Publications by authors named "Vincent Pitard"

33 Publications

Restoration of T-cell Effector Function, Depletion of Tregs, and Direct Killing of Tumor Cells: The Multiple Mechanisms of Action of a-TIGIT Antagonist Antibodies.

Mol Cancer Ther 2021 01 4;20(1):121-131. Epub 2020 Dec 4.

iTeos Therapeutics, Gosselies, Belgium and Cambridge, Massachusetts.

TIGIT is an immune checkpoint inhibitor expressed by effector CD4 and CD8 T cells, NK cells, and regulatory T cells (Tregs). Inhibition of TIGIT-ligand binding using antagonistic anti-TIGIT mAbs has shown potential to restore T-cell function and therapeutic efficacy in murine tumor models when combined with an anti-PD(L)-1 antibody. In the current work, we demonstrate broader TIGIT expression than previously reported in healthy donors and patients with cancer with expression on γδ T cells, particularly in CMV-seropositive donors, and on tumor cells from hematologic malignancies. Quantification of TIGIT density revealed tumor-infiltrating Tregs as the population expressing the highest receptor density. Consequently, the therapeutic potential of anti-TIGIT mAbs might be wider than the previously described anti-PD(L)-1-like restoration of αβ T-cell function. CD155 also mediated inhibition of γδ T cells, an immune population not previously described to be sensitive to TIGIT inhibition, which could be fully prevented via use of an antagonistic anti-TIGIT mAb (EOS-448). In PBMCs from patients with cancer, as well as in tumor-infiltrating lymphocytes from mice, the higher TIGIT expression in Tregs correlated with strong antibody-dependent killing and preferential depletion of this highly immunosuppressive population. Accordingly, the ADCC/ADCP-enabling format of the anti-TIGIT mAb had superior antitumor activity, which was dependent upon Fcγ receptor engagement. In addition, the anti-TIGIT mAb was able to induce direct killing of TIGIT-expressing tumor cells both in human patient material and in animal models, providing strong rationale for therapeutic intervention in hematologic malignancies. These findings reveal multiple therapeutic opportunities for anti-TIGIT mAbs in cancer therapeutics.
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http://dx.doi.org/10.1158/1535-7163.MCT-20-0464DOI Listing
January 2021

Understanding human γδ T cell biology toward a better management of cytomegalovirus infection.

Immunol Rev 2020 11 22;298(1):264-288. Epub 2020 Oct 22.

ImmunoConcEpT UMR 5164, CNRS, Bordeaux University, Bordeaux, France.

Cytomegalovirus (CMV) infection is responsible for significant morbidity and mortality in immunocompromised patients, namely solid organ and hematopoietic cell transplant recipients, and can induce congenital infection in neonates. There is currently an unmet need for new management and treatment strategies. Establishment of an anti-CMV immune response is critical in order to control CMV infection. The two main human T cells involved in HCMV-specific response are αβ and non-Vγ9Vδ2 T cells that belong to γδ T cell compartment. CMV-induced non-Vγ9Vδ2 T cells harbor a specific clonal expansion and a phenotypic signature, and display effector functions against CMV. So far, only two main molecular mechanisms underlying CMV sensing have been identified. Non-Vγ9Vδ2 T cells can be activated either by stress-induced surface expression of the γδT cell receptor (TCR) ligand annexin A2, or by a multimolecular stress signature composed of the γδTCR ligand endothelial protein C receptor and co-stimulatory signals such as the ICAM-1-LFA-1 axis. All this basic knowledge can be harnessed to improve the clinical management of CMV infection in at-risk patients. In particular, non-Vγ9Vδ2 T cell monitoring could help better stratify the risk of infection and move forward a personalized medicine. Moreover, recent advances in cell therapy protocols open the way for a non-Vγ9Vδ2 T cell therapy in immunocompromised patients.
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http://dx.doi.org/10.1111/imr.12922DOI Listing
November 2020

Characterization of a Unique γδ T-Cell Subset as a Specific Marker of Cytomegalovirus Infection Severity.

J Infect Dis 2021 Feb;223(4):655-666

Univ. Bordeaux, CNRS, ImmunoConcEpT, Bordeaux, France.

Cytomegalovirus (CMV) is a major infectious cause of death and disease after transplantation. We have previously demonstrated that the tissue-associated adaptive Vδ2neg γδ T cells are key effectors responding to CMV and associated with recovery, contrasting with their innatelike circulating counterparts, the Vγ9posVδ2pos T cells that respond to phosphoantigens but not to CMV. A third Vγ9negVδ2pos subgroup with adaptive functions has been described in adults. In the current study, we demonstrate that these Vγ9negVδ2pos T cells are also components of the CMV immune response while presenting with distinct characteristics from Vδ2neg γδ T cells. In a cohort of kidney transplant recipients, CMV seropositivity was the unique clinical parameter associated with Vγ9negVδ2pos T-cell expansion and differentiation. Extensive phenotyping demonstrated their substantial cytotoxic potential and activation during acute CMV primary infection or reinfection. In vitro, Vγ9negVδ2pos T cells responded specifically to CMV-infected cells in a T-cell receptor-dependent manner and through strong interferon γ production. Finally, Vγ9negVδ2pos T cells were the only γδ T-cell subset in which expansion was tightly correlated with the severity of CMV disease. To conclude, our results identify a new player in the immune response against CMV and open interesting clinical perspectives for using Vγ9negVδ2pos T cells as an immune marker for CMV disease severity in immunocompromised patients.
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http://dx.doi.org/10.1093/infdis/jiaa400DOI Listing
February 2021

ASS1 Overexpression: A Hallmark of Sonic Hedgehog Hepatocellular Adenomas; Recommendations for Clinical Practice.

Hepatol Commun 2020 Jun 11;4(6):809-824. Epub 2020 Apr 11.

BaRITOn Bordeaux Research in Translational Oncology University of Bordeaux INSERM UMR1053 Bordeaux France.

Until recently, 10% of hepatocellular adenomas (HCAs) remained unclassified (UHCA). Among the UHCAs, the sonic hedgehog HCA (shHCA) was defined by focal deletions that fuse the promoter of Inhibin beta E chain with GLI1. Prostaglandin D2 synthase was proposed as immunomarker. In parallel, our previous work using proteomic analysis showed that most UHCAs constitute a homogeneous subtype associated with overexpression of argininosuccinate synthase (ASS1). To clarify the use of ASS1 in the HCA classification and avoid misinterpretations of the immunohistochemical staining, the aims of this work were to study (1) the link between shHCA and ASS1 overexpression and (2) the clinical relevance of ASS1 overexpression for diagnosis. Molecular, proteomic, and immunohistochemical analyses were performed in UHCA cases of the Bordeaux series. The clinico-pathological features, including ASS1 immunohistochemical labeling, were analyzed on a large international series of 67 cases. ASS1 overexpression and the shHCA subgroup were superimposed in 15 cases studied by molecular analysis, establishing ASS1 overexpression as a hallmark of shHCA. Moreover, the ASS1 immunomarker was better than prostaglandin D2 synthase and only found positive in 7 of 22 shHCAs. Of the 67 UHCA cases, 58 (85.3%) overexpressed ASS1, four cases were ASS1 negative, and in five cases ASS1 was noncontributory. Proteomic analysis performed in the case of doubtful interpretation of ASS1 overexpression, especially on biopsies, can be a support to interpret such cases. ASS1 overexpression is a specific hallmark of shHCA known to be at high risk of bleeding. Therefore, ASS1 is an additional tool for HCA classification and clinical diagnosis.
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http://dx.doi.org/10.1002/hep4.1514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7262286PMC
June 2020

Butyrophilin-2A1 Directly Binds Germline-Encoded Regions of the Vγ9Vδ2 TCR and Is Essential for Phosphoantigen Sensing.

Immunity 2020 03 9;52(3):487-498.e6. Epub 2020 Mar 9.

Institute for Virology and Immunobiology, University of Würzburg, Würzburg, Germany. Electronic address:

Vγ9Vδ2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1's P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to Vγ9Vδ2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for Vγ9Vδ2 TCR-mediated responses. Surface plasmon resonance experiments established Vγ9Vδ2 TCRs used germline-encoded Vγ9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to Vγ9Vδ2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the Vγ9Vδ2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner.
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http://dx.doi.org/10.1016/j.immuni.2020.02.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7083227PMC
March 2020

Uncovering the Anticancer Potential of Murine Cytomegalovirus against Human Colon Cancer Cells.

Mol Ther Oncolytics 2020 Mar 29;16:250-261. Epub 2020 Jan 29.

University of Bordeaux, CNRS, ImmunoConcEpT, UMR 5164, 33076 Bordeaux, France.

Human cytomegalovirus (HCMV) components are often found in tumors, but the precise relationship between HCMV and cancer remains a matter of debate. Pro-tumor functions of HCMV were described in several studies, but an association between HCMV seropositivity and reduced cancer risk was also evidenced, presumably relying on recognition and killing of cancer cells by HCMV-induced lymphocytes. This study aimed at deciphering whether CMV influences cancer development in an immune-independent manner. Using immunodeficient mice, we showed that systemic infection with murine CMV (MCMV) inhibited the growth of murine carcinomas. Surprisingly, MCMV, but not HCMV, also reduced human colon carcinoma development . , both viruses infected human cancer cells. Expression of human interferon-β () and nuclear domain () were induced in MCMV-infected, but not in HCMV-infected human colon cancer cells. These results suggest a decreased capacity of MCMV to counteract intrinsic defenses in the human cellular host. Finally, immunodeficient mice receiving peri-tumoral MCMV therapy showed a reduction of human colon cancer cell growth, albeit no clinical sign of systemic virus dissemination was evidenced. Our study, which describes a selective advantage of MCMV over HCMV to control human colon cancer, could pave the way for the development of CMV-based therapies against cancer.
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http://dx.doi.org/10.1016/j.omto.2020.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052516PMC
March 2020

Inactivation of Proprotein Convertases in T Cells Inhibits PD-1 Expression and Creates a Favorable Immune Microenvironment in Colorectal Cancer.

Cancer Res 2019 Oct 29;79(19):5008-5021. Epub 2019 Jul 29.

Université Bordeaux, Bordeaux, France.

Proprotein convertases (PC) activate precursor proteins that play crucial roles in various cancers. In this study, we investigated whether PC enzyme activity is required for expression of the checkpoint protein programmed cell death protein 1 (PD-1) on cytotoxic T lymphocytes (CTL) in colon cancer. Although altered expression of the PC secretory pathway was observed in human colon cancers, only furin showed highly diffuse expression throughout the tumors. Inhibition of PCs in T cells using the general protein-based inhibitor α1-PDX or the pharmacologic inhibitor Decanoyl-Arg-Val-Lys-Arg-chloromethylketone repressed PD-1 and exhausted CTLs via induction of T-cell proliferation and apoptosis inhibition, which improved CTL efficacy against microsatellite instable and microsatellite stable colon cancer cells. , inhibition of PCs enhanced CTL infiltration in colorectal tumors and increased tumor clearance in syngeneic mice compared with immunodeficient mice. Inhibition of PCs repressed PD-1 expression by blocking proteolytic maturation of the Notch precursor, inhibiting calcium/NFAT and NF-κB signaling, and enhancing ERK activation. These findings define a key role for PCs in regulating PD-1 expression and suggest targeting PCs as an adjunct approach to colorectal tumor immunotherapy. SIGNIFICANCE: Protein convertase enzymatic activity is required for PD-1 expression on T cells, and inhibition of protein convertase improves T-cell targeting of microsatellite instable and stable colorectal cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/19/5008/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-0086DOI Listing
October 2019

Sensing of cell stress by human γδ TCR-dependent recognition of annexin A2.

Proc Natl Acad Sci U S A 2017 03 7;114(12):3163-3168. Epub 2017 Mar 7.

ImmunoConcept, CNRS UMR 5164, University of Bordeaux, F-33000 Bordeaux, France;

Human γδ T cells comprise a first line of defense through T-cell receptor (TCR) recognition of stressed cells. However, the molecular determinants and stress pathways involved in this recognition are largely unknown. Here we show that exposure of tumor cells to various stress situations led to tumor cell recognition by a Vγ8Vδ3 TCR. Using a strategy that we previously developed to identify antigenic ligands of γδ TCRs, annexin A2 was identified as the direct ligand of Vγ8Vδ3 TCR, and was found to be expressed on tumor cells upon the stress situations tested in a reactive oxygen species-dependent manner. Moreover, purified annexin A2 was able to stimulate the proliferation of a Vδ2 γδ T-cell subset within peripheral blood mononuclear cells and other annexin A2-specific Vδ2 γδ T-cell clones could be derived from peripheral blood mononuclear cells. We thus propose membrane exposure of annexin A2 as an oxidative stress signal for some Vδ2 γδ T cells that could be involved in an adaptive stress surveillance.
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http://dx.doi.org/10.1073/pnas.1621052114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373368PMC
March 2017

TCR-dependent sensitization of human γδ T cells to non-myeloid IL-18 in cytomegalovirus and tumor stress surveillance.

Oncoimmunology 2015 May 3;4(5):e1003011. Epub 2015 Feb 3.

Bordeaux University; CIRID ; Bordeaux, France ; CNRS , UMR 5164 , Bordeaux, France.

Human γδ T cells contribute to tissue homeostasis under normal conditions and participate in lymphoid stress surveillance against infection and tumors. However, the molecular mechanisms underlying the recognition of complex cell stress signatures by γδ T cells are still unclear. Tumor cells and human cytomegalovirus (HCMV)-infected cells are known targets of γδ T cells. We show here that many tumor and CMV-infected cells express caspase-1 inflammasomes and release interleukin (IL)-18. Engagement of the T-cell receptor (TCR) on Vδ2 γδ T cells controlled the direct innate immune sensing of IL-18 that enhanced cytotoxicity and interferon gamma (IFNγ) production. This TCR-dependent sensitization to IL-18 was mediated by the upregulation of the innate IL-18 receptor β chain (IL-18Rβ) expression. These findings shed light on inflammasomes as a unified stress signal of tumor and infected cells to alert γδ T cells. Moreover, uncovering the TCR-mediated sensitization of γδ T cells to inflammatory mediators establishes a molecular link between the innate and adaptive immune functions of γδ T cells that could fine tune the commitment of antigen-experienced γδ T cells to inflammatory responses.
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http://dx.doi.org/10.1080/2162402X.2014.1003011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4485801PMC
May 2015

Direct and Indirect Effects of Cytomegalovirus-Induced γδ T Cells after Kidney Transplantation.

Front Immunol 2015 21;6. Epub 2015 Jan 21.

Université de Bordeaux , Bordeaux , France ; UMR 5164, Centre National de la Recherche Scientifique , Bordeaux , France.

Despite effective anti-viral therapies, cytomegalovirus (CMV) is still associated with direct (CMV disease) and indirect effects (rejection and poor graft survival) in kidney transplant recipients. Recently, an unconventional T cell population (collectively designated as Vδ2(neg) γδ T cells) has been characterized during the anti-CMV immune response in all solid-organ and bone-marrow transplant recipients, neonates, and healthy people. These CMV-induced Vδ2(neg) γδ T cells undergo a dramatic and stable expansion after CMV infection, in a conventional "adaptive" manner. Similarly, as CMV-specific CD8+ αβ T cells, they exhibit an effector/memory TEMRA phenotype and cytotoxic effector functions. Activation of Vδ2(neg) γδ T cells by CMV-infected cells involves the γδ T cell receptor (TCR) and still ill-defined co-stimulatory molecules such as LFA-1. A multiple of Vδ2(neg) γδ TCR ligands are apparently recognized on CMV-infected cells, the first one identified being the major histocompatibility complex-related molecule endothelial protein C receptor. A singularity of CMV-induced Vδ2(neg) γδ T cells is to acquire CD16 expression and to exert an antibody-dependent cell-mediated inhibition on CMV replication, which is controlled by a specific cytokine microenvironment. Beyond the well-demonstrated direct anti-CMV effect of Vδ2(neg) γδ T cells, unexpected indirect effects of these cells have been also observed in the context of kidney transplantation. CMV-induced Vδ2(neg) γδ T cells have been involved in surveillance of malignancy subsequent to long-term immunosuppression. Moreover, CMV-induced CD16+ γδ T cells are cell effectors of antibody-mediated rejection of kidney transplants, and represent a new physiopathological contribution to the well-known association between CMV infection and poor graft survival. All these basic and clinical studies paved the road to the development of a future γδ T cell-based immunotherapy. In the meantime, γδ T cell monitoring should prove a valuable immunological biomarker in the management of CMV infection.
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http://dx.doi.org/10.3389/fimmu.2015.00003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4301015PMC
February 2015

Role of Campylobacter jejuni gamma-glutamyl transpeptidase on epithelial cell apoptosis and lymphocyte proliferation.

Gut Pathog 2014 12;6:20. Epub 2014 Jun 12.

Bacteriology Laboratory, University of Bordeaux, F-33000 Bordeaux, France ; INSERM U853, F-33000 Bordeaux, France ; INSERM U853, Bacteriology Laboratory, Université de Bordeaux (site Carreire), F-33076 Bordeaux, France.

Background: A gamma-glutamyl transpeptidase (GGT) is produced by up to 31% of strains of Campylobacter jejuni isolates. C. jejuni GGT is close to Helicobacter pylori GGT suggesting a conserved activity but unlike the latter, C. jejuni GGT has not been studied extensively. In line with the data available for H. pylori, our objectives were to purify C. jejuni GGT from the bacteria, and to evaluate its inhibitory and proapoptotic activities on epithelial cells and human lymphocytes.

Methods: C. jejuni GGT was purified from culture supernatants by chromatography. After verification of the purity by using mass spectrometry of the purified enzyme, its action on two epithelial cell lines and human lymphocytes was investigated. Cell culture as well as flow cytometry experiments were developed for these purposes.

Results: This study demonstrated that C. jejuni GGT is related to Helicobacter GGTs and inhibits the proliferation of epithelial cells with no proapoptotic activity. C. jejuni GGT also inhibits lymphocyte proliferation by causing a cell cycle arrest in the G0/G1 phase. These effects are abolished in the presence of a specific pharmacological inhibitor of GGT.

Conclusion: C. jejuni GGT activity is comparable to that of other Epsilonproteobacteria GGTs and more generally to Helicobacter bilis (inhibition of epithelial cell and lymphocyte proliferation, however with no proapoptotic activity). It could therefore be considered as a pathogenicity factor and promote, via the inhibition of lymphocyte proliferation, the persistence of the bacteria in the host. These observations are consistent with a role of this enzyme in the pathophysiology of chronic infections associated with C. jejuni.
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http://dx.doi.org/10.1186/1757-4749-6-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4080688PMC
July 2014

Cytomegalovirus-responsive γδ T cells: novel effector cells in antibody-mediated kidney allograft microcirculation lesions.

J Am Soc Nephrol 2014 Nov 17;25(11):2471-82. Epub 2014 Apr 17.

University of Bordeaux, Mixte Research Unit 5164, Bordeaux, France; National Center for Scientific Research (CNRS), Bordeaux, France; and

Human cytomegalovirus infection in transplant recipients has been associated with adverse renal allograft outcome and with a large γδ T-cell response, but whether both mechanisms are connected is unknown. We previously showed that most expanded circulating cytomegalovirus-responsive γδ T cells express the Fcγ-receptor CD16, suggesting that γδ T cells may participate in allograft lesions mediated by donor-specific antibodies through antibody-dependent cellular cytotoxicity. Here, we show that cytomegalovirus-specific CD16(pos) γδ T cells can perform antibody-dependent cellular cytotoxicity against stromal cells coated with donor-specific antibodies in vitro. In vivo, graft-infiltrating γδ T cells localized in close contact with endothelial cells only in patients who experienced cytomegalovirus infection and were more frequent within peritubular capillaries and glomeruli from antibody-mediated acute rejections than within those from T cell-mediated acute rejections. Finally, a persistently increased percentage of circulating cytomegalovirus-induced γδ T cells correlated inversely with the 1-year eGFR only in kidney recipients with donor-specific antibodies. Collectively, these data support the conclusion that cytomegalovirus-induced γδ T cells are involved in, and may serve as a clinical biomarker of, antibody-mediated lesions of kidney transplants. Moreover, these findings offer a new physiopathologic link between cytomegalovirus infection and allograft dysfunction in recipients with donor-specific antibodies.
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http://dx.doi.org/10.1681/ASN.2013101052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4214528PMC
November 2014

Anti-metastatic potential of human Vδ1(+) γδ T cells in an orthotopic mouse xenograft model of colon carcinoma.

Cancer Immunol Immunother 2013 Jul 26;62(7):1199-210. Epub 2013 Apr 26.

Centre National de la Recherche Scientifique (CNRS) UMR 5164, Université Bordeaux Ségalen, 33076, Bordeaux, France.

The role of human intraepithelial Vδ1(+) γδ T cell cytotoxic effectors in the immune surveillance against metastatic colon cancer has never been addressed, despite their reported capacity to infiltrate colon carcinomas and to kill colonic cancer cells in vitro. We previously showed that Vδ1(+) γδ T cells are enriched in blood in response to cytomegalovirus (CMV) infection, and that such increase may be protective against epithelial cancers. The objective of the present study was to investigate whether CMV-induced Vδ1(+) γδ T lymphocytes could inhibit the propagation of human colon tumors in vivo, in order to evaluate their immunotherapeutic potential in this context. Even though metastases are an important cause of death in various cancers including colorectal cancer (CRC), the anti-metastatic effect of immune effectors has been poorly analyzed. To this purpose, we set up a reliable model of metastatic colon cancer through orthotopic implantation of luciferase-expressing human HT29 cells in immunodeficient mice. Using bioluminescence imaging to follow the outcome of colonic cancer cells, we showed that a systemic treatment with CMV-induced Vδ1(+) γδ T cells could not only inhibit primary colon tumor growth but also the emergence of secondary tumor foci in the lungs and liver. Finally, our data lead to propose that Vδ1(+) γδ T lymphocytes may directly influence the appearance of metastases independently from their control of primary tumor size. These findings, which extend our previous work, pave the road for the potential manipulation of Vδ1(+) γδ T lymphocytes in novel anti-CRC immunotherapeutic protocols.
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http://dx.doi.org/10.1007/s00262-013-1402-1DOI Listing
July 2013

Cytomegalovirus and tumor stress surveillance by binding of a human γδ T cell antigen receptor to endothelial protein C receptor.

Nat Immunol 2012 Sep 12;13(9):872-9. Epub 2012 Aug 12.

Birmingham Cancer Research UK Cancer Centre, School of Cancer Sciences, University of Birmingham, Birmingham, UK.

T cells bearing γδ T cell antigen receptors (TCRs) function in lymphoid stress surveillance. However, the contribution of γδ TCRs to such responses is unclear. Here we found that the TCR of a human V(γ)4V(δ)5 clone directly bound endothelial protein C receptor (EPCR), which allowed γδ T cells to recognize both endothelial cells targeted by cytomegalovirus and epithelial tumors. EPCR is a major histocompatibility complex-like molecule that binds lipids analogously to the antigen-presenting molecule CD1d. However, the V(γ)4V(δ)5 TCR bound EPCR independently of lipids, in an antibody-like way. Moreover, the recognition of target cells by γδ T cells required a multimolecular stress signature composed of EPCR and costimulatory ligand(s). Our results demonstrate how a γδ TCR mediates recognition of broadly stressed human cells by engaging a stress-regulated self antigen.
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http://dx.doi.org/10.1038/ni.2394DOI Listing
September 2012

A functional screening identifies five microRNAs controlling glypican-3: role of miR-1271 down-regulation in hepatocellular carcinoma.

Hepatology 2013 Jan;57(1):195-204

University Bordeaux, Physiopathologie du cancer du foie, U1053, F-33000 Bordeaux, France.

Unlabelled: Hepatocellular carcinoma (HCC) is the major primary liver cancer. Glypican-3 (GPC3), one of the most abnormally expressed genes in HCC, participates in liver carcinogenesis. Based on data showing that GPC3 expression is posttranscriptionally altered in HCC cells compared to primary hepatocytes, we investigated the implication of microRNAs (miRNAs) in GPC3 overexpression and HCC. To identify GPC3-regulating miRNAs, we developed a dual-fluorescence FunREG (functional, integrated, and quantitative method to measure posttranscriptional regulations) system that allowed us to screen a library of 876 individual miRNAs. Expression of candidate miRNAs and that of GPC3 messenger RNA (mRNA) was measured in 21 nontumoral liver and 112 HCC samples. We then characterized the phenotypic consequences of modulating expression of one candidate miRNA in HuH7 cells and deciphered the molecular mechanism by which this miRNA controls the posttranscriptional regulation of GPC3. We identified five miRNAs targeting GPC3 3'-untranslated region (UTR) and regulating its expression about the 876 tested. Whereas miR-96 and its paralog miR-1271 repressed GPC3 expression, miR-129-1-3p, miR-1291, and miR-1303 had an inducible effect. We report that miR-1271 expression is down-regulated in HCC tumor samples and inversely correlates with GPC3 mRNA expression in a particular subgroup of HCC. We also report that miR-1271 inhibits the growth of HCC cells in a GPC3-dependent manner and induces cell death.

Conclusion: Using a functional screen, we found that miR-96, miR-129-1-3p, miR-1271, miR-1291, and miR-1303 differentially control GPC3 expression in HCC cells. In a subgroup of HCC, the up-regulation of GPC3 was associated with a concomitant down-regulation of its repressor miR-1271. Therefore, we propose that GPC3 overexpression and its associated oncogenic effects are linked to the down-regulation of miR-1271 in HCC.
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http://dx.doi.org/10.1002/hep.25994DOI Listing
January 2013

Antibody-dependent anti-cytomegalovirus activity of human γδ T cells expressing CD16 (FcγRIIIa).

Blood 2012 Feb 16;119(6):1418-27. Epub 2011 Dec 16.

Université de Bordeaux, Bordeaux, France.

Human cytomegalovirus (HCMV) infection is an important cause of morbidity and mortality in transplant recipients. Long-term protective immunity against HCMV requires both sustained specific T-cell response and neutralizing IgG production, but the interplay between these effector arms remains poorly defined. We previously demonstrated that γδ T cells play a substantial role as anti-HCMV T-cell effectors. The observation that CD16 (FcγRIIIA) was specifically expressed by the majority of HCMV-induced γδ T cells prompted us to investigate their cooperation with anti-HCMV IgG. We found that CD16 could stimulate γδ T cells independently of T-cell receptor (TCR) engagement and provide them with an intrinsic antibody-dependent cell-mediated cytotoxic (ADCC) potential. Although CD16(+)γδ T cells did not mediate ADCC against HCMV-infected cells, in accordance with the low level of anti-HCMV IgGs recognizing infected cells, they produced IFNγ when incubated with IgG-opsonized virions. This CD16-induced IFNγ production was greatly enhanced by IL12 and IFNα, 2 cytokines produced during HCMV infection, and conferred to γδ T cells the ability to inhibit HCMV multiplication in vitro. Taken together, these data identify a new antiviral function for γδ T cells through cooperation with anti-HCMV IgG that could contribute to surveillance of HCMV reactivation in transplant recipients.
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http://dx.doi.org/10.1182/blood-2011-06-363655DOI Listing
February 2012

Control of Plasmodium falciparum erythrocytic cycle: γδ T cells target the red blood cell-invasive merozoites.

Blood 2011 Dec 1;118(26):6952-62. Epub 2011 Nov 1.

Centre National de la Recherche Scientifique, Unité Mixte de Recherche, Bordeaux, France.

The control of Plasmodium falciparum erythrocytic parasite density is essential for protection against malaria, because it prevents pathogenesis and progression toward severe disease. P falciparum blood-stage parasite cultures are inhibited by human Vγ9Vδ2 γδ T cells, but the underlying mechanism remains poorly understood. Here, we show that both intraerythrocytic parasites and the extracellular red blood cell-invasive merozoites specifically activate Vγ9Vδ2 T cells in a γδ T cell receptor-dependent manner and trigger their degranulation. In contrast, the γδ T cell-mediated antiparasitic activity only targets the extracellular merozoites. Using perforin-deficient and granulysin-silenced T-cell lines, we demonstrate that granulysin is essential for the in vitro antiplasmodial process, whereas perforin is dispensable. Patients infected with P falciparum exhibited elevated granulysin plasma levels associated with high levels of granulysin-expressing Vδ2(+) T cells endowed with parasite-specific degranulation capacity. This indicates in vivo activation of Vγ9Vδ2 T cells along with granulysin triggering and discharge during primary acute falciparum malaria. Altogether, this work identifies Vγ9Vδ2 T cells as unconventional immune effectors targeting the red blood cell-invasive extracellular P falciparum merozoites and opens novel perspectives for immune interventions harnessing the antiparasitic activity of Vγ9Vδ2 T cells to control parasite density in malaria patients.
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http://dx.doi.org/10.1182/blood-2011-08-376111DOI Listing
December 2011

Modeling of congenital erythropoietic porphyria by RNA interference: a new tool for preclinical gene therapy evaluation.

J Gene Med 2010 Aug;12(8):637-46

INSERM U876, Bordeaux, France.

Background: Congenital erythropoietic porphyria (CEP) is a severe autosomal recessive disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. We recently demonstrated the definitive cure of a murine model of CEP by lentiviral vector-mediated hematopoietic stem cell (HSC) gene therapy. In the perspective of a gene therapy clinical trial, human cellular models are required to evaluate the therapeutic potential of lentiviral vectors in UROS-deficient cells. However, the rare incidence of the disease makes difficult the availability of HSCs derived from patients.

Methods: RNA interference (RNAi) has been used to develop a new human model of the disease from normal cord blood HSCs. Lentivectors were developed for this purpose.

Results: We were able to down-regulate the level of human UROS in human cell lines and primary hematopoietic cells. A 97% reduction of UROS activity led to spontaneous uroporphyrin accumulation in human erythroid bone marrow cells of transplanted immune-deficient mice, recapitulating the phenotype of cells derived from patients. A strong RNAi-induced UROS inhibition allowed us to test the efficiency of different lentiviral vectors with the aim of selecting a safer vector. Restoration of UROS activity in these small hairpin RNA-transduced CD34(+) cord blood cells by therapeutic lentivectors led to a partial correction of the phenotype in vivo.

Conclusions: The RNAi strategy is an interesting new tool for preclinical gene therapy evaluation.
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http://dx.doi.org/10.1002/jgm.1478DOI Listing
August 2010

Common features of gammadelta T cells and CD8(+) alphabeta T cells responding to human cytomegalovirus infection in kidney transplant recipients.

J Infect Dis 2009 Nov;200(9):1415-24

Centre Hospitalier Universitaire de Bordeaux, Bordeaux, France.

Background: Kidney transplant recipients infected with cytomegalovirus (CMV) undergo a persistent gammadelta T cell expansion in their peripheral blood. The anti-CMV function of these cells was previously demonstrated by their ability to kill CMV-infected cells in vitro.

Methods: To gain insight into the role of gammadelta T cells within the antiviral immune network, we compared the expansion kinetics of these T cells with that of CMV pp65-specific CD8(+) alphabeta T cells in the peripheral blood of twenty-one kidney transplant recipients.

Results: Both the percentage and the absolute number of pp65-specific CD8(+) T cells and gammadelta T cells showed a concomitant increase and persistence in most of the kidney transplant recipients with CMV infection. Both cell subsets exhibited an effector/memory phenotype (CD28(-), CD27(-), and CD45RA(+)) that predominated for the entire follow-up period.

Conclusions: In conclusion, CMV-specific CD8(+) alphabeta T cells and gammadelta T cells share common expansion kinetics and a common effector phenotype, suggesting that these cell types act similarly in response to CMV infection.
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http://dx.doi.org/10.1086/644509DOI Listing
November 2009

Cytomegalovirus-induced gammadelta T cells associate with reduced cancer risk after kidney transplantation.

J Am Soc Nephrol 2010 Jan 27;21(1):181-8. Epub 2009 Aug 27.

Centre National de la Recherche Scientifique Unité Mixte de Recherche 5164, Université Victor Segalen Bordeaux 2, Bordeaux, France.

An increase in the number of blood gammadelta T cells follows cytomegalovirus (CMV) infection in kidney transplant recipients. These cells react against CMV-infected cells and tumor epithelial cells in vitro. We hypothesized that these CMV-induced gammadelta T cells play a protective role against cancer in kidney transplant recipients. We performed a longitudinal case-control study involving 18 recipients who developed cancer between 2 and 6 yr after transplantation and 45 recipients who did not. The median percentage of gammadelta T cells among total lymphocytes in patients with malignancies was significantly lower compared with that in control patients at 6, 12, and 18 mo before the diagnosis of cancer. Patients with a gammadelta T cell percentage of more than 4% were protected from cancer. An increase of the Vdelta2(neg) gammadelta T cell subset significantly associated with lower incidence of cancer only in recipients who experienced pre- or postgraft CMV infection. Finally, a retrospective follow-up of 131 recipients for 8 yr revealed that CMV-naive recipients had an approximately 5-fold higher risk of cancer compared with CMV-exposed patients. In summary, these results suggest a protective role of CMV exposure against cancer in kidney transplant recipients.
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http://dx.doi.org/10.1681/ASN.2008101072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2799280PMC
January 2010

Antitumor activity of gammadelta T cells reactive against cytomegalovirus-infected cells in a mouse xenograft tumor model.

Cancer Res 2009 May 21;69(9):3971-8. Epub 2009 Apr 21.

Laboratoire d'Immunologie et d'Immunogénétique, Université Bordeaux 2 and Centre National de la Recherche Scientifique UMR 5164, CHU Bordeaux, Bordeaux, France.

gammadelta T cells recognize stress-induced autoantigens and contribute to immunity against infections and cancer. Our previous study revealed that Vdelta2-negative ((neg)) gammadelta T lymphocytes isolated from transplant recipients infected by cytomegalovirus (CMV) killed both CMV-infected cells and HT29 colon cancer cells in vitro. To investigate the antitumor effects of Vdelta2(neg) clones in vivo, we generated hypodermal HT29 tumors in immunodeficient mice. Concomitant injections of Vdelta2(neg)clones, in contrast to Vdelta2(+) cells, prevented the development of HT29 tumors. Vdelta2(neg) clones expressed chemokine C-C motif receptor 3 (CCR3) and migrated in vitro in response to chemokines secreted by HT29 cells, among which were the CCR3 ligands macrophage inflammatory protein-1delta and monocyte chemoattractant protein-4. More importantly, a systemic i.p. treatment with Vdelta2(neg) clones delayed the growth of HT29 s.c. tumors. The effect of in vivo gammadelta T-cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-CCR3 antibody. gammadelta T-cell passive immunotherapy was dependent on the cytotoxic activity of the gammadelta effectors toward their targets because Vdelta2(neg) clones were not able to inhibit the growth of A431 hypodermal tumors. Our findings suggest that CMV-specific Vdelta2(neg) cells could target in vivo cancer cells, making them an attractive candidate for antitumor immunotherapy.
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http://dx.doi.org/10.1158/0008-5472.CAN-08-3037DOI Listing
May 2009

Long-term expansion of effector/memory Vdelta2-gammadelta T cells is a specific blood signature of CMV infection.

Blood 2008 Aug 6;112(4):1317-24. Epub 2008 Jun 6.

Université Bordeaux 2, Bordeaux, France.

The ability of human gammadelta T cells to develop immunologic memory is still a matter of debate. We previously demonstrated the involvement of Vdelta2- gammadelta T lymphocytes in the response of immunosuppressed organ recipients to cytomegalovirus (CMV). Here, we demonstrate their ability to mount an adaptive immune response to CMV in immunocompetent subjects. Vdelta2- gammadelta T-cell peripheral blood numbers, repertoire restriction, and cytotoxicity against CMV-infected fibroblasts were markedly increased in CMV-seropositive, compared with CMV-seronegative, healthy persons. Whereas Vdelta2- gammadelta T cells were found as naive cells in CMV- patients, they virtually all exhibited the cytotoxic effector/memory phenotype in CMV+ patients, which is also observed in transplanted patients challenged with CMV. This long-term complete remodeling of the Vdelta2- gammadelta T-cell population by CMV predicts their ability to exhibit an adaptive anti-CMV immune response. Consistent with this, we observed that the secondary response to CMV was associated with a faster gammadelta T-cell expansion and a better resolution of infection than the primary response. In conclusion, the increased level of effector-memory Vdelta2- gammadelta T cells in the peripheral blood is a specific signature of an adaptive immune response to CMV infection of both immunocompetent and immunosuppressed patients.
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http://dx.doi.org/10.1182/blood-2008-01-136713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2515135PMC
August 2008

Modulation of lymphocyte proliferation induced by gastric MALT lymphoma-associated Helicobacter pylori strains.

Helicobacter 2008 Jun;13(3):167-73

INSERM U853, Bordeaux, F 33076 France, and Université Victor Segalen Bordeaux 2, Bordeaux, F33076, France.

Background: Helicobacter pylori infection leads to different chronic diseases, suggesting that this bacterium can evade the host immune defense system. The ability to control lymphocyte proliferation may be a mechanism leading to the development of gastric pathologies. Our aim was to characterize the effects of mucosa-associated lymphoid tissue (MALT) associated H. pylori strains on lymphocyte proliferation.

Materials And Methods: We measured the in vitro proliferation of human lymphocytes originally from blood or tonsil samples in the presence or absence of viable bacteria or lysates.

Results: We showed that MALT lymphoma-associated strains are not likely to be directly responsible for anarchical B-cell proliferation in vitro. On the other hand, proliferation of prestimulated T lymphocytes was abolished in vitro by the presence of all H. pylori strains, whether associated with MALT lymphoma or not.

Conclusion: Inhibition of T-cell proliferation may be of major importance in the gastric colonization and in the persistence of the infection. Furthermore, this inhibition may favor anarchical B-cell proliferation in vivo and predispose the host to gastric MALT lymphoma, whereas MALT-associated H. pylori strains do not appear to possess a specific capability to directly stimulate B-lymphocyte proliferation.
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http://dx.doi.org/10.1111/j.1523-5378.2008.00596.xDOI Listing
June 2008

Shared reactivity of V{delta}2(neg) {gamma}{delta} T cells against cytomegalovirus-infected cells and tumor intestinal epithelial cells.

J Exp Med 2005 May;201(10):1567-78

UMR 5164, Centre National de la Recherche Scientifique, IFR 66, Université Bordeaux 2, 33076 Bordeaux, France.

Long-lasting expansion of Vdelta2(neg) gammadelta T cells is a hallmark of cytomegalovirus (CMV) infection in kidney transplant recipients. The ligands of these cells and their role remain elusive. To better understand their immune function, we generated gammadelta T cell clones from several transplanted patients. Numerous patient Vdelta1(+), Vdelta3(+), and Vdelta5(+) gammadelta T cell clones expressing diverse Vgamma chains, but not control Vgamma9Vdelta2(+) T clones, displayed strong reactivity against CMV-infected cells, as shown by their production of tumor necrosis factor-alpha. Vdelta2(neg) gammadelta T lymphocytes could also kill CMV-infected targets and limit CMV propagation in vitro. Their anti-CMV reactivity was specific for this virus among herpesviridae and required T cell receptor engagement, but did not involve major histocompatibility complex class I molecules or NKG2D. Vdelta2(neg) gammadelta T lymphocytes expressed receptors essential for intestinal homing and were strongly activated by intestinal tumor, but not normal, epithelial cell lines. High frequencies of CMV- and tumor-specific Vdelta2(neg) gammadelta T lymphocytes were found among patients' gammadelta T cells. In conclusion, Vdelta2(neg) gammadelta T cells may play a role in protecting against CMV and tumors, probably through mucosal surveillance of cellular stress, and represent a population that is largely functionally distinct from Vgamma9Vdelta2(+) T cells.
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http://dx.doi.org/10.1084/jem.20041851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2212929PMC
May 2005

Expression of MHC class I receptors confers functional intraclonal heterogeneity to a reactive expansion of gammadelta T cells.

Eur J Immunol 2005 Jun;35(6):1896-905

CNRS-UMR 5164, CIRID, IFR66, Université Bordeaux 2, Bordeaux, France.

NK cell receptors for MHC class I molecules (MHC-NKR) can be expressed by T cell subsets. The restricted repertoire and phenotypic characteristics of MHC-NKR(+) T cells indicate that expression of MHC-NKR is acquired upon antigenic challenge and might promote expansion of T cells. Previous studies performed on in vitro generated alphabeta T cell clones concluded that MHC-NKR expression was not a clonal attribute. Here, we examined a massive monoclonal expansion of a non-leukemic gammadelta T cell population found in the peripheral blood of a lung-transplanted patient who suffered from a cytomegalovirus infection. Despite their monoclonality, these T cells displayed a heterogeneous and stable in vivo Ig- and lectin-like MHC-NKR phenotype. Twenty percent of the cells displayed a CD94(+)NKG2A(+) phenotype, and 10% were labeled with an anti-CD158b1/b2/j monoclonal antibody. A CD158b/j(+) gammadelta T cell clone derived in vitro from patient's peripheral blood lymphocytes was shown to express the activating form CD158j (KIR2DS2), which once cross-linked stimulated the clone cytolytic function and costimulated the TCR-induced production of cytokines, independently of the killer-activating receptor-associated protein (KARAP). In conclusion, heterogeneity of MHC-NKR expression confers a functional intraclonal diversity that may participate to induction of specific gammadelta T cell effector functions or proliferation upon pathogen challenge.
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http://dx.doi.org/10.1002/eji.200425837DOI Listing
June 2005

Increase in activated CD8+ T lymphocytes expressing perforin and granzyme B correlates with disease activity in patients with systemic lupus erythematosus.

Arthritis Rheum 2005 Jan;52(1):201-11

CNRS-UMR5164 and IFR66, Université de Bordeaux 2, Place Amélie Raba Léon, 33076 Bordeaux, France.

Objective: Cytotoxic T lymphocyte-mediated killing using granzyme B has recently been proposed to be a preferential and selective source of autoantigens in systemic autoimmune diseases, including systemic lupus erythematosus (SLE), while other reports have indicated that cytolytic activity in SLE patients was decreased. The aim of this study was to examine the phenotypic and functional status of the CD8+ T cells in SLE patients.

Methods: Phenotype analysis of CD8+ T cells was carried out using flow cytometry. The cytotoxic potential of CD8+ T cells and its consequences were examined in redirected-killing experiments. SLE patients with quiescent disease (n = 41) were compared with SLE patients with active disease (n = 20), normal individuals (n = 36), and control patients with vasculitis (n = 14). Cytotoxic CD8+ T cell differentiation was examined by coculture with differentiated dendritic cells (DCs) in the presence of SLE patient sera.

Results: Patients with disease flares were characterized by higher proportions of perforin- and/or granzyme B-positive lymphocytes with a differentiated effector phenotype (CCR7- and CD45RA+). The frequency of these cells in peripheral blood correlated with clinical disease activity as assessed by the SLE Disease Activity Index. These cells generated high amounts of soluble nucleosomes as well as granzyme B-dependent unique autoantigen fragments. Finally, the activation of DCs with serum from a patient with active lupus induced granzyme B expression in CD8+ T lymphocytes.

Conclusion: DCs generated in the presence of sera from SLE patients with active disease could promote the differentiation of CD8+ effector T lymphocytes that are fully functional and able to generate SLE autoantigens. Our data disclose a new and pivotal role of activated CD8+ T lymphocytes in SLE pathogenesis.
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http://dx.doi.org/10.1002/art.20745DOI Listing
January 2005

Coordinated expression of Ig-like inhibitory MHC class I receptors and acquisition of cytotoxic function in human CD8+ T cells.

J Immunol 2004 Dec;173(12):7223-9

Centre d'Immunologie de Marseille-Luminy, Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de la Méditerranée, Marseille, France.

MHC class I-specific inhibitory receptors are expressed by a subset of memory-phenotype CD8(+) T cells. Similar to NK cells, MHC class I-specific inhibitory receptors might subserve on T cells an important negative control that participates to the prevention of autologous damage. We analyzed here human CD8(+) T cells that express the Ig-like MHC class I-specific inhibitory receptors: killer cell Ig-like receptor (KIR) and CD85j. The cell surface expression of Ig-like inhibitory MHC class I receptors was found to correlate with an advanced stage of CD8(+) T cell maturation as evidenced by the reduced proliferative potential of KIR(+) and CD85j(+) T cells associated with their high intracytoplasmic perforin content. This concomitant regulation might represent a safety mechanism to control potentially harmful cytolytic CD8(+) T cells, by raising their activation threshold. Yet, KIR(+) and CD85j(+) T cells present distinct features. KIR(+)CD8(+) T cells are poor IFN-gamma producers upon TCR engagement. In addition, KIR are barely detectable at the surface of virus-specific T cells during the course of CMV or HIV-1 infection. By contrast, CD85j(+)CD8(+) T cells produce IFN-gamma upon TCR triggering, and represent a large fraction of virus-specific T cells. Thus, the cell surface expression of Ig-like inhibitory MHC class I receptors is associated with T cell engagement into various stages of the cytolytic differentiation pathway, and the cell surface expression of CD85j or KIR witnesses to the history of qualitatively and/or quantitatively distinct T cell activation events.
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http://dx.doi.org/10.4049/jimmunol.173.12.7223DOI Listing
December 2004

Potential role of soluble CD40 in the humoral immune response impairment of uraemic patients.

Immunology 2003 Sep;110(1):131-40

UMR-CNRS 5540, Université Bordeaux 2, Bordeaux, France.

CD40/CD154 interaction is essential for both humoral and cellular immune response. We investigated whether this interaction could be altered in patients with kidney failure who are known to present an impaired immune response. To that aim, we measured the levels of the soluble form of CD40 (sCD40), which is known to interfere with CD40/CD154 interaction, in 43 chronic renal failure patients, 162 hemodialysed patients, and 83 healthy donors. Uraemic and haemodialysed patients presented a three- and fivefold increase, respectively, of the antagonist soluble form of CD40 in their serum, when compared to healthy subjects. Serum sCD40 levels correlated with those of creatinine in uraemic non-haemodialysed patients. While sCD40 is widely excreted in urine of healthy individuals, it is not eliminated by dialysis sessions on classic membranes. The return to a normal kidney function in nine haemodialysed patients who received renal transplantation, leads to a rapid decrease of serum sCD40 levels. This natural sCD40 exhibited multimeric forms and was able to inhibit immunoglobulin production by CD154-activated B lymphocytes in vitro. Furthermore, the positive correlation we observed between the serum levels of sCD40 and the deficient response to hepatitis B vaccination in uraemic patients suggests that sCD40 also compromises the humoral response in vivo.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1783029PMC
http://dx.doi.org/10.1046/j.1365-2567.2003.01716.xDOI Listing
September 2003

Membrane-anchored CD40 is processed by the tumor necrosis factor-alpha-converting enzyme. Implications for CD40 signaling.

J Biol Chem 2003 Aug 16;278(35):32801-9. Epub 2003 Jun 16.

CNRS UMR 5540, IFR 66, Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux, France.

The soluble form of CD40 (sCD40), which co-exists with the membrane-anchored form (mCD40), is a natural antagonist of mCD40/CD154 interaction. However, the mechanism leading to the production of sCD40 has never been investigated. Here, we show that the engagement of mCD40 on the surface of B lymphocytes by anti-CD40 antibody led to enhanced sCD40 release associated with decreased amounts of mCD40. This sCD40 production was not affected by vesicular traffic inhibitors but was completely blocked by a broad-spectrum synthetic metalloproteinase (MP) inhibitor (GM6001) or a membrane-anchored MP-specific inhibitor (dec-RVKR-cmk). Recombinant MP disintegrin tumor necrosis factor-alpha converting enzyme (TACE) cleaved the purified CD40 ectodomain/Fc chimeric protein in vitro, giving rise to an sCD40 form similar to that shed from B cell cultures. Moreover, spontaneous production of sCD40 by mCD40-transfected human embryonic kidney cells (constitutively expressing TACE) was enhanced by the overexpression of TACE and abrogated by co-transfection with a dominant-negative TACE mutant. These results provide strong evidence that sCD40 production is an active process regulated by the engagement of mCD40 and its proteolytic cleavage by TACE or a related MP disintegrin. Given the antagonistic activity of sCD40 on the CD40/CD154 interaction, this shedding mechanism might represent an important negative feedback control of CD40 functions.
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http://dx.doi.org/10.1074/jbc.M209993200DOI Listing
August 2003