KLRK1

Detailed Information

NKG2D is an activating cell surface receptor encoded by the Klrk1 gene and is predominantly expressed on cytotoxic immune cells. It is abundantly present on all NK cells, NKT cells, and subsets of γδ T cells. In humans, naïve CD8⁺ T cells also express NKG2D; however, in mice, CD8⁺ T cells only upregulate NKG2D expression upon activation. Typically, CD4⁺ T cells do not express NKG2D even after activation, although under specific pathological conditions in humans, such as Crohn’s disease, juvenile lupus, and cytomegalovirus (CMV) infection, its expression can be induced.

The molecular structure of NKG2D enables it to bind a diverse array of MHC class I-like ligands. These NKG2D ligands (NKG2DLs) generally exhibit low expression under homeostatic conditions but are strongly upregulated during cellular stress, including infection or oncogenic transformation. In humans, the ligands of NKG2D include MICA, MICB, and six members of the ULBP (UL16-binding protein) family.

NKG2D functions as a homodimer of two transmembrane proteins linked by disulfide bonds. Its intracellular domain is short and lacks intrinsic signaling capability. In mice, NKG2D transduces downstream signals via adaptor proteins DAP10 and DAP12, whereas in humans, NKG2D exclusively associates with DAP10.

Two isoforms of NKG2D have been identified in mice: the short isoform (NKG2D-S) and the long isoform (NKG2D-L), which differ by 13 amino acids due to alternative splicing of Klrk1 transcripts. This difference leads to distinct signaling capabilities: NKG2D-L interacts solely with DAP10, while NKG2D-S can associate with both DAP10 and DAP12. In humans, only the NKG2D-L isoform is present, explaining why human NKG2D signaling relies solely on DAP10.

DAP10 and DAP12 initiate distinct downstream signaling pathways. DAP10 contains a YINM motif that recruits the p85 subunit of PI3K and can also bind Grb2, which is tightly associated with Vav1. DAP12 contains an immunoreceptor tyrosine-based activation motif (ITAM), which is phosphorylated by Src family kinases upon NKG2D engagement, facilitating recruitment and activation of the tyrosine kinases Syk and Zap70.

T cells and naïve NK cells predominantly express the NKG2D-L isoform and are thus thought to mediate PI3K-driven processes such as costimulation, cytotoxicity, and cell survival. In contrast, NKG2D-S is induced in activated NK cells in mice, enhancing cytotoxicity and cytokine production via Syk/Zap70 signaling.

NKG2D Ligands Overview

There are eight identified NKG2D ligands (NKG2DLs): MICA, MICB, and ULBP1–6. Among these, MICA and MICB were the first discovered and remain the most extensively studied in the context of immunotherapy. MICA and MICB share approximately 80% homology, whereas members of the ULBP family share less than 25% homology with MICA.

The MICA and MICB genes, located within the MHC region, exhibit high polymorphism, and their alleles are expressed in a codominant fashion. Over 100 MICA alleles and 40 MICB alleles have been documented. The ULBP family comprises nearly 20 alleles. This extensive ligand diversity for a single receptor—NKG2D—raises important questions. The answer lies in host-pathogen co-evolution over millions of years, where the "cat-and-mouse" dynamics between host immune surveillance and pathogen immune evasion mechanisms are central themes in immunogenetics. Among pathogens, herpesviruses are thought to be a major evolutionary force driving the diversification of NKG2D ligands.

Figure 1. The critical role of NKG2D/NKG2D-L axis in NK-AML

cells recognition. (Wu Z, et al., 2021)

Most MICA and MICB proteins encoded by these alleles possess three extracellular domains, a transmembrane region, and a cytoplasmic tail. A notable exception is the MICA008 allele—the most prevalent variant across different populations, which contains an insertion in exon 5 that causes a frameshift. This results in a truncated protein with a partial transmembrane domain and no cytoplasmic tail. Despite this, MICA008 can still be stably expressed on the cell surface. Due to their polymorphism, MICA and MICB are potential targets for alloimmune responses. Anti-MICA/B antibodies have been detected in the sera of patients experiencing rejection after kidney, heart, or lung transplantation.

Dual Role of NKG2D/NKG2DL in Tumor Immunity

The critical role of the NKG2D/NKG2DL pathway in tumor progression control and immune recognition has been validated in numerous experimental models and clinical studies. These findings emphasize the importance of NKG2D and its ligands in tumor immunology, while also highlighting ongoing controversies regarding their potential in cancer therapy.

The function of NKG2D varies between NK cells and other immune subsets. Studies indicate that activation signals via the NKG2D/NKG2DL axis can override inhibitory receptor signals, positioning NKG2D as a master switch for NK cell activation. Thus, this signaling pathway mediates direct cytolytic effects through NK cells. However, its role in CD8⁺ T cells remains contentious. Raulet's pioneering work demonstrated that NKG2D functions as a costimulatory molecule in CD8⁺ T cells, enhancing their effector functions.

As a multifunctional receptor, NKG2D can bind a wide array of ligands directly on target cells, independently of antigen presentation, and initiate immune effector responses or costimulation. This results in the release of cytotoxic granules, including perforin and granzymes, which mediate tumor cell killing.

The expression of NKG2DLs on tumor cells is induced transcriptionally in response to cellular or genomic stress. These ligands are frequently expressed on tumors of epithelial origin, including ovarian carcinoma, colorectal cancer, and leukemia, but are rarely detectable in normal adult tissues. Furthermore, exposure to DNA-damaging agents, certain cytokines, or proliferation-inducing agents can upregulate NKG2DL expression. MICA/B expression has been observed at low levels in normal tissues such as gastrointestinal epithelium. However, during malignant transformation or in response to other stressors—such as oxidative stress and viral infection—NKG2DLs are upregulated.

Clinical data support a correlation between NKG2D expression and cancer status. For instance, NKG2D expression is significantly higher in mononuclear cells from early-stage gastric cancer patients than those from late-stage patients. Additionally, the percentage of NKG2D⁺ NK cells is reduced in cancer patients, suggesting that downregulation of NKG2D may be a key mechanism underlying NK cell dysfunction in cancer. One major mechanism of NKG2D downregulation is the tumor cell–mediated release of soluble NKG2DLs. These are generated through proteolytic shedding from the tumor cell surface, mediated by disulfide isomerase ERp5, as well as members of the ADAM (a disintegrin and metalloproteinase) and MMP (matrix metalloproteinase) families. These soluble ligands act as immunosuppressive factors that facilitate immune evasion by malignant cells, particularly in advanced-stage cancer. Studies have shown that high concentrations of soluble NKG2DLs can inhibit tumor immunity and NK cell activity by downregulating NKG2D expression or through MICA/B shedding. Emerging data also suggest that soluble NKG2DLs may limit the efficacy of immune checkpoint blockade therapies.