Combination Of Cytokines Research Articles

Phenomics Demonstrates Cytokines Additive Induction of Epithelial to Mesenchymal Transition.

Epithelial to mesenchymal transition (EMT) is highly plastic with a programme where cells lose adhesion and become more motile. EMT heterogeneity is one of the factors for disease progression and chemoresistance in cancer. Omics characterisations are costly and challenging to use. We developed single cell phenomics with easy to use wide-field fluorescence microscopy. We analyse over 70,000 cells and combined 53 features. Our simplistic pipeline allows efficient tracking of EMT plasticity, with a single statistical metric. We discriminate four high EMT plasticity cancer cell lines along the EMT spectrum. We test two cytokines, inducing EMT in all cell lines, alone or in combination. The single cell EMT metrics demonstrate the additive effect of cytokines combination on EMT independently of cell line EMT spectrum. The effects of cytokines are also observed at the front of migration during wound healing assay. Single cell phenomics is uniquely suited to characterise the cellular heterogeneity in response to complex microenvironment and show potential for drug testing assays.

Emerging Insights into Memory Natural Killer Cells and Clinical Applications.

Natural killer (NK) cells are innate lymphocytes that can rapidly mount a response to their targets by employing diverse mechanisms. Due to their functional attributes, NK cells have been implicated in anti-viral and anti-tumour immune responses. Although traditionally known to mount non-specific, rapid immune responses, in recent years, the notion of memory NK cells with adaptive features has gained more recognition. Memory NK cells emerge in response to different stimuli, such as viral antigens and specific cytokine combinations. They form distinct populations, accompanied by transcriptional, epigenetic and metabolic reprogramming, resulting in unique phenotypic and functional attributes. Several clinical trials are testing the efficacy of memory NK cells due to their enhanced functionality, bioenergetic profile and persistence in vivo. The therapeutic potential of NK cells is being harnessed in viral infections, with wider applications in the cancer field. In this review, we summarise the current state of research on the generation of memory NK cells, along with their clinical applications in viral infection and cancer.

CXCL9, CXCL10, and CCL19 synergistically recruit T lymphocytes to skin in lichen planus.

Lichen planus (LP) is a chronic, debilitating, inflammatory disease of the skin and mucous membranes that affects 1%-2% of Americans. Its molecular pathogenesis remains poorly understood, and there are no FDA-approved treatments. We performed single-cell RNA sequencing on paired blood and skin samples (lesional and nonlesional tissue) from 7 patients with LP. We discovered that LP keratinocytes and fibroblasts specifically secrete a combination of CXCL9, CXCL10, and CCL19 cytokines. Using an in vitro migration assay with primary human T cells, we demonstrated that CCL19 in combination with either of the other 2 cytokines synergistically enhanced recruitment of CD8+ T cells more than any individual cytokine. Moreover, exhausted T cells in lesional LP skin secreted CXCL13, which, along with CCL19, also enhanced recruitment of T cells, suggesting a feed-forward loop in LP. Finally, LP blood revealed decreased circulating naive CD8+ T cells compared with that in healthy volunteers, consistent with recruitment to skin. Molecular analysis of LP skin and blood samples increased our understanding of disease pathogenesis and identified CCL19 as a new therapeutic target for treatment.

THE THERAPEUTIC POTENTIAL OF PRECONDITIONED MESENCHYMAL STEM CELLS IN ATHEROSCLEROSIS

Atherosclerosis (AS) is a multifactorial and inflammatory disease the cause of which is the formation of lipid and cholesterol plaques on the inner wall of blood vessels. Experimental and clinical studies have shown that the pathogenesis of atherosclerosis is, in particular, connected with inflammatory reactions. Recent studies reported that preconditioned mesenchymal stem cells (MSCs) possess an enhanced immunomodulatory potential. The purpose of the study was to investigate the therapeutic potential of preconditioned MSCs in the development of atherosclerosis. MSCs were isolated from the compact bone of mice by standard protocol. The characterization was conducted using flow cytometry, CFU assay and three-lineage differentiation. Cells were preconditioned with different combinations of cytokines and the optimal concentrations were determined by ELISA and by analysis of co-culturing with lymphocytes. The cytokine-preconditioned MSCs were administrated to mice with induced atherosclerosis. The lipids analysis, measuring of mouse spleens’ weights, ELISA and histological analysis of the aorta sections were done after 8-th weeks. We isolated MSCs and then confirmed the possession of the immunomodulatory properties. Preconditioning with the TNF-α cytokine effectively increased the immunomodulatory properties of MSCs compared to untreated MSCs in vitro. The results of in vivo study that the use of TNF-α-preconditioned MSCs reduced the development of atherosclerosis in mice compared to untreated MSCs: it increased the level of Treg cells in the spleen of mice; significantly reduced the levels of TNF-α and IFN-γ in blood serum; significantly reduced the spleen weights of mice; slightly decreased the total cholesterol levels and also increased HDL levels. Administration of TNF-α-preconditioned MSCs reduced the size of atherosclerotic plaques in the aorta of mice. The results of this study demonstrated the effect of cytokine-preconditioned MSCs in the inhibition of the development of atherosclerosis in a mouse model. However, for application as a therapy for suffered patients additional studies are needed. Besides, using of TNF-α preconditioned MSCs can be considered in other preclinical studies of the different diseases.

IL-15/IL-15Ra Synergies with IL-12 to Induce Functional CD8 T Cells and NK Cells During Chronic SHIV Infection.

Cytokines are key mediators of immune regulation, orchestrate communication between immune cells, and play a pivotal role in shaping the immune landscape during chronic infection and cancer. The therapeutic potential of IL-15/IL-15Rα and IL-12 has been explored individually in various immunotherapeutic strategies, though not as a combination. Therefore, we investigated whether the combination of IL-15/IL-15Rα and IL-12 treatment would enhance the potency and quality of either NK cells, SIV-specific CD8 T cells, or both, compared with single cytokine treatment. Our findings reveal that in vitro IL-15/IL-15Rα and IL-15/IL-15Rα plus IL-12 treatment results in an expansion of functional CD8 T cells and NK cells from uninfected and chronically infected macaques with simian/human immunodeficiency virus. Additionally, the cytokine combination significantly reduced CCR5 expression on total CD4 T cells, limiting the number of viral targets. This study supports the potential utilization of combined IL-15/IL-15Rα plus IL-12 treatment for chronic viral infections and cancer.

In Vitro Insights into the Dietary Role of Glucoraphanin and Its Metabolite Sulforaphane in Celiac Disease.

Sulforaphane is considered the bioactive metabolite of glucoraphanin after dietary consumption of broccoli sprouts. Although both molecules pass through the gut lumen to the large intestine in stable form, their biological impact on the first intestinal tract is poorly described. In celiac patients, the function of the small intestine is affected by celiac disease (CD), whose severe outcomes are controlled by gluten-free dietary protocols. Nevertheless, pathological signs of inflammation and oxidative stress may persist. The aim of this study was to compare the biological activity of sulforaphane with its precursor glucoraphanin in a cellular model of gliadin-induced inflammation. Human intestinal epithelial cells (CaCo-2) were stimulated with a pro-inflammatory combination of cytokines (IFN-γ, IL-1β) and in-vitro-digested gliadin, while oxidative stress was induced by H2O2. LC-MS/MS analysis confirmed that sulforaphane from broccoli sprouts was stable after simulated gastrointestinal digestion. It inhibited the release of all chemokines selected as inflammatory read-outs, with a more potent effect against MCP-1 (IC50 = 7.81 µM). On the contrary, glucoraphanin (50 µM) was inactive. The molecules were unable to counteract the oxidative damage to DNA (γ-H2AX) and catalase levels; however, the activity of NF-κB and Nrf-2 was modulated by both molecules. The impact on epithelial permeability (TEER) was also evaluated in a Transwell® model. In the context of a pro-inflammatory combination including gliadin, TEER values were recovered by neither sulforaphane nor glucoraphanin. Conversely, in the context of co-culture with activated macrophages (THP-1), sulforaphane inhibited the release of MCP-1 (IC50 = 20.60 µM) and IL-1β (IC50 = 1.50 µM) only, but both molecules restored epithelial integrity at 50 µM. Our work suggests that glucoraphanin should not merely be considered as just an inert precursor at the small intestine level, thus suggesting a potential interest in the framework of CD. Its biological activity might imply, at least in part, molecular mechanisms different from sulforaphane.

Circulating interleukin-8 and osteopontin are promising biomarkers of clinical outcomes in advanced melanoma patients treated with targeted therapy

BackgroundCirculating cytokines can represent non-invasive biomarkers to improve prediction of clinical outcomes of cancer patients. Here, plasma levels of IL-8, CCL4, osteopontin, LIF and BDNF were determined at baseline (T0), after 2 months of therapy (T2) and, when feasible, at progression (TP), in 70 melanoma patients treated with BRAF and MEK inhibitors. The association of baseline cytokine levels with clinical response, progression-free survival (PFS) and overall survival (OS) was evaluated.MethodsCytokine concentrations were measured using the xMAP technology. Their ability to discriminate between responding (Rs) and non-responding (NRs) patients was assessed by Receiver Operating Characteristics analysis. PFS and OS were estimated with the Kaplan–Meier method. The Cox proportional hazard model was used in the univariate and multivariate analyses to estimate crude and adjusted hazard ratios with 95% confidence intervals.ResultsCCL4 and LIF were undetectable in the majority of samples. The median osteopontin concentration at T0 and T2 was significantly higher in NRs than in Rs. The median T0 and T2 values of IL-8 were also higher in NRs than in Rs, although the statistical significance was not reached. No differences were detected for BDNF. In 39 Rs with matched T0, T2, and TP samples, osteopontin and IL-8 significantly decreased from T0 to T2 and rose again at TP, while BDNF levels remained unchanged. In NRs, none of the cytokines showed a significant decrease at T2. Only osteopontin demonstrated a good ability to discriminate between Rs and NRs. A high IL-8 T0 level was associated with significantly shorter PFS and OS and higher risk of progression and mortality, and remained an independent negative prognostic factor for OS in multivariate analysis. An elevated osteopontin T0 concentration was also significantly associated with worse OS and increased risk of death. Patients with high IL-8 and high osteopontin showed the lowest PFS and OS, and in multivariate analysis this cytokine combination remained independently associated with a three- to six-fold increased risk of mortality.ConclusionCirculating IL-8 and osteopontin appear useful biomarkers to refine prognosis evaluation of patients undergoing targeted therapy, and deserve attention as potential targets to improve its clinical efficacy.

Good manufacturing practice-grade generation of CD19 and CD123-specific CAR-T cells using piggyBac transposon and allogeneic feeder cells in patients diagnosed with B-cell non-Hodgkin lymphoma and acute myeloid leukemia.

The non-viral production of CAR-T cells through electroporation of transposon DNA plasmids is an alternative approach to lentiviral/retroviral methods. This method is particularly suitable for early-phase clinical trials involving novel types of CAR-T cells. The primary disadvantage of non-viral methods is the lower production efficiency compared to viral-based methods, which becomes a limiting factor for CAR-T production, especially in chemotherapy-pretreated lymphopenic patients. We describe a good manufacturing practice (GMP)-compliant protocol for producing CD19 and CD123-specific CAR-T cells based on the electroporation of transposon vectors. The lymphocytes were purified from the blood of patients undergoing chemotherapy for B-NHL or AML and were electroporated with piggyBac transposon encoding CAR19 or CAR123, respectively. Electroporated cells were then polyclonally activated by anti-CD3/CD28 antibodies and a combination of cytokines (IL-4, IL-7, IL-21). The expansion was carried out in the presence of irradiated allogeneic blood-derived mononuclear cells (i.e., the feeder) for up to 21 days. Expansion in the presence of the feeder enhanced CAR-T production yield (4.5-fold in CAR19 and 9.3-fold in CAR123). Detailed flow-cytometric analysis revealed the persistence of early-memory CAR-T cells and a low vector-copy number after production in the presence of the feeder, with no negative impact on the cytotoxicity of feeder-produced CAR19 and CAR123 T cells. Furthermore, large-scale manufacturing of CAR19 carried out under GMP conditions using PBMCs obtained from B-NHL patients (starting number=200x10e6 cells) enabled the production of >50x10e6 CAR19 in 7 out of 8 cases in the presence of the feeder while only in 2 out of 8 cases without the feeder. The described approach enables GMP-compatible production of sufficient numbers of CAR19 and CAR123 T cells for clinical application and provides the basis for non-viral manufacturing of novel experimental CAR-T cells that can be tested in early-phase clinical trials. This manufacturing approach can complement and advance novel experimental immunotherapeutic strategies against human hematologic malignancies.

Quantification of Cytokine-Induced Cell Death in Human Colonic Organoids Using Live Fluorescence Microscopy.

Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD). This can contribute to defects in intestinal barrier function, exacerbation of inflammation, and disease immunopathogenesis. Cytokines and death receptor ligands are partially responsible for this increase in IEC death. IBD-relevant cytokines, such as TNF-α and IFN-γ, are cytotoxic to IECs both independently and in combination. This protocol describes a simple and practical assay to quantify cytokine-induced cytotoxicity in CD patient-derived colonic organoids using a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software. We also demonstrate how to use the Bliss independence mathematical model to calculate a coefficient of perturbagen interaction (CPI) based on organoid cytotoxicity. The CPI can be used to determine if interactions between cytokine combinations or other types of perturbagens are antagonistic, additive, or synergistic. This protocol can be implemented to investigate the cytotoxic activity of cytokines and other perturbagens using patient-derived colonic organoids.

Current Strategies to Improve Chimeric Antigen Receptor T (CAR-T) Cell Persistence.

Chimeric antigen receptor T (CAR-T) cell therapy has transformed the field of immunology by redirecting T lymphocytes toward tumor antigens. Despite successes in attaining high remission rates as high as 90%,the performance of CAR therapy is limited by the survival of T cells. T cell persistence is crucial as it sustains immune response against malignancies, playing a critical role in cancer treatment outcomes. This review explores various approaches to improve CAR-T cell persistence, focusing on the choice between autologous and allogeneic cell sources, optimization of culture conditions for T cell subsets, metabolite adjustments to modify T cell metabolism, the use of oncolytic viruses (OVs), and advancements in CAR design. Autologous CAR-T cells generally exhibit longer persistence but are less accessible and cost-effective than their allogeneic counterparts. Optimizing culture conditions by promotingTSCMand TCMcell differentiation has also demonstrated increased persistence,as seen with the use of cytokine combinations like IL-7 and IL-15.Metabolic adjustments, such as using 2-deoxy-D-glucose (2-DG) and L-arginine, have enhanced the formation of memory T cells, leading to improved antitumor activity. OVs, when combined with CAR-T therapy, can amplify CAR-T cell penetration and persistence in solid tumors, although further clinical validation is needed. Advances in CAR design from second to fifth generations have progressively improved T cell activation and survival, with fifth-generation CARs demonstratingstrong cytokine-mediated signaling and long-lasting persistence.Understanding the underlying mechanisms behind these strategies is essential for maximizing the potential of CAR-T therapy in treating cancer. Further research is needed to improve safety and efficacy and seamlessly integrate the discussed strategies into the manufacturing process.

Herpes simplex virus-mediated skin infections: cytokines and its interplay

The skin, as the first physical barrier for invading pathogens, also serves as an immunologically active organ. Breaching the skin barrier is thus essential for pathogens to enter the body. The skin contains various immune responsive cells that initiate both the innate and adaptive immune response upon invasion. Activated immune cells help to regulate cytokine response and their differentiation to promote or suppress the crucial immune response against invading pathogens. Human herpes simplex viruses (HHSVs) are the oldest pathogen that can escape immune surveillance of the human host by a well-developed escape mechanism within ganglia, as their evolutionary strategy. In primary infection, a non-specific defense of the host initiates the response against the invading virion. The initial direct antiviral action of the host is regulated by activated macrophages, via the release of cytokines like tumor necrosis factor (TNF), and type-1 interferon (IFN-1). The host-derived cytokines including IFN-12, TNF, and IFN-1 in turn induce natural killer (NK) cells to release IFN-γ. Their positive feedback with synergistic interactions collectively releases nitric oxide (NO) and reactive oxygen species (ROS) against the invading virus. Simultaneously, the combination of cytokines, macrophages, and other cells activates the immune system to eliminate the pathogen. However, the virus has also evolved various mechanisms to counter the host defense strategies. This review will highlight virus-mediated skin infections, especially by HSV, and portray a detailed role of virus-induced cytokines in host-immunity to challenge the invading virion during mucocutaneous HSV infection. Further, this review will discuss the viral-interference on host defense to provide a simplistic overview of the complications of cutaneous HSV infection.

Whole-blood culture-derived cytokine combinations for the diagnosis of tuberculosis.

The diagnosis of tuberculosis (TB) disease and TB infection (TBI) remains a challenge, and there is a need for non-invasive and blood-based methods to differentiate TB from conditions mimicking TB (CMTB), TBI, and healthy controls (HC). We aimed to determine whether combination of cytokines and established biomarkers could discriminate between 1) TB and CMTB 2) TB and TBI 3) TBI and HC. We used hemoglobin, total white blood cell count, neutrophils, monocytes, C-reactive protein, and ten Meso Scale Discovery analyzed cytokines (interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, interferon (IFN)-ɣ, and tumor necrosis factor (TNF)-α) in TruCulture whole blood tubes stimulated by lipopolysaccharides (LPS), zymosan (ZYM), anti-CD3/28 (CD3), and unstimulated (Null) to develop three index tests able to differentiate TB from CMTB and TBI, and TBI from HC. In 52 persons with CMTB (n=9), TB (n=23), TBI (n=10), and HC (n=10), a combination of cytokines (LPS-IFN-ɣ, ZYM-IFN-ɣ, ZYM-TNF-α, ZYM-IL-1β, LPS-IL-4, and ZYM-IL-6) and neutrophil count could differentiate TB from CMTB with a sensitivity of 52.2% (95% CI: 30.9%-73.4%) and a specificity of 100 % (66.4%-100%). Null- IFN-ɣ, Null-IL-8, CD3-IL-6, CD3-IL-8, CD3-IL-13, and ZYM IL-1b discriminated TB from TBI with a sensitivity of 73.9% (56.5% - 91.3%) and a specificity of 100% (69.2-100). Cytokines and established biomarkers failed to differentiate TBI from HC with ≥ 98% specificity. Selected cytokines may serve as blood-based add-on tests to detect TB in a low-endemic setting, although these results need to be validated.

BCG priming followed by a novel interleukin combination activates Natural Killer cells to selectively proliferate and become anti-tumour long-lived effectors

The short-lived nature and heterogeneity of Natural Killer (NK) cells limit the development of NK cell-based therapies, despite their proven safety and efficacy against cancer. Here, we describe the biological basis, detailed phenotype and function of long-lived anti-tumour human NK cells (CD56highCD16+), obtained without cell sorting or feeder cells, after priming of peripheral blood cells with Bacillus Calmette-Guérin (BCG). Further, we demonstrate that survival doses of a cytokine combination, excluding IL18, administered just weekly to BCG-primed NK cells avoids innate lymphocyte exhaustion and leads to specific long-term proliferation of innate cells that exert potent cytotoxic function against a broad range of solid tumours, mainly through NKG2D. Strikingly, a NKG2C+CD57-FcεRIγ+ NK cell population expands after BCG and cytokine stimulation, independently of HCMV serology. This strategy was exploited to rescue anti-tumour NK cells even from the suppressor environment of cancer patients’ bone marrow, demonstrating that BCG confers durable anti-tumour features to NK cells.

Would three BMPs at low concentration be better than one at high concentration? An experimental study with rat osteoprogenitor cells.

Bone morphogenetic proteins (BMPs) are used in clinical practice for stimulation of bone formation, but often evoke serious complications. Recent studies demonstrated that BMPs involved in early stages of bone formation are species specific. In cattle dominate BMP7, growth differentiation factor 5 (GDF5) and NEL-like protein 1 (NELL1) while in rats BMP2, BMP5 and BMP6. The purpose of the study was to compare the action of the species specific BMPs on the osteoprogenitor cells. Thus, rat osteoprogenitor cells were exposed to one BMP in a high dose and three of them at 1/3 of the former. Isolated rat osteoprogenitor cells were treated in culture with different concentrations of BMP2, BMP5 and BMP6 or with lower concentration of combinations of these cytokines. Activity of alkaline phosphatase, calcium deposition and mRNA level for transcription factor SP7 (osterix) and tissue non-specific alkaline phosphatase (TNAP) served as indicators of BMPs effect. BMPs stimulated all studied parameters in comparison with control cultures, but no statistically significant differences were observed between the action of a large dose of one cytokine and a combination of cytokines given at lower concentrations. Three BMPs used in a low dose exert similar effect as the one used at high dose. Since the BMPs stimulate different receptors and activate different signaling pathways the use of the mixture of properly chosen BMPs at low concentration may give better results than the single one at high concentration and may avoid untoward effects.

Development of an alcoholic liver disease model for drug evaluation from human induced pluripotent stem cell-derived liver organoids.

Alcoholic liver disease (ALD) poses a significant health challenge, so comprehensive research efforts to improve our understanding and treatment strategies are needed. However, the development of effective treatments is hindered by the limitation of existing liver disease models. Liver organoids, characterized by their cellular complexity and three-dimensional (3D) tissue structure closely resembling the human liver, hold promise as ideal models for liver disease research. In this study, we use a meticulously designed protocol involving the differentiation of human induced pluripotent stem cells (hiPSCs) into liver organoids. This process incorporates a precise combination of cytokines and small molecule compounds within a 3D culture system to guide the differentiation process. Subsequently, these differentiated liver organoids are subject to ethanol treatment to induce ALD, thus establishing a disease model. A rigorous assessment through a series of experiments reveals that this model partially recapitulates key pathological features observed in clinical ALD, including cellular mitochondrial damage, elevated cellular reactive oxygen species (ROS) levels, fatty liver, and hepatocyte necrosis. In addition, this model offers potential use in screening drugs for ALD treatment. Overall, the liver organoid model of ALD, which is derived from hiPSC differentiation, has emerged as an invaluable platform for advancing our understanding and management of ALD in clinical settings.

Quality Assessment of Metabolic Fitness during T Cell and NK Cell Expansion

Abstract CAR-T cells have revolutionized cancer treatment, and other cell types like CAR-NK cells are also showing promising results. Engineered cells generally need to be expanded ex vivo before being transfused into patients. Growth conditions, including expansion platform, cell culture media, and cytokine composition, time of expansion, etc., can have a strong effect on the characteristics of the final products and need to be monitored to establish the appropriate development process. Recent developments highlight the significance of cell metabolism in influencing immune responses and the anti-tumor efficacy of cell therapies. In the present study, we utilized Seahorse XF technology and assays to characterize T cell and NK cell metabolic fitness during cell expansion. We cultured human T cells and NK cells under different expansion conditions including culture media, culture devices, and cytokine combinations, and sampled cells at different time points for quality assessment of metabolic fitness. Using the new Seahorse XF assay alongside immunophenotypic characterization using Novocyte Flow Cytometry, we obtained robust information on mitochondrial and glycolytic energy production, metabolic poise, and the bioenergetic capacity of the expanded cells, allowing us to identify conditions that resulted in increased metabolic fitness of the cell population that can contribute to defining cell expansion conditions during immunotherapy development.

Noncovalently particle-anchored cytokines with prolonged tumor retention safely elicit potent antitumor immunity.

Preclinical studies have shown that immunostimulatory cytokines elicit antitumor immune responses but their clinical use is limited by severe immune-related adverse events upon systemic administration. Here, we report a facile and versatile strategy for noncovalently anchoring potent Fc-fused cytokine molecules to the surface of size-discrete particles decorated with Fc-binding peptide for local administration. Following intratumoral injection, particle-anchored Fc cytokines exhibit size-dependent intratumoral retention. The 1-micrometer particle prolongs intratumoral retention of Fc cytokine for over a week and has minimal systemic exposure, thereby eliciting antitumor immunity while eliminating systemic toxicity caused by circulating cytokines. In addition, the combination of these particle-anchored cytokines with immune checkpoint blockade antibodies safely promotes tumor regression in various syngeneic tumor models and genetically engineered murine tumor models and elicits systemic antitumor immunity against tumor rechallenge. Our formulation strategy renders a safe and tumor-agnostic approach that uncouples cytokines' immunostimulatory properties from their systemic toxicities for potential clinical application.

Abstract 7540: Impact of IL-2 and IL-7 on human regulatory T cell suppression and pembrolizumab-mediated reversal of human T cell exhaustion

Abstract Inhibitory pathways within the tumour microenvironment limit the anti-cancer immune response. Two central mechanisms limiting the T cell response in the tumour are suppression of effector T cells responses by regulatory T cells, and chronic exposure to tumour antigens that may bestow T cell unresponsiveness or exhaustion. Restoring exhausted T cell responsiveness through blockade of inhibitory immune checkpoints such as the PD-1/PDL-1 interaction is an established therapeutic modality leading to durable responses and improved survival rates in some patients.We have modelled suppressive mechanisms in vitro using purified human T cells and investigated the impact of the cytokines IL-2 and IL-7. A comparison of in vitro assay systems for T cell exhaustion including T cell/allogeneic dendritic cells, a recall response to a viral antigen, prolonged stimulation of T cells to generate an ‘exhausted’ phenotype followed by a secondary stimulation with allogeneic DCs, revealed in some donors, the greatest impact of pembrolizumab was with exhausted T cells, a finding consistent with our studies using a dissociated tumour cell response with primary cells from patients with lung or colon carcinomas. Quantifying a range of cytokines (e.g. IFNγ, TNFα and IL-10) and flow cytometry activation markers (e,g, CD69, CD25, CD71, OX40 and CD137) established that in general pembrolizumab was effective at restoring the IFNγ response, although some donors were not responsive, and there was a more modest impact on other readouts indicating not all T cell functionality is restored. We therefore tested the impact of IL-2 and IL-7 on the reversal of the ‘exhausted’ phenotype and identified further improved functionality and donor responsiveness. We subsequently tested the impact of these cytokines in another ‘inhibitory’ context, that of regulatory T cell suppression; we identified that high-levels of IL-2 overcame regulatory T cell suppression and IL-7 led to an overall enhancement of response.These findings highlight how multiple suppressive mechanisms may need to be overcome to maximally restore anti-cancer immune responses, and that the combination of cytokines, such as IL-2 and IL-7, and checkpoint inhibitors, such as pembrolizumab, may enhance T cell responses. Citation Format: Gokhan Tut, Neale Harrison, Leila Sallie, Emma Welsh, Ashley Pegg, Arshpreet Kaur, Zhi Li, Ines Morano, Hujo Chan, Jamie Cowley, Catherine Brady, John Gordon, Mark Bryant, Omar Qureshi, Nicholas M. Barnes. Impact of IL-2 and IL-7 on human regulatory T cell suppression and pembrolizumab-mediated reversal of human T cell exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7540.

Modern Methods of Obtaining Immune Dendritic Cells With Anti-Tumor Potential

Dendritic cells (DCs) initiate and shape both innate and adaptive immune responses. They specialize in presenting antigens to naïve T cells, thereby directing T cell immune responses and contributing significantly to the maintenance of antitumor immunity. In both human and animal bodies, these cells are present in limited quantities, posing challenges in their procurement. Hence, the quest for obtaining DCs with antitumor properties in vitro from progenitor cells for clinical or experimental use remains pertinent. This research aims to consolidate existing studies on deriving immune DCs from progenitor cells for application in anticancer therapy. Analysis of published reports reveals that monocytes from peripheral blood, mononuclear cells from bone marrow, and cord blood can serve as precursor cells of immune DCs. Protocols for generating immature DCs from progenitor cells involve the addition of various combinations of cytokines to the culture, including granulocyte-macrophage colony-stimulating factor, interleukin-4, and other cytokines. The extensive range of cytokines and conditions influencing the differentiation and functional activity of DCs results in considerable heterogeneity in the phenotypic and functional characteristics of these cells. Sources of tumor antigen for DC-based vaccines encompass tumor lysates, indivi­dual tumor proteins, peptides, and tumor cells in a state of immunogenic apoptosis. This paper delves into the use of maturation factors and cryopreservation as integral stages in obtaining immune DCs. A comprehensive understanding of the parameters involved in obtaining immune DCs is imperative for the development of DC-based vaccines to unleash their full antitumor potential.

The emerging role of effector functions exerted by tissue-resident memory T cells.

The magnitude of the effector functions of memory T cells determines the consequences of the protection against invading pathogens and tumor development or the pathogenesis of autoimmune and allergic diseases. Tissue-resident memory T cells (TRM cells) are unique T-cell populations that persist in tissues for long periods awaiting re-encounter with their cognate antigen. Although TRM cell reactivation primarily requires the presentation of cognate antigens, recent evidence has shown that, in addition to the conventional concept, TRM cells can be reactivated without the presentation of cognate antigens. Non-cognate TRM cell activation is triggered by cross-reactive antigens or by several combinations of cytokines, including interleukin (IL)-2, IL-7, IL-12, IL-15 and IL-18. The activation mode of TRM cells reinforces their cytotoxic activity and promotes the secretion of effector cytokines (such as interferon-gamma and tumor necrosis factor-alpha). This review highlights the key features of TRM cell maintenance and reactivation and discusses the importance of effector functions that TRM cells exert upon being presented with cognate and/or non-cognate antigens, as well as cytokines secreted by TRM and non-TRM cells within the tissue microenvironment.