Naive CD4+ T cells from lupus-prone Fas-intact MRL mice display TCR-mediated hyperproliferation due to intrinsic threshold defects in activation

Autoreactive T cell activation is a consistent feature of murine lupus; however, the mechanism of such activation remains unclear. We hypothesized that naive CD4+ T cells in lupus have a lower threshold of activation through their TCR-CD3 complex that renders them more susceptible to stimulation with self-Ags. To test this hypothesis, we compared proliferation, IL-2 production, and single cell calcium signaling of naive CD4+ T cells isolated from Fas-intact MRL/+(Fas-lpr) mice with H-2k-matched B10.BR and CBA/CaJ controls, following anti-CD3 stimulation in the presence or absence of anti-CD28. We also assessed the responsiveness of naive CD4+ T cells isolated from Fas-intact MRL and control mice bearing a rearranged TCR specific for amino acids 88-104 of pigeon cytochrome c to cognate and low affinity peptide Ags presented by bone marrow-matured dendritic cells. TCR transgenic and wild-type CD4+ T cells from MRL mice displayed a lower threshold of activation than control cells, a response that was class II MHC dependent. The rise in intracellular calcium in MRL vs controls was enhanced and prolonged following anti-CD3 triggering, suggestive of proximal defects in TCR-engendered signaling as the mechanism for the observed hyperactivity. These findings were observed as early as 1-2 mo postweaning and, based on analysis of F1 T cells, appeared to be dominantly expressed. This genetically altered threshold for activation of MRL T cells, a consequence of a proximal defect in CD3-mediated signal transduction, may contribute to the abrogation of T cell tolerance to self-Ags in lupus.     

Fas expression on antigen-specific T cells has costimulatory, helper, and down-regulatory functions in vivo for cytotoxic T cell responses but not for T cell-dependent B cell responses

Fas-mediated apoptosis is an important contributor to contraction of Ag-driven T cell responses acting only on activated Ag-specific T cells. The effects of targeted Fas deletion on selected cell populations are well described however little is known regarding the consequences of Fas deletion on only activated Ag-specific T cells. We addressed this question using the parent-into-F(1) (P-->F(1)) model of acute or chronic (lupus-like) graft-vs-host disease (GVHD) as a model of either a CTL-mediated or T-dependent B cell-mediated response, respectively. By transferring Fas-deficient lpr donor T cells into Fas-intact F(1) hosts, the in vivo role of Ag-specific T cell Fas can be determined. Our results demonstrate a novel dichotomy of Ag-specific T cell Fas function in that: 1) Fas expression on Ag-activated T cells has costimulatory, helper, and down-regulatory roles in vivo and 2) these roles were observed only in a CTL response (acute GVHD) and not in a T-dependent B cell response (chronic GVHD). Specifically, CD4 T cell Fas expression is important for optimal CD4 initial expansion and absolutely required for help for CD8 effector CTL. Donor CD8 T cell Fas expression played an important but not exclusive role in apoptosis and down-regulation. By contrast, CD4 Fas expression played no detectable role in modulating chronic GVHD induction or disease expression. These results demonstrate a novel role for Ag-specific T cell Fas expression in in vivo CTL responses and support a review of the paradigm by which Fas deficiency accelerates lupus in MRL/lpr lupus-prone mice.     

Blockade of programmed death-1 in young (New Zealand black x New Zealand white)F1 mice promotes the activity of suppressive CD8+ T cells that protect from lupus-like disease

The programmed death-1 (PD-1)/programmed death-1 ligand 1 (PD-L1) pathway regulates both stimulatory and inhibitory signals. In some conditions, PD-1/PD-L1 inhibits T and B cell activation, induces anergy, and reduces cytotoxicity in CD8(+) T cells. In other conditions, PD-l/PD-L1 has costimulatory effects on T cells. We recently showed that induction of suppressive CD8(+)Foxp3(+) T cells by immune tolerance of lupus-prone (New Zealand black × New Zealand white)F(1) (BWF(1)) mice with the anti-DNA Ig-based peptide pConsensus (pCons) is associated with significantly reduced PD-1 expression on those cells. In this study, we tested directly the role of PD-1 by administering in vivo neutralizing Ab to PD-1 to premorbid BWF(1) and healthy control mice. Anti-PD-1-treated mice were protected from the onset of lupus nephritis for 10 wk, with significantly improved survival. Although the numbers of T cells declined in aging control mice, they were maintained in anti-PD-1-treated mice, including CD8(+)Foxp3(+) T cells that suppressed syngeneic CD4(+)CD25(-) T cell proliferation and IFN-γ production, reduced production of IgG and anti-dsDNA IgG, induced apoptosis in syngeneic B cells, and increased IL-2 and TGF-β production. The administration of anti-PD-1 Ab to BWF(1) mice after induction of tolerance with pCons abrogated tolerance; mice developed autoantibodies and nephritis at the same time as control mice, being unable to induce CD8(+)Foxp3(+) T suppressor cells. These data suggest that tightly regulated PD-1 expression is essential for the maintenance of immune tolerance mediated by those CD8(+)Foxp3(+) T cells that suppress both T(h) cells and pathogenic B cells. PD-1 regulation could represent a target to preserve tolerance and prevent autoimmunity.     

Anergy in memory CD4+ T cells is induced by B cells

Induction of tolerance in memory T cells has profound implications in the treatment of autoimmune diseases and transplant rejection. Previously, we reported that the presentation of low densities of agonist peptide/MHC class II complexes induced anergy in memory CD4(+) T cells. In the present study, we address the specific interaction of different types of APCs with memory CD4(+) T cells. A novel ex vivo anergy assay first suggested that B cells induce anergy in memory T cells, and an in vivo cell transfer assay further confirmed those observations. We demonstrated that B cells pulsed with defined doses of Ag anergize memory CD4 cells in vivo. We established that CD11c(+) dendritic cells do not contribute to anergy induction to CD4 memory T cells, because diphtheria toxin receptor-transgenic mice that were conditionally depleted of dendritic cells optimally induced anergy in memory CD4(+) T cells. Moreover, B cell-deficient muMT mice did not induce anergy in memory T cells. We showed that B2 follicular B cells are the specific subpopulation of B cells that render memory T cells anergic. Furthermore, we present data showing that anergy in this system is mediated by CTLA-4 up-regulation on T cells. This is the first study to demonstrate formally that B cells are the APCs that induce anergy in memory CD4(+) T cells.     

B cells are required for lupus nephritis in the polygenic, Fas-intact MRL model of systemic autoimmunity.

B cells are required for both the expression of lupus nephritis and spontaneous T cell activation/memory cell accumulation in MRL-Faslpr mice (MRL/lpr). Autoimmunity in the MRL/lpr strain is the result of Fas-deficiency and multiple background genes; however, the precise roles of background genes vs Fas-deficiency have not been fully defined. Fas-deficiency (i.e., the lpr defect) is required in B cells for optimal autoantibody expression, raising the possibility that the central role for B cells in MRL/lpr mice may not extend to MRL/+ mice and, thus, to lupus models that do not depend on Fas-deficiency ("polygenic lupus"). To address this issue, B cell-deficient, Fas-intact MRL/+ mice (JHd-MRL/) were created; and disease was evaluated in aged animals (>9 mo). The JHd-MRL/+ animals did not develop nephritis or vasculitis at a time when the B cell-intact littermates had severe disease. In addition, while activated/memory CD4+ and CD8+ T cells accumulated in B cell-intact mice, such accumulation was substantially inhibited in the absence of B cells. This effect appeared to be restricted to the MRL strain because it was not seen in B cell-deficient BALB/c mice (JHd-BALB) of similar ages. The results indicate that B cells are essential in promoting systemic autoimmunity in a Fas-independent model. Therefore, B cells have an important role in pathogenesis, generalizable to lupus models that depend on multiple genes even when Fas expression is intact. The results provide further rationale for B cell suppression as therapy for systemic lupus erythematosus.     

Cellular immune responses in mice infected with the intestinal nematode Trichuris muris.

CBA and B10.BR mice show variation in immune response to the intestinal nematode Trichuris muris. CBA mice develop strong resistance, eliminating worms from the intestine; B10BR mice are permissive and develop chronic infections. It is already known that resistance and permissiveness reflect differential T helper responses. The data reported here show that resistant CBA mice express good antigen-specific lymphocyte proliferative responses to infection, whereas cells from B10.BR mice are relatively anergic, although still responsive to Concanavalin A (ConA). The possibility that the altered proliferative responsiveness seen in infected B10.BR mice reflected quantitative or qualitative changes in T helper cells was examined by comparing cytokine production and expression of cell surface markers (CD4, CD8, and CD28) in mesenteric lymph node cells and spleen cells from both strains and comparing these with the characteristics of cells from resistant CBA mice and from CBA mice that had been rendered permissive to infection by a combination of irradiation and corticosteroid treatment. As expected, cells from B10.BR mice produced high levels of interferon-gamma (IFN-gamma), whereas those from CBA mice released high levels of IL-5, whether stimulated with adult worm somatic antigens, excretory/secretory antigens, or ConA. Immunosuppressed CBA mice produced high levels of both IFN-gamma and IL-5 throughout the experiment. FACS analysis revealed a decrease of CD4+ and an initial increase in CD8+ cells in all infected mice. No major changes occurred in the relative proportion of CD28(+) cells. Further evaluation of the CD28 costimulatory molecule, measured as mean fluorescence intensity, displayed down-regulation in permissive and immunosuppressed mice. The data obtained suggest that lymphocyte unresponsiveness and permissiveness to T. muris infection may be associated with a down-regulation or an initially reduced expression of costimulatory CD28 molecules.     

Two distinct mechanisms account for the immune response (Ir) gene control of the T cell response to pigeon cytochrome c

Previous experiments have demonstrated that the immune response of MHC congenic mice to pigeon cytochrome c is under Ir gene control. Expression of I-E-encoded gene products influences both the magnitude and fine specificity of the Th cell response to pigeon cytochrome c and phylogenetic derivatives. Results of those experiments implicate both determinant selection and repertoire selection as mechanisms of Ir gene control in this system. In this report we have compared the TCR expressed in pigeon cytochrome c-reactive Th cells from B10.A(I-Ek), B10.A(5R) (I-Eb), and B10.S(9R) (I-Es) mice. The B10.A(5R) strain is a low responder to pigeon cytochrome c, but in response to moth cytochrome c this strain produces T cells which respond to pigeon or moth cytochrome c on B10.A APC. These cells are phenotypically identical to the predominant clonal phenotype seen in the B10.A response to pigeon cytochrome c. In this report, we show that the B10.A and B10.A(5R) pigeon cytochrome c-reactive T cells express essentially identical T cell receptors. These results, coupled with recent studies reporting a relatively low affinity for I-Eb molecules by pigeon cytochrome c peptides compared with moth cytochrome c peptides, strongly argue that the immune response defect in the B10.A(5R) strain is due to a defect in Ag presentation (determinant selection). In contrast, B10.A and B10.S(9R) strains are high responders to pigeon cytochrome c. Both strains produce T cell clones which are capable of responding to cytochrome c presented by either B10.A or B10.S(9R) APC in vitro. We show that, even in T cells with this MHC restriction degeneracy, the TCR expressed in the two strains are different. Because the APC of both strains can clearly present the cytochrome c Ag, we conclude that the differential expression of the TCR in the responses is due to a T cell repertoire selection difference in the two strains. Thus, for the response to one Ag in three MHC congenic strains, there exists evidence that both determinant selection and repertoire selection can be mechanisms of Ir gene control of an immune response.     

Intercellular adhesion molecule 1 is critical for activation of CD28-deficient T cells

Presentation of Ag to T lymphocytes in the absence of the requisite costimulatory signals leads to an Ag-specific unresponsiveness termed anergy, whereas Ag presentation in conjunction with costimulation leads to clonal expansion. B7/CD28 signaling has been shown to provide this critical costimulatory signal and blockade of this pathway may inhibit in vitro and in vivo immune responses. Although T cells from CD28-deficient mice are lacking in a variety of responses, they nonetheless are capable of various primary and secondary responses without the induction of anergy expected in the absence of costimulation. This suggests that there may be alternative costimulatory pathways that can replace CD28 signaling under certain circumstances. In this paper, we show that ICAM-1becomes a dominant costimulatory molecule for CD28-deficient T cells. ICAM-1 costimulates anti-CD3-mediated T cell proliferation and IL-2 secretion in CD28-deficient murine T cells. Furthermore, splenocytes from ICAM-1-deficient mice could not activate CD28-deficient T cells and splenocytes lacking both ICAM and CD28 fail to proliferate in response to anti-CD3-induced T cell signals. This confirms that not only can ICAM-1 act as a CD28-independent costimulator, but it is the dominant, requisite costimulatory molecule for the activation of T cells in the absence of B7/CD28 costimulation.     

Lupus-prone mice have an abnormal response to thioglycolate and an impaired clearance of apoptotic cells

Deficiency of complement in humans and mice is associated with the development of lupus and with abnormal repair of inflammatory and immune complex-mediated tissue injury. Here we ask whether similar defects in the resolution of inflammation are found in mice prone to spontaneous lupus. We compared the response to an i.p. injection of thioglycolate between two lupus-prone strains (MRL/Mp and NZB/W) and two non lupus-prone strains of mice (C57BL/6 and BALB/c). In all four strains the influx of polymorphonuclear neutrophils (PMN) was similar. However, by 96 h clearance of PMN in the control strains was complete, whereas in the autoimmune-prone strains PMN were still detectable. The number of mononuclear cells recruited was markedly reduced in the lupus-prone strains compared with the controls, and their phenotype was different. The lupus-prone strains had significantly fewer elicited macrophages that were CD11b-high and Ly6C-negative. In lupus-prone mice at 24 h there was a significantly increased number of apoptotic PMN free in the peritoneum, accompanied by a reduced percentage of macrophages containing apoptotic bodies, suggesting a defect in their uptake. An impaired ability of resident peritoneal macrophages from lupus-prone mice to engulf apoptotic cells was demonstrated by in vivo and in vitro cell clearance assays. These observations indicate that lupus-prone strains have an abnormal inflammatory response to thioglycolate and an intrinsic impairment in apoptotic cell uptake. These findings have implications for the initiation of autoimmunity, as lupus autoantigens are expressed on dying cells, and impaired disposal of these could enhance the development of autoimmunity.     

The cyclin-dependent kinase inhibitor p27kip1 is required for transplantation tolerance induced by costimulatory blockade

The cyclin-dependent kinase (CDK) inhibitor p27kip1 is an important negative regulator of the cell cycle that sets a threshold for mitogenic signals in T lymphocytes, and is required for T cell anergy in vitro. To determine whether p27(kip1) is required for tolerance in vivo, we performed cardiac allograft transplantation under conditions of combined CD28/CD40L costimulatory blockade. Although this treatment induced long-term allograft survival in wild-type recipients, costimulatory blockade was no longer sufficient to induce tolerance in mice lacking p27kip1. Rejected allografts from p27kip1-/- mice contained more CD4+ T lymphocytes and exhibited more tissue damage than allografts from tolerant, wild-type mice. Infiltrating p27kip1-deficient T cells, but not wild-type T cells, exhibited nuclear expression of cyclins E and A, indicating uncontrolled T cell cycle progression in the graft. The failure of tolerance in p27kip1-/- mice was also accompanied by markedly increased numbers of allospecific, IFN-gamma-producing cells in the periphery, and occurred despite apparently normal regulatory T cell activity. These data demonstrate that the CDK inhibitor p27kip1 enforces the costimulatory requirement for the expansion and differentiation of alloimmune effector T lymphocytes in vivo, and point to CDKs as novel targets for immunosuppressive or tolerance-inducing therapies.     

CD4+ T cells from (New Zealand Black x New Zealand White)F1 lupus mice and normal mice immunized against apoptotic nucleosomes recognize similar Th cell epitopes in the C terminus of histone H3

We have previously reported that peptide 88-99 of histone H4 represents a minimal T cell epitope recognized by Th cells from nonautoimmune BALB/c (H-2(d/d)) mice immunized with nucleosomes. In this study, we tested a panel of overlapping peptides spanning the whole sequences of H4 and H3 for recognition by CD4(+) T cells from unprimed (New Zealand Black (NZB) x New Zealand White (NZW))F(1) lupus mice (H-2(d/z)). None of the 11 H4 peptides was recognized by CD4(+) T cells from (NZB x NZW)F(1) mice. In contrast, these cells proliferated and secreted IL-2, IL-10, and IFN-gamma upon ex vivo stimulation with H3 peptides representing sequences 53-70, 64-78, and 68-85. Peptides 56-73 and 61-78 induced the production of IFN-gamma and IL-10, respectively, without detectable proliferation, suggesting that they may act as partial agonist of the TCR. Th cells from unprimed BALB/c mice and other lupus-prone mice such as SNF(1) (H-2(d/q)) and MRL/lpr (H-2(k/k)) mice did not recognize any peptides present within the H3 region 53-85. We further demonstrated that immunization of normal BALB/c mice with syngeneic liver nucleosomes and spleen apoptotic cells, but not with nonapoptotic syngeneic cells, induced Th cell responses against several peptides of the H3 region 53-85. Moreover, we found that this conserved region of H3, which is accessible at the surface of nucleosomes, is targeted by Abs from (NZB x NZW)F(1) mice and lupus patients, and contains motifs recognized by several distinct HLA-DR molecules. It might thus be important in the self-tolerance breakdown in lupus.     

Blockade of CD86 signaling facilitates a Th2 bias at the maternal-fetal interface and expands peripheral CD4+CD25+ regulatory T cells to rescue abortion-prone fetuses

Intervention in B7 (CD80/CD86)/B7-ligand (CD28/CTLA-4) pathways is an effective way of preventing unwanted immune responses, such as allograft rejection. Pregnancy maintenance represents maternal tolerance to the fetal allograft, which is accompanied by a type 2 helper cell (Th2) bias at the maternal-fetal interface. Here, the costimulatory signal of CD86 was selectively blocked, and that of CD80 was kept unimpaired by administration of anti-murine CD86 monoclonal antibody at the early gestational stage in abortion-prone CBA/JxDBA/2 matings and normal pregnant CBA/JxBALB/c matings. It was demonstrated that in vivo blockade of CD86 costimulation could suppress maternal immune attack to the fetus by shifting cytokines from Th1 predominance to Th2 bias at the maternal-fetal interface, and expanding peripheral CD4+CD25+ regulatory T cells, which play an important role in the development and maintenance of maternal-fetal tolerance. Furthermore, the expression of CD28 and its ligands CD80/CD86 on peripheral lymphocytes was down-regulated, whereas that of CTLA-4 was up-regulated, which might facilitate the suppressive effect of CD4+CD25+ regulatory T cells on the alloreactive T cells. The maternal-fetal immunotolerance induced by CD86 blockade decreased fetal resorption in CBA/JxDBA/2 matings, but did not affect normal pregnant CBA/JxBALB/c matings. These results suggest that selective blockade of CD86 costimulation leads to maternal immune tolerance to embryo antigen, and might contribute to a rational immunoregulatory regimen for recurrent spontaneous abortion.     

Nasal anti-CD3 antibody ameliorates lupus by inducing an IL-10-secreting CD4+ CD25- LAP+ regulatory T cell and is associated with down-regulation of IL-17+ CD4+ ICOS+ CXCR5+ follicular helper T cells

Lupus is an Ab-mediated autoimmune disease. One of the potential contributors to the development of systemic lupus erythematosus is a defect in naturally occurring CD4(+)CD25(+) regulatory T cells. Thus, the generation of inducible regulatory T cells that can control autoantibody responses is a potential avenue for the treatment of systemic lupus erythematosus. We have found that nasal administration of anti-CD3 mAb attenuated lupus development as well as arrested ongoing lupus in two strains of lupus-prone mice. Nasal anti-CD3 induced a CD4(+)CD25(-)latency-associated peptide (LAP)(+) regulatory T cell that secreted high levels of IL-10 and suppressed disease in vivo via IL-10- and TFG-beta-dependent mechanisms. Disease suppression also occurred following adoptive transfer of CD4(+)CD25(-)LAP(+) regulatory T cells from nasal anti-CD3-treated animals to lupus-prone mice. Animals treated with nasal anti-CD3 had less glomerulonephritis and diminished levels of autoantibodies as measured by both ELISA and autoantigen microarrays. Nasal anti-CD3 affected the function of CD4(+)ICOS(+)CXCR5(+) follicular helper T cells that are required for autoantibody production. CD4(+)ICOS(+)CXCR5(+) follicular helper T cells express high levels of IL-17 and IL-21 and these cytokines were down-regulated by nasal anti-CD3. Our results demonstrate that nasal anti-CD3 induces CD4(+)CD25(-)LAP(+) regulatory T cells that suppress lupus in mice and that it is associated with down-regulation of T cell help for autoantibody production.     

Autoreactive T cells revealed in the normal repertoire: escape from negative selection and peripheral tolerance

Self-reactive T cells are known to be eliminated by negative selection in the thymus or by the induction of tolerance in the periphery. However, developmental pathways that allow self-reactive T cells to inhabit the normal repertoire are not well-characterized. In this investigation, we made use of anti-small nuclear ribonucleoprotein particle (snRNP) Ig transgenic (Tg) mice (2-12 Tg) to demonstrate that autoreactive T cells can be detected and activated in both normal naive mice and autoimmune-prone MRL lpr/lpr mice. In contrast, autoreactive T cells of nonautoimmune Tg mice are tolerized by Tg B cells in the periphery. In adoptive transfer studies, autoreactive T cells from MRL lpr/lpr mice can stimulate autoantibody synthesis in nonautoimmune anti-snRNP Tg mice. Transferred CD4 T cells migrate to regions of the spleen proximal to the B cell follicles, suggesting that cognate B cell-T cell interactions are critical to the autoimmune response. Taken together, our studies suggest that anti-snRNP B cells are important APCs for T cell activation in autoimmune-prone mice. Additionally, we have demonstrated that anti-snRNP B cell anergy in nonautoimmune mice may be reversed by appropriate T cell help.     

Galectin-9 Ameliorates Clinical Severity of MRL/lpr Lupus-Prone Mice by Inducing Plasma Cell Apoptosis Independently of Tim-3

Galectin-9 ameliorates various murine autoimmune disease models by regulating T cells and macrophages, although it is not known what role it may have in B cells. The present experiment shows that galectin-9 ameliorates a variety of clinical symptoms, such as proteinuria, arthritis, and hematocrit in MRL/lpr lupus-prone mice. As previously reported, galectin-9 reduces the frequency of Th1, Th17, and activated CD8⁺ T cells. Although anti-dsDNA antibody was increased in MRL/lpr lupus-prone mice, galectin-9 suppressed anti-dsDNA antibody production, at least partly, by decreasing the number of plasma cells. Galectin-9 seemed to decrease the number of plasma cells by inducing plasma cell apoptosis, and not by suppressing BAFF production. Although about 20% of CD19^−/low CD138⁺ plasma cells expressed Tim-3 in MRL/lpr lupus-prone mice, Tim-3 may not be directly involved in the galectin-9-induced apoptosis, because anti-Tim-3 blocking antibody did not block galectin-9-induced apoptosis. This is the first report of plasma cell apoptosis being induced by galectin-9. Collectively, it is likely that galectin-9 attenuates the clinical severity of MRL lupus-prone mice by regulating T cell function and inducing plasma cell apoptosis.     

Blockade of costimulation through B7/CD28 inhibits experimental autoimmune uveoretinitis, but does not induce long-term tolerance

It has been reported that costimulation blockade can result in T cell anergy. We investigated the effects of blocking costimulatory molecules in vivo on the development of experimental autoimmune uveoretinitis (EAU), a model for autoimmune uveitis in humans that is induced in mice by immunization with the retinal Ag interphotoreceptor retinoid binding protein. B10.A mice immunized with a uveitogenic regimen of interphotoreceptor retinoid-binding protein were treated with Abs to B7.1 and B7.2 for 2 wk. Evaluation of EAU and immunological responses 1 wk later showed that disease had been abrogated, and cellular responses were suppressed. To determine whether the costimulation blockade resulted in tolerance, adult-thymectomized mice immunized for uveitis and treated with anti-B7 or anti-CD28 were rechallenged for uveitis induction 5 wk after the initial immunization. Although confirmed to be disease free after the initial immunization, both anti-B7- and anti-CD28-treated mice developed severe EAU and elevated cellular responses after the rechallenge, equivalent to those of control mice. We conclude that in this model costimulatory blockade in vivo prevents the development of autoimmune disease, but does not result in long-term tolerance. The data are compatible with the interpretation that B7/CD28 blockade prevents generation of effector, but not of memory, T cells.     

A defect in deletion of nucleosome-specific autoimmune T cells in lupus-prone thymus: role of thymic dendritic cells

To study central tolerance to the major product of ongoing apoptosis in the thymus, we made new lines of transgenic (Tg) mice expressing TCR of a pathogenic autoantibody-inducing Th cell that was specific for nucleosomes and its histone peptide H4(71-94). In the lupus-prone (SWR x NZB)F1 (SNF1) thymus, introduction of the lupus TCR transgene caused no deletion, but marked down-regulation of the Tg TCR and up-regulation of endogenous TCRs. Paradoxically, autoimmune disease was suppressed in the alphabetaTCR Tg SNF1 mice with induction of highly potent regulatory T cells in the periphery. By contrast, in the MHC-matched, normal (SWR x B10. D2)F1 (SBF1), or in the normal SWR backgrounds, marked deletion of transgenic thymocytes occurred. Thymic lymphoid cells of the normal or lupus-prone mice were equally susceptible to deletion by anti-CD3 Ab or irradiation. However, in the steady state, spontaneous presentation of naturally processed peptides related to the nucleosomal autoepitope was markedly greater by thymic dendritic cells (DC) from normal mice than that from lupus mice. Unmanipulated thymic DC of SNF1 mice expressed lesser amounts of MHC class II and costimulatory molecules than their normal counterparts. These results indicate that apoptotic nucleosomal autoepitopes are naturally processed and presented to developing thymocytes, and a relative deficiency in the natural display of nucleosomal autoepitopes by thymic DC occurs in lupus-prone SNF1 mice.     

CTLA-4 is not required for induction of CD8(+) T cell anergy in vivo.

Recent studies of T cell anergy induction have produced conflicting conclusions as to the role of the negative regulatory receptor, CTLA-4. Several in vivo models of tolerance have implicated the interaction of CTLA-4 and its ligands, B7.1 and B7.2, as an essential step in induction of anergy, while results from a number of other systems have indicated that signals from the TCR/CD3 complex alone are sufficient to induce T cell unresponsiveness. One explanation for this disparity is that the requirements for anergy induction depend closely on the details of the system: in vivo vs in vitro, route of stimulus administration, naive vs memory cells, CD4(+) vs CD8(+) cells, etc. To test this possibility, we established an in vivo anergy model using mice transgenic for the 2C TCR on a recombination-activating gene-2-deficient background, that either express or lack the CTLA-4 molecule. This system provides us with a very homogeneous pool of naive Ag-specific CD8(+) T cells, allowing us to control some of the conditions mentioned above. We found that T cells from CTLA-4-deficient mice were anergized by injections of soluble antigenic peptide as efficiently as were CTLA-4-expressing cells. These results indicate that CTLA-4 is not universally required for in vivo T cell anergy induction and may point to distinctions between regulation of peripheral tolerance in CD4(+) and CD8(+) T cells.     

B cell depletion with anti-CD79 mAbs ameliorates autoimmune disease in MRL/lpr mice

MRL/lpr mice develop a spontaneous systemic lupus erythematosus-like autoimmune syndrome due to a dysfunctional Fas receptor, with contributions from other less well-defined genetic loci. The removal of B cells by genetic manipulation not only prevents autoantibody formation, but it also results in substantially reduced T cell activation and kidney inflammation. To determine whether B cell depletion by administration of Abs is effective in lupus mice with an intact immune system and established disease, we screened several B cell-specific mAbs and found that a combination of anti-CD79alpha and anti-CD79beta Abs was most effective at depleting B cells in vivo. Anti-CD79 therapy started at 4-5 mo of age in MRL/lpr mice significantly decreased B cells (B220(+)CD19(+)) in peripheral blood, bone marrow, and spleens. Treated mice also had a significant increase in the number of both double-negative T cells and naive CD4(+) T cells, and a decreased relative abundance of CD4(+) memory cells. Serum anti-chromatin IgG levels were significantly decreased compared with controls, whereas serum anti-dsDNA IgG, total IgG, or total IgM were unaffected. Overall, survival was improved with lower mean skin scores and significantly fewer focal inflammatory infiltrates in submandibular salivary glands and kidneys. Anti-CD79 mAbs show promise as a potential treatment for systemic lupus erythematosus and as a model for B cell depletion in vivo.     

Role of IL-10 in allergen-specific immunotherapy and normal response to allergens

Induction of specific unresponsiveness (tolerance/anergy) in peripheral T cells by interleukin-10 (IL-10) and recovery by cytokines from the tissue microenvironment represent two key steps in specific immunotherapy of allergy and in natural exposure to allergens in healthy individuals. IL-10 elicits anergy in T cells by selective inhibition of the CD28 costimulatory pathway and controls suppression and development of antigen-specific immunity.