Extended Data Fig. 4: IL23R signaling confers a selective advantage on eTreg cells.

(a) Schematic illustration of Tregs in Foxp3^Cre-YFP/+ Il23r^fl/fl female mice. (b,c) FACS-gating strategy (b) and bar graphs (c) displaying Il23r mRNA expression levels as assessed by qPCR in YFP⁺ and YFP⁻ Treg cells from LNs of Foxp3^Cre-YFP/+Il23r^fl/fl female mice. Data depict one experiment with n = 2 (YFP^-) or n = 3 (YFP⁺). (d-h) Foxp3^Cre-YFP/+Il23r^fl/fl female (d,e,f,h) and Foxp3^Cre-YFP/+ non-floxed (g) mice were inoculated i.d. with B16 cells or left untreated. Treg cells in steady state LNs or tumor and tdLNs on day 9 or 14 post-inoculation were analyzed by flow cytometry. Data shown from one out of two independent experiments with n = 5-6. (d) Heatmap depicting marker expression among Treg cell clusters (tumor). (e, f) Frequency plots of Il23r KO and WT of total Treg cells in tdLNs (e) or steady state LNs (f). (g) Contour plots showing YFP⁺ Treg cells on day 14. (h) Spiral plot displaying differential marker expression between Il23r KO and WT Treg cells (tdLNs). (i) Bar graph displaying Foxp3-expression in YFP^-/YFP⁺Treg cells (tdLNs) of Foxp3^Cre-YFP/+non-floxed mice on day 14 post-inoculation. Data from 1 experiment with n = 3. (j) Contour plots displaying IL23R expression as assessed by flow cytometry in human Tregs from steady state PBMCs (gated on CD45⁺ CD3⁺ CD4⁺CD25⁺CD27⁺ FOXP3⁺ cells) or 2 days anti-CD3/CD28 + IL⁻2 stimulated FACS sorted (CD45⁺CD3⁺CD4⁺CD27⁺CD25⁺CD127⁻ from steady state PBMCs) Tregs (gated on CD45⁺ CD3⁺ CD4⁺CD25⁺CD27⁺ FOXP3⁺ cells). Data shown from one experiment with n = 2. (k) Il23r^fl/fl and Foxp3^Cre-YFPIl23r^fl/fl mice were inoculated i.d. with B16 cells. Treg cells in the tdLNs were analyzed by flow cytometry on day 14 post-inoculation. Violin plots displaying median expression (MFI) of cytokines. Data from one experiment with n = 6-12. (l,m,n) Murine Treg cells were ex vivo stimulated with IFN-γ + IL-6, + /- IL-23 for 5 days and analysed by flow cytometry. Boxplots showing the MFI of Foxp3 (l) and total cell numbers (m). (l,m) n = 5.(n) Histograms (left) and boxplots showing normalized MFIs of pSTAT3/pSTAT5 (right). Combined result from two independent experiments with n = 8. (o) Murine Treg cells were ex vivo stimulated with anti-CD3/CD28 + IL-2 for 5 days and +/-IL-23 for 30 min. Histograms (left) and boxplots showing normalized MFIs (flow cytometry). Representative result from two independent experiments with n = 5 (control) or n = 6 (IL-23). (c,i,k,l,n,o) Data are displayed as mean +/- SEM. (c,h,i,k,l,n,o) Statistical significance was determined using, two-tailed t-test (c,h), t-tests (i,k,l) or the two-tailed Mann-Whitney U-test (n,o).

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