Fig. 4: GFAT1 deficiency decreases the viability and proliferation of αβ-thymocytes but increases γδ-thymocyte cell numbers. | Nature Communications

Fig. 4: GFAT1 deficiency decreases the viability and proliferation of αβ-thymocytes but increases γδ-thymocyte cell numbers.

From: Dietary glucosamine overcomes the defects in αβ-T cell ontogeny caused by the loss of de novo hexosamine biosynthesis

Fig. 4

Thymocytes from male and female age-matched WT and GFAT1T−/− mice were used for the following experiments. a-b Thymocytes were stained for CD4, CD8, CD44, CD25, TCRβ, γδTCR and analyzed by flow cytometry. Representative plots with bar graph of the proportion of subsets (a) or absolute cell numbers (b) expressing surface TCRβ (αβ-lineage) or γδTCR (γδ-lineage) are shown (a, b, n = 8–12 mice). c 0-, 1-, 3-, 5- and 9-week mice were stained for Lin, CD4, CD8α, CD25, CD44, TCRβ and γδTCR followed by flow cytometric analysis of αβ- (express TCRβ) or γδ-lineage subsets among DN thymocytes. The percentage of each lineage is plotted relative to the mouse age (n = 1–6 mice). d–f Thymocytes from e18/19 and 7-week-old mice were harvested and stained with γδTCR, CD24, CD73, Vγ1.1, Vγ2 and Vγ3 followed by flow cytometric analysis. Partition analysis of immature (CD24−CD73− & CD24+CD73−), maturing (CD24+CD73+) and mature (CD24−CD73+) γδ-thymocyte subsets (d). Shown is a representative plot of three experiments with similar results. Percentage is indicated in each quadrant. Bar graphs represent proportion (e) and total number (f) of mature CD24−CD73+γδTCR+ γδ-thymocytes expressing either Vγ1.1, Vγ2 or Vγ3 (e, f, n = 3–9 mice). g Thymocytes from 5-week-old mice were labeled with CFSE and cultured ex vivo for the indicated hours then harvested and stained as in (c), followed by flow cytometry. Gating was set on live thymocytes and either TCRβ+-DN4 (upper panel) or γδTCR+ DN cells (lower panel). Shown is one representative experiment out of three with similar results. h Thymocytes were cultured ex vivo for 24 h or 48 h and stained for Lin, CD4, CD8α, CD25, CD44, Annexin V, and TCRβ. Graph shows viability of TCRβ+-DN4 cells (gated on TCRβ+ DN4 cells) (h) or γδTCR+ DN cells (gated on DN thymocytes expressing γδTCR) (I); h, i, n = 4 mice each. See also Supplementary Fig. 4. For all graphs in (a–c, e, f, h, i), data are mean ± SD, **p < 0.01, ***p < 0.001 using two-sided Student’s t test. Source data are available for (a–f, h, i).

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