Chimeric antigen receptor T-cell immunotherapy represents a breakthrough in treating specific cancer types. However, a critical limitation in its application is the current shortage of donor cells. Attempts to recreate the thymic microenvironment, facilitating the differentiation of therapeutic T-cells from stem cells, hold significant promise for clinical advancements. Nonetheless, replicating crucial elements of T-cell development—dependent on the thymus’s three-dimensional (3D) architecture and its complex matrix composition, including stiffness and intricate cell–cell and cell–matrix interactions—remains unattainable with existing thymic models. To address this, we engineered biomaterials integrated with key thymic components such as Delta-like 4, vascular cell adhesion molecule 1, and proteins derived from collagen. These components are instrumental in directing T-cell development while inhibiting alternative lineage differentiation. Our work led to the identification of a hydrogel formulation that results in the production of both CD4+CD8b+ progenitor (Pro) T-cells and mature, functional CD3+CD8b+ T-cells. The resultant T-cells are not only functional, with the capability for cytokine production, but also mark the establishment of the first hydrogel-based platform for producing T-cells from induced pluripotent stem cell-derived hematopoietic stem and Pro cells. This system uniquely supports essential cell-to-cell and cell-to-extracellular matrix interactions within a 3D context.
Get full access to this article
View all access options for this article.
References
1.
IrvineDJ, MausMV, MooneyDJ, et al.The future of engineered immune cell therapies. Science, 2022; 378(6622):853–858; doi: 10.1126/science.abq6990
2.
SternerRC, SternerRM. CAR-T cell therapy: Current limitations and potential strategies. Blood Cancer J, 2021; 11(4):69; doi: 10.1038/s41408-021-00459-7
3.
CappellKM, KochenderferJN. Long-term outcomes following CAR T cell therapy: What we know so far. Nat Rev Clin Oncol, 2023; 20(6):359–371; doi: 10.1038/s41571-023-00754-1
4.
MelenhorstJJ, ChenGM, WangM, et al.Decade-long leukaemia remissions with persistence of CD4+ CAR T cells. Nature, 2022; 602(7897):503–509; doi: 10.1038/s41586-021-04390-6
5.
AparicioC, AcebalC, González-VallinasM. Current approaches to develop “off-the-shelf” chimeric antigen receptor (CAR)-T cells for cancer treatment: A systematic review. Exp Hematol Oncol, 2023; 12(1):73; doi: 10.1186/s40164-023-00435-w
6.
MichelsA, HoN, BuchholzCJ. Precision medicine: In vivo CAR therapy as a showcase for receptor-targeted vector platforms. Molecular Therapy, 2022; 30(7):2401–2415; doi: 10.1016/j.ymthe.2022.05.018
7.
Zúñiga-PflückerJC. T-cell development made simple. Nat Rev Immunol, 2004; 4(1):67–72; doi: 10.1038/nri1257
8.
SchmittTM, Zúñiga-PflückerJC. Induction of T cell development from hematopoietic progenitor cells by Delta-like-1 in vitro. Immunity, 2002; 17(6):749–756; doi: 10.1016/S1074-7613(02)00474-0
9.
MohtashamiM, ShahDK, KianizadK, et al.Induction of T-cell development by Delta-like 4-expressing fibroblasts. Int Immunol, 2013; 25(10):601–611; doi: 10.1093/intimm/dxt027
10.
EdgarJM, MichaelsYS, ZandstraPW. Multi-objective optimization reveals time- and dose-dependent inflammatory cytokine-mediated regulation of human stem cell derived T-cell development. NPJ Regen Med, 2022; 7(1):11; doi: 10.1038/s41536-022-00210-1
11.
MichaelsYS, EdgarJM, MajorMC, et al.DLL4 and VCAM1 enhance the emergence of T cell–competent hematopoietic progenitors from human pluripotent stem cells. Sci Adv, 2022; 8(34):eabn5522; doi: 10.1126/sciadv.abn5522
12.
Trotman-GrantAC, MohtashamiM, De Sousa CasalJ, et al.DL4-μbeads induce T cell lineage differentiation from stem cells in a stromal cell-free system. Nat Commun, 2021; 12(1):5023; doi: 10.1038/s41467-021-25245-8
13.
StephanSB, TaberAM, JileaevaI, et al.Biopolymer implants enhance the efficacy of adoptive T-cell therapy. Nat Biotechnol, 2015; 33(1):97–101; doi: 10.1038/nbt.3104
14.
HuQ, LiH, ArchibongE, et al.Inhibition of post-surgery tumour recurrence via a hydrogel releasing CAR-T cells and anti-PDL1-conjugated platelets. Nat Biomed Eng, 2021; 5(9):1038–1047; doi: 10.1038/s41551-021-00712-1
15.
ShahNJ, MaoAS, ShihT-Y, et al.An injectable bone marrow–like scaffold enhances T cell immunity after hematopoietic stem cell transplantation. Nat Biotechnol, 2019; 37(3):293–302; doi: 10.1038/s41587-019-0017-2
16.
ChinMHW, NormanMDA, GentlemanE, et al.A hydrogel-integrated culture device to interrogate T cell activation with physicochemical cues. ACS Appl Mater Interfaces, 2020; 12(42):47355–47367; doi: 10.1021/acsami.0c16478
17.
SafaeeH, BakooshliMA, DavoudiS, et al.Tethered jagged-1 synergizes with culture substrate stiffness to modulate notch-induced myogenic progenitor differentiation. Cel Mol Bioeng, 2017; 10(5):501–513; doi: 10.1007/s12195-017-0506-7
18.
KretschmerM, MamistvalovR, SprinzakD, et al.Matrix stiffness regulates notch signaling activity in endothelial cells. J Cell Sci, 2023; 136(2):jcs260442; doi: 10.1242/jcs.260442
19.
OliveiraJT, ReisRL. Hydrogels from polysaccharide-based materials: Fundamentals and applications in regenerative medicine. In: Natural-Based Polymers for Biomedical Applications. Elsevier; 2008; pp. 485–514; doi: 10.1533/9781845694814.4.485
20.
LinC-H, SuJJ-M, LeeS-Y, et al.Stiffness modification of photopolymerizable gelatin-methacrylate hydrogels influences endothelial differentiation of human mesenchymal stem cells. J Tissue Eng Regen Med, 2018; 12(10):2099–2111; doi: 10.1002/term.2745
21.
XueX, HuY, WangS, et al.Fabrication of physical and chemical crosslinked hydrogels for bone tissue engineering. Bioact Mater, 2022; 12:327–339; doi: 10.1016/j.bioactmat.2021.10.029
22.
GoncharovaV, SerobyanN, IizukaS, et al.Hyaluronan expressed by the hematopoietic microenvironment is required for bone marrow hematopoiesis. J Biol Chem, 2012; 287(30):25419–25433; doi: 10.1074/jbc.M112.376699
23.
LinsMP. Thymic extracellular matrix in the thymopoiesis: Just a supporting? BioTech, 2022; 11(3):27; doi: 10.3390/biotech11030027
24.
ShuklaS, LangleyMA, SinghJ, et al.Progenitor T-cell differentiation from hematopoietic stem cells using Delta-like-4 and VCAM-1. Nat Methods, 2017; 14(5):531–538; doi: 10.1038/nmeth.4258
25.
IriguchiS, YasuiY, KawaiY, et al.A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy. Nat Commun, 2021; 12(1):430; doi: 10.1038/s41467-020-20658-3
26.
GendronS, CoutureJ, AoudjitF. Integrin α2β1 inhibits Fas-mediated apoptosis in T lymphocytes by protein phosphatase 2A-dependent activation of the MAPK/ERK pathway. Journal of Biological Chemistry, 2003; 278(49):48633–48643; doi: 10.1074/jbc.M305169200
27.
CastagnaroS, ChrisamM, CesconM, et al.Extracellular collagen VI has prosurvival and autophagy instructive properties in mouse fibroblasts. Front Physiol, 2018; 9:1129; doi: 10.3389/fphys.2018.01129
28.
HansellCF, EspeelP, StamenovićMM, et al.Additive-free clicking for polymer functionalization and coupling by tetrazine–norbornene chemistry. J Am Chem Soc, 2011; 133(35):13828–13831; doi: 10.1021/ja203957h
GopinathanJ, NohI. Click chemistry-based injectable hydrogels and bioprinting inks for tissue engineering applications. Tissue Eng Regen Med, 2018; 15(5):531–546; doi: 10.1007/s13770-018-0152-8
31.
DelplaceV, NickersonPEB, Ortin‐MartinezA, et al.Nonswelling, ultralow content inverse electron‐demand Diels–Alder hyaluronan hydrogels with tunable gelation time: Synthesis and in vitro evaluation. Adv Funct Materials, 2020; 30(14):1903978; doi: 10.1002/adfm.201903978
32.
RutzS, MordmüllerB, SakanoS, et al.Notch ligands Delta-like1, Delta-like4 and Jagged1 differentially regulate activation of peripheral T helper cells. Eur J Immunol, 2005; 35(8):2443–2451; doi: 10.1002/eji.200526294
33.
RizwanM, LingC, GuoC, et al.Viscoelastic notch signaling hydrogel induces liver bile duct organoid growth and morphogenesis. Adv Healthcare Materials, 2022; 11(23):2200880; doi: 10.1002/adhm.202200880
34.
SuraiyaAB, HunML, TruongVX, et al.Gelatin-based 3D microgels for in vitro T lineage cell generation. ACS Biomater Sci Eng, 2020; 6(4):2198–2208; doi: 10.1021/acsbiomaterials.9b01610
35.
LeeY, BalikovD, LeeJ, et al.In Situ forming gelatin hydrogels-directed angiogenic differentiation and activity of patient-derived human mesenchymal stem cells. Ijms, 2017; 18(8):1705; doi: 10.3390/ijms18081705
36.
YaoM, LiJ, ZhangJ, et al.Dual-enzymatically cross-linked gelatin hydrogel enhances neural differentiation of human umbilical cord mesenchymal stem cells and functional recovery in experimental murine spinal cord injury. J Mater Chem B, 2021; 9(2):440–452; doi: 10.1039/D0TB02033H
37.
AsnaghiMA, BarthlottT, GullottaF, et al.Thymus extracellular matrix‐derived scaffolds support graft‐resident thymopoiesis and long‐term in vitro culture of adult thymic epithelial cells. Adv Funct Mater, 2021; 31(20):2010747; doi: 10.1002/adfm.202010747
38.
SeetCS, HeC, BethuneMT, et al.Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids. Nat Methods, 2017; 14(5):521–530; doi: 10.1038/nmeth.4237
39.
Vallmajo‐MartinQ, BroguiereN, MillanC, et al.PEG/HA hybrid hydrogels for biologically and mechanically tailorable bone marrow organoids. Adv Funct Materials, 2020; 30(48):1910282; doi: 10.1002/adfm.201910282
40.
KatoK, LianL-Y, BarsukovIL, et al.Model for the complex between protein G and an antibody Fc fragment in solution. Structure, 1995; 3(1):79–85; doi: 10.1016/S0969-2126(01)00136-8
41.
BiktasovaAK, DudimahDF, UzhachenkoRV, et al.Multivalent forms of the notch ligand DLL-1 enhance antitumor T-cell immunity in lung cancer and improve efficacy of EGFR-Targeted therapy. Cancer Res, 2015; 75(22):4728–4741; doi: 10.1158/0008-5472.CAN-14-1154
42.
YangB, LiuT, GaoG, et al.Fabrication of 3D GelMA scaffolds using agarose microgel embedded printing. Micromachines (Basel), 2022; 13(3):469; doi: 10.3390/mi13030469
43.
AlbigAR, BecentiDJ, RoyTG, et al.Microfibril-associate glycoprotein-2 (MAGP-2) promotes angiogenic cell sprouting by blocking notch signaling in endothelial cells. Microvasc Res, 2008; 76(1):7–14; doi: 10.1016/j.mvr.2008.01.001
44.
ShuklaS. Controlled generation of progenitor T-Cells from hematopoietic stem cells and pluripotent stem cells. PhD [dissertation]. University of Toronto: Toronto; 2017. Available from: https://hdl.handle.net/1807/95681.
LanghansSA. Three-dimensional in vitro cell culture models in drug discovery and drug repositioning. Front Pharmacol, 2018; 9:6; doi: 10.3389/fphar.2018.00006
47.
RodriguesJ, HeinrichMA, TeixeiraLM, et al.3D in vitro model (R)evolution: Unveiling tumor–stroma interactions. Trends Cancer, 2021; 7(3):249–264; doi: 10.1016/j.trecan.2020.10.009
48.
TissinoE, BittoloT, BenedettiD, et al.The VLA-4 integrin is constitutively activated in a fraction of CD49d-expressing chronic lymphocytic leukemia via autonomous BCR-mediated signaling. Blood, 2019; 134(Supplement_1):849–849; doi: 10.1182/blood-2019-124313
49.
MaurettiA, RossiF, BaxNAM, et al.Spheroid three-dimensional culture enhances Notch signaling in cardiac progenitor cells. MRS Communications, 2017; 7(3):496–501; doi: 10.1557/mrc.2017.82
50.
LinT, MatsuzakiG, YoshidaH, et al.CD3 − CD8 + intestinal intraepithelial lymphocytes (IEL) and the extrathymic development of IEL. Eur J Immunol, 1994; 24(5):1080–1087; doi: 10.1002/eji.1830240511
51.
KadivarM, PeterssonJ, SvenssonL, et al.CD8αβ+ γδ T Cells: A novel T cell subset with a potential role in inflammatory bowel disease. J Immunol, 2016; 197(12):4584–4592; doi: 10.4049/jimmunol.1601146
52.
TalayeroP, ManceboE, Calvo-PulidoJ, et al.Innate lymphoid cells groups 1 and 3 in the epithelial compartment of functional human intestinal allografts. Am J Transplant, 2016; 16(1):72–82; doi: 10.1111/ajt.13435
53.
Olivares-VillagómezD, Van KaerL. Intestinal intraepithelial lymphocytes: Sentinels of the mucosal barrier. Trends Immunol, 2018; 39(4):264–275; doi: 10.1016/j.it.2017.11.003
54.
JinG-Z, ParkJ-H, WallI, et al.Isolation and culture of primary rat adipose derived stem cells using porous biopolymer microcarriers. Tissue Eng Regen Med, 2016; 13(3):242–250; doi: 10.1007/s13770-016-0040-z
55.
LevatoR, PlanellJA, Mateos-TimonedaMA, et al.Role of ECM/peptide coatings on SDF-1α triggered mesenchymal stromal cell migration from microcarriers for cell therapy. Acta Biomater, 2015; 18:59–67; doi: 10.1016/j.actbio.2015.02.008
56.
ChoiG, ChaHJ. Recent advances in the development of nature-derived photocrosslinkable biomaterials for 3D printing in tissue engineering. Biomater Res, 2019; 23(1):18; doi: 10.1186/s40824-019-0168-8
57.
ParakA, PradeepP, Du ToitLC, et al.Functionalizing bioinks for 3D bioprinting applications. Drug Discov Today, 2019; 24(1):198–205; doi: 10.1016/j.drudis.2018.09.012
58.
MandalA, CleggJR, AnselmoAC, et al.Hydrogels in the clinic. Bioengineering & Transla Med, 2020; 5(2):e10158; doi: 10.1002/btm2.10158
59.
AgarwalS, WeidnerT, ThalheimerFB, et al.In vivo generated human CAR T cells eradicate tumor cells. OncoImmunology, 2019; 8(12):e1671761; doi: 10.1080/2162402X.2019.1671761
60.
AgarwallaP, OgunnaikeEA, AhnS, et al.Bioinstructive implantable scaffolds for rapid in vivo manufacture and release of CAR-T cells. Nat Biotechnol, 2022; 40(8):1250–1258; doi: 10.1038/s41587-022-01245-x
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
