Sections to Skip for Ch 2 Figure 2.3 - Enzyme digestion of Ab’s Monoclonal Antibodies Fig 2.12 & 2.13

Chapter 2 Antibody Structure and the Generation of B-Cell Diversity Molecular and structural basis of antibody diversity How B cells develop and function in the body How B cells are activated and participate in adaptive immunity

Location and production of Immunoglobulins Antibodies are specific for individual epitopes Membrane bound form is present on a B-cell Ag binding to B cell stimulates it to secrete Ab B cell has all the same BCR on its surface Epitope is the the area where the Antibody binds in the antigen

Heavy (5 classes), Light (2 classes), Constant and Variable Antibody structure Fab Fc Four parts to remember Heavy and light, Constant and Variable Y shape made up of four polypeptide chains Each arm made up of paired chains that are disulfide bonded Stem - paired c-terminal heavy chains, disulfide bonded 1. Variable region: Each Ab molecule has a different amino acid sequence in the V region. Within each Ab molecule, the amino acid sequence of the VH is different from VL 2. Constant Region: -amino acid sequence is the identical or almost identical -heavy chain (5 different constant regions): , , , ,  - light chain (2 different constant regions): ,  3. Variable Domain: three regions vary to a greater extent that the rest of the variable domain - hypervariable regions or complementarity determining regions (CDRs) Heavy (5 classes), Light (2 classes), Constant and Variable Disulfide bonds Skip figure 2.3 (Fab- fragment antigen binding) (Fc- fragment crystallizable)

     pentamers N-linked carbohydrate Hinge region Disulfide bonds Structure Very similar Y shaped structure - All linked by disulfide bonds N-linked carbohydrate Different isotypes based on difference in the Heavy chain - termed using lower case Greek letters IgM and IgE don’t have hinge All monomers in the membrane bound form Soluble form - except IgA (monomers and dimers) and IgM (pentamers) Each of the segments represent a Ig Domain N-linked carbohydrate Hinge region Disulfide bonds Multimeric forms Monomers/dimers Immunoglobulin Isotypes or Classes

Figure 2-5 Immunoglobulin domain ~100 amino acids Each heavy or light chain made of 2 or more segments called domains - About 100 amino acids Very stable - withstand harsh conditions - pH, salt, some proteases Two types of Ig domains - variable and constant Variable - always at the N-terminal end of heavy or light chain - VL and VH together make the Antigen Binding Site Heavy chain of IgG has four domains 1 Variable and 3 constant (VH and CH1 etc) 3D structure show the three globular regions - 2 Fab and 1 Fc ~100 amino acids Two types - variable and constant Antigen Binding site - VH and VL VH of IgG - four domains

Figure 2-6 Anti-parallel B-strands Loop region

Hypervariable (CDR) Regions of Antibodies Compare many different Ab sequences and you find hot spots of variability in the the amino acid sequences - hypervariable regions (HV) Variability is measured by the ratio of number of amino acids found in a position over the frequency of the most common aa. CDR1, CDR2, and CDR3

110 AA light-chain V domain Amino Acid Sequence Variability in the V domain 110 AA light-chain V domain HV domains- hypervariable FR domains- framework Variability calculation

Mechanisms of Epitope Recognition Linear and discontinuous epitopes Multivalent Antigens Polymeric Antibodies Epitope binding mechanisms

Poliovirus VP1- blue, contains several epitopes (white) that can be recognized by humans Physical properties of Antigens Epitope - part of the antigen bound by Ab Mulivalent - antigen with more than one epitope, or more than one copy of an epitope Variety of structures and sizes recognized by Ab’s Linear vs Discontinuous epitopes Affinity - binding strength of an antibody for its epitope

Figure 2-9

Figure 2-26 part 1 of 2

Figure 2-30 Monomers disulfide bonded to the J-chain Dimeric in mucosal lymphoid tissue Monomeric made by B-cells in lymph nodes/spleen

Figure 2-8 Poliovirus VP1- blue, contains several epitopes (white) that can be recognized by humans Three different proteins VP1 - blue

Pockets Molecule DNA Grooves Extended surfaces Lysozyme Antibodies bind a Range of Structures Pockets Molecule DNA Grooves Extended surfaces Lysozyme Antibodies can bind to a wide range of chemical structures and proteins Allergies to small molecules e.g penicillin SLE - systemic lupus erythematosus

Examples of Problematic Ab binding to various structures Pocket: Penicillin - antibiotic that IgE can bind to and initiate inflammatory response Groove: DNA - Systemic Lupus Erythematosus (Lupus) Extended Surface: Lysozyme - Hen egg component -Drug side effect Autoimmune disease Egg allergies

Antibody Structure Summary Produced by B-cells Y shape, Four polypeptide chains, Ig domains Constant and Variable regions 5 classes - IgG, IgM, IgD, IgA, IgE CDR, Hypervariable regions Epitope recognition

Generation of Ig diversity in B cells Unique organization - Only B cells can express Ig protein - Gene segments k l-Light chain , , , ,  - Heavy Chain present on three chromosomes

The Gene Rearrangement Concept Germline configuration Gene segments need to be reassembled for expression Sequentially arrayed Occurs in the B-cells precursors in the bone marrow (soma) A source of diversity BEFORE exposure to antigen

Gene rearrangements during B-cell development V-variable, J-joining, D-diversity gene segments; L-leader sequences l - 30 V & 4 pairs J & C (light chain) chs22 k – 40 V & 5 J & 1 C (50% have 2x V) (light chain) chs2 H – 65 V & 27 D & 6 J chs14 Figure 2-14 Summary of all the gene segments 4 parts to make each chain V, J, D, and L

Figure 2-15 part 1 of 2 J-joining CDR1 and CDR2 CDR3 Reassembly process is called recombination - process where gene segments are cut and spliced together V-region of Light chain is encoded by the combination of one V and one J segment C region is encoded by one C gene Variability is in V gene segments is in the sequences encoding the first and second hypervariable regions - the third hypervariable region is encoded by the junction of V and J. CDR1 and CDR2 CDR3

Figure 2-15 part 2 of 2 D-diversity CDR1 and CDR2 CDR3 An additional gene segment needs to be reassembled to make a functional Heavy chain. - this is called the D segment Requires two recombination events CDR1 and CDR2 CDR3

* Only one light chain loci gives rise to one functional polypeptide! Random Recombination of Gene segments is one factor contributing to diversity (200* + 120*) X 10,530 = 3,369,600 Ig molecules * Only one light chain loci gives rise to one functional polypeptide!

Mechanism of Recombination Recombination signal sequences (RSSs) direct recombination V and J (L chain) V D J (H chain) RSS types consist of: - nonamer (9 base pairs) - heptamer (7 base pairs) - Spacer - Two types: [7-23-9] and [7-12-9] RSS features: - recognition sites for recombination enzymes - recombination occurs in the correct order (12/23 rule)

Mechanism of Somatic Recombination V(D)J recombinase: all the protein components that mediate the recombination steps RAG complex: Recombination Activating Genes (RAG-1 and RAG-2) encode RAG proteins only made in lymphocytes, plus other proteins Recombination only occurs through two different RSS bound by two RAG complexes (12/23 rule) DNA cleavage occurs to form a single stranded hairpin and a break at the heptamer sequences Enzymes that cut and repair the break introduce Junctional Diversity

Junctional Diversity Recombination +

Figure 2-19

Figure 2-18 part 2 of 3 Junctional Diversity Nucleotides introduced at recombination break in the coding joint corresponding to CDR3 of light and heavy chains - V and J of the light chain - (D and J) or (V and DJ) of the heavy chain P nucleotides generate short palindromic sequences N nucleotides are added randomly - these are not encoded Junctional Diversity contribute 3 x107 to overall diversity!

Generation of BCR (IgD and IgM) Rearrangement of VDJ of the heavy chain brings the gene’s promoter closer to C and C Both IgD and IgM are expressed simultaneously on the the surface of the B cell as BCR - ONLY isotypes to do this Alternative splicing of the primary transcript RNA generates IgD and IgM Naïve B cells are early stage B cells that have yet to see antigen and produce IgD and IgM

Alternative Splicing of Primary Transcript to generate IgM or IgD Figure 2-21

Summary Biosynthesis of IgM in B cells

Mature B cell Figure 2-23 Long cytoplasmic tails interact with intracellular signaling proteins Disulfide-linked Transmembrane proteins - invariant Dual-function help the assembled Ig reach the cell surface from the ER signal the B cell to divide and differentiate

Principle of Single Antigen Specificity Each B cell contains two copies of the Ig locus (Maternal and Paternal copies) Only one is allowed to successfully rearrange - Allelic Exclusion All Igs on the surface of a single B cell have identical specificity and differ only in their constant region Result: B cell monospecificity means that a response to a pathogen can be very specific

DNA hybridization of Ig genes can diagnose B-cell leukemias The control cells are mainly neutrophils and when the DNA is digested the V and C regions are on different pieces of DNA - patient is primarily clonal B cells (tumor) with a single type of rearrangement. Also, Demonstrates that the tumor cells are B cells.

Generation of B cell diversity in Ig’s before Antigen Encounter Random combination of V and J (L chain) and V, D, J (H chain) regions Junctional diversity caused by the addition of P and N nucleotides Combinatorial association of Light and Heavy chains (each functional light chain is found associated with a different functional heavy chain and vice versa)

Developmental stages of B cells 1. Development before antigen 2. Development after antigen Mature naive B cell (expressing BCR - IgM and IgD) Immature B cell plasma cell (expressing BCR and secretes Antibodies)

Processes occurring after B cells encounter antigen Processing of BCR versus Antibody Plasma cells switch to secreted Ab Difference occurs in the c-terminus of the heavy chain Primary transcript RNA is alternatively processed to yield transmembrane or secreted Ig’s Somatic Hypermutation 1. Point mutations introduced to V regions 2. 106 times higher mutation rate 3. Usually targets the CDR Affinity maturation - mutant Ig molecules with higher affinity are more likely to bind antigen and their B cells are preferentially selected Isotype switching

RNA processing to generate BCR or Antibody

Somatic Hypermutation

Isotype switching IgM is the first Ab that is secreted in the IR IgM is pentameric and each H chain can bind complement proteins Isotypes with better effector functions are produced by activated B cells Rearrangement of DNA using SWITCH regions - all C genes preceded by switch sequence (except  - start from the  gene and any other C gene (plus sequential) Regulated by cytokines secreted by T cells

Figure 2-31 part 2 of 2 Immunoglobulin classes C regions determine the class of antibody and their effector function Divided into Subclasses based on relative abundance in serum Each class has multiple functions

IgM and IgG can bind complement IgG crosses placenta Receptors for constant regions (Fc Receptors) - IgG (FcG receptors): mac, neutrophils, eosinophils, NK cells, others - IgE (FcE receptors): mast cells, basophils, others

Initial Immune Response mediated by IgM (plasma cells in lymph nodes, spleen, and bone marrow and circulate in blood/lymph) Low affinity binding to antigen via multiple binding sites Exposure of constant region Activate complement Hypermutation and affinity maturation Two binding sites sufficient for strong binding Phagocytose Kill directly Isotype switching to IgG

Higher affinity binding to antigen IgG Extravasation, Higher affinity binding to antigen IgG (lymph nodes, spleen, and bone marrow) Circulates in blood and lymph (most abundant Ab in internal fluids) Recruit phagocytes Multiple effector functions Neutralize antigens Activate complement

Plasma cells in lymph nodes, spleen, bone marrow Secreted into Blood Monomeric IgA Plasma cells in lymph nodes, spleen, bone marrow Secreted into Blood Effector functions Mainly neutralization Minor opsonization and activation of complement Dimeric IgA lymphoid tissue associated with mucosal surfaces Secreted into Gut lumen & body secretions

Plasma cells in lymph nodes or germinal centers IgE Plasma cells in lymph nodes or germinal centers Bind strongly to Mast cells Cross-linking of receptor bound Ab releases histamine and other activators Inflammation - Expulsion of large pathogens - Allergies IgE is produced by plasma cells located in lymph nodes draining the site of antigen entry or locally, at the sites of allergic reactions, by plasma cells derived from germinal centers developing within the inflamed tissue. IgE differs from other antibody isotypes in being located predominantly in tissues, where it is tightly bound to the mast-cell surface through the high-affinity IgE receptor known as Fc[var epsilon]RI.

IgM is the first antibody produced in an immune response against a pathogen made by plasma cells in the lymph nodes, spleen, and bone marrow. Initial immune response IgM will bind to antigen with low affinity which is why you need multiple binding sites - upon binding the sites are available to bind complement which

Figure 2-32 This last step is why a plasma cell cannot switch.

Summary: Generation of B-cell diversity Diversity before Antigen exposure (Antigen Independent) - Random Recombination - Junctional Diversity - Combinatorial association Diversity after Antigen exposure (Antigen Dependent) - Switch to secreted Ab - Somatic Hypermutation - Affinity Maturation - Isotype Switching Immunoglobulin Classes - Properties - Effector functions