1. HIV-infected patients, and have proliferative defects
Activated Th1 and Th2-committed central memory CD4+ T Cells are over-represented in
HIV-infected patients, and have proliferative defects 
S. Perez-Patrigeon1, E. Espinosa2, A. Mondragón-Eguiluz1, G. Olvera García2, U. Orbe2
1 Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico
2 Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas, Mexico City, Mexico
Figure 1.- Activated pre-Th1 cells and activated pre-Th2 cells are over-represented within CD4+ TCM of HIV infected patients
Figure 4.- % CD38+ pre-Th1 of CD4+ TCM cells correlated with proliferative response of CD4+ T cells to CMV
Background
The mechanisms linking chronic immune activation and CD4+ T cell depletion in HIV infection remain poorly understood. In chronic HIV infection, loss of remnant critical levels of effector memory CD4+ T cells has been related to a failure of central memory CD4+ T cells (TCM) to replenish this cell pool by proliferation and differentiation [1-4]. We hypothesized that TCM cells from HIV-infected patients would show an activation-driven commitment to differentiate to effector memory cells instead of proliferating upon TCR stimulation.
CD38+ pre-Th1*
16.8%
CD38+ pre-Th2
2.5%
Pre-Th2 CD38-
6.2%
Pre-Th1 CD38-
2.6%
Other CD38+
45.1%
Other CD38-
17.1%
46.1%
CD38+ pre-Th1
4.9%
4.6%
CD38+ pre-Th2**
12.2%
8.4%
35.5%
0.5 1
1.5 2
2.5 3
3.5
Percent Ki67+ of total CD4+ T cells 5 10 15 20 25 30 35 40
HIV+
HIV-
R=0.76, p=0.005
Materials and methods
Using flow cytometry, CD4+ TCM were identified among PBMCs of 6 untreated HIV infected patients and 5 controls. Pre-Th1 and pre-Th2 effector-committed cells were identified as CXCR5- and either CXCR3+ or CCR4+, respectively. Activation was determined by CD38 expression. Proliferative responses to SEB and HIV and CMV peptides were determined by Ki67 induction. Groups were compared with Mann-Whitney’s U test. Variables within groups were compared using Wilcoxon’s signed rank test. Correlations between variables were determined by linear regression.
Results
CD4+ TCM cells from patients had higher %activated Th1-committed cells and activated Th2-committed cells than controls (p= 0.037, p=0.025 correspondingly) (Fig.1). CD4+ TCM cells with a phenotype indicating pre-Th2 differentiation commitment had a decreased polyclonal proliferative response to SEB compared with uncommitted TCM cells (p=0.043 in patients, p=0.046 in controls) (Fig.2), while pre-Th1 CD4+ TCM cells from controls proliferated less frequently than uncommitted cells in response to SEB (p=0.028) (Fig. 3).
% CD38+ Pre-Th1 of TCM CD4+
Figure 5.- %CD38+ pre Th2 of TCM CD4+ cells correlated with proliferative response of CD4+ T cells to CMV
Total CD4+ TCM of Healthy donors
Total CD4+ TCM of HIV infected Patients .5 1
1.5 2
2.5 3
3.5
Percent Ki-67+ of total CD4+ T cells 4 5 6 7 8 9
HIV+
HIV-
R=0.78, p=0.005
Pre Th1
Pre Th2
Other
CD38+
Gating strategy
Figure 2.- Pre-Th2 CD4+ TCM proliferated less frequently than uncommitted cells upon polyclonal stimulus
PBMCs
Gate: lymphocytes
Gate :CD4+ T cells
Gate: CD4+ T cells
Gate: Tcm CD4+
Committed
Gate: non-committed
Gate: CXCR3+ CCR4-
Gate: CXCR3- CCR4+
CD4-APC-Cy7
Ki-67-FITC
Forward scatter
Side scatter
CCR7-PE-Cy-7
CD45RA-PE-Texas-Red
CXCR5-PerCP-Cy5.5
CD38-PerCP-Cy5.5 5 10 15 20 25 30 35 40
% Ki-67+cells
HIV+
HIV-
*p=0.043
*p=0.046
# events
# events
% CD38+ pre-Th2 of TCM CD4+
Conclusions
Activated central memory CD4+ T cells with a phenotype indicating commitment to differentiate to Th1 and Th2 effector memory cells are over-represented among CD4+ TCM cells from HIV+ patients. Their lower proliferative responses to TCR engagement could render CD4+ TCM less capable of regenerating the CD4+ T cell pool.
The correlation between the frequency of activated and committed CD4+ TCM cells and anti-CMV response suggests that activation-associated commitment in HIV infection is partially driven by CMV infection.
Pre-Th2
Uncommitted
Proliferative responses of Tcm CD4+ with pre-Th2 (CXCR3- CCR4+) and uncommitted (CXCR5-) phenotype to SEB.
Figure 3.- Pre-Th1 CD4+ TCM proliferated less frequently than uncommitted cells upon polyclonal stimulus only in healthy controls
References
1. S. F. Sieg, C. V. Harding and M. M. Lederman, "HIV-1 infection impairs cell cycle progression of CD4(+) T cells without affecting early activation responses", Journal/J Clin Invest, vol. 108, no.5, pp , 2001
 2. M. Guadalupe, E. Reay, S. Sankaran, et al., "Severe CD4+ T-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy", Journal/J Virol, vol. 77, no.21, pp , 2003
3. S. Mehandru, M. A. Poles, K. Tenner-Racz, et al., "Primary HIV-1 infection is associated with preferential depletion of CD4+ T lymphocytes from effector sites in the gastrointestinal tract", Journal/J Exp Med, vol. 200, no.6, pp , 2004
4 . A. Okoye, M. Meier-Schellersheim, J. M. Brenchley, et al., "Progressive CD4+ central memory T cell decline results in CD4+ effector memory insufficiency and overt disease in chronic SIV infection", Journal/J Exp Med, vol. 204, no.9, pp , 2007 .
CXCR3-PE
*p=0.028
Uncommitted
Pre-Th1 5 10 15 20 25 30 35 40
% of Ki-67+cells
CCR4-AF647
HIV-
HIV+
Contact
Proliferative responses of TCM CD4+ with pre-Th1 (CXCR3+ CCR4-) phenotype and uncommitted (CXCR5-) phenotype to SEB.