Kenneth Brasel

Murine Plasmacytoid Pre-Dendritic Cells Generated from Flt3 Ligand-Supplemented Bone Marrow Cultures Are Immature APCs

The putative counterparts of human plasmacytoid pre-dendritic cells (pDCs) have been described in vivo in mouse models and very recently in an in vitro culture system. In this study, we report that large numbers of bone marrow-derived murine CD11c+B220+ pDCs can be generated with Flt3 ligand (FL) as the sole exogenous differentiation/growth factor and that pDC generation is regulated in vivo by FL because FL-deficient mice showed a major reduction in splenic pDC numbers. We extensively analyzed bone marrow-derived CD11c+B220+ pDCs and described their immature APC phenotype based on MHC class II, activation markers, and chemokine receptor level of expression. CD11c+B220+ pDCs showed a nonoverlapping Toll-like receptor pattern of expression distinct from that of classical CD11c+B220− dendritic cells and were poor T cell stimulators. Stimulation of CD11c+B220+ pDCs with oligodeoxynucleotides containing certain CpG motifs plus CD40 ligand plus GM-CSF led to increased MHC class II, CD80, CD86, and CD8α expression levels, to a switch in chemokine receptor expression that affected their migration, to IFN-α and IL-12 secretion, and to the acquisition of priming capacities for both CD4+ and CD8+ OVA-specific TCR-transgenic naive T cells. Thus, the in vitro generation of murine pDCs may serve as a useful tool to further investigate pDC biology as well as the potential role of these cells in viral immunity and other settings.

Polyethylene Glycol-Modified GM-CSF Expands CD11b high CD11c high But Not CD11b low CD11c high Murine Dendritic Cells In Vivo: A Comparative Analysis with Flt3 Ligand

Dendritic cells (DC) are potent APCs that can be characterized in the murine spleen as CD11bhighCD11chigh or CD11blowCD11chigh. Daily injection of mice of Flt3 ligand (FL) into mice transiently expands both subsets of DC in vivo, but the effect of administration of GM-CSF on the expansion of DC in vivo is not well defined. To gain further insight into the role of GM-CSF in DC development and function in vivo, we treated mice with polyethylene glycol-modified GM-CSF (pGM-CSF) which has an increased half-life in vivo. Administration of pGM-CSF to mice for 5 days led to a 5- to 10-fold expansion of CD11bhighCD11chigh but not CD11blowCD11chigh DC. DC from pGM-CSF-treated mice captured and processed Ag more efficiently than DC from FL-treated mice. Although both FL- and pGM-CSF-generated CD11bhighCD11chigh DC were CD8α−, a greater proportion of these DC from pGM-CSF-treated mice were 33D1+ than from FL-treated mice. CD11blowCD11chigh DC from FL-treated mice expressed high levels of intracellular MHC class II. DC from both pGM-CSF- and FL-treated mice expressed high levels of surface class II, low levels of the costimulatory molecules CD40, CD80, and CD86 and were equally efficient at stimulating allogeneic and Ag-specific T cell proliferation in vitro. The data demonstrate that treatment with pGM-CSF in vivo preferentially expands CD11bhighCD11chigh DC that share phenotypic and functional characteristics with FL-generated CD11bhighCD11chigh DC but can be distinguished from FL-generated DC on the basis of Ag capture and surface expression of 33D1.

Paradoxical role of programmed death-1 ligand 2 in Th2 immune responses in vitro and in a mouse asthma model in vivo

Programmed death‐1 ligand 2 (PD‐L2) is a ligand for programmed death‐1 (PD‐1), a receptor that plays an inhibitory role in T cell activation. Since previous studies have shown up‐regulation of PD‐L2 expression by Th2 cytokines, and asthma is driven by a Th2 response, we hypothesized that PD‐L2 might be involved in regulation of the immune response in this disease. We have found that lungs from asthmatic mice had sustained up‐regulation of PD‐1 and PD‐L2, with PD‐L2 primarily on dendritic cells. Although addition of PD‐L2‐Fc in vitro led to decreased T cell proliferation and cytokine production, administration of PD‐L2‐Fc in vivo in a mouse asthma model resulted in elevated serum IgE levels, increased eosinophilic and lymphocytic infiltration into bronchoalveolar lavage fluid, higher number of cells in the draining lymph nodes, and production of IL‐5 and IL‐13 from these cells. Although PD‐1 was expressed on regulatory T cells, PD‐L2‐Fc did not affect regulatory T cell activity in vitro. This study provides in vivo evidence of an exacerbated inflammatory response following PD‐L2‐Fc administration and indicates a potential role for this molecule in Th2‐mediated diseases such as asthma.

Structure-Function Analysis of FLT3 Ligand-FLT3 Receptor Interactions Using a Rapid Functional Screen

FLT3 ligand (FLT3L) stimulates primitive hematopoietic cells by binding to and activating the FLT3 receptor (FLT3R). We carried out a structure-activity study of human FLT3L in order to define the residues involved in receptor binding. We developed a rapid method to screen randomly mutagenized FLT3L using a FLT3R-Fc fusion protein to probe the relative binding activities of mutated ligand. Approximately 60,000 potential mutants were screened, and the DNA from 59 clones was sequenced. Thirty-one single amino acid substitutions at 24 positions of FLT3L either enhanced or reduced activity in receptor binding and cell proliferation assays. Eleven representative proteins were purified and analyzed for receptor affinity, specific activity, and physical properties. Receptor affinity and bioactivity were highly correlated. FLT3L affinity for receptor improved when four individual mutations that enhance FLT3L receptor affinity were combined in a single molecule. A model of FLT3L three-dimensional structure was generated based on sequence alignment and x-ray structure of macrophage colony-stimulating factor. Most residues implicated in receptor binding are widely dispersed in the primary structure of FLT3L, yet they localize to a surface patch in the tertiary model. A mutation that maps to and is predicted to disrupt the proposed dimerization interface between FLT3L monomers exhibits a Stokes radius that is concentration-dependent, suggesting that this mutation disrupts the FLT3L dimer.