Expression of the activation antigen CD69 predicts functionality of in vitro expanded peripheral blood mononuclear cells (PBMC) from healthy donors and HIV-infected patients

Abstract

Gene therapy for AIDS necessitates harvest and expansion of PBMC from HIV-infected patients. We expanded PBMC from healthy blood donors and HIV-infected patients for up to 14 days using four expansion protocols: 3 days of phytohaemagglutinin (PHA) stimulation, continuous PHA stimulation, 3 days of stimulation with anti-CD3 and anti-CD28, and continuous stimulation with anti-CD3 and anti-CD28. Functionality of PBMC was evaluated prior to and after expansion using standard proliferation assay. Phenotype and lymphocyte subset activation defined by expression of CD69 and CD25 were determined using flow cytometry. PBMC from healthy donors and HIV-infected patients were readily expanded. The best expansion was obtained using stimulation for 3 days. After expansion, functionality of PBMC measured as proliferative response was partly conserved. PBMC expanded with stimulation for 3 days exhibited more preserved functionality than PBMC stimulated continuously (P < 0.03). The mean proliferative response in each of the four different expansion protocols correlated with the mean values of CD69 expression. The proliferative responses from patients and healthy donors expanded with PHA stimulation for 3 days correlated with CD69 expression on CD4 cells (r = 0.68, P < 0.01) and on CD8 cells (r = 0.59, P < 0.03). Furthermore, expression of CD69 reliably predicted which patients and donors had highly conserved functionality after in vitro expansion. Finally, PBMC expanded with PHA stimulation for 3 days were examined for apoptosis. Only a minor fraction was primed for apoptosis, and this fraction could be significantly reduced by addition of IL-2 to the culture medium (P < 0.05). In conclusion, the feasibility of expanding PBMC from HIV patients was demonstrated. Expanded PBMC had conserved functionality. Finally, after in vitro expansion, expression of the activation antigen CD69 reliably predicted functionality of PBMC.

Fig. 1

Correlation between mean proliferative responses (10³ ct/min) and mean stimulation-induced increase in expression of CD69 on CD8 cells (% CD69) in cells expanded with four different expansion protocols: (1) 3 days of phytohaemagglutinin (PHA) stimulation, (2) continuous PHA stimulation, (3) 3 days of stimulation with anti-CD3 and anti-CD28, (4) continuous stimulation with anti-CD3 and anti-CD28.

Fig. 2

Patients and healthy donors were divided into two groups containing those with highly conserved proliferative responses (i.e. > 40 × 10³ ct/min, 10 cases) and those with poorly conserved proliferative responses (i.e. ≤ 40 × 10³ ct/min, six cases). The figure shows the mean stimulation-induced increase in percentage of PBMC that expressed CD69 in the two groups (bars). The mean stimulation-induced increase in CD69 expression for the group with a highly conserved proliferative response was 50% while the mean stimulation-induced increase for the group with a poorly conserved proliferative response was 31%. This difference was statistically significant (P < 0.04). •, Individual HIV-infected patients/healthy blood donors.

Fig. 3

DNA measurements were performed on PBMC from two patients (•) and three healthy donors (▪) to determine the fraction of apoptotic cells. DNA measurements were performed with PBMC grown in culture medium and PBMC grown in culture medium without IL-2 for the last 24 h. The mean fraction of apoptotic cells is shown as bars. The fraction of apoptotic cells increased by 1.96-fold when IL-2 was removed (P < 0.05).