Research ArticleTransplantation

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

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Science Translational Medicine  20 Dec 2017:
Vol. 9, Issue 421, eaag3214
DOI: 10.1126/scitranslmed.aag3214
  • Fig. 1 Increasing UCB cell dose impairs short-term progenitor cell engraftment.

    (A) Experimental scheme. (B) Fold changes in CD34+ and CD34+CD45RACD90+ hematopoietic stem cell–enriched (HSC-e) cells [calculated as cell number harvested from bone marrow (BM) of two femurs and two tibiae in relation to the number injected on day 0; see table S1 for the cellular composition of the purified CD34+ fraction and unfractionated umbilical cord blood (UCB) cells and table S2 for the cell number transplanted on day 0]. Dots represent individual mice, with horizontal lines denoting the mean values (n = 5 mice per group from one experiment). Two-tailed Kruskal-Wallis and Mann-Whitney U tests with Bonferroni correction (Padj = 0.992) were used to determine the significance level. *P < 0.05; **P < 0.01; and ***P < 0.001. (C) Heat map representation of the percentage of human cell subsets (gated on 7-AADCD45+HLA-ABC+) detected in the mouse BM (see table S3 for phenotypic definition and table S4 for P values).

  • Fig. 2 Systemically released inflammatory factors directly affect HSC-e cell fate.

    (A) Concentrations of human factors detected in the mouse serum after 14 days. Seven factors whose corresponding receptors were differentially expressed by HSCs are indicated as bold. (B) Forty LinCD34+CD38CD45RACD49f+ cells were cultured in the serum-free control condition with the addition of each test factor. After 7 days, changes in the numbers of CD34+CD45RACD90+ (defined as HSC-e) and the remaining CD34+ (progenitor) and CD34 (differentiated) cells were calculated for each test condition against control. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin. (C) Average values of fold change in relation to control when interferon-inducible cytokine (IP-10) (orange), monocyte chemotactic protein 1 (MCP-1) (green), interferon-γ (IFNγ) (blue), and tumor necrosis factor–α (TNFα) (purple) were individually tested at three different doses (ng/ml) indicated by different shades for each factor. Two-tailed Kruskal-Wallis test and Mann-Whitney U test with Bonferroni correction (Padj = 0.983) were performed to compare each test condition to the control group. Data are from four or five independent experiments (n = 3 within each experiment). Two to five pooled UCB units were used per experiment. *P < 0.05; **P < 0.01; ***P < 0.001; and not significant (n.s.), P ≥ 0.05.

  • Fig. 3 TNFα inhibits CD34+ and HSC-e cell expansion via TNFR1.

    (A and B) Fold expansion of (A) CD34+ and (B) CD34+CD45RACD90+ HSC-e cells from 7-day serum-free culture of UCB CD34+ cells in control or control + TNFα (1 ng/ml). Asterisk denotes statistical significance by two-tailed Mann-Whitney U test. Shown are means ± SD from nine independent experiments. (C and D) Expansion of (C) CD34+ and (D) HSC-e cells after 7 days. Cells were cultured in the presence of TNFα with the addition of etanercept, human immunoglobulin G (hIgG) control, TNF receptor (TNFR)–blocking antibodies (Ab), or mouse IgG1 control (5 μg/ml). Asterisk denotes statistical significance by Kruskal-Wallis test and Mann-Whitney U test with Bonferroni correction (Padj = 0.993) for comparing each test condition to the control. Shown are means ± SD from at least two independent experiments. *P < 0.05; **P < 0.01; and ***P < 0.001.

  • Fig. 4 TNFα directly induces cell death and prolongs cell cycle progression in HSCs.

    (A) Continuous observation of single LinCD34+CD38CD45RACD90+CD49f+ HSCs by time-lapse imaging. Shown is time (hours) cells took to complete the first (generation 0), second (generation 1), and third (generation 2) division. A total of 15 cells did not divide by the end of the 5-day movie when cultured with TNFα. Data are from two independent experiments. (B) Ki67 and Hoechst staining were used to assess the proportion of HSCs in each phase of the cell cycle (G0, Ki67 2n DNA content; G1, Ki67+ 2n DNA content; S-G2-M, Ki67+ > 2n DNA content) over the course of 3-day culture of LinCD34+CD38CD45RACD90+CD49f+ HSCs. Shown are means ± SD of the percentage of cells in G0 from three independent experiments. Two-tailed Mann-Whitney U test was used to determine the significance level. *P < 0.05; **P < 0.01; and ***P < 0.001. (C) Heat map representation of the activation of seven intracellular signaling intermediates in LinCD34+CD38CD45RACD90+CD49f+ HSCs upon stimulation of control or TNFα only cultures by single-cell mass cytometry. Values are calculated as the difference of median arcsinh (signal intensity/5) of the indicated condition compared with the time 0 control and are shown in table S8. (D) Representative histograms of signal intensity of phosphorylated nuclear factor κB (pNFκB) and inhibitor of NFκB-α (IκBα) over time after stimulation of TNFα in LinCD34+CD38CD45RACD90+CD49f+ HSCs. Histogram color corresponds to the arcsinh difference calculated in (C).

  • Fig. 5 Donor-derived activated memory T cells secrete TNFα.

    (A) Representative spanning-tree progression analysis of density-normalized events (SPADE) analysis of human T cell subsets detected in the BM after transplantation of 3 × 106 UCB cells. The tree plot was constructed using 23 cell surface antigens. Shown are expression patterns of CD45, CD3, CD4, CD8, CCR7, and CD45RA. Putative cell populations were annotated manually by black lines encircling node sets expressing the indicated surface marker. The size of each node in the tree indicates the number of cells contained within each node. The color represents the arcsinh-transformed value of the global median intensity of the corresponding marker expression level of the cells in each node. CD3, CD4, and CD8 were used to cluster the T cell populations. CCR7 and CD45RA were used to further identify the naïve (CCR7+CD45RA+), effector memory (CCR7CD45RA), and central memory (CCR7+CD45RA) phenotypes within the CD4+ and CD8+ T cell populations. (B) SPADE representation of intracellular TNFα level in cell subsets identified as described in (A). Color scales represent arcsinh-transformed values of the global median intensity of TNFα expression levels, allowing direct comparison between samples (n = 2 mice per group from one experiment).

  • Fig. 6 TNFα-neutralizing agent enhances short-term engraftment of phenotypically and functionally defined primitive cells in vivo.

    (A) Experimental scheme. i.p., intraperitoneally. (B) Percentage of CD34+ and CD34+CD45RACD90+ HSC-e cells detected in the BM after 17 days. Dots represent individual mice, with horizontal lines denoting the mean values (n = 6 or 8 mice per group from two independent experiments). (C) Heat map representation of the average percentage of human cell populations (gated on 7-AADCD45+HLA-ABC+) detected in the BM by day 17. (D) Heat map representation of human factor levels detected in the mouse serum after 17 days (from one experiment). Two-tailed Kruskal-Wallis and Mann-Whitney U tests with Bonferroni correction (Padj = 0.992) were used to determine the significance level. *P < 0.05; **P < 0.01; and ***P < 0.001.

  • Fig. 7 Blocking IL-6 signaling does not recapitulate the effects of TNFα neutralization.

    (A) Experimental scheme. (B) Percentage of CD34+ and CD34+CD45RACD90+ HSC-e cells and (C) total progenitor numbers quantified by colony-forming cell (CFC) assays detected in the BM after 17 days. PBS, phosphate-buffered saline. Dots represent individual mice, with horizontal lines denoting the mean values (n = 5 mice per group from two independent experiments). Two-tailed Kruskal-Wallis and Mann-Whitney U tests with Bonferroni correction (Padj = 0.992) were used to determine the significance level. *P < 0.05; **P < 0.01; and ***P < 0.001.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/421/eaag3214/DC1

    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 panel 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).

    Movie S1. Time-lapse imaging of human LinCD34+CD38CD45RACD90+CD49f+ HSCs in the presence of 3F.

    Movie S2. Time-lapse imaging of human LinCD34+CD38CD45RACD90+CD49f+ HSCs in the presence of 3F + TNFα (1 ng/ml).

    Movie S3. The inhibition of NFκB signaling immobilized HSCs.

    Reference (56)

  • 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)

    [Download PDF]

    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.

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