2014 “ Towards an HIV Cure ” symposium Melbourne

1. 2014 “Towards an HIV Cure” symposiumMelbourne TLR2 stimulation promotes HIV-1 infection of CD4 T cells by increasing the susceptibility of CCR6- T cells to infection Jean-François Bolduc, M.Sc. CHU de Québec Research Centre and Université Laval’s Faculty of Medecine

2. Background • Following infection, HIV-1 induces structural and immunological disruptions leading to bacterial translocation and release of microbial components in the bloodstream. • Bacterial components may affect gut-homing CD4 T cells such as CCR6- and CCR6+, which are enriched in the gut mucosa and highly permissive to HIV-1 infection. Gosselin, A. et al. Peripheral Blood CCR4+CCR6+ and CXCR3+CCR6+ CD4+ T Cells Are Highly Permissive to HIV-1 Infection. The Journal of Immunology 184, 1604–1616 (2010). Monteiro, P. et al. Memory CCR6+CD4+ T Cells Are Preferential Targets for Productive HIV Type 1 Infection Regardless of Their Expression of Integrin 7. The Journal of Immunology 186, 4618–4630 (2011). Shan, L. & Siliciano, R. F. Unraveling the relationship between microbial translocation and systemic immune activation in HIV infection. The Journal of Clinical Investigation 124, 2368–2371 (2014). In this study, we investigated the effect of TLR2 ligation on the susceptiblity of CCR6-/+ CD4 T cells to HIV-1 infection.

3. Methodology Primary human resting CD4 T cells were purified by magnetic separation and stimulated for 72h with anti-CD3/CD28 antibodies +/- TLR2 ligand.

4. Methodology Primary human activated CD4 T cells were infected for 24h with the complete R5 HIV-1-based reporter virus NL4-3-Bal-IRES-HSA. HSA (heat-stable antigen) (CD24) Murine originproteinexpressedat the surface of the cellsproductivelyinfectedwithfullycompetent virions Imbeault, M., Lodge, R., Ouellet, M. & Tremblay, M. J. Efficient magnetic bead-based separation of HIV-1-infected cells using an improved reporter virus system reveals that p53 up-regulation occurs exclusively in the virus-expressing cell population. Virology 393, 160–167 (2009).

5. Methodology CFSE proliferation assays and flow cytometry analysis have been performed to define and explain the effect of TLR2 ligation on the susceptibility of CCR6-/+ CD4 T cells.

6. Results Compared to CD3/CD28 stimulation, TLR2 ligation increases the % of infected (HSA+) CD4 T cells and viral p24 production.

7. Results TLR2 ligation does not affect uninfected CD4 T cell proliferation.

8. Results TLR2 ligation does not affect uninfected CD4 T cell distribution.

9. Results TLR2 ligation increases the susceptibility of CCR6- cells to HIV-1 infection to levels observed in the CCR6+ subset, while CD3/CD28 stimulation by itself increases infection of CCR6+ cells.

10. Results CD4/CCR5/α4 cell surface expression is higher on CCR6+ than on CCR6- CD4 T cells, but is not affected by TLR2 ligation.

11. Conclusion • TLR2 stimulation couldplay a significantrole in early HIV-1 infection by abolishing post-entry barriers. • The effect of TLR2 ligation on HIV-1 infection susceptibilityis more pronounced on CCR6- compared to CCR6+ CD4 Tcellssubset. • Theseresultshighlight the possible importance of early interactions between HIV-1 and gutmicrobiota and could lead to noveltherapiesseeking to block those interactions.

12. Acknowledgements Director Postdoctoral fellows Scientific editor Adjunct Professors Research assistant Ph.D. student Lab technician

13. Questions