Modulation of CD4(+) T cell-dependent specific cytotoxic CD8(+) T cells differentiation and proliferation by the timing of increase in the pathogen load

Abstract

Background: Following infection with viruses, bacteria or protozoan parasites, naïve antigen-specific CD8(+) T cells undergo a process of differentiation and proliferation to generate effector cells. Recent evidences suggest that the timing of generation of specific effector CD8(+) T cells varies widely according to different pathogens. We hypothesized that the timing of increase in the pathogen load could be a critical parameter governing this process.

Methodology/principal findings: Using increasing doses of the protozoan parasite Trypanosoma cruzi to infect C57BL/6 mice, we observed a significant acceleration in the timing of parasitemia without an increase in mouse susceptibility. In contrast, in CD8 deficient mice, we observed an inverse relationship between the parasite inoculum and the timing of death. These results suggest that in normal mice CD8(+) T cells became protective earlier, following the accelerated development of parasitemia. The evaluation of specific cytotoxic responses in vivo to three distinct epitopes revealed that increasing the parasite inoculum hastened the expansion of specific CD8(+) cytotoxic T cells following infection. The differentiation and expansion of T. cruzi-specific CD8(+) cytotoxic T cells is in fact dependent on parasite multiplication, as radiation-attenuated parasites were unable to activate these cells. We also observed that, in contrast to most pathogens, the activation process of T. cruzi-specific CD8(+) cytotoxic T cells was dependent on MHC class II restricted CD4(+) T cells.

Conclusions/significance: Our results are compatible with our initial hypothesis that the timing of increase in the pathogen load can be a critical parameter governing the kinetics of CD4(+) T cell-dependent expansion of pathogen-specific CD8(+) cytotoxic T cells.

Figure 1. Trypomastigote-induced parasitemia in C57BL/6 mice challenged with different doses of trypomastigotes of T. cruzi.

C57BL/6 mice were infected i.p. with 10²,10³, 10⁴ or 10⁵ bloodstream trypomastigotes of the Y strain of T. cruzi. Parasitemia was followed daily from days 0 to 14 after challenge. The results represent the mean of 5–6 mice±SD. At the peak of infection, the parasitemia of mice infected with each different dose was compared by One-way Anova and Tukey HSD tests. The results of the comparisons were as follows: i) 10²×10³, Non-Significant (NS); ii) 10²×10⁴, P<0.01; iii) 10²×10⁵, P<0.01; iv) 10³×10⁴, P<0.05; v) 10³×10⁵, NS; vi) 10⁴×10⁵, NS. Results are representative of two independent experiments.

Figure 2. Infection in WT C57BL/6 or CD8 KO mice challenged with different doses of T. cruzi.

Groups of WT C57BL/6 or CD8 KO were infected i.p. with 10²,10³, 10⁴ or 10⁵ bloodstream trypomastigotes of the Y strain of T. cruzi. (A) Course of infection, estimated by the number of trypomastigotes per mL of blood. Results represent the mean values of 4–5 mice±SD. The parasitemias of WT C57BL/6 or CD8 KO mice were compared by One-way Anova. Asterisks denote statistically significant differences (P<0.05). (B) Kaplan-Meier curves for survival of WT C57BL/6 or CD8 KO infected mice with different doses of parasites. Statistical analyses were performed using LogRank test comparing the different mouse groups. Initially, we compared groups of WT C57BL/6 infected with different doses. The results of the comparison showed no statistically significant differences among them. Subsequently, we compared WT C57BL/6 or CD8 KO infected with each different dose of parasites. The results of the comparison showed statistically significant differences in between C56BL/6 or CD8 KO challenged with each parasite dose (P<0.0001, in all cases). Finally, statistical analyses were performed comparing the groups of CD8 KO infected with different doses. The results of the comparison were as follows: i) CD8 KO 10²×CD8 KO 10³ (P = 0.0025); ii) CD8 KO 10²×CD8 KO 10⁴ (P = 0.0046); iii) CD8 KO 10²×CD8 KO 10⁵ (P = 0.0016); iv) CD8 KO 10³×CD8 KO 10⁴ (P = 0.01); v) CD8 KO 10³×CD8 KO 10⁵ (P = 0.0035); vi) CD8 KO 10⁴×CD8 KO 10⁵ (P = 0.0082).

Figure 3. Kinetics of specific CD8⁺ T-cell mediated immune responses following challenge with T. cruzi.

Groups of C57BL/6 mice were challenged or not i.p. with 10²,10³, 10⁴ or 10⁵ bloodstream trypomastigotes of the Y strain of T. cruzi. Panels A to E - At the indicated days, the in vivo cytotoxic activity against target cells coated with peptide VNHRFTLV was determined as described in the Methods Section. The results represent the mean of 4 mice±SD per group. Asterisks denote statistically significant differences when we compared T. cruzi challenged with control mice (P<0.05). Panel F- At the indicated days, IFN-γ producing spleen cells specific to the peptide VNHRFTLV were estimated by the ELISPOT assay. The results represent the mean number of peptide-specific spot forming cells (SFC) per 10⁶ splenocytes±SD (n = 4). Results are representative of two or more independent experiments.

Figure 4. Kinetics of CD8⁺ T-cell mediated immune responses specific for sub-dominant epitopes in C57BL/6 mice.

Groups of C57BL/6 mice were challenged or not i.p. with 10²,10³, 10⁴ or 10⁵ bloodstream trypomastigotes of the Y strain of T. cruzi. At the indicated days, the in vivo cytotoxic activity against target cells coated with peptide TsKb-18 or TsKb-20 was determined as described in the Methods Section. The results represent the mean of 4 mice±SD per group. Asterisks denote statistically significant differences when we compared T. cruzi challenged with control mice (P<0.05). ND = Not done. Results are representative of two or more independent experiments.

Figure 5. Specific cytotoxicity in C57BL/6 mice challenged with irradiated or non-irradiated trypomastigotes of T. cruzi.

Groups of C57BL/6 mice were challenged or not i.p. with 10³ or 10⁴ irradiated or non-irradiated bloodstream trypomastigotes of the Y strain of T. cruzi. A) Fifteen days after challenge, the in vivo cytotoxic activity against target cells coated with peptide VNHRFTLV was determined. The results represent the mean of 4 mice±SD per group. B) Fifteen days after challenge, IFN-γ producing spleen cells specific to the peptide VNHRFTLV were estimated by the ELISPOT assay. The results represent the mean number of SFC per 10⁶ splenocytes±SD (n = 4). Asterisks denote statistically significant differences (P<0.05) when we compared mice challenged with irradiated or non-irradiated trypomastigotes of T. cruzi. Results are representative of two independent experiments.

Figure 6. Specific cytotoxicity in WT or genetically deficient mice challenged with T. cruzi.

Groups of WT C57BL/6 (n = 4), WT 129 mice (n = 4), MHC-II KO (n = 4), perforin KO (n = 8), CD4 KO (n = 4), IL-12 KO (n = 4), and IFN-I receptor KO (n = 4) were challenged or not i.p. with 10⁵ bloodstream trypomastigotes of the Y strain of T. cruzi. Ten days after challenge, the in vivo cytotoxic activity against target cells coated with peptide VNHRFTLV was determined. The results represent the mean of the above indicated number of mice±SD per group. The in vivo cytotoxicity was compared by One-way Anova and Tukey HSD tests. The results of the comparisons were as follows: i) WT C57BL/6×MHC-II KO (P<0.01); ii) WT C57BL/6×Perforin KO (P<0.01); iii) WT C57BL/6×CD4 KO (P<0.01); iv) WT C57BL/6×IL-12 KO (NS); v) WT 129×IFN-I receptor KO (NS). Results are representative of two or more independent experiments.