Background: The recent failure of checkpoint-blockade therapies for glioblastoma multiforme (GBM) in late-phase clinical trials has directed interest towards adoptive cellular immunotherapies (ACT). In this open-label, first-in-human trial, we have assessed the safety and therapeutic potential of cytomegalovirus (CMV)-specific ACT in an adjuvant setting for patients with primary GBM, with an ultimate goal to prevent or delay recurrence and prolong overall survival. Methods: Twenty-eight patients with primary GBM were recruited to this prospective study, 25 of whom were treated with in vitro-expanded autologous CMV-specific T cells. Participants were monitored for safety, progression-free survival (PFS), overall survival (OS) and immune reconstitution. Results: No participants showed evidence of ACT-related toxicities. Of 25 evaluable participants, ten were alive at the completion of follow-up, while five were disease free. Reconstitution of CMV-specific T-cell immunity was evident and CMV-specific ACT may trigger bystander effect leading to additional T-cell responses to non-viral tumour-associated antigens through epitope spreading. Long-term follow-up of participants treated before recurrence showed significantly improved OS when compared to those who progressed before ACT (median 23 months, range 7–65 vs. median 14 months, range 5–19; p=0.018). Gene expression analysis of the ACT products indicated that a favourable T-cell gene signature was associated with improved long-term survival. Conclusion: Data presented in this study demonstrate that CMV-specific ACT can be safely used as an adjuvant therapy for primary GBM and, if offered before recurrence, this therapy may improve overall survival of GBM patients.Trial registration: anzctr.org.au: ACTRN12615000656538Funding Source:National Health & Medical Research Council (Australia) Trial registration: anzctr.org.au: ACTRN12615000656538 Funding Source: Philanthropic funding &National Health & Medical Research Council (Australia)
Corey Smith, Katie E. Lineburg, J. Paulo Martins, George Ambalathingal, Michelle A. Neller, Beth Morrison, Katherine K. Matthews, Sweera Rehan, Pauline Crooks, Archana Panikkar, Leone Beagley, Laetitia Le Texier, Sriganesh Srihari, David Walker, Rajiv Khanna
Although the control of bone-resorbing osteoclasts through osteocyte-derived RANKL is well defined, little is known about the regulation of osteoclasts by osteocyte death. Indeed, several skeletal diseases, such as bone fracture, osteonecrosis, and inflammation are characterized by excessive osteocyte death. Herein we show that osteoclasts sense damage-associated molecular patterns (DAMPs) released by necrotic osteocytes via macrophage-inducible C-type lectin (Mincle), which induced their differentiation and triggered bone loss. Osteoclasts showed robust Mincle expression upon exposure to necrotic osteocytes in vitro and in vivo. RNA sequencing and metabolic analyses demonstrated that Mincle activation triggers osteoclastogenesis via ITAM-based calcium signaling pathways, skewing osteoclast metabolism toward oxidative phosphorylation. Deletion of Mincle in vivo effectively blocked the activation of osteoclasts after induction of osteocyte death, improved fracture repair, and attenuated inflammation-mediated bone loss. Furthermore, in patients with osteonecrosis, Mincle was highly expressed at skeletal sites of osteocyte death and correlated with strong osteoclastic activity. Taken together, these data point to what we believe is a novel DAMP-mediated process that allows osteoclast activation and bone loss in the context of osteocyte death.
Darja Andreev, Mengdan Liu, Daniela Weidner, Katerina Kachler, Maria Faas, Anika Grüneboom, Ursula Schlötzer-Schrehardt, Luis E. Muñoz, Ulrike Steffen, Bettina Grötsch, Barbara Killy, Gerhard Krönke, Andreas M. Luebke, Andreas Niemeier, Falk Wehrhan, Roland Lang, Georg Schett, Aline Bozec
Emerging data indicate that complement and neutrophils contribute to the maladaptive immune response that fuels hyper-inflammation and thrombotic microangiopathy, thereby increasing COVID-19 mortality. Here, we investigated how complement interacts with the platelet/neutrophil extracellular traps (NETs)/thrombin axis, using COVID-19 specimens, cell-based inhibition studies and NETs/human aortic endothelial cell (HAEC) co-cultures. Increased plasma levels of NETs, tissue factor (TF) activity and sC5b-9 were detected in patients. Neutrophils of patients yielded high TF expression and released NETs carrying active TF. Treatment of control neutrophils with COVID-19 platelet-rich plasma generated TF-bearing NETs that induced thrombotic activity of HAEC. Thrombin or NETosis inhibition or C5aR1 blockade attenuated platelet-mediated NET-driven thrombogenicity. COVID-19 serum induced complement activation in vitro, consistent with high complement activity in clinical samples. Complement C3 inhibition with compstatin Cp40 disrupted TF expression in neutrophils. In conclusion, we provide a mechanistic basis for a pivotal role of complement and NETs in COVID-19 immunothrombosis. This study supports strategies against SARS-CoV-2 that exploit complement or NETosis inhibition.
Panagiotis Skendros, Alexandros Mitsios, Akrivi Chrysanthopoulou, Dimitrios C. Mastellos, Simeon Metallidis, Petros Rafailidis, Maria Ntinopoulou, Eleni Sertaridou, Victoria Tsironidou, Christina Tsigalou, Maria G. Tektonidou, Theocharis Konstantinidis, Charalampos Papagoras, Ioannis Mitroulis, Georgios Germanidis, John D. Lambris, Konstantinos Ritis
Although broadly protective, stem-targeted Abs against the influenza A virus hemagglutinin (HA) have been well studied, very limited information is available on Abs that broadly recognize the head domain. We determined the crystal structure of the HA protein of the avian H7N9 influenza virus in complex with a pan-H7, non-neutralizing, protective human Ab. The structure revealed a B cell epitope in the HA head domain trimer interface (TI). This discovery of a second major protective TI epitope supports a model in which uncleaved HA trimers exist on the surface of infected cells in a highly dynamic state that exposes hidden HA head domain features.
Jinhui Dong, Iuliia Gilchuk, Sheng Li, Ryan Irving, Matthew T. Goff, Hannah L. Turner, Andrew B. Ward, Robert H. Carnahan, James E. Crowe Jr.
Although IKK-β has previously been shown as a negative regulator of IL-1β secretion in mice, this role has not been demonstrated in humans. Genetic studies of NF-κB signalling in humans with inherited diseases of the immune system have not demonstrated the relevance of the NF-κB pathway in suppression of IL-1β expression. Here, we report an infant displaying clinical pathology comprising neutrophil-mediated auto-inflammation and recurrent bacterial infections. Whole-exome sequencing revealed a de novo heterozygous missense mutation in NFKBIA, resulting in a L34P IκBα variant, that severely repressed NF-κB activation and downstream cytokine production. Paradoxically, IL-1β secretion was elevated in the patient’s stimulated leukocytes, in her induced-pluripotent stem cell-derived macrophages, and in murine bone marrow-derived macrophages containing the L34P mutation. The patient’s hyper-IL-1β secretion correlated with activated neutrophilia and liver fibrosis with neutrophil accumulation. Hematopoietic stem cell transplantation reversed neutrophilia, restored a resting state in neutrophils, and normalized IL-1β release from stimulated leukocytes. Additional therapeutic blockade of IL-1 ameliorated liver damage, whilst decreasing neutrophil activation and associated IL-1β secretion. Our studies reveal a previously unrecognized role of human IκBα as an essential regulator of canonical NF-κB signalling in the prevention of neutrophilic-dependent auto-inflammatory diseases. We showed that IκBα controls IL-1β secretion through a mechanism of self-limiting post-transcriptional regulation. These findings also highlight a therapeutic potential for IL-1 inhibitors to treat complications arising from systemic NF-κB inhibition.
Enrica Ee Kar Tan, Richard Hopkins, Chrissie K. Lim, Saumya Jamuar, Christina Ong, Koh Cheng Thoon, Mark J.A. Koh, Eun Myoung Shin, Derrick Wen Quan Lian, Madhushanee Weerasooriya, Christopher Z.W Lee, Andreas Alvin Purnomo Soetedjo, Chang Siang Lim, Veronica B. Au, WM Edmond Chua, Hui Yin Lee, Leigh Ann Jones, Sharmy Jennifer James, Nivashini Kaliaperumal, Jeffrey Kwok, Ee Shien Tan, Biju Thomas, Lena Ho, Lynn Wu, Anna-Marie Fairhurst, Florent Ginhoux, Adrian K.K. Teo, Yongliang Zhang, Kok Haur Ong, Weimiao Yu, Byrappa Venkatesh, Vinay Tergaonkar, Bruno Reversade, Keh-Chuang Chin, Ah Moy Tan, Woei Kang Liew, John E. Connolly
Antibodies targeting human leukocyte antigen (HLA)/major histocompatibility complex (MHC) proteins limit successful transplantation and transfusion, and their presence in blood products can cause lethal transfusion-related acute lung injury (TRALI). It is unclear which cell types are bound by these ‘anti-leukocyte’ antibodies to initiate an immunologic cascade resulting in lung injury. We therefore conditionally removed MHC class I (MHC I) from likely cellular targets in antibody-mediated lung injury. Only the removal of endothelial MHC I reduced lung injury and mortality, related mechanistically to absent endothelial complement fixation and lung platelet retention. Restoration of endothelial MHC I rendered MHC I-deficient mice susceptible to lung injury. Neutrophil responses, including neutrophil extracellular trap (NET) release, were intact in endothelial MHC I-deficient mice, whereas complement depletion reduced both lung injury and NETs. Human pulmonary endothelial cells showed high HLA class I expression, and post-transfusion complement activation was increased in clinical TRALI. These results indicate that the critical source of antigen for ‘anti-leukocyte’ antibodies is in fact the endothelium, which reframes our understanding of TRALI as a rapid-onset vasculitis. Inhibition of complement activation may have multiple beneficial effects of reducing endothelial injury, platelet retention, and NET release in conditions where antibodies trigger these pathogenic responses.
Simon J. Cleary, Nicholas Kwaan, Jennifer J. Tian, Daniel R. Calabrese, Beñat Mallavia, Mélia Magnen, John R. Greenland, Anatoly Urisman, Jonathan P. Singer, Steven R. Hays, Jasleen Kukreja, Ariel M. Hay, Heather L. Howie, Pearl Toy, Clifford A. Lowell, Craig N. Morrell, James C. Zimring, Mark R. Looney
As a hallmark of immunological ageing, the low-grade, chronic inflammation with accumulation of effector-memory T cells contributes to the increased susceptibility of many ageing-related diseases. While the proinflammatory state of aged T cells indicates a dysregulation of immune homeostasis, whether and how ageing drives regulatory T (Treg) cell ageing and alters their function is not fully understood due to a lack of specific ageing markers. Here, by a combination of cellular, molecular and bioinformatic approaches, we discover that Treg cells senesce more severely than conventional T (Tconv) cells during ageing. We found Treg cells from aged mice were less efficient than young Treg cells to suppress Tconv cell function in an inflammatory-bowel-disease model and to prevent Tconv cell ageing in the irradiation-induced ageing model. Furthermore, we revealed that DCAF1 (DDB1 and CUL4 associated factor 1) was downregulated in aged Treg cells and was critical to restrain Treg cell ageing via glutathione S-transferase P (GSTP1) regulated reactive-oxygen-species (ROS). Importantly, interfering with GSTP1 and ROS pathways reinvigorated the proliferation and function of aged Treg cells. Therefore, our studies uncover an important role of DCAF1-GSTP1-ROS axis in Treg cell senescence, which leads to uncontrolled inflammation and immunological ageing.
Zengli Guo, Gang Wang, Bing Wu, Wei-Chun Chou, Liang Cheng, Chenlin Zhou, Jitong Lou, Di Wu, Lishan Su, Junnian Zheng, Jenny Pan-Yun Ting, Yisong Y. Wan
Arginase 1 (Arg1), which converts L-arginine into ornithine and urea, exerts pleiotropic immunoregulatory effects. However, the function of Arg1 in inflammatory bowel disease (IBD) remains poorly characterized. Here, we found that Arg1 expression correlated with the degree of inflammation in intestinal tissues from IBD patients. In mice, Arg1 was upregulated in an IL-4-/IL-13- and intestinal microbiota-dependent manner. Tie2-Cre+/-Arg1fl/fl mice lacking Arg1 in hematopoietic and endothelial cells recovered faster from experimental colitis than Arg1-expressing littermates. This correlated with decreased vessel density, compositional changes in intestinal microbiota, diminished infiltration by myeloid cells and an accumulation of intraluminal polyamines that promote epithelial healing. The pro-resolving effect of Arg1-deletion was reduced by an L-arginine-free diet, but rescued by simultaneous deletion of other L-arginine-metabolizing enzymes such as Arg2 or Nos2, demonstrating that protection from colitis requires L-arginine. Fecal microbiota transfers from Tie2-Cre+/-Arg1fl/fl mice into wild-type recipients ameliorated intestinal inflammation while transfers from wild-type littermates into Arg1-deficient mice prevented an advanced recovery from colitis. Thus, an increased availability of L-arginine as well as altered intestinal microbiota and metabolic products account for the accelerated resolution from colitis in the absence of Arg1. Consequently, the metabolism of L-arginine may serve as target for clinical intervention in IBD patients.
Julia Baier, Maximilian Gänsbauer, Claudia Giessler, Harald Arnold, Mercedes Muske, Ulrike Schleicher, Soeren Lukassen, Arif B. Ekici, Manfred Rauh, Christoph Daniel, Arndt Hartmann, Benjamin Schmid, Philipp Tripal, Katja Dettmer, Peter J. Oefner, Raja Atreya, Stefan Wirtz, Christian Bogdan, Jochen Mattner
Background: Pediatric SARS-CoV-2 infection can be complicated by a dangerous hyperinflammatory condition termed multisystem inflammatory syndrome in children (MIS-C). The clinical and immunologic spectrum of MIS-C and its relationship to other inflammatory conditions of childhood have not been studied in detail. Methods: We retrospectively studied confirmed cases of MIS-C at our institution from March to June 2020. The clinical characteristics, laboratory studies and treatment response were collected. Data were compared with historic cohorts of Kawasaki disease (KD) and macrophage activation syndrome (MAS). Results: Twenty-eight patients fulfilled the case definition of MIS-C. Median age at presentation was 9 years (range 1 month to 17 years); 50% of patients had pre-existing conditions. All patients had laboratory confirmation of SARS-CoV-2 infection. Seventeen patients (61%) required intensive care, including 7 patients (25%) requiring inotrope support. Seven patients (25%) met criteria for complete or incomplete KD and coronary abnormalities were found in 6 cases. Lymphopenia, thrombocytopenia, and elevation in inflammatory markers, D-dimer, B-type natriuretic peptide, IL-6 and IL-10 levels were common but not ubiquitous. Cytopenias distinguished MIS-C from KD and the degree of hyperferritinemia and pattern of cytokine production differed between MIS-C and MAS. Immunomodulatory therapy given to MIS-C patients included IVIG (71%), corticosteroids (61%) and anakinra (18%). Clinical and laboratory improvement were observed in all cases, including 6 cases that did not require immunomodulatory therapy. No mortality was recorded in this cohort. Conclusion: MIS-C encompasses a broad phenotypic spectrum with clinical and laboratory features distinct from Kawasaki disease and macrophage activation syndrome. Funding: This work was supported by the National Institute of Health / National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) K08-AR074562 (PYL), K08-AR AR073339 (LAH), R01-AR065538, R01-AR073201 and P30-AR070253 (PAN); National Institute of Allergy and Infectious Diseases 5T32AI007512-34 (JL, JR, TB, AAN and RWN); Rheumatology Research Foundation Investigator Awards (PYL and LAH) and Medical Education Award (JSH); Boston Children’s Hospital Faculty Career Development Awards (PYL and LAH), the McCance Family Foundation (JWN), and the Samara Jan Turkel Center (JC, RPS, MBS).
Pui Y. Lee, Megan Day-Lewis, Lauren A. Henderson, Kevin Friedman, Jeffrey Lo, Jordan E. Roberts, Mindy S. Lo, Craig D. Platt, Janet Chou, Kacie J. Hoyt, Annette L. Baker, Tina Banzon, Margaret H. Chang, Ezra Cohen, Sarah de Ferranti, Audrey Dionne, Saddiq Habiballah, Olha Halyabar, Jonathan S. Hausmann, Melissa Hazen, Erin Janssen, Esra Meidan, Ryan W. Nelson, Alan A. Nguyen, Robert P. Sundel, Fatma Dedeoglu, Peter A. Nigrovic, Jane W. Newburger, Mary Beth F. Son
BACKGROUND. The anti-tuberculosis vaccine Bacillus Calmette-Guérin (BCG) reduces overall infant mortality. Induction of innate immune memory, also termed trained immunity, contributes towards protection against heterologous infections. Since immune cells display oscillations in numbers and function throughout the day, we investigated the effect of BCG administration time on the induction of trained immunity. METHODS. 18 volunteers were vaccinated with BCG at 6pm and compared with 36 age- and sex-matched volunteers vaccinated between 8-9 am. Peripheral blood mononuclear cells were stimulated with Staphylococcus aureus and Mycobacterium tuberculosis before, as well as two weeks and three months after BCG vaccination. Cytokine production was measured to assess the induction of trained immunity and adaptive responses, respectively. Additionally, the influence of vaccination time on induction of trained immunity was studied in an independent cohort of 302 individuals vaccinated between 8am-12pm with BCG. RESULTS. Compared to evening vaccination, morning vaccination elicited both a stronger trained immunity and adaptive immune phenotype. In a large cohort of 302 volunteers, early morning vaccination resulted in a superior cytokine production capacity compared with later morning. A cellular, rather than soluble, substrate of the circadian effect of BCG vaccination was demonstrated by the enhanced capacity to induce trained immunity in vitro in morning compared to evening isolated monocytes. CONCLUSIONS. BCG vaccination in the morning induces stronger trained immunity and adaptive responses compared to evening vaccination. Future studies should take vaccine administration time into account when studying specific and non-specific effects of vaccines: early morning should be the preferred moment of BCG administration. FUNDING Spinoza grant of the Netherlands Organization for Scientific Research, ERC Advanced Grant (TRAIN-OLD nr. 833247), Danish National Research Foundation (DNRF108).
L. Charlotte J. de Bree, Vera P. Mourits, Valerie A.C.M. Koeken, Simone J.C.F.M. Moorlag, Robine Janssen, Lukas Folkman, Daniele Barreca, Thomas Krausgruber, Victoria Fife-Gernedl, Boris Novakovic, Rob J.W. Arts, Helga Dijkstra, Heidi Lemmers, Christoph Bock, Leo A.B. Joosten, Reinout van Crevel, Christine S. Benn, Mihai G. Netea