Figure 4. Pre-Treatment of Rag1^−/− C57BL/6 mice with M2a macrophages boosts iTreg-Th17 cell axis development in the spleen and MLN.

(A) Weight change analysis of Rag1^−/− C57BL/6 mice pre-treated with M0, M1, M2a macrophages or not pre-treated. Each line in the left and center panels represents an individual recipient mouse (none n=73, M0 n=11, M1 n=13, M2 n=52). (B) Kaplan-Meier survival curves of mice in (A). (C) Quantification of donor CD4⁺ T cells from SP and MLN for each pre-condition treatment (None n=47, M0 n=8, M1 n=12, M2 n=32). (D) Representative flow cytometry analysis of CD4 and EGFP (Foxp3) expression to assess the frequency of iTreg cells in MLN for each group. Numbers in quadrants are averages. (E–F) Frequency (E) and number (F) of iTreg cells in the SP and MLN. (G) Comparison of the frequency of iTreg cells in MLN and days where mice were taken for all groups. (H–I) Colitis scores (H) and representative H&E stained sections (I) from mice where tissues were taken for histology (none n=15, M0 n=7, M1 n=12, M2 n=26). (J) Representative flow cytometry analysis of IL-17A and IFN-γ expression to assess the frequency of IFN-γ⁺, IL-17A⁺ and IFN-γ⁺ IL-17A⁺ cells in MLN for each group. Numbers in quadrants are averages. (K–L) Frequency (K) and number (L) of CD4⁺ IL-17A⁺ T cells in the SP and MLN for each group. (M–N) Frequency (M) and number (N) of CD4⁺ IFN-γ⁺ T cells in the SP and MLN for each group. (O–P) Frequency (O) and number (P) of CD4⁺ IFN-γ⁺ IL-17A⁺ T cells in the SP and MLN for each group. (Q) Linear regression analysis comparing the frequency of MLN iTreg and CD4⁺IL-17A⁺ T cells from each group. (E, G, K, O, Q) Each symbol represents a mouse, and small horizontal bars represent the mean. Data are from 3–17 independent experiments, 1–5 mice per experiment. *p< 0.05, **p<0.005, ***p<0.0005; Mann- Whitney test.