Co-phagocytosis of VEGFA with HER2-overexpressing cancer cells induced by HER2-VEGFA BsAb improves antitumor responses

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Abstract

We conceived of a type of antitumor mechanism of action by which a soluble target in the tumor microenvironment, such as a tumor-driving growth factor, can be phagocytized along with cancer cells via antibody-dependent cellular phagocytosis (ADCP) using an antibody bispecific for the soluble target and a solid target overexpressed on the cancer cell surface. We explored this concept through engineering bispecific antibodies (BsAbs) co-targeting human epidermal growth factor receptor-2 (HER2) and vascular endothelial growth factor A (VEGFA) in an scFv-IgG format (VHS). We showed that the HER2-VEGFA BsAbs but not the parental antibodies alone or in combination induced co-phagocytosis of VEGFA and HER2-overexpressing cancer cells by tumor-associated macrophages via ADCP. In both immunocompromised and immunocompetent mice with aggressive tumors, the BsAbs demonstrated greater anti-metastasis activity and produced a greater survival benefit than the parental antibodies alone or in combination, in a manner dependent on Fcγ receptors on the macrophages. Our results provide proof of the concept that HER2-VEGFA BsAbs achieve enhanced antitumor activity by leveraging HER2 overexpressed on the cancer cell surface to induce co-phagocytosis of VEGFA. Our findings warrant clinical testing of the strategy to treat metastasis and recurrence of HER2-overexpressing solid tumors that respond to anti-VEGFA therapy.

Authors

Yang Lu, Songbo Qiu, Zhen Fan

Thyroid hormone promotes fetal neurogenesis

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Abstract

Maternal low thyroxine (T4) serum levels during the first trimester of pregnancy correlate with cerebral cortex volume and mental development of the progeny, but why neural cells during early fetal brain development are vulnerable to maternal T4 levels remains unknown. In this study, using iPSCs obtained from a boy with a loss-of-function mutation in MCT8—a transporter previously identified as critical for thyroid hormone uptake and action in neural cells—we demonstrate that thyroid hormones induce transcriptional changes that promote the progression of human neural precursor cells along the dorsal projection trajectory. Consistent with these findings, single-cell, spatial, and bulk transcriptomics from MCT8-deficient cerebral organoids and cultures of human neural precursor cells underscore the necessity for optimal thyroid hormone levels for these cells to differentiate into neurons. The controlled intracellular activation of T4 signaling occurs through the transient expression of the enzyme type 2 deiodinase, which converts T4 into its active form, T3, alongside the coordinated expression of thyroid hormone nuclear receptors. The intracellular activation of T4 in NPCs results in transcriptional changes important for their division mode and cell cycle progression. Thus, T4 is essential for fetal neurogenesis, highlighting the importance of adequate treatment for mothers with hypothyroidism.

Authors

Federico Salas-Lucia, Sergio Escamilla, Amanda Charest, Hanzi Jiang, Randy Stout, Antonio C. Bianco

GPRC5B preserves a mature β-cell state in obesity by controlling MafA expression

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Abstract

In vitro studies have implicated orphan receptor GPRC5B in β-cell survival, proliferation and insulin secretion, but its relevance for glucose homeostasis in vivo is largely unknown. Using tamoxifen-inducible, β-cell-specific GPRC5B knockout mice (Ins-G5b-KOs) we show here that loss of GPRC5B does not affect β-cell function in the lean state, but results in strongly reduced insulin secretion and disturbed glucose tolerance in mice subjected to high fat diet for 16 weeks. Flow cytometry and single-cell expression analyses in islets from obese mice show a reduced β-cell abundance and a less mature β-cell phenotype in Ins-G5b-KOs. Expression of β-cell-specific transcription factor MafA is reduced both on the RNA and protein level, as are transcripts of MafA target genes. Mechanistically, we show that phosphorylation of cAMP response element-binding protein (CREB), a major regulator of MafA expression, is reduced in islets of obese Ins-G5b-KOs, and that this phenotype precedes the downregulation of MafA and MafA target genes. Taken together, GPRC5B helps to maintain mature β-cell function in obesity through cAMP/CREB-dependent regulation of MafA expression.

Authors

Tianpeng Wang, Remy Bonnavion, Janett Piesker, Stefan Günther, Nina Wettschureck

Cleavage of N-terminus of polycystin-1 increases calcium permeability of polycystin-1/2 receptor channel complexes

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Abstract

Mutations on genes encoding polycystin-1 (PC1) and -2 (PC2) cause autosomal-dominant polycystic kidney disease. How these two proteins work together to exert anti-cystogenesis remains elusive. PC1 resembles adhesion G-protein coupled receptors and undergoes autocleavage in the extracellular N-terminus to expose a hidden “stalk” region, which is hypothesized to act as a “tethered agonist”. Here, we showed that wildtype PC1 and PC2 formed functional heteromeric channel complexes in Xenopus oocytes with different biophysical properties from PC2 homomeric channels. Deletion of PC1 N-terminus, which exposed the stalk, increased calcium permeability in PC1/PC2 heteromers that required the presence of stalk. Extracellular application of synthetic stalk peptide increased calcium permeation in stalkless PC1/PC2. Application of Wnt9B protein increased calcium permeability in PC1/PC2, but not in heteromers containing cleavage-resistant mutant PC1. Wnt9B interacted with N-terminal leucine-rich repeat (LRR) of PC1. Pretreatment with LRR blunted the increase in calcium permeability by Wnt9B. Thus, PC1 and PC2 form receptor-channel complexes that is activated by exposure of the stalk region following ligand binding to the PC1 N-terminus. The stalk peptide acts as a tethered agonist to activate PC1/PC2 by impacting ion selectivity of the complexes.

Authors

Runping Wang, Danish Idrees, Mohammad Amir, Biswajit Padhy, Jian Xie, Chou-Long Huang

Type I interferons enhance human dorsal root ganglion nociceptor excitability and induce TRPV1 sensitization

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Abstract

Type I interferons (IFNs) are critical cytokines for antiviral defense and are linked to painful diseases like rheumatoid arthritis, lupus, and neuropathic pain in humans. IFN-α therapy can cause myalgia, headache, joint and abdominal pain. Studies in rodent models demonstrate that direct action of IFNs on sensory neurons in the dorsal root ganglion (DRG) promotes hyperexcitability but rodent behavioral data on IFNs are conflicting, with reports of both pro- and anti-nociceptive actions. We sought to clarify the action of IFN-α and IFN-β on human DRG (hDRG) nociceptors. We found that IFN receptor subunits IFNAR1 and IFNAR2 are expressed by these neurons and their engagement induces canonical STAT1 signaling and non-canonical MAPK activation as measured by increased phosphorylation of the cap-binding protein eIF4E by MNK1/2 kinases. Using patch clamp electrophysiology, Ca2+-imaging, and multi-electrode arrays we demonstrate that IFN-α and -β increase the excitability of hDRG neurons with acute and long-term exposure. Type I IFNs prolong the duration of capsaicin responses, an effect that is blocked by inhibition of MNK1/2 with eFT508, a specific inhibitor of these kinases. This study supports the conclusion that type I IFNs induce hyperexcitability and TRPV1-sensitization when they interact with IFNAR1/2 in hDRG nociceptors.

Authors

Úrzula Franco-Enzástiga, Keerthana Natarajan, Felipe Espinosa, Rafael Granja-Vazquez, Hemanth Mydugolam, Theodore J. Price

Conserved interactions with stromal and immune cells coordinate de novo B-cell lymphopoiesis in fetal intestines

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Abstract

Recent findings suggest that the small intestine (SI) is a novel site for B cell lymphopoiesis during fetal and neonatal life. However, the unique and/or conserved features that enable B cell development at this site remain unclear. To investigate the molecular and cellular scaffolds for B cell lymphopoiesis in mouse and human fetal intestines we leveraged single-cell RNA sequencing, in situ immunofluorescence, spatial transcriptomics and high-dimensional spectral flow cytometry. We found that SI mesenchymal and stromal cells expressed higher levels of chemokines known to recruit common lymphoid progenitors. Importantly, local lymphatic endothelial cells expressed IL7 and TSLP in proximity to IL7R+ precursor B cells, likely promoting their differentiation in the SI. Notably, we found that fetal-derived lymphoid tissue inducer (LTi) cells were required for B cell development and localization in the SI, but not fetal liver. These findings identify a lymphoid tissue development-independent role for this immune cell in B cell development. Collectively, our data reveal a conserved intestinal B cell niche in mice and humans, challenging traditional models of lymphopoiesis. The identification of a requisite cellular/molecular scaffold for fetal B cell development allows future studies to test the importance of this de novo B cell lymphopoiesis to long-term immunity.

Authors

Kimberly A. Carroll, Weihong Gu, Long Phan, Eduardo Gonzalez Santiago, Wenjia Wang, George C. Tseng, Liza Konnikova, Shruti Sharma

Development and Preclinical Evaluation of Next-generation ΔsigH-based Live Candidate Vaccines

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Abstract

To radically diminish TB incidence and mortality by 2035, as set out by the WHO End TB Strategy, there is a desperate need for improved TB therapies and a more effective vaccine against the deadly pathogen Mycobacterium tuberculosis (Mtb). Aerosol vaccination with the MtbΔsigH mutant protects two different species of NHPs against lethal TB challenge by invoking vastly superior T and B cell responses in the lungs through superior antigen-presentation and interferon-conditioning. Since the Geneva consensus on essential steps towards the development of live mycobacterial vaccines recommends that live TB vaccines must incorporate at least two independent gene knock outs, we have now generated several rationally designed, double (DKO)- and triple (TKO) knock-out mutants in Mtb, each containing the ΔsigH deletion. Here, we report preclinical studies in the rhesus macaque model of aerosol infection and SIV/HIV co-infection, aimed at assessing the safety of these MtbΔsigH - based DKOs and TKOs. We found that most of these mutant strains are attenuated in both immunocompetent and SIV-co-infected macaques and combinatorial infection with these generated strong cellular immune responses in the lung, akin to MtbΔsigH. Aerosol infection with these KO strains elicited inducible Bronchus Associated Lymphoid Tissue (iBALT), which is a correlate of protection from TB.

Authors

Garima Arora, Caden W. Munson, Mushtaq Ahmed, Vinay Shivanna, Annu Devi, Venkata S.R. Devireddy, Basil Antony, Shannan Hall-Ursone, Olga D. Gonzalez, Edward Dick Jr., Chinnaswamy Jagannath, Xavier Alvarez, Smriti Mehra, Shabaana A. Khader, Dhiraj K. Singh, Deepak Kaushal

Perivascular inflammation in the progression of aortic aneurysms in Marfan syndrome

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Abstract

Inflammation plays important roles in the pathogenesis of vascular diseases. We here show the involvement of perivascular inflammation in aortic dilatation of Marfan syndrome (MFS). In the aorta of MFS patients and Fbn1C1041G/+ mice, macrophages markedly accumulated in periaortic tissues with increased inflammatory cytokine expression. Metabolic inflammatory stress induced by a high-fat diet (HFD) enhanced vascular inflammation predominantly in periaortic tissues and accelerated aortic dilatation in Fbn1C1041G/+ mice, both of which were inhibited by low-dose pitavastatin. HFD feeding also intensifies structural disorganization of the tunica media in Fbn1C1041G/+ mice, including elastic fiber fragmentation, fibrosis, and proteoglycan accumulation, along with increased activation of TGF-β downstream targets. Pitavastatin treatment mitigated these alterations. For non-invasive assessment of PVAT inflammation in a clinical setting, we developed an automated analysis program for CT images using machine learning techniques to calculate the perivascular fat attenuation index of the ascending aorta (AA-FAI), correlating with periaortic fat inflammation. The AA-FAI was significantly higher in patients with MFS compared to patients without hereditary connective tissue disorders. These results suggest that perivascular inflammation contributes to aneurysm formation in MFS and might be a potential target for preventing and treating vascular events in MFS.

Authors

Hiroyuki Sowa, Hiroki Yagi, Kazutaka Ueda, Masaki Hashimoto, Kohei Karasaki, Qing Liu, Atsumasa Kurozumi, Yusuke Adachi, Tomonobu Yanase, Shun Okamura, Bowen Zhai, Norifumi Takeda, Masahiko Ando, Haruo Yamauchi, Nobuhiko Ito, Minoru Ono, Hiroshi Akazawa, Issei Komuro

IL-23 drives uveitis by acting on a population of tissue-resident entheseal T cells

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Abstract

Recurrent acute anterior uveitis is a frequent extra-articular manifestation of the axial spondyloarthropathies (AxSpA); chronic inflammatory diseases affecting the spine, enthesis, peripheral joints, skin, and gastrointestinal tract. Pathology in AxSpA has been associated with local tissue-resident populations of interleukin (IL)-23 responsive lymphoid cells. Here we characterize a population of ocular T cell defined by CD3+CD4-CD8-CD69+gdTCR+IL-23R+ that reside within the anterior uvea as an ocular entheseal analogue of the mouse eye. Localised cytokine expression demonstrates that uveal IL-23R+ IL-17A-producing cells are both necessary and sufficient to drive uveitis in response to IL-23. This T cell population is also present in humans, occupying extravascular tissues of the anterior uveal compartment. Consistent with the concept of IL-23 as a unifying mediator in AxSpA, we present evidence that IL-23 can also act locally on tissue resident T cells in the anterior compartment of the eye at sites analogous to the enthesis to drive ocular inflammation.

Authors

Robert Hedley, Amy Ward, Colin J. Chu, Sarah E. Coupland, Serafim Kiriakidis, Peter C. Taylor, Stephanie G. Dakin, ORBIT Research Consortium, Christopher D. Buckley, Jonathan Sherlock, Andrew D. Dick, David A. Copland

A dendritic cell population responsible for transglutaminase 2-mediated gluten antigen presentation in celiac disease

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Abstract

In celiac disease (CeD), a gluten-dependent autoimmune disorder, transglutaminase 2 (TG2) deamidates selected glutamine residues in gluten peptides, while HLA-DQ2 presents deamidated antigens to inflammatory T cells. The cellular sources of pathogenic TG2 and DQ2 are unclear. Using chemical biology tools, we show that intestinal CD103+ dendritic cells (DCs) couple cell-surface TG2 to the endocytic LRP1 receptor to simultaneously deamidate gluten antigens and concentrate them in lysosomes. In DQ2-transgenic mice, CD103+ DCs loaded with deamidated antigens migrate from intestinal lamina propria and Peyer’s patches into mesenteric lymph nodes, where they engage T cells. In turn, gluten antigen presentation upregulates intestinal TG2 activity. The tool (HB-230) used to establish a role of CD103+ DCs in gluten antigen presentation and TG2 activation in mice also revealed that the TG2/LRP1 pathway is active in human CD14+ monocytes. Within this population of circulating monocytes, a DC subset with the gut-homing β7-integrin marker is elevated in CeD patients with active disease compared to non-celiac controls or patients on a gluten-free diet. Our findings not only inform the cellular basis for gluten toxicity in CeD but they also highlight the immunologic role of an enigmatic protein of growing therapeutic relevance in CeD and other immune disorders.

Authors

Fu-Chen Yang, Harrison A. Besser, Hye Rin Chun, Megan Albertelli, Nielsen Q. Fernandez-Becker, Bana Jabri, Chaitan Khosla