Figure 4. OvaFla expression in intestinal epithelial cells (IECs) results in OT-I cross-priming that is independent of NLRC4 but partially dependent on gasdermin D.

(A) Schematic depicting the production and analysis workflow of chimeric bm1⁺OvaFla mice (left). At the right, an illustration of either wild-type (WT) OvaFla mice (left of the dashed line) or Nlrc4^–/– OvaFla mice (right of the dashed line) following lethal irradiation and reconstitution with bone marrow from B6.SJL mice. (B) Quantification of OT-Is as a percent of total CD8⁺ T cells (left), the total number of OT-Is (middle), and the total number of CD62L^–CD44⁺ OT-Is (right) in the spleen. (C) Quantification of OT-Is as a percent of total CD8⁺ T cells (left), the total number of OT-Is (middle), and the total number of CD62L^–CD44⁺ OT-Is (right) in the mesenteric lymph nodes. Tissues were harvested and analyzed at day 5 post tamoxifen chow start. (B–C) Data are pooled from three biological replicates, and each dot represents an individual mouse. Data shown as mean ± SD. Significance calculated using one-way ANOVA and Tukey’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001). Only p values between WT and other experimental groups are shown. See Figure 4—source data 1 for exact p values.

Figure 4—figure supplement 1. OvaFla expression in intestinal epithelial cells (IECs) of bm1⁺OvaFla mice results in NAIP–NLRC4 expression and OT-I cross-priming that is independent of NLRC4 but partially dependent on gasdermin D.

(A) Quantification of IL-18 ELISA performed on serum from the mice shown in Figure 4 at day 5 post tamoxifen chow start. (B) Representative histograms of CellTrace Violet dilution for the indicated OvaFla mouse lines. (C) Percent of OT-Is that are CD62L^–CD44⁺ in the spleen (left) and mesenteric lymph nodes (right) of the mice shown in Figure 4. (D) Percent of OT-Is from the mesenteric lymph nodes of the mice in Figure 4 that are IFNγ⁺TNFα⁺ following a 5 hr ex vivo stimulation with phorbol myristate acetate (PMA) (1 μg/mL) and ionomycin (1 μg/mL). Tissues were harvested and analyzed at day 5 post tamoxifen chow start. Samples with fewer than 20 OT-Is were excluded from CD62L, CD44, and cytokine calculations. (A) Data are pooled from two biological replicates. (C) Data are pooled from three biological replicates. Each dot represents an individual mouse. Data shown as mean ± SD. Significance calculated using one-way ANOVA and Tukey’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001). Only p values between wild-type (WT) and other experimental groups are shown. See Figure 4—figure supplement 1—source data 1 for exact p values.

Figure 4—figure supplement 2. K^b donor bone marrow is required for OT-I proliferation and activation in bm1⁺ OvaFla bone marrow chimeras.

(A) Schematic depicting the production and analysis workflow of chimeric bm1⁺OvaFla mice that were given either B6 H-2K^b or H-2K^bm1 bone marrow. (B) Representative flow plots demonstrating the absence of OT-Is in the mice given H-2K^bm1 bone marrow, as depicted in A. (C) Quantification of the total number of OT-Is (top) and the OT-Is as a percent of total CD8⁺ T cells (bottom) in the spleen (left) and mesenteric lymph nodes (right) of bm1⁺ wild-type (WT) and bm1⁺ Nlrc4^– OvaFla mice as depicted in A. Data are from a single experiment, and each dot represents an individual mouse. Data shown in C as mean ± SD.