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Since the start of the COVID-19 pandemic, in a short span of 3 years, vaccination against SARS-CoV-2 has resulted in the end of the pandemic. Patients with inborn errors of immunity (IEI) are at an increased risk for SARS-CoV-2 infection; however, serious illnesses and mortality, especially in primary antibody deficiencies (PADs), have been lower than expected and lower than other high-risk groups. This suggests that PAD patients may mount a reasonable effective response to the SARS-CoV-2 vaccine. Several studies have been published regarding antibody responses, with contradictory reports. The current study is, perhaps, the most comprehensive study of phenotypically defined various lymphocyte populations in PAD patients following the SARS-CoV-2 vaccine. In this study, we examined, following two vaccinations and, in a few cases, prior to and following the 1st and 2nd vaccinations, subsets of CD4 and CD8 T cells (Naïve, TCM, TEM, TEMRA), T follicular helper cells (TFH1, TFH2, TFH17, TFH1/17), B cells (naïve, transitional, marginal zone, germinal center, IgM memory, switched memory, plasmablasts, CD21low), regulatory lymphocytes (CD4Treg, CD8Treg, TFR, Breg), and SARS-CoV-2-specific activation of CD4 T cells and CD8 T cells (CD69, CD137), SARS-CoV-2 tetramer-positive CD8 T cells, and CD8 CTL. Our data show significant alterations in various B cell subsets including Breg, whereas only a few subsets of various T cells revealed alterations. These data suggest that large proportions of PAD patients may mount significant responses to the vaccine.

Toxoplasma gondii is an intracellular parasite that can activate the NLRP1 inflammasome leading to macrophage pyroptosis in Lewis rats, but the underlying mechanism is not well understood. In this study, we performed a genome-wide CRISPR screen and identified the dense granule proteins GRA35, GRA42, and GRA43 as the Toxoplasma effectors mediating cell death in Lewis rat macrophages. GRA35 localizes on the parasitophorous vacuole membrane, where it interacts with the host E3 ubiquitin ligase ITCH. Inhibition of proteasome activity or ITCH knockout prevented pyroptosis in Toxoplasma-infected Lewis rat macrophages, consistent with the "NLRP1 functional degradation model." However, there was no evidence that ITCH directly ubiquitinates or interacts with rat NLRP1. We also found that GRA35-ITCH interaction affected Toxoplasma fitness in IFNγ-activated human fibroblasts, likely due to ITCH's role in recruiting ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane. These findings identify a new role of host E3 ubiquitin ligase ITCH in mediating effector-triggered immunity, a critical concept that involves recognizing intracellular pathogens and initiating host innate immune responses.IMPORTANCEEffector-triggered immunity represents an innate immune defense mechanism that plays a crucial role in sensing and controlling intracellular pathogen infection. The NLRP1 inflammasome in the Lewis rats can detect Toxoplasma infection, which triggers proptosis in infected macrophages and eliminates the parasite's replication niche. The work reported here revealed that host E3 ubiquitin ligase ITCH is able to recognize and interact with Toxoplasma effector protein GRA35 localized on the parasite-host interface, leading to NLRP1 inflammasome activation in Lewis rat macrophages. Furthermore, ITCH-GRA35 interaction contributes to the restriction of Toxoplasma in human fibroblasts stimulated by IFNγ. Thus, this research provides valuable insights into understanding pathogen recognition and restriction mediated by host E3 ubiquitin ligase.

Scientific knowledge evolves in small steps, with occasional backsteps to correct inaccuracies, all occurring within a competitive environment. This perspective for the first time looks at the history of latency-related RNA (LR-RNA) that was later renamed latency-associated transcript (LAT). At the 1986 International Herpesvirus Workshop (IHW) meeting in Leeds, England, Daniel L Rock and Anthony B Nesburn first reported the discovery of human herpes virus 1 (HSV-1) latency-related (LR) RNA that is antisense to ICP0. Less than a month after the IHW meeting, a paper was submitted to Science magazine and 8 months later appeared in print thanking "D. Rock for suggesting RNA complementary to the ICP0 message may be present in latently infected cells". This perspective is not a review of the LAT literature but intends to clarify the timeline of LAT discovery and subsequent breakthroughs such as reactivation, apoptosis, CD8+ T cell exhaustion, and LAT expression in different cell types detected during latency. While many review articles have been written about LAT since 1987, the most comprehensive and balanced review about LAT was written by Dr. David Bloom's group. In this overview, I will discuss our original collaboration with Dr. Dan Rock and subsequent work that our group performed, which is still ongoing. Finally, I will discuss the controversies associated with LAT from its inception to current times. [ABSTRACT FROM AUTHOR]

The cytokine context present in the reproductive tract of cows is closely involved in normal uterine functions, including the estrous cycle and the establishment and maintenance of pregnancy. However, the roles of some cytokines in the uterus, and their relation with reproductive performance remain to be elucidated. Thus, this study aimed to examine the protein expression of several cytokines such as TNFα, IL-6, IL-8, IFNγ, IL-4, and TGF-β3 in endometrial biopsies previous to conception, to evaluate the possible association with delayed conception in dairy cows. Protein expression levels were evaluated by immunohistochemistry. Results showed that the protein expression levels of TNFα, IL-6, IL-4 and TGF-β3 were not associated with the parturition-conception interval, whereas the high protein expression levels of IFNγ were associated with the parturition-conception interval. Finally, the low protein expression of IL-8 showed a statistical tendency to be associated with delayed conception. This is the first report about the protein expression of IFN-γ in the endometrium of dairy cows and also, this cytokine could enhance the favorable conditions to achieve an early pregnancy. [ABSTRACT FROM AUTHOR]

Background: PD-L1 expression on cancer cells is an important mechanism of tumor immune escape, and immunotherapy targeting the PD-L1/PD1 interaction is a common treatment option for patients with melanoma. However, many patients do not respond to treatment and novel predictors of response are emerging. One suggested modifier of PD-L1 is the p53 pathway, although the relationship of p53 pathway function and activation is poorly understood. Methods: The study was performed on human melanoma cell lines with various p53 status. We investigated PD-L1 and proteins involved in IFNγ signaling by immunoblotting and mRNA expression, as well as membrane expression of PD-L1 by flow cytometry. We evaluated differences in the ability of NK cells to recognize and kill target tumor cells on the basis of p53 status. We also investigated the influence of proteasomal degradation and protein half-life, IFNγ signaling and p53 activation on biological outcomes, and performed bioinformatic analysis using available data for melanoma cell lines and melanoma patients. Results: We demonstrate that p53 status changes the level of membrane and total PD-L1 protein through IRF1 regulation and show that p53 loss influences the recently discovered SOX10/IRF1 regulatory axis. Bioinformatic analysis identified a dependency of SOX10 on p53 status in melanoma, and a co-regulation of immune signaling by both transcription factors. However, IRF1/PD-L1 regulation by p53 activation revealed complicated regulatory mechanisms that alter IRF1 mRNA but not protein levels. IFNγ activation revealed no dramatic differences based on TP53 status, although dual p53 activation and IFNγ treatment confirmed a complex regulatory loop between p53 and the IRF1/PD-L1 axis. Conclusions: We show that p53 loss influences the level of PD-L1 through IRF1 and SOX10 in an isogenic melanoma cell model, and that p53 loss affects NK-cell cytotoxicity toward tumor cells. Moreover, activation of p53 by MDM2 inhibition has a complex effect on IRF1/PD-L1 activation. These findings indicate that evaluation of p53 status in patients with melanoma will be important for predicting the response to PD-L1 monotherapy and/or dual treatments where p53 pathways participate in the overall response. [ABSTRACT FROM AUTHOR]

Central nervous system (CNS) injuries induce cell death and consequently the release of myelin and other cellular debris. Microglia as well as hematogenous macrophages actively collaborate to phagocyte them and undergo their degradation. However, myelin accumulation persists in the lesion site long after the injury with detrimental effects on axonal regeneration. This might be due to the presence of inhibitors of phagocytosis in the injury site. As we recently published that some proinflammatory stimuli, like interferon-γ (IFNγ) and lipopolysaccharide (LPS), inhibit myelin phagocytosis in macrophages, we have now studied the signaling pathways involved. A phagocytosis assay in Raw264.7 macrophages and N13 microglia cell lines with labeled myelin was developed with the pHrodo reagent that emits fluorescence in acidic cellular compartments (e.g.lysosomes). Pharmacological inhibition of Janus kinases (Jak) with Brepocitinib restored myelin phagocytosis and rescued the expression of genes related to phagocytosis, like triggering receptor expressed on myeloid cells 2 (TREM2), induced by IFNγ or LPS. In addition, while pharmacological inhibition of the signal transducer and activator of transcription 3 (STAT3) rescued myelin phagocytosis and the expression of phagocytosis related genes in the presence of LPS, it did not have any effect on IFNγ-treated cells. Our results show that Jak pathways participate in the inhibition of myelin phagocytosis by IFNγ and LPS. They also indicate that the resolution of inflammation is important for the clearance of cellular debris by macrophages and subsequent regenerative processes. [ABSTRACT FROM AUTHOR]

Polypous rhinosinusitis is a chronic inflammatory disease of the nasal mucosa and paranasal sinuses. Recombinant interferon has antiproliferative activity and corrects the deficiency of endogenous regulatory molecules, which allows us to consider this class of immunotropic drugs as a promising component of conservative immunotherapy for polyposis rhinosinusitis. Purpose of the study: To select the optimal composition of the experimental composition of interferons and evaluate their pharmacokinetic parameters in laboratory animals.For laboratory evaluation of the effectiveness of the auxiliary components of the proposed composition in creating a local effect, Wistar rats in the amount of 30 animals were selected, to which the proposed form of the drug (IFN1) was administered once intranasally. The control group included 30 animals that were alternately administered intranasally with a similar dose of IFNα2b and IFNγ dissolved in water for injection (IFN2). Quantitative determination of interferon concentration in blood samples was carried out using the enzyme immunoassay method. To assess the dependence of changes in concentrations in the blood of animals on the time elapsed after their administration, standard pharmacokinetic models were used. Next, the integral from the initial moment of time to infinity was determined, which corresponded to the area under the pharmacokinetic curve and made it possible to calculate a number of pharmacokinetic characteristics.The following indicators were obtained: 1) Area under the pharmacokinetic curve (AUCt) ng/ml/min – IFN1 = 683.0; IFN2 = 1707.7. 2) Absorption constant (Kp) – IFN1 = 0.13096; IFN2 = 0.03836. 3) Suction constant (Kel) – IFN1 = 0.00177; IFN2 = 0.00317. 4) Clearance (Cl) ml/min – IFN1 = 129.35; IFN2 = 51.73.Based on the differences in the values of pharmacokinetic parameters (AUCt) for the IFN1 and IFN2 preparations, it can be concluded that the use of a composition based on chitosan, succinic acid and DMSO leads to a pronounced retention of interferon in the nasal mucosa, which provides a pronounced local therapeutic effect and allows for fewer systemic adverse reactions. This composition is suitable for further clinical studies of the effectiveness of immunotherapy for polyposis rhinosinusitis.

Abstract Background PD-L1 expression on cancer cells is an important mechanism of tumor immune escape, and immunotherapy targeting the PD-L1/PD1 interaction is a common treatment option for patients with melanoma. However, many patients do not respond to treatment and novel predictors of response are emerging. One suggested modifier of PD-L1 is the p53 pathway, although the relationship of p53 pathway function and activation is poorly understood. Methods The study was performed on human melanoma cell lines with various p53 status. We investigated PD-L1 and proteins involved in IFNγ signaling by immunoblotting and mRNA expression, as well as membrane expression of PD-L1 by flow cytometry. We evaluated differences in the ability of NK cells to recognize and kill target tumor cells on the basis of p53 status. We also investigated the influence of proteasomal degradation and protein half-life, IFNγ signaling and p53 activation on biological outcomes, and performed bioinformatic analysis using available data for melanoma cell lines and melanoma patients. Results We demonstrate that p53 status changes the level of membrane and total PD-L1 protein through IRF1 regulation and show that p53 loss influences the recently discovered SOX10/IRF1 regulatory axis. Bioinformatic analysis identified a dependency of SOX10 on p53 status in melanoma, and a co-regulation of immune signaling by both transcription factors. However, IRF1/PD-L1 regulation by p53 activation revealed complicated regulatory mechanisms that alter IRF1 mRNA but not protein levels. IFNγ activation revealed no dramatic differences based on TP53 status, although dual p53 activation and IFNγ treatment confirmed a complex regulatory loop between p53 and the IRF1/PD-L1 axis. Conclusions We show that p53 loss influences the level of PD-L1 through IRF1 and SOX10 in an isogenic melanoma cell model, and that p53 loss affects NK-cell cytotoxicity toward tumor cells. Moreover, activation of p53 by MDM2 inhibition has a complex effect on IRF1/PD-L1 activation. These findings indicate that evaluation of p53 status in patients with melanoma will be important for predicting the response to PD-L1 monotherapy and/or dual treatments where p53 pathways participate in the overall response. Graphical Abstracts

Antigen (AG)-specific T cell activity was compared in two groups of patients: those who underwent COVID-19 in 2021 during the circulation of the SARS-CoV-2 delta virus strain (43 individuals); and those who underwent COVID-19 in 2022 (Omicron strain, 23 individuals). The diagnosis was confirmed by PCR analysis of nasopharyngeal and oropharyngeal swabs. The 23 individuals following COVID-19 caused by SARS-CoV-2 (omicron) were part of a cohort of 41 volunteers who were examined multiple times during 2021–2023: during vaccination, after vaccination, before revaccination, and subsequently after illness (6–8 times in total). Due to this, it was possible to compare the indices of specific humoral and cellular immunity in the same patients 1–2 months before and after breakthrough infection. Detection of AG-specific T cells and assessment of their activity by AG-stimulated IFNγ production was carried out by our own previously developed method (Patent RU № 2780369 C1). For stimulation of memory T effectors in vitro, the same antigens were used to determine the concentration of antibodies against SARS-CoV-2 by ELISA method. A total of about 300 blood samples from healthy subjects and patients after COVID-19 were analyzed. Each sample was tested against 3 SARS-CoV-2 antigens and in 2 stimulation modes. A qualitative assessment algorithm for AG-specific T cell activity has been proposed that can be used to monitor the state of cellular immunity in a population in which SARS-CoV-2 virus continues to circulate and to create insights into what level of T cell activation is sufficient to prevent or reduce the severity of SARS-CoV-2 infection. Unvaccinated COVID-19 (SARS-CoV-2, Delta) survivors lacked AG-specific T cells to RBD SARS-CoV-2, but T cell specificity to full-length S glycoprotein at the same level, qualitatively assessed as low in 52% of the group, persisted for up to six months. In previously unvaccinated COVID-19 vaccinees, this duration of persistence of AG-specific T cells in circulating blood was achieved only after revaccination. Hybrid immunity, which we traced as a result of vaccination after COVID-19 (Delta strain) or as a breakthrough infection (SARS-CoV-2, Omicron), is characterized by the highest indices of memory T cell activity (43–46% of the group — normal activity of AG-specific cells, 30–43% — high activity) to all used antigens and the longest duration of preservation of indices at this level. Further investigation of the level of antiviral immunity after COVID-19 may be important for predicting the outcome of new waves of SARS-CoV-2 infection.

CD3+CD20+T lymphocytes are a population of T cells that, along with standard T cell markers, express the atypical membrane molecule CD20 (a traditional B cell marker). These cells were identified not so long ago and are currently being actively studied. Normally, they constitute up to 3-5% of the CD3+T cell compartment in human peripheral blood, and are also found in primary and secondary lymphoid organs, cerebrospinal fluid, brain tissue and liver. In healthy individuals, CD3+CD20+T cells are heterogeneous and contain a lower proportion of CD4+ cells, but produce higher levels of GM-CSF, IFNγ, IL-17, TNFα, IL-4, IL-10, adhesion molecules and chemokine receptors than CD3+CD20-T cells, indicating a highly activated proinflammatory phenotype with properties potentially promoting their pathogenic infiltration into the CNS. Recent studies have established the pathogenic behavior of CD3+CD20+T cells in a wide range of diseases, including hematological and non-hematological CD20+T cell malignancies and HIV, as well as autoimmune pathologies, in particular multiple sclerosis, a disabling inflammatory neurodegenerative disease that is accompanied by damage to the myelin sheath nerve fibers. CD20 positive T cells are detected in patients with multiple sclerosis in the peripheral blood, cerebrospinal fluid (occur at a frequency similar to that of B cells and show a correlation with disease severity) and white matter of the brain. CD20 positive T lymphocytes in the peripheral blood of patients with multiple sclerosis have been shown to produce high levels of IFNγ and IL-17А, which are two proinflammatory cytokines involved in the pathogenesis of this disease. It is possible that CD20+T cells represent a separate subpopulation of Th17 cells, the so-called Th1-polarized Th17, which are the product of redifferentiation of Th17 cells into Th1 and combine the phenotypic characteristics of both populations. And the expression of CD20 T cells may be a valuable marker that determines the target subpopulation of such pathogenic T cells, as well as serve as a target for therapy of autoimmune diseases.

Simple Summary: IFNγ is a multifunctional cytokine produced not only by activated lymphocytes but also in response to cancer immunotherapies; it has both antitumor and tumor-promoting effects. In ovarian cancer cells, the tumor-promoting effects of IFNγ are mediated by increased expression of the proto-oncogene Bcl3, the immune checkpoint PD-L1 and the proinflammatory cytokine IL-8. Recent studies have shown that IFNγ induces the expression of Bcl3, which then promotes the expression of PD-L1 and IL-8 in ovarian cancer cells, resulting in their increased proliferation and migration. In this review, we summarize the recent findings on the IFNγ tumor-promoting functions and on the mechanisms by which IFNγ induces Bcl3, PD-L1 and IL-8 expression in cancer cells, with a special focus on ovarian cancer. We also highlight the importance of a better understanding of these mechanisms to optimize the development of combinatorial approaches in cancer immunotherapies. IFNγ, a pleiotropic cytokine produced not only by activated lymphocytes but also in response to cancer immunotherapies, has both antitumor and tumor-promoting functions. In ovarian cancer (OC) cells, the tumor-promoting functions of IFNγ are mediated by IFNγ-induced expression of Bcl3, PD-L1 and IL-8/CXCL8, which have long been known to have critical cellular functions as a proto-oncogene, an immune checkpoint ligand and a chemoattractant, respectively. However, overwhelming evidence has demonstrated that these three genes have tumor-promoting roles far beyond their originally identified functions. These tumor-promoting mechanisms include increased cancer cell proliferation, invasion, angiogenesis, metastasis, resistance to chemotherapy and immune escape. Recent studies have shown that IFNγ-induced Bcl3, PD-L1 and IL-8 expression is regulated by the same JAK1/STAT1 signaling pathway: IFNγ induces the expression of Bcl3, which then promotes the expression of PD-L1 and IL-8 in OC cells, resulting in their increased proliferation and migration. In this review, we summarize the recent findings on how IFNγ affects the tumor microenvironment and promotes tumor progression, with a special focus on ovarian cancer and on Bcl3, PD-L1 and IL-8/CXCL8 signaling. We also discuss promising novel combinatorial strategies in clinical trials targeting Bcl3, PD-L1 and IL-8 to increase the effectiveness of cancer immunotherapies. [ABSTRACT FROM AUTHOR]

Tumor blood vessels are highly leaky in structure and have poor blood perfusion, which hampers infiltration and function of CD8T cells within tumor. Normalizing tumor vessels is thus thought to be important in promoting the flux of immune T cells and enhancing ant-tumor immunity. However, how tumor vasculature is normalized is poorly understood. Metformin (Met) combined with ant-PD-1 therapy is known to stimulate proliferation of and to produce large amounts of IFNγ from tumor-infiltrating CD8T lymphocytes (CD8TILs). We found that the combination therapy promotes the pericyte coverage of tumor vascular endothelial cells (ECs) to improve blood perfusion and that it suppresses the hyperpermeability through the increase of VE-cadherin. Peripheral node addressin(PNAd) and vascular cell adhesion molecule (VCAM)-1, both implicated to promote tumor infiltration of CD8T cells, were also increased. Importantly, tumor vessel normalization, characterized as the reduced 70-kDa dextran leakage and the enhancement ofVE-cadherin and VCAM-1, were canceled by anti-CD8 Ab or anti-IFNγ Ab injection to mice. The increased CD8TILs were also abrogated by anti-IFNγ Ab injection. In vascular ECs, flow cytometry analysis revealed that pSTATl expression was found to be associated with VE-cadherin expression. Moreover, in vitro treatment with Met and IFNγ enhanced VE-cadherin and VCAM-1 on human umbilical vein endothelial cells (HUVECs). The Kaplan-Meier method revealed a correlation of VE-cadherin or VCAM-1 levels with overall survival in patients treated with immune checkpoint inhibitors. These data indicate that IFNγ-mediated cross talk of CD8TILs with tumor vessels is important for creating a better tumor microenvironment and maintaining sustained antitumor immunity. [ABSTRACT FROM AUTHOR]

Introduction: Sudden sensorineural hearing loss (SSNHL) is a common emergency symptom in otolaryngology that requires immediate diagnosis and treatment. SSNHL has a multifactorial etiology, and its pathophysiologic mechanisms may be associated with inflammatory and metabolic changes that may affect the cochlear microenvironment or its nervous component, thus triggering the process or hindering hearing recovery. Therefore, the aim of this study was to assess metabolic and inflammatory changes to identify systemic parameters that could serve as prognostic factors for hearing recovery in patients with SSNHL. Materials and methods: Thirty patients with a sudden hearing loss of at least 30 dB in three contiguous frequencies were enrolled in this study. Patients were followed up for 4 months and peripheral blood samples were collected at 7 days (V1), 30 days (V2) and 120 days (V3). Interleukins (IL)-1F7, IL-2, IL-4, IL-5, IL-6, IL-10, interferon γ (IFN-γ), tumor necrosis factor α (TNF-α) and adiponectin were quantified in serum. In addition, lipid and glycemic profiles as well as concentration of creatinine, uric acid, fructosamine, peroxide, total proteins and albumin were analyzed. Patients underwent weekly ear-specific hearing tests with standard pure tone thresholds for frequencies of 250-8,000 Hz, speech recognition threshold and word recognition score. Results: Patients with SSNHL were divided into a group of patients who did not achieve hearing recovery (n = 14) and another group who achieved complete and significant recovery (n = 16). Most serologic parameters showed no significant changes or values indicating clinical changes. However, IFN-γ levels decreased by 36.3% between V1 and V2. The cytokine TNF-α showed a statistically significant decrease from V1 to V3 (from 22.91 to 10.34 pg./mL). Adiponectin showed a decrease from 553.7 ng/mL in V1 to 454.4 ng/mL in V3. Discussion: Our results show that serologic cytokine levels change in the acute phase of manifestation of SSNHL and establish a parallel between systemic changes and improvements in hearing, especially TNF-α, which showed differences in hearing recovery. The use of IFN-γ, TNF-α and adiponectin may elucidate the clinical improvement in these patients. [ABSTRACT FROM AUTHOR]

Bone marrow is a dynamic organ composed of stem cells that constantly receive signals from stromal cells and other hematopoietic cells in the niches of the bone marrow to maintain hematopoiesis and generate immune cells. Perturbation of the bone marrow microenvironment by infection and inflammation affects hematopoiesis and may affect immune cell development. Little is known about the effect of malaria on the bone marrow stromal cells that govern the hematopoietic stem cell (HSC) niche. In this study, we demonstrate that the mesenchymal stromal CXCL12-abundant reticular (CAR) cell population is reduced during acute malaria infection. The reduction of CXCL12 and interleukin-7 signals in the bone marrow impairs the lymphopoietic niche, leading to the depletion of common lymphoid progenitors, B cell progenitors, and mature B cells, including plasma cells in the bone marrow. We found that interferon-γ (IFNγ) is responsible for the upregulation of Sca1 on CAR cells, yet the decline in CAR cell and B cell populations in the bone marrow is IFNγ-independent. In contrast to the decline in B cell populations, HSCs and multipotent progenitors increased with the expansion of myelopoiesis and erythropoiesis, indicating a bias in the differentiation of multipotent progenitors during malaria infection. These findings suggest that malaria may affect host immunity by modulating the bone marrow niche. [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV)-based gene therapies have shown promise as novel treatments for rare genetic disorders such as hemophilia A and spinal muscular atrophy. However, cellular immune responses mediated by cytotoxic (CD8+) and helper (CD4+) T cells may target vector-transduced cells as well as healthy immune cells, impacting safety and efficacy. In this study, we describe the optimization and reproducibility of interferon-γ (IFNγ)–based and interleukin-2 (IL-2)–based enzyme-linked immunosorbent spot (ELISpot) assays for measuring T cell responses against AAV peptide antigens. For method optimization, peripheral blood mononuclear cells (PBMCs) were isolated from healthy human donors and stimulated with commercially available major histocompatibility complex (MHC) class I or II–specific peptides as positive controls. Peptide pools were designed from published AAV8 and AAV9 capsid protein sequences and then used to assess the presence of AAV-specific T cell responses. Our results demonstrate a measurable increase in IFNγ and IL-2–producing cells after AAV peptide presentation. Furthermore, there was an observed difference in the magnitude and specificity of response to peptide pools based on AAV serotype and donor. Finally, using individual peptides, we identified a region of the AAV9 capsid protein that can elicit an immunogenic response. This work shows the applicability of ELISpot in assessing anti-AAV immune responses and provides insight into how novel recombinant AAV vectors could be designed to reduce immunogenic potential. [ABSTRACT FROM AUTHOR]

Simple Summary: Despite the recent studies emphasizing the importance of the interferon gamma receptor (IFNγR) pathway in T cell-mediated cytotoxicity against solid tumors, full human ex vivo immune checkpoint drug screening remains a challenge. We employed an engineered gamma interferon activation site response element luciferase reporter (GAS-Luc2) for immune checkpoint drug screening in diverse ex vivo T cell–solid tumor cell co-culture systems. Three GAS-Luc2 reporter tumor cell lines endogenously expressing various immune checkpoints were engineered to produce quantifiable bioluminescence signal in the presence of an immune checkpoint inhibitor where activated T cells release IFNγ. These reporter cell lines also detected paracrine IFNγ signaling for immune checkpoint-targeted ADCC drug screening. Advancement into an artificial antigen-presenting cell line (aAPC) significantly enhanced T cell signaling for improved performance in these ex vivo immune checkpoint drug screening platforms. 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. [ABSTRACT FROM AUTHOR]

Background: Leishmania is an intracellular flagellate protozoan parasite that causes a wide range of clinical diseases in humans. The basis of immunological resistance against leishmaniasis depends on Thl reactions and is within the time period of cytokine function. Methods: In this study, human anti-IL17 antibody and IFNγ-producing promastigote were produced to be used in leishmanization. A sequence of light and heavy chains' gene of anti-IL17 antibody and human IFNγ (hIFNγ) was obtained from the NCBI database and synthesized in the ECORV reaction site in the plasmid pGH, which it's called pGH–hIFNγ–antiIL17. The synthesized part using the restriction enzyme ECORV was extracted from the plasmid and after purification by electroporation was transferred to Iranian lizard Leishmania (I.L.L). Evaluation of structural presence in the I.L.L genome at the level of DNA and mRNA was assessed. The expressions of hIFNγ and anti-IL17 were evaluated and confirmed using ELISA and western blot analysis. The hIFNγ secreted from the culture medium was collected at high concentrations of 124.36 ± 6.47 pg/mL. Results: Targeted gene replacement into the I.L.L genome was successfully performed for the first time using the pGH–hIFNγ–antiIL17 plasmid in an identical replacement process. Stabilized recombinant DNA contains a target gene that has no toxicity to the parasite. Conclusions: The effective achievement of producing a recombinant gene was done for the first time by replacing the I.L.L-CPC gene with plasmid pGH-hIFNγ-antiIL17 by targeted gene replacement. This cab can regulate the production of hIFNγ and anti-IL17. This makes it a viable choice for eliminating leishmania. [ABSTRACT FROM AUTHOR]

In sporadic amyotrophic lateral sclerosis (sALS), IL-17A- and granzyme-positive cytotoxic T lymphocytes (CTL), IL-17A-positive mast cells, and inflammatory macrophages invade the brain and spinal cord. In some patients, the disease starts following a trauma or a severe infection. We examined cytokines and cytokine regulators over the disease course and found that, since the early stages, peripheral blood mononuclear cells (PBMC) exhibit increased expression of inflammatory cytokines IL-12A, IFN-γ, and TNF-α, as well as granzymes and the transcription factors STAT3 and STAT4. In later stages, PBMCs upregulated the autoimmunity-associated cytokines IL-23A and IL-17B, and the chemokines CXCL9 and CXCL10, which attract CTL and monocytes into the central nervous system. The inflammation is fueled by the downregulation of IL-10, TGFβ, and the inhibitory T-cell co-receptors CTLA4, LAG3, and PD-1, and, in vitro, by stimulation with the ligand PD-L1. We investigated in two sALS patients the regulation of the macrophage transcriptome by dimethyl fumarate (DMF), a drug approved against multiple sclerosis and psoriasis, and the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway inhibitor H-151. Both DMF and H-151 downregulated the expression of granzymes and the pro-inflammatory cytokines IL-1β, IL-6, IL-15, IL-23A, and IFN-γ, and induced a pro-resolution macrophage phenotype. The eicosanoid epoxyeicosatrienoic acids (EET) from arachidonic acid was anti-inflammatory in synergy with DMF. H-151 and DMF are thus candidate drugs targeting the inflammation and autoimmunity in sALS via modulation of the NFκB and cGAS/STING pathways.

Diagnosis of specific T cell immunity to the antigenic determinants of SARS-CoV-2 in patients seems to be an increasingly important task due to accumulation of data about the role of T cell immune response in course of coronavirus infection and clearance of SARS-CoV-2 in case of secondary infection. Previously, we designed the recombinant CorD_PS antigen for evaluation of T cell antiviral immunity, containing conservative and immunogenic sequences of structural proteins of the SARS-CoV-2 coronavirus. E. coli CorD_PS producing strain with stable expression of the recombinant CorD_PS antigen was obtained. Aim of the present work is to design a laboratory technology for the production of recombinant antigen CorD_PS, to control the quality of the obtained chimeric protein and to study its immunological activity. Development of the cultivation conditions for E. coli CorD_PS cells was carried out in conical flasks in LB-M_Km medium at 37 °C, then scaled in a fermenter with a volume of 30 liters. Expression was induced by the addition of IPTG. Expression was controlled in culture lysates in 12% SDS-PAGE. The resulting biomass was lysed using an ultrasonic disintegrator with followed by centrifugation. The compositions of lysing and solubilizing buffers were selected, as well as conditions for refolding of the recombinant protein. Cation exchange (SP-sepharose), hydrophobic (Butylsepharose) and exclusive (Sephacryl S-200 HR) chromatography were used sequentially to purify the dissolved protein. Protein impurities in the preparation were determined by reverse-phase HPLC and electrophoresis in 12% SDS-PAGE, residual lipopolysaccharides were determined using a gel-thrombin variant of the LAL test, residual proteins of the producer strain were determined by enzyme immunoassay, residual DNA of the producer strain was determined by binding with PicoGreen dye. Specificity was controlled by indirect enzyme immunoassay. The evaluation of cytokine production by CD4+T lymphocytes in response to their stimulation by recombinant antigen ex vivo was performed on a flow cytofluorimeter. The biomass yield during cultivation of E. coli CorD_PS in a 30L fermenter was up to 20 g/L for 4 hours of 0.1 mM IPTG induction. Sequential washing of inclusion bodies from bacterial cellular components and their subsequent solubilization in a buffer containing 8 M urea allowed to obtain a solution of denatured antigen with a concentration of 10 mg/mL. The efficiency of refolding by dilution was 75%. After three stages of chromatographic purification, protein samples with a concentration of 1.2-1.4 mg/mL, HPLC purity of 98.43%, corresponding to key quality parameters according to the OFS.1.7.1.0007.15, were obtained. The recombinant antigen showed specific binding to a sample of the First WHO International Standard and sample Company Reference Standard No. 3 of anti- SARS-CoV-2 human immunoglobulins in a specified concentration range. CD4+T lymphocytes effectively responded to recombinant antigen treatment by increasing IFNγ production. The optimal concentration of recombinant coronavirus antigen was 5 μg/mL. The developed technological process makes it possible to obtain 5-7 grams of coronavirus recombinant CorD_PS antigen in one cultivation cycle in 30 liters of fermentation medium with key quality parameters according to the OFS.1.7.1.0007.15. As a result of specific immunological activity studies of the recombinant coronavirus antigen CorD_PS, the concept of its possible use as a diagnostic tool for determining the formation of a T cell immune response was confirmed.

Natural killer (NK) cells are crucial components of innate immunity, known for their potent tumor surveillance abilities. Chimeric antigen receptors (CARs) have shown promise in cancer targeting, but optimizing CAR designs for NK cell functionality remains challenging. CAR-NK cells have gained attention for their potential to reduce side effects and enable scalable production in cancer immunotherapy. This study aimed to enhance NK cell anti-tumor activity by incorporating PD1-synthetic Notch (synNotch) receptors. A chimeric receptor was designed using UniProt database sequences, and 3D structure models were generated for optimization. Lentiviral transduction was used to introduce PD1-Syn receptors into NK cells. The expression of PD1-Syn receptors on NK cell surfaces was assessed. Engineered NK cells were co-cultured with PDL1+ breast cancer cells to evaluate their cytotoxic activity and ability to produce interleukin-12 (IL-12) and interferon-gamma (IFNγ) upon interaction with the target cells. This study successfully expressed the PD1-Syn receptors on NK cells. CAR-NK cells secreted IL-12 and exhibited target-dependent IFNγ production when engaging PDL1+ cells. Their cytotoxic activity was significantly enhanced in a target-dependent manner. This study demonstrates the potential of synNotch receptor-engineered NK cells in enhancing anti-tumor responses, especially in breast cancer cases with high PDL1 expression.

BackgroundInterferon-gamma (IFNγ) exerts potent growth inhibitory effects on a wide range of cancer cells through unknown signaling pathways. We pursued complementary screening approaches to characterize the growth inhibition pathway.MethodsWe performed chemical genomics and whole genome targeting CRISPR/Cas9 screens using patient-derived melanoma lines to uncover essential nodes in the IFNγ-mediated growth inhibition pathway. We used transcriptomic profiling to identify cell death pathways activated upon IFNγ exposure. Live imaging experiments coupled with apoptosis assays confirmed the involvement of these pathways in IFNγ-mediated cell death.ResultsWe show that IFNγ signaling activated ERK. Blocking ERK activation rescued IFNγ-mediated apoptosis in 17 of 23 (~ 74%) cell lines representing BRAF, NRAS, NF1 mutant, and triple wild type subtypes of cutaneous melanoma. ERK signaling induced a stress response, ultimately leading to apoptosis through the activity of DR5 and NOXA proteins.ConclusionsOur results provide a new understanding of the IFNγ growth inhibition pathway, which will be crucial in defining mechanisms of immunotherapy response and resistance.

BackgroundAmbient air pollutant (AAP) exposure is associated with adverse pregnancy outcomes, such as preeclampsia, preterm labor, and low birth weight. Previous studies have shown methylation of immune genes associate with exposure to air pollutants in pregnant women, but the cell-mediated response in the context of typical pregnancy cell alterations has not been investigated. Pregnancy causes attenuation in cell-mediated immunity with alterations in the Th1/Th2/Th17/Treg environment, contributing to maternal susceptibility. We recruited women (n = 186) who were 20 weeks pregnant from Fresno, CA, an area with chronically elevated AAP levels. Associations of average pollution concentration estimates for 1 week, 1 month, 3 months, and 6 months prior to blood draw were associated with Th cell subset (Th1, Th2, Th17, and Treg) percentages and methylation of CpG sites (IL4, IL10, IFNγ, and FoxP3). Linear regression models were adjusted for weight, age, season, race, and asthma, using a Q value as the false-discovery-rate-adjusted p-value across all genes.ResultsShort-term and mid-term AAP exposures to fine particulate matter (PM2.5), nitrogen dioxide (NO2) carbon monoxide (CO), and polycyclic aromatic hydrocarbons (PAH456) were associated with percentages of immune cells. A decrease in Th1 cell percentage was negatively associated with PM2.5 (1 mo/3 mo: Q

Bovine Th2 cells have usually been characterized by IL4 mRNA expression, but it is unclear whether their IL4 protein expression corresponds to transcription. We found that grass-fed healthy beef cattle, which had been regularly exposed to parasites on the grass, had a low frequency of IL4+ Th2 cells during flow cytometry, similar to animals grown in feedlots. To assess the distribution of IL4+ CD4+ T cells across tissues, samples from the blood, spleen, abomasal (draining), and inguinal lymph nodes were examined, which revealed limited IL4 protein detection in the CD4+ T cells across the examined tissues. To determine if bovine CD4+ T cells may develop into Th2 cells, naïve cells were stimulated with anti-bovine CD3 under a Th2 differentiation kit in vitro. The cells produced primarily IFNγ proteins, with only a small fraction (<10%) co-expressing IL4 proteins. Quantitative PCR confirmed elevated IFNγ transcription but no significant change in IL4 transcription. Surprisingly, GATA3, the master regulator of IL4, was highest in naïve CD4+ T cells but was considerably reduced following differentiation. To determine if the differentiated cells were true Th2 cells, an unbiased proteomic assay was carried out. The assay identified 4212 proteins, 422 of which were differently expressed compared to those in naïve cells. Based on these differential proteins, Th2-related upstream components were predicted, including CD3, CD28, IL4, and IL33, demonstrating typical Th2 differentiation. To boost IL4 expression, T cell receptor (TCR) stimulation strength was reduced by lowering anti-CD3 concentrations. Consequently, weak TCR stimulation essentially abolished Th2 expansion and survival. In addition, extra recombinant bovine IL4 (rbIL4) was added during Th2 differentiation, but, despite enhanced expansion, the IL4 level remained unaltered. These findings suggest that, while bovine CD4+ T cells can respond to Th2 differentiation stimuli, the bovine IL4 pathway is not regulated in the same way as in mice and humans. Furthermore, Ostertagia ostertagi (OO) extract, a gastrointestinal nematode in cattle, inhibited signaling via CD3, CD28, IL4, and TLRs/MYD88, indicating that external pathogens can influence bovine Th2 differentiation. In conclusion, though bovine CD4+ T cells can respond to IL4-driven differentiation, IL4 expression is not a defining feature of differentiated bovine Th2 cells. [ABSTRACT FROM AUTHOR]

Primary Sjögren's syndrome (pSS) is characterized by its autoimmune nature. This study investigates the role of the IFNγ SNP rs2069705 in modulating the susceptibility to pSS. Differential expression of IFNγ and BAFF was analyzed using the GEO database's mRNA microarray GSE84844. Genotyping of the IFNγ SNP rs2069705 was conducted via the dbSNP website. The JASPAR tool was used for predicting transcription factor bindings. Techniques such as dual-luciferase reporter assays, Chromatin immunoprecipitation, and analysis of a pSS mouse model were applied to study gene and protein interactions. A notable increase in the mutation frequency of IFNγ SNP rs2069705 was observed in MNCs from the exocrine glands of pSS mouse models. Bioinformatics analysis revealed elevated levels of IFNγ and BAFF in pSS samples. The model exhibited an increase in both CD20+ B cells and cells expressing IFNγ and BAFF. Knocking down IFNγ resulted in lowered BAFF expression and less lymphocyte infiltration, with BAFF overexpression reversing this suppression. Activation of the Janus kinase (JAK)/STAT1 pathway was found to enhance transcription in the BAFF promoter region, highlighting IFNγ's involvement in pSS. In addition, rs2069705 was shown to boost IFNγ transcription by promoting interaction between its promoter and STAT4. SNP rs2069705 in the IFNγ gene emerges as a pivotal element in pSS susceptibility, primarily by augmenting IFNγ transcription, activating the JAK/STAT1 pathway, and leading to B-lymphocyte infiltration in the exocrine glands. NEW & NOTEWORTHY: The research employed a combination of bioinformatics analysis, genotyping, and experimental models, providing a multifaceted approach to understanding the complex interactions in pSS. We have uncovered that the rs2069705 SNP significantly affects the transcription of IFNγ, leading to altered immune responses and B-lymphocyte activity in pSS. [ABSTRACT FROM AUTHOR]

Pregnancy-specific β1-glycoprotein (PSG), one of the most important proteins of pregnancy, has a pronounced immunosuppressive effect. Short peptides of PSG, the so-called SLiMs (short linear motifs), are promising molecules for mild immunosuppression. We studied in vitro effect of short PSG peptides (YACS, YQCE, YVCS, and YECE) on differentiation and cytokine profile of human T-regulatory lymphocytes (Treg). T helpers isolated from the peripheral blood and polarized into the Treg phenotype with a T-cell activator (anti-CD2/3/28) and the cytokines IL-2 and transforming grown factor β (TGFβ) were used. PSG peptides were shown to have no direct modulatory effect on Treg differentiation in a culture of CD4+ cells polarized to the Treg phenotype. At the same time, PSG peptides had no effect on the viability and number of CD4+ cells in the in vitro culture. PSG peptides also had no effect on the levels of TNFα, IL-8, IL-2, macrophage inflammatory protein 1β, IL-17, IL-10, IL-6, granulocyte-macrophage CSF, monocyte chemoattractant protein 1, IL-13, IL-5, IL-7, IL-12(p70), IL-1β, granulocyte CSF, IL-4, but decreased IFNγ levels. The observed ability of the YQCE peptide to reduce the production of this proinflammatory Th1 cytokine by T helper cells can be interpreted as a positive effect. Our findings can be used for further development of safe peptide drugs based on SLiMs sequences. [ABSTRACT FROM AUTHOR]

We developed pulmonary emphysema and a type 2 airway inflammation overlap mouse model. The bronchoalveolar lavage (BAL) interleukin 13 (IL‐13), IL‐4, and IL‐5 levels in the overlap model were higher than in the pulmonary emphysema model and lower than in the type 2 airway inflammation model, but IL‐33 level in the lung was higher than in other models. IL‐33 and interferon‐γ (IFNγ) in lungs may control the severity of a type 2 airway inflammation in lung. [ABSTRACT FROM AUTHOR]

In COVID-19, cytokine release syndrome can cause severe lung tissue damage leading to acute respiratory distress syndrome (ARDS). Here, we address the effects of IFNγ, TNFα, IL-1β and IL-6 on the growth arrest of alveolar A549 cells, focusing on the role of the IFN regulatory factor 1 (IRF1) transcription factor. The efficacy of JAK1/2 inhibitor baricitinib has also been tested. A549 WT and IRF1 KO cells were exposed to cytokines for up to 72 h. Cell proliferation and death were evaluated with the resazurin assay, analysis of cell cycle and cycle-regulator proteins, LDH release and Annexin-V positivity; the induction of senescence and senescence-associated secretory phenotype (SASP) was evaluated through β-galactosidase staining and the quantitation of secreted inflammatory mediators. While IL-1 and IL-6 proved ineffective, IFNγ plus TNFα caused a proliferative arrest in A549 WT cells with alterations in cell morphology, along with the acquisition of a secretory phenotype. These effects were STAT and IRF1-dependent since they were prevented by baricitinib and much less evident in IRF1 KO than in WT cells. In alveolar cells, STATs/IRF1 axis is required for cytokine-induced proliferative arrest and the induction of a secretory phenotype. Hence, baricitininb is a promising therapeutic strategy for the attenuation of senescence-associated inflammation. [ABSTRACT FROM AUTHOR]

NK cells play a decisive role in controlling hCMV infection by combining innate and adaptive-like immune reactions. The hCMV-derived VMAPRTLFL (LFL) peptide is a potent activator of NKG2C+ NK cells. Proposed here is an autologous system of LFL stimulation without T lymphocytes and exogenous cytokines that allows us to evaluate NK-cell hCMV-specific responses in more native settings. In this model, we evaluated LFL-induced IFNγ production, focusing on signaling pathways and the degranulation and proliferation of NK cells orchestrated by microenvironment cytokine production and analyzed the transcriptome of expanded NK cells. NK cells of individuals having high anti-hCMV-IgG levels, in contrast to NK cells of hCMV-seronegative and low-positive donors, displayed increased IFNγ production and degranulation and activation levels and enhanced proliferation upon LFL stimulation. Cytokine profiles of these LFL-stimulated cultures demonstrated a proinflammatory shift. LFL-induced NK-cell IFNγ production was dependent on the PI3K and Ras/Raf/Mek signaling pathways, independently of cytokines. In hCMV-seropositive individuals, this model allowed obtaining NK-cell antigen-specific populations proliferating in response to LFL. The transcriptomic profile of these expanded NK cells showed increased adaptive gene expression and metabolic activation. The results complement the existing knowledge about hCMV-specific NK-cell response. This model may be further exploited for the identification and characterization of antigen-specific NK cells. [ABSTRACT FROM AUTHOR]

Allergic contact dermatitis (ACD), a prevalent inflammatory skin disease, is elicited upon repeated skin contact with protein-reactive chemicals through a complex and poorly characterized cellular network between immune cells and skin resident cells. Here, single-cell transcriptomic analysis of the murine hapten-elicited model of ACD reveals that upon elicitation of ACD, infiltrated CD4+ or CD8+ lymphocytes were primarily the IFNγ-producing type 1 central memory phenotype. In contrast, type 2 cytokines (IL4 and IL13) were dominantly expressed by basophils, IL17A was primarily expressed by δγ T cells, and IL1β was identified as the primary cytokine expressed by activated neutrophils/monocytes and macrophages. Furthermore, analysis of skin resident cells identified a sub-cluster of dermal fibroblasts with preadipocyte signature as a prominent target for IFNγ+ lymphocytes and dermal source for key T cell chemokines CXCL9/10. IFNγ treatment shifted dermal fibroblasts from collagen-producing to CXCL9/10-producing, which promoted T cell polarization toward the type-1 phenotype through a CXCR3-dependent mechanism. Furthermore, targeted deletion of Ifngr1 in dermal fibroblasts in mice reduced Cxcl9/10 expression, dermal infiltration of CD8+ T cell, and alleviated ACD inflammation in mice. Finally, we showed that IFNγ+ CD8+ T cells and CXCL10-producing dermal fibroblasts co-enriched in the dermis of human ACD skin. Together, our results define the cell type-specific immune responses in ACD, and recognize an indispensable role of dermal fibroblasts in shaping the development of type-1 skin inflammation through the IFNGR-CXCR3 signaling circuit during ACD pathogenesis.

IntroductionSudden sensorineural hearing loss (SSNHL) is a common emergency symptom in otolaryngology that requires immediate diagnosis and treatment. SSNHL has a multifactorial etiology, and its pathophysiologic mechanisms may be associated with inflammatory and metabolic changes that may affect the cochlear microenvironment or its nervous component, thus triggering the process or hindering hearing recovery. Therefore, the aim of this study was to assess metabolic and inflammatory changes to identify systemic parameters that could serve as prognostic factors for hearing recovery in patients with SSNHL.Materials and methodsThirty patients with a sudden hearing loss of at least 30 dB in three contiguous frequencies were enrolled in this study. Patients were followed up for 4 months and peripheral blood samples were collected at 7 days (V1), 30 days (V2) and 120 days (V3). Interleukins (IL)-1F7, IL-2, IL-4, IL-5, IL-6, IL-10, interferon γ (IFN-γ), tumor necrosis factor α (TNF-α) and adiponectin were quantified in serum. In addition, lipid and glycemic profiles as well as concentration of creatinine, uric acid, fructosamine, peroxide, total proteins and albumin were analyzed. Patients underwent weekly ear-specific hearing tests with standard pure tone thresholds for frequencies of 250–8,000 Hz, speech recognition threshold and word recognition score.ResultsPatients with SSNHL were divided into a group of patients who did not achieve hearing recovery (n = 14) and another group who achieved complete and significant recovery (n = 16). Most serologic parameters showed no significant changes or values indicating clinical changes. However, IFN-γ levels decreased by 36.3% between V1 and V2. The cytokine TNF-α showed a statistically significant decrease from V1 to V3 (from 22.91 to 10.34 pg./mL). Adiponectin showed a decrease from 553.7 ng/mL in V1 to 454.4 ng/mL in V3.DiscussionOur results show that serologic cytokine levels change in the acute phase of manifestation of SSNHL and establish a parallel between systemic changes and improvements in hearing, especially TNF-α, which showed differences in hearing recovery. The use of IFN-γ, TNF-α and adiponectin may elucidate the clinical improvement in these patients.

Cervical cancer is the most common malignant tumor of the female genital organs. In general, the prognosis in patients with advanced cervical cancer is unfavorable. The option of choice for stage IV of the disease and relapses is systemic platinum-containing chemotherapy. Its effectiveness is about 20-26%, life expectancy is 12 to 13 months. Undoubtedly, the search and development of new methods of treating this disease is an extremely urgent task. Immuno-oncology has emerged as a potential new strategy to improve the treatment outcomes of patients with malignant neoplasms. Much attention in research is focused on the opportunity of using interleukin-2, tumor necrosis factor (TNF) and interferon-gamma (IFNγ) for tumor immunotherapy, since these cytokines play a special role in antitumor protection. Due to the active search for new hybrid molecules based on TNFα, some domestically developed recombinant antitumor drugs are implicated into modern practice of clinical oncologists, in particular, “Refnot” (TNFα-thymosin-α1) and “Ingaron” (IFNγ preparation). We analyzed the result of combined treatment (standard polychemotherapy at the 1st line augmented with cytokinotherapy including Refnot + Ingaron therapy) in one patient with recurrent cervical cancer. According to the control examination, upon completion of the polychemotherapy course, a complete tumor response was registered according to the Recist 1.1 criteria. The patient continued to receive cytokines as supporting therapy. Currently, according to control quarterly examinations, a complete regression of recurrent tumor persists from the end of polychemotherapy to 28 months of observation. One should note that when monitoring the state of the immune system during therapy with Refnot and Ingaron, we noted an increase in absolute and relative numbers of T cells to normal levels along with higher cytotoxic and antitumor potential of NK cells without increasing their number. The patient well tolerates the therapy, improved quality of life is documented, and there are no clinically significant side effects. Hence, the therapy with “Refnot” (TNFα-thymosin-α1) and “Ingaron” (IFNγ) in this clinical case proved to be a safe method of maintenance therapy with a positive therapeutic effect thus allowing effective control of recurrent cervical cancer for more than 2 years, as well as significantly improve quality of life of the patient. This type of therapy may be recommended for usage in clinical oncology.

In a recent report in Nature , Goto et al. reveal a novel immune-evasion mechanism adopted by early colorectal cancer (CRC) cells that is based on the transcription factor sex determining region Y (SRY)-box transcription factor 17 (SOX17). Leveraging colorectal adenoma and cancer models to perform comprehensive transcriptomic/chromatin analyses, this work shows that SOX17 generates immune-silent leucine-rich repeat-containing G protein-coupled receptor 5– (LGR5–) tumor cells, which suppress interferon gamma (IFNγ) signaling and promote immune escape. [ABSTRACT FROM AUTHOR]

• Stratification of groups according to asymptomatic COVID19-survivor status and long-COVID with and without cognitive impairment. • Cortical thickness increase in prefronto-temporal areas, most pronounced in long-COVID subgroup with cognitive impairment. • Elevated levels of IL-10, IFNγ, and sTREM2 in long-COVID patients, especially when suffering from cognitive impairment. As the heterogeneity of symptoms is increasingly recognized among long-COVID patients, it appears highly relevant to study potential pathophysiological differences along the different subtypes. Preliminary evidence suggests distinct alterations in brain structure and systemic inflammatory patterns in specific groups of long-COVID patients. To this end, we analyzed differences in cortical thickness and peripheral immune signature between clinical subgroups based on 3 T-MRI scans and signature inflammatory markers in n = 120 participants comprising healthy never-infected controls (n = 30), healthy COVID-19 survivors (n = 29), and subgroups of long-COVID patients with (n = 26) and without (n = 35) cognitive impairment according to screening with Montreal Cognitive Assessment. Whole-brain comparison of cortical thickness between the 4 groups was conducted by surface-based morphometry. We identified distinct cortical areas showing a progressive increase in cortical thickness across different groups, starting from healthy individuals who had never been infected with COVID-19, followed by healthy COVID-19 survivors, long-COVID patients without cognitive deficits (MoCA ≥ 26), and finally, long-COVID patients exhibiting significant cognitive deficits (MoCA < 26). These findings highlight the continuum of cortical thickness alterations associated with COVID-19, with more pronounced changes observed in individuals experiencing cognitive impairment (p < 0.05, FWE-corrected). Affected cortical regions covered prefrontal and temporal gyri, insula, posterior cingulate, parahippocampal gyrus, and parietal areas. Additionally, we discovered a distinct immunophenotype, with elevated levels of IL-10, IFNγ, and sTREM2 in long-COVID patients, especially in the group suffering from cognitive impairment. We demonstrate lingering cortical and immunological alterations in healthy and impaired subgroups of COVID-19 survivors. This implies a complex underlying pathomechanism in long-COVID and emphasizes the necessity to investigate the whole spectrum of post-COVID biology to determine targeted treatment strategies targeting specific sub-groups. [ABSTRACT FROM AUTHOR]

The most crucial disadvantage of DNA‐based vaccines is their low immunogenicity; therefore, finding an effectual adjuvant is essential for their development. Herein, immunostimulatory effects of IFNγ cytokine and a CD40 ligand (CD40L) costimulatory molecule are evaluated as combined with an antigen, and also linked to an antigen in mice. For this purpose, after preparation of the HIV‐1 Nef, IFNγ, and CD40L DNA constructs, and also their recombinant protein in an Escherichia coli expression system, nine groups of female BALB/c mice are immunized with different regimens of DNA constructs. About 3 weeks and also 3 months after the last injection, humoral and cellular immune responses are assessed in mice sera and splenocytes. Additionally, mice splenocytes are exposed to single‐cycle replicable (SCR) HIV‐1 virions for evaluating their potency in the secretion of cytokines in vitro. The data indicate that the linkage of IFNγ and CD40L to Nef antigen can significantly induce the Th‐1 pathway and activate cytotoxic T lymphocytes compared to other regimens. Moreover, groups receiving the IFNγ‐Nef and CD40L‐Nef fusion DNA constructs show higher secretion of IFNγ and TNF‐α from virion‐infected lymphocytes than other groups. Therefore, the IFNγ‐Nef and CD40L‐Nef fusion DNA constructs are suggested to be a potential option for development of an efficient HIV‐1 vaccine. [ABSTRACT FROM AUTHOR]

Background and Objectives: In this study, we applied one-step real time rt-PCR technology type II INF signature to blood and nasopharyngeal (NPS) swabs of acute early recovery children < 1 years hospitalized for bronchiolitis with laboratory-confirmed RSV infection. Materials and Methods: A prospective observational case–control study was conducted in 2021–2022. The study took place in Children Hospital "Regina Margherita", Torino Italy. The study included 66 infants, of which 30 patients were hospitalized for bronchiolitis due to RSV infection and 36 age-matched controls. Inclusion criteria included a positive RSV test for infants with bronchiolitis. We collected peripheral blood and nasopharyngeal swabs for relative quantification of type II Interferon signature by One-Step Multiplex PCR real time. Results: IFN levels were downregulated in the peripheral blood of bronchiolitis patients; these data were not confirmed in the nasopharyngeal swab. There was no correlation between NPS and the type II IFN score in peripheral blood. Conclusions: our study shows for the first time that type II IFN score was significant reduced in peripheral blood of infants with bronchiolitis by RSV compared to age-matched healthy controls; in the NPS swab this resulted downregulation was not statistically significant and the type II IFN score in the NPS swab can be used as marker of resolution of infection or improvement of clinical conditions. [ABSTRACT FROM AUTHOR]

Aging affects the number and function of gamma-delta (γδ) T cells in a tissue-specific manner, modifying the risk for inflammatory disease. These aging-related γδT-cell variations in gingival tissues that could increase the risk for inflammation and periodontal disease remain unknown. Here we sought to identify quantitative and qualitative variations in gingival γδT cells associated with aging that could have an impact in oral immunoinflammatory responses. For this, gingival tissues from young (4 mo) and aged (24 mo) male and female mice were collected and analyzed by flow cytometry. Cell suspensions were stimulated and stained with eFluor450 (cell viability), anti-CD45 (hematopoietic cells), anti-CD3 (lymphocytes), anti-TCRγδ (γδT cells), anti–IL-15rα (cell proliferation), and anti–Notch-3 (senescence marker). Detection of intracellular cytokines IL-17A and interferon γ (IFNγ) was performed. Gingival expression of specific γ- and δ-chains and cytokines was evaluated by quantitative reverse transcription polymerase chain reaction. A significantly higher number of IL-17A–producing γδT cells and IL-17A expression levels were observed in gingival tissues from aged females but not males. Similarly, the number of gingival Notch-3+ γδT cells increased with aging only in females. IL-15rα was not detected in gingival γδT cells. Chains γ1, 2, 4, 5, 6, and 7 as well as δ1, 2, 4, and 6 were detected. Detection levels of all γ chains except γ1 as well as δ1 and δ2 changed with aging in males, females, or both. Interestingly, number of IL-17A–producing conventional T cells similarly increased with aging only in females. Both sexes showed increased IFNγ+ conventional T-cell numbers with aging; however, it reached significance only in females. In conclusion, the number of gingival IL-17A–producing γδT cells and IL-17A expression increase naturally with aging specifically in females. This sexual dimorphism in gingival γδT and conventional Th17 cell numbers and phenotypes suggests distinct aging-related mechanisms of periodontitis in males and females. [ABSTRACT FROM AUTHOR]

Le syndrome d'activation macrophagique (SAM) est l'une des complications graves de la maladie de Still tant chez l'enfant que chez l'adulte. Dans l'état actuel des connaissances, sa pathogénie implique l'activation d'acteurs de l'immunité à la fois innée et acquise, notamment les monocytes, les cellules NK et certaines populations lymphocytaires CD8 positives. L'interleukine 18 (IL-18) et l'interféron γ (IFN-γ) semblent jouer un rôle central. La détection d'un SAM doit être une préoccupation constante au cours de la maladie de Still. Les signes d'alerte sont l'apparition d'une cytopénie d'une ou plusieurs lignées sanguines, l'augmentation progressive des enzymes hépatiques, des LDH, des triglycérides et de la ferritine sérique, la chute du fibrinogène et l'activation des voies de coagulation. Le diagnostic de SAM peut être facilité par l'utilisation de scores spécifiques du SAM (H ou M scores) ou des critères de classification. Le traitement du SAM varie selon sa sévérité et peut comprendre les glucocorticoïdes par voie intraveineuse à forte dose, les immunoglobulines polyvalentes par voie intraveineuse, les inhibiteurs de la voie de l'IL-1 tel que l'anakinra, la ciclosporine, voire des inhibiteurs de la voie de l'IFN-γ. L'utilisation de l'étoposide (VP-16) est réservée aux formes les plus graves. Macrophage activation syndrome (MAS) is one of the serious complications of Still's disease in both children and adults. According to current knowledge, its pathogenesis involves the activation of both innate and acquired immune players, notably monocytes, NK cells and specific CD8-positive lymphocyte populations. Interleukin 18 (IL-18) and interferon γ (IFN-γ) appear to play a central role. The detection of MAS should be a constant concern in Still's disease. Warning signs include the occurrence of cytopenia of one or more cell lines, a progressive increase in liver enzymes, LDH, triglycerides and serum ferritin, a fall in fibrinogen and activation of coagulation pathways. Diagnosis of MAS can be facilitated by the use of specific MAS scores (H or M scores) or classification criteria. MAS treatment varies according to its severity, and may include high-dose intravenous glucocorticoids, intravenous polyvalent immunoglobulins, inhibitors of the IL-1 pathway such as anakinra, ciclosporin, or even inhibitors of the IFN-γ pathway. Etoposide (VP-16) is reserved for the most severe forms. [ABSTRACT FROM AUTHOR]

Abstract We developed pulmonary emphysema and a type 2 airway inflammation overlap mouse model. The bronchoalveolar lavage (BAL) interleukin 13 (IL‐13), IL‐4, and IL‐5 levels in the overlap model were higher than in the pulmonary emphysema model and lower than in the type 2 airway inflammation model, but IL‐33 level in the lung was higher than in other models. IL‐33 and interferon‐γ (IFNγ) in lungs may control the severity of a type 2 airway inflammation in lung.

ABSTRACT Toxoplasma gondii is an intracellular parasite that can activate the NLRP1 inflammasome leading to macrophage pyroptosis in Lewis rats, but the underlying mechanism is not well understood. In this study, we performed a genome-wide CRISPR screen and identified the dense granule proteins GRA35, GRA42, and GRA43 as the Toxoplasma effectors mediating cell death in Lewis rat macrophages. GRA35 localizes on the parasitophorous vacuole membrane, where it interacts with the host E3 ubiquitin ligase ITCH. Inhibition of proteasome activity or ITCH knockout prevented pyroptosis in Toxoplasma-infected Lewis rat macrophages, consistent with the “NLRP1 functional degradation model.” However, there was no evidence that ITCH directly ubiquitinates or interacts with rat NLRP1. We also found that GRA35-ITCH interaction affected Toxoplasma fitness in IFNγ-activated human fibroblasts, likely due to ITCH’s role in recruiting ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane. These findings identify a new role of host E3 ubiquitin ligase ITCH in mediating effector-triggered immunity, a critical concept that involves recognizing intracellular pathogens and initiating host innate immune responses.IMPORTANCEEffector-triggered immunity represents an innate immune defense mechanism that plays a crucial role in sensing and controlling intracellular pathogen infection. The NLRP1 inflammasome in the Lewis rats can detect Toxoplasma infection, which triggers proptosis in infected macrophages and eliminates the parasite’s replication niche. The work reported here revealed that host E3 ubiquitin ligase ITCH is able to recognize and interact with Toxoplasma effector protein GRA35 localized on the parasite-host interface, leading to NLRP1 inflammasome activation in Lewis rat macrophages. Furthermore, ITCH-GRA35 interaction contributes to the restriction of Toxoplasma in human fibroblasts stimulated by IFNγ. Thus, this research provides valuable insights into understanding pathogen recognition and restriction mediated by host E3 ubiquitin ligase.

Older kidney transplant recipients demonstrate increased rates of infection, and lower rates of rejection, compared with younger kidney transplant recipients. However, the mechanism behind this observation remains unknown. To develop a multifaceted view of age-associated immune dysfunction, we determined the function and phenotype of T cells predisposing to vulnerability to infection on a molecular level. Overlapping peptide pools representing the dominant CMV antigens were used to stimulate PBMC collected from 51 kidney transplant recipients, using cytokine secretion to determine specificity and intensity of response. Staphylococcal endotoxin B (SEB) was analyzed in parallel. To define immune cell subsets, we used single cell RNA sequencing (scRNAseq) to evaluate cellular surface markers and gene expression. We found increased frequency of SEB- and CMV-specific T cells was associated with freedom from infection, especially in older patients. Spatialized t-SNE analysis revealed decreased frequency of naïve T cells, increased frequency of TEMRA cells, and decreased frequency of IFNγ secreting T cells in patients with infection. Application of scRNAseq analysis revealed increased frequency of terminally differentiated T cells expressing NK-associated receptors and inhibitory markers. These findings offer unique insight into the mechanism behind vulnerability to infection in the kidney transplant recipient, revealing a specific T cell subtype of impaired antigen response and terminal effector phenotype as markers of T cell senescence.

The immunological factors can play an important role as predictive and prognostic biomarkers in oncopathology. Recently, non-conventional innate-like γδT-lymphocytes have received a lot of attention as a promising effector cell population for cancer immunotherapy. This study describes structural and functional subpopulations of γδT lymphocytes involved in antitumor immunity in patients with malignant and benign tumors of the nasal cavity and paranasal sinuses. The aim of the study was to estimate γδT cell subsets composition and functions in patients with neoplasms of nasal cavity and paranasal sinuses in order to characterize cellular immunity in tumor-associated pathological process.The peripheral venous blood was obtained from 21 patients (13 men and 8 women, average age of 63.0 (56.0-69.0) y. o.) with neoplasms of nasal cavity and paranasal sinuses, and 10 healthy donors. Lymphoid cells phenotype and production of intracellular cytokines were investigated using monoclonal antibodies and flow cytometry, production of extracellular cytokines was measured using enzyme-linked immunosorbent assay kits.The increase of total γδT cells number in patients with squamous cell carcinoma accompanied by changes in Vγ2+/Vγ1+T cells ratio in peripheral blood of both patients’ groups with malignant and benign nasal cavity and paranasal sinuses tumors were revealed as compared to healthy donors. The upregulated γδT cell response to phosphoantigen induction in combination with reduced indices of stimulations were shown in the both patients groups but cytokine profile was different, i.e., the elevated IFNγ production has been determined in patients with squamous cell carcinoma. However, in patients with inverted papilloma, redistribution of γδT cell subsets has been associated with IL-17-producing γδT cells. Moreover, the percent of IFNγ+γδT lymphocytes did correlate with IFNγ concentration in cell culture supernatants of patients with malignant nasal cavity and paranasal sinuses neoplasms (R = 0.61; p < 0.05).The revealed data suggest an involvement of γδT lymphocytes in malignant and benign tumor pathogenesis and may provide a fundamental basis for further detection of possible tumor-associated inflammation and malignization predictors.

Abstract Background Interferon-gamma (IFNγ) exerts potent growth inhibitory effects on a wide range of cancer cells through unknown signaling pathways. We pursued complementary screening approaches to characterize the growth inhibition pathway. Methods We performed chemical genomics and whole genome targeting CRISPR/Cas9 screens using patient-derived melanoma lines to uncover essential nodes in the IFNγ-mediated growth inhibition pathway. We used transcriptomic profiling to identify cell death pathways activated upon IFNγ exposure. Live imaging experiments coupled with apoptosis assays confirmed the involvement of these pathways in IFNγ-mediated cell death. Results We show that IFNγ signaling activated ERK. Blocking ERK activation rescued IFNγ-mediated apoptosis in 17 of 23 (~ 74%) cell lines representing BRAF, NRAS, NF1 mutant, and triple wild type subtypes of cutaneous melanoma. ERK signaling induced a stress response, ultimately leading to apoptosis through the activity of DR5 and NOXA proteins. Conclusions Our results provide a new understanding of the IFNγ growth inhibition pathway, which will be crucial in defining mechanisms of immunotherapy response and resistance.

BackgroundAutism, a childhood behavioral disorder, belongs to a large suite of diseases, collectively referred to as autism spectrum disorders (ASD). Though multifactorial in etiology, approximately 10% of ASD are associated with atopic dermatitis (AD). Moreover, ASD prevalence increases further as AD severity worsens, though these disorders share no common causative mutations. We assessed here the link between these two disorders in the standard, valproic acid mouse model of ASD. In prior studies, there was no evidence of skin involvement, but we hypothesized that cutaneous involvement could be detected in experiments conducted in BALB/c mice. BALB/c is an albino, laboratory-bred strain of the house mouse and is among the most widely used inbred strains used in animal experimentation.MethodsWe performed our studies in valproic acid (VPA)-treated BALB/c hairless mice, a standard mouse model of ASD. Mid-trimester pregnant mice received a single intraperitoneal injection of either valproic acid sodium salt dissolved in saline or saline alone on embryonic day 12.5 and were housed individually until postnatal day 21. Only the brain and epidermis appeared to be affected, while other tissues remain unchanged. At various postnatal time points, brain, skin and blood samples were obtained for histology and for quantitation of tissue sphingolipid content and cytokine levels.ResultsAD-like changes in ceramide content occurred by day one postpartum in both VPA-treated mouse skin and brain. The temporal co-emergence of AD and ASD, and the AD phenotype-dependent increase in ASD prevalence correlated with early appearance of cytokine markers (i.e., interleukin [IL]-4, 5, and 13), as well as mast cells in skin and brain. The high levels of interferon (IFN)γ not only in skin, but also in brain likely account for a significant decline in esterified very-long-chain N-acyl fatty acids in brain ceramides, again mimicking known IFNγ-induced changes in AD.ConclusionBaseline involvement of both AD and ASD could reflect concurrent neuro- and epidermal toxicity, possibly because both epidermis and neural tissues originate from the embryonic neuroectoderm. These studies illuminate the shared susceptibility of the brain and epidermis to a known neurotoxin, suggesting that the atopic diathesis could be extended to include ASD.

We have recently shown that IFNγ, produced during cancer therapy, induces expression of the Bcl3 proto‐oncogene in ovarian cancer (OC) cells, resulting in their increased proliferation, migration, and invasion, but the mechanisms are unknown. Here, we demonstrate that the IFNγ‐induced Bcl3 expression is dependent on JAK1 and STAT1 signaling, and on p65 NFκB. Furthermore, the IFNγ‐induced Bcl3 expression is associated with an increased occupancy of Ser‐727 phosphorylated STAT1 and acetylated histone H3 at the Bcl3 promoter. Our data indicate that Bcl3 promotes expression of the pro‐inflammatory chemokine interleukin‐8 (IL‐8) in OC cells. These findings identify Bcl3 as a novel target of IFNγ/JAK1/STAT1 signaling and suggest that targeting the JAK1/STAT1 pathway may suppress IFNγ‐induced Bcl3 expression in OC.

The prevalence of thermal trauma, the high risk of infectious and non-infectious short- and long- term complications, and the limited effectiveness of the therapeutic approaches used are prerequisites for the search and pathogenetic justification of new therapies, among which the endogenous homeostasis regulator with pleiotropic properties melatonin attracts attention.The aim of the work is to investigate the immunological aspects of intraperitoneal use of melatonin (MT) in experimental thermal trauma (TT).The work was performed on 158 rats of the Wistar line, grade III TT and a relative area of 3.5% were simulated by skin immersion in water at 98-99 °C for 12 s. MT was administered intraperitoneally daily at a dose of 10 mg/kg for 5 days. The quantitative composition of blood cells was evaluated on a hematological analyzer. Plasma concentrations of IL-4, TNFa, IFNg, and CRP were determined on an automatic enzyme immunoassay using rat-specific test systems, and MT by capillary electrophoresis.With experimental TT, against the background of a progressive increase in the number of leukocytes in the blood from 5 to 20 days due to neutrophils, monocytes, basophils, the number of lymphocytes decreases. With TT, the concentration of CRP increases in serum on days 5 and 10. The content of TNFa, IL-4 increases on days 5, 10 and 20 in the absence of significant changes in the concentration of IFNg. The concentration of serum MT does not change significantly. Intraperitoneal use of MT in TT leads to a partial restoration of the number of lymphocytes in the blood on day 5. Evaluation of the cytokine profile in serum revealed a decrease in the concentration of TNFa on days 10 and 20, no significant changes in the concentration of IL-4 and IFNg were recorded, the concentration of CRP decreased on day 5. The concentration of serum MT increases by 5 days.With TT on the 5th, 10th, 20th day of the experiment, the number of neutrophils, monocytes, basophils in the blood increases, decreases – lymphocytes, the serum content of CRP, TNFa, IL-4 increases, the content of IFNg and melatonin does not change. Intraperitoneal use of MT in TT partially restores the number of lymphocytes in the blood, the concentration of CRP, TNFa. A decrease in serum concentrations of TNFa and CRP in TT under the conditions of MT use suggests a limitation of the acute phase response as a consequence of the antioxidant, anti-inflammatory effect of MT, which can accelerate healing and reduce the area of the lesion of TT.

ABSTRACTAmong cancer immunotherapy, which has received great attention in recent years, cancer vaccines can potentially prevent recurrent tumors by using the exquisite power and specificity of the immune system. Specifically, whole tumor cell vaccines (WTCVs) based on surgically resected tumors have been considered to elicit robust anti-tumor immune responses by exposing various tumor-associated antigens to host immunity. However, most tumors have little immunogenicity because of immunoediting by continuous interactions with host immunity; thus, preparing WTCVs based on patient-derived non-modified tumors cannot prevent tumor onset. Hence, the immunogenicity of tumor cells must be improved for effective WTCVs. In this study, we indicate the importance of the interferon regulatory factor 7 (Irf7) axis, including Irf7 and its downstream factors, within tumor cells in regulating immunogenicity. Indeed, WTCVs that augmented the Irf7 axis have exerted remarkable recurrence-preventive effects when vaccinated after tumor inactivation by radiation. Most notably, vaccination with murine colon cancer cells that enhanced the Irf7 axis prevented the development of challenged tumors in all mice and resulted in a 100% survival rate during the observation period. Furthermore, the mechanism leading to vaccine effectiveness was mediated by interferon-gamma-producing B cells. This study provides novel insights into how to enhance tumor immunogenicity and use WTCVs as recurrence prophylaxis.

Physiologically aged lungs are prone to senescence-associated pulmonary diseases (SAPD). This study aimed to determine the mechanism and subtype of aged T cells affecting alveolar type II epithelial (AT2) cells, which promote the pathogenesis of senescence-associated pulmonary fibrosis (SAPF). Cell proportions, the relationship between SAPD and T cells, and the aging- and senescence-associated secretory phenotype (SASP) of T cells between young and aged mice were analyzed using lung single-cell transcriptomics. SAPD was monitored by markers of AT2 cells and found to be induced by T cells. Furthermore, IFNγ signaling pathways were activated and cell senescence, SASP, and T cell activation were shown in aged lungs. Physiological aging led to pulmonary dysfunction and TGF-ß1/IL-11/MEK/ERK (TIME) signaling-mediated SAPF, which was induced by senescence and SASP of aged T cells. Especially, IFNγ was produced by the accumulated CD4+ effector memory T (TEM) cells in the aged lung. This study also found that physiological aging increased pulmonary CD4+ TEM cells, IFNγ was produced mainly by CD4+ TEM cells, and pulmonary cells had increased responsiveness to IFNγ signaling. Specific regulon activity was increased in T cell subclusters. IFNγ transcriptionally regulated by IRF1 in CD4+ TEM cells promoted the epithelial-to-mesenchymal transition by activating TIME signaling and cell senescence of AT2 cells with aging. Accumulated IRF1+CD4+ TEM produced IFNγ in lung with aging and anti-IRF1 primary antibody treatment inhibited the expression of IFNγ. Aging might drive T cell differentiation toward helper T cells with developmental trajectories and enhance cell interactions of pulmonary T cells with other surrounding cells. Thus, IFNγ transcribed by IRF1 in CD4+ effector memory T cells promotes SAPF. IFNγ produced by CD4+ TEM cells in physiologically aged lungs could be a therapeutic target for preventing SAPF. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: The loss of pericytes surrounding the retinal vasculature in early diabetic retinopathy underlies changes to the neurovascular unit that lead to more destructive forms of the disease. However, it is unclear which changes lead to loss of retinal pericytes. This study investigated the hypothesis that chronic increases in one or more inflammatory factors mitigate the signalling pathways needed for pericyte survival. Methods: Loss of pericytes and levels of inflammatory markers at the mRNA and protein levels were investigated in two genetic models of diabetes, Ins2Akita/+ (a model of type 1 diabetes) and Leprdb/db (a model of type 2 diabetes), at early stages of diabetic retinopathy. In addition, changes that accompany gliosis and the retinal vasculature were determined. Finally, changes in retinal pericytes chronically incubated with vehicle or increasing amounts of IFNγ were investigated to determine the effects on pericyte survival. The numbers of pericytes, microglia, astrocytes and endothelial cells in retinal flatmounts were determined by immunofluorescence. Protein and mRNA levels of inflammatory factors were determined using multiplex ELISAs and quantitative reverse transcription PCR (qRT-PCR). The effects of IFNγ on the murine retinal pericyte survival-related platelet-derived growth factor receptor β (PDGFRβ) signalling pathway were investigated by western blot analysis. Finally, the levels of cell death-associated protein kinase C isoform delta (PKCδ) and cleaved caspase 3 (CC3) in pericytes were determined by western blot analysis and immunocytochemistry. Results: The essential findings of this study were that both type 1 and 2 diabetes were accompanied by a similar progression of retinal pericyte loss, as well as gliosis. However, inflammatory factor expression was dissimilar in the two models of diabetes, with peak expression occurring at different ages for each model. Retinal vascular changes were more severe in the type 2 diabetes model. Chronic incubation of murine retinal pericytes with IFNγ decreased PDGFRβ signalling and increased the levels of active PKCδ and CC3. Conclusions/interpretation: We conclude that retinal inflammation is involved in and sustains pericyte loss as diabetic retinopathy progresses. Moreover, IFNγ plays a critical role in reducing pericyte survival in the retina by reducing activation of the PDGFRβ signalling pathway and increasing PKCδ levels and pericyte apoptosis. [ABSTRACT FROM AUTHOR]

Despite the important role of gut microbiota in the maturation of the immune system, little is known about its impact on the development of T-cell responses to vaccination. Here, we immunized C57BL/6 mice with a prime-boost regimen using DNA plasmid, the Chimpanzee Adenovirus, and the modified Vaccinia Ankara virus expressing a candidate HIV T-cell immunogen and compared the T-cell responses between individuals with an intact or antibiotic-depleted microbiota. Overall, the depletion of the gut microbiota did not result in significant differences in the magnitude or breadth of the immunogen-specific IFNγ T-cell response after vaccination. However, we observed marked changes in the serum levels of four cytokines after vaccinating microbiota-depleted animals, particularly a significant reduction in IL-22 levels. Interestingly, the level of IL-22 in serum correlated with the abundance of Roseburia in the large intestine of mice in the mock and vaccinated groups with intact microbiota. This short-chain fatty acid (SCFA)-producing bacterium was significantly reduced in the vaccinated, microbiota-depleted group. Therefore, our results indicate that, although microbiota depletion reduces serum levels of IL-22, the powerful vaccine regime used could have overcome the impact of microbiota depletion on IFNγ-producing T-cell responses. [ABSTRACT FROM AUTHOR]

Innate CD8 T cells are proinflammatory effector T cells that achieve functional maturation in the thymus prior to their export into and maturation in peripheral tissues. Innate CD8 T cells produce the Th1 cytokine IFNγ but depend on the Th2 cytokine IL-4 for their generation. Thus, innate CD8 T cells can permute the intrathymic cytokine milieu by consuming a Th2 cytokine but driving a Th1 cytokine response. The cellular source of IL-4 is the NKT2 subset of invariant NKT (iNKT) cells. Consequently, NKT2 deficiency results in the lack of innate CD8 T cells. Whether NKT2 is the only iNKT subset and whether IL-4 is the only cytokine required for innate CD8 T cell generation, however, remains unclear. Here, we employed a mouse model of NKT1 deficiency, which is achieved by overexpression of the cytokine receptor IL-2Rβ, and assessed the role of other iNKT subsets and cytokines in innate CD8 T cell differentiation. Because IL-2Rβ-transgenic mice failed to generate both NKT1 and innate CD8 T cells, we postulated an in vivo requirement for IFNγ-producing NKT1 cells for innate CD8 T cell development. In-depth analyses of IL-2Rβ-transgenic mice and IFNγ-deficient mice, however, demonstrated that neither NKT1 nor IFNγ was required to induce Eomes or to drive innate CD8 T cell generation. Instead, in vivo administration of recombinant IL-4 sufficed to restore the development of innate CD8 T cells in NKT1-deficient mice, affirming that intrathymic IL-4, and not IFNγ, is the limiting factor and key regulator of innate CD8 T cell generation in the thymus. [ABSTRACT FROM AUTHOR]