High Affinity HIV-1 Specific CTL Become Exhausted in Early HIV-1 High Affinity HIV-1 Specific CTL Become Exhausted in Early HIV-1 InfectionInfection

Abstract C177 Abstract C177

Introduction Introduction

ReferencesReferences

•CD8 PD-1 and LAG-3 expression are positively correlated with viral load (p=0.017; p=0.0001)

•HIV-1 specific T cells are significantly more exhausted than non-specific CD8+ T cells (p<0.0001). Except B*57 TW10 responses, when considering PD-1 expression

•High affinity HIV specific CD8+ are significantly more exhausted (p<0.01) than CD8+ T cells of lower affinity

ResultsResults

Questions Questions• Does T cell exhaustion occur in early HIV infection?

• Are HIV specific CD8+ T cells exhausted and does the restricting HLA influence the CTL exhaustion?

• Does the affinity of the HIV specific CD8+ response influence exhaustion?

High affinity CTL responses express significantly more exhaustion markers then wild- type affinity CTL responses

Conclusions Conclusions

•High affinity specific CTL (detected with CD8null tetramer) are significantly more exhausted then wild-type affinity CD8 responses for all HLA types tested (p<0.01)

AcknowledgementsAcknowledgementsWe would like to thank all SPARTAC trials participants

BackgroundThe magnitude of the Cytotoxic T Lymphoyte (CTL) response in early HIV infection plays an important role in determining plasma viral load; consequently CTL functional impairment or exhaustion in early infection could influence clinical outcome. Here we present evidence that exhaustion occurs in high affinity HIV-specific CTL during early HIV infection.

MethodsPeripheral blood mononuclear cells (PBMC's) drawn from HIV participants enrolled into the SPARTAC trial of short course therapy in primary infection were HLA-typed and analysed by IFN ELISPOTS to determine their CTL responses (n=148). Participants responding to epitopes for HLA A*0201 ILKEPVHGV, B*2705 KRWIILGLNK, B*5701 TSTLQEQIGW and B*5701 KAFSPEVIPMF (n=35) were analysed by FACS. We stained with CD8wild-type and CD8null tetramers, to detect high affinity CTL responses, and also used exhaustion markers PD-1, TIM-3 and LAG-3.

ResultsThere was a significant positive correlation between plasma viral load and percentage of PD-1 positive CD8 T cells and LAG-3 positive CTL (p=0.0017: r2= 0.05718 and p=0.0001: r2= 0.1272). HIV-specific CTL had significantly higher levels of PD-1 (p<0.001), LAG-3 (p<0.001) and TIM-3 (p<0.001) than non-HIV specific CTL, independent of viral load. High affinity HIV-specific CTL responses expressed significantly higher levels of PD-1 (p<0.001), LAG-3 (p<0.001) and TIM-3 (p<0.001) than HIV-specific CD8 with lower wild-type affinity.

ConclusionsHigh affinity HIV-specific CTL express significantly higher levels of exhaustion markers, PD-1, TIM-3 and LAG-3 than HIV-specific CTL with lower wild-type affinity for the same epitope in early HIV infected participants. High affinity CTL exhaustion may contribute to limited viral control in HIV infection.

•The presence of particular Class I HLA alleles are associated with lower plasma viral loads and improved clinical progression. The presence of B*57 and B*27 alleles are associated with enhanced HIV control while A*02 has a neutral impact

•It is as yet unclear why some Class I HLA molecules are associated with better viral control then others, however it may be influenced the quality of the anti- HIV Cytotoxic T lymphocyte (CTL) response which they elicit

•The quality of the CTL response is influenced by the affinity of the CTL T Cell Receptor (TCR) for its cognate HLA/epitope complex. CTL with TCR that have a higher affinity for for the HLA/epitope complex are more able to clear antigen, then CTL with lower affinity 1,2

•CTL responses to virus antigen can also become functionally impaired, or exhausted 3,4. CTL exhaustion is defined by the inability of a CTL to produce cytokines or proliferate in response to environmental stimulus. CTL exhaustion can be characterized by the expression of surface markers such as PD-1, TIM-3 and LAG-3 5,6, 7

•As the CTL responses in early infection play an important role determining plasma viral load, and thus clinical outcome, it is important to determine if T cell exhaustion occurs during early HIV infection

0 2 4 6 8 10 12 14 16 18 20100

1000

10000

100000

1000000

10000000

% of PD-1 Positive CD8 Cells0 10 20 30 40

100

1000

10000

100000

1000000

10000000

% of LAG-3 Positive CD8 Cells

P=0.017

r2=0.0572P=0.0001

r2=0.1272

0 103 104 105

<APC-A>: PD-1

0

20

40

60

80

100

% o

f Ma

x

0 102 103 104 105

<PE-A>: TIM-3

0

20

40

60

80

100

% o

f Ma

x

0 102 103 104 105

<FITC-A>: LAG-3

0

20

40

60

80

100

% o

f Ma

x

PD-1

TIM-3 LAG-3

0

10

20

30

40

50

60

70

80

***

***

0

10

20

30

40

50

60

***

Results Results Results Results

0

5

10

15

20

25

30

35

40

45

***

** **

0

25

50

75

100

Exhaustion No Exhaustion

** ** ** **

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

Methods Methods• Peripheral blood mononuclear cells (PBMC's) were drawn from HIV participants enrolled into the

SPARTAC trial (Short Pulse Anti-Retroviral Therapy At HIV Seroconversion).

• At baseline, when all patients were treatment naïve, each individuals CTL responses were determined by IFN ELISPOTS and HLA typed to 4 digits (n=148)

0 103 104 105

<Qdot 605-A>: Tetramer

0

103

104

105

<P

E-C

y5-A

>: C

D8

70.8 5.76

0.09623.3

0 103 104 105

<Qdot 605-A>: Tetramer

0

103

104

105

<P

E-C

y5-A

>: C

D8

77.5 1.14

0.1321.3

0 103 104 105

<Qdot 605-A>: Tetramer

0

103

104

105

<P

E-C

y5-A

>: C

D8

77.9 0

022.1

Methods Methods (a) Negative (b) Null Tetramer (c) Wild-Type Tetramer

Representative FACS staining of SPARTAC participant with CD8wild-type and CD8null gated on Live, CD3+ ,CD8+ lymphocytes (a) Negative Control (b) CD8nul Tetramer (C) CD8wild-type tetramer

Representative FACS staining of SPARTAC participant gated on Live, CD3+, CD8+ lymphocytes stained with (d) PD-1 (e) TIM-3 (f) Lag-3. Blue line- Negative control; Red line- Isotype control; Green line- Positive

• Participants who responded to appropriate peptides, A*0201 ILKEPVHGV (n=13), B*2705 KRWIILGLNK (n=7), B*5701 TSTLQEQIGW (n=10) and B*5701 KAFSPEVIPMF (n=5) were stained with CD8wild-type and CD8null tetramers

• CD8wild-type and CD8null tetramers detect specific CTL with TCR avidity with KD of ≥ 80 uM and ≥ 50 uM respectively. Additionally T cell exhaustion markers PD-1, TIM-3 and LAG-3 were used to stain HIV specific and non-specific CD8 T cells

• CD8null events are also represented on CD8wild-type plots. Thus CD8wild-type responses are determined by correcting for the CD8null population

PD-1 and LAG-3 Expression on total CD8 T cells is positively correlated with viral load in Early HIV infection

Tetramer

Tetramer

Tetramer

CD8

CD8

CD8

• PD-1 and LAG-3 expression on the total CD8 T cell population was positively correlated with plasma HIV viral load (p=0.017 and p=0.0001 respectively)

• Expression of Tim-3 did not correlate with plasma HIV viral load (p=0.9321- data not shown)

Correlation between (a) PD-1 expression and (b) LAG-3 expression with plasma viral load

•PD-1 expression is significantly higher on A*02 IV9, B*27 KK10 and B*57 KF11 responses then tetramer negative CD8 cells (p<0.0001)

•PD-1 expression on B*57 KF11 and B*57 TW10 specific CTL was significantly lower then that on B*27 KK10 and A*02 IV9 CTL (p<0.01)

•There is no significant difference in PD-1 expression between B*57 TW10 specific CTL and the tetramer negative CD8 population

PD-1, TIM-3 and LAG-3 expression is significantly higher on HIV specific CTL then none HIV specific CTL

•TIM-3 expression is significantly higher on A*02 IV9, B*27 KK10,B*57 KF11and B*57 TW10 responses then tetramer - CD8 cells from HIV+ individuals (p<0.0001)

•TIM-3 is expression is significantly lower on both B*57 KF11 and TW10 CTL then A*02 IV9 and B*27 KK10 (p<0.0001)

•LAG-3 expression is significantly higher on HIV specific CTL responses then the tetramer negative CD8 population (p<000.1) However, there is no significant difference in LAG-3 expression between HIV specific responses across the HLA types tested

(c) Percentage PD-1 expression on A*02 IV9, B*27 KK10, B*57 KF11 and B*57 TW10 Hiv specific CD8+ T cells

(d) Percentage TIM-3 expression on A*02 IV9, B*27 KK10, B*57 KF11 and B*57 TW10 Hiv specific CD8+ T cells

(e) Percentage Lag-3 expression on A*02 IV9, B*27 KK10, B*57 KF11 and B*57 TW10 Hiv specific CD8+ T cells

All samples gated on Live, CD3+, CD8+ cells and detected with CD8wild-type tetramers

*** P<0.0001

** P<0.01

Negative

Isotype Control

Positive

(f) Exhaustion (blue) defined as the expression either PD-1, TIM-3 or LAG-3. No Exhaustion (Red) Tetramer positive but negative for any marker of exhaustion. CD8null events are also represented on CD8wild-type plots CD8wild-type responses are

determined by correcting for the CD8null population

** P<0.01

(c) (d)

(e)

(a) (b)

n=13 n=7 n=10 n=5

1 Speiser et al., J. Immunol 1992 ; 2 Alexander-Miller et al., Proc. Natl. Acad. Sci. USA 1996; 3Zajac et al., 1998 J. Exp. Med; 4 Gallimore et al., 1998 J. Exp. Med

5 Day et al., Nature; 6 Jones et al., J. Exp. Med. 2008; 7 Blackburn et al., Nature Immunology 2008

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.Stephen Hickling1,3,5 and Matthias Hoffmann1,2, Elizabeth Hamlyn3 , Nicola Robinson1, Helen Brown1, Stuart Sims1, Andy Sewell4, Angela Maclean3,5 John Frater1, Rodney Phillips1,5, and the SPARTAC trial investigators