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Reduced in vitro IgG secretion following in vivo injection of interferon (wellferon R) in multiple sclerosis… O’Gorman, Maurice R. G. 1985

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REDUCED IN VITRO IgG SECRETION FOLLOWING IN VIVO INJECTION OF INTERFERON (WELLFERON R ) IN MULTIPLE SCLEROSIS PATIENTS By Maurice R.G. 0'Gorman Hon. B.Sc. University of Western Ontario A THESIS SUBMITTED IN PARTIAL FULLFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF The Faculty of Graduate Studies Pathology Department, University of B r i t i s h Columbia We accept t h i s thesis as conforming to the required standard University of B r i t i s h Columbia MASTER OF SCIENCE i n A p r i l 1985 Raymond Gerard 0'Gorman In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree t h a t permission f o r ex t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f P a t h o l o g y  The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date A p r i l , 1985 DE-6 (3/81) - i i -May 9, 1985 Abstract An i n v i t r o IgG secretion assay was developed to investigate the regulation of the humoral immune response i n humans. Pokeweed mitogen (PWM), a plant l e c t i n derived from Phytolacca americana stimulates human peripheral blood mononuclear c e l l s (PBMNC) to divide and resting B-lymphocytes to d i f f e r e n t i a t e into immunoglobulin secreting c e l l s (ISC). This d i f f e r e n t i a t i o n requires that both monocytes and T-lymphocytes be present i n the culture system. The amount of IgG secreted by these d i f f e r e n t i a t e d B-lymphocytes i n response to PWM appears to be the net re s u l t of a balance between the functional a c t i v i t y of the regulatory T-helper and T-suppressor c e l l s . A l t e rations, q u a l i t a t i v e or quantitative i n any of these leukocyte subsets could conceivably a l t e r the amount of IgG secreted by the B-lymphocyte subpopulation. We have employed t h i s assay to investigate the immune status i n a group of chronic progressive multiple s c l e r o s i s (MS) patients and to assess the immunoregulatory e f f e c t s of interferon (Wellferon R, INF) administered i n vivo to t h i s selected group. Their mononuclear c e l l s (MNC) were studied i n t h i s PWM induced IgG secretion assay before INF treatment and again after 7 days of d a i l y sub-cutaneous i n j e c t i o n s (5 X 10 6 u/day). Twenty patients received the interferon (INF) preparation and eighteen received normal s a l i n e . The study was carried out i n a double b l i n d manner and the code was broken only after i n d i v i d u a l r e s u l t s had been calculated. Supervisor: D.W. Paty . Supervisor Pro tempore: J.J.-F. Oger - i i i -The l e v e l of IgG secreted by PBMNC i n response to PWM was measured i n : 1) patients before treatment; 2) healthy volunteers; and 3) placebo and INF treated patients. We found that the l e v e l of IgG secreted into the supernatants a f t e r 7 days was s i g n i f i c a n t l y higher i n the MS group than i n the control group. We attribute t h i s to an a l t e r a t i o n of the normal immune response i n MS patients. Following one week of d a i l y subcutaneous i n j e c t i o n s , the l e v e l of IgG secreted by the MNC is o l a t e d from INF treated patients was dramatically reduced compared to; 1) the l e v e l observed p r i o r to treatment and 2) the amount of IgG secreted by the MNC is o l a t e d from the placebo treated patients. This i s the f i r s t report on the a b i l i t y of INF administered i n vivo to modulate the i n v i t r o humoral immune response i n humans. Using techniaues to separate and study the function of lymphocyte subsets we have searched for the subset affected by i n vivo INF i n j e c t i o n and found that the INF induced decrease i n IgG secretion seems to be mediated by an e f f e c t on B c e l l s . These studies centered on IgG secretion. In order to investigate suppressor c e l l function we measured the suppression induced by i n v i t r o Con A stimulated lymphocytes on the p r o l i f e r a t i v e response of mitogen stimulated heterologous lymphocytes. Confirming previous reports of decreased suppressor a c t i v i t y i n MS patients we found the l e v e l of suppression s i g n i f i c a n t l y lower i n the patients studied vs the controls. However t h i s Con A induced suppressor a c t i v i t y was not affected by i n vivo i n j e c t i o n of INF. Table of Contents Pages i ; T i t l e Page i i - i i i , Abstract i v - Table of Contents v- v i - L i s t of Tables v i i - L i s t of Figures 1 - 2 8 ; Introduction 1 - 9 ; Immunology and Multiple Sclerosis 10 -.19; Pokeweed Mitogen Induced IgG Secretion i n v i t r o A model of the i n vivo Humoral Immune Response 20 - 28; Immunomodulatory Effects of Interferon 2 9 - 3 7 ; Materials and Methods 3 7 - 6 0 ; Results 6 1 - 6 8 ; Discussion 69 - 85; Bibliograghy - V -L i s t of Tables Table I; Monoclonal Ab to Human PBMNC Surface Antigens, page 43. Table I I ; IgG Secreted i n Culture by MNC Stimulated with PWM, page 44. Table I I I ; IgG Secreted i n Culture by Unstimulated MNC Isolated from Control Subjects and Treated and Untreated MS Patients, page 45. Table IV; Level of PWM Induced IgG Secretion i n MS and Control Individuals, page 46. Table V; Effect of i n vivo Injection of Interferon on the Amount of IgG Secreted i n Culture by MNC Stimulated with PWM, page 47. Table VI; Effect of Interferon Injection on the Level of PWM Induced IgG Secretion i n PBMNC Cultures Isolated from MS Patients, page 48. Table VII; Suppressor A c t i v i t y i n Controls and i n MS Patients, page 50. Table V I I I ; Con A Induced Suppressor A c t i v i t y i n MS Patients Before Treatment and After 1 Week of Daily Injections of Interferon or Placebo, page 53. Table IX; IgG Secretion i n Reconstituted MNC Cultures Stimulated with PWM; Effect of i n vivo Interferon Injection on the T H and B- Lymphocyte Subpopulations, page 54. Table X; IgG Secretion i n Reconstituted MNC Cultures Stimulated with PWM: Effect of i n vivo Interferon Injection on NAC and PAC, page 55. - vi -Table XI; IgG Secretion by PWM Stimulated Cultures of MNC and Monocyte Depleted MNC, page 58. Table XII; Purity of B, T C e l l Separation, page 59. Table X I I I ; E f f i c i e n c y of 0KT8 Removal, page 60. VI 1 L i s t of Figures Figure 1; Con A Induced Suppressor C e l l Assay, page 51. Figure 2; Con A Induced Suppression vs Level of IgG Secreted, page 56. - 1 -Introduction Immunology and Multiple Sclerosis M u l t i p l e Sclerosis (MS) i s a chronic inflammatory disease of the c e n t r a l nervous system (CNS). The c l i n i c a l course of the disease i s e x t r a o r d i n a r i l y variable but generally presents i t s e l f as a series of relapses and remissions or as a chronic progressive disease. The pathological reactions responsible for the observed symptomatology i s p e r i - a x i a l demyelination affecting the central nervous system. The mechanism responsible for the observed destruction of myelin and oligodendrocytes however remains a mystery. Ch a r a c t e r i s t i c lesions of an MS brain are well demarcated fo c a l zones of demyelination or "plaques" (32). Plaques are d i s t r i b u t e d throughout the white matter of the brain and spinal cord with a p r e d i l e c t i o n for certain areas within the CNS, especially the p e r i v e n t r i c u l a r area. (For reviews see (2,134)). The plaques may vary i n age, s i z e and shape and are quite often located perivascularly. Despite many publications r e f e r r i n g to the "early l e s i o n " i n MS, very l i t t l e i s known of the i n i t i a l stage of plaque evolution. The lack of knowledge i n t h i s area stems from the chronic nature of the disease. This has forced pathologists to investigate early lesions by studying e i t h e r the edge of a plaque or the unaffected white matter. I t could be noted here that within the macroscopically normal appearing white matter, A l l e n et a l . (2) observed that 72% of the h i s t o l o g i c a l sections which they studied were abnormal, the most frequently observed abnormality being d i f f u s e g l i o s i s . - 2 -Older plaques are grey due to loss of myelin and have an altered consistency as a result of g l i o s i s (134). Within the plaque, demyelinated axons, numerous ast r o c y t i c processes and very few oligodendroglia are observed. Unlike established plaques, early lesions do not have well demarcated edges. This i s due to oedema and the presence of inflammatory c e l l i n f i l t r a t e s at the s i t e of demyelination (134). The c e l l i n f i l t r a t e s which often surround small blood vessels consist of lymphocytes, macrophage, and plasma c e l l s . The d i s t r i b u t i o n of these c e l l s within the plaques and normal appearing white matter has recently been investigated. Hauser et a l . , (67) observed that l a bearing c e l l s (activated T - c e l l s , B c e l l s and monocytes) are present throughout the normal appearing white matter. Using monoclonal Abs directed against s p e c i f i c lymphocyte subsets, Traugott (185) observed large numbers of 0KT4+ c e l l s (T-helper lymphocytes) at the le s i o n margins and extending well i n t o the adjacent white matter. The 0KT8+ c e l l s (T-suppressor/cytotoxic lymphocytes) were pr e f e r e n t i a l l y d i s t r i b u t e d around small blood vessels at the lesi o n margins. Within the centre of the lesions Ia+ macrophage were most numerous. Prineas et a l . (135) also observed plasma c e l l s i n the unaffected white matter and around the perivascular compartment of t y p i c a l chronic lesions. The proximity of T4+ c e l l s to Ia+ macrophage and plasma c e l l s i n the absence of the 0KT8+ T-suppressor c e l l s within the normal appearing white matter suggests that there may be a lack of cont r o l i n down-regulating immune function i n the CNS of MS patients. - 3 -The etiology of MS remains an enigma. One hypothesis suggests that MS i s an autoimmune disease directed against an as yet unidentified antigen present on myelin or oligodendrocytes. Another hypothesis suggests MS i s due to a chronic v i r a l i n f e c t i o n within the CNS. The two hypotheses are not mutually exclusive and one could hypothesize that an abnormal immune response to a chronic v i r a l i n f e c t i o n i n genetically susceptible individuals results i n the autoimmune destruction of myelin. There i s a plethora of data documenting abnormal immune phenomena i n the serum and cerebral spinal f l u i d (CSF) of MS patients (for reviews see r e f s , 78,124). There are two such abnormalities currently recognized as di a g n o s t i c a l l y important i n MS: 1) intrathecal IgG synthesis or secretion, and 2) the presence of oligoclonal bands of IgG detected i n the CSF and occasionally i n the serum, when such samples are subjected to electrophoresis. Increased intrathecal IgG secretion i s present i n 74% (124) to 92% (78,180) of patients with c l i n i c a l l y d e f i n i t e MS. Elevated l e v e l s of IgG i n the CSF can i n addition to intrathecal IgG synthesis, be the r e s u l t of a damaged blood brain bar r i e r (bbb) or high serum IgG (78). The l a t t e r two factors would resu l t i n the d i f f u s i o n of IgG from the blood to the CSF since the concentration of IgG i s higher i n the blood. The concentration of IgG i n MS serum however i s usually normal (98) and the blood brain b a r r i e r i s only affected i n approximately 20% of MS patients (179). As albumin i s synthesized i n the l i v e r only, a comparison between the amount of IgG and the amount of albumin i n the CSF allows one to d i f f e r e n t i a t e between intrathecal IgG production and bbb damage. Normal - A -albumin levels i n the CSF i n the face of elevated IgG l e v e l s i n the CSF i s i n d i c a t i v e of intrathecal IgG secretion. There have been many indices proposed to measure in t r a t h e c a l l y secreted IgG (124) but the most popular index for diagnostic purposes i s that proposed by Tourtellotte (181). This index i s a measure of the amount of IgG secreted i n t r a t h e c a l l y per day and takes into account both the eff e c t s of high serum IgG and a defective bbb. The values obtained by t h i s method demonstrate abnormally high l e v e l s i n approximately 65% of a l l MS patients and 92% of c l i n i c a l l y d e f i n i t e MS patients (180). When the CSF or brain eluates of MS patients are subject to electrophoresis discrete bands of IgG (oli g o c l o n a l bands) appear. About 90% of MS patients' CSF reveal these discrete o l i g o c l o n a l bands of IgG i n the gamma area of CSF electrophoresis (105). Although these bands are of diagnostic importance, t h i s phenomena i s not s p e c i f i c for MS (for review, see r e f . 112). Oligoclonal banding has been demonstrated i n a variety of neurological inflammatory conditions associated with p a r t i c u l a r l y intense or chronic antigenic stimulus (112). One theory i s that these o l i g o c l o n a l bands of IgG present i n the CSF, brain and occasionally (27%) i n the serum (105) of MS patients represent the products of a few clones of activated B-lymphocytes, and that the immunoglobulins which make up these bands are associated with the e t i o l o g i c a l agent (90,96). Support for the l a t t e r concept i s suggested by data obtained i n the study of subacute sclerosing panencephalitis where the ol i g o c l o n a l IgG i s s p e c i f i c for the e t i o l o g i c a l agent, namely measles virus (97,190,191). In the CSF of MS patients measles virus Ab are found more often than i n controls. This observation was f i r s t noted by Adams et a l . (1) - 5 -i n 1962 and has been amply confirmed (42,158,159). Antibodies to other viruses are also found more often i n the CSF of MS patients than i n other neurological controls (120,158). The presence or absence of these Ab does not correlate with disease a c t i v i t y (42,120). A d d i t i o n a l l y , antibodies to more than one virus have been found simultaneously increased (12,158). These l a t t e r observations coupled with the i d e n t i f i c a t i o n of anti-oligodendrocyte Ab and elevated IgG secretion i n the CSF suggests that t h i s immune response i s non s p e c i f i c and that possibly an abnormality e x i s t s i n the regulation of B - c e l l function. That a s p e c i f i c Ag does e x i s t and i s the target i n the demyelination process cannot be ruled out however since i t has been reported that a s p e c i f i c immune response (shown as an increase of a s p e c i f i c antibody) can be accompanied by many non-specific immune responses ( i e , increased l e v e l s of non-relevant antibodies) (124). I t has also been suggested that the oligoclonal bands represent a secondary phenomenon occurring as a resu l t of the destruction of myelin i n the CNS (78). Obviously the pathogenesis of the bands i s obscure and i t remains to be resolved whether a s p e c i f i c Ag i s involved or i f the ol i g o c l o n a l bands that are observed i n the CNS of MS patients are the re s u l t of a deregulation i n normal immunological control mechanisms. Of p a r t i c u l a r interest to our group i s the demonstration of abnormal immune regulation i n the peripheral blood of MS patients. The immune system can be grossly divided into two main components, e f f e c t o r c e l l s and regulatory c e l l s . The former include B-cells which secrete immunoglobulin, cytotoxic T-cells which mediate c y t o t o x i c i t y against allogeneic targets, K-cells (K=Killer) responsible for antibody mediated - 6 -c e l l u l a r c y t o t o x i c i t y and NK (Natural K i l l e r ) c e l l s which mediate c y t o t o x i c i t y against tumor c e l l s and virus infected c e l l s . The l e v e l of a c t i v i t y of these effector c e l l s appears to be the r e s u l t of the functional state of the regulatory T-cells. The regulatory c e l l s are composed i n part of helper or inducer T-cells and suppressor T- c e l l s , the functions of which are s e l f explanatory. These c e l l s bear d i f f e r e n t surface markers roughly associated with t h e i r function and which permit t h e i r enumeration. Commercially available monoclonal Ab's directed against these c e l l subset s p e c i f i c surface markers (See Table 1) have recently enabled investigators to i s o l a t e these subsets and analyse t h e i r respective functions i n v i t r o . There are numerous studies demonstrating altered numbers (18,28,76,136,1A6,160) and function (5,7,13,61,118,198) of suppressor/cytotoxic c e l l s i n the peripheral blood of MS patients. Before the advent of monoclonal Abs, B and T c e l l s were quantitated and is o l a t e d by the presence of surface Ig and by binding to sheep red blood c e l l s (SRBC, i . e . E-rosetting) respectively. The number of surface Ig po s i t i v e c e l l s (B-cells) appears to be normal i n the blood of MS patients, however the number of E-rosetting (T-cells) c e l l s i s reduced (99,122,186). Oger et a l . (122) and Utermohlen et a l . (189), observed that the avidl y rosetting T-cells (a subset of a l l r o s e t t i n g T-cells) were reduced i n active MS. The T-suppressor/cytotoxic lymphocyte subset bear more receptors for SRBC than does the T-inducer subset, suggesting that t h i s suppressor T- c e l l subset i s s e l e c t i v e l y a l tered. The regulatory T-helper and T-suppressor subsets can also be roughly divided i n t o two groups based on the presence of c e l l surface receptors for the Fc fragment of either IgG (TG-cells) which mediate - 7 -suppression or IgM (TM-cells) which represent the inducer c e l l subset (114). The TG subset reportedly fluctuates with disease a c t i v i t y (76,77,186). Using the currently available monoclonal Ab's (see Table 1) recent studies have demonstrated a reduction i n the l e v e l of c i r c u l a t i n g T s - c e l l s (0KT8, 0KT5, Leu 2),(10,18,28,68,130,146) i n MS patients measured either as a percentage of a l l peripheral blood lymphocytes or as a r a t i o of T-helper to T-suppressor c e l l s ( i . e . 0KT4:0KT8). This observation has been confirmed i n our laboratory (131) however the extent of the reduction i n the number c e l l s bearing the suppressor phenotypes remains to be determined. Changes i n suppressor c e l l numbers vary with d i f f e r e n t forms of the disease (131,136). I t appears that approximately 40% of the chronic progressive patients and 70% of patients i n acute relapse have a decreased number of 0KT8+ c e l l s whereas stable MS patients have normal values (68,124,130,157). The mechanism responsible for the observed decrease i n the number of lymphocytes bearing suppressor subset s p e c i f i c surface markers i s not known. That these c e l l s are destroyed or p r e f e r e n t i a l l y l o s t to the CSF does not agree with the rapid variations i n the proportion of T8+ c e l l s observed, and the number of T8+ c e l l s i n the CNS hardly accounts for the decreased l e v e l s i n the blood (68,124). A more l i k e l y explanation for the reduction of suppressor c e l l numbers i s a modulation i n the expression of the c e l l surface markers (8,131). An autoantibody binding to the surface of the suppressor c e l l subset could conceivably a l t e r the binding of the T s - c e l l s p e c i f i c monoclonal Abs. I f t h i s were the case, the suppressor c e l l would s t i l l be present however i t would not be - 8 -recognized by conventional methods (8). Support for t h i s mechanism stems from the occurence of anti-lymphocyte Ab which have been detected i n the serum of some MS patients (89,100,107,164). Studies of the percentages of 0KT8+ c e l l s and Leu 2a+ c e l l s (separate markers both i d e n t i f y i n g suppressor/cytotoxic lymphocytes) suggest that the 0KT8 marker fluctuates much more than the Leu 2a marker, (which does not appear to vary with c l i n i c a l changes i n the disease) (131). From these results i t seems l i k e l y that the observed apparent reduction i n suppressor c e l l s i n MS patients results from modulation of the surface Ag's (124). Not only are the T suppressor c e l l markers reduced, suppressor c e l l function i s also altered i n MS. I t appears that functional a l t e r a t i o n s p a r a l l e l the c l i n i c a l course of the disease (7,13,18,68,76,77,146). Con A induced suppressor c e l l a c t i v i t y has consistently been demonstrated to be reduced i n patients with MS (7,13,61). Several groups using d i f f e r e n t assays to measure suppression have noted aberrant regulation i n MS (77,118,198). Low doses of i r r a d i a t i o n abrogates Ts function (169). Increased IgG production i n pokeweed mitogen stimulated cultures of ir r a d i a t e d T-cells plus B - c e l l s above the l e v e l IgG produced by non-irradiated T-cells plus B - c e l l s i s i n d i c a t i v e of active suppression. Huddlestone took advantage of t h i s phenomena to. demonstrate increased suppressor a c t i v i t y immediately following c l i n i c a l relapse (77). Similarly other groups have observed increased PWM induced IgG secretion i n the PBMNC cultures of MS patients vs healthy control i n d i v i d u a l s and speculated that t h i s was due to either decreased suppressor c e l l a c t i v i t y or excessive helper a c t i v i t y (10,62,82,93,123). - 9 -As suppressor c e l l s are thought to be involved i n the maintenance of s e l f tolerance, and as these c e l l s have been shown to fluctuate with the c l i n i c a l course of MS i t i s conceivable that once the i n i t i a l pathological event occurs, be i t v i r a l or otherwise, the aberrant immune regulation observed could contribute to the relapsing and remitting nature of t h i s disease. Other immunological phenomenon reported to be altered during the active phase of the disease include: decreased NK c e l l a c t i v i t y (109) along with decreased responsiveness to INF (119), and increased c e l l u l a r c y t o t o x i c i t y by K c e l l s as measured by ADCC (53,110). I t remains to be resolved whether the observed a l t e r a t i o n s i n the immune systems of MS patients are the primary cause of the disease or the secondary r e s u l t of a primary pathological reaction. Regardless of which mechanism proves to be responsible for the i n i t i a t i o n of the disease, i t i s possible that the abberrant immune regulation observed i s contributory to the s e l f perpetuating nature of multiple s c l e r o s i s . Therefore future in v e s t i g a t i o n should explore the notion of abnormal immune regulation i n response to common v i r a l or s e l f Ag's as a mechanism responsible for the relapsing and remitting nature of t h i s disease. - l O -I I . Pokeweed Mitogen Induced IgG Secretion In V i t r o : A Model of the In  Vivo Humoral Immune Response. Immunoglobulin (Ig) production and secretion i s actuated by the B-lymphocyte subset. B-lymphocytes can be defined as c e l l s derived from lymphoid hematopoeitic precursors i n the f e t a l l i v e r and adult bone marrow (55) endowed with the a b i l i t y to synthesize immunoglobulin (195). B-cells are very heterogeneous, ranging from p r i m i t i v e precursors to f u l l y d ifferentiated plasma c e l l s , with concomitant changes i n the surface markers expressed (188), i n patterns of r e c i r c u l a t i o n and homing in t o secondary lymphoid organs (95,173), i n thei r l i f e s p a n (111), i n class of Ig produced (201), and i n th e i r a b i l i t y to respond by p r o l i f e r a t i o n and/or Ig secretion after interaction with the appropriate mitogen or antigen (Ag). B-cell s can be defined h i s t o l o g i c a l l y according to t h e i r state of maturation and the following c l a s s i f i c a t i o n i s generally accepted. 1) Precursor B-cells or pre B- c e l l s : No detectable surface immunoglobulin however cytoplasmic IgM i s detectable and these c e l l s w i l l d i f f e r e n t i a t e into mature B-cells (65). 2) Resting B-cells: Small (7-9 urns) Ig-positive c e l l s that are competent to synthesize Ig molecules some of which w i l l be deposited on the c e l l membrane to serve as antigen-binding receptors (54,113). 3) Activated B-cells: Large blasts or plasma c e l l s which h i s t o l o g i c a l l y demonstrate well developed rough endoplasmic reticulum and golgi apparatus. These large c e l l s develop a f t e r the acti v a t i o n of resting B-cells and are able to synthesize Ab - 11 -molecules at a high rate and export them e x t r a c e l l u l a r l y i n large quantities (108). Resting B - c e l l s can be activated to divide and/or produce immunoglobulin. I t i s the mechanism by which the B-cells are triggered which d i f f e r e n t i a t e s i n v i t r o pokeweed mitogen induced B - c e l l d i f f e r e n t i a t i o n from Ag induced d i f f e r e n t i a t i o n i n v i t r o and i n vivo. The Ag-specific binding receptor on the B - c e l l surface i s i d e n t i c a l to the Ab molecule that the c e l l i s capable of secreting after a c t i v a t i o n . Selective binding of Ag to c e l l ( s ) bearing receptors i s the key to s p e c i f i c a c t i v a t i o n of B - c e l l s . Once the s p e c i f i c binding has occurred the c e l l ( s ) (only a very small fraction of immunocompetent B-cells) becomes activated to expand c l o n a l l y and to d i f f e r e n t i a t e into a state where i t can secrete large amounts of s p e c i f i c Ab. Mitogen a c t i v a t i o n of lymphocytes on the other hand d i f f e r s from the above Ag s p e c i f i c a c t i v a t i o n i n that the number of B-cells activated i s several orders of magnitude higher than the number of precursor c e l l s reported for each Ag-specific clone (37,113) and the m u l t i p l i c i t y of antibody s p e c i f i c i t i e s found a f t e r stimulation i s also much larger. The large v a r i a t i o n i n the s p e c i f i c i t y of the Ab secreted strongly suggests that PWM triggers B - c e l l d i f f e r e n t i a t i o n independently of the Ig s p e c i f i c receptor s i t e . Nothing i s known of the physicochemical structure of the mitogen receptor. Although these differences i n the triggering of resting B - c e l l s e x i s t s , i t appears that once the i n i t i a l event occurs, the mechanism responsible for p r o l i f e r a t i o n and d i f f e r e n t i a t i o n i s the same i n both systems. Once activated, mitogen stimulated immunocompetant c e l l s perform equally, w e l l a l l the functions that antigen-activated c e l l s - 12 -perform (3,63,64). For the above reasons mitogen induced d i f f e r e n t i a t i o n of r e s t i n g B-cells into immunoglobulin secreting c e l l s (ISC) has been used extensively to unravel the complex mechanism of lymphocyte a c t i v a t i o n . Once stimulated, resting B-cells generally divide and d i f f e r e n t i a t e i n t o ISC. C e l l d i v i s i o n however i s not always accompanied by immunoglobulin secretion (151) and conversely extensive Ig secretion i s not necessarily associated with c e l l d i v i s i o n (115,162). From these observations one could postulate that p r o l i f e r a t i o n and d i f f e r e n t i a t i o n of r e s t i n g B-cells into ISC after stimulation are two separate phenomena (49,71). When measuring B - c e l l a c t i v a t i o n therefore one must be aware that d i f f e r e n t methods may measure dif f e r e n t c e l l functions. B - c e l l activation i s currently measured as: a) a p r o l i f e r a t i v e response, measured by the uptake of radiolabelled DNA precursors (e.g. Thymidine). b) an enumeration of Ig secreting c e l l s , measured by e i t h e r intracytoplasmic staining (36,54) or the reverse hemolytic plaque assay (84). c) a quantitative assessment of the immunoglobulin secreted into the supernatant, measured either by ELISA (195b) or radioimmunoassay (197). The above assays are usually used to measure non-specific B-lymphocyte act i v a t i o n . Assays have been developed which do measure Ag-specific Ab responses (Rev. i n 55). - 13 -The remainder of t h i s review w i l l concentrate on the a c t i v a t i o n and regulation of B - c e l l d i f f e r e n t a t i o n i n PWM stimulated human peripheral blood mononuclear c e l l s (PBMNC). Pokeweed mitogen, a plant l e c t i n isolated from Phytolacca  americana, stimulates T-cells to divide (63) and induces the polyclonal a c t i v a t i o n of resting B-cells manifested by t h e i r p r o l i f e r a t i o n and d i f f e r e n t i a t i o n into ISC (204). PWM binds to the B - c e l l surface although t h i s i s by i t s e l f i n s u f f i c i e n t to induce d i f f e r e n t i a t i o n (54). T-cells (47,80,83) as well as macrophages- (43,153,154,155) are required for the PWM induced d i f f e r e n t i a t i o n of resting B-cells i n t o ISC. I t was o r i g i n a l l y argued that macrophages were not required for B - c e l l d i f f e r e n t i a t i o n i n t h i s system (47,79,162) however i t i s now generally accepted that they are required for the generation of ISC. The most probable cause for the c o n f l i c t i n g results stems from the e f f i c a c y of macrophage depletion (22). Contamination by only 1-2% macrophages i n high density cultures i s s u f f i c i e n t to restore the polyclonal B - c e l l response to PWM (153). Most notably i t appears that macrophages have two d i s t i n c t functions i n t h i s system. The f i r s t requires only the presence of i n t a c t c e l l s and the second involves active secretion of lymphokines, most l i k e l y IL-1 (155). Both these functions are required for the complete d i f f e r e n t i a t i o n of resting B-cells into ISC (155). Only i n t a c t macrophages however are required for the PWM induced p r o l i f e r a t i v e response of the PBMNC. Interestingly, the accessory c e l l role of the macrophages and t h e i r secretory products i s not genetically r e s t r i c t e d i n t h i s system as allogeneic macrophages are able to f u l l y reconstitute both the PWM induced generation of ISC (153) and the mitotic responsiveness (43). - 14 -In macrophage reconstitution experiments i t i s i n t e r e s t i n g that the optimal response exceeds the response observed i n the normal c e l l c u l t ures, however once a c r i t i c a l percentage of macrophages has been exceeded the l e v e l of response observed i s s i g n i f i c a n t l y reduced (153-155). Macrophage and/or t h e i r secretory products cannot however reconstitute the PWM induced generation of ISC i f T-lymphocytes are not present i n the culture system (47,79,83). Using the plaaue forming c e l l (PFC) response as a measure of B - c e l l d i f f e r e n t i a t i o n i t was observed that depletion of the T-cell subpopulation i n both peripheral blood and t o n s i l l a r mononuclear c e l l cultures markedly reduced B - c e l l d i f f e r e n t i a t i o n following PWM stimulation. The response was f u l l y reconstituted by adding back T-cells (47,83). T-cell supernatants on the other hand could reconstitute the response of the T - c e l l depleted t o n s i l l a r lymphocyte cultures but not that of the c e l l s from the peripheral blood suggesting a more complex T-cell requirement i n the PWM induced response i n the PBMNC cultures (47). The l e v e l of the response induced by PWM and measured either as the number of ISC or the amount of Ig secreted, varies markedly from one i n d i v i d u a l to the next. The va r i a t i o n i n the responsiveness of the c e l l s from any given individual when assayed over a f a i r l y long period of time however varies much less by comparison (83). Some i n d i v i d u a l s are consistently "high responders" while others are consistently "low or non-responders". I t appears that the l e v e l of responsiveness i n t h i s system i s not due to an incapacity of the B - c e l l subpopulation to d i f f e r e n t i a t e but i s a result of the balance between the helper and suppressor influences i n the regulatory T - c e l l subsets (9,70,124). In - 15 -man these regulatory T - c e l l subsets have been recognized for a number of years. They have been i d e n t i f i e d by hetero-and auto-immune antisera (142), d i f f e r e n t i a l Fc receptor binding (114), and most recently by the production of monoclonal Abs (137) which recognize subset s p e c i f i c surface markers. The recent production of these monoclonal Ab's s p e c i f i c for T - c e l l subsets at fu n c t i o n a l l y different stages of maturation provides powerful to o l s for the i d e n t i f i c a t i o n and i s o l a t i o n of these regulatory T-cells subsets (See Table 1) The T-cell subsets defined by the surface markers 0KT4, T M (Fc receptor for IgM) and Leu 3a contain the helper/inducer a c t i v i t y necessary for the PWM induced d i f f e r e n t i a t i o n of B- c e l l s (114,138). T - c e l l subsets i d e n t i f i e d by the surface markers 0KT5, 0KT8, T G (Fc receptor for IgG) and Leu 2a (15-30% of peripheral blood T-cells) suppress the generation of plasma c e l l s when added together with T-helper c e l l s , B-cells and PWM. Isolation of these subsets using mAbs (81,137,156,174,205) and other less conventional means (47,70,83,114,193,199) have shown that i n addition to T-B c e l l i n t e r a c t i o n s , precise interactions among funct i o n a l l y d i s t i n c t T - c e l l subsets ultimately determine the net outcome of the T-cell immunoregulatory influence on B - c e l l s . The role of the suppressor T-cell subset i n terms of influencing the l e v e l of response observed after PWM stimulation was investigated i n separate laboratories using d i f f e r e n t methods to i s o l a t e the suppressor T - c e l l s . One involved i s o l a t i n g the Tg-cell subset from high responders and non-responders by virtue of the presence of the surface receptor for the Fc fragment of IgG ( T r ) . Addition of the T» c e l l - 16 -subsets i n allogeneic co-cultures demonstrated that the c e l l s (T<0 from non-responders suppressed the PWM response to a much greater extent than the T n c e l l s i s o l a t e d from high responders (69). In another assay the T - c e l l subsets were p u r i f i e d by virtue of the mutally exclusive c e l l surface markers 0KT4 and 0KT8 (T H and T<~ c e l l s respectively) (156). This group observed a s i g n i f i c a n t correlation between the amount of IgG produced i n whole MNC cultures and the l e v e l of suppression induced by the T8 c e l l s i s o l a t e d from the same i n d i v i d u a l ; i . e . , the addition of 0KT8+ c e l l s i s o l a t e d from non-responders to a pool of high responder B - c e l l s , monocytes and T helper c e l l s suppressed PWM induced IgG secretion to a much greater extent than did the addition of 0KT8+ c e l l s i s o l a t e d from high responding individuals. Further support for the role of 0KT8+ c e l l s i n a c t i v e l y regulating the l e v e l of response induced by PWM i s derived from studies which demonstrate that the removal of t h i s subset r e s u l t s i n s i g n i f i c a n t l y enhanced responses (81). I t would appear from these results that the l e v e l of response i s dependent on the amount of suppressor immunoregulatory c e l l function that i s present or has been spontaneously induced as opposed to the absolute number of c e l l s i n the T-suppressor compartment (10,156,193). I t i s becoming increasingly evident that the currently recognized T - c e l l subsets are composed of more than one homogeneous population (59,147,175,193,205). Therefore although the absolute number of c e l l s within the subsets recognized by MAbs may not be responsible for the v a r i a t i o n s observed i n the l e v e l of IgG secreted i n response to PWM one must consider the p o s s i b i l i t y that sub-subsets within the T<- c e l l subset may not be equally represented i n responder vs non-responder i n d i v i d u a l s . - 17 -The mechanism of suppression actuated by the regulatory T-cells i s l i k e l y to be very complex. I t i s well documented that i r r a d i a t i o n (114,169) as well as other methods of preventing i n v i t r o blastogenic responses (49) i n h i b i t the a c t i v i t y of suppressor c e l l s and re s u l t i n increased l e v e l s of responsiveness to PWM i n PBMNC cultures. I t appears that while T H c e l l s are resistant to f a i r l y high l e v e l s of i r r a d i a t i o n (5,000 R) (48), T s c e l l s are sensitive at lower doses (1,500 - 2,000 R) (48). One can conclude from these results that T-helper c e l l s can function i n the absence of blastogenesis while T ^ - c e l l a c t i v i t y i s clo s e l y linked with c e l l d i v i s i o n (49). In PWM stimulated cultures of PBMNC i t would appear that the 0KT8+ Tg c e l l s regulate IgG production by acting at the l e v e l of the T H c e l l . In a complex investigation of the T-T and T-B interactions involved i n the PWM induced B - c e l l d i f f e r e n t i a t i o n to ISC, Thomas et a l (174-177) observed the following: (1) T-helper functions are contained exclu s i v e l y within the 0KT4+ subset and contrary to previous reports t h i s subset i s radiosensitive except at high T:B r a t i o s ( i . e . > 1); and (2) The 0KT8+ subset i s devoid of helper function but expresses a suppressor function that requires the presence of the radiosensitive 0KT4+ subset to function. Interestingly, investigation of patients with T - c e l l leukemias whose blas t s suppress B - c e l l d i f f e r e n t i a t i o n revealed that these leukemic c e l l s also require the presence of radiosensitive normal T-cells to induce the suppression of B - c e l l d i f f e r e n t i a t i o n (29b). These r e s u l t s indicate that i n PWM stimulated cultures of PBMNC, 0KT8+ c e l l s act at the l e v e l of the T H c e l l subpopulation to i n h i b i t the d i f f e r e n t i a t i o n of B-lymphocytes. At the present time the precise mechanism by which the - 18 -0KT8+ c e l l s suppress 0KT4+ dependent B - c e l l d i f f e r e n t i a t i o n i s not known however the following mechanisms can be readily envisioned. 0KT8+ T-cells may interact directy with preformed helper factors or compete with helper factors for t h e i r effect(s) on B-cel l s (177). This p o s s i b i l i t y i s supported by the results of Palacios et a l . (125) who showed that concanavalin A (Con A) activated suppressor T-cells could absorb out the T-cell growth factor IL-2. Furthermore Jones' group demonstrated that the 0KT8+ population regulated the growth of B - c e l l s , T H c e l l s , T<- c e l l s and monocytes <81). I t seems possible therefore that a f t e r PWM stimulation 0KT8+ c e l l s might regulate the l e v e l of response i n PBMNC cultures by the absorption of growth factors from d i f f e r e n t c e l l types. Thomas et a l (177) however demonstrated that 0KT8+ c e l l s did not suppress B- c e l l d i f f e r e n t i a t i o n i n the presence of preformed helper factors. As these helper factors were generated from allogeneic culture one must consider that perhaps PWM may be more potent than helper factor i n activating the 0KT8+ suppressor/effector function. Other mechanisms by which the 0KT8+ population may inter a c t with other subpopulations include the production of suppressor factors that act d i r e c t l y on the T, c e l l s or a l t e r n a t i v e l y act on non-T-cells ( i . e . n monocytes) that may play a role i n 0KT4+ dependent helper factor production. Obviously further studies of d i s t i n c t immunoregulatory c e l l subsets are required i n order to elucidate the precise c e l l u l a r i nteractions involved i n the regulation of PWM induced IgG secretion i n humans. We can conclude that i n the PWM induced d i f f e r e n t i a t i o n of resting B - c e l l s i n t o ISC both intact monocytes, T-cells and t h e i r secretory 1. - 19 -products are required. The l e v e l of response appears to be dependent on the functional state of the radiosensitive suppressor T-lymphocyte subpopulation. Exploring the f i n a l mechanism of regulation i n t h i s system should be rewarding because as we have seen e a r l i e r aberrant IgG secretion within the CNS as well as abnormal suppressor c e l l functions i n the peripheral blood are the main b i o l o g i c a l abnormalities recognized i n MS patients to date. - 20 -I I I . Immunomodulatory Effects of Interferon In 1980 an int e r n a t i o n a l group of s c i e n t i s t s met at the National I n s t i t u t e of Health i n Bethesda, Maryland to define what was meant by "i n t e r f e r o n " , to c l a s s i f y the various interferons that had been discovered, and to devise a system for t h e i r orderly nomenclature. The committee decided^on the following d e f i n i t i o n for interferon: "To Qualify as an interferon a factor must be a protein which exerts virus non-specific, a n t i v i r a l a c t i v i t y at least i n homologous c e l l s through c e l l u l a r metabolic processes involving synthesis of both RNA and p r o t e i n " (170). The interferons were c l a s s i f i e d according to t h e i r antigenic s p e c i f i c i t i e s and the type designations are now alpha (<*), beta (G) and gamma (%) corresponding to previous designations of leukocyte, f i b r o b l a s t and type I I (immune) interferons respectively. In addition to the c l a s s i c a l a n t i - v i r a l and anti-tumor a c t i v i t i e s , i n t e r f e r o n s (IFN) possess immunomodulatory properties (for review see r e f . A l ) . We w i l l focus on the immune modulating e f f e c t s of those INF produced and secreted by the Nawalma lymphoblastoid c e l l l i n e , namely alpha IFN and a small amount of beta-IFN. Conventionally the interferons produced by lymphoblastoid c e l l s have been regarded as forming a separate category. However j u s t as the IFN genes present i n lymphoblastoid c e l l s are the same as i n c i r c u l a t i n g B-lymphocytes, t h e i r products should be the INF of the appropriate types namely alpha and beta (51). Cloning of the alpha IFN genes i n bacteria by recombinant DNA techniques have revealed 9 d i s t i n c t leukocyte (alpha) INF subtypes that - 21 -are coded for by at least 10 genes (58). Studies using monoclonal Abs suggest that two forms of the beta-INF e x i s t (74). In l i g h t of the remarkable heterogeneity within the INF produced, one must consider that each subtype could p o t e n t i a l l y exert a s p e c i f i c b i o l o g i c a l e f f e c t , or a l t e r n a t i v e l y that synergistic effects could occur between d i f f e r e n t subtypes. Most of the e a r l i e r studies on the immunomodulatory eff e c t s of INF used only crude preparations of p a r t i a l l y p u r i f i e d INF's, which made i t d i f f i c u l t to separate the immunomodulatory eff e c t s of INF from those of other b i o l o g i c a l l y active substances (eg. lymphokines) contaminating the preparations. Considering these two points one must be very cautious when inte r p r e t i n g the results of e a r l i e r studies of the immunomodulatory e f f e c t s of INF. Murine Experiments i n v i t r o Most investigations of immune modulating ef f e c t s of INF involve the addit i o n of INF to i n v i t r o assay systems. In human and murine systems both alpha and beta INF have been shown to: enhance NK c e l l a c t i v i t y (91) and the generation of cytotoxic T-lymphocytes (72,207), i n h i b i t DNA synthesis i n lymphocytes stimulated with mitogens or antigens (166) and to modify immunoglobulin synthesis (24,29,29b,32,33,57,66,94,128,152b, 166,168,194) as well as protein synthesis (87). The modulating effects of interferon on B - c e l l d i f f e r e n t i a t i o n i n murine systems are well documented and w i l l be reviewed i n the following section. G i s l e r et a l . (57) were the f i r s t to report that INF could a f f e c t B - c e l l function. Concentrations of murine L - c e l l INF induced by Newcastle Disease Virus (NDV) greater than 3,000 units per ml added to - 22 -mouse spleen c e l l culture resulted i n a reduced plaque forming c e l l (pfc) response against sheep red blood c e l l s (SRBC) as compared to cultures with control or mock interferon preparations. When lower doses of the INF preparation were added to spleen c e l l cultures obtained from low responding mice, the pfc response against SRBC was s i g n i f i c a n t l y increased. Pretreatment of separated B-lymphocytes p r i o r to Ag exposure resulted i n a decrease i n the number of pfc observed thus suggesting that t h i s subset was d i r e c t l y affected by the INF preparation. In a s i m i l a r study Booth et a l . (24) noted that r e l a t i v e l y high doses of L - c e l l interferon induced by reovirus type I I I added to mouse spleen c e l l cultures reduced the number of pfc detected against SRBC compared to control cultures not exposed to INF. This groups also demonstrated that the presence of low doses of t h i s INF preparation enhanced the pfc response against SRBC i n cultures obtained from low responding mice. The i n v i t r o immune modulating effects of these INF preparations appeared to be species s p e c i f i c as rabbit interferon had no effe c t on the mouse spleen c e l l cultures. B) Murine Experiments i n vivo The timing between INF in j e c t i o n and immune stimulation i s c r i t i c a l i n determining the effects observed. Mouse L - c e l l interferon induced by NDV (1.5 x 10 5 units (29b) and 1.8 x 10 5 units (33) divided into 4 doses and injected over 4 hours) was injected into mice 4-48 hours p r i o r to Ag stimulation (SRBC). Similar results were recorded by both groups; - 23 -i . e . a marked reduction i n the number of Ab producing spleen c e l l s observed. This effect was again species s p e c i f i c and human interferon had no effect. I f however the interferon preparation was injected 48-96 hours following Ag i n j e c t i o n enhancement of the Ab response was observed (29b). This l a t t e r group (29b) also investigated the ef f e c t of the i n vivo interferon i n j e c t i o n on the i n v i t r o Ab response of spleen c e l l cultures stimulated with a T-cell independent mitogen derived from S.  tiphimurium (lipopolysaccharide (LPS)). They noted a s i g n i f i c a n t reduction i n the PFC response to LPS i n splenic cultures obtained from mice treated with i n t e r f e r o n , again suggesting that the B-lymphocyte subpopulation i s d i r e c t l y affected by interferon. V i r e l i z i e r e t a l . (194) injected animals with Sendai virus as a interferon inducer (peak c i r c u l a t i n g alpha interferon l e v e l s of 18,000 units/ml of sera 12 hours a f t e r inoculation) and noted a 97% reduction i n the number of Ab forming spleen c e l l s when the Ag (SRBC) was injected 12 hours after the virus inoculation. Interferon containing serum i n h i b i t e d the i n v i t r o plaque formation against SRBC i n spleen c e l l cultures i n a dose dependent fashion. C) Human Experiments i n v i t r o Here also effects of human interferon preparations on the antibody response of peripheral blood mononuclear c e l l s are dependent on the Ag, and the type, dosage and timing of the INF used. Two groups using Cantell type interferon obtained from the Finnish Red Cross investigated i t s effects on the Ab response of human peripheral - 24 -blood mononuclear c e l l s (PBMNC)(66,128). Both groups noted a time dependent effect on the Ab response. Parker (128) observed that i f INF (100 and 500 units/ml) was added to PBMNC cultures 24 hours p r i o r to Ag (HRBC), then washed away, a s i g n i f i c a n t increase i n the number of Ab producing c e l l s was observed. I f INF was present throughout the culture period (10-500 units/ml) the number of Ab forming c e l l detected was reduced. In a simi l a r study Harfast (66) noted that i f c e l l s were pretreated with INF (100 units/ml up to 5,000 units/ml) the amount of IgG secreted i n response to PWM was frequently increased, often by more than 100% over that obtained by untreated PBMNC. In contrast i f INF was present throughout the 7 day culture period reduced production of IgG was observed. Rodriguez et a l . (152b) using p u r i f i e d human alpha int e r f e r o n (10-100 units/ml)(Sigma Chemical Co., St. Louis, Mo.) added together with PWM to human PBMNC noted that the l e v e l of Ig (IgG and IgM) produced i n culture increased. Preincubation of the PBMNC for 24 hours with the human alpha INF preparations (10-100 units/ml) also resulted i n an increased l e v e l of Ig secreted. The use of recombinant interferon i s expected to c l a r i f y the issue. Shalaby et a l . (166) recently reported that three d i f f e r e n t alpha INF subtypes when present throughout the culture period (100-1,000 units/ml) enhanced the Ab response of human PBMNC to SRBC. Yen et a l . (206) compared the eff e c t s of two d i f f e r e n t preparations of p a r t i a l l y p u r i f i e d human alpha-INF (Cantell type) and 2 d i f f e r e n t species of human recombinant alpha interferon (INF alpha A and INF alpha D) on the l e v e l of Ab secreted by PBMNC i n response to PWM. One of the - 25 -p a r t i a l l y p u r i f i e d alpha INF preparations decreased IgG production at a l l concentrations tested (10-10,000 units/ml) when present throughout the 7 day culture period. The other preparation increased IgG production at concentrations of 10 to 30 units/ml. Concentrations above 300 units/ml decreased the l e v e l of IgG detected. Interestingly both species of recombinant human alpha interferon enhanced IgG secretion at a l l the concentrations tested up to 10,000 units/ml. D) Human Experiments i n vivo -In contrast to the thorough i n v i t r o data on the immunomodulating eff e c t s of INF, there are currently no reports of interferon administered i n vivo a f f e c t i n g i n v i t r o or i n vivo Ab responses i n humans. Hopefully we w i l l be able to shed some l i g h t on t h i s area as we are currently investigating the i n v i t r o effects of INF injected i n vivo. Mechanisms of Immunomodulatory A c t i v i t y The influence of INF on Ab production i s obviously well documented but the mechanisms involved remain to be defined. One of the well documented effects of INF administered i n vivo i s the notable decrease i n the number of c i r c u l a t i n g T-lymphocytes. This reduction occurs without a l t e r i n g the r a t i o of T H to T^ c e l l s (75,103). Reports of INF a l t e r i n g the number of c i r c u l a t i n g B-lymphocytes are d i f f i c u l t to analyse and remain largely unresolved to date, due mainly to the d i f f e r e n t treatment regimens employed (75). I t would appear from the results obtained i n investigations of i s o l a t e d lymphocyte subsets ( i n both human and murine systems) that the B - 26 -lymphocyte i s directly affected (24,29,57,66,152b). Other than noting the c e l l subset affected however these investigations accomplished l i t t l e i n terms of defining the effects of interferon at the subcellular le v e l . Rodriguez et a l . (152b) studied the production of, and response to, helper factors (HF) produced by PWM stimulated radioresistant T c e l l s . Human alpha interferon (Sigma)(10-100 units/ml) did not enhance the production of HF by T-cells. Preincubation of B-cells with 50 and 100 units/ml of alpha-INF increased the amount of Ig secreted by these c e l l s in response to HF and PWM. This -result suggests that human alpha INF enhances the B-cell response to T-cell derived growth factor. This would explain the increased Ab response often observed following preincubation of PBMNC with INF. Inhibition of Ig secretion by INF could result from decreased DNA and/or protein synthesis. Inhibition of either c e l l function would reduce the B-cell's capacity to respond to mitogen or Ag but does not explain enhancement of the response. Enhancement of the humoral immune response could possibly involve changes in soluble suppressor substances. Interferon has reportedly induced changes in the c e l l membrane. Some of these changes involve increases in the number of Fc receptor for IgG (50,65b) and augmentation in the expression of class 1 major histocompability antigens (169b). Changes involved in surface structures which are intimately involved in the immune function could alter c e l l to c e l l interactions and contribute to the increases and decreases in Ab production following INF treatment. Inherant i n the discussion of the mechanisms involved in the modulation of Ig production by INF are the effects observed when INF i s added at different times relative to the - 27 -developing immune response. The modulating effects of INF observed change with the time course of INF treatment, suggesting that at di f f e r e n t times INF may be affecting d i f f e r e n t c e l l functions. Although the B-lymphocyte i s strongly implicated as being d i r e c t l y affected by INF, one cannot ignore the fact both alpha and beta INF have been shown to enhance the natural k i l l i n g effect of human PBMNC (91) and enhance the a c t i v i t y of cytotoxic T-cells (207). Other si m i l a r investigations (3,128) have demonstrated that INF affects the T-lymphocyte subset. For example, Heron et a l . (72) demonstrated that pretreatment of PBMNC (or of highly p u r i f i e d T-cells alone) with human alpha INF led to a d i s t i n c t l y enhanced l y s i s of the s p e c i f i c target c e l l . Summary In summary one can conclude that i n addition to the c l a s s i c a l a n t i - v i r a l e ffects of alpha and beta-INF these substances also possess immunomodulatory a c t i v i t i e s , s p e c i f i c a l l y i n a l t e r i n g the Ig secretion response i n v i t r o . However the modulating a c t i v i t i e s of the INF preparations documented to date are not straightforward. This i s not surprising considering that there are d i f f e r e n t subtypes of INF and that the d i f f e r e n t subtypes may possess d i f f e r e n t a c t i v i t i e s (206). Time course and concentration of INF used and the s e n s i t i z i n g Ag or mitogen used have a l l been shown to influence the results obtained. I t i s obvious therefore that various modulating effects of p u r i f i e d alpha-INF preparations (in addition to contaminating lymphokines) w i l l occur depending ultimately on the inherant heterogeneity of the subtypes i n the sample. I - 28 -We have investigated the effect of lymphoblastoid interferon (Wellferon ) administered i n vivo on i n v i t r o IgG secretion and Con A induced suppression i n the PBMNC obtained from a group of chronic progressive MS patients. - 29 -MATERIALS AND METHODS A) Standard IgG Secretion Assay i ) Interferon R The interferon (Wellferon ) was obtained from Burroughs Wellcome Research Laboratories. I t i s produced i n Namalwa c e l l s , a lymphoblastoid c e l l l i n e of B - c e l l o r i g i n and which are currently the largest single source of interferon for c l i n i c a l use (51). Sendai virus i s used for induction and the crude interferon preparations are p u r i f i e d to greater than 80%. The Namalwa interferon consists of at least eight active components (51). I t has been shown that Namalwa c e l l s induced by Sendai virus produce both alpha and beta INF, the l a t t e r comprising approximately 10-20% of the t o t a l (38b). i i ) Patients and Controls A t o t a l of 38 patients with the chronic progressive form of multiple s c l e r o s i s (MS) were included i n the study. The average age was A4.5 _+ 2 years at the time of the f i r s t t e s t . There were 20 female and 18 male patients. In a double blind procedure 20 patients were injected 6 R subcutaneously with 5 x 10 units of interferon (Welferon ) per day and 18 received a s i m i l a r volume of placebo. Both groups were chosen as to be equally represented on the basis of age, sex and the Kurtzke d i s a b i l i t y scale (88). Each week four new patients were introduced into the study and blood was drawn before the i n j e c t i o n of INF and again 7 days af t e r the f i r s t i n j e c t i o n . With each new group of patients entered into the study, one healthy control volunteer was also studied to determine the baseline l e v e l s of response. - 30 -Patients* blood was obtained with t h e i r consent and used for both the IgG secretion assay and the ConA induced suppressor assay. i i i ) MNC Isolation Whole blood was drawn from the antecubital veins of consenting ind i v i d u a l s into vacutainer tubes containing preservative free heparin sodium. The blood was dilute d 1:1 with Hank's balance s a l t solution (no Mg++, no Ca++) (HBSS) (Gibco, Grand Island N.Y.) and underlayed with ficoll-hypaque (Pharamcia Fine Chemicals Piscataway, New Jersey). The mononuclear c e l l s (PBMNC) were subsequently i s o l a t e d by density gradient centrifugation according to the method of Boyum (26). Once isolat e d the PBMNC were washed at least A x i n cold HBSS and once i n cold standard culture media which consisted of RPMI 16A0 with 10% f e t a l c a l f serum (Flow Labs), 0.1 mg/ml gentamycine, 2 mM L-glutamine and the pH adjusted with sodium bicarbonate. The c e l l s were subsequently resuspended to 1 x 10 c e l l s per ml of culture media. i v ) MNC Cultures C e l l cultures were set up i n 1 ml volumes i n 12 x 75 mm p l a s t i c Falcon tubes with or without PWM (Gibco) at a f i n a l d i l u t i o n of 1:300. The cultures were kept at 37°C with 5% C0 2 i n a i r i n a humidified incubator for 7 days at which time the c e l l free supernatants were harvested and stored at -90°C u n t i l assayed for IgG content. v) ELISA Assay to Quantitate IgG The IgG content of the supernatants was measured by an ELISA assay modified from that developed by Voller and Bidwell (195b). - 31 -Ninety s i x well polyvinyl "Immulon" m i c r o t i t r e plates (Dynatech) were pre-coated with 110 uls / w e l l of a f f i n i t y p u r i f i e d goat anti-human IgG (Cappel) gamma chain s p e c i f i c (10 ug/ml) i n a carbonate-bicarbonate buffer (pH 9.6) at 4°C at least overnight. (For an appendix of the buffers and washing solutions refer to ref 195b). The plates were washed free of excess Ab with PBS and 0.05% Tween 20 (v/v) and any remaining s o l u t i o n was discarded by f l i c k i n g the plates and tapping them s o l i d l y on paper towel. To prevent non-specific binding, a blocking s o l u t i o n of 110 u l of PBS with 0.1% BSA was added to each w e l l . After 1-2 hr the plates were washed as described above and the culture supernatants and standards were added to the appropriate wells. The standards consisted of human IgG s e r i a l l y diluted 2 f o l d , from 1,000 ng/ml to 1.95 ng/ml with RPM1 and 0.1% BSA. The culture supernatants were also d i l u t e d with RPMl (0.1% BSA) so as to generate values which correspond to the most accurate portion of the standard curve. These samples were l e f t i n the wells for 1 hr at room temperature. The plates were then washed and 110 u l of a l k a l i n e phosphatase conjugated goat-anti-human IgG (1:1,000 d i l u t i o n of stock, Tago) was added to each well for 1 hr i n the dark. The substrate, p-nitrophenyl phosphate (Sigma 104-105), dissolved i n a diethanolamine buffer was then added to the plates. The color developed at room temperature and after approximately 45 min the absorbance (405 nm) was read on a multi-well spectrophotometer (Dynatech MR 600) which was interf a c e d with an Apple Computer and printer. The l e v e l of absorbance i s proportional to the amount of human IgG bound to the plates thus allowing for the construction of the standard curve. The computer, with the readings (done i n t r i p l i c a t e s ) from each w e l l , i s programmed to - 32 -display and print the standard curve and subsequently derive the unknown IgG concentrations from the standard curve. B) Con A Induced Suppressor Assay i ) Suppressor Generating Phase Note: This assay was conducted on the same 38 patients as above. The patients' PBMNC were assayed before treatment and again a f t e r 7 days of e i t h e r interferon or placebo injections. The mononuclear c e l l s were i s o l a t e d , washed and resuspended to 1 x 10^ c e l l s / m l i n the same cult u r e medium as i n the IgG secretion assay. At t h i s point the c e l l s were stimulated with 3 ug/ml concanavalin A (Con A) i n tissue culture flasks (Falcon) stored upright at 37°C with 5% Cu*2 i n a i r . These c e l l s w i l l subseauently be referred to a "S" c e l l s for "suppressor". Control or "C" c e l l s are set up i n an i d e n t i c a l fashion without Con A. After a period of three days both cultures ( i e "S" and "C" c e l l s ) were treated with mitomycin C (50 ug/ml, Sigma) for 30 min at 37°C. This period i s referred to as the "suppressor generating" phase of the assay. i i ) Responder Phase Af t e r the mitomycin C treatment the c e l l s were washed free of t h i s substance and resuspended to 1 x 10^ viable c e l l s per ml of standard c u l t u r e media. To the appropriate-wells of a round bottom m i c r o - t i t r e plate (Limbro), 1 x 10 5 (100 uls) "S" or "C" c e l l s were added along with 1 x 10 5 fresh allogeneic untreated responder c e l l s or "R" c e l l s . The responder c e l l s were then stimulated with Con A (3 ug/ml) for 3 days - 33 -at 37°C with 5% C0 2 i n a i r . This period i s referred to as the "responder phase". At the end of the responder phase the c e l l s were pulsed for 6 hr with 1 uCi/well of t r i t i a t e d thymidine. The c e l l s were then harvested onto glass f i b r e f i l t e r s v i a a home made c e l l harvester and dried. S c i n t i l l a t i o n f l u i d was added and the v i a l s were counted i n a s c i n t i l l a t i o n counter (LS 9,000, Becton Dickenson). The results were expressed as counts per minute (CPM). This assay measures the non-specific suppression induced by Con A stimulated PBMNC on the p r o l i f e r a t i v e response of fresh heterologous PBMNC stimulated with Con A. The suppression thus generated i s calculated as a percentage based on the difference i n CPM between the suppressor culture ("S" c e l l s + "R" c e l l s ) and the control cultures ("C" c e l l s + "R"-cells) using the following formula % Suppression = 1 - S(- background) + R(- background) + Con A, C(- background) + R(- background) + Con A x 100 C) IgG Secretion i n C e l l Subset Reconstitution Assays i ) Plastic-adhering C e l l (monocyte, macrophage) Reconstitution . a) Donors. Donors included 6 MS patients, 3 of which were treated with R 6 Wellferon (5 x 10 u/day for at least 1 month) and 3 of which were treated with placebo. The patients were not informed of which treatment they were receiving. PBMNC were is o l a t e d as previously described, washed 3x i n cold HBSS, l x i n cold culture media, and resuspended to 2 x 10 6 c e l l s per ml of c u l t u r e media. - 34 -b) Monocyte Is o l a t i o n The monocytes were separated from the mononuclear c e l l s by vir t u e of t h e i r p l a s t i c adhering properties. Twenty m i l l i o n PBMNC were added to 25 cm tissue culture f l a s k s (Falcon) and placed h o r i z o n t a l l y at 37°C for one hr. After t h i s period the non-adherent c e l l s (NAC) were recovered and the removal of plastic-adhering c e l l s (PAC) was repeated two more times. The non-adherant c e l l s (NAC) (monocyte depleted PBMNC) were resuspended to 1 x 10 6 cells/ml of culture media. The p l a s t i c adhering c e l l s , (PAC) i e . monocytes or macrophage were recovered from the f i r s t incubation by scraping the flasks with a rubber policeman; washed once and resuspended to 1 x 10 6 c e l l s / m l . The eff i c a c y of the PAC depletion was assessed by the i n a b i l i t y of PAC depleted MNC to respond to PWM and also by FACS analysis. FACS IV (Fluorescent Activated C e l l Sorter) Analysis : For routine analysis 1 x 10 6 PBMNC were l a b e l l e d with a saturating amount of murine monoclonal antibody conjugated with fluoresceine isothiocyanate for 30 min on i c e . The c e l l s were subsequently washed three times i n cold Hank's balanced s a l t s o l u t i o n and run through the FACS IV (Becton Dickenson). Ten thousand c e l l s are analyzed from within the predetermined lymphocyte scatter peak for the amount of fluorescence. Florescence i s expressed as percent p o s i t i v e fluorescence by subtracting the fluorescence of the control c e l l s . c) Cultures. The following cultures were set up. INF treated patients' NAC reconstituted with 20% INF treated patients' PAC or with 20% of the - 35 -placebo treated patients' PAC and also the cultures of placebo patients' NAC reconstituted with either INF treated PAC or placebo treated PAC. The cultures were set up i n duplicate 1 ml volumes i n 12 x 75 mm Falcon culture tubes. One culture received PWM (1:300 d i l u t i o n ) the other received only culture media. The negative control consisted of unreconstituted monocyte depleted PBMNC cultures. After 7 days of incubation supernatants were harvested as described e a r l i e r . i i ) T-helper (T u) c e l l Reconstitution Assay. n a) Donors Donors consisted of 4 MS patients, two of which had received Wellferon interferon injections for at least 1 month and of two which had received placebo. The PBMNC were isolated and washed as previously discussed. b) T-B lymphocyte separation; E-rosette separation T-cells were separated from the PBMNC by E-rosette separation. B r i e f l y PBMNC, 1 x 10 7/ml, were mixed with a 1% solution (v/v) of packed neuraminidase treated SRBC (30 min at 37°C with 0.75% v/v neuraminidase). These c e l l s were thoroughly mixed, then allowed to incubate at 37°C for 15 min, spun at 1,000 RPM for 10 min and then further incubated for an hour at 4°C. The rosetted c e l l s (ie PBMNC bound by SRBC) were then centrifuged through f i c o l l hypaque at 400 X g for 30 min.. The c e l l s at the interface are referred to as E~ ( i e . not binding sheep erythrocytes) and are composed mainly of B-cells and monocytes. Contaminating T-cells were removed from t h i s population by repeating the E rosette procedure. - 36 -The p e l l e t (after the f i c o l l hypaque density centrifugation step) contains the c e l l s referred to as E + ( i e . positive for binding sheep erythrocytes) and i s composed mainly of T-cells and SRBC. The SRBC are removed by l y s i s with d i s t i l l e d water (the solution i s rapidly brought back to an isotonic state with the proper concentration of s a l i n e ) , followed by 3 washes. The purity of the E + rosette procedure was assessed by two methods; 1, by culturing the E~ c e l l s and E + c e l l s alone and with PWM, and 2, by flow cytofluorometry using the FACS 4. (Becton Dickenson), as discussed i n the previous section. c) Tu-cell Isolation —TT The T H c e l l subset was separated from the E + - c e l l population by a modification of the panning technique employed by Tsoi et a l . (187). B r i e f l y 1 x 10 7 p u r i f i e d T-cells ( E + - c e l l s ) were incubated with 500 u l of a 1:50 d i l u t i o n of the 0KT8 monoclonal Ab for 30 min at 4°C. This Ab recognizes a s p e c i f i c surface marker present on a subset of suppressor T-lymphocytes. The lymphocytes treated with the Ab were washed 3x i n cold HBSS and reconstituted to a volume of 3 ml. This c e l l suspension was added to petridishes previously coated with 160 ug of goat-anti mouse Ig and 75 ug of goat anti-human Ig (the l a t t e r removes any surface Ig p o s i t i v e ( B - c e l l s ) c e l l s which could have been contaminating the E + - c e l l s ) . These plates bind the c e l l s l a b e l l e d with the monoclonal Ab. After approximately 2 hours at 4°C the non-adherent c e l l s are recovered, washed once i n cold culture media and resuspended to 1 x 10^ c e l l s / m l . This process removes the population of T<- c e l l s that express the 0KT8 Ag and leaves the non-adherent c e l l s enriched for T H - c e l l s . - 37 -d) Culture. Five hundred thousand E~ (B-cells and monocytes) from each patient were cultured with 5 x 10 5 T H-enriched c e l l s of each of the other patients and also with the i r autologous T H - c e l l s . The c e l l cultures were set up i n duplicate one ml volumes i n 12 x 75 mm p l a s t i c Falcon culture tubes. One culture was stimulated with a 1:300 d i l u t i o n of stock PWM the other culture received a s i m i l a r volume of RPMI and served as a control. Other controls consisted of E~ and T H - c e l l s cultured alone and stimulated with PWM, the absence of Ig secretion being an i n d i c a t i o n of subset purity. After 7 days at 37°C i n 5% CCv, i n a i r the c e l l free supernatants were harvested and stored at -90°C u n t i l assayed. Results A) IgG Secretion Assay The amount of IgG secreted by 1 x 10 6 peripheral blood mononuclear c e l l s (PBMNC) stimulated with and without PWM was measured i n 38 MS patients and 21 healthy control volunteers. The MNC's obtained from the MS patients were examined before treatment and af t e r one week of daily subcutaneous i n j e c t i o n s of either int e r f e r o n or placebo. These results were compared with the l e v e l of IgG secreted by PWM stimulated MNC obtained from healthy control volunteers. We f i r s t compared the mean l e v e l of IgG secreted by the PWM stimulated MNC of the 38 MS patients to the l e v e l secreted by the MNC of the 21 healthy control volunteers (See Table I I ) . The l e v e l of IgG secreted by the PWM stimulated MNC obtained from - 38 -the MS patients was 2373 +_ 233 ng/ml (mean +_ SEM, n = 38). The l e v e l of IgG secreted by the PWM stimulated MNC obtained from the control group was 1313 +_ 293 ng/ml (n = 21). Using a two t a i l e d student t-test t h i s difference was s i g n i f i c a n t (t = 2.99, p<.01,). The mean l e v e l of IgG secreted by the unstimulated MS cultures was 271 +_ 42 ng/ml and the l e v e l secreted by the unstimulated control cultures was 183 _+ 15 ng/ml. The difference i s s i g n i f i c a n t (p < .05, t = 2.2) (see Table I I I ) . We next compared the l e v e l of IgG secreted by 1 x 10^ MNC of the 38 MS patients before treatment and again after one week of daily subcutaneous injections (see Table V). The le v e l s of IgG secreted by the MNC of the placebo and interferon groups were not d i f f e r e n t p r i o r to treatment. (Placebo, 2542 _+ 339 ng/ml n = 18 vs Interferon, 2221 + 326 ng/ml, n = 20). In the placebo treated group one week of da i l y subcutaneous injections did not s i g n i f i c a n t l y a l t e r the amount IgG secreted, (2542 + 339 ng/ml before, 2176 + 419 ng/ml a f t e r ) . However the l e v e l of IgG secreted by the MNC i s o l a t e d from the 20 in t e r f e r o n treated patients was dramatically reduced a f t e r 1 week of d a i l y subcutaneous injections (2221 _+ 326 ng/ml before, 560 _+ 75 ng/ml a f t e r ) . The change was highly s i g n i f i c a n t as assessed by the student t - t e s t (t=5.2 p < .001). The modulating effect of the interferon i n j e c t i o n s on PWM induced IgG secretion became very apparent i f the l e v e l of response was broken down i n t o either a high (> 1000 ng/ml) or low response (see Table VI). P r i o r to INF injections 17 of the 20 (85%) MS patients were high responders. After 1 week of d a i l y injections there were only 3 of the 20 (15%) PWM stimulated MNC cultures that secreted greater than 1,000 ng/ml - 39 -o f IgG. In the placebo group 15 of the 18 (83%) were high responders. A f t e r one week o f i n j e c t i o n 12 of the 18 (67%) remained high responders. B) Con A Induced Suppressor C e l l Assay The Con A suppressor c e l l assay i s a measure of the a b i l i t y of Con A stimulated mitomycin-treated lymphocytes to suppress the p r o l i f e r a t i v e response of heterologous Con A stimulated lymphocytes. This inducible suppressor c e l l response measured as a percent suppression was examined i n the peripheral blood of 33 chronic progressive MS patients and 18 healthy controls (some of the r e s u l t s were discarded due to technical d i f f i c u l t i e s ) . The results are presented i n Table VII. The mean l e v e l of suppression induced i n the MS group was 7+^5% (mean +/- SEM) and 27 _+ 4% i n the control group. Using the student t-t e s t t h i s difference was s i g n i f i c a n t (t = 3.2, p < .01). Eighteen of the 33 MS patients (54%) had no detectable suppressor c e l l responses (ie < 0). The l e v e l of suppression ranged from minus 65% to plus 49% suppression. In the c o n t r o l group 17 of the 18 (94%) persons tested had detectable suppressor c e l l responses ranging from 2 to 54% suppression. (For i n d i v i d u a l r e s u l t s see Fig. 1) Additionally the inducible suppressor c e l l response was measured i n the MS group one week after having received d a i l y subcutaneous i n j e c t i o n s o f e i t h e r 5 x 10 6 units o f lymphoblastoid interferon or of placebo (see Table V I I I ) . The injections did not a l t e r the l e v e l o f suppression detected. In the interferon treated group the l e v e l o f suppression before treatment was 11% and after treatment the l e v e l o f suppression induced remained 11 + 6%. - 40 -C) IgG Secretion i n MNC Subset Mixing Experiments i ) E + - E~ Mixing Experiment. The PBMNC isolate d from 4 MS patients (2 inter f e r o n treated, 2 placebo treated) were separated into E~ (B-cells and monocytes) and E + ( T - c e l l s ) . The c e l l s expressing the 0KT8 Ag (T-suppressor c e l l s ) were removed from the E + population leaving the E + subset enriched for T-helper c e l l s (for purity see Table X I I I ) . The T-helper enriched c e l l subset obtained from each of the patients was mixed with the autologous E~ c e l l subset and each other patients* E~ c e l l subpopulation ( i e . INF-treated patients' E~ were reconstituted with i , autologous T H enriched subpopulation i i , i n vivo INF treated heterologous T H enriched MNC, and i i i , heterologous i n vivo placebo treated T H enriched MNC. The c e l l mixtures were then stimulated with PWM and after seven days the amounts of IgG secreted i n t o the supernatant were calculated (see Table IX). The T-helper c e l l s obtained from a high responding placebo treated patient could not support a high response (ie >1000 ng/ml IgG) when mixed with the E~ c e l l s of interferon treated patients. S i m i l a r l y T-helper c e l l s obtained from INF treated patients could not reconstitute a s i g n i f i c a n t l e v e l of PWM induced IgG secretion when mixed with int e r f e r o n treated E~ subpopulation and stimulated with PWM. This r e s u l t suggests that the E~ subpopulation of the interferon injected patients could not support PWM induced IgG secretion. The T u c e l l s obtained from the interferon treated patients did n support a high response (1077 ng/ml) when mixed with placebo treated patients E" c e l l s . - Al -The purity of the c e l l subset separation procedures was assessed both functionally and a n a l y t i c a l l y . Functional purity was analysed by the absence of IgG secretion i n p u r i f i e d c e l l subsets following incubation with PWM. None of the subsets ( i . e . E~ + PWM or E + + PWM) when cultured alone with the mitogen secreted IgG above background l e v e l s , i n d i c a t i n g a sa t i s f a c t o r y separation protocol (data not shown). The subsets were also assessed a n a l y t i c a l l y by monoclonal antibody s t a i n i n g and processing through the fluorescent activated c e l l sorter (see Table XII). Greater than 85% of the c e l l s contained i n the E + ( T - c e l l ) subpopulation expressed the Leu-1 Ag (pan T-cell) marker and les s than 7% of the c e l l s expressed surface immunoglobulin ( s l g ) . This subpopulation of T-cells was subject to a panning procedure which included the removal of s l g po s i t i v e by goat anti-human IgA, IgG and IgM bound to the p e t r i dish. However the purity of the f i n a l c e l l preparation was not analyzed due to the li m i t e d number of c e l l s obtained. The "panning" procedure removed greater than 80% of the 0KT8+ c e l l s (see Table X I I I ) . i i ) P l a s t i c Adhering C e l l (PAC) and Non-Adhering C e l l (NAC) Mixing Experiment Monocytes adhere to p l a s t i c (192). The procedure y i e l d s p l a s t i c adhering c e l l s (PAC) and non adherent c e l l s (NAC). Our goal was to investigate whether or not the monocytes obtained from interferon treated patients di f f e r e d functionally from the monocytes obtained from placebo treated patients. This was done by mixing the monocytes (PAC) with - 42 -autologous and heterologous NAC, and then measuring the l e v e l of IgG secreted i n response to PWM stimulation. The r e s u l t s are presented i n Table X. The data presented are the results obtained from 6 MS patients, 3 treated with INF and 3 treated with a placebo. There was no s i g n i f i c a n t difference i n the l e v e l of IgG secreted by the PWM stimulated NAC c e l l s regardless of the source of the monocytes with which they were reconstituted. The levels of IgG secreted by the NAC were not d i f f e r e n t whether reconstitution was done with monocytes obtained from the interferon treated patients (1906 _+ 554 ng/ml) or with monocytes obtained from the placebo treated patients (1708 +_ 650 ng/ml). The monocyte reconstituted (PAC) interferon treated NAC secreted only 581 +_ 201 ng/ml of IgG i n response to PWM stimulation. The placebo treated NAC on the other hand reconstituted with monocytes obtained from the same patients secreted 3252 +_ 379 ng/ml of IgG i n response to PWM. This difference i s highly s i g n i f i c a n t (n = 12, t = 6.23, p <.001). I t would appear that the accessory c e l l role of the monocyte investigated i n t h i s assay i s not affected by the i n vivo interferon i n j e c t i o n s . Additionally i t appears as though the function of one of the lymphocyte subsets contained within the NAC subpopulation (T-cells or B-cells) i s affected. I t has been well documented that monocytes are required for the PWM induced d i f f e r e n t i a t i o n of resting B-lymphocytes i n t o IgG secreting c e l l s . The purity of the monocyte depletion was controlled by the i n a b i l i t y of the PWM stimulated NAC c e l l s to secrete a s i g n i f i c a n t amount of IgG (See Table XI). The monocyte depleted cultures did not secrete - 43 -IgG above the background l e v e l . (Background = 282 _+ 39 ng/ml, monocyte depleted MNC + PWM = 338 + 103 ng/ml). - 44 -Table I Monocloncal Ab to Human PBMNC Surface Antigens Antibody Description Range a % of PBMNC Anti T-1 (Ortho) Pan T-cell Anti T-3 (Ortho) markers 75 + 11% Anti Leu 2a (BD) T-cytotoxic/suppressor 26 + 8% Anti 0KT5 (Ortho) Anti 0KT8 (Ortho) Anti Leu 3 (BD) T-helper/Inducer 45 + 10% Anti T4 (Ortho) a. Samples: Normal peripheral blood mononuclear c e l l suspension is o l a t e d by f i c o l l hypaque density gradient centrifugation (26). Detection System: Indirect immunofluorescence with FACS IV analysis (scatter gates set on the lymphocytic peak). The normal range i s expressed as a percent of peripheral blood lymphocytes. - 45 -Table I I IgG Secreted i n Culture by MNC Stimulated with PWM (mean + SEM) Population Number of IgG Secreted Range Individuals (ng/ml) a (ng/ml) MS 38 2373 + 233* 139-5109 Control 21 1313 + 266 102-4504 Abbreviations: MNC-mononuclear c e l l s , PWM-pokeweed mitogen IgG-immunoglobulin G, SEM-standard error of the mean * d i f f e r e n t from control population, p < .01, ( t - t e s t ) . a. amount of IgG secreted into 1 ml by 10 6 PBMNC stimulated with a 1:300 d i l u t i o n of PWM. - 46 -Table I I I IgG Secreted i n Culture by Unstimulated MNC Isolated from Control Subjects and Treated and Untreated MS Patients Donor Group Level of IgG Secretion (and Number) (ng/ml) (a) MS (38) 271 + 42 Control (21) 183 + 15* MS before PLA (18) b 216 + 26 MS af t e r PLA (18) 237 + 30 MS before INF (20) c 321 + 75 MS af t e r INF (20) 399 + 110 a) mean _+ SEM. b) PLA = placebo treated; daily injections of normal saline for 1 week. c) INF = interferon treated; daily injections of 5 x 10 6 IU of interferon for 1 week. * Different from MS; t = 2.2, p <.05; Two t a i l e d student t - t e s t . - 47 -Table IV LEVEL OF PWM INDUCED IgG SECRETION IN MS AND CONTROL INDIVIDUALS MS CONTROL LOW RESPONDERS3 6 11 HIGH RESPONDERS0 32 10 a LOW RESPONDERS - secrete less than 1000 ng/ml IgG D HIGH RESPONDERS - secrete greater than 1000 ng/ml IgG X 2 = 7.13; p < .01 - 48 -Table V Effect of i n vivo Injection of Interferon on the Amount of IgG Secreted i n Culture by MNC Stimulated with PWM (mean + SEM) Group IgG Secreted IgG Secreted Before Injection After Injection (ng/ml) (ng/ml) Placebo (18) a 2542 + 339 2176 + 419 Interferon (20 ) D 2221 + 326 560 + 75** (5xl0 6units/day 1 week) **diferent from interferon before i n j e c t i o n , t = 4.97, p < .001, ( t - t e s t ) a) Placebo = placebo treated, as i n Table I I I . b) Interferon = interferon treated, as i n Table I I I . t - 49 -Table VI Effect of Interferon Injection on the Level of PWM Induced IgG Secretion In PBMNC Cultures Isolated From MS Patients MS PATIENTS INF TREATED* PLA TREATED** BEFORE AFTER BEFORE AFTER LOW RESPONDERS a 17 3 6 HIGH RESPONDERS D 17 3 15 12 Low Responders - MNC secreted <1000 ng/ml i n response to PWM High Responders - MNC secreted > 1000 ng/ml i n response to PWM. Number of indi v i d u a l s whose PBMNC secreted either greater or less than 1000 ng/ml of IgG i n response to PWM stimulation. X 2 = 9.1, p <.01 X 2 = .6, p > .1 - 50 -Table VII Donor Group and number Suppressor A c t i v i t y i n Controls and i n MS Patients Suppressor A c t i v i t y R C e l l s + Con A + C c e l l s R C e l l s + Con A + S c e l l s Sup-presion (b) (%) MS (33) Control (18) 35523 + 1700(a) 35A23 + 2581 3A305 + 2A3A 28582 + 2796 7 + 5 25 + A c a) CPM + SEM b) Mean _+ SEM, Percent Suppression = 1- Mean CPM of R c e l l s + Con A + S c e l l s x 100 Mean CPM of R c e l l s + Con A + C C e l l s c) Different from MS; t = 2.81, p<.6l - 51 -Fi g . 1 Concanavalin A induced suppressor c e l l response i n 33 chronic progressive multiple s c l e r o s i s patients and 18 healthy controls. The median i s indicated for both groups. The resu l t s are expressed as percent suppression calculated from the following formula: % S = 1 - mean CPM of R c e l l s + S c e l l s + Con A mean CPM of R c e l l s + C c e l l s + Con A X 100 - 52 -CO CO CL. co 6^ 60 50 40 30 20 10 0 -10 -20 -30 -40 -50 -60 -70 MS (n = 33) CONTROL (n = 18) median - 60 • • 50 81 40 • —median 30 20 C 3 • 10 CO CO U J s 0 SUPPR -10 — -20 - -30 — -40 -50 -60 -70 - 53 -Table VIII Con A Induced Suppressor A c t i v i t y i n MS Patients Before Treatment and After 1 week of Daily Injections of Interferon 3 or Placebo 0 Donor Group Suppressor A c t i v i t y and Number "R" C e l l s "R" Cell s + Con A + Con A Suppression + "S" C e l l s (%) INF before (18) INF a f t e r (18) PLA before (15) PLA a f t e r (15) 34719 + 2195(C) 30651 + 1874 37467 + 2831 35825 + 2572 32136 + 2809 ' 26859 + 2316 39490 + 4245 34965 + 3414 1 1 + 6 (d) 11 + 6 - 4 + 7 2 + 6 a) INF = In vivo injections of 5 x 10^ units of lymphoblastoid (Wellferon R ) interferon per day for 1 week. b) PLA = In vivo i n j e c t i o n of a s i m i l a r volume of normal saline per day for 1 week. * c) CPM + SEM d) Mean _+ SEM, for calculation of percent suppression, see Table VII. e) Some samples were not processed due to technical d i f f i c u l t i e s , i . e . out of 38 MS patients' samples, 5 had to be discarded. - 54 -Table IX IgG Secreted i n Reconstituted MNC Cultures Stimulated With PWM: Effect of i n vivo Interferon Injection on the TH and B-lymphocyte Subpopulations (mean _+ SEM) Source of E"-cells ( B - c e l l s , monocytes) Source of Tj-j-cells IgG Secreted (ng/ml) Range (ng/ml) Interferon* Interferon 316 + 51° 176-419 Interferon** Placebo 283 + 32 244-344 Placebo Interferon 506 + 202 212-1077 Placebo Placebo 988 + 489 273-2431 5 x 10 5 E - were mixed with 5 x 10 5 TH i n 1 ml of culture medium and stimulated with PWM (1:300 f i n a l d i l u t i o n ) . Each result i s the mean of 2 reconstitution experiments. [INF E" + INF T M] vs [PLA E" + INF T M ] ; t = .9, p < .05 [INF E- + PLA T M] vs [PLA E" + PLA T M ] ; t = 1.4, p < .05 - 55 -Table X IgG Secretion i n Reconstituted MNC Cultures Stimulated with PWM: Effect of i n vivo Interferon Injection on NAC and PAC (mean _+ SEM) Source of NAC Source of PAC IgG Secreted (ng/ml) PLA NAC INF - PAC 3A92 + A3Ab PLA NAC PLA - PAC 2931 + 727 INF NAC INF - PAC 637 + 291 INF NAC PLA - PAC A86 + 298 Abbreviations: IgG-immunoglobulin-G, MNC-mononuclear c e l l s , PWM-pokeweed mitogen, PLA-NAC-placebo treated non-adherant c e l l s PLA-PAC- " " 11 p l a s t i c adherent c e l l s INF-PAC interferon treated p l a s t i c adherent c e l l s a - A x 105 non-adherent c e l l s (NAC) were mixed with 1 x 10,5 p l a s t i c adhering c e l l s (PAC) i n 1 ml of culture medium and stimulated with PWM (1:300 f i n a l d i l u t i o n ) D - Results are expressed as the mean _+ SEM of the amount IgG secreted into the culture supernatant. c - Each r e s u l t i s the mean of three reconstitution experiments. - 56 -Fi g . 2 The l e v e l of Con A induced suppression vs the amount of PWM induced IgG secretion i n the PBMNC isolated from the same person. The r e s u l t s of 12 MS patients and 12 controls are included. Note there i s no co r r e l a t i o n between these two i n v i t r o immunological parameters even though the l e v e l of response obtained i n each assay i s c l e a r l y influenced by a common lymphocyte subset, namely the 0KT8 suppressor c e l l s . Note: Level of IgG vs %Con A Suppression, Correlation C o e f f i c i e n t s , r. MS r = .36 Control r = -.02 - 57 -50 40 30 20 CO co CO 65 10 0 r-10 -20 is= 2D 23 3.0 3.5 Log i Q LEVEL OF IgG SECRETED (ng/mlj A - MS • - CONTROL - 58 -Table XI IgG Secreted by PWM Stimulated Cultures of MNC and Monocyte Depleted MNC Donor and MNC with no MNC Monocyte Depleted Treatment mitogen + PWM MNC (NAC) + PWM 1. INF 30A + 17 6377 + 1992 1A9 + 3 2. INF 182 + 2 165 + 7 351 + 8 3. INF 319 + 13 28A + 7 237 + 1 A. PLA 273 + 6 4060 + 890 776 + 2A2 5. PLA A50 + 2A 3783 + 363 . 177 + A 6. PLA 163 + 1 157 + 3 NA Mean +_ SEM 282 + 39 2A71 + 985 338 + 103 One m i l l i o n were incubated alone or with PWM (1:300 f i n a l d i l u t i o n ) ; 1 x 10 6 monocyte depleted MNC (NAC) were also cultured with PWM. - 59 -Table XII Purity of B, T C e l l Separation Exp # Cel l Subset % Leu-1 + %+ve sl g • (Rabbit antihuman IgG,A,M) 1 E + (T-cells) E" ( B - c e l l s , 90 4 7 80 monocytes) MNC 75 22 2 E + E" MNC 90 7 75 6 80 16 One m i l l i o n c e l l s from each subset were incubated with a 1:100 d i l u t i o n of Leu-1 FITC (50 ul) and a 1:50 d i l u t i o n (50 ul) of rabbit anti-human IgG,M,A - FITC for 30 minutes on i c e . The c e l l s were analyzed on the FACS. The percent fluorescence was assessed by subtracting the fluorescence of the negative control c e l l s from the test samples. The scatter gates were set on the lymphocyte peak. 1 - 60 -Table XIII Efficiency of 0KT8 Removal C e l l Subset % 0KT8 MNC MNC minus 0KT8+ c e l l s 25.1 A.A 83% of the T8 positive c e l l s were removed 0KT8+ c e l l s 67.0 The c e l l s bearing the 0KT8 marker were removed from the MNC by a panning procedure. B r i e f l y the MNC (1 x 10 7) were incubated on ice with 500 u l of a 1:50 d i l u t i o n of 0KT8. The c e l l s were then washed free of non-binding 0KT8 and added to p e t r i dishes pre-coated with goat anti-mouse IgG. The non-adherent c e l l s were then recovered and stained with a 1:50 d i l u t i o n of goat anti-mouse IgG labelled with fluorescein. The percent po s i t i v e c e l l s were analyzed on the FACS as discussed above. - 61 -Discussion We have measured the l e v e l s of IgG secreted by 10 6 PWM stimulated peripheral blood mononuclear c e l l s obtained from chronic progressive, multiple s c l e r o s i s patients and normal subjects. The MNC isolat e d from the MS group secreted s i g n i f i c a n t l y more IgG i n response to PWM (p < .01) than did the MNC of the control group. This observation has previously been documented (10,62,123,178). Several investigators have studied the underlying cause of t h i s elevated, humural immune response, and have noted reduced suppressor a c t i v i t y (62,82) enhanced T-helper c e l l a c t i v i t y (93) as well as altered B-lymphocyte function (62,93). I t has also been documented that the number of lymphocytes expressing suppressor functions i s reduced during active disease. Thymus derived lymphocytes (T-cells) that bear the surface receptors for the Fc fragment of IgG (T^-cells) can upon int e r a c t i o n with IgG immune complexes suppress the PWM induced d i f f e r e n t i a t i o n of the B-lymphocyte (114). Huddlestone and Oldstone's group observed that the percentage of T G c e l l s i s reduced during the active course of the disease (76). Using monoclonal Ab's s p e c i f i c for c e l l - s u r f a c e markers present on T-suppressor lymphocytes (namely 0KT5+ and 0KT8+) (142,146), many investigators have noted a decrease i n the porportion of MNC bearing these surface markers during active disease (18,130,146,136). Although the absolute number of 0KT8+ c e l l s added to i n v i t r o cultures c l e a r l y influences IgG secretion, there appears to be no c o r r e l a t i o n between the proportion of 0KT8+ c e l l s within ficoll-hypaque p u r i f i e d MNC and the l e v e l of PWM induced IgG secreted by these c e l l s for both MS patients and controls (10). So although as a group the MNC - 62 -is o l a t e d from MS patients ex h i b i t elevated levels of PWM induced IgG secretion and decreased l e v e l s of c e l l s expressing the suppressor phenotype, there appears to be no correla t i o n i n i n d i v i d u a l patients between these two parameters. Analysis of the response of PWM stimulated PBMNC have revealed large fluctuations i n the l e v e l of response (10,69,83,156). Individual patients reproducibly secrete either high or low amounts of IgG i n response to PWM (defined as < or > 1000 ng/ml IgG secreted by 1 x 10 6 MNC/ml). In our investigation involving 38 MS patients and 21 controls we found that i n the MS group 84% were high responders as compared to 48% i n the normal control group (Table IV). In a series of c e l l subset mixing experiments which were designed to investigate the regulatory T,, and T_ c e l l subsets involved i n the n o PWM induced d i f f e r e n t i a t i o n of B-lymphocytes, the group i n Chicago (156) observed that the 0KT8+ c e l l s i s o l a t e d from a low responding i n d i v i d u a l suppressed the PWM induced d i f f e r e n t i a t i o n of a pool of B and T H lymphocytes to a much greater extent that did the same number of 0KT8+ c e l l s i s o l a t e d from a high responder. This observation suggests that the functional state of the 0KT8 lymphocytes may be altered without a concomittant a l t e r a t i o n i n the number of c e l l s . This would explain why there i s no co r r e l a t i o n between the number of 0KT8+ c e l l s and the l e v e l of PWM induced IgG secretion observed i n i n d i v i d u a l patients and controls. Additionally there i s an over representation of high responding ind i v i d u a l s within the MS population suggesting that there may be an a l t e r a t i o n i n the functional state of t h e i r 0KT8+ lymphocytes. - 63 -In a more dir e c t manner we have studied the suppressor function i n these MS patients; a non-specific inducible suppressor c e l l assay. This assay measures the a b i l i t y of Con A, mitomycin C treated MNC to suppress the Con A stimulated p r o l i f e r a t i v e response of heterologous thymus-derived lymphocytes ( T - c e l l s ) . The l e v e l of suppression induced i n the MNC isolated from the MS patients was s i g n i f i c a n t l y lower (p .01) than the l e v e l induced i n the control MNC. There was no negative suppression observed i n the control group whereas 33% of the MS patients PBMNC showed a negative suppressor response. We have been unable to determine the underlying cause of t h i s phenomenon. Reduced Con A induced suppression has been noted by several groups (61,118,203). Other assays of T-suppressor function have also shown decreased suppressor c e l l a c t i v i t y during attacks of MS (6,13,61,118,198). The l e v e l of suppression observed i n i n d i v i d u a l cultures did not correlate with the l e v e l of IgG secreted and t h i s was the same for both the control and MS group ( f i g . 2). This observation i s paradoxical for the following reasons. I t i s documented that the number of T-suppressor c e l l s i s reduced i n active MS. Reinforcing t h i s observation i s the reduced suppresor c e l l a c t i v i t y and increased T - c e l l dependent IgG secretion that i s observed i n v i t r o i n the MNC isolated from MS patients with active disease. However what appears to be a simple phenomenon i s actually auite complex because there i s no co r r e l a t i o n between the proportion of 0KT8+ c e l l s i n ficoll-hypaque separated MNC's and i , the amount of PWM induced IgG secreted (10) or i i , the l e v e l of Con A induced suppression. Add i t i o n a l l y the l e v e l of IgG secreted by PWM stimulated MNC does not - 64 -r e f l e c t the l e v e l of suppression induced by the Con A activated MNC of the same i n d i v i d u a l . This paradox e x i s t s even though i t i s well known that 0KT8 lymphocytes are intimately involved i n both the Con A (10) assay system as well as the PWM induced IgG secretion assay (10,156). The apparent lack of c o r r e l a t i o n between lymphocyte function and numbers l i k e l y stems from the fact that the 0KT8+ subset i s not homogeneous based either on the expression of 0KT8 (177b), and other c e l l surface markers (137, 177b) or c e l l subset function (0KT8 + c e l l s contain both cytotoxic and suppressor c e l l functions (145, 193)). For example the 0KT8 monoclonal Ab defines approximately 30% of the peripheral T-cells and the ent i r e 0KT5 + population as well as the 0KT4" 0KT5", T-cell subset (145). The l a t t e r lymphocyte subpopulation has not been defined by monoclonal Ab and may contain an important suppressor feedback regulator population (145). The precise r o l e of t h i s feedback regulator population as well as i t s regulatory mechanisms remain to be defined. I t seems therefore that further investigation of the T-suppressor c e l l subset i s required before any correlations can be made between the quantitative pathology and the functional abnormalities that are observed i n active MS. The MS patients discussed e a r l i e r were patients involved i n a c l i n i c a l interferon t r i a l at the MS c l i n i c i n the Health Sciences Centre Hospital. The MNC's of each patient were assessed i n two i n v i t r o immunological assays p r i o r to the study and again 7 days a f t e r receiving d a i l y subcutaneous in j e c t i o n s of either 5 x 10^ units of lymphoblastoid interferon or a si m i l a r volume of placebo. Both we and the patients were unaware of the treatment u n t i l a f t e r the results were calculated. - 65 -The i n vivo interferon treatment had a profound effect on the i n  v i t r o IgG secretion assay. The mean l e v e l of IgG secreted by 10 6 PWM stimulated MNC was reduced from 2,221 +_ 325 ng/ml to 560 +_ 75 ng/ml (p .001) i n the 20 patients studied. The amount of IgG secreted by the PWM stimulated MNC isolat e d from 18 placebo treated did not change s i g n i f i c a n t l y a fter 1 week of d a i l y injections (2542 ng/ml before and 2176 ng/ml a f t e r ) . In t h i s assay there i s a very wide range i n the l e v e l of response obtained i n both normal and MS patients. In any given i n d i v i d u a l however the v a r i a t i o n i n the l e v e l of response over a f a i r l y long period of time i s much less than the variations observed i n any given group (83). P r i o r to i n j e c t i o n , 85% of the patients i n the interferon treated group, were "high" responders ( i . e . >1000 ng/ml). After 1 week of d a i l y subcutaneous i n j e c t i o n only 15% of the patients remained high responders (In the placebo group 83% were high responders p r i o r to any i n j e c t i o n and 67% were high responders a f t e r 1 week of daily i n jections of s a l i n e ) . We conclude that the i n vivo interferon injections had a dramatic effect on t h i s i n v i t r o humoral immune response. We also studied the Con A induced suppressor c e l l response i n these two groups of patients before and after i n j e c t i o n of either interferon or placebo. There was no change i n the l e v e l of Con A inducible suppression i n e i t h e r the placebo or interferon treated patients a f t e r one week of d a i l y subcutaneous in j e c t i o n s . I t has been suggested that the 0KT8+ c e l l subpopulation contains the suppressor a c t i v i t y induced by Con A (10). As mentioned e a r l i e r the functional state of t h i s 0KT8+ subpopulation has also been implicated i n - 66 -regulating the l e v e l of IgG secreted by PWM stimulated MNC (156). Since the i n vivo treatment had no ef f e c t on the i n v i t r o Con A induced suppressor c e l l assay and marked effect on the PWM induced IgG secretion assay we would suggest that the interferon was not modulating the a c t i v i t y of t h i s suppressor c e l l subpopulation. Which subset(s) was/were affected by the i n vivo interferon i n j e c t i o n ? We investigated t h i s question by comparing the functional a c t i v i t y of interferon and placebo treated B - c e l l s , T-cells and monocytes i n various c e l l subset mixing experiments. We compared the functional a c t i v i t y of E~ c e l l s (B lymphocytes and monocytes) i s o l a t e d from inte r f e r o n treated patients with the a c t i v i t y of E~ c e l l s i s o l a t e d from placebo treated patients i n t h e i r a b i l i t y to reconstitute an i n v i t r o humoral response when mixed with the T u c e l l s isolated from both n placebo and inteferon treated patients. The E~ c e l l s obtained from the interferon treated patients could not generate a response greater than 419 ng/ml. On the other hand the E~ c e l l s obtained from the placebo treated patients secreted greater than 1000 ng/ml when mixed with e i t h e r the interferon or placebo treated T., c e l l s . n This suggests that perhaps the B-lymphocyte function may have been d i r e c t l y altered by interferon. The E~ c e l l subpopulation i s composed of both B-cells and monocytes so the function of the l a t t e r subpopulation was examined. Monocytes i s o l a t e d from INF treated patients were compared with those isolated from placebo treated patients i n t h e i r a b i l i t y to reconstitute an i n v i t r o humoral response when mixed with MNC that had previously been deprived of monocytes ( i . e . NAC) both from INF and PLA treated patients. - 67 -No difference was found i n the l e v e l of response between the monocyte depleted MNC ( i . e . NAC) reconstituted with either placebo treated or with interferon treated monocytes ( i . e . PAC). To summarize: INF treated E~ c e l l s did not secrete Ig when INF treated macrophage were functionally i n t a c t . This would suggest that an a l t e r a t i o n i n the B-lymphocyte contributed to the observed decrease i n the l e v e l of IgG secreted by PBMNC i n response to PWM. The l e v e l of response reconstituted with placebo treated T u c e l l s n was not s i g n i f i c a n t l y higher than that obtained with interferon treated T,, c e l l s , however we cannot exclude the p o s s i b i l i t y that INF also n a f f e c t s the T u c e l l . This i s the f i r s t report of INF administration i n vivo a f f e c t i n g the humoral immune response i n v i t r o i n humans. Harfast et a l (66) and Rodriguez et a l . previously reported that interferon added i n v i t r o modulated PWM induced IgG secretion. They observed that the presence of 100-5,000 units of human leukocyte interferon present throughout the 7 day culture period s i g n i f i c a n t l y reduced the amount of IgG secreted i n culture i n response to PWM. This result i s i n agreement with Parker's group (128) who noted a suppressed PFC response when human leukocyte interferon was added simultaneously with the Ag (horse red blood c e l l s ) . In contrast to these results pretreatment of the MNC p r i o r to mitogen or antigen stimulation resulted i n a s i g n i f i c a n t l y enhanced IgG secretion compared to untreated controls (66,128). Harfast's group noted that the increased IgG secretion was the direct e f f e c t of the interferon pretreatment on the B-lymphocyte subset. Shalaby's group (166) s i m i l a r l y investigated the effects of three bacteria-derived human recombinant - 68 -alpha interferon subtypes on a s p e c i f i c i n v i t r o humoral immune response. A l l the interferons tested enhanced the PFC formation when present throughout the entire 9 day culture period i n contrast to the previous reports where the presence of interferon throughout the culture period resulted i n either decreased Ig secretion (66) or a decrease i n the number of s p e c i f i c antibody PFC (166). Another group investigating d i f f e r e n t subtypes of recombinant human interferons (206) observed that d i f f e r e n t interferon subtypes had in h i b i t o r y and enhancing effects on the l e v e l of IgG secreted by human MNC. They noted ad d i t i o n a l l y that the concentration of the diff e r e n t INF subtypes influenced t h e i r immunomodulatory a c t i v i t y . I t i s not surprising therefore that c o n f l i c t i n g r e s u l t s are obtained between dif f e r e n t laboratories using different interferon preparations at varying concentrations and who addi t i o n a l l y use d i f f e r e n t Ag or mitogen. What i s important i s that interferon appears to have immune modulatory effects and additionally that the dif f e r e n t subtypes and concentrations used appear to have different properties i n modulating immune responses. 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