Fig. 6: Immunization or leishmanization confer comparable host protection against L. major WT infection. | Nature Communications

Fig. 6: Immunization or leishmanization confer comparable host protection against L. major WT infection.

From: A second generation leishmanization vaccine with a markerless attenuated Leishmania major strain using CRISPR gene editing

Fig. 6

a Schematic representation of the experimental approach. b Gating strategies and multiparameter flow-cytometry based analysis for cytokine secreting T cells from pooled ears of naïve, healed and LmCen−/− immunized group of mice. c Multiparameter analysis for multiple cytokine secreting Live CD3+CD4+CD44+ T cells after 20 h of in-vitro re-stimulation with freeze-thaw L. major antigen (LmFTAg) from pooled ears (2 ears) of naïve control (n = 2), healed (n = 3) and LmCen−/− immunized (n = 3) group of mice plus naive splenic APCs. Results (mean ± SEM) are representative of one experiment. Statistical analysis done by Mann–Whitney two-tailed test (nsp −0.9). d Pie charts analysis of the cytokine profile of Live CD3+CD4+CD44+ T cells. e Gating strategies and zebra plots of immune response in the ear of non-immunized, healed or LmCen−/− immunized mice following needle challenge with wild type L. major-parasites. PBS injected naïve mice is control group. f Ear-derived cells were analyzed and represented as the percentage of IFN-γ-producing Live CD4+CD44Hi-Ly6C+T-bet+ -T cells. Results (mean ± SEM) are representative of one of two independent experiments with pooled ears (2 ears) samples from healed (n = 6), LmCen−/− immunized (n = 6) and age-matched naïve control (n = 6) mice. Statistical analysis done by unpaired one-tailed t-test (nsp −0.31, *p −0.02 and **p −0.005). g Ear IFN-γ expression were measured by RT-PCR analysis from healed (n = 6), LmCen−/− immunized (n = 6) and age-matched naïve control (n = 6) mice following 20 h post needle challenge with wildtype L. major-parasites. Results (mean ± SEM) are representative of one of two independent experiments with pooled ears (2 ears) samples (n = 6 mice per group). Statistical analysis done by unpaired two-tailed t-test (nsp −0.48; **p < 0.009). Five weeks of post-challenge both ear h and draining lymph nodes i parasite load were determined in healed (n = 5), LmCen−/− immunized (n = 6) and age-matched naïve control (n = 8) mice by serial dilution. Results are represented as geometric means with 95% Cl of total 5–8 mice in each group. Data are representative of one experiment. Statistical analysis done by non-parametric Mann–Whitney two-tailed test (nsp < 0.71; **p < 0.004).

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