A Niche for Human and Mouse Type 3 Innate Lymphoid Cells Kerim Hoorweg, Priyanka Narang, Zhi Li, Anne Thuery, Natalie Papazian, David R. Withers, Mark C. Coles and Tom This information is current as Cupedo of September 28, 2021. J Immunol 2015; 195:4257-4263; Prepublished online 16 September 2015; doi: 10.4049/jimmunol.1402584

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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 © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Stromal Cell Niche for Human and Mouse Type 3 Innate Lymphoid Cells

Kerim Hoorweg,*,1 Priyanka Narang,†,1 Zhi Li,† Anne Thuery,† Natalie Papazian,* David R. Withers,‡ Mark C. Coles,† and Tom Cupedo*

Adaptive immunity critically depends on the functional compartmentalization of secondary lymphoid organs. Mesenchymal stro- mal cells create and maintain specialized niches that support survival, activation, and expansion of T and B cells, and integrated analysis of and their niche has been instrumental in understanding adaptive immunity. Lymphoid organs are also home to type 3 innate lymphoid cells (ILC3), innate effector cells essential for barrier immunity. However, a specialized stromal niche for ILC3 has not been identified. A novel lineage-tracing approach now identifies a subset of murine fetal lymphoid tissue organizer cells that gives rise exclusively to adult marginal reticular cells. Moreover, both cell types are conserved from mice to humans and coloc- alize with ILC3 in secondary lymphoid tissues throughout life. In sum, we provide evidence that fetal stromal organizers give rise to Downloaded from adult marginal reticular cells and form a dedicated stromal niche for innate ILC3 in adaptive lymphoid organs. The Journal of Immunology, 2015, 195: 4257–4263.

esenchymal stromal cells are essential for the devel- adaptive immunity, highlighting the essential role of stromal cell– opment of secondary lymphoid organs (reviewed in derived microenvironments in the immune system (9–13). + M Ref. 1). In adult mice, mesenchymal stromal cells Rorgt type 3 innate lymphoid cells (ILC3) are a part of a http://www.jimmunol.org/ maintain the specialized microenvironments in lymphoid tissues family of innate-like immune cells with important roles in barrier where T cells encounter their cognate Ag presented by dendritic immunity to enteric pathogens, surveillance, and intestinal cells and B cells generate high-affinity Abs during homeostasis (reviewed in Ref. 14). Rorgt+ ILC3 reside in mucosal reactions (2). To date, several specialized subsets of stromal cells lymphoid and nonlymphoid tissues, where they are characterized have been identified. T zone fibroblastic reticular cells (TRCs) by expression of the natural cytotoxicity receptor NKp46 in mice provide structural support for the migration of T cells and deliver and NKp44 in humans (15–18). In addition, ILC3 are also found survival and recruitment signals to the migrating T cells in the in peripheral lymphoid organs as natural cytotoxicity receptor- form of IL-7 and CCL19/CCL21 (3). Follicular dendritic cells negative cells (15, 16). Even though ILC3 are found in most (FDCs) are located in follicles and are essential for gen- lymphoid tissues, it has yet to be established whether ILC3 reside by guest on September 28, 2021 eration of high-affinity Abs by presenting immune complexes to in specialized microdomains, analogous to T and B cells. If so, this B cells and for supporting B cell migration and homeostasis would provide novel insights into ILC3 function and regulation. through expression of BAFF and nonclassical CD40 ligands (4–8). During murine embryogenesis, a specialized population of stro- Functional deficiency of either TRC or FDC severely impacts mal cells termed lymphoid tissue organizer (LTo) cells interacts with a subset of ILC3 also known as lymphoid tissue inducer (LTi) cells, at prospective node (LN) anlagen (19–22). This in- *Department of Hematology, Erasmus University Medical Center, 3000 CA Rotter- dam, the Netherlands; †Department of Biology, Centre for Immunology and Infec- teraction induces activation of LTo cells through -b tion, Hull York Medical School, University of York, York YO10 5DD, United receptor (LTbR) and TNFR signaling (22). Activated LTo cells ‡ Kingdom; and Medical Research Council Centre for Immune Regulation, College express ICAM-1, VCAM-1, and mucosal cell adhesion of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom molecule-1 (MAdCAM-1), and secrete the three homeostatic 1K.H. and P.N. contributed equally to this study. , CXCL13, CCL21, and CCL19, that are essential for Received for publication October 10, 2014. Accepted for publication August 22, stromal cell–driven organization of the developing LN. Whereas 2015. LTi cell–LTo cell interactions are crucial for mouse LN and This work was supported by an Erasmus Medical Center grant (to T.C.), ZenithII Peyer’s patch development, splenic white pulp development is LTi Grant 93512004 awarded by the Netherlands Genomics Initiative (to T.C.), Medical cell independent (23–25). Research Council Grant G0601156 (to M.C.C.), Human Frontier Science Program Grant RGP0006/2009-C (to T.C. and M.C.C.), and the People Programme (Marie In adult LNs, a stromal cell population exists that phenotypically Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007- resembles fetal stromal LTo cells (26, 27). These marginal reticular 2013 under Research Executive Agency Grant Agreement 289720. cells (MRC) express VCAM-1, ICAM-1, and MAdCAM-1 (26), Address correspondence and reprint requests to Dr. Mark C. Coles or Dr. Tom and cell lines generated from MRC produce CXCL13 upon LTbR Cupedo, Department of Biology, Centre for Immunology and Infection, Wentworth Way, University of York, York YO10 5DD, U.K. (M.C.C.) or Erasmus Medical stimulation (28). In vivo, MRC can differentiate into FDC after Center, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands (T.C.). E-mail ad- immune activation (29). Due to their phenotypic resemblance to dresses: [email protected] (M.C.C.) or [email protected] (T.C.) fetal LTo cells, it was postulated that MRC represent direct Abbreviations used in this article: E, embryonic day; FDC, follicular ; descendants of the fetal stromal organizer cells (30). However, ILC, ; LN, ; LTi, lymphoid tissue inducer; LTo, lymphoid tissue organizer; LTbR, lymphotoxin-b receptor; MAdCAM-1, mucosal stromal cell lineage relationships between LTo and MRC remain addressin -1; MRC, marginal ; qPCR, quantitative experimentally unaddressed. PCR; RANKL, receptor activator for NF-kB ligand; TRC, zone fibroblastic In this study, we set out to identify and characterize a specialized reticular cell. stromal cell niche for ILC3 in human and mouse secondary Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 lymphoid organs. We show that LTo and MRC are conserved from www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402584 4258 STROMAL NICHE FOR LYMPH NODE ILC3 mice to humans and that they colocalize with ILC3. Using a novel RNA from human tissues was extracted using the RNA-XS kit (Machery lineage-tracing approach, we provide evidence that a subset of Nagel), followed by reverse transcription with random hexamer primers. A fetal LTo cells gives rise exclusively to MRC, suggesting that the Neviti Thermal Cycler (Applied Biosystems) and DyNAmo Flash SYBR Green qPCR kit (Finnzymes) were used for quantitative PCR, with the LTo–MRC lineage provides a lifelong stromal framework for addition of MgCl2 to a final concentration of 4 mM. All reactions were human and mouse ILC3. done in duplicate and are normalized to the expression of GAPDH. Rel- ative expression was calculated by the cycling threshold method as 22Δt. Materials and Methods Statistical analysis Mice Significance values were calculated using an unpaired two-tailed Student Il7Cre (31), and Rosa26 eYFP mice were bred and maintained in a specific t test. The p values , 0.05 were considered statistically significant. pathogen-free facility at the University of York under Home Office license. All animal research was approved by the University of York Ethical Re- view Process and conducted in accordance with ARRIVE guidelines. Results Superovulated female Rosa26eYFP mice were mated with IL-7 Cre males, Identification of human fetal stromal organizer cells and day of plug was defined as day 0.5. The stromal cells driving lymphoid development have Human tissue been identified in mice, but it is unknown whether similar cells have Use of all human tissues was approved by the Medical Ethical Commis- been conserved in humans. To identify putative fetal stromal LTo sion of the Erasmus University Medical Center Rotterdam and was con- cells in humans, we analyzed the stromal composition of devel- tingent on informed consent. Fetal lymph nodes and were obtained oping human LNs during first-trimester pregnancy. At 9 wk ges- from elective abortions, and gestational age was determined by ultrasonic tation, which is prior to the appearance of T cells in the circulation Downloaded from measurement of the skull or femur and ranged from 8 to 22 wk. Adult hepatic LNs were obtained during multiorgan donation procedures. (32), mesenteric LNs develop within the mesentery (33). At this age, individual LNs are undetectable using dissection microscopes Immunohistochemistry (33), but, upon isolation of total first-trimester mesenteries, we 2 2 Murine tissues were paraformaldehyde fixed and passed through sucrose detected CD45 CD31 mesenchymal stromal cells that expressed gradient before embedding in OCT (Tissue-Tek Sakura). The 8- to 10-mm– ICAM-1 and VCAM-1 at intermediate or high levels. In addition, thick cryosections were stained with Abs against GFP (Invitrogen), gp38, these stromal cells expressed LTbR and RANKL, very reminis- http://www.jimmunol.org/ CD21/35, VCAM-1 (eBioscience), Desmin (AbCam), a-smooth muscle actin (Sigma-Aldrich), and the relevant secondary Abs (Molecular Probes). cent of murine LTo cells (Fig. 1A) (19, 22). To verify that these Sections were mounted in ProlongGold Antifade Reagent (Invitrogen). For human stromal cells are indeed associated with lymphoid tissues, total LN overviews, 5 3 7 individual images were merged by using tile we subsequently isolated individual LNs from second-trimester scan under original magnification 340 by Zeiss M710 confocal micro- mesenteries (Fig. 1B). Similar populations of ICAM-1– and scope. Cryosections (6 mm) of human tissues were fixed in acetone for VCAM-1–expressing cells were present in the fetal LNs, and these 5 min and air dried for an additional 10 min. After rehydration, sections were blocked with 1% (w/v) blocking reagent (tyramide signal amplifi- cells also coexpressed LTbR. Having established the presence of cation kits; Molecular Probes) and 10% (v/v) normal human and donkey a phenotypic stromal organizer population, we asked whether serum, followed by treatment with a streptavidin-biotin blocking kit these cells had the ability to function as LTo cells. First, as LTo (Vector Laboratories). Sections were incubated with primary Abs for interact with LTi cells, the spatial distribution of stromal cells and by guest on September 28, 2021 30 min at room temperature, followed by 30-min incubation with Alexa 2 Rorgt+CD3 LTi cells in human fetal developing LNs was de- Fluor–labeled Abs (Molecular Probes) and 5% normal human serum. + 2 Biotinylated primary Abs were incubated overnight at 4˚C and visualized termined (Fig. 1C). Both Rorgt CD3 LTi cells and stromal cells using Tyramide Signal Amplification Kits (Molecular Probes), according expressing VCAM-1 and MadCAM-1 as well as RANKL and to the manufacturer’s instructions. Sections were embedded in Vectashield Podoplanin were preferentially located in the outer cortex of the (Vector Labs). Abs used were as follows: rabbit anti-human CD20 (Epi- + ε LN. The RANKL LTo-like cells were closely associated with the tomics), rabbit anti-human CD3 (Epitomics), mouse anti-human podo- + planin (Covance), rabbit anti-human Lyve-1 (Abcam), mouse anti-human Rorgt LTi cells (Fig. 1D). Second, the functional hallmark of MAdCAM-1 (HBT), mouse anti-human VCAM-1-bio (eBioscience), murine LTo cells is their production of the homeostatic chemo- mouse anti-human receptor activator for NF-kB ligand (RANKL)-bio kines CXCL13, CCL21, and CCL19. We used the characteristic (eBioscience), rat anti-human Rorgt (eBioscience), and goat anti-human expression of RANKL to purify these cells by flow cytometry and CXCL13-bio (R&D Systems). 2 compared transcripts by qPCR to sorted CD45 Flow cytometry and cell sorting CD312 RANKL-Podoplanin+ stromal cells, which contain both Single-cell suspensions of murine tissues were prepared from LNs by dicing TRC and FDC (Fig. 1E). Transcript analysis revealed that, similar them into small pieces and then disaggregating with Liberase CI/DNase 1 to the equivalent murine subset (3), TRC/FDC expressed tran- (Roche Diagnostics), blocked with Fc Block (eBioscience), and then stained scripts for CCL19 and CCL21, most likely in TRC, and transcripts with gp38 (BioLegend), CD31, anti-CD45 biotin (eBioscience), and for CXCL13, most likely from FDC (Table I). In line with mouse streptavidin Pacific Blue (Invitrogen). Stained cells were sorted using cells, human RANKL+ stromal cells contained transcripts for all a Moflo high-speed cell sorter (Beckman Coulter). For flow cytometric analysis of human fetal LNs, the following Abs were used: Alexa Fluor- three homeostatic chemokines, as follows: CCL19, CCL21, and 488–conjugated mouse anti-podoplanin (BioLegend), PE-conjugated CXCL13, a hallmark of stromal organizer cells, as well as for IL7. mouse anti-human RANKL (eBioscience), PE-Cy5.5–conjugated mouse Collectively, these data identify VCAM-1+ICAM-1+RANKL+ anti-CD45 (eBioscience), PE-Cy7–conjugated mouse anti-human CD34 stromal cells as human equivalents to murine LTo cells. (BD Biosciences), and PE-Texas Red–conjugated mouse anti-human CD31 (ImmunoTools). A FACSAria (BD Biosciences) was used for cell Stromal organizers in the fetal human sorting. Data were analyzed with FlowJo software (Tree Star). Having identified stromal LTo cells in human fetal LNs, we Quantitative RT-PCR analysis then assessed whether a putative stromal organizer population RNA was extracted from murine tissues using the RNeasy Mini kit (Qiagen). could also be identified in developing human spleens. In fetal RNA was converted to cDNA using Superscript III First Strand cDNA spleens of 15 wk gestation, primordial white pulp was apparent by synthesis kit (Invitrogen). Quantitative PCR (qPCR) was then performed for perivascular clusters of B cells surrounding arterioles displaying each sorted cell subset in triplicate on ABI 7300 Real Time PCR system using primers to various genes specific to stromal cell subsets (mice or strong CXCL13 labeling. At this gestational age, only few scattered humans) (Table I). Gene expression was quantified relative to the endog- T cells were present (Fig. 2A, left panel). At 18 wk gestation, enous housekeeping gene HPRT using 7000 System software from ABI. B cell clusters had increased in number and size and were now The Journal of Immunology 4259

interspersed with T cells (Fig. 2A, right panel). At both gesta- tional ages, no segregation of T and B cells was observed, in accordance with previous reports describing the appearance of delineated splenic B cell follicles from 24 wk gestation onward (34–37). We hypothesized that putative stromal organizer cells should be contained within the perivascular clusters and might share phenotypic characteristics with LN organizers. In contrast to fetal LNs, we did not detect any RANKL-expressing cells in fetal spleens. In contrast, MAdCAM-1 was abundantly expressed at all ages analyzed, albeit not by stromal cells, but by endothelial cells contained within the fetal red pulp (Fig. 2B). However, from 18 wk gestation onward, a subset of nonhemato- poietic cells, located exclusively within the primordial white pulp, initiated expression of MAdCAM-1 (Fig. 2B, right panel). These MAdCAM-1+ cells were neither vascular nor lymphatic endothe- lium, as shown by the lack of CD31 and Lyve-1 expression, re- spectively (Fig. 2C, left panel). Moreover, in line with LN organizers,

the MAdCAM-1+ stromal cells coexpressed VCAM-1 (Fig. 2C, right Downloaded from panel). To confirm the temporal regulation of MAdCAM-1 expres- sion on splenic stromal cells, flow cytometric analysis of fetal spleens was performed (Fig. 2D). In support of the histological data, a dis- tinct population of MAdCAM-1+ stromal cells was present at 20 wk gestation, yet virtually absent at 14 wk gestation (Fig. 2D). In ad-

dition to MAdCAM-1, these stromal cells were also uniformly http://www.jimmunol.org/ positive for VCAM-1 (Fig. 2E). Fetal splenic organizers transcribe LTo-associated genes As the MAdCAM-1+ VCAM-1+ stromal cells that localize spe- cifically to the developing white pulp showed phenotypic resem- blance to LN LTo cells, we sought to gain mechanistic insight into a possible organizing function of these cells. Therefore, CD452CD312MAdCAM-12 and CD452CD312MAdCAM-1+

cells were sorted to purity and analyzed by qPCR for genes in- by guest on September 28, 2021 volved in lymphoid tissue organization (Fig. 3A). Even though the MadCAM-1–negative populations contain the majority of all splenic reticular cells, including erythroblasts, both populations contained transcripts for LTBR and CXCL13. The T zone–specific transcripts CCL21 and CCL19 were almost exclusively expressed in MAdCAM-1+ cells, suggesting involvement of this stromal cell population in organization of developing T cell areas in the spleen. These data indicate that MAdCAM-1+ stromal cells in fetal spleen share phenotypic and functional characteristics with LN LTo cells and that these cells could be involved in formation and organi- zation of splenic T cell areas. We have previously reported the presence of a small but distinct population of Rorgt+ ILC3 in the fetal spleen (33), but their phenotype and in situ location are undetermined. Because in LNs we observed a striking colocalization between stromal LTo cells and ILC, we analyzed fetal spleen sections for Rorgt+CD32 cells in spatial relation to the MAdCAM-1+ stromal cells (Fig. 3B). In + FIGURE 1. Stromal LTo cells in human fetal LNs. Fetal human LNs and situ, Rorgt ILC3 appeared evenly distributed throughout the red mesenteries were analyzed for presence of cells resembling murine LTo and white pulp of the developing spleen. Within the white pulp, cells. (A) Expression of VCAM-1, ICAM-1, LTbR, and RANKL on CD452 ILC3 often colocalized with MAdCAM-1+ stromal cells, al- 2 CD31 stromal cells from first-trimester mesenteries (n = 3; age range: 8– though this colocalization was far less exclusive compared with 10 wk gestation). (B) Expression of VCAM-1, ICAM-1, and LTbRon 2 2 the developing LNs. Detailed analysis of the ILC3 phenotype CD45 CD31 stromal cells dissected from second-trimester LNs (n =5; revealed that fetal splenic IL7Ra+CD117+ ILC3 were pheno- C + + age range: 8–10 wk gestation). ( ) Localization of VCAM-1 and RANKL typically very similar to ILC3 from fetal and adult LNs (15): stromal cells directly underneath the Podoplanin-expressing subcapsular expression of NKp44 and NKp46 was absent, yet approximately sinus (original magnification 3100). (D) Colocalization of Rorgt+ cells and RANKL+ cells in fetal LNs (original magnification 3100/3250) (n =5). half of the cells expressed NKp30 (Fig. 3C; fetal splenic NK cells (E) Transcript analysis by qPCR of CD452CD312 RANKL+ and RANKL2 as comparison). Collectively, these data indicate that ILC3 are stromal cells purified from second-trimester LNs (n = 8) compared with present in human developing spleens, resemble LN resident total CD45+ cells (n =4).(C–E) Second trimester; age range 15–22 wk ILC3, but are not exclusively localized to regions containing gestation. *p =0.005. stromal organizers. 4260 STROMAL NICHE FOR LYMPH NODE ILC3

Table I. Primer sequences

Gene Forward Primer Reverse Primer gapdh 59-GTCGGAGTCAACGGATT-39 59-AAGCTTCCCGTTCTCAG-39 tnfsf11 59-GTGCAAAAGGAATTACAACA-39 59-CGGTGGCATTAATAGTGAG-39 59-CCTCCAGACAGAATGAAGTT-39 59-AGGGTCCACACACACAAT-39 59-GTACAGCCAAAGGAAGATTC-39 59-GGGGATGGTGTCTTGTC-39 59-AGCCTGCTGGTTCTCTG-39 59-TGCAGCCATCCTTGAT-39 il7 59-CCTCCCCTGATCCTTGTTCT-39 59-CGAGCAGCACGGAATAAAAA-39 GAPDH 59-GTCGGAGTCAACGGATT-39 59-AAGCTTCCCGTTCTCAG-39 TNFSF11 59-GTGCAAAAGGAATTACAACA-39 59-CGGTGGCATTAATAGTGAG-39 CXCL13 59-CCTCCAGACAGAATGAAGTT-39 59-AGGGTCCACACACACAAT-39 CCL21 59-GTACAGCCAAAGGAAGATTC-39 59-GGGGATGGTGTCTTGTC-39 CCL19 59-AGCCTGCTGGTTCTCTG-39 59-TGCAGCCATCCTTGAT-39 IL7 59-CCTCCCCTGATCCTTGTTCT-39 59-CGAGCAGCACGGAATAAAAA-39

MRC are a distinct stromal subset in adult human LNs hepatic LNs harvested during multiorgan donation procedures, The identification of human fetal LTo cells prompted us to de- B cell follicles were evident by Podoplanin staining on FDCs. In termine whether MRC, the phenotypic equivalents to fetal LTo in the interfollicular area, were marked by Downloaded from adult mouse LNs, were also conserved in humans. In resting adult MAdCAM-1 and VCAM-1 (Fig. 4A). MRC-like cells expressing high levels of VCAM-1 and MAdCAM-1 as well as RANKL and Podoplanin were found directly underlying the Lyve-1+ subcap- sular sinus, on the apical side of B cell follicles and prominently in interfollicular areas (Fig. 4A, 4B). As mouse MRC can se- crete CXCL13, immunohistochemistry was performed to detect

CXCL13 protein on human MRC, and these experiments showed http://www.jimmunol.org/ that adult LN-derived RANKL+ stromal cells expressed CXCL13 protein (Fig. 4C). Finally, we assessed whether the fetal colocal- ization of RANKL+ stromal cells and Rorgt+ ILC3 was main- tained into adulthood. Fig. 4D shows that Rorgt+ ILC3 reside mainly in the interfollicular areas of adult human LNs (38), in close proximity to RANKL+ MRC. These data establish that the phenotype and localization of human MRC are phenotypically similar to human fetal LTo cells and correspond to that of murine MRC. Moreover, these findings establish the continuous presence by guest on September 28, 2021 of ILC–stromal cell colocalization in specialized niches of both fetal and adult human LNs. IL7 marks a subset of murine fetal LTo cells The conserved presence in human LNs of fetal LTo cells and adult MRC, and the colocalization of both of these stromal subsets with Rorgt+ ILC3, raised the question as to whether there might be a precursor–progeny relationship between LTo and MRC. If so, this would imply the presence of a lifelong stromal niche for ILC3 in LNs. To gain insight into the precursor–progeny relationship be- tween fetal organizers and MRC, we set out to identify reporter mice marking stromal organizer cells in mouse fetal LNs. IL-7 is expressed by LTo cells in fetal Peyer’s patches and LN anlagen and functions by enhancing LTa1b2 expression on LTi cells (39). To determine whether IL-7 could be used to genetically mark fetal LTo cells, we analyzed the fetal LN anlagen of Il7-Cre BAC transgenic Rosa26eYFP F1 mice where Cre has replaced the first exon of the Il7 gene and eYFP is expressed under the control of the inducible ubiquitous Rosa26 promoter (31). Expression of Il7 indelibly marks cells that have expressed this at suffi- cient levels to allow Cre-mediated recombination of the Rosa locus (40). Il7-cre+ Rosa26eYFP+ embryos were sectioned by FIGURE 2. Stromal organizer cells in fetal human spleen. Fetal human vibratome into 200- to 400-mm sections, and the cervical region spleens were analyzed for the presence of putative stromal organizer cells. (A) was imaged. As seen in Fig. 4A, YFP+ cells were found scattered Appearance of perivascular lymphocyte clusters in fetal human spleens of + B + around the Lyve-1 jugular lymph sac as early as embryonic day 15–18wkgestation.( ) Appearance of MAdCAM-1 cells in developing white + pulp at week 18. (C)MAdCAM-1+Lyve-12CD312 stromal cells in spleens of (E) 14.5. These YFP cells gradually condensed in mesenchymal 18 wk gestation. (D) Flow cytometric analysis of fetal spleens showing the clusters at E15.5 and E16.5 (Fig. 5A) (41). At E17.5, an orga- + appearance of CD452CD312 MAdCAM-1+ stromal cells (n =3).(E)Ex- nized mass of YFP cells can be seen along the boundary of the pression of VCAM-1 on splenic stromal cells subsets at 20 weeks gestation. jugular lymph sac (Fig. 5A), which matures into a LN during The Journal of Immunology 4261

Fetal YFP+ LTo cells give rise to MRC IL-7–driven expression of Cre in I/Vhigh fetal stromal organizer cells in the Il7-Cre+ Rosa26eYFP+ mice allowed us to perform lineage tracing of IL-7–expressing stromal organizers to provide evidence for a possible precursor–progeny relationship between this subset of fetal stromal organizers and MRC in adult LNs. Detailed characterization of LNs isolated from adult Il7-cre+ Rosa26eYFP+ mice by revealed that YFP marked two distinct nonhematopoietic cell types (Fig. 5C). First, Lyve-1+ lymphatic endothelial cells were brightly YFP positive (Fig. 5C, 5D), in line with previous work describing a role for IL-7 pro- duction by lymphatic endothelial cells during LN restructuring (42). Second, a subset of VCAM-1+ stromal cells beneath the subcapsular lymphatic sinus expressed YFP (Fig. 5C, 5D). VCAM-1 is not expressed by the Lyve-1+ endothelium, allowing for the discrimination of stromal and endothelial cells based on these two markers (Fig. 5C). This characteristic localization suggested that the cells marked in the IL-7 reporter are indeed MRC, and this was confirmed by the coexpression Podoplanin Downloaded from (gp38) (Fig. 5D). CD35-expressing FDC were YFP negative (Fig.5D).Strikingly,YFP+ cells did not express MadCAM-1 (Fig. 5E), underscoring the fact that YFP marks a subset of MRC in adult LNs, which is in line with a recent study showing that in steady state LNs FDC do not derive from MRC (29). As TRC are known to transcribe Il7, the absence of YFP in these http://www.jimmunol.org/ cells was unexpected and is most likely due to absence of reg- ulatory elements in the BAC. Although this renders the Il7-YFP mice less suitable for lineage-tracing TRC, they clearly show that the fetal YFP+ stromal organizers did not give rise to adult TRC. To identify the YFP+ cells on a functional level, we pu- by guest on September 28, 2021

FIGURE 3. LTo and ILC in human fetal spleen. (A) Comparison of transcript levels for LTBR, CXCL13, CCL21,andCCL19 in CD452 CD312 MAdCAM-12 and MAdCAM-1+ stromal cells as well as total CD45+ cells from fetal human spleens (n . 3; age 14–22 wk). (B)Lo- calization of Rorgt+CD32 ILC3 in fetal spleens (arrowheads indicate Rorgt+ cells) (original magnification 3100/3250) (n = 3; age 14–22 wk). (C) Phenotype of fetal splenic lineage2CD117+CD127+Rorgt+ ILC3 compared with fetal splenic CD56+CD32 NK cells (n = 3; age 14–22 wk). ***p = 0.0002. progressing gestation. These data show that, in Il7-cre+ Rosa26eYFP+ mice, stromal cells in the developing LNs are marked by YFP. To determine the nature of the stromal cells expressing YFP, mesenteric LNs were isolated from Il7-cre+ Rosa26eYFP+ mice at E16.5 and analyzed by flow cytometry for YFP expression in CD452CD312 stromal cells (Fig. 5B). YFP was virtually undetectable in stromal cells lacking expression of FIGURE 4. Stromal MRC in adult human LNs. Human noninflamed A ICAM-1 and VCAM-1 or in LTo cells expressing these adhesion adult LNs were analyzed for presence of MRC. ( ) Stromal cells expressing VCAM-1 and MAdCAM-1 in adult human LNs. (B) Stromal molecules at intermediate levels. In contrast, approximately half high cells expressing RANKL in adult human LNs (original magnification of the ICAM-1/VCAM-1(I/V) stromal LTo cells were marked 3100/3250). (C) CXCL13 expression by stromal cells at the follicle by YFP, indicating that during fetal LN development a subset of boundary (original magnification 3100/3250). (D) Roryt+ cells in the stromal LTo cells transcribes sufficiently high levels of Il7, and interfollicular areas of adult human LNs colocalize with RANKL+ stro- therefore Cre, to recombine the Rosa locus, allowing for lineage mal cells (arrowheads indicate Rorgt+ cells; original magnification 3100) tracing of this population. (n . 4). 4262 STROMAL NICHE FOR LYMPH NODE ILC3

Discussion In this study, we provide evidence that adult MRC derive from a subset of fetal ICAM-1highVCAM-1high LTo cells, that both fetal LTo and adult MRC are conserved from mouse to human, and that the LTo-MRC lineage colocalizes with LN-resident Rorgt+ ILC3 throughout life. LN development and function are critically dependent on functionally distinct stromal cell networks (1, 43). In contrast to the well-defined functions of TRC and FDC, the function of MRC is largely unknown. MRC were identified based on their resem- blance to stromal organizer cells that interact with LTi cells in fetal LN anlagen, and it was proposed that these cells might have organizing functions in adult LNs (26, 30). We have previously described the presence of fetal LTi cells in human developing LNs and the presence of ILC3 in adult human LNs and spleen (15, 33). In this study, we present data showing first that fetal human LNs also contain stromal LTo cells that share phenotypic and func- tional characteristics with their mouse counterparts, and second that adult human LNs contain distinct stromal MRC. In both fetal Downloaded from and adult LNs, Rorgt+ ILC3 were found in close association with LTo/MRC. In developing human spleens, we also identified a stromal cell population with possible organizing potential. MAdCAM-1+ stromal cells appear in the perivascular lymphocyte clusters of the human spleen during second trimester and show specific transcription of the T zone chemokines CCL19 and http://www.jimmunol.org/ CCL21. In line with mouse data, these splenic organizers do not exclusively colocalize with Rorgt+ ILC, suggesting that the ILC3- independent organization of the murine spleen might be conserved in humans. Finally, we sought to gain insight into a possible precursor–progeny relationship between fetal LTo and adult MRC. If so, this would establish a mesenchymal cell lineage providing a lifelong stromal niche for ILC in LNs. Using Il7 lineage trace

mice (31), we were able to perform lineage tracing of a population by guest on September 28, 2021 of fetal stromal organizer cells. Our analysis revealed that IL7- YFP is selectively induced in a subset of fetal LTo cells that coexpress high levels of ICAM-1 and VCAM-1. As both these adhesion molecules are downstream of the LTbR, it is tempting to speculate that commitment to the MRC lineage is restricted to

stromal cells that were activated by LTa1b2-expressing ILC3. In adult LNs of IL-7 lineage trace mice, a subset of MRC is YFP positive, whereas both FDC and TRC are not marked by the IL7 FIGURE 5. Lineage tracing of fetal LTo cells. (A) Condensation of lineage tracer. This suggests that at least part of the MRC found in YFP+ mesenchyme in the jugular lymph sac (jls) region of embryonic IL7- adult LNs are direct descendants of a population of fetal stromal + + cre Rosa26eYFP mice at the indicated embryonic age in days (n = 3). organizers. In contrast, steady-state TRC and FDC are derived (B) Flow cytometric analysis of CD452CD312 stromal cells from + + from IL-7–negative mesenchymal precursors. Such precursor cells E16.5 mesenteric LNs from IL7-cre Rosa26eYFP mice (n = 2). (C)No 2 high high + are either the YFP ICAM-1 VCAM-1 cells that we ob- coexpression of VCAM-1 and Lyve-1 in LNs from adult IL7-cre 2 Rosa26eYFP+ mice. (D) LNs from adult IL7-cre+ Rosa26eYFP+ mice served in the developing LNs, or additional distinct YFP stromal show coexpression of YFP and Lyve-1 and of YFP and VCAM-1 or gp38. populations. This again is in line with recent data describing the CD35+ FDC do not express YFP (n = 5). (E) Representative flow cytometry development of MRC into FDC after immune stimulation, but not plot of CD452CD312gp38+ LNs from adult IL7-cre+ Rosa26eYFP+ mice at steady state (29). labeled with MAdCAM-1. (F) Transcript analysis of sorted CD452CD312 The evidence for a precursor–progeny relationship between 2 gp38+YFP+ and YFP stromal cells and total CD45+ cells from adult LNs stromal organizers and MRC provided in this work suggests that + + of IL7-cre Rosa26eYFP mice (n = 2–4). these cells could also be functionally related. In line with the function of fetal organizers, one can envision a role for MRC rified YFP-expressing CD452CD312 stromal cells and CD452 either in maintaining lymphoid tissue organization or in restoring CD312 stromal TRC/FDC from adult LNs of IL7-cre+ lymphoid tissue architecture after insult. Additional strategies Rosa26eYFP mice for transcript analysis (Fig. 5F). The YFP+ aimed at the targeted ablation of MRC are needed to address these cells contained transcripts for Tnfsf11 (encoding Rankl), Cxcl13, issues. Ccl21, and Ccl19, positively identifying them as MRCs The findings presented in this study raise the possibility of the (Fig. 5D). Total LN CD45+ cells are shown as negative control. existence of a specialized innate niche in otherwise primarily Collectively, these data indicate that part of the I/Vhigh stromal adaptive secondary lymphoid tissues. The physiological function LTo cells found early in LN development can mature into of ILC3 in lymphoid tissues is still unclear, and an integrated view a subset of MRCs in the adult LNs. of the cross communication between specialized stromal cells and The Journal of Immunology 4263

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