Development of follicular dendritic cells in lymph nodes depends on retinoic acid mediated signaling

Specialized stromal cells occupy and help define B- and T cell domains, which is crucial for proper functioning of our immune system. Signaling through lymphotoxin and TNF-receptors is crucial for development of different stromal subsets which are thought to arise from a common precursor. However, mechanisms that control the selective generation of the different stromal phenotypes are not known. Here we show that in mice, retinoic acid mediated signaling is important for the differentiation of precursors towards the Cxcl13pos follicular dendritic cell (FDC) lineage, while blocking lymphotoxin mediated Ccl19pos fibroblastic reticular cell (FRC) lineage differentiation. Consequently, we see at day of birth Cxcl13posCcl19neg/low and Cxcl13neg/lowCcl19pos cells within neonatal lymph nodes. Furthermore, ablation of retinoic acid receptor signaling in stromal precursors early after birth reduces Cxcl13 expression, while in addition, complete blockade of retinoic acid signaling prevents formation of FDC networks in lymph nodes.


Introduction
Lymph nodes are strategically located to enable quick and effective interactions between antigen presenting cells and lymphocytes leading to antigen specific immune responses.
The stromal cell compartment can be subdivided into various endothelial and mesenchymal subsets (Mueller & Germain, 2009). Until recently, the major different mesenchymal stromal subsets included fibroblastic reticular cells (FRCs), marginal reticular cells (MRCs) and follicular dendritic cells (FDCs) (Chang & Turley, 2015;Koning & Mebius, 2012). Additional mesenchymal stromal subtypes were identified using single cell sequencing (Rodda et al., 2018) and have shed light on the transcriptional profile of lymph node stromal cells during homeostasis and upon immune activation. Despite this increased knowledge, it remains unknown which signaling events drive the differentiation from precursor cells into these various stromal clusters.
From previous studies we know that precursors for stromal cells, including FDCs, are already present in mesenteric lymph nodes of newborn mice (Cupedo, Jansen, Kraal, & Mebius, 2004). Recently, we have shown that nestin pos precursors give rise to the various endothelial and mesenchymal derived lymph node stromal cells (Koning et al., 2016).
More specific studies on the origin of FDCs provided evidence for the presence of local mesenchymal FDC-precursors both in spleen and lymph nodes (Castagnaro et al., 2013;Jarjour et al., 2014;Krautler et al., 2012). The presence of B cells is essential for the development of FDCs and has been shown to rely on lymphotoxin and TNF receptor signaling (Endres et al., 1999;Fu, Huang, Wang, & Chaplin, 1998).
The presence of perivascular precursors for FDCs in the spleen appeared to be independent of lymphotoxin signaling (Krautler et al., 2012) suggesting that a sequence of signaling events is needed for their differentiation and final maturation to become FDCs.
Here we provide evidence that post-natal retinoic acid signaling critically controls development of FDCs. We identify endothelial cells as additional cellular source for the production of retinoic acid thereby highlighting the importance of endothelial and non-endothelial stromal cells for proper development of lymph node niches. Cell specific inhibition of retinoic acid signaling in nestin pos precursor cells reduced Cxcl13 expression but not FDC development. However, upon pharmacological inhibition of retinoic acid signaling we could show that the appearance of FDCs critically depends on retinoic acid signaling during neonatal lymph node development.

Differential stimulation via receptors for retinoic acid and lymphotoxin induces distinct gene expression profiles in mesenchymal cells in vitro.
Lymph node development during embryogenesis involves direct crosstalk between lymphoid tissue inducer (LTi) cells and different types of stromal organizer cells (Bovay et al., 2018;Mebius, 2003;Onder et al., 2017) and requires signaling mediated by retinoic acid as well as lymphotoxin (Honda et al., 2001;S. A. van de Pavert et al., 2009). Mesenchymal cells at the presumptive lymph node site are initially exposed to retinoic acid after which LTα1β2 expressing LTi cells will trigger LTβR-mediated signaling in these cells, allowing their maturation towards stromal organizer cells (S. A. van de Pavert et al., 2009). To determine whether the activity of these signaling pathways either in sequence or on their own could contribute to the different fates of stromal cell subsets present in adult lymph nodes we set up in vitro cultures of early passage E13.5 embryonic mesenchymal cells enriched for mesenchymal stem cells (E13.5 MSC) as confirmed by their tri-lineage differentiation capacity ( fig. 1A).
Transcript analysis upon stimulation with retinoic acid alone showed strong upregulation of the β isoform of the receptor (RARβ), which is specifically involved in the induction of Cxcl13 during the initiation phase of lymph node development (S. A. van de Pavert et al., 2009). Indeed, stimulation with retinoic acid induced Cxcl13 expression in mesenchymal precursors while LTβR stimulation alone did not lead to significant upregulation of Cxcl13, although low expression levels could be detected, as shown before (Dejardin et al., 2002) LTβR stimulation (Crowe et al., 1994) initially leads to the activation of the classical Nf-κB pathway (Dejardin et al., 2002;Siebenlist, Franzoso, & Brown, 1994) while upon prolonged triggering, also the alternative Nf-κB pathway becomes activated resulting in the upregulation of Ccl19, Ccl21 and Il-7 via nuclear localization of relB:p52 dimers (Dejardin et al., 2002;Vondenhoff et al., 2009). As expected, LTβR stimulation of mesenchymal precursors resulted in a significant upregulation of Ccl19, Ccl21 and Il-7 transcripts, which are all expressed by FRC in the T cell zone  1D and E). We also observed no difference in the activation of the non-canonical Nf-kB pathway, since there was similar nuclear relB translocation, while the DNA binding capacity of nuclear relB and p52 was also not affected (suppl. fig. 1). We observed that other LTβR mediated molecules such as VEGF-C and MAdCAM-1 (Vondenhoff et al., 2009) were not affected upon retinoic acid pre-incubation ( fig. 1F).
Owing to these observations, we hypothesized that retinoic acid stimulation serves as a fate determining signal that allows the differentiation of stromal precursor cells towards a follicular dendritic cell signature by preventing their differentiation into T cell zone FRCs upon subsequent LTβR triggering.

Supplementary figure 1. Related to Figure 1. Retinoic Acid signaling prevents upregulation of FRC related genes
(A) Immunofluorescence staining for RelB (in red) and nuclei (in blue) in cytospins of mesenchymal precursors stimulated with or without retinoic acid (6hrs) followed by agonistic anti-LTβR mAb (6hrs). B) TransAM analysis of nuclear p52 and relB after stimulation of mesenchymal precursors with retinoic acid (6hrs) followed by agonistic anti-LTβR mAb (3hrs) or retinoic acid (6hrs) alone or anti-LTβR mAb (3hrs) alone.

Localization of pre-FDCs within post-natal developing lymph nodes.
Retinoic acid and LTβR mediated signaling takes place before birth during lymph node development. Therefore stromal cells that have either received initially only retinoic acid mediated signaling, versus stromal cells that received their first signals via LTβR are potentially already present within lymph nodes at day of birth. To address this we monitored mRNA expression of Cxcl13, induced by retinoic acid mediated signaling, as well as Ccl19, expressed in the majority of stromal precursors (Chai et al., 2013) and induced upon LTβR signaling. Upon analysis of wk0 lymph nodes, we observed expression of Ccl19 mRNA as well as Cxcl13 mRNA within cells throughout the lymph node. Although the majority of cells expressed both molecules, cells with high expression of Cxcl13 mRNA appeared to have low amounts of Ccl19 mRNA, and vice versa ( fig. 2A).
We additionally monitored the expression of Mfge8 mRNA, expressed by FDC precursors within the spleen, and Trance (Tnfsf11), expressed by lymphoid tissue organizer (LTO) cells as well as MRCs (Jarjour et al., 2014;Katakai et al., 2004). We From this data we can conclude that the cells that received retinoic acid mediated signaling as initial trigger during lymph node development and therefore expressed

Multiple cellular sources of retinoic acid within lymph nodes.
To determine whether retinoic acid mediated signaling still occurs after birth and thereby could further contribute to the developing FDC network, we addressed whether cellular sources of retinoic acid were present within neonatal lymph node.
Retinoic acid synthesis depends on the 2-step conversion of vitamin A into retinaldehyde and retinoic acid respectively. The final and irreversible step of this conversion involves members of the aldehyde dehydrogenase enzyme family; Aldh1a1, Aldh1a2, Aldh1a3 and expression of these enzymes identifies cells that produce retinoic acid (Kedishvili, 2016). It is known that stromal cells as well as dendritic cells in adult lymph nodes express Aldh1 enzymes Molenaar et al., 2011), but the expression during early post-natal lymph node development has not been studied before. Therefore, we sorted the three main stromal subsets, FRCs, BECs and LECs as well as dendritic cells (CD11c high MHCII high ) from pLN of week 1 and week 2 old mice, to examine the expression of Aldh1 enzymes. Dendritic cells are known to express high levels of Aldh1a2 (Zhang et al., 2016) and transcriptomic analysis indeed showed that dendritic cells expressed the highest level of Aldh1a2 at both timepoints in pLN but lacked both Aldh1a1 and Aldh1a3 expression. Sorted FRCs expressed both Aldh1a1 and Aldh1a3 but not Aldh1a2. The expression of Aldh1a1 was higher in week 2 sorted FRCs. To our surprise, we found that BECs also expressed Aldh1a1, while LECs express Aldh1a3 ( fig. 3A and supplementary fig 3).
Since lymph node BECs are not recognized as Aldh1 expressing cells, we wanted to verify the expression by protein levels. We therefore performed immunofluorescence Together, these data indicate that during early post-natal lymph node development, Aldh1 enzymes are expressed by multiple cell types that all could serve as cellular sources for retinoic acid that potentially mediate further FDC development.

Postnatal abrogation of retinoic acid signaling in nestin-precursors does not prevent FDC formation.
The multicellular distribution of retinoic acid producing enzymes imposes an impossibility to specifically assign the importance of these cellular sources for postnatal FDC development since the functional consequences of cell specific Aldh1 deletion in one cell subset, could be rescued by the expression of another Aldh1 enzyme in the same cell type (or by the expression of the same and other enzymes in another subset. However, cell specific ablation of retinoic acid receptor signaling in precursor cells could be used to study the importance of retinoic acid signaling for further postnatal FDC development. Although the precursor for FDCs within neonatal lymph nodes have not been identified so far, we have shown in the past that nestin expressing cells can give rise to FDCs (Koning et al., 2016). We reasoned that ablation of retinoic acid receptor signaling in these cells shortly after birth would impact further FDC development. We performed cell specific ablation of retinoic acid receptor signaling in nestin expressing cells by crossing Nes-Cre ERT2 mice with RAR-DN mice in order to abrogate retinoic acid signaling specifically in nestin expressing cells upon tamoxifen treatment. Animals were treated from day 5 after birth for 5 consecutive days and lymph nodes were isolated when the animals were 2 or 3 weeks old.
Upon transcriptomic analysis of whole lymph nodes, we observed no difference in the mRNA expression of RARβ (data not shown) most likely because only a small subset of cells was targeted. mRNA expression of Cxcl13 however was reduced in peripheral lymph nodes of wk2 old animals ( fig. 4A). This was mainly attributed to a reduction of Cxcl13 expression in axillary and brachial lymph nodes since similar levels of Cxcl13 were found in iLN of mice that were Nes-Cre ERT2 negative.

Early postnatal blockade of RAR signaling prevents FDC development.
In order to block all retinoic acid signaling and address whether retinoic acid mediated signaling is involved in FDC development postnatally, we performed pharmacological inhibition of retinoic acid receptor signaling using oral application of BMS493 (Mizee et al., 2013;S.A. van de Pavert et al., 2014). Hereto, we inhibited retinoic acid receptor signaling from postnatal day 4 onwards for 7 or 10 consecutive days.
As expected, transcript levels of RAR-β were down-regulated in peripheral as well as mesenteric lymph nodes upon BMS treatment at both timepoints indicating effective blockade of retinoic acid signaling ( fig. 5A, suppl fig 5A). As a consequence, mRNA levels of Cxcl13 were also reduced and we hardly observed Cxcl13 protein expression in B cell areas and subcapsular sinuses of lymph nodes ( fig. 5B and C). The absence of Cxcl13 expression and FDC networks severely affected Blymphocyte organization. In both peripheral and mesenteric lymph nodes of BMS treated mice, the number of B cell follicles that had formed at day 10 was lower compared to controls (suppl. fig. 5 C & D).
Within the spleen, transcripts for RAR-β were also reduced as a result of BMS493 treatment. At day 7 this resulted in a reduced transcript level of Cxcl13 while 10 days after treatment, Cxcl13 transcript levels were not different from control treated mice (suppl. fig. 5E). Immunofluorescence analysis revealed development of FDC networks in spleens from BMS treated mice (suppl fig. 5F), suggesting different signaling requirements for FDC development in spleen compared to lymph nodes.
To reduce, and not completely block, retinoic acid mediated signaling, we provided suboptimal doses of BMS493 starting at day 4 postnatal for 14 consecutive days.
This treatment resulted in lower RAR-β and Cxcl13 transcript levels ( fig. 5F) and we could detect low levels of Cxcl13 protein in the B cell follicles as well as FDC networks although they appeared smaller (data not shown). Whole lymph node imaging using light sheet microscopy, revealed that the total lymph node volume as well as the B cell follicle volume in suboptimal BMS493 treated peripheral lymph nodes were lower compared to control treated lymph nodes leading to a lower percentage the of LN volume that is occupied by B cell follicles (ratio of B cell area/total LN area) ( fig. 5F and suppl fig. 5H-I). We observed similar results when we blocked FDC development for 7 days and left the mice untreated for 21 days ( fig. 5G and suppl. fig. 5J).
In summary, these data show the requirement of retinoic acid receptor signaling for postnatal development of FDCs within lymph nodes.

Figure 5 Inhibiting retinoic acid receptor signaling prevents CXCL13 expression and development of FDCs in lymph nodes
A) Heatmap showing mRNA expression levels of listed genes in peripheral lymph nodes upon treatment for 7 and 10 days with DMSO or BMS493, starting at day 4 after birth. B-C) Immunofluorescence analysis of CXCL13 protein expression (in red) in lymph nodes of DMSO (control) and BMS treated mice at day 7 (B) and day 10 (C) after start of treatment. D-E) Immunofluoresence analysis (D) and quantification (E) of FDCs (8C12 in blue) in lymph nodes of DMSO (control) and BMS treated animals at day 10 after treatment. F-G) mRNA expression levels of RARβ and CXCL13 and volume ratio of B cell follicle volume over total lymph node volume in peripheral lymph nodes of mice that were treated for 14 days with suboptimal doses of BMS493 vs DMSO (F) or for 7 days with BMS493 vs DMSO and left untreated for 21 days (G) starting at day 4 after birth. The data represent mean ± SEM; n = 3 or more, *, p < 0.05; **, p < 0.01; ***, p < 0.001; unpaired student's t test. Squares and triangles in figures represent data of individual mice. Previous research has shown that lymphotoxin as well as TNFα mediated signaling is a prerequisite for the presence and maintenance of mature FDCs (Endres et al., 1999;Fu et al., 1998). Lymphotoxin signaling is not exclusively required for the differentiation and subsequent maintenance of FDCs as also FRCs and MRCs depend on signaling via this pathway (Roozendaal & Mebius, 2010). This suggests that additional signaling events are needed for precursors to develop into the various stromal subsets. Since retinoic acid plays a crucial role in the initiation of lymph node development before lymphotoxin mediated signaling takes place we hypothesized that retinoic acid signaling could be important for the early differentiation of mesenchymal precursors towards different stromal subsets as well.

A-C) Immunofluorescence staining for B cells (green) and T cells (red) in
Indeed, our results from in vitro stimulated mesenchymal-derived precursors showed that retinoic acid prevented the expression of transcripts that are normally induced upon LTβR signaling through activation of the non-canonical Nf-κB pathway, and that are associated with FRCs (Dejardin et al., 2002). It has been shown before that administration of retinoic acid leads to reduced Nf-κB activity in vivo without affecting p65 DNA binding capacity (Austenaa et al., 2004). We here show that retinoic acid selectively inhibits transcripts associated with T cell stroma, whose expression depends on the activation of the non-canonical Nf-κB pathway, without affecting relB or p52 DNA binding capacity. Even more so, Cxcl13 expression in mesenchymal precursors was unaltered upon sequential stimulation with retinoic acid and LTβR, suggesting a role for retinoic acid in favoring differentiation towards B cell stroma.
By blocking retinoic acid signaling in vivo, we were able to prevent the development of FDCs in lymph nodes, which has been shown to depend on lymphotoxin and TNFα mediated signaling. Together, our data points to a model in which mesenchymal precursor cells for FDCs are sequentially triggered, first by retinoic acid and subsequently by lymphotoxin and TNFα to finally differentiate towards FDCs.
This fate determining process potentially takes place before birth, since Within lymph nodes, expression of Aldh1 enzymes has been reported in gut derived DCs, lymph node stromal cells in mLN and epithelial cells in Peyer's patches (Molenaar et al., 2011;Suzuki et al., 2010;Zhang et al., 2016). In addition, we identified endothelial cells, both high endothelial venules (HEVs) and lymphatic endothelial cells (LECs) as cells that transiently express Aldh1 enzymes most prominently during a period in which lymph nodes expand and stromal subsets are being formed. The observed decrease in Aldh1 expression after this period of expansion suggests that the differentiation of mesenchymal precursors towards the B cell stromal lineage occurs within a postnatal timeframe, which fades away in the absence of inflammatory stimulus once FDC networks are established. Whether endothelial cells re-express Aldh1 enzymes upon activation when FDC networks remodel and new FDCs are needed (Jarjour et al., 2014) has not been studied.
It is unknown how Aldh1 expression in neonatal lymph nodes is induced but (hematopoietic) cell subsets like the neo-migratory DCs (Zhang et al., 2016) could be involved. These cells express Aldh1 enzymes themselves and induce maturation of HEVs as their arrival in gut as well as skin draining lymph nodes leads to increased amounts of PNAd expression on HEVs. This migration depends on commensal fungi and absence of microbiota clearly affects their presence in peripheral lymph nodes (Zhang et al., 2016). Whether neo-migratory DCs are involved in inducing Aldh1 expression on HEVs and LECs and whether retinoic acid produced by these DCs are in fact instrumental for this effect is unknown. However, the blockade of retinoic acid signaling that prevented FDC development in the experiments presented here occurred before the entry of neo-migratory DCs in skin draining lymph nodes, which is around 2 weeks after birth, suggesting that they are not involved (Zhang et al., 2016).
While the first description of FDCs has been a long time ago already (Mitchell & Abbot, 1965), the discovery of their precursors in lymphoid organs has remained elusive until recently. Whereas in the spleen specific precursors for FDCs have been documented in several reports (Castagnaro et al., 2013;Krautler et al., 2012), progenitor-progeny relationships for lymph nodes are not so clear yet. Lineage tracing studies showed MRCs at least in part to be precursors for FDCs (Jarjour et al., 2014). Other models have shown that upon neonatal lineage tracing of lymphoid tissue organizer cells, a subset of these cells give rise to MRCs, but not to FDCs (Hoorweg et al., 2015). In addition, as mentioned by Jarjour et al. other precursors for FDCs may exist (Jarjour et al., 2014). In the spleen, precursors for FDCs can be identified based on the expression of Mfge8 and Cxcl13 and these cells are perivascular located. Here we show that also in neonatal lymph nodes, Mfge8 pos Cxcl13 pos cells can already be identified at day of birth throughout the lymph node and over time, these cells start to localize in B cell follicles as well as in the subcapsular sinus. In adult lymph nodes, the majority of these cells can be detected by the expression of CD35, a marker for FDCs indicating that lymph node FDC precursors can be identified by similar markers as well. However, in adult lymph nodes, we did not detect Mfge8 pos CXCL13 pos expressing cells surrounding blood vessels. This could mean that either precursors for FDCs are not located perivascular or that they do not express these markers upon homeostasis.
Our results allow for a lineage tree model in which precursors upon encounter with retinoic acid and subsequent stimulation with lymphotoxin will become a functional FDC, while they differentiate towards FRC in the absence of retinoic acid mediated signaling.

BMS treatment
For in vivo blockade of retinoic acid signaling, mice were treated with the pan-RAR antagonist BMS493 (5mg/kg; Tocris Bioscience) or vehicle (DMSO) in corn oil as described before (S.A. van de Pavert et al., 2014). Mice were treated for 7 or 10 days via oral gavage twice daily with 10-12 hrs intervals starting at day 4 after birth. After 7 or 10 days, mice were sacrificed for analysis. For temporal blockade of retinoic acid signaling mice were treated the same way for 7 days and left untreated for another 21 days after which they were sacrificed for analysis. For suboptimal blockade of retinoic acid signaling, mice received 2,5mg/kg BMS493 or vehicle (DMSO) for 14 days. Per experimental group (BMS493 vs vehicle) per time point, all animals within 1 litter either receive BMS493 or DMSO, to prevent contamination of of the treatment within one litter. Litters were randomly allocated in experimental groups.

Tamoxifen treatment of Nes-cre ERT2 x RAR DN403 mice
Tamoxifen (Sigma) was dissolved in ethanol first and subsequently preheated corn oil (42 0 C, Sigma) was added and strongly mixed for 1 hr in the darkness. Final concentration was 10mg/kg. For postnatal induction, tamoxifen solution was applied i.p. to the mothers (100 µl/ day) starting at p4 (Schultheiss et al., 2013). Animals were sacrificed at day 14 (p14) or day 21 (p21).

Single cell suspensions
E13.5 mesenchymal cells were prepared as described (S. A. van de Pavert et al., 2009). In short, the head, extremities and organs were removed from E13.5 embryos and the remainings were enzymatically digested with Blendzyme 2 (0,5 mg/ml; Single cell suspensions of lymph nodes were generated as described (Fletcher et al., 2011). In short, upon isolation, lymph nodes were pierced with a 25g needle and place in ice-cold RPMI1640 (Invitrogen). LNs were subsequently digested using an enzyme mixture of 0.2 mg/ml collagenase P (Roche), 0.8 mg/ml Dispase II (Roche) and 0.1 mg/ml DNase I (Roche) in RPMI medium (Invitrogen) without serum. With 5 min intervals, contents were gently mixed. After 15 min, suspensions were gently resuspended, supernatant collected in ice-cold PBS (2% FCS and 5mM EDTA) and centrifuged (5 min 300g, 4 0 C ). Fresh enzyme mix was added to the tubes and digestion was repeated as described. After 15 min, supernatant was collected in the same tube with ice-cold PBS and fresh enzyme mix was added to the remaining fragments. Every 5 min, suspensions were mixed vigorously till no fragments remained. Supernatant was collected in the same collection tube and centrifuged (5min, 300g, 4 0 C).

In vitro stimulation and mRNA transcript analysis
To determine transcript levels, 1x10 5 mesenchymal cells were seeded in 24-wells plate and allowed to adhere for 2-4 hrs in 1:1 (vol/vol) mesencult proliferation medium and DMEM-F12 medium supplemented with 10 % FCS and 2 % antibiotics with glutamine. After 2-4 hrs, medium was replaced with DMEM-F12, 5% FCS with 2% antibiotics and glutamine. The next day, stimulations with retinoic acid or agonistic anti-LTβR mAb were performed in duplo in DMEM-F12, 2% FCS with 2% antibiotics and glutamine. For sequential stimulation experiments, cells were cultured for 6 hrs either in medium alone or in the presence of retinoic acid (100 nM, Fluka, Sigma-Aldrich, Zwijndrecht, The Netherlands, dissolved in ethanol), after which the cells were washed and stimulated with or without agonistic anti-LTβR mAb for another 6 hrs (2μg/ml, clone 4H8-WH2, produced in Carl Ware's laboratory) in duplo.
Cells were subsequently lysed, after which mRNA was isolated from total RNA using the mRNA capture kit (Roche) and cDNA was synthesized using the Reverse transcriptase kit (Promega Benelux, Leiden, The Netherlands) according to manufacturer's protocol.
To determine transcript levels within whole mount lymph nodes, lymph nodes were harvested, lysed and homogenized in TRIzol Reagent (Life Technologies). RNA was isolated by precipitation with isopropanol according to the manufacturer's protocol and cDNA was synthesized from total RNA using RevertAid First Strand cDNA Synthesis Kit (Fermentas Life Sciences) according to the manufacturer's protocol.
Quantitative RT-PCR was performed in duplo on an ABI Prism 7900HT Sequence Detection System (PE Applied Biosystems) or StepOnePlus Real-Time PCR System (ThermoFisher Scientific). Total volume of the reaction mixture was 10 µl, containing cDNA, 300nM of each primer and SYBR Green Mastermix (PE Applied Biosystems).
From a set of 8 housekeeping genes, the two most stable were selected (Cyclo and HPRT). The comparative Ct method (∆Ct) was used to indicate relative changes in mRNA levels between samples. Relative mRNA levels of unstimulated cells or control treated tissues were set at 1,0.

Flow cytometric analysis and cell sorting
To determine cell surface expression, 1x10 5 cells were cultured as indicated. Cells were stimulated with retinoic acid (100 nM

RelB translocation and TransAM-analysis
Nuclear translocation of relB was determined on cytospins of cells stimulated for 6 hrs with retinoic acid followed by agonistic anti-LTβR mAb stimulation for 6 hrs. Cells were fixed with 4% paraformaldehyde for 5 min and subsequently permeabilized with 0.2% Triton-X and stained with anti-relB (Cell Signaling Technology, Danvers, USA) followed by goat-anti-rabbit Alexa 546 (Invitrogen). Cells were embedded in Vinol mounting media (Air Products, Allentown, USA) supplemented with DAPI (Invitrogen) to visualize nuclei. Analysis was performed on a Leica DM6000 fluorescence microscope (Leica Microsystems) equipped with LAS AF software.
For TransAM analysis, cells were stimulated for 6hrs with retinoic acid (100 nM), followed by 3hrs stimulation with the agonistic anti-LTβR mAb, after which nuclear extracts were prepared with the Nucbuster protein extraction kit (Thermo Scientific, Rockford, USA). DNA binding capacity of the Nf-κB subunits relB and p52 were determined with the Nf-κB TransAM family kit (Active Motif, Rixensart, Belgium) according to manufacturer's protocol.

Fluorescent in situ hybridization
Lymph nodes were fixed for 2 hrs in 4% paraformaldehyde at RT. Samples were cryoprotected and subsequently embedded in OCT compound (Sakura Finetek Europe) and stored at -80°C until sectioning all under RNAse free conditions. Cryostat sections (7µm) were collected on superfrost plus adhesive slides (VWR, the Netherlands) and multiplex in situ hybridizations were performed according to manufacturer's protocol (Molecular Instruments and described in (Choi et al., 2018)).
In short, slides were washed with 5x SSCT, pre hybridized in hybridization buffer at 37 0 C in a humidified chamber. Probes were added at 0.4 pmol/100 µl in probe hybridization buffer at 37 0 C and hybridized overnight in a humidified chamber, while covered with parafilm. Next day, slides were washed in decreasing concentrations of probe wash buffer in 5x SSCT (100%, 75%, 50%, 25% respectively) and washed in 5x SSCT at room temperature. Next, samples were pre-incubated with amplification buffer for 30 min. Hairpin solutions were prepared with 6 pmol snap-cooled hairpins/100 µl and added to the slides. Samples were incubated overnight at room temperature in a humidified chamber. Next day, excess hairpins were removed by washing with 5x SSCT. In case of additional antibody staining, antibodies and/or nuclear labeling were added during these washing steps. Finally, slides were embedded in mounting medium and stored at 4 0 C till analysis.

Immunofluorescence
E18.5 embryos were fixed in 0.4% formaldehyde overnight at 4 o C, lymph nodes (postnatal day 0 -6 weeks of age) and spleens from C57BL/6 J and Nes-CRE ERT2 x DN RAR 403 mice were fixed in 4% formaldehyde for 10 min. Samples were cryoprotected and subsequently embedded in OCT compound (Sakura Finetek Europe) and stored at -80°C until sectioning. Cryostat sections (7µm) collected on gelatin coated glasses were fixed in ice-cold acetone for 10 minutes and blocked with 10% NMS in PBS prior to antibody staining. Immunofluorescence staining was performed in PBS, supplemented with 0.1% (wt/vol) Bovine Serum Albumin (BSA).

Sections were embedded in Vinol + DAPI and analyzed on a Leica SP8Confocal
Laser Scanning Microscope or a Leica DM6000 (both Leica Microsystems Nederland b.v., Rijswijk, The Netherlands).
For volumetric analysis of B cell follicles and lymph node size, individual B cell follicles as well as the whole lymph node were masked in Imaris Software and volumes were extracted using Imaris Vantage module. The ratio of B cell follicles within total lymph nodes was calculated as the total volume of B cell follicles within the lymph node volume.
The following anti-mouse antibodies were used; unlabeled anti-MECA325, anti- Unconjugated antibodies were detected with species specific secondary reagents.
Biotinylated anti-CXCL13 was visualized with signal amplification using a TSA ™ Kit with HRP-streptavidin and Alexa Fluor 546 tyramide (Invitrogen).

Statistical Analysis
Statistical analysis was performed as described in the figure legends. The letter 'n' in the figure legends, refers to the number of individual samples, or independent in vitro experiments. For all BMS493 animal experiments, sample size was estimated using a power analysis to verify that the sample size gave a value of > 0.9 if P was > 0.05.
For all other experiments, no statistical method was used to predetermine sample size, experiment blinding upon analysis was not used.