Session Time: 6:00pm-7:00pm
Presentation Time: 6:00pm-7:00pm
Location: Hall D1
In order to establish a protocol to generate large numbers of human alloantigen-reactive regulatory T cells (arTreg) with potent allospecific suppressive capacity for therapeutic application, we compared immature monocyte-derived dendritic cells (mono-DC), LPS-matured DC (LPS-DC), cytokine-treated DC (cyto-DC), CD40L-treated DC (CD40L-DC) and CD40L-expanded B (CD40L-sBc) for their ability to expand arTreg.
Materials & Methods:
To generate mono-DC, peripheral blood immunobead-selected CD14+ cells were cultured with GM-CSF and IL-4 for 7 days. LPS, cytokine cocktail (TNFα, IL-2β and PGE2) or CD40L was added for the last day of culture to generated LPS-DC, cyto-DC and CD40L-DC, respectively. To generate CD40L-sBc, CD20+ cells were cultured and expanded with irradiated 3T3-CD40L cells for 10 days in the presence of IL-4. Each batch of different APCs was generated from the same donor, irradiated and co-cultured with flow-sorted peripheral blood CD4+CD25+CD127– Treg and IL-2 for 12 days. To test their function, the expanded arTreg were co-cultured with autologous Teff from the same donor at different ratios under stimulation with allo-sBc. To ascertain and compare the clonality of arTreg expanded by the different types of allo-APCs, aliquots of sBc-, mono-DC and LPS-DC- expanded Treg were subjected to T cell receptor clonotypic analysis.
Matured DC expressed higher levels of co-stimulatory molecules (CD80 and CD86) than immature DC and CD40L-sBc. Treg cultured with matured DC were expanded ~65 fold, while those cultured with immature DC or CD40L-sBc were expanded 10- to 30- fold. Expression of Foxp3 was comparable between Treg expanded by sBc and the various DCs. In addition, Treg expanded by the different APC were comparable superior suppressors of effector T cell proliferation. TCR repertoire analysis of the expanded arTreg showed that over 80% of the clonotypes were unique to each stimulatory APC type.
Matured allogeneic human mono-DC are superior to immature DC and CD40L-sBc in their ability to expand functionally suppressive Foxp3+ Treg. Unique repertoires of human arTreg are expanded using different APC, which may have implications for their in vivo efficacy.
CITATION INFORMATION: Zhang H, Zhao Y, Tang Q, Thomson A. Expansion, Function and Clonotypic Analysis of Human Alloreactive Treg Stimulated with Different Dendritic Cell Populations or CD40L-Stimulated B Cells. Am J Transplant. 2017;17 (suppl 3).
To cite this abstract in AMA style:Zhang H, Zhao Y, Tang Q, Thomson A. Expansion, Function and Clonotypic Analysis of Human Alloreactive Treg Stimulated with Different Dendritic Cell Populations or CD40L-Stimulated B Cells. [abstract]. Am J Transplant. 2017; 17 (suppl 3). https://atcmeetingabstracts.com/abstract/expansion-function-and-clonotypic-analysis-of-human-alloreactive-treg-stimulated-with-different-dendritic-cell-populations-or-cd40l-stimulated-b-cells/. Accessed August 14, 2020.
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