Lymph node stromal cell subsets — Emerging specialists for tailored tissue-specific immune responses

The effective priming of adaptive immune responses depends on the precise dispatching of lymphocytes and antigens into and within lymph nodes (LNs), which are strategically dispersed throughout the body. Over the past decade, a growing body of evidence has advanced our understanding of lymph node stromal cells (LNSCs) from viewing them as mere accessory cells to seeing them as critical cellular players for the modulation of adaptive immune responses. In this review, we summarize current advances on the pivotal roles that LNSCs play in orchestrating adaptive immune responses during homeostasis and infection, and highlight the imprinting of location-specific information by micro-environmental cues into LNSCs, thereby tailoring tissue-specific immune responses.


Introduction
Lymph nodes (LNs) as part of the secondary lymphoid organs (SLOs) are dispersed throughout the body in close proximity to vascular and lymphatic branching points.Their primary function is to act as hubs of constant immune surveillance in which lymph-and blood-borne constituents are continuously filtered and checked by cells of the adaptive immune system.In humans, LN position and number show slight interindividual variations, while mice possess 22 L Ns that develop exactly at the same anatomical sites (Hoorweg and Cupedo, 2008;Van den Broeck et al., 2006).Each LN is responsible for draining the surrounding tissue at its specific anatomic localization, therefore the influx of antigen-presenting cells migrating from the tissue to the draining LN after antigen uptake represents a unique tissue-derived cell population.In addition, the antigen-pool is often exclusive to the respective draining site, e.g.tissue-specific antigens, food-borne antigens or antigens of microbial origin.Lately, more and more studies have uncovered how much the micro-environment matters for the generation of distinct tissue-resident immune cell subsets.However, transcriptome analyses revealed similarities in circulating lymphocytes isolated from spleen and LNs, indicating that there are many common immunomodulatory functions inherent to SLOs (Miragaia et al., 2019;Zhao et al., 2020).How much impact the micro-environment has on the functional properties of LNs at distinct locations is currently subject of ongoing investigations.In parallel to the exploration of the tissue-specific shaping of immune cell niches, the micro-milieu within the LN itself has garnered interest.Lymph node stromal cells (LNSCs) are responsible for stabilizing and compartmentalizing the LN and have long been viewed as a simple scaffold facilitating immune cell migration and interaction.Recently, the startling heterogeneity of this understudied cell population Abbreviations: Aire, autoimmune regulator; Aldh1a2, aldehyde dehydrogenase 2; BAFF, B cell activating factor; BECs, blood endothelial cells; celLN, celiac lymph node; CRCs, CXCL12-expressing reticular cells; CSF1, colony stimulating factor 1; DC, dendritic cell; FDCs, follicular dendritic cells; FRCs, fibroblastic reticular cells; FSCs, fibroblastic stromal cells; GF, germ-free; HEV, high endothelial venules; IFN, interferon; IL, interleukin; kDA, kilodaltons; ILCs, innate lymphoid cells; LCMV, lymphocytic choriomeningitis virus; LECs, lymphatic endothelial cells; IFRCs, interfollicular FRCs; LNs, lymph nodes; LNSCs, lymph node stromal cells; LT, lymphtoxin; LTβR, lymphotoxinβ receptor; medRCs, medullary FRCs; MAdCAM1, mucosal addressin cell adhesion molecule 1; MHC, major histocompatibility complex; mLNs, mesenteric LNs; MRCs, marginal reticular cells; MyD88, myeloid differentiation response protein 88; PDPN, podoplanin; pLN, peripheral LN; PP, Peyer`s Patches; PTAs, peripheral tissue-restricted antigens; RA, retinoic acid; RALDH, retinal dehydrogenase; RANKL, receptor activator of NF-κB ligand; resDCs, resident DCs; SCs, stromal cells; scRNAseq, single-cell RNA sequencing; SCS, subcapsular sinus; SLOs, secondary lymphoid organs; SPF, specific pathogen-free; TBRC, T-B cell border FRCs; TNF, tumor necrosis factor; TRCs, T cell zone FRCs; Tregs, regulatory T cells.
* Corresponding author at: Department Experimental Immunology, Helmholtz Centre for Infection Research, Inhoffenstr.7, 38124 Braunschweig, Germany.E-mail address: jochen.huehn@helmholtz-hzi.de (J.Huehn). 1 Authors contributed equally.has been revealed, followed by the discovery of various immunomodulatory functions carried out by LNSCs.Moreover, LNSCs are also involved in the initiation of LN development during embryogenesis and early postnatal stages, and recent findings on this topic were excellently summarized elsewhere (Krishnamurty and Turley, 2020;Onder and Ludewig, 2018).In the present review, we aim to summarize current advances in our understanding of how LNSC subsets influence immune cells during homeostasis and infection as well as to which degree these sessile specialists are affected themselves by their LN's anatomic location and micro-environmental factors.

LN micro-architecture
A general infrastructure is shared among all murine LNs.The LN is mantled by a capsule, under which the subcapsular sinus (SCS) acts as a conduit for the afferent lymph.Beneath the SCS lies the LN cortex, which is interspersed with multiple B cell follicles.A centrally oriented paracortex disembogues into the medulla, from which the efferent lymphatics reconnect to circulation.All LNs possess a hilus, from which a main artery enters the medulla to branch of into an articulate network, forming high endothelial venules (HEVs), which intersperse the LN with a higher vascular density in the LN periphery (Jafarnejad et al., 2019;Kelch et al., 2015) (Fig. 1).
The LNSC network tightly regulates immune cell trafficking in these distinct zones.LNSCs can be broadly separated into three subsets, CD45 − CD31 − PDPN + fibroblastic reticular cells (FRCs), CD45 − CD31 + PDPN + lymphatic endothelial cells (LECs) and CD45 − CD31 + PDPN − blood endothelial cells (BECs).FRCs can be further subdivided into the major subsets of marginal reticular cells (MRCs), follicular dendritic cells (FDCs), T cell zone FRCs (TRCs) and medullary FRCs (medRCs).The capsule contains a layer of CD34 + stromal cells (SCs), whose function has not been well characterized, yet (Baumhueter et al., 1994;Rodda et al., 2018).Besides the capsule, CD34 + SCs can also be found in the adventitia of larger vessels, named "adventitial SCs" (Sitnik et al., 2016).LECs situated in the SCS facilitate lymphocyte and dendritic cell (DC) migration across the sinus-cortex interface (Jalkanen and Salmi, 2020), permit the passage of <70 kilodaltons (kDA) lymph-borne antigens into the conduit system (Gretz et al., 2000;Rantakari et al., 2015), while molecules <500 kDA can pass through LECs via transcytosis (Kahari et al., 2019).LECs are lining lymphatic vessels and are therefore also abundantly found in the medullary sinuses in the medulla (Jalkanen and Salmi, 2020).MRCs, characterized by the expression of CXCL13, mucosal addressin cell adhesion molecule 1 (MAdCAM1) and receptor activator of NF-κB ligand (RANKL), line the  FRCs whose function so far remains enigmatic.CD34 + FRCs are also found in the adventitia of larger vessels where they are termed "adventitial SCs".Inside the LN, adventitial SCs surround the vessels entering the LN, which have a defined adventitia (not depicted).2) Below the capsule, the SCS is surrounding the inner parts of the LN.LECs line the SCS and incoming antigen is filtered and distributed across the LN.LECs guide immune cells arriving with the afferent lymph via CCL21 secretion and maintain the local macrophage niche by providing CSF-1.Moreover, LECs line the lymphatics of the medullary sinuses throughout the medulla (not depicted).3) MRCs are located below the capsule in close proximity to the B cell follicles.They produce CXCL13 and RANKL and are capable of replenishing FDCs after FDC ablation during infection.4) In the cortex, CXCL13 + FDCs are residing in the B cell follicles and present antigen to B cells.5) In the light zone of germinal centers, FDCs foster B cell affinity maturation, while CXCL12-producing CRCs are inducing B cell proliferation.6) In the paracortex, TRCs guide T cells and DCs via the expression of CCL19 and CCL21.Moreover, they build a conduit system to efficiently channel lymph throughout the paracortex.7) HEVs are dispersed throughout the LN, facilitate immune cell entry by the expression of adhesion molecules and are composed of an inner layer of BECs, followed by pericytes and lastly a layer of perivascular FRCs.8) In the medulla, where the lymph is collected before LN egress, medRC are producing APRIL, BAFF and IL-6 to sustain the local plasma cell population.BEC, blood endothelial cell; CRC, CXCL12-expressing reticular cell; CSF-1, colony stimulating factor 1; DC, dendritic cell; FDC, fibroblastic dendritic cell; FRC, fibroblastic reticular cell; HEV, high endothelial venule; LEC, lymphatic endothelial cell; LN, lymph node; medRC, medullary FRC; MRC, marginal reticular cell; SC, stromal cell; SCS, subcapsular sinus; TRC, T cell zone FRC.
outer edge of the cortical area and are in close proximity to B cell follicles, where they are thought to give rise to FDCs during B cell follicle development (Jarjour et al., 2014) and drive B cell follicle formation during infection (Dubey et al., 2019).CD21/35 + CXCL13 + FDCs are located inside the B cell follicles, where they present antigens to B cells and support germinal center formation (Pikor et al., 2020;Rodda et al., 2015).In germinal centers, FDCs are positioned in the light zone, while CXCL12 + CRCs are present in the dark zone, where they conjointly foster B cell affinity maturation and proliferation (Bannard et al., 2013;Rodda et al., 2015).CCL19/21 + TRCs are situated in the interfollicular area of the cortex and throughout the paracortex, where they form a conduit system to facilitate lymph flow and foster T cell and DC interaction (Fletcher et al., 2015).In the medulla, cell density decreases and a meshwork of LECs lining the medullary sinuses collects efferent lymph, which is sampled by macrophages (Jalkanen and Salmi, 2020).Here, CXCL12 + medRCs act as a source of plasma cell survival factors, guiding their migration (Huang et al., 2018).The LNs' capillary system forms specialized HEVs, consisting of an inner layer of BECs adjacent to pericytes and an outer layer of perivascular FRCs.Leucocyte entry into the LN parenchyma is assisted by CCL21 chemotactic gradients and expression of the adhesion molecules GlyCAM-1, ICAM-1, MAdCAM1 and CD34 on BECs (Girard et al., 2012).The transcriptome of HEVs is distinct from the surrounding capillary endothelium, which underlines their high degree of specialization.Moreover, it was shown that the HEV transcriptome is highly similar across the mesenteric LNs (mLNs), peripheral LNs (pLN) and Peyer`s Patches (PP), indicating that HEV function is not influenced by the anatomic location of the LNs (Lee et al., 2014).
With the advent of single-cell RNA sequencing (scRNAseq) several recent studies have demonstrated that the current subset classification is likely not sufficiently representing LNSC heterogeneity.In LECs isolated from human LNs, six subsets were identified, some of which are located in the SCS and the medullary sinuses and express neutrophil chemoattractants (Takeda et al., 2019).The existence of multiple LEC subsets was recently also observed in murine LNs (Xiang et al., 2020).FRCs may represent the most heterogeneous LNSC population.In addition to FDCs, MRCs, TRCs and perivascular cells, five additional subsets were initially described in FRCs isolated from murine pLN (Rodda et al., 2018).Also, as many as 14 subsets could be identified among non-endothelial LNSCs sorted from both pLN and mLNs (Pezoldt et al., 2018).There, besides subsets expressing Ccl19, Inmt, Cxcl9 and Nr4a1, which most likely orchestrate the LN's reticular network, several CD34 + subsets were also identified (Pezoldt et al., 2018).Since CD34 + cells are frequently referred to as 'adventitial' SCs, the term of fibroblastic stromal cells (FSCs) was introduced to represent all subsets among CD45 − CD31 − PDPN + cells identified by scRNA-seq, including the highly heterogeneous FRC subsets as well as the CD34 + subsets (Pezoldt et al., 2018;Takeuchi et al., 2018).In addition to these studies, further light was recently shed on the heterogeneity of FRCs with the help of Ccl19-EYFP reporter mice.Here, in addition to FDCs, MRCs, TRCs and perivascular cells, two subsets of medRCs, two population of FRCs located at the T-B cell border (named T-B cell border FRCs, TBRCs) and a subset of interfollicular FRCs (IFRCs) were identified (Perez-Shibayama et al., 2020).Moreover, the generation of Cxcl13-Cre/TdTomato reporter mice enabled the dissection of the cellular composition of the B cell follicle reticular cell network.In total, seven subsets were identified, including two populations of FDCs (termed germinal center light zone FDCs and dark zone FDCs), two medRC subsets and a subset of MRCs, TBRCs and IFRCs (Pikor et al., 2020).

LNSC function during steady-state conditions
Due to the compartmentalization of LNs, LNSCs carry out several functions spanning from lymph filtering, over immune cell entry and guidance to the secretion of survival factors and a supporting role in peripheral tolerance and the mounting of immune responses, which will be discussed in detail in the following sections.

LNSCs and the conduit system
The lymphatic vascular network formed by LECs mediates the transport of soluble antigens and antigen-loaded DCs from the tissue to the draining LNs, which enables the transmission of the immunological state of the drained tissue into the LN (Kedl and Tamburini, 2015).This conduit network collects lymph from the tissue and transports the fluid in an unidirectional manner to the SCS (Forster et al., 2012).Upon entry into the SCS, the constituents of the lymph are separated according to their size.Small particles with a low molecular weight, including chemokines, peptides, cytokines and small metabolites, directly enter a conduit system of collagen cores sheathed by FRCs (Gretz et al., 2000).The accurate separation is enforced by the tight plasmalemma vesicle associated protein diaphragm, which is directly connected to the FRC collagen conduit at the LN sinus (Rantakari et al., 2015;Sixt et al., 2005).The content of the conduit network is either sampled by FDCs in the follicles to elicit humoral responses through B cells or by DCs adjacent to FRCs to prime T cell responses (Roozendaal et al., 2009;Sixt et al., 2005).As the FRC network sheathing the collagen conduit system resides in close proximity to the content of the lymph, it might be able to sample constituents of the lymph rapidly and therefore modulate T cell differentiation in concert with DCs.Besides antigen import from the tissue, the conduit system can also be utilized by B cells to export secreted IgM antibodies out of the LN (Thierry et al., 2018).

LNSCs and lymphocyte trafficking
The initiation of antigen-specific T cell responses requires a tight interaction of naïve T cells and antigen-loaded DCs.How can T cells and DCs, arriving at LNs via different gateways, overcome their spatial separation and finally meet each other in the paracortical areas of the LN? Upon their arrival at the LN, tissue-derived CCR7 + DCs first have to traverse the cell barrier posed by the SCS floor to reach the LN paracortex, a migratory step driven by CCR7 along the CCL21 gradient (Forster et al., 2008).LECs lining the ceiling of the SCS, but not those lining the floor, produce ACKR4 (also known as CCRL1), which scavenges CCL21 from the SCS lumen, thereby creating a CCL21 gradient across the sinus floor to enable the emigration of CCR7 + DCs from the afferent lymph to enter into the LN parenchyma (Ulvmar et al., 2014).Subsequently, CCR7 + DCs further migrate into the T cell zone following the chemotactic cues CCL19 and CCL21, which are secreted by FRCs located in the T cell zone (Braun et al., 2011).CLEC-2 expression by DCs also facilitates their crawling along the endothelial cells lining afferent lymphatic vessels and along FRCs lining the reticular network towards the T cell zone through the podoplanin (PDPN):CLEC2 signaling axis (Acton et al., 2012).Meanwhile, HEVs produce CCL21 and transcytose CCL19 from the parenchyma to facilitate circulating CCR7 + T cell recruitment and ingress into the parenchyma (Baekkevold et al., 2001;Stein et al., 2000).CCL19 and CCL21 produced by FRCs in the T cell zone further induce the migration of CCR7 + T cells towards them, where they finally encounter antigen-bearing DCs (Forster et al., 2008).In contrast to T cells, B cell trafficking within the LNs additionally relies on CXCR5-mediated homing towards CXCL13-rich B cell follicles, where they encounter FDCs (Bajenoff et al., 2006;Wang et al., 2011).

LNSCs and immune homeostasis
At steady state, FRCs in the T cell zone are the major source of the survival factor interleukin (IL)-7, which supports naïve T cell homeostasis, and inhibition of IL7 signaling leads to impaired T cell survival and homing to LNs (Link et al., 2007).FRCs can also support B cell maturation, survival and proliferation in B cell follicles via the production of B cell activating factor (BAFF) (Cremasco et al., 2014).LECs help maintain immune cellularity in the SCS and foster the homeostasis of the CD169 + SCS macrophage pool via secretion of colony stimulating factor 1 (CSF1) (Mondor et al., 2019).MRCs located in the LN cortex at the outer edges of B cell follicles can also support macrophage and innate lymphoid cell (ILC) homeostasis via expression of RANKL (Camara et al., 2019;Perez-Shibayama et al., 2019).
LNSCs also partake in immune responses via multiple mechanisms.In response to interferon (IFN)γ and tumor necrosis factor (TNF), FRCs and LECs can upregulate the expression of inducible nitric oxide synthase (NOS2) and produce nitric oxide to restrict T cell proliferation in a cell-contact-dependent manner (Lukacs-Kornek et al., 2011;Siegert et al., 2011).Sustained expression of the major histocompatibility complex (MHC) II in FRCs serves as another mechanism of T cell restraint.Under homeostatic conditions, FRCs express MHCII albeit to a low extent (Dubrot et al., 2014).Upon inflammation, MHCII expression is elevated by IFNγ-mediated upregulation of the class II transactivator (Dubrot et al., 2014).Despite endogenous expression, the majority of MHCII molecules on FRCs are potentially captured from DCs by transfer of MHCII complexes in a cell-contact-dependent manner (Dubrot et al., 2014).The presentation of peptide-MHCII complexes by FRCs to naïve CD4 + T cells induces CD4 + T cell dysfunction (Abe et al., 2014;Dubrot et al., 2014).In contrast to mechanisms of restraint, it has recently been reported that FRCs can also benefit T cell fitness during their activation.Upon receiving signals from activated T cells, FRCs upregulate immune-stimulatory molecules such as IL6 (Brown et al., 2019).FRC-derived IL-6 promotes IL-2 together with TNF production and leads to chromatin remodeling in newly activated CD8 + T cells, thereby enhancing CD8 + T cell survival, metabolism and their capacity to differentiate into tissue-resident memory populations (Brown et al., 2019).In addition, FRCs in the mLNs form a niche for group 1 ILC homeostasis via IL15 expression (Gil-Cruz et al., 2016).

LNSCs and immune tolerance
Immune tolerance against self and commensal antigens is pivotal for preventing autoimmunity and overt inflammation, and emerging studies reveal a role of LNSCs in peripheral tolerance.Under steady-state conditions, LNSCs, particularly FRCs and LECs, express a range of peripheral tissue-restricted antigens (PTAs) and directly present them to naïve CD8 + T cells in the context of MHCI, finally leading to the deletion of self-reactive T cells (Fletcher et al., 2010;Lee et al., 2007).This process is independent of the autoimmune regulator (Aire), but rather depends on the transcription factor Deaf1, thus it is different from medullary thymic epithelial cell-mediated deletion of self-reactive thymocytes (Cohen et al., 2010;Fletcher et al., 2010;Yip et al., 2009).Different LNSC subsets express distinct groups of PTAs and have their own characteristic antigen display (Cohen et al., 2010;Fletcher et al., 2010).For instance, LECs are the only LNSC population expressing the melanocyte-specific protein tyrosinase, mediating the deletion of tyrosinase-specific T cells (Fletcher et al., 2010).LECs can further restrict self-reactive CD8 + T cells by engaging PD-1 on T cells via PDL1 expression (Cohen et al., 2014).Recently, also LNSC-mediated tolerance of self-reactive CD4 + T cells has been reported.FRCs and LECs are capable of capturing self-antigen-MHCII complexes from DCs, thereby limiting CD4 + T cell proliferation and survival in an antigen-specific manner (Dubrot et al., 2014).Additionally, LECs can serve as an antigen reservoir and transfer the self-antigen β-galactosidase to DCs, which subsequently present it to naïve T cells and induce CD4 + T cell tolerance (Rouhani et al., 2015).
Furthermore, LNSCs indirectly contribute to immune tolerance by modulating regulatory T cells (Tregs), a subpopulation of T cells that is essential for the maintenance of peripheral tolerance.Self-antigen-MHCII presentation by LNSCs supports the non-proliferative maintenance of antigen-specific Tregs (Baptista et al., 2014), and very recently, it has been reported that the continuous presentation of self-antigens by LNSCs is critical for the generation of antigen-specific Tregs, thereby restricting the formation of T follicular helper cells and germinal center

Yersinia pseudotuberculosis
At day 3 post infection, the number of FRCs in mLNs is significantly reduced (Pezoldt et al., 2018) FRCs display an activated phenotype with increased MHCII expression until 4 weeks post infection, a time point when Yersiniae were cleared from mLNs (Pezoldt et al., 2018)  B cell responses (Nadafi et al., 2020).In addition, it was demonstrated that LNSCs contribute to the efficient de novo generation of Tregs in gut-draining LNs, namely mLNs and the celiac lymph node (celLN) (Cording et al., 2014;Pezoldt et al., 2018).FRCs isolated from mLNs express high levels of the retinoic acid (RA)-synthesizing enzyme retinal aldehyde dehydrogenase 2 (Aldh1a2), and thereby contribute to a tolerogenic Treg-inducing micro-environment (Cording et al., 2014).Besides this, it could be further revealed that mLN SCs can directly modulate incoming LN-resident DCs (resDCs) and instruct them with Treg-inducing properties, thereby contributing to the maintenance of intestinal tolerance (Pezoldt et al., 2018).

LNSCs in infection and inflammation
Upon infection, LNs often experience hypertrophy to accommodate the clonal expansion of activated T and B cells.After the resolution of the infection, LNs return to their homeostatic size as lymphocytes egress and contract.These dramatic changes in size are mainly mediated by LNSCs.Under homeostatic conditions, PDPN (also known as gp38) regulates the actomyosin contractility of FRCs, which controls physical tension throughout the FRC reticular network (Astarita et al., 2015).Upon infection, increased amounts of naïve lymphocytes and antigen-loaded migratory DCs flow into the LN parenchyma.The enhanced CLEC2 expression on infiltrating DCs, which is induced by antigen-uptake and inflammation in the tissue, binds PDPN on FRCs, thereby inhibiting PDPN-mediated contractility (Acton et al., 2014;Astarita et al., 2015).Consequently, FRCs stretch and elongate, releasing the tension of the reticular network and resulting in a swelling of the LN.In return, the loosening reticular and conduit networks support increased lymphocyte motility and facilitate DC-mediated T cell activation and proliferation (Astarita et al., 2015).
The influx of lymphocytes into inflamed LNs induces LNSC expansion.LNSC subsets display distinct expansion kinetics in response to different pathogens (Abe et al., 2014;Gregory et al., 2017), and SC responses can be sustained by B cells in an antigen-independent manner (Gregory et al., 2017).Additionally, inflammation induces a robust, yet transient transcriptional program in LNSCs, with an upregulated expression of genes encoding chemokines and molecules involved in the acute-phase response together with the antigen-processing and antigen-presentation machinery (Gregory et al., 2017;Malhotra et al., 2012).Remarkably, the amplified FRC reticular network induced by the primary infection is capable of reducing the magnitude of LNSC responses to subsequent heterologous infections (Gregory et al., 2017).Additionally, the extent of LNSC expansion can be modulated by micro-environmental cues.For instance, inflammation-induced cytokines, including IL-17, IL-4 and IL1β, can promote FRC proliferation (Benahmed et al., 2014;Dubey et al., 2016;Majumder et al., 2019).
Consequently, LNSCs respond differently in the context of diverse infections or inflammatory settings.In order to escape immune surveillance, some pathogens such as lymphocytic choriomeningitis virus (LCMV), can directly target and infect LNSCs, finally inducing damage to the conduit network and resulting in an altered LN architecture (Table 1).
The effects of the LNSC response to infections extend beyond expanding LNs for lymphocyte accommodation.LNSCs can modulate a magnitude of immune responses towards infections.The selective ablation of FRCs results in failure to mount proper T and B cell responses against influenza virus (Cremasco et al., 2014).Lymphtoxin (LT)/lymphotoxinβ receptor (LTβR) signaling is pivotal for the maturation of LNSCs.Loss of LTβR on LNSCs results in impaired immunocompetence and increased susceptibility to acute LCMV infections (Chai et al., 2013).A recent study revealed that the lack of LTβR signaling in early life results in a long-lastingly altered SC composition in mLNs and consequently impaired virus-specific antibody responses against rotavirus (Li et al., 2019).Besides the supportive role in mounting immune responses, LNSCs can also restrain excessive inflammation during enteropathogenic infections.FRC recognition of pathogen entry via myeloid differentiation response protein 88 (MyD88) signaling downregulates IL15 expression in FRCs, thereby restricting exaggerated ILC activation and subsequent inflammatory cell damage (Gil-Cruz et al., 2016).

Impact of the LN micro-environment on the phenotype of SCs
One factor that stands out in the context of LN genesis and the maintenance of SC structural integrity is LT/LTβR signaling.In the mature LN, many SC subsets express LTβR and rely on the expression of LT in lymphocytes and myeloid cells for their survival (Kumar et al., 2015).After inhibition of LTβR in mature LNs, CD35 expression is eliminated and FDC networks collapse (Gommerman et al., 2002).FRCs in general are diminished after CD4 + T cell depletion, emphasizing this T cell population as a critical source of LT (Zeng et al., 2012).Moreover, although TRCs can generate a basic T cell infrastructure, they cannot reach full immunocompetence in the absence of LTβR signaling (Chai et al., 2013).For HEV integrity continued LTβR-signaling is essential (Browning et al., 2005;Liao and Ruddle, 2006;Veerman et al., 2019), and in the absence of CD11c + DCs lymphocyte homing to the LNs is dramatically reduced and HEVs revert to an immature HEV phenotype with a marked downregulation of HEV-specific markers, which was at least partly due to the absence of DC-mediated LT-signaling (Moussion and Girard, 2011).Very recently, it was shown that a transient attenuation of LTβR signaling in utero leads to a permanent alteration of the mLNs SC composition, suggesting a long-lasting impact of early-life LTβR signaling on LNSC phenotype (Li et al., 2019).Aside from these general mechanisms affecting SC maintenance in all LNs as well as LTβR-specific remodeling processes during infection, LT signaling is decisive during the onset of LN organogenesis.Although LN development is not subject of the present review, the following interesting aspect should be briefly discussed: LTβR -/-mice lack PPs and all LNs (Futterer et al., 1998), while LTβ -/-mice lack PPs and most LNs, but retain mLNs and cervical LNs, indicating location-specific differences in LN development dependent on LT signaling (Alimzhanov et al., 1997;Koni et al., 1997).
Since 22 murine LNs always develop at defined anatomical sites, the mouse represents an ideal model to study the influence of the surrounding micro-environment on a sessile cell population with longevity, such as the LNSCs in individual LNs.Although scRNAseq analysis demonstrated a similar SC subset compositions in mLNs and pLN (Perez-Shibayama et al., 2020;Pezoldt et al., 2018;Pikor et al., 2020;Rodda et al., 2018), scRNA-seq and RNAseq analysis also reveal a distinct overall transcriptome of mLN FRCs and pLN FRCs (Malhotra et al., 2012;Pezoldt et al., 2018).Interestingly, it was recently shown that even the FRCs transcriptomes of individual LNs of the mLN chain are discrete  (Majumder et al., 2019) FDCs expand in number via direct differentiation from MRCs into mature FDCs, and express high amounts of CXCL13, BAFF and IL6 (Jarjour et al., 2014;Wu et al., 2009) CXCL13 secretion of FRCs in the perifollicular zone delineates new transient boundaries of the growing follicles (Mionnet et al., 2013) IL6 production of FDCs promotes germinal center reactions, somatic hypermutation and IgG production (Wu et al., 2009) (Esterhazy et al., 2019).To determine whether LNSCs can stably retain their location-specific properties, LN transplantation experiments can be applied (Mebius et al., 1993).After surgically resecting the endogenous intestinal or skin-draining LN, a transplanted LN can readily engraft at the cleared site (Hammerschmidt et al., 2008).During engraftment, the hematopoietic compartment is replaced by cells from the recipients' draining tissue and the circulation.However, LN-resident cells, predominantly SCs of donor origin, remain (Hammerschmidt et al., 2008).
It was observed that the FSC transcriptome of transplanted mLNs to a skin-draining site did not completely adapt a pLN FSC phenotype several weeks after transplantation (Pezoldt et al., 2018).Akin to this, it has been reported that the transplanted LNs stably retain functional properties, such as MAdCAM-1 expression on HEVs originating from mLNs, whereas skin-draining pLN SCs do not gain MAdCAM1 expression if transplanted to the mesenteries (Ahrendt et al., 2008;Hammerschmidt et al., 2008).Together, these data indicate that once matured, the LN's FSC compartment can retain part of its characteristic features for a prolonged period of time even upon translocation to a different anatomical site.Interestingly, the conservation of mLN SC function with regard to MAdCAM-1 expression is only observed if an intact mLN is transplanted.When single-cell suspensions of SCs from skin-draining LNs were transplanted into the mesenteries using a collagen sponge, the establishing LN HEVs indeed express MAdCAM1 (Buettner et al., 2015).These findings suggest that a newly forming LN functionally adapts to the new tissue draining site and can be influenced by micro-environmental factors.
Another interesting aspect to study is the proximity of gut-draining LNs to the intestinal microbiota and therefore the microbe's capacity to shape LNSC subset composition, phenotype and function.FRCs from mLNs, which express high levels of Aldh1a2, act in concert with CD103 + DCs to contribute to the elevated RA levels in mLNs (Hammerschmidt et al., 2008;Malhotra et al., 2012;Molenaar et al., 2011).Retinal dehydrogenase (RALDH) enzymatic activity has been shown to be a vital constituent for the neonatal to adult addressin switch in HEVs in mLNs and pLN, which does not occur in germ-free (GF) mice (Zhang et al., 2016).Transplantation of mLNs either from GF or colonized specific pathogen-free (SPF) mice into the skin-draining popliteal fossa of SPF mice enables the assessment of the contribution of microbiota on LNSC phenotype (Cording et al., 2014;Pezoldt et al., 2018).RNAseq analysis of FSCs isolated from these transplanted LNs as well as endogenous mLNs from GF and SPF mice revealed that the microbial colonization status shapes the FSC transcriptome, although the anatomical location of the LN affects the FSC transcriptome to a much greater degree (Pezoldt et al., 2018).Furthermore, by transplanting mLNs from differently aged SPF donor mice into a skin-draining site, the first ten days after birth were identified as the critical time window at which mLN SCs are stably imprinted with tolerogenic properties (Pezoldt et al., 2018).In addition to the 'naturally existing' neonatal window, when mLN SCs are imprinted by microbiota with tolerogenic properties, the microbiota-mediated stabilization of the functionality of mLN SCs can theoretically also be introduced later in life.Microbiota introduced into adult GF mice by co-housing with SPF mice was also capable of stably imprinting tolerogenic properties into mLN SCs (Pezoldt et al., 2018).
In addition to the microbiota-dependent imprinting of tolerogenic properties into mLN SCs, vitamin A has been identified as another microenvironmental factor that is stably imprinting tolerogenic properties into gut-draining LNs (Cording et al., 2014).The celLN, which is draining the liver as well as the upper gastrointestinal tract, is located in a vitamin A-rich micro-environment since the liver constitutes the major storage organ for dietary vitamin A (Blaner et al., 2016).Several studies Fig. 2. Lymph node stromal cell phenotype and function in the context of microenvironmental niches.LNSC phenotype and function are dependent on signaling pathways that are LN-intrinsic and shared as a common feature between LNs as a SLO.This is likely supplemented by unique cues that are received from a particular LN-draining site. 1) Incoming leucocytes, including B cells, T cells and DCs, provide LT to SC subsets interspersed throughout the LN.TRCs require LTβR signaling for their continued functionality and FDC networks collapse after interruption of LTβR signaling.2) Microbiota can illicit transcriptional changes in mLN FRCs, and their presence induces HEV maturation via CD103 have demonstrated the unique tolerogenic properties of the celLN (Cording et al., 2014;Hauet-Broere et al., 2003;Nutsch et al., 2016;Pezoldt et al., 2018), and transplanted celLN from mice fed with vitamin A-deficient diet lost their superior tolerogenic properties in the non-tolerogenic skin-draining site, while transplanted celLN from mice fed with standard diet retained their tolerogenic properties (Cording et al., 2014).These data suggest a vitamin A-mediated imprinting of tolerogenic properties into celLN SCs, yet whether this imprinting also takes place during early life is still unclear.

LNSCs tailor tissue-specific immune responses
LNSCs are influenced by their micro-environment, but besides being receptive to micro-environmental cues, these cells are also capable of tailoring tissue-specific immune responses (Fig. 2).Adoptively transferred T cell receptor-transgenic naïve T cells could upregulate the expression of gut-homing molecules α 4 β 7 and CCR9 upon antigenspecific priming in mLNs transplanted into the skin-draining popliteal fossa, while pLN transplanted into the gut mesenteries failed to foster the generation of gut-homing T cells (Hammerschmidt et al., 2008;Molenaar et al., 2009).Thus, SCs not only maintain expression of location-specific adhesion molecules, but also modulate T cell differentiation in a location-specific manner.Additionally, LNSCs show location-specific tolerogenic properties.As mentioned above, gut-draining LNs (mLNs and celLN) foster high de novo Treg induction (Cording et al., 2014).When gut-draining LNs and pLNs were transplanted into the skin-draining popliteal fossa and gut mesenteries, respectively, gut-draining celLN and mLNs engrafted at the non-tolerogenic site still fostered efficient de novo Treg induction, while pLNs transplanted to the tolerogenic gut mesenteries failed to support high Treg induction (Cording et al., 2014;Pezoldt et al., 2018).Together, these data demonstrate that gut-draining LNs stably retain their efficient Treg-inducing capacity, a feature maintained by the LNSC compartment.Furthermore, in an intranasal tolerance induction model, the surgical removal of cervical LNs and their subsequent replacement with pLNs abrogated intranasal tolerance induction (Wolvers et al., 1999), further demonstrating the importance of stably imprinted functional properties of LNSCs for the modulation of tissue-specific immune responses.

Conclusion
Quite recently, LNSCs were primarily appreciated as structural constituents defining and organizing the highly compartmentalized architecture of LNs.Advances in the past decade have expanded our understanding of the functionality of LNSCs.Besides forming a scaffold, LNSCs produce chemokines to direct the migration of lymphocytes and DCs into and within LNs.Furthermore, they produce survival factors to promote the survival of incoming immune cells to provide high quality accommodations.LNSCs can also sense the tension of the environment, and either restrain or promote lymphocyte activity to maintain immune homeostasis.The advances in scRNA-seq have further revealed characteristic gene expression signatures of unique LNSC subsets.Remarkably, LNSCs retain location-specific information and tailor tissue-specific immune responses to a degree that was largely ignored, so far.Upon infection, LNSCs are not static, but are dynamically regulated in response to cellular and molecular cues, thereby coordinating adaptive immunity to control the infection.Additionally, LNSCs can contribute to peripheral tolerance towards self-or microbiota-derived antigens.Recent studies highlighted the concept of the neonatal window of opportunity for the establishment of immune tolerance.Indeed, there is also a neonatal window of opportunity for LNSCs to be stably imprinted with tolerogenic properties by micro-environmental factors.In conclusion, the latest advances and continued efforts towards a precise understanding of the role of LNSCs in coordinating immune responses under steady-state conditions and during infection will provide a novel perspective for the design of vaccines, therapies and immunomodulatory strategies against various immune-mediated diseases.

Fig. 1 .
Fig. 1.Lymph node structure and stromal cell subset localization.Inside the LN there are various spatially segregated niches, which are occupied by highly specialized SC subsets. 1) Enveloping the capsule of the LN is a layer of CD34 + FRCs whose function so far remains enigmatic.CD34 + FRCs are also found in the adventitia of larger vessels where they are termed "adventitial SCs".Inside the LN, adventitial SCs surround the vessels entering the LN, which have a defined adventitia (not depicted).2) Below the capsule, the SCS is surrounding the inner parts of the LN.LECs line the SCS and incoming antigen is filtered and distributed across the LN.LECs guide immune cells arriving with the afferent lymph via CCL21 secretion and maintain the local macrophage niche by providing CSF-1.Moreover, LECs line the lymphatics of the medullary sinuses throughout the medulla (not depicted).3) MRCs are located below the capsule in close proximity to the B cell follicles.They produce CXCL13 and RANKL and are capable of replenishing FDCs after FDC ablation during infection.4) In the cortex, CXCL13 + FDCs are residing in the B cell follicles and present antigen to B cells.5) In the light zone of germinal centers, FDCs foster B cell affinity maturation, while CXCL12-producing CRCs are inducing B cell proliferation.6) In the paracortex, TRCs guide T cells and DCs via the expression of CCL19 and CCL21.Moreover, they build a conduit system to efficiently channel lymph throughout the paracortex.7) HEVs are dispersed throughout the LN, facilitate immune cell entry by the expression of adhesion molecules and are composed of an inner layer of BECs, followed by pericytes and lastly a layer of perivascular FRCs.8) In the medulla, where the lymph is collected before LN egress, medRC are producing APRIL, BAFF and IL-6 to sustain the local plasma cell population.BEC, blood endothelial cell; CRC, CXCL12-expressing reticular cell; CSF-1, colony stimulating factor 1; DC, dendritic cell; FDC, fibroblastic dendritic cell; FRC, fibroblastic reticular cell; HEV, high endothelial venule; LEC, lymphatic endothelial cell; LN, lymph node; medRC, medullary FRC; MRC, marginal reticular cell; SC, stromal cell; SCS, subcapsular sinus; TRC, T cell zone FRC.
-derived LPS induces disruption of CCL21 and CXCL13, resulting in enhanced virulence of the pathogen (St John and Abraham, 2009) expression of genes encoding chemokines and molecules involved in the acutephase response and the antigen-processing and antigen-presentation machinery(Malhotra et al., 2012) PolyI:C A subset of LNSCs shows unique up-regulation of PTAs and Aire(Fletcher et al., 2010) Immunization (continued on next page) M.Zou et al.

Table 1
Lymph node stromal cell phenotype in infection and inflammation.