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Aims
• Gene rearrangement and class switching of B-cell Igs.
• T cell receptors- What are they and where do they fit into immunology?– Gene rearrangement to generate diversity
• Readings: Abbas & Lichtman, Chapters 4 & 7
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Human Immunoglobulin Loci
Abbas & Lichtman’s Basic Immunology 4-9
Heavy Chain
Light Chain
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Heavy Chain Synthesis
• Variable (VH) segments
• Diversity (DH) segments
• Joining (JH) segments
• Conserved (CH) segments
V1 V2 Vn5’ 3’Germline DNA
J1-nD1-n
CCCCC
Similar to Abbas & Lichtman’s Basic Immunology 4-10
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Heavy Chain Synthesis
• Intervening DNA between the D and J segments is looped and cut out.
5’ 3’
V1 V2 Vn5’ 3’
Germline DNAJ1-nD1-n
CCCCC
CCCCCV1 V2 Vn
Rearranged B cell DNA
Similar to Abbas & Lichtman’s Basic Immunology 4-10
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Heavy Chain Synthesis
• Intervening DNA between the V and D segments is looped and cut out.
5’ 3’CCCCCV1 V2 Vn
5’ 3’CCCCCV1V2
Rearranged B cell DNA
Rearranged B cell DNA
Similar to Abbas & Lichtman’s Basic Immunology 4-10
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Heavy Chain Synthesis
• The gene is transcribed resulting in a primary RNA containing the VHDHJH segment, the remaining J segments and only the C and C segments.
5’ 3’CCCCCV1V2
5’ 3’CCV2
Rearranged B cell DNA
Primary RNA transcript
Similar to Abbas & Lichtman’s Basic Immunology 4-10
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Heavy Chain Synthesis
• The transcript is processed and the introns and C sequences are removed. The resulting mRNA contains the C segment and will code for an IgM.
5’ 3’CCV2
Primary RNA transcript
5’ 3’CV2
mRNA transcript
Similar to Abbas & Lichtman’s Basic Immunology 4-10
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Heavy Chain Synthesis
• The mRNA is translated by ribosomes into chain.
mRNA transcript
NH2 COOHCV2
heavy chain polypeptide
5’ 3’CV2
Adapted from Abbas & Lichtman’s Basic Immunology 4-12
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Light Chain Synthesis
• Variable (Vk) segments
• Joining (Jk) segments
• Conserved (Ck) segment
• 2 isotypes (40%) and (60%)
V1 V2 V3 Vn C
J1-n
3’5’Germline DNA
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Light Chain Synthesis
• Intervening DNA between the V and J segments is looped and cut out.
V1 V2 V3 Vn C
J1-n
3’5’
Germline DNA
V1 V2V3 C 3’5’
Rearranged B cell DNA
Similar to Abbas & Lichtman’s Basic Immunology 4-10
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Light Chain Synthesis
• The B cell transcribes a segment of DNA into a primary RNA transcript that contains a long intervening sequence of J segments and introns.
V3 C 3’5’Primary RNA transcript
V1 V2V3 C 3’5’
Rearranged B cell DNA
Similar to Abbas & Lichtman’s Basic Immunology 4-10
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Light Chain Synthesis
• This RNA transcript is processed into mRNA by splicing the exons together.
V3 C 3’5’Primary RNA transcript
V3 C 3’5’mRNA transcript
Similar to Abbas & Lichtman’s Basic Immunology 4-10
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Light Chain Synthesis
• The mRNA is translated by ribosomes into k chains.
• Combines with heavy chain to make IgM.
V3 C 3’5’mRNA transcript
V3 C COOHNH2
chain polypeptide
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Light Chain Synthesis
V3 C COOHNH2
chain polypeptide
NH2 COOHCV2
heavy chain polypeptide
IgM
Abbas & Lichtman’s Basic Immunology 4-12
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mRNA Splicing
Primary heavy chain RNA transcript
5’ 3’CCV2
5’ 3’CV2
5’ 3’CV2
IgD mRNA
IgM mRNA
Abbas & Lichtman’s Basic Immunology 4-12
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Ig Class Change
Abbas & Lichtman’s Basic Immunology 7-10
• All classes of immunoglobulin use the same set of variable region genes (ie V-D-J).
• All that changes is the constant region of the heavy chain.
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Ig Class Change
Adapted from Abbas & Lichtman’s Basic Immunology 7-10
• Involves a switch sequence upstream of the constant region gene segments.
• Recombination occurs between the Sμ recombination region and a downstream switch region (S).
• The intervening region containing genes for IgM and IgD in this instance is looped out and then cut, with deletion of the intervening regions and joining of the two switch regions.
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Ig Class Change
Abbas & Lichtman’s Basic Immunology 7-9
• Class change occurs upon B cell activation and maturation.
• Requires signals from ____________________________ (CD3+, CD4+, CD8-).– Binding of receptors
(CD40 ligand)
– Cytokines
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Mechanisms of Antibody Diversity
• Combinatorial diversity– Multiple germ line V genes recombining with J and D
segments.
• Junctional diversity– Imprecision in V-J and V-D-J recombinations (N-
region diversity)– N-nucleotide additions (N-region diversity)
• Note addition is done without a template.
– Somatic point mutation
• Assorted heavy and light chains
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Combinatorial Diversity
• Possible combinations of V-D-J and V-J germ line gene segments.
Abbas & Lichtman’s Basic Immunology 4-11
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Junctional Diversity (N- region diversity)
• Changes in nucleotide sequence at junctions of V, D, and J segments.
• Two types of changes– Exonuclease removal of
nucleotides.
– Random addition of nucleotides by terminal deoxyribonucleotidyl transferase (TdT).
C
V segment J segment
C G G TGA T AT G G
Germ line DNA
Arg Ser TyrTrp
Protein
C G G TGA T AT G G
Recombined DNA
C
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Junctional Diversity (N- region diversity)
• Changes in nucleotide sequence at junctions of V, D, and J segments.
• Two types of changes– Exonuclease removal of
nucleotides.
– Random addition of nucleotides by terminal deoxyribonucleotidyl transferase (TdT).
C
V segment J segment
C G G TGA T AT G G
Germ line DNA
Arg ValTrp
Protein
C G G TTG A CT G G
Recombined DNA
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Junctional Diversity (N- region diversity)
• Changes in nucleotide sequence at junctions of V, D, and J segments.
• Two types of changes– Exonuclease removal of
nucleotides.
– Random addition of nucleotides by terminal deoxyribonucleotidyl transferase (TdT).
C
V segment J segment
C G G TGA T AT G G
Germ line DNA
Arg Leu ValTrp
Protein
C T
C G G ATC G TT G G
Recombined DNA
T A C
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Surface vs. Secreted
• Which poly A site is transcribed determines whether the protein is surface or secreted.
Primary RNA transcript
5’ 3’CV2
5’ 3’CV2
Soluble IgM mRNA
sIgM
Ts
Poly A
Tm
Poly A
Ts
5’ 3’CV2
Membrane IgM mRNA
Ts Tm
mIgM
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Membrane Bound Ig
• IgM or IgD • Associated with a
heterodimer of Ig and Ig.
• Activation of the BCR results in activation of Ig and Ig leading to cytoplasmic signaling and B cell activation
B cell receptor (BCR) complex
Ig Ig
Ig Ig
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Antibody Kinetics
• __________________________ is the tendency of antibody to form stable complexes with an antigen
+ Ag
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Antibody Kinetics
• _________________________ is the strength in which a multivalent antibody binds a multivalent antigen.
Y Y
Y Y Y
IgM
Y
IgG
Ag
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T Cell Receptor
• Present on a majority of T cells both CD4+ and CD8+.
• Heterodimeric molecule.– Linked by a disulfide bond.
• Generated by 4 sets of genes– and genes are expressed in a majority of
peripheral T cells.
– and are expressed on a minority of thymic and peripheral T cells (10%).
• Binds to the peptide-MHC complex.
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T Cell Receptor
• 2 chains• 2 chains
• Associated with CD3– 2 chains– 1 chain– 1 chain
• 2 chains
Adapted from Roitt’s Immunology 4-6
Variable domain
Conserved domain
CD3
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T-Cell Receptor Loci
Adapted from Abbas & Lichtman’s Basic Immunology 4-9
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T Cell Receptor Diversification
V1 V2 Vn5’ 3’
Germline DNA
J1-n
C
V1 V2 Vn5’ 3’
J11-nD1
C
Germline DNA
J21-n
C
D2
•Variable (VH) segments
•Diversity (DH) segments
•Joining (JH) segments
•Conserved (CH) segments
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T Cell Receptor Diversification
V1 V2 Vn5’ 3’
Germline DNA
J1-n
C
V1 V2 Vn5’ 3’
J11-nD1
C
Germline DNA
J21-n
C
D2
•Recombination between V, D, and J gene segments.· and chains contain V and J gene segments.· and chains contain V, D, and J gene segments.·Similar to Ig.
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Comparison of TCR and Ig
Abbas & Lichtman’s Basic Immunology 4-1
• Ig binds to proteins, polysaccharides, Lipids, and nucleic acids, while TCR only binds peptides in MHC.
Forms of Antigen
Recognized
Diversity
Antigen Recognition
Mediated by
Signaling functions
Mediated by
Effector functions
Mediated by
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Antigen Recognition
Abbas & Lichtman’s Basic Immunology 4-7
• Ig binds antigen all by itself, while TCR needs CD4 or CD8 to simultaneously bind.
Antigen Binding
Region
Structure of antigen
recognized
Affinity of antigen
binding
On-rate and Off-rate
Accessory molecules
Involved in binding
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Next Time
• T cell maturation
• Cell-mediated immune responses (CMI)
• Humoral immune responses
• B cell activation
• Readings: Abbas & Lichtman, Chapters 5 & 6
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Objectives
1. Describe the structure and synthesis of the various immunoglobulin Heavy and Light chains, and T-cell receptors.
1. V, D, J, C Segments
2. Describe the process of immunoglobulin class change.
3. Describe the mechanisms of antibody diversity.1. Combinitorial vs Junctional2. Surface vs Secreted
4. Know the difference between affinity and avidity.5. Compare and contrast immunoglobulins and T-cell
receptors.