Diversity in receptor and Immunoglobulin [Autosaved].pptx

Generation of diversity in
receptors
Dr. Tilothama Bhotra
RDWU

GENOME REARRANGEMENTS DURING B-LYMPHOCYTE DIFFERENTIATION
• Overview of B Lymphocyte development & maturation
*stages
* expression of antigen receptors
• Mechanisms of generation of antibody diversity w.r.t. its
receptors
* Variable number of VDJ regions
*Combination of heavy chain and light chain genes
*Junctional Diversity
*Somatic Hypermutation (only B cells)

B and T Cell Development and maturation

EXPRESSION OF ANTIGEN RECEPTORS

Both theories are partially correct
Generation of Diversity
1. Variable number of VDJ regions
2. Combination of heavy chain and light chain genes
3. Junctional Diversity
4. Somatic Hypermutation (only B cells)

The heavy (H) plus kappa (κ) or lambda (λ) chain combinations of BCRs (H-κ/λ) and the alpha
(α) and beta (β) or gamma (γ) and delta (δ) chain combinations of TCRs (αβ or γδ) are
encoded by roughly three hundred different gene segments, yet produce an estimated 5 x
10^7 to 10^9 surface receptors (B+T).
The segments are scattered on human chromosomes 2, 14, and 22.

• The genes that encode diverse antigen receptors of B and T lymphocytes are generated by
the rearrangement in individual lymphocytes of different variable (V) region gene
segments with diversity (D) and joining (J) gene segments.
• A novel rearranged exon for each antigen receptor gene is generated by fusing a specific
distant upstream V gene segment to a downstream segment on the same chromosome. This
specialized process of site-specific gene rearrangement is called V(D)J recombination.
• These analyses showed that the polypeptide chains of many different antibodies of the same
isotype shared identical sequences at their C-terminal ends (corresponding to the constant
domains of antibody heavy and light chains) but differed considerably in the sequences at
their N-terminal ends
• Each antibody chain is actually encoded by at least two genes, one variable and the other
constant, and that the two are physically combined at the level of DNA or of messenger
RNA (mRNA) to eventually give rise to functional Ig proteins.
• Susumu Tonegawa demonstrated that the structure of Ig genes in the cells of an antibody-
producing tumor, called a myeloma or plasmacytoma, is different from that in embryonic
tissues or in nonlymphoid tissues not committed to Ig production.
• These differences arise because DNA segments encoding Ig heavy and light chains are
separated within the inherited loci and are brought together and joined only in developing
B cells but not in other tissues or cell types.
• Similar rearrangements were found to occur during T cell development in the loci encoding
the polypeptide chains of TCRs.
Immunoglobulin genes and its organization

•Functional antigen receptor genes are created only in developing B and T lymphocytes after
DNA rearrangement brings randomly chosen V, (D), and J gene segments into contiguity.
•The process of V(D)J recombination at any Ig or TCR locus involves the rearrangement of
one V gene segment, one D segment (only in Ig heavy chain or TCR β chain loci), and one J
segment in each lymphocyte to form a single V(D)J exon that will code for the variable
region of an antigen receptor protein .
• In the Ig light chain and TCR α and γ loci, which lack D segments, a single rearrangement
event joins a randomly selected V gene segment to a J segment that is also randomly
selected.
• The Ig H and TCR β and δ loci contain D segments, and at these loci two sequential
rearrangement events are needed, first joining a D to a J and then a V segment to the fused
DJ segment.

Generation of Diversity in B and T Cells
• The diversity of the B and T cell repertoires is created by
random combinations of germline gene segments being
joined together and by the addition or deletion of sequences
at the junctions between these segments.
• Combinatorial diversity
• Different combinations of gene segments united by V(D)J
recombination produce different antigen receptors. After
synthesis of antigen receptor proteins, combinatorial diversity
is further enhanced by the juxtaposition of two different,
randomly generated V regions (i.e., VH and VL in Ig molecules
and Vα and Vβ in TCR molecules). Therefore, the total
combinatorial diversity is theoretically the product of the
combinatorial diversity of each of the two associating chains.

Junctional diversity
The largest contribution to the diversity of antigen receptors is made by the removal or
addition of nucleotides at the junctions of the V and D, D and J, or V and J segments at the
time these segments are joined.
• One way in which this can occur is if endonucleases remove nucleotides from the germline
sequences at the ends of the recombining gene segments. In addition, new nucleotide
sequences, not present in the germline, may be added at junctions.
• As described earlier, coding segments (e.g., V and J gene segments) that are cleaved by Rag-1
form hairpin loops whose ends are often cleaved asymmetrically by the enzyme Artemis so that
one DNA strand is longer than the other. The shorter strand has to be extended with nucleotides
complementary to the longer strand before the ligation of the two segments. The longer strand
serves as a template for the addition of short lengths of nucleotides called P nucleotides, and
this process introduces new sequences at the V-D-J junctions.
•Another mechanism of junctional diversity is the random addition of up to 20 non-template-
encoded nucleotides called N nucleotides.
•N region diversification is more common in Ig heavy chains and in TCR β and γ chains than in Ig
κ or λ chains. This addition of new nucleotides is mediated by the enzyme terminal
deoxynucleotidyl transferase (TdT).

Because of junctional diversity, antibody and TCR molecules show the greatest
variability at the junctions of V and C regions, which form the third hypervariable
region, or CDR3.
Although the theoretical limit to the number of Ig and TCR proteins that can be produced
is enormous, the actual number of antigen receptors on B or T cells expressed in each
individual at any one point in time is probably on the order of only 107. This may
reflect the fact that most receptors, which are generated by random DNA recombination,
do not pass the selection processes needed for maturation.
A clinical application of our knowledge of junctional diversity is the determination of the
clonality of lymphoid tumors that arise from B or T cells. This laboratory test is used to
identify monoclonal tumors of lymphocytes and to distinguish tumors from polyclonal
proliferations. Because every lymphocyte clone expresses a unique antigen receptor CDR3
region, the sequence of nucleotides at the V(D)J recombination site serves as a specific
marker for each clone. Thus, by determining the sequence of the junctional regions of
Ig or TCR genes in different B or T cell proliferations, one can establish whether these
lesions arose from a single clone (indicating a tumor) or independently from different clones
(implying nonneoplastic proliferation of lymphocytes). The same method may be used to
identify small numbers of tumor cells in the blood or tissues.

somatic hypermutation
• Additional antibody diversity is generated in rearranged variable-region gene units by a
process called somatic hypermutation.
• As a result of somatic hypermutation, individual nucleotides in VJ or VDJ units are
replaced with alternatives, thus potentially altering the specificity of the encoded
immunoglobulins.
• Occurs only within germinal centers, structures that form in secondary lymphoid organs
within a week or so of immunization with an antigen that activates a T-cell-dependent B-
cell response.
• Somatic hypermutation is targeted to rearranged V regions located within a DNA sequence
containing about 1500 nucleotides, which includes the whole of the VJ or VDJ segment.
• Somatic hypermutation occurs at a frequency approaching 103 per base pair per generation.
• This rate is at least a hundred thousand-fold higher (hence the name hypermutation) than
the spontaneous mutation rate, about 108/bp/generation, in other genes.
• Since the combined length of the H-chain and L-chain variable-region genes is about 600 bp,
one expects that somatic hypermutation will introduce at least one mutation per every
two cell divisions in the pair of VH and VL genes that encode an antibody.
• Most of the mutations are nucleotide substitutions rather than deletions or insertions.
Somatic hypermutation introduces these substitutions in a largely, but not completely,
random fashion. Recent evidence suggests that certain nucleotide motifs and palindromic
sequences within VH and VL may be especially susceptible to somatic hypermutation.
• Somatic hypermutations occur throughout the VJ or VDJ segment, but in mature B cells
they are clustered within the CDRs of the VH and VL sequences,where they are most likely
to influence the overall affinity for antigen. Following exposure to antigen, those B cells with
higher-affinity receptors will be preferentially selected for survival.
• This result of this differential selection is an increase in the antigen affinity of a population
of B cells. The overall process, called affinity maturation, takes place within germinal
centers,

Gene rearrangement
• Light chain DNA rearrangement
• Variable-region gene rearrangements occur in an ordered sequence during B-cell
maturation in the bone marrow.
• The heavy-chain variable-region genes rearrange first, then the light-chain
variable-region genes. At the end of this process, each B cell contains a single
functional variable region DNA sequence for its heavy chain and another for its
light chain.
• The process produces mature, immunocompetent B cells; each such cell is
committed to produce antibody with a binding site encoded by the particular
sequence of its rearranged V genes.
• Further changes as in the immunoglobulin class (isotype) expressed by a B cell,
will not affect the cell’s antigenic specificity.
• Rearranged genes contain the following regions in order from the 5’ to 3’ end: a
short leader (L) exon, a noncoding sequence (intron), a joined VJ gene segment, a
second intron, and the constant region. Upstream from each leader gene segment
is a promoter sequence.
• The rearranged light chain sequence is transcribed by RNA polymerase from the L
exon through the C segment to the stop signal, generating a light-chain primary
RNA transcript. The introns in the primary transcript are removed by RNA
processing enzymes, and the resulting light-chain messenger RNA then exits from
the nucleus. The light-chain mRNA binds to ribosomes and is translated into the
light-chain protein.
• The leader sequence at the amino terminus pulls the growing polypeptide chain
into the lumen of the rough endoplasmic reticulum and is then cleaved, so it is not
present in the finished light-chain protein product.

• Heavy-Chain DNA Undergoes V-D-J Rearrangements
• Requires two separate rearrangement events within the variable region: a DH
gene segment first joins to a JH segment; the resulting DH-JH segment then
moves next to and joins a VH segment to generate a VHDHJH unit that encodes
the entire variable region.
• In heavy-chain DNA, variable-region rearrangement produces a rearranged
gene consisting of the following sequences starting from the 5 end: a short L
exon, an intron, a joined VDJ segment, another intron, and a series of C gene
segments and a promoter sequence located a short distance upstream from
each heavy-chain leader sequence.
• Then RNA polymerase binds to the promoter sequence and transcribe the entire
heavy-chain gene, including the introns.
• Initially, both Cµ and Cδ gene segments are transcribed. Differential
polyadenylation and RNA splicing remove the introns and process the primary
transcript to generate mRNA including either the Cµ or the Cδ transcript. These
two mRNAs are then translated, and the leader peptide of the resulting nascent
polypeptide is cleaved, generating finished µ and δ chains. The production of
two different heavy-chain mRNAs allows a mature, immunocompetent B cell to
express both IgM and IgD with identical antigenic specificity on its surface.

Mechanism of DNA rearrangement
Recombination Signal Sequences Direct Recombination
• DNA sequencing studies revealed the presence of unique recombination signal
sequences (RSSs) flanking each germ-line V, D, and J gene segment.
• One RSS is located 3 to each V gene segment, 5 to each J gene segment, and
on both sides of each D gene segment. These sequences function as signals for
the recombination process that rearranges the genes.
• Each RSS contains a conserved palindromic heptamer and a conserved AT-rich
nonamer sequence separated by an intervening sequence of 12 or 23 base pairs.
• The intervening 12- and 23-bp sequences correspond, respectively, to one and
two turns of the DNA helix; for this reason the sequences are called one-turn
recombination signal sequences and two-turn signal sequences.
Gene Segments Are Joined by Recombinases
• V-(D)-J recombination, which takes place at the junctions between RSSs and
coding sequences, is catalyzed by enzymes collectively called V(D)J
recombinase.
• In 1990 David Schatz, Marjorie Oettinger, and David Baltimore first reported the
identification of two recombination-activating genes, designated RAG-1 and
RAG-2, whose encoded proteins act synergistically and are required to mediate
V-(D)-J joining.
• The RAG-1 and RAG-2 proteins and the enzyme terminal deoxynucleotidyl
transferase (TdT) are the only lymphoid-specific gene products that have been
shown to be involved in V-(D)-J rearrangement.

• The recombination of variable-region gene segments consists of the following steps,
catalyzed by a system of recombinase enzymes (Figure 5-7):
• Recognition of recombination signal sequences (RSSs) by recombinase enzymes,
followed by synapsis in which two signal sequences and the adjacent coding
sequences (gene segments) are brought into proximity
• Cleavage of one strand of DNA by RAG-1 and RAG-2 at the junctures of the signal
sequences and coding sequences. A reaction catalyzed by RAG-1 and RAG-2 in
which the free 3-OH group on the cut DNA strand attacks the phosphodiester bond
linking the opposite strand to the signal sequence, simultaneously producing a hairpin
structure at the cut end of the coding sequence and a flush, 5-phosphorylated, double-
strand break at the signal sequence
• Cutting of the hairpin to generate sites for the addition of P-region nucleotides,
followed by the trimming of a few nucleotides from the coding sequence by a single
strand endonuclease
• Addition of up to 15 nucleotides, called N-region nucleotides, at the cut ends of the
V, D, and J coding sequences of the heavy chain by the enzyme terminal
deoxynucleotidyl transferase
• Repair and ligation to join the coding sequences and to join the signal sequences,
catalyzed by normal doublestrand break repair (DSBR) enzymes
• Recombination results in the formation of a coding joint, falling between the coding
sequences, and a signal joint, between the RSSs. The transcriptional orientation of
the gene segments to be joined determines the fate of the signal joint and intervening
DNA. When the two gene segments are in the same transcriptional orientation, joining
results in deletion of the signal joint and intervening DNA as a circular excision product
(Figure 5-8). Less frequently, the two gene segments have opposite orientations. In
this case joining occurs by inversion of the DNA, resulting in the retention of both the
coding joint and the signal joint (and intervening DNA) on the chromosome. In the
human locus, about half of the V gene segments are inverted with respect to J and
their joining is thus by inversion.

Heptamer and nonamer are recombination signal sequence and required for recombination
Once the receptor is
expressed after VJ/VDJ
joining the recombination
machinery will stop working

Rearrangement mechanisms can lead to two different organizations of the genome
according to the orientation of the forward/reverse V-gene segment and the
downstream J-segment.

•Ku proteins: heterodimer; binds to upper and lower
part of the synapsis; helps the proteins in
recombination; helps to bring some DNA-dependent
protein kinase, Artemis. Cutting and joining happens.
•TdT polymerase adds random nucleotides without a
template at 3’end.
•DNA ligase, XRCC4 are DNA recombination
proteins.
They make a cut and eliminate in the next cycle.

3 domains of RAG-1: NBD, flexible hinge, Zn2+ dependent endonuclease domain.
NBD binds to the nonamer of V and J domain. After the cut the KU protein
comes, then TDT.

•RAG complex cleaves after TC at D and TA at J with free 3’-OH and 5’-P ends that mak a hairpin loop
structure.
•Tdt adds random polynucleotides; Complemenatrity of the bases makes it double stranded; TdT disjoins.
•DNA polymerase and ligase function in DNA synthesis.
•P-pallindromic sequence; N-nuceotide(random);
•NP addition in light chain only at one site and in heavy chain its two sites.

Activation induced cytidine deaminase
Theories: Mutational hotspots; high rate of proliferation of B cells
In primary response light chain has very few mutations , but heavy chains has more.
With shift to secondary and tertiary response the rate of mutation increases.
B cell undergoes affinity maturation: series of mutation at CDRs leads to positive selection of
high affinity antigen receptors in the secondary immune response. Boosters lead to production
of high affinity antibodies by memory cells.

Expression of Ig genes
• As in the expression of other genes, post-transcriptional processing
of immunoglobulin primary transcripts is required to produce
functional mRNAs. The primary transcripts produced from
rearranged heavy-chain and light-chain genes contain intervening
DNA sequences that include noncoding introns and J gene segments
not lost during V-(D)-J rearrangement.
• In addition, as noted earlier, the heavy-chain C-gene segments are
organized as a series of coding exons and noncoding introns.
• Each exon of a CH gene segment corresponds to a constant-region
domain or a hinge region of the heavy-chain polypeptide.
• The primary transcript must be processed to remove the
intervening DNA sequences, and the remaining exons must be
connected by a process called RNA splicing.
• Short, moderately conserved splice sequences, or splice sites, which
are located at the intron exon boundaries within a primary
transcript, signal the positions at which splicing occurs. Processing
of the primary transcript in the nucleus removes each of these
intervening sequences to yield the final mRNA product. The mRNA
is then exported from the nucleus to be translated by ribosomes
into complete H or L chains.

Heavy-Chain Primary Transcripts Undergo Differential
RNA Processing
• Processing of an immunoglobulin heavy-chain primary transcript can
yield several different mRNAs, which explains how a single B cell can
produce secreted or membrane bound forms of a particular
immunoglobulin and simultaneously express IgM and IgD.
• The immunoglobulin genes are expressed only in B-lineage cells, and even
within this lineage, the genes are expressed at different rates during
different developmental stages.
• As with other eukaryotic genes, three major classes of cis regulatory
sequences in DNA regulate transcription of immunoglobulin genes:
• Promoters: relatively short nucleotide sequences, extending about
200 bp upstream from the transcription initiation site, that promote
initiation of RNA transcription in a specific direction
• Enhancers: nucleotide sequences situated some distance upstream or
downstream from a gene that activate transcription from the promoter
sequence in an orientation-independent manner
• Silencers: nucleotide sequences that down-regulate transcription,
operating in both directions over a distance.

Alpha chain locus is very similar to light chain locus and Beta chain locus is similar to heavy
chain locus

Homologous recombination between nonamer sequences results in formation of a loop which
finally gets deleted

Allelic Exclusion Ensures a Single Antigenic Specificity
• B cells, like all somatic cells, are diploid and contain both
maternal and paternal chromosomes.
• It expresses the rearranged heavy-chain genes from one
chromosome and the rearranged light-chain genes from only one
chromosome. The process by which this is accomplished, called
allelic exclusion, ensures that functional B cells never contain
more than one VHDHJH and one VLJL unit.
• essential for the antigenic specificity of the B cell, because the
expression of both alleles would render the B cell multispecific.
• The phenomenon of allelic exclusion suggests that once a
productive VH-DH-JH rearrangement and a productive VL-JL
rearrangement have occurred, the recombination machinery is
turned off, so that the heavy- and light-chain genes on the
homologous chromosomes are not expressed.

•G.D.Yancopoulos and F.W.Alt have proposed a model to account for allelic exclusion
•They suggest that once a productive rearrangement is attained, its encoded
protein is expressed and the presence of this protein acts as a signal to prevent
further gene rearrangement.
•According to their model, the presence of heavy chains signals the maturing B cell
to turn off rearrangement of the other heavy-chain allele and to turn on
rearrangement of the light-chain genes.
•If a productive rearrangement occurs, light chains are produced and then pair
with heavy chains to form a complete antibody molecule.
• The presence of this antibody then turns off further light-chain rearrangement.
•If rearrangement is nonproductive for both alleles, rearrangement of the λ-chain
genes begins. If neither allele rearranges productively, the B cell presumably
ceases to mature and soon dies by apoptosis.

TCR development
Ref: Abbas et al

Stages of T Cell Maturation
• During T cell maturation, there is a precise order in which TCR genes are rearranged and in which
the TCR and CD4 and CD8 coreceptors are expressed.
• In the mouse fetal thymus, surface expression of the γδ TCR occurs first, 3 to 4 days after precursor
cells first arrive, and the αβ TCR is expressed 2 or 3 days later. In human fetal thymuses,γδ TCR
expression begins at about 9 weeks of gestation, followed by expression of the αβ TCR at 10 weeks.
• Double-Negative Thymocytes
Pre-TCR signals contribute to the largest proliferative expansion during T cell development, also initiate
recombination at the TCR α chain locus and drive the transition from the double-negative to the double-
positive stage of thymocyte development, inhibit further rearrangement of the TCR β chain locus on
the unrearranged allele. This results in β chain allelic exclusion
• Pre-T Cell Receptor If a productive (i.e., in-frame) rearrangement of the TCR β chain gene occurs in
a given double-negative T cell, the TCR β chain is expressed on the cell surface in association with
an invariant protein called pre-Tα, along with CD3 and ζ proteins to form the pre-TCR complex
• Double-Positive Thymocytes At the next stage of T cell maturation, thymocytes express both CD4
and CD8 and are called double-positive thymocytes: TCR α gene expression in the double-positive
stage leads to the formation of the complete αβ TCR, which is expressed on the cell surface in
association with CD3 and ζ proteins.
• Double-positive cells that successfully undergo selection processes go on to mature into CD4+ or
CD8+ T cells, which are called single-positive thymocytes.

Heavy Chain Isotype (Class) Switching
• In T-dependent responses, some of the progeny of activated IgM- and IgD-expressing B cells undergo heavy chain isotype
(class) switching and produce antibodies with heavy chains of different classes, such as γ, α, and ε.
• Some isotype switching occurs in B cells in extrafollicular foci, driven by extrafollicular helper T cells, but the process continues
to occur in germinal centers, driven by Tfh cells in the light zone.
• B cells change the isotypes of the antibodies they produce by changing the constant regions of the heavy chains, but the
specificity of the antibodies (which is determined by the variable regions) remains unaltered.
• Isotype switching in response to different types of microbes is regulated by cytokines produced by the helper T cells that are
activated by these microbes. Switching from the original IgM to IgG isotypes is a prominent aspect of T-dependent antibody
responses against many bacteria and viruses.
• In addition, B cells in different anatomic sites switch to different isotypes, in part because of the cytokines produced at these
sites. Specifically, B cells in mucosal tissues switch to IgA, which is the antibody class that is most efficiently transported
through epithelia into mucosal secretions, where it prevents microbes from entering through the epithelia.
• CD40 signals work together with cytokines to induce isotype switching. CD40 engagement induces the expression of the
enzyme AID, which is crucial for both isotype switching and affinity maturation.
• The molecular mechanism of isotype switching is a process called switch recombination, in which the Ig heavy chain DNA in B
cells is cut and recombined such that a previously formed VDJ exon that encodes the V domain is placed adjacent to a
downstream C region and the intervening DNA is deleted. These DNA recombination events involve nucleotide sequences
called switch regions, which are located in the introns between the J and C segments at the 5′ ends of each CH locus, other
than the δ gene. Switch regions are 1 to 10 kilobases long, contain numerous tandem repeats of GC-rich DNA sequences, and
are found upstream of every heavy chain gene. Upstream of each switch region is a small exon called the I exon (for initiator of
transcription) preceded by an I region promoter. Signals from cytokines induce transcription from a particular I region
promoter reading through the I exon, switch region, and adjacent CH exons. These transcripts are known as germline
transcripts. They are not translated into proteins but are required for isotype switching to proceed. Germline transcripts are
found at both the µ locus and the downstream heavy chain locus to which an activated B cell is being induced to switch. At
each participating switch region, the germline transcript facilitates the generation of DNA double-stranded breaks, as
described later. The DNA break in the upstream (µ) switch region is joined to the break in the downstream selected switch
region. As a result, the rearranged VDJ exon just upstream of the µ switch region in the IgM-producing B cell recombines with
the Ig heavy chain gene located immediately after the transcriptionally active downstream switch region.

• The key enzyme required for isotype switching and somatic hypermutation is
AID. AID expression is induced in activated B cells mainly by CD40 signals from
Tfh cells. The enzyme removes an amino group from cytosines in single-stranded
DNA templates, converting cytosine (C) residues to deaminated uracil (U)
residues. AID is targeted to switch at certain GC containing tetranucleotide
motifs. Switch regions are rich in these motifs, and cytokine-induced
transcription through these regions makes them accessible to AID.
• These GC-rich regions contribute to increased stalling of RNA polymerase II,
which, when stalled, efficiently recruits AID to form stable DNA-RNA hybrids
involving the template strand of DNA, thus freeing up the nontemplate strand,
which forms an open single-stranded DNA loop called an R-loop. The generation
of single-stranded DNA by R-loop formation is critical because AID can target only
single-stranded DNA. Nicks are generated on both strands contribute to double
stranded breaks both in the Sµ “donor” switch region and in the downstream
“acceptor” switch region that is involved in a particular isotype switch event. The
double stranded breaks in the two switch regions are joined together (ligated) by
use of the machinery involved in double-stranded break repair by
nonhomologous end joining. In this process, the DNA between the two switch
regions is deleted, and the net result is that the original rearranged V region DNA
is fused to a new constant region.

Affinity Maturation:
Somatic Mutation of Ig Genes and Selection of High-Affinity B Cells
• Affinity maturation is the process that leads to increased affinity of antibodies for a particular antigen
as a T dependent humoral response progresses, and it is the result of somatic mutation of Ig genes
followed by selective survival of the B cells that produce the antibodies with the highest affinities.
Helper T cells and CD40:CD40L interactions are required for somatic mutation to be initiated, and, as a
result, affinity maturation is observed only in antibody responses to T-dependent protein antigens
• In proliferating germinal center B cells in the dark zone, rearranged Ig V genes undergo point mutations
at an extremely high rate: up to 10 amino acid substitutions. The mutations are clustered in the V
regions, mostly in the antigenbinding complementarity-determining regions (CDRs) (Fig. 12.17), and the
presence of mutations correlates with increasing affinities of the antibodies for the antigen that
induced the response.
• B cells that bind antigens in germinal centers with high affinity are selected to survive: The early
response to antigen results in the production of antibodies, some of which form complexes with
residual antigen and may activate complement. Follicular dendritic cells express receptors for the Fc
portions of antibodies and for products of complement activation, including C3b and C3d. These
receptors bind and display antigens that are complexed with antibodies and complement products.
Antigen may also be displayed in free form in the germinal center. Meanwhile, germinal center B cells
that have undergone somatic mutation migrate into the FDC-rich light zone of the germinal center.
These B cells die by apoptosis unless they are rescued by recognition of antigen.
• Only B cells with high-affinity receptors for the antigen are able to bind the antigen when it is present
at low concentrations, and these B cells survive preferentially because of several mechanisms. First,
antigen recognition by itself induces expression of anti-apoptotic proteins of the Bcl-2 family. Second,
high-affinity B cells will preferentially endocytose and present the antigen and interact with the
limiting numbers of Tfh cells in the germinal center. These helper T cells may signal via CD40L to
promote the survival of the B cells with which they interact.
• As more antibody is produced, more of the antigen is eliminated and less is available in the germinal
centers. Therefore, the B cells that will be able to specifically bind this antigen and to be rescued from
death need to express antigen receptors with higher and higher affinity for the antigen. As a result, as
the antibody response to an antigen progresses, the B cells that are selected to survive in germinal
centers produce Ig of increasing affinity for the antigen. This selection process results in affinity
maturation of the antibody response

Clonal selection theory
• Clones of lymphocytes with different specificities are present in unimmunized
individuals and are able to recognize and respond to foreign antigens (Fig. 1.3).
• This fundamental concept is called clonal selection. It was clearly enunciated by
Macfarlane Burnet in 1957, as a hypothesis to explain how the immune system could
respond to a large number and variety of antigens.
• According to this hypothesis, which is now a proven feature of adaptive immunity,
antigen-specific clones of lymphocytes develop before and independent of exposure to
antigen.

Immunologic memory
• Exposure of the immune system to a foreign antigen enhances its ability to
respond again to that antigen. Responses to second and subsequent
exposuresto the same antigen, called secondary immune responses, are
usually more rapid, greater in magnitude, and often qualitatively different from
the first, or primary, immune response to that antigen.
• Immunologic memory occurs because each exposure to an antigen generates
long-lived memory cells specific for the antigen. There are two reasons why the
secondary response is typically stronger than the primary immune response—
memory cells accumulate and become more numerous than the naïve
lymphocytes specific for the antigen that exist at the time of initial antigen
exposure, and memory cells react more rapidly and vigorously to antigen
challenge than do naive lymphocytes.
• Memory enables the immune system to mount heightened responses to
persistent or recurring exposure to the same antigen and thus to combat
infections by microbes that are prevalent in the environment and are
encountered repeatedly.

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