GSK3 inhibition prevents lethal GVHD in mice Guy Klamer, Sylvie Shen, Emma Song, Alison M. Rice, Robert Knight, Robert Lindeman, Tracey A. O’Brien, Alla Dolnikov  Experimental Hematology  Volume 41, Issue 1, Pages 39-55.e10 (January 2013) DOI: 10.1016/j.exphem.2012.09.005 Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 1 BIO prevents lethal GVHD in NSG mice transplanted with human PB MNCs. (A) Kaplan-Meier survival curves. (B) Weight tracking of mice from experiment depicted as donor 2 in (A); p ≤ 0.01 for end-point weights (control: cull, day +51: BIO-treated). (C) Histopathological analysis (hematoxylin and eosin [H&E] stain) of BM harvested at the end of the experiment. Experiment depicted in (A) as donor 3. Histology represents histology observed across BM from three mice per group. 40× magnification. (D) Total BM cellularity of mice at the end point of experiment. Experiment depicted in (A) as donor 3. n ≥ 4 for each group. (E) Histopathological analysis (H&E stain of liver samples harvested from transplanted mice at the end of experiment, i.e., lethal GVHD or survival). Two representative mice from (A) donor 2 illustrated. Long arrow = donor MNC infiltration; short arrow = apoptotic body. (F) Total number of apoptotic bodies in liver, and megakaryocytes in BM, observed per field as viewed under a ×40 magnification objective lens; 30 fields from three mice per group were chosen randomly. (G) Western blot analysis of β-catenin in MNCs harvested from mice 6 hours after 30 mg/kg BIO treatment. BIO treatment occurred on day +13 in one mouse to allow sufficient engraftment and expansion of donor cells for Western blot analysis. (E–G) Data from experiments using intraperitoneal BIO 30 mg/kg (days 0–3) and 3 mg/kg (days 5–17 with drug-free days every 5th day. Vehicle control was 5% (30 mg/kg BIO) and 0.5% (3 mg/kg BIO) DMSO/phosphate-buffered saline. Experimental Hematology 2013 41, 39-55.e10DOI: (10.1016/j.exphem.2012.09.005) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 1 BIO prevents lethal GVHD in NSG mice transplanted with human PB MNCs. (A) Kaplan-Meier survival curves. (B) Weight tracking of mice from experiment depicted as donor 2 in (A); p ≤ 0.01 for end-point weights (control: cull, day +51: BIO-treated). (C) Histopathological analysis (hematoxylin and eosin [H&E] stain) of BM harvested at the end of the experiment. Experiment depicted in (A) as donor 3. Histology represents histology observed across BM from three mice per group. 40× magnification. (D) Total BM cellularity of mice at the end point of experiment. Experiment depicted in (A) as donor 3. n ≥ 4 for each group. (E) Histopathological analysis (H&E stain of liver samples harvested from transplanted mice at the end of experiment, i.e., lethal GVHD or survival). Two representative mice from (A) donor 2 illustrated. Long arrow = donor MNC infiltration; short arrow = apoptotic body. (F) Total number of apoptotic bodies in liver, and megakaryocytes in BM, observed per field as viewed under a ×40 magnification objective lens; 30 fields from three mice per group were chosen randomly. (G) Western blot analysis of β-catenin in MNCs harvested from mice 6 hours after 30 mg/kg BIO treatment. BIO treatment occurred on day +13 in one mouse to allow sufficient engraftment and expansion of donor cells for Western blot analysis. (E–G) Data from experiments using intraperitoneal BIO 30 mg/kg (days 0–3) and 3 mg/kg (days 5–17 with drug-free days every 5th day. Vehicle control was 5% (30 mg/kg BIO) and 0.5% (3 mg/kg BIO) DMSO/phosphate-buffered saline. Experimental Hematology 2013 41, 39-55.e10DOI: (10.1016/j.exphem.2012.09.005) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 1 BIO prevents lethal GVHD in NSG mice transplanted with human PB MNCs. (A) Kaplan-Meier survival curves. (B) Weight tracking of mice from experiment depicted as donor 2 in (A); p ≤ 0.01 for end-point weights (control: cull, day +51: BIO-treated). (C) Histopathological analysis (hematoxylin and eosin [H&E] stain) of BM harvested at the end of the experiment. Experiment depicted in (A) as donor 3. Histology represents histology observed across BM from three mice per group. 40× magnification. (D) Total BM cellularity of mice at the end point of experiment. Experiment depicted in (A) as donor 3. n ≥ 4 for each group. (E) Histopathological analysis (H&E stain of liver samples harvested from transplanted mice at the end of experiment, i.e., lethal GVHD or survival). Two representative mice from (A) donor 2 illustrated. Long arrow = donor MNC infiltration; short arrow = apoptotic body. (F) Total number of apoptotic bodies in liver, and megakaryocytes in BM, observed per field as viewed under a ×40 magnification objective lens; 30 fields from three mice per group were chosen randomly. (G) Western blot analysis of β-catenin in MNCs harvested from mice 6 hours after 30 mg/kg BIO treatment. BIO treatment occurred on day +13 in one mouse to allow sufficient engraftment and expansion of donor cells for Western blot analysis. (E–G) Data from experiments using intraperitoneal BIO 30 mg/kg (days 0–3) and 3 mg/kg (days 5–17 with drug-free days every 5th day. Vehicle control was 5% (30 mg/kg BIO) and 0.5% (3 mg/kg BIO) DMSO/phosphate-buffered saline. Experimental Hematology 2013 41, 39-55.e10DOI: (10.1016/j.exphem.2012.09.005) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 2 BIO prevents BM GVHD manifestation in transplanted mice. (A) Total number of human CD45+ MNCs and CD3+ T cells harvested on day +13 from the spleens (top) and (PB) of DMSO and BIO-treated mice. n ≥ 4 for each group. (B) Percentage (top) and total numbers (bottom) of human CD45+ MNCs in PB of mice. Day 17–21 data represent averaged data from postmortem examinations of lethal GVHD. ∗p ≤ 0.05. (C) Total number of human CD25+ T cells recovered from spleens of mice on day +13. (D) Total number of human CD45+ MNCs and CD3+ T cells recovered from BM of mice on day 13. n ≥ 4 for each group. (E) Cytolytic activity of human CD45+ MNCs harvested from spleens of mice on day +13 (MNC vs. U937 MLC, see Materials and Methods). Data replicated in two independent experiments. Experimental Hematology 2013 41, 39-55.e10DOI: (10.1016/j.exphem.2012.09.005) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 2 BIO prevents BM GVHD manifestation in transplanted mice. (A) Total number of human CD45+ MNCs and CD3+ T cells harvested on day +13 from the spleens (top) and (PB) of DMSO and BIO-treated mice. n ≥ 4 for each group. (B) Percentage (top) and total numbers (bottom) of human CD45+ MNCs in PB of mice. Day 17–21 data represent averaged data from postmortem examinations of lethal GVHD. ∗p ≤ 0.05. (C) Total number of human CD25+ T cells recovered from spleens of mice on day +13. (D) Total number of human CD45+ MNCs and CD3+ T cells recovered from BM of mice on day 13. n ≥ 4 for each group. (E) Cytolytic activity of human CD45+ MNCs harvested from spleens of mice on day +13 (MNC vs. U937 MLC, see Materials and Methods). Data replicated in two independent experiments. Experimental Hematology 2013 41, 39-55.e10DOI: (10.1016/j.exphem.2012.09.005) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 2 BIO prevents BM GVHD manifestation in transplanted mice. (A) Total number of human CD45+ MNCs and CD3+ T cells harvested on day +13 from the spleens (top) and (PB) of DMSO and BIO-treated mice. n ≥ 4 for each group. (B) Percentage (top) and total numbers (bottom) of human CD45+ MNCs in PB of mice. Day 17–21 data represent averaged data from postmortem examinations of lethal GVHD. ∗p ≤ 0.05. (C) Total number of human CD25+ T cells recovered from spleens of mice on day +13. (D) Total number of human CD45+ MNCs and CD3+ T cells recovered from BM of mice on day 13. n ≥ 4 for each group. (E) Cytolytic activity of human CD45+ MNCs harvested from spleens of mice on day +13 (MNC vs. U937 MLC, see Materials and Methods). Data replicated in two independent experiments. Experimental Hematology 2013 41, 39-55.e10DOI: (10.1016/j.exphem.2012.09.005) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 3 BIO inhibits T-cell activation and effector function in vitro. (A) Left: Representative flow cytometry figures of CD25 vs. CFSE division tracking analysis. Right: Pooled data from 10 individual UCB-derived T-cell samples. (B) One representative Ki-67/propidium iodide (PI)–based cell cycle analysis. G0 = Ki-67–negative gate, G1 = PIlow, G2+ = PIhigh. PI-based cell cycle analysis was performed in four independent experiments (pooled data: p ≤ 0.05). (C) Left: Activated T-cell marker expression on UCB-derived T cells stimulated with PHA, CD3/CD28 microbeads, or an allo-source of UCB MNCs. Donor sample sizes (left to right) = 9, 1, 1, 3. Right: CD25 expression on PB-derived T cells stimulated with PHA. Donor sample size = 1. ∗p ≤ 0.05. Experimental Hematology 2013 41, 39-55.e10DOI: (10.1016/j.exphem.2012.09.005) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 4 BIO modulates secretion of Th1/Th2 effector molecules. (A) RT q-PCR analysis (left) and gene expression microarray analysis (right) of T-cell effector and activation molecules. n = 2 for TNFβ, n = 3 for others. ∗p ≤ 0.05. NA refers to a fold-change that could not be detected because the expression signal was <0 in the treated sample. (B) Top: Western blot analysis of TNFα and TNFβ in cytoplasm of UCB-derived T cells. PB2 = PHA + BIO 2 μM. The numbers under the Western blots resemble fold-change of actin compared to control. Bottom: Flow cytometry–based enzyme-linked immunosorbent assay (ELISA) analysis of TNFα in UCB-derived T-cell culture supernatant. ∗p ≤ 0.05. (C) Flow cytometry–based ELISA analysis of Th1/Th2 cytokine levels in supernatant of MLC. UCB MNCs vs. allo-UCB MNCs. Data replicated in 3 independent experiments. ∗p ≤ 0.01. (D) RT q-PCR analysis of IL-10 in resting UCB-derived T cells. ∗p ≤ 0.05. (E) Total CD4+CD25+CD127−FoxP3+ UCB-derived Treg numbers in 4-day culture. ∗p ≤ 0.05. (F) CFSE-based high-resolution division tracking analysis of gated Tregs on day 4. Experimental Hematology 2013 41, 39-55.e10DOI: (10.1016/j.exphem.2012.09.005) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 5 BIO inhibits STAT1 and STAT3 activation in vitro and in vivo. (A) Top: Western blot analysis of STAT/p-STAT in human CD45+ MNCs harvested from spleens of mice on day 13. Bottom: Averaged protein quantification of STAT/p-STAT expression from all mice. n = 2 for control and n = 4 for BIO-treated mice. (B) Western blot analysis of STAT/p-STAT in resting and PHA-activated UCB-derived T cells. (–1) refers to 50 ng/mL IL-2 and (−2) refers to 250 ng/ml IL-2, in culture, respectively. (C) Western blot analysis of β-catenin in resting and PHA-activated UCB-derived T cells ± BIO. (D) RT q-PCR analysis of STAT1/3 target genes in human CD45+ MNCs harvested from the spleens of mice on day 13. Data averaged from 2 mice per group. ∗p ≤ 0.05. (E) CD25+ expression on gated UCB-derived CD4+ (dark histogram) and CD8+ (light histogram) T cells ± PHA ± pyridone 6. Representative of two independent experiments. Division tracking analysis of UCB-derived T cells ± PHA ± pyridone 6. (F) Representative of two independent experiments. Experimental Hematology 2013 41, 39-55.e10DOI: (10.1016/j.exphem.2012.09.005) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 6 BIO inhibits MYC signaling in activated T cells in vitro. (A) Gene expression microarray results (Database for Annotation, Visualization and Integrated Discovery) of cell cycle–related genes that were modulated by BIO. (B) C-MYC target genes that were modulated by BIO in PHA-activated T cells. Gene expression microarray data. (−) and (+) refer to down-regulation and up-regulation of mRNA expression, respectively. C-MYC target genes were identified using an online database (www.myccancergene.org). (C) Western blot analysis of C-MYC and YB-1 in UCB-derived T cells ± PHA ± BIO for 4 days. Replicated in two independent experiments. PB2 = PHA + BIO 2 μM. Experimental Hematology 2013 41, 39-55.e10DOI: (10.1016/j.exphem.2012.09.005) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Supplementary Figure E1 (A) PB platelet (top) and red blood cell (RBC) (bottom) counts from mice treated with DMSO or BIO throughout the experiment depicted in Figure 1A as donor 3. Day 17–21 data represent averaged data from postmortem examinations of lethal GVHD. (B) Top: PB counts of DMSO-treated and BIO-treated mice at the experiment end point. Bottom: PB counts of healthy, nontransplanted, nonirradiated 10-week-old NSG mice. (C) Kaplan-Meier survival curve of mice from humanized mouse model of GVHD. (D) Total number of donor MNCs harvested from spleens and PB after 2, 4, and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (E) Total number of donor MNCs harvested from BM 4 and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (F) Levels of IL-5 and IFNγ in serum of DMSO and BIO-treated mice on day +13. TNFα, IL-2, IL-10, and IL-4 were not detected by multiplex. (G) Human CD45+ MNC (huCD45) percentage in BM on day 13 and at the experiment end point. (H) The effects of BIO treatment on hematopoietic regeneration after sublethal (2.5 Gy) irradiation in nonobese diabetic severe combined immune-deficient mice. BIO dose: 30 mg/kg intraperitoneal twice per week for 2 weeks. Mice culled on day 18. Hematopoietic lineage markers analyzed were Sca1− primitive hematopoietic progenitor cells, CD11b myelomonocytes, Gr1-granulocytes, CD45R, B cells, CD49B− T-cells, and natural killer cells. Experiment and analysis performed by Dr. Robert Knight. All data: n ≥ 3 per group. (I) Engraftment of human CD45+ MNCs in PB and BM of CD34+ HSC engrafted NSG mice after 13 weeks. (J) Multilineage differentiation (CD19+ and CD33+) of human CD34+ HSCs in BM after 13 weeks. (K) Percentage of CD4+ (top) and CD8+ (bottom) T cells in cultures ± PHA ± BIO. PB2 = PHA + BIO 2 μM. (L) Annexin V/7-amino-actinomycin D staining of purified CD3+ UCB T cells treated for 4 days; n = 3. Representative flows shown for donor 1. (M) Flow cytometry figures illustrating CD25 expression on CD4+ and CD8+ PHA-activated UCB-derived T cells. (N) Gene expression microarray analysis data: IL2RA(CD25)/IL-2 signaling target genes and nuclear factor–κB activating genes that were down-regulated by BIO in UCB-derived PHA-activated T cells. (O) Gene expression microarray analysis data: GZMB, co-stimulatory molecules, and chemokine/cytokine and their receptors that were down-regulated by BIO in PHA-activated T cells. (P) Gene expression microarray analysis data: Genes that were down-regulated by BIO in PHA-activated UCB-derived T cells in donor 1 and donor 2, and that were up-regulated by PHA in UCB-derived resting T cells. (Q) Gene expression microarray analysis data: Wnt/β-catenin target genes that were up-regulated by BIO in PHA-activated UCB-derived T cells. (R) A β-catenin DNA reporter construct was transfected into HEK293 cells. Cells were treated for 21 hours before green fluorescent protein expression was analyzed by flow cytometry. One representative experiment (n = 3). Experimental Hematology 2013 41, 39-55.e10DOI: (10.1016/j.exphem.2012.09.005) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Supplementary Figure E1 (A) PB platelet (top) and red blood cell (RBC) (bottom) counts from mice treated with DMSO or BIO throughout the experiment depicted in Figure 1A as donor 3. Day 17–21 data represent averaged data from postmortem examinations of lethal GVHD. (B) Top: PB counts of DMSO-treated and BIO-treated mice at the experiment end point. Bottom: PB counts of healthy, nontransplanted, nonirradiated 10-week-old NSG mice. (C) Kaplan-Meier survival curve of mice from humanized mouse model of GVHD. (D) Total number of donor MNCs harvested from spleens and PB after 2, 4, and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (E) Total number of donor MNCs harvested from BM 4 and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (F) Levels of IL-5 and IFNγ in serum of DMSO and BIO-treated mice on day +13. TNFα, IL-2, IL-10, and IL-4 were not detected by multiplex. (G) Human CD45+ MNC (huCD45) percentage in BM on day 13 and at the experiment end point. (H) The effects of BIO treatment on hematopoietic regeneration after sublethal (2.5 Gy) irradiation in nonobese diabetic severe combined immune-deficient mice. BIO dose: 30 mg/kg intraperitoneal twice per week for 2 weeks. Mice culled on day 18. Hematopoietic lineage markers analyzed were Sca1− primitive hematopoietic progenitor cells, CD11b myelomonocytes, Gr1-granulocytes, CD45R, B cells, CD49B− T-cells, and natural killer cells. Experiment and analysis performed by Dr. Robert Knight. All data: n ≥ 3 per group. (I) Engraftment of human CD45+ MNCs in PB and BM of CD34+ HSC engrafted NSG mice after 13 weeks. (J) Multilineage differentiation (CD19+ and CD33+) of human CD34+ HSCs in BM after 13 weeks. (K) Percentage of CD4+ (top) and CD8+ (bottom) T cells in cultures ± PHA ± BIO. PB2 = PHA + BIO 2 μM. (L) Annexin V/7-amino-actinomycin D staining of purified CD3+ UCB T cells treated for 4 days; n = 3. Representative flows shown for donor 1. (M) Flow cytometry figures illustrating CD25 expression on CD4+ and CD8+ PHA-activated UCB-derived T cells. (N) Gene expression microarray analysis data: IL2RA(CD25)/IL-2 signaling target genes and nuclear factor–κB activating genes that were down-regulated by BIO in UCB-derived PHA-activated T cells. (O) Gene expression microarray analysis data: GZMB, co-stimulatory molecules, and chemokine/cytokine and their receptors that were down-regulated by BIO in PHA-activated T cells. (P) Gene expression microarray analysis data: Genes that were down-regulated by BIO in PHA-activated UCB-derived T cells in donor 1 and donor 2, and that were up-regulated by PHA in UCB-derived resting T cells. (Q) Gene expression microarray analysis data: Wnt/β-catenin target genes that were up-regulated by BIO in PHA-activated UCB-derived T cells. (R) A β-catenin DNA reporter construct was transfected into HEK293 cells. Cells were treated for 21 hours before green fluorescent protein expression was analyzed by flow cytometry. One representative experiment (n = 3). Experimental Hematology 2013 41, 39-55.e10DOI: (10.1016/j.exphem.2012.09.005) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Supplementary Figure E1 (A) PB platelet (top) and red blood cell (RBC) (bottom) counts from mice treated with DMSO or BIO throughout the experiment depicted in Figure 1A as donor 3. Day 17–21 data represent averaged data from postmortem examinations of lethal GVHD. (B) Top: PB counts of DMSO-treated and BIO-treated mice at the experiment end point. Bottom: PB counts of healthy, nontransplanted, nonirradiated 10-week-old NSG mice. (C) Kaplan-Meier survival curve of mice from humanized mouse model of GVHD. (D) Total number of donor MNCs harvested from spleens and PB after 2, 4, and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (E) Total number of donor MNCs harvested from BM 4 and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (F) Levels of IL-5 and IFNγ in serum of DMSO and BIO-treated mice on day +13. TNFα, IL-2, IL-10, and IL-4 were not detected by multiplex. (G) Human CD45+ MNC (huCD45) percentage in BM on day 13 and at the experiment end point. (H) The effects of BIO treatment on hematopoietic regeneration after sublethal (2.5 Gy) irradiation in nonobese diabetic severe combined immune-deficient mice. BIO dose: 30 mg/kg intraperitoneal twice per week for 2 weeks. Mice culled on day 18. Hematopoietic lineage markers analyzed were Sca1− primitive hematopoietic progenitor cells, CD11b myelomonocytes, Gr1-granulocytes, CD45R, B cells, CD49B− T-cells, and natural killer cells. Experiment and analysis performed by Dr. Robert Knight. All data: n ≥ 3 per group. (I) Engraftment of human CD45+ MNCs in PB and BM of CD34+ HSC engrafted NSG mice after 13 weeks. (J) Multilineage differentiation (CD19+ and CD33+) of human CD34+ HSCs in BM after 13 weeks. (K) Percentage of CD4+ (top) and CD8+ (bottom) T cells in cultures ± PHA ± BIO. PB2 = PHA + BIO 2 μM. (L) Annexin V/7-amino-actinomycin D staining of purified CD3+ UCB T cells treated for 4 days; n = 3. Representative flows shown for donor 1. (M) Flow cytometry figures illustrating CD25 expression on CD4+ and CD8+ PHA-activated UCB-derived T cells. (N) Gene expression microarray analysis data: IL2RA(CD25)/IL-2 signaling target genes and nuclear factor–κB activating genes that were down-regulated by BIO in UCB-derived PHA-activated T cells. (O) Gene expression microarray analysis data: GZMB, co-stimulatory molecules, and chemokine/cytokine and their receptors that were down-regulated by BIO in PHA-activated T cells. (P) Gene expression microarray analysis data: Genes that were down-regulated by BIO in PHA-activated UCB-derived T cells in donor 1 and donor 2, and that were up-regulated by PHA in UCB-derived resting T cells. (Q) Gene expression microarray analysis data: Wnt/β-catenin target genes that were up-regulated by BIO in PHA-activated UCB-derived T cells. (R) A β-catenin DNA reporter construct was transfected into HEK293 cells. Cells were treated for 21 hours before green fluorescent protein expression was analyzed by flow cytometry. One representative experiment (n = 3). Experimental Hematology 2013 41, 39-55.e10DOI: (10.1016/j.exphem.2012.09.005) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Supplementary Figure E1 (A) PB platelet (top) and red blood cell (RBC) (bottom) counts from mice treated with DMSO or BIO throughout the experiment depicted in Figure 1A as donor 3. Day 17–21 data represent averaged data from postmortem examinations of lethal GVHD. (B) Top: PB counts of DMSO-treated and BIO-treated mice at the experiment end point. Bottom: PB counts of healthy, nontransplanted, nonirradiated 10-week-old NSG mice. (C) Kaplan-Meier survival curve of mice from humanized mouse model of GVHD. (D) Total number of donor MNCs harvested from spleens and PB after 2, 4, and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (E) Total number of donor MNCs harvested from BM 4 and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (F) Levels of IL-5 and IFNγ in serum of DMSO and BIO-treated mice on day +13. TNFα, IL-2, IL-10, and IL-4 were not detected by multiplex. (G) Human CD45+ MNC (huCD45) percentage in BM on day 13 and at the experiment end point. (H) The effects of BIO treatment on hematopoietic regeneration after sublethal (2.5 Gy) irradiation in nonobese diabetic severe combined immune-deficient mice. BIO dose: 30 mg/kg intraperitoneal twice per week for 2 weeks. Mice culled on day 18. Hematopoietic lineage markers analyzed were Sca1− primitive hematopoietic progenitor cells, CD11b myelomonocytes, Gr1-granulocytes, CD45R, B cells, CD49B− T-cells, and natural killer cells. Experiment and analysis performed by Dr. Robert Knight. All data: n ≥ 3 per group. (I) Engraftment of human CD45+ MNCs in PB and BM of CD34+ HSC engrafted NSG mice after 13 weeks. (J) Multilineage differentiation (CD19+ and CD33+) of human CD34+ HSCs in BM after 13 weeks. (K) Percentage of CD4+ (top) and CD8+ (bottom) T cells in cultures ± PHA ± BIO. PB2 = PHA + BIO 2 μM. (L) Annexin V/7-amino-actinomycin D staining of purified CD3+ UCB T cells treated for 4 days; n = 3. Representative flows shown for donor 1. (M) Flow cytometry figures illustrating CD25 expression on CD4+ and CD8+ PHA-activated UCB-derived T cells. (N) Gene expression microarray analysis data: IL2RA(CD25)/IL-2 signaling target genes and nuclear factor–κB activating genes that were down-regulated by BIO in UCB-derived PHA-activated T cells. (O) Gene expression microarray analysis data: GZMB, co-stimulatory molecules, and chemokine/cytokine and their receptors that were down-regulated by BIO in PHA-activated T cells. (P) Gene expression microarray analysis data: Genes that were down-regulated by BIO in PHA-activated UCB-derived T cells in donor 1 and donor 2, and that were up-regulated by PHA in UCB-derived resting T cells. (Q) Gene expression microarray analysis data: Wnt/β-catenin target genes that were up-regulated by BIO in PHA-activated UCB-derived T cells. (R) A β-catenin DNA reporter construct was transfected into HEK293 cells. Cells were treated for 21 hours before green fluorescent protein expression was analyzed by flow cytometry. One representative experiment (n = 3). Experimental Hematology 2013 41, 39-55.e10DOI: (10.1016/j.exphem.2012.09.005) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Supplementary Figure E1 (A) PB platelet (top) and red blood cell (RBC) (bottom) counts from mice treated with DMSO or BIO throughout the experiment depicted in Figure 1A as donor 3. Day 17–21 data represent averaged data from postmortem examinations of lethal GVHD. (B) Top: PB counts of DMSO-treated and BIO-treated mice at the experiment end point. Bottom: PB counts of healthy, nontransplanted, nonirradiated 10-week-old NSG mice. (C) Kaplan-Meier survival curve of mice from humanized mouse model of GVHD. (D) Total number of donor MNCs harvested from spleens and PB after 2, 4, and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (E) Total number of donor MNCs harvested from BM 4 and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (F) Levels of IL-5 and IFNγ in serum of DMSO and BIO-treated mice on day +13. TNFα, IL-2, IL-10, and IL-4 were not detected by multiplex. (G) Human CD45+ MNC (huCD45) percentage in BM on day 13 and at the experiment end point. (H) The effects of BIO treatment on hematopoietic regeneration after sublethal (2.5 Gy) irradiation in nonobese diabetic severe combined immune-deficient mice. BIO dose: 30 mg/kg intraperitoneal twice per week for 2 weeks. Mice culled on day 18. Hematopoietic lineage markers analyzed were Sca1− primitive hematopoietic progenitor cells, CD11b myelomonocytes, Gr1-granulocytes, CD45R, B cells, CD49B− T-cells, and natural killer cells. Experiment and analysis performed by Dr. Robert Knight. All data: n ≥ 3 per group. (I) Engraftment of human CD45+ MNCs in PB and BM of CD34+ HSC engrafted NSG mice after 13 weeks. (J) Multilineage differentiation (CD19+ and CD33+) of human CD34+ HSCs in BM after 13 weeks. (K) Percentage of CD4+ (top) and CD8+ (bottom) T cells in cultures ± PHA ± BIO. PB2 = PHA + BIO 2 μM. (L) Annexin V/7-amino-actinomycin D staining of purified CD3+ UCB T cells treated for 4 days; n = 3. Representative flows shown for donor 1. (M) Flow cytometry figures illustrating CD25 expression on CD4+ and CD8+ PHA-activated UCB-derived T cells. (N) Gene expression microarray analysis data: IL2RA(CD25)/IL-2 signaling target genes and nuclear factor–κB activating genes that were down-regulated by BIO in UCB-derived PHA-activated T cells. (O) Gene expression microarray analysis data: GZMB, co-stimulatory molecules, and chemokine/cytokine and their receptors that were down-regulated by BIO in PHA-activated T cells. (P) Gene expression microarray analysis data: Genes that were down-regulated by BIO in PHA-activated UCB-derived T cells in donor 1 and donor 2, and that were up-regulated by PHA in UCB-derived resting T cells. (Q) Gene expression microarray analysis data: Wnt/β-catenin target genes that were up-regulated by BIO in PHA-activated UCB-derived T cells. (R) A β-catenin DNA reporter construct was transfected into HEK293 cells. Cells were treated for 21 hours before green fluorescent protein expression was analyzed by flow cytometry. One representative experiment (n = 3). Experimental Hematology 2013 41, 39-55.e10DOI: (10.1016/j.exphem.2012.09.005) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Supplementary Figure E1 (A) PB platelet (top) and red blood cell (RBC) (bottom) counts from mice treated with DMSO or BIO throughout the experiment depicted in Figure 1A as donor 3. Day 17–21 data represent averaged data from postmortem examinations of lethal GVHD. (B) Top: PB counts of DMSO-treated and BIO-treated mice at the experiment end point. Bottom: PB counts of healthy, nontransplanted, nonirradiated 10-week-old NSG mice. (C) Kaplan-Meier survival curve of mice from humanized mouse model of GVHD. (D) Total number of donor MNCs harvested from spleens and PB after 2, 4, and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (E) Total number of donor MNCs harvested from BM 4 and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (F) Levels of IL-5 and IFNγ in serum of DMSO and BIO-treated mice on day +13. TNFα, IL-2, IL-10, and IL-4 were not detected by multiplex. (G) Human CD45+ MNC (huCD45) percentage in BM on day 13 and at the experiment end point. (H) The effects of BIO treatment on hematopoietic regeneration after sublethal (2.5 Gy) irradiation in nonobese diabetic severe combined immune-deficient mice. BIO dose: 30 mg/kg intraperitoneal twice per week for 2 weeks. Mice culled on day 18. Hematopoietic lineage markers analyzed were Sca1− primitive hematopoietic progenitor cells, CD11b myelomonocytes, Gr1-granulocytes, CD45R, B cells, CD49B− T-cells, and natural killer cells. Experiment and analysis performed by Dr. Robert Knight. All data: n ≥ 3 per group. (I) Engraftment of human CD45+ MNCs in PB and BM of CD34+ HSC engrafted NSG mice after 13 weeks. (J) Multilineage differentiation (CD19+ and CD33+) of human CD34+ HSCs in BM after 13 weeks. (K) Percentage of CD4+ (top) and CD8+ (bottom) T cells in cultures ± PHA ± BIO. PB2 = PHA + BIO 2 μM. (L) Annexin V/7-amino-actinomycin D staining of purified CD3+ UCB T cells treated for 4 days; n = 3. Representative flows shown for donor 1. (M) Flow cytometry figures illustrating CD25 expression on CD4+ and CD8+ PHA-activated UCB-derived T cells. (N) Gene expression microarray analysis data: IL2RA(CD25)/IL-2 signaling target genes and nuclear factor–κB activating genes that were down-regulated by BIO in UCB-derived PHA-activated T cells. (O) Gene expression microarray analysis data: GZMB, co-stimulatory molecules, and chemokine/cytokine and their receptors that were down-regulated by BIO in PHA-activated T cells. (P) Gene expression microarray analysis data: Genes that were down-regulated by BIO in PHA-activated UCB-derived T cells in donor 1 and donor 2, and that were up-regulated by PHA in UCB-derived resting T cells. (Q) Gene expression microarray analysis data: Wnt/β-catenin target genes that were up-regulated by BIO in PHA-activated UCB-derived T cells. (R) A β-catenin DNA reporter construct was transfected into HEK293 cells. Cells were treated for 21 hours before green fluorescent protein expression was analyzed by flow cytometry. One representative experiment (n = 3). Experimental Hematology 2013 41, 39-55.e10DOI: (10.1016/j.exphem.2012.09.005) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Supplementary Figure E1 (A) PB platelet (top) and red blood cell (RBC) (bottom) counts from mice treated with DMSO or BIO throughout the experiment depicted in Figure 1A as donor 3. Day 17–21 data represent averaged data from postmortem examinations of lethal GVHD. (B) Top: PB counts of DMSO-treated and BIO-treated mice at the experiment end point. Bottom: PB counts of healthy, nontransplanted, nonirradiated 10-week-old NSG mice. (C) Kaplan-Meier survival curve of mice from humanized mouse model of GVHD. (D) Total number of donor MNCs harvested from spleens and PB after 2, 4, and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (E) Total number of donor MNCs harvested from BM 4 and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (F) Levels of IL-5 and IFNγ in serum of DMSO and BIO-treated mice on day +13. TNFα, IL-2, IL-10, and IL-4 were not detected by multiplex. (G) Human CD45+ MNC (huCD45) percentage in BM on day 13 and at the experiment end point. (H) The effects of BIO treatment on hematopoietic regeneration after sublethal (2.5 Gy) irradiation in nonobese diabetic severe combined immune-deficient mice. BIO dose: 30 mg/kg intraperitoneal twice per week for 2 weeks. Mice culled on day 18. Hematopoietic lineage markers analyzed were Sca1− primitive hematopoietic progenitor cells, CD11b myelomonocytes, Gr1-granulocytes, CD45R, B cells, CD49B− T-cells, and natural killer cells. Experiment and analysis performed by Dr. Robert Knight. All data: n ≥ 3 per group. (I) Engraftment of human CD45+ MNCs in PB and BM of CD34+ HSC engrafted NSG mice after 13 weeks. (J) Multilineage differentiation (CD19+ and CD33+) of human CD34+ HSCs in BM after 13 weeks. (K) Percentage of CD4+ (top) and CD8+ (bottom) T cells in cultures ± PHA ± BIO. PB2 = PHA + BIO 2 μM. (L) Annexin V/7-amino-actinomycin D staining of purified CD3+ UCB T cells treated for 4 days; n = 3. Representative flows shown for donor 1. (M) Flow cytometry figures illustrating CD25 expression on CD4+ and CD8+ PHA-activated UCB-derived T cells. (N) Gene expression microarray analysis data: IL2RA(CD25)/IL-2 signaling target genes and nuclear factor–κB activating genes that were down-regulated by BIO in UCB-derived PHA-activated T cells. (O) Gene expression microarray analysis data: GZMB, co-stimulatory molecules, and chemokine/cytokine and their receptors that were down-regulated by BIO in PHA-activated T cells. (P) Gene expression microarray analysis data: Genes that were down-regulated by BIO in PHA-activated UCB-derived T cells in donor 1 and donor 2, and that were up-regulated by PHA in UCB-derived resting T cells. (Q) Gene expression microarray analysis data: Wnt/β-catenin target genes that were up-regulated by BIO in PHA-activated UCB-derived T cells. (R) A β-catenin DNA reporter construct was transfected into HEK293 cells. Cells were treated for 21 hours before green fluorescent protein expression was analyzed by flow cytometry. One representative experiment (n = 3). Experimental Hematology 2013 41, 39-55.e10DOI: (10.1016/j.exphem.2012.09.005) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Supplementary Figure E1 (A) PB platelet (top) and red blood cell (RBC) (bottom) counts from mice treated with DMSO or BIO throughout the experiment depicted in Figure 1A as donor 3. Day 17–21 data represent averaged data from postmortem examinations of lethal GVHD. (B) Top: PB counts of DMSO-treated and BIO-treated mice at the experiment end point. Bottom: PB counts of healthy, nontransplanted, nonirradiated 10-week-old NSG mice. (C) Kaplan-Meier survival curve of mice from humanized mouse model of GVHD. (D) Total number of donor MNCs harvested from spleens and PB after 2, 4, and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (E) Total number of donor MNCs harvested from BM 4 and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (F) Levels of IL-5 and IFNγ in serum of DMSO and BIO-treated mice on day +13. TNFα, IL-2, IL-10, and IL-4 were not detected by multiplex. (G) Human CD45+ MNC (huCD45) percentage in BM on day 13 and at the experiment end point. (H) The effects of BIO treatment on hematopoietic regeneration after sublethal (2.5 Gy) irradiation in nonobese diabetic severe combined immune-deficient mice. BIO dose: 30 mg/kg intraperitoneal twice per week for 2 weeks. Mice culled on day 18. Hematopoietic lineage markers analyzed were Sca1− primitive hematopoietic progenitor cells, CD11b myelomonocytes, Gr1-granulocytes, CD45R, B cells, CD49B− T-cells, and natural killer cells. Experiment and analysis performed by Dr. Robert Knight. All data: n ≥ 3 per group. (I) Engraftment of human CD45+ MNCs in PB and BM of CD34+ HSC engrafted NSG mice after 13 weeks. (J) Multilineage differentiation (CD19+ and CD33+) of human CD34+ HSCs in BM after 13 weeks. (K) Percentage of CD4+ (top) and CD8+ (bottom) T cells in cultures ± PHA ± BIO. PB2 = PHA + BIO 2 μM. (L) Annexin V/7-amino-actinomycin D staining of purified CD3+ UCB T cells treated for 4 days; n = 3. Representative flows shown for donor 1. (M) Flow cytometry figures illustrating CD25 expression on CD4+ and CD8+ PHA-activated UCB-derived T cells. (N) Gene expression microarray analysis data: IL2RA(CD25)/IL-2 signaling target genes and nuclear factor–κB activating genes that were down-regulated by BIO in UCB-derived PHA-activated T cells. (O) Gene expression microarray analysis data: GZMB, co-stimulatory molecules, and chemokine/cytokine and their receptors that were down-regulated by BIO in PHA-activated T cells. (P) Gene expression microarray analysis data: Genes that were down-regulated by BIO in PHA-activated UCB-derived T cells in donor 1 and donor 2, and that were up-regulated by PHA in UCB-derived resting T cells. (Q) Gene expression microarray analysis data: Wnt/β-catenin target genes that were up-regulated by BIO in PHA-activated UCB-derived T cells. (R) A β-catenin DNA reporter construct was transfected into HEK293 cells. Cells were treated for 21 hours before green fluorescent protein expression was analyzed by flow cytometry. One representative experiment (n = 3). Experimental Hematology 2013 41, 39-55.e10DOI: (10.1016/j.exphem.2012.09.005) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Supplementary Figure E1 (A) PB platelet (top) and red blood cell (RBC) (bottom) counts from mice treated with DMSO or BIO throughout the experiment depicted in Figure 1A as donor 3. Day 17–21 data represent averaged data from postmortem examinations of lethal GVHD. (B) Top: PB counts of DMSO-treated and BIO-treated mice at the experiment end point. Bottom: PB counts of healthy, nontransplanted, nonirradiated 10-week-old NSG mice. (C) Kaplan-Meier survival curve of mice from humanized mouse model of GVHD. (D) Total number of donor MNCs harvested from spleens and PB after 2, 4, and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (E) Total number of donor MNCs harvested from BM 4 and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (F) Levels of IL-5 and IFNγ in serum of DMSO and BIO-treated mice on day +13. TNFα, IL-2, IL-10, and IL-4 were not detected by multiplex. (G) Human CD45+ MNC (huCD45) percentage in BM on day 13 and at the experiment end point. (H) The effects of BIO treatment on hematopoietic regeneration after sublethal (2.5 Gy) irradiation in nonobese diabetic severe combined immune-deficient mice. BIO dose: 30 mg/kg intraperitoneal twice per week for 2 weeks. Mice culled on day 18. Hematopoietic lineage markers analyzed were Sca1− primitive hematopoietic progenitor cells, CD11b myelomonocytes, Gr1-granulocytes, CD45R, B cells, CD49B− T-cells, and natural killer cells. Experiment and analysis performed by Dr. Robert Knight. All data: n ≥ 3 per group. (I) Engraftment of human CD45+ MNCs in PB and BM of CD34+ HSC engrafted NSG mice after 13 weeks. (J) Multilineage differentiation (CD19+ and CD33+) of human CD34+ HSCs in BM after 13 weeks. (K) Percentage of CD4+ (top) and CD8+ (bottom) T cells in cultures ± PHA ± BIO. PB2 = PHA + BIO 2 μM. (L) Annexin V/7-amino-actinomycin D staining of purified CD3+ UCB T cells treated for 4 days; n = 3. Representative flows shown for donor 1. (M) Flow cytometry figures illustrating CD25 expression on CD4+ and CD8+ PHA-activated UCB-derived T cells. (N) Gene expression microarray analysis data: IL2RA(CD25)/IL-2 signaling target genes and nuclear factor–κB activating genes that were down-regulated by BIO in UCB-derived PHA-activated T cells. (O) Gene expression microarray analysis data: GZMB, co-stimulatory molecules, and chemokine/cytokine and their receptors that were down-regulated by BIO in PHA-activated T cells. (P) Gene expression microarray analysis data: Genes that were down-regulated by BIO in PHA-activated UCB-derived T cells in donor 1 and donor 2, and that were up-regulated by PHA in UCB-derived resting T cells. (Q) Gene expression microarray analysis data: Wnt/β-catenin target genes that were up-regulated by BIO in PHA-activated UCB-derived T cells. (R) A β-catenin DNA reporter construct was transfected into HEK293 cells. Cells were treated for 21 hours before green fluorescent protein expression was analyzed by flow cytometry. One representative experiment (n = 3). Experimental Hematology 2013 41, 39-55.e10DOI: (10.1016/j.exphem.2012.09.005) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Supplementary Figure E1 (A) PB platelet (top) and red blood cell (RBC) (bottom) counts from mice treated with DMSO or BIO throughout the experiment depicted in Figure 1A as donor 3. Day 17–21 data represent averaged data from postmortem examinations of lethal GVHD. (B) Top: PB counts of DMSO-treated and BIO-treated mice at the experiment end point. Bottom: PB counts of healthy, nontransplanted, nonirradiated 10-week-old NSG mice. (C) Kaplan-Meier survival curve of mice from humanized mouse model of GVHD. (D) Total number of donor MNCs harvested from spleens and PB after 2, 4, and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (E) Total number of donor MNCs harvested from BM 4 and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (F) Levels of IL-5 and IFNγ in serum of DMSO and BIO-treated mice on day +13. TNFα, IL-2, IL-10, and IL-4 were not detected by multiplex. (G) Human CD45+ MNC (huCD45) percentage in BM on day 13 and at the experiment end point. (H) The effects of BIO treatment on hematopoietic regeneration after sublethal (2.5 Gy) irradiation in nonobese diabetic severe combined immune-deficient mice. BIO dose: 30 mg/kg intraperitoneal twice per week for 2 weeks. Mice culled on day 18. Hematopoietic lineage markers analyzed were Sca1− primitive hematopoietic progenitor cells, CD11b myelomonocytes, Gr1-granulocytes, CD45R, B cells, CD49B− T-cells, and natural killer cells. Experiment and analysis performed by Dr. Robert Knight. All data: n ≥ 3 per group. (I) Engraftment of human CD45+ MNCs in PB and BM of CD34+ HSC engrafted NSG mice after 13 weeks. (J) Multilineage differentiation (CD19+ and CD33+) of human CD34+ HSCs in BM after 13 weeks. (K) Percentage of CD4+ (top) and CD8+ (bottom) T cells in cultures ± PHA ± BIO. PB2 = PHA + BIO 2 μM. (L) Annexin V/7-amino-actinomycin D staining of purified CD3+ UCB T cells treated for 4 days; n = 3. Representative flows shown for donor 1. (M) Flow cytometry figures illustrating CD25 expression on CD4+ and CD8+ PHA-activated UCB-derived T cells. (N) Gene expression microarray analysis data: IL2RA(CD25)/IL-2 signaling target genes and nuclear factor–κB activating genes that were down-regulated by BIO in UCB-derived PHA-activated T cells. (O) Gene expression microarray analysis data: GZMB, co-stimulatory molecules, and chemokine/cytokine and their receptors that were down-regulated by BIO in PHA-activated T cells. (P) Gene expression microarray analysis data: Genes that were down-regulated by BIO in PHA-activated UCB-derived T cells in donor 1 and donor 2, and that were up-regulated by PHA in UCB-derived resting T cells. (Q) Gene expression microarray analysis data: Wnt/β-catenin target genes that were up-regulated by BIO in PHA-activated UCB-derived T cells. (R) A β-catenin DNA reporter construct was transfected into HEK293 cells. Cells were treated for 21 hours before green fluorescent protein expression was analyzed by flow cytometry. One representative experiment (n = 3). Experimental Hematology 2013 41, 39-55.e10DOI: (10.1016/j.exphem.2012.09.005) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Supplementary Figure E1 (A) PB platelet (top) and red blood cell (RBC) (bottom) counts from mice treated with DMSO or BIO throughout the experiment depicted in Figure 1A as donor 3. Day 17–21 data represent averaged data from postmortem examinations of lethal GVHD. (B) Top: PB counts of DMSO-treated and BIO-treated mice at the experiment end point. Bottom: PB counts of healthy, nontransplanted, nonirradiated 10-week-old NSG mice. (C) Kaplan-Meier survival curve of mice from humanized mouse model of GVHD. (D) Total number of donor MNCs harvested from spleens and PB after 2, 4, and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (E) Total number of donor MNCs harvested from BM 4 and 6 days post-transplantation of mice treated with DMSO or BIO. Donor 3 cells used for experiment; n ≥ 3 per data point. (F) Levels of IL-5 and IFNγ in serum of DMSO and BIO-treated mice on day +13. TNFα, IL-2, IL-10, and IL-4 were not detected by multiplex. (G) Human CD45+ MNC (huCD45) percentage in BM on day 13 and at the experiment end point. (H) The effects of BIO treatment on hematopoietic regeneration after sublethal (2.5 Gy) irradiation in nonobese diabetic severe combined immune-deficient mice. BIO dose: 30 mg/kg intraperitoneal twice per week for 2 weeks. Mice culled on day 18. Hematopoietic lineage markers analyzed were Sca1− primitive hematopoietic progenitor cells, CD11b myelomonocytes, Gr1-granulocytes, CD45R, B cells, CD49B− T-cells, and natural killer cells. Experiment and analysis performed by Dr. Robert Knight. All data: n ≥ 3 per group. (I) Engraftment of human CD45+ MNCs in PB and BM of CD34+ HSC engrafted NSG mice after 13 weeks. (J) Multilineage differentiation (CD19+ and CD33+) of human CD34+ HSCs in BM after 13 weeks. (K) Percentage of CD4+ (top) and CD8+ (bottom) T cells in cultures ± PHA ± BIO. PB2 = PHA + BIO 2 μM. (L) Annexin V/7-amino-actinomycin D staining of purified CD3+ UCB T cells treated for 4 days; n = 3. Representative flows shown for donor 1. (M) Flow cytometry figures illustrating CD25 expression on CD4+ and CD8+ PHA-activated UCB-derived T cells. (N) Gene expression microarray analysis data: IL2RA(CD25)/IL-2 signaling target genes and nuclear factor–κB activating genes that were down-regulated by BIO in UCB-derived PHA-activated T cells. (O) Gene expression microarray analysis data: GZMB, co-stimulatory molecules, and chemokine/cytokine and their receptors that were down-regulated by BIO in PHA-activated T cells. (P) Gene expression microarray analysis data: Genes that were down-regulated by BIO in PHA-activated UCB-derived T cells in donor 1 and donor 2, and that were up-regulated by PHA in UCB-derived resting T cells. (Q) Gene expression microarray analysis data: Wnt/β-catenin target genes that were up-regulated by BIO in PHA-activated UCB-derived T cells. (R) A β-catenin DNA reporter construct was transfected into HEK293 cells. Cells were treated for 21 hours before green fluorescent protein expression was analyzed by flow cytometry. One representative experiment (n = 3). Experimental Hematology 2013 41, 39-55.e10DOI: (10.1016/j.exphem.2012.09.005) Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions