Evaluation of potential immunity against the novel severe acute respiratory syndrome (SARS) coronavirus that emerged in 2019 (SARS-CoV-2) is essential for health, as well as social and economic recovery. Generation of antibody response to SARS-CoV-2 (seroconversion) may inform on acquired immunity from prior exposure, and antibodies to the SARS-CoV-2 spike protein receptor binding domain (S-RBD) are speculated to neutralize virus infection. Some serology assays rely solely on SARS-CoV-2 nucleocapsid protein (N-protein) as the antibody detection antigen; however, whether such immune responses correlate with S-RBD response and COVID-19 immunity remains unknown. Here, we generated a quantitative serological enzyme-linked immunosorbent assay (ELISA) using recombinant S-RBD and N-protein for the detection of circulating antibodies in 138 serial serum samples from 30 RT-PCR confirmed SARS-CoV-2 hospitalized patients, as well as 464 healthy and non-COVID-19 serum samples that were collected between June 2017 and June 2020. Quantitative detection of IgG antibodies to the two different viral proteins showed a moderate correlation. Antibodies to N-protein were detected at a rate of 3.6% in healthy and non-COVID-19 sera collected during the pandemic in 2020, whereas 1.6% of these sera were positive for S-RBD. Approximately 86% of individuals positive for S-RBD binding antibodies exhibited neutralizing capacity, but only 74% of N-protein positive individuals exhibited neutralizing capacity. Collectively, our studies show that detection of N-protein binding antibodies does not always correlate with presence of S-RBD neutralizing antibodies, and cautions against the extensive use of N-protein based serology testing for determination of potential COVID-19 immunity.
Kathleen M. McAndrews, Dara P. Dowlatshahi, Jianli Dai, Lisa M. Becker, Janine Hensel, Laura M. Snowden, Jennifer M. Leveille, Michael R. Brunner, Kylie Holden, Nikolas S. Hopkins, Alexandria Harris, Jerusha J. Kumpati, Michael A. Whitt, J. Jack Lee, Luis Ostrosky-Zeichner, Ramesha Papanna, Valerie LeBleu, James Allison, Raghu Kalluri
Age-associated systemic, chronic inflammation is partially attributed to increased self (auto)-reactivity, resulting from disruption of central tolerance in the aged, involuted thymus. This involution causally results from gradually decreased expression of the transcription factor FOXN1 in thymic epithelial cells (TECs), while exogenous FOXN1 in TECs can partially rescue age-related thymic involution. Given the findings that TECs induced from FOXN1-overexpressing embryonic fibroblasts can generate an ectopic de novo thymus under the kidney capsule and intra-thymically injected naturally young TECs can lead to middle-aged thymus regrowth, we attempted to extend these two findings by combining them as a novel thymic rejuvenation strategy with two types of promoter-driven (Rosa26CreERT and FoxN1Cre) Cre-mediated FOXN1-reprogrammed embryonic fibroblasts (FREFs). We engrafted these two-types of FREFs directly into the aged murine thymus. We found significant regrowth of the native aged thymus with rejuvenated architecture and function in both males and females, exhibiting increased thymopoiesis and reinforced thymocyte negative selection, along with reduced senescent T cells and auto-reactive T cell-mediated inflammation in old mice. Therefore, this strategy has preclinical significance and presents a strategy to potentially rescue decreased thymopoiesis and perturbed negative selection to significantly, albeit partially, restore defective central tolerance and reduce subclinical autoimmune symptoms in the elderly.
Jiyoung Oh, Weikan Wang, Rachel Thomas, Dong-Ming Su
Tumor-associated macrophages (TAMs) affect cancer progression and therapy. Ovarian carcinoma often metastasizes to the peritoneal cavity. Here, we found two peritoneal macrophage subsets in mice bearing ID8 ovarian cancer based on the Tim-4 (T-cell immunoglobulin and mucin domain containing 4) expression. Tim-4+ TAMs were embryonically originated and locally sustained while Tim-4- TAMs were replenished from circulating monocytes. Tim-4+ TAMs, but not Tim-4- TAMs, promoted tumor growth in vivo. Relative to Tim-4- TAMs, Tim-4+ TAMs manifested high oxidative phosphorylation and adapted mitophagy to alleviate oxidative stress. High levels of arginase-1 in Tim-4+ TAMs contributed to potent mitophagy activities via weakened mTORC1 activation due to low arginine resultant from arginase-1-mediated metabolism. Furthermore, genetic deficiency of autophagy element FIP200 resulted in Tim-4+ TAM loss via ROS-mediated apoptosis, and elevated T cell-immunity and ID8 tumor inhibition in vivo. Moreover, human ovarian cancer-associated CRIg (complement receptor of the Immunoglobulin superfamily) positive macrophages were transcriptionally, metabolically, and functionally similar to murine Tim-4+ TAMs. Thus, targeting CRIg+ (Tim-4+) TAMs may potentially treat ovarian cancer patients with peritoneal metastasis.
Houjun Xia, Shasha Li, Xiong Li, Weichao Wang, Yingjie Bian, Shuang Wei, Sara Grove, Weimin Wang, Linda Vatan, J. Rebecca Liu, Karen McLean, Ramandeep Rattan, Adnan R. Munkarah, Jun-Lin Guan, Ilona Kryczek, Weiping Zou
Allergic disorders, characterized by Th2 immune responses to environmental substances, are increasingly common in children in Western societies. Multiple studies indicate that breastfeeding, early complementary introduction of food allergens, and antibiotic avoidance in the first year of life reduces allergic outcomes in at-risk children. Why the benefit of these practices is restricted to early life is largely unknown. We identified a preweaning interval during which dietary antigens are assimilated by the colonic immune system. This interval is under maternal control via temporal changes in breast milk, coincides with an influx of naive T cells into the colon, and is followed by the development of a long-lived population of colonic peripherally derived Tregs (pTregs) that can be specific for dietary antigens encountered during this interval. Desynchronization of mothers and offspring produced durable deficits in these pTregs, impaired tolerance to dietary antigens introduced during and after this preweaning interval, and resulted in spontaneous Th2 responses. These effects could be rescued by pTregs from the periweaning colon or by Tregs generated in vitro using periweaning colonic antigen-presenting cells. These findings demonstrate that mothers and their offspring are synchronized for the development of a balanced immune system.
Kathryn A. Knoop, Keely G. McDonald, Paige E. Coughlin, Devesha H. Kulkarni, Jenny K. Gustafsson, Brigida Rusconi, Vini John, I. Malick Ndao, Avraham Beigelman, Misty Good, Barbara B. Warner, Charles O. Elson, Chyi-Song Hsieh, Simon P. Hogan, Phillip I. Tarr, Rodney D. Newberry
A tumor blood vessel is a key regulator of tissue perfusion, immune cell trafficking, cancer metastasis, and therapeutic responsiveness. mTORC1 is a signaling node downstream of multiple angiogenic factors in the endothelium. However, mTORC1 inhibitors have limited efficacy in most solid tumors, in part due to inhibition of immune function at high doses used in oncology patients and compensatory PI3K signaling triggered by mTORC1 inhibition in tumor cells. Here we show that low-dose RAD001/everolimus, an mTORC1 inhibitor, selectively targets mTORC1 signaling in endothelial cells (ECs) without affecting tumor cells or immune cells, resulting in tumor vessel normalization and increased antitumor immunity. Notably, this phenotype was recapitulated upon targeted inducible gene ablation of the mTORC1 component Raptor in tumor ECs (RaptorECKO). Tumors grown in RaptorECKO mice displayed a robust increase in tumor-infiltrating lymphocytes due to GM-CSF–mediated activation of CD103+ dendritic cells and displayed decreased tumor growth and metastasis. GM-CSF neutralization restored tumor growth and metastasis, as did T cell depletion. Importantly, analyses of human tumor data sets support our animal studies. Collectively, these findings demonstrate that endothelial mTORC1 is an actionable target for tumor vessel normalization, which could be leveraged to enhance antitumor immune therapies.
Shan Wang, Ariel Raybuck, Eileen Shiuan, Sung Hoon Cho, Qingfei Wang, Dana M. Brantley-Sieders, Deanna Edwards, Margaret M. Allaman, James Nathan, Keith T. Wilson, David DeNardo, Siyuan Zhang, Rebecca Cook, Mark Boothby, Jin Chen
Dysregulated sensing of self nucleic acid is a leading cause of autoimmunity in multifactorial and monogenic diseases. Mutations in Wiskott-Aldrich syndrome protein (WASp), a key regulator of cytoskeletal dynamics in immune cells, cause autoimmune manifestations and increased production of type-I interferons by innate cells. Here we show that complexes of self-DNA and autoantibodies (DNA-IC) contribute to elevated interferon levels via activation of the cGAS-STING pathway of cytosolic sensing. Mechanistically, lack of endosomal F-actin nucleation by WASp causes a delay in endolysosomal maturation and prolongs the transit time of ingested DNA-IC. Stalling in maturation-defective organelles facilitates leakage of DNA-IC into the cytosol, promoting activation of the TBK1-STING pathway. Genetic deletion of STING, STING and cGAS chemical inhibitors abolish interferon production and rescue systemic activation of interferon stimulated genes in vivo. These data unveil the contribution of cytosolic self-nucleic acid sensing in WAS and underscore the importance of WASp-mediated endosomal actin remodelling to prevent innate activation.
Giulia Maria Piperno, Asma Naseem, Giulia Silvestrelli, Roberto Amadio, Nicoletta Caronni, Karla Evelia Cervantes Luevano, Nalan Liv, Judith Klumperman, Andrea Colliva, Hashim Ali, Francesca Graziano, Philippe Benaroch, Hans Haecker, Richard N. Hanna, Federica Benvenuti
Purpose: There is a rapidly evolving portfolio of immune therapeutic modulators, but the relative incidence of immune targets in human gliomas is unknown. In order to prioritize available immune therapeutics, immune profiling across glioma grades was conducted followed by preclinical determinations of therapeutic effect in immune competent mice harboring gliomas. Methods: CD4+ and CD8+ T cells and CD11b+ myeloid cells were isolated from the blood of healthy donors and the blood and tumors of newly diagnosed and recurrent glioma patients and profiled for the expression of immune modulatory targets with an available therapeutic. Preclinical murine models of glioma were used to assess therapeutic efficacy of agents targeting the most frequently expressed immune targets. Immune effector function was analyzed in the setting of glioma induced immune suppression. Results: In glioma patients, the adenosine-CD73-CD39 immune suppressive pathway was most frequently expressed, followed by PD-1. CD73 expression was upregulated on immune cells by 2-hydroxygluterate in IDH1 mutant glioma patients. In multiple murine glioma models, including those that express CD73, adenosine receptor inhibitors demonstrated a modest therapeutic response; however, the addition of other inhibitors of the adenosine pathway did not further enhance this therapeutic effect. Although adenosine receptor inhibitors could recover immunological effector functions in T cells after the engagement of this pathway, immune recovery was impaired in the presence of gliomas, indicating that irreversible immune exhaustion limits the effectiveness of inhibitors of the adenosine pathway in glioma patients. Conclusions: This study illustrates vetting steps that should be considered prior to clinical trial implementation for immunotherapy resistant cancers including testing an agents ability to restore immunological function in the context of intended use.
Martina Ott, Karl-Heinz Tomaszowski, Anantha Marisetty, Ling-Yuan Kong, Jun Wei, Maya Duna, Katia Blumberg, Xiaorong Ji, Carmen B Jacobs, Gregory N. Fuller, Lauren A. Langford, Jason T. Huse, James P. Long, Jian Hu, Shulin Li, Jeffrey S. Weinberg, Sujit Prabhu, Raymond Sawaya, Sherise D. Ferguson, Ganesh Rao, Frederick F. Lang, Michael A. Curran, Amy B. Heimberger
TLR7 has been linked to the pathogenesis of glomerulonephritis, but its precise roles are not clear. In this study, we evaluated the roles of TLR7 in IgA nephropathy (IgAN). TLR7 proteins were abundant in CD19+ B cells infiltrated in the kidneys of patients with IgAN. The intensities of both intrarenal TLR7 and CD19 proteins were closely associated with kidney function (estimated glomerular filtration rate [eGFR] and serum creatinine concentration) and renal histopathology (tubular atrophy, leukocyte infiltration, tubulointerstitial fibrosis, and global glomerulosclerosis) in patients with IgAN. Meanwhile, TLR7 mRNA levels were significantly increased in peripheral blood B cells of patients with IgAN. TLR7+CD19+ B cells expressed inflammatory cytokines (IL-6 and IL-12) in kidneys and produced high levels of IgA1 and galactose deficient-IgA1 (Gd-IgA1) in peripheral blood of patients with IgAN. Mechanistically, TLR7 activated B cells to produce high levels of Gd-IgA1 via the TLR7-GALNT2 axis in IgAN. Protein levels of GALNT2 were increased by overexpression of TLR7, while they were reduced by TLR7 knockdown in B cells. GALNT2 overexpression augmented Gd-IgA1 production in B cells derived from patients with IgAN. Taken together, high TLR7 expression in B cells has dual roles in the development and progression of IgAN, by facilitating renal inflammation and Gd-IgA1 antibody synthesis.
Nuoyan Zheng, Kaifeng Xie, Hongjian Ye, Yu Dong, Bing Wang, Ning Luo, Jinjin Fan, Jiaqing Tan, Wei Chen, Xueqing Yu
Current management of childhood leukemia is tailored based on disease risk determined by clinical features at presentation. Whether properties of the host immune response impact disease risk and outcome is not known. Here we combine mass cytometry, single cell genomics and functional studies to characterize the bone marrow immune environment in children with B-cell acute lymphoblastic leukemia, and acute myelogenous leukemia at presentation. T cells in leukemia marrow demonstrate evidence of chronic immune activation and exhaustion/dysfunction, with attrition of naïve T cells and TCF1+ stem-like memory T cells and accumulation of terminally-differentiated effector T cells. Marrow-infiltrating natural killer cells also exhibit evidence of dysfunction, particularly in myeloid leukemia. Properties of immune cells identified distinct immune phenotype-based clusters correlating with disease risk in acute lymphoblastic leukemia. High-risk immune signatures were associated with expression of stem-like genes on tumor cells. These data provide a comprehensive assessment of the immune landscape of childhood leukemias and identify targets potentially amenable to therapeutic intervention. These studies also suggest that properties of the host response with depletion of naïve T cells and accumulation of terminal-effector T cells may contribute to the biologic basis of disease risk. Properties of immune microenvironment identified here may also impact optimal application of immune therapies, including T cell-redirection approaches in childhood leukemia.
Jithendra Kini Bailur, Samuel S. McCachren, Katherine E. Pendleton, Juan C. Vasquez, Hong S. Lim, Alyssa Duffy, Deon Doxie, Akhilesh Kaushal, Connor J.R. Foster, Deborah DeRyckere, Sharon M. Castellino, Melissa L. Kemp, Peng Qiu, Madhav Dhodapkar, Kavita Dhodapkar
Clostridioides difficile is a leading cause of nosocomial infection responsible for significant morbidity and mortality with limited options for therapy. Secreted C. difficile toxin B (TcdB) is a major contributor to disease pathology and select TcdB-specific Abs may protect against disease recurrence. However, the high frequency of recurrence suggests that the memory B cell response, essential for new Ab production following C. difficile re-exposure, is insufficient. We therefore isolated TcdB-specific memory B cells from individuals with a history of C. difficile infection and performed single-cell deep sequencing of their Ab genes. Herein, we report that TcdB-specific memory B cell-encoded antibodies showed somatic hypermutation but displayed limited isotype class switch. Memory B cell-encoded monoclonal antibodies generated from the gene sequences revealed low to moderate affinity for TcdB and a limited ability to neutralize TcdB. These findings indicate that memory B cells are an important factor in C. difficile disease recurrence.
Hemangi B. Shah, Kenneth Smith, Edgar J. Scott, II, Jason L. Larabee, Judith A. James, Jimmy D. Ballard, Mark L. Lang
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