Transfer of antigen presenting cells in vivo is a method used by immunologists to examine the potency of antigen presentation by a selected population of cells. This method is most commonly used to analyze presentation of protein antigens to MHC class I or II restricted T cells, but it can also be used for studies of nonconventional antigens such as CD1-presented lipids. In a recent study focusing on CD1d-restricted glycolipid antigen presentation to Natural Killer T cells, we compared antigen presenting properties of splenic B cells, CD8αPos dendritc cells (DCs) and CD8αNeg DCs (Arora et al., 2014). This protocol describes the detailed method used for isolation of these cell populations, and their transfer into recipient mice to analyze their antigen presenting properties.
As a percentage of total mononuclear cells, an average spleen contains approximately 1-3% myeloid dendritic cells (DCs). In absolute numbers, this translates to approximately 0.6-1.8 x 106 DCs. To enhance the number of DCs in spleen, mice were injected subcutaneously with cells from a cultured melanoma cell line (B16.Flt3L) which has been engineered to express the fms-related tyrosine kinase 3 ligand (Flt3L) (Mach et al., 2000). This protein is a growth factor homologous to colony stimulating factor-1 and plays a critical role in the differentiation of hematopoietic stem cells. Administration of this protein into mice as a purified protein results in the expansion of both CD8αPos and CD8αNeg DC subsets in multiple organs. Similar expansion is also seen in mice that have been implanted with tumor cells overexpressing this protein (Mach et al., 2000). In our experience, up to 60% of the total mononuclear cells in a spleen from a mouse with a palpable B16.Flt3L tumor can be CD11c positive dendritic cells, thereby giving a total yield of up to 5 x 107 DCs per mouse. A schematic illustrating the cell enrichment protocol is included in Figure 1, and representative data on purity of cell populations obtained with this protocol is shown in Figure 2.
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