Page 241 - 2020_01-Haematologica-web

MyD88/IRAK4 in donor T cells potentiates GVHD
A
B
CDE
Figure 4. Single TLR blockade is not sufficient to mitigate lethal graft-versus-host disease (GvHD). (A) mRNA from CD4+ T cells, CD8+ T cells, and macrophages of naïve WT B6 mice was subjected to quantitative polymerase chain reaction of MyD88-related TLRs and cytokine receptors. Data from three similar experiments were combined and shown as means±Standard Error (SE). (B) Flow cytometric analysis of TLR2 (top panels) and TLR7 on T cells (bottom panels) were performed. Representative histograms from one of three similar experiments were shown. (C) 5x104 T cells were stimulated with CD3/28 mAb-coated beads in the presence or absence of TLR-ligands for three days (n=12/group). Absolute numbers of T cells after stimulation from four similar experiments were combined and shown as means±SE. (D and E) 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, TLR2-/-, or TLR7-/- B6 donors. Survivals (D) and clinical GvHD scores (E) from two independent experiments are combined and shown (n=10/group).
AB
CDEF
Figure 5. IRAK4 inhibitor PF-06650833 ameliorates graft-versus-host disease (GvHD). (A and B) Sorted 5x104 T cells from B6 mice were incubated with Dynabeads coated with anti-CD3/CD28 mAbs in the presence or absence of PF-06650833 at a concentration of 20 mM for 96 hours. Representative dot plots (A) and frequency (B), means±Standard Error (SE), of IFN-γ producing T cells from two similar experiments are combined (n=8/group). (C and D) T cells were labeled with CellTrace Violet and stimulated with Dynabeads coated with anti-CD3/CD28 mAbs for 72 hours. Representative histograms (C) and MFI of CellTrace Violet (D) (means±SE) from one of two similar experiments were shown (n=4/group). (E and F) Mice were transplanted as in Figure 1C. A group of mice were intravenously injected with 12.0 mg/kg PF-06650833 daily from day 0 to day 20 after BMT. Survival curves (E) and clinical GvHD scores (F) from two independent experiments are combined (n=10/group). Ctrl: control.
haematologica | 2020; 105(1)
231