Characterization of CTL Dysfunction Markers

To characterize potential markers associated with CTL dysfunction in response to LRA-reactivated cells, we exposed CTL1 and CTL2 to cognate HIV-1 antigen over nine weeks. To do so, we stimulated CTL with a 1:1 mixture of irradiated autologous B-cell lines (BCLs) pulsed with cognate HIV peptide (10 μg/ml) and irradiated PBMCs from three healthy HIV-seronegative donors weekly. We used flow cytometry to evaluate variations in the expression of the inhibitory receptors PD-1, TIM-3, LAG-3, and the immune-metabolic marker CD39. Briefly, cells were taken from the culture at weeks 0, 5, 7, and 9 and stained with a Live/Dead probe (APC-Cy7, Invitrogen) and surface markers CD3 (Alexa700, BD Biosciences), CD4 (APC-Cy7, BD Biosciences), CD8 (V500, BD Biosciences), PD-1 (BV421, BD Biosciences), TIM-3 (Alexa 647, BD Biosciences), LAG-3 (PE, BD Biosciences), and CD39 (FITC, BD Biosciences) and incubated at room temperature for 25 min. Samples were washed twice with 1X PBS, fixed in 1% formaldehyde, and acquired on an LSR Fortessa. Data were analyzed with FlowJoV (Tree Star Inc.). Patterns of co-expression of PD-1, LAG-3, TIM-3, and CD39 were analyzed using Pestle and SPICE v5 software (55 (link)).