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. 2019 Jul 19;10:1629. doi: 10.3389/fimmu.2019.01629

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Copyright © 2019 Mengrelis, Lau, Rowell, Solanki, Norris, Ross, Ono, Outram and Crompton.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Hh signaling is active in spleen γδ cells and influences spleen γδ subset distribution and homeostasis. (A) QRT-PCR analysis on CD3+γδ+, CD3+CD4+CD8-, and CD3+CD4-CD8+ populations (purified by FACS) from spleen from 4 week-old WT mice. Expression of Smo, Ptch1, and Gli1 are shown (relative to Hprt). Bar charts show mean ± SEM from three independent experiments (biological replicates). (B) Representative histogram shows GFP-fluorescence, gated on CD3+γδ+ cells from GBS-GFP-tg spleen, giving the percentage of GFP+ cells in the marker shown. Representative contour plot shows anti-CD24 staining against GFP-fluorescence from GBS-GFP-tg spleen, giving the percentage of cells in the quadrants. Bar chart shows the mean ± SEM percentage of GFP+ cells, gated on CD3+γδ+ cells, and gated on CD3+γδ+CD24+ cells from GBS-GFP-tg (n = 3) spleen. (C–G) Adult WT mice were injected ip with 40μg/day of the Smo-inhibitor (Smo-inh) PF-04449913 (Pfizer) (n = 13) or vehicle control (DMSO) (n = 13) daily for 14 days and spleen analyzed by flow cytometry. (C) Representative dot plot shows anti-CD3 and anti-γδ staining on spleen cells from control and Smo-inhibitor treated mice, giving the percentage of CD3+γδ+ cells in the region shown. Scatter plots show the percentage and number of CD3+γδ+ cells in the spleen. (D) Scatter plots show the percentage and number of CD27+CD44- cells, gated on CD3+γδ+ from control and Smo-inhibitor treated mice. (E) Scatter plots show the percentage and number of CD27+CD44+ cells, gated on CD3+γδ+ in the spleen from control and Smo-inhibitor treated mice. (F) Representative dot plot shows anti-NK1.1 staining vs. SSC, gated on CD3+γδ+ spleen cells from control and Smo-inhibitor treated mice, giving the percentage of cells in the region shown. Scatter plots show the percentage and number of NK1.1+ cells, gated on CD3+γδ+ in the spleen from control and Smo-inhibitor treated mice. (G) Representative histogram shows anti-Vγ2 staining, gated on CD3+γδ+ cells in the spleen from control and Smo-inhibitor treated mice. Scatter plots show the percentage and number of Vγ2+ cells, gated on CD3+γδ+ cells in the spleen from control and Smo-inhibitor treated mice. In all scatter plots in this figure, each point represents an individual mouse and bars show mean ± SEM. ^*p < 0.05; ^**p < 0.01.