Quantitative proteomics reveals subset-specific viral recognition in dendritic cells.

Dendritic cell (DC) populations consist of multiple subsets that are essential orchestrators of the immune system. Technological limitations have so far prevented systems-wide accurate proteome comparison of rare cell populations in vivo. Here, we used high-resolution mass spectrometry-based proteomics, combined with label-free quantitation algorithms, to determine the proteome of mouse splenic conventional and plasmacytoid DC subsets to a depth of 5,780 and 6,664 proteins, respectively. We found mutually exclusive expression of pattern recognition pathways not previously known to be different among conventional DC subsets. Our experiments assigned key viral recognition functions to be exclusively expressed in CD4(+) and double-negative DCs. The CD8alpha(+) DCs largely lack the receptors required to sense certain viruses in the cytoplasm. By avoiding activation via cytoplasmic receptors, including retinoic acid-inducible gene I, CD8alpha(+) DCs likely gain a window of opportunity to process and present viral antigens before activation-induced shutdown of antigen presentation pathways occurs.

FLT3-ligand treatment of humanized mice results in the generation of large numbers of CD141+ and CD1c+ dendritic cells in vivo.

We established a humanized mouse model incorporating FLT3-ligand (FLT3-L) administration after hematopoietic cell reconstitution to investigate expansion, phenotype, and function of human dendritic cells (DC). FLT3-L increased numbers of human CD141(+) DC, CD1c(+) DC, and, to a lesser extent, plasmacytoid DC (pDC) in the blood, spleen, and bone marrow of humanized mice. CD1c(+) DC and CD141(+) DC subsets were expanded to a similar degree in blood and spleen, with a bias toward expansion of the CD1c(+) DC subset in the bone marrow. Importantly, the human DC subsets generated after FLT3-L treatment of humanized mice are phenotypically and functionally similar to their human blood counterparts. CD141(+) DC in humanized mice express C-type lectin-like receptor 9A, XCR1, CADM1, and TLR3 but lack TLR4 and TLR9. They are major producers of IFN-λ in response to polyinosinic-polycytidylic acid but are similar to CD1c(+) DC in their capacity to produce IL-12p70. Although all DC subsets in humanized mice are efficient at presenting peptide to CD8(+) T cells, CD141(+) DC are superior in their capacity to cross-present protein Ag to CD8(+) T cells following activation with polyinosinic-polycytidylic acid. CD141(+) DC can be targeted in vivo following injection of Abs against human DEC-205 or C-type lectin-like receptor 9A. This model provides a feasible and practical approach to dissect the function of human CD141(+) and CD1c(+) DC and evaluate adjuvants and DC-targeting strategies in vivo.

Nonplasmacytoid, high IFN-α-producing, bone marrow dendritic cells.

Plasmacytoid dendritic cells (pDC) are the producers of type I IFNs in response to TLR9 ligands. However, we have found that when bone marrow is depleted of pDC, the IFN-α produced in response to TLR9 ligands is not fully removed. We assign the source of this non-pDC IFN-α as a newly described DC type. It displays the high IFN-α producing activity of pDC but to a more limited range of viruses. Unlike pDC, the novel DC display high T cell stimulation capacity. Moreover, unlike mouse pDC, they are matured with GM-CSF and are less prone to apoptosis upon activation stimuli, including viruses. We propose that these DC constitute a novel bone marrow inflammatory DC type, ideally geared to linking innate and adaptive immune responses in bone marrow via their potent IFN-α production and high T cell stimulatory capacity.

Monitoring Dendritic Cell Activation and Maturation.

Since the 1997 discovery that the first identified human homolog of Drosophila Toll could activate the innate immune system, the innate arm of immunity has rapidly taken on a new light as an important player in the recognition of pathogens and damaged self. The recognition of danger by dendritic cells (DC) is a crucial step in activating the adaptive immune system. Different DC express varied subsets of pattern recognition receptors (PRR), enabling both overlap and exclusivity in the recognition of danger signals by DC. PRR-mediated DC maturation and activation can be measured by changes in the surface expression of costimulatory as well as coinhibitory molecules, changes in size and shape of the DC and by their production of different cytokines.

Monitoring dendritic cell activation and maturation.

Since the 1997 discovery that the first identified human homologue of Drosophila Toll could activate the innate immune system, the innate arm of immunity has rapidly taken on a new light as an important player in the recognition of pathogens and damaged self. The recognition of danger by dendritic cells (DC) is a crucial step in activating the adaptive immune system. Different DC express varied subsets of pattern recognition receptors (PRR), enabling both overlap and exclusivity in the recognition of danger signals by DC. PRR-mediated DC maturation and activation can be measured by changes in the surface expression of costimulatory molecules and changes in size and shape of the DC and by their production of different cytokines.

The generation of plasmacytoid and conventional dendritic cells with M-CSF.

Mice lacking the ligand for Flt-3 (CD135) have a massive deficit of dendritic cells (DC) in all organs. This phenotype of FL (FL) knockout mice suggested that FL was the archetypal DC poietin in the steady state. However, FL knockout mice also have reduced numbers of common lymphoid progenitors (CLP) and common myeloid progenitors (CMP) so it is possible that FL deficiency may limit the ability of other growth factors to drive DC development by limiting the pool of progenitor cells available. We found that DC development could be driven from BM cells of FL knockout mice using the myeloid growth factor M-CSF. The M-CSF-driven DC (MDC) developed independently of FL and resembled the DC types present in the spleen in the steady state.

M-CSF: a novel plasmacytoid and conventional dendritic cell poietin.

The critical importance of plasmacytoid dendritic cells (pDCs) in viral infection, autoimmunity, and tolerance has focused major attention on these cells that are rare in blood and immune organs of humans and mice. The recent development of an Flt-3 ligand (FL) culture system of bone marrow cells has led to the simple generation of large numbers of pDCs that resemble their in vivo steady-state counterparts. The FL system has allowed unforeseen insight into the biology of pDCs, and it is assumed that FL is the crucial growth factor for these cells. Surprisingly we have found that a cell type with high capacity for interferon-alpha (IFN-alpha) production in response to CpG-containing oligonucleotides, a feature of pDCs, develop within macrophage-colony-stimulating factor (M-CSF)-generated bone marrow cultures. Analysis of this phenomenon revealed that M-CSF is able to drive pDCs as well as conventional DCs (cDCs) from BM precursor cells in vitro. Furthermore, application of M-CSF to mice was able to drive pDCs and cDCs development in vivo. It is noteworthy that using mice deficient in FL indicated that the M-CSF-driven generation of pDCs and cDCs in vitro and in vivo was independent of endogenous FL.

Fms-like tyrosine kinase 3 ligand administration overcomes a genetically determined dendritic cell deficiency in NOD mice and protects against diabetes development.

A dendritic cell (DC) imbalance with a marked deficiency in CD4- 8+ DC occurs in non-obese diabetic (NOD) mice, a model of human autoimmune diabetes mellitus. Using a NOD congenic mouse strain, we find that this CD4- 8+ DC deficiency is associated with a gene segment on chromosome 4, which also encompasses non-MHC diabetes susceptibility loci. Treatment of NOD mice with fms-like tyrosine kinase 3 ligand (FL) enhances the level of CD4- 8+ DC, temporarily reversing the DC subtype imbalance. At the same time, fms-like tyrosine kinase 3 ligand treatment blocks early stages of the diabetogenic process and with appropriately timed administration can completely prevent diabetes development. This points to a possible clinical use of FL to prevent autoimmune disease.