Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for multiple disorders, but deficiency and dysregulation of T cells limit its utility. Here we report a biomaterial-based scaffold that mimics features of T cell lymphopoiesis in the bone marrow. The bone marrow cryogel (BMC) releases bone morphogenetic protein-2 to recruit stromal cells and presents the Notch ligand Delta-like ligand-4 to facilitate T cell lineage specification of mouse and human hematopoietic progenitor cells. BMCs subcutaneously injected in mice at the time of HSCT enhanced T cell progenitor seeding of the thymus, T cell neogenesis and diversification of the T cell receptor repertoire. Peripheral T cell reconstitution increased ~6-fold in mouse HSCT and ~2-fold in human xenogeneic HSCT. Furthermore, BMCs promoted donor CD4+ regulatory T cell generation and improved survival after allogeneic HSCT. In comparison to adoptive transfer of T cell progenitors, BMCs increased donor chimerism, T cell generation and antigen-specific T cell responses to vaccination. BMCs may provide an off-the-shelf approach for enhancing T cell regeneration and mitigating graft-versus-host disease in HSCT.
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The authors acknowledge discussions with H. Seeherman (Bioventus), R. Yusuf (Dana Farber Cancer Institute) and the Harvard Catalyst Biostatistical Consulting Program funded by the National Institutes of Health (UL1 TR002541). N.J.S. was supported by the Cancer Research Institute Postdoctoral Fellowship. The work was supported by the National Institutes of Health through grants U19 HL129903 and R01 EB023287, and by the Blavatnik Biomedical Accelerator Program at Harvard University.
Integrated supplementary information
(a) Bioactivity of the pooled released BMP-2 measured using alkaline phosphatase enzyme activity in MC3T3-E1 pre-osteoblast cells, as compared to BMP-2 never incorporated into BMC (Native BMP-2) and medium with no added BMP-2 (Growth medium) (b) Bioactivity of BMP-2 quantified at discrete time intervals after release using alkaline phosphatase enzyme activity in MC3T3-E1 pre-osteoblast cells. (c) In vitro bioactivity of Notch ligand DLL-4 measured using a colorimetric assay. (d) Representative fluorescence microscopy images of citrine expression in a CHO-K1+2xHS4-UAS-H2B-Citrine-2xHS4 cH1+hNECD-Gal4esn c9 Notch-reporter cell line at different time intervals on dual BMCs (top row) and blank BMCs (bottom row) Fold expansion and viability of (e) mouse and (f) human hematopoietic cells after 7 days of in vitro culture. Data in a-f are mean ± s.d. of n = 5 and are representative from 3 independent experiments. (*P < 0.05, ** P < 0.01, ***P < 0.001, analysis of variance (ANOVA) with a Tukey post hoc test).
(a) Representative flow cytometric profiles of pre- and post- lineage depleted bone marrow cells used for transplantation (5 independent experiments) (b) Images of the edge of BMCs extracted from the subcutaneous tissue at pre-determined time-intervals post-transplant identifying the margins of the BMCs with collagen (blue-green) and cells (black) and in some sections alginate (red) is observed using Safranin-O staining (10x objective magnification). (c) Representative flow cytometric profiles of the bone marrow and BMC (Dual and BMP-2 only) at Day 28 post-transplant. Donor GFP, myeloid, HSC, LMPP, CLP and myeloid progenitors are identified. (d) Host mesenchymal stromal cells in the BMC and endogenous bone marrow and representative flow cytometry plots. Sca-1+ progenitors are represented as a fraction of CD45- cells. CD44, CD73, CD29, CD105 and CD106 expressing cells are represented as a fraction of Sca-1+ progenitors. (e) Quantification of bone alkaline phosphatase (bALP) and Oil-red-O (ORO) in bone and BMC at Day 20 after subcutaneous injection (n = 6–7). (f) Colony-forming unit assays using bone marrow cells from transplant only and dual BMC treated mice at Days 10, 35 and 70 post-transplant. (g) The concentrations of homing factor SDF-1α and lymphoid progenitor supporting cytokine IL-7 in harvested BMCs. Post-HSCT mice treated with a BMP-2 BMC, and post-HSCT mice treated with a Dual BMC were analyzed and compared to cytokine concentrations in the bone marrow of the same group. Data in b, d-g are mean ± s.d. of n = 5, n = 4, n=7, n=4 and n = 4 respectively and are representative from 2 independent experiments. Data in c are from n = 10 and are representative from 2 independent experiments (*P < 0.05, ** P < 0.01, ***P < 0.001, analysis of variance (ANOVA) with a Tukey post hoc test)
(a) Representative FACS gating strategy for measuring post-HSCT immune cell reconstitution in C57BL/6J mice transplanted with GFP+ donor hematopoietic stem and progenitor cells from 5 independent experiments. (b) Reconstitution of B-cells and Myeloid cells in vivo. B6 mice were irradiated with 1 x 1000 cGy L-TBI dose and were subsequently transplanted with 5 x 105 lineage depleted syngeneic GFP BM cells within 48 hours after L-TBI. The peripheral blood of post-HSCT mice with no BMC (Transplant only), post-HSCT mice treated with a BMP-2 BMC, and post-HSCT mice treated with a Dual BMC were analyzed and measured numbers were compared with pre-radiation immune cell concentrations. (c) Representative FACS plots after post-HSCT of donor and host chimerism in thymocytes (DP, SP4, SP8) and in the splenocytes (CD4+, CD8+, B220+) at Day 28 post transplant in BM-treated and transplant only mice. (d) Representative flow cytometry plots of host (CD 45.2) and donor (CD 45.1) chimerism in sublethally irradiated mice 28 days post-transplant. In (c) and (d) B6 mice were irradiated with 500 cGy SL-TBI and subsequently transplanted with 5 x 105 lineage-depleted bone marrow cells within 48 hours post-radiation. One group was treated with the BMC. Data in b, represent the mean ± s.d. from 5 mice per group at each time point. Data in b, c and d are representative of two independent experiments. (*P < 0.05, ** P < 0.01, ***P < 0.001, analysis of variance (ANOVA) with a Tukey post hoc test).
Pre-B CFUs quantified from the bone marrow of NSG-BLT mice with and without BMC treatment at two time points post transplant Data are mean ± s.d. of n = 4 and are from a single donor in one experiment. (*P < 0.05, ** P < 0.01, ***P < 0.001, analysis of variance (ANOVA) with a Tukey post hoc test).
Supplementary Figure 5 Extended flow cytometry characterization of BMC-generated T cells and culture-generated T-cell progenitors.
(a) Representative flow cytometric profiles of FoxP3+ cells among CD4+ cells and isotype used to identify Treg cells in the thymus and spleen (3 independent experiments). (b) Representative FACS profiles of sorted HSCs (Lin-ckit+Sca-1+) and CD44/CD25 expressing T-cell progenitors 14 Days after co-culture with OP9-DL1 cells (2 independent experiments). (c) Representative flow cytometric profiles of ckit and isotype used to identify ETPs in the thymus.
B6 mice were irradiated with 1 x 1000 cGy L-TBI dose and were subsequently transplanted with 5 x 105 lineage depleted syngeneic GFP BM cells within 48 hours after L-TBI and treated as described in the figure. (a) Total thymocytes quantified at 32- and 42-days post-transplant. (b) Thymus weight quantified between 12- and 42-days post-HSCT. (c) mTEC, cTEC, Fibroblasts and endothelial cells were quantified 22-days poster-HSCT. (d) Total number of early T-lineage progenitors (ETP; CD44+CD25−c-kit+), DN2 (CD44+CD25−), DN3 (CD44+CD25−), DP, SP4, SP8 thymocyte subsets compared across different treatment conditions at 22-days post HSCT. The thymus in post-HSCT mice with no BMC (Transplant only), post-HSCT mice treated with a BMP-2 BMC, and post-HSCT mice treated with a Dual BMC were harvested and weighed and were compared with that of non-radiated mice. All groups in a, c, d are compared with transplant only control. 10-days post-HSCT, BMCs were explanted and surgically placed in the subcutaneous pocket of a second set of B6 mice that were irradiated with 500 cGy SL-TBI. Values are represented as absolute numbers. Data in a, b, c, d, f represent the mean ± s.d. from 5 mice per group at each time point and are representative of at least two independent experiments. (*P < 0.05, ** P < 0.01, ***P < 0.001, analysis of variance (ANOVA) with a Tukey post hoc test).
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