Fig 2: Sorting strategy for Nanostring arrays. Lymphocytes were identified by forward -scatter area (FSC-A) and side scatter area (SSC-A) gates, followed by two singlets gates (FSC-A vs. FSC-W and SSC-A vs. SSC-W) followed by live/dead identification using the infra-red fluorescent viability dye. Alive CD45+ cells were plotted for CD11b+ vs dump channel (anti-NK1.1/Ly6G/CD3e/CD45R/B220) to remove NK cell, granulocyte, T cell and B cells lineages, respectively. The CD11b+ cells were further gated on MHCII. The MHCII- population was sub-gated on Ly6C as MHCII-CD64+Ly6chigh (monocytes), MHCII-CD64+Ly6cinter and MHCII-CD64+Ly6clow (macrophages). Cells were sorted from twenty C57BL/6 mice (n = 20) to obtain sufficient RNA to perform the NanoString expression profiles.

Fig 3: Single-cell RNA sequencing. Single-cell RNA sequencing (scRNA-seq) revealed differences in conjunctival immune cell populations between B6 and Pinkie. (A) UMAP of 19 distinct immune cell clusters in the conjunctiva generated from single-cell transcriptomic profiles of CD45+ cells using Seurat package V4.1.0. (B) UMAP comparing conjunctival immune cell clusters obtained by scRNA seq of CD45+ cells obtained from 8 mice female C57BL/6J and 8 Pinkie age 16 weeks. The percentage of the cells in each cluster is shown in parentheses and the cell count and percentage for clusters are provided in Table 1. (C) Heatmap of the top 50 differentially expressed genes in C57BL/6 and Pinkie conjunctival immune cells. Color of the heatmap based on the natural log of the normalized RNA expression; (D) Violin plots showing expression of IL-17a with expression in each cluster in the plots to the right. (E) Violin plots of γδT cell IL-17 signature genes Ltb, Cxcr6, Rorc, and IL1rf that have significantly higher expression in Pinkie vs. C57BL/6 (B6). ****p < 0.0001.

Fig 4: Two-cell formation of IL-12 triggers downstream IFNγ in response to L. major infection(A) Schematic of experimental design. Bone marrow chimeras were generated by reconstituting irradiated CD45.1+ p40−/− with bone marrow cells from CD45.2+ p40−/−, CD45.2+ p35−/−, or CD45.1+CD45.2+ WT donor mice. Chimeric mice were infected in the footpad dermis with 106 dsRed L. major parasites following reconstitution. 35 days after infection, tissues were harvested and restimulated for intracellular cytokine staining and flow cytometric analysis. Gating strategy used for these panels is shown in Figure S3A.(B) Percentage and of CD44hiIFNγ+CD4+ T cells in the spleen of p40−/− host chimeras measured 35 days after infection (p40−/−→p40−/−, n = 4; p35−/−→p40−/−, n = 6; WT→p40−/−, n = 3).(C) Number of CD44hiIFNγ+CD4+ T cells in the spleen of p40−/− host chimeras reconstituted with p40−/− (circles, blue), p35−/− (squares, green), or WT (triangles, black) donor bone marrow 35 days after infection (unpaired t test, p40−/−→p40−/−, n = 4; p35−/−→p40−/−, n = 6; WT→p40−/−, n = 3).(D) Same as in (B), except CD8+ T cells (p40−/−→p40−/−, n = 4; p35−/−→p40−/−, n = 6; WT→p40−/−, n = 3).(E) Same as in (C), except CD8+ T cells (unpaired t test, p40−/−→p40−/−, n = 4; p35−/−→p40−/−, n = 6; WT→p40−/−, n = 3).(F) Percentage of CD44hiIFNγ+CD4+ T cells in lymphocytes harvested from the footpad lesion tissue of p40−/− host chimeras 35 days after infection (p40−/−→p40−/−, n = 4; p35−/−→p40−/−, n = 6; WT→p40−/−, n = 3).(G) Number of CD44hiIFNγ+CD4+ T cells in lymphocytes harvested from the footpad lesion of p40−/− host chimeras reconstituted with p40−/− (circles, blue), p35−/− (squares, green), or WT (triangles, black) donor bone marrow 35 days after infection (unpaired t test, p40−/−→p40−/−, n = 4; p35−/−→p40−/−,n= 6; WT→p40−/−, n = 3).(H) Same as in (F), except CD8+ T cells (p40−/−→p40−/−, n = 4; p35−/−→p40−/−, n = 6; WT→p40−/−, n = 3).(I) Same as in (G), except CD8+ T cells (unpaired t test, p40−/−→p40−/−, n = 4; p35−/−→p40−/−, n = 6; WT→p40−/−, n = 3).(J and K) Cells from each tissue were harvested 35 days after infection and restimulated with soluble Leishmania antigen (SLA). Concentrations are shown of IFNγ in supernatant by tissues collected from p40−/−→p40−/− (blue), p35−/−→p40−/− (green), and WT→p40−/− (black) after restimulation for (J) 24 and (K) 48h (unpaired t test, p40−/−→p40−/−, n = 4; p35−/−→p40−/−, n = 6; WT→p40−/−, n = 3).Data in this figure are representative of two independent experiments where n refers to the number of biological replicates. (C), (E), (G), and (I)–(K) are displayed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Anything not marked is considered not statistically significant.

Fig 5: Two cell-derived IL-12 activity is functionally different than one-cell derived IL-12 activityBone marrow chimeras were generated by reconstituting irradiated CD45.1+ p40−/− mice with bone marrow cells from CD45.2+ p40−/−, CD45.2+ p35−/−, or CD45.1+CD45.2+ WT donor mice. Chimeric mice were infected in the footpad dermis with 106 dsRed L. major parasites following reconstitution.(A) Biweekly measurement of footpad thickness at the midline following establishment of visible lesion for p40−/− host chimeras reconstituted with p40−/− (circles, blue), p35−/− (squares, green), or WT (triangles, black) through the duration of infection (p40−/−→p40−/−, n = 4; p35−/−→p40−/−, n = 6; WT→p40−/−, n = 3).(B) Footpad thickness at the midline of p40−/− host chimeras reconstituted with p40−/− (circles, blue), p35−/− (squares, green), or WT (triangles, black) 35 days after L. major infection (unpaired t test, p40−/−→p40−/−, n = 4; p35−/−→p40−/−, n = 6; WT→p40−/−, n = 3).(C–H) dsRed L. major parasite quantified by limiting dilution from (C) footpad lesion, (D) dLN, (E) non-dLN (ndLN), (F) spleen, (G) liver, and (H) lung tissue of p40−/− host chimeras reconstituted with p40−/− (circles, blue), p35−/− (squares, green), or WT (triangles, black) 35 days after infection (unpaired t test, p40−/−→p40−/−, n = 4; p35−/−→p40−/−, n = 6; WT→p40−/−, n = 3).Data in this figure are representative of two independent experiments where n refers to the number of biological replicates. All data are displayed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Anything not marked is considered not statistically significant.