System Biology analysis of innate and adaptive immune responses during HIV infection

Model of T cell memory persistence and exhaustion Naive Ag+APC Effector TEM (Pfp, Gr.B, FasL, TNF) Ag stim. IL-2, IL-7, TCR (pfoxo3a) Self renewal persistence Memory (TCM &TEM) TCM Hyperimmune activation Chronic persistent Ag (HIV) Increased inflammation Decrease in helper cytokines Pre-mature death of Memory B cells Dysfunctional Memory T cells Pre-mature death of TCM What are the mechanisms?

Model of T cell memory persistence and exhaustion Naive Ag+APC Effector TEM (Pfp, Gr.B, FasL, TNF) Ag stim. IL-2, IL-7, TCR (pfoxo3a) Self renewal persistence Memory (TCM &TEM) TCM Hyperimmune activation Chronic persistent Ag (HIV) Increased inflammation Decrease in helper cytokines Pre-mature death of Memory B cells? Dysfunctional Memory T cells (PD-1) and? Pre-mature death of TCM (pfoxo3a) What are the mechanisms?

System Biology It is the systematic use of genomic, proteomic, and metabolomic technologies for the construction of network-based models of biological processes. It is the study and identification of targets in an integrated network rather than in isolation. It is the deciphering the effect of a given molecule or pathway in immune system in the context of these many networks.

Global system analysis of innate immunity Ex vivo purified innate immune cells (monocytes, DCs, NK cells macrophages) Healthy, HIV chronic, Resistant to HIV infection, Susceptible to HIV infection, Elite controllers TLR triggered innate cells Virally infected innate cells Yellow Fever, Pox virus, Adeno viruses, HIV and their mutants

We are looking for biological correlates of resistance to HIV infection We compare gene expression of CMCs collected from HIV resistant women (R) against new negatives (NN). Sample Count New Negatives 13 Resistant 22 In Collaboration with Blake Ball, Frank Plummer and Rafick Sekaly

Pathways Analysis Using GSEA Canonical pathways and gene sets from literature Enrichment in NN samples Pathway pfdr Interferon inducible <0.0001 Inflammation <0.0001 TLR <0.0001 IFNA <0.0001 LPS stimulation induced genes <0.0001 TOLL Pathway <0.0001

Pathways Analysis Using GSEA Transcription Factor Targets Enrichment in NN samples Pathway pfdr IRF7 0.002 IRF 0.004 ISRE 0.018

Increased expression in IRF7 signaling in patients susceptible to infection NN Resistant

Fold change Susceptible/Resistant Increase expression in interferon induced genes in Cervical mononuclear cells (CMC) from susceptible sex workers 3 2 1 0 IFI44L IFI6 IFIH1 IFIT1 IFIT2 IFIT3 IFIT5 N=9

Pathways Analysis Using GSEA Canonical pathways and gene sets from literature Enrichment in R samples Pathway pfdr OXIDATIVE PHOSPHORYLATION 0.001

Global System analysis of innate immune cells Identify specific innate transcriptional signatures for multiple viruses and vaccines Vaccine vectors Ad5, poxvirus, YF, HIV TLR triggering TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7 Generation of cell specific response data base Monocytes, mdc, pdc, T cells Generation of tissue specific data base PBMC, vaginal lavages, gut, lymph nodes

Strategy of DC infection and maturation PBMC Isolation mdc Mature DC pdc Infection with viruses, viral vaccines Triggering with TLR Transcriptional profiling

Distinct transcriptional signatures in different Pox viruses and their mutants in infected mdcs Count Color Key Row Z-Score

Fold change over Mock 7 6 5 4 3 2 1 0-1 -2 chemokines and cytokine expression levels in MVAC- C, NYVAC-C and NYVAC mutants CXCL10 CXCL13 CXCL16 CXCL9 IL12A IL15 LITAF TNF

Fold change over Mock IFN expression levels in MVAC-C, NYVAC-C and NYVAC mutants 20 15 10 5 0-5 IFNB1 IFNA10 IFNA16 IFNA2 IFNA21

Fold change over Mock Enhanced expression of pathogen sensing molecules 4 3 2 1 0-1 -2 RIG-1 TLR7 TLR9 IRF1 IRF7 IRF8 MYD88

Fold change over control Induction of IRF genes by macrophages was enhanced by mutant NYVAC-C 6 5 4 3 2 1 0-1 IRAK2 IRF1 IRF2BP2 IRF2BP2 IRF5 IRF7 IRF7 IRF8-2 -3

Summary Differential expression in Early, and late chemokine genes Gens associated with trafficking of T cells, B cells, NK cells and neutrophils cytokines which activate T cells (IL- 15) Genes of the IFN-a and IFN-b machinery

GSEA analysis shows the increased enrichment of immune pathways in NYVACC-KC DM compared to NYVACC

YF specific innate immune signature

YF vaccine induces the production of IL-7 in mdc and pdc

Distinct transcriptional signatures in mdc from healthy and HIV infected subjects HIV Healthy

Deliverables Generation of innate immune response data base Vaccine vectors Ad5, poxvirus, YF, HIV TLR triggering TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7 Generation of adaptive immune response data base HIV, YF, CMV, EBV Generation of cell specific response data base Monocytes, mdc, pdc, T cells Generation of tissue specific data base PBMC, vaginal lavages, gut, lymph nodes

Global system analysis of Adaptive Immunity: Dysfunction during HIV infection

Determine the global transcriptional signatures of HIV and CMV specific CD8+ T cells in HIV infected subjects Primary HIV Chronic HIV Elite controllers HIV HIV HIV CMV CMV CMV Gene array analysis N3-12

Cluster analysis of HIV- and CMV-specific tetramers in HIV infection CMV HIV

Cluster analysis of HIV- and CMV-specific tetramers in chronic infection HIV CMV

Cluster analysis of HIV- and CMV-specific tetramers in elite controllers CMV HIV

Common and unique gene expression levels during HIV infection up-regulated down-regulated

HIV specific CD8+ T cells in chronic stages express negative regulators and are exhausted Fold change of HIV vs CMV 3 2 1 0-1 -2-3 -4-5 -6 P<0.05

CD160 CD160 is expressed on NK, NKT, intraepthelial T cells, and low frequency of peripheral CD4+ and CD8+ T cells CD160 has been shown to significantly enhances TCR induced IL-2 production, proliferation and cytotoxic activities Recently, CD160 has been shown to inhibit TCR induced proliferation and cytokine production The ligand for CD160 is HVEM

n = 63 Untreated (except for STs) Acute: Infected < 6 months Chronics: Infected > 6 months ST: undetectable viremia EC: Undetectable viremia LTNP: low detectable viremia Patient population Time from infection n CD4 CD8 Log VL copies Acute 2.8 (1.6-5.6) 12 360 (257-640) 728 (491-1990) 5.2 (4.13-6.59) 145285 (13578-3917615) Chronic 11.7 (6.4-51.5) 9 494 (296-858) 717 (410-1081) 4.9 (4.0-5.39) 71910 (10020-244029) ST 86.2 (18.4-1199.5) 12 600 (222-915) 732 (332-1120) 1.7 50 EC 134.6 (15.9-189.9) 12 706 (187-1040) 776 (303-1514) 1.7 (1.7-2.99) 50 (50-987) LTNP 147.2 (34.1-220.5) 9 440 (250-788) 941 (300-2498) 3.7 (3.02-4.54) 4228 (1588-34461) Healthy 9

PD-1 CD160 is upregulated during chronic HIV infection on both total and HIV-specific CD8 T cells CD8 PD-1 A Tetramer CD160 CD160 MFI 1320 MFI 5466

CD160 is upregulated during chronic HIV infection on total CD8 T cells

CD160 and PD-1 levels are upregulated during chronic HIV infection on total CD8 T cells

CD160 and PD-1 expression levels are upregulated during chronic HIV infection on HIV-specific CD8 T cells

PD-1+ CD160+ double compartment defines a new exhausted subset during chronic HIV on antigen specific CD8 T cells

CD160 Unstimulated SEB CMV peptide HIV peptide IFN TNF CD107a Cytokines

PD-1+ CD160+ CD8+ T cells are less polyfunctional than SP PD-1 CD160- CD160+

Tetramer A*022 CMV B*07 CMV B*08 NEF B*08 p24 0.77 0.31 1.07 0.01 Isotype control 6.1% 5.26% 5.64% 0.5% HVEM 0.7% 0.51% 0.56% 0.02% PD-L1 4.28% 6.29% 2.62% 0.15% HVEM + PD-L1 CFSE

Blocking CD160 interaction with its ligands increases HIV-specific T cell proliferation

Blocking CD160 interaction with its ligands increases CMV-specific T cell proliferation

PD-1+ CD160+ double compartment is a distinct subset and is transcriptionally different than PD-1+ single subset

Summary Novel compartment More dysfunctional Specific phenotype Identifies chronic and not acute

Acknowledgements R.P. Sekaly Y. Peretz J. Van Grevenhyngue Lydie Trautmann Y. Shi Collaborators Blake Ball Frank Plummer Gepi Pantaleo Rick Koup Bruce Walker Ali Filali JP Gaulet J.P. Routy M.R. Boulassel McGill University Health Center, Montreal Réseau SIDA et MI du FRSQ Montreal