Figure 6. Increased proportion of DCs in MyD88-KO CD11b⁺Gr-1⁺ cells from GVHD hosts. (A, B) The composition of CD11b⁺Gr-1⁺ cells from spleen of allo GVHD hosts on day 7 post-transplantation. PMN-MDSCs (CD11c⁻CD11b⁺CD244⁺Ly6G^hiLy6C^int) and M-MDSCs (CD11c⁻CD11b⁺CD124⁺Ly6G^lowLy6C^hi) including neutrophils (CD11c⁻CD11b⁺CD244⁻Ly6G^hiLy6C^int) and monocytes (CD11c⁻CD11b⁺CD124⁻Ly6G^lowLy6C^hi). (C) Flow cytometric analysis of MHC class II, CD80, and CD86 on CD11c⁺CD11b⁺Gr-1⁺ cells isolated from spleen of WT and MyD88 KO BM GVHD hosts on day 7 post-transplantation. The Geom. MFI was measured and plotted. (D) Proportion of annexin V⁺ cells in CD11b⁺Gr-1⁺CD11c⁺ cells from spleen of allo GVHD hosts on day 7 post-transplantation. (E) Ag-presentation assay. Equal numbers of CFSE-labeled J15 CD8⁺ T cells (CD45.1⁺) were co-cultured with Gr-1⁺ cells (1×10⁵ cells) isolated from spleen in WT or MyD88 KO BM GVHD hosts on day 7 post-transplantation, in the presence of H60 peptide (1 µM) or VSV control peptide (1 µM) for 3 days. The proliferation of J15 CD8⁺ T cells (CD45.1⁺) elicited by H60 peptide was measured by CFSE dilution on day 3 by flow cytometry, and the proportion of proliferation is presented as mean±standard deviation. (F) Immunosuppression assay using Gr-1⁺ cells isolated from spleen in WT or MyD88-KO BM allo GVHD hosts on day 7 post-transplantation. CFSE-labeled CD45.1⁺ T cells (1×10⁵) were co-cultured with equal numbers of Gr-1⁺ cells. T cells only or mixed cells (1:1) were incubated in the absence or presence of anti-CD3 (2 µg/ml)/CD28 (1 µg/ml). The proliferation of CD45.1⁺ T cells was measured by CFSE dilution after 3 days by flow cytometry. The proportions of proliferated CD45.1⁺ CD4⁺ and CD45.1⁺ CD8⁺ T cells were indicated and plotted. Data (B-F) represent 3 independent experiments.

^*p<0.05; ^**p<0.01; ^***p<0.001 (WT vs. KO).