Mouse lysozyme-M knockout mice reveal how the self-determinant hierarchy shapes the T cell repertoire against this circulating self antigen in wild-type mice

We have studied T cell tolerance to defined determinants within ML-M using wild-type (WT; ML-M(+/+)) and LysMcre (ML-M(-/-)) C3H (H-2(k)) mice to determine the relative contribution of ML-M-derived epitopes vs those from other self Ags in selection of the ML-M-specific T cell repertoire. ML-M was totally nonimmunogenic in WT mice, but was rendered immunogenic in LysMcre mice. Most of the response to ML-M in LysMcre mice was directed to the immunodominant determinant region 105-119. This determinant is spontaneously displayed (without adding exogenous ML-M) by macrophages of WT, but not LysMcre, mice and is stimulatory for peptide 105-119 (p105-119)-primed T cells. Moreover, neonatal tolerization of LysMcre mice with p105-119 or ML-M abrogated the T cell response to subsequent challenge with ML-M or p105-119. Furthermore, p95-109 and p110-125 of ML-M were immunogenic in LysMcre mice, but not in WT mice, thereby representing subdominant, tolerance-inducing epitopes of ML-M. As expected, the T cell repertoire to cryptic ML determinants in WT mice was also intact in LysMcre mice. Furthermore, the pattern of response to the related homologue of ML-M, hen eggwhite lysozyme, was similar in these two groups of mice. Thus, several codominant T cell determinants within ML-M contribute significantly to tolerance induction, and the anti-cryptic T cell repertoire to ML-M was positively selected on non-ML-M self ligands. These results reveal that the induction of self tolerance to a multideterminant protein follows the quantitative hierarchy of self-determinant expression and are of relevance in understanding the pathogenesis of autoimmunity.     

The self-directed T cell repertoire against mouse lysozyme reflects the influence of the hierarchy of its own determinants and can be engaged by a foreign lysozyme.

The T cell repertoire is shaped by the processes of positive and negative selection. We have previously shown that mice are tolerant to a native self-Ag, mouse lysozyme (ML), but they respond vigorously when challenged with different ML peptides ("cryptic" self-determinants). In this study, we have addressed the issue of the physiological significance of both the hierarchy (dominance/crypticity) of self-determinants within ML and the anti-cryptic, self (ML)-directed T cell repertoire. Our results demonstrate that there are several ML peptides that bind well to MHC but are totally nonimmunogenic when tested for proliferative T cell response and cytokine secretion: a subset of these peptides presumably represent the originally dominant self-determinants of ML, which have rendered the T cells tolerant during thymic selection. Other ML peptides, which bind well to MHC and are immunogenic, correspond to the cryptic determinants of ML: T cells against cryptic ML determinants escape tolerance induction. Thus, the mature T cell repertoire against ML bears the direct imprint of the hierarchy of self (ML)-determinants. Interestingly, hen egg white lysozyme could prime T cells in vivo that were cross-reactive with certain cryptic ML determinants, and vice versa, without requiring any coimmunization with the foreign lysozyme and ML peptide(s). Moreover, repeated, deliberate priming and expansion of T cells by hen egg white lysozyme immunization concomitantly enhanced T cell response to such cross-reactive ML determinants. This reciprocal self-foreign determinant cross-reactivity may play a previously unrecognized, but crucial, role in the expansion and diversification of self-reactive clones in the autoimmune response.     

The regulatory C-terminal determinants within mycobacterial heat shock protein 65 are cryptic and cross-reactive with the dominant self homologs: implications for the pathogenesis of autoimmune arthritis

The 65-kDa mycobacterial heat shock protein (Bhsp65) has been invoked in the pathogenesis of both adjuvant arthritis (AA) in the Lewis rat (RT.1(l)) and human rheumatoid arthritis. Arthritic Lewis rats in the late phase of AA show diversification of the T cell response to Bhsp65 C-terminal determinants (BCTD), and pretreatment of naive Lewis rats with a mixture of peptides representing these neoepitopes affords protection against AA. However, the fine specificity and physiologic significance of the BCTD-directed T cell repertoire, and the role of homologous self (rat) hsp65 (Rhsp65), if any, in spreading of the T cell response to Bhsp65 have not yet been examined. We observed that T cells primed by peptides comprising BCTD can adoptively transfer protection against AA to the recipient Lewis rats. However, these T cells can be activated by preprocessed (peptide) form of BCTD, but not native Bhsp65, showing that BCTD are cryptic epitopes. The BCTD-reactive T cells can be activated by the naturally generated (dominant) C-terminal epitopes of both exogenous and endogenous Rhsp65 and vice versa. Furthermore, certain individual peptides constituting BCTD and their self homologs can also induce protection against AA. These results support a model for the diversification of T cell response to Bhsp65 during the course of AA involving up-regulation of the display of cryptic BCTD coupled with spontaneous induction of T cell response to the cross-reactive dominant C-terminal epitopes of Rhsp65. The identification of disease-regulating cryptic determinants in Ags implicated in arthritis provides a novel approach for immunotherapy of rheumatoid arthritis.     

Sercarzian immunology--In memoriam. Eli E. Sercarz, 1934-2009

During his long career as a principal investigator and educator, Eli Sercarz trained over 100 scientists. He is best known for developing hen egg white lysozyme (HEL) as a model antigen for immunologic studies. Working in his model system Eli furthered our understanding of antigen processing and immunologic tolerance. His work established important concepts of how the immune system recognizes antigenic determinants processed from whole protein antigens; specifically he developed the concepts of immunodominance and crypticity. Later in his career he focused more on autoimmunity using a variety of established animal models to develop theories on how T cells can circumvent tolerance induction and how an autoreactive immune response can evolve over time. His theory of "determinant spreading" is one of the cornerstones of our modern understanding of autoimmunity. This review covers Eli's entire scientific career outlining his many seminal discoveries.     

Suppressive effect of glutamic acid decarboxylase 65-specific autoimmune B lymphocytes on processing of T cell determinants located within the antibody epitope

Type 1 diabetes is a T cell-mediated disease in which B cells serve critical Ag-presenting functions. In >95% of type 1 diabetic patients the B cell response to the glutamic acid decarboxylase 65 (GAD65) autoantigen is exclusively directed at conformational epitopes residing on the surface of the native molecule. We have examined how the epitope specificity of Ag-presenting autoimmune B cell lines, derived from a type 1 diabetic patient, affects the repertoire of peptides presented to DRB1*0401-restricted T cell hybridomas. The general effect of GAD65-specific B cells was to enhance Ag capture and therefore Ag presentation. The enhancing effect was, however, restricted to T cell determinants located outside the B cell epitope region, because processing/presentation of T cell epitopes located within the autoimmune B cell epitope were suppressed in a dominant fashion. A similar effect was observed when soluble Abs formed immune complexes with GAD65 before uptake and processing by splenocytes. Thus, GAD65-specific B cells and the Abs they secrete appear to modulate the autoimmune T cell repertoire by down-regulating T cell epitopes in an immunodominant area while boosting epitopes in distant or cryptic regions.     

The T cells specific for the carboxyl-terminal determinants of self (rat) heat-shock protein 65 escape tolerance induction and are involved in regulation of autoimmune arthritis

Immunization of Lewis rats with heat-killed Mycobacterium tuberculosis H37Ra leads to development of polyarthritis (adjuvant-induced arthritis; AA) that shares several features with human rheumatoid arthritis (RA). Immune response to the 65-kDa mycobacterial heat-shock protein (Bhsp65) is believed to be involved in induction of AA as well as in experimental modulation of this disease. However, the understanding of several critical aspects of the pathogenesis of AA in the Lewis rat has severely been hampered by the lack of information both regarding the level as well as epitope specificity of tolerance to the mammalian self (rat) homologue of Bhsp65, 65-kDa rat heat-shock protein (Rhsp65), and about the functional attributes of the T cell repertoire specific for this self protein. In this study, we established that tolerance to Rhsp65 in the Lewis rat is incomplete, and that the residual T cells primed upon challenge with this self hsp65 are disease regulating in nature. We also have defined the T cell epitopes in the C-terminal region within Rhsp65 that contribute predominantly to the immune reactivity as well as the AA-protective effect of this self protein. Furthermore, the T cells primed by peptides comprising these C-terminal determinants can be efficiently restimulated by the naturally generated epitopes from endogenous Rhsp65, suggesting that self hsp65 might also be involved in natural remission from acute AA. These novel first experimental insights into the self hsp65-directed regulatory T cell repertoire in AA would help develop better immunotherapeutic approaches for autoimmune arthritis.     

Immune responses induced in HHD mice by multiepitope HIV vaccine based on cryptic epitope modification

CD8+ T cells play an important role in early HIV infection. However, HIV has the capacity to avoid specific CTL responses due to a high rate of mutation under selection pressure. Although the HIV proteins, gag and pol, are relatively conserved, these sequences generate low-affinity MHC-associated epitopes that are poorly immunogenic. Here, we applied an approach that enhanced the immunogenicity of low-affinity HLA-A2.1-binding peptides. The first position with tyrosine (P1Y) substitution enhanced the affinity of HLA-A2.1-associated peptides without altering their antigenic specificity. More importantly, P1Y variants efficiently stimulated in vivo native peptide-specific CTL that also recognized the corresponding naturally processed epitope. The potential to generate CTL against any low-affinity HLA-A2.1-associated peptide provides us with the necessary technique for identification of virus cryptic epitopes for development of peptide-based immunotherapy. Therefore, identification and modification of the cryptic epitopes of gal and pol provides promising candidates for HIV immunotherapy dependent upon efficient presentation by virus cells. Furthermore, this may be a breakthrough that overcomes the obstacle of immune escape caused by high rates of mutation. In this study, bioinformatics analysis was used to predict six low-affinity cryptic HIV gag and pol epitopes presented by HLA-A*0201. A HIV compound multi-CTL epitope gene was constructed comprising the gene encoding the modified cryptic epitope and the HIV p24 antigen, which induced a strong CD8+ T cell immune response regardless of the mutation. This approach represents a novel strategy for the development of safe and effective HIV prophylactic and therapeutic vaccines.     

A threshold for central T cell tolerance to an inducible serum protein

We report an inducible system of self Ag expression that examines the relationship between serum protein levels and central T cell tolerance. This transgenic approach is based on tetracycline-regulated expression of a secreted form of hen egg lysozyme, tagged with a murine hemoglobin (Hb) epitope. In the absence of the tetracycline-regulated transactivator, serum levels of the chimeric protein are extremely low (< or = 0.1 ng/ml) and the mice show partial tolerance to both Hb(64-76) and lysozyme epitopes. In the presence of the transactivator, expression increases to 1.5 ng/ml and the mice are completely tolerant. Partial tolerance was further investigated by crossing these mice to strains expressing transgenic TCRs. At the lowest Ag levels, 3.L2tg T cells (specific for Hb(64-76)/I-E(k)) escape the thymus and approximately 10% of CD4(+) splenocytes express the 3.L2 TCR. In contrast, 3A9 T cells (specific for hen egg lysozyme(46-61)/I-A(k)) are completely eliminated by negative selection. These data define a tolerogenic threshold for negative selection of Ag-specific T cells by circulating self proteins that are 100-fold more sensitive than previously demonstrated. They suggest that partial tolerance at extremely low levels of self Ag exposure is the result of a restricted repertoire of responding T cells, rather than a simple reduction in precursor frequency; tolerogenic thresholds are T cell specific.     

Induction of determinant spreading and of Th1 responses by in vitro stimulation with HER-2 peptides

Immunization with tumor antigens induces cellular and humoral immune responses. These responses by T cells are specific for defined epitopes (determinants) in the molecule of the immunizing tumor antigen. Extension of such responses to self-antigens requires induction of autoimmunity to the tumor. As with systems of autoimmune disease, expression of T cell autoimmunity is charaterized by diversification of responses from the inducer determinant to other responder (cryptic) determinants. Since similar strategies may be useful for therapy of human cancers, we investigated whether the induction of response to a HER-2 peptide F7 (776–789) induces enhanced reactivity of other HER-2 peptides. We found that stimulation with F7 can expand a response to another epitope F13 (884–899) in both an ovarian cancer patient with progressive disease and a healthy donor who shared HLA-DR11. This response was characterized mainly by increased interferon γ secretion, and proliferation, but was not observed with another donor who shared HLA-DR14 and HLA-DQ5 with the patient. Since repeated vaccination with the same epitope may lead to a decline of primary cell reactivity caused by apoptosis spreading the response to other epitopes, the tumor antigen may provide an approach for maintaining an inflammatory Th1 response during cancer vaccination. Received: 10 April 2000 / Accepted: 12 July 2000     

Goodpasture autoantibodies unmask cryptic epitopes by selectively dissociating autoantigen complexes lacking structural reinforcement: novel mechanisms for immune privilege and autoimmune pathogenesis

Rapidly progressive glomerulonephritis in Goodpasture disease is mediated by autoantibodies binding to the non-collagenous NC1 domain of alpha3(IV) collagen in the glomerular basement membrane. Goodpasture epitopes in the native autoantigen are cryptic (sequestered) within the NC1 hexamers of the alpha3alpha4alpha5(IV) collagen network. The biochemical mechanism for crypticity and exposure for autoantibody binding is not known. We now report that crypticity is a feature of the quaternary structure of two distinct subsets of alpha3alpha4alpha5(IV) NC1 hexamers: autoantibody-reactive M-hexamers containing only monomer subunits and autoantibody-impenetrable D-hexamers composed of both dimer and monomer subunits. Goodpasture antibodies only breach the quaternary structure of M-hexamers, unmasking the cryptic epitopes, whereas D-hexamers are resistant to autoantibodies under native conditions. The epitopes of D-hexamers are structurally sequestered by dimer reinforcement of the quaternary complex, which represents a new molecular solution for conferring immunologic privilege to a potential autoantigen. Dissociation of non-reinforced M-alpha3alpha4alpha5(IV) hexamers by Goodpasture antibodies is a novel mechanism whereby pathogenic autoantibodies gain access to cryptic B cell epitopes. These findings provide fundamental new insights into immune privilege and the molecular mechanisms underlying the pathogenesis of human autoimmune Goodpasture disease.     

A role for collagen IV cross-links in conferring immune privilege to the Goodpasture autoantigen: structural basis for the crypticity of B cell epitopes

The detailed structural basis for the cryptic nature (crypticity) of a B cell epitope harbored by an autoantigen is unknown. Because the immune system may be ignorant of the existence of such "cryptic" epitopes, their exposure could be an important feature in autoimmunity. Here we investigated the structural basis for the crypticity of the epitopes of the Goodpasture autoantigen, the alpha3alpha4alpha5 noncollagenous-1 (NC1) hexamer, a globular domain that connects two triple-helical molecules of the alpha3alpha4alpha5 collagen IV network. The NC1 hexamer occurs in two isoforms as follows: the M-isoform composed of monomer subunits in which the epitopes are accessible to autoantibodies, and the D-isoform composed of both monomer and dimer subunits in which the epitopes are cryptic. The D-isoform was characterized with respect to quaternary structure, as revealed by mass spectrometry of dimer subunits, homology modeling, and molecular dynamics simulation. The results revealed that the D-isoform contains two kinds of cross-links as follows: S-hydroxylysyl-methionine and S-lysyl-methionine cross-links, which stabilize the alpha3alpha5-heterodimers and alpha4alpha4-homodimers, respectively. Construction and analysis of a three-dimensional model of the D-isoform of the alpha3alpha4alpha5 NC1 hexamer revealed that crypticity is a consequence of the following: (a) sequestration of key residues between neighboring subunits that are stabilized by domain-swapping interactions, and (b) by cross-linking of subunits at the trimer-trimer interface, which stabilizes the structural integrity of the NC1 hexamer and protects against binding of autoantibodies. The sequestrated epitopes and cross-linked subunits represent a novel structural mechanism for conferring immune privilege at the level of quaternary structure. Perturbation of the quaternary structure may be a key factor in the etiology of Goodpasture disease.     

Immunogenicity and arthritogenicity of recombinant CB10 in B10.RIII mice

Two major T cell determinants are recognized by I-Ar-specific T cells in CII, the immunodominant CII610-618 (GPAGT AGA R) within CB10 and the subdominant CII445-453 (GPAGP AGE R) within CB8. Although the determinants differ by only two residues, CB8 is capable of inducing collagen-induced arthritis (CIA), while CB10 is not. We, therefore, investigated the structural differences between the two determinants that are critical to inducing arthritis. When the CB10 determinant was mutated to that of CB8 using recombinant techniques, the resulting mutant rCB10T614P,A617E product became arthritogenic. Conversely, when the CB8 determinant was mutated to that of CB10, the resulting mutant CB8P449T,E452A was no longer arthritogenic. Comparison of the epitope specificity of the autoantibodies induced by wild-type CB10 and mutant rCB10T614P, A617E revealed no qualitative differences. T cells from mice immunized with either CB10 or mutant rCB10 produced predominantly Th1 cytokines when cultured with the immunizing Ag. In contrast, when cultured with mouse CII, T cells from mice immunized with the nonarthritogenic CB10 produced predominantly Th2 (IL-4 and IL-10) cytokines whereas the arthritogenic mutant rCB10 induced predominantly Th1 (IFN-gamma) cytokines. We conclude that the T cell cytokine response most critical for the induction of CIA is that induced against the corresponding homologous murine T cell determinant and, further, that the structural differences between the T cell determinants in CB8 and -10 are important in breaking self tolerance and inducing autoimmune response.     

T cell studies in a peptide-induced model of systemic lupus erythematosus

We have previously reported that immunization with a peptide mimetope of dsDNA on a branched polylysine backbone (DWEYSVWLSN-MAP) induces a systemic lupus erythematosus-like syndrome in the nonautoimmune BALB/c mouse strain. To understand the mechanism underlying this breakdown in self tolerance, we examined the role of T cells in the response. Our results show that the anti-foreign and anti-self response induced by immunization is T cell dependent and is mediated by I-E(d)-restricted CD4(+) T cells of the Th1 subset. In addition, generation of the critical T cell epitope requires processing by APCs and depends on the presence of both DWEYSVWLSN and the MAP backbone. The breakdown in self tolerance does not occur through cross-reactivity between the T cell epitope of DWEYSVWLSN-MAP and epitopes derived from nuclear Ags. In this induced-model of SLE, therefore, autoreactivity results from the activation of T cells specific for foreign Ag and of cross-reactive anti-foreign, anti-self B cells. Despite the fact that tissue injury is mediated by Ab, the critical initiating T cell response is Th1.     

Characterization of antibodies to chicken riboflavin carrier protein. Immunoneutralizing ability of antibodies to a sequence-specific region of the protein.

The properties of antibodies generated in rabbits against native riboflavin carrier protein (cRCP), riboflavin carrier protein that had been denatured/renatured by SDS treatment (SDS-RCP) or disulphide-bond-reduced then S-carboxymethylated (Carb-RCP) were studied. SDS-RCP could displace native RCP in radioimmunoassay (r.i.a.), whereas Carb-RCP could not. By using antibodies raised in five different rabbits against native cRCP, 125I-labelled Carb-RCP could bind between 0 and 30% of the native antibodies. Antibodies raised against native RCP appear to be largely directed towards specific conformational determinants of RCP. Carb-RCP displaced native RCP in an r.i.a. using antibodies raised against SDS-RCP. SDS denaturation presumably unmasks cryptic epitopes in native RCP. Carb-RCP was a weak immunogen and elicited, presumably, antibodies to sequential epitope/epitopes. When injected into pregnant mice the antibodies caused neutralization of RCP, leading to termination of pregnancy, indicating highly conserved sequential epitopes in chicken and rodent RCP. Antibodies raised against Carb-RCP or native RCP reacted with CNBr fragments of native RCP, further confirming the presence of sequence-specific antibodies elicited by Carb-RCP.     

Mutant mouse lysozyme carrying a minimal T cell epitope of hen egg lysozyme evokes high autoantibody response

Self proteins including foreign T cell epitope induce autoantibodies. We evaluated the relationship between the size of foreign Ag introduced into self protein and the magnitude of autoantibody production. Mouse lysozyme (ML) was used as a model self protein, and we prepared three different ML derivatives carrying T cell epitope of hen egg white lysozyme (HEL) 107-116, i.e, heterodimer of ML and HEL (ML-HEL), chimeric lysozyme that has residue 1-82 of ML and residue 83-130 of HEL in its sequence (chiMH), and mutant ML that has triple mutations rendering the most potent T cell epitope of HEL (sequence 107-116). Immunization of BALB/c mice with these three ML derivatives induced anti-ML autoantibody responses, whereas native ML induced no detectable response. In particular, mutML generated a 10(4) times higher autoantibody titer than did ML-HEL. Anti-HEL107-116 T cell-priming activities were almost similar among the ML derivatives. The heterodimerization of mutant ML and HEL led to significant reduction of the autoantibody response, whereas the mixture did not. These results show that size of the nonself region in modified self Ag has an important role in determining the magnitude of the autoantibody response, and that decrease in the foreign region in a modified self protein may cause high-titered autoantibody response.     

Regulation of the effector stages of experimental autoimmune encephalomyelitis via neuroantigen-specific tolerance induction. II. Fine specificity of effector T cell inhibition.

Ag-specific tolerance induced by the i.v. administration of splenocytes coupled with mouse spinal cord homogenate, containing a mixture of myelin Ag, dramatically inhibits development and expression of clinical and histologic signs of both active and adoptive forms of relapsing experimental autoimmune encephalomyelitis (R-EAE) in the SJL/J host. Here we examined the dose-dependency, route of tolerogen administration, and fine neuroantigen specificity of inhibition of adoptive R-EAE. Expression of clinical R-EAE induced by a polyclonal population of bovine myelin basic protein (MBP)-specific effector T cells was dramatically inhibited in a dose-dependent manner following the i.v., but not s.c. or i.p., injection of MBP-coupled splenocytes. The exquisite Ag specificity of the inhibition was evident by the observation that splenocytes coupled with intact bovine MBP or species variants of MBP homologous with bovine MBP within the major encephalitogenic region (amino acids 84-104), but not with proteolipid protein or mouse kidney homogenate, were able to suppress disease expression. Splenocytes coupled with the MBP84-104 peptide, containing a nested set of the major SJL/J encephalitogenic epitopes, completely inhibited peptide-specific T cell responses, but only partially inhibited the expression of disease transferred by T cells specific for intact MBP, suggesting the participation of T cell responses specific for additional MBP determinants in disease pathogenesis. However, splenocytes coupled with previously identified minor SJL/J encephalitogenic epitopes (MBP91-104 or MBP17-27), or with the Lewis rat major encephalitogenic epitope (MBP68-86), did not suppress disease expression. Collectively, the results demonstrate that MBP84-104-specific T cells and T cells specific for an as yet unidentified MBP epitope(s) contribute to the pathology of R-EAE. In addition, the results demonstrate that peptide-specific tolerance induction appears to have potential for the treatment of T cell-mediated inflammatory diseases.     

Subdominant/cryptic CD8 T cell epitopes contribute to resistance against experimental infection with a human protozoan parasite

During adaptive immune response, pathogen-specific CD8(+) T cells recognize preferentially a small number of epitopes, a phenomenon known as immunodominance. Its biological implications during natural or vaccine-induced immune responses are still unclear. Earlier, we have shown that during experimental infection, the human intracellular pathogen Trypanosoma cruzi restricts the repertoire of CD8(+) T cells generating strong immunodominance. We hypothesized that this phenomenon could be a mechanism used by the parasite to reduce the breath and magnitude of the immune response, favoring parasitism, and thus that artificially broadening the T cell repertoire could favor the host. Here, we confirmed our previous observation by showing that CD8(+) T cells of H-2(a) infected mice recognized a single epitope of an immunodominant antigen of the trans-sialidase super-family. In sharp contrast, CD8(+) T cells from mice immunized with recombinant genetic vaccines (plasmid DNA and adenovirus) expressing this same T. cruzi antigen recognized, in addition to the immunodominant epitope, two other subdominant epitopes. This unexpected observation allowed us to test the protective role of the immune response to subdominant epitopes. This was accomplished by genetic vaccination of mice with mutated genes that did not express a functional immunodominant epitope. We found that these mice developed immune responses directed solely to the subdominant/cryptic CD8 T cell epitopes and a significant degree of protective immunity against infection mediated by CD8(+) T cells. We concluded that artificially broadening the T cell repertoire contributes to host resistance against infection, a finding that has implications for the host-parasite relationship and vaccine development.     

Spontaneous and prostatic steroid binding protein peptide-induced autoimmune prostatitis in the nonobese diabetic mouse

Chronic nonbacterial prostatitis is a poorly defined syndrome of putative autoimmune origin. To further understand its pathogenesis, we have analyzed autoimmune prostatitis in the NOD mouse, a strain genetically prone to develop different organ-specific autoimmune diseases. Spontaneous development of autoimmune prostatitis in the NOD male, defined by lymphomonuclear cell infiltration in the prostate gland, is well-established by approximately 20 wk of age and is stably maintained afterward. Disease development is indistinguishable in NOD and NOR mice, but is markedly delayed in IFN-gamma-deficient NOD mice. A T cell response to the prostate-specific autoantigen prostatic steroid-binding protein (PSBP) can be detected in NOD males before development of prostate infiltration, indicating lack of tolerance to this self Ag. The intraprostatic inflammatory infiltrate is characterized by Th1-type CD4(+) T cells, which are able to transfer autoimmune prostatitis into NOD.SCID recipients. We characterize here experimental autoimmune prostatitis, detected by intraprostatic infiltrate and PSBP-specific T cell responses, induced in 6- to 8-wk-old NOD males by immunization with synthetic peptides corresponding to the C1 subunit of PSBP. Three PSBP peptides induce in NOD mice vigorous T and B cell responses, paralleled by a marked lymphomononuclear cell infiltration in the prostate. Two of these peptides, PSBP(21-40) and PSBP(61-80), correspond to immunodominant self epitopes naturally processed in NOD mice after immunization with PSBP, whereas peptide PSBP(91-111) represents a cryptic epitope. These model systems address pathogenetic mechanisms in autoimmune prostatitis and will facilitate testing and mechanistic analysis of therapeutic approaches in this condition.     

Cytolytic CD8+ T cells directed against a cryptic epitope derived from a retroviral alternative reading frame confer disease protection

Cytolytic CD8(+) T cells (CTL) are key to the immune response that controls virus infections and mediates disease protection. The ability of CTL to induce apoptosis of infected cells and/or limit viral replication is determined by recognition of processed viral peptide epitopes on the surface of the target cell. An understudied source of MHC class I-presented peptides is the aptly named "cryptic epitopes," defined by their nontraditional methods of generation, including derivation from alternative reading frames (ARFs). Although ARF-encoded epitopes have now been documented in a few systems, their potential functional relevance in vivo has been debated. In this study, we demonstrate the physiological significance of an ARF-derived CTL epitope in a retrovirus-induced disease model. We show that disease-susceptible CD8-deficient mice reconstituted with CTL specific for the retroviral ARF-derived SYNTGRFPPL epitope controlled an infection by the LP-BM5 retrovirus isolate, evidently at the level of viral clearance, resulting in protection of these mice from disease. These data indicate that ARF-derived epitopes are indeed relevant inducers of the immune system and demonstrate the importance of atypically generated peptides as functional Ag with a physiologic role in disease protection.