Immunology (Lippincott Illustrated Reviews Series) 2nd Edition
Chapter 8: Generation of Immune Diversity: Lymphocyte Antigen Receptors
Epitope specificity of immunoglobulin molecules produced by B cells and of T-cell receptors is determined before they encounter antigen. Moreover, several possible epitope-binding specificities greatly exceeds several genes within the human genome. This presents a paradox: How does the immune system generate a diverse array of antigen-specific molecules from a limited number of genes? The immune system’s genetic solution is both fascinating and elegant.
II. PROPERTIES OF LYMPHOCYTE ANTIGEN RECEPTORS
Domains located at the amino terminus of immunoglobulin heavy and light chains (variable or VH and VL regions) produced by different B cells are highly variable in amino acid sequence. In contrast, other regions, termed C or constant regions, are limited in variability for immunoglobulins of the same isotype produced by different B cells. Light chains have a single constant domain (CL, also designated as Ck for kappa chains or Cλ for lambda chains), and heavy chains contain multiple constant regions CH1, CH2, CH3, and for some CH4 domains. Heavy or light chain DNA gene segments for both variable and constant regions are rearranged, transcribed into RNA, and translated into a single heavy or light chain polypeptide. Individuals codominantly inherit maternal and paternal sets of alleles for light chain and heavy chain loci. A single B cell or plasma cell may express only the kappa (VkCk) or the lambda (VλCλ) light chain alleles, either maternal or paternal, to the exclusion of all others (Fig. 8.1). Likewise, a single B cell may express only the maternal or paternal VHCH heavy chain alleles but not both. The restriction of VLCL and VHCH expression to a single member of each of the involved chromosome pairs is termed allelic exclusion. However, the combined contributions of all of the B cells mean that both maternal and paternal allotypes (allelic forms) are expressed within any particular individual.
The same principles apply to the genes encoding the αβ and γδ T-cell receptors, which also include light chains (α or γ) and heavy chains (β or δ). Each chain consists of a variable region at its amino terminus and a constant region at its carboxy terminus. The variable regions of the chains are determined by the rearrangement of the DNA encoding them and the production of an mRNA transcript, including both the variable and constant regions. Splicing of the mRNA that is then translated into the polypeptide chains unites the variable and constant regions.
Allelic exclusion. Immunoglobulin light and heavy gene clusters are located on different chromosome pairs, each pair having a maternally derived and a paternally derived chromosome. Each B cell and its progeny use only one parental chromosome to encode its light (#2 or #22) and heavy (#14) chains. A given B cell uses these same variable region gene clusters throughout its lifetime for the immunoglobulins it produces to the exclusion of all others.
III. DNA REARRANGEMENT
Immunologists estimated that each person has the ability to produce a range of individual antigen-specific receptors capable of binding as many as 1015 different epitopes. DNA chromosomal rearrangement is responsible for a significant portion of epitope-specific diversity among T and B cell receptors. Rearrangement occurs at both the DNA and RNA levels by the deletion of nucleotides, followed by reannealing, to bring together gene segments that were previously separated (Fig. 8.2). Inversion of certain DNA sequences, notably within Vk, leads to rearranged nucleotide sequences of the same length as the original (see Fig. 8.2). Additional variation comes from junctional diversity (Fig. 8.3) as “exposed” ends of gene segments (V, D, and J genes) undergoing rearrangement are modified through the addition or removal of nucleotides by deoxynucleotidyl transferase (TdT) before the genes are linked together. Thus, even if the same two genes were to be linked together, the nucleotide sequence at their junctions may be different, and the amino acid sequences encoded would differ.
Chromosome rearrangement. Segments of DNA encoding a series of genes are rearranged by the deletion of intervening DNA. Joining of the remaining segments (a process called annealing) then brings together genes that were originally separated on the chromosome. In addition, DNA rearrangement is sometimes accomplished by the inversion of DNA segments, changing the linear sequence of genes without the removal of intervening DNA.
Junctional diversity. Terminal deoxynucleotidyl transferase (TdT) can add or remove exposed ends of DNA before annealing, producing additional variation in nucleotide sequence.
IV. T-CELL RECEPTORS
As we noted earlier, T cells express distinct, epitope-specific cell-surface receptors (TCRs) that are heterodimers composed of either αβ or γδ light–heavy chain pairs. Each polypeptide contains a single variable region domain and a single constant region domain. T cells express and display T-cell receptor (TCR) complexes composed of an αβ or a γδ TCR (but never both) heterodimer pair, associated CD3 (γ, δ, and ε and a CD247 ζ-chain homodimer), and CD4 or CD8 molecules. Associated transmembrane molecules, such as CD4 or CD8, stabilize the interaction of the TCR with a specific peptide-MHC (pMHC) combination. Others, such as those in the CD3 complex, participate in signal transduction events after TCR-ligand engagement (Fig. 8.4). The short cytoplasmic tail of the TCR lacks signaling sequence or immunoreceptor tyrosine activation motifs (ITAMs). The CD3 and CD247 molecules supply these. Unlike antibodies, TCRs cannot bind free epitopes. They can bind only enzymatically cleaved fragments of larger polypeptides that are presented as pMHC complexes.
T-cell receptors (TCRs). T cells express either αβ or γδ TCR heterodimers. The CD3 complex associates with the TCR to transduce a signal to the interior of the cell when the TCR engages a peptide-MHC complex.
A. Gene clusters encoding T-cell receptors
Gene clusters encoding α and δ chains of the TCR on chromosome 14 are arranged such that the entire δ chain gene cluster (Dδ, Jδ, and Cδ) lies within the a gene cluster between the Dα and Jα chain genes (Fig. 8.5). Genes encoding β and δ chains are located in separate clusters on chromosome 7.
Gene clusters encoding TCR chains. Gene clusters encoding TCR light chains are located on chromosomes 14 (α chain) and 7 (γ chain), and heavy chains on chromosomes 7 (β chain) and 14 (δ chain).
The first level of TCR diversity comes from DNA recombination to produce the variable regions. The selection of V, D, and J genes for rearrangement appears to be random from cell to cell: 2475 α chain sequences (45 Vα 3 55 Jα) 3 1200 β chain sequences (50 Vβ 3 2 Dδ 3 12 Jβ). Random association of α and β chains yields nearly 3 million different epitope-binding sites for αβ chains. For γδ TCRs, 25 γ chain sequences (5 Vγ 3 5 Jγ) 3 24 δ chain sequences (2 Vδ 3 3 Dδ 3 4 Jδ) yields 600 γδ TCR epitope-binding site possibilities. Junctional diversity, a process mediated by TdT (see Fig. 8.3), contributes a second level of diversity by the insertion or deletion of up to 20 nucleotides at the time of recombination. Thus, the total number of possible TCR specificities increases by many orders of magnitude.
B. Variable regions: Rearrangement of V, D, and J genes
TCRs are generated by recombination enzymes or recombinases (e.g., Rag-1 and Rag-2) that mediate genetic rearrangement and recombination, processes similar to those seen for immunoglobulin (see sections 8.V.B and 8.V.C, on pages 100, 101, and 103). Each T cell produces αβ or γδ TCR heterodimers, never both. Gene rearrangement begins by the excision and deletion of DNA between Vα and Jα (light, Fig. 8.5) or Vδ and Dδ (heavy) chain genes. Because the entirety of the δ chain genetic material lies within the α light chain sequence (between Vα and Jα), initiation of recombination by a chain genes deletes δ chain DNA. Conversely, initiation of recombination by δ chain (between Vδ, Dδ, and Dδ) DNA precludes a light chain DNA recombination. For the details of this process, see Figure 8.6.
C. Uniting variable and constant regions
TCR constant region genes (already rearranged into VJ or VDJ units) are united with their respective light chain VJ (Cα or Cγ) by transcription into mRNA, followed by splicing to delete intervening mRNA. The united VJC (Cα or Cγ) or VDJC (Cβ or Cδ) transcripts are then translated into proteins that are the joint product of the rearranged genes.
D. Random combinations of light and heavy chains
Genetic rearrangement and junctional diversity randomly create TCR chains that vary among, but not within, individual T cells. Each developing T cell randomly produces a unique light–heavy chain (αβ or γδ) combination with unique specificity. The theoretical number of possible combinations produced within the body may be estimated to be the product of several possible light chains and several possible heavy chains (Fig. 8.7). Immunologists estimate that 1 to 5 million epitope-binding combinations are possible for TCRs. However, unlike immunoglobulin genes, TCRs do not undergo subsequent changes equivalent to the isotype switching and somatic hypermutation that occur in—immunoglobulins to further increase their diversity (see Sections 8.V.E and 8.V.G, on pages 102 and 105).
Rearrangement to form an αβ TCR. For the α chain, DNA is deleted to join randomly selected V and J genes. An mRNA transcript is then produced containing united VJ genes and a constant gene. This transcript is then spliced to unite the VJ and C genes to form mRNA that can be directly translated to a polypeptide-containing conjoined VJC segments. A similar process occurs for the β chain with the addition of D genes to form a VDJ variable region. γδ TCRs are synthesized in a similar manner.
Formation of TCR peptide-MHC binding regions.
V. B-CELL RECEPTORS
Immunoglobulin gene rearrangements occur in the early stages of B cell precursor differentiation and prior to antigen exposure. These gene rearrangements, along with allelic exclusion, allow for the construction of variable regions that recognize a great portion of the antigenic universe. At any given time, a single B cell produces immunoglobulins of only one specificity and one isotype, formed from the association of light and heavy chains, and inserted into the plasma membrane (Fig 8.8). The rearranged DNA encoding immunoglobulins within B cells is transcribed into primary RNA, intervening sequences are edited out of mRNA, and polypeptides are assembled in the Golgi apparatus and targeted to either the membrane in B cells or for secretion by plasma cells.
B-cell receptors (BCR or immunoglobulin). BCRs are composed of two identical immunoglobulin light (k or λ) and two identical heavy chains. Cell surface associated Igα and Igβ chains transmit signals to the interior of the cell when ligands are bound by the BCR.
A. Gene clusters encoding B-cell receptors
Gene clusters encoding k light chains are found on chromosome 2, whereas those encoding λ light chains are on chromosome 22. The heavy chain gene cluster is located on chromosome 14. Potential antigen-binding combinations are greater than 26 million. Details of this process are shown in Figure 8.9.
B. Light chains
As we have seen, immunoglobulin light chains contain two regions or domains, a variable (VL) domain and a constant (CL) domain. Any given B lymphocyte will generate only identical light chain proteins of either the k or λ type (VkCk or VλCλ), never of both types or a combination of the two.
1. Variable regions: Rearrangement of V and J genes: To generate a light chain variable region of the k or λ type, one of about 100 variable (Vk or Vλ) gene segments recombines with one of four to five joining (Jλ) segments at the DNA levels to create a conjoined VJ pair. The intervening DNA is then removed and degraded. The choice of which V and which J gene to include occurs randomly for each cell. Thus, across a large number of B cells, several hundred different VJ units can be generated.
Gene clusters encoding the BCR chains. Clusters of genes encoding the BCR light chains (k and λ) are located on chromosomes 2 and 22, respectively. Each cluster includes a series of V genes, a series of J genes, and one or more constant (C) genes. The single BCR heavy chain cluster is located on chromosome 14. It includes a series of V genes, a series of D genes, a series of J genes, and a series of constant (C) genes.
2. Uniting variable and constant regions: This occurs at the mRNA level, where a transcript including both the variable (now a VJ unit) region and a constant region is generated. The transcript is then spliced to unite the two regions to produce an mRNA transcript that can be translated directly into a single polypeptide. Details of this process are shown in Fig 8.10.
Rearrangement to form BCR light chains. The rearrangements to form k and λ chains are illustrated. DNA is deleted to join a randomly selected V gene with a randomly selected J gene. An mRNA transcript is then produced that contains the united VJ genes and a constant gene. This transcript is subsequently spliced to unite the VJ and C genes to form an mRNA that can be translated directly to a polypeptide with conjoined VJC segments.
Rearrangement to form BCR heavy chains. Intervening DNA is deleted between randomly selected D and J genes to form as DJ sequence, followed by a second random deletion to form a VDJ unit. An mRNA transcript is produced and spliced to unite the VDJ genes together with the μ or δ genes (the remaining constant region genes are used at a later stage in B cell development) that can be directly translated into IgM or IgD heavy chains. Naïve B cells simultaneously express both IgM and IgD with identical epitope specificity on their cell surfaces.
C. Heavy chains
A single cluster of genes encode the immunoglobulin heavy chain. Heavy chains contain a single variable (VH) and three or four constant (CH1, CH2, CH3, and sometimes CH4) region domains.
1. Variable regions: Rearrangement of V, D, and J genes: To generate a heavy chain variable region, one of about 200 heavy chain variable (VH) genes is combined with one of several diversity (DH) genes and one of numerous joining (JH) genes. The intervening DNA is removed and degraded (see details in Fig. 8.11). A DNA deletion unites randomly selected D and J to form DJ, and second deletion unites a randomly selected V gene with the DJ to form a VDJ unit. The choice of which V, which D, and which J gene to include is a random one for each cell. Thus, across a large number of B cells, many thousands of different VDJ units can be generated.
2. Uniting variable and constant regions occurs, as with light chains, at the mRNA level. An mRNA transcript containing the separated variable (VDJ) and constant regions is produced and then spliced to bring them together, forming a transcript that can be directly translated into a single polypeptide (Fig. 8.11).
D. Heavy and light chain combinations
As with TCRs, antigen-binding variability in immunoglobulins is also determined by the combination (random from cell to cell) of light and heavy chain variable regions. An individual B cell synthesizes immunoglobulin of a single specificity (one particular combination of VL region and VH region), and millions of such combinations are theoretically possible (Fig. 8.12).
E. Isotype switch: Mechanism
Because of its particular combination of VL and VH regions and the effect of allelic exclusion, an individual B cell synthesizes immunoglobulin of only a single specificity. Unstimulated B cells synthesize and display monomeric IgM (and IgD) on their cell surfaces. Upon stimulation, B cells may change the isotype, but not the epitope specificity, of the immunoglobulins they produce. This process, known as the isotype switch, influences the ultimate nature of the humoral immune response.
The intracellular machinery of the stimulated B cell produces immunoglobulins of only a single isotype at a time. Immunoglobulins may be considered the “ballistic missiles” of the adaptive immune system, their V regions forming a specific “warhead” and their constant regions constituting the “rocket” portion of the molecule. Although a single B cell can manufacture a single type of “warhead,” it can be placed on different “rockets” or constant regions. The isotype ultimately determines whether an antibody activates complement or is secreted into a lumen, secreted on a mucous membrane, or immobilized by certain tissues of the body. Isotype switching permits the adaptive immune system to produce antibodies with identical specificity that are capable of initiating various different immune responses. As will be discussed in later chapters, T cells are usually required to activate and stimulate B cells to proliferate, switch isotype, and differentiate into immunoglobulin-secreting plasma cells.
F. Isotype switch: Consequence
The initial or primary antibody response to an epitope is dominated by production of the IgM isotype. Not all B cells initially stimulated by antigen (primary response) become plasma cells, synthesizing and secreting immunoglobulins for the remainder of their lifespan. Some stimulated B cells, memory B cells, are held in reserve against future exposures to antigen. In response to restimulation by the appropriate epitopes, T cell cytokines (e.g., IL-4, IFN-γ, IL-5), and other signals, memory B cells synthesizing IgM can undergo further DNA rearrangement to change the class or isotype of the immunoglobulin through isotype switching. These memory B cells undergo further DNA rearrangement to juxtapose their rearranged VDJ gene regions to different heavy chain C region genes (Fig. 8.13A) and thereby alter the mRNA transcript and ultimately the immunoglobulin isotype (e.g., IgG, IgA, or IgE) that is produced.
Formation of TCR epitope-binding regions by the combination of light and heavy chain variable regions. Each B cell produces a single light chain variable region and a single heavy chain variable region. However, among a population of B cells, several possible combinations of light and heavy chains creates a large number of different epitope-binding sites.
Rearrangements to produce particular isotypes may occur through excision of large DNA segments or through deletion of smaller DNA segments (Fig. 8.13B). As a result, if you were to follow the B cell response to a given epitope over time and repeated stimulation, you would observe that it is typically dominated initially by IgM-producing cells, then by IgG-producing cells, with the eventual appearance of IgA- and IgE-producing B cells as well (Fig. 8.14). The isotype switch is restricted to B cells; there is no equivalent process in T cells.
The isotype switch. A. Following initial activation after contact with its specific epitope (plus other interactions), B cells cease production of IgD, and most of them differentiate into plasma cells that concentrate on secretion of IgM. However, some B cells become memory B cells and remain quiescent for future use. At this point, they express only IgM on their surface. However, if reactivated by a new contact with their specific epitope and appropriate interactions with T cells, they can begin to undergo additional rearrangements of their heavy chain genes at the DNA level to juxtapose their VDJ units with other constant genes. The VDJ units are not altered by these rearrangements. Whatever constant gene is brought adjacent to the VDJ determines what heavy chain gene will be produced. Again, most of these cells will differentiate into plasma cells secreting immunoglobulins of the newly generated isotype, but some will again be reserved as memory cells for subsequent use.
(figure continues on following page.)
B. Isotype switches can occur in multiple ways. Some isotype switches may involve the deletion of large tracts of DNA to bring VDJ segments together with distant constant genes. In other cases, serial reactivations of memory B cells may result in a series of shorter deletions as IgM memory B cells may switch and become IgG memory B cells, then be reactivated again and switch to yet another isotype.
G. Somatic hypermutation
Upon subsequent epitope exposure, memory B cells may switch their expressed isotype, and they may also accumulate small point mutations in the DNA encoding their VL or HL regions during the rapid proliferation that follows restimulation (Fig 8.15). This process, somatic hypermutation, provides additional variation that “fine-tunes” the antibody responses to antigens that are frequently or chronically present. Some mutations may increase the binding affinity of the antibody for its epitope, and this increase in affinity causes those cells to proliferate more rapidly after binding to antigen. As a result, the interaction of antibody with a given epitope becomes tighter and more effective over time, a process called affinity maturation. Like the isotype switch, somatic hypermutation occurs only in B cells, not in T cells.
Consequences of the isotype switch. Repeated or constant stimulation by the same epitope drives B cells from IgM expression to other isotype. The cell’s epitope specificity is not altered by isotype switching.
Somatic hypermutation. B cells undergo multiple round of rapid proliferation on antigenic stimulation. Cells carrying mutations that result in tighter binding are stimulated to proliferated come to dominate the response. Thus antibodies with ever-increase affinity for that epitope are produced, a process called affinity maturation.
• Both heavy and light chain chains of immunoglobulins and T cell receptors contain variable regions that are extremely diverse and constant regions that are relatively consistent.
• The restriction of VLCL and VHCH expression to a single member of the chromosome pair in any given B cell or T cell is termed allelic exclusion. The presence of both maternal and paternal allotypes (allelic forms) is observed within a particular individual.
• The variable regions of immunoglobulins and T cell receptors are formed by the rearrangement (at the DNA level) of multiple genes that are then transcribed into a single mRNA transcript that includes both the variable and constant regions. The variable and constant regions are then brought together by splicing of the mRNA to produce a transcript that can be directly translated into a single polypeptide.
• DNA chromosomal rearrangement is responsible for a significant portion of epitope-specific diversity for T and B cell receptors. Rearrangement occurs at both the DNA and RNA levels by the removal of nucleotides (deletions) followed by reannealing or by inversion of certain DNA sequences. Additional variation comes from junctional diversity.
• Recombination enzymes or recombinases mediate the genetic rearrangement and recombination that generates the variable regions of TCR and immunoglobulin chains.
• TCR and immunoglobulin gene rearrangements occur in the early stages of T cell and B cell precursor differentiation, prior to exposure of the cells to antigen.
• Gene clusters encoding α and δ chains of the TCR are arranged such that all of the δ chain genes (Dδ, Jδ, and Cδ) lie between the Dα and Jα 1 Cα α chain gene.
• Gene clusters encoding immunoglobulin k light, λ light, and heavy chains are found on different chromosomes.
• The initial or primary antibody response to epitopes is dominated by production of the IgM isotype.
• Memory B cells, in response to subsequent restimulation by antigen and interaction with T cells, undergo further DNA rearrangement to juxtapose their rearranged VDJ genes next to different heavy chain C region genes, thereby altering the immunoglobulin isotype (e.g., IgG, IgA, or IgE) produced. This is known as the isotype switch.
• Somatic hypermutation is the process whereby memory B cells are stimulated by subsequent exposures to the same epitope. Small point mutations occur in the DNA encoding their VL or HL regions during the rapid proliferation that follows restimulation.
• Affinity maturation is the process whereby the binding of antibodies to a given antigen becomes better over multiple exposures. It is caused by the accumulations of small mutations that may affect the antigen-binding sites and the positive selection of those cells carrying mutations that result in tighter binding.
8.1. In a patient who later developed an allergy to a certain antigen, the initial response to the antigen consisted of immunoglobulin of the IgM class. However, over time, antigen-specific IgE came to be predominant. This change from an IgM to an IgE response is caused by
A. affinity maturation.
B. allelic exclusion.
C. isotype switching.
D. junctional diversity.
E. somatic hypermutation.
The answer is C. Isotype switching is a process in which rearranged VDJ genes within a memory B cell become juxtaposed through DNA excision from an upstream (5') C region gene with a different C region gene farther downstream (3'). Affinity maturation of antibody for its epitope is independent of isotype. For B cells that have “selected” their maternal or paternal immunoglobulin variable region genes, there are no “do-overs.” Both junctional diversity and somatic hypermutation involve the antigen-binding site for immunoglobulin and do not appear to influence a switch from one isotype to another.
8.2. A 2-year-old child exposed to an antigen for the first time already possesses a B cell with immunoglobulin specific for that antigen. This finding is best explained by
A. antigen-independent immunoglobulin gene
B. antigen stimulation of T cell cytokine production.
C. maternally derived antibodies to that antigen.
D. memory B cells that recognize the antigen.
E. somatic hypermutation of immunoglobulins.
The answer is A. Determination of antibody specificity occurs prior to and independent from an individual’s first encounter with antigen. This process begins developmentally during prenatal and neonatal life. This process is independent of soluble factors (cytokines) produced by T cells and occurs independently of maternal immune function. By definition, memory B cells have previously encountered antigen. Somatic hypermutation occurs only after previous exposure to antigen.
8.3. Serum immunoglobulins containing both maternally and paternally derived Vk light chains are found within an individual. A given B cell, however, expresses only maternally derived or paternally derived Vk chains but never both. This finding is the result of
A. allelic exclusion.
B. antibody diversity.
C. isotype switching.
D. junctional diversity.
E. random VD and VDJ joining.
The answer is A. A given B cell or plasma cell expresses a single maternal or paternal allele of a chromosome pair. This process, known as allelic exclusion, applies to both heavy and light chain genes. An additional exclusion allows for the expression of only a k (chromosome 2) or λ (chromosome 22) gene, never both within the same cell. Allelic exclusion has only a slight impact on genetic variation. Isotype switching, junctional diversity, and random V(D)J joining occur after allelic exclusion
8.4. The role of terminal deoxynucleotidyl transferase (TdT) in development of antibody diversity is to
A. add/remove nucleotides of V, D, and J genes.
B. fuse VD and J segments together in heavy chains.
C. increase binding affinity of antibody for antigen.
D. join CL to CH1, CH2, CH3, or CH4 domains.
E. transfer VL alleles from maternal to paternal chromosomes.
The answer is A. TdT adds or removes nucleotides when the ends of V, (D), and/or J gene segments are exposed. This process, known as junctional diversity, occurs during DNA rearrangement. This process occurs in addition to the fusion of VDJ segments of the heavy chain and occurs prior to a B cell’s exposure to antigen. The light chain constant region (CL) never joins with constant region (CH) domains of the heavy chain to make a polypeptide. A crossover between maternal and paternal VL alleles is an exceedingly rare event, and TdT is not involved.
8.5. When a memory B cell is restimulated by its specific antigen, small point mutations that accumulate in the DNA encoding variable regions of both light and heavy chains may result in
A. antigen-stimulated VDJ joining and new antigen specificity.
B. change from production of IgM to IgG.
C. DNA chromosomal rearrangement and altered antigen specificity.
D. inactivation of either the maternal or paternal VL and VH allele.
E. generation of antibody with increased binding
affinity for its epitope.
The answer is E. Accumulation of point mutations that affect light and heavy chain variable regions may increase binding affinity for antigen, by “fine-tuning” the antigen-binding site of the resulting immunoglobulin molecule. This is known as affinity maturation. These point mutations occur after allelic exclusion and VDJ joining. They do not affect DNA rearrangement, and they do not appear to affect isotype switching.