Higher Interferon-γ Research Articles

QuantiFERON-CMV and monitor predict cytomegalovirus, mortality, and graft-versus-host disease in transplant recipients.

Cytomegalovirus (CMV) is the most prevalent infection in recipients of hematopoietic stem cell transplant (HSCT). QuantiFERON-CMV (QF-CMV) and QuantiFERON-Monitor (QFM) assays were used to test whether immune-competent adult allogeneic HSCT recipients with CMV-specific T cells can control CMV infection or reactivation. Our data demonstrated a significant correlation between CMV infection measured by CMV-antigenemia test and QF-CMV results, graft versus host disease (GvHD), and mortality rates. The QF-CMV test revealed that CMV-specific T cells with higher interferon-γ (IFN-γ) release were correlated with lower CMV infection rates. There was a significant negative association between QF-CMV results, GvHD, and mortality rates. Data showed that a one-unit rise in IFN-γ was linked with a 12.7% reduction in GvHD and a 20.7% reduction in the mortality odds ratio. In addition, a negative correlation was found between QF-M results and CMV infection, with the QFM test predicting protection against CMV infection by 1.9%. This is one of the few studies establishing the QF-CMV test's predictive value for GvHD and mortality, its use to monitor HSCT patients for pre-emptive therapy, and the use of the QFM test to predict CMV infection and mortality in HSCT patients. Thus, these assays could be utilized to optimize preventive and pre-emptive therapy procedures to reduce transplant recipient adverse effects and posttransplant therapy costs.

Clinical Differences between Posner-Schlossman Syndrome Patients with Intermittent Intraocular Pressure Elevation and Glaucomatous Damage

Introduction: Although there is abundant evidence that Posner-Schlossman syndrome (PSS) can lead to secondary glaucoma, data on the clinical differences between PSS patients with secondary glaucoma and those with intermittent intraocular pressure (IOP) elevation are sparse. Methods: This retrospective observational study included 52 patients (52 eyes) diagnosed with PSS and admitted to Zhongnan Hospital of Wuhan University between January 2019 and February 2022. Demographic characteristics and clinical features were gathered from admission records. Patients were divided into two groups: 27 cases with intermittent IOP elevation (group A) and 25 cases with secondary glaucoma (group B and C). Of the secondary glaucoma cases, 18 were further divided into the topical IOP-lowering medications group (group B) and 7 into the glaucoma surgery group (group C). Clinical characteristics of different groups were compared. Results: Compared to the intermittent IOP elevation group, PSS patients with secondary glaucoma had a longer course of disease, a higher incidence of iris depigmentation, lower best corrected visual acuity, lower endothelial cell density, and higher interferon-γ (IFN-γ) concentration and cytomegalovirus (CMV) deoxyribonucleic acid (DNA) copy number in the aqueous humor (all p < 0.05). Group C presented a higher CMV DNA copy number in the aqueous humor than groups A and B (p < 0.05). Compound trabeculectomy proved effective in group C, with a functional filter bleb and well-controlled IOP without disease progression after 1 year of follow-up. Conclusion: Distinctive characteristics existed between PSS patients with secondary glaucoma and those with intermittent IOP elevation. Compound trabeculectomy appears to be an effective treatment option when IOP cannot be controlled through topical medications.

Abstract PS10-04: Tucatinib favourably modulates the immune microenvironment and synergises with anti-PD1 therapy in a trastuzumab resistant HER2+ murine model

Abstract Background: The efficacy of PD-(L)1 inhibitors in patients with trastuzumab-resistant advanced HER2+ breast cancer is poor. Although many HER2 targeted therapies are used clinically, their effect on the tumor immune microenvironment (TME) and whether this contributes to efficacy is not understood. Tucatinib is a potent, highly selective, HER2 small molecule tyrosine kinase inhibitor with proven clinical benefit in the advanced setting. Methods: We used two immunocompetent, HER2+ murine cancer models (trastuzumab-sensitive H2N113 and trastuzumab-resistant fo5) to investigate the effects of tucatinib on tumor growth kinetics, as well as tucatinib anti-tumor efficacy in combination with trastuzumab and PD-1 checkpoint blockade. Effects of tucatinib on the tumor infiltrating lymphocytes were analysed using flow cytometry. To identify genes that were modulated by tucatinib treatment, we performed bulk RNA sequencing on fo5 tumors treated with vehicle or tucatinib. We evaluated healthy donor peripheral blood T cells treated with tucatinib for 96 hours and analysed secreted cytokine levels using cytometric bead array. Results: Treatment of CD4+ and CD8+ T cells with tucatinib together with TCR stimulation resulted significantly higher levels of IFNγ and TNFα compared with stimulated controls (p &amp;lt; 0.0001), suggesting tucatinib can directly modulate human T cell function. In both murine models, tucatinib significantly inhibited tumor growth in a dose-dependent manner and was observed at doses of 25 mg/kg, 50 mg/kg and 100 mg/kg (p &amp;lt; 0.05 between each dose). Median survival was 16 days in the vehicle group vs 50 days with 100 mg/kg of tucatinib (p &amp;lt; 0.0001). Ex vivo analysis of tumors by flow cytometry showed increased infiltration of NK cells (p=0.01) and, CD8+ T cells with high PD-1 (p = 0.001), TIM-3 (p = 0.009), IFNγ (p = 0.003) and Ki67 (p = 0.0003) expression in Tucatinib treated mice compared to vehicle treated mice. Concomitant with these changes there was a significant reduction in numbers of neutrophils (p = 0.0085) and MHC-II low expressing macrophage populations (p &amp;lt; 0.0001) following tucatininb treatment with an increase in frequency of MHC-II expressing dendritic cells (p &amp;lt; 0.0001) and macrophages (p &amp;lt; 0.0001) suggesting an increase in anti-tumor immunity. Similarly, in the fo5 model tucatinib treated tumors had significantly higher interferon-γ (IFNγ) produced by CD8+ T cells (p = 0.005). Gene expression profile analysis shows significant enrichment in pathways associated with immune activation, including antigen binding and presentation (p = 0.0002), adaptive immune responses (p = 0.0002) including IFNγ (p = 0.0002) and IFNα (p = 0.0002). In this model, tucatinib in combination with trastuzumab demonstrated significantly better anti-tumor activity (p = 0.03) and survival (p &amp;lt; 0.0001) compared with tucatinib alone, with 33% of mice achieving complete tumour regressions. Tucatinib in combination with PD-1 inhibition also demonstrated significantly greater anti-tumor efficacy compared to tucatinib alone (p = 0.0079) with increased survival (p = 0.05) and 50% of mice achieving complete tumor regression. Conclusions: This study suggests an anti-tumor immune response may be an important component of the efficacy of tucatinib. This is supported by in vitro data demonstrating tucatinib stimulated human peripheral T cells and in vivo data showing favorable effects on the TME mediated by tucatinib treatment. These changes were associated with improved efficacy when tucatinib was combined with PD-1 inhibition or trastuzumab in the setting of trastuzumab resistance. These findings suggest that the combination of tucatinib and PD-1 inhibition is a rational combination that warrants investigation in the clinical setting, particularly for trastuzumab resistant HER2+ breast tumors. Citation Format: Ran Li, Sneha Sant, Emmaline Brown, Franco Caramia, Ann Byrne, Kylie Clarke, Michael Neeson, Paul J Neeson, Phillip K Darcy, Scott Peterson, Sherene Loi. Tucatinib favourably modulates the immune microenvironment and synergises with anti-PD1 therapy in a trastuzumab resistant HER2+ murine model [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS10-04.

CNOT4 enhances the efficacy of anti-PD-1 immunotherapy in a model of non-small cell lung cancer.

The use of immune checkpoint inhibitors that target programmed cell death-1 (PD-1) has been proposed for the treatment of advanced non-small cell lung cancer (NSCLC). However, in clinical trials, cumulative response rates to anti-PD-1 treatment were approximately 20% in patients with NSCLC. CCR4-NOT transcription complex, subunit 4 (CNOT4) is a RING finger protein with E3 ubiquitin ligase activity. We previously reported that CNOT4 may act as a tumor suppressor in NSCLC. Here, we examined whether CNOT4 can enhance the efficacy of anti-PD-1 immunotherapy in a model of NSCLC. The association of CNOT4 and overall survival was analyzed using datasets from The Cancer Genome Atlas (TCGA). Tumor models were established by subcutaneously implanting tumor cells line (A549 cell) into mice. CNOT4 was observed to be positively associated with relapse-free survival and overall survival in patients with NSCLC. CNOT4 overexpression suppressed tumor growth invivo and enhanced the effect of anti-PD-1 immunotherapy, which was accompanied by increased CD3+ and CD8+ T lymphocyte infiltration and higher interferon-γ and tumor necrosis factor-α levels. In conclusion, CNOT4 may enhance the efficacy of anti-PD-1 immunotherapy and may have potential as a prognostic marker for NSCLC, or as a combinational target with anti-PD-1 treatment for patients with NSCLC.

Double deficiency of toll-like receptors 2 and 4 alters long-term neurological sequelae in mice cured of pneumococcal meningitis

Toll-like receptor (TLR) 2 and 4 signalling pathways are central to the body’s defence against invading pathogens during pneumococcal meningitis. Whereas several studies support their importance in innate immunity, thereby preventing host mortality, any role in protecting neurological function during meningeal infection is ill-understood. Here we investigated both the acute immunological reaction and the long-term neurobehavioural consequences of experimental pneumococcal meningitis in mice lacking both TLR2 and TLR4. The absence of these TLRs significantly impaired survival in mice inoculated intracerebroventricularly with Streptococcus pneumoniae. During the acute phase of infection, TLR2/4-deficient mice had lower cerebrospinal fluid concentrations of interleukin-1β, and higher interferon-γ, than their wild-type counterparts. After antibiotic cure, TLR2/4 double deficiency was associated with aggravation of behavioural impairment in mice, as shown by diurnal hypolocomotion throughout the adaptation phases in the Intellicage of TLR-deficient mice compared to their wild-type counterparts. While TLR2/4 double deficiency did not affect the cognitive ability of mice in a patrolling task, it aggravated the impairment of cognitive flexibility. We conclude that TLR2 and TLR4 are central to regulating the host inflammatory response in pneumococcal meningitis, which may mediate diverse compensatory mechanisms that protect the host not only against mortality but also long-term neurological complications.

PD-1 Expression and Function of T-Cell Subsets in TILs From Human Lung Cancer.

On the basis of the autologous tumor-infiltrating lymphocytes (TILs) or genetically modified TILs for adoptive cell therapy have received more attention. Programmed cell death protein 1 (PD-1) expression on the T cells exert complex response during the tumor immune response. But the composition and function of PD-1T-cell subsets in TILs from human lung cancer still limited. In blood and TILs from human lung cancer patients, we confirmed that PD-1 is expressed in higher levels in CD4T-cell subsets than in CD8T-cell subsets. To further analyze the function of PD-1T cells in TILs, we observed the cytokine production in different T-cell subsets. We found that higher interferon-γ and granzyme B production in CD4/CD8PD-1T-cell subsets in TILs than in peripheral blood mononuclear cells (PBMCs); except for PD-1Tscm, higher tumor necrosis factor-α production was observed in PD-1T-cell subsets in TILs than in PBMCs; the expression level of interleukin-17 were lower in PD-1T cells in TILs than in PBMCs; and perforin expression was significantly reduced in CD4PD-1T cells subsets in TILs compared with peripheral blood. Clarify elucidating the composition and function of PD-1T-cell subsets in TILs will have great value in clinical application for evaluating the sensitivity to PD-1 blockade and selecting the promising candidate T-cell subsets in TILs for combination immunotherapy in human lung cancer.

An immunogenic phenotype in paternal antigen‐specific CD8+ T cells at embryo implantation elicits later fetal loss in mice

Central to pregnancy success is a state of T cell tolerance to paternal antigens, which is initiated at conception. The role and regulation of specific phenotypes of CD8+ T cells in mediating pregnancy tolerance is not clear. This study aimed to investigate the impact on pregnancy outcome of altering the cytokine environment during maternal CD8+ T cell priming in early pregnancy. Transgenic Act-mOVA male mice were mated to C57BL/6 (B6) females to generate fetuses expressing ovalbumin (OVA) as a model paternal antigen. OVA-reactive CD8+ OT-I T cells were activated in vitro with OVA in the presence of either transforming growth factor-β1 (TGFB1) plus interleukin-10 (IL10), or IL2, to mimic normal or dysregulated uterine conditions, respectively, and transferred into pregnant mice on gestational day 3.5. OT-I T cells activated with TGFB1 and IL10, like naive OT-I T cells, did not alter embryo implantation or fetal viability. In contrast, OT-I T cells activated with IL2 caused extensive fetal loss manifesting in mid-gestation. IL2-activated OT-I T cells expressed less FOXP3 and higher interferon-γ (IFNG) than cells activated with TGFB1 and IL10. Fetal loss did not occur in females mated with B6 males, demonstrating the antigen specificity of fetal loss, and was not abrogated by maternal genetic C1q deficiency indicating a mechanism independent of antibody-mediated cytotoxicity. These data indicate that alternative phenotypes generated in maternal CD8+ T cells at the time of priming with paternal antigens can impact pregnancy outcome, such that inappropriate activation of CD8+ T cells before implantation is capable of causing antigen-specific fetal loss later in pregnancy.

Nuclear matrix binding protein SMAR1 regulates T-cell differentiation and allergic airway disease.

Asthma is a complex airway allergic disease involving the interplay of various cell types, cytokines, and transcriptional factors. Though many factors contribute to disease etiology, the molecular control of disease phenotype and responsiveness is not well understood. Here we report an essential role of the matrix attachment region (MAR)-binding protein SMAR1 in regulating immune response during allergic airway disease. Conditional knockout of SMAR1 in T cells rendered the mice resistant to eosinophilic airway inflammation against ovalbumin (OVA) allergen with low immunoglobulin E (IgE) and interleukin-5 (IL-5) levels. Moreover, a lower IgE/IgG2a ratio and higher interferon-γ (IFN-γ) response suggested aberrant skewing of T-cell differentiation toward type 1 helper T cell (Th1) response. We show that SMAR1 functions as a negative regulator of Th1 and Th17 differentiation by interacting with two potential and similar MAR regions present on the promoters of T-bet and IL-17. Thus, we present SMAR1 as a regulator of T-cell differentiation that favors the establishment of Th2 cells by modulating Th1 and Th17 responses.

Differential activation of cord blood and peripheral blood natural killer cells by cytokines

Background aimsNatural killer (NK) cells play important roles in the clearance of infection and transformed cells. Cord blood (CB) is currently used as a source of hematopoietic stem cells for transplantation and is a potential source of NK cells for immunotherapy. We previously showed that CB NK cells are immature and less cytotoxic as compared with peripheral blood (PB) NK cells. We aimed to identify which cytokines, among interleukin (IL)-2, IL-12, IL-15 and IL-18 and their combinations, could fully activate CB NK cells as compared with PB NK cells. MethodsWe performed a comprehensive analysis of phenotype and functionality of cytokine-activated NK cells. ResultsOur results show that the lower responsiveness of CB NK cells to IL-2 is associated with lower levels of expression of IL-2 receptors and decreased phosphorylation of STAT5 as compared with PB NK cells. Activation of CB NK cells with IL-15+18 led to the most robust proliferative response and higher interferon-γ and tumor necrosis factor-α secretion, whereas activation with IL-15+2 promoted enhanced cytotoxicity. PB NK cells responded significantly better to IL-2 than to CB NK cells but were also fully activated with other cytokine treatments including IL-15, IL-15+2 or IL-15+18. It was also possible to use cytokines to generate memory-like NK cells, with sustained ability to produce interferon-γ, from both CB and PB. ConclusionsCB NK cells are fully functional on activation with IL-15+2 or IL-15+18 rather than IL-2 alone as observed for PB NK cells. These cytokines should be considered in the future to activate CB NK cells for therapeutic purposes.

Diminished immune response to vaccinations in obesity: Role of myeloid-derived suppressor and other myeloid cells

Obesity is a chronic inflammatory condition associated with an increased production of cytokines and exacerbated immune response. However, obese subjects are susceptible to infections and respond poorly to vaccines. This study evaluated the immune responses of obese mice and the underlying mechanisms by exploring the roles of myeloid cells. Diet-induced obese (DIO) mice were prepared from C57BL/6J mice fed a high-calorie and high-fat diet for 12 weeks. Humoral and cellular immune responses of DIO mice to a hepatitis B vaccine containing the hepatitis B surface antigen (HBsAg) were assessed in sera and via a lymphoproliferative assay, respectively. The effects of CD11b(+)GR1(+) myeloid-derived suppressor cells (MDSC) and CD11b(+)GR1(-) non-MDSC on T cell proliferation and cytokine production were compared via a cell culture system. The production of cytokines, expression of activation and exhaustion markers, and proportions of apoptotic T cells were estimated with flow cytometry. Increased T and B lymphocyte proliferation and higher interferon-γ and tumor necrosis factor-α levels were detected in spleen cells and liver non-parenchymal cell cultures of DIO mice compared to controls (p<0.05). However, antibody to HBsAg (anti-HBs) levels and HBsAg-specific T cell proliferation were significantly lower in DIO mice compared to controls (p<0.05). The addition of MDSC, but not non-MDSC, induced a decrease in HBsAg-specific T cell proliferation, lower cytokine production, decrease in T cell activation, and increase in T cell exhaustion and apoptosis (p<0.05). MDSC play an important role in mediating impaired antigen-specific immunity.

Dietary Supplementation with White Button Mushrooms Augments the Protective Immune Response to Salmonella Vaccine in Mice1,2

We previously showed that dietary white button mushrooms (WBMs) enhanced natural killer cell activity and that in vitro WBM supplementation promotes maturation and function of dendritic cells (DCs). The current study investigated whether WBM consumption would enhance pathogen-specific immune response using a Salmonella vaccination and infection animal model. C57BL/6 mice were fed diets containing 0%, 2%, or 5% WBM for 4 wk before oral vaccination with live attenuated Salmonella typhimurium SL1479. Four weeks after immunization, mice were orally infected with virulent Salmonella typhimurium SL1344. Immunization increased animal survival and, among immunized mice, the 2% WBM group had a higher survival rate than the other groups. Next, we fed mice 2% WBMs to determine the immunological mechanism underlying the WBM-potentiated protective effect. We found that WBM supplementation increased Salmonella-specific blood immunoglobulin (Ig) G and fecal IgA concentrations. WBM-fed mice also had a higher IgG2a and unchanged IgG1 production, leading to an elevated IgG2a:IgG1 ratio and indicating an enhanced T helper 1 response. Consistent with these results, WBM-fed mice had higher interferon-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-17A production and unchanged IL-4 production in their splenocytes after polyclonal (anti-CD3/CD28) or antigen-specific stimulation. Furthermore, WBM-fed mice had more DCs in the spleen, and these DCs expressed higher levels of activation markers CD40 and major histocompatibility complex-II. These mice also produced more IL-12 and TNF-α postimmunization. Together, these results suggest that WBMs may improve Salmonella vaccine efficacy through an enhanced adaptive immune response.

Gene therapy with interleukin-10 receptor and interleukin-12 induces a protective interferon-γ-dependent response against B16F10-Nex2 melanoma

Antitumor immune responses are associated with proinflammatory cytokines, whereas tumor-developing animals generally have increased the production of immunosuppressive cytokines. Here, we show that splenocytes from C57Bl/6 mice resistant to low doses of B16F10-Nex2 melanoma cells produced twofold or higher interferon-γ (IFN-γ)/interleukin-10 (IL-10) ratios, whereas cells from tumor-bearing animals produced predominantly IL-10. IL-10-knockout (IL-10KO) mice were significantly more resistant to B16F10-Nex2 development, producing increased amounts of IL-12 and IFN-γ. To neutralize IL-10 in vivo, aiming at cancer therapy, recombinant eukaryotic plasmid expressing the soluble extracellular region of the murine IL-10 receptor α-chain was constructed (pcDNA3-sIL-10R). Plasmid-treated melanoma-challenged animals showed extended survival time, the protective response was IFN-γ dependent and enhanced by co-immunization with a plasmid expressing IL-12. Dendritic cells (DCs) from IL-10KO mice, primed with B16F10-Nex2 antigens (TAg), secreted increased amounts of T-helper 1-type cytokines and increased the expression of surface activation markers. Vaccination of C57Bl/6 mice with TAg-activated IL-10KO DCs, as well as with TAg-primed DCs from C57Bl/6 mice transfected with pcDNA3-sIL10R plasmid, significantly increased animal survival. In conclusion, an IFN-γ-dependent protective response was induced against B16F10-Nex2 cells by neutralization of IL-10 with pcDNA3-sIL10R plasmid. This effect was enhanced by association with IL-12 gene therapy (80% protection), and could be mediated by TAg-primed DCs.

Clinical, Parasitologic, and Immunologic Evolution in Dogs Experimentally Infected with Sand Fly-Derived Leishmania chagasi Promastigotes

Experimental infection of dogs with Leishmania infantum has yielded heterogeneous clinical, parasitologic, and immunologic results. We studied dogs infected with 10(5) or 10(4) sand fly-derived promastigotes delivered by the intradermal (ID) or intravenous (IV) routes. Total mortality over 1 year post-infection reached 23.8%. The mortality and proportion of sustained polysymptomatic dogs was highest in the IV-10(5) group. The early appearance of polysymptoms was associated with an increased risk of progression to death. Dissemination of the parasite to lymph nodes was faster, and the subsequent infectivity to sand flies higher, in the IV compared with ID-infected dogs. Parasite-specific IgG1 or IgG2 production was similar among the groups, but higher interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) expression was associated with polysymptomatic dogs. On the basis of the data obtained from this study, a sample size analysis using different endpoints for future vaccine trials is described.

Mechanisms behind flare of renal lupus during murine pregnancy

The outcome of pregnancy in systemic lupus erythematosus is still controversial. The authors recently reported the disappearance of the manifestation of the skin disease but a diminished survival rate in lupus-prone animals undergoing several pregnancies. It was postulated that lupus-prone animals must have subclinical renal symptoms at an early age and that immune and hormonal changes during pregnancy exacerbate immune reactions in the kidneys, leading to a shortened life span. Here, the authors analysed changes at day 14 of pregnancy in lupus-prone LPR (MRL/lpr) mice and MRL controls regarding cytokines, regulatory T (Treg) cells and deposition of immunocomplexes. Worsened kidney function was observed during pregnancy, even in the absence of lupus signs. This was accompanied by renal inflammation and higher interferon-γ and interleukin-10 levels. C3 and immunoglobulin G deposition was enhanced in kidney and placenta from lupus-prone pregnant animals. Pregnancy enhanced the levels of Treg cells in control animals but not in lupus-prone animals. As pregnancy-induced Treg cells were shown to be specific for paternal antigens it is not to be expected that these Treg cells can help to destroy autoreactive cells. The authors conclude that early subclinical kidney disease in lupus-prone animals exacerbates during pregnancy. Albeit obtained with an experimental animal model, their data are potentially of importance for lupus patients of reproductive age.