Program: Oral and Poster Abstracts
Type: Oral
Session: 201. Granulocytes, Monocytes and Macrophages I
Knowing the identity of the precursors of these DCs may facilitate the ex-vivo or in-vivo generation of DCs via the homeostatic pathway, potentially yielding DCs with optimal T cell priming ability. We (Xiao et al. Stem Cell Rep. 2015) and others (Lee et al. J. Exp. Med. 2015) have recently identified a population with DC progenitor potential in human bone marrow and cord blood, respectively. This population can be isolated on basis of a CD34+c-KIT+FLT3+IL3Rαhigh phenotype and is furthermore Lin-CD10-CD11b-CD45RA+CD38+. We have shown that this population is highly enriched for or identical to a common progenitor (P) of macrophages (M), osteoclasts (O) and DCs (D) and termed it MODP. We also identified the progenitor directly upstream from the MODP that still has granulocyte (G) differentiation potential and termed it GMODP.
We hypothesized that DCs generated from GMODP or MODP under homeostatic conditions would have superb T-cell priming capacity. To examine this, the progenitors were sorted by flow cytometry from human bone marrow or cord blood and cultured with Flt3 ligand, M-CSF and IL-3 to generate DCs. We also tested the effect of a mensenchymal stem cell (MSC) feeder layer. Within 2-3 weeks of culture, 1000 DC progenitors generated approximately 150,000-250,000 DCs. Co-culture with MSC increased DC output significantly, at least 2 fold. The progenitor-derived DCs could be discerned into CD141+conventional (c)DC, CD1c+cDC and CD303+plasmacytoid (p)DC. To study T-cell priming capacity of progenitor-derived DCs, we set up an in vitro DC-T co-culture assay. CD141+cDC, CD1c+cDC and CD303+pDC were generated from GMODP or MODP of HLA-A2+ donors, flow cytometrically purified, activated with lipopolysaccharide and loaded with MART-126-35 peptide that represents a melanoma-derived tumor antigen. Primary T cells from peripheral blood of unrelated donors were retrovirally transduced to express a T cell antigen receptor (TCR) ab specific for the HLA-A2/MART-126-35 peptide complex. The ability of the DCs to prime a T-cell response was read out by antigen-specific CD8+ T cell proliferation. All DC subsets were able to induce MART-1 specific T cell proliferation, with the CD1c+cDCs being most potent and the CD303+pDC being least potent.
In conclusion: We have established a culture method to derive DCs with T-cell priming ability from a newly identified DC progenitor. These results are of value for improvement of DC-based immunotherapy.
Disclosures: No relevant conflicts of interest to declare.
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