FIG. 3.

Depletion of CD4⁺ CD25⁺ T cells prior to or during nonlethal acute infection with Brazil strain T. cruzi has no effect on the magnitude of antigen-specific immune responses. Following infection with 1 × 10³ Brazil strain T. cruzi tissue culture trypomastigotes, peripheral blood samples were taken from mice depleted only prior to infection (deplete-infect, n = 7), depleted both prior to and during infection (deplete-infect-deplete, n = 7), infected only (infect, n = 7), and noninfected (“naïve,” n = 3). (A) PBLs were simultaneously stained with anti-CD8α FITC-conjugated antibodies and class I MHC H2-k^b tetramers loaded with either TSKb18 or TSKb20, conjugated to the fluorophores APC or PE, respectively. Naïve mice were included to determine nonspecific background of tetramer-staining. PBLs were fixed and analyzed on a Cyan (Dako Cytomation) flow cytometer. Error bars represent standard deviation. (B) Thirty-two days after infection, an in vivo CTL assay was performed. Spleen cells from naïve B6 mice were separately pulsed with TSKb20 or CZKb9 peptides, or no peptide (unpulsed), then labeled with CFSE, and adoptively transferred to one mouse of each indicated group, as described in the Material and Methods. Splenocytes were recovered from naïve and acutely infected recipient mice 12 h posttransfer, fixed, and analyzed using a flow cytometer (Dako Cytomation). Numbers at the lower left and upper right indicate specific killing of TSKb20 and CZKb9-pulsed target cells, respectively. (C) Splenocytes from naïve, infected only, deplete-infect-depleted, and deplete-infected mice (n = 1, each) 32 days postinfection were cultured in the presence of indicated peptide, media only (“no stim,” negative control), or PMA/ionomycin (“PMA,” positive control) for 5 h in media containing Brefeldin-A. To determine the frequency of CD8⁺ T cells that produced IFN-γ, cells were stained with fluorescently labeled anti-CD8α and anti-IFN-γ antibodies, as stated in the Material and Methods.