Background

1. PBMCs Gate: lymphocytes Gate :CD4+ T cells Side scatter Activated Th1 and Th2-committedcentral memory CD4+ T Cells are over-represented in HIV-infected patients, and have proliferative defects Ki-67-FITC Forward scatter CD4-APC-Cy7 CD38+pre-Th1* 16.8% CD38+ pre-Th1 4.9% CD38+pre-Th2 2.5% CD38+pre-Th2** 12.2% Gate: CD4+ T cells Other CD38- 17.1% CD45RA-PE-Texas-Red Other CD38- 35.5% CCR7-PE-Cy-7 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 Gate: non-committed Gate: TcmCD4+ Ki-67-FITC CXCR5-PerCP-Cy5.5 Gate: CXCR3- CCR4+ Gate: CXCR3+ CCR4- Committed Pre-Th2 CD38- 4.6% Other CD38+ 45.1% Ki-67-FITC Ki-67-FITC Pre-Th2 CD38- 6.2% Other CD38+ 46.1% Figure 1.- Activated pre-Th1 cells and activated pre-Th2 cells are over-represented within CD4+ TCM of HIV infected patients Pre-Th1 CD38- 8.4% Figure 4.- % CD38+ pre-Th1 of CD4+ TCM cells correlated with proliferative response of CD4+ T cells to CMV Pre-Th1 CD38- 2.6% 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. 3.5 CD38-PerCP-Cy5.5 CD38-PerCP-Cy5.5 3 2.5 2 Percent Ki67+of total CD4+T cells R=0.76, p=0.005 1.5 1 HIV- 0.5 HIV+ 0 0 5 10 15 20 25 30 35 40 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+ TCMcells 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 ofTCM CD4+ 3.5 3 2.5 Figure 5.- %CD38+ pre Th2 of TCM CD4+ cells correlated with proliferative response of CD4+ T cells to CMV HIV- 2 Percent Ki-67+of total CD4+ T cells HIV+ Total CD4+ TCM of Healthy donors Total CD4+ TCM of HIV infected Patients 1.5 1 R=0.78, p=0.005 .5 0 0 1 2 3 4 5 6 7 8 9 Gating strategy Figure 2.- Pre-Th2 CD4+ TCM proliferated less frequently than uncommitted cells upon polyclonal stimulus # events # events % CD38+pre-Th2 of TCMCD4+ *p=0.043 • 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. *p=0.046 40 35 30 25 % Ki-67+cells HIV- 20 HIV+ 15 Pre-Th2 Uncommitted 10 5 Proliferative responses of Tcm CD4+ with pre-Th2 (CXCR3- CCR4+) and uncommitted (CXCR5-) phenotype to SEB. 0 Figure 3.- Pre-Th1 CD4+ TCMproliferated 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. 757-764, 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. 11708-11717, 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. 761-770, 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. 2171-2185, 2007 . CXCR3-PE *p=0.028 40 CCR4-AF647 35 30 25 HIV- % of Ki-67+cells 20 HIV+ 15 10 5 0 Pre-Th1 Uncommitted Contact espinosa@iner.gob.mx,ppsyago@gmail.com Proliferative responses of TCMCD4+ with pre-Th1 (CXCR3+ CCR4-) phenotype and uncommitted (CXCR5-) phenotype to SEB.