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

2. 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

3. 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

4. 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)

5.      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

6. 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

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

8. 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

9. 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

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

11. 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

12. Figure 2-9

13. Figure 2-26 part 1 of 2

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

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

16. 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

17. 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

18. 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

19. 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

20. 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

21. 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

22. 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

23. 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

24. * 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!

25. 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)

26. 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

27. Junctional
Diversity
Recombination +

28. Figure 2-19

29. 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!

30. 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

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

32. Summary Biosynthesis of IgM in B cells

33. 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

34. 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

35. 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.

36. 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)

37. 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)

38. 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
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

39. RNA processing to generate BCR or Antibody

40. Somatic Hypermutation

41. 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

43. 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

44. 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

45. 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

46. 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

47. 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

48. 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.

49. 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

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

51. 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