Figure 3

Effect of MyD88-deficiency in TCD-BM on the expansion, and in vivo and in vitro suppressive function of CD11b⁺Gr-1⁺ MDSCs. Lethally irradiated F1 recipients were given 5 × 10⁶ WT or MyD88KO TCD-BM cells plus 1 × 10⁶ purified WT T cells isolated from WT B6 donors (n=5, each group, allo WT and allo KO). (a, b) On day 13 thereafter, the spleen, mesenteric lymph node (MLN) and small intestine (SI) were harvested. (a) Expression levels of CD11b⁺Gr-1⁺ were measured via flow cytometric analysis. The proportions and absolute numbers of CD11b⁺Gr-1⁺ cells are shown. Data are presented as means±s.e.m. and are representative of duplicate experiments. (b) Proportions of Ly6G^hiLy6C^low and Ly6G^lowLy6C^hi populations in the gated CD11b⁺ cells are shown. Data are presented as means±s.e.m. and are representative of duplicate experiments. (c) CD11b⁺Gr-1⁺ cells were isolated from the spleens of allogeneic recipients transplanted with WT TCD-BM and T cells and were processed for May-Grünwald and Giemsa-staining after cytospin preparation (n=3). The percentages of cells in isolated populations exhibiting monocytic (MO; indicated as red arrowhead) and polymorphonuclear (PMN; indicated as black arrowhead) features are shown. (d) CD11b⁺Gr-1⁺ cells isolated from naive B6 mice and allogeneic recipients of T cells plus WT or MyD88KO TCD-BM (n=3, naive, allo WT and allo KO) were compared in terms of the expression of Arginase-1 (Arg-1) and inducible nitric oxide synthase (iNOS) via quantitative RT-PCR. (e) Peripheral blood mononuclear cells (PBMCs) were isolated from GVHD hosts transplanted with WT or MyD88KO TCD-BM (allo WT and allo KO, respectively) on day 13 post transplantation (n=3 per group), and analyzed in terms of the counts of T (CD3⁺), B (B220⁺), myeloid (CD11b⁺) cells of H-2^d-negative donor origin. F4/80-staining was included in the analysis of CD11b⁺Gr-1⁺ cells in PBMCs and spleens. Representative flow cytometry dot plots of frequencies and the corresponding cell numbers are shown. Data are presented as means±s.e.m. The data shown in a-d are representative of two independent experiments. (f, g) Purified T cells (2 × 10⁵) were cultured with irradiated F1 splenocytes (2 × 10⁵) in the presence of purified WT or MyD88KO CD11b⁺Gr-1⁺ cells (WT MDSCs or KO MDSCs, 2 × 10⁵ or 0.2 × 10⁵). The percentages of CFSE and Annexin V on the CD4⁺ (f) and CD8⁺ (g) T cells were determined after 4 days of co-culture (n=3). (h, i) Suppression assays were performed according to the same procedure as described in f and g above, using CD11b⁺Gr-1⁺ cells (2 or 0.2 × 10⁵) purified from GVHD recipients transplanted with 5 × 10⁶ WT (allo WT MDSCs) or MyD88KO TCD-BM cells (allo KO MDSCs) on day 13 post transplantation (n=5). The percentages of CFSE and Annexin V-positive CD4⁺ (h) and CD8⁺ (i) T cells were determined. All of the data are presented as means±s.e.m. Data in f–i represent two independent experiments that yielded similar results. BMT, bone marrow transplantation; CFSE, carboxyfluorescein succinimidyl ester; 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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