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
Paris Est Créteil, Inserm U955, Créteil, France
3Henri Mondor Hospital, APHP, Créteil, France
2Université
XVIII International AIDS Conference | July 18-23 2010 | Vienna, Austria
CD8 T cell differentiation and functional
maturation
Differentiation
IFNg
Cytotoxicity
Proliferation
Naïve
Central
memory
CD45RA+
CCR7+
CD28+
CD27+
Effector
Memory
Effector
memory
1
Effector
memory
2
CD45RA-/CD45RO+
Effector
Terminal
effector
CD45RA+
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)

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)
HYPOTHESIS AND OBJECTIVE
We hypothesized that the expansion of Treg in HIVinfected 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 POPULATION
HIV-1+cART+ subjects (n=14), CD4 >350 cells/ml, VL < 50 HIV RNA copies/ml
STUDY DESIGN
PMNC
D0, flow cytometry
(CD45RA/CCR7/CD27/CD28/CD3/CD8)
Treg depletion,
anti-CD25 DynaBeads
proportions of CD8 T subsets
PMNC,Treg-
PMNC, Treg+
CFSE staining, polyclonal stimulation: immobilized anti-CD3 (5 mg/ml)
D5, flow cytometry
(CFSE/CD45RA/CCR7/CD27/CD28/CD3/CD8)
COMPARISON
proportions and proliferation rates of CD8 T subsets
CD8 T SUBSETS PHENOTYPING :
GATING STRATEGY
CD8 T CELL SUBSETS:
PROLIFERATION RATES
Gated CD3+CD8+ Ly
Gated CD8 T subset
Treg+
Treg-
N
E
M
CFSElow
81%
CFSElow
68 %
CD45RA
TE
CD27
1. CD27+CD45RA+ Naïve, N
2. CD27+CD45RA- Memory, M
3. CD27- CD45RA- Effector, E
4. CD27- CD45RA+ Terminal Effector, TE
CFSE
CFSE-stained Treg+ and Treg- PMNC were
stimulated with 5mg/ml immobilised anti-CD3
% CFSElow cells was determined on D5
Polyclonal stimulation in the presence of Treg
results in a decreased rate of CD8 T cell proliferation
**
100
% CFSElow CD8 T
90
80
70
60
50
40
Treg+
Treg+
CD8
TregTregCD8
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 lower
proliferation rates within E and TE subsets , while M CD8 are not
significantly affected
*
**
*
100
80
60
Treg+
Treg-
40
20
0
N
CD27+45RA+
CD27+45RAM
CD27-45RAE
CD27-45RA+
TE
%CFSE low CD8 T
%CFSE low CD8 T
100
**
50
0
E
TE
* p<0.05, **p<0.01, Student’s T-test
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
Treg inhibit the differentiation of polyclonally stimulated naive CD8 T
cells into CD27-CD45RA- effectors
60
% of CD8 T cells
ns
50
*
40
*
30
ns
20
D0
D5,Treg+
D5,Treg-
10
0
CD27+RA+
N
CD27+RA-
M
Cd27-RA-
E
CD27-RA+
TE
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)
Analysis of memory CD8 T cells: CM/EM
Gated CD3+CD8+ Ly
Gated CD45RA-CD27+CD8 T
EM1
CD45RA
CD28
CM
M
CD27
EM2
CM
CCR7
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
Polyclonal stimulation in the presence of Treg results in a
significant increase of CM (CCR7+) CD8 T cells
70
% of M CD8 T cells
60
50
40
NA
** *
D5,Treg+
30
D5, Treg-
20
10
0
CM
CCR7+
EM1
R7-28+
EM2
R7-28-
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)
Polyclonal stimulation in the presence of Treg results in a
significant increase of CM (CCR7+) CD8 T cells
**
% of M CD8 T cells
60
*
50
D0
40
Treg+
30
Treg-
20
10
0
NS
NS
Treg+
Treg-
D5, anti-CD3
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)
HIV+ specific
CD8
T are
mostly of EM2 phenotype
CD8
T cell
differentiation
Treg
IFNg
Cytotoxicity
Proliferation
Naïve
Central
memory
CD45RA+
CCR7+
CD28+
CD27+
Effector
Memory
Effector
memory
1
Effector
memory
2
CD45RA-/CD45RO+
Effector
Terminal
effector
CD45RA+
Conclusions

Increased frequency of Treg in HIV infection
significantly decreases the rate of CD8 T cell
proliferation, affecting specifically E and TE
subsets.

Polyclonal stimulation of CD8 T cells in the
presence of Treg results in decreased
differentiation of effectors and in accumulation of
CM cells.

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?
ACKNOWLEDGEMENTS
Henri Mondor Hospital, APHP,
Université Paris Est Créteil, Inserm U955,
Créteil, France
Dr. Matthieu Carrière
Dr. Mohammad-Ali Jenabian
Dr. Christine Lacabaratz
Pr. Jean-Daniel Lelièvre
Pr. Yves Lévy
PHC Rila 2009 (Bulgarian Ministry of Education and
Sience / Ministry of Foreign and European Affairs,
France; Sidaction - France; ELTA’90 Ltd - Bulgaria
National Reference Laboratory of Immunology,
National Center of Infectious and Parasitic
Diseases, Sofia, Bulgaria
Dr. Maria Muhtarova
Dr. Draganka Stankulova
Antoaneta Mihova
Pr. Hristo Taskov