Exhaustion of activated CD8+ T cells predicts disease progression in primary HIV-1 infection G.E. Martin*, N. Pantazis*, M. Hoffmann*, S. Hickling, J. Hurst, J. Meyerowitz, C.B. Willberg, N. Robinson, H. Brown, M. Fisher, S. Kinloch, A. Babiker, J. Weber, N. Nwokolo, J. Fox, S. Fidler¶, R. Phillips¶, J. Frater¶, SPARTAC and CHERUB Investigators *, ¶ These authors contributed equally to this work genevieve.martin@ndm.ox.ac.uk The presenting author has no conflict of interest to declare Abstract no. WEAA0105LB

BACKGROUND: T cell exhaustion during HIV infection T cell dysfunction, or exhaustion, occurs during HIV infection[1-4] and may contribute to disease outcomes. Co-inhibitory receptor expression is associated with functional T cell exhaustion. PD-1, Tim-3, Lag-3 Linked to activation Role of persistent antigen We know that - the rate at which HIV-infected individuals develop AIDS is highly variable - T cell immunity plays an important role. T cell dysfunction, or exhaustion, is a hallmark of chronic viral infections including HIV and may contribute to disease outcomes. Upon antigen exposure T cells up-regulate inhibitory receptors (INCL PD-1, TIM-3 and LAG-3) Function acute setting, limit T cell responses. In the context of persistent antigen exposure, this expression is associated with a number of functional defects – decreased proliferation, cytokine production and effector functions – that characterise T cell exhaustion. Day CL et al (2006), Nature, 443:350-354 Blackburn SF et al (2008), Nature Immunology, 10:29-37 Jones RB et al (2008), Journal of Experimental Medicine, 205:2763-2779 Trautmann L et al (2006), Nature Medicine, 12:1198-1202 Image: Chen L & Flies DB (2013), Nature Reviews Immunology, 13(4):227-42

The expression of markers of T cell exhaustion (including PD-1, Tim-3 and Lag-3) has been linked to the size of the HIV reservoir[1] and viral rebound after therapy cessation[2]. It is not known if T cell exhaustion during early HIV infection impacts on disease outcomes. CD4 T cells CD8 T cells PD-1 Tim-3 Lag-3 Proportion with undetectable HIV-1 plasma viral load (<400) We do know that exhaustion marker expression is linked to reservoir size. Recent work from within our group on SPARTAC trial (which I will also talk about today) Orientate to slide and explain axes Expression of PD-1, Tim-3 and Lag-3 on both pre-therapy predicted time to viral rebound following TI. These are survival curves that show time on the x-axis and the proportion of individuals without rebound on the y axis Individuals were split based on expression of markers – blue is low Can see that on both CD4 and CD8 T cells, blue line has longer without viral rebound. We think it probably is, but it has not been demonstrated that T cell exhaustion is linked to clinical outcomes, particularly in early infection Time since treatment interruption (weeks) Chomont N et al (2009), Nature Medicine, 15:893-900 Hurst J et al(2015), Nature Communications, 6:8495

METHODS Measurement of PD-1, Lag-3 and Tim-3 on CD8 T cells pre-ART Assessment of impact on disease progression (CD4 count <350 cells/μL or initiation of long-term ART) subset (n=122) of UK participants SPARTAC trial was a RCT of short-course ART in primary HIV infection (PHI). [1] 3 arms: 12 weeks ART 48 weeks ART No therapy SPARTAC RCT; 2003-2007 in 8 countries Subset Disease progression The expression of PD-1, Lag-3 and Tim-3 was measured on CD8 T cells from individuals with primary HIV infection (PHI; within 6 months of infection) prior to the initiation of antiretroviral therapy (ART). Also measured CD38 - known relationship between activation and progression in chronic infection The impact of these markers on disease progression (defined here as the trial primary endpoint time to CD4 T cell count <350 cells/μL or initiation of long-term ART) within SPARTAC was assessed, with analyses adjusted for appropriate covariates (ART received, baseline CD4+ T cell count and viral load [VL]). Fidler S et al (2013), NEJM, 368:207-217

The same was not seen for Tim-3 and Lag-3 RESULTS KAPLAN MEIERS PD-1 expression on bulk and CD38+ CD8 T cells predicted clinical progression The same was not seen for Tim-3 and Lag-3 Tim-3 Lag-3 SPEND MORE TIME ON THIS These plots show the relationship between time and clinical progression (remind) in SPARTAC participants. Focus intially on left hand panel Participants have been split into quartiles based on PD-1 expression levels. Dose- effect. More PD-1 is faster disease progression Right hand panel shows this on CD38+ CD8 T cells, this is also seen. The same was not seen for TIM-3 and Lag-3

This figure from the original SPARTAC analysis shows the progression to endpoint base on time from seroconversion to randomisation. Demonstrates an intriguing effect of time from seroconversion to randomisation on disease progression – earlier randomisation progressed faster. Fidler S et al (2013), NEJM, 368:207-217

Randomised ≤12 weeks from seroconversion Bulk CD8 T cells The impact of PD-1 on disease progression was most evident in participants sampled within 12 weeks of seroconversion SLOW Because of this finding - I will show you this same analysis stratified by time from seroconversion. We looked at the relationship between PD-1 expression and clinical progression in individuals who who were within 12 weeks and >12 weeks of seroconversion. We looked at this on bulk (top panels) or CD38+ (bottom panels) CD8 T cells. These plots are split at the median, rather than on quartiles with the solid line showing individuals with low PD-1 levels When we split the analysis this way, we see the ability to predict disease progression is only seen when PD-1 is measured <12 weeks of seroconversion (left panels). CD38+ CD8 T cells

Unadjusted HR (95% CI; p value) Adjusted HR (95% CI; p value) ART CD4 T cell count Viral load Bulk CD8s PD-1 1.68 (0.98 - 2.86; 0.057) 1.75 (1.01 - 3.01; 0.045) 1.76 (1.01 - 3.09; 0.047) 0.99 (0.53 - 1.83; 0.96) Tim-3 0.99 (0.73 - 1.33; 0.928) 0.98 (0.73 - 1.31; 0.88) 1.00 (0.73 - 1.35; 0.98) 0.91 (0.66 - 1.25; 0.55) Lag-3 1.30 (0.95 - 1.77; 0.10) 1.35 (1.00 - 1.84; 0.054) 1.46 (1.05 - 2.04; 0.024*) 1.07 (0.73 - 1.58; 0.72) CD38+ CD8s 2.43 (1.16 - 5.12; 0.019) 2.71 (1.28 - 5.74; 0.009*) 2.06 (0.96 - 4.42; 0.064) 0.93 (0.37 - 2.33; 0.88) 1.81 (1.14 - 2.88; 0.012) 1.87 (1.17 - 2.99; 0.009*) 2.05 (1.25 - 3.34; 0.004*) 1.55 (0.91 - 2.65; 0.11) 1.62 (0.99 - 2.66; 0.057) 1.84 (1.10 - 3.09; 0.020*) (1.17 - 3.61; 0.012*) (0.49 - 2.03; 1.00) Associations with endpoint are more marked on CD38+ CD8s than bulk CD8 T cells Associations with endpoint survive adjustment for ART received and CD4 count, but not VL The use of cox models allow for adjustment for appropriate covariates. I know there is a lot of data here – will talk you through the main findings Table shows the relationship between each marker and disease progression (REDEFINE). Show unadjusted, adjusted for ART (which trial arm), basline CD4 and VL First observation Second observation – does not survive adjustment for baseline viral load * Indicates significance at the 0.05 level after adjustment for multiple comparisons

PD-1 Tim-3 Lag-3 Bulk CD8s CD38+ CD8s To us, this observation raised the question of whether this represents exhaustion or just activation. PD-1, Tim-3 and Lag-3 expression on bulk and CD38+ CD8s correlated with VL

Exploration of expression on memory subsets and in association with T-bet, Eomes and CD39 Measurement of PD-1, Lag-3 and Tim-3 on CD8 T cells pre-ART subset (n=16) of participants One of our concerns was that this finding could be all due to activation, no exhaustion. So, sampling limitations – another primary infection cohort. The way we answered this question was to turn to a different primary infection cohort. Introduction to HEATHER HEATHER is an observational cohort of individuals who commenced treatment during primary HIV infection

Expression of PD-1 during PHI is highest on effector memory (TEM) CD8 T cells Expression of PD-1 highest on EM. Same with Tim-3 and Lag-3

PD-1, Tim-3 and Lag-3 expression on CD8s during PHI is related to known surrogates of functional exhaustion To further define if PD-1, Tim-3 and Lag-3 expression in this context represented exhaustion – looked at 2 surrogates of exhaustion ! Combination of transcription factors – shown in murine models and human context to represent functional exhaustion. CD39 – HIV to represent exhaustion TbetdimEomeshi CD8 T cells are exhausted[1] CD39 expression marks exhausted CD8 T cells[2] Buggert M et al (2014), PLoS Pathogens, 10:e1004251 Gupta PK et al (2015), PLoS Pathogens, 10:e1005177

Summary of findings Expression of markers of T cell exhaustion, PD-1, Tim-3 and Lag-3, measured in early HIV infection are associated with clinical progression Greatest effect on CD38+ CD8 T cells More marked within 12 weeks of seroconversion Expression of these markers during PHI is highest on TEM CD8 T cells, and is associated with known surrogates of exhaustion Viraemia during early HIV infection may drive exhaustion of effector CD8 T cells, impacting upon subsequent disease outcomes

We thank the participants of SPARTAC and HEATHER Acknowledgments We thank the participants of SPARTAC and HEATHER CHERUB Collaboration * § ¶ ♢ SPARTAC Sarah Fidler * Nikos Pantazis ‡, § Martin Fisher Sabine Kinloch § Wolfgang Stöhr ‡, § Abdel Babiker ‡, § Rodney Phillips ♢ Jonathan Weber * SPARTAC Trial Investigators HEATHER John Frater ♢ Kholoud Porter ‡, § Jodi Meyerowitz ♢ Nneka Nwokolo Julie Fox ¶ CHERUB Steering Committee HEATHER Investigators University of Oxford John Frater Rodney Phillips Matthias Hoffmann Stephen Hickling Jacob Hurst Nicola Robinson Helen Brown Klenerman Group Christian Willberg Thank: Participants John/Sarah CHERUB Funders Work presented here has recently been accepted for publication as Hoffman M*, Pantazis N* and Martin GE* et al (2016), PLoS Pathogens [in press] ‡ Funded by: