Novel Murine Dendritic Cell Lines: A Powerful Auxiliary Tool for Dendritic Cell Research

Abstract

Research in vitro facilitates discovery, screening, and pilot experiments, often preceding research in vivo. Several technical difficulties render Dendritic Cell (DC) research particularly challenging, including the low frequency of DC in vivo, thorough isolation requirements, and the vulnerability of DC ex vivo. Critically, there is not as yet a widely accepted human or murine DC line and in vitro systems of DC research are limited. In this study, we report the generation of new murine DC lines, named MutuDC, originating from cultures of splenic CD8α conventional DC (cDC) tumors. By direct comparison to normal WT splenic cDC subsets, we describe the phenotypic and functional features of the MutuDC lines and show that they have retained all the major features of their natural counterpart in vivo, the splenic CD8α cDC. These features include expression of surface markers Clec9A, DEC205, and CD24, positive response to TLR3 and TLR9 but not TLR7 stimuli, secretion of cytokines, and chemokines upon activation, as well as cross-presentation capacity. In addition to the close resemblance to normal splenic CD8α cDC, a major advantage is the ease of derivation and maintenance of the MutuDC lines, using standard culture medium and conditions, importantly without adding supplementary growth factors or maturation-inducing stimuli to the medium. Furthermore, genetically modified MutuDC lines have been successfully obtained either by lentiviral transduction or by culture of DC tumors originating from genetically modified mice. In view of the current lack of stable and functional DC lines, these novel murine DC lines have the potential to serve as an important auxiliary tool for DC research.

TLR3-Mediated CD8+ Dendritic Cell Activation Is Coupled with Establishment of a Cell-Intrinsic Antiviral State

“…We previously reported that our mouse CD8 + DC lines retained all major phenotypic and functional features of their in vivo counterparts, including characteristic cell-surface marker expression, responsiveness to TLR stimuli, patterns of cytokine and chemokine production, cross-presentation capacity, and T cell-stimulatory properties (33). This was further confirmed by studying the expression of 257 genes identified as cDCs, pDCs, CD8…”

“…Wild-type (WT) (MuTu1940), Tlr3 2/2 , and Ifnar1 2/2 CD8 + DC lines were established and cultured as described previously (33,34). Splenic CD8 + DCs were isolated from Flt3L-transgenic mice (35) or WT C57BL/6J mice using CD8 + DC purification kit (130-091-169; Miltenyi Biotec).…”

β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

“…4H). Furthermore, ICG- 001 treatment of MutuDC1940 cells, a DC-derived cell line that has many characteristics of CD8α + DC(30), also resulted in strong downregulation of IRF8 (Fig. 4I).…”

Combinational Immunotherapy with Allo-DRibble Vaccines and Anti-OX40 Co-Stimulation Leads to Generation of Cross-Reactive Effector T Cells and Tumor Regression

“…The mutu-1940 DC cell line was used as the APC and alumina nanoparticles were used to facilitate intracellular delivery of isolated SLiPs31. Mutu-1940 DC was an established cell line from CD1c-SV40 large T antigen transgenic C57BL/6 mice32 and it was shown to closely resemble endogenous CD8 + conventional DCs (cDC). When CFSE labeled naïve OT-I T cells were stimulated by SLiPs-loaded mutu-1940 DCs, OVA-specific T-cell proliferation was observed when SLiPs was purified from B78H1-OVA but not B78H1-GFP tumor cells.…”

“…The B78H1 cell line is a MHC-I-deficient subclone of B16 melanoma; B78H1-D b , B78H1-K b , B78H1-D b K b stable cell lines were transfected with plasmids expressing MHC-Ia molecules, these B78H1 cell lines and CT26 were kindly provided by Hyam I. Levitsky (Johns Hopkins University, Baltimore, MD); B78H1-R-OVA-GFP and B78H1-GFP stable cell lines were transfected with plasmids expressing short-lived GFP-R-OVA fusion protein or GFP alone16. Mutu-1940 DCs from C57BL/6 mice were kindly provided by Dr. Hans Acha-Orbea (University of Lausanne, Epalinges, Switzerland)32. Listeria mono cytogenes that express the endogenous 4T1 tumor antigen AH1 (Delta-actA-Lm-AH1-A5) were kindly provided by Dr. Keith Bahjat (Earle A. Chiles Research Institute, Portland, OR).…”