1. 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
The presenting author has no conflict of interest to declare
Abstract no. WEAA0105LB

2. 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:
Blackburn SF et al (2008), Nature Immunology, 10:29-37
Jones RB et al (2008), Journal of Experimental Medicine, 205:
Trautmann L et al (2006), Nature Medicine, 12:
Image: Chen L & Flies DB (2013), Nature Reviews Immunology, 13(4):227-42

3. 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:
Hurst J et al(2015), Nature Communications, 6:8495

4. 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; 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:

5. 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

6. 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:

7. 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

8. 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 ( ; 0.057)
1.75 ( ; 0.045)
1.76
( ; 0.047)
0.99 ( ; 0.96)
Tim-3
0.99 ( ; 0.928)
0.98 ( ; 0.88)
1.00
( ; 0.98)
0.91
( ; 0.55)
Lag-3
1.30 ( ; 0.10)
1.35 ( ; 0.054)
1.46
( ; 0.024*)
1.07
( ; 0.72)
CD38+ CD8s
2.43
( ; 0.019)
2.71
( ; 0.009*)
2.06
( ; 0.064)
0.93
( ; 0.88)
1.81
( ; 0.012)
1.87
( ; 0.009*)
2.05
( ; 0.004*)
1.55
( ; 0.11)
1.62
( ; 0.057)
1.84
( ; 0.020*)
( ; 0.012*)
( ; 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

9. 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

10. 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

11. 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

12. 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:e
Gupta PK et al (2015), PLoS Pathogens, 10:e

13. 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

14. 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: