Soluble IL-2 Research Articles

A biomarker-guided, prospective, phase 2 trial of pre-emptive graft-versus-host disease therapy using anti-thymocyte globulin

Background aimsIntensified immunosuppressive prophylaxis for graft-versus-host disease (GVHD) may be toxic and therefore warranted only in patients at high risk of developing GVHD. In patients who underwent allogeneic hematopoietic cell transplant at the authors’ center, high serum soluble IL-2 receptor alpha (sIL-2Rα) and low IL-15 levels on day 7 post-transplant were found to predict a high risk of developing clinically significant GVHD (sGVHD), defined as grade 2–4 acute GVHD or moderate to severe chronic GVHD. MethodsThis was a prospective, phase 2 trial in which high-risk patients (serum sIL-2Rα >4500 ng/L or IL-15 <31 ng/L) received rabbit anti-thymocyte globulin (ATG) 3 mg/kg on day 8 post-transplant. Controls consisted of patients who had their sIL-2Rα/IL-15 levels measured but did not participate in the trial. A total of 68 trial patients and 143 controls were accrued to this study. The primary endpoint was incidence of sGVHD. ResultsThere was a reduction in sGVHD in high-risk trial patients (received day 8 ATG) compared with high-risk controls (did not receive day 8 ATG) (sub-hazard ratio [SHR] = 0.48, P < 0.05). There was no significant difference between the groups in overall survival or relapse; however, there was a greater incidence of non-GVHD-associated non-relapse mortality in high-risk trial patients (SHR = 3.73, P < 0.05), mostly related to infections. This may be due in part to the biomarkers ineffectively stratifying GVHD risk. ConclusionsPre-emptive ATG therapy is both feasible and effective at reducing sGVHD without increasing relapse. Further mitigation strategies are needed to reduce the risk of infection associated with intensified GVHD prophylaxis. This study was registered at ClinicalTrials.gov (NCT01994824).

FDG PET/computed tomography can detect region-specific neuronal changes following antiretroviral therapy in HIV-infected patients.

FDG PET/computed tomography can detect region-specific neuronal changes following antiretroviral therapy in HIV-infected patients.

Cord Blood NK Cells Engineered to Express a Humanized CD123-Targeted Chimeric Antigen Receptor (CAR) and IL-15 As Off-the-Shelf Therapy for Acute Myeloid Leukemia

The outcomes of patients with AML remain disappointing, with only fewer than half of all adults (and <10% of adults 60 years or older) achieving long-term remissions. Thus, novel therapies are urgently needed. Chimeric antigen receptors (CARs) have been used extensively to redirect the specificity of T cells in patients with lymphoid malignancies. These infusions have been primarily restricted to the autologous setting, but generation of autologous products for each patient is logistically cumbersome and not scalable. A banked allogeneic product could overcome these limitations; but, allogeneic T-cells (even if HLA-matched) carry a major risk of graft-versus-host disease (GVHD). Natural killer (NK) cells provide an attractive alternative to T-cells for CAR therapy as they do not cause GVHD. Cord blood (CB) is a readily available “off-the-shelf” source of allogeneic NK cells that can be expanded to large, highly functional doses for CAR therapy.CD123 (IL3-receptor α chain) is a potential immunotherapeutic target in AML due to its overexpression on leukemic stem cells and AML blasts, rather than normal hematopoietic stem cells. Thus, we reprogrammed CB-NK cells to target CD123+ve tumor cells by retroviral transduction using synthetic constructs producing either murine-derived (iC9-2A-CD123CAR-CD28-CD3z-2A-IL15) or humanized (iC9-2A-hCD123CAR-CD28-CD3z-2A-IL15) scFv domains. We humanized the ScFv derived from murine antibody clone 26292 to avoid human anti-mouse antibody responses, which could potentially limit the persistence and efficacy of the CAR-NK cells when infused in patients. These constructs also express exogenous soluble IL-15 to support NK cellular survival and proliferation, as well as an inducible caspase-9 (iC9) suicide gene as a safety precaution to ablate CAR+ve NK cells if needed.Retroviral transduction with murine (CD123CAR) and humanized (hCD123CAR) (n=12) resulted in a median CAR positivity of 75% and 76%, respectively, 14 days post transduction and viability of >90%. Notably, CAR expression was stable over time. iC9/CAR.123/IL15+ and iC9/hCAR.123/IL15+ NK cells expanded for 2 weeks showed no signs of exhaustion, such as downregulation of eomesodermin and T-bet, or upregulation of KLRG1, and exhibited a mature phenotype, similar to ex vivo expanded non-transduced NK cells (NT-NK) using 22 markers. In a 4 hour 51Cr release assay, murine CD123CAR+ve and hCD123CAR+ve NK cells were equally effective at killing CD123+ve AML cell lines (THP-1 and MOLM-14), and exhibited significantly higher cytotoxicity when compared to NT-NK cells at all tested effector to target cell ratios (20:1; 10:1; 5:1 and 1:1). Notably, CAR123 and hCAR123-transduced NK cells were as equally efficient as NT-NK cells in killing CD123 negative targets such as K562 (positive control) and KARPAS (negative control), indicating that the enhanced killing of CD123+ targets by the transduced cells is mediated by the CAR receptor and not due to a non-specific enhancement in NK cytotoxicity. In addition, CD123CAR+ve and hCD123CAR+ve NK cells showed significantly higher production of IFN-γ, TNF-α, and CD107a degranulation in response to MOLM14 and THP in comparison to NT-NK cells. Indeed, using confocal microscopy, we showed significantly higher accumulation of CAR molecules at the immunologic synapse (IS) formed between CD123CAR+ve and hCD123CAR+ve CB-NK cells and CD123+ve targets compared to the diffuse presence of CARs at the IS with K562 target cells, indicating that CAR molecules participate in IS formation in a CD123-dependent manner.We validated elimination of iC9/CAR.123/IL-15 transduced NK via activation of iC9 in vitro. The addition of 10nM of the small molecule dimerizer AP1903 (Bellicum Pharmaceuticals, Inc) to cultures of iC9/CAR123/IL-15 or iC9/hCAR123/IL-15-transduced NK cells induced apoptosis of transduced NK cells within 4 hours but had no effect on the viability of NT-NK cells. Studies to test the in vivo anti-tumor efficacy of iC9/CAR123/IL-15 or iC9/hCAR123/IL-15-transduced NK cells in an NSG mouse model of MOLM14 are under way.In summary, our data demonstrate the capacity of CD123CAR+ve and hCD123CAR+ve CB-NK cells to kill CD123+ targets and support the use of CD123-specific CAR NK cells in the clinic for patients with relapsed AML as a means to decrease disease burden, possibly prior to consolidative therapies such as allogeneic transplantation. DisclosuresNo relevant conflicts of interest to declare.

Combination Biomarkers for Risk Stratification in Patients With Chronic Heart Failure Biomarkers Prognostication in HF

Current guidelines recommend measuring natriuretic peptide biomarkers to establish prognosis in patients with chronic heart failure with reduced ejection fraction (HFrEF). We assessed whether a combination biomarkers approach improve prognostication in patients with stable HFrEF. An observational cohort study recruited 202 patients with stable HFrEF at a single center, tertiary care hospital undergoing elective cardiac resynchronization therapy device placement from 2013 to 2015. Twenty-four biomarkers were analyzed individually and in combination using Cox proportion hazard regression model for major adverse cardiac events (ie, death, cardiac transplant, left ventricular assist device placement), and major adverse cardiac events plus HF hospitalizations. The single best biomarker for predicting major adverse cardiac events is peripheral mid-regional pro-adrenomedullin (C statistic = 0.771 ± 0.045) compared to current guideline recommended N-terminal pro b-type natriuretic peptide (C=0.668 ± 0.046). The best combined biomarkers for predicting major adverse cardiac events are blood urea nitrogen, coronary sinus C-reactive protein, peripheral mid-regional pro-atrial natriuretic peptide and peripheral soluble IL-1 receptor-like 1 (C = 0.767 ± 0.036). In this observational cohort, the combined biomarkers (blood urea nitrogen, C-reactive protein, mid-regional pro-atrial natriuretic peptide and soluble IL-1 receptor-like 1) or the single biomarker (mid-regional pro-adrenomedullin) was superior to N-terminal pro B-type natriuretic peptide, the current guideline recommended biomarker in predicting cardiovascular outcomes in patients with HFrEF. Larger studies are needed to validate these findings and examine whether single or combined biomarkers improve HFrEF prognostication.

Allogeneic Vδ1 gamma delta T cells engineered with glypican-3 (GPC3)-specific CAR expressing soluble IL-15 have enhanced antitumor efficacy against hepatocellular carcinoma in preclinical models.

e14511 Background: Autologous αβ chimeric antigen receptor (CAR) T cell therapy has shown promising clinical results in hematologic malignancies but limited success in solid tumors. Allogeneic αβ T cell therapy may overcome several challenges faced by autologous therapy but carries the risk of graft-versus-host disease (GvHD) and does not readily recognize multiple tumor-associated antigens. Gamma delta (γδ) T cells are highly cytolytic effectors that can recognize and kill tumor cells in an MHC-unrestricted manner without causing GvHD. The Vδ1 subset is preferentially localized in peripheral tissue and is critical for tumor immunosurveillance. Engineering Vδ1 T cells with CARs can further enhance antitumor activity and represents an attractive and safe approach to treating solid tumors. However, their clinical use has been hindered by the limited number of circulating Vδ1 T cells. Here, we describe the development of the first allogeneic Vδ1 T cells that have been expanded from healthy donor PBMCs and genetically modified to secrete IL-15 (sIL15) and express a CAR targeting glypican-3 (GPC3), a rational target for hepatocellular carcinoma (HCC). Methods: Vδ1 T cells in healthy donor PBMCs were activated by a Vδ1-specific monoclonal antibody and transduced with 41BBζ or 41BBζ-sIL15 GPC3-CARs prior to cell expansion, αβ T cell depletion and cryopreservation. In vitro characterization included: 1) co-culture assays with GPC3-expressing HCC targets HepG2 and PLC/PRF/5, 2) phenotypic analysis by flow cytometry, and 3) cytokine production by multiplexed immunoassay. For in vivo assessment of tumor control, immunodeficient NSG mice were subcutaneously injected with HepG2 cells and treated with a single dose of 41BBζ or 41BBζ-sIL15 GPC3-CAR Vδ1 T cells. Additionally, tissues were harvested 7 days post transfer and analyzed by flow cytometry for Vδ1 T cell tissue homing and proliferation, or at end of study and analyzed for GvHD by immunohistochemistry. Results: Vδ1 T cells expanded over 10,000-fold and routinely reached &gt;80% purity. Expanded Vδ1 T cells showed a primarily naïve-like phenotype (CD45RA+CD27+) with minimal exhaustion receptor expression and displayed robust proliferation, cytokine production, and cytotoxic activity against HCC cell lines expressing low and high GPC3 levels in vitro. In a HepG2 mouse model, GPC3-CAR Vδ1 T cells primarily accumulated and proliferated in the tumor, and a single dose was able to efficiently control tumor burden without causing GvHD. Importantly, 41BBζ-sIL15 GPC3-CAR Vδ1 cells displayed enhanced tumor-specific proliferation that resulted in better tumor control without any toxicity. Conclusions: Our results show that expanded Vδ1 T cells engineered with GPC3-CAR and sIL-15 represent a promising platform for safe and effective off-the-shelf treatment of HCC.

Activation of the acute inflammatory phase response in idiopathic nephrotic syndrome: association with clinicopathological phenotypes and with response to corticosteroids.

BackgroundData on the activation of the acute inflammatory response and its clinicopathological associations in idiopathic nephrotic syndrome (INS) are scarce and discordant.ObjectiveTo analyse the associations between the activation of the inflammatory response, the clinicopathological characteristics of disease and the response to treatment with steroids in patients with INS.MethodsA total of 101 patients with INS due to minimal change disease (MCD; n = 44), focal segmental glomerulosclerosis (FSGS; n = 33) and membranous nephropathy (MN; n = 24) and 50 healthy controls were included. At diagnosis, we measured the levels of haemopexin (Hx), haptoglobin (Hgl), interleukin-6 (IL-6), soluble urokinase-type plasminogen activator receptor (suPAR), tumour necrosis factor-α (TNF-α), soluble IL-1 receptor, interferon-γ and C-reactive protein. We analysed their clinicopathological associations. In MCD and FSGS patients, we determined the association between the levels of these variables and steroid resistance.ResultsThe levels of Hx, Hgl, TNF-α, suPAR and IL-6 were higher in patients with INS than in healthy controls, and were not associated with proteinuria, estimated glomerular filtration rate or serum albumin. In MCD and FSGS patients, Hx, Hgl, IL-6 and TNF-α levels were similar and significantly higher than in MN patients. In patients with MCD and FSGS, multivariate analyses identified FSGS and the levels of Hx, Hgl or IL-6 as independent predictors of steroid resistance.ConclusionsThe activation of the inflammatory response in patients with INS is heterogeneous and more prevalent in MCD or FSGS patients than in those with MN. In MCD and FSGS, elevated levels of Hx, Hgl or IL-6 are independently associated with steroid resistance.

Blocking IL-10 signaling with soluble IL-10 receptor restores in vitro specific lymphoproliferative response in dogs with leishmaniasis caused by Leishmania infantum.

rIL-10 plays a major role in restricting exaggerated inflammatory and immune responses, thus preventing tissue damage. However, the restriction of inflammatory and immune responses by IL-10 can also favor the development and/or persistence of chronic infections or neoplasms. Dogs that succumb to canine leishmaniasis (CanL) caused by L. infantum develop exhaustion of T lymphocytes and are unable to mount appropriate cellular immune responses to control the infection. These animals fail to mount specific lymphoproliferative responses and produce interferon gamma and TNF-alpha that would activate macrophages and promote destruction of intracellular parasites. Blocking IL-10 signaling may contribute to the treatment of CanL. In order to obtain a tool for this blockage, the present work endeavored to identify the canine casIL-10R1 amino acid sequence, generate a recombinant baculovirus chromosome encoding this molecule, which was expressed in insect cells and subsequently purified to obtain rcasIL-10R1. In addition, rcasIL-10R1 was able to bind to homologous IL-10 and block IL-10 signaling pathway, as well as to promote lymphoproliferation in dogs with leishmaniasis caused by L. infantum.

High levels of soluble CD25 in COVID-19 severity suggest a divergence between anti-viral and pro-inflammatory T-cell responses.

ObjectivesWe aimed to gain an understanding of the paradox of the immunity in COVID‐19 patients with T cells showing both functional defects and hyperactivation and enhanced proliferation.MethodsA total of 280 hospitalised patients with COVID‐19 were evaluated for cytokine profiles and clinical features including viral shedding. A mouse model of acute infection by lymphocytic choriomeningitis virus (LCMV) was applied to dissect the relationship between immunological, virological and pathological features. The results from the mouse model were validated by published data set of single‐cell RNA sequencing (scRNA‐seq) of immune cells in bronchoalveolar lavage fluid (BALF) of COVID‐19 patients.ResultsThe levels of soluble CD25 (sCD25), IL‐6, IL‐8, IL‐10 and TNF‐α were higher in severe COVID‐19 patients than non‐severe cases, but only sCD25 was identified as an independent risk factor for disease severity by multivariable binary logistic regression analysis and showed a positive association with the duration of viral shedding. In agreement with the clinical observation, LCMV‐infected mice with high levels of sCD25 demonstrated insufficient anti‐viral response and delayed viral clearance. The elevation of sCD25 in mice was mainly contributed by the expansion of CD25+CD8+ T cells that also expressed the highest level of PD‐1 with pro‐inflammatory potential. The counterpart human CD25+PD‐1+ T cells were expanded in BALF of COVID‐19 patients with severe disease compared to those with modest disease.ConclusionThese results suggest that high levels of sCD25 in COVID‐19 patients probably result from insufficient anti‐viral immunity and indicate an expansion of pro‐inflammatory T cells that contribute to disease severity.

Serologic markers of Epstein-Barr virus reactivation are associated with increased disease activity, inflammation, and interferon pathway activation in patients with systemic lupus erythematosus

SLE is a clinically heterogeneous disease characterized by an unpredictable relapsing-remitting disease course. Although the etiology and mechanisms of SLE flares remain elusive, Epstein-Barr virus (EBV) reactivation is implicated in SLE pathogenesis. This study examined the relationships between serological measures of EBV reactivation, disease activity, and interferon (IFN)-associated immune pathways in SLE patients. Sera from adult SLE patients (n = 175) and matched unaffected controls (n = 47) were collected and tested for antibodies against EBV-viral capsid antigen (EBV-VCA; IgG and IgA), EBV-early antigen (EBV-EA; IgG), cytomegalovirus (CMV; IgG), and herpes simplex virus (HSV-1; IgG). Serological evidence of EBV reactivation was more common in SLE patients compared to controls as demonstrated by seropositivity to EBV-EA IgG (39% vs 13%; p = 0.0011) and EBV-VCA IgA (37% vs 17%; p = 0.018). EBV-VCA, CMV1, and HSV-1 IgG seropositivity rates did not differ between SLE patients and controls. Furthermore, concentrations of EBV-VCA (IgG and IgA) and EBV-EA (IgG) were higher in SLE patients. SLE patients with high disease activity had increased concentrations of EBV-VCA IgA (mean ISR 1.34 vs. 0.97; p = 0.041) and EBV-EA IgG levels (mean ISR 1.38 vs. 0.90; p = 0.007) compared with those with lower disease activity. EBV reactivation was associated with enhanced levels of the IFN-associated molecule IP-10 (p < 0.001) and the soluble mediators BLyS (p < 0.001) and IL-10 (p = 0.0011). In addition, EBV-EA IgG responses were enriched in two previously defined patient clusters with robust expression of IFN and inflammatory or lymphoid and monocyte responses. Patients in these clusters were also more likely to have major organ involvement, such as renal disease. This study supports a possible role for EBV reactivation in SLE disease activity.

Monitoring neutrophil-to-lymphocyte ratio in patients with coronavirus disease 2019 receiving tocilizumab

Monitoring neutrophil-to-lymphocyte ratio in patients with coronavirus disease 2019 receiving tocilizumab

Monocytic myeloid-derived suppressor cells reflect tuberculosis severity and are influenced by cyclooxygenase-2 inhibitors.

Myeloid‐derived suppressor cells (MDSCs) increase in tuberculosis (TB) and may be targets for host‐directed therapy (HDT). In this study, we use flow cytometry to analyze the effects of cyclooxygenase‐2 inhibitors (COX‐2i) on monocytic (M)‐MDSCs in blood from TB patients attending a clinical trial of COX‐2i. The effects of COX‐2i on M‐MDSCs and mycobacterial uptake were also studied by an in vitro mycobacterial infection model. We found that M‐MDSC frequencies correlated with TB disease severity. Reduced M‐MDSC (P = 0.05) and IDO (P = 0.03) expression was observed in the COX‐2i group. We show that peripheral blood‐derived M‐MDSCs successfully internalized Mycobacterium bovis and that in vitro mycobacterial infection increased COX‐2 (P = 0.002), PD‐L1 (P = 0.01), and Arginase‐1 (P = 0.002) expression in M‐MDSCs. Soluble IL‐1β, IL‐10, and S100A9 were reduced in COX‐2i‐treated M‐MDSCs cultures (P < 0.05). We show novel data that COX‐2i had limited effect in vivo but reduced M‐MDSC cytokine production in vitro. The relevance of COX‐2i in a HDT strategy needs to be further explored.

Low Utility of the H-Score and HLH-2004 Criteria to Identify Patients with Secondary Hemophagocytic Lymphohistiocytosis after CAR-T Cell Therapy for Relapsed/Refractory Diffuse Large B-Cell Lymphoma

Introduction: Secondary hemophagocytic lymphohistiocytosis (HLH) is an aggressive life-threatening activation of the immune system triggered by an underlying condition. Use of chimeric antigen receptor therapy (CAR-T) to treat relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL) is associated with cytokine release syndrome (CRS), Immune Effector Cell Associated Neurotoxicity Syndrome (ICANS) and development of secondary HLH. Application of HLH scoring systems such as the H-score or HLH-2004 criteria to identify CAR-T triggered HLH is not validated in this setting. Inability to promptly recognize the development of secondary HLH in CAR-T patients and to distinguish it from CRS may lead to delay in HLH specific therapy and increased mortality. We analyzed the utility of applying the H-score and the HLH-2004 criteria to identify possible patients with HLH post-CAR-T for R/R DLBCL. Methods: A single center retrospective analysis of 75 patients with R/R DLBCL following CAR-T was performed. Using a peak ferritin of 500 mcg/L or higher within 30 days of CAR-T administration, 43 out of 75 patients were identified at risk for HLH. The H-score and HLH-2004 criteria were applied retrospectively. Measurement of NK activity was not available for any patients and soluble IL2 was collected intermittently. The mean H-score was calculated, and two sample t-test performed to evaluate for a difference in the mean H-score between low versus high grade CRS, low versus high grade ICANS, as well as presence versus absence of cytopenias at days 30, 90 and 180. Low grade CRS/ICANS was defined as 1, and high grade as 2 or higher. CRS was graded per Lee 2014 criteria and ICANS per CTCAEv.4. The 43 patients were then subdivided by H-score threshold of 169 into H-score Low (&amp;lt; 169) and H-score High (≥ 169). The threshold of 169 was used given a prior validation study showing optimal sensitivity and specificity for identifying acquired HLH in adult patients with an H-score equal to or higher than 169. Both Progression Free Survival (PFS) and Overall Survival (OS) were defined as time from CAR-T infusion until an event of last follow up. Kaplan-Meier curves were produced to describe the distribution of PFS and OS between two subpopulations of low and high H-Score. Results: Median peak ferritin was 1571.8 mcg/L (range: 375 mcg/L - &amp;gt;50,000 mcg/L, table 1). Median H-score was 135 (range 61 -250). Four patients were treated with HLH directed therapy receiving steroids with either tocilizumab, siltuximab and/or anakinra. Of these 4 patients, only 2 met five HLH-2004 criteria. All 4 patients had H-scores above 169 (209, 211, 228 and 250). Of these 4 patients, one died from complications thought secondary to HLH. Fourteen patients (14/43, 32%) had an H-score &amp;gt;169. Ten patients did not require any HLH directed therapy and had no clinical evidence of HLH. The mean H-score was not different between patients with low vs high grade CRS (148.2 vs 141.8, p=0.7) or low vs high grade ICANS (145.9 vs 142.6, p=0.8). No statistical correlation was seen between H-score and cytopenias at any time point. There was no significant difference in PFS or OS between the H-score low vs high subgroups (p=0.7, p=0.1 respectively, figure 1 and 2). Patients with higher H-score tended to have longer length of hospitalization for CAR-T (Pearson correlation coefficient 0.289). Conclusions: In patients with R/R DLBCL post CAR-T, the use of either the H-score or application of HLH-2004 criteria had low utility in identifying possible HLH in patients who screened in based on peak ferritin within 30 days of CAR-T administration. While apparent differences between the H-score high and low categories may not reach statistical significance due to the small number of patients, our exploratory analysis does not support the use of H-Score to evaluate for HLH post-CAR-T. The immediate post-CAR-T period is characterized by a high inflammatory state, which likely results in high H-scores. Results from our study suggest a need for further characterization of HLH following CAR-T and the role for a CAR-T specific HLH scoring system to distinguish secondary HLH from CAR-T related inflammation in this specific patient population. Disclosures Hardy: American Gene Technologies: Other: DSMB Member; Kite/Gilead: Other: Advisory Board Member; Incyte Corporation: Other: Advisory Board Member.

Prevention of Heme-Induced Human Endothelial Cell Activation By Hemopexin in Vitro

Hemoglobin (Hb) is one of the most abundant proteins in the human body. When red blood cells rupture, cell-free Hb may initiate adverse pathophysiological reactions. Pathophysiology triggered by cell-free Hb plays an important role in modifying the phenotype of sickle cell disease (SCD). SCD is caused by a single nucleotide mutation of the β-globin gene resulting in Hemoglobin-S (HbS) instead of the normal HbA found in healthy individuals. Polymerization of HbS shortens the lifespan of sickle red blood cells and promotes intra- and extravascular hemolysis. In cell-free Hb ferrous Hb (Fe2+) is oxidized into ferric Hb (Fe3+) promoting the dissociation and transfer of heme into lipid compartments where it triggers lipid peroxidation and generation of cytotoxic and pro-inflammatory reaction products. These processes promote endothelial cell activation and damage. The endogenous plasma protein hemopexin exhibits the highest binding affinity for heme and binds heme in a 1:1 binding ratio. Heme bound to hemopexin is rendered relatively non-reactive and is delivered safely to hepatocytes for endocytosis and degradation. To investigate the endothelial-protective function of hemopexin based on its ability to scavenge heme, we exposed human umbilical vein endothelial cells (HUVEC) in vitro to heme(NaOH) in the presence or absence of different hemopexin doses. As a read-out, different markers for endothelial cell activation were analyzed by either flow cytometry or multiplexed particle-based flow cytometry (Luminex). Briefly, confluent HUVEC were preincubated with hemopexin at different concentrations for 5 min before stimulation with heme(NaOH) for 25 min. Following stimulation cells were analyzed by flow cytometry for expression of membrane bound P-Selectin, a robust marker of endothelial cell activation. Alternatively, heme(NaOH) stimulation of hemopexin-preincubated HUVEC was conducted for 16 h and cell culture supernatants were analyzed by Luminex for three additional well-characterized plasma markers of endothelial cell activation: pro-inflammatory cytokine IL-8, cell adhesion molecule VCAM-1 and glycoprotein Von Willebrand factor (vWF). In the absence of hemopexin, heme(NaOH) consistently induced robust cell surface expression of P-Selectin and elevated levels of soluble IL-8, VCAM-1 and vWF. However, hemopexin completely blocked the stimulatory potential of heme as HUVEC exposed to pre-formed heme:hemopexin complexes showed unchanged P-Selectin expression levels compared to negative control samples. We found that hemopexin reduced heme(NaOH)-mediated P-selectin expression on HUVEC in a dose-dependent fashion. Once an equimolar ratio between heme and hemopexin was reached, P-selectin expression was abolished as shown in figure 1. In addition to P-Selectin, hemopexin also had a strong effect to reduce the heme-induced expression of IL-8, VCAM-1 and vWF to background levels. Thus, the presented data underlines on the one hand the stimulatory capacity of heme(NaOH) on endothelial cells and demonstrates on the other hand the potential of hemopexin to efficiently neutralize free heme. In a stoichiometric fashion, hemopexin potently prevents the pro-inflammatory effect of heme on endothelial cells. Hence, our study suggests a protective role of hemopexin for endothelial cells exposed to elevated levels of cell-free heme due to intravascular hemolysis. Additional experiments are required to elucidate the effect of hemopexin on the endothelium in more detail. Combined with our other lines of data, our results further support the investigation of hemopexin as a potential therapeutic agent in the treatment of sickle cell disease. Disclosures Adam: CSL Behring AG: Current Employment. Gentinetta:CSL Behring: Current Employment. Diditchenko:CSL Behring AG: Current Employment. Schaub:CSL Behring AG: Current Employment. Kato:CSL Behring AG: Current Employment. Brinkman:CSL Behring: Current Employment. Zuercher:CSL Behring AG: Current Employment.

Immune Function and Mechanism of Costimulating Molecules PD-1 and OX40 in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a T lymphocyte-mediated autoimmune disease, although its immune mechanism has not been fully studied. In this study, healthy controls (HC), osteoarthritis patients (OA), and RA patients were enrolled, and mice were evenly divided into control, collagen-induced arthritis (CIA), PD-1 Fc/CIA (PD-1 Fc membrane fusion protein administered to CIA mice), OX40 Fc/CIA (OX40 Fc membrane fusion protein administered to CIA mice), and PD-1 Fc + OX40 Fc/CIA groups. The expressions of programmed death-1 (PD-1) and OX40 in CD4+ T lymphocytes and the levels of sPD-1, immunoglobulin, and proinflammatory factors in patients and mice were measured. The results showed that the expression levels of PD-1 and OX40 in CD4+ T lymphocytes separated from the peripheral blood and synovial fluid of RA patients and the spleen of CIA mice were observably elevated. The levels of soluble PD-1, interleukin (IL)-2, IL-4, IL-5, IL-17, and interferon-γ (IFN-γ) in RA patients obviously increased. In animal experiments, PD-1 Fc not only increased the serum levels of immunoglobulin G (IgG), IgG1, and IgG2a in CIA mice, but also increased the levels of IL-4, IL-2, IL-5, IL-17, and IFN-γ in mouse spleen cells and joint tissues, which, however, were reversed by OX40 Fc. In conclusion, OX40 inhibition could reverse the progression of RA caused by PD-1 blocking, and PD-1 might be a potential target for RA. Clinical Trials.gov ID: HGH2018012203.

Patients with obstructive sleep apnea have suppressed levels of soluble cytokine receptors involved in neurodegenerative disease, but normal levels with airways therapy

PurposeObstructive sleep apnea (OSA) results in systemic intermittent hypoxia. By one model, hypoxic stress signaling in OSA patients alters the levels of inflammatory soluble cytokines TNF and IL6, damages the blood brain barrier, and activates microglial targeting of neuronal cell death to increase the risk of neurodegenerative disorders and other diseases. However, it is not yet clear if OSA significantly alters the levels of the soluble isoforms of TNF receptors TNFR1 and TNFR2 and IL6 receptor (IL6R) and co-receptor gp130, which have the potential to modulate TNF and IL6 signaling.MethodsPicogram per milliliter levels of the soluble isoforms of these four cytokine receptors were estimated in OSA patients, in OSA patients receiving airways therapy, and in healthy control subjects. Triplicate samples were examined using Bio-Plex fluorescent bead microfluidic technology. The statistical significance of cytokine data was estimated using the nonparametric Wilcoxon rank-sum test. The clustering of these high-dimensional data was visualized using t-distributed stochastic neighbor embedding (t-SNE).ResultsOSA patients had significant twofold to sevenfold reductions in the soluble serum isoforms of all four cytokine receptors, gp130, IL6R, TNFR1, and TNFR2, as compared with control individuals (p = 1.8 × 10−13 to 4 × 10−8). Relative to untreated OSA patients, airways therapy of OSA patients had significantly higher levels of gp130 (p = 2.8 × 10−13), IL6R (p = 1.1 × 10−9), TNFR1 (p = 2.5 × 10−10), and TNFR2 (p = 5.7 × 10−9), levels indistinguishable from controls (p = 0.29 to 0.95). The data for most airway-treated patients clustered with healthy controls, but the data for a few airway-treated patients clustered with apneic patients.ConclusionsPatients with OSA have aberrantly low levels of four soluble cytokine receptors associated with neurodegenerative disease, gp130, IL6R, TNFR1, and TNFR2. Most OSA patients receiving airways therapy have receptor levels indistinguishable from healthy controls, suggesting a chronic intermittent hypoxia may be one of the factors contributing to low receptor levels in untreated OSA patients.

Increased Circulating Levels of Inflammatory Markers in A Cohort of Adults with Youth-Onset Type 2 Diabetes Mellitus

Background: Young-onset type 2 diabetes (T2DMY) is considered to have a more aggressive disease phenotype, unfavourable long-term outcomes, and a close association with low-grade chronic inflammation. The aim of this study was to explore the circulating inflammatory profile in a cohort of adults with T2DMY and assess its potential associations. Methods: T2DMY adults (n=65) and non-diabetic controls (n=40) underwent detailed anthropometric and biochemical characterisation. Serum levels of six pro-inflammatory biomarkers (soluble E-selectin, IL-1β, IFN-γ, IL-6, IL-8 and angiopoietin like protein-4) were measured by Magnetic Luminex Assays and were compared between study groups. Potential associations of these biomarkers with anthropometric/biochemical parameters and microvascular complications were also tested in the T2DMY group. Results: T2DMY patients had significantly higher circulating levels of all measured inflammatory markers compared to controls, even after adjusting for age and gender (E-selectin: 65.9 ± 4.5 vs. 37.9 ± 8.2 ng/ml; IFN-γ: 220.9 ± 7.4 vs. 160.6 ± 13.5 pg/ml; IL1-β: 50.1 ± 1.8 vs. 38.0 ± 3.3 pg/ml; IL-6: 17.5 ± 0.7 vs. 11.4 ± 1.1 pg/ml; IL-8: 18.1 ± 1.5 vs. 11.7 ± 2.4 pg/ ml; ANGPTL-4: 162.8 ± 9.8 vs. 109.0 ± 15.6 ng/ml; all p-values<0.05). Significant positive correlations were found between fat mass and IL-6 and ANGPTL-4; waist-to-hip ratio and E-selectin, IFN-γ, IL-1β and IL-6; and HbA1c and E-selectin, IFN-γ, IL-1β and IL-6. Within the T2DMY group, E-selectin was significantly elevated in patients with combined albuminuria and retinopathy compared with those without these complications (88.97 ± 43.59 vs 59.38 ± 25.28 ng/ml; p=0.003). Conclusions: T2DMY patients exhibit an adverse profile of key circulating pro-inflammatory biomarkers, which appears associated with worse anthropometric/biochemical parameters and microvascular complications. The role of these biomarkers as predictors of T2DMY and disease progression should be further elucidated.

The Biological Context of C-Reactive Protein as a Prognostic Marker in Renal Cell Carcinoma: Studies on the Acute Phase Cytokine Profile.

High serum levels of the acute phase protein C-reactive protein (CRP) are associated with an adverse prognosis in renal cancer. The acute phase reaction is cytokine-driven and includes a wide range of inflammatory mediators. This overall profile of the response depends on the inducing event and can also differ between patients. We investigated an extended acute phase cytokine profile for 97 renal cancer patients. Initial studies showed that the serum CRP levels had an expected prognostic association together with tumor size, stage, nuclear grading, and Leibovich score. Interleukin (IL)6 family cytokines, IL1 subfamily mediators, and tumor necrosis factor (TNF)α can all be drivers of the acute phase response. Initial studies suggested that serum IL33Rα (the soluble IL33 receptor α chain) levels were also associated with prognosis, although the impact of IL33Rα is dependent on the overall cytokine profile, including seven IL6 family members (IL6, IL6Rα, gp130, IL27, IL31, CNTF, and OSM), two IL1 subfamily members (IL1RA and IL33Rα), and TNFα. We identified a patient subset characterized by particularly high levels of IL6, IL33Rα, and TNFα alongside an adverse prognosis. Thus, the acute phase cytokine reaction differs between renal cancer patients, and differences in the acute phase cytokine profile are associated with prognosis.

TREM-1 and TREM-2 Expression on Blood Monocytes Could Help Predict Survival in High-Grade Glioma Patients.

Objective In recent years, the role of the modern inflammatory markers TREM-1 (triggering receptors expressed on myeloid cells) and HMGB1 (high mobility group box 1 protein) in tumorigenesis has begun to be studied. Their role in gliomas is not clear. The aim of our study was to find the role of inflammation in gliomas. Patients and Methods. In 63 adult patients with gliomas and 31 healthy controls, the expressions of TREM-1 and TREM-2 on CD14+ blood cells (method: flow cytometry) and the levels of soluble sTREM-1, HMGB1, IL-6, and IL-10 (Elisa tests) were analyzed. Results Cox proportional hazard analysis showed that a TREM-1/TREM-2 ratio was associated with reduced overall survival (HR = 1.001, P = 0.023). Patients with a TREM-1/TREM-2 ratio above 125 survived significantly shorter than patients with a TREM-1/TREM-2 ratio below 125. The percentage of CD14+ TREM-1+ cells was strongly associated with a plasma IL-6/IL-10 ratio (positively) and with IL-10 (negatively). Conversely, we found a higher percentage of CD14+ TREM-2+ monocytes in better surviving patients; these cells could downregulate the exaggerated inflammation and potentiate the phagocytosis in the tumor. The serum levels of HMGB1 negatively correlated with the percentage of CD14+ TREM-1+ cells and with the TREM-1/TREM-2 ratio. The positive correlation between the serum levels of a late proinflammatory cytokine HMGB1 with the percentage of TREM2+ CD14+ monocytes can be explained as an effort for suppression of systemic inflammation by anti-inflammatory acting CD14+ TREM-2+ cells. Conclusion We showed that the TREM-1/TREM-2 ratio (expression on the surface of blood monocytes) could help predict prognosis in patients with gliomas, especially in high-grade gliomas, and that systemic inflammation has an impact on the patient's overall survival. This is the first study that showed that TREM expression on monocytes in peripheral blood could help predict prognosis in patients with gliomas.

Acupuncture inhibited airway inflammation and group 2 innate lymphoid cells in the lung in an ovalbumin-induced murine asthma model.

Group 2 innate lymphoid cells (ILC2s) are known to serve important functions in the pathogenesis of allergic airway inflammation. Studies have shown that acupuncture has an anti-inflammatory effect in the airways. However, how acupuncture treatment affects innate immunity, especially with regard to the function of ILC2s in ovalbumin (OVA)-induced allergic airway inflammation, is poorly understood. BALB/c mice were injected and subsequently challenged with OVA ± treated with manual acupuncture. At the end of the experimental course, lung function was assessed by measurement of airway resistance (RL) and lung dynamic compliance (Cdyn). Cytokine levels were detected by enzyme-linked immunosorbent assay (ELISA). ILC2 proportions in the lung were analyzed by flow cytometry. The results showed that airway inflammation and mucus secretion were significantly suppressed by acupuncture treatment. RL decreased while Cdyn increased after acupuncture treatment. There was an apparent decrease in the bronchoalveolar lavage fluid (BALF) concentrations of interleukin (IL)-5, IL-13, IL-9, IL-25 and IL-33 and an increase in soluble IL-33 receptor (sST2) levels compared with untreated asthmatic mice. Acupuncture also reduced the lin-CD45+KLRG1+ST2+ cell proportion in the lung. In conclusion, this study has demonstrated that acupuncture treatment alleviates allergic airway inflammation and inhibits pulmonary ILC2 influx and IL-5, IL-9 and IL-13 production. The inhibition of ILC2s by acupuncture may be associated with the IL-33/ST2-signaling pathway and IL-25 levels, thereby offering protection from the respiratory inflammation associated with asthma.

IL33 Is a Key Driver of Treatment Resistance of Cancer.

Recurrence and treatment resistance are major causes of cancer-associated death. There has been a growing interest in better understanding epithelial-mesenchymal transition, stemness of cancer cells, and exhaustion and dysfunction of the immune system for which numerous genomic, proteomic, microenvironmental, and immunologic mechanisms have been demonstrated. However, practical treatments for such patients have not yet been established. Here we identified IL33 as a key driver of polyploidy, followed by rapid proliferation after treatment. IL33 induction transformed tumor cells into polyploid giant cells, showing abnormal cell cycle without cell division accompanied by Snail deregulation and p53 inactivation; small progeny cells were generated in response to treatment stress. Simultaneously, soluble IL33 was released from tumor cells, leading to expansion of receptor ST2-expressing cells including IL17RB+GATA3+ cells, which promoted tumor progression and metastasis directly and indirectly via induction of immune exhaustion and dysfunction. Blocking IL33 with a specific mAb in murine IL33+ metastatic tumor models abrogated negative consequences and successfully elicited antitumor efficacy induced by other combined treatments. Ex vivo assays using tumor tissues and peripheral blood mononuclear cells of patients with cancer validated the clinical relevancy of these findings. Together, these data suggest that targeting the IL33-ST2 axis is a promising strategy for diagnosis and treatment of patients likely to be resistant to treatments in the clinical settings. SIGNIFICANCE: These findings indicate that the functional role of IL33 in cancer polyploidy contributes to intrinsic and extrinsic mechanisms underlying treatment failure.