key: cord-0803235-sktdrd8q
authors: Lütge, Mechthild; Pikor, Natalia B.; Ludewig, Burkhard
title: Differentiation and activation of fibroblastic reticular cells
date: 2021-05-27
journal: Immunol Rev
DOI: 10.1111/imr.12981
sha: cef2aae7f190d3a8995e5f081a531504943917c7
doc_id: 803235
cord_uid: sktdrd8q

Secondary lymphoid organs (SLO) are underpinned by fibroblastic reticular cells (FRC) that form dedicated microenvironmental niches to secure induction and regulation of innate and adaptive immunity. Distinct FRC subsets are strategically positioned in SLOs to provide niche factors and govern efficient immune cell interaction. In recent years, the use of specialized mouse models in combination with single‐cell transcriptomics has facilitated the elaboration of the molecular FRC landscape at an unprecedented resolution. While single‐cell RNA‐sequencing has advanced the resolution of FRC subset characterization and function, the high dimensionality of the generated data necessitates careful analysis and validation. Here, we reviewed novel findings from high‐resolution transcriptomic analyses that refine our understanding of FRC differentiation and activation processes in the context of infection and inflammation. We further discuss concepts, strategies, and limitations for the analysis of single‐cell transcriptome data from FRCs and the wide‐ranging implications for our understanding of stromal cell biology.

Innate and adaptive immune responses are initiated and coordinated in secondary lymphoid organs (SLOs) that are strategically positioned throughout the body to sample antigens from pathogens, commensal organisms, tumors, or any other environmental sources.

In addition, innate immunological signals from inflamed tissues or the body surfaces are relayed to SLOs to amplify or atone immune reactions. Diligent processes in SLOs guide the cellular interactions that generate protective immunity while minimizing immunopathological damage in the tissues. The decision-making processes that determine the strength, breadth, and specificity of adaptive immune responses occur in dedicated niches within SLOs. Specialized lymphoid organ fibroblasts, commonly referred to as fibroblastic reticular cells (FRCs), form the scaffold of SLOs and determine the microenvironmental conditions for lymphocyte activation and differentiation. 1 signals from a dedicated sampling site, (b) sample and distribute antigen to lymphocytes and antigen-presenting cells, and (c) promote the efficient activation and interaction of cognate T and B lymphocytes. 5 To this end, all SLOs adopt a similar structural patterning with an antigen-sampling zone filled with specialized myeloid cell subsets, and dedicated B cell follicles and adjoining T cell regions to initiate and direct lymphocyte priming and differentiation. Activated lymphocytes must then converge within the B cell follicle or the T cell zone to achieve, for example, the generation of high-affinity antibody responses or effector CD8 + T cells, respectively. This series of "combinatorial decision processes" guiding myeloid cell and lymphocyte homing and compartmentalization is orchestrated by chemokines secreted by FRCs. 6,7 CXCL13-expressing FRCs govern B cell clustering and follicle formation, 8, 9 while CCL19/CCL21-expressing FRC orchestrate dendritic cell and T cell homing and homeostasis. [10] [11] [12] Within each zone, specialized FRC subsets coordinate the directed movement of myeloid cells and activated lymphocytes or the display of antigens to harmoniously secure efficient adaptive immunity.

Several terms have been used to describe the fibroblastic stromal cells underpinning SLOs, including myofibroblasts, pericytes, and FRCs. 13 In 1968, the term reticular cells was first used to describe the elongated fibroblastic cells surrounding thin filamentous processes, a reticulum, traversing the B cell follicle of the lymph node, 14 and the spleen. 15 This topological property is also conserved by FRCs in Peyer's patches, 16 and the formation of a reticulum is a feature not only of fibroblastic cells in the B cell follicle but also in the T cell zone. 13, 17 More recently, FRCs have been characterized by the expression of podoplanin (PDPN, also referred to as gp38) and lack of the endothelial marker CD31 (platelet endothelial cell adhesion molecule-1, PECAM-1). 11 Although PDPN + CD31 − FRCs have often been associated with the T cell zone, 11, 18, 19 the term FRC is now well-accepted to broadly demarcate immune-interacting fibroblastic stromal cells within SLOs. Additional topological and functional attributes characterize distinct FRC subsets, such as those in the B cell follicle or the T cell zone.

While CCL19-secreting FRCs, as well as a subset of CXCL13secreting, antigen-presenting cells termed follicular dendritic cells (FDCs), have prevailed in our knowledge of FRC subsets, in recent years, major advances in genetic models paired with the advent of single-cell transcriptomics have created novel means to resolve FRC heterogeneity and function. New, highly specialized FRC subsets have been defined and implicated in the fine-tuned coordination of lymphocyte migration and priming. In this review, we will discuss recent findings delineating FRC differentiation and activation obtained by genetic targeting in mouse models and discuss important strategies and limitations of high dimensional data analysis for the understanding of FRC biology.

The most basic and still frequently used approach to identify FRCs has been based on the expression of PDPN and an extracellular matrix protein that is recognized by the antibody ER-TR7. 11, 20 However, PDPN-negative FRCs populate several sites in lymph nodes, 21 and PDPN expression in the spleen is largely restricted to the T cell zone. 22, 23 Thus, tracking changes in the relative abundance, phenotype, or transcriptional profile of non-hematopoietic CD31 -PDPN + cells only offers a very limited insight into the nature of FRCs in the classical SLOs (lymph nodes, splenic white pulp, or Peyer's patches) or non-classical SLOs such as fat-associated lymphoid clusters (FALCs). Genetic targeting of particular cell types in vivo using specific promoters to drive the expression of real-time reporters or the Cre recombinase has offered novel means to elaborate origin, phenotype, and function of FRCs.

In contrast to T and B cells, FRC-secreted factors are not readily or sensitively assayed by traditional methods such as flow cytometry or histology. Over the last 10 years, several strains of reporter mice and lineage tracing models expressing Cre recombinase and/or fluorescent proteins (eg, the enhanced yellow fluorescent protein, EYFP) under the promoter of key FRC signature genes have been developed, revolutionizing FRC immunobiology (Table 1 ). Two such transgenic mouse models that take advantage of FRC chemokine expression are the Ccl19-Cre 24 spleen at E19.5 23 ) thereby targeting FRCs in lymph nodes, 21, 24, 28 the splenic white pulp, 29 and Peyer's patches. 30 Similarly, in lymph node anlagen, the Cxcl13-Cre transgene is expressed at E14 in mesenchymal stromal cells, marking all major lymph node FRC subsets in adult mice. 25 In contrast, Cxcl13-expressing, TdTomato + FRCs are largely confined to the B cell follicles. 25, 31 Targeting of this transgene in the splenic white pulp and Peyer's patches remains to be studied in detail.

In addition to these signature chemokine-based FRC-targeting models, a number of transgenic strains permit the lineage tracing of a particular FRC repertoire. The DM2 BAC transgenic model harbors a multiple reporter construct containing the diphtheria toxin receptor (DTR), firefly luciferase, and the fluorescent reporter mCherry under the control of the fibroblast activation proteinα (Fap) promoter. 32 The absence of a Cre recombinase-driven labeling of transgene-targeted cells does not make this particular model suitable for lineage tracing but rather acts as a direct reporter of current FAP expression. The BAC-encoded DTR permits the inducible deletion of FAP-expressing cells following DT administration. In lymph nodes and Peyer's patches, FAP expression is primarily restricted to FRCs in the T cell zone but is detected in a low frequency of cells in the splenic white pulp. 33 The Col6a1-Cre crossed to the R26 mT/mG reporter allows for targeting and lineage tracing of cells expressing, or having expressed Col6a1. 34 This model predominantly targets FDCs and marginal zone reticular cells (MRCs) in Peyer's patches but does not readily target FRCs in the splenic white pulp or lymph nodes. 35 The Cd21-Cre R26R-tdRFP mouse model was designed as a reporter and lineage tracing model of FDCs, based on the high expression of CD21 (murine complement receptor 2) by these cells. 36 Ccl19-iEYFP additionally encodes for a TdTomato reporter under the control of the Ccl19-promoter to distinguish current from past promoter activity. These models have been used to identify a common FAP-expressing progenitor of lymph node FRCs 39 and to delineate differentiation trajectories of splenic white pulp FRCs that were found to originate from multipotent periarterial, CCL19-expressing FRC progenitors. 23 Additional tools to study clonal relationships include multicolor tagging systems based on Brainbow models. 40, 41 The combinatorial expression of multiple fluorescent proteins under the control of the Rosa26 promoter permits the Cre-induced lineage tracing or fate mapping of individual clones within a targeted cell population. This system has been used to study the developmental origin and differentiation dynamics of FDCs in lymph nodes, 42 and FRCs in the splenic white pulp 23 and Peyer's patches. 27 Collectively, these genetic models have aided in elucidating patterns of FRC differentiation across SLOs by marking progenitorprogeny relationships, enriching known or rare cell populations for single-cell RNA-sequencing, and demonstrably establishing the function of FRC subsets as will be discussed in the following sections. In addition, the broad and constitutive targeting of fibroblasts also makes many of these Cre recombinase-driven lineage tracing models suitable for targeting stromal cells in inflammatory lesions or the tumor microenvironment, 32,43-45 making it possible to study immune-interacting stromal cells outside of lymphoid tissues.

In recent years, single-cell profiling has become a fast-growing field with a vast number of technological advances accompanied by a flood of new computational tools and algorithms for single-cell data analysis. 46 This rapid progress opens a multitude of new opportunities, but at the same time poses challenges for standardization, and consequently for data interpretation and reproducibility. 47, 48 Key variables that ultimately determine the quality and reproducibility of the analysis include sequencing depth, total cell number, and clustering strategy. 49, 50 However, these parameters themselves depend on whether heterogeneous cell types are being analyzed, such as in whole organ analyses, or whether the heterogeneity across subsets or activation states of a single-cell type is being performed. 51 Thus, a robust and meaningful clustering depth, assignment, and validation strongly determine the biological interpretation of cellular heterogeneity using single-cell transcriptomics.

The FRC landscape consists of specialized subsets that form distinct niches within SLOs corresponding to the T cell zone, B cell follicle, antigen-sampling regions, and sites of lymphocyte entry ( Figure 1 ). 1, 5 As such, FRCs can be broadly categorized as T cell zone reticular cell Instead, it is often favorable to run a number of algorithms with different parameters to gain confidence about cluster robustness and potential "over-" or "under-clustering". 47 As a general rule of thumb, reducing technical noise and resolving the heterogeneity of highly similar or rare cell populations requires a sufficiently high cell input number, and in the case of FRCs, can be facilitated by enriching for desired subsets using available reporter models 51 (see Table 1 ). Once cluster robustness is tested, the list of each cluster's marker genes should reflect the spatial positioning and immune cell-interacting partners of FRC subsets. Notably, the final choice of clustering depth can vary and should be adapted to the research question and a resolution that can be validated. [56] [57] [58] For validation, overlapping expression of unbiased, computed cluster markers should be verified against known signature genes and spatial orientation by confocal microscopy 59,60 ( Figure 2A ).

While histological cluster validation can be challenging and requires highly expressed marker genes and good antibodies, it achieves a reliable description of FRC subset heterogeneity as shown in a number of recent studies. 23, 27, 31, 53, 54 The molecular properties of FRC subsets as revealed by single-cell transcriptomics will be discussed in more detail in section 4. 

PRC Cd34, Ly6a phenotype of FRC subsets in order to secure niche-specific, optimal "catering" conditions.

TRCs are involved in various crucial processes to establish special- 

PRCs, as their name suggests, are perivascular fibroblasts that fulfill reticular cell functions such as chemokine expression and conduit formation. 13 As perivascular cells ensheath the length of the vascular tree, single-cell transcriptomic analyses demarcate PRCs as CD34-and LY6A (stem cell antigen 1)-expressing cells surrounding blood vessels in each SLO. 23, 27, 53, 54 Further detailed studies are needed to resolve the molecular identity of PRCs along the vascular tree as has been done for PRCs in the CNS 90 or for lymph node blood endothelial cells. 91 Moreover, while the precursor potential of CD34 + PRCs has been demonstrated during development, 23 it remains unclear whether only a subset of PRCs exhibits precursor potential during SLO differentiation and whether the same or different PRC subsets maintain self-renewing properties in already formed SLOs, especially in the context of inflammation-induced remodeling.

In lymph nodes, the medulla is a site of lymphocyte exit and myeloid cell accumulation, penetrated by large blood vessels and lymphatic sinuses. The lymph node vascular tree is rooted in the medulla with large arteries and veins traversing this region, 91 is required for FRC lineage commitment, 24,25 and a further "signal 2" drives subset differentiation 80, 106 (Figure 3 ).

During embryonic development, splenic precursors emerge as mesenchymal condensation within the dorsal mesogastrium at Figure 3A ). The timed, conditional deletion of LTβR from Ccl19-iEYFP-targeted FRCs revealed a block in fate-mapped FRC subset differentiation, whereas mural cell specification and sustenance of a multipotent periarterial progenitor was found to be LTβR-independent. Based on these findings a two-signal program was proposed as model for splenic FRC subset specification. In this model, LTβR-dependent activation of mLTo cells serves as "signal 1" to commit to an FRC lineage, while further FRC subset specialization depends on secondary signals that likely reflect the immune microenvironment and extrinsic imprints from interacting cellular partners ( Figure 3B ). As the sustenance of adult reticular progenitors is independent of LTβR signaling, a yet undefined preceding signal is required to establish and maintain the periarteriolar progenitor niche. 23 Further cell-targeted genetic perturbation studies are required to delineate the nature of this initiating signal in the splenic white pulp.

Depending on the bodily region, lymph node organogenesis in mice is initiated between E12 and E17 and is driven by sequential acti- 25, 111 and adipocyte precursor cells. 112 In line with this finding, it was shown that the genetic abrogation of the canonical NFκB pathway in Ccl19-Cre-expressing cells inhibits FRC differentiation, 113 and constant LTβR signaling throughout postnatal life is required for FRC differentiation. 114 Together these studies reinforce the notion that LTβR and NFκB signaling act as major switch for FRC subset differentiation, equivalent to the "signal 1" suggested for splenic FRCs.

A recent study described YAP and TAZ, effectors of Hippo signaling, as additional factors that determine FRC lineage commitment prior to LTβR engagement 115 identifying this pathway as an initial signal preceding "signal 1" in FRC differentiation ( Figure 3B ). Since G-protein-coupled receptors, mechanical forces and Wnt signaling can feed into the YAP/TAZ signaling pathway, 116 further studies are warranted to explore the cellular and molecular triggers of the Hippo pathway in lymph node FRC precursors.

Peyer's patch organogenesis is initiated at E16.5 when LTi cells ac- FDCs using a multicolor fate-mapping system in lymph nodes. 42 Although pharmacological studies suggest that the continued engagement of key pathways such as the LTβR and TNFR pathway are important to maintaining postnatal FRC subset identity, 98, 114 to what extent these progenitor-progeny relationships are maintained or reshaped in established SLOs over a lifetime of pathogen surveillance remains to be determined.

In the course of the encounter with pathogens, lymphoid microenvironments increase in size through the recruitment and proliferation of immune cells leading to profound changes in the reticular cell network. 2 In addition to FRC proliferation, 74,120 topological changes in FRC network organization occur to support germinal center responses, 31 lymphangiogenesis, 121 Concomitantly, FRCs are involved in modulating adaptive immune responses via the expression of peripheral tissue antigens, 3 inhibitory molecules such as programmed death-ligand 1 (PD-L1), 54, 125 or metabolites such as nitric oxide or prostaglandin E2/cyclooxygenase-2 (PGE 2 /COX-2) in mice, [126] [127] [128] Although there are some inconsistencies in FRC subset characterization across studies (owing to differences in clustering resolution or cell input number), tremendous progress has been made in defining the molecular and functional FRC landscape in SLOs in murine models. It will be important to further elaborate FRC-driven regulation of lymphocyte activation and regulation as pioneered by Fletcher and colleagues who identified molecular mechanisms underlying FRC-restricted T cell activation as potential therapeutic strategies for CAR-T cells. 129 Clearly, future technological advances such as spatial transcriptomics 132 will facilitate the study of human FRCs, bridging knowledge and insights from the murine setting. As SLO FRCs can be regarded as the prototypical immune-interacting fibroblasts, a profound understanding of FRC activation and differentiation will meaningfully guide research on therapeutically targeting stromal cell-underpinned niches in autoimmunity and cancer.

This study received financial support from the Swiss National Science Foundation/Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (grant 180011 to NBP and grants 177208 and 182583 to BL) and Novartis Foundation for Biomedical

Research to (20C217 to NBP). The funders had no role in preparation of the manuscript.

Therapeutics AG, St. Gallen, Switzerland.

Burkhard Ludewig https://orcid.org/0000-0002-7685-573X

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