This study aimed to enhance anti-tumor immune responses to pancreatic cancer via antibody-based blockade of IL-6 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Mice bearing subcutaneous or orthotopic pancreatic tumors were treated with blocking antibodies to IL 6 and/or CTLA-4. In both tumor models, dual IL-6 and CTLA-4 blockade significantly inhibited tumor growth. Additional investigations revealed that dual therapy induced an overwhelming infiltration of T cells into the tumor as well as changes in CD4+ T cell subsets. Dual blockade therapy elicited CD4+ T cells to secrete increased IFN-γ in vitro. Likewise, in vitro stimulation of pancreatic tumor cells with IFN-γ profoundly increased tumor cell production of CXCR3 specific chemokines, even in the presence of IL-6. In vivo blockade of CXCR3 prevented orthotopic tumor regression in the presence of the combination treatment, demonstrating a dependence on the CXCR3 axis for anti-tumor efficacy. Both CD4+ and CD8+ T cells were required for the anti-tumor activity of this combination therapy, as their in vivo depletion via antibodies impaired outcomes. These data represent the first report of IL-6 and CTLA 4 blockade as a means to regress pancreatic tumors with defined operative mechanisms of efficacy. Given these results, this therapeutic combination has potential for immediate clinical translation.
Michael Brandon Ware, Maggie Phillips, Christopher McQuinn, Mohammad Y. Zaidi, Hannah M. Knochelmann, Emily Greene, Brian S. Robinson, Cameron J. Herting, Thomas A. Mace, Zhengjia Chen, Chao Zhang, Matthew R. Farren, Amanda N. Ruggieri, Jacob S. Bowers, Reena Shakya, Alton Brad Farris, Gregory Young, William E. Carson III, Bassel El-Rayes, Chrystal M. Paulos, Gregory B. Lesinski
Multiple randomized, controlled clinical trials have yielded discordant results regarding the efficacy of convalescent plasma in outpatients, with some showing an approximate two-fold reduction in risk and others showing no effect. We quantified binding and neutralizing antibody levels in 492 of the 511 participants from the C3PO trial of a single unit of COVID-19 convalescent plasma (CCP) vs. saline infusion. In a subset of 70 participants, peripheral blood mononuclear cells were obtained to define the evolution of B and T cell responses through day 30. Binding and neutralizing antibody responses were measurably higher one hour post-infusion in recipients of CCP compared to saline plus multivitamin, but levels achieved by the native immune system by day 15 were much higher than seen immediately after CCP administration. Infusion of CCP did not block generation of the host antibody response or skew B or T cell phenotype or maturation. Activated CD4+ and CD8+ T cells were associated with more severe disease outcome. These data show that CCP leads to a measurable boost in anti-SARS-CoV-2 antibodies, but that the boost is modest and may not be sufficient to alter disease course.
John F. McDyer, Mahzad Azimpouran, Valerie L. Durkalski-Mauldin, Robert G. Clevenger, Sharon D. Yeatts, Xutao Deng, William Barsan, Robert Silbergleit, Nahed El Kassar, Iulia Popescu, Dimiter Dimitrov, Wei Li, Emily J. Lyons, Sophia C. Lieber, Mars Stone, Frederick K. Korley, Clifton W. Callaway, Larry J. Dumont, Philip J. Norris
Sosuga virus (SOSV) is a recently discovered paramyxovirus with a single known human case of disease. There has been little laboratory research on SOSV pathogenesis or immunity, and no approved therapeutics or vaccines are available. Here, we report the discovery of human monoclonal antibodies (mAbs) from the circulating memory B cells of the only known human case and survivor of SOSV infection. We isolated six mAbs recognizing the functional attachment protein hemagglutinin-neuraminidase (HN) and 18 mAbs against the fusion (F) protein. The anti-HN mAbs all target the globular head of the HN protein and can be organized into 4 competition-binding groups that exhibit epitope diversity. The anti-F mAbs can be divided into pre- or postfusion conformation-specific categories and further into 8 competition-binding groups. The only antibody in the panel that did not display neutralization activity was the single, postfusion-specific anti-F mAb. Most of the anti-HN mAbs were more potently neutralizing than the anti-F mAbs, with mAbs in one of the HN competition-binding groups possessing ultra-potent (<1 ng/mL) half maximal inhibitory (IC50) virus neutralization values. These findings provide insight into the molecular basis for human antibody recognition of paramyxovirus surface proteins and the mechanisms of SOSV neutralization.
Helen M. Parrington, Nurgun Kose, Erica Armstrong, Laura S. Handal, Summer Diaz, Joseph Reidy, Jinhui Dong, Guillaume B.E. Stewart-Jones, Punya Shrivastava-Ranjan, Shilpi Jain, César G. Albariño, Robert H. Carnahan, James E. Crowe
Pulmonary fibrosis is potentiated by a positive feedback loop involving the extracellular sialidase enzyme NEU3 causing release of active TGF-β1, and TGF-β1 upregulating NEU3 by increasing translation without affecting mRNA levels. In this report, we elucidate the TGF-β1 upregulation of translation mechanism. In human lung fibroblasts, TGF-β1 increased levels of proteins, including NEU3, by increasing translation of the encoding mRNAs without significantly affecting levels of these mRNAs. 180 of these mRNAs share a common 20 nucleotide motif. Deletion of this motif from NEU3 mRNA eliminated the TGF-β1 upregulation of NEU3 translation, while insertion of this motif in two mRNAs insensitive to TGF-β1 caused TGF-β1 to upregulate their translation. RNA-binding proteins including DDX3 bind the RNA motif, and TGF-β1 regulates their protein levels and/or binding to the motif. We found that DDX3 is upregulated in the fibrotic lesions in pulmonary fibrosis patients, and inhibiting DDX3 in fibroblasts reduced TGF-β1 upregulation of NEU3 levels. In the mouse bleomycin model of pulmonary fibrosis, injections of the DDX3 inhibitor RK-33 potentiated survival and reduced lung inflammation, fibrosis, and lung tissue levels of DDX3, TGF-β1, and NEU3. These results suggest that inhibiting a mRNA-binding protein that mediates TGF-β1 upregulation of translation can reduce pulmonary fibrosis.
Wensheng Chen, Darrell Pilling, Richard H. Gomer
HIV-1 usually utilize CCR5 as the co-receptor and rarely switches to CXCR4-tropic until late stage of infection. CCR5+CD4+ T cells are the major virus-producing cells in viremic patients as well as SIV-infected non-human primates. The differentiation of CCR5+CD4+ T cells is associated with the availability of IL15, which increases during acute HIV-1 infection. Here, we report that CCR5 is expressed by CD4+ T cells exhibiting effector or effector memory phenotype with high expression levels of the IL2/IL15 receptor common beta and gamma chains. IL15 but not IL7 improves the survival of CCR5+CD4+ T cells, drives their expansion, and facilitates HIV-1 infection in vitro and in humanized mice. Our study suggests that IL15 plays confounding roles in HIV-1 infection, and future studies on the IL15-based boosting of anti-HIV-1 immunity should carefully exam the potential effects on the expansion of HIV-1 reservoirs in CCR5+CD4+ T cells.
Yuhao Li, Hongbo Gao, Kolin M. Clark, Liang Shan
Helicobacter pylori colonization of the gastric niche can persist for years in asymptomatic individuals. To deeply characterize the host–microbiota environment in H. pylori–infected (HPI) stomachs, we collected human gastric tissues and performed metagenomic sequencing, single-cell RNA-Seq (scRNA-Seq), flow cytometry, and fluorescent microscopy. HPI asymptomatic individuals had dramatic changes in the composition of gastric microbiome and immune cells compared with noninfected individuals. Metagenomic analysis uncovered pathway alterations related to metabolism and immune response. scRNA-Seq and flow cytometry data revealed that, in contrast to murine stomachs, ILC2s are virtually absent in the human gastric mucosa, whereas ILC3s are the dominant population. Specifically, proportion of NKp44+ ILC3s out of total ILCs were highly increased in the gastric mucosa of asymptomatic HPI individuals, and correlated with the abundance of selected microbial taxa. In addition, CD11c+ myeloid cells and activated CD4+ T cells and B cells were expanded in HPI individuals. B cells of HPI individuals acquired an activated phenotype and progressed into a highly proliferating germinal-center stage and plasmablast maturation, which correlated with the presence of tertiary lymphoid structures within the gastric lamina propria. Our study provides a comprehensive atlas of the gastric mucosa–associated microbiome and immune cell landscape when comparing asymptomatic HPI and uninfected individuals.
Chiara Sorini, Kumar P. Tripathi, Shengru Wu, Shawn M. Higdon, Jing Wang, Liqin Cheng, Sanghita Banerjee, Annika Reinhardt, Taras Kreslavsky, Anders Thorell, Lars Engstrand, Juan Du, Eduardo J. Villablanca
Inhibitors of the DNA damage signaling kinase ATR increase tumor cell killing by chemotherapies that target DNA replication forks but also kill rapidly proliferating immune cells including activated T cells. Nevertheless, ATR inhibitor (ATRi) and radiotherapy (RT) can be combined to generate CD8+ T cell–dependent antitumor responses in mouse models. To determine the optimal schedule of ATRi and RT, we determined the impact of short-course versus prolonged daily treatment with AZD6738 (ATRi) on responses to RT (days 1–2). Short-course ATRi (days 1–3) plus RT caused expansion of tumor antigen–specific, effector CD8+ T cells in the tumor-draining lymph node (DLN) at 1 week after RT. This was preceded by acute decreases in proliferating tumor-infiltrating and peripheral T cells and a rapid proliferative rebound after ATRi cessation, increased inflammatory signaling (IFN-β, chemokines, particularly CXCL10) in tumors, and an accumulation of inflammatory cells in the DLN. In contrast, prolonged ATRi (days 1–9) prevented the expansion of tumor antigen–specific, effector CD8+ T cells in the DLN, and entirely abolished the therapeutic benefit of short-course ATRi with RT and anti–PD-L1. Our data argue that ATRi cessation is essential to allow CD8+ T cell responses to both RT and immune checkpoint inhibitors.
Frank P. Vendetti, Pinakin Pandya, David A. Clump, Sandra Schamus-Haynes, Meysam Tavakoli, Maria diMayorca, Naveed M. Islam, Jina Chang, Greg M. Delgoffe, Jan H. Beumer, Christopher J. Bakkenist
B cells within secondary lymphoid tissues (SLTs) encompass a diversity of activation states and multiple maturation processes that reflect antigen recognition and transition through the germinal center (GC) reaction, in which mature B cells differentiate into memory and antibody-secreting cells (ASCs). Here, utilizing single-cell RNA-seq, we identify a range of distinct activation and maturation states of tonsillar-derived B cells. In particular, we identify a previously uncharacterized CCL4/CCL3 chemokine-expressing B-cell population with an expression pattern consistent with BCR/CD40 activation. Furthermore, we present a computational method leveraging regulatory network inference and pseudotemporal modeling to identify upstream transcription factor modulation along a GC to ASC axis of transcriptional maturation. Our dataset provides valuable insight into diverse B-cell functional profiles and will be a useful resource for further studies into the B-cell immune compartment.
Diego A. Espinoza, Carole Le Coz, Emylette Cruz Cabrera, Neil Romberg, Amit Bar-Or, Rui Li
Anti-CD36 antibodies have been suggested to induce transfusion-related acute lung injury (TRALI) upon blood transfusion, particularly in Asian populations. However, little is known about the pathological mechanism of anti-CD36-mediated TRALI, and potential therapies haven’t yet been identified. Here, we developed a murine model of anti-CD36-mediated TRALI to address these questions. We found that administration of mouse monoclonal antibody against CD36 (mAb GZ1) or human anti-CD36 IgG, but not GZ1 F(ab’)2 fragments, induced severe TRALI in Cd36+/+ male mice. Pre-depletion of recipient monocytes or complement, but not neutrophils or platelets, prevented the development of murine TRALI. Moreover, plasma C5a levels after TRALI induction by anti-CD36 were increased more than 3-fold, implying a critical role of complement C5 activation in the mechanism of Fc-dependent anti-CD36-mediated TRALI. Administration of GZ1 F(ab’)2, antioxidant (NAC) or C5-blocker (mAb BB5.1) before TRALI induction completely protected mice from anti-CD36-mediated TRALI. Although no significant amelioration in TRALI was observed when mice were injected with GZ1 F(ab’)2 after TRALI induction, significant improvement was achieved when mice were treated post-induction with NAC or anti-C5. Importantly, anti-C5 treatment completely rescued mice from TRALI, suggesting the potential role of existing anti-C5 drugs in the treatment of patients with TRALI caused by anti-CD36.
Da-Wei Chen, Tian Kang, Xiu-Zhang Xu, Wen-Jie Xia, Xin Ye, Yong-Bin Wu, Yao-Ri Xu, Jing Liu, Hui Ren, Jing Deng, Yang-Kai Chen, Hao-Qiang Ding, Muhammad Aslam, Wioleta M. Zelek, B. Paul Morgan, Rick Kapur, Sentot Santoso, Yong-Shui Fu
Mesenchymal stem cells (MSCs) possess strong immunoregulatory functions, one aspect of which is recruiting monocytes from peripheral vessels to local tissue by secreting MCP1. However, the regulatory mechanisms of MCP1 secretion in MSCs are still unclear. Recently, N6-methyladenosine (m6A) modification was reported to be involved in the functional regulation of MSCs. In this study, we demonstrated that methyltransferase-like 16 (METTL16) negatively regulated MCP1 expression in MSCs through m6A modification. Specifically, the expression of METTL16 in MSCs decreased gradually and was negatively correlated with the expression of MCP1 after coculture with monocytes. Knocking down METTL16 markedly enhanced MCP1 expression and the ability to recruit monocytes. Mechanistically, knocking down METTL16 decreased MCP1 mRNA degradation, which was mediated by the m6A reader YTH N6-methyladenosine RNA binding protein 2 (YTHDF2). We further revealed that YTHDF2 specifically recognized m6A sites on MCP1 mRNA in the CDS region and thus negatively regulated MCP1 expression. Moreover, an in vivo assay showed that MSCs transfected with METTL16 siRNA showed a stronger ability to recruit monocytes. These findings reveal a potential mechanism by which the m6A methylase METTL16 regulates MCP1 expression through YTHDF2-mediated mRNA degradation and suggest a potential strategy to manipulate MCP1 expression in MSCs.
Zhaoqiang Zhang, Zhongyu Xie, Jiajie Lin, Zehang Sun, Zhikun Li, Wenhui Yu, Yipeng Zeng, Guiwen Ye, Jinteng Li, Feng Ye, Zepeng Su, Yunshu Che, Peitao Xu, Chenying Zeng, Peng Wang, Yanfeng Wu, Huiyong Shen
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