Functional Myeloid-Derived Suppressor Cell Subsets Recover Rapidly after Allogeneic Hematopoietic Stem/Progenitor Cell Transplantation Qingdong Guan,

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Functional Myeloid-Derived Suppressor Cell Subsets Recover Rapidly after Allogeneic Hematopoietic Stem/Progenitor Cell Transplantation Qingdong Guan, Anna R. Blankstein, Karla Anjos, Oleksandra Synova, Marie Tulloch, Angeline Giftakis, Bin Yang, Pascal Lambert, Zhikang Peng, Geoff D.E. Cuvelier, Donna A. Wall Biology of Blood and Marrow Transplantation Volume 21, Issue 7, Pages 1205-1214 (July 2015) DOI: 10.1016/j.bbmt.2015.04.015 Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

Biology of Blood and Marrow Transplantation 2015 21, 1205-1214DOI: (10 Biology of Blood and Marrow Transplantation 2015 21, 1205-1214DOI: (10.1016/j.bbmt.2015.04.015) Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Gating strategy of MDSC subsets. (A) CD45+ cells; (B) CD33+ cells; (C) CD15− cells and CD15+ cells; (D) CD14+HLA-DR−/low cells, M-MDSCs; and (E) CD66+ cells, G-MDSCs. Biology of Blood and Marrow Transplantation 2015 21, 1205-1214DOI: (10.1016/j.bbmt.2015.04.015) Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Time course of recovery of MDSC subsets in patients after allo-HSCT. WBCs were isolated, and the phenotype of MDSC subsets were analyzed through flow cytometry. The recovery of WBCs (A), percentage (B) and number (C) of M-MDSCs, and percentage (D) and number (E) of G-MDSCs. Shaded area is the median with IQRs of 12 healthy donors. Biology of Blood and Marrow Transplantation 2015 21, 1205-1214DOI: (10.1016/j.bbmt.2015.04.015) Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Recovery of MDSC subsets in patients accepting myeloablative (MA) or nonmyeloablative (NMA) chemotherapy regimens. (A) M-MDSC recovery in patients receiving NMA, (B) G-MDSC recovery in patients receiving NMA, (C) M-MDSC recovery in patients receiving MA, and (D) G-MDSC recovery in patients receiving MA. Shaded area is the median with IQRs of 12 healthy donors. Biology of Blood and Marrow Transplantation 2015 21, 1205-1214DOI: (10.1016/j.bbmt.2015.04.015) Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 Time course of recovery of T lymphocytes, B lymphocytes, and DN T cells in patients after allo-HSCT. WBCs were stained with fluorescence-labeled antibodies against CD3 and CD19 for T and B lymphocytes and for CD3, CD4, and CD8 for DN T cells and then analyzed by flow cytometry. (A) Recovery of T lymphocytes, (B) recovery of B lymphocytes, and (C) recovery of DN T cells. Shaded area is the median with IQRs of 12 healthy donors. Biology of Blood and Marrow Transplantation 2015 21, 1205-1214DOI: (10.1016/j.bbmt.2015.04.015) Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 Relationship of G-MDSCs with grades II to IV acute GVHD. (A) G-MDSC numbers at preconditioning between patients who developed high grade acute GVHD later and patients who developed low grade GVHD or without GVHD. (B) Ratio of G-MDSC number at days 27 to 29 over the baseline at the preconditioning. *P < .05. Biology of Blood and Marrow Transplantation 2015 21, 1205-1214DOI: (10.1016/j.bbmt.2015.04.015) Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 6 Function assay of MDSC subsets in patients after allo-HSCT. (A) Representative figures of CD4+ T lymphocyte proliferation (gated on CD4+ T cells). (B) Inhibition rate of MDSC subsets on lymphocyte proliferation. (C) Representative figure of percentages of CD4+IFN-γ+ Th1 cells. (D) Percentage of CD4+IFN-γ+ Th1 cells. (E) Representative figure of percentages of CD4+CD25+Foxp3+ Tregs (gated on CD4+ T cells). (F) Percentages of CD4+CD25+Foxp3+ Tregs in the coculture. The results represent 1 of 2 patient experiments. *P < .05, **P < .01, ***P < .001. Biology of Blood and Marrow Transplantation 2015 21, 1205-1214DOI: (10.1016/j.bbmt.2015.04.015) Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 7 Expression of Arg-1 and iNOS in MDSC subsets of patients after allo-HSCT. The expression of Arg-1 and iNOS in MDSC subsets were detected by intracellular staining of Arg-1 and iNOS and analyzed by flow cytometry. (A) Expression levels of Arg-1 in MDSC subsets. (B) Expression levels of iNOS in MDSC subsets. *P < .05, **P < .01, ***P < .001. Biology of Blood and Marrow Transplantation 2015 21, 1205-1214DOI: (10.1016/j.bbmt.2015.04.015) Copyright © 2015 American Society for Blood and Marrow Transplantation Terms and Conditions