MDSC Isolation and T Cell Suppression Assay

Peripheral blood mononuclear cells (PBMC) from a healthy donor were stained with arboxyfluorescein diacetate succinimidyl ester [Vybrant® CFDA SE (CFSE) Cell Tracer Kit, #V12883, Molecular probes by life technologies] according to the manufacturer's manual allowing lymphocyte proliferation traceability by their simultaneous stimulation with 100 U/ml IL-2 (R&D Systems) and 1 μg/ml purified NA/LE Mouse anti-human CD3 (BD Pharmingen, #555336, Clone HIT3a).

PMN-MDSC were freshly isolated from parasitemic participant's PBMC (autoMACS®Pro Separator, Miltenyi Biotec), gathered by positive selection after mouse anti-human CD66b-FITC (BD Pharmingen, #555724) and anti-FITC MicroBeads (Miltenyi Biotec, #130-048-701) staining, as described before (59). Subsequently, the highly purified PMN-MDSC were seeded in a round bottom 96-well plate in different proportions to a fixed amount of 6 × 10⁴ CFSE labeled PBMC per well (1:2, 1:4, 1:8, 1:16) and incubated at 37°C and 5% CO₂. Complete media (RPMI1640 supplemented with 10% autologous serum, 1% L-glutamine, and 1% Penicillin-Streptomycin) without additional cells was added to stimulated and non-stimulated labeled PBMC as positive and negative control, respectively. As controls for the contribution of polymorphonuclear cells (PMN) to suppression, PMN from the participants isolated with an erythrocyte lysis step from the high-density fraction of the Ficoll were seeded in the same manner as PMN-MDSC.

After 4 days of incubation, cells were harvested and stained with mouse IgG1, κ anti-human CD4-PE (Biolegend, #300508, Clone RPA-T4) and mouse IgG1, κ anti-human CD8a-APC (Biolegend, #300912, Clone HIT8a). Shortly before measurement, Propidium Iodide (BD Pharmingen, #51-66211E) was added to determine cell viability. CFSE fluorescence intensity was analyzed by flow cytometry to determine proliferation of CD4⁺ and CD8⁺ T cells (Supplementary Figures 2D,E). Proliferation was defined as the ratio of percentage of T cell proliferated following addition of PMN-MDSC or PMN to percentage of T cell proliferation without co-culture (58).