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. 2018 May 25;9:1138. doi: 10.3389/fimmu.2018.01138

Figure 2.

Figure 2

Retinoic acid-related orphan receptor gamma t (RORγt)-dependent T helper (Th17) cells are dispensable for acute intestinal graft-versus-host disease (GvHD). (A) Naïve CD4+ T cells were isolated from the spleen by negative selection using magnetic microbeads and in vitro polarized into inflammatory Th17 cells by co-culturing anti-CD3/anti-CD28 activated T cells in the presence of anti-IFNγ antibodies and either without (−) or with (+) recombinant interleukin (IL)-1β, IL-6, and IL-23. After 5 days, T cells were harvested, RNA was isolated, and transcribed into cDNA followed by quantitative real-time PCR (qPCR) analyses of Il23r transcript levels. Gene expression levels detected within T cells cultured under drift conditions were arbitrarily set down to 1 and all other gene expression levels were normalized to the expression level of this control. Data are combined from two individual experiments and were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons posttest. **p < 0.01 was considered significant. Data are shown as mean ± SEM. (B–E) For GvHD induction, BALB/c mice were irradiated (day 0), transplanted with T cell-depleted bone marrow (BM) (day 1) and subsequently injected with allogeneic splenic CD3+ T cells of wild-type (WT) (n = 18) or Rorc−/− (n = 23) mice (day 2). As a control, one group of mice received T cell-depleted BM alone [no T cells (noT); n = 6]. (B) Clinical symptoms of GvHD mice were scored three times a week. Pooled data from three independent experiments are shown and were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparisons posttest. (C) Between day 28 and day 30, colonic inflammation of WT (n = 12) or Rorc−/− (n = 11) T cell receiving mice was assessed by colonoscopy and representative endoscopic images are shown. Mice were sacrificed the day after colonoscopy. (D) Histopathological scoring of the colitis activity within the distal colon of WT (n = 9) or Rorc−/− (n = 10) T cell receiving mice was performed. One representative hematoxylin and eosin-stained histological cross-section of the distal colon per group is shown. Scale bars: 200 µm. Shown results represent pooled data from two individual experiments. (E) Gene expression levels detected within colonic tissue of WT or Rorc−/− T cell receiving mice of Tnf (WT n = 16; Rorc−/− n = 12) and Il1b (WT n = 17; Rorc−/− n = 14) around day 30 were analyzed by qPCR. Data display pooled data from at least three individual experiments. Data were analyzed by Student’s t-test and are shown as mean ± SEM.