Fundamentals of Microbiology & Immunology (MIM 425)
Lectures #7B: The Generation of Diversity in the Immune Response
Amy Kenter, PhD star1@uic.edu; Tel. 6-5293
PART III:
B. Affinity Maturation
Over the course of a humoral immune response, affinity maturation occurs in the germinal
centers, whereby affinity of an antibody for its specific antigen is increased by somatic
hypermutation and antigen selection of high affinity clones. In somatic hvpermutation,
individual nucleotides in VJ or VDJ units are replaced with alternative bases, thus adjusting the
specificity of and potentially increasing the affinity of the encoded Ig. In antigen selection of
high affinity clones, clonal selection occurs by the same mechanisms as above, where antigenantibody complexes possessing a moderate fit are the most favored and the most positively
stimulated. (see Janeway fig.4.9)
C. Why are 5 classes of Ig necessary?
There are 5 different classes of immunoglobulins (Ig) IgG, IgA, IgD, IgM, and IgE.
Different antibody classes have different functions including:
(see Janeway fig.4.16; 4.17)
• Complement (C’) fixation: This is important for the initiation of the inflammatory response.
This involves the constant region of IgG1 which binds complement and initiates the classical
pathway and inflammatory response, as well as IgG3 and IgM. IgA, IgD, IgE are incapable of
this function.
• Placental transfer of immunity via IgG. IgM, IgD, IgA, and IgE are incapable of this
function.
• Binding of Staphylococcus protem A and Streptococcus protein G (only IgG can do this
(although not the IgG3 subclass)). This is very important in fighting bacterial infections. IgG
interacts with proteins on the surface of the bacteria, forming an immune complex, which goes
on to initiate the humoral and inflammatory response. IgM, IgD, IgA, and IgE are incapable of
this function.
Where are immunoglobulins found in the body? In the serum:
• IgG1 is the most predominant antibody.
• There are moderate amounts of IgM.
• IgA, IgD, and IgE are virtually impossible to detect in the serum.
• A large rise in IgE is responsible for allergic hypersensitivity.
Fc receptors recognize antibody and draw them to the surface of cells (see Janeway
fig.9.20).
Many immune cells (neutropliils, phagocytes, macrophages, granulocytes, eosinophyls), have Fc
receptors which are specific for the constant region (i.e. the tail part) of immunoglobulins. Upon
binding of the Fc receptor by Ig the receptor transduces a signal through its transmembrane
domain to a protein kinase on the cytoplasmic side, inducing a second messenger cascade. In
summary, the presence of a particular kind of Ig can have a great effect on the functioning of the
immune system, inflammatory response, and hematopoetic system.
D. Immunoglobulin switches its isotype
Today we will consider how Ig maintain their antigen binding specificity (same variable region)
while diversifying their effector function. (see Janeway fig.4.16; 4.17).
There are multiple constant regions for the H chain ( see Janeway fig. 4.18)
RNA processing causes a switch from IgM to IgD expression
IgM and IgD are simultaneously expressed on the surface of mature B cells found in the spleen.
How can this occur since the VDJ region is located upstream of the C gene and is not
contiguous with the C gene? Constant regions have multiple exons. For example, C has 4
domains: CH1, hinge, CH2-CH4. A long RNA transcript stretching from upstream of VDJ all
the way through C and then through C is generated. There is then a choice of splicing site,
depending on the differentiation state of the B cell:
•
If the long transcript is spliced at a particular poly adenylation site (pA1), then the C
region will be translated, leading to IgM.
•
If the long transcript is spliced at the pA2 Site, then through RNA splicing, the C region
will be spliced out and the C will be translated, leading to IgD.
(see Janeway fig. 4.19)
Differential RNA processing explains the progression from membrane-bound to secreted
antibody.
Ig exists either on the surface of B cells as an antigen receptor (BCR) or it is secreted from
plasma cells. How does the cell facilitate the transition between membrane associated Ig and
being secreted? This decision occurs via RNA splicing which results in the addition of either a
membrane exon or an alternate secretory tail exon. The molecular control of this RNA splicing is
not entirely understood. Transmembrane and secreted forms of IgM and IgD are derived from
the same gene by alternative RNA splicing.
The C gene is composed of several exons and introns. Membrane anchored or secreted 
chains have identical aa sequence up to their carboxyl termini, where they diverge. The
membrane anchored form is derived from RNA which splices out the end of the fourth C exon.
To make secreted  chains either this splice must be suppressed or RNA synthesis must be
terminated at the first poly A addition site so that transcripts never reach the membrane-anchorcoding mini-exons.
Differential RNA processing allows the synthesis and secretion of IgM and IgD Abs by a single
plasma cell. (see Janeway fig.4.21)
Antibody class switch beyond IgD involves DNA recombination in the switch regions of the
H chain
Every B cells begins life expressing IgM. The form of heavy chain expressed is always
determined by the C region. IgM is composed of a VDJ region located upstream of the C gene.
Later in the immune response the same VDJ may be expressed in IgG, IgA or IgE. This change
is known as the isotype switch and is stimulated by cytokines released by T cells.
We previously discussed the generation of different variable regions via VDJ recombination into
the C locus. Here we discuss how multiple constant regions assemble with a single VDJ
region. There are 7 (mouse) or 8 (human) constant regions on the heavy chain locus. In other
words, for a given human variable region, there are multiple constant regions possible:

In the human Ig locus, there is a duplication of the constant region locus (as opposed to mice
with only a single copy of the locus). The IgH gene locus contains the constant region genes
which span a region 200 kb to the 3’-side of the JH locus. (see Janeway fig.4.20)
How does the V region, which is contiguous with C assemble with other constant regions?
There is a second DNA rearrangement on heavy chain locus which transposes the variable
regions next to the appropriate constant region via deletion looping out mechanism.
Between the V region and C there is switch DNA (S) which is highly repetitive. In the case of
switch region for the C gene it is a simple tandem repeat of (GACCT)5 – (GGGGT) which is
repeated. Switch regions are found upstream from all constant regions (except C).
(see Janeway fig.4.20)
Cytokines affect Ig expression
Isotype switching is controlled by cytokines (which overlap between the humoral and
inflammatory response). Lymphokines can direct sterile transcription of switch regions.
A transcriptional promoter is located upstream of each switch region which is stimulated by a
specific cytokine. For example:
• Lipopolysaccharide (LPS) derived from the cell walls of gram negative bacteria, activates
mouse sp1enic B cells, causing transcription but no translation (i.e. it is a sterile transcription) of
the S3 and S2b regions but not the S1 region.
LPS + IL-4 causes S3 and S2b promoters to be silenced and instead there is sterile
transcription of the S1 and S regions.
1) II-4 (made by TH cells) is important for the transcription of C1 and C. In other words
induces the  and the  switches.
2) TGF (made by TH and Tc cells) is important for the transcription of C
3) -IFN (made by TH and Tc and NK cells) is important for the transcription of S 3 and Sa .
These cytokines can be definitively related to immunodeficiency, i.e. IgG deficiency can stem
from an IL-4 gene mutation.
When things go wrong during the development of the B cell
Errors in either VDJ recombination, somatic hypermutation or isotype class switch
recombination can lead to chromosomal translocations. Specific translocations are associated
with B cell malignancies, including leukemias, a malignancy which grows as single cells, and
lymphomas, which grows as a tumor mass. There are many checkpoints to prevent errant
recombination, but errors, especially translocations, still occur. For instance, although we hope
that the S region recombines with the S region, it may recombine with a site on another
chromosome entirely, creating a balanced translocation (all pieces are conserved when two
chromosomes swap pieces, no chromosomal pieces lost). For example, a balanced translocation
between the c-myc gene (oncogene) on chromosome 8 and the Ig gene on chromosome 14
causes Burkitt’s lymphoma. Below is a list of some errors in the development of B cell, which
can lead to a variety of clinical problems. The status of the rearrangement on the heavy chain and
light chains can be used towards diagnosing various clinical conditions.
MUTIPLE CHOICE
(a)
When a B cell shifts from the synthesis of JgM to IgA
molecules, it is said to have undergone __________.
1.
2.
3.
4.
5.
somatic hypermutation
isotype class switch
VDJ joining
RNA processing
None of the above
(b)
_____________ context.
Unrearranged antibody genes are said to be in their
1.
2.
3.
4.
5.
germline
original
primordial
mature
all of the above
(c)
into contiguous alignment.
_________________brings the VL and JL gene segments
1.
2.
3.
4.
5.
VDJ joining
Somatic hypermutation
Isotype class switch
RNA splicing
None of the above
(d)The s and m mRNAs differ only in their ___________ ends.
1.
2.
3.
4.
5.
coding regions
introns
mRNAs
5’
3’
(e)The ____________ model suggests that any L chain may associate with any H chain.
1.
2.
3.
4.
5.
clonal deletion
combinatorial association
clonal selection
VDJ joining
Isotype switching
ANSWERS
a) 2; b) 1; c) 1; d) 5; e)2