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3741 A Multimodal Single Cell Atlas of Human Tonsils Reveals New Insights into T and B Cell Differentiation

Program: Oral and Poster Abstracts
Session: 203. Lymphocytes and Acquired or Congenital Immunodeficiency Disorders: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science, genomics, immune mechanism, immunology, Biological Processes, molecular biology
Monday, December 12, 2022, 6:00 PM-8:00 PM

Sergio Aguilar1*, Ramon Massoni1*, Paula Soler-Vila, PhD2*, Juan C Nieto1*, Marc Elosua-Bayes1*, Domenica Marchese1*, Marta Kulis, PhD2*, Amaia Vilas-Zornoza, PhD3,4*, Marco Matteo Bühler2,5,6*, Sonal Rashmi1*, Clara Alsinet1*, Ginevra Caratù1*, Catia Moutinho1*, Sara Ruiz1*, Patricia Lorden1*, Giulia Lunazzi1*, Dolors Colomer, PhD2,3,6,7*, Gerard Frigola, MD8*, Will Blevins1*, Sara Palomino9*, Gomez-Cabrero David, PhD9,10*, Xabier Agirre, PhD3,4*, Marc Weniger11*, Federico Marini12,13*, Francisco Javier Cervera Paz14*, Peter M Baptista14*, Isabel Vilaseca15*, Felipe Prósper, MD, PhD3,4,16*, Ralf Küppers, PhD11*, Elías Campo, PhD MD3,7,17,18, Holger Heyn1,19*, Ivo Gut1,19* and José I. Martín-Subero, PhD2,3,7,20*

1CNAG-CRG, Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain
2Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
3Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Spain
4Hemato-Oncology Program, Center for Applied Medical Research (CIMA), University of Navarra, IDISNA, Universidad de Navarra, Pamplona, Spain
5Department of Pathology and Molecular Pathology, University Hospital Zurich, Zurich, Switzerland
6Hematopathology Section, Pathology department, Hospital Clinic, Barcelona, Spain
7Departament de Fonaments Clínics, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain
8Hematopathology Section, Pathology Department, Hospital Clínic of Barcelona, Barcelona, Spain
9Translational Bioinformatics Unit (TransBio), Navarrabiomed, Navarra Health Department (CHN), Public University of Navarra (UPNA), Navarra Institute for Health Research (IdiSNA), Pamplona, Spain
10Bioscience Program, Biological and Environmental Sciences and Engineering Division (BESE), King Abdullah University of Science and Technology KAUST, Thuwal, Saudi Arabia
11Institute of Cell Biology (Cancer Research), Medical Faculty, University of Duisburg-Essen, Essen, Germany
12Center for Thrombosis and Hemostasis, Johannes Gutenberg University Medical Center Mainz, Mainz, Germany
13Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
14Department of Otorhinolaryngology, University of Navarra, Pamplona, Spain
15Otorhinolaryngology Head-Neck Surgery Department, Hospital Clínic, IDIBAPS Universitat de Barcelona, Barcelona, Spain
16Departamento de Hematología, Clínica Universidad de Navarra, University of Navarra, Pamplona, Spain
17Hematopathology Section, Pathology Department, Hospital Clinic, Barcelona, Spain
18IDIBAPS, Barcelona, Spain
19Universitat Pompeu Fabra (UPF), Barcelona, Spain
20Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain

Palatine tonsils are secondary lymphoid organs under constant exposure to antigens via the upper respiratory tract which makes them a compelling organ for adaptive immunity development. They represent an important niche for lymphocyte maturation, where they differentiate into a wide variety of functional states, tailoring immune responses to different insults. However, although some efforts have been made to characterise the lymphocyte maturation process in human tonsils at single cell resolution, these studies lacked cell numbers and multimodal, i.e multiomics, information to comprehensively capture the cellular heterogeneity of the process. Here we generated a comprehensive human tonsil atlas of >357,000 cells profiled across five different data modalities, such as transcriptome, epigenome, proteome, adaptive immune repertoire and spatial location. We identified 121 cell subtypes and states across lineages and characterised the maturation process of both B and T lymphocytes. The high number of profiled cells allowed the detection of rare cell subtypes, such as precursor B and T cells, the latter representing <0.01% of detected cells, supporting an ongoing maturation of lymphoid cells outside the bone marrow and thymus, respectively. Beyond these rare precursor lymphocytes, the tonsil is the niche for multiple steps of mature T and B cell differentiation.

With regard to mature T cells, we described 37 different T cell (TC) subpopulations being CD4 TC the more representative population with 58,783 cells, which illustrated the maturation process of this lineage. We identified an early bifurcation following antigen recognition by naïve CD4 TC, resulting in two distinct precursor populations of T follicular helper (fh) and non-Tfh cells. Tfh precursor cells continued their differentiation trajectory towards terminally differentiated populations, with functions in the production of mature B cells (BC) and in the enhancement of antibody secretion. On the other branch of the CD4 TC differentiation trajectory, our data revealed three transcriptional Treg subtypes, with functions in the suppression of antitumor responses, COVID-19 and the control of BC antibody production, respectively. The multimodal study design further enabled the interrogation of regulatory circuits driving TC specialization, describing a potential BCL6 superenhancer specifically active in follicular TC. Intriguingly, the same region has been previously linked to BCL6 gene activation in Germinal Center (GC) BC, indicating its function as follicle-specific rather than cell type-specific cis-regulatory element.

Mature B cells corresponded to the greatest population of the dataset, with >70% cells assigned to the BC lineage allowing the fine-grained description of the BC maturation process. We identified 8 distinct naïve BC (NBC) subpopulations and comprehensively characterized the activation pathway upon first antigen recognition, further expanding previous findings focused on MYC. Additionally, by the combination of gene expression and chromatin accessibility profiles we disentangled the transcription factor (TF) hierarchy associated with the Light zone (LZ) to Dark Zone (DZ) reentry. We identified the activity NF-kB family genes as one of the first determinants of the TF hierarchy that drives this specialization, which seems to be shared in LZ cells reentering the DZ as well as activated NBCs that enter the DZ for the first time. However, this regulation was not observed in LZ GCBCs that further differentiate into plasma cells (PCs). This final maturation step towards an antibody secreting phenotype was also characterized, identifying the first stages of PC precursor cells derived from either memory or LZ-GCBC and their transcriptional dynamics was also spatially mapped in the tonsillar tissue. Charting the regulatory landscape in PCs we validated the presence of described PC TFs, but we also discovered SIX5 as a novel potential TF associated with the maturation, but not the commitment, of PCs. Moreover, we identified a higher activity of the regulatory network of SIX5 in multiple myeloma.

This study presents a multimodal single cell view of the TC and BC maturation process taking place in the tonsil. We not only present our analysis as a resource but we discover a yet unappreciated cellular heterogeneity as well as novel gene regulatory mechanisms driving naive towards highly specialized lymphoid cells.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH