AbstractHookworm infection remains a significant public health concern, particularly in low- and middle-income countries, where mass drug administration has not stopped reinfection. Developing a vaccine is crucial to complement current control measures, which necessitates a thorough understanding of host immune responses.

摘要钩虫感染仍然是一个重要的公共卫生问题，特别是在中低收入国家，大规模药物管理局尚未停止再感染。开发疫苗对于补充当前的控制措施至关重要，这需要彻底了解宿主的免疫反应。

By leveraging controlled human infection models and high-dimensional immunophenotyping, here we investigated the immune remodeling following infection with 50 Necator americanus L3 hookworm larvae in four naïve volunteers over two years of follow-up and compared the profiles with naturally infected populations in endemic areas.

通过利用受控的人类感染模型和高维免疫分型，我们在两年的随访中研究了四名幼稚志愿者感染50只美洲钩虫L3钩虫幼虫后的免疫重塑，并将其与流行地区自然感染人群进行了比较。

Increased plasmacytoid dendritic cell frequency and diminished responsiveness to Toll-like receptor 7/8 ligand were observed in both controlled and natural infection settings. Despite the increased CD45RA+ regulatory T cell (Tregs) frequencies in both settings, markers of Tregs function, including inducible T-cell costimulatory (ICOS), tumor necrosis factor receptor 2 (TNFR2), and latency-associated peptide (LAP), as well as in vitro Tregs suppressive capacity were higher in natural infections.

在受控和自然感染环境中均观察到浆细胞样树突状细胞频率增加和对Toll样受体7/8配体的反应性降低。尽管在两种情况下CD45RA+调节性T细胞（Tregs）频率增加，但Tregs功能的标志物，包括诱导型T细胞共刺激（ICOS），肿瘤坏死因子受体2（TNFR2）和潜伏期相关肽（LAP），以及体外Tregs抑制能力在自然感染中较高。

Taken together, this study provides unique insights into the immunological trajectories following a first-in-life hookworm infection compared to natural infections..

综上所述，与自然感染相比，这项研究为首次钩虫感染后的免疫轨迹提供了独特的见解。。

IntroductionHuman hookworm infection continues to cause significant health and economic burdens in low- and middle-income countries1. To reduce the morbidity caused by hookworm infection, mass drug administration (MDA) with albendazole or mebendazole has been implemented2. However, MDA does not provide the necessary protection against reinfection to break the transmission cycle.

引言人类钩虫感染继续在中低收入国家造成重大的健康和经济负担1。为了降低钩虫感染引起的发病率，已经实施了阿苯达唑或甲苯咪唑的大规模药物管理（MDA）2。然而，MDA不能提供必要的保护，防止再次感染以打破传播周期。

Furthermore, concerns about treatment failure with mebendazole and the possible emergence of drug resistance have been raised3, highlighting the need for a vaccine.The rational design of vaccines requires a thorough understanding of the immune response of the host. Controlled human infection models serve as unique and promising means to study the host’s immune response, owing to the known time of infection and inoculum dose, which are difficult to determine in naturally occurring infections in endemic areas.

此外，人们对甲苯咪唑治疗失败和可能出现耐药性的担忧已经提出3，突出了对疫苗的需求。疫苗的合理设计需要彻底了解宿主的免疫反应。受控制的人类感染模型是研究宿主免疫反应的独特而有前途的手段，因为已知的感染时间和接种剂量在流行地区的自然感染中很难确定。

The combination of controlled human infection models with high-dimensional immunophenotyping has revealed new insights into host-pathogen interactions, such as in malaria4,5. Although controlled human hookworm infection (CHHI) studies have involved more than 200 participants in total6, a high-dimensional analysis of the immune response to hookworm has not been performed.In CHHI studies conducted to date, eosinophilia, the production of total and hookworm-specific immunoglobulin (Ig) E and IgG, as well as cytokine production by immune cells in culture supernatants have been reported, indicating the activation of CD4+T cells and mixed T helper 1 (TH1) or 2 (TH2) cytokine responses7,8.

受控人类感染模型与高维免疫分型的结合揭示了对宿主-病原体相互作用的新见解，如疟疾4,5。尽管受控人类钩虫感染（CHHI）研究总共涉及200多名参与者6，但尚未对钩虫的免疫反应进行高维分析。在迄今为止进行的CHHI研究中，已经报道了嗜酸性粒细胞增多，总的和钩虫特异性免疫球蛋白（Ig）E和IgG的产生，以及培养上清液中免疫细胞产生的细胞因子，表明CD4+T细胞和混合T辅助细胞的活化1（TH1）或2（TH2）细胞因子反应7,8。

Here, we performed a high-dimensional mapping of cellular immunological responses in CHHI study participants who were experimentally infected with one dose of 50 Necator americanus L3 larvae and followed for two .

在这里，我们对CHHI研究参与者进行了细胞免疫反应的高维作图，这些参与者通过实验感染了一剂50只Necator americanus L3幼虫，随后进行了两次。

Cell culture

细胞培养

Cells were thawed, washed in RPMI 1640 supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM pyruvate, 2 mM glutamate, and 10% FCS, and adjusted to a concentration of 5 × 106 cells/ml. Co-stimuli αCD28 (BD Bioscience) and αCD49d (BD Bioscience) were added at a concentration of 1 µg/ml.

将细胞解冻，在补充有100U/ml青霉素，100g/ml链霉素，1mM丙酮酸，2mM谷氨酸和10%FCS的RPMI 1640中洗涤，并调节至浓度为5×106个细胞/ml。以1μg/ml的浓度加入共刺激αCD28（BD Bioscience）和αCD49d（BD Bioscience）。

Cells were then stimulated with 5 µg/ml antigen extract, 200 ng/ml staphylococcal enterotoxin B (SEB; Sigma-Aldrich), and 10% FCS/RPMI for 24 h at 37 °C under 5% CO2. At the last four hours of stimulation, 10 µg/ml brefeldin A (Sigma-Aldrich) was added..

然后用5μg/ml抗原提取物，200 ng/ml葡萄球菌肠毒素B（SEB；Sigma-Aldrich）和10%FCS/RPMI在37℃，5%CO 2下刺激细胞24小时。在刺激的最后四个小时，加入10µg/ml布雷菲德菌素A（Sigma-Aldrich）。。

Flow cytometry antibody staining

流式细胞仪抗体染色

After stimulation, cells were then washed twice in phosphate-buffered saline (PBS), stained for viable cells with LIVE/DEADTM Fixable Aqua (Thermofisher), and fixed with 1.9% paraformaldehyde (Sigma-Aldrich) in PBS. Subsequently, cells were washed in FACS buffer (0.5% BSA in PBS, Roche, and 2 mM EDTA, Sigma-Aldrich) and then permeabilized with eBioscienceTM Permeabilization Buffer (ThermoFisher).

刺激后，将细胞在磷酸盐缓冲盐水（PBS）中洗涤两次，用LIVE/DEADTM Fixable Aqua（Thermofisher）染色活细胞，并用PBS中的1.9%多聚甲醛（Sigma-Aldrich）固定。随后，将细胞在FACS缓冲液（PBS中的0.5%BSA，Roche和2mM EDTA，Sigma-Aldrich）中洗涤，然后用eBioscienceTM透化缓冲液（Thermofisher）透化。

Cells were stained in a 96-well V-bottom plate with 50 μL of flow cytometry panel antibody mixture (Table S3) diluted in eBioscience™ permeabilization buffer (ThermoFisher) with 1% human Fc receptor blocker (eBioscience) at 4 °C for 30 min. Cells were then resuspended in the FACS buffer before acquisition..

将细胞在96孔V型底板中用50μL流式细胞术面板抗体混合物（表S3）染色，该混合物在含有1%人Fc受体阻滞剂（eBioscience）的eBioscience™透化缓冲液（ThermoFisher）中稀释，在4℃下30分钟。然后在采集前将细胞重悬于FACS缓冲液中。。

Cell acquisition

细胞采集

Stained cells were acquired with a FACSCanto II flow cytometer (BD Biosciences). The compensation matrix was set using single-stained compensation beads (BDTM CompBead). FCS files were analyzed with the FlowJo v10 software (BD Life Sciences), where positions of gates were guided with fluorescence-minus-one (FMO) or unstimulated controls..

用FACSCanto II流式细胞仪（BD Biosciences）获得染色的细胞。使用单染色补偿珠（BDTM CompBead）设置补偿矩阵。使用FlowJo v10软件（BD Life Sciences）分析FCS文件，其中门的位置由荧光减一（FMO）或未受刺激的对照引导。。

Polyclonal responses to PMA/Ionomycin and R-848Cell culturePBMC were thawed as described above. For assessment of intracellular cytokine expression via spectral cytometry, cells were washed in RPMI 1640 (ThermoFisher), supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM pyruvate, 2 mM glutamate, and 10% FCS (Greiner Bio-One), and adjusted to a concentration of 1 × 106 cells/ml.

如上所述解冻对PMA/离子霉素和R-848细胞培养物PBMC的多克隆反应。为了通过光谱细胞术评估细胞内细胞因子的表达，将细胞在补充有100U/ml青霉素，100g/ml链霉素，1mM丙酮酸，2mM谷氨酸和10%FCS（Greiner Bio-One）的RPMI 1640（ThermoFisher）中洗涤，并调节至1×106个细胞/ml的浓度。

Cells were then resuspended in 100 µl RPMI + 10% FCS and stimulated for 6 h with PMA (100 ng/ml, Sigma-Aldrich) and ionomycin (1 µg/ml, Sigma-Aldrich) at 37 °C under 5% CO2. After 4 h of stimulation, 10 µg/ml Brefeldin A (Sigma-Aldrich) was added.Flow cytometry antibody stainingAfter stimulation, cells were washed twice in phosphate-buffered saline (PBS), stained for viable cells with LIVE/DEADTM Fixable Blue (Thermofisher), washed again twice in FACS buffer (PBS supplemented with 0.5% BSA, Roche) and 2 mM EDTA (Sigma-Aldrich), and incubated with Human TruStain FcX™ (BioLegend) and True-Stain Monocyte Blocker™ (BioLegend) according to the manufacturer’s instruction for 5 min at room temperature (RT).

然后将细胞重悬于100μlRPMI++10%FCS中，并用PMA（100 ng/ml，Sigma-Aldrich）和离子霉素（1μg/ml，Sigma-Aldrich）在37℃，5%CO 2下刺激6小时。刺激4小时后，加入10µg/ml布雷菲德菌素A（Sigma-Aldrich）。流式细胞术抗体染色刺激后，将细胞在磷酸盐缓冲盐水（PBS）中洗涤两次，用LIVE/DEADTM Fixable Blue（Thermofisher）染色活细胞，在FACS缓冲液（补充有0.5%BSA的PBS，Roche）和2mM EDTA（Sigma-Aldrich）中再次洗涤两次，并根据制造商的说明在室温（RT）下与Human TruStain FcX™（BioLegend）和True Stain Monocyte Blocker™（BioLegend）孵育5分钟。

The antibody surface cocktail, prepared in Brilliant Stain Buffer Plus (BD Biosciences) was added to the cells and incubated for 30 min at RT. The list of antibodies can be found in Table S3. Cells were then washed twice in FACS buffer and afterward fixed and permeabilized with the eBioscienceTM Foxp3 Transcription Factor Staining Buffer Set (ThermoFisher) for 30 min at 4 °C.

将在Brilliant Stain Buffer Plus（BD Biosciences）中制备的抗体表面混合物加入细胞中，并在室温下孵育30分钟。抗体列表可在表S3中找到。然后将细胞在FACS缓冲液中洗涤两次，然后用eBioscienceTM Foxp3转录因子染色缓冲液组（ThermoFisher）在4℃固定并透化30分钟。

Subsequently, cells were washed twice with the Permeabilization buffer from the eBioscienceTM Foxp3 Transcription Factor Staining Buffer Set before being incubated with Human TruStain FcX™ and True-Stain Monocyte Blocker™ for 5 min at 4 °C and then stained with the intracellular/intranuclear antibody cocktail for.

随后，将细胞用来自eBioscienceTM Foxp3转录因子染色缓冲液组的透化缓冲液洗涤两次，然后与Human TruStain FcX™和True Stain Monocyte Blocker™在4℃温育5分钟，然后用细胞内/核内抗体混合物染色。

Data availability

数据可用性

Source data for figures are provided with this paper. Processed mass cytometry data is stored in Zenodo under restricted access (https://zenodo.org/records/10889855). Raw mass and flow cytometry data are available from corresponding authors upon request through a data transfer agreement. Restrictions on access are due to patient confidentiality issues; even pseudonymized data can be related back to individuals in some cases. Source data are provided with this paper..

本文提供了数字的源数据。经过处理的大规模细胞计数数据在受限访问下存储在Zenodo中(https://zenodo.org/records/10889855)。原始质量和流式细胞仪数据可根据要求通过数据传输协议从通讯作者处获得。访问限制是由于患者保密问题；在某些情况下，甚至假名数据也可能与个人相关。本文提供了源数据。。

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Download referencesAcknowledgementsWe would like to thank the Flow Cytometry Core Facility at Leiden University Medical Center for assistance with our flow and mass cytometry experiments. This study is part of the EDCTP2 programme supported by the European Union. The Controlled Human Hookworm Infection in Leiden (CHHIL) trial was funded by the Dioraphte Foundation and by NWO Spinoza prize of M.Y.

下载参考文献致谢我们要感谢莱顿大学医学中心的流式细胞术核心设施对我们的流式和大规模细胞术实验的帮助。这项研究是欧盟支持的EDCTP2计划的一部分。莱顿控制人类钩虫感染（CHHIL）试验由Dioraphte基金会和M.Y.的NWO斯宾诺莎奖资助。

M.D.M. is funded by the Indonesian Endowment Fund for Education (LPDP, Reference No. S-1598/LPDP.3/2016). B.G.D. is a Senior Research Associate of the Fonds de la Recherche Scientifique (F.R.S-FNRS).Author informationAuthor notesThese authors contributed equally: Mikhael D. Manurung, Friederike Sonnet.These authors jointly supervised this work: Meta Roestenberg, Mariateresa Coppola, Maria Yazdanbakhsh.Authors and AffiliationsLeiden University Center for Infectious Diseases (LU-CID), Leiden University Medical Center, Leiden, The NetherlandsMikhael D.

M、 D.M.由印度尼西亚教育捐赠基金（LPDP，参考号S-1598/LPDP.3/2016）资助。B、 G.D.是科学研究基金会（F.R.S-FNRS）的高级研究员。作者信息作者注意到这些作者做出了同样的贡献：Mikhael D.Manurung，Friederike十四行诗。这些作者共同监督了这项工作：Meta Roestenberg，Mariateresa Coppola，Maria Yazdanbakhsh。作者和附属机构莱顿大学传染病中心（LU-CID），莱顿大学医学中心，荷兰莱顿Smikhael D。

Manurung, Friederike Sonnet, Marie-Astrid Hoogerwerf, Jacqueline J. Janse, Yvonne Kruize, Laura de Bes-Roeleveld, Marion König, Simon P. Jochems, Meta Roestenberg, Mariateresa Coppola & Maria YazdanbakhshCentre for Molecular Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, AustraliaAlex LoukasLaboratory of Immunology-Vaccinology, FARAH, University of Liège, Liège, BelgiumBenjamin G.

马努龙，弗里德里克·十四行诗，玛丽·阿斯特里德·胡润，杰奎琳·J·詹斯，伊冯·克鲁兹，劳拉·德·贝斯·罗莱维德，马里恩·科尼格，西蒙·P·乔希姆斯，梅塔·罗斯滕贝格，玛利亚·特雷萨·科波拉和玛利亚·亚兹丹巴赫·施切特，澳大利亚热带健康与医学研究所，詹姆斯·库克大学，凯恩斯，澳大利亚莱克斯·洛卡斯免疫学疫苗学实验室，法拉，利日大学，利日，BelgiumBenjamin G。

DewalsDepartment of Parasitology, Faculty of Medicine, University of Indonesia, Jakarta, IndonesiaTaniawati SupaliAuthorsMikhael D. ManurungView author publicationsYou can also search for this author in.

印度尼西亚雅加达印度尼西亚大学医学院寄生虫学系印度尼西亚亚尼亚瓦蒂SupaliAuthorsMikhael D.Manrungview作者出版物您也可以在中搜索这位作者。

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PubMed Google ScholarContributionsConceptualization: M. Roestenberg, M. Yazdanbakhsh. Funding Acquisition: M. Roestenberg, M. Yazdanbakhsh. Clinical trial setup: M.A. Hoogerwerf, J.J. Janse, M.Roestenberg. Clinical trial samples collection: M.D. Manurung, Y. Kruize, J.J. Janse, T.

PubMed谷歌学术贡献概念：M.Roestenberg，M.Yazdanbakhsh。资金收购：M.Roestenberg，M.Yazdanbakhsh。临床试验设置：M.A.Hoogerwerf，J.J.Janse，M.Roestenberg。临床试验样本收集：M.D.Manurung，Y.Kruize，J.J.Janse，T。

Supali. Materials and reagents: B.G. Dewals, A. Loukas. Mass cytometry experiments: M.D. Manurung, F. Sonnet, M. König. Flow cytometry experiments: F. Sonnet, M. Coppola, L. de Bes. Data analysis and visualization: M.D. Manurung. Writing: M.D. Manurung, F. Sonnet, S.P. Jochems, M. Coppola, and M. Yazdanbakhsh.

苏帕利。材料和试剂：B.G.Dewals，A.Loukas。大规模细胞计数实验：M.D.Manurung，F.Sonnet，M.König。流式细胞术实验：F.Sonnet，M.Coppola，L.DeBes。数据分析和可视化：M.D.Manurung。写作：M.D.马努龙、F.十四行诗、S.P.乔希姆斯、M.科波拉和M.亚兹丹巴赫什。

All authors critically reviewed and approved the final version for publication.Corresponding authorCorrespondence to.

所有作者都严格审查并批准了最终版本。对应作者对应。

Maria Yazdanbakhsh.Ethics declarations

玛丽亚·亚兹丹巴赫什（MariaYazdanbakhsh）。道德宣言

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Reprints and permissionsAbout this articleCite this articleManurung, M.D., Sonnet, F., Hoogerwerf, MA. et al. Controlled human hookworm infection remodels plasmacytoid dendritic cells and regulatory T cells towards profiles seen in natural infections in endemic areas.

转载和许可本文引用本文Manurung，M.D.，Sonnet，F.，Hoogerwerf，MA。等人控制的人类钩虫感染将浆细胞样树突状细胞和调节性T细胞重塑为流行地区自然感染中的特征。

Nat Commun 15, 5960 (2024). https://doi.org/10.1038/s41467-024-50313-0Download citationReceived: 11 January 2023Accepted: 08 July 2024Published: 16 July 2024DOI: https://doi.org/10.1038/s41467-024-50313-0Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

Nat Commun 155960（2024）。https://doi.org/10.1038/s41467-024-50313-0Download引文接收日期：2023年1月11日接收日期：2024年7月8日发布日期：2024年7月16日OI：https://doi.org/10.1038/s41467-024-50313-0Share本文与您共享以下链接的任何人都可以阅读此内容：获取可共享链接对不起，本文目前没有可共享的链接。复制到剪贴板。

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