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.     

Low-dose peptide tolerance therapy of lupus generates plasmacytoid dendritic cells that cause expansion of autoantigen-specific regulatory T cells and contraction of inflammatory Th17 cells

Subnanomolar doses of an unaltered, naturally occurring nucleosomal histone peptide epitope, H4(71-94), when injected s.c. into lupus-prone mice, markedly prolong lifespan by generating CD4+25+ and CD8+ regulatory T cells (Treg) producing TGF-beta. The induced Treg cells suppress nuclear autoantigen-specific Th and B cells and block renal inflammation. Splenic dendritic cells (DC) captured the s.c.-injected H4(71-94) peptide rapidly and expressed a tolerogenic phenotype. The DC of the tolerized animal, especially plasmacytoid DC, produced increased amounts of TGF-beta, but diminished IL-6 on stimulation via the TLR-9 pathway by nucleosome autoantigen and other ligands; and those plasmacytoid DC blocked lupus autoimmune disease by simultaneously inducing autoantigen-specific Treg and suppressing inflammatory Th17 cells that infiltrated the kidneys of untreated lupus mice. Low-dose tolerance with H4(71-94) was effective even though the lupus immune system is spontaneously preprimed to react to the autoepitope. Thus, H4(71-94) peptide tolerance therapy that preferentially targets pathogenic autoimmune cells could spare lupus patients from chronically receiving toxic agents or global immunosuppressants and maintain remission by restoring autoantigen-specific Treg cells.     

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.     

T cell tolerance to germline-encoded antibody sequences in a lupus-prone mouse

The BCR V region has been implicated as a potential avenue of T cell help for autoreactive B cells in systemic lupus erythematosus. In principle, either germline-encoded or somatically generated sequences could function as targets of such help. Preceding studies have indicated that class II MHC-restricted T cells in normal mice attain a state tolerance to germline-encoded Ab diversity. In this study, we tested whether this tolerance is intact in systemic lupus erythematosus-prone (New Zealand Black x SWR)F1 mice (SNF1). Using a hybridoma sampling approach, we found that SNF1 T cells were tolerant to germline-encoded Ab sequences. Specifically, they were tolerant to germline-encoded sequences derived from a lupus anti-chromatin Ab that arose spontaneously in this strain. This was true both for diseased and prediseased mice. Thus, there does not appear to be a global defect in T cell tolerance to Ab V regions in this autoimmune-prone strain either before or during autoimmune disease.     

Molecular and structural properties of three autoimmune IgG monoclonal antibodies to histone H2B

In systemic autoimmune diseases such as lupus the immune system produces autoantibodies to nuclear antigens including DNA and histone molecules. In the present study, we describe three monoclonal IgG antibodies that have been obtained from lupus-prone MRL/lpr mice. These three antibodies react with the amino terminus of histone H2B, a region of the molecule that is accessible in chromatin. Using a series of overlapping H2B synthetic peptides and structural analogues, we have mapped the different epitopes recognized by these antibodies. We have also sequenced the combining sites (variable regions) of the antibodies and modeled their interactions with the corresponding epitopes. Overall, the data suggest that the mechanisms of interaction with antigen are different for each of the three antibodies, even though they all react with the amino-terminal domain of the histone H2B molecule. The results also suggest that the binding between these antibodies and histone H2B is different from that between most antibodies and conventional protein antigens since the heavy chain complementarity-determining region 3 appears to play only a limited role in the three antibodies tested. The study of the interaction between self-antigens and spontaneously occurring autoantibodies may help us elucidate the mechanisms driving the expansion of self-reactive lymphocytes.     

Very low-dose tolerance with nucleosomal peptides controls lupus and induces potent regulatory T cell subsets

We induced very low-dose tolerance by injecting lupus prone (SWR x NZB)F1 (SNF1) mice with 1 mug nucleosomal histone peptide autoepitopes s.c. every 2 wk. The subnanomolar peptide therapy diminished autoantibody levels and prolonged life span by delaying nephritis, especially by reducing inflammatory cell reaction and infiltration in kidneys. H4(71-94) was the most effective autoepitope. Low-dose tolerance therapy induced CD8+, as well as CD4+ CD25+ regulatory T (Treg) cell subsets containing autoantigen-specific cells. These adaptive Treg cells suppressed IFN-gamma responses of pathogenic lupus T cells to nucleosomal epitopes at up to a 1:100 ratio and reduced autoantibody production up to 90-100% by inhibiting nucleosome-stimulated T cell help to nuclear autoantigen-specific B cells. Both CD4+ CD25+ and CD8+ Treg cells produced and required TGF-beta1 for immunosuppression, and were effective in suppressing lupus autoimmunity upon adoptive transfer in vivo. The CD4+ CD25+ T cells were partially cell contact dependent, but CD8+ T cells were contact independent. Thus, low-dose tolerance with highly conserved histone autoepitopes repairs a regulatory defect in systemic lupus erythematosus by generating long-lasting, TGF-beta-producing Treg cells, without causing allergic/anaphylactic reactions or generalized immunosuppression.     

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.     

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.     

V region sequences of an idiotypically connected family of pathogenic anti-DNA autoantibodies

The H and L chain V region sequences of nine anti-DNA mAb that are representative of a pathogenic population of autoantibodies produced by the nephritis prone (SWR x NZB)F1 (SNF1) mice, were determined. These nine anti-DNA autoantibodies were idiotypically connected members of a cross-reactive Id family called the Id564 cluster. Moreover, these autoantibodies were all cationic in charge, IgG2b in isotype, and their H chain C regions had the normal SWR parent's allotype. Although derived from two different SNF1 animals, these pathogenic autoantibodies possessed highly homologous Leader-VH sequences that could account for their idiotypic cross-reactivity. Furthermore, the VH region sequences of these anti-DNA antibodies contained numerous basic residues that could impart their cationic charge. The Leader-VH sequences of these autoantibodies were also highly homologous to that of an anti-NP antibody-related germ-line gene of C57BL/6 mice, called VH-23. Among these nine pathogenic autoantibodies, three sets of clonally related anti-DNA antibodies could be identified. Thus the Id564 cluster of cationic anti-DNA autoantibodies of SNF1 mice are encoded by highly related VH genes, and this idiotypically connected population of pathogenic autoantibodies are selected to undergo an oligoclonal expansion in the lupus-prone SNF1 mice.     

Accelerated macrophage apoptosis induces autoantibody formation and organ damage in systemic lupus erythematosus

Increased monocyte/macrophage (Mphi) apoptosis occurs in patients with systemic lupus erythematosus (SLE) and is mediated, at least in part, by an autoreactive CD4(+) T cell subset. Furthermore, autoreactive murine CD4(+) T cells that kill syngeneic Mphi in vitro induce a lupus-like disease in vivo. However, it is unclear whether increased Mphi apoptosis in SLE per se is sufficient to accelerate/promote autoimmunity. We have investigated whether increased Mphi apoptosis in vivo, induced by the administration of clodronate liposomes, can exacerbate the autoimmune phenotype in NZB x SWR (SNF(1)) lupus-prone mice, and induce autoantibody production in haplotype-matched BALB/c x DBA1 (DBF(1)) non-lupus-prone mice. Lupus-prone mice SNF(1) mice that were treated with clodronate liposomes, but not mice treated with vehicle, developed significant increases in autoantibodies to dsDNA, nucleosomes, and the idiotypically related family of nephritic Abs Id(LN)F(1), when compared with untreated SNF(1) mice. Furthermore, clodronate treatment hastened the onset of proteinuria and worsened SNF(1) lupus nephritis. When compared with vehicle-treated controls, clodronate-treated non-lupus-prone DBF(1) mice developed significantly higher levels of anti-nucleosome and Id(LN)F(1) Abs but did not develop lupus nephritis. We propose that Mphi apoptosis contributes to the pathogenesis of autoantibody formation and organ damage through both an increase in the apoptotic load and impairment in the clearance of apoptotic material. This study suggests that mechanisms that induce scavenger cell apoptosis, such as death induced by autoreactive cytotoxic T cells observed in SLE, could play a pathogenic role and contribute to the severity of the disease.     

Heavy chain revision in MRL mice: a potential mechanism for the development of autoreactive B cell precursors

Abs reactive to DNA and DNA/histone complexes are distinguished by the presence of positively charged amino acids, such as arginine, in the heavy chain complementarity-determining region 3. The presence of these amino acids partly results from atypical V(H)-D-J(H) rearrangements such as D-D fusions and D inversions. Previous results in our laboratory demonstrated that newborn autoimmune MRL/MpJ-+/+ mice undergo these unusual recombinations more frequently when compared with normal C3H/HeJ controls. In addition, the heavy chain junctions in newborn MRL mice demonstrated a preferred usage of V(H)-proximal D genes and distal J(H) genes suggestive of secondary gene rearrangements. In this study we explore the possibility that adult MRL B220(+)IgM(-) pre B cells, which have not yet undergone Ag selection, exhibit similar rearrangement patterns. Indeed, MRL pre-B cells possessed more atypical rearrangements (D-D fusions) than those of C3H/HeJ mice. However, the biased use of upstream D genes and downstream J(H) genes observed in the newborn MRL mice was not present in the pre-B cell library. These results suggest that the heavy chain rearrangement process persists later during B cell life in lupus-prone mice and lead us to propose a model of heavy chain receptor revision in the periphery of autoimmune mice.     

Atorvastatin inhibits autoreactive B cell activation and delays lupus development in New Zealand black/white F1 mice

Systemic lupus erythematosus is a multisystem autoimmune disease characterized by a wide range of immunological abnormalities that underlie the loss of tolerance. In this study we show that administration of atorvastatin to lupus-prone NZB/W F(1) mice resulted in a significant reduction in serum IgG anti-dsDNA Abs and decreased proteinuria. Histologically, the treatment was associated with reduced glomerular Ig deposition and less glomerular injury. Disease improvement was paralleled by decreased expression of MHC class II on monocytes and B lymphocytes and reduced expression of CD80 and CD86 on B lymphocytes. Consequent upon this inhibition of Ag presentation, T cell proliferation was strongly impaired by atorvastatin in vitro and in vivo. A significant decrease in MHC class II expression was also observed in the target organ of lupus disease (i.e., the glomerulus). Serum cholesterol in atorvastatin-treated lupus mice fell to the level found in young NZB/W mice before disease onset. This is the first demonstration that atorvastatin can delay the progression of a spontaneous autoimmune disease and may specifically benefit patients with systemic lupus erythematosus.     

Suppression of experimental autoimmune uveitis in rats by the oral administration of the uveitopathogenic S-antigen fragment or a cross-reactive homologous peptide.

The oral administration of S-antigen fragment (a synthetic peptide designated as peptide M and known to be uveitopathogenic for rat, guinea pig, and monkey) to Lewis rats prior to challenge with an emulsion of peptide M and CFA resulted in either a total or partial suppression of experimental autoimmune uveitis (EAU), a T cell-mediated autoimmune disease studied as a model for human uveitis and experimental autoimmune pinealitis (EPA). Both the clinical and histopathologic manifestations of the disease were suppressed in a dose-dependent manner. Pinealitis associated with EAU was also suppressed by the oral administration of peptide M. Additionally, ingestion of a fragment of baker's yeast (Saccharomyces cerevisiae) histone H3, which has five consecutive amino acids identical to peptide M and which has been found to be uveitopathogenic in Lewis rats, induced tolerance to either peptide M or synthetic histone H3 peptide. In addition, the proliferative response to peptide M was inhibited in peptide M-fed rats. The suppression of EAU and in vitro lymphocyte proliferative responses to peptide M were observed to be antigen specific, since oral feeding of a control protein (BSA) exerted no suppressive effect. Furthermore, the T cells isolated from the spleen and lymph nodes of animals rendered tolerant by oral administration of peptide M can transfer protection against EAU adoptively. These results demonstrate that the oral administration of an autoantigen or its homologous peptide initiates an antigen-specific cellular mechanism which may ameliorate EAU.     

Production of a recombinant cholera toxin B subunit-insulin B chain peptide hybrid protein by Brevibacillus choshinensis expression system as a nasal vaccine against autoimmune diabetes

Mucosally induced tolerance is an attractive strategy for preventing or reducing autoimmune diseases. Here, we produced a recombinant CTB fusion protein linked with autoantigen T cell epitope of insulin B chain peptide 9-23 (C19S) at levels up to 200 mg/L culture media in Brevibacillus choshinensis secretion-expression system. Receptor-competitive assay showed that the CTB-insulin peptide binds to GM1 receptor almost equivalent degree as the native form of CTB. Non-obese diabetes (NOD) mice that spontaneously develop an insulin-dependent diabetes were nasally immunized with CTB-insulin peptide (5 microg) for three times. The nasal treatment significantly reduced the development of insulin-dependent diabetes and peptide specific DTH responses after systemic immunization with the insulin peptide B 9-23(C19S) in CFA. Nasal administration of as high as 50 microg of the peptide alone demonstrated a similar level of the disease inhibition. In contrast, all mice given 5 microg of the insulin peptide alone or 5 microg of insulin peptide with 25 microg of the free form of CTB did not lead to the suppression of diabetes development and DTH responses. Because molecular weight of the insulin peptide is about one tenth of that of the CTB-insulin peptide, the results demonstrate that the recombinant hybrid of autoantigen and CTB increased its tolerogenic potential for nasal administration by up 100-fold on molar base of autoantigen peptide. Taken together, nasally-induced tolerance by administration of the recombinant B. choshinensis-derived hybrid protein of CTB and autoantigen T cell-epitope peptide could be useful mucosal immunetherapy for the control of T cell-mediated autoimmune diseases.     

Histone autoantigens in murine lupus. Definition of a major epitope within an accessible region of chromatin.

In SLE and in the (NZB x NZW)F1 murine model of this disease, IgG autoantibodies are frequently produced to DNA and histones. In the present study, we define a linear epitope on histone H2B that is recognized by (NZB x NZW)F1 mice. IgG antibodies from anti-H2B positive (but not anti-H2B negative) mice bound strongly to a peptide containing the first 15 N-terminal amino acids, a region that is exposed in chromatin. Competitive inhibition studies showed that the binding of autoantibodies to H2B in ELISA as well as the binding to soluble H2B was substantially blocked by this peptide. Studies with smaller peptides mapped the epitope to residues 3-12. Individual mice recognized different residues within this region, and a sequence search did not reveal proteins other than H2B that could elicit this spectrum of antibodies. Interestingly, these autoantibody specificities were not a component of those induced in preautoimmune mice by immunization with H2B/RNA complexes or with H2B peptide 1-30 containing the autoantigenic sequence. These findings argue that recognition of a specific N-terminal region of self histone contributes to the anti-H2B autoantibody response in lupus. Autoreactive B cells with specificity for this sequence seem to develop only after the autoimmune process has been initiated.     

Production of high affinity autoantibodies in autoimmune New Zealand Black/New Zealand white F1 mice targeted with an anti-DNA heavy chain

Lupus-prone, anti-DNA, heavy (H) chain "knock-in" mice were obtained by backcrossing C57BL/6 mice, targeted with a rearranged H chain from a VH11(S107)-encoded anti-DNA hybridoma (D42), onto the autoimmune genetic background of New Zealand Black/New Zealand White (NZB/NZW) F1 mice. The targeted female mice developed typical lupus serologic manifestations, with the appearance of transgenic IgM anti-DNA autoantibodies at a young age (2-3 mo) and high affinity, somatically mutated IgM and IgG anti-DNA Abs at a later age (6-7 mo). However, they did not develop clinical, lupus-associated glomerulonephritis and survived to at least 18 mo of age. L chain analysis of transgenic anti-DNA Abs derived from diseased NZB/NZW mouse hybridomas showed a very restricted repertoire of Vkappa utilization, different from that of nonautoimmune (C57BL/6 x BALB/c)F1 transgenic anti-DNA Abs. Strikingly, a single L chain was repetitively selected by most anti-DNA, transgenic NZB/NZW B cells to pair with the targeted H chain. This L chain had the same Vkappa-Jkappa rearrangement as that expressed by the original anti-DNA D42 hybridoma. These findings indicate that the kinetics of the autoimmune serologic manifestations are similar in wild-type and transgenic lupus-prone NZB/NZW F1 mice and suggest that the breakdown of immunologic tolerance in these mice is associated with the preferential expansion and activation of B cell clones expressing high affinity anti-DNA H/L receptor combinations.     

B cell tolerance and positive selection in lupus

Systemic lupus erythematosus is considered a prototype of systemic autoimmune diseases; however, despite considerable advances in recent years in the understanding of basic mechanisms in immunology, little progress has been made in elucidating the etiology and pathogenesis of this disease. This even holds for inbred mice, such as the lupus-prone New Zealand Black/New Zealand White F(1) mice, which are all genetically programmed to develop lupus at a predetermined age. This frustrating state of affairs calls for a fundamental change in our scientific thinking and the opening of new directions in lupus research. In this study, we suggest that intrinsic B cell tolerance mechanisms are not grossly impaired in lupus-prone mice, but that an unusually strong positive selection event recruits a small number of autoreactive B cells to the germinal centers. This event could be facilitated by nucleic acid-protein complexes that are created by somatic changes in the susceptible animal.