Cyp26b1 is not required for nT_reg cell development and suppressive function.

<p>T cells were isolated from spleens of <i>Cyp26b1</i>^fl/fl and <i>Cyp26b1</i>^−/− mice. (<b>A</b>) CD4⁺Foxp3⁺CD25⁺ nT_reg cell frequencies were determined by flow cytometry. (<b>B</b>) Purified CD4⁺CD25⁺ nT_reg were co-cultured with CFSE-labeled CD4⁺CD25⁻ conventional T (T_c) cells at increasing ratios and suppression of T_c cells was measured by flow cytometry. Data in (<b>A</b>) are from one representative experiment of 3 independent experiments (n = 3−4 per experiment); Data in (<b>B</b>) are from a single experiment.</p

Cyp26b1 limits iT_reg and T_H17 cell differentiation <i>in vitro</i>.

<p>CD4⁺ T cells were isolated from <i>Cyp26b1</i>^fl/fl and <i>Cyp26b1</i>^−/− mice and cultured in iT_reg cell- or T_H17 cell-promoting conditions, in either serum-containing media or in serum-free media. (<b>A</b>) Gene expression of <i>Cyp26b1</i> (normalized relative to <i>Actb</i>) was measured by qRT-PCR. (<b>B</b>) Frequencies of IL-17a⁺ T_H17 cells and (<b>C</b>) Foxp3⁺ CD25⁺ iTreg cells were determined by flow cytometry. Data in (<b>A</b>) represent mean±SEM of 4 independent experiments; Data in (<b>B</b>) and (<b>C</b>) are from one representative experiment of 4 independent experiments.</p

<i>Hic1</i> expression in intestinal ILCs is vitamin A dependent and is required for intestinal immune homeostasis.

<p>(A) ILCs (lin^neg CD90⁺ CD127⁺ cells) were analyzed by flow cytometry for <i>Hic1</i>^{<i>Citrine</i>} reporter expression from the intestinal lamina propria (LP). Data representative of 2 independent experiments (B) <i>Hic1</i> reporter expression in intestinal LP ILCs (lin^neg CD90⁺ CD127⁺ cells) from <i>Hic1</i>^{<i>Citrine</i>} mice fed a control diet, <i>Hic1</i>^Citrine mice fed a vitamin A deficient (VAD) diet, and controls fed a control diet was analyzed by flow cytometry. Data are representative of 2 independent experiments (<i>n</i> = 4–5 per group). (C, D) Intestinal LP cells from <i>Hic1</i>^<i>fl/fl</i> and <i>Hic1</i>^<i>Rorc</i> mice at steady state were analyzed by flow cytometry to enumerate populations of ILC3s (RORγt⁺) and ILC2s (GATA3⁺). Data are from 3 independent experiments (<i>n</i> = 6–7 per group) *, P < 0.05; **, P < 0.01; Mann-Whitney test. Error bars indicate SEM.</p

Cyp26b1 is dispensable for normal lymphoid development.

<p><i>Cyp26b1</i> was specifically deleted in T cells. Thymus, spleen and mesenteric lymph nodes (mesLNs) from <i>Cyp26b1</i>^fl/fl and <i>Cyp26b1</i>^−/− mice were analyzed for CD4⁺ and CD8⁺ cell frequencies by flow cytometry. Data are from one representative experiment of 2 independent experiments (n = 3–4 per experiment).</p

Cyp26b1-deficient T cells fail to promote intestinal inflammation following adoptive transfer into <i>Rag1^−/−</i> mice.

<p>CD4⁺CD45RB^highCD25⁻ naïve effector T cells from <i>Cyp26b1</i>^fl/fl or <i>Cyp26b1</i>^−/− mice were transferred i.p. into <i>Rag1</i>^−/− mice. (<b>A</b>) Weight loss was monitored over the entire disease course. (<b>B</b>) Histological sections of proximal colons stained with hematoxylin and eosin were scored for pathology and colons were scored for gross pathology. Cells from spleens and mesLNs were polyclonally stimulated overnight and stained for (<b>C</b>) IL-17a and (<b>D</b>) Foxp3 and CD25, then measured by flow cytometry. Gene expression of (<b>E</b>) <i>Cd4</i>, (<b>F</b>) <i>Il17a</i>, (<b>G</b>) <i>Ifng</i> and <i>Tnfa</i>, and (<b>H</b>) <i>Foxp3</i> (normalized relative to <i>Actb</i>) in proximal colons was measured by qRT-PCR. Data in (<b>A–H</b>) are representative of one of 2 independent experiments (n = 7−9 per experiment).</p

ILC3-intrinsic HIC1 is required for immunity to <i>Citrobacter rodentium</i> infection.

<p><i>Hic1</i>^<i>Rorc</i> and <i>Hic1</i>^<i>fl/fl</i> mice were orally inoculated with <i>C</i>. <i>rodentium</i>. (A) Weight loss (percentage of initial weight) was calculated for each mouse over course of infection. (B, C) Bacterial loads (CFU/g) from fecal pellets (B) and liver (C) were measured at 11 days post inoculation (p.i.). (D) Quantitative RT-PCR was performed to determine expression of <i>Il17a</i>, <i>Il22</i> and <i>Reg3g</i> from distal colon tissue 11 days p.i. (E, F) H&E stained histological sections of colon (E) and liver (F) from 11 days p.i. Scale bar represents 100μm. Black arrows indicate inflammatory infiltrate. (A–D) Data are pooled from 3 independent experiments (<i>n</i> = 13–14 per group). *, P < 0.05; **, P < 0.01; Mann-Whitney test. Error bars indicate SEM. nd, none detected.</p

ILC3-intrinsic HIC1 is required for immunity to <i>Citrobacter rodentium</i> in T cell-depleted mice.

<p><i>Hic1</i>^<i>Rorc</i> and <i>Hic1</i>^<i>fl/fl</i> mice were treated with a depleting anti-CD4 antibody and then orally inoculated with <i>C</i>. <i>rodentium</i>. (A) Colonic mRNA expression of <i>Cd4</i> in control and anti-CD4 antibody treated <i>Hic1</i>^<i>Rorc</i> and <i>Hic1</i>^<i>fl/fl</i> mice. (B) Weight loss (percentage of initial weight) was calculated for each mouse over course of infection. (C, D) Bacterial loads (CFU/g) from fecal pellets (C) and liver (D) were measured at 11 days post inoculation (p.i.). (E) Quantitative RT-PCR was performed to determine expression of <i>Il17a</i>, <i>Il22</i> and <i>Reg3g</i> from distal colon tissue 11 days p.i. (F, G) H&E stained histological sections of colon (F) and liver (G) from 11 days p.i. Scale bar represents 100μm. Black arrows indicate inflammatory infiltrate. Data from one experiment (<b><i>n</i></b> = 4–6 per group). *, P < 0.05; Mann-Whitney test. Error bars indicate SEM. nd, none detected.</p

Hematopoietic deficiency of HIC1 results in susceptibility to <i>Citrobacter rodentium</i> infection.

<p><i>Hic1</i>^<i>Vav</i> and <i>Hic1</i>^<i>fl/fl</i> mice were orally inoculated with <i>C</i>. <i>rodentium</i>. (A) Weight loss (percentage of initial weight) was calculated for each mouse over course of infection. (B, C) Bacterial loads (CFU/g) from fecal pellets (B) and liver (C) were measured at 11 days post inoculation. (D) Quantitative RT-PCR was performed to determine expression of <i>Il17a</i>, <i>Il22</i> and <i>Reg3g</i> from distal colon tissue 11 days post inoculation. Data are pooled from 2 independent experiments (<i>n</i> = 8–9 per group). *, P < 0.05; **, P < 0.01; Mann-Whitney test. Error bars indicate SEM. nd, none detected.</p

Deficiency in Cyp26b1 does not alter expression of intestinal homing molecules on T cells.

<p>(<b>A</b>) CD4⁺ T cells were isolated from <i>Cyp26b1</i>^fl/fl and <i>Cyp26b1</i>^−/− mice and stimulated with α-CD3/CD28 and IL-2 with or without 10 nM AtRA and α4β7 integrin expression was measured by flow cytometry. (<b>B</b>) CD4⁺ T cells were isolated from <i>Cyp26b1</i>^fl/fl and <i>Cyp26b1</i>^−/− mice were transiently stimulated with α-CD3/CD28 and IL-2 in the presence or absence of 10 nM AtRA to induce CCR9 expression measured by flow cytometry. (<b>C</b>) Gene expression of <i>Batf</i>, <i>Itga4</i>, <i>Itgb7</i>, and <i>Ccr9</i> was measured in isolated <i>Cyp26b1</i>^fl/fl and <i>Cyp26b1</i>^−/− T cells polarized under T_H17- and T_reg-promoting conditions (normalized relative to <i>Actb</i>). Data in (<b>A</b>) are from a single experiment; Data in (<b>B–C</b>) are representative of 3 independent experiments.</p

<i>Hic1</i> expression in T cells and dendritic cells is not required for immunity to <i>Citrobacter rodentium</i> infection.

<p>(A–C) <i>Hic1</i>^<i>CD4</i> and <i>Hic1</i>^<i>fl/fl</i> mice were orally inoculated with <i>C</i>. <i>rodentium</i>. (A) Weight loss (percentage of initial weight) was calculated for each mouse over course of infection. (B) Bacterial loads (CFU/g) from fecal pellets were measured over course of infection. (C) Quantitative RT-PCR was performed to determine expression of <i>Il17a</i>, <i>Il22</i> and <i>Reg3g</i> from distal colon tissue 14 days post inoculation. (D–F) <i>Hic1</i>^<i>CD11c</i> and <i>Hic1</i>^<i>fl/fl</i> mice were orally inoculated with <i>C</i>. <i>rodentium</i>. (D) Weight loss (percentage of initial weight) was calculated for each mouse over course of infection. (E) Bacterial loads (CFU/g) from fecal pellets were measured over course of infection. (F) Quantitative RT-PCR was performed to determine expression of <i>Il17a</i>, <i>Il22</i> and <i>Reg3g</i> from distal colon tissue 11 days post inoculation. (A-C) Data are pooled from 3 independent experiments (<i>n</i> = 7–11 per group). (D-F) Data are pooled from 2 independent experiments (<i>n</i> = 4–5 per group) *, P < 0.05; Mann-Whitney test. Error bars indicate SEM. ns, not significant.</p