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S. Matsuoka et al.
portions of Foxp3+ cells among donor CD4+ T cells were significantly greater in the spleen of recipients of MyD88- /- T cells than those in controls (Figure 3G and H). Taken together, donor T cells require MyD88 signaling to differ- entiate into Th1, Th17, and Tc1, while it is dispensable for Th2 and Treg differentiation after allo-SCT.
IRAK4 inhibition, but not single TLR deficiency, mitigates lethal graft-versus-host disease
MyD88 transduces signals from both TLR and IL-1 fam- ily cytokines, such as IL-1, IL-18 and IL-33. Although roles of IL-1, IL-18, and IL-33 in GvHD have been well studied, role of T-cell TLR remained to be clarified. First, we exam- ined expression of MyD88-related TLR and IL-1 family cytokine receptors on CD4+ and CD8+ T cells. Q-PCR demonstrated that resting CD4+ and CD8+ T cells expressed TLR2 and TLR7, but to a lesser extent to the levels in macrophages (Figure 4A). In addition, CD4+ T cells also expressed IL-1R, IL-18R and IL-33R, and CD8+ T cells did IL-18R. Flow cytometric analysis confirmed expression of TLR2 and TLR7 both on CD4+ and CD8+ T cells (Figure 4B). Expression of TLR2 and TLR7 on T cells were functional, such as TLR2 ligand, Pam3CSK4, and TLR7 ligands, ssRNA40 and R848 enhanced T-cell proliferative response to CD3/CD28 stimulation in vitro, indicating that TLR2 and TLR7 on T cells have co-stimu-
latory function in T-cell responses (Figure 4C). In contrast, LPS, flagellin, or CpG-ODN did not enhance T-cell expan- sion. Next, we evaluated the role of TLR2 and TLR7 in GvHD by transplanting TLR2-/- or TLR7-/- T cells plus WT TCD-BM. Mortality and morbidity of GvHD in recipients of TLR2-/- or TLR7-/- T cells were identical to those of WT T cells, suggesting that inhibition of single TLR signaling was insufficient to ameliorate GvHD (Figure 4D and E).
Next, we tested if pharmacological inhibition of MyD88 signaling could ameliorate GvHD, using IRAK4 inhibitor PF-06650833 that inhibits signal transduction through MyD88. First, we found that addition of PF-06650833 to culture significantly suppressed T-cell production of IFN-γ after CD3/CD28 stimulation without affecting T-cell pro- liferative response in vitro (Figure 5A-D). PF-06650833 administered daily for three weeks after allogeneic BMT significantly ameliorated morbidity and mortality of GvHD, indicating that MyD88/IRAK4 is a potential phar- macological therapeutic target of GvHD (Figure 5E and F).
MyD88 signaling is dispensable for graft-versus-leukemia activity of donor T cells
Considering the significant reduction of GvHD in the absence of MyD88 signaling in donor T cells, it is of inter- est to evaluate the impact of MyD88 signaling in donor T cells on GvL effect after allogeneic BMT. Lethally irradiat-
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Figure 3. MyD88 deficient donor T cells skew from Th1, Tc1 and Th17 toward T regulatory cell (Treg) differentiation after allogeneic bone marrow transplantation (alloBMT). Mice were transplanted as in Figure 1C. (A-H) Donor T cells harvested from spleens on day +7 were stimulated with PMA/ionomycin and intracellular IFN-γ (A-B), IL-17 (C-D), and IL-4 (E-F) were stained. Representative dot plots (A, C, E), frequencies (B, D, F; top panels) means±Standard Error (SE) and absolute num- bers (B, H, E; bottom panels) (means±SE) of cytokine producing donor T cells are shown. Data from two similar experiments were combined (n=10/group). Representative dot plots (G), frequencies (H; top panels) (means±SE), and absolute numbers (H; bottom panels) (means±SE) of donor CD4+Foxp3+ Tregs from a rep- resentative experiment of three similar experiments are shown (n=5/group). *P<0.05; **P<0.01.
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