MOD#83
Monday, May 12, 2003, 1 PM
Dr. Mathew
Kevin Stancoven for Jeff Brace
Page 1 of 5
GENETICS OF IMMUNOGLOBULINS
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Objectives:
o Define the following immunologic terms:
 isotype, allotype and idiotype
o Discuss the major stages in B cell development
o Discuss major theories and molecular mechanisms of Generation of Diversity
o Discuss - Class Switching and Affinity maturation
Isotype, Allotype, & Idiotype
o Isotype: Refers to different classes of immunoglobulins
 Isotype differences are originally defined by antigenic determinant located in the
constant domains
 There are 5 major isotype classes of immunoglobulins
 Example: IgG & IgA are different isotypes.
o Allotype: Refers to differences between two alleles of the same constant genes
 Allotypic differences are defined by antigenic determinants located in constant
domains
 If you compare 2 human’s IgG gamma chains, they will be slight differences
(allotypes)
 Important in transfusion reactions and transplant reactions
o Idiotype: Refers to antigenic variations within an individual of a given species
 Idiotypic differences are located at the antigen binding site (CDR)
 CDR: Complementarity-determining region
 Antibodies that have idiotype differences only, they are only different at their
antigen binding region (CDR)
B Cell Development & Differentiation
o There are several stages in the B cell differentiation pathway
o Differentiation of B cell is part antigen independent & part antigen dependent (influenced
by T cells)
o B cells made is bone marrow and development is antigen independent
o Later on, B cell development is dependent on antigens and T cells
 No T cells (AIDS): humoral immune response becomes ineffective because B
cells don’t develop correctly
o Pro-B cell: The earliest B-lineage cells (Progenitor B-cells)
 From pluripotent stem cell
 DJ and VDJ gene rearrangement
o Pre-B cell: Express μ chain & surrogate light chains VpreB and λ5
 Pre-B cell receptor is expressed on the surface
o Immature B cell: IgM molecule is expressed on cell surface
Pathway of B cell Development
MOD#83
Monday, May 12, 2003, 1 PM
Dr. Mathew
Kevin Stancoven for Jeff Brace
Page 2 of 5
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B Cell Development & Differentiation
o Mature B cell: Mature B cells express both IgM and IgD on the cell surface
 Once mature B cell interacts with antigen, it will become a plasma cell
o Plasma Cell: Terminal differentiated form of B cells which secretes only one isotype of
antibody
o Memory B cells: Some B cells do not differentiate into plasma cells, but remain as long
lived memory cells after interaction with antigen
 They respond in an accelerated fashion to antigen stimulation compared to
Virgin B cells (B cell that has never interacted with an antigen)
 Retain high affinity for specific antigen – principle behind immunity
 The second time an antigen interacts with the B cells, the immune
response is very rapid & effective, and the antigen (bacteria, virus) is
quickly killed
 Memory B cells play an important role in secondary or anemnestic antibody
response
Genomic Organization of Human Ig Genes
o There are three loci in the genome that code for Ig molecules
o Kappa chain locus – chr.2
o Lambda chain locus - chr.22
o Heavy chain locus - chr.14
o Both kappa & lambda have multiple V (variable) genes and J (joining segment) genes
o In addition to V & J, heavy chains also have D (diversity segment) genes
ORGANIZATION OF Ig GENES
o Lambda light chains (chromosome 2)
 30 variable genes, 4 joining genes, 4 constant genes
o Kappa light chains (chromosome 22)
 40 variable genes, 5 joining genes, 1 constant gene
o Heavy chains (chromosome 14)
 65 variable genes, 6 joining genes, 1 constant gene, 27 diversity genes
o All the genes on the same chromosome are linked genes
 All the genes on chromosome 2 that code for lambda light chains are linked
o Immunoglobulins have “leader sequence” (L1, L2, etc…) that direct the Ig to stay
membrane bound
 Secreted Ig do not have leader sequences, and they are released from the cell
ORGANIZATION OF Ig HEAVY CHAIN CONSTANT GENES
o Dr. Mathew said we should know the organization of the heavy-chain constant genes
o In human heavy chains, the variable genes come first, then diversity genes, joining genes,
and lastly constant genes
o Human heavy chain constant genes:
 Cμ, Cδ, Cγ3, Cγ1, ψCε, Cα1, Cγ2, Cγ4, Cε, Cα2
 ψCε is a pseudogene that is non-functional
 Cγ3 will give rise to IgG3
o Mouse heavy chain constant genes:
 Cγ3, Cγ1, Cγ2α, Cγ2δ, Cε, Cα
o When immunoglobulin class switching occurs, each gene may be deleted and subsequent
(non-deleted) genes are spliced together
 This determines the classes of immunoglobulin – covered later
GENERATION OF ANTIBODY DIVERSITY
o There are billions of antigens in the universe & a human individual can express as many
as ten billion or more different antibody repertoire
o The immune system can recognize & bind any foreign antigen that enters the body
MOD#83
Monday, May 12, 2003, 1 PM
Dr. Mathew
Kevin Stancoven for Jeff Brace
Page 3 of 5
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Billions of genes to code for each antibody type will not be an effective system
 Remember that we have only about 35,000 functional genes
 Dr. Mathew compared this to communism in the USSR, if our body used all of
its genetic information to code for antibodies, the rest of our body systems
would not work properly
 Our body has developed to be able to recognize and bind any of the billions of
antigens that may enter our body – and only used a minimal amount of genetic
information in the process
o Three theories were developed to explain antibody diversity:
 Germline Theory
 Minigene Theory
 Somatic Mutation Theory
Germline Theory
o According to germline theory, there is a separate gene for each different antibody chain
o All genetic information for generation of diversity is in the germline genome and no other
mechanisms are required
o In this case, we would need billion of genes just for antibodies
Minigene Theory
o The minigene theory postulates that the genes that code for variable domains are
segmented, and these gene segments can join in various combinations
o Each different combination leads to a variation in the structure of antigen binding site
o The use of recombinant DNA technology identified multiple gene segments (V, D, J) in
both light & heavy chain loci
 It has been established that V region genes combine with the J & D gene
segments to code for the variable domain
 Both the J & D gene segments code for amino acids within the CDR3 domains
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By using the above table:
 Any Kappa light chain may have any one of the 40 variable segments and any
one of the 5 joining segments
 5*40=200 different kappa chains
 Any Lambda light chain may have any one of the 30 variable segments and any
one of the 4 joining segments
 4*30=120 different lambda chains
MOD#83
Monday, May 12, 2003, 1 PM
Dr. Mathew
Kevin Stancoven for Jeff Brace
Page 4 of 5
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Any heavy chain may have any one of the 65 variable segments, any one of the
6 joining segments, and any one of the 27 diversity segments
 65*6*27=10530 different heavy chains
Somatic Mutation Theory
o This postulates that there is a novel hypermutation mechanism operates at the
immunoglobulin loci that introduces point mutations into the variable regions of both
light & heavy chains
o Somatic hypermutation occurs after VJ or VDJ joining has occurred and continues
throughout the life of the B cell
o Consequently, more somatic mutations are observed in the IgG than in IgM
 IgM is produced first in response to an antigen
 IgG is produced after a week or so after an antigen exposure and IgG levels stay
high months after the exposure
o Those B cells that express somatic variants with increased affinity for the antigen are
preferentially stimulated to proliferate and mature into AFC (plasma cells)
 This phenomenon is called Affinity maturation
 The B cells that express somatic variants with decreased affinity for the antigen
are not stimulated & do not proliferate
Generation of Junctional Diversity
o Recombination at V, J, & D gene segments are not precise
 This generates further variation at the joining region - Junctional diversity
o Junctional diversity generated by 2 mechanisms:
o P nucleotides:
 Junctional diversity is increased by endonuclease cleavage of DNA loops at the
break point that creates P-nucleotides (generated from palindromic sequence)
 Break the DNA and joined back together at palindromic sequences
 The DNA is rejoined by recombinase
 Recombinase activating genes: RAG-1 & RAG-2
o N nucleotides:
 N-nucleotides (non-template encoded) are added at the break point by the
enzyme TdT (Terminal deoxynucleotidyl transferase)
 When the DNA is broken, TdT will add nucleotides at the junction
 Increases the diversity – adding nucleotides not in a factor of 3 will change the
translation of the whole reading frame because amino acids are determined by
sets of 3 nucleotides
 Similar to an insertion mutation that we talked about last week in
Robbins
Generation of Variable Region Genes
o Complete variable regions are generated by somatic recombination of separate gene
segments
o Light chain variable regions are constructed from two gene segments: V & J
 The constant region (C) is joined to VJ by RNA splicing
o Heavy chain variable regions are constructed from three gene segments: V, D, & J
o First the D & J gene segments join, then the V gene segment joins to the DJ
 The constant region is joined by RNA splicing
Generation of Light & Heavy Chains
o This slides shows the process of going from genes to the immunoglobulins
o Recombination, transcription, splicing, translation
Immunoglobulin Class Switching
o Mature B cells express both IgM & IgD at the cell surface
 μ and δ mRNAs are produced by differential spicing of heterogeneous nuclear
RNA (hnRNA)
MOD#83
Monday, May 12, 2003, 1 PM
Dr. Mathew
Kevin Stancoven for Jeff Brace
Page 5 of 5
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 μ mRNA needed for IgM
 δ mRNA needed for IgD
o Upon stimulation by antigen, B cells proliferate & continues to differentiate
o During this time the same VDJ rearranged segment is joined to another constant gene
 Thus B cell may switch from expressing IgM to any other Isotype
 This is called class switching or isotype switching
o Class switching will cause a plasma cell that is producing one type of immunoglobulin
(IgM) to begin making a different class of immunoglobulin (IgG)
o Isotype switching involves recombination between switch regions and the DNA between
the switch regions are deleted
o Class switching may occur in response to particular cytokines (IL-4) or in response to a
particular antigen/pathogen
 Reasons not completely understood
Isotype Switching
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Using the above flow chart, a plasma cell that is secreting IgA can never switch to
produce IgG, because the DNA information needed to produce IgG has already been
deleted
o This depends of the order of the genes on the DNA:
 Cμ, Cδ, Cγ3, Cγ1, ψCε, Cα1, Cγ2, Cγ4, Cε, Cα2
Production of Transmembrane & Secreted IgM
o Normally, the antibody is present on the cell membrane
o When the antibody interacts with an antigen, differential splicing cuts out the genetic
information that codes for the transmembrane region
o With no transmembrane region, the antibody is able to be secreted