Tumor immune killing depends not only on whether immune cells are present, but also on the balance between inhibitory and activating signals. NKG2A restricts NK cell and CD8⁺ T cell function through HLA-E, whereas NKG2D recognizes tumor stress ligands and enhances immune killing. Together, these two axes influence tumor immune escape and therapeutic response.

Keywords: NKG2A; NKG2D; CD94; HLA-E; NK cells; CD8⁺ T cells; immune checkpoint; tumor immune escape; DAP10; DAP12; MICA; MICB; ULBP; immunotherapy

1 Molecular Composition and Immunological Positioning of NKG2A and NKG2D

1.1 Molecular Composition of NKG2A

NKG2A is encoded by the KLRC1 gene and belongs to the C-type lectin-like receptor family. NKG2A usually does not recognize ligands alone, but forms a CD94/NKG2A heterodimer with CD94 to jointly recognize the non-classical MHC class I molecule HLA-E.

(1) CD94/NKG2A heterodimer

CD94 mainly participates in forming the ligand-recognition structure, while NKG2A provides inhibitory signal transduction capacity. Together, they constitute an HLA-E-recognizing receptor and serve as an important inhibitory checkpoint on NK cells and some CD8⁺ T cells.

(2) Intracellular ITIM motif

The intracellular region of NKG2A contains immunoreceptor tyrosine-based inhibitory motifs (ITIMs). After HLA-E binding, ITIMs are phosphorylated and recruit phosphatases such as SHP-1 and SHP-2, thereby weakening cytotoxic activation signals.

(3) Expressing cells

NKG2A is mainly expressed on NK cells and can also be found on some CD8⁺ T cells, tissue-resident-like T cells, and cytotoxic lymphocytes under chronic stimulation. Increased NKG2A expression usually suggests that immune cells may be regulated by inhibitory checkpoints.

1.2 Molecular Composition of NKG2D

NKG2D is encoded by the KLRK1 gene and also belongs to the C-type lectin-like receptor family, but functionally it is an activating immune receptor. Unlike NKG2A, NKG2D does not need to form a heterodimer with CD94. Instead, it enhances cytotoxic function in NK cells and some T cells by recognizing ligands on tumor cells or stressed cells.

(1) NKG2D receptor

NKG2D can be expressed on NK cells, CD8⁺ T cells, γδ T cells, and some NKT cells. In NK cells, NKG2D can directly participate in cytotoxic activation. In CD8⁺ T cells, NKG2D more often functions as a co-stimulatory receptor that enhances killing responses together with TCR signaling.

(2) DAP10 adaptor protein

Human NKG2D mainly transmits signals through DAP10. DAP10 can connect downstream molecules such as PI3K and Grb2/Vav, promoting immune synapse formation, cytoskeletal rearrangement, degranulation, and release of cytotoxic molecules.

(3) DAP12-related signaling

DAP12 contains an ITAM motif and can mediate stronger activating signals. The relationship between DAP12 and NKG2D is not completely consistent across species and cellular contexts. Therefore, DAP10 and DAP12 should not be simply equated in writing or experimental interpretation. In human NKG2D research, DAP10 is usually the more central adaptor protein.

1.3 Immunological Positioning of NKG2A and NKG2D

NKG2A and NKG2D respectively represent the “inhibitory brake” and “activating recognition” in immune killing. NKG2A reduces NK cell and CD8⁺ T cell responses through HLA-E, while NKG2D enhances attacks against abnormal cells by recognizing stress ligands. Together, they determine whether immune cells can cross the killing threshold.

(1) NKG2A restricts immune killing

When tumor cells or antigen-presenting cells express high levels of HLA-E, CD94/NKG2A signaling can inhibit NK cell degranulation, IFN-γ production, and target cell killing.

(2) NKG2D enhances abnormal cell recognition

When tumor cells undergo DNA damage, replication stress, heat shock, oxidative stress, or malignant transformation, NKG2D ligands such as MICA, MICB, and ULBPs can be upregulated, enhancing immune recognition.

(3) The two axes jointly determine immune status

If the HLA-E/NKG2A axis is enhanced while NKG2D ligands are reduced or shed, tumor cells are more likely to evade immune killing. If NKG2A-mediated inhibition is relieved and NKG2D ligands are sufficiently exposed, NK cell and CD8⁺ T cell killing capacity is more likely to recover.

2 CD94/NKG2A-HLA-E Inhibitory Signaling Axis

2.1 HLA-E Is the Core Ligand of the NKG2A Axis

HLA-E is a non-classical MHC class I molecule that can present short peptides derived from sources such as MHC I signal peptides and is recognized by the CD94/NKG2A complex. Compared with classical HLA-A, HLA-B, and HLA-C, HLA-E has lower polymorphism and is more suitable as an immune checkpoint ligand that broadly regulates NK cell and CD8⁺ T cell function.

(1) HLA-E upregulation

After tumor cells upregulate HLA-E, binding to CD94/NKG2A can increase, transmitting inhibitory signals to immune cells. Under this condition, even if NK cell or CD8⁺ T cell infiltration is present in tumor tissue, actual killing function may still be restricted.

(2) HLA-E and abnormal antigen presentation

Some tumors evade immune recognition by altering classical MHC I expression, while still retaining or upregulating HLA-E to restrict NK cell responses through NKG2A signaling. This makes the HLA-E/NKG2A axis an important complementary pathway in tumor immune escape research.

(3) Significance of HLA-E detection

HLA-E expression level can be used to determine whether tumors have the ligand basis to initiate the NKG2A inhibitory axis. If only NKG2A is detected without HLA-E, the functional significance of this inhibitory checkpoint can be overestimated.

2.2 Downstream Inhibitory Signaling of NKG2A

After CD94/NKG2A binds HLA-E, the intracellular ITIM motifs of NKG2A are phosphorylated and recruit SHP-1 and SHP-2. Through dephosphorylation, SHP-1 and SHP-2 weaken downstream signals from activating receptors, reducing immune cell degranulation, cytotoxic molecule release, and inflammatory factor production.

(1) Inhibition of degranulation

NKG2A signaling can reduce CD107a externalization, indicating suppressed degranulation capacity of NK cells or CD8⁺ T cells.

(2) Reduced cytotoxic molecule release

Granzyme B and Perforin are important execution molecules for cytotoxic killing. When NKG2A signaling is enhanced, this type of killing output may decrease.

(3) Inhibition of cytokine production

The NKG2A axis can reduce production of effector factors such as IFN-γ, further weakening local antitumor immune responses.

2.3 Experimental Interpretation of the NKG2A Axis

The NKG2A-HLA-E axis should not be judged by a single expression result. More reliable interpretation should include receptor expression, ligand expression, and functional output. If NKG2A blockade or HLA-E reduction enhances immune cell degranulation, Granzyme B release, IFN-γ production, and tumor cell killing, it more strongly indicates that this axis has functional inhibitory effects.

(1) Receptor side

Detect the proportion of NKG2A⁺ cells among NK cells or CD8⁺ T cells to determine whether the inhibitory receptor basis is present.

(2) Ligand side

Detect HLA-E expression in tumor cells or tissues to determine whether an effective ligand source exists.

(3) Functional side

Detect CD107a, Granzyme B, Perforin, IFN-γ, and tumor cell killing rate to determine whether the NKG2A axis truly restricts immune killing.

3 NKG2D Ligand Recognition and DAP10/DAP12 Activating Signaling

3.1 NKG2D Recognizes Tumor Stress Ligands

NKG2D ligands mainly include MICA, MICB, and ULBP family members. These ligands are usually expressed at low levels in normal tissues, but can be upregulated under DNA damage, viral infection, heat shock, oxidative stress, malignant transformation, and proliferative stress, enabling the immune system to recognize abnormal cells.

(1) MICA and MICB

MICA and MICB are the most common ligands in NKG2D research. Increased surface expression usually indicates that tumor cells are more easily recognized by NK cells or NKG2D⁺ cytotoxic T cells.

(2) ULBP family

The ULBP family can also be recognized by NKG2D. Different ULBP members show marked expression differences depending on tissue origin, tumor type, and stress-inducing conditions. Research should not use detection of only one ligand to summarize the entire NKG2D ligand axis.

(3) Soluble ligands

MICA, MICB, and related ligands can be released as soluble ligands through proteolytic cleavage or as membrane-bound ligands carried by exosomes and other extracellular vesicles. These forms may reduce ligand availability on tumor cells or regulate NKG2D expression on immune cells, thereby weakening immune killing. Soluble ligands may reduce recognizable signals on the tumor cell surface and induce downregulation of NKG2D on immune cells, thereby weakening immune killing.

3.2 DAP10-Mediated Activating Signaling

Human NKG2D mainly transduces signals through DAP10. The intracellular region of DAP10 can recruit PI3K, Grb2/Vav, and related signaling molecules, promoting cytoskeletal rearrangement, immune synapse stability, degranulation, and cytotoxic molecule release. Increased NKG2D expression only indicates that immune cells have potential recognition capacity. Whether true activation occurs should be judged together with ligand status, adaptor proteins, and functional readouts.

(1) PI3K-related signaling

DAP10 can connect PI3K-related pathways, promoting immune cell activation and metabolic support and making cytotoxic responses more likely to occur.

(2) Grb2/Vav-related signaling

Grb2/Vav participates in cytoskeletal and immune synapse regulation, helping immune cells form effective contact with target cells.

(3) Functional output

After NKG2D activation, enhanced degranulation, increased Granzyme B and Perforin release, elevated IFN-γ production, and increased tumor cell death should be observed.

3.3 DAP12-Related Issues

DAP12 contains an ITAM motif and can mediate strong activating signals, but its relationship with NKG2D needs to be interpreted according to species and cell background. In human NKG2D research, DAP10 is usually the core adaptor protein. DAP12-related signaling is more commonly discussed in mouse NKG2D isoforms or specific experimental systems.

(1) DAP10 and DAP12 should not be conflated

DAP10 is more associated with co-stimulation and PI3K-related signaling, whereas DAP12 is more associated with ITAM-mediated activating signaling. Their downstream signal properties are different.

(2) DAP12 involvement cannot be inferred only from NKG2D expression

Whether DAP12 participates should be judged according to species, cell type, NKG2D isoform, and adaptor protein detection results.

(3) Human experimental systems should prioritize DAP10

In human NK cells and most human tumor immunology models, DAP10 is usually the main adaptor protein for interpreting NKG2D function.

4 Balance Between NKG2A- and NKG2D-Mediated Immune Activation

4.1 Inhibitory and Activating Signals Jointly Determine the Killing Threshold

Whether NK cells and CD8⁺ T cells kill tumor cells depends on the integrated result of inhibitory and activating receptor signals. The NKG2A-HLA-E axis raises the killing threshold, while the NKG2D-stress ligand axis lowers the killing threshold. These two axes are not isolated pathways, but jointly determine killing intensity within the same immune cells and tumor microenvironment.

Figure 1. Balanced regulation of NK-cell cytotoxicity by the HLA-E/NKG2A inhibitory axis and the NKG2D activating axis

4.2 Why NKG2A or NKG2D Should Not Be Interpreted Alone

High NKG2A expression suggests that immune cells may be suppressed. However, if tumor cells lack HLA-E, this axis may not be the main limiting factor. High NKG2D expression suggests that immune cells have activation potential. However, if MICA, MICB, or ULBPs are insufficient on the tumor cell surface, or if abundant soluble ligands induce NKG2D downregulation, NKG2D expression itself does not represent effective killing.

(1) NKG2A should be interpreted together with HLA-E

Only when both receptor and ligand are present and functional suppression is observed can the NKG2A axis be considered functionally relevant.

(2) NKG2D should be interpreted together with ligand status

High NKG2D expression does not equal strong activation. It is necessary to analyze whether NKG2D ligands are sufficiently exposed on tumor cells.

(3) Final interpretation should return to killing function

Receptor and ligand expression provide only the mechanistic basis. CD107a, Granzyme B, Perforin, IFN-γ, and target cell death are key readouts for judging immune function.

5 NKG2A/NKG2D Signaling and Tumor Immune Escape

5.1 HLA-E Upregulation Mediates Inhibitory Escape

Tumor cells can upregulate HLA-E to enhance binding to CD94/NKG2A, thereby reducing the ability of NK cells and CD8⁺ T cells to attack tumor cells. This escape mechanism differs from the PD-1/PD-L1 axis and more directly affects the killing threshold of NK cells and some cytotoxic T cells.

(1) Restriction of NK cell killing

NK cells use integrated inhibitory and activating receptor signals to determine whether a target cell is abnormal. Increased HLA-E can restrict NK cell degranulation and cytotoxic molecule release through NKG2A signaling.

(2) Restriction of CD8⁺ T cell effector function

Some tumor-infiltrating CD8⁺ T cells can express NKG2A. HLA-E-NKG2A signaling can further inhibit cytotoxic function beyond TCR recognition.

(3) Coexistence with other immune checkpoints

The NKG2A axis can coexist with immunosuppressive pathways such as PD-1, TIGIT, and LAG-3, forming a multilayered inhibitory network in the tumor microenvironment.

5.2 NKG2D Ligand Regulation Mediates Immune Escape

NKG2D ligands should help the immune system recognize stressed or transformed cells, but tumors can weaken this axis through multiple mechanisms. Ligand downregulation, reduced surface expression, soluble ligand release, and immunosuppressive factors in the tumor microenvironment can all reduce NKG2D-mediated immune recognition.

(1) Reduced surface ligands

Tumor cells can reduce surface expression of MICA, MICB, or ULBPs, lowering the chance of recognition by NK cells.

(2) Soluble ligand release

After shedding of ligands such as MICA and MICB, surface recognition signals on tumor cells decrease, and NKG2D downregulation on immune cells can be induced.

(3) Microenvironmental suppression

TGF-β, chronic inflammation, metabolic stress, and immunosuppressive cells can reduce NKG2D expression or weaken its signal output.

5.3 Dual-Pathway Imbalance Promotes Immune Tolerance

Enhanced NKG2A signaling and weakened NKG2D signaling can jointly promote tumor immune escape. The former strengthens inhibitory “do not attack” signals, while the latter weakens activating “recognize abnormality” signals. When both occur simultaneously, immune cells have difficulty fully recognizing tumors and are also restricted by inhibitory checkpoints, ultimately showing reduced degranulation, insufficient cytotoxic molecule release, decreased IFN-γ production, and lower tumor killing efficiency.

6 NKG2A- and NKG2D-Targeted Immunotherapy Research

6.1 NKG2A Blockade Strategy

The goal of NKG2A blockade is to relieve HLA-E-mediated inhibitory signaling and restore killing function in NK cells and CD8⁺ T cells. This strategy is suitable for models with high HLA-E expression, increased proportions of NKG2A⁺ immune cells, and the presence of NK cell or cytotoxic T cell infiltration but insufficient killing.

(1) Releasing the inhibitory checkpoint

Blocking NKG2A can reduce the restriction imposed by HLA-E on NK cells and CD8⁺ T cells, making it easier for immune cells to complete degranulation and cytotoxic output.

(2) Combination with the PD-1/PD-L1 pathway

NKG2A and PD-1/PD-L1 involve different receptor–ligand systems and inhibitory mechanisms, but their target cell populations overlap, as both can regulate subsets of tumor-infiltrating CD8⁺ T cells. Combined analysis helps explain why blockade of a single checkpoint may be insufficient.

(3) Functional validation

If CD107a, Granzyme B, IFN-γ, and tumor killing rate increase after NKG2A blockade, this suggests that the inhibitory axis may have therapeutic intervention value.

6.2 NKG2D Enhancement Strategy

The core of NKG2D enhancement is to increase the recognizability of tumor cells or enhance cytotoxic function triggered through NKG2D in immune cells. This direction is often combined with NK cell therapy, CAR-NK, γδ T cells, DNA damage stress, and ligand shedding inhibition research.

(1) Increasing surface ligand expression

Radiotherapy, DNA-damaging drugs, epigenetic regulation, or cellular stress can increase NKG2D ligand exposure on some tumor cells.

(2) Reducing ligand shedding

Inhibiting shedding of MICA/MICB and related ligands helps maintain recognition signals on the tumor cell surface and reduces negative regulation of NKG2D by soluble ligands.

(3) Enhancing effector cell recognition

Strategies such as NKG2D-CAR, NK cell expansion, or cytokine preactivation can enhance immune cell recognition and killing of NKG2D ligand-positive tumor cells.

6.3 Combination Therapy Logic

NKG2A blockade and NKG2D enhancement are complementary. The former releases the inhibitory brake, while the latter enhances activating recognition. If a tumor has both high HLA-E expression and low surface expression of NKG2D ligands, NKG2A blockade alone may be insufficient to form strong killing. If NKG2D ligands are abundant but NKG2A inhibitory signaling is strong, NKG2D enhancement may also be offset by the inhibitory axis. Therefore, combination strategies should be designed based on the expression and functional status of HLA-E, NKG2A, NKG2D, and their ligands.

7 Products Related to NKG2A/NKG2D Research

NKG2A and NKG2D respectively represent an inhibitory checkpoint and an activating stress-recognition axis in tumor immunity. Enhanced NKG2A-HLA-E signaling raises the immune killing threshold, whereas exposure of NKG2D ligands helps strengthen tumor recognition. Establishing a continuous evaluation system based on “receptor expression-ligand status-signal output-killing function” is useful for explaining tumor immune escape mechanisms and provides a basis for NK cell, CD8⁺ T cell, and combination immunotherapy research.

For more related articles, please see below:

[1] Killer T Cell-Mediated Apoptosis Pathway: Recognition, Effector Molecule Release, and Target Cell Death Mechanisms

[2] Immune Regulatory Targets in Therapeutic Monoclonal Antibodies: Mechanisms of the BAFF and PD-1 Pathways