Large Scale Expansion of Human Dendritic Cells with Regulatory Function from Peripheral Blood Monocytes Employing Leukapheresis and Rapamycin for Clinical Trials.

Regulatory T cells (Treg) exert pivotal roles on the induction and maintenance of peripheral tolerance. Adoptive transfer of Treg is a promising strategy to control acute GVHD. However, in vitro expansion of Treg is costly, laborious and time-consuming (more than 3 weeks). As an alternative source, dendritic cells (DC) with regulatory function (DCreg) seem to be attractive, since DC are potent antigen presenting cells capable of regulating the initiation of various immune responses and can be generated in a relatively short term culture from peripheral blood (PB) monocytes. In the present study, we examined the feasibility of generating DCreg on a large scale from PB monocytes obtained by leukapheresis. PB mononuclear cells (range 1.8×10⁹ to 3.1×10⁹) were obtained from 5 healthy volunteers with informed consent by leukapheresis for 1–2 hours using a COBE Spectra apparatus. Immature DC were generated from isolated CD14⁺ monocytes by culturing with GM-CSF and IL4 with or without various concentrations of rapamycin for 5–7days. For maturation, immature DC were cultured with the addition of TNFα for 2 days. The yield of immature DC cultured with rapamycin (10ng/ml) was 8.4±3.6×10⁷, constituting 4.2±1.8% of the initial PB mononuclear cells. FACS analysis showed that DC cultured with rapamycin (DC-rapa) had immature DC phenotype with CD14⁻, CD1a⁺, CD83⁻. But the surface expression of CD86, CD40, HLA-DR was much lower compared with control DC (DC-ctr) cultured without rapamycin. The reduction of the expression of CD86, CD40, HLA-DR was dependent of rapamycin dose and resistant to maturational stimulation of TNFα. DC-rapa showed very weak allostimulatory activity in a mixed lymphocyte culture (MLC) compared with DC-ctr, corresponding to the reduced expression of HLA-DR and costimulatory molecules. To further determine the types of cells involved, CSFE-labelled non-isolated CD4⁺ T cells were cultured with DC-rapa or DC-ctr for 7 days and the proliferating cells were identified by FACS analysis. DC-rapa inhibited the proliferation of CD4⁺CD25⁻ alloreactive T cells by approximately 60% while the inhibitory effect of DC-rapa on CD4⁺ CD25⁺ cells was minimal. Neither the irradiation of DC nor the addition of IL2 altered the inhibition. Stimulation with both anti-CD3 and ani-CD28 moonoclonal antibodies coated on microbeads reversed the inhibition completely, suggesting the importance of the signals via T cell receptors and costimulatory molecules in the inhibition by DC-rapa. Double staining with anti-CD25 and anti-Foxp3 monoclonal antibodies revealed that DC-rapa increased the percentage and number of CD25⁺Foxp3⁺ Treg by approximately 2.6 times compared with DC-ctr during 7-day culture. In conclusion, these results demonstrated that immature DC induced with rapamycin have a potent immunoregulatory function including preferential expansion of Treg. To the best of our knowledge, this is the first demonstration that immunoregulatory DC can be generated on a large scale in relatively short term culture with rapamycin following leukapheresis. These findings have an important clinical implication to facilitate DC-based immunotherapy for acute GVHD.