Skip to main content
. 2018 May 25;9:1138. doi: 10.3389/fimmu.2018.01138

Figure 5.

Figure 5

Retinoic acid-related orphan receptor gamma t-independent intestinal graft-versus-host disease (GvHD) is controlled by basic leucine zipper transcription factor ATF-like (BATF). (A) Transcript levels of Batf in colonic tissue of allo-HSCT BALB/c mice with severe signs of intestinal GvHD following transplantation of allogeneic CD3+ T cells derived from of Batf−/− and Rorc−/− mice were measured by quantitative real-time PCR (qPCR) at day 15 (Batf−/− n = 12; Rorc−/− n = 2) and day 30 (Batf−/− n = 12; Rorc−/− n = 4). Data represent pooled data from two independent experiments. Gene expression levels detected in colonic tissues of no T cells (noT) controls at day 15 were arbitrarily set down to 1 and all other gene expression levels were normalized to the expression level detected within noT controls. (B) Transcript levels of Rorc in colonic tissue of irradiated BALB/c mice transplanted with T cell depleted bone marrow alone (noT, n = 11) or followed by injection of allogeneic CD3+ T cells of wild-type (WT) (n = 12) mice were measured by qPCR at day 30. (C) Transcript levels of RORC in colonic tissue biopsies from allo-HCT patients were measured by qPCR. Samples were grouped into indicated categories based on the absence (−GvHD; n = 30) or presence (+GvHD; n = 22) of GvHD-associated histopathological lesions or by the absence (−apoptosis; n = 32) or presence (+apoptosis; n = 20) of GvHD-related epithelial cell apoptosis. Data are shown as normalized relative RORC expression levels calculated from a standard curve. (D–G) Experimental GvHD induction was achieved as described in Figures 1 and 2. To this end, allogeneic hematopoietic stem cell transplantation BALB/c mice were transplanted with allogeneic CD3+ T cells derived from WT (n = 4), Rorc−/− (n = 3), or Rorc−/−Batf−/− (n = 3) mice or received no T cells (noT) as a control (n = 4). (D) Mice were assessed three times a week for signs and severity of systemic GvHD. Data were analyzed by two-way ANOVA testing followed by Bonferroni’s multiple comparisons posttest. ****p < 0.0001 was considered significant, whereas p > 0.05 indicates no significance (ns). (E) At day 28, colitis severity was determined by colonoscopy and representative endoscopic images are shown. (F) Expression levels of Ifng and Csf2 transcripts within colonic tissues of Rorc−/− (n = 7) or Rorc−/−Batf−/− (n = 3) T cell receiving mice were analyzed by qPCR. Data are shown from at least one experiment. (G) Colonic lamina propria mononuclear cells of indicated donor T cell receiving mice were isolated and the frequencies of granulocyte-macrophage colony-stimulating factor (GM-CSF+) or IFNγ+ CD4+ donor T cells were assessed by flow cytometry employing intracellular cytokine staining techniques. Data of panels (A,E) were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons posttest. **p < 0.01, ***p < 0.001, and ****p < 0.0001 was considered significant. Data of panels (B,C,F,G) were analyzed by Student’s t-test. *p < 0.05 and **p < 0.01 was considered significant. All data are shown as mean ± SEM.