Endogenous IL-10 Research Articles

ST2 + T-Regulatory Cells in Renal Inflammation and Fibrosis after Ischemic Kidney Injury.

Inflammation is a major cause of kidney injury. The Interleukin (IL)-1 family cytokine IL-33 is released from damaged cells and modulates the immune response through its receptor ST2 expressed on many cell types, including regulatory T-cells (Tregs). While a proinflammatory role of IL-33 has been proposed, exogenous IL-33 expanded Tregs and suppressed renal inflammation. However, the contribution of endogenous IL-33/ST2 for the role of Tregs in the resolution of kidney injury has not been investigated. We used murine renal ischemia-reperfusion injury and kidney organoids to delineate the role of the ST2 and amphiregulin (AREG) specifically in Treg cells using targeted deletion. Bulk and single-cell RNA sequencing was performed on flow-sorted Tregs from spleen and CD4 T-cells from post-ischemic kidneys respectively. The protective role of ST2-sufficient Tregs was analyzed using a novel co-culture system of syngeneic kidney organoids and Treg cells under hypoxic conditions. Bulk RNA-sequencing of splenic and single-cell-RNA-sequencing of kidney T-cells showed that ST2+ Tregs are enriched for genes related to Treg proliferation and function. Genes for reparative factors such as AREG were also enriched in ST2+ Tregs. Treg-specific deletion of ST2 or AREG exacerbated kidney injury and fibrosis in the unilateral ischemia reperfusion injury model. In co-culture studies, WT but not ST2-deficient Tregs preserved the hypoxia-induced loss of kidney-organoid viability, which was restored by AREG supplementation. Our study identified the role of the IL-33/ST2 pathway in Tregs for resolution of kidney injury. The transcriptome of ST2+ Tregs was enriched for reparative factors including AREG. Lack of ST2 or AREG in Tregs worsened kidney injury. Tregs protected kidney organoids from hypoxia in ST2 and AREG-dependent manner. Thus Treg-based approaches could be of benefit for resolution of renal injury.

IL-13 protects from C. difficile colitis

ObjectivesClostridioides difficile infection (CDI) is the leading hospital-acquired infection in North America. We have previously discovered that antibiotic disruption of the gut microbiota decreases intestinal IL-33 and IL-25 and increases susceptibility to CDI. We further found that IL-33 promotes protection through type 2 Innate Lymphoid Cells (ILC2s), which produce IL-13. However, the contribution of IL-13 to disease has never been explored. MethodsWe used a validated model of CDI in mice, in which we neutralized via blocking antibodies, or administered recombinant protein, IL-13 to assess the role of this cytokine during infection using weight and clinical scores. Fluorescent activated cell sorting (FACS) was used to characterize myeloid cell population changes in response to IL-13 manipulation. ResultsWe found that administration of IL-13 protected, and anti-IL-13 exacerbated CDI. Additionally, we observed alterations to the monocyte/macrophage cells following neutralization of IL-13 as early as day three post infection. We also observed elevated accumulation of myeloid cells by day four post-infection following IL-13 neutralization. Neutralization of the decoy receptor, IL-13Rα2, resulted in protection from disease, likely through increased available endogenous IL-13. ConclusionsOur data highlight the protective role of IL-13 in protecting from more severe CDI and the association of poor responses with a dysregulated monocyte-macrophage compartment. These results increase our understanding of type 2 immunity in CDI and may have implications for treating disease in patients.

Unleashing Natural IL18 Activity Using an Anti-IL18BP Blocker Induces Potent Immune Stimulation and Antitumor Effects.

Recombinant cytokines have limited anticancer efficacy mostly due to a narrow therapeutic window and systemic adverse effects. IL18 is an inflammasome-induced proinflammatory cytokine, which enhances T- and NK-cell activity and stimulates IFNγ production. The activity of IL18 is naturally blocked by a high-affinity endogenous binding protein (IL18BP). IL18BP is induced in the tumor microenvironment (TME) in response to IFNγ upregulation in a negative feedback mechanism. In this study, we found that IL18 is upregulated in the TME compared with the periphery across multiple human tumors and most of it is bound to IL18BP. Bound IL18 levels were largely above the amount required for T-cell activation in vitro, implying that releasing IL18 in the TME could lead to potent T-cell activation. To restore the activity of endogenous IL18, we generated COM503, a high-affinity anti-IL18BP that blocks the IL18BP:IL18 interaction and displaces precomplexed IL18, thereby enhancing T- and NK-cell activation. In vivo, administration of a surrogate anti-IL18BP, either alone or in combination with anti-PD-L1, resulted in significant tumor growth inhibition and increased survival across multiple mouse tumor models. Moreover, the anti-IL18BP induced pronounced TME-localized immune modulation including an increase in polyfunctional nonexhausted T- and NK-cell numbers and activation. In contrast, no increase in inflammatory cytokines and lymphocyte numbers or activation state was observed in serum and spleen. Taken together, blocking IL18BP using an Ab is a promising approach to harness cytokine biology for the treatment of cancer.

Abstract 4072: Unleashing natural IL-18 activity using an anti-IL-18BP blocker antibody induces potent immune stimulation and anti-tumor effects

Abstract IL-18 is an inflammasome induced proinflammatory cytokine that augments T and NK cell activity and stimulates IFNγ production. The activity of IL-18 is naturally blocked by a high affinity endogenous binding protein (IL-18BP). IL-18BP is induced in the tumor microenvironment (TME) in response to IFNγ upregulation in a negative feedback mechanism. By evaluating 88 human tumor specimens and serum samples we were able to show that IL-18 is upregulated in the TME (median 11.2ng/gr) compared to serum samples (median 0.3ng/ml). Moreover, we showed that most of the IL-18 is bound by IL-18BP. IL-18BP-bound IL-18 levels were largely above the amount required for T cell activation in vitro (1.2ng/gr), implying that blocking IL-18BP has the potential to release IL-18 in tumors above the minimum range required for immune system stimulation. Next, to assess whether tumor endogenous IL-18 levels released by IL-18BP blockade are sufficient to provoke anti-tumor responses, COM503, a high affinity (<1pM) anti-IL-18BP Ab, was generated and examined in T and NK cell-based assays. In a co-culture assay of tumor cells with ex-vivo stimulated human tumor infiltrating CD8+ lymphocytes, COM503 was able to displace IL-18 from a pre-formed complex and enhance IFNγ (197%, p<0.01) and TNFα (84% p<0.01) secretion. Additionally, COM503 induced human NK cell activation as reflected by increased IFNγ secretion (26-fold, p<0.001). Finally, in human ex vivo dissociated tumor cells assay, COM503 mediated an increase in Granzyme B (25%), IFNγ (38%), TNFα (58%) and IL-12 (50%) production. In vivo, administration of an anti-mouse IL-18BP Ab resulted in potent anti-tumor responses and increased survival across multiple mouse tumor models. In orthotopic E0771 tumor model, anti-IL-18BP Ab induced significant tumor growth inhibition (91% TGI, p<0.0001), as well as pronounced TME-localized immune modulation. This modulation included an increase in CD8+ T cells expansion (108.5%, p=0.015) and activation, specifically in polyfunctional effector IFNγ+GrB+CD8+ T cells (259%, p=0.02) and IFNγ+TNFα+NK cells (77%, p=0.001). Similarly, anti-tumor effects were shown in MC38OVAdim model (58% TGI, p<0.001), accompanied by a robust TME-localized immune modulation including increased CD8+ T cells expansion (85%, p=0.009) and IFNγ secretion (76%, p=0.052). In contrast to immune modulation in the TME, no increase in inflammatory cytokines and lymphocyte numbers or activation state was observed in serum and spleen. Taken together, our data suggest that IL-18 is upregulated in the TME, mostly bound by IL-18BP, and could be exploited to induce pronounced TME-localized immune modulation. Anti-IL-18BP Ab approach has a leading edge in inhibiting tumor growth while avoiding peripheral toxicity associated with administration of a cytokine. COM503 is currently undergoing IND-enabling studies. Citation Format: Assaf Menachem, Zoya Alteber, Gady Cojocaru, Tal Fridman Kfir, Dan Blat, Olga Leiderman, Moran Galperin, Lital Sever, Nadav Cohen, Keren Cohen, Roy Zvi Granit, Sandra Vols, Masha Frenkel, Lior Faigenbloom, Liron Soffer, Karin Meyer, Keren Menachem, Hadas Galon Tilleman, Dina Morein, Itamar Borukhov, Amir Toporik, Michal Perpinial Shahor, Evgeny Tatirovsky, Pierre Ferre, Eran Ophir. Unleashing natural IL-18 activity using an anti-IL-18BP blocker antibody induces potent immune stimulation and anti-tumor effects [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 4072.

The ST2+ Treg/amphiregulin axis protects from immune-mediated hepatitis.

The alarmin IL-33 has been implicated in the pathology of immune-mediated liver diseases. IL-33 activates regulatory T cells (Tregs) and type 2 innate lymphoid cells (ILC2s) expressing the IL-33 receptor ST2. We have previously shown that endogenous IL-33/ST2 signaling activates ILC2s that aggravate liver injury in murine immune-mediated hepatitis. However, treatment of mice with exogenous IL-33 before induction of hepatitis ameliorated disease severity. Since IL-33 induces expression of amphiregulin (AREG) crucial for Treg function, we investigated the immunoregulatory role of the ST2+ Treg/AREG axis in immune-mediated hepatitis. C57BL/6, ST2-deficient (Il1rl1-/-) and Areg-/- mice received concanavalin A to induce immune-mediated hepatitis. Foxp3Cre+ x ST2fl/fl mice were pre-treated with IL-33 before induction of immune-mediated hepatitis. Treg function was assessed by adoptive transfer experiments and suppression assays. The effects of AREG and IL-33 on ST2+ Tregs and ILC2s were investigated in vitro. Immune cell phenotype was analyzed by flow cytometry. We identified IL-33-responsive ST2+ Tregs as an effector Treg subset in the murine liver, which was highly activated in immune-mediated hepatitis. Lack of endogenous IL-33 signaling in Il1rl1-/- mice aggravated disease pathology. This was associated with reduced Treg activation. Adoptive transfer of exogenous IL-33-activated ST2+ Tregs before induction of hepatitis suppressed inflammatory T-cell responses and ameliorated disease pathology. We further showed increased expression of AREG by hepatic ST2+ Tregs and ILC2s in immune-mediated hepatitis. Areg-/- mice developed more severe liver injury, which was associated with enhanced ILC2 activation and less ST2+ Tregs in the inflamed liver. Exogenous AREG suppressed ILC2 cytokine expression and enhanced ST2+ Treg activation in vitro. In addition, Tregs from Areg-/- mice were impaired in their capacity to suppress CD4+ T-cell activation in vitro. Moreover, application of exogenous IL-33 before disease induction did not protect Foxp3Cre+ x ST2fl/fl mice lacking ST2+ Tregs from immune-mediated hepatitis. In summary, we describe an immunoregulatory role of the ST2+ Treg/AREG axis in immune-mediated hepatitis, in which AREG suppresses the activation of hepatic ILC2s while maintaining ST2+ Tregs and reinforcing their immunosuppressive capacity in liver inflammation.

IL-10 Differentially Promotes Mast Cell Responsiveness to IL-33, Resulting in Enhancement of Type 2 Inflammation and Suppression of Neutrophilia.

Mast cells (MCs) play critical roles in the establishment of allergic diseases. We recently demonstrated an unexpected, proinflammatory role for IL-10 in regulating MC responses. IL-10 enhanced MC activation and promoted IgE-dependent responses during food allergy. However, whether these effects extend to IgE-independent stimuli is not clear. In this article, we demonstrate that IL-10 plays a critical role in driving IL-33-mediated MC responses. IL-10 stimulation enhanced MC expansion and degranulation, ST2 expression, IL-13 production, and phospho-relA upregulation in IL-33-treated cells while suppressing TNF-α. These effects were partly dependent on endogenous IL-10 and further amplified in MCs coactivated with both IL-33 and IgE/Ag. IL-10's divergent effects also extended invivo. In a MC-dependent model of IL-33-induced neutrophilia, IL-10 treatment enhanced MC responsiveness, leading to suppression of neutrophils and decreased TNF-α. In contrast, during IL-33-induced type 2 inflammation, IL-10 priming exacerbated MC activity, resulting in MC recruitment to various tissues, enhanced ST2 expression, induction of hypothermia, recruitment of eosinophils, and increased MCPT-1 and IL-13 levels. Our data elucidate an important role for IL-10 as an augmenter of IL-33-mediated MC responses, with implications during both allergic diseases and other MC-dependent disorders. IL-10 induction is routinely used as a prognostic marker of disease improvement. Our data suggest instead that IL-10 can enhance ST2 responsiveness in IL-33-activated MCs, with the potential to both aggravate or suppress disease severity depending on the inflammatory context.

Endogenous IL-27 during toxoplasmosis limits early monocyte responses and their inflammatory activation by pathological T cells.

The molecule IL-27 is critical in limiting the immune response to the parasite Toxoplasma gondii. In the absence of IL-27, a lethal, overactive immune response develops during infection. However, when exactly in the course of infection this molecule is needed was unclear. By selectively inhibiting IL-27 during this parasitic infection, we discovered that IL-27 was only needed during, but not prior to, infection. Additionally, IL-27 is only needed in the active areas in which the parasite is replicating. Finally, our work found that a previously unstudied cell type, monocytes, was regulated by IL-27, which contributes further to our understanding of the regulatory networks established by this molecule.

Multiplexed miRNA and Protein Analysis Using Digital Quantitative PCR in Microwell Arrays.

Proteins and microRNAs (miRNAs) act in tandem within biological pathways to regulate cellular functions, and their misregulation has been correlated to numerous diseases. Because of their interconnectedness, both miRNAs and proteins must be evaluated together to obtain accurate insights into the molecular pathways of pathogenesis. However, few analytical techniques can measure both classes of biomolecules in parallel from a single biological sample. Here, microfluidic digital quantitative PCR (dqPCR) was developed to simultaneously quantify miRNA and protein targets in a multiplexed assay using a single detection chemistry. This streamlined analysis was achieved by integrating base-stacking PCR and immuno-PCR in a microfluidic array platform. Analyses of let-7a (miRNA) and IL-6 (protein) were first optimized separately to identify thermocycling and capture conditions amenable to both biomolecules. Singleplex dqPCR studies exhibited the expected digital signals and quantification cycles for both analytes over a range of concentrations. Multiplexed analyses were then conducted to quantify both let-7a and IL-6 with high sensitivity (LODs ∼ 3 fM) over a broad dynamic range (5-5000 fM) using only standard PCR reagents. This multiplexed dqPCR was then translated to the analysis of HEK293 cell lysate, where endogenous let-7a and IL-6 were measured simultaneously without sample purification or pretreatment. Collectively, these studies demonstrate that the integration of BS-PCR and immuno-PCR achieves a sensitive and streamlined approach for multiplexed analyses of miRNAs and proteins, which will enable researchers to gain better insights into disease pathogenesis in future applications.

Biological and clinical roles of IL-18 in inflammatory diseases.

Several new discoveries have revived interest in the pathogenic potential and possible clinical roles of IL-18. IL-18 is an IL-1 family cytokine with potent ability to induce IFNγ production. However, basic investigations and now clinical observations suggest a more complex picture. Unique aspects of IL-18 biology at the levels of transcription, activation, secretion, neutralization, receptor distribution and signalling help to explain its pleiotropic roles in mucosal and systemic inflammation. Blood biomarker studies reveal a cytokine for which profound elevation, associated with detectable 'free IL-18', defines a group of autoinflammatory diseases in which IL-18 dysregulation can be a primary driving feature, the so-called 'IL-18opathies'. This impressive specificity might accelerate diagnoses and identify patients amenable to therapeutic IL-18 blockade. Pathogenically, human and animal studies identify a preferential activation of CD8+ T cells over other IL-18-responsive lymphocytes. IL-18 agonist treatments that leverage the site of production or subversion of endogenous IL-18 inhibition show promise in augmenting immune responses to cancer. Thus, the unique aspects of IL-18 biology are finally beginning to have clinical impact in precision diagnostics, disease monitoring and targeted treatment of inflammatory and malignant diseases.

IL-10 protects against OPC ferroptosis by regulating lipid reactive oxygen species levels post stroke

Accumulation of reactive oxygen species (ROS), especially on lipids, induces massive cell death in neurons and oligodendrocyte progenitor cells (OPCs) and causes severe neurologic deficits post stroke. While small compounds, such as deferoxamine, lipostatin-1, and ferrostatin-1, have been shown to be effective in reducing lipid ROS, the mechanisms by which endogenously protective molecules act against lipid ROS accumulation and subsequent cell death are still unclear, especially in OPCs, which are critical for maintaining white matter integrity and improving long-term outcomes after stroke. Here, using mouse primary OPC cultures, we demonstrate that interleukin-10 (IL-10), a cytokine playing roles in reducing neuroinflammation and promoting hematoma clearance, significantly reduced hemorrhage-induced lipid ROS accumulation and subsequent ferroptosis in OPCs. Mechanistically, IL-10 activated the IL-10R/STAT3 signaling pathway and upregulated the DLK1/AMPK/ACC axis. Subsequently, IL-10 reprogrammed lipid metabolism and reduced lipid ROS accumulation. In addition, in an autologous blood injection intracerebral hemorrhagic stroke (ICH) mouse model, deficiency of the endogenous Il-10, specific knocking out Il10r or Dlk1 in OPCs, or administration of ACC inhibitor was associated with increased OPC cell death, demyelination, axonal sprouting, and the cognitive deficits during the chronic phase of ICH and vice versa. These data suggest that IL-10 protects against OPC loss and white matter injury by reducing lipid ROS, supporting further development of potential clinical applications to benefit patients with stroke and related disorders.

The autoinflammation-associated NLRC4V341A mutation increases microbiota-independent IL-18 production but does not recapitulate human autoinflammatory symptoms in mice.

Autoinflammation with infantile enterocolitis (AIFEC) is an often fatal disease caused by gain-of-function mutations in the NLRC4 inflammasome. This inflammasomopathy is characterized by macrophage activation syndrome (MAS)-like episodes as well as neonatal-onset enterocolitis. Although elevated IL-18 levels were suggested to take part in driving AIFEC pathology, the triggers for IL-18 production and its ensuing pathogenic effects in these patients are incompletely understood. Here, we developed and characterized a novel genetic mouse model expressing a murine version of the AIFEC-associated NLRC4V341A mutation from its endogenous Nlrc4 genomic locus. NLRC4V341A expression in mice recapitulated increased circulating IL-18 levels as observed in AIFEC patients. Housing NLRC4V341A-expressing mice in germfree (GF) conditions showed that these systemic IL-18 levels were independent of the microbiota, and unmasked an additional IL-18-inducing effect of NLRC4V341A expression in the intestines. Remarkably, elevated IL-18 levels did not provoke detectable intestinal pathologies in NLRC4V341A-expressing mice, even not upon genetically ablating IL-18 binding protein (IL-18BP), which is an endogenous IL-18 inhibitor that has been used therapeutically in AIFEC. In addition, NLRC4V341A expression did not alter susceptibility to the NLRC4-activating gastrointestinal pathogens Salmonella Typhimurium and Citrobacter rodentium. As observed in AIFEC patients, mice expressing a murine NLRC4V341A mutant show elevated systemic IL-18 levels, suggesting that the molecular mechanisms by which this NLRC4V341A mutant induces excessive IL-18 production are conserved between humans and mice. However, while our GF and infection experiments argue against a role for commensal or pathogenic bacteria, identifying the triggers and mechanisms that synergize with IL-18 to drive NLRC4V341A-associated pathologies will require further research in this NLRC4V341A mouse model.

Phase I Open-Label Single-Arm Study of Sscart-19 in Adult Patients with Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia in China

Background: Although CAR-T therapy has shown amazing efficacy in the treatment of hematologic malignancies in recent years, it is also accompanied by high-frequency of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), especially in acute lymphoblastic leukemia patients. Therefore, it is not recommended to enroll patients with active CNS involvement in CAR-T therapy. ssCART-19 is an autologous CD19-specific chimeric antigen receptor T-cell with short hairpin RNA-IL-6 gene silencing element. ssCART-19 can simultaneously express CAR structure and IL-6 silencing element. CAR structure can recognize and bind CD19 positive tumor cells, enhance T cell amplification, and finally exert T cell killing activity, such as releasing cytokines and perforin. Post-transcriptional expression of short hairpin RNA-IL-6 gene silencing element can recognize mRNA of IL-6, inhibit the expression of endogenous IL-6 in CAR-T cells through RNA interference mechanism, and then reduce the stimulation or activation intensity of downstream immune cells (mainly monocytes), with the control of the overall release intensity of cytokines, it reduces the incidence of severe CRS and ICANS, at the end reduces the clinical risk of CAR-T therapy. Methods: The trial of ssCART-19 in adult patients with R/R B-cell ALL (NCT04825496) is a single-arm, open-label Phase 1 study conducted in China. Patients underwent leukapheresis to obtain T cells for ssCART-19 manufacturing. Patients received fractionated infusions split over 3 days (10%; 30%; 60%) after lymphodepleting chemotherapy (3 days fludarabine and cyclophosphamide). Adverse events (AEs) were graded according to CTCAE, v5.0; CRS and ICANS were graded according to ASTCT Consensus Grading. The overall response rate(ORR) of complete response (CR) and CR with incomplete hematological recovery (CRi) within 3 months, duration of remission, relapse-free survival, and OS were assessed as secondary endpoints. Results : ssCART-19 was administered at 3 doses levels (DL) of 1×10 6/kg, 5×10 6/kg, or 10×10 6/kg. As of June 30th 2023 data cutoff (the median follow-up 15.9 months), 13 patients enrolled and underwent leukapheresis, 12 patients were received ssCART-19 treatment, 5 patients in DL1, 6 patients in DL2, and 1 patients in DL3. ssCART-19 was manufactured using serum-free culture system and no manufacture failure was reported. All 12 patients diagnosed with B-cell ALL were relapsed (75.0%) and refractory (25.0%) to multiple lines of prior therapy (100.0% patients had received 2 or more therapies). Five patients (41.7%) had more than 50% blasts in the bone marrow. All 12 patients did not have DLT events within 28 days. The most common ssCART-19-related AEs were transient neutropenia (100.0%), lymphopenia (100.0%), leukopenia (100.0%), anaemia (100.0%) and thrombocytopenia (83.3%). Adverse events of special interest (AESI) were CRS and ICANS. Among the 12 patients, 9 patients (75.0%) developed CRS, and 3 patients (25.0%) were grade 3 CRS. Notably, none of the 12 patients were occurred ICANS. No death cases were reported due to CRS or ICANS. A 83.3% overall response rate was achieved in efficacy-evaluable patients (n=12), with 7 complete response (CR, 58.3%), and 3 CR with incomplete hematological recovery (CRi,25.0%). The median duration of remission, relapse-free survival and OS have not been reached. Among them, 6 patients were in ongoing remission for over 12 months. Notably, one of the 12 patients (S012) had central nervous system infiltration with grade 2 before ssCART-19 infusion(Figure 1). After ssCART-19 therapy, the patient had no ICANS and got continuous remission. Robust CAR T-cell expansion occurred in all patients with a median time to T max of 12.5 days (range 9-14d), and peak copy number (C max) of 52495 (194-348000) copies/μg DNA. Conclusions ： ssCART-19 CAR-T cell therapy can effectively reduce the incidence of severe CRS and ICANS, especially control the occurrence of ICANS. And ssCART-19 achieved a high rate of remission in adult patients with R/R B-cell ALL. The ORR within 3 months were 83.3%. And, ssCART-19 shows good clinical efficacy in the treatment of leukemia patients with central nervous system infiltration. Thus, with the combination of short hairpin RNA-IL-6 gene silencing element, ssCART-19 can greatly improve the safety of CAR-T therapy, especially in central nervous system infiltration patients.

SAT020 Is Testosterone The Male Sex Hormone Responsible For Increased Male Mortality In Melanoma? In-vitro Studies On Two Human Melanoma (BLM, 1205Lu) Cell Models

Abstract Disclosure: P. Ramaraj: None. Epidemiological data pointed to increased male mortality in melanoma and clinical studies pointed to better protection of menstruating females in melanoma than post-menopausal women and men of any age. Our in-vitro studies with progesterone (P) on human melanoma (BLM and 1205Lu) cell lines showed a significant decrease in cell growth along with a decrease in the secretion of proinflammatory cytokine IL-8 by these cells. These in-vitro studies correlated with the hormonal data which showed that progesterone levels in menstruating females ranged between 1000 - 1500 ng/dl, compared to post-menopausal women 20 - 100 ng/dl and men 27-90 ng/dl, suggesting low progesterone levels meant no protection in melanoma. This observation prompted us to hypothesize that lack of progesterone in males could be a cause for increased male mortality. This hypothesis also raised the question of the role of male sex hormones (AD-androstenedione and T-testosterone) in increased male mortality. In order to address these questions in-vitro experiments were planned. First, to indirectly induce endogenous IL-8 by pretreating the cells with endothelin and then adding steroid hormones (AD, T, P). Secondly, to add IL-8 directly to the cells and then add steroid hormones. Finally, to suppress endogenous IL-8 with curcumin and then add T to one set of cells and IL-8 + T to another set of cells. For the first experiment, the result was a marginal increase in cell growth (67%) in cells pretreated with endothelin followed by T-treatment compared to straight T-treated cells (60% cell growth) without endothelin pretreatment. For the second experiment, there was a slight increase in cell growth in IL-8-added cells, but it was not statistically significant. For the final experiment, the cells treated with IL-8 + T after curcumin pretreatment showed a partial restoration of cell growth (49%) compared to cells (40% cell growth) treated with plain T after curcumin treatment, suggesting T was not able to control or regulate IL-8 action like progesterone. These in-vitro experiments suggested that in males 2 reasons 1) a deficiency of progesterone and 2) inefficiency of T to control endogenous IL-8 could possibly result in increased IL-8 levels. This increased IL-8 level in males could lead to increased cell growth and metastasis in melanoma leading to death. So, a situation arising out of male reproductive endocrine physiology could probably be responsible for increased male mortality in melanoma. Presentation: Saturday, June 17, 2023

The role of predictive and prognostic values of inflammatory markers in acute pancreatitis: a narrative review

Pancreatitis is an inflammatory condition affecting the pancreas and is classified into 2 types, acute and chronic, which can manifest in various forms. This review article summarizes the role of predictive and prognostic values of inflammatory markers in the pathogenesis of acute pancreatitis, mainly focused on preclinical and clinical studies. It includes serum amyloid A (SAA), monocyte chemotactic protein-1 (MCP-1), erythrocyte sedimentation rate (ESR), interleukin-6 (IL-6), C-reactive protein (CRP), IL-10, myeloperoxidase, pentraxin 3, and plasminogen activator inhibitor 1. SAA3 plays a crucial role in developing acute pancreatitis by triggering a receptor-interacting protein 3–dependent necroptosis pathway in acinar cells. Targeting SAA3 could be a potential strategy for treating acute pancreatitis. The recruitment of monocytes/macrophages and the activation of the systemic MCP-1 signaling pathway play a role in the progression of pancreatitis, and blocking MCP-1 may have a suppressive effect on the development of pancreatic fibrosis. The ESR can predict severe acute pancreatitis with slightly lower accuracy than CRP. When ESR and CRP levels are combined at 24 hours, they predict severe acute pancreatitis accurately. IL-6 plays a crucial role in activating the Janus kinase/signal transducers and activators of the transcription pathway, exacerbating pancreatitis and contributing to the initiation and progression of pancreatic cancer. Endogenous IL-10 plays a crucial role in controlling the regenerative phase and limiting the severity of fibrosis and glandular atrophy induced by repeated episodes of acute pancreatitis in mice. The predictive and diagnostic roles of these inflammatory factors in pancreatitis were introduced in detail in this review.

Influence of different feed additives on the level of dactylogyrus infection in carp years

A comparative study of the effect of various preparations on the level of infection of Cyprinus carpio yearlings with dactylogyrus was carried out. The studies were carried out in spring under aquatic conditions. The fish were divided into 7 groups and fed 6 times a day. The control group was given pure food, and the experimental group was given the addition of various preparations. The study was carried out in 2 stages. At the first stage, the experimental fish were fed with Roncoleukin, a structural and functional analog of endogenous IL-2, and the enterosorbent agent, Polyfepam; Bacillus subtilis “SUB-PRO” and biologically active additive “Ecoflor”. The infestation of carps was assessed by the extensiveness of invasion and the index of abundance of parasites. The experiments performed showed differences in the level of infection depending on the drug used. According to the effect on the infection of juvenile carp with dactygyrus, the studied preparations can be conditionally divided into 2 groups: increasing and decreasing the intensity of invasion by the end of the 7th day of the experiment. The first group includes “Polyfepam” and “AQUATAN”, and the rest of the drugs to the second. On the 7th day of the experiment, the lowest infestation was recorded in the group of carp, which were added to the Antibak feed, and the highest — with the addition of AQUATAN. On day 14, in all experimental groups, the parasite abundance index exceeded the control values obtained before the start of the experiment. A significant increase in the intensity of invasion was noted in the experimental group, which received a drug based on ellagitannins with food. In other groups, this indicator increased slightly.

A phase 1/2 study of AU-007, a monoclonal antibody (mAb) that binds to IL-2 and inhibits CD25 binding, in patients with advanced solid tumors: Interim results.

e14507 Background: AU-007 is a computationally designed mAb that binds to IL-2 on its CD25 binding epitope. AU-007 bound IL-2 (A/IL-2) cannot bind to trimeric (CD25, CD122, CD132) IL-2 receptors (IL-2R) on Tregs, vascular endothelium, or eosinophils, but IL-2’s binding to dimeric IL-2Rs (CD122, CD132) on T effector and NK cells is unhindered. AU-007 thus redirects endogenous or exogenous IL-2 (aldesleukin) towards T effector and NK cell activation, while diminishing immunosuppressive Treg activation and vascular leak. Unique in the IL-2 field, AU-007 can redirect endogenous IL-2 generated from A/IL-2 driven T effector cell expansion in vivo, converting a Treg-mediated autoinhibitory loop into an immune stimulating loop. A/IL-2 is expected to prolong the 85-minute T1/2 of IL-2, allowing endogenous IL-2 (as A/IL-2) or low dose aldesleukin to initiate an anti-tumor response. Methods: This first-in-human Phase 1 study consists of 3 dose escalation arms, each starting with one 1+2 cohort followed by 3+3 cohorts. Arm 1A evaluates escalating doses of monotherapy AU-007 (intravenous, every 2 weeks [Q2W]). Arm 1B evaluates AU-007 (Q2W) plus 1 loading dose of subcutaneous, low dose aldesleukin with the 1st AU-007 dose. The AU-007 dose will be fixed with escalating aldesleukin doses in sequential cohorts. Arm 1C evaluates AU-007 plus escalating doses of concomitant subcutaneous, low dose aldesleukin, both Q2W. The dose-limiting toxicity (DLT) evaluation period is the first 28 days of the 1st cycle. Tumor assessments by computed tomography scan occur with each 8-week cycle. The AU-007 and aldesleukin dose and schedule for Phase 2 expansion will be based on safety, efficacy, pharmacokinetics (PK), and pharmacodynamics (PD). Results: As of 01 February 2023, 8 patients have been enrolled into the first 3 Cohorts of Arm 1A (0.5, 1.5, and 4.5 mg/kg AU-007). AU-007 was well tolerated with no DLTs and all treatment-related adverse events were Grade 1. These occurred in 3 patients, 1 at each dose level. The 4th Cohort of Arm 1A (9 mg/kg AU-007) and the 1st Cohort of Arm 1B (4.5 mg/kg AU-007 + 1 aldesleukin dose of 15,000 IU/kg) are now being evaluated. Three of 4 tumor evaluable patients had a best response of stable disease and 2 continue treatment. Two patients discontinued with objective progression and one with clinical progression. Initial PK data (0.5 and 1.5 mg/kg) demonstrate dose proportional AU-007 serum concentrations with typical characteristics of an IgG1 human mAb. All 7 patients with available PD data demonstrate overall decreasing Tregs (% change and absolute) and eosinophils, which both express the trimeric IL-2 receptor. Conclusions: AU-007 monotherapy at doses up to 4.5 mg/kg Q2W is safe and well tolerated, with initial signs of immune modulation consistent with AU-007’s mechanism of action. These findings warrant continued escalation and combination with aldesleukin. Clinical trial information: NCT05267626 .

Establishing a novel and sensitive assay for bioactivity determination of anti-CD25 antibodies

Anti-CD25 antibodies have been approved for renal transplantation and has been used prior to and during transplantation by the Food and Drug Administration (FDA). However, no reported bioassays have been reflected the mechanism of action (MOA) of anti-CD25 antibodies. Here, we describe the development and validation of a reporter gene assay (RGA) based on the engineered C8166-STAT5RE-Luc cells expressing endogenous IL-2 receptors and a STAT5-inducible element-driven firefly luciferase in C8166 cell lines. The RGA was fully validated according to the International Conference on the Harmonization of Technical Requirements for the Registration of Pharmaceuticals for the Human Use-Q2 (ICH-Q2). After optimization, the assay showed excellent specificity, linearity, accuracy, precision, and robustness. Due to the MOA relatedness and the excellent assay performance, the RGA is suitable for exploring the critical quality attributes (CQAs), release inspection, comparability and stability of anti-CD25 mAbs.

The role of death receptor signaling pathways in mouse Sertoli cell avoidance of apoptosis during LPS- and IL-18-induced inflammatory conditions

Lipopolysaccharide (LPS) triggers infectious acute inflammation, and interleukin (IL)-18 is an inflammasome-mediated cytokine. We previously demonstrated that endogenous IL-18 induces testicular germ cell apoptosis during acute inflammation when plasma IL-18 levels are high. Additionally, high-dose recombinant IL-18 (rIL-18) induced Leydig cell apoptosis. The blood-testis barrier formed by Sertoli cells protects testicular germ cells from both exogenous and endogenous harmful substances. However, the impact of LPS and IL-18 on Sertoli cells remained unclear. We stimulated TM4 cells, a mouse Sertoli cell line, with LPS (200 or 1000 ng/mL) or rIL-18 (0.1–100 ng/mL) at levels that induced Leydig cell apoptosis in our previous study and assessed caspase 3 cleavage and the mRNA expression of inflammatory cytokines and markers of apoptotic pathways (Tnfr1, Fasl, Fas, Fadd) after stimulation. Il6 mRNA was increased by LPS stimulation. Tnfα mRNA was increased by 200 ng/mL LPS but not 1000 ng/mL LPS. Fas was increased, but Fasl was decreased, by LPS. LPS had little influence on Tnfr1 or Fadd mRNA expression and did not induce apoptosis. Il18 mRNA was not increased, and Il18r1 was significantly decreased following LPS treatment. Treatment with rIL-18 increased Il18r1 mRNA and induced inflammation, but decreased Tnfr1 and had little influence on apoptosis, as indicated by Tnfα, Fasl, Fas, Fadd and cleaved caspase 3. These results suggested that Sertoli cells do not easily undergo apoptosis despite strong inflammatory stimuli. Additionally, Sertoli cells may resist inflammation and play a larger role in protecting testicular homeostasis than other component cells of the testis.

Negative Feedback Expansion of Tregs Caused by Endogenous IL-2 Limits the Activity of IL-2-based Therapies

Stimulating effector T-cells (Teffs) without inducing regulatory T-cells (Tregs) has been the primary goal of IL-2-based therapies for cancer. Recently, modified IL-2 designed for differential T-cell expansion for the treatment of cancer has failed in the clinic. We propose that treatments based on exogenous administrations of modified IL-2 are inherently undermined by a negative feedback loop, caused by IL-2 secreted endogenously from activated effector T-cells. This endogenous IL-2 secretion subsequentially induces Treg expansion and inhibits the immune response that is essential for cancer clearance. Here, we demonstrate that treatments utilizing exogenous modified IL-2 indeed induce Treg expansion. To circumvent this negative feedback, we computationally designed a novel monoclonal humanized antibody (AU-007) that binds human IL-2 with pM affinity at a predefined epitope and completely blocks IL-2 binding to CD25 that is highly expressed on Tregs, without hindering IL-2 binding to CD122/CD132 dimer receptor expressed over effector cells. This epitope-specific, high-affinity antibody controls endogenous IL-2 and prevents it from expanding Tregs while allowing it to expand Teffs. We show that controlling endogenous IL-2 using AU-007 abrogates the negative feedback loop and replaces it with a positive feedback loop that enhances the expansion of NK cells and Teffs, an effect considered favorable for cancer immunotherapy.

Abstract 5287: Is testosterone the male sex hormone responsible for increased male mortality in melanoma? In-vitro studies based on human melanoma (BLM) cell model

Abstract Clinical studies showed that menstruating females were better protected in melanoma than post-menopausal women and men of any age. But, these studies did not correlate with the steroid status of females. Our in-vitro study with progesterone (P), a female sex hormone showed significant inhibition of human melanoma cell growth. A literature survey showed that progesterone levels ranged between 1000-1500 ng/dl in menstruating females compared to post-menopausal women (20-100 ng/dl) and men (27-90 ng/dl), suggesting increased progesterone levels could offer protection to the menstruating females. Moreover, Elisarray showed that progesterone action was mediated by specific suppression of proinflammatory cytokine IL-8. So, it was proposed that a low level of progesterone in males leading to non-suppression of IL-8 in melanoma, could be responsible for increased melanoma cell growth and male mortality. This hypothesis prompted us to check the effect of male sex hormones androstenedione (AD) and testosterone (T) on melanoma cell growth and IL-8 secretion. Both AD and T decreased IL-8 secretion along with a decrease in cell growth. However, when progesterone was added along with androgens, there was a significant decrease in cell growth and in IL-8 secretion. This raised the question of why male mortality was high when androgens also decreased cell growth and IL-8 secretion. In order to address this question, it was decided to induce endogenous IL-8 in melanoma cells by pre-incubating with endothelin (50ng/ml) and then add steroids. AD and P individually decreased IL-8 secretion and in combination significantly decreased IL-8 secretion and cell growth even in endothelin pretreated cells when compared to their respective control cells. But, T was not able to suppress endogenous IL-8 induced by endothelin and in fact, cell growth also was slightly increased compared to straight T-treated cells. Conclusion: So, in males 2 reasons 1) a deficiency of progesterone and 2) inefficiency of T to suppress endogenous IL-8 could possibly result in increased IL-8 levels. This increased IL-8 level in males could lead to increased cell growth and metastasis in melanoma (IL-8 had already been shown to stimulate melanoma cell growth and metastasis by us and others) leading to death. So, a situation arising out of male reproductive endocrine physiology could probably be responsible for increased male mortality in melanoma. Citation Format: Pandurangan Ramaraj. Is testosterone the male sex hormone responsible for increased male mortality in melanoma? In-vitro studies based on human melanoma (BLM) cell model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5287.