Panel A: A timeline diagram shows the experimental design where C 57 B L slash 6 J W T mice were colonized with H. hepaticus (H h) on days 0, 2, and 4, followed by H h-specific nave C D 4 positive T cell transfer on day 7, and intraperitoneal injections of B 7 co-stimulation blockade on days 7, 11, and 14. Analysis was conducted on day 17. Panel B: A bar graph shows the frequency of endogenous C D 4 positive T cells in the mesenteric lymph nodes (m L N) of W T mice treated with I g G 2 a slash 2 b or C D 80 slash C D 86. The y-axis represents the percentage of C D 4 positive T cells, and the x-axis shows the treatment groups. Panel C: A bar graph shows the frequency of endogenous C D 4 positive T cells in the large intestine (L I) of W T mice treated with I g G 2 a slash 2 b or C D 80 slash C D 86. The y-axis represents the percentage of C D 4 positive T cells, and the x-axis shows the treatment groups. Panel D: Bar graphs show the frequency and cell number of endogenous R O R gamma t negative Treg cells in the m L N and L I of the mice. The y-axes represent the percentage and cell number of R O R gamma t negative Treg cells, and the x-axes show different mouse genotypes. Panel E: A timeline diagram shows the experimental design where C 57 B L slash 6 J W T or transgenic mice were orally gavaged with H. hepaticus (H h) on days 0, 2, and 4, followed by H h-specific nave C D 4 positive T cell transfer on day 7, and intraperitoneal injections of C T L A 4-I g or anti-C T L A 4 on days 5, 10, 13, and 16. Analysis was conducted on day 18. Panel F: Bar graphs show the ratio between endogenous R O R gamma t positive and R O R gamma t negative Treg cells by frequency and cell number in the m L N of mice treated with I g G 1 F c or C T L A 4-I g. The y-axes represent the ratio of R O R gamma t positive to R O R gamma t negative Treg cells, and the x-axes show the treatment groups. Panel G: Bar graphs show the ratio between endogenous R O R gamma t positive and R O R gamma t negative Treg cells by frequency and cell number in the L I of mice treated with I g G 1 F c or C T L A 4-I g. The y-axes represent the ratio of R O R gamma t positive to R O R gamma t negative Treg cells, and the x-axes show the treatment groups. Panel H: Bar graphs show the frequency and cell number of H h-specific and endogenous R O R gamma t positive Treg cells in the m L N of C d 80 minus minus C d 86 minus minus; C D 80 T g fl slash fl mice and C l e c 9 a C r e C d 80 minus minus C d 86 minus minus; C D 80 T g f l slash f l mice. The y-axes represent the percentage and cell number of R O R gamma t positive Treg cells, and the x-axes show different mouse genotypes. Panel I: Bar graphs show the frequency and cell number of H h-specific and endogenous R O R gamma t positive Treg cells in the L I of C d 80 minus minus C d 86 minus minus; C D 80 T g f l slash f l mice and C l e c 9 a C r e C d 80 minus minus C d 86 minus minus; C D 80 T g f l slash f l mice. The y-axes represent the percentage and cell number of R O R gamma t positive Treg cells, and the x-axes show different mouse genotypes. Panel J: Bar graphs show the frequency and cell number of endogenous R O R gamma t negative Treg cells in the m L N of the mice. The y-axes represent the percentage and cell number of R O R gamma t negative Treg cells, and the x-axes show different mouse genotypes. Panel K: Bar graphs show the frequency and cell number of endogenous R O R gamma t negative Treg cells in the L I of the mice. The y-axes represent the percentage and cell number of R O R gamma t negative Treg cells, and the x-axes show different mouse genotypes.
Experimental designs and immune profiling of RORγt + Treg cells. (A) C57BL/6J WT mice were colonized with Hh on days 0, 2, and 4, followed by Hh-specific naïve CD4+ T cell transfer retro-orbitally on day 7; mice were i.p. injected with blockade of costimulatory molecules B7-1 (CD80) and B7-2 (CD86) on days 7, 11, and 14. Mice were euthanized for analysis on day 17. (B and C) Frequency of endogenous CD4+ T cells in the (B) mLN and (C) LI of WT mice treated with IgG2a/2b (n = 11) or αCD80/αCD86 (n = 11) as shown in Fig. 1, A–C. (D) Frequency and cell number of endogenous RORγt− Treg cells in the mLN (left two panels) and LI (right two panels) of the mice in Fig. 1, D–I. (E) C57BL/6J WT or transgenic mice as indicated were orally gavaged with Hh on days 0, 2, and 4, followed by Hh-specific naïve CD4+ T cell transfer retro-orbitally on day 7; mice were i.p. injected with CTLA4-Ig or anti-CTLA4 on days 5, 10, 13, and 16. Mice were euthanized for analysis on day 18. (F and G) Ratio between endogenous RORγt+ and RORγt− Treg cells by frequency (left panels) or cell number (right panels) in the (F) mLN and (G) LI of the mice treated with IgG1 Fc (n = 15) or CTLA4-Ig (n = 15) as shown in Fig. 2, A–C. (H and I) Frequencies (left panels in H or I) and cell number (right panels in H or I) of Hh-specific (top panels in H or I) or endogenous (bottom panels in H or I) RORγt+ Treg cells in the (H) mLN and (I) LI of Cd80−/−Cd86−/−; CD80Tgfl/fl mice (n = 4), and Clec9aCreCd80−/−Cd86−/−; CD80Tgfl/fl mice (n = 5). (J and K) Frequency (left panel) and cell number (right panel) of endogenous RORγt− Treg cells in the (J) mLN and (K) LI of the mice in Fig. S1, H and I. The data in Fig. S1 are pooled from three independent experiments. The data are shown as means ± SEM; the statistics shown in B, C, F, G, H, I, J, and K were obtained by unpaired Student’s t test (two-tailed); the statistics shown in D were obtained by ordinary one-way ANOVA with Tukey’s multiple comparisons test. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.