Recombinant Interleukin-2 Research Articles

Immuno-inflammatory in vitro hepatotoxicity models to assess side effects of biologicals exemplified by aldesleukin.

Hepatotoxicity induced by immunotherapeutics is an appearing cause for immune-mediated drug-induced liver injury. Such immuno-toxic mechanisms are difficult to assess using current preclinical models and the incidence is too low to detect in clinical trials. As hepatotoxicity is a frequent reason for post-authorisation drug withdrawal, there is an urgent need for immuno-inflammatory in vitro models to assess the hepatotoxic potential of immuno-modulatory drug candidates. We developed several immuno-inflammatory hepatotoxicity test systems based on recombinant human interleukin-2 (aldesleukin). Co-culture models of primary human CD8+ T cells or NK cells with the hepatocyte cell line HepaRG were established and validated with primary human hepatocytes (PHHs). Subsequently, the HepaRG model was refined by increasing complexity by inclusion of monocyte-derived macrophages (MdMs). The main readouts were cytotoxicity, inflammatory mediator release, surface marker expression and specific hepatocyte functions. We identified CD8+ T cells as possible mediators of aldesleukin-mediated hepatotoxicity, with MdMs being implicated in increased aldesleukin-induced inflammatory effects. In co-cultures of CD8+ T cells with MdMs and HepaRG cells, cytotoxicity was induced at intermediate/high aldesleukin concentrations and perforin was upregulated. A pro-inflammatory milieu was created measured by interleukin-6 (IL-6), c-reactive protein (CRP), interferon gamma (IFN-γ), and monocyte chemoattractant protein-1 (MCP-1) increase. NK cells responded to aldesleukin, however, only minor aldesleukin-induced cytotoxic effects were measured in co-cultures. Results obtained with HepaRG cells and with PHHs were comparable, especially regarding cytotoxicity, but high inter-donor variations limited meaningfulness of the PHH model. The in vitro test systems developed contribute to the understanding of potential key mechanisms in aldesleukin-mediated hepatotoxicity. In addition, they may aid assessment of immune-mediated hepatotoxicity during the development of novel immunotherapeutics.

Tension-activated nanofiber patches delivering an anti-inflammatory drug improve repair in a goat intervertebral disc herniation model.

Conventional microdiscectomy treatment for intervertebral disc herniation alleviates pain but does not repair the annulus fibrosus, resulting in a high incidence of recurrent herniation and persistent dysfunction. The lack of repair and the acute inflammation that arise after injury can further compromise the disc and result in disc-wide degeneration in the long term. To address this clinical need, we developed tension-activated repair patches (TARPs) for annulus fibrosus repair and local delivery of the anti-inflammatory factor anakinra (a recombinant interleukin-1 receptor antagonist). TARPs transmit physiologic strain to mechanically activated microcapsules embedded within the patch, which release encapsulated bioactive molecules in direct response to spinal loading. Mechanically activated microcapsules carrying anakinra were loaded into TARPs, and the effects of TARP-mediated annular repair and anakinra delivery were evaluated in a goat model of annular injury in the cervical spine. TARPs integrated with native tissue and provided structural reinforcement at the injury site that prevented aberrant disc-wide remodeling resulting from detensioning of the annular fibrosus. The delivery of anakinra by TARP implantation increased matrix deposition and retention at the injury site and improved maintenance of disc extracellular matrix. Anakinra delivery additionally attenuated the inflammatory response associated with TARP implantation, decreasing osteolysis in adjacent vertebrae and preserving disc cellularity and matrix organization throughout the annulus fibrosus. These results demonstrate the therapeutic potential of TARPs for the treatment of intervertebral disc herniation.

Low-dose IL-2 Treatment Rescues Cognitive Deficits by Repairing the Imbalance Between Treg and Th17 Cells at the Middle Alzheimer's Disease Stage.

Multiple studies highlight the role of effector and regulatory CD4+T cells in the pathophysiology of Alzheimer's disease, and foster low-dose IL-2 treatment which induces regulatory CD4+T (Treg) cells expansion and activation as a promising strategy for its treatment. However, studies demonstrating discrepant Treg functions in AD have been reported. In addition, a compromised immune system associated with aging may substantially impact on these processes. Here, we report that there is an altered balance of activity between Treg cells and IL-17-producing helper T (Th17) cells in periphery and brain of APP/PS1 mice along the disease progression. A dramatic loss of the healthy balance of activity between Treg and Th17 cells was found at the middle disease stage. While peripheral low-dose recombinant human IL-2 administration could selectively modulate the abundance of Treg cells and repair the imbalance between Treg and Th17 subsets at the middle disease stage. We further show that modulation of peripheral immune balance through low-dose IL-2 treatment reduces the neuro-inflammation and increases numbers of plaque-associated microglia, accompanied by marked reduction of Aβ plaque deposition and slower cognitive declines in APP/PS1 mice at the middle disease stage. Our study highlights the therapeutic potential of repurposed IL-2 for innovative immunotherapy based on modulation of the homeostasis of CD4+T cell subsets in Alzheimer's disease at the middle disease stage.

A unique role for IL-13 in inducing esophageal eosinophilia through MID-1 and STAT6.

Eosinophilic esophagitis (EoE) is associated with allergen-driven inflammation of the esophagus and an upregulated Th2 cytokine signature. Recombinant interleukin (IL)-13 (rIL-13) administration to mice induces some of the hallmark features of EoE, including increased eotaxin expression and eosinophil recruitment. Inflammation in EoE has previously been shown to depend on the expression of TRAIL and MID-1, which reduced protein phosphatase 2A (PP2A) activity. The relationship between IL-13 and TRAIL signalling in esophageal eosinophilia is currently unknown. To investigate the interaction between IL-13-driven eosinophil infiltration and TRAIL or MID-1 in the esophagus. We administered rIL-13 to wild type (WT), TRAIL-deficient (Tnsf10-/-) or STAT6-deficient (STAT6-/-) mice and targeted MID-1 with small interfering RNA. rIL-13 administration to mice increased TRAIL and MID-1 expression in the esophagus while reducing PP2A activity. TRAIL deficient, but not STAT6 deficient mice demonstrated increased MID-1 expression and PP2A reduction upon IL-13 challenge which correlated with eosinophil infiltration into the esophagus. Silencing MID-1 expression with siRNA completely ablated IL-13 induced eosinophil infiltration of the esophagus, restored PP2A activity, and reduced eotaxin-1 expression. IL-13-driven eosinophil infiltration of the esophagus induced eosinophilia and eotaxin-1 expression in a STAT6-dependent and MID-1-dependent manner. This study highlights a novel mechanism employed by IL-13 to perpetuate eosinophil infiltration.

Activation of the Interleukin-18 Signaling Pathway via Direct Receptor Dimerization in the Absence of Interleukin-18.

Interleukin 18 (IL-18) is a key cytokine involved in the activation of T and NK cells, which are major effector cells in tumor killing. However, recombinant IL-18 showed limited efficacy in clinical trials. A recent study showed the lack of efficacy was largely due to the existence of IL-18BP, a soluble decoy receptor for IL-18. It was shown that engineered IL-18 variants that maintained pathway activation, but avoided IL-18BP binding, could exert potent antitumor effects. In this study, we demonstrated an alternative strategy to activate IL-18 signaling through direct receptor dimerization. These results provide evidences that the IL-18 pathway can be activated by directly bridging the receptors and, therefore, bypassing the IL-18BP-mediated inhibition.

Recombinant Interleukin-1 Receptor Antagonist Is an Effective First-Line Treatment Strategy in New-Onset Systemic Juvenile Idiopathic Arthritis, Irrespective of HLA-DRB1 Background and IL1RN Variants.

Human leukocyte antigen (HLA)-DRB1*15:01 has been recently associated with interstitial lung disease (LD), eosinophilia, and drug reactions in systemic juvenile idiopathic arthritis (sJIA). Additionally, genetic variants in IL1RN have been linked to poor response to anakinra. We sought to reproduce these findings in a prospective cohort study of patients with new-onset sJIA treated with anakinra as first-line therapy. HLA and IL1RN risk alleles were identified via whole-genome sequencing. Treatment responses and complications were compared between carriers versus noncarriers. Seventeen of 65 patients (26%) carried HLA-DRB1*15:01, comparable with the general population, and there was enrichment for HLA-DRB1*11:01, a known risk locus for sJIA. The rates of clinical inactive disease (CID) at 6 months, 1 year, and 2 years were generally high, irrespective of HLA-DRB1 or IL1RN variants, but significantly lower in carriers of an HLA-DRB1*11:01 allele. One patient, an HLA-DRB1*15:01 carrier, developed sJIA-LD. Of the three patients with severe drug reactions to biologics, one carried HLA-DRB1*15:01. The prevalence of eosinophilia did not significantly differ between HLA-DRB1*15:01 carriers and noncarriers at disease onset (6.2% vs 14.9%, P = 0.67) nor after the start of anakinra (35.3% vs 37.5% in the first 2 years of disease). We observed high rates of CID using anakinra as first-line treatment irrespective of HLA-DRB1 or IL1RN variants. Only one of the 17 HLA-DRB1*15:01 carriers developed sJIA-LD, and of the three patients with drug reactions to biologics, only one carried HLA-DRB1*15:01. Although thorough monitoring for the development of drug hypersensitivity and refractory disease courses in sJIA, including sJIA-LD, remains important, our data support the early start of biologic therapy in patients with new-onset sJIA irrespective of HLA-DRB1 background or IL1RN variants.

298 Interleukin-6 Family Members Improve Pre-Implantation Bovine Blastocyst Composition

Abstract Inner cell mass (ICM) size is a predictor of bovine blastocyst quality and post-transfer pregnancy success. In vitro produced (IVP) embryos have reduced ICM cell numbers compared with in vivo produced embryos, and we propose this is one reason pregnancy success is compromised in IVP embryos. Our recent work found that human recombinant interleukin-6 (IL6) supplementation during in vitro embryo culture increased ICM cell numbers. We hypothesized that other human recombinant members of the IL6 cytokine family, specifically interleukin-11 (IL11) and leukemia inhibitory factor (LIF), will have a similar response. In vitro maturation, fertilization and culture procedures were used to produce embryos that were treated with 100 ng/mL recombinant human recombinant IL6, IL11, LIF, or carrier [control; 1% (wt/vol) SOF-BSA] beginning at day 6 post-fertilization (25 embryos/50 mL drop; 3 drops/treatment; 5 replicates). A subset of day 8 blastocysts (n = 28 to 29) was fixed and processed for cell counting. Analyses were performed by least-squares ANOVA using the general linear model. No treatment differences in overall blastocyst rates, trophectoderm (TE) cell numbers, and total cell numbers were observed at day 8. Treatment with IL6 (P = 0.09) and LIF (P = 0.06) tended to increase ICM cell numbers when compared with the control, whereas IL11 was not different from the control. Treatment with IL6 increased the ICM:TE ratio (P < 0.05) but the other cytokines did not influence this ratio. Several treatment by stage interactions were observed. In regular day 8 blastocysts (n = 10 to 14), none of the treatments affected ICM numbers or the ICM:TE ratio, but LIF increased TE number (P < 0.05) and IL6 increased total cell number (P < 0.05). When grouping expanded and hatched blastocysts together (n = 14 to 18), each treatment increased ICM cell numbers and IL6 increased the ICM:TE ratio (P < 0.05). Furthermore, both IL11 (P = 0.08) and LIF (P = 0.08) tended to increase the ICM:TE ratio. Only LIF increased total cell numbers (P < 0.05). None of the other treatments affected total cell numbers. These observations suggest that IL6, IL11, and LIF are capable of influencing ICM cell numbers in IVP bovine embryos, but they may possess different abilities in affecting TE and overall cell numbers. These findings differ from previous findings from our lab. We suspect that transitioning from bovine recombinant preparations with undefined purity and bioactivity to using purified human recombinant proteins with high bioactivity allowed us to detect these responses for each cytokine. Collectively, this work implicates several members of the IL6 cytokine family as embryokines that may be used to improve bovine IVP embryo quality. Funding for this work was provided by USDA-NIFA (2017-67015-2646 and 2021-67015-34485) and by NIH (R21-OD026516-01).

Anti-BCMA Dual Epitope-Binding CAR-Marrow Infiltrating Lymphocytes (MILs) Could Offer a Potent Innovative Immunotherapeutic Tool Against Multiple Myeloma

Introduction: Chimeric antigen receptor (CAR) T cells are a promising new treatment for multiple myeloma (MM). Marrow-infiltrating lymphocytes (MILs) derived from MM patient's bone marrow are another potential innovative T cell source for CAR-based cell therapy, capable of robustly and specifically targeting myeloma cells through polyclonal targets. MILs possess polyclonal central memory T cell populations, which could be as effective MM killings via polyantigenic cytotoxic T cells with greater recognitions of broad range of tumor antigens via the polyclonal memory TCR as well as the CAR pathway with strong recognition of tumor antigens. Nanobodies are currently the smallest stable antibody molecules having high affinity for antigen, allowing them to strongly penetrate tumor tissue and even directly to the blood-brain barrier while remaining low toxicity and immunogenicity. With the peculiar properties, nanobodies have been widely used as the antigen biding domain of CAR-T cell. In this study, we investigated anti-B-cell maturation antigen (BCMA) dual epitope-binding CAR-MILs against MM. Methods: CD3 + T cells isolated from bone marrow mononuclear cells of MM patients were stimulated with Ex Vivo CD3/CD28 in the presence of recombinant human IL-2. The nanobodies VHH01 and VHH02 were provided as dual epitope-binding for BCMA (Senlang, China). After expansion and activation for 2 days, anti-BCMA dual epitope-binding CAR was transduced to the activated MILs and, they were stimulated with Ex Vivo CD3/CD28 in the presence of recombinant human IL-2, IL-7, IL-15, and IL-21. Expanded and activated BCMA dual epitope-binding CAR-MILs (BCMA CAR-MILs) were harvested on days 7, 14, and 21 for further experiments. Results: The transduction efficiency of BCMA CAR-MILs was approximately 34%, and the expansion of BCMA CAR-MILs was > 100 folds over 21-day culture period. Additionally, BCMA CAR-MILs showed a dramatically increased proportion of CD8 + T cells, while the proportion of CD4 + T cells decreased. Moreover, BCMA CAR-MILs were enriched with a higher proportion of central memory T cells (Tcm) (80%), while the proportions of regulatory T cells (Tregs) and checkpoint molecules (TIGIT, TIM3, and CD73) were not so high. The BCMA CAR-MILs showed potent cytotoxicity, particularly high levels of CD107a degranulation against BCMA-expressing target cancer cell lines, such as Daudi, Raji, RPMI8226, and U266B1 cells, and autologous CD138 + primary myeloma cells. Conclusion: This study suggested that BCMA dual epitope-binding CAR-MILs were easily expanded and activated using bone marrow mononuclear cells from MM patients with high transduction efficiency and induced a potent anti-myeloma immune response, which may offer an alternative innovative immunotherapeutic approach against MM patients in the future.

Preferential Efficacy of Remission Maintenance with HDC/IL-2 in Age Groups of Patients with Chemoresponsive or Normal Karyotype AML

Background Relapse in complete remission (CR) remains a leading cause of morbidity and mortality in acute myeloid leukemia (AML). Previous phase 3 trial data show that combinatorial immunotherapy with histamine dihydrochloride and low-dose interleukin-2 (HDC/IL-2) reduces relapse incidence in the post-consolidation phase, in particular among younger adult AML patients (<60 years old) in CR1 (Brune et al., Blood 108:88). Recent post-hoc analyses identified subgroups of responding patients including (1) those achieving CR1 after the first induction course of chemotherapy (referred to as chemoresponsive AML) and (2) those carrying leukemic cells of normal karyotype (NK-AML), i.e. devoid of structural or numerical chromosomal aberrations. Here we report analyses of treatment efficacy in a phase 3 trial within age groups of patients with chemoresponsive AML or NK-AML. Methods Three hundred and twenty AML patients (18-84 years old, median 57) who were ineligible for allogeneic stem cell transplantation were randomly assigned to receive HDC/IL-2 or no treatment (control) for remission maintenance in a phase 3 trial. Patients were recruited in Europe, Israel, Oceania, and USA. Treatment was initiated in CR after the completion of consolidation chemotherapy and comprised 10 consecutive 3-week cycles of HDC/IL-2 for 18 months (mo) with 3- or 6-week rest periods. In each cycle, patients received HDC at 0.5 mg and human recombinant low-dose IL-2 (aldesleukin; 16,400 IU/kg) s.c. twice daily (n=160) or no treatment (control; n=160). After 18 months of treatment, all patients were monitored for >18 additional mo (median follow-up 48 mo). Results Among study subjects in CR1 203/261 (78%) of evaluable patients attained CR after the first induction cycle (chemoresponsive AML) and 128/220 (58%) had NK-AML. Within both subgroups, analysis of efficacy by age at diagnosis implied preferred benefit of HDC/IL-2 vs. control in patients 40-59 years old (Figure 1) with independent efficacy in patients 40-49 and 50-59 years old (Table 1). The treatment arm was not significantly superior over the control arm for LFS or OS in the youngest patients (18-39 years old) or in patients >60 years old (P>0.2, logrank test, for all comparisons (LFS and OS)) within subgroups of chemosensitive AML or NK-AML. Conclusions We speculate that the observed benefit of HDC/IL-2 in patients responding swiftly to initial chemotherapy may be explained by more efficient immune-mediated elimination of leukemic cells at a lower burden of leukemia. The preferential efficacy of HDC/IL-2 in NK-AML points towards efficacy in subgroups of leukemic mutations that are less common in the youngest group of patients. The reason for the lack of significant efficacy in patients >60 years old, despite achievement of CR1 after the initial chemotherapy and despite having NK-AML, remains unknown but may be explained by fading anti-leukemic immunity in old patients. Overall, these findings may be considered for the selection of patients suitable for immunotherapy with HDC/IL-2 in an emerging landscape of strategies for relapse prevention in AML.

IL-27 Promotes Neutrophil Extracellular Trap Generation in Sickle Cell Disease

Introduction: Neutrophil Extracellular Traps (NETs) are neutrophil-derived damage-associated molecular patterns (DAMPs) composed of decondensed chromatin decorated with citrullinated histones and neutrophil proteases. Recently, we have shown that caspase-4-dependent activation (cleavage) of the pore-forming protein, Gasdermin-D (GSDMD) in neutrophils promotes NETs generation leading to lung injury in SCD. IL-27, an anti-inflammatory cytokine with some pro-inflammatory properties is significantly elevated in the plasma of SCD patients, however, it remains debatable whether IL-27 signaling in neutrophils serves to promote or inhibit NETs generation in SCD. Methods: Citrated blood samples from SCD or race matched control humans were incubated with saline, hemin (20 mM), or hemin (20 mM) + recombinant human IL-27 (200 ng/mL) for 15 minutes at room temperature. Platelet-poor-plasma from the three treatment groups were incubated with sytox green and fluorescent Abs against neutrophil elastase (NE) and histones for in situ staining of extracellular DNA, NE and histones, respectively. Imaging flow cytometry was used to detect circulating NETs (cNETs) as 1-2 mm fragments triple-positive for extracellular DNA, NE, and histones. Results: Incubation with hemin led to significant increase in cNETs concentration in SCD but not control human blood, which was further increased following incubation of SCD human blood with hemin in presence of IL-27. Conclusion: Our current findings suggest that IL-27 signaling promotes NETs generation by neutrophils in SCD patient blood. Currently, studies are underway to identify the molecular mechanism underlying IL-27-dependent NETs generation in SCD.

Conditioning Impacts on Regulatory T-Cells (Tregs) in the Microenvironment of AML: Observations from the Phase 1 Cynk-001-AML-001 Trial

Introduction: Evidence shows that NK cells can exhibit potent anti-tumor activity against acute myeloid leukemia (AML). CYNK-001 is a CD56+CD3- enriched, off-the-shelf, allogeneic natural killer (NK) cell therapy expanded from placental CD34 cells. The use of recombinant IL-2 (rhIL-2) therapy has induced regression of tumors by stimulating the activation and proliferation of NK cells. Low dose RhIL-2 has been shown to selectively expand and enhance the expression of maturation markers on the surface of NK cells, making them more potent killers in vitro and in vivo. CYNK-001-AML-001 is a Phase I multi-dose study evaluating the safety, tolerability, and persistence of CYNK-001 with or without rhIL-2 in adults with primary or secondary acute myeloid leukemia (AML) in morphologic complete remission (CR) with minimal residual disease (MRD) or relapsed/refractory (R/R) AML Aim: To determine the safety and tolerability of rhIL-2 when added to CYNK-001 and assess the impact of rhIL-2 on CYNK-001 activity and persistence. Methods This Phase I, single-arm, dose escalation clinical trial (NCT04310592) enrolled patients 18-80 years with R/R AML or MRD-positive CR. Escalating doses of CYNK-001 cells were administered following a standard 3+3 design, with 2 separate arms (MRD+ and R/R AML). Cohort 4a enrolled MRD+ AML subjects, while cohorts 4b and 5b enrolled R/R AML subjects. Cohort 4a and 4b utilized a dose of 1.8 billion CYNK-001 x 3 doses of CYNK-001 without rhIL-2. Cohort 5b utilized same dose of CYNK-001 along with rhIL-2. The decision to administer rhIL-2 in cohorts 6a, 6b, 7a and 7b would be based on review of safety, efficacy, and translational data in Cohorts 5b. In cohort 5b rhIL-2 was administered at a dose of 6M IU on days 0,2,4,7,9,11 and 14. Cohort 4b was used as a comparator. Results Five patients in cohort 5b received rhIL-2. Treatment was well tolerated with no dose limiting toxicities (DLT's) being observed. Ten adverse events ≥ grade 3 were experienced. The commonest grade 3 AE's were raised AST (2/10) and ALT (2/10). Two grade 4 AE's were reported including febrile neutropenia and a pre-existing GVHD. In cohort 4b, 3 out of 6 patients achieved an objective response (3x MLFS) at day 28. No patient in cohort 5b achieved a response. While there was an apparent higher baseline blast count in cohort 5b which may have contributed to the differences, translational data demonstrated a surge in Tregs without enhancement of T helper or cytotoxic cells or NK cell reconstitution. In cohort 4a, one subject who received rhIL-2 (6MIU/dose) in error showed an increase in Tregs similar to cohort 5b. Conclusion Treatment with CYNK-001 in combination with rhIL-2 was well tolerated with no dose-limiting toxicities observed. However, this approach does not enhance CD4 helper T cells, CD8 cytotoxic cells or NK cell reconstitution, while stimulated proliferation of Tregs. CYNK-001 proliferation and persistence was not enhanced with rhIL-2 infusion. Based on these data, rIL-2 was excluded from subsequent cohorts in this study.

An “Off-the-Shelf” CD2 Universal CAR-T Therapy Combined with a Long-Acting IL-7 for T-Cell Malignancies

INTRODUCTION: T-cell malignancies are associated with frequent relapse and high morbidity in children and adults, partly due to the lack of effective or targeted treatment options. Despite the use of multidrug chemotherapy, <50% of adults and <75% of children with T-ALL survive longer than five years. While CAR-T cell therapy is remarkable in treating B-cell malignancies, its use in T-cell malignancies is restricted due to fratricide, risk of immunodeficiency, and immune escape. METHODS: To broaden the use of CAR-T cells in pan T-cell malignancies, we developed an allogenic “universal” CD2-targeting CAR-T (UCART2), in which both CD2 and the T-cell receptor alpha subunit are both deleted by CRISPR/Cas9 editing to prevent both fratricide and GvHD. In vitro and in vivo efficacy of UCART2, alone or in combination with rhIL-7-hyFc (efineptakin alfa), a long-acting IL-7, was evaluated using CD2+ HH CBR-GFP T-ALL cell line and primary CD2+ T-ALL PDXs. To determine the impact of CD2 antigen loss on CAR-T cell function, CRISPR-mediated CD2 KO was performed in CD19 targeting CAR-T cells (UCART19ΔCD2). Single-cell secretome analyses on target-stimulated UCART19 and UCART19ΔCD2 were performed using the Isolight platform (IsoPlexis). The in vivo efficacy of UCART19 and UCART19ΔCD2 was evaluated in a CD19+ NALM6 GFP-CBR xenograft model. RESULTS: UCART2 demonstrated efficacy against T-ALL and CTCL in vitro and prolonged the survival of tumor-engrafted NSG mice in vivo. While CD2 deletion in UCART19 had little impact on in vitro killing efficacy, single-cell secretome analysis revealed that CD2 deletion in UCART19 reduced frequencies of the effector cytokines, including Granzyme-B and IFN-γ. We also observed that UCART19ΔCD2 (CD2-) had reduced anti-tumor efficacy compared to UCART19 (CD2+) in vivo in a CD19+ NALM6 GFP-CBR xenograft model. Of note, the reduced efficacy resulting from CD2 deletion was reversed when combined with rhIL-7-hyFc, a long-acting recombinant human interleukin-7. Treatment with rhIL-7-hyFc enhanced UCART2 expansion and persistence in vivo(Figure 1A) resulting in increased survival of NSG mice engrafted with both the primary patient T-ALL model (Figure 1B) and in a tumor re-challenge model in vivo. CONCLUSIONS: We have developed an allogenic “universal” CD2-targeting CAR-T cell UCART2, which is effective against T-ALL and CTCL/T-NHL. We found that CD2 deletion in CAR-T cells resulted in reduced secretion of effector cytokines and reduced anti-tumor activity in vivo, indicating that CD2 is critical for CAR-T function. However, when combined with rhIL-7-hyFc (efineptakin alfa), a long-acting recombinant IL-7, UCART2 induced durable complete responses in both primary and tumor rechallenge experiments in vivo. Our data suggest that UCART2, in combination with rhIL-7-hyFc, could be a suitable approach for treating T-cell malignancies.

TRPM2-L Participates in the Interleukin-6 Pathway to Enhance Tumor Growth in Prostate Cancer by Hypoxia-Inducible Factor-1α.

Interleukin-6 (IL-6) can promote cell proliferation in prostate cancer (PCa). Full-length transient receptor potential melastatin 2 (TRPM2-L) is highly expressed in PCa. However, the association between IL-6 and TRPM2-L in PCa is unclear. Here, human PCa cell lines, PC-3 and DU-145, were treated with 10 μg/mL tocilizumab, an IL-6 receptor (IL-6R) inhibitor, and the TRPM2-L protein expression in cells was significantly decreased. Cells were stably transfected with TRPM2 short-interfering RNA (siRNA) and cell survival clearly declined. Recombinant IL-6 treatment weakened the effects of TRPM2-siRNA on cell survival. TRPM2-L binds directly to IL-6R in PC-3 and DU-145 cells. The protein expression of hypoxia-inducible factor-1α was suppressed by reduction with TRPM2-L in PC-3 and DU-145 cells. Human umbilical vein endothelial cells (HUVECs) were indirectly cocultured with PCa cells, and the invasion and angiogenic activity of HUVECs were enhanced after coculture with PCa cells. However, TRPM2-L reduction in PCa cells significantly decreased the invasion and angiogenic activity of HUVECs compared to the control coculture. In vivo, xenograft tumors were induced using PC-3 cells. Tocilizumab treatment or TRPM2-L reduction clearly suppressed tumor growth. Meanwhile, the injection of mouse recombinant IL-6 weakened the antitumor effects of TRPM2-L reduction. These data demonstrate that the IL-6/TRPM2-L axis in PCa tumor growth is important, and interference of the IL-6/TRPM2-L axis may be a novel approach for PCa therapy.

Airway epithelial cells and macrophages trigger IL-6-CD95/CD95L axis and mediate initial immunopathology of COVID-19

Airway epithelial cells (AEC) infected with SARS-CoV-2 may drive the dysfunction of macrophages during COVID-19. We hypothesized that the direct interaction of AEC with macrophages mediated by CD95/CD95L or indirect interaction mediated by IL-6 signaling are key steps for the COVID-19 severe acute inflammation. The interaction of macrophages with apoptotic and infected AEC increased CD95 and CD163 expression, and induced macrophage death. Macrophages exposed to tracheal aspirate with high IL-6 levels from intubated patients with COVID-19 or to recombinant human IL-6 exhibited decreased HLA-DR expression, increased CD95 and CD163 expression and IL-1β production. IL-6 effects on macrophages were prevented by both CD95/CD95L antagonist and by IL-6 receptor antagonist and IL-6 or CD95 deficient mice showed significant reduction of acute pulmonary inflammation post-infection. Our findings show a non-canonical CD95L-CD95 pathway that simultaneously drives both macrophage activation and dysfunction and point to CD95/CD95L axis as therapeutic target.

Exogenous IL-25 ameliorates airway neutrophilia via suppressing macrophage M1 polarization and the expression of IL-12 and IL-23 in asthma

BackgroundSevere asthma is associated with substantial mortality and has unmet therapeutic need. A subset of severe asthma is characterized by neutrophilic airway inflammation. Classically activated (or M1) macrophages which express IL-12 and IL-23 are associated with airway neutrophilia in asthma. Exogenous IL-25 was reported to suppress intestinal inflammation in animal models of inflammatory bowel diseases via suppressing IL-12 and IL-23 production. We hypothesize that IL-25 ameliorates airway neutrophilia via inhibiting macrophage M1 polarization and the expression of IL-12 and IL-23 in asthma.MethodsIn a mouse model of neutrophil-dominant allergic airway inflammation, the effect of mouse recombinant IL-25 on airway inflammation were assessed by H&E staining and bronchoalveolar lavage (BAL) cell counting. The percentage of M1 macrophages in lung tissue and BAL cells were analyzed by flow cytometry. Quantitative PCR and immunostaining were performed to measure the expression of Il12, Il23, and inflammatory cytokines. Mechanistic experiments were performed in primary culture of macrophages from mouse lungs. The expression of IL-12, IL-23 and IL-25 in sputum was analyzed in a cohort of severe asthma and subjects with eosinophilic or non-eosinophilic asthma.ResultsIntranasal administration of IL-25 markedly decreased the number of neutrophils in BAL cells in a murine model of neutrophil-dominant allergic airway inflammation. Moreover, exogenous IL-25 decreased the number of M1 macrophages, and reduced the expression of IL-12, IL-23 in the lungs of the mouse model. Exogenous IL-25 also inhibited the expression of inflammatory cytokines IL-1β, IFN-γ, TNF-α and IL-17 A. In vitro, IL-25 suppressed IL-12 and IL-23 expression in lipopolysaccharide (LPS)-stimulated primary culture of mouse pulmonary macrophages. Mechanistically, IL-25 inhibited LPS-induced c-Rel translocation to nucleus via STAT3-dependent signaling. In a cohort of severe asthma, IL-25 protein levels in sputum were significantly lower than control subjects. The transcript levels of IL-12 and IL-23 were increased whereas IL-25 transcripts were decreased in sputum cells from subjects with non-eosinophilic asthma compared to eosinophilic asthma.ConclusionsIL-25 expression is downregulated in subjects with severe or non-eosinophilic asthma. Exogenous IL-25 ameliorates airway neutrophilia, at least in part, via inhibiting macrophage M1 polarization and the expression of IL-12 and IL-23.

DMOG protects against murine IL-33-induced pulmonary type 2 inflammation through HIF-1 pathway in innate lymphoid cells

One of the traditional methods of treating allergy is to avoid the allergen, protocol that has long been used in high altitude clinics. It has been hypothesized that the therapeutic effect of high altitude on allergy is due to allergen avoidance, exposure to sunlight and reduced stress. However, the contribution of environmental elements like low oxygen pressure and hypoxia remains underexplored. In this study, we examined the role of hypoxia in the development of type 2 lung inflammation. Mice were administered with papain or recombinant IL-33 intra-nasally to induce type 2 lung inflammation. Some of them were treated additionally with the prolyl hydroxylase (PHD) inhibitor DMOG, which mimics hypoxia. DMOG treatment exhibited an inhibitory effect on the lung inflammation induced by papain or IL-33, operating in a manner independent of T and B cells. The anti-inflammatory effect of DMOG was accompanied by a downregulation of IL-5 and IL-13 in innate lymphoid cells (ILCs), which was abolished in HIF-1α deficient mice. Collectively, our findings suggest that DMOG's modulatory effect on IL-5 and IL-13 operates through the HIF-1 pathway, resulting in a reduction in type 2 lung inflammation. These findings underscore the role of the PHD-HIF pathway in IL-5 and IL-13 expression in lung ILCs and pharmacological inhibition of PHD might be a novel therapeutic candidate for type 2 lung inflammation.

Anakinra authorized to treat severe coronavirus disease 2019; Sepsis breakthrough or time to reflect?

The European Medicines Agency (EMA) and the United States Food and Drug Administration (FDA) announced conditions for using recombinant human interleukin-1 receptor antagonist (rhIL-1ra) to treat hospitalized patients with Coronavirus disease 2019 (COVID-19) and risk for progression. These decisions followed publication of the suPAR-guided Anakinra treatment for Validation of the risk and early Management OF seveRE respiratory failure by COVID-19 (SAVE- MORE) phase 3 clinical trial that yielded positive results. We conducted a literature review and theoretical analysis of IL-1 blockade as a therapy to treat COVID-19. Using a stepwise analysis, we assessed clinical applicability of the SAVE-MORE results and evaluated conceptual support for interleukin-1 suppression as a suitable approach to COVID-19 treatment. This therapeutic approach was then examined as an example of inflammation-suppressing measures used to treat sepsis. Anakinra use as a COVID-19 therapy seems to rely on a view of pathogenesis that incorrectly reflects human disease. Since COVID-19 is an example of sepsis, COVID-19 benefit due to anti-inflammatory therapy contradicts an extensive history of unsuccessful clinical study. Repurposing rhIL-1ra to treat COVID-19 appears to exemplify a cycle followed by inflammation-suppressing sepsis treatments. A landscape of treatment failures is interrupted by a successful clinical trial. However, subsequent confirmatory study fails to replicate the positive data. We suggest further experimentation is not a promising pathway to discover game-changing sepsis therapies. A different kind of approach may be necessary.

Proinflammatory IFNγ Is Produced by but Not Required for the Generation of Eomes+ Thymic Innate CD8 T Cells.

Innate CD8 T cells are proinflammatory effector T cells that achieve functional maturation in the thymus prior to their export into and maturation in peripheral tissues. Innate CD8 T cells produce the Th1 cytokine IFNγ but depend on the Th2 cytokine IL-4 for their generation. Thus, innate CD8 T cells can permute the intrathymic cytokine milieu by consuming a Th2 cytokine but driving a Th1 cytokine response. The cellular source of IL-4 is the NKT2 subset of invariant NKT (iNKT) cells. Consequently, NKT2 deficiency results in the lack of innate CD8 T cells. Whether NKT2 is the only iNKT subset and whether IL-4 is the only cytokine required for innate CD8 T cell generation, however, remains unclear. Here, we employed a mouse model of NKT1 deficiency, which is achieved by overexpression of the cytokine receptor IL-2Rβ, and assessed the role of other iNKT subsets and cytokines in innate CD8 T cell differentiation. Because IL-2Rβ-transgenic mice failed to generate both NKT1 and innate CD8 T cells, we postulated an in vivo requirement for IFNγ-producing NKT1 cells for innate CD8 T cell development. In-depth analyses of IL-2Rβ-transgenic mice and IFNγ-deficient mice, however, demonstrated that neither NKT1 nor IFNγ was required to induce Eomes or to drive innate CD8 T cell generation. Instead, in vivo administration of recombinant IL-4 sufficed to restore the development of innate CD8 T cells in NKT1-deficient mice, affirming that intrathymic IL-4, and not IFNγ, is the limiting factor and key regulator of innate CD8 T cell generation in the thymus.

Anakinra and hepatotoxicity in pediatric rheumatology: a case series

BackgroundAnakinra is a recombinant interleukin-1 (IL-1) receptor antagonist used in systemic juvenile idiopathic arthritis (sJIA), refractory Kawasaki disease (KD) and cryopyrin-associated autoinflammatory syndrome (CAPS). Anakinra associated hepatotoxicity, while rare, has been described in several cases in daily practice. ​In this case series the authors describe three pediatric patients with this side effect in the setting of severe macrophage activation syndrome (MAS) in KD and sJIA.Case presentationThe first patient was a 12-year-old boy who presented with fever, maculo-papular exanthema and polyarthralgia. Tonsillitis, distal limb induration and tender cervical lymph nodes were observed. Erythrocyte-sedimentation rate (ESR), C-reactive protein (CRP), ferritin (11,975 ng/mL), D-dimers (5,98 mg/L FEU) and soluble CD25 (3645 pg/mL) levels were elevated. Exclusion of sepsis / toxic shock syndrome warranted introduction of IV methylprednisolone and immunoglobulin (IG IV), with partial response. A MAS secondary to KD was assumed, and anakinra 2 mg/kg/day was introduced. Twenty days later he developed new-onset nausea and severe cyto-cholestasis, normalizing after 2 months of drug discontinuation. Posterior onset of polyarthritis and evanescent lead to a final diagnosis of sJIA. The second patient was a 2-year-old boy with a 10-day history of fevers, generalized rash, hepatosplenomegaly and strawberry tongue. Leucocytosis with neutrophilia and elevated CRP were observed. Initial treatment with IVIG in the setting of incomplete KD was ineffective. Mild anaemia, leukopenia and very high serum ferritin (maximum 26,128 ng/mL) ensued. Presumptive sJIA associated MAS was treated with IV methylprednisolone and anakinra 2 mg/kg/day, with prompt response. Four weeks later transaminitis was detected, and temporary anakinra suspension led to normalisation of laboratorial values. The third case related to a 4-year-old boy presenting with fever, maculopapular rash and cervical lymphadenopathy. CRP and ESR were elevated, and KD was diagnosed. IVIG and methylprednisolone were initiated with clinical worsening, warranting for anakinra introduction at 2 mg/kg/day. After three weeks, liver enzymes progressively elevated, resolving on 2 weeks of anakinra discontinuation.ConclusionsTo the best of our knowledge, this is the first case series describing anakinra associated hepatotoxicity in pediatric patients with rheumatic diseases other than sJIA, bringing additional insight to therapeutic monitoring in patients undergoing this treatment.

Production of recombinant human long-acting IL-18 binding protein: inhibitory effect on ulcerative colitis in mice.

Interleukin-18 binding protein (IL-18BP) is a natural IL-18 inhibitor in vivo, which can effectively neutralize IL-18 and inhibit the inflammatory signaling pathway induced by IL-18, thus playing an anti-inflammatory role. Traditional production methods primarily rely on eukaryotic animal cell expression systems, which often entail complex processes, lower yields, and increase production costs. In this study, we present a novel approach for expressing IL-18BP fusion protein using the Escherichia coli (E. coli) system. The N-terminal segment of IL-18BP was fused with the small ubiquitin-related modifier (SUMO) tag, enabling soluble expression, while the C-terminal segment was fused with the human IgG1 Fc fragment to prolong its in vivo lifespan. Through screening, we obtained a high-expression engineering strain from a single colony and developed optimized protocols for fermentation and purification of the recombinant SUMO-IL-18BP-Fc protein. The SUMO tag was subsequently cleaved using SUMO protease, and the purified recombinant human IL-18BP-Fc (rhIL-18BP-Fc) exhibited a purity exceeding 90% with a yield of 1 g per liter of bacterial solution. The biological activities and underlying mechanisms of rhIL-18BP-Fc were evaluated using cell lines and a mouse model. Our results demonstrated that rhIL-18BP-Fc effectively inhibited IL-18-stimulated IFN-γ production in KG-1a cells in vitro and ameliorated DSS-induced ulcerative colitis in mice. In conclusion, we successfully employed the SUMO fusion system to achieve high-level production, soluble expression, and prolonged activity of rhIL-18BP-Fc in E. coli. These findings lay the groundwork for future large-scale industrial production and pharmaceutical development of rhIL-18BP-Fc protein. KEY POINTS: • Effective expression, fermentation, and purification of bioactive rhIL-18BP-Fc protein in E. coli. • The rhIL-18BP-Fc protein has a great potential for the therapy of ulcerative colitis by inhibiting the expression of inflammatory factors.