Sylvie Garcia

A novel flow cytometric assay for quantitation and multiparametric characterization of cell-mediated cytotoxicity

Cell-mediated cytotoxicity is a crucial mechanism involved in several fundamental immunological processes such as protection against intracellular pathogens or termination of an immune response. This phenomenon is classically evaluated by the 51Cr release assay, which requires a radioactive isotope and does not permit the characterization of cells involved in the cytotoxic reaction. We describe a new flow cytometry method, developed in the context of CD95-mediated cell death, which allows the precise quantitation of cell-mediated cytotoxicity and the detection of intracellular events involved in the cytotoxic process. This assay uses a combination of two dyes, i.e. 5- (and 6-) carboxyfluorescein diacetate succinimydyl ester (CFSE) to label effector cells and 7-amino actinomycin D (7-AAD) to stain apoptotic target cells. We show that this assay is more sensitive than the 51Cr release assay and makes it possible to quantitate the percentage of cell lysis and, concomitantly, to immunophenotype target cells. It also facilitates the analysis of some events of the apoptotic pathway such as caspase activation or the expression of mitochondrial molecules. This new assay should contribute to a better understanding of the mechanisms involved in cell-mediated cytotoxicity in normal and pathological situations.

Composite cell sheets: a further step toward safe and effective myocardial regeneration by cardiac progenitors derived from embryonic stem cells.

Background— The safety and efficacy of myocardial regeneration using embryonic stem cells are limited by the risk of teratoma and the high rate of cell death. Methods and Results— To address these issues, we developed a composite construct made of a sheet of adipose tissue–derived stroma cells and embryonic stem cell–derived cardiac progenitors. Ten Rhesus monkeys underwent a transient coronary artery occlusion followed, 2 weeks later, by the open-chest delivery of the composite cell sheet over the infarcted area or a sham operation. The sheet was made of adipose tissue–derived stroma cells grown from a biopsy of autologous adipose tissue and cultured onto temperature-responsive dishes. Allogeneic Rhesus embryonic stem cells were committed to a cardiac lineage and immunomagnetically sorted to yield SSEA-1+ cardiac progenitors, which were then deposited onto the cell sheet. Cyclosporine was given for 2 months until the animals were euthanized. Preimplantation studies showed that the SSEA-1+ progenitors expressed cardiac markers and had lost pluripotency. After 2 months, there was no teratoma in any of the 5 cell-treated monkeys. Analysis of >1500 histological sections showed that the SSEA-1+ cardiac progenitors had differentiated into cardiomyocytes, as evidenced by immunofluorescence and real-time polymerase chain reaction. There were also a robust engraftment of autologous adipose tissue–derived stroma cells and increased angiogenesis compared with the sham animals. Conclusions— These data collected in a clinically relevant nonhuman primate model show that developmentally restricted SSEA-1+ cardiac progenitors appear to be safe and highlight the benefit of the epicardial delivery of a construct harboring cells with a cardiomyogenic differentiation potential and cells providing them the necessary trophic support.

A CCR4 antagonist combined with vaccines induces antigen-specific CD8+ T cells and tumor immunity against self antigens.

Regulatory T cells (Tregs) may impede cancer vaccine efficacy in hematologic malignancies and cancer. CCR4 antagonists, an emergent class of Treg inhibitor, have been shown to block recruitment of Tregs mediated by CCL22 and CCL17. Our aim was to demonstrate the ability of a CCR4 antagonist (a small chemical molecule identified in silico) when combined with vaccines to break peripheral tolerance controlled by Tregs, a prerequisite for the induction of CD8(+) T cells against self Ags. Immunization of transgenic or normal mice expressing tumor-associated self Ags (Her2/neu, OVA, gp100) with a CCR4 antagonist combined with various vaccines led to the induction of effector CD8(+) T cells and partial inhibition of tumor growth expressing self Ags in both prophylactic and therapeutic settings. The CCR4 antagonist was more efficient than cyclophosphamide to elicit anti-self CD8(+) T cells. We also showed that the population of Tregs expressing CCR4 corresponded to memory (CD44(high)) and activated (ICOS(+)) Tregs, an important population to be targeted to modulate Treg activity. CCR4 antagonist represents a competitive class of Treg inhibitor able to induce functional anti-self CD8(+) T cells and tumor growth inhibition when combined with vaccines. High expression of CCR4 on human Tregs also supports the clinical development of this strategy.

Towards a clinical use of human embryonic stem cell-derived cardiac progenitors: a translational experience

AIM There is now compelling evidence that cells committed to a cardiac lineage are most effective for improving the function of infarcted hearts. This has been confirmed by our pre-clinical studies entailing transplantation of human embryonic stem cell (hESC)-derived cardiac progenitors in rat and non-human primate models of myocardial infarction. These data have paved the way for a translational programme aimed at a phase I clinical trial. METHODS AND RESULTS The main steps of this programme have included (i) the expansion of a clone of pluripotent hESC to generate a master cell bank under good manufacturing practice conditions (GMP); (ii) a growth factor-induced cardiac specification; (iii) the purification of committed cells by immunomagnetic sorting to yield a stage-specific embryonic antigen (SSEA)-1-positive cell population strongly expressing the early cardiac transcription factor Isl-1; (iv) the incorporation of these cells into a fibrin scaffold; (v) a safety assessment focused on the loss of teratoma-forming cells by in vitro (transcriptomics) and in vivo (cell injections in immunodeficient mice) measurements; (vi) an extensive cytogenetic and viral testing; and (vii) the characterization of the final cell product and its release criteria. The data collected throughout this process have led to approval by the French regulatory authorities for a first-in-man clinical trial of transplantation of these SSEA-1(+) progenitors in patients with severely impaired cardiac function. CONCLUSION Although several facets of this manufacturing process still need to be improved, these data may yet provide a useful platform for the production of hESC-derived cardiac progenitor cells under safe and cost-effective GMP conditions.

IL-2 coordinates IL-2–producing and regulatory T cell interplay

Regulation of IL-2–producing CD4+ T cell numbers is controlled by a quorum-sensing feedback loop as regulatory T cells sense the IL-2 produced.

Potential deleterious effect of anti-viral cytotoxic lymphocyte through the CD95 (FAS/APO-1)-mediated pathway during chronic HIV infection.

The potential deleterious effect through a CD95-based pathway of anti-viral cytotoxic lymphocyte (CTL) during HIV-infection was studied. The present paper reports that a Nef specific CTL line derived from an HIV-infected person is able to kill not only Nef-expressing target cells but also CD95+ compliant Jurkat cells. The two mechanisms of cytotoxicity, i.e. perforin-vs-CD95-dependent were differentiated according to their respective Ca(2+)-dependence. The existence of the dual killing machinery in the anti-HIV CTL line was correlated with the coexpression in these cells of perforin and CD95-L molecules. A model of AIDS pathogenesis involving the deleterious effect through the CD95 pathway of the viral specific CTL response is discussed.

T Cell Apoptosis in HIV Infection: Mechanisms and Relevance for AIDS Pathogenesis

Human immunodeficiency virus (HIV) infection results in the progressive destruction of CD4 T lymphocytes, generally associated with disease progression. Despite years of investigation, the mechanisms responsible for the deletion of this lymphocyte subset are still not elucidated (Levy 1993; Weiss 1993; Gougeon 1995). CD4 cell destruction can be mediated directly by virus replication as a consequence of viral gene expression or indirectly by priming of uninfected cells to apoptosis when triggered by different agents. For example, Tat, a viral transcription factor, was shown to affect transciption of genes involved in cell survival. Tat was found to upregulate Bcl-2 expression, protecting cells from apoptosis (Zauli et al. 1995). In contrast, establishment of stable Tat-expressing cell lines or addition of exogenous Tat have been reported to sensitize cells to CD95-, anti-T-cell receptor (TCR)- and anti-CD4-induced apoptosis (Li et al. 1995; Westendorp et al. 1995). Vpr gene, required for productive infection of non-dividing cells (Hattori et al. 1990), was also recently found to induce apoptosis by blocking the cell cycle in the G2 phase (Bartz et al. 1996; Steward et al. 1997). The cytopathic effect of HIV in CD4 T cell cultures, manifested by ballooning of cells and formation of syncytia, was shown to be associated with apoptosis (Laurent-Crawford et al. 1991; Terai et al. 1991).

Population variation in NAIP functional copy number confers increased cell death upon Legionella pneumophila infection

The NAIP gene encodes an intracellular innate immunity receptor that senses flagellin. The genomic region containing NAIP presents a complex genomic organization and includes various NAIP paralogs. Here, we assessed the degree of copy number variation of the complete NAIP gene (NAIPFull) in various human populations and studied the functional impact of such variation on host cell fate using Legionella pneumophila as an infection model. We determined that African populations have a NAIPFull duplication at a higher frequency than Europeans and Asians, with an increased transcription of the gene. In addition, we demonstrated that a higher amount of the NAIPFull protein dramatically increases cell death upon infection by L. pneumophila, a mechanism that may account for increased host resistance to infection. We postulate that the NAIPFull gene duplication might have been evolutionary maintained, or even selected for, because it may confer an advantage to the host against flagellated bacteria.

Splenic Retention of Plasmodium falciparum Gametocytes To Block the Transmission of Malaria

ABSTRACT Plasmodium falciparum is transmitted from humans to Anopheles mosquito vectors via the sexual erythrocytic forms termed gametocytes. Erythrocyte filtration through microsphere layers (microsphiltration) had shown that circulating gametocytes are deformable. Compounds reducing gametocyte deformability would induce their splenic clearance, thus removing them from the blood circulation and blocking malaria transmission. The hand-made, single-sample prototype for microsphiltration was miniaturized to a 96-well microtiter plate format, and gametocyte retention in the microsphere filters was quantified by high-content imaging. The stiffening activity of 40 pharmacological compounds was assessed in microtiter plates, using a small molecule (calyculin) as a positive control. The stiffening activity of calyculin was assessed in spleen-mimetic microfluidic chips and in macrophage-depleted mice. Marked mechanical retention (80% to 90%) of mature gametocytes was obtained in microplates following exposure to calyculin at concentrations with no effect on parasite viability. Of the 40 compounds tested, including 20 antimalarials, only 5 endoperoxides significantly increased gametocyte retention (1.5- to 2.5-fold; 24 h of exposure at 1 μM). Mature gametocytes exposed to calyculin accumulated in microfluidic chips and were cleared from the circulation of macrophage-depleted mice as rapidly as heat-stiffened erythrocytes, thus confirming results obtained using the microsphiltration assay. An automated miniaturized approach to select compounds for their gametocyte-stiffening effect has been established. Stiffening induces gametocyte clearance both in vitro and in vivo. Based on physiologically validated tools, this screening cascade can identify novel compounds and uncover new targets to block malaria transmission. Innovative applications in hematology are also envisioned.

Endogenous TCR recombination in TCR Tg single RAG-deficient mice uncovered by robust in vivo T cell activation and selection.

Recombination activating gene (RAG)-deficient TCR (T Cell Receptor) Tg (transgenic) mice are routinely used as sources of monoclonal T cells. We found that after the transfer of T cells from a RAG-2-deficient 5CC7 TCR Tg mice into allogeneic hosts we recovered a population of T cells expressing diverse αβ-TCRs. In fact, in the thymus and spleen of the 5CC7 RAG-2-deficient donor mice, we detected rare T cells expressing non-Tg TCR chains. Similar observations were obtained using T cells from two other TCR transgenic strains, namely RAG-2-deficient aHY and RAG-1-deficient OT-1 mice. The sequences of the endogenous TCR transcripts suggested that gene recombination could occur, albeit quite inefficiently, in the RAG-deficient mice we used. In agreement, we evidenced rare TCR Vα and Vβ-chain transcripts in non-Tg RAG-2-deficient mice. Since in these non-Tg RAG-deficient mice no mature T cells could ever be found, our findings suggested a role for the TCR Tg in rescuing rare recombined endogenous chains. Robust T-cell activation by the allogeneic environment favored the selection and expansion of the rare cells expressing endogenous TCRs. Potential mechanisms involved in the recombination of the endogenous TCR chains in the different strains of RAG-deficient mice used, and in particular the possibility of RAG-1 hypomorphism due to an incomplete knocking out procedure, are discussed. Our findings have important experimental implications for studies using TCR-Tg RAG-deficient cells as monoclonal T cell populations.