1. Dale and Betty Bumpers Vaccine Research Center National Institute of Allergy and Infectious Diseases National Institutes of Health Richard A. Koup Vaccine Research Center Lymph node structure and HIV-1 infection: T cell immunopathogenesis

2. Background HIV replication is active in lymph nodes throughout the course of HIV infection HIV leads to an initial hypertrophy, followed by involution of lymphoid tissues (Tim Schacker) SIV infection in rhesus macaques has a similar pathogenesis to HIV infection in humans (Guido Silvestri) and can be used to study the immuno- pathogenesis of HIV infection in lymph nodes In this talk I will concentrate on using acute and early SIV infection of rhesus macaques to model HIV pathogenesis in lymph nodes

3. Janeway’s Immunobiology (8 th Edition) The Lymph Node has a Complicated Structure

4. Simplified LN Structure Paracortical Area (T cells) Lymphoid Follicle (B cells) Germinal Center (B and T cells) Light Zone (T/B cell interaction) Dark Zone (B cell proliferation)

5. Simplified LN Structure – HIV Infection UninfectedInfected - Early Germinal Center Hypertrophy (increased germinal center T cells) Loss of non- germinal center T cells Infected - Late

6. Outline Virus replication in LN during acute/early HIV/SIV infection Changes in CD4 T cell populations in LNs during acute/early HIV/SIV replication Underlying mechanisms Non-T cell consequences

7. SIV as a model for HIV infection Nature 434:1093-7, 2005

8. Plasma Viral Load (Log 10 ) Acute Infection: Plasma Viral Loads

9. CD4 T Cells, % of CD3 + Total CD4 Cell Dynamics

10. 0 20 40 Mesenteric LN 0 20 40 PBMC 037101417037101417 DaysPost-infection CD4 Memory, % of CD3 + Memory CD4 T Cell Dynamics Early expansion at d. 3 Loss of ~80% of cells by d. 17

11. Cell-Associated Viral Loads

12. These data do not take into account structural localization within the LN Where is the virus replicating with respect to the paracortical T cell zone and the light zone of the germinal center? What is happening to CD4 T cell frequency in these areas during acute and early SIV infection? Paracortical T cell zone Light zone of the germinal center

13. Surface markers can distinguish CD4 T cells from these different areas Paracortical T cell zone Light zone of the germinal center CD95 CD28 CD8 CD4 CD3 Aqua FSC-A FSC-H PD-1 CCR7 010 3 4 5 0 2 3 4 5 70.8 19.5

14. Where does SIV replicate? PD-1 CCR7 0 0.5 1.0 1.5 Early SIV (>2 months) 0 1 2 3 4 5 Acute SIV (3-21 days) SIV Gag DNA (copies/cell) p=0.0078 p=0.0156 010 3 4 5 0 2 3 4 5 ICOS CD150 Paracortical T cell zone Light zone of the germinal center Would expect to see depletion of CD4 T cells in germinal centers

15. SIV: Relative accumulation of CD4 T cells in GCs 010 2 3 4 5 0 2 3 4 5 29.1 12.9 47.8 PD-1 CCR7 CCR7 high PD-1 low CCR7 high/low PD-1 dim T FH % of CM CD4 T cells SIV acute SIV early SIV -

16. CD4 PD1 Ki-67 Michael Gerner, Ron Germain Light zone: T - B cell interaction Dark zone: B cell proliferation Accumulation of GC CD4 T cells during SIV infection: Abundant GCs with retained architecture

17. Mechanism? UninfectedInfected - Early ?

18. No correlation between VLs and percent GC T cells during SIV infection Percent GC T cells in LN Viral Loads

19. GC T cells % of CM CD4 T cells Accumulation of GC T cells is associated with general immune activation (sCD14) p=0.0013 p=0.0004 p=0.0164 sCD14 (x10 6 pg/ml) SIV acute SIV early (high % of T FH ) SIV - SIV early (low % of T FH )

20. SIV: Relative accumulation of GC T cells IL-6 signaling drives the up-regulation of Bcl-6 and enhanced T cell responses that are seen during chronic LCMV infection in mice. Does IL-6 production drive the accumulation of GC T cells during early SIV infection in monkeys?

21. SIV - SIV + (early) p=0.0215 Increase in the IL-6/IL-6R axis is associated with GC T cell accumulation during SIV infection Plasma IL-6 (pg/ml) p=0.0136 Percent GC T cells in LN IL6Ra on GC T cells (MFI)

22. Mechanism? UninfectedInfected - Early IL-6 2) Altered phospho STAT (3>1) 3) GC T cell differentiation 4) Immune activation 1) Petrovas et al, J. Clin. Invest., 2012

23. SIV - SIV chronic (low % GC T cells) SIV chronic (high % GC T cells) p=0.015 010 2 3 4 5 0 2 3 4 5 86.1 010 3 4 5 0 2 3 4 5 28.7 010 3 4 5 0 3 4 5 9.84 6.4922.3 61.3 010 3 4 5 0 3 4 5 15.8 0.721.87 81.5 Aqua SSC CD20 CD3 PNA IgG PBMC LN PNA high IgG low PNA high IgG high SIV - SIV + low T SIV + high T GC T cells are T FH that influence B cells differentiation and antibody production SIV chronic (low % GC T cells) SIV chronic (high % GC T cells) gp120 p=0.051 SIV-specific IgG (titer, x10 4 ) 0 2.5 5 7.5 10

24. Conclusions HIV/SIV replication is profound during acute infection This leads to a massive depletion of memory CD4 T cells in the LN and gut CD4 T cell depletion from the gut leads to microbial translocation and general immune activation (discussed elsewhere) Immune activation promotes differentiation of T cells in the LN which move to and accumulate in the GCs (lymphoid hypertrophy) – Non-pathogenic SIV controls immune activation Continued immune activation ultimately leads to fibrosis of the lymph nodes

25. Simplified LN Structure – HIV Infection UninfectedInfected - Early Germinal Center Hypertrophy (increased germinal center T cells) Loss of non- germinal center T cells Infected - Late Direct infection: CD4 depletion Immune activation: GC hypertrophy followed by fibrosis

26. Dale and Betty Bumpers Vaccine Research Center National Institute of Allergy and Infectious Diseases National Institutes of Health Costas Petrovas Takuya Yamamoto Kristin Boswell Joseph Casazza Rob Paris David Ambrozak Immunology Laboratory NIAID/NCI Collaborators Michael Gerner Ron Germain Human Immunology Section Netanya Sandler Daniel Douek ImmunoTechnology Section Mario Roederer Lab Animal Medicine John-Paul Todd Srinivas Rao