{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,13]],"date-time":"2026-03-13T15:55:23Z","timestamp":1773417323198,"version":"3.50.1"},"reference-count":35,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2025,5,11]],"date-time":"2025-05-11T00:00:00Z","timestamp":1746921600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Swiss National Science Foundation","award":["310030_204226"],"award-info":[{"award-number":["310030_204226"]}]},{"name":"Swiss National Science Foundation","award":["UM1 AI164561"],"award-info":[{"award-number":["UM1 AI164561"]}]},{"name":"Swiss National Science Foundation","award":["1R01AI179476-01"],"award-info":[{"award-number":["1R01AI179476-01"]}]},{"DOI":"10.13039\/100000060","name":"NIAID","doi-asserted-by":"publisher","award":["310030_204226"],"award-info":[{"award-number":["310030_204226"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"NIAID","doi-asserted-by":"publisher","award":["UM1 AI164561"],"award-info":[{"award-number":["UM1 AI164561"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"NIAID","doi-asserted-by":"publisher","award":["1R01AI179476-01"],"award-info":[{"award-number":["1R01AI179476-01"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>Follicles (Fs)\/Germinal Centers (GCs) in tonsils and lymph nodes are dynamic microenvironments where diverse immune cell populations interact for the development of antibody responses against pathogens. The accurate in situ phenotypic analysis of these immune cells is a prerequisite for the comphehensive understanding of GC development. In this study, we explore unsupervised clustering approaches for distinguishing cell populations within F\/GCs using marker expression data. We evaluate multiple clustering algorithms and find that k-means clustering provides the most effective separation of distinct cell subsets. Additionally, we investigate the predictive potential of common GC markers (CD3, CD4, CD20 and BCL6) for PD-1 expression, an important immune checkpoint regulator. Our analysis demonstrates that PD-1 expression can be reliably inferred using these markers, suggesting potential applications for automated cell classification in immunological studies. This approach enhances our ability to analyze immune cell heterogeneity and may contribute to improved understanding of GC dynamics in health and disease. Our findings support the use of computational clustering for high-dimensional immune profiling.<\/jats:p>","DOI":"10.3390\/biology14050530","type":"journal-article","created":{"date-parts":[[2025,5,12]],"date-time":"2025-05-12T12:18:06Z","timestamp":1747052286000},"page":"530","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Unsupervised Clustering of Cell Populations in Germinal Centers Using Multiplexed Immunofluorescence"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1284-5284","authenticated-orcid":false,"given":"Simon","family":"Burgermeister","sequence":"first","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, Institute of Pathology, Lausanne University Hospital, University of Lausanne, CH-1011 Lausanne, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-7668-7439","authenticated-orcid":false,"given":"Michail","family":"Orfanakis","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, Institute of Pathology, Lausanne University Hospital, University of Lausanne, CH-1011 Lausanne, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9398-3143","authenticated-orcid":false,"given":"Spiros","family":"Georgakis","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, Institute of Pathology, Lausanne University Hospital, University of Lausanne, CH-1011 Lausanne, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-6267-0851","authenticated-orcid":false,"given":"Cloe","family":"Brenna","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, Institute of Pathology, Lausanne University Hospital, University of Lausanne, CH-1011 Lausanne, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8614-1661","authenticated-orcid":false,"given":"Helen","family":"Lindsay","sequence":"additional","affiliation":[{"name":"Biomedical Data Science Center, Lausanne University Hospital, Lausanne University, CH-1011 Lausanne, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9435-0110","authenticated-orcid":false,"given":"Craig","family":"Fenwick","sequence":"additional","affiliation":[{"name":"Service of Immunology and Allergy, Department of Medicine, Lausanne University Hospital, Lausanne University, CH-1011 Lausanne, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3651-2721","authenticated-orcid":false,"given":"Giuseppe","family":"Pantaleo","sequence":"additional","affiliation":[{"name":"Service of Immunology and Allergy, Department of Medicine, Lausanne University Hospital, Lausanne University, CH-1011 Lausanne, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3867-0232","authenticated-orcid":false,"given":"Raphael","family":"Gottardo","sequence":"additional","affiliation":[{"name":"Biomedical Data Science Center, Lausanne University Hospital, Lausanne University, CH-1011 Lausanne, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5067-5810","authenticated-orcid":false,"given":"Constantinos","family":"Petrovas","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, Institute of Pathology, Lausanne University Hospital, University of Lausanne, CH-1011 Lausanne, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,5,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1002\/cac2.12023","article-title":"Overview of multiplex immunohistochemistry\/immunofluorescence techniques in the era of cancer immunotherapy","volume":"40","author":"Tan","year":"2020","journal-title":"Cancer Commun."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1007\/s00281-022-00974-0","article-title":"Highly multiplexed spatial profiling with CODEX: Bioinformatic analysis and application in human disease","volume":"45","author":"Kuswanto","year":"2022","journal-title":"Semin. Immunopathol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"100197","DOI":"10.1016\/j.modpat.2023.100197","article-title":"Multiplex Immunohistochemistry and Immunofluorescence: A Practical Update for Pathologists","volume":"36","author":"Harms","year":"2023","journal-title":"Mod. Pathol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Hickey, J.W., Tan, Y., Nolan, G.P., and Goltsev, Y. (2021). Strategies for Accurate Cell Type Identification in CODEX Multiplexed Imaging Data. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.727626"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1038\/s41413-024-00359-z","article-title":"Spatial multiplexed immunofluorescence analysis reveals coordinated cellular networks associated with overall survival in metastatic osteosarcoma","volume":"12","author":"Lacinski","year":"2024","journal-title":"Bone Res."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"524","DOI":"10.1016\/j.cell.2019.03.016","article-title":"B Cell Responses: Cell Interaction Dynamics and Decisions","volume":"177","author":"Cyster","year":"2019","journal-title":"Cell"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"e8","DOI":"10.4110\/in.2024.24.e8","article-title":"Cytokines in Follicular Helper T Cell Biology in Physiologic and Pathologic Conditions","volume":"24","author":"Choi","year":"2024","journal-title":"Immune Network"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1132","DOI":"10.1016\/j.immuni.2019.04.011","article-title":"Follicular Helper Cell Biology: A Decade of Discovery and Diseases","volume":"50","author":"Shane","year":"2019","journal-title":"Immunity"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1038\/nrc1589","article-title":"Mechanisms of B-cell lymphoma pathogenesis","volume":"5","year":"2005","journal-title":"Nat. Rev. Cancer"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1271","DOI":"10.1038\/nm.4411","article-title":"Defining total-body AIDS-virus burden with implications for curative strategies","volume":"23","author":"Estes","year":"2017","journal-title":"Nat. Med."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"33455","DOI":"10.1073\/pnas.2018488117","article-title":"IBEX: A versatile multiplex optical imaging approach for deep phenotyping and spatial analysis of cells in complex tissues","volume":"117","author":"Radtke","year":"2020","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Georgakis, S., Ioannidou, K., Mora, B.B., Orfanakis, M., Brenna, C., Muller, Y.D., Estrada, P.M.D.R., Sharma, A.A., Pantaleo, G., and de Leval, L. (2025). Cellular and molecular determinants mediating the dysregulated germinal center immune dynamics in systemic lupus erythematosus. Front. Immunol., 16.","DOI":"10.3389\/fimmu.2025.1530327"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Moysi, E., Estrada, P.M.D.R., Torres-Ruiz, F., Reyes-Ter\u00e1n, G., Koup, R.A., and Petrovas, C. (2021). In Situ Characterization of Human Lymphoid Tissue Immune Cells by Multispectral Confocal Imaging and Quantitative Image Analysis; Implications for HIV Reservoir Characterization. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.683396"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"364","DOI":"10.1016\/j.immuni.2012.07.011","article-title":"Histo-cytometry: A method for highly multiplex quantitative tissue imaging analysis applied to dendritic cell subset microanatomy in lymph nodes","volume":"37","author":"Gerner","year":"2012","journal-title":"Immunity"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Bai, Y., Zhu, B., Rovira-Clave, X., Chen, H., Markovic, M., Chan, C.N., Su, T.H., McIlwain, D.R., Estes, J.D., and Keren, L. (2021). Adjacent Cell Marker Lateral Spillover Compensation and Reinforcement for Multiplexed Images. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.652631"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1226","DOI":"10.1038\/s41592-019-0582-9","article-title":"ilastik: Interactive machine learning for (bio)image analysis","volume":"16","author":"Berg","year":"2019","journal-title":"Nat. Methods"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"379","DOI":"10.1016\/j.immuni.2024.01.006","article-title":"An atlas of cells in the human tonsil","volume":"57","author":"Nieto","year":"2024","journal-title":"Immunity"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"979","DOI":"10.12688\/f1000research.73600.2","article-title":"Doublet identification in single-cell sequencing data using scDblFinder","volume":"10","author":"Germain","year":"2022","journal-title":"f1000research"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1038\/s41587-023-01767-y","article-title":"Dictionary learning for integrative, multimodal and scalable single-cell analysis","volume":"42","author":"Hao","year":"2024","journal-title":"Nat. Biotechnol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1038\/s41586-020-2649-2","article-title":"Array programming with NumPy","volume":"585","author":"Harris","year":"2020","journal-title":"Nature"},{"key":"ref_21","first-page":"2825","article-title":"Scikit-learn: Machine Learning in Python","volume":"12","author":"Pedregosa","year":"2011","journal-title":"J. Mach. Learn. Res."},{"key":"ref_22","unstructured":"Ali, M. (2020). PyCaret: An Open Source, Low-Code Machine Learning Library in Python, PyCaret. Available online: https:\/\/www.pycaret.org."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1109\/MCSE.2007.55","article-title":"Matplotlib: A 2D graphics environment","volume":"9","author":"Hunter","year":"2007","journal-title":"Comput. Sci. Eng."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"3021","DOI":"10.21105\/joss.03021","article-title":"seaborn: Statistical data visualization","volume":"6","author":"Waskom","year":"2021","journal-title":"J. Open Source Softw."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1109\/TVCG.2016.2599030","article-title":"Vega-Lite: A Grammar of Interactive Graphics","volume":"23","author":"Satyanarayan","year":"2017","journal-title":"IEEE Trans. Vis. Comput. Graph."},{"key":"ref_26","unstructured":"Guyon, I., Luxburg, U.V., Bengio, S., Wallach, H., Fergus, R., Vishwanathan, S., and Garnett, R. (2017). A Unified Approach to Interpreting Model Predictions. Advances in Neural Information Processing Systems 30 (NeurIPS 2017), Curran Associates, Inc."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Orfanakis, M., Molyvdas, A., and Petrovas, C. (2024). In Situ Characterization of Human Follicular Helper CD4 T Cells. Intracellular Pathogens: Methods and Protocols, Springer.","DOI":"10.1007\/978-1-0716-3890-3_18"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"381","DOI":"10.1016\/j.coi.2013.03.003","article-title":"Manipulating the PD-1 pathway to improve immunity","volume":"25","author":"Kamphorst","year":"2013","journal-title":"Curr. Opin. Immunol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1016\/j.it.2010.07.001","article-title":"The elusive identity of T follicular helper cells","volume":"31","author":"Yu","year":"2010","journal-title":"Trends Immunol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"471","DOI":"10.1016\/j.immuni.2016.09.001","article-title":"Germinal Center B Cell Dynamics","volume":"45","author":"Mesin","year":"2016","journal-title":"Immunity"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1708","DOI":"10.1038\/s41596-019-0156-4","article-title":"High-dimensional cell-level analysis of tissues with Ce3D multiplex volume imaging","volume":"14","author":"Li","year":"2019","journal-title":"Nat. Protoc."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Wang, V.G., Liu, Z., Martinek, J., Foroughi Pour, A., Zhou, J., Boruchov, H., Ray, K., Palucka, K., and Chuang, J.H. (2024). Computational immune synapse analysis reveals T-cell interactions in distinct tumor microenvironments. Commun. Biol., 7.","DOI":"10.1038\/s42003-024-06902-2"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"e2016855118","DOI":"10.1073\/pnas.2016855118","article-title":"Acquisition of optimal TFH cell function is defined by specific molecular, positional, and TCR dynamic signatures","volume":"118","author":"Padhan","year":"2021","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"e0176022","DOI":"10.1128\/jvi.01760-22","article-title":"Lymph-Node-Based CD3+ CD20+ Cells Emerge from Membrane Exchange between T Follicular Helper Cells and B Cells and Increase Their Frequency following Simian Immunodeficiency Virus Infection","volume":"97","author":"Samer","year":"2023","journal-title":"J. Virol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"528","DOI":"10.1126\/science.1136736","article-title":"Imaging of Germinal Center Selection Events during Affinity Maturation","volume":"315","author":"Allen","year":"2007","journal-title":"Science"}],"container-title":["Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-7737\/14\/5\/530\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:30:51Z","timestamp":1760031051000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-7737\/14\/5\/530"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,5,11]]},"references-count":35,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2025,5]]}},"alternative-id":["biology14050530"],"URL":"https:\/\/doi.org\/10.3390\/biology14050530","relation":{},"ISSN":["2079-7737"],"issn-type":[{"value":"2079-7737","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,5,11]]}}}