Figure 4

Injection of CD11b⁺Gr-1⁺ MDSCs from WT mice, but not MyD88KO mice, regulates the development of acute GVHD in recipients of MyD88KO donor TCD-BM. (a) CD11b⁺Gr-1⁺ cells were isolated from either WT or MyD88KO mice. This population comprises ⩾95% of the CD11b⁺Gr-1⁺ MDSCs fraction by flow cytometric analysis. Each May-Grünwald and Giemsa-stained cytospin preparation is shown. (b) Lethally irradiated F1 recipients were given 5 × 10⁶ MyD88KO TCD-BM cells plus 1 × 10⁶ purified WT T cells from allogeneic B6 donors. Mice exhibiting GVHD that had received MyD88KO TCD-BM cells were injected with 1.0 × 10⁶ CD11b⁺Gr-1⁺ MDSCs isolated from WT or MyD88KO mice (n=12; allo KO+WT MDSCs or allo KO+KO MDSCs, respectively) at days 3, 5, and 7 post transplantation. In parallel, GVHD was induced with WT or MyD88KO TCD-BM plus WT T cells, but without MDSC supplementation (n=9; allo WT or allo KO, respectively). Data from two similar experiments are combined. The Kaplan–Meier survival curves are shown (allo KO+WT MDSCs vs. allo KO+KO MDSCs by the Wilcoxon rank-sum test). The data are presented as means±s.e.m. (c) Small bowels were collected for histological examination on day 13 after transplantation. Pathological scores were compared between mice receiving WT MDSCs and MyD88KO MDSCs, as shown in Figure 1d. Representative pathological findings of severe lesions are indicated as arrows. The middle figures (× 12.5) are the areas within the black squares, and the right figures (× 200 images) are representative of regions with severe damage. Data are presented as means±s.e.m. (d) The absolute numbers of total cells in the spleens, mesenteric lymph nodes (MLN) and small intestine (SI) were assessed from the allogeneic recipients of 1.0 × 10⁶ CD11b⁺Gr-1⁺ MDSCs on day 13 post transplantation. (e–g) The frequencies and absolute numbers of donor T cells (e) with donor-type CD4⁺ (f) and CD8⁺ (g) T cells analyzed via flow cytometry are shown. (h) Expression of CD11b⁺Gr-1⁺ was measured using FACS analysis. The frequencies and absolute numbers of CD11b⁺Gr-1⁺ cells are shown. Data are presented as means±s.e.m. Data in c–h are representative of duplicate experiments (n=6 per experiment). BMT, bone marrow transplantation; FACS, fluorescence-activated cell sorting; GVHD, graft-vs.-host disease; KO, knockout; MDSC, myeloid-derived suppressor cell; TCD-BM, T-cell depleted bone marrow; WT, wild type.

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