lpr mice: a role for self Ia-reactive T cells

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The basis of autoimmunity in MRL-lpr/lprmice: a role for self Ia-reactive T cells Yvonne J. Rosenberg, Alfred D. Steinberg and Thomas J. Santoro Murine models of systemic lupus e~thematosus (SLE) have significantly contributed to our understanding of human autoimmunity. One such strain, theMRL-lpr/lpr, spontaneouslydevelopsan autoimmune diseasemanifestedclinically by arthritis, vasculitis, immune-complexglomerulonephritis and autoantibody production1-3. In this article Yvonne Rosenberg and her colleaguessuggesta theoreticalbasisfor the developmentof autoimmunity in MRL-lpr/lpr mice. Mice which are homozygous for the lpr gene also exhibit profound lymphoproliferation characterized by expansion of a T-cell subset bearing a dull Lyt-1 +2 - phenotype ~'5. Paradoxically, in the face of massive T-cell growth in vivo, lpr spleen and lymph node cells display markedly impaired interleukin 2 (IL2) production and proliferate poorly in response to mitogenic lectins 6'7. It is currently believed that abnormalities in T-cell function play a fundamental etiologic role in the autoimnmune disease of MRL-lpr/Ipr mice. This is supported by studies which show (i) that marrow pre-T stern cells from affected MRL-lpr/Ipr mice can mature in a congenic MRL- +/+ thymus and induce a disease which is identical to that usually seen in the lpr/lpr mouseS; and (ii) that neonatal thymectomy of MRL-lpr/lpr mice prevents the development of both lymphadenopathy and autoimmunity 9. However, the precise pathways which lead to derangements in the T-cell pool and the mechanism by which such aberrations are sustained remain unclear. Our proposal of a theoretical basis for the development of autoimmunity in MRL-lpr/lpr mice stems both from data gleaned in short-term in-vitro cultures of cells from MRL-lpr/lpr mice l°'ll and from studies using long-term T-cell lines derived from spleen and lymph nodes of this strain 12. Such lines grow spontaneously in vitroand exhibit a greater diversity of effector functions than previously described using fresh cells. Since the autoimmune state is clearly complex, derangements at both the cellular level and the level of soluble mediators will be discussed in terms of their potential pathogenetic roles. Our idea is that there exists a T-cell subset which recognizes self Ia determinants and is stimulated to produce lymphokines. These, in turn, signal further Ia expression on non-T cells and proliferation of the T-cell pool. This circuit results ultimately in lymphadenopathy and stimulation of autoantibody and IgG production by B cells (see Fig. 1). T h e T - c e l l b a s i s for a u t o i m m u n i t y - a role for I a - r e a c t i v e cells?

Despite the greatly increased number o f T cells in t h e lymph nodes of MPd-lpr/lpr mice, most of these cells are in a resting state 13, suggesting that the majority of lymphocytes accumulate as a result of migration rather than in situ proliferation. The proliferative signals rebponsible for the Section on Cellular Immunology, Arthritis and Rheumatism Branch, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20205, USA. © 1984, Elsevier Science Publishers B.V., Amsterdam 0167 - ff919/84/$02.00

extensive growth of the dull Lyt-1 +2 - T cells characteristic of these mice, therefore, appear to be delivered prior to their appearance in the lymph node, possibly in the spleen or thymus. What are these inducing signals? Although responses in autologous mixed lymphocyte reactions have been reported to be impaired in the MRLlpr/Ipr ~4, the recent finding that many T-cell lines derived from these mice demonstrate reactivity to self Ia antigens 15raises the intriguing possibility that autologous Ia may be a relevant initiating 'antigen' in the etiology and pathogenesis Of MRL-lpr/lpr disease. Indeed, the critical role of Ia antigens in vivo with regard to disease expression has been demonstrated in relation to tumor rejection a6 and experimentally induced autoimmune diseases ~7. However, since Ia-reactive T cells are commonly found during the establishment of antigen-specific MHCrestricted T-cell clones from many strains ~8, any consideration of a role for seif ia-reactive T cells il~ the development of autoimmunity presupposes that the conditions for Ia presentation are quantitatively and/or qualitatively different in autoimmune-prone mice compared to those present in immunologically normal strains. In this connection, it appears that the unusual conditions necessary to support continued triggering and expansion of an Ia-reactive T-cell pool do exist in such mice. Thus, it has been reported that MRL-lpr/lpr mice possess greatly increased numbers of Ia + macrophages (Md?) 19'2°. In peritoneal exudate cells, this represents a 10- to 80-fold increase in the number ofIa ÷ while increases in the spleen and thymus can be largely accounted for by increased cellularity. Whether the amount of Ia expressed per cell is also increased is not clear. It is assumed that Ia expression on B cells will also be increased and that such cells can act as stimulators for Ia-reactive T cells. Moreover, a mechanism to sustain continued high Ia levels is also known to operate in mice which bear the lpr gene. T cells from the thymus and spleen of MRL-lpr/lpr mice, but not from the enlarged lymph node, are unique in their ability to elaborate a macrophage Ia-positive recruiting factor, (MIRF) in the absence of deliberate exposure to exogenous antigen'9. That the active cytokine in M I R F is immune interferon (IFNy) is indicated by the observation that identical effects on Ia expression by macrophages and B cells have been achieved using purified IFNy in vitro21-23. Based on the above findings, a positive feedback circuit is postulated to exist in which a population of MRL-lpr/lpr T cells responds to autologous Ia with elaboration of lymphokines. These soluble mediators, in turn, signal both increased Ia expression on non-T cells and prolifera-

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Fig. 1 Schemedepictingpossiblecellular interactions and cytokine prodnction leading to autoimmunityin MRL-lpr/lpr mice. .la ~ a

CSF

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1

IFN'7

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AUTOANTIBODY POLYC/ONAL B-CELLACTIVATION tion of the T-cell pool. The precise nature of the determinants being recognized is not clear. T cells m a y have the capacity to bind to and to recognize Ia determinants either alone or in association with autoantigens and/or environmental antigens presented by autologous presenting cells. If the expanded dull Lyt-1 ÷ T-cell population has a skewed specificity repertoire or activation requirements different from other T cells, selective expansion of this subset would be facilitated. Finally, an alteration in any part of the circuit m a y result in organ-specific differences in the lymphocyte populations. For example, a deficiency in the appropriate Ia-stimulating cells necessary for activation o f T cells m a y be the reason for the resting state of lymph node T cells. The possibility that migrant cells in the lymph node are functionally terminal is less likely since lymph node T cells, although unable to produce I F N spontaneously, are able to do so following in-vitro culture with concanavalin A (Con A) (T. J . Santoro, unpublished observations). Furthermore, a paucity of the appropriate Ia + presenting cells m a y provide an explanation for the difficulty encountered in establishing long-term T-cell lines derived from MRL-Ipr/lpr lymph nodes (our unpublished observations) relative to those from the spleen, an organ which contains a relatively large n u m b e r of activated and proliferating cells ~s.

Derangements in cytokine production in MRL-lpr/lpr mice: an explanation for cellular abnormalities Excessive Ia positivity of macrophages or B cells m a y

Increased Ia expression by macrophages and B cells stimulate T ceilswhich recognize self-Ia determinants either alone or in association with autoantigens. Activated T cells, by virtue of newly expressed receptors, produce and/or respond to a variety of cytokines, for example IFNy, colony-stimulating factor (CSF), IL2, a B-cell proliferating factor (BPF), and a T-cell replacing factor (TRF). In the case of IFNy, elevated levels result from both the spontaneous synthesis by MRL-lpr/lprT cells and the absence of a suppressor cell capable of regulating its production. Increased numbers of Ia-bearing cells are the consequence of the enhanced activity of IFN and its Ia-inducing properties. CSF, known to be produced by MRL-lpr/IprT-celllines, is thought to act on macrophages to promote ILl release. As in immunologically normal mice, ILl is presumed to play some role in the production of IL2 by competent T cells. The dull Lyt 1 +2 - T cellspossibly both produce IL2 and proliferate in response to their own growth factor. At least two types of T-cell-derived factors act on a population of B cells, resulting in autoantibody production and large increases in the numbers of Ig-reacting cells:one promotes B-cellgrowth (BPF); a second functions as a differentiation signal (IKI~) whlctl apparcnn)' k:ad~: t~J Ig ~c~ tion. Finally, activated B ceils may perpetuate the cycle by further stimulating the Iareactive T ceils to release lymphokines.

either be an inherent defect or the consequence of continuous stimulation by IFN. In the case of MRL-lpr/lpr mice, in contrast to most strains, the ability o f T cells to spontaneously synthesize an Ia-inducing factor 19 and the finding of serologically detectable levels of I F N in unstimulated animals (A. D. Steinberg et al., unpublished observations) strongly suggest that Ia expression is constantly being induced. In addition, whereas I F N is primarily produced by L y t - 1 - 2 + T cells in spleen cell cultures from normal mice 24, it is predominantly a product of Lyt-1 *2 - T cells in MRL-lpr/lpr mice ~°. Thus, cells with the same phenotype as those which are responsible for the massive lymphoproliferation seen in the lpr mouse are capable of I F N production. Accordingly, constitutive IFN synthesis is also seen in Lyt-1 +2 T-cell lirtes obtained from this strain (Y. J . Rosenberg and T. J . Santoro, unpublished observations). Another finding which links abnormalities in I F N activity to the development of autoimmunity is the observation that the regulatory networks which dictate the level of this mediator appear to be malfunctioning in autoimmune-prone mice. Preliminary experiments have shown that T cells capable of suppressing I F N y synthesis are absent in MRL-lpr/lpr mice zS. Thus, I F N levels, and consequently Ia expression, are high because of both spontaneous synthesis and a breakdown in the ability to regulate its production. Since a subset of Lyt- 1 +2 +3 + T cells necessary for antigen-specific suppression appear to be absent in MRL-lpr/lpr m i c C 6, it is possible that the

66 defect in IFN regulation also results from a deficiency in this same subpopulation. Whether IFN production is prematurely elevated due to spontaneous synthesis or a lack of suppression during the neonatal period is unclear. It is also conceivable that lpr-bearing mice intrinsically develop an abnormally large number of Ia* -stimulating cells which indirectly result in elevated levels of IFNy. Although the genotype of the thymus is not the determining factor in the expression of autoimmunity 8, neonatal thymectomy does prevent the disease 9, indicating that the capacity of MRL-lpr/Ipr T cells to spontaneously produce IFN is genetically programmed for expression as early as the pre-thymic stage. In immunologically normal mice IFN synthesis appears to require the production of IL2 27. However, experiments in MRL-lpr/lpr mice have shown that in Con A-stimulated spleen cell cultures, which display minimal IL2 activity, normal levels of IFN), are demonstrated, suggesting an independence of these two lymphokine activities 1°. Recently a similar dissociation between IFN production and proliferation has been reported following stimulation of antigen- specific T-cell clones 28. Although it has been suggested that defects in IL2 activity are important in the development of routine lupus 7, results from this laboratory have indicated that normal amounts of IL2 can be produced by MRL-lpr/lpr cells. Thus, we have found that levels of IL2 comparable to those seen in normal mice can be achieved by culturing MRL-lpr/lpr T cells with both Con A and the co-mitogen phorbol myristate acetate ( P M A ) ' . Moreover, such T cells require minimal levels of IL2 to proliferate optimally ~9, suggesting a dissociation between the amount of IL2 produced and the growth potential of cells in the same culture. Finally, long-term T-cell lines derived from fourmonth-old MRL-lpr/lpr mice can constitutively produce IL212. Based on the above we suggest that IL2 is produced in vivo but that a high turnover rate renders it undetectable. The hyporesponsiveness of fresh spleen or lymph node cells could then be a consequence of a concentration-dependent down regulation of IL2 receptors, with possible re-expression following culture in vitro. Indeed, Con A-stimulated lymph node cells become responsive to IL2 when precultured for three days in vitro prior to mitogen addition 12. The explanation that T cells express a high density of IL2 receptors which constantly absorb IL2, however, cannot be excluded. The status of interleukin 1 (ILl) activity in cultures from MRL-lpr/lpr mice is incompletely understood. Although production of this monokine has been reported to be normal, experiments in this laboratory have shown that inducible IL 1 activity is grossly deficient in the setting of frank autoimmunity (T. J. Santoro et al., unpublished observations). The latter finding may be unrelated to the progression of autoimmune disease in the lpr mouse since Ia ÷ B cells have been shown to stimulate lymphokine release from T-cell clones in the absence of ILl 30 B cells and autoimmunity In MRL-Ipr/lpr mice, uncontrolled T-cell activation leads to extensive B-cell stimulation. This results in the production of autoantibodies to many autologous antigens and a marked, non-specific increase in Ig-secreting

Immunology Today, vol. 5, No. 3, 1984

cells 1-3's.The gready enhanced helper cell activity, which accompanies the increased numbers of dull Lyt-1 * cells, is reflected in the predominance ofT-cell-dependent IgG isotypes of the secreted antibody and Ig ~1. A B-ceU differentiation factor derived from these T cells has been shown to selectively enhance IgG production 32. In addition to help for Ig secretion provided by long-term T-cell lines, it appears that supernatants from these lines can also provide signals for B-cell growth 33. Thus, in this environment, unlike that in most strains, MRL-lpr/lpr B cells are constantly provided with both proliferative and maturational signals. Moreover, B cells which express Ia may also provide the inductive stimuli for T cells to proliferate and to produce the lymphokines required to perpetuate the cycle. The MRL-lpr/lpr mouse: an in-vivo autologous mixed lymphocyte reaction In summary, we suggest that unregulated production of IFN is central to the development of autoimmunity in MgL-lpr/lpr mice (Fig. 1). The uncontrolled IFN activity results in high Ia expression on Md~ and B cells. The Iabearing cells, in turn, stimulate T cells to release lymphokines. These soluble mediators act as helper factors for B cells (B-cell proliferating factor, T-cell replacing factor) Mdp (colony-stimulating factor), other T cells (IL2), and as a source of IFN to continue the cycle. In this way, the MRL-lpr/lpr mouse manifests a continuously ongoing autologous mixed ,~y--p~'~, ,,- .,u~-. ~. . .~. action directed exclusively towards help and without the capacity for suppression. A circuit of this nature provides autologous Ia-reactive T cells with an important role in the induction and propagation of autoimmunity and could be of value in predicting possible manipulations in vivo aimed at preventing or ameliorating disease.

Acknowledgements The authors wish to thank Drs Ronald Schwartz and Dan Longo for many helpful discussions and for a review of the paper. W e are grateful to Ms Betty Roupe-Grittin for aid in the preparation of this manuscript.

References 1 Murphy, E. D. and Roths, J. B. (1980) Mouse News Lett. 58, 51 2 Steinberg, A. D., Huston, D. P., Taurog, J. D. etal.(1981)Immunol. Rev. 55, 121 3 Theofilopoulos, A. N. and Dixon, F. J. (1982) Am. J. Pathol. 108, 321 4 Lewis, D. E., Giorgi, J. v. and Warner, N. L. (1981) Nature (London) 289, 298 5 Morse, H. C , III, Davidson, W. F., Yetter, R. A. et aL (1982) J. Immunol. 129, 2612 6 Altman, A., Tbeofilopoulos, A. N., Weiner, R. etal. (1981)d( Ex# Med. 154, 791 7 Wofsy, D., Murphy, E. D., Roths, J. B. et al. (1981).]. Exp. Med. 154, 1671 8 Theofdopoulos, A. N., Balderas, R. S., Shawler, D. L. et aL (1981)J. Exp. Med. 153, 1405 9 Steinberg, A. D., Roths, J. B., Murphy, E. D. et al. (1980)J. Immunol. 125, 871 10 Santoro, T. J., Benjamin, W. R., Oppenhelm, J. J. and Steinberg, A. D. (1983)J. ImmunoL 231,265 11 Santoro, T.J., Luger, T. A., Raveche, E.S. etal. (1983)Eur.J. lmmunol. 13, 601 12 Rosenberg, Y. J., Steinberg, A. D. and Santoro, T. J. (submitted for publication) 13 Raveche, E. S., Steinberg, A. D., DeFranco, A. L. and Tjio, J. H. (1982)J. IrnmunoL 129, 1219 14 Glimcher, L. H., Steinberg, A. D., House, S. B. and Green, I. (1980)J. Imrnunol. 125, t832 15 Rosenberg, Y. J. and Santoro, T. J. (manuscript in preparation)

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16 Greene, M. I., Perry, L. L. and Benacerraf, B. (1979)Am.J. PathoL 95, 159 17 Steinman, L., Rosenbaum, J. T., Sriram, S. and McDevitt, H. O. (1981) Proe. Natl Acad. SoL USA 78, 7111 18 Imperiale, M. J., Faherty, D. A., Sproviero, J. F. and Zauderer, M. (1982)~ Immunol. 129, 1843 19 Lu, C. Y. and Unanue, E. R. (I982) Clin. ImmunoL ImmunolpathoL 25, 213 20 Kelley, V. (1983) Fed. Proc. Fed. Am. Soc. Exp. Biol. 41, 481 (Abstr.) 21 Steeg, P. S., Moore, R. N., Johnson, H. M. and Oppenheim, J. J. (1982)J. Exp. Med. 156, 1780 22 King, D. P. and Jones, P. P. (1983)J. ImmunoL 131,315 23 Wong, G. H. W., Clark-Lewis, I., McKimm-Breschkin, J. L. et aL (1983)J. lmmunoL 131, 788 24 Sonnenfeld, G., Mandel, A. D. and Merigan, T. C. (1979) Immunology 36, 883

H]I.°.,..

New

25 Santoro, T. J. and Steinberg, A. D. (manuscript in preparation) 26 Gershon, R. K., Horowitz, M., Kemp, J. D. et al. (1978) in Genetic Control of Autoimmune Dispose (Rose, N. R., Bignazzi, P. E. and Warner, N. L., eds.), p. 151, Elsevier/North-Holland, New York 27 Farrar, W. L.,Johnson, H. M. and Farrar, J . J . (1981)J. Immunol. 126, 1120 28 Hecht, T. T., Longo, D. L. and Matis, L. A . J . Immunol. (in press) 29 Santoro, T. J., Raveche, E. S., Luger, T. A. et al. (manuscript submitted for publication) 30 Kappler, J., White, J., Wegmann, D. et aL 1982 Proc. NatlAcad. Sci. USA 79, 3604 31 Theoft!opoulos, A. N., Slawler, D. L., Eisenberg, R. A. and Dixon, F. J. (1980)J. Exp. med. 151,446 32 Prud'homme, G. J., Balderas, R. S., Dixon, F. J. and Theofilopoulos, A. N. (1983),}'. Exp. Med. 157, 1815 33 Rosenberg, Y. J. (manuscript in preparation)

directions in research

Helper T lymphocytes and isotype expression Two recent articles, by Teale 1 and Lanzavecchia 2, have shown that individual clones of helper T (TH) lymphocytes and clones of alloreactive T cells are able to trigger the expression of all classes of immunoglobulins (Igs). These confirm some earlier reports 3-5but contradict other conclusions 6-8 that for Ig expression (isotype, allotype or idiotype), help from two types o f T cell is needed: Tin, a carrier-specific, major histocompatibility ( M H C ) restricted cell: and T~o. reco~nizin~ an I~ determinant. The experiments of Rosenberg and Asofsky8 show, for instance, that IgG but not IgM expression requires signals from a Tin-cell population that is absent in mice suppressed at birth for Ig production. Teale 1, using classical T-cell culture technology, has generated a number of TH-cell clones specific for mouse keyhole limpet hemocyanin (KLH). These were used in vivo to stimulate B cells in a modified splenic fragment assay so that the isotypes produced by individual B-cell clones could be assesed. These experiments indicate that a single TH-cell clone can generate individual B-cell clones secreting a number of different isotypes. These results were obtained with 2,4-dinitrophenol (DNP)-specific memory B cells as well as with B cells from unprimed animals. The cloned TH cells could stimulate the same frequency of antigen-specific B cells as did the polyclonal population of TH cells provided by carrier-primed recipients in the conventional splenic fragment assay. Although no evidence for isotype-specific T cells was found, some clones appear restricted in the isotypes they generated: one of the clones, for instance, mediated the production of only IgM, IgG~ and IgA. These results confirm the experiments ofTh~ze et al. 3 and Seman et al. 4 performed with poly(Glu 6° Ala 3° Tyr 1°) (GAT)-specific TH-cell clones. In vitro most of these clones in the presence of D N P - G A T and DNP-primed B cells generated all classes and subclasses of Ig (IgM, IgG3, IgG~, IgGzb , IgG2a and IgA). In these experiments the necessity of Tin-type T cells for isotype expression was completely excluded since all T cells were removed from the DNP-primed B-cell population used. In Teale's experiments the participation of Tin-type T cells, such as isotype-specific Tn cells remains possible because they could be present in

the spleens of recipient animals used in the splenic fragment assay. With fowl gamma globulin (FGG)specific clones Cammisuli and Schreier 9 found that IgG anti-azobenzene arsonate (ARS) antibodies could be produced in the presence of A R S - F G G and ARS-primed B cells. Tees and Schreier 1°, using anti-sheep red blood cell (SRBC)-specific TH-cell clones, were not able to obtain an in-vivo IgG SRBC-specific response with unprimed B cells from nu/nu mice. Teale I shows that the KLH-soeeific clones can trigger an anti-DNP response with B cells from unprimed animals, suggesting that Tees' and Schreier's results may be influenced by the experimental conditions used and cannot be taken as an indication that Tu-cell clones cannot drive different C H gene expression. Lanzavecchia 2 used human alloreactive T-cell clones isolated from a secondary mixed lymphocyte reaction and cloned by limiting dilution in the presence of irradiated stimulator cells and interleukin 2 (IL2). All the dones tested provided help in vitro for the production of all the isotypes, including IgE. The frequency of the B cells activated was about 1 in 8 for IgG, 1 in 12 for IgM, 1 in 28 for IgA, and 1 in 2 100 for IgE. The data do not provide evidence for a high frequency of switches among activated B cells and the B cells activated did not require either the antigen or direct interaction with TH cells: these triggering requirements are typical of memory B cells that have already been activated and may have switched in vivo. These experiments confirm the results of Coutinho et al. 5 with C3H/HeJ alloreactive T-cell clones directed against minor antigen(s) of C3H/Tif splenocytes. These clones can activate a large set of B cells which can produce all isotypes, although a predominance of IgG 1 and IgG2a subclass expression was observed. Since alloreactive T cells can recognize minor antigens in the Ia context they are comparable with conventional TH cells. However, whether the results obtained with alloreactive T cell clone can be fully extended to conventional T H cells remains to be established. If individual TH-cell clones and alloreactive T-cell clones permit the expansion and secretion of B cells producing different isotypes, then isotype-specific helper T cells (Tin) are not a prerequisite for a response of a given © 1984,ElsevierSciencePublishersB.V.,Amsterdam 0167- 4919/8~d$02.00