Fig 2: Assessment of cerebral pathology in heme pathway KO parasite-infected mice.a Evans blue extravasation in the brain of mice infected with PbWT and heme pathway PbKO parasites. b Quantification of Evans blue in the brain samples of mice infected with PbWT (n = 6) and heme pathway PbKO (n = 9) parasites. (mean ± SD; ***P < 0.001, unpaired t-test; two-sided). c H&E staining of the brain sections prepared from PbWT and PbKO parasite-infected mice. Black arrows—intracerebral and petechial hemorrhages, blue arrows—thrombosed blood vessels and brown arrows—gross demyelination. Images were captured using 10x objective. Scale bar = 50 µm. n = 3–5 independent experiments. d IgG extravasation in the brain sections of PbWT and PbKO parasite-infected mice. Black arrows—areas showing IgG immunoreactivity. Images were captured using 10x objective. Scale bar = 50 µm. n = 3 independent experiments. e H&E staining of the brain sections indicating (black arrows) occluded vasculatures containing luminal and abluminal leukocytes, and parasite-derived Hz. Images were captured using 60x objective. Scale bar = 10 µm. n = 3 independent experiments. f Immunofluorescence analysis of parasite accumulation in the brain sections of PbWT and PbKO parasite-infected mice. n = 3 independent experiments. g Immunofluorescence analysis of CD3+ cells in the blood vessels of PbWT and PbKO parasite-infected mice. n = 2 independent experiments. h Immunofluorescence analysis of ß-APP staining in the brain sections of PbWT and PbKO parasite-infected mice. n = 2 independent experiments. f–h Images were captured using 20x objective. Scale bar = 20 µm. Source data are provided as a Source Data file.

Fig 3: The BCG->naive mice showed more CD4+ in spleen and peripheral blood (A-H). FACS: the proportion of CD3+ cells in spleen and peripheral blood for each group (A-B); the proportion of CD4+ and CD8+ cells in spleen and peripheral blood for each group (C-D); bars denote the proportion of CD3+ and CD4+to total lymphocytes found in the spleen and peripheral blood of each group (E-H). *p< 0.05, **p< 0.01; n = 6/group.

Fig 4: CRISPR/Cas9 gene editing and TIRs expression after 72 h CD8+ T cell stimulation. (A) Upper panel. Mean Fluorescence Intensity (MFI) of LAG-3, PD-1, and TIM-3 in MH CD8+ T cells after 72 h stimulation with anti-CD3/CD28 antibodies. Values correspond to the fold induction of CD3/CD28 stimulated MH CD8+ T cells over the unstimulated. Lower panel. Representative dot plot curves for PD-1, LAG-3, and TIM-3 intensity. Black line (straight and dotted) corresponds to N.T.; red line (straight and dotted) corresponds to 3KO. Bars are representative of three independent experiments with MH CD8+ T cells derived from three different animals. (B) Upper panel. Mean Fluorescence Intensity (MFI) of PD-1, LAG-3, and TIM-3 in OT-1 CD8+ T cells after 72 h stimulation with splenocytes incubated with OVA peptide. Values correspond to the fold induction of OVA stimulated OT-1 CD8+ T cells over the unstimulated. Lower panel. Representative dot plot curves for PD-1, LAG-3, and TIM-3 intensity. Black line (straight and dotted) corresponds to N.T.; red line (straight and dotted) corresponds to 3KO. Bars are representative of six independent experiments with OT-1 CD8+ T cells derived from six different animals. N.T., not transfected; T.C., transfection control; 3KO, Triple Knock Out for PD-1, LAG-3, and TIM-3 genes; N.S., not stimulated. Statistical analysis: one-way ANOVA with post-hoc test. * p < 0.05.

Fig 5: Effect of 3KO OT-1 CD8+ adoptive T cell transfer (ATT) on B16 tumor growth and survival. (A) Tumor growth after ATT of not transfected, transfection control, and triple CRISPR/Cas9 edited T cells. After ATT B16 tumors were measured every 2–3 days over a period of 16 days and mice were sacrificed after 16 days or when a tumor volume of 1500 mm3 was reached (experimental endpoints). (B) Survival rate after B16 xenograft. Percent of survival was calculated over a period of 16 days and statistical analysis was performed on a number of animals >6 for each group. (C) When one predefined experimental endpoint was reached, spleen and tumor were isolated, smashed through a cell strainer, and stained for CD3, CD8, Vß5, PD-1, LAG-3, and TIM-3. Bars represent the percentage of CD8+Vß5+ over total CD3+ T cells. Bars are representative of =6 animals per group. (D) Weight measurement after ATT every 2–3 days over a period of 16 days. N.T, not transfected; T.C. transfection control; 3KO, triple CRIPR/Cas9 edited OT-1 CD8+ T cells. Statistical analysis: one-way ANOVA with post-hoc test. * p < 0.05.