EXPRESSION OF THE ACTIVATION ANTIGEN CD69 PREDICTS FUNCTIONALITY OF IN-VITRO EXPANDED PERIPHERAL-BLOOD MONONUCLEAR-CELLS (PBMC) FROM HEALTHY DONORS AND HIV-INFECTED PATIENTS

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 stim ulation, 3 days of stimulation with anti-CD3 and anti-CD28, and contin uous stimulation with anti-CD3 and anti-CD28. Functionality of PBMC wa s evaluated prior to and after expansion using standard proliferation assay. Phenotype and lymphocyte subset activation defined by expressio n of CD69 and CD25 were determined using flow cytometry. PBMC from hea lthy 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 co nserved. PBMC expanded with stimulation for 3 days exhibited more pres erved functionality than PBMC stimulated continuously (P < 0.03). The mean proliferative response in each of the four different expansion pr otocols correlated with the mean values of CD69 expression. The prolif erative responses from patients and healthy donors expanded with PHA s timulation 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 hi ghly conserved functionality after in vitro expansion. Finally, PBMC e xpanded with PHA stimulation for 3 days were examined for apoptosis. O nly 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 pa tients was demonstrated. Expanded PBMC had conserved functionality. Fi nally, after in vitro expansion, expression of the activation antigen CD69 reliably predicted functionality of PBMC.