Technical Advance: Ascorbic Acid Induces Development of Double-Positive T Cells From Human Hematopoietic Stem Cells in the Absence of Stromal Cells

J Leukoc Biol. 2014 Dec;96(6):1165-75. doi: 10.1189/jlb.1TA0214-121RR. Epub 2014 Aug 25.

Abstract

The efficacy of donor HSCT is partly reduced as a result of slow post-transplantation immune recovery. In particular, T cell regeneration is generally delayed, resulting in high infection-related mortality in the first years post-transplantation. Adoptive transfer of in vitro-generated human T cell progenitors seems a promising approach to accelerate T cell recovery in immunocompromised patients. AA may enhance T cell proliferation and differentiation in a controlled, feeder-free environment containing Notch ligands and defined growth factors. Our experiments show a pivotal role for AA during human in vitro T cell development. The blocking of NOS diminished this effect, indicating a role for the citrulline/NO cycle. AA promotes the transition of proT1 to proT2 cells and of preT to DP T cells. Furthermore, the addition of AA to feeder cocultures resulted in development of DP and SP T cells, whereas without AA, a preT cell-stage arrest occurred. We conclude that neither DLL4-expressing feeder cells nor feeder cell conditioned media are required for generating DP T cells from CB and G-CSF-mobilized HSCs and that generation and proliferation of proT and DP T cells are greatly improved by AA. This technology could potentially be used to generate T cell progenitors for adoptive therapy.

Keywords: DLL4 protein; T cell precursor; adoptive cellular immunotherapy; transplantation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, Differentiation, T-Lymphocyte / analysis
  • Antigens, Differentiation, T-Lymphocyte / biosynthesis
  • Antioxidants / pharmacology
  • Ascorbic Acid / pharmacology*
  • CD4 Antigens / analysis*
  • CD8 Antigens / analysis*
  • Cell Division / drug effects
  • Cells, Cultured
  • Citrulline / metabolism
  • Coculture Techniques
  • Filgrastim
  • Gene Expression Profiling
  • Gene Rearrangement, T-Lymphocyte
  • Granulocyte Colony-Stimulating Factor / pharmacology
  • Hematopoietic Stem Cell Mobilization
  • Hematopoietic Stem Cells / cytology
  • Hematopoietic Stem Cells / drug effects*
  • Humans
  • Immunomagnetic Separation
  • Integrins / analysis
  • Lymphopoiesis / drug effects*
  • Nitric Oxide / metabolism
  • Primary Cell Culture / methods
  • Receptors, Chemokine / analysis
  • Recombinant Proteins / pharmacology
  • Stromal Cells
  • T-Lymphocyte Subsets / immunology*
  • omega-N-Methylarginine / pharmacology

Substances

  • Antigens, Differentiation, T-Lymphocyte
  • Antioxidants
  • CD4 Antigens
  • CD8 Antigens
  • Integrins
  • Receptors, Chemokine
  • Recombinant Proteins
  • Granulocyte Colony-Stimulating Factor
  • omega-N-Methylarginine
  • Citrulline
  • Nitric Oxide
  • Ascorbic Acid
  • Filgrastim