Expression of CCR6 and CXCR6 by Gut-Derived CD4+/CD8α+ T-Regulatory Cells, Which Are Decreased in Blood Samples From Patients With Inflammatory Bowel Diseases.

BACKGROUND & AIMS: Faecalibacterium prausnitzii, a member of the Clostridium IV group of the Firmicutes phylum that is abundant in the intestinal microbiota, has anti-inflammatory effects. The relative level of F prausnitzii is decreased in fecal samples from patients with inflammatory bowel diseases (IBDs) compared with healthy individuals. Reduced F prausnitzii was correlated with relapse of Crohn's disease after surgery. We identified, in human colonic mucosa and blood, a population of T regulatory type 1-like T regulatory (TREG) cells that express CD4 and CD8α (DP8α T cells) and are specific for F prausnitzii. We aimed to determine whether they are altered in patients with IBD. METHODS: We isolated DP8α T cells from human colon lamina propria and blood samples and used flow cytometry to detect markers of cells that are of colon origin. We quantified DP8α cells that express colon-specific markers in blood samples from 106 patients with IBD, 12 patients with infectious colitis, and 35 healthy donors (controls). We identified cells that respond to F prausnitzii. Cells were stimulated with anti-CD3, and their production of interleukin 10 was measured by enzyme-linked immunosorbent assay. We compared the frequency and reactivity of cells from patients vs controls using the 2-sided Student t test or 1-way analysis of variance. RESULTS: Circulating DP8α T cells that proliferate in response to F prausnitzii express the C-C motif chemokine receptor 6 (CCR6) and C-X-C motif chemokine receptor 6 (CXCR6). These cells also have features of TREG cells, including production of IL-10 and inhibition of T-cell proliferation via CD39 activity. The proportion of circulating CCR6+/CXCR6+ DP8α T cells was significantly reduced (P < .0001) within the total population of CD3+ T cells from patients with IBD compared with patients with infectious colitis or controls. A threshold of <7.875 CCR6+/CXCR6+ DP8α T cells/10,000 CD3+ cells discriminated patients with IBD from those with infectious colitis with 100% specificity and 72.2% sensitivity. CONCLUSIONS: We identified a population of gut-derived TREG cells that are reduced in blood samples from patients with IBD compared with patients with infectious colitis or controls. These cells should be studied further to determine the mechanisms of this reduction and how it might contribute to the pathogenesis of IBD and their prognostic or diagnostic value.

CD4CD8αα lymphocytes, a novel human regulatory T cell subset induced by colonic bacteria and deficient in patients with inflammatory bowel disease.

How the microbiota affects health and disease is a crucial question. In mice, gut Clostridium bacteria are potent inducers of colonic interleukin (IL)-10-producing Foxp3 regulatory T cells (Treg), which play key roles in the prevention of colitis and in systemic immunity. In humans, although gut microbiota dysbiosis is associated with immune disorders, the underlying mechanism remains unknown. In contrast with mice, the contribution of Foxp3 Treg in colitis prevention has been questioned, suggesting that other compensatory regulatory cells or mechanisms may exist. Here we addressed the regulatory role of the CD4CD8 T cells whose presence had been reported in the intestinal mucosa and blood. Using colonic lamina propria lymphocytes (LPL) and peripheral blood lymphocytes (PBL) from healthy individuals, and those with colon cancer and irritable bowel disease (IBD), we demonstrated that CD4CD8αα (DP8α) T lymphocytes expressed most of the regulatory markers and functions of Foxp3 Treg and secreted IL-10. Strikingly, DP8α LPL and PBL exhibited a highly skewed repertoire toward the recognition of Faecalibacterium prausnitzii, a major Clostridium species of the human gut microbiota, which is decreased in patients with IBD. Furthermore, the frequencies of DP8α PBL and colonic LPL were lower in patients with IBD than in healthy donors and in the healthy mucosa of patients with colon cancer, respectively. Moreover, PBL and LPL from most patients with active IBD failed to respond to F. prausnitzii in contrast to PBL and LPL from patients in remission and/or healthy donors. These data (i) uncover a Clostridium-specific IL-10-secreting Treg subset present in the human colonic LP and blood, (ii) identify F. prausnitzii as a major inducer of these Treg, (iii) argue that these cells contribute to the control or prevention of colitis, opening new diagnostic and therapeutic strategies for IBD, and (iv) provide new tools to address the systemic impact of both these Treg and the intestinal microbiota on the human immune homeostasis.

A spliced antigenic peptide comprising a single spliced amino acid is produced in the proteasome by reverse splicing of a longer peptide fragment followed by trimming.

Peptide splicing is a novel mechanism of production of peptides relying on the proteasome and involving the linkage of fragments originally distant in the parental protein. Peptides produced by splicing can be presented on class I molecules of the MHC and recognized by CTLs. In this study, we describe a new antigenic peptide, which is presented by HLA-A3 and comprises two noncontiguous fragments of the melanoma differentiation Ag gp100(PMEL17) spliced together in the reverse order to that in which they appear in the parental protein. Contrary to the previously described spliced peptides, which are produced by the association of fragments of 3-6 aa, the peptide described in this work results from the ultimate association of an 8-aa fragment with a single arginine residue. As described before, peptide splicing takes place in the proteasome by transpeptidation involving an acyl-enzyme intermediate linking one of the peptide fragment to a catalytic subunit of the proteasome. Interestingly, we observe that the peptide causing the nucleophilic attack on the acyl-enzyme intermediate must be at least 3 aa long to give rise to a spliced peptide. The spliced peptide produced from this reaction therefore bears an extended C terminus that needs to be further trimmed to produce the final antigenic peptide. We show that the proteasome is able to perform the final trimming step required to produce the antigenic peptide described in this work.

HLA anchor optimization of the melan-A-HLA-A2 epitope within a long peptide is required for efficient cross-priming of human tumor-reactive T cells.

The uptake and long-term cross-presentation of tumor Ag long peptides (LP) by dendritic cells (DC) make them attractive cancer vaccine candidates. However, it remains to be established whether LP can prime long-lived tumor-reactive CTL and whether other cell types are able to cross-present them. Using HLA-A2 healthy donor and melanoma patient-derived PBMC, we studied the in vitro cross-priming potential of Melan-A 16-40 LP bearing the HLA-A2-restricted epitope 26-35 or its analog 26-35(A27L) and compared it to the priming capacity of the short analog. We then addressed LP priming capacity in vivo using HLA-A2 mice. We also studied LP cross-presentation by monocyte-derived DC, plasmacytoid DC, monocytes, and B cells. We showed that the modified LP gave rise to high and sustained cross-presentation by monocyte-derived DC. This led to cross priming in vitro and in vivo and to expansion of long-lived tumor-reactive cytotoxic T cells. In contrast, the LP containing the natural 26-35 epitope primed specific T cells poorly, despite its long-lived cross-presentation, and T cells primed against the short analog were short-lived. We further showed that LP cross-presentation is restricted to monocytes and conventional DC. These results document for the first time, to our knowledge, the strong immunogenicity of a human tumor Ag LP. Of note, they underscore that this property is critically dependent on sufficient HLA binding affinity and/or TCR ligand potency of the cross-presented epitope. We conclude that LP fulfilling this requirement should be used as tumor vaccines, together with DC maturating agents, especially the Melan-A 16-40(A27L) LP, for the treatment of HLA-A2(+) melanoma patients.

Human gut microbiota-reactive DP8α Tregs prevent acute graft-versus-host disease in a CD73-dependent manner.

Graft-versus-host disease (GvHD) is a life-threatening complication frequently occurring following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Since gut microbiota and regulatory T cells (Tregs) are believed to play roles in GvHD prevention, we investigated whether DP8α Tregs, which we have previously described to harbor a T cell receptor specificity for the gut commensal Faecalibacterium prausnitzii, could protect against GvHD, thereby linking the microbiota and its effect on GvHD. We observed a decrease in CD73+ DP8α Treg frequency in allo-HSCT patients 1 month after transplantation, which was associated with acute GvHD (aGvHD) development at 1 month after transplantation, as compared with aGvHD-free patients, without being correlated to hematological disease relapse. Importantly, CD73 activity was shown to be critical for DP8α Treg suppressive function. Moreover, the frequency of host-reactive DP8α Tregs was also lower in aGvHD patients, as compared with aGvHD-free patients, which could embody a protective mechanism responsible for the maintenance of this cell subset in GvHD-free patients. We also showed that human DP8α Tregs protected mice against xenogeneic GvHD through limiting deleterious inflammation and preserving gut integrity. Altogether, these results demonstrated that human DP8α Tregs mediate aGvHD prevention in a CD73-dependent manner, likely through host reactivity, advocating for the use of these cells for the development of innovative therapeutic strategies to preclude aGvHD-related inflammation.

Microbiota-induced regulatory T cells associate with FUT2-dependent susceptibility to rotavirus gastroenteritis.

The FUT2 α1,2fucosyltransferase contributes to the synthesis of fucosylated glycans used as attachment factors by several pathogens, including noroviruses and rotaviruses, that can induce life-threatening gastroenteritis in young children. FUT2 genetic polymorphisms impairing fucosylation are strongly associated with resistance to dominant strains of both noroviruses and rotaviruses. Interestingly, the wild-type allele associated with viral gastroenteritis susceptibility inversely appears to be protective against several inflammatory or autoimmune diseases for yet unclear reasons, although a FUT2 influence on microbiota composition has been observed. Here, we studied a cohort of young healthy adults and showed that the wild-type FUT2 allele was associated with the presence of anti-RVA antibodies, either neutralizing antibodies or serum IgA, confirming its association with the risk of RVA gastroenteritis. Strikingly, it was also associated with the frequency of gut microbiota-induced regulatory T cells (Tregs), so-called DP8α Tregs, albeit only in individuals who had anti-RVA neutralizing antibodies or high titers of anti-RVA IgAs. DP8α Tregs specifically recognize the human symbiont Faecalibacterium prausnitzii, which strongly supports their induction by this anti-inflammatory bacterium. The proportion of F. prausnitzii in feces was also associated with the FUT2 wild-type allele. These observations link the FUT2 genotype with the risk of RVA gastroenteritis, the microbiota and microbiota-induced DP8α Treg cells, suggesting that the anti-RVA immune response might involve an induction/expansion of these T lymphocytes later providing a balanced immunological state that confers protection against inflammatory diseases.

HLA-E/ß2 microglobulin overexpression in colorectal cancer is associated with recruitment of inhibitory immune cells and tumor progression.

The host immune response plays a major role in colorectal carcinoma (CRC) progression. A mechanism of tumor immune escape might involve expression of the human leucocyte antigen (HLA)-E/ß2m on tumor cells. The inhibitory effect of HLA-E/ß2m on CD8+ cytotoxic T lymphocytes and natural killer (NK) cells is mediated by the main HLA-E receptor CD94/NKG2A. As the pathophysiological relevance of this mechanism in CRC remains unknown, this prompted us to examine, in situ, in a series of 80 CRC (i) the HLA-E and ß2m coexpression by tumor cells, (ii) the density of CD8+, cytotoxic, CD244+ and NKP46+ intraepithelial tumor-infiltrating lymphocyte (IEL-TIL) and (iii) the expression of CD94/NKG2 receptor on IEL-TIL. These data were then correlated to patient survival. We provided (i) the in situ demonstration of HLA-E/ß2m overexpression by tumor cells in 21% of CRC characterized by an overrepresentation of signet ring cell carcinomas, mucinous carcinomas and medullary carcinomas, (ii) the significant association between HLA-E/ß2m overexpression by tumor cells and increased density of CD8+ cytotoxic, CD244+ and CD94+ IEL-TIL and (iii) finally, the unfavorable prognosis associated with HLA-E/ß2m overexpression by tumor cells. Our findings show that HLA-E/ß2m overexpression is a surrogate marker of poor prognosis and point to a novel mechanism of tumor immune escape in CRC in restraining inhibitory IEL-TIL.

Human gut microbiota-reactive DP8α regulatory T cells, signature and related emerging functions.

In mice, microbiota-induced Tregs both maintain intestinal homeostasis and provide resistance to immuno-pathologies in the adult. Identifying their human functional counterpart therefore represents an important goal. We discovered, in the human colonic lamina propria and blood, a FoxP3-negative IL-10-secreting Treg subset, which co-expresses CD4 and CD8α (hence named DP8α) and displays a TCR-reactivity against Faecalibacterium prausnitzii, indicating a role for this symbiotic bacterium in their induction. Moreover, supporting their role in intestinal homeostasis, we previously reported both their drastic decrease in IBD patients and their protective role in vivo against intestinal inflammation, in mice. Here, we aimed at identifying the genomic, phenotypic and functional signatures of these microbiota-induced Tregs, towards delineating their physiological role(s) and clinical potential. Human F. prausnitzii-reactive DP8α Treg clones were derived from both the colonic lamina propria and blood. RNA-sequencing, flow cytometry and functional assays were performed to characterize their response upon activation and compare them to donor- and tissue-matched FoxP3+ Treg clones. DP8α Tregs exhibited a unique mixed Tr1-like/cytotoxic CD4+ T cell-profile and shared the RORγt and MAF master genes with mouse gut microbiota-induced FoxP3+ Tregs. We revealed their potent cytotoxic, chemotactic and IgA-promoting abilities, which were confirmed using in vitro assays. Therefore, besides their induction by a Clostridium bacterium, DP8α Tregs also partake master genes with mouse microbiota-induced Tregs. The present identification of their complete signature and novel functional properties, should be key in delineating the in vivo roles and therapeutic applications of these unique human microbiota-induced Tregs through their study in pathological contexts, particularly in inflammatory bowel diseases.

Human CD4+CD8α+ Tregs induced by Faecalibacterium prausnitzii protect against intestinal inflammation.

Abundance of Faecalibacterium prausnitzii, a dominant bacterium of the human microbiota that exhibits antiinflammatory effects, is decreased in patients with inflammatory bowel diseases (IBD). In humans, colonic lamina propria contains IL-10-secreting, Foxp3- Tregs characterized by a double expression of CD4 and CD8α (DP8α) and a specificity for F. prausnitzii. This Treg subset is decreased in IBD. The in vivo effect of DP8α cells has not been evaluated yet to our knowledge. Here, using a humanized model of a NSG immunodeficient mouse strain that expresses the HLA D-related allele HLA-DR*0401 but not murine class II (NSG-Ab° DR4) molecules, we demonstrated a protective effect of a HLA-DR*0401-restricted DP8α Treg clone combined with F. prausnitzii administration in a colitis model. In a cohort of patients with IBD, we showed an independent association between the frequency of circulating DP8α cells and disease activity. Finally, we pointed out a positive correlation between F. prausnitzii-specific DP8α Tregs and the amount of F. prausnitzii in fecal microbiota in healthy individuals and patients with ileal Crohn's disease.

Tumor-reactive CD4+ CD8αß+ CD103+ αßT cells: a prevalent tumor-reactive T-cell subset in metastatic colorectal cancers.

High level of T-cell infiltration in colorectal carcinomas (CRCs) is a good prognostic indicator, but the tumor reactivity of this infiltrate (tumor infiltrating lymphocytes [TIL]) is poorly documented. This study examined the presence, phenotype and functional features of tumor-reactive lymphocytes in human CRC. Freshly dissociated TIL and T cell lines were isolated from CRC samples and from some paired normal colonic mucosa. Four tumor cell lines were obtained. Autologous tumor reactivity of CRC TIL and tumor-reactive cell features were analyzed. We demonstrate the presence among CRC TIL of variable fractions (up to 18%) of double positive CD4(+) CD8αß(+) (DP) αß T cells. Interestingly, a high proportion (16-20%) of this TIL subset displayed tumor reactivity, whilst this was the case for no or few single positive TIL. Low levels of DP TIL were found in most CRC samples and in normal colonic mucosa, but these cells were higher in metastatic CRC. Furthermore, we showed that DP TIL were polyclonal, restricted by HLA class-I, proliferated poorly and secreted higher amounts of IL-4 and IL-13 than single positive T cells, on cognate or CD3 stimulation. DP CRC TIL also expressed CD103, confirming their mucosal origin. Increased frequencies of tumor-reactive DP TIL in metastatic CRC suggest that these cells play a role in the metastatic process of this cancer. Based on their high secretion of IL-4 and IL-13 and on previously described roles of these cytokines in cancers, we postulate that DP TIL could favor CRC growth or metastasis and/or downmodulate immune responses to these tumors.

alpha v beta3-dependent cross-presentation of matrix metalloproteinase-2 by melanoma cells gives rise to a new tumor antigen.

A large array of antigens that are recognized by tumor-specific T cells has been identified and shown to be generated through various processes. We describe a new mechanism underlying T cell recognition of melanoma cells, which involves the generation of a major histocompatibility complex class I-restricted epitope after tumor-mediated uptake and processing of an extracellular protein--a process referred to as cross-presentation-which is believed to be restricted to immune cells. We show that melanoma cells cross-present, in an alpha v beta3-dependent manner, an antigen derived from secreted matrix metalloproteinase-2 (MMP-2) to human leukocyte antigen A*0201-restricted T cells. Because MMP-2 activity is critical for melanoma progression, the MMP-2 peptide should be cross-presented by most progressing melanomas and represents a unique antigen for vaccine therapy of these tumors.

Frequent occurrence of high affinity T cells against MELOE-1 makes this antigen an attractive target for melanoma immunotherapy.

We recently showed that the infusion of tumor infiltrating lymphocytes specific for the MELOE-1 antigen was associated with a prolonged relapse-free survival for HLA-A2(+) melanoma patients who received tumor infiltrating lymphocytes therapy. Here, we characterized the MELOE-1/A2-specific T-cell repertoire in healthy donors and melanoma patients to further support an immunotherapy targeting this epitope. Using tetramer enrichment followed by multicolor staining, we found that MELOE-1-specific T cells were present in the blood of healthy donors and patients at similar frequencies (around 1 in 1x10(5) CD8(+) cells). These cells mainly displayed a naïve phenotype in 4/6 healthy donors and 3/6 patients, whereas high proportions of memory cells were observed in the remaining individuals of both groups. There was a recurrent usage of the Valpha12.1 chain for 17/18 MELOE-1-specific T-cell clones derived from healthy donors or patients, associated with diverse Vbeta chains and V(D)J junctional sequences. All clones derived from melanoma patients (9/9) were reactive against the MELOE-1(36-44) peptide and against HLA-A2(+) melanoma cell lines. This study documents the existence of a large TCR repertoire specific for the MELOE-1/A2 epitope and its capacity to give rise to antitumor CTL that supports the development of immunotherapies targeting this epitope.

Concomitant decrease of double-positive lymphocyte population CD4CD8αα and Faecalibacterium prausnitzii in patients with colorectal cancer.

INTRODUCTION AND AIMS: Changes in the composition of the gut microbiota in patients with colorectal cancer (CRC) compatible with a contribution of the gut microbiota in carcinogenesis have been reported. In particular, a decrease Faecalibacterium prausnitzii has been identified. A CD4CD8αα, double-positive lymphocyte population (DP8α) has recently been demonstrated in the human colon and blood with regulatory functions and specificity for F. prausnitzii. Here, we aimed to detect dysbiosis in the fecal microbiome of patients with CRC by metagenomic analysis, and to look for changes in the levels of DP8α circulating T cells specific for F. prausnitzii in these patients. PATIENTS AND METHODS: Patients with CRC and control subjects were prospectively included. None had received antibiotics in the previous month or any anti-tumor treatment. A stool sample was collected for each participant, and analyzed by shotgun sequencing. The DP8α T cell population was identified and quantified on fresh whole blood by flow cytometry with anti-CD45, anti-CD3, anti-CD4 and anti-CD8α co-labeling. RESULTS: Twenty-one patients with CRC and 20 controls subjects were included. We found that mean relative abundance of five species was significantly decreased in CRC patients compared with controls, including F. prausnitzii, Barnesiella intestinihominis, Alistipes finegoldii, Bacteroides eggerthii and Eubacterium siraeum. We also found that the DP8α T cell population was significantly decreased in the blood of CRC patients compared with controls. CONCLUSION: In our work, we showed that a reduced abundance of F. prausnitzii in CRC patients was associated to a significant decrease in the circulating DP8α Treg population, suggesting a potential involvement of reduced activity of DP8α T cells in colonic carcinogenesis. These findings open new diagnostic and therapeutic strategies for CRC.

Long-lasting cross-presentation of tumor antigen in human DC.

DC cross-present exogenous antigens on MHC class I molecules, a process required for the onset of anti-tumor immune responses. In order to study the cross-presentation of tumor antigens by human DC, we compared the pathways of cross-presentation of long peptides requiring internalization and intracellular processing with the direct presentation of short peptides, which does not require intracellular processing. We found that, after brief incubations with DC, short peptides were presented to CD8(+) T cells with higher efficiencies than long peptides. After longer times of chase in the absence of peptide, however, the efficiency of presentation of the two types of peptides was reversed. After 2-3 days, DC pulsed with long peptides still activated T cells efficiently, while DC pulsed with short peptides failed to do so. Long-lasting presentation of the long peptides was, at least in part, due to a stored persistent pool of antigen, which was still available for loading on MHC class I molecules after several days of chase. These results show that the use of long synthetic peptides allows the efficient, long-lasting, presentation of tumor antigens, suggesting that long peptides represent an interesting approach for active anti-tumor vaccination.

An additional ORF on meloe cDNA encodes a new melanoma antigen, MELOE-2, recognized by melanoma-specific T cells in the HLA-A2 context.

We characterized a new melanoma antigen derived from one of the multiple open reading frames (ORFs) of the meloe transcript. The meloe gene is overexpressed in melanomas as compared to other cancer cell lines and normal tissues. The corresponding transcript is rather unusual, in that it does not contain a long unique ORF but multiple short ORFs. We recently characterized a tumor epitope derived from a polypeptide (MELOE-1) encoded by the ORF(1230-1370) and involved in relapse prevention of melanoma patients treated with autologous tumor infiltrating lymphocytes (TIL). Here we show that the ORF(285-404) encodes a polypeptide called MELOE-2 that also generated a HLA-A2 epitope recognized by a melanoma-specific T cell clone derived from the same TIL population from which we derived the MELOE-1-specific T cell clone. We also showed that HLA-A2 melanoma cells were spontaneously recognized by the MELOE-2-specific T cell clone, and we detected the presence of MELOE-2 reactive T cells in another TIL population infused to a patient who remained relapse-free after TIL treatment. These results demonstrate that translation of meloe transcript in melanoma cells can produce at least two immunogenic polypeptides, MELOE-1 and MELOE-2, from two distinct ORFs that could be relevant target for melanoma immunotherapy.

Increased frequency of nonconventional double positive CD4CD8 alphabeta T cells in human breast pleural effusions.

Breast cancer remains a leading cause of cancer-related death within the female population. Immunotherapy is expected to provide additional therapeutic benefits but has met so far limited success. This may be due in part to the poor understanding of immune responses to breast cancer. Although CD4(+) and CD8(+) T lymphocytes infiltrate these tumors, the phenotype and functions of these cells remain ill defined. This study was designed to investigate further about these questions, taking advantage of multiparameter flow cytometry on lymphocytes derived from peripheral blood, solid tumors, metastatic lymph nodes and pleural effusions samples of patients with breast cancer. Results showed that, in addition to conventional CD4(+) and CD8(+) alphabeta T cells, individual tumors and most pleural effusions contained significant fractions of unconventional double positive (DP) CD4(+)CD8(+) alphabeta T cells. These DP T cells displayed the phenotype and cytotoxic potential of effector/memory activated CD8(+) T cells but differed essentially from these cells by a high production of IL-5 and IL-13. The increased frequency of DP T cells in advanced breast cancer and their high lytic potential and original cytokine profile suggest that this T-cell subset may play a specific role in the regulation of immune responses to human breast cancer.

A fast and efficient HLA multimer-based sorting procedure that induces little apoptosis to isolate clinical grade human tumor specific T lymphocytes.

HLA multimers are now widely used to stain and sort CD8 T lymphocytes specific for epitopes from viral or tumoral antigens presented in an HLA class I context. However, the transfer of this technology to a clinical setting to obtain clinical grade CD8 T lymphocytes that may be used in adoptive cell transfer (ACT) is hindered by two main obstacles: the first obstacle is the use of streptavidin or derived products that are not available in clinical grade to multimerize HLA/peptide monomers and the second is the reported high degree of apoptosis that eventually occurs when T cell receptors are crosslinked by HLA multimers. In the present report, we describe new HLA multimers composed of immunomagnetic beads covalently coupled to a mAb specific for the AviTag peptide and coated with HLA/peptide monomers bearing the non biotinylated AviTag at the COOH terminus of the HLA heavy chain. Thus, all the components of this new reagent can be obtained in clinical grade. We compared these new multimers with the previously described multimers made with streptavidin beads coated with biotinylated HLA/peptide monomers, in terms of sorting efficiency, recovery of functional T cells, apoptosis and activation. We provide evidence that the new multimers could very efficiently sort pure populations of T lymphocytes specific for three different melanoma antigens (Melan-A, gp100 and NA17-A) after a single peptide stimulation of melanoma patients' PBMC. The recovered specific T cells were cytotoxic against the relevant melanoma cell-lines and, in most cases, produced cytokines. In addition, in marked contrast with streptavidin-based multimers, our new multimers induced very little apoptosis or activation after binding specific T lymphocytes. Altogether, these new multimers fulfill all the necessary requirements to select clinical grade T lymphocytes and should facilitate the development of ACT protocols in cancer patients.

A new tyrosinase epitope recognized in the HLA-B*4002 context by CTL from melanoma patients.

Melanoma reactive CTL were obtained by stimulating PBL from a melanoma patient in remission since 1994 following adjuvant TIL immunotherapy, with the autologous melanoma cell line. They were cloned by limiting dilution. One CTL clone recognized melanoma cell lines expressing tyrosinase and the B*4002 molecule, either spontaneously or upon transfection. We demonstrated that this clone recognizes the tyrosinase-derived nonapeptide 316-324 (ADVEFCLSL) and the overlapping decapeptide 315-324 (SADVEFCLSL). We derived two distinct additional specific CTL clones from this same patient that were also reactive against B*4002 melanoma cell lines, suggesting a relative diversity of this specific repertoire in this patient. Stimulating PBMC derived from four additional B*4002 melanoma patients with the tyrosinase 316-324 nonapeptide induced the growth of specific cells for two of the patients, demonstrating the immunogenicity of this new epitope. Our data show that this nonapeptide is a new tool that could be used to generate melanoma-specific T cells for adoptive immunotherapy or serve as a peptide vaccine for HLA-B*4002 melanoma patients.

Faecalibacterium prausnitzii Skews Human DC to Prime IL10-Producing T Cells Through TLR2/6/JNK Signaling and IL-10, IL-27, CD39, and IDO-1 Induction.

The human colonic mucosa contains regulatory type 1-like (Tr1-like, i.e., IL-10-secreting and Foxp3-negative) T cells specific for the gut Clostridium Faecalibacterium prausnitzii (F. prausnitzii), which are both decreased in Crohn's disease patients. These data, together with the demonstration, in mice, that colonic regulatory T cells (Treg) induced by Clostridium bacteria are key players in colon homeostasis, support a similar role for F. prausnitzii-specific Treg in the human colon. Here we assessed the mechanisms whereby F. prausnitzii induces human colonic Treg. We demonstrated that F. prausnitzii, but not related Clostridia, skewed human dendritic cells to prime IL-10-secreting T cells. Accordingly, F. prausnitzii induced dendritic cells to express a unique array of potent Tr1/Treg polarizing molecules: IL-10, IL-27, CD39, IDO-1, and PDL-1 and, following TLR4 stimulation, inhibited their up-regulation of costimulation molecules as well as their production of pro-inflammatory cytokines IL-12 (p35 and p40) and TNFα. We further showed that these potent tolerogenic effects relied on F. prausnitzii-induced TLR2/6 triggering, JNK signaling and CD39 ectonucleotidase activity, which was induced by IDO-1 and IL-27. These data, together with the presence of F. prausnitzii-specific Tr1-like Treg in the human colon, point out to dendritic cells polarization by F. prausnitzii as the first described cellular mechanism whereby the microbiota composition may affect human colon homeostasis. Identification of F. prausnitzii-induced mediators involved in Tr1-like Treg induction by dendritic cells opens therapeutic avenues for the treatment of inflammatory bowel diseases.

Double positive CD4+CD8+ T cells are part of the adaptive immune response against Candida albicans.

Although multiple immune cells participate in the innate and adaptive immune response against Candida albicans, the elucidation of cellular and inflammation kinetics may be a promising strategy to decipher events propitious to infection eradication. We used an in vitro Candida-human leucocyte coculture approach to study the dynamics of rare CD4+CD8+ double positive T lymphocytes (DP T) produced in response to this fungus. Our results highlight the presence of two phenotypically distinct subsets of DP T cells: CD4hiCD8lo and CD4loCD8hi, and that the different ratio of these cells correlates with infection outcome. The ratio of CD4hiCD8lo over CD4loCD8hi by day 6 was significantly higher in controlled infections and decreased when infection persisted due to a significant increase in the proportion of CD4loCD8hi. When infection was controlled, CD4hiCD8lo T cells secreted IFNγ, TNFα, IL-4 and IL-10 cytokines two days after challenge. By day 2, under conditions of persistent infection, CD4hiCD8lo and CD4loCD8hi T cells secreted significant levels of IL-4 and IL-10, respectively, compared to uninfected cultures. Frequency kinetics and original cytokine profiles detailed in this work indicate that DP T cells could participate in the adaptive immune response to C. albicans.