PBMC Stimulation and Cytokine Quantification

Cryopreserved PBMC were thawed in complete media. A portion of the PBMC was depleted of CD8⁺T cells (CD8depl PBMC) using the Dynal CD8 Positive Isolation Kit (Invitrogen, Carlsbad CA). Total PBMC or CD8depl PBMC were plated in the presence of soluble Leishmania antigens from L. major parasites (SLA, 2.5 μg/mL, generous gift of Dr. Frank Neva) for 6 days at 37°C, 5% CO₂. Pokeweed mitogen (PWM, 5 μg/mL, Sigma) was used as a positive control. Cell-free supernatant was collected from each well, triplicate subjects pooled, and used to quantify cytokines using the Q-Plex Human Cytokine–IR Array (Quansys Biosciences, Logan, UT) according to manufacturer’s protocol [23 (link)]. For LPA, cells were pulsed as previously reported [24 (link)].