IL-2R Alpha Research Articles

OP36 Lusvertikimab, a first-in-class IL7 receptor antagonist, in moderate to severe Ulcerative Colitis: results of a multicenter, randomized, placebo-controlled phase II study

Abstract Background OSE127 lusvertikimab (L) is a first-in-class antagonist of IL-7R alpha chain developed for the treatment of ulcerative colitis (UC). The study objectives were to evaluate the efficacy and safety of L in patients (pts) with ulcerative colitis (UC) in a multicentre, randomised, double-blind, placebo-controlled phase 2 study CoTikiS (NCT04882007). Methods Adults with moderately to severely active UC (modified Mayo score [MMS] 4-9) and inadequate response to conventional and/or failure to advanced therapies were randomised 1:1:1 in 3 treatment groups (placebo, L450 mg or L850 mg) administered intravenously at week 0 (W0), W2 and W6. L450mg was dropped at a prespecified futility analysis after 58 patients. It was later showed not futile. The primary endpoint (PE) was the comparison between groups in changes of the MMS from baseline at W10. Key secondary endpoints were clinical remission, endoscopic improvement, endoscopic remission and changes in endoscopic activity by the UC Endoscopic Index of Severity (UCEIS). Adverse events (AEs) and laboratory abnormalities were assessed for safety. Results There were no differences in demographic and disease characteristics between treatment groups (placebo n=49, L450 mg n=35 or L850 mg n=51). The PE was achieved: L450, L850 and pooled dose L450/L850 significantly reduced MMS at W10 as compared to placebo. Key secondary endpoints were also improved by L450, L850 and pooled L450/L850 as compared to placebo. Significantly greater percentages of patients were in clinical remission (22%, 13.0% and 16.2% respectively vs 4.4% for placebo), had an endoscopic remission (34.7%, 19.4%, and 25% vs 12.6 % for placebo) and endoscopic improvement (44.6%, 24.3%, and 31.9% vs 12.6% for placebo) with L450. The UCEIS score was significantly improved with L450. L450 and L850 were well tolerated. The most frequent adverse events for L450, L850 and placebo were lymphopenia (5.6%, 3.9% and 2% respectively), anaemia (2.8%, 0% and 4.1%), pyrexia (5.6%, 0%, 2%), UC exacerbation (2.8%, 3.9%, 6.1%) and Covid 19 (2.8%, 0%, 4.1%). Lymphopenia was of mild severity, transient and did not result in drug interruption, drug withdrawal or increased infection rate. No clinical or biological specific safety signal was reported. Conclusion Lusvertikimab, a pure IL-7 receptor antagonist, demonstrated its clinical and endoscopic efficacy in moderate to severe UC compared to placebo at week 10 with no clinically relevant safety signal. These positive findings support the further clinical development for treatment of UC to elaborate its potential place in the therapeutic armentarium of UC.

DUPILUMAB: A NEW ALLY FOR THE TREATMENT OF EOSINOPHILIC ESOPHAGITIS?

INTRODUCTION: Eosinophilic esophagitis is a chronic, immune-mediated disease of the gastrointestinal tract, resulting from the interaction between genetic, environmental and immunological factors of the affected patient. The clinical picture is characterized by esophageal dysfunction, which may present as dysphagia, precordial pain, upper abdominal pain or food impaction. Treatment aims to improve clinical symptoms and prevent disease progression and subsequent complications. In this context, immunobiologicals are emerging and promising therapies, with Dupilumab being the first specific treatment approved by the FDA. It is an IgG4 monoclonal antibody that targets IL-4R alpha and, from there, reduces protein transphorylation, transcription and the T helper 2-regulated response. The objective of this systematic review is to evaluate the efficacy of this drug in the treatment of eosinophilic esophagitis, in comparison with the options already available on the market. METHOD: PubMed searches were performed using the keywords “eosinophilic esophagitis dupilumab” and “dupilumab treatment esophagitis eosinophilic”. Initially, 60 results were obtained, but after exclusion by title, abstract and full text, 14 articles were included. RESULT: all articles analyzed demonstrated improvement in patients using Dupilumab, with 13 of them indicating a reduction in eosinophil counts, 8 showing clinical improvement in symptoms, 8 indicating histological improvement, 7 indicating improvement in the endoscopic pattern, according to the Endoscopic Reference for Eosinophilic Esophagitis, and 4 indicating a reduction in the Dysphagia Symptom Questionnaire (DQQ) score. DISCUSSION: eosinophilic esophagitis is a rare disease, but with potential for progression, negatively interfering with the patient's quality of life. Therefore, since current drug therapy is nonspecific, the use of Dupilumab presents itself as an innovative and optimistic solution for the future, because it changes the understanding of disease management, expands therapeutic options and improves prognosis. CONCLUSION: the use of Dupilumab in the treatment of eosinophilic esophagitis has proven to be effective. However, for the treatment to be well established with regard to the doses used, frequency of administration and longitudinal monitoring, further studies are still required.

No Effects of Omega-3 Supplementation on Kynurenine Pathway, Inflammation, Depressive Symptoms, and Stress Response in Males: A Placebo-Controlled Trial.

Background: Increased inflammation and heightened physiological stress reactivity have been associated with pathophysiology of depressive symptoms. The underlying biological mechanisms by which inflammation and stress may influence neurogenesis are changes in the kynurenine (KYN) pathway, which is activated under stress. Supplementation with n-3 polyunsaturated fatty acids (n-3 PUFAs) has anti-inflammatory properties and can increase stress resilience. Whether n-3 PUFAs alter KYN stress response is unknown. Objectives: This placebo-controlled study investigated the effect of n-3 PUFAs on KYN metabolism, inflammation, depressive symptoms, and mood. Moreover, stress-induced changes following a laboratory stressor have been assessed. Methods: In this placebo-controlled study, 47 healthy male adults received either 4 g n-3 PUFAs per day (Omega-3 group) or a placebo (Placebo group) for 12 weeks. Results: A significant group-by-time interaction was found for the inflammatory markers gp130 (F = 7.07, p = 0.011), IL-6R alpha (F = 10.33, p = 0.003), and TNF_RI (F= 10.92, p = 0.002). No significant group-by-time interactions were found for KYN metabolites, depressive symptoms, and mood (except for Hedonic tone (F = 6.50, p = 0.014)), nor for stress-induced changes in KYN metabolites and mood following a laboratory stressor. Conclusions: Overall, increased n-3 PUFA levels in healthy men ameliorate inflammatory markers but do not ameliorate KYN metabolism, depressive symptoms, mood, or KYN metabolism and mood following a stress induction. This study was registered at ClinicalTrials.gov with the identifier NCT05520437 (30/08/2022 first trial registration).

Comparative Analysis Molecular Simulation IL6R Alpha with TCZ and HIL6: Mechanism in Inflammatory Responses

Comparative Analysis Molecular Simulation IL6R Alpha with TCZ and HIL6: Mechanism in Inflammatory Responses

735 - A case series of dupilumab for vitiligo and alopecia areata in the setting of AD

Abstract Introduction/Background Dupilumab is a human monoclonal antibody of the IgG4 subclass and binds to the IL-4R alpha subunit, causing downstream inhibition of IL-4 and IL-13 signaling and thus downregulating the T-helper-2 (Th2) cytokine response.1,2,3 Approved by the Food and Drug Administration in 2017, dupilumab is FDA-approved to treat atopic dermatitis (moderate-to-severe), asthma (moderate-to-severe), chronic rhinosinusitis with nasal polyposis, and eosinophilic ­esophagitis. Vitiligo is a common autoimmune depigmenting skin disorder that is prevalent in 1.38% of US adults, and 2.16% in adolescents in the US, with a range of 0.4-2% in most populations.4,5,6 It occurs when the immune system of the body attacks melanocytes, skin cells, that produce melanin, and has been linked to the Koebner phenomenon which occurs when trauma induces lesions, with events including scratching as would be noted in AD.7 AD has also been linked to Vitiligo, especially in children under the age of 12 years.8,9,10 There are many available therapies for vitiligo, topical, systemic, phototherapy, and surgical types being most commonly used.11,12,13 Objectives To address specific features of AA/ Vitiligo/ AD overlap that would support benefit of dupilumab prescribing. Methods An IRB-exempt review was conducted of patient charts for individuals who received dupilumab who had alopecia areata or vitiligo associated with AD. Results Six patients with AD and alopecia universalis (AU), and one patient with AD, vitiligo, and alopecia areata (AA) were identified for review. The patients treated for AU included a 5 year-old Asian female, a 12 year-old white female, a 14 year-old African American male, A 15 year-old Hispanic female, a 26 year-old African American female, and a 78 year-old Hispanic female. The five year-old regrew hair for 4 months but had rapid loss when forced to discontinue due to insurance reasons. The 12 year-old female who had AU for 8 years and had no regrowth. The 15 year-old, 26 year-old, and 78 year-old had rapid regrowth of hair in addition to AD improvement, however, the 15 year-old required addition of an oral JAK inhibitor to retain hair growth. The vitiligo/AA patient is a 61 year-old female patient who had 50% BSA confetti lesion vitiligo affecting the chest, back, abdomen, arms, and legs. She had rapid disease stabilization (2 months) and at 1 year had 90% facial and 70% extremity repigmentation with dupilumab and topical 1.5% ruxolitinib. The same patient had 40% scalp hair loss which fully resolved upon repigmentation of the scalp. Response was noted rapidly but plateaued at 12 months. Conclusions The overlap of AD with AA and Vitiligo points to a shared pathogenesis of the conditions. One of the likely reasons for this is the Koebner phenomenon, which has been poorly characterized. We hypothesize that the Koebner phenomenon is triggered through an IL-4/ IL-13 mechanism, i.e. for specific individuals IL-4 and IL-13 hyper-reactivity can be a Koebner-based trigger. Additionally, AA overlaps with AD is poorly characterized. However, the linkage has been recognized in recent AAD guidelines addressing AD comorbidities.14 We hypothesize that IL-4/ IL-13 hyper-reactivity in the skin can act similarly in AU. There is in AA an overlap of TH1-CXCL9/10 expression and interferon gamma overexpression in addition to TH2- including IL-13 overexpression.15 Therefore, the blockade of IL-4/ IL-13 may be effective through multiple mechanisms of activity. There is already notable Phase 2a data supporting dupilumab usage in AA.16 Our experience demonstrates sustained hair growth in half of patients treated. AA/ Vitiligo overlap appears to respond well to dupilumab. This is supported by a recent case report demonstrating benefit of dupilumab in programmed cell death inhibitor-1 induced vitiligo with associated refractory pruritus.17 Liu et al have observed in vitro that rising IL-4 levels were linked to increased vitiligo risk.18 On the other hand, some reports of vitiligo after initiating dupilumab therapy do exist, with some new-onset and worsening described. These cases are limited but bear consideration. In our patient, disease stabilization was noted in a generalized confetti-vitiligo patient, but a topical JAK inhibitor was used adjunctively.19,20,21,22,23 Given the need for long-term maintenance, the safety of dupilumab is an attractive feature. Therefore, further exploration of dupilumab therapy for vitiligo/AA/AD, vitiligo/AD/Koebner+, and AU/ AD is needed. This is particularly important to address in patients under the age of 12 years who currently have no approved systemic medications for vitiligo and AU.24 When AU, vitiligo, and combinations of the two are comorbid with AD, there is an expectation of circulating IL-4/13 elevations and localized IL-4/13 elevation that support a potential role for dupilumab therapy in these conditions. Given that AD is associated with vitiligo of childhood, and severe alopecia areata is linked to AD, systemic therapy would be necessary in these individuals.

Targeting of CD25+ Activated T Cells in Gvhd-Associated Marrow Suppression and Cytopenias

Introduction: Secondary cytopenias occurring after hematopoietic cell transplant (HCT) are an observed complication of graft-versus-host disease (GVHD). Causes include peripheral immune mediated destruction (hemolytic anemia or thrombocytopenia), usually associated with autoantibody production, and peripheral destruction associated with microangiopathy (TMA). However, cases have been observed where marrow production of the affected lineage is abnormal. Immune-mediated cases are treated with increased immunosuppression, e.g., steroids or calcineurin inhibitors (CNI), and TMA with complement inhibition. To better understand the mechanisms and treatment of hypoproductive cytopenias in GVHD, we combined analyses of hematopoietic stem and progenitor cells (HSPC) and peripheral blood and marrow activated T cells. We describe 3 cases of GVHD-associated cytopenias marked by HSPC depletion and presence of CD25 (IL-2R alpha subunit)-positive activated T cells in the marrow that responded to combinations of immunosuppression containing basiliximab (basil) anti-CD25 monoclonal antibody blockade. Methods/Results: We describe combined HSPC and T cell analyses in 3 patients (P) with severe GVHD-associated cytopenias. P1 was at 5 years post-HCT for acute lymphoblastic leukemia, P2 at 2 years post-HCT for severe aplastic anemia (SAA), and P3 at 1.5 months post-liver transplant for alcohol use disorder-associated cirrhosis and hepatocellular carcinoma ( Table 1). Because of refractory cytopenias, all were being considered for HCT. However, marrow analyses revealed that each had normal frequencies of phenotypic HSC, with variable decreases in cellularity and frequencies of committed progenitors ( Table 2). Chimerism analyses showed that 100% of CD34+ cells in P1 and P2 were of donor origin, and in P3 of host origin. All 3 marrows had activated CD3+ T cells per expression of CD69 and/or CD38, and frequency of CD25 on >10% of T cells. In addition, P1 had evidence of IgG bound to committed progenitor populations. The treatment strategy was to block IL-2 signaling with basil (induction of 20 mg on day 1, 4, 7, 14, 21, 28, followed by alternate week and then monthly dosing for 2-9 months), combined with suppression of IL-2 production with steroid and/or CNI. After induction, repeat analyses of the blood and marrow T-cells confirmed complete blockade of CD25. Therapy was also individualized for specific clinical conditions or as noted on marrow evaluation. P1 received prednisone, CNI, and rituximab for a presumed anti-HSPC autoantibody. At 9 months he was weaned off all therapy and remains on no immunosuppressive treatment 2 years later. P2 did not receive CNI due to chronic renal failure but received steroids and later sirolimus. At ~18 months, his cytopenias recurred, but subsequently responded to basil re-induction and steroids. P3 has received prednisone, CNI, and sirolimus indefinitely for prevention of liver rejection and/or hepatocellular carcinoma relapse. P1 had no viral reactivation; P2 had CMV pneumonia with his second course responsive to antiviral therapy, completion of basil, and tapering of steroids; and P3 had CMV reactivation responsive to antiviral therapy and EBV viremia. P3 has a recent diagnosis of EBV+ intrahepatic B cell lymphoma. In all cases, blood counts normalized except P2 who has anemia due to CKD. Discussion: HSPC analyses in these patients with GVHD and cytopenias demonstrate that peripheral changes are not due to loss of HSC, but instead to quantitative and/or qualitative defects in committed progenitor populations, likely mediated by activated CD25+ T cells. In addition, the presence of IgG on HSPCs in P1 demonstrates a novel role for antibody-mediated humoral suppression in some cases. The presence of HSC indicates that there may be no need for re-transplant. Previous studies of SAA have shown activated T cells to be drivers of aplasia and is the basis for immune suppressive therapy (anti-thymocyte globulin and CNI). Our analyses of patients with GVHD-associated cytopenias demonstrates that targeting of IL-2 signaling via combined CD25 receptor blockade and pharmacologic inhibition of IL-2 production can correct the marrow defects and cytopenias, albeit with need for attention to viral infections. Targeting CD25 in patients with evidence of CD25+ activated T cells is a rational approach to the difficult problem of GVHD-associated marrow suppression and cytopenias.

Upper airway disease diagnosis as a predictive biomarker of therapeutic response to biologics in severe asthma.

Asthma is a heterogeneous disease sharing airway instability but with different biology, risk factors, and response-to-therapy patterns. Biologics have revolutionized the one-size-fits-to-all approach to personalized medicine in severe asthma (SA), which relies on the identification of biomarkers that define distinct endotypes. Thus, blood eosinophils and, to some extent, exhaled nitric oxide (FeNO) can predict the response to approved anti-type 2 (T2) biologics (anti-IgE, anti-IL-5, and anti-IL-4R alpha), whereas age at onset and comorbidities such as anxiety/depression, obesity, reflux, and upper airway disease (UAD) also influence therapeutic responses in SA. In this article, focusing on the predictive value of biomarkers for the therapeutic response to biologics in SA, we first summarize the level of prediction achieved by T2 biomarkers (blood eosinophils, FeNO) and then review whether data support the predictive value of upper airway diagnosis on such outcomes. Post hoc analysis of most studies with T2 biologics suggests that chronic rhinosinusitis with nasal polyps (CRSwNP) and, to a lower extent, allergic rhinitis may help in predicting clinical response. Considering that T2 biologics are now also approved for the treatment of severe CRSwNP, diagnosis of upper airway disease is a key step in determining eligibility for such therapy.

Ectodomain shedding of proteins important for SARS-CoV-2 pathogenesis in plasma of tobacco cigarette smokers compared to electronic cigarette vapers: a cross-sectional study.

The impact of tobacco cigarette (TCIG) smoking and electronic cigarette (ECIG) vaping on the risk of development of severe COVID-19 is controversial. The present study investigated levels of proteins important for SARS-CoV-2 pathogenesis present in plasma because of ectodomain shedding in smokers, ECIG vapers, and non-smokers (NSs). Protein levels of soluble angiotensin-converting enzyme 2 (ACE2), angiotensin (Ang) II (the ligand of ACE2), Ang 1-7 (the main peptide generated from Ang II by ACE2 activity), furin (a protease that increases the affinity of the SARS-CoV-2 spike protein for ACE2), and products of ADAM17 shedding activity that predict morbidity in COVID-19 (IL-6/IL-6R alpha (IL-6/IL-6Rα) complex, soluble CD163 (sCD163), L-selectin) were determined in plasma from 45 NSs, 30 ECIG vapers, and 29 TCIG smokers using ELISA. Baseline characteristics of study participants did not differ among groups. TCIG smokers had increased sCD163, L-selectin compared to NSs and ECIG vapers (p < 0.001 for all comparisons). ECIG vapers had higher plasma furin compared to both NSs (p < 0.001) and TCIG smokers (p < 0.05). ECIG vaping and TCIG smoking did not impact plasma ACE2, Ang 1-7, Ang II, and IL-6 levels compared to NSs (p > 0.1 for all comparisons). Further studies are needed to determine if increased furin activity and ADAM17 shedding activity that is associated with increased plasma levels of sCD163 and L-selectin in healthy young TCIG smokers may contribute to the future development of severe COVID-19 and cardiovascular complications of post-acute COVID-19 syndrome.

Targeting interleukin 4 receptor alpha on tumor-associated macrophages reduces the pro-tumor macrophage phenotype

Tumor-associated macrophages (TAMs) are an abundant tumor-promoting cell type in the tumor microenvironment (TME). Most TAMs exhibit a pro-tumor M2-like phenotype supportive of tumor growth, immune evasion, and metastasis. IL-4 and IL-13 are major cytokines that polarize macrophages to an M2 subset and share a common receptor, IL-4 receptor alpha (IL-4R alpha). Treatment of human ex vivo polarized M2 macrophages and M2 macrophage precursors with IL-4R alpha antagonist antibody Dupilumab (DupixentⓇ) reduces M2 macrophage features, including a shift in cell surface marker protein expression and gene expression. In animal models of prostate cancer, both pharmacologic inhibition of IL-4R alpha and genetic deletion of IL-4R alpha utilizing an Il4ra -/- mouse model result in decreased CD206 on TAMs. These data support IL-4R alpha as a target to reduce the pro-tumor, M2-like macrophage phenotype as a novel adjunct cancer therapy.

P1425: ACTIVATED AND EXPANDED NATURAL KILLER CELLS TRANSDUCED WITH AN NKG2D CHIMERIC ANTIGEN RECEPTOR AGAINST ACUTE MYELOID LEUKEMIA

Background: Acute Myeloid Leukemia (AML) is a hematological malignancy incurable for almost all patients. Chimeric Antigen Receptor (CAR) therapy is showing promising results in other hematological disorders but remains challenging in AML since no specific antigens have been described yet. Using NKG2D as a CAR, a natural NK receptor with 8 ligands overexpressed in several tumors, could surmount AML targeting limitations. T cells are considered the gold standard immune effector cells for CAR therapy, but they show toxicities that could be overcome using other cells such as activated and expanded natural killer cells (NKAE), that can be used in an allogeneic context with no GvHD, have natural anti-tumor properties and a short lifespan within the organism. In this project we analyze the in vitro efficacy and security of peripheral blood NKAE cells lentivirally transduced with an NKG2D-41BB-CD3z CAR. Aims: The primary objective of this project is to assess the efficacy and security of a cell immunotherapy against AML based on the use of NKAE cells transduced with an NKG2D CAR. Methods: NKAE cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors (HD) and/or AML patients after 7 days of coculture with the irradiated cell line k562-mb21-41BBL by magnetic immunodepletion, when they were transduced with lentiviral vectors carrying NKG2D-41BB-CD3z CAR construct. CAR expression was measured up to 15 days post-transduction by flow cytometry. Cytotoxic activity against AML cell lines was performed by Propidium Iodide and Annexin V staining. Toxicity was evaluated by Europium-TDA assays. Results: AML can be targeted with an NKG2D CAR since all patients studied expressed at least one NKG2DL (Fig.1). Primary NK cells can be lentivirally transduced with an NKG2D CAR, showing a modest but stable CAR expression up to 15 days after transduction (23% ±4,7% NKG2D+, 33,35% ±4,25% GFP+). CAR-NKAE cells perform robust cytotoxicity towards MOLM-13 AML cell line after 24h of coculture at an effector:target ratio of 1:1, exerting a quasi-total lysis of AML cells (Fig.2A). This represents a significant increase in anti-leukemia effect compared to untransduced NKAE (92,6% ±0,3% vs 72,2% ±10%, p= 0,0138). Surface expression of relevant molecules for NK activity showed that NKp30, NKp44 (natural cytotoxicity receptors), CD69 (early activation marker), CD25 (IL2R alpha chain), FasL (mediates FasL-mediated cytotoxicity), NKp80 (C-type lectin-like surface-activating receptor) and TRAIL (TNF-Related Apoptosis Inducing Ligand) were more expressed by transduced NKAE (Fig.2B). Cytokine release profile revealed a higher production of IL-6, IL-17A, sFasL and IFNg by CAR-NKAE (Fig.2C). We did not observe relevant toxic effects of none of the cells against healthy PBMCs and very low toxicities were found against NL-20 lung cell line, but there were no differences between NKAE and CAR-NKAE. Image:Summary/Conclusion: Our preliminary results show that primary NK cells can be lentivirally transduced with a second generation NKG2D CAR, exerting a robust anti-leukemia activity towards AML cell lines. Transduced cells show a more cytotoxic immunophenotype as well as a higher release of cytolytic molecules and proinflammatory and proapoptotic proteins, that could be responsible of the increased anti-tumor response. Hence, NKG2D-CAR transduced NKAE cells could be a suitable approach to treat AML. Other costimulatory domains are being studied in order to assess which one triggers a higher cytotoxic activity, thus allowing decreasing the effector:target ratios and therefore, on-target off-tumor effect.

Cytokine profile and brain biopsy in a case of childhood-onset central nervous system vasculitis in Noonan syndrome-like disorder due to a novel CBL variant

The authors describe a 5-year-old girl who developed a Noonan syndrome-like disorder as a result of the CBL c.1194C>G/p.His398Gln variant, including headache, papilledema, intracranial hypertension, hyperproteinorrhachia, leucorrhachia, and brain inflammation and vasculitis with CD3 positive lymphocyte infiltration. The patient responded partially to corticosteroids, acetazolamide, and ventriculoperitoneal valve placement. The serum cytokine profile revealed persistently elevated levels of IL-1 RA, IL-2R alpha, IL-6, IL-18, MCP-1, and MCP-3. Cyclophosphamide was used as a bridge to allogeneic hematopoietic stem cell transplantation in this case.

Salivary biomarkers in children with juvenile idiopathic arthritis and healthy age-matched controls: a prospective observational study

Monitoring the immune system’s regulation and signaling using saliva could be of interest for clinicians and researchers. Saliva, a biofluid with close exchange with serum, is influenced by circadian variance and oral factors such as masticatory function. This study investigated the detectability and concentration of cytokines and chemokines in saliva in children with juvenile idiopathic arthritis (JIA) as well as saliva flow and the influence of orofacial pain on saliva flow. Of the 60 participants (7–14 years old) enrolled, 30 had a diagnosis of JIA and active disease, and 30 were sex- and age-matched healthy controls. Demographic data and three validated questions regarding presence of orofacial pain and dysfunction were recorded. Stimulated whole saliva was collected and analyzed using a customized R&D bead-based immunoassay with 21 targeted biomarkers. Fourteen of these were detectable and showed similar levels in both children with JIA and controls: TNF-alpha, TNFRSF1B, MMP-2, MMP-3, IL-1alpha, IL-1beta, IL-6R alpha, IL-8, S100A8, CCL2, CCL3, IL-10, CCL11, and CXCL9. In addition, there was no difference in salivary flow rate between groups, but there was an association between orofacial pain and reduced saliva flow rate for both groups.Trial registration: ClinicalTrials.gov Protocol id: 2010/2089-31/2.

Trichuris muris infection drives cell-intrinsic IL4R alpha independent colonic RELMα+ macrophages.

The intestinal nematode parasite Trichuris muris dwells in the caecum and proximal colon driving an acute resolving intestinal inflammation dominated by the presence of macrophages. Notably, these macrophages are characterised by their expression of RELMα during the resolution phase of the infection. The RELMα+ macrophage phenotype associates with the presence of alternatively activated macrophages and work in other model systems has demonstrated that the balance of classically and alternatively activated macrophages is critically important in enabling the resolution of inflammation. Moreover, in the context of type 2 immunity, RELMα+ alternatively activated macrophages are associated with the activation of macrophages via the IL4Rα. Despite a breadth of inflammatory pathologies associated with the large intestine, including those that accompany parasitic infection, it is not known how colonic macrophages are activated towards an alternatively activated phenotype. Here, we address this important knowledge gap by using Trichuris muris infection, in combination with transgenic mice (IL4Rαfl/fl.CX3CR1Cre) and IL4Rα-deficient/wild-type mixed bone marrow chimaeras. We make the unexpected finding that education of colonic macrophages towards a RELMα+, alternatively activated macrophage phenotype during T. muris infection does not require IL4Rα expression on macrophages. Further, this independence is maintained even when the mice are treated with an anti-IFNγ antibody during infection to create a strongly polarised Th2 environment. In contrast to RELMα, PD-L2 expression on macrophages post infection was dependent on IL4Rα signalling in the macrophages. These novel data sets are important, revealing a surprising cell-intrinsic IL4R alpha independence of the colonic RELMα+ alternatively activated macrophage during Trichuris muris infection.

Tumour immune evasion mechanism in non-small cell lung cancer by inducing Blimp-1 in CD4+CD25+Foxp-3+ T regulatory cells

Abstract Immunotherapy improves the life expectancy of patients with lung cancer by targeting the immune inhibitory T cell surface markers resulting in the activation of anti-tumor T cell survival and suppression of tumor cell growth. The transcription factor repressor Positive Regulatory Domain Containing Protein 1 (encoded in humans by the Prdm1 gene) also known as B lymphocytes-induced maturation protein-1 (Blimp-1) affects the homeostasis and function of CD4+ and CD8+ T cells as well as T regulatory cells (T regs). We recently reported that Blimp-1 is induced in the lung tumoral region of patients with lung ADC. To further investigate whether Blimp-1 could be a candidate gene involved in the regulation of the anti-tumor immune-response in lung cancer, we started to investigate peripheral blood mononuclear cells (PBMCs) isolated from healthy control subjects and NSCLC patients for Blimp-1 expression in immunosuppressive CD4+CD25+Foxp3+ T regs. The PBMCs were cultured with and without antiCD3/CD28 antibodies, without and with TGF-beta, to mimic the tumor microenvironment, for 4 days and subsequently analyzed by flow cytometry using antibodies against Blimp-1, Foxp3, CD4 and CD25(IL-2R alpha chain). Here we found, by trend, induced CD4+Blimp1+CD25+Foxp3+ Tregs in unstimulated PBMCs from patients with lung cancer. Moreover, TGF-beta further induced these Blimp1+CD4+CD25+Foxp3+ Tregs in PBMCs obtained both from healthy controls and from NSCLC patients. Taken together these data indicate that the tumor microenvironment induces Blimp-1 in immunosuppressive CD4+CD25+Foxp3+ Tregs via TGF-beta. Thus, current immunotherapy should be combined to TGF-beta inhibitors to target CD4+CD25+Blimp1+Foxp-3+ T regulatory cells.

Atopic Eczema: Pathophysiological Findings as the Beginning of a New Era of Therapeutic Options.

Atopic eczema (AE) is a chronic inflammatory disease hallmarked by intense pruritus and eczematous lesions. It depicts one of the most common skin diseases affecting a major part of children and several percentages of adults.Both pathogenesis and pathophysiology are based on complex orchestrated interactions of skin barrier defects, immunological changes, the environment, and an abundance of other contributing factors. Frequently, AE displays the starting point for other allergic diseases such as allergic asthma and rhinoconjunctivitis. Additionally, the risk of developing food allergy is increased. Furthermore, the disease is accompanied by a susceptibility to bacterial, fungal, and viral infections. The development of new therapies received great impetus by an ample research of the pathophysiological mechanisms, leading to a new era in the treatment of severe atopic eczema due to targeted treatments, e.g. the IL-4R alpha specific monoclonal antibody dupilumab.This article provides an overview of the causative and pathophysiological characteristics, the clinical and diagnostic aspects as well as current and future therapeutical possibilities focusing allergic aspects contributing to the course of the disease.

FRI0113 THE BASELINE SOLUBLE GP130 IS ASSOCIATED WITH THE RESPONSE OF RHEUMATOID ARTHRITIS PATIENTS TO SARILUMAB

Background:IL-6 contributes significantly to the chronic inflammatory process of rheumatoid arthritis (RA). Sarilumab (SRL), a human anti-human IL-6 receptor alpha monoclonal antibody that blocks the signaling originated by the IL-6/IL-6R complex like tocilizumab,is an effective treatment. However, predictors of the response to sarilumab are still required.Objectives:We aimed to combine IL-6, soluble IL-6R (sIL-6R) and gp130 (sgp130) levels to identify groups of sarilumab responses.Methods:This research is a retrospective study. a total of 32 RA patients with SRL therapy in our department from February 1 in 2018 to December 31 in 2019 were included. Serum and clinical data from 32 RA patients were collected before treatment and until the last visit. Follow-up period was up to one year after starting SRL treatment. Serum were tested for IL-6 (Human IL-6 Quantikine ELISA Kit, R&amp;D systems), sIL-6R (Human soluble IL-6R alpha Quantikine ELISA Kit, R&amp;D systems) and sgp130 (Human soluble gp130 Quantikine ELISA Kit, R&amp;D systems), using specific ELISAs according to the manufacturer’s instructions. Hierarchical cluster analysis (JMP14.3.0) was used to establish the relationship between IL-6, sIL-6R and sgp130. We evaluated the efficacy of SRL treatment on the last visit using European League Against Rheumatism (EULAR) response criteria in the groups of patients. The other statistical analyses were performed with EZR 1.41, and p Values less than 0.05 were considered significant.Results:The median age of patients was 70.5 (IQR: 66.5-74.3) years and the median of disease duration was 7.3 (1.7-15.3) years. Nine (28.1%) patients were biologics and Jakinibs naive. the median follow-up periods were 24 (12-26) weeks. The baseline DAS28 was median 4.39 (3.77 - 5.43), and CDAI was 21.1 (11.7-29.5). When comparing responders and non-responders, there were no significant differences in any of the baseline parameters and cytokines. Four statistical significant clusters of RA patients (i.e., Group1, Group2, Group3 and tocilizumab use group before SRL) were defined by serum concentrations of IL-6, sIL-6R and spg130 at baseline. The levels of IL-6 expressed as median in Group1 patients were 25.6 (14.4–72.2) pg/ml, in Group2 5.9 (3.3–11.3) pg/ml, and in Group3 70.2 (45.4–86.1) pg/ml (p &lt; 0.002, significant difference only between Group2 and Group3). The levels of sIL-6R expressed as median in Group1 patients were 38.7 (34.7-45.1) ng/ml, in Group2 35.1 (24.8-41.9) ng/ml, and in Group3 35.7 (34.2-39.8) ng/ml (p = 0.5477). The levels of sgp130 expressed as median in Group1 patients were 272.6 (263.0-277.2) ng/ml, in Group2 223.1 (221.0-228.0) ng/ml, and in Group3 204.6 (192.0-207.6) ng/ml (p &lt; 0.00003, significant difference between the three groups respectively). There were no significant differences in any of the baseline clinical features and laboratory findings between the three groups. Out of the 8 patients in Group1 had a good or moderate response to SRL. Conversely, the percentage of patients with no response to SRL was higher in Group3 than in Group1 and Group2.Conclusion:RA patients could be easily stratified prior to the rapeutic intervention with sgp130 related to the IL-6 signal reguration. Group1 patients, who had the best response to SRL, had the highest level of sgp130.Table 1.Comparison of baseline serum IL-6, sIL-6R and sgp130 of each groups of patientsTCZ use before SRLGroup 1Group 2Group 3P valuen=3N=9N=8N=9IL-6,pg/mL69.8,77.6,592.6Median[IQR]25.6[14.4-72.2]5.9[3.3-11.3]70.2[45.4-86.1]&lt;0.002csIL-6R,ng/mL390.5,413.2,481.7Median[IQR]38.7[34.7-45.1]35.1[24.8-41.9]35.7[34.2-39.8]0.547sgp130,ng/mL205.6,219.2,239.8Median[IQR]273[263-277]223[221-228]205[192-208]&lt;0.001abc*a, b and c mean that statically significant difference between subgroups as a: group1 vs. 2, b: group 1 vs. 3, c: group 2 vs. 3.Disclosure of Interests:Takahiro Yoshikawa: None declared, Tetsuya Furukawa: None declared, Masao Tamura: None declared, Teppei Hashimoto: None declared, Mai Morimoto: None declared, Naoto Azuma: None declared, Kiyoshi Matsui Grant/research support from: Asahi Kasei Pharma, Astellas Pharma (research grants), Speakers bureau: Bristol-Myers Squibb (lecture fees)

Super2-expressing CAR T cells generate improved anti-tumor responses against solid tumors

Abstract Chimeric antigen receptors (CAR) augment tumor specific immune responses by redirecting all T cells towards recognition of tumor antigen. Although CAR T cell treatment shows promising therapeutic effects in leukemia patients, current CAR therapies are inadequate against solid tumors, in part from exclusion or functional exhaustion in the suppressive microenvironment. Exogenously administered Interleukin 2 (IL-2) improves efficacy and expansion of CAR T cell therapy. However, IL-2 administration is limited by high toxicity and low half-life. Super2 is an engineered IL-2 variant that binds with high affinity to the beta and gamma chains of the IL-2 receptor even without IL-2R alpha chain expression. We found that Super2 leads to increased phosphorylation of STAT-5 and more effectively activates lymphocyte populations compared to endogenous IL-2 in vitro. We hypothesized that engineering CAR T cells to express Super2 will improve their functionality within the tumor microenvironment. CAR T cell-intrinsic Super2 may also increase activation of the endogenous tumor-reactive T cells resulting in enhanced tumor control and the establishment of long-term memory even after loss of CAR T cells. To test this hypothesis, we designed CAR T cells that produce Super2 constitutively or in response to CAR stimulation. We found that Super2-expressing CAR T cells increased tumor infiltrating leukocytes (TILs) compared to CAR T cell treatment alone in immune competent murine models. Additionally, Super2-expressing CAR T cells delayed B16F10 melanoma growth in comparison to CAR T cell alone. We conclude that Super2-expressing CAR T cells provide improved protection against solid tumors.

Expression of the IL-2R in Human Podocytes and the Effect of Activation on Autophagy and Apoptosis

Background Interleukin 2 (IL-2) treatment is associated with proteinuria. Materials and Methods: A conditionally immortalized human podocyte cell line was used to investigate expression of the podocyte specific marker podocin, IL-2R alpha (IL-2Rα), apoptosis marker Bax, and autophagy markers LC3I AND LC3II, determined by quantitative immunoblotting, following 24, 48, and 72 hours of IL-2 stimulation, comparing them to unstimulated cells. Results: Podocin was expressed at all time points. IL-2Rα expression was increased after 24 and 72 hrs (p = 0.0014, p = 0.0139) and decreased after 48 hours (p = 0.0445). Bax, LC3I, and LC3II were increased after 24 hrs (p = 0.0094, p = 0.0016, p = 0.0004) and 48 hrs (p = 0.0072, p = 0.0024, p = 0.0087). Conclusion: Human podocytes express the IL-2R and activation results in increased autophagy and apoptosis.

CD73 Rather Than CD39 Is Mainly Involved in Controlling Purinergic Signaling in Calcified Aortic Valve Disease.

The study aimed to compare composition of peripheral blood T-cell subsets and assess their surface expression of CD39 and CD73 ectonucleotidases in patients with severe and moderate aortic stenosis (AS) as well as to evaluate involvement of T-cell-mediated immune processes in valve calcification. The study was performed with 38 patients suffering from severe calcified aortic stenosis (SAS), 33 patients with MAS, and 30 apparently healthy volunteers (HVs). The relative distribution and percentage of T-cell subsets expressing CD39 and CD73 were evaluated by flow cytometry. T helper (Th) and cytotoxic T-cell subsets (Tcyt) were identified by using CD3, CD4, and CD8 antibodies. Regulatory T cells (Tregs) were characterized by the expression of CD3, CD4, and high IL-2R alpha chain (CD25high) levels. CD45R0 and CD62L were used to assess differentiation stage of Th, Tcyt, and Treg subsets. It was found that MAS and SAS patients differed in terms of relative distribution of Tcyt and absolute number of Treg. Moreover, the absolute number of Tcyt and terminally differentiated CD45RA-positive effector T-cells (TEMRA) subset was significantly higher in SAS vs. MAS patients and HVs. However, the absolute and relative number of naïve Th and the absolute number of Treg were significantly higher in MAS vs. SAS patients; the relative number of naïve Tregs was significantly (p < 0.01) decreased in SAS patients. It was shown that CD73 expression was significantly higher in SAS vs. MAS patients noted in all EM, CM, TEMRA, and naïve Th cell subsets. However, only the latter were significantly increased (p = 0.003) in patients compared with HVs. SAS vs. MAS patients were noted to have significantly higher percentage of CD73+ EM Tcyt (p = 0.006) and CD73+ CM Tcyt (p = 0.002). The expression of CD73 in patients significantly differed in all three Treg populations such as EM (p = 0.049), CM (p = 0.044), and naïve (p < 0.001). No significant differences in CD39 expression level was found in MAS and SAS patients compared with the HV group. Overall, the data obtained demonstrated that purinergic signaling was involved in the pathogenesis of aortic stenosis and calcification potentially acting via various cell types, wherein among enzymes, degrading extracellular ATP CD73 rather than CD39 played a prominent role.

Abstract 2360: Targeting IL-4R alpha on tumor-associated macrophages as a therapeutic strategy for prostate cancer

Abstract Prostate cancer tumor growth and disease progression are highly influenced by non-cancerous host cells within the tumor microenvironment including immune cells. High prevalence of tumor-associated macrophages (TAMs) in tumor biopsies is associated with poorer outcomes. TAMs comprise as much as 50 percent of prostate cancer tumors making them an abundant and critical target for treating prostate cancer. Macrophages adopt different functions in response to signaling molecules in their environment. The majority of prostate cancer TAMs resemble alternatively-activated M2 macrophages which, in healthy individuals, are primarily involved in wound healing. M2 TAMs behave similarly to wound-healing M2s by promoting cancer progression and immune suppression. Macrophages are converted to M2s when the signaling molecules interleukin-4 (IL-4) or IL-13 bind IL-4 Receptor Type I or II. Both of these receptor complexes contain the IL-4 receptor alpha subunit and signal through STAT6 to regulate downstream gene transcription and conversion to M2. Due to the central role of IL-4R alpha in tumor-promoting M2s, we hypothesize that disrupting IL-4R alpha signaling will undermine the tumor-promoting capabilities of M2-like TAMs. Using human macrophages cultured in vitro, we detect IL-4R alpha expression in non-malignant human macrophages by qPCR and immunoblot, critical data that is absent in the current literature. Additionally, using the FDA-approved antibody dupilumab, we show that antagonizing IL-4R alpha prevents STAT6 phosphorylation both before and after macrophages have been converted to M2s. Additionally, we show that dupilumab mitigates the M2 characteristics of these macrophages by nanostring analysis of M2-associated genes and ELISA of immunosuppressive cytokines. These data provide evidence that targeting IL-4R alpha on human macrophages impairs tumor-promoting M2 functions. To assess IL-4R alpha expression on TAMs in vivo, we used the HiMyc and TRAMP transgenic mouse models. These mice develop spontaneous prostate cancer tumors and are more accurate representations of the primary tumor microenvironment than injected tumor models (i.e. subcutaneous and orthotopic models). Using nanostring analysis and immunohistochemistry of prostates from 8-week-, 6-month-, and 12-month-old mice, we assessed immune cell infiltration levels, M2 characteristics of TAMs, and IL-4R alpha expression of TAMs. This data provides evidence for the M2-like characteristics of TAMs and IL-4R alpha as a viable TAM target. Subsequent experiments will include assessing IL-4R alpha expression on TAMs in human tumors using prostate cancer tissue microarrays. Moreover, we will continue to investigate the therapeutic potential of antagonizing IL-4R alpha through in vivo studies. In conclusion, these data implicate targeting IL-4R alpha as a promising therapeutic strategy for targeting TAMs in prostate cancer. Citation Format: Amber E. De Groot, Kayla Myers, Sarah R. Amend, Kenneth J. Pienta. Targeting IL-4R alpha on tumor-associated macrophages as a therapeutic strategy for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2360.