Supplementary Material for:

Enhanced human hematopoietic stem and progenitor cell engraftment by blocking donor T cell–mediated TNFα signaling

Weijia Wang, Hisaki Fujii, Hye Jin Kim, Karin Hermans, Tatiana Usenko, Stephanie Xie, Zhi-Juan Luo, Jennifer Ma, Cristina Lo Celso, John E. Dick, Timm Schroeder, Joerg Krueger, Donna Wall, R. Maarten Egeler, Peter W. Zandstra*

*Corresponding author. Email: peter.zandstra{at}utoronto.ca

Published 20 December 2017, Sci. Transl. Med. 9, eaag3214 (2017)
DOI: 10.1126/scitranslmed.aag3214

This PDF file includes:

• Materials and Methods
• Fig. S1. Significant weight loss was observed starting from day 17 when 5 × 10⁶ UCB cells were transplanted.
• Fig. S2. Human cell subsets detected in the mouse BM.
• Fig. S3. Transplantation of UCB resulted in systemic inflammation as indicated by an array of human inflammatory factors detected in mouse sera within 2 weeks.
• Fig. S4. Flow cytometry–based HSC-e cell assay.
• Fig. S5. TNFα reduced the percentage of CD34⁺ and CD34⁺CD45RA⁻CD90⁺ HSC-e cells in culture.
• Fig. S6. Flow cytometric analysis of TNFR1 and TNFR2 receptor expression.
• Fig. S7. TNFα-induced cell death.
• Fig. S8. TNFα inhibited HSC cell cycle entry.
• Fig. S9. Intracellular signaling activation analysis by single-cell mass cytometry.
• Fig. S10. Higher UCB dose led to reduced numbers of HSPCs engrafted in the BM, whereas no significant weight loss and GVHD symptoms were observed at 3 × 10⁶ UCB cell dose.
• Fig. S11. System-wide analysis of human cell subsets present in the BM by single-cell mass cytometry.
• Fig. S12. Body weight and GVHD were monitored over the course of 17 days.
• Fig. S13. Neutralizing TNFα increased the engrafted numbers of CD34⁺ and CD34⁺CD45RA⁻CD90⁺ HSC-e cells in the BM 17 days after transplantation.
• Fig. S14. Numbers of human (CD45⁺HLA-ABC⁺) cell populations detected in the BM after 17 days.
• Fig. S15. Human (CD45⁺HLA-ABC⁺) cell subsets detected in the BM 17 days after transplantation by flow cytometry.
• Fig. S16. Etanercept treatment resulted in faster and enhanced reconstitution of human myeloid and megakaryocytic cells in the PB.
• Fig. S17. Etanercept treatment had no significant effect on short-term engraftment of T cell–depleted UCB cells.
• Fig. S18. Etanercept treatment led to changes in the composition of donor T and dendritic cell subsets.
• Fig. S19. Blocking IL-6 receptor did not recapitulate the effects of TNFα blockade.
• Fig. S20. Human (CD45⁺HLA-ABC⁺) cell subsets detected in the BM 17 days after transplantation by flow cytometry.
• Fig. S21. Blocking TNFα increased the percentage of CD34⁺CD38⁻CD45RA⁻CD90⁺CD49f⁺ HSCs present in the BM 17 days after transplantation.
• Fig. S22. Etanercept treatment had no significant effect on short-term engraftment of human CD34⁺ cells.
• Fig. S23. Etanercept treatment had no negative impact on long-term HSCs.
• Fig. S24. Serum concentrations of 29 human factors with the treatment of etanercept.
• Fig. S25. Dose-response experiment demonstrating the numerical advantage of using unselected UCB cells when combined with etanercept.
• Fig. S26. Donor T cell–mediated TNFα signaling impairs the survival, division, and short-term engraftment of transplanted stem and progenitor cells.
• Table S1. Cellular composition of the purified CD34⁺ fraction and unfractionated UCB cells.
• Table S2. Numbers of CD34⁺ and CD34⁺CD45RA⁻CD90⁺ cells transplanted on day 0.
• Table S3. Phenotypic definition of human cell subsets assessed by flow cytometry.
• Table S4. P values determined by Kruskal-Wallis and Mann-Whitney U tests with Bonferroni correction (P_adj = 0.992) for multiple comparisons among groups (corresponds to fig. S2).
• Table S5. List of differentially overexpressed receptors in HSCs (the elevated serum factors are shaded).
• Table S6. Data summary of time-lapse videos.
• Table S7. Antibody panel for measuring intracellular signaling events using single-cell mass cytometry.
• Table S8. Calculated median arcsinh (signal intensity/5) differences in stimulated CD34⁺CD38⁻CD45RA⁻CD90⁺CD49f⁺ cells relative to unstimulated cells (at time 0).
• Table S9. Antibody pane for measuring intracellular cytokine levels using single-cell mass cytometry.
• Table S10. Antibody panel used to assess the human cell phenotypes in mouse BM by flow cytometry.
• Table S11. P values determined by Kruskal-Wallis and Mann-Whitney U tests with Bonferroni correction (P_adj = 0.992) for multiple comparisons among groups (corresponds to fig. S15).
• Table S12. P values determined by Kruskal-Wallis and Mann-Whitney U tests with Bonferroni correction (P_adj = 0.992) for multiple comparisons among groups (corresponds to fig. S16).
• Table S13. P values determined by Kruskal-Wallis and Mann-Whitney U tests with Bonferroni correction (P_adj = 0.992) for multiple comparisons among groups (corresponds to fig. S20).
• Reference (56)

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Other Supplementary Material for this manuscript includes the following:

• Movie S1. (.avi format). Time-lapse imaging of human Lin⁻CD34⁺CD38⁻CD45RA⁻CD90⁺CD49f⁺ HSCs in the presence of 3F.
• Movie S2. (.avi format). Time-lapse imaging of human Lin⁻CD34⁺CD38⁻CD45RA⁻CD90⁺CD49f⁺ HSCs in the presence of 3F + TNFα (1 ng/ml).
• Movie S3. (.mp4 format). The inhibition of NFκB signaling immobilized HSCs.