IL-6 Receptor Complex Research Articles

Screening and analysis for autophagy-related lncRNA in fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Long non-coding RNA (lncRNA) has become a key epigenetic regulator that regulates gene expression and affects a variety of biological processes. LncRNA plays an important role in the occurrence and development of rheumatoid arthritis (RA). The study on lncRNA in peripheral blood cells of RA patients has been reported. However, there is no study on autophagy regulation by lncRNA in RA patients. This study aims to provide a new direction for the diagnosis and treatment of RA via screening the changes of lncRNAs in RA fibroblast-like synoviocytes (RA-FLSs) before and after autophagy and finding the key lncRNAs targeting RA-FLSs autophagy. Synovial tissues of 6 RA patients after knee and hip joint surgery were obtained, and RA-FLSs were cultured to the 5th generation for further experiments (tissue culture method). After treatment with mTOR inhibitor PP242, the expression of LC3-II was detected by Western blotting. Total RNAs of 3 cases of RA-FLSs before and after treatment with mTOR inhibitor PP242 were extracted by TRIzol and screened by Agilent Human ceRNA Microarray 2019 (4×180 K, design ID: 086188) chip. The lncRNAs with significantly changed expression levels were selected (difference multiple≥2.0, P<0.05). Bioinformatics technology was used to analyze the gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of the differentially expressed lncRNAs, and to explore the possible role of differentially expressed lncRNAs in the pathogenesis of RA. Subsequently, ENST00000584721.1 and ENST00000615939.1 were identified in all the 6 samples of RA-FLSs using real-time RT-PCR, which were selected by previous chip screen combined with GO enrichment analysis and KEGG analysis. RA-FLSs were successfully isolated and cultured from the synovial tissues of the patient's knee or hip joint. After 6 RA-FLSs were treated with PP242, the expression level of autophagy marker protein LC3-II was increased (P<0.05). PP242 induced autophagy in RA-FLSs. LncRNA sequencing analysis showed that a total of 591 lncRNAs were significantly changed, of which 428 were up-regulated and 163 were down-regulated.Go analysis showed that these differentially expressed lncRNAs were associated with negative regulation of Th17 function, T cell related cytokines production, and TGF-β receptor signaling pathway, as well as IL-6 receptor complex formation and type I TGF-β receptor binding. KEGG analysis showed that autophagy pathway, TGF-β, TNF-α, IL-17 signaling pathway, and Th17, Th1, Th2, osteoclast differentiation pathway were the most abundant signal pathways of differentially expressed lncRNAs during autophagy. The expression of ENST00000584721.1 was up-regulated (P<0.05) and the expression ofENST00000615939.1 was down-regulated (P<0.05) in RA-FLSs undergoing autophagy, by using real-time RT-PCR validation which was in consistent with the microarray results. The reliability of microarray screening differential genes was confirmed. GO analysis showed that ENST00000584721.1 and ENST00000615939.1 were related to ribosome transport and autophagy assembly, respectively. KEGG analysis showed that ENST00000584721.1 was related to mitogen-activated protein kinase (MAPK) signaling pathway, and ENST00000615939.1 was related to FoxO signaling pathway. Differentially expressed lncRNAs in RA-FLSs have been identified with microarray analysis. In RA, differential expression of lncRNAs is involved in the autophagy of RA-FLSs. The underlying mechanisms based on bioinformatics analysis include regulating the secretion of cytokines, such as IL-6, TGF-β, TNF-α and IL-17, participating in the immune cell differentiation, such as Th17, Th1, Th2 cells and osteoclasts, as well as regulating the autophagy pathway, MAPK, FoxO, and other signaling pathways. It has been verified that the expression of ENST0000584721.1 is up-regulated and ENST0000615939.1 is down-regulated after autophagy of RA FLSs, which provides a good experimental basis for further study on the mechanism of lncRNA in RA-FLSs autophagy.

1034TiP ARTISTRY-6: Nemvaleukin alfa monotherapy in patients with advanced mucosal and cutaneous melanoma

Despite improved outcomes for melanoma patients with the introduction of checkpoint inhibitors (CPIs), ∼50% of patients do not respond. A subset of responders ultimately progress and have limited treatment options, underscoring a high unmet need for novel treatments with durable benefit. Patients with mucosal melanoma exhibit response rates and progression-free survival times ∼2 times lower than those with cutaneous melanoma. Nemvaleukin alfa (nemvaleukin, ALKS 4230) is a novel, engineered cytokine that selectively binds the intermediate-affinity interleukin-2 receptor complex to preferentially activate CD8+ T and NK cells with minimal expansion of regulatory T-cells.

Pharmacokinetics and Pharmacodynamic Effects of Nemvaleukin Alfa, a Selective Agonist of the Intermediate-Affinity IL-2 Receptor, in Cynomolgus Monkeys.

Nemvaleukin alfa (nemvaleukin, ALKS 4230) is a novel cytokine created by the fusion of circularly permuted interleukin-2 (IL-2) to the IL-2Rα subunit of the IL-2 receptor (IL-2R) complex that confers selectivity for the intermediate-affinity IL-2R expressed on CD8+ T cells and natural killer (NK) cells. The pharmacokinetics and selective pharmacodynamic properties of nemvaleukin have been demonstrated using in vitro and in vivo mouse models. The pharmacokinetic/pharmacodynamic effects of nemvaleukin on immune cell subtypes were evaluated in cynomolgus monkeys after intravenous and subcutaneous administration to inform dose selection and predict pharmacodynamic effects in humans. Male drug-naïve cynomolgus monkeys (N = 15) were administered either single-dose (0.3 mg/kg i.v.; 0.3 mg/kg or 1.0 mg/kg s.c.) or repeated doses (0.1 mg/kg i.v. on days 1-5 or 0.5 mg/kg s.c. on days 1 and 4) of nemvaleukin. Serial blood samples were collected for pharmacokinetic assessment, immunophenotyping by flow cytometry, and profiling of serum cytokines. Repeat-dose subcutaneous administration of nemvaleukin with less frequent dosing resulted in total systemic exposure and trough serum concentrations comparable to those seen with intravenous administration, with lower peak serum concentrations. Transient elevation of interferon-γ and IL-6 peaked at 2 and 8 hours after intravenous and subcutaneous administration, respectively. Selective expansion of immunoprotective central memory, effector memory, terminal effector CD8+ T cells, and CD56+ NK cells, and minimal expansion of immunosuppressive CD4+CD25+FoxP3+ regulatory T cells was observed after both intravenous and subcutaneous administration. These data support the ongoing clinical evaluation of intravenous and subcutaneous nemvaleukin. SIGNIFICANCE STATEMENT: Administration of the novel interleukin-2 receptor agonist nemvaleukin alfa to cynomolgus monkeys resulted in selective expansion of immune effector cells, including CD8+ T and natural killer cells with minimal effects on immunosuppressive CD4+ regulatory T cells, confirming the design of nemvaleukin and highlighting its potential as a cancer immunotherapy. Subcutaneous administration of nemvaleukin achieved systemic exposure and immunostimulatory effects similar to those observed after more frequent intravenous dosing and may represent a practical alternative in a clinical setting.

Enhanced Spontaneous Anti-Tumor Cytotoxicity of Natural Killer Cells in Cutaneous T-Cell Lymphoma Patients

Cutaneous T-cell lymphoma (CTCL) is characterized by the expansion of malignant CD4+ T cells in the skin. There are two main subtypes of CTCL, mycosis fungoides (MF) and Sézary syndrome (SS). Previous studies have demonstrated defects in cell-mediated immunity, including altered cytokine profiles and decreased neutrophil function, and patients often have recurring bacterial and viral infections. Recent studies have shown increased expression of interleukin (IL)-15 in malignant CD4+ T cells in CTCL patients (Mishra et al., Cancer Discovery, 2016). Since IL-15 can enhance NK cell differentiation and activation, we hypothesized that NK cells from CTCL patients might have phenotypic and functional alterations due to these changes in homeostatic IL-15 levels.To evaluate the absolute number of NK cells in CTCL patients, CD56+Lineage- lymphocytes were quantitated by flow cytometry. SS patients had significantly fewer NK cells as compared to normal donors (mean ± SEM of absolute cell numbers in normal donors=0.2442 ± 0.02, n=51; SS=0.1072 ± 0.02, n=9; MF=0.2161 ± 0.01, n=112; unpaired t-test normal vs. SS, p=0.007). NK cell counts were significantly associated with overall survival, with the short-term risk of death increasing by 87.5% for every increase in 0.1 of absolute NK cell counts on univariate analysis utilizing the Cox proportional hazards model (Figure 1A; p=0.0413). We then examined the cytotoxic capacity of NK cells (CD56+Lineage-) freshly purified from both MF and SS patients by standard chromium release assay. There was a significantly higher level of specific lysis of tumor cells (K562 target cells) by NK cells from CTCL patients as compared to normal donors (Figure 1B, at 50:1 ratio mean ± SEM of percent NK cell cytotoxicity in normal vs CTCL = 27.47 ± 4.76, n=13 vs. 52.54 ± 4.88, n=12, unpaired t-test, p=0.001). Upon further transcript analysis of NK cell cytolytic mediators from CTCL patients compared to normal donors, we observed significantly increased levels of perforin (mean ± SEM of relative RNA in normal vs CTCL = 91.39 ± 9.18, n=3 vs. 485.7 ± 43.15, n=7, unpaired t-test, p=0.0004) and granzyme B (143 ± 23.79, n=3 vs. 431 ± 68.13, n=7 unpaired t-test, p=0.03). In addition, we observed increased levels of interferon-gamma and Fas Ligand (5.149 ± 0.4775, n=3 vs.13.93 ± 1.52, n=7; unpaired t-test p=0.007), indicating NK cells from CTCL patients exhibited consistently elevated activation patterns as compared to normal donor NK cells. We then examined the expression pattern of the IL-15 receptor complex on NK cells in CTCL patients as compared to normal donors by RNA sequencing. There was a significant elevation in IL-15Rα (mean ± SEM of relative RNA in normal vs CTCL = 0.8677 ± 0.1018, n=3 vs. 1.772 ± 0.2055, n=7; p=0.03) and IL-15Rγ (57.02 ± 7.525, n=3 vs. 154.7 ± 12.14, n=7; p=0.001), while IL-15Rβ was also elevated (128 ± 23.68, n=3 vs. 223.2 ± 25.43, n=7; p=0.056). Next we evaluated protein levels of phosphorylated signal transducer and activator of transcription 5 (p-STAT5) in fresh whole blood as the surrogate marker of IL-15 signaling. Preliminary findings demonstrate an increase in p-STAT5 in CTCL patients as compared to normal donors (mean ± SEM of fluorescent intensity in normal donors vs CTCL patients =1506 ± 90.82, n=5 vs. 1270 ± 50.59, n=4; unpaired t-test; p=0.07).These data suggest that NK cell activation is enhanced in patients with CTCL, including MF patients with disease localized to the skin. Further investigations aimed at understanding the disconnect between NK cell numbers, activation, and disease control in CTCL patients are warranted. [Display omitted] DisclosuresPorcu:Kiowa: Research Funding; Miragen: Research Funding; Celgene: Research Funding; Tetralogic: Research Funding; Innate Pharma: Research Funding; Kura: Research Funding; Galderma: Research Funding; Cell Medica: Research Funding. William:Miragen: Consultancy, Honoraria.

DOP01 Efficacy and safety of the IL-6 trans-signalling inhibitor olamkicept: a phase 2 randomized, placebo-controlled trial in moderately to severely active Ulcerative Colitis

Abstract Background Total inhibition of IL-6 or its receptor represents a potent anti-inflammatory therapy with considerable side effects. Selective targeting IL-6 trans-signalling may have safety advantages that differentiates it from current pan-IL-6 inhibitors. We evaluated the efficacy and safety of olamkicept, a soluble gp130-Fc fusion protein that binds to the IL-6 and soluble IL-6 receptor complex, as induction therapy for active ulcerative colitis (UC). Methods This multi-national, randomized, double-blind, placebo-controlled phase 2 trial (NCT03235752) enrolled patients with active UC (full Mayo score ≥5, rectal bleeding (RB) score ≥1, endoscopy score (ES) ≥2) with an inadequate response to at least conventional therapy, in a 1:1:1 ratio to receive either placebo, olamkicept 300 mg or 600 mg biweekly for 12 weeks. Primary efficacy endpoint was clinical response (decrease in Mayo score from baseline ≥3 and ≥30%, including RB ≤1 or RB decrease ≥1) at week 12. Secondary endpoints were mucosal healing (ES 0 or 1) and clinical remission (Mayo score ≤2, with no subscore &amp;gt;1 and RB=0). The efficacy endpoints were analysed by logistic regression. All p-values were 2-sided without adjustment for multiplicity. Results Of 91 treated patients (30 in placebo, 31 in olamkicept 300 mg group and 30 in 600 mg group), 88 patients (29:30:29) were evaluable for efficacy. Baseline disease and demographic characteristics were similar among the groups (Table 1). Most patients (94.5%) were bio-naïve. The percentage of patients achieving clinical response at week 12 was significantly greater for olamkicept 600 mg than placebo (58.6% vs 34.5%, P=0.032). Clinical remission at week 12 occurred in 0% (placebo), 6.7% (olamkicept 300 mg) and 20.7% (olamkicept 600 mg, P&amp;lt;0.001) of patients. Mucosal healing at week 12 occurred in 3.4%, 10% and 34.5% (P&amp;lt;0.001) of patients, respectively (Figure 1). Incidence of treatment emergent adverse events (TEAEs) was similar across the groups. The most common TEAEs included upper respiratory tract infection, increased AST levels, and increased urine bilirubin levels, which were mild to moderate and mostly transient. Serious adverse events (SAEs) were reported in 6.7%, 3.2% and 3.3% of patients, respectively. There were no deaths, or other severe AEs associated with current IL-6 inhibitors, such as perforations, severe infections, neutropenia or thrombocytopenia. Conclusion Biweekly 600 mg olamkicept induction therapy demonstrated clinical efficacy with respect to achieving clinical response, clinical remission and mucosal healing in patients with active UC. Olamkicept was well tolerated with a favourable safety profile. The positive results of this phase 2 study support further development of olamkicept in IBD.

The Timing and Abundance of IL-2Rβ (CD122) Expression Control Thymic iNKT Cell Generation and NKT1 Subset Differentiation.

Invariant NKT (iNKT) cells are thymus-generated innate-like T cells, comprised of three distinct subsets with divergent effector functions. The molecular mechanism that drives the lineage trifurcation of immature iNKT cells into the NKT1, NKT2, and NKT17 subsets remains a controversial issue that remains to be resolved. Because cytokine receptor signaling is necessary for iNKT cell generation, cytokines are proposed to contribute to iNKT subset differentiation also. However, the precise roles and requirements of cytokines in these processes are not fully understood. Here, we show that IL-2Rβ, a nonredundant component of the IL-15 receptor complex, plays a critical role in both the development and differentiation of thymic iNKT cells. While the induction of IL-2Rβ expression on postselection thymocytes is necessary to drive the generation of iNKT cells, surprisingly, premature IL-2Rβ expression on immature iNKT cells was detrimental to their development. Moreover, while IL-2Rβ is necessary for NKT1 generation, paradoxically, we found that the increased abundance of IL-2Rβ suppressed NKT1 generation without affecting NKT2 and NKT17 cell differentiation. Thus, the timing and abundance of IL-2Rβ expression control iNKT lineage fate and development, thereby establishing cytokine receptor expression as a critical regulator of thymic iNKT cell differentiation.

CD44s is a negative regulator of IL‐2 mediated T‐cell proliferation

T lymphocytes receive co-stimulatory and inhibitory signals from the extracellular matrix to regulate their proliferation but the underlying machinery of these interactions is unclear. T-cell activation involves antigen recognition through a T cell receptor, the engagement of co-stimulatory receptors and production of cytokines such as IL-2. IL-2 is a pleotropic cytokine which signals through the trimeric IL-2 receptor complex leading to proliferation and differentiation. During T cell activation, the matrix receptor CD44 is upregulated. CD44 is primarily envisioned as a receptor involved in T lymphocyte trafficking through interactions with hyaluronan (HA), a ubiquitous component of the extracellular matrix. Herein, we observe the standard isoform of CD44 (CD44s) negatively influences proliferation of the IL-2 dependent T cell line, CTLL-2. CD44s exerts the influence on proliferation through an IL-2-pSTAT5 independent pathway. Furthermore, using an inducible system to control CD44s expression, strong induction of CD44s results in CTLL-2 death through an unknown mechanism. Surprisingly, alternative splicing of CD44 variants (CD44v) which only differ in the extra-cellular-domain stalk region of the protein, rescues the death phenotype. These observations suggest CD44s may limit a T cell's response to IL-2 through inhibition of an IL-2 mediated growth pathway resulting in cell death and that CD44 alternative splicing relieves the inhibition.

Dynamic Immune/Inflammation Precision Medicine: The Good and the Bad Inflammation in Infection and Cancer

Normal or “good” inflammation process starts from a local cellular response against injury or any infectious agent, with the activation of neutrophils, macrophages, Langerhans cells, dendritic cells, and innate immune cells. Cytokines and chemokines are produced to amplify the local inflammatory process followed by the migration of immune cells to the regional lymph nodes where adaptive immune response is initiated. Systemic inflammation enhances the biological response to mobilize additional cells from central and peripheral immune/hematopoietic system. Local mechanisms to limit inflammation are initiated and lead to healing. During the normal inflammatory process, there is a balance between the production of inflammatory chemokines/cytokines such as Tumor Necrosis Factor (TNF)-α, interleukin (IL)-6 and IL-1 and the production of compounds that limit inflammation and have an immune suppressive effect, such as IL-10 and Transforming Factor (TGF) β. IL-6 and IL-6/soluble IL-6 Receptor (R) complex stimulate liver cells to produce inflammatory proteins, which represents the systemic inflammation response. The magnitude and the duration of the systemic inflammatory response are linked to the cause, under genetic and epigenetic control. Significant inflammation as seen in septic shock, in severe forms of infections or in certain active cancers, represents the “bad inflammation”, correlated with a poor prognosis. In addition, the persistence of a chronic smoldering inflammation may lead to pathological situations which are observed in the majority of inflammatory, degenerative, dysmetabolic, or dysimmune diseases and cancer. Chronic smoldering inflammation is a cross between different pathological situations possibly linked. In addition, within the tumor microenvironment, inflammatory process results from different cellular mechanisms modulated by metabolic and vascular changes. On the contrary, a limited and balanced inflammation initiates the normal immune response, including the adaptive response which amplifies any immunotherapy, including vaccines. Immune checkpoint inhibitors and chimeric antigen receptor (CAR) T-cells are associated with cytokine release syndrome, a clinical risk leading to the use of anti-cytokine drugs. Nowadays, it is time to monitor the dynamic inflammatory process for a better immune precision medicine in both infections and cancer.

Dupilumab en el tratamiento del asma

Dupilumab es un anticuerpo monoclonal humano contra receptores de interleucina (IL)-4 e IL-4/IL-13. Estas son citocinas clave en la génesis de la inflamación tipo 2, predominante en los pacientes con asma. Los ensayos clínicos que evalúan la eficacia de dupilumab incluyen tres ensayos pivotales controlados versus placebo fase 2b o 3 de 24 a 52 semanas en pacientes ≥ 12 años, con asma moderada a grave (no controlada con dosis media a alta de corticosteroides inhalados) o asma grave (dependiente de corticosteroides orales). En estos estudios, la suma de dupilumab al tratamiento se toleró adecuadamente y redujo la tasa de exacerbaciones graves, mejoró la función pulmonar, así como el control del asma y la calidad de vida, de igual forma redujo las dosis de corticosteroides sistémicos orales sin afectar el control. Dupilumab mostró eficacia en varios subgrupos de pacientes, aunque aquellos con inflamación tipo 2 mostraron un beneficio más destacado. Dupilumab está indicado (y es una opción terapéutica valiosa) en pacientes ≥ 12 años de edad que tienen asma de moderada o grave con inflamación tipo 2/fenotipo eosinofílico, descontrolada a pesar de los tratamientos convencionales o en aquellos con dependencia a corticosteroides sistémicos orales para el control.

Mir21 modulates inflammation and sensorimotor deficits in cervical myelopathy: data from humans and animal models.

Degenerative cervical myelopathy is a common condition resulting from chronic compression of the spinal cord by degenerating structures of the spine. Degenerative cervical myelopathy present a wide range of outcomes, and the biological factors underlying this variability are poorly understood. Previous studies have found elevated MIR21-5p in the sub-acute and chronic neuroinflammatory environment after spinal cord injury. As chronic spinal cord neuroinflammation is a major feature of degenerative cervical myelopathy, we hypothesized that MIR21-5p may be particularly relevant to disease pathobiology, and could serve as a potential biomarker. A prospective cohort study of 69 human degenerative cervical myelopathy patients (36 male:33 female) between the ages of 30 and 78 years was performed to identify the relationship between MIR21-5p expression, symptom severity and treatment outcomes. Results from this study identified a positive correlation between elevated plasma MIR21-5p expression, initial symptom severity and poor treatment outcomes. Subsequent validation of these relationships using a mouse model of degenerative cervical myelopathy identified a similar elevation of MIR21-5p expression at 6 and 12 weeks after onset, corresponding to moderate to severe neurological deficits. To further determine how MIR21-5p affects cervical myelopathy pathobiology, this mouse model was applied to a Mir21 knockout mouse line. Deletion of the Mir21 gene preserved locomotor function on rotarod and forced swim tests, but also resulted in increased nociception based on tail flick, Von Frey filament and electrophysiological testing. Critically, Mir21 knockout mice also had reduced spinal cord inflammation, demonstrated by the reduction of Iba1+ microglia by ∼50% relative to wild-type controls. In vitro experiments using primary microglial cultures confirmed that MIR21-5p expression was greatly increased after exposure to lipopolysaccharide (pro-inflammatory), Il4 (anti-inflammatory) and hypoxia. Mir21 knockout did not appear to alter the ability of microglia to respond to these stimuli, as expression of key pro- and anti-inflammatory response genes was not significantly altered. However, target prediction algorithms identified the IL6/STAT3 pathway as a potential downstream target of MIR21-5p, and subsequent in vitro testing found that expression of components of the IL6 receptor complex, Il6ra and Il6st, were significantly higher in Mir21 knockout microglia. In aggregate, these data show that Mir21 plays a role in the progression of motor deficits and neuroinflammatory modulation in degenerative cervical myelopathy. Given this role in neuroinflammation, and its association with poor patient outcomes, MIR21-5p represents a potential therapeutic target and a new marker for prognostication.

Localization of interleukin-6 signaling complex in epithelialized apical lesions of endodontic origin.

We aimed to determine the immunolocalization patterns of the interleukin (IL)-6 signaling complex in epithelialized and non-epithelialized apical lesions of endodontic origin (ALEOs). Epithelialized (n = 8) and non-epithelialized (n = 7) ALEOs were obtained from teeth with indication of extraction in patients with clinical diagnosis of apical periodontitis. All tissues were subjected to routine processing for histopathologic examination and primary antibodies for IL-6, IL-6 receptor (R), and glycoprotein (gp)-130 were used for immunohistochemistry and double immunofluorescence co-localization. IL-6, IL-6R, and gp-130 were immunolocalized in endothelial cells and mononuclear leukocytes in a diffuse pattern within the connective tissue of epithelialized and non-epithelialized ALEOs. In the epithelialized lesions, two different patterns were identified: IL-6 signaling complex was localized within the proliferating epithelium in a diffuse intracellular pattern and in a cell membrane localization pattern within the mature epithelial lining, showing a decreased intensity towards the surface layers. IL-6, IL-6R, and gp-130 localized to mononuclear inflammatory cells, vascular endothelial cells, and immature proliferating epithelia in a diffuse pattern and in mature lining epithelia in a localized cell membrane pattern, supporting a role for epithelial proliferation during cyst formation. Additional cell membrane co-localization of IL-6 receptor complex suggests classic signaling involvement in addition to trans-signaling.

Immunomodulatory Effects of IL-2 and IL-15; Implications for Cancer Immunotherapy.

Simple SummaryEmerging knowledge of the two type I cytokine family members IL-2 and IL-15 has led to critical therapeutic implications for cancer treatment. Here we discuss the distinct roles of IL-2 and IL-15 in activating various functions of T and NK cells with a particular focus on the signals that contribute to the resistance of immune suppressive factors within the tumor microenvironment. We furthermore highlight efforts to modify these cytokines to amplify their antitumor efficacy while minimizing toxicity. Finally, we summarize the clinical applications of IL-2 and IL-15 in metastatic cancer. The type I cytokine family members interleukin-2 (IL-2) and IL-15 play important roles in the homeostasis of innate and adaptive immunity. Although IL-2 and IL-15 receptor complexes activate similar signal transduction cascades, triggering of these receptors results in different functional activities in lymphocytes. While IL-2 expands regulatory T cells and CD4+ helper T cells, IL-15 supports the development of central memory T cells and NK cells. Recent data have provided evidence that IL-2 and IL-15 differ in their ability to activate T and NK cells to resist various forms of immune suppression. The diverse roles of these two cytokines have on immune cells lead to critical therapeutic implications for cancer treatment. In this review, we discuss the distinct roles of IL-2 and IL-15 in activating various functions in T and NK cells with a particular focus on the signals that participate in the resistance of tumor-derived immune suppressive factors. Furthermore, we summarize current clinical applications of IL-2 and IL-15 in metastatic malignancies, either as monotherapy or in combination with other agents, and highlight the future trends for research on these cytokine-based immunotherapies.

Abstract 2222: Engineered variants of Neo-2/15 potently expand CAR-T cells and promote antitumor activity in lymphoma and solid tumor mouse models

Abstract Chimeric antigen receptor (CAR) T cells have achieved remarkable success as therapies for B cell malignancies, but failure might occur due to insufficient CAR-T cell expansion. Furthermore, CAR-T cells have been less effective in treating epithelial cancers, which is believed to be due to poor CAR-T cell accumulation at the tumor site. Interleukin-2 (IL-2) is a potent signaling molecule that induces T cell proliferation and survival. Therefore, it has been suggested that IL-2 co-administration might be effective in promoting CAR-T expansion, persistence, and tumor infiltration. Natural IL-2 acts by binding to the trimeric IL-2 receptor complex consisting of the IL-2Rα, IL-2Rβ and common-γ subunits. Although the IL-2Rα subunit is not directly involved in the signaling cascade, when present, it greatly increases the binding (and signaling) potency of IL-2. An obstacle to applying IL-2 to CAR-T therapy is the expression of IL-2Rα on many target-cells, such as immunosuppressive regulatory T cells (Treg) and endothelial cells. Consequently, high doses of IL-2 cause severe toxicities, including vascular leak syndrome, while low doses are therapeutically suboptimal due to preferential expansion of Treg cells. In addition, as a therapeutic, IL-2 has poor pharmacokinetics, necessitating frequent dosing or continuous infusion. Neoleukin-2/15 (Neo-2/15), a recently reported de novo IL-2 and IL-15 mimetic, effectively agonizes the IL-2 receptor IL-2Rβγ and has no dependency (or interaction) with IL-2Rα. Therefore, Neo-2/15 can activate T cells with high potency regardless of IL-2Rα expression. Here, we studied the combination of CAR-T cells with two reengineered versions of Neo-2/15. In the first strategy, NL-201, a half-life-extended version of Neo-2/15, was dosed weekly (300μg/kg) in a B cell lymphoma xenograft model. NL-201 induced potent CD19 CAR-T cell expansion and prolonged mice survival. NL-201 also induced potent ROR1 CAR-T cell expansion in a solid tumor syngeneic model of mice engrafted with the mutant K-ras/p53-/- tumor line overexpressing hROR1. NL-201 significantly increased the frequency of CAR-T cells at the tumor site compared to IL-2 and also dramatically increased the CD8:Treg ratio in the tumor. In the second approach, Neo-2/15 was targeted to engineered CAR-T cells by fusing the molecule to a DARPin domain specific for a transduction marker co-expressed with the CAR. In a B cell tumor xenograft model, targeted Neo-2/15 effectively increased CAR-T cell expansion and prolonged survival compared to treatment with CAR-T cells and non-targeted Neo-2/15. Together, these results show that engineered versions of Neo-2/15 induce robust expansion of CAR-T cells and can enhance their antitumor activity. Citation Format: Isabel Leung, Megan Templeton, Alfredo Rubio, Carl Walkey, Daniel-Adriano Silva, Stanley Riddell. Engineered variants of Neo-2/15 potently expand CAR-T cells and promote antitumor activity in lymphoma and solid tumor mouse models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2222.

Abstract 2433: Multiomic plasma profiling identifies potential signatures of disease progression in early-stage NSCLC

Abstract Blood-based markers can be used to non-invasively predict cancer progression after treatment. Here, cell-free DNA (cfDNA) and plasma proteins were evaluated to explore biological signatures of progression in non-small cell lung cancer (NSCLC). Baseline plasma samples (n=24; 16 progressors, 8 non-progressors) were from patients diagnosed in 2004 with stage I-III NSCLC, collected prior to surgical resection, and retrospectively analyzed. Six patients were treated with neoadjuvant therapies, one with adjuvant therapy, and 17 with surgery alone. Progression was defined as a relapse event or death by any cause. Whole-genome sequencing was performed to characterize cfDNA fragments, which reflect nucleosome protection and chromatin state. Transcriptional activation for protein-coding genes was inferred by modeling fragment distribution around each transcription start site. Univariate comparisons of gene activation between progressors and non-progressors and Cox proportional hazard ratios (HRs) were calculated by grouping patients above or below the median of the marker of interest. This analysis revealed IL-1RN, the gene encoding for the IL-1RA antagonist to the IL-1 receptor complex, as the gene most negatively correlated with progression-free survival (PFS) (r = -0.76, p &amp;lt; 0.0001; Cox HR = 13.77, p &amp;lt; 0.001). This gene was also significantly more active in progressors than in non-progressors (p &amp;lt; 0.005). The binding activity of ~500 transcription factors was also inferred by measuring chromatin accessibility across the genome, revealing SOX-9 to be significantly associated with progression (p &amp;lt; 0.0001, FDR = 1.1%) and the factor most negatively correlated with PFS (r = -0.72, p &amp;lt; 0.001, FDR = 16.9%). Both IL-1RN and SOX-9 have been previously reported to affect survival in NSCLC patients.In addition, the abundances of ~450 proteins including cytokines, receptors, and enzymes were measured. Six proteins were identified as differentially abundant between progression groups. Among these, IL-1α was more abundant in progressors vs. non-progressors (effect size = 0.92, p &amp;lt; 0.05). Notably, IL-1RA abundance did not differ between these groups. Both IL-1α (r = -0.61, p &amp;lt; 0.01; Cox HR = 3.78, p &amp;lt; 0.05) and IL-1RA (r = -0.75, p &amp;lt; 0.001; Cox HR = 1.13, p = 0.78) were negatively correlated with PFS in progressors. Finally, all features and analytes were integrated to identify biological signatures that may be shared among proteins and cfDNA. These signatures differed significantly (p &amp;lt; 0.05) between progressors and non-progressors, suggesting differences in cytokine signaling. The multiomics platform described here integrates biological signals with computational featurization to reveal clinically relevant signatures. Specifically, findings from a small cohort of early-stage NSCLC patients demonstrated the potential of this platform to reveal signatures of progression in NSCLC. Citation Format: Francesco Vallania, Hayley Warsinske, Peter Ulz, Tzu-Yu Liu, Karen Assayag, Krishnan K. Palaniappan, Mitch Bailey, Irving Wang, David E. Weinberg, Riley Ennis, C Jimmy Lin, Anne-Marie Martin, Nancy Krunic. Multiomic plasma profiling identifies potential signatures of disease progression in early-stage NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2433.

Defining the Threshold IL-2 Signal Required for Induction of Selective Treg Cell Responses Using Engineered IL-2 Muteins.

Among all T and NK cell subsets, regulatory T (Treg) cells typically respond to the lowest concentrations of IL-2 due to elevated surface expression of the IL-2R alpha chain (IL2RA; CD25) and the high affinity IL-2 receptor (IL-2R) complex. This enhanced sensitivity forms the basis for low-dose (LD) IL-2 therapy for the treatment of inflammatory diseases, where efficacy correlates with increased Treg cell number and expression of functional markers. Despite strong preclinical support for this approach, moderate and variable clinical efficacy has raised concerns that adequate Treg selectivity still cannot be achieved with LD IL-2, and/or that doses are too low to stimulate effective Treg-mediated suppression within tissues. This has prompted development of IL-2 variants with greater Treg selectivity, achieved through attenuated affinity for the signaling chains of the IL-2R complex (IL2RB or CD122 and IL2RG or CD132) and, consequently, greater reliance on high CD25 levels for full receptor binding and signaling. While certain IL-2 variants have advanced to the clinic, it remains unknown if the full range of IL-2R signaling potency and Treg-selectivity observed with low concentrations of wildtype IL-2 can be sufficiently recapitulated with attenuated IL-2 muteins at high concentrations. Using a panel of engineered IL-2 muteins, we investigated how a range of IL-2R signaling intensity, benchmarked by the degree of STAT5 phosphorylation, relates to biologically relevant Treg cell responses such as proliferation, lineage and phenotypic marker expression, and suppressor function. Our results demonstrate that a surprisingly wide dynamic range of IL-2R signaling intensity leads to productive biological responses in Treg cells, with negligible STAT5 phosphorylation associating with nearly complete downstream effects such as Treg proliferation and suppressor activity. Furthermore, we show with both in vitro and humanized mouse in vivo systems that different biological responses in Treg cells require different minimal IL-2R signaling thresholds. Our findings suggest that more than minimal IL-2R signaling, beyond that capable of driving Treg cell proliferation, may be required to fully enhance Treg cell stability and suppressor function in vivo.

Dupilumab: A Review in Chronic Rhinosinusitis with Nasal Polyps.

Dupilumab (Dupixent®) is a fully human IgG4 monoclonal antibody against the interleukin (IL)-4receptor α (IL-4Rα) subunit, which is shared by the type I IL-4 and the type II IL-4/IL-13 receptor complexes. By binding to and blocking this subunit, dupilumab inhibits IL-4 and IL-13, which are the major drivers of human type 2 inflammatory disease [e.g. asthma, atopic dermatitis and chronic rhinosinusitis with nasal polyps (CRSwNP)]. Dupilumab, administered subcutaneously, is the first biological therapy to be approved for the treatment of adults with inadequately controlled CRSwNP in the EU and the USA. In two placebo-controlled, multinational, phase III studies of 24 and 52weeks' duration, the addition of dupilumab (300mg every 2weeks) to the intranasal corticosteroid treatment of adults with severe, inadequately controlled CRSwNP was generally well tolerated and improved nasal polyp size, sinus opacification and health-related quality of life (HR-QOL), relieved the major symptoms of CRSwNP (nasal congestion or obstruction, nasal discharge and loss of smell) and reduced the use of systemic corticosteroids and the need for nasal polyp surgery. Improvements in nasal polyp size, sinus opacification and nasal congestion or obstruction were achieved regardless of the presence of comorbid asthma or NSAID-exacerbated respiratory disease, or a history of previous nasal polyp surgery, with patients with comorbid asthma also demonstrating improvements in lung function and asthma control regardless of their baseline eosinophil count. Thus, add-on subcutaneous dupilumab is a valuable treatment option for adults with inadequately controlled CRSwNP.

Interleukin-6: An Important Mediator of Allograft Injury.

Interleukin-6 (IL-6) is a cytokine with critical innate and adaptive immunity functions. Its diverse immunological and physiological actions include direction of immune cell differentiation, initial response to invading pathogens and ischemic injury, sustained plasma cell growth, and immunoglobulin production. IL-6 transcriptional dysregulation is commonly seen in patients with autoimmune or inflammatory disorders. Emerging information suggests that IL-6 transcription is upregulated in patients with kidney and heart transplant rejection and may account for perpetuation of inflammatory responses in the allograft, leading to allograft rejection and vasculopathy. IL-6-directed therapeutics include monoclonal antibodies directed at IL-6, the IL-6 receptor (IL-6R), and Janus kinase inhibitors. IL-6-mediated signaling to cell targets is unique, involving classic signaling (IL-6->IL-6R) cell membrane receptors, transsignaling (IL-6->soluble IL-6R->gp130) which activates any cell, and the recently discovered IL-6/IL-6R transpresentation in which antigen-presenting cells synthesize and express IL-6/IL-6R complexes, which are transported through the cell membrane subsequently interacting with gp130 to costimulate T cells. Currently, there are new trials in autoimmunity and heart and kidney transplantation to determine effectiveness of inhibiting IL-6/IL-6R to ameliorate chronic allograft rejection and coronary allograft vasculopathy. Therapeutic trials aimed at prevention of ischemia/reperfusion injury to allografts based on animal data should be considered.

IL-4 enhances interferon production by virus-infected human mast cells

IL-4 enhances interferon production by virus-infected human mast cells

Signaling through the interleukin-4 and interleukin-13 receptor complexes regulates cholangiocyte TMEM16A expression and biliary secretion.

TMEM16A is a Ca2+-activated Cl- channel in the apical membrane of biliary epithelial cells, known as cholangiocytes, which contributes importantly to ductular bile formation. Whereas cholangiocyte TMEM16A activity is regulated by extracellular ATP-binding membrane purinergic receptors, channel expression is regulated by interleukin-4 (IL-4) through an unknown mechanism. Therefore, the aim of the present study was to identify the signaling pathways involved in TMEM16A expression and cholangiocyte secretion. Studies were performed in polarized normal rat cholangiocyte monolayers, human Mz-Cha-1 biliary cells, and cholangiocytes isolated from murine liver tissue. The results demonstrate that all the biliary models expressed the IL-4Rα/IL-13Rα1 receptor complex. Incubation of cholangiocytes with either IL-13 or IL-4 increased the expression of TMEM16A protein, which was associated with an increase in the magnitude of Ca2+-activated Cl- currents in response to ATP in single cells and the short-circuit current response in polarized monolayers. The IL-4- and IL-13-mediated increase in TMEM16A expression was also associated with an increase in STAT6 phosphorylation. Specific inhibition of JAK-3 inhibited the increase in TMEM16A expression and the IL-4-mediated increase in ATP-stimulated currents, whereas inhibition of STAT6 inhibited both IL-4- and IL-13-mediated increases in TMEM16A expression and ATP-stimulated secretion. These studies demonstrate that the cytokines IL-13 and IL-4 regulate the expression and function of biliary TMEM16A channels through a signaling pathway involving STAT6. Identification of this regulatory pathway provides new insight into biliary secretion and suggests new targets to enhance bile formation in the treatment of cholestatic liver disorders.NEW & NOTEWORTHY The Ca2+-activated Cl- channel transmembrane member 16A (TMEM16A) has emerged as an important regulator of biliary secretion and hence, ductular bile formation. The present studies represent the initial description of the regulation of TMEM16A expression in biliary epithelium. Identification of this regulatory pathway involving the IL-4 and IL-13 receptor complex and JAK-3 and STAT-6 signaling provides new insight into biliary secretion and suggests new therapeutic targets to enhance bile formation in the treatment of cholestatic liver disorders.

IL-6 trans-signaling drives a STAT3-dependent pathway that leads to structural alterations of the peritoneal membrane.

IL-6 is a vital inflammatory factor in the peritoneal cavity of patients undergoing peritoneal dialysis (PD). The present study examined the effect of IL-6 trans-signaling on structural alterations of the peritoneal membrane. We investigated whether the epithelial-to-mesenchymal transition (EMT) process of human peritoneal mesothelial cells (HPMCs) and the production of proangiogenic factors were controlled by IL-6 trans-signaling. Its role in the peritoneal alterations was detected in a mouse model. The morphology of HPMCs and levels of cytokines in PD effluent were also explored. Stimulation of HPMCs with the IL-6 and soluble IL-6 receptor complex (IL-6/S) promoted the EMT process of HPMCs depending on the STAT3 pathway. In a coculture system of HPMCs and human umbilical vein endothelial cells, IL-6/S mediated the production of VEGF and angiopoietins so as to downregulate the expression of endothelial junction molecules and finally affect vascular permeability. Daily intraperitoneal injection of high glucose-based dialysis fluid induced peritoneal fibrosis, angiogenesis, and macrophage infiltration in a mouse model, accompanied by phosphorylation of STAT3. Blockade of IL-6 trans-signaling prevented these peritoneum alterations. The fibroblast-like appearance of HPMCs ex vivo was upregulated in patients undergoing prevalent PD accompanied by increasing levels of IL-6, VEGF, and angiopoietin-2 in the PD effluent. Taken together, these findings identified a critical link between IL-6 trans-signaling and structural alterations of the peritoneal membrane, and it might be a potential target for the treatment of patients undergoing PD who have developed peritoneal alterations.