At a glance: DC cell subsets from peripheral blood
|Plasmacytoid DCs (pDCs)||CD303 (BDCA-2), CD304 (BDCA-4/Neuropilin-1), CD123, CD4, CD45RA, CD141 (BDCA-3)||Upon pathogen encounter, produce large amounts of IFNs|
|CD1c+ myeloid DCs (cDC2 or MDC2)||0.6% of peripheral blood mononuclear cells (PBMCs)||CD1c, CD11c, CD123, CD13, CD33, CD32, CD64, FcεRI, CD2, CD45RO, CD141. A small proportion also express CD14 and CD11b||Produce IL-12 upon TLR3 or TLR8 stimulation leading to TH1 CD4+ T cell polarization and priming of naive CD8+ T cells|
|CD141+ myeloid DCs (cDC1 or MDC1)||0.05% of PBMCs||Clec9a, XCR1, CD11c, CD13, CD33|
Three subsets of DCs can be identified in peripheral blood: plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) or conventional DC (cDCs), which are further subdivided into two subsets based on the expression of the surface markers CD1c (BDCA-1) and CD141 (BDCA-3).
pDCs produce large amounts of type I IFNs, IFN-α, and IFN-β in response to binding nucleic acids typical of viruses and bacteria on toll-like receptors (TLR). TLR7 recognizes single-stranded RNA, and TLR9 recognizes CpG DNA. IFNs have pleiotropic effects on several immune cells including T cells, NK cells, and mDCs, which makes pDCs critical responders to viral infections. However, the antigen uptake capacity of pDCs is inferior to mDCs and at steady-state, pDCs can induce tolerance rather than immune response (PMID: 28536413, 25852695).
Human pDCs are defined as CD303 (BDCA-2)+, CD304 (BDCA-4/Neuropilin-1)+, CD123+, CD4+, CD45RA+, CD141 (BDCA-3)dim, CD1c (BDCA-1)–, and CD2–. They lack expression of lineage markers (CD3, CD14, CD16, CD19, CD20, CD56) and express neither myeloid markers, such as CD13 and CD33, nor Fc receptors, such as CD16, CD64, or FcεRI (PMID: 16920966, 11602645, 11913066).
CD1c+ mDCs, also called cDC2 or MDC2, account for 0.6% of PBMCs. They produce IL-12 upon TLR3 or TLR8 stimulation with poly (I:C) or R848, which leads to polarization of TH1 CD4+ T cells and priming of naive CD8+ T cells. The CD1c (BDCA-1) antigen is a member of the CD1 protein family that are structurally related to MHC class I proteins and mediate the presentation of non-peptide antigens to T cells.
The CD1c antigen is specifically expressed on DCs that are CD11chigh CD123low. CD1c+ mDCs have monocytic morphology and express myeloid markers such as CD13 and CD33, as well as Fc receptors, such as CD32, CD64, and FcεRI. Furthermore, they are Lin (CD3, CD16, CD19, CD20, CD56)–, CD2+, CD45RO+, CD141 (BDCA-3)low, CD303 (BDCA-2)–, and CD304 (BDCA-4/Neuropilin-1)–. A minor proportion of CD1c+ mDCs expresses CD14 and CD11b. CD1c is also expressed by circulating B cells and by CD1a+ DCs generated ex vivo from monocytes or hematopoietic precursor cells.
CD141+ mDCs, sometimes also called cDC1 or MDC1, are a very rare subset of blood DCs representing less than 0.05% of total PBMCs. They share several functional and phenotypical features with CD1c+ mDCs, such as IL-12 secretion and TLR8 expression, but are also characterized by IFN-λ secretion upon activation. CD141+ mDCs exhibit better cross-presentation of antigens derived from dead cells thanks to expression of the necrotic receptor Clec9a. This receptor and the chemokine (C motif) receptor 1 (XCR1) are exclusive markers of this subset. XCR1 is also expressed by the corresponding mouse subset CD8α+ DCs.CD141 (BDCA-3)+ mDCs are Clec9a+, XCR1+, CD11cdim, CD123–, CD1c (BDCA-1)–, and do not express lineage markers such as CD3, CD14, CD16, CD19, CD20, or CD56. They express myeloid markers, including CD13 and CD33, and are monocytic in appearance. In contrast to CD1c+ mDCs, CD141+ mDCs do not express CD2 and Fc receptors such as CD32, CD64, and FcεRI. They also differ in terms of toll-like receptor expression, cytokine production, and T helper cell polarization. The CD141 (BDCA-3) antigen is expressed at much lower levels on CD1c+ mDCs, pDC, monocytes, and granulocytes in blood.
At a glance: Kits and reagents for the isolation of various DC subsets from PBMCs
|Cell subset||Isolation strategy||Comments||Automation||Product|
|Pan DCs||Depletion of non-target cells||Isolation of all untouched DC subpopulations||Yes*||Pan-DC Enrichment Kit, human|
|pDCs||Depletion of non-target cells||Isolation of untouched pDCs||Yes*||Plasmacytoid Dendritic Cell Isolation Kit II, human|
|CD1c (BDCA-1)+ myeloid DCs||Depletion of B cells followed by positive selection of target cells||Yes*||CD1c (BDCA-1)+ Dendritic Cell Isolation Kit, human|
|CD141 (BDCA-3)++ myeloid DCs||Positive selection of target cells||Yes*||CD141 (BDCA-3) MicroBead Kit, human|
|*Automation options range from fully automated benchtop solutions such as the autoMACS® Pro Separator to high-throughput platforms such as the MultiMACS™ Cell24 Separator Plus or MultiMACS X.|
Pre-enrichment of untouched Pan-DCs from human PBMCs. Cells were isolated using the Pan-DC Enrichment Kit, human, a MidiMACS™ Separator and an LS Column, and then fluorescently stained with CD303 (BDCA-2)-FITC, CD141 (BDCA-3)-APC, CD1c (BDCA-1)-PE, and CD20-PerCP. Flow cytometry analysis was done on the MACSQuant® Analyzer, where cell debris, dead cells, and B cells were excluded based on scatter signals, propidium iodide fluorescence, and CD20 expression.
At a glance: Markers for the detection of DCs from peripheral blood by flow cytometry
|Mo-DCs||pDCs||DC1c+ cDCs||CD141+ cDCs|
|CD14–||CD303 (BDCA-2)+||CD172 (SIRP-α)+||CD141 (BDCA-3)+|
|CD11b+||CD304 (BDCA-4)+||CD1c (BDCA-1)+||CLEC9a (DNGR-1)+|
|CD209 (DC-SIGN)high||CD123 (IL-3R)high||CD11b+||XCR1+|
|CD83–||ILT7+||CD123 (IL-3R)int||CD123 (IL-3R)–|
At a glance: Reagents for stimulation and antigen loading of DCs from peripheral blood
|Supplement||Human Flt-3 ligand|
|Stimulation||Antigen-specific activation of DCs; available in research, premium, and MACS® GMP grades||PepTivator Peptide Pools|
|Stimulation||TLR ligand||TLR3 Agonists|
|Stimulation||TLR ligand||TLR7/8 Agonists|
|Stimulation||TLR ligand||TLR9 Agonists|
Miltenyi Biotec offers high-quality cytokines and stimulation reagents that ensure consistent results from the cell culture and stimulation of DCs. After isolation, enriched DCs are cultured with recombinant cytokines, such as Human IL-3, Human GM-CSF, and Human Flt-3 Ligand. They can then be activated via exposure to antigens and/or toll-like receptor (TLR) ligands.As antigen-presenting cells, DCs can be pulsed with antigens to activate T cells in an antigen-specific way. Effective priming of naive T cells, as well as fast and efficient re-stimulation of CD4+ and CD8+ T cells in an MHC class– and haplotype-independent way, can be achieved with PepTivator® Peptide Pools. These 15-mer peptides with 11-amino-acid overlaps cover the complete sequence of an antigen, and Miltenyi Biotec offers extensive panels of virus- or tumor-specific antigens.
Optimal stimulation of DCs is necessary for robust immune responses that lead to desired T cell activation. Several studies aim to find the right stimulation for DCs to induce such a response. The ability of a DC to respond to a specific stimulus depends on the composition of pathogen recognition receptors, in particular toll-like receptors (TLRs), which recognize highly conserved structural motifs as signatures of invading pathogens. Miltenyi Biotec offers a broad selection of products for DC activation via TLR ligands.
TLR3 recognizes virally derived double- or single-stranded RNA leading to activation of immune cells such as DCs and B cells. The natural ligands for TLR3 can be mimicked by synthetic RNAs (oligoribonucleotides or ORNs) like polyinosinic-polycytidylic acid, poly (I:C).
TLR7 and TLR8 are involved in responses to viral infection. They recognize single-stranded RNAs as their natural ligand, as well as small synthetic molecules. Miltenyi Biotec provides high-quality TLR7/8 agonist ORNs for the activation of TLR7 only, TLR8 only, or TLR7 and TLR8 simultaneously, each with respective controls.TLR9 recognizes synthetic oligonucleotides containing CpG motifs (CpG ODNs) that mimic the immune stimulatory effect of bacterial DNA, promoting TH1 and pro-inflammatory cytokine induction to facilitate maturation and activation of DCs and other antigen-presenting cells. Miltenyi Biotec offers four classes of TLR9 agonists: A, B, C and P. The A-class and P-class agonists are recommended for pDC activation.
At a glance: Kits and reagents for the generation of Mo-DCs
|Supplement for differentiation||Combined with Human GM-CSF for differentiation||Human IL-4|
|Supplement for differentiation||Combined with Human IL-4 for differentiation||Human GM-CSF|
|Supplement for differentiation||Optimized mix of Human GM-CSF and Human IL-4 for generation of Mo-DCs||CytoBox Mo-DC, human|
|Culture medium||Medium supplemented with Human GM-CSF and Human IL-4||Mo-DC Differentiation Medium, human|
|Culture medium||Optimized medium supplemented with L-glutamine and TNF-α||Mo-DC Maturation Medium, human|
|Evaluation of in vitro Mo-DC generation||Pre-mixed staining antibody cocktail to assess differentiation by flow cytometry||Mo-DC Differentiation Inspector, human|
|Induction of DC maturation||Human CD40-Ligand|
|induction of DC maturation||Human TNF-α|
|Induction of DC maturation||Human IL-6|
|Induction of DC maturation||Human IL-1β|
Although primary DCs open new exciting avenues to investigate DCs for basic and clinical applications, for decades human DCs have been generated in vitro from peripheral blood monocytes using GM-CSF and IL-4 to drive differentiation over 5–7 days. For products for the isolation of monocytes, see cell separation reagents.
These monocyte-derived DCs (Mo-DCs) have the characteristics of immature DCs and can be matured by culturing them in the presence of microbial, pro-inflammatory, or T cell–derived stimuli. Mo-DCs have been tested in a broad range of immunotherapy-based protocols, primarily for cancer research.