Fig. 5: Inoculation of healed mice with killed L. major parasites induces Treg expansion and lesion reactivation.

A C57Bl/6 Foxp3-GFP mice were infected via intradermal ear injections with 1 million dsRed⁺ L. major parasites. 60 days post infection, mice were challenged with PBS or 5 × 10⁶ heat-killed wild-type or PEPCK^-/- L. major parasites into the footpad. The ears were prepared for intravital microscopy or flow cytometry analysis 14 days post challenge. B Representative micrographs of the lesions post challenge. Blue: collagen (SHG), red: dsRed⁺ L. major, Green: Foxp3-GFP cells. C Lesion scores of ears post challenge. Statistical analysis of the last timepoint: Two-tailed unpaired Student’s t test. Mean +/− SD. Data combined from three independent experiments. D Pictures of albino C57Bl/6 ears 14 days post challenge, red circles highlighting lesion area. E Parasite burden from ears of healed mice challenged with PBS, or 5 × 10⁶ heat-killed wild-type or PEPCK^−/− L. major parasites into the footpad. Each dot represents data from one ear. Two-tailed unpaired Student’s t test, ns not significant. F Phenotypic analysis of Tregs in lesion skin. Gray: Tregs extracted from PBS-challenged mice, Red and Yellow: Tregs extracted from mice challenged with WT or PEPCK^−/− heat-killed L. major, respectively. G Absolute cell counts of PEPCK-specific CD45⁺CD3⁺CD4⁺Foxp3⁺ cells in the ears and draining lymph nodes. Each dot represents a single ear or draining lymph node. n = 2 biologically independent mice for h.k. L. major challenge, 3 for PBS-challenged mice, and 4 for h.k. PEPCK^−/− L. major challenge. Two-tailed unpaired Student’s t test, ns not significant. Mean +/− SD. Data combined from two independent experiments. H Percent and MFI* (Absolute expression, MFI × %IFNγ⁺) of IFNγ production in CD45⁺CD3⁺CD4⁺ Tbet⁺ T cells in the ears and draining lymph nodes. Each dot represents a single ear/ draining lymph node/spleen, n = 2 biologically independent mice for PBS or h.k. L. major challenge, and 3 for h.k. PEPCK^−/− L. major challenge. Two-tailed unpaired Student’s t test, ns not significant. Mean +/− SD. Source data are provided as a Source Data file.