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GENERATION OF ANTIBODY DIVERSITY*
*Special Thanks to Steven J. Norris, Ph.D
Jeffrey K. Actor, Ph.D. Jeffrey K. Actor, Ph.D. Pathology and Laboratory MedicinePathology and Laboratory Medicine
The University of TexasThe University of Texas--Houston Medical SchoolHouston Medical School
~1015 to 1018 Different Antibodies
and T cell Receptors Produced!
Antibody Structure and Function
Variable DomainsAntigen-BindingActivity
Constant DomainsFunctional Activities
Generation of antigen-binding diversity occurs before antigen exposure through VDJ rearrangement
VDJ rearrangement
Surface IgM/IgD expressing B cells
Exposure to Ag,helper T cells
Development of plasma cells, secretion of Ag-specific antibodies
Isotype Switching -after antigen exposure
Recombination Events Begin with the Heavy Chain Ig Locus
• B cells rearrange their DNA sequences.• Expresses IgM and IgD in B cells initially
–
and
constant region genes• Other heavy chain isotypes expressed after
antigen exposure (IgG1, IgG2, IgG3, and IgG4, IgA1, IgA2, IgE).– Encoded by 1 , 2 , 3 , 4 , 1 , 2 ,
• Found in germline (unrearranged) configuration in all other cell types.
Abbrev. Meaning Number Size Function
V Variable ~50 ~95 aa
D Diversity ~20 ~3-6 aa Form part of Variable Domain
J Joining ~6 ~13 aa
C Constant 9* ~110 aa /Domain
Form the Constant Regions
Immunoglobulin heavy chain gene segments
*One locus for each isotype
Germline DNA
L V1 L V2 L VN D1-26 J1-6 C C C3 C4 CC1 C2C2C1
Heavy Chain Locus and VDJ Rearrangement
D-J joiningV-D joining
Looping Out of DNA During Rearrangement
V(D)J recombinase = RAG1 and RAG2
C
C
Expression of Surrogate Light Chains Prior to Light Chain Expression
After successful rearrangement, the
chain is transported to the surface of the cell along with the surrogate light chain proteins, VpreB and 5. The presence of this complex on pre-B cells triggers the initiation of light chain rearrangement.
55
Genetic organization and recombination events.
Germline DNA
Rearranged DNA
Primary RNATranscript
mRNA
Somatic Recombination(D-J joining)
Somatic Recombination(V-D-J Joining)
Transcription,RNA Splicing
Translation, ProcessingVH
CJH
DH
Similarly ProcessedLight Chain
Mature Heavy Chain (IgM)
D2L V1 L V2 L VN D1 J4 C C C3 C4 CC1 C2C2C1D3 J5J6
L V1 L V2 J4 C C C3 C4 CC1C2C1D3 J5J6 C2
L V1 L V2 L VN D1-26 J1-6 C C C3 C4 CC1 C2C2C1
V2 J4D3L C
AAAAAC
Light Chain Loci• Two separate loci – kappa () and lambda () on two
different chromosomes• Code for kappa and lambda light chains • Have V and J gene segments (no D segments)• After successful heavy chain rearrangement, kappa
gene rearrangement occurs• If kappa rearrangement is successful, the resulting
immature B cell expresses IgM with kappa light chains
• If kappa rearrangement is not successful, lambda gene rearrangement occurs
• Successful lambda rearrangement results in expression of IgM with lambda light chains
Light Chain Loci
VDJ
4005
3004
Kappa Gene Rearrangement
D-J joining
Result - functional IgM!
5
Coexpression of IgM and IgD - Alternate Splicing
Coexpression IgM and IgD: Alternate Splicing
Stages of B Cell Development
Mechanisms of Ag-Binding Diversity• Each individual can produce B and T cells with 1015
to 1018 different specificities!• We make antibodies and T cell receptors that react
with almost any compound, including those that are synthetic and have never occurred in nature
• Most of this diversity is generated during V(D)J rearrangement, which occurs prior to antigen exposure
• The diverse group of B and T cells produced is called a “repertoire”
• Of this repertoire, <1% of B and T cells will respond to any single antigen or infectious agent
CDRs (L1, L2, L3, H1, H2, H3) form the Ag-binding pocket (Paratope) and determines Ag-binding specificity
Basis of Diversity – Variation within Complementarity Determining Regions
V D J
Five Mechanisms of Antibody Diversity
• Availability of multiple V gene segments• Combinatorial diversity (different VDJ
and VJ combinations)• Assortment of heavy and light chains• Junctional and insertional diversity• Somatic hypermutation
1. Multiple V regions
• Heavy chain locus - ~50 V regions• Kappa and lambda loci – 40 V regions
each• Encode the CDR1 and CDR2
2. Combinatorial diversity (different VDJ and VJ combinations)
• Different V, D, and J combinations are selected randomly during B cell development
• Number of V genes X D genes X J genes = number of possibilities
• 50 X 20 X 6 = 6000 heavy chain combos• ~160-200 VJ combinations in kappa and
lambda loci• Affects the diversity of CDRs
3. Assortment of different H and L chains
• Egg Drop Soup• Won Ton Soup• Hot and Sour Soup
• Kung Pao Chicken • Egg Foo Young• Sweet and Sour
Pork• Mongolian Beef• Moo Shoo Pork• Shrimp Chow Mein
3 X 6 = 18 different combinations
Ig H and L chain combinations
• 6000 H chain VDJ combinations
• 200 kappa chain VJ combinations
• 160 lambda chain VJ combinations
H and L combinations =6000 x 200 + 6000 x 160 ~2 x 106 possibilities
4. Junctional and insertional diversity
• V(D)J recombination sites not precise, but instead are ‘sloppy’
• Recombination a few base pairs one direction or another will change amino acid sequence
• N-region addition – random insertion of nucleotides to DJ or VD junctions of heavy chain by terminal deoxynucleotide transferase
• Affects sequence of CDRs– At VJ and VDJ junctions
Junctional and insertional diversity
5. Somatic hypermutation
• Point mutations occurring in the V regions• Mutation rate in heavy and light chain V
regions ~10,000 times higher than background following B cell activation
• Only form of Ab diversity that occurs after antigenic stimulation
• Results in affinity maturation, ie. selection of mutants that have a higher affinity for the antigen in secondary responses
Isotype Switching• Definition – change in the heavy chain isotype
expressed by a given B cell.• Constant region downstream of V region is
expressed.• ‘Naive’ B cells can only express IgM and IgD;
plasma cells that develop from naive B cells express first IgM, then switch to other isotypes.
• V region and thus antigen binding specificity doesn’t change during isotype switching.
• Isotype switching is stimulated by Ag exposure + cytokines produced by T cells.
Mechanism – deletion of DNA between switch regions
Isotype Class Switching.
Rearranged DNAin B cell
Deletion of InterveningConstant Region
Sequences
Translation,Processing
VH JHDHL C
AAA
Mature Heavy Chain (IgG1)
L VH JH C C C3 C4 CC1C2C1DH C2
Mature Heavy Chain (IgE)
Mature Heavy Chain (IgA2)
VH JHDHL C1
AAAVH JHDHL C2
AAA
IFN- IL-4IL-13 IL-5
Influence of T cell Cytokines
mRNA
L VH JH C4 CC1C2C1DH C2 L VH JH C C1DH C2 L VH JHDH C2
Transcription
Membrane vs. Secreted Ig Expression
• B cells express characterized by membrane- bound immunoglobulins
• Plasma cells characterized by secreted immunoglobulins
• Membrane-bound Ig has a hydrophobic tail that anchors it to the cytoplasmic membrane; this ‘tail’ is lacking from secreted Ig
• Results from differential termination of transcription – secreted form transcript is shorter, lacks region encoding the membrane (M) exon
Membrane vs. Secreted Ig Expression
Summary• Antigen binding diversity occurs during VDJ
rearrangement, BEFORE antigen exposure.
• The ~1015 different antigen binding site combinations result from five different mechanisms: multiple gene segments, combinatorial diversity, junctional and insertional diversity, expression of different H and L chain pairs, and somatic hypermutation.
A six-year old black male received scheduled immunizations against Diphtheria, tetanus and pertussis throughout the first two years of his life. Upon boosting, he demonstrated increased antibody affinity to binding diphtheria antigens. The mechanism that affects immunoglobulin antigen binding affinity which occurs after formation of a mature B cell is:A. Isotype switching B. Combinatorial diversity C. Junctional diversity D. Somatic hypermutationE. V gene segment diversityOption D (Somatic hypermutation) is correct. Generation of antigen-binding diversity results from recombination of the variable (V), diverse (D), and joining (J) gene segments during B cell development, with somatic mutation occurring after antigenic stimulation to allow increases to occur in affinity. There are five sources of antibody diversity: 1) presence of multiple V gene segments; 2) Combinatorial diversity, resulting from random recombination of V, D, and J segment combinations; 3) junctional and insertional diversity, resulting in changes in the V-D and D-J junctions; 4) coexpression of different H and L chain pairs; and 5) somatic hypermutation.
Regulation of Ig Expression
• Enhancer element – a region of DNA near the J gene segments that increases transcription from the Ig gene promoters
• V(D)J joining brings the enhancer element close to the promoter increased transcription
• Differentiation into plasma cells increases Ig expression ~1,000 fold
Effect of Enhancers
Enhancer Sequence
Weak Mucho gusto!
+
Ig Gene Superfamily
• Immunoglobulins and T cell receptors are found in vertebrates, not in invertebrates
• Ig and TCR most likely evolved from related proteins involved in cell-cell interactions
• These proteins are members of the Ig gene superfamily
All members of Ig gene superfamily have domain structure
• Sequence similarity• 100-110 amino acids per domain• Beta-pleated sheet structure• Intrachain disulfide bond (Cys-Cys)
Example –Ig light chain
Members of the Ig gene superfamily include:
Big Bang Theory
• Recombinase proteins RAG1 and RAG2 central to the VDJ rearrangement process
• RAG1 and RAG2 resemble bacterial recombinases
• Big bang theory – RAG genes were acquired from bacteria or fungi, resulting in the development of Ig and TCR systems in an early vertebrate ancestor
Stages of B Cell Development
Kuby, Immunology
Clinical Vignette – from Rosen and Geha, “Case Studies in Immunology”
Case: Hyper IgM Immunodeficiency – Dennis Fawcett has recurrent sinus infections due to a defect in CD40 production and isotype switching, which precludes production of immunoglobulins other than IgM.
Rosen and Geha, Fig. 3.3
Defect in signalling protein, CD40
X
Antibody Structure and FunctionHow is it Formed?
Ag-binding diversity• Multiple V segments• V(D)J joining• Heavy, light chain assortment• Junctional/insertional diversity• Somatic mutation
Functional diversity• Biological activity related to Ig isotype• Isotype switching occurs after antigen exposure