Activity Of Interferon-gamma Research Articles

GAS-Luc2 Reporter Cell Lines for Immune Checkpoint Drug Screening in Solid Tumors.

Recent studies highlight the integral role of the interferon gamma receptor (IFNγR) pathway in T cell-mediated cytotoxicity against solid but not liquid tumors. IFNγ not only directly facilitates tumor cell death by T cells but also indirectly promotes cytotoxicity via myeloid phagocytosis in the tumor microenvironment. Meanwhile, full human ex vivo immune checkpoint drug screening remains challenging. We hypothesized that an engineered gamma interferon activation site response element luciferase reporter (GAS-Luc2) can be utilized for immune checkpoint drug screening in diverse ex vivo T cell-solid tumor cell co-culture systems. We comprehensively profiled cell surface proteins in ATCC's extensive collection of human tumor and immune cell lines, identifying those with endogenously high expression of established and novel immune checkpoint molecules and binding ligands. We then engineered three GAS-Luc2 reporter tumor cell lines expressing immune checkpoints PD-L1, CD155, or B7-H3/CD276. Luciferase expression was suppressed upon relevant immune checkpoint-ligand engagement. In the presence of an immune checkpoint inhibitor, T cells released IFNγ, activating the JAK-STAT pathway in GAS-Luc2 cells, and generating a quantifiable bioluminescent signal for inhibitor evaluation. These reporter lines also detected paracrine IFNγ signaling for immune checkpoint-targeted ADCC drug screening. Further development into an artificial antigen-presenting cell line (aAPC) significantly enhanced T cell signaling for superior performance in these ex vivo immune checkpoint drug screening platforms.

Early application of IFNγ mediated the persistence of HBV in an HBV mouse model

The antiviral activity of interferon gamma (IFNγ) against hepatitis B virus (HBV) was demonstrated both in vivo and in vitro in a previous study. IFNγ can suppress HBV replication by accelerating the decay of replication-competent nucleocapsids of HBV. However, in this study, we found that the direct application of the mouse IFNγ (mIFNγ) expression plasmid to the liver of an HBV hydrodynamic injection (HI) mouse model led to the persistence of HBV, as indicated by sustained HBsAg and HBeAg levels in the serum as well as an increased percentage of the HBsAg positive mice, whereas the level of HBV DNA in the serum and the expression of HBcAg in the liver were inhibited at the early stage after HI. Meanwhile, we found that the productions of both HBcAb and HBsAb were suppressed after the application of mIFNγ. In addition, we found that HBV could be effectively inhibited in mice immunized with HBsAg expression plasmid before the application of mIFNγ. Furthermore, mIFNγ showed antiviral effect and promoted the production of HBsAb when the mice subjected to the core-null HBV plasmid. These results indicate that the application of mIFNγ in the HBV HI mouse model, the mice showed defective HBcAg-specific immunity that impeded the production of HBcAb and HBsAb, finally allowing the persistence of the virus. Moreover, IFNγ-induced negative immune regulatory factors also play an important role in virus persistence.

Abstract 5100: Multimodal real world data reveals immunogenomic drivers of acquired and primary resistance to immune checkpoint blockade

Abstract Background: Why some patients fail or have short lived response to immune checkpoint blockade (ICB) immunotherapy remains largely unknown. While baseline molecular assessments have provided clues to prognostic factors, insights into resistance drivers remains elusive. This is partially due to the difficulty in getting access to post progression samples from patients that were either primary resistant or developed acquired resistance after an initial response to ICB. Thus, the tumor-intrinsic and -extrinsic features that are selected for during progression and potentially drive primary and acquired resistance to immunotherapy remain underexplored. Methods: To compare clinical features and immunogenomic drivers of acquired and primary resistance to ICB across major cancers, we analysed and annotated de-identified patient records in the Tempus real-world database. We built an immuno-oncology cohort consisting of >2500 multimodal (DNA, RNA and clinical outcome data) pre-treatment baseline with >1500 post-treatment tumour biopsy samples from mainly NSCLC, TNBC, HNC and Bladder cancer patients. We used bulk RNA-seq data to estimate activation of the hallmark oncogenic pathways and immune cell composition and used panel DNA-seq data (>500 genes) to quantify mutation selection at the gene and pathway levels using dndscv. Results: Compared to acquired, primary resistant patients tended to have a higher observation of liver lesions at progression. Post-ICB, acquired resistant NSCLC and HNC patients showed a significantly inflamed tumour microenvironment (TME) characterised by higher estimation of infiltration of T cells and myeloid cells and higher activation of interferon gamma (IFNg) signalling as compared to primary resistant patients. In addition, in post-ICB acquired resistance in NSCLC we observed selection for mutations in genes involved in known immunomodulatory pathways, including loss-of-function mutations in B2M. Consistently, acquired resistance patients showed stronger selection for mutations in Hedgehog and Notch pathways as compared to primary resistance patients across NSCLC, HNC and TNBC post-ICB. Conclusions: Acquired and primary ICB resistant patients have distinct clinical and molecular features at progression. Their tumours’ TME is fundamentally different with acquired resistance TMEs being infiltrated with immune cells albeit escaped post progression. In addition, ICB selects mutations that potentially activate pathways such as Notch and Hedgehog. This multi-modal Real-World Data with post therapy biopsies has given insights for patient selection strategies and provides rational into combination treatment options for acquired resistant patients. Citation Format: Mohamed Reda Keddar, Sebastian Carrasco Pro, Kathleen Burke, Ana Camelo Stewart, Mark Cobbold, Ross Stewart, Sajan Khosla, Ben Sidders, Scott Hammond, Douglas C. Palmer, Jonathan Dry, Martin L. Miller. Multimodal real world data reveals immunogenomic drivers of acquired and primary resistance to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5100.

Abstract 75: Luciferase reporter cell lines allow simultaneous incorporation of tumor cells, innate immune cells, and adaptive immune cells for in-depth immune checkpoint studies

Abstract Despite the considerable success of immune checkpoint therapies targeting T cells, a sizable proportion of patients experience resistance or relapse due to the immunosuppressive nature of the tumor microenvironment. Myeloid cells, a major component that suppresses effector lymphocytes, have emerged as an alternative and promising therapeutic target. However, there is a deep lack of widely accessible immunological models capable of representing the intricate three-way interaction between tumor cells, T cells, and myeloid cells. To address this need, we conducted a comprehensive protein profiling of human tumor and immune cell lines available at ATCC for various established and novel immune checkpoint molecules. Cell lines with high endogenous expression of the immune checkpoint proteins, such as programmed death-ligand 1 and 2 (PD-L1 and PD-L2), cluster of differentiation 155 (CD155), B7 homolog 3 (B7-H3), sialic acid-binding Ig-like lectin 10 (Siglec-10), or signal-regulatory protein alpha (SIRPα), were selected and constructed into luciferase reporter cell lines. For tumor reporter cell lines, a gamma interferon activation site (GAS) response element was placed upstream of the luciferase gene in the lentiviral vector, enabling the activation of the JAK-STAT signaling pathway within tumor cells to induce luciferase expression. In myeloid reporter cell lines, a nuclear factor kappa B (NF-κB) response element replaced GAS to monitor the activation of the NF-κB signaling pathway. In the presence of corresponding immune checkpoint inhibitors that enhance T cell-mediated anti-tumor activity, these reporter cell lines produce a bioluminescent signal based on luciferase expression. This signal can be easily detected and quantified to assess the efficacy of the inhibitor. Our data revealed that bioluminescence intensity in the tumor and myeloid reporter cell lines increased by >100-fold in a dose-dependent manner in response to interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) stimulation, respectively, and by >50-fold in response to the conditioned media collected from activated primary T cells. Furthermore, in co-culture assays involving various combinations of immune cell and tumor cell types with corresponding immune checkpoint inhibitors, these reporter cell lines demonstrated a significant increase in bioluminescence intensity. In conclusion, these newly established luciferase reporter cell lines offer an excellent ex vivo model for cancer immunotherapy. These cell lines naturally express immune checkpoint proteins, enabling the sensitive and reproducible monitoring of combinatorial responses from various immune cell types. Citation Format: Hyeyoun Chang, Alicia C. Walker, John G. Foulke, Luping Chen, Fang Tian, Zhizhan Gu. Luciferase reporter cell lines allow simultaneous incorporation of tumor cells, innate immune cells, and adaptive immune cells for in-depth immune checkpoint studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 75.

AMPKα2 promotes tumor immune escape by inducing CD8+ T-cell exhaustion and CD4+ Treg cell formation in liver hepatocellular carcinoma

BackgroundAdenosine monophosphate-activated protein kinase (AMPK) is associated with the development of liver hepatocellular carcinoma (LIHC). AMPKα2, an α2 subunit of AMPK, is encoded by PRKAA2, and functions as the catalytic core of AMPK. However, the role of AMPKα2 in the LIHC tumor immune environment is unclear.MethodsRNA-seq data were obtained from the Cancer Genome Atlas and Genotype-Tissue Expression databases. Using the single-cell RNA-sequencing dataset for LIHC obtained from the China National Genebank Database, the communication between malignant cells and T cells in response to different PRKAA2 expression patterns was evaluated. In addition, the association between PRKAA2 expression and T-cell evolution during tumor progression was explored using Pseudotime analysis, and the role of PRKAA2 in metabolic reprogramming was explored using the R “scMetabolis” package. Functional experiments were performed in LIHC HepG2 cells.ResultsAMPK subunits were expressed in tissue-specific and substrate-specific patterns. PRKAA2 was highly expressed in LIHC tissues and was associated with poor patient prognosis. Tumors with high PRKAA2 expression displayed an immune cold phenotype. High PRKAA2 expression significantly promoted LIHC immune escape. This result is supported by the following evidence: 1) the inhibition of major histocompatibility complex class I (MHC-I) expression through the regulation of interferon-gamma activity in malignant cells; 2) the promotion of CD8+ T-cell exhaustion and the formation of CD4+ Treg cells in T cells; 3) altered interactions between malignant cells and T cells in the tumor immune environment; and 4) induction of metabolic reprogramming in malignant cells.ConclusionsOur study indicate that PRKAA2 may contribute to LIHC progression by promoting metabolic reprogramming and tumor immune escape through theoretical analysis, which offers a theoretical foundation for developing PRKAA2-based strategies for personalized LIHC treatment.

In Silico Analysis of Genes Associated with the Pathogenesis of Odontogenic Keratocyst.

Identify key genes in the pathogenesis of sporadic OK through in silico analysis. The GSE38494 technical sheet on OK was analyzed using GEOR2. Their functional and canonical signaling pathways were enriched in the NIH-DAVID bioinformatic platform. The protein-protein interaction network was constructed by STRING and analyzed with Cytoscape-MCODE software v 3.8.2 (score > 4). Post-enrichment analysis was performed by Cytoscape-ClueGO. A total of 768 differentially expressed genes (DEG) with a fold change (FC) greater than 2 and 469 DEG with an FC less than 2 were identified. In the post-enrichment analysis of upregulated genes, significance was observed in criteria related to the organization of the extracellular matrix, collagen fibers, and endodermal differentiation, while the downregulated genes were related to defensive response mechanisms against viruses and interferon-gamma activation. Our in silico analysis showed a significant relationship with mechanisms of extracellular matrix organization, interferon-gamma activation, and response to viral infections, which must be validated through molecular assays.

Cellular and transcriptional impacts of Janus kinase and/or IFN-gamma inhibition in a mouse model of primary hemophagocytic lymphohistiocytosis.

Primary hemophagocytic lymphohistiocytosis (pHLH) is an inherited inflammatory syndrome driven by the exuberant activation of interferon-gamma (IFNg)-producing CD8 T cells. Towards this end, ruxolitinib treatment or IFNg neutralization (aIFNg) lessens immunopathology in a model of pHLH in which perforin-deficient mice (Prf1-/-) are infected with Lymphocytic Choriomeningitis virus (LCMV). However, neither agent completely eradicates inflammation. Two studies combining ruxolitinib with aIFNg report conflicting results with one demonstrating improvement and the other worsening of disease manifestations. As these studies used differing doses of drugs and varying LCMV strains, it remained unclear whether combination therapy is safe and effective. We previously showed that a ruxolitinib dose of 90 mg/kg lessens inflammation in Prf1-/- mice infected with LCMV-Armstrong. To determine whether this dose controls inflammation induced by a different LCMV strain, we administered ruxolitinib at 90mg/kg to Prf1-/- mice infected with LCMV-WE. To elucidate the impacts of single agent versus combination therapy, Prf1-/- animals were infected with LCMV, treated or not with ruxolitinib, aIFNg or both agents, and analyzed for disease features and the transcriptional impacts of therapy within purified CD8 T cells. Ruxolitinib is well-tolerated and controls disease regardless of the viral strain used. aIFNg, administered alone or with ruxolitinib, is most effective at reversing anemia and reducing serum IFNg levels. In contrast, ruxolitinib appears better than aIFNg, and equally or more effective than combination therapy, at lessening immune cell expansion and cytokine production. Each treatment targets distinct gene expression pathways with aIFNg downregulating IFNg, IFNa, and IL-6-STAT3 pathways, and ruxolitinib downregulating IL-6-STAT3, glycolysis, and reactive oxygen species pathways. Unexpectedly, combination therapy is associated with upregulation of genes driving cell survival and proliferation. Ruxolitinib is tolerated and curtails inflammation regardless of the inciting viral strain and whether it is given alone or in combination with aIFNg. When administered at the doses used in this study, the combination of ruxolitinb and aIFNg appears no better than treatment with either drug alone in lessening inflammation. Further studies are warranted to elucidate the optimal doses, schedules, and combinations of these agents for the treatment of patients with pHLH.

Abstract 6643: Immune checkpoint reporter cell lines based on the protein profiling of ATCC cell lines for cancer immunotherapy drug screening

Abstract The success of immune checkpoint inhibitors in the treatment of various types of cancers and their continued growth in the market have driven burgeoning interests in developing more drugs in this category. However, the intrinsic complexity of the immunological models and the variable drug responses among different cancer types have become the most prominent challenges. To facilitate large scale research projects and drug discovery of immune checkpoint inhibitors, we conducted a comprehensive protein profiling of ATCC’s vast portfolio of human tumor and immune cell lines for several established and novel immune checkpoint molecules. Based on this protein profiling data, we generated immune checkpoint reporter cancer cell lines with high expression of endogenous immune checkpoint molecule ligands (PD-L1, CD155, and B7-H3). The reporter system contains a gamma interferon activation site (GAS)-response element upstream of the luciferase gene, preventing luciferase expression when the immune checkpoint molecule ligand binds to its corresponding receptor that suppresses T cell-mediated antitumor activity. In the presence of a relevant immune checkpoint inhibitor, a luciferase expression-based bioluminescent signal is produced, which can be readily detected and quantitated to evaluate the efficacy, potency, and dynamics of the inhibitor. Our data showed that the bioluminescence in the reporter cancer cells increased approximately 100-250 folds in a dose-dependent manner in response to interferon gamma stimulation, which mimics the signaling from activated CD8+ cytotoxic T cells. The bioluminescence increased approximately 50-100 folds in response to CD8+ primary T cell-conditioned media stimulation. In particular, we observed up to 5-fold increase in luciferase signal from our PD-L1 reporter cell line in response to co-culture with CD8+ primary T cells in the presence of an anti-PD-L1 blocking antibody in a dose-dependent manner. The luciferase expression and endogenous immune checkpoint molecule ligand expression were well maintained after the cell lines had reached >30 population doubling level. These results highlight the robustness and responsiveness of the reporter system for the assessment of T cell-mediated immune responses triggered by checkpoint inhibitors. These immune checkpoint reporter cancer cell lines yield exceptional in vitro and ex vivo assay sensitivity and reproducibility, while simplify the complex immunological model by providing physiologically relevant expression of immune checkpoint molecule ligands, in comparison to similar assays with an artificial checkpoint ligand overexpression system. Citation Format: Hyeyoun Chang, Kevin Tyo, John Foulke, Luping Chen, Zhizhan Gu, Fang Tian. Immune checkpoint reporter cell lines based on the protein profiling of ATCC cell lines for cancer immunotherapy drug screening. [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 6643.

<i>In vitro</i> activity of human recombinant interferon gamma against SARS-CoV-2 virus

The development of drugs against SARS-CoV-2 continues to be crucial for reducing the spread of infection and associated mortality. The aim of the work is to study the neutralization of the SARS-CoV-2 virus with interferon gamma preparations in vitro. The activity of recombinant human interferon gamma for intramuscular and subcutaneous administration of 500,000 IU and for intranasal administration of 100,000 IU against the SARS-CoV-2 virus in vitro was studied. The methodological approach of this study is based on the phenomenon of a decrease in the number of plaques formed under the action of a potential antiviral drug. The antiviral activity of recombinant interferon gamma has been experimentally confirmed, both in preventive and therapeutic application schemes. The smallest number of plaques was observed with the preventive scheme of application of the tested object at concentrations of 1000 and 333 IU/ml. The semi-maximal effective concentration (EC50) with the prophylactic regimen was 24 IU/ml. The preventive scheme of application of the tested object turned out to be more effective than therapeutic one, which is probably explained by the launch of the expression of various interferon-stimulated genes that affect to a greater extent the steps of virus entry into the cell and its reproduction. Further study of the effect of drugs based on recombinant interferon gamma on the reproduction of the SARS-CoV-2 virus for clinical use for prevention and treatment is highly relevant.

Immunomodulatory Activity of Begonia Medicinalis Ethanolic Extract in Experimental Animals

Benalu batu (B. medicinalis) is a plant endemic to Central Sulawesi that has been reported to possess anticancer, antioxidant, and antiviral activities. The mechanism of action of these activities is still unclear. This study aimed to evaluate the immunostimulatory effect of ethanol extract of B. medicinalis on an in vivo model by measuring the macrophage phagocytotic activity and cytokine production of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) on male mice and rats, respectively. The extract was obtained by the maceration method for 3 × 24 h using 70% ethanol. The doses of the extract were 60, 120, and 240 mg/kg body weight (bw). The percentage of macrophage phagocytosis and the TNF-α, and IFN-γ levels were measured on the eighth day, one hour after intraperitoneal injection of Staphylococcus aureus ATCC 25923. The result showed that ethanol extract of B. medicinalis activated macrophage phagocytosis in a dose-dependent manner, with a significant increase in cytokine expression of TNF-α and IFN-γ. The optimal dose was 240 mg/kg bw, with a higher percentage of phagocytic activity and higher levels of TNF-α and IFN-γ than Stimuno® as a positive control and other dosage treatments. This study suggests that B. medicinalis ethanol extract has the effect of an immunomodulator and provides a scientific basis for traditional usage of this herb plant.

Multi-omic and spatial dissection of immunotherapy response groups in non–small cell lung cancer (NSCLC).

8544 Background: Immune checkpoint inhibitors (ICI) have shown durable benefit in a subset of non-small cell lung cancer (NSCLC) patients. The composition of the tumour microenvironment (TME) is becomingly increasingly recognised as an important factor to predict response to therapy. Methods: Here, we applied digital spatial profiling of the tumour and stromal compartments from a 2nd line NSCLC ICI-treated cohort (n = 41 patient) and standard of care (SOC), platinum treated NSCLC cohort (n = 47), to identify tissue-based signatures of response to therapy. Results: We demonstrate by mIHC that the interaction of CD68+ macrophages with PD1+, FoxP3+ cells is significantly enriched in ICI refractory tumours (p = 0.012). Patients sensitive to ICI therapy expressed higher levels of IL2 receptor alpha (CD25, p = 0.028) within the tumour compartments, which corresponded with the increased expression of IL2 mRNA (p = 0.001) within their stroma. Immuno-inhibitory markers CTLA-4 (p = 0.021) and IDO-1 (p = 0.023) were supressed in ICI-responsive patients. Tumour CD44 (p = 0.02) was depleted in the response group and corresponded inversely with significantly higher stromal expression of one of its ligands, SPP1 (osteopontin, p = 0.008). Analysis of dysregulated transcripts indicated the potential inhibition of stromal interferon-gamma (IFNγ) activity, estrogen-receptor and Wnt-1 signalling activity within the tumour cells of ICI responsive patients. Cox survival analysis indicated tumour CD44 expression was associated with poorer prognosis (HR = 1.61, p = 0.01), consistent with its depletion in ICI sensitive patients. Similarly, stromal CTLA-4 (HR = 1.78, p = 0.003) and MDSC/M2 macrophage marker ARG1 (HR = 2.37, p = 0.01) were associated with poorer outcome while BAD (HR = 0.5, p = 0.01) appeared protective. The SOC cohort paralleled similar roles for immune checkpoints and pro-apoptotic markers, with LAG3 (HR = 3.81, p = 0.04) indicating poorer outcome, and BIM (HR = 0.16, p = 0.014) with improved outcome. Interestingly, stromal mRNA for E-selectin (HR = 652, p = 0.001), CCL17 (HR = 70, p = 0.006) and MTOR (HR = 1065, p = 0.008) were highly associated with poorer outcome in ICI treated patients, indicating pro-tumourigenic features in the tumour microenvironment that may facilitate ICI resistance. Conclusions: Through multi-modal approaches, we have dissected the characteristics of NSCLC treatment groups and provide evidence for the role of several markers including IL2, CD25, CD44 and SPP1 in the efficacy of current generations of ICI therapy.

MO169: Three Doses of SARS‑COV‑2 BNT162B2 MRNA Vaccine Results in an Enhanced Immunologic B- And T-Cell Response in Haemodialysis Patients

BACKGROUND AND AIMSThe immune system is affected by uremia. Haemodialysis (HD) patients have an increased risk of acquiring infections due to many healthcare contacts and have a suboptimal response to vaccination and a high mortality from COVID-19 infection. Accumulating data indicate that two doses of vaccines are not enough, and most HD-patients have now received a third dose. The aim of the study was to describe the antibody and T-cell response to three doses of SARS‑CoV‑2 BNT162b2 mRNA vaccination and change over time.METHODInitially, 50 patients (mean age 69.4 years and 62% men) with end-stage kidney disease (ESKD) and haemodialysis treatment, at the dialysis outpatient clinic, Uppsala Academic Hospital, Sweden were enrolled into the study. Administration of SARS‑CoV‑2 BNT162b2 mRNA vaccine began on 28 Decemeber 2020. In September 2021, the patients received their third vaccine dose. During the study four patients died, four received a kidney transplant and two did not receive the third vaccine dose. A total of 41 (82%) patients received three doses of vaccine and were followed up until 3 months after the third dose. The antibody response was measured at four timepoints; 7–15 weeks and 6–8 months after the second dose, 3 weeks and 3 months after the third dose, and the T-cell response at three timepoints; 7–15 weeks after the second dose, 3 weeks and 3 months after the third dose. SARS-CoV-2 IgG antibody test (Abbott Architect) was performed against Spike antigen (anti-S) positive both after COVID-19 infection and vaccination (quantitative method used in routine diagnostics at Laboratory of Clinical Microbiology, Uppsala) and T-cell reactivity testing against the Spike protein using ELISPOT technology measuring interferon-gamma activity was performed at ABC-labs, Solna.RESULTSAfter two doses, IgG antibodies (IgG abs) to anti-S were detected in 37 (74%) of 50 patients, 5 (10%) had a borderline response and 8 (16%) were negative. T-cell response were detected in 29 (58%) of 50 patients and in 21 (42%) no response was detected. Before the third dose IgG abs to anti-S were detected in 24 (52%) of 46 patients, 3 (7%) had a borderline response and 19 (41%) were negative. Three weeks after the third dose IgG abs to anti-S were detected in 39 (95%) of 41 patients, and 2 (5%) were negative. T-cell responses were detected in 35 (85%) of 41 patients and in 6 (15%) no response. Three months after the third dose IgG ab to anti-S were still detected in 38 (95%) of 40 patients, and 2 (5%) were negative. Changes in IgG ab to anti-S and T-cell response over time in patients who received all three doses of vaccine and were followed up until 3 months after the latest dose (n = 40 and 37) are displayed in Figures 1 and 2 (preliminary data).CONCLUSIONThese results highlight the need for at least three doses of the SARS‑CoV‑2 BNT162b2 mRNA vaccine. It also indicates that the effect of the vaccine decreases slower after dose 3 than after dose 2 since almost all patients had a measurable immune response 3 months after dose 3. However, not all patients develop an immunological response. In a clinical setting, it is justified to measure the antibody response after vaccination to identify patients that are not protected and where one needs to take other measures to protect them from infection and/or to give early treatment in case of symptoms.FIGURE 1:Changes in IgG antibodies to anti-S in patients who received three doses of SARS-CoV-2 BNT162b2 mRNA vaccine and were followed up until 3 months after the latest dose (n = 40).FIGURE 2:Changes T-cell response patients who received three doses of SARS-CoV-2 BNT162b2 mRNA vaccine and were followed up until 3 months after the latest dose (n = 37).

The genetics of pediatric cutaneous autoimmunity: The sister diseases vitiligo and alopecia areata

Autoimmunity, the reaction of the host against self, is a complex pattern of immunologic response that allows the immune system to react to “normal” tissue. Many such diseases exist in the skin, but in particular, two stand out as visible manifestations of autoimmune cutaneous attack. These are vitiligo, the immune attack on the melanocyte, and alopecia areata, the immune attack of the hair unit. These two illnesses include the prototypes of "simple" attacks on specific components of the skin. Other such illnesses are psoriasis—a complex series of immunologic responses resulting in a characteristic pattern of keratinocyte hyperproliferation—and lichen planus, which is similarly associated with a typical cutaneous skin disease pattern, but these will not be included in this review. In comparison, autoimmune polyendocrinopathies have defined pathways of disease based on single-gene mutations that can result in a variety of autoimmune skin features including vitiligo and alopecia areata. This contribution reviews known patterns of autoimmunity in childhood skin disease with a particular focus on single-gene disorders where they exist, and the more common multifactorial genes that contribute to this breach of the immune system's regulatory checks and balances. Genetic mechanisms that will be covered include innate and adaptive immunity, major histocompatibility complex II class associations, genetic polymorphisms, and major histocompatibility complex I class associations that bridge autoinflammatory and autoimmunity states, as well as alterations in oxidative metabolism promoting tissue destruction. End-organ tissue destruction appears to occur via interferon gamma and cytotoxic mediated activity and is a promising new therapeutic target. Janus kinase inhibitors also have the benefit clinically of both affecting the immune cascade at the cytotoxic T cell level and allowing triggering of melanocyte and hair follicular activity through the signal transducer and activator of transcription pathway, representing an attractive therapeutic with dial activity, among other cytokines implicated in end-organ destruction. Transplacental autoimmunity will not be covered herein. Greater insight into mechanisms of pediatric cutaneous autoimmunity in vitiligo and alopecia areata has yielded better therapeutic options and interventions. Understanding the basis of autoimmune triggering in our patients, that is, personalized medicine, may allow for targeted therapeutics with a goal of induction of remission, regeneration, or rejuvenation of the end-organ with the least toxic side effect profile.

Mutation of Signal Transducer and Activator of Transcription 5 (STAT5) Binding Sites Decreases Milk Allergen αS1-Casein Content in Goat Mammary Epithelial Cells.

αS1-Casein (encoded by the CSN1S1 gene) is associated with food allergy more than other milk protein components. Milk allergy caused by αS1-casein is derived from cow milk, goat milk and other ruminant milk. However, little is known about the transcription regulation of αS1-casein synthesis in dairy goats. This study aimed to investigate the regulatory roles of signal transducer and activator of transcription 5 (STAT5) on αS1-casein in goat mammary epithelial cells (GMEC). Deletion analysis showed that the core promoter region of CSN1S1 was located at −110 to −18 bp upstream of transcription start site, which contained two putative STAT5 binding sites (gamma-interferon activation site, GAS). Overexpression of STAT5a gene upregulated the mRNA level and the promoter activity of the CSN1S1 gene, and STAT5 inhibitor decreased phosphorylated STAT5 in the nucleus and CSN1S1 transcription activity. Further, GAS site-directed mutagenesis and chromatin immunoprecipitation (ChIP) assays revealed that GAS1 and GAS2 sites in the CSN1S1 promoter core region were binding sites of STAT5. Taken together, STAT5 directly regulates CSN1S1 transcription by GAS1 and GAS2 sites in GMEC, and the mutation of STAT5 binding sites could downregulate CSN1S1 expression and decrease αS1-casein synthesis, which provide the novel strategy for reducing the allergic potential of goat milk and improving milk quality in ruminants.

Microvascular Inflammation: An Incremental Path to Refining the Diagnosis of Antibody-mediated Rejection in Heart Transplantation

Microvascular Inflammation: An Incremental Path to Refining the Diagnosis of Antibody-mediated Rejection in Heart Transplantation

Different Response to Immunotherapy According to Melanoma Histologic Subtype.

Superficial spreading melanoma (SSM) and nodular melanoma (NM) are the most common melanoma histologic subtypes and are characterized by different biological features. We retrospectively analyzed all consecutive patients with advanced melanoma, treated with anti-PD-1 and/or anti-CTLA-4 at our center, with data available on primary tumor subtype. The primary objective was to assess the association between histologic subtype and patients' outcomes. In addition, we analyzed whole-exome and whole-transcriptome sequencing data of a cohort of advanced melanoma to identify genes and related pathways, characterized by significant differences between NMs and SSMs. Twenty-one patients with NM and 39 with SSM, treated with anti-PD-1(53/60) as monotherapy or combined with anti-CTLA-4 (7/60), were analyzed. All known clinical-pathologic prognostic factors were well balanced between NM and SSM groups, except for the ECOG-PS score. The overall response rate was 52.4% (95% confidence interval, 29.8-74.3) in the NMs group versus 20.5% (9.3-36.5) in the SSMs group (P-value=0.02). The median progression-free survival and overall survival were, respectively, 13.9 and 44.5 months in the NMs group versus only 3.2 and 12 months in SSMs group (progression-free survival P-value=0.032; overall survival P-value=0.002). Multivariable analysis adjusting for the ECOG-PS, confirmed similar results. Whole-exome and whole-transcriptome data of 28 NMs and 21 SSMs were analyzed. No significant differences were observed in terms of both TMB and frequency of mutation in any gene. A total of 266 genes were overexpressed in NMs as compared with SSMs, and enrichment-analysis revealed a significant enrichment (false discovery rate<0.05) of genes belonging to immune-related pathways involved in antigens presentation mechanisms, response to interferon gamma and neutrophil activation. We provided clinical evidences suggesting a relevant association between melanoma histologic subtype and response to immunotherapy.

Peri-Transplant Alemtuzumab Levels Predict Risk of Secondary Graft Failure and Inversely Impact CXCL9 Levels after RIC HCT (A Correlative Biology Study to BMT-CTN 1204 RICHI)

Peri-Transplant Alemtuzumab Levels Predict Risk of Secondary Graft Failure and Inversely Impact CXCL9 Levels after RIC HCT (A Correlative Biology Study to BMT-CTN 1204 RICHI)

Kynurenine Pathway Activation and Deviation in Fibrosing Chronic Graft-Versus-Host Disease

Background: Fibrosing chronic graft-versus-host disease (cGVHD) is a debilitating complication after allogeneic stem cell transplantation (allo-SCT), and its pathophysiology is poorly understood. Kynurenine and its metabolites were shown to associate with both, interferon-gamma (IFNg) activation and fibrosis. This study investigates the interplay between members of the IFNγ pathway and Kynurenin-derived metabolites in cGVHD.Methods: Using a liquid chromatography tandem mass spectrometry approach on sera obtained on day+100 (n=430) and/or at onset of cGVHD symptoms (n=196) of our prospectively collected biobank after alloSCT, we measured concentrations of Kyn pathway metabolites (kynurenin (Kyn), anthranilic acid (AA), kynurenic acid (KA), 3-hydroxykynurenine (3-HK), and 3-hydroxy-anthranilic acid (3-HAA). We also measured C-X-C chemokine ligand 9 (CXCL9), interleukin (IL-)18, tryptophane (Trp) and indoleamine-2,3-dioxygenase (IDO) by ELISA.Results: We observed increased CXCL9 and IDO levels, and increased activity of the Kynurenine pathway in both non-severe and severe cGVHD. Interestingly, severe fibrosing cGVHD differed from any other form of cGVHD in a significant pathway shift toward AA and KA. This resulted in a reduced 3-HAA/AA ratio. A score consisting of low 3-HAA/AA ratio and high KA serum levels at onset of mild cGVHD symptoms predicted risk specifically of severe fibrosing cGVHD. This altered pathway in severe fibrosing cGVHD correlated with reduced activity of the Vitamin-B2-dependent kynurenine monooxygenase, low vitamin B6, and increased interleukin-18. Our results are consistent with a triple-hit model for fibrosing cGVHD including low activity of kynurenine monooxygenase (KMO, Vitamin B2 and B6 dependent) in the context of high IL18 serum levels and CXCL9-induced IDO activation (Figure 1).Conclusion: Chronic GVHD associates with an IFNg signature and increased activity of the Kynurenin pathway. KA and 3-HAA/AA defined the first molecular distinction between fibrosing and non-fibrosing cGVHD. The mechanism may involve Vitamin B2/B6 deficiency, and high CXCL9 and IL18. The Kyn metabolite signature is a candidate biomarker for severe fibrosing cGHVD and provides a rationale for translational trials on prophylactic Vitamin B2/B6 supplementation for cGVHD prevention.Figure 1: Triple hit model of fibrosing cGVHD (Hypothesis):Severe fibrosing cGVHD associates with a strong CXCL9-induced activation of IDO. The result of this activation is influenced by a reduced activity of kynurenine monooxygenase (KMO), either due to genetic polymorphisms or lack of Vitamin B2, and further aggravated by lack of Vitamin B6. The resulting metabolic deviation increases AA concentrations and reduces the 3-HAA/AA ratio. Finally, high IL-18 associates with increased serum KA. These three hits result in a metabolic situation with high KA and a low ratio 3-HAA/AA that predicts risk of fibrosing cGVHD. [Display omitted] DisclosuresMueller-Tidow: Janssen Cilag: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Bioline: Consultancy, Research Funding.

Investigation of Interferon Gamma Activity Using Bioinformatics Methods.

Breast cancer grows from the breast tissue and is a severe health problem worldwide. Genetics is believed to be the primary cause of all cases of breast cancer via gene mutation. Bioinformatics methodology has been used to determine the sequences and structures of bioactive substances. This study aimed to analyze the function and structure of the Interferon Gamma (IFNγ) in healthy controls and patients with breast cancer using bioinformatics methods. Blood samples were collected from 75 patients with breast cancer and 25 healthy subjects as control samples. The results showed transition mutation (30%) and transversion mutation (70%) in patients with breast cancer. Moreover, missense mutations (84%) and silent mutations (16%) were detected by BLAST. In addition, the amino acid of the IFNγ protein consisting of alpha-helical, β-sheet, and coil of secondary structure was determined in this study using BioEdit. The results of the physicochemical properties of the IFNγ protein reflect the function, stability, molecular weight, isoelectric point, and instability index of the IFNγ protein using ProtParam. Moreover, the results of mutation affected the percentage of alpha-helix, β-turns, and coil in breast cancer patients compared to healthy groups with reference of NCBI using PSIpred program. Additionally, the PHYRE2 server and RasMol program showed a tertiary structure of the IFNγ protein in breast cancer patients. Furthermore, the STRING program revealed the poly IFNγ protein interacted with other proteins to perform its functions normally. From the recorded data in the current study, it was concluded that IFNγ is considered a marker for patients with breast cancer.

A myxovirus resistance like protein involved in CgIFNLP mediated immune response of oyster Crassostrea gigas

The myxovirus resistance (Mx) proteins belong to interferon (IFN)-induced dynamin GTPase and play a pivotal role in the inhibition of replication of numerous viruses. In the present study, an Mx homologue (designated as CgMx1) was identified from oyster Crassostrea gigas. The open reading frame (ORF) of CgMx1 cDNA was of 1689 bp encoding a peptide of 562 amino acid residues. There was an N-terminal dynamin GTPase domain in the predicted peptide, which consisted of a tripartite GTP-binding motif (GDXXSGKS, DLPG and T/NKXD). The deduced amino acid sequence of CgMx1 shared 30–39% similarity with other Mx family members. And CgMx1 was clustered with Mx from H. discus, and then assigned into the invertebrate branch of the phylogenetic tree. The mRNA transcripts of CgMx1 were constitutively distributed in all the tested tissues, with the highest level in haemocytes (1342.45-fold of labial palps, p < 0.05). The mRNA expression of CgMx1 in haemocytes was significantly up-regulated to the highest level at 6 h (13.14-fold, p < 0.001) after poly (I:C) treatment and at 24 h (66.28-fold, p < 0.001) after recombinant IFN-like protein (rCgIFNLP) stimulation, respectively. CgMx1 protein was found to distribute in both the cytoplasm and nucleus of haemocytes. In the oysters with CgIFNLP and signal transducer and activator of transcription (CgSTAT) silenced by RNAi, the mRNA expression of CgMx1 decreased significantly in the haemocytes at 12 h after poly (I:C) stimulation, which was 0.02-fold and 0.04-fold of that in EGFP-RNAi oysters (p < 0.001), respectively. Meanwhile, EMSA assay revealed that CgSTAT was able to transactivate CgMx1 promoter through directly binding to its interferon-stimulated response element (ISRE) and gamma interferon activation site (GAS). The above results indicated that CgMx1 participated in the immune response of C. gigas through the signal pathway mediated by CgIFNLP and CgSTAT.