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Hidradenitis suppurativa (HS) is a chronic, debilitating inflammatory skin disease with a poorly understood immunopathogenesis. Here, we report that HS lesional skin is characterized by the expansion of innate lymphocytes and T cells expressing CD2, an essential activation receptor and adhesion molecule. Lymphocytes expressing elevated CD2 predominated with unique spatial distribution throughout the epidermis and hypodermis in the HS lesion. CD2 + cells were mainly innate lymphocytes expressing the NK cell marker, CD56, and CD4 + T cells. Importantly, these CD2 + cells interacted with CD58 (LFA3) expressing epidermal keratinocytes and fibroblasts in the hypodermis. Granzyme A bright NKT cells (CD2 + CD3 + CD56 bright ) clustered with α-SMA expressing fibroblasts juxtaposed to epithelialized tunnels and fibrotic regions of the hypodermis. Whereas NK cells (CD2 + CD56 dim ) were perforin + , granzymes A + and B + , and enriched adjacent to hyperplastic follicular epidermis and tunnels of HS showing presence of apoptotic cells. The cytokines IL-12, IL-15, and IL-18, which enhance NK cell maturation and function were significantly elevated in HS. Ex vivo HS skin explant cultures treated with CD2:CD58 interaction-blocking anti-CD2 monoclonal antibody attenuated secretion of inflammatory cytokines/chemokines and suppressed inflammatory gene signature. Additionally, CD2:CD58 blockade altered miRNAs involved in NK/NKT differentiation and/or function. In summary, we show that a cellular network of heterogenous NKT and NK cell populations drives inflammation and is critical in the pathobiology of HS, including tunnel formation and fibrosis. Finally, CD2 blockade is a viable immunotherapeutic approach for the effective management of HS., Competing Interests: Competing interests statement:E.B. and D.B. are co-founders of ITB-MED. Patent application number: PCT/US2023/022364.

In solid tumors, the exhaustion of natural killer (NK) cells and cytotoxic T cells in the immunosuppressive tumor microenvironment poses challenges for effective tumor control. Conventional humanized mouse models of hepatocellular carcinoma patient-derived xenografts (HCC-PDX) encounter limitations in NK cell infiltration, hindering studies on NK cell immunobiology. Here, we introduce an improved humanized mouse model with restored NK cell reconstitution and infiltration in HCC-PDX, coupled with single-cell RNA sequencing (scRNA-seq) to identify potential anti-HCC treatments. A single administration of adeno-associated virus carrying human interleukin-15 reinstated persistent NK cell reconstitution and infiltration in HCC-PDX in humanized mice. scRNA-seq revealed NK cell and T cell subpopulations with heightened PDCD1 and TIGIT levels. Notably, combination therapy with anti-PD-1 and anti-TIGIT antibodies alleviated HCC burden in humanized mice, demonstrating NK cell-dependent efficacy. Bulk-RNA sequencing analysis also revealed significant alterations in the tumor transcriptome that may contribute to further resistance after combination therapy, warranting further investigations. As an emerging strategy, ongoing clinical trials with anti-PD-1 and anti-TIGIT antibodies provide limited data. The improved humanized mouse HCC-PDX model not only sheds light on the pivotal role of NK cells but also serves as a robust platform for evaluating safety and anti-tumor efficacy of combination therapies and other potential regimens, complementing clinical insights., Competing Interests: Declaration of interests Q.C. is the scientific cofounder of two biotech companies., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Natural killer (NK) cells are a promising allogeneic immunotherapy option due to their natural ability to kill tumor cells, and due to their apparent safety. This study describes the development of a GMP-compliant manufacturing protocol for the local production of functionally potent NK cells tailored for high-risk acute myeloid leukemia (AML) and neuroblastoma (NBL) patients. Moreover, the quality control strategy and considerations for product batch specifications in early clinical development are described. The protocol is based on the CliniMACS Prodigy platform and Natural Killer Cell Transduction (NKCT) (Miltenyi Biotec). NK cells are isolated from leukapheresis through CD3 depletion and CD56 enrichment, followed by a 12-hour activation with IL-2 and IL-15 cytokines. Three CliniMACS Prodigy processes demonstrated the feasibility and consistency of the modified NKCT process. A three-step process without expansion, however, compromised the NK cell yield. T cells were depleted effectively, indicating excellent safety of the product. Characterization of the NK cells before and after cytokine activation revealed a notable increase in the expression of activation markers, particularly CD69, consistent with enhanced functionality. Intriguingly, the NK cells exhibited increased killing efficacy against patient-derived CD33 + AML blasts and NBL cells in vitro, suggesting a potential therapeutic benefit in AML and NBL., (© 2024. The Author(s).)

Limited oxygen (hypoxia) in solid tumors poses a challenge to successful immunotherapy with natural killer (NK) cells. NK cells have impaired cytotoxicity when cultured in hypoxia (1% oxygen) but not physiologic (>5%) or atmospheric oxygen (20%). We found that changes to cytotoxicity were regulated at the transcriptional level and accompanied by metabolic dysregulation. Dosing with interleukin-15 (IL-15) enhanced NK cell cytotoxicity in hypoxia, but preactivation with feeder cells bearing IL-21 and 4-1BBL was even better. Preactivation resulted in less perturbed metabolism in hypoxia; greater resistance to oxidative stress; and no hypoxia-induced loss of transcription factors (T-bet and Eomes), activating receptors, adhesion molecules (CD2), and cytotoxic proteins (TRAIL and FasL). There remained a deficit in CD122/IL-2Rβ when exposed to hypoxia, which affected IL-15 signaling. However, tri-specific killer engager molecules that deliver IL-15 in the context of anti-CD16/FcγRIII were able to bypass this deficit, enhancing cytotoxicity of both fresh and preactivated NK cells in hypoxia.

Bronchopulmonary dysplasia (BPD) is a severe lung disease in preterm infants, the abnormal proliferate and differentiate ability of type II epithelial cells (AEC II) is the key to the pathological basis of BPD. Mechanisms regarding abnormal AEC II in BPD remain unclear. The present work investigated the role and mechanisms of invariant natural killer T (iNKT) cells in lung disorder in BPD using public datasets, clinical samples, a hyperoxia-induced BPD mouse model and AEC II-iNKT cells transwell co-culture system. Firstly, we found that the NKT cells development factor IL-15 increased over time in patients with BPD in public databases, and clinically collected peripheral blood NKT cells in patients with BPD were increased. Subsequently, the percentage of iNKT cells increased in hyperoxia group compared with normoxia group, with the highest at P7, accompanied by increased activation with abnormal lung development. The administration of anti-CD1d neutralizing antibody to inhibit iNKT cells could alleviate the abnormal lung development of hyperoxia group mice, while α-GalCer administration could aggravate lung injury in hyperoxia group mice, and adoptive transfer of iNKT cells could aggravate the abnormal lung development in hyperoxia group mice. In addition, to further verify the role of iNKT cells on AEC II, AEC II-iNKT cells co-culture system was established. The presence of iNKT cells could aggravate the abnormal expression of SP-C and T1α under hyperoxia. Meanwhile, RNA-seq analysis showed that ferroptosis-related genes were highly expressed in AEC II co-cultured with iNKT cells under hyperoxia. We further validated the effect of the presence of iNKT cells under hyperoxia environment on AEC II ferroptosis levels, suggested that iNKT cells promote AEC II ferroptosis under hyperoxia, accompanied by decreased expression of SP-C and T1α. Our study found that the recruitment of iNKT cells in the lung may be an important cause of alveolarization disorder in BPD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Expression of IL-15 on the surface of human graft endothelial cells (ECs) bound to the IL-15Rα subunit can increase the activation of CTLs, potentiating allograft rejection. Our previous work showed that surface expression of this protein complex could be induced by alloantibody-mediated complement activation through increased IL-1β synthesis, secretion, and autocrine/paracrine IL-1-mediated activation of NF-κB. In this article, we report that cultured human ECs express eight differently spliced IL-15 transcripts. Remarkably, IL-1β does not alter the expression level of any IL-15 transcript but induces surface expression independently of RNA polymerase II-mediated transcription while requiring new protein translation. Mechanistically, IL-1β causes an NF-κB-mediated reduction in the level of microRNA Let-7c-3p, thereby relieving a block of translation of IL-15 surface protein. Let7c-3p anti-miR can induce EC surface expression of IL-15/IL-15Rα in the absence of complement activation or of IL-1, enabling IL-15 transpresentation to boost CD8 T cell activation. Because of the complexity we have uncovered in IL-15 regulation, we recommend caution in interpreting increased total IL-15 mRNA or protein levels as a surrogate for transpresentation., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Background: The benefits of exercise are well known; however, many of the underlying molecular mechanisms are not fully understood. Skeletal muscle secretes myokines, which mediate muscle-organ crosstalk. Myokines regulate satellite-cell proliferation and migration, inflammatory cascade, insulin secretion, angiogenesis, fatty oxidation, and cancer suppression. To date, the effects of different exercise modes (namely, aerobic and resistance exercise) on myokine response remain to be elucidated. This is crucial considering the clinical implementation of exercise to enhance general health and wellbeing and as a medical treatment., Methods: A systematic search was undertaken in PubMed, MEDLINE, CINAHL, Embase, SPORTDiscus, and Web of Science in April 2023. Eligible studies examining the effects of a single bout of exercise on interleukin15 (IL-15), irisin, secreted protein acidic and rich in cysteine (SPARC), oncostatin M (OSM), and decorin were included. A random-effects meta-analysis was also undertaken to quantify the magnitude of change., Results: Sixty-two studies were included (n = 1193). Overall, exercise appeared to induce small to large increases in myokine expression, with effects observed immediately after to 60 min post-exercise, although these were mostly not statistically significant. Both aerobic and resistance exercise resulted in changes in myokine levels, without any significant difference between training modes, and with the magnitude of change differing across myokines. Myokine levels returned to baseline levels within 180 min to 24 h post-exercise. However, owing to potential sources of heterogeneity, most changes were not statistically significant, indicating that precise conclusions cannot be drawn., Conclusion: Knowledge is limited but expanding with respect to the impact of overall and specific effects of exercise on myokine expression at different time points in the systemic circulation. Further research is required to investigate the effects of different exercise modes at multiple time points on myokine response., Competing Interests: Competing interests The authors declare that they have no competing interests., (Copyright © 2024. Production and hosting by Elsevier B.V.)

Hypobaric hypoxia (HH) limits oxygen supply to tissues and increases metabolic demands, especially during exercise. We studied the influence of HH exposure on the subcutaneous adipose tissue (SAT) thickness and circulating metabolic-related cytokines levels after a resistance training (R T ) program. Twenty trained men participated in a traditional hypertrophy R T for 8 weeks (three sessions/week) under intermittent terrestrial HH (2320 m) or normoxia (N, 690 m) conditions. Before, at week 6, and after the R T , SAT, and vastus lateralis (VL) muscle thickness were measured by ultrasound. Blood samples were taken to analyse serum cytokines (IL-6, IL-15, irisin, and myostatin) by multiplex immunoassay. Our findings revealed a moderate reduction in IL-6 and irisin in HH following the R T (ES < -0.64; p < 0.05). Additionally, R T in HH promoted serum IL-15 release (ES = 0.890; p = 0.062), which exhibited a trivial inverse association with the reductions observed on SAT (-17.69%; p < 0.001) compared with N. R T in HH explained ~50% of SAT variance ( p < 0.001). These results highlight the benefit of stressor factors linked to R T in HH on SAT through the modulation of serum metabolic cytokine profiles, suggesting a potential effect on overall body composition.

Advances in imaging, segmentation and tracking have led to the routine generation of large and complex microscopy datasets. New tools are required to process this 'phenomics' type data. Here, we present 'Cell PLasticity Analysis Tool' (cellPLATO), a Python-based analysis software designed for measurement and classification of cell behaviours based on clustering features of cell morphology and motility. Used after segmentation and tracking, the tool extracts features from each cell per timepoint, using them to segregate cells into dimensionally reduced behavioural subtypes. Resultant cell tracks describe a 'behavioural ID' at each timepoint, and similarity analysis allows the grouping of behavioural sequences into discrete trajectories with assigned IDs. Here, we use cellPLATO to investigate the role of IL-15 in modulating human natural killer (NK) cell migration on ICAM-1 or VCAM-1. We find eight behavioural subsets of NK cells based on their shape and migration dynamics between single timepoints, and four trajectories based on sequences of these behaviours over time. Therefore, by using cellPLATO, we show that IL-15 increases plasticity between cell migration behaviours and that different integrin ligands induce different forms of NK cell migration., Competing Interests: Competing interests T.W.F.S. is the sole proprietor of Quorumetrix Solutions and provided custom scientific data processing and visualization services used in this study., (© 2024. Published by The Company of Biologists Ltd.)

Development of novel therapeutic strategies that reactivate latent HIV and sensitize reactivated cells to apoptosis is crucial towards elimination of the latent viral reservoir. Among the clinically relevant latency reversing agents (LRA) under investigation, the γc-cytokine IL-15 and the superagonist N-803 have been shown to reactivate latent HIV ex vivo and in vivo. However, their clinical benefit can be hindered by IL-15 promoting survival of infected cells. We previously identified a small molecule, HODHBt, that sensitizes latently infected cells to death upon reactivation with γc-cytokines through a STAT-dependent pathway. In here, we aimed to identify and evaluate FDA-approved compounds that could also sensitize HIV-infected cells to apoptosis. Using the Connectivity Map (CMap), we identified the retinol derivative 13-cis-retinoic acid (Isotretinoin) causes similar transcriptional changes as HODHBt. Isotretinoin enhances IL-15-mediated latency reversal without inducing proliferation of memory CD4 T cells. Ex vivo analysis of PBMCs from ACTG A5325, where Isotretinoin was administered to ART-suppressed people with HIV, showed that Isotretinoin treatment enhances IL-15-mediated latency reversal. Furthermore, we showed that a combination of IL-15 with Isotretinoin promotes the reduction of translation-competent reservoirs ex vivo. Mechanistically, combination of IL-15 and Isotretinoin increases caspase-3 activation specifically in HIV-infected cells but not uninfected cells. Our results suggest that Isotretinoin can be a novel approach to target and eliminate translation-competent HIV reservoirs., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Howard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Changes in mechanosensitive ion channels following radiation have seldom been linked to therapeutic sensitivity or specific factors involved in antitumor immunity. Here, in this study, we found that the mechanical force sensor, Piezo2, was significantly upregulated in tumor cells after radiation, and Piezo2 knockout in tumor cells enhanced tumor growth suppression by radiotherapy. Specifically, loss of Piezo2 in tumor cells induced their IL-15 expression via unleashing JAK2/STAT1/IRF-1 axis after radiation. This increase in IL-15 activates IL-15Rα on tumor-infiltrating CD8+ T cells, thereby leading to their augmented effector and stem cell-like properties, along with reduced terminal exhausted feature. Importantly, Piezo2 expression was negatively correlated with CD8 infiltration, as well as with radiosensitivity of patients with rectum adenocarcinoma receiving radiotherapy treatment. Together, our findings reveal that tumor cell-intrinsic Piezo2 induces radioresistance by dampening the IRF-1/IL-15 axis, thus leading to impaired CD8+ T cell-dependent antitumor responses, providing insights into the further development of combination strategies to treat radioresistant cancers., (© 2024 Miao et al.)

Natural killer (NK) cells are the main innate antitumor effector cells but their function is often constrained in the tumor microenvironment. It has been reported that the E3 ligase FBXO38 accelerates PD-1 degradation in tumor-infiltrating T cells to unleash their cytotoxic function. In this study, we found that the transcriptional levels of FBXO38 in intratumoral NK cells of patients with cancer and tumor-bearing mice were significantly lower than in peritumoral NK cells. Conditional knockout of FBXO38 in NK cells accelerated tumor growth and increased tumor metastasis. FBXO38 deficiency resulted in impaired proliferation and survival of tumor-infiltrating NK (TINK) cells. Mechanistically, FBXO38 deficiency enhanced TGF-β signaling, including elevating expression of Smad2 and Smad3, which suppressed expression of the transcription factor Eomes and further reduced expression of surface IL15Rβ and IL15Rγc on NK cells. Consequently, FBXO38 deficiency led to TINK cell hyporesponsiveness to IL15. Consistent with these observations, FBXO38 mRNA expression was positively correlated with the proliferation of TINK cells in multiple human tumors. To study the therapeutic potential of FBXO38, mice bearing human tumors were treated with FBXO38 overexpressed human primary NK cells and showed a significant reduction in tumor size and prolonged survival. In conclusion, our results suggest that FBXO38 sustains NK-cell expansion and survival to promote antitumor immunity and have potential therapeutic implications as they suggest FBXO38 could be harnessed to enhance NK cell-based cancer immunotherapy., (©2024 American Association for Cancer Research.)

Microglia play a major role in the immune defense system of the central nervous system and are activated in many neurological diseases. The immunomodulatory cytokine interleukin (IL)-15 is known to be involved in microglia response and inflammatory factors release. Neoprzewaquinone A (NEO) is an active compound isolated from Salvia miltiorrhiza Bunge. Our previous study has shown that NEO significantly inhibit the proliferation of IL-15-treated Mo7e cells. However, the role of NEO in the structure and function of IL-15-treated human microglial cells (HMC3) remains unclear. Thus, our study aimed to quantitatively analyze the beneficial effects of NEO on HMC3 cells following IL-15 treatment. The cell viability, phagocytosis, migration and energy metabolism were evaluated by Cell Counting Kit-8 (CCK8), scratch assay, pHrodo™ Red Zymosan BioParticles™ Conjugate, and Agilent Seahorse XF Cell Mito Test. Cephalothin (CEP) was selected as a positive drug because it has obvious inhibitory effect on IL-15 and IL-15Rɑ. Our results showed that IL-15 stimulated the proliferation, migration and phagocytosis of HMC3 cells in a time-dependent manner. Interestingly, NEO exhibited significant suppressive effects on these IL-15-induced changes, which were even superior to those observed with the CEP. Moreover, IL-15 treatment did not significantly alter energy metabolism, including glycolysis and mitochondrial respiration. NEO and CEP alone effectively reduced glycolysis, non-mitochondrial respiration, basal respiration, ATP turnover, respiration capacity, and H + leak in HMC3 cells. Furthermore, NEO displayed a partial regulatory effect on mitochondrial function in IL-15-treated HMC3 cells. Our study confirms the effectively inhibition of NEO on IL-15-induced microglial activation and provides valuable insights into the therapeutic prospects of NEO in neuropsychiatric disorders associated with IL-15 and microglia., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Systemically administered cytokines are potent immunotherapeutics but can cause severe dose-limiting toxicities. To overcome this challenge, cytokines have been engineered for intratumoral retention after local delivery. However, despite inducing regression of treated lesions, tumor-localized cytokines often elicit only modest responses at distal untreated tumors. In the present study, we report a localized cytokine therapy that safely elicits systemic antitumor immunity by targeting the ubiquitous leukocyte receptor CD45. CD45-targeted immunocytokines have lower internalization rates relative to wild-type counterparts, leading to sustained downstream cis and trans signaling between lymphocytes. A single intratumoral dose of αCD45-interleukin (IL)-12 followed by a single dose of αCD45-IL-15 eradicated treated tumors and untreated distal lesions in multiple syngeneic mouse tumor models without toxicity. Mechanistically, CD45-targeted cytokines reprogrammed tumor-specific CD8 + T cells in the tumor-draining lymph nodes to have an antiviral transcriptional signature. CD45 anchoring represents a broad platform for protein retention by host immune cells for use in immunotherapy., (© 2024. The Author(s).)

Immunotherapy has emerged as a promising approach for cancer treatment, with oncolytic adenoviruses showing power as immunotherapeutic agents. In this study, we investigated the immunotherapeutic potential of an adenovirus construct expressing CXCL9, CXCL10, or IL-15 in clear cell renal cell carcinoma (ccRCC) tumor models. Our results demonstrated robust cytokine secretion upon viral treatment, suggesting effective transgene expression. Subsequent analysis using resistance-based transwell migration and microfluidic chip assays demonstrated increased T-cell migration in response to chemokine secretion by infected cells in both 2D and 3D cell models. Flow cytometry analysis revealed CXCR3 receptor expression across T-cell subsets, with the highest percentage found on CD8+ T-cells, underscoring their key role in immune cell migration. Alongside T-cells, we also detected NK-cells in the tumors of immunocompromised mice treated with cytokine-encoding adenoviruses. Furthermore, we identified potential immunogenic antigens that may enhance the efficacy and specificity of our armed oncolytic adenoviruses in ccRCC. Overall, our findings using ccRCC cell line, in vivo humanized mice, physiologically relevant PDCs in 2D and patient-derived organoids (PDOs) in 3D suggest that chemokine-armed adenoviruses hold promise for enhancing T-cell migration and improving immunotherapy outcomes in ccRCC. Our study contributes to the development of more effective ccRCC treatment strategies by elucidating immune cell infiltration and activation mechanisms within the tumor microenvironment (TME) and highlights the usefulness of PDOs for predicting clinical relevance and validating novel immunotherapeutic approaches. Overall, our research offers insights into the rational design and optimization of viral-based immunotherapies for ccRCC., Competing Interests: Oncolytic viruses encoding for cytokines CXCL9, CXCL10 and IL-15 have been licensed to Ximbio/CancerTools.org for commercialization to other academic or commercial researchers. V.S. is currently employed by AstraZeneca. V.C is a co-founder and shareholder at VALO Therapeutics. Other authors report there are no competing interests to declare., (© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Background: Immune checkpoint ligand-receptor interactions appear to be associated with multiple myeloma (MM) progression. Simultaneously, previous studies showed the possibility of PD-1 and TIM-3 expression on T cells upon stimulation with common γ-chain family cytokines in vitro and during homeostatic proliferation. The aim of the present work was to study the impact of homeostatic proliferation on the expansion of certain T cell subsets up-regulating PD-1 and TIM-3 checkpoint molecules., Methods: The expression of CD25, CD122, CD127 common γ-chain cytokine receptors, phosphorylated signal transducer and activator of transcription-5 (pSTAT5) and eomesodermin (EOMES) was comparatively assessed with flow cytometry in PD-1- and TIM-3-negative and positive T cells before the conditioning and during the first post-transplant month in peripheral blood samples of MM patients., Results: Substantial proportions of PD-1- and TIM-3-positive T lymphocytes expressed common γ-chain cytokine receptors and pSTAT5. Frequencies of cytokine receptor expressing cells were significantly higher within TIM-3 + T cells compared to PD-1 + TIM-3 - subsets. Considerable proportions of both PD-1-/TIM-3-negative and positive CD8 + T cells express EOMES, while only moderate frequencies of CD4 + PD-1 + /TIM-3 + T cells up-regulate this transcription factor. Besides, the surface presence of CD25 and intranuclear expression of EOMES in CD4 + T cells were mutually exclusive regardless of PD-1 and TIM-3 expression. The stimulation with common γ-chain cytokines up-regulates PD-1 and TIM-3 during the proliferation of initially PD-1/TIM-3-negative T cells but fails to expand initially PD-1 + and TIM-3 + T cell subsets in vitro ., Conclusions: Both PD-1 and TIM-3 expressing T cells appear to be able to respond to homeostatic cytokine stimulation. Differences in common γ-chain cytokine receptor expression between PD-1 + and TIM-3 + T cells may reflect functional dissimilarity of these cell subsets. Checkpoint blockade appears to alleviate lymphopenia-induced proliferation of PD-1 + T cells but may raise the possibility of immune-mediated adverse events., Competing Interests: The authors declare that they have no conflicts of interest to report regarding the present study., (© 2024 The Authors.)

We present a TALEN-based workflow to generate and maintain dual-edited (IL-15 +/+ /TGFβR2 -/- ) iPSCs that produce enhanced iPSC-derived natural killer (iNK) cells for cancer immunotherapy. It involves using a cell lineage promoter for knocking in (KI) gene(s) to minimize the potential effects of expression of any exogenous genes on iPSCs. As a proof-of-principle, we KI IL-15 under the endogenous B2M promoter and show that it results in high expression of the sIL-15 in iNK cells but minimal expression in iPSCs. Furthermore, given that it is known that knockout (KO) of TGFβR2 in immune cells can enhance resistance to the suppressive TGF-β signaling in the tumor microenvironment, we develop a customized medium containing Nodal that can maintain the pluripotency of iPSCs with TGFβR2 KO, enabling banking of these iPSC clones. Ultimately, we show that the dual-edited IL-15 +/+ /TGFβR2 -/- iPSCs can be efficiently differentiated into NK cells that show enhanced autonomous growth and are resistant to the suppressive TGF-β signaling., Competing Interests: Declaration of interests A.-P.C., P.G., L.L., P.A., H.H., H.-M.C., D.Z., A.R., D.T., A.A., and W.L. are employees of and hold stock or stock options in Cytovia Therapeutics. J.E. is a consultant to Cytovia Therapeutics and received funding support from Cytovia Therapeutics., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Infectious, inflammatory and autoimmune conditions present differently in males and females. SARS-CoV-2 infection in naive males is associated with increased risk of death, whereas females are at increased risk of long COVID 1 , similar to observations in other infections 2 . Females respond more strongly to vaccines, and adverse reactions are more frequent 3 , like most autoimmune diseases 4 . Immunological sex differences stem from genetic, hormonal and behavioural factors 5 but their relative importance is only partially understood 6-8 . In individuals assigned female sex at birth and undergoing gender-affirming testosterone therapy (trans men), hormone concentrations change markedly but the immunological consequences are poorly understood. Here we performed longitudinal systems-level analyses in 23 trans men and found that testosterone modulates a cross-regulated axis between type-I interferon and tumour necrosis factor. This is mediated by functional attenuation of type-I interferon responses in both plasmacytoid dendritic cells and monocytes. Conversely, testosterone potentiates monocyte responses leading to increased tumour necrosis factor, interleukin-6 and interleukin-15 production and downstream activation of nuclear factor kappa B-regulated genes and potentiation of interferon-γ responses, primarily in natural killer cells. These findings in trans men are corroborated by sex-divergent responses in public datasets and illustrate the dynamic regulation of human immunity by sex hormones, with implications for the health of individuals undergoing hormone therapy and our understanding of sex-divergent immune responses in cisgender individuals., (© 2024. The Author(s).)

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by persistent immune cell activation and the overproduction of autoantibodies, affecting various organs such as joints, kidneys, and skin. Interleukin-15 (IL-15) is a pleiotropic cytokine that modulates immune cells of the innate and adaptive immune systems, playing a crucial role in the development of inflammatory and protective immune responses. However, the role of IL-15 in SLE pathogenesis and the therapeutic effects of IL-15 blockade on SLE remain unknown. In this study, we conducted flow cytometry analysis and identified a significant increase in the frequencies of IL-15 + and IL-15R + cells in peripheral blood CD4 + T cells, CD8 + T cells, dendritic cells (DCs), monocytes, and natural killer (NK) cells of patients with SLE compared to healthy controls (HCs). Besides, we found elevated levels of serum IL-15 in SLE patients compared to HCs. Furthermore, we evaluted the effectiveness of IL-15 mAb treatment in a chronic graft-versus-host disease (cGVHD) mouse model of SLE. We observed that the IL-15 mAb treatment effectively reduced the frequencies of CD4 + CD44 hi CD62L lo PD-1 + CD153 + senescent CD4 + T cells, B220 + CD11c + T-bet + age-associated B cells (ABCs), Tfh cells, and germinal center (GC) B cells, alleviated lupus-associated manifestations such as serum anti-double-stranded DNA antibody (anti-dsDNA) and kidney injury in the SLE mouse model of cGVHD. These findings provide compelling preclinical evidence suggesting the pathogenic role of IL-15 in SLE and the therapeutic potential of IL-15 blockade in the treatment of SLE., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Intraepithelial lymphocytes are the first line of defence of the human intestinal immune system. Besides, their composition is altered on patients with coeliac disease (CD), so they are considered as biomarkers with utility on their diagnose and/or monitoring. Our aim is to address their variability through the human gastrointestinal tract in health and characterized them in further depth in the coeliac duodenum., Methods: Intraepithelial lymphocytes were isolated from human gastric, duodenal, ileal and colonic biopsies, then stained with specific antibodies and acquired by flow cytometry., Results: Our results confirmed that the profile of Intraepithelial lymphocytes change through the length of the human gastrointestinal tract. Besides and given the central role that Interleukin-15 (IL-15) elicits on CD pathogenesis; we also assessed the expression of its receptor revealing that there was virtually no functional IL-15 receptor on duodenal Intraepithelial lymphocytes. Nevertheless and contrary to our expectations, the active IL-15 receptor was not increased either on Intraepithelial lymphocytes from CD patients., Conclusions: IL-15 might require additional stimulus to activate intraepithelial lymphocytes. These findings may provide novel tools to aid on a CD diagnosis and/or monitoring, at the time that provide the bases to perform functional studies in order of getting a deeper insight in the specific function that Intraepithelial lymphocytes elicit on CD pathogenesis., (© 2024 The Author(s). European Journal of Clinical Investigation published by John Wiley & Sons Ltd on behalf of Stichting European Society for Clinical Investigation Journal Foundation.)

Natural killer (NK) cells often become dysfunctional during tumor progression, but the molecular mechanisms underlying this phenotype remain unclear. To explore this phenomenon, we set up mouse lymphoma models activating or not activating NK cells. Both tumor types elicited type I interferon production, leading to the expression of a T cell exhaustion-like signature in NK cells, which included immune checkpoint proteins (ICPs). However, NK cell dysfunction occurred exclusively in the tumor model that triggered NK cell activation. Moreover, ICP-positive NK cells demonstrated heightened reactivity compared to negative ones. Furthermore, the onset of NK cell dysfunction was swift and temporally dissociated from ICPs induction, which occurred as a later event during tumor growth. Last, NK cell responsiveness was restored when stimulation was discontinued, and interleukin-15 had a positive impact on this reversion. Therefore, our data demonstrate that the reactivity of NK cells is dynamically controlled and that NK cell dysfunction is a reversible process uncoupled from the expression of ICPs.

IL-15 is a homeostatic cytokine for human T and NK cells. However, whether other cytokines influence the effect of IL-15 is not known. We studied the impact that IL-10, TGF-β, IL-17A, and IFN-γ have on the IL-15-induced proliferation of human T cells and the expression of HLA class I (HLA-I) molecules. Peripheral blood lymphocytes (PBLs) were labeled with CFSE and stimulated for 12 days with IL-15 in the absence or presence of the other cytokines. The proportion of proliferating T cells and the expression of cell surface HLA-I molecules were analyzed using flow cytometry. The IL-15-induced proliferation of T cells was paralleled by an increase in the expression of HC-10-reactive HLA-I molecules, namely on T cells that underwent ≥5-6 cycles of cell division. It is noteworthy that the IL-15-induced proliferation of T cells was potentiated by IL-10 and TGF-β but not by IL-17 or IFN-γ and was associated with a decrease in the expression of HC-10-reactive molecules. The cytokines IL-10 and TGF-β potentiate the proliferative capacity that IL-15 has on human T cells in vitro, an effect that is associated with a reduction in the amount of HC-10 reactive HLA class I molecules induced by IL-15.

The formation of memory T cells is a fundamental feature of adaptative immunity, allowing the establishment of long-term protection against pathogens. Although emerging evidence suggests that metabolic reprogramming is crucial for memory T cell differentiation and survival, the underlying mechanisms that drive metabolic rewiring in memory T cells remain unclear. Here, we found that up-regulation of the nuclear receptor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) instructs the metabolic reprogramming that occurs during the establishment of central memory CD8 + T cells. PPARβ/δ-regulated changes included suppression of aerobic glycolysis and enhancement of oxidative metabolism and fatty acid oxidation. Mechanistically, exposure to interleukin-15 and expression of T cell factor 1 facilitated activation of the PPARβ/δ pathway, counteracting apoptosis induced by antigen clearance and metabolic stress. Together, our findings indicate that PPARβ/δ is a master metabolic regulator orchestrating a metabolic switch that may be favorable for T cell longevity.

Skeletal muscle has gained recognition as an endocrine organ releasing myokines upon contraction during physical exercise. These myokines exert both local and pleiotropic health benefits, underscoring the crucial role of muscle function in countering obesity and contributing to the overall positive effects of exercise on health. Here, we found that exercise activates muscle p38γ, increasing locomotor activity through the secretion of interleukin-15 (IL-15). IL-15 signals in the motor cortex, stimulating locomotor activity. This activation of muscle p38γ, leading to an increase locomotor activity, plays a crucial role in reducing the risk of diabetes and liver steatosis, unveiling a vital muscle-brain communication pathway with profound clinical implications. The correlation between p38γ activation in human muscle during acute exercise and increased blood IL-15 levels highlights the potential therapeutic relevance of this pathway in treating obesity and metabolic diseases. These findings provide valuable insights into the molecular basis of exercise-induced myokine responses promoting physical activity.

Glioblastoma (GBM) is an aggressive brain cancer with limited therapeutic options. Natural killer (NK) cells are innate immune cells with strong anti-tumor activity and may offer a promising treatment strategy for GBM. We compared the anti-GBM activity of NK cells engineered to express interleukin (IL)-15 or IL-21. Using multiple in vivo models, IL-21 NK cells were superior to IL-15 NK cells both in terms of safety and long-term anti-tumor activity, with locoregionally administered IL-15 NK cells proving toxic and ineffective at tumor control. IL-21 NK cells displayed a unique chromatin accessibility signature, with CCAAT/enhancer-binding proteins (C/EBP), especially CEBPD, serving as key transcription factors regulating their enhanced function. Deletion of CEBPD resulted in loss of IL-21 NK cell potency while its overexpression increased NK cell long-term cytotoxicity and metabolic fitness. These results suggest that IL-21, through C/EBP transcription factors, drives epigenetic reprogramming of NK cells, enhancing their anti-tumor efficacy against GBM., Competing Interests: Declaration of interests M.S., M. Daher, R.B., H.R., S.A., Y.L., N.U., E.L., E.J.S., K.R., and The University of Texas MD Anderson Cancer Center have an institutional financial conflict of interest with Takeda Pharmaceutical. R.B., E.J.S., K.R., and The University of Texas MD Anderson Cancer Center have an institutional financial conflict of interest with Affimed GmbH. K.R. participates on the Scientific Advisory Board for Avenge Bio, Virogin Biotech, Navan Technologies, Caribou Biosciences, Bit Bio Limited, Replay Holdings, oNKo Innate, and The Alliance for Cancer Gene Therapy ACGT. K.R. is the Scientific founder of Syena. E.J.S. has served on the Scientific Advisory Board for Adaptimmune, Axio, Celaid, FibroBiologics, Navan Technologies, New York Blood Center and Novartis. M Daher participates on the Scientific Advisory Board for Cellsbin. M.S., H.S., E.J.S., and K.R. have filed for a patent (20230074303); “Cell immunotherapy for the treatment of cancer.”, (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Lytic Epstein-Barr virus (EBV) infection plays a major role in the pathogenesis of nasopharyngeal carcinoma (NPC). For patients with recurrent or metastatic NPC and resistant to conventional therapies, adoptive cell therapy using EBV-specific cytotoxic T cells (EBV-CTLs) is a promising option. However, the long production period (around 3 to 4 weeks) and low EBV-CTL purity (approximately 40% of total CD8 T cells) in the cell product limits the application of EBV-CTLs in clinics. Thus, this study aimed to establish a protocol for the rapid production of EBV-CTLs., Methods: By culturing peripheral blood mononuclear cells (PBMCs) from EBV-seropositive donors with EBV-specific peptides and interleukin (IL)-2, IL-15, and interferon α (IFN-α) for 9 days, we identified that IL-15 can enhance IL-2-mediated CTL activation and significantly increase the yield of CTLs., Results: When IFN-α was used in IL-2/IL-15-mediated CTL production from days 0 to 6, the productivity of EBV-CTLs and EBV-specific cytotoxicity significantly were reinforced relative to EBV-CTLs from IL-2/IL-15 treatment. Additionally, IFN-α-induced production improvement of virus-specific CTLs was not only the case for EBV-CTLs but also for cytomegalovirus-specific CTLs., Conclusion: We established a novel protocol to rapidly expand highly pure EBV-CTLs from PBMCs, which can produce EBV-CTLs in 9 days and does not require feeder cells during cultivation., (© 2024. The Author(s).)

Despite the remarkable success of chimeric antigen receptor (CAR) T therapy in hematological malignancies, its efficacy in solid tumors remains limited. Cytokine-engineered CAR T cells offer a promising avenue, yet their clinical translation is hindered by the risks associated with constitutive cytokine expression. In this proof-of-concept study, we leverage the endogenous interferon (IFN)-γ promoter for transgenic interleukin (IL)-15 expression. We demonstrate that IFN-γ expression is tightly regulated by T cell receptor signaling. By introducing an internal ribosome entry site IL15 into the 3' UTR of the IFN-γ gene via homology directed repair-mediated knock-in, we confirm that IL-15 expression can co-express with IFN-γ in an antigen stimulation-dependent manner. Importantly, the insertion of transgenes does not compromise endogenous IFN-γ expression. In vitro and in vivo data demonstrate that IL-15 driven by the IFN-γ promoter dramatically improves CAR T cells' antitumor activity, suggesting the effectiveness of IL-15 expression. Last, as a part of our efforts toward clinical translation, we have developed an innovative two-gene knock-in approach. This approach enables the simultaneous integration of CAR and IL-15 genes into TRAC and IFN-γ gene loci using a single AAV vector. CAR T cells engineered to express IL-15 using this approach demonstrate enhanced antitumor efficacy. Overall, our study underscores the feasibility of utilizing endogenous promoters for transgenic cytokines expression in CAR T cells., Competing Interests: Declaration of interests The study was funded by Shenzhen Celconta Life Science Co., Ltd. Two patents related to this study have been filed, and Shenzhen Celconta Life Science Co., Ltd. holds the right of the patents., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

NK cells are cytotoxic innate immune cells involved in antitumor immunity, and they provide a treatment option for patients with acute myeloid leukemia (AML). In this issue of the JCI, Cubitt et al. investigated the role of CD8α, a coreceptor present on approximately 40% of human NK cells. IL-15 stimulation of CD8α- NK cells induced CD8α expression via the RUNX3 transcription factor, driving formation of a unique induced CD8α (iCD8α+) population. iCD8α+ NK cells displayed higher proliferation, metabolic activity, and antitumor cytotoxic function compared with preexisting CD8α+ and CD8α- subsets. Therefore, CD8α expression can be used to define a potential dynamic spectrum of NK cell expansion and function. Because these cells exhibit enhanced tumor control, they may be used to improve in NK cell therapies for patients with AML.

Background: Natural killer (NK) cells are being extensively studied as a cell therapy for cancer. These cells are activated by recognition of ligands and antigens on tumor cells. Cytokine therapies, such as IL-15, are also broadly used to stimulate endogenous and adoptively transferred NK cells in patients with cancer. These stimuli activate the membrane protease ADAM17, which cleaves various cell-surface receptors on NK cells as a negative feedback loop to limit their cytolytic function. ADAM17 inhibition can enhance IL-15-mediated NK cell proliferation in vitro and in vivo. In this study, we investigated the underlying mechanism of this process., Methods: Peripheral blood mononuclear cells (PBMCs) or enriched NK cells from human peripheral blood, either unlabeled or labeled with a cell proliferation dye, were cultured for up to 7 days in the presence of rhIL-15±an ADAM17 function-blocking antibody. Different fully human versions of the antibody were generated; Medi-1 (IgG1), Medi-4 (IgG4), Medi-PGLALA, Medi-F(ab') 2 , and TAB16 (anti-ADAM17 and anti-CD16 bispecific) to modulate CD16A binding. Flow cytometry was used to assess NK cell proliferation and phenotypic markers, immunoblotting to examine CD16A signaling, and IncuCyte-based live cell imaging to measure NK cell antitumor activity., Results: The ADAM17 function-blocking monoclonal antibody (mAb) Medi-1 markedly increased early NK cell activation by IL-15. By using different engineered versions of the antibody, we demonstrate involvement by CD16A, an activating Fcγ receptor and well-described ADAM17 substrate. Hence, Medi-1 when bound to ADAM17 on NK cells is engaged by CD16A and blocks its shedding, inducing and prolonging its signaling. This process did not promote evident NK cell fratricide or dysfunction. Synergistic signaling by Medi-1 and IL-15 enhanced the upregulation of CD137 on CD16A + NK cells and augmented their proliferation in the presence of PBMC accessory cells or an anti-CD137 agonistic mAb., Conclusions: Our data reveal for the first time that CD16A and CD137 underpin Medi-1 enhancement of IL-15-driven NK cell activation and proliferation, respectively, with the latter requiring PBMC accessory cells. The use of Medi-1 represents a novel strategy to enhance IL-15-driven NK cell proliferation, and it may be of therapeutic importance by increasing the antitumor activity of NK cells in patients with cancer., Competing Interests: Competing interests: MF, JSM and BW are inventors on the patent application WO2022266341A1 (Targeted adam17 blocker compounds, anti-adam17 antibodies, methods of making, and methods of using)., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Natural killer (NK) cells hold promise in cancer treatment due to their ability to spontaneously lyse cancer cells. For clinical use, high quantities of pure, functional NK cells are necessary. Combining adherence-based isolation with specialized media showed the unreliability of the isolation method, but demonstrated the superiority of the NK MACS ® medium, particularly in suboptimal conditions. Neither human pooled serum, fetal calf serum (FCS), human platelet lysate, nor chemically defined serum replacement could substitute human AB serum. Interleukin (IL-)2, IL-15, IL-21, and combined CD2/NKp46 stimulation were assessed. IL-21 and CD2/NKp46 stimulation increased cytotoxicity, but reduced NK cell proliferation. IL-15 stimulation alone achieved the highest proliferation, but the more affordable IL-2 performed similarly. The RosetteSep™ human NK cell enrichment kit was effective for isolation, but the presence of peripheral blood mononuclear cells (PBMCs) in the culture enhanced NK cell proliferation, despite similar expression levels of CD16, NKp46, NKG2D, and ICAM-1. In line with this, purified NK cells cultured in NK MACS ® medium with human AB serum and IL-2 demonstrated high cytotoxicity against primary glioblastoma stem cells.

Adoptive cellular therapy (ACT) using memory-like (ML) natural killer (NK) cells, generated through overnight ex vivo activation with IL-12, IL-15, and IL-18, has shown promise for treating hematologic malignancies. We recently reported that a multifunctional fusion molecule, HCW9201, comprising IL-12, IL-15, and IL-18 domains could replace individual cytokines for priming human ML NK cell programming ("Prime" step). However, this approach does not include ex vivo expansion, thereby limiting the ability to test different doses and schedules. Here, we report the design and generation of a multifunctional fusion molecule, HCW9206, consisting of human IL-7, IL-15, and IL-21 cytokines. We observed > 300-fold expansion for HCW9201-primed human NK cells cultured for 14 days with HCW9206 and HCW9101, an IgG1 antibody, recognizing the scaffold domain of HCW9206 ("Expand" step). This expansion was dependent on both HCW9206 cytokines and interactions of the IgG1 mAb with CD16 receptors on NK cells. The resulting "Prime and Expand" ML NK cells exhibited elevated metabolic capacity, stable epigenetic IFNG promoter demethylation, enhanced antitumor activity in vitro and in vivo, and superior persistence in NSG mice. Thus, the "Prime and Expand" strategy represents a simple feeder cell-free approach to streamline manufacturing of clinical-grade ML NK cells to support multidose and off-the-shelf ACT., (© 2024. The Author(s).)

Metabolic disorders are highly prevalent in modern society. Exercise mimetics are defined as pharmacological compounds that can produce the beneficial effects of fitness. Recently, there has been increased interest in the role of eugenol and transient receptor potential vanilloid 1 (TRPV1) in improving metabolic health. The aim of this study was to investigate whether eugenol acts as an exercise mimetic by activating TRPV1. Here, we showed that eugenol improved endurance capacity, caused the conversion of fast-to-slow muscle fibers, and promoted white fat browning and lipolysis in mice. Mechanistically, eugenol promoted muscle fiber-type transformation by activating TRPV1-mediated CaN signaling pathway. Subsequently, we identified IL-15 as a myokine that is regulated by the CaN/nuclear factor of activated T cells cytoplasmic 1 (NFATc1) signaling pathway. Moreover, we found that TRPV1-mediated CaN/NFATc1 signaling, activated by eugenol, controlled IL-15 levels in C2C12 myotubes. Our results suggest that eugenol may act as an exercise mimetic to improve metabolic health via activating the TRPV1-mediated CaN signaling pathway., Competing Interests: TH, XC, JH, PZ, YL, AW, HY, BY, DC, ZH No competing interests declared, (© 2023, Huang, Chen et al.)

Natural killer (NK) cells play a crucial role in immune response against viral infections and tumors. However, further investigation is needed to better understand the key molecules responsible for determining the fate and function of NK cells. In this study, we made an important discovery regarding the involvement of the Hippo kinases Mst1 and Mst2 as novel regulators in maintaining mouse NK cell homeostasis. The presence of high Mst1 and Mst2 (Mst1/2) activity in NK cells is essential for their proper development, survival and function in a canonical Hippo signaling independent mode. Mechanistically, Mst1/2 induce cellular quiescence by regulating the processes of proliferation and mitochondrial metabolism, thereby ensuring the development and survival of NK cells. Furthermore, Mst1/2 effectively sense IL-15 signaling and facilitate the activation of pSTAT3-TCF1, which contributes to NK cell homeostasis. Overall, our investigation highlights the crucial role of Mst1/2 as key regulators in metabolic reprogramming and transcriptional regulation for mouse NK cell survival and function, emphasizing the significance of cellular quiescence during NK cell development and functional maturation., (© 2024. The Author(s).)

NK cells have been shown to exhibit inflammatory and immunoregulatory functions in a variety of healthy and diseased settings. In the context of chronic viral infection and cancer, distinct NK cell populations that inhibit adaptive immune responses have been observed. To understand how these cells arise and further characterize their immunosuppressive role, we examined in vitro conditions that could polarize human NK cells into an inhibitory subset. TGF-β1 has been shown to induce regulatory T cells in vitro and in vivo; we therefore investigated if TGF-β1 could also induce immunosuppressive NK-like cells. First, we found that TGF-β1/IL-15, but not IL-15 alone, induced CD103+CD49a+ NK-like cells from peripheral blood NK cells, which expressed markers previously associated with inhibitory CD56+ innate lymphoid cells, including high expression of GITR and CD101. Moreover, supernatant from ascites collected from patients with ovarian carcinoma also induced CD103+CD49a+ NK-like cells in vitro in a TGF-β-dependent manner. Interestingly, TGF-β1/IL-15-induced CD103+CD56+ NK-like cells suppressed autologous CD4+ T cells in vitro by reducing absolute number, proliferation, and expression of activation marker CD25. Collectively, these findings provide new insight into how NK cells may acquire an inhibitory phenotype in TGF-β1-rich environments., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Background: Inflammatory cytokines play key pathogenic roles in liver fibrosis. IL-15 is a proinflammatory cytokine produced by myeloid cells. IL-15 promotes pathogenesis of several chronic inflammatory diseases. However, increased liver fibrosis has been reported in mice lacking IL-15 receptor alpha chain (IL-15Rα), suggesting an anti-fibrogenic role for IL-15. As myeloid cells are key players in liver fibrosis and IL-15 signaling can occur independently of IL-15Rα, we investigated the requirement of IL-15 and IL-15Rα in liver fibrosis., Methods: We induced liver fibrosis in Il15 -/- , Il15ra -/- and wildtype C57BL/6 mice by the administration of carbon tetrachloride (CCl 4 ). Liver fibrosis was evaluated by Sirius red and Mason's trichrome staining and α-smooth muscle acting immunostaining of myofibroblasts. Gene expression of collagens, matrix modifying enzymes, cytokines and chemokines was quantified by RT-qPCR. The phenotype and the numbers of intrahepatic lymphoid and myeloid cell subsets were evaluated by flow cytometry., Results: Both Il15 -/- and Il15ra -/- mice developed markedly reduced liver fibrosis compared to wildtype control mice, as revealed by reduced collagen deposition and myofibroblast content. Il15ra -/- mice showed further reduction in collagen deposition compared to Il15 -/- mice. However, Col1a1 and Col1a3 genes were similarly induced in the fibrotic livers of wildtype, Il15 -/- and Il15ra -/- mice, although notable variations were observed in the expression of matrix remodeling enzymes and chemokines. As expected, Il15 -/- and Il15ra -/- mice showed markedly reduced numbers of NK cells compared to wildtype mice. They also showed markedly less staining of CD45 + immune cells and CD68 + macrophages, and significantly reduced inflammatory cell infiltration into the liver, with fewer pro-inflammatory and anti-inflammatory monocyte subsets compared to wildtype mice., Conclusion: Our findings indicate that IL-15 exerts its profibrogenic role in the liver by promoting macrophage activation and that this requires trans-presentation of IL-15 by IL-15Rα., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Cloutier, Variya, Akbari, Rexhepi, Ilangumaran and Ramanathan.)

Antigen-driven human effector-memory CD8+ T cells expressing low levels of the CD8β chain have been previously described. However, little is known on a possible antigen-independent trigger. We have examined the impact that IL-15 has on the expression of CD8β on purified human naïve CD8+ T cells after CFSE labeling and culture with IL-15. As expected, IL-15 induced naïve CD8+ T cells to proliferate and differentiate. Remarkably, the process was associated with a cell-cycle dependent down-modulation of CD8β from the cell surface, leading to the generation of CD8αβ low and CD8αβ - (i.e., CD8αα) T cells. In contrast, expression of the CD8α chain remained steady or even increased. Neither IL-2 nor IL-7 reproduced the effect of IL-15. Determination of mRNA levels for CD8α and CD8β isoforms by qPCR revealed that IL-15 promoted a significant decrease in mRNA levels of the CD8β M-4 isoform, while levels of the M-1/M-2 isoforms and of CD8α increased. Noteworthy, CD8+ T cell blasts obtained after culture of CD8+ T cells with IL-15 showed a cell-cycle dependent increase in the level of the tyrosine kinase Lck, when compared to CD8+ T cells at day 0. This study has shown for the first time that IL-15 generates CD8αα + αβ low and CD8αα + αβ - T cells containing high levels of Lck, suggesting that they may be endowed with unique functional features., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Esgalhado, Reste-Ferreira, Weinhold, Uhrberg, Cardoso and Arosa.)

Cytokines are co-administrated with vaccines or co-expressed in the vaccine virus genome to improve protective efficacy by stimulating immune responses. Using glycosylphosphatidylinositol (GPI) anchoring by attachment to the target cytokine, we constructed recombinant Marek's disease virus (MDV) vaccine strain 301B/1 (v301B/1-rtg-IL-15) that expresses chicken interleukin-15 (IL-15) as the membrane-bound form at the cell surface. We evaluated the vaccine efficacy of v301B/1-rtg-IL-15 given as a bivalent Marek's disease (MD) vaccine in combination with turkey herpesvirus (HVT) against a very virulent plus MDV strain 648A challenge. The efficacy was compared with that of conventional bivalent MD vaccine, as a mixture with HVT plus parental v301B/1 or v301B/1-IL-15, which expresses a natural form of IL-15. The membrane-bound IL-15 expression did not interfere with the virus growth of recombinant v301B/1-rtg-IL-15. However, the MD incidence in birds vaccinated with v301B/1-rtg-IL-15 was higher than that of birds given the conventional bivalent MD vaccine containing parental v301B/1 virus, although the v301B/1-rtg-IL-15 vaccinated group showed increased natural killer cell activation at day 5 postvaccination, the same day as challenge. Overall, the protection of v301B/1-rtg-IL-15 was not improved from that of v301B/1 against very virulent plus MDV challenge.

The surface receptor CD8α is present on 20%-80% of human (but not mouse) NK cells, yet its function on NK cells remains poorly understood. CD8α expression on donor NK cells was associated with a lack of therapeutic responses in patients with leukemia in prior studies, thus, we hypothesized that CD8α may affect critical NK cell functions. Here, we discovered that CD8α- NK cells had improved control of leukemia in xenograft models compared with CD8α+ NK cells, likely due to an enhanced capacity for proliferation. Unexpectedly, we found that CD8α expression was induced on approximately 30% of previously CD8α- NK cells following IL-15 stimulation. These induced CD8α+ (iCD8α+) NK cells had the greatest proliferation, responses to IL-15 signaling, and metabolic activity compared with those that sustained existing CD8α expression (sustained CD8α+) or those that remained CD8α- (persistent CD8α-). These iCD8α+ cells originated from an IL-15Rβhi NK cell population, with CD8α expression dependent on the transcription factor RUNX3. Moreover, CD8A CRISPR/Cas9 deletion resulted in enhanced responses through the activating receptor NKp30, possibly by modulating KIR inhibitory function. Thus, CD8α status identified human NK cell capacity for IL-15-induced proliferation and metabolism in a time-dependent fashion, and its presence had a suppressive effect on NK cell-activating receptors.

Chimeric antigen receptor (CAR) T cells have demonstrated outstanding therapeutic success in hematological malignancies. Yet, their efficacy against solid tumors remains constrained due to inadequate infiltration of cytotoxic T and CAR-T cells in the tumor microenvironment (TME), a factor correlated with poor prognosis in patients with solid tumors. To overcome this limitation, we engineered CAR-T cells to secrete CXCL10 and IL15 (10 × 15 CAR-T), which sustain T cell viability and enhance their recruitment, thereby amplifying the long-term cytotoxic capacity of CAR-T cells in vitro. In a xenograft model employing NUGC4-T21 cells, mice receiving 10 × 15 CAR-T cells showed superior tumor reduction and extended survival rates compared to those treated with second-generation CAR-T cells. Histopathological evaluations indicated a pronounced increase in cytotoxic T cell accumulation in the TME post 10 × 15 CAR-T cell treatment. Therefore, the synergistic secretion of CXCL10 and IL15 in these CAR-T cells enhances T cell recruitment and adaptability within tumor tissues, improving tumor control. This approach may offer a promising strategy for advancing CAR-T therapies in the treatment of solid tumors., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Zhen-Wu-Tang (ZWT), a conventional herbal mixture, has been recommended for treating lupus nephritis (LN) in clinic. However, its mechanisms of action remain unknown. Here we aimed to define the immunological mechanisms underlying the effects of ZWT on LN and to determine whether it affects renal tissue-resident memory T (T RM ) cells. Murine LN was induced by a single injection of pristane, while in vitro T RM cells differentiated with IL-15/TGF-β. We found that ZWT or mycophenolate mofetil treatment significantly ameliorated kidney injury in LN mice by decreasing 24-h urine protein, Scr and anti-dsDNA Ab. ZWT also improved renal pathology and decreased IgG and C3 depositions. In addition, ZWT down-regulated renal Desmin expression. Moreover, it lowered the numbers of CD8 + T RM cells in kidney of mice with LN while decreasing their expression of TNF-α and IFN-γ. Consistent with in vivo results, ZWT-containing serum inhibited T RM cell differentiation induced by IL-15/TGF-β in vitro. Mechanistically, it suppressed phosphorylation of STAT3 and CD122 （IL2/IL-15Rβ）expression in CD8 + T RM cells. Importantly, ZWT reduced the number of total F4/80 + CD11b + and CD86 + , but not CD206 + , macrophages in the kidney of LN mice. Interestingly, ZWT suppressed IL-15 protein expression in macrophages in vivo and in vitro. Thus, we have provided the first evidence that ZWT decoction can be used to improve the outcome of LN by reducing CD8 + T RM cells via inhibition of IL-15/IL-15R /STAT3 signaling., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The autoimmunity-promoting cytokine, Interleukin-15 (IL-15), is often claimed to be a key pathogenic cytokine in alopecia areata (AA). Yet, rhIL-15 promotes human hair follicle (HF) growth ex vivo. We have asked whether the expression of IL-15 and its receptor (IL-15R) isoforms is altered in human AA and how IL-15 impacts on human HF immune privilege (HF-IP) in the presence/absence of interferon-γ (IFNγ), the well-documented key AA-pathogenic cytokine, as well as on hair regrowth after experimental AA induction in vivo. Quantitative immunohistomorphometry showed the number of perifollicular IL-15 + T cells in AA skin biopsies to be significantly increased compared to healthy control skin, while IL-15, IL-15Rα, and IL-15Rγ protein expression within the hair bulb were significantly down-regulated in AA HFs. In organ-cultured human scalp HFs, rhIL-15 significantly reduced hair bulb expression of MICA, the key "danger" signal in AA pathogenesis, and increased production of the HF-IP guardian, α-MSH. Crucially, ex vivo, rhIL-15 prevented IFNγ-induced HF-IP collapse, restored a collapsed HF-IP by IL-15Rα-dependent signaling (as documented by IL-15Rα-silencing), and protected AA-preventive immunoinhibitory iNKT10 cells from IFNγ-induced apoptosis. rhIL-15 even promoted hair regrowth after experimental AA induction in human scalp skin xenotransplants on SCID/beige mice in vivo. Our data introduce IL-15 as a novel, functionally important HF-IP guardian whose signaling is constitutively defective in scalp HFs of AA patients. Our data suggest that selective stimulation of intrafollicular IL-15Rα signaling could become a novel therapeutic approach in AA management, while blocking it pharmacologically may hinder both HF-IP restoration and hair re-growth and may thus make HFs more vulnerable to AA relapse., Competing Interests: Declaration of Competing Interest None declared. For the record, RP and JG are employees of, and JC and AG consult for a company that develops hair growth-modulatory agents (www.cutaneon.com)., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Natural killer (NK) cells are a critical first line of defense against viral infection. Rare mutations in a small subset of transcription factors can result in decreased NK cell numbers and function in humans, with an associated increased susceptibility to viral infection. However, our understanding of the specific transcription factors governing mature human NK cell function is limited. Here we use a non-viral CRISPR-Cas9 knockout screen targeting genes encoding 31 transcription factors differentially expressed during human NK cell development. We identify myocyte enhancer factor 2C (MEF2C) as a master regulator of human NK cell functionality ex vivo. MEF2C-haploinsufficient patients and mice displayed defects in NK cell development and effector function, with an increased susceptibility to viral infection. Mechanistically, MEF2C was required for an interleukin (IL)-2- and IL-15-mediated increase in lipid content through regulation of sterol regulatory element-binding protein (SREBP) pathways. Supplementation with oleic acid restored MEF2C-deficient and MEF2C-haploinsufficient patient NK cell cytotoxic function. Therefore, MEF2C is a critical orchestrator of NK cell antiviral immunity by regulating SREBP-mediated lipid metabolism., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Type 2 diabetes (T2D) is characterized by muscle metabolic dysfunction that exercise can minimize, but some patients do not respond to an exercise intervention. Myokine secretion is intrinsically altered in patients with T2D, but the role of myokines in exercise resistance in this patient population has never been studied. We sought to determine if changes in myokine secretion were linked to the response to an exercise intervention in patients with T2D. The participants followed a 10-week aerobic exercise training intervention, and patients with T2D were grouped based on muscle mitochondrial function improvement (responders versus non-responders). We measured myokines in serum and cell-culture medium of myotubes derived from participants pre- and post-intervention and in response to an in vitro model of muscle contraction. We also quantified the expression of genes related to inflammation in the myotubes pre- and post-intervention. No significant differences were detected depending on T2D status or response to exercise in the biological markers measured, with the exception of modest differences in expression patterns for certain myokines (IL-1β, IL-8, IL-10, and IL-15). Further investigation into the molecular mechanisms involving myokines may explain exercise resistance with T2D; however, the role in metabolic adaptations to exercise in T2D requires further investigation.

Sepsis-induced myocardial dysfunction (SIMD) is associated with poor prognosis and increased mortality in patients with sepsis. Cytokines are important regulators of both the initiation and progression of sepsis. Interleukin-15 (IL-15), a pro-inflammatory cytokine, has been linked to protective effects against myocardial infarction and myocarditis. However, the role of IL-15 in SIMD remains unclear. We established a mouse model of SIMD via cecal ligation puncture (CLP) surgery and a cell model of myocardial injury via lipopolysaccharide (LPS) stimulation. IL-15 expression was prominently upregulated in septic hearts as well as cardiomyocytes challenged with LPS. IL-15 pretreatment attenuated cardiac inflammation and cell apoptosis and improved cardiac function in the CLP model. Similar cardioprotective effects of IL-15 pretreatment were observed in vitro. As expected, IL-15 knockdown had the opposite effect on LPS-stimulated cardiomyocytes. Mechanistically, we found that IL-15 pretreatment reduced the expression of the pro-apoptotic proteins cleaved caspase-3 and Bax and upregulated the anti-apoptotic protein Bcl-2. RNA sequencing and Western blotting further confirmed that IL-15 pretreatment suppressed the activation of nuclear factor kappa B (NF-κB) signaling in mice with sepsis. Besides, the addition of NF-κB inhibitor can significantly attenuate cardiomyocyte apoptosis compared to the control findings. Our results suggest that IL-15 pretreatment attenuated the cardiac inflammatory responses and reduced cardiomyocyte apoptosis by partially inhibiting NF-κB signaling in vivo and in vitro, thereby improving cardiac function in mice with sepsis. These findings highlight a promising therapeutic strategy for SIMD.

Background: NK cells can be genetically engineered to express a transgenic T-cell receptor (TCR). This approach offers an alternative strategy to target heterogenous tumors, as NK:TCR cells can eradicate both tumor cells with high expression of HLA class I and antigen of interest or HLA class I negative tumors. Expansion and survival of NK cells relies on the presence of IL-15. Therefore, autonomous production of IL-15 by NK:TCR cells might improve functional persistence of NK cells. Here we present an optimized NK:TCR product harnessed with a construct encoding for soluble IL-15 (NK:TCR/IL-15), to support their proliferation, persistence and cytotoxic capabilities., Methods: Expression of tumor-specific TCRs in peripheral blood derived NK-cells was achieved following retroviral transduction. NK:TCR/IL-15 cells were compared with NK:TCR cells for autonomous cytokine production, proliferation and survival. NK:BOB1-TCR/IL-15 cells, expressing a HLA-B*07:02-restricted TCR against BOB1, a B-cell lineage specific transcription factor highly expressed in all B-cell malignancies, were compared with control NK:BOB1-TCR and NK:CMV-TCR/IL-15 cells for effector function against TCR antigen positive malignant B-cell lines in vitro and in vivo., Results: Viral incorporation of the interleukin-15 gene into engineered NK:TCR cells was feasible and high expression of the TCR was maintained, resulting in pure NK:TCR/IL-15 cell products generated from peripheral blood of multiple donors. Self-sufficient secretion of IL-15 by NK:TCR cells enables engineered NK cells to proliferate in vitro without addition of extra cytokines. NK:TCR/IL-15 demonstrated a marked enhancement of TCR-mediated cytotoxicity as well as enhanced NK-mediated cytotoxicity resulting in improved persistence and performance of NK:BOB1-TCR/IL-15 cells in an orthotopic multiple myeloma mouse model. However, in contrast to prolonged anti-tumor reactivity by NK:BOB1-TCR/IL-15, we observed in one of the experiments an accumulation of NK:BOB1-TCR/IL-15 cells in several organs of treated mice, leading to unexpected death 30 days post-NK infusion., Conclusion: This study showed that NK:TCR/IL-15 cells secrete low levels of IL-15 and can proliferate in an environment lacking cytokines. Repeated in vitro and in vivo experiments confirmed the effectiveness and target specificity of our product, in which addition of IL-15 supports TCR- and NK-mediated cytotoxicity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 van Hees, Morton, Remst, Wouters, Van den Eynde, Falkenburg and Heemskerk.)

Myokines, released from the muscle, enable communication between the working muscles and other tissues. Their release during physical exercise is assumed to depend on immune-hormonal-metabolic interactions concerning mode (endurance or resistance exercise), duration, and intensity. This meta-analysis aims to examine the acute changes of circulating myokines inducing immunoregulatory effects caused by a bout of resistance exercise and to consider potential moderators of the results. Based on this selection strategy, a systematic literature search was conducted for resistance exercise intervention studies measuring interleukin (IL-) 6, IL-10, IL-1ra, tumor necrosis factor (TNF-) α, IL-15, IL-7, transforming growth factor (TGF-) β1, and fractalkines (FKN) before and immediately after resistance exercise in healthy individuals. Random-effects meta-analysis was performed for each myokine. We identified a moderate positive effect of resistance exercise for IL-6 and IL-1ra. Regarding IL-15 and TNF-α, small to moderate effects were found. For IL-10, no significant effect was observed. Due to no data, meta-analyses for IL-7, TGF-β1, and FKN could not be performed. No moderators (training status, type of exercise, risk of bias, age, sex, time of day, exercise volume, exercise intensity, exercise dose) of the results were detected for all tested myokines. Taken together, this systematic review and meta-analysis showed immediate positive effects of an acute resistance exercise session on IL-6, IL-1ra, TNF-α, and IL-15 levels., (© 2024 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Purpose: Chimeric antigen receptor (CAR) and T-cell receptor (TCR) T-cell therapies are effective in a subset of patients with solid tumors, but new approaches are needed to universally improve patient outcomes. Here, we developed a technology to leverage the cooperative effects of IL15 and IL21, two common cytokine-receptor gamma chain family members with distinct, pleiotropic effects on T cells and other lymphocytes, to enhance the efficacy of adoptive T cells., Experimental Design: We designed vectors that induce the constitutive expression of either membrane-tethered IL15, IL21, or IL15/IL21. We used clinically relevant preclinical models of transgenic CARs and TCRs against pediatric and adult solid tumors to determine the effect of the membrane-tethered cytokines on engineered T cells for human administration., Results: We found that self-delivery of these cytokines by CAR or TCR T cells prevents functional exhaustion by repeated stimulation and limits the emergence of dysfunctional natural killer (NK)-like T cells. Across different preclinical murine solid tumor models, we observed enhanced regression with each individual cytokine but the greatest antitumor efficacy when T cells were armored with both., Conclusions: The coexpression of membrane-tethered IL15 and IL21 represents a technology to enhance the resilience and function of engineered T cells against solid tumors and could be applicable to multiple therapy platforms and diseases. See related commentary by Ruffin et al., p. 1431., (©2023 American Association for Cancer Research.)

Chronic myeloid leukemia (CML) is a clonal myeloproliferative hematological disease characterized by the chimeric breakpoint-cluster region/Abelson kinase1 (BCR::ABL1) oncoprotein; playing a pivotal role in CML molecular pathology, diagnosis, treatment, and possible resistance arising from the success and tolerance of tyrosine kinase inhibitor (TKI)-based therapy. The transcription factor STAT5 constitutive signaling, which is influenced by the cytokine signaling network, triggers BCR::ABL1-based CML pathogenesis and is also relevant to acquired TKI resistance. The unsuccessful therapeutic approaches targeting BCR::ABL1, in particular third-line therapy with ponatinib, still need to be further developed with alternative combination strategies to overcome drug resistance. As treatment with the STAT5 inhibitor pimozide in combination with ponatinib resulted in an efficient and synergistic therapeutic approach in TKI-resistant CML cells, this study focused on identifying the underlying amplification of ponatinib response mechanisms by determining different cytokine expression profiles in parental and ponatinib-resistant CML cells, in vitro . The results showed that expression of interleukin (IL) 1B , IL9 , and IL12A-B was increased by 2-fold, while IL18 was downregulated by 2-fold in the ponatinib-resistant cells compared to sensitive ones. Importantly, ponatinib treatment upregulated the expression of 21 of the 23 interferon and IL genes in the ponatinib-resistant cells, while treatment with pimozide or a combination dose resulted in a reduction in the expression of 19 different cytokine genes, such as for example, inflammatory cytokines, IL1A-B and IL6 or cytokine genes associated with supporting tumor progression, leukemia stem cell growth or poor survival, such as IL3 , IL8 , IL9 , IL10 , IL12 , or IL15 . Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis results showed that the genes were mainly enriched in the regulation of receptor signaling through the Janus kinase/signal transducer and activator of transcription pathway, cytokine-cytokine receptor interaction, and hematopoietic cell lineage. Protein-protein interaction analysis showed that IL2 , IL6 , IL15 , IFNG , and others appeared in the top lists of pathways, indicating their high centrality and importance in the network. Therefore, pimozide could be a promising agent to support TKI therapies in ponatinib resistance. This research would help to clarify the role of cytokines in ponatinib resistance and advance the development of new therapeutics to utilize the STAT5 inhibitor pimozide in combination with TKIs.

Introduction: The clinical success of chimeric antigen receptor-modified T cells (CAR-T cells) for hematological malignancies has not been reproduced for solid tumors, partly due to the lack of cancer-type specific antigens. In this work, we used a novel combinatorial approach consisting of a versatile anti-FITC CAR-T effector cells plus an FITC-conjugated neuroblastoma (NB)-targeting linker, an FITC-conjugated monoclonal antibody (Dinutuximab) that recognizes GD2., Methods: We compared cord blood (CB), and CD45RA-enriched peripheral blood leukapheresis product (45RA) as allogeneic sources of T cells, using peripheral blood (PB) as a control to choose the best condition for anti-FITC CAR-T production. Cells were manufactured under two cytokine conditions (IL-2 versus IL-7+IL-15+IL-21) with or without CD3/CD28 stimulation. Immune phenotype, vector copy number, and genomic integrity of the final products were determined for cell characterization and quality control assessment. Functionality and antitumor capacity of CB/45RA-derived anti-FITC CAR-T cells were analyzed in co-culture with different anti-GD2-FITC labeled NB cell lines., Results: The IL-7+IL-15+IL-21 cocktail, in addition to co-stimulation signals, resulted in a favorable cell proliferation rate and maintained less differentiated immune phenotypes in both CB and 45RA T cells. Therefore, it was used for CAR-T cell manufacturing and further characterization. CB and CD45RA-derived anti-FITC CAR-T cells cultured with IL-7+IL-15+IL-21 retained a predominantly naïve phenotype compared with controls. In the presence of the NB-FITC targeting, CD4+ CB-derived anti-FITC CAR-T cells showed the highest values of co-stimulatory receptors OX40 and 4-1BB, and CD8+ CAR-T cells exhibited high levels of PD-1 and 4-1BB and low levels of TIM3 and OX40, compared with CAR-T cells form the other sources studied. CB-derived anti-FITC CAR-T cells released the highest amounts of cytokines (IFN-γ and TNF-α) into co-culture supernatants. The viability of NB target cells decreased to 30% when co-cultured with CB-derived CAR-T cells during 48h., Conclusion: CB and 45RA-derived T cells may be used as allogeneic sources of T cells to produce CAR-T cells. Moreover, ex vivo culture with IL-7+IL-15+IL-21 could favor CAR-T products with a longer persistence in the host. Our strategy may complement the current use of Dinutuximab in treating NB through its combination with a targeted CAR-T cell approach., Competing Interests: MR has received honoraria for participation in advisory boards from Amplicell and stock options from Orgenesis. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 García-García, G. Sánchez, Ivanova, Pastora, Alcántara-Sánchez, García-Martínez, Martín-Antonio, Ramírez and González-Murillo.)

Aims: Elevated levels of adipokine chemerin have been identified in oral squamous cell carcinoma (OSCC) and found to be associated with metastasis to the cervical lymph nodes. The underlying mechanism through which chemerin affects OSCC progression is unclear. The aims of this study were firstly to determine chemerin levels and cytokine concentrations in serum from patients with OSCC and in OSCC cell cultures, and secondly to observe chemerin effects on OSCC cell cytokine secretion, migration, and invasion in vitro., Methods: Serum samples were collected from 20 patients diagnosed with OSCC, including groups with (LN+) and without (LN-) cervical lymph node metastasis. A Luminex liquid suspension assay was used to quantify serum concentrations of 27 types of cytokines. Correlations between chemerin and cytokines (i.e., IL-6, IL-15, GM-CSF, RANTES, TNF-α, and VEGF) were analyzed. ELISAs (enzyme-linked immunosorbent assays) were used to determine concentrations of chemerin and selected cytokines in serum and in supernatants of OSCC cell cultures (SCC9 and SCC25 cell lines). OSCC cells were stimulated with human recombinant chemerin, STAT3 inhibitor, or IL-6 together with TNF-α neutralizing antibodies. Phosphorylated STAT3 protein levels were measured with western blot analysis. OSCC cell migration and invasion were investigated with Transwell assays., Results: Compared to the LN- group, OSCC patients with cervical lymph node metastasis had higher levels of IL-6 (P = 0.006), IL-15 (P = 0.020), GM-CSF (P = 0.036), RANTES (P = 0.032), TNF-α (P = 0.005), VEGF (P = 0.006), and chemerin (P = 0.001). Patients' serum chemerin levels correlated directly with IL-6, GM-CSF, TNF-α, and VEGF levels in OSCC patients. Exogenous recombinant chemerin treatment promoted secretion of IL-6 and TNF-α via activation of STAT3 in OSCC cells. Chemerin induced OSCC-cell migration and invasion, and these effects were reduced by IL-6 and TNF-α neutralizing antibodies., Conclusion: Our findings indicate that chemerin may play a role in advancing OSCC progression by increasing production of IL-6 and TNF-α, perhaps via a mechanism involving STAT3 signaling., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)