Pub Date : 2024-09-12DOI: 10.1101/2024.09.07.611809
Epshita A. Islam, Jamie E. Fegan, Joseph J. Zeppa, Sang K. Ahn, Dixon Ng, Elissa G. Currie, Jessica Lam, Trevor F. Moraes, Scott D. Gray-Owen
Neisseria gonorrhoeae, which causes the sexually transmitted infection gonorrhea and N. meningitidis, a leading cause of bacterial meningitis and septicemia, are closely related human-restricted pathogens that inhabit distinct primary mucosal niches. While successful vaccines against invasive meningococcal disease have been available for decades, the rapid rise in antibiotic resistance has led to an urgent need to develop an effective gonococcal vaccine. Several surface antigens are shared among these two pathogens, making cross-species protection an exciting prospect. However, the type of vaccine-mediated immune response required to achieve protection against respiratory versus genital infection remains ill defined. In this study, we utilize well established mouse models of female lower genital tract colonization by N. gonorrhoeae and upper respiratory tract colonization by N. meningitidis to examine the performance of transferrin binding protein B (TbpB) vaccines formulated with immunologically distinct vaccine adjuvants. We demonstrate that vaccine-mediated protection is influenced by the choice of adjuvant, with Th1/2-balanced adjuvants performing optimally against N. gonorrhoeae, and both Th1/2-balanced and Th2-skewing adjuvants leading to a significant reduction in N. meningitidis burden. We further establish a lack of correlation between protection status and the humoral response or bactericidal titre. Combined, this work provides supports the feasibility for a single vaccine formulation to achieve pan-neisserial coverage.
{"title":"Adjuvant-dependent impacts on vaccine-induced humoral responses and protection in preclinical models of nasal and genital colonization by pathogenic Neisseria","authors":"Epshita A. Islam, Jamie E. Fegan, Joseph J. Zeppa, Sang K. Ahn, Dixon Ng, Elissa G. Currie, Jessica Lam, Trevor F. Moraes, Scott D. Gray-Owen","doi":"10.1101/2024.09.07.611809","DOIUrl":"https://doi.org/10.1101/2024.09.07.611809","url":null,"abstract":"Neisseria gonorrhoeae, which causes the sexually transmitted infection gonorrhea and N. meningitidis, a leading cause of bacterial meningitis and septicemia, are closely related human-restricted pathogens that inhabit distinct primary mucosal niches. While successful vaccines against invasive meningococcal disease have been available for decades, the rapid rise in antibiotic resistance has led to an urgent need to develop an effective gonococcal vaccine. Several surface antigens are shared among these two pathogens, making cross-species protection an exciting prospect. However, the type of vaccine-mediated immune response required to achieve protection against respiratory versus genital infection remains ill defined. In this study, we utilize well established mouse models of female lower genital tract colonization by N. gonorrhoeae and upper respiratory tract colonization by N. meningitidis to examine the performance of transferrin binding protein B (TbpB) vaccines formulated with immunologically distinct vaccine adjuvants. We demonstrate that vaccine-mediated protection is influenced by the choice of adjuvant, with Th1/2-balanced adjuvants performing optimally against N. gonorrhoeae, and both Th1/2-balanced and Th2-skewing adjuvants leading to a significant reduction in N. meningitidis burden. We further establish a lack of correlation between protection status and the humoral response or bactericidal titre. Combined, this work provides supports the feasibility for a single vaccine formulation to achieve pan-neisserial coverage.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"19 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211216","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 : 2024-09-12DOI: 10.1101/2024.09.06.611736
Thais Boccia, Weikang Pan, Victor Fattori, Rodrigo Cervantes-Diaz, Michael S. Rogers, Ivan Zanoni, Alex G. Cuenca
Trained immunity enhances responsiveness of the innate immune system upon restimulation. Although adjuvants are used to enhance immune responses, we showed that repeated administration of alum, termed adjuvant conditioning (AC), establishes an immunosuppressive environment that delays allogeneic graft rejection by expanding myeloid-derived suppressor cells (MDSCs). Here, we show that AC-induced MDSCs suppress antigen specific adaptive responses both in vitro and in vivo, and that the immunosuppression is abolished in the absence of NLRP3 and IL-1 signaling. Allogeneic pancreatic islets transplanted into AC-treated NLRP3-/- mice are not protected, demonstrating that AC requires NLRP3 signaling. Finally, AC also has an immunosuppressive effect on human PBMCs. Overall, our data show that AC establishes an immunosuppressive milieu via the NLRP3/IL-1 axis, leading to trained immunosuppression, or trained tolerance. Our findings give a potent mandate to explore the possibility to target the NLRP3/IL-1 pathway as a new promising strategy to condition transplant recipients and promote allograft tolerance.
训练免疫可增强先天性免疫系统在再次刺激时的反应能力。虽然佐剂可用于增强免疫反应，但我们发现，反复给予明矾（称为佐剂调节（AC））可建立免疫抑制环境，通过扩大髓源性抑制细胞（MDSCs）来延迟异体移植排斥反应。在这里，我们发现 AC 诱导的 MDSCs 在体外和体内都能抑制抗原特异性适应性反应，而且在缺乏 NLRP3 和 IL-1 信号传导的情况下，这种免疫抑制会消失。异体胰岛移植到经过 AC 处理的 NLRP3-/- 小鼠体内不会受到保护，这表明 AC 需要 NLRP3 信号传导。最后，AC 对人类 PBMCs 也有免疫抑制作用。总之，我们的数据表明，AC 可通过 NLRP3/IL-1 轴建立免疫抑制环境，导致训练有素的免疫抑制或训练有素的耐受。我们的研究结果有力地推动了探索以 NLRP3/IL-1 通路为靶点作为调节移植受者和促进异体移植耐受的新策略的可能性。
{"title":"Adjuvant conditioning shapes the adaptive immune response and promotes trained immunotolerance via NLRP3/IL-1","authors":"Thais Boccia, Weikang Pan, Victor Fattori, Rodrigo Cervantes-Diaz, Michael S. Rogers, Ivan Zanoni, Alex G. Cuenca","doi":"10.1101/2024.09.06.611736","DOIUrl":"https://doi.org/10.1101/2024.09.06.611736","url":null,"abstract":"Trained immunity enhances responsiveness of the innate immune system upon restimulation. Although adjuvants are used to enhance immune responses, we showed that repeated administration of alum, termed adjuvant conditioning (AC), establishes an immunosuppressive environment that delays allogeneic graft rejection by expanding myeloid-derived suppressor cells (MDSCs). Here, we show that AC-induced MDSCs suppress antigen specific adaptive responses both in vitro and in vivo, and that the immunosuppression is abolished in the absence of NLRP3 and IL-1 signaling. Allogeneic pancreatic islets transplanted into AC-treated NLRP3-/- mice are not protected, demonstrating that AC requires NLRP3 signaling. Finally, AC also has an immunosuppressive effect on human PBMCs. Overall, our data show that AC establishes an immunosuppressive milieu via the NLRP3/IL-1 axis, leading to trained immunosuppression, or trained tolerance. Our findings give a potent mandate to explore the possibility to target the NLRP3/IL-1 pathway as a new promising strategy to condition transplant recipients and promote allograft tolerance.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211217","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 : 2024-09-11DOI: 10.1101/2024.09.06.611697
Andrea J Manrique-Rincon, Ben Foster, Stuart Horswell, David A Goulding, David J Adams, Anneliese O Speak
Immunotherapy has revolutionised the treatment of multiple cancer types, however, these treatments only work for a proportion of patients and biomarkers to predict response are lacking. One correlate of response is the reinvigoration of a subset of CD8 T cells that have an exhausted phenotype and impaired functionality. In order to develop new therapies, reproducible models are required to identify candidate target genes that enables the reversal of key hallmarks of T cell exhaustion. Here we describe the development of an in vitro model by chronically stimulating T cells with their cognate antigen and performed an in depth temporal phenotypic characterisation. This model recapitulates many of the critical hallmarks of exhaustion, including increased expression of canonical exhaustion surface markers, impaired proliferation, reduced cytokine production, decreased release of cytotoxic granules, and metabolic alterations, including dysfunctional mitochondria. These exhaustion hallmarks were validated using an in vivo model and a gene signature identified which robustly define the shared in vitro and in vivo exhausted state. Critically, this signature is also observed in tumour infiltrating T cells from multiple human tumour types, validating the translational potential of this model for discovering and triaging new therapies.
免疫疗法为多种癌症的治疗带来了革命性的变化，然而，这些疗法只对一部分患者有效，而且缺乏预测反应的生物标志物。反应的一个相关因素是表型衰竭、功能受损的 CD8 T 细胞亚群重新焕发活力。为了开发新的疗法，需要建立可重复的模型来确定候选靶基因，从而逆转 T 细胞衰竭的关键特征。在这里，我们描述了通过用T细胞的同源抗原长期刺激T细胞而建立的体外模型，并进行了深入的时间表型表征。该模型再现了衰竭的许多关键特征，包括典型衰竭表面标志物表达增加、增殖受损、细胞因子产生减少、细胞毒性颗粒释放减少以及代谢改变（包括线粒体功能障碍）。利用体内模型对这些衰竭特征进行了验证，并确定了一个基因特征，该特征有力地定义了体外和体内共同的衰竭状态。重要的是，在多种人类肿瘤类型的肿瘤浸润 T 细胞中也观察到了这一特征，从而验证了该模型在发现和分辨新疗法方面的转化潜力。
{"title":"Simulating CD8 T Cell Exhaustion: A Comprehensive Approach","authors":"Andrea J Manrique-Rincon, Ben Foster, Stuart Horswell, David A Goulding, David J Adams, Anneliese O Speak","doi":"10.1101/2024.09.06.611697","DOIUrl":"https://doi.org/10.1101/2024.09.06.611697","url":null,"abstract":"Immunotherapy has revolutionised the treatment of multiple cancer types, however, these treatments only work for a proportion of patients and biomarkers to predict response are lacking. One correlate of response is the reinvigoration of a subset of CD8 T cells that have an exhausted phenotype and impaired functionality. In order to develop new therapies, reproducible models are required to identify candidate target genes that enables the reversal of key hallmarks of T cell exhaustion. Here we describe the development of an in vitro model by chronically stimulating T cells with their cognate antigen and performed an in depth temporal phenotypic characterisation. This model recapitulates many of the critical hallmarks of exhaustion, including increased expression of canonical exhaustion surface markers, impaired proliferation, reduced cytokine production, decreased release of cytotoxic granules, and metabolic alterations, including dysfunctional mitochondria. These exhaustion hallmarks were validated using an in vivo model and a gene signature identified which robustly define the shared in vitro and in vivo exhausted state. Critically, this signature is also observed in tumour infiltrating T cells from multiple human tumour types, validating the translational potential of this model for discovering and triaging new therapies.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"26 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142226773","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 : 2024-09-11DOI: 10.1101/2024.09.10.612185
Pyae P. Kyawe, Ping Liu, Zhaozhao Jiang, Evan S. Bradley, Thomas Cicuto, Melanie I Trombly, Neal Silverman, Katherine Fitzgerald, William M. McDougall, Jennifer P. Wang
Influenza A virus (IAV) is a respiratory pathogen with a segmented negative-sense RNA genome that can cause epidemics and pandemics. The host factors required for the complete IAV infectious cycle have not been fully identified. Here, we examined select host factors that were identified by independent CRISPR screens as candidate contributors to IAV infectivity. We performed CRISPR-mediated knockout of cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS) as well as CRISPR-mediated overexpression of beta-1,4 N-acetylgalactosaminyltransferase 2 (B4GALNT2) and adenosine deaminase acting on RNA 1 (ADAR1) in the human bronchial epithelial A549 cell line and evaluated IAV infectivity. We confirmed that the knockout of CMAS or overexpression of B4GALNT2 restricts IAV infection by diminishing binding to the cell surface but has no effect on vesicular stomatitis virus infection. While ADAR1 overexpression does not significantly inhibit IAV replication, it has a pro-viral effect with coxsackie B virus (CVB) infection. This pro-viral effect is not likely secondary to reduced type I interferon (IFN) production, as the induction of the IFN-stimulated genes ISG15 and CXCL10 is negligible in both parent and ADAR1-overexpressing A549 cells following CVB challenge. In contrast, ISG15 and CXCL10 production is robust and equal for parent and ADAR1-overexpressing A549 cells challenged with IAV. Taken together, these data provide insight into how host factors identified in CRISPR screens can be further explored to understand the dynamics of pro- and anti-viral factors.
{"title":"CRISPR editing of candidate host factors that impact influenza A virus infection","authors":"Pyae P. Kyawe, Ping Liu, Zhaozhao Jiang, Evan S. Bradley, Thomas Cicuto, Melanie I Trombly, Neal Silverman, Katherine Fitzgerald, William M. McDougall, Jennifer P. Wang","doi":"10.1101/2024.09.10.612185","DOIUrl":"https://doi.org/10.1101/2024.09.10.612185","url":null,"abstract":"Influenza A virus (IAV) is a respiratory pathogen with a segmented negative-sense RNA genome that can cause epidemics and pandemics. The host factors required for the complete IAV infectious cycle have not been fully identified. Here, we examined select host factors that were identified by independent CRISPR screens as candidate contributors to IAV infectivity. We performed CRISPR-mediated knockout of cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS) as well as CRISPR-mediated overexpression of beta-1,4 N-acetylgalactosaminyltransferase 2 (B4GALNT2) and adenosine deaminase acting on RNA 1 (ADAR1) in the human bronchial epithelial A549 cell line and evaluated IAV infectivity. We confirmed that the knockout of CMAS or overexpression of B4GALNT2 restricts IAV infection by diminishing binding to the cell surface but has no effect on vesicular stomatitis virus infection. While ADAR1 overexpression does not significantly inhibit IAV replication, it has a pro-viral effect with coxsackie B virus (CVB) infection. This pro-viral effect is not likely secondary to reduced type I interferon (IFN) production, as the induction of the IFN-stimulated genes ISG15 and CXCL10 is negligible in both parent and ADAR1-overexpressing A549 cells following CVB challenge. In contrast, ISG15 and CXCL10 production is robust and equal for parent and ADAR1-overexpressing A549 cells challenged with IAV. Taken together, these data provide insight into how host factors identified in CRISPR screens can be further explored to understand the dynamics of pro- and anti-viral factors.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"17 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211219","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 : 2024-09-11DOI: 10.1101/2024.09.06.611551
Maegan L Brockman, Triniti A Scruggs, Lanfang Wang, Gabriella Kabboul, John W Calvert, Rebecca D Levit
Background: Neutrophils are the most rapid and abundant immune cells to infiltrate the myocardium following myocardial ischemia/reperfusion injury (MI/R). Neutrophil heterogeneity has not been well characterized in MI/R, and studies have shown conflicting results regarding the impact of neutrophil depletion on cardiac injury. We thus aim to study the impact of neutrophils with enriched type I interferon signature and the role of STING (stimulator of interferon genes) signaling in neutrophils on cardiac reperfusion injury. Methods: We utilized single-cell RNA sequencing to study neutrophil heterogeneity in response to MI/R. We generated a neutrophil-specific STING knockout mouse to assess the role of neutrophil STING in a model of MI/R. We examined cardiac function following injury via echocardiography and assessed the immune cell trajectory following injury utilizing flow cytometry. Results: We identified a population of neutrophils with enriched type I interferon signaling and response to type I interferon following MI/R. We found that genetic deletion of neutrophil-specific STING led to worsened cardiac function following MI/R. Further investigation of the immune response by flow cytometry revealed decreased neutrophil infiltration into the myocardium and a shift in macrophage polarization. Conclusions: Our findings suggest that neutrophil-specific STING is cardioprotective in MI/R, partly due to its effects on downstream immune cells. These results demonstrate that early alterations or therapeutic interventions can influence key events in the resolution of inflammation following MI/R.
背景：中性粒细胞是心肌缺血/再灌注损伤（MI/R）后浸润心肌最快速、最大量的免疫细胞。中性粒细胞在心肌缺血/再灌注损伤中的异质性尚未得到很好的描述，有关中性粒细胞耗竭对心脏损伤影响的研究结果也相互矛盾。因此，我们旨在研究具有丰富 I 型干扰素特征的中性粒细胞对心脏再灌注损伤的影响以及中性粒细胞中 STING（干扰素基因刺激器）信号转导的作用。研究方法我们利用单细胞 RNA 测序技术研究了中性粒细胞对 MI/R 反应的异质性。我们产生了一种中性粒细胞特异性 STING 基因敲除小鼠，以评估中性粒细胞 STING 在 MI/R 模型中的作用。我们通过超声心动图检查了损伤后的心脏功能，并利用流式细胞术评估了损伤后的免疫细胞轨迹。结果：我们发现中性粒细胞群在 MI/R 后具有丰富的 I 型干扰素信号传导和对 I 型干扰素的反应。我们发现，遗传性删除中性粒细胞特异性 STING 会导致 MI/R 后心脏功能恶化。流式细胞术对免疫反应的进一步研究表明，中性粒细胞对心肌的浸润减少，巨噬细胞的极化发生了变化。结论：我们的研究结果表明，嗜中性粒细胞特异性 STING 对心肌梗死/急性心肌梗死具有心脏保护作用，部分原因是其对下游免疫细胞的影响。这些结果表明，早期改变或治疗干预可影响 MI/R 后炎症消退的关键事件。
{"title":"The Cardioprotective Role of Neutrophil-Specific STING in Myocardial Ischemia/Reperfusion Injury","authors":"Maegan L Brockman, Triniti A Scruggs, Lanfang Wang, Gabriella Kabboul, John W Calvert, Rebecca D Levit","doi":"10.1101/2024.09.06.611551","DOIUrl":"https://doi.org/10.1101/2024.09.06.611551","url":null,"abstract":"Background: Neutrophils are the most rapid and abundant immune cells to infiltrate the myocardium following myocardial ischemia/reperfusion injury (MI/R). Neutrophil heterogeneity has not been well characterized in MI/R, and studies have shown conflicting results regarding the impact of neutrophil depletion on cardiac injury. We thus aim to study the impact of neutrophils with enriched type I interferon signature and the role of STING (stimulator of interferon genes) signaling in neutrophils on cardiac reperfusion injury. Methods: We utilized single-cell RNA sequencing to study neutrophil heterogeneity in response to MI/R. We generated a neutrophil-specific STING knockout mouse to assess the role of neutrophil STING in a model of MI/R. We examined cardiac function following injury via echocardiography and assessed the immune cell trajectory following injury utilizing flow cytometry. Results: We identified a population of neutrophils with enriched type I interferon signaling and response to type I interferon following MI/R. We found that genetic deletion of neutrophil-specific STING led to worsened cardiac function following MI/R. Further investigation of the immune response by flow cytometry revealed decreased neutrophil infiltration into the myocardium and a shift in macrophage polarization. Conclusions: Our findings suggest that neutrophil-specific STING is cardioprotective in MI/R, partly due to its effects on downstream immune cells. These results demonstrate that early alterations or therapeutic interventions can influence key events in the resolution of inflammation following MI/R.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"11 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211218","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 : 2024-09-11DOI: 10.1101/2024.09.06.611687
Michaela Gregorova, Marianna Santopaolo, Lucy C Garner, Divya Diamond, Narayan Ramamurthy, Vi Tran, Nguyet Nguyen Minh, Eben Jones, Mike Nsubuga, Curtis Luscombe, Hoa Vo Thi My, Ho Quang Chanh, Nguyen Thi Xuan Chau, Dong Thi Hoai Tam, Duyen Huynh Thi Le, Cao Thi Tam, Paul Klenerman, Sophie Yacoub, Laura Rivino
Dengue is a mosquito-borne virus infection affecting half of the world's population for which therapies are lacking. The role of T and NK-cells in protection/immunopathogenesis remains unclear for dengue. We performed a longitudinal phenotypic, functional and transcriptional analyses of T and NK-cells in 124 dengue patients using flow cytometry and single-cell RNA-sequencing. We show that T/NK-cell signatures early in infection discriminate patients who will progress to severe dengue (SD) from those who do not. In patients with overweight/obesity these signatures are exacerbated compared to healthy weight patients, supporting their increased susceptibility to SD. In SD, CD4+/CD8+ T-cells and NK-cells display increased co-inhibitory receptor expression and decreased cytotoxic capacity compared to non-SD. Furthermore, type-I Interferon signalling is downregulated in SD, suggesting defective virus-sensing mechanisms may underlie NK/T-cell dysfunction. We propose that dysfunctional "professional killer" T/NK-cells underpin dengue pathogenesis. Our findings pave the way for the evaluation of immunomodulatory therapies for dengue.
登革热是一种由蚊子传播的病毒感染，影响着全球一半的人口，目前尚无治疗方法。T细胞和NK细胞在登革热保护/免疫发病机制中的作用仍不清楚。我们利用流式细胞术和单细胞 RNA 序列对 124 名登革热患者的 T 细胞和 NK 细胞进行了纵向表型、功能和转录分析。我们发现，在感染早期，T/NK 细胞特征可区分会发展为重症登革热（SD）的患者和不会发展为重症登革热（SD）的患者。与体重健康的患者相比，超重/肥胖患者的这些特征更加明显，这证明他们更容易感染 SD。与非 SD 患者相比，SD 患者的 CD4+/CD8+ T 细胞和 NK 细胞的协同抑制受体表达增加，细胞毒性能力下降。此外，Ⅰ型干扰素信号在SD中下调，这表明病毒感应机制缺陷可能是NK/T细胞功能失调的原因。我们认为，"专业杀手 "T/NK细胞功能失调是登革热发病机制的基础。我们的发现为评估登革热的免疫调节疗法铺平了道路。
{"title":"Early NK-cell and T-cell dysfunction marks progression to severe dengue in patients with obesity and healthy weight","authors":"Michaela Gregorova, Marianna Santopaolo, Lucy C Garner, Divya Diamond, Narayan Ramamurthy, Vi Tran, Nguyet Nguyen Minh, Eben Jones, Mike Nsubuga, Curtis Luscombe, Hoa Vo Thi My, Ho Quang Chanh, Nguyen Thi Xuan Chau, Dong Thi Hoai Tam, Duyen Huynh Thi Le, Cao Thi Tam, Paul Klenerman, Sophie Yacoub, Laura Rivino","doi":"10.1101/2024.09.06.611687","DOIUrl":"https://doi.org/10.1101/2024.09.06.611687","url":null,"abstract":"Dengue is a mosquito-borne virus infection affecting half of the world's population for which therapies are lacking. The role of T and NK-cells in protection/immunopathogenesis remains unclear for dengue. We performed a longitudinal phenotypic, functional and transcriptional analyses of T and NK-cells in 124 dengue patients using flow cytometry and single-cell RNA-sequencing. We show that T/NK-cell signatures early in infection discriminate patients who will progress to severe dengue (SD) from those who do not. In patients with overweight/obesity these signatures are exacerbated compared to healthy weight patients, supporting their increased susceptibility to SD. In SD, CD4+/CD8+ T-cells and NK-cells display increased co-inhibitory receptor expression and decreased cytotoxic capacity compared to non-SD. Furthermore, type-I Interferon signalling is downregulated in SD, suggesting defective virus-sensing mechanisms may underlie NK/T-cell dysfunction. We propose that dysfunctional \"professional killer\" T/NK-cells underpin dengue pathogenesis. Our findings pave the way for the evaluation of immunomodulatory therapies for dengue.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"6 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211221","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 : 2024-09-11DOI: 10.1101/2024.09.11.608426
Ana-Maria Nitulescu, Weijie Du, Viktor Glaser, Jonas Kath, Robert Greensmith, Nanna S Mikkelsen, Maik Stein, Rasmus Bak, Michael Kaminski, Dimitrios Laurin Wagner
Non-viral gene editing via CRISPR-Cas12a offers an alternative to Cas9-based methods, providing better targeting of AT-rich regions, simplified guide RNA manufacturing, and high specificity. However, the efficacy of editing outcomes is subject to various factors, with tem-plate format playing a crucial role. Currently, the predominant non-viral template format for inducing homology-directed repair (HDR) after nuclease-induced DNA breaks is double-stranded DNA (dsDNA), which is toxic when transfected at high doses. Previous studies have demonstrated that using single-stranded DNA (ssDNA) with flanking double-stranded Cas-target-sequences (CTS) as a repair template for Cas9-mediated gene editing can miti-gate this toxicity and increase knock-in efficiency. Here, we investigate CTS design for As-Cas12a Ultra by exploring PAM orientation and binding requirements of the Cas12a-crRNA complex. Additionally, we rule out in-vitro ssDNase activity of AsCas12a Ultra under cell-physiological Mg2+ conditions. Finally, we showcase the advantage of using ssDNA with double-stranded CTS end modifications (ssCTS) at high doses for delivering clinically relevant transgenes of varying sizes into three T-cell receptor-CD3 complex genes (TRAC, CD3ζ, CD3ϵ), achieving up to 90% knock-in rates for a 0.8kb insert at the CD3ϵ locus. Overall, AsCas12a Ultra and ssCTS donors represent a platform for highly efficient knock-in in primary human T cells with minimal toxicity.
{"title":"Single-stranded HDR templates with truncated Cas12a binding sequences improve knock-in efficiencies in primary human T cells","authors":"Ana-Maria Nitulescu, Weijie Du, Viktor Glaser, Jonas Kath, Robert Greensmith, Nanna S Mikkelsen, Maik Stein, Rasmus Bak, Michael Kaminski, Dimitrios Laurin Wagner","doi":"10.1101/2024.09.11.608426","DOIUrl":"https://doi.org/10.1101/2024.09.11.608426","url":null,"abstract":"Non-viral gene editing via CRISPR-Cas12a offers an alternative to Cas9-based methods, providing better targeting of AT-rich regions, simplified guide RNA manufacturing, and high specificity. However, the efficacy of editing outcomes is subject to various factors, with tem-plate format playing a crucial role. Currently, the predominant non-viral template format for inducing homology-directed repair (HDR) after nuclease-induced DNA breaks is double-stranded DNA (dsDNA), which is toxic when transfected at high doses. Previous studies have demonstrated that using single-stranded DNA (ssDNA) with flanking double-stranded Cas-target-sequences (CTS) as a repair template for Cas9-mediated gene editing can miti-gate this toxicity and increase knock-in efficiency. Here, we investigate CTS design for As-Cas12a Ultra by exploring PAM orientation and binding requirements of the Cas12a-crRNA complex. Additionally, we rule out in-vitro ssDNase activity of AsCas12a Ultra under cell-physiological Mg2+ conditions. Finally, we showcase the advantage of using ssDNA with double-stranded CTS end modifications (ssCTS) at high doses for delivering clinically relevant transgenes of varying sizes into three T-cell receptor-CD3 complex genes (TRAC, CD3ζ, CD3ϵ), achieving up to 90% knock-in rates for a 0.8kb insert at the CD3ϵ locus. Overall, AsCas12a Ultra and ssCTS donors represent a platform for highly efficient knock-in in primary human T cells with minimal toxicity.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"64 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211220","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 : 2024-09-10DOI: 10.1101/2024.09.05.611435
Honghua He, Jihong Zhong, Qinghua Li, Chen Deng, Xin Yuan, Kaixiang Zhang, Lirong Nie, Nali Cai
Objective: To elucidate the causal relationship between immune cells and diffuse large B-cell lymphoma (DLBCL), we conducted a Mendelian randomization analysis. Methods: Mendelian randomization (MR) leverages genetic variants as instruments to infer causal effects from observational data. Here, we performed a two-sample MR analysis to assess the causal impact of 731 immune cell types on DLBCL. We employed various MR techniques, including the weighted median estimator (WME) and inverse variance weighting (IVW), and conducted sensitivity analyses to ensure result robustness. Additionally, reverse MR analysis was performed to explore the potential causal relationship between DLBCL and immune cells. Results: We identified seventeen immune features with causal links to DLBCL, categorized across various cellular groups: four in B cells, two in T cell maturation stages, six in Tregs, four in the TBNK group, and one in dendritic cells (DCs). Sensitivity analyses confirmed the absence of heterogeneity, horizontal pleiotropy, and bias in our findings. Reverse causal analysis revealed a causal association between DLBCL and one of the seventeen immune cell types identified. Conclusions: Our MR analysis of seventeen immune cell types uncovers the complex interactions between the immune system and DLBCL, providing crucial insights into the tumor microenvironment and potential avenues for targeted immunotherapy.
{"title":"Immune Cell Influence on Diffuse Large B-Cell Lymphoma: A Mendelian Randomization Analysis","authors":"Honghua He, Jihong Zhong, Qinghua Li, Chen Deng, Xin Yuan, Kaixiang Zhang, Lirong Nie, Nali Cai","doi":"10.1101/2024.09.05.611435","DOIUrl":"https://doi.org/10.1101/2024.09.05.611435","url":null,"abstract":"Objective: To elucidate the causal relationship between immune cells and diffuse large B-cell lymphoma (DLBCL), we conducted a Mendelian randomization analysis.\u0000Methods: Mendelian randomization (MR) leverages genetic variants as instruments to infer causal effects from observational data. Here, we performed a two-sample MR analysis to assess the causal impact of 731 immune cell types on DLBCL. We employed various MR techniques, including the weighted median estimator (WME) and inverse variance weighting (IVW), and conducted sensitivity analyses to ensure result robustness. Additionally, reverse MR analysis was performed to explore the potential causal relationship between DLBCL and immune cells.\u0000Results: We identified seventeen immune features with causal links to DLBCL, categorized across various cellular groups: four in B cells, two in T cell maturation stages, six in Tregs, four in the TBNK group, and one in dendritic cells (DCs). Sensitivity analyses confirmed the absence of heterogeneity, horizontal pleiotropy, and bias in our findings. Reverse causal analysis revealed a causal association between DLBCL and one of the seventeen immune cell types identified.\u0000Conclusions: Our MR analysis of seventeen immune cell types uncovers the complex interactions between the immune system and DLBCL, providing crucial insights into the tumor microenvironment and potential avenues for targeted immunotherapy.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"410 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211264","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 : 2024-09-10DOI: 10.1101/2024.09.06.611470
Esther L Jones, Benjamin Demarco, Madelon ML de Jong, Han Cai, Sarah Hill, Ryan E. Glass, Gemma Harris, Saba Nayar, Benjamin A Fisher, Audrey Gerard, Jelena S Bezbradica, Lynn B Dustin
Sjögren's disease (SjD) causes localised and systemic inflammation due to autoantibody production against intracellular proteins, such as TRIM21/Ro52. TRIM21 is an E3 ubiquitin ligase which binds antibody Fc domains on opsonised pathogens, which have escaped extracellular immunity and entered cytosols; TRIM21 ubiquitinates these, driving their proteasomal degradation. How and why TRIM21 becomes an autoantigen remains unclear. We show that TRIM21 is released upon lytic cell death (pyroptosis/necroptosis) but not apoptosis. Released TRIM21 binds circulating antibody Fc domains, and forms large immune complexes (ICs). These are further enhanced with TRIM21/Ro52 seropositive SjD plasma antibodies, where interactions are mediated via both Fc and F(ab')2 domains. TRIM21-ICs are taken up by macrophages, which in high interferon environments drive pro-inflammatory responses, antigen presentation, and inflammatory and metabolic transcriptional changes. Whilst many cytosolic proteins are released by dead cells, due to its high affinity for antibodies, TRIM21 can generate large ICs. This may perpetuate inflammation and antigen presentation, causing TRIM21 to be highly autoimmunogenic.
斯约格伦病（SjD）会引起局部和全身性炎症，这是由于针对细胞内蛋白（如 TRIM21/Ro52）的自身抗体产生所致。TRIM21 是一种 E3 泛素连接酶，它能与蛋白溶解的病原体上的抗体 Fc 结构域结合，这些病原体逃过了细胞外免疫，进入了细胞溶质；TRIM21 对这些病原体进行泛素化，促使其蛋白酶体降解。TRIM21如何以及为何成为自身抗原仍不清楚。我们的研究表明，TRIM21会在细胞溶解性死亡（热凋亡/坏死）时释放，但不会在细胞凋亡时释放。释放的 TRIM21 与循环抗体 Fc 结构域结合，形成大型免疫复合物（IC）。这些复合物与 TRIM21/Ro52 血清阳性 SjD 血浆抗体结合后会进一步增强，其中的相互作用是通过 Fc 和 F(ab')2 结构域介导的。TRIM21-IC 被巨噬细胞吸收，在高干扰素环境中，巨噬细胞会驱动促炎反应、抗原呈递以及炎症和代谢转录变化。虽然死亡细胞会释放出许多细胞膜蛋白，但由于 TRIM21 与抗体的高亲和力，它能产生大量的 IC。这可能会使炎症和抗原呈递永久化，导致 TRIM21 成为高度自身免疫原。
{"title":"Autoantigen TRIM21 (Ro52) assembles pro-inflammatory immune complexes following lytic cell death","authors":"Esther L Jones, Benjamin Demarco, Madelon ML de Jong, Han Cai, Sarah Hill, Ryan E. Glass, Gemma Harris, Saba Nayar, Benjamin A Fisher, Audrey Gerard, Jelena S Bezbradica, Lynn B Dustin","doi":"10.1101/2024.09.06.611470","DOIUrl":"https://doi.org/10.1101/2024.09.06.611470","url":null,"abstract":"Sjögren's disease (SjD) causes localised and systemic inflammation due to autoantibody production against intracellular proteins, such as TRIM21/Ro52. TRIM21 is an E3 ubiquitin ligase which binds antibody Fc domains on opsonised pathogens, which have escaped extracellular immunity and entered cytosols; TRIM21 ubiquitinates these, driving their proteasomal degradation. How and why TRIM21 becomes an autoantigen remains unclear. We show that TRIM21 is released upon lytic cell death (pyroptosis/necroptosis) but not apoptosis. Released TRIM21 binds circulating antibody Fc domains, and forms large immune complexes (ICs). These are further enhanced with TRIM21/Ro52 seropositive SjD plasma antibodies, where interactions are mediated via both Fc and F(ab')2 domains. TRIM21-ICs are taken up by macrophages, which in high interferon environments drive pro-inflammatory responses, antigen presentation, and inflammatory and metabolic transcriptional changes. Whilst many cytosolic proteins are released by dead cells, due to its high affinity for antibodies, TRIM21 can generate large ICs. This may perpetuate inflammation and antigen presentation, causing TRIM21 to be highly autoimmunogenic.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"12 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211224","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 : 2024-09-10DOI: 10.1101/2024.09.08.611922
Itay Raphael, Anzar A Mujeeb, Elisabet Ampudia-Mesias, ReidAnn E Sever, Brandon McClellan, Stephen C Frederico, Chaim T Sneiderman, Apoorva Mirji, Ali Daba, Francisco Puerta-Martinez, Michal Nisnboym, Wilson B Edwards, Michael Graner, Christopher Moertel, Maria G Castro, Gary Kohanbash, Michael Olin
High-grade gliomas are a major health challenge with poor prognosis and high morbidity. Immune-checkpoint inhibitors (ICI) have emerged as promising therapeutic options for several malignancies yet show little efficacy against central nervous system (CNS) tumors. CD200 is a newly recognized immune checkpoint that modulates immune homeostasis. CD200 protein is expressed by a variety of cells, including immune cells and stromal cells, and is overexpressed by many tumors. The shedding of CD200 from tumor cells can create an immunosuppressive environment that dampens anti-tumor immunity by modulating cytolytic activity and cytokine expression both within and outside the tumor microenvironment (TME). While it is well-accepted that CD200 induces a pro-tumorigenic environment through its ability to suppress the immune response, we sought to determine the role of glioma-specific expression of CD200. We show that CD200 is expressed across glioma types, is shed from tumor cells, and increases over time in the serum of patients undergoing immunotherapy. Using CD200 knockout (KO) glioma models, we demonstrated that glioma cell-derived CD200 promotes tumor growth in vivo and in vitro. Notably, CD200 KO gliomas are spontaneously rejected by their host, a process that required a fully functional immune system, including NK and T-cells. Moreover, we report that glioma-derived or brain-injected soluble CD200 contributes to the suppression of antigen-specific CD8 T-cells in the draining lymph nodes (dLNs). Our work provides new mechanistic insights regarding CD200-mediated immunosuppression by gliomas.
{"title":"CD200 depletion in glioma enhances antitumor immunity and induces tumor rejection","authors":"Itay Raphael, Anzar A Mujeeb, Elisabet Ampudia-Mesias, ReidAnn E Sever, Brandon McClellan, Stephen C Frederico, Chaim T Sneiderman, Apoorva Mirji, Ali Daba, Francisco Puerta-Martinez, Michal Nisnboym, Wilson B Edwards, Michael Graner, Christopher Moertel, Maria G Castro, Gary Kohanbash, Michael Olin","doi":"10.1101/2024.09.08.611922","DOIUrl":"https://doi.org/10.1101/2024.09.08.611922","url":null,"abstract":"High-grade gliomas are a major health challenge with poor prognosis and high morbidity. Immune-checkpoint inhibitors (ICI) have emerged as promising therapeutic options for several malignancies yet show little efficacy against central nervous system (CNS) tumors. CD200 is a newly recognized immune checkpoint that modulates immune homeostasis. CD200 protein is expressed by a variety of cells, including immune cells and stromal cells, and is overexpressed by many tumors. The shedding of CD200 from tumor cells can create an immunosuppressive environment that dampens anti-tumor immunity by modulating cytolytic activity and cytokine expression both within and outside the tumor microenvironment (TME). While it is well-accepted that CD200 induces a pro-tumorigenic environment through its ability to suppress the immune response, we sought to determine the role of glioma-specific expression of CD200. We show that CD200 is expressed across glioma types, is shed from tumor cells, and increases over time in the serum of patients undergoing immunotherapy. Using CD200 knockout (KO) glioma models, we demonstrated that glioma cell-derived CD200 promotes tumor growth in vivo and in vitro. Notably, CD200 KO gliomas are spontaneously rejected by their host, a process that required a fully functional immune system, including NK and T-cells. Moreover, we report that glioma-derived or brain-injected soluble CD200 contributes to the suppression of antigen-specific CD8 T-cells in the draining lymph nodes (dLNs). Our work provides new mechanistic insights regarding CD200-mediated immunosuppression by gliomas.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"61 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142226774","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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