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Fig.14

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 thelight-chain (L-chain) loci. Cells are allowed to progress to the next stage when a productiverearrangement has been achieved. Each rearrangement has about a one in three chance of beingsuccessful, but if the first attempt is nonproductive, development is suspended and there is achance for one or more further attempts. The scope for repeated rearrangements is greater atthe light-chain loci (see Fig. 7.16), so that fewer cells are lost between the pre-B and immature B

cell stages than in the pro-B to pre-B transition.Figure 7.15

Rearranged immunoglobulin gene is expressed immediately as protein by the developing B cell

In early pro-B cells, heavy-chain gene rearrangement is not yet complete and no functional

protein is expressed, as shown in the top panel. As soon as a productive heavy-chain generearrangement has taken place, chains are expressed by the cell in a complex with two other

chains, ?5 and VpreB, which together make up a surrogate light chain. The whole immunoglobulin-like complex is known as the pre-B-cell receptor (second panel). It is also associated with twoother protein chains, Iga (CD79a) and Ig (CD79), in the cell. These associated chains signal the Bcell to halt heavy-chain gene rearrangement, and drive the transition to the large pre-B cell stageby inducing proliferation. The progeny of large pre-B cells stop dividing and become small pre-Bcells, in which light-chain gene rearrangements commence. Successful light-chain gene

rearrangement results in the production of a light chain that binds the chain to form a completeIgM molecule, which is expressed together with Iga and Ig at the cell surface, as shown in the

third panel. Signaling via these surface IgM molecules is thought to trigger the cessation oflightchain gene rearrangement.


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Fig.15


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Fig.16

Figure 7.16

Nonproductive lightchain 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 gene rearrangement. Light-chain

rescue is illustrated for the human ? locus. If the first rearrangement is nonproductive, a 5' V?gene segment can recombine with a 3' J? gene segment to remove the out-of-frame join and

replace it. In principle, this can happen up to five times on each chromosome, because there arefive functional J? gene segments in humans. If all rearrangements of ?-chain genes fail to yield aproductive light-chain join, ?-chain gene rearrangement may succeed (see Fig. 7.14).

Fig.17

Figure 7.17

Allelic exclusion in individual B cells

Most species have genetic polymorphisms of the constant

regions of their immunoglobulin heavy- and light-chaingenes; these are known as allotypes (see Section 4-20). In

rabbits, for example, all of the B cells in an individualhomozygous for the a allele of the immunoglobulin heavy-chain locus (Igh) will express immunoglobulin of allotype a,

whereas in an individual homozygous for the b allele all the Bcells make immunoglobulin of allotype b. In a heterozygousanimal, which carries the a allele on one of the Igh chromo-

somes and the b allele on the other, individual B cells can beshown to carry either a-type or b-type immunoglobulin, but

not both. This allelic exclusion reflects productiverearrangement of only one of the two parental Igh alleles.


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Fig.18

Figure 7.18

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-lineagedevelopment, and have been included because of their proven importance in the developmentalsequence, largely on the basis of studies in mice. Their individual contributions to B-cell

development are discussed in the text, with the exception of the Octamer Transcription Factor,Oct-2, which binds the octamer ATGCAAAT found in the heavy-chain promoter and elsewhere, andGATA-2, which is one example of the many transcription factors that are active in several

hematopoietic lineages. The pax-5 gene product, known as B-lineage-specific activator protein(BSAP), is involved in regulating the expression of several of the other proteins listed. The tight

temporal regulation of the expression of these proteins, and of the immunoglobulin genesthemselves, would be expected to impose a strict sequence on the events of B-cell differentiation.


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Fig.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

The immunoglobulin heavy-chain locus is illustrated. First panel: in germline DNA, stem cells, andnonlymphoid cells, the chromatin containing the immunoglobulin genes is in a closed conformation.Second panel: in the early pro-B cell, lineage-specific proteins bind to the Ig enhancer elements

(e); for the heavy-chain locus these are in the J-C intron and 3' to the C exons. The DNA is now inan open conformation, and low-level transcription from promoters (P) upstream of the D and J

gene segments occurs. Third panel: the rearrangement of D to J that follows the initiation oftranscription of the D and J gene segments leads to a low level of transcription from promoters

located upstream of the D gene segments. For some D-J joins in the mouse this may result in theexpression of a truncated heavy-chain (D) at levels sufficient to abort further development, butin most cases it is followed by initiation of low-level transcription of an upstream V gene segment.

Fourth panel: subsequent rearrangement of a V gene segment brings its promoter under theinfluence of the heavy-chain enhancers, leading to enhanced production of a heavy-chain mRNA

in large pre-B cells and their progeny. S represents the switch signal sequence for isotypeswitching (see Section 4-16).


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Fig.20

Figure 7.20Signals through the ?:d 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 expressneither CD4 nor CD8, the developing thymocytes express both a ?:d T-cell receptor and the pre-T-cell receptor composed of the TCR chain and the pre-Ta chain (pTatop panel). At this stage,

if the thymocyte receives signals through the ?:d receptor, the cell commits to the ?:d lineage,switching off expression of the -chain gene and thus the pre-Ta receptor (middle left panel).This cell then matures into a ?:d T cell and migrates out of the thymus into the peripheral

circulation (bottom left panel). On the other hand, if the developing thymocyte receives a signalthrough the pre-TCR (middle right panel) then the cell switches off expression of the ?:d TCR,

deletes the d chain genes as a preliminary to rearranging the TCRa- chain locus, and goes on toexpress a mature a: T-cell receptor (bottom right panel).


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Fig.21

Figure 7.21The rearrangement of T-cell receptor ? and d genes in the mouse proceeds in waves of cells

expressing different V? and Vd gene segments

At about 2 weeks of gestation, the C?1 locus is expressed with its closest V gene (V?5; also knownas V?3). After a few days V?5-bearing cells decline (upper panel) and are replaced by cellsexpressing the next most proximal gene, V?6. Both these rearranged ? chains are expressed with

the same rearranged d-chain gene, as shown in the lower panels, and there is little junctionaldiversity in either the V? or the Vd chain. As a consequence, most of the ?:d T cells produced in

each of these early waves have the same specificity, although the antigen recognized in each case

is not known. The V?5-bearing cells become established selectively in the epidermis, whereas theV?6-bearing cells become established in the epithelium of the reproductive tract. After birth, the

a: T-cell lineage becomes dominant and, although ?:d T cells are still produced, they are a muchmore heterogeneous population, bearing receptors with a great deal of junctional diversity.


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Fig.22


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Figure 7.22

The stages of gene rearrangement in a: T cells

The sequence of gene rearrangements is shown, together with an indication of the stage at whichthe events take place and the nature of the cell-surface receptor molecules expressed at eachstage. The -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 segmentsrearrange before V gene segments rearrange to DJ (second and third panels). It is possible tomake up to four attempts to generate a productive rearrangement at the -chain locus, as there

are four D gene segments and two sets of J gene segments (not shown). The productivelyrearranged gene is expressed initially within the cell and then at low levels on the cell surface. Itassociates with pTa, a surrogate 33 kDa a chain that is equivalent to ?5 in B-cell development, andthis pTa: heterodimer forms a complex with the CD3 chains (fourth panel). The expression of thepre-T-cell receptor signals the developing thymocytes via the tyrosine kinase Lck to halt -chaingene 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 a chain is nowable to undergo rearrangement. The first a-chain gene rearrangement deletes all d D, J, and C

gene segments on that chromosome, although these are retained as a circular DNA, proving thatthese are nondividing cells (bottom panel). This permanently inactivates the d-chain gene.

Rearrangements at the a-chain locus can proceed through several cycles, because of the largenumber of Va and Ja gene segments, so that productive rearrangements almost always occur.When a functional a chain is produced that pairs efficiently with the chain, the CD3low CD4+

CD8+ thymocyte is ready to undergo selection for its ability to recognize self peptides inassociation with self MHC molecules.

Figure 7.23The 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 thymocytedevelopment as determined by cell-surface marker expression. The proteins listed here are aselection of those known to be associated with early T-lineage development, and have been

included because of their proven importance in the developmental sequence, largely on the basisof studies in mice. Their individual contributions to T-cell development are discussed in the text.

Figure 7.24

Multiple successive rearrangement events can rescue nonproductive T-cell receptor a-chain

gene rearrangements

The multiplicity of V and J gene segments at the a-chain locus allows successive rearrangement

events to leapfrog over previously rearranged VJ segments, deleting any intervening gene

segments. The a-chain rescue pathway resembles that of the immunoglobulin ? light-chain genes(see Section 7-10), but the number of possible successive rearrangements is greater. a-chain Generearrangement continues until either a productive rearrangement leads to positive selection or the

cell dies.


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Fig.23

Fig.24


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