Interferon-γ Secretion Research Articles

Efficacy of a novel bivalent vaccine containing porcine circovirus type 2d and Mycoplasma hyopneumoniae against a dual PCV2d and Mycoplasma hyopneumoniae challenge.

Information on efficacy of a novel bivalent vaccine containing porcine circovirus type 2d (PCV2d) and Mycoplasma hyopneumoniae. To evaluate bivalent vaccine for efficacy under experimental conditions. Clinically healthy 35 weaned piglets at 18 days of age were used. A 2.0 mL dose of bivalent vaccine was administered intramuscularly to pigs at 21 days of age in accordance with the manufacturer's instructions. The pigs were challenged at 42 days of age either intranasally with PCV2d, or intratracheally with M. hyopneumoniae, or with both. Vaccinated-challenged pigs improved the growth performance compared to pigs that were unvaccinated and then, challenged. Vaccinated-challenged pigs elicited a significant amount of protective immunity for PCV2d-specific neutralizing antibodies and interferon-γ secreting cells (IFN-γ-SC) as well as for M. hyopneumoniae-specific IFN-γ-SC compared to unvaccinated/challenged pigs. Induction of systemic cellular and humoral immune responses from bivalent vaccination reduced the viral and mycoplasmal loads in the blood and larynx. Vaccination and challenge simultaneously reduced both lung and lymphoid lesion severity when compared to unvaccinated-challenged pigs. The results of this study demonstrated that the evaluated bivalent PCV2d and M. hyopneumoniae vaccine was efficacious in protecting pigs from the most predominant PCV2d genotype in the field today, as evaluated with a dual PCV2d and M. hyopneumoniae challenge under experimental conditions.

Impact of delayed PBMC processing on functional and genomic assays

Peripheral blood mononuclear cells (PBMCs) are commonly isolated from whole blood samples in clinical trials. Isolated PBMCs can be cryopreserved for use in downstream assays such as flow cytometry, single-cell RNA sequencing (scRNA-seq) and enzyme-linked immunosorbent spot (ELISpot) assays to aid understanding of disease biology and treatment effects, and biomarker identification. However, due to logistical practicalities, delays from blood collection to PBMC processing may exceed 24 h, which can potentially affect PBMC function and, ultimately, downstream assay results.Whole blood samples from 20 healthy adults were collected and incubated at 20–25 °C for 2–48 h before PBMC processing. PBMC viability was measured, and flow cytometry immunophenotyping, scRNA-seq and ELISpot were performed following increasing PBMC processing delays. The RosetteSep™ granulocyte depletion kit was used to evaluate the impact of granulocyte contamination following processing delay. Processed scRNA-seq reads were used to identify cell clusters based on marker genes. scRNA-seq data was further used to determine gene expression correlation and pathway activity score in major PBMC cell types (T cells, B cells, natural killer cells, monocytes and dendritic cells) between PBMC preparations subjected to shorter (2–4 h) and longer (8–48 h) processing delays. ELISpot assays evaluated the impact of processing delays on the number of interferon-γ (IFN-γ) secreting cells from ex vivo stimulated PBMCs.PBMC viability was reduced after a 48-h processing delay. Flow cytometry showed that granulocyte contamination of PBMCs increased after 24 h. Cluster analysis of scRNA-seq data identified 23 immune cell type gene expression clusters that were not significantly changed upon granulocyte depletion. Gene expression correlations across the major PBMC cell types were < 0.8 after 24 h of delay compared with 2 or 4 h of delay. Inflammatory, proliferation and signaling pathway activities increased, whereas IFN-γ and metabolic pathway activities decreased with increasing PBMC processing delays. The number of IFN-γ secreting cells trended towards a reduction as PBMC processing delays increased.PBMC processing delays should be minimised when designing clinical trials to reduce outcome variability in downstream assays. Ideally clinical trial sites should have on-site PBMC processing capabilities or be located close to such facilities.

2-Methoxyestradiol-3,17-O,O-bis-sulfamate inhibits store-operated Ca2+ entry in T lymphocytes and prevents experimental autoimmune encephalomyelitis

Ca2+ signaling is one of the essential signaling systems for T lymphocyte activation, the latter being an essential step in the pathogenesis of autoimmune diseases such as multiple sclerosis (MS). Store-operated Ca2+ entry (SOCE) ensures long lasting Ca2+ signaling and is of utmost importance for major downstream T lymphocyte activation steps, e.g. nuclear localization of the transcription factor ‘nuclear factor of activated T cells’ (NFAT). 2-Methoxyestradiol (2ME2), an endogenous metabolite of estradiol (E2), blocks nuclear translocation of NFAT. The likely underlying mechanism is inhibition of SOCE, as shown for its synthetic sulfamate ester analogue 2-ethyl-3-sulfamoyloxy-17β-cyanomethylestra-1,3,5(10)-triene (STX564).Here, we demonstrate that another synthetic bis-sulfamoylated 2ME2 derivative, 2-methoxyestradiol-3,17-O,O-bis-sulfamate (2-MeOE2bisMATE, STX140), an orally bioavailable, multi-targeting anticancer agent and potent steroid sulfatase (STS) inhibitor, antagonized SOCE in T lymphocytes. Downstream events, e.g. secretion of the pro-inflammatory cytokines interferon-γ and interleukin-17, were decreased by STX140 in in vitro experiments.Remarkably, STX140 dosed in vivo completely blocked the clinical disease in both active and transfer experimental autoimmune encephalomyelitis (EAE) in Lewis rats, a T cell-mediated animal model for MS, at a dose of 10 mg/kg/day i.p., whereas neither 2ME2 nor Irosustat, a pure STS inhibitor, showed any effect. The STS inhibitory activity of STX140 is therefore not responsible for its activity in this model.Taken together, inhibition of SOCE by STX140 resulting in full antagonism of clinical symptoms in EAE in the Lewis rat, paired with the known excellent bioavailability and pharmaceutical profile of this drug, open potentially new therapeutic avenues for the treatment of MS.

PCSK3 Overexpression in Sjögren's Syndrome Patients May Be Regulated by rs4932178 SNP in Its Promoter Region and Correlates with IFN-γ Gene Expression.

The PCSK3 gene encodes for the protease enzyme Furin, which promotes proteolytic maturation of important regulators of the immune response, and also enhances the secretion of interferon-γ (IFN). Several studies have suggested its possible involvement in the pathogenesis of chronic inflammatory diseases. We investigated the PCSK3 gene expression level in peripheral blood mononuclear cells isolated from Sjögren's Syndrome (SS) patients and healthy controls and we evaluated a possible correlation with IFN-γ gene expression. Moreover, we also explored the variability of two PCSK3 genetic polymorphisms (rs4932178 and rs4702) to evaluate a possible association between these polymorphisms and the expression levels of this gene. We observed, by RT-qPCR, that the PCSK3 expression level was significantly higher in SS patients compared to the controls (p = 0.028), and we confirmed a positive correlation between PCSK3 and IFN-γ expression levels (p < 0.001). Moreover, we reported that the variant homozygous genotype of rs4932178 SNP is associated with a higher expression of the PCSK3 gene (p = 0.038) and with the SS susceptibility (p = 0.016). Our data suggest that Furin could play a role in SS development, also promoting IFN-γ secretion.

Impact of shipping temperature on cell viability and T cell responses to bacterial antigens.

Background: Interferon-γ (IFN-γ) secretion by T cells is a key correlate of immune protection against many pathogens including tuberculosis and the neglected tropical disease melioidosis. Clinical studies in tropical regions of immune responses to pathogens and vaccine monitoring studies require the collection of samples in resource-limited rural areas and subsequent shipment to central laboratories for downstream assays and long-term storage. Here, we studied the impact of two different shipping temperatures on the viability, composition and function of peripheral blood mononuclear cells (PBMC) using multi-colour flow cytometry and IFN-γ enzyme-linked immunospot assay (IFN-γ ELISpot), in order to provide guidance on sample shipment conditions for future clinical studies. Methods: Paired peripheral blood mononuclear cell (PBMC) samples from recovered melioidosis patients were stored in liquid nitrogen (-196°C) and then shipped from Bangkok, Thailand to Oxford, UK at either -80°C (dry ice) or -196°C (dry shipper). After thawing, cell viability and composition were assessed by flow cytometry and antigen specific responses to Burkholderia pseudomallei (BP) were measured using IFN-γ ELISpot. Results: We observed modest lowering of viability in the majority of samples and a reduction in IFN-γ responses to BP which correlated to a decrease of monocytes and natural killer cells in samples shipped at -80°C compared to -196°C. Despite being lower in magnitude antigen-specific responses remained detectable in the majority of samples. Conclusions: Here we demonstrate that shipment of cryopreserved PBMC at -196°C has a benefit on cell viability, recovery and T cell responses to bacterial antigens, although useful information can still be obtained from samples shipped at -80°C, thus providing important guidance for sample management in future clinical trials.

Abstract 1192: Branched-chain keto acids exert an immune-suppressive and neurodegenerative microenvironment in CNS leptomeningeal lymphoma

Abstract Leptomeningeal disease is a devastating complication characterized by the rapid onset of debilitating neurological symptoms and a markedly poor prognosis. It is diagnosed in 5-15% of hematological cancers. Here, we aim to identify the metabolic and immune remodeling of the tumor leptomeningeal microenvironment in Non-Hodgkin lymphoma through comprehensive multi-omic analysis of patient specimens, in vivo models, and in vitro functional studies. Cerebrospinal fluid (CSF) from leptomeningeal lymphoma (LML) patients and tumor-free controls were collected and analyzed using single-cell RNA sequencing (scRNA-Seq), metabolomics, proteomics, and lipidomics. Patients’ CSF cellular components were highly enriched with heterogeneous B-cell populations. Few T-lymphocytes in LML patients showed expression of activation or proliferation markers. Poor prognosis was associated with significant infiltration of macrophages and a lack of dendritic cells. These findings were confirmed to be unique to LML using immune-competent animal models where tumor cells were injected intrathecally, and into lymph nodes. Moreover, LML patients’ CSF showed an accumulation of branched-chain keto-acids (BKAs), which are well-known neurotoxins, metabotoxins, and acidogens. Absolute quantification of individual BKAs was confirmed by high-performance liquid chromatography and showed a 30-fold increase in LML patients’ CSF. To investigate the effect of BKAs accumulation on T-lymphocytes, we examined their viability, proliferation, and activation with BKAs treatment in the context of physiological CSF. Cell trace violet-labeled T lymphocytes showed a significant reduction in proliferation and viability with increasing doses of BKAs. Additionally, a significant decline in the secretion of TNF-α, interferon-γ, granzyme B, and IL-2 was found using ELISA assays, indicating a reduction in T-lymphocyte activation. The proteomic and lipidomic analysis of patients’ CSF also revealed a significant downregulation in proteins and lipids vital for neuronal development, synaptic organization, and myelin sheath integrity. Similarly, scRNA-Seq of the leptomeningeal layer collected from the LML mouse model revealed compromised leptomeningeal integrity. Neurological scoring of the LML mouse model showed disease progression to be associated with rapid neurological decline. To investigate the direct effect of BKAs on the leptomeningeal and neuronal integrity, we tested different concentrations of BKAs on murine primary neuronal cells and human leptomeningeal cells (HMC). MTT assays showed a significant reduction in HMC metabolic activity after 72 hours and in neuronal metabolic activity after 7 days. In conclusion, our data unveil the immunosuppressive and neurodegenerative role of BKAs in the leptomeningeal tumor microenvironment. Citation Format: Mariam Lotfy Khaled, Gerald Wallace, Brittany Evernden, Zhihua Chen, Hasan Alhaddad, Yuan Ren, Oscar Ospina, Maclean Hall, Ann Chen, Timothy J. Robinson, John Koomen, Shari Pilon-thomas, Peter Forsyth, Inna Smalley. Branched-chain keto acids exert an immune-suppressive and neurodegenerative microenvironment in CNS leptomeningeal lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1192.

Codon Pair Deoptimization (CPD)-Attenuated PRRSV-1 Vaccination Exhibit Immunity to Virulent PRRSV Challenge in Pigs.

Commercially used porcine respiratory and reproductive syndrome (PRRS) modified live virus (MLV) vaccines provide limited protection with heterologous viruses, can revert back to a virulent form and they tend to recombine with circulating wild-type strains. Codon pair deoptimization (CPD) is an advanced method to attenuate a virus that overcomes the disadvantages of MLV vaccines and is effective in various virus vaccine models. The CPD vaccine against PRRSV-2 was successfully tested in our previous study. The co-existence of PRRSV-1 and -2 in the same herd demands protective immunity against both viruses. In this study, live attenuated PRRSV-1 was constructed by recoding 22 base pairs in the ORF7 gene of the E38 strain. The efficacy and safety of the CPD live attenuated vaccine E38-ORF7 CPD to protect against virulent PRRSV-1 were evaluated. Viral load, and respiratory and lung lesion scores were significantly reduced in animals vaccinated with E38-ORF7 CPD. Vaccinated animals were seropositive by 14 days post-vaccination with an increased level of interferon-γ secreting cells. In conclusion, the codon-pair-deoptimized vaccine was easily attenuated and displayed protective immunity against virulent heterologous PRRSV-1.

Curcumin Enhances the Efficacy of Docetaxel by Promoting Anti-Tumor Immune Response in Head and Neck Squamous Cell Carcinoma

This study evaluated the feasibility of curcumin and docetaxel (DTX) combination therapy for head and neck squamous cell carcinoma (HNSCC). Animal assay demonstrated DTX has certain limitations in improving immunosuppressive microenvironment. Treatment with curcumin overcame this inhibition and reduced tumor progression. Curcumin synergized DTX showed significantly greater reduction in tumor burden than either treatment alone via down-regulation of MDSCs, M2 macrophages and up-regulation of CD8+ T cells, NK cells, M1 macrophages. Meanwhile, the secretion of CXCL1 was decreased in tumor. Conversely, the secretion of interferon-γ and tumor necrosis factor-α were increased. Our study provided a promising therapeutic strategy for HNSCC.

Supramolecular assembly of a trivalent peptide hydrogel vaccine for cancer immunotherapy.

Vaccination shows great promise in cancer immunotherapy. However, the induction of robust and broad therapeutic CD8 T cell immunity against tumors is challenging due to the essential heterogenicity of tumor antigen expression. Recently, bioinspired materials have reshaped the field of cancer nanomedicine. Herein, a bioinspired nanofibrous trivalent peptide hydrogel vaccine was constructed using the spontaneous supramolecular co-assembly of three antigenic epitope-conjugated peptides, which could mimic the fibrillar structure and biological function of the extracellular matrix and naturally occurring protein assembly. The hydrogel vaccine could be accurately and flexibly adjusted to load each antigenic peptide at a defined ratio, which facilitated the antigen presentation of dendritic cells and significantly improved the initiation of CD8 T cell response and the secretion of interferon-γ (IFN-γ). C57BL/6 mice were immunized with the trivalent peptide hydrogel vaccine, where it elicited a high broad-spectrum antitumor CD8 T cell response that significantly inhibited the growth of B16 tumors in the absence of additional immunoadjuvants or delivery systems. In summary, the supramolecular assembly of triple antigenic epitope-conjugated peptides offers a simple, customizable, and versatile approach for the development of cancer vaccines with remarkable therapeutic efficacy, thereby providing a highly versatile platform for the application of personalized multivalent tumor vaccines. STATEMENT OF SIGNIFICANCE: (1) We report a feasible, versatile and bioinspired approach to manufacture a multivalent peptide-based hydrogel cancer vaccine in the absence of additional adjuvants, which closely mimics immune niches, co-delivers antigen epitopes, greatly promotes antigen presentation to DCs and their subsequent homing to dLNs and elicits a broad-spectrum antitumor CD8 T cell response, resulting in significant inhibition of B16 tumor growth. (2) This feasible and efficient co-assembly strategy provides an attractive platform for engineering a range of multivalent vaccines at defined ratios to further enhance antigen-specific T cell responses. This approach may also be used for personalized immunotherapy with neo-epitopes.

SN-38, an active metabolite of irinotecan, enhances anti-PD-1 treatment efficacy in head and neck squamous cell carcinoma.

Anti-programmed cell death 1 (anti-PD-1) therapy shows definite but modest activity in patients with advanced/metastatic head and neck squamous cell carcinoma (HNSCC). Preliminary evidence suggests that SN-38, an activated form of irinotecan that increases expression of the transcription factor FoxO3a, can suppress programmed cell death ligand-1 (PD-L1) expression in breast and ovarian tumor models. We analyzed the SN-38-mediated activation of natural killer cells in vitro and explored the efficacy of SN-38 in combination with anti-PD-1 for treatment in vivo. In vitro, SN-38 enhanced the expression of FoxO3a and reduced the expression of c-Myc and PD-L1 dose-dependently in tumor cells. Low-dose SN-38 increased interferon-γ secretion by NK cells and promoted NK cell-mediated cytotoxicity in tumor cells. In vivo studies revealed that at non-cytotoxic drug concentrations, SN-38 significantly enhanced anti-PD-1 activity in suppressing murine tumor growth. We found increased NK cell and CD8+ T-cell infiltration in post-treatment tumors. RNA-seq analysis indicated that SN-38 increased the enrichment of immune cells and biological function genes related to the immune responses. SN-38 is a potentially beneficial adjunct to checkpoint inhibitor therapy in HNSCC. Further studies exploring its mechanism of action and possible applications are necessary. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

PIL-12 delivered by polymer based nanovector for anti-tumor genetherapy

Finding more effective and safe non-viral vectors to transfer genes into cancer cells has become the key of immune gene therapy for cancer. Herein a triblock compound MPEG2000–PDLLA4000–MPEG2000 modified by cationic liposome DOTAP was used as a non-viral vector DOTAP/MPEG2000–PDLLA4000–MPEG2000 (DMPM) to effectively transfer interleukin (IL)-12 plasmid (pIL-12) into tumor tissue. IL-12 produced by transfected tumor cells successfully inducing lymphocyte proliferation and promoting interferon-γ (IFN-γ) secretion, which resulted in tumor cells death. The ability of DMPM to transfer pIL-12 and the immune effect induced by IL-12 in cells had been explored. The anti-tumor effect, mechanism and safety of pIL-12/DMPM in mice cancer model were investigated in this study. Our results showed that the pIL-12 transferred by DMPM was highly expressed both in CT26 cells and B16-F10 cells. IL-12 expressed in the culture supernatant of transfected tumor cells stimulated lymphocyte proliferation and promoted IFN-γ secretion. The experimental result confirmed that pIL-12/DMPM therapy significantly reduced tumor growth in mice model. We designed the nanocomposite DMPM to deliver pIL-12 for cancer treatment and explored its therapeutic efficacy and the underlying anti-tumor mechanism. Our study suggested pIL-12 loaded by DMPM complex would be an effective strategy for cancer treatment.

Correlation of high serum interleukin-6 with clinical outcome and T cell response in patients with unresectable hepatocellular carcinoma treated with atezolizumab and bevacizumab.

596 Background: We elucidated the clinical and immunologic implications of serum interleukin (IL)-6 levels in patients with unresectable hepatocellular carcinoma (HCC) treated with atezolizumab and bevacizumab (Ate/Bev). Methods: We prospectively enrolled 165 patients with unresectable HCC (Discovery cohort: 84 patients from three centers; Validation cohort: 81 patients from one center). Baseline blood samples were analyzed using a flow cytometric bead array. The tumor immune microenvironment was analyzed using RNA sequencing. Results: In the Discovery cohort, clinical benefit (CB) was defined as a complete or partial response or stable disease for ≥ 6 months. Among various blood-based biomarkers, the serum IL-6 level was significantly higher in patients without CB than in those with CB (mean 11.56 vs. 5.05 pg/mL, P=0.02). The optimal cutoff value for high IL-6 was determined as 18.49 pg/mL using maximally selected rank statistics, and 15.2% of patients were identified to have high IL-6 levels at baseline. In both the Discovery and Validation cohorts, patients with high baseline IL-6 levels had a reduced response rate and worse progression-free and overall survival after Ate/Bev compared with that of patients with low IL-6 levels. In the multivariable Cox regression analysis, the clinical implication of high IL-6 levels persisted even after adjustment for various confounding factors. Patients with high IL-6 levels showed reduced interferon-γ and tumor necrosis factor-α secretion from CD8+ T cells. Moreover, excess IL-6 suppressed cytokine production and CD8+ T-cell proliferation. Finally, patients with high IL-6 levels exhibited a non-T-cell-inflamed immunosuppressive tumor microenvironment. Conclusions: High baseline IL-6 levels can be associated with poor clinical outcomes and impaired T-cell function in patients with unresectable HCC after Ate/Bev treatment.

Ocrelizumab Impairs the Phenotype and Function of Memory CD8+ T Cells: A 1-Year Longitudinal Study in Patients With Multiple Sclerosis.

Depleting CD20+ B cells is the primary mechanism by which ocrelizumab (OCRE) is efficient in persons with multiple sclerosis (pwMS). However, the exact role of OCRE on other immune cell subsets directly or indirectly remains elusive. The purpose of this study is to characterize the dynamics of peripheral immune cells of pwMS on OCRE. We collected blood samples from 38 pwMS before OCRE onset (T0) and at 6 and 12 months (T6, T12) after initiation. To cover the immune cell diversity, using mass cytometry time of flight, we designed a 38-parameter panel to analyze B, T, and innate immune cell markers and CNS migratory markers. In parallel, viral-specific CD8+ T-cell responses were assessed by the quantification of interferon-γ secretion using the enzyme-linked immunospot assay on cytomegalovirus, Epstein-Barr virus, and influenza stimulations. Beside B-cell depletion, we observed a loss in memory CD8+CD20+ and central memory CD8+ T cells but not in CD4+CD20+ T cells already at T6 and T12 (p < 0.001). The loss of memory CD8+ T cells correlated with a lower CXCR3 expression (p < 0.001) and CNS-related LFA-1 integrin expression (p < 0.001) as well as a reduced antiviral cellular immune response observed at both time points (p < 0.001). Of note, we did not observe major changes in the phenotype of the other cell types studied. Seven of 38 (18.4%) patients in our cohort presented with infections while on OCRE; 4 of which were switched from dimethyl fumarate. Finally, using a mixed linear model on mass cytometry data, we demonstrated that the immunomodulation induced by previous disease-modifying therapies (DMTs) was prolonged over the period of the study. In addition to its well-known role on B cells, our data suggest that OCRE also acts on CD8+ T cells by depleting the memory compartment. These changes in CD8+ T cells may be an asset in the action of OCRE on MS course but might also contribute to explain the increased occurrence of infections in these patients. Finally, although more data are needed to confirm this observation, it suggests that clinicians should pay a special attention to an increased infection risk in pwMS switched from other DMTs to OCRE.

High serum IL-6 correlates with reduced clinical benefit of atezolizumab and bevacizumab in unresectable hepatocellular carcinoma

We elucidated the clinical and immunologic implications of serum IL-6 levels in patients with unresectable hepatocellular carcinoma (HCC) treated with atezolizumab and bevacizumab (Ate/Bev). We prospectively enrolled 165 patients with unresectable HCC (discovery cohort: 84 patients from three centres; validation cohort: 81 patients from one centre). Baseline blood samples were analysed using a flow cytometric bead array. The tumour immune microenvironment was analysed using RNA sequencing. In the discovery cohort, clinical benefit 6 months (CB6m) was defined as complete or partial response, or stable disease for ≥6 months. Among various blood-based biomarkers, serum IL-6 levels were significantly higher in participants without CB6m than in those with CB6m (mean 11.56 vs. 5.05pg/ml, p= 0.02). Using maximally selected rank statistics, the optimal cut-off value for high IL-6 was determined as 18.49pg/ml, and 15.2% of participants were found to have high IL-6 levels at baseline. In both the discovery and validation cohorts, participants with high baseline IL-6 levels had a reduced response rate and worse progression-free and overall survival after Ate/Bev treatment compared with those with low baseline IL-6 levels. In multivariable Cox regression analysis, the clinical implications of high IL-6 levels persisted, even after adjusting for various confounding factors. Participants with high IL-6 levels showed reduced interferon-γ and tumour necrosis factor-α secretion from CD8+ T cells. Moreover, excess IL-6 suppressed cytokine production and proliferation of CD8+ T cells. Finally, participants with high IL-6 levels exhibited a non-T-cell-inflamed immunosuppressive tumour microenvironment. High baseline IL-6 levels can be associated with poor clinical outcomes and impaired T-cell function in patients with unresectable HCC after Ate/Bev treatment. Although patients with hepatocellular carcinoma who respond to treatment with atezolizumab and bevacizumab exhibit favourable clinical outcomes, a fraction of these still experience primary resistance. We found that high baseline serum levels of IL-6 correlate with poor clinical outcomes and impaired T-cell response in patients with hepatocellular carcinoma treated with atezolizumab and bevacizumab.

The Immunoregulatory Effect of Aconite Treatment on H22 Tumor-Bearing Mice via Modulating Adaptive Immunity and Natural Killer-Related Immunity.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and, in its advanced stages, has a 5-year survival rate of only 3% to 5%. Despite novel mechanisms and treatments being uncovered over the past few years, effective strategies for HCC are currently limited. Previous studies have proven that aconite can suppress tumor growth and progression and prevent the recurrence and metastasis of multiple cancers, but the underlying molecular mechanisms are largely unknown. In this study, different doses of aconite were applied to mice bearing subcutaneous HCC tumors. It was found that aconite had a therapeutic effect on H22 tumor-bearing mice in a dose-dependent manner by reducing tumor volumes and prolonging survival times, which could be attributed to the immunoregulatory effect of aconite. Furthermore, results showed that high-dose administration of aconite could enhance adaptive immunity and natural killer (NK) cell-mediated immunity by regulating the secretion of interferon-γ, upregulating T cells and NK cells, and modulating the expression of the NK cytotoxicity biomarker CD107a and the inhibitory receptor TIGIT. This study revealed a novel mechanism through which aconite exerts antitumor effects, not merely through apoptosis induction pathways, providing more sound evidence that aconite has the potential to be developed into an effective anti-HCC agent.

Pharmacology, pharmacokinetics, and toxicity characterization of a novel anti-CD73 therapeutic antibody IBI325 for cancer immunotherapy

It is an intriguing approach to target the ecto-5′-nucleotidase CD73 to confer synergetic beneficial survival in cancer patients, along with clinically established immunotherapy targets. In this study, a fully human, subnanomolar affinity CD73 antibody IBI325 was developed using the yeast display platform. Compared with Oleclumab, IBI325 was equivalent in hCD73 affinity and more potent in cell-bound and soluble CD73 enzymatic inhibition, and no hook effects were observed. Correspondingly, adenosine monophosphate-mediated immune suppression was reversed by IBI325, and significant T cell proliferation and release of cytokines were observed. Also, IBI325 enhanced the T cell recall response by inducing interferon-γ secretion. The antitumor efficacy of IBI325 was investigated in a hPBMC-reconstituted NOG mouse model, and a hCD73 knock-in mouse model. Consequently, IBI325 induced a significant tumor regression by inducing intratumoral immune cell expansion, and a combo therapy of IBI325 and aPD-1 was superior in efficacy than aCD73 or aPD-1 monotherapy. Additionally, the binding epitopes of CD73 to IBI325 were distinct from previously reported aCD73 therapeutics. IBI325 displayed acceptable pharmacokinetics and sufficient tolerable safety profiles to support clinical development. In conclusion, the pharmacology, pharmacokinetics, and toxicity profiles of IBI325 with complete CD73 inhibition were characterized, and encouraging preclinical outcomes were reported.

Effect of intravenous lipid infusion on biomarkers of insulin resistance and immune functions of dry and nonpregnant dairy cows

During the transition period, dairy cows often experience negative energy balance, which can induce metabolic and immunological disturbances. Previous work has shown that there is a relationship between the dysfunction of immune cells and the increase in blood nonesterified fatty acid (NEFA) concentration. Nevertheless, it is difficult to determine the exact effect of NEFA on the immune system, as other metabolic and hormonal perturbations occur simultaneously during the transition period. In the present study, we have determined the effect of NEFA on immune functions using an experimental model designed to assess the effects independently of energy balance, as well as hormonal and metabolic changes due to parturition. Six dry and nonpregnant cows were infused with either sterile water (control treatment) or a lipid emulsion (Intralipid 20%, Frenesius Kabi, lipid treatment) at a rate of 1 mL/kg per hour for 6 h according to a crossover design. Blood concentrations of NEFA, β-hydroxybutyrate (BHB), and glucose were measured every hour throughout the infusion period, and 1 and 18 h after the end of infusion. Proliferation and interferon-γ secretion of lymphocytes, phagocytosis, and oxidative burst of neutrophils and blood insulin concentration were evaluated before, during, and at the end of the infusion. For NEFA, BHB, and glucose, treatment × time interactions were present. When compared with the control condition, NEFA and BHB levels were greater in the plasma of cows infused with lipids from 1 h after the start of infusion until 1 h after the end of infusion. Glucose level also increased in response to lipid infusion from 2 h of infusion until 1 h after the end of treatment. For sterile water and lipid infusions, respectively, maximal concentrations were 0.06 ± 0.10 mM and 1.39 ± 0.10 mM for NEFA, 0.70 ± 0.05 mM and 1.06 ± 0.05 mM for BHB, and 4.56 ± 0.27 mM and 6.90 ± 0.27 mM for glucose. For all blood metabolites, there were no differences between treatments 18 h postinfusion. Lipid infusion significantly increased blood insulin concentration at 3 and 6 h of infusion. However, it returned to its basal concentration 18 h after the end of the infusion. Lymphoproliferation declined as early as 3 h after the start of the lipid infusion. At 3 and 6 h of infusion, lipid treatment significantly reduced INF-γ concentration in the culture cell supernatant. The lipid infusion did not affect neutrophil phagocytosis. Nevertheless, the efficacy of the response was affected by a reduction of neutrophils' oxidative burst. These results confirm that NEFA inhibits immune functions independently of energy balance and other changes that occur during the transition period. They also indicate that high blood lipid concentration causes insulin resistance.

Regulation of Notch Signaling Pathway to Innate Lymphoid Cells in Patients with Acute Myocardial Infarction

ABSTRACT The Notch signaling pathway is an important regulator in fate decisions and immune responses of innate lymphoid cells (ILCs). However, the function of Notch signaling in ILCs in acute coronary syndrome is still not fully elucidated. Thirty-one unstable angina pectoris (UAP) patients, 21 acute myocardial infarction (AMI) patients, and 20 controls were included in this study. Peripheral blood mononuclear cells (PBMCs) were isolated. The mRNA expression levels of Notch receptors and ligands were measured by real-time PCR, while ILC subsets were measured by flow cytometry. Lin− cells were purified and stimulated with γ-secretase inhibitor (GSI). ILC subsets, transcription factors, and secreted cytokines were assessed. Notch receptor and ligand mRNA levels were elevated in PBMCs and peripheral lin− cells from AMI patients. There was no significant difference in total lin−CD45+CD161+CD127+ ILC frequency among three groups. The CRTH2−CD117− ILC1 subset was down-regulated, while the CRTH2+ ILC2 subset was up-regulated in AMI patients. The CRTH2−CD117+ ILC3 subpopulation was comparable among the three groups. ILC1% was negatively correlated with Notch1 and Notch2 in AMI patients. Inhibition of Notch signaling pathway by GSI induced elevations in ILC1 frequency, T-bet mRNA expression, and interferon-γ secretion and reduced ILC2 frequency, GATA3 mRNA levels, and interleukin-5/interleukin-13 production by lin− cells from AMI patients. The current data indicated that activation of Notch signaling pathway might contribute to ILC1-to-ILC2 shift in peripheral blood in AMI patients.

Differentiation and Immunological Function of MDSC-Derived Dendritic Cells.

Dendritic cells (DCs) play a key role in initiating and regulating immune responses, and in addition to their roles in vivo, DCs are used as natural adjuvants for various tumor vaccines. In vitro, monocytes can be used to induce DCs, but in tumor patients, due to insufficient bone marrow hematopoiesis, extramedullary hematopoiesis and tumor-associated myeloid cells expand, and monocytes mainly exist in the form of myeloid-derived suppressor cells (MDSCs). The purpose of this experiment was to explore the differences in the differentiation and immune function of DCs induced by MDSCs in tumor patients. In a mouse model, we used normal mouse bone marrow cell-derived DCs as control cells, and in a tumor-bearing model, we induced MDSCs in the spleen to generate DCs (MDSC-DCs). Through flow cytometry, we found that the production of MDSC-DCs was significantly higher than that of control mice, and the secretion of interferon-γ of MDSC-DCs was significantly reduced. Through OVA antigen presentation experiments, we found that the antigen presentation ability of MDSC-DCs was significantly decreased. Through adoptive treatment of tumor-bearing mice cells, we found that the antitumor immune function of MDSC-DCs was significantly reduced. After that, we explored the mechanism of the decrease of immune function activity of MDSC-DCs. We determined that the surface markers of MDSC-DCs were changed by flow cytometry. Through flow sorting and RNA sequencing, we found that some pathways and key gene expression in MDSC-DCs were changed. In conclusion, this study found that the immune function of MDSC-DCs decreased and explored the mechanism of the decreased immune function activity.

Autophagy-related 9 homolog B regulates T-cell-mediated immune responses in oral lichen planus

ObjectivesThis study aimed to explore the impacts of autophagy-related 9 homolog B (ATG9B)-mediated autophagy on T-cell immune responses in oral lichen planus. DesignATG9B expression was detected in lesions and local T cells by immunohistochemical analysis and immunofluorescence assay. The effects of ATG9B-mediated autophagy on T-cell immune responses were explored after ATG9B-overexpression or ATG9B-knockdown lentivirus transfection. A coculture system of activated T cells and lipopolysaccharide-induced keratinocytes was used to simulate the main cell crosstalk in oral lichen planus. ResultsThe expression of ATG9B upregulated in lesions and local T cells of oral lichen planus, especially in non-erosive oral lichen planus, suggesting that ATG9B may be a diagnostic factor for oral lichen planus. Notably, ATG9B-knockdown T cells of oral lichen planus demonstrated autophagy suppression, enhanced proliferation, and attenuated apoptosis, whereas overexpression of ATG9B showed opposite effects on T cells. In the coculture system of T cells and keratinocytes, ATG9B-knockdown T cells of oral lichen planus, but not ATG9B-overexpression T cells, promoted the proliferation and apoptosis of their cocultured keratinocytes. Additionally, exogenous insulin-like growth factor 1 (IGF1) significantly reversed the apoptosis rates of keratinocytes cocultured with T cells expressing abnormal ATG9B. Furthermore, ATG9B-overexpression T cells showed decreased secretion of interferon-γ and tumor necrosis factor-α in the coculture system. ConclusionsThis study revealed the regulatory roles of ATG9B-mediated T-cell autophagy on T-cell immune responses and crosstalk between T cells and keratinocytes in of oral lichen planus.