Treg exert differential effects on the proliferation and differentiation of CD8 T cell subsets in chronic HIV-1

infection M. Nikolova1, M. Muhtarova1, M. Younas2, J.D. Lelievre2,3, H.

Taskov1, Y. Levy2,3 1National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria

2Université Paris Est Créteil, Inserm U955, Créteil, France 3Henri Mondor Hospital, APHP, Créteil, France

XVIII International AIDS Conference | July 18-23 2010 | Vienna, Austria

CD45RA+

Centralmemory

Effectormemory

1

Effector Terminal effector

Effector memory

2

Naïve Memory Effector

CD8 T cell differentiation and functional maturation

CD45RA-/CD45RO+CCR7+CD28+CD27+

CD45RA+

IFNg Cytotoxicity

Proliferation

Differentiation

Treg are expanded in acute and chronic HIV-1 infection, and inhibit effector CD8 T cell responses in vitro (Weiss L. et al, Blood 2004; Kinter A. et al, J. Exp. Med 2004)

Background & Rationale

CD8 T memory/effector subset balance determines the control of chronic viral infections

HIV infection is characterized by a decreased proliferative capacity of CM CD8 T cells and incomplete differentiation of HIV-specific effector T cells (Appay V. et al, J. Exp. Med 2000; Champagne P. et al, Nature 2001) We have previously shown that Treg (CD4+CD25highFoxP3+) influence M/E CD8 subset balance in HIV- donors: Treg inhibit the proliferation of effectors and the differentiation of memory CD8 T upon polyclonal and antigen-specific in vitro stimulation (Nikolova M. et al, Blood, 2009)

HYPOTHESIS AND OBJECTIVE

We hypothesized that the expansion of Treg in HIV-infected patients might contribute to the dysbalance between effector and memory CD8 T cells

Our objective was to investigate the effects of Treg on the proliferation and maturation of different CD8 T cell subsets in chronic HIV-1 infection

STUDY POPULATIONHIV-1+cART+ subjects (n=14), CD4 >350 cells/ml, VL < 50 HIV RNA copies/ml

STUDY DESIGN

D5, flow cytometry (CFSE/CD45RA/CCR7/CD27/CD28/CD3/CD8)

COMPARISONproportions and proliferation rates of CD8 T

subsets

PMNC

Treg depletion, anti-CD25 DynaBeads

PMNC, Treg+ CFSE staining, polyclonal stimulation: immobilized anti-CD3 (5

mg/ml)

D0, flow cytometry (CD45RA/CCR7/CD27/CD28/CD3/CD8)proportions of CD8 T subsets

PMNC,Treg-

1. CD27+CD45RA+ Naïve, N2. CD27+CD45RA- Memory, M 3. CD27- CD45RA- Effector, E4. CD27- CD45RA+ Terminal Effector, TE

CD8 T SUBSETS PHENOTYPING :GATING STRATEGY

CD8 T CELL SUBSETS: PROLIFERATION RATES

CFSElow

68 %

CFSE

Treg+Treg-

Gated CD8 T subset

CFSE-stained Treg+ and Treg- PMNC were stimulated with 5mg/ml immobilised anti-CD3 % CFSElow cells was determined on D5

N

ME

CD27

Gated CD3+CD8+ Ly

CFSElow

81%TE

CD

45R

A

Proliferation rates of Treg+ and Treg- CD8 T (n=14, D5 anti-CD3),mean 72 vs. 81 % CFSElow CD8 T

** p<0.01, Student’s T-test

Polyclonal stimulation in the presence of Treg

results in a decreased rate of CD8 T cell proliferation

40

50

60

70

80

90

100

Treg+ Treg-Treg+ CD8

Treg- CD8

% C

FSElo

w C

D8

T

**

0

50

100

0

20

40

60

80

100

CD27+45RA+ CD27+45RA- CD27-45RA- CD27-45RA+

Treg+Treg-

* **

Proliferation rates of Treg+ and Treg- CD8 T subsets (D5, anti-CD3); av. 46% vs. 74% E, (P< 0.05) and 48% vs. 85% TE, (P< 0.01)

have proliferated

N

%C

FSE

low

CD

8 T

Polyclonal stimulation in the presence of Treg results in lower proliferation rates within E and TE subsets , while M CD8

are not significantly affected

M E TE E TE

%C

FSE

low

CD

8 T

* **

* p<0.05, **p<0.01, Student’s T-test

Distribution of CD8 T subsets before and after polyclonal stimulation in the absence or in the presence of Treg; 19 and 27 % E CD8 were observed in the presence and in the absence of Treg, respectively

(* p<0.05, n=14, Student’s T-test)

Treg inhibit the differentiation of polyclonally stimulated

naive CD8 T cells into CD27-CD45RA- effectors

0

10

20

30

40

50

60

CD27+RA+ CD27+RA- Cd27-RA- CD27-RA+

D0D5,Treg+D5,Treg-

N M E TE

% o

f CD

8 T

cells

**

ns

ns

Analysis of memory CD8 T cells: CM/EM

CD27

CD

45R

A

M

Gated CD3+CD8+ Ly Gated CD45RA-CD27+CD8 T

CCR7

CD

28

Analysis of CD28/CCR7 co-expression on M (CD27+CD45RA-) CD8 T cells after polyclonal stimulation (D5, anti-CD3) in the absence or in

the presence of Treg

CM

CMEM1

EM2

0

10

20

30

40

50

60

70

CCR7+ R7-28+ R7-28-

NAD5,Treg+D5, Treg-

Polyclonal stimulation in the presence of Treg results in a significant increase of CM (CCR7+) CD8

T cells

Composition of the M CD8 T cell subset before and after polyclonal stimulation, in the absence or in the presence of Treg.

(n=14, **p<0.05, **p<0.05 in comp. to D0, Student’s T-test)

% o

f M C

D8

T ce

lls

** *

CM

EM1 EM2

NS Treg+ Treg-0

10

20

30

40

50

60

Proportions of CM cells within the memory (CD27-CD45RA+) CD8 T cell subset before and after 5 days anti-CD3 stimulation in the

presence or in the absence of Treg: Average % of CM CD8 were 15.5 vs. 24 and 16 respectively.

(n=14, *p<0.05, **p<0.01, Student’s T-test)

Polyclonal stimulation in the presence of Treg results in a significant increase of CM (CCR7+) CD8

T cells

NS

***%

of M

CD

8 T

cells

D0

Treg+

Treg-

D5, anti-CD3

Centralmemory

Effectormemory

1

Effector Terminal effector

Effector memory

2

Naïve Memory Effector

CD45RA-/CD45RO+ CD45RA+CD45RA+CCR7+CD28+

Treg

CD8 T cell differentiation HIV+ specific CD8 T are mostly of EM2 phenotype

IFNg CytotoxicityProliferation

CD27+

Polyclonal stimulation of CD8 T cells in the presence of Treg results in decreased

differentiation of effectors and in accumulation of CM cells.

Conclusions Increased frequency of Treg in HIV

infection significantly decreases the rate of CD8 T cell proliferation, affecting specifically E and TE subsets.

Globally, these results indicate that the expansion of Treg in the settings of HIV infection and generalized immune activation may contribute to the dysbalance between M and E antigen-specific CD8 T cells

The questions to answer

Subset-specific effects of Treg at the level of HIV-specific CD8 T cells

Subset-specific effects of Treg at the level of other virus-specific CD8 T cells

Mechanisms of Treg subset-specific effects in HIV infection… PD1/PDL1?

National Reference Laboratory of Immunology, National Center of Infectious

and Parasitic Diseases, Sofia, Bulgaria

Henri Mondor Hospital, APHP, Université Paris Est Créteil, Inserm

U955, Créteil, France Dr. Matthieu Carrière

Dr. Mohammad-Ali Jenabian Dr. Christine LacabaratzPr. Jean-Daniel Lelièvre

Pr. Yves Lévy

Dr. Maria MuhtarovaDr. Draganka Stankulova

Antoaneta Mihova Pr. Hristo Taskov

ACKNOWLEDGEMENTS

PHC Rila 2009 (Bulgarian Ministry of Education and Sience / Ministry of Foreign and European

Affairs, France; Sidaction - France; ELTA’90 Ltd - Bulgaria