Impact of IL-21 on Natural Killer cell proliferation and function - a mathematical and functional assessment.

Natural killer (NK) cells are currently in use as immunotherapeutic agents for cancer. Many different cytokines are used to generate NK cells including IL-2, IL-12, IL-15 and IL-18 in solution and membrane bound IL-21. These cytokines drive NK cell activation through the integration of STAT and NF-κB pathways, which overlap and synergize, making it challenging to predict optimal cytokine combinations. We integrated functional assays for NK cells cultured in a variety of cytokine combinations with feature selection and mechanistic regression models. Our regression model successfully predicts NK cell proliferation for different cytokine combinations and indicates synergy between STAT3 and NF-κB transcription factors. Use of IL-21 in solution in the priming, but not post-priming phase of NK cell culture resulted in optimal NK cell proliferation, without compromising cytotoxicity or IFN-γ secretion against hepatocellular carcinoma cell lines. Our work provides a mathematical framework for interrogating NK cell activation for cancer immunotherapy.

Harnessing natural killer cell effector function against cancer.

Natural killer (NK) cells are cytotoxic innate lymphoid cells that participate in anti-tumour and anti-viral immune responses. Their ability to rapidly destroy abnormal cells and to enhance the anti-cancer function of dendritic cells, CD8+ T cells, and macrophages makes them an attractive target for immunotherapeutic strategies. The development of approaches that augment NK-cell activation against cancer is currently under intense preclinical and clinical research and strategies include chimeric antigen receptor NK cells, NK-cell engagers, cytokines, and immune checkpoint inhibitors. In this review, we highlight recent advances in NK-cell therapeutic development and discuss their potential to add to our armamentarium against cancer.

MASLD and the Development of HCC: Pathogenesis and Therapeutic Challenges.

Metabolic-dysfunction-associated steatotic liver disease (MASLD, previously known as non-alcoholic fatty liver disease (NAFLD)) represents a rapidly increasing cause of chronic liver disease and hepatocellular carcinoma (HCC), mirroring increasing rates of obesity and metabolic syndrome in the Western world. MASLD-HCC can develop at an earlier stage of fibrosis compared to other causes of chronic liver disease, presenting challenges in how to risk-stratify patients to set up effective screening programmes. Therapeutic decision making for MASLD-HCC is also complicated by medical comorbidities and disease presentation at a later stage. The response to treatment, particularly immune checkpoint inhibitors, may vary by the aetiology of the disease, and, in the future, patient stratification will be key to optimizing the therapeutic pathways.

Cattle killer immunoglobulin-like receptor expression on leukocyte subsets suggests functional divergence compared to humans.

Cattle, sheep, and goats are the only species outside primates known to have an expanded and diversified family of killer immunoglobulin-like receptors (KIR). Primate KIR are expressed on the surface of NK and T cells and bind MHC-I to control activation. However, the surface expression, ligands and function of bovid KIR remain unknown. Cattle botaKIR2DL1 is the only functional KIR of the same DL-lineage as the expanded KIR in primates and we examined if leukocyte expression patterns were consistent with human. We raised a specific mouse anti-botaKIR2DL1 monoclonal antibody and assessed its utility in flow cytometry, ELISA, and western blot. Unlike primates, cattle DL-lineage KIR (botaKIR2DL1) is present on B cells and monocytes in addition to T cells and low-level expression on NK cells. Expression decreases after in vitro PBMC stimulation with IL-2. This suggests that botaKIR2DL1 has different functions, and potentially ligands, compared to primate KIR.

XPO1 inhibition sensitises CLL cells to NK cell mediated cytotoxicity and overcomes HLA-E expression.

The first-in-class inhibitor of exportin-1 (XPO1) selinexor is currently under clinical investigation in combination with the BTK inhibitor ibrutinib for patients with chronic lymphocytic leukaemia (CLL) or non-Hodgkin lymphoma. Selinexor induces apoptosis of tumour cells through nuclear retention of tumour suppressor proteins and has also recently been described to modulate natural killer (NK) cell and T cell cytotoxicity against lymphoma cells. Here, we demonstrate that XPO1 inhibition enhances NK cell effector function against primary CLL cells via downregulation of HLA-E and upregulation of TRAIL death receptors DR4 and DR5. Furthermore, selinexor potentiates NK cell activation against CLL cells in combination with several approved treatments; acalabrutinib, rituximab and obinutuzumab. We further demonstrate that lymph node associated signals (IL-4 + CD40L) inhibit NK cell activation against CLL cells via upregulation of HLA-E, and that inhibition of XPO1 can overcome this protective effect. These findings allow for the design of more efficacious combination strategies to harness NK cell effector functions against CLL.

Operando NMR metabolomics of a microfluidic cell culture.

In this work we demonstrate the use of microfluidic NMR for in situ culture and quantitative analysis of metabolism in hepatocellular carcinoma (HCC) cell lines. A hydrothermal heating system is used to enable continuous in situ NMR observation of HCC cell culture over a 24 h incubation period. This technique is nondestructive, non-invasive and can measure millimolar concentrations at microlitre volumes, within a few minutes and in precisely controlled culture conditions. This is sufficient to observe changes in primary energy metabolism, using around 500-3500 cells per device, and with a time resolution of 17 min. The ability to observe intracellular responses in a time-resolved manner provides a more detailed view of a biological system and how it reacts to stimuli. This capability will allow detailed metabolomic studies of cell-culture based cancer models, enabling quantification of metabolic reporgramming, the metabolic tumor microenvironment, and the metabolic interplay between cancer- and immune cells.

Testing to sustain hepatitis C elimination targets in people who inject drugs: A network-based model.

Little is known about the level of testing required to sustain elimination of hepatitis C (HCV), once achieved. In this study, we model the testing coverage required to maintain HCV elimination in an injecting network of people who inject drugs (PWID). We test the hypothesis that network-based strategies are a superior approach to deliver testing. We created a dynamic injecting network structure connecting 689 PWID based on empirical data. The primary outcome was the testing coverage required per month to maintain prevalence at the elimination threshold over 5 years. We compared four testing strategies. Without any testing or treatment provision, the prevalence of HCV increased from the elimination threshold (11.68%) to a mean of 25.4% (SD 2.96%) over the 5-year period. To maintain elimination with random testing, on average, 4.96% (SD 0.83%) of the injecting network needs to be tested per month. However, with a 'bring your friends' strategy, this was reduced to 3.79% (SD 0.64%) of the network (p < .001). The addition of contact tracing improved the efficiency of both strategies. In conclusion, we report that network-based approaches to testing such as 'bring a friend' initiatives and contact tracing lower the level of testing coverage required to maintain elimination.

Antiviral Responses of Tissue-resident CD49a+ Lung Natural Killer Cells Are Dysregulated in Chronic Obstructive Pulmonary Disease.

Rationale: Tissue-resident natural killer (trNK) cells have been identified in numerous organs, but little is known about their functional contribution to respiratory immunity, in particular during chronic lung diseases such as chronic obstructive pulmonary disease (COPD). Objectives: To investigate the phenotype and antiviral responses of trNK cells in murine cigarette smoke-induced experimental COPD and in human lung parenchyma from COPD donors. Methods: Mice were exposed to cigarette smoke for 12 weeks to induce COPD-like lung disease. Lung trNK cell phenotypes and function were analyzed by flow cytometry in both murine and human disease with and without challenge with influenza A virus. Measurements and Main Results: In the mouse lung, CD49a+CD49b+EOMES+ and CD49a+CD49b-EOMESlo NK cell populations had a distinct phenotype compared with CD49a- circulating NK cells. CD49a+ NK cells were more extensively altered earlier in disease onset than circulating NK cells, and increased proportions of CD49a+ NK cells correlated with worsening disease in both murine and human COPD. Furthermore, the presence of lung disease delayed both circulating and trNK cell functional responses to influenza infection. CD49a+ NK cells markedly increased their NKG2D, CD103, and CD69 expression in experimental COPD after influenza infection, and human CD49a+ NK cells were hyperactive to ex vivo influenza infection in COPD donors. Conclusions: Collectively, these results demonstrate that trNK cell function is altered in cigarette smoke-induced disease and suggests that smoke exposure may aberrantly prime trNK cell responsiveness to viral infection. This may contribute to excess inflammation during viral exacerbations of COPD.

UK-Wide Multicenter Evaluation of Second-line Therapies in Primary Biliary Cholangitis.

BACKGROUND & AIMS: Thirty-to-forty percent of patients with primary biliary cholangitis inadequately respond to ursodeoxycholic acid. Our aim was to assemble national, real-world data on the effectiveness of obeticholic acid (OCA) as a second-line treatment, alongside non-licensed therapy with fibric acid derivatives (bezafibrate or fenofibrate). METHODS: This was a nationwide observational cohort study conducted from August 2017 until June 2021. RESULTS: We accrued data from 457 patients; 349 treated with OCA and 108 with fibric acid derivatives. At baseline/pre-treatment, individuals in the OCA group manifest higher risk features compared with those taking fibric acid derivatives, evidenced by more elevated alkaline phosphatase values, and a larger proportion of individuals with cirrhosis, abnormal bilirubin, prior non-response to ursodeoxycholic acid, and elastography readings >9.6kPa (P < .05 for all). Overall, 259 patients (OCA) and 80 patients (fibric acid derivatives) completed 12 months of second-line therapy, yielding a dropout rate of 25.7% and 25.9%, respectively. At 12 months, the magnitude of alkaline phosphatase reduction was 29.5% and 56.7% in OCA and fibric acid groups (P < .001). Conversely, 55.9% and 36.4% of patients normalized serum alanine transaminase and bilirubin in the OCA group (P < .001). The proportion with normal alanine transaminase or bilirubin values in the fibric acid group was no different at 12 months compared with baseline. Twelve-month biochemical response rates were 70.6% with OCA and 80% under fibric acid treatment (P = .121). Response rates between treatment groups were no different on propensity-score matching or on sub-analysis of high-risk groups defined at baseline. CONCLUSION: Across the population of patients with primary biliary cholangitis in the United Kingdom, rates of biochemical response and drug discontinuation appear similar under fibric acid and OCA treatment.

Targeting KIR as a novel approach to improve CAR-NK cell function.

Chimeric antigen receptor (CAR) NK cells are demonstrating promising activity in clinical trials and possess a favorable safety profile compared to CAR-T cells. The Killer cell Immunoglobulin-like Receptors (KIR) have a critical role in the control of NK cell function, and recently, this family of activating and inhibitory receptors have been targeted to improve CAR-NK function. These strategies include the utilisation of inhibitory KIR to reduce trogocytosis-associated NK cell fratricide, the downregulation of inhibitory KIR on CAR-NK cells to alleviate HLA mediated suppression, the selection of CAR-NK cell donors enriched for activating KIR, and the use of activating KIR intracellular domains within novel CAR constructs. These pre-clinical studies demonstrate the potential utility of targeting the KIR to improve CAR-NK cell efficacy and patient outcomes.

Disruption of the NKG2A:HLA-E Immune Checkpoint Axis to Enhance NK Cell Activation against Cancer.

Ligation of the inhibitory receptor NKG2A by its ligand HLA-E negatively regulates the activation of natural killer (NK) cells, as well as subsets of CD8+ T cells and innate T cell populations. NKG2A has recently become a novel immune checkpoint target for the treatment of cancer and direct antibody mediated blockade of NKG2A function is currently under assessment in two phase 3 clinical trials. In addition to direct targeting, the NKG2A:HLA-E axis can also be disrupted indirectly via multiple different targeted cancer agents that were not previously recognised to possess immunomodulatory properties. Increased understanding of immune cell modulation by targeted cancer therapies will allow for the design of rational and more efficacious drug combination strategies to improve cancer patient outcomes. In this review, we summarise and discuss the various strategies currently in development which either directly or indirectly disrupt the NKG2A:HLA-E interaction to enhance NK cell activation against cancer.

KIR2DS2 Expression Identifies NK Cells With Enhanced Anticancer Activity.

NK cells are promising cellular therapeutics against hematological and solid malignancies. Immunogenetic studies have identified that various activating killer cell Ig-like receptors (KIRs) are associated with cancer outcomes. Specifically, KIR2DS2 has been associated with reduced incidence of relapse following transplant in hematological malignancies and improved outcomes in solid tumors, but the mechanism remains obscure. Therefore, we investigated how KIR2DS2 expression impacts NK cell function. Using a novel flow cytometry panel, we show that human NK cells with high KIR2DS2 expression have enhanced spontaneous activation against malignant B cell lines, liver cancer cell lines, and primary chronic lymphocytic leukemia cells. Surface expression of CD16 was increased on KIR2DS2high NK cells, and, accordingly, KIR2DS2high NK cells had increased activation against lymphoma cells coated with the clinically relevant anti-CD20 Abs rituximab and obinutuzumab. Bulk RNA sequencing revealed that KIR2DS2high NK cells have upregulation of NK-mediated cytotoxicity, translation, and FCGR gene pathways. We developed a novel single-cell RNA-sequencing technique to identify KIR2DS2+ NK cells, and this confirmed that KIR2DS2 is associated with enhanced NK cell-mediated cytotoxicity. This study provides evidence that KIR2DS2 marks a population of NK cells primed for anticancer activity and indicates that KIR2DS2 is an attractive target for NK-based therapeutic strategies.

Trans-ancestral fine-mapping of MHC reveals key amino acids associated with spontaneous clearance of hepatitis C in HLA-DQß1.

Spontaneous clearance of acute hepatitis C virus (HCV) infection is associated with single nucleotide polymorphisms (SNPs) on the MHC class II. We fine-mapped the MHC region in European (n = 1,600; 594 HCV clearance/1,006 HCV persistence) and African (n = 1,869; 340 HCV clearance/1,529 HCV persistence) ancestry individuals and evaluated HCV peptide binding affinity of classical alleles. In both populations, HLA-DQß1Leu26 (p valueMeta = 1.24 × 10-14) located in pocket 4 was negatively associated with HCV spontaneous clearance and HLA-DQß1Pro55 (p valueMeta = 8.23 × 10-11) located in the peptide binding region was positively associated, independently of HLA-DQß1Leu26. These two amino acids are not in linkage disequilibrium (r2 < 0.1) and explain the SNPs and classical allele associations represented by rs2647011, rs9274711, HLA-DQB1∗03:01, and HLA-DRB1∗01:01. Additionally, HCV persistence classical alleles tagged by HLA-DQß1Leu26 had fewer HCV binding epitopes and lower predicted binding affinities compared to clearance alleles (geometric mean of combined IC50 nM of persistence versus clearance; 2,321 nM versus 761.7 nM, p value = 1.35 × 10-38). In summary, MHC class II fine-mapping revealed key amino acids in HLA-DQß1 explaining allelic and SNP associations with HCV outcomes. This mechanistic advance in understanding of natural recovery and immunogenetics of HCV might set the stage for much needed enhancement and design of vaccine to promote spontaneous clearance of HCV infection.

Impact of the COVID-19 pandemic on routine surveillance for adults with chronic hepatitis B virus (HBV) infection in the UK.

Background: To determine the impact of the COVID-19 pandemic on the population with chronic Hepatitis B virus (HBV) infection under hospital follow-up in the UK, we quantified the coverage and frequency of measurements of biomarkers used for routine surveillance (alanine transferase [ALT] and HBV viral load). Methods: We used anonymized electronic health record data from the National Institute for Health Research (NIHR) Health Informatics Collaborative (HIC) pipeline representing five UK National Health Service (NHS) Trusts. Results: We report significant reductions in surveillance of both biomarkers during the pandemic compared to pre-COVID-19 years, both in terms of the proportion of patients who had ≥1 measurement annually, and the mean number of measurements per patient. Conclusions: These results demonstrate the real-time utility of HIC data in monitoring health-care provision, and support interventions to provide catch-up services to minimise the impact of the pandemic. Further investigation is required to determine whether these disruptions will be associated with increased rates of adverse chronic HBV outcomes.

Selinexor Enhances NK Cell Activation Against Malignant B Cells via Downregulation of HLA-E.

Selinexor is an FDA approved selective inhibitor of the nuclear export protein exportin-1 (XPO1) and causes specific cancer cell death via nuclear accumulation of tumor suppressor proteins. Design of rational studies for the use of selinexor in combination with other therapeutic agents, such as immunotherapies, requires a fundamental understanding of the effects of selinexor on the immune system. One important emerging area of immunotherapy are natural killer (NK) cell based therapeutics. NK cell function is tightly regulated by a balance of signals derived from multiple activating and inhibitory receptors. Thus in cancer, up-regulation of stress ligands recognised by activating receptors or down-regulation of HLA class I recognised by inhibitory receptors can result in an anti-cancer NK cell response. Changes in XPO1 function therefore have the potential to affect NK cell function through shifting this balance. We therefore sought to investigate how selinexor may affect NK cell function. Selinexor pre-treatment of lymphoma cells significantly increased NK cell mediated cytotoxicity against SU-DHL-4, JeKo-1 and Ramos cells, concurrent with increased CD107a and IFNγ expression on NK cells. In addition, selinexor enhanced ADCC against lymphoma cells coated with the anti-CD20 antibodies rituximab and obinutuzumab. In probing the likely mechanism, we identified that XPO1 inhibition significantly reduced the surface expression of HLA-E on lymphoma cell lines and on primary chronic lymphocytic leukemia cells. HLA-E binds the inhibitory receptor NKG2A and in accordance with this, selinexor selectively increased activation of NKG2A+ NK cells. Our data reveals that selinexor, in addition to its direct cytotoxic activity, also activates an anti-cancer immune response via disruption of the inhibitory NKG2A:HLA-E axis.

The role of NK cells in oncolytic viral therapy: a focus on hepatocellular carcinoma.

Natural killer (NK) cells have a key role in host anti-tumour immune responses via direct killing of tumour cells and promotion of adaptive immune responses. They are therefore attractive targets to promote the anti-tumour efficacy of oncolytic viral therapies. However, NK cells are also potent components of the host anti-viral immune response, and therefore have the potential for detrimental anti-viral responses, limiting the spread and persistence of oncolytic viruses. Oncolytic viruses are currently being investigated for the treatment of hepatocellular carcinoma (HCC), a leading cause of cancer-related death with a high unmet clinical need. In this review, we highlight the role of NK cells in oncolytic virus therapy, their potential for improving treatment options for patients with HCC, and discuss current and potential strategies targeting NK cells in combination with oncolytic viral therapies.

Injecting network structure determines the most efficient strategy to achieve Hepatitis C elimination in people who inject drugs.

Transmission of Hepatitis C (HCV) continues via sharing of injection equipment between people who inject drugs (PWID). Network-based modelling studies have produced conflicting results about whether random treatment is preferable to targeting treatment at PWID with multiple partners. We hypothesise that differences in the modelled injecting network structure produce this heterogeneity. The study aimed to test how changing network structure affects HCV transmission and treatment effects. We created three dynamic injecting network structures connecting 689 PWID (UK-net, AUS-net and USA-net) based on published empirical data. We modelled HCV in the networks and at 5 years compared prevalence of HCV 1) with no treatment, 2) with randomly targeted treatment and 3) with treatment targeted at PWID with the most injecting partnerships (degree-based treatment). HCV prevalence at 5 years without treatment differed significantly between the three networks (UK-net (42.8%) vs. AUS-net (38.2%), p < 0.0001 and vs. USA-net (54.0%), p < 0.0001). In the treatment scenarios UK-net and AUS-net showed a benefit of degree-based treatment with a 5-year prevalence of 1.0% vs. 9.6% p < 0.0001 and 0.15% vs. 0.44%, p < 0.0001. USA-net showed no significant difference (29.3% vs. 29.2%, p = 0.0681). Degree-based treatment was optimised with low prevalence, moderate treatment coverage conditions whereas random treatment was optimised in low treatment coverage, high prevalence conditions. In conclusion, injecting network structure determines the transmission rate of HCV and the most efficient treatment strategy. In real-world injecting network structures, the benefit of targeting HCV treatment at individuals with multiple injecting partnerships may have been underestimated.