Separating the Wheat from the Chaff among HLA-DQ Eplets.

In transplantation, anti-HLA Abs, especially targeting the DQ locus, are well-known to lead to rejection. These Abs identified by Luminex single Ag assays recognize polymorphic amino acids on HLA, named eplets. The HLA Eplet Registry included 83 DQ eplets, mainly deduced from amino acid sequence alignments, among which 66 have not been experimentally verified. Because eplet mismatch load may improve organ allocation and transplant outcomes, it is imperative to confirm the genuine reactivity of eplets to validate this approach. Our study aimed to confirm 29 nonverified eplets, using adsorption of eplet-positive patients' sera on human spleen mononuclear cells and on transfected murine cell clones expressing a unique DQα- and DQß-chain combination. In addition, we compared the positive beads patterns obtained in the two commercially available Luminex single Ag assays. Among the 29 nonverified DQ eplets studied, 24 were confirmed by this strategy, including the 7 DQα eplets 40E, 40ERV, 75I, 76 V, 129H, 129QS, and 130A and the 17 DQß eplets 3P, 23L, 45G, 56L, 57 V, 66DR, 66ER, 67VG, 70GT, 74EL, 86A, 87F, 125G, 130R, 135D, 167R, and 185I. However, adsorption results did not allow us to conclude for the five eplets 66IT, 75S, 160D, 175E, and 185T.

Tissue-plasminogen activator effects on the phenotype of splenic myeloid cells in acute inflammation.

Tissue-plasminogen activator (tPA) is a serine protease well known for its fibrinolytic function. Recent studies indicate that tPA could also modulate inflammation via plasmin generation and/or by receptor mediated signalling in vitro. However, the contribution of tPA in inflammatory processes in vivo has not been fully addressed. Therefore, using tPA-deficient mice, we have analysed the effect of lipopolysaccharide (LPS) challenge on the phenotype of myeloid cells including neutrophils, macrophages and dendritic cells (DCs) in spleen. We found that LPS treatment upregulated the frequency of major histocompatibility class two (MHCII+) macrophages but also, paradoxically, induced a deep downregulation of MHCII molecule level on macrophages and on conventional dendritic cells 2 (cDC2). Expression level of the CD11b integrin, known as a tPA receptor, was upregulated by LPS on MHCII+ macrophages and cDC2, suggesting that tPA effects could be amplified during inflammation. In tPA-/- mice under inflammatory conditions, expression of costimulatory CD86 molecules on MHCII+ macrophages was decreased compared to WT mice, while in steady state the expression of MHCII molecules was higher on macrophages. Finally, we reported that tPA deficiency slightly modified the phenotype of DCs and T cells in acute inflammatory conditions. Overall, our findings indicate that in vivo, LPS injection had an unexpectedly bimodal effect on MHCII expression on macrophages and DCs that consequently might affect adaptive immunity. tPA could also participate in the regulation of the T cell response by modulating the levels of CD86 and MHCII molecules on macrophages.

Targeting NMDA Receptors at the Neurovascular Unit: Past and Future Treatments for Central Nervous System Diseases.

The excitatory neurotransmission of the central nervous system (CNS) mainly involves glutamate and its receptors, especially N-methyl-D-Aspartate receptors (NMDARs). These receptors have been extensively described on neurons and, more recently, also on other cell types. Nowadays, the study of their differential expression and function is taking a growing place in preclinical and clinical research. The diversity of NMDAR subtypes and their signaling pathways give rise to pleiotropic functions such as brain development, neuronal plasticity, maturation along with excitotoxicity, blood-brain barrier integrity, and inflammation. NMDARs have thus emerged as key targets for the treatment of neurological disorders. By their large extracellular regions and complex intracellular structures, NMDARs are modulated by a variety of endogenous and pharmacological compounds. Here, we will present an overview of NMDAR functions on neurons and other important cell types involved in the pathophysiology of neurodegenerative, neurovascular, mental, autoimmune, and neurodevelopmental diseases. We will then discuss past and future development of NMDAR targeting drugs, including innovative and promising new approaches.

Roles of the tissue-type plasminogen activator in immune response.

The COVID-19 pandemic has once again brought to the forefront the existence of a tight link between the coagulation/fibrinolytic system and the immunologic processes. Tissue-type plasminogen activator (tPA) is a serine protease with a key role in fibrinolysis by converting plasminogen into plasmin that can finally degrade fibrin clots. tPA is released in the blood by endothelial cells and hepatocytes but is also produced by various types of immune cells including T cells and monocytes. Beyond its role on hemostasis, tPA is also a potent modulator of inflammation and is involved in the regulation of several inflammatory diseases. Here, after a brief description of tPA structure, we review its new functions in adaptive immunity focusing on T cells and antigen presenting cells. We intend to synthesize the recent knowledge on proteolysis- and receptor-mediated effects of tPA on immune response in physiological and pathological context.

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

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.

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

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.

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

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.

[Mechanisms of radiation-induced lymphopenia and therapeutic impact]. / Mécanismes de la lymphopénie radio-induite et implications thérapeutiques.

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.

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

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.

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

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 in vitro 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.

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

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.

The ADAMTS131239-1253 peptide is a dominant HLA-DR1-restricted CD4+ T-cell epitope.

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, 13th 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 in silico 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 ADAMTS131239-1253 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 ADAMTS131239-1253 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 ADAMTS131239-1253-loaded HLA-DR tetramers.

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

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.

[Nectins and nectin-like receptors DNAM-1 and CRTAM: new ways for tumor escape]. / Les récepteurs de nectines/nectines-like DNAM-1 et CRTAM - Immuno-surveillance ou échappement tumoral ?

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.

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

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.

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.

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-ß, 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.

CD49d expression as a promising biomarker to monitor natalizumab efficacy.

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.

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

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.

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

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.

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

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.