Immunobiologie [Animal Physiology and Immunobiology]

Etude des premières étapes des réponses immunes : rôle des cellules dendritiques [Study of the first steps of immune responses: role of dendritic cells]
Since their discovery in 1973 by Zanvil Cohn and Ralph Steinman, dendritic cells (DCs) have gained increased interest among immunologists, as these cells display unique properties aimed at inducing an immune response against antigens that invade the organism. They perform multiple tasks with high efficiency, and appear to determine the specificity, magnitude and nature (Th1/Th2) of the immune response.In the mouse, two subclasses of dendritic cells have been described that differ by their CD8á expression and their localization in lymphoid organs.We have assessed the function of both subclasses in vivo by injecting purified DC, pulsed extracorporeally with antigen, into the footpads of syngeneic mice and analyzing the immune response of lymph node cells. Our data show that both DC subsets efficiently prime antigen-specific T cells in vivo, and direct the development of distinct T helper cell populations. Administration of CD8á- DC induces a Th2-type response, whereas injection of CD8á+ DC leads to Th1 differentiation. Th1 priming is dependent on IL-12 production by DCs.These findings suggest that the nature of the DC that presents the antigen to naive T cells may dictate the class selection of the adaptive immune response. Experiments are under way to assess the role of various DC populations in activating the recently discovered Th17 cell population, which plays a role in autoimmune and allergic inflammation. More recently, a number of reports have shown that, in addition to their well known stimulatory properties, DCs may play a major role in peripheral tolerance. It is still unclear whether a distinct subtype or activation status of DC exists which promotes the differentiation of suppressor rather than effector T cells from naive precursors. We tested whether the naturally occurring CD4+CD25+ regulatory T cells may control immune responses induced by DCs in vivo. We found that the development of IFN-a-producing cells and cytotoxic T cells was strongly enhanced in the absence of regulatory T cells. By contrast, Th2 priming was downregulated in the same conditions. Collectively, these observations suggest that the function of mature DCs is under the tight control of the naturally occurring CD4+CD25+ cells in vivo and that regulatory T cells influence the character of T cell differentiation by selectively dampening Th1-type responses and CTLs. [Since their discovery in 1973 by Zanvil Cohn and Ralph Steinman, dendritic cells (DCs) have gained increased interest among immunologists, as these cells display unique properties aimed at inducing an immune response against antigens that invade the organism. They perform multiple tasks with high efficiency, and appear to determine the specificity, magnitude and nature (Th1/Th2) of the immune response.In the mouse, two subclasses of dendritic cells have been described that differ by their CD8á expression and their localization in lymphoid organs.We have assessed the function of both subclasses in vivo by injecting purified DC, pulsed extracorporeally with antigen, into the footpads of syngeneic mice and analyzing the immune response of lymph node cells. Our data show that both DC subsets efficiently prime antigen-specific T cells in vivo, and direct the development of distinct T helper cell populations. Administration of CD8á- DC induces a Th2-type response, whereas injection of CD8á+ DC leads to Th1 differentiation. Th1 priming is dependent on IL-12 production by DCs.These findings suggest that the nature of the DC that presents the antigen to naive T cells may dictate the class selection of the adaptive immune response. Experiments are under way to assess the role of various DC populations in activating the recently discovered Th17 cell population, which plays a role in autoimmune and allergic inflammation. More recently, a number of reports have shown that, in addition to their well known stimulatory properties, DCs may play a major role in peripheral tolerance. It is still unclear whether a distinct subtype or activation status of DC exists which promotes the differentiation of suppressor rather than effector T cells from naive precursors. We tested whether the naturally occurring CD4+CD25+ regulatory T cells may control immune responses induced by DCs in vivo. We found that the development of IFN-a-producing cells and cytotoxic T cells was strongly enhanced in the absence of regulatory T cells. By contrast, Th2 priming was downregulated in the same conditions. Collectively, these observations suggest that the function of mature DCs is under the tight control of the naturally occurring CD4+CD25+ cells in vivo and that regulatory T cells influence the character of T cell differentiation by selectively dampening Th1-type responses and CTLs.]

Métabolisme cellulaire et régulation des réponses immunes [Cellular metabolism and regulation of immune responses]
To gain insight into the genetic basis of inflammation, our laboratory has recently undertaken the identification of genes selectively expressed by DCs, a cell population known to play a major role in the control of inflammatory responses. Using a modified differential display strategy, we have identified a series of transcripts representing genes specifically upregulated in DCs. One of the transcripts has been found to represent the murine homolog of a bacterial gene coding for a nicotinamide phosphoribosyl transferase (NAmPRTase), an enzyme involved in NAD biosynthesis. We have recently demonstrated that similarly to its microbial counterpart, the murine protein is a NAmPRTase, catalyzing the condensation of nicotinamide with 5-phosphoribosyl-1-pyrophosphate to yield nicotinamide mononucleotide, an intermediate in the biosynthesis of Nicotinamide adenine dinucleotide (NAD). This observation led us to further explore the possible relationship between NAD metabolism and the control of immune responses. Nicotinamide adenine dinucleotide (NAD) has been known for several decades to play a major role as a coenzyme in numerous oxidation-reduction reactions. Recently, the distinct role of NAD as a precursor for molecules involved in regulatory processes has also been recognized. NAD can indeed serve as a substrate for covalent protein modification catalysed by enzymes identified as ADP-ribosyl transferases. During ADP ribosylation, the ADP-ribose moiety of NAD is enzymatically transferred onto an acceptor protein, a reaction known to profoundly affect the target protein effector function. Numerous proteins endowed with ADP-ribosyl tyransferase capacity have been identified in mammals including mono and poly (ADP-ribosyl) transferases (respectively mARTs and PARPs) and sirtuins. This novel class of NAD-dependent protein deacetylases is involved in gene and has recently attracted great interest following the demonstration that they may play a role in cell longevity in both yeast and C. elegans. Elevated expression of the NAmPRTase in cells of the innate immune system has led us to postulate an important role for NAD-dependent enzymes in the control of the inflammatory response, an hypothesis presently under investigation in our laboratory. [To gain insight into the genetic basis of inflammation, our laboratory has recently undertaken the identification of genes selectively expressed by DCs, a cell population known to play a major role in the control of inflammatory responses. Using a modified differential display strategy, we have identified a series of transcripts representing genes specifically upregulated in DCs. One of the transcripts has been found to represent the murine homolog of a bacterial gene coding for a nicotinamide phosphoribosyl transferase (NAmPRTase), an enzyme involved in NAD biosynthesis. We have recently demonstrated that similarly to its microbial counterpart, the murine protein is a NAmPRTase, catalyzing the condensation of nicotinamide with 5-phosphoribosyl-1-pyrophosphate to yield nicotinamide mononucleotide, an intermediate in the biosynthesis of Nicotinamide adenine dinucleotide (NAD). This observation led us to further explore the possible relationship between NAD metabolism and the control of immune responses. Nicotinamide adenine dinucleotide (NAD) has been known for several decades to play a major role as a coenzyme in numerous oxidation-reduction reactions. Recently, the distinct role of NAD as a precursor for molecules involved in regulatory processes has also been recognized. NAD can indeed serve as a substrate for covalent protein modification catalysed by enzymes identified as ADP-ribosyl transferases. During ADP ribosylation, the ADP-ribose moiety of NAD is enzymatically transferred onto an acceptor protein, a reaction known to profoundly affect the target protein effector function. Numerous proteins endowed with ADP-ribosyl tyransferase capacity have been identified in mammals including mono and poly (ADP-ribosyl) transferases (respectively mARTs and PARPs) and sirtuins. This novel class of NAD-dependent protein deacetylases is involved in gene and has recently attracted great interest following the demonstration that they may play a role in cell longevity in both yeast and C. elegans. Elevated expression of the NAmPRTase in cells of the innate immune system has led us to postulate an important role for NAD-dependent enzymes in the control of the inflammatory response, an hypothesis presently under investigation in our laboratory.]

Développement et fonction des cellules ''T helper'' dans la production d'anticorps in vivo [Development and function of helper T cells for antibody production in vivo]
CD4+ T lymphocytes play a central role in antibody responses to T cell dependent antigens by activating B lymphocytes, promoting their survival and allowing affinity maturation and isotype switch. CD4+ T cells also control cell-mediated immune responses as well as allergic reactions. It is still unclear whether CD4+ T lymphocytes helping B cells represent a specialised sub-population or if they belong to a larger population having multiple functions. Recent data from the literature and vaccination studies suggest that CD4+ T cell-dependent antibody secretion by B lymphocytes could be dissociated from production of Th1 and Th2 cytokines. The aim of our project is to identify and characterize CD4+ T lymphocytes specialised in helping antibody production by B lymphocytes. As T helper cell for antibody secretion cannot be distinguished from naive T cells on a cytokine production-base data, we developed an in vitro test to directly detect their capacity to sustain an IgG class switch in B cell cooperation tests. This experimental setting allowed us to compare the B cell help activity mediated by distinct CD4+ T cell populations obtained following antigen inoculation in transgenic mice. Our results suggest that B helper T cells differ from both Th1 and Th2 cells and could therefore represent a 'new' CD4+ T cell population (ThB) distinct from the 'classical' Th1 and Th2 cytokine producer cells. In vivo induction of ThB function (leading to an optimal humoral response) in the absence of a cytokine-mediated inflammatory response could be of great potential in the development of vaccine strategies. We therefore studied the ThB cell activation requirements following a dendritic cell (DC)-based immunisation protocol in mice. Interestingly, we observed that DC kept in an 'immature' state were fully capable of promoting antigen specific antibody secretion in vivo, despite defective IFN induction. Eperiments are under way to assess the role of various factors (cytokines, transcription factors,') in the differentiation of naïve T cells towards the ThB cell type. These studies may lead to novel vaccination strategies inducing optimal antibody responses while minimizing local inflammatory reactions. [CD4+ T lymphocytes play a central role in antibody responses to T cell dependent antigens by activating B lymphocytes, promoting their survival and allowing affinity maturation and isotype switch. CD4+ T cells also control cell-mediated immune responses as well as allergic reactions. It is still unclear whether CD4+ T lymphocytes helping B cells represent a specialised sub-population or if they belong to a larger population having multiple functions. Recent data from the literature and vaccination studies suggest that CD4+ T cell-dependent antibody secretion by B lymphocytes could be dissociated from production of Th1 and Th2 cytokines. The aim of our project is to identify and characterize CD4+ T lymphocytes specialised in helping antibody production by B lymphocytes. As T helper cell for antibody secretion cannot be distinguished from naive T cells on a cytokine production-base data, we developed an in vitro test to directly detect their capacity to sustain an IgG class switch in B cell cooperation tests. This experimental setting allowed us to compare the B cell help activity mediated by distinct CD4+ T cell populations obtained following antigen inoculation in transgenic mice. Our results suggest that B helper T cells differ from both Th1 and Th2 cells and could therefore represent a 'new' CD4+ T cell population (ThB) distinct from the 'classical' Th1 and Th2 cytokine producer cells. In vivo induction of ThB function (leading to an optimal humoral response) in the absence of a cytokine-mediated inflammatory response could be of great potential in the development of vaccine strategies. We therefore studied the ThB cell activation requirements following a dendritic cell (DC)-based immunisation protocol in mice. Interestingly, we observed that DC kept in an 'immature' state were fully capable of promoting antigen specific antibody secretion in vivo, despite defective IFN induction. Experiments are under way to assess the role of various factors (cytokines, transcription factors,') in the differentiation of naïve T cells towards the ThB cell type. These studies may lead to novel vaccination strategies inducing optimal antibody responses while minimizing local inflammatory reactions.]

Cellules T régulatrices [Regulatory T cells]
Interest in regulatory T cells has exploded since the discovery by Sakaguchi and colleagues of a minor population of CD4+ CD25+ T cells which plays a central role in the prevention of autoimmune reactions in vivo. 'Natural regulatory T cells' develop in the thymus, are present in naïve animals and their depletion causes the development of a spectrum of autoimmune. By contrast, 'adaptive or induced' regulatory T cells develop in peripheral lymphoid tissues. TGF-; and IL-10 may be involved in the conversion of CD4+ CD25- T cells into CD4+ CD25+ FoxP3+ which display suppressive activity. Our laboratory has undertaken studies to evaluate the role of natural regulatory T cells in the control of humoral and cellular responses. Our results show elimination of natural regulatory T cells (which constitutively express CD25) led to increased antigen-specific antibody production but did not affect T-independent B cell responses, suggesting that CD4+ CD25+ T exert a feedback mechanism on antibody secretion by selectively dampening the T cell help for B cell activation. Similarly, natural regulatory T cells downregulate activation of Th1 (but not Th2) 'type responses, as demonstrated by a strong increase in IFN production and CTL activity in mice immunized with antigen-pulsed DCs and depleted of CD25+ cells (see above discussion). Although active suppression mediated by the naturally occurring CD4+ CD25+ regulatory T cells is crucial for peripheral tolerance and prevention of autoimmunity, it might also, in some circumstances, be considered as an obstacle to successful vaccination, in particular in cancer patients (see below). In the course of studies aimed at evaluating the function in vivo of the inhibitory receptor CTLA-A4, we found that treatment of mice with anti-CTLA4 mAb (4F10, kindly provided by J. Bluestone) at the time of priming induced the development of a population of ICOShi CD4+ T cells producing IL-10 and expressing FoxP3. These induced regulatory T cells seem to be distinct from the naturally occurring Treg and inhibit primary and memory Th1 responses. Experiments are under way to test the role of IL-10 and the enzyme indoleamine 2, 3 dioxygenase in the CTLA-4-induced down-regulation of Th1-type responses. [Interest in regulatory T cells has exploded since the discovery by Sakaguchi and colleagues of a minor population of CD4+ CD25+ T cells which plays a central role in the prevention of autoimmune reactions in vivo. 'Natural regulatory T cells' develop in the thymus, are present in naïve animals and their depletion causes the development of a spectrum of autoimmune. By contrast, 'adaptive or induced' regulatory T cells develop in peripheral lymphoid tissues. TGF-; and IL-10 may be involved in the conversion of CD4+ CD25- T cells into CD4+ CD25+ FoxP3+ which display suppressive activity. Our laboratory has undertaken studies to evaluate the role of natural regulatory T cells in the control of humoral and cellular responses. Our results show elimination of natural regulatory T cells (which constitutively express CD25) led to increased antigen-specific antibody production but did not affect T-independent B cell responses, suggesting that CD4+ CD25+ T exert a feedback mechanism on antibody secretion by selectively dampening the T cell help for B cell activation. Similarly, natural regulatory T cells downregulate activation of Th1 (but not Th2) 'type responses, as demonstrated by a strong increase in IFN production and CTL activity in mice immunized with antigen-pulsed DCs and depleted of CD25+ cells (see above discussion). Although active suppression mediated by the naturally occurring CD4+ CD25+ regulatory T cells is crucial for peripheral tolerance and prevention of autoimmunity, it might also, in some circumstances, be considered as an obstacle to successful vaccination, in particular in cancer patients (see below). In the course of studies aimed at evaluating the function in vivo of the inhibitory receptor CTLA-A4, we found that treatment of mice with anti-CTLA4 mAb (4F10, kindly provided by J. Bluestone) at the time of priming induced the development of a population of ICOShi CD4+ T cells producing IL-10 and expressing FoxP3. These induced regulatory T cells seem to be distinct from the naturally occurring Treg and inhibit primary and memory Th1 responses. Experiments are under way to test the role of IL-10 and the enzyme indoleamine 2, 3 dioxygenase in the CTLA-4-induced down-regulation of Th1-type responses.]

Immunothérapie du cancer [Cancer immunotherapy]
A major factor limiting the efficacy of cancer immunotherapy is the development of mechanisms allowing tumors to resist or escape immune rejection. We are studying these mechanisms in vivo in preclinical models in order to define strategies to boost immunotherapy by overcoming tumoral immune resistance. We focus on the role of regulatory T lymphocytes, myeloid suppressor cells, indoleamine 2,3-dioxygenase, and other potential immunosuppressive molecules. Natural regulatory T cells. Our current results show that depletion of natural regulatory T cells (by anti-CD25 mAb treatment) induces rejection of P815 mastocytoma in 70-to-80% of mice. These observations suggest that natural regulatory T cells inhibit the spontaneous tumor-specific immune response, a hypothesis consistent with the infiltration of cells displaying the phenotype of natural T regulatory T cells in P815. We are characterizing the specificity, phenotype and function of Treg infiltrating the tumor at various time points (using FACS, confocal microscopy, RT-PCR, in vitro coculture) in order to test whether distinct subpopulations , i.e. natural or induced, infiltrate a growing P815 tumor. Indoleamine 2, 3-dioxygenase (IDO) In addition to tumor cells, antigen-presenting-cells have been shown to produce IDO, in particular upon engagement with CTLA-4 receptor on regulatory T cells. We are evaluating the impact of IDO expressed by tumor cells versus immune cells, using tumor cells expressing various amounts of IDO (available at ICP) and WT or IDO-KO mice (kindly provided by A. Mellor). We have evidence that regulatory T cell populations may induce IDO production by DC in vitro and in vivo Myeloid suppressor cells The comparison of cell populations which infiltrate progressing versus regressing P815 tumors led us to identify a population of CD11c+ CD11b+ Gr1+ cells which infiltrates regressing tumors. By contrast, a population of CD11c- CD11b+ Gr1+ cells is detected in progressing P815 tumors. The latter cells, which have been observed in other cancer models and also in some cancer patients, are thought to bear immunosuppressive properties and have been called Myeloid Suppressor Cells (MSC). We will characterize the phenotype and function of both subpopulations, and in particular analyse their production of cytokines known to influence the outcome of tumor growth. [A major factor limiting the efficacy of cancer immunotherapy is the development of mechanisms allowing tumors to resist or escape immune rejection. We are studying these mechanisms in vivo in preclinical models in order to define strategies to boost immunotherapy by overcoming tumoral immune resistance. We focus on the role of regulatory T lymphocytes, myeloid suppressor cells, indoleamine 2,3-dioxygenase, and other potential immunosuppressive molecules. Natural regulatory T cells. Our current results show that depletion of natural regulatory T cells (by anti-CD25 mAb treatment) induces rejection of P815 mastocytoma in 70-to-80% of mice. These observations suggest that natural regulatory T cells inhibit the spontaneous tumor-specific immune response, a hypothesis consistent with the infiltration of cells displaying the phenotype of natural T regulatory T cells in P815. We are characterizing the specificity, phenotype and function of Treg infiltrating the tumor at various time points (using FACS, confocal microscopy, RT-PCR, in vitro coculture) in order to test whether distinct subpopulations , i.e. natural or induced, infiltrate a growing P815 tumor. Indoleamine 2, 3-dioxygenase (IDO). In addition to tumor cells, antigen-presenting-cells have been shown to produce IDO, in particular upon engagement with CTLA-4 receptor on regulatory T cells. We are evaluating the impact of IDO expressed by tumor cells versus immune cells, using tumor cells expressing various amounts of IDO (available at ICP) and WT or IDO-KO mice (kindly provided by A. Mellor). We have evidence that regulatory T cell populations may induce IDO production by DC in vitro and in vivo Myeloid suppressor cells The comparison of cell populations which infiltrate progressing versus regressing P815 tumors led us to identify a population of CD11c+ CD11b+ Gr1+ cells which infiltrates regressing tumors. By contrast, a population of CD11c- CD11b+ Gr1+ cells is detected in progressing P815 tumors. The latter cells, which have been observed in other cancer models and also in some cancer patients, are thought to bear immunosuppressive properties and have been called Myeloid Suppressor Cells (MSC). We will characterize the phenotype and function of both subpopulations, and in particular analyse their production of cytokines known to influence the outcome of tumor growth.]