Memory CCR6CD4 T-Cells are Selectively Imprinted with a Transcriptional Program Favorable to Productive HIV-1 Infection

1. Memory CCR6+CD4+ T-Cells are Selectively Imprinted with a Transcriptional Program Favorable to Productive HIV-1 Infection Good Afternoon I would like to thank the organizers for giving me the opportunity to present our work.Good Afternoon I would like to thank the organizers for giving me the opportunity to present our work.

2. It is well known that HIV-1 productively infects and persists in a very small fraction of memory CD4+ T-cells.s Therefore, our goals are to identify phenotypic and functional markers of discrete CD4+ T-cells subsets permissive vs resistant to HIV infection, And to identify the molecular mechanisms of HIV permissiveness vs resistance in primary T-cells.It is well known that HIV-1 productively infects and persists in a very small fraction of memory CD4+ T-cells.s Therefore, our goals are to identify phenotypic and functional markers of discrete CD4+ T-cells subsets permissive vs resistant to HIV infection, And to identify the molecular mechanisms of HIV permissiveness vs resistance in primary T-cells.

3. Th17 cells play an important role in mucosal homeostasis and act as a bridge between innate and adaptive immunity. On the opposite, Th17 cells play a deleterious role in HIV pathogenesis by their ability to produce pro-inflammatory cytokines and to support viral replication Several groups demonstrated that Th17 cells are depleted from the GALT upon HIV and SIV infection. These alterations lead to microbial translocation from the gut, which is a cause for chronic immune activation and disease progression.Th17 cells play an important role in mucosal homeostasis and act as a bridge between innate and adaptive immunity. On the opposite, Th17 cells play a deleterious role in HIV pathogenesis by their ability to produce pro-inflammatory cytokines and to support viral replication Several groups demonstrated that Th17 cells are depleted from the GALT upon HIV and SIV infection. These alterations lead to microbial translocation from the gut, which is a cause for chronic immune activation and disease progression.

4. The chemokine receptor CCR6 is a marker for Th17 cells and a regulator of T-cell trafficking into the GALT Peyer�s Patches Our laboratory demonstrated by real time PCR that the highest levels of integrated HIV DNA were detected in CCR6+ T-cells from 6 HIV infected patients, These results provide evidence that CCR6 is a marker for memory CD4+ T cells highly permissive to HIV infection in vivo. The chemokine receptor CCR6 is a marker for Th17 cells and a regulator of T-cell trafficking into the GALT Peyer�s Patches Our laboratory demonstrated by real time PCR that the highest levels of integrated HIV DNA were detected in CCR6+ T-cells from 6 HIV infected patients, These results provide evidence that CCR6 is a marker for memory CD4+ T cells highly permissive to HIV infection in vivo.

5. Imprinting of CD4+ T-cells with gut-homing potential: Mucosal dendritic cells ? Retinoic acid (RA) production ? up-regulation of integrin ?4?7 ? Migration into the gut (Mora et al., Immunity, 2004 ; Manicassamy et al., Nat Immunol, 2009) Integrin ?4?7 and HIV / SIV: New binding receptor for HIV gp120 (Arthos et al., Nat Immunol, 2008) Identifies a subset of memory CD4+ T-cells producing IL-17 that is preferentially infected and depleted during acute SIV infection (Kader et al., Mucosal Immunol, 2009; Wang et al., Mucosal Immunol., 2009) Peripheral blood ?4?7+ CD4+ T-cells are depleted during primary HIV infection (Krzysiek et al, Blood, 2001) The integrin a4b7 is a critical regulator for T-cell trafficking into the GALT. Its expression is induced on T-cells by retinoic acid produced by mucosal dendritic cells. Recent studies identified the integrin a4b7 as a new binding receptor for HIV gp120. And a marker for Th17 cells that are preferentially infected and depleted during acute SIV infection� It is also known that peripheral blood a4B7+ CD4+ T cells are depleted during primary HIV infection.The integrin a4b7 is a critical regulator for T-cell trafficking into the GALT. Its expression is induced on T-cells by retinoic acid produced by mucosal dendritic cells. Recent studies identified the integrin a4b7 as a new binding receptor for HIV gp120. And a marker for Th17 cells that are preferentially infected and depleted during acute SIV infection� It is also known that peripheral blood a4B7+ CD4+ T cells are depleted during primary HIV infection.

6. We investigated: whether memory T-cells co-expressing CCR6 and integrin ?7 are selective HIV targets whether RA-induced imprinting for gut-homing selectively increases CCR6+ T-cell permissiveness to infection In this context, in the present work we investigated: - Whether XXX and - Whether XXX In this context, in the present work we investigated: - Whether XXX and - Whether XXX

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8. The 4 subsets were then exposed to an R5 HIV strain. HIV-p24 levels were quantified by ELISA at different time points post infection. The highest levels of HIV replication were detected in CCR6+ T cells expressing or not B7. Similar results were obtained when levels of integrated HIV-DNA were quantified by real time PCR. The 4 subsets were then exposed to an R5 HIV strain. HIV-p24 levels were quantified by ELISA at different time points post infection. The highest levels of HIV replication were detected in CCR6+ T cells expressing or not B7. Similar results were obtained when levels of integrated HIV-DNA were quantified by real time PCR.

9. The integrin a4B7 is expressed on T-cells at low levels and under an inactive state. The upregulation and transition toward an activated form is mediated by RA. We therefore investigated the effects of ATRA on CCR6+ and CCR6- T cell permissiveness to HIV infection. Our results showed that ATRA significantly upregulated a4B7 expression on both CCR6+ and CCR6- T cell subsets. In contrast, ATRA significantly upregulated CCR5 expression on CCR6+ but not CCR6- T cells. These results suggested the possibility that ATRA selectively increases permissiveness to R5 HIV replication in CCR6+ T cells only. The integrin a4B7 is expressed on T-cells at low levels and under an inactive state. The upregulation and transition toward an activated form is mediated by RA. We therefore investigated the effects of ATRA on CCR6+ and CCR6- T cell permissiveness to HIV infection. Our results showed that ATRA significantly upregulated a4B7 expression on both CCR6+ and CCR6- T cell subsets. In contrast, ATRA significantly upregulated CCR5 expression on CCR6+ but not CCR6- T cells. These results suggested the possibility that ATRA selectively increases permissiveness to R5 HIV replication in CCR6+ T cells only.

10. To test this possibility, cells were exposed to a R5 HIV strain and integrated HIV-DNA levels were quantified by real time PCR. We observed a significantly higher ability of ATRA-treated CCR6+ compared CCR6- T cells to support HIV integration. To determine whether ATRA act on the permissiveness of CCR6+ T cells to HIV infection at entry but at post-entry levels as well, cells were exposed to a VSVG-pseudotyped HIV, which enters cells by endocytosis, independently of the HIV receptor and co-receptors. Our results demonstrated significantly higher levels of proviral DNA in ATRA-treated CCR6+ compared with CCR6- T cells. Together, these results demonstrated that ATRA preferentially increases HIV permissiveness in CCR6+ T cells at entry and post-entry levels.To test this possibility, cells were exposed to a R5 HIV strain and integrated HIV-DNA levels were quantified by real time PCR. We observed a significantly higher ability of ATRA-treated CCR6+ compared CCR6- T cells to support HIV integration. To determine whether ATRA act on the permissiveness of CCR6+ T cells to HIV infection at entry but at post-entry levels as well, cells were exposed to a VSVG-pseudotyped HIV, which enters cells by endocytosis, independently of the HIV receptor and co-receptors. Our results demonstrated significantly higher levels of proviral DNA in ATRA-treated CCR6+ compared with CCR6- T cells. Together, these results demonstrated that ATRA preferentially increases HIV permissiveness in CCR6+ T cells at entry and post-entry levels.

11. Indeed, we observed higher production of TNF-a and higher NF-kB activity in CCR6+ compared to CCR6- T-cells. However, ATRA did not selectively increase TNF-a production or NF-kB activity in CCR6+ T-cells. Therefore other molecular mechanisms may explain the preferential increase in HIV permissiveness induced by ATRA in CCR6+ T-cellsIndeed, we observed higher production of TNF-a and higher NF-kB activity in CCR6+ compared to CCR6- T-cells. However, ATRA did not selectively increase TNF-a production or NF-kB activity in CCR6+ T-cells. Therefore other molecular mechanisms may explain the preferential increase in HIV permissiveness induced by ATRA in CCR6+ T-cells

12. TCR signaling and cell activation (Lck, ZAP-70, PTPN13, MAP3K4, TANK) Lineage polarization profiles (IL-22, IL-26, CCL20, IL-5, IL-9) Regulation of gene transcription (RORC, RORA, PPARG, ARNTL, KLF2, NLF2, ATF5, E2F2, RUNX1) Immunological synapse formation (CXCR6, TNFRSF18) Finally, we performed genome-wide analysis of gene expression using the microarray technology in CCR6+ and CCR6- T-cells stimulated via CD3/CD28 in the presence or absence of ATRA. Our preliminary analysis demonstrated that both CCR6+ and CCR6- T cells respond to ATRA treatment by modulating distinct sets of genes Among transcripts upregulated by ATRA in CCR6+ versus CCR6- T-cells, we identified gene sets associated with TCR signaling and cell activation, lineage polarization profiles, regulation of gene transcription, and immunological synapse formation Experiments are running in the lab using RNA interference and chemical inhibitors to identify among these transcripts new molecular determinants of HIV permissiveness in CCR6+ T-cells Finally, we performed genome-wide analysis of gene expression using the microarray technology in CCR6+ and CCR6- T-cells stimulated via CD3/CD28 in the presence or absence of ATRA. Our preliminary analysis demonstrated that both CCR6+ and CCR6- T cells respond to ATRA treatment by modulating distinct sets of genes Among transcripts upregulated by ATRA in CCR6+ versus CCR6- T-cells, we identified gene sets associated with TCR signaling and cell activation, lineage polarization profiles, regulation of gene transcription, and immunological synapse formation Experiments are running in the lab using RNA interference and chemical inhibitors to identify among these transcripts new molecular determinants of HIV permissiveness in CCR6+ T-cells

13. CCR6+ memory T-cells expressing or not the gut-homing integrin ?7 are highly permissive to HIV replication. However, CCR6+ T-cells co-expressing integrin ?7 and CCR5 might have an extraordinary ability to disseminate HIV from the portal sites of entry ATRA selectively enhances permissiveness to HIV-replication in CCR6+ T-cells via entry (CCR5 upregulation) and yet unidentified post-entry mechanisms CCR6 is a marker for memory CD4+ T-cells imprinted with a transcriptional program favorable to HIV replication The identification of HIV dependency factors in CCR6+ T-cells will open the path for the design of new therapeutic strategies to limit HIV replication in these cells while maintaining their role in mucosal immunity In conclusion, our results demonstrated that XXX. However, XXX We also demonstrated that XXXX thus, providing evidence that XXXX Considering these findings, we believe that XXXX In conclusion, our results demonstrated that XXX. However, XXX We also demonstrated that XXXX thus, providing evidence that XXXX Considering these findings, we believe that XXXX

15. Acknowledgements I would like to finish by acknowledging my mentor and my colleagues for their contribution to this work. I also wish to thank Dr Sekaly and people in his lab, together with the technicians in the Flow Cytometry core facility for their scientific and technical support. The access to HIV-infected patient samples was made possible by our collaboration with dedicated clinicians, and via the FRSQ-SIDA Infectious Diseases Network in Qu�bec, Canada Finally, I address our appreciation to HIV-infected and uninfected subjects who enrolled in our study I would like to finish by acknowledging my mentor and my colleagues for their contribution to this work. I also wish to thank Dr Sekaly and people in his lab, together with the technicians in the Flow Cytometry core facility for their scientific and technical support. The access to HIV-infected patient samples was made possible by our collaboration with dedicated clinicians, and via the FRSQ-SIDA Infectious Diseases Network in Qu�bec, Canada Finally, I address our appreciation to HIV-infected and uninfected subjects who enrolled in our study