cDC1s are required for optimal expansion, chemokine/cytokine receptor expression, and gut homing of Cryptosporidium-specific CD4 ⁺ T cells. (A–E) 1 day prior to infection with 5 × 10⁴ maCp-ova-gp61 oocysts, 1 × 10⁶ CD45.1⁺ Nur77-GFP reporter SMARTA T cells were labeled with CTV and transferred into WT B6 mice or age/sex-matched Irf8+32^−/− mice. Mice were sacrificed at 1, 4, and 6 dpi and priming of SMARTA T cells was interrogated using flow cytometry. (A and B) Representative flow plots from the mesLN of infected mice at 1, 4, and 6 dpi pre-gated on SMARTA T cells showing Nur77-GFP expression compared to cell division, with summary in B showing means with SEM. (C–E) Representative flow plots from mesLN SMARTAs showing CTV versus CXCR3 (C) and LPAM-1 (D) with summary in E. Gating for SMARTAs in A–E: Singlets, Live⁺, NK1.1⁻, CD19⁻, EpCAM⁻, CD90.2⁺, CD8a⁻, CD4⁺, TCR Vβ8.3⁺, CD45.1⁺. For A–E, data is from one experiment representative of two independent experiments, n = 3 mice/group, bar graphs are plotted as mean with SEM. Statistical significance was determined by t test. *P ≤ 0.05, **P ≤ 0.01. (F–I) 1 day prior to infection with 5 × 10⁴ maCp-ova-gp61 oocysts, 2 × 10⁴ CD45.1⁺ SMARTA T cells were transferred into WT B6 mice or age/sex-matched Irf8+32^−/− mice. At 10 dpi, mesLN, SILP, and IEL were isolated and analyzed by flow cytometry. (F) Representative flow plots showing the presence or absence of SMARTA T cells in the mesLN (top), SILP (middle), or IEL (bottom). Flow plots are from one experiment representative of four independent experiments. (G) Summary of F showing means with SEM. For F and G, data are from two independent experiments representative of four independent experiments, n = 3–4 mice/group. Bar graphs show mean with SEM. Statistical significance was determined by two-way ANOVA and multiple comparisons. *P ≤ 0.05, ****P ≤ 0.0001. (H and I) Percentage of mesLN SMARTA T cells staining positive for IL-18Ra with representative flow plots in H and summary in I. Gating for SMARTAs in G–I: Singlets, Live⁺, NK1.1⁻, CD19⁻, EpCAM⁻, CD90.2⁺, CD8a⁻, CD4⁺, CD44-hi, CD45.1⁺. For H and I, data are from one experiment representative of two independent experiments, n = 3 mice/group. Bar graphs show mean with SEM. Statistical significance was determined by two-way ANOVA and multiple comparisons. *P ≤ 0.05.