Increased Cytokine Secretion Research Articles

Micro-fragmented adipose tissue cellular composition varies by processing device and analytical method

Autologous adipose-derived biologics are of clinical interest based on accessibility of adipose tissue, a rich source of progenitor and immunomodulatory cells. Micro-fragmented adipose tissue (MFAT) preserves the cellular niche within intact extracellular matrix, potentially offering benefit over enzymatically-liberated stromal vascular fraction (SVF), however lack of standardized analyses complicate direct comparison of these products. In this study, MFAT from LipoGems® and AutoPose™ Restore systems, which utilize different washing and resizing methods, was analyzed for cellular content using different techniques. Flow cytometry was performed on SVF, with or without culture, and on the adherent cell population that naturally migrated from undigested MFAT. Cytokine release during culture was also assessed. SVF contained more diverse progenitor populations, while MFAT outgrowth contained lower cell concentrations of predominantly mesenchymal stromal cells (MSC). MSCs were significantly higher in MFAT from the AutoPose System for all analyses, with increased cytokine secretion characterized by high levels of anti-inflammatory and low to non-detectable inflammatory cytokines. These results demonstrate that cellularity depends on MFAT processing methods, and different techniques can be employed to evaluate graft cellularity. Comparisons of cell concentrations determined via these methods could be used to better interpret inter-study variability.

Immunomodulatory effects of carbon ion radiotherapy in patients with localized prostate cancer

PurposeRadiotherapy is one of the main local treatment modalities for prostate cancer, while immunosuppressive effect induced by radiotherapy is an important factor of radiation resistance and treatment failure. Carbon ion radiotherapy (CIRT) is a novel radiotherapy technique and the immunomodulatory effect of CIRT provides the possibility of overcoming radioresistance and improving efficacy. The aim of this study was to assess the immune response evoked by CIRT in localized prostate cancer patients.MethodsThirty-two patients were treated by CIRT combined with or without hormone therapy and peripheral blood samples were collected before and after CIRT. Investigation of peripheral immune cell frequency, proliferation, and cytokine expression was conducted by flow cytometry, real-time quantitative PCR and ELISA.ResultsThere were no significant differences in the frequencies of CD3 + , CD4 + , CD8 + T cells and NK cells after CIRT. CD4/CD8 ratio increased whereas B cells decreased. All lymphocyte subsets except regulatory T cells (Tregs) displayed increased proliferation and T cells exhibited increased functionality after CIRT, characterized by modestly increased cytokine secretion of TNF. Moreover, higher frequencies of Tregs were shown. Neither monocytic myeloid-derived suppressor cells (MDSCs) nor early MDSCs changed after CIRT. TGF-β1 gene expression decreased while IL-6 showed a non-significant trend towards a decrease. Both IL-10 gene expression and plasma TGF‐β1 level were unchanged.ConclusionCIRT demonstrates the potential to elicit immune activation in localized prostate cancer patients, based on sparing lymphocytes, increased lymphocyte proliferation, enhanced T-cell functionality, together with limited induction of immunosuppressive cells and reduced expression of immunosuppressive cytokines.

Extracellular Vesicles of Mesenchymal Stem Cells Are More Effectively Accessed through Polyethylene Glycol-Based Precipitation than by Ultracentrifugation.

Extracellular vesicles (EVs) have been identified as cell-cell communication agents, and EVs derived from mesenchymal stem cells (MSCs) exhibit therapeutic effects similar to those of the cells of origin. Precipitation methods have been used extensively for EV harvests, such as UC- (ultracentrifugation-) or PEG- (polyethylene glycol-) based methods, and the difference in EVs derived from MSCs by UC and PEG is not fully understood. We harvested EVs from amniotic fluid MSCs (AF-MSCs) by UC- or PEG-based precipitation methods and conducted a comparison study of those EVs derived by the two methods: output, RNA, and protein expression of EVs and EV biological reaction in a THP-1-cell model of LPS induction, which was considered an infection model. There was no difference in morphology, size, or specific marker-positive ratio of PEG-EVs and UC-EVs, but PEG obtained more EV particles, protein, and RNA than the UC method. In our THP-1 model of LPS induction, MSC-EVs did not lead to a change in protein expression but inhibited the LPS-induced increase in cytokine secretion. UC-EVs were more effective for TNF-α inhibition, and PEG-EVs were more effective for IL10 inhibition. Thus, our findings provide evidence that PEG-based precipitation is a more efficient mesenchymal stem cell-extracellular vesicle-derived method than UC.

Consumption of Artificial Sweetener Acesulfame Potassium Increases Preterm Risk and Uterine Contraction with Calcium Influx Increased via Myosin Light Chain Kinase–Myosin Light Chain 20 Related Signaling Pathway

The consumption of artificial sweeteners has been rapidly increasing, with potentially hazardous effects on human reproduction. This study aims to explore the effect of Acesulfame Potassium (Ace K) and its potential mechanism to induce uterine contraction through in vitro, ex vivo, in vivo, and clinical observation studies. Used ex vivo and in vitro studies to analyze its effect on uterine contraction and involved signaling pathway. Used the long-term, high-dose exposure to examine Ace K's affection for contractive-related protein expression. By involving a cohort of 613 participants, to assess the dose-responsiveness of Ace K consumption and calculate the odd ratio of Ace K consumption and the relationship with preterm risk. Animal studies show increasing uterine contraction, cytokine secretion, and altered contraction-related protein expression. Human data show that higher consumption of Ace K may be related to early delivery. Long-term high-dose exposure to Ace K can induce uterine hypercontraction, increase cytokine secretion, and alters contraction-related protein expression. These findings suggest that women who suffer from uterine hypercontraction causes painfulness should pay more attention to the zero- or low-calorie soft drinks or food products containing Ace K.

A novel anti-CD47-targeted blockade promotes immune activation in human soft tissue sarcoma but does not potentiate anti-PD-1 blockade.

The treatment options for metastatic soft tissue sarcomas (STSs) are limited. In most cases, immunotherapy with immune checkpoint inhibitors has not been successful so far. Macrophages dominate the immune landscape of STSs; thus, combinatorial strategies aiming at both tumor-infiltrating lymphocytes and macrophages may represent a particularly relevant treatment approach for metastatic or recurrent STSs. In this cohort study, 66 patients who underwent surgery for STSs were enrolled. Tumor cells and tumor-infiltrating immune cells were analyzed using flow cytometry and immunohistochemistry. In cell suspensions obtained from surgical resections, human T cells were activated by superparamagnetic polymer beads and cultured at a concentration of 0.3 × 106/µl in the absence or presence of therapeutic monoclonal antibodies (anti-PD-1, anti-CD47, and anti-PD-1 + anti-CD47). Supernatants from cell suspensions were analyzed using multiplex Luminex cytokine bead-based immunoassays. The most profound response to anti-CD47 therapy was observed in an undifferentiated pleiomorphic sarcoma which also displayed high expression of CD47 in the tumor microenvironment. Both anti-PD-1 and anti-CD47 therapies drastically increased the production of pro-inflammatory cytokines in the tumor microenvironment of STSs, but co-administration of both agents did not further increase cytokine secretion. Furthermore, all patient samples treated with a combination of both anti-PD-1 and anti-CD47 antibodies showed a dramatic reduction in cytokine secretion. Our findings suggest that anti-PD-1 and anti-CD47 therapies do not enhance each other, and the combined application of anti-PD-1 and anti-CD47 agents in vitro limits rather than potentiates their efficacy.

Multi-omic comparisons between CFBE41o- cells stably expressing wild-type CFTR and F508del-mutant CFTR

BackgroundCystic fibrosis (CF) is characterized by chronic inflammation and excessive cytokines secretion in the lung. Isogenic human CF bronchial epithelial (CFBE41o-) cell lines stably expressing wt-CFTR (WTBE) or F508del mutant (CFBE) are widely used tools in understanding responses to stimuli or drugs and CF pathogenesis in vitro. However, the intrinsic cellular differences in culture are unknown. MethodsWe performed integrative analyses of these isogenic cells at the protein, mRNA, and chromatin levels in the submerged and air-liquid interface (ALI) conditions to determine cell intrinsic effects of mutant versus complemented CFTR expression. ResultsCFBE and WTBE cells displayed different cytokine secretion patterns, including IL-6, IL-8, CXCL1, CXCL10, and CCL5. The ALI culture dramatically increased cytokine secretion in both cells. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) result showed different chromatin landscapes upon polarization and CFBE cells, compared to WTBE cells, exhibited higher genome-wide chromatin accessibility under both culture methods. At the transcriptome level, differentially expressed genes identified by mRNA sequencing between two cell lines were highly concentrated in immunity-related pathways. ConclusionsThis multilayered study shows that expression of wild-type CFTR has an epithelial cell intrinsic effect on the cell's epigenome and transcriptome particularly in immunity relevant activities. These data will serve as a resource for the CF community and may serve as epithelial biomarkers for CFTR mRNA therapy.

Abstract 2884: Optimized CD28 bispecific antibodies for targeted activation of T cells within the tumor microenvironment

Abstract Tumor-targeted CD28 bispecific antibodies (bsAbs) are designed to co-stimulate T cells specifically within the tumor microenvironment. By bridging T cells to malignant cells expressing a selected tumor-associated antigen (TAA), CD28 bsAbs deliver the so-called signal 2 to T cells unleashing their full cytotoxic potential. Contrary to bivalent CD28 monoclonal antibody (mAb) superagonists, the activation of CD28 with monovalent CD28 bispecifics requires the binding of a TAA expressed by the cancer cells to induce CD28 clustering. Preclinical studies have shown the benefit of adding costimulatory CD28 bsAbs for the treatment of solid tumors, boosting the efficacy of bispecific T cell engagers (CD3 bsAbs) or PD-(L)1 checkpoint inhibitors. Using our proprietary fully human κλ body antibody platform, which relies on a common heavy chain and on two distinct light chains driving specificity and affinity (i.e., one kappa and one lambda), we have identified a panel of agonist anti-CD28 kappa arms. Using our existing lambda anti-TAA arms (e.g., targeting CD19, MSLN, HER2, EGFR), an array of TAA-CD28 κλ bodies targeting different tumors were generated. The capacity of these CD28 κλ bodies to provide signal 2 to T cells was assessed both in vitro and in vivo in combination with CD3 bsAbs. In the presence of cancer cells and corresponding TAA-CD3 bsAbs, the CD28 κλ bodies enabled the activation of primary human CD4 and CD8 T cells, leading to T cell proliferation and secretion of cytokines such as IL-2 and IFNγ, and granzyme B. Importantly, when used alone or in combination with an untargeted CD3 bsAb (that is, in the absence of signal 1), CD28 κλ bodies showed no sign of T cell activation, potentially limiting peripheral toxicity. The resulting change in the T cell activation status led to a potent in vitro T cell-dependent cellular cytotoxicity (TDCC) against TAA-expressing cancer cell lines. CD28 κλ bodies targeting gastrointestinal tumors were tested in combination with CD3 bsAbs in xenogeneic tumor models and synergistically improved the antitumoral activity of T cell engagers, leading to an enhanced tumor control. Thus, via the identification of optimal agonist anti CD28 arms, CD28 bsAbs targeting multiple TAAs were designed for tumor-specific activation of the immune system. The resulting TAA-CD28 κλ bodies can enhance the antitumor response induced by CD3-retargeting bsAbs via the induction of T cell proliferation and activation, increased cytokine secretion and boosted anti-tumoral cytotoxicity. Other anti-TAA arms are being explored, to expand the panel of TAAs that could be easily paired with our anti-CD28 platform arms. Citation Format: Sara Majocchi, Pauline Lloveras, Coline Burnet-Merlin, Lise Nouveau, Nicolas Pleche, Pauline Malinge, Claudia Batista, Christelle Daviet, Maud Charreton, Guillemette Pontini, Franck Gueneau, Valery Moine, Giovanni Magistrelli, Limin Shang, Walter Ferlin, Krzysztof Masternak. Optimized CD28 bispecific antibodies for targeted activation of T cells within the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2884.

Abstract 571: Inclusion of a Dap10 costimulatory domain decreases cytokine release syndrome in chimeric antigen-expressing gamma delta T cells

Abstract Adoptive transfer of T cells is a promising anti-tumor therapy for many cancers. To enhance tumor recognition by T cells, chimeric antigen receptors (CAR) consisting of signaling domains fused to receptors that recognize tumor antigens can be expressed in T cells. While CAR T cells can successfully treat hematopoietic malignancies, CAR T cells have not shown success in treating solid tumors. There are many factors that may enhance CAR T cell efficacy and safety for solid tumor treatment, including addition of costimulatory domains to increase T cell functions and switching the T cell type used from alpha beta to gamma delta T cells. We developed a novel CAR (chimeric PD-1, chPD1) that targets the programmed death 1 receptor (PD-1) ligands expressed on many types of solid tumors. The aim of this study was to compare anti-tumor efficacy and safety of chPD1-expressing gamma delta and alpha beta T cells in murine breast and ovarian cancer models. Inclusion of different costimulatory domains, CD28, 4-1BB, and Dap-10, in the chPD1 receptor was also compared. There were no differences in T cell viability and proliferation when comparing costimulatory domains or T cell type. Additionally, there were no significant differences in killing of murine breast and ovarian cancer cell lines. ChPD1-CD28 T cells had increased secretion of proinflammatory and cytokine release syndrome (CRS)-inducing cytokines, including IFN-gamma, IL-2, IL-6, GM-CSF, and nitric oxide, compared to chPD1-41BB and chPD1-Dap10 receptors. Additionally, alpha beta chPD1 T cells had increased cytokine secretion of inflammatory cytokines compared to chPD1 gamma delta T cells. Furthermore, while gamma delta and alpha beta T cells expressing chPD1 receptors with a Dap10 domain had similar anti-tumor efficacy in vivo, chPD1-Dap10 gamma delta T cells had the lowest serum levels of inflammatory cytokines IFN-gamma, IL-6, and GM-CSF. Therefore, inclusion of Dap10 domains in gamma delta T cells may be a safer and more efficacious option for CAR T cell therapy of solid tumors. Citation Format: Amorette E. Barber. Inclusion of a Dap10 costimulatory domain decreases cytokine release syndrome in chimeric antigen-expressing gamma delta T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 571.

Hematopoietic Progenitor Kinase 1 in Tumor Immunology: A Medicinal Chemistry Perspective.

Hematopoietic progenitor kinase 1 (HPK1), a hematopoietic cell-restricted member of the serine/threonine Ste20-related protein kinases, is a negative regulator of the Tcell receptor, B cell receptor, and dendritic cells. Loss of HPK1 kinase function increases cytokine secretion and enhances Tcell signaling, virus clearance, and tumor growth inhibition. Therefore, HPK1 is considered a promising target for tumor immunotherapy. Several HPK1 inhibitors have been reported to regulate Tcell function. In addition, HPK1-targeting PROTACs, which can induce the degradation of HPK1, have also been developed. Here, we provide an overview of research concerning HPK1 protein structure, function, and inhibitors and propose perspectives and insights for the future development of agents targeting HPK1.

A first-in-human phase 1 trial of nx-1607, a first-in-class oral CBL-B inhibitor, in patients with advanced solid tumor malignancies.

TPS2691 Background: Casitas B-lineage lymphoma proto-oncogene B (CBL-B) is an E3 ubiquitin ligase expressed in multiple immune cell lineages, which in contrast to cell surface immune checkpoints, acts as a regulator of both T and NK cell activation. Inhibition of CBL-B enhances T-cell response, increases response to suboptimal priming, and restores response in exhausted T cells. Thus CBL-B is a promising immune-oncology target and may overcome challenges seen with other T-cell directed therapies. NX-1607 is an oral small molecule inhibitor of CBL-B that has demonstrated anti-tumor activity and long-term survival in murine models as both a single agent and in combination with programmed cell death protein-1 (PD-1) antibodies. Further, NX-1607 elicits dose-dependent increases in cytokine secretion and proliferation in T-cell receptor-stimulated primary human T cells with enhanced tumor antigen-specific T-cell and NK cell anti-tumor responses. Thus, NX-1607 may be effective as a single agent or it may significantly enhance efficacy of other anti-tumor agents. Methods: NX-1607-101 is a first-in-human, multicenter, open-label, Phase 1 dose escalation and expansion trial evaluating NX-1607 in a variety of indications including platinum-resistant epithelial ovarian cancer (EOC), gastric cancer, squamous cell carcinoma of the head and neck (HNSCC), metastatic melanoma, non-small cell lung cancer (NSCLC), metastatic castration-resistant prostate cancer (mCRPC), malignant pleural mesothelioma (MPM), triple-negative breast cancer (TNBC), locally advanced or metastatic urothelial cancer, cervical cancer, microsatellite stable colorectal cancer (MSS CRC), and diffuse large B-cell lymphoma with Richter transformation (DLBCL-RT). The main objective is to establish the safety and tolerability of NX-1607, characterize PK/PD, and determine the recommended Phase 1b dose. NX-1607 will be given orally once daily at doses ranging from 5 to 100 mg in up to 6 dose levels. Dose escalation will proceed using an accelerated modified Fibonacci dose escalation design that transitions to a standard 3 + 3 design based on protocol-specific criteria. Up to 8 expansion cohorts in Phase 1b will be composed of patients with subsets of advanced cancers. Key eligibility criteria include patients with metastatic or unresectable disease that have progressed after prior therapy and for whom standard therapy with proven clinical benefit does not exist, is no longer effective, or is not appropriate. Prior treatment with immune checkpoint inhibitors or CAR-T cells with washout is allowed, but a history of active autoimmune disease is not. Up to 336 patients (60 in Phase 1a, 276 in Phase 1b using a Simon 2-stage design) will be enrolled at approximately 20 sites in the UK and US and treated until disease progression or unacceptable toxicity. Dose escalation is ongoing. Clinical trial information: NCT05107674.

Immune-enhancing efficacy of Curtobacterium proimmune K3 lysates isolated from Panax ginseng beverages in cyclophosphamide-induced immunosuppressed mice

• Curtobacterium proimmune K3 lysates (Ly) was isolated from Panax ginseng beverages. • Ly secreted greatly immune-related cytokines in mouse splenocytes. • Ly increased production of cytokines in CTX-induced immunosuppressed mice. • Ly phosphoraylated NF‐κB (p65-IκBα) and MAPK (JNK, ERK and p38) signaling pathways. • Ly ameliorated the gut microbiota in the immunosuppressed mice. In this study, the effects of Curtobacterium proimmune K3 lysates, isolated from Panax ginseng beverages, on splenocytes and cyclophosphamide (CTX)-induced immunosuppressed mice were investigated. Treatment with C. proimmune K3 lysates stimulated splenocyte proliferation and increased cytokine secretion in vitro and splenocytes ex vivo . Treatment with C. proimmune K3 lysates alleviated CTX-induced immunosuppression by modulating innate immunity in vivo , by recovering immune organ index and natural killer cell cytotoxicity, as well as by stimulating the adaptive immune system, via restoration of splenic lymphocyte proliferation, delayed-type hypersensitivity response, and IgG and IgA content. Moreover, C. proimmune K3 lysates treatment remarkably enhanced cytokine production in the spleen and thymus, compared with CTX-immunosuppressed mice. Our results suggest C. proimmune K3 lysates hold potential as a novel immunomodulatory agent for use in therapeutic purposes or as an ingredient in functional foods.

Transcriptional Analysis of Total CD8+ T Cells and CD8+CD45RA- Memory T Cells From Young and Old Healthy Blood Donors.

Memory CD8+ T cells accumulate with aging, while the naïve T cell compartment decreases, leading to an increased susceptibility to infections and a decreased vaccine efficiency. To get deeper insights into the underlying mechanisms, this study aims to determine the age-dependent expression profile of total versus memory CD8+ T cells from young and old donors. Total CD8+ and CD8+CD45RA- memory T cells isolated from young (<30 years) and old (>60 years) donors were stimulated with anti-CD3 and anti-CD28 antibodies for 48h before analyzing the cytokine secretion and activation markers by flow cytometry and changes in the expression profiles using RNA sequencing. Gene ontology (GO) term enrichment analyses were performed for up-regulated and uniquely expressed transcripts identified in the T cell populations of both age groups. Total and memory CD8+ T cells from old donors expressed significantly higher CD25 levels and have an increased cytokine secretion. While approximately 1,500 up-regulated transcripts were identified in all groups, CD8+CD45RA- memory T cells of old donors had approximately 500 more uniquely expressed transcripts. Four GO terms related to the JAK-STAT pathway were identified for up-regulated transcripts in the total CD8+ T cells of old donors, whereas CD8+CD45RA- memory T cells GO terms related to adjacent pathways, like JNK and MAPK/ERK, were found. Additionally, the unique transcripts of CD8+CD45RA- memory T cells of old donors were related to the JNK, MAPK and IL-12 pathways. For both T cell populations of the old donors, cytokine and JAK-STAT pathway transcripts were up-regulated. Thus, an age-dependent effect was observed on the transcriptomes of total and memory CD8+ T cells. The CD8+ CD45RA- memory T cells from old donors maintained the increased cytokine secretion of the total CD8+ T cell population and the increased JAK-STAT pathway transcripts, which have an impact on inflammation and senescence.

T cell-mediated elimination of cancer cells by blocking CEACAM6–CEACAM1 interaction

ABSTRACT Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), a cell surface receptor, is expressed on normal epithelial tissue and highly expressed in cancers of high unmet medical need, such as non-small cell lung, pancreatic, and colorectal cancer. CEACAM receptors undergo homo- and heterophilic interactions thereby regulating normal tissue homeostasis and angiogenesis, and in cancer, tumor invasion and metastasis. CEACAM6 expression on malignant plasma cells inhibits antitumor activity of T cells, and we hypothesize a similar function on epithelial cancer cells. The interactions between CEACAM6 and its suggested partner CEACAM1 on T cells were studied. A humanized CEACAM6-blocking antibody, BAY 1834942, was developed and characterized for its immunomodulating effects in co-culture experiments with T cells and solid cancer cells and in comparison to antibodies targeting the immune checkpoints programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), and T cell immunoglobulin mucin-3 (TIM-3). The immunosuppressive activity of CEACAM6 was mediated by binding to CEACAM1 expressed by activated tumor-specific T cells. BAY 1834942 increased cytokine secretion by T cells and T cell-mediated killing of cancer cells. The in vitro efficacy of BAY 1834942 correlated with the degree of CEACAM6 expression on cancer cells, suggesting potential in guiding patient selection. BAY 1834942 was equally or more efficacious compared to blockade of PD-L1, and at least an additive efficacy was observed in combination with anti-PD-1 or anti-TIM-3 antibodies, suggesting an efficacy independent of the PD-1/PD-L1 axis. In summary, CEACAM6 blockade by BAY 1834942 reactivates the antitumor response of T cells. This warrants clinical evaluation.

Fetuin-A secretion from β-cells leads to accumulation of macrophages in islets, aggravates inflammation and impairs insulin secretion.

Elevated fetuin-A levels, chemokines and islet-resident macrophages are crucial factors associated with obesity-mediated type 2 diabetes (T2D). Here, the aim of the study was to investigate the effect of MIN6 (a mouse insulinoma cell line)-derived fetuin-A (also known as AHSG) in macrophage polarization and decipher the effect of M1 type pro-inflammatory macrophages in commanding over insulin secretion. MIN6 and islet-derived fetuin-A induced expression of the M1 type macrophage markers Emr1 (also known as Adgre1), Cd68 and CD11c (Itgax) (∼1.8 fold) along with increased cytokine secretion. Interestingly, suppression of fetuin-A in MIN6 successfully reduced M1 markers by ∼1.5 fold. MIN6-derived fetuin-A also induced chemotaxis of macrophages in a Boyden chamber chemotaxis assay. Furthermore, high-fat feeding in mice showed elevated cytokine and fetuin-A content in serum and islets, and also migration and polarization of macrophages to the islets, while β-cells failed to meet the increased insulin demand. Moreover, in MIN6 culture, M1 macrophages sharply decreased insulin secretion by ∼2.8 fold. Altogether our results support an association of fetuin-A with islet inflammation and β-cell dysfunction, owing to its role as a key chemoattractant and macrophage polarizing factor.

Nanobody-Engineered Natural Killer Cell Conjugates for Solid Tumor Adoptive Immunotherapy.

Cancer immunotherapy based on natural killer (NK) cells is demonstrated to be a promising strategy. However, NK cells are deficient in ligands that target specific tumors, resulting in limited antitumor efficacy. Here, a glycoengineering approach to imitate the chimeric antigen receptor strategy and decorate NK cells with nanobodies to promote NK-based immunotherapy in solid tumors is proposed. Nanobody 7D12, which specifically recognizes the human epidermal growth factor receptor (EGFR) that is overexpressed on many solid tumors, is coupled to the chemically synthesized DBCO-PEG4 -GGG-NH2 by sortase A-mediated ligation to generate DBCO-7D12. The NK92MI cells bearing azide groups are then equipped with DBCO-7D12 via bioorthogonal click chemistry. The resultant 7D12-NK92MI cells exhibit high specificity and affinity for EGFR-overexpressing tumor cells in vitro and in vivo by the 7D12-EGFR interaction, causing increased cytokine secretion to more effectively kill EGFR-positive tumor cells, but not EGFR-negative cancer cells. Importantly, the 7D12-NK92MI cells also show a wide anticancer spectrum and extensive tumor penetration. Furthermore, mouse experiments reveal that 7D12-NK92MI treatment achieves excellent therapeutic efficacy and outstanding safety. The authors' works provide a cell modification strategy using specific protein ligands without genetic manipulation and present a potential novel method for cancer-targeted immunotherapy by NK cells.

Innate immune responses after stimulation with Toll-like receptor agonists in ex vivo microglial cultures and an in vivo model using mice with reduced microglia

BackgroundPast experiments studying innate immunity in the central nervous system (CNS) utilized microglia obtained from neonatal mouse brain, which differ developmentally from adult microglia. These differences might impact our current understanding of the role of microglia in CNS development, function, and disease.MethodsCytokine protein secretion was compared in ex vivo P3 and adult microglial cultures after exposure to agonists for three different toll-like receptors (TLR4, lipopolysaccharide [LPS]; TLR7, imiquimod [IMQ]; and TLR9, CpG Oligodeoxynucleotide [CpG-ODN] 1585). In addition, changes in inflammatory gene expression in ex vivo adult microglia in response to the TLR agonists was assessed. Furthermore, in vivo experiments evaluated changes in gene expression associated with inflammation and TLR signaling in brains of mice with or without treatment with PLX5622 to reduce microglia.ResultsEx vivo adult and P3 microglia increased cytokine secretion when exposed to TLR4 agonist LPS and to TLR7 agonist IMQ. However, adult microglia decreased expression of numerous genes after exposure to TLR 9 agonist CpG-ODN 1585. In contrast, in vivo studies indicated a core group of inflammatory and TLR signaling genes increased when each of the TLR agonists was introduced into the CNS. Reducing microglia in the brain led to decreased expression of various inflammatory and TLR signaling genes. Mice with reduced microglia showed extreme impairment in upregulation of genes after exposure to TLR7 agonist IMQ.ConclusionsCultured adult microglia were more reactive than P3 microglia to LPS or IMQ exposure. In vivo results indicated microglial influences on neuroinflammation were agonist specific, with responses to TLR7 agonist IMQ more dysregulated in mice with reduced microglia. Thus, TLR7-mediated innate immune responses in the CNS appeared more dependent on the presence of microglia. Furthermore, partial responses to TLR4 and TLR9 agonists in mice with reduced microglia suggested other cell types in the CNS can compensate for their absence.

TLR5 signalling is hyper-responsive in porcine cystic fibrosis airways epithelium

Excessive lung inflammation and airway epithelium damage are hallmarks of cystic fibrosis (CF) disease. It is unclear whether lung inflammation is related to an intrinsic defect in the immune response or to chronic infection. We aimed to determine whether TLR5-mediated response is defective in the CF airway epithelium. We used a newborn CF pig model to study intrinsic alterations in CF airway epithelium innate immune response. Airway epithelial cells (AECs) were stimulated with flagellin or lipopolysaccharide to determine responses specific for TLR5 and TLR4, respectively. We observed a significant increase in cytokine secretion when CF AECs were stimulated with flagellin compared to wild type (WT) AECs. These results were recapitulated when AECs were treated with an inhibitor of CFTR channel activity. We show that TLR5-signalling is altered in CF lung epithelium at birth. Modulation of TLR5 signalling could contribute to better control the excessive inflammatory response observed in CF lungs.

The upregulation of miR-204-3p in LPS-induced acute lung injury aggravated pulmonary endothelial cells apoptosis via targeting sulfatase 2.

Acute lung injury (ALI) results from the injury of alveolar epithelial cells and pulmonary capillary endothelial cells, with a high mortality rate ranging from 29% to 42%. Therefore, more efficient therapeutic strategies for ALI are necessary. Numerous studies revealed that miRNAs play a role in the regulation of ALI. Lipopolysaccharide (LPS) can induce an inflammatory response and has been widely applied in the establishment of the mouse ALI model. Here, we reported that miR-204-3p expression was upregulated by LPS treatment with increased cytokine secretion. LPS treatment promoted cell apoptosis, accompanied by abnormal cell structure and unobvious alveolar structure. These effects could be prevented by down-regulation of miR-204-3p, and promoted by miR-204-3p overexpression. Sulfatase 2 (SULF2) appeared to be the target of miR-204-3p predicted by TargetScan. Downregulation of miR-204-3p enhanced the protein level of SULF2, indicating that SULF2 was a target of miR-204-3p, which could negatively regulate the expression of SULF2. Thus, targeting miR-204-3p may be a potential therapeutic strategy for ALI.

BAY 60-6583 Enhances the Antitumor Function of Chimeric Antigen Receptor-Modified T Cells Independent of the Adenosine A2b Receptor.

Chimeric antigen receptor (CAR) T cells are powerful in eradicating hematological malignancies, but their efficacy is limited in treating solid tumors. One of the barriers is the immunosuppressive response induced by immunomodulatory signaling pathways. Pharmacological targeting of these immunosuppressive pathways may be a simple way to improve the efficacy of CAR T cells. In this study, anti-CD133 and anti-HER2 CAR T cells were generated from healthy donors, and combination therapy using CAR T cells and small molecules targeting adenosine receptors was performed in vitro and in vivo with the goal of probing for potential synergistic antitumor activities. The adenosine A2b receptor agonist, BAY 60-6583, was found to significantly increase cytokine secretion of CD133-or HER2-specific CAR T cells when co-cultured with the respective target tumor cells. The in vitro cytotoxicity and proliferation of CAR T cells were also enhanced when supplied with BAY 60-6583. Furthermore, the combination with this small molecule facilitated the anti-HER2 CAR T cell-mediated elimination of tumor cells in a xenograft mouse model. However, the enhanced antitumor activities could not be suppressed by knockout of the adenosine A2b receptor in CAR T cells. Furthermore, mass spectrometry and computational methods were used to predict several potential alternative targets. Four potential targets (pyruvate kinase M (PKM), Talin-1, Plastin-2, and lamina-associated polypeptide 2) were captured by a photo-affinity probe, of which PKM and Talin-1 were predicted to interact with BAY 60-6583. Overall, our data suggest that BAY 60-6583 upregulates T cell functions through a mechanism independent of the adenosine A2b receptor.

The Neuroinflammatory and Neurotoxic Potential of Palmitic Acid Is Mitigated by Oleic Acid in Microglial Cells and Microglial-Neuronal Co-cultures.

Neuroinflammation has been implicated in the pathogenesis of neurodegeneration and is now accepted as a common molecular feature underpinning neuronal damage and death. Palmitic acid (PA) may represent one of the links between diet and neuroinflammation. The aims of this study were to assess whether PA induced toxicity in neuronal cells by modulating microglial inflammatory responses and/or by directly targeting neurons. We also determined the potential of oleic acid (OA), a monounsaturated fatty acid, to counteract inflammation and promote neuroprotection. We measured the ability of PA to induce the secretion of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), the induction of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling pathways, as well as the phosphorylation of c-Jun, and the expression of inducible nitric oxide synthase (iNOS). Finally, to determine whether PA exerted an indirect neurotoxic effect on neuronal cells, we employed a microglia-neuron co-culture paradigm where microglial cells communicate with neuronal cells in a paracrine fashion. Herein, we demonstrate that PA induces the activation of the NF-κB signalling pathway and c-Jun phosphorylation in N9 microglia cells, in the absence of increased cytokine secretion. Moreover, our data illustrate that PA exerts an indirect as well as a direct neurotoxic role on neuronal PC12 cells and these effects are partially prevented by OA. These results are important to establish that PA interferes with neuronal homeostasis and suggest that dietary PA, when consumed in excess, may induce neuroinflammation and possibly concurs in the development of neurodegeneration.