B7.2−/− Mature Dendritic Cells Generate T-Helper 2 and Regulatory T Donor Cells in Fetal Mice after In Utero Allogeneic Bone Marrow Transplantation Swati.

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B7.2−/− Mature Dendritic Cells Generate T-Helper 2 and Regulatory T Donor Cells in Fetal Mice after In Utero Allogeneic Bone Marrow Transplantation Swati Bhattacharyya, Morton J. Cowan Biology of Blood and Marrow Transplantation Volume 11, Issue 9, Pages 657-671 (September 2005) DOI: 10.1016/j.bbmt.2005.05.012 Copyright © 2005 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Proliferation of allogeneic (BALB/c) CD3+ splenocytes in response to varying numbers of B6 mDCs from WT and knockout mice. A, Mature DCs from WT B6 donors. B, Mature DCs from B7.1−/− B6 donors. C, Mature DCs from B7.2−/− B6 donors. Biology of Blood and Marrow Transplantation 2005 11, 657-671DOI: (10.1016/j.bbmt.2005.05.012) Copyright © 2005 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Fluorescence-activated cell-sorting analysis of donor H2Kb+CD4+ T cells in blood 6 weeks after IUT with lin− BM plus B7.2−/− mDCs. A, Dot-plot analysis of CD3 versus CD4 of H2Kb+ gated cells. BALB/c and B6 blood were used as negative and positive controls, respectively. B, Cytokine expression (PE-conjugated interferon γ, IL-2, IL-10, and IL-4) in H2Kb+CD4+ splenocytes from an IUT recipient. C, CD25 expression in H2Kb+CD4+ splenocytes after IUT. Biology of Blood and Marrow Transplantation 2005 11, 657-671DOI: (10.1016/j.bbmt.2005.05.012) Copyright © 2005 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Inhibition of the MLR by CD4+CD25+ and CD4+CD25− T-regulatory cells. CD4+CD25+ and CD4+CD25− T cells were evaluated as regulatory cells to inhibit the allogeneic MLR between BALB/c splenocyte responders and irradiated B6 splenocyte stimulators. The experiment was repeated twice. A, Donor CD4+CD25− versus CD4+CD25+ T-cell fractions were cocultured in the MLR. B, Effect of anticytokine antibodies (anti–IL-10, anti–IL-4, and anti–TGF-β) on in vitro proliferation of allogeneic (BALB/c) CD3+ splenocytes when stimulated with donor H2Kb+CD4+CD25− T cells. C, Effect of anticytokine antibodies (anti–IL-10, anti–IL-4, and anti–TGF-β) on in vitro proliferation of allogeneic (BALB/c) CD3+ splenocytes when stimulated with donor H2Kb+CD4+CD25+ T cells. Biology of Blood and Marrow Transplantation 2005 11, 657-671DOI: (10.1016/j.bbmt.2005.05.012) Copyright © 2005 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 Characterization of donor cell engraftment in pooled tissues (spleen and thymus) of IUT recipients of mDCs from B7.2−/− B6 and WT lin− BM from B6 donors. The percentage and subtype of donor cells were evaluated by 5-color flow cytometry analysis and sorting. A, Donor (H2Kb+) cells in spleen at the time of harvest: dot plot shows immunophenotyping of donor (H2Kb+) cells in spleen, CD4-APC, and CD25/peridinin chlorophyll protein (PerCP)/Cy5. B, Further characterization of donor (H2Kb+CD4+CD25+) cells in spleen by immunophenotyping for CTLA-4/PE. C, Donor (H2Kb+) cells in the thymus at the time of harvest showing immunophenotyping of donor (H2Kb+) cells in the thymus for CD4-APC and CD25-PerCP-Cy5. D, Further characterization of donor (H2Kb+CD4+CD25+) cells in spleen by immunophenotyping for CTLA-4/PE. The donor cells in the thymus were also analyzed for dendritic cell and T-cell phenotype. E, Donor (H2Kb+) cells in the thymus at the time of harvest. The dot plot shows CD3-APC versus H2Kb-FITC. F, Thymic CD3-APC gated cells plotted for CD4-PE versus CD8-APC-Cy7. G, Thymic donor dendritic cells in which H2Kb+ cells were evaluated for CD11c-PerCP-Cy5 versus MHC II/PE. H, Cells displaying high CD11c and MHC II were gated and plotted for CD80-APC and CD86-APC-Cy7. Biology of Blood and Marrow Transplantation 2005 11, 657-671DOI: (10.1016/j.bbmt.2005.05.012) Copyright © 2005 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 Donor cell engraftment in IUT recipients that underwent subsequent megadose transplantation. The percentage and subtype of donor cells were evaluated by flow cytometry and PCR. A, Donor (H2Kb+) cells in blood 6 weeks after mega-BMT in IUT recipients with lin− BM plus B7.2−/− mDCs. B, Lineage analysis of donor (H2Kb+) cells in blood 6 weeks after mega-BMT in IUT recipients with lin− BM and B7.1−/− mDCs. C, PCR-amplified H2Kb products in WT B6 control splenocytes (lane 1) and in thymus (lanes 2 and 7), spleen (lanes 3 and 8), blood (lanes 4 and 9), and bone marrow (lanes 5 and 10) of IUT recipients of lin− BM plus B7.1−/− mDCs (lanes 2–5) and lin− BM plus B7.2−/− mDCs (lanes 7–10). Biology of Blood and Marrow Transplantation 2005 11, 657-671DOI: (10.1016/j.bbmt.2005.05.012) Copyright © 2005 American Society for Blood and Marrow Transplantation Terms and Conditions