Fundamentals of Immunology & Microbiology (MIM 425)
Lectures #6: The Generation of Diversity in the Immune Response
Amy Kenter, PhD star1@uic.edu; Tel. 6-5293
Objective
How is possible for the body to produce millions of different antigen receptors? In this
next section we will explore additional genetic strategies used by the B cell to “generate
immunological diversity.” The overall structure of the immunoglobulin gene loci have
been described and the mechanism of VDJ joining has been reviewed. We will now
integrate this information into an overall perspective on the various strategies the immune
system uses to generate Ab diversity.
PART I:
A. Summary: Strategies for the generation of diversity of Ag:Ab interactions
1. V genes are composed of multiple gene segments
i) Lk chain V genes are generated from V and J segments ( 30 Vk/4J = 30x4=150)
ii) H chain V genes are generated from V, D and J segments (50 VH/30 D/ 4 J=
50x30=1500x4=6000)
2. The process of assembling VJ and VDJ gene segments is called VDJ joining. This
process is sloppy and generates additional diversity at the junctions of the joined DNA
segments. This raises the potential diversity to 2x10e7 Ag binding sites.
3. Ab gene segment joining is developmentally regulated to ensure that B cells express
monospecific BCR. This phenomenon is referred to as allelic exclusion
4. Combinatorial Association of 150 L chains with 6000 H chain equals 9x10e5
5. Ag driven somatic hypermutation fine tunes Ab responses after immunization. There
is a progressive increase in the binding affinity of Ab for Ag. This is referred to as
affinity maturation.
B. Generation of Ab Diversity by V(D)J Joining
Organization of Heavy and Light Chain Loci in the Genome:
Ig genes are assembled from gene segments
L chain V regions are constructed from two gene segments, V and J (the joining
segment). The C region is encoded in a separate exon and is joined to the VJ region by
RNA splicing of the L chain mRNA. As in all extracellular proteins, Ig L chain V genes
contain a leader (L) segment upstream of the V region gene. The L region directs protein
into the cell’s secretory pathways and is then removed in a post-translational
modification. (See Janeway fig.4.2)
H chain V regions are constructed from three gene segments; V, diversity (D) and J
segments. First, D and J are joined, then V is joined to D. The heavy chain C region
genes are encoded in several exons. The C region exons are joined to the VDJ segment
by RNA splicing. (See Janeway fig.4.2)
Variable regions genes are present in multiple copies
In human and mouse there are two independent L chain loci and one H chain locus.
In human, the Llocus contains about 40 V regions organized in a cluster followed by
four C region genes each of which are paired with a J region gene. The L locus has a
single C region gene and a cluster of about 30 V segments and 5 J region segments.
Similarly, in the H chain locus, the 50 V and 30 D and 6 J segments are each clustered
separately followed by an array of eight C region gene segments.
(See Janeway fig.4.3; 4.4)
Rearrangement of V, D, J gene segments is guided by flanking sequences in DNA
The mechanism of VDJ joining is similar for the L and H chain genes. Recombination
occurs by looping-out and deletion of the DNA between the two gene segments which are
rearranging.
(see Janeway Fig. 4.6)
The joining of D to J and V to D is mediated by non-coding regions of flanking DNA.
Conserved sequence is found adjacent to the points at which recombination occurs. (see
Fig. 4.5).
The sequences consist of a conserved block of seven nucleotides termed a heptamer
which is always contiguous with the coding sequence. This is followed by a spacer of 12
or 23 nucleotides. The spacer is followed by a second block of nine nucleotides, termed
the nonamer. The spacer sequence is not conserved but its length is conserved and
corresponds to one or two turns of the DNA helix. The heptamer-spacer-nonamer is
referred to as the recombination signal sequence (RSS). Recombination only occurs
between RSSs on the same chromosome. Recombination can only link a gene segment
flanked with a 12 base pair spacer to one with a 23 base pair spacer. This is referred to as
the 12/23 rule. In this way in the H chain locus, the D and J can be linked and the V and
D can be linked but the V and J can not be directly joined. (See Janeway fig.4.5).
A double strand break occurs in the two RSSs which are to recombine. The ends of the
heptamer sequence are joined precisely in a head-to-head fashion to form a signal joint
which forms a circlular piece of DNA which is lost when the cell divides. (see Janeway
4.6).
(See Janeway fig.4.7; 4.8)
The joining the V and D is imprecise and causes much additional diversity.
Specialized enzymes are required for VDJ joining. RAG-1 and RAG-2 genes are
responsible for the initial cleavage of the RSS and constitute the VDJ recombinase.
When the initial cleavage of the RSS occurs a hairpin is formed. The hairpin must be
opened for recombination to ensue. The hairpin can be opened in a number of ways
which leads to the addition of “P” nucleotides. (see Janeway 4.7; 4.8).
The second source of additional nucleotides are called “N” nucleotides. This is because
they are not templated. An enzyme, terminal deoxynucleotidyl transferase (TdT) is
responsible for their addition. N nucleotides are absent from the L chain because TdT is
not expressed when L chain rearrangement occurs. H chains rearranged in the fetus also
are devoid of N nucleotides since the TdT enzyme is not expressed in the fetus. The
absence of N nucleotides in the fetus has important consequences for the development of
the fetal VH repertoire. Since the total number of added residues is random the added
nucleotides often disrupt the reading frame of the V gene. These rearrangements are
called nonproductive.
(see Janeway 4.8).
T cell receptor is rearranged by VDJ joining
(see Janeway Figures 4.11-4.15).