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MyD88/IRAK4 in donor T cells potentiates GVHD
vivo was also equivalent (Figure S2A and B). T-cell prolifer- ation was much more robust in vivo compared to in vitro, probably due to the presence of potent inflammatory milieu induced by TBI. On the other hand, annexin V+ apoptotic donor CD8+ T cells were significantly increased in the recipients of MyD88-/- T cells on day +7, suggesting that MyD88 is an intrinsic survival factor of donor T cells after allo-BMT (Figure 2G).
To assess the role of MyD88 in donor T-cell differentia- tion after allo-BMT, cytokine production was evaluated in donor T cells isolated on day +7 after BMT. MyD88-/- CD4+
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T cells produced significantly less IFN-γ and IL-17, and MyD88-/- donor CD8+ T cells produced less INF-γ than their WT counterpart, indicating that MyD88 signaling is critical for Th1, Th17, and Tc1 differentiation (Figure 3A- D). In contrast, IL-4 production from MyD88-/- donor CD4+ T cells tend to be greater than that from WT CD4+ T cells (Figure 3E and F). Impaired Th1/Tc1, not Th2, dif- ferentiation in MyD88-/- T cells was also confirmed in vitro after CD3/CD28 stimulation (Figure S2C-F). Furthermore, flow cytometric analysis on day +7 demonstrated that absolute numbers of donor CD4+ Foxp3+ Tregs and pro-
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Figure 2. MyD88 is not essential for T-cell proliferation and survival after allogeneic bone marrow transplantation (alloBMT). (A,B,E-H) Mice were transplanted as in Figure 1C. Absolute numbers of donor CD4+ and CD8+ T cells (A) and donor chimerism (B) in the spleen on day +7. Data from four independent experiments were combined and shown as means±Standard Error (SE) (n=17-18/group). (C and D) CellTrace Violet-labeled T cells from WT or MyD88-/- B6 mice were injected and dilu- tion of CellTrace Violet in donor CD4+ and CD8+ T cells in the spleen were assessed on days 3 and 4. Representative histograms (C) and MFI of Cell Trace Violet (D) (means±SE) from two experiments were combined (n=4-6 group). (E and F) Recipients were intraperitoneally injected with 1 mg BrdU on day +6 and BrdU taken up by donor T cells was evaluated two hours later. Representative dot plots (E) and proportions of BrdU positive cells among donor T cells (F) (means±SE). Data from two experiments were combined (n=10/group). (G) Proportion of Annexin V+ cells among donor T cells in the spleen on day +7. Data from a representative experiment of two similar experiments were combined and shown as means±SE (n=9-11 / group). *P<0.05; ***P<0.005.
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