A novel and efficient approach to high-throughput production of HLA-E/peptide monomer for T-cell epitope screening.

Over the past two decades, there has been a great interest in the study of HLA-E-restricted αß T cells during bacterial and viral infections, including recently SARS-CoV-2 infection. Phenotyping of these specific HLA-E-restricted T cells requires new tools such as tetramers for rapid cell staining or sorting, as well as for the identification of new peptides capable to bind to the HLA-E pocket. To this aim, we have developed an optimal photosensitive peptide to generate stable HLA-E/pUV complexes allowing high-throughput production of new HLA-E/peptide complexes by peptide exchange. We characterized the UV exchange by ELISA and improved the peptide exchange readout using size exclusion chromatography. This novel approach for complex quantification is indeed very important to perform tetramerization of MHC/peptide complexes with the high quality required for detection of specific T cells. Our approach allows the rapid screening of peptides capable of binding to the non-classical human HLA-E allele, paving the way for the development of new therapeutic approaches based on the detection of HLA-E-restricted T cells.

Cross-Reactive Donor-Specific CD8+ Tregs Efficiently Prevent Transplant Rejection.

To reduce the use of non-specific immunosuppressive drugs detrimental to transplant patient health, therapies in development aim to achieve antigen-specific tolerance by promoting antigen-specific regulatory T cells (Tregs). However, identification of the natural antigens recognized by Tregs and the contribution of their dominance in transplantation has been challenging. We identify epitopes derived from distinct major histocompatibility complex (MHC) class II molecules, sharing a 7-amino acid consensus sequence positioned in a central mobile section in complex with MHC class I, recognized by cross-reactive CD8+ Tregs, enriched in the graft. Antigen-specific CD8+ Tregs can be induced in vivo with a 16-amino acid-long peptide to trigger transplant tolerance. Peptides derived from human HLA class II molecules, harboring the rat consensus sequence, also activate and expand human CD8+ Tregs, suggesting its potential in human transplantation. Altogether, this work should facilitate the development of therapies with peptide epitopes for transplantation and improve our understanding of CD8+ Treg recognition.

Molecular mechanisms of lineage decisions in metabolite-specific T cells.

Mucosal-associated invariant T cells (MAIT cells) recognize the microbial metabolite 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) presented by the MHC class Ib molecule, MR1. MAIT cells acquire effector functions during thymic development, but the mechanisms involved are unclear. Here we used single-cell RNA-sequencing to characterize the developmental path of 5-OP-RU-specific thymocytes. In addition to the known MAIT1 and MAIT17 effector subsets selected on bone-marrow-derived hematopoietic cells, we identified 5-OP-RU-specific thymocytes that were selected on thymic epithelial cells and differentiated into CD44- naive T cells. MAIT cell positive selection required signaling through the adapter, SAP, that controlled the expression of the transcription factor, ZBTB16. Pseudotemporal ordering of single cells revealed transcriptional trajectories of 5-OP-RU-specific thymocytes selected on either thymic epithelial cells or hematopoietic cells. The resulting model illustrates T cell lineage decisions.

Histo-blood group antigen-binding specificities of human rotaviruses are associated with gastroenteritis but not with in vitro infection.

Human strains of rotavirus A (RVAs) recognize fucosylated glycans belonging to histo-blood group antigens (HBGAs) through their spike protein VP8*. Lack of these ligands due to genetic polymorphisms is associated with resistance to gastroenteritis caused by P[8] genotype RVAs. With the aim to delineate the contribution of HBGAs in the process, we analyzed the glycan specificity of VP8* proteins from various P genotypes. Binding to saliva of VP8* from P[8] and P[4] genotypes required expression of both FUT2 and FUT3 enzymes, whilst binding of VP8* from the P[14] genotype required FUT2 and A enzymes. We further defined a glycan motif, GlcNAcß3Galß4GlcNAc, recognized by P[6] clinical strains. Conversion into Lewis antigens by the FUT3 enzyme impaired recognition, explaining their lower binding to saliva of Lewis positive phenotype. In addition, the presence of neutralizing antibodies was associated with the presence of the FUT2 wild type allele in sera from young healthy adults. Nonetheless, in vitro infection of transformed cell lines was independent of HBGAs expression, indicating that HBGAs are not human RV receptors. The match between results from saliva-based binding assays and the epidemiological data indicates that the polymorphism of human HBGAs controls susceptibility to RVAs, although the exact mechanism remains unclear.

Carcinoma-associated fucosylated antigens are markers of the epithelial state and can contribute to cell adhesion through CLEC17A (Prolectin).

Terminal fucosylated motifs of glycoproteins and glycolipid chains are often altered in cancer cells. We investigated the link between fucosylation changes and critical steps in cancer progression: epithelial-to-mesenchymal transition (EMT) and lymph node metastasis.Using mammary cell lines, we demonstrate that during EMT, expression of some fucosylated antigens (e.g.: Lewis Y) is decreased as a result of repression of the fucosyltransferase genes FUT1 and FUT3. Moreover, we identify the fucose-binding bacterial lectin BC2L-C-Nt as a specific probe for the epithelial state.Prolectin (CLEC17A), a human lectin found on lymph node B cells, shares ligand specificities with BC2L-C-Nt. It binds preferentially to epithelial rather than to mesenchymal cells, and microfluidic experiments showed that prolectin behaves as a cell adhesion molecule for epithelial cells. Comparison of paired primary tumors/lymph node metastases revealed an increase of prolectin staining in metastasis and high FUT1 and FUT3 mRNA expression was associated with poor prognosis. Our data suggest that tumor cells invading the lymph nodes and expressing fucosylated motifs associated with the epithelial state could use prolectin as a colonization factor.

MHC-derived allopeptide activates TCR-biased CD8+ Tregs and suppresses organ rejection.

In a rat heart allograft model, preventing T cell costimulation with CD40Ig leads to indefinite allograft survival, which is mediated by the induction of CD8+CD45RClo regulatory T cells (CD8+CD40Ig Tregs) interacting with plasmacytoid dendritic cells (pDCs). The role of TCR-MHC-peptide interaction in regulating Treg activity remains a topic of debate. Here, we identified a donor MHC class II-derived peptide (Du51) that is recognized by TCR-biased CD8+CD40Ig Tregs and activating CD8+CD40Ig Tregs in both its phenotype and suppression of antidonor alloreactive T cell responses. We generated a labeled tetramer (MHC-I RT1.Aa/Du51) to localize and quantify Du51-specific T cells within rat cardiac allografts and spleen. RT1.Aa/Du51-specific CD8+CD40Ig Tregs were the most suppressive subset of the total Treg population, were essential for in vivo tolerance induction, and expressed a biased, restricted Vß11-TCR repertoire in the spleen and the graft. Finally, we demonstrated that treatment of transplant recipients with the Du51 peptide resulted in indefinite prolongation of allograft survival. These results show that CD8+CD40Ig Tregs recognize a dominant donor antigen, resulting in TCR repertoire alterations in the graft and periphery. Furthermore, this allopeptide has strong therapeutic activity and highlights the importance of TCR-peptide-MHC interaction for Treg generation and function.

Soluble HLA-I/peptide monomers mediate antigen-specific CD8 T cell activation through passive peptide exchange with cell-bound HLA-I molecules.

Accumulating evidence that serum levels of soluble class I HLA molecules (sHLA-I) can, under various pathological conditions, correlate with disease stage and/or patient survival, has stimulated interest in defining whether sHLA-I can exert immunological functions. However, despite a mounting number of publications suggesting the ability of sHLA-I to affect immune effectors in vitro, the precise underlying mechanism still remains controversial. In this article, we address potential functions of both classical and nonclassical sHLA-I, using soluble recombinant HLA-I/peptide monomers, and clearly demonstrate their ability to trigger Ag-specific activation of CD8 T cells in vitro. Furthermore, we provide strong evidence that this behavior results from the passive transfer of peptides from monomers to T cell-bound HLA-I molecules, allowing for fratricide representation and activation. Hence, we proposed a unifying model of T cell activation by HLA-I/peptide monomers, reappraising the potential involvement of sHLA-I molecules in the immune response.

Impact of TCR reactivity and HLA phenotype on naive CD8 T cell frequency in humans.

The impact of MHC phenotype on the shaping of the peripheral naive T cell repertoire in humans remains unknown. To address this, we compared the frequency and antigenic avidity of naive T cells specific for immunodominant self-, viral, and tumor Ags presented by a human MHC class I allele (HLA-A*02, referred to as A2) in individuals expressing or not this allele. Naive T cell frequencies varied from one Ag specificity to another but were restrained for a given specificity. Although A2-restricted T cells showed similar repertoire features and antigenic avidities in A2+ and A2- donors, A2 expression had either a positive, neutral, or negative impact on the frequency of A2-restricted naive CD8 T cells, depending on their fine specificity. We also identified in all donors CD4 T cells specific for A2/peptide complexes, whose frequencies were not affected by MHC class I expression, but nevertheless correlated with those of their naive CD8 T cell counterparts. Therefore, both selection by self-MHC and inherent TCR reactivity regulate the frequency of human naive T cell precursors. Moreover this study also suggests that T cell repertoire shaping by a given self-MHC allele is dispensable for generation of immunodominant T cell responses restricted by this particular allele.

Structural bases for the affinity-driven selection of a public TCR against a dominant human cytomegalovirus epitope.

Protective T cell responses elicited along chronic human CMV (HCMV) infections are sometimes dominated by CD8 T cell clones bearing highly related or identical public TCR in unrelated individuals. To understand the principles that guide emergence of these public T cell responses, we have performed structural, biophysical, and functional analyses of an immunodominant public TCR (RA14) directed against a major HLA-A*0201-restricted HCMV Ag (pp65(495-503)) and selected in vivo from a diverse repertoire after chronic stimulations. Unlike the two immunodominant public TCRs crystallized so far, which focused on one peptide hotspot, the HCMV-specific RA14 TCR interacts with the full array of available peptide residues. The conservation of some peptide-MHC complex-contacting amino acids by lower-affinity TCRs suggests a shared TCR-peptide-MHC complex docking mode and supports an Ag-driven selection of optimal TCRs. Therefore, the emergence of a public TCR of an oligoclonal Ag-specific response after repeated viral stimulations is based on a receptor displaying a high structural complementarity with the entire peptide and focusing on three peptide hotspots. This highlights key parameters underlying the selection of a protective T cell response against HCMV infection, which remains a major health issue in patients undergoing bone marrow transplantation.

Selection of high-avidity CD8 T cells correlates with control of hepatitis C virus infection.

UNLABELLED: Both strong antigenic avidity and acquisition of proper effector functions contribute to the efficacy of antiviral T cell responses. To correlate these parameters with the outcome of hepatitis C virus (HCV) infection, we characterized HCV-specific CD8 T cell lines isolated after immunomagnetic sorting of peripheral blood mononuclear cells from human leukocyte antigen A*02 (HLA-A*02) individuals with various HCV serological statuses, using recombinant HLA-A*0201 multimers loaded with three immunodominant HCV genotype 1-derived epitopes. CD8 T cells specific for these three epitopes were derived from most HLA-A*0201 individuals, regardless of their HCV serology or clinical outcome. Donors recovered from genotype 1 HCV infection were enriched for high-avidity T cells with enhanced interferon gamma (IFN-gamma), tumor necrosis factor alpha, and cytotoxic T lymphocyte responses, when compared with seronegative donors and seropositive patients infected with irrelevant HCV genotypes. Patients chronically infected with genotype 1 strain yielded almost exclusively low-avidity T cells, whose hyporesponsiveness was primarily attributable to low T cell receptor (TCR) avidity rather than intrinsic functional defects. CONCLUSION: This study suggests that strong IFN-gamma responses associated with efficient viral clearance primarily result from Ag-driven selection/survival of HCV-specific T cells expressing high-avidity TCR. It also suggests a link between the quality of the initial HCV-specific T cell repertoire and susceptibility to chronic infection.

Staphylococcal enterotoxin-like toxins U2 and V, two new staphylococcal superantigens arising from recombination within the enterotoxin gene cluster.

To test the hypothesis that the Staphylococcus aureus enterotoxin gene cluster (egc) can generate new enterotoxin genes by recombination, we analyzed the egc locus in a broad panel of 666 clinical isolates of S. aureus. egc was present in 63% of isolates, confirming its high prevalence. The archetypal organization of the egc locus, consisting of five enterotoxin genes plus two pseudogenes, was found in 409 of 421 egc-positive strains. The egc locus was incomplete in a few strains and occasionally harbored an insertion sequence and transposase genes. These strains may represent evolutionary intermediates of the egc locus. One strain with an atypical egc locus produced two new enterotoxins, designated SElV and SElU2, generated by (i) recombination between selm and sei, producing selv, and (ii) a limited deletion in the varphient1-varphient2 pseudogenes, producing selu2. Recombinant SElV and SElU2 had superantigen activity, as they specifically activated the T-cell families Vbeta 6, Vbeta 18, and Vbeta 21 (SElV) and Vbeta 13.2 and Vbeta 14 (SElU2). Immunoscope analysis showed a Gaussian CDR3 size distribution of T-cell receptor Vbeta chain junctional transcripts of expanded Vbeta subsets in toxin-stimulated cultures, reflecting a high level of polyclonality. These data show that egc is indeed capable of generating new superantigen genes through recombination.

Impact of CD8-MHC class I interaction in detection and sorting efficiencies of antigen-specific T cells using MHC class I/peptide multimers: contribution of pMHC valency.

Recombinant soluble multimeric forms of MHC class I molecules loaded with antigenic peptides (pMHC) have turned out to be particularly useful to detect and isolate specific T cells. These applications both rely on the oligomerization of pMHC monomers in order to compensate for their inherent low affinity for the TCR. In this study, we have evaluated the precise contribution of CD8-pMHC interaction on the specificity and sorting efficiency of pMHC multimers according to their degree of oligomerization. To this end several wild-type versus mutated pMHC complexes (A*0201, B*0701, B*0801, B*3501) carrying point mutations known to reduce (245V mutants) or to abrogate (227,8KA mutants) CD8-pMHC interaction and showing various degrees of valency have been used. We show that irrespective of the HLA allele tested, 245V mutation strongly improves the binding specificity and immunomagnetic sorting efficiency of pMHC multimers. Our results also indicate that the contribution of CD8 to the binding of pMHC multimers to specific CD8+ T cells is inversely correlated to the degree of pMHC oligomerization. Consequently, efficient staining or specific sorting of high-affinity T cells (i.e. CD8 independent) can only be achieved using 227,8KA pMHC complexes with low-order oligomerization.

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.

Production of recombinant human trimeric CD137L (4-1BBL). Cross-linking is essential to its T cell co-stimulation activity.

The interaction between 4-1BB ligand (CD137L), a member of the tumor necrosis factor superfamily, and its receptor 4-1BB provides a co-stimulatory signal for T lymphocyte proliferation and survival. However, the structure of 4-1BBL has not been thoroughly investigated, and none of the human recombinant 4-1BBL molecules available have been described as capable of co-stimulating T cells. The present work provides a model of the three-dimensional structure of the tumor necrosis factor homology domain of 4-1BBL and describes the production of a recombinant human soluble 4-1BBL whose originality lies in that it contains the whole extracellular tail preceding the tumor necrosis factor homology domain and an AviTag peptide (AviTag-4-1BBL) allowing enzymatic biotinylation and multimerization via streptavidin. We provide evidence that this chimeric protein exists as a homotrimer, whereas commercial FLAG-tagged 4-1BBL does not. This resulted in a much higher affinity for 4-1BB (1.2 nM) as compared with FLAG-4-1BBL (55.2 nM). We demonstrate that the single extracellular cysteine residue in the tail (Cys-51) could form a disulfide bond, both in our recombinant protein and in physiologically expressed 4-1BBL. The mutation of this cysteine residue exerted no effect on trimerization but increased the dissociation rate of AviTag-4-1BBL from 4-1BB. In its soluble form, AviTag-4-1BBL did not stimulate purified T cells but dramatically inhibited proliferation of peripheral blood mononuclear cells stimulated with anti-CD3 mAb. In contrast, a very significant co-stimulatory effect was observed on purified T cells once AviTag-4-1BBL was immobilized onto streptavidin beads. In addition, we show that the cross-linking of two trimeric AviTag-4-1BBL molecules was the minimum step required to elicit significant costimulatory activity.

Adoptive transfer of tumor-reactive Melan-A-specific CTL clones in melanoma patients is followed by increased frequencies of additional Melan-A-specific T cells.

In this study, we report the adoptive transfer of highly tumor-reactive Melan-A-specific T cell clones to patients with metastatic melanoma, and the follow-up of these injected cells. These clones were generated from HLA-A*0201 patients by in vitro stimulations of total PBMC with the HLA-A*0201-binding Melan-A peptide analog ELAGIGILTV. Ten stage IV melanoma patients were treated by infusion of these CTL clones with IL-2 and IFN-alpha. The generated T cell clones, of effector/memory phenotype were selected on the basis of their ability to produce IL-2 in response to HLA-A*0201 Melan-A-positive melanoma lines. Infused clones were detected, by quantitative PCR, in the blood of three patients for periods ranging from 7 to 60 days. Six patients showed regression of individual metastases or disease stabilization, and one patient experienced a complete response, but no correlation was found between the detection of the infused clones in PBMC or tumor samples and clinical responses. Nonetheless, frequencies of Melan-A/A2-specific lymphocytes, measured by tetramer labeling, increased after treatment in most patients. In one of these patients, who showed a complete response, this increase corresponded to the expansion of new clonotypes of higher avidity than those detected before treatment. Together, our results suggest that infused CTL clones may have initiated an antitumor response that may have resulted in the expansion of a Melan-A-specific CTL repertoire.

Search for correlation of CD8 T cell response to Epstein-Barr virus with clinical status in rheumatoid arthritis: a 15 month followup pilot study.

OBJECTIVE: To assess in a longitudinal 15 month followup study the CD8 T cell response to immunodominant Epstein-Barr virus (EBV) antigens of 17 patients with rheumatoid arthritis (RA); and to seek an association between these responses and both clinical activity/severity of RA and a qualitative PCR for EBV in peripheral blood. METHODS: At each patient's visit every 3 months: (1) RA activity was assessed for Disease Activity Score (DAS-28); (2) a qualitative PCR for EBV was performed; (3) CD8 T cell response to EBV epitopes was screened in peripheral blood, using an autopresentation assay of 13 EBV peptides previously identified as immunodominant targets in RA synovia. Activation of anti-EBV CD8 T cells was evaluated by measuring the release of tumor necrosis factor-a. RESULTS: The semiquantitative CD8 T cell response to EBV roughly paralleled RA clinical activity in only 4/17 patients. No clear association could be found between positive PCR for EBV (performed at least once in 10/17 patients) and RA activity/severity or fatigue. Reactivity was not qualitatively broader in samples where PCR for EBV proved positive, and most often focused on one or 2 EBV antigens. However, these antigens differed between patients, as did the magnitude of CD8 T cell response to immunodominant antigens at different timepoints for the same patient. CONCLUSION: The CD8 T cell response to EBV paralleled clinical activity in only 4/17 patients. Our pilot study does not support the hypothesis that this CD8 response contributes to RA activity/flares, although the quantitative variations in the pattern of this reactivity over time confirmed that control of EBV manifestations was difficult in most patients with RA.