Immunosuppressive Protein Research Articles

Efficacy of interleukin 10 gene hydrofection in pig liver vascular isolated 'in vivo' by surgical procedure with interest in liver transplantation.

AimLiver transplantation is the only curative strategy for final stage liver diseases. Despite the great advances achieved during the last 20 years, the recipient immune response after transplantation is not entirely controlled. This results in high rates of acute cell rejection and, approximately, 10% of early mortality. Therapeutic treatment could be improved by efficiently transfecting genes that encode natural immunosuppressant proteins, employing safe procedures that could be transferred to clinical setting. In this sense, interleukin 10 plays a central role in immune tolerance response by acting at different levels.MethodshIL10 gene was hydrofected by retrograde hydrodynamic injection in pig liver with complete vascular exclusion mediated by an ‘in vivo’ surgical procedure. Levels of IL10 DNA, RNA and protein were determined within liver tissue 1 and 10 days after the injection and, more frequently, also the interleukin-10 protein in peripheral blood.ResultsThe procedure was safe for the animals and neither hemodynamic parameters nor liver function determinations showed relevant alterations. The hIL10 hydrofection in watertight liver mediated efficient gene transfer and this was transcribed and translated to protein, achieving up to 110 pg/ml of IL10 in peripheral blood. This value is close to that considered able to reduce the activity of TNFα by half (IL10 IC50 for TNFα = 124 pg/ml).ConclusionsResults of this work suggest that IL10 liver hydrofection with vascular exclusion in vivo is a safe and transferable procedure that mediates plasma protein levels with potential clinical interest in immune modulation after transplantation.

Drug-induced PD-L1 expression and cell stress response in breast cancer cells can be balanced by drug combination

The impact of chemotherapy on tumor-immune system interaction can be either beneficial or harmful, which is represented by the immunogenic cell death (ICD) paradigm or overexpression of the immunosuppressive protein – programmed death ligand 1 (PD-L1). In this study we explore the impact of steroid receptor coactivator inhibitor, other targeted anti-cancer compounds and traditional chemotherapeutic agents on the expression of PD-L1 in four breast cancer (BC) cell lines. Our results show that these agents induce PD-L1 expression, yet the magnitude of this induction varies substantially across the different compounds. In addition, we utilized the E0771 ER + BC cells as a model to examine in greater detail the relationship between pharmacological pressure, cell stress and the induction of PD-L1. Our results imply that drug induced PD-L1 expression occurs in the broader context of cell-stress, without conferring acquired drug-resistance. Furthermore, a balance between BC cytotoxicity, induction of cell-stress and the overexpression of PD-L1 can be achieved through the selection of appropriate combinations of anti-cancer compounds. Therefore, we propose that drug combination can be employed not only for increasing the direct kill of cancer cells, but also as a strategy to minimize the activation of immunosuppressive and cancer cell pro-survival program responses during drug treatment.

Pregnancy Zone Protein Is Associated with Airway Infection, Neutrophil Extracellular Trap Formation, and Disease Severity in Bronchiectasis.

Rationale: PZP (pregnancy zone protein) is a broad-spectrum immunosuppressive protein believed to suppress T-cell function during pregnancy to prevent fetal rejection. It has not previously been reported in the airway.Objectives: To characterize PZP in the bronchiectasis airway, including its relationship with disease severity.Methods: Label-free liquid chromatography/mass spectrometry was performed for sputum protein profiling of patients with bronchiectasis confirmed by high-resolution computed tomography. Results for patients with and without Pseudomonas aeruginosa infection were compared. Sputum and serum PZP was measured by validated ELISA. Airway infection status was established by culture and 16S ribosomal RNA sequencing. Immunofluorescence, ELISA, and electron microscopy were used to identify the cellular source of PZP in neutrophils treated with multiple stimuli.Measurements and Main Results: Elevated PZP was identified by label-free liquid chromatography/mass spectrometry as being associated with P. aeruginosa infection. In a validation study of 124 patients, sputum but not serum concentrations of PZP were significantly associated with the Bronchiectasis Severity Index, the frequency of exacerbations, and symptoms. Airway infection with Proteobacteria such as P. aeruginosa was associated with higher concentrations of PZP. PZP in sputum was directly related to airway bacterial load. Neutrophils induced to form neutrophil extracellular traps (NETs) with phorbol myristate acetate released high concentrations of PZP in vitro, and fluorescence microscopy confirmed the presence of PZP in NETs, whereas fluorescence and electron microscopy localized PZP to the cytoplasm and nuclei of neutrophils. Effective antibiotic therapy reduced sputum PZP.Conclusions: PZP is released into NETs. We report a novel link between airway infection, NET formation, and disease severity in bronchiectasis during chronic airway inflammation.

Human acute myeloid leukemia blast-derived exosomes in patient-derived xenograft mice mediate immune suppression

Exosomes are virus-size membrane-bound vesicles of endocytic origin present in all body fluids. Plasma of AML patients is significantly enriched in exosomes, which carry a cargo of immunosuppressive molecules and deliver them to recipient immune cells, suppressing their functions. However, whether these exosomes originate from leukemic blasts or from various normal cells in the bone marrow or other tissues is unknown. In the current study, we developed an AML PDX model in mice and studied the molecular cargo and immune cell effects of the AML PDX exosomes in parallel with the exosomes from plasma of the corresponding AML patients. Fully engrafted AML PDX mice produced exosomes with characteristics similar to those of exosomes isolated from plasma of the AML patients who had donated the cells for engraftment. The engrafted leukemic cells produced exosomes that carried human proteins and leukemia-associated antigens, confirming the human origin of these exosomes. Furthermore, the AML-derived exosomes carried immunosuppressive proteins responsible for immune cell dysfunctions. Our studies of exosomes in AML PDX mice serve as a proof of concept that AML blasts are the source of immunosuppressive exosomes with a molecular profile that mimics the content and functions of the parental cells.

Abstract 2370: Synergistic effect of carboplatin on farletuzumab (FAR) tumor cell killing via downregulation of the humoral immunosuppressive CA125 protein and enhanced antibody dependent cellular cytotoxicity (ADCC)

Abstract Farletuzumab (FAR) is a humanized monoclonal antibody (mAb) that targets folate receptor alpha (FRA), a cell surface protein highly overexpressed on a large number of cancers. Clinical evidence suggests that FAR enhances the anti-tumor effects of carboplatin and taxane in ovarian cancer patients whose tumors express low CA125 levels. In light of the recent evidence that CA125 has a direct effect on suppressing the humoral immune mechanisms [antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC)] of FAR (1, 2) and the findings that chemotherapeutic agents can enhance the ADCC activity of certain mAbs, we tested the potential synergistic effects that carboplatin may have on FAR immune effector function. Here we show the immunosuppressive effects that CA125 has on FAR-mediated tumor cell killing in vivo and demonstrate that carboplatin is able to suppress cell surface CA125 expression leading to enhanced FAR ADCC on FRA-expressing ovarian tumor cells. CA125 has been previously found to suppress FAR-mediated tumor cell killing in vitro. To determine its effect in vivo, we tested single agent FAR against isogenic tumor cells in mouse xenografts. OVCAR-3 is a FRA positive ovarian tumor cell line that expresses high levels of CA125 on its cell surface. A CA125 shRNA knockdown line (OV-KD) has been previously generated and shown to be more susceptible to FAR ADCC than its parental counterpart. Xenograft studies using these lines found that FAR had significant single agent tumor killing in the OV-KD xenografts in contrast to OVCAR-3 xenografts. We next tested FAR in vivo killing when used in combination with carboplatin. These data showed that carboplatin had a synergistic effect when combined with FAR treatment as compared to single agent treatment alone. To determine if this mechanism involved enhanced ADCC, we conducted similar studies in vitro whereby OVCAR-3 cells were treated with a sublethal concentration of carboplatin for 72 hrs, washed and then tested for FAR ADCC killing. These data found that FAR had enhanced ADCC activity on carboplatin treated cells as compared to untreated cells. Finally, to explore the potential mechanism by which this effect may occur, we examined the steady-state levels of both the mannose-6-phosphate receptor (M6PR), reported to be upregulated by chemotherapeutic drugs leading to enhanced ADCC via higher granzyme B uptake and the CA125 immunosuppressive factor. Our data found that both M6PR and CA125 expression were significantly suppressed following carboplatin exposure. These data support a mechanism by which carboplatin may enhance the humoral immune response for FAR and potentially other anti-tumor mAbs via downregulation of the immunosuppressive CA125 protein. 1. OncoTarget 8:66747-57, 20172. 2. EJI 201847707, 2018 Citation Format: J. Bradford Kline, Keiji Furuuchi, James Fulmer, Jennifer McDonough, Luigi Grasso, Nicholas C. Nicolaides. Synergistic effect of carboplatin on farletuzumab (FAR) tumor cell killing via downregulation of the humoral immunosuppressive CA125 protein and enhanced antibody dependent cellular cytotoxicity (ADCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2370.

Abstract 1174: Tumor-specific EphA2 receptor tyrosine kinase inhibits anti-tumor immunity by recruiting suppressive myeloid populations in non-small cell lung cancer

Abstract Background: Given the success of both targeted and immunotherapies against cancer, there is increasing utility for identifying targeted agents that also promote anti-tumor immunity. EphA2 is a receptor tyrosine kinase that contributes to tumor growth and metastasis in various cancer types and has been identified as a viable target for non-small cell lung cancer (NSCLC). Here, we examine how tumor-specific EphA2 affects the infiltration of different immune populations, the activation or inhibition of those populations, and the cytokine and chemokine milieu in mouse models of NSCLC. Methods: We generated two murine NSCLC cell lines, one derived from Lewis Lung Carcinoma and the other derived from a primary lung tumor harboring a KRAS mutation, as well as p53 and LKB1 loss of function alterations created using CRISPR-Cas9 gene editing. Effects of EphA2 overexpression in these cells were evaluated in both in vitro assays and in vivo models via subcutaneous tumor implantation and tail vein-injected orthotopic tumor formation in immunocompetent, syngeneic mice. Tumor immune infiltrate was assessed by flow cytometry, and cytokine and chemokine RNA expression levels were evaluated using Nanostring’s PanCancer Immune Profiling Panel. Results: Although EphA2 overexpression in these cell lines did not display significant proliferative advantage in vitro in MTT and colony formation assays, it did confer a growth advantage in vivo. Analysis of lung tumor immune infiltrate revealed decreased NK and T cells in the EphA2-overexpressing tumors, as well as increased myeloid populations, such as macrophages and monocytes. Furthermore, T cell activation markers, such as CD25, CD44, and CD69, in both CD4 and CD8 T cells were decreased in the EphA2-overexpressing tumors, while the percentage of regulatory T cells was increased. These changes in T cell activity were accompanied by increased presence of monocyte-derived CD11b+ Ly6C+ Ly6G- cells and Gr1- F4/80+ MHCII+ tumor-associated macrophages (TAMs). Higher infiltration of these myeloid populations coincided with increased monocyte-attracting chemokine and receptor expression, including CCL2, CCL7, CCL8, CCL12, and CCR2 and increased expression of immunosuppressive proteins, including TGFβ and arginase 1. Conclusion: Our studies suggest tumor-specific EphA2 inhibits infiltration and activation of key lymphocytic populations, especially T cells, while recruiting monocytes and likely promoting their transformation into myeloid-derived suppressor cells (MDSCs) and TAMs. Further functional studies are needed to validate the role of these myeloid cells. Elucidation of EphA2’s role in regulating immune recruitment and function will advance our understanding of tumor immune evasion and the potentially beneficial consequences of targeting EphA2 in cancer on the tumor microenvironment. Citation Format: Eileen Shiuan, Wenqiang Song, Shan Wang, Mark Boothby, Jin Chen. Tumor-specific EphA2 receptor tyrosine kinase inhibits anti-tumor immunity by recruiting suppressive myeloid populations in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1174.

Therapy Aimed to Suppress the Production of the Immunosuppressive Protein Progesterone Induced Blocking Factor (PIBF) May Provide Palliation and/or Increased Longevity for Patients With a Variety of Different Advanced Cancers - A Review.

Progesterone induced blocking factor (PIBF) is a unique protein that is not present in normal cells, but is found predominantly in rapidly growing cells of the fetal placental unit or cancer cells. There is a larger "parent" form that is a nuclear protein involved in cell to cell regulation, allowing tumor cells to proliferate and invade tissues. The parent compound is cleaved into smaller intracytoplasmic isoforms that can suppress cellular immune response, especially, but not limited to natural killer cells. The progesterone receptor antagonist mifepristone can suppress messenger RNA for PIBF, but can also suppress the intracytoplasmic protein. Treating cancer cell lines, intact animals with a variety of spontaneous cancers, and people with various cancers with mifepristone, has been found to inhibit cancer growth, and provide both palliation of symptoms and longevity possibly by suppressing this unique immunomodulatory protein.

Effect of Competitive Training on Antiviral Immune Activity in Collegiate Gymnasts

Regular moderate exercise has numerous health benefits, including positive effects on immune function, although periods of intense training may increase susceptibility to respiratory tract pathogens such as influenza virus. PURPOSE: To determine the effect of intense athletic training on the antiviral immune response profile and to examine the effect of ingestion of eggs on this response. METHODS: Female participants from a Division I varsity gymnastics team were recruited and either consumed whole eggs daily (E; n = 7) or maintained their normal diets (NE; n = 6) for the duration of the study. Blood was collected at three time points: before the high intensity pre-season training began (T1), one month into pre-season (T2), and two months after T2 at the end of pre-season (T3). Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood, cultured ex vivo, and challenged with influenza A/PR/8/34 for 24 hours. Cell supernatants were analyzed using a multiplex cytokine array assay. RESULTS: Statistical analyses revealed lower levels of cytokines involved in both innate (IL-1β, IL-6, TNFα) and T cell-mediated immunity (IFNγ, IL-2, IL-7) produced in response to virus stimulation at T2 (6.8, 87.3, 351, 731, 82.0, and 3.2 pg/mL, respectively) compared to T1 (12.1, 317, 534, 2469, 311, and 5.5 pg/mL, respectively) and T3 (17.4, 268, 712, 3018, 212, and 5.1 pg/mL, respectively; p < 0.05, paired t-tests). PBMCs from gymnasts who became ill during the study period (n = 3) produced less IFNα (427 vs. 1442 pg/mL) at T2 and more sCD40L (11.6 vs. 5.8 pg/mL) at T3 than those who remained healthy (p < 0.05, independent t-tests). When egg consumption was examined, a mixed ANOVA with repeated measures revealed a significant treatment by time interaction and follow-up post hoc tests identified differences in inflammatory cytokine production at T3 (12.3 vs. 23.3 and 496 vs. 963 pg/mL for IL-1β and IL-6, respectively, in E vs. NE; p < 0.05). CONCLUSION: These preliminary data suggest that, during intense training, both innate and adaptive antiviral defenses are impaired but that egg consumption may attenuate training-associated inflammation. Increases in immunosuppressive proteins, such as sCD40L, may also increase susceptibility to illness in athletes. Funding was provided by the Iowa Egg Council and Egg Nutrition Center.

The CD14+HLA-DRlo/neg Monocyte: An Immunosuppressive Phenotype That Restrains Responses to Cancer Immunotherapy.

Recent successes in cancer immunotherapy have been tempered by sub-optimal clinical responses in the majority of patients. The impaired anti-tumor immune responses observed in these patients are likely a consequence of immune system dysfunction contributed to by a variety of factors that include, but are not limited to, diminished antigen presentation/detection, leukopenia, a coordinated network of immunosuppressive cell surface proteins, cytokines and cellular mediators. Monocytes that have diminished or no HLA-DR expression, called CD14+HLA-DRlo/neg monocytes, have emerged as important mediators of tumor-induced immunosuppression. These cells have been grouped into a larger class of suppressive cells called myeloid derived suppressor cells (MDSCs) and are commonly referred to as monocytic myeloid derived suppressor cells. CD14+HLA-DRlo/neg monocytes were first characterized in patients with sepsis and were shown to regulate the transition from the inflammatory state to immune suppression, ultimately leading to immune paralysis. These immunosuppressive monocytes have also recently been shown to negatively affect responses to PD-1 and CTLA-4 checkpoint inhibition, CAR-T cell therapy, cancer vaccines, and hematopoietic stem cell transplantation. Ultimately, the goal is to understand the role of these cells in the context of immunosuppression not only to facilitate the development of targeted therapies to circumvent their effects, but also to potentially use them as a biomarker for understanding disparate responses to immunotherapeutic regimens. Practical aspects to be explored for development of CD14+HLA-DRlo/neg monocyte detection in patients are the standardization of flow cytometric gating methods to assess HLA-DR expression, an appropriate quantitation method, test sample type, and processing guidances. Once detection methods are established that yield consistently reproducible results, then further progress can be made toward understanding the role of CD14+HLA-DRlo/neg monocytes in the immunosuppressive state.

PD-1 checkpoint therapy promotes pro-survival phenotype in regulatory T cells in a model of claudin-low breast cancer

Abstract Results We have previously shown in our claudin-low genetically engeneired mouse model (GEMM) that Tregs are increased in the tumor microenvironment (TME), and express high levels of PD-1. We hypothesized that one cause for the lack of activity of α-PD-1 therapy in GEMMs and patients with claudin-low tumors was the activation of PD-1 expressing Tregs in the TME. To evaluate this hypothesis, Tregs were isolated from claudin-low tumors and functionally evaluated ex vivo. In vitro, PD-1 blockade decreased the suppressive activity of Tregs at high Treg:Tnaive cell ratios, but no difference was found in suppression at physiologically relevant ratios. We compared the transcriptional profiles of Tregs isolated from tumor bearing mice with or without α-PD-1 therapy using RNA-Seq. There were 27 signifcantly differentially regulated genes in Tregs from mice treated with α-PD-1 compared to untreated. These genes were associated with survival pathways using IPA with JUN, FOS (P < 0.001) and BCL2 (P = 0.02) upregulated in Tregs exposed to α-PD-1 treatment. Based on these data, we hypothesized that α-PD-1 treatment on Tregs results in a pro-survival phenotype. We found in vitro that Tregs in the presence of α-PD-1 proliferated significantly more than untreated Tregs (P < 0.0001). Importantly, PD-1 blockade did not significantly alter the expression of immunosuppressive proteins such as CTLA-4, CD25, TGFβ, or Il-10 as measured by flow cytometry. Conclusions We found that PD-1 blockade increases proliferation of Tregs in the T11 model, while not significantly altering suppressive capabilities thus leading to enhanced immunosuppression in the TME. Activation of Tregs by α-PD-1 therapy may be responsible for hyperacute tumor flares post therapy.

Selective HDAC6 inhibitors improve anti-PD-1 immune checkpoint blockade therapy by decreasing the anti-inflammatory phenotype of macrophages and down-regulation of immunosuppressive proteins in tumor cells

Histone deacetylases (HDACs) are involved in diverse cellular regulatory mechanisms including non-canonical functions outside the chromatin environment. Several publications have demonstrated that selective HDAC inhibitors (HDACi) can influence tumor immunogenicity and the functional activity of specific immune cells. In particular, the selective inhibition of HDAC6 has been reported to decrease tumor growth in several malignancies. However, there is still no clarity about the cellular components mediating this effect. In this study, we evaluated the HDAC6i Nexturastat A as a priming agent to facilitate the transition of the tumor microenvironment from “cold” to “hot”, and potentially augment immune check-point blockade therapies. This combination modality demonstrated to significantly reduce tumor growth in syngeneic melanoma tumor models. Additionally, we observed a complete neutralization of the up-regulation of PD-L1 and other immunosuppressive pathways induced by the treatment with anti-PD-1 blockade. This combination also showed profound changes in the tumor microenvironment such as enhanced infiltration of immune cells, increased central and effector T cell memory, and a significant reduction of pro-tumorigenic M2 macrophages. The evaluation of individual components of the tumor microenvironment suggested that the in vivo anti-tumor activity of HDAC6i is mediated by its effect on tumor cells and tumor-associated macrophages, and not directly over T cells. Overall, our results indicate that selective HDAC6i could be used as immunological priming agents to sensitize immunologically “cold” tumors and subsequently improve ongoing immune check-point blockade therapies.

Immunosuppressive effects of tick protein RHcyst-1 on murine bone marrow-derived dendritic cells

BackgroundTicks, as blood-feeding arthropod vectors, have evolved their own unique mechanism to suppress host immune responses and evade immune defenses in order to complete blood-feeding. The immunoregulatory effect of tick bioactive molecules on hosts has been widely reported, and the cystatin family has been identified as one of the major immunomodulators. In previous studies, we obtained a novel tick salivary bioactive protein named RHcyst-1, which belongs to the type 1 cystatin family. Here, we demonstrated the effects of RHcyst-1 on the host immune response mainly on dendritic cell (DC) function. Understanding the function of tick-derived bioactive molecule may help to clarify the mechanisms of how ticks escape the host immune response and help to control ticks and tick-borne disease transmission.MethodsBone marrow-derived DCs (BMDCs) were generated and induced by GM-CSF and IL-4 with or without RHcyst-1 addition. Flow cytometry was used to analyze the differentiation and maturation of BMDCs and T cell cytokine production. Quantitative real-time PCR (qRT-PCR) and western blot were used to measure changes in expression within STAT and p38 MAPK signaling pathways.ResultsFlow cytometry analysis revealed that RHcyst-1 inhibited the differentiation of BMDCs, but had no effect on the maturation of BMDCs. T cells co-cultured with DCs treated with RHcyst-1 produced significantly less TNF-α, IFN-γ and IL-2 than the control group. Further analysis showed that the mRNA level and phosphorylation of p38, ERK and STAT were significantly changed after RHcyst-1 added to bone marrow monocytes during the differentiation stage.ConclusionsOur results suggest that RHcyst-1 is one of the major immunosuppressive proteins of BMDC function from blood-feeding ticks.

The Immune Response to Influenza is Suppressed by the Synthetic Food Additive and Nrf2 Activator, tert‐butylhydroquinone (tBHQ)

Influenza virus infections have been a persistent threat to society, causing up to 650,000 respiratory deaths annually world‐wide. The United States alone suffered 80,000 influenza‐related deaths during the 2017/18 flu season. Although vaccine coverage has increased in the U.S., this has not correlated with a decline in influenza‐related infections or deaths. Accordingly, there is significant interest in the identification of environmental factors which may contribute to influenza burden and/or reduce vaccine efficacy. Previous work from our laboratory demonstrated that a widely‐used food additive, ‐tert‐butylhydroquinone (tBHQ), impairs human CD4+ T cell activation and murine TH1 cell polarization (important in anti‐viral defense) ex vivo. These effects occurred through activation of the stress‐activated transcription factor, nuclear factor erythroid 2‐related factor 2 (Nrf2), as evidenced using Nrf2‐deficient human and murine models. Under basal conditions, Nrf2 is repressed in the cytosol by its repressor protein, Kelch‐like ECH‐associated protein 1 (Keap1), where it is quickly degraded via the 26S proteasome pathway. Upon the introduction of cell stress, such as the electrophilic stress induced by tBHQ, the interactions between Nrf2 and Keap1 are disrupted such that Nrf2 is no longer degraded, and Nrf2 accumulates in the nucleus where it upregulates cytoprotective genes. Studies from our lab and others have demonstrated immunomodulatory roles for Nrf2, including effects on macrophages, dendritic cells, and T cells. The tBHQ‐mediated decrease in Th1 polarization led us to hypothesize that consumption of the Nrf2 activator, tBHQ, would impair the T cell‐mediated immune response to acute influenza infection. To test this hypothesis, mice on either a low‐dose tBHQ or control diet were infected with influenza A/PR/8/34 (H1N1). tBHQ exposure impaired CD8+ T cell infiltration in the lung, CD4+/CD8+ T cell activation and effector function, the numbers of influenza‐specific T cells in the draining lymph nodes, and viral clearance. Interestingly, although we did not observe a decrease in the number of TH1 cells with tBHQ exposure at this timepoint (10 days post‐infection), there was an increase in the expression of the immunosuppressive proteins, CTLA‐4 and IL‐10. Of importance, the impaired immune response during primary infection correlated with more severe weight loss and prolonged infection during subsequent infection with a related strain of influenza (x31), indicating that tBHQ impaired the heterosubtypic memory response. Notably, an impaired heterosubtypic memory response would be expected to reduce vaccine efficacy. Overall, this study shows that tBHQ, at a dose relevant to human diet, impairs the primary and memory immune responses to influenza infection.Support or Funding InformationThis work was supported by NIH grants ES024966 and GM092715.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Delphinidin Chloride and Its Hydrolytic Metabolite Gallic Acid Promote Differentiation of Regulatory T cells and Have an Anti-inflammatory Effect on the Allograft Model.

Regulatory T cells (Tregs) control the reactivity of other T cells to prevent excessive inflammatory responses. They also plays a role in preventing autoimmune diseases; but when they are overproduced, they decreased vital immunity, which can lead to invasion of external pathogens. Therefore, it is most important in preventing the development of immune diseases to maintain the homeostasis of these cells. Delphinidin chloride is an anthocyanidin and known to have anti-oxidant activities. However, its structure is very unstable and easily decomposed. One of these degradation products is gallic acid, which also has anti-oxidant effects. In this study, we examined the effect of these materials on Tregs in controlling immune response. It was found that these materials further promote differentiation into Tregs, and TGF-β and IL-2 related signals are involved in this process. Furthermore, it was verified that a variety of immunosuppressive proteins were secreted more, and the function of induced Tregs was also increased. Finally, in the allograft model, we could find a decrease in activated T cells when these materials were treated because they increased differentiation into Tregs. Therefore, these two materials are expected to become new candidates for the treatment of diseases caused by excessive activation of immune cells, such as autoimmune diseases. PRACTICAL APPLICATION: Delphinidin, a kind of anthocyanin rich in pigmented fruits, and its hydrolytic metabolite, gallic acid, are known to have antimicrobial and anti-oxidant properties. In this experiment, it was shown that delphinidin and gallic acid had an effect of increasing the differentiation of regulatory T cells, and the effect of suppressing the function of memory T cells was also observed. Due to these functions, delphinidin and gallic acid might have the potential to be used as immune suppressive agents in organ transplant and autoimmune disease patients or be a model for food development associated with the immune system.

Hypoxia-inducible factor-1α and nuclear factor-κB play important roles in regulating programmed cell death ligand 1 expression by epidermal growth factor receptor mutants in non-small-cell lung cancer cells.

Some driver gene mutations, including epidermal growth factor receptor (EGFR), have been reported to be involved in expression regulation of the immunosuppressive checkpoint protein programmed cell death ligand 1 (PD‐L1), but the underlying mechanism remains obscure. We investigated the potential role and precise mechanism of EGFR mutants in PD‐L1 expression regulation in non‐small‐cell lung cancer (NSCLC) cells. Examination of pivotal EGFR signaling effectors in 8 NSCLC cell lines indicated apparent associations between PD‐L1 overexpression and phosphorylation of AKT and ERK, especially with increased protein levels of phospho‐IκBα (p‐IκBα) and hypoxia‐inducible factor‐1α (HIF‐1α). Flow cytometry results showed stronger membrane co‐expression of EGFR and PD‐L1 in NSCLC cells with EGFR mutants compared with cells carrying WT EGFR. Additionally, ectopic expression or depletion of EGFR mutants and treatment with EGFR pathway inhibitors targeting MEK/ERK, PI3K/AKT, mTOR/S6, IκBα, and HIF‐1α indicated strong accordance among protein levels of PD‐L1, p‐IκBα, and HIF‐1α in NSCLC cells. Further treatment with pathway inhibitors significantly inhibited xenograft tumor growth and p‐IκBα, HIF‐1α, and PD‐L1 expression of NSCLC cells carrying EGFR mutant in nude mice. Moreover, immunohistochemical analysis revealed obviously increased protein levels of p‐IκBα, HIF‐1α, and PD‐L1 in NSCLC tissues with EGFR mutants compared with tissues carrying WT EGFR. Non‐small‐cell lung cancer tissues with either p‐IκBα or HIF‐1α positive staining were more likely to possess elevated PD‐L1 expression compared with tissues scored negative for both p‐IκBα and HIF‐1α. Our findings showed important roles of phosphorylation activation of AKT and ERK and potential interplay and cooperation between NF‐κB and HIF‐1α in PD‐L1 expression regulation by EGFR mutants in NSCLC.

Isolation and characterization of exosomes from IDH mutant gliomas.

152 Background: Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are found in > 70% of intermediate grade gliomas. Gain-of-function mutations in IDH1 promote diffuse glioma formation through epigenetic reprogramming of a number of genes, including immune-related genes. Tumor-derived exosomes (TEX) accumulate in the tumor microenvironment (TME) and in body fluids of patients. TEX serve as a communication system between tumor and normal cells. Circulating TEX carry a complex molecular and genetic cargo and interfere with functions of immune cells. This study characterizes TEX produced by IDH mutant glioma spheres and TEX from plasma of patients with IDH mutant gliomas. Methods: TEX produced by patient-derived IDH1(mut) or IDH1(wt) glioma spheres or TEX in patient’s plasma were isolated by mini size exclusion chromatography (mini-SEC). TEX morphology, size, numbers and molecular profile were characterized and the crossing of the blood-brain barrier (BBB) and biodistribution was investigated by in vivo imaging of TEX. Interactions of TEX with endothelial cells, astrocytes and immune cells were demonstrated by confocal microscopy. Results: TEX isolated from supernatants of IDH1(mut) or IDH1(wt) glioma spheres by mini-SEC carried TSG101 and showed the typical size and vesicular morphology of exosomes. BCA protein assays and qNano analysis revealed an elevated exosome production by IDH mutant cells ( p< 0.01). TEX carried immunosuppressive proteins (FasL, CTLA-4 and TRAIL) and IDH mutant exosomes carried higher levels of these proteins and of adenosine pathway components CD39 and CD73. Labeled TEX injected in the white matter of mice crossed the BBB and were detectable in distant organs 24h after injection. TEX were rapidly internalized by endothelial cells and astrocytes, but not by T cells, which interacted with TEX by surface signaling. These results were confirmed by investigating TEX isolated from glioma patient’s plasma. Conclusions: The data suggest that TEX play an important role in IDH mutant gliomas and drive tumor progression in part by inducing suppression of immune cells. Future efforts will focus on characterizing immunosuppressive effects of TEX derived from IDH mutant cells vs gliomas without IDH mutations.

Treatment Combining CD200 Immune Checkpoint Inhibitor and Tumor-Lysate Vaccination after Surgery for Pet Dogs with High-Grade Glioma.

Recent advances in immunotherapy have included inhibition of immune checkpoint proteins in the tumor microenvironment and tumor lysate-based vaccination strategies. We combined these approaches in pet dogs with high-grade glioma. Administration of a synthetic peptide targeting the immune checkpoint protein, CD200, enhanced the capacity of antigen-presenting cells to prime T-cells to mediate an anti-glioma response. We found that in canine spontaneous gliomas, local injection of a canine-specific, CD200-directed peptide before subcutaneous delivery of an autologous tumor lysate vaccine prolonged survival relative to a historical control treated with autologous tumor lysate alone (median survivals of 12.7 months and 6.36 months, respectively). Antigen-presenting cells and T-lymphocytes primed with this peptide suppressed their expression of the inhibitory CD200 receptor, thereby enhancing their ability to initiate immune reactions in a glioblastoma microenvironment replete with the immunosuppressive CD200 protein. These results support consideration of a CD200 ligand as a novel glioblastoma immunotherapeutic agent.

Glycodelin as a Serum and Tissue Biomarker for Metastatic and Advanced NSCLC.

A major part of non-small cell lung cancer (NSCLC) patients treated with mono- or multimodal concept develop therapy resistance. Despite the abundance of biomarkers investigated in the past, there is still a need for valid NSCLC biomarkers. Glycodelin, an immunosuppressive endometrial protein, has been shown to be also expressed in NSCLC. Here, we investigated its potential as a biomarker in metastatic and advanced stage NSCLC. Glycodelin gene and protein expression were measured in 28 therapy-naïve resected tumors as well as in corresponding brain (n = 16) and adrenal gland (n = 12) metastasis by qPCR and IHC. Moreover, we correlated glycodelin gene expression of cryoconserved therapy-naïve biopsies (n = 55) of advanced stage patients with glycodelin serum concentrations and patient survival. Using follow-up samples of the patients, we monitored glycodelin serum concentrations during therapy. Glycodelin expression correlated between primary tumor and distant metastases within the same patients. The gene expression of glycodelin in therapy-naïve biopsies also correlated with the serum concentrations of the patients (r = 0.60). Patients with elevated serum concentrations showed a tendency in lower overall survival (p = 0.088) and measuring of glycodelin indicated a progression of the disease earlier compared to clinical diagnostic. Taken together, we demonstrate that glycodelin is a promising prognostic and follow-up biomarker for metastatic and advanced NSCLC.

Bion-1301 Blocks APRIL-Induced Anti-Apoptotic Signaling, Immune Suppressive Phenotype, and Chemokine Production Associated with Multiple Myeloma

A proliferation-inducing ligand (APRIL) is produced by multiple accessory and myeloid cells in the bone marrow niche. Through binding to its receptors B-cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI), APRIL plays an important role in the development and maintenance of cells derived from the B cell lineage. Both APRIL and its receptors have been identified as promotors of multiple myeloma (MM), a disease that is characterized by malignant proliferation of plasma cells in the bone marrow. We have shown that BION-1301, a first-in-class APRIL-targeting humanized antibody, blocks APRIL-induced effects on MM cell survival and the tumor microenvironment (TME).Despite recent approval of new therapies for MM, this type of cancer remains incurable as patients relapse and eventually become refractory to the current therapeutic regimens. We hypothesize that the presence of high levels of APRIL in the bone marrow of patients with MM establishes a protected environment favoring tumor cell survival and proliferation. The effects of APRIL were studied on tumor cell lines alone or in co-culture with osteoclasts, which are thought to produce APRIL in the bone marrow niche. We further hypothesize that engaging APRIL with BION-1301 sensitizes MM cells for other MM-targeting therapeutic agents.First, stimulation of MM cells with APRIL led to rapid phosphorylation of proteins in the NFκB pathway, that was completely blocked by the BION-1301 parental antibody hAPRIL.01A. In addition, by validating expression of known NFκB target genes, APRIL appears to protect MM cells from drug-induced toxicity by upregulation of anti-apoptotic genes (e.g. MCL-1), since APRIL enhanced survival of MM cells that were treated with anti-MM drugs, such as dexamethasone. Importantly, addition of hAPRIL.01A blocked this pro-survival effect, highlighting the potential of BION-1301 to enhance MM sensitivity towards MM-targeting agents.We confirmed the immune suppressive potential of APRIL by showing that APRIL upregulates known immunosuppressive proteins such as IL-10 and PD-L1, indicating that BION-1301 could increase sensitivity to therapies such as daratumumab that depend on the immune system.Finally, in vitro APRIL increased secretion of the chemokines MIP-1α and MIP-1β by MM cell lines, which are known to stimulate osteoclast differentiation and activation. This suggests a positive feedback loop in which APRIL, produced mainly by osteoclasts in the bone marrow, induces chemokine production by MM cells, and these chemokines in turn stimulate osteoclasts. Importantly, when hAPRIL.01A is added to block APRIL, chemokine production drops, indicating that in addition to sensitizing MM tumor cells for drug treatment, BION-1301 also suppresses osteoclast activity.Altogether, we demonstrate in vitro that APRIL induces anti-apoptotic signaling, triggers expression of an immune suppressive phenotype, and could potentially modulate the tumor microenvironment through the production of key chemokines. Blocking APRIL with BION-1301 may dampen these effects, illustrating the potential of BION-1301, as a therapeutic agent for MM, particularly in combination therapies. BION-1301 is currently in a phase 1 clinical trial for MM. DisclosuresBakker:Aduro Biotech: Employment. Habraken:Aduro Biotech: Employment. Dulos:Aduro Biotech: Employment. Van Elsas:Aduro Biotech Europe: Employment. Vink:Aduro Biotech Europe: Employment. Eenennaam:Aduro Biotech Europe: Employment, Equity Ownership.

Influence of cryptosporidiosis agent on biochemical and immunological parameters of blood of calves

Cows, young cattle and calves of black-and-bark breeds, spontaneously affected by the causative agent Cryptosporidium parvum, were selected for the research. Blood for research was taken from calves in the morning before feeding from the jugular vein. Biochemical parameters of blood serum were determined using a biochemical analyzer of the closed type. Changes in the activity of serum enzymes in the initial stages of the disease are not specific, they indicate a compensatory reaction of liver cells and biliary tract. Effect of Cryptosporidium in calves body accompanied by severe disorders that affect the metabolism of proteins, carbohydrates and enzymes. These changes lead to structural changes in the organs at the cellular level. Serum infested animals significantly reduced total protein content of 25.5% (P < 0.001), albumin – by 14.6% (P < 0.01), the concentration of glucose in – 21.2% (P < 0.01), carotene content – 12.6% (P < 0.05), the level of calcium and phosphorus – 17.5 and 18% (P < 0.05), respectively, and increased total bilirubin content by 23% (P < 0.001), indicating significant violations of the protein synthesis and detoxification processes in the liver and the tension in the metabolism of their body. Reduced glucose concentration in blood serum is due to the fact that in the body of sick animals there have been increased costs for maintaining the energy needs of its own organism. In conducting studies for 35 days in animals in the experimental group noted an increase in the concentration of circulating immune complexes by 10.5% relative to control. Increasing the concentration of circulating immune complexes in serum of calves, patients with cryptosporidiosis, indicates the presence of specific interaction antigen-antibody and decrease the activity of the humoral link of the immune system. In the determination of immunosuppressive proteins – serum cords in serum of calves, it was found that at 7, 14 days their concentration was within the physiological limits. Increasing the concentration of circulating immune complexes in serum of calves, patients with cryptosporidiosis, indicates the presence of specific interaction antigen-antibody and decrease the activity of the humoral link of the immune system.