Figure 9.

Pirb is expressed on a subset of memory CD4⁺IL17a⁺cells. Flow cytometry strategy for gating on CD3⁺CD4⁺ naive and memory T cells. Splenocytes from (A) WT (top row), Pirb^-/- (bottom row), and (B) IL17a GFP reporter mice were analyzed for PIRA/B expression on T-cell subsets. Plots were pregated on CD3⁺CD4⁺ T cells. (C) Flow cytometry of the expression of PIRA/B and CD69 on CD3⁺CD4⁺CD44⁺ T cells isolated from colonic lamina propria of Il10^-/- and Pirb^-/-Il10^-/- at 10 weeks of age (left). Quantification of colonic lamina propria CD3⁺CD4⁺CD44⁺CD69⁺CD103⁺ T cells (right) (Il10^-/-, n = 9; Pirb^-/-Il10^-/-, n = 9). (D) Quantification of colonic lamina propria CD3⁺CD4⁺CD44⁺CD69⁺CD103⁺ T cells from Rag^-/- mice that received either 400,000 CD4⁺ CD45RB^hi WT or Pirb^-/- T cells (WT, n = 4; Pirb^-/-, n = 7). (E) Flow cytometry of the expression of PIRA/B and IL17a on CD3⁺CD4⁺CD44⁺ T cells isolated from colonic lamina propria of WT IL17a GFP and Pirb^-/- IL17a GFP reporter mice at 10 weeks of age (left). Quantification of CD3⁺CD4⁺CD44⁺IL17a⁺ T cells (right) (WT, n = 5; Pirb^-/-, n = 5). (F) Heat map of differentially expressed genes based on RNA sequencing data (GSE130446) of a selected subset of genes known to be involved in resident memory T cells and Th17 cells comparing between former Th17 (exTh17) resident memory T cells, Th17 resident memory T cells, lymph node naïve CD4⁺ T cells, and lymph node memory CD4⁺ T cells. (G) Representative plots of donor WT (CD45.2⁺) and Pirb^-/- (CD45.1⁺CD45.2⁺) CD4⁺ T cells circulating in the peripheral blood of a Rag^-/- recipient mouse. (H) Percentage of donor WT (CD45.2⁺) and Pirb^-/- (CD45.1⁺CD45.2⁺) CD4⁺ T cells circulating in the peripheral blood in Rag^-/- recipient mice at different time points (WT, n = 10; Pirb^-/-, n = 10). (I) Percentage of donor WT (CD45.2⁺) and Pirb^-/- (CD45.1⁺CD45.2⁺) CD4⁺ T cells that accumulated in different tissues in Rag^-/- recipient mice (WT, n = 9; Pirb^-/-, n = 9). (J) Representative plots of colonic CD3⁺CD4⁺CD44⁺ CD103⁺ T cells that have been derived from donor WT (CD45.2⁺) and Pirb^-/- (CD45.1⁺CD45.2⁺) CD4⁺ T cells injected in a Rag^-/- recipient mouse. (K) Percentage (left) and cell counts (right) of colonic CD3⁺CD4⁺CD44⁺ CD103⁺ T cells that have been derived from donor WT (CD45.2⁺) and Pirb^-/- (CD45.1⁺CD45.2⁺) CD4⁺ T cells injected in a Rag^-/- recipient mouse (WT, n = 9; Pirb^-/-, n = 9). Data are presented as means ± SEM. Statistical analysis was performed using 2-way analysis of variance (P < .05) followed by the (H, I, and K) Sidak multiple comparison test or (C–E) unpaired t test. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. Data shown encompass 3 independent experiments. LPMC, lamina propria mononuclear cell.