Panel A: A volcano plot showing differentially expressed genes K l r b 1 b and S p i 1 in L T i-like I L C 3 s and e T A C s respectively. The x-axis represents log 2 fold change (F C) and the y-axis represents minus log 10 of p-value (P). Panel B: Flow cytometry gating strategies for T cell subsets (C D 4 positive and C D 8 positive) and antigen-presenting cell (A P C) populations, including conventional dendritic cells (c D C s), macrophages, and R O R gamma t positive A P C s (e T A C s and I L C 3 s) in the mesenteric lymph nodes (m L N). Panel C: Bar graph quantifying C T L A 4-I g binding to A P C and T cell subsets in cells isolated from m L N of mice. The x-axis lists different cell subsets and the y-axis shows the percentage of Human-I g G-P E positive cells. Panel D: Bar graph quantifying C T L A 4-I g binding to A P C and T cell subsets in cells isolated from the large intestine (L I) of mice. The x-axis lists different cell subsets and the y-axis shows the percentage of Human-I g G-P E positive cells. Panel E: Bar graphs showing frequencies and cell numbers of c D C s, macrophages, and R O R gamma t positive A P C s in the m L N of mice treated with I g G 1 F c and C T L A 4-I g. The x-axis lists different treatments and the y-axis shows the percentage or cell number of specific cell subsets. Panel F: Bar graphs showing frequencies and cell numbers of c D C s, macrophages, and R O R gamma t positive A P C s in the L I of mice treated with I g G 1 F c and C T L A 4-I g. The x-axis lists different treatments and the y-axis shows the percentage or cell number of specific cell subsets.
Flow cytometry gating strategies and profiling of APC subsets and T cells. (A) Volcano plot showing Klrb1b and Spi1 as differentially expressed genes in LTi-like ILC3s and eTACs, respectively, of the scRNA-seq dataset generated from the healthy mouse mLN samples as published (Lyu et al., 2022). FC, fold change. (B) Flow cytometry gating strategies for T cell subsets (CD4+ and CD8+) and APC populations, including cDCs, macrophages, and RORγt+ APCs (eTACs and ILC3s) in the mLN; similar strategies were applied for gating T cells and APC populations in the LI. (C and D) Quantification of CTLA4-Ig binding to APC and T cell subsets in the cells isolated from the (C) mLN or (D) LI of the mice followed by incubation with PBS (n = 7), IgG1 Fc (n = 7), and CTLA4-Ig (n = 8). (E and F) Frequencies of total, CD80+, CD86+, or cell numbers of cDCs, macrophages, or RORγt+ APCs in the (E) mLN and (F) LI of mice treated with IgG1 Fc (n = 10) and CTLA4-Ig (n = 10). The data in Fig. S2 are pooled from two independent experiments. The data are shown as means ± SEM; the statistics shown in E and F were obtained by unpaired Student’s t test (two-tailed). ns, not significant; *P < 0.05; **P < 0.01. cDCs, conventional dendritic cells.