Interleukin-2 Receptor Beta Research Articles

Recurrent Visceral Leishmaniasis in a Case With Interleukin-12 Receptor Beta-1 Deficiency.

In this study, we present the case of a children who was followed up for recurrent visceral leishmaniasis and diagnosed with IL-12Rβ1 deficiency. A female patient who received Bacille Calmette-Guérin (BCG) vaccine 2 months after birth and developed visceral leishmaniasis at the age of 91 months was subsequently diagnosed with IL-12Rβ1 deficiency. The patient's diagnosis and treatment process were examined retrospectively. IL-12Rβ1 deficiency is an autosomal recessive disease characterized by susceptibility to recurrent and/or severe infections caused by weakly pathogenic mycobacteria and salmonella. Infections with other intramacrophagic organisms may also occur, although rarely. Based on this information, it is believed that the mutation in the IFN-γ/IL-12 axis in our patient predisposed her to recurrent Leishmania infections. This study adds to the limited literature on IL12RB1 deficiency as a cause of VL. Patients diagnosed with VL should be evaluated immunologically, as recurrent Leishmania infections may occur in those with IL-12Rβ1 defects.

O35 THE SYNERGISM BETWEEN HIGH GLUCOSE AND INTERLEUKIN-1beta PROMOTES INFLAMMASOME ACTIVATION AND THE RELEASE OF SMALL EXTRACELLULAR VESICLES IN HUMAN AORTIC SMOOTH MUSCLE CELLS

Background and objective: Hyperglycemia and inflammatory cytokines like interleukin (IL)-1beta play a key role in the vascular complications associated to diabetes melllitus (DM), such as atherosclerosis and hypertension. Understanding the pathophysiological mechanisms involved, including altered intercellular communication, is essential to identify potential therapeutical targets. We investigated the synergism of IL-1beta and high glucose on: (1) the activation of nucleotide-binding-oligomerization-domain, leucine-rich-repeat, and pyrin-domain-containing-3 (NLRP3)-inflammasome, an innate immunity component linked to vascular disease, and (2) the production and cargo of small extracellular vesicles (sEVs) as key players in vascular intercellular communication. Methods: Human aortic smooth muscle cells (HASMC) were challenged for 18 h with IL-1beta (10 ng/ml) in the presence of normal (5,5 mmol/L) or high glucose (22 mmol/L). Protein levels were quantified by Western blot, while the formation of ASC-like specks, reflecting NLRP3 activation, was assessed by indirect immunofluorescence. sEVs were determined by nanotracking analysis and endothelial senescence by the beta-galactosidase activity assay. Results: In HASMC cultures, IL-1beta favored NLRP3-inflammasome priming, as shown by NF-kappaB (phospho-p65) activation while NLRP3 and pro-IL-1beta levels were increased. The cytokine equally promoted NLRP3-inflammasome complex assembly, caspase-1 activation, and a subsequent release of mature IL-1beta in an auto-inflammatory loop. High glucose alone did not promote these effects, but it exacerbated some actions of IL-1beta and potentiated the release of HASMC-derived sEVs induced by IL-1beta, increasing their cargo content in NLRP3-inflammasome components. Moreover, these sEVs induce autocrine NLRP3-inflammasome activation in naïve HASMC and paracrine senescence in human endothelial cells. The IL-1 receptor blocker anakinra (1 mg/ml) blunted the IL-1beta effect, as well as their exacerbation by high glucose. Conclusion: High glucose exacerbates the activation of NLRP3-inflammasome by IL-1beta and alters intercellular communication by modifying the number and content of sEVs, which can expand inflammation in neighboring tissues. By preventing such synergy, biological drugs blocking IL-1beta receptors arise as potential pharmacological tools to attenuate DM associated vascular abnormalities.

Inflammatory Response Genes' Polymorphism Associated with Risk of Rheumatic Heart Disease.

Rheumatic heart disease (RHD) caused by group A streptococcus infection is one of the most important reasons of cardiovascular morbidity and mortality in low- and middle-income countries. Aberrant host immune response modulated by polymorphisms in inflammatory response genes plays an important role in RHD pathogenesis. This study aimed to determine risk-associated polymorphic variants in inflammatory response genes in Caucasian RHD patients. A total of 251 Caucasian RHD patients and 300 healthy donors were recruited for this study, and 27 polymorphic sites in 12 genes (TLR1, TLR2, TLR4, TLR6, IL1B, IL6R, IL6, IL10, IL12RB1, IL12B, TNF and CRP) were analyzed using allele-specific PCR. It was demonstrated that the polymorphic variants rs1800871 and rs1800872 in the IL10 gene, rs 1130864, rs3093077 and rs1205 in the CRP gene, rs375947 in the IL12RB1 gene, rs 5743551 and rs5743611 in the TLR1 gene, and rs3775073 in the TLR6 gene can modify RHD risk in a gender- and age-dependent manner. The obtained results can be used to determine the personalized risk of RHD in healthy donors during medical examination or screening, as well as to develop appropriate early prevention strategies targeting RHD in the risk groups.

IL-12 drives the differentiation of human T follicular regulatory cells.

T follicular regulatory (Tfr) cells can counteract the B cell helper activity of T follicular helper (Tfh) cells and hinder the production of antibodies against self-antigens or allergens. A mechanistic understanding of the cytokines initiating the differentiation of human regulatory T (Treg) cells into Tfr cells is still missing. Herein, we report that low doses of the pro-Tfh cytokine interleukin-12 (IL-12) drive the induction of a Tfr cell program on activated human Treg cells while also preserving their regulatory function. Mechanistically, we found that IL-12 led to STAT4 (signal transducer and activator of transcription 4) phosphorylation and binding to IL-12-driven follicular signature genes. Patients with inborn errors of immunity in the IL12RB1 gene presented with a strong decrease in circulating Tfr cells and produced higher levels of anti-actin autoantibodies in vivo. Overall, this study unveils IL-12 as an inducer of Tfr cell differentiation in vivo and provides an approach for the in vitro generation of human Tfr-like cells.

Primary immune regulatory disorders (PIRD): expanding the mutation spectrum in Turkey and identification of sixteen novel variants

Human Inborn Errors of Immunity (IEIs) encompass a clinically and genetically heterogeneous group of disorders, ranging from mild cases to severe, life-threatening types. Among these, Primary Immune Regulatory Disorders (PIRDs) constitute a subset of IEIs characterized by diverse clinical phenotypes, prominently featuring severe atopy, autoimmunity, lymphoproliferation, hyperinflammation, autoinflammation, and susceptibility to malignancies. According to the latest report from the International Union of Immunological Societies (IUIS), PIRDs arise from mutations in various genes including LYST, RAB27A, AP3B1, AP3D1, PRF1, UNC13D, STX11, STXBP2, FAAP24, SLC7A7, RASGRP1, CD70, CTPS1, RLTPR, ITK, MAGT1, PRKCD, TNFRSF9, SH2DIA, XIAP, CD27 (TNFRSF7), FAS (TNFRSF6), FASLG (TNFSF6), CASP10, CASP8, FADD, LRBA, STAT3, AIRE, ITCH, ZAP70, TPP2, JAK1, PEPD, FOXP3, IL2RA, CTLA4, BACH2, IL2RB, DEF6, FERMT1, IL10, IL10RA, IL10RB, NFAT5, TGFB1, and RIPK1 genes. We designed a targeted next-generation sequencing (TNGS) workflow using the Ion AmpliSeq™ Primary Immune Deficiency Research Panel to sequence 264 genes associated with IEIs on the Ion S5™ Sequencer. In this study, we report the identification of 38 disease-causing variants, including 16 novel ones, detected in 40 patients across 15 distinct PIRD genes. The application of next-generation sequencing enabled rapid and precise diagnosis of patients with PIRDs.

Next-generation sequencing of host genetics risk factors associated with COVID-19 severity and long-COVID in Colombian population

Coronavirus disease 2019 (COVID-19) was considered a major public health burden worldwide. Multiple studies have shown that susceptibility to severe infections and the development of long-term symptoms is significantly influenced by viral and host factors. These findings have highlighted the potential of host genetic markers to identify high-risk individuals and develop target interventions to reduce morbimortality. Despite its importance, genetic host factors remain largely understudied in Latin-American populations. Using a case–control design and a custom next-generation sequencing (NGS) panel encompassing 81 genetic variants and 74 genes previously associated with COVID-19 severity and long-COVID, we analyzed 56 individuals with asymptomatic or mild COVID-19 and 56 severe and critical cases. In agreement with previous studies, our results support the association between several clinical variables, including male sex, obesity and common symptoms like cough and dyspnea, and severe COVID-19. Remarkably, thirteen genetic variants showed an association with COVID-19 severity. Among these variants, rs11385942 (p < 0.01; OR = 10.88; 95% CI = 1.36–86.51) located in the LZTFL1 gene, and rs35775079 (p = 0.02; OR = 8.53; 95% CI = 1.05–69.45) located in CCR3 showed the strongest associations. Various respiratory and systemic symptoms, along with the rs8178521 variant (p < 0.01; OR = 2.51; 95% CI = 1.27–4.94) in the IL10RB gene, were significantly associated with the presence of long-COVID. The results of the predictive model comparison showed that the mixed model, which incorporates genetic and non-genetic variables, outperforms clinical and genetic models. To our knowledge, this is the first study in Colombia and Latin-America proposing a predictive model for COVID-19 severity and long-COVID based on genomic analysis. Our study highlights the usefulness of genomic approaches to studying host genetic risk factors in specific populations. The methodology used allowed us to validate several genetic variants previously associated with COVID-19 severity and long-COVID. Finally, the integrated model illustrates the importance of considering genetic factors in precision medicine of infectious diseases.

Abstract 6312: Orthogonal IL-2/IL-2Rβ signaling selectively enhances and sustains a synthetic effector state via a novel mechanism and outperforms constitutive armoring approaches

Abstract CAR T cell therapy has demonstrated remarkable clinical efficacy against blood malignancies. However, prominent barriers, including limited proliferation, CAR T cell persistence, and poor T cell effector function, hinder its potential, especially in solid tumors. Various engineering approaches attempt to address these issues by armoring CAR T cells with factors that enhance the function and maintenance of T cells in the anti-tumor response. We previously described a human orthogonal IL-2/IL-2R system consisting of a pegylated, IL-2 mutein (STK- 009) that does not significantly activate the wild type receptor and a mutated IL-2 Receptor Beta (oRβ) that does not significantly respond to its native ligand, wild type IL-2. This system provides a “private IL-2 signal” in engineered oRβ-expressing cells while avoiding signaling in bystander immune cells, reducing toxicities. We have demonstrated STK-009/oRβ’s ability to selectively expand oRβ-expressing CAR T cells (SYNCAR T cells) targeting heme (SYNCAR-001, CD19) and solid tumor (SYNCAR-002, GPC3) targets at will. Importantly, the system enhanced anti-tumor control driving deep and durable responses in both bulky lymphoma and hepatocellular carcinoma (HCC) tumor models. Additionally, in a non-human primate model, STK-009 did not potentiate wild type T or NK cells and exhibited no evidence of toxicity. Currently, the combination of STK-009 + SYNCAR-001 is undergoing Phase I clinical trials (NCT05665062). Here, we conducted a direct comparison of several armoring approaches including the SYNCAR platform, the overexpression of c-Jun, an IL-15/IL-15Rα fusion, and IL-18 in human CAR T cells targeting GPC3. To assess their effects in overcoming CAR T cell dysfunction, cells were subjected to continuous antigen exposure in vitro. STK-009 treated SYNCAR-002 T cells displayed an enhanced proliferative capacity, sustained effector cytokine secretion, and an increased activation state when compared to other armoring technologies and PBS treatment. In a HCC tumor model, treatment with STK-009/SYNCAR-002 demonstrated superior tumor control with minimal toxicities. Interestingly, IL-18 overexpression also controlled tumors, but was accompanied by uncontrolled CAR T cell expansion and toxicity resembling severe GVHD. scRNAseq analyses revealed STK-009-treated SYNCAR-002 T cells simultaneously activated effector genes and repressed an exhaustion gene signature. This analysis also identified a novel mechanism by which STK-009/oRβ signaling induces a superior synthetic effector state via transcriptional repression of the stress response/proteosome. These findings support the advantages of an orthogonal platform that selectively drives and sustains potent T cell effector function without safety concerns associated with constitutively expressed armored approaches. Citation Format: Ethan Jung, Helena Silva, Marie Semana, Ivan Cheng, Somya Singh, Michele Bauer, Mohammed Ali, Henry Rosas, Ryan Burgess, George Zeng, Woei Chang, Navneet Ratti, Deepti Rokkam, Patrick J. Lupardus, Martin Oft, Paul-Joseph Aspuria. Orthogonal IL-2/IL-2Rβ signaling selectively enhances and sustains a synthetic effector state via a novel mechanism and outperforms constitutive armoring approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6312.

Genetic susceptibility to Tuberculosis in an infant with BCG-osis

Mendelian susceptibility to mycobacterial diseases (MSMD) is a group of innate immunity defects characterized by inborn susceptibility to intracellular pathogens. These are due to defects in the interleukin 12-23-interferon gamma y axis. Twenty-three genetic mutations have been identified in this axis, of which mutation in the IL12RB1 gene is the most common genetic defect of MSMD identified. An 11-month-old infant presented with features of axillary lymphadenitis and skin lesions both of which were positive for acid-fast bacilli on Ziehl– Neelsen stain and GeneXpert. Keeping a high index of suspicion of primary immunodeficiency, the child was evaluated further and was diagnosed as a case of MSMD with IL12RB1 gene mutation.

Identification of Biomarker IL2Rβ in Ankylosing Spondylitis via Multi-Chip Integration Analysis of Gene Differential Expression.

Ankylosing spondylitis (AS) is a chronic inflammatory autoimmune disease that affects axial joints such as the spine. Early diagnosis is essential to improve treatment outcomes. The purpose of this study is to uncover underlying genetic diagnostic features of AS. We downloaded gene expression data from the Gene Expression Omnibus (GEO) database for three studies of groups of healthy and AS samples. After preprocessing and normalizing the data, we employed linear models to identify significant differentially expressed genes (DEGs) and further integrated the differential genes to acquire reliable differential transcriptional markers. Gene functional enrichment analysis was conducted to obtain enriched pathways and regulatory gene interactions were extracted from pathways to further elucidate pathway networks. Seventy-three reliably differentially expressed genes (DEGs) were integrated by differential analysis. Utilizing the regulatory relationships of the 21 Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway genes that were enriched in the analysis, a regulatory network of 622 genes was constructed and its topological properties were further analyzed. Functional enrichment analysis found 73 DEGs that were strongly associated with immune pathways like Th17, Th1 and Th2 cell differentiation. Using KEGG combined with DEGs, six hub genes (KLRD1, HLA-DRB3, HLA-DRB5, IL2Rβ, CD247, and CXCL10) were suggested from the network. Of these, the IL2Rβ gene was significantly differentially expressed compared with the normal control. IL2Rβ (Interleukin-2 receptor beta) is strongly associated with the onset and progression of autoimmune joint diseases, and may be used as a potential biomarker of AS. This study offers new characteristics that can help in the diagnosis and individualized therapy of AS.

Abstract A077: A novel, tumor-targeted immunocytokine comprising an anti-PD-L1 Affimer® fused to IL-15 exhibits potent anti-tumor activity

Abstract Affimer® biotherapeutics are novel 15kDa scaffold proteins based on the human protease inhibitor Stefin A. Affimer® proteins lack disulfide bonds and post translational modifications including glycosylation. Large, diverse phage display libraries have been created which incorporate peptide loops into the scaffold backbone. We have previously identified high-affinity competitive binders to human programmed death-ligand 1 (PD-L1) using phage display which block interaction with programmed death 1 (PD-1). Lead anti-PD-L1 Affimer® proteins were formatted by fusing to human Fc or as in-line-fusions to a serum albumin binding Affimer® to extend pharmacokinetic half-life. Here we describe novel immunocytokines that consist of half-life extended Affimer® antagonist to PD-L1 fused to human Interleukin 15 (IL-15) receptor alpha sushi domain and human IL-15 (PDL1-IL15 Affimer® fusion proteins). PD-L1 is upregulated on tumor cells, macrophages, and dendritic cells in the tumor microenvironment. PD-L1 engagement with PD-1 on T cells inhibits T cell proliferation, transcriptional activation, and cytokine release, resulting in suppressed anti-tumor response. IL-15 signals through the IL-2 receptor beta chain and the common gamma chain (IL-2Rβ/γ) and induces differentiation and proliferation of CD8+ T cells and Natural Killer (NK) cells; it does not stimulate regulatory T cells (Tregs). Immunostimulatory cytokines have therapeutic potential but are often associated with toxicity when administered systemically. PDL1-IL15 Affimer® fusion proteins have the potential to bring the immunomodulatory cytokine directly to the tumor and reverse the immunosuppressive tumor microenvironment by enhancing T cell activation, while also introducing a checkpoint inhibitor to augment the anti-tumor immune response. We characterized binding and activity of PDL1-IL15 Affimer® fusion proteins in in vitro assays. PDL1-IL15 Affimer® fusion proteins bind both PD-L1 and IL-2Rβ/γ and exhibit PD-1:PD-L1 blocking activity and IL-2Rβ/γ activation. PDL1-IL15 Affimer® fusion proteins reversed T cell exhaustion in a human mixed lymphocyte reaction assay: PDL1-IL15 Affimer® fusion proteins showed greater activity than either the PD-L1 or the IL-15 component alone. Furthermore, PDL1-IL15 Affimer® fusion proteins enhanced immune cell killing of tumor cells in co-culture assays of human peripheral blood mononuclear cells and tumor cells. In summary, data suggests that PDL1-IL15 Affimer® fusion proteins can reverse T cell exhaustion and induce immune cell activation to elicit an anti-tumor effect. Ongoing in vivo work aims to investigate the efficacy and tolerability profile of PDL1-IL15 Affimer® fusion proteins to direct future development of this therapeutic. In addition, the data will support wider utility of the Affimer® platform to develop new ways of targeting the tumor while reducing systemic toxicity. Citation Format: Victoria Juskaite, Deepa Avisetti, David H Jones, Ahmad Sobri, Gregory Billenness, Hanna Buist, Tik Nga Tong, Norhakim Yahya, Fiona McLaughlin. A novel, tumor-targeted immunocytokine comprising an anti-PD-L1 Affimer® fused to IL-15 exhibits potent anti-tumor activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A077.

Interleukin-6 plasma levels are elevated in patients with acute myocardial infarction and predict death or recurrent infarction within 1 year - a case-control study

Abstract Background Acute myocardial infarction (AMI) harbours marked inflammatory components. While cytokines of the same family have been causally related to patient outcome, the role of interleukin (IL)-6 in AMI remains incompletely understood. Recent trials suggest benefits of inhibiting IL-6 in patients with ST-segment elevation myocardial infarction (STEMI). In patients at high atherosclerotic risk, IL-6 blockade reduces markers of inflammation and thrombosis. Purpose This study aimed to assess the levels of IL-6 in AMI patients and their predictive value. Methods 160 cases (recurrent AMI or death within 1 year) and 160 matched controls were selected from the prospective, multicentric SPUM-ACS Cohort. All patients had an AMI (STEMI or NSTEMI) as initial event and underwent percutaneous coronary intervention (PCI) within 24h of symptom onset. Blood samples were drawn before PCI and biomarker plasma levels were assessed (mesoscale). Comparisons were made with age- and sex-matched healthy blood donors (HD). IL-6 levels are presented as medians and interquartile ranges. Cases, controls and HD were compared using the Kruskal-Wallis test. Correlations of baseline variables to IL-6 levels within cases and controls were assessed by linear regression analyses. Patients were divided into those with higher or lower IL-6 levels according to the median IL-6 level. Kaplan-Meier (KM) analyses for the combined endpoint of death or recurrent AMI were performed. Results The overall median IL-6 level was 21.3pg/ml. Patients with higher IL-6 had more previous AMI (n=22, 14%) than patients with lower IL-6 (n=12, 7%, p=0.045). Patients with increased IL-6 had more previous PCI (n=30, 20%) than patients with lower IL6 (n=19, 11%, p=0.045). Both cases and controls showed higher levels of IL-6 than HD (cases: 25.7pg/ml [14.8–81.6], controls: 20.0pg/ml [11.3–40.1], HD: 5.9pg/ml [3.6–9.9]; cases/controls vs. HD: p&amp;lt;0.0001). Among AMI patients, cases showed higher IL-6 levels than controls (p=0.030) (Figure 1). Within cases and controls, IL-6 was related to IL-1beta (r=0.628, p&amp;lt;0.001), IL-1alpha (r=0.174, p=0.002) and IL-1 receptor antagonist (r=0.422, p&amp;lt;0.001). IL-6 was related to neutrophil/lymphocyte ratio (r=0.147, p=0.016), but not to high-sensitivity C-reactive protein (r=0.087, p=0.142) or N-terminal pro B-type natriuretic peptide (r=0.091, p=0.118). IL-6 was related to Killip Class upon presentation (r=0.190, p&amp;lt;0.001). KM analyses showed significantly higher rates of death or recurrent AMI within 1 year for patients with higher IL-6 (log-rank p=0.006, Figure 2), with early divergence of the curves. Conclusions IL-6 is markedly higher in patients with AMI compared to healthy controls and predicts death or recurrent AMI within 1 year. IL-6 might be a valuable target for early anti-inflammatory therapy to patients with AMI. A deeper understanding of factors influencing IL-6 levels may help select those patients that will benefit most from such treatment.Figure 1:Cases vs. Controls vs. HDFigure 1:Kaplan-Meier Analysis

Molecular reshaping of phage-displayed Interleukin-2 at beta chain receptor interface to obtain potent super-agonists with improved developability profiles

Interleukin-2 (IL-2) engineered versions, with biased immunological functions, have emerged from yeast display and rational design. Here we reshaped the human IL-2 interface with the IL-2 receptor beta chain through the screening of phage-displayed libraries. Multiple beta super-binders were obtained, having increased receptor binding ability and improved developability profiles. Selected variants exhibit an accumulation of negatively charged residues at the interface, which provides a better electrostatic complementarity to the beta chain, and faster association kinetics. These findings point to mechanistic differences with the already reported superkines, characterized by a conformational switch due to the rearrangement of the hydrophobic core. The molecular bases of the favourable developability profile were tracked to a single residue: L92. Recombinant Fc-fusion proteins including our variants are superior to those based on H9 superkine in terms of expression levels in mammalian cells, aggregation resistance, stability, in vivo enhancement of immune effector responses, and anti-tumour effect.

Association of IL10RA, IL10RB, and IL22RA Polymorphisms/Haplotypes with Susceptibility to and Clinical Manifestations of SLE.

Systemic lupus erythematosus (SLE) is characterized by an imbalance between proinflammatory and anti-inflammatory mediators. Single-nucleotide polymorphisms (SNPs) in genes coding IL10RA, IL10RB, and IL22RA could affect their expression or function and disrupt immune homeostasis. We aimed to analyze the associations of IL10RA, IL10RB, and IL22RA polymorphisms/haplotypes with patients' susceptibility to and clinical manifestations of SLE. Our study included 103 SLE patients and 99 healthy controls. The genotypes of the selected polymorphisms within IL10RA (rs10892202, rs4252270, rs3135932, rs2228055, rs2229113, and rs9610), IL10RB (rs999788, rs2834167, and rs1058867), and IL22RA (rs3795299 and rs16829204) genes were determined by TaqMan® Assays. IL10RB rs1058867 G allele carriers were significantly more frequent among the controls than among the SLE patients (76.8% vs. 61.2%; p = 0.017, OR = 0.477, 95% CI: 0.258-0.879). The IL10RB CAA haplotype was more frequent among the SLE patients than in the control group (42.7% vs. 30.7%; p = 0.027). The IL22RA rs3795299 C allele and rs16829204 CC genotype were associated with Hashimoto thyroiditis in the SLE patients (n = 103; p = 0.002 and p = 0.026, respectively), and in all the included participants (n = 202, p < 0.000 and p = 0.007, respectively), and the IL22RA CC haplotype was more frequent in the SLE patients with Hashimoto thyroiditis (p = 0.047) and in the overall participants with Hashimoto thyroiditis (n = 32, p = 0.004). The IL10RA, IL10RB, and IL22RA polymorphisms/haplotypes could be associated with SLE susceptibility and various clinical manifestations, and the IL22RA CC haplotype could be associated with Hashimoto thyroiditis.

Endogenous interleukin-22 prevents cardiac rupture after myocardial infarction in mice.

Myocardial infarction (MI) can result in fatal myocardial rupture or heart failure due to adverse remodeling and dysfunction of the left ventricle. Although recent studies have shown that exogenous interleukin (IL)-22 shows cardioprotective effect after MI, the pathophysiological significance of endogenous IL-22 is unknown. In this study, we investigated the role of endogenous IL-22 in a mouse model of MI. We produced MI model by permanent ligation of the left coronary artery in wild-type (WT) and IL-22 knock-out (KO) mice. The post-MI survival rate was significantly worse in IL-22KO mice than in WT mice due to a higher rate of cardiac rupture. Although IL-22KO mice exhibited a significantly greater infarct size than WT mice, there was no significant difference in left ventricular geometry or function between WT and IL-22KO mice. IL-22KO mice showed increase in infiltrating macrophages and myofibroblasts, and altered expression pattern of inflammation- and extracellular matrix (ECM)-related genes after MI. While IL-22KO mice showed no obvious changes in cardiac morphology or function before MI, expressions of matrix metalloproteinase (MMP)-2 and MMP-9 were increased, whereas that of tissue inhibitor of MMPs (TIMP)-3 was decreased in cardiac tissue. Protein expression of IL-22 receptor complex, IL-22 receptor alpha 1 (IL-22R1) and IL-10 receptor beta (IL-10RB), were increased in cardiac tissue 3 days after MI, regardless of the genotype. We propose that endogenous IL-22 plays an important role in preventing cardiac rupture after MI, possibly by regulating inflammation and ECM metabolism.

Murine cytotoxic CD4+ T cells in the tumor microenvironment are at a hyper-maturation stage of Th1 CD4+ T cells sustained by IL-12.

The roles of tumor-infiltrating CD4+Foxp3- T cells are not well characterized due to their plasticity of differentiation, and varying levels of activation or exhaustion. To further clarify this issue, we used a model featuring subcutaneous murine colon cancer and analyzed the dynamic changes of phenotype and function of the tumor-associated CD4+ T-cell response. We found that, even at a late stage of tumor growth, the tumor-infiltrating CD4+Foxp3- T cells still expressed effector molecules, inflammatory cytokines and molecules that are expressed at reduced levels in exhausted cells. We used microarrays to examine the gene-expression profiles of different subsets of CD4+ T cells and revealed that the tumor-infiltrating CD4+Foxp3- T cells expressed not only type 1 helper (Th1) cytokines, but also cytolytic granules such as those encoded by Gzmb and Prf1. In contrast to CD4+ regulatory T cells, these cells exclusively co-expressed natural killer receptor markers and cytolytic molecules as shown by flow-cytometry studies. We used an ex vivo killing assay and proved that they could directly suppress CT26 tumor cells through granzyme B and perforin. Finally, we used pathway analysis and ex vivo stimulation to confirm that the CD4+Foxp3- T cells expressed higher levels of IL12rb1 genes and were activated by the IL-12/IL-27 pathway. In conclusion, this work finds that, in late-stage tumors, the tumor-infiltrating lymphocyte population of CD4+ cells harbored a sustained, hyper-maturated Th1 status with cytotoxic function supported by IL-12.

IL-12 drives the differentiation of human T follicular regulatory cells

Abstract T follicular regulatory (Tfr) cells can counteract the B cell-helper activity of T follicular helper (Tfh) cells and hinder the production of antibodies against self-antigens or allergens. A mechanistic understanding of the cues initiating the differentiation of T regulatory (Treg) cells into Tfr cells, which is instrumental for the therapeutic manipulation of diseases associated with a Tfr cell imbalance, is still missing. Despite their opposed roles, Tfr and Tfh cell differentiation appears to be controlled by partially overlapping signals, including TCR stimulation and costimulatory molecules. However, it is still unknown if cytokines that have been shown to control the biology of human Tfh cells, including IL-12 and activin A, can influence the differentiation of Tfr cells. To address this question, we evaluated the impact of these two cytokines on the in vitro differentiation of Tfr cells. Herein, we report a role for IL-12 in driving, on activated Treg cells, the induction of molecules that belong to the Tfr cell program, including CXCR5, PD-1, BCL6 and ICOS meanwhile preserving Tfr regulatory function. Conversely, activin A presented a largely negligible effect on the in vitro differentiation of Tfr-like cells and only modestly strengthened the IL-12-driven differentiation of Tfr-like cells. Importantly, patients with inborn errors of immunity in IL12RB1 gene presented with a severe decrease in circulating Tfr cells and produced higher levels of anti-actin autoantibodies in vivo. Overall, this study unveils IL-12 as an inducer of Tfr cell differentiation in vivo and provides a novel approach for the in vitro generation of human Tfr-like cells. AI123738

Genetic haplotypes associated with immune response to Leishmania infantum infection in dogs.

Leishmaniasis is a zoonotic parasitic disease, and the main reservoir of the parasite is the dog, although recent years have seen an increase in other mammalian species. In the Mediterranean region, where it is an endemic disease, it is caused by the species Leishmania infantum. The Ibizan hound, an autochthonous breed of this region, appears to have a genetic resistance to parasitic infection, whereas other canine breeds, such as the Boxer, are susceptible to infection. These differences are related to the differentiated activation of the immune response, with the Ibizan hound activating the Th1 immune response, whereas the Boxer breed triggers the Th2 immune response. Cytokine levels and genomic haplotypes of several genes involved in the immune response were analysed in twenty-eight Ibizan hound (resistant canine breed model) and twenty-four Boxer (susceptible canine breed) without clinical signs in the Mediterranean region. Cytokine levels were analysed by ELISA commercial kits and haplotypes were studied using CanineHD DNA Analysis BeadChip including 165,480 mapped positions. The results show 126 haplotypes associated with differential immune response in dogs. Specifically, haplotypes in IL12RB1, IL6R, CIITA, THEMIS, NOXA1, HEY2, RAB38, SLC35D2, SLC28A3, RASEF and DAPK1 genes are associated with serum levels of IFN-γ, IL-2, IL-8, and IL-18. These results suggest that the resistance or susceptibility to Leishmania infantum infection could be a consequence of haplotypes in several genes related to immune response. Future studies are needed to elucidate the relationship of these haplotypes with immune response and gene expression regulation.

Abstract CT125: A Phase 1 study to evaluate the safety and tolerability of a combination autologous CD19 CAR T cell therapy (SYNCAR-001) and orthogonal IL-2 (STK-009) in subjects with relapsed or refractory CD19 expressing hematologic malignancies (NCT05665062)

Abstract Background: Chimeric antigen receptor T cell therapy (CAR T) has demonstrated remarkable clinical efficacy in hematological malignancies. However, compromised T cell effector function, proliferation, and persistence can limit CAR T from reaching their full curative potential. Interleukin-2 (IL-2) is a potent stimulator of T cells, however therapeutic use of IL-2 is limited by systemic toxicity due its pleiotropy. Therefore, to provide a selective IL-2 signal to engineered T cells, we have developed a human orthogonal ligand/receptor system consisting of a half-life extended pegylated IL-2 mutein (STK-009) and a mutated IL-2 Receptor Beta (hoRb) that responds to STK-009 but not wild type IL-2. SYNCAR-001 is an autologous CAR T co-expressing a CD19 CAR and hoRb on the T cell surface. In mouse models of refractory lymphoma, STK-009 treatment led to expansion and activation of SYNCAR-001 cells with a maintenance of stem cell memory and effector T cell phenotypes. When added to SYNCAR-001, STK-009 increased complete response rate and durable responses in a dose dependent manner. In non-human primate studies, STK-009 alone demonstrated no significant biological activity in IL-2 sensitive populations (T cells or NK cells) and was tolerable without toxicity. Methods: This is a first-in-human, open-label, dose escalation study of combination SYNCAR-001 + STK-009 in adults with relapsed or refractory (r/r) CD19+ hematologic malignancies. The objectives of this study are to evaluate the safety, preliminary efficacy, pharmacokinetics, immunogenicity, and pharmacodynamics of SYNCAR-001 + STK-009. Dose escalation follows a standard 3+3 design with STK-009 being escalated while SYNCAR-001 is held at a single fixed dose. A dose extension will enroll a cohort of patients treated at a selected dose level and indication based on dose escalation findings. SYNCAR-001 is dosed intravenously (IV) once at Day 0 and STK-009 is dosed subcutaneously (SC) over the course of the study. Participants receive a single safety lead-in dose of STK-009 prior to lymphodepletion and subsequent SYNCAR-001 infusion. After SYNCAR-001 initiation, STK-009 is dosed SC weekly for 12 weeks and then monthly for 3 months. Eligible participants include individuals with r/r chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and selected r/r B-cell non-Hodgkin lymphoma (NHL subtypes of large B cell, mantle cell, and indolent lymphoma). Prior CD19 CAR use is excluded. The primary endpoint of safety includes outcomes such as adverse events and dose-limiting toxicities. Secondary endpoints include assessments of response, pharmacokinetics, and immunogenicity. Exploratory endpoints include assessment of immune cell populations, and relevant gene/protein expression, as well as persistence, phenotype, and functionality of SYNCAR-001 in the peripheral blood and/or bone marrow in response to STK-009. Citation Format: Maria Lia Palomba, Matthew G. Mei, Paolo F. Caimi, Matthew Cortese, Marco Davila, Greg Yadnik, Nestor Huang, David Mou, Martin Oft, Paul-Joseph Aspuria, Anita Mehta-Damani, Navneet Ratti, Naiyer Rizvi, Alex Azrilevich, Ran Reshef. A Phase 1 study to evaluate the safety and tolerability of a combination autologous CD19 CAR T cell therapy (SYNCAR-001) and orthogonal IL-2 (STK-009) in subjects with relapsed or refractory CD19 expressing hematologic malignancies (NCT05665062) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT125.

Evaluation of Single Nucleotide Polymorphisms (SNPs) Associated with Genetic Resistance to Bovine Paratuberculosis in Marchigiana Beef Cattle, an Italian Native Breed.

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of paratuberculosis (PTB), a widespread chronic enteritis of ruminants. The progression of the infection depends on the containment action of innate and cell-mediated immunity (CMI), and it is related to environmental and genetic factors. In particular, PTB susceptibility seems to be associated with specific genes coding for immune regulators involved in the cell-mediated response during the infection. The aim of this preliminary study was to verify, in Italian beef cattle, an association between MAP infectious status and the presence of single nucleotide polymorphisms (SNPs) in candidate genes. To the best of our knowledge, this is the first investigation conducted on a native beef cattle breed, known as Marchigiana, reared in Central Italy. The present research, based on a longitudinal study, aimed to identify and correlate phenotypic and genetic profiles characteristic of the subjects potentially able to contrast or contain PTB. In a MAP-infected herd, ELISA, IFN-γ tests, qPCR, and cultures were performed at a follow-up, occurring within a period ranging from three to six years, to evaluate the individual state of infection. Animals testing positive for at least one test were considered infected. DNA samples of 112 bovines, with known MAP statuses, were analyzed to verify an association with SNPs in the genes encoding gamma-interferon (BoIFNG), interleukin receptor 10 (IL10RA), interleukin receptor 12 (IL12RB2), and toll-like receptors (TLR1, TLR2, TLR4). Regarding statistical analysis, the differences among target genes and pairs of alleles in the analyzed groups of animals, were evaluated at a significance level of p < 0.05. For IL10RA and for IL12RB2 genes, relevant differences in genotypic frequencies among the considered cattle groups were observed. For all candidate genes studied in this investigation, SNP genotypes already associated with PTB resistance were found more frequently in our population, suggesting potential resistance traits in the Marchigiana breed.

Network-Based Assessment of Minimal Change Disease Identifies Glomerular Response to IL-7 and IL-12 Pathways Activation as Innovative Treatment Target.

Background: Minimal change disease (MCD), a major cause of nephrotic syndrome, is usually treated by corticosteroid administration. MCD unresponsiveness to therapy and recurrences are nonetheless frequently observed, particularly in adults. To explore MCD-related pathogenetic mechanisms and to identify novel drug targets ultimately contributing to novel therapeutic avenues with a certain specificity for MCD, we compared glomerular transcriptomes from MCD with membranous nephropathy (MN) patients and healthy controls. Methods: Renal biopsies from adult patients with MCD (n = 14) or MN (n = 12), and non-diseased controls (n = 8) were selected from the Norwegian Kidney Biopsy Registry. RNA for 75 base-pair paired-end RNASeq were obtained from laser capture micro-dissected (LCM) glomeruli from FFPE sections. Transcriptional landscapes were computed by combining pathway-centered analyses and network science methodologies that integrate multiple bioinformatics resources. Results: Compared to normal glomeruli, cells from MCD displayed an inflammatory signature apparently governed by the IL1 and IL7 systems. While enrichment of IL1 production and secretion was a shared feature of MCD and MN compared to normal tissue, responses involving IL7 pathway activation were unique to MCD. Indeed, IL7R expressed by glomeruli was the most upregulated gene of the interleukin family in MCD versus normal controls. IL7 pathway activation was paralleled by significant enrichment in adaptive immune system processes and transcriptional regulation and depletion in pathways related to energy metabolism and transcription. Downregulation of these organ function-related themes again occurred predominately in MCD and was significantly less pronounced in MN. Immunofluorescence and immunohistochemistry, respectively, confirmed the expression of phosphorylated IL-7 receptor alpha (IL7RA, CD127) and IL12 receptor beta 1 (IL12RB1) proteins. Conclusions: Gene expression profiling of archival FFPE-biopsies identifies MCD-specific signatures with IL7RA and IL12RB1 as novel targets for MCD treatment.