Antigen-responsive CD4+ T cells from C3H mice chronically infected with Leishmania amazonensis are impaired in the transition to an effector phenotype

Abstract

C3HeB/FeJ mice challenged with Leishmania major develop a polarized Th1 response and subsequently heal, whereas Leishmania amazonensis challenge leads to chronic lesions with high parasite loads at 10 weeks postinfection. In this study, a comparison of draining lymph node cells from L. amazonensis- and L. major-infected mice at 10 weeks postinfection showed equivalent percentages of effector/memory phenotype CD44hi CD4+ T cells producing interleukin-2 (IL-2) and proliferating after antigen stimulation. However, these cells isolated from L. amazonensis-infected mice were not skewed toward either a Th1 or Th2 phenotype in vivo, as evidenced by their unbiased Th1/Th2 transcription factor mRNA profile. In vivo antigen stimulation with added IL-12 failed to enhance gamma interferon (IFN-gamma) production of CD4+ T cells from L. amazonensis-infected mice. Antigen stimulation of CD4+ T cells from L. amazonensis-infected mice in vitro in the presence of IL-12 resulted in production of only 10 to 15% of the IFN-gamma produced by T cells from L. major-infected mice under identical conditions. These results suggest that the CD4+ T-cell response during chronic L. amazonensis infection is limited during the transition from an early activated CD4+ T-cell population to an effector cell population and demonstrate that these T cells have an intrinsic defect beyond the presence or absence of IL-12 during antigen stimulation.

FIG. 1.

Both L. amazonensis- and L. major-infected mice have equivalent percentages of CD44^hi CD62L^lo CD4⁺ T cells in the DLN. (A) Lesion development in C3H mice infected with either L. amazonensis or L. major. Data are represented as the mean ± standard deviation of the mean of one representative experiment with eight mice per group. (B) Parasite burden in the feet of L. amazonensis- or L. major-infected mice at 10 weeks postinfection. Data are represented as the mean ± the standard error of three separate experiments. *, statistically significant difference at P < 0.05 as determined by Fisher's PLSD test. (C) DLN cells were harvested from mice infected with either L. amazonensis or L. major for 10 weeks or from age-matched uninfected (naïve) controls and stained for flow cytometry as described in Materials and Methods. The dot plots are based on a live CD4⁺ gate and are representative of four separate experiments.

FIG. 2.

Ag-specific CD44^hi CD4⁺ T-cell population from mice chronically infected with L. amazonensis is capable of producing IL-2 and proliferating. (A) DLN cells were harvested from infected mice at 10 weeks postinfection; stimulated for 24 h with 50 μg/ml of Ag; stained with fluorescent antibodies against CD4, CD44, and IL-2; and then analyzed by flow cytometry (see Materials and Methods). Cells from L. amazonensis-infected mice were stimulated with L. amazonensis Ag, and cells from L. major-infected mice were stimulated with L. major Ag. Data are represented as the mean ± standard error of three separate experiments. (B) DLN cells were harvested at 10 weeks postinfection, labeled with CFSE, cultured with (Ag Stim) or without (No Stim) 50 μg/ml of their respective Ag for 4 days, stained with fluorescent antibodies against CD4 and CD44, and then analyzed by flow cytometry. Cells from uninfected mice were stimulated with L. amazonensis Ag; cells from infected mice were stimulated as described for panel A. Data are represented as the mean ± the standard error of five separate experiments and are expressed as the percentage of the total CD44^hi CD4⁺ T cells present in culture that are proliferating. *, statistically significant difference between No Stim and Ag Stim within a group at P < 0.05 as determined by a paired t test. (C) Cells were cultured and assayed as described for panel B. All dot plots are for cells simulated with 50 μg/ml Ag, are based on a live CD4⁺ gate, and are representative of five separate experiments. Quadrant statistics are percentages and are calculated based on a live CD4⁺ gate.

FIG. 3.

CD44^hi CD4⁺ T cells from L. amazonensis-infected mice do not have a skewed Th1/Th2 response. At 10 weeks postinfection, CD44^hi CD4⁺ T cells from the DLN of Leishmania-infected or control mice were sorted and analyzed ex vivo for (A) T-bet and (B) GATA-3 mRNA expression via real-time RT-PCR. Data are represented as the mean ± standard error of four separate experiments. (C) DLN cells were harvested at 10 weeks postinfection; stimulated for 24 h with 50 μg/ml of their respective Ag as described in the legend to Fig. 2A; stained with fluorescent antibodies against CD4, CD44, and either IFN-γ or IL-4; and then analyzed by flow cytometry. Data are represented as the mean ± standard error of six separate experiments. *, statistically significant difference between indicated groups at P < 0.05 as determined by Fisher's PLSD test.

FIG. 4.

CD4⁺ T cells present in L. amazonensis-infected mice respond to Ag but exhibit limited IL-12 responsiveness in vivo. Mice that had been infected in the left hind footpad with L. amazonensis for 10 weeks were injected in the right (uninfected) hind footpad with either PBS, L. amazonensis Ag, or Ag and IL-12. L. major-infected mice were challenged in their uninfected footpad with L. major Ag. After 48 h post-Ag challenge, LN cells draining the site of Ag challenge were harvested and (A) analyzed via flow cytometry for the percentage of CD44^hi CD4⁺ T cells present in the LN after Ag challenge. *, statistically significant difference between the indicated treatments at P < 0.05 as determined by a paired t test. (B) LN cells draining the site of Ag challenge were stimulated for 3 days in the presence (Ag Stim) or absence (No Stim) of 50 μg/ml of their respective Ag as described in Fig. 2A; supernatants were assayed for IFN-γ via ELISA. *, statistically significant difference between No Stim and Ag Stim within a group at P < 0.05 as determined by a paired t test; **, statistically significant difference from all other groups at P < 0.05 as determined by Fisher's PLSD test. (C) CD4⁺ T cells were purified from the LN draining the site of Ag challenge and analyzed via real-time RT-PCR for T-bet and GATA-3 mRNA expression. Data are expressed as the ratio of T-bet to GATA-3 mRNA after each target had been normalized to GAPDH. *, statistically significant difference between Ag and Ag plus IL-12 within a group at P < 0.05 as determined by a paired t test; **, statistically significant difference from all other groups at P < 0.05 as determined by Fisher's PLSD test. All data are represented as the mean ± standard error of two (C) to three (A and B) separate experiments.

FIG. 5.

CD4⁺ T cells from L. amazonensis-infected mice have limited responsiveness to IL-12 in vitro. At 10 weeks postinfection, 1 × 10⁵ CD4⁺ T cells from the DLN of infected mice were stimulated with 50 μg/ml of their respective Ag as described in Fig. 2A and cocultured with 1 × 10⁶ mitomycin C-treated splenocytes from naïve mice for 5 days under either (A) neutral (no polarizing cytokines or antibodies) or (B) Th1 (rIL-12 and anti-IL-4) conditions. Supernatants were assayed for IFN-γ via ELISA. Data are represented as the mean ± standard error of four separate experiments. *, statistically significant difference at P < 0.05 as determined by Fisher's PLSD test.