Pub Date : 2024-08-11DOI: 10.1101/2024.08.10.607257
Xin Gao, Hayley A McNamara, Jiwon Lee, Adrian F Lo, Deepyan Chatterjee, Dominik Spensberger, Daniel Fernandez-Ruiz, Kevin Walz, Ke Wang, Hannah G Kelly, Kai Pohl, Patricia E Carreira, Andrea Do, Le Xiong, Lynette Beattie, Alexandra J Spencer, Daniel HD Gray, Friedrich Frischknecht, Melanie Rug, Ian A. Cockburn
Our understanding of T-cell-dependent humoral responses has been largely shaped by studies involving model antigens such as recombinant proteins and small viruses 1,2. In these contexts, B cells internalize the entire antigen or pathogen, to present a range of antigen to helper CD4+ T cells to initiate the humoral response. However, this model does not account for large pathogens such as parasites that are too large to be taken up by individual B cells, and the mechanisms by which B cells acquire and present antigens from large complex pathogens to T cells remain poorly understood. Here we used Plasmodium, the causative parasite of malaria, as a model to investigate the requirements for T cell help for B cells targeting the Plasmodium surface circumsporozoite protein (CSP). Upon Plasmodium sporozoite (SPZ) immunization, CSP-specific B cells can form a synapse-like structure with SPZs and take up CSP and non-CSP surface antigens. As a result, CSP-specific B cells can receive help from CD4+ T cells specific to antigens that are located on the surface but not cytosol of the Plasmodium SPZ. Therefore, B cells can obtain help, not only from T cells with the same protein specificity, but also from T cells specific for spatially linked antigens. This flexibility in T cell help, may enhance the initiation and maintenance of immune responses to complex pathogens.
我们对依赖于 T 细胞的体液反应的认识主要是通过对重组蛋白和小病毒等模型抗原的研究形成的1,2。在这些情况下，B 细胞内化整个抗原或病原体，向辅助性 CD4+ T 细胞呈现一系列抗原，从而启动体液反应。然而，这种模式无法解释寄生虫等大型病原体，因为单个 B 细胞无法吸收这些病原体，而且人们对 B 细胞从大型复杂病原体中获取并向 T 细胞呈现抗原的机制仍然知之甚少。在这里，我们以疟疾的致病寄生虫疟原虫为模型，研究了T细胞帮助B细胞靶向疟原虫表面环孢子虫蛋白（CSP）的要求。疟原虫孢子虫（SPZ）免疫后，CSP特异性B细胞可与SPZ形成突触样结构，并吸收CSP和非CSP表面抗原。因此，CSP 特异性 B 细胞可从 CD4+ T 细胞那里获得帮助，这些细胞可特异性地获得位于疟原虫 SPZ 表面而非细胞膜上的抗原。因此，B 细胞不仅能从具有相同蛋白质特异性的 T 细胞那里获得帮助，还能从对空间上相关的抗原具有特异性的 T 细胞那里获得帮助。T 细胞帮助的这种灵活性可能会增强对复杂病原体的免疫反应的启动和维持。
{"title":"B cells targeting parasites capture spatially linked antigens to secure T cell help","authors":"Xin Gao, Hayley A McNamara, Jiwon Lee, Adrian F Lo, Deepyan Chatterjee, Dominik Spensberger, Daniel Fernandez-Ruiz, Kevin Walz, Ke Wang, Hannah G Kelly, Kai Pohl, Patricia E Carreira, Andrea Do, Le Xiong, Lynette Beattie, Alexandra J Spencer, Daniel HD Gray, Friedrich Frischknecht, Melanie Rug, Ian A. Cockburn","doi":"10.1101/2024.08.10.607257","DOIUrl":"https://doi.org/10.1101/2024.08.10.607257","url":null,"abstract":"Our understanding of T-cell-dependent humoral responses has been largely shaped by studies involving model antigens such as recombinant proteins and small viruses 1,2. In these contexts, B cells internalize the entire antigen or pathogen, to present a range of antigen to helper CD4+ T cells to initiate the humoral response. However, this model does not account for large pathogens such as parasites that are too large to be taken up by individual B cells, and the mechanisms by which B cells acquire and present antigens from large complex pathogens to T cells remain poorly understood. Here we used Plasmodium, the causative parasite of malaria, as a model to investigate the requirements for T cell help for B cells targeting the Plasmodium surface circumsporozoite protein (CSP). Upon Plasmodium sporozoite (SPZ) immunization, CSP-specific B cells can form a synapse-like structure with SPZs and take up CSP and non-CSP surface antigens. As a result, CSP-specific B cells can receive help from CD4+ T cells specific to antigens that are located on the surface but not cytosol of the Plasmodium SPZ. Therefore, B cells can obtain help, not only from T cells with the same protein specificity, but also from T cells specific for spatially linked antigens. This flexibility in T cell help, may enhance the initiation and maintenance of immune responses to complex pathogens.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"12 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141946476","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-11DOI: 10.1101/2024.08.11.607476
Calvin Xu, Andreas Obers, Minyi Qin, Alice Brandli, Joelyn Wong, Xin Huang, Allison Clatch, Aly Fayed, Graham Starkey, Rohit DCosta, Claire L Gordon, Lynette Beattie, Laura K Mackay, Dale I Godfrey, Hui-Fern Koay
Unconventional T cells, including mucosal-associated invariant T (MAIT), natural killer T (NKT), and gamma-delta T (γδT) cells, comprise distinct T-bet+, IFN-γ+ and RORγt+, IL-17+ subsets which play differential roles in health and disease. NKT1 cells are susceptible to ARTC2-mediated P2X7 receptor (P2RX7) activation, but the effects on other unconventional T-cell types are unknown. Here, we show that MAIT, γδT, and NKT cells express P2RX7 and are sensitive to P2RX7-mediated cell death. Mouse peripheral T-bet+ MAIT1, γδT1, and NKT1 cells, especially in liver, co-express ARTC2 and P2RX7, which can be further upregulated by retinoic acid. Blocking ARTC2 or inhibiting P2RX7 protected MAIT1, γδT1, and NKT1 cells from cell death, enhanced their survival in vivo, and increased the number of IFN-γ-secreting cells without affecting IL-17 production. Importantly, this revealed the existence of IFN-γ and IL-4 co-producing unconventional T-cell populations normally lost upon isolation due to ARTC2/P2RX7-induced death. Administering extracellular NAD in vivo activated this pathway, depleting P2RX7-sensitive unconventional T cells. Our study reveals ARTC2/P2RX7 as a common regulatory axis modulating the unconventional T-cell compartment, affecting the viability of IFN-γ- and IL-4-producing T cells, offering important insights to facilitate future studies into how these cells can be regulated in health and disease.
{"title":"Selective regulation of IFN-γ and IL-4 co-producing unconventional T cells by purinergic signalling","authors":"Calvin Xu, Andreas Obers, Minyi Qin, Alice Brandli, Joelyn Wong, Xin Huang, Allison Clatch, Aly Fayed, Graham Starkey, Rohit DCosta, Claire L Gordon, Lynette Beattie, Laura K Mackay, Dale I Godfrey, Hui-Fern Koay","doi":"10.1101/2024.08.11.607476","DOIUrl":"https://doi.org/10.1101/2024.08.11.607476","url":null,"abstract":"Unconventional T cells, including mucosal-associated invariant T (MAIT), natural killer T (NKT), and gamma-delta T (γδT) cells, comprise distinct T-bet+, IFN-γ+ and RORγt+, IL-17+ subsets which play differential roles in health and disease. NKT1 cells are susceptible to ARTC2-mediated P2X7 receptor (P2RX7) activation, but the effects on other unconventional T-cell types are unknown. Here, we show that MAIT, γδT, and NKT cells express P2RX7 and are sensitive to P2RX7-mediated cell death. Mouse peripheral T-bet+ MAIT1, γδT1, and NKT1 cells, especially in liver, co-express ARTC2 and P2RX7, which can be further upregulated by retinoic acid. Blocking ARTC2 or inhibiting P2RX7 protected MAIT1, γδT1, and NKT1 cells from cell death, enhanced their survival in vivo, and increased the number of IFN-γ-secreting cells without affecting IL-17 production. Importantly, this revealed the existence of IFN-γ and IL-4 co-producing unconventional T-cell populations normally lost upon isolation due to ARTC2/P2RX7-induced death. Administering extracellular NAD in vivo activated this pathway, depleting P2RX7-sensitive unconventional T cells. Our study reveals ARTC2/P2RX7 as a common regulatory axis modulating the unconventional T-cell compartment, affecting the viability of IFN-γ- and IL-4-producing T cells, offering important insights to facilitate future studies into how these cells can be regulated in health and disease.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"92 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141969887","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-11DOI: 10.1101/2024.08.11.607460
Deepak Kaushik, Aysika Das, Claudia Silva, Charlotte D'Mello, Luiz Gustavo N Almeida, Nazanin Ghasemi, Paola Neri, Antoine Dufour, Nizar Jacques Bahlis, Mengzhou Xue, Voon Wee Yong
Monocytes and monocyte-derived macrophages have important roles in the initiation and progression of multiple sclerosis (MS). These cells undergo metabolic reprogramming to generate immunophenotypes that promote leukocyte infiltration, axonal degeneration and demyelination, worsening MS pathology. The mechanisms that dictate metabolic programs in monocytes and macrophages in MS remain unclear. We previously reported that extracellular matrix metalloproteinase inducer (EMMPRIN, CD147), a glycoprotein that acts as a chaperone of monocarboxylate transporter 4 (MCT4), assisted with glycolysis-driven pro-inflammatory phenotype in macrophages in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Using newly-generated CCR2CreERT2:EMMPRINfl/fl (CCR2:EMMP) mice, we report that presymptomatic deletion of EMMPRIN in CCR2+ monocytes prevented or reduced clinical disability of EAE. This was correspondent with decreased infiltration of leukocytes into the CNS. Single cell RNA-seq of blood monocytes from EAE and proteomics analysis of macrophages from CCR2:EMMP-/- mice revealed significant alterations in metabolic programs, particularly reduced glycolysis and elevated mitochondrial electron transport and fatty acid oxidation, which were linked to their reduced pro-inflammatory traits. Our findings implicate EMMPRIN as a key regulator of metabolic pathways that exacerbate pro-inflammatory functions of monocytes in MS.
{"title":"EMMPRIN confers metabolic advantage for monocytes and macrophages to promote disease in a model of multiple sclerosis","authors":"Deepak Kaushik, Aysika Das, Claudia Silva, Charlotte D'Mello, Luiz Gustavo N Almeida, Nazanin Ghasemi, Paola Neri, Antoine Dufour, Nizar Jacques Bahlis, Mengzhou Xue, Voon Wee Yong","doi":"10.1101/2024.08.11.607460","DOIUrl":"https://doi.org/10.1101/2024.08.11.607460","url":null,"abstract":"Monocytes and monocyte-derived macrophages have important roles in the initiation and progression of multiple sclerosis (MS). These cells undergo metabolic reprogramming to generate immunophenotypes that promote leukocyte infiltration, axonal degeneration and demyelination, worsening MS pathology. The mechanisms that dictate metabolic programs in monocytes and macrophages in MS remain unclear. We previously reported that extracellular matrix metalloproteinase inducer (EMMPRIN, CD147), a glycoprotein that acts as a chaperone of monocarboxylate transporter 4 (MCT4), assisted with glycolysis-driven pro-inflammatory phenotype in macrophages in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Using newly-generated CCR2CreERT2:EMMPRINfl/fl (CCR2:EMMP) mice, we report that presymptomatic deletion of EMMPRIN in CCR2+ monocytes prevented or reduced clinical disability of EAE. This was correspondent with decreased infiltration of leukocytes into the CNS. Single cell RNA-seq of blood monocytes from EAE and proteomics analysis of macrophages from CCR2:EMMP-/- mice revealed significant alterations in metabolic programs, particularly reduced glycolysis and elevated mitochondrial electron transport and fatty acid oxidation, which were linked to their reduced pro-inflammatory traits. Our findings implicate EMMPRIN as a key regulator of metabolic pathways that exacerbate pro-inflammatory functions of monocytes in MS.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"11 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141946475","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-10DOI: 10.1101/2024.08.10.607461
Noor Momin, Steffen Pabel, Arnab Rudra, Nina Kumowski, I-Hsiu Lee, Kyle Mentkowski, Masahiro Yamazoe, Laura Stengel, Charlotte G. Muse, Hana Seung, Alexandre Paccalet, Cristina Gonzalez-Correa, Emily B. Jacobs, Jana Grune, Maximilian J. Schloss, Samuel Sossalla, Gregory Wojtkiewicz, Yoshiko Iwamoto, Patrick McMullen, Richard N. Mitchell, Patrick T. Ellinor, Daniel G. Anderson, Kamila Naxerova, Matthias Nahrendorf, Maarten Hulsmans
Atrial fibrillation (AFib) and the risk of its lethal complications are propelled by fibrosis, which induces electrical heterogeneity and gives rise to reentry circuits. Atrial TREM2+ macrophages secrete osteopontin (encoded by Spp1), a matricellular signaling protein that engenders fibrosis and AFib. Here we show that silencing Spp1 in TREM2+ cardiac macrophages with an antibody-siRNA conjugate reduces atrial fibrosis and suppresses AFib in mice, thus offering a new immunotherapy for the most common arrhythmia.
{"title":"Therapeutic Spp1 silencing in TREM2+ cardiac macrophages suppresses atrial fibrillation","authors":"Noor Momin, Steffen Pabel, Arnab Rudra, Nina Kumowski, I-Hsiu Lee, Kyle Mentkowski, Masahiro Yamazoe, Laura Stengel, Charlotte G. Muse, Hana Seung, Alexandre Paccalet, Cristina Gonzalez-Correa, Emily B. Jacobs, Jana Grune, Maximilian J. Schloss, Samuel Sossalla, Gregory Wojtkiewicz, Yoshiko Iwamoto, Patrick McMullen, Richard N. Mitchell, Patrick T. Ellinor, Daniel G. Anderson, Kamila Naxerova, Matthias Nahrendorf, Maarten Hulsmans","doi":"10.1101/2024.08.10.607461","DOIUrl":"https://doi.org/10.1101/2024.08.10.607461","url":null,"abstract":"Atrial fibrillation (AFib) and the risk of its lethal complications are propelled by fibrosis, which induces electrical heterogeneity and gives rise to reentry circuits. Atrial TREM2+ macrophages secrete osteopontin (encoded by Spp1), a matricellular signaling protein that engenders fibrosis and AFib. Here we show that silencing Spp1 in TREM2+ cardiac macrophages with an antibody-siRNA conjugate reduces atrial fibrosis and suppresses AFib in mice, thus offering a new immunotherapy for the most common arrhythmia.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"43 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141946477","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-10DOI: 10.1101/2024.08.08.607190
Kathryn M Moore, Stephanie L Foster, Elizabeth J Elrod, Katharine A Floyd, M. Elliott Williams, Meenakshi Kar, Jacob Vander Velden, Madison Ellis, Ansa Malik, Bushra Wali, Stacey Lapp, Amanda Metz, Steven E Bosinger, Robert A Seder, Rama Rao Amara, Vineet D Menachery, Jacob E Kohlmeier, Arash Grakoui, Mehul S Suthar
Waning immunity and the emergence of immune evasive SARS-CoV-2 variants jeopardize vaccine efficacy leading to breakthrough infections. We have previously shown that innate immune cells play a critical role in controlling SARS-CoV-2. To investigate the innate immune response during breakthrough infections, we modeled breakthrough infections by challenging low-dose vaccinated mice with a vaccine-mismatched SARS-CoV-2 Beta variant. We found that low-dose vaccinated infected mice had a 2-log reduction in lung viral burden, but increased immune cell infiltration in the lung parenchyma, characterized by monocytes, monocyte-derived macrophages, and eosinophils. Single cell RNA-seq revealed viral RNA was highly associated with eosinophils that corresponded to a unique IFN-γ biased signature. Antibody-mediated depletion of eosinophils in vaccinated mice resulted in increased virus replication and dissemination in the lungs, demonstrating that eosinophils in the lungs are protective during SARS-CoV-2 breakthrough infections. These results highlight the critical role for the innate immune response in vaccine mediated protection against SARS-CoV-2.
{"title":"Eosinophils protect against SARS-CoV-2 following a vaccine breakthrough infection","authors":"Kathryn M Moore, Stephanie L Foster, Elizabeth J Elrod, Katharine A Floyd, M. Elliott Williams, Meenakshi Kar, Jacob Vander Velden, Madison Ellis, Ansa Malik, Bushra Wali, Stacey Lapp, Amanda Metz, Steven E Bosinger, Robert A Seder, Rama Rao Amara, Vineet D Menachery, Jacob E Kohlmeier, Arash Grakoui, Mehul S Suthar","doi":"10.1101/2024.08.08.607190","DOIUrl":"https://doi.org/10.1101/2024.08.08.607190","url":null,"abstract":"Waning immunity and the emergence of immune evasive SARS-CoV-2 variants jeopardize vaccine efficacy leading to breakthrough infections. We have previously shown that innate immune cells play a critical role in controlling SARS-CoV-2. To investigate the innate immune response during breakthrough infections, we modeled breakthrough infections by challenging low-dose vaccinated mice with a vaccine-mismatched SARS-CoV-2 Beta variant. We found that low-dose vaccinated infected mice had a 2-log reduction in lung viral burden, but increased immune cell infiltration in the lung parenchyma, characterized by monocytes, monocyte-derived macrophages, and eosinophils. Single cell RNA-seq revealed viral RNA was highly associated with eosinophils that corresponded to a unique IFN-γ biased signature. Antibody-mediated depletion of eosinophils in vaccinated mice resulted in increased virus replication and dissemination in the lungs, demonstrating that eosinophils in the lungs are protective during SARS-CoV-2 breakthrough infections. These results highlight the critical role for the innate immune response in vaccine mediated protection against SARS-CoV-2.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"141 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141969525","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-10DOI: 10.1101/2024.08.10.606309
Antonia O. Cuff, Ee Von Woon, Thomas Bainton, Brendan Browne, Phoebe M. Kirkwood, Frances Collins, Douglas A Gibson, Philippa Saunders, Andrew W. Horne, Mark R. Johnson, David A. MacIntyre, Victoria Male
Innate lymphoid cells (ILC) are prominent in the human uterine mucosa and play physiological roles in pregnancy. ILC3 are the second-most common ILC subset in the uterine mucosa, but their role remains unclear. Here we define two subsets of lineage-negative CD56+ CD117+ CRTH2- uterine ILC3, distinguished by their expression of CD127. The CD127- subset is most numerous and active during menstruation and immediately after parturition, suggesting a role in repair of the uterine mucosa (called endometrium outside of pregnancy); the CD127+ subset is most numerous and active immediately after menstruation, as the endometrium regenerates. In healthy endometrium, ILC3 are spatially associated with glandular epithelial and endothelial cells, which both express receptors for the ILC3-derived cytokines, IL-22 and IL-8. In the eutopic endometrium of people with endometriosis, ILC3 are located further from glandular epithelial and endothelial cells suggesting that these cells may be less exposed to ILC3 products, potentially with negative consequences for endometrial regeneration. Our findings highlight the dynamic nature of ILC3 in the uterine mucosa and suggest their primary role is in repair and regeneration. An improved understanding of uterine ILC3 will inform future research on endometrial health and disease.
{"title":"Dynamic roles of ILC3 in endometrial repair and regeneration","authors":"Antonia O. Cuff, Ee Von Woon, Thomas Bainton, Brendan Browne, Phoebe M. Kirkwood, Frances Collins, Douglas A Gibson, Philippa Saunders, Andrew W. Horne, Mark R. Johnson, David A. MacIntyre, Victoria Male","doi":"10.1101/2024.08.10.606309","DOIUrl":"https://doi.org/10.1101/2024.08.10.606309","url":null,"abstract":"Innate lymphoid cells (ILC) are prominent in the human uterine mucosa and play physiological roles in pregnancy. ILC3 are the second-most common ILC subset in the uterine mucosa, but their role remains unclear. Here we define two subsets of lineage-negative CD56+ CD117+ CRTH2- uterine ILC3, distinguished by their expression of CD127. The CD127- subset is most numerous and active during menstruation and immediately after parturition, suggesting a role in repair of the uterine mucosa (called endometrium outside of pregnancy); the CD127+ subset is most numerous and active immediately after menstruation, as the endometrium regenerates. In healthy endometrium, ILC3 are spatially associated with glandular epithelial and endothelial cells, which both express receptors for the ILC3-derived cytokines, IL-22 and IL-8. In the eutopic endometrium of people with endometriosis, ILC3 are located further from glandular epithelial and endothelial cells suggesting that these cells may be less exposed to ILC3 products, potentially with negative consequences for endometrial regeneration. Our findings highlight the dynamic nature of ILC3 in the uterine mucosa and suggest their primary role is in repair and regeneration. An improved understanding of uterine ILC3 will inform future research on endometrial health and disease.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"78 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141969906","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-10DOI: 10.1101/2024.08.10.607414
Natalie B Hagan, Charles Inaku, Nikesh Kunder, Tayleur White, Thierry Iraguha, Anna Meyer, Kristen Pauken, Jason Matthew Schenkel
Leukocytes migrate through the blood and extravasate into organs to surveil the host for infection or cancer. Recently, we demonstrated that intravenous (IV) anti-CD45.2 antibody labeling allowed for precise tracking of leukocyte migration. However, the narrow labeling window can make this approach challenging for tracking rare migration events. Here, we show that altering antibody administration route and fluorophore can significantly extend the antibody active labeling time. We found that while both IV and intraperitoneal (IP) anti-CD45.2 antibody labeled circulating leukocytes after injection, they had different kinetic properties that impacted labeling time and intensity. Quantification of circulating antibody revealed that while unbound IV anti-CD45.2 antibody rapidly decreased, unbound IP anti-CD45.2 antibody increased over one hour. Using in vitro and in vivo serial dilution assays, we found that Alexa Fluor 647 (AF647) and Brilliant Blue 700 (BB700) dyes had the greatest labeling sensitivity compared to other fluorophores. However, IP antibody injection with anti-CD45.2 BB700, but not AF647, resulted in continuous blood leukocyte labeling for over 6 hours. Finally, we leveraged IP anti-CD45.2 BB700 antibody to track slower migrating leukocytes into tumors. We found that IP anti-CD45.2 antibody injection allowed for the identification of ~seven times as many tumor-specific CD8+ T cells that had recently migrated from blood into tumors. Our results demonstrate how different injection routes and fluorophores affect anti-CD45.2 antibody leukocyte labeling and highlight the utility of this approach for defining leukocyte migration in the context of homeostasis and cancer.
白细胞通过血液迁移并渗入器官，以监测宿主的感染或癌症情况。最近，我们证明了静脉注射抗 CD45.2 抗体可精确追踪白细胞迁移。然而，由于标记窗口较窄，这种方法对于追踪罕见的迁移事件具有挑战性。在此，我们展示了改变抗体给药途径和荧光基团可显著延长抗体的活性标记时间。我们发现，虽然静脉注射和腹腔注射（IP）的抗-CD45.2抗体在注射后都能标记循环白细胞，但它们具有不同的动力学特性，会影响标记时间和标记强度。循环抗体定量显示，未结合的 IV 抗 CD45.2 抗体迅速减少，而未结合的 IP 抗 CD45.2 抗体在一小时内增加。通过体外和体内系列稀释试验，我们发现与其他荧光团相比，Alexa Fluor 647（AF647）和亮蓝 700（BB700）染料的标记灵敏度最高。然而，IP 抗体注射抗 CD45.2 BB700（而非 AF647）可持续标记血液白细胞超过 6 小时。最后，我们利用 IP 抗 CD45.2 BB700 抗体追踪进入肿瘤的迁移速度较慢的白细胞。我们发现，注射 IP 抗 CD45.2 抗体可识别出七倍于最近从血液迁移到肿瘤的肿瘤特异性 CD8+ T 细胞。我们的研究结果表明了不同的注射途径和荧光团对抗CD45.2抗体白细胞标记的影响，并强调了这种方法在平衡和癌症背景下定义白细胞迁移的实用性。
{"title":"In vivo antibody labeling route and fluorophore dictate labeling efficiency, sensitivity, and longevity","authors":"Natalie B Hagan, Charles Inaku, Nikesh Kunder, Tayleur White, Thierry Iraguha, Anna Meyer, Kristen Pauken, Jason Matthew Schenkel","doi":"10.1101/2024.08.10.607414","DOIUrl":"https://doi.org/10.1101/2024.08.10.607414","url":null,"abstract":"Leukocytes migrate through the blood and extravasate into organs to surveil the host for infection or cancer. Recently, we demonstrated that intravenous (IV) anti-CD45.2 antibody labeling allowed for precise tracking of leukocyte migration. However, the narrow labeling window can make this approach challenging for tracking rare migration events. Here, we show that altering antibody administration route and fluorophore can significantly extend the antibody active labeling time. We found that while both IV and intraperitoneal (IP) anti-CD45.2 antibody labeled circulating leukocytes after injection, they had different kinetic properties that impacted labeling time and intensity. Quantification of circulating antibody revealed that while unbound IV anti-CD45.2 antibody rapidly decreased, unbound IP anti-CD45.2 antibody increased over one hour. Using in vitro and in vivo serial dilution assays, we found that Alexa Fluor 647 (AF647) and Brilliant Blue 700 (BB700) dyes had the greatest labeling sensitivity compared to other fluorophores. However, IP antibody injection with anti-CD45.2 BB700, but not AF647, resulted in continuous blood leukocyte labeling for over 6 hours. Finally, we leveraged IP anti-CD45.2 BB700 antibody to track slower migrating leukocytes into tumors. We found that IP anti-CD45.2 antibody injection allowed for the identification of ~seven times as many tumor-specific CD8+ T cells that had recently migrated from blood into tumors. Our results demonstrate how different injection routes and fluorophores affect anti-CD45.2 antibody leukocyte labeling and highlight the utility of this approach for defining leukocyte migration in the context of homeostasis and cancer.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"66 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141946478","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-10DOI: 10.1101/2024.08.09.605851
Patrick Ross, Hijab Fatima, Daniel P Leaman, Jessica Matthias, Kathryn Spencer, Michael B Zwick, Scott C Henderson, Emily M. Mace, Charles Daniel Murin
Natural Killer (NK) cells utilize effector functions, including antibody-dependent cellular cytotoxicity (ADCC), for the clearance of viral infection and cellular malignancies. NK cell ADCC is mediated by Fc𝛾RIIIa (CD16a) binding to the fragment crystallizable (Fc) region of immunoglobulin G (IgG) within immune complexes on a target cell surface. While antibody-induced clustering of CD16a is thought to drive ADCC, the molecular basis for this activity has not been fully described. Here we use MINFLUX nanoscopy to map the spatial distribution of stoichiometrically labeled CD16a across the NK cell membrane, revealing the presence of pairs of CD16a molecules with intra-doublet distance of approximately 17 nm. NK cells activated on supported lipid bilayers by Trastuzumab results in an increase of synaptic regions with greater CD16a density. Our results provide the highest spatial resolution yet described for CD16a imaging, offering new insight into how CD16a organization could influence ADCC activity, for example through self-association or with binding partners influencing the degree of ADCC signaling. MINFLUX holds great promise to further unravel the molecular details driving CD16a-based activation of NK cells.
{"title":"Spatial localization of CD16a at the human NK cell ADCC lytic synapse","authors":"Patrick Ross, Hijab Fatima, Daniel P Leaman, Jessica Matthias, Kathryn Spencer, Michael B Zwick, Scott C Henderson, Emily M. Mace, Charles Daniel Murin","doi":"10.1101/2024.08.09.605851","DOIUrl":"https://doi.org/10.1101/2024.08.09.605851","url":null,"abstract":"Natural Killer (NK) cells utilize effector functions, including antibody-dependent cellular cytotoxicity (ADCC), for the clearance of viral infection and cellular malignancies. NK cell ADCC is mediated by Fc𝛾RIIIa (CD16a) binding to the fragment crystallizable (Fc) region of immunoglobulin G (IgG) within immune complexes on a target cell surface. While antibody-induced clustering of CD16a is thought to drive ADCC, the molecular basis for this activity has not been fully described. Here we use MINFLUX nanoscopy to map the spatial distribution of stoichiometrically labeled CD16a across the NK cell membrane, revealing the presence of pairs of CD16a molecules with intra-doublet distance of approximately 17 nm. NK cells activated on supported lipid bilayers by Trastuzumab results in an increase of synaptic regions with greater CD16a density. Our results provide the highest spatial resolution yet described for CD16a imaging, offering new insight into how CD16a organization could influence ADCC activity, for example through self-association or with binding partners influencing the degree of ADCC signaling. MINFLUX holds great promise to further unravel the molecular details driving CD16a-based activation of NK cells.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"14 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141969526","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.09.606346
Alexandria C Wells, Djalma Souza Lima-Junior, Verena M Link, Margery Smelkinson, Siddharth R Krishnamurthy, Liang Chi, Elisha Segrist, Claudia A Rivera, Ana Teijeiro, Nicolas Bouladoux, Yasmine Belkaid
Maintenance of tissue integrity is a requirement of host survival. This mandate is of prime importance at barrier sites that are constitutively exposed to the environment. Here, we show that exposure of the skin to non-inflammatory xenobiotics promotes tissue repair; more specifically, mild detergent exposure promotes the reactivation of defined retroelements leading to the induction of retroelement-specific CD8+ T cells. These T cell responses are Langerhans cell dependent and establish tissue residency within the skin. Upon injury, retroelement-specific CD8+ T cells significantly accelerate wound repair via IL-17A. Collectively, this work demonstrates that tonic environmental exposures and associated adaptive responses to retroelements can be coopted to preemptively set the tissue for maximal resilience to injury.
保持组织的完整性是宿主生存的必要条件。这项任务对于持续暴露于环境中的屏障部位至关重要。在这里，我们展示了皮肤暴露于非炎症性异种生物会促进组织修复；更具体地说，暴露于温和的洗涤剂会促进确定的逆源因子重新激活，从而诱导逆源因子特异性 CD8+ T 细胞。这些 T 细胞反应依赖于朗格汉斯细胞，并在皮肤内建立组织驻留。受伤后，逆源特异性 CD8+ T 细胞通过 IL-17A 显著加速伤口修复。总之，这项研究表明，强直性环境暴露和对逆源因子的相关适应性反应可被用于预先设置组织，使其对损伤具有最大的复原力。
{"title":"Adaptive immunity to retroelements promotes barrier integrity","authors":"Alexandria C Wells, Djalma Souza Lima-Junior, Verena M Link, Margery Smelkinson, Siddharth R Krishnamurthy, Liang Chi, Elisha Segrist, Claudia A Rivera, Ana Teijeiro, Nicolas Bouladoux, Yasmine Belkaid","doi":"10.1101/2024.08.09.606346","DOIUrl":"https://doi.org/10.1101/2024.08.09.606346","url":null,"abstract":"Maintenance of tissue integrity is a requirement of host survival. This mandate is of prime importance at barrier sites that are constitutively exposed to the environment. Here, we show that exposure of the skin to non-inflammatory xenobiotics promotes tissue repair; more specifically, mild detergent exposure promotes the reactivation of defined retroelements leading to the induction of retroelement-specific CD8+ T cells. These T cell responses are Langerhans cell dependent and establish tissue residency within the skin. Upon injury, retroelement-specific CD8+ T cells significantly accelerate wound repair via IL-17A. Collectively, this work demonstrates that tonic environmental exposures and associated adaptive responses to retroelements can be coopted to preemptively set the tissue for maximal resilience to injury.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"13 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141946482","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-09DOI: 10.1101/2024.08.09.607337
Desiree I. Frecot, Simone Blaess, Teresa R. Wagner, Philipp D. Kaiser, Bjoern Traenkle, Madeleine Fandrich, Meike Jakobi, Armin M. Scholz, Stefan Nueske, Nicole Schneiderhan-Marra, Cécile Gouttefangeas, Manfred Kneilling, Bernd J. Pichler, Dominik Sonanini, Ulrich Rothbauer
Purpose: Human OX40 (hOX40/CD134), a member of the TNF receptor superfamily, is mainly expressed on activated T lymphocytes. Triggered by its ligand OX40L (CD252), it provides costimulatory signals that support the differentiation, proliferation and long-term survival of T cells. Besides being a relevant therapeutic target, hOX40 is also an important biomarker for monitoring the presence or infiltration of activated T cells within the tumor microenvironment (TME), the inflammatory microenvironment (IME) in immune-mediated diseases (IMIDs) and the lymphatic organs. Here, we developed novel single domain antibodies (nanobodies, Nbs) targeting hOX40 to monitor the activation status of T cells by in vivo molecular imaging. Methods: Nbs against hOX40 (hOX40-Nbs) were selected from an immunized Nb-library by phage display. The identified hOX40-Nbs were characterized in vitro, including determination of their specificity, affinity, stability, epitope recognition and their impact on OX40 signaling and T cell function. A lead candidate was site-specifically conjugated with a fluorophore via sortagging and applied for noninvasive in vivo optical imaging (OI) of hOX40-expressing cells in a xenograft mouse model. Results: Our selection campaign revealed four unique Nbs that exhibit strong binding affinities and high stabilities under physiological conditions. Epitope binning and domain mapping indicated the targeting of at least two different epitopes on hOX40. When analyzing their impact on OX40 signaling, an agonistic effect was excluded for all validated Nbs. Incubation of activated T cells with hOX40-Nbs did not affect cell viability or proliferation patterns, whereas differences in cytokine release were observed. In vivo OI with a fluorophore-conjugated lead candidate in experimental mice with hOX40-expressing xenografts demonstrated its specificity and functionality as an imaging probe. Conclusion: Considering the need for advanced probes for noninvasive in vivo monitoring of T cell activation dynamics, we propose, that our hOX40-Nbs have a great potential as imaging probes for noninvasive and longitudinal in vivo diagnostics. Quantification of OX40+ T cells in TME or IME will provide crucial insights into the activation state of infiltrating T cells, offering a valuable biomarker for assessing immune responses, predicting treatment efficacy, and guiding personalized immunotherapy strategies in patients with cancer or IMIDs.
目的：人类 OX40（hOX40/CD134）是 TNF 受体超家族的成员，主要在活化的 T 淋巴细胞上表达。在其配体 OX40L（CD252）的触发下，它可提供支持 T 细胞分化、增殖和长期存活的成本刺激信号。除了是一个相关的治疗靶点外，hOX40 还是一个重要的生物标记物，可用于监测肿瘤微环境（TME）、免疫介导疾病（IMIDs）中的炎症微环境（IME）和淋巴器官中活化 T 细胞的存在或浸润情况。在此，我们开发了靶向 hOX40 的新型单域抗体（纳米抗体，Nbs），通过体内分子成像监测 T 细胞的活化状态。方法通过噬菌体展示从免疫Nb库中筛选出针对hOX40的Nbs（hOX40-Nbs）。对鉴定出的 hOX40-Nbs 进行了体外表征，包括确定其特异性、亲和性、稳定性、表位识别及其对 OX40 信号传导和 T 细胞功能的影响。通过sortagging技术将一个候选先导基因与荧光团定点连接，并在异种移植小鼠模型中应用于hOX40表达细胞的非侵入性体内光学成像（OI）：我们的筛选活动发现了四种独特的 Nbs，它们在生理条件下表现出很强的结合亲和力和很高的稳定性。表位分选和域映射表明，它们至少靶向了 hOX40 上的两个不同表位。在分析它们对 OX40 信号传导的影响时，所有已验证的 Nbs 都排除了激动效应。用 hOX40-Nbs 培养活化的 T 细胞不会影响细胞活力或增殖模式，但观察到细胞因子的释放存在差异。在表达 hOX40 的异种移植实验小鼠体内使用荧光团结合的候选先导物进行活体 OI，证明了其作为成像探针的特异性和功能性。结论：考虑到体内无创监测 T 细胞活化动态对先进探针的需求，我们认为我们的 hOX40-Nbs 作为成像探针在体内无创和纵向诊断方面具有巨大潜力。对TME或IME中的OX40+ T细胞进行定量分析，将有助于深入了解浸润T细胞的活化状态，为评估癌症或IMID患者的免疫反应、预测治疗效果和指导个性化免疫疗法策略提供有价值的生物标志物。
{"title":"Making the effect visible – OX40 targeting nanobodies for in vivo imaging of activated T cells","authors":"Desiree I. Frecot, Simone Blaess, Teresa R. Wagner, Philipp D. Kaiser, Bjoern Traenkle, Madeleine Fandrich, Meike Jakobi, Armin M. Scholz, Stefan Nueske, Nicole Schneiderhan-Marra, Cécile Gouttefangeas, Manfred Kneilling, Bernd J. Pichler, Dominik Sonanini, Ulrich Rothbauer","doi":"10.1101/2024.08.09.607337","DOIUrl":"https://doi.org/10.1101/2024.08.09.607337","url":null,"abstract":"Purpose: Human OX40 (hOX40/CD134), a member of the TNF receptor superfamily, is mainly expressed on activated T lymphocytes. Triggered by its ligand OX40L (CD252), it provides costimulatory signals that support the differentiation, proliferation and long-term survival of T cells. Besides being a relevant therapeutic target, hOX40 is also an important biomarker for monitoring the presence or infiltration of activated T cells within the tumor microenvironment (TME), the inflammatory microenvironment (IME) in immune-mediated diseases (IMIDs) and the lymphatic organs. Here, we developed novel single domain antibodies (nanobodies, Nbs) targeting hOX40 to monitor the activation status of T cells by in vivo molecular imaging. Methods: Nbs against hOX40 (hOX40-Nbs) were selected from an immunized Nb-library by phage display. The identified hOX40-Nbs were characterized in vitro, including determination of their specificity, affinity, stability, epitope recognition and their impact on OX40 signaling and T cell function. A lead candidate was site-specifically conjugated with a fluorophore via sortagging and applied for noninvasive in vivo optical imaging (OI) of hOX40-expressing cells in a xenograft mouse model.\u0000Results: Our selection campaign revealed four unique Nbs that exhibit strong binding affinities and high stabilities under physiological conditions. Epitope binning and domain mapping indicated the targeting of at least two different epitopes on hOX40. When analyzing their impact on OX40 signaling, an agonistic effect was excluded for all validated Nbs. Incubation of activated T cells with hOX40-Nbs did not affect cell viability or proliferation patterns, whereas differences in cytokine release were observed. In vivo OI with a fluorophore-conjugated lead candidate in experimental mice with hOX40-expressing xenografts demonstrated its specificity and functionality as an imaging probe. Conclusion: Considering the need for advanced probes for noninvasive in vivo monitoring of T cell activation dynamics, we propose, that our hOX40-Nbs have a great potential as imaging probes for noninvasive and longitudinal in vivo diagnostics. Quantification of OX40+ T cells in TME or IME will provide crucial insights into the activation state of infiltrating T cells, offering a valuable biomarker for assessing immune responses, predicting treatment efficacy, and guiding personalized immunotherapy strategies in patients with cancer or IMIDs.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141946479","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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