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Cellular abnormalities seen in the autoimmune MRL-lpr mouse strain Kaminski, Debra Ann 1989

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CELLULAR ABNORMALITIES S E E N IN THE AUTOIMMUNE MRL-/pr M O U S E STRAIN By D E B R A A N N KAMINSKI B .Sc , The University of British Columbia, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY We accept this thesis conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April, 1989 © Debra Ann Kaminski, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of jtf/g?d6(6£0& j The University of British Columbia Vancouver, Canada DE-6 (2/88) A B S T R A C T The MRL-+ and MRL-/pr mouse strains spontaneously develop autoimmune disease similar to human SLE . The autosomal recessive Ipr gene causes extensive lymphoproliferation of a unique DN T cell. The transfer of the Ipr gene to non-autoimmune mice [B6-/pr] has revealed other functions of the Ipr gene, among them autoAb production. The study of disease in MRL and Ipr mouse models affords an opportunity to determine the cellular basis of immune dysregulation seen in ensuing disease. The observation that Ipr mice elicit a decreased ability to respond to mitogen or alloantigen activation was initially investigated. Coincident with the age-dependent hyporesponsiveness, an increase in the number of cells bearing the tumour-specific marker recognized by the YE19.1.3 McAb was demonstrated. These YE19.1.3+ cells were separated from cells not bearing the marker, and those bearing the marker were unable to respond to conA activation. The YE19.1.3- cells could respond to conA, but demonstrated an age related decrease in responsiveness. These results were demonstrated in both MRL-/pr and B6-/pr mice. Lastly, the involvement of thymus education in the development of lymphadenopathy was investigated by making B6-/pr->B6-/pr and ->B6 chimeras. The percent of YE19.1.3+ cells and mitogen reactivity of these cells and the YE19.1.3- cells were comparable to B6-/pr controls in ->B6-/pr mice; the percent of YE19.1.3+ cells for ->B6 mice was greatly reduced, but mitogen responsiveness had not changed for these cells, hence the cells could not respond, demonstrating an intrinsic defect in YE19.1.3+ cells that cannot be overcome by education in a normal thymus. No age related hyporesponsiveness to conA was seen for the YE19.1.3- cells from ->B6 ii mice. Several aspects of certain A P C s in Ipr mice were examined. The assays used demonstrated both qualitative and quantitative abnormalities seen in MRL-+, MRL-/pr and B6-/pr mice. First of all, quantification by esterase staining implied that with age, MRL-lpr P E C s contained very high numbers of 1710s which resulted in less than optimal conditions for responder T cells to proliferate to conA or alloAg presentation. B6-/pr P E C s had normal numbers of esterase positive PECs . When B6-/pr mice were old, their P E C s augmented T cell responses, suggesting a qualitative rather than a quantitative defect in these mice when compared to MRL-lpr mice. Unfortunately, quantification using the McAb F4/80 could not support or refute the above results. 111 production from either Ipr animal strain did not appear to be much different from H-2 compatible controls. The studies performed on DC-enriched populations suggested that peculiar differences between MRL-lpr and B6-/pr DCs exist. MRL-lpr mice were tested for in vitro TsC function. It had previously been shown that preculture allowed for responsiveness by MRL-lpr NWT, thus experimentation was done and confirmed earlier results. Mixing experiments suggested that there may be some in vitro TsC function, but culture with a McAb specific for TsCs and TsFs [B16G] could not support those results. The above observations were not seen with B6-/pr NWT. Lastly, in vivo T sC function with respect to auto a-DNA production was assessed. MRL-+, MRL-/pr and B6-/pr mice were treated once, at the age of two months, with the immunotoxin B16G-HP to remove TsCs . Control C B A and B6 mice were also treated. This treatment led to elevated auto a-DNA production in all experimental mice; due to the variability in autoAb production for individual mice, significant differences between the treated and untreated groups could only be shown for B6-/pr mice, one iii week after treatment. The same treatment in control mice failed to induce auto a-DNA IgG. iv TABLE O F C O N T E N T S Page A B S T R A C T ii LIST O F FIGURES vi ABBREVIATIONS viii ACKNOWLEDGEMENTS x GENERAL INTRODUCTION 1 MATERIALS AND METHODS 8 C H A P T E R ONE : Analysis of DN Ipr T cell development and function. I. INTRODUCTION 19 II. RESULTS 25 III. DISCUSSION 50 C H A P T E R TWO: Analysis of A P C function. I. INTRODUCTION 64 II. RESULTS 70 III. DISCUSSION 92 C H A P T E R THREE : Analysis of TsC function in vitro and in vivo. I. INTRODUCTION 109 II. RESULTS 111 III. DISCUSSION 134 APPENDIX 150 LITERATURE CITED 153 v LIST O F F IGURES Page Figure 1: Proliferative responses to ConA. 26 Figure 2: MLR to alloAg presentation. 29 Figure 3a: YE19.1.3 surface Ag expression determined by F A C S analysis: H-2K mice. 32 Figure 3b: YE19.1.3 surface Ag expression determined by F A C S analysis: H-2B mice. 34 Figure 4: Proliferative response to conA of the YE19.1.3+ and YE19.1.3- populations of cells isolated by F A C S from a) MRL-/pr mice, b) B6-/pr mice. 37 Figure 5: Panning and cytoxicity to enrich for YE19.1.3+ cells. Analysis of proliferation to conA by cells from a) MRL-/pr mice, b) B6-/pr mice. 40 Figure 6: YE19.1.3 surface Ag expression determined by F A C S analysis: B6-/pr->B6-/pr and ->B6 Chimeras. 44 Figure 7: Proliferative response to conA of the YE19.1.3+ and YE19.1.3- populations of cells isolated from B6-/pr ->B6-/pr and ->B6 chimeric mice by a) FACS , b) panning. 46 Figure 2-1: A P C function for proliferation: P E C titrations in a) MRL-/pr mice and b) B6-/pr mice. 71 Figure 2-2: A P C function in the MLR: Spleen cell stimulators. 74 Figure 2-3a: Quantification of mas in P E C s by esterase staining: H-2K mice. 77 vi Page Figure 2-3b: Quantification of mos in P E C s by esterase staining: H-2B mice. 79 Figure 2-4: 111 production by splenic and peritoneal mos. 82 Figure 2-5: A P C function for proliferation in MRL-/pr mice: DC titrations. 85 Figure 2-6: A P C function in the MLR: DC stimulators, H-2K. 87 Figure 2-7: A P C function in the MLR: DC stimulators, H-2B. 90 Figure 3-1: Proliferative responses after two day preculture of NWT. 112 Figure 3-2: Mixing experiments. 114 Figure 3-3: In vitro culture with B16G. 117 Figure 3-4: In vivo TsC deletion therapy: Effect on auto 123 a - D N A production. vii ABBREVIATIONS Ab, antibody; Ad, adherent cell; Ag, antigen; AMLR, autologous mixed lymphocyte reaction; ANA, antinuclear antibody; APC , antigen presenting cell; a- , an antibody to - ; BCDF, B cell differentiation factor; bm, bone marrow cell; BSA, bovine serum albumin; B6, C57BI6/J; ->B6-/pr [B6], a chimera made by reconstituting a B6-/pr [B6] mouse with B6-/pr bone marrow after lethal irradiation; CBA, CBA/J; c\ complement; conA, concanavalin A; C P M , counts per minute of radioactivity; CTL, cytotoxic T lymphocyte; CsA, cyclosporine A; CSF , colony stimulating factor; CSFR , C S F receptor; DCs, dendritic cells; c -DMEM, complete Dulbecco's modified eagle medium; DMSO, dimethyl sulphoxide; DN, double negative cells CD4-CD8-; DNA, deoxyribonucleic acid; DiEA, diethanolamine; DTH, delayed type hypersensitivity; ELISA, enzyme linked immunosorbent assay; FACS , fluorescence activated cell sorter; F C S , fetal calf (bovine) serum; FITC, fluorescene isothiocyanate; GvH, graft versus host; HP, hematoporphyrin; H E P E S , N-2-Hydroxyethylpiperazine N'-2-Ethanesulphonic acid; id, idiotype; Ig, immunoglobulin; 111, interleukin 1; II2, interleukin 2; II2R, II2 receptor; II3, interleukin 3; y-\FN, gamma interferon; LN, lymph node; LPS , lipopolysaccharide; LTRC, low toxicity rabbit complement; McAb, monoclonal antibody; 2-me, 2-mercaptoethanol; MLR, mixed lymphocyte reaction; me, macrophage; MRL-+, MRL/Mp-+/+; MRL-/pr, MRI_/Mp-/pr//pr ; NAd, nonadherent cell; NK, natural killer cell; NWT, nylon wool T cells isolated from spleen; O- , old; PBS , phospate buffered saline; PBST, PBS-tween 20; PBSCT , PBS-casein-tween 20; PECs , peritoneal exudate cells; PG , prostaglandin; PLL, poly-L-lysine; PMA, phorbol myristic acid; viii substrate used in ELISA assays; PP , Peyer's patches; RT, room temperature; S LE or lupus, systemic lupus erythematosus; TcR, T cell receptor; ThC, T helper cell; TLI, total lymphoid irradiation; TR, transferrin receptor; ^H-TdR, tritiated thymidine deoxyribonucleotide; Tx, thymectomy; TsC, T suppressor cell; TsF, T suppressor factor; WBI, whole body irradiation; Y- , young ix A C K N O W L E D G E M E N T S The acquisition of knowledge is but a lifelong task made easier by our predecessors, peers and friends. There are so many to whom I am grateful, that the list would seem endless, but none as much as my supervisor, Dr. J . Doug Waterfield, without whom, his guidance and continual support, I may not have obtained this degree. Thank you Doug. As always, love Debbie x G E N E R A L INTRODUCTION In 1976, Murphy and Roths, at The Jackson Laboratory, developed the MRL congenic, inbred mouse strains, MRL/MP-+/+ [MRL-+]* and MRUMP-lpr/lpr [MRL-lpr]. In 1960 crosses were initiated with the intention to transfer the achrondroplasia mutation from the AKR/J strain to a mouse breed lacking early incidence of leukemia. New complications arose which were not overcome until backcrosses to the LG/J strain were performed. Subsequently, Murphy and Roths carried out rigid inbreeding on this mouse and the MRL mouse substrains were created. From the series of crosses and intercrosses between LG/J [H-2D+F], AKR/J [H-2K], C3H/Di [H-2K], and C57BL/6J [H-2B] followed by the strict inbreeding, the genome of the MRL mouse has been estimated to be comprised of seventy-five, 12.6,12.1 and 0.3 percent respectively of the above mouse genomes, and the haplotype has been designated as H-2K [derived from AKR, not C3H (3)] (1.2). During the twelfth generation of inbreeding, a mutation arose in some of the offspring which caused generalized, massive LN enlargement early in life. Breeding experiments with F1, F2 and reciprocal backcrosses demonstrated that a single, autosomal, recessive gene was responsible for the iymphoproliferation seen, hence this gene was termed Ipr. To create a congenic Ipr counterpart to the normal MRL strain which failed to develop the lymphadenopathy, the Ipr gene was transferred by cross-intercross matings creating MRL-+ and MRL-/pr, and by 1980 the genome of these two strains differed by less than 0.1 percent (1,2). * See abbreviations, p. viii. 1 T h e congen i c M R L mice are a lb ino with a large body s i z e [from LG/J] (1). They deve l op an au to immune d i s e a s e resembl ing human S L E , cha rac te r i zed by B ce l l hyperactivity. E leva ted numbers of p laque forming B ce l l s , su r face Ig posi t ive B ce l ls , and A b secret ing p l a s m a ce l l s have been de tec ted (4). In addit ion, B ce l l s s how an a ge re lated d e c r e a s e in r e spons i venes s to mi togen act ivat ion (5,13) sugges t ing an inc rease in mature and act ivated B ce l l s with t ime. E l eva ted B ce l l activity l eads to high titres of Ig and autoAb, most notably to D N A and nuc lear mater ia l , a nd IC d i s e a s e deve l ops result ing in g lomerulonephr i t i s , ult imately, the c a u s e of death (3). Other s ymp toms ev ident in M R L mice that a re representat ive of human S L E inc lude heavy prote inurea and lowered c' leve ls [both c o n s e q u e n c e s of the g lomerulonephr i t is] , e nhan c ed s ymp toms in fema le m i ce [consistent with the p redom inance of lupus in women] , arthritis, o c ca s i ona l myocard ia l infarction, modera te assoc ia t i on of the d i s e a s e with S jogren 's s ynd rome [another ch ron i c au to immune d i sorder that affects primari ly the exocr ine g l ands but may ex tend to s y s t em i c d i s e a s e and lympho id neop las i a (6)], s o m e centra l ne rvous s y s t em invo lvement, and rheumato id factor product ion (3). Thus , the congen i c M R L m o u s e subst ra ins have prov ided ind ispens ib le mode l s for the study of human S L E . In M R L - + mice, A N A beg in to appea r a round five months of a g e (2) and S L E d i s e a s e deve l ops principal ly in the s e c ond yea r of life (3,5). Thus , MRL -+ m ice have been ca tegor i zed a s a late-life lupus stra in. F ema l e m i ce d ie s omewha t ear l ier than ma le s with fifty percent mortal ity at s even teen months , ma les at twenty-two months . In their c ongen i c 2 counterparts, the autosomal recessive Ipr gene seems to function in two ways: first, it causes an accelerated or early-life disease, with Ab and autoAb production beginning around two months and a much reduced lifespan, with fifty percent mortality around five months, again with females succumbing slightly sooner. Therefore, MRL- Ipr mice provide scientists with an early-life lupus strain. Second, relating to the derivation of its name, the Ipr gene leads to the lymphoproliferation of a unique T cell which becomes noticeable in the peripheral lymphoid organs also around the second month of life (3). Progressively, the number of these T cells increases to the extent that LNs can be as much as one-hundred times the weight of age and sex-matched MRL-+ LN weight by four months of age (1). A greatly reduced lifespan and the extensive lymphadenopathy [of which, incidently, there is no human counterpart] exemplify how profound the effects of the Ipr gene are in mice with predisposing autoimmune disease. Since disease presentation in MRL mice is largely manifested by increased B cell activity, and a T cell expansion in the Ipr counterpart, numerous studies have been designed to establish causal immune relationships in the pathology and etiology of disease, and have revealed gross cellular defects in the immune system of these mice. With respect to other cellular immune functions, numerous discrepancies have evolved, depending on the experimental design. Thus, the function of the following remain to be clearly defined: a. ThC: normal (7), increased (5,8) and decreased (9-12) b. TsC: normal (5,7,13,14), increased (8,11,12,15-17) c. CTL: normal (7,18), decreased (8,19-21) 3 d. tolerance induction: normal (14), resistent (22,23) e. NK: increased (2,24), decreased (24,26). Some studies reveal abnormalities that appear more definitive: a. DTH: decreased (5,26-28) b. AMLR: decreased (19,21,29,30) c. T cells: reduced responsiveness to: i) mitogen (15,44,45), ii) alloAg (15,21,26,44), arid iii) 112(44,46). d. APC s : several peculiarities (3,31-43,69). Furthermore, the production of many lymphokines has been studied, some with conflicting results, further complicating the understanding of the disease process: II2 (8,44,130), y-IFN (39,41,48), II3 (45,53,54), 111 (36,39,40,55), C S F (47) and B C D F (57). The determination that Ipr was a single autosomal recessive gene enabled easy genetic manipulations for it to be transfered to other inbred strains of mice. By early 1980, Murphy and Roths had successfully done so to eight other strains, each demonstrating different degrees of lymphoproliferation and lifespan (2). Such strains have permitted analyses of the influence of different background genes on Ipr expression (2,58-64). The B6-/pr mouse strain has been used to determine Ipr gene function in the absence of S L E background genes in this thesis. The grossly, visible lymphadenopathy seen in this mouse strain predominates in the cervical LNs, unlike the generalized lymphadenopathy seen in all LNs 4 in the MRL-/pr mouse strain; however, in both strains, the spleen is greatly distended with age (64). The lifespan of the B6-/pr mouse is considerably longer than that seen for the MRL-/pr mouse, with fifty percent mortality around one year, and death results from breathing obstruction caused by the enlargement of the cervical LNs (4). An interesting observation is that these mice are larger in size to age and sex-matched, congenic B6 mice. Experimentation has determined that in the B6-/pr strain, the Ipr gene causes the production of numerous types of autoAbs, including a-double stranded DNA , a-IgG or rheumatoid factor, a-thymocyte, a-gp70 and high levels of serum Ig and IC, as is seen in the MRL mouse strain, showing that autoAb production need not be obligatorily linked to S L E background gene expression (58,59,63,64,66). In addition to autoAbs, natural Abs to exogenous antigens are also elevated [to BSA, KLH, DNP-BSA, DNP-KLH, and TMP-HEA] (63). Despite the high serum levels of all types of Abs, B6-/pr mice only develop a very mild renal disease, proposed to be reduced from the quality and composition of the IC deposited in the kidney, hence the reason why these mice do not develop lupus per se nor die from the general complications of lupus disease (4). Not only can serological similarities be shown for these two strains, but also, many of the abnormal cellular properties seen in the MRL-/pr mouse can be extended to the B6-/pr strain, although differences have been noted and are no doubt due to background gene influence (9,21,25,28,49,52,53,65,67,70-75). For example, in the absence of S L E background genes, sex hormone influence on Ipr gene expression is more 5 dramatic than that seen with MRL-/pr mice. Female B6-/pr mice develop lymphadenopathy, and have Ab production, to a much greater extent than B6-/pr males and earlier (59). Female B6 mice have B cell hyperactivity in the absence of the Ipr gene (59), thus in the presence of it, the sex influence is greatly enhanced. This emphasizes that the true function of the Ipr gene may be masked in different genetic backgrounds, hence the development of other mouse strains with Ipr has been necessary to reveal its function(s). Limiting dilution analyses (18,47) and studies using McAbs (further referenced in chapters one and two), designed to determine populations of cells responsible for the above observed peculiarities, have failed to pinpoint any single subpopulation as the sole cause of d isease and, rather, emphasize that a combination of cellular immune and regulatory events are involved. Nevertheless, certain subsets of cells, defined phenotypically, have been shown to be functionally defective and/or phenotypically abnormal. Genetic studies definitely show that the predisposition to lupus disease in MRL mice can be attributed to a hematopoietic system defect [stem cell or lymphoid precursor], that the Ipr gene acts as an accelerator, and that non-genetic factors influence disease expression (3,4). Moreover, molecular studies are in the initial stages and thus far have failed to reveal significant alterations as putative causal agents leading to disease expression (4,68). So, despite elaborate descriptions of what may be occurring and models of how in these mice [at the genetic, cellular, humoral, somewhat at the molecular level, as well as pathologically and histologically] it still remains elucive why the disease develops. 6 In the three chapters of this thesis, I will further evaluate the cellular abnormalities seen and attempt to explain what relevance these may have. 7 MATERIALS AND METHODS Mice: The mice used in this study were Balb/cJ, CBA, B6, B6-/pr, MRL-+, MRL-/pr. Mice were originally obtained from The Jackson Laboratory, Bar Harbour, Maine. These animals were bred and maintained in our animal facilities, Department of Oral Biology, UBC, on a diet of Purina Mouse Chow and water and on a twelve/twelve hour light/dark schedule. Media and Reagents: a) Medium: High glucose, sodium bicarbonate buffered, L-glutamine [200 mM] and penicillin-streptomycin [100 International Units-100Lig/ml] supplemented, Dulbecco's Modified Eagle Medium was purchased from the Media Prepartion Service Centre at the Terry Fox Laboratory, Vancouver, B.C. To make c-DMEM, the following were added [final concentration]: FCS (GIBCO, Grand Is., New York) [ten percent]; sodium pyruvate solution (GIBCO) [1 mM]; 2-me (Sigma Chemical Company, St. Louis, Missouri) [5x10"5M]; HEPES (GIBCO) [1 mM], b) Mitogens: LPS from E. coli was purchased from Sigma and conA from Pharmacia Fine Chemicals, Pisatataway, N.J. c) Nylon Wool Columns: Source: Nylon wool was purchased from Fenwal Laboratories, Division of Travenol Laboratories Incorporated, Deerfield, Illinois. Two types were used: i) Leuko-Pak leukocyte filter, code 4C2401, or ii) Scrubbed nylon fibre, code 4C2906. Preparation: Type i) Nylon wool from two or three filter units was removed and placed in approximately one and 8 one half litres of distilled water in a two litre erlenmeyer flask, covered with tin foil, brought to a boil and boiled for ten minutes. The water was decanted, the nylon wool rinsed three times with distilled water and then resuspended in one and one half litres of distilled water and put on to boil again; this proceedure was repeated for a total of six times boiling. After the final rinsing, the excess water was squeezed out, the nylon wool was spread out on foil and left to dry in a flow hood for two to three days. Once dry, the nylon wool was stored in a covered plastic container. Type ii) For this nylon wool, the washing procedure was the same after the nylon wool was first autoclaved in 0.2N hydrochloric acid for twenty minutes, left to cool and then rinsed three times with distilled water. Packing of Columns: The columns were made from ten cc syringes (Becton Dickinson, #5604) packed to six to seven mis with 0.7 grams of teased apart, loosely folded and packed, washed nylon wool. These were then placed into sterilization pouches (Chex-all II instant sealing, gas or steam sterilization, Propper Manufacturing Company, Long Is. City, N.Y.) and autoclaved at one-hundred-twenty degrees C for twenty minutes. d) Plates: cell culture: All assays were plated in 96 flat-bottomed welled plates purchased from Corning Glass Works, Corning, New York. panning: petri dishes, 60mm, Falcon #3002, were purchased from Becton Dickinson. ELISA: 96 flat-bottomed well plates, Falcon #3915, were purchased from Becton Dickinson. e) Density gradients: B S A was bought from Sigma. f) ELISA: PLL, DNA [sonicated to reduce the size to approximately 3000 bases or less as determined by agarose gel electrophoresis] and pNPP substrate tablets, were obtained from Sigma. DiEA and Tween 20 were purchased from Fisher Scientific, Fair Lawn, N.J. Casein was bought 9 from BDH Chemicals, Poole, England. Plates were read on a Titertek Multiscan, Flow Laboratories, Mississauga, Ontario, at absorbance 405 nm. g) Abs: a-Lyt2 [rat lgG2a] recognizes the CD8 determinant on T cells and was prepared from ammonium sulphate precipitation of culture supernatant from the cell line obtained from Dr. F. Takei, Terry Fox Laboratory, Vancouver, B.C. a-mouse IgG-alkaline phophatase [goat] was purchased from Tago Incorporated, Burlingame, California, a-rat IgG-FITC [goat] was purchased from Dimensions Laboratories, Miss issauga Ontario. a-rat Ig [rabbit, Cappel Ab] was obtained from Organon Teknika, West Chester, PA. a-rat IgM-FITC [goat] was purchased from Cooper Biomedicals Incorporated, Malver, Pennsylvania. a-Thy1.2 [rat IgM] had been made several years ago in our laboratory from a cell line that Dr. J.D. Waterfield originally obtained from Drs. P. Lake and A. Mitchison. a-Thy1.2-FITC [mouse IgM] was obtained from ICN Biomedicals Canada Limited, Montreal, Quebec. B16G [rat lgG2b] recognizes T sCs and TsFs and was prepared as a fifty percent ammonium sulphate cut ascites in Dr. J .G . Levy's laboratory, Department of Microbiology, UBC . B16G-HP: To link HP (Sigma), a light activatable toxin, to B16G the following was done: 0.4 ml of HP at one mg/ml in D M S O (Sigma) was added to ten mis P B S and then two mis of this was added to B16G to reach a final concentration of 0.714 mg/ml in four mis total. This was allowed to react overnight at four degrees C in the dark and then was extensively dialysed in the dark. F4/80 [rat lgG2b] recognizes monocytes and mes and was a gift to Dr. N. Deslauriers, from Dr. S. Gordon, Oxford, England, which she kindly shared with us. GK1.5 [rat lgG2b] cell line was a gift from Dr. M. Bevan, Scripps Clinic and Research Foundation, La Jolla, California. The Ab recognizes the CD4 determinant on T cells it was prepared from ammonium sulphate 1 0 precipitation of culture supernatant. YEW. 1.3 [rat lgG2b] recognizes a determinant present on the DN Ipr T cells and was a gift from Dr. F. Takei. It came as an ammonium sulphate preparation of ascitic fluid. Serum samples from control and experimental mice were obtained biweekly either by eye or tail bleeding. Blood was left to clot at thirty-seven degrees C for thirty minutes and then overnight at four degrees C. Serum samples were then aliquotted into fifteen u.l amounts and stored at minus twenty degrees C until use. LTRC was purchased from Cedarlane Laboratories, Hornby, Ontario. Cell preparations: a) Spleen cell suspensions were obtained by teasing cells from the excised spleens of mice. The solution obtained was pipetted vigorously and cellular aggregates were allowed to settle out and were removed. Cel ls were washed three times in cold PBS , counted on a hemacytometer, and resuspended to the appropriate concentration in c -DMEM. b) P E C s were obtained by washing the peritoneal cavity of mice with ten mis of ice cold PBS . Cells once harvested were washed three times with cold PBS . When treated to remove T cells, the procedure was according to that recorded for cytotoxicity. P E C s were then irradiated with 3000 rad from a Gammacel l 220 ( 6 0 C o , Atomic Energy of Canada, in the Department of Chemistry, UBC). c) NWT: Columns were removed from the sterile pouches asceptically [in a class I culture containment hood] and mounted in a clamp on a ring stand. Seven mis c -DMEM was loaded onto the column. Using a metal spatula, the sides of the column were tapped to remove air bubbles and completely wet the nylon wool with medium. Another seven 11 to ten mis c -DMEM were allowed to pass through the column which was, after running dry, then placed in a autoclaved beaker, covered with aluminum foil. The beaker containing the column was then placed into a five percent carbon dioxide incubator at thirty-seven degrees C for thirty to ninety minutes. While the columns were incubating, single spleen cell suspensions were prepared at a concentration of 1x10** cells/ml. One ml of the spleen cell suspension was added drop by drop [approximately one drop per second] to the column and washed into the column with one ml c -DMEM, drop by drop. The loaded column was then placed back into the beaker and this was put into the incubator for one to two hours. NAd cells were collected by passing ten mis c -DMEM, drop by drop over the column. Cel ls collected were greater than ninety-five percent T cells as determined by F A C S analysis. d) Panning : Panning was performed according to a modification of the method developed by Dr. F. Takei (personal communication). 1x10** NWT were treated with ammonium chloride solution to lyse erythrocytes, washed three times in P B S and resuspended to 2-5x10 7cells/ml in c -DMEM. Saturating amounts of GK1.5 and a-l_yt2 McAbs were added to the cells which were then incubated on ice for forty-five minutes. Cel ls were washed three times with PBS , 1x10** were resuspended in two and one half mis c -DMEM and incubated for forty minutes at four degrees C on a petri dish that had been precoated with a-rat Ig at 100 ug/ml, and washed three times with PBS . The NAd cells were removed and this procedure was repeated two more times. The final NAd cells were the DN cells, enriched for YE19.1.3+ cells and the first Ad cells were equivalent to 'normal' peripheral T cells and enriched for YE19.1.3- cells. Enrichment by panning yielded a two to three fold enrichment in YE19.1.3+ cells as 1 2 determined by F A C S analysis. e) Cytotoxicity: Cytotoxic Abs were added at the appropriate titre [determined experimentally] to a cell sample and incubated for thirty minutes at RT in c -DMEM. The cells were then washed once. LTRC in c -DMEM was filter sterilized and added to the cells, according to the manufacturer's recommendation, and this mixture was incubated for forty-five minutes in a thirty-seven degrees C water bath. After this incubation, the cells were washed three times and used for studies. f) F A C S Analysis: Cell populations were analysed on a F A C S IV-40, Becton Dickinson, in the Department of Neurology, University Hospital, UBC Site, by Technician Dan Zecchini. Cell preparation: NWT were incubated with an appropriate concentration of primary Ab in c -DMEM for forty minutes on ice. After washing three times with PBS , an appropriate concentration of secondary, FITC-labelled Ab in c -DMEM was added and the cells were incubated on ice for another thirty to forty minutes. Cells were washed with PBS three times, resuspended, and analysed. The percentage of positive cells was determined by comparing the crossover point between the above labelled cells to cells labelled with the secondary Ab alone. Positively and negatively labelled cells were obtained by setting suitable gates on the F A C S and collecting separate populations. Populations obtained this way were greater than niney-five percent pure. g) Enriched Dendritic Cell Preparation: Enriched DC populations were prepared by discontinuous B S A density gradients and selective adherence with minor modifications from the method developed by G . Sunshine (183). Single spleen cell suspensions [approximately 1 x10 8 cells] were pelleted and resuspended in two mis thirty-five percent BSA 13 in PBS . A discontinuous gradient, from the bottom to the top, of thirty-five, twenty-nine, twenty-six, twenty-three and ten percent BSA was then made in ultracentrifuge tubes; tubes were spun at ten-thousand rpm for thirty minutes at four degrees C. The band at the twenty-three and ten percent BSA interface was immediately harvested and washed three times. These cells were resuspended in two and a half mis c -DMEM and plated on a sterile 60 mm petri dish. After two hours incubation in a thirty-seven degrees C, five percent carbon dioxide incubator, the NAd cells were removed, after gentle agitation, and five mis c -DMEM was added to the Ad cells which were then left to incubate over night [approximately eighteen hours]. The NAd population after the overnight incubation constituted the DC-enriched population. Before being used in assays, these cells were iradiated [3000 rad], as the P E C s were. h) Cel l preparation for esterase staining: Twenty-five JLXI of cell suspensions, prepared as mentioned above, at a concentration of approximately 1x10^/ml in PBS , were dropped onto clean slides and allowed to air dry completely before being used for staining. Assays: a) Proliferation: Anywhere from 1-5x10^ NWT from different mice were plated in each well [0.2 ml] either with or without conA at 1.25 u.g/ml on day zero. On day two, cultures were labelled with one u.Ci -TdR/well and incubated for another eighteen hours. Then, day three, the cultures were harvested [mash harvester] on Whatman glass microfibre filters (Whatman Laboratory Products Inc., Clifton, New Jersey). After drying for at least twenty minutes, samples were counted in three mis scintillation cocktail [forty percent methanol, sixty percent 14 toluene, plus 0.0042 percent Liqueflor (NEN Research Products, Boston, Massachusetts)] for one minute on a Liquid Scintillation Counter PW4700, Phillips, Holland. Counts per minute plus standard deviation were calculated from triplicate cultures. In chapters one and two when P E C s or DCs were added to cultures, the cells were isolated as described and added to cultures as indicated for individual experiments. For two day incubation experiments, chapter three, NWT were cultured at 1x10^/ml in c -DMEM on 60 mm petri dishes under standard culture conditions and then tested, after culture, for conA responsiveness. For mixing experiments, chapter three, predicited values were determined by adding together the values obtained for the different concentrations of cells cultured individually. When B16G was added to cultures, chapter three, fifty and one-hundred u.g/ml was used as this was the optimal working concentration determined for in vitro studies (A. Tench-Stammers, personal communication). b) Mixed Lymphocyte Reactions: Responder populations were 1 x10 6 NWT from different mouse strains per 0.2 ml well. Stimulator populations consisted of 1 x10 6 spleen cells irradiated with 3000 rad. Cultures were labelled with one u.Ci 3H-TdR/wel l on day three and harvested on day four; C P M were determined by the same procedure as for the proliferation assay. c) IL1 assay: The cell line: D10.G4.1 was a gift from Dr. N. Reiner, Vancouver General Hospital, Vancouver, B.C., who originally obtained it from Dr. C. Janeway, Yale University, Connecticut, USA. The cells were maintained in c -DMEM supplemented with II2 from the supernatant of PMA-stimulated EL4 cells. The cells were allostimulated with irradiated [3000 rad] B6 spleen cells approximately every three to four weeks and 15 not used in the 111 assay until at least two weeks after allostimulation, nor later than four weeks afterward. Prior to use in the assay, the cells were washed three times and left to starve in c-DMEM without an II2 source over night. On the day of the assay, viable cells were harvested by ficoll-paque (Pharmacia) treatment and washed three times with PBS. The recombinant 111 standard control was a gift from Dr. P. Conlon, Immunex Corporation, Seattle, Washington. D10.G4.1 assay: D10.G4.1 cells are useful because in the presence of suboptimal concentrations of conA (0.25 - 0.50 u.g/ml), they are very sensitive to and proliferate with minute quantities of 111. 1 - 2 x 1 0 4 cells/well were plated on day zero, in the presence of conA and dilutions of 111 samples. The rest of the assay procedure was the same as for the proliferation assay. d) Esterase staining: Staining was performed according to a protocol developed in Dr. N. DesLauriers laboratory, Laval, Quebec. First, stock solutions of a-napthyl acetate and phosphate buffer [without sodium chloride] were prepared: one gram a-napthyl acetate, fifty mis acetone and fifty mis distilled water comprised the a-napthyl acetate stock solution. A 0.1 M solution of phosphate buffer was made by dissolving 2.05 grams monobasic sodium hypophosphate with 19.09 grams dibasic sodium hypophosphate-heptahydrate in eight-hundred mis distilled water; this solution was then adjusted to pH 7.3. These two stock solutions were stored at four degrees C until use. To prepare a working substrate, two mis of the stock a-napthyl acetate solution was mixed with fifteen mis of the phosphate buffer solution, fifteen mis distilled water and twenty milligrams fast red TR salt (Sigma). Slides were incubated in this mixture for thirty to forty minutes at RT, washed in tap water and left to air dry. The stained slides were then observed under the microscope. 16 e) E L I S A a s s a y . T h e amoun t of e a c h al iquot to e a ch we l l w a s fifty u.1. P la tes we r e p recoa ted with P L L , fifty jag/ml in P B S overn ight at four d eg r ee s C . T h e next day, p lates we re w a s h e d three t imes with P B S . D N A at twenty u.g/ml w a s incubated on the p lates for one hour at th i r ty-seven deg r ee s C and then the p lates we re w a s h e d three t imes with P B S . P la tes we re b locked with ca se i n [two percent in P B S ] for one hour at th i r ty-seven deg r ee s C and then w a s h e d three t imes with P B S T [tween at 0.05 percent] . A b s amp l e s at se lec ted di lut ions in P B S C T [case in at one percent] we re p lated and incubated at R T for one hour and then w a s h e d with P B S T . a -mouse IgG label led with a lka l ine phospha ta se at a di lut ion of one in e ight - thousand w a s then added to the p lates wh i ch we re incubated at R T for one hour. Next, the p lates we re w a s h e d three t imes with P B S T a n d p N P P substrate in D i E A buffer w a s al iquotted to e a c h wel l . O n c e deve l oped [overnight incubation], the p lates we re read at 4 0 5 nm on the titertek. A b s o r b a n c e va lues we re ca l cu la ted from repl icates of four. Cont ro l s inc lude 0 - M R L - / p r se rum [fifty percent a m m o n i u m su lphate precipitated] and non-auto immune, two to four month o ld , untreated Ba lb/c m o u s e s e r um . Data calculations: A b s o r b a n c e 4 0 5 nm va lues for e a c h s e r um s amp l e we re ave raged . T h e s e va l ues we re s tanda rd i zed to the posi t ive contro l s e rum that w a s u sed for all E L I S A a s s a y s ; va lues we re read off of the l inear portion of the posit ive contro l s e r um [curve ob ta ined f rom Cr i cke t Graph] and thus are e xp r e s sed a s 'control equ iva lents ' . Con se cu t i v e ab so rbance 4 0 5 nm va lues we re plotted aga ins t t ime a n d ana l y s ed statist ical ly. The stat ist ical tests u s ed we re con f i dence interval for the di f ference be tween two mean s [parametric] and the Mann-Wh i tney test [non-parametr ic]. M i ce from the s a m e contro l /exper imenta l g roups we re a s s a y e d on the s a m e day to obta in the 1 7 final results. Chimera preparation: Tibias and femurs were removed from two to five month old male mice. The ends of these were cut off and then they were flushed with cold PBS to remove all cells. These bm cells were pipetted to make a single cell suspension and washed three times. To remove T cells the bm cells were treated with oc-Thy1.2 plus c' as outlined for cytotoxicity. To make chimeras, treated bm cells were resuspended to 5 x 1 0 7 cells/ml and 0.2 ml was injected by the tail vein into lethally irradiated [900 rad] recipient B6 and B6-/pr mice. 111 production by spleen cells and PECs: 1 x 1 0 6 spleen cells or 5x10^ P E C s [treated to remove T cells], in one ml of c -DMEM per two ml well, were incubated for twenty-four hours in the presence of twenty u.g L P S per ml. After twenty-four hours, 0.8 ml of the supernatant of each well was removed and 0.8 ml of c -DMEM was added. Cel ls were left for another twenty-four hours [forty-eight hours total] and one-half ml of the supernatant was harvested and either used immediately, or frozen at minus twenty degrees C until use in the 111 assay. B16G-HP treatment of mice: 0.1 ml of B16G-HP solution was injected by the tail vein into mice being treated. Animals were kept in the dark for two hours and then exposed to light [22.5 mW/cm 2 twenty-four cm above the animals] for four hours. 1 8 C H A P T E R O N E INTRODUCTION In addition to the Ipr gene acceleration of lupus disease in MRL-Ipr mice, there is the expansion of a unique population of T cells under the influence of the Ipr gene. These cells initially populate the P P as early as two weeks and subsequently are seen in the LNs, spleen [by eight weeks] and then, lastly, may be seen in the thymus (76). By two months of age, the accumulation of these cells in the peripheral LNs becomes palpably and visibly apparent, especially in the axillary LNs (2). In B6-/pr mice, LN enlargement does not parallel that seen in MRL-/pr mice. Rather, the cervical LNs are primarily affected and become outwardly infiltrated by four months (64). Despite the increasingly gross distortion in the peripheral lymphoid organs with age, the source of these cells remains elucive. That they derive from a radio-sensitive progenitor is evident from irradiation studies, which show that sublethal WBI or TLI leads to the elimination of the outgrowing population and eradication of the disease symptoms associated with the Ipr gene (2,65,77,78). Chimera studies of MRL-/pr bm transfers into irradiated MRL-+ mice have shown that a GvH disease develops and leads to rapid death before any detection of lymphoproliferation (2,79,80). However, when conditions were created which prevented GvH, transfer of Ipr disease with Ipr bm was seen (89). That B cell abnormalities are also seen (63,64) would suggest a lymphoid restricted defect exists. All attempts at transfering disease with peripheral lymphoid cells have failed (2,79,81) so the 'developed' Ipr cells are unable to cause disease. Studies on the ultrastructure of the bm 19 demonstrate evidence of active lymphopoiesis in association with a distinctive stromal cell (82). Thus, this may be the actual site of origin of these cells. Cell cycle analyses on bm and LN cells in MRL-/pr mice have shown little difference in frequency between their proliferating cells and those of MRL-+ and normal mice (83). However, the absolute numbers of proliferating cells in the LN of the MRL-/pr mouse would be greatly increased due to the lymphadenopathy. It has been suggested that in situ proliferation could contribute significantly to the increased numbers of these cells (101), so this may be another location where they arise. Thymus influence in the expression of lymphoproliferative lupus disease of MRL-/pr mice is readily apparent from the following studies: a) neonatal Tx abrogates lymphoproliferative disease (2,41,84,85) and reconstitution even with a normal thymus restores it (86). That only neonatal Tx abrogates subsequent disease emphasizes the necessity for early establishment of the T cell repertoire in the disease; b) B6-nu/nu-lpr/lpr [B6-nu-lpr] mice fail to develop the expanded Ipr T cell, but do so if grafted with a congenic thymus (63,87). A controversial, but still plausible, mechanism suggested in the nu mouse is that T cells may develop extrathymically, as with age, increasing numbers of T cells are seen in the lymphoid organs of these mice (63). In the B6-nu-lpr mouse though, this pathway of T cell development is not efficient enough to cause lymphoproliferative disease in these animals, unless grafted with a thymus, and further stresses the requirement for a full complement of T cells, generated by the thymus, for the expansion of Ipr T cells by the Ipr gene; 2 0 c) thymus influence via T cells specifically is shown in studies using in vivo treatment with a-Thy 1 (88) or a -L3T4 (128) which abolish disease. Thymus influence is not the only mechanism operating as splenectomy only during a restricted time period led to improvement of disease and transfer of MRL-/pr spleen to thymectomized animals restored disease (90). Furthermore, sex hormones play a role which is readily apparent in B6-/pr female mice which develop a more severe disease than the males and earlier (59), an influence which is largely masked in MRL-/pr mice by the background S LE genes. Complementing the sex influence observations are in vivo manipulations which showed amelioration of Ipr disease by androgen treatment (84). So, the Ipr gene acts in a complex manner to cause the outgrowth of a unique population of T cell and accelerated disease in MRL-/pr mice. A vast amount of evidence has accumulated characterizing the unique Ipr T cells that expand and populate the periphery in Ipr mice. Detailed studies performed on the phenotype of these cells demonstrate they are H-2K+ (91), dull Thy1+ (91,92) and dull Ly1+ [CD5+] (91,92). They have been designated DN as they lack surface expression of L3T4 [CD4] (93) and Lyt 2 [CD8] (91,92,94). That they are T cells is confirmed by TcR alpha, beta and gamma rearrangements (95-98) [with some abnormalities in transcripts seen (97,98)] and low level expression of TcR protein (96,98). Anomalies with the TcR/CD3 protein complex exist: an abnormally sized TcR protein species has been immunoprecipitated (99) and p21 of the CD3 complex is permanently phosphorylated on a tyrosine 21 residue (100), an event normally triggered by T cell activation. Lack of Ig gene rearrangements (92) and no surface Ig expression (91,92) would also support the T cell nature of these cells. Some traits of the Ipr cells common to other T cells are: ThB- [an early thymocyte marker] (92), H11 + [a T cell marker] (92), Mel14+ [for T cell homing and is reduced upon activation] (1 Of), Ly-24+ [is PGP-1 , recognized by the 9F3 McAb (102-105), on immature T cells for homing, and acquired by mature T cells upon activation], Lp1+ [an Ag involved in T and B cell interactions necessary for humoral immune responses] (106), II2R- (20) and TR- (94) [is present on proliferating T cells (129)]. They also express some markers common to T and B cells: Ly6.2 [on Lyt2+ T, pre-B and B cells] (70,107,109), and YE19.1.3 [a tumour specific determinant found on EL4 T lymphoma cells, NS-1 myeloma cells, and a B cell line] (94). Paradoxically, Ipr T cells express many markers normally seen on pre-B and B cells: 14D10 [a 55-60 kDa protein also recognized by the McAbs 2C2 (92) and 100C5 (110)] (71), Lp2 [which also shows similar reactivity with 2C2 and 100C5 plus another McAb 6B2 (92)], 3A1 [similar to the above] (92), and Ly5 [is B220, a member of the T200 or leukocyte common Ag family] (92,113,114). Lastly, they also express high levels of PC.1, characteristic of 2 2 plasma cells (115). Other peculiarities of these cells are: altered surface carbohydrate properties in that they demonstrate: a) variable binding to T cell lectins [norma: PNA, increased: LPA, MPA, ConA, reduced: HPA, SBA, BSA-1, PHA-L] (91); b) B cell lectin binding properties: [WGA, Pa-2, Spud, BSI and Limulin-CRP] (116); and c) unusual Paul-Bunnell (117,118) and Forssman (118) Ags, the former being a glycolipid and the latter a glycoprotein; altered potassium channels (119,138) and a lower electronegative surface charge than other T cells (91). Oncogene studies thus far have revealed elevated levels of c-myb [typical for thymocytes] (73,74,120,121), increased levels of c-raf (121) and slightly increased levels of c-myc [important for proliferation (127)] (122). Lastly, the majority of the cells are in the Go-G1 phase of the cell cycle [characteristic of resting T cells] (83,114). On first approximation, the cells accumulating in the peripheral lymphoid organs seem to be strange with respect to phenotype, by expressing pre-B and B cell markers in addition to T cell markers. However, purification studies on the minor [three to five percent] DN thymocyte populations in normal mice and subsequent surface marker analyses have revealed that DN thymocytes in normal mice also bear many of the determinants seen on the Ipr DN T cells (123-126). Recently, Budd et al, when looking at the thymi of Ipr and normal mice, were able to phenotypically define both normal and abnormal T cells developing in the 2 3 Ipr thymus and, conversely, found a Ipr counterpart in the normal thymus (126). This suggests that normal T cell development occurs in Ipr mice and that the Ipr DN T cells are included, but, for some reason, by some still unknown mechanism, perhaps incomplete differentiation, these cells are prevented from maturing properly or from being eliminated in the thymus, and hence accumulate in all of the lymphoid organs of Ipr mice. Subsequent studies of Ipr DN cells in comparison to phenotypically similar cells isolated from normal fetal and adult mice have suggested that the Ipr DN cells are functionally similar to day sixteen fetal thymocytes (67). Originally, numerous studies on whole populations of cells from the spleen and LNs from MRL-lpr mice demonstrated profound abnormalities in T cell function as outlined in the general introduction. However, whole population studies such as these cannot reveal definitive conclusions on any particular subset of the accumulating Ipr DN cells. Recently, a McAb, YE19.1.3, was found to react almost exclusively with Ipr DN cells (94). The McAb was originally selected for its reactivity with EL-4, a T cell lymphoma, and cross-reactivity was found only on two other B cell lines, as mentioned earlier. We have used this McAb to select for and study the in vitro functional capacity of the population of Ipr DN T cells to which it binds in MRL-/pr and B6-/pr mice. Chimeras were made to assess the role of the Ipr gene in the development of the T cell repertoire. We questioned the role of the thymus in these chimeras, B6 or B6-/pr mice lethally irradiated and reconstituted with Ipr bm cells, by following the. development of YE19.1.3+ Ipr DN T cells and analysing their function in vitro. 2 4 C H A P T E R O N E R E S U L T S Aae associated hyporesponsiveness to ConA: The observation that Ipr mice demonstrate an age dependent decrease in responsiveness to conA was used as the starting point for studies. NWT from MRL-/pr, congenic, age-matched MRL-+ and H-2 compatible C B A mice were cultured at an optimal conA concentration and assayed for their proliferative capacity in a standard three-day experiment. Young and old mice were used to see the effect of age related disease expression on the proliferative response. It can be seen in figure 1 that the control, non-autoimmune C B A strain NWT proliferated well in response to conA; the MRL-+ mouse NWT, with background genes for SLE , had a reduced capacity to respond. This effect was enhanced by the Ipr gene in the MRL-/pr mice and the effect worsened with age, as seen by the negligible response by the old MRL-/pr mouse NWT. In a second system, using the B6-/pr mouse, the effect of the Ipr gene in the absence of the S LE background genes can be determined. In figure 1, where NWT from B6 and B6-/pr mice of differing ages were assayed for their proliferative ability in response to conA, the same trend that was seen in the MRL system was repeated here; B6, non-autoimmune control, mice NWT responded well to the mitogen and the young Ipr mice NWT had a slightly reduced response which worsened with age to the point of background levels [which correlated with outward appearance of disease]. Eventhough background S L E genes caused a reduced response to conA, as seen by the response of MRL-+ NWT, the recessive autosomal Ipr gene also accounted for a substantial reduction in response seen in 25 Figure 1: Proliferative responses to conA: NWT responder cells [5x10 5] from the listed strains were cultured in the presence of 1.25Lig/ml conA to assess proliferation. Assays were set up on day zero, labelled on day two with tritiated thymidine, and harvested on day three. Radioactivity incorporporation was determined by a scintillation counter. The results are given as mean plus or minus standard deviation of triplicate cultures. Control cultures, where no conA was added, were all less than 500 cpm. CBA, and 'Y' mice were three months old, B6 and 'O' mice were six months old and the MRL-+ mouse was twelve months old. All mice were female. 2 6 0 5 0 0 0 0 1 0 0 0 0 0 CPM MRL-/pr and B6-/pr mice. Aae-dependent decrease in responsiveness to alloAas: In order to study a more physiological immune response, MRL-/pr, MRL-+ and B6-/pr NWT were assayed in MLRs . 1 x10 6 NWT purified from the H-2K responders, C B A control, were cultured with irradiated [3000 rad] B6 spleen cells at a ratio of one for four days; radioactivity incorporation was assessed and compared. Similarly, H-2B responders were cultured with C B A stimulators and their capacity to respond was assessed. Figure 2 demonstrates that the non-autoimmune, C B A and B6 NWT, were able to respond well when stimulated by alloAgs, as was seen when they were cultured with mitogen. The MRL-+ NWT responded almost as well as the C B A NWT. The MRL-/pr mice NWT demonstrated a good response when the mice were young and that response was completely abrogated when the mice were five months of age; similar results to the MRL-/pr NWT were seen for the B6-/pr system. Thus, the alloAg stimulation results paralleled what was seen for mitogen stimulation. The YE19.1.3 population of cells: The reduced ability of Ipr mice NWT to respond to conA may have been due to a T cell defect or an A P C defect. Many have suggested that the phenomenon is due to the outgrowth of an odd Ipr Tce l l (18,47,52,132). Studies in Ipr mice in the past used unseparated populations of cells. Due to the peculiar phenotype of these odd Ipr cells, many researchers have used different surface markers and their unique patterns of expression on these Ipr cells to enrich or purify them and subsequently study their function. Fortunately, we had access to the YE19.1.3 McAb, which we used for purification studies of the YE19.1.3+ 2 8 Figure 2: MLR to alloaAg presentation: NWT [1 x10 6 ] from the strains indicated were cultured in the presence of irradiated stimulator spleen cells to assess the MLR response. Assays were established on day zero, labelled on day three and harvested on day four. Results are given as mean plus or minus standard deviation of triplicate cultures. Control values, without any stimulators, were below 1300 cpm. CBA, B6 and T mice were all two months old and 'O' mice were six months old. H-2K cells were all male and H-2B all female. 2 9 CPM Ipr cells. The YE19.1.3 McAb was originally isolated by its reactivity with the T cell lymphoma, EL4. The McAb binds to a surface molecule of approximate molecular weight of 230,000 under non-reducing conditions and 115,000 under reducing conditions. It was shown to have high reactivity for the DN Ipr cell (94). First, the reactivity of this McAb with NWT populations from different mouse strains was determined. NWT from CBA, MRL-+, MRL-/pr, B6 and B6-/pr mice were labelled indirectly with YE19.1.4 [first with YE19.1.3, then labelled with a second, FITC-conjugated, a-rat IgG] and analysed by FACS . The results are illustrated in figures 3a and 3b. First, all cells from non-autoimmune controls as well as the MRL-+ congenic strain showed a lack of reactivity with YE19.1.3. MRL-/pr and B6-/pr cells before the onset of disease also demonstrated low reactivity. Once older, significant reactivity patterns were seen for both Ipr mice NWT. These F A C S profiles clearly revealed that the surface phenotype of lymphocytes from NWT in Ipr mice was different from congenic and H-2 compatible mouse strains in that they bear a marker recognized by the YE19.3.1 McAb. The age study demonstrated that the proportion of cells bearing this marker increased with age in the Ipr strains. Analysis of conA responsiveness: Purification and enrichment of  YE19.1.3+ cells: The YE19.1.3 McAb allowed us a means of investigating the suggestion that the outgrowth of the odd population was responsible for the decrease in responsiveness seen to mitogens and alloAgs (18,47,52,137). To study this issue, we asked whether enriched YE19.1.3+ 31 Figure 3a: YEW. 1.3 surface Ag expression determined by FACS analysis. H-2K mice. 1 x 1 0 6 NWT from the mice indicated were stained indirectly with YE19.1.3 McAb and analysed on the FACS . Fluorescent gains were the same for all samples analysed. Fluorescence intensity and relative cell number were in log scale. 1 x10 4 cells were analysed with red cells and cellular debris gated out. Controls were cells labelled with the secondary Ab alone. Numbers are the total percent of cells stained with the YE19.1.3 McAb. The age of the mice were: C B A five weeks, MRL-+ one year, Y MRL-/pr four weeks and O MRL-/pr four months. All mice were female. 3 2 control YE19.1.3 fluorescence intensity 33 Figure 3b: YE 19.1.3 surface Ag expression determined by FACS analysis. H-2B mice. 1 x 1 0 6 NWT from the mice indicated were stained indirectly with the YE19.1.3 McAb and analysed by F A C S as stated for Figure 3a. B6 and Y B6-/pr mice were two and a half to three months old and O B6-/pr mice were five months old. All mice were female. 3 4 B6 Y B6-lpr O B6-lpr control YE19.1.3 A V. I V Au. 8 0 fluorescence intensity or YE19.1.3- cells were capable of responding in the proliferation [conA] assay. To separate these populations we used the YE19.1.3 McAb and FACS , indirect panning or cytotoxicity. We used NWT enriched spleen cells as our starting population; we reasoned that it would be difficult to recover enough 'normal' cells from the LN from older mice due to the magnitude of odd Ipr cells infiltrating those organs with age. FACS: NWT from Ipr mice were indirectly labelled with YE19.T.3 and cells were separated on the basis of bright or dull fluorescence. The two fractions of cells were collected and assayed for responsiveness to conA. To ensure that A P C s were not a limiting factor, irradiated, a-Thy 1.2 + c' treated P E C s from H-2 compatible, non-autoimmune mice were added at an optimal dose [previously determined] to some cultures. In figures 4a and 4b we see that the YE19.1.3+ cells from MRL-/pr and B6-/pr mice were incapable of proliferating under these conditions, whereas the YE19.1.3- cells could. Due to the increase in response seen when A P C s were added, A P C s were limiting in the F A C S purified populations of cells. Interestingly, the B6-/pr mouse was at an age well into disease [six months] and its YE19.1.3- 'normal' cells were unable to respond well [marginally better than the unseparated population of NWT]. Exogenous A P C s from normal B6 mice were added to the cultures and only a minor increase in response was seen. The MRL-/pr mouse was at the age of disease onset [two months old] and its 'normal' YE19.1.3- cells showed a very good response to conA [improved by C B A PECs] . Perhaps an age related defect is present in the 'normal' population of splenic T cells as well. This observation is further supported by the results shown in figure 5, where the reverse situation existed: O MRL-/pr 'normal' cells [NAd] 3 6 Figure 4: Proliferative response to conA of the YE 19.1.3+ and YEW. 1.3-populations of cells isolated by FACS from a) MRL-lpr mice, b) B6-lpr mice. 5 x 1 0 7 NWT were labelled indirectly with the YE19.1.3 McAb and sorted by F A C S at a gate setting to obtain greater than or equal to ninety-five percent purity of sorted cells. YE19.1.3+ and - cells were collected separately and 2.5x10 5 were assayed for their ability to proliferate to conA as outlined in figure 1. a) P E C s [2.5x10 4] from two month old C B A mice were treated to remove T cells and irradiated. Control cultures, where no conA was added, were all less than 600 cpm. Two month old male MRL-lpr mice were used, b) Controls [without conA] were all less than 300 cpm. Irradiated and a-Thy1 and c' treated P E C s added were from three month old B6 mice. Six month old B6-/pr mice were used. The results are given as mean plus or minus standard deviation of triplicate cultures. 3 7 Z D . • -1 QC YE19.1.3 + unseparated 10000 20000 +ConA+CBA PECs +ConA 30000 ->—I—*-40000 50000 -"—'—I 6 0 0 0 0 CPM CPM 38 responded marginally better than the unseparated population of MRL-/pr NWT and Y B6-/pr 'normal 'T cells [Ad] respondeded very well to conA. Panning: Because the Ipr cells have clearly been shown not to express L3T4 and Lyt 2, we decided to obtain our populations by an easier, less time-consuming and inexpensive method. NWT were incubated with GK1.5 and a-Lyt 2 McAbs. After washing, they were panned on 60 mm petri dishes precoated with a-rat Ig to remove the cells bearing those markers. The NAd [YE19.1.3+ enriched] versus the Ad [YE19.1.3- enriched] cells were analyzed for their ability to respond to conA as was done for the F A C S purified cells. The results seen in figure 5a and 5b were similar to what was seen for the F A C S isolation in figures 4a and 4b. NAd populations responded somewhat better than YE19.1.3+ cells isolated by F A C S and this may reflect two possibilities: either the YE19.1.3+ cells were not equal to the DN populations isolated by panning or the DN cells were contaminated by 'normal' cells. Exogenous A P C s were not added to the cultures and did not appear to be limiting when considering the B6-/pr Ad population response. They may, however, have been limiting in the NAd populations. If so, as demonstrated in figures 4a and 4b when P E C s were added to the cultures, the predicted response for the NAd, YE19.1.3+ enriched, populations would be a response that would only approach that seen for the unseparated populations of NWT. The efficiency of the panning procedure was assessed by F A C S and clearly an enrichment for the YE19.1.3+ population was demonstrated [data not shown]. Cytotoxicity: The treatment of NWT with McAbs to the surface determinants CD4 and CD8 and then with c' allowed for selective 3 9 Figure 5: Panning and cytoxicity to enrich for YEW. 1.3+ cells. Analysis of proliferation to conA by cells from a) MRL-lpr mice b) B6-lprmice. For panning, 1 x10 8 NWT, labelled with GK1.5 and a-Lyt2, were gently layered onto dishes precoated with cc-rat Ig. After appropriate incubations, NAd cells were layered gently onto new precoated dishes similarly two more times. The final NAd population [YE19.1.3+ enriched] and the first Ad population [YE19.1.3- enriched] were used in the proliferation assay. For cytotoxicity, NWT were treated with GK1.5 and a-Lyt2 plus c\ This procedure only enriched for the YE19.1.3+ cells. For the assay, 5 x 1 0 5 NWT were cultured in the presence of conA to determine proliferation as outlined for figure 1. a) Controls [without conA] were all less than 1000 cpm. Male MRL- Ipr mice were nine months old. b) Controls [without conA] were all less than 1000 cpm. Female BS-lpr mice were four and one half months old. The results are given as mean plus or minus standard deviation of triplicate cultures. 4 0 0 1 1 ' 1 ' 1— 2000 4000 6000 - 1 1 1 8000 10000 41 enrichment of the DN Ipr and YE19.13+ cells. The results seen after this treatment (figures 5a and 5b) further reinforced our initial observations seen with the panning data (also in figures 5a and 5b): DN cells from MRL-/pr or B6-/pr mice were unable to respond to conA very well. From the enrichment studies, then, it is clear that YE19.1.3+ cells or DN cells were unable to respond to mitogen even in the presence of normal A P C s and the 'normal' non-/pr T cell demonstrated an age related decrease in responsiveness to conA. Since these two observations seen were true for both MRL-/pr and B6-/pr NWT, this indicates that the properties related to the Ipr DN and 'normal' T cells seen are associated with the expression of the Ipr gene and are not due to induced S L E genes as B6-/pr mice do not have these genes. Thymus involvement: Chimera studies: Numerous studies have shown the thymus influence and T cell dependence influencing lymphoproliferative disease by the Ipr gene, particularly in MRL-/pr mice (63,84-86). By crossing the Ipr gene onto normal mouse strains, the effects of the Ipr gene in the absence of S L E background genes have been determined and have shown that Ipr causes not only lymphadenopathy, but also autoAb production (58,59,63,64); however, autoAbs in other Ipr mice remain primarily of the IgM class, never approach the extremes seen in MRL-/pr mice, and do not cause IC glomerulonephritis. Thus, S L E background genes are important in the pathology of disease and the effects caused by the S LE genes are greatly enhanced by the Ipr gene. To question the role of the thymus in the ontogeny of YE19.1.3+ and -4 2 populations of T cells, chimeras were created under the conditions where there were no extrathymic or thymic influences by S L E background or Ipr genes. The influence a normal B6 thymus would have on the development and function of DN Ipr cells was investigated; control B6-lpr ->B6-/pr and experimental B6-/pr ->B6 chimeras were established. Chimeras at greater than three months of age were sacrificed, NWT were prepared and the cells were analysed by indirect F A C S for the proportion of YE19.1.3+ cells. The F A C S profiles in figure 6 show that the number of YE19.1.3+ cells in the ->B6-/pr chimera was very similar to the B6-/pr control mouse in figure 3b. In the ->B6 chimera, very few YE19.1.3+ cells were present. Thus, it appears that Ipr cells required a Ipr environment to develop in. In addition, as was done for both the MRL-/pr and B6-/pr strains [by F A C S or panning], cell populations were isolated and tested for their ability to proliferate in response to conA. In figure 7a, the ->B6-/pr chimera unseparated NWT responded similarly to the age matched B6-/pr mouse NWT (3,840 +/- 703 cpm compared to 5,394 +/- 633 cpm). Separated subsets again showed that the YE19.1.3+ cells from ->B6-/pr mice could not respond to conA and the YE19.1.3-cells proliferated marginally better than the unseparated population. In the ->B6 mouse, the unseparated NWT responded almost as well as an age matched B6 mouse NWT (16,911+/- 4,554 cpm compared to 25,510 +/-348 cpm), and much better than the ->B6-/pr NWT. However, when separated into individual subsets, neither of these populations could respond very well. Because no A P C s were added, to exclude the possibility that they were limiting by being gated out during the F A C S procedure, no conclusive statements can be said about the response to conA by the 4 3 Figure 6: YEW. 1.3 surface Ag expression determined by FACS analysis: Chimeras. 1 x10^ NWT from the mice indicated were stained indirectly with the YE19.1.3 McAb, and analysed on the FACS as outlined for figure 3a. B6 mice were three months old, chimeras were seven and a half months old. 44 fluorescence Intensity Figure 7: Proliferative response to conA of the YE 19.1.3+ and YE 19.1.3-populations of cells isolated from ->B6lprand ->B6 chimeric mice by a) FACS, b) panning, a) FACS . Cells were prepared and collected as described for figure 4 and 2 .5x10 5 were assayed for their ability to proliferate to conA as outlined in figure 1. Controls [without conA] were all less than 300 cpm. Chimeras were three and one half to four months old. The proliferation of control mice of the same age were: B6, 25,510+/-348 cpm and B6-/pr, 5,394+/-633 cpm. The total percent of YE19.1.3+cel ls were: ->B6-/pr, seventy-nine; ->B6, six. b) Panning. Cel ls were prepared as outlined for figure 5. For the assay, 5x10^ NWT were cultured in the presence of conA to determine proliferation as outlined for figure 1. Controls [without conA] were all less than 2,000 cpm. Chimeras were seven and a half months old. Three month old control mice gave the following proliferative responses: B6, 28.093+/-927 cpm and B6-/pr, 19,007+/-2,135 cpm, and five month old B6-/pr, 7,673+/-1 ,051 cpm. The asterisk denotes no data for the untreated ->B6 NWT. There weren't enough cells to measure the response of 5 x 1 0 5 NWT, however, 1 x10 5 NWT gave a response of 6,659 cpm. The total percent of YE19.1.3+ cells was: ->B6-/pr, fifty and ->B6, one. The results are given as mean plus or minus standard deviation of triplicate cultures. 4 6 YE19.1.3- cells from this experiment. The total number of YE19.1.3+ cells in this ->B6 chimera was around six percent and that for the ->B6-/pr chimera was seventy-nine percent. It was reasoned, as APC studies were being performed at the same time (chapter two), that since B6-lpr mice failed to demonstrate an A P C defect, any response seen would not be influenced by the APCs present. Thus, at first, the lack of response seen by the YE19.1.3- cells in the ->B6 mice seemed somewhat peculiar. When panning was done subsequently (figure 7b), however, it became clear that APCs were most likely limiting in the FACS purified populations of cells from the chimeric mice. In figure 7b, the chimeras were seven and a half months old. The ->B6-/pr chimera unseparated NWT and Ad [YE19.1.3- enriched] cells responses were similar to those seen for the FACS separation of cells in figure 7a, both being slightly less than the response seen for an O B6-/pr control mouse (7,673 +/-1,051 cpm). The NAd [YE19.1.3+ enriched] cells from both chimeras seemed to mount a minor response, somewhat more than what was seen for the FACS separated cells in figure 7a. However, this response failed to exceed the response seen for the unseparated ->B6-/pr NWT, and the same phenomenon was also seen when panning was done on both MRL-/pr and B6-/pr mice (figures 5a and 5b). This, again, probably reflects slightly different populations being isolated by the FACS and panning procedures. FACS gives highly purified populations of cells. Since the responses seen for cytotoxicity and panning were the same (figure 5), the marginal increased proliferation in panning most likely reflects the functional capacity of different YE19.1.3- DN T cells. The most striking feature of this experiment is the response seen from the 48 ->B6 chimera Ad [YE19.1.3- enriched] cells; it was the same as the response seen for control B6 NWT (29,753 +/- 692 cpm compared to 28,093 +/- 927 cpm). What this response implies is that, one, in the F A C S experiment (figure 7a), A P C s were limiting and ,two, that the potentially age-related response of 'normal' Ipr cells in MRL-lpr and B6-/pr mice is associated with the Ipr environment or is secondary to the development of other primary Ipr gene cellular defects. The percentage of YE19.1.3+ cells in the ->B6 mouse was one and ->B6-/pr was fifty. Eventhough the Ipr gene confers the potential to influence T cell development, under the influence of a normal environment to develop, not only the thymus but everywhere, this potential remains largely inert. The few YE19.1.3+ cells that do develop maintain the characteristics of MRL-lpr and B6-/pr YE19.1.3+ cells, thus nothing can change the state of the cells. The 'normal' cells from the Ipr host appeared to grow and function normally in the normal B6 host, which suggests that the age related decrease in function seen with 'normal' Ipr cells in MRL-lpr and B6-/pr mice is secondary to some other defect which cannot be corrected by normal APC s . 4 9 C H A P T E R O N E DISCUSSION Confirmation of published data: The commencement of studies on the T cell compartment in MRL-lpr mice was inspired by the observation, made by ourselves (figs 1 and 2,45,130,131) and many, that MRL-lpr spleen and LN cells show an age-related hyporesponsiveness to mitogen (15,44) and alloAg activation (15,26,44). These studies were performed initially with MRL-/pr mice and subsequently with B6-/pr mice, and stood as the groundwork for the commencement of further studies. Our results showed that Ipr mice cannot mount as good responses as non-autoimmune, H-2 compatible, age-matched strains of mice and that this decreased responsiveness worsens with age [agreeing with published data]. Secondly, F A C S analyses using the YE19.1.3 McAb clearly demonstrated that Ipr cells exclusively bind the YE19.1.3 McAb and that the number of cells bearing this marker increases with age [concomitant with mitogen and alloAg hyporesponsiveness]. These findings were extended to the B6-/pr strain. The diminishing responsiveness seen in Ipr mice (figures 1 and 2) and the increase in YE19.1.3+ cells (figures 3a and 3b) promoted the suggestion that a causal relationship existed. In other words, many have argued that there must be something wrong with Ipr DN T cells, and as their numbers augment in the periphery, they dilute out the 'good' or responsive cells, hence reducing the overall responsiveness seen (18,47,52,132). Indeed, limiting dilution analyses on the precursor frequency of T cell subsets have revealed that the absolute numbers of certain functional T cells are in a normal range, but their frequency is 5 0 largely reduced (18,47). In addition, DN cells freshly isolated from Ipr mice have been shown to be functionally inert (67,133-135); however, studies such as this, on whole populations of cells, fail to reveal the specific subsets of cells responsible for the phenomena seen and it has been shown that normal cells are contained in the populations of Ipr DN cells (126). With the rapid advancement in studies performed on pure populations of subsets of cells, sometimes delineating specific functions for these cells, it is now apparent that drawing decisive conclusions requires extensive purification procedures. This issue was pointed out by comparing the responses seen by YE19.1.3+ cells purified by F A C S (figures 4a, 4b, and 7a) to those seen by the NAd, YE19.1.3+ enriched, cells obtained by panning (figures 5a, 5b and 7b) and DN, YE19.1.3+ enriched, cells selected for by cytotoxicity (figures 5a and 5b). F A C S purified YE19.1.3+ cells demonstrated a complete lack of response; enriched cells, by panning or cytotoxicity responded minimally, but significantly more that the F A C S isolated cells. Our purified populations of YE19.1.3+ cells clearly were unable to proliferate to mitogen activation. This is not surprising in light of recent evidence on purified populations of DN Ipr cells. Many characteristics of DN lymphocytes from Ipr mice would compare them to immature thymocytes. Phenotypically, their lack of II2R expression (20,133), low expression of the TcR (96,98,124,126), expression of pre-B and B cell markers (70,71,92,107,109,110,113) and elevated levels of c-myb (73,74,120,121) are characteristic of immature T cells (123-126,136, 137,139). Their lack of ThB (92) would suggest that they have progressed beyond a recent immigrant to the thymus, very immature, and their lack of 51 CD4 (93) and CD8 (91,92,94), CD4+ or CD8+ T cells representing the majority of mature T cells, would suggest that they still have a long way in development to go. That they are unable to respond to mitogen activation [that is, via proliferation, II2R expression and II2 production (133) ] or to stimulation by Abs to surface molecules which mature T cells can be activated by [for example, by the TcR, CD3, CD2, Thy1 or Ly6.2 (134) ] would further support their immmature nature (140). In fact, due to the expression of B220, lack of II2R and low TcR, this suggests that the Ipr cell is blocked at a stage of development prior to MHC restriction and tolerance induction in thymus ontogeny, as studies using in vitro thymus organ culture in the presence of a-TcR Ab showed that mature T cells failed to develop, but there was no effect on the existence of immature thymocytes expressing a low level of the TcR (137). As activation is required for immature T cells to progress in development, it becomes important to consider that Roth, et al, (141) state that the binding of ligand to its receptor may be the first step in the activation of cells. If there is an abnormality in the TcR, for example because of decreased number or perhaps a lowered affinity for ligand due to some structural mutation, this may be the stop for further progression of signal transduction and thymus selection [MHC restriction/ tolerance induction]. An altered form of the TcR has been isolated from DN Ipr T cells (99). Furthermore, changes in cell surface carbohydrates during ontogeny (142-144) have been revealed and deemed important for specific interactions such as intercellular contacts or as receptors for growth factors. MRL-/pr mice display many alterations in cell surface carbohydrates [as explained in the introduction to chapter one] which would further imply a reason for an arrest in development. However, 5 2 further studies comparing DN thymocyte and mature DN cell surface carbohydrate composition to that seen on Ipr DN cells are required before any conclusions can be drawn about their surfaces. Another possibility alluded to by Sprent et al (145) is that during T ontogeny, many cells enter the thymus, a large number are produced, a few leave, yet one never sees necrosis in sections made of the thymus gland. This led Sprent to suggest that T cells use the program for self-destruction called "apoptosis" [that all cells have] which allows for their elimination in the thymus, perhaps via soluble mediators. If Ipr cells had a dysfunctional program due to the action of the Ipr gene, this would be a mechanism whereby they would evade elimination in the thymus, should they be end cells. Alternatively, if this program for self-destruction could only be activated during a certain stage of development, for example requiring certain surface markers to be expressed, and these Ipr cells haven't differentiated to this state, are stuck in an immature state prior to this time as suggested above, then this could be a mechanism how such cells fail to be eliminated. That some of these DN Ipr T cells can be triggered to mature by the use of specific agents (114,146), or by culture (45,72,132) suggests that at least some of these are not destined to die, but, rather, are arrested in development for whatever reason. Paradoxically, the DN Ipr cells appear to be stuck in an activated state. The protein p21 of the TcR/CD3 complex, is constitutively phosphorylated on a tyrosine residue, an event normally only induced upon T cell activation (100). They express an elevated level of c-myc (122), characteristic of active, proliferating cells (127); they express Ly24 5 3 [Pgp1], a surface molecule induced on mature T cells upon activation (102-105). They cannot be activated via the Thy1 or Ly6.2 alternative pathways of activation, nor by stimulation through the TcR/CD3 complex (134) . They express high levels of potassium channels (138), normally seen only in activated T cells (147). However, these potassium channels are different from the type induced upon and required for activation (119). Numerous other characteristics would also suggest that they are not fully activated yet: they express Mel14, which is normally decreased upon activation (101); they are in the Go-G1 phase of the cell cycle (83,114) and they express neither the II2R or the TR (20,94), characteristic of proliferating cells. Only if conventional activation mechanisms are bypassed, such as by the use of calcium ionophore and phorbol ester, can one get freshly isolated DN Ipr T cells to respond (67,133). The highly increased concentration of ionophore required suggests that perhaps defects in the calcium side of the activation pathway are disrupted. Phorbol ester activation of protein kinase C was shown to be normal (134), but the formation of inositol phosphates by these cells has been shown to be defective (148). Also MRL-lpr DN T cells demonstrated much greater activity of TcR modification and turnover than that seen for MRL-+ T cells (135) suggesting a defect in TcR mediated signal transduction required for activation, other than protein kinase C. These defects could provide a mechanism by which the DN Ipr T cells are arrested in development during ontogeny and suggest that they have been activated to differentiate, but cannot due to defects in intracellular mechanisms. That some cells can develop in culture (45,72,114,132,146) would confirm the heterogeneity 5 4 of t hese ce l l s . That s o m e that do deve l op e xp re s s the C D 8 A g and ga in cytolyt ic activity wou ld prov ide ev i dence for more 'mature', l i neage d i rec ted ce l l s . Cu l ture of immature D N ce l l s i so lated f rom adult t hymus in the p r e sence of c o n A supernatant over t ime a l l ows for the deve l opmen t of Me l14- , B220- , Ly24+, Ly1 dul l , II2 R- D N ce l l s . T h e s e ce l l s largely e xp r e s s autoreact iv i ty and w h e n st imulated exp re s s the C D 4 A g a n d p roduce II2 a n d II3 (A. Och i , persona l commun ica t ion) . S u c h ce l l s prov ide ev i dence for s o m e pe rhaps normal maturat ional p r o ce s se s , not s e e n in the he te rogenous populat ion of Ipr D N T ce l ls , a nd further suppor t the idea that Ipr D N T ce l l s a re on the deve lopmenta l pathway, but s tuck in the p rocess . Further s tud ies a re requi red to determine where , or into what deve lopmenta l pa thway the YE19 .1 .3+ subpopu la t ion of D N Ipr T ce l l s wou ld fit in the s c h e m e of T ce l l ontogeny. It may be usefu l to look at these ce l l s at the mo lecu la r level [transcripts a n d genes ] to de l ineate spec i f i c deta i ls a nd to do protein ana l y s e s on the Ab- i so lab le ce l l sur face proteins, for e xamp l e with respec t to ca rbohydra te moiet ies. Mo re importantly, c ompa r i s on s to s imi lar popu la t ions of ce l l s f rom normal mice, if poss ib le , a re necessa ry . A l l s tud ies thus far have c ompa r ed the Ipr D N T ce l l s to D N thymocy tes rather than to per iphera l D N T ce l l s . Recen t s tud ies in our laboratory sugges t that YE19 .1 . 3+ D N T ce l l s a re a very minor c omponen t of mature T ce l l s in the sp l een . It will be interest ing to study these ce l l s further [if enough , in numbers , c a n be obtained] and c ompa r e t hem to those present in d i s e a s e d mice. B reak ing new a round: W h e n w e move on and regard the abil ity of the 5 5 cells not bearing the YE19.1.3 marker, we see better reactivity than the unseparated population of NWT cells (figures 4a, 4b, 5a, and 5b). However, the proliferative responses never approach those seen in non-autoimmune H-2 compatible strains, and appear to demonstrate an age related decrease in responsiveness too, suggesting a defect may exist even in the 'normal' Ipr populations of T cells as well. Although this was not studied further, much recent evidence on Ipr mice, and humans with SLE , supports this notion: i) F A C S analyses readily demonstrate a change in the overall T cell populations present in the lymphoid organs with age, with the percentage of Lyt2+ cells being greatly reduced and the L3T4+ increased [data not shown] (13). ii) Miescher et al (98) demonstrated that fresh Ipr mature T cells [single positives] display a low density of the TcR. iii) Santoro et al (9) demonstrated that the CD4+ T cells in Ipr mice were defective in their ability to produce II2 and II3 upon conA stimulation, at an age when the B220+ [Ipr DN T cell] cells were limiting. This was shown for B6-/pr mice as well. iv) In a different study (128), Santoro et al administered an <x-L3T4 Ab in vivo, which demonstrated that lymphoproliferation was heavily dependent on the CD4+ subset; peripheral lymphoid organs were close to normal weights and there was a great reduction in the total number of B220+ cells in addition to decreased levels of pathogenic [IgG class] autoabs. Studies performed in normal mice using the above type of protocol [a-L3T4 Ab in vivo] have shown that there is an increase in the percentage of DN cells in the spleen (150) and in PP (151). v) The CD4+ subset of cells has recently been shown to comprise 5 6 two mutually exclusive subsets of cells identified by the markers 2H4 and 4B4 in the human (152-161). 4B4+ cells are inducers of help and 2H4 cells are inducers of suppression. Studies in Humans with S L E have demonstrated that these populations of cells are in abnormal proportions (156,159). Experiments have suggested that cells with similar functions as the 2H4+ cells are necessary for the AMLR, and this is of interest as MRL-/pr mice demonstrate a diminished AMLR response (19,29,30,111). This issue is discussed further in chapter three. vi) Hefeneider et al with limiting dilution studies showed normal responsiveness and frequencies of several T cell populations, revealing that fully functional cells exist (47). However, limiting dilution analyses require the incubation and expansion of clones that add additional artifacts; first of all, the clones may not necessarily be representative of the cell populations on the whole seen in vivo and secondly, incubation frees the cells from in vivo effects that may govern a different cell reactivity in vivo than that seen in vitro (45,72,132). Therefore, an in vivo influence may not be readily assessed from these types of studies which, in this case, can only detect functional cells. On this note, I remark that in vitro work often gives misleading results and must, therefore, be interpretted with caution. vii) Ron et al (23) tried in vivo priming of LN T cells with adjuvant and found that this response was decreased and could not be restored in older MRL-/pr mice. This hints that there may be an abnormal CD4+ population or that this population may be suppressed, or, because of accessory cell requirement in T cell activation, that there may be a defect in an A P C . 5 7 What this is all leading to is that the odd, expanded population of T cells in Ipr mice may not necessarily be causative agents in disease, but, rather, may result from some other cellular abnormality. In fact, because Budd and others (126) have shown that a similar counterpart to this cell in Ipr mice can be isolated from normal mice, it seems that this cell population doesn't warrant all of the credit for the malfunctions seen in Ipr mice. Instead, it appears that these cells develop as the result of other primary cellular defects. Chimeras: The thymus gland provides a special ized microenvironment for the education/development of functional T cells (3). In Ipr mice, the role of the thymus in the pathogenesis of disease has been dissected by numerous means. Specifically, thymus involvement in Ipr disease has been shown by the following few examples: 1) only neonatal Tx of Ipr mice prevents disease (84,85) and 2) reconstitution even with a normal thymus restores disease (86); 3) B6-nu-lpr mice do not demonstrate Ipr disease until grafted with a thymus (63,87); 4) in vivo administration of a-Thy1 or a-L3T4 Ab prevents lymphoprolifer-ation and accelerated disease (88,128); and 5) Ipr cells can be detected [phenotypically] in the thymus, and may increase in that gland with age (76). That only neonatal Tx abrogates disease emphasizes the necessity of early establishment of the T cell repertoire in the disease process. This also suggests that extrathymic mechanisms for the generation of T cells may be functional in Ipr mice because once T cells are established in the periphery, Ipr d isease cannot be changed by Tx. This would also be 5 8 supported by the fact that a spleen graft can also induce disease in neonatally thymectomized animals (90). Conversely, at restricted times splenectomy can improve disease. That a normal thymus can restore disease suggests that the thymic epithelium is not involved in the generation of the Ipr DN T cells. Since B6-nu-lpr mice can develop disease when grafted with a normal thymus, this similarly demonstrates that the Ipr gene expression is not required per se in the thymus gland constituents [thymic epithelium]. Because regiments of Ab treatment not specific for the Ipr T cell can abrogate disease, they suggest other cellular malfunctions exist [for example in CD4+ cells as discussed earlier] as primary causes in disease development. None of these studies excluded the possibility of other extrathymic Ipr influences in the development of Ipr DN T cells as the Ipr gene was present in aJl cells of the body of those mice. In order to determine what role thymus education and extrathymic involvement there is in the development of the YE19.1.3+ cell and this cell's functional capacity, we created B6-/pr->B6 chimeras by conventional means and examined what developed. To have a better understanding of chimeras, there are a few points which must be clarified. After one lethal dose of WBI there is rapid depletion of lymphoid and bm cells in a mouse (163,164). The thymus epithelium and non-gland constitution is only transiently affected (165); recovery is very rapid after bm reconstitution (166). Mos and DCs are seen very early post irradiation (145). Repopulation of the thymus by lymphoid cells is a biphasic phenomenon; at five to seven days, the thymus is repopulated by radioresistant host T cells; by day fourteen, these radioresistant T cells have been replaced by prothymocytes from 59 donor bm (163,164,166-168). From this point, as is seen normally in unmanipulated mice, there is continual low-level inflow of precursors into the thymus of extrathymic [largely bm] origin (169,170,171), as thymic precursors have limited self-renewal capacity (171). In addition, it was determined by others when +/+ ->lpr chimeras were made, they demonstrated less than ten percent repopulation by residual host bm at seven months post- transplantation after lethal irradiation and reconstitution, emphasizing the efficiency of the procedure (66). Before discussing the cellular results seen in the chimeric mice, several observations must be mentioned. First, five to ten percent of the lethally irradiated mice died, even though they were reconstituted with bm. These were of both ->B6 and ->B6-/pr types. This indicates that in fact, lethal irradiation had been performed, but for some reason, reconstitution failed in a minor population of mice. Second, lymphoproliferation in the control ->B6-/pr mice was similar to that seen in unmanipulated B6-/pr mice, but was not seen in the ->B6 mice; spleens of ->B6 mice contained approximately 5 x 1 0 7 cells, similar to unmanipulated B6 mice. Spleens of chimeric ->B6 mice were also granular, unlike those of B6 mice. In a rare few ->B6 mice, the lymphoid organs, for some reason, were completely expanded with the YE19.1.3+ DN T cell population. Third, the lifespan of ->B6 mice was greatly extended beyond that of ->B6-/pr mice; at twelve months post-reconstitution, few [five percent] ->B6-/pr mice remained, whereas all ->B6 mice were still alive. The lymphoproliferation seen in ->B6-/pr (figure 6) mice paralleled 6 0 that seen for B6-/pr mice (figure 3b) and so did the reactivity of the YE19.1.3+ cells (figures 7a and 7b compared to figures 4b and 5b), indicating a successful positive control. F A C S analysis on the ->B6 chimeras revealed the expansion of the YE19.1.3+ cells in the NWT to be maximally twelve percent of the total number of cells [this number being fifty percent or better for the B6-/pr or ->B6-/pr chimera]. If, the Ipr gene were unable to operate in a normal host unless all cells of the body contained this gene [and therefore its potential capacity to be expressed in any cell population], one would expect not to see the development of this population of cells by simply transfering bm into a syngeneic host lacking this gene. Because some YE19.1.3+ cells did arise, the Ipr gene can act in the hematopoietic precursor population, but not nearly as well as when the whole body has this gene. The limited expansion of the YE19.1.3+ cells suggests that the Ipr gene operates at some other hematopoietic cell level, whose development is greatly retarded in a normal host. The reduced production of the 'other' cells allows for the expansion of only a few YE19.1.3+ cells, because if all that were required for lymphoproliferation were the endogenous expression of the Ipr gene, then one would expect to see massive lymphadenopathy. Thus adequate 'turn on' signalling by whatever means, is lacking when the bm cells are the only ones containing the Ipr gene. More importantly, when stimulated by mitogen, the YE19.1.3+ cells isolated from ->B6 chimeras were still unable to respond (figure 7a), like all other Ipr YE19.1.3+ cells (figures 4a and 4b), which provides evidence for a precursor defect. Since the Ad [YE19.1.3- enriched] cells panned from the ->B6 chimeras (figure 7b) were able to respond well, this provides further evidence that the defect seen in the 'normal' T cells is also related to other primary cell with Ipr gene 61 expression and is learned in ontogeny in an Ipr environment; perhaps it is due to class II positive accessory cells in the thymus, since a normal thymus grafted in neonatally thymectomized MRL-/pr animals allowed for Ipr disease expression, but a normal thymus could not cause Ipr disease in its own host environment. To this end, unusual stromal cells have been detected in the bm of Ipr mice (82) and are sites of active lymphopoiesis. It would follow then, that the Ipr environment would be required for this unique stromal cell to develop, with other genes modifying the degree of rapidity with which this cell develops; S LE background genes in the combination with the Ipr gene cause very accelerated disease, B6 background genes allow slower progression of disease and so on in other strains with the Ipr gene (3, 58-62). Since YE19.1.3+ DN cells have been isolated from the periphery of normal mice and constitute a very small population (126, ourselves, unpublished observations), they probably would have gone unnoticed without the aid of the abnormal disease state induced by Ipr. Further studies should concentrate on comparisons of this population of cells seen in normal, MRL-/pr, B6-/pr, and chimera mice. Some chimera studies in both MRL-/pr and B6-/pr mice have shown that transfer of Ipr bm into +/+ mice caused a GvH-like disease with severe wasting within three to five months, even if mature T cells had been removed from the bm. The converse, +/+ bone marrow into Ipr recipients, led to prolonged survival of the mice and suppression of the lymphoproliferation. The investigators concluded that lymphoid precursors are inherently abnormal and that non-lymphoid environmental factors had little influence on disease state (3). More recent studies, similar to ours, support our results in that no GvH like disease developed 6 2 (80). Prior results may have been due to the use of mice that were not completely congenic. In summary, the observation that Ipr mice elicit a decreased ability to respond to mitogen or alloAg activation was initially investigated. Coincident with the age-dependent hyporesponsiveness, an increase in the number of cells bearing the tumour-specific marker recognized by the YE19.1.3 McAb was demonstrated. These YE19.1.3+ cells were separated from cells not bearing the marker, and those bearing the marker were unable to respond to conA activation. The YE19.1.3- cells could respond to conA, but demonstrated an age related decrease in responsiveness. These results were demonstrated in both MRL-/pr and B6-/pr mice. Lastly, the involvement of thymus education in the development of lymphadenopathy was investigated by making B6-/pr->B6-/pr and ->B6 chimeras. The percent of YE19.1.3+ cells and mitogen reactivity of these cells and the YE19.1.3- cells were comparable to B6-/pr controls in ->B6-/pr mice; the percent of YE19.1.3+ cells for ->B6 mice was greatly reduced but mitogen responsiveness had not changed for these cells, hence the cells could not respond, demonstrating an intrinsic defect in YE19.1.3 cells that cannot be overcome by education in a normal thymus. No age related hyporesponsiveness to conA was seen for the YE19.1.3- cells from ->B6 mice. 6 3 C H A P T E R T W O I N T R O D U C T I O N A P C s a re fundamenta l to the initiation and deve l opmen t of a ce l lu lar immune re sponse . The funct ion of the A P C is to exp re s s A g on its su r face in the context of the appropr iate M H C mo lecu le to spec i f i c T ce l l s in order for them to mount a ce l lu lar immune response . Spec i f i c ce l l s represent ing su ch A P C s inc lude mononuc l ea r phagocy tes [including mos] , ep ide rma l l angerhans ce l l s a nd Kupffer ce l l s [t issue mas], non-phagocyt i c D C s and B ce l l s . In this chapter , w e shal l concent ra te on the mo and the D C A P C s . T h e ear l iest and most we l l - charac ter i zed A P C s we re the mononuc l ea r phagocy tes , in the mature state known a s m o s . M o s der ive f rom the hematopo ie t i c bm whe re they deve lop into immature monocy te s and s oon after, enter the c i rcu lat ion. They leave the c i rcu lat ion, migrate into t i s sues whe re they mature. W h e n they mature, they e xp re s s sur face mo lecu l e s and receptors, and deve lop spec i f i c characer i s t i c s by wh i ch they c a n be identif ied (172,173). M o s e xp re s s the immuno log ica l markers : F c R [for b ind ing Ig], C 3 R [for b ind ing c' c omponen t C3b] (174), C S F - 1 R [for b inding C S F - 1 ] (175) and c l a s s II M H C ant igens (176). A useful su r face marker, a s i de f rom the many that now have been recogn i zed on mos , s u ch a s M a d , 2, and 3, is the determinant recogn i zed by the M c A b F4/80 (177). T h e F4/80 determinant is found in h igh concentrat ion only on monocy tes and mos ; however , o n ce the mo ha s been act ivated, F4/80 exp ress i on de c r ea se s . A l so , s igni f icant d i f ferences in exp ress i on of this sur face protein have been found 64 depending on the type of intraperitoneal stimulation, culture conditions and state of maturation of the mo. The specificity and sensitivity of F4/80, compared with other surface markers mentioned, makes it a McAb of choice for mo studies (177-180). Due to the phagocytic nature of the mos and the role they play in host defense, they have been graced with an array of enzymes which have been particularly useful in their characterization. A few of these include lysozyme, peroxidase and 5' nucleotidase. A preferred enzyme used in mo study is non-specific esterase, as staining for it provides very reliable results. When the substrates a-napthyl acetate or butyrate are used, all monocytes and mos stain brightly throughout their cytoplasm. However, slight variations in overall reaction are seen depending upon the develop-mental stage, culture conditions and functional state of the mos being studied (172). The second type of A P C we studied was the non-phagocytic DC. Due to the many similar properties that mos and DCs share, it is believed that mos and DCs originate from a common monocytic precursor of bm origin. Although little is known about the kinetics and sites of DC production and movement between different organs, i.e. they have not been detected in fresh bm samples, evidence from rat bm culture studies show that DCs develop from precursors with properties not much different from those of mos (181), which lends support to the above hypothesis. DCs were first described as interdigitating reticulum cells, probably due to their appearance. DCs are low-density, irregularly shaped cells 6 5 with long dendritic processes [hence the name] and irregularly shaped nuclei. They constitute a very minor population in all lymphoid organs; in particular for the interest of this chapter, in the spleen, they are found at less than one percent (183). In addition to being non-phagocytic [thus lacking the appropriate organelles/ substructures /machinery (108,182)], they differ from mos also by lacking conventional mo markers: FcR, C3bR, M a d and F4/80. Not only are their surface markers different, DCs have a rapid turnover rate (184) and have a different in vivo distribution (185) from mos. They are steriod- and radio-sensitive, and like other hematopoeitic cells, they express T200 (184,186). They are strongly and constitutively (186) class II+ and, as a result are efficient at presenting Ag (183) and at stimulating MLRs (182,183,188,189). Numerous investigators have sought to determine dysfunctions in the A P C compartment of the immune response in MRL-/pr mice. The majority of earlier studies focussed on the mo's ability to process, degrade and present Ags and the ability to remove IC from serum as these were among the first defects seen in S LE patients (37,190). When we first look at Ag presentation, we see that a plethora of studies demonstrated that the P E C population has a greatly increased percentage and number of la+ mos in older MRL-/pr mice (3,36-39). Some reports indicated that la+ splenic adherent cells increased with age (40), but others stated they actually decreased in number with age (34). Even so, la+ premononuclear cells appear in the spleen of MRL-/pr and MRL-+ mice with the same frequency and absolute number (3). As for the actual number of la molecules on the surface of each cell, nothing has been determined. Theofilopolous and Dixon determined that the Ipr gene must 6 6 be in the homozygous form in MRL mice to see an increase of la+ mos with age and that neonatal Tx had no effect on this observation (3). Kelley and Roths were unable to demonstrate the same increase in la+ mos in P E C s of B6-/pr mice (38). Lu and Unanue were able to show that la+ mos could be induced by repeatedly injecting supernatants derived from non-mitogen stimulated MRL-/pr spleen cells [not LN cells] into normal mice; thus, they deduced that this phenomenon was caused by a MIRF-like substance [y-IFN] being secreted by MRL-/pr 'proliferating T cells (39). MRL-/pr mice have even been shown to express aberrant class II M H C Ags (42). For a T cell response to Ag to occur, not only must there be adequate presentation, but also sufficient second signal, 111. So, in addition to abnormalities in the la+ mo/APC compartment of MRL-/pr mice, 111 production has been studied at length. Results have indicated somewhat confusing results however; decreased 111 production by P E C mos of MRL-/pr mice compared to MRL-+ and H-2K controls (36,55), minimally decreased production not only by P E C mos, but also by two hour splenic adherent cells and normal levels of 111 production have all been reported (39). Thus, conclusive results concerning A P C ability to produce 111 are still lacking. When regarding the ability of mos in the reticuloendothelial system to remove undesirable materials, such as IC, investigators have found that there are reduced numbers of FcRs on MRL-/pr P E C mos. This defect accounts for the low binding and diminished phagocytosis of opsonized rbcs (32-34,207). In addition, these effects are seen in MRL-+ mice and are recognized before the clinical onset of disease in MRL-/pr mice, thus 6 7 suggest an inherent defect in these mos. The same effects have been seen with hepatic non-parenchymal cells in MRL-lpr mice (32,33). These studies parallel findings in patients with S L E as well (32,193). The MRL-lpr mouse strain demonstrably has a defect in the T cell compartment of the cellular immune response, which, with age, obscures other cellular abnormalities in the immune system (chapter one). Regardless, as mentioned above, numerous investigators have been able to describe defects associated with A P C populations. Aside from the la+ expression and 111 production, another suggestion has been that suppressor mos exist in the adherent population of A P C s which are responsible for the poor immune response seen (194). However, mixing experiments by others would disclaim this suggestion (40). Furthermore, Abs to mos may influence the proclaimed 'APC defects' seen (196,197). A s far as the DC component of the A P C compartment of the immune response, little work has been done in MRL-/pr mice. One study has reported that splenic MRL-/pr DCs are unable to function as well as the splenic MRL-+ DCs (198). All of these above mentioned studies allude to the existence of an A P C defect(s) in MRL-/pr mice. Earlier research done in this laboratory established a defect in MRL-/pr mos, a response that could be largely corrected by substituting normal, H-2 compatible A P C s for MRL-/pr A P C s (130). The research put forth in this chapter was designed to further explore this observation. The questions asked were: is there a quantitative defect [numbers of APCs , too many being suppressive], a 6 8 qualitative defect [in 111 production by mos] or a defect in a specific population of APCs , specifically in DCs? 69 CHAPTER TWO RESULTS APC function for proliferation in MRL-lpr mice: titrations of PEC  mos. Aside from the obvious T cell defect causing hyporesponsiveness to conA, we decided to explore the possibility that there may be a defect in an APC population. We had an indication that some defect might be occuring at the APC level in addition to the obvious T cell defect because in the MLR assays, we always saw that, with age, MRL-/pr spleen cells stimulated B6 responders much less than CBA spleen cells. However, what wasn't clear was whether this reduced response was due to some complex, in vivo influence, such as suppression or activation or simply due to a defective population of APCs. When a-Thy1 and c' treated PECs were used as stimulators in a MLR, no response was seen [data not shown]. Thus, a different approach to study the problem was necessary. Using a different method to assess a potential APC abnormality, a-Thy1 and c' treated PECs from normal and diseased animals were.added, in a dose response manner, to H-2 compatible, normal CBA mouse NWT in a conA response. PECs were treated to remove T cells to exclude the possibility that they were the reason for the observations made. In figure 2-1 a it can seen that: i) the normal NWT responded well at any dose of normal PECs added, ii) when high numbers of MRL-+ PECs were added, normal NWT were unable to respond as well as when fewer numbers of MRL-+ PECs were added, the latter responses being similar to the responses seen when normal CBA PECs were added, and iii) at high numbers, Y MRL-/pr PECs greatly reduced the ability of normal CBA NWT to respond to conA, an effect not easily overcome. O MRL-/pr PECs 70 Figure 2-1: APC function for proliferation: PEC titrations in a) MRL-lpr mice, b) B6-lpr mice. 5 x 10 5 C B A NWT were cultured in the presence of 1.25Lig conA/ml. Irradiated and a-Thy1 and c' treated P E C s from CBA, MRL-+ and MRL/pr mice were added at the ratios indicated. After forty-eight hours in culture, tritiated thymidine was added. After another eighteen hours, cultures were harvested and radioactivity incorporation was assessed. Negative controls [responders without conA and irradiated PECs] gave background counts less than 5000 cpm. C B A NWT cultured with conA alone gave a response of 124,710+/-2,296 cpm. All mice were three months old and male, b) 2 .5x10 5 B6 NWT were cultured with conA and B6 or B6-/pr P E C s [treated, at the ratios indicated] and assessed for proliferation under the same conditions described for (a). Negative control cultures were less than 500 cpm. B6 NWT cultured with 1.25 jig conA/ml gave counts of 38,148+/-2,586 cpm. B6 and Y B6-/pr mice were six weeks old, O B6-/pr was six months old. Results represent the mean plus or minus the standard deviation of triplicate cultures. 71 ratio C B A NWT : APC ratio B6 NWT : A P C reduced the proliferative response of C B A NWT even more than the Y MRL-/pr P E C s did [data not shown]. To establish what role the Ipr gene plays in the absence of background S L E genes in A P C function, the B6-/pr mouse strain was used for study in comparison to the normal B6 mouse strain. P E C titration experiments were performed with B6 NWT responders using oc-Thy1 and c' treated B6 and B6-/pr PECs . In figure 2-1 b, Y B6-lpr P E C s allowed for the same response as B6 PECs . Either of these two populations of P E C s added [at the doses tested] increased the responses over the B6 NWT plus conA control, except for at the very highest doses, where the response was inhibited. O B6-/pr P E C s allowed for an increased response over that seen when normal B6 P E C s or Y B6-/pr P E C s were used. Overall, then, it appears that MRL-+ A P C s demonstrated a reduced ability to help normal T cell respond to mitogen, an effect that was enhanced in MRL-/pr mice by the Ipr gene, whereas B6-/pr PECs , which contain only the Ipr gene, failed to show an inability to help, but, rather, demonstrated a marginally better ability to help when they were from older mice. A P C function in the MLR. To observe how well H-2K and H-2B Ipr cells could stimulate allogeneically, in figure 2-2, the results of a MLR with normal NWT responders stimulated with Ipr and normal irradiated spleen cells is shown. Compared to normal stimulators, MRL-+ and Y MRL-/pr spleen cells demonstrated a reduced ability to stimulate, B6 NWT being the responders. Y and O B6-/pr spleen cells seemed to 7 3 Figure 2-2: APC function in the MLR: Spleen cell stimulators. 1 x 1 0 6 B6 NWT were cultured in the presence of 1 x10 6 irradiated spleen cells from CBA, MRL-+ and MRL-/pr mice. Also, 1 x10 6 C B A NWT were cultured in the presence of 1x10^ irradiated spleen cells from B6 and B6-/pr mice. After three days in culture, tritiated thymidine was added and after four days, total, the incorporation of radioactivity was determined. Results represent the mean plus or minus the standard deviation of triplicate cultures. CBA, B6, MRL-+ and 'Y' mice were six weeks old, 'O' mice were five to six months old. 7 4 I f ) T T T T T 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0 1 2 0 0 0 1 4 0 0 0 1 6 0 0 0 1 8 0 0 0 2 0 0 0 0 CPM stimulate C B A NWT just as well as the B6 spleen cells did. Therefore, a similar A P C defect in alloAg stimulation duplicated the P E C A P C activity seen in the P E C titration experiments (figures 2-1 a and 2-1 b), a defect in MRL-+ made more severe by the Ipr gene, but the Ipr gene by itself failed to demonstrate an influence. Because MRL-lpr cells were inhibitory at high numbers in the response to conA (figure 2-1), this suggested that either the proportion of mes was much greater than other A P C s [as even the CBA, and B6, mos in high numbers were suppressive], they were defective at Ag presentation or lymphokine production or both, or a subset of important A P C s were defective. Each of these possibilities was explored further. Quantification of mos. Esterase staining. One approach that was taken to determine the number of mos present in the different mouse P E C populations was the use of the nonspecific esterase stain. P E C s were harvested from each mouse strain, treated with cc-Thy1 and c\ either cytospun or air-dried onto glass slides and then stained. The results in figure 2-3 demonstrate: i) that cells stained to varying degrees, making interpretation of the results somewhat difficult and very subjective, ii) that overall it may be noted that the total amount of 'red' cells seen in the Old MRL-lpr (a, #4) sample was much greater than that seen in the MRL-+ (a, #3), young (a, #3) or C B A (a, #1) cell samples, and iii) the amount of mos present in the B6 (b, #1) and BQ-lpr (b, #2-4) cell samples were approximately the same, suggesting that the effect seen when MRL-lpr P E C s are added to C B A NWT in the conA response (figure 2-1) may simply have been due too many mos being present and thus 7 6 Figure 2-3a: Quantification of mos in PECs by esterase staining: H-2K mice. PECs , harvested from CBA, B6, MRL-+ and Ipr mice were left to air dry on slides and subsequently stained for esterase activity. The magnification is two-hundred times. The age of the mice were as follows: 1) CBA, eight months; 2) MRL-+, six months; 3) MRL-/pr, three weeks; and 4) MRL-/pr, three months. 77 Figure 2-3b: Quantification of mes in PECs by esterase staining: H-2B mice. P E C s were prepared and stained as described for figure 2-3a. Magnification is two-hundred times. The age of the mice are: 1) B6, months; 2) and 3) B6-/pr, three months; and 4) B6-/pr, six and one half months. 7 9 suppressive. Therefore the reduced responses seen in Figure 2-1 may have been due to a numbers effect, however, this staining procedure would not be suitable for quantitating cells due to the inherent properties associated with it. F4/80 McAAb analysis. A second approach to assess the numbers of mos present in P E C populations was used. The McAb, F4/80 which reacts specifically with a determinant on monocytes and mos (177-180), was used with F A C S analysis. This determinant decreases in concentration on the surface on mos once they are activated (177). P E C s were harvested from the different animals, treated and labelled with F4/80 and analyzed by FACS . The results obtained, however, were inconclusive [data not shown] and failed to clarify the esterase results. A P C ability to produce IL1. PECs , a-Thy1 plus c' treated, and spleen cell populations were stimulated with LPS . Supernatants were harvested and analysed for the presence of IL1 [at a ten percent dilution] by their ability to stimulate the proliferation of an IL1-dependent cell line, D10.G4.1. The results seen in figure 2-4, suggest that neither the P E C s nor the spleen cells were deficient in their ability to produce IL1. There was no significant difference among all of the H-2K or the H-2B cells tested. Thus the reduced responses seen in figure 2-1 when MRL-+ and MRL-/pr A P C s were added cannot be due to a lack of 111. DCs as A P C s in the proliferative response to conA. Since it is well known that DCs, a subset of APCs , provide very potent activity in mitogenic and allogeneic responses (182,183,189), conA titrations and 81 Figure 2-4: 111 production by splenic and peritoneal mos. 2 x 1 0 5 P E C s [a-Thy1 and c' treated], in one ml, and 1 x10 6 spleen cells, in one ml, were cultured in the presence of 20u.g LPS/ml. Supernatants were harvested and tested for their ability to stimulate the proliferation of the 111-dependent cell line D10.G4.1. 2 x 1 0 4 D10.G4.1 cells were cultured with 1.25jig conA/ml and the supernatant samples [diluted to ten percent] for forty-eight hours. After this time they were labelled with tritiated thymidine and radioactivity incorporation was determined. Control cultures [without any supernatant] gave counts less than 4,000 cpm. Results represent the mean plus or minus the standard deviation of triplicate cultures. CBA, B6, MRL-+ and 'Y' mice were six to eight weeks old, 'O' mice were five months old. 8 2 CPM MLRs using DC-enriched populations of cells were studied to delineate the functional capacity of such cells from each mouse strain. DCs were enriched from the spleen and subsequently used in culture. As there is no way of assessing the numbers of DCs present in the enriched populations obtained, a conA titration was performed to compare the results seen when using the two types of APCs. In assessing the ability of the different H-2K DC populations at helping normal CBA NWT respond to conA (figure 2-5b), no difference among any of the DC populations can be seen at all doses tested, except for a decrease seen when MRL-lpr DCs were at the very highest dose. As these DCs were only enriched populations, there was the possibility that contaminating splenic mos may have been present, hence the cause of the results seen. In fact, when esterase staining was done on the DC-enriched cells, mos were found to be present [data not shown]. The presence of mos in the splenic enriched DCs suggested that not only are PEC mos dysfunctional, but so are splenic mos. In figure 2-5a, a PEC titration is shown for comparison. Clearly, the populations of enriched DCs allow for a perhaps marginal increase in proliferative response to conA under the conditions used, suggesting that they are functionally normal for all H-2K strains tested. DCs used in MLRs: To assess the ability of the DC enriched populations at stimulating in the MLR, varying numbers of DCs were tested. For lack of enough cells, responses of twenty, ten and five to one responder NWT to DC were compared to a ratio of one for responder NWT to spleen cells [like that for figure 2-2]. In figure 2-6a, the typical MLR response using spleen cell stimulators, as was described in figure 2-2, is seen. In figure 2-6b, at a dose of five to one, CBA DCs stimulated better 84 Figure 2-5: APC function for proliferation in MRL-lpr mice: DC titrations. 5x10^ C B A NWT were cultured in the presence of 1.25u.g conA/ml and a) with irradiated P E C s [cc-Thy1 and c' treated] or b) irradiated DCs at the dilutions indicated and assessed for proliferation as described in figure 2-1 a. Control cultures of NWT without conA, or A P C s alone were less than 700 cpm and NWT plus conA alone was 148,874 +/-13,733 cpm. Results represent the mean plus or minus the standard deviation of triplicate cultures. C B A mice were three to five months old , MRL-+ and MRL-lpr mice were were five to six months old. 8 5 CO 00 1 1 0 1 0 0 1 1 0 1 0 0 Ratio CBA NWT : APC Ratio CBA NWT : APC F igure 2-6: APC function in the MLR: DC stimulators, H-2K. 1 x 1 0 6 B 6 N W T were cu l tured in the p r e sence of a) 1x10^ irradiated sp l een ce l ls , or b) i rradiated D C s at the concent ra t ions ind icated, f rom C B A , M R L - + a n d MRL-lpr m ice . Prol i ferat ion w a s dete rmined a s desc r i bed for f igure 2-2. Nega t i ve cu l tures g a ve coun ts less than 450 c p m . Resu l t s represent the mean p lus or m inus the s tandard dev iat ion of tr ipl icate cu l tures. B 6 mice we re three months o ld and C B A , MRL -+ and Y MRL- /pr m i ce we re s ix w e e k s o ld a n d O MRL - /pr , s ix months o ld . 87 a 0 Y MRL-lpr SC • O MRL-lpr SC 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 • 1 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 CPM b • O MRL-lpr DC 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 CPM 88 than all other H-2K DCs, which showed approximately equal abilities to stimulate. This trend was also seen at a dose of ten to one NWT to DC. In figures 2-7a and 2-7b the H-2B stimulator DCs in the MLR are depicted. From the dose response MLRs, it can be seen that Y B6-/pr DCs stimulated almost as well as B6 DCs, but with disease onset and age, that stimulatory capacity was lost. However, it may have been that there were reduced numbers of DCs present in the B6-/pr spleen with age so that if too high a dilution were used, the function of the DCs would not be seen. Compare figures 7a and 7b, ten to one with five to one of responder NWT to DCs. From the DC studies, it appears that MRL-+ and MRL-/pr DCs are normal, but that B6-/pr DCs demonstrated an age-related defect in function. Since the DC populations were enriched and contained mos, in MRL-+ and MRL-/pr mice, there may have been a DC defect, but it may have been obscured by the mos present. Further studies are needed to decipher the possibilities. 89 Figure 2-7: APC function in the MLR: DC stimulators, H-2B. 1 x10 6 C B A NWT were cultured in the presence of irradiated DCs, at the concentrations indicated, from B6 and B6-/pr mice, a) and b) denote separate experiments using the same conditions. Proliferation was determined as described for figure 2-2. Positive control, irradiated spleen cell stimulators from B6 and young and old B6-/pr mice all gave counts around 7,000 cpm. C B A NWT alone gave cpm of less than 500. Results represent the mean plus or minus the standard deviation of triplicate cultures. In (a) CBA, B6, and Y B6-/pr mice were six weeks old, in (b) they were eleven weeks old and in both (a) and (b) O B6-/pr mice were six months old. In a) B6-/pr mice were female, in b) they were male. 9 0 M B6 DC 0 YB6-lprDC • OB6-lprDC 0 2000 4000 6000 8000 10000 12000 14000 16000 CPM E3 YB6-lprDC • OB6-lprDC • r i i i i i i i i | I F I I I I i f ? | • i t i t f f f f i | i i i i f f i t i ; t i i i T n i i | m T T i i i r | i l 0 10000 20000 30000 40000 50000 60000 CPM 91 C H A P T E R TWO DISCUSSION Previous experimentation in this laboratory demonstrated that age associated mitogenic (130) and Ag specific hyporesponsiveness seen in MRL mice (131) was not only due to a T cell defect, but also caused by a mo/APC malfunction, one not associated with the Ipr gene, nor the production of 111 [and its requirements] and not due to IC blockage of A P C function. In addition, probably associated with this primary mo defect, MRL A P C s were unable to respond well in a primary MLR. Because Y MRL-/pr NWT were able to respond to antigenic and mitogenic responses, but not well, we chose to analyse this further. As was done in earlier studies, comparisons to B6-/pr cells were also done to see the effects of the Ipr gene in the absence of background S L E genes. P E C titrations: When the original experiments were performed (130), a ratio of responder NWT cells to P E C s of ten to one was always used in the conA responses, which always demonstrated a much reduced response when using MRL-/pr P E C s instead of C B A P E C s as A P C s . To better understand the T cell-mo effect occurring, a titration of a constant number of C B A NWT to varying numbers of H-2K [CBA, MRL-+, or MRL-/pr] P E C s was performed to see if at some point MRL-/pr P E C responses would approach those seen when using C B A PECs . Figure 2-1 a shows that high numbers of mos became suppressive or inhibitory for proliferative responses [CBA P E C s added at the highest dose]. The titration figures demonstrated that S LE background genes have an influence on the peritoneal A P C populations, an effect that worsened in the presence of the Ipr gene. Repeatedly, only when in small quantity [high ratio of NWT to 9 2 PECs] did MRL-lpr P E C s support'normal' mitogenic responses, not statistically different from when C B A P E C s were added. Surprisingly, this effect was seen even when the MRL-/pr P E C s were obtained from a MRL-lpr animal six weeks old, before the onset of clinical disease expression which, with respect to the mononuclear phagocyte system, is demonstrated by increased numbers of mos in the peritoneal cavity, with a high proportion of these being la+ (36,38,39). Because the C B A NWT could proliferate to conA without the addition of exogenous APCs , A P C s must have been present in the NWT preparations. This was confirmed by esterase staining [data not shown]. The reduced response by MRL-+ P E C s confirmed earlier results seen in this lab (130), that MRL-+ express a defect unrelated to the Ipr gene. Because the presence of fewer MRL-lpr P E C s allowed for an increased response to occur [which approached that seen with C B A P E C s at a responder to P E C ratio of 320 in figure 1a] this suggested that the reduced response seen was either due to defective or suppressive mos. At this point, what that influence was remained uncertain, but the Ipr gene in combination with the S L E background genes enhanced the above A P C abnormality. When the titration experiments were extended to the B6-/pr system in figure 2-1 b, a slightly different picture was seen. In figure 2-1 b, B6-/pr P E C s from young mice helped responses similarly to B6 PECs . However, after the age of four months, [the onset of clinical disease (131)], the B6-/pr P E C s supported an enhanced proliferative response over that seen when B6 and Y B6-/pr P E C s were used. This was interesting as it further implicated that the results seen with the MRL-/pr P E C s [figure •i 9 3 1 a] were due to an additive effect of the S L E background genes in association with the Ipr gene and that the Ipr gene acting alone was insufficient to cause a mo defect for mitogenic proliferative responses. As well, figure 2-1 b showed that in the presence of other defective genes such as background S LE genes, the true function of the Ipr gene in the P E C mo population may be masked. This was studied further. When spleen cells were used as stimulators of MLRs, figure 2-2, the results of figures 2-1 a and 2-1 b were reinforced; MRL-+ spleen cells demonstrated a reduced ability to stimulate and this ability was diminished in the presence of the Ipr gene, and B6-lpr spleen cells could stimulate well as B6 spleen cells. Numerous possibilities could have explained the MRL-/pr P E C titration observations made. One argument could have been that TsCs were present in the P E C populations and they were responsible for the reduced response seen. However, the T cells present were eliminated with oc-Thy 1 plus c' treatment before P E C use. In fact, this treatment enhanced the suppressive effect [data not shown]. The effect seen, therefore, could not have been due to TsCs . Quantification: As the absolute numbers of mos in the P E C populations were unknown, and since it was shown that high concentrations of mos were suppressive to T proliferative responses (figures 2-1 a and 2-1 b), the numbers of mos present were assessed by esterase staining and F4/80 McAb analysis. 9 4 Nonspecific esterase staining was chosen to analyse cell populations for the presence of mos as this enzyme has been proven to be one of the most reliable markers for the identification of mos when a-napthyl-acetate or-butyrate are used as the substrate. Although T cells may stain positive, the pattern of staining between the two types of cells varies. Some T cells show one or a few dots of positive staining since, in T cells, the enzyme is localized in granules in the cytoplasm; in mononuclear phagocytes, the staining occurs diffusely throughout the cytoplasm (172). A problem that was encountered using the esterase stain was that, as has been previously noted (172), the intensity of staining varied. This variability made the exact quantification of positive cells difficult. The range of positivity in staining has been attributed to the different developmental stages and functional states of these cells (172). What was clear from figures 2-3a and 2-3b though, was that with age, the proportion of esterase positive cells seen in older MRL-/pr mice was greatly increased compared to non-autoimmune, H-2K C B A control cells. This data correlated well with published data concerning the age associated increase in number of peritoneal la+ mos in MRL-lpr mice (3,36,38,39). One of these studies mentioned that the absolute numbers and percentages of la+ mos seen in the peritoneal cavity of MRI-/pr mice increased with age when compared to MRL-+ mice, but that in the spleen the numbers were comparable to normal mice (38). This observation would suggest that different populations of mos may exist in different anatomical locations in the mouse, for example, spleen versus thymus versus peritoneal cavity (39,40,195). One study demonstrated that the 9 5 percentage of la+ mos in the spleen was less in the Ipr than in the MRL-+ mouse (40) when such cells originally began in equal numbers in both MRL-lpr and MRI-+ mice (3). The former study also showed that la+ splenic adherent cells actually decreased with age in these mice, however, a comparison to a non-autoimmune, H-2K compatible control was not shown (40). It may be that the peritoneal cavity acts as a 'reservoir' for these 'defective' mo. This observation, of increasing esterase positive cells in the peritoneum of MRL-lpr mice with age brings up a few points. First of all, as the number of la+ mos and esterase positive P E C s in Y MRL-lpr mice was comparable with those seen in C B A animals (figure 2-3a), the reason for the 'suppressive' effect seen on the C B A NWT proliferative response by Y MRL-lpr P E C s cannot be due to too many mos being present. This point reinforces the observation that MRL-+ mice APCs , expressing S L E background genes, demonstrated a defect, and suggests that at a young age in MRL-/pr mice these gene effects will be aparent, but when the mouse gets older, the backgroud effects are blurred by the Ipr gene effects. The normal esterase activity demonstrated in Y MRL-/pr mice fails to exclude other mo defects with respect to the MLR. The possibility that some of the mos may have been, by some means, unable to effectively present, as a result of having inappropriate la expression (42), was not excluded, as absolute numbers of la molecules on the surface of these cells have not yet been determined. Or, as has been proposed by Rosenberg et al (36) [to be due to y-IFN] these la+ mos may be already activated and thus 'paralyzed', since splenic and peritoneal mos typically express very little la and the proportion of mos expressing la is very little. Once activated, 9 6 from T cell products [lymphokines] and T cell-mo interactions, an increase in the number of la molecules on the surface and an increase in the numbers of la+ mos is seen (199). Another possibility still lies in some unknown presentation defect unrelated to surface la expression. The last study showed that y-IFN increased the number of la+ mos, which was great for presentation, but really had no effect on a primary proliferative response by responding T cells (199). It must emphasized, however, that y-IFN does many things to mos (200,201) in addition to increasing c lass II Ag expression. In light of recent evidence on la molecules seen in autoimmune disease (202,203), evidence for MHG/HLA disease associations is increasing. The titration data with the H-2K system and autoimmune MRL mice showed a pronounced defect (figures 2-1 a and 2-5a) and the autoimmune-free H-2B system failed to show this profound abnormality (figure 2-1 b); these results would lend support to the above theory. Some have suggested that the lymphoid hyperplasia may lie in an abnormal la molecule in the MRL-/pr mouse, in that small numbers of (lA^ lE^) have been demonstrated and were derived from the LG/J ancestor (42). Perhaps the expression of this abnormal la molecule could contribute to the reduced capacity of both MRL mice to present Ag and may explain the background S L E gene effects seen. This would not, however, explain the age-related increase in dysfunction seen in the MRL-/pr mos, or increased in function seen in B6-/pr mos; both are probably due to the influence of the Ipr gene. As it has been established that the culture of mos in the absence of 9 7 exogenous y-IFN leads to decreased expression of surface la molecules after twenty-four hours (162) and, hence, a reduced ability to present Ag, P E C s were incubated for a couple of days and then tested for their ability to help non-autoimmune responders in a mitogenic response. The results demonstrated no difference in the capacity of fresh and incubated [one or two days] MRL-lpr P E C s in helping C B A NWT to proliferate [data not shown]. These results would argue that although y-IFN may be playing a role in vivo to increase the number of la+ mos, and/or the percentage of la molecules on the surface of the mos, the reduced ability of MRL-/pr P E C s to help mitogen stimulation of C B A NWT is not due to an overstimulation or activation of these mo by y-IFN, as by twenty-four hours in culture, the effects of y-IFN have been alleviated. In addition, this would argue that circulating Abs to mos (196,197) or IC are not involved in the abnormalities seen, and further supports previous experimentation done in this laboratory (131). An intrinsic defect in mos was not excluded by such an experiment, thus further experimentation is required. The issue doesn't seem to be one of la Ag expression on mature mos. MRL-/pr mice cannot combat a Listeria monocytogenes infection, a particulate intracellular bacterium known to stimulate la Ag expression on mos (38). An inability to fight infection, normally achieved through opsonization of the bacteria, may reflect on a phagocytosing defect. PGE1 at pharmacological doses (5,31,38) can cure MRL-/pr mice of a Listeria infection. PGE1 can also inhibit lymphokine induced increase in la expression on mos (205). That PGE1 can also cure MRL-/pr mice of their their autoimmune disease would lead one to conclude that the effect of 9 8 PGE1 is not primarily on the mo population, but must act, rather, at a different level, perhaps by preventing the development of defective mos or T subpopulations of cells, i.e. at the stromal level in the bm and thymus. Indeed, C F U committed to mo development are very sensitive to low concentrations and are specifically inhibited by PGE1 (206). On the other hand, high amounts of Abs, autoAbs and ICs are present in the system of MRL-lpr mice (3) and MRL-/pr mice show a defect in IC clearing (2,37). It is also known that IC bind c\ and that fragments of C3 [C3b, C3bi and C3c] stimulate the synthesis of P G E 2 by mos which acts as an autoregulatory molecule (37). Interesting enough, the PGE1 treatment failed to alter serum levels of ANA and a-DNA Abs (5). P G E 2 may simply be in low quantity in MRL-/pr mice with age because of an abnormal population of mos, which cannot produce it. Thus, a logical conclusion would be that, in conjunction with the outgrowth of the abnormal T cells (chapter 1), there is also the outgrowth of a defective mo population. A defect inherent in the development of mos in MRL-lpr mice is supported by others (40,207). In this respect, it would be interesting to see how BQ-lpr mice would combat a Listeria infection and what the effects of PGE1 as a treatment would be, not only on the development of the infection, but also on their disease state. As the B6-/pr mo function seemed relatively normal, at least when monitored by the ability of these cells to help mitogenic proliferation in figure 2-1 b and stimulate alloreactivity in figure 2-2, and numbers of mos in P E C s also seemed normal (figure 2-3b), predictions would be that these mice would easily combat the infection. Support for this would come from the fact that although B6-/pr mice produce autoAbs, they do not develop autoimmune disease per se in that they fail to demonstrate IC glomerulonephritis (4); hence, B6-/pr mice do not have 9 9 the same phagocytosing defect as MRL-lpr mice. This further emphasizes the effect of the Ipr gene effects in the absence of S L E background gene effects. If B6-/pr mice did show an inability to fight the Listeria infection, those results might give some insight as to the function of the Ipr gene product. F4/80 McAb analysis was chosen to quantitate mos as there was a small, but readily available source of the McAb and due to the specificity of F4/80 (178). F4/80 is a rat McAb which precipitates a cell surface glycoprotein of Mr 160,000. It is present on both monocytes and mos, and is readily detectable on various sources of mos (177). A s a monocyte develops from a monoblast/promonocyte into a blood monocyte, the concentration of surface F4/80 Ag increases (178,201). F4/80 is in highest concentration on mature mos, however, is much reduced on the surface of activated mos. In addition, culture of mos causes, besides adherence and spreading, reduced expression of the F4/80 molecule unless the mos have been thioglycollate elicited (177). Unfortunately, under the conditions used, the F4/80 McAb analysis gave inconclusive results, thus, further studies are required. Superficially, it appeared that the S LE genes served to increase the number of F4/80+ cells in the peritoneum of MRL-/pr mice and that the Ipr gene acted to reduce the numbers of these cells with age in both the MRL-/pr and B6-/pr strains of mice. If the percentage of F4/80+ cells remains high when MRL-+ and MRL-/pr animals get older, this would suggest that there are increasing numbers of mos in the unactivated state as activated mos down regulate F4/80+ expression. If the percentage of 1 00 F4/80+ mos decreased, the reduction would probably be due to either activated cells or cells too immature, before F4/80 expression, but after esterase activity expression [which occurs very early in the development of mos (173)]. Since the number of F4/80+ cells in the peritoneum of MRL-lpr mice seemed to correlate inversely with age, and, from figure 2-3a, the number of esterase positive mos in the peritoneum correlated proportionally with age, the prediction is that these cells would be in the activated state. Recent studies by Dang-Vu et al (35), and others (39,69), would support this notion. This will remain speculation until further experimentation is done. 111 Production: Feasibly, as has been extensively studied by many yet without an accepted opinion (36,39,40,55,131), MRL-/pr mos could be defective in the production and secretion of second signal required for T cell activation, 111. In figure 2-4, there seemed to be no difference in ability to make and secrete 111 among the populations of P E C s or spleen cells in each H-2 group. However, one must take into consideration that with the O MRL-lpr and B6-/pr mice, the different populations of cells are diluted out by the 'odd'T cells as the animals grow older (chapter one). In effect, as the mouse gets older, there are decreased percentages of all types of cells, eventhough the splenomegaly has arisen from extensively increased numbers of aJi cells, but much more so from elevated numbers of the 'odd' T cells (18,47). So, for a small aliquot of spleen cells, because of the percentage decrease, fewer numbers of A P C s are present, hence fewer mos. As a result, there would have to be increased production of 111 per cell to maintain the same overall production. To discern if this were in fact true, splenic adherent cells from the different mice could be 101 obtained and then stimulated for 111 production to see how they compare. A s the P E C populations were cc-Thy1 and c' treated, and thus mo enhanced, it seems safe to conclude that there was no difference between the different mice in each group with respect to 111 production by peritoneal mos. Should these differences in 111 production between the spleen cells and P E C s be real, then this provides good evidence for a difference in these two sources of mos in MRL-lpr mice. Interestingly, Boswell et al (211), studied 111 gene expression in mos isolated from MRL-+ and MRL-lpr kidney and found that compared to MRL-+ mos, Ipr mos produced a ten fold increase of IH-alpha and 111-beta transcripts. In addition, a 1200 nucleotide, as opposed to the typical 1600 nucleotide, transcript of 111 -beta was found; this mRNA was expressed in high quantities in those mice demonstrating nephritis. 111 was detected in the glomeruli of such mice and not in non-diseased mice. Mos isolated from the glomeruli secreted 111 in culture. That renal mos, from mice displaying nephritis, produced elevated levels of 111 is interesting and important for understanding kidney pathology as a disease process in these mice; these mos were probably elicited from the inherent IC processes in the kidney. The continued presence of these mos, thus, is incidental to their inability to remove IC (2,37), hence, continued tissue damage and release of inflammatory mediators maintains their presence. From the results in figure 2-4, a mo defect in the ability to secrete 111 seems to be nonexistent, or, as mentioned above, there may be overproduction of 111. Because 111 stimulates P G E 2 production by mos in 1 0 2 an autoregulatory fashion (37), an elevated response in 111 production causing P G E 2 release could explain a 'suppressed' phenomenon as seen in vitro, but then that would be unsupportive of earlier predictions that a mo defect in P G production exists. On the other hand, elevated 111 production may lead to too much help which would also be suppressive. Slightly increased 111 production would support the increased proliferation seen when B6-/pr P E C s were used in the titration in figure 2-1 b. The overproduction of 111 appears to be critical to the autoimmune disease process as described by Boswell (211). The role of secreted 111 for T cell proliferation seems to be that of enhancing the overall response rather that being essential for the response to occur (212), so elevated production of 111 would necessarily cause disease exacerbation. Therefore, mo dysfunction in 111 production may also contribute to the differences in proliferation seen in figures 2-1 a and 2-1 b. Dendritic Cell function: The last area of concentration of the A P C studies was the inquiry into whether other populations of A P C s demonstrate unusual immunological activities. Little information on the DC population of A P C s in MRL-/pr and B6-/pr mice is available. One report published studied the A P C and stimulatory capacity of MRL-/pr versus MRL-+ DCs and concluded that7pr DCs were defective, however, a comparison to normal mice was lacking (198). Using a discontinuous BSA density gradient technique and subsequent selective adherence (183), DC-enriched populations from the spleen were isolated. Mos and DCs are of similar density and thus migrate to the same position on the BSA gradient (182). The selective adherence procedure 1 0 3 further enriches for DCs. The last step in the protocol was to remove FcR+ cells by resetting, but proved to be very tricky [and probably further complicated by the fact that MRL mos show a reduced ability to phagocytose opsonized rbcs and latex beads (32,33)]. As the quantity of DCs in the spleen is less than one percent (183,185), recovery of adequate numbers of cells to count and use with any degree of significance would have been greatly reduced by the last step of purification and would have required the use of many animals making the acquisition of DCs very labour intensive. Due to the above considerations, the last step was avoided. The problem with using a DC enriched cell preparation was that there was no way to quantitate the number of DCs present. In addition, because mos were also present in the preparation, the effects seen have to be interpretted with caution. Since P E C s contain no DCs (185), the P E C titration experiments demonstrated what effect increasing numbers of mos have on the NWT response to conA (figures 2-1 a and 2-1 b). Even though the absolute requirement for DCs in a conA response remains unknown (182), DCs at as low a concentration as one percent of the responding population are able to stimulate maximal proliferative responses and the increase in numbers of DCs fails to show a change in those responses (183). Although a McAb to DCs has been developed, 33D1 (186), the use of this McAb has shown equivocal results (R.M. Meloche, personal communication). Thus, using 33D1 may or may not prove beneficial in assessing DC numbers. Figure 2-5b illustrates the H-2K titration response of the DCs, using 104 C B A NWT as responders to conA. The P E C titration (figure 2-5a) shows a response like that seen in figure 2-1 a. When first observing the DC titration, at higher NWT to DC ratios (ten and forty to one), the responses were the same for all DCs. All DCs appeared equally functional under these experimental conditions. What it also suggests is that the concentration of DCs must be such that it is one percent or better ~ at least for the ratio of ten to one in this figure and probably for forty to one, as even though the response at forty to one was slightly less than that seen for ten to one, it was not statistically different. At the lower dose of NWT to DCs, two and one half to one, the proliferative response is reduced. Since, from the P E C titrations, high numbers of P E C s [mos] caused a lower proliferative response, and the enriched populations of DCs had mos in them [determined by esterase staining, but not shown], the explanation for this effect would be due to the presence of too many mos which at high concentrations are suppressive, as was seen in the P E C titration figures, or perhaps by overproduction of 111, as previously discussed. In light of this, it would also seem that at the higher NWT to DC ratios (ten and forty to one) the mos present in the DC enriched populations [obviously in fewer numbers than at the two and one half to one ratio] had little effect on the DCs ' abilities to stimulate proliferative responses. The presence of DCs in a primary proliferative response is essential; DCs are necessary to initiate the response and mos further act as accesory cells to perpetuate the response (182); mos are unable to replace DCs in this type of analysis. Mos also, by virtue of their ability to produce 111, allow for an augmentation of the T cell response (212). So, as 1 0 5 discussed earlier, if MRL-lpr splenic mos have the capacity to secrete large amounts of 111, then an abnormality in the Ipr DCs may not be seen in this experiment due to the enhanced T cell response from the extra 111 being produced. The only way to determine this would be to obtain mo-free populations of DCs. More indirectly, one could do a C B A DC titration experiment with C B A NWT and add back varying numbers of MRL-lpr P E C s or splenic mos. Predictions would be that the increased numbers of mos would cause a much more reduced response compared to C B A P E C s due to the nature of the Ipr mos. The second point worth discussing with the H-2K titration of DCs is that at forty to one NWT to DCs, the optimal conA response of C B A NWT was not greatly enhanced above NWT responding to conA alone. This is in disagreement with published data of MRL-lpr DCs (198) and suggests that C B A NWT contain DCs which allow for the good proliferative response seen and that the response is not largely influenced by the increase in numbers of the other DCs added. Nonetheless, DCs from all of the mice tested caused the same results, suggesting no defect exists. In figure 2-6 the capacity of DC-enriched populations to stimulate a MLR (b) was compared with the spleen cell stimulatory capacity (a). C B A DCs demonstrated the normal response and all of the MRL DCs demonstrated decreased stimulatory capacity. C B A had no defect in DCs nor mos, thus the mos present in the C B A enriched DC population did not influence the stimulatory response. As MRL-+ and MRL-/pr both had dysfunctional mos present, it may well be that their DCs were normal, but the response seen was influenced by the mos present. As the DC 106 stimulated MLR response was the same for each of the MRL mice, it is probable that there were adequate numbers of DCs present for the MLR to occur, since DCs are required for a primary proliferative MLR response to occur (182). It may have been, that the MRL background S L E genes caused one level of defect in the DCs, leading to the first overall reduction in stimulatory capacity as seen when comparing C B A to MRL-+ DCs [supported by the other study (198)], and that a further Ipr gene defect would have obscured the initial defect by the enhanced ability of splenic mos to secrete 111, which ultimately would have caused an increase in response by the responding T cells. Purification studies are reqired to discern which of the possibilities exists. When the MLR was extended to consider the effect of the Ipr gene on DC function by including B6-/pr DCs as stimulators (figures 2-7a and 2-7b), the response seen was slightly different. When B6-/pr mice were young, DCs were able to stimulate a response similar to that seen with B6 DCs. With age, it appeared that B6-/pr DCs lost their ability to stimulate. As was previously shown with P E C titration experiments (figure 2-1 b), the B6-/pr P E C s showed no 'suppression' defect. Thus, the DC stimulation may therefore show the real Ipr defect. It may, in contrast, show a dilution effect; that is, the absolute numbers of mos present in the DC enriched populations may far outnumber the DCs, so that DCs present are suboptimal for MLR stimulation at the dilutions tested [compare figure 2-7a with 2-7b]. The DC is required for the primary MLR in vitro. If the numbers of DCs were suboptimal for stimulation, then a reduced response would be predicted, as was seen. 1 0 7 Clearly, the DC studies performed on the MRL-/pr and B6-/pr mice require further analyses with more stringent conditions before conclusions may be drawn. In summary, in this chapter several aspects of certain A P C s in Ipr mice were examined. The assays used demonstrated both qualitative and quantitative abnormalities seen in MRL-+, MRL-/pr and B6-/pr mice. First of all, quantification by esterase staining implied that with age, MRL-/pr P E C s contained very high numbers of mos which resulted in less than optimal conditions for responder NWT to proliferate to conA or alloAg presentation. B6-/pr P E C s had normal numbers of esterase positive cells. When B6-/pr mice were old, their P E C s augmented T cell responses, suggesting a qualitative rather than a quantitative defect in these mice when compared to MRL-/pr mice. Unfortunately, quantification using the McAb F4/80 could not support or refute the above results. 111 production from either Ipr animal strain did not appear to be much different from H-2 compatible controls. The studies performed on DC-enriched populations suggested that peculiar differences between MRL-/pr and B6-/pr DCs exist. 1 0 8 C H A P T E R T H R E E INTRODUCTION Many theories as to why autoimmune diseases, such as SLE , develop exist, each with supportive experimental information which prompted them and not without controversy (37). One of these theories, diminished/ abnormal T S C function is evaluated in this chapter. It is well understood that a complex sequence of interactions and a number of cells are involved in the initiation, progression and termination of an immune response. Some TSC s are negative regulator cells which act at the terminating stage of an elicited immune response; others act to prevent self-responding cells from attacking and degenerating self. Hence, a lesion created by the absence, loss or elimination of any particular autoAg specific TsC could feasibly lead to an autoimmune phenomenon. A generalized TsC defect however, is more controversial, harder to explain and, thus, unlikely (37). The status of T sC function in MRL-/pr mice has long been sought. One of the earliest studies, by Gershon (11), demonstrated that the cells required for Ag-specific suppression were absent in MRL-/pr mice. Subsequently, others (36) review that different Ag-specific T sCs were also missing in these mice, suggesting specific defects in certain cells rather than a generalized TsC defect. Further evidence to support this notion is provided by early studies done by Murphy and Roths (2,13) who found that the spleen of MRL-/pr mice contained elevated numbers of I-J+ cells; however, these studies failed to reveal the functional status of these cells. A more recent study in our lab (214), showing that the 109 functional yield and activity of TsFs isolated from MRL-lpr spleen was comparable to that found in C B A spleen, would lend evidence to the presumption of defects in, or absence of, monoclonal T sC populations rather than in all of the TsCs . To analyse a particular TsC defect, the problem can be approached in two ways. If a T sC clone with specific regulatory properties is available, the clone can be added into a system to see the regulatory effects on the cells in question. This is specific and would require that such a clone be readily available. In addition, an assay system to detect the level of 'added' regulation would be needed. On the other hand, T sC could be eliminated and then TsC lesion in question could be monitored. This would require a means to specifically deplete TsCs and an assay to determine the level of 'depleted' regulation. For the studies performed in this chapter, a technique to eliminate TsCs in mice was well established and readily available. The technique is known as T sC deletion therapy using a McAb, B16G, directed to all T sCs and TsFs (213-217), coupled to a light-activatable toxin, HP (218,219). The lesion chosen for analysis in both normal and autoimmune mice was autoAb production. In particular, a-DNA autoAb production of the IgG class was measured. Pathogenic Abs to DNA are a hallmark of S L E disease (221) [secondary to B cell hyperactivity] and are easily assayed by ELISA (222-226). Mice were treated with B16G-HP and monitored for auto a -DNA , by ELISA technique, and for survival. In addition, other in vitro assays, unrelated to the B16G-HP study, were used to assess in vitro T sC function. 1 10 C H A P T E R T H R E E R E S U L T S Two Dav Incubation of NWT: To assess TsC function in vitro, a few experiments were done while the in vivo, B16G-HP experiment was in progress. NWT from CBA, MRL-/pr and MRL-+ mice were incubated for one or two days or used fresh and assayed for proliferation in response to conA. In figure 3-1 it can be seen that for both C B A and MRL-+ NWT, the ability to respond decreased with days of preculture, whereas the MRL-/pr NWT response was elevated by two days preculture. This observation supported earlier observations in our laboratory of II3 production by MRL-/pr NWT only after two days preculture (45) and those of Kelley et al (132) where two day incubations allowed cells to express II2R, both which seemed to be due to alleviation of some in vivo down regulatory influence. This phenomenon, noted in a subsequent experiment, was lost in O MRL-/pr mice and [not shown]. When B6-/pr NWT were treated in the same way, no change was seen [data not shown]. When PECs , not treated to remove T cells, were incubated under the same conditions above and added back to NWT, no changes the responses were seen either [not shown]. Therefore, two day incubation of NWT alleviated some suppressive in vivo influence in Y MRL-/pr mice. Mixing Experiments: Another means of assessing active suppression is by mixing experiments. NWT isolated from normal C B A and B6 mice were cultured in the presence of increasing numbers of their own or H-2 compatible diseased mice NWT and assessed for the overall proliferative response to conA. Figure 3-2 shows the results for the H-2K mice. When MRL-+ NWT were added to C B A NWT in increasing numbers (figure 3-2b), 111 Figure 3-1: Proliferative responses after two day preculture of NWT. NWT from CBA, MRL-+ and MRL-lpr mice were cultured for one or two days. 2.5x10^ NWT, freshly isolated or incubated, were cultured in the presence of 1.25u.g conA/ml to assess proliferation. After two days in culture, tritiated thymidine was added and after another day, radioactivity incorporation was determined. Control cultures [no conA] gave counts of less than 900 cpm. Results represent the mean plus or minus standard deviation of triplicate cultures. All mice were male and two to three months old. 1 1 2 days of preculture 113 Figure 3-2: Mixing experiments. 2 .5x10 5 C B A NWT were cultured with varying amounts of a) C B A NWT, b) MRL-+NWT, or c) MRL-/pr NWT in the presence of 1.25u.g conA/ml to determine what effect each of the added NWT would have on the prol i ferate response of the original 2.5x10^ C B A NWT. Proliferation was assessed as described for figure 3-1. Predicted values, P, for each condition, a, b, and c, were determined by adding the proliferation seen by the individual cultures when cultured separately. Control cultures [no conA] were less than 750 cpm. Results represent the mean plus or minus standard deviation of triplicate cultures. C B A were three months old, MRL-+ six months old, and MRL-lpr eight months old. All mice were male. 1 14 NWT added NWT added NWT added proliferative responses seen were similar to those seen when CBA NWT were added (figure 3-2a), that being proliferative responses close to predicted values. However, when MRL-lpr NWT were added (figure 3-2c), the proliferative response was significantly reduced, much less than the predicted value, especially when high numbers were added, suggesting active suppression. When mixing experiments were performed with H-2B mice there was no evidence for generalized suppression as all proliferative responses were close to predicted values [data not shown]. Again, in MRL-lpr mice, there appeared to be some suppression. B16G in vitro: B16G added to MLR cultures. B16G is a McAb, developed in Dr. Levy's laboratory, which recogizes a determinant on all TsCs and their TsFs (213-217). It was reasoned that if TsCs were active or being induced, culture with this McAb would alleviate the effects seen, as has been experimentally shown in other systems (A. Tench-Stammers, personal communication). B6 NWT were stimulated by irradiated CBA or MRL-/pr spleen cells in the absence of or presence of B16G or an irrelevant, class matched McAb, 690, and the proliferation was determined. In figure 3-3 it can be seen that the responses by B6 NWT were unchanged in the presence of B16G or 690 when stimulated by either of the spleen cells. As the responses seen were all the same for the individual stimulators, MRL-/pr spleen cells were unable to induce TsCs in the MLR. The reduced response seen compared to the CBA stimulators probably reflects inherent APC defects as discussed in chapter two. B16G-HP in vivo: suppressor deletion experiments. To determine what role TsCs play in vivo in the production of auto a-DNA IgG, TsC 116 Figure 3-3: In vitro culture with B16G. 1 x 1 0 6 B6 NWT were cultured in the presence of the cc-TsC McAb, B16G at 50u.g/ml, or 690, an irrelevant, subclass matched control McAb at the same concentration and 1x10** irradiated spleen cells from C B A or MRL-lpr mice. After three days in culture, tritiated thymidine was added and after four days, radioactivity incorporation was determined. Responders or stimulators alone gave cpm less than 500. Results represent the mean plus or minus standard deviation of triplicate cultures. B6 and C B A mice were female and six weeks old and MRL-/pr mice were male and eleven weeks old. 1 17 CPM deletion treatment was performed in MRL-+, MRL-lpr, and B6-/pr mice. C B A and B6 mice were also treated as controls. All mice were approximately two months old. They were each bled one week prior [week -one] to the treatment date [week zero], one week subsequently [week +one] and then every two weeks thereafter for a period of up to six months to be monitored for autoAb production to DNA by ELISA. Mice were also monitored for survival and demonstrated no significant difference between treated and untreated mice in all groups [data not shown]. The Ab profiles of all mice are depicted in figures 3-4 a though j [absorbance measurements against time]. The absorbance measurements have all been standardized to a positive control serum and read as such. The negative control was serum collected and pooled from Balb/c mice, two to four months old. Figures 3-4, a and b: CBA mice. None of the treated C B A mice (b) showed any real autoAb production. One of the untreated mice (91, a) had quite a high peak of autoAbs for a non-autoimmune mouse (absorbance of 0.14), but this peak lasted only for one measurement and only happened once. In total, four untreated C B A mice (91, 92, 93, 96, a) showed one measurement elevated peaks [maximum absorbances from 0.037 to 0.064 as compared to average autoAb production of 0.019 absorbance], two mice on greater than one occasion (92, 93, a), but these levels were negligible when compared to the levels of autoAbs produced by MRL-/pr mice (figures 3-4, g and h). Figures 3-4, c and d: B6 mice. One B6 mouse in both groups (2, 22, c and d respectively) demonstrated the ability to produce somewhat 119 elevated autoAbs which lasted for only one measurement [0.061 and 0.056 for 2 and 22 respectively compared to average absorbances of 0.032 for both groups]. Only one treated mouse, 26 (d), demonstrated the ability to produce somewhat elevated autoAbs for nine measurements. These measurements, as was seen with C B A mice (figures 3-4, a and b), were negligible when compared to MRL-/pr mice (figures 3-4, g and h). Figures 3-4, e and f: MRL-+ mice. In both groups of MRL-+ mice there were individual mice that produced high titres of autoAbs in such a manner that the autoAb production increased very dramatically in a short period of time, within a few measurements (73, 77, 79, e, and 85, f). Two untreated mice (72, 76, e) showed elevated autoAb production greater than 0.10 absorbance by the end of the experiment. In the treated MRL-+ group (f), two mice (86, 86) also demonstrated higher levels of autoAbs by the end of the experiment. Furthermore, three other mice in the treated group (83, 89, 810, f) demonstrated raised levels of autoAbs by week fifteen and these levels rose moderately [0.15 to 0.30 absorbance] and remained moderately elevated for six measurements. Interestingly, mouse 89 (f), treated, also had an earlier [week three] sustained [three measurements] minor peak [approximately an absorbance of 0.10], shortly after treatment. Overall, after week fifteen, the autoAb production by treated MRL-+ mice (f) was more variable than that seen in the untreated group (e). Figures 3-4, g and h: MRL-lpr mice. Due to the dilution of test sera [1/1000], it appeared there were no readings for the first few weeks of each group, but in reality, autoAb production was already at a significant level [approximately at 0.15 compared with 0.024 absorbance for the 120 negative control]. In both the treated (g) and untreated (h) groups there were mice which produced extreme quantities of autoAbs very rapidly: some by week seven (63, 67, 68, h), others by week thirteen (58, 65, g and h respectively) and still others later on (62, 56, h and g respectively). The major difference between these two groups of mice was that the treated mice (h) produced much higher levels of autoAbs, much earlier, than the untreated mice (g), yet due to the variability of autoAb production by different mice, analyses failed to reveal statistical differences between the two groups. Figures 3-4, iandj: B6-lpr mice. In both the treated (j) and untreated (i) B6-/pr mice groups, similar profiles of autoAb production were revealed. With some mice, autoAb production increased and continued as such throughout the experiment (35, 38, i and 42, 46, 47, j). Other mice demonstrated peaks of autoAb production (31, 32, 33, 34, 37, i and 41, 43, 44, 48, j). The major difference between the treated (j) and untreated (i) groups was that in the treated group, more mice reached higher levels of autoAb production earlier than the untreated mice and these levels stayed higher. In fact, due to the variability in autoAb production by individual mice, the only measurement where a significant difference in autoAb production could be demonstrated was at the second measurement, one week after treatment, using the statistical tool of confidence intervals for the difference between the means of all mice combined, treated versus nontreated [parametric], also by the [nonparametric] Mann-Whitney Test. Overall, one in vivo treatment of mice with a McAb to T sCs was 121 sufficient to have an effect on autoAb production in MRL-+, MRL-lpr and B6-/pr mice, but had little effect on normal B6 and CBA mice. 122 Figure 3-4: Suppressor Tcell deletion therapy; Effect on autoAb production. The following mice [numbers] were treated with B16G-HP to remove T sC at week zero: b) C B A males [ten], d) B6 males [eight], f) MRL-+ females [ten], h) MRL-lpr males [eight], and j) B6-/pr females [nine]. The nontreated mice were as follows: a) C B A females [eight], c) B6 females [nine], e) MRL-+ females [seven], g) MRL-/pr males [eight], and i) B6-/pr females [eight]. Each mouse was bled one week before [week -1] the treatment date, one week after [week +1] the treatment date and subsequently thereafter every two weeks. Serum samples were analysed by ELISA for autoAbs to DNA. In figures a, b, c, and d, the serum samples were diluted to two percent; in e and f they were diluted to one percent; in g and h they were diluted to 0.1 percent; and in i and j they were diluted to 0.25 percent, to obtain detectable autoAb production. The negative control serum was taken from two to four month old Balb/c mice and was diluted to two percent. Absorbance measurements from all serum samples were converted to the same positive control value, thus are all standardized. The statistical tests, confidence interval for the difference between means [parametric] and the Mann-Whitney test [non-parametric] were used to study the resultant Ab profiles. 1 2 3 o.l4n 0.00 | i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i -2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 week week 0.070 -0.065 -0.060 -0.055 -0.050 -0.045 -0.040-0.035 -0.030-0.025 -0.020 -0.015-0.010-0.005 -0.000 2 4 - a — 6 - * 7 - A 8 + neg. cont. CD C\J • i — | — i — i — i — i — i — r 2 0 2 4 6 - I — i — | — i — i — i — i — i — i — • — i — i — i — i — i — i — i — i — i — • — i 8 1 0 12 14 16 18 20 22 24 26 28 week E c m o 0) o c co o w n < 0.070 -• 0.065 -0.060 -0.055 -0.050 -0.045 -0.040 -0.035 -0.030-0.025 -0.020 -0.015-0.010-0.005 -0.000 -D 21 22 ~a 23 -o 24 25 -o 26 -ft 27 -ft 28 neg. cont. i—r 2 0 - l—'—i—•—i—•— i—•—i— 1 — i— 1 —i—'—i— 1 —I—>—l— 1 —I— 1 —I 6 8 10 12 14 16 18 20 22 24 26 28 week Absorbance 405nm o o o o o o p p p o w i o c n o o i o c n o <D CD -vl a> Ol -t^  CO IV) CO CD 1 2 8 Absorbance 405nm o o I ro o ro -. CTi -co -o o p o o o o ro cn o co o _1_ o co 01 o O _J (D <D XT ro co ro o ro ro ro ro 01 ro oo 3 CD <p O O 3 oo oo oo oo oo oo 0 0 oo cn 0 0 oo CO oo ro oo 129 Absorbance 405nm CO 130 Absorbance 405nm zr 131 Absorbance 405nm O 3 CD cp o o 3 8 co co CO cn 8 CO l\3 co 132 Absorbance 405nm 1 3 3 C H A P T E R T H R E E DISCUSSION While the in vivo T sC deletion experiment was in progress, a few experiments were performed to first reconfirm published data and next to and assess, if possible, T sC status in MRL-/pr mice. The aim was to determine if MRL-lpr and B6-/pr mice demonstrate in vitro T sC function. Two dav incubation experiment. The two day incubation experiment (figure 3-1) confirmed previous work done in this lab (45), as well as work done by others (72,132), in that, two day incubation allowed proliferative responsiveness of MRL-/pr NWT. This, in our laboratory was attributed to de novo II3 production; others imparted II2R expression to the response. Regardless of the mechanism(s), two day preculture clearly revealed that some in vivo down-regulatory mechanism(s) had been relieved in the responding MRL-/pr cells, thus allowing these NWT to proliferate [and perform a variety of functions]. That such activity was seen only for MRL-/pr NWT would suggest that S LE background genes [in MRL-+ mice] or the Ipr gene alone [in B6-/pr mice] do not elicit this suppression effect at the age of the mice tested. Since older mice failed to demonstrate the suppressive influence, that suggests greater than one mechanism was operating to generate the suppression seen and with age, could not be alleviated [data not shown]. More complete age related studies are required to support this suggestion. Mixing experiments. Upon first approximation, mixing experiments are an ideal way to assess TsC activity. In figure 3-2, the mixing of C B A NWT with C B A NWT (a) or MRL-+ NWT with C B A NWT (b) revealed responses 1 34 as would be expected. However, when MRL-lpr NWT were mixed with C B A NWT (c), it seemed that high numbers of Ipr NWT were suppressive. A few points must be noted regarding these results. First, as these mice had not been treated to induce suppression in vivo, it wouldn't be expected in vitro. Second, that only MRL-lpr NWT demonstrated some suppressive influence on C B A NWT would support experiment 3-1 data, and that of others which have demonstrated evidence for elevated TsCs (2,13). However, it must be noted that the MRL-lpr NWT used were from a very old mouse, and demonstrated negligible proliferative activity on their own (c, (MRL-/p/)-R). This low response was partially attributed to a mo influence in chapter two, so what may exist, rather than a TsC effect, could be a mo effect. As the MRL-+ mo defect wasn't as substantial as the one seen for MRL-/pr mos, it follows that it would be more difficult to demonstrate that defect in this type of experiment; this reasoning was supported by the fact that even with the MRL-/pr NWT, the influence was seen only at high doses of MRL-/pr NWT added (c). A prediction would be that if much younger MRL-/pr mice were used under the same conditions, the results seen would be less dramatic, if at all significant. Due to the numerous immunological cellular dysfunctions in MRL-/pr mice, extensive planning and laborious purification would be necessary to delineate a T sC influence. When mixing experiments were done with B6 and B6-/pr NWT, no suppression was seen, [data not shown]. In addition, as B6-/pr mice demonstrated rather normal mo function (chapter two), these results complement chapter two data. 1 3 5 B16G in vitro. Next, B16G was added to MLR cultures to determine if T sCs were being induced in the responding population by the stimulating population. B16G is a McAb which recognizes a determinant on T sCs and TsFs, thus by including B16G in culture, the function of TsCs and their factors should be blocked (personal communication A.T.S.). 690 is an irrelevant McAb of the same subclass as B16G and served as a negative control. That either McAb alone with the responding NWT caused some proliferation was expected as these McAbs were ascities preparations. That B16G failed to alter the C B A stimulatory response would imply that T sCs were not induced in the MLR. Due to the variability in response seen by MRL-/pr spleen cell stimulators, the response seen was not statistically different from the control. Because the MRL-/pr mouse used for stimulation was very young [ten weeks] not a large difference in response would be expected even if there were one [as predicted for figure 3-2], as at this age, the disease state of the animal has not advanced very far clinically, and cellular functions are still somewhat intact (3). Also, as mentioned for figure 3-2, the predominanat mo defect would block any possible T sC influence, as shown by the overall reduced response of B6 NWT to MRL-/pr spleen cell stimulators. Again, studies using older mice NWT need to be done before definitive answers will be reached. Thus the overall in vitro assessment of T sC function in T cell responses studied was clouded by the issue of a mo defect in MRL-/pr mice. Nonetheless, in vivo down regulation was suggested by the two day incubation experiment (figure 3-1). B16G-HP in vivo: The focus of this chapter was to assess what 1 36 regulatory influence T sCs ultimately have on B cell populations in diseased mice as monitored by autoAb production, a - D N A IgG. To do this , a well-established method known as TsC deletion therapy (218,219), developed in Dr. Levy's laboratory, was used to eliminate TsCs, serum was harvested, and a-DNA IgG were determinined by ELISA method. B16G-HP was the immunotoxin used to eliminate T sCs in vivo. HP is a lightractivatable toxin, has been used previously for cancer therapy, which when coupled to specific McAb provides a very potent, specific and efficient means of eliminating cells bearing the determinant recognized by the McAb. B16G-HP has been used very successfully for tumour regression (218,219). Rationales for the treatment regiment using B16G-HP and for studying auto a-DNA IgG production are presented in the appendix. B cells and SLE disease. Before discussing the results for the B16G-HP experiment, it is important to understand the relationship between B cell activity and S L E disease. Hyper B cell activity is the common denominator among all murine and human forms of lupus (3,4). B cell function in S L E mice has been extensively studied (4) and has been shown to be elevated via numerous criteria. Two measures of abnormalities have been associated with B cell hyperactivity in murine SLE : one, excessive proliferation and differentiation of B cells upon addition of lymphokine and, two, excessive levels of BCDF , but a normal B cell response to the lymphokine. Much of the elevated B cell proliferation is imparted by the Ipr gene, as evidenced by comparison of enhanced 137 disease in MRL-lpr mice to a late-onset disease seen in MRL-+ mice. However, the Ipr gene need not be the only accelerating factor of disease. Mice having some SLE background genes, if subjected to certain exogenous stimuli, can develop early lethal disease like that seen in MRL-/pr mice. Also, when treated chronically with LPS, normal mice homozygously bearing the Ipr gene developed rapid fatal disease. Continued LPS stimulation in normal mice was of little consequence (4). These results imply that a loss of immunoregulation, as by chronic B cell stimulation, in a lupus predisposed host would be sufficient for early accelerated disease expression. Emphasis here is on the fact that death occurs from IC glomerulonephritis, largely due to a-DNA IgG IC deposits in the kidney, which ultimately caused renal failure. Thus, by removing down regulation by eliminating TsC with B16G-HP treatment, instead of over-riding a regulatory influence by continued positive stimulation of B cells, some effect was expected. Analysis ofB16G-HP results. In view that chronic stimulation of normal mice by LPS didn't cause SLE disease like that seen in MRL-/pr mice (4), it was not surprising to see that no B6 or CBA mice when treated once with B16G-HP developed disease or high titres of a-DNA IgG autoAbs. Chronic removal of TsCs in normal mice may lead to SLE-like IC glomerulonephritis, but more likely would cause a severe combined autoimmunite disease as both T cell and B cell activity would become elevated and mos have only limited regulatory capacity (227). It is interesting that one untreated CBA mouse, 91 (figure 3-4a), showed quite a high autoAb titre, seven times greater than the average production by all members of the CBA group, a titre which approached the levels produced in 138 MRL-/pr mice around the onset of clinical disease of two months (figures 3-4, g and h). Such an episode shows that autoimmune responses may occur, but since it was very short-lived [one measurement], it demonstrates the normal immune system's capacity to bring autoimmunity under control. That one treated B6 mouse (26, figure 3-4d) had elevated levels, compared to the group, of a-DNA IgG for approximately nine measurements is peculiar. In the B6 group treated and non-treated mice were of different sex [male and female respectively], thus the two groups may or may not be directly compared. In fact, B6 female mice have demonstrated minor autoAb production (59). As similar autoAb profiles existed for both groups, it seems likely that sex differences were not playing a role [as in Ipr disease, female sex hormones exacerbate the disease (59) and those of males reduce disease expression (84) and in this experiment the males were treated]. The prolonged, elevated autoAb production in mouse 26 (figure 3-4d) may have been a real effect. Even if the observation seen was real, it still wasn't significant when compared with the level of a-DNA IgG produced by MRL-lpr mice (figures 3-4, g and h). Nevertheless, it revealed how TsC deletion could cause autoimmune phenomena and also reiterates that one treatment to remove TsCs in a non-predisposed host will have little effect. Due to the overall similarities in time course autoAb production.for treated and untreated mice for the remainder of groups (MRL-+ figures 3-4e and f, MRL-lpr figures 3-4 g and h, and B6-/pr figures 3-4 i and j) it is clear that a single treatment to TsCs was insufficient to cause major 1 39 changes. What can be seen visually, however, are minor influences. Specifically, in all three groups, larger numbers of mice produced more-elevated amounts of a-DNA IgG, but variability in autoAb produced by individual mice at any one time prevented statistically significant differences. One exception was the time point of one week after treatment for B6-/pr mice [using the statistical tests: confidence interval for the difference between two means and the Mann-Whitney test]. If there were any strain that would have been predicted to have been affected, it would have been this group. The reason for this prediction is based on the effects of L P S treatment in B6-/pr mice (4). That the a-TsC treatment affected change within one week of treatment demonstrates the power of the treatment in these mice, and, predictions for future chronic TsC removal would be that of L P S treatment, extreme, rapid, fatal disease. The results of this B16G-HP treatment become important for providing evidence that exogenous [environmental] influences on individuals with genetic predisposition to disease [remember one Ipr gene consequence is the production of autoAbs of the IgM class] can elicit the phenotype of disease [whereas it may not have happened at all] earlier [if it did occur] and perhaps more severe. The treated MRL-+ mice (figure 3-4f), with a different genetic predisposition, rather than earlier disease, late disease was still seen, but somewhat more severe with respect to autoAb production. With MRL-/pr mice (figures 3-4, g and h), a combination of the two minor effects seen in the MRL-+ and B6-/pr mice appeared to be the situation. What is clear about S LE disease in these mice strains is that it must 1 40 be caused by greater than one immune defect acting in concert; mice with the Ipr gene alone fail to develop disease (4), mice with S L E background genes develop mild disease later, and mice with both types of genes develop severe disease earlier (3). Also, factors, either immunological in nature or not, can influence disease outcome. That some consequence was seen in all of the diseased mice treated provides support for T sC defects in S L E and perhaps in other autoimmune disease. In addition, that one treatment allowed change demonstrates the delicacy or fragility of the immune system in these mice. Many speculations could be made as to the reasons why or what may be happening, but due to the relatively minor differences seen, speculations will remain as such, to provide hypotheses, until further experimentation is done. Nonetheless, B16G-HP immunotherapy may provide valuable insight into the mechanisms causing S L E disease and autoAb production. Relation of autoAbs to SLE disease. Despite a few examples of autoAbs directly being involved in disease pathogenesis, it is becoming clear that the inherent properties of autoAbs, as molecules themselves or at the molecular level, are secondarily related to the disease which develops (149,187,228,229). Studies by Kof lerand others using monoclonal autoAbs derived from lupus mice have shown that the germline genes, usage of the germline gene repertoire, use of gene segments and somatic mutation are all similar for the production of normal Abs and autoAbs. Their studies revealed that autoAbs of a given specificity may be produced by restricted use of specific VH gene families and expanded in 141 \ response to specific autoAgs instead of being polyclonally activated (68). That the preimmune Ab repertoire is directed against self suggests that auto reactivity is the norm (231). Cohen and Cooke, although lacking experimental evidence, suggest that since we all produce natural autoAbs of the IgM class, they must serve some protective function, for example in preventing immune responses to self (232). Only when autoAbs switch to the IgG subclass do they have the potential to become pathogenic and on that note, others point out that when additional abnormalities operate in concert with autoAb production, that's when the development of disease is seen (233,234). The observation that IgM and IgG a -DNA autoAbs display broad, yet limited or specific, cross reactivity has led to the suggestion that DNA is not the immunogen (231,235). Indeed DNA is not a very good immune stimulator (235). In a review by Theofilopolous and Dixon (3) i) limiting dilution analysis showed similar frequency of a -DNA IgM cells in normal and lupus mice, ii) normal and autoimmune mice share common a -DNA ids, and iii) a- id Abs raised in normal mice cross reacted with MRL-lpr serum ids. All of these observations reinforce the commonality of a -DNA Abs. Nonetheless, in certain circumstances, such as lupus, these Abs aid in the pathogenesis of disease, and the Ipr gene augments a -DNA production as well as disease severity. Relation of TsC to SLE disease. TsC activity in MRL-lpr mice has been shown to be normal or elevated (7,8,11,12,15). However, due to the cellular abnormalities present in the immune system of these mice, which 1 4 2 increase with age, the results must be interpretted with caution. Extensive purification of cellular populations, in addition to proper controls, must be done in order to delineate specific defects and, in the above-mentioned references, were missing. Nonetheless, these studies did elucidate immune system peculiarities and potential T sC irregularities in MRL-lpr mice. Another phenomenon, the AMLR, defined as the proliferative response of T cells to self class II antigen on autologous non-T cells (29,155), has been shown to be deficient in MRL-lpr mice, with the responder population deemed responsible for the lack of response seen (3,29,30). The A M L R has been considered very important in the generation of T suppressor-inducer signals and, hence, the down regulation of immune responses stimulated by self MHC (152). In the human, a functional CD4+ suppressor-inducer population has been defined on the basis of lack of expression of a surface molecule 4B4 and expression of the surface molecule 2H4, hence CD4+ 4B4- 2H4+. 4B4, [is CDw29 (153), 135 kDa (160)] is a molecule related to the integrin family of adhesion molecules (154), and presumably functions in a similar way. 2H4, [or Lp 220, 220 kDa] is a member of the T200 or leukocyte common antigen/CD45 family, but is designated CD45R due to its restricted distribution of B, NK, and T cell subsets (153). 2H4 may be involved in cell-cell interactions (154,157,158,161), signal transmission (154,161), cell adhesion or homing (158), as its relation to cellular function also remains to be determined. CD4+ 4B4+ 2H4- cells are the helper-inducer subset and respond to PWM stimulation of Ig synthesis, 1 4 3 Ag-specific Ab production and soluble Ag stimulation (156). CD4+ 4B4-2H4+ cells, the suppressor-inducer subset, as mentioned above with respect to the AMLR, proliferate maximally to autologous non-T cells expressing class II antigens (156). Upon activation, these cells acquire increased levels of the 2H4 molecule and subsequently turn on CD8+ cells to effect suppression (152,155,156). In S L E patients with renal disease, the percentage of CD4+ 2H4+ cells in the peripheral blood is substantially reduced, and in some cases these cells are functionally defective (159), and the CD8+ population of cells is increased (156,159). A direct correlation of a reduced level of 2H4+ cells and renal disease has been demonstrated (155), implicating the necessity of such a population of cells for maintenance of responses to self. As the suppressor-inducer population responds to autologous non-T (156) and in the AMLR the stimulator cell seems to be a mo, a mo defect (chapter two) would contribute to a reduced or ablated AMLR seen in MRL-/pr mice (3,29,30). In the same auto-regulatory sense, auto cx-ids are also necessary for down regulating immune responses and some auto a-id Abs are lacking in MRL-/pr mice (236). Thus, more than one mechanism or defect may be operating to prevent the regulation of Abs to DNA in MRL-/pr mice (191). Other in vivo immunomodulations performed to alter murine lupus. Numerous other immunomanipulations have been examined for their influence on S LE disease in MRL-/pr or other Ipr and lupus mice. One of the earlier modifications was the inbreeding of the xid gene, for X-linked immunodeficiency, into MRL-/pr mice (237,238). xid causes the depletion of a mature subset of B cells and the impairment of some 144 T-dependent Ab responses. Despite the improvement of renal disease and overall autoAb production, a-DNA production was still pronounced. When xid was introduced into C3H-gld [generalized lymphadenopathy, a gene which causes similar phenotypic disease to that seen with Ipr (61)] and C3H-/pr mice, it was almost able to dissociate the B and T cell manifestations of Ipr disease as it wiped out most of the B cell abnormalities caused by Ipr (239). When considered together, the above results are interesting as they demonstrate the profound Ipr gene influence in the presence of background S LE genes in causing not only a T cell disease, but also a B cell disease with respect to auto a-DNA production in MRL-/pr mice. When considered as a therapeutic intervention, this method would be of little use in humans. Another treatment performed some time ago was that of radiation therapy, low dose, 300 rad, WBI or TLI [multiple small doses, 200 rad] (65,77). Both suppressed autoimmune disease in MRL-/pr mice implicating a radiosensitive cell(s) in the disease process. What first comes to mind is selective TsC defects, that prevent regulation of the 'odd 'T (chapter one), and B cells [i.e. a-DNA producing], are being eliminated so that other mechanisms [CTL, AMLR] can be activated and lead to the regulation of the 'unregulated' populations. In this sense, the 'odd 'T resembles a situation much like that seen with tumour growth, the main difference being that these cells are not malignant (3,220). Their increase in numbers would necessarily be seen as a shift in immune status. It would follow then, that since they arise early in the development of disease [as with tumour growth] there probably is a specific immune response against them. But, also seen with tumour 1 4 5 growth, their persistence would lead to the generation of T sCs to prevent continued immune responsiveness against them [as such a response in essence would be autoimmune]. However, radiation therapy must have removed a precursor necessary for disease expression as, B16G-HP treatment to remove TsCs caused, if anything, elevated auto a-DNA production rather than a reduction. This suggests that some T sC function was working to prevent other cellular abnormalities from completely rendering the host immunologically incompetent. Thus, radiation treatment revealed a necessary T cell involvement of disease which doesn't appear to be at the effector TsC level. These bits of information complement the earlier discussed observations of seemingly lack of Ab molecule involvement in causing disease (68) and suggest that the absence of a ThC necessary for the induction of effector T sC may be the missing link. In MRL-lpr, mice several in vivo therapies with McAbs have been done to intervene with B or T cell function. 1) Monoclonal a-DNA treatment led to a-id Abs being produced, but to no change in autoimmune status (240), reinforcing the suggestion that autoAb production is secondary to some other primary cellular defect. a-DNA treatment in normal mice did not cause disease either (241). 2) Monoclonal a-Sm treatment allowed increased survival, reduced renal damage, but no change in auto a-DNA was seen (230). a-Sm is a subset of Abs which react with a ribonucleoprotein isolated from a patient called Smith, are present only in S LE and in about twenty-five percent of MRL-/pr mice (242). This study with a-Sm implies that 1 46 several dysregulated immune functions, such as autoAbs, contribute to the overall disease process and the alleviation of one or more dysregulated autoAbs helps relieve the extent of disease, but can only correct the specific defect which it is directed to. 3) McAbs to determinants present on the 'odd' population of T cells (chapter one), specifically to Mel 14 [homing receptor] or 6B2 [B220 or Ly5] redirected or reduced respectively, lymphadenopathy, but again, no change in a-DNA was seen (101). In this study no mention of the influence of these treatment on survival was mentioned and likely had none which may be interpretted to mean that the lymphoproliferation is a consequence of other more important primary cellular defects [as discussed in chapters one and two] which need to be regulated before disease process will change. On this note, the use of YE19.1.3 in vivo would probably result in similar changes in MRL-/pr mice. 4) a-Thy 1.2 [a McAb to all T cells] treatment in MRL-/pr mice, early in disease, as a one time treatment failed to alter disease significantly. However, when chronic treatment was performed, lymphoproliferation, autoAb production and renal disease were reduced and survival increased, emphasizing the T cell dependence of disease (88). 5) Monoclonal a-L3T4 [to remove CD4+ cells] treatment has been performed in NZB/W mice, another lupus strain, after the establishment of disease and allowed for improvement of disease all round including a-DNA production, substantiating the suggestion of CD4+ cells in the disease process, in these mice (243). When this McAb was administered to normal mice as a Fab fragment, it caused a minor reduction of CD4+ cells and allowed for specific immune tolerance to be developed (244). When normal animals were treated with the whole McAb, 147 CD4+ cells did decrease in number in the spleen, LNs, PP and blood, and in the spleen, an increase in CD8+ and DN cells was seen (150,151). Thus, oc-L3T4 treatment appears to be specific and not cause any adverse effects in normal mice. Chronic a-L3T4 treatment in MRL-lpr disease led to improvements of disease much like that seen for NZB/W mice in that a-DNA IgG was greatly reduced. There was no effect on IgM a-DNA (128). 6) McAb treatment to the II2R, to class II molecules, and a-id to a-DNA have been done in NZB/W mice, as well, and have demonstrated alleviated renal disease and overall improvement of autoimmune disease. All treatments suggested a role for activated ThC in the pathogenesis of disease; activated ThCs display H2Rs and secrete lymphokines which induce class II expression on APCs (192,204,245). At this point it is important to consider experiments which have used CsA as therapy, an antifungal agent with potent suppressing ability with respect to immune function. CsA acts to inhibit ThCs, probably by preventing II2 mRNA expression, without an effect on TsCs; however, the complete range of functions by CsA remains unclear at this time (246). In MRL-/pr mice, CsA treatment improved disease state without a reduction in autoAb production (247,248) and served to exemplify the action of SLE and the Ipr genes on B cells in the absence of T cell help. Each of the above treatments dissects out contributions of different cellular functions to SLE disease. That McAb treatment in vivo can serve to either enhance or ablate specific cellular functions, perhaps treatment of MRL-/pr mice with B16G would be a better therapy to see if TsCs could be activated and down regulate disease. Whatever is done, further 148 experimentation is needed to determine the exact involement of the TsC loop in immunoregulation in MRL-/pr mice. In summary, MRL-/pr mice were tested for in vitro TsC function. It had previously been shown that preculture allowed for responsiveness by MRL-lpr NWT, thus experimentation was done and confirmed earlier results. Mixing experiments suggested that there may be some in vitro TsC function, but culture with a McAb specific for TsCs and TsFs [B16G] could not support those results. The above observations were not seen with B6-/pr NWT. Lastly, in vivo TsC function with respect to auto a-DNA production was assessed. MRL-+, MRL-/pr and B6-/pr mice were treated once, at the age of two months, with the immunotoxin B16G-HP to remove TsCs. Control CBA and B6 mice were also treated. This treatment led to elevated auto a-DNA production in all experimental mice; due to the variability in autoAb production for individual mice, significant differences between the treated and untreated groups could only be shown for B6-/pr mice, one week after treatment. The same treatment in control mice failed to induce auto a-DNA IgG. By further studying the cellular defects, potentially in CD4+ cells, in APCs and with respect to TsC function, we hope to determine what role, or lack there of, each plays in eliciting autoimmune disease in MRL-/pr mice. Understanding the mechanisms which lead to B cell hyperactivity and autoAb production will help in designing therapeutic intervention and perhaps the development of cures for not only SLE, but other autoimmune diseases as well. 149 APPENDIX Rationale for the treatment regiment using B16G-HP: The in vivo use of B16G-HP was rather 'blind' as it was unclear if: 1) the conditions used were such that there would be an observable effect 2) one treatment would be enough 3) the age of the mice was appropriate. If there was an effect, it was unclear a) how profound it would be b) if it would be early or late. 1) Of primary interest in this study were the effects such a treatment would have in MRL-lpr mice. As the conditions used were established for a tumour regression model, it seemed reasonable that they would be sufficient in MRL-/pr mice. Tumour rejection is prevented by elevated numbers of TsCs. Thus the treatment regiment used for tumour regression was such that it was able to abolish elevated numbers of TsCs (218,219). As the DN T cells in MRL-lpr mice are not malignant, (3,220) and it has been suggested that there are increased numbers of TsCs in the spleen of these animals (2,13), it was reasoned that the amount of immunotoxin used would be sufficient to remove circulating TsCs. None of the negative controls were performed as in the tumour regression model they proved to have no effect. 2) As the immune system of MRL-lpr mice is predisposed to immunoregulatory abnormalities, it was considered to be fragile and thus 150 would be easily disturbed, hence one treatment was deemed sufficient. 3) Since MRL-lpr mice manifest clinical disease around eight weeks of age (2,3), it was felt that this time would prove to be the best age at which to treat the mice. At this age when autoAbs in MRL-lpr mice become signigicantly elevated (3,220). Rationales for autoAb assessment. Auto a-DNA IgG production was the immune function chosen to be monitored for several reasons. First, a-DNA Ab production is always associated with lupus disease, in humans and mice, thus a readily detectable characteristic would be under study (221). Second, IgM autoAb production ensues in B6-/pr and B6-lpr-nu mice (63) before clinical disease expression. a-DNA IgM is not associated with the morbidity and mortality of the disease, which result from IC glomerulonephritis (3). Although the characteristics of a-DNA Abs have been well defined (reviewed in 231), and it remains unclear what a-DNA Ab traits are necessary for their pathogenicity, a direct correlation between serum a-DNA IgG and clinical disease exists (221), thus is of utmost interest to resolve. Third, Steinberg [at the Nineteenth International Leukocyte Culture Conference, May 1988] has found that B cell hyperactivity in lupus is peculiar in that it appears to be oligoclonal rather than polyclonal. Thus TsC function may parallel this B cell oligoclonality and polyclonal removal of TsCs could affect polyclonal B cell hyperactivity. Any effect developed would give insight to the disease process. Lastly, a-DNA Abs are easily detected by ELISA. ELISA is a rapid, sensitive (222), specific (225), stable (226), inexpensive technique which is not affected by non-lg serum components which can bind DNA 151 (222). ELISA is easily measureable, and can be, if desired, used to distinguish between the different isotypes of a-DNA (224). Early problems encountered with ELISA technique included high background, false positives, and poor reproducibility (223). However, such problems have been largely overcome by experimental manipulations. The major drawback of ELISA is that the absolute amount of Ab present in serum is not easily determined (224). As for the types of a-DNA Abs that develop, and can be detected, with lupus disease, Theofilopolous and Dixon (3) review that there are four: 1) ones that react with double-stranded DNA only and are rare, 2) ones that react with single-stranded DNA only, 3) ones that react with both single- and double-stranded DNA, which also show cross reactivity with other substances such as phospholipids, and 4) those that react with Z-DNA. The ELISA used in these experiments probably detects the first three types of a-DNA Abs, if not all four, as the DNA used in the assays was calf thymus DNA purchased from Sigma and sonicated to fragments mainly of the size of one- to three-thousand bases as determined by agarose gel electrophoresis [data not shown]. Breaking up the DNA as such allowed for better activity from serum samples, presumably due to the generation of free single-stranded DNA ends which would allow for type (2) Abs to be detected. 152 LITERATURE CITED 1. Murphy, E.D. and J.B. 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