**8. NK cell-activating and inhibitory receptors**

NK cells express two major types of receptors, inhibitory and activating receptors, that may belong to one of the following receptor categories:


NK cell receptors can be either MHC class I-dependent or MHC class I-independent receptors (**Table 2**).

#### **8.1. MHC class I-dependent receptors**

#### *8.1.1. Killer cell immunoglobulin-like receptors*

Killer cell immunoglobulin-like receptors (KIRs) are a family of type I transmembrane glycoproteins belonging to the immunoglobulin superfamily (IgSF) receptors and grouped together with other receptors of the same IgSF. KIR genes are found in a cluster on human chromosome 19q13.4 within the 1 Mb leukocyte receptor complex (LRC) [28].

KIR molecules are also expressed by some T-cell subtypes. The ligands for several KIRs are subsets of MHC I molecules.

#### *8.1.1.1. Dominant receptors on NK cells*

NK cell functions are regulated by a balance between activating and inhibitory signals [29]. Their receptors recognizing the same MHC I or other ligands are polymorphic and highly homologous and can induce two opposite signals, but one of them dominates signal transduction. Usually, the presence of MHC I molecules on a cell generates a dominant negative signal, but some ligands induced by abnormal or virus-encoded cell damage can stimulate activating receptors and generate a dominant positive signal. Additionally, the absence of MHC I molecules is not sufficient to induce a dominant activating signal, especially in normal cells. From the molecular point of view, it is the length of the cytoplasmic domain that determines the function of NK cells. Thus, long-tailed receptors (L) are associated with an inhibitory function upon ligand binding *via* an immune tyrosine-based inhibitory motif (ITIM), while short ones (S) lack the ITIM and instead associate with the TYRO protein tyrosine kinase-binding protein to transduce activating signals. In the rare situation where an NK cell co-expresses an inhibitory and an activating KIR (KAR) with the same specificity, the inhibitory receptors block activation signals at an early step [30].

**7. "Missing self" hypothesis**

4 Natural Killer Cells

NK cells lack antigen-specific receptors, but their activation can be blocked by an inhibitory signal generated by their recognition of MHC I alleles on host cells. However, the absence of MHC molecules I triggers an activating signal [19]. Nevertheless, the recognition of MHC I molecules would be one of the major causes of the tumor escape from NK cell immune surveillance and activation. Therefore, among the current therapeutic strategies is the use of

NK cells express two major types of receptors, inhibitory and activating receptors, that may

NK cell receptors can be either MHC class I-dependent or MHC class I-independent receptors

Killer cell immunoglobulin-like receptors (KIRs) are a family of type I transmembrane glycoproteins belonging to the immunoglobulin superfamily (IgSF) receptors and grouped together with other receptors of the same IgSF. KIR genes are found in a cluster on human chromosome

KIR molecules are also expressed by some T-cell subtypes. The ligands for several KIRs are sub-

NK cell functions are regulated by a balance between activating and inhibitory signals [29]. Their receptors recognizing the same MHC I or other ligands are polymorphic and highly homologous and can induce two opposite signals, but one of them dominates signal transduction. Usually, the presence of MHC I molecules on a cell generates a dominant negative signal, but some ligands induced by abnormal or virus-encoded cell damage can stimulate activating receptors and generate a dominant positive signal. Additionally, the absence of MHC I molecules is not sufficient to induce a dominant activating signal, especially in normal cells. From the molecular point of view, it is the length of the cytoplasmic domain that determines the function of NK cells. Thus,

(i) Immunoglobulin superfamily (IgSF) - activating or inhibitory - receptors.

monoclonal antibodies that target the NK cell inhibitory receptors.

**8. NK cell-activating and inhibitory receptors**

(ii) C-type lectin family - activating or inhibitory - receptors.

19q13.4 within the 1 Mb leukocyte receptor complex (LRC) [28].

(iii) Natural cytotoxicity - activating - receptors (NCRs).

belong to one of the following receptor categories:

**8.1. MHC class I-dependent receptors**

sets of MHC I molecules.

*8.1.1.1. Dominant receptors on NK cells*

*8.1.1. Killer cell immunoglobulin-like receptors*

(**Table 2**).



Adapted from Ref. [20] and completed from Refs. [21–26]. Costimulatory NK cell receptors are not presented here.\* An unusual activating KIR with L cytoplasmic and hybrid D0-D2 structure domains, displaying very weak inhibitory potential [27]. BAT3, B-associated transcript 3; B7-H6, B7 homolog 6; HLA, human leukocyte antigen; HLA-Bw4-I80, HLA-Bw4 molecules containing an isoleucine in position 80; ILTs, immunoglobulin-like transcripts; ILRs, immunoglobulinlike receptors; HA, hemagglutinin; IgSF, immunoglobulin superfamily; KAR, killer cell-activating receptors; KIR, killer cell immunoglobulin-like receptor; LLT1, lectin like transcript-1; CTLR, C-type lectin-like receptor; MICA/MICB, major histocompatibility complex class I-related chain A/B; KLRG1, co-inhibitory receptor killer cell lectin like receptor G1; NCR, natural cytotoxicity receptor; NK cell, natural killer cell; NDHN, Newcastle disease hemagglutinin-neuraminidase; NKAT, natural killer-associated transcript; ULBP, UL-16-binding proteins.

**Table 2.** Inhibitory and activating human NK cell receptors.

#### *8.1.1.2. Classification of KIR molecules*

**NK cell receptors Ligands**

CTLRs CD94-

NKG2C

CD94- NKG2E/H

Adapted from Ref. [20] and completed from Refs. [21–26]. Costimulatory NK cell receptors are not presented here.\*

unusual activating KIR with L cytoplasmic and hybrid D0-D2 structure domains, displaying very weak inhibitory potential [27]. BAT3, B-associated transcript 3; B7-H6, B7 homolog 6; HLA, human leukocyte antigen; HLA-Bw4-I80, HLA-Bw4 molecules containing an isoleucine in position 80; ILTs, immunoglobulin-like transcripts; ILRs, immunoglobulinlike receptors; HA, hemagglutinin; IgSF, immunoglobulin superfamily; KAR, killer cell-activating receptors; KIR, killer cell immunoglobulin-like receptor; LLT1, lectin like transcript-1; CTLR, C-type lectin-like receptor; MICA/MICB, major histocompatibility complex class I-related chain A/B; KLRG1, co-inhibitory receptor killer cell lectin like receptor G1; NCR, natural cytotoxicity receptor; NK cell, natural killer cell; NDHN, Newcastle disease hemagglutinin-neuraminidase;

KIR2DL KIR2DL4/KIR103 (CD158d)\*

KIR2DS (1) KIR2DS1 (CD158h)

(2) KIR2DS2 (CD158j)

(3) KIR2DS3/NKAT7

(4) KIR2DS4 (CD158i)

(5) KIR2DS5 (CD158g)

KIR3DS KIR3DS1/NKB1 (CD158e2) Ligand still needs

CD159c HLA-E loaded

CD159a HLA-E loaded

NKG2D CD314 MICA, MICB,

NCR1 NKp46 (CD335) Viral HA and

NCR2 NKp44 (CD336) Viral HA NCR3 NKp30 (CD337) BAT3, B7-H6 on

HLA-G, HLA-Bw4

HLA-Cw2, HLA-Cw4, HLA-Cw5, HLA-Cw6 HLA-Cw1, HLA-Cw3, HLA-Cw7, HLA-Cw8 With no detectable avidity for C1, C2, or any other HLA class I epitope HLA-C (weak) With no detectable avidity for C1, C2, or any other HLA class I epitope

to be identified (HLA-Bw4-I80, HLA-Bw4-T80, allotype HLA-B\*2705?)

with HLA-A, HLA-B, HLA-C, or HLA-G leader peptide

with HLA-A, HLA-B, HLA-C, or HLA-G leader peptide

ULBP1, ULBP2, ULBP3, ULBP4

human tumor cells

An

NDHN

IgSF receptors

Activating NK cell receptors/KARs

6 Natural Killer Cells

MHC class I-dependent receptors

MHC class I-independent receptors

IgSF receptors

NKAT, natural killer-associated transcript; ULBP, UL-16-binding proteins.

**Table 2.** Inhibitory and activating human NK cell receptors.

KIR molecules are classified into two types according to the number of extracellular Ig-like domains (D), 2D and 3D, and by whether they have a long (L) or short (S) cytoplasmic domain (**Figure 1**) [31].

#### *8.1.2. CD94-NKG2 C-type lectin receptor complex*

C-type lectin receptors are characterized by calcium-dependent carbohydrate recognition domain (CRD) and the presence of one or more C-type lectin-like (CTLD) domains. They play a crucial role in enabling NK cells to discriminate between self and nonself [32, 33]. The CD94- NKG2 C-type lectin receptors have been found to be expressed predominantly on the surface of a majority of NK cells and on subsets of CD8<sup>+</sup> T cells [34] and to be involved in NK cellmediated recognition of MHC I molecules [35]. They are encoded by the NK gene complex (NKC) on human chromosome 12 (12p13.3–12p13.4). NKG2 receptors recognize the nonclassical MHC class I HLA-E molecule and can provide either an activating signal through their noncovalently association with the immunoreceptor tyrosine-based activation motif (ITAM) containing DNAX adaptor protein of 12 kDa (DAP12) or an inhibitory signal when they contain an immunoreceptor tyrosine-based inhibitory motif (ITIM).

The cell surface molecule CD94 is a common invariant chain in five different disulfidelinked heterodimeric transmembrane glycoprotein complexes, including CD94-NKG2A, CD94-NKG2B, CD94-NKG2C, CD94-NKG2E, and CD94-NKG2H [36].

NKG2F is expressed in the cytosol and therefore does not form heterodimers with CD94. NKG2D (apparently not belonging to the NKG2 family) is expressed on the cell surface of NK cells, γδ T cells, and subsets of CD4+ and CD8+ T cells as a homodimer. It lacks an ITIM sequence and is

**Figure 1.** Structure of inhibitory and activating KIRs. DAP12, DNAX adaptor protein of 12 kDa; FcεRI-γ, high-affinity immunoglobulin epsilon receptor subunit gamma; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; KIR, killer cell immunoglobulin-like receptor. This Figure was illustrated using image fragments from Servier Medical Art.

not associated with CD94. Its signaling is achieved by association with two dimers of DAP10, a transmembrane adaptor molecule containing a tyrosine-based signaling motif (YINM), carrying phosphatidylinositol-3 kinase-binding sites in their cytoplasmic tails and recruiting growth factor receptor-bound protein 2 (Grb2) [37].

Both NKG2A and NKG2B are alternative spliced products from a single gene. They can dimerize with CD94 to form inhibitory receptors through their cytoplasmic domains, which contain two ITIMs. Conversely, CD94-NKG2C, CD94-NKG2E, and CD94-NKG2H dimers and homodimer-forming NKG2D and the orphan receptor NKG2F activate NK cells [37–39].

#### **8.2. MHC class I-independent receptors**

There are at least three activating and two inhibitory MHC class I-independent receptors.

#### *8.2.1. Natural cytotoxicity receptors*

Natural cytotoxicity receptors (NCRs) are composed by a heterogeneous group of molecules belonging to IgSF and include NKp46 (NCR1), NKp44 (NCR2), and NKp30 (NCR3) activating receptors targeting most tumor cell lines. They are characterized by a type I transmembrane domain containing a positively charged amino acid residue and a short cytoplasmic tail. All these transmembrane type I receptors are expressed almost exclusively by NK cells. Binding of one or more of these receptors with a specific ligand leads to the increased NK cell activation and cytotoxicity [40]. It has been reported that these receptors can initiate tumor targeting by recognition of heparan sulfate on cancer cells [41].

#### *8.2.2. C-type lectin receptors*

C-type lectin receptors include mostly killer cell lectin-like receptor subfamily G member 1 (KLRG1) and KLRB1 (also known as NK1.1, NKR-P1A, or CD161), which inhibit the cytotoxicity of NK cells and therefore prevent tissue damage. NKR-P1A is encoded by the KLRB1 gene and recognizes lectin like transcript-1 (LLT1) as a functional ligand. Its signaling in NK cells has previously been known to involve the activation of acid sphingomyelinase, which represent the catabolic pathway for N-acyl-sphingosine generation as a second messenger for the induction of apoptosis, proliferation, and differentiation [42]. KLRG1 is expressed by antigen-experienced (memory) CD4+ and CD8+ T cells and by a large proportion of NK cells and naive phenotype CD4+ and CD8+ T cells in umbilical cord blood, as well as in a substantial subset of γδ T cells [43]. KLRG1 can bind three of the classical cadherins (E, N, and R), which are ubiquitously expressed in vertebrates and mediate cell-cell adhesion by homotypic and heterotypic interactions [44]. It has also been postulated to be a marker of senescence [45].
