Reduced Graft-versus-Host Disease in C3-Deficient Mice Is Associated with Decreased Donor Th1/Th17 Differentiation  Qing Ma, Dan Li, Roza Nurieva, Rebecca Patenia, Roland Bassett, Wei Cao, Andrei M. Alekseev, Hong He, Jeffrey J. Molldrem, Michael H. Kroll, Richard E. Champlin, George E. Sale, Vahid Afshar-Kharghan  Biology of Blood and Marrow Transplantation  Volume 18, Issue 8, Pages 1174-1181 (August 2012) DOI: 10.1016/j.bbmt.2012.05.014 Copyright © 2012 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 GVHD mortality and morbidity in WT and C3−/− recipient mice. (A) Lethally irradiated WT or C3−/− mice received TCD bone marrow cells and splenocytes from Balb/c mice. The control were WT mice received only TCD BM cells. Survival was monitored daily for 8 weeks. Distributions of time to death were estimated using the Kaplan-Meier method, and groups were compared using the log-rank test. The results of four independent experiments (5 mice/group/experiment) were summarized. P = .0008; n = 20 for each group. (B) Average daily weight of recipient WT and C3−/− mice as a surrogate measure of GVHD. (C) Representative tissue sections from GVHD organs on day 7 posttransplantation stained with hematoxylin and eosin. In WT mice (top panel), arrows indicate the histopathological changes in the skin (inflammatory cells infiltration and apoptotic cells in hair follicles), intestine (necrosis/apoptosis in the hyperplastic crypts), liver (lymphoplasmacytic infiltration of portal tracts, segmental loss of bile duct epithelial cells and areas of focal necrosis of hepatocytes), and lung (thickened alveolar wall with inflammatory infiltration and necrotic cells). In C3−/− recipient mice (lower panel), inflammatory cell infiltration was minimal with normal structure of skin, small intestine, liver, and alveoli in the lung. (D) Average GVHD scores of skin, intestine, liver, and lung in WT and C3−/− recipient mice. Results are shown as mean ± SD (t test; n = 3 in each group). *P < .05. Biology of Blood and Marrow Transplantation 2012 18, 1174-1181DOI: (10.1016/j.bbmt.2012.05.014) Copyright © 2012 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Donor-derived T cells in WT and C3−/− recipient mice. (A) Donor-derived T cells in the secondary lymphoid organs. Spleen, lymph nodes, and Peyer's patches were harvested from WT and C3−/− recipient mice on day 7 posttransplantation (n = 3 in each group from 3 independent experiments). Cells recovered from these tissues were stained with CD3, CD4, CD8, and H-2Dd antibodies. The numbers of donor-derived T cells (H-2Dd+) and CD4+H-2Dd+ and CD8+H-2Dd+ subsets were determined by FACS. (B-D) Donor-derived IL-17– and IFN-γ–producing cells in WT and C3−/− recipient mice. Cells from spleen, mesenteric, and peripheral lymph nodes were collected from WT mice (n = 4) and C3−/− mice (n = 5) at 7 days posttransplantation. Cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin in the presence of Golgi-stop for 5 hours, and then stained with CD4, IL-17, IFN-γ, and H-2Dd antibodies. The percentage (B and C) and absolute number (D) of IL-17+, IL-17+IFN-γ+, and IFN-γ+ cells from donor-derived CD4+ cells (CD4+H-2Dd+) was determined by FACS from 4 independent experiments. In (B) the data are representative contour plots of intracellular IL-17 and IFN-γ staining gated on CD4+H-2Dd+ cells, and the quadrant gates were decided based on isotype controls. In (C) results are shown as bar graphs representing mean ± SD. P values were calculated using a t test comparing WT and C3−/− mice. *P < .05. Biology of Blood and Marrow Transplantation 2012 18, 1174-1181DOI: (10.1016/j.bbmt.2012.05.014) Copyright © 2012 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Donor-derived T cells in WT and C3−/− recipient mice. (A) Donor-derived T cells in the secondary lymphoid organs. Spleen, lymph nodes, and Peyer's patches were harvested from WT and C3−/− recipient mice on day 7 posttransplantation (n = 3 in each group from 3 independent experiments). Cells recovered from these tissues were stained with CD3, CD4, CD8, and H-2Dd antibodies. The numbers of donor-derived T cells (H-2Dd+) and CD4+H-2Dd+ and CD8+H-2Dd+ subsets were determined by FACS. (B-D) Donor-derived IL-17– and IFN-γ–producing cells in WT and C3−/− recipient mice. Cells from spleen, mesenteric, and peripheral lymph nodes were collected from WT mice (n = 4) and C3−/− mice (n = 5) at 7 days posttransplantation. Cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin in the presence of Golgi-stop for 5 hours, and then stained with CD4, IL-17, IFN-γ, and H-2Dd antibodies. The percentage (B and C) and absolute number (D) of IL-17+, IL-17+IFN-γ+, and IFN-γ+ cells from donor-derived CD4+ cells (CD4+H-2Dd+) was determined by FACS from 4 independent experiments. In (B) the data are representative contour plots of intracellular IL-17 and IFN-γ staining gated on CD4+H-2Dd+ cells, and the quadrant gates were decided based on isotype controls. In (C) results are shown as bar graphs representing mean ± SD. P values were calculated using a t test comparing WT and C3−/− mice. *P < .05. Biology of Blood and Marrow Transplantation 2012 18, 1174-1181DOI: (10.1016/j.bbmt.2012.05.014) Copyright © 2012 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Recipient-derived lymphoid DCs in WT and C3−/− recipient mice. Cells from spleen and lymph nodes were collected from WT (n = 5) and C3−/− (n = 5) recipient mice 2 days posttransplantation, and stained with CD11c, CD8α, and H-2Db antibodies. The percentage of CD8α+ and CD8α− DCs (CD11c+H-2Db+) were determined by FACS. (A) Percentage of recipient-derived CD8α+ DCs (CD11c+H-2Db+) shown in the representative contour plots of CD8α and CD11c expression gated on H-2Db+ cells. The quadrant gates were determined based on isotype controls. (B) Results shown as bar graphs representing mean ± SD of 4 independent experiments. P values were calculated using a t test comparing WT and C3−/− mice. *P < .05. Biology of Blood and Marrow Transplantation 2012 18, 1174-1181DOI: (10.1016/j.bbmt.2012.05.014) Copyright © 2012 American Society for Blood and Marrow Transplantation Terms and Conditions