1. CD4+ memory stem cells (T SCM ) in pathogenic and non- pathogenic SIV infections Guido Silvestri, MD Yerkes National Primate Research Center Emory University School of Medicine Emory Center for AIDS Research (CFAR) Emory Vaccine Center

2. The pattern of Infected CD4+ T Cell is a key determinant of AIDS pathogenesis Damage to LN architecture & lymphoid niche Loss of CD4 T cell homeostasis/CD4 depletion Chronic immune activation Pattern of in vivo infected cells Features & efficacy of host immune responses Size and dynamics of the “latent” reservoir Picker. J Exp Med 2007; Letvin et al. Science 2007; Roederer et al. J Exp Med 2007; Brenchley et al. Immunity 2010; Chomont et al., Nat Med 2009; Paiardini et al. Nat Med 2011; Brenchley et al. Blood 2012; Autran et al., Clin Inf Dis 2012; Chahroudi et al., Science 2012; Saez-Cirion et al. PLoS Path 2013

3. Comparative AIDS Research AIDS NO YES YES Blood CD4 depletion Rare YES YES Viral Load HIGH HIGH HIGH SM RM HIV Preserved gut & LN integrity YES NO NO Microbial translocation NO YES YES Chronic immune activation NO YES YES

4. Restricted infection of CD4+ T CM cells in SIV-infected sooty mangabeys Level of infected cells in vivo Level of infected cells following in vitro infection Paiardini et al, Nat Med 2011

5. LN anatomic distribution of productively infected cells in RMs, humans, and SMs. (A) SIV/HIV vRNA + cells/mm 2 in LNs of chronically infected RMs, humans, and SMs. (B) SIV/HIV vRNA + in B-cell follicles (C) Percentage of LN area that consists of B-cell follicles. Brenchley et al, Blood 2012 Higher levels of Tfh infection in SIV-infected RMs and HIV-infected humans than SIV-infected SMs

6. Progressive Infection and AIDS Chronic, generalized Immune Activation How HIV and SIV infection cause AIDS: Lessons from natural SIV infections Virus replication in central memory CD4+ T cells

7. Naïve CellEffector (short-lived, activated) Memory cell pool Proliferation and clonal expansion in response to antigenic stimulation Contraction phase Effector Memory : Resident in periphery. Able to respond quickly to re-exposure. CD62L- CCR7- Central Memory : Resident in LNs, spleen. Able to self-renew and differentiate into Tem. CD62L+ CCR7+ Memory stem cell antigen Complexity of the memory CD4+ T cell pool

8. What is a T memory stem cell (Tscm)? 1-3% of circulating and LN-based CD4+ and CD8+ T cells. Identified by classical naïve T cell markers (CD45R+CD45RO- CD62L+CCR7+CD127+CD27+CD28+), but also CD95 & CD122. Express more LFA-1 and CXCR3 than naïve cells, but less than T CM (and less Bcl-2 than naïve cells but more than T CM ). Least differentiated memory subset, with high proliferative potential, and capable of self-renewal. Multipotent: able to generate both T CM and T EM Gattinoni et al., Nat Med 2009; Turtle et al., Immunity 2009; Muranski et al., Immunity 2011; Gattinoni et al., Nat Med 2011; Luckey CJ, Weaver CT. Cell Stem Cell, 2012; Roederer et al., J Clin Invest, 2012

9. A.A. T SCM CD8 FSC-H CD95 SSC-A CCR7 Dead/14/16/20 CD27 CD45RACD28CD4 CD122 FSC-ACD3FSC-A Identification of CD4+T SCM in healthy rhesus macaques and sooty mangabeys. E.E. C. B.B. D.

10. A.A. E.E. C. B.B. D. F. Identification of CD4+T SCM in healthy rhesus macaques and sooty mangabeys.

11. B. C. CD4+ T SCM express levels of CXCR3 and CD11a similar to other memory CD4+ T cells subsets, and levels of Bcl-2 that are intermediate between naïve and T CM.

12. A.A. Higher levels of CCR5 expression on CD4+T SCM of healthy RMs as compared to SMs. C. B.B. D. E.E. F.F. CD4 SM RM CCR5

13. Selective preservation of CD4+T SCM cells during pathogenic SIV infection of RMs B.D. A. p= n.s.

14. SIV infection of RMs is associated with a significant depletion of CCR5+CD4+T SCM. A. B.B. C.

15. SIV infection of RMs is associated with increased proliferation of CD4+T SCM that significantly inversely correlates with the levels of CD4+T CM A. B. C. D.

16. A. B. C. Dynamics of CD4+T SCM during non-pathogenic infection of SM D.

17. Robust levels of CD4+T SCM infection in vivo are observed in SIV-infected RMs but not in SIV-infected SMs

18. CD4+ T memory stem cells in pathogenic and non-pathogenic SIV infections CD4+ T SCM are numerically preserved during BOTH pathogenic and nonpathogenic SIV infections. However, SIV-infected RM show (i) a selective depletion of CD4+CCR5+ T SCM ; and (ii) higher levels of CD4+ T SCM proliferation that correlate significantly with the level of CD4+ T CM depletion. Robust levels of direct virus infection of CD4+ T SCM are found only in SIV-infected RM, with 6 out of 7 SIV-infected SM showing no evidence of CD4+ T SCM infection.

19. CD4+ T memory stem cells in pathogenic and non-pathogenic SIV infections RM SM T SCM T CM CCR5+Ki67+ T SCM T CM SIV AIDS T SCM T CM

20. Increasing contribution of Tscm to HIV reservoir over time (M. Lichterfeld) Lichterfeld, unpublished

21. Hypothesis: can reservoirs be eliminated when there is no virus in T SCM cells? T SCM T CM T EM ART T SCM T CM T EM Persistent T SCM (and T CM ) reservoirs Absence of T SCM (and T CM ) reservoirs

22. Acknowledgments Silvestri Lab Emily Cartwright Vandy Vanderford Steven Bosinger Ann Chahroudi Ankita Chowdhury Maud Mavigner Kiran Mir Tim Hayes Luca Micci Tayebeh Hashempour Charlene Wang Alex Ortiz (NIH) Nichole Klatt (UW) Diane Carnathan Paul Carnathan SBRI -- Seattle Donald Sodora Emory University/Yerkes Mirko Paiardini Colleen McGary Cynthia Derdeyn James Else Eric Hunter Vincent Marconi Ray Schinazi NIH/NIAID/NCI Jake Estes Jason Brenchley Daniel Douek Univ. of Pennsylvania Ron Collman Mike Betts Beatrice Hahn Univ. of Ulm Frank Kirchhoff Ragon Institute/HMS Mathias Lichterfeld Case Western Michael Lederman Institut Pasteur Michaela Muller-Trutwin University of Pittsburgh Ivona Pandrea Cristian Apetrei Los Alamos National Labs Alan Perelson Supported by NIH/NIAID, Emory CFAR, Georgia Research Alliance