COURSE TITLE: IMMUNOLOGY/ IMMUNOCHEMISTRY (3 UNITS)

2019/2020 Second Semester

Module 3_Lecture 2: Immunoglobulins- 24/04/2020 (2hours)

Lecturer: Dr. O. Oduwole

• Gene organization and assembly:

✓ The immunoglobulin repertoire (subtype) is encoded for by multiple germline gene
segments that undergo somatic diversification in developing B‐cells. Although the
basic components needed to generate an immunoglobulin repertoire are inherited, an
individual’s mature antibody repertoire is essentially formed during their lifetime by
alteration of the inherited germline genes.
✓ The first evidence that immunoglobulin genes are rearranged by somation
recombination was reported by Hozumi and Tonegawa (1976). Because somatic
recombination involves rearrangement of DNA in somatic rather than gamete cells,
the newly recombined genes are not inherited.
✓ As a result, the primary immunoglobulin repertoire will differ slightly from one
individual to the next, and will be further modified during an individual’s lifetime by
their exposure to different antigens.
The immunoglobulin variable gene segments and loci:

✓ The variable light and heavy chain loci in humans contain multiple gene segments, which
are joined, using somatic recombination, to produce the final V region exon.
✓ Exons are the region that carry information for a functional protein (genetic code)
✓ The human heavy chain variable region is constructed from the joining of three gene
segments, V (variable), D (diversity), and J (joining), whereas the light chain variable
gene is constructed by the joining of two gene segments, V and J.
✓ The human VH genes have been mapped to chromosome 14, although orphan IgH genes
have also been identified on chromosomes 15 and 16.
✓ The human VH locus, as for other antibody gene segments, is highly polymorphic, and
has likely evolved through the repeated duplication, deletion, and recombination of DNA.
✓ Polymorphisms found within the germline repertoire are due to the insertion or deletion
of gene segments or the occurrence of different alleles of the same segment.
✓ A number of pseudogenes, are also present in immunoglobulin loci.
✓ There are approximately 100 human VH genes, which can be grouped into seven families
based on sequence homology. Members of a given family show approximately 80%
sequence homology at the nucleotide level.
✓ The functional heavy chain repertoire is formed from approximately 40 functional VH
genes, 23 DH genes and 6 JH genes.
✓ For the light chain, the human lambda locus maps to chromosome 22, with approximately
30 functional Vλ genes and 5 functional Jλ gene segments.
✓ The Vλ genes can be grouped into 10 families. The human kappa locus on chromosome 2
is composed of a total of approximately 40 functional Vk genes and 5 functional Jk
genes. However, the kappa locus contains a large duplication of most of the Vk genes,
and most of the Vk genes in this distal cluster, although functional, are seldom used.
✓ The numbers of V genes vary between individuals as a result of polymorphisms.

V,(D), J recombination and combinatorial diversity:

✓ V(D)J recombination is a highly regulated and ordered event.
✓ The light chain exon is constructed from a single V‐to‐J gene segment join.
✓ The heavy chain locus, a D segment is first joined to a J segment, and then the V segment
is joined to the combined DJ sequence.
✓ The rearranged DNA is transcribed, the RNA transcript is spliced to bring together the V
region exon and the C region exon, and lastly the spliced mRNA is translated to produce
the final immunoglobulin protein.
✓ Numerous unique immunoglobulin genes can be made by joining different combinations
of the V, D, and J segments at the heavy and light chain loci.
✓ The creation of diversity in the immunoglobulin repertoire through this joining of various
gene segments is known as combinatorial diversity.
 ✓ Additional diversity is created when different heavy chains are paired with different
lambda or kappa light chains.

Gene conversion and repertoire diversification:

✓ Although mice and humans use combinatorial and junctional diversity as a mechanism to
generate a diverse repertoire, in many species, including birds, cattle, swine, sheep,
horses, and rabbits, V(D)J recombination results in assembly and expression of a single
functional gene.
✓ These animals achieve repertoire diversification by gene conversion, a process in which
pseudo V genes are used as templates to be copied into the assembled variable region
exon.
✓ The animals can also achieve further diversification by somatic hypermutation. “Somatic
hypermutation (or SHM) is a cellular mechanism by which the immune system adapts
to the new foreign elements that confront it (e.g. microbes), as seen during class
switching”.
✓ The process of gene conversion was originally identified in chickens, in which immature
B‐cells have the same variable region exon.
✓ During B‐cell development in the bursa of Fabricius, rapidly proliferating B‐cells
undergo gene conversion to diversify the immunoglobulin repertoire.
✓ Stretches of sequences from germline variable region pseudogenes, located upstream of
the functional V genes, are introduced into the VL and VH regions. This process takes
place in the ileal Peyer’s patches of cattle, swine, and horses, and in the appendix of
rabbits.
✓ These gut‐associated lymphoid tissues are the mammalian equivalent of the bursa in these
species.

Class switch recombination

Antigen‐stimulated IgM expressing B‐cells in germinal centers of secondary lymphoid organs,
such as the spleen and lymph nodes, undergo class switch recombination.
Class switch recombination (CSR): is a process whereby the IgH constant region exon of a
given antibody is exchanged for an alternative exon, giving rise to the expression of antibodies
with the same antigen specificity but of differing isotypes, and therefore of differing effector
functions as described earlier.
✓ CSR occurs through a deletional DNA recombination event at the IgH locus, which has
been extensively studied in mice.
✓ Constant region exons for IgD, IgG, IgE, and IgA isotypes are located downstream of the
IgM (Cμ- constant region of the IgM) exon, and CSR occurs between switch or S
regions.
✓ S regions are repetitive sequences, which are often G‐rich on the non-template strand,
that are found upstream of each CH exon except Cδ (constant region of the IgD).
✓ Breaks are introduced into the DNA of two S regions and fusion of the S regions leads to
a rearranged CH locus, in which the variable exon is joined to an exon for a new constant
region.
✓ The DNA between the two switch (exchange) regions is excised and forms an episomal
circle.
✓ Finally, alternative splicing of the primary RNA transcript generated from the rearranged
DNA gives rise to either membrane bound or secreted forms of the immunoglobulin.
✓ Before recombination occurs between switch regions, transcription is initiated from a
promoter found upstream of an exon that precedes all CH genes capable of undergoing
CSR, the intervening (I) exon. These germline transcripts include I, S, and C region
exons, and do not appear to code for any functional protein.
✓ . The precise mechanism responsible for CSR is still nor well understood.
✓ The joining of S regions may be mediated by association with transcriptional promoters,
enhancers, chromatin factors, DNA repair proteins,

Assignment:
Draw a diagram of the V(D)J recombination.

1) Mansel Haeney: 'Introduction to clinical immunology’, London Butterworths 1985

2) Roitt's Essential Immunology. Thirteenth Edition. Peter J. Delves, Seamus J. Martin, Dennis R.

Burton, Ivan M. Roitt. 13th edition. | Chichester, West Sussex ; Hoboken, [NJ] : John Wiley

& Sons, Inc., 2017.