Fig. 2: Mouse cytokine-responsive CD4−/CD8− mucosal T cells respond to microbial stimuli. | Nature Communications

Fig. 2: Mouse cytokine-responsive CD4−/CD8− mucosal T cells respond to microbial stimuli.

From: A conserved population of MHC II-restricted, innate-like, commensal-reactive T cells in the gut of humans and mice

Fig. 2

See also Supplemental Fig. 4. Mouse immune cells were isolated from various tissues and analyzed by flow cytometry and/or stimulated in vitro. A Flow cytometric analysis of colonic lamina propria leukocytes from IL-23RGFP/+ mice gated TCRβ + lin-iTCR- (MR1tet-CD1dtet-TCRγδ-CD11c-CD11b-B220-) showing IL-7R and IL-23R co-expressing6 cells (left). The gated IL-7R+/IL-23R+ population is shown for CD4 and CD8a expression followed by quantification of the percent of the IL7R+/IL23R+ population is either CD4+, CD8a+ or CD4/CD8a double negative (DN). Quantification of gated TCRβ+ in- CD4, CD8a, or DN cells that have the IL-7R/IL-23R positive phenotype. Each point represents one individual mouse (n = 6), representative of three independent experiments. One-way ANOVA with Tukey’s multiple comparison test. B Example flow cytometry plot shows TCRβ + lin-iTCR-CD4/CD8− cells expressing IL-7R also express the IL-18R (left). IL-7R and NK1.1 are not co-expressed in DN T cells (right). Plots are from different experiments but representative of two experiments each, n = 5. C Example FACS plot (colon) and quantification of DN phenotype in mouse colon, lung, liver, mesenteric lymph node (MLN), spleen, and thymus. Each point represents one individual mouse. n = 5 for colon, n = 6 for all others, representative of two independent experiments. One-way ANOVA with Dunnett’s multiple comparisons test comparing each mean to colon. D Example FACS plot (colon) and quantification of T cells innate-like phenotype (IL-7R+/PLZF+) in mouse colon, lung, liver, MLN, spleen, and thymus. Each point represents one individual mouse. n = 5 for colon, n = 6 for all others, representative of two independent experiments. One-way ANOVA with Dunnett’s multiple comparisons tests comparing each mean to colon. E FACS sorted TCRβ + lin-iTCR-CD4/CD8− cells were isolated from mouse colon and stimulated in vitro with either aCD3/aCD28 or IL-23 or rested in complete media for 24 h when RNA was extracted and qPCRs performed for Cd3e, Zbtb16 (PLZF), Zbtb7b (ThPOK), Il22, Il17a, Csf2 (GM-CSF), Ifng, and Areg. Genes were normalized to Hprt. Each point represents one mouse. Data represent two combined experiments, n = 9 in total except for Zbtb7b and Areg n = 3. Kruskal–Wallis with a Dunn multiple comparison test. F Quantification of FACS data from DN TCRβ+lin− cells from colon LPLs from mice aged 4, 20, and 50 weeks. n = 5, representative of two independent experiments. One-way ANOVA with Tukey’s multiple comparison test. G Quantification of FACS data of IL-7R expression on the DN T-cell population of 4, 20, and 50-week-old mice. Each point represents an individual mouse. n = 5, representative of two independent experiments. Mean with standard deviation shown, one-way ANOVA with Tukey’s multiple comparison test. H Quantification of FACS data of DN TCRβ + lin− cells from colon LPLs from specific pathogen-free (SPF) or germ-free (GF) mice. Each point represents an individual mouse. n = 6, representative of three independent experiments. Unpaired t test. I Absolute cell numbers of the colonic DN cell population of SPF and GF mice. Each point represents and individual mouse. n = 6, representative of three independent experiments with unpaired t test. Mean with standard deviation shown for all graphs. Source data are provided as a Source Data file.

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