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S. Matsuoka et al.
panied by inflammatory-cell infiltration, was significantly less severe in recipients of MyD88-/- T cells (Figure 1E-G), although liver GvHD was the same as in controls. The thymic GvHD characterized by the loss of CD4+CD8+ double positive (DP) thymocytes was also less severe in MyD88-/- T-cell recipients than in controls (Figure 1H-J). The role of donor T-cell MyD88 signaling in GvHD was not strain dependent, as MyD88 deficiency in donor T cells ameliorates GvHD in the B6 into BALB/c model (Figure 1K).
Absence of MyD88 signaling impairs donor Th1/Tc1/Th17 differentiation
Induction of GvHD is primarily dependent on donor T-cell reactivity to host alloantigens.14 To evaluate the
effects of MyD88 signaling in donor T-cell activation in vivo, donor T-cell expansion was evaluated in the spleen early after BMT. Numbers of CD8+ T cells were signifi- cantly less in MyD88-/- T-cell recipients than those in con- trols seven days after BMT (Figure 2A). Both groups showed complete donor T-cell chimerism, ruling out mixed chimerism as a cause of reduced numbers of donor T cells (Figure 2B). Analysis of cell division using CellTrace Violet-labeled T cells showed equivalent cell division between WT and MyD88-/- T cells at both 72 and 96 hours after BMT (Figure 2C and D). Donor T-cell proliferation determined by BrdU uptake on day 6 also showed equiv- alent T-cell uptake of BrdU in both groups (Figure 2E and F). Proliferative response of WT and MyD88-/- T cells to CD3/CD28 stimulation in vitro and after allogeneic SCT in
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Figure 1. MyD88 signaling in donor T cells exaggerates graft-versus-host disease (GvHD). (A and B) Lethally irradiated B6D2F1 mice were transplanted with 5x106 bone marrow (BM) cells plus 5x106 splenocytes from wild-type (WT) (n=21) or MyD88-/- (n=21) B6 donors on day 0. Survival (A) and clinical GvHD scores (B) from four independent experiments are combined. (C-H) Lethally irradiated B6D2F1 mice were transplanted with 5x106 T-cell-depleted bone marrow cells (TCD-BM) cells from WT B6 mice plus 1x106 purified T cells from WT or MyD88-/- B6 donors. Survival (C) and clinical GvHD scores (D) from five independent experiments are com- bined (n=25-26 / group). (E) Representative Hematoxylin & Eosin (H&E) images of the small intestine, colon, and liver harvested 6-8 weeks after BM transplantation (BMT). (F) Pathological GvHD scores of the liver and total pathological scores in the gut which is the sum of the scores of the small intestine and colon. Data from three independent experiments are combined and shown as means ± Standard Error (SE) (n=8-14/group). (G) Numbers of Paneth cells morphologically identified as cells containing eosinophilic granules at crypt base of the small intestines (white arrow heads in Figure 1E) on day +7 after BMT. Data from two similar experi- ments were combined and shown as means ± SE (n=12 / group). (H-J) CD4+CD8+ double positive thymocytes were assessed 6-8 weeks after BMT. Representative dot plots (H), frequencies (I) (means±SE), and absolute numbers (J) (means±SE) of CD4+CD8+ thymocytes from one of two similar experiments were shown (n=5/ group). (K) BALB/c mice recipients were transplanted with 5x106 TCD-BM cells from WT B6 mice plus 1x106 purified T cells from WT or MyD88-/- B6 donors following total body irradiation on day 0 (n=11/group). Data from two similar experiments were combined. Bar, 50 mm. **P<0.01; ***P<0.005.
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