Three-Dimensional Visualization of the Mouse Organization in Health and

This information is current as Magali Irla, Jeanne Guenot, Gregg Sealy, Walter Reith, Beat of October 1, 2021. A. Imhof and Arnauld Sergé J Immunol published online 17 December 2012 http://www.jimmunol.org/content/early/2012/12/16/jimmun ol.1200119 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 17, 2012, doi:10.4049/jimmunol.1200119 The Journal of Immunology

Three-Dimensional Visualization of the Mouse Thymus Organization in Health and Immunodeficiency

Magali Irla,1 Jeanne Guenot, Gregg Sealy, Walter Reith,1 Beat A. Imhof,1 and Arnauld Serge´1

Lymphoid organs exhibit complex structures tightly related to their function. Surprisingly, although the thymic medulla constitutes a specialized microenvironment dedicated to the induction of tolerance, its three-dimensional topology remains largely elusive because it has been studied mainly in two dimensions using thymic sections. To overcome this limitation, we have developed an automated method for full organ reconstruction in three dimensions, allowing visualization of intact mouse lymphoid organs from a collection of immunolabeled slices. We validated full organ reconstruction in three dimensions by reconstructing the well- characterized structure of skin-draining lymph nodes, before revisiting the complex and poorly described corticomedullary orga- nization of the thymus. Wild-type thymi contain ∼200 small medullae that are connected to or separated from a major medullary Downloaded from compartment. In contrast, thymi of immunodeficient Rag22/2 mice exhibit only ∼20 small, unconnected medullary islets. Upon total body irradiation, medullary complexity was partially reduced and then recovered upon bone marrow transplantation. This intricate topology presents fractal properties, resulting in a considerable corticomedullary area. This feature ensures short distances between cortex and medulla, hence efficient migration, as assessed by mathematical models. Remarkably, this junction is enriched, particularly in neonates, in medullary thymic epithelial cells expressing the . The emergence of a major medullary compartment is induced by CD4+ via CD80/86 and lymphotoxin-a signals. This http://www.jimmunol.org/ comprehensive three-dimensional view of the medulla emphasizes a complex topology favoring efficient interactions between developing T cells and autoimmune regulator–positive medullary thymic epithelial cells, a key process for induction. The Journal of Immunology, 2013, 190: 000–000.

lymphocytes play critical roles in cell-mediated immu- by cTECs. They then migrate into the medulla in a CCR7- nity and are generated in the thymus by sequential dependent manner (5). The medulla promotes negative selec- T maturation and selection events. The thymus is divided tion, which consists in eliminating autoreactive SP thymocytes into a cortex, supporting the earliest events in thymocyte devel- that carry TCRs exhibiting high affinities for self-antigen–MHC complexes (6). The mTECs play a critical role in this process by

opment, and a medulla, in which thymocytes undergo final se- by guest on October 1, 2021 lection processes (1, 2). The thymus is composed of a highly expressing a wide range of tissue-restricted self-antigens repre- organized three-dimensional (3D) network of cortical and med- senting peripheral tissues (7). The expression of these Ags is ullary thymic epithelial cells (cTECs and mTECs, respectively) regulated in part by the autoimmune regulator (Aire) (8). Thymic (3). The cTECs promote positive selection, leading to survival of selection events ultimately lead to the emergence of a T cell double-positive (DP) CD4+CD8+ thymocytes carrying TCRs population carrying a randomly generated TCR repertoire that capable of interacting with self-MHC (4). Positively selected has been purged of autoreactive specificities. Mature naive thymocytes become CD8+ or CD4+ single-positive (SP), depend- T cells are exported to the periphery, where they circulate and ing on their interactions with MHC class I or II expressed migrate into secondary lymphoid organs, such as lymph nodes (LNs). In LNs, T cells are localized in the deep cortex (T-zone), whereas B cells are found in the outer cortex, arranged in fol- Department of Pathology and Immunology, Faculty of Medicine, University of Ge- neva, 1211 Geneva, Switzerland licles (B-zones) (9, 10). 1M.I., W.R., B.A.I., and A.S. contributed equally to this work. Lymphoid organ structures were originally unraveled by classical histological techniques. Their complex and highly Received for publication January 10, 2012. Accepted for publication October 31, 2012. organized pattern was documented using tissue section coun- This work was supported by the Swiss National Science Foundation (Ambizione terstaining or immunofluorescence staining, with Abs revealing PZ00P3_131945 to M.I., 31003A_127255 to W.R., and 31003AB_135701 to B.A.I.), specific cell types. A pioneering study (11) suggested that the 3D a Jules Thorn grant (to M.I.), and Oncosuisse funding (ME8335 to B.A.I.). geometry of the thymic medulla displays more convoluted Address correspondence and reprint requests to Dr. Magali Irla and Dr. Arnauld Serge´ structures than previously thought, with irregular foci and ex- at the current addresses: Centre d’Immunologie de Marseille-Luminy, Aix-Marseille Universite´, 13288 Marseille, France (M.I.) or Centre de Recherche en Cance´rologie de tensive branching. Another approach based on serial sections Marseille, Aix-Marseille Universite´, 13273 Marseille, France (A.S.). E-mail addresses: recently described the 3D organization of the bone marrow (12). [email protected] (M.I.) and [email protected] (A.S.) More recently, advanced imaging techniques, such as two-photon The online version of this article contains supplemental material. microscopy, allowed a deeper comprehension of the 3D organi- Abbreviations used in this article: 3D, three-dimensional; Aire, autoimmune regula- zation of lymphoid organs. Ex vivo LN explants and in vivo tor; BMT, bone marrow transfer; CMJ, corticomedullary junction; cTEC, cortical thymic epithelial cell; DP, double-positive; em., emission; ex., excitation; FOR3D, surgically exposed LNs and thymic slices were dynamically full organ reconstitution in three dimensions; K8, keratin 8; K14, keratin 14; LN, imaged by two-photon microscopy (13–15). However, because lymph node; LT, lymphotoxin; mTEC, medullary thymic epithelial cell; SP, single- tissue penetration remains limited to ∼400 mm, the 3D topology positive; TBI, total body irradiation; WT, wild-type. of entire lymphoid organs remains largely elusive, particularly Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 for the thymus.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200119 2 HEALTHY AND IMMUNODEFICIENT THYMIC 3D TOPOLOGY

We have developed a user-friendly method allowing visualiza- Axiocam MRm camera, and shift-free filter-sets: DAPI [excitation (ex.) tion of the 3D architecture of lymphoid organs from fluorescence BP365/50–emission (em.) BP455/50], Alexa Fluor 488 (ex. BP470/40–em. microscopy of immunolabeled tissue sections with multiple Abs. BP530/50), Cy3 (ex. BP565/30–em. BP620/60), and Alexa Fluor 647 (ex. BP640/30–em. BP690/50). A picture of each section was saved with a This technique was first validated for skin-draining LNs and then Pannoramic Viewer (3DHistech). The highest possible magnifications, applied to the thymus, in healthy and immunodeficient conditions, 83 for LN sections, 23 for entire thymic sections, and 103 for high res- using the Rag22/2 mouse model, which is almost devoid of me- olution for visualizing Aire+ cells, led to pixel widths of 2.6 mm, 6.5 mm, m dulla, and during thymic recovery upon total body irradiation and 1.3 m, respectively. (TBI) and bone marrow transfer (BMT). We provide novel insight Computational analysis into the organization, development, and function of the thymic medulla, which is composed of a large medulla surrounded by Full organ reconstruction in three dimensions (FOR3D) is automatically performed through the following steps. a hundred islets. Unlike Rag22/2 thymi, this organization remains preserved upon TBI. This complex topology provides a significant Section registration corticomedullary junction (CMJ) that is enriched, particularly in Sections were first registered, using ImageJ (National Institutes of Health) + neonates, in Aire mTECs, implicated in the induction of T cell and the dedicated plugin StackReg (21) (Fig. 1B, Supplemental Video 1B). tolerance. We show that CD4+ thymocytes constitute the main Intensity equalization cellular actor mediating this 3D organization. This effect is me- diated by lymphotoxin (LT) a signaling, which is upregulated in This and the following steps are performed automatically by starting the SP thymocytes by the CD80/86 costimulatory molecules. This rendering_3D function in Matlab (Mathworks). Labeling variations may intricate medullary geometry is expected to favor the encounter of result in artifactual disparities in fluorescence intensity between sections, + as denoted within the collection of uncorrected sections. With the as- Downloaded from autoreactive SP thymocytes with Aire mTECs and thus the effi- sumption of a nearly parabolic profile over depth, as generated by a ciency of T cell tolerance induction. pseudospheroidal organ, intensities could be corrected for possible abrupt variations arising between images (see dashed boxes in image collections before and after equalization, Fig. 1B, 1C, respectively; Supplemental Materials and Methods Video 1C). We performed a least-square fit of the mean intensity profile Mice Imean with a second-order equation (Supplemental Fig. 1B). Frame inten- sities were then corrected by normalizing according to the fit results Ifit:

C57BL/6 mice (Charles River) were maintained in a conventional animal http://www.jimmunol.org/ 2/2 b 2/2 2/2 a2/2  } facility. Rag2 (16), 2m (17), H2-Aa (18), LT (19), and I ðZÞ 2/2 I ðx; y; zÞ¼Iðx; y; zÞ fit : CD80/86 (20) mice were maintained under specific pathogen–free eq I ðZÞ conditions. Unless mentioned, male and female mice were used at the age mean of 6–12 wk for adults and 10 d for neonates. All experiments were ap- The exponent a allows tuning this normalization. Because organs are proved by and performed in accordance with guidelines from the Swiss not totally spherical, a was set empirically to 0.2, according to the degree veterinary authorities. of homogeneity among slices. This setting resulted in homogeneous var- iations of intensity over depth. Bone marrow chimeric mice C57BL/6 mice were lethally irradiated (900 rad), sacrificed after 3 d or Data filtering and subsampling 7 injected i.v. with 10 bone marrow cells obtained from wild-type (WT) Images were smoothed by median filtering (Fig. 1D, Supplemental Video by guest on October 1, 2021 donors. 1D) to better reveal macrocellular structures. Voxel size is set by section TEC isolation and flow cytometry thickness and microscope settings, typically generating an axial resolution 23 larger than the lateral resolution. Hence, thymic images with an initial Individual thymi were digested with collagenase D (1 mg/ml)/DNase I (2 pixel width of 6.5 mm were subsampled by 2-fold, leading to a final pixel mg/ml; Roche). Cells were passed through a 70-mm mesh to remove de- width of 13 mm. Filtered images were combined into a single 3D array. bris. Hematopoietic cells were depleted with anti-CD45 magnetic beads Gaussian filtering over 3D neighbors further reduced frame-to-frame dis- + (Miltenyi Biotec) by AutoMACS. CD452 cells were incubated at 4˚C with crepancies (Fig. 1E, Supplemental Video 1E). To visualize Aire cells (Fig. Fc-block (anti-CD16/CD32; Pharmingen), costained with anti-EpCAM 5F), FOR3D was performed at higher resolution, using images captured at + (clone G8.8; eBioscience) and anti-Ly51 (clone 6C3; Pharmingen), and 103 magnification, with a pixel size of 1.3 mm. Aire cell detection was then fixed and stained for intracellular Aire (clone 5H12; eBioscience) improved by background subtraction. using BD Cytofix/Cytoperm and Perm/Wash buffers (Pharmingen). Rendering in 3D Immunofluorescence staining Thresholds were set for each channel according to fluorescence intensity, Organs were dissected, and fat tissue was removed to avoid interference using the Matlab functions graythresh and opthr (Fig. 1F, Supplemental with 3D reconstruction. Frozen tissues embedded in O.C.T. (Sakura Video 1F), with particular attention to the K14 staining, used to discrim- Finetek) were cut into 20-mm-thick sections. Sections were fixed with 2% inate medulla from cortex. Users may also set thresholds. Surfaces were paraformaldehyde. Abs were diluted in hybridization buffer (1% BSA; determined using the Matlab function isosurface (Fig. 1G), with partial 0.01% Triton; and 0.1 M Tris buffer, pH 7.4). LN sections were costained transparency to allow visualizing inside organ structures. A 3D rotation for 30 min with Alexa Fluor 647–conjugated anti-IgD (clone 11-26; video was obtained by viewing the organ from various orientations (Fig. eBioscience) and Alexa Fluor 488–conjugated anti-CD3 (clone 500A2; 1H, Supplemental Video 2). Invitrogen). Thymic sections were costained for 30 min with rabbit anti- keratin 14 (clone AF64; Covance Research) and either biotin-conjugated Individual volume computation rat anti-keratin 8 (clone C51; ProGene) or Alexa Fluor 488–conjugated We quantified 3D structures by identifying individual objects within the a anti-Aire (clone 5H12; eBioscience) or hamster anti-LT (clone AF.B3; organ. Matlab allowed definition of an isosurface, which was often split into Pharmingen). Thymic slices were then incubated for 10 min in blocking several distinct objects. These regions were measured individually using buffer (3% BSA in 0.01% Triton and 0.1 M Tris buffer) before incubation Imaris (Bitplane) (Fig. 1I, Supplemental Video 3). for 30 min with Cy3-conjugated anti-rabbit (Invitrogen), Alexa Fluor 488– conjugated streptavidin (Invitrogen), or Alexa Fluor 546 anti-hamster (Invitrogen). Sections were counterstained with 1 ml/ml DAPI and Results mounted with Mowiol (Calbiochem). Reconstructing lymphoid organs by automated FOR3D Automated image acquisition analysis Slices were scanned using either an LSM 510 confocal or the automated Fig. 1 and Supplemental Video 1 depict the steps used by FOR3D. epifluorescence microscope Mirax (Zeiss) (Fig. 1A, Supplemental Video Serial sections from WT mouse thymi were immunostained and 1A). The Mirax system uses a Plan-Apochromat 203/0.8 objective, a Zeiss images acquired over the entire sections. Images were first aligned The Journal of Immunology 3

FIGURE 1. Overview of the FOR3D process. This function block diagram depicts the oper- ations iteratively applied to images acquired by fluorescence microscopy of immunolabeled thymic sections, costained with Abs against the mTEC-specific marker K14 (red) and the cTEC-specific marker K8 (green) and counter- stained for nuclei (DAPI, blue). Each block corresponds to a main task (A–I), with software used and eventual subfunction indicated below

(left column). Note that equalize_stack (C), fil- Downloaded from ter_stack (D, E), and rendering_3D (F–H)are homemade functions, whereas medfilt2, smooth3, graythresh, isosurface, and view are original Matlab functions. Each step is illustrated by representative results: collection of all images (middle column; scale bar, 10 mm) and a rep- resentative one (right column; scale bar, 1 mm). http://www.jimmunol.org/ The last steps (G–I) combine all images within a single 3D thymic lobe (axes graduated in millimeters). Dashed boxes (B, C) denote sec- tions typically corrected by the intensity equal- ization step. The thresholded image (F) was color coded with partial transparency, in agreement with the isosurface (G) and 3D (H). In the last 3D view, obtained by Imaris (I), individual medullae

are color coded according to their volume, from by guest on October 1, 2021 red (small medullae) to yellow (large central medulla), as indicated by the color bar.

relative to each other, and fluorescence intensity was equalized; were homogenously distributed in the deep cortex. Using FOR3D, images were smoothed, combined as a 3D volume, and thresh- we reconstructed inguinal LNs in 3D (Fig. 2B, Supplemental Video olded to define an isosurface enclosing each labeled structure. The 4). This procedure permitted quantification of B- and T-zone vol- fully automated FOR3D process uses the homemade Matlab umes and numbers. The percentages of volume occupied by the B- function rendering_3D, which first displays a graphical user in- and T-zones in the LNs were 18 6 10% and 71 6 25%, respec- terface listing default parameters (Supplemental Fig. 1A). Starting tively (mean LN volume: 2.4 6 0.7 mm3, n = 4) (Fig. 2C). We analysis is hence straightforward and user friendly, provides 3D observed an average number of 12.5 6 3 B-zones per LN. Col- movies, and allows volume quantification of specific structures. lectively, these data confirm the highly structured 3D organization FOR3D was first assessed by 3D reconstruction of skin-draining of B- and T-zones of mouse inguinal LNs. Performing FOR3D on LNs, highly compartmentalized structures involved in the initiation LNs allowed us to validate this process, demonstrating its useful- and regulation of adaptive immune responses. Whole inguinal LNs ness for reconstructing lymphoid organs in 3D. m from adult WT mice were sectioned into 20- m sections, yielding 2/2 ∼60 per LN. Sections were stained with Abs against the B- and T- FOR3D of WT and Rag2 immunodeficient thymi: zone markers IgD and CD3. Nuclei were counterstained with DAPI visualization of the corticomedullary organization (Fig. 2A). We observed that inguinal LNs exhibit well-defined B- Cortical and medullary regions of the thymus play critical roles in and T-zones. In agreement with previous studies (10), B cells were early events of thymocyte development and self-tolerance induc- arranged in follicles within the outer cortex. In contrast, T cells tion, respectively. To gain better insight into the 3D topological 4 HEALTHY AND IMMUNODEFICIENT THYMIC 3D TOPOLOGY Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 2. FOR3D of WT inguinal LNs, WT and Rag22/2 thymic lobes. (A) Representative image of an inguinal WT LN section costained with the T cell–specific marker CD3 (green) and the B cell–specific marker IgD (red) and counterstained with DAPI (blue). Scale bar, 1 mm. (B) FOR3D of a WT inguinal LN depicting B-zones (red) and T-zone (green). (C) B-zone volumes were measured by FOR3D. (D) Representative image of a WT thymic section, costained for K14 (red) and K8 (green) and counterstained with DAPI (blue). Scale bar, 1 mm. (E) FOR3D of a WT thymic lobe depicting the subcapsular region (blue), the cortical compartment (green), and the medullary compartment (red). Arrowhead and arrow respectively indicate medullary islets con- nected or not connected to the main medulla. (F) Medulla volumes were measured by FOR3D. The dashed circle denotes the largest medulla of each lobe. (G) Representative image of a Rag22/2 thymic section, stained as described for WT thymi (D). Scale bar, 1 mm. (H) FOR3D of a Rag22/2 thymic lobe depicting the subcapsular region (blue), the cortical compartment (green), and the medullary compartment (red). (I) Individual medulla volumes were measured by FOR3D. In (B), (E), and (H), all axes are graduated in millimeters. In (C), (F), and (I), horizontal lines represent the geometric mean and SD, derived from 4 LNs and 3 WT or 3 Rag22/2 thymic lobes, respectively, as indicated by different colors. Statistical significance, from unpaired Student t test p value between WT and Rag22/2 medullary volumes: ***p , 0.001. B, B-zones; C, cortex; M, medulla; SC, subcapsule; T, T-zone. organization of WT mouse thymus, whole lobes were sectioned for allows visualization from different angles and definitive determi- FOR3D analysis. To delineate the structure of the fully developed nation of whether or not medullary structures are connected. A organ, we focused on the adult thymus, yielding ∼100 sections. striking feature of the medullary topology was the systematic Sections were stained with Abs against the cTEC-specific marker observation in all thymi of a large central medulla, either subtly keratin 8 (K8) and the mTEC-specific marker keratin 14 (K14) to connected to (Fig. 2E, arrowhead) or surrounded by smaller islets detect the cortex and medulla, respectively. Nuclei, labeled with (Fig. 2E, arrow). Overall, the medulla exhibits an intricate orga- DAPI, are particularly dense in the subcapsular region. Fig. 2D nization, which was further examined by performing a fractal illustrates a representative WT thymic section exhibiting a well- analysis. This mathematical theory is dedicated to describing defined CMJ. In agreement with previous studies (11, 22, 23), complex topologies with noninteger dimensions. For instance, medullary regions varied considerably in size and shape. To better crumpled paper can be interpreted as intermediate between a sur- apprehend this complexity, we reconstructed thymic lobes by face (D = 2) and a volume (D = 3). Fractal analysis determined FOR3D (Fig. 2E; Supplemental Videos 2, 3, 5). The 3D rotation that the medulla dimension is 2.25 6 0.2 (n = 3, Supplemental The Journal of Immunology 5

Fig. 2C), emphasizing its complexity. In contrast, the cortex did gesting that several islets became connected owing to volumetric not exhibit the signature of fractal objects (D = 3, as for a perfect constraints. sphere). FOR3D also allowed volume quantification of medullary BMT from WT donors to irradiated WT mice allowed gradual compartments as well as of total thymic lobes. The percentage of recovery of thymopoiesis and thymic organization over time. This medullary volume was 9.7 6 3.7% (mean lobe volume: 25.9 6 8.8 recovery was assessed by flow cytometry, showing the sequential mm3, n = 3). Furthermore, as performed for B-zones in LNs, this reappearance of DP thymocytes by 1 wk after BMT, followed by procedure permitted the quantification of individual medullary the development of CD4+ and CD8+ SP thymocytes after 2 and 3 islets (Fig. 2F). We observed, on average, 171.7 6 32.5 medullary wk (Fig. 3A, 3B). FOR3D revealed that the increase in thymic size islets per thymic lobe. Individual medullary volumes were dis- after 1 wk concerned mainly the cortex (Fig. 3E, Supplemental tributed logarithmically over several orders of magnitude, with Videos 8–10), occurring consistently with the reappearance of DP a geometric mean volume of 1.6 1024 mm3 (geometric SD 9.97). thymocytes. This increase in cortical volume continues 2 wk after This variability was intrinsic to each analyzed lobe. BMT because of the accumulation of DP thymocytes (Fig. 3B, We further deciphered the corticomedullary organization of the 3E). The central medullary compartment (Fig. 3G, dashed circles) thymus in immunodeficient conditions. We applied our method and islets started to increase in size only after 2 wk, concomitantly to Rag22/2 mice (16), which exhibit an early block in thymocyte with the reappearance of SP thymocytes. This change was asso- development at the DN3 stage and are consequently devoid of SP ciated with a decrease in the medullary fractal dimension to values thymocytes. Formation of the medulla is controlled by the pres- similar to those in WT conditions (Df 2.2, Supplemental Fig. ence of SP thymocytes (24, 25). Accordingly, Rag22/2 mice have 2C), relating the complexity of the medullary shape directly to the defective medulla formation (26, 27). A representative image of development of SP thymocytes. At 3 wk after BMT, the thymic a Rag22/2 thymic section costained for K14 and K8 and coun- volume became comparable to what it was before TBI, whereas Downloaded from terstained with DAPI is shown in Fig. 2G. Using FOR3D, we the CMJ area progressively increased (Fig. 3E). Because FOR3D reconstructed Rag22/2 thymic lobes in 3D (Fig. 2H, Supplemental allowed us to segment the thymus into two compartments, cortex Video 6). Rag22/2 thymi are 10-fold smaller than WT thymi and and medulla, we computed the migration distance required for contain, on average, only 20.7 6 9.3 small, isolated medullary thymocytes located anywhere within the cortex to reach the CMJ. islets, occupying 0.21 6 0.13% of the thymic volume (n =3) This distance was reduced upon TBI, consistently with an exten- without any large central medulla. Moreover, no fractal feature sively affected cortex, but recovered to a basal value after BMT http://www.jimmunol.org/ was observed, denoting a simple topology. Individual medullary (Fig. 3E). volumes were reduced by 2.5-fold, compared with those in WT thymi, with a geometric mean volume of 6.5 1025 mm3 (geo- The complex medullary topology favors the encounter of SP metric SD 3.8, Fig. 2I). Collectively, these results demonstrate the thymocytes with the CMJ complex and highly structured 3D organization of the medulla in We investigated the impact of a fractal medulla by comparing 2/2 healthy conditions, which is largely lost in Rag2 immunode- experimental measures obtained from WT (Fig. 4A) and immu- ficient mice, directly correlating the complex medullary organi- nodeficient thymi (Rag22/2 and upon TBI) (Fig. 4B, 4C, re- zation with the development of SP thymocytes. spectively) with two mathematical models. In one model, the by guest on October 1, 2021 medulla was represented by a single sphere (Fig. 4D), whereas in 3D medullary topology upon thymic recovery after TBI and the other, the medullary compartment was modeled by a set of BMT randomly located spheres (Fig. 4E). Computing migration dis- Although lymphoablative treatments such as chemotherapy and tances from cortical locations to the CMJ led to a rather narrow TBI severely affect thymic cellular composition and architecture distribution for WT thymi, with an average distance of 114 6 42 (28, 29), their impact on the 3D corticomedullary organization mm(n = 3, Fig. 4A). Distances were overall increased in Rag22/2 remains unknown. We used FOR3D to investigate how the thymic thymi (Fig. 4B), consistently with a noticeably reduced medulla, topology evolves after TBI and subsequent BMT. In WT mice, and in contrast decreased in thymi after TBI (Fig. 4C), harboring 3 d after a lethal dose of TBI, DP thymocytes were massively a reduced cortex. The spherical medulla model (Fig. 4D) led to eliminated. As previously reported (28), the small remnant pop- a broad distance distribution. In contrast, the random pattern, ulation corresponded mainly to DN, CD4+, and CD8+ SP thy- composed of several objects (Fig. 4E), led to a distribution similar mocytes (Fig. 3A, 3B). Although TBI led to severe thymic atrophy to that obtained experimentally. This comparison illustrates that characterized by an 8-fold reduction in volume, staining with K14 the complex medullary topology allows shorter distances between and K8 Abs revealed a well-defined corticomedullary organization the cortex and the CMJ than that afforded by a simple spherical (Fig. 3C, 3D). The massive deletion of DP thymocytes led to medulla. FOR3D also allowed direct measurement of the CMJ a severe 10-fold reduction in cortical volume and a less pro- area. Rag22/2 thymi, being almost devoid of medulla, afford a nounced, but noticeable, 3-fold decrease in medullary volume, negligible CMJ area (0.42 6 0.23 mm2), compared with the WT accompanied by a 2-fold decrease in the CMJ area (Fig. 3E). CMJ area (40.5 6 10.6 mm2), whereas thymi after TBI display an FOR3D revealed that medullary islets were 10-fold smaller and intermediate area (22.3 6 8.8 mm2). Compared with the WT 4-fold less numerous after TBI than in WT thymi (Fig. 3D–G, medullary volume, a sphere provides the smallest possible area Supplemental Fig. 2, Supplemental Video 7). The medulla ap- (14.7 6 4.2 mm2) (Fig. 4F). The random model, being also peared densely compacted within a small cortex (Fig. 3F). Con- composed of spheres, leads to a comparable value. However, both sistently, the medullary fractal dimension increased to 2.48 6 areas are 3- to 5-fold lower than the experimentally measured 0.07 after TBI (Supplemental Fig. 2C), indicating that the topol- fractal CMJ area. Hence, a fractal medulla ensures a substantial ogy becomes less complex in irradiated mice. The logarithmic CMJ area, together with short distances from any cortical location, distribution of islet volumes was homogenously distributed for all thus favoring efficient thymocyte migration. Immunodeficient medullae in WT thymi, including the largest. In contrast, in irra- conditions such as Rag22/2 and irradiated thymi present defects diated animals the largest medulla (Fig. 3G, dashed circles) was in these two parameters, defining a new signature of their defi- ∼100 times larger than the remainder of the distribution, sug- ciency. 6 HEALTHY AND IMMUNODEFICIENT THYMIC 3D TOPOLOGY Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. FOR3D of WT thymic lobes after TBI and following BMT. (A) Thymocytes isolated from untreated mice (WT), irradiated mice 3 d after TBI and mice reconstituted 1, 2, or 3 wk after WT BMT, were analyzed for CD4 and CD8 expression by flow cytometry. (B) Histograms show the numbers of DN, DP, CD4+, and CD8+ thymocytes for the indicated conditions. TBI denotes 3 d after TBI; w1, w2, and w3 denote 1, 2, and 3 wk after WT BMT, respectively. (C) Representative images of thymic sections costained for K14 (red) and K8 (green) and counterstained with DAPI (blue), with same scale for each condition. Scale bar, 1 mm. (D) FOR3D of thymic lobes depicting the subcapsular region (blue), the cortical compartment (green), and the medullary compartment (red) for each condition at the same scale. All axes are graduated in millimeters. (E) Histograms show quantification of cortical and medullary volumes as well as CMJ area for the indicated conditions. (F) FOR3D of thymic lobes for each condition, with medullae color coded according to their volume, from red (smallest) to yellow (largest). Scale bar, 1mm.(G) Medullary volumes were measured by FOR3D. Horizontal lines represent the geometric mean and SD, derived from three thymic lobes for each condition (see Supplemental Fig. 2), as indicated by different colors. Dashed circles denote the largest medulla of each lobe. In (B), (E), and (G), means and standard deviations are derived from three mice per condition. Statistical significance, from one-way ANOVA with post hoc Bonferroni test: *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 4. Distance from cortex to medulla in experimental and modelized thymi. (A) The FOR3D results obtained on a thymic lobe (see Fig. 1) allow by guest on October 1, 2021 delimitation of the cortex (white) and medulla (black), illustrated by the representative binary image (top panel) corresponding to the middle of the thymus. The Euclidean distance from every cortical location to the nearest medullary position was computed and represented as a distance map (pseudocolors, middle panel) and histogram (bottom panel), with the average value d indicated by the red dashed line. For comparison, the same computation was performed for two immunodeficient conditions as well as for two mathematical models: Rag22/2 thymi (B), thymi from TBI mice (C), theoretical spherical thymi exhibiting a single and concentric medulla (D), and theoretical spherical thymi with small medullae randomly located in 3D (E). Sizes of the modelized medullae in (D) and (E) were set to the mean total volume experimentally determined in WT conditions (5.5 mm3). Scale bar, 1 mm. (F) The histograms depict the mean distance from cortical localization to the CMJ (left) and the CMJ area (right), as measured by FOR3D for the indicated experimental conditions or mathematical models. Means and standard deviations are derived from three measures. Statistical significance, from one-way ANOVA with post hoc Bonferroni test for comparison with WT: **p , 0.01, ***p , 0.001.

The CMJ is enriched in Aire+ mTECs, particularly in neonates observed for a majority (∼60%) of medullae (Supplemental Fig. The thymic medulla is specialized for the deletion of autoreactive 3A). This enrichment was further analyzed by computing the per- centage of Aire staining relative to the distance from the CMJ SP thymocytes. The mTECs play a pivotal role in this process by + expressing peripheral self-antigens regulated in part by the tran- external border. The gradual enrichment in Aire cells follows a scription factor Aire. It has been suggested that Aire+ mTECs are sharp gradient, culminating in a 2-fold increase at the CMJ in located at the CMJ, in addition to the medulla (30, 31). A pref- neonates and to a lower extent in adults (Fig. 5C). This gradient is erential localization at the CMJ was notably reported by in situ firmly established within a few micrometers from the CMJ border. + hybridization (32), but this specific pattern has not yet been The increased density in Aire mTECs in neonates compared with quantified. Furthermore, a recent study indicated that Aire is adult mice suggests that the frequency of these cells decreases in + particularly important during the perinatal period for preventing adults. To confirm, we quantified the frequency of Aire mTECs the development of multiorgan autoimmunity (33). Because we within the total mTEC population in neonates and adults by flow + observed that the medulla exhibits a complex topology leading to cytometry (Fig. 5D, 5E). The frequency of Aire mTECs was 23 a large CMJ area, we quantified the density of Aire+ mTECs lo- higher in neonates than in adults, in accordance with the quantifi- cated at the CMJ and in the medulla in neonates and adult thymic cation of Aire+ cell density observed by immunohistology (Fig. 5B, sections by costaining for K14 and Aire (Fig. 5A). The CMJ re- 5C). Preferential localization of Aire+ cells at the CMJ was further gion was defined as an external fringe 80 mm in width surrounding confirmed by FOR3D of thymi costained for K14 and Aire (Fig. the medulla. The density of Aire+ mTECs is higher at the CMJ 5E; Supplemental Videos 11, 12). Computing in 3D the distance of than in the medulla at both stages of development (Fig. 5B). Aire+ cells from the CMJ (Fig. 5F; Supplemental Fig. 3B, 3C) led Moreover, both regions exhibit a higher density at the neonate to a distribution biased toward lower values, denoting that Aire+ stage than in the adult. Enrichment in Aire+ cells at the CMJ was cells are preferentially located at the border rather than in the inner 8 HEALTHY AND IMMUNODEFICIENT THYMIC 3D TOPOLOGY

FIGURE 5. Aire+ mTECs are enriched at the CMJ in the thymus. (A) Thymic sections from neonate or adult WT mice were costained for K14 (red) and Aire (green) and imaged by confocal microscopy. M denotes the medulla, and CMJ denotes the corticomedullary junction, which was defined by an 80-mm-thick zone surrounding the medullae. Scale bar, 100 mm. (B) The density of Aire+ cells in the center of medullae and at the CMJ was measured using Metamorph. Each symbol represents an individual confocal image. (C) The per- centage of area exhibiting Aire fluorescence intensity above threshold was averaged over all pixels, in either the medulla (M) or the cortex (C), being at the same distance from the CMJ outer border, which was defined as the limit of K14 staining (vertical dashed line at 0). Downloaded from Curves represent the mean and SD (plain and dashed lines, respectively), derived from 12 confocal images for neonates and adults. (D) Representative flow cy- tometry profiles for the expression of EpCAM and Aire by CD452EpCAM+Ly512/lo mTECs from neo- nate and adult WT mice. Numbers represent the per- centages of cells within the indicated gates. (E) The http://www.jimmunol.org/ histogram summarizes quantifications of the percen- tages of Aire+ mTECs observed in total mTECs. (F) FOR3D of neonate (top) and adult (bottom) WT thymic lobes costained for K14 (red) and Aire (green) and counterstained with DAPI (blue) depicts Aire+ cell 3D localization within and at the border of the medulla. (G) The distribution of distances from Aire+ cells within the medulla to the CMJ was computed in 3D for neo-

nate and adult thymi and compared with a spherical by guest on October 1, 2021 medulla modelized with homogenously distributed cells. Mean values d are indicated by dashed lines. In (B) and (E), means and standard deviations are derived from three mice per condition. Statistical significance, from unpaired Student t test p value: *p , 0.05, **p , 0.01, ***p , 0.001.

medulla. Importantly, the CMJ constitutes the first region that SP and WT thymi, medullary volumes and fractal dimensions (Fig. thymocytes encounter when migrating to the medulla and is where 6A, 6B, 6F; Supplemental Fig. 4; Supplemental Video 13) were also autoreactive thymocytes are deleted. The substantial CMJ area, similar. Notably, a central compartment accounted for the majority because of the complex medullary topology, together with the local of the medullary volume (asterisk in Fig. 6A, 6B). In contrast, H2- enrichment in Aire+ cells, enhances the crucial importance of this Aa2/2 thymi lacked any large medullary compartment, leading to privileged interface for the induction of T cell tolerance. a noticeably reduced volume (Fig. 6C, 6F; Supplemental Fig. 4;

+ Supplemental Video 14). Isolated islets displayed various sizes, The emergence of the central medulla is promoted by CD4 with the largest one accounting for only 0.08 6 0.07 mm3 (20.2 6 a thymocytes via CD80/86 and LT signaling 1.4%, n = 3) of the total volume, compared with 2.0 6 1.5 mm3 It is well described that the formation of the thymic medulla is (73.6 6 33.1%, n = 3) for WT mice. We further investigated the controlled by the development of SP thymocytes (24, 34–36). We molecular mediators responsible for this complex architecture. SP investigated the subset of SP thymocytes responsible for this thymocytes interact with mTEC via several signaling axes of the complex 3D medullary organization. Thymic lobes from WT, TNF family, such as CD40, receptor activator of NF k-B (RANK), 2/2 + 2/2 b2m (lacking CD8 thymocytes), and H2-Aa (lacking and LTb receptor (LTbR) (25, 37, 38). Previous studies suggested CD4+ thymocytes) mice were sectioned, costained for K8 and that only the LTa1b2–LTbR axis was correlated with a defect in K14, and reconstructed by FOR3D (Fig. 6A–C). Thymic lobe medullary architecture (23, 39). We analyzed the 3D architecture in 2/2 2/2 2/2 volumes were comparable for all conditions (Fig. 6F). In b2m LTa thymi and found a topology similar to that in H2-Aa The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 6. CD4+ thymocytes are required for 3D medullary organization through CD80/86 and LTa signals. (A–E) Representative images of thymic sections, costained for K14 (red) and K8 (green) and counterstained with DAPI (blue) (upper panel). FOR3D rendering using Matlab (middle panel) and 2/2 2/2 2/2 Imaris, with medullae color coded according to their volume, from red (smallest) to yellow (largest) (lower panel) of a WT, b2m , H2-Aa ,LTa ,or CD80/862/2 thymus. Same scale used for each condition. Scale bar, 1 mm. All axes are graduated in millimeters. The asterisk denotes the largest medulla 2/2 for WT and b2m mice. (F) Histograms show quantification of volumes for thymic lobe, total medulla, mean medullary islet, and largest medulla for the indicated conditions. Mean and SD are derived from three thymic lobes (see Supplemental Fig. 4). Statistical significance, from one-way ANOVAwith post hoc Bonferroni test: *p , 0.05. (G) Thymic sections from WT or CD80/862/2 mice were costained for K14 (blue), CD4 (green), and LTa (red) and imaged by confocal microscopy with identical settings for each condition. LTa staining was evaluated by computing the mean value of the fluorescence intensity (n = 3 medullae for WT mice and n = 6 medullae for CD80/862/2 mice). Scale bar, 40 mm. thymi, characterized by isolated islets without any large central a medulla mostly devoid of any major compartment, as for H2- compartment (Fig. 6D, Supplemental Video 15). Islet and total Aa2/2 and LTa2/2 mice (Fig. 6E, 6F; Supplemental Video 16). medullary volumes displayed values intermediate between those of Because LTa2/2 and CD80/862/2 thymi exhibit similar pheno- H2-Aa2/2 and WT thymi (Fig. 6F). In H2-Aa2/2,asinLTa2/2, types, we hypothesized that CD80/86 could regulate LTa expres- the fractal dimension was increased to a value closer to 3, denoting sion. We thus compared LTa expression in WT and CD80/862/2 a topology of reduced complexity (Supplemental Fig. 4C). We medullae by immunofluorescence. We observed a significant de- previously reported that Ag-specific interactions between CD4+ crease of LTa protein expression in CD4+ SP thymocytes from thymocytes and mTECs determine the cellularity of mature Aire+ CD80/862/2 medullae (Fig. 6G), showing that LTa is upregulated mTECs (31). In this context of cellular interactions, we therefore upon CD28–CD80/86 engagement. Altogether, these results indi- hypothesized a possible involvement of the CD28–CD80/86 co- cate that CD4+ thymocytes are required for the establishment of stimulatory pathway in controlling the 3D medullary organization. a proper medullary topology with a major compartment through the FOR3D indeed revealed that CD80/862/2 thymi also presented CD80/86 and LTa signaling pathways. 10 HEALTHY AND IMMUNODEFICIENT THYMIC 3D TOPOLOGY

Discussion sents not only the privileged site of homing and export of thy- Reconstructing the 3D architecture of lymphoid organs provides mocytes from the vasculature (11, 42) but also the interface where a better understanding of the topology of their specific compart- SP thymocytes reach the medulla. We found that the medulla ments. It also allows quantification of defined structures in terms of exhibits a fractal topology, which is characterized by islet volumes number and volume, such as B cell follicles in LNs or medullary spanning over several orders of magnitude. This feature substan- islets in the thymus, as shown in this article. Furthermore, by tially increases the surface of the CMJ interface by almost 103. allowing 3D rotation, this approach demonstrated that certain Fractal geometry also ensures that the average distance from any compartments are connected through highly curved structures, location within the cortex to the nearest medulla remains rea- whereas others are disconnected. This geometric distribution is sonably low, as experimentally measured for the medullary ge- difficult to assess using two-dimensional sections, particularly for ometry by FOR3D. For comparison, in a nonfractal model with thymic medullary islets, which exhibit a highly complex topology. a single central medulla, this average distance increases substan- Diagrams depicting the medullary geometry of the thymus in tially. A random medulla model exhibits a more complex geom- textbooks or academic Web sites oversimplify its organization, etry and hence also ensures that any location in the cortex remains underestimating its complexity by depicting either a central sufficiently close to the medulla. Considering pseudo-Brownian m 2 compartment or a collection of small medullae. Our results firmly motion with an average motility of 7 m /min, as measured by establish the existence of an intricate central compartment that is two-photon microscopy (13, 14), the distance for a thymocyte either subtly connected or not connected to a hundred smaller randomly located in the cortex to reach the CMJ is covered in ∼ 6 6 peripheral islets, with a broad distribution of volumes. The co- 7.8 1 h, instead of 194 12 h for the spherical medulla model. Of note, thymocytes undergo partially directed, instead of existence of individual medullae is in agreement with the obser- Downloaded from vation that mTEC islets develop from single precursors (40). strictly Brownian, motion, being guided by stromal network and Thymic function can be affected by numerous conditions, such chemotactic cues such as CCL19 and CCL21 (2, 43). Directed as SCIDs or therapeutic treatments such as chemotherapy and TBI. motion, compared with erratic motion, results in a reduced mi- We investigated the 3D corticomedullary organization in Rag22/2 gration time. However, thymocytes also undergo sequestration in specific niches in a -dependent manner, leading to an immunodeficient mice and during thymic recovery after TBI and 2/2 overall residency time of a few days (42, 44). Even if linear or

BMT. Rag2 mice exhibit an early defect in T cell development http://www.jimmunol.org/ random behaviors, together with transient arrests, directly have an and thus lack adequate mTEC development (26). We found that impact on motion duration, this will similarly affect cell motion the thymus of these animals is devoid of the large central medulla for experimental and theoretical configurations. This effect will observed in healthy animals and exhibits only ∼20 small, un- hence not qualitatively have a bearing on the conclusion that a connected islets with a noncomplex topology. These observations complex medulla shape ensures reduced distance and duration for indicate that the intricate 3D organization of the medulla is tightly thymocytes to encounter the CMJ. Fractal geometry, together with controlled by the development of SP thymocytes. We further ap- the enrichment in Aire+ cells at the CMJ, stresses the importance plied FOR3D to decipher to which extent the corticomedullary of harboring a substantial CMJ area for favoring fast and efficient organization is affected upon TBI and subsequent BMT. A severe interactions between SP thymocytes and mTECs and should thus thymic atrophy was observed after lethal TBI, which was char- by guest on October 1, 2021 be crucial for adequately educating thymocytes and avoiding au- acterized by a substantial decrease in cortical and, to a lesser toimmune disorders. extent, medullary volumes. Despite these massive effects, the We investigated the cellular and molecular mediators dictating fundamental corticomedullary organization characterized by this intricate 3D medullary topology. Formation and homeostasis of a large central medulla and surrounding islets was surprisingly the medulla are known to rely on signaling crosstalk between conserved. This finding could be explained by the fact that before mTECs and SP thymocytes (24, 25). We show that proper 3D TBI, mTECs have been already educated through interactions medullary organization is specifically controlled by CD4+ thy- with SP thymocytes, allowing preservation of their overall orga- mocytes, which are necessary and sufficient for inducing the 2/2 nization, which was not the case in Rag2 mice. However, the emergence of the major medulla, hence generating a consequent number of islets and medullary complexity were reduced after CMJ area. We further deciphered the signals mediating this effect TBI. Upon BMT, the cortical volume started to increase already of CD4+ thymocytes on medullary organization. We first used the after 1 wk, correlating with the reappearance of DP thymocytes, LTa2/2mouse model, because studies on two-dimensional sec- whereas the medullary volume increased after 2 wk, correlating tions suggested that this could influence medullary ar- with the development of SP thymocytes. These rearrangements also chitecture (23, 39). Of interest, the absence of LTa led to an correlate with a recovery in topological complexity, as assessed intermediate phenotype between that of WT mice and that of mice by fractal dimensions. Overall, a major difference between the two lacking CD4+ thymocytes. LTa2/2 thymi were also characterized immunodeficient conditions studied in this article is the quasi- by the lack of a large central medulla, leading to a lower reduction 2/2 absence of medulla in Rag2 thymi, whereas TBI essentially of medullary volume. This finding suggests that additional mo- affects the cortex. Nevertheless, in both cases, the exquisite intri- lecular mediators, which remain to be identified, could be ex- cacy between cortex and medulla is affected, which can be directly pected to contribute to the effect induced by CD4+ thymocytes. related to defects in thymocyte development and migration. Because SP thymocytes interacting with mTECs control their In healthy conditions, SP thymocytes migrating from the cortex development, we investigated the implication of costimulatory to the medulla encounter the CMJ, which is enriched in Aire+ molecules, expressed by mTECs (27, 31). Previous studies ruled mTECs. Remarkably, we found that this region is particularly out a direct effect of the CD40L–CD40 axis on the medullary dense in Aire+ mTECs in neonates, compared with adults. This architecture (45). Nevertheless, the role of the CD28–CD80/86 finding is consistent with the fact that Aire was described as im- axis had not been investigated so far. In this article, we show portant during the perinatal period for preventing the emergence that CD80/86 are required for upregulating LTa expression in of autoimmune disorders (33). Aire+ mTECs, by expressing pe- CD4+ thymocytes, leading to the establishment of a proper 3D ripheral self-antigens, play a critical role in the deletion of T cells medullary organization, notably through the emergence of a large bearing self-reactive TCRs (25, 41). Therefore, the CMJ repre- central medulla. Therefore, during thymic development, growing The Journal of Immunology 11 mTEC islets (40) are expected to join together, leading to the Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin. Science 264: 703–707. emergence of large structures and ultimately to a major medulla. 20. Borriello, F., M. P. Sethna, S. D. Boyd, A. N. Schweitzer, E. A. Tivol, D. Jacoby, FOR3D should be relevant for deciphering the 3D organization T. B. Strom, E. M. Simpson, G. J. Freeman, and A. 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