Summary Tumor-infiltrating CD8 T cells were found to frequently express the inhibitory receptor NKG2A, particularly in immune-reactive environments and after therapeutic cancer vaccination. High dimensional cluster analysis demonstrated that NKG2A marks a unique immune effector subset preferentially co-expressing the tissue-resident CD103 molecule, but not immune checkpoint inhibitors. To examine if NKG2A represented an adaptive resistance mechanism to cancer vaccination, we blocked the receptor with an antibody and knocked out its ligand Qa-1b, the conserved ortholog of HLA-E, in four mouse tumor models. The impact of therapeutic vaccines was greatly potentiated by disruption of the NKG2A/Qa-1b axis, even in a PD-1 refractory mouse model. NKG2A blockade therapy operated through CD8 T cells, but not NK cells. These findings indicate that NKG2A-blocking antibodies might improve clinical responses to therapeutic cancer vaccines.

The implementation of immune checkpoint inhibitors to the oncology clinic signified a new era in cancer treatment. After the first indication of melanoma, an increasing list of additional cancer types are now treated with immune system targeting antibodies to PD-1, PD-L1 and CTLA-4, alleviating inhibition signals on T cells. Recently, we published proof-of-concept results on a novel checkpoint inhibitor, NKG2A. This receptor is expressed on cytotoxic lymphocytes, including NK cells and subsets of activated CD8+ T cells. Blocking antibodies to NKG2A unleashed the reactivity of these effector cells resulting in tumor control in multiple mouse models and an early clinical trial. Monalizumab is inhibiting this checkpoint in human beings and future clinical trials will have to reveal its potency in combination with other cancer treatment options.

The success of checkpoint blockade therapy revolutionized cancer treatment. However, we need to increase the fraction of responding patients and overcome acquired resistance to these therapies. Recently, the inhibitory receptor NKG2A received attention as a new kid on the block of immune checkpoints. This receptor is selectively expressed on cytotoxic lymphocytes, including natural killer cells and CD8 T cells, and NKG2A+ T cells are preferentially residing in tissues, like the tumor microenvironment. Its ligand, histocompatibility leucocyte antigen E (HLA-E), is a conserved nonclassical HLA class I molecule that binds a limited peptide repertoire and its expression is commonly detected in human cancer. NKG2A blockade as a standalone therapy appears poorly effective in mouse tumor models, however, in the presence of activated T cells, for example, induced by PD-1/PD-L1 blockade or cancer vaccines, exerts strongly enhanced efficacy. Clinical trials demonstrated safety of the humanized NKG2A-blocking antibody, monalizumab, and first results of phase II trials demonstrate encouraging durable response rates. Further development of this axis is clearly warranted.

The surface inhibitory receptor NKG2A forms heterodimers with the invariant CD94 chain and is expressed on a subset of activated CD8 T cells. As antibodies to block NKG2A are currently tested in several efficacy trials for different tumor indications, it is important to characterize the NKG2A+ CD8 T cell population in the context of other inhibitory receptors. Here we used a well‐controlled culture system to study the kinetics of inhibitory receptor expression. Naïve mouse CD8 T cells were synchronously and repeatedly activated by artificial antigen presenting cells in the presence of the homeostatic cytokine IL‐7. The results revealed NKG2A as a late inhibitory receptor, expressed after repeated cognate antigen stimulations. In contrast, the expression of PD‐1, TIGIT and LAG‐3 was rapidly induced, hours after first contact and subsequently down regulated during each resting phase. This late, but stable expression kinetics of NKG2A was most similar to that of TIM‐3 and CD39. Importantly, single‐cell transcriptomics of human tumor‐infiltrating lymphocytes (TILs) showed indeed that these receptors were often coexpressed by the same CD8 T cell cluster. Furthermore, NKG2A expression was associated with cell division and was promoted by TGF‐β in vitro, although TGF‐β signaling was not necessary in a mouse tumor model in vivo. In summary, our data show that PD‐1 reflects recent TCR triggering, but that NKG2A is induced after repeated antigen stimulations and represents a late inhibitory receptor. Together with TIM‐3 and CD39, NKG2A might thus mark actively dividing tumor‐specific TILs.

We previously demonstrated that high HLA-E expression in ovarian carcinoma and NSCLC may neutralize the survival benefit of T-cell infiltration, suggesting NKG2A could represent an immune checkpoint. Here, we demonstrate higher frequencies of NKG2A+CD8+ T cells in TIL samples obtained from human HNSCC biopsies in patients with measurable immune reactivity to HPV16-viral antigens. CyTOF analysis suggested that this T-cell subset harbored an CD103+ effector phenotype. In mouse tumor models, therapeutic cancer vaccines increased the frequency of NKG2A+-positive CD8+ TIL up to 80%. Expression of Qa-1, the mouse homologue of HLA-E, on tumor cells also increased after cancer vaccinations. In vitro studies revealed that upregulation of Qa-1 was mediated by T cell-derived IFNγ. Blockade of this inhibiting NKG2A-Qa-1 axis by antibody improved the T-cell mediated rejection of tumors and, in addition, genetic knockdown of Qa-1 in three different tumor models recapitulated this finding. Blocking of NKG2A as a stand-alone treatment was not effective, indicating that NKG2A constitutes an adaptive resistance mechanism in response to CD8 T-cell activation. In conclusion, NKG2A is enriched on CD8+ TIL and functions as an immune checkpoint that restrains therapeutic efficacy of cancer vaccines. Citation Format: Thorbald van Hall, Nadine van Montfoort, Linda Borst, Michael J. Korrer, Young Kim, Pascale André, Nicolai Wagtmann, Sytse Piersma, Sjoerd H. van der Burg. The inhibitory receptor NKG2A acts as a checkpoint on CD8 T cells in the context of cancer vaccines [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A97.