Human CD90 identifies Th17/Tc17 T cell subsets that are depleted in HIV-infected patients.

By revisiting CD90, a GPI-anchored glycoprotein, we show that CD90 is expressed by a subset of CD4(+) and CD8(+) human T cells. CD4(+)CD90(+) cells share similarities with Th17 cells because they express the Th17-specific transcription factor RORC2 and produce IL-17A. CD4(+)CD90(+) cells are activated memory T cells that express the gut mucosal markers CCR6, CD161, and the α(4) and ß(7) integrins. Compared with CD90-depleted CCR6(+) memory Th17 cells, CD4(+)CD90(+) cells express higher levels of IL-22 and proinflammatory cytokines (IL-6, TNF-α and GM-CSF), but they produce lower levels of IL-21 and no IL-9. Analyses of CD8(+)CD90(+) cells reveal that they express RORC2 and are able to produce higher levels of IL-17A, IL-22, and CCL20 compared with CD90-depleted CD8(+) cells. These data show that CD90 identifies Th17 and Tc17 cells with a peculiar cytokine profile. Studies of circulating CD90(+) cells in HIV patients show that CD90(+) cells are decreased with an imbalance of the CD4(+)CD90(+)/regulatory T cell ratio in nontreated patients compared with treated patients and healthy donors. Overall, human CD90 identifies a subset of Th17 and Tc17 cells within CD4(+) and CD8(+) T cells, respectively, which are depleted during HIV infection.

A human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes.

Human coronaviruses are associated with upper respiratory tract infections that occasionally spread to the lungs and other organs. Although airway epithelial cells represent an important target for infection, the respiratory epithelium is also composed of an elaborate network of dendritic cells (DCs) that are essential sentinels of the immune system, sensing pathogens and presenting foreign antigens to T lymphocytes. In this report, we show that in vitro infection by human coronavirus 229E (HCoV-229E) induces massive cytopathic effects in DCs, including the formation of large syncytia and cell death within only few hours. In contrast, monocytes are much more resistant to infection and cytopathic effects despite similar expression levels of CD13, the membrane receptor for HCoV-229E. While the differentiation of monocytes into DCs in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 requires 5 days, only 24 h are sufficient for these cytokines to sensitize monocytes to cell death and cytopathic effects when infected by HCoV-229E. Cell death induced by HCoV-229E is independent of TRAIL, FasL, tumor necrosis factor alpha, and caspase activity, indicating that viral replication is directly responsible for the observed cytopathic effects. The consequence of DC death at the early stage of HCoV-229E infection may have an impact on the early control of viral dissemination and on the establishment of long-lasting immune memory, since people can be reinfected multiple times by HCoV-229E.

Sensitivity of human pleural mesothelioma to oncolytic measles virus depends on defects of the type I interferon response.

Attenuated measles virus (MV) is currently being evaluated as an oncolytic virus in clinical trials and could represent a new therapeutic approach for malignant pleural mesothelioma (MPM). Herein, we screened the sensitivity to MV infection and replication of twenty-two human MPM cell lines and some healthy primary cells. We show that MV replicates in fifteen of the twenty-two MPM cell lines. Despite overexpression of CD46 by a majority of MPM cell lines compared to healthy cells, we found that the sensitivity to MV replication did not correlate with this overexpression. We then evaluated the antiviral type I interferon (IFN) responses of MPM cell lines and healthy cells. We found that healthy cells and the seven insensitive MPM cell lines developed a type I IFN response in presence of the virus, thereby inhibiting replication. In contrast, eleven of the fifteen sensitive MPM cell lines were unable to develop a complete type I IFN response in presence of MV. Finally, we show that addition of type I IFN onto MV sensitive tumor cell lines inhibits replication. These results demonstrate that defects in type I IFN response are frequent in MPM and that MV takes advantage of these defects to exert oncolytic activity.

Natural oncolytic activity of live-attenuated measles virus against human lung and colorectal adenocarcinomas.

Lung and colorectal cancers are responsible for approximately 2 million deaths each year worldwide. Despite continual improvements, clinical management of these diseases remains challenging and development of novel therapies with increased efficacy is critical to address these major public health issues. Oncolytic viruses have shown promising results against cancers that are resistant to conventional anticancer therapies. Vaccine strains of measles virus (MV) exhibit such natural antitumor properties by preferentially targeting cancer cells. We tested the ability of live-attenuated Schwarz strain of MV to specifically infect tumor cells derived from human lung and colorectal adenocarcinomas and demonstrated that live-attenuated MV exhibits oncolytic properties against these two aggressive neoplasms. We also showed that Schwarz MV was able to prevent uncontrollable growth of large, established lung and colorectal adenocarcinoma xenografts in nude mice. Moreover, MV oncolysis is associated with in vivo activation of caspase-3 in colorectal cancer model, as shown by immunohistochemical staining. Our results provide new arguments for the use of MV as an antitumor therapy against aggressive human malignancies.

A chimeric measles virus with a lentiviral envelope replicates exclusively in CD4+/CCR5+ cells.

We generated a replicating chimeric measles virus in which the hemagglutinin and fusion surface glycoproteins were replaced with the gp160 envelope glycoprotein of simian immunodeficiency virus (SIVmac239). Based on a previously cloned live-attenuated Schwarz vaccine strain of measles virus (MV), this chimera was rescued at high titers using reverse genetics in CD4+ target cells. Cytopathic effect consisted in the presence of large cell aggregates evolving to form syncytia, as observed during SIV infection. The morphology of the chimeric virus was identical to that of the parent MV particles. The presence of SIV gp160 as the only envelope protein on chimeric particles surface altered the cell tropism of the new virus from CD46+ to CD4+ cells. Used as an HIV candidate vaccine, this MV/SIVenv chimeric virus would mimic transient HIV-like infection, benefiting both from HIV-like tropism and the capacity of MV to replicate in dendritic cells, macrophages and lymphocytes.

Initial depletion of regulatory T cells: the missing solution to preserve the immune functions of T lymphocytes designed for cell therapy.

We investigated the causes of the altered functionality of T cells cultured under conditions designed for cell and gene therapy and the strategies to prevent their defects. We first showed that human T cells cultured for 6 days with anti-CD3 +/- anti-CD28 antibodies and interleukin-2 presented a 50% decrease of their proliferative responses to allogeneic or recall antigens. Similarly, day-6 cultured murine T cells completely lost their capacity to reject allogeneic skin grafts and to provoke graft-versus-host disease (GVHD) when infused into irradiated semi-allogeneic mice. Interestingly, injection of higher amounts of cultured T cells restored GVHD induction. Moreover, depletion of CD25+ cells prior to T-cell cultures can prevent these deficiencies both in mice and humans. Therefore, we demonstrated that culture conditions used for T-cell therapy preferentially activated and expanded regulatory T cells (Treg's). Thus, we showed that dividing cells sorted from T-cell cultures strongly suppressed the proliferation of autologous T cells in response to allogeneic stimulation. An increased detection of Foxp3 at mRNA and protein levels in the cultures confirmed the Treg expansion. Overall, we demonstrate that T-cell cultures promote Treg expansion over effector T cells, leading to deleterious immune functions, and that this imbalance can be prevented by an initial depletion of CD25+ cells.

Lentiviral transduction of human hematopoietic cells by HIV-1- and SIV-based vectors containing a bicistronic cassette driven by various internal promoters.

BACKGROUND: Lentiviral gene transfer into hematopoietic cells has been mostly optimized with vectors carrying a single reporter gene. For many clinical applications, lentiviral vectors should contain more than one gene because transduced cells should be enriched by a selectable marker or killed for safety reasons after use. Thus, we compared various vectors containing a bicistronic cassette driven by different ubiquitous promoters for their ability to transduce human T-lymphocytes, CD34+-cells, and dendritic cells (DCs) derived from CD34+-cells or monocytes. METHODS: We designed HIV or SIV constructs containing a bicistronic cassette composed of two reporter genes (thy1/GFP) linked by an internal ribosome entry site sequence and driven by the cytomegalovirus (CMV) or elongation factor 1alpha (EF1alpha) promoters. The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) was or not inserted within the constructs, the Vpx accessory protein was or not used for SIV vectors. Target cells were infected at the same multiplicity of infection, transduction efficiency was analyzed both by flow cytometry and vector integration. RESULTS: For T-cells, HIV-based vectors/WPRE+ in which the thy1/GFP cassette was driven by the EF1alpha promoter were more efficient than SIV-based vectors. For CD34+-cells and CD34+-derived DCs, better thy1/GFP expression was achieved when the CMV promoter drove the cassette inserted into HIV-based vectors/WPRE+. Conversely, for monocyte-derived DCs, the cassette yielded better thy1/GFP expression when inserted into SIV-based vectors/WPRE+ and driven by the CMV or EF1alpha promoters, the use of Vpx significantly improving the expression levels. CONCLUSIONS: Our results provide guidelines for improving the transduction of T-cells, CD34+-cells or DCs with lentiviral bicistronic vectors designed for clinical applications.

Efficient transduction and selection of human T-lymphocytes with bicistronic Thy1/HSV1-TK retroviral vector produced by a human packaging cell line.

BACKGROUND: T-cells expressing the HSV1-TK suicide gene can be used for the control of graft-versus-host disease following allogeneic stem cell transplantation. To develop clinical trials based on such a strategy, we have generated under good manufacturing procedures a novel 'split genome' human packaging cell line (1704 cells). METHODS: To minimize the risk of generating replication-competent retroviruses, pol was truncated to remove sequences overlapping with env. To improve retroviral infection and selection of transduced T-cells, high titers of GALV-pseudotyped retroviral particles harboring a bicistronic Thy1-IRES-TK vector coding for the CD90 GPI-anchored membrane molecule were produced by 1704 cells. RESULTS: Using 1704 cell supernatant and an optimized transduction protocol, approximately 50% of primary T-cells were transduced and could then be purified (approximately 95%) using clinical-grade immunomagnetic beads directed against CD90. Over 96% of these OKT3/IL-2-activated CD90(+)-selected T-cells were killed by ganciclovir. Cell proliferation and cytokine production of transduced T-cells and HLA-restricted cytotoxicity of transduced T-cell clones were identical to those of their non-transduced counterparts cultured under the same conditions. CONCLUSIONS: GALV-pseudotyped retroviral particles harboring a bicistronic Thy1-IRES-TK vector allow efficient transduction and rapid selection of human T-cells under conditions applicable for clinical trials using the new human 1704 packaging cell line.