Lecture 9: NK Cells Flashcards
What are NK cells?
Innate like lymphocytes, which do not express polymorphic clonal receptors like T and B cells
What NK functions are similar to CD8 T cells?
- production of perforin and granzyme b to lyse target cells
- production of IFN-y
- kill target cells via FAS/FAS-L interactions
In what ways are NK cells different from CD8 T cells?
- NK cells are not antigen restricted
- do not require antigen presentation via MHC for activation
What is the main role of NK cells?
Kill infected cells or kill cancer cells as they arise in the body
How are NK cells controlled?
By a balance between activatory receptors and inhibitory receptors
How does missing self activation work?
If NK cells detect missing MHCI receptors on the cell surface (loss from viral infection or cancer), the cells are identified as non-self and killed
How does self-induced self activation work?
Stress ligands can be upregulated in cells, which can be bound by NK cell surface proteins to push activation signalling which becomes strong enough to override inhibitory signals received from binding MHC
What different receptors define NK cells in mice and humans?
Mice: expression of NK1.1 in C57Bl6 mice, and Nkp46 in other strains
Humans: expression of CD56
What are examples of activatory receptors?
NKG2D, NKp46/44/30, and CD16
What are examples of inhibitory receptors?
NKG2A and KIR receptors
What are key features of CD56bright NK cells?
- rare subset in the blood (5%)
- abundant in certain tissues
- rapidly produce immunomodulatory cytokines
What are key features of CD56dim NK cells?
- dominant subset in peripheral blood
- play a cytotoxic effector role by releasing granules
- NK cells that go in and kill target cells
What are key features of adaptive NK cells?
- long lived NK cells which lack CD56
- rapidly proliferate and produce cytokines upon viral re-exposure
- kill target cells with the help of antibodies
Where are NK cells derived from?
Secondary lymphoid organs from common lymphoid progenitors (same as B and T cells)
What happens as HSCs develop into multipoint progenitor cells (MPPs)?
Cells gain CD45, so start to commit to becoming immune cells
What happens as MPPs develop into common lymphoid progenitors (CLPs)?
Cells gain CD10 and lose CD34 expression, cell can still become pro-B, pre-T or NK precursor
What happens as CLPs develop into NK progenitors (NKPs)?
Cell starts to commit to becoming an NK cell, so upregulates CD122, NKG2D and CD7
Acquisition of IL-1R1 marks early commitment to becoming NKP, once CD122 is unregulated they are irreversibly committed
What happens as NKPs develop into immature NK cells (iNK)?
Upregulation of activatory molecules NKG2D and NKp46/44/30
What happens as iNKs develop into fully committed mature NK cells?
CD56 is expressed
What experiment was used to test IL-15R importance and what was the outcome?
- transferred NK cells from IL-15R KO mice into either WT mice or IL-15R mice
- use congenial markers to identify NK cells and survival in both sets
- NK cells survived in WT mice but died in KO mice
= IL-15R is required to maintain NK cell numbers
= IL-15R doesn’t function on NK cell
What is IL-15 transpresentation?
- IL15 is not secreted, but is loaded onto a receptor in the ER which is put onto the cell surface
- is presented to NK cells through cell-cell interactions and allows corresponding singaling cascade to occur
What are the 2 main effector pathways of effector mechanisms?
Granule mediated cytotoxicity and receptor mediated cytotoxicity
How is the immune synapse formed?
NK from immune synapse with target cells via integral binding (LFA-1)
How does LFA-1 prevent granule diffusion away from the target cells?
- lytic granules are transported along microtubules and fuse with the plasma membrane to be released into the synaptic cleft
- LFA-1 creates tight junction between cells and holds them in close proximity, preventing diffusion of cytolytic granules out of immune synapse
- perforin punctures holes, lets granzyme in which interacts with pathways such as caspase activation, mitochondrial dysfunction, and caspase independent apoptosis
How do perforin and granzyme kill the target cell?
- neutral pH of extracellular environment allows perforin to insert into the plasma membrane
- interaction with Ca2+ ions allows colocalisation of perforin monomers to create a channel
- granzyme passes through the channel and induces target cell death
What 3 mechanisms do NK cells use to prevent damaging themselves during granule mediated cytotoxicity?
SB9, CD107a, and Cathepsin B
What is SB9?
A granzyme B inhibitor found in the cytoplasm of NK cells, which acts as a substrate for granzyme B resulting in conformational changes and deactivation of the enzymatic activity
What is CD107a?
A degranulation marker which is unregulated to the plasma membrane during degranulation and inhibits the binding of perforin to the NK cell plasma membrane
What is cathepsin B?
Cleaves perforin and prevents insertion into the plasma membrane, as it is expressed at the plasma membrane during degranulation
What are the 3 main receptor mediated cytotoxic pathways?
Fas/FasL, TRAIL/TRAIL R and TNF
What happens in the Fas/FasL pathway?
- FasL binds to Fas
- Induces activation of caspase 8/10
- Results in caspase cascade leading to in programmed cell death and apoptosis
What happens in the TAIL/TRAIL R pathway?
- triggers caspase activation
- triggers caspase cascade
- results in cell death through apoptosis or inhibition of the mitochondria
How does TNF induce cell death?
TNF can bind to TNFR-1 or 2 which induces apoptosis
How does antibody dependent cellular cytotoxicity work?
- Antibodies bind to target antigens on the cell surface (eg. bacterial proteins or viral antigen)
- Fc receptors on NK cells use CD16 to bind to the Fc region of the antibody antigen complex
- Cross linking of CD16 signals to NK cells to release intracellular stores of perforin and granzyme
- perforin punctures holes allowing granzyme to enter and induce apoptosis
Why do we need NK cells?
- same function as CD8 T cells but are much faster to respond
- respond to missing self
- do not require antigen presentation via MHC
What are then main routes of NK immunotherapies?
- Chemokines: NK-specific activation
- Checkpoint inhibition: immune checkpoint blockade
- Antibody mediated interventions: neutralising tumour ligands, or blocking inhibitory receptors so they can’t inhibit NK cell function
- Adoptive transfer of modified cells: engineered CAR NK cells
- Cytokines: induce NK cell activation (IL-2, IL-15)
- Tri-specific NK engagers: trigger activator domain on NK cells and bring them into close proximity of tumour cells
What are CARs and what are they used for?
- Chimeric antigen receptors, often used with T cells and Nk cells to improve immunotherapy function
- Combines various intracellular signalling domains with the fragment of antigen binding (FAB) from an Ig = creates super receptor
What do CARs do?
Lower the activation threshold for NK cells and allow them to bypass many of the inhibitory receptors that would otherwise impede their function in the tumour microenvironment
What are chemokines?
Chemotactic cytokines which bind to and signal through G protein coupled receptors
What are 4 main families of chemokines?
CC, CXC, CX3C, and XC
What is the purpose of the atypical chemokine receptors?
Removes chemokines after an immune response to prevent contact inflammation
What is CCL2?
- CCL2 is an inflammatory cytokine which is upregulated during inflammation
- binds to CCR2 to promote response, and also atypical receptor ACKR2
Why do tumours produce a lot of CCL2?
Expressed early in tumour production with high potential to draw in macrophages - if tumours bring macrophages to the microenvironment, can turn them into tumour associated macrophages and promote angiogenesis, increase tumour growth ands increase blood supply
