Expansion, Function and Clonotypic Analysis of Human Alloreactive Treg Stimulated with Different Dendritic Cell Populations or CD40L-Stimulated B Cells.
1Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA
2Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA
3Department of Surgery, UCSF, San Francisco, CA
Meeting: 2017 American Transplant Congress
Abstract number: C276
Keywords: Antigen presentation, B cells, T cell clones, Tolerance
Session Information
Session Name: Poster Session C: Tolerance/Immune Regulation
Session Type: Poster Session
Date: Monday, May 1, 2017
Session Time: 6:00pm-7:00pm
Presentation Time: 6:00pm-7:00pm
Location: Hall D1
Purpose:
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.
Results:
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.
Conclusions:
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 May 17, 2025.« Back to 2017 American Transplant Congress