Lecture 5 - NK cells Flashcards
Lineage
- The “Common Lymphoid Progenitor” in the bone marrow gives rise to all lymphocytes (including T, B and NK cells)
- NK cells discovered in 1970s – large lymphocyte with granular cytoplasm
- NK cells lack antigen specific receptors (T cell receptors on T cells, B cell receptors on B cells)
- NK cells express various types of innate receptors (respond to cellular surface ligands)
Innate Lymphoid Cells (ILC)
• Lymphocytes derived from common lymphocyte progenitor
- Expression of transcription factor Id2 repress T and B cell development
- Absence of T and B cell receptors and co-receptors
- Express receptor for IL-7
• Migrate to peripheral tissues (dermis, small intestine, Lung)
- 3 types of ILC
ILC 1
simulated by IL-12 & IL-18 from Macrophages and DC. Produce INFɣ for defence against viruses and intracellular pathogens. NK cells are a subset
of ICL1, however ILC1 requires IL-7 for growth and development and NK require IL-15 (NK cells in blood and periphery).
ILC 2
stimulated by Thymic stromal lymphopoietin (TSLP) and IL-33. Produce IL-4, IL-5 and IL-13. Protect against parasites.
ILC 3
stimulated by IL-1β, IL-23. Produce IL-17 and IL-22. Defence against bacteria and fungi.
Natural killer (NK) cells
- Lymphoid cell (subtype of ILC1)
- Kill infected, stressed or tumour cells
- Release toxic granules (similar to CTL)
- They are ready for action once they leave the bone marrow (no further differentiation required)
- 5 – 15% of mononuclear cells in Blood
- Express germline DNA encoded receptors
- (T and B cell receptors are derived from somatic recombination)
- In humans - identified by expressing CD16 (binds Fc region of IgG) and CD56 (adhesion molecule), as well as the absence of CD3
CD3-, CD56+
- Cytotoxic NK cells (blood) = CD3-, CD56lo, CD16+
* Immunomodulatory NK cells (LN) = CD3- , CD56hi, CD16-
Role of NK cells
•Part of the innate immune system (unlike T and B lymphocytes)
•NK cells eliminate both virally infected, stressed
cells and tumours
- Direct contact - cytotoxicity by release of toxic
granules (perforin and granzymes), apoptosis - Indirectly - produce IFN-ɣ and TNF
Indirect protection (NK cells)
• Activity of NK cells increased 20-100 fold by IFN-α, IFN-β or IL-12 produced by macrophages and DC
- Response to many pathogens but especially virus
infections
• NK cells also produce large amounts of INFɣ and TNF
- Activates macrophages, stimulates DC, induces CD4 to TH1 (Cytotoxic T cells and IgG)
• Serve to contain virus infection before Cytotoxic T cells or neutralizing antibodies of adaptive immune system starts
Direct contact (NK cells)
• NK cells eliminate both virally infected and cancer cells by direct contact
- NK cells recognize reduced MHC class I molecules via activating and inhibitory receptors
- Antibody Dependent Cell Mediated Cytotoxicity (ADCC) (NK cells can attach to antibodies (via Fc receptor on surface of cells)
- Direct cytotoxicity by release of perforin and granzymes (Similar to CD8 cytotoxic T cells)
- NK cells also can induce apoptosis via Fas ligand or tumour necrosis factor related apoptosis inducing ligand (TRAIL)
Apoptosis
- Damaged, old or stressed cells need to be removed.
- The mechanism for this is called apoptosis
- Cells often express receptors (Fas and other death receptors) when stressed that once bound to the ligand initiate apoptosis
- NK cells have Fas ligand (CD95L) on their surface, that binds to Fas (CD95) on target cell
- Tumour necrosis factor related apoptosis inducing ligand (TRAIL) binding to death receptors (DR4 & DR5) on target cell
Activating and Inhibitory Receptors
- NK cells can distinguish health cells from infected and stressed cells by surface molecules
- By activating and inhibitory receptors on NK cell surface
- NK cell activity is regulated by a balance between signals from these receptors
Activating receptors
- Stimulate protein kinases that phosphorylate down stream signalling substrates
- Activating receptors usually bind molecules that are expressed as a result of infection or malignancy (stress induced self)
Inhibitory receptors
- Stimulate phosphatases that counter the kinases
- When inhibitory receptors engaged the signal stops the activating signal
- Inhibitory receptors usually bind surface molecules that have high expressing and loss of these molecules indicate dysfunction (missing self)
Receptors on NK cells
- Balance of activating and inhibitory signals determines outcome of target cell
- Many different receptors on NK cells respond to a variety of ligands on target cell surfaces (Many receptors, particularly inhibitory receptors, bind MHC class 1)
- Some of these receptors are very polymorphic and so different individuals will respond differently (similar to the MHC)
- Different NK cells express receptors to a larger or less degree than other NK cells within an individual (So NK cell populations, within a person, respond differently)
NK cell receptors
- Killer immunoglobulin-like receptors (KIR)
- Killer lectin-like receptors (KLR)
- NKG2 and CD94
Killer immunoglobulin-like receptors (KIR)
- Major receptors in humans
- Polymorphic
- Activating and inhibitory
Killer lectin-like receptors (KLR)
- Ly49 receptors in mice
- Polymorphic
- Activation and inhibitory
NKG2 and CD94
- Both lectin-like receptors
- Form heterodimer together
- Interact with HLA-E in humans and Qa1 in mice
- HLA-E is not polymorphic but binds fragments of other HLA class 1 – detects reduced HLA class 1 (NKG2A, C, E and F)
- NKG2D activating receptor binds MIC-A, MIC-B and RAET1 family which are induced by stress
Inhibitory and Activating Cytoplasmic tails
- Immunoreceptor tyrosine-based inhibition motif (ITIM)
* Immunoreceptor tyrosine-based activation motif (ITAM)
Immunoreceptor tyrosine-based inhibition motif (ITIM)
- When ligands bind (extracellular) the tyrosine in the ITIM becomes phosphorylated
- Binds intracellular tyrosine phosphatases (SHP-1 and SHP-2)
- Phosphatases (SHP-1 and SHP-2) become localised at cell membrane
- These inhibit signalling by other receptors by removing phosphates from tyrosine residues of other signalling molecules
- Long tails for KIR
Immunoreceptor tyrosine-based activation motif (ITAM)
- Cytoplasmic tail associated with signalling protein called DAP12 (containing ITAM)
- When ligation occurs ITAM becomes phosphorylated and activates tyrosine kinases (Syk or ZAP-70)
- Further signalling lead to release of toxic granules
Killer Immunoglobulin-like Receptors (KIR)
- Major receptors on NK cells in humans
- Members of immunoglobulin superfamily
- 17 genes or pseudogenes
- Encoded on Chromosome 19
- Highly polymorphic
- Number and type of KIR genes present
- Allelic variation within each KIR gene
KIR structure & nomenclature
• Extracellular Ig domains (D)
- 2D (binds HLA-C)
- 3D (binds HLA-A & B)
• Cytoplasmic tail
- Long (L) - inhibitory
- Short (S) - activating
• For example
- KIR3DL1
- KIR2DS2
KIR genotypes
• NK cell action depends on balance of activating and inhibitory signals
- Activating KIR provide responsiveness
- Inhibitory KIR provide self-tolerance
- KIR bind to HLA-A, B and C (classical MHC class 1)
- KIRs are members of the immunoglobulin superfamily which are located on chromosome 19q13.4 in the leukocyte receptor complex (LRC)
KIR genes
- KIR contains 17 genes
- P in the name indicates a psuedogene
- Different individuals will have different sets of KIR genes forming “haplotypes”
- “A” haplotype is absence of any of the B haplotypes genes
- “B” is has more activating KIR than A
NK cells and Cancer
- NK cells kill many types of tumour cells
- Also respond to the absence of class I MHC molecules because the recognition of class I MHC molecules delivers inhibitory signals to NK cells.
- Some tumours lose expression of class I MHC molecules, perhaps as a result of selection against class I MHC–expressing cells by CTLs.
NK cells and Cancer (loss of MHC class I)
• Loss of class I MHC molecules makes the tumours particularly good targets for NK cells.
• Some tumours also express MIC-A, MIC-B, and ULB, which are ligands for the NKG2D activating receptor on NK cells.
NK cells targeted to tumour cells
• In addition, NK cells can be targeted to IgG antibody–coated tumour cells by Fc receptors (FcγRIIIor CD16).
• The tumouricidal capacity of NK cells is increased by
cytokines, including type1 interferon (IFN-αβ), IL-15, and IL-12, and the anti-tumour effects of these cytokines are partly attributable to stimulation of NK cell activity.
How do Innate Lymphoid cells (ILC) differ from T and B
lymphocytes?
- Expression of transcription factor Id2 repress T and B cell development
- Absence of T and B cell receptors and co-receptors
- Express receptor for IL-7
What role do NK cells play in the immune system – what do they protect against?
- NK cells respond to cellular surface ligands
- Kill infected, stressed or tumour cells
- Release toxic granules (similar to CTL)
- Express germline DNA encoded receptors
How would identify an NK cell by flow-cytometry?
Labelling lymphocytes with fluorescent antibodies specific for CD16, CD56 and CD3, followed by flow cytometry analysis, enables the identification of NK cells and distinguishes them from NK-T cells
Describe the indirect mechanism by which NK cells destroy target cells
- produce IFN-ɣ and TNF
- this activates macrophages, stimulates DC, induces CD4 to TH1 (Cytotoxic T cells and IgG)
What is the “missing self” hypothesis?
According to the ‘missing self’ hypothesis, one function of NK cells is to recognize and eliminate cells that fail to express self major histocompatibility complex (MHC) class I molecules
How do activating and inhibitory receptors work in identifying target cells to kill?
- Activating receptors usually bind molecules that are expressed as a result of infection or malignancy (stress induced self)
- Inhibitory receptors usually bind surface molecules that have high expressing and loss of these molecules indicate dysfunction (missing self)
List and describe the types of receptors used by NK cells.
- Killer immunoglobulin-like receptors (KIR)
- Killer lectin-like receptors (KLR)
- NKG2 and CD94
What does KIR stand for?
Killer immunoglobulin-like receptors
How would you describe KIR2DS1 what haplotype would a person have this KIR be?
2D - binds HLA-C
S - Short (activation)
this is a B haplotype and is more activating
Describe the direct contact mechanisms by which NK cells destroy target cells
- NK cells recognize reduced MHC class I molecules via activating and inhibitory receptors
- Antibody Dependent Cell Mediated Cytotoxicity (ADCC)
• NK cells can attach to antibodies (via Fc receptor) on surface of cells
