Immunology_Janeway

Immunology_Janeway. and immune defects associated with deletions of chromosome 22q11. The nude mutation in mice is due to a defect in the gene for Foxn1, a transcription factor required for terminal epithelial cell differentiation; the name nude was given to this mutation because it also causes hairlessness. Rare cases of a defect in the human FOXN1 gene (which is on chromosome 17) have been associated with T-cell immunodeficiency, absence of a thymus, congenital alopecia, and nail dystrophy.


Immunology_Janeway

Immunology_Janeway. In mice, the thymus continues to develop for 3–4 weeks after birth, whereas in humans it is fully developed at birth. The rate of T-cell production by the thymus is greatest before puberty. After puberty, the thymus begins to shrink, and the production of new T cells in adults is reduced, although it does continue throughout life. In both mice and humans, removal of the thymus after puberty is not accompanied by any notable loss of T-cell function or numbers. Thus, it seems that once the T-cell repertoire is established, immunity can be sustained without the production of significant numbers of new T cells; the pool of peripheral T cells is instead maintained by long-lived T cells and also by division of some mature T cells. 8-11 Commitment to the T-cell lineage occurs in the thymus following Notch signaling.


Immunology_Janeway

Immunology_Janeway. 8-11 Commitment to the T-cell lineage occurs in the thymus following Notch signaling. T lymphocytes develop from a lymphoid progenitor in the bone marrow that also gives rise to B lymphocytes. Some of these progenitors leave the bone marrow and migrate to the thymus. In the thymus, the progenitor cell receives a signal from thymic epithelial cells that is transduced through a receptor called Notch1 to switch on specific genes. Notch signaling is widely used in animal development to specify tissue differentiation; in lymphocyte development, the Notch signal instructs the precursor to commit to the T-cell lineage rather than the B-cell lineage. Notch signaling is required throughout T-cell development and is also thought to help regulate other T-cell lineage choices, including the α:β versus γ:δ choice.


Immunology_Janeway

Immunology_Janeway. Notch signaling in thymic progenitor cells is essential to initiate the T-cellspecific gene expression program and commitment to the T-cell lineage (Fig. 8.18). First, Notch signaling induces the expression of two transcription factors, T-cell factor-1 (TCF1) and GATA3, each of which is required for T-cell development. Together, TCF1 and GATA3 initiate expression of several T-lineage-specific genes, such as those encoding components of the CD3 complex, as well as Rag1, a gene required for T-cell receptor and B-cell receptor gene rearrangements (see Fig. 8.18). However, TCF1 and GATA3 are not sufficient to induce the entire program of T-cell-specific gene expression. A third transcription factor, Bcl11b, is required to induce T-lineage commitment by restricting progenitor cells from adopting alternative fates; this final phase of T-cell commitment is a necessary prerequisite for activating the complete T-cell gene expression program.


Immunology_Janeway

Immunology_Janeway. 8-12 T-cell precursors proliferate extensively in the thymus, but most die there. T-cell precursors arriving in the thymus from the bone marrow spend up to a week differentiating there before they enter a phase of intense proliferation. In a young adult mouse the thymus contains about 108 to 2 × 108 thymocytes. About 5 × 107 new cells are generated each day; however, only about 106 to 2 × 106 (roughly 2–4%) of these leave the thymus each day as mature T cells. Despite the disparity between the number of T cells generated in the thymus and the number leaving, the thymus does not continue to grow in size or cell number. This is because about 98% of the thymocytes that develop in the thymus also die in the thymus by apoptosis (see Section 1-14). Cells undergoing apoptosis are recognized and ingested by macrophages, and apoptotic bodies, which are the residual condensed chromatin of apoptotic cells, are seen inside macrophages throughout the thymic cortex (Fig. 8.19). This apparently


Immunology_Janeway

Immunology_Janeway. Fig. 8.17 The epithelial cells of the thymus form a network surrounding developing thymocytes. In this scanning electron micrograph of the thymus, the developing thymocytes (the spherical cells) occupy the interstices of an extensive network of epithelial cells. Photograph courtesy of W. van Ewijk. Fig. 8.18 The stages of α:β T-cell development in the mouse thymus correlate with the program of gene rearrangement, and the expression of cell-surface proteins, signaling proteins, and transcription factors.


Immunology_Janeway

Immunology_Janeway. Lymphoid precursors are triggered to proliferate and become thymocytes committed to the T-cell lineage through interactions with Notch ligands expressed on the thymic stroma. T-cell commitment requires Notch signaling to induce the expression of TCF1 and GATA3, which in turn induce the expression of Bcl11b. This gene expression program begins in the double-negative (DN1) cells that express CD44 and Kit. Cells become irreversibly committed to the T-cell lineage at the subsequent (DN2) stage, which is marked by expression of CD25, the α chain of the IL-2 receptor. After this, the DN2 (CD44+CD25+) cells begin to rearrange the β-chain locus, becoming CD44low and Kitlow as this occurs, and they become DN3 cells. The DN3 cells are arrested in the CD44lowCD25+ stage until they productively rearrange the β-chain locus; the in-frame β chain then pairs with a surrogate chain called pTα to form the pre-T-cell receptor (pre-TCR), which is expressed on the cell surface and triggers entry into the


Immunology_Janeway

Immunology_Janeway. the β-chain locus; the in-frame β chain then pairs with a surrogate chain called pTα to form the pre-T-cell receptor (pre-TCR), which is expressed on the cell surface and triggers entry into the cell cycle. Expression of small amounts of pTα:β on the cell surface in association with CD3 signals the cessation of β-chain gene rearrangement and triggers rapid cell proliferation, which causes the loss of CD25. The cells are then known as DN4 cells. Eventually, the DN4 cells cease to proliferate and CD4 and CD8 are expressed. The small CD4+CD8+ double-positive cells begin efficient rearrangement at the α-chain locus. The cells then express low levels of an α:β T-cell receptor and the associated CD3 complex and are ready for selection. Most cells die by failing to be positively selected or as a consequence of negative selection, but some are selected to mature into CD4 or CD8 single-positive cells and eventually to leave the thymus. Maturation of CD4+CD8+ double-positive cells into CD4 or


Immunology_Janeway

Immunology_Janeway. a consequence of negative selection, but some are selected to mature into CD4 or CD8 single-positive cells and eventually to leave the thymus. Maturation of CD4+CD8+ double-positive cells into CD4 or CD8 single-positive cells is regulated by transcription factors ThPOK and Runx3, respectively. KLF2 is first expressed at the single-positive stage; if it is absent, thymocytes exhibit a defect in emigrating to peripheral lymphoid tissues, due in part to their failure to express receptors involved in trafficking, such as the sphingosine 1-phosphate (S1P) receptor, S1PR1 (see Fig. 8.32). The individual contributions to T-cell development of the other proteins are discussed in the text.


Immunology_Janeway

Immunology_Janeway. Fig. 8.19 Developing T cells that undergo apoptosis are ingested by macrophages in the thymic cortex. The left panel shows a section through the thymic cortex and part of the medulla in which cells have been stained for apoptosis with a red dye. The thymic cortex is to the right in the photograph. Apoptotic cells are scattered throughout the cortex but are rare in the medulla. The right panel shows at higher magnification a section of thymic cortex that has been stained red for apoptotic cells and blue for macrophages. The apoptotic cells can be seen within macrophages. Magnifications: left panel, ×45; right panel, ×164. Photographs courtesy of J. Sprent and C. Surh. profligate waste of thymocytes is a crucial part of T-cell development because it reflects the intensive screening that each thymocyte undergoes for the ability to recognize self peptide:self MHC complexes and for self-tolerance.


Immunology_Janeway

Immunology_Janeway. 8-13 Successive stages in the development of thymocytes are marked by changes in cell-surface molecules. Like developing B cells, developing thymocytes pass through a series of distinct stages. These are marked by changes in the status of the T-cell receptor genes and in the expression of the T-cell receptor, and by changes in the expression of cell-surface proteins such as the CD3 complex (see Section 7-7) and the co-receptor proteins CD4 and CD8 (see Section 4-18). These surface changes reflect the state of functional maturation of the cell, and particular combinations of cell-surface proteins are used as markers for T cells at different stages of differentiation. The principal stages are summarized in Fig. 8.20. Two distinct lineages of T cells—α:βand γ:δ, which have different types of T-cell receptor chains—are produced early in T-cell development. Later, α:β T cells develop into two distinct functional subsets—CD4 T cells and CD8 T cells.


Immunology_Janeway

Immunology_Janeway. When progenitor cells first enter the thymus from the bone marrow, they lack most of the surface molecules characteristic of mature T cells, and their receptor genes are not rearranged. These cells give rise to the major population of α:β T cells and the minor population of γ:δ T cells. If injected into the peripheral circulation, these lymphoid progenitors can even give rise to B cells and NK cells, although it is uncertain whether individual thymic progenitor cells retain this multipotency, or whether the progenitor cell population consists of a mixture of cells, only some of which are fully committed to the αβor γδ T-cell lineage.


Immunology_Janeway

Immunology_Janeway. Interactions with the thymic stroma trigger an initial phase of differentiation along the T-cell lineage pathway, followed by cell proliferation and the expression of the first cell-surface molecules specific for T cells, for example, CD2 and (in mice) Thy-1. At the end of this phase, which can last about a week, the thymocytes bear distinctive markers of the T-cell lineage but do not express any of the three cell-surface markers that define mature T cells. These are the CD3:T-cell receptor complex and the co-receptors CD4 or CD8. Because of the absence of CD4 and CD8, such cells are called double-negative thymocytes (see Fig. 8.20).


Immunology_Janeway

Immunology_Janeway. Fig. 8.20 Two distinct lineages of thymocytes are produced in the thymus. CD4, CD8, and T-cell receptor complex molecules (CD3, and the T-cell receptor α and β chains) are important cell-surface molecules for identifying thymocyte subpopulations. The earliest cell population in the thymus does not express any of these proteins, and because these cells do not express CD4 or CD8, they are called ‘double-negative’ thymocytes. These cells include precursors that give rise to two T-cell lineages: the minority population of γ:δ T cells (which lack CD4 or CD8 even when mature), and the majority α:β T-cell lineage. The development of prospective α:β T cells proceeds through stages in which both CD4 and CD8 are expressed by the same cell; these are known as ‘double-positive’ thymocytes. These cells enlarge and divide. Later, they become small resting double-positive cells that express low levels of the T-cell receptor. Most thymocytes die within the thymus after becoming small double-positive


Immunology_Janeway

Immunology_Janeway. enlarge and divide. Later, they become small resting double-positive cells that express low levels of the T-cell receptor. Most thymocytes die within the thymus after becoming small double-positive cells, but those cells whose receptors can interact with self peptide:self MHC molecular complexes lose expression of either CD4 or CD8 and increase the level of expression of the T-cell receptor. The outcome of this process is the ‘single-positive’ thymocytes, which, after maturation, are exported from the thymus as mature single-positive CD4 or CD8 T cells.


Immunology_Janeway

Immunology_Janeway. In the fully developed thymus, only ~60% of the double-negative thymocytes are immature T cells. The double-negative thymocyte pool (about 5% of all thymocytes) also includes two populations of more mature T cells that belong to minority lineages, including T cells expressing γ:δ T-cell receptors (see Section 8-16) and T cells bearing α:β T-cell receptors of very limited diversity (iNKT cells; see Section 6-19). In this and subsequent discussions, we reserve the term ‘double-negative thymocytes’ for the immature thymocytes that do not yet express a complete T-cell receptor molecule. These cells give rise to both γ:δ and α:β T cells (see Fig. 8.20), although most of them develop along the α:β pathway.


Immunology_Janeway

Immunology_Janeway. The α:β pathway is shown in more detail in Fig. 8.18. The double-negative stage can be further subdivided into four stages on the basis of expression of the adhesion molecule CD44, CD25 (the α chain of the IL-2 receptor), and Kit, the receptor for SCF (see Section 8-1). At first, double-negative thymocytes express Kit and CD44 but not CD25 and are called DN1 cells; in these cells, the genes encoding both chains of the T-cell receptor are in the germline configuration. As thymocytes mature, they begin to express CD25 on their surface and are called DN2 cells; later, expression of CD44 and Kit is reduced, and they are called DN3 cells.


Immunology_Janeway

Immunology_Janeway. Rearrangement of the T-cell receptor β-chain locus begins in DN2 cells with some Dβ to Jβ rearrangements and continues in DN3 cells with Vβ to DJβ rearrangement. Cells that fail to make a successful rearrangement of the β-chain locus remain at the DN3 (CD44lowCD25+) stage and soon die, whereas cells that make productive β-chain gene rearrangements and express the β-chain protein lose expression of CD25 once again and progress to the DN4 stage, in which they proliferate. The functional significance of the transient expression of CD25 is unclear: T cells develop normally in mice in which the IL-2 gene has been deleted by gene knockout (see Appendix I, Section A-35). By contrast, Kit is quite important for the development of the earliest double-negative thymocytes, in that mice lacking Kit have a much smaller number of double-negative T cells. In addition, continuous Notch signaling is important for progression through each stage of T-cell development. A second essential factor is IL-7,


Immunology_Janeway

Immunology_Janeway. a much smaller number of double-negative T cells. In addition, continuous Notch signaling is important for progression through each stage of T-cell development. A second essential factor is IL-7, which is produced by the thymic stroma. In the absence of IL-7, IL-7 receptor α, γ-c, or the IL-7 receptor signaling protein Jak3, a severe block in T-cell development occurs in both mice and humans. In fact, the human primary immunodeficiency disease characterized by defects in T cells and NK cells, X-linked SCID (severe combined immunodeficiency disease), is caused by a genetic deficiency leading to the absence of γ-c protein expression.


Immunology_Janeway

Immunology_Janeway. In DN3 thymocytes (see Fig. 8.18), the expressed β chains pair with a surrogate pre-T-cell receptor α chain called pTα (pre-T-cell α), which allows the assembly of a complete pre-T-cell receptor (pre-TCR) that is analogous in structure and function to the pre-B-cell receptor. The pre-TCR is expressed on the cell surface in a complex with the CD3 molecules that provide the signaling components of T-cell receptors (see Section 7-7). As with the preB-cell receptor, the assembly of the CD3:pre-T-cell receptor complex causes constitutive signaling that does not require interaction with a ligand. Recent structural evidence shows that the pre-TCR forms dimers in a manner similar to pre-BCR dimerization. The pTα Ig domain makes two important contacts. It associates with the constant-region Ig domain of the Vβsubunit to form the pre-TCR itself. A distinct surface of the pre-Tα then binds to a Vβ domain from another pre-TCR molecule, forming a bridge between two different pre-TCRs. The region


Immunology_Janeway

Immunology_Janeway. of the Vβsubunit to form the pre-TCR itself. A distinct surface of the pre-Tα then binds to a Vβ domain from another pre-TCR molecule, forming a bridge between two different pre-TCRs. The region of contact with the Vβ involves residues that are highly conserved across many Vβ families. In this way, expression of the pre-TCR induces ligand-independent dimerization, which leads to cell proliferation, the arrest of further β-chain gene rearrangement, and the expression of both CD8 and CD4. These double-positive thymocytes make up the vast majority of thymocytes. Once the large double-positive thymocytes have ceased to proliferate and have become small double-positive cells, the α-chain locus begins to rearrange.


Immunology_Janeway

Immunology_Janeway. thymic cortical epithelial cells and the receptors of developing T cells has a crucial role in positive selection, as we will see later in this chapter. After positive selection, developing T cells migrate from the cortex to the medulla. The medulla contains fewer lymphocytes, and those present are predominantly the newly matured single-positive T cells that will eventually leave the thymus. The medulla plays a role in negative selection. The antigen-presenting cells in this environment include dendritic cells that express co-stimulatory molecules, which are generally absent from the cortex. In addition, specialized medullary epithelial cells present peripheral antigens for the negative selection of T cells reactive for these self antigens. 8-15 T cells with α:βor γ:δ receptors arise from a common progenitor.


Immunology_Janeway

Immunology_Janeway. T cells bearing γ:δ receptors differ from α:β T cells in that they are found primarily in epithelial and mucosal sites and lack expression of the CD4 and CD8 co-receptors; in comparison with α:β T cells, relatively little is known about the ligands recognized by the γ:δ T-cell receptors, which are thought not to be MHC restricted (see Section 4-20). Recall from Section 5-11 that different genetic loci are used to make these two types of T-cell receptors. The γ and δ loci are the first to undergo rearrangement, followed shortly thereafter by the β locus. In addition, the δ locus is contained within the α locus, so rearrangements at the α locus eliminate the δ coding sequences on the chromosome. While the mechanism regulating commitment of individual precursor cells to the α:β versus the γ:δ lineage is still not understood, there is some plasticity in this process. This can be deduced from the pattern of gene rearrangements found in thymocytes and in mature γ:δ and α:β T cells. Mature


Immunology_Janeway

Immunology_Janeway. lineage is still not understood, there is some plasticity in this process. This can be deduced from the pattern of gene rearrangements found in thymocytes and in mature γ:δ and α:β T cells. Mature γ:δ T cells can contain rearranged β-chain genes, although 80% of these are nonproductive, and mature α:β T cells often contain rearranged, but mostly out-of-frame, γ-chain genes.


Immunology_Janeway

Immunology_Janeway. 8-16 T cells expressing γ:δ T-cell receptors arise in two distinct phases during development. Although γ:δ T cells arise from the same progenitors as α:β T cells, most mature γ:δ T cells are components of the innate rather than the adaptive immune system. When their maturation in the thymus is complete, the cells have acquired a defined effector function that can be rapidly elicited following their activation. After emigration from the thymus, most γ:δT cells home to mucosal and epithelial sites in the body, and take up stable residence in these locations.


Immunology_Janeway

Immunology_Janeway. In mice, the majority of γ:δ T cells in the body arise during embryonic development and the early neonatal period. In the fetal thymus, the first T cells to develop are γ:δ T cells that all express T-cell receptors assembled from the same Vγ and Vδ regions (Fig 8.22). These cells populate the epidermis; the T cells become wedged among the keratinocytes and adopt a dendritic-like form that has given them the name of dendritic epidermal T cells (dETCs) (Fig. 8.23). DETCs provide surveillance of the skin and respond to infection and injury by producing cytokines and chemokines. These factors induce inflammation to enhance pathogen clearance, and they promote wound healing to repair lesions in the skin. In steady-state conditions, dETCs also produce growth factors that help maintain epidermal growth and survival.


Immunology_Janeway

Immunology_Janeway. Following the dETC cells, a second subset of γ:δ T cells develops in the fetal thymus. These cells home to mucosal epithelia of tissues such as the reproductive tract and the lung, and also to the dermis of the skin. This subset is programmed to produce inflammatory cytokines such as IL-17 when stimulated, and is thought to play a role in responses to infection and injury. Like the dETCs, these IL-17-producing γ:δ T (Tγ:δ-17) cells express T-cell receptors that are essentially invariant, being composed of a single Vγ–Vδ combination. However, the two subsets, dETCs and the fetal Tγ:δ-17 cells, express T-cell receptors that use distinct Vγ gene segments—Vγ5 in the dETCs and Vγ6 in the Tγ:δ-17 cells. As fetal thymocytes do not express the enzyme TdT, there are no N-nucleotides contributing additional diversity at the junctions between V, D, and J gene segments of the T-cell receptors in these two fetally derived


Immunology_Janeway

Immunology_Janeway. V 2–7D1 D2 J2 C V˜4J˜1 C˜1 Stem cells in adult Starting during late fetal development ° ° ° ° ° V 2–7D1 D2 J2 C° ° ° ° ° V˜7J˜ C˜ ˜:° cells home to lymph nodes and spleen and are programmed to secrete IFN-˜ ˜:° cells home to intestinal epithelium and are programmed to secrete IFN-˜ 107 106 105 104 103 15 16 17 18 19 1 2 V1 D2 J2 C V6 J1 C1 C1 Numbers of thymocytes skin epidermis uterus skin, dermis lung spleen, lymph nodes liver intestinal epithelium Days of gestation Birth Weeks of age V1 D2 J2 C V 5 J1 V 2–7D1 D2 J2 C V˜4J˜1 C˜1 Stem cells predominant in fetus and newborn, rare in adult Starting day 17 of development Stem cells in fetus Days 14–18 of development Stem cells in fetus Days 16–19 of development V˜5 V˜6 V˜4 and V˜1 V˜1,2,4 and V˜7 ° ° ° ° ˜ ˜ ˜ ˜ ˜:° thymocytes ° ° ° °˜ ˜ ° ° ° ° ° V°6D 1D 2J 2 C V˜1J˜ C˜ ° ° ° ° ˜:° T cells become established in skin epidermis and are programmed to secrete keratinocyte growth factor, and infammatory cytokines and chemokines ˜:° T cells


Immunology_Janeway

Immunology_Janeway. ° ° ° ° V°6D 1D 2J 2 C V˜1J˜ C˜ ° ° ° ° ˜:° T cells become established in skin epidermis and are programmed to secrete keratinocyte growth factor, and infammatory cytokines and chemokines ˜:° T cells become established in mucosal epithelia, including reproductive tract, lung, and skin dermis, and are programmed to secrete IL-17 ˜:° cells home to lymph nodes, spleen, lung, and skin dermis and are programmed to secrete IL-17 ˜:° cells home to lymph nodes, spleen, lung, and liver and are programmed to secrete IL-4 and IFN-˜


Immunology_Janeway

Immunology_Janeway. Fig. 8.22 The rearrangement of T-cell receptor γ and δ genes in the mouse proceeds in waves of cells expressing different Vγ and Vδ gene segments. At about 2 weeks of gestation in the mouse, the Cγ1 locus is expressed with its closest V gene (Vγ5). After a few days, Vγ5-bearing cells decline in numbers in the thymus (first row of panels) and are replaced by cells expressing the next most proximal gene, Vγ6. Both these rearranged γ chains are expressed with the same rearranged δ-chain gene, as shown in the lower panels, and there is little junctional diversity in either the Vγ or the Vδ chain. As a consequence, most of the γ:δ T cells produced in each of these early waves share the same specificity, although the antigen recognized in each case is not known. The Vγ5-bearing cells become established selectively in the epidermis; they are programmed to secrete keratinocyte growth factor and inflammatory cytokines and chemokines. In contrast, Vγ6-bearing cells become established in the


Immunology_Janeway

Immunology_Janeway. established selectively in the epidermis; they are programmed to secrete keratinocyte growth factor and inflammatory cytokines and chemokines. In contrast, Vγ6-bearing cells become established in the lung, the dermis of the skin, and the epithelium of the reproductive tract, and are programmed to secrete IL-17. The next wave of γδ development begins on day 17 of gestation, and produces two different populations. One population rearranges and expresses the Vγ4 chain, which pairs with heterogeneous delta chains. These Vγ4-bearing cells are the second subset of Tγ:δ-17 (IL-17-secreting) cells, and home to lymph nodes, spleen, lung, and the dermis of the skin. The second population in this wave expresses Vγ1, and homes to lymph nodes, spleen, and liver. Some of these cells are paired with Vγ6 chains and are programmed to secrete IL-4 and IFN-γ, and represent γ:δ NKT cells. Finally, the last wave of γ:δ T-cell development begins late during fetal development, and persists into adulthood.


Immunology_Janeway

Immunology_Janeway. chains and are programmed to secrete IL-4 and IFN-γ, and represent γ:δ NKT cells. Finally, the last wave of γ:δ T-cell development begins late during fetal development, and persists into adulthood. This last wave includes a heterogeneous population of cells bearing Vγ1, Vγ2, and Vγ4 chains paired with many different delta chains. These cells home to lymphoid organs and are programmed to secrete IFN-γ. The other population in this last wave are cells bearing the Vγ7 chain paired with heterogeneous delta chains. These γ:δ cells home to the intestinal epithelium and are programmed to secrete IFN-γ as well as antimicrobial compounds. Although γ:δ T cells continue to be produced after birth, at this stage the α:β T-cell lineage becomes the dominant population developing in the thymus.


Immunology_Janeway

Immunology_Janeway. Fig. 8.23 Dendritic epidermal T cells reside within the epithelial layer, forming an interdigitating network with Langerhans cells. This face-on view of a murine epidermal sheet shows Langerhans cells (green) and dendritic epidermal T cells (dETCs; red) forming an interdigitating network within the layers of the epidermis. The epidermal epithelial cells are not visible in this fluorescence image. The branching dendritic-like form of these γ:δ T cells is the source of their name. Although the ligands for all γ:δ T-cell receptors are not known, some γ:δ T cells recognize nonclassical MHC molecules (see Sections 6-16 and 6-17), which can be induced in epithelia by stresses such as UV damage or pathogens. Thus, dETCs may serve as sentinels of such damage, producing cytokines that activate the innate immune response and, in turn, adaptive immunity. Courtesy of Adrian Hayday.


Immunology_Janeway

Immunology_Janeway. subsets of γ:δ T cells. Why certain V, D, and J gene segments are selected for rearrangement at particular times during embryonic development remains incompletely understood.


Immunology_Janeway

Immunology_Janeway. dETCs and the Vγ6-positive Tγ:δ-17 cells develop exclusively from the early wave of hematopoietic stem cells derived from the fetal liver (see Fig. 8.22). Consequently, these two γ:δ T-cell subsets arise only for a brief period of time in the fetal thymus, and then never again. A second phase of γ:δ T-cell development is initiated in the fetal thymus just before birth. This phase persists at a low level in the adult thymus throughout life, and produces several subsets of cells, each with distinct effector functions and tissue homing properties. Like the dETCs and fetal Tγ:δ-17 cells, these later-developing γ:δ T cells can be generally classified by their usage of distinct Vγ–Vδ regions in their T-cell receptors (see Fig. 8.22), although the receptor sequences within each population are more diverse due to the presence of N-region nucleotides added by TdT.


Immunology_Janeway

Immunology_Janeway. One population of these later-developing γ:δ T cells is programmed to secrete IL-17 when activated; these represent a second subset of Tγ:δ-17 cells that express a different Vγ region than do the fetal Tγ:δ-17 cells. Specifically, these later-developing Tγ:δ-17 cells express T-cell receptors using the Vγ4 region. This Tγ:δ-17 subset is found in all lymphoid organs, as well as in the skin dermis and the intestinal epithelium, where the cells provide rapid inflammatory signals in response to bacterial and parasitic infections. In addition, γ:δ T cells using the Vγ7 region in their T-cell receptors also develop in this second phase. The Vγ7-positive γ:δ T cells home specifically to the intestinal epithelium. In that location, the cells are poised to respond to gut microbes that breach the epithelial barrier, and are important producers of antibacterial compounds as well as IFN-γ.


Immunology_Janeway

Immunology_Janeway. In contrast to the γ:δT-cell subsets that reside in barrier tissues such as the skin and intestinal epithelium, γ:δ T cells are also found in lymphoid organs. The majority of the lymphoid-resident γ:δ T cells arise during the late fetal–early neonatal period as well as thereafter, and represent a more diverse population expressing the Vγ1 region. Vγ1-positive T cells are composed of two major groups—an IFN-γplus IL-4-producing subset that homes to the liver as well as several lymphoid organs, and an IFN-γ-producing subset that homes to all lymphoid organs. The former population of cells, which can be identified by their expression of a unique TCRδ chain (Vδ6) that is paired to Vγ1, is remarkably similar to the α:β T-cell receptor-expressing subset of iNKT cells, and the cells are therefore often referred to as γ:δ NKT cells. Unlike the mucosal and epithelial resident γ:δ T-cell populations, whose importance in tissue homeostasis, repair, and innate defense against infections has been


Immunology_Janeway

Immunology_Janeway. often referred to as γ:δ NKT cells. Unlike the mucosal and epithelial resident γ:δ T-cell populations, whose importance in tissue homeostasis, repair, and innate defense against infections has been well established, the functions of γ:δ T cells within secondary lymphoid organs is still not well understood.


Immunology_Janeway

Immunology_Janeway. 8-17 Successful synthesis of a rearranged β chain allows the production of a pre-T-cell receptor that triggers cell proliferation and blocks further β-chain gene rearrangement.


Immunology_Janeway

Immunology_Janeway. We now return to the development of α:β T cells. The rearrangement of the βand α-chain loci closely parallels the rearrangement of immunoglobulin heavy-chain and light-chain loci during B-cell development (see Sections 8-2 through 8-5). As shown in Fig. 8.24, the β-chain gene segments rearrange first, with the Dβ gene segments rearranging to Jβ gene segments, and this is followed by Vβ to DJβ rearrangement. If no functional β chain can be synthesized from this rearrangement, the cell will not be able to produce a pre-T-cell receptor and will die. However, unlike B cells with nonproductive heavy-chain gene rearrangements, thymocytes with nonproductive β-chain VDJ rearrangements can be rescued by further rearrangement, which is possible because of the two clusters of Dβ and Jβ gene segments upstream of two Cβ genes (see Fig. 5.13).


Immunology_Janeway

Immunology_Janeway. The likelihood of a productive VDJ join at the β locus is therefore somewhat higher than the 55% chance for a productive immunoglobulin heavy-chain gene arrangement. Once a productive β-chain gene rearrangement has occurred, the β chain is expressed together with the invariant pTα and the CD3 molecules (see Fig. 8.24) and is transported in this complex to the cell surface. The β:pTα complex is a functional pre-T-cell receptor analogous to the μ:VpreB:λ5 pre-Bcell receptor complex in B-cell development (see Section 8-3). Expression of


Immunology_Janeway

Immunology_Janeway. Fig. 8.24 The stages of gene rearrangement in α:β T cells. The sequence of gene rearrangements is shown, together with an indication of the stage at which the events take place and the nature of the cell-surface receptor molecules expressed at each stage. The β-chain locus rearranges first, in CD4–CD8– double-negative thymocytes expressing CD25 and low levels of CD44. As with immunoglobulin heavy-chain genes, D to J gene segments rearrange before V gene segments rearrange to DJ (second and third panels). It is possible to make up to four attempts to generate a productive rearrangement at the β-chain locus, because there are four D gene segments with two sets of J gene segments associated with each TCRβ chain locus (not shown). The productively rearranged gene is expressed initially within the cell and then at low levels on the cell surface. It associates with pTα, a surrogate 33-kDa α chain that is equivalent to λ5 in B-cell development, and this pTα:β heterodimer forms a complex with


Immunology_Janeway

Immunology_Janeway. the cell and then at low levels on the cell surface. It associates with pTα, a surrogate 33-kDa α chain that is equivalent to λ5 in B-cell development, and this pTα:β heterodimer forms a complex with the CD3 chains (fourth panel). The expression of the pre-T-cell receptor signals the developing thymocytes to halt β-chain gene rearrangement and to undergo multiple cycles of division. At the end of this proliferative burst, the CD4 and CD8 molecules are expressed, the cell ceases cycling, and the α chain is now able to undergo rearrangement. The first α-chain gene rearrangement deletes all δ D, J, and C gene segments on the chromosome, although these are retained as a circular DNA, indicating that these are nondividing cells (bottom panel). This permanently inactivates the δ-chain gene. Rearrangements at the α-chain locus can proceed through several cycles, because of the large number of Vα and Jα gene segments, so that productive rearrangements almost always occur.


Immunology_Janeway

Immunology_Janeway. When a functional α chain is produced that pairs efficiently with the β chain, the CD3lowCD4+CD8+ thymocyte is ready to undergo selection for its ability to recognize self peptides in association with self-MHC molecules.


Immunology_Janeway

Immunology_Janeway. the pre-T-cell receptor at the DN3 stage of thymocyte development induces signals that cause the phosphorylation and degradation of RAG-2, thus halting β-chain gene rearrangement and ensuring allelic exclusion at the β locus. These signals also induce the DN4 stage, in which rapid cell proliferation occurs, and eventually the co-receptor proteins CD4 and CD8 are expressed. The pre-T-cell receptor signals constitutively via the cytoplasmic protein kinase Lck, an Src-family tyrosine kinase (see Fig. 7.12), but seems not to require a ligand on the thymic epithelium. Lck subsequently associates with the co-receptor proteins. In mice genetically deficient in Lck, T-cell development is arrested before the CD4+CD8+ double-positive stage, and no α-chain gene rearrangements can be made.


Immunology_Janeway

Immunology_Janeway. The role of the expressed β chain in suppressing further β-locus rearrangement can be demonstrated in mice containing a rearranged TCRβ transgene: these mice express the transgenic β chain on virtually 100% of their T cells, and rearrangement of their endogenous β-chain genes is strongly suppressed. The importance of pTα has been shown in mice deficient in pTα, in which there is a hundredfold decrease in α:β T cells and an absence of allelic exclusion at the β locus.


Immunology_Janeway

Immunology_Janeway. During the proliferation of DN4 cells triggered by expression of the pre-T-cell receptor, the RAG-1 and RAG-2 genes are repressed (see Fig. 8.18). Hence, no rearrangement of the α-chain locus occurs until the proliferative phase ends, at which time RAG-1 and RAG-2 are transcribed again, and the functional RAG-1:RAG-2 complex accumulates. This ensures that each cell in which a β-chain gene has been successfully rearranged gives rise to many CD4+CD8+ thymocytes. Once the cells stop dividing, each of them can independently rearrange its α-chain genes, so that a single functional β chain can be associated with many different α chains in the progeny cells. During the period of α-chain gene rearrangement, α:β T-cell receptors are first expressed and selection by self peptide:self MHC complexes on the thymus cells can begin.


Immunology_Janeway

Immunology_Janeway. The progression of thymocytes from the double-negative to the double-positive and finally to the single-positive stage is accompanied by a distinct pattern of expression of proteins involved in DNA rearrangement, signaling, and T-cell-specific gene expression (see Fig. 8.18). TdT, the enzyme responsible for the insertion of N-nucleotides, is expressed throughout T-cell receptor gene rearrangement; N-nucleotides are found at the junctions of all rearranged α and β genes. Lck and another tyrosine kinase, ZAP-70, are both expressed from an early stage in thymocyte development. As well as its key role in signaling from the pre-T-cell receptor, Lck is also important for γ:δ T-cell development. In contrast, gene knockout studies (see Appendix I, Section A-35) show that ZAP-70, although expressed from the double-negative stage onward, is not essential for pre-T-cell receptor signaling, as double-negative thymocytes also express the related Syk kinase, which is capable of fulfilling this


Immunology_Janeway

Immunology_Janeway. from the double-negative stage onward, is not essential for pre-T-cell receptor signaling, as double-negative thymocytes also express the related Syk kinase, which is capable of fulfilling this role. Instead, ZAP-70 is required later, to promote the development of single-positive thymocytes from double-positive thymocytes; at this stage, Syk is no longer expressed. Fyn, an Src-family kinase similar to Lck, is expressed at increasing levels from the double-positive stage onward. It is not essential for the development of α:β thymocytes as long as Lck is present, but is required for the development of iNKT cells (see Section 8-26).


Immunology_Janeway

Immunology_Janeway. 8-18 T-cell α-chain genes undergo successive rearrangements until positive selection or cell death intervenes. The T-cell receptor α-chain genes are comparable to the immunoglobulin κ and λ light-chain genes in that they do not have D gene segments and are rearranged only after their partner receptor chain has been expressed. As with the light-chain genes, repeated attempts at α-chain gene rearrangement are possible, as illustrated in Fig. 8.25. The presence of multiple Vα gene segments, and about 60 Jα gene segments spread over some 80 kilobases of DNA, allows many successive Vα to Jα rearrangements to take place at both α-chain alleles. This means that T cells with an initial nonproductive α-gene rearrangement are much more likely to be rescued by a subsequent rearrangement than are B cells with a nonproductive light-chain gene rearrangement.


Immunology_Janeway

Immunology_Janeway. One key difference between B and T cells is that the final assembly of an immunoglobulin leads to the cessation of gene rearrangement and initiates the further differentiation of the B cell, whereas rearrangement of the Vα gene segments continues in T cells unless there is signaling by a self peptide:self MHC complex that positively selects the receptor (see Section 8-19 below). This means that many T cells have in-frame rearrangements on both chromosomes and so can produce two types of α chains. This is possible because expression of the T-cell receptor is not in itself sufficient to shut off gene rearrangement. Continued rearrangements on both chromosomes can allow several different α chains to be produced successively as well as simultaneously in each developing T cell and to be tested for self peptide:self MHC recognition in partnership with the same β chain. This phase of gene rearrangement lasts for 3 or 4 days in the mouse and ceases only when positive selection occurs as a


Immunology_Janeway

Immunology_Janeway. for self peptide:self MHC recognition in partnership with the same β chain. This phase of gene rearrangement lasts for 3 or 4 days in the mouse and ceases only when positive selection occurs as a consequence of receptor engagement, or when the cell dies. One can predict that if the frequency of positive selection is sufficiently low, roughly one in three mature T cells will express two productively rearranged α chains at the cell surface. This has been confirmed for both human and mouse T cells. Thus, in the strict sense, T-cell receptor α-chain genes are not subject to allelic exclusion.


Immunology_Janeway

Immunology_Janeway. T cells with dual specificity might be expected to give rise to inappropriate immune responses if the cell is activated through one receptor yet can act upon target cells recognized by the second receptor. However, only one of the two receptors is likely to be able to recognize peptide presented by a self MHC molecule, and so the T cell will have only a single functional specificity. This is because once a thymocyte has been positively selected by self peptide:self MHC recognition, α-chain gene rearrangement ceases. Thus, the existence of cells with two α-chain genes productively rearranged and two α chains expressed at the cell surface does not truly challenge the idea that a single functional specificity is expressed by each cell.


Immunology_Janeway

Immunology_Janeway. Fig. 8.25 Multiple successive rearrangement events can rescue nonproductive T-cell receptor α-chain gene rearrangements. The multiplicity of V and J gene segments at the α-chain locus allows successive rearrangement events to ‘leapfrog’ over previously rearranged VJ segments, deleting any intervening gene segments. The α-chain rescue pathway resembles that of the immunoglobulin κ light-chain genes (see Section 8-5), but the number of possible successive rearrangements is greater. α-chain gene rearrangement continues until either a productive rearrangement leads to positive selection or the cell dies. Summary.


Immunology_Janeway

Immunology_Janeway. Summary. The thymus provides a specialized and architecturally organized microenvironment for the development of mature T cells. Precursors of T cells migrate from the bone marrow to the thymus, where they interact with environmental cues, such as ligands for the Notch receptor, that drive commitment to the T lineage. Developing thymocytes develop along one of several T-cell lineages: the most prominent subsets in the thymus are γ:δ T cells, conventional α:β T cells, and α:β T cells with receptors of very limited diversity, such as iNKT cells.


Immunology_Janeway

Immunology_Janeway. T-cell progenitors develop along the γ:δ or the α:β T-cell lineages. Early in ontogeny, the production of γ:δ T cells predominates over α:β T cells, and these cells populate several peripheral tissues, including the skin, the intestine, and other mucosal and epithelial surfaces. These subsets predominantly develop from fetal liver, rather than bone marrow, stem cells. Later, more than 90% of thymocytes express α:β T-cell receptors. In developing thymocytes, the γ, δ, and β genes are the first to rearrange. Cells of the α:β lineage that rearrange a functional beta chain form a pre-T-cell receptor that signals thymocyte proliferation, α-chain gene rearrangement, and CD4 and CD8 expression. Most steps in T-cell development take place in the thymic cortex, whereas the medulla contains mainly mature T cells. Positive and negative selection of T cells.


Immunology_Janeway

Immunology_Janeway. Positive and negative selection of T cells. Up to the stage at which an α:β receptor is produced, T-cell development is independent of MHC proteins or antigen. From this point onward, developmental decisions in the α:β T-cell lineage depend on the interaction of the receptor with peptide:MHC ligands it encounters in the thymus, and we now consider this phase of T-cell development.


Immunology_Janeway

Immunology_Janeway. T-cell precursors committed to the α:β lineage at the DN3 stage undergo vigorous proliferation in the subcapsular region and progress to the DN4 stage. These cells then rapidly transit through an immature CD8 single-positive stage and become double-positive cells that express low levels of the T-cell receptor and both the CD4 and CD8 co-receptors as they move deeper into the thymic cortex. These double-positive cells have a life-span of only about 3–4 days unless they are rescued by engagement of their T-cell receptor. The rescue of double-positive cells from programmed cell death and their maturation into CD4 or CD8 single-positive cells is the process known as positive selection. Only about 10–30% of the T-cell receptors generated by gene rearrangement will be able to recognize self peptide:self MHC complexes and thus function in self MHC-restricted responses to foreign antigens (see Chapter 4); those that have this capability are selected for survival in the thymus. Double-positive


Immunology_Janeway

Immunology_Janeway. MHC complexes and thus function in self MHC-restricted responses to foreign antigens (see Chapter 4); those that have this capability are selected for survival in the thymus. Double-positive cells also undergo negative selection: T cells whose receptors recognize self peptide:self MHC complexes too strongly undergo apoptosis, thus eliminating potentially self-reactive cells. In this section, we examine the interactions between developing double-positive thymocytes and different thymic components and discuss the mechanisms by which these interactions shape the mature T-cell repertoire.


Immunology_Janeway

Immunology_Janeway. 8-19 Only thymocytes whose receptors interact with self peptide:self MHC complexes can survive and mature.


Immunology_Janeway

Immunology_Janeway. Early experiments using bone marrow chimeras (see Appendix I, Section A-32) and thymic grafting provided evidence that MHC molecules in the thymus influence the MHC-restricted T-cell repertoire. However, mice transgenic for rearranged T-cell receptor genes provided the first conclusive evidence that the interaction of the T cell with self peptide:self MHC complexes is necessary for the survival of immature T cells and their maturation into naive CD4 or CD8 T cells. For these experiments, the rearranged αand β-chain genes were cloned from a T-cell clone (see Appendix I, Section A-20) whose origin, antigen specificity, and MHC restriction were known. When such genes are introduced into the mouse genome, they are expressed early during thymocyte development. As a consequence of expressing functional transgene-encoded TCRα-and β-chain proteins in developing T cells, the rearrangement of endogenous T-cell receptor genes is inhibited, albeit to different degrees. In general, endogenous


Immunology_Janeway

Immunology_Janeway. functional transgene-encoded TCRα-and β-chain proteins in developing T cells, the rearrangement of endogenous T-cell receptor genes is inhibited, albeit to different degrees. In general, endogenous β-chain gene rearrangement is inhibited completely but that of α-chain genes is inhibited only incompletely. The result is that most of the developing thymocytes in TCR transgenic mouse lines express the T-cell receptor encoded by the transgenes.


Immunology_Janeway

Immunology_Janeway. By introducing T-cell receptor transgenes specific for a known peptide:MHC complex, the effect of allelic variations in MHC molecules on the maturation of thymocytes with receptors of known specificity can be studied directly, without the need for immunization and analysis of effector function. Such studies showed that thymocytes bearing a particular T-cell receptor could develop to the double-positive stage in thymuses that expressed different MHC molecules from those present in the mouse from which the original T-cell clone was isolated. However, these transgenic thymocytes developed into mature CD4 or CD8 single-positive thymocytes only if the thymus expressed the same self MHC molecule as that on which the original T-cell clone was selected (Fig. 8.26).


Immunology_Janeway

Immunology_Janeway. Such experiments also discovered the fate of T cells that fail positive selection. Rearranged receptor genes from a mature T cell specific for a peptide presented by a particular MHC molecule were introduced into a recipient mouse lacking that MHC molecule, and the fate of thymocytes was investigated by staining with antibodies specific for the transgenic receptor. Antibodies against other molecules, such as CD4 and CD8, were used at the same time to mark the stages of T-cell development. It was found that cells that fail to recognize the MHC molecules present on the thymic epithelium never progress further than the double-positive stage and die in the thymus within 3 or 4 days of their last division. 8-20 Positive selection acts on a repertoire of T-cell receptors with inherent specificity for MHC molecules.


Immunology_Janeway

Immunology_Janeway. Positive selection acts on a repertoire of T-cell receptors whose specificity is determined by randomly generated combinations of V, D, and J gene segments (see Section 5-7). Despite this, T-cell receptors exhibit a bias toward recognition of MHC molecules even before positive selection. If the specificity of the unselected repertoire were completely random, only a very small proportion of thymocytes would be expected to recognize any MHC molecule. However, an inherent specificity of T-cell receptors for MHC molecules has been detected by examining mature T cells that represent the unselected receptor repertoire. Such T cells can be produced in vitro from fetal thymuses that lack expression of MHC class I and MHC class II molecules by triggering generalized ‘positive selection’ using antibodies that bind to the Vβ chain of T-cell receptors and to the CD4 co-receptor. When such antibody-selected CD4 T cells are examined, roughly 5% can respond to any one MHC class II genotype. Because


Immunology_Janeway

Immunology_Janeway. that bind to the Vβ chain of T-cell receptors and to the CD4 co-receptor. When such antibody-selected CD4 T cells are examined, roughly 5% can respond to any one MHC class II genotype. Because these cells developed without selection by MHC molecules, this reactivity must reflect an inherent MHC-specificity encoded in the germline V gene segments. This specificity should significantly increase the proportion of receptors that can be positively selected by any individual’s MHC molecules.


Immunology_Janeway

Immunology_Janeway. The germline-encoded reactivity seems to be due to specific amino acids in the CDR1 and CDR2 regions of T-cell receptor Vβ and Vα regions. The CDR1 and CDR2 regions are encoded in the germline V gene segments and are highly


Immunology_Janeway

Immunology_Janeway. Fig. 8.26 Positive selection is demonstrated by the development of T cells expressing rearranged T-cell receptor transgenes. In mice transgenic for rearranged α:β T-cell receptor genes, the maturation of T cells depends on the MHC haplotype expressed in the thymus. If the transgenic mice express the same MHC haplotype in their thymic stromal cells as the mouse from which the rearranged TCRα-chain and TCRβ-chain genes originally developed (both MHCa, top panel), then the T cells expressing the transgenic T-cell receptor will develop from the double-positive stage (pale green) into mature T cells (dark green), in this case mature CD8+ single-positive cells. If the MHCa-restricted TCR transgenes are genetically crossed into a different MHC background (MHCb, yellow, bottom panel), then developing T cells expressing the transgenic receptor will progress to the double-positive stage but will fail to mature further. This failure is due to the absence of an interaction between the transgenic


Immunology_Janeway

Immunology_Janeway. T cells expressing the transgenic receptor will progress to the double-positive stage but will fail to mature further. This failure is due to the absence of an interaction between the transgenic T-cell receptor with MHC molecules on the thymic cortex, and thus no signal for positive selection is delivered, leading to apoptotic death by neglect.


Immunology_Janeway

Immunology_Janeway. Fig. 8.27 The MHC molecules that induce positive selection determine co-receptor specificity. In mice transgenic for T-cell receptors restricted by an MHC class I molecule (top panel), the mature T cells that develop all have the CD8 (red) phenotype. In mice transgenic for receptors restricted by an MHC class II molecule (bottom panel), all mature T cells have the CD4 (blue) phenotype. In both cases, normal numbers of immature, double-positive thymocytes (half blue, half red) are found. The specificity of the T-cell receptor determines the outcome of the developmental pathway, ensuring that the only T cells that mature are those equipped with a co-receptor that is able to bind the same self MHC molecule as the T-cell receptor.


Immunology_Janeway

Immunology_Janeway. variable (see Section 5-8). But among this variability, certain amino acids are conserved and common to many V segments. Analysis of numerous crystal structures has revealed that when the T-cell receptor binds a peptide:MHC complex, specific amino acids of the Vβ region interact with a particular part of the MHC protein. For example, in many human and mouse Vβ regions, the CDR2 has a tyrosine at position 48, and this interacts with a region in the middle of the α1 helix of MHC class I and class II proteins. Two other amino acids commonly found in other Vβ regions (tyrosine at 46 and glutamic acid at 54) interact with the same region of MHC. T cells expressing Vβ genes with mutations at any of these positions showed reduced positive selection, demonstrating that the interaction of such V regions with MHC molecules contributes to T-cell development.


Immunology_Janeway

Immunology_Janeway. 8-21 Positive selection coordinates the expression of CD4 or CD8 with the specificity of the T-cell receptor and the potential effector functions of the T cell.


Immunology_Janeway

Immunology_Janeway. At the time of positive selection, the thymocyte expresses both CD4 and CD8 co-receptor molecules. By the end of thymic selection, mature α:β T cells ready for export to the periphery have stopped expressing one of these co-receptors. The majority of these cells belong to the conventional CD4 or CD8 T-cell lineages. Less abundant subsets, such as iNKT cells and a subset of regulatory T cells expressing CD4 and high levels of CD25, also develop in the thymus from CD4+CD8+ cells. Moreover, almost all mature T cells that express CD4 have receptors that recognize peptides bound to self MHC class II molecules and are programmed to become cytokine-secreting helper T cells. In contrast, most of the cells that express CD8 have receptors that recognize peptides bound to self MHC class I molecules and are programmed to become cytotoxic effector cells. Thus, positive selection also determines the cell-surface phenotype and functional potential of the mature T cell, selecting the appropriate


Immunology_Janeway

Immunology_Janeway. and are programmed to become cytotoxic effector cells. Thus, positive selection also determines the cell-surface phenotype and functional potential of the mature T cell, selecting the appropriate co-receptor for efficient antigen recognition and the appropriate program for the T cell’s eventual functional differentiation in an immune response.


Immunology_Janeway

Immunology_Janeway. Experiments with mice transgenic for rearranged T-cell receptor genes show clearly that the specificity of the T-cell receptor for self peptide:self MHC complexes determines which co-receptor a mature T cell will express. If the transgenes encode a T-cell receptor specific for antigen presented by self MHC class I molecules, mature T cells that express the transgenic receptor are CD8 T cells. Similarly, in mice made transgenic for a receptor that recognizes antigen with self MHC class II molecules, mature T cells that express the transgenic receptor are CD4 T cells (Fig. 8.27).


Immunology_Janeway

Immunology_Janeway. The importance of MHC molecules in this selection is illustrated by the human immunodeficiency diseases caused by mutations that lead to an absence of MHC molecules on lymphocytes and thymic epithelial cells. People who lack MHC class II molecules have CD8 T cells but only a few, highly abnormal CD4 T cells; a similar result has been obtained in mice in which MHC class II expression has been eliminated by targeted gene disruption (see Appendix I, Section A-35). Likewise, mice and humans that lack MHC class I molecules lack CD8 T cells. Thus, MHC class II molecules are absolutely required for CD4 T-cell development, whereas MHC class I molecules are similarly required for CD8 T-cell development.


Immunology_Janeway

Immunology_Janeway. In mature T cells, the co-receptor functions of CD8 and CD4 depend on their respective abilities to bind invariant sites on MHC class I and MHC class II molecules (see Section 4-18). Co-receptor binding to an MHC molecule is also required for normal positive selection, as shown for CD4 in the experiment discussed in the next section. In thymocytes, nearly all of the Lck is associated with CD4 and CD8 co-receptors, providing a mechanism to ensure that signaling is initiated only in thymocytes bearing T-cell receptors that bind to MHC molecules. Thus, positive selection depends on engagement of both the antigen receptor and co-receptor by an MHC molecule, and this signal determines the survival of single-positive cells that express only the appropriate co-receptor. Commitment to either the CD4 or CD8 lineage is coordinated with receptor specificity, and it seems that the developing thymocyte integrates signals from both the antigen receptor and the co-receptor. Co-receptor-associated


Immunology_Janeway

Immunology_Janeway. CD4 or CD8 lineage is coordinated with receptor specificity, and it seems that the developing thymocyte integrates signals from both the antigen receptor and the co-receptor. Co-receptor-associated Lck signals are most effectively delivered when CD4 rather than CD8 is engaged as a co-receptor, and these Lck signals play a large part in the decision to become a mature CD4 cell.


Immunology_Janeway

Immunology_Janeway. T-cell receptor signaling regulates this choice of the CD4 versus the CD8 lineage by controlling the expression of two transcription factors, ThPOK and Runx3 (see Fig. 8.18). The role of ThPOK was identified through a naturally occurring loss-of-function mutation in mice that lacked CD4 T-cell development. In mice lacking ThPOK, MHC class II-restricted thymocytes are redirected toward the CD8 lineage. ThPOK is not expressed in pre-selection double-positive thymocytes, but strong T-cell receptor signaling at this stage of development induces its expression. ThPOK, in turn, reinforces its own expression and represses expression of Runx3; together, the expression of ThPOK and absence of Runx3 lead to CD4 commitment and the ability to express cytokine genes typical of CD4 cells. If T-cell signaling is of insufficient strength or duration, however, ThPOK is not induced, and Runx3 is allowed to be expressed. This leads to silencing of CD4 expression, maintenance of CD8 expression, and the


Immunology_Janeway

Immunology_Janeway. is of insufficient strength or duration, however, ThPOK is not induced, and Runx3 is allowed to be expressed. This leads to silencing of CD4 expression, maintenance of CD8 expression, and the expression of genes typical of CD8 T cells, namely, genes that encode proteins involved in target-cell killing.


Immunology_Janeway

Immunology_Janeway. While the majority of double-positive thymocytes that undergo positive selection develop into either CD4 or CD8 single-positive T cells, the thymus also generates less numerous populations of other T-cell subsets with specialized functions; these will be discussed further in Section 8-26. 8-22 Thymic cortical epithelial cells mediate positive selection of developing thymocytes. Thymus transplantation studies indicate that stromal cells are important for positive selection. These cells form a web of cell processes that make close contacts with the double-positive T cells undergoing positive selection (see Fig. 8.17), and T-cell receptors can be seen clustering with MHC molecules at the sites of contact. Direct evidence that thymic cortical epithelial cells mediate positive selection comes from an ingenious manipulation of mice whose MHC class II genes have been eliminated by targeted gene disruption (Fig. 8.28).


Immunology_Janeway

Immunology_Janeway. Fig. 8.28 Thymic cortical epithelial cells mediate positive selection. In the thymus of normal mice (first panels), which express MHC class II molecules on epithelial cells in the thymic cortex (blue) as well as on medullary epithelial cells (orange) and bone marrow-derived cells (yellow), both CD4 (blue) and CD8 (red) T cells mature. Double-positive thymocytes are shown as half red and half blue. The second panels represent mutant mice in which MHC class II expression has been eliminated by targeted gene disruption; in these mice, few CD4 T cells develop, although CD8 T cells develop normally. In MHC class II-negative mice containing an MHC class II transgene engineered so that it is expressed only on the epithelial cells of the thymic cortex (third panels), normal numbers of CD4 T cells mature. In contrast, if a mutant MHC class II molecule with a defective CD4binding site is expressed (fourth panel), positive selection of CD4 T cells does not take place. This indicates that the


Immunology_Janeway

Immunology_Janeway. mature. In contrast, if a mutant MHC class II molecule with a defective CD4binding site is expressed (fourth panel), positive selection of CD4 T cells does not take place. This indicates that the cortical epithelial cells are the critical cell type mediating positive selection and that the MHC class II molecule needs to be able to interact with the CD4 protein.


Immunology_Janeway

Immunology_Janeway. Mutant mice that lack MHC class II molecules do not normally produce CD4 T cells. To test the role of the thymic epithelium in positive selection, an MHC class II gene was placed under the control of a promoter that restricted the gene’s expression to thymic cortical epithelial cells. This was then introduced as a transgene into the MHC class II-mutant mice, and CD4 T-cell development was restored. A variant of this experiment showed that, to promote the development of CD4 T cells, the MHC class II molecule on the thymic cortical epithelium must be able to interact effectively with CD4. Thus, when the MHC class II transgene expressed in the thymus contains a mutation that prevents binding of the MHC to CD4, very few CD4 T cells develop. Equivalent studies of CD8 interaction with MHC class I molecules showed that co-receptor binding is also necessary for the positive selection of CD8 cells.


Immunology_Janeway

Immunology_Janeway. The critical role of the thymic cortical epithelium in positive selection raises the question whether there is anything distinctive about the antigen-presenting properties of these cells. The thymic stromal cells may simply be in closest proximity to the developing thymocytes, as there are very few macrophages and dendritic cells in the cortex to perform the antigen presentation. In addition, however, thymic epithelium differs from other tissues in the expression of key proteases that are involved in MHC class I and II antigen processing (see Section 6-8). Cortical epithelial cells express cathepsin L as opposed to the more widely expressed cathepsin S, and mice deficient in cathepsin L have severely impaired CD4 T-cell development. Thymic epithelial cells from mice lacking cathepsin L exhibit a relatively high proportion of MHC class II molecules on their surface that retain the class II invariant chain-associated peptide (CLIP) (see Fig. 6.11). Cortical epithelial cells also express


Immunology_Janeway

Immunology_Janeway. a relatively high proportion of MHC class II molecules on their surface that retain the class II invariant chain-associated peptide (CLIP) (see Fig. 6.11). Cortical epithelial cells also express a unique proteasome subunit, β5T, whereas other cells express β5 or β5i. Mice deficient in β5T have severely impaired CD8 T-cell development. Because mice that lack either cathepsin L or β5T still have normal levels of MHC on the surface of their thymic cortical cells, it would seem that it is the peptide repertoire displayed by the MHC molecules on cortical epithelial cells that is responsible for altering CD8 T-cell development, although the mechanism is still unclear.


Immunology_Janeway

Immunology_Janeway. 8-23 T cells that react strongly with ubiquitous self antigens are deleted in the thymus. When the T-cell receptor of a mature naive T cell is strongly ligated by a peptide:MHC complex displayed on a specialized antigen-presenting cell in a peripheral lymphoid organ, the T cell is activated to proliferate and produce effector T cells. In contrast, when the T-cell receptor of a developing thymocyte is similarly ligated by a self peptide:self MHC complex in the thymus, it dies by apoptosis (Fig. 8.29). The response of immature T cells to stimulation by antigen is the basis of negative selection. Elimination of immature T cells in the thymus prevents their potentially harmful activation later, should they encounter the same self peptides when they are mature T cells.


Immunology_Janeway

Immunology_Janeway. Negative selection has been demonstrated using TCR-transgenic mice expressing T-cell receptors specific for self peptides derived from proteins encoded on the Y chromosome, and thus expressed only in male mice. Thymocytes bearing these receptors disappear from the developing T-cell population in male mice at the double-positive stage of development, and no single-positive cells bearing the transgenic receptors mature. By contrast, in female mice, which lack the male-specific peptide, T cells bearing the transgenic receptors mature normally. Negative selection to male-specific peptides has also been demonstrated in nontransgenic mice and also occurs by deletion of T cells.


Immunology_Janeway

Immunology_Janeway. TCR transgenic mice were very useful for the classic experiments above, but they express a functional T-cell receptor earlier during development than normal mice and have a very high frequency of cells reactive to any particular peptide. A more realistic system for evaluating negative selection involves the transgenic expression of only the β chain of a T-cell receptor reactive to a given peptide antigen. In such mice, the β chain pairs with endogenous α chains, yet the frequency of peptide-reactive T cells is sufficient for detection using peptide:MHC tetramers (see Appendix I, Section A-24). These and other more physiologic approaches showed that clonal deletion can occur at either the double-positive or the single-positive stage, presumably depending on where the T cell encounters the antigen that causes deletion.


Immunology_Janeway

Immunology_Janeway. These experiments illustrate the principle that self peptide:self MHC complexes encountered in the thymus purge the mature T-cell repertoire of cells bearing self-reactive receptors. One obvious problem with this scheme is that many tissue-specific proteins, such as pancreatic insulin, would not be expected to be expressed in the thymus. However, it is now clear that many such ‘tissue-specific’ proteins are expressed by certain stromal cells in the thymic medulla; thus, intrathymic negative selection could apply even to proteins that are otherwise restricted to tissues outside the thymus. The expression of some, but not all, tissue-specific proteins in the thymic medulla is controlled by a gene called AIRE (autoimmune regulator). AIRE is expressed in medullary stromal cells (Fig. 8.30), interacts with many proteins involved in transcription, and seems to lengthen transcripts that would otherwise terminate earlier. Mutations in AIRE give rise to the human autoimmune disease known as


Immunology_Janeway

Immunology_Janeway. interacts with many proteins involved in transcription, and seems to lengthen transcripts that would otherwise terminate earlier. Mutations in AIRE give rise to the human autoimmune disease known as autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) or autoimmune polyglandular syndrome type I, highlighting the important role of intrathymic expression of tissue-specific proteins in maintaining tolerance to self. Negative selection of developing T cells involves interactions with ubiquitous and tissue-restricted self antigens, and can take place in both the thymic cortex and the thymic medulla (see Fig. 8.29).


Immunology_Janeway

Immunology_Janeway. It is unlikely that all possible self proteins are expressed in the thymus. Thus, negative selection in the thymus may not remove all T cells reactive to self antigens that appear exclusively in other tissues or are expressed at different stages


Immunology_Janeway

Immunology_Janeway. Fig. 8.29 Negative selection of thymocytes can occur in the cortex or the medulla. When the T-cell receptor (TCR) on a developing thymocyte is strongly stimulated by recognition of self peptide:self MHC complexes (red cell), the thymocyte is induced to die, a process known as negative selection. Negative selection can occur in the cortex when a CD4+CD8+ double-positive thymocyte has strong reactivity to peptide:MHC complexes found on cortical epithelial cells (left panel). Negative selection can also occur in the medulla when an immature CD4 or CD8 single-positive thymocyte receives strong T-cell receptor signaling following recognition of peptide:MHC complexes on medullary epithelial cells (middle panel) or on bone marrow-derived macrophages or dendritic cells (right panel). AIRE expression in the thymus Fig. 8.30 AIRE is expressed in the medulla of the thymus and promotes the expression of proteins normally expressed in peripheral tissues.


Immunology_Janeway

Immunology_Janeway. AIRE expression in the thymus Fig. 8.30 AIRE is expressed in the medulla of the thymus and promotes the expression of proteins normally expressed in peripheral tissues. Expression of AIRE by thymic medullary cells is limited to the medullary region of the thymus, where it is expressed in a subset of epithelial-like cells. The expression of the thymic medullary epithelial marker MTS10 is shown in red. AIRE expression is shown in green by immunofluorescence, and is present in only a fraction of the medullary epithelial cells. Photograph courtesy of R.K. Chin and Y.-X. Fu. in development. There are, however, several mechanisms operating in the periphery that can prevent mature T cells from responding to tissue-specific antigens; these are discussed in Chapter 15, when we consider the problem of autoimmune responses and their avoidance. 8-24 Negative selection is driven most efficiently by bone marrow-derived antigen-presenting cells.


Immunology_Janeway

Immunology_Janeway. 8-24 Negative selection is driven most efficiently by bone marrow-derived antigen-presenting cells. As discussed above, negative selection occurs throughout thymocyte development, both in the thymic cortex and in the medulla, and so is likely to be mediated by antigen presentation by several different cell types (see Fig. 8.29). There does seem to be a hierarchy in the effectiveness of cells in mediating negative selection. At the top are bone marrow-derived dendritic cells and macrophages. These are antigen-presenting cells that also activate mature T cells in peripheral lymphoid tissues, as we shall see in Chapter 9. The self antigens presented by these cells are therefore the most important source of potential autoimmune responses, and T cells responding to such self peptides must be eliminated in the thymus.


Immunology_Janeway

Immunology_Janeway. In addition, both the thymocytes themselves and the thymic epithelial cells can cause the deletion of self-reactive cells. The medullary epithelial cells expressing AIRE, and thus presenting a wide range of self-antigens, are one population that has been shown to directly induce thymocyte negative selection. More generally, in patients undergoing bone marrow transplantation from an unrelated donor, where all the thymic macrophages and dendritic cells are of donor type, negative selection mediated by thymic epithelial cells is of critical importance in maintaining tolerance to the recipient’s own antigens. 8-25 The specificity and/or the strength of signals for negative and positive selection must differ.


Immunology_Janeway

Immunology_Janeway. T cells undergo both positive selection for self MHC restriction and negative selection for self-tolerance by interacting with self peptide:self MHC complexes expressed on stromal cells in the thymus. An unresolved issue is how the interaction of the T-cell receptor with self peptide:self MHC complexes distinguishes between these opposite outcomes. First, more receptor specificities must be positively selected than are negatively selected. Otherwise, all the cells that were positively selected in the thymic cortex would be eliminated by negative selection, and no T cells would ever be produced. Second, the consequences of the interactions that lead to positive and negative selection must differ: cells that recognize self peptide:self MHC complexes on cortical epithelial cells are induced to mature, whereas those whose receptors might confer strong and potentially damaging autoreactivity are induced to die.


Immunology_Janeway

Immunology_Janeway. Currently, the choice between positive and negative selection is thought to hinge on the strength of self peptide:self MHC binding by the T-cell receptor, an idea known as the affinity hypothesis (Fig. 8.31). Low-affinity interactions rescue the cell from death by neglect, leading to positive selection; high-affinity interactions induce apoptosis and thus negative selection. Because more complexes are likely to bind weakly than strongly, this model explains the positive selection of a larger repertoire of cells than are negatively selected. Using T-cell receptor transgenic thymocytes, it was shown that variants of the antigenic peptide could induce positive selection in thymic organ cultures or in vivo. Peptide variants that induced positive selection had a lower affinity for the T-cell receptor than did antigenic peptide. How this quantitative difference in receptor affinity translates into a qualitatively distinct cell fate is still an area of active investigation. Many of the


Immunology_Janeway

Immunology_Janeway. receptor than did antigenic peptide. How this quantitative difference in receptor affinity translates into a qualitatively distinct cell fate is still an area of active investigation. Many of the biochemical signals induced by low-affinity interactions are weaker or of shorter duration than those from high-affinity interactions. However, low-affinity interactions lead to sustained


Immunology_Janeway

Immunology_Janeway. Fig. 8.31 The affinity model of T-cell positive and negative selection. Random TCRα and β chain gene rearrangements generate a large pool of immature thymocytes expressing a varied repertoire of specificities. The T-cell receptors on many of these cells fail to have sufficient binding strength to the self peptide:self MHC complexes on thymic epithelium and so receive no signals. These cells die by neglect. Another fraction of immature thymocytes are positively selected because their T-cell receptors bind with sufficient strength to the self peptide:self MHC complexes on thymic epithelium to generate T-cell receptor-dependent survival signals. From this cohort of positively selected thymocytes, negative selection removes those thymocytes whose receptors have excessively strong reactivity to self peptides complexed with self MHC molecules (resulting in clonal deletion), thereby establishing self-tolerance of the mature T-cell population. A small subset of positively selected cells


Immunology_Janeway

Immunology_Janeway. to self peptides complexed with self MHC molecules (resulting in clonal deletion), thereby establishing self-tolerance of the mature T-cell population. A small subset of positively selected cells receiving signals slightly weaker than those inducing negative selection differentiate into regulatory T cells (Tregs), a process referred to as agonist selection.


Immunology_Janeway

Immunology_Janeway. activation of the protein kinase Erk, whereas high-affinity interactions lead to only transient activation of Erk, suggesting that differential activation of this or other MAPKs might determine the outcome of thymic selection. Indeed, experiments showed that developing T cells need to engage low-affinity ligands for more than 24 hours for positive selection to occur. 8-26 Self-recognizing regulatory T cells and innate T cells develop in the thymus. Additional populations besides the conventional CD4+ and CD8+ α:β T cells discussed above emerge from the thymus; they are numerically minor but functionally important. Two of these subsets, the T cells (see Section 9-23) reg and the iNKT cells (see Section 6.18), have been well studied, and found to each have unique developmental requirements.


Immunology_Janeway

Immunology_Janeway. Thymically derived T cells are a subset of CD4+ T cells that function to main- reg tain self-tolerance. These cells arise from CD4+CD8+ thymocytes, as do conventional T cells. During their maturation, they upregulate the transcription factor FoxP3. T cell development also depends on IL-2 receptor signaling, a cytokine signal that is not required for the development of conventional T cells. The repertoire of T-cell receptors expressed on T cells is thought to be composed of receptors with high affinity for self MHC:self peptide complexes. Evidence supporting this conclusion comes from studies showing that some lines of TCR transgenic mice generate large numbers of T cells when the mice also express the antigen for this T-cell receptor. In addition, studies using mice expressing a fluorescent reporter that monitors T-cell receptor signal strength have shown that T cells express high levels of the fluorescent reporter, both during their development and after their export from the thymus,


Immunology_Janeway

Immunology_Janeway. reporter that monitors T-cell receptor signal strength have shown that T cells express high levels of the fluorescent reporter, both during their development and after their export from the thymus, indicating that they likely express T-cell receptors with high affinity for self. This process of positive selection following high-affinity T-cell receptor interactions with self peptide:self MHC complexes has been termed agonist selection—in other words, agonist selection refers to interactions of a T-cell receptor with a self peptide:self MHC that would normally activate a mature T cell expressing that T-cell receptor.


Immunology_Janeway

Immunology_Janeway. A second specialized subset of T cells that develops from CD4+CD8+ thymocyte precursors is a lineage known as invariant NKT cells (iNKT cells), based on their expression of the NK1.1 receptor commonly found on NK cells. iNKT cells are activated as part of the early response to many infections; they differ from the major lineage of α:β T cells in recognizing CD1 molecules rather than MHC class I or MHC class II molecules (see Section 6-18). Unlike other T cells, iNKT cells require for their development a T-cell receptor interaction with CD1 molecules expressed on thymocytes and a signal through the adaptor protein SAP. iNKT cells, like γ:δ T cells, acquire a defined effector program during their development in the thymus. Therefore, these cells exhibit a memory-cell phenotype when they leave the thymus and


Immunology_Janeway

Immunology_Janeway. After 3–4 days in the medulla, CD4 and CD8 T cells upregulate SIP1 and exit the thymus into the blood blood vessel SIPSIPR1 CD4 and CD8 T cells entering the medulla are not fully mature Fig. 8.32 Thymocyte emigration is induced by signaling through the sphingosine 1-phosphate receptor, S1PR1. CD4 and CD8 single-positive thymocytes that have successfully survived positive and negative selection are found in the medulla but are not yet fully mature. At the termination of the maturation process, which takes 3–4 days, the CD4 and CD8 single-positive thymocytes upregulate the sphingosine 1-phosphate (S1P) receptor, known as S1PR1. S1PR1 is a G-proteincoupled receptor that promotes chemotaxis of the cells toward the ligand S1P. Due to the high levels of S1P in the blood, single-positive thymocytes are induced to leave the thymus by entering the blood, where they become part of the recirculating naive T-cell population.


Immunology_Janeway

Immunology_Janeway. MOVIE 8.1 migrate to peripheral lymphoid tissues and mucosal surfaces. iNKT cells have been suggested to develop in response to ‘agonist’ signaling. Recent studies have revealed that CD1-binding lipid antigens produced by the commensal microbes in the gut are an important source of these agonist ligands, and that the composition of the gut microbiome regulates the development of iNKT cells early in life. Since agonist stimulation of immature T cells is also known to cause clonal deletion, it is not yet clear which activating interactions lead to clonal deletion in the thymus and which lead to selection of Treg cells or the nonconventional iNKT cells. 8-27 The final stage of T-cell maturation occurs in the thymic medulla.


Immunology_Janeway

Immunology_Janeway. After surviving positive and negative selection, thymocytes complete their maturation in the thymic medulla and then emigrate to peripheral lymphoid organs. Their final maturation results in changes to the T-cell receptor signaling machinery. Whereas an immature double-positive or single-positive thymocyte stimulated through the T-cell receptor will undergo apoptosis, a mature single-positive thymocyte responds by proliferating. The final maturation stage takes less than 4 days, and functionally competent T cells then emigrate from the thymus into the bloodstream (Fig. 8.32). Emigration requires recognition of the lipid molecule sphingosine 1-phosphate (S1P) by the G-proteincoupled receptor S1PR1, which is expressed by thymocytes during their final maturation. S1P is present in high concentration in blood and lymph, and mature thymocytes seem to be drawn toward it. Mature thymocytes also express CD62L (L-selectin), a lymph-node homing receptor that facilitates the localization of


Immunology_Janeway

Immunology_Janeway. in blood and lymph, and mature thymocytes seem to be drawn toward it. Mature thymocytes also express CD62L (L-selectin), a lymph-node homing receptor that facilitates the localization of mature naive T cells to peripheral lymphoid organs after their emigration from the thymus.


Immunology_Janeway

Immunology_Janeway. 8-28 T cells that encounter sufficient quantities of self antigens for the first time in the periphery are eliminated or inactivated.


Immunology_Janeway

Immunology_Janeway. Many autoreactive T cells are purged during their development in the thymus. As discussed in Section 8-23, this negative selection process is facilitated by the AIRE protein, which promotes the expression of many tissue-specific antigens in thymic medullary epithelial cells. Nonetheless, not all self antigens are expressed in the thymus, and some autoreactive T cells complete their maturation and migrate to the periphery. Our understanding of the fates of autoreactive T cells in the periphery comes mainly from the study of mice transgenic for self-reactive T-cell receptors. In some cases, T cells reacting to self antigens in the periphery are eliminated. This usually follows a brief period of activation and cell division, and so is known as activation-induced cell death. In other cases, the self-reactive cells may be rendered anergic. When studied in vitro, these anergic T cells prove refractory to signals delivered through the T-cell receptor.


Immunology_Janeway

Immunology_Janeway. The question immediately arises: if the encounter of a mature naive lymphocyte with a self antigen leads to cell death or anergy, why does this not also happen to a mature lymphocyte that recognizes a pathogen-derived antigen? The answer is that infection sets up inflammation, which induces the expression of co-stimulatory molecules on the antigen-presenting dendritic cells and the production of cytokines promoting lymphocyte activation. The outcome of an encounter with antigen in these conditions is the activation, proliferation, and differentiation of the lymphocyte to effector-cell status. In the absence of infection or inflammation, dendritic cells still process and present self antigens, but in the absence of co-stimulatory and other signals, any interaction of a mature lymphocyte with its specific antigen seems to result in a tolerance-inducing (tolerogenic) signal from the antigen receptor. Summary.


Immunology_Janeway

Immunology_Janeway. The stages of thymocyte development up to the expression of the pre-T-cell receptor—including the decision between commitment to either the α:β or the γ:δ lineage—are all independent of interactions with peptide:MHC antigens. With the successful rearrangement of α-chain genes and expression of the T-cell receptor, α:β thymocytes undergo further development that is determined by the interactions of their T-cell receptors with self peptides presented by the MHC molecules on the thymic stroma. CD4+CD8+ double-positive thymocytes whose receptors interact with self peptide:self MHC complexes on thymic cortical epithelial cells are positively selected, and will eventually mature into CD4 or CD8 single-positive cells. T cells that react too strongly with self antigens are deleted in the thymus, a process driven by bone marrow-derived antigen-presenting cells and AIRE-expressing epithelial cells in the medullary region of the thymus. The outcome of positive and negative selection is the


Immunology_Janeway

Immunology_Janeway. a process driven by bone marrow-derived antigen-presenting cells and AIRE-expressing epithelial cells in the medullary region of the thymus. The outcome of positive and negative selection is the generation of a mature conventional T-cell repertoire that is both MHC-restricted and self-tolerant. Some non-conventional T-cell lineages undergo ‘agonist’ selection following strong T-cell receptor signaling. Precisely how the recognition of self peptide:self MHC ligands by the T-cell receptor leads to either positive or negative selection remains an unsolved problem.


Immunology_Janeway

Summary to Chapter 8.

Immunology_Janeway. Summary to Chapter 8.


Immunology_Janeway

Immunology_Janeway. In this chapter we have learned about the formation of the B-cell and T-cell lineages from an uncommitted hematopoietic stem cell. The somatic gene rearrangements that generate the highly diverse repertoire of antigen receptors—immunoglobulin for B cells, and the T-cell receptor for T cells— occur in the early stages of the development of T cells and B cells from a common bone marrow-derived lymphoid progenitor. Mammalian B-cell development takes place in fetal liver and, after birth, in the bone marrow; T cells also originate from stem cells in the fetal liver or the bone marrow, but undergo most of their development in the thymus. Much of the somatic recombination machinery, including the RAG proteins that are an essential part of the V(D)J recombinase, is common to both B and T cells. In both B and T cells, gene rearrangements begin with the loci that contain D gene segments, and proceed successively at each locus. The first step in B-cell development is the rearrangement of the


Immunology_Janeway

Immunology_Janeway. In both B and T cells, gene rearrangements begin with the loci that contain D gene segments, and proceed successively at each locus. The first step in B-cell development is the rearrangement of the locus for the immunoglobulin heavy chain, and for T cells the β chain. In each case, the developing cell is allowed to proceed to the next stage of development only if the rearrangement has produced an in-frame sequence that can be translated into a protein expressed on the cell surface: either the pre-B-cell receptor or the pre-T-cell receptor. Cells that do not generate successful rearrangements for both receptor chains die by apoptosis. The course of conventional B-cell development is summarized in Fig. 8.14, and that of α:βT cells in Fig. 8.33.


Immunology_Janeway

Immunology_Janeway. Once a functional antigen receptor has appeared on the cell surface, the lymphocyte is tested in two ways. Positive selection tests for the potential usefulness of the antigen receptor, whereas negative selection removes self-reactive cells from the lymphocyte repertoire, rendering it tolerant to the antigens of the body. Positive selection is particularly important for T cells, because it ensures that only cells bearing T-cell receptors that can recognize antigen in combination with self MHC molecules will continue to mature. Positive selection also coordinates the choice of co-receptor expression. CD4 becomes expressed by T cells harboring MHC class II-restricted receptors, and CD8 by cells harboring MHC class I-restricted receptors. This ensures the optimal use of these receptors in responses to pathogens. For B cells, positive selection seems to occur at the final transition from immature to mature B cells, which occurs in peripheral lymphoid tissues. Tolerance to self antigens is


Immunology_Janeway

Immunology_Janeway. to pathogens. For B cells, positive selection seems to occur at the final transition from immature to mature B cells, which occurs in peripheral lymphoid tissues. Tolerance to self antigens is enforced by negative selection at different stages throughout the development of both B and T cells, and positive selection likewise seems to represent a continuous process.


Immunology_Janeway

Immunology_Janeway. Fig. 8.33 A summary of the development of human α:β T cells. The state of the T-cell receptor genes, the expression of some essential intracellular proteins, and the expression of some cell-surface molecules are shown for successive stages of α:β T-cell development. Note that because the T-cell receptor genes do not undergo further changes during antigen-driven development, only the phases during which they are actively undergoing rearrangement in the thymus are indicated. The antigen-dependent phases of CD4 and CD8 cells are depicted separately, and are detailed in Chapter 9.


Immunology_Janeway

Immunology_Janeway. TERMINALDIFFERENTIATIONTERMINALANTIGENDEPENDENTDIFFERENTIATIONANTIGENDEPENDENTStem cell Early double-negative thymocyte Late double-negative thymocyte Early double-positive thymocyte Late double-positive thymocyte Naive CD4 T cell Memory CD4 T cell Effector CD4 T cell Naive CD8 T cell Memory CD8 T cell Effector CD8 T cell ˜-chain gene rearrangements T cells °-chain gene rearrangements Intra-cellular proteins Surface marker proteinsANTIGENINDEPENDENTpre-T receptor D–J rearranged V–DJ rearranged V–J rearranged Germline Germline CD34? CD2 HSA CD44hi RAG-1 RAG-2 TdT Lck ZAP-70 RAG-1 RAG-2 TdT Lck ZAP-70 RAG-1 RAG-2 Lck ZAP-70 Lck ZAP-70 LKLF Lck ZAP-70 CD25 CD44lo HSA PT˜CD4 CD8 HSA CD4 CD62L CD45RA CD5 CD69 CD4 CD8 HSA CD4 CD45RO CD44 CD4 CD45RO CD44hi Fas FasL (type 1) CD8 CD45RA CD8 CD45RO CD44 FasL Fas CD8 CD44hi TH17: IL-17 TH1: IFN-°TH2: IL-4 IFN-°granzyme perforin Germline Germline T-cell receptor Questions.


Immunology_Janeway

Immunology_Janeway. Questions. 8.1 True or False: B-cell development is not affected in mice that are lacking the cytokine receptor common γ chain (γ-c). 8.2 Fill-in-the-Blanks: B-cell development is regulated by the expression of various transcription factors that enable gene rearrangement and the successful progression into a new developmental stage. For example, during the _________ stage, Rag-1 and Rag-2 expression is induced by __________, which permits the successful D to J rearrangement and then V to DJ rearrangement of the heavy-chain locus. As a consequence, a functional __________ is expressed, and upon signaling, the cell is instructed to perform _________ and progress toward the next developmental step and rearrange the light-chain locus. 8.3 True or False: Self antigen recognition is needed in order to cross-link the pre-B-cell receptor, which in turn allows this complex to signal and permit the transition from pro-B cell to pre-B cell.


Immunology_Janeway

Immunology_Janeway. 8.4 Matching: Match the B-cell stage with the proper description: A. Early pro-B cell i. V-DJ rearranging (heavy chain) B. Small pre-B cell ii. D-J rearranging (heavy chain) C. Immature B cell iii. Expressing pre-B-cell receptor D. Late pro-B cell iv. V-J rearranging (light chain) E. Large pre-B cell v. Surface IgM 8.5 Short Answer: How does the process of allelic exclusion prevent the rearrangement of the second heavy-chain locus, and why is this important? 8.6 Short Answer: How can one large pre-B cell give rise to multiple B cells with different antigen specificities? 8.7 Matching: Match the following terms to the appropriate definition: A. Receptor editing i. Result of persistent autoreactivity after failure of successful receptor editing B. Isotypic exclusion ii. Selection of either the κ or the λ light chain C. Clonal deletion iii. Result of a peripheral encounter of a weakly cross-linking or low-valence antigen


Immunology_Janeway

Immunology_Janeway. B. Isotypic exclusion ii. Selection of either the κ or the λ light chain C. Clonal deletion iii. Result of a peripheral encounter of a weakly cross-linking or low-valence antigen D. Anergy iv. Process by which the light-chain locus is rearranged in order to produce a nonautoreactive receptor E. Immunological v. B cells that have affinity for ignorance a self-antigen but for various reasons do not respond to it 8.8 True or False: All CD4 and CD8 double-negative thymocytes are immature T cells. 8.9 Matching: Match the correct expression of CD44 and CD25 and the T-cell receptor locus rearrangement status with the appropriate DN T-cell stage: A. DN1 i. CD44+CD25+, D to J TCRβ-chain locus rearrangement B. DN2 ii. CD44+CD25–, germline T-cell receptor locus C. DN3 iii. CD44lowCD25+, V to DJ β-chain locus rearrangement


Immunology_Janeway

Immunology_Janeway. A. DN1 i. CD44+CD25+, D to J TCRβ-chain locus rearrangement B. DN2 ii. CD44+CD25–, germline T-cell receptor locus C. DN3 iii. CD44lowCD25+, V to DJ β-chain locus rearrangement D. DN4 iv. CD44–CD25–, functional β-chain rearrangement 8.10 Fill-in-the-Blanks: Successful rearrangement of the ______ during the DN__ stage permits the formation of the pre-T-cell receptor, which is analogous in structure and function to the pre-B-cell receptor. The TCRβ chain associates itself with the _______, which allows ligand-independent cross-linking of the pre-T-cell receptor, causing __________, the arrest of further ________ gene rearrangement, and the expression of both _______. As with the B-cell light-chain locus, the _________ can undergo multiple rearrangements to produce a functional protein. 8.11 Matching: Match the following subsets of murine γ:δ T cells with the appropriate description:


Immunology_Janeway

Immunology_Janeway. 8.11 Matching: Match the following subsets of murine γ:δ T cells with the appropriate description: A. Dendritic i. Can be divided in two groups: epidermal IFN-γ and IL-4 producing, and IFN-γ T cells producing subsets B. Vγ4+ ii. Cells that home to the reproductive tract, lung, and dermis; upon stimulation these can produce inflammatory cytokines C. Vγ6+ T cells iii. A population of later developing γ:δ T cells programmed to secrete IL-17 when activated, and can be found in all lymphoid organs, as well as the dermis D. Vγ1+ T cells iv. Cells that, as a response to a pathogen or a wound, can induce inflammation, promote wound healing, and produce growth factors; also characterized by their T-cell receptors’ use of the Vγ5 segment E. Vγ7+ T cells v. Specifically home to the intestinal epithelium 8.12 Multiple Choice: Which of the following options correctly describes a difference between the B-cell receptor and the T-cell receptor?


Immunology_Janeway

Immunology_Janeway. A. VDJ rearrangement of the T-cell receptor β chain occurs first in T-cell development, as opposed to the B-cell receptor, which undergoes VDJ rearrangement after light-chain VJ rearrangement. B. T cells do not require the formation of a pre-T-cell receptor in order to advance their development, as opposed to B cells, which require signaling through the pre-B-cell receptor in order to undergo allelic exclusion and continue development. C. Expression of the B-cell receptor stops further light-chain rearrangement and enforces strict allelic exclusion, while expression of the T-cell receptor does not restrict further rearrangements of the alpha chain until there is signaling through peptide:MHC binding, resulting in many T cells that express two different TCRα chains. D. TCRα chains cannot undergo successive rearrangements, as opposed to B-cell receptors, which go through the process of receptor editing.


Immunology_Janeway

Immunology_Janeway. D. TCRα chains cannot undergo successive rearrangements, as opposed to B-cell receptors, which go through the process of receptor editing. 8.13 Multiple Choice: Which of the following correctly describes T regulatory cells (Treg cells)? A. Treg cells are a subset of CD8+ T cells that express cytotoxic activity against cells infected by intracellular pathogens. B. The Treg T-cell receptor is characterized by its weak affinity for self MHC so that self-tolerance can be mediated. C. Treg cells express FoxP3. D. In many cases, autoimmunity is the product of overactive T cells. General references. Loffert, D., Schaal, S., Ehlich, A., Hardy, R.R., Zou, Y.R., Muller, W., and Rajewsky, K.: Early B-cell development in the mouse—insights from mutations introduced by gene targeting. Immunol. Rev. 1994, 137:135–153.