Loss of CD96 Expression as a New Biomarker for T-cell Senescence in HIV-1 Infection

Transcription

1 Loss of CD96 Expression as a New Biomarker for T-cell Senescence in HIV-1 Infection Mohamed El-Far, PhD CHUM research center (CRCHUM) University of Montreal, QC Canada 6th International Workshop on HIV & Aging Washington, DC, USA 5-6 October 2015

2 Loss of CD96 Expression as a New Biomarker for T-cell Senescence in HIV-1 Infection We declare that we have no conflict of interest

3 Canadian Cohort of HIV Why do we need a cure for + Slow progressors (CCHSP) HIV? *Slow progressor: HIV + study participant meeting any of the above definitions and having no signs of AIDS **Antiretroviral treatment

4 CD4 T-cell decline in HIV-infected slow progressors

5 Loss of immunological and virological control in SPs CD4 counts/mm NVC # HIV RNA copies/ml plasma CD4 cells/mm 3 at baseline (1273) HIV RNA copies/ml at baseline (1092) Years since infection (single points represent clinic visits) 0 2% 27% 10% Total number of subjects Number of subjects losing control EC VC NVC

6 Transcriptional analysis of PBMCs from subjects losing control Total PBMCs from n=5 Average loss of CD4 211 cells Average increase in VL 20 fold V1 (Before loss of control) V2 (After loss of control) Microarray analysis 1.3 fold change p< genes down-regulated 83 genes up-regulated

7 CD96 negatively regulates cytokine production by NK cells CD96 T cell-activated increased late expression (TACTILE) is expressed by both NK and T-cells Initially thought to enhance NK killing capacity through binding to the nectin receptor CD155 (PVR) Fuchs, A. et al J. Immunol Abundant expression of IFNγ Chan et al., 2014 Nat. immunol. 15(5): Martinet and Smyth, Nature Rev. Immunol

8 Potential role of CD96 in negative regulation of cell activation Bernhardt. Nat. Immunol

9 Down-regulation of CD96 on CD8 + T-cells in HIV infection IFNγ Perforin Eriksson et al., PLoS One. 2012;7(12):e51696.

10 Down-regulation of CD96 on SP T-cells upon loss of control CD4 T-cells CD8 T-cells Frequency of Max Visit 2 (Loss control) Visit 1 (Control) Frequency of Max Visit 2 (Loss of control) Visit 1 (Control) CD96 CD96

11 Down-regulation of CD96 on CD4 + and CD8 + T-cells in HIV infection NK cells Kruskal-Wallis p= CD4 + T-cells Kruskal-Wallis p=0.004 CD8 + T-cells Kruskal-Wallis p< p<0.01 p<0.05 p<0.001 p<0.05 p<0.001 MFI of CD96 MFI of CD96 MFI of CD96 HIV neg HIV + HIV + (EC) (TP) HIV neg HIV + HIV + (EC) (TP) HIV neg HIV + HIV + (EC) (TP)

12 Down-regulation of CD96 on CD8 + T-cells in HIV infection HIV neg HIV + HIV + (EC) (TP) 5.8% 47.3% 10.9% 32.1% 27.1% 10.9% CD3 + CD8 + CD45RA neg 5.4% 21% 14.8% 15.5% 25.4% 6.3% CD3 + CD8 + CD45RA + Frequency of CD96 bri Total Memory CD8 CD8 + CD45RA + Total Memory CD8 CD8+ CD45RA + Kruskal-Wallis p< HIV neg p<0.001 p<0.05 p<0.05 HIV + (EC) HIV + (TP) Frequency of CD96 bri Kruskal-Wallis p< p<0.001 p<0.05 NS HIV neg HIV + HIV + (EC) (TP) Frequency of CD96 dim Kruskal-Wallis p< NS p<0.001 p<0.05 HIV neg HIV + HIV + (EC) (TP) Frequency of CD96 dim Kruskal-Wallis p< p<0.001 p<0.05 NS HIV neg HIV + HIV + (EC) (TP)

13 Low CD96 expression is associated with a more differentiated phenotype CD8 + CD45RA + CD8 + CD45RA neg CD8 Naïve (CCR7 + CD27 + ) CD8 TEMRA (CCR7 neg CD27 neg ) CD8 CM (CCR7 + CD27 + ) CD8 EM (CCR7 neg CD27 neg ) Wilcoxon p< Wilcoxon p< Wilcoxon p< Wilcoxon p< Frequency of Naive Frequency of TEMRA Frequency of T CM Frequency of T EM CD96 bri CD96 dim CD96 bri CD96 dim CD96 bri CD96 dim CD96 bri CD96 dim CD96 bri CD45RA + CD96 dim CD45RA + CD96 bri CD45RA neg CD96 dim CD45RA neg

14 Low CD96 expression is associated with a senescent phenotype CD8 + CD45RA + p< CD8 + CD45RA + p< CD8 + CD45RA neg p< CD8 + CD45RA neg p< CD27 + CD28 + DP CD27 neg CD28 neg DN CD27 + CD28 + DP CD27 neg CD28 neg DN CD96 bri CD96 dim CD96 bri CD96 dim CD96 bri CD96 dim CD96 bri CD96 dim Frequency of CD38+ cells Total CD45RA + CD8 p=0.001 CD96 bri CD96 dim Total CD45RA neg CD8 Total CD45RA neg CD8 p< p= Frequency of CD38 + cells MFI of CD57 + MFI of CD57 + Total CD45RA + CD8 p=0.001 CD96 bri CD96 dim CD96 bri CD96 dim CD96bri CD96 dim

15 Down-regulation of CD96 is associated with increased activation and increased PD-1 expression Spearman r= p=0.009 Spearman r= p=0.05 CD96 MFI on Memory CD CD38 MFI on Memory CD8 CD96 MFI on Memory CD PD-1 MFI on Memory CD8 HIV + subjects (TP and EC, n=10/group)

16 Poor replicative capacity of CD96 dim CD8 + and CD4 + T-cells CD8+CD45RA neg CD8+CD45RA neg CD96+ CD8+CD45RA neg CD96 neg CD8 CD4 FSCA Counts Counts FSCA Counts Counts CD96 CFSE CFSE CD4+CD45RA neg CD4+CD45RA neg CD96+ CD4+CD45RA neg CD96 neg CD96 CFSE CFSE

17 Conclusions Loss of immunological control (CD4 decline) and virological control (increased VL) is associated with significant down-regulation of CD96 expression on T-cells, mainly CD8 Down-regulation of CD96 on CD8 T-cells is associated with a more differentiated phenotype (increased frequencies of effector and terminally differentiated cells) Cells down-regulating CD96 expression in HIV infection are enriched with CD27 neg CD28 neg Double negative phenotype These cells express significantly high levels of CD57, typical phenotype of senescent cells These cells were previously shown to express high levels of perforin and IFNγ However, T-cells with the CD96 low senescent phenotype have inferior capacity to proliferate compared to CD96 + cells

18 Future work Validating CD28 and CD27 down-regulation at the transcriptional level (permanent replicative senescence) Measuring the telomere and/or assessing the telomerase activity in CD96 bri versus CD96 dim CD8 + memory T-cells Characterizing the exhaustion phenotype of CD8 + memory T-cells within the CD96 bri and CD96 dim subpopulations Investigating CD96 signaling in T-cells by over-expression of CD96 and triggering with anti- CD96 antibodies using bead systems (in the presence of anti-cd3/anti-cd28 antibodies) Investigating the impact of CD96 knockdown with RNA interference on T-cell activation, cytokine production, proliferation and senescence in culture Transcriptional profiling of CD96 bri and CD96 dim ex vivo from memory CD8 T-cells from HIVinfected subjects by microarray to identify a signature associated with T-cell senescence

19 Why do we need a cure Thanks for HIV? CRCHUM Dr Cecile Tremblay Dr Petronela Ancuta Pascale Kouassi Mohamed Sylla Stephanie Matte Annie Chamberland Odalis Asin-Milan Ahmed Fouda Annie Gosselin Tevy-Suzy Tep Sylvia Pouvreau Thomas Fabre McGill University Dr Nicole Bernard Dr Jean-Pierre Routy University of Montreal Dr Gerardo Ferbyre Patients

20 Backups

21 regulation of T cells: Too Much of Good Profound loss of metabolic, functional and cytolytic capacities KLR G-1?? Tim-3 Recurring exposure and activation/inhibition PD-1 CTLA- 4 CD4/8 LAG- 3 IDO Specific up-regulation during HIV infection Expression correlates with disease progression (phase of infection, viral load, CD4 counts, CD4/CD8 ratio) 1- IL-2 2- Proliferation 3- TNFα 4- INFγ

22 EC VC NVC

23 Subject ID Time of observation CD4 decline/year P value VL Inc/Year P value Started ART EC ± ± NO VC ± < ± NO ± ± < NO ± ± < NO ± < ± Yes ± < ± Yes NVC ± < ± Yes ± < ± Yes ± < ± < NO ± ± NO ± ± NO ± < ± < Yes ± < ± Yes ± < ± Yes ± ± Yes