Generation of Feline Dendritic Cells Derived from Peripheral Blood Monocytes for In Vivo Use

Abstract

Dendritic cells (DCs) are professional antigen-presenting cells that can prime T cells and polarize the cellular immune response. Because Th1-type immune responses have been connected to success in combating viral infection, a promising therapeutic application of DCs would be their differentiation in vitro and injection back into the host to boost an immune response in infected animals. This study was aimed both at developing a protocol to cultivate feline DCs in the absence of exogenous proteins for their use in vivo and at investigating what might be the most appropriate stimulus to induce their maturation in vitro and finding correlates of maturation. We generated DCs from peripheral blood monocytes in the presence of feline interleukin-4 and granulocyte-macrophage colony stimulating factor, and after 5 days their maturation was induced with either lipopolysaccharide, human recombinant tumor necrosis factor alpha, poly(I:C), or activated feline platelets. After 48 h, their CD14, CD1a, major histocompatibility complex class II, and B7.1 surface expression was analyzed in parallel with their ability to uptake antigen or prime a mixed leukocyte reaction. The results presented show that feline DCs cultured in autologous plasma differentiate and are able to mature in the presence of stimuli similar to the ones currently used for other species. The present work sets the grounds for future use of DCs obtained by the protocol described for in vivo vaccination and immunotherapy of feline immunodeficiency virus-infected cats.

Evaluation of Feline Monocyte-Derived Dendritic Cells Loaded with Internally Inactivated Virus as a Vaccine against Feline Immunodeficiency Virus

“…We have previously described the generation of feline MDCs in conditions established to avoid any contact of the cells with foreign protein, implying the use of autologous plasma and feline cytokines. We have also shown that the only maturation stimulus tested leading to full feline MDC maturation in the cat was LPS and that the most suitable surface markers to follow maturation were MHC-II and B7.1 expression levels, while the best functional assay was considered to be MLR (2,3).…”

“…We then used the same virus isolate to challenge vaccinated cats. Although the feline DC maturation stage is not as straightforward to define as that for human DCs, where well-defined markers allow us to distinguish between mature and immature cells, correlates of feline MDC maturation, such as MHC-II and B7.1 expression levels and the ability to induce MLR, have been described previously (3). In the present study, after MDCs were loaded with antigen, the maturation stimulus used in vitro was LPS for all MDCs, because previous work has shown LPS is the only stimulus known to induce the ability of MDCs to start MLR in the feline model.…”

“…PBMCs were obtained by gradient centrifugation over a Ficoll-Paque layer for 30 min at 550 ϫ g. MDCs were generated from a maximum of 35 ml of blood at any one time, as described previously (3). Cells isolated were washed in apyrogenic saline, counted, and resuspended in medium at 3 ϫ 10 6 cells/ml.…”

Characterization of Monocyte-Derived Dendritic Cells from Cats Infected with Feline Immunodeficiency Virus

“…All cats were shown to be positive for FIV antibody and negative for feline leukemia virus Cell preparation: The methods for the preparation of DCs and macrophages were modified based on previously described protocols [4,13,27]. Briefly, heparinized whole blood was layered onto Ficoll/Hypaque (Lymphoprep; Nycomed Pharma AS, Oslo, Norway) and centrifuged at 560 g for 45 min at room temperature.…”

“…Monocytes incubated with granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) acquire the properties of DCs. Moreover, several studies have been performed to establish a method for generating feline DCs [4,13,27]. Similar to human and murine DCs, feline DCs have been generated from monocytes cultured with GM-CSF and IL-4, and they also possessed the characteristic properties of DCs.…”