Generation of Functional, Antigen-Specific CD8+ Human T Cells from Cord Blood Stem Cells Using Exogenous Notch and Tetramer-TCR Signaling

Abstract

In vitro differentiation of mouse and human stem cells into early T cells has been successfully demonstrated using artificial Notch signaling systems. However, generation of mature, antigen-specific, functional T cells, directly from human stem cells has remained elusive, except when using stromal co-culture of stem cells retrovirally transfected with antigen-specific T cell receptors (TCRs). Here we show that human umbilical cord blood (UCB)-derived CD34+CD38−/low hematopoietic stem cells (HSCs) can be successfully differentiated into functional, antigen-specific cytotoxic CD8+ T cells without direct stromal co-culture or retroviral TCR transfection. Surface-immobilized Notch ligands (DLL1) and stromal cell conditioned medium successfully induced the development of CD1a+CD7+ and CD4+CD8+ early T cells. These cells, upon continued culture with cytomegalovirus (CMV) or Influenza-A virus epitope-loaded HLA-A*0201 tetramers, resulted in the generation of a polyclonal population of CMV-specific or Influenza-specific CD8+ T cells respectively. Upon further activation with antigen-loaded target cells, these antigen-specific, stem cell-derived T cells exhibited cytolytic functionality, specifically CD107a surface mobilization, IFNγ production, and Granzyme B secretion. Such scalable, in vitro generation of functional, antigen-specific human T cells from human stem cells could eventually provide a readily available cell source for adoptive transfer immunotherapies and also allow better understanding of human T cell development.

Engineering cord blood to improve engraftment after cord blood transplant

“…− /low UCBderived HSCs can be differentiated to early T cells using surface-immobilized Notch ligands, which when stimulated with HLA-A*0201 tetramers for CMV and Influenza-A produced functional and viral specific CD8 + T cells (98).…”

Technical Advance: Ascorbic acid induces development of double-positive T cells from human hematopoietic stem cells in the absence of stromal cells

“…However, more signals seem to be necessary for proper TCR␣␤ up-regulation and the development of SP T cells in the feeder-free culture, for example, the presence of MHC molecules. MHC-tetramers, anti-CD28, anti-CD3, and OP-9/ DL1 conditioned medium have been required for the generation of antigen-specific cytotoxic CD8 ϩ cells in the feeder-free system [42]. This suggests that our current system could possibly be used for the generation of antigen-specific T cells.…”

Biomaterial Strategies for Immunomodulation

“…However, these approaches are not viable in patients for whom autologous T cells are not available (such as patients with lymphoma or those who are immune-deficient). For such patients, robust and reproducible in vitro generation of functional, transplantable T cells from embryonic stem or adult stem cells will be necessary (176). With this in mind, Roy and coworkers (140) functionalized magnetic microbeads with the notch ligand DLL4 using streptavidin--biotin binding and antibody--antigen coupling.…”

“…Thy1.2 + early T cells were successfully generated from mouse bone marrow hematopoietic stem cells using DLL4 functionalized beads. In a similar approach, stem cells were differentiated into antigen-specific CD8 + human T cells using DLL1-coated plates and cytomegalovirus or human leukocyte antigen (HLA) A*201 tetramers loaded with influenza A virus epitopes (176). The resultant T cell population was a polyclonal population that was specific for cytomegalovirus or influenza; additionally, these cells were found to exhibit cytolytic functionality against infected cells and to have high IFN-γ production and Granzyme B secretion (176).…”