Antigen-specific T-cell Response Analysis

The number of T. cruzi antigen-responsive IFN-γ- and IL-2-secreting T-cells was determined by ex vivo ELISPOT assays, using a commercial kit (ELISPOT Human IFN-γ or IL-2 ELISPOT Set; BD Biosciences, USA), as described elsewhere (23 (link), 25 (link)). PBMC were stimulated with 10 µg/mL of a T. cruzi-derived lysate preparation, 10 µg/mL T. cruzi-derived recombinant proteins, 10 µg/mL Montenegro Leishmania antigen, 20 ng/mL Phorbol 12-myristate 13-acetate (Sigma) plus 500 ng/mL ionomycin (Sigma), or media alone for 16–20 hr at 37°C and 5% CO₂. Spot forming cells (SFCs) were automatically enumerated using ImmunoSpot analyzer (CTL). The mean number of spots in triplicate wells was obtained for each condition, and the number of specific IFN-γ and IL-2-secreting T-cells was calculated by subtracting the value of the wells containing media alone from the antigen-stimulated spot count. Responses were considered positive if (1) a minimum of 10 SFC/4 × 10⁵ PBMC were present per well and (2) this number was at least twice the value of wells with media alone (26 (link)).