The fibroblast: An emerging key player in thymic T cell selection

Abstract Fibroblasts have recently attracted attention as a key stromal component that controls the immune responses in lymphoid tissues. The thymus has a unique microenvironment comprised of a variety of stromal cells, including fibroblasts and thymic epithelial cells (TECs), the latter of which is known to be important for T cell development because of their ability to express self‐antigens. Thymic fibroblasts contribute to thymus organogenesis during embryogenesis and form the capsule and medullary reticular network in the adult thymus. However, the immunological significance of thymic fibroblasts has thus far only been poorly elucidated. In this review, we will summarize the current views on the development and functions of thymic fibroblasts as revealed by new technologies such as multicolor flow cytometry and single cell–based transcriptome profiling. Furthermore, the recently discovered role of medullary fibroblasts in the establishment of T cell tolerance by producing a unique set of self‐antigens will be highlighted.

the subcapsular region and differentiate into CD4 + CD8 + (double positive, DP) thymocytes in the outer cortex. DP thymocytes that have completed gene rearrangement express the rearranged TCR on the cell surface. Upon interaction between the TCR and selfpeptide/MHC complexes, the cells with functional TCR are induced to differentiate into CD4 + CD8 − (CD4 single positive, CD4SP) or CD4 − CD8 + (CD8SP) thymocytes (positive selection), while cells expressing self-reactive TCR are deleted (negative selection). 6 The positive selection of such a diverse TCR repertoire depends on the ability of cTECs to produce and present a unique set of self-peptides via MHC molecules. [7][8][9][10][11][12] Positively selected SP thymocytes migrate from the cortex to the medulla, attracted by chemokines produced by mTECs. [13][14][15][16] In the medulla, mTECs express a large number of highly diverse antigens called tissue-restricted antigens (TRAs) that represent almost all of the tissues in the entire body. 17 SP thymocytes that recognize such TRAs, thus establishing selftolerance in T cells. 15,16,[19][20][21][22][23][24] In addition to TECs, which control T cell differentiation and selection, a variety of non-TEC stromal cells support the thymic microenvironment. The blood vasculature is an important parenchymal component of the thymus that supplies oxygen and nutrients and provides entry and exit points for T cells as well as other immune cells. The cortex contains a network of capillaries, while the CMJ and medulla are enriched with arterioles and postcapillary venules. 25,26 These are made up of functionally distinct endothelial cells that control the influx of bloodborne molecules as well as ETP ingress and mature T cell egress. [27][28][29] The thymus also contains mesenchymal cells that originate from neural crest (NC) cells. These NC-derived mesenchymal cells are important for the differentiation and expansion of TECs during embryogenesis. In the postnatal thymus, the mesenchymal cells are predominantly found as fibroblasts in the capsule and medulla, and also as vascular mural cells ( Figure 1A). However, despite their abundance in the thymus, the immunological significance of thymic fibroblasts in the postnatal thymus has been both less explored and understood than that of TECs.
Fibroblasts have been generally considered to be ordinary cells without specific features, distributed in tissues throughout the body.
However, recent studies have revealed the functional heterogeneity of fibroblasts under various physiological and pathological conditions, [30][31][32] including immune responses in secondary lymphoid organs 33,34 or upon viral infection. 35 This review will provide a current state-of-the-art overview of thymic fibroblasts, focusing on historical studies and recently reported findings on their nature and immunological functions.

| Overview of thymic mesenchymal cells
In the adult thymus, NC-derived mesenchymal cells are predominantly found in the capsule and medulla. 36,37 The capsule of the mouse thymus comprises a monolayer of fibroblasts (capsular fibroblasts, capFbs) that covers the surface of the thymic parenchyma. 25 The human thymus is covered by a capsule from which interlobular septa arise and divide the parenchyma into lobes. 38,39 There are some sparsely distributed fibroblasts in the cortex, but their structural features are not presently known. In the medulla, NC-derived cells are found as medullary fibroblasts (mFbs) and vascular mural cells. mFbs form the reticular network 40 as well as the blood vessel adventitial layer. 41 Mural cells are subdivided into pericytes and vascular smooth muscle cells (VSMCs), both of which are embedded in the basement membrane and ensheath the endothelial tubes. Pericytes and VSMCs are distinguished by the absence or the presence of contractility. 42,43 Although the definition of these cells has been different or even deemed controversial in different studies, in this review we define pericytes as non-contractile cells and VSMCs as contractile cells expressing α-smooth muscle actin (α-SMA).
Traditionally, monoclonal antibodies such as ER-TR7 and MTS-15   have been used for the detection of thymic fibroblasts. ER-TR7 reacts with an unidentified intracellular epitope of fibroblasts, 44 while MTS-15 binds glycosphingolipid on the fibroblast surface. [45][46][47] It was shown that in flow cytometry approximately one-half of PDGFRα + thymic fibroblasts are MTS-15 + . 47 Although these antibodies recognize not only fibroblasts but also endothelial cells and mural cells, studies using these antibodies have led to the discovery of more specific molecular markers of thymic fibroblasts.
As summarized in Table 1, several proteins have been reported as thymic stromal cell subset markers, including fibroblasts. PDGFRα and PDGFRβ are markers widely used for detecting thymic fibroblasts. [48][49][50] PDGFRα is highly expressed in capFbs and mFbs, while PDGFRβ is prominent in pericytes and VSMCs. capFbs and mFbs also express podoplanin (Pdpn, also called gp38) and CD34 at high levels. 40,41 Pericytes and VSMCs can be distinguished from fibroblasts by their high expression of Mcam (CD146) and integrin α7 Most of our understanding of thymic fibroblasts has come from studies using the thymus of animals such as mice and rats. Even though human thymus samples can be obtained from aborted fetuses or neonatal cardiac surgery, these specimens are generally not readily available in many countries. Therefore, previous studies on human thymic fibroblasts have been limited mainly to histological observations using fixed thymus specimens. Recently, however, some have attempted to clarify the functional classification and agerelated changes of human thymic fibroblasts using new technologies such as single-cell transcriptomics. These studies will be discussed in detail later (Section 2.5).
In order to understand how these thymic fibroblasts develop and are localized within the thymus, it is necessary to have a close look at the organogenesis of the thymus.

| Mesenchymal cells in organogenesis of the thymus
The thymus originates from the 3rd pharyngeal pouch, a temporary embryonic structure composed of evaginated endodermal epithelial cells. 52 The epithelial cells are surrounded by NC-derived mesenchymal cells, which support pouch patterning, organogenesis of the thymus as well as parathyroid grand, and differentiation of the epithelial cells into TECs. Along with the proliferation of TECs and organization of epithelial parenchyma, the surrounding mesenchymal cells form the capsule that covers the surface, while a fraction of these cells invaginate into the thymus across the epithelial layers to establish an intrathymic network of fibroblasts. 49,53 Along with this migration, mesoderm-derived progenitor cells enter into the thymus and differentiate into blood vessel endothelial cells in order to form a vascular network. 54 Thus, the thymic epithelial, mesenchymal, and endothelial cells spatially and functionally interact in a coordinated manner in order to organize the thymic microenvironment.
Kernfeld et al performed single-cell RNA sequencing (RNA-seq) of whole cell types from embryonic thymus, including mesenchymal cells and TECs. 66 Figure 3 shows the uniform manifold approximation and projection (UMAP) clustering of their data (GSE107910). Igf1, Fgf7, Fgf10, and Aldh1a2 (a gene encoding an enzyme for retinoic acid biosynthesis) were specifically expressed in mesenchymal cells (cluster 2), suggesting the non-redundant role of mesenchymal cells as a source of these key factors. Bmp4 is expressed in both mesenchymal cells and TECs (cluster 1), consistent with a previous report that the deletion of Bmp4 in both NC-derived cells and endoderm-derived cells (but not either one alone) resulted in defects in thymus organogenesis. 62 The Wnt ligand Wnt4 is reported to induce the expression of FoxN1 in TECs 63 and is highly expressed in TECs, but only slightly in mesenchymal cells, suggesting a role for the Wnt pathway in fetal TEC differentiation, mainly in an autocrine manner. This single-cell study also revealed that the thymic mesenchyme strongly expresses Delta-like non-canonical Notch ligand 1 (Dlk1, also called Pref1), which is reported to support thymocyte cellularity in organ culture. 67 Fetal thymic mesenchymal cells are thought to be a heterogeneous mixture of cells with different characteristics rather than a homogeneous population, and are remotely located in the outer and inner sites of the thymus. However, the mechanism underlying their heterogeneity has yet to be elucidated.
In the following Sections 2.3 to 2.5, recent advances in the identification and characterization of fibroblast subsets as well as other mesenchymal cells in the adult thymus by flow cytometry and transcriptome analyses will be reviewed.

| Flow cytometry of the thymic fibroblast subsets
Since several different markers are co-expressed in different cell types ( FSP1 (S100A4) S100a4 Note: Abbreviations: capFb, capsular fibroblast; cTEC, cortical thymic epithelial cell; EC, endothelial cell; mFb, medullary fibroblast; mTEC, medullary thymic epithelial cell; PC, pericyte; VSMC, vascular smooth muscle cell.  protocols to efficiently dissociate cells from thymus tissue and to distinguish different stromal cell populations using multicolor cytometry. 45,46,69,70 To date, the collagenase extracted from Clostridium histolyticum has been widely used for dissociating thymic stromal cells. Liberase research grade enzymes, a blend of purified collagenase and other proteases, are able to dissociate thymic epithelial cells in higher yield than using crude collagenase products, 71 and are now most widely used as the standard protocol. Although they cleave certain cell surface epitopes and thereby weaken the staining intensity, Liberase enzymes are also useful for preparing non-epithelial thymic stromal cells including fibroblasts, endothelial cells, and vascular mural cells in high yield and quality. Recently, we developed a gradual method of digestion using the Liberase enzyme that allows for the fractionation of thymic cells based on their location within the thymus. 72 This location-based fractionation method allows the physical separation of remotely localized thymic fibroblast subsets, capFbs, and mFbs. We identified a cell-surface protein, dipeptidyl peptidase-4 (DPP4, also called CD26), which is highly expressed in capFbs but not in mFbs, and consequently established a method to separate capFbs (DPP4 + Pdpn + ) and mFbs (DPP4 − Pdpn + ) by flow cytometry (Figure 2A) as well as histological staining ( Figure 1C).
Among the thymic stromal cells, mural cells are relatively difficult to dissociate. When dissociated with collagenase D, a crude collagenase preparation, the yield of Pdpn − CD146 + mural cells, including pericytes and VSMCs is very low or even undetectable compared to the yield when Liberase TM is used ( Figure 2B). This might be due to the excessive cellular damage caused by contaminating components such as endotoxin in the crude enzyme preparations, and possibly explains the reason why these cells have not been readily detected in previous studies (see Section 2.5). Even with Liberase, it is still possible that some unnoticed stromal cell types are lost during enzymatic digestion.
Collectively, however, by using cell dissociation with Liberase and multicolor flow cytometry, it has now been made possible to determine and isolate almost all of the types of stromal cells that compose thymic microenvironment.

| Population-based transcriptome profiling of thymic fibroblasts
In order to characterize the nature and function of thymic fibroblasts, many studies have sought to reveal their gene expression profiles. However, it has been difficult to delineate a unified gene expression pattern for thymic fibroblasts, because the markers used to isolate the cell subsets vary from study to study.
Patenaude and Perreault performed whole transcriptome analysis by RNA-seq of thymic mesenchymal cells (Lineage − EpCAM − CD31 − Sca1 + ). 73 Their results revealed that Sca1 + thymic mesenchymal cells exhibit a higher expression of genes involved in epithelial interaction, apoptotic cell clearance, and T-cell progenitor expansion, compared to their bone or skin counterparts, suggesting a pivotal role for these cells in the thymic microenvironment.
However, since the Sca1 + mesenchymal cell population is a mixture of the fibroblasts, pericytes, and VSMCs, the cell subsets which express each of the key genes remain to be determined.  In the following sections, we will focus on the thymic fibroblast subsets capFb and mFb, summarizing how they develop and regulate T cell development, referring to the studies with transcriptome data as well as genetically modified mouse models. Whether DPP4 is involved in the function of the thymic capsule remains to be elucidated. Figure 5A shows the KEGG pathway enrichment analysis of the transcriptome data of the thymic fibroblast subsets. 72 The Wnt signaling pathway was found to be significantly enriched in capFbs compared with mFbs. Indeed, capFbs express many Wnt family ligands and regulators (Wnt2, Wnt5a, Wnt9a, Wnt11, Sfrp2, and Sfrp4) at higher levels than mFbs as well as other thymic stromal cells ( Figure 5B). Thymic mesenchymal cells (Pdpn + Ly51 − ) are a major thymic source of retinoic acid, which exerts an inhibitory effect on TEC proliferation. 65 The transcriptome data show that the genes encoding retinoic acid-producing enzymes Aldh1a1, Aldh1a2, and Aldh1a3 are strongly expressed in capFbs and/or mesothelial cells: Aldh1a1 in both, Aldh1a2 in mesothelial cells, and Aldh1a3 in capFbs ( Figure 4D and data not shown). Mice with TECs unable to respond to retinoic acid display an aberrant cTEC phenotype, including increased proliferation and the accumulation of an immature population, with a subsequent reduction in thymic cellularity. 91 Thus, the retinoic acid produced in the outermost layer of the thymus acts as a regulator of TECs and is important for normal T cell development. suggesting that CD248 may exert effects in the outermost niches of the thymus. CD248-deficient mice display age-dependent decline of thymus size and thymocyte cellularity, and, in particular, a marked reduction of DN3 thymocytes. 97 The proliferation of DN3 thymocytes that occurs in the subcapsular zone may be regulated by the CD248 expressed in capFbs. It was also shown that CD248-deficient mice exhibit delayed recovery of thymus size and vascularization following infection-induced atrophy. Although the mechanism remains unclear, CD248 may promote re-vascularization and thymocyte growth during postinfection regeneration.

| Control of T cell development by capFbs
How capFbs and mesothelial cells control the outermost barrier of the thymus and thymus integrity needs to be clarified in future.
The interplay between capFbs and subcapsular cTECs may also be important for supporting T cell development in the subcapsular zone and outer cortex, but determining its physiological significance and molecular basis still remains a challenge.

| Development of mFbs
On histological analysis, mFbs are detected as a reticular structure interwoven with but also clearly separated from the network of mTECs. 40,72 A population of mFbs expressing CD34 forms adventitial layers that surround mural cells and endothelial cells, and thus referred to as adventitial cells. 41 In flow cytometry analysis combined with the location-based fractionation method, mFbs are found to be

| Gene expression in mFbs
As a result of KEGG pathway analysis, the genes for TNF signaling and NF-κB signaling as well as antigen processing and presentation were found to be significantly enriched in mFbs compared with capFbs ( Figure 5A). These findings suggest that TNF signaling and NF-κB activation pathways play important roles in development and/or function of mFbs and that mFbs are differentiated such that they have a higher antigen presentation capacity than capFbs. Also, a set of genes, including certain collagens (Col6a5, Col6a6), matrix metalloprotease-9 (Mmp9), metabolic enzymes (Hmgcs2, Ltc4s, and Qprt), and TGFβ-binding proteins (Ltbp1 and Ltbp2) are predominantly expressed in mFbs among all of the thymic stromal cell types.
mFbs form a conduit-like structure that resembles the one formed by fibroblastic reticular cells (FRCs) in the lymph nodes. 40,98 To determine the functional cue for mFbs, the transcriptome was compared between mFbs and lymph node FRCs. 72

| LTβR-dependent maturation of mFbs
Single-cell RNA-seq analysis of mouse thymic stromal cells demonstrated that the genes highly expressed in mFbs, such as Serpine2 and Apod, are prominently detected in clusters 2 and 4 ( Figure 4C,D).
Certain mFb-associated genes, including Mmp9, Ltbp1, and Col6a5, are detectable in cluster 4 but not cluster 2. These cluster 4-specific F I G U R E 6 Comparison of the whole transcriptome between thymic mFbs and lymph node FRCs. RNA-seq data of mFbs, mesenteric lymph node (mLN) FRCs, and skin-draining lymph node (sLN) FRCs (GEO accession no. GSE147357) were used. 72  genes are expressed in mFbs from adult but not neonatal mice, 72 suggesting that clusters 4 and 2 represent mature and immature mFbs, respectively. In addition, most of these mature mFb-associated genes are expressed under the control of the lymphotoxin signal.
The TNF superfamily ligand lymphotoxin (LTα 1 β 2 ) is predominantly expressed by developing SP thymocytes in the thymus and binds to the lymphotoxin β receptor (LTβR) expressed in thymic stromal cells to induce intracellular signal transduction. The LTβR is expressed at the highest level in mFbs among the thymic stromal cells. 72 In mFbs from LTβR-deficient mice, the expression of a large fraction of mFb-associated genes was diminished. Indeed, LTβRdeficient mFbs displayed a reduced expression of mFb-associated proteins such as Pdpn, ICAM-1, and VCAM-1. 41,111 Thus, the LTβR signal critically controls the functional maturation of mFbs. It is known that the LTβR signal is required for the maturation of lymph node FRCs, 112 offering an analogy that shows that these distinct fibroblast subsets share common signaling pathways for maturation.

| Self-antigen expression by mFbs for the induction of immune tolerance
Early studies showed that fibroblasts are capable of presenting selfantigens to induce the positive selection of thymocytes, suggesting that the ability to mediate positive selection is not limited to the thymic epithelium. 113,114 However, a subsequent series of studies revealed that positive selection requires proteasomes and lysosomal proteases that are uniquely expressed in cTECs. [7][8][9][10][11][115][116][117] Also, the major stromal cells that interact with preselected DP thymocytes are cTECs, 118 128,129 Similarly, in the thymus, mFb-specific antigens might also be transferred to and presented by thymic DCs so as to induce T cell tolerance, since a substantial portion (about half) of mTEC-derived self-antigens are indirectly presented by thymic DCs. 130,131 Indeed, it was demonstrated that the cytoplasmic proteins produced in thymic fibroblasts can be transferred to thymic DCs. 72 This mechanism explains how LTβR deficiency in fibroblasts results in the production of autoantibodies against mFb-specific antigens.

| Regulation of mTEC development by mFbs
It is also possible that mFbs indirectly promote T cell tolerance by controlling mTECs, since the fibroblast-specific deletion of the LTβR causes a reduction in the number of mTECs. 72

F I G U R E 7
LTβR-dependent genes in mFbs include TRAs. To define the TRAs, the Shannon entropy score was calculated using the gene expression profiles (GEO accession no. GSE10246). 147 The full source code for analysis is available in GitHub (https://github.com/nitta takes hi/Immun olRev_Fig7). Genes with an entropy score of less than 3.5 are defined as TRA genes. Although these TRA genes were extracted from comprehensive transcriptome data by unbiased mathematical calculation, they may also contain genes that encode functional proteins in mFbs and fibroblast lineage-specific proteins. (A) The gene expression data on the mFbs from Ltbr ΔFb mice (Twist2-Cre Ltbr flox/flox (n = 4)) compared to those from control mice (C57BL/6 (n = 2) and Ltbr flox/flox (n = 4)) are from an RNA-seq dataset (GEO accession no. GSE147357). 72 TRA genes with a mean RPKM >10 in the control mFbs and the ratio of RPKM (Ltbr ΔFb /control) > 0. 5

| Regulation of T cell migration by mFbs
The LTβR signal induces the expression of Pdpn in mFbs. Pdpn is a mucin type glycoprotein expressed in various types of stromal cells and in particular is highly expressed in FRCs in the lymph nodes.
In the thymus, Pdpn is expressed in capFbs and mFbs as well as a fraction of TECs (Table 1). [133][134][135] The extracellular domain of Pdpn binds to various proteins that are secreted by or displayed on other cells. 136 Pdpn + mFbs form conduit-like structures in the medulla and bind the chemokine CCL21 produced by mTECs. 40 In the absence of Pdpn, CCL21 fails to efficiently localize in the medulla, a failure which is accompanied by both inefficient migration and generation of Tregs in the medulla. A similar phenotype is observed in mice lacking CCL21 or CCR7, the receptor for CCL21, suggesting a role for Pdpn-immobilized CCL21 on mFbs in thymic Treg generation.
Two very recent reports demonstrated that CCL21 is displayed on the surface of mFbs and pericytes around blood vessels. 137,138 Cell-surface binding of CCL21 is mediated by the heparan sulfate strongly expressed by these cells, and consistent with this, the EXT family genes, Ext1 and Ext2, that encode the glycosyl transferases for heparan sulfate biosynthesis are highly expressed in mFbs and pericytes. 138  has not yet been elucidated.
As shown by transcriptome analyses, mFbs themselves express certain chemokine genes, such as Cxcl14 and Cx3cl1, but not Ccl21. 72 Whether and how these mFb-specific chemokines contribute to cell migration in the thymus and exert immunological functions are still presently unknown.

| FIB ROB L A S TS IN AG E-REL ATED THYMIC INVOLUTION AND ADIP OS IS
The thymus undergoes an age-related progressive atrophy called involution that is characterized by qualitative and quantitative changes in stromal cells as well as their replacement with adipocytes. 3,139,140 In particular, mTECs exhibit a marked decrease in cellularity and an alteration in gene expression patterns with aging. 46,141 In contrast, the frequency of thymic fibroblasts increases with aging, so the ratio of fibroblasts to TECs is markedly increased in aged mice. 46 It was shown that TECs in aged mice can give rise to fibroblasts and further into adipocytes, by a process called epithelial-to-mesenchymal transition (EMT). 142 contain mesenchymal stem cells. 145 These thymosphere-forming cells were shown to be capable of giving rise to fibroblasts and adipocytes under appropriate culture conditions. It was also shown in an early study that mesenchymal stromal cells isolated from human thymus are able to differentiate in vitro into adipocytes. 146 Nevertheless, at present, there is no conclusive evidence as to which TECs or thymic fibroblasts are responsible for the age-related adiposis of the thymus. This is an important issue for understanding the contribution of the entire repertoire of thymic stromal cells, including fibroblasts, to age-related thymic atrophy, as well as for exploring the possible technologies that would allow thymic regeneration.

| CON CLUDING REMARK S
With the recent advance of large-scale datasets of stromal cells across multiple organs, we now stand at a new beginning for a comprehensive understanding of cellular characteristics and interactions in the im-

CO N FLI C T O F I NTE R E S T
There is no conflict of interest to declare.