Fig. 4.

Enhanced neutrophil generation by IDO-KO BM cells in GVHD hosts in vivo and in vitro. (A) Detection of neutrophils in the Ly6G^hi cells generated in GVHD hosts on day 7 posttransplantation. Representative CD115 and CD244 FACS profiles of Ly6G^hi (CD45.2^-) cells are shown. Percentages of PMN-MDSCs and neutrophils in Ly6G^hi cells and the numbers are plotted. (B and C) Comparison of the transcriptome of the IDO-KO and WT Gr-1⁺ cells with those of mouse neutrophils and monocytes retrieved from ImmGen (Immunological Genome Project; https://www.immgen.org/). (B) The DEGs up-regulated in IDO-KO (labeled as IDO KO > WT; red) and down-regulated in IDO-KO (labeled as IDO-KO < WT; green) were plotted (monocyte versus neutrophil scatter plot). Some representative genes are marked with their names. (C) Counts of up- and down-regulated genes in IDO-KO for monocyte and neutrophil areas (below and above, respectively, the diagonal of the scatter plot in B) were tested for significance by Fisher’s exact test. (D) Fractions and numbers of PMN-MDSCs and neutrophils among Ly6G^hi cells generated in vitro from the TCD-BM of IDO-KO and WT mice. Representative CD115 and CD244 FACS profiles of Ly6G^hi cells are shown. (E) T cell phenotype of BALB.B GHVD hosts treated with a neutrophil-depleting or control Ab. Splenic T cells were analyzed on day 14 posttransplantation. Frequencies of IFN-γ⁺ or IL-17⁺ cells in CD4⁺ and CD8⁺ T cells, Foxp3⁺CD25⁺ Tregs, and H60-tetramer⁺ CD8 T cells are plotted. Data (A, D, and E) represent three independent experiments (n = 3 per group per experiment) and are presented as means ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001 as determined by Student’s t test). ns, not statistically significant.