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Recent experimental data have shown that mice could be immunized efficiently, in particular against cancer, by the injection of antigen-loaded dendritic cells (DC) or macrophages (MPH). In the present work, these two antigen-presenting cells (APC) were prepared in humans from circulating mononuclear cells (MNC). MPH were obtained from MNC that were cultured in hydrophobic plastic bags and purified by elutriation. DC were from the culture of adherent elutriation-purified monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The two APC were prepared in parallel from the same donors and their phenotype and antigen-presenting capacity were compared. DC differed from MPH by a higher expression of HLA-DR and CD23 and a lower expression of CD14, CD64 and of adhesion molecules. DC and MPH were comparably effective in (a) enhancing the mitotic response of autologous lymphocytes to immobilized anti-CD3 (accessory function); (b) presenting melanoma peptides to specific cytotoxic T lymphocyte (CTL) clones; and (c) stimulating the generation of CTL directed against a myxovirus influenza peptide. However, DC were more effective than MPH in inducing the mitotic response of allogeneic peripheral blood leucocytes (PBL), possibly because of their higher expression of HLA class II molecules. In conclusion, DC and MPH prepared from blood MNC did not differ substantially in their ability to activate HLA class I-restricted T-cell responses by exogenous peptide presentation. [ABSTRACT FROM AUTHOR]

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.

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., Competing Interests: SL and TC are employed by Biofortis Merieux Nutrisciences. The remaining authors declare that the research was conducted in absence of any commercial or financial relationship that could be construed as a potential conflict of interest., (Copyright © 2023 Godefroy, Barbé, Le Moullac-Vaidye, Rocher, Breiman, Leuillet, Mariat, Chatel, Ruvoën-Clouet, Carton, Jotereau and Le Pendu.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jotereau, Alameddine, Teusan, Pédron, Jouand, Altare and Godefroy.)

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.

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., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

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., (Copyright © 2019 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

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.

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 (T REG ) 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 T REG 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 T REG 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., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

In studies in murine models, active suppression by IL-10-secreting Foxp3 regulatory T cells (Tregs) has emerged as an essential mechanism in colon homeostasis. However, the role of the equivalent subset in humans remains unclear, leading to suggestions that other subsets and/or mechanisms may substitute for Foxp3 Tregs in the maintenance of colon homeostasis. We recently described a new subset of CD4CD8αα T cells reactive to the gut bacterium Faecalibacterium prausnitzii and endowed with regulatory/suppressive functions. This subset is abundant in the healthy colonic mucosa, but less common in that of patients with inflammatory bowel disease (IBD). We discuss here the physiological significance and potential role of these Tregs in preventing inflammation of the gut mucosa and the potential applications of these discoveries for IBD management.

Matrix metalloproteinase-2 (MMP-2) is involved in several physiological mechanisms, including wound healing and tumor progression. We show that MMP-2 directly stimulates dendritic cells (DCs) to both upregulate OX40L on the cell surface and secrete inflammatory cytokines. The mechanism underlying DC activation includes physical association with Toll-like receptor-2 (TLR2), leading to NF-κB activation, OX40L upregulation on DCs, and ensuing TH2 differentiation. Significantly, MMP-2 polarizes T cells toward type 2 responses in vivo, in a TLR2-dependent manner. MMP-2-dependent type 2 polarization may represent a key immune regulatory mechanism for protection against a broad array of disorders, such as inflammatory, infectious, and autoimmune diseases, which can be hijacked by tumors to evade immunity., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

Antitumor vaccination using synthetic long peptides (SLP) is an additional therapeutic strategy currently under development. It aims to activate tumor-specific CD8(+) CTL by professional APCs such as DCs. DCs can activate T lymphocytes by MHC class I presentation of exogenous antigens - a process referred to as "cross-presentation". Until recently, the intracellular mechanisms involved in cross-presentation of soluble antigens have been unclear. Here, we characterize the cross-presentation pathway of SLP Melan-A16-40 containing the HLA-A2-restricted epitope26-35 (A27L) in human DCs. Using confocal microscopy and specific inhibitors, we show that SLP16-40 is rapidly taken up by DC and follows a classical TAP- and proteasome-dependent cross-presentation pathway. Our data support a role for the ER-associated degradation machinery (ERAD)-related protein p97/VCP in the transport of SLP16-40 from early endosomes to the cytoplasm but formally exclude both sec61 and Derlin-1 as possible retro-translocation channels for cross-presentation. In addition, we show that generation of the Melan-A26-35 peptide from the SLP16-40 was absolutely not influenced by the proteasome subunit composition in DC. Altogether, our findings propose a model for cross-presentation of SLP which tends to enlarge the repertoire of potential candidates for retro-translocation of exogenous antigens to the cytosol.

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.