TGF-ß signalling limits effector function capacity of NK cell anti-tumour immunity in human bladder cancer.

BACKGROUND: Natural killer (NK) cells are important innate immunity players and have unique abilities to recognize and eliminate cancer cells, particularly in settings of antibody-opsonization and antibody-dependant cellular cytotoxicity (ADCC). However, NK cell-based responses in bladder cancers to therapeutic antibodies are typically immunosuppressed, and these immunosuppressive mechanisms are largely unknown. METHODS: Single cell RNA sequencing (scRNA-seq) and high-dimensional flow cytometry were used to investigate the phenotype of tumour-infiltrating NK cells in patients with bladder cancer. Further, in vitro, and in vivo models of this disease were used to validate these findings. FINDINGS: NK cells within bladder tumours displayed reduced expression of FcγRIIIa/CD16, the critical Fc receptor involved in ADCC-mediated cytotoxicity, on both transcriptional and protein levels. Transcriptional signatures of transforming growth factor (TGF)-ß-signalling, a pleiotropic cytokine known for its immunosuppressive and tissue residency-inducing effects, were upregulated in tumour-infiltrating NK cells. TGF-ß mediated CD16 downregulation on NK cells, was further validated in vitro, which was accompanied by a transition into a tissue residency phenotype. This CD16 downregulation was also abrogated by TGF-ßR signalling inhibition, which could also restore the ADCC ability of NK cells subject to TGF-ß effects. In a humanized mouse model of bladder cancer, mice treated with a TGF-ß inhibitor exhibited increased ADCC activity compared to mice treated only with antibodies. INTERPRETATION: This study highlights how TGF-ß-rich bladder cancers inhibit NK cell-mediated ADCC by downregulating CD16. TGF-ß inhibition represents new avenues to reverse immunosuppression and enhance the tumoricidal capacity of NK cells in bladder cancer. FUNDING: The Guimaraes Laboratory is funded by a US Department of Defense-Breast Cancer Research Program-Breakthrough Award Level 1 (#BC200025), a grant (#2019485) awarded through the Medical Research Future Fund (MRFF, with the support of the Queensland Children's Hospital Foundation, Microba Life Sciences, Richie's Rainbow Foundation, Translational Research Institute (TRI) and UQ), and a grant (#RSS_2023_085) funded by a Metro South Health Research Support Scheme. J.K.M.W. is funded by a UQ Research Training Program PhD Scholarship and N.O. is funded by a NHMRC Postgraduate Scholarship (#2021932).

Highlight of 2023: Unlocking the therapeutic potential of natural killer cells-advances in adaptive functions, cellular engineering and immunotherapy.

Natural killer (NK) cells possess potent cytotoxicity against infected and cancerous cells and hold promise in the development of new immunotherapies. This article for the Highlights of 2023 Series focuses on current advances in NK cell biology in cancerous and infectious settings and highlights opportunities for therapeutic interventions, including engineered NK cell therapies and advancements in feeder cell technologies.

Exploring NK cell receptor dynamics in paediatric leukaemias: implications for immunotherapy and prognosis.

Objectives: Immunotherapies targeting natural killer (NK) cell receptors have shown promise against leukaemia. Unfortunately, cancer immunosuppressive mechanisms that alter NK cell phenotype prevent such approaches from being successful. The study utilises advanced cytometry to examine how cancer immunosuppressive pathways affect NK cell phenotypic changes in clinical samples. Methods: In this study, we conducted a high-dimensional examination of the cell surface expression of 16 NK cell receptors in paediatric patients with acute myeloid leukaemia and acute lymphoblastic leukaemia, as well as in samples of non-age matched adult peripheral blood (APB) and umbilical cord blood (UCB). An unsupervised analysis was carried out in order to identify NK cell populations present in paediatric leukaemias. Results: We observed that leukaemia NK cells clustered together with UCB NK cells and expressed relatively higher levels of the NKG2A receptor compared to APB NK cells. In addition, CD56dimCD16+CD57- NK cells lacking NKG2A expression were mainly absent in paediatric leukaemia patients. However, CD56br NK cell populations expressing high levels of NKG2A were highly represented in paediatric leukaemia patients. NKG2A expression on leukaemia NK cells was found to be positively correlated with the expression of its ligand, suggesting that the NKG2A-HLA-E interaction may play a role in modifying NK cell responses to leukaemia cells. Conclusion: We provide an in-depth analysis of NK cell populations in paediatric leukaemia patients. These results support the development of immunotherapies targeting immunosuppressive receptors, such as NKG2A, to enhance innate immunity against paediatric leukaemia.

In situ single-cell profiling sheds light on IFI27 localisation during SARS-CoV-2 infection.

The utilization of single-cell resolved spatial transcriptomics to delineate immune responses during SARS-CoV-2 infection was able to identify M1 macrophages to have elevated expression of IFI27 in areas of infection.

Leveraging spatial omics for the development of precision sarcoma treatments.

Sarcomas are rare and heterogeneous cancers that arise from bone or soft tissue, and are the second most prevalent solid cancer in children and adolescents. Owing to the complex nature of pediatric sarcomas, the development of therapeutics for pediatric sarcoma has seen little progress in the past decades. Existing treatments are largely limited to chemotherapy, radiation, and surgery. Limited knowledge of the sarcoma tumor microenvironment (TME) and of well-defined target antigens in the different subtypes necessitates an alternative investigative approach to improve treatments. Recent advances in spatial omics technologies have enabled a more comprehensive study of the TME in multiple cancers. In this opinion article we discuss advances in our understanding of the TME of some cancers enabled by spatial omics technologies, and we explore how these technologies might advance the development of precision treatments for sarcoma, especially pediatric sarcoma.

Molecular mechanisms of tumour necrosis factor signalling via TNF receptor 1 and TNF receptor 2 in the tumour microenvironment.

Tumour necrosis factor (TNF) is a primary mediator of inflammatory processes by facilitating cell death, immune cell activation and triggering of inflammation. In the cancer context, research has revealed TNF as a multifaceted cytokine that can be both pro- or anti-tumorigenic depending on what context is observed. We explore the plethora of ways that TNF and its receptors manipulate the functional and phenotypic characteristics in the tumour microenvironment (TME) on both tumour cells and immune cells, promoting either tumour elimination or progression. Here, we discuss the latest cutting-edge TNF-focused biologics currently in clinical translation that modifies the TME to derive greater immune responses and therapeutic outcomes, and further give perspectives on the future of targeting TNF in the context of cancer by emerging technological approaches.

Copper chelation suppresses epithelial-mesenchymal transition by inhibition of canonical and non-canonical TGF-ß signaling pathways in cancer.

BACKGROUND: Metastatic cancer cells exploit Epithelial-mesenchymal-transition (EMT) to enhance their migration, invasion, and resistance to treatments. Recent studies highlight that elevated levels of copper are implicated in cancer progression and metastasis. Clinical trials using copper chelators are associated with improved patient survival; however, the molecular mechanisms by which copper depletion inhibits tumor progression and metastasis are poorly understood. This remains a major hurdle to the clinical translation of copper chelators. Here, we propose that copper chelation inhibits metastasis by reducing TGF-ß levels and EMT signaling. Given that many drugs targeting TGF-ß have failed in clinical trials, partly because of severe side effects arising in patients, we hypothesized that copper chelation therapy might be a less toxic alternative to target the TGF-ß/EMT axis. RESULTS: Our cytokine array and RNA-seq data suggested a link between copper homeostasis, TGF-ß and EMT process. To validate this hypothesis, we performed single-cell imaging, protein assays, and in vivo studies. Here, we used the copper chelating agent TEPA to block copper trafficking. Our in vivo study showed a reduction of TGF-ß levels and metastasis to the lung in the TNBC mouse model. Mechanistically, TEPA significantly downregulated canonical (TGF-ß/SMAD2&3) and non-canonical (TGF-ß/PI3K/AKT, TGF-ß/RAS/RAF/MEK/ERK, and TGF-ß/WNT/ß-catenin) TGF-ß signaling pathways. Additionally, EMT markers of MMP-9, MMP-14, Vimentin, ß-catenin, ZEB1, and p-SMAD2 were downregulated, and EMT transcription factors of SNAI1, ZEB1, and p-SMAD2 accumulated in the cytoplasm after treatment. CONCLUSIONS: Our study suggests that copper chelation therapy represents a potentially effective therapeutic approach for targeting TGF-ß and inhibiting EMT in a diverse range of cancers.

Oncogenic drivers dictate immune control of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a genetically heterogeneous, aggressive hematological malignancy induced by distinct oncogenic driver mutations. The effect of specific AML oncogenes on immune activation or suppression is unclear. Here, we examine immune responses in genetically distinct models of AML and demonstrate that specific AML oncogenes dictate immunogenicity, the quality of immune response and immune escape through immunoediting. Specifically, expression of NrasG12D alone is sufficient to drive a potent anti-leukemia response through increased MHC Class II expression that can be overcome with increased expression of Myc. These data have important implications for the design and implementation of personalized immunotherapies for patients with AML.

Weaponizing natural killer cells for solid cancer immunotherapy.

Enhancing natural killer (NK) cell-based innate immunity has become a promising strategy for immunotherapy against hard-to-cure solid cancers. Monoclonal antibody (mAb) therapy has been used to activate NK-cell-mediated antibody-dependent cellular cytotoxicity (ADCC) towards solid cancers. Cancer cells, however, can subvert immunosurveillance using multiple immunosuppressive mechanisms, which may hamper NK cell ADCC. Mechanisms to safely enhance ADCC by NK cells, such as utilizing temporary inhibition of receptor endocytosis to increase antibody presentation from target to effector cells can now be used to enhance NK-cell-mediated ADCC against solid tumors. This review summarizes and discusses the recent advances in the field and highlights current and potential future use of immunotherapies to maximize the therapeutic efficacy of innate anticancer immunity.

CIS controls the functional polarization of GM-CSF-derived macrophages.

The cytokine granulocyte-macrophage-colony stimulating factor (GM-CSF) possesses the capacity to differentiate monocytes into macrophages (MØs) with opposing functions, namely, proinflammatory M1-like MØs and immunosuppressive M2-like MØs. Despite the importance of these opposing biological outcomes, the intrinsic mechanism that regulates the functional polarization of MØs under GM-CSF signaling remains elusive. Here, we showed that GM-CSF-induced MØ polarization resulted in the expression of cytokine-inducible SH2-containing protein (CIS) and that CIS deficiency skewed the differentiation of monocytes toward immunosuppressive M2-like MØs. CIS deficiency resulted in hyperactivation of the JAK-STAT5 signaling pathway, consequently promoting downregulation of the transcription factor Interferon Regulatory Factor 8 (IRF8). Loss- and gain-of-function approaches highlighted IRF8 as a critical regulator of the M1-like polarization program. In vivo, CIS deficiency induced the differentiation of M2-like macrophages, which promoted strong Th2 immune responses characterized by the development of severe experimental asthma. Collectively, our results reveal a CIS-modulated mechanism that clarifies the opposing actions of GM-CSF in MØ differentiation and uncovers the role of GM-CSF in controlling allergic inflammation.

Nanobiomaterials to modulate natural killer cell responses for effective cancer immunotherapy.

Natural killer (NK) cells have emerged as a major target for cancer immunotherapies, particularly as cellular therapy modalities because they have relatively less toxicity than T lymphocytes. However, NK cell-based therapy suffers from many challenges, including problems with its activation, resistance to genetic engineering, and large-scale expansion needed for therapeutic purposes. Recently, nanobiomaterials have emerged as a promising solution to control the challenges associated with NK cells. This focused review summarises the recent advances in the field and highlights current and future perspectives of using nanobiomaterials to maximise anticancer responses of NK cells for safe and effective immunotherapy. Finally, we provide our opinion on the role of smart materials in activating NK cells as a potential cellular therapy of the future.

The cancer cell-derived extracellular vesicle glycocode in immunoevasion.

Recent evidence suggests that cancer cell-derived extracellular vesicles might facilitate immunoevasion. Glycans are known to play a key role in immunomodulation, especially when tethered to biological membranes. However, the extracellular vesicle glycocode in cancer immunoevasion remains a largely unexplored area with promising potential for new putative diagnostic and therapeutic applications.

TGFß and CIS Inhibition Overcomes NK-cell Suppression to Restore Antitumor Immunity.

Antibodies targeting "immune checkpoints" have revolutionized cancer therapy by reactivating tumor-resident cytotoxic lymphocytes, primarily CD8+ T cells. Interest in targeting analogous pathways in other cytotoxic lymphocytes is growing. Natural killer (NK) cells are key to cancer immunosurveillance by eradicating metastases and driving solid tumor inflammation. NK-cell antitumor function is dependent on the cytokine IL15. Ablation of the IL15 signaling inhibitor CIS (Cish) enhances NK-cell antitumor immunity by increasing NK-cell metabolism and persistence within the tumor microenvironment (TME). The TME has also been shown to impair NK-cell fitness via the production of immunosuppressive transforming growth factor ß (TGFß), a suppression which occurs even in the presence of high IL15 signaling. Here, we identified an unexpected interaction between CIS and the TGFß signaling pathway in NK cells. Independently, Cish- and Tgfbr2-deficient NK cells are both hyperresponsive to IL15 and hyporesponsive to TGFß, with dramatically enhanced antitumor immunity. Remarkably, when both these immunosuppressive genes are simultaneously deleted in NK cells, mice are largely resistant to tumor development, suggesting that combining suppression of these two pathways might represent a novel therapeutic strategy to enhance innate anticancer immunity.

CIS and TGF-ß regulatory pathways influence immunity to bacterial infection.

Immunotherapy has revolutionized cancer therapy by reactivating tumour-resident cytotoxic lymphocytes. More recently, immunotherapy has emerged to restore immunity against infectious agents, including bacterial infections. Immunotherapy primarily targets inhibitory pathways in T cells, however interest in other effector populations, such as natural killer (NK) cells, is growing. We have previously discovered that NK cell metabolism, proliferation and activation can be neutralized through the immunosuppressive transforming growth factor (TGF)-ß pathway by inducing plasticity of NK cells and differentiation into innate lymphoid cell (ILC)1-like subsets. NK cells are also regulated through cytokine-inducible SH2-containing protein (CIS), which is induced by interleukin (IL)-15 and is a potent intracellular checkpoint suppressing NK cell survival and function. Targeting these two distinct pathways to restore NK cell function has shown promise in cancer models, but their application in bacterial infection remains unknown. Here, we investigate whether enhancement of NK cell function can improve anti-bacterial immunity, using Salmonella Typhimurium as a model. We identified conversion of NK cells to ILC1-like for the first time in the context of bacterial infection, where TGF-ß signalling contributed to this plasticity. Future study should focus on identifying further drivers of ILC1 plasticity and its functional implication in bacterial infection model. We further describe that CIS-deficient mice displayed enhanced pro-inflammatory function and dramatically enhanced anti-bacterial immunity. Inhibition of CIS may present as a viable therapeutic option to enhance immunity towards bacterial infection.

Towards efficient immunotherapy for bacterial infection.

The emergence of multiantibiotic-resistant bacteria, often referred to as superbugs, is leading to infections that are increasingly difficult to treat. Further, bacteria have evolved mechanisms by which they subvert the immune response, meaning that even antibiotic-sensitive bacteria can persist through antibiotic therapy. For these reasons, a broad range of viable therapeutic alternatives or conjunctions to traditional antimicrobial therapy are urgently required to reduce the burden of disease threatened by antibiotic resistance. Immunotherapy has emerged as a leading treatment option in cancer, and researchers are now attempting to apply this to infectious disease. This review summarizes and discusses the recent advances in the field and highlights current and future perspectives of using immunotherapies to treat bacterial infections.

Enhancing Natural Killer Cell Targeting of Pediatric Sarcoma.

Osteosarcoma, Ewing sarcoma (EWS), and rhabdomyosarcoma (RMS) are the most common pediatric sarcomas. Conventional therapy for these sarcomas comprises neoadjuvant and adjuvant chemotherapy, surgical resection of the primary tumor and/or radiation therapy. Patients with metastatic, relapsed, or refractory tumors have a dismal prognosis due to resistance to these conventional therapies. Therefore, innovative therapeutic interventions, such as immunotherapy, are urgently needed. Recently, cancer research has focused attention on natural killer (NK) cells due their innate ability to recognize and kill tumor cells. Osteosarcoma, EWS and RMS, are known to be sensitive to NK cell cytotoxicity in vitro. In the clinical setting however, NK cell cytotoxicity against sarcoma cells has been mainly studied in the context of allogeneic stem cell transplantation, where a rapid immune reconstitution of NK cells plays a key role in the control of the disease, known as graft-versus-tumor effect. In this review, we discuss the evidence for the current and future strategies to enhance the NK cell-versus-pediatric sarcoma effect, with a clinical focus. The different approaches encompass enhancing antibody-dependent NK cell cytotoxicity, counteracting the NK cell mechanisms of self-tolerance, and developing adoptive NK cell therapy including chimeric antigen receptor-expressing NK cells.

MAIT cells regulate NK cell-mediated tumor immunity.

The function of MR1-restricted mucosal-associated invariant T (MAIT) cells in tumor immunity is unclear. Here we show that MAIT cell-deficient mice have enhanced NK cell-dependent control of metastatic B16F10 tumor growth relative to control mice. Analyses of this interplay in human tumor samples reveal that high expression of a MAIT cell gene signature negatively impacts the prognostic significance of NK cells. Paradoxically, pre-pulsing tumors with MAIT cell antigens, or activating MAIT cells in vivo, enhances anti-tumor immunity in B16F10 and E0771 mouse tumor models, including in the context of established metastasis. These effects are associated with enhanced NK cell responses and increased expression of both IFN-γ-dependent and inflammatory genes in NK cells. Importantly, activated human MAIT cells also promote the function of NK cells isolated from patient tumor samples. Our results thus describe an activation-dependent, MAIT cell-mediated regulation of NK cells, and suggest a potential therapeutic avenue for cancer treatment.

Natural Killer Cells and Type 1 Innate Lymphoid Cells in Hepatocellular Carcinoma: Current Knowledge and Future Perspectives.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1) are specific innate lymphoid cell subsets that are key for the detection and elimination of pathogens and cancer cells. In liver, while they share a number of characteristics, they differ in many features. These include their developmental pathways, tissue distribution, phenotype and functions. NK cells and ILC1 contribute to organ homeostasis through the production of key cytokines and chemokines and the elimination of potential harmful bacteria and viruses. In addition, they are equipped with a wide range of receptors, allowing them to detect "stressed cells' such as cancer cells. Our understanding of the role of innate lymphoid cells in hepatocellular carcinoma (HCC) is growing owing to the development of mouse models, the progress in immunotherapeutic treatment and the recent use of scRNA sequencing analyses. In this review, we summarize the current understanding of NK cells and ILC1 in hepatocellular carcinoma and discuss future strategies to take advantage of these innate immune cells in anti-tumor immunity. Immunotherapies hold great promise in HCC, and a better understanding of the role and function of NK cells and ILC1 in liver cancer could pave the way for new NK cell and/or ILC1-targeted treatment.