NK Cells

Question 1

What are NK Cells?

Answer 1

Natural killer ( NK ) cells constitute the first line of
defence against cancer and form a subset of the innate lymphoid cell ( ILC ) family, ILC1 , with cytotoxic properties resembling CD8+ T cells - controlling viral, bacterial infection and tumor growth.

5-25% of lymphocytes in blood.

Question 2

Hvad genkender NK Celler? Og har de unik antigen specifikke receptorer?

Answer 2

- Nej.
In contrast to adaptive immune T cells, ILCs lack rearranged unique antigen-specific receptors and, instead, express germline-encoded receptors that recognize common ligands upregulated in cells under stress caused by infection, DNA damage, or malignant transformation, such as MHC class I chain-related gene A and B ( MICA/ B) and UL16-binding proteins ( ULBPs1- 6).

Question 3

Hvordan modbalancerer NK Celler deres aktiverende receptorer?

Answer 3

To counterbalance activating receptors, NK cells express killer inhibitory receptors ( KIR ), primarily directed against self MHC class I molecules to prevent autoreactivity. NK cells can also be shaped by the tumor microenvironment to express checkpoint inhibitors, such as programmed cell death protein-1 ( PD - 1), lymphocyte activating gene 3 ( LAG- 3), and T cell Ig mucin receptor 3 ( TIM- 3 ).

Question 4

Hvad muliggør CD16 i NK Celler?

Answer 4

Mediate antibody dependent cell-mediated cytotoxicity ( ADCC ) via expression of CD16.

Question 5

NK Cells stammer fra?

Answer 5

Hematopoietic Stemm Cells (HSC) –> Common Lymphoid Pregoenitor (CLP) –> Common Helper-ILC Precursor (CHILP) –> NK Cells (ILC-1)+ ILC-1, ILC-2, ILC-3.
- ILC-1,-2,-3 er non cytotoksiske og komplementerer T helper cells vha. pro-inflammatoriske cytokiner, som tilsammen aktiverer CTL’s (Cytotoxic T Lymphocytes)

Question 6

Hvilke typer af NK celler findes der?

Answer 6

1) Cytotoxic NK Cells (CD56+, CD16++)
2) Cytokine producing NK Cells, IFN-g (CD56++, CD16low)

Det er selvfølgelig et spektruM!

Question 7

Hvilke funktioner har de foskellige NK Celler?

Answer 7

NK Cells: Genkende inficerede celler samt tumorer og dræbe disse vha. Granzym, perforiner og Interferon Gamma.
ILC-1: Understøtter T hjælper celler vha. cytokiner og igangsætter CTL (Cd8+ T celler) og T celle responses.

Question 8

Hvad er CD16?

Answer 8

• Cluster of Differentiation 16 er en Fc receptor nødvendig for ADCC hos NK Cells. CD16 binder antistoffer (IgG i deres Fc del)

Question 9

Hvordan aktiveres NK Celler?

Answer 9

• NK Celler reguleres via en balance af inhiberende og aktiverende signaler fra cellerne den interaggerer med. Denne balance afgør om en NK celle aktiveres eller ej.

Question 10

Hvilke aktiverende receptorer/ligander til NK Celler kender du?

Answer 10

• aKIR (Binder MHC-I)
• NKG2D (binder MICA/B)
• CD16 (Binder FC delen af IgG antistoffer)
• NCR (Natural Cytotoxicity Receptor)
• 2B4 (med dens CD48 ligand)

Question 11

Hvilke inhiberende receptorer/ligander til NK Celler kender du?

Answer 11

• MHC-I er en kritisk inhiberende ligand - modvirker reaktion mod “self”.
• iKIR (Binder MHC-I, HLA molekyler)
• NKG2A (Binder HLA-E)

Question 12

Kan aktiverende signaler for NK celler selv signal transducere??

Answer 12

Nej, ofte har de korte cytoplasmiske haler uden signal transducerende egenskaber.
- De associerer generelt med adapter molekyler med ITAMS der, når de fosforyleres, aktiverer downstream signalering.

Question 13

Kan aktiverende signaler for NK celler signal transducere?

Answer 13

Nej, ofte har de korte cytoplasmiske haler uden signal transducerende egenskaber.
- De associerer generelt med adapter molekyler gennem ladede aminosyrer som indeholder ITAMS der, når de fosforyleres, aktiverer downstream signalering. Der resulterer i cytokine eller lytiske granulae frigørelse.

Question 14

Kan inhiberende signaler for NK celler selv signal transucere?

Answer 14

• Ja, det har ofte lange cytoplasmiske haler med ITIMs (immunoreceptor tyrosine-based inhibition motifs). Der når de aktiveres fosforyleres af Lck og Fyn. Herefter kan SHP-1,-2 og SHIP-1,-2 fosforyleres og aktiveres og forhindre yderligere downstream signalerer ved at defosforylere ZAP70/PI3K

Question 15

Hvad binder KIRs?

Answer 15

HLA - A, B, C på target cells.

Question 16

Hvad binder KIRs?

Answer 16

HLA - A, B, C (MHC-I) på target cells.

Både aKIR og iKIR binder MHC-I, NK celler kan altså både blive aktiverede og inhiberede af MHC-I

Question 17

NKG2 familien består af? Og hvilke er aktiverende og inhiberende?

Answer 17

NKG2A: Inhiberende!
NKG2C: Aktiverende.
NKG2D: Stærkt aktiverende.

Question 18

Hvor udtrykkes MICA og hvad binder de til?

Answer 18

gastric and glandular epithelial cells
in the form of intracellular MICA ( 44 ). These proteins
are induced only by stress in virally infected cells and
tumors, with MICA widely reported in cancers of the
lung, breast, kidney, prostate, ovary, colon, melanoma,
and leukemia

• MICA/B er ligander for NKG2D receptoren.

Question 19

Hvad er DNAM-1?

Answer 19

• DNAX accessory molecule 1 (DNAM-1)
• Kontinuerligt udtrykt aktiverende receptor på NK Celler og CTLs bruges til hårdføre tumor celler der er resistente til NK Celle medieret cytotoxicitet.

Question 20

Hvordan kan en tumor undgår drab af NK celler?

Answer 20

• Tumorer kan lave opløseligt MICA/B som snyder NKG2D receptoren på NK celler.
• Tumoren kan udskille TGF-beta som kan nedregulere NKG2D receptor og NCR receptor.

Question 21

How can you take advantage of receptor/ligand interactions to increase NK Cell Tumor control/recognition?

Answer 21

1) Antibody- Dependent Cell- Mediated
Cytotoxicity - Øge recognition af tumorer via mAbs
2) Checkpoint Blockade Therapy - In NK cells, an exhausted phenotype is indicated by higher expression of inhibitory receptors including KIR3DL1 , KIR2DL3 , and PD- 1 , and a lower expression of activating receptors such as NKG2D and NKp46. - Frigør inhiberende checkpoints så som PD-1, NKG2A eller iKIRs ved at binde et antistof til liganden så receptoren ikke aktiveres.
3) Allogeneic NK cell therapy - NK cells mismatched for iKIR-MHC I with the recipient, have iKIRs that do not recognize donor MHC I and thus are not inhibited, freeing them to recognize stress ligands on transformed tumor cells.
4) Antibody - BiKE and TRiKE - dual and triple antibody linking of tumor to NK Cells to enhance ADCC.

Question 22

Hvad er CAR? Og CAR NK Cells?

Answer 22

• Chimeric antigen receptor (CAR) - giver mulighed for at genkende et specifict antigen.
• Øger NK cellernes tumor specificitet og genkendelse.

Question 23

NK Cells have two major functions - which are?

Answer 23

1) Cytoxicity (CD56+, CD16++)

2) Cytokine production, IFN-g (CD56++, CD16low)

Question 24

Where can you find NK Cells?

Answer 24

They travel around in the body like (activated) T cells. That is, in blood, lymph and in tissue. The whole body more or less.

Question 25

Hvad afgør om en NK Celle aktiveres?

Answer 25

• Balancen mellem aktiverende og inhiberende signaler.
• Især missing self/lack of MHC-I er aktiverende.
• Kræver ofte mindst 2 aktiverende receptorer (NKG2D eller IgG) og resulterer i cytotoxicitet, cytokiner eller begge.

Question 26

Hvis en celle er bundet af et antistof, skal NK Cellen så bruge andre aktiverende signaler?

Answer 26

• Nej, har en celle Antistoffer bundet til sig, er det nok til at aktivere en NK celle igennem dens CD16 (Fc receptor)

Question 27

Hvilke NK Celle receptorer binder MHC I, og hvilken effekt har det?

Answer 27

• NKG2A eller iKIR
  1) NKG2A binder MHC-I (HLA-E) og giver et inhiberende signal.
  2) KIRs binder til specifikke HLA-A,-B, -C og-G alleler og er ofte inhiberende. De kan dog også være aktiverende.

Question 28

NK Celle uddannelse sker på hvilken måde?

Answer 28

• En balance mellem aktiverende og inhiberende signaler -> Licensing.
• Sker i bone marrow.
• Mere negativ signal i licensing = flere positive signaler i balanceringen og nemmere aktivering i vævet.

1) Umoden NK Celle udtrykker iKIR som genkender self-HLA-C, dette aktiverer SHP-1 phosphotase som forhindrer NK Celle aktivering ved at de-p Vav1.
2) Signaler fra aktiverende receptorer (eks. 2B4 eller NKG2D) fosforylerer Vav1.
3) En dynamisk balance mellem de-p-Vav1 og p-Vav1 uddanner cellen.
4) Cellen går til periferien hvor balancen er opretholdt indtil den møder inhiberende ligander (fra sunde celler) eller aktiverende ligander fra en syg celle som aktiverer den ved at danne flere p-Vav-1 end de-p-Vav1

• Because of the variation in HLA-A,-B and -C motifs, we have differences all of us. Meaning some will have more inhibitory signals than others, and others will have less inhibitory signals –> Donor muligheder. Kunne angribe raskt væv, det ser vi dog ikke, vi ved ikke hvorfor.
• You are only licensed if you get a negative signal (could be MHC-I or another). The more negative signals they get, the more positive signals they will get in the balance, and the easier it till be to activate them when needed.
• You can also have unlicensed NK Cells, if you don’t have a build up of negative and positive signals in a balance. They will just have to have more positive signals.

Question 29

Har NK Celler både NKG2A og iKIRs?

Answer 29

• Umodne NK Celler udtrykker først NKG2A:CD94 og derefter iKIRs.
• Er der ingen KIRs der binder MHC-1, så opretholder cellen ekspression af CD94:NKG2A som sin eneste inhiberende receptor for MHC-I (kan være godt i tumorer da disse vil være mere aktive!).
• Er der iKIRs der binder MHC-I, nedregulerer cellen ofte, men ikke altid, NKG2A.

Question 30

Udtrykker NK Celler kun en type receptorer?

Answer 30

• Nej, NK celler udtrykker en diverse kombination af receptorer som er forskellige, men ikke nødvendighvis unikke (ingen somatisk hypermutation eller hypervariabilitet).

Question 31

NKG2D er hvilken type NK Cell receptor, og hvilke ligander binder den? Og hvornår ses disse ligander?

Answer 31

• En aktiverende NK Celle receptor som binder ligander induceret af:
  1) Celle stress
  2) Infektion
  3) Transformation

Question 32

Hvad kan være en NKG2D shortcoming?

Answer 32

1) Tumorer/cancer kan producere opløselig MICA/NKG2D ligand som aktiverer NK og CD8+ T celler. Undgår selv at blive dræbt.
2) Kronisk inflammation kan udtrykke NKG2D ligander på overfladen og udarte sig i kronisk inflammation eller autoimmunitet
3) Vira eller bakterier kan indsætte NKG2D ligander på indersiden af membranen istedet for ydersiden.

Question 33

Har NK Celler hukommelse?

Answer 33

• Ikke på samme måde som det adaptive immun system. MEN.
• Ved CMV infektioner ses det at aktiverede effektor NK cellerne ændrer phenotype. (Får bl.a. flere NKG2C, en aktiverende receptor.) –> kaldes Trained Immunity

Question 34

Hvad er trained immunity?

Answer 34

• Det at efter en infektion, får innate immunceller, NK Celler, en epigenetisk reprogrammering der ændrer deres phenotype til at have flere effector funktioner.

Question 35

Hvad præsenterer HLA-E? Og hvad binder CD94:NKG2A?

Answer 35

• Leader-peptide sequences fra HLA-A,-B og -C

(Den første del af HLA-A,-b og -C som derefter kommes i HLA-E “kløften”)

• CD94:NKG2A binder HLA-E (MHC-I)

Question 36

Hvad er de to overordnede strategier for NK Cell immunoterapi?

Answer 36

• Prepare NK Cells ex vivo and transplant them - for example with CAR NK cells
• Change/act in the microenviroment around a tumor in vivo.

Both could rely on blocking of inhibitory receptors or activating activatory receptors.

Question 37

Nævn 3 strategier for aktivering af NK Celler i cancer

Answer 37

1) Addition of activating cytokines, yet they have seen to be ineffective.
2) Histone demethylation or deacetylating agents (activation) of proinflammatory genes
3) Administration of mAbs triggering activation or inhibiting checkpoints or binding the tumor to the NK Cells via BiKE’s or TRiKE’s. CD16 (Fc receptor) can induce Antibody-dependent cell-mediated cytotoxicity (ADCC) alone.

Question 38

How is it with number of NK Cells and the tumour microenvironment?

Answer 38

• NK Cell infiltration has been detected in multiple solid tumors and - High NK Cell infiltration = high survivability. But they normally comprise of only minor populations. AND it might now have been the proper markers for NK Cells, as other cells express these as well.
• It seems however that the NK Cells mostly reside outside the tumor nest (TN) and are more present in the perifery (parenchym) and therefore lack the direct contact needed for ADCC.

Question 39

How does the tumour microenvironment affect NK Cell function?

Answer 39

• It seems to inhibit the ADCC NK Cell phenotype via Tregs, supresive cytokines produced by other immune cells and by inhibitory mechanisms from the tumour itself.