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Aims Gene rearrangement and class switching of B-cell Igs.
Lecture #9 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
Heavy Chain Light Chain Abbas & Lichtman’s Basic Immunology 4-9
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Heavy Chain Synthesis Variable (VH) segments Diversity (DH) segments
Vn 5’ 3’ Germline DNA J1-n D1-n Ce Ca Cg Cd Cm Variable (VH) segments Diversity (DH) segments Joining (JH) segments Conserved (CH) segments Similar to Abbas & Lichtman’s Basic Immunology 4-10
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Heavy Chain Synthesis 5’ 3’ V1 V2 Vn Germline DNA J1-n D1-n Ce Ca Cg Cd Cm Rearranged B cell DNA Intervening DNA between the D and J segments is looped and cut out. Similar to Abbas & Lichtman’s Basic Immunology 4-10
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Heavy Chain Synthesis 5’ 3’ Ce Ca Cg Cd Cm V1 V2 Vn Rearranged B cell DNA Intervening DNA between the V and D segments is looped and cut out. Similar to Abbas & Lichtman’s Basic Immunology 4-10
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Heavy Chain Synthesis 5’ 3’ Ce Ca Cg Cd Cm V1 V2 Rearranged B cell DNA Primary RNA transcript The gene is transcribed resulting in a primary RNA containing the VHDHJH segment, the remaining J segments and only the Cm and Cd segments. Similar to Abbas & Lichtman’s Basic Immunology 4-10
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Heavy Chain Synthesis 5’ 3’ Cd Cm V2 Primary RNA transcript mRNA transcript The transcript is processed and the introns and Cd sequences are removed. The resulting mRNA contains the Cm segment and will code for an IgM. Similar to Abbas & Lichtman’s Basic Immunology 4-10
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Heavy Chain Synthesis mRNA transcript NH2 COOH Cm V2 m heavy chain polypeptide 5’ 3’ The mRNA is translated by ribosomes into m chain. Adapted from Abbas & Lichtman’s Basic Immunology 4-12
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Light Chain Synthesis Variable (Vk) segments Joining (Jk) segments
Vn Ck J1-n 3’ 5’ Germline DNA Variable (Vk) segments Joining (Jk) segments Conserved (Ck) segment 2 isotypes l (40%) and k (60%)
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Light Chain Synthesis Germline DNA V1 V2 V3 Vn Ck J1-n 3’ 5’ Rearranged B cell DNA V1 V2 V3 Ck 3’ 5’ Intervening DNA between the V and J segments is looped and cut out. Similar to Abbas & Lichtman’s Basic Immunology 4-10
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Light Chain Synthesis V3 Ck 3’ 5’ Primary RNA transcript V1 V2 Rearranged B cell DNA The B cell transcribes a segment of DNA into a primary RNA transcript that contains a long intervening sequence of J segments and introns. Similar to Abbas & Lichtman’s Basic Immunology 4-10
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Light Chain Synthesis V3 Ck 3’ 5’ Primary RNA transcript V3 Ck 3’ 5’ mRNA transcript This RNA transcript is processed into mRNA by splicing the exons together. Similar to Abbas & Lichtman’s Basic Immunology 4-10
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Light Chain Synthesis V3 Ck 3’ 5’ mRNA transcript COOH NH2 k chain polypeptide The mRNA is translated by ribosomes into k chains. Combines with heavy chain to make IgM.
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Light Chain Synthesis IgM NH2 COOH m heavy chain polypeptide
Ck COOH NH2 k chain polypeptide Cm V2 m heavy chain polypeptide IgM Abbas & Lichtman’s Basic Immunology 4-12
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mRNA Splicing 5’ 3’ IgD mRNA Primary heavy chain RNA transcript
Cd Cm V2 IgD mRNA IgM mRNA Abbas & Lichtman’s Basic Immunology 4-12
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Ig Class Change 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. Abbas & Lichtman’s Basic Immunology 7-10
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Ig Class Change Involves a switch sequence upstream of the constant region gene segments. Recombination occurs between the Sμ recombination region and a downstream switch region (Sg). 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. Adapted from Abbas & Lichtman’s Basic Immunology 7-10
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Ig Class Change Class change occurs upon B cell activation and maturation. Requires signals from ____________________________ (CD3+, CD4+, CD8-). Binding of receptors (CD40 ligand) Cytokines Abbas & Lichtman’s Basic Immunology 7-9
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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)
V segment J segment G T A Germ line DNA Arg Ser Tyr Trp Protein Recombined DNA 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).
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Junctional Diversity (N- region diversity)
V segment J segment G T A Germ line DNA Arg Val Trp Protein Recombined DNA 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).
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Junctional Diversity (N- region diversity)
V segment J segment G T A Germ line DNA Arg Leu Val Trp Protein Recombined DNA 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).
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Surface vs. Secreted Primary RNA transcript 5’ 3’ Cm V2 Soluble IgM mRNA sIgM Ts Poly A Tm Membrane IgM mRNA mIgM Which poly A site is transcribed determines whether the protein is surface or secreted.
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Membrane Bound Ig IgM or IgD
Lecture #9 Membrane Bound Ig IgM or IgD Associated with a heterodimer of Iga and Igb. Activation of the BCR results in activation of Iga and Igb leading to cytoplasmic signaling and B cell activation B cell receptor (BCR) complex Iga Igb Figure 8.8 B-cell activation is similar to T-cell activation. If membrane Ig becomes cross-linked (e.g. by a T-independent antigen), tyrosine kinases, including Lck, Lyn, Fyn and Blk, become activated. They phosphorylate the ITAM domains in the Igα and Igβ chains of the receptor complex. These can then bind another kinase, Syk, which activates phospholipase C. This acts on membrane PIP2 to generate IP3 and diacyl glycerol (DAG) which activates protein kinase C. Signals from the other kinases are transduced to activate nuclear transcription factors.
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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 IgM 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 a and b genes are expressed in a majority of peripheral T cells. g and d 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 a chains 2 b chains Associated with CD3 2 z chains
Variable domain Conserved domain CD3 2 a chains 2 b chains Associated with CD3 2 e chains 1 d chain 1 g chain 2 z chains Adapted from Roitt’s Immunology 4-6
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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
Vn 5’ 3’ Germline a DNA J1-n Ca J11-n D1 Cb1 Germline b DNA J21-n Cb2 D2 Variable (VH) segments Diversity (DH) segments Joining (JH) segments Conserved (CH) segments
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T Cell Receptor Diversification
J1-n 5’ 3’ V1 V2 Ca Vn Germline a DNA J11-n J21-n D1 D2 5’ 3’ V1 V2 Vn Cb1 Cb2 Germline b DNA Recombination between V, D, and J gene segments. a and g chains contain V and J gene segments. b and d chains contain V, D, and J gene segments. Similar to Ig.
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Comparison of TCR and Ig
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 Abbas & Lichtman’s Basic Immunology 4-1
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Antigen Recognition 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 Abbas & Lichtman’s Basic Immunology 4-7
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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 Describe the structure and synthesis of the various immunoglobulin Heavy and Light chains, and T-cell receptors. V, D, J, C Segments Describe the process of immunoglobulin class change. Describe the mechanisms of antibody diversity. Combinitorial vs Junctional Surface vs Secreted Know the difference between affinity and avidity. Compare and contrast immunoglobulins and T-cell receptors.
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