The KIR Receptor Complex

The adaptive immune response recognises infection through presentation of pathogen derived peptides in association with MHC to the host T cells. One of the mechanisms which pathogens use to evade this immune response is to down regulate their MHC cell surface expression. Natural Killer (NK) cells are able to detect altered expression of MHC through a number of cell surface receptors leading to target cell lysis. These receptors include the killer immunoglobulin like receptors (KIR), which are also expressed on some effector T cells. In humans, the KIR gene cluster is located on chromosome 19. KIR genes are both polygenic and polymorphic. The KIR gene cluster codes for 15 expressed KIR genes and 2 pseudo genes.

The ligands for KIR receptors are HLA class I molecules. These include HLA-C locus antigens with either Asn (Group 1 HLA-C antigens) or Lys (Group 2 HLA-C antigens) at position 80, the HLA-Bw4 epitope and some HLA-A antigens.

KIR receptors binding to HLA class I are either inhibitory or stimulatory with the overall effect of NK cell interaction with the target cell dependent on the balance between these inhibitory and stimulatory signals. It is thought that the inhibitory KIR’s bind class I with greater affinity than the corresponding activating KIR with the effect that under normal circumstances the inhibitory signal prevails. The ‘missing self’ hypothesis holds that NK cell alloreactivity occurs when the ligand for inhibitory KIR receptors is down regulated or ‘missing’, leading to activation. This however requires that KIR receptors engage their cogent HLA class I molecules during maturation to acquire effector function. NK cells that express only inhibitory KIRs for absent HLA class I molecules are hypo responsive in the non-transplant setting.

Inhibitory KIR receptors possess long cytoplasmic tails with immunoreceptor tyrosine based inhibitory motifs (ITIMs). Activating KIR receptors have short cytoplasmic tails that pair with adaptor molecules with immunoreceptor tyrosine based activating motif (ITAMs). The nomenclature for KIR receptors therefore includes an ‘L’ (long tail) for inhibitory KIR’s and an ‘S’ (short tail) for activating KIR’s. The nomenclature also includes ‘P’ for pseudo genes. The inhibitory and activating KIR receptors share sequence and structural similarities in their extracellular domains. KIR’s have either 2 or 3 extracellular immunoglobulin domains and this is reflected in their nomenclature as either ‘2D’ or ‘3D’, giving KIR receptors nomenclature such as KIR2DL1, KIR2DS2 and KIR3DL1, where the final digit indicates the order in which the genes were described.

Different KIR genes have been identified – KIR2DL1-3, KIR2DL5, KIR3DL1-3 are inhibitory, KIR2DS1-5 and KIR3DS1 are activating, KIR2DP1 and KIR3DP1 are pseudo genes and KIR2DL4 has both inhibitory and activating properties. KIR2DL2 and KIR2DL3 recognize HLA-C1 with an Asn80 residue. KIR2DL1 recognizes HLA-C2 alleles with Lys80 residue. KIR3DL1 is the receptor for HLA-B alleles sharing the Bw4 specificity. Finally, KIR3DL2 was shown to function as a receptor for HLA-A3/-A11 alleles when bound to Epstein–Barr virus (EBV) peptides.

The KIR genes assemble into haplotypes with two haplotypes described, ‘A’ and ‘B’. The ‘A’ haplotype has only one activating KIR (2DS4), while the ‘B’ haplotype has a higher number of activating KIRs and generally possess more KIRs than the ‘A’ haplotype.

MICA/MICB

The major histocompatibility complex class I related chain (MIC) was first described in the 1990’s. The genes are located centromeric to the HLA class I B gene. The MIC gene family consists of seven members MICA–MICG. The only two MIC genes which are expressed are MICA and MICB, the others are pseudo genes.

MICA and MICB genes are polymorphic but not as much as the classical HLA class I genes. A total of 107 MICA alleles and over 47 MICB alleles have been described as of Nov 2018. MICA has six exons separated by five introns. Exon 1 encodes the leader peptide, exons 2–4 encode the three extracellular domains, exon 5 encodes the transmembrane domain and exon 6 encodes the cytoplasmatic tail. MICA genes are in linkage disequilibrium with HLA-B alleles

Unlike HLA class I where the polymorphic residues are located mainly in the region that forms the peptide binding groove, polymorphism in MIC is more dispersed throughout the α2 and α3 domains. There is also polymorphism in the trans-membrane region. Many MIC antigens have the same extracellular domains with the only differences lying in the trans-membrane regions.

MICA and MICB antigens are constitutively expressed on epithelial cells, especially those of the gastrointestinal tract and on fibroblasts, monocytes, dendritic cells and on endothelial cells. They are not constitutively expressed on lymphocytes. They are however up regulated in stressed cells and act as a marker of cell stress.

The structure of MICA is similar to that of HLA class I but has some sticking differences. Like HLA class I, MICA has three extracellular domains (α1, 2 and 3), a transmembrane region and a cytoplasmic domain. Unlike HLA class I, the MICA protein does not associate with β2 microglobulin. The MICA α1 and 2 domains form a platform that is analogous to the platform formed by HLA class I α1 and 2 domains. In HLA class I, this platform forms the peptide binding groove. The MICA molecule however has extensive disordering of sections of the alpha helix in the α2 domain resulting in a very shallow groove, incapable of binding peptide. The MICA α1 and 2 platform domains do not interact with the α3 domain except for being linked together through a short linker chain. This allows for some flexibility in the structure.

MIC antigens serve as ligands receptors on NK cells and on some T cells. The MICA molecule interacts with NK cells, γδ T cells, and αβ CD8+ T cells, which express NKG2D, a common activating NK cell receptor. The NKG2D receptor forms a complex with MICA by binding orthogonal to the alpha helices of the platform α1 and 2 domains.

NKG2D Genetic Organisation and Antigen Structure

Natural Killer (NK) cells are one of the major forms of lymphocytes, comprising approximately 15% of all circulating lymphocytes. NK cells are involved primarily in the innate immune response but also contribute to the adaptive immune response. NK cells are characterised by cell surface expression of a number of receptors including Killer cell Immunoglobulin like Receptor (KIR) and NKG2D. NKG2D is also expressed on γδ T cells and on αβ CD8+ T cells.

NKG2D is a major recognition receptor for the detection and elimination of cells either as a result of infection or genomic stress such as in cancer. In NK cells, NKG2D serves as an activating receptor, able to trigger cytotoxicity. The function of NKG2D on CD8+ T cells is to send co-stimulatory signals to activate the T cells. The ligand for the NKG2D receptor is the MICA molecule. The NKG2D receptor forms a complex with MICA by binding orthogonal to the alpha helices of the platform α1 and 2 domains.

The NKG2D gene (also known as KLRK1), is located in the natural killer complex (NKC) on chromosome 12. The NKG2D gene is highly conserved with only a few alleles described. In humans, the NKG2D gene has 10 exons. Exons 2–4 encode the intracellular and transmembrane domain. Exons 5–8 encode the ligand-binding, membrane-bound domain which protrudes into extracellular space.

The NKG2D molecule is a member of a C-type lectin-like family receptor called CD94/NKG2. It is a transmembrane anchored receptor expressed as a disulphide-linked homodimer on the cell surface, with a molecular weight of ~42 kDa. Each NKG2D homodimer associates with two DAP10 homodimers to form a hexameric structure (DAP10 and DAP12 are signalling subunits which are highly conserved in evolution and associate with a large family of receptors in many cell types).

Signals triggered by the NKG2D receptor binding to MICA ligands are transmitted through the associated DAP10 dimer.

In the alloimmune response, CD56+ NK cells expressing granzyme have been shown to accumulate in kidney biopsies of patients undergoing acute rejection. One proposed mechanism of action is that DSAs are able to bind to the endothelium and to recruit NK cells that produce IFNγ and trigger antibody dependent cellular cytotoxicity (ADCC). Expression of NKG2D on NK cells and CD8+ T cells is modulated by cytokines. IL-2, IL-7, IL-12, IL-15 and IFN-α upregulate NKG2D expression, whereas TGFβ, IFNβ1, IL-21, IL-4, IL-12 and IFNγ downmodulate NKG2D. NKG2D has been shown to be upregulated as part of the alloimmune response.

Minor Histocompatibility Antigens

HLA presents the major genetic barrier to stem cell transplantation. However, evidence that other genetic systems are involved includes GvHD and some degree of rejection even when transplanting with HLA identical siblings. A non-HLA system which is thought to contribute to this is the minor histocompatibility antigen (MiHA) system. Minor histocompatibility antigens comprise of peptides derived from proteins in which some degree of polymorphism exists such that there may be differences between the patient and donor repertoires. These peptides can be presented to the immune system by both HLA class I and II antigens.

The best characterised minor antigens are the Y chromosome derived HY peptide and the autosomal HA1 to HA5 peptides. Minor histocompatibility antigens such as HA1 and HA2 have restricted tissue distribution and are present normally only on haematopoietic cells. Others such as HY are more ubiquitously distributed, expressed for instance on gut epithelium. HA1 and HA2 are expressed on leukemic cells and some tumour cells, making them potential targets for cellular therapy. In mice, allogeneic stem cell transplantation donor CD8+ T cells specific for a MiHA found in the recipient has been shown to inhibit the division of leukemic cells. However, there is a risk in developing GVHD if the T cells are specific for MiHAs expressed ubiquitously on epithelial cells. Immune cell restricted MiHAs such as MiHA HB-1, are ideal targets for graft-versus- leukemia (GVL) since not all nucleated cells would be targeted by responding T cells.