Limited role for CD4+ T-cell help in the initial priming of Trypanosoma cruzi-specific CD8+ T cells

Abstract

Immune control of the protozoan parasite Trypanosoma cruzi requires the activation of both CD4+ and CD8+ T cells. We recently identified two T. cruzi trans-sialidase peptides that are targets of approximately 30% of all CD8+ T cells during acute T. cruzi infection in mice. To determine whether CD4+ T cells are required for generation of these dominant CD8+ T-cell responses, major histocompatibility complex class II (MHC II)-deficient mice were infected with the Brazil strain of T. cruzi and examined for the generation of antigen-specific CD8+ T cells. Strong trans-sialidase TSKB18- and TSKB20-specific CD8+ T-cell responses were generated in both the presence and the absence of CD4+ help. However, the magnitudes of the immunodominant TSKB20-specific CD8+ T-cell responses detectable using class I MHC-peptide tetramers were consistently lower in the blood and spleens of MHC II-deficient mice. Spleen cells from infected MHC II-deficient mice produced gamma interferon after in vitro stimulation with T. cruzi peptides at levels similar to those in wild-type mice, and MHC II-deficient mice displayed strong T. cruzi peptide-specific cytotoxic T-lymphocyte activity in vivo. Thus, primary CD8+ T-cell responses in experimental T. cruzi infection are generated in the absence of CD4+ T cells, providing further evidence that T. cruzi directly activates and licenses antigen-presenting cells. Nevertheless, unhelped CD8+ T cells in T. cruzi-infected mice fail to reach the frequencies achieved in the presence of CD4 T-cell help and are unable to prevent acute-phase death of these mice.

FIG. 1.

Antigen-specific CD8⁺ T cells develop in the absence of CD4⁺ T-cell help. Blood from uninfected naïve mice (filled triangles), infected B6.129 wild-type mice (filled circles), and infected MHC II KO mice (empty circles) was sampled at the indicated time points after infection and stained with H-2K^b tetramers bearing the TSKB20 (A) or TSKB18 (B) T. cruzi peptide. Values represent the means ± SD of tetramer-positive cells among CD8⁺ T cells. Statistically significant differences (P < 0.05) in the frequencies of peptide-specific CD8⁺ T cells between MHC II KO and wild-type mice are indicated by asterisks. (C) TSKB20/K^b and TSKB18/K^b staining of blood from naïve mice, infected B6.129 mice, infected B6 mice, and infected MHC II KO mice at 19 days postinfection. Cells shown are gated on CD4⁻ CD11b⁻ B220⁻ lymphocytes. Data are representative of two experiments (n = 3 to 5 mice per group).

FIG. 2.

Antigen-specific CD8⁺ T cells are present in peripheral tissues of wild-type and MHC II KO mice. Lymphocytes were recovered from tissues of B6.129 wild-type mice (black bars) and MHC II KO mice (gray bars) 28 days after T. cruzi infection and were stained with TSKB20 (top)- and TSKB18 (bottom)-MHC I tetramers. Bars represent the mean frequencies of CD8⁺ tetramer-positive lymphocytes for three mice per group; error bars represent SD. Asterisks denote significant differences in the frequencies of CD8⁺ tetramer-positive lymphocytes between B6/129 wild-type mice and MHC II KO mice (P < 0.05).

FIG. 3.

Antigen-specific IFN-γ-producing cells are generated in the absence of CD4⁺ T-cell help following T. cruzi infection. Spleen cells were harvested from MHC II KO mice (black bars), wild-type mice (gray bars), and uninfected naïve mice (white bars) at days 15, 21, and 28 (D15, D21, and D28) postinfection. Frequencies of IFN-γ-producing CD8⁺ T cells were determined by intracellular cytokine staining after overnight in vitro stimulation with T. cruzi peptides, as described in Materials and Methods. Values represent the means ± SD of IFN-γ⁺ cells among CD8⁺ T cells (n = three mice per group) and are representative of two experiments. Statistically significant differences in IFN-γ production between B6.129 and MHC II KO mice are denoted by asterisks.

FIG. 4.

T. cruzi-infected MHC II KO mice maintain peptide-specific CTL activity. Spleen cells pulsed with T. cruzi peptides (no peptide, TSKB18, or TSKB20) were labeled with CFSE (low, medium, and high concentrations, respectively) and transferred to naïve mice, infected MHC II KO mice, and infected B6.129 wild-type mice as described in Materials and Methods. Numbers represent the percent specific killing of the target cells loaded with the indicated peptide at day 15 postinfection and were calculated as described in Materials and Methods. Histograms are gated on CFSE⁺ lymphocytes. Data are representative of two experiments (n = three mice per group). np, no peptide pulse; T20, TSKB20; T18, TSKB18.