Thymus machinery for T-cell selection

How thymic epithelial cells control the T-cell repertoire

Abstract

An immunocompetent and self-tolerant pool of naive T cells is formed in the thymus through the process of repertoire selection. T cells that are potentially capable of responding to foreign antigens are positively selected in the thymic cortex and are further selected in the thymic medulla to help prevent self-reactivity. The affinity between T-cell antigen receptors expressed by newly generated T cells and self-peptide–major histocompatibility complexes displayed in the thymic microenvironments plays a key role in determining the fate of developing T cells during thymic selection. Recent advances in our knowledge of the biology of thymic epithelial cells have revealed unique machinery that contributes to positive and negative selection in the thymus. In this article, we summarize recent findings on thymic T-cell selection, focusing on the machinery unique to thymic epithelial cells.

Introduction

The thymus produces T cells and selects newly produced T cells to generate a functionally competent and self-tolerant T-cell pool. After migrating into the microenvironment of the thymic cortex, hematopoietic lymphoid progenitors are induced to develop into T-cell antigen receptor (TCR)-expressing CD4⁺CD8⁺ double-positive (DP) thymocytes. The V(D)J rearrangement of TCRα and TCRβ genomic loci in early thymocytes allows the formation of great diversity in the pre-selected repertoire of TCR recognition specificities in newly generated cortical DP thymocytes, which are individually selected for their fate according to their TCR recognition specificities. DP thymocytes that express TCRs interacting at low affinity with self-peptide-associated major histocompatibility complex class I (MHC-I) or MHC class II (MHC-II) molecules displayed in the thymic cortex are rescued from cell death to survive and differentiate into CD4⁺CD8^– or CD4^–CD8⁺ single-positive (SP) thymocytes, a process called positive selection in the thymus (1–3).

Positively selected thymocytes begin expressing the chemokine receptor CCR7 and migrate to the thymic medulla, where a subpopulation of medullary thymic epithelial cells (mTECs) produce CCR7 ligands, including CCL21 (4, 5), thereby attracting the positively selected thymocytes. In the thymic medulla, a distinct subpopulation of mTECs produces a wide range of self-molecules, including ‘tissue-specific’ self-proteins (i.e. proteins that are otherwise only found in specific peripheral tissues) and displays the vast majority of self-antigens, in cooperation with dendritic cells (DCs) (6–8).

Medullary thymocytes that express TCRs with high affinity for self-peptide–MHC (self-pMHC) complexes displayed in the medullary microenvironment are either deleted by apoptosis or destined to become regulatory T cells and other agonist-selected ‘unconventional’ T-cell populations (3, 9, 10). Negative selection in the thymus conventionally refers to the process of self-antigen-mediated deletion of developing thymocytes through apoptosis, including deletion of the high-affinity self-reactive thymocytes in the medulla. The lineage deviation in the medulla, which gives rise to regulatory and other ‘unconventional’ T cells by high-affinity TCR interactions, could be viewed as a form of positive selection of unconventional T cells, but could alternatively be viewed as a form of negative selection of thymocytes to exclude self-reactive cells from the ‘conventional’ effector T-cell pool.

Here, we start by discussing the importance of TCR affinity thresholds in thymocyte selection. Focusing on recent advances, we then detail the machinery that is utilized in the cortex and in the medulla to screen developing T cells and enable the optimal repertoire to be available for peripheral immune responses.

TCR affinity

The affinity between TCRs and their peptide–MHC ligands determines the fate of developing thymocytes. Early studies showed that differences in ligand concentration and ligating TCR valency as well as differences among agonist and antagonist peptide ligands affect life-or-death determination in immature thymocytes (11–14). The findings that agonist peptide ligation promotes differentiation into unconventional T-cell lineages, including regulatory T cells, pointed to the view that the quality of TCR signals triggered in developing thymocytes not only determines life-or-death selection in generating a conventional T-cell pool but also contributes to determining a way to direct cells towards unconventional T-cell subpopulations (15, 16).

It is now understood that the affinity between TCRs expressed by developing thymocytes and self-pMHC complexes displayed in the thymus is the major parameter that determines the fate of newly generated T cells. The TCR–ligand affinity determines life or death (i.e. positive and negative selection) as well as lineage direction to become functionally distinct cells (e.g. conventional CD4⁺ helper and CD8⁺ killer T cells). A low-affinity interaction between TCR and pMHC promotes thymocyte maturation to give rise to functionally competent T cells (i.e. positive selection), whereas a high-affinity interaction causes the deletion of self-reactive T cells (i.e. negative selection) or the generation of unconventional T cells (1, 17).

The narrow TCR–pMHC affinity range sets the threshold for life or death in developing thymocytes, contributing to the enrichment of functionally potent and self-protective T cells while excluding potentially harmful self-reactive T cells from the mature T-cell pool (18, 19). It is considered that the affinity threshold determines the dependence on intra-thymocyte signals through Themis, Tespa1, a voltage-gated sodium-ion channel and miR-181a (a non-coding small RNA), all of which are expressed in pre-selected DP thymocytes and are important for promoting positive selection rather than negative selection (20–24).

Within the TCR–ligand affinity range that promotes positive selection, the ligand affinity during positive selection influences later functional capability in positively selected T cells (25–28), as was recently discussed elsewhere (29). Recent studies have indicated that CD8⁺ T cells differ in functional capability depending on their ontogeny during the fetal, neonatal and adult periods (30) and that the TCR–ligand affinity during positive selection appears higher in neonatal mice than in adult mice (31). These observations suggest that the magnitude of TCR–ligand affinity optimal for thymic positive selection, which affects subsequent antigen responsiveness in T cells, diminishes through postnatal ontogeny and ageing.

Cortex machinery

In the thymic cortex, cortical TECs (cTECs) architecturally and functionally provide the microenvironment for T-lymphoid progenitor cells to become T cells, for example by supplying T-lineage-inducing molecules including DLL4 and IL-7 (32, 33). The cTECs also provide a unique microenvironment, for example by expressing MHC-I and MHC-II molecules, for newly generated T cells to undergo positive selection (34, 35). Recent studies have revealed that cTECs are heterogeneous in mediating these functions (35, 36) and are capable of optimizing T-cell positive selection by further providing unique machinery for protein degradation (Fig. 1).

Fig. 1.

Cortical protein degradation and medullary gene expression in the thymus.

MHC-II-associated self-peptide production in cTECs

Peptides associated with MHC-II molecules are generated by proteolysis within the lumen of the endosomal–lysosomal system. Cathepsin L, a lysosomal endopeptidase abundant in cTECs, is involved in the processing of MHC-II invariant chain (Ii) and in the generation of MHC-II-associated self-peptides that promote cTEC-mediated positive selection of CD4⁺ T cells (37–39). Another endosomal peptidase, thymus-specific serine protease (TSSP, also known as Prss16), is also abundant in cTECs. Studies using MHC-II-restricted TCR transgenic mice showed that TSSP contributes to the production of positively selecting MHC-II-associated self-peptides (40–42). TSSP is also reported to play a role in the production of negatively selecting MHC-II-associated self-peptides, influencing the severity of autoimmune inflammation in type 1 diabetes in the NOD mouse strain (43–45).

Both cathepsin L and TSSP are abundant in cTECs but are also expressed in various cell types other than cTECs. Their co-abundance in combination with other proteases may create a unique endosomal–lysosomal proteolytic microenvironment in cTECs, which may contribute to the production of unique MHC-II-associated self-peptides that optimize positive selection of CD4⁺ T cells.

Unlike most cells, in which autophagy is activated by nutrient starvation, cTECs are constitutively active in autophagy-mediated degradation of cytoplasmic proteins (46, 47). Autophagy in cTECs contributes to the production of MHC-II-associated self-peptides, including cytoplasmic protein-derived peptides, thereby optimizing the repertoire selection of CD4⁺ T cells (47, 48).

Stabilization of surface MHC-II in cTECs

March family E3 ubiquitin ligases regulate the degradation and turnover of MHC-II molecules. The cTECs predominantly express March8, the activity of which is attenuated by the immunoglobulin superfamily cell surface protein CD83, which is also expressed in cTECs. CD83 stabilizes surface MHC-II molecules in cTECs by slowing March8-mediated MHC-II turnover (49, 50). CD83-deficient mice are defective in MHC-II stabilization in cTECs and in CD4⁺ T-cell generation in the thymus (49–51), whereas these defects are rescued by a deficiency in March8 (49, 50). CD83- and March8-mediated stabilization of surface MHC-II molecules in cTECs may contribute to maintaining persistent TCR engagement, which is important for positive selection of CD4⁺ T cells in the thymus (2, 52).

MHC-I-associated self-peptide production in cTECs

Peptides associated with MHC-I molecules are generated through proteasome-mediated degradation of cytoplasmic proteins. The cTECs express a unique form of proteasome, termed the thymoproteasome, which is characterized by the inclusion of the catalytic subunit β5t, also known as Psmb11 (53, 54). The expression of β5t and β5t-containing thymoproteasomes is highly specific to cTECs (55, 56). In comparison with other forms of proteasomes, β5t-containing thymoproteasomes exhibit altered proteolytic specificity, producing a unique set of MHC-I-associated peptides (57, 58). In β5t-deficient mice, CD8⁺ T cells are reduced in numbers, altered in TCR repertoire and defective in fine-tuning TCR responsiveness (28, 59–61). Thus, thymoproteasome-dependent positive selection in the thymic cortex is important for promoting positive selection of CD8⁺ T cells. The possible mechanisms underlying how β5t-containing thymoproteasome works in the positive selection of CD8⁺ T cells are discussed elsewhere (62–64).

Human genome variations in β5t

In humans, β5t is also specifically expressed in cTECs (65). More than 150 kinds of genomic variations, including single nucleotide polymorphisms in the β5t-coding sequence, have been registered in the National Center for Biotechnology Information (NCBI) public database. Among those variants, the rs34457782 single nucleotide polymorphism that alters the 49th amino acid of the human β5t pro-protein from glycine to serine (G49S) is detected at an appreciable allele frequency in various human populations (66). The 49th amino acid is located at the carboxyl terminus of the pro-peptide that is cleaved to generate catalytically active β5t protein, the amino terminus of which begins at the 50th threonine.

The G49S variant in either human β5t or mouse β5t has inefficient processing of the pro-peptide to generate mature β5t protein (66). In mice that are manipulated to carry this variation in the genome, heterozygotes show reduced β5t expression in cTECs and homozygotes further exhibit reduction in the numbers of CD8⁺ T cells in the thymus and the periphery (66). In cohort studies in humans, however, no severe health problems have been noticed in many heterozygous and a small number of homozygous human individuals (66). A study has reported that homozygosity in this variation is associated with an elevated risk of Sjögren’s syndrome, an autoimmune disease (67). Further analysis of the health of humans with this genomic variation, particularly of the homozygotes, would help improve our understanding of the thymoproteasome-dependent positive selection of CD8⁺ T cells in humans.

Medulla machinery

It is the privilege of positively selected thymocytes to migrate to the thymic medulla (68, 69). In the medullary microenvironment, the thymocytes must interact with a variety of antigen-presenting cells, including mTECs and hematopoietic DCs, in order to establish self-tolerance in T cells (Fig. 1). Only cells that survive medullary selection may be released from the thymus to the circulation as mature naive T cells.

Promiscuous gene expression in mTECs

More than 85% of all genes encoded in the genome, including many tissue-restricted self-antigen genes, are detectable in mTECs (70–73). This characteristic, termed promiscuous gene expression (pGE), in mTECs contributes to the display of self-antigens, including tissue-restricted self-antigens, to newly generated T cells and therefore the establishment of self-tolerance in T cells.

The nuclear protein Aire is an epigenetic regulator that magnifies pGE in mTECs through interactions with multiple partner proteins (74–78). It has been shown that Aire enhances gene transcription by releasing RNA polymerase II at silenced loci, promoting promiscuousness in gene expression (79). It has also been shown that Aire limits transcription by repressing Brg1-mediated chromatin accessibility, which is implicated as a mechanism to avoid the deleterious effects of ectopically expressed genes (80). The development and function of mTECs, including pGE, is regulated by signals mediated via tumor necrosis factor superfamily members RANK, CD40 and LTβR (81–83)—the ligands of which are provided by positively selected thymocytes and by other thymic hematopoietic cells (84–86)—and by transcription factors RelB, NFκB2, Fezf2 and Blimp1 (87–92).

Hematopoietic antigen-presenting cells

The thymic medulla is enriched in hematopoietic antigen-presenting cells, including DCs and B cells, which contribute to the establishment of self-tolerance in T cells (93–97). Thymic DCs are classified into CD8α⁺Sirpα^– conventional DCs (cDC1s), CD8α^–Sirpα⁺ cDCs (cDC2s) and plasmacytoid DCs (pDCs). It has been shown that cDC1s are intrathymically generated, whereas cDC2s and pDCs are derived from the peripheral circulation into the thymus (98–100).

The cDC1s efficiently mediate the cross-presentation of mTEC-derived pGE antigens and efficiently promote the generation of regulatory T cells (101). The scavenger receptor CD36 expressed by cDC1s facilitates the transfer of mTEC-derived self-antigens to cDC1s (102). The chemokine CCL21Ser, which is produced by an LTβR-dependent mTEC subpopulation (103), promotes the recruitment of cDC1 progenitors to the thymus (104). The chemokine XCL1, which is produced by a distinct Aire-dependent mTEC subpopulation, supports the interplay between mTECs and DCs to promote the generation of regulatory T cells (105).

Thymic tuft cells

The mTEC population is functionally and morphologically heterogeneous, including cystic ciliated cells, neurosecretory cells, concentric Hassall’s corpuscles and cholinergic chemosensory cells (106–108). Recent studies have highlighted a subpopulation of mTECs that resemble tuft cells of the small intestine (109, 110). Like tuft cells in the gut, the thymic tuft cells are dependent on the transcription factor Pou2f3 for their generation, and express chemosensory receptors and the pro-inflammatory cytokine IL-25 (109, 110). Unlike intestinal tuft cells, however, thymic tuft cells are generated as a post-Aire mTEC subpopulation and express both MHC-I and MHC-II molecules (109). The functions of thymic tuft cells are not clear, although reports suggest their roles in supporting type 2 invariant natural killer T cells (iNKT2s) to form an IL-4-rich microenvironment and/or in controlling type 2 innate lymphoid cells (ILC2s) in the thymic medulla.

Medullary thymocyte traffic

Multiple chemokines expressed by mTECs and other cells in the thymic medulla are important for regulating the traffic of developing thymocytes. Positively selected thymocytes, which are generated in the thymic cortex, begin expressing CCR7 and migrate to the thymic medulla in response to CCR7 ligands produced by mTECs (68, 69, 111, 112). A recent study has pointed to the non-redundant importance of CCL21Ser among several different CCR7 ligands (113). The CCR7–CCL21Ser chemokine axis essentially contributes to the establishment of self-tolerance in T cells in the thymic medulla (113). The medullary migration of positively selected thymocytes is additionally regulated by chemokines CXCL12 and CCL25 as well as Sema3E–PlexinD1 guidance proteins (114–117). Chemokines CCL17 and CCL22 and their receptor CCR4 are also suggested to contribute to the medullary migration of positively selected thymocytes (118, 119).

The egress of mature thymocytes from the thymic medulla to the circulation is regulated by the interaction between sphingosine-1-phosphate receptor 1 (S1P1) expressed by mature thymocytes and S1P, which is abundant in the circulation (120). The role of CCR7 chemokine signals in thymic emigration during the newborn period has also been described (121). A recent study has suggested that CCR7 expression on iNKT cells defines iNKT precursor cells that emigrate from the thymus (122). Another study has pointed to a role for type 2 IL-4 receptor-mediated signals in mTECs in the thymic egress of mature thymocytes (123).

Conclusions

The cortical and medullary microenvironments in the thymus coordinate to sequentially support the formation of a self-protective T-cell pool, by providing distinct yet unique machinery in cTECs and mTECs (Fig. 1). The thymic cortex offers a unique protein degradation machinery both in the cytoplasm and in the endosomal–lysosomal lumen of cTECs to promote the display of a unique set of self-peptides that are important for positive selection of functionally competent T cells. In contrast, the thymic medulla is equipped with unique gene expression machinery in the nuclei of mTECs, enabling the display of an unusually promiscuous set of self-peptides that contribute to the establishment of self-tolerance in newly generated T cells.

These machineries in cTECs and mTECs appear mechanistically and anatomically distinct from each other, in protein degradation and in gene expression, respectively. However, these sets of machinery share a common importance in displaying the body’s unique self to developing thymocytes for the formation of a functionally competent yet self-tolerant T-cell pool. The coordination of these sets of machinery for cTEC-dependent positive selection and mTEC-dependent self-tolerance is central to the development of the functional immune system.

As summarized in this article, recent advances in TEC biology have revealed new mechanisms that contribute to T-cell repertoire formation in the thymus. In parallel, studies have uncovered tremendous diversity in TECs (Fig. 2). Still, these findings have been largely achieved by the analysis of cTECs and mTECs isolated from the thymus by enzymatic digestion method, which can yield only a small fraction of cTECs and mTECs (124, 125). The biology in the majority of TECs has yet to be examined. Further advances in thymus biology may accelerate our understanding of T-cell repertoire formation and immune system development.

Fig. 2.

Multiple tasks coordinated by cortex and medulla in the thymus.

Funding

This work was supported by the Intramural Research Program of the US National Institutes of Health, the National Cancer Institute and the Center for Cancer Research, and by grants from MEXT-JSPS (16H02630 to Y.T., 17K08884 to I.O. and 17K15727 to K.K.), Ichiro Kanehara Foundation (to I.O.), Daiichi Sankyo Foundation of Life Science (to I.O.) and Glaxo Smith Kline Japan (to K.K.).

Conflicts of interest statement: The authors declared no conflicts of interest.