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 (TCR)Vβ 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.