Interleukin-6 Receptor Blockade Research Articles

Interleukin 1β triggers synaptic and memory deficits in Herpes simplex virus type-1-infected mice by downregulating the expression of synaptic plasticity-related genes via the epigenetic MeCP2/HDAC4 complex

Extensive research provides evidence that neuroinflammation underlies numerous brain disorders. However, the molecular mechanisms by which inflammatory mediators determine synaptic and cognitive dysfunction occurring in neurodegenerative diseases (e.g., Alzheimer’s disease) are far from being fully understood. Here we investigated the role of interleukin 1β (IL-1β), and the molecular cascade downstream the activation of its receptor, to the synaptic dysfunction occurring in the mouse model of multiple Herpes simplex virus type-1 (HSV-1) reactivations within the brain. These mice are characterized by neuroinflammation and memory deficits associated with a progressive accumulation of neurodegenerative hallmarks (e.g., amyloid-β protein and tau hyperphosphorylation). Here we show that mice undergone two HSV-1 reactivations in the brain exhibited increased levels of IL-1β along with significant alterations of: (1) cognitive performances; (2) hippocampal long-term potentiation; (3) expression synaptic-related genes and pre- and post-synaptic proteins; (4) dendritic spine density and morphology. These effects correlated with activation of the epigenetic repressor MeCP2 that, in association with HDAC4, affected the expression of synaptic plasticity-related genes. Specifically, in response to HSV-1 infection, HDAC4 accumulated in the nucleus and promoted MeCP2 SUMOylation that is a post-translational modification critically affecting the repressive activity of MeCP2. The blockade of IL-1 receptors by the specific antagonist Anakinra prevented the MeCP2 increase and the consequent downregulation of gene expression along with rescuing structural and functional indices of neurodegeneration. Collectively, our findings provide novel mechanistic evidence on the role played by HSV-1-activated IL-1β signaling pathways in synaptic deficits leading to cognitive impairment.

Serum rescues surgical manipulation induced intestinal barrier permeability greater than interleukin 1 receptor antagonist conditioned serum in large animal model

Surgical manipulation induced intestinal barrier permeability has been previously demonstrated in rodent models and may prove crucial in the development of postoperative complications such as postoperative ileus and sepsis. However, the observation of this phenomena in a large animal model, and further understanding of mechanisms as well as potential therapeutic approaches has yet to be fully explored. The enteric glial cell population may prove to be a critical participant as they sense the intestinal environment and communicate rapidly with surrounding cell populations. Blockade of glial IL-1 receptor has proven successful in preventing postoperative ileus, a possible sequela of barrier permeability, in a rodent model. Therefore, we hypothesized that a large animal model would demonstrate surgical manipulation-induced intestinal barrier permeability, which could be prevented by luminal injection of interleukin 1 receptor antagonist conditioned serum (IRAP). Ischemia was first induced in the distal jejunum of 6–8-week-old pigs to mimic the disease process that would require intestinal surgery. Jejunum proximal and distal to the ischemic loops was then manipulated by “milking” the intestinal contents and applying friction to the serosa with sterile gauze with and without the injection of 1ml of nonconditioned porcine serum or IRAP into the proximal or distal loop. Forty-five minutes after manipulation, the tissues were collected for immunofluorescence histology and mounted on Ussing chambers to measure transepithelial electrical resistance (TEER) which directly corresponds to mucosal barrier resistance. TEER values for the manipulated proximal intestine were significantly more permeable than control tissue (p<0.0001). In addition, preliminary fluorescent quantification has indicated an increase in glial marker, glial fibrillary acidic protein, in the manipulated tissue (p =0.0499). TEER values for manipulated distal tissue trended towards increased permeability compared to control tissue (p=0.1995). Addition of IRAP significantly increased the resistance of the proximal loop compared to manipulation alone (p=0.0003). Interestingly, addition of control porcine serum significantly increased the resistance of both the proximal and distal loops compared to manipulation alone, and to a greater extent than IRAP (p= <0.0001 and 0.0008 respectively).While the effects of IRAP on interleukin 1 signaling may play a role in prevention of surgical manipulation induced intestinal barrier permeability, it appears than non-conditioned serum may offer therapeutic benefits. Ongoing studies will focus on determining the mechanism of this therapeutic effect on the intestinal epithelia and glia population. NIH K01 OD 028207, NIH-NICHD R01 HD095876 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Macrophage secretory IL-1β promotes docetaxel resistance in head and neck squamous carcinoma via SOD2/CAT-ICAM1 signaling.

Docetaxel (DTX) combined with cisplatin and 5-fluorouracil has been used as induction chemotherapy for head and neck squamous cell carcinoma (HNSCC). However, the development of acquired resistance remains a major obstacle to treatment response. Tumor-associated macrophages are associated with chemotherapeutic resistance. In the present study, increased infiltration of macrophages into the tumor microenvironment (TME) was significantly associated with shorter overall survival and increased resistance to chemotherapeutic drugs, particularly DTX, in patients with HNSCC. Macrophage coculture induced expression of intercellular adhesion molecule 1 (ICAM1), which promotes stemness and the formation of polyploid giant cancer cells, thereby reducing the efficacy of DTX. Both genetic silencing and pharmacological inhibition of ICAM1 sensitized HNSCC to DTX. Macrophage secretion of IL-1β was found to induce tumor expression of ICAM1. IL-1β neutralization and IL-1 receptor blockade reversed DTX resistance induced by macrophage coculture. IL-1β activated superoxide dismutase 2 and inhibited catalase, thereby modulating intracellular levels of ROS and inducing ICAM1 expression. Arsenic trioxide (ATO) reduced macrophage infiltration into the TME and impaired IL-1β secretion by macrophages. The combinatorial use of ATO enhanced the in vivo efficacy of DTX in a mouse model, which may provide a revolutionary approach to overcoming acquired therapeutic resistance in HNSCC.

Addition of interleukin-6 receptor blockade to carfilzomib-based desensitization in a highly sensitized nonhuman primate model

Sensitized patients, those who had prior exposure to foreign human leukocyte antigens, are transplanted at lower rates due to challenges in finding suitable organs. Desensitization strategies have permitted highly sensitized patients to undergo kidney transplantation, albeit with higher rates of rejection. This study assesses targeting plasma cell and interleukin (IL)-6 receptor for desensitization in a sensitized nonhuman primate kidney transplantation model. All animals were sensitized using two sequential skin transplants from maximally major histocompatibility complex-mismatched donors. Carfilzomib (CFZ)/tocilizumab (TCZ) desensitization (N=6) successfully decreased donor-specific antibody (DSA) titers and prevented the expansion of B cells compared to CFZ monotherapy (N=3). Dual desensitization further delayed, but did not prevent humoral rebound, as evidenced by a delayed increase in post-kidney transplant DSA titers. Accordingly, CFZ/TCZ desensitization conferred a significant survival advantage over CFZ monotherapy. A trend toward increased T follicular helper cells was also observed in the dual therapy group along the same timeline as an increase in DSA and subsequent graft loss. Cytomegalovirus reactivation also occurred in the CFZ/TCZ group but was prevented with ganciclovir prophylaxis. In accordance with prior studies of CFZ-based dual desensitization strategies, the addition of IL-6 receptor blockade resulted in desensitization with further suppression of posttransplant humoral response compared to CFZ monotherapy.

M1 macrophages evoke an increase in polymeric immunoglobulin receptor (PIGR) expression in MDA-MB468 breast cancer cells through secretion of interleukin-1β

High expression of polymeric immunoglobulin receptor (PIGR) in breast cancer is associated with increased 5-year survival rate. However, the factors influencing PIGR expression in breast cancer have not been elucidated. The aim of this study was to determine the role of macrophages and cytokines affecting expression of PIGR in two breast cancer cell lines. M1, M2 macrophage conditioned media (CM) and recombinant human cytokines were used to determine factors which increased PIGR expression in MCF7 (HTB-22) and MDA-MB468 (HTB-132) breast cancer cell lines. The level of PIGR expression in the cells and PIGR secretory component were evaluated by real-time quantitative polymerase chain reaction and Western blotting. M1 macrophage CM induced a dose-dependent increase in PIGR mRNA expression in MDA-MB468 cells, up to 20-fold. The level of PIGR expression in MCF7 cells was very low and not affected by M1 and M2 CM. Interferon gamma (IFN-γ) and interleukin (IL)-1β also increased PIGR expression in MDA-MB468 and MCF7 cells. However, IL-1β was demonstrated to increase in M1 macrophages, while IFN-γ was not. The role of IL-1β secreted from M1 macrophages in increasing expression of PIGR was confirmed by IL-1 receptor blockade, indicating that IL-1β was the major M1 macrophage-derived cytokine that enhanced PIGR expression. Elevated PIGR expression in breast cancer in vivo may reflect the polarization state of tumor-associated immune cells.

A real-world study of interleukin-6 receptor blockade in patients with neuromyelitis optica spectrum disorder.

Neuromyelitis optica spectrum disorder (NMOSD) is a relapsing autoimmune disease that can cause permanent neurological disabilities. However, the interleukin-6 (IL-6) signaling pathway is a promising therapeutic target for relapse prevention. Therefore, this study evaluated the long-term effectiveness of tocilizumab, a humanized anti-IL-6 receptor antibody, for NMOSD. We enrolled 65 patients with NMOSD who received regular intravenous administration of tocilizumab (8mg/kg) between October 2017 and January 2022. Then, we retrospectively collected data on the clinical characteristics and baseline glial fibrillary acidic protein (GFAP) and neurofilament light chain levels. The primary outcome was the annualized relapse rate (ARR). Risk factors were assessed using a multivariable logistic regression model. During the median follow-up of 34.1 (interquartile range: 25.5-39.3) months, 23% (15/65) of patients relapsed during tocilizumab treatment, but the median ARR decreased from 1.9 (range 0.12-6.29) to 0.1 (range 0-1.43, p < 0.0001). A prolonged infusion interval (> 4weeks, odds ratio [OR]: 10.7, 95% confidence interval [CI]: 1.6-71.4, p = 0.014) and a baseline plasma GFAP level of > 220pg/mL (OR: 20.6, 95% CI 3.3-129.4, p = 0.001) were risk factors for future relapses. During treatment, the median Expanded Disability Status Scale score significantly decreased in aquaporin-4 antibody-positive and -negative patients, but the pain did not considerably improve. There were no severe safety concerns. Tocilizumab treatment significantly reduced the relapse rate in patients with NMOSD. However, prolonged infusion intervals and high baseline plasma GFAP levels may increase the relapse risk during tocilizumab therapy.

Abstract P3061: A Monoclonal Antibody Blocking Interleukin-1 Receptor Accessory Protein Reduces Acute Viral Myocarditis Severity

IL-1, IL-33 and IL-36 have complementary, pro-inflammatory roles during the immune response that promote autoimmune diseases, particularly myocarditis. While antibodies that neutralize circulating cytokines exist, we hypothesized that blockade of their shared co-receptor, IL-1 receptor accessory protein (IL1RAP), would exhibit a more potent and broader anti-inflammatory profile. The CAN10 antibody blocks IL1RAP and is currently under development for treating severe autoimmune and inflammatory diseases. To investigate if IL1RAP blockade is an effective treatment of cardiac inflammation, we induced autoimmune viral myocarditis in 8-week-old Balb/c male mice with Coxsackievirus B3 and treated them with mCAN10, an anti-mouse IL1RAP monoclonal antibody. Mice were euthanized at day 10, the peak of acute inflammation in the heart. IL1RAP blockade reduced the severity of myocarditis at this timepoint when compared to isotype, saline or IL-1 receptor antagonist (IL1RA) treatment (the gold standard in IL-1 receptor blockade), without affecting viral clearance from the heart. Spectral flow cytometry of heart immune populations revealed that mCAN10 significantly reduced the number of infiltrating leukocytes of broad populations, especially effector CD4 + and CD8 + T cells, inflammatory Ly6C + CCR2 + monocytes, neutrophils and eosinophils. We also observed early echocardiographical parameters suggesting improved heart function, including ejection fraction, fractional shortening and interventricular septum diameter. Interestingly, mCAN10-treated mice demonstrated reduced proliferation of bone marrow precursors of inflammatory myeloid and lymphoid lineage leukocytes. Splenic immune cell populations were not affected, indicating that mCAN10 does not cause global immune suppression in these animals. Altogether, our data show that a single monoclonal antibody targeting three inflammatory cytokine systems, IL-1, IL-33 and IL-36, potently reduces acute viral myocarditis severity in a broader fashion that is not recapitulated by IL-1α/β blockade, and shows translational promise for therapeutics of inflammatory disease. Phase I clinical studies of the CAN10 antibody are in preparation.

Effects of the Interleukin-6 Receptor Blocker Sarilumab on Metabolic Activity and Differentiation Capacity of Primary Human Osteoblasts.

Interleukin (IL-) 6 is a key factor in the inflammatory processes of rheumatoid arthritis. Several biologic agents target the IL-6 signaling pathway, including sarilumab, a monoclonal antibody that blocks the IL-6 receptor and inhibits IL-6-mediated cis- and trans-signaling. A careful analysis of the IL-6 signaling blockade should consider not only inflammatory processes but also the regenerative functions of IL-6. The purpose of this study was to investigate whether inhibition of the IL-6 receptors affects differentiation of human primary osteoblasts (hOB). The effects of sarilumab on viability and the differentiation capacity in unstimulated osteoblasts as well as after stimulation with various IL-6 and sIL6-R concentrations were determined. Sarilumab treatment alone did not affect the differentiation or induction of inflammatory processes in hOB. However, the significant induction of alkaline phosphatase activity which was observed after exogenous IL-6/sIL-6R costimulation at the highest concentrations was reduced back to baseline levels by the addition of sarilumab. The IL-6 receptor blockade also decreased gene expression of mediators required for osteogenesis and bone matrix maintenance. Our results demonstrate that concomitant administration of the IL-6 receptor blocker sarilumab can inhibit IL-6/sIL-6R-induced osteogenic differentiation.

Interleukin-6 Receptor Blockade can Increase the Risk of Nonalcoholic Fatty Liver Disease: Indications From Mendelian Randomization.

Background: Interleukin-6 receptor (IL-6R) blockade has been approved for inflammation-associated diseases and whether it is effective in treating non-alcoholic fatty liver disease (NAFLD) is still unknown. Methods: A target-based Mendelian randomization was performed to appraise whether inhibiting the IL-6 signaling pathway via IL-6R blockade can reduce the risk of NAFLD. The previously established genetic proxy SNP rs2228145 was mainly used to appraise the therapeutic effects and the genetic-predicted circulating IL-6 level was treated as the exposure with ∼30,000 samples. The genetic association between SNP rs2228145 (A > C) and NAFLD was obtained from non-FinnGen GWAS (1,483 cases and 17,781controls) and FinnGen GWAS (894 cases and 217,898 controls). The causal effects were estimated using a Wald ratio method and were combined using a fixed-effects meta-analysis. Furthermore, the SNP rs12048091 was employed as another proxy in the sensitivity analysis. Results: The positive control analysis suggested the SNP rs2228145 can mimic the effects of IL-6R blockade where inhibiting IL-6 signaling can reduce the risk of rheumatoid arthritis [OR = 0.68 (0.58, 0.80)] and coronary heart disease [OR = 0.75 (0.68, 0.84)]. This Mendelian randomization analysis suggested that IL-6R blockade can adversely increase the risk of NAFLD in the non-FinnGen GWAS [OR = 1.99 (1.27, 3.13)] while not significant in the FinnGen consortium. The fixed-effects meta-analysis indicated inhibiting the IL-6 signaling pathway can reduce the risk of NAFLD [OR = 1.80 (1.26, 2.57)]. When including SNP rs12048091 as the genetic instrument, the meta-analysis using two genetic variants also indicated a similar effect on NAFLD [OR = 1.83 (1.32, 2.53)]. There was no heterogeneity in the whole analysis. Conclusion: Our Mendelian randomization suggested inhibiting the IL-6 signaling pathway via IL-6R blockade might increase the risk of NAFLD, suggesting IL-6R should play a protective role in NAFLD.

Resolvin E1 attenuates doxorubicin-induced endothelial senescence by modulating NLRP3 inflammasome activation

Endothelial cell senescence contributes to chronic inflammation and endothelial dysfunction, while favoring cardiovascular disorders and frailty. Senescent cells acquire a pro-inflammatory secretory phenotype that further propagates inflammation and senescence to neighboring cells. Cell senescence can be provoked by plethora of stressors, including inflammatory molecules and chemotherapeutic drugs. Doxorubicin (Doxo) is a powerful anthracycline anticancer drug whose clinical application is constrained by a dose-limiting cardiovascular toxicity. We here investigated whether cell senescence can contribute to the vascular damage elicited by Doxo. In human umbilical vein endothelial cells (HUVEC) cultures, Doxo (10–100 nM) increased the number of SA-β-gal positive cells and the levels of γH2AX, p21 and p53, used as markers of senescence. Moreover, we identified Doxo-induced senescence to be mediated by the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome, a key player of the immune innate system capable of releasing interleukin (IL)-1β. In fact, IL-1β itself mimicked the stimulatory action of Doxo on both NLRP3 activation and cellular senescence, while the pharmacological blockade of IL-1 receptors markedly attenuated the pro-senescence effects of Doxo. In search of additional pharmacological strategies to attenuate Doxo-induced endothelial senescence, we identified resolvin E1 (RvE1), an endogenous pro-resolving mediator, as capable of reducing cell senescence induced by both Doxo and IL-1β by interfering with the increased expression of pP65, NLRP3, and pro-IL-1β proteins and with the formation of active NLRP3 inflammasome complexes. Overall, RvE1 and the blockade of the NLRP3 inflammasome-IL-1β axis may offer a novel therapeutic approach against Doxo-induced cardiovascular toxicity and subsequent sequelae.

Amino Acid Metabolism and Protein Turnover in Lean and Obese Humans During Exercise-Effect of IL-6 Receptor Blockade.

Interleukin-6 (IL-6) is implicated in skeletal muscle wasting and in regulating skeletal muscle hypertrophy in the healthy state. This work aimed to determine the role of IL-6 in regulating systemic protein and amino acid metabolism during rest, exercise, and recovery in lean and obese humans. In a nonrandomized, single-blind design, 12 lean and 9 obese individuals were infused first with 0.9% saline (Saline), secondly with the IL-6 receptor antibody tocilizumab (Acute IL-6R ab), and 21 days later with saline while still under tocilizumab influence (Chronic IL-6R ab). Outcome measures were determined before, during, and after 90 minutes of exercise at 40% Wattmax by isotope dilution technique, using primed continuous infusion of L-[ring-D5]phenylalanine and L-[D2]tyrosine. Main outcomes measures included systemic protein turnover and plasma amino acid concentrations. We saw no effect of acute or chronic IL-6 receptor blockade on protein turnover. In lean individuals, chronic IL-6 receptor blockade increased plasma concentrations of total amino acids (rest Δ + 186 μmol/L; 95% CI, 40-332; recovery Δ + 201 μmol/L; 95% CI, 55-347) and essential amino acids (rest Δ + 43 μmol/L; 95% CI, 12-76; recovery Δ + 45 μmol/L; 95% CI, 13-77) independently of exercise but had no such effect in obese individuals (total amino acids rest Δ + 63 μmol/L; 95% CI, -170 to 295, recovery Δ - 23 μmol/L, 95% CI, -256 to 210; essential amino acids rest Δ + 26 μmol/L; 95% CI, -21 to 73, recovery Δ + 11 μmol/L; 95% CI, -36 to 58). IL-6 receptor blockade has no effect on protein turnover in fasting lean and obese humans during rest, exercise, and recovery. Chronic IL-6 receptor blockade increases total and essential amino acid concentrations only in lean individuals.

Host‐directed anti‐mycobacterial activity of colchicine, an anti‐gout drug, via strengthened host innate resistance reinforced by the IL‐1β/PGE2 axis

To diversify and expand possible tuberculosis (TB) drug candidates and maximize limited global resources, we investigated the effect of colchicine, an FDA-approved anti-gout drug, against Mycobacterium tuberculosis (Mtb) infection because of its immune-modulating effects. We evaluated the intracellular anti-Mtb activity of different concentrations of colchicine in murine bone marrow-derived macrophages (BMDMs). To elucidate the underlying mechanism, RNA sequencing, biological and chemical inhibition assays, and Western blot, quantitative real-time PCR, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical analyses were employed. Finally, type I interferon-dependent highly TB-susceptible A/J mice were challenged with virulent Mtb H37Rv, and the host-directed therapeutic effect of oral colchicine administration on bacterial burdens and lung inflammation was assessed 30 days post-infection (2.5mg·kg-1 every 2days). Colchicine reinforced the anti-Mtb activity of BMDMs without affecting cell viability, indicating that colchicine facilitated macrophage immune activation upon Mtb infection. The results from RNA sequencing, NLRP3 knockout BMDM, IL-1 receptor blockade, and immunohistochemistry analyses revealed that this unexpected intracellular anti-Mtb activity of colchicine was mediated through NLRP3-dependent IL-1β signalling and Cox-2-regulated PGE2 production in macrophages. Consequently, the TB-susceptible A/J mouse model showed remarkable protection, with decreased bacterial loads in both the lungs and spleens of oral colchicine-treated mice, with significantly elevated Cox-2 expression at infection sites. The repurposing of colchicine against Mtb infection in this study highlights its unique function in macrophages upon Mtb infection and its novel potential use in treating TB as host-directed or adjunctive therapy.

Use of Contrast-Enhanced Ultrasound Sonography in Giant Cell Arteritis: A Proof-of-Concept Study.

C-Reactive protein and erythrocyte sedimentation rate are crucial parameters used to monitor giant cell arteritis (GCA). Given that tocilizumab is approved for the treatment of GCA, these parameters are less sensitive because of the effects of interleukin-6 receptor blockade. Thus, the optimal method for monitoring GCA patients undergoing tocilizumab therapy, especially patients exhibiting a persistent thickened vessel wall in large vessels, remains unclear. Contrast-enhanced ultrasonography (CEUS) can increase the visibility of tissue perfusion by slow blood flow, which cannot be detected by power color doppler. We used CEUS to investigate patients with active and inactive GCA of the large vessels (active large vessel arteritis [aLVV]/inactive large vessel arteritis [iLVV]) who were not administered tocilizumab in this proof-of-concept study. After injection of the ultrasound contrast agent, the contrasted area (CA) of large vessels in a transverse section was calculated twice: first when the lumen was contrasted completely and once again 4-8 s later. We investigated the value of increase in CA that exhibited the best sensitivity and specificity for aLVV. Twenty-four patients were included in this study: 15 with aLVV and 9 with iLVV. The CA increased from 32.2 ± 16.8 to 52.5 ± 21.3 mm2 (p < 0.0001) in aLVV. The mean CA remained unchanged in iLVV. The best cutoff value to differentiate between aLVV and iLVV was a ≥25% increase in CA with a sensitivity and specificity of 91.7% and 100%, respectively. Our study indicates that CEUS can detect aLVV with high sensitivity and specificity. Incorporation of CEUS into routine clinical practice might result in a good method for monitoring disease activity in LVV in GCA patients. The limitation of our study was the small number of patients and the lack of investigator blinding to clinical data.

169TiP Tocilizumab, ipilimumab, and nivolumab for the treatment of advanced melanoma, non-small cell lung cancer, or urothelial carcinoma

169TiP Tocilizumab, ipilimumab, and nivolumab for the treatment of advanced melanoma, non-small cell lung cancer, or urothelial carcinoma

IL-6 receptor blockade does not slow β cell loss in new-onset type 1 diabetes.

BackgroundIL-6 receptor (IL-6R) signaling drives development of T cell populations important to type 1 diabetes pathogenesis. We evaluated whether blockade of IL-6R with monoclonal antibody tocilizumab would slow loss of residual β cell function in newly diagnosed type 1 diabetes patients.MethodsWe conducted a multicenter, randomized, placebo-controlled, double-blind trial with tocilizumab in new-onset type 1 diabetes. Participants were screened within 100 days of diagnosis. Eligible participants were randomized 2:1 to receive 7 monthly doses of tocilizumab or placebo. The primary outcome was the change from screening in the mean AUC of C-peptide collected during the first 2 hours of a mixed meal tolerance test at week 52 in pediatric participants (ages 6–17 years).ResultsThere was no statistical difference in the primary outcome between tocilizumab and placebo. Immunophenotyping showed reductions in downstream signaling of the IL-6R in T cells but no changes in CD4 memory subsets, Th17 cells, Tregs, or CD4+ T effector cell resistance to Treg suppression. A DC subset decreased during therapy but regressed to baseline once therapy stopped. Tocilizumab was well tolerated.ConclusionTocilizumab reduced T cell IL-6R signaling but did not modulate CD4+ T cell phenotypes or slow loss of residual β cell function in newly diagnosed individuals with type 1 diabetes.Trial RegistrationClinicalTrials.gov NCT02293837.FundingNIH National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and National Institute of Allergy and Infectious Diseases (NIAID) UM1AI109565, UL1TR000004 from NIH/National Center for Research Resources (NCRR) Clinical and Translational Science Award (CTSA), NIH/NIDDK P30DK036836, NIH/NIDDK U01DK103266, NIH/NIDDK U01DK103266, 1UL1TR000064 from NIH/NCRR CTSA, NIH/National Center for Advancing Translational Sciences (NCATS) UL1TR001878, UL1TR002537 from NIH/CTSA; National Health and Medical Research Council Practitioner Fellowship (APP1136735), NIH/NIDDK U01-DK085476, NIH/CTSA UL1-TR002494, Indiana Clinical and Translational Science Institute Award UL1TR002529, Vanderbilt Institute for Clinical and Translational Research UL1TR000445. NIH/NCATS UL1TR003142, NIH/CTSA program UL1-TR002494, Veteran Affairs Administration, and 1R01AI132774.

Early Cytomegalovirus Reactivation after Allogenic Bone Marrow Transplantation Is Associated with the Loss of Recipient-Derived Humoral Immunity and Is Reduced By IL-6 Inhibition

Background:Interleukin-6 (IL-6) promotes the differentiation of pathogenic T cells and represents a dominant pathway of cytokine-dysregulation and graft-versus-host disease (GVHD). Cytomegalovirus (CMV) reactivation is a common and life-threatening infectious complication following allogenic bone marrow transplantation (BMT). The role of T cell immunity is long-established, and our group recently demonstrated that CMV strain-specific immunoglobulins (Ig) are critical in preventing CMV reactivation in preclinical models (Martins JP et al, Science 2019).Methods and Results:In a phase I/II clinical trial (Kennedy GA et al, Lancet Onc. 2014), we observed unexpectedly low rates of CMV reactivation following IL-6 receptor (IL-6R) blockade with tocilizumab (TCZ) compared to historical control (8/35 vs 20/43, P < 0.05). We used our preclinical models of murine CMV (MCMV) reactivation to pursue the mechanisms. We transplanted bone marrow (BM) and T cells from naïve B6 donors into CMV-latently infected B6D2F1 recipients and monitored MCMV reactivation. Ablation of IL-6R in donor T cells (using B6 CD4 cre x IL-6R fl/fl transgenic mice; IL-6R -/-) significantly reduced MCMV reactivation 5 weeks after BMT, as measured by plasma viremia and infectious viral loads in the liver (Fig 1A).Next, we examined the contribution of donor T and B cells to the suppression of MCMV reactivation by IL-6R inhibition. At 2 - 3 weeks after BMT and prior to MCMV reactivation, cytokine (IFNɣ/TNF) secretion from CD4 + T cells (in response to MCMV infected DC) was not detectable; MCMV m38 tetramer + CD8 T cells were present at very low frequencies (< 0.1% of CD8 T cells) which were not altered by IL-6R ablation. The frequency of CXCR-5 +PD-1 + T FH cells was comparable or lower in IL-6R -/- vs wild-type (WT) T cells after BMT. Transplant of BM from B6.μMT mice (unable to generate mature B cells and plasma cells) did not increase MCMV viremia (Fig 1B), excluding a role for donor B cells. Thus, early MCMV reactivation in the presence of IL-6 is independent of MCMV-specific T cells and donor B cells.To study the effects of IL-6 on recipient-derived humoral immunity we quantified MCMV-specific IgG in plasma after BMT and found that levels were significantly higher in recipients of IL-6R -/- T cells (Fig 1C). Furthermore, MCMV-IgG levels in plasma correlated with MCMV viremia (r = - 0.72, P < 0.0001) and viral loads in liver (r = - 0.68, P < 0.0001). MCMV-IgG2a, which can only be generated by B6D2F1 recipients, was significantly higher in recipients of IL-6R -/- T cells, confirming that differences in humoral responses were of recipient-origin. To define relevant mechanisms, we examined the kinetics of IgG after BMT by monitoring the loss of murine IgG (transferred on day 0) in plasma (Fig 1D). Recipients of IL-6R -/- T cells showed significantly slower loss of IgG than recipients of WT T cells, with or without GVHD prophylaxis with cyclosporine (CSA). Thus, ablation of IL-6 signaling in donor T cells is associated with reduced loss of recipient IgG and protection from MCMV reactivation, an outcome which seems independent of the effects of IL-6 on GVHD.We correlated our findings with data from a recent randomized, placebo-controlled, double-blind phase III clinical trial of TCZ administration on Day -1 of BMT (Kennedy GA et al, Blood 2021). TCZ reduced CMV reactivation in at risk seropositive BMT recipients of volunteer unrelated donor grafts regardless of acute GVHD (all at risk recipients, TCZ vs. control: 12/26 vs 21/28, P = 0.03; at risk without grade 2-4 GVHD, TCZ vs control: 6/18 vs 9/13, P = 0.03). TCZ did not alter the frequency of B cells or the frequency and function of HCMV-specific CD8 + T cells, quantified by HCMV-pMHC tetramer-staining and HCMV peptide-specific cytokine (IFNγ/TNF) secretion. In contrast, levels of HCMV-specific IgG at day +30 in HCMV-seropositive recipients were significantly higher in the TCZ versus control group (Fig 1E). Consistent with our preclinical studies, the level of HCMV-IgG significantly and inversely correlated with early HCMV reactivation (within 35 days) after transplant.Conclusion:These data confirm the importance of recipient-derived humoral immunity in controlling early CMV reactivation in clinical BMT recipients. Critically, we demonstrate the ability of IL-6R blockade to maintain protective humoral responses until effective donor-derived adaptive immunity can be generated. [Display omitted] DisclosuresBoeckh: Merck: Consultancy, Research Funding; SymBio Pharmaceuticals: Consultancy; Helocyte: Consultancy; Evrys Bio: Consultancy; Moderna: Consultancy; Gilead: Consultancy, Research Funding; AlloVir: Consultancy; GSK: Consultancy. Hill: Generon corporation: Consultancy; NapaJun Pharma: Consultancy; Compass Therapeutics: Research Funding; Syndax Pharmaceuticals: Research Funding; Applied Molecular Transport: Research Funding; iTeos Therapeutics: Consultancy, Research Funding; Roche: Research Funding; Neoleukin Therapeutics: Consultancy.

Ascorbate Deficiency Is Associated with Severity of Cytokine Release Syndrome Following Therapy with Chimeric Antigen Receptor T-Cells

The therapy of hematologic malignancies has been revolutionized by the development of therapies using chimeric antigen receptor modified T-cells (CAR-T). However, CAR-T cell therapy is often associated with cytokine release syndrome (CRS), characterized by fevers, hypotension, and hypoxia, as well as immune effector cell-associated neurotoxicity syndrome (ICANS). Severe cases of CRS can result in significant morbidity and mortality. Although disease features such as tumor burden may predict for more severe CRS and ICANS, other clinical features and biomarkers predictive of CRS and ICANS remain lacking, with the exception of measurements of serum cytokines following cell infusion (PMID: 27076371, PMID: 24553386). Experimental models have implicated elaboration of inflammatory cytokines such as IL-1 and IL-6 by monocytes in the pathogenesis of CRS and ICANS (PMID: 29808007, PMID: 29808005). Accordingly, the severity and duration of CRS and ICANS can be mitigated in part by IL-6 receptor blockade with tocilizumab and treatment with corticosteroids such as dexamethasone. Recent work has implicated ascorbate in the regulation of the activity of TET enzymes in hematopoietic cells (PMID: 28825709, PMID: 28823558). Given that TET2 deficiency has been associated with increased elaboration of inflammatory cytokines such as IL-6 and IL-1 by macrophages (PMID 3026882, PMID: 28104796, PMID: 28636844), we reasoned that ascorbate deficiency might predict for more pronounced cytokine release in patients leading to more severe CRS or ICANS.We identified 13 patients receiving CAR-T cell therapy for hematologic malignancies at the University of Texas Southwestern. Plasma specimens were collected from patients at baseline prior to receipt of lymphodepleting chemotherapy and/or at two weeks following CAR-T cell infusion. Given the poor reliability of clinical ascorbate measurements due to oxidation, we used an optimized protocol incorporating a C13-labeled ascorbate internal standard to obtain highly precise serum measurements using liquid-chromatography mass spectrometry. The incidence and severity of CRS and ICANS was classified using standardized grading criteria as per the American Society for Transplantation and Cellular Therapy.We measured serum ascorbate in 7 baseline and 12 post CAR-T cell infusion specimens obtained from 13 patients, with a median age of 65 (range 53 to 77). The cohort included eight patients with diffuse large B-cell lymphoma and two patients with mantle cell lymphoma receiving CD19-targeted CAR-T cells, as well as three patients with multiple myeloma receiving BCMA-targeted CAR-T cells. Eight patients developed grade one CRS, three patients developed grade two CRS, and two patients did not develop CRS. One patient developed grade one ICANS, one developed grade two ICANS, and one developed grade three ICANS. Eight patients received dexamethasone for CRS or ICANS, and eight patients received tocilizumab. Five patients only received one dose of tocilizumab, while two received two doses and one received three doses.Taking all pre- and post-CAR-T cell infusion ascorbate measurements into account, a significant correlation was found between having low serum ascorbate levels and a higher maximal grade of CRS or ICANS (Figure 1A, r 2=-0.64, p=0.0039). Post-infusion ascorbate measurements also demonstrated a significant correlation between low serum ascorbate levels and higher maximal CRS or ICANS (Figure 1B, r 2=-0.78, p=0.0035), while there was no correlation between pre-infusion ascorbate measurements and CRS or ICANS. Finally, we noted a significant decrease in serum ascorbate levels when comparing pre-infusion to post-infusion specimens (Figure 1C, p=0.048), including five paired specimens. There was no significant correlation between serum ascorbate levels and the number of doses of tocilizumab or dexamethasone administered.Low serum ascorbate levels may be associated with an increased risk for developing severe CRS and ICANS following CAR-T cell therapy. Although follow-up studies with a larger cohort of patient are necessary to substantiate this correlation, these data provide preliminary evidence that serum ascorbate levels may serve as a useful biomarker to predict severity of CRS and ICANS. Furthermore, they suggest ascorbate supplementation as a promising future strategy to mitigate these common complications of CAR-T cell therapy. [Display omitted] DisclosuresKansagra: Alynylam, Celgene/BMS, Cota Health, GSK, Janssen, Karyopharm, Oncopeptide, Pfizer, Takeda, Sanofi: Membership on an entity's Board of Directors or advisory committees. Anderson: Celgene, BMS, Janssen, GSK, Karyopharm, Oncopeptides, Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Awan: Abbvie: Consultancy; Dava Oncology: Consultancy; Johnson and Johnson: Consultancy; Incyte: Consultancy; BMS: Consultancy; Astrazeneca: Consultancy; ADCT therapeutics: Consultancy; Pharmacyclics: Consultancy; Janssen: Consultancy; Beigene: Consultancy; Merck: Consultancy; Gilead sciences: Consultancy; Cardinal Health: Consultancy; Verastem: Consultancy; MEI Pharma: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Kite pharma: Consultancy; Genentech: Consultancy. Madanat: Stem line pharmaceutical: Honoraria; Blue Print Pharmaceutical: Honoraria; Onc Live: Honoraria; Geron Pharmaceutical: Consultancy. Patel: Agios: Membership on an entity's Board of Directors or advisory committees; PVI: Honoraria; Celgene-BMS: Membership on an entity's Board of Directors or advisory committees. Sweetenham: EMA Wellness: Membership on an entity's Board of Directors or advisory committees.

The IL-6R and Bmi-1 axis controls self-renewal and chemoresistance of head and neck cancer stem cells

Despite major progress in elucidating the pathobiology of head and neck squamous cell carcinoma (HNSCC), the high frequency of disease relapse correlates with unacceptably deficient patient survival. We previously showed that cancer stem-like cells (CSCs) drive tumorigenesis and progression of HNSCC. Although CSCs constitute only 2–5% of total tumor cells, CSCs contribute to tumor progression by virtue of their high tumorigenic potential and their resistance to chemo-, radio-, and immunotherapy. Not only are CSCs resistant to therapy, but cytotoxic agents actually enhance cancer stemness by activating transcription of pluripotency factors and by inducing expression of Bmi-1, a master regulator of stem cell self-renewal. We hypothesized therapeutic inhibition of interleukin-6 receptor (IL-6R) suppresses Bmi-1 to overcome intrinsic chemoresistance of CSCs. We observed that high Bmi-1 expression correlates with decreased (p = 0.04) recurrence-free survival time in HNSCC patients (n = 216). Blockade of IL-6R by lentiviral knockdown or pharmacologic inhibition with a humanized monoclonal antibody (Tocilizumab) is sufficient to inhibit Bmi-1 expression, secondary sphere formation, and to decrease the CSC fraction even in Cisplatin-resistant HNSCC cells. IL-6R inhibition with Tocilizumab abrogates Cisplatin-mediated increase in CSC fraction and induction of Bmi-1 in patient-derived xenograft (PDX) models of HNSCC. Notably, Tocilizumab inhibits Bmi-1 and suppresses growth of xenograft tumors generated with Cisplatin-resistant HNSCC cells. Altogether, these studies demonstrate that therapeutic blockade of IL-6R suppresses Bmi-1 function and inhibits cancer stemness. These results suggest therapeutic inhibition of IL-6R might be a viable strategy to overcome the CSC-mediated chemoresistance typically observed in HNSCC patients.

Blocking endogenous IL-6 impairs mobilization of free fatty acids during rest and exercise in lean and obese men

SummaryLack of interleukin-6 (IL-6) leads to expansion of adipose tissue mass in rodents and humans. The exact underlying mechanisms have not been identified. In this placebo-controlled, non-randomized, participant-blinded crossover study, we use the IL-6 receptor antibody tocilizumab to investigate the role of endogenous IL-6 in regulating systemic energy metabolism at rest and during exercise and recovery in lean and obese men using tracer dilution methodology. Tocilizumab reduces fatty acid appearance in the circulation under all conditions in lean and obese individuals, whereas lipolysis (the rate of glycerol appearance into the circulation) is mostly unaffected. The fact that fatty acid oxidation is unaffected by IL-6 receptor blockade suggests increased re-esterification of fatty acids. Glucose kinetics are unaffected. We find that blocking endogenous IL-6 signaling with tocilizumab impairs fat mobilization, which may contribute to expansion of adipose tissue mass and, thus, affect the health of individuals undergoing anti-IL-6 therapy (Clinicaltrials.gov: NCT03967691).

Exosomes from primed MSCs can educate monocytes as a cellular therapy for hematopoietic acute radiation syndrome

BackgroundAcute radiation syndrome (ARS) is caused by acute exposure to ionizing radiation that damages multiple organ systems but especially the bone marrow (BM). We have previously shown that human macrophages educated with exosomes from human BM-derived mesenchymal stromal cells (MSCs) primed with lipopolysaccharide (LPS) prolonged survival in a xenogeneic lethal ARS model. The purpose of this study was to determine if exosomes from LPS-primed MSCs could directly educate human monocytes (LPS-EEMos) for the treatment of ARS.MethodsHuman monocytes were educated by exosomes from LPS-primed MSCs and compared to monocytes educated by unprimed MSCs (EEMos) and uneducated monocytes to assess survival and clinical improvement in a xenogeneic mouse model of ARS. Changes in surface molecule expression of exosomes and monocytes after education were determined by flow cytometry, while gene expression was determined by qPCR. Irradiated human CD34+ hematopoietic stem cells (HSCs) were co-cultured with LPS-EEMos, EEMos, or uneducated monocytes to assess effects on HSC survival and proliferation.ResultsLPS priming of MSCs led to the production of exosomes with increased expression of CD9, CD29, CD44, CD146, and MCSP. LPS-EEMos showed increases in gene expression of IL-6, IL-10, IL-15, IDO, and FGF-2 as compared to EEMos generated from unprimed MSCs. Generation of LPS-EEMos induced a lower percentage of CD14+ monocyte subsets that were CD16+, CD73+, CD86+, or CD206+ but a higher percentage of PD-L1+ cells. LPS-EEMos infused 4 h after lethal irradiation significantly prolonged survival, reducing clinical scores and weight loss as compared to controls. Complete blood counts from LPS-EEMo-treated mice showed enhanced hematopoietic recovery post-nadir. IL-6 receptor blockade completely abrogated the radioprotective survival benefit of LPS-EEMos in vivo in female NSG mice, but only loss of hematopoietic recovery was noted in male NSG mice. PD-1 blockade had no effect on survival. Furthermore, LPS-EEMos also showed benefits in vivo when administered 24 h, but not 48 h, after lethal irradiation. Co-culture of unprimed EEMos or LPS-EEMos with irradiated human CD34+ HSCs led to increased CD34+ proliferation and survival, suggesting hematopoietic recovery may be seen clinically.ConclusionLPS-EEMos are a potential counter-measure for hematopoietic ARS, with a reduced biomanufacturing time that facilitates hematopoiesis.