ARTICLE OPEN Understanding repertoire sequencing data through a multiscale computational model of the germinal center Rodrigo García-Valiente 1,2,3,15 , Elena Merino Tejero 1,2,3,15 , Maria Stratigopoulou 4,5 , Daria Balashova 1,2,3 , Aldo Jongejan 1,2,3 , Danial Lashgari 1,2,3 , Aurélien Pélissier 6,7 , Tom G. Caniels 5,8 , Mathieu A. F. Claireaux 5,8 , Anne Musters 9,10 , Marit J. van Gils 5,8 , María Rodríguez Martínez 6 , Niek de Vries 9,10 , Michael Meyer-Hermann 11,12 , Jeroen E. J. Guikema 4,13 , Huub Hoefsloot 14 and Antoine H. C. van Kampen 1,2,3,14 ✉ Sequencing of B-cell and T-cell immune receptor repertoires helps us to understand the adaptive immune response, although it only provides information about the clonotypes (lineages) and their frequencies and not about, for example, their affinity or antigen (Ag) specificity. To further characterize the identified clones, usually with special attention to the particularly abundant ones (dominant), additional time-consuming or expensive experiments are generally required. Here, we present an extension of a multiscale model of the germinal center (GC) that we previously developed to gain more insight in B-cell repertoires. We compare the extent that these simulated repertoires deviate from experimental repertoires established from single GCs, blood, or tissue. Our simulations show that there is a limited correlation between clonal abundance and affinity and that there is large affinity variability among same-ancestor (same-clone) subclones. Our simulations suggest that low-abundance clones and subclones, might also be of interest since they may have high affinity for the Ag. We show that the fraction of plasma cells (PCs) with high B-cell receptor (BcR) mRNA content in the GC does not significantly affect the number of dominant clones derived from single GCs by sequencing BcR mRNAs. Results from these simulations guide data interpretation and the design of follow-up experiments. npj Systems Biology and Applications (2023)9:8 ; https://doi.org/10.1038/s41540-023-00271-y INTRODUCTION The germinal center (GC) plays a crucial role in the adaptive immune response 1–3 . GCs are microanatomical structures found in secondary lymphoid organs and are formed when an adaptive response is initiated. These structures are responsible for a process called affinity maturation during which the affinity and specificity of the BcR for the Ag is improved over the course of several weeks. The GC reaction begins with the activation of a limited number of antigen (Ag)-specific B cells that start to proliferate (clonal expansion) to form the so-called GC dark zone (DZ), as defined by histology staining. During the proliferation of these B cells, now called centroblasts (CBs), their BcR is changed due to somatic hypermutations (SHMs), which increase or decrease the binding affinity of the BcR for the Ag. The CBs differentiate to centrocytes (CCs) and migrate to the GC light zone (LZ) where they collect Ag presented by follicular dendritic cells (FDCs) and, subsequently, interact with T-follicular helper (Tfh) cells to become positively selected to return to the DZ to undergo further rounds of proliferation and SHM. Memory B cells (MBCs) and PCs are output cells (OCs) from the GC. In general, MBCs are of lower affinity than PCs, and are produced mostly at the initial state of the GC reaction, while higher affinity PCs are produced at later stages 4 although this might be related to the nature of the Ag 5 . Mammals have an immense immune repertoire comprising B cells and T cells with unique BcRs and T-cell receptors (TcRs) to combat the large variety of Ags. The B-cell repertoire has been estimated to include about 10 15 members for the naive repertoire although a much smaller fraction of mature B cells is maintained in our body 6 . The diversity of BcR results from several processes that include their development in the bone marrow through somatic recombination of V(D)J genes that encode the receptor and induce junctional diversity, and pairing of different BcR heavy and light chains 7 . Finally, additional diversity is created by SHMs in the GC. The BcR is a heterotetramer composed of two immunoglobulin heavy chains (IgHs) and two immunoglobulin light chains (IgL). Each chain harbors three complementary determining regions (CDRs 1–3) that encompass the most variable parts of the Ab and are responsible for Ag binding. The four BcR framework regions (FWRs 1–4) mostly provide structural support for the CDRs 8–10 . Immune receptor repertoires in blood or tissue can be profiled using next-generation sequencing technologies 11–14 . These BcR and TcR repertoire-sequencing experiments have been applied for a broad range of applications, including vaccinology, infection, and (auto)immune disorders 15–21 . Typically, the pre-processing of repertoire-sequencing data results in a set of clones and their abundancies in the measured samples. A clone represents a 1 Amsterdam UMC location University of Amsterdam, Epidemiology and Data Science, Meibergdreef 9, Amsterdam, The Netherlands. 2 Amsterdam Public Health, Methodology, Amsterdam, The Netherlands. 3 Amsterdam Infection and Immunity, Inflammatory Diseases, Amsterdam, The Netherlands. 4 Cancer Center Amsterdam, Amsterdam, The Netherlands. 5 Amsterdam UMC location University of Amsterdam, Medical Microbiology and Infection Prevention, Meibergdreef 9, Amsterdam, The Netherlands. 6 IBM Research Zurich, 8803 Rüschlikon, Switzerland. 7 Department of Biosystems Science and Engineering, ETH Zurich, 4058 Basel, Switzerland. 8 Amsterdam Infection and Immunity, Infectious Diseases, Amsterdam, The Netherlands. 9 Amsterdam UMC location University of Amsterdam, Experimental Immunology, Meibergdreef 9, Amsterdam, The Netherlands. 10 Amsterdam Rheumatology & Immunology Center, Amsterdam, The Netherlands. 11 Department for Systems Immunology and Braunschweig Integrated Centre of Systems Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany. 12 Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Braunschweig, Germany. 13 Amsterdam UMC location University of Amsterdam, Pathology, Lymphoma and Myeloma Center Amsterdam, Meibergdreef 9, Amsterdam, The Netherlands. 14 Biosystems Data Analysis, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands. 15 These authors contributed equally: Rodrigo García-Valiente, Elena Merino Tejero. ✉ email: a.h.vankampen@amsterdamumc.nl www.nature.com/npjsba Published in partnership with the Systems Biology Institute 1234567890():,;