Benoı̂t Salomon

CD28 induces immunostimulatory signals in dendritic cells via CD80 and CD86.

Bidirectional signaling along the B7–CTLA-4 coreceptor pathway enables reciprocal conditioning of T cells and dendritic cells. Although T cells can instruct dendritic cells to manifest tolerogenic properties after CTLA-4 engagement of B7, such a B7-mediated signaling is not known to occur in response to CD28. Here we show that mouse dendritic cells were induced by soluble CD28 to express interleukin 6 and interferon-γ. Production of interleukin 6 required B7-1 (CD80), B7-2 (CD86) and p38 mitogen-activated protein kinase and prevented interferon-γ-driven expression of immunosuppressive tryptophan catabolism. In vivo, an adjuvant activity of soluble CD28 was demonstrated as enhanced T cell-mediated immunity to tumor and self peptides and protection against microbial and tumor challenge. Thus, different ligands of B7 can signal dendritic cells to express functionally distinct effector responses.

Ex Vivo-Expanded CD4+CD25+ Immunoregulatory T Cells Prevent Graft-versus-Host-Disease by Inhibiting Activation/Differentiation of Pathogenic T Cells

CD4+CD25+ immunoregulatory T cells (Tregs) can be administered to inhibit graft-vs-host disease (GVHD) while preserving graft-vs-leukemia activity after allogeneic bone marrow transplantation in mice. Preclinical studies suggest that it is necessary to infuse as many Tregs as conventional donor T cells to achieve a clinical effect on GVHD. Thus, it would be necessary to expand Tregs ex vivo before transplantation. Two strategies have been proposed: expansion of Tregs stimulated by anti-CD3/CD28-coated microbeads for polyclonal activation or by host-type allogeneic APCs for selecting Tregs specific for host Ags. In this study, we describe the mechanisms by which ex vivo-expanded Tregs act on donor T cells to prevent GVHD in mice. We demonstrate that expanded Tregs strongly inhibited the division, expansion, and differentiation of donor T cells, with a more pronounced effect with Tregs specific for host Ags. These latter cells permit the efficient and durable control of GVHD and favor immune reconstitution.

IL-4 confers NK stimulatory capacity to murine dendritic cells: a signaling pathway involving KARAP/DAP12-triggering receptor expressed on myeloid cell 2 molecules.

Dendritic cells (DC) regulate NK cell functions, but the signals required for the DC-mediated NK cell activation, i.e., DC-activated NK cell (DAK) activity, remain poorly understood. Upon acute inflammation mimicked by LPS or TNF-α, DC undergo a maturation process allowing T and NK cell activation in vitro. Chronic inflammation is controlled in part by Th2 cytokines. In this study, we show that IL-4 selectively confers to DC NK but not T cell stimulatory capacity. IL-4 is mandatory for mouse bone marrow-derived DC grown in GM-CSF (DCGM/IL-4) to promote NK cell activation in the draining lymph nodes. IL-4-mediated DAK activity depends on the KARAP/DAP12-triggering receptor expressed on myeloid cell 2 signaling pathway because: 1) gene targeting of the adaptor molecule KARAP/DAP12, a transmembrane polypeptide with an intracytoplasmic immunoreceptor tyrosine-based activation motif, suppresses the DCGM/IL-4 capacity to activate NK cells, and 2) IL-4-mediated DAK activity is significantly blocked by soluble triggering receptor expressed on myeloid cell 2 Fc molecules. These data outline a novel role for Th2 cytokines in the regulation of innate immune responses through triggering receptors expressed on myeloid cells.

Regulatory and Effector T Cell Activation Levels Are Prime Determinants of In Vivo Immune Regulation

Little is known about the in vivo conditions in which CD4+CD25+ regulatory T cells (Treg) exert their suppressive effect in nonlymphopenic mice. To this end, we analyzed Treg-mediated suppression of expansion and cytokine production at different levels of Ag-specific CD4+CD25− T cell activation. Using Ab-mediated depletion of endogenous Treg, we show that basal immunosuppression is dependent on effector T cell activation. These polyclonal Treg, which were poorly activated in our immunization conditions, were effective in weak but not high T cell activation context. In contrast, the same immunization conditions led to proliferation of cotransferred Ag-specific Treg. Those efficiently inhibited T cell proliferation and cytokine production even in strong T cell activation context. Interestingly, Treg selectively suppressed expansion or cytokine production depending on the experimental approach. The importance of the immune context for efficient suppression is further supported by the observation that Treg depletion exacerbated diabetes of NOD mice only at the early stage of the disease. Overall, our study suggests that Treg-mediated suppression depends on the relative activation of Treg and effector T cells in vivo. This balance may be a critical factor in the regulation of immune responses.

A truncated herpes simplex virus thymidine kinase phosphorylates thymidine and nucleoside analogs and does not cause sterility in transgenic mice.

Dividing eukaryotic cells expressing the herpes simplex virus type 1 thymidine kinase (TK) gene are sensitive to the cytotoxic effect of nucleoside analogs such as acyclovir or ganciclovir (GCV). Transgenic mice with cell-targeted expression of this conditional toxin have been used to create animals with temporally controlled cell-specific ablation. In these animal models, which allow the study of the physiological importance of a cell type, males are sterile. In this study, we showed that this phenomenon is due to testis-specific high-level expression of short TK transcripts initiated mainly upstream of the second internal ATG of the TK gene. This expression is DNA methylation independent. To obtain a suicide gene that does not cause male infertility, we generated and analyzed the properties of a truncated TK (delta TK) lacking the sequences upstream of the second ATG. We showed that when expressed at sufficient levels, the functional properties of delta TK are similar to those of TK in terms of thymidine or GCV phosphorylation. This translated into a similar GCV-dependent toxicity for delta TK- or TK-expressing cells, both in vitro and in transgenic mice. However, delta TK behaved differently from TK in two ways. First, it did not cause sterility in delta TK transgenic males. Second, low-level delta TK RNA expression did not confer sensitivity to GCV. The uses of delta TK in cell-specific ablation in transgenic mice and in gene therapy are discussed.

A POPULATION OF INTERSTITIAL CELLS IN THE ANTERIOR PITUITARY WITH A HEMATOPOIETIC ORIGIN AND A RAPID TURNOVER : A RELATIONSHIP WITH FOLLICULO-STELLATE CELLS?

The non-hormone secreting folliculo-stellate (FS) cells in the anterior pituitary (AP) appear heterogeneous. Some of these cells have been described as having a neuroectodermal origin and being glial, while some others have been suggested to be monocytic or dendritic cells (DC). We have analyzed here the hematopoietic origin of interstitial cell populations in the AP. In the rat AP, the relative densities of S100+ FS cells and major histocompatibility complex (MHC) class II-expressing DC-like cells show a parallel increase in the postnatal period between the age of 3 weeks to 2 months. We first looked for the presence of donor derived cells in the AP of lethally irradiated bone marrow (BM)-transplanted rats. Donor derived myeloid cells carrying the n haplotype of the MHC class I antigen (RT1.An) reacting with the OX27 moAb, could not be detected in the AP three months after transplantation. It appeared, however, that OX27+ DC-like cells a-priori were virtually absent from the rat AP. Therefore this transplantation model was not suitable for our studies. We then turned to a model of transgenic mice expressing a suicide gene in subpopulations of dendritic cells. Mice were lethally irradiated and received a BM transplant from the transgenic animals, with or without a treatment with ganciclovir (GCV) that specifically kills the dividing cells expressing the suicide gene. This model has already been used to identify and delete mainly dendritic cell populations, viz N418+ and ER-BMDM1+ dendritic cells in the marginal zones of the spleen and in the thymic medulla. We observed in the AP a 30% reduction of the ER-BMDM1+ FS-like cells and a 50-100% reduction of interstitial cells expressing the F4/80, Mac-1 and MOMA-1 markers in the mice receiving the transgenic BM and treated with GCV, compared to control mice that were not treated with GCV or that received non-transgenic BM. When a treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF) was initiated during the GCV treatment, we observed an even stronger reduction of the above-mentioned interstitial cell populations. These data indicate that in the mouse AP a population of stellate cells exists with a hematopoietic origin, that expresses markers of myeloid cells, and that has a rapid turnover.

Conditional ablation of dendritic cells in mice: comparison of two animal models.

Dendritic cells (DC) knockout in mice should be a powerful mean to appreciate the role of these cells in physiological or pathological situations. In order to generate such an animal model, we used a stategy based on the DC-specific expression of a suicide gene in transgenic mice. We used the herpes simplex virus type 1-thymidine kinase (HSV1-TK) which allows conditional ablation of dividing HSV1-TK expressing cells by converting the non toxic ganciclovir (GCV) into a toxic metabolite1,2. DC expression of HSV1-TK in transgenic mice was attempted with the HIV-LTR promoter which had been previously shown to preferentially direct the expression of a CAT transgene in Langerhans cells3. We generated LTR-TK transgenic mice expressing the HSV1-TK gene under the control of the HIV-LTR promoter. We showed a low but preferential expression of the transgene in DC, leading to DC depletion in spleen and thymus following GCV administration4. This depletion was often associated with a thymic atrophy and a wasting syndrome. To rule out the possibility that a transgene expression leakiness in different tissues could be responsible for these pathological findings, we analysed the effects of GCV treatment in syngenic normal mice engrafted with transgenic bone marrow cells. In this situation, HSV1-TK expression is limited to hematopoietic cells.

The Role of Dendritic Cells in the Transport of HIV to Lymph Nodes Analysed in Mouse

The migration route of dendritic cells (DC) from peripheral tissues to lymph nodes (1) and their capacity to pass HIV infection to T cells (2–4) support a critical role for DC in the early events of HIV infection. It is assumed, but not yet demonstrated, that DC are the first HIV target cells in the mucosa and that their migration to draining lymph nodes will result in the transmission of HIV to T lymphocytes. Interestingly, this can be studied in a murine model, although murine cells are not susceptible to HIV infection. Indeed, (i) murine DC pre-incubated with HIV are able to transfer it to human T lymphocytes as efficiently as human DC (4); (ii) murine DC can be manipulated in vitro, re-injected, and their in vivo migration can be analysed.

Prevention of Graft-Versus-Host disease in mice using a suicide gene expressed in T-lymphocytes

Alloreactive T cells present in a bone marrow transplant are responsible for graft-versus-host disease (GVHD), but their depletion is associated with impaired engraftment, immunosuppression, and loss of the graft-versus-leukemia effect. We developed a therapeutic strategy against GVHD based on the selective destruction of these alloreactive T cells, while preserving a competent T-cell pool of donor origin. We generated transgenic mice expressing in their T lymphocytes the Herpes simplex type 1 thymidine kinase (TK) suicide gene that allows the destruction of dividing T cells by a ganciclovir treatment. T cells expressing the TK transgene were used to generate GVHD in irradiated bone marrow grafted mice. We show that a short 7-day ganciclovir treatment, initiated at the time of bone marrow transplantation, efficiently prevented GVHD in mice receiving TK-expressing T cells. These mice were healthy and had a normal survival. They maintained a T-cell pool of donor origin that responded normally to in vitro stimulation with mitogens or third party alloantigens, but were tolerant to recipient alloantigens. Our experimental system provides the proof of concept for a therapeutic strategy of GVHD prevention using genetically engineered T cells.