CD69 expression discriminates MHC-dependent and -independent stages of thymocyte positive selection

In the thymus, phenotypically and functionally mature single positive cells are generated from immature CD4+8+ precursors by a process known as positive selection. Although this event is known to involve alphabetaTCR ligation by peptide/MHC complexes expressed on thymic stromal cells, it is clear that positive selection is a multistage process involving transition through an intermediate CD4+8+69+ phase as well as subsequent postselection phases. By analyzing the development of preselection CD4+8+69- and intermediate CD4+8+69+ thymocytes in the presence of MHC class I-deficient, MHC class II-deficient, and MHC double-deficient thymic stromal cells, we investigated the role of MHC molecules at three distinct points during positive selection. Although the initiation of positive selection is critically dependent upon MHC interactions, we find the that later stages of maturation, involving the differentiation of CD4+8- and CD4-8+ cells from CD4+8+69+ thymocytes, occur in the absence of MHC molecules. Moreover, an analysis of the postselection proliferation of newly generated CD4+8- and CD4-8+ thymocytes shows that this also occurs independently of MHC molecules. Thus, our data provide direct evidence that, although positive selection is a multistage process initiated by TCR-MHC interactions, continuation of this process and subsequent postselection events are independent of ongoing engagement of the TCR.     

Peptide specificity of thymic selection of CD4+CD25+ T cells.

The CD4(+)CD25(+) regulatory T cells can be found in the thymus, but their need to undergo positive and negative selection has been questioned. Instead, it has been hypothesized that CD4(+)CD25(+) cells mature following TCR binding to MHC backbone, to low abundant MHC/peptide complexes, or to class II MHC loaded with peripheral autoantigens. In all these circumstances, processes that are distinct from positive and negative selection would govern the provenance of CD4(+)CD25(+) cells in the thymus. By comparing the development of CD4(+)CD25(-) and CD4(+)CD25(+) cells in mice expressing class II MHC molecules bound with one or many peptide(s), we show that the CD4(+)CD25(+) cells appear during natural selection of CD4(+) T cells. The proportion of CD4(+)CD25(+) cells in the population of CD4(+) thymocytes remains constant, and their total number reflects the complexity of selecting class II MHC/peptide complexes. Hence, thymic development of CD4(+)CD25(+) cells does not exclusively depend on the low-density, high-affinity MHC/peptide complexes or thymic presentation of peripheral self-Ags, but, rather, these cells are selected as a portion of the natural repertoire of CD4(+) T cells. Furthermore, while resistant to deletion mediated by endogenous superantigen(s), these cells were negatively selected on class II MHC/peptide complexes. We postulate that while the CD4(+)CD25(+) thymocytes are first detectable in the thymic medulla, their functional commitment occurs in the thymic cortex.     

Specialisation of thymic epithelial cells for positive selection of CD4+8+ thymocytes.

Following their migration into the thymus, hemopoeitic stem cell precursors enter a complex developmental pathway involving proliferation, differentiation and alphabetaT-cell receptor (alphabetaTCR)-mediated selection procedures, in order to generate mature T-cell populations ready for export to the periphery. Thus, a critical stage during intrathymic T-cell development involves the generation of functionally mature CD4+8- and CD4-8+ cells from immature CD4+8- precursor thymocytes, a poorly understood process referred to as positive selection. While interactions between the alphabetaTCR and MHC-peptide complexes are known to be essential for the initiation of positive selection, additional unknown signals are also required. Using an in vitro reaggregate thymic organ culture system which allows comparison of the abilities of various cell types to induce maturation of CD4+8+ precursors, we provide evidence that both MHC-peptide complexes and specialised accessory molecules must be provided by thymic epithelium for efficient mediation of positive selection. Moreover, analysis of positive selection in the presence of thymic and non-thymic stromal cells expressing MHC class II molecules with the same limited peptide array suggests that this unique ability of thymic epithelium to mediate positive selection of CD4+8- cells is not solely due to presentation of a specialised peptide repertoire, but is dependent upon provision of specialised accessory interactions.     

Expression of CD1 and class I MHC antigens by human thymocytes

The acquisition of surface class I MHC molecules is associated with the maturation of thymocytes. Here, surface expression of class I MHC and CD1, which represents a family of MHC-related molecules, was analyzed on various human immature and mature thymocyte subpopulations. Class I expression was inversely related to the expression of CD1. The majority of CD4+ CD8+ cortical type thymocytes expressed low levels of class I MHC Ag, the previously described CD4+ CD8+ thymocyte subpopulation with low CD8 expression exhibited intermediate levels of class I MHC, whereas most of the single positive CD4 and CD8 thymocytes displayed high levels of class I MHC. Biochemical comparison of CD1 and class I showed that thymic class I molecules were post-translationally modified by phosphorylation, whereas CD1 was not phosphorylated. Furthermore, our studies suggested that in addition to CD1/CD8 complexes, thymocytes bear CD8/class I complexes. Chemical cross-linking and peptide mapping studies clearly identified the CD8-associated protein on thymic clones as the class I MHC molecule.     

Cutting edge: thymic selection and autoreactivity are regulated by multiple coreceptors involved in T cell activation.

Immune responses are shaped by several processes that promote responses to pathogens and hinder responses to self. One mechanism that contributes to this polarization in response is negative selection, in which thymocytes that can respond to self-peptide/MHC complexes are deleted from the T cell repertoire. I found here that several coreceptors known to contribute to mature T cell activation also participate in negative selection. Interestingly, these molecules appeared to act in a cooperative fashion. Blocking the contribution of these molecules in fetal thymus organ culture not only prevented negative selection in the CD4+ lineage, but also induced the appearance of autoreactive thymocytes. This is the first demonstration that blocking coreceptor interactions during thymic development can produce autoreactive T cells. The contribution of negative selection to the mature T cell repertoire and to autoimmunity is discussed in light of these results.     

TCR signaling for initiation and completion of thymocyte positive selection has distinct requirements for ligand quality and presenting cell type

Thymocyte selection involves signaling by TCR engaging diverse self-peptide:MHC molecule ligands on various cell types in the cortex and medulla. Here we separately analyze early and late stages of selection to better understand how presenting cell type, ligand quality, and the timing of TCR signaling contribute to intrathymic differentiation. TCR transgenic CD4+CD8+ thymocytes (double positive (DP)) from MHC-deficient mice were stimulated using various presenting cells and ligands. The resulting CD69high cells were isolated and evaluated for maturation in reaggregate cultures with wild-type or MHC molecule-deficient thymic stroma with or without added hemopoietic dendritic cells (DC). Production of CD4+ T cells required TCR signaling in the reaggregates, indicating that transient recognition of self-ligands by DP is inadequate for full differentiation. DC bearing a potent agonist ligand could initiate positive selection, producing activated thymocytes that matured into agonist-responsive T cells in reaggregates lacking the same ligand. DC could also support the TCR signaling necessary for late maturation. These results argue that despite the negative role assigned to DC in past studies, neither the peptide:MHC molecule complexes present on DC nor any other signals provided by these cells stimulate only thymocyte death. These findings also indicate that unique epithelial ligands are not necessary for positive selection. They provide additional insight into the role of ligand quality in selection events and support the concept that following initiation of maturation from the DP state, persistent TCR signaling is characteristic of and perhaps required by T cells.     

A novel role for autophagy in T cell education

During T cell development in the thymus, scanning of peptide/major histocompatibility (MHC) molecule complexes on the surface of thymic epithelial cells ensures that only useful (self-MHC restricted) and harmless (self-tolerant) thymocytes survive. In recent years, a number of distinct cell-biological features of thymic epithelial cells have been unraveled that may have evolved to render these cells particularly suited for T cell selection, e.g., cortical epithelial cells use unique proteolytic enzymes for the generation of MHC/peptide complexes, whereas medullary epithelial cells "promiscuously" express otherwise tissue-restricted self-antigens. We recently showed that macroautophagy in thymic epithelial cells contributes to CD4 T cell selection and is essential for the generation of a self-tolerant T cell repertoire. We propose that the unusually high constitutive levels of autophagy in thymic epithelial cells deliver endogenous proteins to MHC class II molecules for both positive and negative selection of developing thymocytes.     

Role of coreceptors in positive selection and lineage commitment.

Recent experiments have re-awakened interest in a stochastic/selective model of positive selection of T lymphocytes. A revised version of the model has been proposed whereby commitment of double-positive thymocytes to either the CD4 or CD8 lineage requires two engagements with MHC molecules: the first, initiating the differentiation program, signals down-regulation of one or the other coreceptor, regardless of the T cell receptor's specificity for MHC class I or II molecules; the second, leading to terminal differentiation, screens the choice of coreceptor by permitting only those cells with matched receptors and coreceptors to proceed. Here we explore the role of coreceptors in the two stages of positive selection by manipulating CD8 expression in MHC class II-deficient mice, crossing them with either CD8-negative animals or animals carrying combinations of CD8 alpha and CD8 beta transgenes. We find that coreceptors are required at both stages of positive selection and that artificial expression of the down-modulated CD8 molecule can quite efficiently rescue cells that have made a 'mistake' in their choice of coreceptor. We also establish that commitment to the CD4 pathway and to the helper phenotype can be linked.     

Soluble factor-mediated differentiation of CD4+CD8+ thymocytes to single positives in vitro

Although the thymic microenvironment provides the necessary elements for T-cell differentiation, the precise role of individual components remains to be determined. In this paper, attempts were made to address the possibility that CD4 or CD8 single-positive (SP) thymocytes could be developed from immature CD4+CD8+ (double-positive; DP) thymocytes in a suspension culture in the presence of soluble factors. We observed that IL-4 and IFN-gamma weakly induced DP cells to differentiate to CD4 cells, but not to CD8. In contrast, IL-2 weakly induced differentiation to CD8. Interestingly, Con A sup strongly induced differentiation to CD8 SP from the purified DP thymocytes prepared from C57BL/6 or LCMV TCRtg mice. In particular, it was found that thymocyte culture with Con A sup generated CD69+DP cells, and the CD69+DP differentiated to CD8 SP under the suspension culture with soluble factors. Thus, Con A sup or combinations of IL-2, IL-4 and IL-7 strongly induced differentiation of CD69+DP to CD8 SP, whereas individual cytokines did not. These results suggest that soluble factors like cytokines play an important role in the generation of SP thymocytes in the absence of thymic stromal cells, at least from a distinctive subpopulation like CD69+DP thymocytes, and perhaps from those of broader range when in conjunction with TCR/MHC interaction.     

A role for CD8 in limiting degeneracy of thymocyte selection.

The introduction of a soluble TCR (sTCR) recognizing class I major histocompatibility complex (MHC) in the fetal thymic microenvironment in vitro produces the selection of thymocytes with enhanced avidity for self class I MHC (8). The sTCR was supposed to impose enhanced avidity for self MHC at an early degenerate phase of TCR-driven selection. This could determine increased reactivity to self at later stages of differentiation when specificity of TCR-ligand interaction augments and the effect of sTCR vanishes. This hypothesis was based on the observed deletion of CD4+8+ thymocytes upon upregulation of TCR and the increase in cell size of some CD8+ cells which are expanded in long-term fetal thymus organ cultures (FTOC) as well as in the periphery of adoptively transferred nude mice. Here we show that the developing alphabeta thymocyte which does not express CD8 at the cell surface has a selective advantage in FTOC with sTCR, thus suggesting that participation of CD8 in self peptide/MHC recognition confers specificity to T-cell selection and results in excessive signaling in thymocytes in spite of the presence of sTCR.     

Autoantigen-independent deletion of diabetogenic CD4+ thymocytes by protective MHC class II molecules

Some MHC class II genes provide dominant resistance to certain autoimmune diseases via mechanisms that remain unclear. We have shown that thymocytes bearing a highly diabetogenic, I-Ag7-restricted beta-cell-reactive TCR (4.1-TCR) undergo negative selection in diabetes-resistant H-2g7/x mice by engaging several different antidiabetogenic MHC class II molecules on thymic (but not peripheral) hemopoietic cells, independently of endogenous superantigens. Here we have investigated 1) whether this TCR can also engage protective MHC class II molecules (I-Ab) on cortical thymic epithelial cells in the absence of diabetogenic (I-Ag7) molecules, and 2) whether deletion of 4.1-CD4+ thymocytes in I-Ab-expressing mice might result from the ability of I-Ab molecules to present the target beta-cell autoantigen of the 4.1-TCR. We show that, unlike I-Ag7 molecules, I-Ab molecules can restrict neither the positive selection of 4.1-CD4+ thymocytes in the thymic cortex nor the presentation of their target autoantigen in the periphery. Deletion of 4.1-CD4+ thymocytes by I-Ab molecules in the thymic medulla, however, is a peptide-specific process, since it can be triggered by hemopoietic cells expressing heterogeneous peptide/I-Ab complexes, but not by hemopoietic cells expressing single peptide/I-Ab complexes. Thus, unlike MHC-autoreactive or alloreactive TCRs, which can engage deleting MHC molecules in the thymic cortex, thymic medulla, and peripheral APCs, the 4.1-TCR can only engage deleting MHC molecules (I-Ab) in the thymic medulla. We therefore conclude that this form of MHC-induced protection from diabetes is based on the presentation of an anatomically restricted, nonautoantigenic peptide to highly diabetogenic thymocytes.     

An essential role for the IL-7 receptor during intrathymic expansion of the positively selected neonatal T cell repertoire

Intrathymic T cell development is a multistage process involving discrete phases of proliferation as well as differentiation. From studies on IL-7 or IL-7Ralpha-deficient mice, it is clear that the IL-7 receptor (IL-7R) plays a critical role during the initial stages of intrathymic CD4-8- precursor development. In contrast, the role of IL-7R in later stages of thymocyte development are unclear. Here, we have used various approaches to investigate directly the role of the IL-7R in thymocyte positive selection and the recently described phase of postselection proliferation. First, we show that positive selection involves selective up-regulation of IL-7Ralpha- and IL-7Rgamma-chains, with the majority of CD4+ and CD8+ cells being IL-7R+. Second, MHC class II+ thymic epithelium-which drives postselection proliferation-expresses IL-7 mRNA. Finally, analysis of positive selection and postselection proliferation in thymocytes from IL-7Ralpha-/- neonates shows that positive selection occurs normally, whereas postselection expansion is drastically reduced. Thus, our data provide the first evidence that, as well as playing a role during early phases of thymic development, IL-7R mediates intrathymic expansion of positively selected thymocytes, which may aid in establishment of the neonatal peripheral T cell pool.     

CCR7 expression in developing thymocytes is linked to the CD4 versus CD8 lineage decision

During thymic development, T cell progenitors undergo positive selection based on the ability of their T cell Ag receptors (TCR) to bind MHC ligands on thymic epithelial cells. Positive selection determines T cell fate, in that thymocytes whose TCR bind MHC class I (MHC-I) develop as CD8-lineage T cells, whereas those that bind MHC class II (MHC-II) develop as CD4 T cells. Positive selection also induces migration from the cortex to the medulla driven by the chemokine receptor CCR7. In this study, we show that CCR7 is up-regulated in a larger proportion of CD4(+)CD8(+) thymocytes undergoing positive selection on MHC-I compared with MHC-II. Mice bearing a mutation of Th-POK, a key CD4/CD8-lineage regulator, display increased expression of CCR7 among MHC-II-specific CD4(+)CD8(+) thymocytes. In addition, overexpression of CCR7 results in increased development of CD8 T cells bearing MHC-II-specific TCR. These findings suggest that the timing of CCR7 expression relative to coreceptor down-regulation is regulated by lineage commitment signals.     

Altered thymocyte development resulting from expressing a deleting ligand on selecting thymic epithelium.

The maturation of CD4+8- and CD4-8+ thymocytes from CD4+8+ thymocytes is dependent on the mandatory interaction of their alpha beta TCR with selecting ligands expressed on thymic epithelial cells (TE). This is referred to as positive selection. The deletion of CD4+8+ thymocytes that express autospecific TCR (negative selection) is mediated primarily by bone marrow-derived cells. Previous studies have shown that TE is relatively ineffective in mediating the deletion of CD4+8- thymocytes expressing autospecific TCR but TE can render them anergic, i.e., nonresponsive, to the self Ag. The mechanism by which anergy is induced in these cells is unknown. In this study, we used thymocytes expressing a transgenic TCR specific for the male Ag presented by H-2Db class I MHC molecules to examine how expression of the deleting ligand by TE affects thymocyte development and phenotype. The development of female TCR-transgenic thymocytes was examined in irradiated male hosts or in female hosts that had received male fetal thymic epithelial implants. It was observed that the development of transgenic-TCR+ thymocytes was affected in mice with male TE. CD4+8+ thymocytes with reduced CD8 expression and markedly enhanced transgenic TCR expression accumulated in mice with male TE. Development of CD4-8+ thymocytes was also affected in these mice in that fewer were present and they expressed an intermediate CD8 coreceptor level. These CD4-8+ thymocytes expressed a high level of the transgenic TCR, retained the ability to respond to anti-TCR antibodies, but were nonresponsive to male APC. However, the maturation of CD4+8- thymocytes, which are also derived from CD4+8+ precursor cells, was relatively unaffected. In an in vitro assay for assessing negative selection, male TE failed to delete CD4+8+ thymocytes expressing the transgenic TCR under conditions where they were efficiently deleted by male dendritic cells. Collectively these results support the conclusion that male TE was inefficient in mediating deletion. Furthermore, expression of the deleting ligand on thymic epithelium interferes with the maturation of functional male-specific T cells and results in the accumulation of CD4+8+ and CD4-8+ thymocytes expressing a lower level of the CD8 coreceptor but a high level of the transgenic TCR.     

Coreceptor signal strength regulates positive selection but does not determine CD4/CD8 lineage choice in a physiologic in vivo model

TCR signals drive thymocyte development, but it remains controversial what impact, if any, the intensity of those signals have on T cell differentiation in the thymus. In this study, we assess the impact of CD8 coreceptor signal strength on positive selection and CD4/CD8 lineage choice using novel gene knockin mice in which the endogenous CD8alpha gene has been re-engineered to encode the stronger signaling cytoplasmic tail of CD4, with the re-engineered CD8alpha gene referred to as CD8.4. We found that stronger signaling CD8.4 coreceptors specifically improved the efficiency of CD8-dependent positive selection and quantitatively increased the number of MHC class I (MHC-I)-specific thymocytes signaled to differentiate into CD8+ T cells, even for thymocytes expressing a single, transgenic TCR. Importantly, however, stronger signaling CD8.4 coreceptors did not alter the CD8 lineage choice of any MHC-I-specific thymocytes, even MHC-I-specific thymocytes expressing the high-affinity F5 transgenic TCR. This study documents in a physiologic in vivo model that coreceptor signal strength alters TCR-signaling thresholds for positive selection and so is a major determinant of the CD4:CD8 ratio, but it does not influence CD4/CD8 lineage choice.     

Opening a window on thymic positive selection: developmental changes in the influence of cosignaling by integrins and CD28 on selection events induced by TCR engagement

How TCR and non-TCR signals are integrated by thymocytes to generate a decision to undergo either positive or negative selection remains incompletely understood. Recent evidence suggests that TCR signal transduction changes its quality during thymocyte maturation, but whether the contributions of various cosignaling or costimulatory pathways to thymocyte selection also are modified during development is unclear. Questions also remain about the possible selective roles of specific costimulatory pathways in induction of differentiation vs death among thymocytes at any given stage of maturity. To address these issues, a quantitative in vitro analysis of initiation of CD4+CD8+ thymocyte differentiation as measured by CD69 up-regulation/coreceptor down-modulation was conducted in parallel with an analysis of induction of death. Using transfected cells varying in their surface display of ICAM-1 or B7.1 along with antibody blocking experiments, we demonstrate here that ICAM-1 provides a selective boost to signaling for differentiation without substantially affecting induction of death among CD4+CD8+ cells, a property that is lost as thymocytes mature further. In contrast, B7 engagement enhances both cell activation and death in parallel. Based on these data, we propose that the high level of ICAM-1 on cortical epithelial cells plays a special role in opening a window between TCR signaling for differentiation vs death, permitting efficient initiation of positive selection on epithelial ligands. In contrast, late CD28-dependent cosignaling on hemopoietic cells in the medulla would help enforce negative selection by augmenting the effects of TCR engagement by low levels of high affinity ligands.     

MHC non-restricted, CD95-independent apoptosis of immature thymocytes induced by thymic epithelial cells

The interaction of thymocytes with thymic epithelial cells in the absence of an exogenous antigen was studied in vitro. Thymic, but not splenic epithelial cells induced apoptosis of thymocytes. A thymic epithelial cell line (TEC) induced apoptosis of thymocytes but not of splenic T-cells. The target population for TEC-induced death were immature CD4(+)8(+) (double positive), but not mature single positive thymocytes. TEC also induced DNA fragmentation in day 18 foetal thymocytes, most of which are CD4(+)8(+) cells. Radiation leukemia virus (RadLV)-transformed thymic lymphoma clones expressing various phenotypes reflected this sensitivity, in that a CD4(+)8(+)3(+) clone apoptosed by thymic epithelial cells or TEC. Other, single positive or double negative clones were resistant. Thymocytes from C3H (H-2(k)), C57BL/6 (H-2(b)) and Balb/C (H-2(d)) mice apoptosed equally in response to either C57BL/6 thymic epithelial cells or TEC (H-2(b) x H-2(d)). Likewise, thymocytes from MRLIpr((-/-)) and B6Ipr((-/-)) mice, which do not express CD95 were also apoptosed by TEC.The data suggest that thymic epithelial cells induce MHC non-restricted, Fas-independent apoptosis of immature thymocytes. This response may reflect a mechanism through which thymocytes expressing TcR with no affinity to self MHC/peptide complexes are eliminated.     

Developmental alterations in thymocyte sensitivity are actively regulated by MHC class II expression in the thymic medulla

Developing thymocytes are positively selected if they respond to self-MHC-peptide complexes, yet mature T cells are not activated by those same self-complexes. To avoid autoimmunity, positive selection must be followed by a period of maturation when the cellular response to TCR signals is altered. The mechanisms that mediate this postselection developmental tuning remain largely unknown. Specifically, it is unknown whether developmental tuning is a preprogrammed outcome of positive selection or if it is sensitive to ongoing interactions between the thymocyte and the thymic stroma. We probed the requirement for MHC class II-TCR interactions in postselection maturation by studying single positive (SP) CD4 thymocytes from K14/A(beta)(b) mice, in which CD4 T cells cannot interact with MHC class II in the thymic medulla. We report here that SP CD4 thymocytes must receive MHC class II signals to avoid hyperactive responses to TCR signals. This hyperactivity correlates with decreased expression of CD5; however, developmental tuning can occur independently of CD5, correlating instead with differences in the distribution of Lck. Thus, the maturation of postselection SP CD4 thymocytes is an active process mediated by ongoing interactions between the T cell and MHC class II molecules. This represents a novel mechanism by which the thymic medulla prevents autoreactivity.     

A dominant role for the thymus and MHC genes in determining the peripheral CD4/CD8 T cell ratio in the rat.

During their development, immature CD4CD8 double positive thymocytes become committed to either the CD4 or CD8 lineage. The final size of the peripheral CD4 and CD8 T cell compartments depends on thymic output and on the differential survival and proliferation of the respective T cell subsets in the periphery. Our results reveal that the development of the distinct peripheral CD4/CD8 T cell ratio between Lewis and Brown Norway rats originates in the thymus and, as shown by the use of radiation bone marrow chimeras, is determined by selection on radio-resistant stromal cells. Furthermore, this difference is strictly correlated with the MHC haplotype and is the result of a reduction in the absolute number of CD8 T cells in Brown Norway rats. These data suggest that the distinct CD4/CD8 T cell ratio between these two rat strains is the consequence of differential interactions of the TCR/CD8 coreceptor complex with the respective MHC class I haplotypes during selection in the thymus.