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.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Development and application of nanomaterials, nanotechnology and nanomedicine for treating hematological malignancies
Journal of Hematology & Oncology Open Access 23 June 2023
Signal Transduction and Targeted Therapy Open Access 24 March 2022
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Datasets supporting the findings of this study are available from the corresponding authors upon reasonable request.
Goronzy, J. J. & Weyand, C. M. Successful and maladaptive T cell aging. Immunity 46, 364–378 (2017).
Liston, A., Enders, A. & Siggs, O. M. Unravelling the association of partial T-cell immunodeficiency and immune dysregulation. Nat. Rev. Immunol. 8, 545–558 (2008).
Blazar, B. R., Murphy, W. J. & Abedi, M. Advances in graft-versus-host disease biology and therapy. Nat. Rev. Immunol. 12, 443–458 (2012).
Krenger, W., Blazar, B. R. & Holländer, G. A. Thymic T-cell development in allogeneic stem cell transplantation. Blood 117, 6768–6776 (2011).
Zlotoff, D. A. et al. Delivery of progenitors to the thymus limits T-lineage reconstitution after bone marrow transplantation. Blood 118, 1962–1970 (2011).
Chaudhry, M. S., Velardi, E., Dudakov, J. A. & Brink, M. R. Thymus: the next (re) generation. Immunol. Rev. 271, 56–71 (2016).
Mohtashami, M., Shukla, S., Zandstra, P. & Zúñiga-Pflücker, J. C. in Synthetic Immunology 95–120 (Watanabe, T. & Takahama, Y., eds, Springer, Tokyo, 2016).
Perales, M.-A. et al. Recombinant human interleukin-7 (CYT107) promotes T-cell recovery after allogeneic stem cell transplantation. Blood 120, 4882–4891 (2012).
Skrombolas, D. & Frelinger, J. G. Challenges and developing solutions for increasing the benefits of IL-2 treatment in tumor therapy. Exp. Rev. Clin. Immunol. 10, 207–217 (2014).
Dudakov, J. A. et al. Interleukin-22 drives endogenous thymic regeneration in mice. Science 336, 91–95 (2012).
Cobbold, M. et al. Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA–peptide tetramers. J. Exp. Med. 202, 379–386 (2005).
Rooney, C. M. et al. Infusion of cytotoxic T cells for the prevention and treatment of Epstein–Barr virus–induced lymphoma in allogeneic transplant recipients. Blood 92, 1549–1555 (1998).
Zakrzewski, J. L. et al. Tumor immunotherapy across MHC barriers using allogeneic T-cell precursors. Nat. Biotechnol. 26, 453 (2008).
Van Coppernolle, S. et al. Functionally mature CD4 and CD8 TCR αβ cells are generated in OP9-DL1 cultures from human CD34+ hematopoietic cells. J. Immunol. 183, 4859–4870 (2009).
Awong, G. et al. Human proT-cells generated in vitro facilitate hematopoietic stem cell–derived T-lymphopoiesis in vivo and restore thymic architecture. Blood 122, 4210–4219 (2013).
Love, P. E. & Bhandoola, A. Signal integration and crosstalk during thymocyte migration and emigration. Nat. Rev. Immunol. 11, 469 (2011).
Radtke, F., MacDonald, H. R. & Tacchini-Cottier, F. Regulation of innate and adaptive immunity by Notch. Nat. Rev. Immunol. 13, 427 (2013).
Serwold, T., Ehrlich, L. I. R. & Weissman, I. L. Reductive isolation from bone marrow and blood implicates common lymphoid progenitors as the major source of thymopoiesis. Blood 113, 807–815 (2009).
Vionnie, W. et al. Specific bone cells produce DLL4 to generate thymus-seeding progenitors from bone marrow. J. Exp. Med. 212, 759–774 (2015).
Smith, K. Y. et al. Thymic size and lymphocyte restoration in patients with human immunodeficiency virus infection after 48 weeks of zidovudine, lamivudine, and ritonavir therapy. J. Infect. Dis. 181, 141–147 (2000).
Wozney, J. M. et al. Novel regulators of bone formation: molecular clones and activities. Science 242, 1528–1534 (1988).
Koshy, S. T., Zhang, D. K., Grolman, J. M., Stafford, A. G. & Mooney, D. J. Injectable nanocomposite cryogels for versatile protein drug delivery. Acta Biomater. 65, 36–43 (2018).
Brainard, D. M. et al. Induction of robust cellular and humoral virus-specific adaptive immune responses in human immunodeficiency virus–infected humanized BLT mice. J. Virol. 83, 7305–7321 (2009).
Douek, D. C. et al. Assessment of thymic output in adults after haematopoietic stem cell transplantation and prediction of T-cell reconstitution. Lancet 355, 1875–1881 (2000).
Smadja, D. M. et al. Bone morphogenetic proteins 2 and 4 are selectively expressed by late outgrowth endothelial progenitor cells and promote neoangiogenesis. Arterioscler. Thromb. Vasc. Biol. 28, 2137–2143 (2008).
Lafage-Proust, M.-H. et al. Assessment of bone vascularization and its role in bone remodeling. Bonekey Rep. 4, 662 (2015).
Kuznetsov, S. A. et al. The interplay of osteogenesis and hematopoiesis: expression of a constitutively active PTH/PTHrP receptor in osteogenic cells perturbs the establishment of hematopoiesis in bone and of skeletal stem cells in the bone marrow. J. Cell Biol. 167, 1113–1122 (2004).
Song, J. et al. An in vivo model to study and manipulate the hematopoietic stem cell niche. Blood 115, 2592–2600 (2010).
Wils, E.-J. et al. Flt3 ligand expands lymphoid progenitors prior to recovery of thymopoiesis and accelerates T cell reconstitution after bone marrow transplantation. J. Immunol. 178, 3551–3557 (2007).
Maillard, I. et al. Notch-dependent T-lineage commitment occurs at extrathymic sites following bone marrow transplantation. Blood 107, 3511–3519 (2006).
Garber, K. Driving T-cell immunotherapy to solid tumors. Nat. Biotechnol. 36, 215–219 (2018).
Jangalwe, S., Shultz, L. D., Mathew, A. & Brehm, M. A. Improved B cell development in humanized NOD-scid IL2Rγ null mice transgenically expressing human stem cell factor, granulocyte-macrophage colony-stimulating factor and interleukin-3. Immun. Inflamm. Dis. 4, 427–440 (2016).
Ripamonti, U. Bone induction by recombinant human osteogenic protein-1 (hOP-1, BMP-7) in the primate Papio ursinus with expression of mRNA of gene products of the TGF-β superfamily. J. Cell. Mol. Med. 9, 911–928 (2005).
Heliotis, M., Lavery, K., Ripamonti, U., Tsiridis, E. & Di Silvio, L. Transformation of a prefabricated hydroxyapatite/osteogenic protein-1 implant into a vascularised pedicled bone flap in the human chest. Int. J. Oral Maxillofac. Surg. 35, 265–269 (2006).
Warnke, P. et al. Growth and transplantation of a custom vascularised bone graft in a man. Lancet 364, 766–770 (2004).
ClinicalTrials.gov. Dendritic cell activating scaffold in melanoma. https://clinicaltrials.gov/ct2/show/NCT01753089
Carragee, E. J., Hurwitz, E. L. & Weiner, B. K. A critical review of recombinant human bone morphogenetic protein-2 trials in spinal surgery: emerging safety concerns and lessons learned. Spine J. 11, 471–491 (2011).
Biffi, R. et al. Use of totally implantable central venous access ports for high-dose chemotherapy and peripheral blood stem cell transplantation: results of a monocentre series of 376 patients. Ann. Oncol. 15, 296–300 (2004).
Li, M. O. & Rudensky, A. Y. T cell receptor signalling in the control of regulatory T cell differentiation and function. Nat. Rev. Immunol. 16, 220 (2016).
Hoffmann, P., Ermann, J., Edinger, M., Fathman, C. G. & Strober, S. Donor-type CD4+CD25+regulatory T cells suppress lethal acute graft-versus-host disease after allogeneic bone marrow transplantation. J.Exp. Med. 196, 389–399 (2002).
Wan, Y. Y. & Flavell, R. A. ‘Yin–Yang’ functions of transforming growth factor-β and T regulatory cells in immune regulation. Immunol. Rev. 220, 199–213 (2007).
Bencherif, S. A. et al. Injectable cryogel-based whole-cell cancer vaccines. Nat. Commun. 6, 7556 (2015).
Palchaudhuri, R. et al. Non-genotoxic conditioning for hematopoietic stem cell transplantation using a hematopoietic-cell-specific internalizing immunotoxin. Nat. Biotechnol. 34, 738 (2016).
Bencherif, S. A. et al. Injectable preformed scaffolds with shape-memory properties. Proc. Natl Acad. Sci. USA 109, 19590–19595 (2012).
Macdonald, M. L. et al. Tissue integration of growth factor–eluting layer-by-layer polyelectrolyte multilayer coated implants. Biomaterials 32, 1446–1453 (2011).
Sprinzak, D. et al. Cis-interactions between Notch and Delta generate mutually exclusive signalling states. Nature 465, 86 (2010).
Nandagopal, N. et al. Dynamic ligand discrimination in the Notch signaling pathway. Cell 172, 869–880 (2018).
Zakrzewski, J. L. et al. Adoptive transfer of T-cell precursors enhances T-cell reconstitution after allogeneic hematopoietic stem cell transplantation. Nat. Med. 12, 1039 (2006).
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.
Magenta Therapeutics, equity and consulting: D.T.S.; Agios Pharmaceuticals, director, equity: D.T.S.; Fate Therapeutics, equity and consulting: D.T.S.; Clear Creek Bio, director, equity and consulting: D.T.S.; FOG Pharma, consulting: D.T.S.; Red Oak Medicines, director, equity, consulting: D.T.S.; Lifevaultbio, director, equity: D.T.S.; Bone Therapeutics, consulting: D.T.S.; Novartis, sponsored research: D.T.S. and D.J.M.; Agnovos, consulting: D.J.M.; Amgen, sponsored research: D.J.M.; Samyang Corp., consulting: D.J.M.; Decibel, sponsored research: D.J.M.; Merck, sponsored research: D.J.M.; Immulus, equity: D.J.M.; Inventors, patent applications (PCT/US2017/016729): N.J.S., A.S.M., T.-Y.S., D.J.M. and D.T.S.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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).
About this article
Cite this article
Shah, N.J., Mao, A.S., Shih, TY. et al. An injectable bone marrow–like scaffold enhances T cell immunity after hematopoietic stem cell transplantation. Nat Biotechnol 37, 293–302 (2019). https://doi.org/10.1038/s41587-019-0017-2
This article is cited by
Development and application of nanomaterials, nanotechnology and nanomedicine for treating hematological malignancies
Journal of Hematology & Oncology (2023)
Immune microenvironment: novel perspectives on bone regeneration disorder in osteoradionecrosis of the jaws
Cell and Tissue Research (2023)
Signal Transduction and Targeted Therapy (2022)
DNA scaffolds enable efficient and tunable functionalization of biomaterials for immune cell modulation
Nature Nanotechnology (2021)
Nature Reviews Immunology (2021)