Dendritic cells Flashcards
Dendritic cells are at the edge between what?
Dendritic cells are at the edge between innate and adaptive immunity as they take a pattern and convert it to a specific sequenced to be expressed as part of adaptive immunity
what family do DC’s belong to?
leukocytes
what are the function of DC’s and what have they evolved to do?
Primary function is to capture and present protein antigens to naïve T-lymphocytes
Evolved to translate innate recognition into adaptive immunity
DC’s are professional…
…APC’s
APCs also macrophages and B-lymphocytes
APC’s - 2 major functions during adaptive immunity?
Capture and process antigens for presentation to T-lymphocytes
Produce signals required for proliferation and differentiation of lymphocytes
what is a unique role of DC’s out of all of APC’s
Among the APCs, dendritic cells are unique in their ability to present antigen to naïve T cells and therefore to induce primary immune responses
DC is the ONLY one capable of stimulating a naïve T cell
what happens when an immature DC captures an antigen?
once antigen is captured from a site of inflammation, immature DC travels to secondary lymphoid organ
here, DC’s present antigen to T cells and B cells. upon interacting, they die but the T and B cells have already been activated
B and T cells migrate to the site of inflammation, on the way producing cytokines that recruit cells from innate immunity such as macrophages
how can DC’s be generated in vitro?
You can generate dendritic cells invitro from monocytes
Monocytes in the presence of GM-CSF and IL-4 make an immature DC, CD14 drops of the surface
Use IL1 6 TNF will make a mature DC
Dendritic cell life cycle
generated from hematopoietic stem cells in the bone marrow
differentiate under the control of a complex network of soluble growth factors produced by bone-marrow stroma and direct cell-cell contact with bone marrow stromal cells
e.g. GM-CSF, IL-3, FLT3L
give rise to circulating precursors that home to tissues where they reside as immature cells
when an immature DC becomes mature, what does it lose?
When an immature DC becomes mature, it loses its endocytic functions, but upregulates all the molecules required for T cell, B cell interaction such as CD86 and CD-40
where are immature DC’s located?
Immature cells are widely distributed in all tissues particularly those which interface the environment
Located throughout epithelium of the skin, the respiratory tract, and the gastrointestinal tract
Comprise only 0.1-1% of cells in different lymphoid and non-lymphoid tissues
where are immature DC’s recruited to, and by what?
Recruited to sites of inflammation in peripheral tissue
- By chemokines - MIP-3α (CCL20), RANTES (CCL5) and MIP-1α (CCL3)
- Immature DCs express chemokine receptors – CCR1, CCR2, CCR5, CCR6 and CXCR1
- DCs accumulate at the site of infection very rapidly – within an hour
what are immature DC’s very efficient at?
antigen capture
Antigenic material includes…
Apoptotic bodies – cell undergone apoptosis Bacterial material Material from virally infected cells Hsp/antigen complexes Immunoglobulin cross-linked material Extracellular fluid Material from healthy cells
HSP
Heat shock proteins – when undergoing heat stress, they form a scaffold in the cell to stop it from denaturing
HSP in tumour cells, when cultured with DCs, allow the DC to present tumour antigens
HSPs have peptides bound to them such that when HSP is taken up the peptide is taken with it
name some antigen uptake pathways
- Receptor mediated endocytosis
- Phagocytosis of particulate material
- Macropinocytosis
Receptor mediated endocytosis
C-type lectin receptors: mannose receptor, DEC-205 - bind to bacteria
Fcγ receptor types I (CD64) and types II (CD32) - bind to antibodies bound to bacteria
CD91 α2-macroglobulin receptor (hsp) - can take up HSPs (tumour peptide delivery)
Phagocytosis of particulate material
Apoptotic and necrotic cell fragments Bacteria including mycobacteria Intracellular parasites such as Leishmania major Viruses Latex beads
Macropinocytosis
Aquaporins – water channels involved in regulating osmotic pressure, may be responsible for constitutive macropinocytosis
Maturation - what is needed?
Dendritic cells need to receive a maturation stimulus to trigger their transition from immature antigen capturing cells to mature antigen presenting cells
Under steady-state conditions only a small number of tissue-resident DCs ‘spontaneously’ mature and migrate to DLNs
Maturation Stimuli
Pathogenic molecules as a consequence of infection:
- lipopolysaccharide (LPS)
- Bacterial DNA
- dsRNA
Balance between pro- and anti-inflammatory signals in local environment
-TNF-α, IL-1, IL-6, IL-10, TGF-β and prostaglandins
T cell derived signals
- CD40L
DC Maturation - what happens?
Down regulation of inflammatory chemokine receptors
-chemokines usually hold DC’s at the site of inflammation, so by downregulating them DC’s can migrate to secondary lymphoid organs
Down regulation of antigen capture
-Loss of endocytic and phagocytic receptors
Change in morphology
- Loss of adhesive structures
- Cytoskeleton remodelling - allows them to move, reduce adhesive molecules
- Acquisition of high cellular motility
Up regulation of receptors for homing to lymphoid tissue
-CCR7 surface receptor
Up regulation of antigen presentation
Up regulation of co-stimulatory molecules
-CD40, CD58, CD80, CD86
Dendritic Cell Migration - what is it regulated by?
Migration of Dendritic cells to the lymphoid tissues is regulated by chemokine - chemokine receptor interactions
Dendritic Cell Migration - what happens?
DCs become responsive to CCL19/MIP-3β and CCL21/ 6Ckine
- CCL19 is expressed in the afferent lymph ducts
- CCL21 is expressed in high endothelial venules of lymph nodes and in the T-cell areas of spleen and lymph nodes
CCL19 and CCL21 guide DCs from the tissue to the T cell areas of lymph nodes
Its moving to an environment where it can present the antigen it has captured
Expression of chemokine receptors by immature vs mature DC’s?
Immature DC receptors CCR1 CCR2 CCR4 CCR5 CCR6 CXCR1 CXCR4
Mature DC
CCR7
Expression of chemokine receptors AND their ligands by immature vs mature DC’s?
CCR1 - MIP-1α (CCL3), RANTES (CCL5), MCP-3, MIP-5 CCR2 - MCPs CCR4 - TARC, MDC CCR5 - MIP-1α, MIP-1β CCR6 - MIP-3α (CCL20) CXCR1 - IL-8 CXCR4 - SDF-1
Mature DC
CCR7 - MIP-3β (CCL19), 6Ckine (CCL21)
role of CCR7?
CCR7 is a surface receptor upregulated during DC maturation
it is specific for driving these cells from the circulation from the tissues to the lymph nodes
factors involved in DC maturation?
- inducing factors
- Cytokines: e.g. GM-CSF, Il-3, FLT3L
- Endothelial migration
DC precursors - properties:
Cytokine Release - IFN alpha, TNF, IL-1
Immature DC - properties: Antigen Capture Endocytosis Phagocytosis High intracellular MHCII (MIICs) High CCR1, CCR5, CCR6 Low CCR7 Low CD54, 58, 80, 86 Low CD40 Low CD83 High CD68 No DC-LAMP Tissue distribution
Mature DC - properties: Antigen Presentation Low endocytosis Low phagocytosis High Surface MHCII Low CCR1, CCR5, CCR6 High CCR7 High CD54, 58, 80, 86 High CD40 High CD83 Low CD68 High DC-LAMP
immature DC into a mature DC:
Pathogens: e.g. LPS, bacterial DNA, viral dsRNA
Cytokines: e.g. TNF, MCM
T cells e.g. DC40L
Structure of lymph node
germinal centres
Interactions in the lymph node
Mature DCs migrate to the T cell zones in the lymph node
Stimulate quiescent, naïve and memory T and B lymphocytes
Selection of rare CD4+ and CD8+ T cells and B cell clones
Induce an immune reaction by priming cytoxic T cells and helper T cells
- Antigens presented in the context of Class II prime T helper cells
- Antigens presented in the context of Class I prime Cytotoxic T cells
DC - T Cell interactions
DC-SIGN
(DC-specific intercellular adhesion molecule (ICAM)-3 grabbing non-integrin)
DC specific ligand for ICAM-3 expressed on naïve T cells
Promotes transient clustering between a DC and a T cell, this allows a DC to screen numerous T cells for a matched TCR
Dectin-1
DC specific type II C-type
Lectin binds T cells promoting their proliferation
CD80 and CD86
Co-stimulatory molecules expressed on mature DCs
Regulate T cell activity
CD40 – CD40L
T cells can activate DCs via CD40L
DC directed B cell activation
Follicular areas of the lymph node
DC can directly signal B cells to proliferate
CD40 : CD40L (T cell) interaction at the DC (menage a trois)
DC secretes cytokines to cause proliferation of B cells and Ig production
DC may hold onto a Tcell for a number of hours before all the appropriate signals have been transferred to activate the T cell
DC interactions with NK cells
Occurs at site of infection - DCs maintain the NK cells in a state of readiness at the site of infection
Pathogen activated DCs can activate NK cells through cell-cell contact and soluble signals
- IFNα, IFN-β, IL-2, IL-12, IL-15 and IL-18
- Leads to NK cell secretion of IFNγ and cytolytic activity
Il-12, IL-18 and IL-15 from DC promote NK proliferation and survival
IFNγ and TNF release from NK cells can mature DCs
Non-MHC dependent
Classical Antigen Presentation
Both endogenous and exogenous peptides are presented by DCs
DCs can present antigen in the context of either MHC class II or MHC class I
Endogenous material is presented on MHC class I molecules
Exogenous material can be presented on both MHC class II (and MHC class I) molecules
Cross-presentation
Dendritic Cells are also able to load exogenous antigens on Class I
How does exogenous material become loaded onto Class I? Mechanism not yet fully understood
Hypothesis: route of antigen internalisation is critical for loading on to MHC class I
Receptor mediated endocytosis and phagocytosis results in presentation on MHC class I e.g. FcR and CD91 mediated internalisation
Dependence on TAP – suggesting cytosolic to ER transport involved
role of cathepsin?
MHC class II presentation
Cathepsin cleaves the invariant chain of MHCII, which in immature DCs is inhibited by cystatin C
DCs can take antigens from the outside and load this onto the proteasome system to present on Class I
What’s the problem with cross-presentation?
Cross-presenting DC are valid targets for CTL, express viral peptides and MHC-I on the cell surface.
DC contain SPI-6 (serine protease inhibitor-6) protect against lysis. Th1 cells induce SPI-6 in DC
BUT ALSO, they can die:
Death of the DC may be a way of self-limiting the response.
Sufficient clonal expansion
Too many CTL may be damaging - Some of the DCs die when there is enough CTL activation
NEEDS TO BE CONTROL
Dendritic cell subsets - how do DC’s differ?
Although all DCs perform essentially the same function to collect, process and present antigen they are a heterogenous group cells
DCs differ in: the signals the respond to the regulatory signals they transmit the precursor cells they derive from their location stage of maturation
Early studies divided dendritic cells into two groups called?
Early studies divided dendritic cells into two groups called DC1 and DC2 based on their ability to induce either Th1 or Th2 responses respectively
The DC1 and DC2 division is an oversimplification
2 diff models
- plasticity
- specialised lineage
how do DC phenotypes differ in tissues than blood?
DC’s tend to have a more activated phenotype in tissues than in blood (increased CD80, 86, 83 and 40 and CCR7).
majority of DC’s located where?
Major population of DC in blood, tissues and lymphoid organs (~1% mononuclear cells)
Plasmacytoid DCs (pDCs)
Plasma-cell like morphology
Minor population of blood DCs
May have rearranged BCR/TCR
Some ability to present antigen, express MHC2, CD80 and 86
High expression of TLR7 and 9
Primary role seems to be in production of Type 1 IFN in response to virus
CD34+/CD11clow/CD45RA+
CD123+ (IL-3R)
Recent evidence suggests that mDC and pDC MAY have a common precursor.
Tissue derived DCs
Langerhans Cells
Found in the skin and skin draining lymph nodes
Express high levels of langerin
Dermal/interstitial DCs
Found in all tissues
Traffic to draining lymph nodes
role of DC’s in tolerance
Some T cells need to be tolerised
DCs play a role in both central and peripheral tolerance
Infection needs to tell ‘tolerising’ DC to become ‘immune activating’
Danger signals
Central tolerance
Mediated by thymic DCs
Thymic DCs Induce the apoptotic death of potentially self-reactive T cells within the thymus
Peripheral tolerance
Mediated by DCs
DCs promote tolerance by killing T cells, paralysing T cells (anergy) or by inducing the generation of antigen specific regulatory T cells
All DCs have the capacity to induce tolerance or immunity, the distinction is depending on their maturation state
Either immature DCs produce tolerance whereas mature DCs immunity
Or Mature DCs need to be activated to produce immunity, quiescent mature DCs maintain self-tolerance
“Danger signals” are required to produce immunity
what are PPR’s?
Pattern Recognition Receptors, which recognise “Danger Signals”
Toll-Like Receptors are a large family of pattern recognition receptors expressed on dendritic cells
Recognise conserved molecular products derived from various classes of pathogen including bacteria, viruses, fungi and protozoa - pathogen associated molecular patterns (PAMPs)
TLR triggering has pleiotrophic effects on DCs
- Promoting survival, chemokine secretion, expression of chemokine receptors, migration, cytoskeletal and shape changes or endocytic re-modelling
role of danger signals?
Danger signals act directly on DCs to drive migration and maturity
Danger signals affect the DC:T cell interaction
role of TLR signalling?
TLR signalling regulates three categories of signals DCs deliver to T cells
Antigen (Signal 1)
Co-stimulation (Signal 2)
Cytokines (Signal 3)
Antigen: Signal 1
In vitro treatment of DCs with LPS increases antigen loading onto MHC class II and surface display of MHCII:peptide complexes
Poly I:C treatment of mouse DC enhances MHCI presentation and cross-presentation.
Co-stimulation: Signal 2
All TLR agonists tested to date increase expression of CD40, CD80 and CD86 in at least one DC sub-set
Cytokines: Signal 3
E.g. CpG and LPS stimulate DCs to produce IL-6 which makes responding T cells refractory to suppression by regulatory T cells
TLR signalling can promote synthesis of IL-1, IL-2 and/or TNF-α by DCs, all of which can contribute to clonal expansion of T cells
TLR ligation can induce production of IL-12p70 which directs differentiation of CD4+ and CD8+ T cells into type 1 effectors
TLR locations and combinations
TLRs found on cell surfaces
TLR1, TLR2, TLR4, TLR5, TLR6
TLRs found in the membranes of the endosomes used to degrade pathogens
TLR3, TLR7, TLR8, TLR9
Many of the TLRs signal in pairs e.g.
TLR1/TLR2 bind bacterial lipopeptides and GPI-anchored proteins in parasites
TLR-6/TL2 pairs bind lipoteichoic acid from gram-positive cell walls and zymosan from fungi
TLR-4/TLR-4 pairs bind lipopolysaccharide from gram-negative cell walls pairs
Th1 / Th2 polarisation
Th1
Key cytokine in polarising immune response is IL-12p70 produced by dendritic cells
IL-12p70 produced in response to DCs detecting intracellular infection
e.g. signalling through endosomally located TLR3 in response to dsRNA in apoptotic cell body
IFN-α produced by pDCs also polarises for Th1
Th2
IL-4 directs CD4 T cells to promote B cell proliferation
In response to DC detecting an extracellular infection
e.g. signalling through cell surface bound TLR5 in response to flagellin
how do viral infections affect DC’s?
May affect the DC ability to act as an APC
Evade the CTL response
BUT cross-presentation of virus taken up by uninfected DCs will induce the CD8 response
