Publications by authors named "Olivier Toutirais"

27 Publications

  • Page 1 of 1

Tissue plasminogen activator worsens experimental autoimmune encephalomyelitis by complementary actions on lymphoid and myeloid cell responses.

J Neuroinflammation 2021 Feb 20;18(1):52. Epub 2021 Feb 20.

UNICAEN, INSERM, GIP Cyceron, Institut Blood and Brain @Caen-Normandie (BB@C), UMR-S U1237, Physiopathology and Imaging of Neurological Disorders (PhIND), Normandie Univ, Caen, France.

Background: Tissue plasminogen activator (tPA) is a serine protease involved in fibrinolysis. It is released by endothelial cells, but also expressed by neurons and glial cells in the central nervous system (CNS). Interestingly, this enzyme also contributes to pathological processes in the CNS such as neuroinflammation by activating microglia and increasing blood-brain barrier permeability. Nevertheless, its role in the control of adaptive and innate immune response remains poorly understood.

Methods: tPA effects on myeloid and lymphoid cell response were studied in vivo in the mouse model of multiple sclerosis experimental autoimmune encephalomyelitis and in vitro in splenocytes.

Results: tPA animals exhibited less severe experimental autoimmune encephalomyelitis than their wild-type counterparts. This was accompanied by a reduction in both lymphoid and myeloid cell populations in the spinal cord parenchyma. In parallel, tPA increased T cell activation and proliferation, as well as cytokine production by a protease-dependent mechanism and via plasmin generation. In addition, tPA directly raised the expression of MHC-II and the co-stimulatory molecules CD80 and CD86 at the surface of dendritic cells and macrophages by a direct action dependent of the activation of epidermal growth factor receptor.

Conclusions: Our study provides new insights into the mechanisms responsible for the harmful functions of tPA in multiple sclerosis and its animal models: tPA promotes the proliferation and activation of both lymphoid and myeloid populations by distinct, though complementary, mechanisms.
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http://dx.doi.org/10.1186/s12974-021-02102-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7897384PMC
February 2021

Natalizumab in Multiple Sclerosis Treatment: From Biological Effects to Immune Monitoring.

Front Immunol 2020 24;11:549842. Epub 2020 Sep 24.

Laboratory of Immunology, Department of Biology, CHU Caen Normandie, Caen, France.

Multiple sclerosis is a chronic demyelinating disease of the central nervous system (CNS) with an autoimmune component. Among the recent disease-modifying treatments available, Natalizumab, a monoclonal antibody directed against the alpha chain of the VLA-4 integrin (CD49d), is a potent inhibitor of cell migration toward the tissues including CNS. It potently reduces relapses and active brain lesions in the relapsing remitting form of the disease. However, it has also been associated with a severe infectious complication, the progressive multifocal leukoencephalitis (PML). Using the standard protocol with an injection every 4 weeks it has been shown by a close monitoring of the drug that trough levels soon reach a plateau with an almost saturation of the target cell receptor as well as a down modulation of this receptor. In this review, mechanisms of action involved in therapeutic efficacy as well as in PML risk will be discussed. Furthermore the interest of a biological monitoring that may be helpful to rapidly adapt treatment is presented. Indeed, development of anti-NAT antibodies, although sometimes unapparent, can be detected indirectly by normalization of CD49d expression on circulating mononuclear cells and might require to switch to another drug. On the other hand a stable modulation of CD49d expression might be useful to follow the circulating NAT levels and apply an extended interval dose scheme that could contribute to limiting the risk of PML.
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http://dx.doi.org/10.3389/fimmu.2020.549842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541830PMC
September 2020

Autoimmune encephalitis mediated by B-cell response against N-methyl-d-aspartate receptor.

Brain 2020 10;143(10):2957-2972

Normandie Univ, UNICAEN, INSERM, U1237, PhIND "Physiopathology and Imaging of Neurological Disorders", Institut Blood and Brain at Caen-Normandie, Cyceron, Caen, France.

Anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis is a neuropsychiatric disease characterized by an antibody-mediated autoimmune response against NMDAR. Recent studies have shown that anti-NMDAR antibodies are involved in the pathophysiology of the disease. However, the upstream immune and inflammatory processes responsible for this pathogenic response are still poorly understood. Here, we immunized mice against the region of NMDA receptor containing the N368/G369 amino acids, previously implicated in a pathogenic response. This paradigm induced encephalopathy characterized by blood-brain barrier opening, periventricular T2-MRI hyperintensities and IgG deposits into the brain parenchyma. Two weeks after immunization, mice developed clinical symptoms reminiscent of encephalitis: anxiety- and depressive-like behaviours, spatial memory impairment (without motor disorders) and increased sensitivity to seizures. This response occurred independently of overt T-cell recruitment. However, it was associated with B220+ (B cell) infiltration towards the ventricles, where they differentiated into CD138+ cells (plasmocytes). Interestingly, these B cells originated from peripheral lymphoid organs (spleen and cervical lymphoid nodes). Finally, blocking the B-cell response using a depleting cocktail of antibodies reduced the severity of symptoms in encephalitis mice. This study demonstrates that the B-cell response can lead to an autoimmune reaction against NMDAR that drives encephalitis-like behavioural impairments. It also provides a relevant platform for dissecting encephalitogenic mechanisms in an animal model, and enables the testing of therapeutic strategies targeting the immune system in anti-NMDAR encephalitis.
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http://dx.doi.org/10.1093/brain/awaa250DOI Listing
October 2020

[Mechanisms of radiation-induced lymphopenia and therapeutic impact].

Bull Cancer 2020 Jul - Aug;107(7-8):813-822. Epub 2020 May 22.

Centre François-Baclesse/ARCHADE, département de radiothérapie, 3, avenue General Harris, 14000 Caen, France; Laboratoire de physique corpusculaire IN2P3/ENSICAEN - UMR6534, Caen, France; Normandie University, UNICAEN, Caen, France. Electronic address:

Radiation induced lymphopenia is frequent and can be severe and durable. Although lymphocytes have long been known as highly radiosensitive cells, it is poorly characterized. Radiation-induced lymphopenia seems to affect lymphocyte subpopulations differently and seems to be influenced by radiation modalities. The depth and duration of lymphopenia depend on the location of the irradiation and the volumes of treatment. Importantly, radiation-induced lymphopenia has been associated with poorer prognosis in several tumor types. The knowledge about radiation-induced lymphopenia might lead to a rethinking of the modalities of radiotherapy and new approaches to restore lymphocytes counts.
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http://dx.doi.org/10.1016/j.bulcan.2020.04.009DOI Listing
August 2020

Presence of T cells directed against CD20-derived peptides in healthy individuals and lymphoma patients.

Cancer Immunol Immunother 2019 Oct 7;68(10):1561-1572. Epub 2019 Sep 7.

Sorbonne Université, Sorbonne Paris Cité, Université Paris Descartes, Université Paris Diderot, Inserm UMRS 1138, "Cancer, Immune Control and Escape" Laboratory, Centre de Recherche des Cordeliers, Paris, France.

Preclinical and clinical studies have suggested that cancer treatment with antitumor antibodies induces a specific adaptive T cell response. A central role in this process has been attributed to CD4 T cells, but the relevant T cell epitopes, mostly derived from non-mutated self-antigens, are largely unknown. In this study, we have characterized human CD20-derived epitopes restricted by HLA-DR1, HLA-DR3, HLA-DR4, and HLA-DR7, and investigated whether T cell responses directed against CD20-derived peptides can be elicited in human HLA-DR-transgenic mice and human samples. Based on in vitro binding assays to recombinant human MHC II molecules and on in vivo immunization assays in H-2 KO/HLA-A2-DR1 transgenic mice, we have identified 21 MHC II-restricted long peptides derived from intracellular, membrane, or extracellular domains of the human non-mutated CD20 protein that trigger in vitro IFN-γ production by PBMCs and splenocytes from healthy individuals and by PBMCs from follicular lymphoma patients. These CD20-derived MHC II-restricted peptides could serve as a therapeutic tool for improving and/or monitoring anti-CD20 T cell activity in patients treated with rituximab or other anti-CD20 antibodies.
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http://dx.doi.org/10.1007/s00262-019-02389-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6805815PMC
October 2019

HLA-Class II Artificial Antigen Presenting Cells in CD4 T Cell-Based Immunotherapy.

Front Immunol 2019 17;10:1081. Epub 2019 May 17.

Inserm U1237, Physiopathology and Imaging of Neurological Disorders, Caen University Hospital, Caen, France.

CD4 T cells differentiate into various T helper subsets characterized by distinct cytokine secreting profiles that confer them effector functions adapted to a variety of infectious or endogenous threats. Regulatory CD4 T cells are another specialized subset that plays a fundamental role in the maintenance of immune tolerance to self-antigens. Manipulating effector or regulatory CD4 T cells responses is a promising immunotherapy strategy for, respectively, chronical viral infections and cancer, or severe autoimmune diseases and transplantation. Adoptive cell therapy (ACT) is an emerging approach that necessitates defining robust and efficient methods for the expansion of antigen-specific T cells then infused into patients. To address this challenge, artificial antigen presenting cells (AAPCs) have been developed. They constitute a reliable and easily usable platform to stimulate and amplify antigen-specific CD4 T cells. Here, we review the recent advances in understanding the functions of CD4 T cells in immunity and in immune tolerance, and their use for ACT. We also describe the characteristics of different AAPC models and the way to improve their stimulating functions. Finally, we discuss the potential interest of these AAPCs, both as fundamental tools to decipher CD4 T cell responses and as reagents to generate clinical grade antigen-specific CD4 T cells for immunotherapy.
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http://dx.doi.org/10.3389/fimmu.2019.01081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533590PMC
September 2020

The ADAMTS13 peptide is a dominant HLA-DR1-restricted CD4 T-cell epitope.

Haematologica 2017 11 27;102(11):1833-1841. Epub 2017 Jul 27.

Institut National de la Santé et de la Recherche Médicale (INSERM), Unité Mixte de Recherche en Santé (UMR S) 1138, Centre de Recherche des Cordeliers, Equipe Immunopathology and Therapeutic Immunointervention, Paris, France.

Acquired thrombotic thrombocytopenic purpura is a rare and severe disease characterized by auto-antibodies directed against "A Disintegrin And Metalloproteinase with Thrombospondin type 1 repeats, 13 member" (ADAMTS13), a plasma protein involved in hemostasis. Involvement of CD4 T cells in the pathogenesis of the disease is suggested by the IgG isotype of the antibodies. However, the nature of the CD4 T-cell epitopes remains poorly characterized. Here, we determined the HLA-DR-restricted CD4 T-cell epitopes of ADAMTS13. Candidate T-cell epitopes were predicted and binding affinities were confirmed in competitive enzyme-linked immunosorbent assays. ADAMTS13-reactive CD4 T-cell hybridomas were generated following immunization of HLA-DR1 transgenic mice (Sure-L1 strain) and used to screen the candidate epitopes. We identified the ADAMTS13 peptide as the single immunodominant HLA-DR1-restricted CD4 T-cell epitope. This peptide is located in the CUB2 domain of ADAMTS13. It was processed by dendritic cells, stimulated CD4 T cells from Sure-L1 mice and was recognized by CD4 T cells from an HLA-DR1-positive patient with acute thrombotic thrombocytopenic purpura. Interestingly, the ADAMTS13 peptide demonstrated promiscuity towards HLA-DR11 and HLA-DR15. Our work paves the way towards the characterization of the ADAMTS13-specific CD4 T-cell response in patients with thrombotic thrombocytopenic purpura using ADAMTS13-loaded HLA-DR tetramers.
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http://dx.doi.org/10.3324/haematol.2015.136671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5664387PMC
November 2017

Increased frequencies of circulating and tumor-resident Vδ1 T cells in patients with diffuse large B-cell lymphoma.

Leuk Lymphoma 2018 01 31;59(1):187-195. Epub 2017 May 31.

b Normandie University, UNICAEN, INSERM U919, Sérine Protéases et Physiopathologie de l'unité Neurovasculaire , Caen , France.

Gamma-delta (γδ) T cells contribute to the innate immune response against cancer. In samples of 20 patients upon DLBCL diagnosis, we found that Vδ1 T cells were the major γδ T cell subset in tumors and PBMCs of patients, while Vδ2 T cells were preponderant in PBMCs of healthy subjects. Interestingly, the germinal center (GC) subtype was associated with an increase in Vδ1 T cells in tumors, whereas the non-GC subtype was associated with a lower frequency of γδ T cells. While circulating Vδ1 T cells of patients or HSs mostly exhibited a naïve phenotype, the majority of tumor Vδ1 T cells showed a central memory phenotype. Resident or circulating γδ T cells from patients were not functionally impaired since they produced high levels of IFN-γ. Collectively, our findings are in favor of γδ T cell activation in tumors and open new perspectives for their modulation in DLBCL immunotherapy.
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http://dx.doi.org/10.1080/10428194.2017.1321751DOI Listing
January 2018

Artificial antigen-presenting cells expressing HLA class II molecules as an effective tool for amplifying human specific memory CD4(+) T cells.

Immunol Cell Biol 2016 08 29;94(7):662-72. Epub 2016 Feb 29.

Inserm U919, Serine Proteases and Pathophysiology of the Neurovascular Unit, Caen, France.

Owing to their multiple immune functions, CD4(+) T cells are of major interest for immunotherapy in chronic viral infections and cancer, as well as for severe autoimmune diseases and transplantation. Therefore, standardized methods allowing rapid generation of a large number of CD4(+) T cells for adoptive immunotherapy are still awaited. We constructed stable artificial antigen-presenting cells (AAPCs) derived from mouse fibroblasts. They were genetically modified to express human leukocyte antigen (HLA)-DR molecules and the human accessory molecules B7.1, Intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-3 (LFA-3). AAPCs expressing HLA-DR1, HLA-DR15 or HLA-DR51 molecules and loaded with peptides derived from influenza hemagglutinin (HA), myelin basic protein (MBP) or factor VIII, respectively, activated specific CD4(+) T-cell clones more effectively than Epstein-Barr virus (EBV)-transformed B cells. We also showed that AAPCs were able to take up and process whole Ag proteins, and present epitopes to specific T cells. In primary cultures, AAPCs loaded with HA peptide allowed generation of specific Th1 lymphocytes from healthy donors as demonstrated by tetramer and intracellular cytokine staining. Although AAPCs were less effective than autologous peripheral blood mononuclear cells (PBMCs) to stimulate CD4(+) T cells in primary culture, AAPCs were more potent to reactivate and expand memory Th1 cells in a strictly Ag-dependent manner. As the availability of autologous APCs is limited, the AAPC system represents a stable and reliable tool to achieve clinically relevant numbers of CD4(+) T cells for adoptive immunotherapy. For fundamental research in immunology, AAPCs are also useful to decipher mechanisms involved in the development of human CD4 T-cell responses.
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http://dx.doi.org/10.1038/icb.2016.25DOI Listing
August 2016

[Nectins and nectin-like receptors DNAM-1 and CRTAM: new ways for tumor escape].

Med Sci (Paris) 2014 May 13;30(5):537-43. Epub 2014 Jun 13.

Inserm U919, GIP (groupe d'intérêt public) Cyceron, université de Caen Basse-Normandie, 14074 Caen, France.

Nectin and nectin-like (Necl) are cell adhesion molecules expressed in various tumors. They were alternatively reported as involved in tumor suppressor or oncogenic functions that led to their use as histological or serological cancer markers. Gene inactivation in lung carcinoma but overexpression in leukemia were reported for Necl-2. DNAM-1 and CRTAM are emerging NK receptors of immune cells that were described to interact with nectin and Necl. DNAM-1, constitutively expressed by CD8(+) T cells, NK or γδ T lymphocytes, is a ligand of Necl-5. It participates to tumor immunosurveillance promoting Necl-5 expressing tumor cell lysis. CRTAM, only expressed after lymphocyte activation, is a ligand of Necl-2. Engagement of CRTAM with Necl-2 has opposite effects depending on the type of lymphocyte. For NK or CD8(+) T cells, it promotes cytotoxicity and IFNγ secretion favoring immunosurveillance. By contrast, CRTAM/Necl-2 interaction triggers cell death of activated TVg9Vd2 γδ T cells favoring immune escape. Nectin and Necl-mediated interactions appear to be crucial for the delicate balance between tumor escape and antitumor response.
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http://dx.doi.org/10.1051/medsci/20143005017DOI Listing
May 2014

CRTAM receptor engagement by Necl-2 on tumor cells triggers cell death of activated Vγ9Vδ2 T cells.

J Immunol 2013 May 25;190(9):4868-76. Epub 2013 Mar 25.

INSERM UMR991 "Foie, Métabolisme et Cancer," F-35033 Rennes, France.

Human Vγ9Vδ2 T cells exert potent in vitro and in vivo antitumor activities, making them promising candidates for immunotherapy strategies. Recognition of tumor cells by Vγ9Vδ2 T cells requires engagement of the TCR and/or NK receptors. Recently, one of the novel NK receptors, the class I-restricted T cell-associated molecule (CRTAM), has been described to promote cytotoxic function of NK cells and to lead to IFN-γ secretion by CD8(+) T cells through interaction with its ligand, Necl-2. A better understanding of the role of CRTAM in Vγ9Vδ2 T cell functions is highly relevant to optimize innate-like T cell-based cancer immunotherapy. In this article, we report that CRTAM is transiently expressed on activated Vγ9Vδ2 T lymphocytes following TCR engagement. However, CRTAM-Necl-2 interaction does not modify the cytotoxic function or IFN-γ secretion of Vγ9Vδ2 T cells. The expression of CRTAM in activated Vγ9Vδ2 T cells is quickly downregulated following interaction with Necl-2 on tumor cells. Of interest, CRTAM is concurrently acquired at the cell surface of Necl-2(+) tumor cells through Vγ9Vδ2 T cell membrane capture. Finally, we highlight that coculture experiments with tumor cells expressing Necl-2 result in significant cell death of CRTAM(+) Vγ9Vδ2 T cells. CRTAM-mediated cell death is dependent on an autophagic process, but not on apoptosis or necroptosis, as attested by the expression of characteristic markers and blocking experiments with specific inhibitors. On the basis of these findings, we propose that Necl-2 on tumor cells represents a new tumor counterattack mechanism and a potential target to improve efficiency of γδ T cell-based immunotherapy.
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http://dx.doi.org/10.4049/jimmunol.1202596DOI Listing
May 2013

Sensitization of ovarian carcinoma cells with zoledronate restores the cytotoxic capacity of Vγ9Vδ2 T cells impaired by the prostaglandin E2 immunosuppressive factor: implications for immunotherapy.

Int J Cancer 2012 Aug 21;131(4):E449-62. Epub 2011 Dec 21.

Université de Rennes 1, Rennes, France.

Epithelial ovarian cancer (EOC) usually spreads into the peritoneal cavity, thereby providing an opportunity for intraperitoneal adoptive immunotherapy with Vγ9Vδ2 T lymphocytes, a T cell subpopulation endowed with high lytic properties against tumor cells. However, previous studies have reported that Vγ9Vδ2 T cells fail to expand from peripheral blood mononuclear cells in one-third of patients with cancer. Here, from a cohort of 37 patients with EOC, a multiple correspondence analysis identified three populations, one of which was not suitable for Vγ9Vδ2 T-cell adoptive therapy. Interestingly, the ineligible patients were identified based on the frequency of Vγ9Vδ2 T cells in their peripheral blood and the patients' age. The average time to tumor recurrence was also found to be significantly different between the three populations, suggesting that the innate immune response is involved in EOC prognosis. A dramatic decrease in the lytic properties of Vγ9Vδ2 T cells occurred following incubation with ascitic supernatant and was found to be associated with reduced perforin/granzyme degranulation. Prostaglandin E2, but not IL-6, IL-10, VEGF or TGF-β, showed immunosuppressive effects in Vγ9Vδ2 T cells. Interestingly, our results emphasize that pretreating ovarian tumor cells with zoledronate partially reverses the immunosuppressive effects of ovarian cancer-associated ascites and restores a high level of lytic activity. These data sustain that optimal Vγ9Vδ2 T-cell adoptive immunotherapy previously requires counteracting the tumor immunosuppressive microenvironment. Altogether, our findings provide a rationale for clinically evaluating Vγ9Vδ2 T-cell adoptive immunotherapy with intraperitoneal carcinomatosis presensitization by zoledronate in patients with EOC.
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http://dx.doi.org/10.1002/ijc.27353DOI Listing
August 2012

CD49d expression as a promising biomarker to monitor natalizumab efficacy.

J Neurol Sci 2012 Mar 1;314(1-2):138-42. Epub 2011 Nov 1.

CHU de Caen, Department of Neurology, Caen, F-14000, France.

Natalizumab (Tysabri™), a monoclonal antibody against the α4-integrin of VLA-4 (CD49d) antigen of leukocytes, is highly effective in multiple sclerosis (MS). The most common reason for treatment failure is the development of neutralizing antibodies (NAbs). According to health authorities Nabs testing is recommended in case of relapse or repeated infusion reactions. However NAbs may develop in clinically asymptomatic patients. In this study we investigated if CD49d expression could serve as a biomarker of natalizumab bioavailability and treatment response. In a cohort of 49 natalizumab treated relapsing-remitting MS, followed over 2 years, CD49d expression was determined on peripheral blood mononuclear cells (PBMCs) before each infusion and compared to NAbs and serum natalizumab levels. In a majority of patients (41/49) the CD49d expression in PBMCs was strongly inhibited (>50%) after the first infusion and maintained at low levels throughout the treatment period. In contrast, in eight patients (16%) there was an early recovery of CD49d expression to pre-treatment levels related to NABs development. While three cases experienced hypersensitivity reactions, three others were identified solely on the basis of an undiminished level of CD49d, with neither infusion reaction nor clinical worsening. These 3 patients had very high levels of NAbs and no detectable serum natalizumab. Two additional patients had early but transient recovery of CD49d expression. These patients had low levels of transient Nabs and returned to significant CD49d inhibition after few natalizumab infusions. We suggest that monitoring of CD49d expression can be used as a surrogate biomarker of natalizumab efficiency. If the CD49d expression is sustained at pre-treatment levels, patients should be tested for persistent NAbs and considered for treatment interruption.
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http://dx.doi.org/10.1016/j.jns.2011.10.005DOI Listing
March 2012

Stroke and the immune system: from pathophysiology to new therapeutic strategies.

Lancet Neurol 2011 May;10(5):471-80

Institut National de la Santé et de la Recherche Médicale (INSERM) U919, Serine Proteases and Pathophysiology of the Neurovascular Unit, UMR CNRS 6232 Ci-NAPs, Cyceron, Université de Caen Basse-Normandie, Caen, France.

Stroke is the second most common cause of death worldwide and a major cause of acquired disability in adults. Despite tremendous progress in understanding the pathophysiology of stroke, translation of this knowledge into effective therapies has largely failed, with the exception of thrombolysis, which only benefits a small proportion of patients. Systemic and local immune responses have important roles in causing stroke and are implicated in the primary and secondary progression of ischaemic lesions, as well as in repair, recovery, and overall outcome after a stroke. However, potential therapeutic targets in the immune system and inflammatory responses have not been well characterised. Development of novel and effective therapeutic strategies for stroke will require further investigation of these pathways in terms of their temporal profile (before, during, and after stroke) and risk-to-benefit therapeutic ratio of modulating them.
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http://dx.doi.org/10.1016/S1474-4422(11)70066-7DOI Listing
May 2011

Aminobisphosphonate-pretreated dendritic cells trigger successful Vgamma9Vdelta2 T cell amplification for immunotherapy in advanced cancer patients.

Cancer Immunol Immunother 2010 Nov 26;59(11):1611-9. Epub 2010 Jun 26.

EE341 Biothérapies Innovantes, Faculté de Médecine, Université de Rennes 1, Rennes, France.

Hepatocellular carcinoma (HCC) and colorectal carcinoma with hepatic metastases (mCRC) are cancers with poor prognosis and limited therapeutic options. New approaches are needed and adoptive immunotherapy with Vgamma9Vdelta2 T lymphocytes represents an attractive strategy. Indeed, Vgamma9Vdelta2 T cells were shown to exhibit efficient lytic activity against various human tumor cell lines, and in vitro Vgamma9Vdelta2 T expansion protocol based on single phosphoantigen stimulation could be easily performed for healthy donors. However, a low proliferative response of Vgamma9Vdelta2 T cells was observed in about half of the cancer patients, leading to an important limitation in the development of Vgamma9Vdelta2 T cell-based immunotherapy. Here, for the first time in the context of cancer patients, Vgamma9Vdelta2 T cell expansions were performed by co-culturing peripheral blood mononuclear cell (PBMCs) with autologous dendritic cells (DCs) pretreated with aminobisphosphonate zoledronate. For patients not responding to the conventional culture protocol, co-culture of PBMC with zoledronate-pretreated DCs induced strong cell expansion and allowed reaching a minimal rate of purity of 70% of Vgamma9Vdelta2 T cells. The potent immunostimulatory activity of zoledronate-treated DCs was associated with higher amount of isopentenyl pyrophosphate (IPP) in the culture and was correlated with better ability to activate Vgamma9Vdelta2 T cells as measured by IFN-gamma production. Moreover, we demonstrated that the cytotoxic level of Vgamma9Vdelta2 T cells against freshly autologous tumor cells isolated from patients could be significantly increased by pretreating the tumor cells with zoledronate. Thus, this method of generating Vgamma9Vdelta2 T cells leads eligible for Vgamma9Vdelta2 T cell adoptive immunotherapy the HCC and mCRC patients.
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http://dx.doi.org/10.1007/s00262-010-0887-0DOI Listing
November 2010

Immunostimulatory properties of dendritic cells after Leishmania donovani infection using an in vitro model of liver microenvironment.

PLoS Negl Trop Dis 2010 Jun 8;4(6):e703. Epub 2010 Jun 8.

Université de Rennes 1, Rennes, France.

Background: Recent advances demonstrated that liver dendritic cells (DCs) promote immunologic hyporesponsiveness that may contribute to hepatic tolerance. Although there has been significant work on the phenotypic and functional roles of such DCs, the impact of liver microenvironment on the immune properties of infected DC is still poorly explored, probably because of the limitations of modelization.

Methodology/principal Findings: Here, we hypothesized that DC tolerogenic properties have an impact on the antimicrobial response, particularly during the infection by the protozoan parasite Leishmania donovani. Indeed, a lymphocytic Th2 environment was reported to favour the growth and proliferation of L. donovani. We first modelized an adequate monocyte-differentiated DC model, either in rat liver epithelial cell- or in a human hepatic non-parenchymal cell-conditioned medium in order to infect them further. We established that DCs differentiated in a hepatic microenvironment displayed a CD14+/CD16+/CD123+ phenotype, secreted low IL-12p70 and had an impaired capacity to stimulate allogeneic T lymphocyte proliferation and IFNgamma secretion. We then infected DCs with L. donovani in the in vitro-defined hepatic microenvironment. The infection of hepatic DCs restored their capacity to stimulate allogeneic T-cell proliferation and to induce lymphocytic secretion of IFNgamma. Such characteristics were recently shown to favour granuloma formation in mice liver.

Conclusions/significance: Our results suggest that the specific immunostimulatory properties of infected hepatic DCs might amplify the granuloma maturation, which warrants the effective control of infection in the liver during visceral leishmaniasis.
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http://dx.doi.org/10.1371/journal.pntd.0000703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2882335PMC
June 2010

HLA-A*0201-restricted CEA-derived peptide CAP1 is not a suitable target for T-cell-based immunotherapy.

J Immunother 2010 May;33(4):402-13

Inserm U614, Faculty of Medicine, Institute for Biomedical Research, University Hospital, Rouen, France.

Carcinoembryonic antigen (CEA) is a potential target for antigen-specific immunotherapy, as it is frequently overexpressed in human carcinomas. Moreover, an epitope derived from CEA, designated CAP1 (YLSGANLNL), has been proposed as naturally processed and presented by tumors in the human leukocyte antigen (HLA)-A*0201 context. Our aim was to fully characterize and assess the clinical relevance of the HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) response against CEA. Stable and potent artificial antigen presenting cells (AAPCs) were used to evaluate T-cell response against CEA. These cells efficiently activate CTLs against tumor-derived epitopes after transduction with the antigenic peptides or full-length proteins. We found that AAPCs genetically modified to express CAP1, the agonist peptide CAP1-6D, or the whole CEA protein were not able to activate CAP1-specific CTLs from HLA-A*0201+ healthy donors or patients with colorectal carcinoma, even after multiple stimulations. In addition, we showed that a CAP1-specific T-cell clone, obtained after multiple stimulations of T cells of a HLA-A*0201+ healthy donor in vitro with autologous antigen presenting cells, recognized CEA(-) HLA-A*0201+ tumors transduced with a minigene encoding CAP1 but failed to react against HLA-A*0201+ tumor cells expressing CEA. Finally, AAPCs expressing the whole CEA protein did not induce any specific CTL response against CEA+ HLA-A*0201+ tumor cells highlighting the potential difficulty of mounting an efficacious T-cell response against this autoantigen. Altogether, our data indicate that CAP1 is not efficiently processed and presented by CEA+ tumor cells, and therefore, is not an appropriate target for T-cell-based immunotherapy.
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http://dx.doi.org/10.1097/CJI.0b013e3181d366daDOI Listing
May 2010

[Tgammadelta lymphocytes in oncology: unconventional killer lymphocytes].

Med Sci (Paris) 2010 Feb;26(2):185-91

EE 341 Biothérapies innovantes, Université de Rennes 1, Faculté de médecine de Rennes, Rennes Cedex 35043, France. veronique.catros@ univ-rennes1.fr

Gamma delta T cells are a distinct subset of CD3+ T cells featuring both T cells receptors that are encoded by Vgamma- and Vdelta- gene segments and characteristics of innate immunity. In human blood, 80% of those express Vgamma9Vdelta2-TCRs that are specific for conserved non peptidic compound, phosphoantigens (PAgs). Vgamma9Vdelta2 T cells recognize in vitro a wide array of transformed cells and are activated in vivo in various tumor. Owing to their ability to directly kill tumor cells and produce inflammatory cytokines (such as IFN-gamma) boosting antitumor properties of other immune effector cells, gamma delta T cells contribute to protective immunity against cancers. These observations, and the recent availability of synthetic clinical grade PAg or pharmacological inducers of PAg (e.g. aminobisphosphonates) able to trigger Vgamma9Vdelta2 T cell, have fostered development of new gammadelta T cell-based therapeutic strategies, which are depicted in this review.
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http://dx.doi.org/10.1051/medsci/2010262185DOI Listing
February 2010

DNAX accessory molecule-1 (CD226) promotes human hepatocellular carcinoma cell lysis by Vgamma9Vdelta2 T cells.

Eur J Immunol 2009 May;39(5):1361-8

EE 341 Biothérapies Innovantes, Faculté de Médecine, Université de Rennes 1, Rennes, France.

Human Vgamma9Vdelta2 T lymphocytes can be activated by nonpeptidic antigens such as the mevalonate pathway-derived isopentenyl pyrophosphate or synthetic phosphoantigen such as bromohydrin pyrophosphate. They display a strong cytotoxic activity against several tumor types, including hepatocellular carcinoma (HCC). Little is known about the mechanisms underlying Vgamma9Vdelta2 T-cell recognition of tumor cells, but there is strong evidence that activating NK receptors play a role in gammadelta T-cell cytotoxicity. In this study, we showed that the two NK receptors DNAX accessory molecule-1 (DNAM-1) and CD96 were expressed by Vgamma9Vdelta2 T cells. The ligands Nectin-like-5 specific of both DNAM-1 and CD96, and also Nectin-2, an additional ligand of DNAM-1, were present on all HCC cell lines analyzed. Furthermore, we demonstrated by mAb-mediated masking experiments that cytotoxicity against HCC cells as well as IFN-gamma production in gammadelta T cells were dependent on DNAM-1. Our experiments indicated that Nectin-like-5 but not Nectin-2 was involved in DNAM-1-dependent gammadelta T-cell functions. We did not reveal a role for CD96 in the killing of HCC cells. Finally, we showed by combined mAb-mediated blockade that DNAM-1 and NKG2D could cooperate in the cell lysis of HCC.
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http://dx.doi.org/10.1002/eji.200838409DOI Listing
May 2009

Activation of tumor-specific T cells by dendritic cells expressing the NY-ESO-1 antigen after transfection with the cationic lipophosphoramide KLN5.

J Gene Med 2008 Jun;10(6):628-36

Université de Rennes 1, Faculté de Médecine, UPRES 3891, Rennes, France.

Background: Genetic modification of human monocyte-derived dendritic cells (DC) with cDNA sequences encoding tumor-associated antigens (TAA) is a promising strategy for cancer immunotherapy. The present study aimed to develop a nonviral gene transfer method based on the use of the cationic lipophosphoramide reagent, KLN-5, as an alternative to the commonly used viral vectors.

Methods: First, the efficiency of KLN5 for gene transfection into DC was investigated using the green fluorescent protein (GFP) reporter gene. The highest transfection efficiency/cell viability ratio was determined by flow cytometry. Next, DC were transfected with a plasmid encoding NY-ESO-1, a TAA expressed in numerous cancers, according to the transfection protocol previously established with the GFP reporter. Transfected DC were then co-cultured with a CD8+ NY-ESO-1 specific HLA-A*02.01 T cell clone to control their ability to correctly process and present the corresponding epitope in the HLA-A*02.01 context. Finally, T cell activation was assessed via flow cytometry-based detection of interferon-gamma production.

Results: An optimal KLN5/plasmid DNA ratio allowing both significant transgene expression and high viability of DC could be determined. Under the established experimental conditions, antigen processing and presentation of the immunodominant (SLLMWITQC(157-165)) epitope in the HLA-A*0201 context was demonstrated by activation of the NY-ESO-1-specific CD8+ T cell clone.

Conclusions: KLN5-based gene transfection into DC allows the efficient induction of TAA presentation and may thus represent a novel attractive nonviral approach for cancer vaccination.
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http://dx.doi.org/10.1002/jgm.1188DOI Listing
June 2008

Soluble HLA-G molecules impair natural killer/dendritic cell crosstalk via inhibition of dendritic cells.

Eur J Immunol 2008 Mar;38(3):742-9

UPRES EA 3889, Faculté de Médecine, Université de Rennes 1, Rennes, France.

HLA-G molecules are known to exert immunosuppressive action on DC maturation and on NK cells, and can in consequence inhibit respectively T cell responses and NK cytolysis. In this study, we show that monocyte-derived DC, differentiated in the presence of GM-CSF and IL-4, are sensitive to soluble (s) HLA-G molecules during LPS/IFN-gamma maturation as demonstrated by the decrease of CD80 and HLA-DR expressions and IL-12 secretion. Moreover, DC pretreated with sHLA-G were found to activate NK/DC crosstalk less than non-treated DC. Early activation of NK cells co-cultured with autologous DC was diminished as assessed by CD69 expression. The IFN-gamma production was impaired whereas a slight inhibition of the NK cell cytotoxicity against Daudi cell line was observed. Since sHLA-G is expressed in grafts or sites of tumour proliferation, its indirect action on NK cells via DC could constitute a pathway of early inhibition for both innate and specific immune responses.
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http://dx.doi.org/10.1002/eji.200736918DOI Listing
March 2008

Vgamma9Vdelta2 T cell-mediated recognition of human solid tumors. Potential for immunotherapy of hepatocellular and colorectal carcinomas.

Cancer Immunol Immunother 2008 Apr 1;57(4):531-9. Epub 2007 Sep 1.

UPRES EA 3891, Faculté de Médecine de Rennes, 2 Avenue du Pr Léon Bernard, CS34317, 35043, Rennes cedex, France.

Introduction: Vgamma9Vdelta2 T lymphocytes are reported to participate in the anti-tumor immune surveillance in human. They are known to recognize phosphoantigens and molecules expressed on cells undergoing neoplasic transformation. In this study, we investigated phenotype and anti-tumor cytotoxicity of ex vivo expanded Vgamma9Vdelta2 T cells in view of adoptive immunotherapy.

Materials And Methods: Experiments were performed with peripheral blood samples from eleven patients [six colorectal carcinoma (CRC), four hepatocellular carcinoma (HCC), one sarcoma] and sixteen healthy donors.

Results/discussion: Ex vivo expansion of Vgamma9Vdelta2 T cells could be achieved by a single dose of phosphoantigen, either bromohydrin pyrophosphate or zoledronate, and supported by exogenous IL-2. After 2 weeks, expanded Vgamma9Vdelta2 T lymphocytes acquired the effector memory phenotype CD45RA(-)CD45RO(high)CD27(-). They expressed NKG2D and CD161 and the proinflammatory CXCR3 and CCR5 chemokine receptors. Vgamma9Vdelta2 T cells displayed a strong lytic activity toward a broad panel of tumor cell lines or primary cultures. Interestingly, HCC and CRC primary cells could be lysed by autologous Vgamma9Vdelta2 T cells whereas autologous normal cells were not sensitive to the lysis. mAbs blocking assays demonstrated that TCR was the most important receptor involved in the lysis of tumor cells. However, NKG2D receptor could deliver a costimulatory signal enhancing the lysis of HCC and CRC tumors expressing MICA/B. Treatment of tumor cells by the mevalonate pathway inhibitor, zoledronate, enhanced the killing of both HCC and CRC. Expansion index of Vgamma9Vdelta2 T cells was in similar levels in healthy donors or in cancer patients and total expansion was suitable for adoptive immunotherapy.

Conclusion: These results provide a rationale for the clinical evaluation of Vgamma9Vdelta2 T lymphocytes in HCC and CRC.
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http://dx.doi.org/10.1007/s00262-007-0391-3DOI Listing
April 2008

In vitro antitumor lymphocyte generation using dendritic cells and innate immunity mechanisms as tumor cell treatments.

Anticancer Res 2007 Jul-Aug;27(4B):2385-92

UPRES EA 3891, Faculté de Médecine de Rennes, France.

Dendritic cells play a central role in the initiation and regulation of acquired and innate immunity, playing an important role in immunosurveillance and antitumor reaction. This reaction is mediated by effector cells and soluble factors. We chose to investigate four dendritic cell loading methods by mimicking innate immunity mechanisms and using whole tumor cell treatments in order to stimulate lymphocytes: sodium hypochlorite, TNFalpha and IFNgamma and IgG opsonization. These methods were compared in an HLA.A2 model of healthy donors and with the M74 melanoma cell line. Treated tumor cell-loaded DC were able to increase proliferation of lymphocytes. Moreover, a CTL population was stimulated, as shown by their specific cytotoxicity against tumor cells (with w6/32 antibody assays), against MelanA/MART-1 loaded T2 cells and using MelanA/MART-1 tetramer. IgG opsonization seemed to be less efficient than other tumor cell treatments. These loaded DC, or the obtained effector cells, could be interesting for therapeutic applications in antitumor cell therapy.
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September 2007

Low TCR avidity and lack of tumor cell recognition in CD8(+) T cells primed with the CEA-analogue CAP1-6D peptide.

Cancer Immunol Immunother 2007 Dec 13;56(12):1979-91. Epub 2007 Jun 13.

Unit of Immunotherapy of Human Tumors, IRCCS Istituto Nazionale Tumori, Via Venezian 1, 20133, Milan, Italy.

The use of "altered peptide ligands" (APL), epitopes designed for exerting increased immunogenicity as compared with native determinants, represents nowadays one of the most utilized strategies for overcoming immune tolerance to self-antigens and boosting anti-tumor T cell-mediated immune responses. However, the actual ability of APL-primed T cells to cross-recognize natural epitopes expressed by tumor cells remains a crucial concern. In the present study, we show that CAP1-6D, a superagonist analogue of a carcinoembriyonic antigen (CEA)-derived HLA-A*0201-restricted epitope widely used in clinical setting, reproducibly promotes the generation of low-affinity CD8(+) T cells lacking the ability to recognized CEA-expressing colorectal carcinoma (CRC) cells. Short-term T cell cultures, obtained by priming peripheral blood mononuclear cells from HLA-A*0201(+) healthy donors or CRC patients with CAP1-6D, were indeed found to heterogeneously cross-react with saturating concentrations of the native peptide CAP1, but to fail constantly lysing or recognizing through IFN- gamma release CEA(+)CRC cells. Characterization of anti-CAP1-6D T cell avidity, gained through peptide titration, CD8-dependency assay, and staining with mutated tetramers (D227K/T228A), revealed that anti-CAP1-6D T cells exerted a differential interaction with the two CEA epitopes, i.e., displaying high affinity/CD8-independency toward the APL and low affinity/CD8-dependency toward the native CAP1 peptide. Our data demonstrate that the efficient detection of self-antigen expressed by tumors could be a feature of high avidity CD8-independent T cells, and underline the need for extensive analysis of tumor cross-recognition prior to any clinical usage of APL as anti-cancer vaccines.
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http://dx.doi.org/10.1007/s00262-007-0342-zDOI Listing
December 2007

Ex vivo expansion of antitumor cytotoxic lymphocytes with tumor-associated antigen-loaded dendritic cells.

Anticancer Res 2005 May-Jun;25(3B):2177-85

UPRES 3891, Faculté de Médecine de Rennes, France.

Cell therapy with lymphocytes is an attractive approach for cancer immunotherapy. Methods to generate ex vivo effector cells directed against whole autologous tumor antigens are under investigation. Our procedure involved stimulation of autologous lymphocytes with antigen-pulsed dendritic cells (DC). Experimental conditions were established with DC, matured with TNFa, LPS and CD40L, from healthy donors and the M74 melanoma cell line. DC were pulsed with either irradiated, apoptotic or necrotic tumor cells or fused with tumor cells. Increase of lymphocyte cytotoxicity and IFNy production were repeatedly observed with tumor cell-loaded DC. Stimulation of tumor-associated antigen-specific lymphocytes was clearly shown. MelanA-MART1 (dominant melanoma-associated antigen) tetramer staining revealed a high frequency of specific T cells. Lymphocytes were able to efficiently lyse MelanA-MART1-pulsed T2 target and MelanA-expressing target cells (M74) after CD56+ cells depletion. We confirmed with other tumor cell lines that this DC-mediated procedure induced activation of cytolytic lymphocytes.
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November 2005

Constitutive expression of TGF-bêta1, interleukin-6 and interleukin-8 by tumor cells as a major component of immune escape in human ovarian carcinoma.

Eur Cytokine Netw 2003 Oct-Dec;14(4):246-55

UPRESA 2261, Faculté des Sciences Biologiques et Médicales de Rennes, France.

Tumors could use several mechanisms to coexist with the host's immune system or to protect themselves from an immune response. Thus, insufficient expression of cell surface molecules on tumor cells, which are important for T cell recognition or activation, could lead to induction of a state of tolerance. Tumor cells could also produce cytokines that would inhibit the immune response and allow tumor progression. Here, we studied, in vitro, the cell surface expression of immunologically important molecules in seven ovarian carcinoma (OVCA) cell lines and the constitutive expression of cytokines. All OVCA cell lines expressed MHC class I molecules, ICAM-1 and LFA-3 adhesion molecules, necessary to induce a specific cytotoxic T-cell response, as well as the CD40 costimulatory molecules. Conversely, the lack of the dominant costimulatory molecules, CD80 (B7.1) and CD86 (B7.2) could be a possible explanation of poor immunogenicity of OVCA tumors. Immunosuppressive TGF-beta1 was detected at the mRNA level in all cell lines but was weakly secreted in supernatants. By contrast, IL-10 was never found. Most of them constitutively produced IL-8 and IL-6, two cytokines known as tumor promoting factors whereas the proinflammatory cytokines TNF-alpha, IL-1beta and GM-CSF were rarely produced. Data from this study could be useful for designing new strategies of immunotherapy to improve immunogenicity and/or limit protumor cytokine production.
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August 2004

Cytotoxic effector cells with antitumor activity can be amplified ex vivo from biopsies or blood of patients with renal cell carcinoma for cell therapy use.

Cancer Immunol Immunother 2003 Nov 15;52(11):699-707. Epub 2003 Jul 15.

UPRES 2261, Faculté de Médecine de Rennes, 2 Avenue du Professeur Léon Bernard, 35043 Rennes, France.

Adoptive immunotherapy with antitumor effector cells is an attractive therapeutic approach in metastatic renal cell carcinoma (RCC). The aim of the work was to enhance in vitro activation of lymphocytes with optimal cytotoxic activity against tumor cells. We evaluated a procedure based on the use of dendritic cells (DCs) loaded with irradiated tumor cells (DC-Tu) to stimulate lymphocytes. Experimental conditions were established with cells from healthy donors and melanoma cell lines. Procedures were then applied to cells from RCC patients. A total of 30 tumor biopsies, 14 proximal lymph nodes, and 17 peripheral blood samples from 30 patients were used. When lymphocytes were stimulated in vitro with DC-Tu, they responded to tumor cells with an increased cytolytic activity for all the assays with donor cells (n=18). For RCC patients, DC-Tu stimulation improved the final cytotoxic activity in only half of the assays (16/31). When significantly enhanced (>10%, n=8), responder cells resulted in a final 43% cytotoxicity against autologous RCC cells. Mechanism of lysis was at least in part class I mediated. Effector cells have no lytic activity against normal renal cells. Percentage of cells with regulatory T-cell phenotype was not found to be enhanced in the DC-Tu stimulated lymphocytes. Individual differences were observed in the characteristics of DCs generated from RCC patients in contrast to that observed in donors and could explain why lymphocyte stimulation was not improved by DC-Tu in half of the RCC assays. T-cell spreading was suitable for a therapeutic use (>10(9) cells) irrespective of the procedure (with or without DC-Tu stimulation) or the tissular origin of lymphocytes from patients. Data show that precursors of selective antitumor effector cells are present in patients with RCC and can be amplified in vitro either with or without DC-Tu stimulation. One of these populations could be chosen for an adoptive transfer immunotherapy.
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http://dx.doi.org/10.1007/s00262-003-0412-9DOI Listing
November 2003