Cytokines and cytotoxic pathways in engraftment resistance to purified allogeneic hematopoietic stem cells Christian Scheffold, Yolanda C. Scheffold,

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Cytokines and cytotoxic pathways in engraftment resistance to purified allogeneic hematopoietic stem cells Christian Scheffold, Yolanda C. Scheffold, Thai M. Cao, Jennifer Gworek, Judith A. Shizuru Biology of Blood and Marrow Transplantation Volume 11, Issue 1, Pages 1-12 (January 2005) DOI: 10.1016/j.bbmt.2004.10.002 Copyright © 2005 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Survival and chimerism analysis of lethally irradiated mice that underwent transplantation across MHC barriers. Titrated doses of MHC-mismatched AKR/J (H2k) HSCs were injected into lethally irradiated B6.WT (H2b) mice. A, Survival of mice that underwent transplantation with 300, 1000, 3000, or 6000 HSCs was compared with that of XRT controls (950 cGy of radiation). Results are pooled from 2 independent experiments. B, Flow cytometric analysis of peripheral blood from B6.WT mice performed 6 weeks after they received 6000 HSCs. Detection of hematopoietic chimerism was performed with donor-specific staining for the class I H2 marker (α-H2k) versus cell lineage markers. Shown are profiles of B- and T-cell chimerism from the peripheral blood of a representative engrafted B6.WT recipient; these are compared with blood profiles from control unmanipulated B6.WT and AKR/J mice. Note that the engrafted mouse is a partial T-cell chimera, whereas the B cells are all donor derived. Biology of Blood and Marrow Transplantation 2005 11, 1-12DOI: (10.1016/j.bbmt.2004.10.002) Copyright © 2005 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Effects of α-ASGM1 polyclonal antibody pretreatment on the survival of lethally irradiated mice that underwent transplantation across MHC barriers. Lethally irradiated B6.WT mice received antibody treatment on day −7 (intravenously) and on day −1 (intraperitoneally) before transplantation of titrated doses of MHC-mismatched AKR/J HSCs. A, Survival of mice that underwent transplantation with 300, 1000, 3000, or 6000 HSCs is compared with that of XRT controls (950 cGy of radiation). B, Flow cytometric analysis of spleen cells 7 days after intravenous and intraperitoneal administration of 50 μg of α-ASGM1 antibody of treated (top) and untreated (bottom) B6.WT mice. Detection of ASGM1-expressing T-cell subsets (CD3, CD4, CD8, and T-cell receptor αβ) and NK cells (NK1.1) was performed by FACS analysis by using α-ASGM1 as a staining reagent. Note the depletion of lymphocyte subsets in the α-ASGM1 antibody-treated mouse. Biology of Blood and Marrow Transplantation 2005 11, 1-12DOI: (10.1016/j.bbmt.2004.10.002) Copyright © 2005 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Transplantation of allogeneic HSCs into recipients lacking expression of the CD8 antigen. Titrated doses of MHC-mismatched AKR/J HSCs were injected into lethally irradiated CD8-deficient mice (B6.CD8α−/−). Shown is the survival of mice that underwent transplantation with 300, 1000, 3000, or 6000 HSCs compared with the survival of XRT controls (950 cGy of radiation). Results are pooled from 2 independent experiments. Biology of Blood and Marrow Transplantation 2005 11, 1-12DOI: (10.1016/j.bbmt.2004.10.002) Copyright © 2005 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 Transplantation of allogeneic HSCs into recipients with defects in the perforin or Fas pathways. Titrated doses of MHC-mismatched AKR/J HSCs were injected into lethally irradiated (A) perforin-deficient (B6.pfp−/−) or (B) FasL-defective (B6.gld) recipients. Survival of mice that underwent transplantation with 300, 1000, 3000, or 6000 HSCs is compared with that of XRT controls (950 cGy of radiation). Results are pooled from 2 independent experiments. Biology of Blood and Marrow Transplantation 2005 11, 1-12DOI: (10.1016/j.bbmt.2004.10.002) Copyright © 2005 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 Transplantation of allogeneic HSCs into recipients defective in a single cytokine. Titrated doses of MHC-mismatched AKR/J HSCs were injected into lethally irradiated C57BL/6 mice defective for (A) IFN-γ, (B) TGF-β, (C) TNF-α, (D) IL-4, or (E) IL-10. Survival of mice that underwent transplantation with 300, 1000, 3000, or 6000 HSCs is compared with that of XRT controls (950 cGy of radiation). Results are pooled from 2 independent experiments for each treatment group. Biology of Blood and Marrow Transplantation 2005 11, 1-12DOI: (10.1016/j.bbmt.2004.10.002) Copyright © 2005 American Society for Blood and Marrow Transplantation Terms and Conditions