Pub Date : 2024-09-10DOI: 10.1101/2024.09.09.612153
Leidy Marian Valencia, Yoandra Martinez-Montano, Jose Alberto Gomez, Roger Sarduy, Arletty Hernandez, Spencer Dominic Proctor, Ayme Fernandez-Calienes, Victor Brito, Yosdel Soto
Atherosclerosis is initiated by the retention of ApoB-containing lipoproteins in the arterial wall, mediated by glycosaminoglycan chains of proteoglycans. At the Center for Molecular Immunology, we are developing the P3R99 monoclonal antibody (mAb) to target this process. This study characterizes new P3R99 mAb variants expressed in CHO-K1 and HEK-293 cell lines. We compared these variants with the parental mAb from NS0 cells using SDS-PAGE, size exclusion and cation exchange chromatography, dynamic light scattering, peptide mapping, far-UV circular dichroism, and PNGase F deglycosylation. All variants exhibited a molecular size of ~150 kDa, ~99% purity, and similar average particle sizes (12.5-13.7 nm). They displayed a high β-sheet content (>40%) and basic amino acids on the surface, with minor differences in peptide maps compared to the parental mAb. Notable differences were found in the content of acidic and basic species and glycosylation profiles. NS0-derived P3R99 had lower G0F content (10.39%), higher G1F (38.29%) and G2F (30.44%) levels, with more terminal galactose (83.07%) and sialylation (15.33%). In contrast, CHO-K1 and HEK-293 variants showed similar glycosylation patterns. Despite these differences, the antigen and atherosclerotic lesion recognition properties of the mAb were unaffected in vitro. Biodistribution studies in Sprague Dawley rats (1 mg, IV, n=3) revealed preferential accumulation of the new P3R99 variants in aortas and reduced LDL arterial retention (1 mg, IP). Passive administration of the mAbs (2 mg every three days, three IV doses, n=6-7) in a Lipofundin 20%-induced atherosclerosis NZW rabbit model also demonstrated preferential accumulation in aortas and reduced atherosclerosis, with 60% of treated rabbits not developing lesions. These results suggest that the P3R99 mAb derived from CHO-K1 and HEK-293 cells retains its antiatherogenic properties despite structural differences from the NS0-derived mAb associated with the different expression systems.
动脉粥样硬化的起因是含载脂蛋白 B 的脂蛋白在蛋白聚糖链的介导下滞留在动脉壁上。在分子免疫学中心，我们正在开发 P3R99 单克隆抗体（mAb）来靶向这一过程。本研究描述了在 CHO-K1 和 HEK-293 细胞系中表达的新型 P3R99 mAb 变体。我们使用 SDS-PAGE、尺寸排阻色谱法和阳离子交换色谱法、动态光散射、肽图、远紫外圆二色性和 PNGase F 脱糖法将这些变体与来自 NS0 细胞的亲本 mAb 进行了比较。所有变体的分子大小约为 150 kDa，纯度约为 99%，平均粒径（12.5-13.7 nm）相似。它们显示出较高的β片段含量（40%）和表面的碱性氨基酸，与亲本 mAb 相比，肽图差异较小。酸性和碱性种类的含量以及糖基化图谱存在显著差异。来源于 NS0 的 P3R99 的 G0F 含量较低（10.39%），G1F（38.29%）和 G2F（30.44%）水平较高，末端半乳糖（83.07%）和糖基化（15.33%）较多。相比之下，CHO-K1 和 HEK-293 变体显示出相似的糖基化模式。尽管存在这些差异，但 mAb 的抗原和动脉粥样硬化病变识别特性在体外未受影响。在 Sprague Dawley 大鼠体内进行的生物分布研究（1 毫克，静脉注射，n=3）显示，新的 P3R99 变体在主动脉中优先蓄积，并减少了低密度脂蛋白的动脉滞留（1 毫克，IP）。在利波芬定 20% 诱导的动脉粥样硬化 NZW 兔子模型中，被动给药 mAbs（每三天 2 毫克，三次静脉注射，n=6-7）也显示了在主动脉中的优先积聚，并减少了动脉粥样硬化，60% 的受治疗兔子没有发生病变。这些结果表明，来自 CHO-K1 和 HEK-293 细胞的 P3R99 mAb 仍具有抗动脉粥样硬化的特性，尽管与 NS0 衍生的 mAb 在结构上存在差异，而且表达系统也不同。
{"title":"Characterization and Antiatherogenic Potential of P3R99 Monoclonal Antibody Against Sulfated Glycosaminoglycans: Physicochemical and Functional Insights","authors":"Leidy Marian Valencia, Yoandra Martinez-Montano, Jose Alberto Gomez, Roger Sarduy, Arletty Hernandez, Spencer Dominic Proctor, Ayme Fernandez-Calienes, Victor Brito, Yosdel Soto","doi":"10.1101/2024.09.09.612153","DOIUrl":"https://doi.org/10.1101/2024.09.09.612153","url":null,"abstract":"Atherosclerosis is initiated by the retention of ApoB-containing lipoproteins in the arterial wall, mediated by glycosaminoglycan chains of proteoglycans. At the Center for Molecular Immunology, we are developing the P3R99 monoclonal antibody (mAb) to target this process. This study characterizes new P3R99 mAb variants expressed in CHO-K1 and HEK-293 cell lines. We compared these variants with the parental mAb from NS0 cells using SDS-PAGE, size exclusion and cation exchange chromatography, dynamic light scattering, peptide mapping, far-UV circular dichroism, and PNGase F deglycosylation. All variants exhibited a molecular size of ~150 kDa, ~99% purity, and similar average particle sizes (12.5-13.7 nm). They displayed a high β-sheet content (>40%) and basic amino acids on the surface, with minor differences in peptide maps compared to the parental mAb. Notable differences were found in the content of acidic and basic species and glycosylation profiles. NS0-derived P3R99 had lower G0F content (10.39%), higher G1F (38.29%) and G2F (30.44%) levels, with more terminal galactose (83.07%) and sialylation (15.33%). In contrast, CHO-K1 and HEK-293 variants showed similar glycosylation patterns. Despite these differences, the antigen and atherosclerotic lesion recognition properties of the mAb were unaffected in vitro. Biodistribution studies in Sprague Dawley rats (1 mg, IV, n=3) revealed preferential accumulation of the new P3R99 variants in aortas and reduced LDL arterial retention (1 mg, IP). Passive administration of the mAbs (2 mg every three days, three IV doses, n=6-7) in a Lipofundin 20%-induced atherosclerosis NZW rabbit model also demonstrated preferential accumulation in aortas and reduced atherosclerosis, with 60% of treated rabbits not developing lesions. These results suggest that the P3R99 mAb derived from CHO-K1 and HEK-293 cells retains its antiatherogenic properties despite structural differences from the NS0-derived mAb associated with the different expression systems.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"20 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211223","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}
Uterine carcinosarcoma (UCS) is a rare gynecologic cancer with unfavorable prognosis. Its biphasic histologic components are known to associated with outcomes. Dissecting the tumor heterogeneity within UCS is crucial to discover molecular biomarkers for patient stratification. The aim of this study was to utilize spatial profiling technology to analyze immune presentation in different histological components within UCS tumors. The study included 23 UCS patients whose tumor sections were subjected to nanoString GeoMx Digital Spatial Profiling (DSP) to profile 30 immune-related proteins in 491 segments. We identified distinct cluster-labeled subtypes showing varied immune marker expressions. The immune presentations within the carcinoma and sarcoma components showed significant differences. Markers indicating M2 macrophages (CD45+CD163+) and epithelial-mesenchymal transition (EMT) (SMA, fibronectin) were highly expressed in the sarcoma components. The immune presentation subtypes within each histological component also differed between patients with recurrence in 5 years and without recurrence over 5 years. The cluster-labeled subtype with high Neutrophil/PMN-MDSC marker expression (CD66b) was associated with longer survival, whereas the subtype with high NK cell marker expressions (CD56) was linked to poor prognosis. Our result suggested that immune features within the carcinoma component of UCS tumors would determine patient outcomes and should be considered as a strategy for patient stratification.
{"title":"Inter- and intra-tumor heterogeneous immune presentation in primary uterine carcinosarcoma determined by digital spatial profiling","authors":"Yingxuan Li, Wei-Chou Lin, Ko-Chen Chen, Wen-Chun Chang, Chin-Jui Wu, Lin-Hung Wei, Ruby Yun-Ju Huang, Bor-Ching Sheu","doi":"10.1101/2024.09.05.611350","DOIUrl":"https://doi.org/10.1101/2024.09.05.611350","url":null,"abstract":"Uterine carcinosarcoma (UCS) is a rare gynecologic cancer with unfavorable prognosis. Its biphasic histologic components are known to associated with outcomes. Dissecting the tumor heterogeneity within UCS is crucial to discover molecular biomarkers for patient stratification. The aim of this study was to utilize spatial profiling technology to analyze immune presentation in different histological components within UCS tumors. The study included 23 UCS patients whose tumor sections were subjected to nanoString GeoMx Digital Spatial Profiling (DSP) to profile 30 immune-related proteins in 491 segments. We identified distinct cluster-labeled subtypes showing varied immune marker expressions. The immune presentations within the carcinoma and sarcoma components showed significant differences. Markers indicating M2 macrophages (CD45+CD163+) and epithelial-mesenchymal transition (EMT) (SMA, fibronectin) were highly expressed in the sarcoma components. The immune presentation subtypes within each histological component also differed between patients with recurrence in 5 years and without recurrence over 5 years. The cluster-labeled subtype with high Neutrophil/PMN-MDSC marker expression (CD66b) was associated with longer survival, whereas the subtype with high NK cell marker expressions (CD56) was linked to poor prognosis. Our result suggested that immune features within the carcinoma component of UCS tumors would determine patient outcomes and should be considered as a strategy for patient stratification.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"33 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211261","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: 10.1101/2024.09.05.611179
Maria C Hesselman, Marius Zeeb, Peter Rusert, Chloé Pasin, Jennifer Mamrosh, Samuel Kariuki, Michèle Sickmann, Masako M Kaufmann, Daniel Schmidt, Nikolas Friedrich, Karin J Metzner, Audrey Rindler, Herbert Kuster, Craig Adams, Ruwayhida Thebus, Michael Huber, Sabine Yerly, Karoline Leuzinger, Matthieu Perreau, Roger Koller, Günter Dollenmaier, Simona Frigerio, Dylan H Westfall, Wenjie Deng, Allan C DeCamp, Michal Juraska, Srilatha Edupuganti, Nyaradzo Mgodi, Hugh Murrell, Nigel Garrett, Kshitij Wagh, James I Mullins, Carolyn Williamson, Penny L Moore, Huldrych F Günthard, Roger D Kouyos, Alexandra Trkola
The identification of HIV-1 Envelope glycoprotein (Env) traits associated with development of neutralization cross-reactivity in natural infection is critical for vaccine design. Here we describe the presence of additional Cysteine (Cys) residues in V1 that are enriched among people with elite neutralization breadth. Using >65,000 V1 sequences from the CATNAP database, the AMP trials and three large longitudinal HIV infection cohorts, the SHCS, ZPHI and CAPRISA studies, we show that Env variants with extra V1 Cys are present at low levels throughout infection and fluctuate in frequency over time within participants. We demonstrate an independent association of extra V1 Cys with elite plasma neutralization, and a strong preference for two versus one extra Cys, suggesting certain Envs introduce an additional disulfide bond for stabilization. We observed high levels of neutralization resistance among Envs from 34 bNAb donors, of which 17.6% had elongated V1 regions with extra Cys. We show that extra V1 Cys moderately increase neutralization resistance in an Env from a V2-Apex bNAb-inducer. Modulation of the accessibility of bNAb epitopes on this Env by extra V1 Cys enhanced epitope shielding of several regions, but increased V2 exposure. This suggests that escape from autologous neutralizing activity drove insertion of the extra V1 Cys, creating a modified antigen that may have favored V2 bNAb induction in this donor. Overall, we identify a rare motif of twin Cys in V1 that confers increased neutralization resistance and Env stabilization, is associated with bNAb induction, and may hold potential for incorporation into future HIV bNAb immunogens.
{"title":"Rare twin cysteine residues in the HIV-1 envelope variable region 1 link to neutralization escape and breadth development","authors":"Maria C Hesselman, Marius Zeeb, Peter Rusert, Chloé Pasin, Jennifer Mamrosh, Samuel Kariuki, Michèle Sickmann, Masako M Kaufmann, Daniel Schmidt, Nikolas Friedrich, Karin J Metzner, Audrey Rindler, Herbert Kuster, Craig Adams, Ruwayhida Thebus, Michael Huber, Sabine Yerly, Karoline Leuzinger, Matthieu Perreau, Roger Koller, Günter Dollenmaier, Simona Frigerio, Dylan H Westfall, Wenjie Deng, Allan C DeCamp, Michal Juraska, Srilatha Edupuganti, Nyaradzo Mgodi, Hugh Murrell, Nigel Garrett, Kshitij Wagh, James I Mullins, Carolyn Williamson, Penny L Moore, Huldrych F Günthard, Roger D Kouyos, Alexandra Trkola","doi":"10.1101/2024.09.05.611179","DOIUrl":"https://doi.org/10.1101/2024.09.05.611179","url":null,"abstract":"The identification of HIV-1 Envelope glycoprotein (Env) traits associated with development of neutralization cross-reactivity in natural infection is critical for vaccine design. Here we describe the presence of additional Cysteine (Cys) residues in V1 that are enriched among people with elite neutralization breadth. Using >65,000 V1 sequences from the CATNAP database, the AMP trials and three large longitudinal HIV infection cohorts, the SHCS, ZPHI and CAPRISA studies, we show that Env variants with extra V1 Cys are present at low levels throughout infection and fluctuate in frequency over time within participants. We demonstrate an independent association of extra V1 Cys with elite plasma neutralization, and a strong preference for two versus one extra Cys, suggesting certain Envs introduce an additional disulfide bond for stabilization. We observed high levels of neutralization resistance among Envs from 34 bNAb donors, of which 17.6% had elongated V1 regions with extra Cys. We show that extra V1 Cys moderately increase neutralization resistance in an Env from a V2-Apex bNAb-inducer. Modulation of the accessibility of bNAb epitopes on this Env by extra V1 Cys enhanced epitope shielding of several regions, but increased V2 exposure. This suggests that escape from autologous neutralizing activity drove insertion of the extra V1 Cys, creating a modified antigen that may have favored V2 bNAb induction in this donor. Overall, we identify a rare motif of twin Cys in V1 that confers increased neutralization resistance and Env stabilization, is associated with bNAb induction, and may hold potential for incorporation into future HIV bNAb immunogens.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"14 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211266","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: 10.1101/2024.09.06.611679
Sana D Karam, Jacob Gadwa, Justin Yu, Miles Piper, Michael W Knitz, Laurel B Darragh, Nicholas A Olimpo, Sophia Corbo, Brooke Neupert, Jessica I Beynor, Alexander Nguyen, Chloe Hodgson, Khalid Nady Mohammed Abdelazeem, Diemmy Nguyen, Anthony Saviola, Mudita Pincha, Christian Klein, Maria Amann
Background: Treatment with immunotherapy can elicit varying responses across cancer types, and the mechanistic underpinnings that contribute to response vs. progression remain poorly understood. However, to date there are few preclinical models that accurately represent these disparate disease scenarios. Methods: Using combinatorial radio-immunotherapy consisting of PD-1 blockade, IL2Rβγ biased signaling, and OX40 agonism we were able to generate preclinical tumor models with conflicting responses, where head and neck squamous cell carcinoma (HNSCC) models responds and pancreatic ductal adenocarcinoma (PDAC) progresses. Results: By modeling these disparate states, we find that regulatory T cells (Tregs) are expanded in PDAC tumors undergoing treatment, constraining tumor reactive CD8 T cell activity. Consequently, the depletion of Tregs restores the therapeutic efficacy of our treatment and abrogates the disparity between models. Moreover, we show that through heterotopic implantations that the site of tumor development defines the response to therapy, as implantation of HNSCC tumors into the pancreas resulted in comparable levels of tumor progression. Conclusions: This work highlights complexity of combining immunotherapies within the tumor microenvironment and further defines the immune and non-immune components of the tumor microenvironment as an intrinsic feature of immune suppression.
背景：使用免疫疗法治疗不同类型的癌症会产生不同的反应，而导致反应与进展的机理基础仍然鲜为人知。然而，迄今为止，很少有临床前模型能准确代表这些不同的疾病情况：方法：使用由 PD-1 阻断、IL2Rβγ 偏倚信号传导和 OX40 激动组成的组合放射免疫疗法，我们能够生成具有相互冲突反应的临床前肿瘤模型，其中头颈部鳞状细胞癌（HNSCC）模型有反应，而胰腺导管腺癌（PDAC）有进展：通过模拟这些不同的状态，我们发现接受治疗的 PDAC 肿瘤中调节性 T 细胞（Tregs）会扩大，从而限制肿瘤反应性 CD8 T 细胞的活性。因此，Tregs的消耗恢复了我们治疗的疗效，并消除了模型之间的差异。此外，我们通过异位移植证明，肿瘤发生的部位决定了对治疗的反应，因为将 HNSCC 肿瘤移植到胰腺会导致肿瘤进展水平相当：这项工作凸显了在肿瘤微环境中结合免疫疗法的复杂性，并进一步明确了肿瘤微环境中的免疫和非免疫成分是免疫抑制的内在特征。
{"title":"Divergent response to radio-immunotherapy is defined by intrinsic features of the tumor microenvironment","authors":"Sana D Karam, Jacob Gadwa, Justin Yu, Miles Piper, Michael W Knitz, Laurel B Darragh, Nicholas A Olimpo, Sophia Corbo, Brooke Neupert, Jessica I Beynor, Alexander Nguyen, Chloe Hodgson, Khalid Nady Mohammed Abdelazeem, Diemmy Nguyen, Anthony Saviola, Mudita Pincha, Christian Klein, Maria Amann","doi":"10.1101/2024.09.06.611679","DOIUrl":"https://doi.org/10.1101/2024.09.06.611679","url":null,"abstract":"Background: Treatment with immunotherapy can elicit varying responses across cancer types, and the mechanistic underpinnings that contribute to response vs. progression remain poorly understood. However, to date there are few preclinical models that accurately represent these disparate disease scenarios.\u0000Methods: Using combinatorial radio-immunotherapy consisting of PD-1 blockade, IL2Rβγ biased signaling, and OX40 agonism we were able to generate preclinical tumor models with conflicting responses, where head and neck squamous cell carcinoma (HNSCC) models responds and pancreatic ductal adenocarcinoma (PDAC) progresses.\u0000Results: By modeling these disparate states, we find that regulatory T cells (Tregs) are expanded in PDAC tumors undergoing treatment, constraining tumor reactive CD8 T cell activity. Consequently, the depletion of Tregs restores the therapeutic efficacy of our treatment and abrogates the disparity between models. Moreover, we show that through heterotopic implantations that the site of tumor development defines the response to therapy, as implantation of HNSCC tumors into the pancreas resulted in comparable levels of tumor progression.\u0000Conclusions: This work highlights complexity of combining immunotherapies within the tumor microenvironment and further defines the immune and non-immune components of the tumor microenvironment as an intrinsic feature of immune suppression.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"73 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211222","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: 10.1101/2024.09.08.611829
Mehri Hajiaghayi, Fatemeh Gholizadeh, Eric Han, Samuel Little, Niloufar Rahbari, Isabella Ardila, Carolina Lopez Naranjo, Kasra Tehranimeh, Steve CC Shih, Peter J. Darlington
Introduction: Adrenergic receptors regulate metabolic, cardiovascular, and immunological functions in response to the sympathetic nervous system. The effect of β2- adrenergic receptor (AR) as a high-expression receptor on different subpopulations of T cells is complex and varies depending on the type of ligand and context. While traditional β2-AR agonists generally suppress T cells, they potentially enhance IL-17A production by Th17 cells. The effects of pharmacological drugs that count as biased agonists of AR like nebivolol are not completely understood. We investigated the impact of nebivolol on human memory CD4+ T (Th1, Th2, Th17) cells and polarized naive Th17 cells highlighting its potential for IL-17A suppression via a non-canonical β2-AR cell-signaling pathway. Methods: The effects of nebivolol were tested on healthy human peripheral blood mononuclear cells, purified memory Th cells, and polarized naive Th17 cells activated with antiCD3/antiCD28/antiCD2 ImmunoCult reagent. IFN‐γ, IL-4, and IL-17A which are primarily derived from Th1, Th2, and Th17 cells respectively, were quantified by ELISA and flow cytometry. IL-10 was measured by ELISA. Gene expression of RORC, ADRB1, ADRB2, and ADRB3 was evaluated by qPCR. The ADRB2 gene was knocked out in memory Th cells using CRISPR/Cas9. Protein expression of phosphorylated-serine133-CREB and phosphorylated-NF-κB p65 was assessed by Western blot. Proliferation was assessed by fluorescent dye loading and flow cytometry. Results: Nebivolol treatment decreased IL-17A and IFN‐γ secretion by activated-memory Th cells and elevated IL-4 levels. Nebivolol reduced the proportion of IL-17A+ Th cells and downregulated RORC expression. Unlike the β2-AR agonist terbutaline, nebivolol inhibited the shift of naive CD4+ T cells towards the Th17 phenotype. IL-10 and proliferation index remained unchanged. Nebivolol-treated β2-knockout memory Th cells showed significant inhibition of β2AR-mediated signaling, evidenced by the absence of IL-17A suppression compared to controls. Phosphorylation of the NF-κB p65 subunit was inhibited by nebivolol, but CREB phosphorylation was not changed, suggesting a selective transcriptional control. Conclusions: The findings demonstrate that nebivolol acts through a β2-AR-mediated signaling pathway, as a distinctive anti-inflammatory agent capable of selectively shifting Th17 cells and suppressing phosphorylation of NF-κB. This highlights nebivolol as a potential for therapeutic interventions in chronic autoimmune conditions with elevated IL-17A levels.
{"title":"β2-Adrenergic Biased Agonist Inhibits the Development of Th17 and the Response of Memory Th17 Cells in an NF-κB-Dependent Manner.","authors":"Mehri Hajiaghayi, Fatemeh Gholizadeh, Eric Han, Samuel Little, Niloufar Rahbari, Isabella Ardila, Carolina Lopez Naranjo, Kasra Tehranimeh, Steve CC Shih, Peter J. Darlington","doi":"10.1101/2024.09.08.611829","DOIUrl":"https://doi.org/10.1101/2024.09.08.611829","url":null,"abstract":"Introduction: Adrenergic receptors regulate metabolic, cardiovascular, and immunological functions in response to the sympathetic nervous system. The effect of β2- adrenergic receptor (AR) as a high-expression receptor on different subpopulations of T cells is complex and varies depending on the type of ligand and context. While traditional β2-AR agonists generally suppress T cells, they potentially enhance IL-17A production by Th17 cells. The effects of pharmacological drugs that count as biased agonists of AR like nebivolol are not completely understood. We investigated the impact of nebivolol on human memory CD4+ T (Th1, Th2, Th17) cells and polarized naive Th17 cells highlighting its potential for IL-17A suppression via a non-canonical β2-AR cell-signaling pathway.\u0000Methods: The effects of nebivolol were tested on healthy human peripheral blood mononuclear cells, purified memory Th cells, and polarized naive Th17 cells activated with antiCD3/antiCD28/antiCD2 ImmunoCult reagent. IFN‐γ, IL-4, and IL-17A which are primarily derived from Th1, Th2, and Th17 cells respectively, were quantified by ELISA and flow cytometry. IL-10 was measured by ELISA. Gene expression of RORC, ADRB1, ADRB2, and ADRB3 was evaluated by qPCR. The ADRB2 gene was knocked out in memory Th cells using CRISPR/Cas9. Protein expression of phosphorylated-serine133-CREB and phosphorylated-NF-κB p65 was assessed by Western blot. Proliferation was assessed by fluorescent dye loading and flow cytometry. Results: Nebivolol treatment decreased IL-17A and IFN‐γ secretion by activated-memory Th cells and elevated IL-4 levels. Nebivolol reduced the proportion of IL-17A+ Th cells and downregulated RORC expression. Unlike the β2-AR agonist terbutaline, nebivolol inhibited the shift of naive CD4+ T cells towards the Th17 phenotype. IL-10 and proliferation index remained unchanged. Nebivolol-treated β2-knockout memory Th cells showed significant inhibition of β2AR-mediated signaling, evidenced by the absence of IL-17A suppression compared to controls. Phosphorylation of the NF-κB p65 subunit was inhibited by nebivolol, but CREB phosphorylation was not changed, suggesting a selective transcriptional control. Conclusions: The findings demonstrate that nebivolol acts through a β2-AR-mediated signaling pathway, as a distinctive anti-inflammatory agent capable of selectively shifting Th17 cells and suppressing phosphorylation of NF-κB. This highlights nebivolol as a potential for therapeutic interventions in chronic autoimmune conditions with elevated IL-17A levels.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"6 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211260","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: 10.1101/2024.09.04.611333
Ananya Gupta, Vibha Taneja, Javier Rangel-Moreno, Abhimanyu Abhimanyu, Mushtaq Ahmed, Nilofer Naqvi, Kuldeep Singh Chauhan, Daniela Trejo-Ponce de Leon, Gustavo Ramírez-Martínez, Luis Jiménez-Alvarez, Cesar Luna-Rivero, Joaquin Zuniga, Deepak Kaushal, Shabaana Khader
Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (Mtb), infects approximately one-fourth of the world’s population. While most infected individuals are asymptomatic, latent TB infection (LTBI) can progress to cause pulmonary TB (PTB). We recently reported an increased accumulation of mast cells (MCs) in lungs of macaques with PTB, compared with LTBI in macaques. MCs respond in vitro to Mtb exposure via degranulation and by inducing proinflammatory cytokines. In the current study, we show the dominant production of chymase by MCs in granulomas of humans and macaques with PTB. Using scRNA seq analysis, we show that MCs found in LTBI and healthy lungs in macaques are enriched in genes involved in tumor necrosis factor alpha, cholesterol and transforming growth factor beta signaling. In contrast, MCs clusters found in PTB express transcriptional signatures associated with interferon gamma, oxidative phosphorylation, and MYC signaling. Additionally, MC deficiency in the mouse model showed improved control of Mtb infection that coincided with reduced accumulation of lung myeloid cells and diminished inflammation at chronic stages. Thus, these collective results provide novel evidence for the pathological contribution of MCs during Mtb infection and may represent a novel target for host directive therapy for TB.
{"title":"Mast cells promote pathology and susceptibility in tuberculosis","authors":"Ananya Gupta, Vibha Taneja, Javier Rangel-Moreno, Abhimanyu Abhimanyu, Mushtaq Ahmed, Nilofer Naqvi, Kuldeep Singh Chauhan, Daniela Trejo-Ponce de Leon, Gustavo Ramírez-Martínez, Luis Jiménez-Alvarez, Cesar Luna-Rivero, Joaquin Zuniga, Deepak Kaushal, Shabaana Khader","doi":"10.1101/2024.09.04.611333","DOIUrl":"https://doi.org/10.1101/2024.09.04.611333","url":null,"abstract":"Tuberculosis (TB), caused by the bacterium <em>Mycobacterium tuberculosis</em> (<em>Mtb</em>), infects approximately one-fourth of the world’s population. While most infected individuals are asymptomatic, latent TB infection (LTBI) can progress to cause pulmonary TB (PTB). We recently reported an increased accumulation of mast cells (MCs) in lungs of macaques with PTB, compared with LTBI in macaques. MCs respond in vitro to <em>Mtb</em> exposure via degranulation and by inducing proinflammatory cytokines. In the current study, we show the dominant production of chymase by MCs in granulomas of humans and macaques with PTB. Using scRNA seq analysis, we show that MCs found in LTBI and healthy lungs in macaques are enriched in genes involved in tumor necrosis factor alpha, cholesterol and transforming growth factor beta signaling. In contrast, MCs clusters found in PTB express transcriptional signatures associated with interferon gamma, oxidative phosphorylation, and MYC signaling. Additionally, MC deficiency in the mouse model showed improved control of <em>Mtb</em> infection that coincided with reduced accumulation of lung myeloid cells and diminished inflammation at chronic stages. Thus, these collective results provide novel evidence for the pathological contribution of MCs during <em>Mtb</em> infection and may represent a novel target for host directive therapy for TB.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"28 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211262","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}
Background: T-cell activation and proliferation are critical for understanding immune responses in both healthy and pathological conditions such as acute graft-versus-host disease (aGVHD). Phytohemagglutinin (PHA) and interleukin-2 (IL-2) are commonly used in in vitro assays to study T-cell dynamics. Our study aimed to determine the optimal concentrations of PHA and IL-2 for promoting T-cell proliferation and survival and to evaluate how these conditions impact T-cell responses in healthy individuals versus aGVHD patients. Methods: Peripheral blood samples were collected from age- and sex-matched healthy individuals (n=10) and aGVHD patients (n=10). CD3+ T-cell were isolated and stimulated with varying concentrations of PHA (1-10μg/ml) and IL-2 (50-500 IU/ml). Cell proliferation was assessed using MTS and CFSE assays, while apoptosis was evaluated with Annexin V/7-AAD staining. Results: We observed enhanced proliferation of healthy individuals at higher PHA concentrations (5-10μg/ml), whereas aGVHD patients exhibited heightened proliferation at lower PHA concentrations (1-2.5μg/ml) at 48 hours. Prolonged exposure to PHA led to decreased proliferation in aGVHD patients, while healthy individuals continued to show increased proliferation at 72 hours with optimal PHA concentrations of 5.0-7.5μg/ml. The CFSE assay confirmed these findings, showing a higher proliferation rate in healthy individuals at elevated PHA concentrations and in aGVHD patients at lower concentrations. IL-2 supplementation (50 IU/ml) significantly enhanced T-cell proliferation and survival, with the optimal concentration supporting robust proliferation over extended culture periods. Conclusion: Our study identifies optimal PHA and IL-2 concentrations for in vitro T-cell studies, with 7.5μg/ml of PHA and 50IU/ml of IL-2 providing robust T-cell proliferation in healthy individuals. In contrast, aGVHD patients' T-cells showed better proliferation at lower PHA concentrations (1.0μg/ml) and similar IL-2 requirements. These results underscore the need for tailored experimental conditions based on patient profiles to effectively study T-cell behavior and improve therapeutic strategies for T-cell dysregulation in aGVHD and other conditions.
{"title":"A Comparative assessment of T-Cell response of Healthy donors and Acute Graft-versus-Host-Disease Patients: Customizing immune monitoring platform","authors":"Mohini Mendiratta, Meenakshi Mendiratta, Sandeep Rai, Ritu Gupta, Sameer Bakhshi, Mukul Aggarwal, Aditya Kumar Gupta, Hridayesh Prakash, Sujata Mohanty, Ranjit K Sahoo","doi":"10.1101/2024.09.05.611044","DOIUrl":"https://doi.org/10.1101/2024.09.05.611044","url":null,"abstract":"Background: T-cell activation and proliferation are critical for understanding immune responses in both healthy and pathological conditions such as acute graft-versus-host disease (aGVHD). Phytohemagglutinin (PHA) and interleukin-2 (IL-2) are commonly used in in vitro assays to study T-cell dynamics. Our study aimed to determine the optimal concentrations of PHA and IL-2 for promoting T-cell proliferation and survival and to evaluate how these conditions impact T-cell responses in healthy individuals versus aGVHD patients.\u0000Methods: Peripheral blood samples were collected from age- and sex-matched healthy individuals (n=10) and aGVHD patients (n=10). CD3+ T-cell were isolated and stimulated with varying concentrations of PHA (1-10μg/ml) and IL-2 (50-500 IU/ml). Cell proliferation was assessed using MTS and CFSE assays, while apoptosis was evaluated with Annexin V/7-AAD staining. Results: We observed enhanced proliferation of healthy individuals at higher PHA concentrations (5-10μg/ml), whereas aGVHD patients exhibited heightened proliferation at lower PHA concentrations (1-2.5μg/ml) at 48 hours. Prolonged exposure to PHA led to decreased proliferation in aGVHD patients, while healthy individuals continued to show increased proliferation at 72 hours with optimal PHA concentrations of 5.0-7.5μg/ml. The CFSE assay confirmed these findings, showing a higher proliferation rate in healthy individuals at elevated PHA concentrations and in aGVHD patients at lower concentrations. IL-2 supplementation (50 IU/ml) significantly enhanced T-cell proliferation and survival, with the optimal concentration supporting robust proliferation over extended culture periods.\u0000Conclusion: Our study identifies optimal PHA and IL-2 concentrations for in vitro T-cell studies, with 7.5μg/ml of PHA and 50IU/ml of IL-2 providing robust T-cell proliferation in healthy individuals. In contrast, aGVHD patients' T-cells showed better proliferation at lower PHA concentrations (1.0μg/ml) and similar IL-2 requirements. These results underscore the need for tailored experimental conditions based on patient profiles to effectively study T-cell behavior and improve therapeutic strategies for T-cell dysregulation in aGVHD and other conditions.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"7 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211263","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 : 2024-09-09DOI: 10.1101/2024.09.06.611661
Hugo D. Perdomo, Ayda Khorramnejad, Nfamara M. Cham, Alida Kropf, Davide Sogliani, Mariangela Bonizzoni
How and to what extent mosquito-virus interaction is influenced by climate change is a complex question of ecological and epidemiological relevance. We worked at the intersection between thermal biology and vector immunology and studied shifts in tolerance and resistance to the cell fusing agent virus (CFAV), a prominent component of the mosquito virome know to contribute to shaping mosquito vector competence, in warm-acclimated and warm-evolved Aedes albopictus mosquitoes. We show that the length of the thermal challenge influences the outcome of the infection with warm-evolved mosquitoes being more tolerant to CFAV infection, while warm-acclimated mosquitoes being more resistant and suffering from extensive fitness costs. These results highlight the importance of considering fluctuations in vector immunity in relation to the length of a thermal challenge to understand natural variation in vector response to viruses and frame realistic transmission models.
{"title":"Prolonged thermal stress enhances mosquito tolerance to viral infection","authors":"Hugo D. Perdomo, Ayda Khorramnejad, Nfamara M. Cham, Alida Kropf, Davide Sogliani, Mariangela Bonizzoni","doi":"10.1101/2024.09.06.611661","DOIUrl":"https://doi.org/10.1101/2024.09.06.611661","url":null,"abstract":"How and to what extent mosquito-virus interaction is influenced by climate change is a complex question of ecological and epidemiological relevance. We worked at the intersection between thermal biology and vector immunology and studied shifts in tolerance and resistance to the cell fusing agent virus (CFAV), a prominent component of the mosquito virome know to contribute to shaping mosquito vector competence, in warm-acclimated and warm-evolved <em>Aedes albopictus</em> mosquitoes. We show that the length of the thermal challenge influences the outcome of the infection with warm-evolved mosquitoes being more tolerant to CFAV infection, while warm-acclimated mosquitoes being more resistant and suffering from extensive fitness costs. These results highlight the importance of considering fluctuations in vector immunity in relation to the length of a thermal challenge to understand natural variation in vector response to viruses and frame realistic transmission models.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"54 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142226802","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 : 2024-09-08DOI: 10.1101/2024.09.06.611632
Melanie Urbanek-Quaing, Yin-Han Chou, Manoj Kumar Gupta, Katja Steppich, Birgit Bremer, Hagen Schmaus, Katja Deterding, Benjamin Maasoumy, Heiner Wedemeyer, Cheng-Jian Xu, Anke R M Kraft, Markus Cornberg
Objective: Chronic hepatitis B virus (HBV) infection results in the exhaustion of HBV-specific T cells and the development of epigenetic imprints that impair immune responses and limit the effectiveness of immune checkpoint inhibitor (ICI) monotherapy, such as αPD-L1. This study aimed to determine whether the DNA methyltransferase inhibitor decitabine (DAC) can reverse these epigenetic imprints and enhance the efficacy of ICI in restoring HBV-specific T cell responses. Methods: We investigated HBV-specific CD4+ and CD8+ T cell responses by 10-day in vitro stimulation of peripheral blood mononuclear cells (PBMCs) from patients with chronic HBV infection. PBMCs were stimulated with HBV core-specific overlapping peptide pools and HLA-A*02-restricted peptides, including core18 and pol455. The immunomodulatory effect of the combination of DAC/αPD-L1 was assessed via flow cytometry. Responder stratification was investigated by comparison of clinical characteristics, ex vivo DNA methylation analysis of PBMCs, and determination of IFNγ plasma levels. Results: Treatment with DAC and αPD-L1 enhanced HBV-specific CD4+ T cell responses in a significant proportion of 53 patients, albeit with variability. The effect was independent of the HBcrAg level. Ex vivo DNA methylation revealed hypermethylation of key genes like IFNG among DAC-responders versus non-responders, supported by altered ex vivo IFNγ plasma levels. Further analysis of HBV-specific CD8+ T cell responses in 22 HLA-A*02-positive patients indicated distinct response patterns between HBV-core18- and HBV-pol455-specific T cells, with pol455-specific CD8+ T cells showing increased susceptibility to DAC/αPD-L1, surpassing αPD-L1 monotherapy response. Conclusions: The combination of DAC and αPD-L1 shows promising effects in improving HBV-specific T cell responses in vitro. Our study highlights the potential of remodeling exhaustion-associated epigenetic signatures to enhance HBV-specific T cell restoration, suggesting a novel immunotherapeutic avenue for chronic HBV infections.
目的：慢性乙型肝炎病毒（HBV）感染会导致HBV特异性T细胞衰竭和表观遗传学印记的形成，从而损害免疫应答并限制免疫检查点抑制剂（ICI）单药治疗（如αPD-L1）的疗效。本研究旨在确定DNA甲基转移酶抑制剂地西他滨（DAC）能否逆转这些表观遗传学印记，并提高ICI在恢复HBV特异性T细胞应答方面的疗效：我们通过对慢性 HBV 感染患者的外周血单核细胞（PBMCs）进行为期 10 天的体外刺激，研究了 HBV 特异性 CD4+ 和 CD8+ T 细胞反应。用 HBV 核心特异性重叠肽池和 HLA-A*02 限制性肽（包括 core18 和 pol455）刺激 PBMC。通过流式细胞术评估了 DAC/αPD-L1 组合的免疫调节效果。通过比较临床特征、PBMCs 体外 DNA 甲基化分析和 IFNγ 血浆水平的测定来研究应答者分层。结果使用 DAC 和 αPD-L1 治疗可增强 53 例患者中相当一部分人的 HBV 特异性 CD4+ T 细胞应答，尽管存在差异。这种效应与 HBcrAg 水平无关。体内外 DNA 甲基化显示，在 DAC 反应者与非反应者中，IFNG 等关键基因的甲基化程度过高，体内外 IFNγ 血浆水平的改变也证实了这一点。对 22 名 HLA-A*02 阳性患者的 HBV 特异性 CD8+ T 细胞反应的进一步分析表明，HBV-core18-和 HBV-pol455 特异性 T 细胞之间的反应模式不同，pol455 特异性 CD8+ T 细胞对 DAC/αPD-L1 的敏感性增加，超过了对αPD-L1 单药的反应。结论DAC 和 αPD-L1 的组合在体外改善 HBV 特异性 T 细胞反应方面显示出良好的效果。我们的研究强调了重塑衰竭相关的表观遗传学特征在增强 HBV 特异性 T 细胞恢复方面的潜力，为慢性 HBV 感染提供了一条新的免疫治疗途径。
{"title":"Enhancing HBV-specific T cell responses through a combination of epigenetic modulation and immune checkpoint inhibition","authors":"Melanie Urbanek-Quaing, Yin-Han Chou, Manoj Kumar Gupta, Katja Steppich, Birgit Bremer, Hagen Schmaus, Katja Deterding, Benjamin Maasoumy, Heiner Wedemeyer, Cheng-Jian Xu, Anke R M Kraft, Markus Cornberg","doi":"10.1101/2024.09.06.611632","DOIUrl":"https://doi.org/10.1101/2024.09.06.611632","url":null,"abstract":"<strong>Objective</strong>: Chronic hepatitis B virus (HBV) infection results in the exhaustion of HBV-specific T cells and the development of epigenetic imprints that impair immune responses and limit the effectiveness of immune checkpoint inhibitor (ICI) monotherapy, such as αPD-L1. This study aimed to determine whether the DNA methyltransferase inhibitor decitabine (DAC) can reverse these epigenetic imprints and enhance the efficacy of ICI in restoring HBV-specific T cell responses.\u0000<strong>Methods</strong>: We investigated HBV-specific CD4<sup>+</sup> and CD8<sup>+</sup> T cell responses by 10-day <em>in vitro</em> stimulation of peripheral blood mononuclear cells (PBMCs) from patients with chronic HBV infection. PBMCs were stimulated with HBV core-specific overlapping peptide pools and HLA-A*02-restricted peptides, including core<sub>18</sub> and pol<sub>455</sub>. The immunomodulatory effect of the combination of DAC/αPD-L1 was assessed via flow cytometry. Responder stratification was investigated by comparison of clinical characteristics, <em>ex vivo</em> DNA methylation analysis of PBMCs, and determination of IFNγ plasma levels. <strong>Results</strong>: Treatment with DAC and αPD-L1 enhanced HBV-specific CD4<sup>+</sup> T cell responses in a significant proportion of 53 patients, albeit with variability. The effect was independent of the HBcrAg level. <em>Ex vivo</em> DNA methylation revealed hypermethylation of key genes like IFNG among DAC-responders versus non-responders, supported by altered <em>ex vivo</em> IFNγ plasma levels. Further analysis of HBV-specific CD8<sup>+</sup> T cell responses in 22 HLA-A*02-positive patients indicated distinct response patterns between HBV-core<sub>18</sub>- and HBV-pol<sub>455</sub>-specific T cells, with pol<sub>455</sub>-specific CD8<sup>+</sup> T cells showing increased susceptibility to DAC/αPD-L1, surpassing αPD-L1 monotherapy response. <strong>Conclusions</strong>: The combination of DAC and αPD-L1 shows promising effects in improving HBV-specific T cell responses <em>in vitro</em>. Our study highlights the potential of remodeling exhaustion-associated epigenetic signatures to enhance HBV-specific T cell restoration, suggesting a novel immunotherapeutic avenue for chronic HBV infections.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"177 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211321","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 : 2024-09-08DOI: 10.1101/2024.09.04.611236
Biana Bernshtein, Julia A Zhiteneva, Jeshina Janardhanan, Chanchal Wagh, Meagan Kelly, Smriti Verma, Wonyeong Jung, Salima Raiyan Basher, Mohammad Ashraful Amin, Shakil Mahamud, Nazmul Hasan Rajib, Fahima Chowdhury, Ashraful Islam Khan, Richelle C. Charles, Peng Xu, Paul Kovac, Subhra Chakraborty, Robert W. Kaminski, Galit Alter, Taufiqur Rahman Bhuiyan, Firdausi Qadri, Edward T Ryan
Shigellosis is the second leading cause of diarrheal death in children younger than five years of age globally. At present, there is no broadly licensed vaccine against shigella infection. Previous vaccine candidates have failed at providing protection for young children in endemic settings. Improved understanding of correlates of protection against Shigella infection and severe shigellosis in young children living in endemic settings is needed. Here, we applied a functional antibody profiling approach to define Shigella-specific antibody responses in young children versus older individuals with culture-confirmed shigellosis in Bangladesh, a Shigella endemic area. We analyzed Shigella-specific antibody isotypes, FcR binding and antibody-mediated innate immune cell activation in longitudinal serum samples collected at clinical presentation and up to 1 year later. We found that higher initial Shigella O-specific polysaccharide (OSP)-specific and protein-specific IgG and FcgR binding levels correlated with less severe disease regardless of patient age, but that individuals under 5 years of age developed a less prominent class switched, FcR-binding, functional and durable antibody response against both OSP and protein Shigella antigens than older individuals. Focusing on the largest cohort, we found that functional S. flexneri 2a OSP-specific responses were significantly induced only in individuals over age 5 years, and that these responses promoted monocyte phagocytosis and activation. Our findings suggest that in a Shigella endemic region, young children with shigellosis harbor a functional antibody response that fails to maximally activate monocytes; such a response may be important in facilitating subsequent innate cell clearance of Shigella, especially via recruitment and activation of polymorphonuclear cells capable of directly killing Shigella.
{"title":"Limited O-specific polysaccharide (OSP)-specific functional antibody responses in young children with Shigella infection in Bangladesh","authors":"Biana Bernshtein, Julia A Zhiteneva, Jeshina Janardhanan, Chanchal Wagh, Meagan Kelly, Smriti Verma, Wonyeong Jung, Salima Raiyan Basher, Mohammad Ashraful Amin, Shakil Mahamud, Nazmul Hasan Rajib, Fahima Chowdhury, Ashraful Islam Khan, Richelle C. Charles, Peng Xu, Paul Kovac, Subhra Chakraborty, Robert W. Kaminski, Galit Alter, Taufiqur Rahman Bhuiyan, Firdausi Qadri, Edward T Ryan","doi":"10.1101/2024.09.04.611236","DOIUrl":"https://doi.org/10.1101/2024.09.04.611236","url":null,"abstract":"Shigellosis is the second leading cause of diarrheal death in children younger than five years of age globally. At present, there is no broadly licensed vaccine against shigella infection. Previous vaccine candidates have failed at providing protection for young children in endemic settings. Improved understanding of correlates of protection against Shigella infection and severe shigellosis in young children living in endemic settings is needed. Here, we applied a functional antibody profiling approach to define Shigella-specific antibody responses in young children versus older individuals with culture-confirmed shigellosis in Bangladesh, a Shigella endemic area. We analyzed Shigella-specific antibody isotypes, FcR binding and antibody-mediated innate immune cell activation in longitudinal serum samples collected at clinical presentation and up to 1 year later. We found that higher initial Shigella O-specific polysaccharide (OSP)-specific and protein-specific IgG and FcgR binding levels correlated with less severe disease regardless of patient age, but that individuals under 5 years of age developed a less prominent class switched, FcR-binding, functional and durable antibody response against both OSP and protein Shigella antigens than older individuals. Focusing on the largest cohort, we found that functional S. flexneri 2a OSP-specific responses were significantly induced only in individuals over age 5 years, and that these responses promoted monocyte phagocytosis and activation. Our findings suggest that in a Shigella endemic region, young children with shigellosis harbor a functional antibody response that fails to maximally activate monocytes; such a response may be important in facilitating subsequent innate cell clearance of Shigella, especially via recruitment and activation of polymorphonuclear cells capable of directly killing Shigella.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"28 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142211270","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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