Efficient Generation of Plasmacytoid Dendritic Cell from Common Lymphoid Progenitors by Flt3 Ligand

Abstract

Dendritic cells (DCs), including conventional DCs (cDCs) and plasmacytoid DCs (pDCs) are critical for initiating and controlling the immune response. However, study of DC, particularly pDC, function is hampered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation preferentially favor the production of cDCs over pDCs. Here, we demonstrated that pDCs could be efficiently generated in vitro from common lymphoid progenitors (CLPs) using Flt3 ligand (FL) in three different culture systems, namely feeder-free, BM-feeder and AC-6-feeder. This was in stark contrast to common DC progenitors (CDPs), in which cDCs were prominently generated under the same conditions. Moreover, the efficiency and function of pDCs generated from these three systems varied. While AC-6 system showed the greatest ability to support pDC development from CLPs, BM-feeder system was able to develop pDCs with better functionality. pDCs could also be expanded in vivo using hydrodynamic gene transfer of FL, which was further enhanced by the combined treatment of FL and IFN-α. Interestingly, IFN-α selectively promoted the proliferation of CLPs and not CDPs, which might contribute to enhanced pDC development. Together, we have defined conditions for in vitro and in vivo generation of pDCs, which may be useful for investigating the biology of pDCs.

<em>In Vitro</em> Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

“…Additionally, the concentration of FL also affects DC development. Specifically, at low doses (25-50 ng/ml) cDCs are preferentially developed, while at high doses (100-200 ng/ml) pDCs are predominantly generated 24 . In addition to AC-6 cell density and FL dosage, careful selection of a batch of FBS that efficiently supports DC development in this culture system is central to the success of the procedure.…”

“…The uniqueness of this culture system is due to the use of AC-6 cells, a stromal cell line, as feeders. This approach has been shown to provide not only the cytokines, such as IL-7, SCF, M-CSF and FL 4 /well in 12-well plate) tends to support cDC development 24 . Therefore, it is recommended to first seed several concentrations of AC-6 cells in order to optimize conditions when scaling up the culture system 23 .…”

“…Moreover, they express relatively high levels of Tcf4 (encodes E2-2) and Rag1, two pDC-specific genes, but lower levels of Id2, a cDC-specific gene, than do cDCs 17 . Even though these pDCs lack Siglec-H and BST2, markers considered characteristic of murine pDCs, they are still able to produce higher levels of IFN-I than do cDCs and upregulate CD86 when infected with vesicular stomatitis virus (VSV) 24 . This suggests that these pDCs, although incompletely differentiated, are still functional.…”

Immunomodulatory Arming Factors—The Current Paradigm for Oncolytic Vectors Relies on Immune Stimulating Molecules

“…Another arming gene that recruits myeloid lineages is the fms-related tyrosine kinase 3 ligand (FLT3L). FLT3L is particularly useful for developing plasmacytoid dendritic cells, as well as the differentiation of hematopoietic cells in bone marrow [54]. The presence of plasmacytoid dendritic cells promotes a pro-inflammatory milieu which activates effector immune populations leading to viremic control [55], which could translate to tumor response via a bystander effect.…”