Colorectal cancer (CRC) progression is driven by dynamic interactions among tumor cells, immune infiltrates, and the gut microbiota. While regulatory T cells (Tregs) may contribute to immune suppression in CRC, the role of non-conventional Tregs remains poorly defined. We identified a non-conventional population of microbiota-induced Tregs in the human colonic mucosa that co-expressed CD4, CD8α, CXCR6, and CCR6, termed DP8α Tregs, that exert potent immunomodulatory properties in different inflammatory settings. Their status and role in CRC, however, have not been investigated. Here, using multiparametric flow cytometry in a prospective cohort of CRC patients, we showed that DP8α Tregs are significantly enriched in tumors compared to paired non-tumoral colonic mucosa. Tumor-infiltrating DP8α Tregs displayed elevated expression of the CD39/CD73 ectonucleotidases, as well as CCR5, consistent with a suppressive phenotype within the tumor microenvironment. Functional co-culture assays further demonstrated that sorting DP8α Tregs from CRC tumors inhibited both CD4 and CD8 T-cell proliferation, an effect largely reversed by pharmacological inhibition of CD39 and CD73, which was associated with reduced IL-2 levels. Together, these findings show that DP8α Tregs enriched in the CRC tumor microenvironment, are able to suppress effector T-cell responses through the purinergic pathway and support further investigation of their contribution to immune regulation in CRC.

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.

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 DP8 alpha 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(+) DP8 alpha 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 DP8 alpha Treg suppressive function. Moreover, the frequency of host-reactive DP8 alpha 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 DP8 alpha Tregs protected mice against xenogeneic GvHD through limiting deleterious inflammation and preserving gut integrity. Altogether, these results demonstrated that human DP8 alpha 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.

Allogeneic stem cell transplantation (allo-HSCT) to treat hematological malignancies can induce life-threatening complications, such as graft-versus-host disease (GvHD). Steroids remain the first-line treatment for acute GvHD (aGvHD). Nevertheless, new treatments are needed to, not only, treat steroid-resistant patients and improve allo-HSCT outcomes, but also reduce steroid complications. Increasing evidences strongly suggest that gut microbiota composition and the activity of regulatory T cells (Tregs) could be involved in GvHD prevention. We have identified a novel FoxP3-negative IL-10-secreting Treg subset, named DP8α, enriched in the colonic mucosa and present in blood. These cells display a TCR-specificity for the gut ClostridiumIV bacterium Faecalibacterium prausnitzii. Sizable fractions of these cells also expressed the membrane-bound ectonucleotidases CD39 and CD73, which are directly involved in their suppressive activity in vitro. In vivo, these enzymes play key regulatory roles by both degrading proinflammatory extracellular ATP and inducing the production of immunosuppressive adenosine. An alteration in this adenosine/purinergic pathway has also been linked to GvHD occurrence. Altogether, these data prompted us to hypothesize that F. prausnitzii-reactive DP8α Tregs, whose suppressive activity is driven by the purinergic pathway, could bridge microbiota dysbiosis and GvHD incidence in allo-HSCT patients. Moreover, decreased levels of Clostridia, especially Faecalibacteriumspp, in patient's stools, have been associated with aGvHD risk, and mice gavaged with Treg-inducing Clostridia displayed milder GvHD and improved survival. Therefore, we addressed a potential role for DP8α Tregs in the prevention of aGvHD. We first analyzed cells from 63 patients with hematological malignancies, who received allo-HSCT, among whom a third developed aGvHD. We quantified their circulating DP8α Tregs and their CD39 and CD73 expression pre- and post-allo-HSCT. While the percentage of CD73-expressing DP8α Tregs within CD3+ T cells before HSCT was not different, a strikingly deficiency of CD73-expressing DP8α Tregs was strongly associated with aGvHD development (p<.001) at 1-month post-transplant, as compared to aGvHD-free patients (Fig.1). Importantly, CD73 expression was not affected on any other T cell subset analyzed. CD73 decrease on DP8α Tregs did not result from corticotherapy since it was 1/ equally observed before and after treatment in aGvHD patients and 2/ not observed following in vitro treatment of PBMCs with methylprednisolone. Moreover, the decrease of CD73-expressing DP8α Tregs in aGvHD patients was observed across all treatment conditions, which were heterogeneous, and therefore appears relevant and likely related to the aGvHD condition itself. Altogether, these data strongly suggest that a CD73-dependent functional alteration of DP8α Tregs could, at least in part, be involved in aGvHD occurrence and/or development. We next evaluated the therapeutic efficacy of CD73+ DP8α Tregs in a pre-clinical model of acute xeno-GvHD induced through human PBMC injection in irradiated NSG recipient mice. In vitro-expanded CD73+ DP8α Treg injected i.v. drastically protected mice against xeno-GvHD (Log-rank, p<.0001; Fig.2) without preventing the development of human chimerism. Moreover, these in vivo experiments, repeated 4 times using different PBMC donors, all confirmed the potent protective role of CD73+ DP8α Tegs in this model. Accordingly, serum inflammatory human cytokines and tissue infiltration by human cells were significantly lower in mice treated with CD73+ DP8α Tregs, as compared to untreated mice. In addition, the colon was rapidly populated with the injected DP8α Tregs and well-preserved in treated mice, while clearly damaged in untreated mice. Altogether, these results strongly support a role for CD73+ DP8α Tregs in aGVHD prevention and advocate for the use of these cells to both predict aGvHD risks and give rise to the development of innovative therapeutic strategies to preclude GvHD-related inflammation. Such therapeutic approaches would be based on the infusion of DP8α Tregs and/or their in vivo stimulation, e.g., with F. prausnitzii-derived antigens or probiotics. Of note, DP8α Tregs display an uniquely high proliferating potential and stable immunosuppressive functions in vitro, a key feature to implement such therapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

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.

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 AIMSChanges 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 METHODSPatients 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.RESULTSTwenty-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.CONCLUSIONIn 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.

Prognosis for hematological malignancies treated with allogeneic stem cell transplantation (allo-HSCT) remains dismal and can often induce potentially deadly complications, such as graft versus host disease (GvHD). Steroids remain the first-line treatment for aGvHD. Nevertheless, new treatments are needed to improve allo-HSCT outcomes and reduce steroid complications. In this context, increasing evidences suggest that gut microbiota composition and the activity of regulatory T cells (Tregs) are involved in GvHD prevention (Elias and Rudensky, 2019, Br. J. Haematol. ; Shono and van den Brink, 2018, Nat. Rev. Cancer). We have identified a novel FoxP3-negative IL-10-secreting Treg subset, named DP8α, enriched in the colon mucosa and present in blood, characterized by its CD3+/CD4+/CD8αLOW/ CCR6+/CXCR6+ phenotype and its TCR-specificity for the gut ClostridiumIV bacterium Faecalibacterium prausnitzii (Sarrabayrouse et al., 2014, PLoS Biol ; Godefroy et al., 2018, Gastroenterology). Sizable fractions of these cells also expressed the membrane-bound ectonucleotidases CD39 and CD73 (gating strategy shown in Figure 1), which are directly involved in their suppressive activity in vitro (Godefroy et al., 2018, Gastroenterology). In vivo, these molecules play key regulatory roles both by degrading extracellular ATP and thus limiting its inflammasome-related proinflammatory role, and by inducing the production of immunosuppressive adenosine. Accordingly, an alteration in adenosine/purinergic signaling has been suggested in GvHD occurrence (Deaglio et al., 2007, J. Exp. Med. ; Wang et al., 2013, PLoS ONE). Thus, we postulated that F. prausnitzii-reactive DP8α Tregs, whose suppressive activity depends on purinergic signaling, could bridge microbiota dysbiosis and GvHD incidence in allo-HSCT patients. In support of this, decreased levels of Clostridium bacteria, especially Faecalibacteriumspp, in patient's stools, have been associated with aGvHD risk (Noor et al., 2019, J Innate Immun). Moreover, mice gavaged with butyrate-producing and Treg-inducing Clostridia displayed milder GvHD and improved survival (Mathewson et al., 2016, Nat. Immunol.). Therefore, we recently analyzed 63 patients with hematological malignancies, who received allo-HSCT, among which 21 developed an aGvHD (pathologies and treatments are listed Table I). We quantified circulating DP8α Tregs and analyzed their expression of CD39 and CD73 in donors, before and after mobilization, and in patients at one week before and 1, 2, and 3 months post allo-HSCT. Strikingly, results clearly showed that aGvHD development was strongly associated with a lack of expression of CD73 by DP8α Tregs (p<.0001) (Fig.2A), but not by any other T cell subsets analyzed. DP8α frequency and their CD39 expression were similar in GvHD+ versus GvHD- patients. Importantly, CD73 decrease did not result from their corticotherapy since it was equally observed before and after treatment (Fig.2B) and since in vitro treatment of PBMCs with methylprednisolone did not alter CD73 expression whatsoever. Therefore, the CD73 decrease observed in aGvHD patients, appears relevant and likely related to the aGvHD condition itself. It is noteworthy that this cohort was rather heterogeneous in terms of hematological diseases, conditioning regimens and prophylaxis treatments (Table I). Nevertheless, this drastic CD73 decrease in aGvHD patients was observed across all these conditions, strongly strengthening its relevancy. Altogether, these data strongly suggest that a CD73-dependent functional alteration of DP8α Tregs are, at least in part, involved in aGvHD occurrence. These results could therefore be used to predict GvHD risk and also give rise to the development of therapeutic strategies. Such innovative treatments could be based on the infusion of DP8α Tregs with appropriate features, since we have shown both the uniquely high proliferating potential and the stable immunosuppressive function of these cells in vitro. Moreover, administration of DP8α target antigens (F. prausnitzii-derived), in the form of peptides, proteins, or even bacterial lysates, such as probiotics, could also be foreseen to either directly in vivo expand these Tregs or indirectly through the induction of tolerogenic dendritic cells (Alameddine et al., 2019, Front Immunol) and subsequent Treg differentiation/priming to limit GvHD-related inflammation. Figure 1 Disclosures Chevallier: Incyte Corporation: Honoraria.

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.

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 & AIMSFaecalibacterium 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.METHODSWe 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.RESULTSCirculating 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.CONCLUSIONSWe 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.

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.

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.

Abstract Background: Cancer cells, including melanoma, can be highly resistant to traditional treatments such as chemotherapy and radiotherapy. As a result, a lot of effort has been put into developing immune therapies to treat cancer patients. The main barrier to design effective immunotherapies is the immunosuppression induced by the tumor. Matrix metalloproteinase-2 (MMP-2) is over-expressed in most cancers including melanoma, and its expression is associated with increased dissemination and poorer survival/prognosis. Here, we not only uncovered an immunosuppressive role of MMP-2 in inducing ineffective/detrimental TH2 immune responses, but also the underlying mechanisms. Methods: Human dendritic cells (DCs) were cultured in the presence of MMP-2 and/or various TLR agonists. DCs responses were monitored using immunostaining, ELISA or cytometric bead array methods. Autologous CD4+ T cells were stimulated using these conditioned tumor-associated antigen (TAA)-pulsed DCs. TAA-specific CD4+ T cells were cloned and characterized using mainly the same techniques as for DCs. Results: Here we showed that MMP-2-conditioned human DCs primed naïve CD4+ T cells to differentiate into an inflammatory TH2 phenotype, i.e. mainly secreting TNFα, IL-4 and IL-13 and expressing GATA3. Accordingly, we detected MMP-2-specific CD4+ T cells displaying the same inflammatory TH2 profile in tumor-infiltrating lymphocytes from melanoma patients. Patients displaying such responses tended to have a poorer survival outcome. We revealed the underlying mechanisms for these T cell skewing: on the one hand, MMP-2 was found to degrade the type-I IFN receptor IFNAR1, thereby preventing STAT1 phosphorylation, which is necessary for IL-12 production. On the other hand, MMP-2 triggers the Toll-like receptor (TLR)-2 on DCs, which leads to NF-κB activation and OX40L expression. Both lack of IL-12 and over-expression of OX40L were found responsible for skewing T cell responses towards a detrimental TH2 phenotype. Conclusions: MMP-2, therefore, acts as an endogenous TH2 “conditioner” and may underlie the prevalence of detrimental TH2 responses in cancer. Our findings also described the responsible MMP-2-dependent mechanisms, which open the way to novel therapeutic strategies and/or targets to skew anti-tumor CD4+ T cell responses towards a more efficient anti-tumor TH1 phenotype to treat cancer patients. Citation Format: Emmanuelle Godefroy, Anne Gallois, Juliana Idoyaga, Francine Jotereau, Nina Bhardwaj. Matrix metalloproteinase-2 dysregulate anti-tumor immunity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-252. doi:10.1158/1538-7445.AM2014-LB-252

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.