Published OnlineFirst May 14, 2020; DOI: 10.1158/1078-0432.CCR-19-2095 CLINICAL CANCER RESEARCH | REVIEW The NKG2A–HLA-E Axis as a Novel Checkpoint in the Tumor Microenvironment Linda Borst, Sjoerd H. van der Burg, and Thorbald van Hall ABSTRACT ◥ The success of checkpoint blockade therapy revolutionized binds a limited peptide repertoire and its expression is commonly cancer treatment. However, we need to increase the fraction of detected in human cancer. NKG2A blockade as a standalone responding patients and overcome acquired resistance to these therapy appears poorly effective in mouse tumor models, how- therapies. Recently, the inhibitory receptor NKG2A received ever, in the presence of activated T cells, for example, induced by attention as a new kid on the block of immune checkpoints. PD-1/PD-L1 blockade or cancer vaccines, exerts strongly This receptor is selectively expressed on cytotoxic lymphocytes, enhanced efficacy. Clinical trials demonstrated safety of the þ including natural killer cells and CD8 T cells, and NKG2A T humanized NKG2A-blocking antibody, monalizumab, and first cells are preferentially residing in tissues, like the tumor micro- results of phase II trials demonstrate encouraging durable environment. Its ligand, histocompatibility leucocyte antigen E response rates. Further development of this axis is clearly (HLA-E), is a conserved nonclassical HLA class I molecule that warranted. Introduction The Similarities and Differences within The unprecedented clinical impact of immune checkpoint block- the NKG2A Family ade therapy induced a new era of cancer treatment (1). After Activation of T cells is the result of T-cell receptor (TCR) ligation introduction in clinical practice of antibodies to CTLA-4 and the with antigen–HLA complexes in combination with costimulatory PD-1/PD-L1 axis, additional checkpoints like TIM-3, TIGIT, and signals, like CD28 and CD27, and cytokines. To avoid unrestricted VISTA and stimulation of activating receptors like CD27, CD40, immune responses that might result in pathology toward host and 4-1BB are evaluated for their antitumor-inducing immunity. tissues, T cells are also equipped with multiple inhibitory receptors, Recently, the immune inhibitory receptor NKG2A draw the atten- which are expressed in a programmed way (11). As revealed, CTLA- – tion as the new kid on the block of checkpoints (2 5). NKG2A 4 and PD-1 inhibitory receptors are also considered as markers of forms heterodimers with the CD94 chain (6) and recognizes the recent T-cell activation. The spaciotemporal dynamics are different nonclassical HLA class I molecule HLA-E. These recent studies for each inhibitory receptor, resulting in distinct profiles of immune demonstrated high expression of NKG2A on natural killer (NK) pathology revealed in knockout mice. The CTLA-4–knockout cells and cytotoxic CD8 T cells in the tumor microenvironment as a mouse displays spontaneous and overt lymphoproliferation, where- result of PD-1 blockade therapy as well as after immune activation as PD-1 and NKG2A knockouts show much milder phenotypes by cancer vaccines (7, 8). First clinical trials in gynecologic cancers (12–15). The cytoplasmic tail of the NKG2A receptor contains two and head and neck carcinoma with the NKG2A-blocking antibody immunoreceptor tyrosine-based inhibition motifs (ITIM) capable monalizumab shows safety and promising clinical responses in of recruiting both SHP-1 (16) and SHP-2 phosphatases, but not the refractory disease, including regressions of target lesions (8, 9). inositol phosphatase SHIP (Fig. 1;refs.17–19). Both ITIMs Interestingly, protein levels of histocompatibility leucocyte antigen are required to mediate the maximal inhibitory signal, but the E (HLA-E), which is the sole ligand of the CD94/NKG2A recep- membrane-distal ITIM is of primary importance rather than the tor (10), are frequently upregulated in many cancers, suggesting membrane-proximal ITIM (20). The partner CD94 has only seven that this axis functions as an acquired resistance mechanism after cytoplasmic amino acids, thus lacks ITIMs and has no role in immune activation in the tumor microenvironment. Here, we downstream signaling. Interestingly, CD94 can also form a hetero- provide an overarching view on the current understanding of dimer with a very close family member of NKG2A (encoded by the NKG2A and HLA-E in cancer and touch upon gaps in our Klrc1 gene), which is named NKG2C (encoded by the Klrc2 gene). knowledge. NKG2C is an activating receptor transducing a stimulating signal via association with DAP12, bearing an immunoreceptor tyrosine- based activation motif (21). The NKG2C protein carries some Department of Medical Oncology, Oncode Institute, Leiden University Medical amino acid differences compared with NKG2A, resulting in a 6-fold Center, Leiden, the Netherlands. lower affinity for the shared ligand HLA-E (22, 23). In NK cells, the Corresponding Authors: Thorbald van Hall, Leiden University Medical Center, two family members are usually not expressed together in the same Albinusdreef 2, Leiden 2333 ZA, the Netherlands. Phone: 3107-1526-6945; Fax: cell and a switch to NKG2C marks more mature NK cells, also 31 71 5264036; E-mail: [email protected]; and Linda Borst, E-mail: “ ” [email protected] referred to as adaptive NK cells (24, 25). Another activating family member, NKG2D (encoded by the Klrk1 gene) is more distantly Clin Cancer Res 2020;26:1–8 related and does not partner with CD94, but rather forms homo- doi: 10.1158/1078-0432.CCR-19-2095 dimers, and is engaged by stress-induced self-proteins, for example, Ó2020 American Association for Cancer Research. MICA and ULBP members (26). AACRJournals.org | OF1 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst May 14, 2020; DOI: 10.1158/1078-0432.CCR-19-2095 Borst et al. Expression and Function of NKG2A in Box 1: The basics of HLA-E Cytotoxic Lymphocytes In contrast to classical MHC class I molecules, HLA-E is Approximately half of peripheral NK cells display the NKG2A high virtually nonpolymorphic with only two functional alleles present receptor and these cells are mostly present in the CD56 fraction, in the human population: the HLA-EÃ01:01 and the HLA-EÃ01:03 which contains the more immature cells. Intratumoral NK cells have variants. These two alleles only differ in a single amino acid at somewhat higher frequencies of NKG2A (7). Interestingly, NKG2A position 107, being arginine (01:01) or glycine (01:03). Position 107 expression on CD8 T cells seems to be highly regulated, as peripheral is located outside the peptide-binding groove of the heavy cells hardly express the receptor, but a substantial fraction of intra- chain (53). The crystal structure of the peptide-binding groove, tumoral T cells, especially those in immune reactive milieu, display single alanine substitutions, and peptide elution, have demonstrat- NKG2A (7, 27). These CD8 T cells often display a late effector memory ed that HLA-E has an optimal structure to bind peptides with two phenotype and lack expression of typical central memory markers primary anchor residues at positions 2 and 9, and secondary anchor CCR7, CD27, and CD28 or late effector marker KLRG1. Interestingly, residues at position 7 and possibly 3 (54–59). Surprisingly, a very we and others found NKG2A expression on CD8 T cells harboring a limited repertoire of peptides was found under homeostatic con- tissue-resident signature, marked by specific integrins like CD103 – ditions and the most prominent are the signal peptides of classical (7, 28 30). Although induction of NKG2A is initiated by TCR MHC class I molecules. The majority of HLA class I alleles share a b triggering, the presence of tissue cytokines like IL15 and TGF might consensus sequence in their signal peptides with the amino acid – b enhance its expression (31 33). TGF is often overtly present in the motif of VMAPRTLLL (Table 1). Positions 7 and 8 vary between tumor microenvironment (34). However, the functional relationship leucine and valine, without affecting binding affinity. Some HLA b between the tissue residence program, NKG2A, and TGF is unclear at alleles, however, contain a substitution at residues p2, 3, or 6, which the moment. impair binding (23). In addition, the immunotolerance molecule Aside from its expression regulation, the NKG2A downstream HLA-G, which is expressed in immune-privileged sites and fre- effects are also not completely unraveled. The inhibitory signals quently in cancers, encodes a unique leader peptide that binds with induced by NKG2A receptor engagement results in decreased capacity fi – high af nity to HLA-E (60, 61). Interestingly, the leader peptide of NK cells and CD8 T cells to lyse target cells (33, 35 39). NKG2A motif is extremely conserved among mammalian species, implying triggering inhibits cytotoxic effector functions on NK cells by disrupt- an important role in immune defense (62) and is even copied by the ing the actin network at the immunologic synapse of activating human cytomegalovirus gpUL40 protein to sustain HLA-E surface receptor NKG2D (40). For CD8 T cells such mode of action is still display on infected cells while shutting down classical HLA class I to be elucidated, although NKG2D or the immunologic synapse of the – fi antigen presentation of host cells (63 65). Importantly, the CD94/ TCR could be lucid candidates (41). In this context, the recent ndings NKG2A receptor exhibits clear preference for peptide-containing on the operational mechanism of PD-1, which was suggested to HLA-E, especially for this consensus leader peptide (56). Under dephosphorylate T-cell costimulatory receptor CD28, illustrate our stress, HLA-E can present a peptide of multidrug resistance– limited understanding at molecular level of these important check- associated protein 7 (ALALVRMLI; ref. 66) and under high cell points (42, 43). CTLA-4 executes its inhibitory effects by competing densities the leader peptide QMRPVSRVL of hSP60 stabilizes with CD28 for the same ligands on antigen presenting cells, thereby HLA-E (67).