NKG7 – regulating endosomal pathways?

The immune system has the capacity to protect the host against infection by pathogens and the development of tumors. Protective immune responses typically involve the production and release of proinflammatory cytokines and cytotoxic granules by lymphocytes. Studies into how immune responses are generated and regulated have resulted in some remarkable advances in the treatment of diseases. For example, the development of checkpoint inhibitors for the treatment of cancer has stemmed from research into how T-cell responses are regulated. A recent article in Nature Immunology by Ng and colleagues¹ has identified natural killer (NK) cell granule protein 7 (NKG7) as a regulator of granule exocytosis and inflammation during infection and in tumor control. The identification of NKG7 as a critical mediator of inflammation in response to diverse immunological challenges suggests that selective targeting of this molecule may provide a novel approach to modulate immune responses in a broad range of disease settings.

Despite being first described more than 25 years ago,² little is known about the function of NKG7 (also known as GMP-17). The observation that NKG7 is an integral membrane protein localized to cytotoxic granules of NK cells and translocates to the cell surface after target cell recognition³ provided the first indication as to its potential role in the immune response. The analysis of human patient samples has provided some correlative evidence that NKG7 is important for the cytotoxic activity of lymphocytes. For example, a population of NKG7⁺ CD4 T cells that express proteins characteristic of cytotoxic cells, including granzyme A and B (GzmA/B), was expanded in a HIV⁺ long-term nonprogressor and in healthy cytomegalovirus-positive patients.⁴ In transplant patients, the expression of NKG7 by cytotoxic CD8 T cells infiltrating renal allografts was linked to acute graft rejection and immunoglobulin A nephropathy.⁵ Thus, a correlation between NKG7 expression and cytotoxic cells has been noted, but a rigorous examination of the role of NKG7 in immune responses has been lacking.

Using a variety of experimental models Ng et al. have established that the expression of NKG7 is crucial for promoting inflammation. The authors initially examined the Cancer Genome Atlas and found that high expression of NKG7 in tumors was associated with better survival of human melanoma patients, providing some evidence that NKG7 might have a role in antitumor immunity. This observation was extended using several NK cell-dependent preclinical cancer models in NKG7-deficient mice. In the absence of NKG7, control of tumor metastasis was impaired, and Nkg7^–/– mice were more sensitive to chemically induced carcinogenesis, demonstrating that NKG7 plays a critical role in antitumor responses. While alterations in NKG7 expression levels in patients with cutaneous or intestinal T-cell lymphomas have been noted,^{6, 7} how these changes impacted on cell functionality was unknown. The research by Ng et al. is significant in that it is the first study to directly demonstrate that NKG7 contributes to the control of tumors.

Ng and colleagues also utilized a murine model of cerebral malaria to examine the role of NKG7 in an inflammatory setting that is detrimental to the host. In this model, end-stage immunopathology is mediated by the recruitment of parasite-specific cytotoxic T cells to the brain.⁸ Significantly, unlike wild-type mice, Nkg7^–/– mice infected with Plasmodium berghei ANKA did not develop severe neurological symptoms and had a significant survival advantage. The improved survival of Nkg7^–/– mice after P. berghei infection correlated with a reduction in the accumulation of activated parasite-specific CD8 T cells to the brain. Previous research has established that cerebral malaria is mediated by antigen-specific CD8 T cells that produce perforin and GzmB, molecules essential for cytotoxic function.⁹ The findings of Ng et al. therefore implicate NKG7 as being important for the generation and function of cytotoxic CD8 T cells.

Having identified NKG7 as an important mediator of inflammation, the authors examined the mechanism by which NKG7 functions. The capacity of both NK cells and CD8 T cells to kill target cells was found to be impaired in the absence of NKG7. Importantly, NKG7-deficient NK cells formed conjugates with target cells as efficiently as WT cells and no obvious differences in synapse formation were noted. Furthermore, the expression of GzmB and perforin were not altered by the absence of NKG7. Thus, lack of NKG7 does not seem to prevent the activation of effectors or inhibit their capacity to interact with target cells. Rather, the defective cytotoxic capacity of NKG7-deficient cells appears to result from reduced translocation and/or release of cytotoxic molecules stored within lytic granules (Figure 1). Lytic granules are composed of a core of cytotoxic proteins including perforin and granzymes surrounded by a lipid bilayer containing glycoproteins including CD107a (LAMP-1) and CD107b (LAMP-2). Upon engagement with a target cell, signaling events in the effector cell result in lytic granules moving toward the immune synapse formed between the target and effector cells.^{10, 11} The fusion of the cytotoxic granule membrane with the plasma membrane of the effector cell results in the release of the granule contents into the immune synapse and ultimately results in the destruction of the target cell. Ng et al. found that NKG7 was localized to intracellular cytotoxic granules in resting CD8 T cells and that after stimulation NKG7 colocalized with CD107a at the cell surface. Furthermore, NK cells or CD8 T cells lacking NKG7 had a reduced capacity to degranulate as measured by cell surface expression of CD107a. Together, these data imply that NKG7 has a role in cytotoxic granule migration toward the immune synapse or perhaps fusion of the granule with the cellular membrane, but the exact mechanism by which NKG7 functions is still to be resolved.

While the data of Ng et al. strongly implicate NKG7 as having a role in the release of cytotoxic granules, several observations made by the authors suggest that NKG7 has additional functions. Adoptive transfer experiments indicated that NKG7 is required for the expansion and trafficking of CD8 T cells after P. berghei infection. Furthermore, while defective CD4 T-cell responses were noted in Nkg7^–/– mice following Leishmania donovani infection, the authors suggested that this is a result of cell extrinsic factors, with alterations in antigen presentation proposed as a potential mechanism of action. These seemingly disparate observations can be reconciled if NKG7 regulates the migration and/or fusion of other intracellular vesicles or organelles, in addition to its role within cytotoxic granules. The fact that NKG7 is expressed by cells that lack cytotoxic activity provides some support for this possibility. For example, a role for NKG7 in endosome function would have the potential to impact on multiple cellular processes. The endosomal network is composed of a series of membrane-bound compartments that are integral to the sorting of protein cargo for degradation, recycling to the cell surface or secretion, with many functions of immune cells relying on endosomal pathways. Professional antigen-presenting cells utilize endosomal pathways for the generation and display of antigens on their cell surface,¹² thus, NKG7 localization at this site could impact on antigen presentation (Figure 1). Similarly, T-cell activation requires the co-ordinated redistribution and polarization of organelles including the Golgi apparatus, endoplasmic reticulum and the multivesicular bodies. Mutations or targeted deletion of proteins regulating the movement and/or polarization of organelles can inhibit T-cell signaling, resulting in defective T-cell responses (for example, see Piotrowski et al.¹³). While major defects in synapse formation between NKG7-deficient NK cells and target cells were not identified, a more subtle defect impacting T-cell activation cannot be ruled out. A thorough investigation into the subcellular localization of NKG7 in cells of the immune system and determining whether NKG7 interacts with other proteins will aid in elucidating how NKG7 mediates the diverse effects observed by Ng and colleagues.

Overall, the study by Ng et al.¹ has established NKG7 as a critical molecule required for the generation of inflammatory responses. A precise mechanistic understanding of how NKG7 promotes inflammation is now required as this will establish whether the specific targeting of this protein can provide a novel means of modulating inflammatory responses.

Conflict of Interest

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTION

Iona S Schuster: Conceptualization; Writing-original draft; Writing-review & editing. Christopher E Andoniou: Conceptualization; Writing-original draft; Writing-review & editing.