Figure 4.

Cell phenotype and effector inflammatory/cytotoxic functions of splenic CD8⁺ T-cells. ccl3^+/+ and ccl3^−/− mice were infected with 100 trypomastigote forms of the Colombian T. cruzi strain and analyzed at 120 dpi. (A) TCR expression in splenocytes. After selection of singlets (FSC-Lin × FSC-Area, R1), dead-cell exclusion (FSC-A × SSC-Lin, R2), TCR histograms (representative figures), graphs show total TCR⁺ cells (%) and TCR^Low cells (%). (B) Presence of Pfn⁺ and IFNγ⁺ CD8⁺ T-cells. After selection of singlets (FSC-Lin × FSC-Area, R1), dead-cell exclusion (FSC-A × SSC-Lin, R2), TCR × CD8 dot plot (gating on TCR⁺CD8⁺ cells, R3), representative Pfn × IFNγ dot plots are shown. Graph shows CD8⁺ IFNγ⁺ T-cells (%). (C) The numbers of CD8⁺ IFNγ⁺ were determined by ex vivo ELISpot stimulating splenocytes with the H-2K^b-resctricted ASP2 VNHRFTLV peptide. Bars represent the in vivo specific cytotoxicity lysis detected in T. cruzi-infected ccl3^+/+ and ccl3^−/− mice. Each experimental group consisted of three NI mice and four to five T. cruzi-infected mice. Representative histogram profiles of in vivo cytotoxicity assay showing the specific lysis of in NI controls and T. cruzi-infected ccl3^+/+ mice of H-2K^b-resctricted VNHRFTLV peptide-labeled CFSE^high cells compared with non-stimulated CFSE^low cells, at 120 dpi. Experiments were repeated twice. Data are presented as means ± SE. **p < 0.01 and ***p < 0.001, comparing T. cruzi-infected and NI mice; ^#p < 0.05 and ^##p < 0.01, comparing T. cruzi-infected ccl3^+/+ and ccl3^−/− mice. (ANOVA Bonferroni posttest, t-Student test).