Pub Date : 2025-03-19DOI: 10.1126/scitranslmed.adt2124
Matthew Lacorcia, Pushpak Bhattacharjee, Abby Foster, Melinda Y. Hardy, Jason A. Tye-Din, John A. Karas, John M. Wentworth, Fergus J. Cameron, Stuart I. Mannering
Type 1 diabetes (T1D) is an autoimmune disease where T cells mediate the destruction of the insulin-producing β cells found within the islets of Langerhans in the pancreas. Autoantibodies to β cell antigens are the only tests available to detect β cell autoimmunity. T cell responses to β cell antigens, which are known to cause T1D, can only be measured in research settings because of the complexity of assays and the large blood volumes required. Here, we describe the β cell antigen–specific T cell assay (BASTA). BASTA is a simple whole-blood assay that can detect human CD4 + T cell responses to β cell antigens by measuring antigen-stimulated interleukin-2 (IL-2) production. BASTA is both more sensitive and specific than the CFSE (carboxyfluorescein diacetate succinimidyl ester)–based proliferation assay. We used BASTA to identify the regions of preproinsulin that stimulated T cell responses specifically in blood from people with T1D. BASTA can be done with as little as 2 to 3 milliliters of blood. We found that effector memory CD4 + T cells are the primary producers of IL-2 in response to preproinsulin peptides. We then evaluated responses to individual and pooled preproinsulin peptides in a cross-sectional study of pediatric patients: without T1D, without T1D but with a first-degree relative with T1D, or diagnosed with T1D. In contrast with other preproinsulin peptides, full-length C-peptide (PI 33–63 ) showed high specificity for T1D [area under the curve (AUC) = 0.86)]. We suggest that BASTA will be a useful tool for monitoring changes in β cell–specific CD4 + T cell responses both in research and clinical settings.
{"title":"BASTA, a simple whole-blood assay for measuring β cell antigen–specific CD4 + T cell responses in type 1 diabetes","authors":"Matthew Lacorcia, Pushpak Bhattacharjee, Abby Foster, Melinda Y. Hardy, Jason A. Tye-Din, John A. Karas, John M. Wentworth, Fergus J. Cameron, Stuart I. Mannering","doi":"10.1126/scitranslmed.adt2124","DOIUrl":"https://doi.org/10.1126/scitranslmed.adt2124","url":null,"abstract":"Type 1 diabetes (T1D) is an autoimmune disease where T cells mediate the destruction of the insulin-producing β cells found within the islets of Langerhans in the pancreas. Autoantibodies to β cell antigens are the only tests available to detect β cell autoimmunity. T cell responses to β cell antigens, which are known to cause T1D, can only be measured in research settings because of the complexity of assays and the large blood volumes required. Here, we describe the β cell antigen–specific T cell assay (BASTA). BASTA is a simple whole-blood assay that can detect human CD4 <jats:sup>+</jats:sup> T cell responses to β cell antigens by measuring antigen-stimulated interleukin-2 (IL-2) production. BASTA is both more sensitive and specific than the CFSE (carboxyfluorescein diacetate succinimidyl ester)–based proliferation assay. We used BASTA to identify the regions of preproinsulin that stimulated T cell responses specifically in blood from people with T1D. BASTA can be done with as little as 2 to 3 milliliters of blood. We found that effector memory CD4 <jats:sup>+</jats:sup> T cells are the primary producers of IL-2 in response to preproinsulin peptides. We then evaluated responses to individual and pooled preproinsulin peptides in a cross-sectional study of pediatric patients: without T1D, without T1D but with a first-degree relative with T1D, or diagnosed with T1D. In contrast with other preproinsulin peptides, full-length C-peptide (PI <jats:sub>33–63</jats:sub> ) showed high specificity for T1D [area under the curve (AUC) = 0.86)]. We suggest that BASTA will be a useful tool for monitoring changes in β cell–specific CD4 <jats:sup>+</jats:sup> T cell responses both in research and clinical settings.","PeriodicalId":21580,"journal":{"name":"Science Translational Medicine","volume":"55 1","pages":""},"PeriodicalIF":17.1,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143653435","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-19DOI: 10.1126/scitranslmed.adn5603
John K. Mich, Jiyun Ryu, Aguan D. Wei, Bryan B. Gore, Rong Guo, Angela M. Bard, Refugio A. Martinez, Emily M. Luber, Jiatai Liu, Yemeserach M. Bishaw, Robert J. Christian, Luiz M. Oliveira, Nicole Miranda, Jan-Marino Ramirez, Jonathan T. Ting, Ed S. Lein, Boaz P. Levi, Franck K. Kalume
Dravet syndrome (DS) is a severe developmental epileptic encephalopathy marked by treatment-resistant seizures, developmental delay, intellectual disability, motor deficits, and a 10 to 20% rate of premature death. Most patients with DS harbor loss-of-function mutations in one copy of SCN1A , which encodes the voltage-gated sodium channel (Na V )1.1 alpha subunit and has been associated with inhibitory neuron dysfunction. Here, we generated a split-intein form of SCN1A and used a dual-vector delivery approach to circumvent adeno-associated virus (AAV) packaging limitations. In addition, we applied previously developed enhancer technology to produce an interneuron-specific gene replacement therapy for DS, called DLX2.0- SCN1A . The split-intein SCN1A vectors produced full-length Na V 1.1 protein, and functional sodium channels were recorded in HEK293 cells in vitro. Administration of dual DLX2.0- SCN1A AAVs to wild-type mice produced full-length, reconstituted human protein by Western blot and telencephalic interneuron–specific and dose-dependent Na V 1.1 expression by immunohistochemistry. These vectors also conferred strong dose-dependent protection against postnatal mortality and seizures in Scn1a fl/+ ;Meox2-Cre and Scn1a +/R613X DS mouse models. Injection of single or dual DLX2.0- SCN1A AAVs into wild-type mice did not result in increased mortality, weight loss, or gliosis as measured by immunohistochemistry. In contrast, expression of SCN1A in all neurons driven by the human SYNAPSIN I promoter caused an adverse effect marked by increased mortality in the preweaning period, before disease onset. These findings demonstrate proof of concept that interneuron-specific AAV-mediated SCN1A gene replacement can rescue DS phenotypes in mouse models and suggest that it could be a therapeutic approach for patients with DS.
{"title":"Interneuron-specific dual-AAV SCN1A gene replacement corrects epileptic phenotypes in mouse models of Dravet syndrome","authors":"John K. Mich, Jiyun Ryu, Aguan D. Wei, Bryan B. Gore, Rong Guo, Angela M. Bard, Refugio A. Martinez, Emily M. Luber, Jiatai Liu, Yemeserach M. Bishaw, Robert J. Christian, Luiz M. Oliveira, Nicole Miranda, Jan-Marino Ramirez, Jonathan T. Ting, Ed S. Lein, Boaz P. Levi, Franck K. Kalume","doi":"10.1126/scitranslmed.adn5603","DOIUrl":"https://doi.org/10.1126/scitranslmed.adn5603","url":null,"abstract":"Dravet syndrome (DS) is a severe developmental epileptic encephalopathy marked by treatment-resistant seizures, developmental delay, intellectual disability, motor deficits, and a 10 to 20% rate of premature death. Most patients with DS harbor loss-of-function mutations in one copy of <jats:italic>SCN1A</jats:italic> , which encodes the voltage-gated sodium channel (Na <jats:sub>V</jats:sub> )1.1 alpha subunit and has been associated with inhibitory neuron dysfunction. Here, we generated a split-intein form of <jats:italic>SCN1A</jats:italic> and used a dual-vector delivery approach to circumvent adeno-associated virus (AAV) packaging limitations. In addition, we applied previously developed enhancer technology to produce an interneuron-specific gene replacement therapy for DS, called DLX2.0- <jats:italic>SCN1A</jats:italic> . The split-intein <jats:italic>SCN1A</jats:italic> vectors produced full-length Na <jats:sub>V</jats:sub> 1.1 protein, and functional sodium channels were recorded in HEK293 cells in vitro. Administration of dual DLX2.0- <jats:italic>SCN1A</jats:italic> AAVs to wild-type mice produced full-length, reconstituted human protein by Western blot and telencephalic interneuron–specific and dose-dependent Na <jats:sub>V</jats:sub> 1.1 expression by immunohistochemistry. These vectors also conferred strong dose-dependent protection against postnatal mortality and seizures in <jats:italic> Scn1a <jats:sup>fl/+</jats:sup> ;Meox2-Cre </jats:italic> and <jats:italic> Scn1a <jats:sup>+/R613X</jats:sup> </jats:italic> DS mouse models. Injection of single or dual DLX2.0- <jats:italic>SCN1A</jats:italic> AAVs into wild-type mice did not result in increased mortality, weight loss, or gliosis as measured by immunohistochemistry. In contrast, expression of <jats:italic>SCN1A</jats:italic> in all neurons driven by the human <jats:italic>SYNAPSIN I</jats:italic> promoter caused an adverse effect marked by increased mortality in the preweaning period, before disease onset. These findings demonstrate proof of concept that interneuron-specific AAV-mediated <jats:italic>SCN1A</jats:italic> gene replacement can rescue DS phenotypes in mouse models and suggest that it could be a therapeutic approach for patients with DS.","PeriodicalId":21580,"journal":{"name":"Science Translational Medicine","volume":"90 1","pages":""},"PeriodicalIF":17.1,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143653436","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-19DOI: 10.1126/scitranslmed.adp0675
Itay Raphael, Zujian Xiong, Chaim T. Sneiderman, Rebecca A. Raphael, Moshe Mash, Lance Schwegman, Sydney A. Jackson, Casey O’Brien, Kevin J. Anderson, ReidAnn E. Sever, Liam D. Hendrikse, Sarah R. Vincze, Aaron Diaz, James Felker, Javad Nazarian, Yael Nechemia-Arbely, Baoli Hu, Udai S. Kammula, Sameer Agnihotri, Jeremy N. Rich, Alberto Broniscer, Jan Drappatz, Taylor J. Abel, Shikhar Uttam, Eugene I. Hwang, Thomas M. Pearce, Michael D. Taylor, Michal Nisnboym, Thomas G. Forsthuber, Ian F. Pollack, Maria Chikina, Dhivyaa Rajasundaram, Gary Kohanbash
The diverse T cell receptor (TCR) repertoire confers the ability to recognize an almost unlimited array of antigens. Characterization of antigen specificity of tumor-infiltrating lymphocytes (TILs) is key for understanding antitumor immunity and for guiding the development of effective immunotherapies. Here, we report a large-scale comprehensive examination of the TCR landscape of TILs across the spectrum of pediatric brain tumors, the leading cause of cancer-related mortality in children. We show that a T cell clonality index can inform patient prognosis, where more clonality is associated with more favorable outcomes. Moreover, TCR similarity groups’ assessment revealed patient clusters with defined human leukocyte antigen associations. Computational analysis of these clusters identified putative tumor antigens and peptides as targets for antitumor T cell immunity, which were functionally validated by T cell stimulation assays in vitro. Together, this study presents a framework for tumor antigen prediction based on in situ and in silico TIL TCR analyses. We propose that TCR-based investigations should inform tumor classification and precision immunotherapy development.
{"title":"The T cell receptor landscape of childhood brain tumors","authors":"Itay Raphael, Zujian Xiong, Chaim T. Sneiderman, Rebecca A. Raphael, Moshe Mash, Lance Schwegman, Sydney A. Jackson, Casey O’Brien, Kevin J. Anderson, ReidAnn E. Sever, Liam D. Hendrikse, Sarah R. Vincze, Aaron Diaz, James Felker, Javad Nazarian, Yael Nechemia-Arbely, Baoli Hu, Udai S. Kammula, Sameer Agnihotri, Jeremy N. Rich, Alberto Broniscer, Jan Drappatz, Taylor J. Abel, Shikhar Uttam, Eugene I. Hwang, Thomas M. Pearce, Michael D. Taylor, Michal Nisnboym, Thomas G. Forsthuber, Ian F. Pollack, Maria Chikina, Dhivyaa Rajasundaram, Gary Kohanbash","doi":"10.1126/scitranslmed.adp0675","DOIUrl":"https://doi.org/10.1126/scitranslmed.adp0675","url":null,"abstract":"The diverse T cell receptor (TCR) repertoire confers the ability to recognize an almost unlimited array of antigens. Characterization of antigen specificity of tumor-infiltrating lymphocytes (TILs) is key for understanding antitumor immunity and for guiding the development of effective immunotherapies. Here, we report a large-scale comprehensive examination of the TCR landscape of TILs across the spectrum of pediatric brain tumors, the leading cause of cancer-related mortality in children. We show that a T cell clonality index can inform patient prognosis, where more clonality is associated with more favorable outcomes. Moreover, TCR similarity groups’ assessment revealed patient clusters with defined human leukocyte antigen associations. Computational analysis of these clusters identified putative tumor antigens and peptides as targets for antitumor T cell immunity, which were functionally validated by T cell stimulation assays in vitro. Together, this study presents a framework for tumor antigen prediction based on in situ and in silico TIL TCR analyses. We propose that TCR-based investigations should inform tumor classification and precision immunotherapy development.","PeriodicalId":21580,"journal":{"name":"Science Translational Medicine","volume":"43 1","pages":""},"PeriodicalIF":17.1,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143653472","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-19DOI: 10.1126/scitranslmed.adr8941
Raphaël Porret, Ana Alcaraz-Serna, Benjamin Peter, Jeremiah Bernier-Latmani, Rebecca Cecchin, Oscar Alfageme-Abello, Laura Ermellino, Morteza Hafezi, Eleonora Pace, M. Fleur du Pré, Erica Lana, Dela Golshayan, Dominique Velin, Justin Eyquem, Qizhi Tang, Tatiana V. Petrova, George Coukos, Melita Irving, Caroline Pot, Giuseppe Pantaleo, Ludvig M. Sollid, Yannick D. Muller
Celiac disease, a gluten-sensitive enteropathy, demonstrates a strong human leukocyte antigen (HLA) association, with more than 90% of patients carrying the HLA-DQ2.5 allotype. No therapy is available for the condition except for a lifelong gluten-free diet. To address this gap, we explored the therapeutic potential of regulatory T cells (T regs ). By orthotopic replacement of T cell receptors (TCRs) through homology-directed repair, we generated gluten-reactive HLA-DQ2.5–restricted CD4 + engineered (e) T effector cells (T effs ) and eT regs and performed in vivo experiments in HLA-DQ2.5 transgenic mice. Of five validated TCRs, TCRs specific for two immunodominant and deamidated gluten epitopes (DQ2.5-glia-α1a and DQ2.5-glia-α2) were selected for further evaluation. CD4 + eT effs exposed to deamidated gluten through oral gavage colocalized with dendritic and B cells in the Peyer’s patches and gut-draining lymph nodes and specifically migrated to the intestine. The suppressive function of human eT regs correlated with high TCR functional activity. eT regs specific for one epitope suppressed the proliferation and gut migration of CD4 + eT effs specific for the same and the other gluten epitope, demonstrating bystander suppression. The suppression requires an antigen-specific activation of eT regs given that polyclonal T regs failed to suppress CD4 + eT effs . These findings highlight the potential of gluten-reactive eT regs as a therapeutic for celiac disease.
{"title":"T cell receptor precision editing of regulatory T cells for celiac disease","authors":"Raphaël Porret, Ana Alcaraz-Serna, Benjamin Peter, Jeremiah Bernier-Latmani, Rebecca Cecchin, Oscar Alfageme-Abello, Laura Ermellino, Morteza Hafezi, Eleonora Pace, M. Fleur du Pré, Erica Lana, Dela Golshayan, Dominique Velin, Justin Eyquem, Qizhi Tang, Tatiana V. Petrova, George Coukos, Melita Irving, Caroline Pot, Giuseppe Pantaleo, Ludvig M. Sollid, Yannick D. Muller","doi":"10.1126/scitranslmed.adr8941","DOIUrl":"https://doi.org/10.1126/scitranslmed.adr8941","url":null,"abstract":"Celiac disease, a gluten-sensitive enteropathy, demonstrates a strong human leukocyte antigen (HLA) association, with more than 90% of patients carrying the HLA-DQ2.5 allotype. No therapy is available for the condition except for a lifelong gluten-free diet. To address this gap, we explored the therapeutic potential of regulatory T cells (T <jats:sub>regs</jats:sub> ). By orthotopic replacement of T cell receptors (TCRs) through homology-directed repair, we generated gluten-reactive HLA-DQ2.5–restricted CD4 <jats:sup>+</jats:sup> engineered (e) T effector cells (T <jats:sub>effs</jats:sub> ) and eT <jats:sub>regs</jats:sub> and performed in vivo experiments in HLA-DQ2.5 transgenic mice. Of five validated TCRs, TCRs specific for two immunodominant and deamidated gluten epitopes (DQ2.5-glia-α1a and DQ2.5-glia-α2) were selected for further evaluation. CD4 <jats:sup>+</jats:sup> eT <jats:sub>effs</jats:sub> exposed to deamidated gluten through oral gavage colocalized with dendritic and B cells in the Peyer’s patches and gut-draining lymph nodes and specifically migrated to the intestine. The suppressive function of human eT <jats:sub>regs</jats:sub> correlated with high TCR functional activity. eT <jats:sub>regs</jats:sub> specific for one epitope suppressed the proliferation and gut migration of CD4 <jats:sup>+</jats:sup> eT <jats:sub>effs</jats:sub> specific for the same and the other gluten epitope, demonstrating bystander suppression. The suppression requires an antigen-specific activation of eT <jats:sub>regs</jats:sub> given that polyclonal T <jats:sub>regs</jats:sub> failed to suppress CD4 <jats:sup>+</jats:sup> eT <jats:sub>effs</jats:sub> . These findings highlight the potential of gluten-reactive eT <jats:sub>regs</jats:sub> as a therapeutic for celiac disease.","PeriodicalId":21580,"journal":{"name":"Science Translational Medicine","volume":"40 1","pages":""},"PeriodicalIF":17.1,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143653438","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-19DOI: 10.1126/scitranslmed.adt0527
Olivia R. Hoffman, Jennifer L. Koehler, Jose Ezekiel Clemente Espina, Anna M. Patterson, Emily S. Gohar, Emanuel M. Coleman, Barry A. Schoenike, Claudia Espinosa-Garcia, Felipe Paredes, Nicholas H. Varvel, Raymond J. Dingledine, Jamie L. Maguire, Avtar S. Roopra
All current drug treatments for epilepsy, a neurological disorder affecting more than 50 million people, merely treat symptoms, and a third of patients with epilepsy do not respond to medication. There are no disease-modifying treatments that may be administered briefly to patients to enduringly eliminate spontaneous seizures and reverse cognitive deficits. Applying network approaches to whole tissue and single-nucleus transcriptomic data collected from mouse models of temporal lobe epilepsy and publicly available transcriptomic data from human temporal lobectomy samples, we confirmed a previously described pattern of rapid and transient induction of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway within days of epileptogenic insult. This was followed by a resurgent activation of the JAK/STAT pathway weeks to months later with the onset of spontaneous seizures. Targeting the first wave of JAK/STAT activation after epileptic insult did not prevent seizures. However, inhibition of the second wave with CP690550 (tofacitinib) over a 2-week period enduringly suppressed seizures, rescued deficits in spatial memory, and alleviated epilepsy-associated histopathological alterations. Seizure suppression lasted for at least 2 months after the final dose. These results indicate that reignition of inflammatory JAK/STAT3 signaling in chronic epilepsy opens a window for disease modification with the US Food and Drug Administration–approved, orally available drug CP690550.
{"title":"Disease modification upon 2 weeks of tofacitinib treatment in a mouse model of chronic epilepsy","authors":"Olivia R. Hoffman, Jennifer L. Koehler, Jose Ezekiel Clemente Espina, Anna M. Patterson, Emily S. Gohar, Emanuel M. Coleman, Barry A. Schoenike, Claudia Espinosa-Garcia, Felipe Paredes, Nicholas H. Varvel, Raymond J. Dingledine, Jamie L. Maguire, Avtar S. Roopra","doi":"10.1126/scitranslmed.adt0527","DOIUrl":"https://doi.org/10.1126/scitranslmed.adt0527","url":null,"abstract":"All current drug treatments for epilepsy, a neurological disorder affecting more than 50 million people, merely treat symptoms, and a third of patients with epilepsy do not respond to medication. There are no disease-modifying treatments that may be administered briefly to patients to enduringly eliminate spontaneous seizures and reverse cognitive deficits. Applying network approaches to whole tissue and single-nucleus transcriptomic data collected from mouse models of temporal lobe epilepsy and publicly available transcriptomic data from human temporal lobectomy samples, we confirmed a previously described pattern of rapid and transient induction of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway within days of epileptogenic insult. This was followed by a resurgent activation of the JAK/STAT pathway weeks to months later with the onset of spontaneous seizures. Targeting the first wave of JAK/STAT activation after epileptic insult did not prevent seizures. However, inhibition of the second wave with CP690550 (tofacitinib) over a 2-week period enduringly suppressed seizures, rescued deficits in spatial memory, and alleviated epilepsy-associated histopathological alterations. Seizure suppression lasted for at least 2 months after the final dose. These results indicate that reignition of inflammatory JAK/STAT3 signaling in chronic epilepsy opens a window for disease modification with the US Food and Drug Administration–approved, orally available drug CP690550.","PeriodicalId":21580,"journal":{"name":"Science Translational Medicine","volume":"37 1","pages":""},"PeriodicalIF":17.1,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143653437","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Hsuan-Yuan Wang, Husam Taher, Craig N. Kreklywich, Kimberli A. Schmidt, Elizabeth A. Scheef, Richard Barfield, Claire E. Otero, Sarah M. Valencia, Ke Zhang, Claire Callahan, Francesco Monticolo, Yueqing Qiao, Roxanne M. Gilbride, Chelsea M. Crooks, Anne Mirza, Kelsey Knight, Matilda J. Moström, Tabitha D. Manuel, Lesli Sprehe, Savannah Kendall, Nathan Vande Burgt, Timothy F. Kowalik, Peter A. Barry, Scott G. Hansen, Jian Shu, Alice F. Tarantal, Cliburn Chan, Daniel N. Streblow, Louis J. Picker, Amitinder Kaur, Klaus Früh, Sallie R. Permar, Daniel Malouli
Congenital cytomegalovirus (cCMV) is the leading infectious cause of neonatal neurological impairment worldwide, but the viral factors enabling vertical spread across the placenta remain undetermined. The pentameric complex (PC), composed of the subunits gH/gL/UL128/UL130/UL131A, has been demonstrated to be important for entry into nonfibroblast cells in vitro. These findings link the PC to broad cell tropism and virus dissemination in vivo, denoting all subunits as potential targets for intervention strategies and vaccine development. To determine the relevance of the PC for congenital transmission in a translational nonhuman primate model, we engineered a rhesus CMV (RhCMV) mutant lacking the orthologs of UL128 and UL130, which demonstrated diminished infection of epithelial cells in vitro. However, intravenous inoculation of either CD4+ T cell–depleted or immunocompetent RhCMV-seronegative pregnant rhesus macaques (RMs) in the early second trimester with the PC-deficient mutant resulted in maternal RhCMV peak plasma viremia similar to inoculations with PC-intact RhCMV, although virus shedding in saliva and urine was limited. Infections with the PC-intact virus induced IgG responses that neutralized RhCMV entry into epithelial cells in tissue culture. These responses were reduced, but not absent, from animals infected with the PC-deficient virus, which also induced IgG responses against gH. Moreover, congenital CMV transmission was confirmed in multiple animals infected with PC-deficient virus by detecting viral DNA in the amniotic fluid, indicating that transplacental transmission in RMs is not contingent on the PC.
{"title":"The pentameric complex is not required for congenital CMV transmission in seronegative rhesus macaques","authors":"Hsuan-Yuan Wang, Husam Taher, Craig N. Kreklywich, Kimberli A. Schmidt, Elizabeth A. Scheef, Richard Barfield, Claire E. Otero, Sarah M. Valencia, Ke Zhang, Claire Callahan, Francesco Monticolo, Yueqing Qiao, Roxanne M. Gilbride, Chelsea M. Crooks, Anne Mirza, Kelsey Knight, Matilda J. Moström, Tabitha D. Manuel, Lesli Sprehe, Savannah Kendall, Nathan Vande Burgt, Timothy F. Kowalik, Peter A. Barry, Scott G. Hansen, Jian Shu, Alice F. Tarantal, Cliburn Chan, Daniel N. Streblow, Louis J. Picker, Amitinder Kaur, Klaus Früh, Sallie R. Permar, Daniel Malouli","doi":"","DOIUrl":"","url":null,"abstract":"<div >Congenital cytomegalovirus (cCMV) is the leading infectious cause of neonatal neurological impairment worldwide, but the viral factors enabling vertical spread across the placenta remain undetermined. The pentameric complex (PC), composed of the subunits gH/gL/UL128/UL130/UL131A, has been demonstrated to be important for entry into nonfibroblast cells in vitro. These findings link the PC to broad cell tropism and virus dissemination in vivo, denoting all subunits as potential targets for intervention strategies and vaccine development. To determine the relevance of the PC for congenital transmission in a translational nonhuman primate model, we engineered a rhesus CMV (RhCMV) mutant lacking the orthologs of UL128 and UL130, which demonstrated diminished infection of epithelial cells in vitro. However, intravenous inoculation of either CD4<sup>+</sup> T cell–depleted or immunocompetent RhCMV-seronegative pregnant rhesus macaques (RMs) in the early second trimester with the PC-deficient mutant resulted in maternal RhCMV peak plasma viremia similar to inoculations with PC-intact RhCMV, although virus shedding in saliva and urine was limited. Infections with the PC-intact virus induced IgG responses that neutralized RhCMV entry into epithelial cells in tissue culture. These responses were reduced, but not absent, from animals infected with the PC-deficient virus, which also induced IgG responses against gH. Moreover, congenital CMV transmission was confirmed in multiple animals infected with PC-deficient virus by detecting viral DNA in the amniotic fluid, indicating that transplacental transmission in RMs is not contingent on the PC.</div>","PeriodicalId":21580,"journal":{"name":"Science Translational Medicine","volume":"17 789","pages":""},"PeriodicalIF":15.8,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.science.org/doi/reader/10.1126/scitranslmed.adm8961","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143602900","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-12DOI: 10.1126/scitranslmed.adp2124
Xingyuan Hu, Xiaoyan Kang, Faming Zhao, Yaoyuan Cui, Yu Fu, Xiaohang Yang, Jingjing Yin, Wenting Li, Junpeng Fan, Bin Yang, Zixuan Fang, Tianyu Qin, Xucui Zhuang, Yiting Liu, Chenzhao Feng, Yunyi Yang, Funian Lu, Li Zhang, Weihao Chen, Miaofang Wu, Ning Du, Xia Sheng, Xin Zhou, Jing Li, Gang Chen, Chaoyang Sun
The benefit of hyperthermic intraperitoneal chemotherapy (HIPEC) in ovarian cancer remains controversial, hindering the development of rational combination therapies based on hyperthermia (HT). This study reports the preliminary results of the neoadjuvant HIPEC (NHIPEC) trial (ChiCTR2000038173), demonstrating enhanced tumor response in high-grade serous ovarian cancer with NHIPEC. Through single-cell RNA sequencing analysis, we identified both homogeneous and heterogeneous cellular responses to HT within the tumor and microenvironment. Epithelial-mesenchymal transition–activated tumor cells and matrix metallopeptidase 11 (MMP-11) + cancer-associated fibroblasts (CAFs) exhibited greater reductions and higher sensitivity to HT. CUT&Tag and RNA sequencing integration unveiled the differential binding programs and transcriptional regulatory mechanisms of HSF1 under normothermia (NT) and HT in tumor cells and CAFs. Furthermore, HT ameliorated the immunosuppressive tumor microenvironment, and in vivo mouse models confirmed the combined antitumor effects of HT and programmed cell death ligand 1 blockade. These findings provide an innovative strategy for rational combination therapy with HT in ovarian cancer.
{"title":"Heterogeneous cellular responses to hyperthermia support combined intraperitoneal hyperthermic immunotherapy for ovarian cancer mouse models","authors":"Xingyuan Hu, Xiaoyan Kang, Faming Zhao, Yaoyuan Cui, Yu Fu, Xiaohang Yang, Jingjing Yin, Wenting Li, Junpeng Fan, Bin Yang, Zixuan Fang, Tianyu Qin, Xucui Zhuang, Yiting Liu, Chenzhao Feng, Yunyi Yang, Funian Lu, Li Zhang, Weihao Chen, Miaofang Wu, Ning Du, Xia Sheng, Xin Zhou, Jing Li, Gang Chen, Chaoyang Sun","doi":"10.1126/scitranslmed.adp2124","DOIUrl":"https://doi.org/10.1126/scitranslmed.adp2124","url":null,"abstract":"The benefit of hyperthermic intraperitoneal chemotherapy (HIPEC) in ovarian cancer remains controversial, hindering the development of rational combination therapies based on hyperthermia (HT). This study reports the preliminary results of the neoadjuvant HIPEC (NHIPEC) trial (ChiCTR2000038173), demonstrating enhanced tumor response in high-grade serous ovarian cancer with NHIPEC. Through single-cell RNA sequencing analysis, we identified both homogeneous and heterogeneous cellular responses to HT within the tumor and microenvironment. Epithelial-mesenchymal transition–activated tumor cells and matrix metallopeptidase 11 (MMP-11) <jats:sup>+</jats:sup> cancer-associated fibroblasts (CAFs) exhibited greater reductions and higher sensitivity to HT. CUT&Tag and RNA sequencing integration unveiled the differential binding programs and transcriptional regulatory mechanisms of HSF1 under normothermia (NT) and HT in tumor cells and CAFs. Furthermore, HT ameliorated the immunosuppressive tumor microenvironment, and in vivo mouse models confirmed the combined antitumor effects of HT and programmed cell death ligand 1 blockade. These findings provide an innovative strategy for rational combination therapy with HT in ovarian cancer.","PeriodicalId":21580,"journal":{"name":"Science Translational Medicine","volume":"14 1","pages":""},"PeriodicalIF":17.1,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143599164","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-12DOI: 10.1126/scitranslmed.adp4754
Leslie A. Nangle, Zhiwen Xu, David Siefker, Christoph Burkart, Yeeting E. Chong, Liting Zhai, Yanyan Geng, Clara Polizzi, Lauren Guy, Lisa Eide, Yao Tong, Sofia Klopp-Savino, Michaela Ferrer, Kaitlyn Rauch, Annie Wang, Kristina Hamel, Steve Crampton, Suzanne Paz, Kyle P. Chiang, Minh-Ha Do, Luke Burman, Darin Lee, Mingjie Zhang, Kathleen Ogilvie, David King, Ryan A. Adams, Paul Schimmel
Interstitial lung disease (ILD) consists of a group of immune-mediated disorders that can cause inflammation and progressive fibrosis of the lungs, representing an area of unmet medical need given the lack of disease-modifying therapies and toxicities associated with current treatment options. Tissue-specific splice variants (SVs) of human aminoacyl-tRNA synthetases (aaRSs) are catalytic nulls thought to confer regulatory functions. One example from human histidyl-tRNA synthetase (HARS), termed HARS WHEP because the splicing event resulted in a protein encompassing the WHEP-TRS domain of HARS (a structurally conserved domain found in multiple aaRSs), is enriched in human lung and up-regulated by inflammatory cytokines in lung and immune cells. Structural analysis of HARS WHEP confirmed a well-organized helix-turn-helix motif. This motif bound specifically and selectively to neuropilin-2 (NRP2), a receptor expressed by myeloid cells in active sites of inflammation, to inhibit expression of proinflammatory receptors and cytokines and to down-regulate inflammatory pathways in primary human macrophages. In animal models of lung injury and ILD, including bleomycin treatment, silicosis, sarcoidosis, chronic hypersensitivity pneumonitis, systemic sclerosis, and rheumatoid arthritis–ILD, HARS WHEP reduced lung inflammation, immune cell infiltration, and fibrosis. In patients with sarcoidosis, efzofitimod treatment resulted in down-regulation of gene expression for inflammatory pathways in peripheral immune cells and stabilization of inflammatory biomarkers in serum after steroid tapering. We demonstrate the immunomodulatory activity of HARS WHEP and present preclinical data supporting ongoing clinical development of the biologic efzofitimod based on HARS WHEP in ILD.
{"title":"A human histidyl-tRNA synthetase splice variant therapeutic targets NRP2 to resolve lung inflammation and fibrosis","authors":"Leslie A. Nangle, Zhiwen Xu, David Siefker, Christoph Burkart, Yeeting E. Chong, Liting Zhai, Yanyan Geng, Clara Polizzi, Lauren Guy, Lisa Eide, Yao Tong, Sofia Klopp-Savino, Michaela Ferrer, Kaitlyn Rauch, Annie Wang, Kristina Hamel, Steve Crampton, Suzanne Paz, Kyle P. Chiang, Minh-Ha Do, Luke Burman, Darin Lee, Mingjie Zhang, Kathleen Ogilvie, David King, Ryan A. Adams, Paul Schimmel","doi":"10.1126/scitranslmed.adp4754","DOIUrl":"https://doi.org/10.1126/scitranslmed.adp4754","url":null,"abstract":"Interstitial lung disease (ILD) consists of a group of immune-mediated disorders that can cause inflammation and progressive fibrosis of the lungs, representing an area of unmet medical need given the lack of disease-modifying therapies and toxicities associated with current treatment options. Tissue-specific splice variants (SVs) of human aminoacyl-tRNA synthetases (aaRSs) are catalytic nulls thought to confer regulatory functions. One example from human histidyl-tRNA synthetase (HARS), termed HARS <jats:sup>WHEP</jats:sup> because the splicing event resulted in a protein encompassing the WHEP-TRS domain of HARS (a structurally conserved domain found in multiple aaRSs), is enriched in human lung and up-regulated by inflammatory cytokines in lung and immune cells. Structural analysis of HARS <jats:sup>WHEP</jats:sup> confirmed a well-organized helix-turn-helix motif. This motif bound specifically and selectively to neuropilin-2 (NRP2), a receptor expressed by myeloid cells in active sites of inflammation, to inhibit expression of proinflammatory receptors and cytokines and to down-regulate inflammatory pathways in primary human macrophages. In animal models of lung injury and ILD, including bleomycin treatment, silicosis, sarcoidosis, chronic hypersensitivity pneumonitis, systemic sclerosis, and rheumatoid arthritis–ILD, HARS <jats:sup>WHEP</jats:sup> reduced lung inflammation, immune cell infiltration, and fibrosis. In patients with sarcoidosis, efzofitimod treatment resulted in down-regulation of gene expression for inflammatory pathways in peripheral immune cells and stabilization of inflammatory biomarkers in serum after steroid tapering. We demonstrate the immunomodulatory activity of HARS <jats:sup>WHEP</jats:sup> and present preclinical data supporting ongoing clinical development of the biologic efzofitimod based on HARS <jats:sup>WHEP</jats:sup> in ILD.","PeriodicalId":21580,"journal":{"name":"Science Translational Medicine","volume":"8 1","pages":""},"PeriodicalIF":17.1,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143599291","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Leslie A. Nangle, Zhiwen Xu, David Siefker, Christoph Burkart, Yeeting E. Chong, Liting Zhai, Yanyan Geng, Clara Polizzi, Lauren Guy, Lisa Eide, Yao Tong, Sofia Klopp-Savino, Michaela Ferrer, Kaitlyn Rauch, Annie Wang, Kristina Hamel, Steve Crampton, Suzanne Paz, Kyle P. Chiang, Minh-Ha Do, Luke Burman, Darin Lee, Mingjie Zhang, Kathleen Ogilvie, David King, Ryan A. Adams, Paul Schimmel
Interstitial lung disease (ILD) consists of a group of immune-mediated disorders that can cause inflammation and progressive fibrosis of the lungs, representing an area of unmet medical need given the lack of disease-modifying therapies and toxicities associated with current treatment options. Tissue-specific splice variants (SVs) of human aminoacyl-tRNA synthetases (aaRSs) are catalytic nulls thought to confer regulatory functions. One example from human histidyl-tRNA synthetase (HARS), termed HARSWHEP because the splicing event resulted in a protein encompassing the WHEP-TRS domain of HARS (a structurally conserved domain found in multiple aaRSs), is enriched in human lung and up-regulated by inflammatory cytokines in lung and immune cells. Structural analysis of HARSWHEP confirmed a well-organized helix-turn-helix motif. This motif bound specifically and selectively to neuropilin-2 (NRP2), a receptor expressed by myeloid cells in active sites of inflammation, to inhibit expression of proinflammatory receptors and cytokines and to down-regulate inflammatory pathways in primary human macrophages. In animal models of lung injury and ILD, including bleomycin treatment, silicosis, sarcoidosis, chronic hypersensitivity pneumonitis, systemic sclerosis, and rheumatoid arthritis–ILD, HARSWHEP reduced lung inflammation, immune cell infiltration, and fibrosis. In patients with sarcoidosis, efzofitimod treatment resulted in down-regulation of gene expression for inflammatory pathways in peripheral immune cells and stabilization of inflammatory biomarkers in serum after steroid tapering. We demonstrate the immunomodulatory activity of HARSWHEP and present preclinical data supporting ongoing clinical development of the biologic efzofitimod based on HARSWHEP in ILD.
{"title":"A human histidyl-tRNA synthetase splice variant therapeutic targets NRP2 to resolve lung inflammation and fibrosis","authors":"Leslie A. Nangle, Zhiwen Xu, David Siefker, Christoph Burkart, Yeeting E. Chong, Liting Zhai, Yanyan Geng, Clara Polizzi, Lauren Guy, Lisa Eide, Yao Tong, Sofia Klopp-Savino, Michaela Ferrer, Kaitlyn Rauch, Annie Wang, Kristina Hamel, Steve Crampton, Suzanne Paz, Kyle P. Chiang, Minh-Ha Do, Luke Burman, Darin Lee, Mingjie Zhang, Kathleen Ogilvie, David King, Ryan A. Adams, Paul Schimmel","doi":"","DOIUrl":"","url":null,"abstract":"<div >Interstitial lung disease (ILD) consists of a group of immune-mediated disorders that can cause inflammation and progressive fibrosis of the lungs, representing an area of unmet medical need given the lack of disease-modifying therapies and toxicities associated with current treatment options. Tissue-specific splice variants (SVs) of human aminoacyl-tRNA synthetases (aaRSs) are catalytic nulls thought to confer regulatory functions. One example from human histidyl-tRNA synthetase (HARS), termed HARS<sup>WHEP</sup> because the splicing event resulted in a protein encompassing the WHEP-TRS domain of HARS (a structurally conserved domain found in multiple aaRSs), is enriched in human lung and up-regulated by inflammatory cytokines in lung and immune cells. Structural analysis of HARS<sup>WHEP</sup> confirmed a well-organized helix-turn-helix motif. This motif bound specifically and selectively to neuropilin-2 (NRP2), a receptor expressed by myeloid cells in active sites of inflammation, to inhibit expression of proinflammatory receptors and cytokines and to down-regulate inflammatory pathways in primary human macrophages. In animal models of lung injury and ILD, including bleomycin treatment, silicosis, sarcoidosis, chronic hypersensitivity pneumonitis, systemic sclerosis, and rheumatoid arthritis–ILD, HARS<sup>WHEP</sup> reduced lung inflammation, immune cell infiltration, and fibrosis. In patients with sarcoidosis, efzofitimod treatment resulted in down-regulation of gene expression for inflammatory pathways in peripheral immune cells and stabilization of inflammatory biomarkers in serum after steroid tapering. We demonstrate the immunomodulatory activity of HARS<sup>WHEP</sup> and present preclinical data supporting ongoing clinical development of the biologic efzofitimod based on HARS<sup>WHEP</sup> in ILD.</div>","PeriodicalId":21580,"journal":{"name":"Science Translational Medicine","volume":"17 789","pages":""},"PeriodicalIF":15.8,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143602918","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Xingyuan Hu, Xiaoyan Kang, Faming Zhao, Yaoyuan Cui, Yu Fu, Xiaohang Yang, Jingjing Yin, Wenting Li, Junpeng Fan, Bin Yang, Zixuan Fang, Tianyu Qin, Xucui Zhuang, Yiting Liu, Chenzhao Feng, Yunyi Yang, Funian Lu, Li Zhang, Weihao Chen, Miaofang Wu, Ning Du, Xia Sheng, Xin Zhou, Jing Li, Gang Chen, Chaoyang Sun
The benefit of hyperthermic intraperitoneal chemotherapy (HIPEC) in ovarian cancer remains controversial, hindering the development of rational combination therapies based on hyperthermia (HT). This study reports the preliminary results of the neoadjuvant HIPEC (NHIPEC) trial (ChiCTR2000038173), demonstrating enhanced tumor response in high-grade serous ovarian cancer with NHIPEC. Through single-cell RNA sequencing analysis, we identified both homogeneous and heterogeneous cellular responses to HT within the tumor and microenvironment. Epithelial-mesenchymal transition–activated tumor cells and matrix metallopeptidase 11 (MMP-11)+ cancer-associated fibroblasts (CAFs) exhibited greater reductions and higher sensitivity to HT. CUT&Tag and RNA sequencing integration unveiled the differential binding programs and transcriptional regulatory mechanisms of HSF1 under normothermia (NT) and HT in tumor cells and CAFs. Furthermore, HT ameliorated the immunosuppressive tumor microenvironment, and in vivo mouse models confirmed the combined antitumor effects of HT and programmed cell death ligand 1 blockade. These findings provide an innovative strategy for rational combination therapy with HT in ovarian cancer.
{"title":"Heterogeneous cellular responses to hyperthermia support combined intraperitoneal hyperthermic immunotherapy for ovarian cancer mouse models","authors":"Xingyuan Hu, Xiaoyan Kang, Faming Zhao, Yaoyuan Cui, Yu Fu, Xiaohang Yang, Jingjing Yin, Wenting Li, Junpeng Fan, Bin Yang, Zixuan Fang, Tianyu Qin, Xucui Zhuang, Yiting Liu, Chenzhao Feng, Yunyi Yang, Funian Lu, Li Zhang, Weihao Chen, Miaofang Wu, Ning Du, Xia Sheng, Xin Zhou, Jing Li, Gang Chen, Chaoyang Sun","doi":"","DOIUrl":"","url":null,"abstract":"<div >The benefit of hyperthermic intraperitoneal chemotherapy (HIPEC) in ovarian cancer remains controversial, hindering the development of rational combination therapies based on hyperthermia (HT). This study reports the preliminary results of the neoadjuvant HIPEC (NHIPEC) trial (ChiCTR2000038173), demonstrating enhanced tumor response in high-grade serous ovarian cancer with NHIPEC. Through single-cell RNA sequencing analysis, we identified both homogeneous and heterogeneous cellular responses to HT within the tumor and microenvironment. Epithelial-mesenchymal transition–activated tumor cells and matrix metallopeptidase 11 (MMP-11)<sup>+</sup> cancer-associated fibroblasts (CAFs) exhibited greater reductions and higher sensitivity to HT. CUT&Tag and RNA sequencing integration unveiled the differential binding programs and transcriptional regulatory mechanisms of HSF1 under normothermia (NT) and HT in tumor cells and CAFs. Furthermore, HT ameliorated the immunosuppressive tumor microenvironment, and in vivo mouse models confirmed the combined antitumor effects of HT and programmed cell death ligand 1 blockade. These findings provide an innovative strategy for rational combination therapy with HT in ovarian cancer.</div>","PeriodicalId":21580,"journal":{"name":"Science Translational Medicine","volume":"17 789","pages":""},"PeriodicalIF":15.8,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143602886","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
京公网安备 11010802042870号
京ICP备2023020795号-1
扫码关注我们
求助内容：
应助结果提醒方式：