A substantial proportion of patients who have recovered from coronavirus disease-2019 (COVID-19) experience COVID-19-related symptoms, even months after hospital discharge. We extensively immunologically characterized patients who recovered from COVID-19. In these patients, T cells were exhausted, with increased PD-1+ T cells, as compared to healthy controls. Plasma levels of IL-1ß, IL-1RA and IL-8, among others, were also increased in patients who recovered from COVID-19. This altered immunophenotype was mirrored by a reduced ex vivo T cell response to both nonspecific and specific stimulation, revealing a dysfunctional status of T cells, including a poor response to SARS-CoV-2 antigens. Altered levels of plasma soluble PD-L1 as well as of PD1 promoter methylation and PD1-targeting miR-15-5p in CD8+ T cells were also observed, suggesting abnormal function of the PD-1/PD-L1 immune checkpoint axis. Notably, ex vivo blockade of PD-1 nearly normalized the aforementioned immunophenotype and restored T cell function, reverting the observed post-COVID-19 immune abnormalities; indeed, we also noted an increased T cell-mediated response to SARS-CoV-2 peptides. Finally, in a neutralization assay, PD-1 blockade did not alter the ability of T cells to neutralize SARS-CoV-2 spike pseudotyped lentivirus infection. Immune checkpoint blockade ameliorates post-COVID-19 immune abnormalities and stimulates an anti-SARS-CoV-2 immune response.
Cristian Loretelli, Ahmed Abdelsalam, Francesca D'Addio, Moufida Ben Nasr, Emma Assi, Vera Usuelli, Anna Maestroni, Andy Joe Seelam, Elio Ippolito, Stefania Di Maggio, Lara Loreggian, Dejan Radovanovic, Claudia Vanetti, Jun Yang, Basset El Essawy, Antonio Rossi, Ida Pastore, Laura Montefusco, Maria Elena Lunati, Andrea M. Bolla, Mara Biasin, Spinello Antinori, Pierachille Santus, Agostino Riva, Gianvincenzo Zuccotti, Massimo Galli, Stefano Rusconi, Paolo Fiorina
Kidneys are critical target organs of COVID-19, but susceptibility and responses to infection remain poorly understood. Here, we combine SARS-CoV-2 variants with genome edited kidney organoids and clinical data to investigate tropism, mechanism, and therapeutics. SARS-CoV-2 specifically infects organoid proximal tubules amongst diverse cell types. Infections produce replicating virus, apoptosis, and disrupted cell morphology, features of which are revealed in the context of polycystic kidney disease. Cross-validation of gene expression patterns in organoids reflect proteomic signatures of COVID-19 in the urine of critically ill patients indicating interferon pathway upregulation. SARS-CoV-2 viral variants Alpha, Beta, Gamma, Kappa, and Delta exhibit comparable levels of replication in organoids. Infection is ameliorated in ACE2-/- organoids and blocked via treatment with de novo designed spike binder peptides. Collectively, these studies clarify the impact of kidney infection in COVID-19 as reflected in organoids and clinical populations, enabling assessment of viral fitness and emerging therapies.
Louisa Helms, Silvia Marchiano, Ian B. Stanaway, Tien-Ying Hsiang, Benjamin A. Juliar, Shally Saini, Yan Ting Zhao, Akshita Khanna, Rajasree Menon, Fadhl Alakwaa, Carmen Mikacenic, Eric D. Morrell, Mark M. Wurfel, Matthias Kretzler, Jennifer L. Harder, Charles E. Murry, Jonathan Himmelfarb, Hannele Ruohola-Baker, Pavan K. Bhatraju, Michael Gale, Jr., Benjamin S. Freedman
Inhibitors of the renin-angiotensin system (RAS) are widely used to treat hypertension. Using mice harboring fluorescent cell lineage tracers, single-cell RNA-seq, and long-term inhibition of RAS in both mice and humans, we found that deletion of renin or inhibition of the RAS leads to concentric thickening of the intrarenal arteries and arterioles. This severe disease is caused by the multiclonal expansion and transformation of renin cells from a classical endocrine phenotype to a matrix-secretory phenotype: the cells surround the vessel walls and induce the accumulation of adjacent smooth muscle cells and extracellular matrix, resulting in blood flow obstruction, focal ischemia, and fibrosis. Ablation of the renin cells via conditional deletion of β1integrin prevents arteriolar hypertrophy, indicating that renin cells are responsible for vascular disease. Given these findings, prospective morphological studies in humans are necessary to determine the extent of renal-vascular damage caused by the widespread use of inhibitors of RAS.
Hirofumi Watanabe, Alexandre G. Martini, Evan A. Brown, Xiuyin Liang, Silvia Medrano, Shin Goto, Ichiei Narita, Lois J. Arend, Maria Luisa S. Sequeira-Lopez, R. Ariel Gomez
BACKGROUND. Although aberrant glycosylation is recognized as a hallmark of cancer, glycosylation in clinical breast cancer (BC) metastasis has not yet been studied. While preclinical studies show that the glycocalyx coating of cancer cells is involved in adhesion, migration, and metastasis, glycosylation changes from primary tumor (PT) to various metastatic sites remain unknown in patients. METHODS. We investigated N-glycosylation profiles in 17 metastatic BC patients from our rapid autopsy program. Primary breast tumor, lymph node metastases, multiple systemic metastases, and various normal tissue cores from each patient were arranged on unique single-patient tissue microarrays (TMAs). We performed mass spectrometry imaging (MSI) combined with extensive pathology annotation of these TMAs, which enabled spatially differentiated cell-based analysis of N-glycosylation patterns in metastatic BC. RESULTS. N-glycan abundance increased during metastatic progression independent of BC subtype and treatment regimen, with high-mannose glycans most frequently elevated in BC metastases, followed by fucosylated and complex glycans. Bone metastasis, however, displayed increased core-fucosylation and decreased high-mannose glycans. Consistently, N-glycosylated proteins and N-glycan biosynthesis genes were differentially expressed during metastatic BC progression, with reduced expression of EpCAM and mannose-trimming enzymes and elevated N-glycan branching and sialylation enzymes in BC metastases versus PT. CONCLUSION. We show for the first time in patients that N-glycosylation of breast cancer cells undergoing metastasis occurs in a metastatic site-specific manner, supporting the clinical importance of high-mannose, fucosylated, and complex N-glycans as future diagnostic markers and therapeutic targets in metastatic BC. FUNDING. United States National Institutes of Health grants NIH R01CA213428, R01CA213492, T32CA193145, Dutch Province Limburg “LINK”, European Union ERA-NET TRANSCAN2-643638.
Klára Ščupáková, Oluwatobi T. Adelaja, Benjamin Balluff, Vinay Ayyappan, Caitlin M. Tressler, Nicole M. Jenkinson, Britt S.R. Claes, Andrew P. Bowman, Ashley M. Cimino-Mathews, Marissa J. White, Pedram Argani, Ron M.A. Heeren, Kristine Glunde
Tumor necrosis factor (TNF) ligation of TNF receptor 1 (TNFR1) promotes either inflammation and cell survival by inhibiting RIPK1’s death-signaling function and activating NF-kB, or causes RIPK1 to associate with the death-inducing signaling complex to initiate apoptosis or necroptosis. The cellular source of TNF that results in RIPK1-dependent cell death remains unclear. To address this, we employed in vitro systems and murine models of T cell-dependent transplant or tumor rejection in which target cell susceptibility to RIPK1-dependent cell death could be genetically altered. We show that TNF released by T cells is necessary and sufficient to activate RIPK1-dependent cell death in target cells and thereby mediate target cell cytolysis, independent of T cell frequency. Activation of the RIPK1-dependent cell death program in target cells by T cell-derived TNF accelerates murine cardiac allograft rejection and synergizes with anti-PD1 administration to destroy checkpoint blockade-resistant, murine melanoma. Together, the findings uncover a distinct immunological role for TNF released by cytotoxic effector T cells following cognate interactions with their antigenic targets. Manipulating T cell TNF and/or target cell susceptibility to RIPK1-dependent cell death can be exploited to either mitigate or augment T cell-dependent destruction of allografts and malignancies to improve outcomes.
Nicholas Chun, Rosalind L. Ang, Mark Chan, Robert L. Fairchild, William M. Baldwin III, Julian K. Horwitz, Jesse D. Gelles, Jerry Edward Chipuk, Michelle A. Kelliher, Vasile I. Pavlov, Yansui Li, Dirk Homann, Peter S. Heeger, Adrian T. Ting
Glucagon, a hormone released from pancreatic α-cells, plays a key role in maintaining euglycemia. New insights into the signaling pathways that control glucagon secretion may stimulate the development of novel therapeutic agents. In this study, we investigated the potential regulation of α-cell function by G proteins of the Gq family. The use of a chemogenetic strategy allowed us to selectively activate Gq signaling in mouse α-cells in vitro and in vivo. Acute stimulation of α-cell Gq signaling led to elevated plasma glucagon levels, accompanied by increased insulin release and improved glucose tolerance. Moreover, chronic activation of this pathway greatly improved glucose tolerance in obese mice. We also identified an endogenous Gq-coupled receptor (vasopressin 1b receptor; V1bR) that is enriched in mouse and human α-cells. Agonist-induced activation of the V1bR strongly stimulated glucagon release in a Gq-dependent fashion. In vivo studies indicated that V1bR-mediated glucagon release plays a key role in the counter-regulatory hyperglucagonemia under hypoglycemic and glucopenic conditions. These data indicate that α-cell Gq signaling represents an important regulator of glucagon secretion, resulting in multiple beneficial metabolic effects. Thus, drugs that target α-cell enriched Gq-coupled receptors may prove useful to restore euglycemia in various pathophysiological conditions.
Liu Liu, Diptadip Dattaroy, Katherine F. Simpson, Luiz F. Barella, Yinghong Cui, Yan Xiong, Jian Jin, Gabriele M. König, Evi Kostenis, Jefferey C. Roman, Klaus H. Kaestner, Nicolai M. Doliba, Jürgen Wess
The PD-1: PD-L1 is a potent inhibitory pathway involved in immune regulation and a potential therapeutic target in transplantation. In this study, we show that overexpression of PD-1 (PD-1 Tg) on T cells promotes allograft tolerance in a fully MHC-mismatched cardiac transplant model when combined with costimulation blockade (CTLA-4-Ig). PD-1 overexpression on T cells also protected against chronic rejection in a single MHC II mismatched cardiac transplant model, while it still allowed the generation of an effective immune response against an Influenza A virus. Notably, Treg cells from PD-1 Tg mice were required for tolerance induction and presented higher ICOS expression than those from wild-type mice. Survival benefit of PD-1 Tg recipients required ICOS signaling and donor PD-L1 expression. These results indicate that modulation of PD-1 expression, in combination with a costimulation blockade, is a promising therapeutic target to promote transplant tolerance.
Thiago J. Borges, Naoka Murakami, Isadora T. Lape, Rodrigo B. Gassen, Kaifeng Liu, Songjie Cai, Joe Daccache, Kassem Safa, Tetsunosuke Shimizu, Shunsuke Ohori, Alison M. Paterson, Paolo Cravedi, Jamil Azzi, Peter Sage, Arlene Sharpe, Xian C. Li, Leonardo V. Riella
Severe respiratory coronavirus 2 (SARS-CoV-2) promotes an imbalanced host response which underlies the development and severity of COVID-19. Infections with viruses are known to modulate transposable elements (TEs) which can exert downstream effects by modulating host gene expression, innate immune sensing, or activities encoded by their protein products. We investigated the impact of SARS-CoV-2 infection on TE expression using RNA-seq data from cell lines and from primary patient samples. Using a bioinformatic tool, Telescope, we showed that SARS-CoV-2 infection led to up- or down-regulation of TE transcripts, a subset of which differed from cells infected with SARS, MERS, RSV, HPIV3 or IAV. Differential expression of key retroelements specifically identified distinct virus families such as coronaviridae, with unique retroelement expression subdividing viral species. Analysis of ChIP-seq data shows that TEs differentially expressed in SARS-CoV-2 infection are enriched for binding sites for TFs involved in immune responses and for pioneer transcription factors. In COVID-19 patient samples, there was a significant TE overexpression in bronchoalveolar lavage fluid and downregulation in peripheral blood mononuclear cells. Thus, while the host gene transcriptome is altered by infection with SARS-CoV-2, the retrotranscriptome may contain the most distinctive features of the cellular response to SARS-CoV-2 infection.
Jez L. Marston, Matthew Greenig, Manvendra Singh, Matthew L. Bendall, Rodrigo R.R. Duarte, Cédric Feschotte, Luis P. Iñiguez, Douglas F. Nixon
Nociceptors, the high-threshold primary sensory neurons that trigger pain, interact with immune cells in the periphery to modulate innate immune responses. Whether they also participate in adaptive and humoral immunity is, however, not known. In this study, we probed if nociceptors have a role in distinct airway and skin models of allergic inflammation. In both models, the genetic ablation and pharmacological silencing of nociceptors substantially reduced inflammatory cell infiltration to the affected tissue. Moreover, we also found a profound and specific deficit in IgE production in these models of allergic inflammation. Mechanistically, we discovered that the nociceptor-released neuropeptide Substance P help triggered the formation of antibody secreting cells and their release of IgE. Our findings suggest that nociceptors, in addition to their contributions to innate immunity, play a key role in modulating the adaptive immune response, particularly B cell antibody class switching to IgE.
Shreya Mathur, Jo-Chiao Wang, Corey R. Seehus, Florence Poirier, Theo Crosson, Yu-Chen Hsieh, Benjamin Doyle, Seungkyu Lee, Clifford J. Woolf, Simmie L. Foster, Sebastien Talbot
Leukemia stem cells (LSCs) promote the disease and seem resistant to therapy and immune control. Why LSCs are selectively resistant against elimination by cytotoxic CD8+ T cells (CTLs) is still unknown. In this study, we demonstrate that LSCs in chronic myeloid leukemia (CML) can be recognized and killed by CD8+ CTLs in vitro. However, Tregs, which preferentially localized close to CD8+ CTLs in CML bone marrow (BM), protected LSCs from MHC-class I dependent CD8+ CTL-mediated elimination in vivo. BM Tregs in CML were characterized by the selective expression of tumor necrosis factor receptor 4 (Tnfrsf4). Stimulation of Tnfrsf4-signaling did not deplete Tregs but reduced the capacity of Tregs to protect LSCs from CD8+ CTL-mediated killing. In the BM of newly diagnosed CML patients, TNFRSF4 mRNA levels were significantly increased and correlated with the expression of the Treg-restricted transcription factor FOXP3. Overall, these results identify Tregs as key regulator of immune escape of LSCs and TNFRSF4 as a potential target to reduce the function of Tregs and boost anti-leukemic immunity in CML.
Magdalena Hinterbrandner, Viviana Rubino, Carina Stoll, Stefan Forster, Noah Schnüriger, Ramin Radpour, Gabriela M. Baerlocher, Adrian F. Ochsenbein, Carsten Riether
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