Fig 1. Protection at sites of Leishmania re-challenge is associated with adaptive CD4⁺ T-cell immunity.

(A-M) Chronically infected (2^o) or naïve (1^o) age-matched controls (AMCs) were challenged via intra-dermal inoculation of 2 x 10⁵ L. major metacyclic promastigotes in both ears and the dermal site of challenge was analyzed at day 4 post-challenge (p.ch.) (A) Experimental design following the style of Butler et. al. (Cell Rep 2017). (B) Representative flow plots of the indicated dermal T cell populations. (C) Absolute number of the indicated T cell populations per ear. (D) Parasite loads in individual ears as determined by limiting dilution assay (LDAs). (E) Gene expression levels of cxcl9 and cxcl10 chemokines in bulk tissue by qRT-PCR. (F, H, J, L) Representative flow plots and (G, I, K, M) the frequency and absolute number of the indicated phagocyte populations per ear. (N-P) Naive congenic recipient mice were irradiated and transferred with 2 x 10⁷ bone marrow derived cells from chronic (2^o BMT) or naïve (1^o BMT) mice prior to challenge. (N) Experimental design. (O) Parasite loads in individual ears as determined by LDAs. (P) Frequencies of the indicated phagocyte populations. (Q) Absolute # of CD4⁺CD90.2⁺IFN-γ⁺ T cells per ear after overnight antigen re-stimulation. In (A-M) n = 3 mice/group/experiment, representative of 3 repeat experiments; in (N-Q) n = 5–8 total mice pooled from two independent experiments. (*) p<0.05, (**) p<0.01, (***) p<0.005, (****) p<0.0001, n.s. = not significant. Statistical analysis was performed using Student’s t-tests or one-way ANOVA with Tukey post-test.