Crossrecognition by CD8 T cell receptor αβ cytotoxic T lymphocytes of peptides in the self and the mycobacterial hsp60 which share intermediate sequence homology

Immunization of C57BL/6 mice with the mycobacterial heat shock protein (hsp) 60 in immunostimulating complexes caused the in vivo activation of autoreactive major histocompatibility complex class I (H-2D^b)-restricted CD8 T cell receptor (TcR) α/β cells. A CD8 TcR α/β clone with specificity for the mycobacterial hsp60 peptide_499–508 was derived from this immunization, which, in addition, recognized syngeneic macrophages which had been stressed by interferon-γ (IFN-γ) stimulation. The stress-induced, self peptide could be extracted from IFN-γ-stressed macrophages by acid elution, suggesting that the IFN-γ-induced self peptide is derived from an endogenous protein. Based on our observation that lysis of stressed target cells by this cytotoxic T lymphocyte (CTL) clone was specifically inhibited by hsp60-specific antisense oligonucleotides, we used synthetic peptides representing amino acid (aa) sequences of the murine hsp60 for target cell sensitization and identification of the relevant self peptide. Synthetic peptides representing 9-mer to 11-mer aa sequences of the murine hsp60 with asparagine in anchor position 4 or 5 as the minimal requirement for H-2D^b binding were tested in CTL assays. The overlapping murine hsp60 peptides_{162–170/171} were stimulatory at a concentration as low as 10–100 pM. Seven other peptides of the murine hsp60 required intermediate peptide concentrations of 10–100 nM for recognition by the CTL clone. Although the murine and mycobacterial hsp60 peptides recognized by this CTL clone showed only intermediate homology (3 identical and 3 similar aa), our data suggest that endogenous hsp60 itself is the source of self peptide(s) presented by IFN-γ-stressed macrophages to the cross-reactive CTL clone with promiscuous specificity. This notion is consistent with the idea of hsp as a link between infection and autoimmunity.     

Self-peptides that bind with low affinity to the diabetes-associated I-A(g7) molecule readily induce T cell tolerance in non-obese diabetic mice

Although non-obese diabetic (NOD) mice spontaneously develop T cell autoimmunity, it is not clear whether this phenomenon results from a defect in tolerance to self-Ag. Furthermore, as autoimmunity has been postulated to result from T cell responses directed toward self-peptides that bind with low affinity to NOD I-A(g7) MHC class II molecules, it is important to determine whether the expression of such peptides induces tolerance. We have constructed NOD transgenic (Tg) mice expressing the Leishmania antigen receptor for C kinase (LACK) Ag in either the thymus or pancreatic beta cells. We identified LACK peptides that were the targets of T cells in LACK-immunized NOD mice while binding to I-A(g7) with low affinity. While CD4(+) T cells from NOD mice secreted IFN-gamma, IL-4, IL-5 and IL-10 in response to LACK, those from LACK-expressing Tg mice secreted reduced levels of cytokines. Experiments using peptide/MHC multimers showed that LACK-expressing Tg mice exhibited self-reactive CD4(+) T cells with impaired proliferation capabilities. Hence, even self-peptides that bind to I-A(g7) with low affinity can induce tolerance in NOD mice. This result is important in light of the commonly held hypothesis that T cells reacting to peptides that bind to MHC with low affinity escape tolerance induction and cause autoimmunity.     

Autoreactive T cells from normal mice recognize mycobacterial 65 kd heat-shock protein from Mycobacterium bovis.

We established a TCR V beta 6+ CD4+ autoreactive T cell line from lymph node cells of unprimed BALB/c mice bred in a specific pathogen-free condition. The T cell line proliferated in the presence of irradiated spleen cells expressing MHC class II I-Ad and/or I-Ed molecules. The proliferative response of the autoreactive T cell line was significantly enhanced in response to 65 kd heat-shock protein (hsp) from Mycobacterium bovis, a member of the hsp60 family, which is highly conserved in both prokaryotes and eukaryotes. Our results suggest that the autoreactive T cells in normal mice may recognize endogenous peptides of self-hsp60 bound to MHC class II gene products and are cross-reactive to mycobacterial 65 kd hsp. The autoreactive hsp-specific T cells may therefore play an important role in the immune surveillance against invasion of various pathogens and tumors by recognizing infected and transformed autologous cells that express high levels of endogenous hsp60.     

Dual overlapping peptides recognized by insulin peptide B:9-23 T cell receptor AV13S3 T cell clones of the NOD mouse

T cells isolated from islets of non-obese diabetic (NOD) mice are enriched for insulin-reactive cells. The great majority of these T cells recognize insulin B chain peptide (B:9-23). B:9-23 reactive T cell clones are diabetogenic and show a dramatic TCR alpha -chain restriction (predominant AV13S3). We have studied the reactivity of five different B:9-23 reactive T cell clones to truncated peptides and alanine substituted analogues of B:9-23. Amongst these AV13S3 T cell clones, one reacted with peptide B:9-16 and four with B:13-23. The two peptides have in common only four amino acids (B:13-16; EALY). Having defined minimal peptide epitopes, we evaluated a mutant insulin sequence (B:13 glutamine) which retains metabolic activity. As predicted, this single amino acid change abrogated T cell reactivity. In addition, we have created a modified I-A(g7)gene with the B:9-23 peptide covalently linked to I-A(g7). Antigen presenting cells transfected with this construct were excellent presenting cells for all clones studied. The definition of dual peptide motifs and creation of bioactive covalent I-A(g7)-B:9-23 should facilitate studies of the pathogenic significance and antigen recognition by B:9-23 reactive diabetogenic T cells.     

Prevention of diabetes in the NOD mouse by a Th1 clone specific for a hsp60 peptide

Peptide-based therapies have been shown to be effective in the prevention of diabetes in the NOD mouse. We have been interested in the T cell response elicited by such therapies and have been studying a T cell clone (C3.5) specific for hsp60 AA 437-460, generated following immunization with the hsp60 437-460 peptide. The C3.5 clone was CD4(+), Vbeta8.3 TCR(+), I-A(g7)restricted and of the Th1 type. The injection of this clone into prediabetic NOD mice prevented the adoptive transfer of the disease and suppressed the development of spontaneous diabetes. This effect was reflected in a reduction in the degree and severity of insulitis in mice injected with this clone. In addition, an antibody response was elicited to the C3.5 clone in mice given multiple injections of the clone. The epitope recognized by C3.5 is located in the N-terminus of the hsp60 AA 437-460 peptide, and this clone was unable to recognize the native hsp60 molecule. These data raise questions concerning the mechanism by which peptide-based therapies prevent autoimmune disease.     

Epitope specificity and MHC restriction of rheumatoid arthritis synovial T cell clones which recognize a mycobacterial 65 kDa heat shock protein.

CD4+ T cell clones specific for the mycobacterial hsp 65 were obtained from synovial fluid of a DR4 homozygous rheumatoid arthritis (RA) patient. A stimulatory epitope was defined using both deletion mutants of the mycobacterial hsp 65 and synthetic peptides and proved to be in a highly conserved region of the molecule. Despite this, however, there was no recognition by these clones of either the recombinant human homologue of mycobacterial hsp 65, P60, nor of a synthetic peptide containing an amino acid sequence from P60 corresponding to the epitope defined in the mycobacterial hsp 65. When the pattern of HLA restriction shown by the hsp-65-specific T cell clones was investigated, all clones tested proved to be restricted by HLA-DP rather than the more usual HLA-DR. Inhibition experiments suggested that this restriction also applied to the polyclonal synovial T cell response to hsp 65, but not to other antigens. Exclusive restriction of T cell recognition of an antigen by HLA-DP has not been reported previously, and strongly suggests that in this case the T cell repertoire for recognizing hsp 65 in the context of DR4 is deficient. Such an association between DR4 and the inability to respond to an immunodominant bacterial antigen may have implications for the pathogenesis of RA.     

Secretory granule autoantigen in insulin-dependent diabetes mellitus is related to 62 kDa heat-shock protein (hsp60).

Recent studies in NOD mice suggest that cellular and humoral responses against beta cell protein(s) cross-reactive with mycobacterial heat-shock protein, hsp60, are implicated in the development of autoimmune diabetes. However, this putative, hsp60-related autoantigen has not yet been identified nor have the preceding events triggering the autoimmunity against it. Our recent studies show that antibodies to the mammalian hsp60 bind specifically to the 62 kDa protein located to insulin secretory granules and mitochondria of pancreatic beta cells of healthy mice [1]. In islets of prediabetic NOD mice affected by insulitis, the cellular distribution of this hsp60-related antigen was found to be altered. In the present report, we have examined whether this endogenous hsp60-related protein of secretory granules serves as an autoantigen in type I diabetes. The results of Western blot analysis indicate that diabetic mice sera show reactivity to a 62 kDa islet cell antigen. The NOD mice sera that were positive in detection of the 62 kDa islet cell antigen were also able to recognize the recombinant human hsp60. Immunogold electron microscopy revealed that diabetic NOD mouse sera, cross-reactive to human recombinant hsp60, recognize the antigen located in secretory granules of beta cells. Double-immunogold labelling demonstrated that antigens recognized by both diabetic NOD mice sera and monoclonal hsp60 antibodies co-localized in the same secretory granules of beta cells. Preincubation of islet cell sections with one type of antibody blocks subsequent binding of the other, indicating that epitopes recognized by both antisera on these proteins are shared. Moreover, preadsorption of diabetic sera with the recombinant human hsp60 abolished labelling of secretory granules. These results indicate that the hsp60-related protein of beta cell secretory granules is an autoantigen in type I diabetes in NOD mice.     

Relative resistance to nasally induced tolerance in non-obese diabetic mice but not other I-A(g7)-expressing mouse strains

I-A(g7) is a unique class II MHC molecule that is clearly associated with autoimmune diabetes in non-obese diabetic (NOD) mice. To determine if I-A(g7) is defective in its ability to deliver tolerogenic signals in vivo, H-2(g7) mice were nasally pretreated with antigen, prior to immunization, to induce antigen-specific regulation. Nasally pretreated NOR (H-2(g7)) and (NON).NOD (H-2(g7)) congenic mice showed responses similar to those of NON (H-2(nb1)), BALB/c (H-2(d)) and B10.PL (H-2(u)) mice-a reduced recall response and a deviated T(h) cytokine profile. However, we found that NOD (H-2(g7)) mice are comparatively resistant to immunological tolerance induced by nasal pretreatment, such that at the usually effective dose no significant reduction was seen in the proliferative recall responses to nominal antigen after immunization. (NOD x BALB/c)F(1) (H-2(g7/d)) and (NOD x NOR)F(1) (H-2(g7)) mice were similarly resistant to nasal-induced tolerance, although significantly higher nasal doses of antigen were able to overcome the resistance in NOD and F(1) mice. Interestingly, activated NOD T cells were resistant to cell death induced by re-stimulation with plate-bound anti-CD3. These results demonstrate that activated T cells in NOD mice are defective in their ability to respond to regulatory signals delivered in vivo or in vitro. Furthermore, NOD T cells have an increased resistance to tolerance induced by I-A(g7)-dependent (antigen) or I-A(g7)-independent (anti-CD3) mechanisms. Thus, while I-A(g7) may contribute to insulin-dependent diabetes mellitus by selecting a particular repertoire of self-reactive T cell clones, additional defects in the peripheral T cells themselves are required to allow the expansion of diabetogenic clones and the development of autoimmune disease.     

Molecular profile of the T cell receptors of regulatory and effector CD4+ T cells recognizing overlapping determinants on glutamic acid decarboxylase (524-543)

Glutamic acid decarboxylase (GAD65) is one of the autoantigens that initiates pathogenic T cell responses against insulin-secreting pancreatic beta cells in Type 1 diabetes (T1D). Previously it was shown that spontaneously arising pathogenic T cell responses in the NOD mouse model are confined to GAD530-543 (p530). However, regulatory T cell subpopulations, which can prevent diabetes, can also be generated, for example, by immunization with GAD524-538 (p524) or GAD524-543. Interestingly, two functionally distinct subpopulations of T cells which recognize overlapping determinants of GAD524-543, p524 and p530, utilize distinct TCR Vbeta families, Vbeta4 for pathogenic, and Vbeta12 for regulatory T cells. We characterized T cell receptors (TCRs) from each subpopulation of T cells and visualized p524-specific TCR/p524/I-A(g7) and p530-specific TCR/p530/I-A(g7) complexes via molecular modeling to help us understand, at a molecular level, the in vivo expansion of p524- or p530-specific T cells in the NOD model of T1D. The absolute restriction in Vbeta usage but not Valpha usage and conserved CDR3beta lengths for both T cell subpopulations demonstrates that the beta chains are main contributors in shaping both p524/I-A(g7) and p530/I-A(g7) restricted TCRs. However, only Vbeta4+ T cells but not Vbeta12+ T cells contain a common motif (DWG) in CDR3beta and may involve all of CDR1beta, CDR2beta, and CDR3beta in the recognition of the C-terminus of p530. These observations imply that the spontaneously arising p530-restricted TCRs may be selected under stringent structural frameworks to bind p530/I-A(g7) with high affinity. Thus, the pathogenic p530-specific T cells may arise from a small pool of autoreactive T cells upon breaking tolerance.     

Heat shock protein 60 and adjuvant arthritis: a model for T cell regulation in human arthritis

Heat shock proteins (hsp) are highly conserved, immune-dominant microbial proteins, whose expression is increased at sites of inflammation. In the experimental model of adjuvant arthritis (AA) immune responses to hsp determine the outcome of disease. AA can be transferred with a single T cell clone specific for a sequence of mycobacterial hsp65 (Mhsp65). Immunization with whole Mhsp65 on the other hand, protects in virtually all forms of experimental arthritis, including AA. This protective effect seems the consequence of the induction of a T cell response directed against self-hsp60. A similar protective effect of self-hsp60-specific T cells seems present in patients with a spontaneous remitting form of juvenile idiopathic arthritis. Next to hsp60, other hsp have similar protective effects in arthritis, while other conserved microbial proteins lack such capacity. Nasal administration of hsp60 peptides induces IL-10-driven regulatory T cells that are highly effective in suppressing arthritis. Thus hsp60, or peptides derived from hsp60, are suitable candidates for immune therapy in chronic arthritis.     

Searching for the Cartilage-associated Mimicry Epitope in Adjuvant Arthritis

Adjuvant arthritis (AA) is a T cell mediated disease which can be induced in genetically susceptible rats by immunization with heat-killed Mycobacterium tuberculosis ( Mt ) suspended in incomplete Freund's adjuvant. The critical mycobacterial T cell epitope for the induction of AA was previously identified as residues 178-186 of the mycobacterial 65 kDa heat shock protein ( Mt. hsp65 178-186 ). It was suggested that the development of AA was due to molecular mimicry between a mycobacterial epitope and a cartilage-associated self-antigen. However, until now such cartilage-associated mimicry epitope has not been identified. In this study we designed a computer search profile to predict mimicry self-epitopes, and investigated whether one or more of these self-epitopes could serve as mimicry epitopes in AA. Although several of these self-epitopes were recognized by arthritogenic T cells, no cross-reactivity was found between T cells specific for these self-epitopes and Mt. hsp65 178-186 specific T cells.     

Searching for the cartilage-associated mimicry epitope in adjuvant arthritis

Adjuvant arthritis (AA) is a T cell mediated disease which can be induced in genetically susceptible rats by immunization with heat-killed Mycobacterium tuberculosis (Mt) suspended in incomplete Freund's adjuvant. The critical mycobacterial T cell epitope for the induction of AA was previously identified as residues 178-186 of the mycobacterial 65 kDa heat shock protein (Mt. hsp65(178-186)). It was suggested that the development of AA was due to molecular mimicry between a mycobacterial epitope and a cartilage-associated self-antigen. However, until now such cartilage-associated mimicry epitope has not been identified. In this study we designed a computer search profile to predict mimicry self-epitopes, and investigated whether one or more of these self-epitopes could serve as mimicry epitopes in AA. Although several of these self-epitopes were recognized by arthritogenic T cells, no cross-reactivity was found between T cells specific for these self-epitopes and Mt. hsp65(178-186) specific T cells.     

T cell proliferative responses of type 1 diabetes patients and healthy individuals to human hsp60 and its peptides

T cell responses to peptide epitopes of the 60 kDa heat shock protein (hsp60) have been shown to play a role in the pathogenesis of type 1 insulin-dependent diabetes mellitus (IDDM) in mice. To test whether hsp60 autoimmunity might be involved in human type 1 diabetes, we studied T cell proliferative responses (stimulation index; SI) to intact human hsp60, to hsp60 peptides and to a recall antigen (tetanus toxoid) in 25 newly diagnosed type 1 diabetes patients, in 22 type 2 (non-insulin-dependent diabetes mellitus, NIDDM) patients, and in 25 healthy blood donors. There were no significant differences between the T cell responses of the three groups to tetanus toxoid. However, the responses to hsp60 of the type 1 diabetes group (median SI=5) were significantly greater (P<0. 01) than those of the type 2 group (median SI=1.67) and of the blood donors (median SI=1.7). Epitope mapping revealed significant responses to at least seven different peptides, with prevalent responses to the p277 peptide previously mapped in NOD mice and to peptide p32. Thus, newly diagnosed type 1 diabetes patients, similar to prediabetic and newly diabetic NOD mice, show heightened autoimmunity to hsp60 and hsp60 peptides.     

Elongated peptides,not the predicted nonapeptide stimulate a major histocompatibility complex class I-restricted cytotoxic T lymphocyte clone with specificity for a bacterial heat shock protein

The peptides recognized by an H-2D^b-restricted CD8 cytotoxic T lymphocyte (CTL) clone which is specific for the 60-kDa mycobacterial heat shock protein (hsp) and cross-reacts with stressed host cells were characterized. None of the nonapeptides from hsp60 conforming to the H-2D^b binding motif were able to sensitize target cells for lysis by this CTL clone. Sequence analysis of the stimulatory fraction from a trypsin digest of hsp60, together with synthetic peptide studies, defined a cluster of overlapping epitopes. Carboxy-terminal extension by at least one amino acid of the nonamer predicted to bind best to H-2D^b was essential for CTL recognition. Two such elongated peptides, a 10-mer and a 12-mer stimulated the clone at similarly low concentrations in the 100 pM range. We assume that these two peptides comply best with the natural epitope. In contrast, the 11-mer was inactive. The stimulatory 10-mer bound to H-2D^b with an efficacy similar to that of the nonapeptide corresponding to the H-2D^b motif, as revealed by peptide induced major histocompatibility complex (MHC) surface expression on RMA-S cells and competitive blocking of epitope recognition by the nonamer. Binding of these carboxy-terminally extended peptides to the MHC groove can be explained by anchoring through the amino acid residue Asn in position 5 of the peptide and by intrusion of the hydrophobic carboxy-terminal Ala (10-mer) or Leu (12-mer), but not Gly (11-mer), into the hydrophobic pocket of the H-2D^b cleft. Because the carboxy-terminal part is thus larger than predicted this region of the peptide may arch up from the binding groove. We assume that recognition of steric components of the MHC/peptide complex broaden the range of epitope specificity for a single T cell receptor. This flexibility not only promotes recognition of several overlapping peptides from a single antigen, but may also increase the chance of cross-reaction with similar peptides from unrelated proteins, including autoantigens. Consistent with this latter assumption, the T cell clone cross-recognizes mycobacterial hsp60 and stressed host cells.     

HLA-DQ8 transgenic and NOD mice recognize different epitopes within the cytoplasmic region of the tyrosine phosphatase-like molecule, IA-2

Type 1 diabetes mellitus is strongly associated with HLA-DQ8 in humans and I-A(g7) in the NOD mouse. The disease is characterized by loss of tolerance to auto-antigens such as GAD, insulin, and the protein tyrosine phosphatase-like molecule, IA-2. We identified T cell epitopes on the intracytoplasmic region of IA-2 by immunizing DQ8/NOD, DQ8/B10, and NOD mice with overlapping 18 mer peptides in CFA. We identified four peptides presented both by DQ8 and NOD, five DQ8 specific peptides, and six NOD specific peptides. Both mouse lines failed to respond to ten peptides. We demonstrated MHC class II and CD4 restriction of proliferative responses using appropriate blocking antibodies. To understand the role of non-MHC genes in the generation of immune response to the islet auto-antigen, we evaluated cytokine secretion following immunization of DQ8 transgenic mice with strongly immunogenic peptides. The NOD background resulted in increased secretion of cytokines. In conclusion, we have identified IA-2 peptides that induce lymphoproliferative responses in DQ8 transgenic and NOD mice and shown that these peptides stimulate production of Th1 and Th2 cytokines.     

Characterization of T-cell receptor gamma delta T cells appearing at the early phase of murine Listeria monocytogenes infection.

It has been shown that T-cell receptor (TcR) gamma delta + CD4- CD8- T cells increase in number and have an important role in early protection in murine Listeria monocytogenes infection. In this report, to characterize further the phenotype of the gamma delta T cells in listeriosis, we analysed V region gene usage and in vitro antigen recognition of the TcR gamma delta T cells in the peritoneal cavity of mice at the early phase after i.p. infection with a sublethal dose of L. monocytogenes. The gamma delta T cells predominantly expressed V delta 6 which has been reported to be expressed by TcR gamma delta-bearing foetal thymocyte hybridomas specific to mycobacterial and self heat-shock protein (hsp) 60. These early appearing CD3+ CD4- CD8- T cells in Listeria-infected mice, which were reported to be TcR gamma delta T cells, increased in proportion and in size by in vitro stimulation with recombinant hsp 60 from Mycobacterium bovis and purified protein derivative from M. tuberculosis but not by stimulation with heat-killed L. monocytogenes. A 65,000 MW molecule was detected in the lysate of viable L. monocytogenes but not in the lysate of heat-killed L. monocytogenes by a monoclonal antibody (mAb) raised against mycobacterial hsp 60. These results suggest that the V delta 6-bearing peripheral gamma delta T cells are activated by recognizing listerial hsp 60 expressed by viable L. monocytogenes. The hsp 60-reactive V delta 6-bearing T cells may have an important role in protection against L. monocytogenes and other parasites that express hsp 60 at high level.     

Inflammation activates self hsp60-specific T cells

Injection of incomplete Freund's adjuvant (IFA) into the footpads of BALB/c mice induced an acute inflammation. Draining popliteal lymph nodes showed major histocompatibility complex (MHC) class II-restricted proliferation when challenged in vitro with recombinant Mycobacterium bovis 65-kDa heat shock protein (hsp65). αβ Tcell receptor-positive, CD4⁺, hsp65-specific T cell lines and clones were generated from these lymph nodes, and 87% of clones responded to a P galactosidase fusion protein containing residues 238–573 of human hsp60. Seventy percent of these hsp60-responsive clones also responded to a synthetic peptide corresponding to residues 412–423 of the mouse hsp60. This peptide also induced significant responses in IFA-primed lymph node cells but not in lymphoid cells from unimmunized mice. These results demonstrate that T cells specific for epitopes in self hsp60 are activated during inflammatory responses induced in the absence of exogenous bacterial hsp65. The findings of this study may provide a basis for understanding the often reported isolation of mycobacterial hsp65-responsive T cells from inflammatory sites of arthritis patients, and the protective effects of preimmunization with hsp65 in experimental models of arthritis.     

Heat shock proteins and autoimmunity in humans

Summary T cells and antibodies against self and non-self hsp are present in both patients and healthy controls. T cells responding to hsp65 can be involved in autoimmune diseases, this was demonstrated for two site-specific animal autoimmune diseases: AA in Lewis rats and diabetes (IDDM) in NOD mice. In human ReA there is evidence for a direct stimulation of joint T cells by antigens of the organisms causing the infection which precedes the joint inflammation. The individual antigens of the triggering bacteria still have to be defined, but hsp65 may be of importance since this is one of the molecules recognized by synovial T cells in ReA patients.In RA there are no clear data implicating an infection in the initiation of joint inflammation, but mycobacteria have been suggested to be involved. We have discussed experimental findings which are in favor of, or in contradiction with, a role of mycobacterial antigens — particularly hsp65 — in the etiology of RA. T cells recognizing hsp65 and other mycobacterial antigens are present in the joint, but there is no indication for a specific involvement of one or a limited set of (myco)bacterial antigens in the pathogenesis of RA.     

Adoptive transfer of islet antigen-autoreactive T cell clones to transgenic NOD.Ea(d)mice induces diabetes indicating a lack of I-E mediated protection against activated effector T cells

Transgenic insertion of the MHC class II Ea(d)gene in NOD mice restores I-E expression and prevents T-cell-mediated autoimmune diabetes (IDDM). The specific molecular and cellular mechanisms responsible for the diabetes resistance of transgenic NOD.Ea(d)mice remain unclear. We adoptively transferred islet antigen-specific T cell clones into NOD and transgenic NOD.Ea(d)mice to evaluate the level of protection provided by I-E expression against activated effector T cells. We have found that neither neonatal or 3-5-week-old I-E-expressing NOD.Ea(d)mice can completely inhibit the diabetogenic activities of activated islet antigen-specific T cell clones. These data indicate that Ealpha protein expression in NOD antigen presenting cells (APC) does not reduce islet autoantigen presentation in the context of I-A(g7)below the threshold required for stimulation of effector/memory diabetogenic T cells. Our results suggest that the mechanism of Ealpha protein-mediated diabetes resistance in NOD mice may be "antigen ignorance," in which the quantity of islet autoantigens presented in the context of I-A(g7)by APC is reduced below the threshold required to activate nai;ve islet antigen-specific T cells.     

The 65-kDa heat-shock protein in the pathogenesis, prevention and therapy of autoimmune arthritis and diabetes mellitus in rats and mice

Conclusions hsp are molecules which are highly conserved from procaryotes to eukaryotes. At a first glance the immune system should treat these molecules as self. However, strong immune reactions to bacterial hsp are observed during infection in mammals.hsp65 plays a role in several autoimmune diseases in animal models. In AA in Lewis rats the involvement of hsp65 has been revealed by T cell clones which induce disease in naive recipients, or by T cell vaccination experiments. T cell clones which show in vivo activity have been used as tools in vitro to define epitopes involved in the disease process. In this manner mycobacterial hsp65 and its epitope peptide 180–188 were deduced for AA in Lewis rats. Similarily the epitope p277 was defined for diabetes in NOD mice.The role of hsp65 in several other autoimmune diseases was seen when animals were pretreated with hsp65 and found to be protected from subsequent induction of autoimmune disease. From the involvement of hsp65 in several different autoimmune diseases, it would appear that hsp65 is somehow a key factor in natural autoimmunity. At a fist glance this is surprising since mycobacterial hsp65 shows 50% amino acid homology with human hsp65, in other words it is half-self.Peptide epitopes, peptide 180–188 in AA in Lewis rats and p277 in IDDM in NOD mice, have been used for peptide vaccination, which represents another possibility for prevention of autoimmune disease. The immunological mechanism which leads to resistance from autoimmune disease involves hsp65 immunity and appears not to be associated with tolerance or non-responsiveness to hsp65, but seems to be due rather to modulation of naturally existing networks of idiotype-anti-idiotype T cells organized around hsp65 as the target antigen.