Interferon-γ-induced GBP1 is an inhibitor of human papillomavirus 18.

BACKGROUND: Human papillomavirus (HPV) infection is an important factor leading to cervical cell abnormalities. 90% of cervical cancers are closely associated with persistent infection of high-risk HPV, with the highest correlation with HPV16 and 18. Currently available vaccines and antivirals have limited effectiveness and coverage. Guanylate binding protein 1 (GBP1) was induced by interferon gamma and involved in many important cellular processes such as clearance of various microbial pathogens. However, whether GBP1 can inhibit human papillomavirus infection is unclear. RESULTS: In this study, we found that GBP1 can effectively degrade HPV18 E6, possibly through its GTPase activity or other pathways, and E6 protein degrades GBP1 through the ubiquitin-proteasome pathway to achieve immune escape. CONCLUSION: Therefore, GBP1 is an effector of IFN-γ anti-HPV activity. Our findings provided new insights into the treatment of HPV 18 infections.

Deletion of the TMEM30A gene enables leukemic cell evasion of NK cell cytotoxicity.

Natural killer (NK) cell immunotherapy has gained attention as a promising strategy for treatment of various malignancies. In this study, we used a genome-wide CRISPR screen to identify genes that provide protection or susceptibility to NK cell cytotoxicity. The screen confirmed the role of several genes in NK cell regulation, such as genes involved in interferon-γ signaling and antigen presentation, as well as genes encoding the NK cell receptor ligands B7-H6 and CD58. Notably, the gene TMEM30A, encoding CDC50A-beta-subunit of the flippase shuttling phospholipids in the plasma membrane, emerged as crucial for NK cell killing. Accordingly, a broad range of TMEM30A knock-out (KO) leukemia and lymphoma cells displayed increased surface levels of phosphatidylserine (PtdSer). TMEM30A KO cells triggered less NK cell degranulation, cytokine production and displayed lower susceptibility to NK cell cytotoxicity. Blockade of PtdSer or the inhibitory receptor TIM-3, restored the NK cell ability to eliminate TMEM30A-mutated cells. The key role of the TIM-3 - PtdSer interaction for NK cell regulation was further substantiated by disruption of the receptor gene in primary NK cells, which significantly reduced the impact of elevated PtdSer in TMEM30A KO leukemic cells. Our study underscores the potential significance of agents targeting the interaction between PtdSer and TIM-3 in the realm of cancer immunotherapy.

Increased levels of villus-derived exosomal miR-29a-3p in normal pregnancy than uRPL patients suppresses decidual NK cell production of interferon-γ and exerts a therapeutic effect in abortion-prone mice.

OBJECTIVE: Recurrent pregnancy loss (RPL) patients have higher absolute numbers of decidual natural killer (dNK) cells with elevated intracellular IFN-γ levels leading to a pro-inflammatory cytokine milieu, which contributes to RPL pathogenesis. The main objective of this study was twofold: first to explore the regulatory effects and mechanisms of villus-derived exosomes (vEXOs) from induced abortion patients or RPL patients at the level of intracellular IFN-γ in dNK cells; second to determine the validity of application of vEXOs in the treatment of unexplained RPL (uRPL) through in vitro experiments and mouse models. METHODS: Exosomes were isolated from villus explants by ultracentrifugation, co-cultured with dNK cells, and purified by enzymatic digestion and magnetically activated cell sorting. Flow cytometry, enzyme-linked immunosorbent assays, and RT-qPCR were used to determine IFN-γ levels. Comparative miRNA analysis of vEXOs from induced abortion (IA) and uRPL patients was used to screen potential candidates involved in dNK regulation, which was further confirmed by luciferase reporter assays. IA-vEXOs were electroporated with therapeutic miRNAs and encapsulated in a China Food and Drug Administration (CFDA)-approved hyaluronate gel (HA-Gel), which has been used as a clinical biomaterial in cell therapy for > 30 years. In vivo tracking was performed using 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine iodide (DiR) labelling. Tail-vein and uterine horn injections were used to evaluate therapeutic effects of the engineered exosomes in an abortion-prone mouse model (CBA/J × DBA/2 J). Placental growth was evaluated based on placental weight. IFN-γ mRNA levels in mouse placentas were measured by RT-qPCR. RESULTS: IFN-γ levels were significantly higher in dNK cells of uRPL patients than in IA patients. Both uRPL-vEXOs and IA-vEXOs could be efficiently internalized by dNK cells, whereas uRPL-vEXOs could not reduce the expression of IFN-γ by dNK cells as much as IA-vEXOs. Mechanistically, miR-29a-3p was delivered by vEXOs to inhibit IFN-γ production by binding to the 3' UTR of IFN-γ mRNA in dNK cells. For in vivo treatment, application of the HA-Gel effectively prolonged the residence time of vEXOs in the uterine cavity via sustained release. Engineered vEXOs loaded with miR-29a-3p reduced the embryo resorption rate in RPL mice with no signs of systemic toxicity. CONCLUSION: Our study provides the first evidence that villi can regulate dNK cell production of IFN-γ via exosome-mediated transfer of miR-29a-3p, which deepens our understanding of maternal-fetal immune tolerance for pregnancy maintenance. Based on this, we developed a new strategy to mix engineered vEXOs with HA-Gel, which exhibited good therapeutic effects in mice with uRPL and could be used for potential clinical applications in uRPL treatment.

TYK2 inhibition with deucravacitinib ameliorates erosive oral lichen planus.

Erosive oral lichen planus (OLP) is a challenging disease. This T cell driven disorder frequently shows a treatment unresponsive course and strongly limits patients' quality of life. The disease lacks FDA or EMA approved drugs for its treatment and the efficacy of the commonly administered treatments (i.e. topical and systemic steroids, steroid sparing agents) is often only partial. Although the etiopathogenesis of the disease still needs to be fully elucidated, recent advances helped to identify interferon-É£ (IFN-É£) as a pivotal cytokine in OLP pathogenesis, thus making the interference with its signalling a therapeutic target. Janus kinase (JAK) inhibitors therefore gained relevance for their inhibitory effect on IFN-É£ signalling. While some drugs such as abrocitinib, upadacitinib, tofacitinib directly interfere with IFN-É£ signalling through blockade of JAK1 and/or JAK2, deucravacitinib, a selective TYK-2 inhibitor indirectly interferes on IFN-É£ activation through interference with interleukin (IL)-12, a potent promotor for Th1/IFN-É£ responses. This mechanism of action makes deucravacitinib a candidate drug for the treatment of OLP. Here we provide initial evidence that deucravacitinib 6 mg daily has a beneficial effect in three patients with oral OLP.

RSAD2 is abundant in atherosclerotic plaques and promotes interferon-induced CXCR3-chemokines in human smooth muscle cells.

In atherosclerotic lesions, monocyte-derived macrophages are major source of interferon gamma (IFN-γ), a pleotropic cytokine known to regulate the expression of numerous genes, including the antiviral gene RSAD2. While RSAD2 was reported to be expressed in endothelial cells of human carotid lesions, its significance for the development of atherosclerosis remains utterly unknown. Here, we harnessed publicly available human carotid atherosclerotic data to explore RSAD2 in lesions and employed siRNA-mediated gene-knockdown to investigate its function in IFN-γ-stimulated human aortic smooth muscle cells (hAoSMCs). Silencing RSAD2 in IFN-γ-stimulated hAoSMCs resulted in reduced expression and secretion of key CXCR3-chemokines, CXCL9, CXCL10, and CXCL11. Conditioned medium from RSAD2-deficient hAoSMCs exhibited diminished monocyte attraction in vitro compared to conditioned medium from control cells. Furthermore, RSAD2 transcript was elevated in carotid lesions where it was expressed by several different cell types, including endothelial cells, macrophages and smooth muscle cells. Interestingly, RSAD2 displayed significant correlations with CXCL10 (r = 0.45, p = 0.010) and CXCL11 (r = 0.53, p = 0.002) in human carotid lesions. Combining our findings, we uncover a novel role for RSAD2 in hAoSMCs, which could potentially contribute to monocyte recruitment in the context of atherosclerosis.

Combination of IFN-gamma with STING agonist and PD-1 immune checkpoint blockade: a potential immunotherapy for gastric cancer.

Suppression of the cGAS-STING pathway is an immune escape mechanism in cancer cells. The critical role of this pathway in gastric cancer (GC) is not fully understood. Herein, we evaluated the effect of the interferon-gamma (IFN-gamma), STING agonist, PD-1 immune checkpoint blockade, and their combination on the cGAS-STING pathway in GC. Expression of cGAS and STING in tumor tissue samples and adjacent normal tissue (ANT) biopsies of fifty new GC patients was evaluated by quantitative real-time PCR (qRT-PCR). Moreover, cGAS and STING expression levels were examined in Peripheral Blood Mononuclear Cells (PBMC) samples of forty GC patients and twenty-five healthy subjects. The apoptosis rate of cancer cells was analyzed by Annexin V-FITC/PI. Cell proliferation was measured by the BrdU assay. Also, IFN-ß levels were evaluated in the supernatants of the treated groups. The cGAS expression was decreased in patients with distant metastasis. Co-cultures treated with IFN-gamma showed an elevated level of cGAS and STING expressions in PBMC and cancer cells. The rate of apoptosis increased in all the treatment groups. In addition, the rate of proliferation in PBMCs increased in different treated groups. The main role of PBMCs in cytotoxicity was determined by a comparative analysis of the viability of cells treated with all treatments, both with and without PBMCs. The production of IFN-ß was elevated in all treated groups. The current study suggests that a combination therapy using IFN-gamma, STING agonist, and anti-PD-1 antibody can provide a promising approach to the treatment of GC.

Causal relationship between inflammatory cytokines and autoimmune thyroid disease: a bidirectional two-sample Mendelian randomization analysis.

Background: Autoimmune thyroid disease (AITD) ranks among the most prevalent thyroid diseases, with inflammatory cytokines playing a decisive role in its pathophysiological process. However, the causal relationship between the inflammatory cytokines and AITD remains elusive. Methods: A two-sample Mendelian randomization (MR) analysis was performed to elucidate the causal connection between AITD and 41 inflammatory cytokines. Genetic variations associated with inflammatory cytokines were sourced from the FinnGen biobank, whereas a comprehensive meta-analysis of genome-wide association studies (GWASs) yielded data on Graves' disease (GD) and Hashimoto thyroiditis. Regarding the MR analysis, the inverse variance-weighted, MR-Egger, and weighted median methods were utilized. Additionally, sensitivity analysis was conducted using MR-Egger regression, MR-pleiotropy residual sum, and outliers. Results: Seven causal associations were identified between inflammatory cytokines and AITD. High levels of tumor necrosis factor-ß and low levels of stem cell growth factor-ß were indicative of a higher risk of GD. In contrast, high levels of interleukin-12p70 (IL-12p70), IL-13, and interferon-γ and low levels of monocyte chemotactic protein-1 (MCP-1) and TNF-α suggested a higher risk of HD. Moreover, 14 causal associations were detected between AITD and inflammatory cytokines. GD increases the levels of macrophage inflammatory protein-1ß, MCP-1, monokine induced by interferon-γ (MIG), interferon γ-induced protein 10 (IP-10), stromal cell-derived factor-1α, platelet-derived growth factor BB, ß-nerve growth factor, IL-2ra, IL-4, and IL-17 in blood, whereas HD increases the levels of MIG, IL-2ra, IP-10, and IL-16 levels. Conclusion: Our bidirectional MR analysis revealed a causal relationship between inflammatory cytokines and AITD. These findings offer valuable insights into the pathophysiological mechanisms underlying AITD.

Transcriptional Changes in Radiation-Induced Lung Injury: A Comparative Analysis of Two Radiation Doses for Preclinical Research.

In a recent stereotactic body radiation therapy animal model, radiation pneumonitis and radiation pulmonary fibrosis were observed at around 2 and 6 weeks, respectively. However, the molecular signature of this model remains unclear. This study aimed to examine the molecular characteristics at these two stages using RNA-seq analysis. Transcriptomic profiling revealed distinct transcriptional patterns for each stage. Inflammatory response and immune cell activation were involved in both stages. Cell cycle processes and response to type II interferons were observed during the inflammation stage. Extracellular matrix organization and immunoglobulin production were noted during the fibrosis stage. To investigate the impact of a 10 Gy difference on fibrosis progression, doses of 45, 55, and 65 Gy were tested. A dose of 65 Gy was selected and compared with 75 Gy. The 65 Gy dose induced inflammation and fibrosis as well as the 75 Gy dose, but with reduced lung damage, fewer inflammatory cells, and decreased collagen deposition, particularly during the inflammation stage. Transcriptomic analysis revealed significant overlap, but differences were observed and clarified in Gene Ontology and KEGG pathway analysis, potentially influenced by changes in interferon-gamma-mediated lipid metabolism. This suggests the suitability of 65 Gy for future preclinical basic and pharmaceutical research connected with radiation-induced lung injury.

Interferon-gamma signaling promotes cartilage regeneration after injury.

Osteoarthritis is a common chronic disease and major cause of disability and chronic pain in ageing populations. In this pathology, the entire joint is involved, and the regeneration of articular cartilage still remains one of the main challenges. Here, we investigated the molecular mechanisms underlying cartilage regeneration in young mice using a full-thickness cartilage injury (FTCI) model. FTCI-induced cartilage defects were created in the femoral trochlea of young and adult C57BL/6 mice. To identify key molecules and pathways involved in the early response to cartilage injury, we performed RNA sequencing (RNA-seq) analysis of cartilage RNA at 3 days after injury. Young mice showed superior cartilage regeneration compared to adult mice after cartilage injury. RNA-seq analysis revealed significant upregulation of genes associated with the immune response, particularly in the IFN-γ signaling pathway and qRT-PCR analysis showed macrophage polarization in the early phase of cartilage regeneration (3 days) in young mice after injury, which might promote the removal of damaged or necrotic cells and initiate cartilage regeneration in response to injury. IFN-γR1- and IFN-γ-deficient mice exhibited impaired cartilage regeneration following cartilage injury. DMM-induced and spontaneous OA phenotypes were exacerbated in IFN-γR1-/- mice than in wild-type mice. Our data support the hypothesis that IFN-γ signaling is necessary for cartilage regeneration, as well as for the amelioration of post-traumatic and age-induced OA.

New insight into arginine and tryptophan metabolism in macrophage activation during tuberculosis.

Arginine and tryptophan are pivotal in orchestrating cytokine-driven macrophage polarization and immune activation. Specifically, interferon-gamma (IFN-γ) stimulates inducible nitric oxide synthase (iNOS) expression), leading to the conversion of arginine into citrulline and nitric oxide (NO), while Interleukin-4 (IL4) promotes arginase activation, shifting arginine metabolism toward ornithine. Concomitantly, IFN-γ triggers indoleamine 2,3-dioxygenase 1 (IDO1) and Interleukin-4 induced 1 (IL4i1), resulting in the conversion of tryptophan into kynurenine and indole-3-pyruvic acid. These metabolic pathways are tightly regulated by NAD+-dependent sirtuin proteins, with Sirt2 and Sirt5 playing integral roles. In this review, we present novel insights that augment our understanding of the metabolic pathways of arginine and tryptophan following Mycobacterium tuberculosis infection, particularly their relevance in macrophage responses. Additionally, we discuss arginine methylation and demethylation and the role of Sirt2 and Sirt5 in regulating tryptophan metabolism and arginine metabolism, potentially driving macrophage polarization.

[Effect of LAG3 deficiency on natural killer cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis].

OBJECTIVE: To investigate the effect of LAG-3 deficiency (LAG3-/-) on natural killer (NK) cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis. METHODS: C57BL/6 mice, each weighing (20 ± 2) g, were divided into the LAG3-/- and wild type (WT) groups, and each mouse in both groups was inoculated with 3 000 E. multilocularis protoscoleces via the hepatic portal vein. Mouse liver and spleen specimens were collected 12 weeks post-infection, sectioned and stained with sirius red, and the hepatic lesions and fibrosis were observed. Mouse hepatic and splenic lymphocytes were isolated, and flow cytometry was performed to detect the proportions of hepatic and splenic NK cells, the expression of CD44, CD25 and CD69 molecules on NK cell surface, and the secretion of interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin (IL)-4, IL-10 and IL-17A. RESULTS: Sirius red staining showed widening of inflammatory cell bands and hyperplasia of fibrotic connective tissues around mouse hepatic lesions, as well as increased deposition of collagen fibers in the LAG3-/-group relative to the WT group. Flow cytometry revealed lower proportions of mouse hepatic (6.29% ± 1.06% vs. 11.91% ± 1.85%, P < 0.000 1) and splenic NK cells (4.44% ± 1.22% vs. 5.85% ± 1.10%, P > 0.05) in the LAG3-/- group than in the WT group, and the mean fluorescence intensity of CD44 was higher on the surface of mouse hepatic NK cells in the LAG3-/- group than in the WT group (t = -3.234, P < 0.01), while no significant differences were found in the mean fluorescence intensity of CD25 or CD69 on the surface of mouse hepaticNK cells between the LAG3-/- and WT groups (both P values > 0.05). There were significant differences between the LAG3-/- and WT groups in terms of the percentages of IFN-γ (t = -0.723, P > 0.05), TNF-α (t = -0.659, P > 0.05), IL-4 (t = -0.263, P > 0.05), IL-10 (t = -0.455, P > 0.05) or IL-17A secreted by mouse hepatic NK cells (t = 0.091, P > 0.05), and the percentage of IFN-γ secreted by mouse splenic NK cells was higher in the LAG3-/- group than in the WT group (58.40% ± 1.64% vs. 50.40% ± 4.13%; t = -4.042, P < 0.01); however, there were no significant differences between the two groups in terms of the proportions of TNF-α (t = -1.902, P > 0.05), IL-4 (t = -1.333, P > 0.05), IL-10 (t = -1.356, P > 0.05) or IL-17A secreted by mouse splenic NK cells (t = 0.529, P > 0.05). CONCLUSIONS: During the course of E. multilocularis infections, LAG3-/- promotes high-level secretion of IFN-γ by splenic NK cells, which may participate in the reversal the immune function of NK cells, resulting in aggravation of hepatic fibrosis.

The Role of Cytokines in Cutaneous T Cell Lymphoma: A Focus on the State of the Art and Possible Therapeutic Targets.

Cutaneous T cell lymphomas (CTCLs), encompassing mycosis fungoides (MF) and Sézary syndrome (SS), present a complex landscape influenced by cytokines and cellular responses. In this work, the intricate relationship between these inflammatory proteins and disease pathogenesis is examined, focusing on what is known at the clinical and therapeutic levels regarding the most well-known inflammatory mediators. An in-depth look is given to their possible alterations caused by novel immunomodulatory drugs and how they may alter disease progression. From this narrative review of the actual scientific landscape, Interferon-gamma (IFN-γ) emerges as a central player, demonstrating a dual role in both promoting and inhibiting cancer immunity, but the work navigates through all the major interleukins known in inflammatory environments. Immunotherapeutic perspectives are elucidated, highlighting the crucial role of the cutaneous microenvironment in shaping dysfunctional cell trafficking, antitumor immunity, and angiogenesis in MF, showcasing advancements in understanding and targeting the immune phenotype in CTCL. In summary, this manuscript aims to comprehensively explore the multifaceted aspects of CTCL, from the immunopathogenesis and cytokine dynamics centred around TNF-α and IFN-γ to evolving therapeutic modalities. Including all the major known and studied cytokines in this analysis broadens our understanding of the intricate interplay influencing CTCL, paving the way for improved management of this complex lymphoma.

Plasma Levels of Interferon Gamma associated with Hearing Loss and Hearing Loss Sensation through the Handicap Questionnaire Inventory for the Elderly Screening Version.

OBJECTIVE: The aim of this study was to verify the association between the auditory handicap found in the Hearing Handicap Inventory for the Elderly-Screening Version (HHIE-S) questionnaire and hearing loss and the plasma levels of inflammatory biomarkers. MATERIALS AND METHODS: Cross-sectional study with 76 participants, 67 (88%) females and 9 (12%) males, with a mean age of 70 years. Tonal threshold audiometry and self-assessment with HHIE-S questionnaire were performed to measure the plasma levels of interleukin-2 (IL-2), IL-4, IL-6, and IL-10; tumor necrosis factor alpha; and interferon gamma (IFN-γ) flow cytometry method. For all data analyzed, the significance level adopted was P < 0.05 and 95% confidence interval. RESULTS: An inverse correlation was observed between the increase in plasma levels of IFN-γ and normal auditory handicap (P = 0.015; rs = -0.280). The severe handicap group showed an increase in the averages I (P = 0.005; rs = 0.350) and II (P = 0.016; rs = 0.368) in the right ear and the light/moderate handicap group increased the means I (P = 0.027; rs = 0.350) and II (P = 0.046; rs = 0.310) of the left ear. A statistically significant association was found between the speech recognition threshold (SRT) test results of the right ear and the severe handicap group (P = 0.002; rs = 0.271). CONCLUSIONS: There was an association between the increase in plasma levels of IFN-γ and normal auditory handicap. Additionally, statistically significant associations were observed between the mild/moderate and severe handicap groups with the increase in hearing means and an increase in SRT associated with the severe handicap group.

Serum cytokine biosignatures for identification of tuberculosis among HIV-positive inpatients.

BACKGROUND: Serum cytokines correlate with tuberculosis (TB) progression and are predictors of TB recurrence in people living with HIV. We investigated whether serum cytokine biosignatures could diagnose TB among HIV-positive inpatients. METHODS: We recruited HIV-positive inpatients with symptoms of TB and measured serum levels of inflammation biomarkers including IL-2, IL-4, IL-6, IL-10, tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). We then built and tested our TB prediction model. RESULTS: 236 HIV-positive inpatients were enrolled in the first cohort and all the inflammation biomarkers were significantly higher in participants with microbiologically confirmed TB than those without TB. A binary support vector machine (SVM) model was built, incorporating the data of four biomarkers (IL-6, IL-10, TNF-α and IFN-γ). Efficacy of the SVM model was assessed in training (n=189) and validation (n=47) sets with area under the curve (AUC) of 0.92 (95% CI 0.88 to 0.96) and 0.85 (95% CI 0.72 to 0.97), respectively. In an independent test set (n=110), the SVM model yielded an AUC of 0.85 (95% CI 0.76 to 0.94) with 78% (95% CI 68% to 87%) specificity and 85% (95% CI 66% to 96%) sensitivity. Moreover, the SVM model outperformed interferon-gamma release assay (IGRA) among advanced HIV-positive inpatients irrespective of CD4+ T-cell counts, which may be an alternative approach for identifying Mycobacterium tuberculosis infection among HIV-positive inpatients with negative IGRA. CONCLUSIONS: The four-cytokine biosignature model successfully identified TB among HIV-positive inpatients. This diagnostic model may be an alternative approach to diagnose TB in advanced HIV-positive inpatients with low CD4+ T-cell counts.

Important Considerations for ELISpot Validation.

The ELISpot assay has a solid place in the immune monitoring field for over 40 years. It is an assay that can assess the function of single immune cells in a straightforward and easy-to-learn approach. Its use in basic research, translational, and clinical work has been documented in countless publications. Harmonization guidelines and invaluable tools for optimal assay performance and evaluation exist. However, the validation of an established ELISpot protocol has been left to diverse opinions about how to interpret and tackle typical validation parameters. This chapter addresses important considerations for ELISpot validation, including the interpretations of validation parameters for a meaningful description of assay performance.

Interferon-γ/IL-2 ELISpot and mRNA Responses to the SARS-CoV2, Feline Coronavirus Serotypes 1 (FCoV1), and FCoV2 Receptor Binding Domains by the T Cells from COVID-19-Vaccinated Humans and FCoV1-Infected Cats.

The receptor binding domain (RBD) of SARS-CoV-2 (SCoV2) has been used recently to identify the RBD sequences of feline coronavirus serotypes 1 (FCoV1) and 2 (FCoV2). Cats naturally infected with FCoV1 have been shown to possess serum reactivities with FCoV1 and SCoV2 RBDs but not with FCoV2 RBD. In the current study, COVID-19-vaccinated humans and FCoV1-infected laboratory cats were evaluated for interferon-gamma (IFNγ) and interleukin-2 (IL-2 ELISpot responses by their peripheral blood mononuclear cells (PBMC) to SCoV2, FCoV1, and FCoV2 RBDs. Remarkably, the PBMC from COVID-19-vaccinated subjects developed IFNγ responses to SCoV2, FCoV1, and FCoV2 RBDs. The most vaccinated subject (five vaccinations over 2 years) appeared to produce hyperreactive IFNγ responses to all three RBDs, including the PBS media control. This subject lost IFNγ responses to all RBDs at 9 months (9 mo) post-last vaccination. However, her IL-2 responses to FCoV1 and FCoV2 RBDs were low but detectable at 10 mo post-last vaccination. This observation suggests that initially robust IFNγ responses to SCoV2 RBD may be an outcome of robust inflammatory IFNγ responses to SCoV2 RBD. Hence, the T-cell responses of vaccine immunity should be monitored by vaccine immunogen-specific IL-2 production. The PBMC from chronically FCoV1-infected cats developed robust IFNγ responses to SCoV2 and FCoV2 RBDs but had the lowest IFNγ responses to FCoV1 RBD. The constant exposure to FCoV1 reinfection may cause the IFNγ responses to be downregulated to the infecting virus FCoV1 but not to the cross-reacting epitopes on the SCoV2 and FCoV2 RBDs.

Performance and Stability of New Class of Fetal Bovine Sera (FBS) and Its Lyophilized Form in ELISpot and FluoroSpot Assays: Applications for Monitoring the Immune Response in Vaccine, and Cell and Gene Immunotherapy in Clinical Trials.

Interferon-gamma (IFNγ) ELISpot and FluoroSpot are widely used assays to detect functional cell responses in immunotherapy clinical studies. Recognized for their importance in vaccine development studies to quantitate immune responses, these assays have more recently risen to the forefront in cell and gene therapy as well as cancer immunotherapy fields where responses against cancer neoantigens are not easily detectable above assay background. Here, we test a new class of fetal bovine serum (FBS), CultraPure FBS, in ex vivo ELISpot and FluoroSpot assays and cultured FluoroSpot assays following in vitro expansion. Several CultraPure FBS lots that have been specially formulated through the process of lyophilization (lyo-FBS) were compared to liquid CultraPure FBS. We stimulated human PBMCs with antigen-specific peptide pools diluted in media supplemented with liquid CultraPure FBS or lyo-FBS and found equivalent cytokine production with negligible to no assay background with both liquid and lyo-FBS formats. Moreover, the lyo-FBS showed lot-to-lot consistency and 90-day refrigerated (4 °C) stability in both ex vivo direct and in vitro cultured assays. In addition, we present here a method using lyo-FBS for the expansion of low-frequency antigen-specific T cells, mimicking the low frequency seen with cancer neoantigens by utilizing a cultured FluoroSpot assay. Our results demonstrate the presence of Granzyme B, interferon-gamma (IFNγ), and tumor necrosis factor (TNF) production by antigen-specific polyfunctional T cells following a 9-day culture using media supplemented with lyo-FBS.

The causal association between circulating cytokines with the risk of frailty and sarcopenia under the perspective of geroscience.

Introduction: Circulating cytokines were considered to play a critical role in the initiation and propagation of sarcopenia and frailty from observational studies. This study aimed to find the casual association between circulating cytokines and sarcopenia and frailty from a genetic perspective by two-sample Mendelian randomization (MR) analysis. Methods: Data for 41 circulating cytokines were extracted from the genome-wide association study dataset of 8,293 European participants. Inverse-variance weighted (IVW) method, MR-Egger, and weighted median method were applied to assess the relationship of circulating cytokines with the risk of aging-related syndromes and frailty. Furthermore, MR-Egger regression was used to indicate the directional pleiotropy, and Cochran's Q test was used to verify the potential heterogeneity. The "leave-one-out" method was applied to visualize whether there was a causal relationship affected by only one anomalous single-nucleotide polymorphisms. Results: Genetic predisposition to increasing levels of interleukin-10 (IL-10), IL-12, and vascular endothelial growth factor (VEGF) was associated with the higher risk of low hand grip strength according to the IVW method [R = 1.05, 95% CI = 1.01-1.10, P = 0.028, false discovery rate (FDR)-adjusted P = 1.000; OR = 1.03, 95% CI = 1.00-1.07, P = 0.042, FDR-adjusted P = 0.784; OR = 1.02, 95% CI = 1.00-1.05, P = 0.038, FDR-adjusted P = 0.567]. Furthermore, genetically determined higher macrophage colony-stimulating factors (M-CSFs) were associated with a lower presence of appendicular lean mass (OR = 1.01, 95% CI = 1.00-1.02, P = 0.003, FDR-adjusted P = 0.103). Monokine induced by interferon-γ (MIG) and tumor necrosis factor-beta (TNF-ß) were associated with a higher risk of frailty (OR = 1.03, 95% CI = 1.01-1.05, P < 0.0001, FDR-adjusted P = 0.012; OR = 1.01, 95% CI = 1.00-1.03, P = 0.013, FDR-adjusted P = 0.259). In this study, we did not find heterogeneity and horizontal pleiotropy between the circulating cytokines and the risk of frailty and sarcopenia. Conclusion: Genetic predisposition to assess IL-10, IL-12, and VEGF levels was associated with a higher risk of low hand grip strength and M-CSF with the presence of appendicular lean mass. The high levels of TNF-ß and MIG were associated with a higher risk of frailty. More studies will be required to explore the molecular biological mechanisms underlying the action of inflammatory factors.

Reciprocal inhibition between TP63 and STAT1 regulates anti-tumor immune response through interferon-γ signaling in squamous cancer.

Squamous cell carcinomas (SCCs) are common and aggressive malignancies. Immune check point blockade (ICB) therapy using PD-1/PD-L1 antibodies has been approved in several types of advanced SCCs. However, low response rate and treatment resistance are common. Improving the efficacy of ICB therapy requires better understanding of the mechanism of immune evasion. Here, we identify that the SCC-master transcription factor TP63 suppresses interferon-γ (IFNγ) signaling. TP63 inhibition leads to increased CD8+ T cell infiltration and heighten tumor killing in in vivo syngeneic mouse model and ex vivo co-culture system, respectively. Moreover, expression of TP63 is negatively correlated with CD8+ T cell infiltration and activation in patients with SCC. Silencing of TP63 enhances the anti-tumor efficacy of PD-1 blockade by promoting CD8+ T cell infiltration and functionality. Mechanistically, TP63 and STAT1 mutually suppress each other to regulate the IFNγ signaling by co-occupying and co-regulating their own promoters and enhancers. Together, our findings elucidate a tumor-extrinsic function of TP63 in promoting immune evasion of SCC cells. Over-expression of TP63 may serve as a biomarker predicting the outcome of SCC patients treated with ICB therapy, and targeting TP63/STAT/IFNγ axis may enhance the efficacy of ICB therapy for this deadly cancer.