Introduction

Antigen (Ag)-specific regulatory T cells (Tregs) have a great therapeutic clinical potential. However, large numbers are needed before Tregs can be used as cellular immunotherapy in kidney transplant patients. Therefore, optimal expansion protocols are needed to generate these cell numbers from peripheral blood isolated Tregs, but are presently lacking. In this study, mature monocyte-derived dendritic cells (moDC) were studied for their capacity to generate alloAg-specific Tregs and compared with PBMC-induced and polyclonal Treg expansion.

Methods

For this purpose highly pure Tregs (CD4⁺CD25^brightCD127^– T cells) and T cells depleted for Tregs (Teff=responder) were obtained using FACS sorting. The Tregs were expanded using ΑCD3/ΑCD28-coated microbeads (polyclonal) or an Ag-specific expansion protocol using HLA-mismatched PBMC or mature moDC as stimulator cells. The different expanded Tregs were stained for FOXP3 and their suppressive capacity was tested in a mixed lymphocyte reaction (MLR) using different ratios of Treg:Teff from 1:5 to 1:320. As a control, suppression was tested in a MLR using fully mismatched PBMC to both the responder as well as stimulator cells (third party).

Results

Mature allogeneic moDC were on average 40-times more potent in inducing Treg expansion when compared to PBMC. For example, at a ratio of 1:10 (DC:Tregs), a similar fold expansion was observed during a period of 11 days, compared to a ratio of 4:1 (PBMC:Tregs), i.e. 16±6 and 14±6, respectively. With a 1:1 DC to Tregs ratio, it was possible to get >100-fold expansion of Tregs. The presence of rapamycine did not change the Treg expansion potency of DC. Remarkably, the presence of exogenous IL-15 but not IL-2 was needed for optimal DC-induced Treg expansion. Stable high FOXP3 expression was observed in all expanded Tregs. Both allogeneic DC and PBMC expanded Tregs were potent suppressors. For example, at a Treg to Teff ratio of 1:320, the allogeneic proliferation was inhibited by 60-70%. The proliferation to third party-stimulators was not inhibited at this ratio of Tregs. Polyclonal-expanded Tregs were less potent suppressors when compared to Ag-specific expanded Tregs and no suppressive activity was observed below a ratio of 1:320.

Conclusions

Mature allogeneic DC are highly efficient cells for expansion of potent alloAg specific Tregs. This observation opens a new avenue for generation of Tregs on a large scale for immunotherapy.

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