Hormonal regulation of B cell development: 17 beta-estradiol impairs negative selection of high-affinity DNA-reactive B cells at more than one developmental checkpoint

There are increasing data suggesting that sex hormones, such as estrogen, have immunomodulatory effects and play a role in disease progression and pathogenesis in patients with the autoimmune disorder systemic lupus erythematosus. We have shown previously that treatment with 17beta-estradiol (E2) induces a lupus phenotype in BALB/c mice that express a transgene-encoded H chain of an anti-DNA Ab. Because E2 treatment interferes with normal tolerance of naive DNA-reactive B cells, we elected to study the effects of hormonal modulation on the regulation of autoreactive B cells at early developmental checkpoints. Single-cell PCR was performed to study the repertoire of DNA-reactive B cell subsets. High-affinity DNA-reactive B cells were rescued at both the immature and transitional B cell stage in E2-treated mice. Interestingly, although low-affinity DNA-reactive B cells survive negative selection in control mice, the frequency of these cells was significantly reduced in the mature pool of E2-treated mice, suggesting that the high-affinity DNA-reactive cells that mature to immunocompetence out-compete the low-affinity population for survival as mature B cells. These data provide evidence that an elevation in serum levels of E2 facilitates the maturation of a pathogenic naive autoreactive B cell repertoire and hampers the maturation of a potentially protective autoreactive B cell repertoire. Furthermore, these data show that both positive and negative selection occur within the transitional B cell stage.     

Defective CD8+ T cell peripheral tolerance in nonobese diabetic mice.

Nonobese diabetic (NOD) mice develop spontaneous autoimmune diabetes that involves participation of both CD4+ and CD8+ T cells. Previous studies have demonstrated spontaneous reactivity to self-Ags within the CD4+ T cell compartment in this strain. Whether CD8+ T cells in NOD mice achieve and maintain tolerance to self-Ags has not previously been evaluated. To investigate this issue, we have assessed the extent of tolerance to a model pancreatic Ag, the hemagglutinin (HA) molecule of influenza virus, that is transgenically expressed by pancreatic islet beta cells in InsHA mice. Previous studies have demonstrated that BALB/c and B10.D2 mice that express this transgene exhibit tolerance of HA and retain only low-avidity CD8+ T cells specific for the dominant peptide epitope of HA. In this study, we present data that demonstrate a deficiency in peripheral tolerance within the CD8+ T cell repertoire of NOD-InsHA mice. CD8+ T cells can be obtained from NOD-InsHA mice that exhibit high avidity for HA, as measured by tetramer (K(d)HA) binding and dose titration analysis. Significantly, these autoreactive CD8+ T cells can cause diabetes very rapidly upon adoptive transfer into NOD-InsHA recipient mice. The data presented demonstrate a retention in the repertoire of CD8+ T cells with high avidity for islet Ags that could contribute to autoimmune diabetes in NOD mice.     

Autoimmune myocarditis does not require B cells for antigen presentation.

T cells constitute the pathogenic effector cell population in autoimmune myocarditis in BALB/c mice. Using mice rendered deficient for B cells by a targeted disruption to the IgM transmembrane domain or by treatment with anti-IgM Ab from birth, we asked whether B cells are a critical APC in the induction of autoimmune myocarditis. B cell-deficient mice immunized with cardiac myosin develop myocarditis comparable in incidence and severity to that in wild-type mice, suggesting that autoreactive T cells that cause myocarditis in BALB/c mice are activated by macrophages or dendritic cells. Since it does not appear that presentation of cryptic epitopes is critical for the breakdown of self tolerance, potentially pathogenic T cells recognizing dominant myosin epitopes must have escaped tolerization. Either anatomic sequestration of cardiac myosin peptide-MHC complexes or subthreshold presentation of cardiac myosin peptides by conventional APC can explain the survival of these autoreactive T cells.     

B cell tolerance checkpoints that restrict pathways of antigen-driven differentiation

Autoreactive B cells can be regulated by deletion, receptor editing, or anergy. Rheumatoid factor (RF)-expressing B lymphocytes in normal mice are not controlled by these mechanisms, but they do not secrete autoantibody and were presumed to ignore self-Ag. Surprisingly, we now find that these B cells are not quiescent, but instead are constitutively and specifically activated by self-Ag. In BALB/c mice, RF B cells form germinal centers (GCs) but few Ab-forming cells (AFCs). In contrast, autoimmune mice that express the autoantigen readily generate RF AFCs. Most interestingly, autoantigen-specific RF GCs in BALB/c mice appear defective. B cells in such GCs neither expand nor are selected as efficiently as equivalent cells in autoimmune mice. Thus, our data establish two novel checkpoints of autoreactive B cell regulation that are engaged only after initial autoreactive B cell activation: one that allows GCs but prevents AFC formation and one that impairs selection in the GC. Both of these checkpoints fail in autoimmunity.     

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.     

Specificity-based negative selection of autoreactive B cells during memory formation

Autoreactive B cells are not completely purged from the primary B cell repertoire, and whether they can be prevented from maturation into memory B cells has been uncertain. We show here that a population of B cells that dominates primary immune responses of BALB/c mice to influenza virus A/PR/8/34 hemagglutinin (HA) are negatively selected in transgenic mice expressing PR8 HA as an abundant membrane-bound Ag (HACII mice). However, a separate population of B cells that contains precursors of memory B cells is activated by PR8 virus immunization and is subsequently negatively selected during the formation of the memory response. Negative selection of PR8 HA-specific B cells altered the specificity of the memory B cell response to a mutant virus containing a single amino acid substitution in a B cell epitope. Strikingly, this skewed reactivity resulted from an increase in the formation of memory B cells directed to non-self-epitopes on the mutant virus, which increased 8-fold in HACII mice relative to nontransgenic mice and precisely compensated for the absence of autoreactive PR8 HA-specific memory B cells. Negative selection of PR8 HA-specific B cells was a dominant process, since B cells from HACII mice could induce negative selection of PR8 HA-specific B cells from BALB/c mice. Lastly, HA-specific memory responses were unaffected by self-tolerance in another lineage of HA-transgenic mice (HA104 mice), indicating that the amount and/or cell type in which self-Ags are expressed can determine their ability to prevent autoreactive memory B cell formation.     

Anergic responses characterize a large fraction of human autoreactive naive B cells expressing low levels of surface IgM

B cell anergy represents an important mechanism of peripheral immunological tolerance for mature autoreactive B cells that escape central tolerance enforced by receptor editing and clonal deletion. Although well documented in mice, the extent of its participation in human B cell tolerance remains to be fully established. In this study, we characterize the functional behavior of strictly defined human naive B cells separated on the basis of their surface IgM (sIgM) expression levels. We demonstrate that cells with lower sIgM levels (IgM(lo)) are impaired in their ability to flux calcium in response to either anti-IgM or anti-IgD cross-linking and contain a significantly increased frequency of autoreactive cells compared with naive B cells with higher levels of sIgM. Phenotypically, in healthy subjects, IgM(lo) cells are characterized by the absence of activation markers, reduction of costimulatory molecules (CD19 and CD21), and increased levels of inhibitory CD22. Functionally, IgM(lo) cells display significantly weaker proliferation, impaired differentiation, and poor Ab production. In aggregate, the data indicate that hyporesponsiveness to BCR cross-linking associated with sIgM downregulation is present in a much larger fraction of all human naive B cells than previously reported and is likely to reflect a state of anergy induced by chronic autoantigen stimulation. Finally, our results indicate that in systemic lupus erythematosus patients, naive IgM(lo) cells display increased levels of CD95 and decreased levels of CD22, a phenotype consistent with enhanced activation of autoreactive naive B cells in this autoimmune disease.     

Impaired Fas signaling pathway is involved in defective T cell apoptosis in autoimmune murine arthritis

Proteoglycan (PG)-induced arthritis (PGIA) is a novel autoimmune murine model for rheumatoid arthritis induced by immunization with cartilage PG in susceptible BALB/c mice. In this model, hyperproliferation of peripheral CD4(+) T cells has been observed in vitro with Ag stimulation, suggesting the breakdown of peripheral tolerance. Activation-induced cell death (AICD) is a major mechanism for peripheral T cell tolerance. A defect in AICD may result in autoimmunity. We report in this study that although CD4(+) T cells from both BALB/c and B6 mice, identically immunized with human cartilage PG or OVA, express equally high levels of Fas at the cell surface, CD4(+) T cells from human cartilage PG-immunized BALB/c mice, which develop arthritis, fail to undergo AICD. This defect in AICD in PGIA may lead to the accumulation of autoreactive Th1 cells in the periphery. The impaired AICD in PGIA might be ascribed to an aberrant expression of Fas-like IL-1beta-converting enzyme-inhibitory protein, which precludes caspase-8 activation at the death-inducing signaling complex, and subsequently suppresses the caspase cascade initiated by Fas-Fas ligand interaction. Moreover, this aberrant expression of Fas-like IL-1beta-converting enzyme-inhibitory protein may also mediate TCR-induced hyperproliferation of CD4(+) T cells from arthritic BALB/c mice. Our data provide the first insight into the molecular mechanism(s) of defective AICD in autoimmune arthritis.     

BAFF/APRIL inhibition decreases selection of naive but not antigen-induced autoreactive B cells in murine systemic lupus erythematosus

BAFF inhibition is a new B cell-directed therapeutic strategy for autoimmune disease. Our purpose was to analyze the effect of BAFF/APRIL availability on the naive and Ag-activated B cell repertoires in systemic lupus erythematosus, using the autoreactive germline D42 H chain (glD42H) site-directed transgenic NZB/W mouse. In this article, we show that the naive Vκ repertoire in both young and diseased glD42H NZB/W mice is dominated by five L chains that confer no or low-affinity polyreactivity. In contrast, glD42H B cells expressing L chains that confer high-affinity autoreactivity are mostly deleted before the mature B cell stage, but are positively selected and expanded in the germinal centers (GCs) as the mice age. Of these, the most abundant is VκRF (Vκ16-104*01), which is expressed by almost all IgG anti-DNA hybridomas derived from the glD42H mouse. Competition with nonautoreactive B cells or BAFF/APRIL inhibition significantly inhibited selection of glD42H B cells at the late transitional stage, with only subtle effects on the glD42H-associated L chain repertoire. However, glD42H/VκRF-encoded B cells were still vastly overrepresented in the GC, and serum IgG anti-DNA Abs arose with only a slight delay. Thus, although BAFF/APRIL inhibition increases the stringency of negative selection of the naive autoreactive B cell repertoire in NZB/W mice, it does not correct the major breach in B cell tolerance that occurs at the GC checkpoint.     

Impaired autoreactive T cell-induced T cell-T cell interaction in aged mice

Self-Ia-reactive (autoreactive) L3T4+ T cell clones have been shown earlier to stimulate the proliferation of syngeneic naive L3T4+ T cells and initiate a T cell-T cell (T-T) interaction leading to the generation of immunoregulatory circuits. Since aging has been shown to be associated with a decline of the immune responsiveness, age-related alterations in the T-T interaction was investigated in the present study. Using several I-Ed-specific autoreactive T cell clones isolated from 2- to 3-month-old (young) DBA/2 mice as stimulators, it was observed that L3T4+ T cells from 22- to 24-month-old (aged) DBA/2 mice, failed to demonstrate a significant response to the autoreactive T cells. In contrast, L3T4+ T cells from young mice responded strongly to the autoreactive T cell clones. The deficient T-T cell interaction in aged mice correlated with an impaired syngeneic mixed lymphocyte reaction in these mice, thereby suggesting that aging induces a defect both in the autoreactive T cells and in T cells which react with the autoreactive T cells. When exogenous recombinant interleukin 2 (rIL-2), recombinant interleukin 4 (rIL-4), or a combination of these was added to the interaction, it was observed that rIL-4 but not rIL-2 enhanced the T-T interaction in young mice. However, rIL-4 or a combination of rIL-2 and rIL-4 failed to correct the defective T-T interaction in aged mice. Since the T cell network is believed to play an important role in the maintenance of normal immune system homeostasis, the present study suggests that age-related alterations in T and B cell functions and increased susceptibility to autoimmune diseases with age may result from a defect in the T cell network regulation.     

Unique signaling properties of B cell antigen receptor in mature and immature B cells: implications for tolerance and activation

Immature B cells display increased sensitivity to tolerance induction compared with their mature counterparts. The molecular mechanisms underlying these differences are poorly defined. In this study, we demonstrate unique maturation stage-dependent differences in B cell Ag receptor (BCR) signaling, including BCR-mediated calcium mobilization responses. Immature B cells display greater increases in intracellular calcium concentrations following Ag stimulation. This has consequences for the induction of biologically relevant responses: immature B cells require lower Ag concentrations for activation than mature B cells, as measured by induction of receptor editing and CD86 expression, respectively. BCR-induced tyrosine phosphorylation of CD79a, Lyn, B cell linker protein, and phospholipase Cgamma2 is enhanced in immature B cells and they exhibit greater capacitative calcium entry in response to Ag. Moreover, B cell linker protein, Bruton's tyrosine kinase, and phospholipase Cgamma2, which are crucial for the induction of calcium mobilization responses, are present at approximately 3-fold higher levels in immature B cells, potentially contributing to increased mobilization of calcium. Consistent with this possibility, we found that the previously reported lack of inositol-1,4,5-triphosphate production in immature B cells may be explained by enhanced inositol-1,4,5-triphosphate breakdown. These data demonstrate that multiple mechanisms guarantee increased Ag-induced mobilization of calcium in immature B cells and presumably ensure elimination of autoreactive B cells from the repertoire.     

Role of Mls-1 locus and clonal deletion of T cells in susceptibility to collagen-induced arthritis in mice

The role of T cell-mediated and humoral immunity to type II collagen has been well documented in collagen-induced arthritis (CIA). Previous work from our laboratory has indicated that genomic deletions of TCR V beta genes may play a role in CIA resistance in mice. This indicated a selectivity of TCR usage by autoreactive T cells in CIA in mice. Certain strains of mice, although having a normal genomic V beta TCR repertoire, can show clonal deletion of peripheral T cells that bear specific V beta gene products in their TCR. These clonally deleted T cells are reactive with self-Ag such as minor lymphocyte stimulation (Mls) Ag. An Mls-congenic strain, BALB.D2.Mlsa, which differs only at the Mls-1 a locus from BALB/c (Mls-1b), was used to examine the effect of clonal deletion of Mls-1a-reactive T cells in CIA. These two strains were crossed to three CIA-susceptible strains, B10.RIII (H-2r, Mls-1b), DBA/1 (H-2q, Mls-1a), and B10.Q (H-2q, Mls-1b), and the crosses were injected with type II collagen. A significantly decreased incidence of arthritis was observed in the (BALB.D2.Mlsa x B10.Q)F1 hybrids, compared with (BALB/c x B10.Q)F1 hybrids, upon immunization with chick type II collagen. The BALB.D2.Mlsa cross mice also had significantly lower levels of antimouse collagen antibodies. Flow cytometric analysis confirmed the clonal deletion of Mls-1a-reactive V beta 8.1, V beta 6, V beta 7, and V beta 9 subsets in the (BALB.D2.Mlsa x B10.Q)F1 hybrids. The study of H-2q/d mice in (BALB.D2.Mlsa x B10.Q) x B10.Q back-crosses demonstrated a significant correlation between CIA resistance and Mls-1a locus. On the other hand, B10.RIII crosses showed only a modest decrease in CIA incidence in the presence of Mls-1a. As expected, all the DBA/1 crosses had an equal incidence of CIA, which was somewhat less than that seen in DBA/1 mice themselves. These studies point out that the Mls-1a locus could play a role in decreasing CIA incidence by clonal deletion of T cells bearing specific V beta TCR, which may be involved in the pathogenesis of CIA. The influence of the clonal deletion of T cells on CIA, and hence the usage of specific V beta TCR by autoreactive anti-type II collagen T cells, however, depends not only on the source of the type II collagen and the MHC class II molecules involved but also on other background genes in mice.     

Identification of a minimal T cell epitope recognized by antinucleosome Th cells in the C-terminal region of histone H4

Autoreactive T cells responding to systemic autoantigens have been characterized in patients and mice with autoimmune diseases and in healthy individuals. Using peptides covering the whole sequence of histone H4, we characterized several epitopes recognized by lymph node Th cells from nonsystemic lupus erythematosus-prone mice immunized with the same peptides, the H4 protein, or nucleosomes. Multiple T epitopes were identified after immunizing H-2d BALB/c mice with H4 peptides. They spanned residues 28-42, 30-47, 66-83, 72-89, and 85-102. Within the region 85-102, a minimal CD4+ T epitope containing residues 88-99 was characterized. Although Abs to peptide 88-99 recognized H4, this peptide does not contain a dominant B cell epitope recognized by anti-H4 Abs raised in BALB/c mice or Abs from NZB/NZW H-2d/z lupus mice. Th cells primed in vivo with H4 responded to H4, but not to peptide 88-99. However, this peptide was able to stimulate the proliferation and IL-2 secretion of Th cells generated after immunization with nucleosomes. H488-99 thus represents a cryptic epitope with regard to H4 and a supradominant epitope presented by nucleosome, a supramolecular complex that plays a key role in lupus. This study shows that in the normal repertoire of naive BALB/c mice, autoreactive Th cells specific for histones are not deleted. The reactivity of these Th cells seems to be relatively restricted and resembles that of Th clones generated from SNF1 ((SWR x NZB)F1; I-Ad/q) lupus mice described earlier.     

Differential impact of T cell repertoire diversity in diabetes-prone or -resistant IL-10 transgenic mice

Expression of IL-10 transgene (tg) in pancreatic beta cells failed to induce autoimmune insulitis and diabetes in (BALB/c x NOD)F1 mice. However, IL-10-expressing tg littermates from backcrosses (N2 and N3) with NOD mice became diabetic at 5 to 10 weeks of age in an MHC-dependent manner. In this study, we tested the possibility that enhancement in frequency of islet antigen (Ag)-specific T cells overrides the protective effects of a diabetes-resistant genetic background and promotes diabetes in IL-10 tg (BALB/c x NOD)F1 mice. For this test, we introduced the IL-10 transgene into tg BDC2.5 mice expressing the islet Ag-specific Vbeta4 T cell repertoire by breeding Ins-IL-10+/BALB/c mice with BDC2.5 mice. The progeny (Ins-IL-10+/BALB/c x BDC2.5+)F1 mice doubly tg for IL-10 and Vbeta4 (BDC2.5) T cell repertoire, developed diabetes at 10 to 18 weeks of age with a much more aggressive T cell infiltrate in the pancreatic islets than in single tg mice. Surprisingly, these diabetic mice were free from acute pancreatitis but had apoptotic beta cells in the islet infiltrate. Conversely, mice tg for Vbeta4 (BDC2.5) T cell repertoire but not IL-10 had no diabetes and no apoptotic beta cells in the islet infiltrate. Therefore, an increase in the frequency of islet-specific T cells apparently overcomes the protection from diabetes by a resistant genetic background. Interestingly, N2 backcross mice doubly tg for Vbeta4 (BDC2.5) T cell repertoire and IL-10, compared to N2 backcross mice tg for IL-10 only, eventually became diabetic but with a delayed onset and reduced incidence of disease. These findings demonstrate that, along with IL-10, an increase in frequency of islet antigen-specific T cells (a) overrides the protective effect of genetic resistance to autoimmune diabetes in F1 mice and (b) delays the onset of an otherwise accelerated diabetes in (Ins-IL-10+/NOD)N2 backcross mice.     

T and B cell recovery in arthritis adoptively transferred to SCID mice: antigen-specific activation is required for restoration of autopathogenic CD4+ Th1 cells in a syngeneic system

T cell homeostasis is a physiological function of the immune system that maintains a balance in the numbers and ratios of T cells at the periphery. A self-MHC/self-peptide ligand can induce weak (covert) signals via the TCR, thus providing an extended lifespan for naive T cells. A similar mechanism is responsible for the restoration of immune homeostasis in severe lymphopenic conditions such as those following irradiation or chemotherapy, or upon transfer of lymphocytes to nu/nu or SCID mice. To date, the genetic backgrounds of donor and recipient SCID mice were unmatched in all autoimmune arthritis transfer experiments, and the recovery of lymphoid cells in the host has not been followed. In this study, we present the adoptive transfer of proteoglycan (PG)-induced arthritis using unseparated and T or B cell-depleted lymphocytes from arthritic BALB/c donors to genetically matched syngeneic SCID recipient mice. We demonstrate that selectively recovered lymphoid subsets determine the clinical and immunological status of the recipient. We found that when T cells were depleted (>98% depleted), B cells did not produce PG-specific anti-mouse (auto) Abs unless SCID mice received a second Ag (PG) injection, which promoted the recovery of Ag-specific CD4(+) Th1 cells. Reciprocally, as a result of B cell recovery, high levels of serum anti-PG Abs were found in SCID mice that received B cell-depleted (>99% depleted) T lymphocytes. Our results indicate a selective and highly effective cooperation between CD4(+) T cells and B lymphocytes that is required for the restoration of pathological homeostasis and development of autoimmune arthritis in SCID mice.     

Control of the autoimmune response by type 2 nitric oxide synthase

Immune defense against pathogens often requires NO, synthesized by type 2 NO synthase (NOS2). To discern whether this axis could participate in an autoimmune response, we immunized NOS2-deficient mice with the autoantigen acetylcholine receptor, inducing muscle weakness characteristic of myasthenia gravis, a T cell-dependent Ab-mediated autoimmune disease. We found that the acetylcholine receptor-immunized NOS2-deficient mice developed an exacerbated form of myasthenia gravis, and demonstrated that NOS2 expression limits autoreactive T cell determinant spreading and diversification of the autoantibody repertoire, a process driven by macrophages. Thus, NOS2/NO is important for silencing autoreactive T cells and may restrict bystander autoimmune reactions following the innate immune response.     

Endogenous H/K ATPase beta-subunit promotes T cell tolerance to the immunodominant gastritogenic determinant

A CD4(+) T cell response to the gastric H/K ATPase beta-subunit (H/Kbeta) is required for the onset of experimental autoimmune gastritis in BALB/c mice. The extent to which endogenous H/Kbeta contributes toward the tolerance of the H/Kbeta-specific T cell repertoire in normal individuals is not known. By comparison of T cell responses in H/Kbeta-deficient (o/o) and H/Kbeta-expressing BALB/c mice, in this work we show that the endogenous H/Kbeta autoantigen plays a major role in the tolerance of pathogenic H/Kbeta-specific T cells. First, T cell-dependent Ab responses to the H/Kbeta Ag were enhanced in H/K ATPase-immunized H/Kbeta-deficient mice compared with wild-type mice. Second, peptide immunization experiments indicated that immune responses to the major gastritogenic epitope of the H/K ATPase, namely H/Kbeta(253-277), were significantly more vigorous in H/Kbeta-deficient mice compared with wild-type mice. Third, unfractionated splenocytes from H/Kbeta-deficient mice, but not H/Kbeta-expressing mice, induced autoimmune gastritis after adoptive transfer to BALB/c nude mice. The enhanced responses to H/Kbeta in H/Kbeta-deficient mice were shown to be intrinsic to CD4(+)CD25(-) T cells rather than a change in status of CD4(+)CD25(+) regulatory T cells. We conclude from these studies that the H/Kbeta-specific T cells in wild-type mice represent the residue of a T cell repertoire, directed toward a single determinant, that has been subjected to partial tolerance induction.     

Progression from acute to chronic disease in a murine parent-into-F1 model of graft-versus-host disease

The parent-into-immunocompetent-F(1) model of graft-vs-host disease (GVHD) induces immune dysregulation, resulting in acute or chronic GVHD. The disease outcome is thought to be determined by the number of parental anti-F(1) CTL precursor cells present in the inoculum. Injection of C57BL/6 (B6) splenocytes into (B6 x DBA/2)F(1) (B6D2F(1)) mice (acute model) leads to extensive parental cell engraftment and early death, whereas injection of DBA/2 cells (chronic model) results in little parental cell engraftment and a lupus-like disease. This study demonstrated that injection of BALB/c splenocytes into (BALB/c x B6)F(1) (CB6F(1)) mice resulted in little engraftment of parental lymphocytes and the development of lupus as expected. Injection of B6 splenocytes into CB6F(1) initiated an initial burst of parental cell engraftment similar to that of B6 into B6D2F(1). However, the acute disease resolved, and the CB6F(1) mice went on to develop chronic GVHD with detectable Abs to ssDNA, dsDNA, and extractable nuclear Ags. Limiting dilution CTL assays determined that B6 splenocytes have CTL precursor frequencies of 1/1000 against both CB6F(1) and B6D2F(1), whereas DBA/2 and BALB/c splenocytes have a CTL precursor frequency of 1/20,000 for their respective F(1)s. The Th cell precursor frequency for B6 anti-DBA/2 was 3-fold higher than that for B6 anti-BALB/c determined by limiting dilution proliferation assays. These results indicate the importance of adequate allospecific helper as well as effector T cells for the induction and maintenance of acute GVHD in this model, and presents an unexpected model in which initial acute GVHD is replaced by the chronic form of disease.     

Mice deprived of exogenous antigenic stimulation develop a normal repertoire of functional T cells.

The development of T cells and the selection of the TCR repertoire in the absence of exogenous antigenic stimulation were investigated. For this purpose germfree BALB/c mice fed an ultrafiltered solution of chemically defined low m.w. nutrients (GF-CD) were used. Previous studies on B cell development and differentiation in GF-CD mice have demonstrated a high reduction in the number of cells secreting Ig of the non-IgM isotypes but an Ig-VH gene usage and a B cell specificity repertoire that is substantially different from that observed in conventional adult mice and more closely resembles that of neonatal conventional mice. In contrast, the present comparison of the various lymphocyte populations in the thymus, lymph nodes, and spleen from GF-CD and conventional mice using flow cytometry analysis revealed no significant differences. Analysis of the TCR-V beta expression on both mature thymocytes and lymph node T cells showed a high degree of similarity between GF-CD and conventional mice. These findings indicate a marked difference in the influence of exogenous antigenic stimulation on the development of B and T cells. Additionally, development in an environment free of exogenous antigenic stimulation allows for full functional maturation of T cells to occur, because MLC showed that GF-CD splenic T cells could mount allogeneic responses in a way similar to T cells generated in a conventional environment. Most importantly, full Th cell function is generated, because activation of GF-CD spleen cells by cross-linking with mAb against CD3 resulted in the induction of cells secreting IFN-gamma and Ig of the non-IgM isotypes, which cannot be detected in GF-CD sera. These findings demonstrate that functional T and B cells develop in mice that have not been exposed to exogenous Ag, and that the TCR repertoire, in contrast to the B cell compartment, is predominantly shaped by endogenously expressed Ag.     

Early cellular events in systemic autoimmunity driven by chromatin-reactive T cells

In vivo exposure of the thymus of normal mice to procainamide-hydroxylamine, a lupus-inducing drug, causes development of chromatin-reactive T cells. Autoantibodies subsequently appear, but their origin and significance are unknown. The current studies were undertaken to determine the specificities of B cells that respond to chromatin-reactive T cells at the initiation of this autoimmune process. Three days after adoptive transfer of 6 x 10(6) chromatin-reactive T cells, B cells with the capacity to secrete IgM anti-chromatin antibodies were detected in 1/10(6) splenocytes, and these became 10- to 50-fold more numerous if either the donor T cells or the recipient had defective Fas due to the lpr allele. Five days later these mice developed IgG anti-chromatin-secreting B cells at a precursor frequency of 3-6 x 10(-5). B cells with dDNA-binding activity isolated from mice primed in vivo to a complex of methylated pigeon cytochrome c and dDNA could stimulate naive, cytochrome c-reactive T cells in vitro, demonstrating that B cells can internalize dDNA-bound proteins through their dDNA immunoblobulin receptor and can functionally present a T cell epitope. However, no capacity of chromatin for binding anti-dDNA antibodies was detected, and IgM dDNA-specific B cells did not expand when challenged with chromatin-reactive T cells in vivo. The rapid and robust expansion of anti-chromatin-secreting B cells indicates that the normal immune repertoire includes nontolerant autoreactive B cells that respond to strong T cell drive and are readily manifested if Fas-mediated activation-induced cell death is inhibited.