P. C. Kung, G. Goldstein and L. Chess. Functional analysis of human T cell subsets defined by monoclonal antibodies. IV. Induction of Suppressor Cells within the OKT4+ population. J . exp. Med. 154:459 (1981). Voller, A., Bidwell, D. & Bartlett, A. Microplate immunoassays for the immune diagnosis of viral infections. In: Manual of Clinical Immunology (eds N. Rose & H. Freedman) p. 506. American Society of Microbiology, Washington DC. (1976) Wasserman, J . , L.V. Von Stedingk, G. Biberfeld, B. Petrini, H Blomgren and E. Baral. The effect of irradiation on T-cell suppression of ELISA-determined Ig production by human blood B-cells in vitro. Clin. exp. Immunol. 38:366 (1979). Wu, L. Y. F., A. Blanco, M. D. Cooper and A. R. Lawton. Ontogeny of B-lymphocyte differentiation induced by Pokeweed mitogen. Cl in. Immunol, and Immunopathol. 5:208 (1976). - 1 2 8 -Chapter 3 IMMUNE FUNCTION AND DISEASE ACTIVITY IN MULTIPLE SCLEROSIS 3:1 Introduction Early theories of the etiology of multiple sclerosis suggested that a viral infection encountered early in life initiated the disease process which was expressed several years later. However the inability to unequivocally link a specific virus or group of viruses to MS despite intensive investigation has led to the development of immunological theories in the cause of MS. The existence of an animal model, chronic experimental allergic encephalomyelit is induced by immunization with basic protein of myelin, (which mimics MS in many of its clinical and pathological features [Lassman and Wisniewski, 1979]) suggests that MS may also be an autoimmune disease. The over-representation of certain HLA antigens in the MS population as compared to the healthy population [reviewed by Oger and Arnason, 1980] and the observation that this phenomena has been reported in other autoallergic diseases (eg. Goodpasture's syndrome), [Rees et al , 1978] provide further support for the hypothesis that autoimmunity may be involved in the cause or progression of MS. Immunological abnormalities in the peripheral blood, brain and cerebrospinal fluid (CSF) compartments of MS patients as reviewed in chapter 1, provides the most convincing -1 2 9 -evidence that immunological mechanisms are involved in the cause and/or progression of MS. The course of MS may take the form of a relapsing remitting disease (that may eventually become progressive) or of a progressive disease from the onset. In the relapsing remitting form of the disease it is necessary to define an attack. An attack or relapse is usually defined as the appearance of a new symptom or the reappearance of a previous symptom lasting more than 24 hours and occurring at any time after the initial attack [McAlpine 1972]. Since there is no laboratory method for assessing disease activity, the determination of a relapse in cases where the symptoms may not be particularly convincing and due perhaps to infection, depression, or fever rests on the experience of the attending neurologists. The use of brain and spinal chord imaging techniques (magnetic resonance imaging) to monitor the course of MS is much more sensitive than clinical cr i ter ia, computer ized axial tomography or electrophysiological methods in detecting the appearance of new "lesions" [Paty et al, 1988]. It is well recognized that while a patient is clinically stable there may be new lesions appearing in "clinically silent areas". It is conceivable that the above phenomenon would pose problems in immune function studies based on clinical disease activity by causing large overlaps in the results obtained between clinically active and clinically silent MS patients. The onset of the progressive form of MS is usually associated with a later age than the remitting form. Reports of the proportion of patients who are progressive from the onset varies from 10 [McAlpine, 1972] to over 25% [Sheperd, 1979]. The variations probably - 1 3 0 -stem from the difficulties in retrospective analysis [Matthews, 1985, from Millar, 1949]. Reports of the proportion of patients who become progressive after a period of the relapsing remitting form are also variable due to the fact that the number increases with increasing length of follow-up [Matthews, 1985]. There are an unpredictable number of patients with the progressive form of MS whose pathological [Farrell et al., 1985] and clinical disease stabilize [Miller et al., 1988]. One of the most consistent pathological findings in MS is the occurrence of intrathecal immunoglobulin synthesis [Walsh et al . , 1983]. This immunoglobulin is separated into oligoclonal bands when the cerebrospinal fluid is electrophoresed [Johnson et al., 1977]. The specificity of the majority of this immunoglobulin is not known. We believe that the abnormalit ies in immunoglobulin synthesis and secretion within the C N S of MS patients are relevant to the disease pathogenesis and any attempt to resolve the pathogenesis of MS will have to incorporate these phenomena. In an attempt to clarify the abnormalities in IgG secretion observed in vivo, we have used the PWM induced IgG secretion as a model of the in vivo humoral immune response and studied in vitro immune regulation in groups of MS which have been strictly classified in terms of clinical disease activity. Patients were classif ied as either relapsing-remitting (RR) or as chronic-progressive (CP). The RR group was further divided according to the attending neurologist into RR-active (RR-A) if they were experiencing an attack or RR-stable (RR-S) if they were not. The - 1 3 1 -RR-A group was further subdivided according to the duration of the clinical relapse at the time of immune function testing. The chronic progressive (CP) MS patients were also subdivided into CP-act ive (CP-A) or CP-stable (CP-S) based on the clinical progression of the disease (assessed by the Extended Kurtzke Disability Status Scale, [Kurtze, 1983]) during the year preceding immune function testing. Changes in immune function associated with changes in clinical disease activity have been documented by several groups, (for review see Chapter 1). In the RR group of MS patients we observed differences in immune function between the subgroups of patients studied. Most recently, Oger et a l . , [1988] reported that patients who developed large lesions as visual ized by magnetic resonance imaging (MRI), developed associated changes in immune function. In the C P - M S patients we have obtained evidence suggesting that abnormal i t ies in in vitro immunoglobul in secret ion are associated only with the clinically active group of C P - M S . Recently Miller et al., [1988] reported that a substantial proportion of C P - M S patients stabilize. A report by Farrell et al., [1985] indicated that the C N S of patients with "burnt out" C P - M S no longer showed signs of inf lammat ion. These results and our observations suggest that immune abnormalities in chronic progressive MS are associated with the progression of clinical disease. Previous reports of in vitro immune regulation in chronic progressive patients are fairly consistent suggesting that a/ the ratio of T helper cells over the number of T suppressor cells is increased [Bach et al., 1980; 1985; Weiner et al., 1984; Mingioli & McFarlin, 1984; Craig et al., 1985], bl suppressor cell function is - 1 3 2 -reduced [Antel et al., 1978; Arnason & Antel., 1978] and c/ PWM induced IgG secretion is elevated compared to healthy control individuals [Goust, Hogan & Amaud, 1982; Oger et al., 1982; Antel et al., 1984; Tjernlund et al., 1984]. We have confirmed and extended all of these findings and additionally we provide the first report that the P B M C of chronic progressive MS patients have a reduced ability to suppress in vitro immunoglobulin synthesis. - 1 3 3 -3:2 Materials and Methods 3:2:i Patients: The MS patients involved in this investigation were seen at the UBC Acute Care Hospital's outpatient clinic; all had clinically definite MS according to Poser's committee criteria [Poser et al.,1983] and had not been treated with anti-inflammatory or immunomodulatory drugs during the previous 2 months. Of all the MS patients visiting the clinic, 43 chronic progressive MS and 24 relapsing remitting MS patients had blood drawn for immunological studies. There were 2 control groups; healthy volunteers (HC) and other neurological disease patients (OND). The latter consisted of patients suffering from non-inflammatory neurological d iseases (Seizure disorders, 6; migraines, 6; tumor,1; dystonia,1; Huntington's chorea, 1; hyster ical aphon ia ,1 ; hyperthyroid, 1; myasthenia gravis, 1; narcolepsy, 1 and sciatica, 1). 3:2:i:a MS patient Subgroups; Relapsing Remitting-Attack; The patients who presented at the UBC MS clinic due to either the appearance of new symptoms or the worsening of a previous symptom as assessed objectively by the neurologist, were classified as relapsing remitting attack (RR-A). These patients were further classified according to the length of time which had passed between the onset of the attack and the immune function test. Group 1 included patients that were studied during the first 3 weeks of a -1 3 4 -relapse, group 2 included patients that were studied between 3 and 10 weeks after the onset of a relapse. Stable patients were classified as such if they had remained free of clinical attacks during the 6 months preceding immune function testing. 3:2:i:b RR-stable MS patients; Prognostic value of the PWM induced IgG secretion assay; Patients who presented at the MS clinic and were classified as stable according to the above criteria had blood drawn and PWM induced IgG secretion measured. The patients were subsequently followed clinically for a 6 month period and were grouped a posteriori into group 1 (if they suffered a relapse) or group 2 (if they remained stable, see fig. 3:1). The results of the PWM induced IgG secretion in both groups were assessed as a possible prognostic indicator. 3:2:i:c Chronic Progressive MS; The charts of MS patients who had been classified as chronic progressive were evaluated without prior knowledge of immune function results for the progression of their disease during the 12 months prior to immune function testing. Patients whose clinical condition had not worsened (ie. lost 1 point on the Extended Kurtzke Disability Status Scale, (EKDSS), [Kurtzke,1984]) were reclassified as chronic progressive-stable (CP-S). The MS patients who had lost at least 1 point on the E K D S S were reclassified as chronic progressive-active (CP-A). - 1 3 5 -3 :2 : i i I m m u n e f u n c t i o n s t u d i e s : 3 : 2 : i i : a P W M i n d u c e d IgG s e c r e t i o n ; This assay was performed on peripheral blood mononuclear cells (PBMC) isolated from whole blood, washed and cultured as reported in chapter 2. Briefly, 1x10 6 PBMC were cultured with PWM in one ml of RPMI 1640 (10% fetal calf serum, 2 mM L-glutamine and 10 mg of Gentamicin sulfate/100 ml of medium, RPMI complete) for 7 or 10 days at 37°C. After this incubation period the supernatants were harvested and stored at -70°C until assayed for IgG content by a sensitive ELISA. 3 : 2 : i i : b C o n A s u p p r e s s o r c e l l a s s a y : Our assay is slightly modified from that reported by Shou et al., [1976]. Mononuclear cells were washed at least four times and 1 x 10 6 cells/ml were resuspended in RPMI 1640 with 10% fetal calf serum, 200 jig gentamicin per 100 ml of medium and 2 mM L-glutamine (standard culture medium). Aliquots of 5 ml were placed in vertically-held 25 c m 2 tissue culture flasks and cultured with Concanavalin A (Con A) at 3 ng/ml ('S' cells) or without Con A ('C cells). After 3 days the cells were treated with mitomycin C (25 mg/ml) for 30 min. at 37°C, washed three times with balanced salt solution and resuspended. Triplicate 100 (0.I samples (105) of either ' C cells or 'S ' cells were then added to 100 uJ (105) of heterologous responder cells ('R' cells) plus Con A (3 |ig/ml) for 3 days at 37°C. One microcurie of tritiated thymidine was added to all cultures for the final 6 h. The percentage suppression was calculated as follows: - 1 3 6 -cpm in "R" + "S" cells %S= 1- x 100 cpm in "R" + "C" cells 3:2:i ix ConA induced suppress ion of PWM induced IgG secretion; This technique is a modification of an assay initially de-veloped by Schwartz et al., [1977]. Briefly, 10 7 PBMC, isolated and washed as above were resuspended in 10 ml of RPMI complete in vertically held 75 c m 2 tissue culture flasks with Con A (6u.g/ml) and cultured for 48 hr. at 37°C. These cells are referred to as suppressor cells or 'S ' cel ls. Control cultures consisted of the same number of cells incubated in culture medium alone and are referred to as ' C cells. After this time period both 'S ' and ' C cells were washed at least 4 times in cold RPMI complete. Graded numbers of 'S ' or ' C cells were then added to 2x10 6 heterologous high responding (ie. 1x10 6 P B M C secreted >1000 ng of IgG in response to PWM after 7 days in culture) P B M C , 'R' cells and stimulated with PWM for 7 days after which the supernatants were harvested, assayed for IgG and the percent suppression calculated according to the following formula; x 100 IgG secreted in " S " + "R" cultures %S= 1-IgG secreted in "C" + "R" cultures - 1 3 7 -3:2:ii:d T cell subset enumeration: One million PBMC isolated and washed as above were labelled with saturating amounts of fluorescein-conjugated mouse monoclonal antibody: Leu1 for CD5, Leu3a for CD4, and Leu2a for CD8. Cells were stained on ice for 30 min and washed. Positivity was read from a FACS IV (Becton Dickinson) with gating on the lymphocyte scatter peak. Results were expressed as percent positive cells and the ratio of the CD4+ to the CD8+ cells was calculated. 3:2:ii:e T helper cell subset labelling and analysis; Al iquots of 1x10 6 P B M C isolated and washed as above were labelled with either of the following combinations of monoclonal Ab; Leu3a-PE (1:25, Becton Dickinson, Mountain View, CA) and 2H4-FITC (1:10, Coulter Immunology, Hialeah, FA) or Leu3a-PE (1:25) and 4B4-FITC (1:20, Coulter Immunology) on ice for 30 min. Controls consisted of un labe led ce l l s . Ana l ys i s was per formed by f low cytofluorographics on a F A C S IV (Becton Dickinson). The scatter gates were set on the mononuclear cell peak and 4x10 4 cells were analyzed in "DP" mode for the amount of red or green fluorescence. Percentage of Leu3a+ve cells expressing the 2H4 antigen was calculated by dividing the number of Leu3a+2H4+ (T suppressor/inducer, Tsi) cells (ie. positive for red and green) by the total number of Leu3a+ve cells. The percentage of Leu3a+ve cells expressing the 4B4 Ag was calculated by dividing the number of Leu3a+4B4+ve (T helper/inducer, Thi) cells (ie. positive for red and green) by the total number of Leu3a+ve cells. A Tsi:Thi ratio was then calculated by dividing the - 1 3 8 -percentage of Leu3a cells expressing the 2H4 Ag by the percentage of Leu3a cells expressing the 4B4 Ag. - 1 3 9 -3:3 Results 3:3:i:a PWM induced IgG secretion in Relapsing Remitting MS patients; RR-attack; The RR MS patients enrolled at the UBC clinic are asked to report to the clinic if they develop either a new symptom or the worsening of a previous symptom. Eleven patients visited the clinic due to the onset of a clinical relapse and had their blood drawn for immune function studies. The level of PWM induced IgG secretion was significantly lower in the recent relapse group (2691 ±956 ng) than the level secreted by the older relapse group (8742±1262 ng, p<.01, Student t-test, see table 3:l). There was a significant correlation (r=.739, p<.01, Fig. 3:2) between the amount of IgG secreted in 10 day cultures and the length of time since onset of attack in this group of patients. We used a cutoff point of 3 weeks to separate the RR-MS patients in recent relapse (seen at the clinic <3 weeks after the onset) from the older relapse patients (seen at the clinic between 3 and 10 weeks). 3:3:i:b PWM induced IgG secret ion in Stable MS as a prognost ic indicator; We measured PWM induced IgG secretion in RR-MS patients who were and had been clinically stable for at least 6 months. After the immune function test the patients were followed for another 6 months and then grouped a posteriori according to whether or not they had developed a clinical attack (see Fig. 3.1); Group 1 had an attack and Group 2 remained stable (table 3:ll). Of the 7 patients who went on the have an attack 6 were initially high responders (ie. secreted - 1 4 0 ->1000 ng). Of the 13 patients in group 2, only 6 were high responders. The amount of IgG secreted by Group 1 patients (2757±429 ng) was higher than the amount secreted by group 2 patients (1750±531 ng) although the difference was not significant by Student t-test. 3:3 : i : c Chronic progressive MS patients; Of the 43 C P - M S patients seen at the clinic, 27 had the PWM induced IgG secretion measured at 7 days and 31 were measured at 10 days. At 7 days the C P - M S patients (2608±278) secreted significantly more IgG than the healthy control group (1306±310 ng, p<.01, Student t-test). The frequency distribution of the response is illustrated in Fig. 3.3. Only 4 of the 27 C P - M S patients secreted less than1000 ng of IgG compared to 19 out of 30 healthy controls (Chi sqare = 13.9, p<.001). In an effort to increase the difference in PWM IgG secretion between C P - M S and healthy controls we lengthened the culture incubation period from 7 to 10 days. However this did not increase the difference in levels of PWM induced IgG secretion observed between MS and controls (see total C P - M S , table 3:lll, and frequency distribution, Fig. 3:4). We separated the C P - M S patients into 2 groups based on the clinical activity of their disease during the preceding 12 months and re-evaluated in terms of the PWM IgG secretion assay (Table 3:111). Patients who had worsened clinically and lost at least one point on the E K D S S were grouped as CP-active (CP-MS-A, n=13), patients who had remained stable for the preceding year were grouped as CP-stable (CP-MS-S, n=18). PWM induced IgG secretion (measured after 10 days) in the C P - M S - A group (6427±1296 ng) was significantly higher than - 1 4 1 -that observed in the healthy control group (3694+708 ng, n=25). PWM induced IgG secretion in the C P - M S - S (4368±836 ng) group did not differ from the healthy control g r o u p . The C P - M S - S group was significantly older (51.2±1.7 vs 40.5±4.0, p<.005, Student t-test) and had a longer disease duration (16.2±2.3 vs 9.8±2.4, p<.05, Student t-test) than the C P - M S - A group. 3:3:ii Con A induced suppression of Con A induced proliferation; Con A induced suppression was lower in the 25 chronic progressive patients tested (4±5%) than in the 18 healthy controls tested (24±4%, for individual results see figure 3:5). This difference was highly significant (p<0.001, Student t-test). 3:3:iii C o n A induced Suppress ion of PWM Induced IgG secretion in CP-MS-A, and controls; Graded numbers of Con A pretreated "S " cells or untreated, " C " cells, obtained from 6 C P - M S - A patients, 6 age and sex matched patients with other neurological diseases (OND), and 6 age and sex matched healthy control volunteers (HC) were added to 2x10 6 PWM stimulated P B M C obtained from heterologous high responders (ie. secrete >1000 ng of IgG after 7 days in culture). The amount of suppression was calculated according to the formula described in Materials and Methods section (see dose response curve, Fig 3:6). Suppression of IgG secretion exerted by the cells obtained from the C P - M S patients was less than the suppression exerted by both control groups at all the suppressor cell numbers tested (Table 3:IV). -1 4 2 -3:3:iii Surface phenotype of T cells in MS patients and c o n t r o l s ; Percentages of cells expressing T cell surface markers CD4, CD5 and CD8 were not different between the two groups (Table 3:V), but the CD4:CD8 ratio was significantly higher in the chronic progressive MS patients (4.1±0.4%) than in the healthy control group (2.9±4%, p <0.05, Student t-test). 3:3:iv Surface phenotype of CD4+ cells in MS patients and c o n t r o l s ; PWM induced IgG secretion and the percentage of T helper cells cells expressing either the T suppressor-inducer phenotype (Tsi, Leu3a+2H4+) or the T helper-inducer phenotype (Thi, Leu3a+4B4+) were measured. Two active MS groups (ie. 6 CP-MS-A , 5 females and 1 male and 7 RR-MS-A, 4 females and 3 males) and 2 control groups (8 OND, 2 females and 6 males and 14 HC, 7 males and 7 females) were included in this study (Table 3:VI). These groups did not differ significantly in age. The T helper cells of the C P - M S - A patients contained significantly fewer cells expressing the Tsi phenotype, more cells expressing the Thi phenotype and a lower Tsi:Thi ratio than either control group. Although the amount of IgG secreted was higher than all other groups tested the difference was not significant. We attribute this to the limited number of individuals tested. The R R - M S -A group did not differ significantly from the control groups in terms of the T helper cell phenotypes or PWM induced IgG secretion. -1 4 3 -3:4 Discuss ion The literature on immune function in multiple sclerosis patients is replete with inconsistencies. One of the main reasons for discrepancies stems from the lack of uniformity in subgrouping MS patients in various stages of disease activity. While some reports have not subgrouped the MS patients at all, others have grouped active MS patients as both chronic progressive and relapsing remitting-attack. We will demonstrate that these 2 groups differ immunologically and should not be grouped (see further). The not infrequent observation that cl inically stable MS patients show pathological changes within the C N S by serial computed tomography or magnetic resonance imaging is also responsible for contributing to the large overlaps observed between active and inactive disease. In our investigation of immune abnormalities in MS we have subgrouped the patients according to very strict criteria as; relapsing remitt ing-stable, relapsing remitting-attack, chronic progressive-stable and chronic progressive-active. In the relapsing remitting-attack patients we observed that the amount of IgG secreted in response to PWM correlated with length of time since the onset of the attack. The observation corroborates a recent study on serial immune function in relapsing remitting MS patients followed by magnetic resonance imaging (MRI), [Oger et al., 1988a]. In patients who had no evidence of MRI lesions there was no change in the level of PWM induced IgG secretion. In the patients who developed large MRI lesions there was a mean reduction in IgG -1 4 4 -secretion of 62%. There was no apparent recovery in the level of IgG secretion when the lesions began to decrease in size. This latter observation differs from our clinical study where IgG secretion was significantly higher in the patients who had been in relapse for a longer period (>3 weeks and <10 weeks). These discrepancies could be explained by the poor correlation found between MRI lesions and clinical attacks. When MRI lesions and clinical attack co-exist, their tempo is different but most often [Willoughby et al., 1988] they do not co-exist. It is uncertain at this point whether the changes in immune function precede or follow the changes in disease activity. We have however generated preliminary data indicating that long lasting reductions of PWM induced IgG secretion follow large MRI lesions which did not have any clinical correlate [Oger et a l . 1988b]. Hopefully with well planned serial studies of immune function in relation to MRI on select groups of MS patients, we will be able to answer the fundamental question of whether changes in immune function are the cause or consequence of CNS pathology. In a group of clinically stable patients, classified as relapsing remitting, we assessed the prognostic value of measuring PWM induced IgG secretion. We measured PWM induced IgG secretion in 20 cl inical ly stable MS and then fol lowed the patients' c l in ical progression for the next 6 months. Of the patients who went on to develop an attack most were initially high responders in the PWM assay compared with less that half of the patients who remained stable. This difference was not significant and one of the difficulties with this type of study is that patients may be clinically stable but have active disease as assessed by MRI [Jacobs et al., 1986; Paty et -1 4 5 -al., 1988a, 1988b]. The results nevertheless indicate a trend which we feel should be pursued with magnetic resonance imaging along with clinical examination as measures of disease activity. Our observations that in the chronic progressive MS patients, concanavalin A induced suppressor activity is reduced and that PWM induced IgG secretion and the ratio of T helper to T suppressor cells is increased over that observed in healthy individuals brings a consensus to the literature on immune function in chronic progressive MS pat ients. PWM induced IgG secretion in the C P - M S patients when measured after 7 days was significantly higher than the level observed in the healthy control group. Most investigators have observed this phenomena [Oger et al., 1982; 1986; Goust et al., 1982, Tjernlund et al., 1984; Antel et al., 1984; O'Gorman et al., 1987] while others have not [Hauser et al. 1985]. In an effort to study the basis of the increased IgG secretion in the MS patients we lengthened the culture incubation period from 7 to 10 days. We then analyzed the 10 day IgG secretion results after reviewing the clinical histories of these C P -MS patients and grouping them as either C P - A or C P - S (burnt-out). The PWM induced IgG secretion response in the C P - M S patients who were not clinically active did not differ from the healthy control group. However the C P - M S patients who had clinically active disease secreted significantly higher levels of IgG compared to the control group. The level of IgG secreted after 10 days in culture in the total group of C P - M S patients was not significantly different from the healthy control group. As the differences in IgG secretion were different after 7 days in culture it may be argued that the differences -1 4 6 -in the level of response are due to differences in the rate of response. We feel however that the lack of significance is a statistical problem due to increased variability with increased length of culture. It has been reported that a certain proportion of chronic progressive MS patients become clinically stable with a variable degree of functional disability [Farrell et a l . , 1985; Miller et al . , 1988]. Our results indicate that the chronic progressive-stable group ("burnt-out") are older and have had a longer duration of disease than the chronic progressive-active patients and we suggest that immune function in these patients returns to normal coinciding with the "burning out" of their clinical disease. This result is consistent with the report of Farrell et al . [1985] indicating that the inflammatory process had subsided in the CNS of chronic progressive "burnt out" MS patients as evidenced by the absence of oligoclonal bands in the C S F and of lymphocyt ic infi ltrates in the brain. We recommend that investigations of immune function in chronic progressive MS patients differentiate between the "burnt out" chronic progressive MS patients and the chronic progressive MS patients who are in the active phase of the disease. We have confirmed that concanavalin A induced suppressor cell activity is reduced in chronic progressive active MS patients. Reduced mitogen induced suppressor cell activity is one of the more consistent in vitro immunological abnormalities reported in active MS patients [Arnason and Antel, 1978; Antel et al., 1978,1979, 1986; Neighbour and Bloom, 1979; Gonzalez et al., 1979; Sheremata et al., 1982; Haahr et al., 1983; Tjernlund et al. 1984; Oger et al., 1985]. This assay involves 2 stages; 1, stimulating P B M C with Ag or mitogens and 2, -1 4 7 -adding these cells to mitogen stimulated autologous or heterologous P B M C and then measuring the suppression of the proliferative response. We modified this suppressor assay by changing the 2nd phase and measured suppression of mitogen induced IgG secretion as opposed to mitogen induced proliferation. A similar assay has been used to measure suppression in healthy controls [Schwartz et al . , 1977] but to our knowledge has not been used to study suppressor cell function in MS. Suppression of IgG secretion exerted by the chronic progressive patients' P B M C was consistently lower than the suppression observed by either the other neurological d isease patients' or the healthy control groups' (matched for both sex and age) at all three concentrations of suppressor cells tested. The relationship between the reduced ability to suppress PWM induced IgG secretion in heterologous PBMC cultures and the increased level of PWM induced IgG secretion in chronic progressive patients P B M C cultures is not clear. It has been suggested that a reduction in CD8+ cells is responsible for the increase in IgG secretion and the decreased suppressor cell activity observed in active MS patients [Rosenkoetter et al., 1984]. The investigation of the percentages of cells expressing subset specific surface markers in MS has been very controversial. However it is generally agreed that the ratio of T helper cel ls over T suppressor cel ls is increased in chronic progressive patients [Bach et al., 1980; 1985; Weiner et al., 1984; Mingioli & McFarlin, 1984; Craig et al., 1985]. We propose that since all three abnormalities are consistently present in chronic progressive MS patients that they are all linked. -1 4 8 -In addition to the major T cell subsets, we measured the percentage of T helper cells expressing either the T suppressor-inducer (Tsi) marker, CD45R (Leu3a+2H4+) or the T helper-inducer (Thi) marker, CDw29 (Leu3a+4B4+) in chronic progressive MS, relapsing-remitting MS in attack, other neurological disease controls and healthy controls. The PBMC of the chronic progressive MS group contained fewer T helper cells expressing the Tsi phenotype than either control group. Reduced numbers of Tsi cells in C P - M S patients has recently been reported by two other groups [Rose et al., 1985; Morimoto et al., 1987]. We also observed that the percentage of T helper cells expressing the Thi phenotype was increased in the C P -MS-A group over that observed in the healthy controls. This has not been reported previously. Interestingly PWM induced IgG secretion was higher in this C P - M S - A group than in either of the control groups. The RR-MS-A group did not differ from the control groups in terms of the markers studied. We wonder if the alterations in the Tsi cells may not be involved in both the reduced suppression and increased PWM induced IgG secretion observed in C P - M S - A patients. The reduction in the Tsi cell subset in MS is at this time the most consistent finding in terms of surface markers in MS. We feel that further evaluation of the function of this subset is warranted. The cause of the reduced number of Tsi cells in MS in not known. Recently, the group in Boston studied the percentage of Tsi cells in the C S F of active MS patients in order to determine if the reduced number of Tsi cells in the peripheral blood could be due their migration into the C N S [Chofflon et al., 1988]. This group observed a reduced number of CD4+2H4+ cells in the C S F compared to the -1 4 9 -peripheral blood. Interestingly, there was an increased number of CD4+4B4+ cells (Thi) in the C S F compared to the peripheral blood. This group concluded that the reduced number of CD4+2H4+ (Tsi) in the peripheral blood of MS patients is not due to migration into the C N S . Recent reports have suggested that activation of the CD4+2H4+ (Tsi) cell subset results in the loss of the expression of this marker and a concomitant gain in the Thi marker measured with either the 4B4 [Morimoto et al., 1986] or UCHL 1 [Abkar et al., 1988 and Serra et al., 1988] monoclonal antibodies. It is possible that the activation of T helper cells in the peripheral blood and C S F of active MS patients is the mechanism responsible for the observed reduction in the percentage of CD4+2H4+ cel ls and the increased number of Leu3a+4B4+ cells. Indeed there are many reports suggesting that activated PBMC are present in both the peripheral blood and C S F of MS patients [Golaz et al., 1983; Hafler et al., 1985; Hofman et al., 1986]. Reports of spontaneous proliferative activity in in vitro P B M C cultures obtained from MS patients [Hughes et al. , 1977; Lisak and Zweiman, 1977; Noronha et al., 1980; Fraser et al., 1979; Brinkman et al., 1984] and increased spontaneous Ig secretion [Hauser et al., 1985] also suggest that the PBMC are stimulated in vivo. In summary we have observed differences in immune function in MS patients studies during different phases of clinical activity. In R R - M S patients PWM induced IgG secretion correlates with the length of time since the onset of a clinical relapse. Clinically stable RR patients may have a slightly poorer prognosis if they are high responders in the PWM induced IgG secretion assay. Chronic progressive patients who have ceased to deteriorate clinically do not -1 5 0 -differ from healthy controls in terms of PWM induced IgG secretion and clinically active chronic-progressive MS patients exhibit in vitro immunoregulatory abnormalities ie. increased PWM induced IgG secretion, reduced ConA induced suppression and abnormal T cell subset phenotypes. - 1 5 1 -THE PROGNOSTIC VALUE OF MEASURING THE LEVEL OF PWM INDUCED  IGG SECRETION IN STABLE MS PATIENTS Immune function studied j 6 months — (FOLLOW UP PERIOD) Fig.3:1. Diagrammatic representation of the procedure used to study the prognostic value of measuring PWM induced IgG secretion in the peripheral blood mononuclear cells of Relapsing Remitting (RR) MS patients during the stable phase of their disease. RR-MS patients stable for at least 6 months prior to immune function testing. Six months after the test the patients were grouped a posteriori according to the presence of a clinical attack. Group 1 suffered a clinical relapse during the 6 month follow up period and Group 2 remained stable. -1 5 2 -Fig. 3:2. The amount of IgG secreted by the PWM stimulated PBMC of RR-MS patients in remission correlates with the length of time since their last attack, r=.739, p<.01. -1 5 3 -Table 3:l. PWM induced IgG secretion in Relapsing Remitting (RR) patients during remiss ion: Patients were separated into 2 groups based on the length of time since since their last relapse Group (n) Amount of IgG secreted mean+SEM (ng/ml) Healthy controls (25) 3694+708 RR Recent Attack (5) (< 3 weeks) 2691 ±956 RR Older Attack (6) (3-10 weeks) 8742±1262 * *-Different from controls and patients with recent relapse by Student t-test, p< .01 -1 5 4 -T a b l e 3:11. PWM induced IgG secretion in stable MS patients with relapsing remitting disease. Patients are sub-grouped according to the results of further follow-up as indicated in fig. 3:1 Level of Response Group (N) HR (>1000 ng of IgG) LR (<1000 ng of IgG) Mean (ng) Group 1 (Attack) 7 6 1 2757±429§ Group 2 (Stable) 13t 6 7 1750±531 §- amount of IgG secreted by 1x106 PWM stimulated PBMC f-these 2 groups were not statistically different by Student t-test -1 5 5 -Frequency distribution of the percentage of Healthy controls or Chronic Progressive MS patients whose PBMNC secrete IgG at a given level Fig 3:3. Frequency distribution of the percentage of healthy control individuals or chronic progress ive MS patients whose PBMC secrete IgG at a given level when stimulated with PWM in vitro for 7 days, (mean level of IgG secreted by the 27 CP-MS is greater than the 30 healthy controls, p<.01, Student t-test). -1 5 6 -Table 3:111. PWM stimulated IgG secretion (10 days) in Controls and 2 groups of MS with chronic progressive disease, CP-Active and CP-Stable. Group N Age Disease IgG secreted mean+SEM Duration mean±SEM Median Healthy Control 25 33.5±1.7 NA 3694±708 CP-Activef 13 40.5±4.0 10±2 6472±12961 CP-Stable (burnt out)§ 18 51.2±1.7ft 16±2§§ 4368±836 All CP MS 31 46.2±1.8 14±2 52501740 2152 5774 4155 4950 f-loss of 1 point on the Extended Kurtzke Disability Status Scale in one year § - no change in the EKDS in 1 year 1f- different from control by Student t-test (p<.025) §§-different from CP-MS-A by Student t-test (p<05) f f-different from CP-MS-A by Student t-test (p<.005) NA- not applicable -1 5 7 -Frequency distribution of the percentage of Healthy controls or Chronic Progressive MS patients whose PBMNC secrete IgG at a given level % of individuals secreting IgG at the level indicated on the ordinate i ""i i 14 15 16 IgG Secreted after 10 days in culture (x 1 0 l 3 ng/ml) HEALTHY C CHRONIC MS Fig. 3:4. Frequency distribution of the percentage of healthy control individuals or chronic progress ive MS patients whose PBMC secrete IgG at a given level when stimulated with PWM in vitro for 10 days. -1 5 8 -Figure 3.5. Concanavalin A induced suppressor cell activity in the PBMC of 25 chronic progressive MS and 18 Healthy controls. Horizontal lines indicate means and the vertical lines indicate the SEM. -1 5 9 -Suppression of PWM induced IgG secretion by increasing numbers of ConA pretreated PBMC obtained from 7 different individuals 0 .1x106 .25x106 .5x106 # of suppressor cells added to 2x106 PWM stimulated PBMC ••- MM •<>- SD ••" WE TJ BM -A- LO CV Fig. 3:6. A dose response effect on the suppression of IgG secretion in PWM stimulated PBMC cultures is observed by adding increasing numbers of ConA pretreated suppressor c e l l s . -1 6 0 -Table 3:IV. Suppression of PWM induced IgG secretion by ConA preactivated PBMC obtained from healthy controls (HC), other neurological disease controls (OND) and chronic progressive multiple sc lerosis (CP-MS) patients. Number of ConA pretreated suppressor cells added to 2x10 6 PWM stimulated PBMC (% Suppression, mean ± SEM) Group .5x10 6 .25x10 6 .1x10 6 HC (6) OND (6) CP-MS (6) 89.2±2.5 a 89.0+2.5° 81.3±3.4c 87.6+1.6d 84.3±2.2e 67.2±8.6 f 82.0±2.6 76.6±2.3 67.7±7.8 c<b,p<.05 c<a,p<.025 f<d,p<.05 (by Student t-test) - 1 6 1 -Table 3:V The percentage of CD4+, DC5+, CD8+ and the CD4:CD8 ratio in chronic-progressive MS patients and healthy controls. %CD5+(Leul) %CD8+(Leu2a) %CD4+(Leu3a) CD4/CD8 Group (n) (mean±SEM) (mean±SEM) (meaniSEM) (mean±SEM) CP-MS (n=30) 70.8±1.7 16.2±1.4 53.6±1.6 4.1±0.4 Controls (n=21) 70.911.6 19.811.7 47.612.6 2.910.4f tsignificantly different from CP-MS (Student t-test, p<.05) -1 6 2 -Table 3:VI. Surface phenotype of CD4+ T helper cells and the level of PWM induced IgG secretion in the PBMC obtained from 2 MS group and 2 control groups. Age Level of PWM induced Tsi Thi Group (N) (years) IgG Secretion (ng/ml) (Leu3a 2H4+) (Leu3a4B4+) Tsi:Thi 7 days 10 days (%) (%) H C a 14 46±4 1615±451 4266±1191 50+4 51+4 1.1±.1 CMC*3 8 45+4 1583±778 4172±1633 57+7 52+5 1.2+.2 RR-MS-A C ! 7 39+4 1567±429 4600+1330 47±5 55+5 0.9+2 CP-MS-A d 6 43±3 2943±955 6288±1370 31±4t 64+6§ 0.5±.n a-HC=healthy controls b-OND= other neurological disease controls c-RR-MS-A= Relapsing Remitting MS in relapse d-CP-MS-A=Chronic progressive MS Active, for further description of patient groups see Materials and methods, t- different from HC and OND, p< .01, by Student t-test §- different from HC, p<.05, by Student t-test ^-different from HC (p<.025) and from OND, p<.005, by Student t-test -1 6 3 -3:5 References Abkar, AN . , Terry, L , Timms, A., Beverley, P C L , and Janossy, G. Loss of CD45R and gain of UCHL1 reactivity is a feature of primed T cells. J . Immunol. 140; 2171 (1988). Antel, J . , Arnason, BGW., and Medof, ME. Suppressor cell function in multiple sclerosis: correlation with clinical disease activity. Ann. Neurol. 5; 338 (1979). Antel, JP . , Peeples, DM., Reder, AT. and Arnason, BGW. Analysis of T regulator cell surface markers and functional properties in multiple sclerosis. J . Neuroimmunol. 6; 93 (1984). Antel, J .P . , Weinrich, M. & Arnason, B.G.W. Mitogen responsiveness and suppressor cell function in multiple sclerosis. Neurology 28, 999 (1978). Arnason, BGW., and Antel, J . (1978). Suppressor cell function in multiple sclerosis. Ann. Immunol. 129C;159. Bach M.A., Phan-Dinh-Tuy, F., Tournier, E., Chartenoud L., Bach, J .F. , Martin, C. & Degos, J .D. Deficient suppressor T cells in active multiple sclerosis. Lancet ii, 1221 (1980). -1 6 4 -Bach, M.A., Martin, C , Cesaro, P., Eizenbaum, J.F. & Degos, J.D. T cell subsets in multiple sclerosis, J . Neuroimmunol. 7, 331 (1985). Brinkman, C J J . , Nillesen, WM., Hommes, OR. Lymphocyte subpopulations in multiple sclerosis: spontaneous and mitogen-induced activity. Clin. Immunol. Immunopath. 31;364 (1984). Chofflon, M., Weiner, HL., and Hafler, DA. Suppressor inducer (CD4+2H4+) T cel ls in multiple sc lerosis cerebrospinal fluid. Neurology 38 (suppl. 1); 196 (1988). Compston, DAS. , and Hughes, P J . Peripheral blood lymphocyte sub-populations and multiple sclerosis. J . Neurology. 6; 105 (1984). Craig, J .C . , Hawkins, S.A., Swallow, M.W., Lyttle, J.A., Patterson, V.H. , Merret, J .D. & Haire, M. Subsets of T lymphocytes in relation to T lymphocyte function in multiple sclerosis. Cl in. exp. Immunol. 61, 548 (1985) . Farrell, MA., Kaufmann, J C E . , Gilbert, J J . , Noseworthy, JH . , Armstrong, HA and Ebers, G C . Oligoclonal bands in multiple sclerosis: Clinical-pathologic correlation. Neurology 35; 212 (1985). Fraser, KB. , Millar, JDH. , Haire, M., McCrae, J . Increased tendency to spontaneous in vitro lymphocyte transformation in clinically active multiple sclerosis. Lancet ii; 715 (1979). -1 6 5 -Golaz, J . , Steck, A., and Moretta, L. Activated T lymphocytes in patients with multiple sclerosis. Neurology 33;1371 (1983). Gonzalez, RL. , Dau, P C , Spitler, S E . Altered regulation of mitogen responsiveness by suppressor cells in multiple sclerosis. Clin. exp. Immunol. 36;78 (1979). Goust, J M . , Hogan, EL. , and Arnaud, P. Abnormal regulation of IgG production in multiple sclerosis. Neurology 32; 228 (1982). Haahr, S., Moller-Larsen, A., Pedersen, E. Immunological parameters in multiple sclerosis patients with special reference to the herpes virus group. Clin. exp. Immunol. 51; 197 (1983). Hafler, DA., Fox, DA., Manning, ME., Schlossman, SF. , Reinhertz, EL., and Weiner, HL. In vivo activated T-lymphocytes in the peripheral blood and cerebral spinal fluid of patients with multiple sclerosis. New Engl. J . Med. 312; 1405 (1985). Hauser, SL . , Ault, KA., Johnson, D., Hoban, C , and Weiner, HL. Increased IgG secretion by unstimulated mononuclear cells in active multiple sclerosis and functional assessment of the T8 subset. Clin. Immunol. Immunopath. 37; 312 (1985). Hofman MF., mon Hanwehr, Rl . , Dinarello, CA., Mizel, SB. , Hinton, D., and Merri l l , J E . Immunoregulatory molecules and IL2 receptors identified in multiple sclerosis brain. J . Immunol. 136;3239 (1986). -1 6 6 -Hughes, R A C , Gray, I., Clifford-Jones, R., Stern, M. Immune response to myelin basic protein in multiple sclerosis. Proc. R. Soc. Med. 70;874 (1977). Jacobs, L , Kinkel, WR., Polachini, I., and Kinkel PR. Correlations of nuclear magnetic resonance imaging, computerized tomography, and clinical profiles in multiple sclerosis. Neurology 36; 27 (1986). Johnson, KP. , Arrigo, S C . , Nelson, B J . , and Ginsberg, A. Agarose e lect rophoresis of cerebrospinal fluid in multiple sc le ros is . Neurology 27; 273 (1977). Kurtzke, J F . Rating neurologic impairment in multiple sclerosis: An expanded disability status scale (EKDSS) . Neurology 33; 1444 (1983). Kurtzke, J F . Disability rating scales in multiple sclerosis. Annals of the NY Acad. Sci . 346; 347 (1984). Lassman , H., and Wisniewski , H M . Chronic relapsing E A E -Cl inicopathological comparison with Multiple Sc leros is . Arch. Neurol. 36; 490 (1979). L isak, R P . , Zweiman, B. In vitro cel l -mediated immunity of cerebrospinal fluid lymphocytes to myelin basic protein in primary demyelinating diseases. New Engl. J . Med. 297;850 (1977). -1 6 7 -McAlpine, D. In: Multiple sclerosis: a reappraisal, 2nd edition. Churchill Livingstone, Edinburgh, p. 197 (1972). Matthews, WB., In: McAlpine's multiple sclerosis. W.B Matthews, ED. Acheson, JR . Batchelor and RO. Weller Eds. Churchill Livingstone, Hong Kong, p. 49 (1985). Millar, JHD. , Disseminated sclerosis: a follow-up of 91 cases. Lancet 2; 556 (1949). Miller, A. Drexler, E., Kei lson, M., Slagle, S. and Bornstein, M. Spontaneous Stabilization in patients with chronic progressive MS. Neurology 38 (Suppl 1); 194 (1988). Mingioli E.S. & McFarlin, D.E. Leukocyte surface antigens in patients with multiple sclerosis. J . Neuroimmunol. 6, 131 (1984). Morimoto, C , Hafler, DA., Weiner, HL., Letvin, NL., Hagan, M., Daley, J . , and Schlossman, SA. Selective loss of the suppressor-inducer T-cell subset in progressive multiple sclerosis: Analysis with anti-2H4 monoclonal antibody. N. Engl. J . Med. 316;67 (1987). Morimoto, C , Letvin, NL., Rudd, C E . , Hagan M., Takeuchi, T., and Schlossman, SF . The role of the 2H4 molecule in the generation of suppressor function in Con A-activated T cells. J . Immunol. 137; 3247 (1986). -1 6 8 -Neighbour, PA and Bloom, BR. Absence of virus-induced lymphocyte suppression and interferon production in multiple sclerosis. Proc. natl. Acad. Sci . USA. 76;476 (1979). Noronha, A B C , Richman, DP. , Arnason, BGW. Detection of in vivo stimulated cerebrospinal fluid lymphocytes by flow cytometry in patients with multiple sclerosis. New Engl. J . Med. 303;713 (1980). O'Gorman, M R G . , Oger, J F . , and Kastrukoff, LK. Reduction of immunoglobul in G secret ion in vitro fol lowing long term lymphoblasto id interferon (Wellferon) treatment in multiple sclerosis patients. Clin. exp. Immunol. 67; 66 (1987). Oger, JF . , and Arnason, BGW. HLA patterns in multiple sclerosis. In: H. Bauer (Ed.), Progress in Multiple Sclerosis Research, Springer-Verlag, Berlin, p.460 (1980). Oger J . , Kastrukoff, L.F., Li, DKB., and Paty, DW. Multiple sclerosis: In relapsing patients, immune functions vary with disease activity as assessed by MRI. Neurology; In press (1988a). Oger J . , O'Gorman, MRG. , Willoughby, E., Li, D., and Paty, DW. Changes in immune function in relapsing multiple sclerosis correlate with disease activity as assessed by magnetic resonance imaging. N.Y. Acad. Sci . In press. (1988b). -1 6 9 -Oger JF . , Kastrukoff, L , O'Gorman, M., and Paty, DW. Progressive multiple sclerosis: abnormal immune functions in vitro and aberrant correlation with enumeration of lymphocyte subpopulat ions. J . Neuroimmunol. 12;37 (1986). Oger, J . , Antel, J P . , Kuo, HH. , and Arnason BGW. Influence of Azathioprine (Imuran) on immune function in multiple sclerosis. Ann. Neurol. 11; 177 (1982). Oger, J F . , O'Gorman, M., Kastrukoff, L , Paty, D. In vitro simultaneous testing of three immune parameters helps differentiate progressive MS from controls. Neurology 35; 313 (1985). Paty, DW., Kastrukoff, L , Morgan, N. and Hiob, L. Suppressor T lymphocytes in multiple sclerosis- analysis of patients with acute relapsing and chronic progressive disease. Ann. Neurol. 14; 445 (1983). Paty, DW., Koopmans, R., Isaac, C. Willoughby, E., Li, DKB. Serial MRI studies in multiple sclerosis: A new method for assessing disease activity in both chronic progressive and relapsing d isease . Neurology 38; (suppl 1); 255 (1988). Paty, DW., Oger, JJF . , Kastrukoff, LF., Hashimoto, SA., Hooge, JP . , Eisen, AA., Eisen, KA., Purves, S J . , Low MD., Brandejs, V., Robertson, WD., and Li, DKB. Magnetic resonance imaging (MRI) in the diagnosis of multiple sclerosis: A prospective study with comparison of clinical -1 7 0 -evaluation, evoked potentials, oligoclonal banding and computerized tomography. Neurology 38; 180 (1988). Poser, C , Paty, DPW., Scheinberg, L , McDonald, I., Davis, FA., Ebers, G C , Johnson KP. , Sibley, WA., Silberberg, DH., and Tourtellotte, WW. New diagnost ic criteria for multiple sc leros is : guidel ines for research protocols. Annals of Neurology 13; 227 (1983). Rees, A J . , Peters, DK., Compston, DAS., and Batchelor, JR. Strong association between HLA-DRw2 and antibody mediated Goodpasture's syndrome. Lancet 1; 966 (1978). Reinhertz, EL., Weiner, HL., Hauser, SL. , Cohen, HA., Distaso, JA and Schlossman, S F . Loss of suppressor T cells in active multiple sclerosis-analysis with monoclonal antibodies. N. Engl. J . Med. 303;125 (1980). Rose, LM. , Ginsberg, AH. , Rothstein, TL., Ledbetter, JA. , Clark, Ea. Selective loss of a subset of T helper cells in active multiple sclerosis. Proc. Natl. Acad. Sci . USA. 82;7389 (1985). Rosenkoetter, M., A. T. Reder, J . J . -F . Oger, and J . P. Antel. T cell regulation of polyclonally induced immunoglobulin secretion in humans. J . Immunol. 132:1779 (1984). Schwartz, S.A., Shou, L., Good, R.A., and Choi, Y . S . Suppression of immunoglobulin synthesis and secret ion by peripheral blood - 1 7 1 -lymphocytes from normal donors. Proc. Natl. Acad. Sc i . USA. 74; 2099 (1977). Serra, HM. , Krowka, J F . , Ledbetter, JA. , and Pilarski, LM. Loss of CD45R (Lp220) represents a post-thymic T cell differentiation event. J . Immunol. 140; 1435 (1988). Shepherd, Dl . Clinical features of multiple sclerosis in north-east Scotland. Acta Neurologica Scand. 60; 218 (1979). Sheremata, W., Rxepelia, A J . , Berger, J.,Sazant, A., and Castro, A. Inhibition of concanavalin A inducible suppressor cell function in multiple sclerosis by adrenocorticotropic hormone. Ann. Neurol. 12; 103A (1982). Shou, L., Schwartz, S.A. & Good, R.A. Suppressor cell activity after concanavalin A treatment of lymphocytes from normal donors. J . exp. Medicine. 143, 1100 (1976). Tjernlund, U., Cesaro, P., Tournier, E., Degos, JD. , Bach, JF . , and Bach, MA. T-cell subsets in multiple sclerosis: a comparative study between cell surface antigens and function. Cl in. Immunol. Immunopath. 32; 185 (1984). Walsh , M J . , Tourtellotte, WW., Potvin, AR. , Potvin, J H . The cerebrospinal fluid in multiple sc leros is ; in Hal lpike, Adams, -1 7 2 -Tourtellotte, Eds. Multiple sclerosis. Pathology, diagnosis and management, p. 275, Chapman and Hall (1983). Weiner, H.L., Hafler, D.A., Fallis, R.J., Johnson, D., Ault, K.A. & Hauser, S .L . Altered blood T-cell subsets in patients with multiple sclerosis. J . Neuroimmunol. 6, 115 (1984). Willoughby, EW., Grochowski, E., Li, D., Oger, J . , Kastrukoff, LK., and Paty, DW. Serial magnetic resonance scanning in multiple sclerosis: A prospective study in relapsing patients. Ann. Neurol. (in press). -1 7 3 -Chapter 4 REDUCTION OF IMMUNOGLOBULIN G SECRETION IN VITRO FOLLOWING LONG TERM LYMPHOBLASTOID INTERFERON ( W E L L F E R O N ® ) TREATMENT IN CHRONIC PROGRESSIVE MULTIPLE SCLEROSIS PATIENTS 4:1 Introduction In addition to its antiviral activities, interferon modifies the immune response. Most of the studies on the effects of interferon on immune function have been carried out in assay systems in vitro where it has been directly added. Human alpha interferon added to human cells in vitro enhances natural killer (NK) cell function and the generation of cytotoxic T lymphocytes [Herberman et a l . , 1981; Zarling, 1981]. Interferon also inhibits DNA synthesis in lymphocytes stimulated by mitogens or antigens [Blomgren, Strender & Cantell , 1974; Lee et a l . , 1982; Shalaby & Week, 1983]. The effects of interferon on Ig synthesis vary with the type of interferon, its concentration and the timing of its addition in relation to the addition of mitogen or antigen [Harfast et al., 1981; Choi, Lim & Sanders, 1981; Parker et al., 1981; Levinson, Dziarski & Hooks, 1982; Fleisher et al., 1982; Rodriguez et al., 1983; Yen & Reem, 1983]. The effect of injection of interferon in vivo has been studied mainly in mice. Murine interferon injected or induced in vivo reduces the number of antibody secreting spleen cells produced in response to -1 7 4 -sheep red blood cells, a T cell dependent antigen [Chester et al., 1973; Brodeur & Merigan, 1975; Virelizier et al., 1977]. With the aim of altering the course of some human diseases, interferon has been used in therapeutic trials where curative therapy is not available [see Krim, 1984 for review]. Our group is currently involved in a double blind study on the effect of interferon on the clinical course of multiple sclerosis (MS). In MS a number of immunological abnormalities have been reported. Natural killer cell activity has been shown to be decreased and this abnormality can be temporarily corrected by the administration in vivo of interferon [Hirsch & Johnson, 1984; Panitch et al., 1984; Rice et al., 1984]. As reported in Chapter 3, peripheral blood mononuclear cells (PBMC) isolated from chronic progressive MS patients secrete more immunoglobulin G in response to pokeweed mitogen (PWM) than PBMC isolated from healthy controls and Con A induced suppression has been shown to be lower than in healthy controls. T lymphocyte markers have also been shown to be abnormal in the peripheral blood of MS patients. The CD8 cell surface marker is reduced and most authors agree that the CD4/CD8 ratio is elevated compared to normal controls. We did not measure the T suppressor-inducer or T suppressor-inducer markers in this group of chronic progressive MS patients because the results indicating abnormalities in these subsets in MS were not available at the initiation of this project, We observed that Con A suppression, PWM induced IgG secretion and T lymphocyte surface markers are all abnormal in this group of chronic progressive MS patients taking part in a double blind clinical trial of (Wellferon®) lymphoblastoid interferon. After the initiation -1 7 5 -of the trial we observed an abrupt reduction of PWM induced IgG secretion in vitro, without a change in Con A induced suppression or in the T cell surface phenotype in the patients receiving interferon injections as compared to patients receiving placebo injections. -1 7 6 -4:2 Materials and Methods 4:2:i Patients: The patients were taking part in a double blind trial of interferon at the MS Clinic at the University of British Columbia. All had clinically definite MS of the progressive type with a disability between 3 and 6 on the Kurtzke scale [Kurtzke, 1961]. Deterioration over the preceding 6 months had been substantiated clinically in each patient. All patients had MRI of the head compatible with the diagnosis. Patients were randomized into placebo (PLA) or interferon (IFN) groups. Both groups were similar in terms of age, sex and Kurtzke index. The lymphocyte data were handled blindly on coded samples. The clinical status of the patients was evaluated blindly. IgG secretion, Con A suppression and T cell surface markers were evaluated on the first 38 trial patients before the start of treatment, after I week, I month and 6 months of treatment and 6 months after the treatment was stopped. Only 30 of these patients completed the first year of the trial and had immunological studies done at each time point. Twenty-one healthy laboratory workers were used as contro ls. 4:2:ii Interferon: Interferon (Wellferon®, Burroughs Wellcome) was administered daily by subcutaneous injection (5 x 1 0 6 units of human -1 7 7 -lymphoblastoid). The placebo consisted of an injection of a similar volume of isotonic saline. 4:2:iii IgG secretion in vitro: This assay was performed as reported in chapters 2 and 3, briefly P B M C were isolated by Ficol l-hypaque density gradient centrifugation from the peripheral blood and washed 3 times in cold HBSS and once cold RPMI complete. Aliquots of 1 X 1 0 6 PBMC were then cultured with PWM (1:300 final dilution) or without PWM for 7 days at 37°C in 5% CO2 in air. After this time the cultures were centrifuged (400xg) for 10 min and the cell free supernatants were harvested and stored at -70°C until they were assayed for IgG content by ELISA. 4:2:iv Con A suppressor cell assay: This assay was performed as in Chapter 3. Briefly the mononuclear cells were washed at least four times and 1 x 10^ cells/ml were resuspended in RPMI 1640 with 10% fetal calf serum, 200 u.g gentamicin per 100 ml and 2 mM L-glutamine (culture medium). Aliquots of 5 ml were placed in vertically-held 25 c m 2 tissue culture flasks and cultured with Con A at 3 u,g/ml ( X ' cells) or without Con A f C cells). After 3 days the cells were treated with mitomycin C (25 mg/ ml) for 30 min at 37°C, washed three times with balanced salt solution and resuspended. Triplicate 100 jil samples (10 5 ) of either X " cells or X " cells were then added to 100 \i\ (10 5) of -1 7 8 -heterologous responder cells f R ' cells) plus Con A (3 u.g/ml) for 3 days at 37°C. One microcurie of tritiated thymidine was added to all cultures for the final 6 h. The percentage suppression was calculated as follows: x 100 4:2:v T cell subset enumeration: One million P B M C were labelled with saturating amounts of fluorescein-conjugated mouse monoclonal antibody: Leu 1 for CD-5, Leu 3a for CD-4, and Leu 2a for CD-8. Cells were stained on ice for 30 min and washed. Positivity was read from a F A C S IV (Becton Dickinson) with gating on the lymphocyte scatter peak. Results were expressed as percent positive cells and the ratio of the CD4+ to the CD8+ cells was calculated. 4:2:vi Lymphocyte subset mixing experiments: 4:2:vi:a E- cells + T helper cells;. Mononuclear cells were separated into red blood cell binding (E+) and non-binding (E -) cells with sheep red blood cells treated with 2-aminoethylisothiouronium bromide according to the method of Saxon et al . [1976]. The OKT8+ cells and surface lg+ cells were subsequently removed from the E+ cell subset by panning. Briefly, the E + cells were labelled with OKT8 for 30 min on ice, washed, cpm in cultures of ("R" and "S " cells) %S= 1 cpm in cultures of ("R" and " C " cells) -1 7 9 -resuspended and added to petri dishes precoated with F(ab')2 fragments of a mixture of goat anti-mouse IgG and anti-human IgG, IgA and IgM. Non-adherent cells were recovered (T helper cells). One hundred thousand E- cells were co-cultured with 1 0 5 heterologous and autologous T helper cells in a final volume of 200 uJ. Following stimulation by PWM and 1 week in culture, the plates were spun for 5 min at 400xg and the cel l free supernatants stored at -70°C until assayed for IgG content. 4:2:v»:b Monocyte deprived PBMC + monocytes; Two x 1 0 7 PBMC in 10 ml of RPMI with 10% F C S were depleted of adherent cells (monocytes) by two cycles of adherence on plastic petri dishes (1 h at 37°C). Adherent cells were recovered from the first cycle by gentle scraping. Eight x105 monocyte-deprived P B M C were reconstituted with 2x10^ heterologous or autologous monocytes in a final volume of 200 jil. This ratio had previously been determined to give optimal IgG secretion in response to PWM after 7 days in culture. Purity of the cell subsets was assessed by a lack of IgG secretion in response to PWM. 4:2:vii Stat ist ics. Comparisons between groups was assessed using Student's t-test or the non parametric test, the Mann Whitney U test. In all cases statistical analysis was performed on raw data and not on log transformed data. -1 8 0 -4:3 Results 4:3:i MS patients and healthy controls: IgG-secretion by P B M C in response to PWM was significantly higher in the 30 MS patients (before they were included in the IFN-trial) than in the 21 healthy controls (2392±270 ng/ml vs 14991293 ng/ml respectively, p<0.05, Student t-test, Fig. 4:1a). Unstimulated cultures did not differ significantly (253±46 ng/ml for MS and 183±15 ng/ml for controls). Con A induced suppression was lower in the 25 (5 cultures were discarded due to technical reasons) MS patients tested (4±5%) than in the 18 controls tested (24+4% Fig. 4:1b). This d i f ference was highly signi f icant (p<0.001, Student t-test). Percentages of cells expressing T cell surface markers CD4, CD5 and CD8 were not different between the two groups (Table 4:l), but the CD4:CD8 ratio was significantly higher in the MS group (4.1+0.4%) than in the control group (2.9±4%, P<0.05, Student t-test). 4:3:ii MS Patients during the interferon trial: 4:3:ii:a Before treatment. The mean IgG secretion response of the 14 patients destined to be treated with interferon (IFN) (2388±416 ng/ml) was similar to the mean response of the 16 patients in the placebo (PLA) group (2396±363 ng/ml, Fig. 4:2). Similarly the level of Con A-induced suppression did not differ (7+7% for IFN and 2±8% for PLA, Fig. 4:3). The percentage of PBMC expressing surface markers CD5, CD4 and CD8 - 1 8 1 -as well as the CD4:CD8 ratios were not different between the two groups (Table 4:l). 4:3:ii:b After 1 week of treatment; Following 1 week of daily injections, IgG secretion by the IFN-group (699±96 ng/ml) was dramatically lower than that secreted by the PLA-group (1756±399, p< 0.01, Student t-test). No significant difference in Con A induced suppression was observed between the groups (2±5% for the IFN group vs 5±6% for the PLA group, Fig. 4:3). Because this assay was not affected by interferon in the face of a marked reduction in IgG secretion, it was not continued. There was no difference in the percentage of CD4 or CD8 positive cells between the two groups (see Table 4:l) but the IFN-group contained a slightly higher percentage of cells expressing the pan T cell surface marker CD5 (72.7±1.7%) than the PLA-group (67.6±1.7%; P < 0.05). There was no difference in the CD4:CD8 ratio (IFN, 3.7±0.5 and PLA, 3.3±0.3). 4:3:ii:c At 1 month and 6 months of treatment; After 1 month of daily injections the IFN-group still secreted less IgG (529±80 ng/ml) than the PLA-group (1748±382 ng/ml; P < 0.01). Cell surface markers were not different. After 6 months of daily injections, the IFN-group still secreted less IgG (617+68 ng/ml) than the PLA-group (1177±275 ng/ml) but the difference was not significant. Again no differences were noted in the surface markers studied. -1 8 2 -4:3:ii:d Six months after the last injection; Following discontinuation of the injections and a 6 month washout period, IgG secretion and T cell surface markers were again studied. The IFN-group and the PLA-group secreted equivalent amounts of IgG (1077±226 ng/ml and 944+227 ng/ml respectively, Fig. 4:2) and there was no difference between the two groups with respect to the T cell surface markers studied. 4:3:iii Subset mixing experiments; E- and T helper cell subsets were separated from two IFN-patients who had secreted high amounts of IgG in the PWM assay but following IFN treatment secreted low amounts, and one PLA-patient who secreted large amounts of IgG (Table 4:ll). The E- cells from both IFN-patients did not secrete IgG above background when mixed with the T cells from the other IFN-patient or the PLA-patient. The E- cells obtained from the PLA-patient secreted IgG above background when mixed with the T helper cells from either IFN-patient. Autologous mixing of the E- and T cells indicated that the PLA-patient remained a high responder, and both IFN-patients remained low responders. The E- cell subset consists predominantly of B cells and monocytes. To further investigate the lack of response in the E- cells obtained from IFN-patients, we assessed the function of the monocytes and of the monocyte-depleted subpopulations isolated from two other low responder IFN- patients and two other high responder PLA- patients (Table 4:lll). Monocytes obtained from IFN-patients were not different from the monocytes from PLA-patients in their ability to reconstitute a high IgG secretion response when mixed with -1 8 3 -monocyte-depleted P B M C from the PLA-patients. Neither PLA or IFN monocytes could, however, reconstitute a significant IgG secretion response when mixed with the monocyte-depleted P B M C obtained from the IFN-patients. -1 8 4 -4:4 Discuss ion Our results indicate that in chronic progressive MS patients, Wellferon causes a profound decrease in PWM induced IgG secretion in vitro after 1 week and 1 month of daily subcutaneous injections. The decrease was still apparent after 6 months of treatment but less striking in comparison to the placebo-treated group. Assessment of the duration of the effect of interferon is made difficult by the progressive lowering in IgG secretion observed in the PLA-group over the course of the trial. This progressive reduction in the PLA-group was not due to the placebo injections as after the 6 month washout period, IgG secretion did not increase. It is unlikely that the IFN-patients were becoming refractory to the modulating effects of the interferon injections as IgG secretion increased to the level observed in the PLA-group when the injections were stopped. The progressive reduction in IgG secretion observed in the PLA-group was probably due to the difficulty in maintaining the consistency of the assay over the extensive period of time required for this trial. Different lots of reagents, decreased activity of PWM (stored at -20°C) and a new technician could all have contributed to the drift observed. Investigation of the mechanism responsible for the interferon-induced reduction in IgG secretion involved measurement of the number and functions of different cell subsets. Repeated measurement of the T cell subsets indicated that the only significant difference observed between the IFN- and PLA-groups was an elevation in the CD5 marker in the IFN-groups at one week. Mixing experiments to -1 8 5 -evaluate the function of different subsets indicated that the E - c e l l s contained the subset affected by interferon injection in vivo. Results of monocyte depleted cultures showed that the monocytes were not the affected fraction of the E- cel ls, thus suggesting that the B lymphocyte could be the subset directly affected. This observation is consistent with the fact that the interferon did not affect Con A suppression. This latter function is believed to be mediated by a T cell subset. These results are also supported by previous experiments where alpha interferon added in vitro was shown to depress IgG secretion, and that the B cell subset was directly affected [Harfast et al., 1981; Fleisher et al., 1982). Before inclusion in the IFN trial IgG secretion, Con A suppression and the T cell surface markers were measured in this group of MS patients and a group of healthy controls. This group of MS patients presented the in vitro immune abnormalities reported in chapter 3; they secreted an amount of IgG higher than controls, Con A induced suppressor cell activity was lower than healthy controls and there was a significant elevation in the CD4/CD8 ratio over the control group. The interferon given in this therapeutic trial was Wellferon® which contains predominantly alpha and some beta interferon and is stabilized by 90% human albumin. We cannot formally exclude the possibility that the effect of IFN was due to some contaminant. It is striking, however, that Peters et al. , [1985] in a preliminary report coincidental to ours [O'Gorman & Oger, 1985] showed that injection in vitro of the same quantity of recombinant alpha interferon in a group of patients with chronic type B hepatitis was followed by reduced - 1 8 6 -PWM-induced IgG secretion in vitro and that this was the result of a direct effect on the B cell subset. -1 8 7 -5000 -4000 -E | ^3000 CD 2000 -1000 8 O °o° 88 -e-e-o 8 o% -I 4 60 - i 45 -30 -_ 15 c o w 0 CO CD CL Q. 3 - 1 «5 CO 1 ° 30 -45 -T T 60 8 o O 9 •r i MS Control MS Control Fig. 4:1. a. IgG production (ng/ml) in response to PWM by PBMC obtained from MS patients and controls (mean+SEM). Differences are significant (p<0.05, Student t-test); b. Con A induced suppression in MS patients and controls ( m e a n ± S E M ) . Differences are highly significant, (p<.001, Student t-test). -1 88-Table 4:l. Percentages of CD4+, CD5+, CD8+ cells and the CD4:CD8 ratios in the MS patients (a) before treatment and the control group, and (b) at various time points following their assignment to the IFN (n=14) or PLA (n=16) groups Group %CD5+(Leu 1) %, CD8+(Leu 2a) (Mean 1 s.e.m.) (Mean ± s.e.m.) %,CD4+ (Leu 3a) (Mean ± s.e.m.) CD4/CD8 (a) CP-MS(n=30) 70.8±1.7 Controls (n=21) 70.9±1.6 16.211.4 19.811.7 53.611.6 47.912.6 4.110.4 2.910.4* (b) PLA Before treatment 73.411.7 IFN PLA Week 1 IFN PLA 1 month IFN PLA 6 months IFN 67.812.9 67.611.7 72.711.7** 71.111.8 75.111.5 68.512.0 65.612.4 PLA 6 months after 69.011.9 the last treatment IFN 70.611.8 17.1+2.0 15.311.8 17.811.8 18.612.0 16.811.8 17.612.3 17.411.8 17.612.1 14.411.2 15.112.1 53.912.3 53.112.2 51.412.1 55.311.8 50.912.4 56.511.9 49.912.3 49.612.4 52.1 12.0 49.812.0 3.81 0.5 4.410.7 3.310.3 3.710.5 3.610.5 4.310.7 3.310.4 3.610.6 3.910.3 4.510.8 * Significant different from chronic progressive MS (p< 0.5, by Student t-test). ** Significant different from PLA group after 1 week of treatment, (p< 0.5, by Student t-test). - 1 8 9 -2500-1 week 1 month 6 months Pre R x | | Duration of Treatment I [Post R Fig. 4:2. IgG production (mean+SEM) in response to PWM by PBMC obtained from 30 MS patients at various time points following their inclusion into IFN (shaded bars) or PLA-treated groups (open bars). * Significant difference (p<.05, Student t-test) -1 9 0 -6 0 -4 0 -c o 20-CD C L a. CO 0 20 - 4 0 60 80 6b o -8-1 99 8 i r i Plac Inf ( Pre R J 8 8 o - Q - T O 1 oo o o I 1 Plac Inf ( 1 week post P* ) F i g . 4:3. C o n A s u p p r e s s i o n in M S p a t i e n t s f o l l o w i n g i n c l u s i o n i n t o t h e I F N - o r P L A - t r e a t e d g r o u p s b e f o r e t r e a t m e n t a n d a g a i n a f te r 1 w e e k of d a i l y s u b c u t a n e o u s i n j e c t i o n s o f W e l l f e r o n ( l y m p h o b l a s t o i d i n t e r f e r o n ) . - 1 9 1 -Table 4:11. Poke weed mitogen induced IgG secretion (ng/ml) in co-cultures of 10 5 E~ cells and 10 5 T helper cells from two IFN-patients and one PLA-patient Source of T helper cells Source of E- cells None IFNI IFN2 PLA None 0 300 300 304 IFNI 309 419 330 230 IFN2 306 176 340 330 PLA 311 503* 1077* 2400* * Significantly higher responses than E" obtained from IFN patients mixed with T helper cells from IFN or PLA-patients (Mann-Whitney, P < 0.05). - 1 9 2 -Table 4:lll. Pokeweed mitogen induced IgG secretion (ng/ml) in co-cultures of 2 x 10 5 monocytes (plastic adherent cells) and 8 x 10 5 monocyte depleted PBMC obtained from two PLA and two IFN patients. Source of monocyte depleted PBMC Source of monocytes IFN1 IFN2 PLA1 PLA2 351 237 776 177 IFN1 238 NA 2967* 4443* IFN2 NA 177 NA NA PLA1 204 171 1490* 3820* PLA2 NA NA NA 3488* NA, Not available. * Significantly higher responses than monocyte depleted PBMC obtained from IFN-patients reconstituted with monocytes from IFN or PLA-patients (Mann Whitney, P<0.01). - 1 9 3 -4 : 5 References Blomgren, H., Strender, H. & Cantell, K. Effect of human leukocyte interferon on the response of lymphocytes to mitogenic stimuli in vitro. Scand. J . Immunol. 3, 697 (1984). Brodeur, B.R. & Merigan, T.C. Mechanisms of the suppressive effect of interferon on antibody synthesis in vivo. J . Immunol. 114, 1323 (1975). Chester, T.S., Paucker K. & Merigan, T.C. Suppression of mouse antibody producing spleen cells by various interferon preparations. Nature 246, 92 (1973). Choi, Y.S. , Lim, K.H. & Sanders, F.K. Effect of interferon alpha on pokeweed mitogen-induced differentiation of human peripheral blood B lymphocytes. Cell. Immunol. 64, 20 (1981). Fleisher, T.A., Attalah, A.M., Tasato, G., Blaese, R.M. & Greene, W.C. Interferon-mediated Inhibition of polyclonal immunoglobulin synthesis. J . Immunol. 129, 1099 (1982). Harfast, B., Huddlestone, J.R., Casali P., Merigan, T.C. & Oldstone, M. B.A. Interferon acts directly on human B lymphocytes to modulate immunoglobulin synthesis. J . Immunol. 127, 2146 (1981). -1 9 4 -Herberman, R.B., Ortaldo, J.R., Menachem, R. & Pestka, S. Augmentation of natural and antibody-dependent cell-mediated cytotoxicity by pure human leukocyte interferon. J . Clin. Immunol. 1, 149 (1981). Hirsch, R.L. & Johnson, K.P. Interferon enhances natural killer cell activity in vitro in multiple Sclerosis (Abst.). Neurology 34 (Supple, 1), 111 (1984). Krim, M. Interferons and their applications: past, present and future. In: Interferons and Their Applications Vol. 71, p. 1. (Eds P.E. Came & W.A. Carter) Springer Verlag, New York (1984). Kurtzke, J.F, On the evaluation of disability in multiple Sclerosis. Neurology (Minneapolis) 11, 686 (1961). Lee, S.H., Kelly S., Chiu H. & Stebbing, N. Stimulation of natural killer cell activity and inhibition of proliferation of various leukemic cells by purified human leukocyte interferon subtypes. Cancer Res. 42, 1312 (1982). Levinson, A.I., Dziarsi, A. & Hooks, J .J . Modulation of polyclonal B-cell differentiation by human leukocyte alpha interferon. Clin. exp. Immunol. 49, 677 (1982). Neighbor, P.A. & Bloom, B.R. Absence of virus-induced lymphocyte suppression and interferon production in multiple sclerosis. Proc. natl Acad. Sci. USA. 76, 476 (1979). - 1 9 5 -O'Gorman, M.R.G, & Oger, J . Reduced in vitro IgG secretion following in vivo injection of interferon in multiple sclerosis patients. Fed. Proc. 44, 1923 (1985). Panitch, HS., Francis, FS., Hooper, C J . , Fukushima, PI., Bleumers, EB., and Johnson, KP. Serial immunological studies in multiple sclerosis. Neurology 34 (Suppl.); 111 (1984). Parker, M.A., Mandel, A.D., Wallace, J.H. & Sonnenfeld, G. Modulation of the human in vitro antibody response by human leukocyte interferon preparations. Cell. Immunol. 58, 464 (1981). Peters, M., Walling, D.W. & Hoofnagle, J.H. The in vivo effects of interferon in man. Fed. Proc. 44, 1923 (1985). Rice, G.P.A., Talbot, P., Woelfel, E.M., Vandvik, B., Braheny, S., Knobler, R.L, Sipe, J . & Oldstone, M.B.A. Immunological observations in patients with multiple sclerosis treated with human alpha interferon (Abst.) Neurol. 34, (Suppl. 1), 112 (1984). Rodriguez, A., Prinz, W.A., Sibbit, W .L , Bankhurst, A.D. & Williams, R.C. JR. Alpha-interferon increases immunoglobulin production in cultured human mononuclear leukocytes. J . Immunol. 130, 1215 (1983). Saxon, A., Feldhaus, J.L. & Robbins, R.A. Single step separation of human T and B cells using AET treated SRBC rosettes. J . Immunol. Methods. 12, 285 (1976). - 1 9 6 -Shalaby, M.R. & Week, P. Bacteria-derived human leukocyte interferons alter in vitro humoral and cellular immune responses. Cell. Immunol. 82, 269 (1983). Virelizier, J.L., Chan, E.L. & Allison, A.C. Immunosuppressive effects of lymphocyte (type II) and leukocyte (type I) interferon on primary antibody responses in vivo and in vitro, Clin. exp. Immunol. 30, 299 (1977). Yen, N. H. & Reem, G.H. Immunoregulatory functions of natural and recombinant alpha interferons. Biol. Interf System, Abstract. 1(2) (1983). Zarling, J.M. Enhancement of human cytotoxic T-cell responses and NK cell activity by purified fibroblast interferon and polyribonucleotide inducers of interferon. In: Mediation of Cellular Immunity in Cancer by Immune Modifiers (eds M.A. Chirigos, M.J. Mastrangelo, M. Mitchell & M, Krim) Raven Press, New York (1981). -1 9 7 -Chapter 5 OVERALL SUMMARY AND HYPOTHESIS My original hypothesis was that, in multiple sclerosis abnormal immune function contributes to the progression of clinical disease activity. In this last section I will summarize the background ev idence documented by several laboratories intimating that overactive immunity is associated with multiple sclerosis. I will then summarize our results regarding this hypothesis and discuss the relevance of these findings. For years scientists [eg. Adams and Imigawa, 1962] have reported that the sera of MS patients contains increased titres of Ab to several antigens including numerous viruses such as measles, herpes simplex virus and Ebstein Barr virus. This observation coupled with the observation that the frequency of viral infections is lower in MS patients compared to the frequency observed in healthy controls [Sibley et al., 1985] suggests that MS patients have a more active immune system than healthy control individuals. Interestingly it has been reported that when MS patients do contract a viral infection, the exacerbation rate during the "at risk" period is almost 3 times as high as during the "not at risk period" [Sibley et al.,1985]. One interpretation of these observations is that in multiple sclerosis an overactive immune response to infection exists. - 1 9 8 -Another line of evidence suggesting that activation of the immune system may be associated with the C N S pathology observed in MS is derived from studies on the animal model of MS, experimental allergic encephalomyelitis (EAE). It has been shown that myelin basic protein specific T cell lines injected into syngeneic rats causes an acute form of EAE . Only preactivated T cells however can transfer the disease. Wekerly [1987] has shown that activated T cells directed against various antigens readily cross the blood brain barrier. Additionally this group has shown that the interaction of activated T cells with glial cells (probably via gamma interferon) can lead to the synthesis and expression of MHC Class II Ag which are critically required for immunogenic presentation of Ag to T cells. Indeed gamma interferon treated astrocytes have been shown to be efficient Ag presenting cel ls able to strongly up regulate the response of syngeneic T cells [Fiertz et al., 1985]. The ability of gamma interferon to induce the expression of MHC Class II Ag on glial cells in vitro may be of relevance in MS. Recently in a pilot trial of gamma interferon it was shown that the patients who received the drug had increased cellular immune responses and significantly more clinical relapses than the patients who received the placebo [Panitch et al.,1987]. This trial was abruptly terminated by the overseeing committee. These observations coupled with the demonstration that the immune system of multiple sclerosis patients is an "activated state" (reviewed in Chapter 1) and the very consistent observation that greater than 90% of all clinically definite multiple sclerosis patients actively secrete immunoglobulin within the C N S , suggests to me that - 1 9 9 -the immune system is activated and that regulation is shifted slightly towards "help" as opposed to "suppression". One mechanism which could lead to and result in the maintainance of an "activated state" is retroviral infection. T cells infected with retrovirus express IL2 receptors and they have the capacity for indefinite growth [Gallo, 1984]. There is some evidence suggesting that retroviral infection may be involved in MS. Infection with retrovirus can lead to T cell subpopulation inbalances and polyclonal B cell activation [Gallo, 1984], in vitro abnormalities commonly reported in multiple sclerosis (see Chapter 1 and 3). It has been reported that the C S F cells of MS patients contain RNA with homology to human T-lymphotrophic virus type-1 (HTLV-1) [Koprowski et al.,1985 and Hauser et al., 1986] and that the sera of these patients contains antibodies reactive with HTLV-1 antigen in concentrations significantly higher than controls [Koprowski et al.,1985]. Others [Gessain et al. , 1986 and Rice et al.,1986] however found that MS patients did not have antibody titres to HTLV 1 or III above those seen in healthy controls. Additionally as mentioned in chapter 1, the C S F and serum of multiple sclerosis patients contains increased titres of antibody to a number of other v i ruses. It has also been reported that T cell clones derived from peripheral blood and C S F of MS patients show prolonged expression of IL2 receptors and prolonged responsiveness to the proliferative effects of IL2 [DeFreitas et al., 1986]. However the exact mechanism for this has not been determined. - 2 0 0 -The discovery of the mitogenic activity of a root extract (Pokeweed mitogen, PWM) derived from the plant Phytolacca americana was accidental. Two technicians in the laboratory of P. Fames were accidently inoculated with an extract of the root and shortly thereafter, examination of their peripheral blood revealed large numbers of immature basophilic lymphocytes and typical plasma cells which persisted for at least 8 weeks [Fames et a l , 1965]. Stimulation of peripheral blood lymphocytes (PBMC) with PWM has become the most widely used model of in vivo immunoglobulin synthesis and secretion. We have used this model to study immune regulation in healthy controls and different groups of multiple sclerosis patients in various phases of clinical disease activity. Our contribution to the hypothesis that abnormalities in immune function contribute to the progression of multiple sclerosis is summarized below. In the first part of this dissertation we compared various immunological parameters involved in the regulation of PWM induced IgG secretion in the P B M C obtained from low responder healthy control volunteers versus high responders. We observed that the PBMC of low responders differed from the high responders' in each of the following in vitro immune parameters studied: their T helper cells contained a higher phenotypic ratio of suppressor-inducer over helper-inducer cells; the proliferative response of their T cells to autologous B cells and monocytes (AMLR) was lower (an indirect measurement of T helper cell activity); and the amount of in vivo radiation sensitive -201 -suppression was higher. Additionally the E- (B cells and monocytes) obtained from the LR individuals secreted less IgG than the HR E- cells when both subsets were mixed with the same HR E+ cells. These results suggest that T suppressor cells, T helper cells (Tsi and Thi), and B cells were all involved in the regulation of PWM induced IgG secretion and that the function of each of these subsets differ in HR and LR individuals. It is therefore conceivable that alterations in any of these subsets would alter the final differentiation process of B cells to immunoglobulin secreting cells. We have not explored the responses and/or production of lymphokines by both T and B cells which could affect the immunoglobul in secret ion response. Conceivably, future studies could explore the differences between high and low responders in the responses and/or production of such factors as ; IL4, IL5, IL6, transforming growth factor-13 and the T cell-replacing factors. In the second part of the dissertation we examined the PWM induced IgG secretion responses of the PBMC obtained from multiple sclerosis patients during various phases of clinical disease activity. In relapsing remitting MS patients who were clinically stable we observed a trend indicating that patients with a high response in the PWM IgG secretion assay were more likely to relapse than patients with a low response. The PWM induced IgG secretion assay may be more useful as prognostic tool if instead of clinical criteria, MRI were used to monitor disease activity as it is now generally agreed that most multiple sclerosis reveal pathology by MRI even though they remain clinically stable [Willoughby et al, In press]. In the relapsing-remitt ing multiple sc leros is patients in clinical relapse we observed that PWM induced IgG secretion response varied with clinical disease activity. Patients who had been suffering a clinical relapse for more than 3 weeks secreted significantly higher amounts of IgG than both healthy controls and patients who had been in relapse for 3 weeks or less. It is difficult to determine whether these change in immune function (ie. reduced IgG secretion) preceded or followed the clinical relapse however we have generated prelimary data suggesting that a reduction in IgG secretion follows the development of pathology as assessed by magnetic resonance imaging [Oger et al. 1988]. The reduction in IgG seen immediately following clinical relapse could be due to the migration of helper cells to the C N S (see further) or the release of immunomodulatory substances from the C N S lesions. I feel that we are beginning to see a trend in the relapsing remitting multiple sclerosis patients suggesting that the PWM induced IgG secretion assay varies in response to disease activity. This trend may be resolved with a well planned serial study involving more objective assessment of disease activity (eg. magnetic resonance imaging). In patients with chronic progressive M S , an unpredictable number with variable degrees of functional disability will for no known reason stabilize and cease to deteriorate clinically. We measured PWM induced IgG secretion in this group and in active chronic progressive multiple sclerosis patients and compared the results with the level of IgG secreted by healthy control individuals. The chronic progressive stable multiple sclerosis patients did not differ from the healthy control group whereas the active chronic progressive MS patients secreted significantly higher levels of IgG than the healthy control group. These results suggest that active disease is associated with abnormal immune function and that as the clinical disease "burns out" the immunological abnormalities return to normal. The chronic progressive stable multiple sclerosis group was older and had a longer duration of disease than the chronic progressive active group. It is possible that either overactive immune function is turned down because the destruction process in C N S burns out or alternatively the over active immune response burns out and causes a cessat ion of white matter destruction. In chronic progressive multiple sclerosis patients we have observed reduced suppressor cell function and abnormal representation of the number of T cells involved in the induction of help versus the number of T helper cells involved in the induction of suppression. We interpret these results in the chronic progressive patients as evidence that the abnormalities in immune regulation involve the T helper cell subset. The final part of this study involved measuring the effect of in vivo lymphoblastoid interferon treatment on the in vitro immune function abnormalities observed in a group of chronic progressive active multiple sclerosis patients. The lymphoblastoid interferon injections caused a dramatic reduction in the level of PWM induced IgG secretion as compared to patients who received a placebo. However T suppressor cell activity and the T cell subset markers studied were not significantly affected. Examination of the subset affected by the in vivo interferon injections revealed that a subset of cells (most likely the B lymphocytes) present in the E- subpopulation - 2 0 4 -and not the monocytes or T cells was directly affected by the in vivo treatment. Although the increased PWM induced IgG secretion observed in chronic progressive MS was reduced following the interferon injections, it is becoming apparent that this treatment did not provide the improvement in functional impairment we had ant ic ipated. In summary I feel that the most important contribution of this work to the understanding of immune abnormalities in the progression of clinical disease is our observation that the immune system is in an activated state as evidenced by the abnormal T helper cell subset ratios and that regulation is abnormally shifted towards help as opposed to suppression as evidenced by the increased level of IgG secreted in response to PWM and the reduced suppressor cell activity. It is apparent throughout this study that there are problems associated with the assessment of disease activity based on clinical criteria. If we are to determine the relevance of the immune abnormalities to the progression of disease we must employ more objective and more accurate methods of measuring disease activity. The multiple sclerosis group at the University of British Columbia is at the forefront of this sort of investigation. We are currently using magnetic resonance imaging to monitor disease activity. For example we have recently observed that the pathology in the C N S of mildly clinically affected relapsing remitting multiple sclerosis patients is much more continuous that previously thought [Isaac et al . , 1988; Willoughby et al., in press]. We are also beginning to understand the evolution of new lesions, it is now apparent that new lesions increase in s ize and then shrink to very small lesions which presumably represent the extent of the demyelinated area and that these changes can occur independently of changes in other new lesions or in chronic stable ones. We have recently reported that immune function changes followed the development of very large, isolated lesions in the CNS of a group of relapsing remitting multiple sclerosis patients [Oger et al., 1988]. These results have been generated on a small group of patients who do not have typical MRI profiles and therefore they must be interpreted with caution. Of utmost significance in immune function studies on cl inical ly stable versus cl inical ly active relapsing remitting multiple sclerosis is the now general consensus that clinical disease activity correlates very poorly with C N S pathology as assessed by magnetic resonance imaging. Additionally we have observed that the MRI activity occurring within the C N S of chronic progressive patients is very much increased over the activity observed in the relapsing remitting patients [Koopmans et a l . , submitted for publication]. In summary we have shown that there are abnormalities in immune function associated with different clinical stages of multiple sclerosis. It is conceivable that abnormal T cell activation is related to disease progression and this could be the result of inheriting an abnormal representation of T cells involved in the induction of suppression. Alternatively an individual could have T cells which are particularly sensit ive to the induction of activation or relatively insensitive to the induction of suppression. It is also possible that there is an abnormality in secondary biochemical events occurring after the induction of activation in the T cells of MS patients. - 2 0 6 -Interestingly the most consistent abnormalities occur in a subgroup of chronic progressive multiple sclerosis patients who have also been shown to have the most active pathology as assessed by magnetic resonance imaging. In order to generate insight into the question of whether the immunological abnormalities observed are a result of and/or cause of C N S pathology we will have to plan an investigation involving the simultaneous and serial measurement of PWM induced IgG secretion, enumeration of T cell activation and subset markers and the measurement of a helper and of a suppression function in conjunction with magnetic resonance imaging on a clinically heterogeneous group of patients and healthy controls. 5:1 References Adams, CWM. , and Imagawa DT. Measles virus antibodies in multiple sclerosis. Proc. Soc. Exp. Biol. Med. 111; 562 (1962). DeFreitas, E C , Sandberg-Wollheim, M., Schonely, K., Boufal, M., and Koprowski, H. Regulation of interleukin 2 receptors on T cells from multiple sclerosis patients. Proc. Natl. Acad. Sc i . USA. 83; 2637 (1986). Fames, P., Barker, BE . , Brownhill, LE . , and Fanger, H. Mitogenic activity in Phytolacca americana (pokeweed). Lancet 1; 170 (1964). Fiercz, W., Enkler, B., Reske, K., Wekerle, H., and Fontana, A. Astrocytes as antigen-presenting cells. I. Induction of la antigen expression on astroytes by T cells via immune interferon and its effect on antigen presentation. J Immunol. 134; 3785. (1985). Gal lo , R C . Human T-cell leukaemia-lymphoma virus and T-cell malignancies in adults. Cancer Surveys 3;113 (1984). Gessain, A., Abel, L., De-The, G., Vernant, J C , Raverdy P., Guillard, A. Lack of antibody to HTLV-1 and HIV in patients with multiple sclerosis from France and French West Indies. Br. MEd J . 293; 424 (1986). - 2 0 8 -Hauser, S L , Aubert, C , Burks, JS . , Kerr, D., Lyon-Caen, O., de-The, G., Brahic, M. Analysis of human T-lymphotropic virus sequences in multiple sclerosis tissue. Nature 322; 176 (1986). Issac, C , Li, D., Genton, M., Jardine, C , Grochowski, E., Palmer, M., Kastrukoff, LF. , Oger, J . , and Paty, DW. Multiple sclerosis: A serial study using MRI in relapsing patients. Neurology (in press). Koopmans, RA., Li, DKB., Jardine, C , Costley, L., Hall, S., Grochowski, E., Oger, J J F . , Kastrukoff, L., and Paty, DW. Chronic prgressive multiple sclerosis: Magnetic resonance imaging assessment of disease activity over a six month period. Submitted for publication. Koprowski, H., DeFreitas, E C , Harper, ME. , Sandberg-Wollheim, M., Sheremata, WA., Robert-Guroff, M., Saxinger, CW., Feinberg, MB., Wong-Staal, F., and Gallo, RC. Multiple sclerosis and human T-cell lymphotropic retroviruses. Nature 318; 154 (1985). Oger J . , O'Gorman, MRG., Willoughby, E., Li, D., and Paty, DW. Changes in immune function in relapsing multiple sclerosis correlate with disease activity as assessed by magnetic resonance imaging. N.Y. Acad. Sci . In press. (1988). Panitch, HS. , Hirsch, RL., Schindler, J . , Johnson, KP . Treatment of multiple sclerosis with gamma interferon: Exacerbations associated with activation of the immune system. Neurology 37;1097. (1987). - 2 0 9 -Rice, G P . , Armstrong, HA., Bulman, DE., Paty, DW., Ebers, G C . Absence of antibody to HTLV I and III in sera of C a n a d i a n patients with multiple sclerosis and chronic myelopathy. Ann. Neurol. 20; 533 (1986). Sibley, WA., Bamford, CR. , Clark, K. Clinical viral infections and multiple sclerosis. The Lancet; 1313 (1985). Wekerly, H., Sun, D., Oropeza-Wekerly, RL., and Meyermann, R. Immune reactivity in the nervous sustem: modulation of T-lymphocyte activation by glial cells. J Exp. Biol. 132; 43 (1987). Willoughby, EW., Grochowski, E., Li, D., Oger, J . , Kastrukoff, LK., and Paty, DW. Serial magnetic resonance scanning in multiple sclerosis: A prospective study in relapsing patients. Ann. Neurol. (in press). 

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