The thymus is an intricate primary lymphoid organ, wherein bone marrow–derived lymphoid progenitor cells are induced to develop into functionally competent T cells that express a diverse TCR repertoire, which is selected to allow for the recognition of foreign Ags while avoiding self-reactivity or autoimmunity. Thymus stromal cells, which can include all non–T lineage cells, such as thymic epithelial cells, endothelial cells, mesenchymal/fibroblast cells, dendritic cells, and B cells, provide signals that are essential for thymocyte development as well as for the homeostasis of the thymic stroma itself. In this brief review, we focus on the key roles played by thymic stromal cells during early stages of T cell development, such as promoting the homing of thymic-seeding progenitors, inducing T lineage differentiation, and supporting thymocyte survival and proliferation. We also discuss recent advances on the transcriptional regulation that govern thymic epithelial cell function as well as the cellular and molecular changes that are associated with thymic involution and regeneration.

The thymus is a primary lymphoid organ in which bone marrow–derived lymphoid progenitor cells, with continuous support from thymic stromal cells, develop into functionally competent T cells. The developmental stages followed by the majority of thymocytes are conventionally classified by the surface expression status of CD4 and CD8, starting with double-negative (DN), immature single-positive (SP), double-positive (DP), and mature SP T cells (Fig. 1). The DN stage can be further classified, as DN1 to DN4 cells, based on the expression of CD44 and CD25, with DN1 cells expressing CD44 but not CD25, followed by appearance of CD25 during DN2 stage, loss of CD44 at DN3 stage, and finally, downregulation of CD25 in DN4 cells. The DN1 population contains a small subset (DN1a/b) that is Kit+ (CD117+), also known as early thymic progenitor cells (ETPs), and is regarded as the earliest progeny of the bone marrow–derived thymic-seeding progenitors (TSPs) (1). Throughout their development, thymocytes need to pass three major selection checkpoints: TCR β-selection at the DN3 to DN4 stages and positive and negative selection at the DP to SP stages (Fig. 1). This brief review will focus on the following: 1) the roles thymic stromal cells, which can include all non-T lineage cells, such as thymic epithelial cells (TEC), endothelial cells, mesenchymal/fibroblast cells, dendritic cells (DCs), and B cells, play during early thymocyte development; 2) transcriptional regulation of key genes involved in this process; and, 3) changes in thymic stromal cells during thymic involution and regeneration.

FIGURE 1.

Schematic overview of the thymic stromal cell microenvironment. TSPs follow blood flow (arrow) to reach the thymus and, with the help from adhesion molecules and chemokines, enter the thymus near the CMJ, at which point they become known as ETP. In the cortex, cTECs provide cytokines, such as IL-7 and SCF, to promote ETP proliferation as well as DLL4-mediated Notch signaling to induce the differentiation of ETP toward the T lineage. Following differentiation from the DN to the DP stage, TCR-dependent positive selection events enable the differentiation of conventional SP cells that migrate to medulla in response to chemokines, such as CCL21, and undergo negative selection, in which mTECs, DCs, and medullary fibroblasts have been shown to play an important role. As a result, mature naive T cells expressing a diverse TCR repertoire that can recognize potential foreign Ags while avoiding self-reactivity are generated and leave thymus into circulation.

FIGURE 1.

Schematic overview of the thymic stromal cell microenvironment. TSPs follow blood flow (arrow) to reach the thymus and, with the help from adhesion molecules and chemokines, enter the thymus near the CMJ, at which point they become known as ETP. In the cortex, cTECs provide cytokines, such as IL-7 and SCF, to promote ETP proliferation as well as DLL4-mediated Notch signaling to induce the differentiation of ETP toward the T lineage. Following differentiation from the DN to the DP stage, TCR-dependent positive selection events enable the differentiation of conventional SP cells that migrate to medulla in response to chemokines, such as CCL21, and undergo negative selection, in which mTECs, DCs, and medullary fibroblasts have been shown to play an important role. As a result, mature naive T cells expressing a diverse TCR repertoire that can recognize potential foreign Ags while avoiding self-reactivity are generated and leave thymus into circulation.

Close modal

Homing of progenitor cells.

Thymus organogenesis in mouse embryos starts at the third pharyngeal pouch around embryonic day (E) 11, when the future thymus coexists with the future parathyroid glands in primordia. The common primordia then detach from the pharynx and migrate caudally and medially, during which thymic anlagen separate from the parathyroid anlagen between E12 and E12.5. The parathyroid rudiment then stays where they separate as the thymic anlagen continue their migration within the thorax and ends up on top of the heart (2).

Initially, the thymus, made up of two separate lobes, is solely composed of endoderm-derived TEC progenitors, which are enveloped by a neural crest cell–derived capsule (3). Homing of the first wave of TSPs to thymic rudiment occurs as early as E11.5 and is mediated primarily by the activities of two pairs of chemokine ligand/receptors, namely CCL25/CCR9 and CCL21/CCR7 (46). Another pair of chemokine ligand/receptor, CXCL12/CXCR4, provides partial compensation in the absence of CCL25/CCR9 or CCL21/CCR7 (6). At E11.5, CCL21 but not CCL25 is detected within the common primordia (7). A day later, Ccl21 mRNA is found primarily in the parathyroid region at remarkably high levels, whereas Ccl25 mRNA was found to be expressed exclusively within the thymic rudiment. In contrast, Cxcl12 mRNA was present in both the thymus and surrounding tissues (8). Based on their expression patterns, CCL21 appears to be the major player to recruit the first wave of TSPs to the region surrounding thymus–parathyroid primordia because of its early and higher expression levels, but it would not attract/maintain TSPs exclusively inside of the thymic anlagen. It is the role of CCL25 to attract and maintain TSPs inside of the thymic anlagen. In contrast, CXCL12 could help to recruit TSP to common primordia and further into thymic anlagen in the absence of CCL21 or CCL25, but its contribution would be relatively minor because of its lower expression levels as well as lack of a gradient between the thymic rudiment and surrounding tissues. This is consistent with the impaired thymopoiesis phenotype observed in E14.5 embryos lacking these three chemokine receptors individually or in various combinations (5, 6). Loss of CCR9/CCL25 signaling alone resulted in the greatest reduction, whereas loss of CXCR4/CXCL12 signaling alone led to the mildest reduction in the number of ETPs. Although simultaneous loss of any two signaling pathways resulted in greater reduction in ETPs than loss of corresponding individual ones, double knockout of Ccr9 and Ccr7 almost eliminated the homing of the first wave of TSPs. It should be noted that homing of first-wave TSPs gradually decreased from E12 until the second wave of TSPs homing into the vascularized thymus (9), likely as a result of the separation of thymic anlagen from parathyroid anlagen and consequently reduction in CCL21 levels surrounding thymic rudiment (2). The importance of the parathyroid anlagen in the homing of the first wave TSPs was also reflected by the reduced thymic colonization of lymphoid progenitors in mice deficient in GCM2, the transcription factor critical for parathyroid gland development (2, 5).

After the thymus becomes vascularized around E15.5 (5), chemokines produced by thymic endothelial cells appear to also play an important albeit less essential role in TSP homing. The expression profiles of chemokines in thymic endothelial cells have not been systematically investigated, but mRNA encoding Ccl25, Cxcl12, and Ccl19, another CCR7 ligand, have all been detected in thymic endothelial cells in neonatal or adult mice (1012). Among them, CCL25/CCR9 signaling plays a dominant role in TSP homing, as its deficiency alone but not that of CCR7 and/or CXCR4 led to a detectable reduction in TSP homing, although additional disruption of CCR7 and/or CXCR4 resulted in further ETP reduction in E17.5 or adult thymus (6, 1315). It should be pointed out that chemokines produced by TEC regulate intrathymic trafficking of developing thymocytes (16). In particular, it has recently been shown that CCL21 protein encoded by the Ccl21a gene in medullary TEC (mTEC) is required for recruiting positively selected thymocytes to the medulla for effective negative selection (17).

In addition to chemokines, adhesion molecules expressed on thymic endothelial cells play a critical role in TSP homing. For example, P-selectin on thymic endothelial cells and its ligand PSGL-1, expressed on bone marrow–derived lymphoid progenitor cells, have been demonstrated to be critical for TSP homing (13, 18). In addition, ICAM-1 and VCAM-1, expressed on thymic endothelial cells, were also shown to be required for efficient adhesion and extravasation of TSPs when assayed using short-term homing assays (13).

Thymic endothelial cells likely also produce molecules other than those discussed above to facilitate TSP homing to the thymus. A recent study found that more than half of P-selectin+ thymic endothelial cells did not express Ly-6C at the cell surface, a phenotype similar to high endothelial venules found in lymph nodes (11). These Ly-6CP-selectin+ endothelial cells were preferentially associated with the perivascular space at the corticomedullary junction (CMJ) region (11), where TSP were reported to enter the thymus (19, 20). Disruption of LTβR signaling, which was initiated by engagement of lymphotoxin/LIGHT produced by positively selected SP thymocytes to LTβR expressed on endothelial cells, resulted in a decrease in Ly-6C and an accompanying increase in Ly-6C+ population among P-selectin+ endothelial cells without significantly affecting the total number of P-selectin+ endothelial cells (11). This led to a reduction in the ETP population. RNA-sequencing analysis revealed, among the molecules discussed above, only an ∼2-fold increase of Vcam1 mRNA and ∼5-fold less of Cxcl12 mRNA in Ly-6CP-selectin+ endothelial cells compared with Ly-6C+P-selectin+ endothelial cells, suggesting molecules other than those discussed above contribute to the activity of Ly-6CP-selectin+ endothelial cells to facilitate TSP homing (11). Significant differences in the expression levels of many genes involved in cell adhesion, extracellular matrix organization, and blood vessel morphogenesis were observed (11), although which of these are additionally involved in TSP homing remains to be identified and elucidated.

Thymocyte survival and proliferation.

In the postnatal thymus, the extremely low frequency (<0.001%) of newly arrived TSPs, now called ETPs, highlights the challenges of thymic entry by these rare cells. Nonetheless, ETPs must survive and proliferate to generate a significant pool of cells that will be induced to fully commit to the T cell lineage and eventually give rise to thymocytes bearing a diverse TCR repertoire. Key early players produced by thymic stromal cells are stem cell factor (SCF)/Kit-ligand and IL-7, which provide survival/proliferation signals. DN1 and DN2 cells express high levels of cell surface Kit. In mice with naturally occurring dominant loss-of-function mutation of Kit or Scf, the numbers of pre–β-selection early thymocytes decreased by 40-fold, although total thymic cellularity at birth decreased only by half, because of compensative proliferation (transit-amplifying cell rescue) during later stages of thymocyte development (21). The reduction in early thymocyte numbers was mediated at least partially by reduced cell proliferation as determined by BrdU incorporation.

Elegant transplant experiments showed that engraftment of an Scf mutant thymus under the kidney capsule of a wild-type (WT) recipient led to impaired thymocyte proliferation, demonstrating that it was primarily the loss of intrathymic SCF that affected the cellularity (21). Intrathymic SCF is primarily produced by thymic endothelial cells and cortical TECs (cTECs), with additional contribution from some mesenchymal/fibroblast cells (22). SCF-expressing endothelial cells were found almost exclusively in the cortex and coexpress high-levels of mRNA of Dll4 and Cxcl12 but not Ccl25 (22). There are two forms of SCF, membrane bound and secreted/soluble. DN1 cells but not DN2 cells were found preferentially associated with endothelial cells expressing membrane-bound SCF. Knocking out membrane-bound SCF specifically in endothelial cells led to a 30-fold reduction in the earliest DN1 cells, a 16-fold reduction in late DN1 cells, and an 8-fold reduction in DN2 cells, whereas a knockout of membrane-bound SCF specifically in TEC led to a more significant loss of DN2 cells (15-fold) than DN1 cells (4-fold), consistent with the proximity of DN1 cells to membrane-bound SCF-expressing endothelial cells (22). It should be noted that, although lymphoid progenitor cells in bone marrow express Kit and endothelial cells in bone marrow express SCF, the authors did not observe any decrease in lymphoid progenitors in bone marrow from these mice (22). Furthermore, it was shown that there was increased apoptosis but no difference in cell division when purified DN1 cells were cocultured in vitro with primary thymic stromal cells deficient in membrane-bound SCF compared with WT stromal cells, which led the authors to conclude that membrane-bound SCF mainly provides survival, rather than proliferative, signals to early DN cells (22). However, the in vitro culture conditions might not be optimal for cell proliferation. Given the significant difference in BrdU incorporation in early thymocytes from naturally occurring Scf mutants compared with that from WT mice thymus (21), it is likely that SCF provides both survival and proliferative signals to early developing thymocytes.

IL-7 is a key lymphopoietic cytokine produced solely by TECs in the thymus (23, 24). The IL-7R is composed of two subunits, the IL-7R unique α-chain (IL7Rα/CD127) and the common γ-chain (γc/CD132), shared with IL-2R, IL-4R, IL-9R, IL-15R, and IL-21R. Cell surface IL-7R expression could be detected in all but DP stages of thymocytes, with DN2 and DN3 cells showing the most responsiveness to IL-7 in an in vitro assay (25, 26). Germline deletion of γc, Il7ra, or Il7, as well as TEC-specific deletion of Il7, all led to >90% reduction in thymic cellularity in adult mice (2731). The more severe phenotype than that seen in mice with a loss of SCF/Kit signaling is consistent with the fact that IL-7 signaling impacts all but the DP stages of thymocyte development, in contrast to the confined effect of SCF/Kit signaling on DN1/2 cells.

More DN cells from IL-7–deficient thymus than WT thymus were found in the G0/G1 phase of the cell cycle and were undergoing apoptosis (32). In addition, reduced cell proliferation was also observed in positively selected IL-7R–deficient thymocytes (33, 34), suggesting that both increased cell death and reduced cell proliferation are responsible for a severe reduction in thymic cellularity in the absence of IL-7 signaling. Mechanistically, IL-7 signaling has been found to regulate the expression of trophic receptors and cell cycle regulators in pre- and post–β-selection DN cells (35). In addition, IL-7 signaling has also been shown to upregulate the expression of anti-apoptotic Bcl2 and Mcl1 mRNA (32, 36). Consequently, forced expression of BCL-2 or knocking out proapoptotic Bim could partially restore thymic cellularity in mice deficient of IL-7 signaling (3739). Besides the survival and proliferative activities, IL-7 signaling has also been proposed to coordinate the proliferation, differentiation, and Tcra recombination during β-selection and to participate in the lineage decision of CD8SP cells (35, 40).

In addition to SCF and IL-7, thymic stromal cells have also been proposed to regulate thymocyte survival and proliferation via Notch, CXCL12/CXCR4, Wnt, Hedgehog and BMP signaling (4146). The role of Notch signaling will be discussed in detail in the following section.

Differentiation.

Notch signaling provides the absolutely essential intrathymic cue that dictates lymphoid progenitors with multilineage potential to differentiate exclusively toward the T cell lineage. In the absence of Notch signaling, the thymus becomes a place where the development of B cells instead of T cells is supported (4749). Although lymphoid progenitors and developing thymocytes express NOTCH1, NOTCH2, and NOTCH3 receptors, whereas thymic stromal cells express Notch ligands JAGGED1, JAGGED2, DLL1, and DLL4, only interactions between NOTCH1/DLL4 are physiologically required for T cell development in mice (5053). DLL4 is expressed at high levels in most cTECs but not mTECs as well as thymic endothelial cells (22, 50). Although the functional significance of DLL4 on cTECs has been clearly demonstrated, the role of DLL4 expressed on thymic endothelial cells has not been directly investigated. It should be noted that knocking out Dll4 in all endothelial cells using Cdh5-CreERT2 led to an 80% reduction in the number of ETP and an even more severe (∼90%) decrease in total thymic cellularity (54). The impaired T cell lymphopoiesis was attributed to disruption of the DLL4-mediated bone marrow niche, which led to an ∼2-fold reduction in the frequency of common lymphoid progenitors, a TSP candidate, in the bone marrow. This interpretation is consistent with the recent finding from our laboratory that prethymic Notch signaling is required for generation of TSP in bone marrow (55). Nonetheless, it does not exclude the possibility that loss of DLL4 on thymic endothelial cells could partially contribute to the severely impaired thymopoiesis observed in these pan-endothelial Dll4 knockout mice (54).

A comprehensive recent study compared the distribution of the above mentioned four Notch ligands between postnatal human and mouse thymus and further examined their temporal changes in human thymus using immunofluorescence on thymic sections (56). The authors observed a significant reduction in DLL4 levels in human postnatal (<18 mo) cTEC compared with fetal (11–19 wk of embryonic development) cTECs. Of note, only low levels of DLL4 were observed on rare cTEC at subcapsular regions as well as on some mTEC; although, thymic endothelial cells, perivascular mesenchymal (CK19CD34+) cells, and medullary myeloid (CD11c+) cells still expressed high levels of DLL4. This is consistent with a previous report showing that only 10–20% of cTECs obtained from the thymus of early postnatal humans expressed detectable levels of cell surface DLL4 as determined by flow cytometry (57). Nonetheless, the high levels and frequencies of DLL4 expression seen in fetal cTECs when thymocyte development is highly active, as well as its high expression levels on endothelial cells and perivascular cells at the CMJ, where TSPs enter the thymus, suggest an important role of DLL4 in human T lymphopoiesis (56).

In contrast, active Notch signaling, as indicated by staining for the cleaved NOTCH1 intracellular domain, was seen throughout the cortex, albeit more at the CMJ and subcapsular regions in postnatal human thymus, suggesting that perhaps ligand(s) other that DLL4 may provide Notch signaling at the inner cortex in the postnatal human thymus (56). JAGGED2 was found to be the most prominent Notch ligand in the cortex of postnatal and adult (6- to 11-y-old) human thymus (56), consistent with a previous report that more than 90% of cTEC expressed JAGGED2 in postnatal samples as determined by flow cytometry (57). Ectopic JAGGED2 expression in bone marrow–derived stromal OP9 cells has been shown capable of activating Notch and promoting human T cell development from cord blood–derived hematopoietic progenitor cells (57). Therefore, JAGGED2 might play a more significant role in human T lymphopoiesis as compared with mouse. Furthermore, JAGGED2 expression exhibited a gradient decreasing from CMJ toward the subcapsular region in postnatal but not adult human thymus, opposite of the gradient increasing from CMJ toward subcapsular region in the postnatal mouse thymus. The physiological significance of this gradient and its difference between human and mouse remains to be fully elucidated. In addition, whereas JAGGED1 was found primarily expressed on mTECs in human fetal and postnatal thymus, consistent with its distribution in mouse postnatal thymus, more cTECs in the subcapsular region expressed JAGGED1 in the human adult thymus. The biological relevance of this temporospatial regulation is unknown. Lastly, no significant difference in DLL1 distribution between human and mouse and its temporal regulation in human thymus was observed, suggesting a dispensable role for DLL1 in vivo, as demonstrated by earlier work using TEC-specific deletion of Dll1 in mice (58). However, further validation regarding the sensitivity and specificity of the primary Abs used was missing, raising a concern as to whether some observations might need to be re-examined.

Positive and negative selection and alternative outcomes.

Thymic stromal cells, TECs in particular, educate thymocytes so that newly generated T cells can exit the thymus and recognize foreign Ags in the context of self-MHC while being tolerant to self-antigen recognition in the periphery (59). In brief, cTECs express a unique β5t-containing thymoproteasome that provides a distinctive set of peptides for MHC class I loading that supports the positive selection of CD8SP while expressing CD83 to stabilize MHC class II (MHCII) expression and expressing TSSP and cathepsin L to generate MHCII-associated self-peptides in promoting positive selection of CD4SP, respectively. In contrast, mTECs, together with DCs and B cells, mediate self-tolerance and negative selection by expressing and presenting tissue-restricted self-antigens.

A special type of mTEC, named thymic tuft cells, that has a similar transcriptional profile to intestinal tuft cells were recently identified (60, 61). Its exact biological role in regulating thymocyte development still needs to be further investigated, as the two research groups observed different immunological phenotypes in the absence of thymic tuft cells. In addition to TECs, endothelial cells, DCs, and B cells, there are other types of thymic stromal cells that play various roles in supporting thymocyte development and/or maintaining homeostasis of the thymic stromal environment itself. Mesenchymal/fibroblast cells have been shown to support the organized thymic architecture and thymic vascular network as well as to promote the proliferation and differentiation of TECs (6266). Recently, it has been reported that a subtype of thymic fibroblasts that is FSP1+ is essential for the homeostasis of mature mTECs (67). More significantly and unexpectedly, a recent study revealed that the generation of medullary gp38+ and CD26 fibroblasts, and their expression of tissue-restricted Ags, relied on lymphotoxin-β signaling, as knocking out lymphotoxin-β receptor specifically in fibroblasts led to a loss of central tolerance and autoimmune phenotype (68). Furthermore, neutrophils that are recruited to the medulla by CXCL5 secreted by Hassall corpuscles–mTEC were shown to produce IL-23 to activate plasmacytoid DCs, which in turn produced IFN-α to support SP maturation (69). Moreover, the thymus is well innervated, and neurotransmitters/neuropeptides have been reported to modulate thymopoiesis (70, 71).

The significance of the transcription factor FoxN1 to thymopoiesis has been appreciated since the identification of its mutation as the genetic cause of the athymic nude (nu/nu) phenotype (72). It was further reinforced by the observation that forced expression of FoxN1 alone was enough to induce mouse embryonic fibroblasts to become functional TECs that support T cell development both in vitro and in vivo when engrafted under the kidney capsule (73). It is interesting to note that the thymus was converted to a bipotent lymphoid organ to produce both T cells and B cells when Foxn1 was replace by Foxn4, which closely resembles the metazoan ancestor gene for Foxn1, through transgenic expression of Foxn4 under the Foxn1 promoter in nude mice (74). The generation of a significant number of immature B cells in this transgenic strain was mainly due to lower and perhaps patchy levels of DLL4 and consequently a higher IL7:DLL4 ratio. However, whether the reduction in Dll4 mRNA resulted from a decrease in Dll4 expression in all cells and/or a decrease in percentage of TECs expressing Dll4 mRNA was not investigated; but it might be biologically relevant, as the immature B cells were found predominantly in the perivascular space, spatially separated from thymocytes.

Chromatin immunoprecipitation sequencing analyses of genome-wide FoxN1-binding sites confirmed that many thymopoietic genes discussed above, such as Dll4, Cxcl12, Ccl25, and Psmb11, are direct transcriptional targets of FoxN1 (75, 76). Consistent with a previous observation (77), Il7 was not found to be a FoxN1 target gene. However, it is surprising that Scf was not found to be a direct FoxN1 transcription target, given that it was not expressed in the TECs from nude thymic anlagen (75) but was upregulated during FoxN1-induced transformation of mouse embryonic fibroblasts to TEC (73). In addition, several other genes, whose expression were previously shown to be downregulated following a decrease in FoxN1 protein level (78), such as MHCII, Cathepsin L, CD40, and Aire, were not found to be high-confidence FoxN1 direct transcriptional targets (76). Therefore, transcriptional regulation of these genes by FoxN1 must be mediated by a yet-to-be-identified transcription factor(s) that itself could be a direct FoxN1 transcriptional target. In contrast, Psmb11/β5t, CD83, and Tssp/Prss16, which are required for efficient positive selection of CD8+ and CD4+ SPs, respectively, were identified as high-confidence FoxN1 direct transcriptional targets, although their regulation by FoxN1 was not previously recognized. Direct transcriptional activation of Psmb11/β5t by FoxN1 was confirmed by another independent study (79). Furthermore, many mitochondria- and Golgi-related genes were found to be direct FoxN1 transcriptional targets, although their physiological activities related to thymopoiesis have not been investigated and remain to be revealed.

Despite the critical role of FoxN1 in creating and maintaining the thymopoietic microenvironment, how FoxN1 expression within the initial thymic anlage becomes turned on at E11 in a tissue-specific manner is not completely understood. Defects in Pax1/Pax9, Eya1, Hox3a, and Six1 resulted in impaired thymus development and therefore may regulate FoxN1 expression (8084). In addition, BMP4 and Wnt signaling have been reported to modulate FoxN1 expression levels in TECs (8587). However, evidence that these transcription factors bind to the FoxN1 promoter and/or enhancer region to initiate and maintain FoxN1 expression in a tissue-specific manner is still lacking. Recently an enhancer region located within its first intron was found to be required for thymus-specific expression of FoxN1 (88). In addition, consistent with previous observations, putative PAX1, SIX1, and SMAD binding sites were identified in this enhancer region. However, the physiological relevance of these putative binding sites remains to be further investigated and may hold the key as to what factor(s) turn on FoxN1 expression and thus bring about thymopoiesis.

The thymus undergoes postpuberty age-associated involution in both mouse and human, and changes in thymic stromal cells, especially a decrease in the numbers of TECs, has been proposed to be the primary cause underlying age-associated involution or atrophy (8992). In addition, a decrease in TEC numbers has also been reported to occur during pregnancy-induced thymic atrophy and in male mice undergoing chemotherapy (92, 93). In contrast, no reduction in TEC numbers were observed in two recent studies examining age-associated or pregnancy-induced thymic involution, respectively (94, 95). This discrepancy resulted mostly from how TEC numbers were measured. All studies that observed a reduction in TEC numbers used flow cytometry following enzymatic digestion to estimate TEC cellularity, which has recently been shown by two independent research groups to dramatically underestimate TEC numbers by at least 10-fold (96, 97). More importantly, the loss of different types of TECs during this procedure was not proportional (96). Therefore, the recovery rate for individual subtypes of TECs might be inconsistent under different biological conditions, and consequently, it is technically unreliable to compare total TEC numbers and composition using flow cytometry–based approaches. In the two recent studies that did not observe a decrease in TEC numbers during thymic involution/atrophy, one elegantly employed immunofluorescence microscopy to count TECs; the other still used flow cytometry but with a new enzymatic digestion protocol, which the authors reported to recover ∼five-times more TECs compared with the enzymatic digestion protocol used in previous studies (95).

When qualitative change in thymic stromal cells during pregnancy-induced involution and postpartum regeneration was examined using bulk RNA-sequencing, the expression levels of FoxN1 and its target genes, such as Dll4, Ccl25, and Cxcl12, were found to be lower in cTEC during involution and rebound significantly during early postpartum regeneration (95). This is consistent with previous observations showing that FoxN1 expression levels gradually decreased during aging (98100). In contrast, forced expression of FoxN1 appeared to rejuvenate an aged thymus (101), suggesting that changes in the expression levels of FoxN1 and its target genes is one of the driving forces behind thymic involution and regeneration. In addition, a change in Il7 mRNA levels was also observed during pregnancy-induced involution and postpartum regeneration as well as age-associated thymic involution (95, 98), implying a role for a reduction in IL7 levels in driving thymic involution. However, what led to the reduction in FoxN1 and Il7 mRNA levels remains to be investigated.

A profound change in cTEC cell size and morphology was recently reported to occur during age-associated involution and transient regeneration of aged thymus after surgical castration (94). Using an elegant and powerful multicolor confetti expression approach under the control of FoxN1-Cre, which labeled almost all TECs with one of the four fluorescence proteins individually or in diverse combinations, enabled the visualization and measurement of cell morphology and size. In young (∼5-wk-old) thymus, long and looping cell projections from individual cTECs form an extensive intracellular labyrinth with 20–30 holes, with each hole filled with 2–10 thymocytes. As a result, each cTEC can support the development of 100–150 thymocytes, consistent with the estimated thymocyte/cTEC ratio of ∼100 for 10-d-old thymus (96). In contrast, cell projections were difficult to detect in aged (12-mo-old) thymus, especially at the subcapsular region, because of shortening and thinning of cell projections. Consequently, the total cell surface area of individual cTEC decreased by ∼50%, whereas cell volume reduced by ∼30% in aged compared with young thymuses. During regeneration after castration, the extended cell projections and complex cell morphology were restored in some but not all cTECs. In contrast, no change in mTEC morphology was observed during involution and regeneration. Of note, no active cell proliferation was observed for cTECs during regeneration of aged thymus, although it was readily detectable for mTECs. Therefore, it was the change in cell morphology and consequently the thymopoietic activity but not the quantity of cTECs that drives thymic involution and regeneration. Given that castration has been shown to increase FoxN1 protein levels in, but not to increase the percentage of, FoxN1-expressing TEC (99), it is tempting to speculate that only FoxN1-expressing cTECs restored their morphology after castration as a result of elevated FoxN1 protein levels through the activities of high-confident FoxN1 target genes involved in mitochondria and Golgi functions.

The thymus is a sophisticated primary lymphoid organ, with its stromal cell components providing all the necessary support to promote thymocyte development. An appreciation of one key stromal cell–dependent event mediated by Notch receptor–ligand interactions enabled us to develop the OP9-DL coculture system to produce T lineage cells in vitro and open the field for further studies of the molecular mechanisms involved in this process (102, 103). We hope that, with further investigation on, and better understanding of, signals provided by thymic stromal cells during the development of distinct subtypes of T cells, we will be able to create more advanced cell culture system(s) capable of generating all types of T cells as desired and enable new avenues for their clinical and therapeutic applications.

We are grateful to Dr. Michele K. Anderson for advice and comments during the preparation of this review.

This work was supported by grants from the Natural Sciences and Engineering Research Council (2016-06592) and the Canadian Institutes of Health Research (154332). J.C.Z.-P. was supported by a Canada Research Chair in Developmental Immunology.

Abbreviations used in this article:

CMJ

corticomedullary junction

cTEC

cortical TEC

DC

dendritic cell

DN

double-negative

DP

double-positive

E

embryonic day

ETP

early thymic progenitor cell

MHCII

MHC class II

mTEC

medullary TEC

SCF

stem cell factor

SP

single-positive

TEC

thymic epithelial cell

TSP

thymic-seeding progenitor

WT

wild-type.

1
Petrie
,
H. T.
,
J. C.
Zúñiga-Pflücker
.
2007
.
Zoned out: functional mapping of stromal signaling microenvironments in the thymus.
Annu. Rev. Immunol.
25
:
649
679
.
2
Gordon
,
J.
,
N. R.
Manley
.
2011
.
Mechanisms of thymus organogenesis and morphogenesis.
Development
138
:
3865
3878
.
3
Manley
,
N. R.
,
E. R.
Richie
,
C. C.
Blackburn
,
B. G.
Condie
,
J.
Sage
.
2011
.
Structure and function of the thymic microenvironment.
Front. Biosci.
16
:
2461
2477
.
4
Moore
,
M. A.
,
J. J.
Owen
.
1967
.
Experimental studies on the development of the thymus.
J. Exp. Med.
126
:
715
726
.
5
Liu
,
C.
,
F.
Saito
,
Z.
Liu
,
Y.
Lei
,
S.
Uehara
,
P.
Love
,
M.
Lipp
,
S.
Kondo
,
N.
Manley
,
Y.
Takahama
.
2006
.
Coordination between CCR7- and CCR9-mediated chemokine signals in prevascular fetal thymus colonization.
Blood
108
:
2531
2539
.
6
Calderón
,
L.
,
T.
Boehm
.
2011
.
Three chemokine receptors cooperatively regulate homing of hematopoietic progenitors to the embryonic mouse thymus.
Proc. Natl. Acad. Sci. USA
108
:
7517
7522
.
7
Liu
,
C.
,
T.
Ueno
,
S.
Kuse
,
F.
Saito
,
T.
Nitta
,
L.
Piali
,
H.
Nakano
,
T.
Kakiuchi
,
M.
Lipp
,
G. A.
Hollander
,
Y.
Takahama
.
2005
.
The role of CCL21 in recruitment of T-precursor cells to fetal thymi.
Blood
105
:
31
39
.
8
Bleul
,
C. C.
,
T.
Boehm
.
2000
.
Chemokines define distinct microenvironments in the developing thymus.
Eur. J. Immunol.
30
:
3371
3379
.
9
Ramond
,
C.
,
C.
Berthault
,
O.
Burlen-Defranoux
,
A. P.
de Sousa
,
D.
Guy-Grand
,
P.
Vieira
,
P.
Pereira
,
A.
Cumano
.
2014
.
Two waves of distinct hematopoietic progenitor cells colonize the fetal thymus.
Nat. Immunol.
15
:
27
35
.
10
Gossens
,
K.
,
S.
Naus
,
S. Y.
Corbel
,
S.
Lin
,
F. M.
Rossi
,
J.
Kast
,
H. J.
Ziltener
.
2009
.
Thymic progenitor homing and lymphocyte homeostasis are linked via S1P-controlled expression of thymic P-selectin/CCL25.
J. Exp. Med.
206
:
761
778
.
11
Shi
,
Y.
,
W.
Wu
,
Q.
Chai
,
Q.
Li
,
Y.
Hou
,
H.
Xia
,
B.
Ren
,
H.
Xu
,
X.
Guo
,
C.
Jin
, et al
.
2016
.
LTβR controls thymic portal endothelial cells for haematopoietic progenitor cell homing and T-cell regeneration.
Nat. Commun.
7
:
12369
.
12
Ueno
,
T.
,
K.
Hara
,
M. S.
Willis
,
M. A.
Malin
,
U. E.
Höpken
,
D. H.
Gray
,
K.
Matsushima
,
M.
Lipp
,
T. A.
Springer
,
R. L.
Boyd
, et al
.
2002
.
Role for CCR7 ligands in the emigration of newly generated T lymphocytes from the neonatal thymus.
Immunity
16
:
205
218
.
13
Scimone
,
M. L.
,
I.
Aifantis
,
I.
Apostolou
,
H.
von Boehmer
,
U. H.
von Andrian
.
2006
.
A multistep adhesion cascade for lymphoid progenitor cell homing to the thymus.
Proc. Natl. Acad. Sci. USA
103
:
7006
7011
.
14
Krueger
,
A.
,
S.
Willenzon
,
M.
Lyszkiewicz
,
E.
Kremmer
,
R.
Förster
.
2010
.
CC chemokine receptor 7 and 9 double-deficient hematopoietic progenitors are severely impaired in seeding the adult thymus.
Blood
115
:
1906
1912
.
15
Zlotoff
,
D. A.
,
A.
Sambandam
,
T. D.
Logan
,
J. J.
Bell
,
B. A.
Schwarz
,
A.
Bhandoola
.
2010
.
CCR7 and CCR9 together recruit hematopoietic progenitors to the adult thymus.
Blood
115
:
1897
1905
.
16
Takahama
,
Y.
2006
.
Journey through the thymus: stromal guides for T-cell development and selection.
Nat. Rev. Immunol.
6
:
127
135
.
17
Kozai
,
M.
,
Y.
Kubo
,
T.
Katakai
,
H.
Kondo
,
H.
Kiyonari
,
K.
Schaeuble
,
S. A.
Luther
,
N.
Ishimaru
,
I.
Ohigashi
,
Y.
Takahama
.
2017
.
Essential role of CCL21 in establishment of central self-tolerance in T cells.
J. Exp. Med.
214
:
1925
1935
.
18
Rossi
,
F. M.
,
S. Y.
Corbel
,
J. S.
Merzaban
,
D. A.
Carlow
,
K.
Gossens
,
J.
Duenas
,
L.
So
,
L.
Yi
,
H. J.
Ziltener
.
2005
.
Recruitment of adult thymic progenitors is regulated by P-selectin and its ligand PSGL-1.
Nat. Immunol.
6
:
626
634
.
19
Lind
,
E. F.
,
S. E.
Prockop
,
H. E.
Porritt
,
H. T.
Petrie
.
2001
.
Mapping precursor movement through the postnatal thymus reveals specific microenvironments supporting defined stages of early lymphoid development.
J. Exp. Med.
194
:
127
134
.
20
Mori
,
K.
,
M.
Itoi
,
N.
Tsukamoto
,
H.
Kubo
,
T.
Amagai
.
2007
.
The perivascular space as a path of hematopoietic progenitor cells and mature T cells between the blood circulation and the thymic parenchyma.
Int. Immunol.
19
:
745
753
.
21
Rodewald
,
H. R.
,
K.
Kretzschmar
,
W.
Swat
,
S.
Takeda
.
1995
.
Intrathymically expressed c-kit ligand (stem cell factor) is a major factor driving expansion of very immature thymocytes in vivo.
Immunity
3
:
313
319
.
22
Buono
,
M.
,
R.
Facchini
,
S.
Matsuoka
,
S.
Thongjuea
,
D.
Waithe
,
T. C.
Luis
,
A.
Giustacchini
,
P.
Besmer
,
A. J.
Mead
,
S. E.
Jacobsen
,
C.
Nerlov
.
2016
.
A dynamic niche provides kit ligand in a stage-specific manner to the earliest thymocyte progenitors.
Nat. Cell Biol.
18
:
157
167
.
23
Moore
,
N. C.
,
G.
Anderson
,
C. A.
Smith
,
J. J.
Owen
,
E. J.
Jenkinson
.
1993
.
Analysis of cytokine gene expression in subpopulations of freshly isolated thymocytes and thymic stromal cells using semiquantitative polymerase chain reaction.
Eur. J. Immunol.
23
:
922
927
.
24
Alves
,
N. L.
,
O.
Richard-Le Goff
,
N. D.
Huntington
,
A. P.
Sousa
,
V. S.
Ribeiro
,
A.
Bordack
,
F. L.
Vives
,
L.
Peduto
,
A.
Chidgey
,
A.
Cumano
, et al
.
2009
.
Characterization of the thymic IL-7 niche in vivo.
Proc. Natl. Acad. Sci. USA
106
:
1512
1517
.
25
Sudo
,
T.
,
S.
Nishikawa
,
N.
Ohno
,
N.
Akiyama
,
M.
Tamakoshi
,
H.
Yoshida
,
S.
Nishikawa
.
1993
.
Expression and function of the interleukin 7 receptor in murine lymphocytes.
Proc. Natl. Acad. Sci. USA
90
:
9125
9129
.
26
Van De Wiele
,
C. J.
,
J. H.
Marino
,
B. W.
Murray
,
S. S.
Vo
,
M. E.
Whetsell
,
T. K.
Teague
.
2004
.
Thymocytes between the beta-selection and positive selection checkpoints are nonresponsive to IL-7 as assessed by STAT-5 phosphorylation.
J. Immunol.
172
:
4235
4244
.
27
Peschon
,
J. J.
,
P. J.
Morrissey
,
K. H.
Grabstein
,
F. J.
Ramsdell
,
E.
Maraskovsky
,
B. C.
Gliniak
,
L. S.
Park
,
S. F.
Ziegler
,
D. E.
Williams
,
C. B.
Ware
, et al
.
1994
.
Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice.
J. Exp. Med.
180
:
1955
1960
.
28
von Freeden-Jeffry
,
U.
,
P.
Vieira
,
L. A.
Lucian
,
T.
McNeil
,
S. E.
Burdach
,
R.
Murray
.
1995
.
Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine.
J. Exp. Med.
181
:
1519
1526
.
29
Cao
,
X.
,
E. W.
Shores
,
J.
Hu-Li
,
M. R.
Anver
,
B. L.
Kelsall
,
S. M.
Russell
,
J.
Drago
,
M.
Noguchi
,
A.
Grinberg
,
E. T.
Bloom
, et al
.
1995
.
Defective lymphoid development in mice lacking expression of the common cytokine receptor gamma chain.
Immunity
2
:
223
238
.
30
DiSanto
,
J. P.
,
W.
Müller
,
D.
Guy-Grand
,
A.
Fischer
,
K.
Rajewsky
.
1995
.
Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor gamma chain.
Proc. Natl. Acad. Sci. USA
92
:
377
381
.
31
Shitara
,
S.
,
T.
Hara
,
B.
Liang
,
K.
Wagatsuma
,
S.
Zuklys
,
G. A.
Holländer
,
H.
Nakase
,
T.
Chiba
,
S.
Tani-ichi
,
K.
Ikuta
.
2013
.
IL-7 produced by thymic epithelial cells plays a major role in the development of thymocytes and TCRγδ+ intraepithelial lymphocytes.
J. Immunol.
190
:
6173
6179
.
32
von Freeden-Jeffry
,
U.
,
N.
Solvason
,
M.
Howard
,
R.
Murray
.
1997
.
The earliest T lineage-committed cells depend on IL-7 for Bcl-2 expression and normal cell cycle progression.
Immunity
7
:
147
154
.
33
Hare
,
K. J.
,
E. J.
Jenkinson
,
G.
Anderson
.
2000
.
An essential role for the IL-7 receptor during intrathymic expansion of the positively selected neonatal T cell repertoire.
J. Immunol.
165
:
2410
2414
.
34
Tani-ichi
,
S.
,
A.
Shimba
,
K.
Wagatsuma
,
H.
Miyachi
,
S.
Kitano
,
K.
Imai
,
T.
Hara
,
K.
Ikuta
.
2013
.
Interleukin-7 receptor controls development and maturation of late stages of thymocyte subpopulations.
Proc. Natl. Acad. Sci. USA
110
:
612
617
.
35
Boudil
,
A.
,
I. R.
Matei
,
H. Y.
Shih
,
G.
Bogdanoski
,
J. S.
Yuan
,
S. G.
Chang
,
B.
Montpellier
,
P. E.
Kowalski
,
V.
Voisin
,
S.
Bashir
, et al
.
2015
.
IL-7 coordinates proliferation, differentiation and Tcra recombination during thymocyte β-selection.
Nat. Immunol.
16
:
397
405
.
36
Opferman
,
J. T.
,
A.
Letai
,
C.
Beard
,
M. D.
Sorcinelli
,
C. C.
Ong
,
S. J.
Korsmeyer
.
2003
.
Development and maintenance of B and T lymphocytes requires antiapoptotic MCL-1.
Nature
426
:
671
676
.
37
Akashi
,
K.
,
M.
Kondo
,
U.
von Freeden-Jeffry
,
R.
Murray
,
I. L.
Weissman
.
1997
.
Bcl-2 rescues T lymphopoiesis in interleukin-7 receptor-deficient mice.
Cell
89
:
1033
1041
.
38
Maraskovsky
,
E.
,
L. A.
O’Reilly
,
M.
Teepe
,
L. M.
Corcoran
,
J. J.
Peschon
,
A.
Strasser
.
1997
.
Bcl-2 can rescue T lymphocyte development in interleukin-7 receptor-deficient mice but not in mutant rag-1-/- mice.
Cell
89
:
1011
1019
.
39
Pellegrini
,
M.
,
P.
Bouillet
,
M.
Robati
,
G. T.
Belz
,
G. M.
Davey
,
A.
Strasser
.
2004
.
Loss of Bim increases T cell production and function in interleukin 7 receptor-deficient mice.
J. Exp. Med.
200
:
1189
1195
.
40
McCaughtry
,
T. M.
,
R.
Etzensperger
,
A.
Alag
,
X.
Tai
,
S.
Kurtulus
,
J. H.
Park
,
A.
Grinberg
,
P.
Love
,
L.
Feigenbaum
,
B.
Erman
,
A.
Singer
.
2012
.
Conditional deletion of cytokine receptor chains reveals that IL-7 and IL-15 specify CD8 cytotoxic lineage fate in the thymus.
J. Exp. Med.
209
:
2263
2276
.
41
Ciofani
,
M.
,
J. C.
Zúñiga-Pflücker
.
2005
.
Notch promotes survival of pre-T cells at the beta-selection checkpoint by regulating cellular metabolism.
Nat. Immunol.
6
:
881
888
.
42
Trampont
,
P. C.
,
A. C.
Tosello-Trampont
,
Y.
Shen
,
A. K.
Duley
,
A. E.
Sutherland
,
T. P.
Bender
,
D. R.
Littman
,
K. S.
Ravichandran
.
2010
.
CXCR4 acts as a costimulator during thymic beta-selection.
Nat. Immunol.
11
:
162
170
.
43
Tussiwand
,
R.
,
C.
Engdahl
,
N.
Gehre
,
N.
Bosco
,
R.
Ceredig
,
A. G.
Rolink
.
2011
.
The preTCR-dependent DN3 to DP transition requires Notch signaling, is improved by CXCL12 signaling and is inhibited by IL-7 signaling.
Eur. J. Immunol.
41
:
3371
3380
.
44
Liang
,
H.
,
A. H.
Coles
,
Z.
Zhu
,
J.
Zayas
,
R.
Jurecic
,
J.
Kang
,
S. N.
Jones
.
2007
.
Noncanonical Wnt signaling promotes apoptosis in thymocyte development.
J. Exp. Med.
204
:
3077
3084
.
45
El Andaloussi
,
A.
,
S.
Graves
,
F.
Meng
,
M.
Mandal
,
M.
Mashayekhi
,
I.
Aifantis
.
2006
.
Hedgehog signaling controls thymocyte progenitor homeostasis and differentiation in the thymus.
Nat. Immunol.
7
:
418
426
.
46
Hager-Theodorides
,
A. L.
,
S. V.
Outram
,
D. K.
Shah
,
R.
Sacedon
,
R. E.
Shrimpton
,
A.
Vicente
,
A.
Varas
,
T.
Crompton
.
2002
.
Bone morphogenetic protein 2/4 signaling regulates early thymocyte differentiation.
J. Immunol.
169
:
5496
5504
.
47
Radtke
,
F.
,
A.
Wilson
,
G.
Stark
,
M.
Bauer
,
J.
van Meerwijk
,
H. R.
MacDonald
,
M.
Aguet
.
1999
.
Deficient T cell fate specification in mice with an induced inactivation of Notch1.
Immunity
10
:
547
558
.
48
Wilson
,
A.
,
H. R.
MacDonald
,
F.
Radtke
.
2001
.
Notch 1-deficient common lymphoid precursors adopt a B cell fate in the thymus.
J. Exp. Med.
194
:
1003
1012
.
49
Han
,
H.
,
K.
Tanigaki
,
N.
Yamamoto
,
K.
Kuroda
,
M.
Yoshimoto
,
T.
Nakahata
,
K.
Ikuta
,
T.
Honjo
.
2002
.
Inducible gene knockout of transcription factor recombination signal binding protein-J reveals its essential role in T versus B lineage decision.
Int. Immunol.
14
:
637
645
.
50
Koch
,
U.
,
E.
Fiorini
,
R.
Benedito
,
V.
Besseyrias
,
K.
Schuster-Gossler
,
M.
Pierres
,
N. R.
Manley
,
A.
Duarte
,
H. R.
Macdonald
,
F.
Radtke
.
2008
.
Delta-like 4 is the essential, nonredundant ligand for Notch1 during thymic T cell lineage commitment.
J. Exp. Med.
205
:
2515
2523
.
51
Hozumi
,
K.
,
C.
Mailhos
,
N.
Negishi
,
K.
Hirano
,
T.
Yahata
,
K.
Ando
,
S.
Zuklys
,
G. A.
Holländer
,
D. T.
Shima
,
S.
Habu
.
2008
.
Delta-like 4 is indispensable in thymic environment specific for T cell development.
J. Exp. Med.
205
:
2507
2513
.
52
Shi
,
J.
,
M.
Fallahi
,
J. L.
Luo
,
H. T.
Petrie
.
2011
.
Nonoverlapping functions for Notch1 and Notch3 during murine steady-state thymic lymphopoiesis.
Blood
118
:
2511
2519
.
53
Suliman
,
S.
,
J.
Tan
,
K.
Xu
,
P. C.
Kousis
,
P. E.
Kowalski
,
G.
Chang
,
S. E.
Egan
,
C.
Guidos
.
2011
.
Notch3 is dispensable for thymocyte β-selection and Notch1-induced T cell leukemogenesis.
PLoS One
6
: e24937.
54
Tikhonova
,
A. N.
,
I.
Dolgalev
,
H.
Hu
,
K. K.
Sivaraj
,
E.
Hoxha
,
Á.
Cuesta-Domínguez
,
S.
Pinho
,
I.
Akhmetzyanova
,
J.
Gao
,
M.
Witkowski
, et al
.
2019
.
The bone marrow microenvironment at single-cell resolution. [Published erratum appears in 2019 Nature 572: E6.]
Nature
569
:
222
228
.
55
Chen
,
E. L. Y.
,
P. K.
Thompson
,
J. C.
Zúñiga-Pflücker
.
2019
.
RBPJ-dependent Notch signaling initiates the T cell program in a subset of thymus-seeding progenitors.
Nat. Immunol.
20
:
1456
1468
.
56
García-León
,
M. J.
,
P.
Fuentes
,
J. L.
de la Pompa
,
M. L.
Toribio
.
2018
.
Dynamic regulation of NOTCH1 activation and Notch ligand expression in human thymus development.
Development
145
: dev165597.
57
Van de Walle
,
I.
,
G.
De Smet
,
M.
Gärtner
,
M.
De Smedt
,
E.
Waegemans
,
B.
Vandekerckhove
,
G.
Leclercq
,
J.
Plum
,
J. C.
Aster
,
I. D.
Bernstein
, et al
.
2011
.
Jagged2 acts as a Delta-like Notch ligand during early hematopoietic cell fate decisions.
Blood
117
:
4449
4459
.
58
Hozumi
,
K.
,
N.
Negishi
,
D.
Suzuki
,
N.
Abe
,
Y.
Sotomaru
,
N.
Tamaoki
,
C.
Mailhos
,
D.
Ish-Horowicz
,
S.
Habu
,
M. J.
Owen
.
2004
.
Delta-like 1 is necessary for the generation of marginal zone B cells but not T cells in vivo.
Nat. Immunol.
5
:
638
644
.
59
Kondo
,
K.
,
I.
Ohigashi
,
Y.
Takahama
.
2019
.
Thymus machinery for T-cell selection.
Int. Immunol.
31
:
119
125
.
60
Bornstein
,
C.
,
S.
Nevo
,
A.
Giladi
,
N.
Kadouri
,
M.
Pouzolles
,
F.
Gerbe
,
E.
David
,
A.
Machado
,
A.
Chuprin
,
B.
Tóth
, et al
.
2018
.
Single-cell mapping of the thymic stroma identifies IL-25-producing tuft epithelial cells.
Nature
559
:
622
626
.
61
Miller
,
C. N.
,
I.
Proekt
,
J.
von Moltke
,
K. L.
Wells
,
A. R.
Rajpurkar
,
H.
Wang
,
K.
Rattay
,
I. S.
Khan
,
T. C.
Metzger
,
J. L.
Pollack
, et al
.
2018
.
Thymic tuft cells promote an IL-4-enriched medulla and shape thymocyte development.
Nature
559
:
627
631
.
62
Jenkinson
,
W. E.
,
E. J.
Jenkinson
,
G.
Anderson
.
2003
.
Differential requirement for mesenchyme in the proliferation and maturation of thymic epithelial progenitors.
J. Exp. Med.
198
:
325
332
.
63
Jenkinson
,
W. E.
,
S. W.
Rossi
,
S. M.
Parnell
,
E. J.
Jenkinson
,
G.
Anderson
.
2007
.
PDGFRalpha-expressing mesenchyme regulates thymus growth and the availability of intrathymic niches.
Blood
109
:
954
960
.
64
Itoi
,
M.
,
N.
Tsukamoto
,
H.
Yoshida
,
T.
Amagai
.
2007
.
Mesenchymal cells are required for functional development of thymic epithelial cells.
Int. Immunol.
19
:
953
964
.
65
Foster
,
K.
,
J.
Sheridan
,
H.
Veiga-Fernandes
,
K.
Roderick
,
V.
Pachnis
,
R.
Adams
,
C.
Blackburn
,
D.
Kioussis
,
M.
Coles
.
2008
.
Contribution of neural crest-derived cells in the embryonic and adult thymus.
J. Immunol.
180
:
3183
3189
.
66
Müller
,
S. M.
,
C. C.
Stolt
,
G.
Terszowski
,
C.
Blum
,
T.
Amagai
,
N.
Kessaris
,
P.
Iannarelli
,
W. D.
Richardson
,
M.
Wegner
,
H. R.
Rodewald
.
2008
.
Neural crest origin of perivascular mesenchyme in the adult thymus.
J. Immunol.
180
:
5344
5351
.
67
Sun
,
L.
,
C.
Sun
,
Z.
Liang
,
H.
Li
,
L.
Chen
,
H.
Luo
,
H.
Zhang
,
P.
Ding
,
X.
Sun
,
Z.
Qin
,
Y.
Zhao
.
2015
.
FSP1(+) fibroblast subpopulation is essential for the maintenance and regeneration of medullary thymic epithelial cells.
Sci. Rep.
5
:
14871
.
68
Nitta
,
T.
,
M.
Tsutsumi
,
S.
Nitta
,
R.
Muro
,
E. C.
Suzuki
,
K.
Nakano
,
Y.
Tomofuji
,
S.
Sawa
,
T.
Okamura
,
J. M.
Penninger
,
H.
Takayanagi
.
2020
.
Fibroblasts as a source of self-antigens for central immune tolerance.
Nat. Immunol.
21
:
1172
1180
.
69
Wang
,
J.
,
M.
Sekai
,
T.
Matsui
,
Y.
Fujii
,
M.
Matsumoto
,
O.
Takeuchi
,
N.
Minato
,
Y.
Hamazaki
.
2019
.
Hassall’s corpuscles with cellular-senescence features maintain IFNα production through neutrophils and pDC activation in the thymus.
Int. Immunol.
31
:
127
139
.
70
Leposavić
,
G.
,
I.
Pilipović
,
M.
Perišić
.
2011
.
Cellular and nerve fibre catecholaminergic thymic network: steroid hormone dependent activity.
Physiol. Res.
60
(
Suppl. 1
):
S71
S82
.
71
Leposavić
,
G. M.
,
I. M.
Pilipović
.
2018
.
Intrinsic and extrinsic thymic adrenergic networks: sex steroid-dependent plasticity.
Front. Endocrinol. (Lausanne)
9
:
13
.
72
Nehls
,
M.
,
D.
Pfeifer
,
M.
Schorpp
,
H.
Hedrich
,
T.
Boehm
.
1994
.
New member of the winged-helix protein family disrupted in mouse and rat nude mutations.
Nature
372
:
103
107
.
73
Bredenkamp
,
N.
,
S.
Ulyanchenko
,
K. E.
O’Neill
,
N. R.
Manley
,
H. J.
Vaidya
,
C. C.
Blackburn
.
2014
.
An organized and functional thymus generated from FOXN1-reprogrammed fibroblasts.
Nat. Cell Biol.
16
:
902
908
.
74
Swann
,
J. B.
,
A.
Weyn
,
D.
Nagakubo
,
C. C.
Bleul
,
A.
Toyoda
,
C.
Happe
,
N.
Netuschil
,
I.
Hess
,
A.
Haas-Assenbaum
,
Y.
Taniguchi
, et al
.
2014
.
Conversion of the thymus into a bipotent lymphoid organ by replacement of FOXN1 with its paralog, FOXN4.
Cell Rep.
8
:
1184
1197
.
75
Calderón
,
L.
,
T.
Boehm
.
2012
.
Synergistic, context-dependent, and hierarchical functions of epithelial components in thymic microenvironments.
Cell
149
:
159
172
.
76
Žuklys
,
S.
,
A.
Handel
,
S.
Zhanybekova
,
F.
Govani
,
M.
Keller
,
S.
Maio
,
C. E.
Mayer
,
H. Y.
Teh
,
K.
Hafen
,
G.
Gallone
, et al
.
2016
.
Foxn1 regulates key target genes essential for T cell development in postnatal thymic epithelial cells.
Nat. Immunol.
17
:
1206
1215
.
77
Zamisch
,
M.
,
B.
Moore-Scott
,
D. M.
Su
,
P. J.
Lucas
,
N.
Manley
,
E. R.
Richie
.
2005
.
Ontogeny and regulation of IL-7-expressing thymic epithelial cells.
J. Immunol.
174
:
60
67
.
78
Nowell
,
C. S.
,
N.
Bredenkamp
,
S.
Tetélin
,
X.
Jin
,
C.
Tischner
,
H.
Vaidya
,
J. M.
Sheridan
,
F. H.
Stenhouse
,
R.
Heussen
,
A. J.
Smith
,
C. C.
Blackburn
.
2011
.
Foxn1 regulates lineage progression in cortical and medullary thymic epithelial cells but is dispensable for medullary sublineage divergence.
PLoS Genet.
7
: e1002348.
79
Uddin
,
M. M.
,
I.
Ohigashi
,
R.
Motosugi
,
T.
Nakayama
,
M.
Sakata
,
J.
Hamazaki
,
Y.
Nishito
,
I.
Rode
,
K.
Tanaka
,
T.
Takemoto
, et al
.
2017
.
Foxn1-β5t transcriptional axis controls CD8+ T-cell production in the thymus.
Nat. Commun.
8
:
14419
.
80
Manley
,
N. R.
,
M. R.
Capecchi
.
1995
.
The role of Hoxa-3 in mouse thymus and thyroid development.
Development
121
:
1989
2003
.
81
Wallin
,
J.
,
H.
Eibel
,
A.
Neubüser
,
J.
Wilting
,
H.
Koseki
,
R.
Balling
.
1996
.
Pax1 is expressed during development of the thymus epithelium and is required for normal T-cell maturation.
Development
122
:
23
30
.
82
Peters
,
H.
,
A.
Neubüser
,
K.
Kratochwil
,
R.
Balling
.
1998
.
Pax9-deficient mice lack pharyngeal pouch derivatives and teeth and exhibit craniofacial and limb abnormalities.
Genes Dev.
12
:
2735
2747
.
83
Xu
,
P. X.
,
W.
Zheng
,
C.
Laclef
,
P.
Maire
,
R. L.
Maas
,
H.
Peters
,
X.
Xu
.
2002
.
Eya1 is required for the morphogenesis of mammalian thymus, parathyroid and thyroid.
Development
129
:
3033
3044
.
84
Zou
,
D.
,
D.
Silvius
,
J.
Davenport
,
R.
Grifone
,
P.
Maire
,
P. X.
Xu
.
2006
.
Patterning of the third pharyngeal pouch into thymus/parathyroid by Six and Eya1.
Dev. Biol.
293
:
499
512
.
85
Balciunaite
,
G.
,
M. P.
Keller
,
E.
Balciunaite
,
L.
Piali
,
S.
Zuklys
,
Y. D.
Mathieu
,
J.
Gill
,
R.
Boyd
,
D. J.
Sussman
,
G. A.
Holländer
.
2002
.
Wnt glycoproteins regulate the expression of FoxN1, the gene defective in nude mice.
Nat. Immunol.
3
:
1102
1108
.
86
Wertheimer
,
T.
,
E.
Velardi
,
J.
Tsai
,
K.
Cooper
,
S.
Xiao
,
C. C.
Kloss
,
K. J.
Ottmüller
,
Z.
Mokhtari
,
C.
Brede
,
P.
deRoos
, et al
.
2018
.
Production of BMP4 by endothelial cells is crucial for endogenous thymic regeneration.
Sci. Immunol.
3
: eaal2736.
87
Swann
,
J. B.
,
B.
Krauth
,
C.
Happe
,
T.
Boehm
.
2017
.
Cooperative interaction of BMP signalling and Foxn1 gene dosage determines the size of the functionally active thymic epithelial compartment.
Sci. Rep.
7
:
8492
.
88
Larsen
,
B. M.
,
J. E.
Cowan
,
Y.
Wang
,
Y.
Tanaka
,
Y.
Zhao
,
B.
Voisin
,
M. G.
Constantinides
,
K.
Nagao
,
Y.
Belkaid
,
P.
Awasthi
, et al
.
2019
.
Identification of an intronic regulatory element necessary for tissue-specific expression of Foxn1 in thymic epithelial cells.
J. Immunol.
203
:
686
695
.
89
Gray
,
D. H.
,
N.
Seach
,
T.
Ueno
,
M. K.
Milton
,
A.
Liston
,
A. M.
Lew
,
C. C.
Goodnow
,
R. L.
Boyd
.
2006
.
Developmental kinetics, turnover, and stimulatory capacity of thymic epithelial cells.
Blood
108
:
3777
3785
.
90
Gui
,
J.
,
X.
Zhu
,
J.
Dohkan
,
L.
Cheng
,
P. F.
Barnes
,
D. M.
Su
.
2007
.
The aged thymus shows normal recruitment of lymphohematopoietic progenitors but has defects in thymic epithelial cells.
Int. Immunol.
19
:
1201
1211
.
91
Chinn
,
I. K.
,
C. C.
Blackburn
,
N. R.
Manley
,
G. D.
Sempowski
.
2012
.
Changes in primary lymphoid organs with aging.
Semin. Immunol.
24
:
309
320
.
92
Hun
,
M. L.
,
K.
Wong
,
J. R.
Gunawan
,
A.
Alsharif
,
K.
Quinn
,
A. P.
Chidgey
.
2020
.
Gender disparity impacts on thymus aging and LHRH receptor antagonist-induced thymic reconstitution following chemotherapeutic damage.
Front. Immunol.
11
:
302
.
93
Laan
,
M.
,
U.
Haljasorg
,
K.
Kisand
,
A.
Salumets
,
P.
Peterson
.
2016
.
Pregnancy-induced thymic involution is associated with suppression of chemokines essential for T-lymphoid progenitor homing.
Eur. J. Immunol.
46
:
2008
2017
.
94
Venables
,
T.
,
A. V.
Griffith
,
A.
DeAraujo
,
H. T.
Petrie
.
2019
.
Dynamic changes in epithelial cell morphology control thymic organ size during atrophy and regeneration.
Nat. Commun.
10
:
4402
.
95
Dumont-Lagacé
,
M.
,
T.
Daouda
,
L.
Depoërs
,
J.
Zumer
,
Y.
Benslimane
,
S.
Brochu
,
L.
Harrington
,
S.
Lemieux
,
C.
Perreault
.
2020
.
Qualitative changes in cortical thymic epithelial cells drive postpartum thymic regeneration.
Front. Immunol.
10
:
3118
.
96
Hirakawa
,
M.
,
D.
Nagakubo
,
B.
Kanzler
,
S.
Avilov
,
B.
Krauth
,
C.
Happe
,
J. B.
Swann
,
A.
Nusser
,
T.
Boehm
.
2018
.
Fundamental parameters of the developing thymic epithelium in the mouse.
Sci. Rep.
8
:
11095
.
97
Sakata
,
M.
,
I.
Ohigashi
,
Y.
Takahama
.
2018
.
Cellularity of thymic epithelial cells in the postnatal mouse.
J. Immunol.
200
:
1382
1388
.
98
Ortman
,
C. L.
,
K. A.
Dittmar
,
P. L.
Witte
,
P. T.
Le
.
2002
.
Molecular characterization of the mouse involuted thymus: aberrations in expression of transcription regulators in thymocyte and epithelial compartments.
Int. Immunol.
14
:
813
822
.
99
Rode
,
I.
,
V. C.
Martins
,
G.
Küblbeck
,
N.
Maltry
,
C.
Tessmer
,
H. R.
Rodewald
.
2015
.
Foxn1 protein expression in the developing, aging, and regenerating thymus.
J. Immunol.
195
:
5678
5687
.
100
O’Neill
,
K. E.
,
N.
Bredenkamp
,
C.
Tischner
,
H. J.
Vaidya
,
F. H.
Stenhouse
,
C. D.
Peddie
,
C. S.
Nowell
,
T.
Gaskell
,
C. C.
Blackburn
.
2016
.
Foxn1 is dynamically regulated in thymic epithelial cells during embryogenesis and at the onset of thymic involution.
PLoS One
11
: e0151666.
101
Bredenkamp
,
N.
,
C. S.
Nowell
,
C. C.
Blackburn
.
2014
.
Regeneration of the aged thymus by a single transcription factor.
Development
141
:
1627
1637
.
102
Schmitt
,
T. M.
,
J. C.
Zúñiga-Pflücker
.
2002
.
Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro.
Immunity
17
:
749
756
.
103
Mohtashami
,
M.
,
D. K.
Shah
,
H.
Nakase
,
K.
Kianizad
,
H. T.
Petrie
,
J. C.
Zúñiga-Pflücker
.
2010
.
Direct comparison of Dll1- and Dll4-mediated Notch activation levels shows differential lymphomyeloid lineage commitment outcomes.
J. Immunol.
185
:
867
876
.

The authors have no financial conflicts of interest.