Background: Dendritic cell therapy is a promising approach to reduced dependence on immunosuppressive drugs and for the promotion of tolerance in transplant recipients. Our aim was to characterize for the first time miniature swine regulatory dendritic cells (DCreg) generated from either peripheral blood monocytes or bone marrow (BM) cells for prospective evaluation in a pre-clinical miniature vascular composite allograft (VCA) model.

Methods: Fresh blood or femoral BM was obtained from partially inbred, MHC-defined miniature swine that have been defined at the MHC genes encoding class I and class II antigens. Control immature DC (imDC) were generated from either CD14+ bead-isolated blood monocytes or BM cells over 7 days of culture in media supplemented with 10% FCS, porcine (p)GM-CSF and pIL-4. To generate DCreg, vitamin D3 (VitD3; 25nM) was added on day 0, and VitD3 and pIL-10 (60 ng/ml) added on day 4. To ascertain the responsiveness of the DCs to TLR4 stimulation, LPS was added to either the control DC (matDC) or the DCreg cultures (DCregLPS) on day 6. Cell surface phenotype was determined by staining the DC with fluorochrome-conjugated antibodies against SLA-DR, CD80/CD86, CD172a, CD1 and CD3 and by comparing mean fluorescence intensity (MFI) and percentage positive cells. The ability of the DCreg to stimulate allogeneic T cell proliferation was determined by carboxyfluorescein succinimidyl ester (CFSE)-mixed leukocyte reaction (MLR).

Results: Swine BM-derived DCreg expressed the myeloid cell antigen CD172a (SIRPa) and CD1, and low levels of MHC II and co-stimulatory molecules (SLA-DR, CD80/86) when compared to imDC and matDC. These DCreg were poor allogeneic T cell stimulators compared to matDC. After LPS challenge (DCregLPS), levels of T cell co-stimulatory molecules remained low and the DCreg remained poor stimulators of allogeneic T cells. Similar results were obtained generating DCreg from blood. However, adequate numbers of DCreg for prospective adoptive cell therapy could be generated more readily from BM of donor animals.

Conclusions: Swine DCreg that resist maturation in response to a potent inflammatory stimulus can be generated efficiently from BM to achieve adequate numbers for adoptive cell therapy.

CITATION INFORMATION: Elgendy T., Kim D., Zahorchak A., Ezzelarab M., Solari M., Thomson A. Comparative Analysis of Bone Marrow versus Blood Monocyte-Derived Regulatory Dendritic Cells for Evaluation in a Miniature Swine Vascular Composite Allotransplantation Model Am J Transplant. 2017;17 (suppl 3).

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