Immunosuppressive effects of triptolide via interleukin-2/receptor signaling.

BACKGROUND: Triptolide (TP) has been confirmed to possess many beneficial functions including anti-inflammation and immunosuppression. OBJECTIVE: The present study aimed to explore the potential involvement of IL-2/IL-2R pathway in the immunosuppressive activities of TP. METHODS: Cultured CTLL-2 cells were utilized to evaluate the potential benefits of TP. Then cell viability was determined by CCK-8 assay, IFN-γ level by ELISA assay, Annexin V-FITC/PI double-staining and CD25 expression by flow cytometry, and protein expression by western blotting. Additionally, rhIL-2-driven lymphocytes following ConA activation were investigated. The interactions of TP with IL-2 and IL-2Rα were investigated by binding assays and molecular dynamics simulations. RESULTS: TP treatment attenuated IFN-γ level and cell viability in both rhIL-2-induced CTLL-2 cells and rhIL-2-driven splenic lymphocytes. TP treatment increased cellular apoptosis/necrosis and cleaved PARP-1 level, while suppressed c-Myc level in rhIL-2-induced CTLL-2 cells. Additionally, TP treatment reduced CD25 expression on CTLL-2 cell surface. Notably, the phosphorylation protein levels in IL-2R signaling pathways were inhibited by TP exposure prior to rhIL-2 stimulation. SPR and BLI assays verified TP that directly bound to rhIL-2 and rmIL-2Rα, respectively. Molecular simulations suggested that TP bound at the interface of IL-2 and IL-2Rα near the hydrophobic patch composed of F62, L92 on IL-2 and L23, I46, V139 on IL-2Rα, resulting in decreased binding free energy between IL-2 and IL-2Rα. CONCLUSIONS: These findings collectively emphasized that TP interfered IL-2/IL-2Rα interactions, down-regulated IL-2Rα expression, and inhibited IL-2R signaling pathways activation, thereby leading to the immune cells being desensitized to rhIL-2 and exhibiting immunosuppressive properties.

Associations between per- and poly-fluoroalkyl substance (PFAS) exposure and immune responses among women in the California Teachers study: A cross-sectional evaluation.

INTRODUCTION: Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants that have been linked to a number of health outcomes, including those related to immune dysfunction. However, there are limited numbers of epidemiological-based studies that directly examine the association between PFAS exposure and immune responses. METHODS: In this cross-sectional study nested in the California Teachers Study cohort, we measured nine PFAS analytes in serum. Of the 9 analytes, we further evaluated four (PFHxS [perfluorohexane sulfonate], PFNA [perfluorononanoic acid], PFOA [perfluorooctanoic acid], PFOS [perfluorooctanesulfonic acid]) that had detection levels of > 80 %, in relation to 16 systemic inflammatory/immune markers and corresponding immune pathways (Th1 [pro-inflammatory/macrophage activation], B-cell activation, and T-cell activation). Study participants (n = 722) were female, completed a questionnaire regarding various health measures and behaviors, and donated a blood sample between 2013-2016. The association between PFAS analytes and individual immune markers and pathways were evaluated by calculating odds ratios (OR) and 95 % confidence intervals (CI) in a logistic regression model. PFAS analytes were evaluated both as a dichotomous exposure (above or below the respective median) and as a continuous variable (per 1 unit increase [ng/mL]). RESULTS: The prevalence of detecting any PFAS analyte rose with increasing age, with the highest PFAS prevalence observed among those aged 75 + years and the lowest PFAS prevalence observed among those aged 40-49 years (study participant age range: 40-95 years). Significant associations with BAFF (B-cell activating factor) levels above the median were observed among participants with elevated (defined as above the median) levels of PFHxS (OR=1.53), PFOA (OR=1.43), and PFOS (OR=1.40). Similarly, there were statistically significant associations between elevated levels of PFHxS and TNFRII (tumor necrosis factor receptor 2) levels (OR=1.78) and IL2Rα (interleukin 2 receptor subunit alpha) levels (OR=1.48). We also observed significant inverse associations between elevated PFNA and sCD14 (soluble cluster of differentiation 14) (OR=0.73). No significant associations were observed between elevated PFNA and any immune marker. Evaluation of PFAS exposures as continuous exposures in association with dichotomized cytokines were generally consistent with the dichotomized associations. CONCLUSIONS: PFAS exposure was associated with altered levels of circulating inflammatory/immune markers; the associations were specific to PFAS analyte and immune marker. If validated, our results may suggest potential immune mechanisms underlying associations between the different PFAS analytes and adverse health outcomes.

Impact of ibrutinib on inflammation in a mouse model of Graves' orbitopathy.

Introduction: Bruton's tyrosine kinase (BTK) and interleukin (IL)-2 Inducible T-cell Kinase (ITK) inhibitors have anti-inflammatory properties. We investigated the therapeutic effect of ibrutinib, an orally bioavailable BTK/ITK inhibitor, in a mouse model of Graves' orbitopathy (GO). Methods: Genetic immunization was performed through intramuscular administration of the recombinant plasmid, pCMV6-hTSHR cDNA, to 8-week-old female BALB/c mice. Serum levels of T3, T4, and thyroid-stimulating hormone receptor (TSHR) antibodies (TRAbs) were quantified using enzyme-linked immunosorbent assay. Histopathological changes in orbital tissues were examined using immunohistochemistry (IHC) staining for TSHR and various inflammatory markers. Following successful genetic immunization, ibrutinib was orally administered daily for 2 weeks in the GO model mice. After treatment, the mRNA and protein expression levels of BTK, ITK, IL-1ß, and IL-6 in orbital tissues were evaluated using real-time PCR and Western blotting. Results: In total, 20 mice were sacrificed to confirm successful genetic immunization. The GO mouse group exhibited significantly increased serum T3, T4, and TRAb levels. IHC revealed increased expression of TSHR, IL-1ß, IL-6, transforming growth factor-ß1, interferon-γ, CD40, CD4, BTK, and ITK in the GO mouse model. The orbital inflammation was significantly attenuated in ibrutinib-treated mice. The mRNA and protein expression levels of BTK, ITK, IL-1ß, and IL-6 in orbital tissue were lower in ibrutinib-treated GO mouse group compared to the phosphate-buffered saline-treated GO mouse group. Conclusion: The GO mouse model demonstrated enhanced BTK and ITK expression. Ibrutinib, a BTK/ITK inhibitor, suppressed the inflammatory cytokine production. These findings highlight the potential involvement of BTK/ITK in the inflammatory pathogenesis of GO, suggesting its role as a novel therapeutic target.

Systemic T-cell activation and interferon-γ activity in indeterminate severe hepatitis (iSH) are reminiscent of hemophagocytic lymphohistiocytosis (HLH): Implications for T-cell and interferon-γ directed therapies.

BACKGROUND: Severe hepatitis cases in children are increasingly recognized, however, the exact etiology remains unknown in a significant proportion of patients. Cases of indeterminate severe hepatitis (iSH) may progress to indeterminate pediatric acute liver failure (iPALF), hence understanding the immunobiology is critical to preventing disease progression. Hemophagocytic lymphohistiocytosis (HLH) is a systemic hyperinflammatory disorder associated with T-cell and macrophage activation with liver injury. OBJECTIVES: We hypothesized a high proportion of patients with iSH demonstrate systemic T-cell activation similar to HLH prior to developing iPALF and that the degree of T-cell activation in iSH might correlate with outcomes. METHODS: From 2019-2022, 14 patients with iSH and 7 patients with PALF of known, non-immune etiology were prospectively enrolled. We compared immune signatures of iSH, HLH, known PALF, and healthy controls. RESULTS: We found that patients with iSH have increased CD8+ T-cell activation and high interferon-γ activity similar to HLH. The amplitude of CD8+ T-cell activation was predictive of iSH progression to iPALF. We also found that in patients with iSH, ferritin had only modest elevation. However, age-normalized plasma soluble interleukin-2 receptor (sIL-2R) to ferritin level ratio can distinguish iSH from known PALF and HLH. As a proof of concept, we report that in three patients with steroid refractory iSH, emapalumab, an IFN-γ blocking antibody used in combination with steroids, improved liver function and may have prevented progression to PALF. CONCLUSIONS: Our data suggests flow-based T-cell activation markers could help in early identification and risk stratification for targeted intervention in patients with iSH.

Serum soluble interleukin-2 receptor alpha may predict tubulointerstitial inflammatory cell infiltration and short-term disease progression in immunoglobin A nephropathy.

This study aims to explore the relationship between serum soluble interleukin-2 receptor alpha (sIL-2Rα) levels and histologic features in immunoglobin A nephropathy (IgAN), and evaluate its predicting values on disease progression and remission status. IgAN patients were included retrospectively. Lee classification, Oxford classification and histological scoring were evaluated. Patients' estimated filtration rate (eGFR) and proteinuria remission status were collected during 6-month follow-up. Logistic regression was used to determine the risk factors and predicting value. Receiver operating characteristic (ROC) curve were used to determine the predicting value for outcome. One hundred seventy-two subjects were included in this study. Individuals in moderate-to-severe tubulointerstitial inflammatory cell infiltration group manifested with significantly elevated serum sIL-2Rα levels than those in non-to-mild group. Serum sIL-2Rα levels were positively correlated with infiltration scores. Serum sIL-2Rα was an independent risk factor for moderate-to-severe inflammatory cell infiltration [sIL-2Rα: OR 1.29 (1.015-1.640, p = 0.038)]. ROC curve analysis regarding predictive value for moderate-to-severe inflammatory cell infiltration of sIL-2Rα suggested area under curve was 0.859 (0.801-0.918, p = 0.000) when sIL-2Rα combined with eGFR < 60 mL/(min·1.73 m2), 24-h proteinuria excretion > 1.0 g, and hemoglobin. It showed good sensitivity (71.6%) and specificity (87.6%). Additionally, sIL-2Rα levels at kidney biopsy were strong predictive factor for kidney function loss 6 months after kidney biopsy [OR 4.161 (1.013-17.088, p = 0.048)]. High serum sIL-2Rα was significantly associated with serious inflammatory cell infiltration in IgAN, and it showed strong predictive value for disease prognosis. Serum sIL-2Rα could be a useful noninvasive biomarker to evaluate the extent of histological injury and disease prognosis in IgAN.

Interleukin-2 immunotherapy reveals human regulatory T cell subsets with distinct functional and tissue-homing characteristics.

Due to its stimulatory potential for immunomodulatory CD4+ regulatory T (Treg) cells, low-dose interleukin-2 (IL-2) immunotherapy has gained considerable attention for the treatment of autoimmune diseases. In this investigator-initiated single-arm non-placebo-controlled phase-2 clinical trial of low-dose IL-2 immunotherapy in systemic lupus erythematosus (SLE) patients, we generated a comprehensive atlas of in vivo human immune responses to low-dose IL-2. We performed an in-depth study of circulating and cutaneous immune cells by imaging mass cytometry, high-parameter flow cytometry, transcriptomics, and targeted serum proteomics. Low-dose IL-2 stimulated various circulating immune cells, including Treg cells with a skin-homing phenotype that appeared in the skin of SLE patients in close interaction with endothelial cells. Analysis of surface proteins and transcriptomes revealed different IL-2-driven Treg cell activation programs, including gut-homing CD38+, skin-homing HLA-DR+, and highly proliferative inflammation-homing CD38+ HLA-DR+ Treg cells. Collectively, these data define the distinct human Treg cell subsets that are responsive to IL-2 immunotherapy.

Tripokin: A multi-specific immunocytokine for cancer immunotherapy.

Antibodies that target the tumor microenvironment can be used to deliver pro-inflammatory payloads, such as cytokines. Cytokines are small proteins able to modulate the activity of the immune system, and antibody-cytokine fusion proteins have been tested in preclinical and clinical settings. In this study, we describe Tripokin, a novel multi-specific fusion protein that combines interleukin-2 and a single amino acid mutant of tumor necrosis factor. The two pro-inflammatory payloads were fused to the L19 antibody, a clinical-grade antibody against the extradomain B of fibronectin. The human payloads were used for clinical applications, while the corresponding murine cytokines were used for preclinical studies. The resulting fusion proteins were produced in mammalian cells and purified to homogeneity. The murine Tripokin product was well tolerated in tumor-bearing mice at three doses of 30 µg in a 2-day interval and promoted rapid tumor eradication in murine models, more efficiently than single-agent immunocytokines. Tripokin induced rapid tumor necrosis and stimulated a robust immune response, impacting innate and adaptive immune pathways. In addition, the combination with immune checkpoint inhibitors further boosted the therapeutic efficacy of our molecule. Tripokin represents a promising clinical candidate for the simultaneous delivery of interleukin-2 and tumor necrosis factor to neoplastic sites.

Efficacy and safety of low-dose interleukin 2 in the treatment of moderate-to-severe bullous pemphigoid: A single center perspective-controlled trial.

BACKGROUND: Regulatory T cells (Tregs) are reduced in the peripheral blood and skin lesions of patients with bullous pemphigoid (BP). Low-dose interleukin 2 (IL-2) therapy can stimulate Tregs specifically, suggesting potential for the treatment of BP. OBJECTIVE: To evaluate the response to low-dose IL-2 therapy in the treatment of moderate-to-severe BP. METHODS: Forty-three patients with moderate-to-severe BP were included. The therapy included systemic corticosteroids with an initial dose of 0.5 mg/kg/d for moderate and 1.0 mg/kg/d for severe disease, respectively, combined with allowed immunosuppressants for the control group, whereas in addition to the same corticosteroid therapy, IL-2 (half million IU) was administered subcutaneously every other day for the treatment group for 8 weeks. The primary outcome was the number of days required to achieve disease control. Secondary outcomes included other clinical responses. RESULTS: The number of days required to achieve disease control with the treatment group was (7.60 ± 3.00), which was shorter than in the control group (10.43 ± 3.06) (P = .008). The total amount of systemic corticosteroids was less, and no serious infections were detected in the treatment group. LIMITATIONS: Single center, open-label study with short duration and small size. CONCLUSION: Our trial supports the potential of low-dose IL-2 therapy for patients with moderate-to-severe BP, which showed earlier treatment responses.

Diagnostic Approach to IgG4-Related Retroperitoneal Fibrosis After Colorectal Cancer Surgery in a Patient With Normal IgG4 Levels: A Case Report.

An asymptomatic 75-year-old man who underwent transverse colon cancer surgery two years previously presented with retroperitoneal fibrosis (RPF) around the ventral sacral and right external iliac artery and vein on abdominal computed tomography (CT) during a routine surveillance visit. We assumed cancer recurrence or immunoglobulin G4 (IgG4)-related disease (RD), but although generic tumor markers and IgG4 levels were normal, soluble interleukin 2 receptor (sIL-2R) was elevated at 569 U/mL (reference: 122-496 U/mL). No diagnosis was made at this time, and the patient was followed up. He subsequently developed edema of both lower extremities. Abdominal enhanced CT showed an enlarged RPF without invasion of surrounding organs and with a delayed contrast effect, and positron emission tomography-CT showed fluorodeoxyglucose accumulation in the same area but a lower standardized uptake value (SUV) than at the time of transverse colon cancer diagnosis. Although generic tumor markers and IgG4 levels remained within the reference range, sIL-2R was further elevated to 1100 U/mL. An open biopsy and histopathology showed a high IgG4/IgG-positive cell ratio and infiltration of IgG4-positive plasma cells. The patient was finally diagnosed with IgG4-RD RPF. In cases of RPF after colorectal cancer surgery, the combined findings of elevated sIL-2R, lack of infiltration into surrounding organs, and lower SUV values ​​than at the cancer site could provide useful information to aid the diagnosis of IgG4-RD RPF.

Combined analysis of host IFN-γ, IL-2 and IP-10 as potential LTBI biomarkers in ESAT-6/CFP-10 stimulated blood.

Background: Accurate diagnosis of latent tuberculosis infected (LTBI) individuals is important in identifying individuals at risk of developing active tuberculosis. Current diagnosis of LTBI routinely relies on the detection and measurement of immune responses using the Tuberculin Skin Test (TST) and interferon gamma release assays (IGRAs). However, IGRA, which detects Mycobacterium tuberculosis specific IFN-γ, is associated with frequent indeterminate results, particularly in immunosuppressed patients. There is a need to identify more sensitive LTBI point of care diagnostic biomarkers. The aim of this study was to assess the validity of early secreted antigen target 6 kDa (ESAT-6) and culture filtrate protein 10 (CFP-10) stimulated plasma to identify additional cytokines and chemokines as potential biomarkers of LTBI. Method: The levels of 27 cytokines and chemokines were measured by Bio-Plex Pro cytokine, chemokine and growth factor assay in ESAT-6 and CFP-10 co-stimulated plasma from 20 LTBI participants with positive IGRA (Quantiferon TB Gold plus) and 20 healthy controls with negative IGRA. Traditional ELISA was used to validate the abundance of the best performing markers in 70 LTBI and 72 healthy participants. All participants were HIV negative. Results: We found that Interleukin 1 receptor antagonist (IL1ra) (p = 0.0056), Interleukin 2 (IL-2) (p < 0.0001), Interleukin 13 (IL-13) (p < 0.0001), Interferon gamma-induced protein 10 (IP-10) (p < 0.0001), and Macrophage inflammatory protein-1 beta (MIP1b) (p = 0.0010) were significantly higher in stimulated plasma of LTBI compared to healthy individuals. Stimulated plasma IL-2 (cutoff 100 pg/mL), IP-10 (cutoff 300 pg/mL) and IL-13 (5 pg/mL) showed potential in diagnosing LTBI with PPV = 100%, 0.89.4%, and 80.9% and NPV = 86.9%, 0.85.7%, and 84.2%, respectively. Conclusion: Our data shows that co-stimulating whole blood with ESAT-6 and CFP-10 may help distinguish LTBI from healthy individuals. We also identified IL-2 and IP-10 as potential biomarkers that could be added to the currently used IFN-γ release assays in detection of LTBI.

Enhancing natural killer cells proliferation and cytotoxicity using imidazole-based lipid nanoparticles encapsulating interleukin-2 mRNA.

mRNA applications have undergone unprecedented applications-from vaccination to cell therapy. Natural killer (NK) cells are recognized to have a significant potential in immunotherapy. NK-based cell therapy has drawn attention as allogenic graft with a minimal graft-versus-host risk leading to easier off-the-shelf production. NK cells can be engineered with either viral vectors or electroporation, involving high costs, risks, and toxicity, emphasizing the need for alternative way as mRNA technology. We successfully developed, screened, and optimized novel lipid-based platforms based on imidazole lipids. Formulations are produced by microfluidic mixing and exhibit a size of approximately 100 nm with a polydispersity index of less than 0.2. They are able to transfect NK-92 cells, KHYG-1 cells, and primary NK cells with high efficiency without cytotoxicity, while Lipofectamine Messenger Max and D-Lin-MC3 lipid nanoparticle-based formulations do not. Moreover, the translation of non-modified mRNA was higher and more stable in time compared with a modified one. Remarkably, the delivery of therapeutically relevant interleukin 2 mRNA resulted in extended viability together with preserved activation markers and cytotoxic ability of both NK cell lines and primary NK cells. Altogether, our platforms feature all prerequisites needed for the successful deployment of NK-based therapeutic strategies.

Evaluating the role of interleukin-2 and interleukin-12 in pediatric patients with concurrent Mycoplasma pneumoniae and Epstein-Barr virus infections.

BACKGROUND: Mycoplasma pneumoniae (MP) frequently causes respiratory infections in children, whereas Epstein-Barr virus (EBV) typically presents subclinical manifestations in immunocompetent pediatric populations. The incidence of MP and EBV co-infections is often overlooked clinically, with the contributory role of EBV in pulmonary infections alongside MP remaining unclear. AIM: To evaluate the serum concentrations of interleukin-2 (IL-2) and interleukin-12 (IL-12) in pediatric patients with MP pneumonia co-infected with EBV and assess their prognostic implications. METHODS: We retrospectively analyzed clinical data from patients diagnosed with MP and EBV co-infection, isolated MP infection, and a control group of healthy children, spanning from January 1, 2018 to December 31, 2021. Serum IL-2 and IL-12 levels were quantified using enzyme-linked immunosorbent assay. Logistic regression was employed to identify factors influencing poor prognosis, while receiver operating characteristic (ROC) curves evaluated the prognostic utility of serum IL-2 and IL-12 levels in co-infected patients. RESULTS: The co-infection group exhibited elevated serum IL-2 and C-reactive protein (CRP) levels compared to both the MP-only and control groups, with a reverse trend observed for IL-12 (P < 0.05). In the poor prognosis cohort, elevated CRP and IL-2 levels, alongside prolonged fever duration, contrasted with reduced IL-12 levels (P < 0.05). Logistic regression identified elevated IL-2 as an independent risk factor and high IL-12 as a protective factor for adverse outcomes (P < 0.05). ROC analysis indicated that the area under the curves for IL-2, IL-12, and their combination in predicting poor prognosis were 0.815, 0.895, and 0.915, respectively. CONCLUSION: Elevated serum IL-2 and diminished IL-12 levels in pediatric patients with MP and EBV co-infection correlate with poorer prognosis, with combined IL-2 and IL-12 levels offering enhanced predictive accuracy.

A novel scoring system based on sIL-2R for predicting IVIG resistance in Chinese children with KD.

OBJECTIVE: This study aimed to develop a novel scoring system utilizing circulating interleukin (IL) levels to predict resistance to intravenous immunoglobulin (IVIG) in Chinese patients with Kawasaki disease (KD). We further compared this scoring system against six previously established scoring methods to evaluate its predictive performance. METHODS: A retrospective analysis was conducted on KD patients who were treated at the cardiovascular medical ward of our institution from January 2020 to December 2022. Six scoring systems (Egami, Formosa, Harada, Kobayashi, Lan and Yang) were analyzed, and a new scoring system was developed based on our data. RESULTS: In our study, 521 KD patients were recruited, 42 of whom (8.06%) were identified as resistant to IVIG. Our study indicated that IVIG-resistant KD patients were at an increased risk for the development of coronary arterial lesions (CALs) (P = 0.001). The evaluation of IVIG resistance using various scoring systems revealed differing levels of sensitivity and specificity, as follows: Egami (38.10% and 88.52%), Formosa (95.24% and 41.13%), Harada (78.57% and 43.22%), Kobayashi (66.67% and 74.95%), Lan (66.67% and 73.49%), and Yang (69.05% and 77.24%). Our novel scoring system utilizing sIL-2R demonstrated the highest sensitivity and specificity of 69.29% and 83.91%, respectively, and calibration curves indicated a favorable predictive accuracy of the model. CONCLUSION: Our newly developed scoring system utilizing sIL-2R demonstrated superior predictive performance in identifying IVIG resistance among Chinese patients with KD.

X-linked severe combined immunodeficiency complicated by disseminated bacillus Calmette-Guérin disease caused by a novel pathogenic mutation in exon 3 of the IL2RG gene: a case report and literature review.

X-linked severe combined immunodeficiency (X-SCID), caused by mutations in the gamma-chain gene of the interleukin-2 receptor (IL2RG), is a prevalent form of SCID characterized by recurrent and fatal opportunistic infections that occur early in life. The incidence of disseminated bacillus Calmette-Guérin (BCG) disease among children with SCID is much higher than in the general population. Here, we report the case of a 4-month-old male infant who presented with subcutaneous induration, fever, an unhealed BCG vaccination site, and hepatosplenomegaly. Metagenomic next-generation sequencing in blood, and the detection of gastric juice and skin nodule pus all confirmed the infection of Mycobacterium tuberculosis. Lymphocyte subset analysis confirmed the presence of T-B+NK immunodeficiency. Whole-exome and Sanger sequencing revealed a novel microdeletion insertion mutation (c.316_318delinsGTGAT p.Leu106ValfsTer42) in the IL2RG gene, resulting in a rare shift in the amino acid sequence of the coding protein. Consequently, the child was diagnosed with X-SCID caused by a novel mutation in IL2RG, complicated by systemic disseminated BCG disease. Despite receiving systemic anti-infection treatment and four days of hospitalization, the patient died three days after discharge. To the best of our knowledge, this specific IL2RG mutation has not been previously reported. In our systemic review, we outline the efficacy of systemic anti-tuberculosis therapy, hematopoietic stem cell transplantation, and gene therapy in children with SCID and BCG diseases caused by IL2RG gene mutation.

Interleukin-2 mediated associations between gut microbiota and acute myeloid leukemia: A population-based mediation Mendelian randomization study.

The relationship between the gut microbiota and acute myeloid leukemia (AML) has been established, but the exact role of interleukin (IL) in mediating this relationship has remained unclear. This study aimed to utilize whether interleukins mediate the relationships between gut microbiota and AML, thereby identifying potential novel targets for future AML treatment. Mendelian randomization (MR) is a method for finding the causality of exposure and outcome. Final instrumental variables were selected based on MR assumptions, and used to judge validity of the results. Our study identified risk and protective factors for AML, and interleukin-related gut microbiota. Finally, mediation MR analyses resulted in Interleukin-2 (IL-2) mediated associations between Clostridiaceae 1, Clostridium sensu stricto 1 and AML, with IL-2 respectively explaining 13.96 % and 12.11 % of the total effect of the aforementioned gut microbiota on AML. Our results successfully identified causal effects between specific gut microbiota, AML, and interleukins, while also elucidating the mediating role of IL-2 in these associations using MR analysis. These findings provide valuable insights into potential therapeutic targets for AML treatment.

Unmasking the Culprits: A Case of Epstein-Barr Virus-Associated Hemophagocytic Lymphohistiocytosis Presenting With Mouth Ulcers and Nosebleeds.

Hemophagocytic lymphohistiocytosis (HLH) is an aggressive syndrome of excessive immune activation. It usually occurs in children, mainly during the first year of life. Primary hemophagocytic lymphohistiocytosis is more common and usually occurs in immunocompromised patients. Secondary hemophagocytic lymphohistiocytosis, on the other hand, is less common, especially in immunocompetent patients. Here, we intend to present a case of a 55-year-old male patient who had no known immune deficiency, presented with epistaxis, and was found to have Epstein-Barr virus (EBV)-induced hemophagocytic lymphohistiocytosis.

Association between IL-2 Receptor and Severe Coronary Artery Calcification in Patients with Coronary Artery Disease.

Background: Coronary artery calcification (CAC) is a crucial marker for coronary atherosclerosis, and the extent of CAC is closely linked to the incidence and progression of cardiovascular diseases. The interleukin-2 (IL-2) receptor (IL-2R), which plays a critical role in mediating the proliferation and differentiation of immune cells, may also be involved in the development of CAC. The study aimed to investigate the relationship between IL-2R and CAC, with the goal of providing new insights into cardiovascular diseases. Methods: In this study, we enrolled 606 patients diagnosed with coronary artery disease to assess CAC. Based on coronary artery calcification score (CACS), patients were divided into two groups: the non-severe CAC group (CACS ≤ 400 Agatston units, AU) and the severe CAC group (CACS > 400 AU). Results: The results showed that IL-2R levels were significantly higher in patients with severe CAC compared to those with non-severe CAC (383 vs. 352 pg/mL, p = 0.002). Moreover, the level of IL-2R was positively correlated with the severity of CAC, independent of other clinical risk factors. According to Receiver Operating Characteristic (ROC) curve, the IL-2R prediction model demonstrated a good capability in distinguishing severe CAC with the Area Under the Curve (AUC) value of 0.726. Conclusions: Our study suggests that IL-2R is independently associated with the occurrence of severe CAC in coronary artery disease (CAD) patients. Additionally, IL-2R may play a crucial role in the development of advanced atherosclerosis. Consequently, therapeutic strategies targeting the IL-2/IL-2R pathway may be effective in preventing or treating CAD.

Bridging therapy-induced phenotypes and genetic immune dysregulation to study interleukin-2-induced immunotoxicology.

Interleukin-2 (IL-2) holds promise for the treatment of cancer and autoimmune diseases, but its high-dose usage is associated with systemic immunotoxicity. Differential IL-2 receptor (IL-2R) regulation might impact function of cells upon IL-2 stimulation, possibly inducing cellular changes similar to patients with hypomorphic IL2RB mutations, presenting with multiorgan autoimmunity. Here, we show that sustained high-dose IL-2 stimulation of human lymphocytes drastically reduces IL-2Rß surface expression especially on T cells, resulting in impaired IL-2R signaling which correlates with high IL-2Rα baseline expression. IL-2R signaling in NK cells is maintained. CD4+ T cells, especially regulatory T cells are more broadly affected than CD8+ T cells, consistent with lineage-specific differences in IL-2 responsiveness. Given the resemblance of cellular characteristics of high-dose IL-2-stimulated cells and cells from patients with IL-2Rß defects, impact of continuous IL-2 stimulation on IL-2R signaling should be considered in the onset of clinical adverse events during IL-2 therapy.

Discovery and development of ANV419, an IL-2/anti-IL-2 antibody fusion protein with potent CD8+ T and natural killer cell-stimulating capacity for cancer immunotherapy.

Novel engineered IL-2 agonists strive to increase the therapeutic window of aldesleukin (human IL-2) by increasing selectivity toward effector over regulatory T cells and reducing dose-limiting toxicities. Here we describe ANV419, an IL-2/anti-IL2 antibody fusion protein designed for selective IL-2 receptor ßγ (IL-2 Rßγ) activation by sterically hindering IL-2 from binding to IL-2 Rα. The fusion protein has an IL-2 connected to the light chain complementarity-determining region (CDR) domain of a humanized antibody that binds to IL-2 at the same epitope as IL-2 Rα. Optimization of the selectivity and pharmacological properties led to the selection of ANV419. ANV419 preferentially expands CD8+ T cells and natural killer (NK) cells over Tregs and can be safely administered at doses that elicit strong pharmacodynamic effects and efficacy in mouse tumor models. Its anti-tumor efficacy was enhanced when combined with programmed cell death protein 1 (PD-1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) checkpoint inhibitors. ANV419 also enhances the NK cell killing capacity and increases tumor growth inhibition when used alongside trastuzumab in a Her-2+ xenograft mouse model. In cynomolgus monkeys, the estimated half-life of ANV419 is 24 h, and doses that induced sustained expansion of effector cells were well tolerated without the severe toxicities typically observed with high-dose IL-2. These data support the clinical development of ANV419 in solid tumors and hematological malignancies as monotherapy and in combination with checkpoint inhibitors or agents that induce antibody-dependent cellular cytotoxicity. ANV419 is currently in Phase 1/2 clinical development and may provide cancer patients with a wider therapeutic window than aldesleukin.

Oxidative refolding by Copper-catalyzed air oxidation consistently increases the homogeneity and activity of a Novel Interleukin-2 mutein.

Interleukin-2 (IL-2) has been used in cancer treatment for over 30 years. However, due to its high toxicity, new mutant variants have been developed. These variants retain some of the biological properties of the original molecule but offer other therapeutic advantages. At the Center of Molecular Immunology, the IL-2 no-alpha mutein, an IL-2 agonist with lower toxicity than wtIL-2, has been designed, produced, and is currently being evaluated in a Phase I/II clinical trial. The mutein is produced in E. coli as an insoluble material that must be refolded in vitro to yield a fully active protein. Controlled oxidation steps are essential in the purification process of recombinant proteins produced in E. coli to ensure the proper formation of the disulfide bonds in the molecules. In this case, the new purification process includes a copper-catalyzed air oxidation step to induce disulfide bond establishment. The optimal conditions of pH, copper, protein and detergent concentration for this step were determined through screening. The produced protein demonstrated a conserved 3D structure, higher purity, and greater biological activity than the obtained by established process without the oxidation step. Four batches were produced and evaluated, demonstrating the consistency of the new process.