immunology B cell effector mechanisms and antibody presentation

1. B Cell Effector Mechanisms And
Antibodies
Presenter: Martin M Ssejjoba
Modulator: Dr. Simon Kimuda

2. Presentation Outline
B Cell Overview
Antibody Structure
Classes of Antibodies
& Functions
B Cell Effector
Mechanisms
Polyclonal &
Monoclonal

3. B cell overview: What are B cells?
• B cell lineage was identified in
1965 (Cooper M.D et al 2015)
• B cells are adaptive immune
lymphocytes capable of
antibody production
• B cell receptor
• Cluster of differentiation
B cell development
Jeffrey L Bennet et al. 2015

4. Antibody structure: In History - 1939
• Tiselius and Kabat
• Immunized rabbits
• Two serum aliquots
• Electrophoresis technique
• Difference in the ɣ - globulin
peak
Kindt, T., Goldsby, R., Osborne, B., Kuby, J. and Kuby, J. (2007). Kuby immunology. New York: W.H.
Freeman.

5. Structure of the Antibody
Kindt, T., Goldsby, R., Osborne, B., Kuby, J. and Kuby, J. (2007). Kuby immunology. New York: W.H. Freeman.

6. Antibody Structure: Basic Description
• 4 polypeptide chains: Light (MW-25000) and Heavy (MW-50000)
• Bonds in the polypeptide chains – Heterodimer
• Non covalent bonds form a dimer of dimers
• Segment of varying amino acid sequence - Variable region; specificity
• Complementarity Determining Region (CDR)
• Segment of relatively constant amino acid sequence – Constant region
• Carbohydrate binding site
• Antibodies are glycoproteins

7. Antibody Structure: Revealed By
• Chemical and enzymatic
methods
Papain enzyme
Pepsin Enzyme
Mercaptoethanol
Kindt, T., Goldsby, R., Osborne, B., Kuby, J. and Kuby, J. (2007). Kuby immunology. New York: W.H. Freeman.

8. Antibody Structure: Sequencing
• Amino terminal – Variable region
• At the constant region of the
heavy chain
• Five heavy chain types were
deduced α, ϒ, δ, μ and ε -
isotypes
• Minor differences in the α and ϒ
– subisotypes
α1, α2
ϒ1, ϒ2, ϒ3 and ϒ4
• Variable region
• At the constant region - there
are 2 basic amino sequences
Kappa - 60%
Lamda - 40%
• Only one light chain is found in
an individual antibody
• Minor differences in the λ light
chains types lead to sub types
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9. Classes of Antibodies
Kindt, T., Goldsby, R., Osborne, B., Kuby, J. and Kuby, J. (2007). Kuby immunology. New York: W.H. Freeman.

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11. Antibody Structure: Determined By Protein
Organization
• Primary structure
Makes up the variable and
constant region
• Secondary structure
Anti parallel β-pleated sheets
• Tertiary structure
Compact with globular domains
• Quaternary structure
Functional domains

12. Antibodies: Membrane Bound versus Secreted
• Secreted form have a hydrophilic amino
acid sequence of various lengths at the
carboxyl-terminus
• Membrane bound antibodies contain
hydrophobic α-helical transmembrane
region and intracellular juxtamembrane
stretch which binds to the negatively
charged phospholipid head groups of
the inner plasma membrane

13. Antibodies: Antigenic Determinants
Allotypes
• Mutiple allele existence on a
gene
• IgA allotypes in IgA2 subclass
• Anti-allotypic antibody
Injection
Pregnancy
Blood transfusion
Adiotypes
• Found at the VH & VL
• Idiotopes summate to idiotype
of the antibody
• Anti-idiotypic antibody
Injection

14. Ig Superfamily
• Shared features in immunoglobulins suggest common ancestry
• Proteins whose corresponding genes derived from a primordial gene
encoding a basic domain structure
• Immunoglobulin fold domain – shared feature
• Gene duplication and diversion led to various heavy and light chain
genes
• Members of the Ig Superfamily include;
Platelet derived factor
Cell adhesion molecules
T cell receptor
T cell accessory proteins

15. Classes of antibodies: Functions
Antibody Function
IgG
• Binds to Fc receptors of phagocytes to enable phagocytosis
• Activates complement pathway (Unlike IgG4)
IgA
Serves an important effector function at mucous membrane surfaces,
which are the main entry sites for most pathogenic organisms
IgE
IgE antibodies mediate the immediate hypersensitivity reactions that are
responsible for the symptoms of hay fever, asthma, hives, and anaphylactic
shock
IgM
• First immunoglobulin class produced in a primary response to an
antigen, and it is also the first immunoglobulin to be synthesized by the
neonate
• Best complement activator
IgD No biological effector function has been identified for IgD

16. B Cell Effector Mechanism
Complement Activation
Neutralization
Opsonization
Antibody Dependent Cell
Mediated Cytotoxicity

17. The Complement System
• Serum at 37 degrees & 56 degrees
• Complement proteins are inactive plasma proteins
• Activated by particular conditions to generate products
• Activation of complement involves a proteolytic cascade
• To remain stably active - Products of complement activation become
covalently attached to;
Microbial cell surfaces
Antibodies bound to microbes
Other antigens
Apoptotic bodies
• Inhibition of complement activation – regulatory proteins

18. Complement
Activation
Abul K. Abbas, MBBS, Andrew
H, Lichtman, MD, PhD, and Shiv
Pillai. Cellular and Molecular
Immunology, 8th Edition. 2015

19. Complement system: The Products
• C3b binds to pathogens – opsonization
• C5a is an important chemotactic protein helping to recruit
inflammatory cells
• C3a and C5a have anaphylatoxin activity, directly triggering
degranulation of mast cells, increasing vascular permeability and
smooth muscle contraction
• C5b initiates formation of the MAC a cytolytic end product of the
complement system

21. Antibody Dependent Cell Mediated
Cytotoxicity (ADCC)
• Cells involved in ADCC - non
specific
• FC receptor
• Mechanisms employed by ADCC
cells on activation
• ADCC invitro
• Measles virus
• Cell-mediated killing of helminths
Kindt, T., Goldsby, R., Osborne, B., Kuby, J. and Kuby, J. (2007). Kuby
immunology. New York: W.H. Freeman.

22. Neutralization
Explained
Abul K. Abbas, MBBS, Andrew H, Lichtman, MD, PhD, and Shiv Pillai. Cellular
and Molecular Immunology, 8th Edition. 2015

23. Polyclonal & Monoclonal Antibodies
• Polyclonal antibodies
Arise from multiple B cell clones
Have a heterogeneous collection of binding sites
• A monoclonal antibody
Derived from a single B cell clone
Is a homogeneous collection of binding sites

24. Polyclonal Vs Monoclonal Antibodies
Kindt, T., Goldsby, R., Osborne, B., Kuby, J. and Kuby, J. (2007). Kuby immunology. New York: W.H. Freeman.

25. Hybridoma Technology: In history
• Production of monoclonal
antibodies was invented by César
Milstein and Georges J. F. Köhler in
1975
• Nobel prize recipients - 1984

26. Monoclonal Antibodies: Clinical Application
• Antibody diagnosing reagents; detecting pregnancy, pathogenic
microorganisms diagnosis, drug blood level measurement etc.
• Monoclonal imaging techniques; Radiolabeled monoclonal
antibodies can be used invivo for detecting or locating tumor antigens
• Immunotoxin therapeutic agents; tumor-specific monoclonal
antibodies coupled to lethal toxins are valuable therapeutic reagents
Kindt, T., Goldsby, R., Osborne, B., Kuby, J. and Kuby, J. (2007). Kuby immunology. New York: W.H. Freeman.

27. Monoclonal vs polyclonal: Comparison
Comparison Polyclonal Monoclonal
Cost of production Inexpensive More expensive
Time taken for production Short Long
Specificity of antibodies
generated
Generate large amounts of
non-specific antibodies
Generate large amounts of
specific antibodies
Epitope recognition
Recognize multiple
epitopes on any one
antigen therefore higher
potential for cross
reactivity
Recognize only a specific
epitope on an antigen
therefore reducing the
probability of cross
reactivity
Stability
Highly stable and tolerant
to pH and buffer changes
Sensitive to pH and buffer
changes

28. Take Home Points
Serology the study of antibody immune response to antigenic
substances
Antibody structure is directly linked to antibody antigenic and
biological functions
B cell effector mechanisms: B cellular immune responses that are
utilized to combat evading pathogens
Hybridoma technology: For the production of monoclonal antibodies

29. Further Reading
• Kindt, T., Goldsby, R., Osborne, B., Kuby, J. and Kuby, J. (2007). Kuby
immunology. New York: W.H. Freeman.
• Abul K. Abbas, MBBS, Andrew H, Lichtman, MD, PhD, and Shiv Pillai.
Cellular and Molecular Immunology, 8th Edition. 2015

30. Thank you for
listening