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1.  CD4CD8αα Lymphocytes, A Novel Human Regulatory T Cell Subset Induced by Colonic Bacteria and Deficient in Patients with Inflammatory Bowel Disease 
PLoS Biology  2014;12(4):e1001833.
Gut bacterium Faecalibacterium prausnitzii activates a newly identified set of human IL-10-producing Treg cells (CD4CD8αα lymphocytes), revealing a mechanism by which commensal microbes contribute to host immunity.
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.
Author Summary
It has become evident that bacteria in our gut affect health and disease, but less is known about how they do this. Recent studies in mice showed that gut Clostridium bacteria and their metabolites can activate regulatory T cells (Treg) that in turn mediate tolerance to signals that would ordinarily cause inflammation. In this study we identify a subset of human T lymphocytes, designated CD4CD8αα T cells that are present in the surface lining of the colon and in the blood. We demonstrate Treg activity and show these cells to be activated by microbiota; we identify F. prausnitzii, a core Clostridium strain of the human gut microbiota, as a major inducer of these Treg cells. Interestingly, there are fewer F. prausnitzii in individuals suffering from inflammatory bowel disease (IBD), and accordingly the CD4CD8αα T cells are decreased in the blood and gut of patients with IBD. We argue that CD4CD8αα colonic Treg probably help control or prevent IBD. These data open the road to new diagnostic and therapeutic strategies for the management of IBD and provide new tools to address the impact of the intestinal microbiota on the human immune system.
PMCID: PMC3979654  PMID: 24714093
2.  Cross-Presentation of Synthetic Long Peptides by Human Dendritic Cells: A Process Dependent on ERAD Component p97/VCP but Not sec61 and/or Derlin-1 
PLoS ONE  2014;9(2):e89897.
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.
PMCID: PMC3937416  PMID: 24587108
3.  Matrix metalloproteinase-2 conditions human dendritic cells to prime inflammatory TH2 cells via an IL-12- and OX40L-dependent pathway 
Cancer cell  2011;19(3):333-346.
Matrix metalloproteinase-2 (MMP-2) is a proteolytic enzyme degrading the extracellular matrix and over-expressed by many tumors. Here, we documented the presence of MMP-2-specific CD4+ T cells in tumor-infiltrating lymphocytes (TILs) from melanoma patients. Strikingly, MMP-2-specific CD4+ T cells displayed an inflammatory TH2 profile, i.e. mainly secreting TNFα, IL-4 and IL-13 and expressing GATA-3. Furthermore, MMP-2-conditioned dendritic cells (DCs) primed naïve CD4+ T cells to differentiate into an inflammatory TH2 phenotype through OX40L expression and inhibition of IL-12p70 production. MMP-2 degrades the type-I IFN receptor, thereby preventing STAT1 phosphorylation, which is necessary for IL-12p35 production. Active MMP-2, therefore, acts as an endogenous type-2 “conditioner” and may play a role in the observed prevalence of detrimental type-2 responses in melanoma.
Several melanoma-associated antigens have been targeted in immunization strategies to treat melanoma patients. However, the therapeutic efficacy of these approaches remains limited, indicating an urgent need for improvement. Because MMP-2 activity is critical for melanoma progression, it represents an interesting target for vaccine therapy. We show that MMP-2 is an immunogenic tumor antigen. However, MMP-2-specific CD4+ T lymphocytes display a suboptimal inflammatory TH2 profile. MMP-2-conditoned DCs prime TH2 responses against several melanoma-associated antigen (MAA), suggesting that MMP-2 can create a TH2 skewing microenvironment in a bystander fashion. Elucidation of the underlying mechanisms opens the way to improving immune responses towards a more effective TH1 response, and highlights the potential of MMP2 as a target antigen in melanoma.
PMCID: PMC3073826  PMID: 21397857
4.  Crystal structures of HLA-A*0201 complexed with Melan-A/MART-126(27L)-35 peptidomimetics reveal conformational heterogeneity and highlight degeneracy of T cell recognition◊ 
Journal of medicinal chemistry  2010;53(19):7061-7066.
There is growing interest in using tumor associated antigens presented by class-I major histocompatibility complex (MHC-I) proteins as cancer vaccines. As native peptides are poorly stable in biological fluids, researchers have sought to engineer synthetic peptidomimetics with greater biostability. Here, we demonstrate that antigenic peptidomimetics of the Melan-A/MART-126(27L)-35 melanoma antigen adopt strikingly different conformations when bound to MHC-I, highlighting the degeneracy of T cell recognition and revealing the challenges associated with mimicking native peptide conformation.
PMCID: PMC2951488  PMID: 20806940
5.  Folding of Matrix Metalloproteinase-2 Prevents Endogenous Generation of MHC Class-I Restricted Epitope 
PLoS ONE  2010;5(7):e11894.
We previously demonstrated that the matrix metalloproteinase-2 (MMP-2) contained an antigenic peptide recognized by a CD8 T cell clone in the HLA-A*0201 context. The presentation of this peptide on class I molecules by human melanoma cells required a cross-presentation mechanism. Surprisingly, the classical endogenous processing pathway did not process this MMP-2 epitope.
Methodology/Principal Findings
By PCR directed mutagenesis we showed that disruption of a single disulfide bond induced MMP-2 epitope presentation. By Pulse-Chase experiment, we demonstrated that disulfide bonds stabilized MMP-2 and impeded its degradation. Finally, using drugs, we documented that mutated MMP-2 epitope presentation used the proteasome and retrotranslocation complex.
These data appear crucial to us since they established the existence of a new inhibitory mechanism for the generation of a T cell epitope. In spite of MMP-2 classified as a self-antigen, the fact that cross-presentation is the only way to present this MMP-2 epitope underlines the importance to target this type of antigen in immunotherapy protocols.
PMCID: PMC2912773  PMID: 20689590
6.  Double Positive CD4CD8 αβ T Cells: A New Tumor-Reactive Population in Human Melanomas 
PLoS ONE  2010;5(1):e8437.
Double positive (DP) CD4CD8 Tαβ cells have been reported in normal individuals as well as in different pathological conditions including inflammatory diseases, viral infections and cancer, but their function remains to be elucidated. We recently reported the increased frequency of DP Tαβ cells in human breast pleural effusions. This manuscript addresses the question of the existence and above all the role of this non-conventional DP sub-population among tumor associated lymphocytes in melanomas.
Methodology/Principal Findings
We analyzed the intratumoral cell infiltrate in solid metastasis (n = 6) and tumor invaded lymph nodes (n = 26) samples from melanomas patients by multiparametric cytometry. Here we documented for the first time significant increased frequency of DP T cells in about 60% of melanoma tumors compared to blood samples. Interestingly, a high proportion of these cells produced TNF-α in response to autologous melanoma cell lines. Besides, they are characterized by a unique cytokine profile corresponding to higher secretion of IL-13, IL-4 and IL-5 than simple positive T cells. In deep analysis, we derived a representative tumor-reactive DP T cell clone from a melanoma patient's invaded lymph node. This clone was restricted by HLA-A*2402 and recognized both autologous and allogeneic tumor cells of various origins as well as normal cells, suggesting that the target antigen was a ubiquitous self antigen. However, this DP T cell clone failed to kill HLA-A*2402 EBV-transformed B cells, probably due to the constitutive expression of immunoproteasome by these cells.
In conclusion, we can postulate that, according to their broad tumor reactivity and to their original cytokine profile, the tumor associated DP T cells could participate in immune responses to tumors in vivo. Therefore, the presence of these cells and their role will be crucial to address in cancer patients, especially in the context of immunotherapies.
PMCID: PMC2797605  PMID: 20052413
7.  MELOE-1 is a new antigen overexpressed in melanomas and involved in adoptive T cell transfer efficiency 
The Journal of Experimental Medicine  2008;205(11):2673-2682.
A cytotoxic T lymphocyte (CTL) clone was derived from a tumor-infiltrating lymphocyte (TIL) population infused to a melanoma patient who remained relapse free for 10 yr after this adoptive transfer. This clone recognized all melanoma cell lines tested and, to a lower extent, melanocytes, in the context of human histocompatibility leukocyte antigen A2 (HLA-A2), but it did not recognize other tumor cell types. The gene coding for the antigen recognized by this clone was identified by the screening of a melanoma complementary DNA expression library. This antigen is overexpressed in melanomas, compared with other cancer cell lines and healthy tissues, and was thus called melanoma-overexpressed antigen (meloe). Remarkably, the structure of meloe was unusual, with multiple short open reading frames (ORFs). The peptide recognized by the CTL clone was encoded by one of these ORFs, called MELOE-1. Using a specific HLA-A2/peptide tetramer, we showed a correlation between the infusion of TILs containing MELOE-1–specific T cells and relapse prevention in HLA-A2 patients. Indeed, 5 out of 9 patients who did not relapse were infused with TILs that contained MELOE-1–specific T cells, whereas 0 out of the 21 patients who relapsed was infused with such TIL-containing lymphocytes. Overall, our results suggest that this new antigen is involved in immunosurveillance and, thus, represents an attractive target for immunotherapy protocols of melanoma.
PMCID: PMC2571940  PMID: 18936238
8.  αvβ3-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 αvβ3-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.
PMCID: PMC2212908  PMID: 15998788
9.  A Processed Pseudogene Codes for a New Antigen Recognized by a Cd8+ T Cell Clone on Melanoma 
The Journal of Experimental Medicine  2000;191(9):1617-1624.
The M88.7 T cell clone recognizes an antigen presented by HLA B*1302 on the melanoma cell line M88. A cDNA encoding this antigen (NA88-A) was isolated using a library transfection approach. Analysis of the genomic gene's sequence identified it is a processed pseudogene, derived from a retrotranscript of mRNA coding for homeoprotein HPX42B. The NA88-A gene exhibits several premature stop codons, deletions, and insertions relative to the HPX42B gene. In NA88-A RNA, a short open reading frame codes for the peptide MTQGQHFLQKV from which antigenic peptides are derived; a stop codon follows the peptide's COOH-terminal Val codon. Part of the HPX42B mRNA's 3′ untranslated region codes for a peptide of similar sequence (MTQGQHFSQKV). If produced, this peptide can be recognized by M88.7 T cells. However, in HPX42B mRNA, the peptide's COOH-terminal Val codon is followed by a Trp codon. As a result, expression of HPX42B mRNA does not lead to antigen production. A model is proposed for events that participated in creation of a gene coding for a melanoma antigen from a pseudogene.
PMCID: PMC2213434  PMID: 10790436
peptide; epitope; processing; tumor immunity; CTL

Results 1-9 (9)