1. Figures replaced or skipped for Ch 2
Figure Enzyme digestion of Ab’s
Flow cytometry - Fig 2.13
Monoclonal Ab Fig 2.12 was replaced

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
Humoral Immunity - immunity dominated by antibodies that can be transferred to another person
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

4. Antibody structure
Fab Fc
Heavy (5 classes), Light (2 classes), Constant (C) and Variable (V)
Disulfide bonds link heavy chains
Fab- fragment antigen binding
Fc- fragment crystallizable

5.      Immunoglobulin Isotypes or Classes pentamers
N-linked carbohydrate
Hinge region
Disulfide bonds
Monomer as BCR
Multimeric forms
k l-Light chain
Ig domain  
Monomers or dimers

6. Figure 2-5 Immunoglobulin (Ig) domain
~100 amino acid domain - very stable
Two types Ig domains- variable and constant
Antigen Binding site - VH and VL
Heavy chain of IgG - four domains - VH, CH1, CH2, CH3

7. Figure 2-6 Anti-parallel -strands - contribute to the structural part
Loop region - in the V domain contribute to variability

8. Hypervariable Regions (CDR) of Antibodies
HV - hypervariable regions
CDR - complementarity determining regions
Framework region -  strand region that has reduced variability

9. Amino Acid Sequence Variability in the V domain
110 amino acid light-chain V domain
Framework regions can have variability but variability is much higher in the HV regions

10. Physical Properties of Antigens-Antibody Binding
Epitope - part of the antigen bound by Ab
Antigen is usually carbohydrate or protein
Lock and Key concept
Variety of structures and sizes recognized by Ab’s
Affinity vs Avidity - terms describing binding strength of an antibody for its epitope

11. Epitopes
Epitope (antigenic determinant) is the part of the antigen bound by Ab
Most antigens have multiple epitopes (multivalent)
Usually carbohydrate or peptide.
Fig. 2.9

12. Mechanisms of Epitope Recognition
Linear and discontinuous epitopes
Multivalent Antigens
Polymeric Antibodies
Affinity vs Avidity - terms describing binding strength of an antibody for its epitope
Epitope binding mechanisms

14. Figure 2-9

15. Figure 2-26 part 1 of 2
First Ab to be made during during an immune response to an antigen
Monomers disulfide bonded together via the J-chain (joining chain)

16. Figure 2-30 Monomers disulfide bonded to the J-chain
Dimeric in mucosal lymphoid tissue - secreted into the gut to prevent pathogens binding to gut cells and can act as an antitoxin
Monomeric made by B-cells in lymph nodes/spleen and is not J-chain dependent

17. Antibody-antigen interaction
Non-covalent binding:
Electrostatic
Hydrogen bonds
Van der Waals force
Hydrophobic forces
Affinity: Strength of interaction between epitope and one antigen-binding site
Avidity: Strength of the sum of interactions between antibody and antigen

18. Figure 2-8
Poliovirus
VP1- blue, contains several epitopes (white) that can be recognized by human antibodies

19. Pockets Grooves Extended surfaces Molecule DNA Lysozyme
Antibodies bind a Range of Structures
Pockets
Grooves
Extended surfaces
Molecule
DNA
Lysozyme

20. Examples of Problematic Ab binding to various structures
Pocket:
Penicillin - can bind to RBC surface proteins to create a foreign epitope. IgE binds to drug-RBC protein complex and initiates an inflammatory response
Groove:
DNA - Systemic Lupus Erythematosus (Lupus) - autoimmune disease in which antibodies are made against DNA and other molecules leading to inflammatory reactions in joints, skin and kidney
Extended Surface:
Lysozyme/Ovalbumin - allergies to hen egg components - more common in children under 5 - “desensitization” protocol can help

21. Haptens Small molecules that are not immunogenic by
themselves, but can bind immunoglobulins or TCRs.
Haptens can induce an immune response when linked to a larger protein.

22. Polyclonal vs Monoclonal Antibodies1 Ag 4 2
Isolate B-cells
Spleen
Myeloma
cells
Hybridoma
Cells 3 1 2 1 2 3 4 + 4 3 1 2 3 4
Isolate serum
Ab-1
Ab-2
Ab-3
Ab-4
Polyclonal
antibodies 1 Ag 4 2
Monoclonal
antibodies
Ab-1, Ab-2
Ab-3, Ab-4 3

23. Crossreactivity
Occurs when an Antiserum is raised against antigen A but also reacts with antigen B
Antigen A and B share epitopes
Antigen A and B have similar
(but not identical) epitopes

24. 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

25. Examples of Commercial uses for Ab’s
Pregnancy test
Rh disease therapy
Antitoxin serum - Antivenin, rabies, etc
Anti-cancer monoclonals - will be covered later in the course

26. Pregnancy test Pregnancy window Control window Input window Reaction
zone
Urine
Mouse Monoclonal
anti-HCG Ab-enzyme conjugate
Immobile Polyclonal
anti-HCG Ab
+ dye substrate
Immobile Goat Anti-mouse Ab
+ dye substrate

27. Rh disease
Rh disease (Erythroblastosis Fetalis) - Hemolytic disease of a newborn - occurs in Rh- woman carrying Rh+ fetus
RhoGAM - human plasma with anti-Rh+ (D antigen) antibodies. Works by binding any fetal RBC’s before the mother is able to produce an immune response and form anti-D IgG

28. Antitoxin serum
Serum can be used in the prophylaxis and/or treatment of rabies, botulism, diphtheria, gas gangrene, snake and spider bites
Antivenin Crotalidae Polyvalent (ACP) - horse serum based antivenom - gold standard for snake bites but can cause “serum sickness”
New types of antitoxins
Fragmented antivenin (CroFab) where only the Fab fragment (sheep-based) is used
Humanized antibodies derived from mouse sources

29. 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

30. 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

31. V-variable, J-joining, D-diversity gene segments; L-leader sequences
Light Chain
Variable - V, J
Constant - C
Heavy Chain
Variable - V, D, J

32. 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

33. V-region of light chain consists of one V and one J segment
C-region is encoded by one C segment
Variability comes from the V segment
CDR1 and CDR2
CDR3

34. D-diversity CDR1 and CDR2 CDR3
Two recombination events needed to combine 3 segments to make the Variable region

35. * 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!

36. 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: [7-12-9], [7-23-9]
RSS features:
- recognition sites for recombination enzymes
- recombination occurs in the correct order
(12/23 rule)

38. 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
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

39. Junctional
Diversity
Recombination +

40. Figure 2-19

41. 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 contributes 3 x107 to overall diversity!

42. 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

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

44. Summary Biosynthesis of IgM in B cells

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

46. 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 l k H * *
Chromosome 22 2 14

47. DNA hybridization of Ig genes can diagnose B-cell leukemias
Peripheral blood from healthy patient is made up of mostly neutrophils
Peripheral blood from a leukemia patient has an abnormally high proportion of B-cells.
Cancer cells are derived from one clonal line of B-cell which has V and C chains rearranged next to each other

48. 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)

49. Concept of Combinatorial Association

50. 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)

51. 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

52. RNA processing to generate BCR or Antibody
MC - membrane coding SC - secretion coding

53. Somatic Hypermutation (random introduction of point mutations)
Mutations occur throughout the V domain - especially CDR
Occurs on both gene copies - but only one expresses protein
AID - Activation induced Cytidine Deaminase
UNG - Uracil-DNA glycosylase

54. Process of Affinity Maturation
Hypermutation leads to different B-cells
Mutant BCRs have various affinities
Higher affinity BCR’s are preferentially selected to mature
IgM
Hypermutation
IgG
IgM

55. 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

56. Switch regions flank each C gene (except delta)
Mu to any other isotype
Sequential switching
AID is important
AID deficiency ---> Hyper IgM syndrome (antibodies not made after IgM and IgD )

58. Stages at which Isotype Switching occurs

59. IgM IgG IgE IgA antigen-independent pre-B cell immature B cell
stem cell
(IgM +)
(IgM +
, IgD +)
antigen-dependent
IgM
isotype switching
IgG
IgE
IgA

60. Does Isotype Switching occur in one B cell?
Activated B cell resides in the Germinal Center
-some individuals will mature directly into plasma cells
Some B cells in the germinal center divide and undergo hypermutation and/or isotype switching
After this stage they cannot divide and the higher affinity ones are selected
These cells can mature to plasma cells
End result: The B cell makes a different antibody isotype but with the same specificity

61. 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

62. 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

63. 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

64. 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

65. 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
IgA most made of any Ab

66. Plasma cells in lymph nodes or germinal centers
IgE
Plasma cells in lymph nodes or germinal centers
Bind strongly to Mast cells via Fc receptor
Cross-linking of receptor bound Ab releases histamine and other activators
Inflammation
- Expulsion of large pathogens
- Allergies

67. IgD
Very low concentration in serum
Primarily found with IgM on naïve mature B cells
Function is not clear

69. Figure 2-32

70. 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