Landscape of T Follicular Helper Cell Dynamics in Human Germinal Centers Emmanuel Donnadieu, Kerstin Bianca Reisinger, Sonja Scharf, Yvonne Michel, Julia Bein, Susanne Hansen, This information is current as Andreas G. Loth, Nadine Flinner, Sylvia Hartmann and of September 28, 2021. Martin-Leo Hansmann J Immunol published online 22 July 2020 http://www.jimmunol.org/content/early/2020/07/21/jimmun ol.1901475 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2020/07/21/jimmunol.190147 Material 5.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published July 22, 2020, doi:10.4049/jimmunol.1901475 The Journal of Immunology Landscape of T Follicular Helper Cell Dynamics in Human Germinal Centers Emmanuel Donnadieu,*,1 Kerstin Bianca Reisinger,†,1 Sonja Scharf,† Yvonne Michel,† Julia Bein,†,‡ Susanne Hansen,† Andreas G. Loth,x Nadine Flinner,{ Sylvia Hartmann,†,‡ and Martin-Leo Hansmann†,‡,{ T follicular helper (Tfh) cells play a very important role in mounting a humoral response. Studies conducted in mouse models have revealed with good kinetic and spatial resolution the dynamics of these cells in germinal centers (GC) and their cross-talk with B cells upon an immune response. However, whether a similar migratory behavior is performed by human Tfh cells is unclear, as technology to track them in situ has been lacking. In this study, we combined traditional immunohistochemistry and real-time fluorescent imaging approaches on fresh human adenoid slices to provide static and dynamic information on Tfh cells. Our data indicate that GC light zones are composed of two distinct areas in terms of Tfh cell distribution and migration. In the outer GC Downloaded from light zones, Tfh cells migrate actively and with a high ability to form dynamic clusters showing intense and rapid reorganization. In these outer regions, Tfh cells demonstrate multiple interactions between each other. Conversely, in central regions of GC light zones, Tfh cells are much more static, forming long-lasting conjugates. These findings reveal for the first time, to our knowledge, the dynamic behavior whereby Tfh cells migrate in human GC and highlight the heterogeneity of GC for Tfh cell motility. The Journal of Immunology, 2020, 205: 000–000. http://www.jimmunol.org/ subset of CD4 T cells, T follicular helper (Tfh) cells a pre-Tfh phenotype with the expression of canonical markers specialize in helping B cells to produce Abs. Mature Tfh such as CXCR5, PD-1, ICOS, and Bcl6. These cells migrate to the A cells localize to follicles in secondary lymphoid organs, T–B junction of secondary lymphoid follicles where the first T–B where they provide help to B cells in germinal centers (GC). interaction takes place. Tfh then move in the GC, a microana- Various help mechanisms have been reported that include contact- tomical structure in which proliferating Ag-specific B cells un- dependent process, such as CD40 ligands, and soluble molecules, dergo Ig affinity maturation, class-switch recombination, and such as IL-21. These contact-dependent and soluble signals support differentiation into long-lived plasma cells and memory B cells. survival and differentiation of GC B cells [for a review, see (1)]. Time-lapse two-photon laser-scanning microscopy experiments by guest on September 28, 2021 Recent work has significantly expanded our understanding of performed in mouse models have clarified how Tfh move and how when Tfh cells are formed and how these cells contribute to they interact with B cells in GC (3–5). A striking feature that help B cells (2). Tfh cell differentiation is initiated when naive emerged in the real-time imaging studies was that GC Tfh cells CD4+ T cells in the T cell zone encounter activated Ag-presenting were highly motile and frequently engage with B cells (3). Two dendritic cells. Activated T cells primed by dendritic cells acquire very distinct T–B conjugates were noticed. Unlike the frequent formation of stable conjugates during initial T cell–B cell inter- action at the T–B junctions, GC Tfh cells interact briefly with *De´partement Immunologie, Inflammation, et Infection, Institut Cochin, INSERM, U1016, CNRS, UMR8104, Universite´ de Paris, F-75014 Paris, France; †Dr. Senckenberg GC B cells. Only a low proportion of GC B cells form stable Institute of Pathology, Goethe University, 60590 Frankfurt am Main, Germany; conjugates with GC Tfh (3, 4). It was shown that these B cells ‡Reference and Consultant Center for Lymph Node and Lymphoma Diagnostics, x engaging Tfh in stable conjugates were capable of collecting and 60590 Frankfurt, Germany; Department of Otolaryngology, Head and Neck Sur- gery, University Hospital, 60590 Frankfurt am Main, Germany; and {Frankfurt presenting more Ag to Tfh cells. These prolonged interactions Institute for Advanced Studies, 60438 Frankfurt, Germany were responsible for promoting the positive selection of B cell 1E.D. and K.B.R. contributed equally to this work. clones (6). ORCIDs: 0000-0002-4985-7254 (E.D.); 0000-0002-5439-3909 (K.B.R.); 0000-0002- Several elements have been shown to control Tfh cell migration 0300-7027 (S.S.); 0000-0003-3424-1091 (S.H.). and interaction with B cells. One can mention the costimulatory Received for publication December 17, 2019. Accepted for publication June 28, receptor ICOS, which regulates Tfh recruitment to follicles but also 2020. regulates cognate T–B interactions in the GC (4, 5). A role of the This work was supported by Deutsche Forschungsgemeinschaft (FOR1961 Control-T chemokine receptor CXCR5, which binds to CXCL13 produced consortium, Grants HA1284/7-2 and HA6145/2-1). by follicular dendritic cells (FDC), stromal cells that build the M.-L.H. and E.D. designed the study; K.B.R., Y.M., S.H., N.F., J.B., and E.D. performed research; A.G.L. provided human samples; S.S. and E.D. analyzed data; architecture of GC light zones, was also demonstrated. S.H. provided insights and advice; M.-L.H. and E.D. wrote the manuscript. These elegant studies, which described the dynamics of Tfh in Address correspondence and reprint requests to Dr. Emmanuel Donnadieu, De´partement intact GC, have all been performed in mouse models that did not Immunologie, Inflammation, et Infection, Institut Cochin, Universite´ de Paris, 22 rue entirely reflect the true situation in humans. For instance, most ´ Mechain, 75014 Paris, France. E-mail address: [email protected] studies relied on the use of a cognate Ag, which usually binds to The online version of this article contains supplemental material. high-affinity BCRs and TCRs. In addition, several important Abbreviations used in this article: 3D, three-dimensional; FDC, follicular dendritic questions remain to be addressed concerning the role of different cell; GC, germinal center; Tfh, T follicular helper. subsets of Tfh cells and whether Tfh cells communicate to cells Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 other than just B cells. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901475 2 IMAGING OF Tfh CELLS IN HUMAN LYMPHOID TISSUES In human lymphoid tissues, our knowledge of Tfh cells is mainly and Alexa Fluor 405–anti-human fibronectin (clone HFN 7.1; Novus Bi- based on flow cytometry experiments or immunostained sections ologicals). All Abs were diluted in RPMI 1640 without Phenol red and m for different lymphoid subpopulations (7). These studies have used at a concentration of 10 g/ml. To concentrate the Abs on the tissue, a stainless steel ring was placed to the agarose surrounding the slice. In some highlighted some common features but also some notable differ- experiments, fresh slices were labeled for 15 min at 37˚C with DAPI and ences between mouse and human Tfh cells (8). In addition, these DRAQ5 to assess the nuclei of dead and live cells. are static evaluations that may not reflect the dynamic process T cells were imaged with a Leica SP8 confocal microscope (Leica ongoing during an immune response. Microsystems) equipped with a 37˚C thermostated chamber. The temper- ature was constant, and oxygen was controlled by self-written appropriate In this study, we tried to combine the advantage of cell differ- software. For dynamic imaging, adenoid slices were secured with a entiation by immunohistochemistry with a dynamic approach in stainless steel slice anchor (Warner Instruments) and perfused at a rate of human fresh lymphoid tissues that we have established in human 0.8 ml/min with a solution of RPMI 1640 without Phenol red, bubbled with tumors (9, 10) and more recently in human adenoids (11). Tissue 95% O2 and 5% CO2. Ten minutes later, images from a first microscopic 3 3 3 slices enabled us to monitor for the first time, to our knowledge, field were acquired with either a 10 ,25 ,or63 objective. For four- dimensional analysis of cell migration, stacks of 10–12 sections (z step = human Tfh cells in space and time. 5 mm) were acquired every 10–20 s for 20 min at depths up to 80 mm.