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Background and purpose Acute myeloid leukemia (AML) is an aggressive disease with suboptimal overall survival, especially in relapsed/refractory patients. The primary goal of salvage therapy in this patient is to achieve optimal disease control, thereby allowing the transition to hematopoietic stem cell transplantation (HSCT), which remains the only curative option for a subset of these patients. Allogeneic KIR ligand-mismatched CD56 + NK/NKT-like cells have demonstrated antileukemic activity and represent a promising platform for the development of novel cellular therapies. Study design Relapsed/refractory non-M3 AML patients who were not HSCT candidates were included in this phase I clinical trial. Patients received the FLAG conditioning regimen followed by three escalating doses (1 × 10⁶, 3 × 10⁶, 5 × 10⁶ cells/kg) of CD56 + NK/NKT-like cells at 5-day intervals. Results A total of 11 patients with a median age of 41.5 years were enrolled in the study. On average, they received three lines of prior chemotherapy and showed 18% blasts in their bone marrow. The infusion of CD56⁺ NK/NKT-like cells was safe, with no serious toxicity or graft-versus-host disease (GVHD) observed in any patient. Following this treatment protocol, five patients (45.4%) achieved complete remission (CR), with or without hematologic count recovery. Four of these patients (36.3%) underwent successful HSCT and remained event-free to the end of the follow-up period. Conclusion Overall, these trials indicated that the FLAG regimen chemotherapy combined with allogeneic KIR ligand-mismatched CD56 + NK/NKT-like cell infusion is safe and may serve as an effective bridge to HSCT in 36.3% of patients with refractory/relapsed non-M3 AML. Graphic Abstract

Natural killer (NK) cells, initially identified for their rapid virus-infected and leukemia cell killing and tumor destruction, are pivotal in immunity. They exhibit multifaceted roles in cancer, viral infections, autoimmunity, pregnancy, wound healing, and more. Derived from a common lymphoid progenitor, they lack CD3, B-cell, or T-cell receptors but wield high cytotoxicity via perforin and granzymes. NK cells orchestrate immune responses, secreting inflammatory IFNγ or immunosuppressive TGFβ and IL-10. CD56 dim and CD56 bright NK cells execute cytotoxicity, while CD56 bright cells also regulate immunity. However, beyond the CD56 dichotomy, detailed phenotypic diversity reveals many functional subsets that may not be optimal for cancer immunotherapy. In this review, we provide comprehensive and detailed snapshots of NK cells’ functions and states of activation and inhibitions in cancer, autoimmunity, angiogenesis, wound healing, pregnancy and fertility, aging, and senescence mediated by complex signaling and ligand-receptor interactions, including the impact of the environment. As the use of engineered NK cells for cancer immunotherapy accelerates, often in the footsteps of T-cell-derived engineering, we examine the interactions of NK cells with other immune effectors and relevant signaling and the limitations in the tumor microenvironment, intending to understand how to enhance their cytolytic activities specifically for cancer immunotherapy.

ObjectiveAs the development and progression of colorectal cancer (CRC) are known to be affected by the immune system, cell subsets such as T cells, natural killer (NK) cells, and natural killer T (NKT) cells are considered interesting targets for immunotherapy and clinical biomarker research. Until now, the role of systemic immune profiles in tumor progression remains unclear. In this study, we aimed to characterize the immunophenotype of circulating T cells, NK cells, and NKT-like cells in patients with CRC, and to subsequently correlate these immunophenotypes to clinical follow-up data.MethodsUsing multiparameter flow cytometry, the subset distribution and immunophenotype of T cells (CD3+CD56−), CD56dim NK cells (CD3−CD56dim), CD56bright NK cells (CD3−CD56bright), and NKT-like (CD3+CD56+) cells were investigated in peripheral blood mononuclear cell (PBMC) samples from 71 CRC patients and 19 healthy donors.ResultsCRC patients showed profound differences in immune cell subset distribution and their immunophenotype compared to healthy donors, as characterized by increased percentage of regulatory T cells, and reduced expression level of the natural cytotoxicity receptors NKp44 and NKp46 on both CD56dim NK cells and NKT-like cells. Finally, we showed in a multivariate analysis that above-median percentage of CD16+ NKT-like cells was independently associated with shorter disease-free survival in CRC patients.ConclusionThe altered phenotype of circulating immune cell subsets in CRC and its association with clinical outcome highlight the potential use of PBMC subsets as prognostic biomarkers in CRC, thereby contributing to better insight into the role of systemic immune profiles in tumor progression.

New Findings What is the central question of this study? Does a ketogenic diet (KD) modulate circulating counts of natural killer (NK) cells, including CD56bright and CD56dim subsets, and their ability to activate (CD69 expression) following in vitro antigen stimulation in response to exhaustive moderate‐intensity exercise? What is the main finding and its importance? The KD amplified the biphasic exercise‐induced NK cell response due to a greater mobilisation of the cytotoxic CD56dim subset but did not alter NK cell CD69 expression. The KD appears to modulate exercise‐induced circulating NK cell mobilisation and egress, but not antigen‐stimulated circulating NK cell activation. We investigated the effect of a 31‐day ketogenic diet (KD) compared with a habitual, carbohydrate (CHO)‐based diet on total circulating natural killer (NK) CD3−CD56+, dim and bright subset count, and antigen‐stimulated CD3−CD56+ cell activation (CD69+) in response to exhaustive running. In a randomised, repeated‐measures, cross‐over study, eight trained, male endurance athletes ingested a 31‐day low‐CHO KD or their habitual diet (HD). On day 31, participants ran to exhaustion at 70% V̇O2max $\\dot{V}_{{\\rm{O}}_{2}{\\rm{max}}}$(∼3.5–4 h, ∼45–50 km). A low‐CHO (<10 g) meal was ingested prior to the KD trial, with fat ingested during exercise. A high‐CHO (2 g kg−1) meal was ingested prior to the HD trial, with CHO (∼55 g h−1) ingested during exercise. Venous blood samples were collected at pre‐exercise, post‐exercise and 1 h post‐exercise. The KD amplified the classical exercise‐induced biphasic CD3−CD56+ cell response by increasing the post‐exercise counts (P = 0.0004), which appeared to be underpinned by the cytotoxic CD3−CD56dim subset (main effect of time point, P < 0.0001). The KD had no effect on NK cells’ expression of CD69 or their geometric mean fluorescence intensity of CD69 expression, either for unstimulated or for antigen‐stimulated NK cells (all P > 0.05). In conclusion, adaptation to a KD may alter the number of circulating NK cells but not their ability to activate to an antigenic challenge.

Microphysiological systems (MPS), also referred to as tissue chips, incorporating 3D skeletal myobundles are a novel approach for physiological and pharmacological studies to uncover new medical treatments for sarcopenia. We characterize a MPS in which engineered skeletal muscle myobundles derived from donor‐specific satellite cells that model aged phenotypes are encapsulated in a perfused tissue chip platform containing platinum electrodes. Our myobundles were derived from CD56+ myogenic cells obtained via percutaneous biopsy of the vastus lateralis from adults phenotyped by age and physical activity. Following 17 days differentiation including 5 days of a 3 V, 2 Hz electrical stimulation regime, the myobundles exhibited fused myotube alignment and upregulation of myogenic, myofiber assembly, signaling and contractile genes as demonstrated by gene array profiling and localization of key components of the sarcomere. Our results demonstrate that myobundles derived from the young, active (YA) group showed high intensity immunofluorescent staining of α‐actinin proteins and responded to electrical stimuli with a ~1 μm displacement magnitude compared with non‐stimulated myobundles. Myobundles derived from older sedentary group (OS) did not display a synchronous contraction response. Hypertrophic potential is increased in YA‐derived myobundles in response to stimulation as shown by upregulation of insulin growth factor (IGF‐1), α‐actinin (ACTN3, ACTA1) and fast twitch troponin protein (TNNI2) compared with OS‐derived myobundles. Our MPS mimics disease states of muscle decline and thus provides an aged system and experimental platform to investigate electrical stimulation mimicking exercise regimes and may be adapted to long duration studies of compound efficacy and toxicity for therapeutic evaluation against sarcopenia. Novel platform to study age‐related differences in skeletal muscle response to electrical stimulation using a 3 dimensional microphysiological system D MPS that incorporates cells derived from well phenotyped adults characterized by age and physical activity.

MUC1 is a tumour-associated antigen expressed by many solid tumours, including non-small-cell lung cancer. TG4010 is a modified vaccinia Ankara expressing MUC1 and interleukin 2. In a previous study, TG4010 combined with chemotherapy showed activity in non-small-cell lung cancer and the baseline value of CD16, CD56, CD69 triple-positive activated lymphocytes (TrPAL) was shown to be potentially predictive of TG4010 efficacy. In this phase 2b part of the phase 2b/3 TIME trial, we further assess TG4010 in combination with first-line chemotherapy and use of the TrPAL biomarker in this setting. In this phase 2b part of a randomised, double-blind, placebo-controlled, phase 2b/3 trial, we recruited previously untreated patients aged 18 years or older with stage IV non-small-cell lung cancer without a known activating EGFR mutation and with MUC1 expression in at least 50% of tumoural cells. Patients were randomly allocated (1:1) by an external service provider to subcutaneous injections of 108 plaque-forming units of TG4010 or placebo from the beginning of chemotherapy every week for 6 weeks and then every 3 weeks up to progression, discontinuation for any reason, or toxic effects, stratified according to baseline value of TrPAL (≤ or > the upper limit of normal [ULN]) and, in addition, a dynamic minimisation procedure was used, taking into account chemotherapy regimen, histology, addition or not of bevacizumab, performance status, and centre. Patients, site staff, monitors, the study funder, data managers, and the statistician were masked to treatment identity. The primary endpoint was progression-free survival, assessed every 6 weeks, to validate the predictive value of the TrPAL biomarker. If patients with TrPAL values of less than or equal to the ULN had a Bayesian probability of more than 95% that the true hazard ratio (HR) for progression-free survival was less than 1, and if those with TrPAL values of greater than the ULN had a probability of more than 80% that the true HR for progression-free survival was more than 1, the TrPAL biomarker would be validated. We did primary analyses in the intention-to-treat population and safety analyses in those who had received at least one dose of study drug and had at least one valid post-baseline safety assessment. Monitors, site staff, and patients are still masked to treatment assignment. This trial is registered with ClinicalTrials.gov, number NCT01383148. Between April 10, 2012, and Sept 12, 2014, we randomly allocated 222 patients (TG4010 and chemotherapy 111 [50%]; placebo and chemotherapy 111 [50%]). In the whole population, median progression-free survival was 5·9 months (95% CI 5·4–6·7) in the TG4010 group and 5·1 months (4·2–5·9) in the placebo group (HR 0·74 [95% CI 0·55–0·98]; one-sided p=0·019). In patients with TrPAL values of less than or equal to the ULN, the HR for progression-free survival was 0·75 (0·54–1·03); the posterior probability of the HR being less than 1 was 98·4%, and thus the primary endpoint was met. In patients with TrPAL values of greater than the ULN, the HR for progression-free survival was 0·77 (0·42–1·40); the posterior probability of the HR being greater than 1 was 31·3%, and the primary endpoint was not met. We noted grade 1–2 injection-site reactions in 36 (33%) of 110 patients in the TG4010 group versus four (4%) of 107 patients in the placebo group. We noted no grade 3 or 4 nor serious adverse events deemed to be related to TG4010 only. Four (4%) patients presented grade 3 or 4 adverse events related to TG4010 and other study treatments (chemotherapy or bevacizumab) versus 11 (10%) in the placebo group. No serious adverse event was related to the combination of TG4010 with other study treatments. The most frequent severe adverse events were neutropenia (grade 3 29 [26%], grade 4 13 [12%] in the TG4010 group vs grade 3 22 [21%], grade 4 11 [10%] in the placebo group), anaemia (grade 3 12 [11%] vs grade 3 16 [15%]), and fatigue (grade 3 12 [11%], grade 5 one [1%] vs grade 3 13 [12%]; no grade 4 events). TG4010 plus chemotherapy seems to improve progression-free survival relative to placebo plus chemotherapy. These data support the clinical value of the TrPAL biomarker in this clinical setting; because the primary endpoint was met, the trial is to continue into the phase 3 part. Transgene, Avancées Diagnostiques pour de Nouvelles Approches Thérapeutiques (ADNA), and OSEO.

Natural killer (NK) cells are critical to both innate and adaptive immunity. However, the development and heterogeneity of human NK cells are yet to be fully defined. Using single-cell RNA-sequencing technology, here we identify distinct NK populations in human bone marrow and blood, including one population expressing higher levels of immediate early genes indicative of a homeostatic activation. Functionally matured NK cells with high expression of CX3CR1 , HAVCR2 (TIM-3), and ZEB2 represents terminally differentiated status with the unique transcriptional profile. Transcriptomic and pseudotime analyses identify a transitional population between CD56 bright and CD56 dim NK cells. Finally, a donor with GATA2 T354M mutation exhibits reduced percentage of CD56 bright NK cells with altered transcriptome and elevated cell death. These data expand our understanding of the heterogeneity and development of human NK cells. Natural killer (NK) cells are important innate immune cells with diverse functions. Here the authors use single-cell RNA-sequencing of purified human bone marrow and peripheral blood NK cells to define five populations of NK cells with distinct transcriptomic profile to further our understanding of NK development and heterogeneity.

Over the past years, the phenotypic and functional boundaries distinguishing the main cell subsets of the immune system have become increasingly blurred. In this respect, CD56 (also known as neural cell adhesion molecule) is a very good example. CD56 is the archetypal phenotypic marker of natural killer cells but can actually be expressed by many more immune cells, including alpha beta T cells, gamma delta T cells, dendritic cells, and monocytes. Common to all these CD56-expressing cell types are strong immunostimulatory effector functions, including T helper 1 cytokine production and an efficient cytotoxic capacity. Interestingly, both numerical and functional deficiencies and phenotypic alterations of the CD56 immune cell fraction have been reported in patients with various infectious, autoimmune, or malignant diseases. In this review, we will discuss our current knowledge on the expression and function of CD56 in the hematopoietic system, both in health and disease.

Invasive aspergillosis causes significant morbidity and mortality in immunocompromised patients. Natural killer (NK) cells are pivotal for antifungal defense. Thus far, CD56 is the only known pathogen recognition receptor on NK cells triggering potent antifungal activity against Aspergillus fumigatus . However, the underlying cellular mechanisms and the fungal ligand of CD56 have remained unknown. Using purified cell wall components, biochemical treatments, and ger mutants with altered cell wall composition, we herein found that CD56 interacts with the A . fumigatus cell wall carbohydrate galactosaminogalactan (GAG). This interaction induced NK-cell activation, degranulation, and secretion of immune-enhancing chemokines and cytotoxic effectors. Supernatants from GAG-stimulated NK cells elicited antifungal activity and enhanced antifungal effector responses of polymorphonuclear cells. In conclusion, we identified A . fumigatus GAG as a ligand of CD56 on human primary NK cells, stimulating potent antifungal effector responses and activating other immune cells.