伊莱斯特女包:Dendritic Cell Research

莱斯特·德·利昂柯特 www.jclehg.com.cn

Dendritic cells (DCs) constitute a diverse set of hematopoietic cell types that play important roles in innate and adaptive immunity. There is great interest in exploiting DCs to develop immunotherapies for cancer, chronic infections and autoimmune disease as well as for induction of transplantation tolerance.

BD continues to expand its instrument and reagent portfolio to enable the enrichment, sorting and analysis of DCs and their different subsets by multicolor flow cytometry.

Explore different dendritic cell lineages and subsets in humans and mice. 

Overview

Dendritic cells (DCs) constitute a diverse set of hematopoietic cell types that play important roles in innate and adaptive immunity. 1-3 They are potent antigen sensing and antigen presenting cells (professional APCs) that are uniquely capable of initiating primary immune responses to foreign antigens while safeguarding tolerance to self antigens. 4 DCs guide the specificity, magnitude and polarity of immune responses. Because of their instrumental role in the immune system and their natural adjuvant properties, there is great interest in exploiting DCs to develop immunotherapies for cancer, chronic infections and autoimmune disease, as well as for induction of transplantation tolerance. 5-7 Accordingly, there is increasing research activity, both in the intricacies of basic DC biology, as well as in murine models of disease and the application of this knowledge to preclinical strategies for the manipulation of DCs in human disease.

Dendritic cell maturation

Immature DCs arise from progenitor cells in the bone marrow and migrate to practically all lymphoid and nonlymphoid tissues throughout the body, including the skin, lungs and intestines. 8,9 A diverse array of transcription factors, signaling molecules, growth factors, cytokines, chemokines and adhesion receptors has been implicated in the differentiation pathway from common DC progenitors to mature DCs. 1,10,11 In addition, through diverse assortments of surface pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), immature DCs receive and process further maturation signals by discerning damage-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs) in their local environments. 1,5,7,12 This sensing of damaged cells or pathogens allows DCs to carry out their sentinel-like functions to maintain the body’s integrity.

Maturing tissue DCs alter their surface chemokine receptor and adhesion molecule profiles according to microenvironmental cues and home in to secondary lymphoid organs in response to chemotactic signals. Within lymphoid tissues, immature resident or incoming nonresident DCs can be further stimulated and differentiate to become mature, functional DCs. Mature DCs have advanced capabilities to process and present antigens in the context of self-MHC antigens to naïve CD4 + or CD8 + T cells. This leads to either initiation of primary immune responses against foreign antigens or downregulation of potential T-cell reactivity directed against self antigens. Mature DCs stimulate naïve T cells through their increased surface expression of peptide-loaded major histocompatibility complex (MHC) antigens, costimulatory (or coinhibitory) receptors and ligands, for example, CD80 and CD86, and the release of cytokines such as IL-6, IL-12p70 or interferons (IFNs). 1,3,10,11 T cells can further tune the nature of mature DCs. Responding T cells may reciprocally regulate DCs, for example, through CD40-CD40L interactions or by T-cell–derived cytokines such as IL-4 or IFN-γ. In this way, T cells may additionally instruct the professional APCs, which can promote different types of T-cell–dependent immunity or tolerance.

Multifunctional roles of dendritic cells

Not only are DCs potent initiators of immune responses, they also play important regulatory roles in determining the type, magnitude and duration of immune responses that ensue. 1,4,10,11 DCs accomplish this by their differential expression of cell surface ligands and receptors as well as by secreting distinct profiles of cytokines, chemokines and inflammatory mediators. For example, DCs that release IL-12p70 may preferentially promote type-1 CD4 + T-helper cells (Th1) or cytolytic CD8 + T cells. Other DC types may promote T-cell–dependent humoral or cell-mediated immune responses characteristic of Th2, Th9, Th17, Th22, T follicular helper (Tfh) or regulatory T (Treg) cells. The issue of exactly which DCs orchestrate these types of T-cell–dependent immune responses, and how they do it, remains open and intensively investigated.

Some studies point to the DC’s maturity level as crucial, whereas others point to the major influence of the pathogen type or the tissue site involved. These are all critical parameters that require careful study. The truth may lie somewhere in between, since there is such a large degree of functional plasticity within the DC pathway. 2-4,8,11,13,14 The essential link that DCs provide between innate and adaptive immunity is also becoming more appreciated. Not only do DCs mature in response to danger signals, thus becoming capable of inducing a productive T-cell response, they also trigger natural responses to invading infectious agents by activating macrophages, natural killer (NK) cells, natural killer T cells (NKT cells), granulocytes and mast cells. 15 The discovery that plasmacytoid DCs (pDCs) are a major source of IFNs, quickly secreting them in response to certain viruses, 10 serves as an important example of the multifunctional role played by DCs in both innate and adaptive immune responses.

Dendritic cell heterogeneity

Multiple types of precursor, immature and mature DCs (for example, Langerhans cells, dermal or interstitial DCs, blood DCs) that differ in origin, morphology, localization, maturation state, phenotype and function 1-4,8,10,11,16 have been described. Despite some cell surface phenotypic differences between the two species, two generally accepted types of DCs have been described in human and mouse model systems that appear to represent different lineages: plasmacytoid DCs (pDCs) and myeloid DCs (mDCs), also known as classical or conventional DCs (cDCs). 1,11,13,14pDCs have a tremendous capacity to produce IFNs but may not present antigens as efficiently as mDCs. 1,3,10 Human pDCs are distinguished by their coexpression of CD123 and CD304 whereas mouse pDCs express CD45R/B220 and Ly-6C. 1,10,11 Two major classes of mDCs have been further classified in the human and mouse species, which are defined by the alternative expression of either IFN regulatory factor 4 (IRF4 + DCs) or IRF-8 (IRF-8 + DCs). 11 IRF4 + DCs in humans characteristically express CD1c, whereas mouse counterparts express either CD4 (lymphoid resident DCs) or CD11b (migratory DCs). The IRF4 + DCs from both species coexpress CD172a/Sirp-α and can efficiently present antigens to naïve CD4 + T cells. Conversely, human IRF8 + DCs typically express CD141, while mouse equivalents express CD8a (lymphoid resident DCs) or CD103 (migratory DCs) with all subsets expressing the XCR1 chemokine receptor, CD370/Clec9a, and capable of presenting antigen to CD4 + T cells and CD8 + T cells. Human and mouse Langerhans cells (LCs) likewise coexpress several distinguishing markers in common including CD207/Langerin, CD326/EpCAM, and CD324/E-Cadherin. 1,11,13,14 DC subsets residing in the dermis and intestines of both species have also been described. 1,11,16-18 For a summary of human and mouse DC counterparts, see Table 1.  

Another class of DCs, inflammatory DCs, may arise from monocytes that may be driven by environmental stimuli to take on the characteristics and functions of DCs. 1 Clearly, provocative interspecies differences as well as similarities in certain functionally related molecules are being described for the various DC subsets including their expressed profiles of TLRs, CLRs, CD1 molecules, chemokine receptors and their cytokine secretion patterns. 1,6,7 Since a combination of factors, including the DC subset and maturation stage, influence resulting T-cell responses, detailed phenotypic analysis combined with functional studies will be one of the useful approaches in further studying the intricacies of DC biology in physiological as well as pathological conditions.

Table 1. Functionality of human DC subsets and their mouse DC counterparts

Human DC Subsets Mouse DC Counterparts Frequency Localization Cytokine Production Upon Stimulation 1
pDC pDC ~1% peripheral blood mononuclear cells (PBMCs) Human blood  
Lymph node T-cell zone  
Tonsil
IFN-I +, IFN-III (IFN-λ) + 
IL-6 +, IL-8 + 
IP-10 (CXCL10) + 
TNF +
CD1c + DCs CD4 + or CD11b + DCs ~1% PBMCs Human blood  
Nonlymphoid tissues:  
skin, liver, lung and gut  
Lymphoid tissues:  
spleen, lymph nodes
IL-1β +, IL-6 +, IL-8 + 
IL-10 +, IL-12 + 
IL-23 + 
TNF + 

IL-15 + (skin)
CD141 + DC CD8 + or CD103 + DCs 0.03% PBMCs  
CD8 + DCs: 20–40% of mouse spleen and lymph node cDCs
Human lymph node, tonsil, spleen, bone marrow  
Human nonlymphoid tissues:  
skin, lung, liver, intestine  

CD8 + DCs: Mouse lymphoid tissues
IFN-I +, IFN-III (IFN-λ) + 
IL-12 + (mouse)  
CXCL-10 (IP-10) + 
TNF +2
LCs  
(Langerhans Cells)
LCs  
(Langerhans Cells)
3-5% epidermal cells Human stratified squamous epithelia,  
draining lymph nodes
IL-15 +
Inflammatory DCs Inflammatory DCs   Inflammatory sites IL-1β +, IL-6 + 
IL-10 +, IL-12 +, IL-23 + 
TNF +

1Cytokine production could vary with the stimulant used, the stimulation conditions or the physiological state of the cell.

2TNF is not typically produced by human CD141 + DCs in response to TLR8 stimulation.

References

  1. Merad M, Sathe P, Helft J, Miller J, Mortha A. The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting.  Annu Rev Immunol. 2013;31:563-604.
  2. Schraml BU, Reis e Sousa C. Defining dendritic cells.  Curr Opin Immunol. 2015;32:13-20.
  3. Collin M, McGovern N, Haniffa M. Human dendritic cell subsets.  Immunology. 2013;140:22-30.
  4. O'Keeffe M, Mok WH, Radford KJ. Human dendritic cell subsets and function in health and disease.  Cell Mol Life Sci. 2015;72:4309-4325.
  5. Apostolopoulos V, Thalhammer T, Tzakos AG, Stojanovska L. Targeting antigens to dendritic cell receptors for vaccine development.  J Drug Deliv. 2013;2013:869718.
  6. Cohn L, Delamarre L. Dendritic cell-targeted vaccines.  Front Immunol. 2014;5:255.
  7. Delamarre L, Mellman I. Harnessing dendritic cells for immunotherapy.  Sem Immunol. 2011;23:2-11.
  8. Breton G, Lee J, Liu K, Nussenzweig MC. Defining human dendritic cell progenitors by multiparametric flow cytometry.  Nat Protoc.2015;10:1407-1422.
  9. Poltorak MP, Schraml BU. Fate mapping of dendritic cells.  Front Immunol. 2015;6:199.
  10. Swiecki M, Colonna M. The multifaceted biology of plasmacytoid dendritic cells.  Nat Rev Immunol. 2015;15:471-485.
  11. Murphy TL, Grajales-Reyes GE, Wu X, et al. Transcriptional control of dendritic cell development.  Annu Rev Immunol. 2016;34:93-119.
  12. Durand M, Segura E. The known unknowns of the human dendritic cell network.  Front Immunol. 2015;6:129.
  13. Dutertre CA, Wang LF, Ginhoux F. Aligning bona fide dendritic cell populations across species.  Cell Immunol. 2014;291:3-10.
  14. Schlitzer A, Ginhoux F. Organization of the mouse and human DC network.  Curr Opin Immunol. 2014;26:90-99.
  15. Reis e Sousa C. Activation of dendritic cells: translating innate into adaptive immunity.  Curr Opin Immunol. 2004;16:21-25.
  16. Malissen B, Tamoutounour S, Henri S. The origins and functions of dendritic cells and macrophages in the skin.  Nat Rev Immunol.2014;14:417-428.
  17. Henri S, Poulin LF, Tamoutounour S, et al. CD207+ CD103+ dermal dendritic cells cross-present keratinocyte-derived antigens irrespective of the presence of Langerhans cells.  J Exp Med. 2010;207:189-206.
  18. Klechevsky E, Morita R, Liu M, et al. Functional specializations of human epidermal Langerhans cells and CD14+ dermal dendritic cells.  Immunity. 2008;29:497-510.
Phenotyping Human DCs Phenotyping Mouse DCs Enrichment and Sorting DC Function

Human dendritic cells

Human dendritic cell (DC) subsets can be characterized by a number of surface and intracellular markers through flow cytometry.

See Table 2 for key markers for phenotypic characterization of various human DC subsets; intracellular markers are indicated in  bold. BD Life Sciences has a broad portfolio of antibody reagents to most of these specificities in multiple formats to allow flexibility in panel design and downstream analysis.


All human DC subsets are identified as lineage  (CD3  CD19  CD20  CD56  CD14 1) and CD45 + MHCII (HLA-DR) + CD11c 2.


Table 2. Human DC subsets

DC Subset Key Markers
Primary Markers Additional Positive Markers Additional Negative Markers Transcription Factors
Plasmacytoid DCs CD123 (IL-3Rα) high 
CD303 (BDCA2/CLEC4C) + 
CD304 (Neuropilin-1/BDCA4) + 
CD85g (ILT7) + 
CD11c low 
CD11b , CD14
CD2 ±, CD4 +, CD45RA + 
CD141 (BDCA3) low 
CD272 (BTLA) + 
CD366 (TIM-3) ± 
CD367 (DCIR/CLEC4A) + 
CD371 (CLEC12A) ± 

TLR7 high, TLR9 high 

FLT3 (CD135) + 
GM-CSFR (CD116) + 

CCR5 +, CXCR4 +
CD1a , CD1c (BDCA1)  
CD16 (FcγRIII)  
CD172a (Sirp-α)  
CD207 (Langerin)  
CD324 (E-Cadherin)  
CD326 (EpCAM)  
CD369 (Dectin-1/CLEC7A)  
CD370 (CLEC9A/DNGR1)  
CLEC6A (Dectin-2)  

XCR1
IRF7 +, IRF8 + 
SpiB +
CD1c + Myeloid DCs CD1c (BDCA1) + 
CD172a (Sirp-α) + 
CLEC6A (Dectin-2) + 
CD11b +/low, CD11c + 
CX3CR1 + 
CD14 low/–
CD4 +, CD13 +, CD26 low, CD33 + 
CD45RO +, CD141 (BDCA3) ± 
CD272(BTLA) + 
CD366 (TIM-3) + 
CD367 (DCIR/CLEC4A) + 
CD369 (Dectin-1/CLEC7A) + 
CD371 (CLEC12A) + 
CD1a + [skin and intestine]  
CD141(BDCA3) + [intestine]  

TLR3 low, TLR4 low,  TLR8 +, TLR10 low 

FLT3 (CD135) + 
GM-CSFR (CD116) +
CD1a , CD16 (FcγRIII)  
CD123 (IL-3Rα)  
CD207 (Langerin)  
CD304 (Neuropilin-1/BDCA4) , CD324 (E-Cadherin)  
CD326 (EpCAM)  
CD370 (CLEC9A/DNGR1)  
ESAM  

XCR1
IRF4 +
CD141 + Myeloid DCs CD141 (BDCA3) high 
CD370 (CLEC9A/DNGR1) + 
NECL2 (CADM1) + 
CD11c +/low 
CD14
CD4 +, CD11b low 
CD26 +, CD162 high 
CD205 (DEC-205) high 
CD272 (BTLA) high 
CD367 (DCIR/CLEC4A) + 
CD366 (TIM-3) + 
CD369 (Dectin-1/CLEC7A) + 
CD371 (CLEC12A) + 

TLR3 +,  TLR8 + 

FLT3 (CD135) + 
GM-CSFR (CD116) + 

XCR1 +
CD1a , CD1c (BDCA1) , CD16 (FcγRIII)
CD172a (Sirp–α)  
CD207 (Langerin)  
CD304 (Neuropilin-1/BDCA4)  
CD324 (E-Cadherin)  
CD326 (EpCAM)
IRF8 + 
BATF3 +
Langerhans Cells CD207 (Langerin) + 
CD324 (E-Cadherin) + 
CD326 (EpCAM) + 
CD11b low, CD11c + 
CD14
CD1a high, CD1c (BDCA1) + 
CD36 + 
CD172a (Sirp-α) + 
CD369 (Dectin-1/CLEC7A) +, CD371 (CLEC12A) + 
CLEC6A (Dectin-2) + 

TLR1 +, TLR2 +, TLR3 low, TLR6 +
CD304 (Neuropilin-1/BDCA4)  
CD367 (DCIR/CLEC4A)  
XCR1
 
CD1a + Dermal DCs CD1a + 
CD64 (FcγRI) + 
CD366 (TIM-3) + 
CD11b +, CD11c + 
CD14
CD1c (BDCA1) + 
CD172a (Sirp-α) + 
CD367 (DCIR/CLEC4A) + 
CD369 (Dectin-1/CLEC7A) + 
CLEC6A (Dectin-2) + 

TLR1–3 +, TLR6 +,  TLR7 +, TLR10 +
CD207 (Langerin)  
CD209 (DC-SIGN/CLEC4L)  
CD324 (E-Cadherin)  
CD326 (EpCAM)
 
CD14 + Dermal DCs CD14 + 
CD209 (DC-SIGN/CLEC4L) + 
CD11b +, CD11c +
CD1c (BDCA1) + 
CD172a (Sirp-α) + 
CD367 (DCIR/CLEC4A) + 
CD369 (Dectin-1/CLEC7A) + 
CLEC6A (Dectin-2) + 

TLR1–3 +, TLR6,  TLR7 + 
CSF–1R (CD115) +
CD1a  
CD207 (Langerin)  
CD324 (E-Cadherin)  
CD326 (EpCAM)  
CD366 (TIM-3)
 

Inflammatory DCs

(Monocyte-Derived DCs)

CD16 (FcγRIII)  + 
CD64 (FcγRI) + 
CD1a + 
CD1c (BDCA1) + 
CD11b +, CD11c + 
CD14 ±
CD172a (Sirp-α) + 
CD206 (MR/CLECL13D) + 
CD209 (DC-SIGN/CLEC4L) + 
CD367 (DCIR/CLEC4A) + 
CD369 (Dectin-1/CLEC7A) + 
CD371 (CLEC12A) + 
CLEC6A (Dectin-2) + 

TLR3 low, TLR4 +,  TLR7 low,  TLR8 + 

CCR2 (CD192) +
CD207 (Langerin)  
CD324 (E-Cadherin)  
CD326 (EpCAM)
 

1CD14 is negative or low on all DC subsets except for CD14 + dermal DCs and inflammatory DCs.

2CD11c is positive on all DC subsets except for plasmacytoid DCs where expression has been reported as low or negative.

TLR7, 8 and 9 are endosomal and require intracellular staining.

The following examples on peripheral blood illustrate DC subset identification based on available BD OptiBuild™ custom reagents

Analysis of human DC populations on a BD LSRFortessa? X-20 cell analyzer

Analysis of human DC populations on a three-laser BD FACSVerse? flow cytometer

Bibliography

  1. Merad M, Sathe P, Helft J, Miller J, Mortha A. The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting.  Ann Rev Immunol. 2013;31:563-604.
  2. Schraml BU, Reis e Sousa C. Defining dendritic cells.  Curr Opin Immunol. 2015;32:13-20.
  3. Collin M, McGovern N, Haniffa M. Human dendritic cell subsets.  Immunology. 2013;140:22-30.
  4. O'Keeffe M, Mok WH, Radford KJ. Human dendritic cell subsets and function in health and disease.  Cell Mol Life Sci. 2015;72:4309-4325.
  5. Apostolopoulos V, Thalhammer T, Tzakos AG, Stojanovska L. Targeting antigens to dendritic cell receptors for vaccine development.  J Drug Deliv. 2013;2013:869718.
  6. Cohn L, Delamarre L. Dendritic cell-targeted vaccines.  Front Immunol. 2014;5:255.
  7. Delamarre L, Mellman I. Harnessing dendritic cells for immunotherapy.  Sem Immunol. 2011;23:2-11.
  8. Breton G, Lee J, Liu K, Nussenzweig MC. Defining human dendritic cell progenitors by multiparametric flow cytometry.  Nat Protoc.2015;10:1407-1422.
  9. Poltorak MP, Schraml BU. Fate mapping of dendritic cells.  Front Immunol. 2015;6:199.
  10. Swiecki M, Colonna M. The multifaceted biology of plasmacytoid dendritic cells.  Nat Rev Immunol. 2015;15:471-485.
  11. Murphy TL, Grajales-Reyes GE, Wu X, et al. Transcriptional control of dendritic cell development.  Annu Rev Immunol. 2016;34:93-119.
  12. Durand M, Segura E. The known unknowns of the human dendritic cell network.  Front Immunol. 2015;6:129.
  13. Dutertre CA, Wang LF, Ginhoux F. Aligning bona fide dendritic cell populations across species.  Cell Immunol. 2014;291:3-10.
  14. Schlitzer A, Ginhoux F. Organization of the mouse and human DC network.  Curr Opin Immunol. 2014;26:90-99.
  15. Reis e Sousa C. Activation of dendritic cells: translating innate into adaptive immunity.  Curr Opin Immunol. 2004;16:21-25.
  16. Malissen B, Tamoutounour S, Henri S. The origins and functions of dendritic cells and macrophages in the skin.  Nat Rev Immunol.2014;14:417-428.
  17. Henri S, Poulin LF, Tamoutounour S, et al. CD207+ CD103+ dermal dendritic cells cross-present keratinocyte-derived antigens irrespective of the presence of Langerhans cells.  J Exp Med. 2010;207:189-206.
  18. Klechevsky E, Morita R, Liu M, et al. Functional specializations of human epidermal Langerhans cells and CD14+ dermal dendritic cells.  Immunity. 2008;29:497-510.
  19. Autenrieth SE, Grimm S, Rittig SM, et al. Profiling of primary peripheral blood- and monocyte-derived dendritic cells using monoclonal antibodies from the HLDA10 Workshop in Wollongong, Australia.  Clin Transl Immunology. 2015;4:e50.
  20. Ohradanova-Repic A, Machacek C, Fischer MB, Stockinger H. Differentiation of human monocytes and derived subsets of macrophages and dendritic cells by the HLDA10 monoclonal antibody panel.  Clin Transl Immunology. 2016;5:e55.
  21. Clark GJ, Kupresanin F, Fromm PD, et al. New insights into the phenotype of human dendritic cell populations.  Clin Transl Immunology.2016;5:e61.
  22. Fromm PD, Kupresanin F, Brooks AE, et al. A multi-laboratory comparison of blood dendritic cell populations.  Clin Transl Immunology.2016;5:e68.

Mouse dendritic cells

Mouse dendritic cell (DC) subsets can be characterized by a number of surface and intracellular markers through flow cytometry.

See Table 3 for key markers for phenotypic characterization of various mouse DC subsets; intracellular markers are indicated in  bold. BD Life Sciences has a broad portfolio of antibody reagents to most of these specificities in multiple formats to allow flexibility in panel design and downstream analysis.


Mouse DC markers are identified as lineage  (CD3  CD19  CD49b  or NK1.1  CD14 ) and CD45 + MHCII + CD11c +.

 

Table 3. Mouse DC subsets

DC Subset Key Markers
Primary Markers Additional Positive Markers Additional Negative Markers Transcription Factors
Plasmacytoid DCs (pDCs) CD45R (B220) + 
CD317 (BST-2) + 
Ly-6C + 
Siglec-H + 
CD11c low 
CD14
CD4 +, CD26 + 
CD172a (Sirp-α) + 
CD209a (DC-SIGN) + 
CD272 (BTLA) + 
CD370 (Clec9a/DNGR1) + 
Gr1 (Ly-6C and Ly-6G) + 
FLT3 (CD135) + 
TLR7 high, TLR9 high
CD11b  
CD24  
CD36 , CD64 (FcγRI) , CD103  
CD205 (DEC-205)  
CD207 (Langerin)  
CD326 (EpCAM)  
DCIR2 (Clec4a4/33D1)  
F4/80  
CX3CR1 , XCR1
IRF7 +, IRF8 + 
BATF3 high 
SpiB + 

Zbtb46
CD4 +CD11b + 
Lymphoid-Resident DCs
CD4 + 
CD11b + 
CD11c high 
CD8a  
CD14
CD24 +, CD26 + 
CD172a (Sirp-α) + 
CD205 (DEC-205) + 
CD209a (DC-SIGN) + 
CD272 (BTLA) low 
DCIR2 (Clec4a4/33D1) + 
ESAM + 
F4/80 + 
FLT3 (CD135) + 
TLR5 +,  TLR7 +, TLR9 + 
CX3CR1 +
CD36  
CD45R (B220)  
CD64 (FcγRI) , CD103  
CD207 (Langerin)  
CD326 (EpCAM)  
CD370 (Clec9a/DNGR1)  
Ly6C  
XCR1
IRF4 + 
Zbtb46 + 
BATF3 high
CD4 CD11b + 
Conventional Migratory DCs
CD11b + 
CD11c + 
CD4  
CD8a  
CD14
CD24 ±, CD26 + 
CD64 (FcγRI) + 
CD172a (Sirp-α) + 
CD209a (DC-SIGN) ± 
CD272 (BTLA) low 
DCIR2 (Clec4a4/33D1) + 
F4/80 + 
Ly-6C ± 
FLT3 (CD135) + 
TLR5 +,  TLR7 +, TLR9 + 
CX3CR1 +
CD36  
CD45R (B220) , CD103  
CD207 (Langerin)  
CD326 (EpCAM)  
CD370 (Clec9a/DNGR1)  
XCR1
IRF4 + 
IRF2 + 
Zbtb46 + 
BATF3 high
CD8a + Conventional  
Lymphoid-Resident DCs
CD8a + 
CD11c high 
CD4  
CD11b  
CD14
CD1d1 +, CD24 +, CD26 + 
CD36 +, CD103 ± 
CD205 (DEC-205) + 
CD207 (Langerin) ± 
CD272 (BTLA) high 
CD370 (Clec9a/DNGR1) + 
NECL2 (CADM1) + 
FLT3 (CD135) + 
TLR3 +, TLR4 +, TLR11 + 
XCR1 +, CX3CR1 ±
CD45R (B220)  
CD64 (FcγRI)  
CD172a (Sirp-α)  
CD209a (DC-SIGN)  
CD326 (EpCAM)  
DCIR2 (Clec4a4/33D1)  
F4/80  
Ly-6C
IRF8 + 
BATF3 + 
Zbtb46 +
CD103 + Conventional  
Migratory DCs
CD103 + 
CD11c high 
CD4  
CD8  
CD11b  
CD14
CD1d1 +, CD24 + 
CD26 +, CD36 + 
CD205 (DEC-205) + 
CD207 (Langerin) + 
CD272 (BTLA) high 
CD370 (Clec9a/DNGR1) + 
NECL2 (CADM1/CD317) + 
FLT3 (CD135) + 
TLR3 +, TLR4 +, TLR11 + 
XCR1 +
CD45R (B220)  
CD64 (FcγRI)  
CD172a (Sirp-α)  
CD209a (DC-SIGN)  
CD326 (EpCAM)  
DCIR2 (Clec4a4/33D1)  
F4/80  
Ly-6C  
CX3CR1
IRF8 + 
BATF3 + 
Zbtb46 +
Langerhans Cells CD207 (Langerin) + 
CD324 (E-Cadherin) + 
CD326 (EpCAM) + 
CD11b + 
CD11c + 
CD14
CD24 + 
CD172a (Sirp-α) + 
CD205 (DEC-205) + 
F4/80 + 
TLR3 +, TLR11 +
CD8a , CD26 , CD36  
CD45R (B220) , CD103  
CD209a (DC-SIGN)  
CD370 (Clec9a/DNGR1)  
DCIR2 (Clec4a4, 33D1)  
Ly-6C  
CX3CR1 , XCR1
 
CD207 + Dermal DCs CD207 (Langerin) + 
CD11b low 
CD11c +
CD103 ± CD45R (B220)  
CD172a (Sirp-α)  
CD326 (EpCAM)
 
CD207  Dermal DCs CD207 (Langerin)  
CD11b ± 
CD11c +
CD172a (Sirp-α) + CD45R (B220)  
CD103  
CD326 (EpCAM)
 
Inflammatory DCs  
(Monocyte–Derived DCs)
CD64 (FcγRI) + 
CD11b + 
CD11c + 
CD14
CD209a (DC-SIGN) + 
CD272 (BTLA) low 
Ly-6C + 
TLR1-6,  TLR7-8 +, TLR10 +
CD8a , CD45R (B220) , CD103  
CD172a (Sirp-α)  
CD207 (Langerin)  
CD326 (EpCAM)
 

The following examples on mouse spleen illustrate DC subset identification based on available BD OptiBuild™ custom reagents

Bibliography

  1. Merad M, Sathe P, Helft J, Miller J, Mortha A. The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting.  Ann Rev Immunol. 2013;31:563-604.
  2. Swiecki M, Colonna M. The multifaceted biology of plasmacytoid dendritic cells.  Nat Rev Immunol. 2015;15:471-485.
  3. Murphy TL, Grajales-Reyes GE, Wu X, et al. Transcriptional control of dendritic cell development.  Ann Rev Immunol. 2016;34:93-119.
  4. Dutertre CA, Wang LF, Ginhoux F. Aligning bona fide dendritic cell populations across species.  Cell Immunol. 2014;291:3-10.
  5. Schlitzer A, Ginhoux F. Organization of the mouse and human DC network.  Cur Opin Immunol. 2014;26:90-99.
  6. Poltorak MP, Schraml BU. Fate mapping of dendritic cells.  Front Immunol. 2015;6:199.
  7. Henri S, Poulin LF, Tamoutounour S, et al. CD207+ CD103+ dermal dendritic cells cross-present keratinocyte-derived antigens irrespective of the presence of Langerhans cells.  J Exp Med. 2010;207:189-206.

Isolation of dendritic cells

Certain research approaches require the use of isolated populations of dendritic cells (DCs) for further characterization, culture or functional studies. Isolation of these very rare cell types, which make up only 0.1–1% of blood mononuclear cells, has been a limitation historically.1 Advances in flow cytometry and magnetic-bead separation technologies have made DC isolation easier. Flow cytometry based cell sorting enables the isolation of single cells for further analysis.

Dendritic cell populations

Isolated DC populations are often used to carry out functional assays, such as co-culture with T cells, or for other downstream applications. 2 BD offers approaches for the successful isolation of both human and mouse DCs.

The BD IMag™ cell separation system is based on a simple yet highly effective direct magnet technology. Positive or negative selection of DCs, for example, is feasible with researchers selecting from a range of IMag-formatted cell surface binding antibodies. Easy-to-use  human and mouse DC enrichments sets, employing a negative selection of non-DC populations in a few short steps, are also available.

Flow cytometric sorting is a very effective way to isolate these extremely rare cells, since it delivers high purity and recovery of cells and is well suited for the isolation of cells defined by a multimarker phenotype. Many researchers have relied on BD FACSAria™ cell sorters to isolate specific DC populations. 3

Isolation of pDCs and mDCs from human PBMCs

Plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) can be isolated from human peripheral blood on the basis of the phenotypes Lin  HLA-DR + CD123 + CD11c  (pDCs) and Lin  HLA-DR + CD123  CD11c +(mDCs). In the example shown, a pre-enrichment step was performed using a  BD IMag Human Dendritic Cell Enrichment set, Cat. No. 558420, on peripheral blood mononuclear cells (PBMCs), negatively selecting erythrocytes, platelets and peripheral leucocytes (that are not DCs), thereby providing a DC-enriched sample for cell surface staining and sorting.

Cells were stained with lineage cocktail FITC, CD123 BV421, HLA-DR PE and CD11c APC. The viability dye FVS780 was used to exclude dead cells. Cells were sorted using a BD FACSAria flow cytometer. The combination of BD IMag-based enrichment and flow cytometric sorting is a convenient time-reducing workflow generating highly pure DC populations normally found at low frequencies in human blood.

BD Application Specialists are available to provide field- or phone-based advice to support you in your sorting applications.

Rapid purification of viable pDCs and mDCs from human peripheral blood

Upper plots: In the example shown, lineage HLA-DR+ cells in whole blood amount to 0.41% of total events, encompassing pDC and mDC populations (0.16% and 0.15% of total events, respectively). Cells were first gated based on light-scatter properties, followed by doublet discrimination. BD Horizon™ Fixable Viability Stain 780 was used for dead cell exclusion. DC subpopulations were then defined as follows: LineageFITC (Cat. No. 340546), HLA-DR PEhigh, CD123 BV421+, CD11c APC (pDC) and lineage FITC, HLA-DR PEhigh, CD123 BV421 and CD11c APC+(mDC). Residual macrophages were negligible as confirmed by staining with CD14 BV786 (data not shown). 
Center plots: The BD IMag Human Dendritic Cell Enrichment Set (Cat. No. 558420) enables rapid (~45 min) enrichment of the lineage HLA-DR+cells from PBMCs amounting to 29.1% of total events, from an original frequency of 0.41%. The associated pDC and mDC subpopulations were each enriched to >10% of total events, representing roughly a 90-fold enrichment with ~99% viability determined using BD Horizon Fixable Viability Stain 780 (FVS780) (data not shown). 
Lower plots: Following BD IMag enrichment, highly purified DC populations can be isolated by flow cytometric cell sorting. The lower contour plots show lineage HLA-DR PEhigh CD123 BV421+ CD11c APC pDCs and lineage HLA-DR PEhigh CD123 BV421 CD11c APC+ mDCs after sorting using a BD FACSAria™ Fusion cell sorter. Post-sort analysis indicated ~99% purity. 
Employment of the BD IMag magnetic enrichment set reduced the sort time for 100,000 mDCs from an estimated 4.5 hours to 5 minutes (default settings for an 85-μm sort setup and four-way purity sort mode).

Single cells for transcriptome analysis


An increasing number of researchers want to characterize the transcriptional state of single cells as a complementary approach to subset definition based on specific surface-marker expression patterns. 4,5

BD flow cytometry platforms such as the BD FACSMelody™, BD FACSAria™ III, BD FACSAria™ Fusion, BD FACSJazz™ and BD Influx™ support accurate single-cell sorting into different types of plates. Single-cell sorting is made possible by the automated deposition unit, and this utility is enhanced when combined with the index-sorting function, which records the flow cytometric data (for example, marker phenotype) and sort location (X and Y coordinates of the sort-collection device) for each sorted event. In this way, results of post-sorting assays, which may include DNA or RNA sequence analysis, can be precisely traced back to the flow characteristics of the specific cell.

References

  1. Freudenthal PS, Steinman RM. The distinct surface of human blood dendritic cells, as observed after an improved isolation method. Proc Natl Acad Sci U S A. 1990;87:7698-7702.
  2. Markey KA, Gartlan KH, Kuns RD, MacDonald KPA, Hill GR. Imaging the immunological synapse between dendritic cells and T cells. J Immunol Methods. 2015;423:40-44.
  3. Breton G, Lee J, Liu K, Nussenzweig MC. Defining human dendritic cell progenitors by multiparametric flow cytometry. Nature protocols. 2015;10:1407-1422 26292072.
  4. Jaitin DA, Kenigsberg E, Keren-Shaul H, et al. Massively parallel single-cell RNA-Seq for marker-free decomposition of tissues into cell types. Science. 2014; 343:776-779.
  5. Vu Manh T-P, Bertho N, Hosmalin A, Schwartz-Cornil I, Dalod M. Investigating evolutionary conservation of dendritic cell subset identity and functions. Front Immunol. 2015;6:260.

Cytokine Detection

Measurement of the types and amounts of cytokines that are secreted by dendritic cells (DCs) provides insight into the nature and magnitude of the DC response. Multiplexed assays are increasingly being used to measure secreted factors, such as cytokines and chemokines.

BD Cytometric Bead Array: multiplexed quantitation

BD™ Cytometric Bead Array (CBA)

The broad dynamic range of fluorescence detection and multiplexed measurement allow for small sample volumes, fewer sample dilutions and substantially less time to establish the value of an unknown vs a conventional enzyme-linked immunosorbent assay (ELISA) approach.

The BD CBA portfolio includes assays for measurement of a variety of soluble factors, including secreted proteins such as cytokines or chemokines, shed or released cell surface markers (for example, sCD14) plus cell signaling molecules such as phosphoproteins within cell lysates.

BD CBA assays for cytokines

BD™ CBA kits

BD™ CBA Flex Sets

For extra sensitivity,  BD™ CBA Enhanced Sensitivity Flex Sets allow the quantitation of specific analytes through very low concentration ranges (less than 1 pg/mL).

Measuring secreted cytokines can also provide information about the activity of different functional DC subsets in a cellular sample. For example, BD™ CBA assays have been used to quantitate cytokines and/or chemokines from virally infected plasmacytoid DCs (pDCs) and monocyte-derived DCs in vitro. 1

Intracellular detection of cytokines

By using a protein transport inhibitor to block secretion, cytokines can be detected in the cell in which they are being produced. Hence, it is possible to distinguish whether cytokine production by an activated cell population is the result of a few cells producing large amounts of cytokine or a large population of cells each producing small quantities of cytokine.

BD’s well-established kits, buffer systems and protocols for intracellular cytokine.

Detection of IFN-α, TNF-α and IL-12 in CpG-stimulated human PBMCs  

Human peripheral blood mononuclear cells (PBMCs) were stimulated with CpG (4 hours, upper panel) or IFN-γ + LPS (16 hours, lower panel) with Brefeldin A added 2 hours after the stimulus. Cells were stained with surface markers (a lineage negative cocktail, HLA-DR, CD123, CD11c), then fixed and permeabilized with BD Cytofix/Cytoperm buffers, followed by intracellular staining for IFN-α, TNF or IL-12. Samples were acquired on a BD™ LSR II system. Data is shown for lineage , HLA-DR + cells.

Transcription factors and other intracellular proteins

Analysis of DC physiology, from early development to effector functions of mature DCs, has revealed intracellular changes associated with developmental status, cellular activation or other functional properties.

BD offers solutions that uniquely enable analysis of these intracellular molecules, to support researchers in deciphering the interconnected pathways regulating DC biology.

Basic principles of intracellular staining

Cells are fixed and permeabilized (symbolized by the dashed-line membrane), stained and then analyzed by flow cytometry. For studies of secreted proteins, cells are first treated with a protein transport inhibitor to allow accumulation of the target protein inside the cell.

Intracellular detection with optimized buffers and antibodies

Multicolor flow cytometry is a powerful technique for the analysis of intracellular molecules. Simultaneous analysis with markers for cell surface DC subsets can reveal information about the differentiation and activation state of distinct populations of DCs. While techniques for cell surface staining are relatively standard, optimal staining for intracellular markers often depends on the biology of the target protein.

To facilitate the detection of intracellular molecules by flow cytometry, BD has developed monoclonal antibodies, specialized buffer systems, fixable viability dyes and kits that are optimized for detection of specific types of intracellular targets.

Analysis of intracellular TLR7 expression by mouse bone-marrow cells

Mouse bone-marrow cells were stained with BD Horizon™ Fixable Viability Stain 450 (FVS450, Cat. No. 562247), and fixed and permeabilized using the BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (Cat. No. 554714). The cells were then stained with APC Rat Anti-Mouse CD45R/B220 (Cat. No. 553092/561880) and FITC Hamster Anti-Mouse CD11c (Cat. No. 553801/557400/ 561045) antibodies, and either PE Mouse IgG1, κ Isotype Control (Cat. No. 554680, dashed-line histograms) or PE Mouse Anti-Mouse TLR7 antibody (Cat. No. 565557, solid-line histograms). The fluorescence histograms showing total cellular TLR7 expression (or Ig Isotype control staining) were derived from either CD11cCD45R/B220 (bottom Plot D) or CD11c+ CD45R/B220+ (bottom Plot E) gated events with the forward (not shown) and side light-scatter (SSC-A) characteristics of intact FVS450 live cell discriminated bone marrow lymphoid cells as indicated (top Plots A–C). Flow cytometric analysis was performed using a BD LSRFortessa™ cell analyzer system.

Detection of transcription factors

The BD Pharmingen™ Transcription Factor Buffer

BD offers several DC-relevant, flow cytometry validated transcription-factor antibodies. For example, in mice, Zbtb46 is expressed in conventional DCs and their precursors, but not in pDCs. Conversely, Spi-B is required for pDC development and function.2

Analysis of Zbtb46 expression

 

Left Panel. Two-color flow cytometric analysis of Zbtb46 expression in mouse splenocytes. Mouse splenocytes were fixed and permeabilized using the BD Pharmingen™ Transcription Factor Buffer Set (Cat. No. 562574/562725). The cells were stained with BD Horizon BV421 Armenian Hamster Anti-Mouse CD11c (Cat. No. 565452) and either PE Rat IgG1, κ Isotype Control (Cat. No. 554685; left plot) or BD Pharmingen PE Rat Anti-Mouse Zbtb46 antibody (Cat. No. 565832; right plot). Two-color flow cytometric contour plots showing the correlated expression of Zbtb46 (or Ig Isotype control staining) vs CD11c were derived from gated events with the forward and side light-scatter characteristics of intact splenocytes.

Right Panel. Multicolor flow cytometric analysis of Zbtb46 expression in mouse splenic conventional DCs. Similarly fixed and permeabilized splenocytes were stained with BD Horizon BV421 Armenian Hamster Anti-Mouse CD11c, Alexa Fluor® 647 Rat Anti-Mouse I-A/I-E (Cat. No. 562367) and either PE Rat IgG1, κ Isotype Control (dashed-line histogram) or PE Rat Anti-Mouse Zbtb46 antibody (solid-line histogram). The fluorescence histogram showing Zbtb46 expression (or Ig Isotype control staining) for conventional dendritic cells (cDCs) was derived from CD11chigh I-A/I-Ehigh gated events with the forward and side light-scatter characteristics of intact splenic leucocytes.

Flow cytometric analysis was performed using a BD FACSCanto™ II flow cytometer system.

Detection of phosphoproteins

BD Phosflow™ antibodies are monoclonal phosphoepitope-specific antibodies validated for flow cytometric detection. The recommended permeabilization buffer for most BD Phosflow antibodies is BD Phosflow™ Perm Buffer III, but alternative permeabilization buffers are also available, for enabling cell surface staining for subpopulation analysis, for example.

A number of BD Phosflow antibody specificities are available for analysis of signaling pathways involved in DC development and activation, such as reagents for pSTAT3 and pSTAT5 which have differing effects on pDC development. The BD Phosflow system has been used to measure phosphoproteins such as pSyk after viral infection of pDCs in vitro,3 and is widely employed within cell-signaling studies for innate and adaptive immunity. Click here

Immunofluorescence and immunohistochemistry

DC morphology and subcellular localization

Using well-defined monoclonal antibodies with fluorescence imaging allows for the identification of DCs within tissues and insights into the mechanistic aspects of DC biology in vitro.

BD’s well-characterized monoclonal antibodies that are directly conjugated to new and bright photostable fluorescent dyes, such as BD Horizon™ BV421 and BD Horizon BV480, enable multicolor imaging beyond conventional three- to four-color microscopy.

Image analysis of CD11c expression in mouse spleen and human tonsil

Immunohistochemical analysis of Langerin (CD207) expression by Langerhans cells within human skin

References

  1. Hoyer S, Gerer KF, Pfeiffer IA, et al. Electroporated antigen-encoding mRNA is not a danger signal to human mature monocyte-derived dendritic cells.  J Immunol Res. 2015;952184.
  2. Sasaki I, Hoshino K, Sugiyama T, et al. Spi-B is critical for plasmacytoid dendritic cell function and development.  Blood. 2012;120:4733-4743.
  3. Lo CC, Schwartz JA, Johnson DJ, et al. HIV delays IFN-α production from human plasmacytoid dendritic cells and is associated with SYK phosphorylation.  PLoS One. 2012;7(5):e37052.

Analysis of human DC populations on a three-laser BD FACSVerse? flow cytometer

Analysis of human DC populations on a BD LSRFortessa? X-20 cell analyzer

Analysis of key mouse DC subsets (7-color core panel) on a BD FACSCelesta? system

Adding drop-ins: 10-color mouse DC panel on the BD FACSCelesta system

Rapid purification of viable plasmacytoid and myeloid DCs from human peripheral blood

Purification of DC subsets from mouse splenocytes

Image analysis of CD11c expression in mouse spleen and human tonsil

Analysis of Zbtb46 expression

Detection of IFN-α, TNF-α and IL-12 in CpG-stimulated human PBMCs

Immunohistochemical analysis of Langerin (CD207) expression by Langerhans cells within human skin

Analysis of intracellular TLR7 expression by mouse bone-marrow cells


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