Panel A: A timeline diagram shows the experimental procedure involving C 57 B L slash 6 J W T mice colonized with H h, followed by treatments with anti-I L-10 R and C T L A 4-I g, and subsequent analysis on day 24. Panel B: Flow cytometry plots display H h-specific C D 4 positive T cells in mesenteric lymph nodes (m L N) and large intestine (L I) of mice treated with I g G 1 F c or C T L A 4-I g. Panel C: Bar graphs show the frequencies of H h-specific R O R gamma t positive Treg cells in m L N and L I. Panel D: Bar graphs depict the frequencies of H h-specific T H 17 cells in m L N and L I. Panel E: A bar graph illustrates the frequencies of T-bet positive H h-specific T H 17 cells in L I. Panel F: A bar graph shows the frequencies of I F N positive H h-specific C D 4 positive T cells in L I. Panel G: Flow cytometry plots display T N F positive H h-specific T H 17 cells in L I. Panel H: A bar graph shows the frequency of T N F positive H h-specific T H 17 cells in L I. Panel I: Images of the large intestine (L I) show typhlocolitis in mice treated with I g G 1 F c or C T L A 4-I g. Panel J: Images of enlarged mesenteric lymph nodes (m L N) in treated mice. Panel K: Histological images of the distal colon stained with H and E show neutrophil infiltration in mice treated with I g G 1 F c or C T L A 4-I g. Panel L: A bar graph shows the cell number of neutrophil infiltration in L I.
CTLA4-Ig enforces microbiota-specific RORγt + Treg cells and protects from gut inflammation. (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 anti-IL-10R on days 0, 6, 12, and 18, and were i.p. injected with CTLA4-Ig on days 5, 10, 13, 16, 19, and 22. Mice were euthanized for analysis on day 24. (B–D) (B) Flow cytometry plots of the Hh-specific CD4+ T cells (mLN, top panel; LI, bottom panel) and frequencies of (C) Hh-specific RORγt+ Treg cells and (D) Hh-specific TH17 cells in the mLN (top panel) and LI (bottom panel) of Hh-colonized WT mice treated with IgG1 Fc (n = 5) or CTLA4-Ig (n = 4). TH17 cells were gated as CD4+ Foxp3− RORγt+ T cells, and this strategy was applied consistently in all experiments. (E and F) Frequencies of (E) T-bet+ Hh-specific TH17 cells and (F) IFNγ+ Hh-specific CD4+ T cells in LI of the WT mice in B. (G and H) (G) Flow cytometry plots and (H) frequency of TNF+ Hh-specific TH17 cells in LI of the WT mice in B. (I–K) Images of (I) LI showing typhlocolitis, (J) enlarged mLNs, and (K) H&E staining of the distal colon in the WT mice in B. Scale bars, 100 μm. (L) Cell number of neutrophil infiltration in LI of the WT mice in B. The data in Fig. 3 are representative of two independent experiments. The data are shown as means ± SEM; the statistics shown in C, D, E, F, H, and L were obtained by unpaired Student’s t test (two-tailed). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.