Cross-Reactivity of Herpesvirus-Specific CD8 T Cell Lines Toward Allogeneic Class I MHC Molecules

Although association between persistent viral infection and allograft rejection is well characterized, few examples of T-cell cross-reactivity between self-MHC/viral and allogeneic HLA molecules have been documented so far. We appraised in this study the alloreactivity of CD8 T cell lines specific for immunodominant epitopes from human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV). CD8 T cell lines were generated after sorting with immunomagnetic beads coated with either pp65495–503/A*0201, BMLF1259–267/A*0201, or BZLF154–64/B*3501 multimeric complexes. Alloreactivity of the CD8 T cell lines against allogeneic class I MHC alleles was assessed by screening of (i) TNF-α production against COS-7 cells transfected with as many as 39 individual HLA class I-encoding cDNA, and (ii) cytotoxicity activity toward a large panel of HLA-typed EBV-transformed B lymphoblastoid cell lines. We identified several cross-reactive pp65/A*0201-specific T cell lines toward allogeneic HLA-A*3001, A*3101, or A*3201. Moreover, we described here cross-recognition of HLA-Cw*0602 by BZLF1/B*3501-specific T cells. It is noteworthy that these alloreactive CD8 T cell lines showed efficient recognition of endothelial cells expressing the relevant HLA class I allele, with high level TNF-α production and cytotoxicity activity. Taken together, our data support the notion that herpes virus-specific T cells recognizing allo-HLA alleles may promote solid organ rejection

Analyse de réponses lymphocytaires T CD8+ dirigées contre le virus de l'hépatite C (corrélation avec le statut clinique)

La majorité des individus infectés par le virus de l'hépatite C (VHC) restent chroniquement infectés et sont prédisposés à une cirrhose ou à un hépatocarcinome. Pour mieux comprendre les mécanismes qui influencent la progression vers l'infection chronique, nous avons fait une analyse fonctionnelle et moléculaire de lymphocytes T (LT) VHC-spécifiques chez des individus séropositifs, ayant ou non résolu leur infection, ainsi que chez des individus séronégatifs. Nous avons isolé par tri immunomagnétique, des LT CD8+ dirigés contre trois épitopes VHC immunodominants restreints par HLA-A*0201. Des LT spécifiques de ces complexes antigéniques ont pu être isolés pour tous les individus. Les patients chroniquement infectés ont montré des réponses T VHC-spécifiques plus faibles que les patients qui avaient résolu leur infection, en termes de production d'IFN-gamma, de cytotoxicité et de tests de dégranulation. Ces différences fonctionnelles seraient liées à une moindre fréquence de LT spécifiques exprimant un TCR de forte affinité chez les patients chroniquement infectés, plutôt qu à des défauts fonctionnels intrinsèques ou à une down-modulation par des récepteurs inhibiteurs. Les individus sains ont montré majoritairement des réponses de faible avidité, analogues à celles observées chez des patients en infection chronique, bien que des populations de forte avidité aient été observées chez certains individus. Nos résultats indiquent qu'un enrichissement en LT de forte avidité est associé à une clairance virale efficace et suggèrent un lien entre la qualité du répertoire lymphocytaire T VHC-spécifique initial et la susceptibilité à l'infection chroniqueUp to 170 million people are infected by hepatitis C virus (HCV), among whom 4/5 remain chronically infected and are predisposed to cirrhosis and/or hepatocarcinoma. To better understand the mechanisms that influence progression to HCV chronic infection versus viral control, we performed an in-depth functional and molecular analysis of highly purified HCV-specific T cells in seropositive donors, having resolved their infection or not, and in HCV-seronegative healthy donors. We managed to isolate from blood samples HCV-specific CD8+ T cells directed against three immunodominant HCV epitopes restricted by HLA-A*0201, using magnetic beads coated with HLA class I/peptide complexes. T cell populations specific to those antigens were isolated from almost all donors, irrespective of their serological status. Chronically infected patients yielded weaker HCV-specific CD8+ responses than those having cleared their infection, as assessed by IFN- production, cytotoxicity and degranulation assays. These functional differences were primarily accounted for by decreased frequency of specific T cells expressing high-affinity TCR in chronically infected patients rather than by intrinsic T cell functional defects or by active downmodulation by inhibitory receptors. Healthy donors yielded mainly low avidity HCV-specific T cell responses, very similar to chronic patients, although high avidity subsets were detected in some individuals. Our results indicate that enrichment for high avidity T cells is associated with successful viral clearance and suggest a link between the quality of the initial HCV-specific T cell repertoire and susceptibility to chronic infectionNANTES-BU Sciences (441092104) / SudocSudocFranceF

Selection of T cell clones expressing high-affinity public TCRs within Human cytomegalovirus-specific CD8 T cell responses.

Assessment of clonal diversity of T cell responses against human CMV (HCMV), a major cause of morbidity in immunodepressed patients, provides important insights into the molecular basis of T cell immunodominance, and has also clinical implications for the immunomonitoring and immunotherapy of HCMV infections. We performed an in-depth molecular and functional characterization of CD8 T cells directed against an immunodominant HLA-A2-restricted epitope derived from HCMV protein pp65 (NLV/A2) in steady state and pathological situations associated with HCMV reactivation. NLV/A2-specific T cells in healthy HCMV-seropositive donors showed limited clonal diversity and usage of a restricted set of TCR Vbeta regions. Although TCRbeta-chain junctional sequences were highly diverse, a large fraction of NLV/A2-specific T cells derived from distinct individuals showed several recurrent (so-called "public") TCR features associated in some cases with full conservation of the TCRalpha chain junctional region. A dramatic clonal focusing of NLV/A2-specific T cells was observed in situations of HCMV reactivation and/or chronic inflammation, which resulted in selection of a single clonotype displaying similar public TCR features in several patients. In most instances the NLV/A2-specific dominant clonotypes showed higher affinity for their Ag than subdominant ones, thus suggesting that TCR affinity/avidity is the primary driving force underlying repertoire focusing along chronic antigenic stimulation

Analysis of the TCR Vb repertoire of pp65_495–502/A*0201- or BZLF1/B*3501-sorted T cell lines, using TCR Vβ-specific mAb.

<p>The percentage of pp65/A*0201- or BZLF1/B*3501-specific T cells stained by the various anti-TCR Vβ mAb is mentioned according to the IMGT nomenclature (Beckman Coulter anti-TCR Vβ name is indicated in bracket). All T cell lines were derived from PBL, either from healthy donors or from RA patients.</p>a<p>Percentage determined by TCR sequencing (ref. 28). T cell lines exhibiting alloreactivity are marked in bold.</p

HLA class I typing of endothelial cells.

<p>ND : Not determined.</p

Screening of CD8 T cell lines enriched in HCMV- or EBV-specific T cells for cross-reactivity to allogeneic MHC molecules.

a<p>CD8 T cell lines were screened on COS-7 cells transfected with individual HLA-encoding cDNA and TNF-α production was measured after a 6h-coculture. All T cell lines were PBL-derived. ND : not determined.</p

Screening of HCMV- or EBV-specific CD8 T cell lines for cross-recognition of allogeneic MHC molecules.

<p>CD8 T cell lines, sorted with recombinant pMHC multimeric complexes specific to HCMV (pp65_495–503/A*0201) or EBV lytic epitopes (BMLF1_259–267/A*0201 or BZLF1_54–64/B*3501), were tested: (A) for cytotoxicity toward a panel of 30 HLA-typed LCL, in a 4-h chromium release assay (Effector-Target ratio 15∶1). HLA-specific killing of LCL was observed for 3 of the 11 pp65/A2-specific T cell lines tested: T cell line from D01 killed A*3101⁺ LCL, T cell line from D03 killed A*3201⁺ LCL, and T cell line from D08 killed A*3001⁺ LCL. The BZLF1/B*3501-sorted T cell line from D15 killed LCL sharing Cw*0602 expression. Only T cell lines that showed alloresponse are displayed. LCL triggering an alloresponse are shown as well as some of the tested LCL which do not elicit a cytotoxic response. Cognate peptide-HLA complexes (pp65_495–503/A*0201 or BZLF1_54–64/B*3501) were used as positive control. Data are presented as the mean percentage lysis and are representative of 3 different experiments. (<b>B</b>) for TNF-α production toward COS-7 cells transfected with plasmids encoding class I HLA alleles. T cells were added 2 days after the transfection, and the TNF-α content of the supernatant, expressed in pg/mL, was estimated 6 h later by testing the toxicity of the supernatants for TNF-α sensitive WEHI-164 clone 13 cells. TNF-α production was observed for the pp65/A*0201-sorted T cell line from D03, that recognized selectively COS cells expressing HLA-A*3201, and for the BZLF1/B*3501 T cell line from D15 that recognized selectively COS cells expressing HLA-Cw*0602. Plasmids encoding class I HLA alleles that elicits a response are shown as well as some of the plasmid encoding class I HLA alleles that do not induce TNF-α secretion. T cell lines that did not produce TNF-α are not represented. Cognate peptide-HLA complexes (pp65_495–503/A*0201 or BZLF1_54–64/B*3501) were used as positive control. One out three independent experiments is shown.</p

Enrichment in pp65_495–503/A*0201- or BZLF1_54–64/B*3501-specific T cells after sorting of CD8 T cells with pMHC magnetic multimers.

<p>(<b>A</b>) CD8 T cells, derived from the PBL from D01 and D03 were sorted with 245V mutated pp65_495–503/A*0201 multimers, then expanded in culture, and stained with PE-conjugated pp65_495–503/A*0201 tetramers and FITC-conjugated anti-CD3. (<b>B</b>) CD8 T cells derived from the PBL from D15 were sorted with 245V mutated BZLF1_54–64/B*3501 multimers, and then expanded in culture. Unsorted and sorted T cells were stained with PE-conjugated BZLF1/B35 tetramers and FITC-conjugated anti-CD3. The percentage of positive cells is indicated in the upper right quadrant.</p