The selection of developing CD4+ and CD8+ T cells is dependent on recognition of antigen (peptide–MHC complexes) in the thymus and is responsible for preserving useful cells and eliminating potentially harmful ones. The immature, or unselected, repertoire of T lymphocytes consists of cells whose receptors may recognize any peptide antigen (self or foreign) displayed by any MHC molecule (also self or foreign). In addition, receptors may theoretically be expressed that do not recognize any pep tide–MHC molecule complex. In every individual, the only useful T cells are the ones specific for foreign peptides presented by that individual's MHC molecules—that is, self MHC molecules. When double-positive thymocytes first express αβ TCRs, these receptors encounter self peptides (the only peptides normally present in the thymus) displayed by self MHC molecules (the only MHC molecules available to display peptides), mainly on thymic epithelial cells in the cortex. The outcome of this recognition is determined primarily by the strength of the encounter between TCRs and self antigen–MHC complexes.
Positive Selection of Thymocytes: Development of the Self MHC–Restricted T-Cell Repertoire
Positive selection is the process in which thymocytes whose TCRs bind with low avidity (i.e., weakly) to self peptide–self MHC complexes are stimulated to survive and to differentiate into either CD4+ T cells or CD8+ T cells (see Fig. 1). Double positive thymocytes are produced without antigenic stimulation and begin to express αβ TCRs. In the thymic cortex, these immature cells encounter epithelial cells that display a variety of self peptides bound to MHC class I and class II molecules. Weak recognition of these self peptide–self MHC complexes promotes the survival of selected T cells; these are the cells that will enter the periphery and recognize peptides displayed by the same (self) MHC molecules. Thymocytes whose receptors do not recognize self MHC molecules are permitted to die by apoptosis because they will not be able to recognize antigens displayed by MHC molecules in the periphery; this phenomenon is called death by neglect (see Fig. 1).
Fig1. An overview of T-cell development in the thymus. Precursors of T cells travel from the bone mar row through the blood to the thymus. The progenitors of αβ T cells are double-negative T cells. In the thymic cortex, these cells begin to express TCRs and CD4 and CD8 coreceptors. Selection processes eliminate self reactive T cells in the cortex at the double-positive (DP) stage and also eliminate single-positive (SP) medullary thymocytes. They promote survival of thymocytes whose TCRs bind self major histocompatibility complex (MHC) molecules with low affinity. Functional and phenotypic differentiation into CD4+CD8− or CD8+CD4− SP T cells occurs in the medulla, and mature T cells are released into the circulation. Some DP cells differentiate into CD4+CD8− regulatory T cells (Treg, see Chapter 15). The development of γδ T cells is not shown.
During the transition from double-positive to single-positive cells, thymocytes whose TCRs recognize self MHC class I become CD8+CD4−, and cells with TCRs that recognize self MHC class II become CD4+CD8−. Thus, these cells become com mitted to the class II–restricted CD4 or class I–restricted CD8 lineage. Two models have been proposed to explain the process of lineage commitment, as a result of which coreceptors are correctly matched with the TCRs that recognize a specific class of MHC molecules. The stochastic or probabilistic model suggests that the commitment of immature T cells toward either lineage depends on the random probability of a double-positive cell differentiating into a single-positive CD4+ or a CD8+ thymocyte. In this model, a newly generated single-positive CD8+ T cell that has a TCR that can recognize self MHC class I and peptide in the thymus with a low affinity survives because it can engage the CD8 coreceptor, but a newly generated CD8+ T cell whose TCR recognizes only self MHC class II and peptide with low affinity does not survive because its coreceptor does not contribute to signaling in this T cell. Similarly, only single-positive CD4+ T cells whose TCRs can recognize self MHC class II and peptide (and not self MHC class I and peptide) with low affinity would survive positive selection in this stochastic model. An alternative and more widely accepted view is that the process of lineage commitment linked to positive selection is driven by specific signals that instruct the double-positive T cell to become CD4+ or CD8+. According to this instructional model, MHC class I– and MHC class II–restricted TCRs each deliver signals of different strengths (or for different durations) that actively induce expression of the correct coreceptor and shut off expression of the other coreceptor. It is known that double-positive cells go through a stage at which they express high CD4 and low CD8. If the TCR on such a cell is MHC class I–restricted, when it sees the appropriate MHC class I and self peptide, it will receive a weaker signal of shorter duration because levels of the CD8 coreceptor are low, and in addition, CD8 associates less well with the LCK tyrosine kinase than CD4 does. These shorter duration signals activate transcription factors such as RUNX3 that maintain the CD8+ T-cell phenotype by regulating the expression of the CD8 gene and by silencing the CD4 gene. Conversely, if the TCR on the cell is MHC class II–restricted, when it sees MHC class II, it will receive a stronger signal of longer duration because CD4 levels are high and CD4 associates relatively well with LCK. These more prolonged signals activate the transcription factor GATA3, which commits cells toward a CD4 fate, and induces the expression of a repressor called ThPoK, which prevents the expression of lineage-defining genes of CD8+ T cells.
Peptides bound to MHC molecules on thymic epithelial cells play an essential role in positive selection. In Chapter 6, we described how MHC molecules that are expressed on the cell surface always contain bound peptides. These MHC-associated peptides on thymic APCs probably serve two roles in positive selection—first, they promote stable cell surface expression of MHC molecules, and second, they may influence the specificities of the T cells that are selected. It is also clear from a variety of experimental studies that some peptides are better than others in supporting positive selection, and different peptides differ in the repertoires of T cells they select. These results suggest that specific antigen recognition, and not just MHC recognition, has some role in positive selection.
In Chapter 6, we also described the unique proteasomal subunit, β5t, that is expressed only in cortical thymic epithelial cells in proteasomes called thymoproteasomes. These functionally altered proteasomes generate unique self peptides that contribute to positive selection on MHC class I expressed on cortical thymic epithelial cells (cTECs). There is evidence from knockout mice that indicates that the generation of unique pep tides in the thymic cortex reduces the likelihood of autoreactivity in the pool of positively selected T cells. Indeed, in rodents, cTECs express the enzyme cathepsin L for antigen processing in lysosomes, while most APCs in the periphery utilize cathepsin S. Cathepsin L generates unique MHC class II–binding peptides in cTECs that are used in positive selection. In mice engineered to lack cathepsin L, cTECs are unable to replace CLIP with other peptides and these mice have a major reduction in CD4+ T cells, reflecting a specific defect in positive selection.
The model of positive selection based on weak recognition of self antigens raises a fundamental question: How does positive selection driven by weak recognition of self antigens produce a repertoire of mature T cells specific for foreign antigens? The likely answer is that positive selection allows many different T-cell clones to survive, and many of these T cells that recognize self peptides with low affinity will, after maturing, recognize foreign peptides with a high enough affinity to be activated and to generate useful immune responses.
Negative Selection of Thymocytes: Central Tolerance
Thymocytes whose receptors recognize peptide–MHC complexes in the thymus with high avidity undergo apoptosis (called negative selection) or differentiate into regulatory T cells (see Fig. 1). Among the double-positive T cells that are generated in the thymus, some may express TCRs that recognize self antigens with high affinity. The peptides present in the thymus are self peptides derived from widely expressed protein antigens as well as from some proteins believed to be restricted to particular tissues. (Recall that microbes that enter through the common routes, i.e., epithelia, are captured and transported to lymph nodes and tend not to enter the thymus.) In immature T cells, a major consequence of high-avidity antigen recognition is the triggering of apoptosis, leading to death or deletion of the cells. Therefore, many of the immature thymocytes that express high-affinity receptors for self antigens in the thymus die, resulting in negative selection of the T-cell repertoire. This process eliminates the potentially most dangerous self-reactive T cells and is one of the mechanisms of self-tolerance, which ensures that the immune system does not respond to many self antigens. Tolerance induced in immature lymphocytes by recognition of self antigens in the generative (or central) lymphoid organs is also called central tolerance, to be contrasted with peripheral tolerance induced in mature lymphocytes by self antigens in peripheral tissues. We will discuss the mechanisms and physiologic importance of immunologic tolerance in more detail in Chapter 15.
The deletion of immature self-reactive T cells may occur in part at the double-positive stage in the cortex but more extensively in newly generated single-positive T cells in the medulla. The thy mic APCs that mediate negative selection at the double-positive stage are cTECs (which also mediate positive selection). Negative selection of single-positive thymocytes may be mediated by bone marrow–derived dendritic cells and macrophages, which are abundant in the medulla, as well as by medullary thymic epithelial cells. Single-positive T cells express CCR7 and are drawn to the thymic medulla by CCL19 and CCL21 expressed by medullary stromal cells and endothelial cells. In the medulla, medullary thymic epithelial cells (mTECs) express a nuclear protein called AIRE (autoimmune regulator) that induces low-level expression of some self antigens that are normally expressed only in specific peripheral organs (so-called tissue-restricted antigens). Less well understood transcriptional mechanisms that sometimes occur in cells in which AIRE has been shut off help different individual mTECs to differentiate into “thymomimetic” cells resembling different peripheral lineages (described in Chapter 15). AIRE and other transcription factors thus ensure that many peripheral anti gens are processed and presented to immature T cells by mTECs, facilitating the deletion (negative selection) of these developing self-reactive T cells. A mutation in the gene that encodes AIRE results in an autoimmune polyglandular syndrome, underscoring the importance of AIRE in mediating central tolerance to tissue specific antigens.
The mechanism of negative selection in the thymus is the induction of death by apoptosis. Unlike the phenomenon of death by neglect, which occurs in the absence of positive selection, in negative selection, active death-promoting signals are generated when the TCR of immature thymocytes binds with high affinity to antigen. TCR signaling may induce expression of a proapoptotic protein called BIM, which probably plays an important role in thymocyte apoptosis during negative selection. It is also clear that although high-avidity antigen recognition by immature T cells triggers apoptosis, the same recognition by mature lymphocytes, in concert with other signals, initiates proliferative T-cell responses. The biochemical basis of this fundamental difference in responses of immature and mature cells is not known.
Recognition of self antigens in the thymus can generate a population of CD4+ regulatory T cells (Treg) that function to prevent autoimmune reactions (see Chapter 15). It is not clear which factors determine the choice between the two alternative fates of immature T cells that recognize self antigens with high avidity—namely, the deletion of immature T cells or the development of regulatory T cells. One possibility is that weak signals induce positive selection of thymocytes, strong signals induce negative selection, and intermediate signals induce differentiation into Treg. But how the level of signals is controlled and how they influence the fate of developing T cells is not known. While CD28 is not required for the development of naive CD4+ and CD8+ T cells, this costimulatory receptor is required for the generation of some Tregs in the thymus.
