Supplementary Materials

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+CD45RACD90+ 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+CD45RACD90+ 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+CD38CD45RACD90+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+CD45RACD90+ 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 (Padj = 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+CD38CD45RACD90+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 (Padj = 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 (Padj = 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 (Padj = 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 LinCD34+CD38CD45RACD90+CD49f+ HSCs in the presence of 3F.
  • Movie S2. (.avi format). Time-lapse imaging of human LinCD34+CD38CD45RACD90+CD49f+ HSCs in the presence of 3F + TNFα (1 ng/ml).
  • Movie S3. (.mp4 format). The inhibition of NFκB signaling immobilized HSCs.