Thymic γδ T cells in the absence of miR-181a/b-1.

<p>(A) Expression analysis of miR-181a in FACS-sorted thymocytes pooled from 5 adult or 8 neonatal TcrdH2BeGFP mice. Expression levels of the indicated cell populations were analyzed by quantitative RT-PCR and normalized to snoRNA 412. Error bars show range of relative expression levels from triplicates. (B) Bar graph shows absolute γδ T cell numbers in miR-181a/b-1^–/–x TcrdH2BeGFP mice (–/–) compared to TcrdH2BeGFP and miR-181a/b-1^+/–x TcrdH2BeGFP controls (ctrl.), pooled data from five independent experiments with each 2–5 mice per group, mean + SD. (C) Expression analysis of miR-181d in FACS-sorted thymocytes pooled from 5 miR-181a/b-1^–/–x TcrdH2BeGFP mice (–/–) and TcrdH2BeGFP controls (ctrl.). One representative experiment of two independent experiments that gave similar results. Expression levels of the indicated cell populations were analyzed by quantitative RT-PCR and normalized to snoRNA 412. Error bars show range of relative expression levels from triplicates. (D–I) FACS analysis of thymic γδ T cells in–/–mice compared to ctrl mice (D, F-I) and mixed bone marrow chimeras (E). (D) Vγ usage of thymic γδ T cells (gated on Tcrβ^–GFP^hi cells). Scatter plot shows pooled data from five experiments with 3–6 mice per group, one dot represents one mouse, mean. (E) Flow cytometric analysis of 1:1 mixed bone marrow chimeras. Scatter plot shows ratios of miR-181a/b-1^–/–(KO) and miR-181a/b-1 sufficient wild type (WT) donor Vγ1⁺ and Vγ4⁺ cells among all lymphocytes, respectively. Data are pooled from two independent experiments with each 3 mice per group, harmonic mean. (F) Scatter plot shows absolute numbers of NK1.1⁺ cells, pooled data from five independent experiments with each 2–5 mice per group. (G) Scatter plot shows absolute numbers of NK1.1⁺ γδ T cells, pooled data from five independent experiments with each 2–5 mice per group. (H) Representative contour plots of cluster B (CD44^hiCD24^–) and cluster A (CD44^–/loCD24⁺) γδ thymocytes (gated on Tcrβ^–GFP^hi cells), numbers indicate mean +/–SD of pooled data from four independent experiments with each 2–5 mice per group. (I) Representative contour plots of CCR6⁺ and NK1.1⁺ cluster B cells, numbers indicate mean +/–SD of pooled data from four independent experiments with each 2–6 mice per group. Statistical analyses were performed using the Mann-Whitney test.</p

Unchanged peripheral lymph node γδ T cell compartment in the absence of miR-181a/b-1.

<p>FACS analysis of γδ T cells in pLN of miR-181a/b-1^–/–x TcrdH2BeGFP mice (–/–) compared to miR-181a/b-1 sufficient controls, TcrdH2BeGFP and miR-181a/b-1^+/–x TcrdH2BeGFP mice (here referred to as ctrl.). (A) Total γδ T cells numbers in pLN of the indicated phenotypes. Scatter plot shows pooled data from five independent experiment with n = 2–5 mice per group, mean. (B) Scatter plot shows absolute numbers of NK1.1⁺ γδ T cells, pooled data from five independent experiments with each 2–5 mice per group, mean. (C) Vγ usage of γδ T cells (gated on Tcrβ^–GFP^hi cells). Bar graph shows pooled data from 5 experiments with 3–6 mice per group, mean + SD. (D + E) Intracellular cytokine staining for IFN-γ and IL-17A gated on γδ T cells. (D) Representative contour plots of two independent experiments with similar outcome, with each n = 2–5 mice per group. Numbers indicate mean +/–SD from pooled data. (E) Bar gaph shows pooled data from the two independent experiments, mean + SD. Statistical analyses were performed using the Mann-Whitney test.</p

γδ NKT cells fill empty iNKT liver niches in miR-181a/b-1 deficient mice.

<p>FACS analysis of liver lymphocytes of miR-181a/b-1^–/–x TcrdH2BeGFP mice (–/–) compared to TcrdH2BeGFP and miR-181a/b-1^+/–x TcrdH2BeGFP controls (here referred to as ctrl.). (A + B) Analysis of αβ and γδ NKT cells in miR181a/b-1 deficient mice compared to controls. (A) Representative contour plots illustrating the gating strategy for the indicated cell populations in (B). (B) Scatter plot shows frequencies of the indicated cell populations among lymphocytes after doublets were excluded, gated as depicted in (A), pooled data from three independent experiments with each 3–4 mice per group, mean. (C) Bar graph shows total γδ NKT cell numbers, pooled data from 3 independent experiments, each n = 3–4 mice per group, mean + SD. (D) Flow cytometric analysis of 1:1 mixed bone marrow chimeras. Scatter plot shows ratios of miR-181a/b-1^–/–(KO) and miR-181a/b-1 sufficient wild type (WT) donor cells among all lymphocytes, αβ vNKT, αβ iNKT and γδ NKT lymphocytes, respectively, pooled data from two independent experiments with each 4 mice per group, harmonic mean. (E) Scatter plot shows frequencies of INFγ⁺ cells among γδ T cells, pooled data from three independent experiments with each 2–5 mice per group, mean. (F) Vγ usage of liver γδ T cells (gated on Tcrβ^–GFP^hi cells). Bar graph shows pooled data from five experiments with 3–6 mice per group, mean + SD. (G) Analysis of liver Vγ1⁺Vδ6.3⁺ γδ T cells. Scatter plot shows frequencies of Vγ1⁺Vδ6.3⁺ cells among γδ T cells, pooled data from five independent experiments with each 2–5 mice per group, mean. Statistical analyses were performed using the Mann-Whitney test.</p

γδ NKT cells replenish empty niches of missing liver αβ iNKT cells independent of TCR specificity for CD1d.

<p>FACS analysis of liver lymphocytes stained for CD1d/PBS-57 tetramer (CD1d tet) binding. Contour plots show representative CD1d tet binding versus γδ-GFP reporter fluorescence from two independent experiments, with each involving 3 mice per group of miR-181a/b-1 deficient (–/–) and miR-181a/b-1 sufficient (ctrl.) mice.</p

Dendritic epidermal T cells develop in the absence of miR-181a/b-1.

<p>(A) Analysis of Vγ5⁺ γδ T cells in the skin of miR-181a/b-1^–/–x TcrdH2BeGFP mice (–/–) compared to TcrdH2BeGFP mice (ctrl.). (left) Representative contour plots illustrating the gating for Vγ5⁺ γδ T cells. (right) Scatter plot shows frequencies of Vγ5⁺ γδ T cells, pooled data from two independent experiments with each n = 3 mice per group, mean. (B) Histological analysis of epidermal sheets from ears of miR-181a/b-1^–/–x TcrdH2BeGFP mice (–/–) compared to TcrdH2BeGFP mice (ctrl.). Epidermal sheets were stained for DETCs (yellow, overlay red and green) with CD3 (red) and TcrdGFP⁺ (green) cells indicate γδ T cells. Original magnification: 20x, scale bars: 20μm.</p