Epitope mapping of the indirect T cell response to allogeneic class I MHC: sequences shared by donor and recipient MHC may prime T cells that provide help for alloantibody production

Indirect allorecognition occurs when T cells recognize donor MHC presented as peptide epitopes by recipient APC, but the precise nature of the epitopes involved remains unclear. Rejection of rat MHC class I-disparate PVG.R8 (RT1.A(a)) grafts by PVG.RT1(u) (RT1.A(u)) recipients is mediated by indirectly restricted CD4 T cells that provide help for the generation of alloantibody. In this study, epitope mapping was performed using a functionally relevant readout (alloantibody production) to identify key peptides that prime an indirect alloimmune response, leading to graft rejection. PVG.RT1(u) rats were immunized with a series of overlapping 15-mer peptides (peptides 1-18) that spanned the alpha1 and alpha2 domains of the RT1.A(a) molecule. Several peptides were able to accelerate both the alloantibody response to the intact RT1.A(a) Ag and PVG.R8 heart graft rejection. An immunodominant epitope was identified within the hypervariable region of the alpha1 domain. Fine mapping of this region with a second series of peptides overlapping by single amino acids confirmed the presence of an eight-amino acid core determinant. Additional "subdominant" epitopes were identified, two of which were located within regions of amino acid homology between the RT1.A(a) and RT1.A(u) molecules and not, as had been expected, within other hypervariable regions. The contribution of self-epitopes to indirect allorecognition was emphasized by the demonstration that i.v. administration of a 15-mer peptide encompassing one of the subdominant self-determinants diminished the recipient's ability to mount an alloantibody response on challenge with intact A(a) alloantigen. Our findings suggest that cryptic self-epitopes recognized by autoreactive T cells may contribute to allograft rejection and should be considered when designing novel strategies for inducing tolerance to alloantigen.     

Tracking of proinflammatory collagen-specific T cells in early and late collagen-induced arthritis in humanized mice

Rheumatoid arthritis is a chronic inflammatory disease associated with certain HLA-DR4 subtypes. The target autoantigen(s) is unknown, but type II collagen (CII) is a candidate, with a single immunodominant DR4-restricted 261-273 T cell epitope (CII(261-273)). In the present study, we have prepared HLA-DR4:CII(261-273) tetramers and analyzed peripheral blood, lymph node, and synovial fluid cells from DR4-transgenic mice with early and late collagen-induced arthritis to draw a fuller picture of the role of CII-reactive Th cells in disease development. Their frequencies increased approximately 20-fold in blood 1-2 wk postimmunization, and even more in acutely arthritic joints. Our data strongly suggest that CII-specific Th cells are necessary, but not sufficient for collagen-induced arthritis. The CII-specific Th cells displayed an activated proinflammatory Th1 phenotype, and their expansion correlated with onset and severity of arthritis and also with anti-CII Ab levels. Surprisingly, shortly after the first clinical signs of arthritis, activated HLA-DR4:CII tetramer(+) cells became undetectable in the synovial fluid and rare in the blood, but persisted in lymph nodes. Consequently, future human studies should focus on patients with early arthritis, and on their synovial cells, to re-evaluate the occurrence and pathogenic importance of CII-specific or other Th cells in rheumatoid arthritis.     

Inefficient cross-presentation limits the CD8+ T cell response to a subdominant tumor antigen epitope

CD8(+) T lymphocytes (T(CD8)) responding to subdominant epitopes provide alternate targets for the immunotherapy of cancer, particularly when self-tolerance limits the response to immunodominant epitopes. However, the mechanisms that promote T(CD8) subdominance to tumor Ags remain obscure. We investigated the basis for the lack of priming against a subdominant tumor epitope following immunization of C57BL/6 (B6) mice with SV40 large tumor Ag (T Ag)-transformed cells. Immunization of B6 mice with wild-type T Ag-transformed cells primes T(CD8) specific for three immunodominant T Ag epitopes (epitopes I, II/III, and IV) but fails to induce T(CD8) specific for the subdominant T Ag epitope V. Using adoptively transferred T(CD8) from epitope V-specific TCR transgenic mice and immunization with T Ag-transformed cells, we demonstrate that the subdominant epitope V is weakly cross-presented relative to immunodominant epitopes derived from the same protein Ag. Priming of naive epitope V-specific TCR transgenic T(CD8) in B6 mice required cross-presentation by host APC. However, robust expansion of these T(CD8) required additional direct presentation of the subdominant epitope by T Ag-transformed cells and was only significant following immunization with T Ag-expressing cells lacking the immunodominant epitopes. These results indicate that limited cross-presentation coupled with competition by immunodominant epitope-specific T(CD8) contributes to the subdominant nature of a tumor-specific epitope. This finding has implications for vaccination strategies targeting T(CD8) responses to cancer.     

Role of APC in the selection of immunodominant T cell epitopes

Following antigenic challenge, MHC-restricted T cell responses are directed against a few dominant antigenic epitopes. Here, evidence is provided demonstrating the importance of APC in modulating the hierarchy of MHC class II-restricted T cell responses. Biochemical analysis of class II:peptide complexes in B cells revealed the presentation of a hierarchy of peptides derived from the Ig self Ag. Functional studies of kappa peptide:class II complexes from these cells indicated that nearly 20-fold more of an immunodominant epitope derived from kappa L chains was bound to class II DR4 compared with a subdominant epitope from this same Ag. In vivo, T cell responses were preferentially directed against the dominant kappa epitope as shown using Ig-primed DR4 transgenic mice. The bias in kappa epitope presentation was not linked to differences in class II:kappa peptide-binding affinity or epitope editing by HLA-DM. Rather, changes in native Ag structure were found to disrupt presentation of the immunodominant but not the subdominant kappa epitope; Ag refolding restored kappa epitope presentation. Thus, Ag tertiary conformation along with processing reactions within APC contribute to the selective presentation of a hierarchy of epitopes by MHC class II molecules.     

T cell epitopes of human myelin oligodendrocyte glycoprotein identified in HLA-DR4 (DRB1*0401) transgenic mice are encephalitogenic and are presented by human B cells.

Myelin oligodendrocyte glycoprotein (MOG) is an Ag present in the myelin sheath of the CNS thought to be targeted by the autoimmune T cell response in multiple sclerosis (MS). In this study, we have for the first time characterized the T cell epitopes of human MOG restricted by HLA-DR4 (DRB1*0401), an MHC class II allele associated with MS in a subpopulation of patients. Using MHC binding algorithms, we have predicted MOG peptide binding to HLA-DR4 (DRB1*0401) and subsequently defined the in vivo T cell reactivity to overlapping MOG peptides by testing HLA-DR4 (DRB1*0401) transgenic mice immunized with recombinant human (rh)MOG. The data indicated that MOG peptide 97-108 (core 99-107, FFRDHSYQE) was the immunodominant HLA-DR4-restricted T cell epitope in vivo. This peptide has a high in vitro binding affinity for HLA-DR4 (DRB1*0401) and upon immunization induced severe experimental autoimmune encephalomyelitis in the HLA-DR4 transgenic mice. Interestingly, the same peptide was presented by human B cells expressing HLA-DR4 (DRB1*0401), suggesting a role for the identified MOG epitopes in the pathogenesis of human MS.     

T cell epitope characterization in tandemly repetitive Trypanosoma cruzi B13 protein

Proteins containing tandemly repetitive sequences are present in several immunodominant protein antigens in pathogenic protozoan parasites. The tandemly repetitive Trypanosoma cruzi B13 protein is recognized by IgG antibodies from 98% of Chagas' disease patients. Little is known about the molecular mechanisms that lead to the immunodominance of the repeated sequences, and there is limited information on T cell epitopes in such repetitive antigens. We finely characterized the T cell recognition of the tandemly repetitive, degenerate B13 protein by T cell lines, clones and PBMC from Chagas' disease cardiomyopathy (CCC), asymptomatic T. cruzi infected (ASY) and non-infected individuals (N). PBMC proliferative responses to recombinant B13 protein were restricted to individuals bearing HLA-DQA1*0501(DQ7), -DR1, and -DR2; B13 peptides bound to the same HLA molecules in binding assays. The HLA-DQ7-restricted minimal T cell epitope [FGQAAAG(D/E)KP] was identified with an overlapping combinatorial peptide library including all B13 sequence variants in T. cruzi Y strain B13 protein; the underlined small residues GQA were the major HLA contact residues. Among natural B13 15-mer variant peptides, molecular modeling showed that several variant positions were solvent (TCR)-exposed, and substitutions at exposed positions abolished recognition. While natural B13 variant peptide S15.9 seems to be the immunodominant epitope for Chagas' disease patients, S15.4 was preferentially recognized by CCC rather than ASY patients, which may be pathogenically relevant. This is the first thorough characterization of T cell epitopes of a tandemly repetitive protozoan antigen and may suggest a role for T cell help in the immunodominance of protozoan repetitive antigens.     

Characterization of a tolerogenic T cell epitope of type II collagen and its relevance to collagen-induced arthritis.

A synthetic peptide representing sequences of type II collagen, (CII 245-270), has previously been used to induce tolerance and suppress arthritis in DBA/1 mice. To determine important residues, a series of peptides, each containing one or two site-directed substitutions, was generated. Mononuclear cells from DBA/1 mice immunized with CII were cultured in the presence of each peptide and the T cell response determined by measuring IFN-gamma in culture supernatant fluids. Substitutions within the region CII 260-270 led to significant decreases in IFN-gamma responses, identifying this sequence as a T cell epitope. To determine the effects of substitutions within this epitope on arthritis, substituted peptides were administered to neonatal mice as tolerogens. Five site-directed substitutions, four of which included the insertion of a residue found in type I collagen to replace its type II counterpart, abrogated the ability of the peptides to induce tolerance and suppress arthritis. These substitutions were located at residues 260, 261, 263, 264, and 266. Two patterns of T cell reactivity were observed. Peptides containing individual substitutions at positions 261, 264, or 266 were capable of generating a significant T lymphokine response, although those containing substitutions at residues 260 or 263 were ineffective Ag. Systematic analysis of the fine structures of T cell determinants important for autoimmune arthritis can lead to strategies for therapeutic intervention.     

T cells that are naturally tolerant to cartilage-derived type II collagen are involved in the development of collagen-induced arthritis

The immunodominant T-cell epitope that is involved in collagen-induced arthritis (CIA) is the glycosylated type II collagen (CII) peptide 256-270. In CII transgenic mice, which express the immunodominant CII 256-270 epitope in cartilage, the CII-specific T cells are characterized by a partially tolerant state with low proliferative activity in vitro, but with maintained effector functions, such as IFN-γ secretion and ability to provide B cell help. These mice were still susceptible to CIA. The response was mainly directed to the glycosylated form of the CII 256-270 peptide, rather than to the nonglycosylated peptide. Tolerance induction was rapid; transferred T cells encountered CII within a few days. CII immunization several weeks after thymectomy of the mice did not change their susceptibility to arthritis or the induction of partial T-cell tolerance, excluding a role for recent thymic emigrants. Thus, partially tolerant CII autoreactive T cells are maintained and are crucial for the development of CIA.     

Modulation of DNA vaccine-elicited CD8+ T-lymphocyte epitope immunodominance hierarchies

Generating broad cellular immune responses against a diversity of viral epitopes is a major goal of current vaccine strategies for human immunodeficiency virus type 1 (HIV-1) and other pathogens. Virus-specific CD8(+) T-lymphocyte responses, however, are often highly focused on a very limited number of immunodominant epitopes. For an HIV-1 vaccine, the breadth of CD8(+) T-lymphocyte responses may prove to be critical as a result of the need to cover a wide diversity of viral isolates in the population and to limit viral escape from dominant epitope-specific T lymphocytes. Here we show that epitope modification strategies can alter CD8(+) T-lymphocyte epitope immunodominance hierarchies elicited by a DNA vaccine in mice. Mice immunized with a DNA vaccine expressing simian immunodeficiency virus Gag lacking the dominant D(b)-restricted AL11 epitope generated a marked and durable augmentation of responses specific for the subdominant D(b)-restricted KV9 epitope. Moreover, anatomic separation strategies and heterologous prime-boost regimens generated codominant responses against both epitopes. These data demonstrate that dominant epitopes can dramatically suppress the immunogenicity of subdominant epitopes in the context of gene-based vaccines and that epitope modification strategies can be utilized to enhance responses to subdominant epitopes.     

Capsid-specific cytotoxic T lymphocytes recognize three distinct H-2D(b)-restricted regions of the BeAn strain of Theiler's virus and exhibit different cytokine profiles

The role of virus-specific cytotoxic T lymphocytes (CTL) in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease, a viral model for multiple sclerosis, is not yet clear. To investigate the specificity and function of CTL generated in response to TMEV infection, we generated a panel of overlapping 20-mer peptides encompassing the entire capsid and leader protein region of the BeAn strain of TMEV. Binding of these peptides to H-2K(b) and H-2D(b) class I molecules of resistant mice was assessed using RMA-S cells. Several peptides displayed significant binding to H-2K(b), H-2D(b), or both. However, infiltrating cytotoxic T cells in the central nervous system of virus-infected mice preferentially lysed target cells pulsed with VP2(111-130/121-140) or VP2(121-130), a previously defined CTL epitope shared by the DA strain of TMEV and other closely related cardioviruses. In addition, at a high effector-to-target cell ratio, two additional peptides (VP2(161-180) and VP3(101-120)) sensitized target cells for cytolysis by infiltrating T cells or splenic T cells from virus-infected mice. The minimal epitopes within these peptides were defined as VP2(165-173) and VP3(110-120). Based on cytokine profiles, CTL specific for these subdominant epitopes are Tc2, in contrast to CTL for the immunodominant epitope, which are of the Tc1 type. Interestingly, CTL function towards both of these subdominant epitopes is restricted by the H-2D molecule, despite the fact that these epitopes bind both H-2K and H-2D molecules. This skewing toward an H-2D(b)-restricted response may confer resistance to TMEV-induced demyelinating disease, which is known to be associated with the H-2D genetic locus.     

Immunoproteasomes down-regulate presentation of a subdominant T cell epitope from lymphocytic choriomeningitis virus

The cytotoxic T cell response to pathogens is usually directed against a few immunodominant epitopes, while other potential epitopes are either subdominant or not used at all. In C57BL/6 mice, the acute cytotoxic T cell response against lymphocytic choriomeningitis virus is directed against immunodominant epitopes derived from the glycoprotein (gp33-41) and the nucleoprotein (NP396-404), while the gp276-286 epitope remains subdominant. Despite extensive investigations, the reason for this hierarchy between epitopes is not clear. In this study, we show that the treatment of cells with IFN-gamma enhanced the presentation of gp33-41, whereas presentation of the gp276-286 epitope from the same glycoprotein was markedly reduced. Because proteasomes are crucially involved in epitope generation and because IFN-gamma treatment in vitro and lymphocytic choriomeningitis virus infection in vivo lead to a gradual replacement of constitutive proteasomes by immunoproteasomes, we investigated the role of proteasome composition on epitope hierarchy. Overexpression of the active site subunits of immunoproteasomes LMP2, LMP7, and MECL-1 as well as overexpression of LMP2 alone suppressed the presentation of the gp276-286 epitope. The ability to generate gp276-286-specific CTLs was enhanced in LMP2- and LMP7-deficient mice, and macrophages from these mice showed an elevated presentation of this epitope. In vitro digests demonstrated that fragmentation by immunoproteasomes, but not constitutive proteasomes led to a preferential destruction of the gp276 epitope. Taken together, we show that LMP2 and LMP7 can at least in part determine subdominance and shape the epitope hierarchy of CTL responses in vivo.     

A quantitative analysis of the variables affecting the repertoire of T cell specificities recognized after vaccinia virus infection

Many components contribute to immunodominance in the response to a complex virus, but their relative importance is unclear. This was addressed using vaccinia virus and HLA-A*0201 as the model system. A comprehensive analysis of 18 viral proteins recognized by CD8(+) T cell responses demonstrated that approximately one-fortieth of all possible 9- to 10-mer peptides were high-affinity HLA-A*0201 binders. Peptide immunization and T cell recognition data generated from 90 peptides indicated that about one-half of the binders were capable of eliciting T cell responses, and that one-seventh of immunogenic peptides are generated by natural processing. Based on these results, we estimate that vaccinia virus encodes approximately 150 dominant and subdominant epitopes restricted in by HLA-A*0201. However, of all these potential epitopes, only 15 are immunodominant and actually recognized in vivo during vaccinia virus infection of HLA-A*0201 transgenic mice. Neither peptide-binding affinity, nor complex stability, nor TCR avidity, nor amount of processed epitope appeared to strictly correlate with immunodominance status. Additional experiments suggested that vaccinia infection impairs the development of responses directed against subdominant epitopes. This suggested that additional factors, including immunoregulatory mechanisms, restrict the repertoire of T cell specificities after vaccinia infection by a factor of at least 10.     

The immunodominant CD8 T cell response to the human cytomegalovirus tegument phosphoprotein pp65(495-503) epitope critically depends on CD4 T cell help in vaccinated HLA-A*0201 transgenic mice

Immunodominance hierarchies operating in immune responses to viral Ags limit the diversity of the elicited CD8 T cell responses. We evaluated in I-A(b+)/A2-HHD-II and HLA-DR1(+)/A2-DR1 mice the HLA-A*0201-restricted, multispecific CD8 T cell responses to the human CMV tegument phosphoprotein pp65 (pp65) Ag. Vaccination of mice with pp65-encoding DNA elicited high IFN-γ(+) CD8 T cell frequencies to the pp65(495-503)/(e6) epitope and low responses to the pp65(320-328)/(e3) and pp65(522-530)/(e8) epitopes. Abrogation of the e6-specific immunity efficiently enhanced e3- and e8-specific T cell responses by a pp65(Δ501-503) DNA vaccine. The immunodominant e6-specific (but not the e3- and e8-specific) CD8 T cell response critically depends on CD4 T cell help. Injection of monospecific DNA- or peptide-based vaccines encoding the e3 or e8 (but not the e6) epitope into mice elicited CD8 T cells. Codelivering the antigenic peptides with different heterologous CD4 T cell helper epitopes enhanced e6-specific (but not e3- or e8-specific) CD8 T cell responses. Similarly, homologous CD4 T cell help, located within an overlapping (nested) pp65(487-503) domain, facilitated induction of e6-specific CD8 T cell responses by peptide-based vaccination. The position of the e6 epitope within this nested domain is not critical to induce the immunodominant, e6-specific CD8 T cell response to the pp65 Ag. Distant CD4 T cell epitope(s) can thus provide efficient help for establishing pp65-e6 immunodominance in vaccinated mice. These results have practical implications for the design of new T cell-stimulating vaccines.     

A reductionist cell-free major histocompatibility complex class II antigen processing system identifies immunodominant epitopes

Immunodominance is defined as restricted responsiveness of T cells to a few selected epitopes from complex antigens. Strategies currently used for elucidating CD4(+) T cell epitopes are inadequate. To understand the mechanism of epitope selection for helper T cells, we established a cell-free antigen processing system composed of defined proteins: human leukocyte antigen-DR1 (HLA-DR1), HLA-DM and cathepsins. Our reductionist system successfully identified the physiologically selected immunodominant epitopes of two model antigens: hemagglutinin-1 (HA1) from influenza virus (A/Texas/1/77) and type II collagen (CII). When applied for identification of new epitopes from a recombinant liver-stage antigen of malaria falciparum (LSA-NRC) or HA1 from H5N1 influenza virus ('avian flu'), the system selected single epitopes from each protein that were confirmed to be immunodominant by their capacity to activate CD4(+) T cells from H5N1-immunized HLA-DR1-transgenic mice and LSA-NRC-vaccinated HLA-DR1-positive human volunteers. Thus, we provide a new tool for the identification of physiologically relevant helper T cell epitopes from antigens.     

Identification of novel subdominant epitopes on the carcinoembryonic antigen recognized by CD4+ T cells of lung cancer patients

The carcinoembryonic Ag (CEA) is an attractive target for immunotherapy because of its expression profile and role in tumor progression. To verify the existence of spontaneous anti-CEA CD4+ T cells in lung cancer patients, we first identified CEA sequences forming naturally processed epitopes, and then used the identified epitopes to test their recognition by CD4+ T cells from the patients. We had previously identified CEA(177-189/355-367) as an immunodominant epitope recognized by CD4+ T cells in association with several HLA-DR alleles. In this study, we identified four additional subdominant CEA sequences (CEA(99-111), CEA(425-437), CEA(568-582), and CEA(666-678)), recognized in association with one or more HLA-DR alleles. Peptide-specific CD4+ T cells produced proinflammatory cytokines when challenged with the native protein and CEA-expressing tumor cells, thus demonstrating that the identified CEA sequences contain naturally processed epitopes. However, CEA is expressed in the thymus and belongs to the CD66 family that comprises highly homologous molecules expressed on hemopoietic cells, raising concerns about tolerance interfering with the in vivo development of anti-CEA immunity. We thus tested the spontaneous reactivity to the identified epitopes of peripheral blood CD4+ T lymphocytes from eight early-stage lung cancer patients bearing CEA-positive tumors. We found GM-CSF- and IFN-gamma-producing CD4+ T cells in two patients. Our data indicate that CD4+ immune responses against CEA develop in neoplastic patients, suggesting that tolerance toward CEA or cross-reactive CD66 homologous molecules might be either not absolute or be overcome in the neoplastic disease.     

In vivo hierarchy of immunodominant and subdominant HLA-A*0201-restricted T-cell epitopes of HBx antigen of hepatitis B virus

A polyepitopic CD8+ T-cell response is critical for the control of hepatitis B virus (HBV) infection. The HBV X protein (HBx) is a multifunctional protein that is important for the viral life cycle and for host-virus interactions. The aim of this study was to analyze the immunogenicity and dominance of various HLA-A*0201-restricted HBx-derived epitopes. For this purpose, we immunized HLA-A*0201-transgenic mice with HBx-derived peptides and DNA. This is a powerful model for studying the induction of HLA-A*0201-restricted immune responses in vivo, as these mice possess a cytotoxic T lymphocyte (CTL) repertoire representative of HLA-A2.1 individuals. We used cytotoxic tests and enzyme-linked immunosorbent spot (ELISPOT) assays to study the induction of specific cytotoxic and interferon (IFN)-gamma-secreting T cells. This allowed us to classify the HBx epitopes according to their T-cell activation capacity. After endogenous processing of the antigen synthesized in vivo after DNA-based immunization, we found that the HBx-specific T-cell response is targeted against one immunodominant epitope. Furthermore, following peptide immunization, we identified six additional novel subdominant T-cell epitopes. Inclusion of well-characterized epitopic sequences of HBx in a new vaccine for chronic HBV infections could help to broaden the T-cell response.     

Different immunodominance of HIV-1-specific CTL epitopes among three subtypes of HLA-A*26 associated with slow progression to AIDS

It is speculated that HLA-A^∗26-restricted HIV-1-specific CTLs can control HIV-1, since HLA-A^∗26 is associated with a slow progression to AIDS. In three major HLA-A^∗26 subtypes, HLA-A^∗2601-restricted, and HLA-A^∗2603-restricted HIV-1 epitopes have been identified, but HLA-A^∗2602-restricted ones have not. We here identified HLA-A^∗2602-restricted HIV-1 epitopes by using reverse immunogenetics and compared the immunodominance of the epitopes among the three subtypes. Out of 110 HIV-1 peptides carrying HLA-A^∗26 anchor residues, only the Gag169-177 peptide, which had been previously identified as an HLA-A^∗2601- and HLA-A^∗2603-restricted immunodominant epitope, induced Gag169-177-specific CD8⁺ T cells from only two of six HLA-A^∗2602⁺ HIV-1-infected individuals. No difference in affinity of this epitope peptide was found among these three HLA-A^∗26 subtypes, indicating that Gag169-177 was effectively presented by HLA-A^∗2602 but recognized as a subdominant epitope in HIV-1-infected HLA-A^∗2602⁺ individuals. These findings indicate different immunodominance of Gag169-177 epitope among 3 HLA-A^∗26 subtypes.     

Immunodominance among EBV-derived epitopes restricted by HLA-B27 does not correlate with epitope abundance in EBV-transformed B-lymphoblastoid cell lines

Using synthetic peptides, the HLA-B27-restricted CTL response to EBV in asymptomatic virus carriers has been mapped to four epitope regions in EBV latent cycle Ags. One of these peptide-defined epitopes (RRIYDLIEL) tends to be immunodominant and is recognized in the context of all three B27 subtypes studied, B*2702, B*2704, and B*2705. The other peptide-defined epitopes induce responses only in the context of one subtype, the immunogenic combinations being RRARSLSAERY/B*2702, RRRWRRLTV/B*2704, and FRKAQIQGL/B*2705. We used immunoaffinity chromatography to isolate the naturally presented viral peptides associated with these MHC class I molecules on the surface of EBV-transformed B-LCL. Using CTL reconstitution assays in conjunction with mass spectrometry, we established that the naturally processed and presented peptides are identical with the previously identified synthetic sequences. Despite the subtype-specific immunogenicity of three of the four epitopes, all four epitope peptides were found in association with each of the three different HLA-B27 subtypes. Indeed, those peptides that failed to induce a response in the context of a particular HLA-B27 subtype were frequently presented at greater abundance by that subtype than were the immunogenic peptides. Furthermore, among the peptides that did induce a response, immunodominance did not correlate with epitope abundance; in fact the immunodominant RRIYDLIEL epitope was least abundant, being present at less than one copy per cell. The relationship of this unexpected finding to the persistence of EBV is discussed.     

A transient post-translationally modified form of cartilage type II collagen is ignored by self-reactive T cells

Lysine residues in type II collagen (CII) are normally hydroxylated and subsequently glycosylated in the chondrocyte. The immunodominant T cell epitope of CII involves such post-translationally modified lysine at position 264 that has been shown to be critical in the pathogenesis of murine collagen-induced arthritis and also in human rheumatoid arthritis. In this study we identified a line of transgenic mice expressing a TCR specific for hydroxylated rat CII epitope. They were crossed with transgenic mice expressing the rat CII epitope, either specifically in cartilage (MMC mice) or systemically (TSC mice), to analyze T cell tolerance to a post-translationally modified form of self-CII. The mechanism of T cell tolerance to the hydroxylated CII epitope in TSC mice was found to involve intrathymic deletion and induction of peripheral tolerance. In contrast, we did not observe T cell tolerance in the MMC mice. Analysis of CII prepared from rat or human joint cartilage revealed that most of the lysine 264 is glycosylated rather than remaining hydroxylated. Therefore, we conclude that the transient post-translationally modified form of cartilage CII does not induce T cell tolerance. This lack of T cell tolerance could increase the risk of developing autoimmune arthritis.     

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.