Fig 2: TLR2 shapes the cellular microenvironment in vivo in the Mtb-infected lung. C57BL6/6J or Tlr2− /− mice were infected with Mtb (~200 CFU). Six-week post infection, mice were euthanized, lungs were harvested, and single-cell suspensions were generated for flow cytometry analysis and scRNAseq. (A) Flow cytometry analysis of total macrophages and macrophage subpopulations as a percent of live CD45+ cells (3–5 biological replicates per group). (B–H) scRNAseq (two biological and two technical replicates). (B) UMAP projection of cell type lineages. (C) Cell density projection on UMAP projections for each condition (WT uninfected, WT infected, Tlr2 −/− uninfected, Tlr2 −/− infected). (D) Odds ratio and FDR-adjusted P value for differential abundance changes between infected WT and Tlr2 −/− samples calculated using a generalized binomial linear model. (E) Number of differentially expressed genes (DE genes) within each cell type between infected WT and Tlr2− /− mice. (F, and G) Volcano plot describing fold change and FDR-adjusted P values for DE genes in recruited macrophages between infected WT and Tlr2− /− mice. Labeled genes are among the genes that contribute most significantly to s-PC1. Average infection signature scores between WT and Tlr2− /− mice within recruited macrophages. (H) Average Hallmark MSigDB signature scores between WT and Tlr2− /− mice within recruited macrophages. (I) Enrichment analysis of DE genes in WT recruited macrophages across three distinct databases.

Fig 3: IL5+ ILC2s are required to maintain M2-like dermal TRMs which play a critical role in non-healing L. major infection.a Representative flow cytometric analysis of ear isolates prepared from naive R5: ROSA26-LSL-tdTomato mice. CD45.2+CD11b-CD90.2+NK1.1--gated cells were analyzed for the expression of TCRβ and TCRγδ, and subdivided into DETCs (green; TCRγδhighTCRβ-), γδT cells (blue; TCRγδintTCRβ-), ILC2s (Red; TCRγδ-TCRβ-), and αβT cells (orange; TCRγδ-TCRβ+). Both CD2/CD3 and tdTomato expression were shown by overlaying color-coded populations in CD2-CD3 axes and histogram respectively. b tdTomato expression in ILC2s and (c) the absolute numbers of indicated cells recovered from ears of R5 (n = 8) vs. R5: ROSA26iDTR (n = 7) mice were measured at 12 days p.i. with 2 × 105 LmSd followed by DT treatment. d, e The same parameters are shown as above in ear isolates prepared from R5 (n = 4) vs. R5: Gata3f/f (n = 4) mice at day 12 p.i. with 2 × 105 LmSd, or (f) WT (n = 8) vs. RT: Gata3f/f (n = 8) vs. R5: Gata3f/f (n = 8) mice adoptively transferred with in 0.5 × 106 in vitro expanded, splenic ILC2s from WT mice, intravenously administered on day 0 p.i. with 2 × 105 LmSd. g Lesion development and pathology score over the course of infection with 103 LmSd metacyclic promastigotes in the ear dermis of Gata3f/f, Il4−/−, and R5: Gata3f/f animals (n = 8). h Parasite burdens were quantified at 12 weeks p.i. Values represent mean ± standard deviation (n = 8). *P < 0.05, **P < 0.01, and ***P < 0.01 by two-sided non-parametric Mann-Whitney test (b–e) and one-way ANOVA with Dunn’s posttest compared with Gata3f/f (f, g). Data are representative of two independent experiments (a–g). Source data are provided as a Source Data file.

Fig 4: Ccl24-cre transgenic mice target MHCII-MRhigh dermal TRMs and TRMs in multiple tissues.a Representative flow cytometric analysis of ear isolates from ROSA26-LSL-tdTomato mice vs. Ccl24-cre: ROSA26-LSL-tdTomato mice infected with LmSd-GFP for 8 days. b Live CD45.2+ singlets were gated into eosinophils, PMNs, inflammatory monocytes, moDCs, and dermal TRMs. The tdTomato+ cells in red were overlayed onto gatings. c Representative confocal image of ear dermis of infected Ccl24-cre: ROSA26-LSL-tdTomato mice which were intravenously injected with efluor450 anti-CD31 Abs and Cy5-ManoceptTM. d Quantification of percent tdTomato positive of dermal TRMs in (b) (n = 6). e Flow cytometric analysis of tdTomato+ cells in Ccl24-cre: ROSA26-LSL-tdTomato mice which were overlayed onto gatings for indicated organs. pgWAT; perigonadal white adipose tissue. The complete gating strategies are shown in Supplementary Fig. 6. Percent tdTomato positive of TRMs are labeled in red. f–j Immunohistochemistry analysis of Alexa488-Laminin Abs stained tissue sections or whole mount from Ccl24-cre: ROSA26-LSL-tdTomato mice which were intravenously injected with efluor450 anti-CD31 Abs and Cy5-ManoceptTM. G glomerulus. Tubules; Renal tubules which were visualized with filtered Cy5-ManoceptTM. Data are representative of more than three independent mouse experiments (a–c). See also Supplementary Fig. 5 and 6. Source data are provided as a Source Data file.

Fig 5: Effector CD8 T cells with an inflammatory, exhausted profile are associated with intrinsic prolidase deficiency. (A) Schematic of bone marrow chimera experiment. CD45.1+ recipients were lethally irradiated and reconstituted for 8 wk with equal amounts of CD45.1+CD45.2+ WT and either CD45.2+ WT or CD45.2+ Pepd−/− BM. (B) Relative proportions of thymic populations and splenic T cell subsets from BM chimeras reconstituted with equal ratios of CD45.1+CD45.2+ WT and either CD45.2+ WT or CD45.2+ Pepd−/− BM, end-gated based on CD45.1 and CD45.2. Data show one experiment representative of four independent repeats. (C) Ratios of proportions of specified CD4 or CD8 cell populations to naive subset within WT or Pepd−/− CD45.2+ splenic CD4 and CD8 T cells from chimeric reconstituted BM, pooled from six experimental replicates. CD4 effector, ****p ≤ 0.0001; CD4 Treg, ***p = 0.00031; CD8 CEM, **p = 0.00131; CD8 effector, ****p ≤ 0.00001. (D) Relative proportions of B cell reconstitution across organs in BM chimeras described in (A), representative of three independent repeats. (E) WT and Pepd−/− CD45.2+ T cells from BM chimeras described in (A) were sorted using flow cytometry and negative enrichment, then labeled with hashtag Abs, pooled, and labeled with 13 ADT T cell markers before 3′ single-cell transcriptomic sequencing. WT and Pepd−/− CD8 populations were identified by WNN-UMAP dimensionality reduction and clustering, with populations of effectors ringed in red. (F) Volcano plot depicting differential gene expression between inflammatory effectors (cluster 4) and SLEC-like effectors (cluster 6) of WNN-clustered CD8 populations from (E). Differentially expressed genes were determined using the Wilcoxon rank sum test and are colored in red at a threshold of log2FC = 0.5 and adjusted p value <0.01. Data for (E) and (F) are from one scRNA-seq experiment. (G) Flow cytometric determination of proportions of WT and Pepd−/− splenic CD45.2+ CD8 subsets of chimeras reconstituted as in (A), pooled from three independent experiments, each with five to six mice per group and significance determined by an unpaired two-tailed t test, adjusting for multiple comparisons. PD1+KLRG1− effector, **p = 0.0055; Ly6c+ naive, ****p ≤ 0.0001.