Human alloreactive-regulatory T cell profile after ex vivo expansion using CD40L-stimulated B cells or monocyte-derived dendritic cells

Abstract

Abstract Alloreactive regulatory T cells (arTregs) expanded with CD40L-stimulated B cells (sBcs) are being tested clinically to induce tolerance and control rejection in liver and kidney transplantation. In research settings, allogeneic monocyte-derived DCs (moDCs) have been shown to expand arTregs. We directly compared the efficacy of sBcs and cytokine-matured moDCs in expanding arTregs, and the phenotype of sBc-stimulated arTregs (sBc-arTregs) and moDC-stimulated arTregs (moDC-arTregs). arTregs expanded twice as much after stimulation with moDCs than with sBcs. Neither sBcs nor moDCs produced much TNFα, IL-1α, IL-1β, IL-6, IL-12P70, or IL-23 that can destabilize Tregs. sBc- and moDC-arTregs expressed similar percentages of Treg-defining markers - FOXP3, HELIOS, CD25, CD27, and CD62L. moDC-arTregs had slightly elevated FOXP3 and CD62L MFI. Compared to primary Tregs, sBc- and moDC-arTregs expressed similar or decreased levels of TBX21, GATA3, and RORC mRNA. However, greater percentages of moDC-arTregs produced IFNγ, IL-4, and IL-17A, and a greater percentage of sBc-arTregs produced IL-17A, but far less than those percentages produced by similarly stimulated conventional CD4+ T cells (Tconvs). The majority of cytokine-expressing arTregs were FOXP3+. Percentages of CXCR3 and CCR4, but not CCR6, increased after sBc and moDC stimulation. Using a 770-gene Nanostring panel and unsupervised clustering, sBc- and moDCs-arTregs were found to be mostly similar, but distinct from primary Tregs and further separated from Tconvs. Thus, arTregs expanded by sBcs or moDCs are phenotypically similar, likely stable, and had features that may enable them to traffic to inflammatory sites, e.g. transplanted organs undergoing alloimmune attack.