Figure 5

Injection of a sublethal dose of LPS into donor mice increases the frequency of CD11b⁺Gr-1⁺ cells, with elevation of the MyD88 expression level in the TCD-BM, and attenuates the severity of acute GVHD. WT or MyD88KO B6 donor mice were subjected to intraperitoneal (i.p.) injection of LPS at 2 mg kg⁻¹ for 3 days (LPS BM). Mice not receiving LPS received an equal amount of the LPS vehicle (PBS i.p., PBS BM). (a) TCD-BM CD11b⁺Gr-1⁺ cells were examined by flow cytometry for each group (n=5). Data are presented as means±s.e.m. (b) MyD88 mRNA expression in TCD-BM was compared between mice injected with LPS and vehicle (n=3). (c) Lethally irradiated F1 recipients were given 5 × 10⁶ LPS- or PBS-treated donor TCD-BM cells plus 2 × 10⁶ purified WT T cells from allogeneic B6 donors (n=9/LPS BM, n=8/PBS BM). Data from two similar experiments are combined. Kaplan–Meier survival curves are shown (LPS BM vs. PBS BM by the Wilcoxon rank-sum test). Data are presented as means±s.e.m. (d) Alleviation of GVHD by transplantation with LPS-pretreated TCD-BM in a miHA-mismatched allo-HSCT model. BALB.B mice were transplanted with LPS- or PBS-pretreated TCD BM cells (5 × 10⁶) plus 5 × 10⁶ purified T cells from allogeneic B6 mice after total body irradiation (n=10 in each group, LPS BM and PBS BM). The corresponding Kaplan–Meier survival curves are shown (LPS BM vs. PBS BM by the Wilcoxon rank-sum test). The clinical severity of acute GVHD was assessed weekly. Data shown represent two independent experiments. (e–i) Lethally irradiated F1 recipients were given 5 × 10⁶ LPS- or PBS-treated donor TCD-BM cells plus 1 × 10⁶ purified WT T cells from allogeneic B6 donors (n=6 per group). (e) The absolute numbers of total cells in the spleens, mesenteric lymph nodes (MLN) and small intestine (SI) were assessed on day 13 post transplantation. (f–h) The frequencies and absolute numbers of donor T cells (f), and donor-type CD4⁺ (g) and CD8⁺ (h) T cells analyzed via flow cytometry are shown. (i) The expression levels of CD11b⁺Gr-1⁺ were measured via FACS. The frequencies and absolute numbers of CD11b⁺Gr-1⁺ cells are shown. The data are presented as means±s.e.m. Data in a, b and e–i are representative of duplicate experiments. BMT, bone marrow transplantation; FACS, fluorescence-activated cell sorting; GVHD, graft-vs.-host disease; KO, knockout; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; TCD-BM, T-cell depleted bone marrow; WT, wild type.

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