Natural Born Killers: NK Cells and CD8+ T Lymphocytes

Question 1

How are CD8 T cells and NK cells similar?

Answer 1

Both Cytotoxic T cells and Natural Killer cells essentially do the same job but as part of different types of immunity

CD8 T cells + NK cells = Both kill infected cells/tumour cells

Question 2

Which cell is a major part of the innate immune system?

Answer 2

NK cells: non-specific (or broadly specific), immediate response

Question 3

What do NK cells do?

Answer 3

→Virus enters cell, virus nucleic acids are copied + transcribed + translated to form new viral proteins, which then assemble to form new virus particles which bud out from the cell to give new viruses
→Immune system must detect self-cells when in this state – when producing viral proteins
→NK cells find out what happens inside their target from looking at cell surface proteins

Question 4

Which immune cell is an integral part of the adaptive immune system?

Answer 4

T cells: highly-specific, delayed response`

Question 5

Where do T cells and NK cells originate?

Answer 5

Both arise from common lymphoid progenitor cell

Both part of lymphocyte lineage

Question 6

What is the role of cytotoxic CD8+ T cells?

Answer 6

Cytotoxic T-cells destroy:

• Infected cells (bacteria/viruses/parasites)
• Tumour cells

Question 7

Cytotoxic T-cells/CD8+ T-cells

Answer 7

→Cytotoxic ADAPTIVE immune cells
→Kill virally-infected targets
→Kill tumour cells
→Controlled by TCR recognition, with CD8 co-receptor
→Highly specific (TCR for specific antigen)

Question 8

Natural Killer Cells

Answer 8

→Cytotoxic INNATE immune cells = broader specificity for target, recognise multiple diff target cells
→Kill virally infected targets
→Kill tumour cells
→Various cell surface Rs - Controlled by a balance of signals between different activating and inhibitory receptors on their surface.
→Broad specificity for target cells

Question 9

What determines whether innate NK cell kills its target?

Answer 9

Balance b/w activating signals/inhibitory signals determines whether NK cell kills its target.

→Inhibitory Rs = Inhibit NK cells from killing
→Activating Rs = Activate NK cells to kill

→Inhibitory signals > Activating signals = inhibits NK cell from killing its target.
→Activating signals > Inhibitory signals = activates NK cell to kill its target.

Question 10

What is the role of MHC Class I molecules?

Answer 10

MHC class I proteins at cell surface form a structure that holds antigenic peptides for surveillance T cells.

Question 11

How are MHC I molecules recognised?

Answer 11

MHC I = recognised by CD8+ cytotoxic T cells

Question 12

How do MHC molecules aid pathogen recognition?

Answer 12

MHC class I present intracellular proteins(short peptide) at cell surface

T-cell recognises this MHC I + peptide + detects self/non-self

MHC I–CD8+ cytotoxic T-cells, CD8 coR also binds to MHC I

Question 13

Which proteins are presented on MHC I molecules?

Answer 13

Intracellular Proteins (healthy/tumour/viral proteins) are processed + presented on MHC class I proteins at cell surface.

→Intracellular antigen(protein) enters proteasome. e.g. viral protein synthesised in cytoplasm
→Proteasome cleaves protein into small peptide fragments
→Peptide fragment enters ER + binds to empty MHC I. →Peptide-MHC I complex migrates to cell surface (secretory pathway). MHC I presents the peptide for TCR to bind. (cytotoxic T-cells = kill virally-infected target cell)

Question 14

Describe the structure of MHC I molecules

Answer 14

MHC I = 2 polypeptides, non-covalently bound

Humans: HLA-A , HLA-B , HLA-C

Peptide-Binding Cleft/Groove = 2 𝛂 helices at sides of groove + ꞵ-pleated sheet(base).
ꞵ2 microglobulin supports PBG.

Question 15

Which chr is the MHC gene complex on?

Answer 15

→chr 6
→3 MHC I genes on chr 6 = HLA-A , HLA-B , HLA-C
→2 x chr6 = 6 MHC I genes total per individual

Each MHC I gene is highly polymorphic - many variants of each HLA-A , HLA-B and HLA-C genes

Question 16

Which cells express MHC I molecules?

Answer 16

all nucleated cells

Question 17

Why don’t we see many pathogens mutating to avoid antigen presentation?

Answer 17

MHC class I proteins are central to antiviral immune responses

• Multiple genes (e.g. 2 copies each of HLA-A, B + C)
• High genetic variability within these genes(polymorphic) = MHC proteins are v genetically diverse

Polymorphisms in MHC proteins maintain ability to fight infections

Question 18

Outline what enables MHC I variability

Answer 18

Polymorphisms in upper peptide-binding part of MHC protein - provide variation in the peptide binding groove.

MHC proteins are v genetically diverse.

Question 19

Where on the MHC molecule do pathogenic peptides bind?

Answer 19

Amino acids in the MHC peptide binding groove create pockets where the bound peptide can “anchor”

Question 20

Where do the genetic variations appear within the MHC?

Answer 20

Genetic variations appear in the peptide-binding groove = determines which peptide the specific MHC variant binds

Question 21

How do polymorphisms provide variation in MHC I molecules?

Answer 21

→Different aa substitutions = different size/shape/charge (+ve/-ve) of MHC pockets = determines which aa’s/peptides can bind to the MHC
→Peptides ANCHOR into these MHC PBG pockets
→Different peptides bind to different MHC alleles

Question 22

What substances do TCRs recognise?

Answer 22

TCR recognise 2 things:

• MHC protein itself (hence compatibility)
• Antigenic peptide presented by MHC protein

TCR binds to both MHC protein and peptide antigen being presented by it.
TCR binds diagonally.

Question 23

How does MHC structure allow T cell recognition?

Answer 23

Binds with a diagonal footprint that cuts across both alpha helices with the peptide in between - allows T cell recognition

Question 24

Why can CD8 and TCRs bind to MHC simultaneously?

Answer 24

Distant binding sites allow CD8 and TCR to bind MHC-I at the same time.

CD8 binds to lower MHC.
Lower MHC is conserved = no genetic variation
+ CD8 sequence is also conserved

Question 25

What is the role of CD8 binding to MHC?

Answer 25

CD8 acts as a co-receptor for MHC-I, and is required for the T cell to make an effective response

Question 26

Where on the MHC I molecule do TCRs bind?

Answer 26

TCR binds to the α1α2 domains

Question 27

Which part of the MHC I molecule do CD8 peptides bind?

Answer 27

CD8 binds to the support domains (α3 and β2m) - very highly conserved region to ensure CD8 binding - Similar situation for CD4 and MHC-II

Question 28

Many virus mechanisms reduce MHC I at cell surface presenting viral peptides = evade the immune response. Outline how different viruses may subvert MHC functions.

Answer 28

Microbes may subvert MHC upregulation by: - Inhibit MHC-I production (adenovirus) - Block peptide-MHC binding in ER (HSV) - Retain MHC-I in ER (adenovirus, HCMV) - Target MHC-I for disposal from ER (HCMV) - Downregulate MHC-I from cell surface (HIV)

Question 29

What are NK cells?

Answer 29

Classical NK cells are large granular lymphocytes that are not T or B cells

Question 30

Describe the protein structures of classic NK cells

Answer 30

- Don't express TCR (CD3) or B cell receptor - Express cell surface marker CD56 - CD3- CD56+

Question 31

What are the 2 major roles of NK cells

Answer 31

Cytotoxic functions and cytokine secretion There are different populations of NK specialised more towards either function Most are specialised for cytotoxic functions

Question 32

Immune system detects this downregulation of MHC I at cell surface using ........

Answer 32

KIR on NK cells = Killer Ig-like receptors(inhibitory receptor/signal) = 'missing self' mechanism →All nucleated cells express MHC I. Missing/Low MHC I = danger signal to immune system →Detect ↓ MHC I by Killer Ig-Like Rs (KIR) →KIR bind to MHC I of healthy cell. KIR sends inhibitory signal to NK cells = do not kill, healthy cell →Virus inside cell = ↓ MHC I = KIR cannot bind = no inhibitory signal to NK cells = no ‘do not kill’ signal = NK cell kills target by releasing lytic granules = ‘missing self’ mechanism. NK cell responds to absence of self-MHC protein

Question 33

How do MHC I molecules recognise NK cells?

Answer 33

Killer Ig-like receptors (KIR) are innate immune receptors that regulate the activity of Natural Killer cells - their binding site is within the MHC PBG

Question 34

What is the function of KIR?

Answer 34

When KIR recognise MHC-I they inhibit NK cells from releasing lytic granules (-ve signal)

Question 35

Why do KIR inhibit NK cytotoxic functions?

Answer 35

Some viruses down-regulate MHC-I as a means to evade cytotoxic T cells, loss of MHC-I is also a common feature of tumour cells Mechanism also protects healthy cells

Question 36

How does a lack of MHC I molecules enable NK cytotoxic effects?

Answer 36

If a target cell does not express MHC-I then there is no KIR inhibition, lytic granules will be released to lyse the target Known as “missing self”

Question 37

Where do KIR bind n MHC?

Answer 37

Inhibitory KIR binds to the same face of MHC-I as the T cell receptor KIR only binds 1 end of MHC PBG = slight specificity (not fine specificity like TCR). KIR are polymorphic KIR recognises subsets of MHC-I alleles but is not specific for 1 MHC-peptide complex

Question 38

Describe the variation in KIR

Answer 38

KIR are also polymorphic, + also, individual KIR genes vary in their presence between individuals. Different MHC-I/KIR combinations show disease associations e.g. in HIV infection. →Diff KIRs recognise diff subsets of MHC I proteins. →1 NK cell expresses multiple KIR + other NK Rs, but each KIR has a diff target set of MHC alleles

Question 39

What is the role of natural Cytotoxicity receptors on NK cells?

Answer 39

These provide activating signals to NK cells, but their range of ligands are not well characterised (+ve signal) →KIRs deliver inhibitory signal to NK cell when recognise MHC I →NK cells have various Rs – some activating Rs, some inhibitory Rs →Some activating Rs = natural cytotoxicity Rs →Diff. NK cells express diff activating Rs →Activating Rs on NK cells deliver activating signals to NK cell = NK cell kills target cell →e.g. NK activating R that binds viral haemagglutinin = triggers activation of NK cell to kill →e.g. NK activating R that binds a ligand expressed on tumour cells = triggers activation of NK cell to kill

Question 40

Target cell death/survival by NK cell depends on .............

Answer 40

Activating/Inhibitory signal balance determines whether NK cell kills/not slide 31 - If Activating Rs ligand binding > Inhibiting Rs ligand binding = activate NK cell = NK cell kills target by lysis - If Inhibiting Rs ligand binding > Activating Rs ligand binding = inhibit NK cell = inhibits NK cell from killing target by lysis - Inhibition by inhibitory KIR R binding MHC I - Activation by activating R binding activating ligand

Question 41

How is NK Cell activity regulated?

Answer 41

NK cell activity is regulated by a balance between inhibitory signals (recognising MHCs) and activating signals from other receptors

Question 42

Why do NK cells kill tumour cells?

Answer 42

- Viruses downregulate MHC I - Tumour cells also downregulate MHC I to evade cytotoxic T-cell response = lose inhibition by KIRs. - + Tumour cell expresses stress proteins = ↑ activating signals + ↓ inhibition = NK cells kill target = target cell lysis

Question 43

What is ADCC?

Answer 43

Antibody-Dependent Cell-Mediated Cytotoxicity is a Super activating signal for NK cells - Fc receptors (CD16)

Question 44

Outline how Fc receptors on NK cells activate a lytic response

Answer 44

- Antibodies (Fv) bind antigens on pathogen(virus) cell surface. - Fc receptors(activating Rs) on NK cells recognise bound antibodies. - NK cell binds Ab Fc portion = activating signal to NK cell = NK cell kills target by lysis NK cells (innate immune response) synergise w Ab response (adaptive immune system) = kill cells that Abs bind to = Antibody-dependent cell-mediated cytotoxicity (ADCC)

Question 45

How do Tumour cells evade immune responses?

Answer 45

Similar to many pathogens, tumor cells can evade the adaptive immune system, by downregulating the expression of MHC class I This consequently makes them more susceptible to NK cells

Question 46

How do cytotoxic (T + NK) cells kill target cells?

Answer 46

- NK cells and T cells carry granules filled with cytotoxic proteins - Release cytotoxic granules at site of contact with target cell. (precisely directed towards infected/tumour cell to not damage other cells)

Question 47

Name 3 proteins found in cytotoxic granules in CD8+ T cells

Answer 47

- Perforin - punches holes in target cell membrane = granule contents(other proteins) enter target cell - Granzymes - serine proteases, triggers apoptosis - Granulysin - antimicrobial, triggers apoptosis

Question 48

What is the role of perforin?

Answer 48

Delivers contents of granules into cytoplasm of target cell

Question 49

How do granzymes cause cytotoxic effects?

Answer 49

Serine proteases | Activate apoptosis once inside target cell cytoplasm

Question 50

How does granulysin activate apoptosis?

Answer 50

Antimicrobial action | Causes apoptosis

Question 51

How do CD8 T-cells trigger apoptosis?

Answer 51

T-cell comes into contact w its target cell = triggers cytoskeleton reorganization = granules are delivered to exact site of T-cell contact via TCR with its target cell (TCR-MHC interaction) Targeted process of lytic granules released directly into the target cell = kills target cell (mins) Cytotoxic T-cell kills target ------------------------------------------ CD8 cells can also trigger apoptosis of target through Fas/FasL protein interaction - Not cytotoxic granule-dependent - Fas ligand (FasL) on T cells engages Fas on target cells to trigger apoptotic pathway - Fas/FasL-triggered apoptosis is used to dispose of unwanted lymphocytes - at the end of immune response to eliminate excess T-cells when no longer required - Slower process (hrs) than cytotoxic granule release (mins) - Loss of Fas = autoimmune lymphoproliferative syndrome (ALPS) - swollen lymph glands

Question 52

Describe the Fas/FasL interaction

Answer 52

Fas ligand (FasL) on T cells engages Fas on target cells to trigger apoptotic pathway

Question 53

What is the use of Fas/FasL apoptosis?

Answer 53

Fas/FasL triggered apoptosis is used to dispose of unwanted lymphocytes

Question 54

What is the consequence of loss of Fas?

Answer 54

Loss of Fas can result in autoimmune lymphoproliferative syndrome (ALPS) due to lack of removal of excess lymphocytes

Question 55

Are lymphocytes innate/adaptive?

Answer 55

Innate immunity lymphocytes: Natural Killer cells | Adaptive immunity lymphocytes: T-cells, B-cells