Unraveling the genetic basis of the causal association between inflammatory cytokines and osteonecrosis.

Background: Previous studies have reported that the occurrence and development of osteonecrosis is closely associated with immune-inflammatory responses. Mendelian randomization was performed to further assess the causal correlation between 41 inflammatory cytokines and osteonecrosis. Methods: Two-sample Mendelian randomization utilized genetic variants for osteonecrosis from a large genome-wide association study (GWAS) with 606 cases and 209,575 controls of European ancestry. Another analysis included drug-induced osteonecrosis with 101 cases and 218,691 controls of European ancestry. Inflammatory cytokines were sourced from a GWAS abstract involving 8,293 healthy participants. The causal relationship between exposure and outcome was primarily explored using an inverse variance weighting approach. Multiple sensitivity analyses, including MR-Egger, weighted median, simple model, weighted model, and MR-PRESSO, were concurrently applied to bolster the final results. Results: The results showed that bFGF, IL-2 and IL2-RA were clinically causally associated with the risk of osteonecrosis (OR=1.942, 95% CI=1.13-3.35, p=0.017; OR=0.688, 95% CI=0.50-0.94, p=0.021; OR=1.386, 95% CI=1.04-1.85, p = 0.026). there was a causal relationship between SCF and drug-related osteonecrosis (OR=3.356, 95% CI=1.09-10.30, p=0.034). Conclusion: This pioneering Mendelian randomization study is the first to explore the causal link between osteonecrosis and 41 inflammatory cytokines. It conclusively establishes a causal association between osteonecrosis and bFGF, IL-2, and IL-2RA. These findings offer valuable insights into osteonecrosis pathogenesis, paving the way for effective clinical management. The study suggests bFGF, IL-2, and IL-2RA as potential therapeutic targets for osteonecrosis treatment.

A novel dual mechanism-of-action bispecific PD-1-IL-2v armed by a "ßγ-only" interleukin-2 variant.

Introduction: Interleukin-2 (IL-2) is one of the first cytokines to be discovered as an immune agonist for cancer immunotherapy. Biased IL-2 variants had been discovered to eliminate Treg activation or enhance the tumor specific T cell cytotoxicity. However, all the biased IL-2 variants pose the risk to overstimulate immune response at a low-dose range. Here, we introduce a novel dual-MOA bispecific PD-1-IL-2v molecule with great anti-tumor efficacy in a high dosed manner. Methods: The novel IL-2 variant was designed by structural truncation and shuffling. The single armed bispecific PD-1-IL-2v molecule and IL-2v were studied by immune cell activations in vitro and in vivo and anti-tumor efficacy in mouse model. Results and discussion: The IL-2 variant in this bispecific antibody only binds to IL-2Rßγ complex in a fast-on/off manner without α, ß or γ single receptor binding. This IL-2v mildly activates T and NK cells without over stimulation, meanwhile it diminishes Treg activation compared to the wild type IL-2. This unique bispecific molecule with "ßγ-only" IL-2v can not only "in-cis" stimulate and expand CD8 T and NK cells moderately without Treg activation, but also block the PD-1/L1 interaction at a similar dose range with monoclonal antibody.

Interleukin-2 enhancer binding factor 2 negatively regulates the replication of duck hepatitis A virus type 1 by disrupting the RNA-dependent RNA polymerase activity of 3D polymerase.

The interaction between viral components and cellular proteins plays a crucial role in viral replication. In a previous study, we showed that the 3'-untranslated region (3'-UTR) is an essential element for the replication of duck hepatitis A virus type 1 (DHAV-1). However, the underlying mechanism is still unclear. To gain a deeper understanding of this mechanism, we used an RNA pull-down and a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay to identify new host factors that interact with the 3'-UTR. We selected interleukin-2 enhancer binding factor 2 (ILF2) for further analysis. We showed that ILF2 interacts specifically with both the 3'-UTR and the 3D polymerase (3Dpol) of DHAV-1 through in vitro RNA pull-down and co-immunoprecipitation assays, respectively. We showed that ILF2 negatively regulates viral replication in duck embryo fibroblasts (DEFs), and that its overexpression in DEFs markedly suppresses DHAV-1 replication. Conversely, ILF2 silencing resulted in a significant increase in viral replication. In addition, the RNA-dependent RNA polymerase (RdRP) activity of 3Dpol facilitated viral replication by enhancing viral RNA translation efficiency, whereas ILF2 disrupted the role of RdRP in viral RNA translation efficiency to suppress DHAV-1 replication. At last, DHAV-1 replication markedly suppressed the expression of ILF2 in DEFs, duck embryo hepatocytes, and different tissues of 1 day-old ducklings. A negative correlation was observed between ILF2 expression and the viral load in primary cells and different organs of young ducklings, suggesting that ILF2 may affect the viral load both in vitro and in vivo.

Long-lasting mRNA-encoded interleukin-2 restores CD8+ T cell neoantigen immunity in MHC class I-deficient cancers.

Major histocompatibility complex (MHC) class I antigen presentation deficiency is a common cancer immune escape mechanism, but the mechanistic implications and potential strategies to address this challenge remain poorly understood. Studying ß2-microglobulin (B2M) deficient mouse tumor models, we find that MHC class I loss leads to a substantial immune desertification of the tumor microenvironment (TME) and broad resistance to immune-, chemo-, and radiotherapy. We show that treatment with long-lasting mRNA-encoded interleukin-2 (IL-2) restores an immune cell infiltrated, IFNγ-promoted, highly proinflammatory TME signature, and when combined with a tumor-targeting monoclonal antibody (mAB), can overcome therapeutic resistance. Unexpectedly, the effectiveness of this treatment is driven by IFNγ-releasing CD8+ T cells that recognize neoantigens cross-presented by TME-resident activated macrophages. These macrophages acquire augmented antigen presentation proficiency and other M1-phenotype-associated features under IL-2 treatment. Our findings highlight the importance of restoring neoantigen-specific immune responses in the treatment of cancers with MHC class I deficiencies.

Tipping the balance toward transplantation tolerance: in vivo therapy using a mutated IL-2.

Immune tolerance to allogenic transplanted tissues remains elusive, and therapeutics promoting CD4+FOXP3+ Tregs are required to achieve this ultimate goal. In this issue of the JCI, Efe and colleagues engineered an Fc domain fused to a human mutein IL-2 (mIL-2-Fc) bearing mutations that confer preferential binding to the high-affinity IL-2 receptor expressed on Tregs. In vivo mIL-2-Fc therapy effectively heightened mouse, monkey, and human Treg numbers, promoted tolerance to minor antigen mismatched skin grafts in mice, and synergized with immunosuppressive drugs used in the clinic. These findings warrant clinical trials that assess the efficacy of mIL-2-Fc in transplantation.

Tumor-activated IL-2 mRNA delivered by lipid nanoparticles for cancer immunotherapy.

Interleukin-2 (IL-2) exhibits the unique capacity to modulate immune functions, potentially exerting antitumor effects by stimulating immune responses, making it highly promising for immunotherapy. However, the clinical use of recombinant IL-2 protein faces significant limitations due to its short half-life and systemic toxicity. To overcome these challenges and fully exploit IL-2's potential in tumor immunotherapy, this study reports the development of a tumor-activated IL-2 mRNA, delivered via lipid nanoparticles (LNPs). Initially, ionizable lipid U-101 derived nanoparticles (U-101-LNP) were prepared using microfluidic technology. Subsequent in vitro and in vivo delivery tests demonstrated that U-101-LNP achieved more effective transfection than the approved ALC-0315-LNP. Following this, IL-2F mRNAs, encoding fusion proteins comprising IL-2, a linker, and CD25 (IL-2Rα), were designed and synthesized through in vitro transcription. A cleavable linker, consisting of the peptide sequence SGRSEN↓IRTA, was selected for cleavage by matrix metalloproteinase-14 (MMP-14). IL-2F mRNA was then encapsulated in U-101-LNP to create U-101-LNP/IL-2F mRNA complexes. After optimization, assessments of expression efficiency, masking, and release characteristics revealed that IL-2F with linker C4 demonstrated superior performance. Finally, the antitumor activity of IL-2F mRNA was evaluated. The results indicated that U-101-LNP/IL-2F mRNA achieved the strongest antitumor effect, with an inhibition rate of 70.3%. Immunohistochemistry observations revealed significant expressions of IL-2, IFN-γ, and CD8, suggesting an up-regulation of immunomodulation in tumor tissues. This effect could be ascribed to the expression of IL-2F, followed by the cleavage of the linker under the action of MMP-14 in tumor tissue, which sustainably releases IL-2. H&E staining of tissues treated with U-101-LNP/IL-2F mRNA showed no abnormalities. Further evaluations indicated that the U-101-LNP/IL-2F mRNA group maintained proper levels of inflammatory factors without obvious alterations in liver and renal functions. Taken together, the U-101-LNP/IL-2F mRNA formulation demonstrated effective antitumor activity and safety, which suggests potential applicability in clinical immunotherapy.

Inducible IL-2 production and IL-2+ cell expansion are landmark events for T-cell activation of teleost.

As a multipotent cytokine, interleukin (IL)-2 plays important roles in activation, differentiation and survival of the lymphocytes. Although biological characteristics and function of IL-2 have been clarified in several teleost species, evidence regarding IL-2 production at the cellular and protein levels is still scarce in fish due to the lack of reliable antibody. In this study, we developed a mouse anti-Nile tilapia IL-2 monoclonal antibody (mAb), which could specifically recognize IL-2 protein and identify IL-2-producing lymphocytes of tilapia. Using this mAb, we found that CD3+ T cells, but not CD3- lymphocytes, are the main cellular source of IL-2 in tilapia. Under resting condition, both CD3+CD4-1+ T cells and CD3+CD4-1- T cells of tilapia produce IL-2. Moreover, the IL-2 protein level and the frequency of IL-2+ T cells significantly increased once T cells were activated by phytohemagglutinin (PHA) or CD3 plus CD28 mAbs in vitro. In addition, Edwardsiella piscicida infection also induces the IL-2 production and the expansion of IL-2+ T cells in the spleen lymphocytes. These findings demonstrate that IL-2 takes part in the T-cell activation and anti-bacterial adaptive immune response of tilapia, and can serve as an important marker for T-cell activation of teleost fish. Our study has enriched the knowledge regarding T-cell response in fish species, and also provide novel perspective for understanding the evolution of adaptive immune system.

Exploring the impact of nano-Se and nano-clay feed supplements on interleukin genes, immunity and growth rate in European Sea Bass (Dicentrarchus labrax).

This study aimed to investigate the effects of adding Nano-Selenium (NSe) and Nano-clay (NC) as feed supplements on European Sea Bass (Dicentrarchus labrax). Two separate experiments were conducted, one with NC and the other with NSe. Each experiment consisted of four sub-groups with varying concentrations of NC or NSe. The expression levels of five immune-related genes (TNF-α, TNF-ß, IL-2, IL-6 and IL-12) were measured using Real-time Quantitative PCR (Rt-PCR) Assay. The results showed an increase in the expression of interleukins (IL-2, IL-6 and IL-12) and pro-inflammatory cytokines (TNF-α and TNF-ß) after exposure to NC and NSe. TNF-α gene expression was significantly higher with both 1 mg and 10 mg concentrations of NC and NSe. TNF-ß gene expression was highest with the 5 mg concentration of NC. The concentrations of 1 mg and 10 mg for NC, and 1 mg, 5 mg, and 10 mg for NSe, led to the highest (p < 0.05) levels of IL-2 expression compared to the control. Similar trends were observed for IL-6 and IL-12 gene expression. Understanding the impact of these concentrations on gene expression, growth rate, biochemical indices, and antioxidant status can provide valuable insights into the potential applications of NC and NSe supplements on European Sea Bass.

Construction of rBCG carrying the IL-2-BZLF1 fusion gene and its immunological function.

In this research, a recombinant Bacillus Calmette Guerin (rBCG) vector vaccine carrying a human IL-2 and EBV BZLF1 fusion gene (IL-2-BZLF1-rBCG) was constructed. The IL-2-BZLF1-rBCG construct was successfully generated and stably expressed the IL-2 and BZLF1 proteins. IL-2-BZLF1-rBCG activated the immune system and promoted the secretion of IFN-γ and TNF-α by CD4+ and CD8+ T cells. IL-2-BZLF1-rBCG activated lymphocytes to effectively kill EBV-positive NPC cells in vitro. Additionally, IL-2-BZLF1-rBCG stimulated the proliferation of NK cells and lymphocytes in vivo, activated related immune responses, and effectively treated EBV-positive NPC. The immune response to and pharmacological effect of IL-2-BZLF1-rBCG were explored in vitro and in vivo to provide a theoretical and experimental basis for the prevention and treatment of EBV-positive tumors with an rBCG vector vaccine. KEY POINTS: • rBCG with human IL-2 and BZLF1 of EB virus was constructed • The IL-2-BZLF1 fusion gene was stably expressed with rBCG • rBCG with IL-2-BZLF1 has an obvious immune response in vitro and in vivo.

Evaluation of gene-gene interaction between the interleukin (IL)-2 and IL-2RA gene polymorphisms in schizophrenia patients in the Turkish Population.

OBJECTIVES: To evaluate the genetic polymorphisms in IL-2 and IL-2RA genes in schizophrenia (SCZ) patients by comparing them with healthy controls. METHODS: A sample of 127 patients with SCZ and 100 healthy volunteers were included in the case-control study. These individuals were consecutively selected from the Malazgirt State Hospital Psychiatry Outpatient Clinic in Mus, Turkey, over the three months from October 2020 to December 2020. The Structured Clinical Interview for DSM-5 Disorders, Clinician Version (SCID-5-CV) was used to confirm the diagnosis according to the DSM-5 criteria. In addition, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to determine gene polymorphisms from DNA material. RESULTS: Our findings indicated significant differences in the IL-2 genotype and allele frequencies between SCZ patients and the healthy control group. Specifically, the frequency of the homozygous GG genotype was notably higher in SCZ patients compared to the control group. Conversely, when comparing the IL-2RA genotype and allele frequencies of SCZ patients with the control group, no statistically significant differences were observed between the 2 groups. When compared to individuals with other genotypes, interaction analysis indicated that carriers of the GG/AG (IL-2/IL-2RA) genotype demonstrated a significantly increased risk of SCZ. CONCLUSION: In light of the analyses, our study indicates that while the IL-2 genotype polymorphism may be considered a risk factor for developing SCZ, the IL-2RA variant was not associated with SCZ among Turkish patients.

Effect of Organic Selenium Supplementation on the Antioxidant Status, Immune Response, and the Relative Expression of IL-2 and IFN-γ Genes in Ewes During the Hot Season.

This study was conducted to evaluate the effects of different doses of selenium (Se) from Sel-Plex© (selenium-enriched Saccharomyces cerevisiae yeast) supplement on the antioxidant status, the antibody titers against the foot-and-mouth disease virus, and the expression of interleukin-2 (IL-2) and interferon-γ (IFN-γ) genes in ewes during the hot season. Six ewes were kept at 25 °C and received basal diet (the negative control group), and 24 ewes were kept at 38 °C for 5 h per day and received no supplement (the positive control), 0.15, 0.30, and 0.45 mg Se/kg. Ewes in the positive control had higher (P<0.001) liver enzyme activity, malondialdehyde (MDA), and cortisol levels, and lower antibody titer than the negative control. The liver enzymes' lowest (P<0.001) activities were observed in ewes receiving 0.30 and 0.45 mg Se/kg. Ewes receiving 0.30 and 0.45 mg Se/kg had lower MDA levels than other treatments. Ewes receiving 0.30 and 0.45 mg Se/kg had higher (P<0.001) total antioxidant capacity levels than those receiving 0.15 mg Se/kg and the positive control. Se-supplemented groups had lower (P<0.001) relative expression of IL-2 and higher (P<0.04) expression of IFN-γ than the positive control. The antibody titer was the same in the positive control and the group receiving 0.15 mg Se/kg. Ewes fed a diet with 0.30 and 0.45 mg Se/kg had higher (P<0.011) antibody titer than the positive control. The Se supplementation can reverse the decrease of antioxidant capacity and immune function caused by heat stress, and 0.3 mg Se/kg from Sel-Plex©is the best dose.

Molecular Engineering of Interleukin-2 for Enhanced Therapeutic Activity in Autoimmune Diseases.

The interleukin-2 (IL-2) cytokine plays a crucial role in regulating immune responses and maintaining immune homeostasis. Its immunosuppressive effects have been harnessed therapeutically via administration of low cytokine doses. Low-dose IL-2 has shown promise in the treatment of various autoimmune and inflammatory diseases; however, the clinical use of IL-2 is complicated by its toxicity, its pleiotropic effects on both immunostimulatory and immunosuppressive cell subsets, and its short serum half-life, which collectively limit the therapeutic window. As a result, there remains a considerable need for IL-2-based autoimmune disease therapies that can selectively target regulatory T cells with minimal off-target binding to immune effector cells in order to prevent cytokine-mediated toxicities and optimize therapeutic efficacy. In this review, we discuss exciting advances in IL-2 engineering that are empowering the development of novel therapies to treat autoimmune conditions. We describe the structural mechanisms of IL-2 signaling, explore current applications of IL-2-based compounds as immunoregulatory interventions, and detail the progress and challenges associated with clinical adoption of IL-2 therapies. In particular, we focus on protein engineering approaches that have been employed to optimize the regulatory T-cell bias of IL-2, including structure-guided or computational design of cytokine mutants, conjugation to polyethylene glycol, and the development of IL-2 fusion proteins. We also consider future research directions for enhancing the translational potential of engineered IL-2-based therapies. Overall, this review highlights the immense potential to leverage the immunoregulatory properties of IL-2 for targeted treatment of autoimmune and inflammatory diseases.

IL2RA rs2104286 and IL2 rs2069762 polymorphisms may be associated with bipolar disorder and its clinical findings.

Study results supported that immuno-inflammatory pathways in the brain and environment contribute to the etiopathogenesis of bipolar disorder (BD), a chronic affective disease. Our study aimed to assess the relationship between BD risk and interleukin 2 (IL2) and interleukin 2 receptor subunit alpha (IL2RA) variants in a Turkish population. Genomic DNA from 86 diagnosed BD patients and 100 healthy blood donors was extracted. IL2RA rs2104286, IL2 rs2069762, and IL2 rs2069763 variants were genotyped using the polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) method. It was compared to the relationship between the genotype distributions of these variants and clinical characteristics. Results were evaluated statistically. A statistically significant difference in the genotype distribution of the IL2RA rs2104286 variant was found between patients and controls. There was no GG genotype in the patient group. The IL2RA rs2104286 AA genotype was more common in the patient group than the controls, and the AG genotype was higher in the controls compared to the patients (p = 0.001, p = 0.001, respectively). The IL2 rs2069762 and IL2 rs2069763 genotype distributions did not differ between the patient and control groups (p > 0.05). We found that the clinical global impression severity (CGI-S) score was higher in those with IL2 rs2069762 TG and GG genotypes. In this study, we showed for the first time that the genotype distribution of IL2RA rs2104286 and IL2 rs2069762 is associated with BD susceptibility and CGI-S score in a Turkish population.

Immune synergistic mechanism of recombinant plasmid adjuvant containing chicken IL-4 and IL-2 fusion genes on chicken coccidia live vaccine.

The recombinant plasmid pCI-IL-4-IL-2-EGFP containing fusion genes of chicken IL-4 and IL-2 can be used as an adjuvant to enhance the anticoccidiosis effect of the chicken coccidia live vaccine. The chickens were divided into 3 groups: blank control group, vaccine + pCI-IL-4-IL-2-EGFP adjuvant coimmunization group, and vaccine-only group to investigate the immune synergy mechanism of recombinant plasmid adjuvant pCI-IL-4-IL-2-EGFP. The expressions of IL-2, IL-4, TNF-α, and IFN-γ in chicken sera and tissues were detected by ELISA and RT-qPCR, and the proliferation of T and B lymphocytes and antigen presenting cells (APC) in chicken immune organs and intestines were detected by acid alpha-naphthalase (ANAE) staining, methyl green pyronine (MGP) staining, and immunofluorescence (IF) staining, respectively. Results showed that the mRNA expression of IL-2, IL-4, IFN-γ and the number of activated T and B lymphocytes were significantly upregulated in the spleen and cecum tonsils of chickens in vaccine + pCI-IL-4-IL-2-EGFP group compared with the vaccine-only group on 7 d after vaccination (P < 0.05). Protein contents of IL-2, IL-4 and TNF-α in vaccine + pCI-IL-4-IL-2-EGFP group were significantly increased compared to vaccine-only group on 28 d of inoculation (P < 0.05). The number of T and B lymphocytes and APC in chickens of the vaccine+ pCI-IL-4-IL-2-EGFP group was significantly higher than that of the vaccine-only group in cecum tonsils, thymus and spleen after 14 and 28 d of inoculation (P < 0.05). All results revealed that pCI-IL-4-IL-2-EGFP adjuvant enhanced the immune response of chicken coccidia live vaccine by upregulating the expression of IL-2, IL-4, TNF-α, and IFN-γ and promoting the proliferation of T, B lymphocytes and APCs in chicken intestines and immune organ sites. Moreover, our study provides a theoretical basis for the clinical application of cytogenic plasmids as adjuvants.

Targeting alternative splicing as a new cancer immunotherapy-phosphorylation of serine arginine-rich splicing factor (SRSF1) by SR protein kinase 1 (SRPK1) regulates alternative splicing of PD1 to generate a soluble antagonistic isoform that prevents T cell exhaustion.

BACKGROUND: Regulation of alternative splicing is a new therapeutic approach in cancer. The programmed cell death receptor 1 (PD-1) is an immunoinhibitory receptor expressed on immune cells that binds to its ligands, PD-L1 and PD-L2 expressed by cancer cells forming a dominant immune checkpoint pathway in the tumour microenvironment. Targeting this pathway using blocking antibodies (nivolumab and pembrolizumab) is the mainstay of anti-cancer immunotherapies, restoring the function of exhausted T cells. PD-1 is alternatively spliced to form isoforms that are either transmembrane signalling receptors (flPD1) that mediate T cell death by binding to the ligand, PD-L1 or an alternatively spliced, soluble, variant that lacks the transmembrane domain. METHODS: We used PCR and western blotting on primary peripheral blood mononuclear cells (PBMCs) and Jurkat T cells, IL-2 ELISA, flow cytometry, co-culture of melanoma and cholangiocarcinoma cells, and bioinformatics analysis and molecular cloning to examine the mechanism of splicing of PD1 and its consequence. RESULTS: The soluble form of PD-1, generated by skipping exon 3 (∆Ex3PD1), was endogenously expressed in PBMCs and T cells and prevents cancer cell-mediated T cell repression. Multiple binding sites of SRSF1 are adjacent to PD-1 exon 3 splicing sites. Overexpression of phosphomimic SRSF1 resulted in preferential expression of flPD1. Inhibition of SRSF1 phosphorylation both by SRPK1 shRNA knockdown and by a selective inhibitor, SPHINX31, resulted in a switch in splicing to ∆Ex3PD1. Cholangiocarcinoma cell-mediated repression of T cell IL-2 expression was reversed by SPHINX31 (equivalent to pembrolizumab). CONCLUSIONS: These results indicate that switching of the splicing decision from flPD1 to ∆Ex3PD1 by targeting SRPK1 could represent a potential novel mechanism of immune checkpoint inhibition in cancer.

An engineered immunocytokine with collagen affinity improves the tumor bioavailability, tolerability, and therapeutic efficacy of IL-2.

The clinical utility of human interleukin-2 (hIL-2) is limited by its short serum half-life, preferential activation of regulatory T (TReg) over immune effector cells, and dose-limiting toxicities. We previously engineered F10 immunocytokine (IC), an intramolecularly assembled cytokine/antibody fusion protein that linked hIL-2 to an anti-IL-2 antibody (denoted F10) that extended IL-2 half-life and augmented the immune effector to TReg ratio. Here, we leveraged molecular engineering to improve the anti-tumor therapeutic efficacy and tolerability of F10 IC by developing an iteration, denoted F10 IC-CBD (collagen binding domain), designed for intratumoral administration and in situ retention based on collagen affinity. F10 IC-CBD retained IL-2 bioactivity exclusively in the tumor and eliminated IL-2-associated toxicities. Furthermore, F10 IC exhibited potent single-agent therapeutic efficacy and synergy with systemic immune checkpoint blockade and elicited an abscopal response in mouse tumors models. This engineered fusion protein presents a prototype for the design of intratumoral therapies.

Distinct use of super-enhancer elements controls cell type-specific CD25 transcription and function.

The IL-2 receptor α chain (IL-2Rα/CD25) is constitutively expressed on double-negative (DN2/DN3 thymocytes and regulatory T cells (Tregs) but induced by IL-2 on T and natural killer (NK) cells, with Il2ra expression regulated by a STAT5-dependent super-enhancer. We investigated CD25 regulation and function using a series of mice with deletions spanning STAT5-binding elements. Deleting the upstream super-enhancer region mainly affected constitutive CD25 expression on DN2/DN3 thymocytes and Tregs, with these mice developing autoimmune alopecia, whereas deleting an intronic region decreased IL-2-induced CD25 on peripheral T and NK cells. Thus, distinct super-enhancer elements preferentially control constitutive versus inducible expression in a cell type-specific manner. The mediator-1 coactivator colocalized with specific STAT5-binding sites. Moreover, both upstream and intronic regions had extensive chromatin interactions, and deletion of either region altered the super-enhancer structure in mature T cells. These results demonstrate differential functions for distinct super-enhancer elements, thereby indicating previously unknown ways to manipulate CD25 expression in a cell type-specific fashion.

Exploring the causal relationship between inflammatory cytokines and migraine: a bidirectional, two-sample Mendelian randomization study.

To further evaluate the causal relationships between inflammatory cytokines and migraine, we conducted a bidirectional, two-sample Mendelian randomization (MR) analysis using genetic data from publicly available genome-wide association studies (GWAS). We used several MR methods, including random-effect inverse-variance weighting (IVW), weighted median, MR-Egger, to test the causal relationships. Sensitivity analyses were also conducted to evaluate the robustness of the results. The results showed that hepatocyte growth factor (HGF) was positively associated with the risk of migraine (odds ratio [OR], 1.004; 95% confidence interval [CI], 1.001-1.008; P = 0.022). In addition, Interleukin-2 (IL-2) was considered a downstream consequence of migraine (OR, 0.012; 95% CI, 0.000-0.0929; P = 0.046). These findings suggest that HGF may be a factor associated with the etiology of migraine, while IL-2 is more likely to be involved in the downstream development of migraine.

Interleukin-2 gene methylation levels and interleukin-2 levels associated with environmental exposure as risk biomarkers for preterm birth.

AIM: To compare interleukin-2 levels (IL-2) and IL-2 gene site 1 methylation levels between preterm newborns (PN) and full-term newborns (FN) and investigate their association with the environmental exposure of their mothers during pregnancy. METHODS: IL-2 and IL-2 gene site 1 methylation levels were assessed in 50 PN and 56 FN. Newborns' mothers filled in questionnaires about their living and occupational environments, habits, diets, and hobbies. RESULTS: The mothers of PN were significantly more frequently agrarian/rural residents than the mothers of FN. PN had significantly higher IL-2 levels, and significantly lower methylation of IL-2 gene site 1 levels than FN. CONCLUSION: IL-2 levels, hypomethylation of the IL-2 gene site 1, and the mother's rural residence (probably due to pesticide exposure) were predictive biomarkers for preterm birth. For the first time, we present the reference values for the methylation of IL-2 gene site 1 in PN and FN, which can be used in the clinical setting and biomonitoring.

Multivalent, asymmetric IL-2-Fc fusions show enhanced selectivity for regulatory T cells.

The cytokine interleukin-2 (IL-2) has the potential to treat autoimmune disease but is limited by its modest specificity toward immunosuppressive regulatory T (Treg) cells. IL-2 receptors consist of combinations of α, ß, and γ chains of variable affinity and cell specificity. Engineering IL-2 to treat autoimmunity has primarily focused on retaining binding to the relatively Treg-selective, high-affinity receptor while reducing binding to the less selective, low-affinity receptor. However, we found that refining the designs to focus on targeting the high-affinity receptor through avidity effects is key to optimizing Treg selectivity. We profiled the dynamics and dose dependency of signaling responses in primary human immune cells induced by engineered fusions composed of either wild-type IL-2 or mutant forms with altered affinity, valency, and fusion to the antibody Fc region for stability. Treg selectivity and signaling response variations were explained by a model of multivalent binding and dimer-enhanced avidity-a combined measure of the strength, number, and conformation of interaction sites-from which we designed tetravalent IL-2-Fc fusions that had greater Treg selectivity in culture than do current designs. Biasing avidity toward IL2Rα with an asymmetrical multivalent design consisting of one α/ß chain-binding and one α chain-binding mutant further enhanced Treg selectivity. Comparative analysis revealed that IL2Rα was the optimal cell surface target for Treg selectivity, indicating that avidity for IL2Rα may be the optimal route to producing IL-2 variants that selectively target Tregs.