Unlocking the therapeutic potential of the NKG2A-HLA-E immune checkpoint pathway in T cells and NK cells for cancer immunotherapy

Abstract

Immune checkpoint blockade, which enhances the reactivity of T cells to eliminate cancer cells, has emerged as a potent strategy in cancer therapy. Besides T cells, natural killer (NK) cells also play an indispensable role in tumor surveillance and destruction. NK Group 2 family of receptor A (NKG2A), an emerging co-inhibitory immune checkpoint expressed on both NK cells and T cells, mediates inhibitory signal via interaction with its ligand human leukocyte antigen-E (HLA-E), thereby attenuating the effector and cytotoxic functions of NK cells and T cells. Developing antibodies to block NKG2A, holds promise in restoring the antitumor cytotoxicity of NK cells and T cells. In this review, we delve into the expression and functional significance of NKG2A and HLA-E, elucidating how the NKG2A-HLA-E axis contributes to tumor immune escape via signal transduction mechanisms. Furthermore, we provide an overview of clinical trials investigating NKG2A blockade, either as monotherapy or in combination with other therapeutic antibodies, highlighting the responses of the immune system and the clinical benefits for patients. We pay special attention to additional immune co-signaling molecules that serve as potential targets on both NK cells and T cells, aiming to evoke more robust immune responses against cancer. This review offers an in-depth exploration of the NKG2A-HLA-E pathway as a pivotal checkpoint in the anti-tumor responses, paving the way for new immunotherapeutic strategies to improve cancer patient outcomes.

General introduction

Tumor immunotherapy holds significant importance in the field of cancer therapy

Immunotherapy as a new milestone differing from existing treatments (surgery, chemotherapy, radiotherapy and molecular targeted therapy) has caught a lot of attention in the combat of cancer.¹ Physiologically, immune checkpoint pathways play an important role in maintaining autoimmune tolerance and regulating peripheral tissue immune responses.² In cancer, however, they may undesirably block antitumor immune responses.^{3 4} Therefore, recently, immune checkpoint blocking therapies (ICB) have been developed as cancer therapy to interfere with these negatively regulating molecules like cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), programmed cell death 1 (PD-1) and its ligand (PD-L1) to allow immune cells to regain their ability to attack tumor cells.^{5 6} Previous studies of immune checkpoints are mostly focused on terminally differentiated effector T cells, but few on natural killer (NK) cells. The overall response rate of first-generation ICB is not high, around 20–30%,^{7 8} and patients with higher tumor mutation burden respond better. Unfortunately, ICB can only restore the function of naturally arising T cells, and whether these are sufficient to eradicate the tumor depends on the patients’ own immune system together with the immunogenicity of the tumor (ie, the number of “foreign” antigens it expresses). Often, the number of patients’ autologous T cells that can recognize tumor antigens is limited, and the overall function of CD8⁺ T cells after repeated chemotherapy is low.⁹ Hence, understanding the mechanisms of resistance to ICB and identifying novel immunotherapeutic targets to overcome the low response rate of first-generation ICB in cancer is necessary.¹⁰

In this review, we will summarize the research progress from preclinical and clinical studies regarding a less explored co-inhibitory pathway NKG2A-HLA-E in oncology, and highlight the current gaps in our knowledge of the interaction between NKG2A and HLA-E. Additionally, we will discuss the in vitro and in vivo strategies of NKG2A blockade to unleash NK cell and CD8⁺ T cell immune responses, and will propose alternative immunotherapeutic targets to be combined with NKG2A blockade that are expressed on both T cells and NK cells.

Introduction to NKG2A receptor and interaction with its ligand HLA-E

NKG2A is encoded by killer cell lectin-like receptor C1 (KLRC1), which is located on chromosome 12. NKG2A molecule, also named CD159a or NK cell receptor A, is an inhibitory receptor in the NKG2 receptor family that is mainly expressed on the surface of NK cells, natural killer T (NKT) cells and some T cells including Th2 cells, αβ CD8⁺ T cells and γδ CD8⁺ T cells (Vδ1 and Vδ2).¹¹ In the peripheral blood of healthy people, about 50% of NK cells and around 5% of CD8⁺ T cells express NKG2A. On the surface of human immune cells, NKG2A and an NK cell surface membrane protein CD94, that is encoded by killer cell lectin-like receptor D1, form a heterodimer complex CD94/NKG2A by a disulfide bond. The CD94/NKG2A heterodimer is expressed prominently by cytotoxic lymphocytes and is able to sense the ligand, HLA-E in humans and Qa-1 (ortholog of HLA-E) in mice on target cells. The non-classical major histocompatibility complex (MHC) class-I molecule, HLA-E, is the predominant ligand for the CD94/NKG2A receptor,¹² which is widely expressed on different cells at low levels and upregulated in many cancer types.¹³ Binding of CD94/NKG2A to its cognate ligand HLA-E inhibits immune activity,¹⁴ and helps to prevent the occurrence and progression of autoimmune diseases.¹⁵

Additionally, CD94/NKG2A as an NK cell inhibitory receptor, may be involved in the functional education of NK cells, setting the balance between inhibitory and stimulatory signals, although this hypothesis was recently challenged.^{5 16} The so-called NK cell education is the process in which the host MHC-I molecules interact with the specific receptors on the surface of NK cells, by which NK cells achieve functional maturity.¹⁷ Educated NK cells have stronger killing and cytokine secretion function against “missing self” (target cells lacking autologous MHC-I molecules), but they do not respond to cells expressing autologous MHC-I molecules because they express MHC-I specific inhibitory receptors to maintain self-tolerance.^{18 19} These inhibitory receptors include classical MHC-I-dependent (ie, KIR in humans, Ly49 in mice) and non-classical MHC-I-dependent (ie, CD94/NKG2A in humans) variants. In addition, they also express MHC-I-independent receptors (ie, CD48, SLAMF6 and Clr-B). Uneducated NK cells developed in an MHC-I deficient environment show reduced recognition function against “missing self”, despite losing the inhibitory MHC-I-associated signal.^{20 21} Although uneducated NK cells are hyporesponsive and require a higher activation threshold compared with their educated counterparts, when they are activated in inflammatory or stimulatory environments, as might occur during infection or cancer, both subsets exhibit similar functionality.^{20 22}

HLA-E is highly conserved, with only two subtypes (E*01:01 and E*01:03), and there is no significant difference in preference of antigen presentation between the two subtypes.²³ HLA-E among others binds epitopes derived from signal peptides (SP) of HLA-A, HLA-B, HLA-C and HLA-G and thereby acts as a “sensor” for changes in expression of HLA,²⁴ allowing detection of HLA downregulation (ie, “missing self”) by microbes or malignant transformation.²⁵ HLA-E presentation of HLA class-I SP to CD94/NKG2A was demonstrated to depend on every component of the HLA class-I loading complex rendering this system highly sensitive to any perturbation in the HLA class-I pathway.^{26 27} Presentation of “functional SP” (ie, SP that enables CD94/NKG2A engagement), however, may be restricted to inflammatory conditions (eg, in the presence of interferons (IFNs), particularly IFN-ɣ), indicating that the CD94/NKG2A-based inhibitory system may operate only secondary to immune activation.^{27 28}

The frequency distribution of functional SP sequence variants differs significantly between individuals as not every HLA allotype produces a functional SP.²⁴ Furthermore, also among functional SP the affinity for CD94/NKG2A may differ.²⁴ Besides presenting SP from HLA class-I, HLA-E can also present pathogen-derived or stress protein-derived peptides. The peptide loaded determines whether HLA-E is a ligand for NKG2A or NKG2C. When HLA class-I derived SP are loaded on HLA-E, the affinity of HLA-E for NKG2A is much higher than that for NKG2C therefore it usually binds to NKG2A,^{29 30} while pathogen peptides often make it a ligand for NKG2C,^{31 32} which delivers a stimulatory signal instead of an inhibitory signal. Moreover, viruses like cytomegalovirus (CMV)³² but probably also some cancers have evolved to present peptides in HLA-E that mimic SP from HLA class-I delivering inhibitory signals via CD94/NKG2A, allowing undetected downregulation of HLA.³³ Thus, depending on the peptide presented, HLA-E may bind inhibitory CD94/NKG2A or activating NK cell receptors such as CD94/NKG2B and CD94/NKG2C abrogating CD94/NKG2A-mediated inhibition.³⁴

Increased interaction of HLA-E expressed by cancer cells and CD94/NKG2A has been shown to limit antitumor responses of CD8⁺ tumor-infiltrating T cells and NK cells in different cancer types (figure 1).³⁵ As we will detail below, the NKG2A-HLA-E axis can function as an acquired resistant mechanism after immune activation.²⁸ Further exploration of the role of the NKG2A-HLA-E pathway may help to increase the efficacy of immunotherapy, especially in patients with cancer.

Figure 1. Interaction between CD94/NKG2A and HLA-E. Overexpression of HLA-E limits acquired T cell responses to tumor and infection. The signal from HLA-E detected by the CD94/NKG2A receptor on CD8⁺ T cells (or NK cells) sends inhibitory signal mediated by two immune receptor tyrosine-based inhibitory motifs (ITIM) from the cytoplasmic tail to inhibit cellular function. HLA, human leukocyte antigen; NK, natural killer; PD-1, programmed cell death 1; PD-L1, programmed death-ligand 1; TCR, T-cell receptor.

NKG2A on NK cells in cancer

NKG2A on NK cells in hematological malignancies

Early studies of NKG2A on the surface of NK cells mainly focused on hematological malignancies. Interestingly, CD94/NKG2A complex was first found to be expressed in NK cell lymphoma.³⁶ However, in most cases, NKG2A was expressed on tumor-infiltrating NK cells, mediating tumor tolerance to immunity. In patient-derived primary myeloma cells, high expression of HLA-E inhibited the degranulation of NKG2A⁺ NK cell subsets.³⁷ In another study blocking or knocking out NKG2A resulted in a significant increase in NK cell-mediated cytotoxicity against multiple myeloma cells.^{38 39} Concordant with the dependence of NKG2A inhibitory signals on HLA, NK cell cytotoxicity was limited against HLA-deficient K562 cells (human lymphoblast cell line) as well as low-expressing HLA-E multiple myeloma cell lines, where NKG2A⁺ NK cells displayed more effective degranulation than NKG2A⁻ NK cells.⁴⁰ These studies indicate that the NKG2A-HLA-E interaction is one of the key factors that determine the NK cell cytotoxicity in multiple myeloma.

The regulation of NKG2A on NK cells has also been extensively studied in leukemia due to its involvement in NK cell reconstitution and education after haploidentical hematopoietic stem cell transplantation, which increasingly aroused the interest of clinicians and researchers.^{41 42} Upregulated NKG2A expression on NK cells from patients with acute myeloid leukemia was associated with failure to achieve remission.⁴³ Notably, NKG2A is a dominant, transcriptionally induced checkpoint that characterizes differentiated memory-like NK cells instead of conventional NK cells.⁴⁴ A recent study also showed that in B cell precursor acute lymphoblastic leukemia (BCP-ALL) NK cells displayed a suppressed phenotype, due to the high expression of NKG2A, which became a single predominant marker distinguishing patients with BCP-ALL and healthy donors.⁴⁵ In contrast, another study identified an unconventional CD56⁻ CD16⁺ NK cell subset with decreased NKG2A expression from 27% of patients with acute myelocytic leukemia, which was associated with an inferior clinical outcome, decreased overall survival and progression-free survival.⁴⁶ This unsuspected association might also be explained, however, by the additional decreased expression of two activating receptors, NKG2D and CD226, on these CD56⁻ CD16⁺ NK cells. In another study in chronic lymphocytic leukemia (CLL), conventional CD56⁺ CD16⁺ NK cells were also confirmed to express NKG2A, while malignant B cells highly expressed HLA-E and little NK cell activating receptors thus likely inhibiting NK cell recognition.⁴⁷ Indeed, the authors demonstrated that blocking NKG2A on NK cells of patients with CLL was sufficient to restore NK cell-mediated direct cytotoxicity against HLA-E-expressing target cells in vitro. Additionally, inhibiting surface expression of HLA-E on malignant B cells via small molecule inhibitors such as selinexor, may be an indirect method to enhance NK cell activation.⁴⁸ In summary, NKG2A is a principal factor for suppressed NK cell phenotype and function in multiple myeloma and leukemia and hence a potential therapeutic target.

NKG2A on NK cells in solid tumors

In addition to being involved in hematological malignancies, NKG2A⁺ NK cells have also been found to infiltrate in a variety of solid tumors. Renal cell carcinoma-infiltrating NK cells overexpressed CD94/NKG2A and their lysis activity was inhibited in a context of specific HLA class-I allotypes.⁴⁹ Similar increased NKG2A expression on NK cells was also found in breast cancer,⁵⁰ lung cancer⁵¹ and hepatocellular carcinoma (HCC).⁵² Furthermore, a higher density of tumorous NKG2A⁺ NK cells that exhibited an exhausted phenotype and was associated with a poor prognosis in gastric and liver cancer.^{53 54} Notably and counterintuitively, non-classical HLA-E is generally increased in solid tumors, despite the fact the classical HLA molecules are often lost or downregulated in tumors.⁵⁵ Like for hematological malignancies, highly expressed HLA-E impaired NK cell activity in neuroblastoma,⁵⁶ glioma⁵⁷ and liver cancer.⁵³ Therefore, we may infer that also in solid tumors elevated expression of NKG2A and HLA-E appears to be a means by which cancer immune recognition is prevented.

NKG2A is differentially expressed on different NK cell subsets. In several solid tumor types it was found that NKG2A was detectable only on CD56-bright NK cells but was faint or negative on CD56-dim NK cells.⁵⁸ In HCC, the proportion of CD56-bright CD16⁻ NK cells with high NKG2A expression was increased, despite a reduction in the number of total NK cells.⁵⁹ In breast cancer, a large proportion of CD56-bright CD16⁺ NK cells with high NKG2A expression infiltrated in the tumor-draining lymph nodes that were the first metastatic site of breast cancer.⁶⁰ This indicates that upregulation of NKG2A in tumor-draining lymph nodes might contribute to metastasis via the lymphatic system.⁶⁰ Additionally, in an HLA-E⁺ metastatic breast cancer mouse model NKG2A (KLRC1)-knockout NK cells demonstrated higher cytotoxicity that stalled tumor progression and increased survival compared with KLRC1-expressing NK cells.⁶¹ Also, in a murine lung cancer model the upregulation of NKG2A combined with the downregulation of activating receptors NKG2D and Ly49l was associated with cancer metastasis.⁶² Concordantly in pancreatic cancer it has been reported that circulating tumor cells (CTC) and NK cells may interact via HLA-E-CD94/NKG2A, thus facilitating tumor metastasis. Mechanistically in vitro and in vivo studies demonstrated that platelet-derived RGS18 promoted the expression of HLA-E on CTC through AKT-GSK3β-CREB signaling, thereby inhibiting the cytotoxicity of NK cells against CTC and promoting hepatic metastasis of pancreatic cancer cells.⁶³ Inversely, these data suggest that differential expression of NKG2A by different NK cell subpopulations may influence tumor recognition and spreading.

NKG2A on T cells in tumor microenvironment

Identification of NKG2A on CD8⁺ T cells in cancer

Although NKG2A is known as an NK cell inhibitory receptor, already before it was found in NK cell lymphoma, it was interestingly first reported to be expressed in T cells of patients with cancer. As early as 1999, scientists from Switzerland discovered that patients with melanoma carried tumor antigen-specific cytotoxic T lymphocytes expressing CD94/NKG2A.⁶⁴ It was also the first time to employ specific antibodies to block CD94 or CD94/NKG2A heterodimer, which demonstrated the inhibitory function of NKG2A on patient-derived cytotoxic MelanA-specific T lymphocytes against a MelanA-expressing melanoma cell line in vitro. A recent in vitro study demonstrated that repeated antigen stimulations induced the expression of NKG2A on mouse CD8⁺ T cells.⁶⁵ However, NKG2A is acquired later than PD-1 as well as other co-inhibitory checkpoints such as CTLA-4, TIGIT and LAG-3 in bladder cancer.^{65 66} CD8⁺ T cell co-culture experiments with dendritic cells (DCs) showed that the late but stable expression kinetics of NKG2A rather resembled that of TIM-3 and CD39⁶⁵, which are considered exhaustion markers of tumor-reactive CD8⁺ tumor-infiltrating lymphocytes.⁶⁷ Together, these findings indicate that NKG2A might be preferentially expressed on tumor-specific exhausted CD8⁺ T cells.⁶⁸ From the late acquirement of NKG2A on exhausted CD8⁺ T cells we might infer that NKG2A-HLA-E pathway may be predominantly activated in the advanced stages of tumor progression instead of tumor-initiating stages.

NKG2A on T cells in solid tumors

Several studies reported the expression of NKG2A on tumor-infiltrating CD8⁺ T cells in breast cancer. Single-cell immune profiling of breast cancer recently identified a distinct inhibitory T cell clone expressing CD39 and NKG2A among all expanded tumor-infiltrating T cell clones.⁶⁹ Similarly, tumor-infiltrating CD8⁺ T lymphocytes expressed a higher percentage of CD94/NKG2A than peripheral blood mononuclear cell in several gynecological malignancies.70,72 Up to 50% of intraepithelial infiltrating CD8⁺ T lymphocytes expressed NKG2A. Importantly, 80% of tumor tissue harbored equal or higher levels of HLA-E than normal epithelia in ovarian and cervical cancers, indicating the ligand of NKG2A was also present to suppress intratumoral cytotoxic T lymphocyte activity and worsen prognosis.⁷¹ Inhibition of NKG2A⁺ CD8⁺ T cell activity by HLA-E-expressing tumor cells was also demonstrated in bladder cancer and could be partly abrogated by NKG2A blockade in an HLA-E-dependent manner in vitro.⁶⁵

Furthermore, in EpCAM⁺ epithelial cancer cells in tumors of gastrointestinal origin that highly expressed HLA-E on tumor cells and tumor-infiltrating DC subsets, CD94/NKG2A was found expressed on effector immune cells such as CD8⁺ T cells, NKT cells and NK cells but not on CD4⁺ T cells.³⁵ Particularly, among CD8⁺ tumor-infiltrating lymphocytes, CD94/NKG2A was exclusively expressed on PD-1^high CD8⁺ T cells, underscoring once more its relation with aggravated dysfunctionality of tumor-infiltrating CD8⁺ T cells.³⁵ Interestingly, mucosal-associated invariant T cells with impaired effector activity in esophageal adenocarcinoma were also CD8⁺ PD-1⁺ NKG2A⁺.⁷³ In colorectal cancer, NKG2A was co-expressed with CD94 mainly on CD8⁺ αβ T cells instead of NK cells.⁷⁴ This tumor-infiltrating NKG2A⁺ CD8⁺ subpopulation was composed of tissue-resident T cells of which the majority was terminally exhausted.⁷⁵ Similarly, in lung cancer tissue-resident memory CD8⁺ T cells expressed NKG2A and displayed an exhausted phenotype.⁷⁶

Regulation of NKG2A and HLA-E in tumor microenvironment

NKG2 family protein-mediated and HLA molecule-mediated regulation

Immunosuppression mediated by the interaction between NKG2A and HLA-E is influenced by other NKG2 family proteins (figure 2). The activating CD94/NKG2C receptor, like CD94/NKG2A, binds HLA-E¹² but, as indicated in introduction has a different peptide preference. Thus, signaling via NKG2C counteracts/balances the immunosuppressive role of NKG2A. Moreover, knocking out the NKG2A-coding gene, KLRC1, increased the expression of NKG2C, which enhanced NK cell cytotoxicity against HLA-E⁺ solid tumors.⁶¹ In addition, NKG2A blockade with anti-NKG2A antibody restrained Epstein-Barr virus(EBV) infected tumor cell expansion, predominantly acted through activating NKG2A⁺ NKG2C⁺ NK cells.⁷⁷ These findings underscore the opposite immune functions of NKG2A and NKG2C. Also activating receptor NKG2D might counteract NKG2A. Unlike the classical NKG2 family proteins (such as NKG2A, NKG2C, NKG2E), NKG2D does not bind to CD94 to form a heterodimer, and its ligand is MICA/B instead of HLA-E. During mouse CMV infection, the increased level of NKG2D ligands expressed on NK cells was correlated with NK cell-mediated fratricide in a perforin-dependent and NKG2D-dependent manner.⁷⁸ In a culture model of human colorectal tumor spheroids with immune cells, an antibody targeting MHC class I polypeptide-related sequence A/B (MICA/B) on tumor cells could promote NK cell infiltration and activation, and enhance tumor cell apoptosis and spheroid destruction.⁷⁹ At the same time, however, NKG2A and HLA-E were also upregulated in response to NK cell infiltration. Therefore, it was suggested that prolonged NKG2D-mediated activation leads to NK cell exhaustion, tipping the balance between engagement of activating and inhibitory NK cell receptors towards immune suppression.⁸⁰ This is further illustrated by the recent finding in patients with hepatitis B virus-related HCC that a higher ratio of NKG2A/NKG2D on circulating NK cells was associated with suppressed NK cell activity in vitro and predicted shorter progression-free survival.⁵² Moreover, the activation of DNA damage response on highly stimulated NK cells leads to an increase of MULT-1 and NKG2D ligands in NK cells, triggering internalization and downregulation of NKG2D, which influences the onset of NK cell exhaustion. Given the upregulation of NKG2A on activated NK cells, this downregulation of NKG2D on exhausted NK cells also pushes towards higher immunosuppression and dysfunctionality.⁸¹

Figure 2. The NKG2A-HLA-E axis in the tumor microenvironment. The inhibitory role of NKG2A on NK cells (violet) and T cells (green) against tumor cells (red) is interfered by multiple factors. First, immunosuppression mediated by the interaction between NKG2A and HLA-E is influenced by other NKG2 family proteins and other HLA molecules, such as the activating CD94/NKG2C complex receptor, non-classical NKG2D receptor triggering NK cell activation, and HLA-G-KIR2DL4 harboring preferential inhibitory function. Second, interferons and interleukins secreted by immune cells in turn affect their antitumor immune function via NKG2A-HLA-E axis. For example, IFN-γ can increase the expression of NKG2A on NK cells and the expression of HLA-E as well as HLA-G on tumor cells to enhance the NKG2A-dependent inhibition of cytotoxic response. Single or combined stimulation of IL-12, IL-2 and IL-15 leads to increased expression of NKG2A, but may not impair NK cell cytotoxicity. Third, other immune cells in the microenvironment affect the antitumor cytotoxicity of NK cells or CD8⁺ T cells. Macrophages contribute to NK cell-mediated cytotoxicity against tumor cells through stimulatory NKG2D receptor,¹³⁴ while CD4⁺ Foxp3⁻ T cells inhibit the activity of NK cells via Qa-1 (murine ortholog of HLA-E) interacting with NKG2A.¹³⁵ CD47 blockade activates CD103⁺ dendritic cells (DCs) which can release IL-12 and CXCL9 to recruit NKG2A downregulated NK cells with higher antitumor immunity.¹¹⁰ Cancer-associated fibroblasts (CAF) increase the CD103⁺ NKG2A⁺ resident memory phenotype of T cells and inhibit PD-1⁺ TIM-3⁺ exhaustion phenotype of CD8⁺ T cells to promote T cell cytotoxicity,¹³⁶ while B cells mainly mediate terminal exhaustion of CD8⁺ T cells through NKG2A-HLA-E.¹³⁷ CTC, circulating tumor cell; HLA, human leukocyte antigen; IFN, interferon; IL, interleukin; NK, natural killer; PD-1, programmed cell death 1.

Cytokine-mediated regulation of NKG2A and HLA-E interaction

Cytokine-mediated regulation of the NKG2A and HLA-E interaction is also important in immune suppression and restoration (figure 2). The most significant effect comes from IFN-γ, which is instrumental for the cytotoxic responses of both CD8⁺ T cells and NK cells against tumor cells. As the dominant regulatory factor in the tumor microenvironment,⁸² IFN-γ was able to upregulate NKG2A on NK cells⁸³ and HLA-E as well as HLA-G on tumor cells⁸⁴ to increase the CD94/NKG2A-dependent inhibition of cytotoxic responses.^{85 86} However, the upregulation of HLA-E by IFN-γ seems to be limited to cells expressing hardly any HLA-E at their surface but rather harbor a significant amount of intracellular HLA-E. Cells constitutively expressing membrane and soluble HLA-E in contrast do not respond to IFN-γ.⁸⁷ As explained in introduction the effect of IFN-γ on HLA-E is likely driven by the upregulation of classical HLA molecules and the resulting loading of HLA-E with HLA-derived SP. Interestingly, in a recent CRISPR screen, loss of IFN-γ sensitized several tumor models to immunotherapy by lifting classical and non-classical MHC class I-mediated NK cell inhibition. In the same report, strong IFN signatures were shown to be associated with poor response to ICB therapy in patients with renal cell carcinoma or melanoma.⁸⁸

While IFN-γ increases the expression of NKG2A and HLA-E on NK cells and tumor cells respectively, interleukins (ILs) can also increase NKG2A expression by NK cells or T cells. An early study found that IL-12 induced the expression of NKG2A and/or CD94 in melanoma-reactive CD8⁺ T cells,⁸⁹ which was inhibited by IL2-mediated CD28/B7 co-stimulation via an NFAT (nuclear factor of activated T cells)-independent component of the calcineurin pathway.⁹⁰ In addition, ex vivo IL-2/IL-15 stimulation could also elevate the expression of NKG2A on NK cells from patients with multiple myeloma without impairing the killing capacity of NK cells against autologous tumor cells.³⁹ Indeed, combined stimulation of IL-2, IL-12 and IL-15 was able to increase the population of CD16⁺ CD56-bright NK cells with high cytotoxicity regardless of the increased expression of NKG2A.⁹¹ Finally, on NK cells O-GlcNAcylation was found to regulate NK cell cytotoxicity by enhancing the expression of inhibitory NKG2A but also that of activating NKG2D and NKp44, as well as the pro-inflammatory cytokines TNF-α, IFN-γ, perforin and granzyme B.⁹²

Blocking NKG2A-HLA-E pathway improves antitumor immunity in tumor microenvironment

Blocking NKG2A to boost antitumor immunity of NK cells and T cells

The immunosuppressive role of NKG2A in tumor microenvironment suggests that blocking NKG2A may be a potential therapeutic approach to improve the immune responses of NK cells and T cells.^{93 94} Several clinical studies have already validated the value of this approach and many are still ongoing (table 1). A first-in-class humanized IgG4 monoclonal antibody (mAb), monalizumab was widely reported to target NKG2A and promote both NK cell and T cell functions.2855 95,97 In patients with leukemia, the specificity of monalizumab in blocking NKG2A was demostrated,⁴⁷ and NKG2A blockade was found sufficient to restore the cytotoxic ability of NK cells in both CLL and acute myelocytic leukemia.⁹⁵ A recent mouse study demonstrated that intratumoral injection of NK cells in tumor-bearing mice could achieve therapeutic effect only when anti-NKG2A mAb was co-administered.⁹⁶ On another note, in an HPV16-induced carcinoma mouse model treated with a long peptide vaccine, NKG2A blockade rather operated through CD8⁺ T cells while NK cells were dispensable. This was due to the strong upregulation of NKG2A on CD8⁺ T cells but not NK cells induced by the vaccine.²⁸ Therefore, NKG2A blockade may enhance both NK cell- and CD8⁺ T cell-mediated antitumor immunity. However, though tumor-infiltrating NK cells in squamous cell carcinoma of head and neck (HNSCC) showed increased NKG2A expression, a recent phase II study of monalizumab in these patients showed limited effect and hence did not meet the primary endpoint.⁹⁸ Likewise, only short-term disease stabilization was observed in patients with advanced gynecologic malignancies after receiving monalizumab therapy.⁹⁹ Monalizumab despite its preclinical potential so far did not fulfill its promise.

Table 1. Finalized and ongoing clinical trials investigating NKG2A blockade.

Study identifier	Study duration	Status	Trial phase	Patient population	Number of cases	Drug targets	Interventions
NCT02643550André et al10	2015–2023	Published	Phase 1/2	Head and neck neoplasms	143	NKG2A+EGFR	Monalizumab+cetuximab
Tinker et al99	2019 - NA	Published	Phase 2	Advanced gynecologic malignancy	58	NKG2A	Monalizumab (IPH2201)
NCT03088059Galot et al98	2017–2025	Published	Phase 2	HNSCC	340	EGFR, CDKs, NKG2A, PD-L1, PARP, GITR	Afatinib, palbociclib, standard of care, IPH2201, durvalumab, niraparib, INCAGN01876
NCT03822351Herbst et al106	2018–2023	Published	Phase 2	Stage III NSCLC	188	PD-L1, PD-L1+CD73/NKG2A	Durvalumab alone or+novel agents (oleclumab, monalizumab)
NCT03794544Cascone et al107	2019–2021	Published	Phase 2	NSCLC	84	PD-L1, PD-L1+CD73/NKG2A/STAT3	Durvalumab alone or+novel agents (oleclumab, monalizumab, danvatirsen)
Geurts et al108	2023 - NA	Published	Phase 2	HER2-positive metastatic breast cancer	11	NKG2A+HER2	Monalizumab+trastuzumab
NCT02671435Patel et al105	2016–2024	Published	Phase 1/2	Advanced solid tumor malignancies, include CRC	383	NKG2A+PD-L1	Monalizumab+durvalumab (MEDI4736)
NCT02921685	2016–2020	NA	Phase 1	Hematologic malignancies	18	NKG2A	Monalizumab (IPH2201)
NCT02557516	2015–2019	Terminated	Phase 1/2	CLL	22	NKG2A+BTK	Monalizumab (IPH2201)+ibrutinib
NCT04307329	2021–2030	Active, not recruiting	Phase 2	Breast cancer	38	NKG2A+HER2	Monalizumab+trastuzumab
NCT06152523	2023–2028	Not yet recruiting	Phase 2	MSI and/or dMMR CRC	43	NKG2A/PD-1 + CTLA-4	Monalizumab/MEDI5257
NCT04590963	2020–2024	Active, not recruiting	Phase 3	HNSCC	370	EGFR, EGFR+NKG2A	Monalizumab+cetuximab, versus placebo+cetuximab
NCT05221840	2022–2030	Recruiting	Phase 3	NSCLC	999	PD-L1+NKG2 A/CD73	Monalizumab+durvalumab or durvalumab+oleclumab
NCT05903092	2023–2025	Recruiting	Phase 2	First-line treatment of extensive stage small cell lung cancer	38	NKG2A+PD-L1 + platinum	Monalizumab+durvalumab (MEDI4736)+platinum-based chemotherapy
NCT03307941	2017–2017	Withdrawn	Phase 1/2	Locally advanced esophageal and gastro-esophageal junction cancer	0	NKG2A, NKG2A+oxaliplatin/5-fluorouracil	Monalizumab alone or+oxaliplatin/5-fluorouracil
NCT04333914	2020–2021	Completed	Phase 2	Advanced or metastatic hematological or solid tumor and SARS-CoV-2 (COVID-19) infection	19	NKG2A, autophagy, C5aR1	Monalizumab or autophagy inhibitor (GNS651) or avdoralimab or standard of care
NCT03833440	2019–2024	Recruiting	Phase 2	NSCLC	120	PD-L1+NKG2A/EGFR/ATR/MET	Durvalumab (MEDI4736)+monalizumab or oleclumab or AZD6738 or savolitinib, or a standard third-line or fourth-line chemotherapy maintenance (docetaxel)
NCT05061550	2022–2028	Recruiting	Phase 2	NSCLC	490	PD-L1+EGFR/NKG2A	Durvalumab+oleclumab/monalizumab or AZD0171 and platinum doublet chemotherapy (CTX), or volrustomig+platinum doublet chemotherapy, or datopotamab deruxtecan (Dato-DXd)+durvalumab and single agent platinum chemotherapy
NCT03801902	2019–2025	Suspended	Phase 1	NSCLC and stage II/III lung cancer	48	Radiotherapy+PD-L1 + NKG2A/EGFR	Radiotherapy+durvalumab alone plus either monalizumab or oleclumab
NCT04145193	2020–2024	Withdrawn	Phase 2	Microsatellite-stable CRC	0	PD-L1, PD-L1+EGFR PD-L1+NKG2A	Standard of care (FOLFOX) alone and durvalumab, durvalumab and oleclumab, durvalumab and monalizumab
NCT04914351	2022–2023	Active, not recruiting	Phase 1	Locally advanced/metastatic solid tumors	17	NKG2A	HY-0102 (anti-NKG2A mAb)
NCT06094777/CTR20233455	2023–2025	Not yet recruiting	Phase 1	Locally advanced/metastatic solid tumors	50	NKG2A	HY-0102 (anti-NKG2A mAb)
NCT04349267	2020–2025	Recruiting	Phase 1/2	Advanced solid tumors	308	NKG2A+PD-1/EGFR	BMS-986315 (antagonist of NKG2A)+nivolumab or cetuximab
NCT06094296	2023–2027	Recruiting	Phase 2	Stage IV or recurrent NSCLC	196	Chemotherapy+PD-1, Chemotherapy+PD-1+NKG2A	Chemotherapy and nivolumab in combination with/without BMS-986315 (antagonist of NKG2A) as first-line treatment
NCT06116136	2024–2029	Not yet recruiting	Phase 1/2	Locally advanced and unresectable or metastatic MSI-H/dMMR gastro-esophageal junction /gastric cancer	32	NKG2A+PD-1	S095029 (anti-NKG2A antibody) in combination with pembrolizumab
NCT06162572	2024–2027	Not yet recruiting	Phase 1/2	Previously untreated advanced NSCLC with high PD-L1 expression	176	IgG4+TIM-3/CD73/NKG2A	Cemiplimab, in combination with either S095018 (anti-TIM-3 antibody), S095024 (anti-CD73 antibody), or S095029 (anti-NKG2A antibody)
NCT05162755	2021–2024	Recruiting	Phase 1	Advanced solid tumor malignanciesmetastatic gastric or CRC	51	NKG2A+PD-1, NKG2A+PD-1 + HER2/EGFR	S095029 (Anti-NKG2A) + Sym021 (Anti-PD-1) with/without Anti-HER2 mAb or anti-EGFR mAbs (futuximab/modotuximab)
EUCTR2020-005902-24-IT	2021–NA	Ongoing	Phase 2	AML or myelodysplastic syndrome undergoing haploidentical transplantation with post-transplantation cyclophosphamide	42	NKG2A	Anti-NKG2A monoclonal antibody (humZ270 mAb, IPH2201)
CTR20221537	2022–NA	Recruiting	Phase 3	Locally advanced (stage III), unresectable NSCLC	170+999	PD-L1+EGFR/CD73	Durvalumab+oleclumab or durvalumab+monalizumab

AMLacute myeloid leukemiaCLLchronic lymphocytic leukemiaCRCcolorectal cancersdMMRmismatch repair-deficientEGFRepidermal growth factor receptorHNSCCsquamous cell carcinoma of head and neckmAbmonoclonal antibodyMSI-Hmicrosatellite instability-highNAnot availableNSCLCnon-small cell lung cancerPD-L1programmed death-ligand 1

Despite the fact that monalizumab is currently the most widely used clinical-grade mAb to block NKG2A (table 1), a next-generation anti-NKG2A mAb, KSQ mAb, may have advantages over monalizumab for the treatment of cancer.⁹⁷ A similar clinical receptor occupancy was achieved by KSQ mAb with a lower dose or less frequent administration compared with monalizumab. Additionally, another interesting study found that dasatinib (ABL tyrosine-kinase inhibitor) could also inhibit NKG2A and enhance NK cell cytotoxicity in chronic myeloid leukemia while imatinib and nilotinib, two other ABL tyrosine-kinase inhibitors could not.¹⁰⁰ Of course, the strongest blocking effect would be achieved by NKG2A knockout. A meaningful preclinical study discovered that NKG2A-null NK cells generated by retroviral transduction of NK cells with an antibody-based NKG2A-ER (endoplasmic reticulum) retention construct harbored higher cytotoxicity than NKG2A⁺ NK cells treated with a traditional anti-NKG2A mAb in vivo.¹⁰¹ This suggests that adoptive NK cell therapy or T cell therapy with NKG2A knockout cells might have potential in the treatment of patients with cancer.

Blocking NKG2A in combination with other immunotherapies

Since NKG2A blockade can improve the immune response in the tumor environment, it is not hard to imagine that a combination of NKG2A blockade with other ICB therapies might further enhance the functions of NK cells or CD8⁺ T cells to achieve better clinical outcome. The identification of a subset of NKG2A⁺ mature NK cells with high level of PD-1 in the ascites of some patients with ovarian cancer implied the potential of combined therapy of NKG2A blockade and PD-1/PD-L1 blockade. In patients with muscle-invasive bladder cancer, the intratumoral co-expression of NKG2A and PD-L1 was associated with an immune-reactive tumor microenvironment enriched with effector cells and immune molecules. This suggested that NKG2A^hi PD-L1^hi tumors might benefit from the combination of NGK2A blockade with PD-L1 inhibitor.¹⁰² Heterogeneous MHC-I murine models confirmed that combining anti-NKG2A mAb and anti-PD-L1 mAb therapies could restore complete immune responses, but depended on both the presence of activated, tumor-infiltrating NK cells and CD8⁺ T cells.¹⁰³ In this murine model, anti-NKG2A mAb mainly restored the degranulation of NK cells, while anti-PD-L1 mAb mainly restored the degranulation of CD8⁺ T cells. Furthermore, a combination of NKG2A blockade and PD-1 blockade could improve the response to radiotherapy in mouse radioresistant tumors while NKG2A/Qa-1b inhibition alone could not.¹⁰⁴ In clinical assessments of the combination, durvalumab (anti-PD-L1 mAb) plus monalizumab were well tolerated but not very effective in a phase I study in various solid tumors,¹⁰⁵ but did show increased objective response rate and prolonged progression-free survival in patients with unresectable stage III non-small cell lung cancer (NSCLC), compared with durvalumab alone.¹⁰⁶ Notably, when applied in the neoadjuvant setting to treat respectable NSCLC, durvalumab in combination with monalizumab induced major pathologic responses (defined as≤10% portion of surviving tumor cells in the tumor after surgery) that were associated with increased effector immune cell infiltration, IFN response, markers of tertiary lymphoid structure formation and systemic functional immune cell activation.¹⁰⁷

In addition to being regulated by other NKG2 family proteins and classical HLA molecules, NKG2A is also closely associated with other activating and inhibitory receptors and ligands. This suggests that besides PD-1 there is potential to block NKG2A in combination with other immune molecule targeting therapies. In HNSCC, tumor-infiltrating NK cells showed increased NKG2A expression, and NK cells, CD4⁺ and CD8⁺ T cells shared an increment of co-stimulatory receptor GITR, hence GITR agonist and NKG2A antagonist might be exploited for immunotherapy.⁹¹ Moreover, the potential of combining NKG2A blockade with small molecule targeting therapies is also being explored. Notably, the combination of monalizumab and trastuzumab (anti-HER2 mAb) could not induce any objective response in metastatic HER2-positive breast cancer.¹⁰⁸ Combination of cetuximab targeting epidermal growth factor receptor together with monalizumab, however, achieved an objective response rate of 31% in patients with HNSCC, and enhanced both NK cell antibody-dependent cell cytotoxicity and T cell activity.¹⁰ It suggests that this combination may be an effective way to prevent tumor progression for some tumors. Furthermore, bortezomib (a proteasome inhibitor used to treat multiple myeloma) enabled NK cell killing of multiple myeloma cells via reducing cell surface HLA-E expression on myeloma cells, and it could increase the infiltration of NKG2A⁺ NK cells. Additional therapeutic NKG2A blockade may further increase the cytolytic ability of NK cells in this setting.¹⁰⁹ Additionally, blocking CD47 facilitated the recruitment of low NKG2A-expressing NK cells into the tumor microenvironment of a HCC murine model, and these cells displayed higher granzyme B, NKG2D, IFN-γ and TNF-α expression, and enhanced antitumor immunity, rendering CD47 another potential target to combine with monalizumab therapy.¹¹⁰ In summary, monotherapy with monalizumab thus far is not very effective but in combination with other forms of (immune) therapy results may be more promising for some cancer types in particular. Currently, NKG2A is being targeted in many different cancer types, combined with a wide array of other treatments including chemotherapeutics and several different checkpoint inhibitors (eg, mAb against PD-1, PD-L1, TIM-3 or CD73). We are eagerly awaiting the results of the ongoing clinical trials (table 1) assessing single NKG2A blockade and combination therapies.

Other co-signaling immune pathways acting on both NK cells and T cells

In addition to NKG2A, several other co-inhibitory immune checkpoints have been found to be expressed on both NK and T cells¹¹¹ (figure 3) that could offer therapeutic targets in combination with NKG2A blockade. Among them, the most widely studied is TIGIT, which is expressed by NK cells, CD8⁺ T cells, regulatory T cells and follicular T helper cells.¹¹² Like NKG2A, TIGIT is an inhibitory receptor on NK cells and has been shown to cause NK cell dysfunction.¹¹³ Similarly, CD8⁺ T cells with a high level of TIGIT expression showed a dysfunctional phenotype¹¹⁴ and TIGIT blockade in combination with PD-1 blockade could restore antitumor immunity of human CD8⁺ T cells¹¹⁵,¹¹⁶. CD96 is another co-inhibitory receptor which shares the ligand CD155 with TIGIT, and can also affect the immune function of both NK cells and T cells.117,119 In addition, co-inhibitory receptor PVRIG (also named CD112R) shares the ligand CD112 with TIGIT, which was reported to be dually expressed on NK cells and T cells.120,122 These three co-inhibitory receptors regulate both NK cells and T cells in multiple types of solid tumor, revealing the complex compensatory mechanism of negative regulation in the tumor immune microenvironment.

Figure 3. Immune co-signaling receptors on both NK cells and T cells. Co-inhibitory CD94/NKG2A heterodimeric receptor is expressed on NK cells and T cells and is often upregulated along with its ligand HLA-E in tumors. NKG2D is a co-stimulatory receptor expressed on both NK cells and T cells. Another co-stimulatory receptor, CD226, shares ligand CD155 with co-inhibitory receptors TIGIT and CD96, and shares ligand CD112 with co-inhibitory receptor PVRIG (CD112R). Co-inhibitory receptor CD161 on NK cells and T cells binds to ligand CLEC2D. Red plus signs indicate stimulatory signals, blue minus signs indicate inhibitory signals. APC, antigen-presenting cell; HLA-E, human leukocyte antigen-E; NK, natural killer; PVRIG, poliovirus receptor-related immunoglobulin domain-containing protein; CLEC2D, C-type lectin domain family 2 member D.

Of course, we cannot ignore the co-stimulatory receptor, CD226, with which the above-mentioned receptors share the same ligands CD155 and CD112 and thus have competitive antagonism. Evidence showed that CD226 was both required by NK cells and T cells for the elimination of tumor cells.¹²³ Meanwhile, CD226 was requisite for the antitumor function of CD8⁺ T cells and able to improve responses to other ICB therapies.^{124 125} Another co-stimulatory receptor that is expressed on both NK cells and T cells is NKG2D, which was indispensable to maintaining the killing capability of CD8⁺ T cells against MHC-I negative tumor cells.¹²⁶ Furthermore, NKG2D-CAR expressing T cells were proven to display efficient antitumor activity against multiple solid tumors.^{127 128} In addition, NKG2D-CAR transduced NK cells also exhibited promising antitumor activity in various cancers.¹²⁹ Taken together, these potential targets of co-stimulatory and co-inhibitory receptors on NK cells and CD8⁺ T cells aid NKG2A in maintaining the immune balance in the tumor environment.

Conclusions and future perspectives

As a newly emerging co-inhibitory immune checkpoint that exists on the surface of both NK cells and T cells, the NKG2A-HLA-E axis plays an important role in regulating the antitumor immune functions of NK cells and CD8⁺ T cells.¹³⁰ Therapeutic targeting of this axis represents a novel opportunity in cancer immunotherapy which simultaneously relieves suppression of NK cell and T cell functions, thereby unleashing their cytotoxic effects and enhancing recognition and clearance of tumors. This strategy is anticipated to offer a new treatment option for a wide range of cancer types. Moreover, combining NKG2A blockade with existing (eg, PD-1) and newly emerging immunotherapeutic drugs (eg, PD-1, TIGIT or GITR mAb)131,133 and/or therapeutic vaccination²⁸ may synergistically enhance immune responses, further improving treatment outcomes. Indeed, current clinical trials suggest that the efficacy of anti-NKG2A monotherapy is suboptimal, and clinically assessed combination therapies thus far appear more promising.

Furthermore, although tumors present peptides in HLA-E allowing for its surface expression and providing a ligand for NKG2A, the identity of these specific peptides remains unknown. Therefore, analyzing the composition of HLA-E-bound peptides using mass spectrometry is now demanded to better understand the mechanism of NKG2A-HLA-E interaction in tumors. Another point to note is that the presentation of functional SP may be restricted to inflammatory conditions, further indicating that the CD94/NKG2A-based inhibitory system mostly operate secondary to immune activation.²⁴ Moreover, NKG2A is upregulated on tumor-specific cytotoxic T lymphocytes following peptide vaccination,²⁸ which also suggests that NKG2A acts in a secondary fashion as an activation marker to dampen T cell responses. We can speculate that this interesting and important finding may mean that tumors that are devoid of inflammation/immune activation could be less prone to respond to single-agent CD94/NKG2A blockade. In this setting, the application of a therapeutic vaccine to activate the immune response with subsequent blocking of NKG2A may be a potential strategy to enhance vaccine-induced antitumor responses. To fully unleash the potential of NKG2A-targeted therapies, the inhibitory landscape affecting NK cells and T cells needs to be further investigated. Nonetheless, NKG2A remains a promising tool in cancer immunotherapy, potentially offering patients broader and more effective treatment options in the near future.