HLA-B7-restricted EBV-specific CD8+ T cells are dysregulated in multiple sclerosis

It was hypothesized that the EBV-specific CD8(+) T cell response may be dysregulated in multiple sclerosis (MS) patients, possibly leading to a suboptimal control of this virus. To examine the CD8(+) T cell response in greater detail, we analyzed the HLA-A2-, HLA-B7-, and HLA-B8-restricted EBV- and CMV-specific CD8(+) T cell responses in a high number of MS patients and control subjects using tetramers. Content in cytolytic granules, as well as cytotoxic activity, of EBV- and CMV-specific CD8(+) T cells was assessed. We found that MS patients had a lower or a higher prevalence of HLA-A2 and HLA-B7, respectively. Using HLA class I tetramers in HLA-B7(+) MS patients, there was a higher prevalence of MS patients with HLA-B*0702/EBV(RPP)-specific CD8(+) T cells ex vivo. However, the magnitude of the HLA-B*0702/EBV(RPP)-specific and HLA-B*0702/CMV(TPR)-specific CD8(+) T cell response (i.e., the percentage of tetramer(+) CD8(+) T cells in a study subject harboring CD8(+) T cells specific for the given epitope) was lower in MS patients. No differences were found using other tetramers. After stimulation with the HLA-B*0702/EBV(RPP) peptide, the production of IL-2, perforin, and granzyme B and the cytotoxicity of HLA-B*0702/EBV(RPP)-specific CD8(+) T cells were decreased. Altogether, our findings suggest that the HLA-B*0702-restricted viral (in particular the EBV one)-specific CD8(+) T cell response is dysregulated in MS patients. This observation is particularly interesting knowing that the HLA-B7 allele is more frequently expressed in MS patients and considering that EBV is associated with MS.     

A similarity in peptide cross-reactivity between alloantigen- and nominal antigen-induced CD8+ T cell responses in vitro

Raising tumor-specific allorestricted T cells in vitro for adoptive transfusion is expected to circumvent host tumor tolerance. However, it has been assumed that alloreactive T cell clones activated in vitro ranges from peptide-specific with high avidity to peptide-degenerate with low avidity. In this study, we examined the peptide specificity and cross-reactivity of T cell responses in vitro to an allogeneic epitope and a nominal epitope with a modified co-culture of lymphocytes and autologous monocytes. After binding to the monocyte via the interaction of its Fc part and the cell surface IgG Fc receptor type I (FcγRI), a fusion protein consisting of the extracellular domains of HLA-A2 molecule and the Fc region of IgG1 (the dimer) introduced a single epitope into the co-culture. The dimer-coated monocytes stimulated the proliferation of autologous CD8⁺ T cells after co-culturing. The CD8⁺ T cell responses were self-HLA-restricted for HLA-A2-positive (HLA-A2+ve) samples and allo-HLA-restricted for HLA-A2-negative (HLA-A2-ve) samples, since the co-cultural bulks stained with HLA-A2 tetramers, human interferon-gamma (IFN-γ) production in response to T cell receptor (TCR) ligands, and cytotoxicity against a panel of target cells exhibited peptide-specific properties. Two HLA-A2-restricted peptides with sequence homology were included, allowing the comparison of cross-reactivity between allo-antigen- and nominal antigen-induced CD8⁺ T cell responses. Interestingly, the allo- and self-HLA-restricted CD8⁺ T cell responses were similar in the peptide cross-reactivity, although the allorestricted T cell response seemed, overall, more intensive and had higher binding affinity to specific tetramer. Our findings indicated the alloreactive T cells raised by the co-culture in vitro were as peptide specific and cross-reactive as the self-HLA-restricted ones.     

Endogenous Processing and Presentation of T-cell Epitopes from Chlamydia trachomatis with Relevance in HLA-B27-associated Reactive Arthritis

Chlamydia trachomatis triggers reactive arthritis, a spondyloarthropathy linked to the human major histocompatibility complex molecule HLA-B27, through an unknown mechanism that might involve molecular mimicry between chlamydial and self-derived HLA-B27 ligands. Chlamydia-specific CD8⁺ T-cells are found in reactive arthritis patients, but the immunogenic epitopes are unknown. A previous screening of the chlamydial genome for putative HLA-B27 ligands predicted multiple peptides that were recognized in vitro by CD8⁺ T-lymphocytes from patients. Here stable transfectants expressing bacterial fusion proteins in human cells were generated to investigate the endogenous processing and presentation by HLA-B27 of two such epitopes through comparative immunoproteomics of HLA-B27-bound peptide repertoires. A predicted T-cell epitope, from the CT610 gene product, was presented by HLA-B27. This is, to our knowledge, the first endogenously processed epitope involved in HLA-B27-restricted responses against C. trachomatis in reactive arthritis. A second predicted epitope, from the CT634 gene product, was not detected. Instead a non-predicted nonamer from the same protein was identified. Both bacterial peptides showed very high homology with human sequences containing the HLA-B27 binding motif. Thus, expression and intracellular processing of chlamydial proteins into human cells allowed us to identify two bacterial HLA-B27 ligands, including the first endogenous T-cell epitope from C. trachomatis involved in spondyloarthropathy. That human proteins contain sequences mimicking chlamydial T-cell epitopes suggests a basis for an autoimmune component of Chlamydia-induced HLA-B27-associated disease.Chlamydia trachomatis is an obligate intracellular parasite that infects the urogenital epithelium. It is a very common pathogen and one frequently inducing reactive arthritis (ReA)¹ (1). Multiple strategies, including down-regulation of major histocompatibility complex (MHC) class I and class II expression (2–4) and persistence, have been developed by the bacteria to evade the immune system. Yet both CD4⁺ and CD8⁺ T-cell responses are activated upon infection (5). In particular, HLA-B27-restricted CD8⁺ T-lymphocytes are found in patients with Chlamydia-induced ReA (6, 7). The role of these cells in the pathogenesis and evolution of ReA to chronic disease is probably mediated by IFN-γ. Secretion of this cytokine is critical for the protective immunity against Chlamydia (8) because it inhibits the bacterial growth (9). However, this is often insufficient to promote complete clearance of C. trachomatis, and actually IFN-γ-induced depletion of the tryptophan pool induces the differentiation of the metabolically active reticular bodies to persistent forms (10), which sustain chronic infection and ReA. The high prevalence of HLA-B27 among patients with Chlamydia-induced ReA (11), especially in its chronic form, suggests a pathogenetic mechanism based on interactive effects of the bacteria and HLA-B27 that seems unrelated to the capacity of C. trachomatis to infect or replicate into HLA-B27-positive cells (12). One such mechanism could be T-cell-mediated autoimmunity elicited by molecular/antigenic mimicry between chlamydial and self-derived HLA-B27 ligands. Antigenic mimicry between chlamydial and homologous α-myosin-derived peptides is crucial to inducing autoimmune myocarditis in mice (13). Breakdown of cytotoxic T-lymphocyte (CTL) tolerance to HLA-B27 was observed in transgenic rats upon exposure to C. trachomatis (14). Cross-reactivity between HLA-B27-restricted Chlamydia-specific CTL with self-derived HLA-B27 epitopes has not been reported. However, a biochemical basis for it was provided by the finding of an endogenously processed and presented peptide from the DNA primase of C. trachomatis with high homology to a self-derived HLA-B27 ligand (15, 16).Because of the likely involvement of HLA-B27 in the pathogenesis of chronically evolving ReA, the role of CD8⁺ T-cell responses in the protective immunity against C. trachomatis and the presence of HLA-B27-restricted T-cells in patients with Chlamydia-induced ReA, the identification of relevant chlamydial epitopes becomes crucial to establish the pathogenetic mechanism of this disease. Unfortunately a direct analysis of chlamydial HLA-B27 ligands expressed on infected cells is exceedingly difficult because of their extremely low amounts, which challenge even the most sensitive techniques of MS. In the case of Chlamydia, the situation is further complicated by the down-regulation of MHC class I expression shortly after infection (3, 4). To our knowledge, only one MHC class I ligand was recently identified, in the mouse system, from Chlamydia muridarum-infected cells using state-of-the-art MS techniques (17). Due in part to this difficulty, alternative approaches, such as expression cloning and synthetic peptide epitope mapping (18, 19) or MHC class I tetramer arrays (20), have been used to identify MHC class I-restricted chlamydial T-cell epitopes in mice. In a previous study (6) predictive algorithms were used to screen the whole genome of C. trachomatis for nonamer peptide sequences containing the HLA-B*2705 binding motif and a high probability of being generated by proteasomal cleavage. This led to identifying multiple sequences that, when used as synthetic peptides in vitro, stimulated CD8⁺ T-cells from patients with Chlamydia-induced ReA. Such cells could also be detected in the synovial fluid of these patients using HLA-B27 tetramers complexed to some of these peptides (7).Although these strategies identify chlamydial sequences that are recognized by CD8⁺ T-cells they do not prove that these peptides are the endogenously processed epitopes that activated the natural T-cell responses to the bacteria in vivo. Because of the intrinsic cross-reactivity of T-cells (21, 22), it is conceivable that synthetic peptides recognized in vitro may be different from the natural epitopes generated by endogenous processing of the chlamydial proteins that elicit the HLA-B27-restricted T-cell responses in ReA patients. To investigate this issue we focused on two predicted epitopes (6). Stable transfectants expressing the corresponding chlamydial proteins fused to green fluorescent protein (GFP) were generated in a B*2705-positive cell line. The endogenous processing and presentation of the predicted epitopes or other peptides from the same bacterial protein were analyzed by comparative immunoproteomics analysis of the B*2705-bound peptide repertoires from transfected and untransfected cells and sequencing of peptides differentially presented on the bacterial protein transfectant.     

Identification of HLA-B27-restricted peptides from the Chlamydia trachomatis proteome with possible relevance to HLA-B27-associated diseases

The association of HLA-B27 with ankylosing spondylitis and reactive arthritis is the strongest one known between an MHC class I Ag and a disease. We have searched the proteome of the bacterium Chlamydia trachomatis for HLA-B27 binding peptides that are stimulatory for CD8(+) cells both in a model of HLA-B27 transgenic mice and in patients. This was done by combining two biomathematical computer programs, the first of which predicts HLA-B27 peptide binding epitopes, and the second the probability of HLA-B27 peptide generation by the proteasome system. After preselection, immunodominant peptides were identified by Ag-specific flow cytometry. Using this approach we have identified for the first time nine peptides derived from different C. trachomatis proteins that are stimulatory for CD8(+) T cells. Eight of these nine murine-derived peptides were recognized by cytotoxic T cells. The same strategy was used to identify B27-restricted chlamydial peptides in three patients with reactive arthritis. Eleven peptides were found to be stimulatory for patient-derived CD8(+) T cells, of which eight overlapped those found in mice. Additionally, we applied the tetramer technology, showing that a B27/chlamydial peptide containing one of the chlamydial peptides stained CD8(+) T cells in patients with Chlamydia-induced arthritis. This comprehensive approach offers the possibility of clarifying the pathogenesis of B27-associated diseases.     

Identification of novel human aggrecan T cell epitopes in HLA-B27 transgenic mice associated with spondyloarthropathy

The pathology of ankylosing spondylitis, reactive arthritis, and other spondyloarthropathies (SpA) is closely associated with the human leukocyte class I Ag HLA-B27. A characteristic finding in SpA is inflammation of cartilage structures of the joint, in particular at the site of ligament/tendon and bone junction (enthesitis). In this study, we investigated the role of CD8+ T cells in response to the cartilage proteoglycan aggrecan as a potential candidate autoantigen in BALB/c-B27 transgenic mice. We identified four new HLA-B27-restricted nonamer peptides, one of them (no. 67) with a particularly strong T cell immunogenicity. Peptide no. 67 immunization was capable of stimulating HLA-B27-restricted, CD8+ T cells in BALB/c-B27 transgenic animals, but not in wild-type BALB/c mice. The peptide was specifically recognized on P815-B27 transfectants by HLA-B27-restricted CTLs, which were also detectable by HLA tetramer staining ex vivo as well as in situ. Most importantly, analysis of the joints from peptide no. 67-immunized mice induced typical histological signs of SpA. Our data indicate that HLA-B27-restricted epitopes derived from human aggrecan are involved in the induction of inflammation (tenosynovitis), underlining the importance of HLA-B27 in the pathogenesis of SpA.     

A long peptide from MELOE-1 contains multiple HLA class II T cell epitopes in addition to the HLA-A*0201 epitope: an attractive candidate for melanoma vaccination

CD4⁺ T cells contribute importantly to the antitumor T cell response, and thus, long peptides comprising CD4 and CD8 epitopes may be efficient cancer vaccines. We have previously identified an overexpressed antigen in melanoma, MELOE-1, presenting a CD8⁺ T cell epitope, MELOE-1_36–44, in the HLA-A*0201 context. A T cell repertoire against this epitope is present in HLA-A*0201+ healthy subjects and melanoma patients and the adjuvant injection of TIL containing MELOE-1 specific CD8⁺ T cells to melanoma patients was shown to be beneficial. In this study, we looked for CD4⁺ T cell epitopes in the vicinity of the HLA-A*0201 epitope. Stimulation of PBMC from healthy subjects with MELOE-1_26–46 revealed CD4 responses in multiple HLA contexts and by cloning responsive CD4⁺ T cells, we identified one HLA-DRβ1*1101-restricted and one HLA-DQβ1*0603-restricted epitope. We showed that the two epitopes could be efficiently presented to CD4⁺ T cells by MELOE-1-loaded dendritic cells but not by MELOE-1+ melanoma cell-lines. Finally, we showed that the long peptide MELOE-1_22–46, containing the two optimal class II epitopes and the HLA-A*0201 epitope, was efficiently processed by DC to stimulate CD4⁺ and CD8⁺ T cell responses in vitro, making it a potential candidate for melanoma vaccination.     

Identification of Special AT-Rich Sequence Binding Protein 1 as a Novel Tumor Antigen Recognized by CD8+ T Cells: Implication for Cancer Immunotherapy

Background

A large number of human tumor-associated antigens that are recognized by CD8⁺ T cells in a human leukocyte antigen class I (HLA-I)-restricted fashion have been identified. Special AT-rich sequence binding protein 1 (SATB1) is highly expressed in many types of human cancers as part of their neoplastic phenotype, and up-regulation of SATB1 expression is essential for tumor survival and metastasis, thus this protein may serve as a rational target for cancer vaccines.

Methodology/Principal Findings

Twelve SATB1-derived peptides were predicted by an immuno-informatics approach based on the HLA-A*02 binding motif. These peptides were examined for their ability to induce peptide-specific T cell responses in peripheral blood mononuclear cells (PBMCs) obtained from HLA-A*02⁺ healthy donors and/or HLA-A*02⁺ cancer patients. The recognition of HLA-A*02⁺ SATB1-expressing cancer cells was also tested. Among the twelve SATB1-derived peptides, SATB1_565–574 frequently induced peptide-specific T cell responses in PBMCs from both healthy donors and cancer patients. Importantly, SATB1_565–574-specific T cells recognized and killed HLA-A*02⁺ SATB1⁺ cancer cells in an HLA-I-restricted manner.

Conclusions/Significance

We have identified a novel HLA-A*02-restricted SATB1-derived peptide epitope recognized by CD8⁺ T cells, which, in turn, recognizes and kills HLA-A*02⁺ SATB1⁺ tumor cells. The SATB1-derived epitope identified may be used as a diagnostic marker as well as an immune target for development of cancer vaccines.     

Low TCR avidity and lack of tumor cell recognition in CD8<Superscript>+</Superscript> T cells primed with the CEA-analogue CAP1-6D peptide

The use of “altered peptide ligands” (APL), epitopes designed for exerting increased immunogenicity as compared with native determinants, represents nowadays one of the most utilized strategies for overcoming immune tolerance to self-antigens and boosting anti-tumor T cell-mediated immune responses. However, the actual ability of APL-primed T cells to cross-recognize natural epitopes expressed by tumor cells remains a crucial concern. In the present study, we show that CAP1-6D, a superagonist analogue of a carcinoembriyonic antigen (CEA)-derived HLA-A*0201-restricted epitope widely used in clinical setting, reproducibly promotes the generation of low-affinity CD8⁺ T cells lacking the ability to recognized CEA-expressing colorectal carcinoma (CRC) cells. Short-term T cell cultures, obtained by priming peripheral blood mononuclear cells from HLA-A*0201⁺ healthy donors or CRC patients with CAP1-6D, were indeed found to heterogeneously cross-react with saturating concentrations of the native peptide CAP1, but to fail constantly lysing or recognizing through IFN- γ release CEA⁺CRC cells. Characterization of anti-CAP1-6D T cell avidity, gained through peptide titration, CD8-dependency assay, and staining with mutated tetramers (D227K/T228A), revealed that anti-CAP1-6D T cells exerted a differential interaction with the two CEA epitopes, i.e., displaying high affinity/CD8-independency toward the APL and low affinity/CD8-dependency toward the native CAP1 peptide. Our data demonstrate that the efficient detection of self-antigen expressed by tumors could be a feature of high avidity CD8-independent T cells, and underline the need for extensive analysis of tumor cross-recognition prior to any clinical usage of APL as anti-cancer vaccines.     

HLA-A*11:01-restricted CD8+ T cell immunity against influenza A and influenza B viruses in Indigenous and non-Indigenous people

HLA-A*11:01 is one of the most prevalent human leukocyte antigens (HLAs), especially in East Asian and Oceanian populations. It is also highly expressed in Indigenous people who are at high risk of severe influenza disease. As CD8⁺ T cells can provide broadly cross-reactive immunity to distinct influenza strains and subtypes, including influenza A, B and C viruses, understanding CD8⁺ T cell immunity to influenza viruses across prominent HLA types is needed to rationally design a universal influenza vaccine and generate protective immunity especially for high-risk populations. As only a handful of HLA-A*11:01-restricted CD8⁺ T cell epitopes have been described for influenza A viruses (IAVs) and epitopes for influenza B viruses (IBVs) were still unknown, we embarked on an epitope discovery study to define a CD8⁺ T cell landscape for HLA-A*11:01-expressing Indigenous and non-Indigenous Australian people. Using mass-spectrometry, we identified IAV- and IBV-derived peptides presented by HLA-A*11:01 during infection. 79 IAV and 57 IBV peptides were subsequently screened for immunogenicity in vitro with peripheral blood mononuclear cells from HLA-A*11:01-expressing Indigenous and non-Indigenous Australian donors. CD8⁺ T cell immunogenicity screening revealed two immunogenic IAV epitopes (A11/PB2_320-331 and A11/PB2_323-331) and the first HLA-A*11:01-restricted IBV epitopes (A11/M_41-49, A11/NS1_186-195 and A11/NP_511-520). The immunogenic IAV- and IBV-derived peptides were >90% conserved among their respective influenza viruses. Identification of novel immunogenic HLA-A*11:01-restricted CD8⁺ T cell epitopes has implications for understanding how CD8⁺ T cell immunity is generated towards IAVs and IBVs. These findings can inform the development of rationally designed, broadly cross-reactive influenza vaccines to ensure protection from severe influenza disease in HLA-A*11:01-expressing individuals.     

Effect of secondary anchor amino acid substitutions on the immunogenic properties of an HLA-A*0201-restricted T cell epitope derived from the Trypanosoma cruzi KMP-11 protein

The TcTLE peptide (TLEEFSAKL) is a CD8⁺ T cell HLA-A*0201-restricted epitope derived from the Trypanosoma cruzi KMP-11 protein that is efficiently processed, presented and recognized by CD8⁺ T cells from chagasic patients. Since the immunogenic properties of wild-type epitopes may be enhanced by suitable substitutions in secondary anchor residues, we have studied the effect of introducing specific mutations at position 3, 6 and 7 of the TcTLE peptide. Mutations (E3L, S6V and A7F) were chosen on the basis of in silico predictions and in vitro assays were performed to determine the TcTLE-modified peptide binding capacity to the HLA-A*0201 molecule. In addition, the functional activity of peptide-specific CD8⁺ T cells in HLA-A2⁺ chagasic patients was also interrogated. In contrast to bioinformatics predictions, the TcTLE-modified peptide was found to have lower binding affinity and stability than the original peptide. Nevertheless, CD8⁺ T cells from chronic chagasic patients recognized the TcTLE-modified peptide producing TNF-α and INF-γ and expressing CD107a/b, though in less extension than the response triggered by the original peptide. Overall, although the amino acids at positions 3, 6 and 7 of TcTLE are critical for the peptide affinity, they have a limited effect on the immunogenic properties of the TcTLE epitope.     

Soluble HLA/peptide monomers cross-linked with co-stimulatory antibodies onto a streptavidin core molecule efficiently stimulate antigen-specific T cell responses

Soluble MHC–peptide complexes, commonly referred to as tetramers, have been shown to induce strong cross-linking of TCR and CD8, resulting in a vigorous activation followed by a rapid non-apoptotic CD8⁺ T cell death. This has limited tetramer use for antigen-specific T cells isolation and cloning, as sorted tetramer positive cells were shown to possess compromised functional integrity. Here we show that the cross-linking of a secondary co-stimulatory signal into oligomeric MHC:peptide complexes prevents such cell death, and in contrast strongly stimulates antigen-specific T cell responses. Such soluble antigen-presenting complexes (sAPCs) containing MHC:peptide complexes linked to either anti-CD27 or anti-CD28 antibodies were capable of priming and expanding HLA-A*0201 restricted CMV specific T cells and also of generating functional HLA-A*0301 restricted BCR/ABL-specific T cell responses. These sAPCs constitute an encouraging alternative method for generating antigen-specific T cells that could be applied to a variety of antigens. A. I. Dodi and P. J. Travers contributed equally to this work. This paper is an original contribution from the meeting which took place on 28 and 29 May 2008 in Nottingham, UK, celebrating the contribution of Prof. I. A. “Tony” Dodi (^†29.1.2008) to the EU project “Network for the identification and validation of antigens and biomarkers in cancer and their application in clinical tumour immunology (ENACT)”.     

Rapid Antigen Processing and Presentation of a Protective and Immunodominant HLA-B*27-restricted Hepatitis C Virus-specific CD8+ T-cell Epitope

HLA-B*27 exerts protective effects in hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections. While the immunological and virological features of HLA-B*27-mediated protection are not fully understood, there is growing evidence that the presentation of specific immunodominant HLA-B*27-restricted CD8+ T-cell epitopes contributes to this phenomenon in both infections. Indeed, protection can be linked to single immunodominant CD8+ T-cell epitopes and functional constraints on escape mutations within these epitopes. To better define the immunological mechanisms underlying HLA-B*27-mediated protection in HCV infection, we analyzed the functional avidity, functional profile, antiviral efficacy and naïve precursor frequency of CD8+ T cells targeting the immunodominant HLA-B*27-restricted HCV-specific epitope as well as its antigen processing and presentation. For comparison, HLA-A*02-restricted HCV-specific epitopes were analyzed. The HLA-B*27-restricted CD8+ T-cell epitope was not superior to epitopes restricted by HLA-A*02 when considering the functional avidity, functional profile, antiviral efficacy or naïve precursor frequency. However, the peptide region containing the HLA-B*27-restricted epitope was degraded extremely fast by both the constitutive proteasome and the immunoproteasome. This efficient proteasomal processing that could be blocked by proteasome inhibitors was highly dependent on the hydrophobic regions flanking the epitope and led to rapid and abundant presentation of the epitope on the cell surface of antigen presenting cells. Our data suggest that rapid antigen processing may be a key immunological feature of this protective and immunodominant HLA-B*27-restricted HCV-specific epitope.     

The Nucleocapsid Protein of Rift Valley Fever Virus Is a Potent Human CD8+ T Cell Antigen and Elicits Memory Responses

There is no licensed human vaccine currently available for Rift Valley Fever Virus (RVFV), a Category A high priority pathogen and a serious zoonotic threat. While neutralizing antibodies targeting the viral glycoproteins are protective, they appear late in the course of infection, and may not be induced in time to prevent a natural or bioterrorism-induced outbreak. Here we examined the immunogenicity of RVFV nucleocapsid (N) protein as a CD8⁺ T cell antigen with the potential for inducing rapid protection after vaccination. HLA-A*0201 (A2)-restricted epitopic determinants were identified with N-specific CD8⁺ T cells from eight healthy donors that were primed with dendritic cells transduced to express N, and subsequently expanded in vitro by weekly re-stimulations with monocytes pulsed with 59 15mer overlapping peptides (OLPs) across N. Two immunodominant epitopes, VT9 (VLSEWLPVT, N_121–129) and IL9 (ILDAHSLYL, N_165–173), were defined. VT9- and IL9-specific CD8⁺ T cells identified by tetramer staining were cytotoxic and polyfunctional, characteristics deemed important for viral control in vivo. These peptides induced specific CD8⁺ T cell responses in A2-transgenic mice, and more importantly, potent N-specific CD8⁺ T cell reactivities, including VT9- and IL9-specific ones, were mounted by mice after a booster vaccination with the live attenuated RVF MP-12. Our data suggest that the RVFV N protein is a potent human T cell immunogen capable of eliciting broad, immunodominant CD8⁺ T cell responses that are potentially protective. Understanding the immune responses to the nucleocapsid is central to the design of an effective RVFV vaccine irrespective of whether this viral protein is effective as a stand-alone immunogen or only in combination with other RVFV antigens.     

Sharing of an HLA-B27-restricted H-Y antigen between rat and mouse

The purpose of this work was twofold: 1 to learn whether rats transgenic for HLA-B27 and the human ₂-microglobulin gene HB2M can mount B27-restricted cytolytic T lymphocyte (CTL) responses to the male H-Y antigen, and 2 to learn whether such CTLs would recognize both rat and mouse H-Y in the context of HLA-B27. Female rats of the B27/HB2M transgenic line 21-4L were primed in vivo with cells from males of the same line. CTL effectors were generated from lymph node cells of these females following culture with irradiated antigen-presenting cells from either male 21-4L rats or male mice of the B27/HB2M transgenic 56-3 line. The CTLs showed male-specific, B27-specific lysis of both rat and mouse targets. Lysis of B27 targets was inhibitable by monoclonal antibodies specific for B27 or rat CD8. Specific lysis of male B27 rat and mouse targets was inhibitable equally by either rat or mouse male B27 cold targets, but not significantly by female or nontransgenic cold targets. The B27-restricted CTLs neither recognized nor were inhibited by B27⁺ or B27^- male or female human targets. These results demonstrate that CD8⁺, B27-restricted, anti-H-Y CTLs recognize and evolutionarily conserved H-Y peptide antigen in both rats and mice. In addition, they establish the transgenic rat as a model system for examining the T-cell response to antigen presented by class I HLA molecules. Correspondence to: J. D. Taurog.     

Effect of regulatory T cells and adherent cells on the expansion of HBcAg-specific CD8 T cells in patients with chronic hepatitis B virus infection

Patients with chronic HBV infection show poor immune response to HBV-specific CD8⁺ T cells. Several studies demonstrate that regulatory T cells (Treg) and dendritic cells (DC) are important to maintain peripheral immune tolerance. In this study, we investigated the effects of CD4⁺CD25⁺Treg and/or the adherent cells (AC) on the proliferation of HBc18-27-specific CD8⁺ T cells (c18-27-CD8Ts) in response to in vitro stimulation. The frequency of c18-27-CD8Ts in four different mixed leukocyte reactions (MLRs) were analyzed using an HLA-A2-HBc18-27 tetramer. The data indicated that the median percentage of c18-27-CD8Ts in four different MLRs were significant difference in patients with chronic HBV infection. Our results showed that Treg and/or AC might suppress the frequency of HBc18-27-specific CD8⁺ T cell proliferation in response to in vitro stimulation in chronic HBV patients, and AC might be more effective than Treg.     

Quantitation of Anaplasma marginale major surface protein (MSP)1a and MSP2 epitope-specific CD4+ T lymphocytes using bovine DRB3*1101 and DRB3*1201 tetramers

Antigen-specific CD4⁺ T cells play a critical role in protective immunity to many infectious pathogens. Although the antigen-specific CD4⁺ T cells can be measured by functional assays such as proliferation or cytokine enzyme-linked immunospot, such assays are limited to a specific function and cannot quantify anergic or suppressed T cells. In contrast, major histocompatiblity complex (MHC) class II tetramers can enumerate epitope-specific CD4⁺ T cells independent of function. In this paper, we report the construction of bovine leukocyte antigen MHC class II tetramers using a novel mammalian cell system to express soluble class II DRA/DRB3 molecules and defined immunodominant peptide epitopes of Anaplasma marginale major surface proteins (MSPs). Phycoerythrin-labeled tetramers were either loaded with exogenous peptide or constructed with the peptide epitope linked to the N terminus of the DRB3 chain. A DRB3*1101 tetramer loaded with MSP1a peptide F2-5B (ARSVLETLAGHVDALG) and DRB3*1201 tetramers loaded with MSP1a peptide F2-1-1b (GEGYATYLAQAFA) or MSP2 peptide P16-7 (NFAYFGGELGVRFAF) specifically stained antigen-specific CD4⁺ T cell lines and clones. Tetramers constructed with the T-cell epitope linked to the DRB3 chain were slightly better at labeling CD4⁺ T cells. In one cell line, the number of tetramer-positive T cells increased to approximately 94% of the CD4⁺ T cells after culture for 21 weeks with specific antigen. This novel technology should be useful to track the fate of antigen-specific CD4⁺ T-cell responses in cattle after immunization or infection with persistent pathogens, such as A. marginale, that modulate the host immune response.     

Novel HLA-B27-restricted Epitopes from Chlamydia trachomatis Generated upon Endogenous Processing of Bacterial Proteins Suggest a Role of Molecular Mimicry in Reactive Arthritis

Reactive arthritis (ReA) is an HLA-B27-associated spondyloarthropathy that is triggered by diverse bacteria, including Chlamydia trachomatis, a frequent intracellular parasite. HLA-B27-restricted T-cell responses are elicited against this bacterium in ReA patients, but their pathogenetic significance, autoimmune potential, and relevant epitopes are unknown. High resolution and sensitivity mass spectrometry was used to identify HLA-B27 ligands endogenously processed and presented by HLA-B27 from three chlamydial proteins for which T-cell epitopes were predicted. Fusion protein constructs of ClpC, Na⁺-translocating NADH-quinone reductase subunit A, and DNA primase were expressed in HLA-B27⁺ cells, and their HLA-B27-bound peptidomes were searched for endogenous bacterial ligands. A non-predicted peptide, distinct from the predicted T-cell epitope, was identified from ClpC. A peptide recognized by T-cells in vitro, NQRA(330–338), was detected from the reductase subunit. This is the second HLA-B27-restricted T-cell epitope from C. trachomatis with relevance in ReA demonstrated to be processed and presented in live cells. A novel peptide from the DNA primase, DNAP(211–223), was also found. This was a larger variant of a known epitope and was highly homologous to a self-derived natural ligand of HLA-B27. All three bacterial peptides showed high homology with human sequences containing the binding motif of HLA-B27. Molecular dynamics simulations further showed a striking conformational similarity between DNAP(211–223) and its homologous and much more flexible human-derived HLA-B27 ligand. The results suggest that molecular mimicry between HLA-B27-restricted bacterial and self-derived epitopes is frequent and may play a role in ReA.     

Peptide-MHC class I tetrameric complexes display exquisite ligand specificity

The production of synthetic MHC-peptide tetramers has revolutionized cellular immunology by revealing enormous CD8(+) T cell expansions specific for peptides from various pathogens. A feature of these reagents, essential for their staining function, is that they bind T cells with relatively high avidity. This could, theoretically, promote cross-reactivity with irrelevant T cells leading to overestimates of epitope-specific T cell numbers. Therefore, we have investigated the fine specificity of CTL staining with these reagents for comparison with functional data. Using a panel of CTL clones with distinct fine specificity patterns for analogs of an HLA-B8-binding EBV epitope, together with B8 tetramers incorporating these peptides, we show a very good correlation between tetramer staining and peptide activity in cytotoxicity assays. Significant staining only occurred with tetramers that incorporate strong stimulatory agonist peptides and not weak agonists that are unlikely to induce full T cell activation at physiological levels of presentation. In almost every case where a peptide analog had >10-fold less activity than the optimal EBV peptide in cytotoxicity assays, the corresponding tetramer stained with >10-fold less intensity than the EBV epitope tetramer. Furthermore, by examining an EBV-specific clonotypic T cell expansion in EBV-exposed individuals, we show similar fine specificity in tetramer staining of fresh peripheral T cells. Collectively, our data demonstrate the exquisite specificity of class I MHC-peptide tetramers, underlining their accuracy in quantifying only those T cells capable of recognizing the low levels of cell surface peptide presented after endogenous Ag processing.     

Effects of lamivudine on the hepatitis B virus specific CD8+ cytotoxic T lymphocyte responses via peptide-MHC tetrameric complexes assay

Summary Lamivudine, an oral nucleoside analogue, inhibits hepatitis B virus (HBV) replication. It has been shown to be able to restore T cell responsiveness and to induce a type 1 T helper cell (Th 1) immunity in chronic HBV patients. To further examine the effects of lamivudine on cytotoxic T Imphocyte (CTL), responses, two HBV antigenic peptide-HLA-A2 tetrameric complexes containing peptides derived from HBV core protein (residues 18–27; FLPSDFFPSV) and polymerase (residue 551–559; YMDDVVLGA) were constructed. These two tetramers were used to serially determine the frequency of HBV antigen-specific CD8⁺ T cells before and during the treatment of lamivudine. The specificity of these tetramers was confirmed by (a) nonstaining of CD8⁺ T cells from HLA-A2-negative HBV patients, (b) having variable frequency data in the different teteramer measurement, and (c) showing peptide-specific CTL activity in the sorted tetramer-stanining CD8⁺T cells. Low frequency of HBV-specific CTLs was measured for both tetramers before lamivudine treatment. However, the number of CD8⁺ T cells specific for HBV core 18–27 increased significantly during lamivudine treatment. In contrast, relatively lower frequency of HBV pol 551–559 specific CD8⁺T cells was persistently measured after lamivudine treatment. These results indicated that the lamivudine treatment could enhance HBV specific CTL responses.