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Janeway CA Jr, Travers P, Walport M, et al. Immunobiology: The Immune System in Health and Disease. 5th edition. New York: Garland Science; 2001.

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Immunobiology: The Immune System in Health and Disease. 5th edition.

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The rearrangement of antigen-receptor gene segments controls lymphocyte development

In this part of the chapter we look in more detail at the steps that lead to a mature lymphocyte expressing a unique antigen receptor on its surface. The binding site of this receptor is formed from the variable regions of two different receptor chains and, in general, lymphocyte development is regulated so that each mature cell produces only one of each of these (for example, one immunoglobulin heavy chain and one light chain in B cells), and thus bears receptors of a single specificity. Production of a complete antigen receptor thus entails two series of gene segment rearrangements, one for each receptor-chain locus. Each series of rearrangements continues until a protein product is made, at which point the cell moves on to the next stage of development. This process is guided by signals that regulate the expression of the transcription factors and enzymes that control the rearrangement process. Such signals are generated by expression of a complete antigen receptor and also by expression of a pre-B- or pre-T-cell receptor in which the first receptor chain to be successfully rearranged is combined with a surrogate second chain; these receptors form complexes with accessory chains that have a signaling function. T-cell precursors develop to express either of two mutually exclusive types of T-cell receptor, the α:β receptor or the γ:δ receptor, and we will describe how this is thought to occur.

7-8. B cells undergo a strictly programmed series of gene rearrangements in the bone marrow

As we saw in Chapter 4, the genes that encode immunoglobulin V regions are initially organized as an array of separate gene segments. These must be rearranged and joined together in the developing B cell to produce a complete V-region sequence. The recombination process is imprecise, however, with the random addition of nucleotides at the joins between gene segments (see Section 4-8). This means that it is a matter of chance whether the juxtaposed J sequence, for example, and the μ constant (C)-region sequence downstream (see Fig. 4.8), can be read in the correct reading frame; each time a V gene segment undergoes rearrangement to a J segment, or to an already rearranged DJ sequence, there is a roughly two in three chance of generating an out-of-frame sequence downstream from the join. Thus, B-cell development has evolved to preserve and multiply those B cells that have made productive joins and to eliminate cells that have not.

In addition, because there are two alleles for each immunoglobulin locus in the diploid genome, each of which can rearrange, the cell must prevent both alleles from making productive joins, lest the cell express two or more receptors of different antigen specificities. This is accomplished by checking for productive joins as soon as an allele has rearranged. When a productive join is made, it generally signals the cell to cease the current phase of rearrangement and progress to the next stage. Assembly of the genes for a complete receptor requires three separate recombination events, which occur at different stages of B-cell development. These are, in the order that they occur: the joining of D to JH and VH to DJH to produce the functional heavy-chain gene, and the joining of VL to JL to produce the functional light-chain gene. The kappa chain locus is generally rearranged before the lambda chain locus, the latter only initiating rearrangement if the kappa locus rearrangements have failed to generate a productive join.

Because only about one in three joins will be successful, and three successful joins are required to express a complete immunoglobulin molecule, a large number of developing B cells are lost because they fail to make a productive rearrangement at one of these stages. The sequence of immunoglobulin gene rearrangements and the points at which nonproductive rearrangements can lead to cell loss are shown in Fig. 7.14. Far fewer cells are expected to be lost because of failure to make productive light-chain gene rearrangements than are lost at the stage of heavy-chain gene rearrangement. This is partly because there are two light-chain loci—κ and λ—that can be rearranged, and partly because the opportunity for successive rearrangement attempts is much greater at each light-chain locus.

Figure 7.14. The steps in immunoglobulin gene rearrangement at which cells can be lost.

Figure 7.14

The steps in immunoglobulin gene rearrangement at which cells can be lost. The developmental program usually rearranges the heavy-chain (H-chain) locus first and then the light-chain (L-chain) loci. Cells are allowed to progress to the next stage when (more...)

7-9. Successful rearrangement of heavy-chain immunoglobulin gene segments leads to the formation of a pre-B-cell receptor that halts further VH to DJH rearrangement and triggers the cell to divide

Immunoglobulin heavy-chain gene rearrangement begins in early pro-B cells with D to JH joining. This typically occurs at both alleles of the heavy-chain locus, at which point the cell becomes a late pro-B cell. The cell then proceeds to rearrange a VH gene segment to the DJH sequence. Most D to JH joins in humans are potentially useful, as almost all human D gene segments can be translated in all three reading frames without encountering a stop codon. Thus, there is no need of a special mechanism for distinguishing successful D-JH joins, and at this early stage there is also no need to ensure that only one allele undergoes rearrangement. Indeed, given the likely rate of failure at later stages, starting off with two successfully rearranged D-J sequences is an advantage.

VH to DJH rearrangement occurs first on only one chromosome. A successful rearrangement means that intact μ chains are produced, VH to DJH rearrangement ceases, and the cell progresses to become a pre-B cell. In at least two out of three cases, however, the first rearrangement is nonproductive, and VH to DJH rearrangement continues on the other chromosome, again with a theoretical one in three chance of being productive. A rough estimate of the chance of generating a pre-B cell is thus something less than 55% (1/3 + (2/3 × 1/3) = 0.55).

The large pre-B cell in which a successful heavy-chain gene rearrangement has just occurred stops VH to DJH rearrangement and begins to divide. This change in the cell's state is thought to occur after the transient expression of the rearranged heavy chain as part of a pre-B-cell receptor, which in some way provides the cell with a signal to halt rearrangement and to start proliferation. Pro-B cells in which rearrangements at both heavy-chain alleles are nonproductive are unable to receive this signal, and are eliminated. A considerable proportion of pro-B cells is therefore lost at this stage.

The pre-B-cell receptor is formed by an association between the μ heavy chain and two proteins that are made in pro-B cells, which pair noncovalently to form a surrogate light chain (Fig. 7.15). One of these is called λ5 because of its close similarity to the C domain of the λ light chain, and the other, called VpreB, resembles a light-chain V domain but has an extra amino-terminal region. The signaling capability of the pre-B-cell receptor depends on its further association with Igα and Igβ, two invariant accessory chains that also attend the mature B-cell receptor (see Section 6-6). Thus λ5, VpreB, the μ heavy chain, and the attendant Igα and Igβ chains together form a pre-B-cell receptor complex, which structurally resembles a mature B-cell receptor complex.

Figure 7.15. A productively rearranged immunoglobulin gene is expressed immediately as a protein by the developing B cell.

Figure 7.15

A productively rearranged immunoglobulin gene is expressed immediately as a protein by the developing B cell. In early pro-B cells, heavy-chain gene rearrangement is not yet complete and no functional μ protein is expressed, as shown in the top (more...)

The pre-B-cell receptor complex is expressed only transiently, perhaps because the production of λ5 stops as soon as pre-B-cell receptors begin to be formed. Although the pre-B-cell receptor is expressed at low levels on the surface of pre-B cells, it is not clear whether it interacts with an external ligand. It may simply be the assembly of the receptor, or even its insertion into the endoplasmic reticulum membrane, where most pre-B-cell receptor molecules are found, that generates the signals required for further development. Nevertheless, its formation is an important checkpoint in B-cell development. In mice that either lack λ5 or possess mutant heavy-chain genes that cannot produce transmembrane heavy chains, the pre-B-cell receptor cannot be formed and B-cell development is blocked after heavy-chain gene rearrangement. Because the normal signals that halt VH to DJH rearrangement are not given, λ5 knockout mice have rearrangements of the heavy-chain genes on both chromosomes in all pre-B cells, so that about 10% of the cells have two productive VDJH rearrangements.

In normal mice, the appearance of the pre-B-cell receptor coincides with inactivation of one of the recombinase subunits, RAG-2, by phosphorylation, which targets it for degradation. In addition, synthesis of the mRNAs for both RAG-2 and the other recombinase subunit, RAG-1, is suppressed, suggesting that this is the mechanism by which further rearrangement at the heavy-chain locus is blocked. Expression of the pre-B-cell receptor is also associated with cell enlargement, followed by a burst of proliferation. These cells then undergo the transition to small resting pre-B cells, in which the RAG-1:RAG-2 recombinase is again produced and the light-chain locus can be rearranged. The pre-B-cell receptor therefore appears to signal to the cell that a complete heavy-chain gene has been formed, that further rearrangements at this locus should be suppressed, and that development can proceed to the next stage. The intracellular tyrosine kinase Btk (see Section 6-10) is thought to play a part in transducing this signal, as in its absence B-cell development is blocked at the pre-B cell stage.

In the mouse, the large pre-B cells divide several times, expanding the population of cells with successful in-frame joins by approximately thirty to sixtyfold before they become resting small pre-B cells. A large pre-B cell with a particular rearranged heavy-chain gene therefore gives rise to numerous progeny. Upon reaching the small pre-B-cell stage, each of these progeny can make a different rearranged light-chain gene. Thus cells with many different antigen specificities are generated from a single pre-B cell. This makes an important contribution to B-cell receptor diversity. A similar course of events occurs in the thymus, allowing a diversity of T-cell receptor α chains to be expressed with a successfully rearranged β chain during T-cell development. (Image clinical_small.jpgX-linked Agammaglobulinemia, in Case Studies in Immunology, see Preface for details)

7-10. Rearrangement at the immunoglobulin light-chain locus leads to cell-surface expression of the B-cell receptor

In mice and humans, the κ light-chain locus tends to rearrange before the λ locus. This was first deduced from the observation that myeloma cells secreting λ light chains generally have both their κ and λ light-chain genes rearranged, whereas in myelomas secreting κ light chains generally only the κ genes are rearranged. This order is occasionally reversed, however, and λ gene rearrangement does not absolutely require the prior rearrangement of the κ genes.

As with the heavy-chain locus, rearrangements at the light-chain locus generally take place at only one allele at a time. Unlike the case of the heavy-chain genes, however, there is scope for repeated rearrangements of unused V and J gene segments at each allele (Fig. 7.16). Several successive attempts at productive rearrangement of a light-chain gene can therefore be made on one chromosome before initiating any rearrangements on the second chromosome.

Figure 7.16. Nonproductive light-chain gene rearrangements can be rescued by further gene rearrangement.

Figure 7.16

Nonproductive light-chain gene rearrangements can be rescued by further gene rearrangement. The organization of the light-chain loci in mice and humans offers many opportunities for rescue of pre-B cells that initially make an out-of-frame light-chain (more...)

The chances of eventually generating an intact light chain are greatly increased by the potential for multiple successive rearrangement events at each allele and by the chance to successfully rearrange either of the two light-chain loci. As a result, most cells that reach the pre-B cell stage succeed in generating progeny that bear intact IgM molecules and can be classified as immature B cells.

Once a light-chain gene has been rearranged successfully, light chains are synthesized and combine with the heavy chain to form intact IgM (see Fig. 7.15). IgM appears at the cell surface together with Igα and Igβ to form the functional B-cell receptor complex. If the newly expressed receptor encounters a strongly cross-linking antigen—that is, if the B cell is strongly self-reactive—development is halted and the cell will not mature further. This is the first negative selection process that B cells undergo. On the other hand, if the IgM is not self-reactive, the cell continues to mature. It is not yet clear how, in the absence of binding to a specific antigen, the B cell senses that a functional immunoglobulin receptor has been expressed, and thereby receives signals for further maturation. A role for Igα in signaling at this stage is indicated by a reduction in B-lineage cells in mice that express Igα with a truncated cytoplasmic domain, which therefore cannot signal to the interior of the cell. In these mice the number of immature B cells in the marrow is reduced fourfold, and the number of peripheral B cells is reduced one hundredfold. This shows that an ability to signal through Igα is particularly important in dictating emigration of B cells from the bone marrow and/or their survival in the periphery once a complete immunoglobulin molecule is expressed.

Thus, by signaling the completion of a productive rearrangement, the synthesis of an immunoglobulin heavy chain or light chain and its assembly into a receptor (either the pre-B-cell receptor or IgM) leads to the further maturation of the B cell and ultimately to the cessation of further rearrangements. The shutdown of rearrangement once a productive join is made is the mechanism underlying allelic exclusion, a term that signifies the expression of only one of the two alleles of a given gene in a diploid cell (Fig. 7.17). Allelic exclusion occurs at both the heavy-chain locus and the light-chain loci. The phenomenon was discovered over 30 years ago, when it provided one of the original pieces of experimental support for the clonal selection theory. A similar mechanism underlies isotypic exclusion, production of a light chain from only one of the two light-chain loci—κ or λ—in each individual cell.

Figure 7.17. Allelic exclusion in individual B cells.

Figure 7.17

Allelic exclusion in individual B cells. Most species have genetic polymorphisms of the constant regions of their immunoglobulin heavy- and light-chain genes; these are known as allotypes (see Section 4-20). In rabbits, for example, all of the B cells (more...)

Allelic exclusion seems to operate without substantial allelic preference, as the alleles at each locus are generally expressed at roughly equal frequencies; chance probably determines which allele rearranges first. In light-chain isotypic exclusion, however, there is a decided preference for which locus is rearranged first, and the ratios of κ-expressing versus λ-expressing mature B cells vary from one extreme to the other in different species. In mice and rats it is 95% κ to 5% λ; in humans it is typically 65:35, and in cats it is 5:95, the opposite of that in mice. These ratios correlate most strongly with the number of functional Vk and Vλ gene segments in the species genome. They also reflect the kinetics and efficiency of gene segment rearrangements. The κ:λ ratio in the mature lymphocyte population is useful in clinical diagnostics, as an aberrant κ:λ ratio indicates the dominance of one clone and the presence of a lymphoproliferative disorder, which may be malignant.

7-11. The expression of proteins regulating immunoglobulin gene rearrangement and function is developmentally programmed

A variety of proteins have a role in the development of B cells, and many of these contribute to regulating and executing the sequential steps in immunoglobulin gene segment rearrangement that mark the different stages in B-cell differentiation. Figure 7.18 lists some of these proteins, and shows how their expression is regulated through the different stages of B-cell development.

Figure 7.18. The temporal expression of several cellular proteins known to be important for B-cell development.

Figure 7.18

The temporal expression of several cellular proteins known to be important for B-cell development. The proteins listed here are a selection of those known to be associated with early B-lineage development, and have been included because of their proven (more...)

Both immunoglobulin gene rearrangement and T-cell receptor gene rearrangement depend on the proteins RAG-1 and RAG-2, products of the recombination-activation genes RAG-1 and RAG-2. These proteins are unique to vertebrates, and thus are at least part of the reason that only vertebrates have rearranging antigen receptor genes and can mount an adaptive immune response. The RAG-1:RAG-2 dimer is a component of the V(D)J recombinase (see Section 4-5) which is active at very early stages of lymphoid development. In both B- and T-cell lineages there is a later and temporary suppression of RAG gene expression after successful rearrangements have been completed at the first of the two rearranging loci (the immunoglobulin heavy-chain locus in B cells and the TCR-β locus in T cells). Thus the RAG proteins are inactive during the burst of cell proliferation that follows these first successful rearrangements (see Sections 7-9 and 7-15), but they are resynthesized later when the cells cease dividing and go on to make rearrangements at the second locus.

Another enzyme, terminal deoxynucleotidyl transferase (TdT), contributes to the diversity of both B-cell and T-cell antigen receptor repertoires by adding N-nucleotides at the joints between rearranged gene segments (see Section 4-8). As with the RAG proteins, TdT is expressed in early lymphoid progenitors but, unlike the RAG-1:RAG-2 enzyme, it is not essential to the rearrangement process. Indeed, at the time in fetal development when the peripheral immune system is first being supplied with T and B lymphocytes, TdT is expressed at low levels, if at all. In adult humans, it is expressed in pro-B cells but its expression declines at the pre-B cell stage, when heavy-chain gene rearrangement is complete and light-chain gene rearrangement has commenced. This timing explains why N-nucleotides are found in the V-D and D-J joints of heavy-chain genes but only in about a quarter of human light-chain joints. N-Nucleotides are rarely found in mouse light-chain V-J joints, showing that TdT is switched off slightly earlier in the development of mouse B cells.

Proteins required for formation of a functional receptor complex are also essential for B-cell development. The invariant proteins Igα (CD79α) and Igβ (CD79β) are components of both the pre-B-cell receptor and the B-cell receptor complexes on the cell surface (see Fig. 7.15). As well as enabling immunoglobulins to be transported to the cell surface, Igα and Igβ transduce signals from these receptors by interacting with intracellular tyrosine kinases through their cytoplasmic tails (see Section 6-6). Igα and Igβ are expressed from the pro-B cell stage until the death of the cell or its terminal differentiation into an antibody-secreting plasma cell. Curiously, mice lacking Igβ have a block in B-cell development at the pro-B cell stage, before VDJH rearrangements are complete. The requirement for Igβ is thought to be due to the assembly of an Igα:Igβ complex with the chaperone protein calnexin in the normal pro-B cell, which may trigger signals needed for further development.

Other signal transduction proteins with a role in B-cell development are included in Fig. 7.18. Bruton's tyrosine kinase (Btk), a Tec-family kinase (see Section 6-10), has received intense scrutiny because mutations in the Btk gene cause a profound B-lineage-specific immune deficiency, Bruton's X-linked agammaglobulinemia (XLA) (Image clinical_small.jpgX-linked Agammaglobulinemia, in Case Studies in Immunology, see Preface for details), in which no mature B cells are produced. In humans, the block in B-cell development caused by mutations at the XLA locus is almost total, interrupting the transition from pre-B cell to immature B cell. A similar, though less severe, defect called X-linked immunodeficiency or xid arises from mutations in the corresponding gene in mice.

Finally, several transcription factors or gene-regulatory proteins are essential for B-cell development, as shown by deficiencies of the B-cell lineage in genetically engineered mice lacking these proteins. At least 10 transcription factors necessary for normal B-lineage development have been described, and there are likely to be others. Many of these, like Ikaros, the absence of which leads to a lack of B cells, are also required for the development of other hematopoietic lineages as well. Essential transcription factors for B-cell development include the products of the E2A gene. These products, which are derived by alternative splicing, induce another transcription factor, the early B-cell factor (EBF), which in turn regulates the transcription of the gene for Igα. In the absence of E2A or EBF even the earliest identifiable stage in B-cell development, D-JH joining, fails to occur. Another important trans-cription factor is the pax-5 gene product, one isoform of which is the B-lineage-specific activator protein (BSAP). This protein is active in late pro-B cells and enables the proper functioning of the heavy-chain enhancers located 3′ of the heavy-chain C-region genes. It also binds to regulatory sites in the genes for λ5, VpreB, and other B-cell specific proteins. In the absence of BSAP, pro-B cells fail to develop further down the B-cell pathway but can be induced to give rise to T cells and various myeloid cell types. Thus BSAP is required for the commitment of the pro-B cell to the B-cell lineage.

It seems likely that these regulatory proteins and others like them together direct the developmental program of B-lineage cells. In particular, the proteins involved in the tissue-specific transcriptional regulation of immunoglobulin genes are likely to be important in regulating the order of events in gene rearrangement. The V(D)J recombinase system is not in itself lineage-specific; it operates in both B- and T-lineage cells and uses the same core enzymes, RAG-1:RAG-2, which recognize the same conserved recombination signal sequences in both immunoglobulin and T-cell receptor genes. Yet rearrangements of T-cell receptor genes do not occur in B-lineage cells, nor do complete rearrangements of immunoglobulin genes occur in T cells. The ordered rearrangement events that do occur are associated with low-level transcription of the gene segments about to be joined. This is probably because key transcription factors such as BSAP bind to the DNA and ‘open’ the chromatin, making it accessible to the recombinase enzymes (Fig. 7.19).

Figure 7.19. Proteins binding to promoter and enhancer elements contribute to the sequence of gene rearrangement and regulate the level of RNA transcription.

Figure 7.19

Proteins binding to promoter and enhancer elements contribute to the sequence of gene rearrangement and regulate the level of RNA transcription. The immunoglobulin heavy-chain locus is illustrated. First panel: in germline DNA, stem cells, and nonlymphoid (more...)

As a consequence of immunoglobulin gene rearrangement, the promoter upstream of the V gene segments is brought nearer to the enhancers associated with the C gene segments. This in turn brings transcription factors that have bound the promoter and enhancer into proximity, resulting in a dramatic increase in transcription of the rearranged segments. Thus, gene rearrangement can be viewed as a powerful mechanism for regulating gene expression, as well as for generating receptor diversity. Several cases of gene rearrangement that brings the rearranged genes under the control of a new promoter are known from prokaryotes and single-celled eukaryotes, but in vertebrates only the immunoglobulin and T-cell receptor genes are known to use gene rearrangement to regulate gene expression.

7-12. T cells in the thymus undergo a series of gene segment rearrangements similar to those of B cells

Developing T cells face a similar challenge to developing B cells. They must assemble a functional gene for each T-cell receptor chain while at the same time ensuring that each T cell expresses receptors of only one specificity. Not surprisingly, T cells follow an almost identical strategy to B cells, in that the receptor is assembled in stages, with each stage being checked for correct assembly. Moreover, as in B cells, a productively rearranged gene is expressed as soon as it is made, and the product(s) are assembled into a receptor complex, in this case either a pre-T-cell receptor or a bona fide T-cell receptor. Expression of this receptor is instrumental in promoting further development, which ultimately results in shutting down further rearrangement at the locus that has just been active.

Despite the similarities with B-cell development, the control of antigen-receptor assembly during development is more complicated for T cells because there are two different kinds of T cells that could be generated from an undifferentiated precursor—α:β T cells or γ:δ T cells. These two types are distinguished by the different genetic loci that are used to make their T-cell receptors, as described in Section 4-13. Thus, the T-cell developmental program must control to which of the two lineages a precursor commits and must also ensure that a fully developed T cell only expresses receptor components of one or the other lineage. Another key difference between B and T cells is that the final assembly of an immunoglobulin leads to cessation of gene rearrangement and initiates the further differentiation of the B cell, whereas in the case of T cells, rearrangement of the Vα gene segments continues unless there is signaling to positively select the receptor.

7-13. T cells with α:β or γ:δ receptors arise from a common progenitor

T cells bearing γ:δ receptors differ from α:β T cells in the types of antigen they recognize, in the pattern of expression of the CD4 and CD8 co-receptors, and in their anatomical distribution in the periphery. The two types of T cell also differ in function, although relatively little is known about the function of γ:δ T cells (see Sections 2-28 and 3-19). The gene rearrangements found in thymocytes and in mature γ:δ and α:β T cells suggest that these two cell lineages diverge from a common precursor after certain gene rearrangements have already occurred (Fig. 7.20). Mature γ:δ T cells can have productively rearranged β-chain genes, and mature α:β T cells often contain rearranged, but mostly (about 80%) out-of-frame, γ-chain genes.

Figure 7.20. Signals through the γ:δ TCR and the pre-T-cell receptor compete to determine the fate of thymocytes.

Figure 7.20

Signals through the γ:δ TCR and the pre-T-cell receptor compete to determine the fate of thymocytes. During differentiation of T cells in the thymus, at the double-negative stage where they express neither CD4 nor CD8, the developing thymocytes (more...)

The β, γ, and δ loci undergo rearrangement almost simultaneously in developing thymocytes. At present, the factors that regulate the lineage commitment of these cells are not known; indeed, commitment to the γ:δ lineage might simply depend on whether productive rearrangements at a γ and a δ gene have occurred in the same cell. In this view, successful rearrangement of a γ and a δ gene leads to the expression of a functional γ:δ T-cell receptor that signals the cell to differentiate along the γ:δ lineage. In most precursors, however, there is a successful rearrangement of a β-chain gene, which results in the production of a functional β-chain protein, before successful rearrangement of both γ and δ has occurred. The β chain pairs with the surrogate α chain, pTα, to create a pre-T-cell receptor (β:pTα), thus arresting further gene rearrangement and signaling the thymocyte to proliferate, to express its co-receptor genes, and eventually to start rearranging the α-chain genes. It is not known whether the TCRβ:pTα receptor recognizes a specific ligand, but it is known that its expression leads to signaling via the cytoplasmic tyrosine kinase Lck and that this is crucial for the further development of an α:β T cell (see Section 7-15). It seems likely that signals through the pre-T receptor also commit the cell to the α:β lineage (see Fig. 7.20). According to this view, the mature γ:δ cells that carry productive β-gene rearrangements committed to the γ:δ rather than α:β lineage because they have received a signal from an assembled γ:δ receptor before having assembled a functional pre-T-cell receptor. An alternative theory is that these cells were diverted into becoming γ:δ cells at a later stage, after receiving signals from a pre-T receptor and progressing to the second phase of RAG enzyme activity. However, it remains uncertain whether further rearrangements at the γ and δ loci can occur at this point. Normally, α-gene rearrangements ensue and these delete the intervening δ-chain gene segments as an extrachromosomal circle (see Section 4-13 and Fig. 4.15). The subsequent maturation of α:β T cells depends on rearrangements at the α locus leading to a functional α:β receptor that is positively selected for its ability to recognize self MHC molecules. The signals that drive a γ:δ T cell to mature are also unknown. Certain γ:δ T cells seem able to develop in the absence of a functioning thymus and are present, for example, in nude mice.

7-14. T cells expressing particular γ- and δ-chain V regions arise in an ordered sequence early in life

We have just described how a precursor T cell that can become either an α:β or a γ:δ T cell becomes directed to one or the other lineage. During the development of the organism, these pathways are not equally used; instead, the generation of the various types of T cells—even the particular V region assembled in γ:δ cells—are developmentally controlled. The first T cells to appear during embryonic development carry γ:δ T-cell receptors (Fig. 7.21). In the mouse, where the development of the immune system can be studied in detail, γ:δ T cells first appear in discrete waves or bursts, with the T cells in each wave populating distinct sites in the adult animal.

Figure 7.21. 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.

Figure 7.21

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, the Cγ1 locus is expressed with its closest (more...)

The first wave of γ:δ T cells populates the epidermis; the T cells become wedged among the keratinocytes and adopt a dendritic-like form which has given them the name of dendritic epidermal T cells (dETCs). The second wave homes to the epithelia of the reproductive tract. Remarkably, given the large number of theoretically possible rearrangements, the receptors expressed by these early waves of γ:δ T cells are essentially homogeneous. All the cells in each wave assemble the same Vg and Vd regions. Each different wave, however, uses a different set of V, D, and J gene segments. Thus, certain V, D, and J gene segments are selected for rearrangement at particular times during embryonic development; the reasons for this limitation are poorly understood. There are no N-nucleotides contributing additional diversity at the junctions between V, D, and J gene segments, reflecting the absence of the enzyme TdT from these fetal T cells.

After these initial waves, T cells are produced continuously rather than in bursts, and α:β T cells predominate, making up more than 95% of thymocytes. The γ:δ T cells produced at this stage are different from those of the early waves. They have a considerably more diverse receptor repertoire, for which several different V gene segments have been used, and the receptor sequences have abundant N-nucleotide additions. Most of these γ:δ T cells, like α:β T cells, are found in peripheral lymphoid tissues rather than in the epithelial sites populated by the early γ:δ T cells.

The developmental changes in V gene segment usage and N-nucleotide addition in murine γ:δ T cells parallel changes in B-cell populations during fetal development, which will be discussed later (see Section 7-28). Their functional significance is unclear, however, and not all of these changes in the pattern of receptors expressed by γ:δ T cells occur in humans. Certainly, the γ:δ T cells that home to the skin of mice, the dendritic epidermal T cells (dETCs), do not seem to have exact human counterparts, although there are γ:δ T cells in the human reproductive and gastrointestinal tracts. The mouse dETCs may serve as sentinel cells which are activated upon local tissue damage or as cells that regulate inflammatory processes (see Section 2-28).

7-15. Rearrangement of the β-chain locus and production of a β chain trigger several events in developing thymocytes

T cells expressing α:β receptors first appear a few days after the earliest γ:δ T cells and rapidly become the most abundant type of thymocyte (see Fig. 7.21). The rearrangement of the β- and α-chain loci during T-cell development follows a sequence that closely parallels the rearrangement of immunoglobulin heavy- and light-chain loci during B-cell development (see Section 7-9). As shown in Fig. 7.22, the β-chain genes rearrange first, with the Dβ gene segments rearranging to Jβ gene segments, and this is followed by Vβ to DJβ gene rearrangement. If no functional β chain can be synthesized from these rearrangements, the cell will not be able to produce a pre-T-cell receptor and will die unless it makes successful rearrangements at both the γ and δ loci (see Section 7-13). However, unlike B cells with nonproductive immunoglobulin heavy-chain gene rearrangements, thymocytes with nonproductive β-chain VDJ rearrangements can be rescued by subsequent rearrangements, which are possible owing to the presence of two clusters of Dβ and Jβ gene segments upstream of two Cβ genes (see Fig. 4.11). This increases the likelihood of a productive VDJ join from 55% for immunoglobulin heavy-chain genes to more than 80% for T-cell receptor β-chain genes.

Figure 7.22. The stages of gene rearrangement in α:β T cells.

Figure 7.22

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. (more...)

Once a productive β-chain gene rearrangement has occurred, the β chain is expressed together with the invariant partner chain pTα and the CD3 molecules (see Fig. 7.22) and is transported to the cell surface. The β:pTα complex is a functional pre-T-cell receptor analogous to the μ:VpreB:λ5 pre-B-cell receptor complex in B-cell development (see Section 7-9). Expression of the pre-T-cell receptor triggers the phosphorylation and degradation of RAG-2, halting β-chain gene rearrangement and thus ensuring allelic exclusion at the β locus. It also induces rapid cell proliferation and eventually the expression of the co-receptor proteins CD4 and CD8. All these events require the cytoplasmic protein tyrosine kinase Lck, which 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 are made.

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

During the proliferative phase triggered by expression of the pre-T-cell receptor, the RAG-1 and RAG-2 genes are also repressed. Hence, no rearrangement of the α-chain locus occurs until the proliferative phase ends, when RAG-1 and RAG-2 genes are transcribed again, and the functional RAG-1:RAG-2 heterodimer accumulates. This ensures that each cell in which a β-chain gene has been successfully rearranged gives rise to many CD4+ CD8+ double-positive thymocytes. Once the cells stop dividing, each of these 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 peptide:MHC complexes in the thymus can begin.

Along the progression of T cells from the double-negative to the double-positive and finally single-positive stage, there is a distinct pattern of expression of molecules involved in rearrangement, signaling, and also transcription factors that most likely control developmental fates as well as the expression of important T-cell loci like those of the T-cell receptor itself. The expression of these molecules through T-cell development is illustrated in Fig. 7.23. The expression of the RAG proteins has already been described. TdT, the protein responsible for insertion of N-nucleotides at gene segment junctions in both B and T cells, is expressed throughout the developmental period in which thymocytes are rearranging T-cell receptor gene segments; N-nucleotides can be found at the junctions of all rearranged α and β genes. Lck, a Src-family tyrosine kinase, and ZAP-70, another tyrosine kinase, are both expressed from an early stage in thymocyte development. Lck plays a key role in signaling of thymocytes at the pre-TCR+ stage, since mice lacking Lck have a profound loss of cells beyond this stage. Lck is also important for γ:δ T-cell development. On the other hand, gene knockout studies (see Appendix I, Section A-47) show that ZAP-70, though expressed from the double-negative stage onward, plays a later role in promoting the development of single-positve thymocytes from double-positive thymocytes. Fyn is expressed at increasing levels from the double-positive stage onward, but is not absolutely essential for thymocyte development as long as Lck is present. The roles these molecules play in mature T cell signaling are described in Chapter 6. Finally, a set of transcription factors guides the development of thymocytes from one stage to the next. A number of important factors have been identified; in some cases gene knockout studies have shown at which stage of development they play essential roles. Ikaros and GATA-3 are expressed in early T-cell progenitors, and in the absence of either, T-cell development is generally disrupted; moreover, these molecules also have roles in the normal functioning of mature T cells. In contrast, Ets-1, though also expressed in early progenitors, does not have an essential role for T-cell development, although mice lacking this factor do not make NK cells. TCF1 (T-cell factor-1) is first expressed during the double-negative stage and in its absence double-negative T cells that make productive β rearrangements do not undergo proliferation as usual in response to the pre-TCR signal. Thus, TCF1-deficient mice fail to make double-positive thymocytes efficiently. LKLF (lung Kruppel-like factor) is first expressed late in thymocyte development at the single-positive stage; if LKLF is absent, single-positive cells do develop, but they are abnormal, displaying a partially activated phenotype. Thus, transcription factors are turned on at various developmental stages and are responsible for normal development through those stages by controlling the expression of important genes. Transcription factors mediate the response to developmental signals by turning on the set of appropriate genes when the factors are activated to bind DNA.

Figure 7.23. The temporal expression of several cellular proteins known to be important for early T-cell development.

Figure 7.23

The temporal expression of several cellular proteins known to be important for early T-cell development. The expression of a set of proteins is depicted with respect to the stages of thymocyte development as determined by cell-surface marker expression. (more...)

7-16. 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 gene has been expressed. As with the immunoglobulin light-chain genes, repeated attempts at rearrangement are possible. Indeed, the presence of multiple Vα gene segments, and around 60 Jα gene segments spread over some 80 kb of DNA allows many successive Vα to Jα rearrangements. 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 (Fig. 7.24).

Figure 7.24. Multiple successive rearrangement events can rescue nonproductive T-cell receptor α-chain gene rearrangements.

Figure 7.24

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’ (more...)

The potential for many successive rearrangements at both alleles of the α-chain locus virtually guarantees that a functional α chain will be produced in every developing T cell. Moreover, many T cells have in-frame rearrangements on both chromosomes and thus can produce two types of α chain. This is possible because, unlike the situation in B cells, expression of the T-cell receptor is not in itself sufficient to shut off gene rearrangement. Thus, rearrangement of α-chain genes continues even after production of a T-cell receptor at the cell surface. Continued rearrangements can allow several different α chains to be produced successively in each developing T cell and to be tested for 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 only ceases when positive selection occurs as a consequence of receptor engagement, or when the cell dies. Thus, in the strict sense, T-cell receptor α-chain genes are not subject to allelic exclusion (see Section 7-10). However, as we will see in the next part of this chapter, only T-cell receptors that are positively selected for self MHC recognition can function in self MHC-restricted responses. The regulation of α-chain gene rearrangement by positive selection therefore ensures that each T cell has only a single functional specificity, even if two different α chains are expressed.

Clearly, the engagement of any particular T-cell receptor with a self MHC:self peptide ligand will depend on the receptor's specificity. Thus, the phase of α-chain gene rearrangement marks an important change in the forces shaping the destiny of the T cell. Up to this point, the development of the thymocyte has been independent of antigen; from this point on, developmental decisions depend on the interaction of the T-cell receptor with its peptide:MHC ligands, as described in the next part of the chapter.


As lymphocytes differentiate from primitive stem cells, they proceed through stages that are marked by the sequential rearrangement of the antigen-receptor gene segments at the different genetic loci. As each complete receptor-chain gene is generated, the protein it encodes is expressed as part of a receptor, and this signals the developing cell to progress to the next developmental step. For B cells, the heavy-chain locus is rearranged first. If rearrangement is successful and a pre-B-cell receptor is made, heavy-chain gene rearrangement ceases and the resulting pre-B cells proliferate, followed by the start of rearrangement at a light-chain locus. If the initial light-chain gene rearrangement is productive, a complete immunoglobulin B-cell receptor is formed, gene rearrangement ceases, and B-cell development proceeds. If it is not, light-chain gene rearrangement continues until either a new productive rearrangement is made or all available J regions have been used up. If a productive rearrangement is not made, the developing B cell dies.

In developing T cells, receptor loci rearrange according to a defined program similar to that in B cells. There is, however, the added complication that individual precursor T cells can follow one of two distinct lines of development. There are four T-cell receptor gene loci—γ, δ, α, and β. Rearrangement at these loci leads to cells bearing either γ:δ T-cell receptors or α:β T-cell receptors. Early in ontogeny, γ:δ T cells predominate, but from birth onward more than 90% of thymocytes express T-cell receptors encoded by rearranged α and β genes. In developing thymocytes, the γ, δ, and β loci rearrange first, and start rearranging virtually simultaneously. Productive rearrangements of both a γ and a δ gene may lead to the production of a functional γ:δ T-cell receptor and the development of a γ:δ T cell. Most cells, however, enter the α:β lineage. In these cells the generation of a β-chain gene before both γ and δ have rearranged leads to the expression of a functional β chain and a pre-T-cell receptor. This receptor, like the pre-B-cell receptor, signals the developing cell to proliferate, to arrest β-chain gene rearrangement, to express CD4 and CD8, and eventually to rearrange the α-chain gene. Rearrangement of the α-chain genes continues in these CD4+ CD8+ double-positive thymocytes until positive selection allows the maturation of a single-positive α:β cell; almost all cells will make productive α-gene rearrangements but the vast majority of thymocytes die at the CD4 CD8 double-positive stage because they are not positively selected.

Thus gene rearrangement follows an ordered sequence in both B and T lineages to produce immature lymphocytes that each bear antigen receptors of a single specificity on their surface. These antigen receptors can now be tested for their antigen-recognition properties and the cells selected accordingly; these selection processes are described in the next part of this chapter.

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Copyright © 2001, Garland Science.
Bookshelf ID: NBK27113


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