Regulatory dendritic cells (DCregs) have shown considerable potential for safe and effective prolongation of allograft survival in pre-clinical models. In humans, DCregs with purity >84% and with <0.3% contaminating T cells can be generated readily from precursor monocytes isolated from apheresis products using CD14-specific immunobeads or elutriation. In preparation for testing of DCregs in clinical organ transplantation, we undertook both small and large-scale (GMP) manufacturing of DCregs generated in rhGM-CSF and rhIL-4 plus vitamin D3 and rhIL-10 during 7-day culture. DCregs cultured from either source exhibited similar low levels of CD80 and CD86, but comparatively high levels of co-inhibitory programed death ligand-1 (PDL-1) resulting in high PD-L1:CD86 MFI ratios (immunobead DCreg: 28±11, elutriated mono DCreg: 38±32) compared to immature DC (iDC) cultured in GM-CSF+IL-4 alone (immunobead iDC: 2.8±1.4, elutriated mono DCreg: 7.1±7). The DCregs resisted phenotypic maturation but unlike control iDC, further upregulated the PD-L1:CD86 MFI ratio in response to LPS stimulation (immunobead DCreg: 40±25, elutriated mono DCreg: 56±50). Whereas control iDC secreted high levels of pro-inflammatory IL-12p70 and TNFα and no IL-10 in response to LPS, the converse was observed for LPS-stimulated DCreg. These DCregs were poor stimulators of naïve and memory allogeneic CD4⁺ and CD8⁺ T cell proliferation, IFNγ, IL-17, or perforin/granzyme B production in CFSE-MLR. However, their T cell stimulatory function was partially restored by addition of PD-1 blocking mAb. Using high-throughput TCR sequencing we determined that naïve T cells displayed highly diverse allogeneic TCR repertoires after DCreg stimulation, unlike memory T cells (with CD8>CD4) which showed less diverse repertoires and more alloreactive TCR oligoclones, that were further expanded by mature DC but to a lesser degree by DCreg.

These demonstrate the feasibility of generating highly-purified human DCregs for clinical application, and reveal their potential for regulation of alloreactive T cell repertoire and function.

CITATION INFORMATION: Zahorchak A, Butterfield L, Macedo C, Hamm D, Metes D, Thomson A. High PD-L1/CD86 MFI Ratio and IL-10 Secretion Characterize Maturation-Resistant Human Regulatory Dendritic Cells (DCregs) Generated for Clinical Testing in Transplantation. Am J Transplant. 2016;16 (suppl 3).

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