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.