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B cell analysis

1. B cell development and Antibody diversification MLT 371

2. Learning Objectives To know the developmental stages of B lymphocytes. To know the function of B lymphocytes To know the different types of antibodies and their functions To know the different structures of Antibodies. To know the theories surrounding Antibody diversification.

3. Introduction to B cell B lymphocytes formed from and name after their site of Origin Bone marrow in Humans and Bursa of Fabricius in birds. They form the basis of humoral immunity by the production of antibodies. Belongs to the adaptive immunity. A cell from lymphoid lineage. Express the B cell receptor (BCR).

4. B Cell Development B cell development begin in the Bone marrow and is completed in the peripheral lymphoid organs (Spleen). They leave the bone marrow into the periphery as immature B cells where complete their maturation as mature B cell. They start of as stem cells, then differentiate to Pro-B cell, Pre-B cell, Immature B cell and finally mature B cells. There are two genes known as Recombination Activating Genes (RAG1 AND RAG2) that play roles in the diversity of the Immunoglobulin Receptor on the B cell.

5. B cell development Recombination of Immunoglobulin receptor genes starts at the Pre- B stage through a process known as somatic recombination. B cells at the immature stage express IgM and at the mature stage expresses IgM and IgD . The Stem cell to the Pre-B cell developmental stages occurs in the bone marrow, whereas at the immature stage B cells leave the bone marrow into the periphery. Negative selection (Deletion/ receptor editing) occurs at the Immature B cell stage.

6. Antibody Production and diversification Antibody production occurs at the activation phase after encountering an antigen and subsequently binding to it at periphery. B cells differentiate to effector cell known as the Plasma cell to produce Antibodies. There are five classes of Antibodies/Immunoglobulin namely IgG, IgA, IgM, IgE and IgD . Each of Class of antibody has specific function that are different from each other.

7. Immunoglobulin/ Antibody structure Antibodies (Ab) are also known as Immunoglobulin (Ig) are Y shaped proteins that are produced by plasma cells to detect foreign substances or antigens such as bacteria and viruses. There are four polypeptide chains: two identical heavy chains and two identical light chains connected by disulfide bonds.  There are five types of Ig heavy chain (in mammal) denoted by the Greek letters: α, δ, ε, γ, and μ. There are two types of Ig Light chain in mammals, namely Lamda (λ) and Kappa (κ).

8. Immunoglobulin/ Antibody structure Ig is made of a variable region (Fab ), and a constant region (Fc) The variable regions varies depending on the different antigen types. The constant region remain constant and does not change Each heavy and light chain in an immunoglobulin molecule contains an amino-terminal variable (V) region (Fab) that consists of 100 to 110 amino acids and differ from one antibody to another.

9. Immunoglobulin/ Antibody structure The remainder of each chain in the molecule – the constant (C) region exhibits limited variation that defines the two light chain subtypes and the five heavy chains subclasses. The amino terminal portions, corresponding to the V regions, bind to antigen; effector functions are mediated by the carboxy-terminal domains (Fc region).

10. Immunoglobulin/ Antibody structure

11. Antibody Classes The Five types of antibodies- IgA, IgG, IgE , IgD , IgM are classified according to the type of heavy chain within the constant region. Structurally IgG, IgD and IgE exist as monomers whilst IgA and IgM exist as dimers and Pentamers respectively. Serum containing antigen-specific antibodies is called antiserum.

12. IgG IgG is the principal immunoglobulin of the secondary response. They are mostly found in adults who have been consistently exposed to foreign antigens. They constitute 80% of the circulating Immunoglobulin in the adult. They have a monomeric structure, and Heavy chain ( γ ). IgG provides long term protection because it persists for months and years after the presence of the antigen that has triggered their production. IgG protect against bacteria viruses, neutralize bacterial toxins, trigger compliment protein systems and bind antigens to enhance the effectiveness of phagocytosis. They provide passive immunity to developing fetus.

13. IgG subclasses IgG can be subdivided into IgG1, IgG2, IgG3 and IgG4 depending on the differences in their constant region. IgG1 is the most abundant and most subclass. IgG1 is important for mediating antibody responses against viral pathogens . IgG2 plays a role in protection against protein antigens but is predominantly responsible for IgG responses against bacterial capsular polysaccharides.

14. IgG Subclasses IgG3 protects against a range of intracellular bacteria, parasites, and viruses. IgG4 antibodies are often formed following repeated or long-term exposure to antigen in a non-infectious setting and may become the dominant subclass. Eg. Allergens Helminth or filarial parasite infections may result in the formation of IgG4 antibodies and high IgG4 titers can be associated with an asymptomatic infection.

15. Major Function of IgG Providing immunity for the newborn because IgG is the only antibody that can cross the placenta. • Fixing complement. • Coating antigen for enhanced phagocytosis (opsonization). • Neutralizing toxins and viruses. • Participating in agglutination and precipitation reactions.

16. Secretory IgA They are dimeric in structure. There are two subclasses, designated IgA1 and IgA2. IgA2 is the predominant form in secretions at mucosal surfaces, whereas IgA1 is mainly found in serum. They (IgA2) exist in secretions such as saliva, breastmilk (Lactation), mucosal lining of GI tract, and sweat. They are the most abundant antibody in the body. They play an important role in the protection and homeostatic regulation of intestinal, respiratory, and urogenital mucosal epithelia.

17. Secretory IgA

18. Functions Secretory IgA The main function of secretory IgA is to patrol mucosal surfaces and act as a first line of defense. It plays an important role in neutralizing toxins produced by microorganisms and helps to prevent bacterial and viral adherence to mucosal surfaces. Complexes of IgA and antigen are easily trapped in mucus and then eliminated by the ciliated epithelial cells of the respiratory or intestinal tract.

19. IgM It can exist both as a pentamer or monomer. They express the μ heavy chain IgM is the first immunoglobulin to be synthesized in a primary response, it is the first to appear after antigenic stimulation and the first to appear in the maturing infant. It is synthesized only as long as antigen remains present because there are no memory cells for IgM . Expressed as a receptor on the surface B cell. Contributes to agglutination and effective complement activation.

20. Functions of IgM Complement fixation Agglutination Opsonization Toxin neutralization. IgM is the most efficient of all immunoglobulins at triggering the classical complement pathway, because a single molecule can initiate the reaction as a result of its multiple binding sites.

21. IgM

22. IgD They exist as a monomer. They have the (δ) Heavy chain. They are rare. They are the Ig expressed as receptors on the surface of B cells. Cells bearing only IgM receptors appear incapable of an IgG response, whereas those with both IgM and IgD receptors are capable of responding to T-cell help and switching to synthesis of IgG, IgA, or IgE . They don’t have any protective function. They have short life span.

23. Structure of IgD

24. IgE They are extremely rare. It is the least abundant immunoglobulin in the serum. They activate mast cells and basophils. They have the (ε) or H chain. After synthesis it attaches to basophils, Langerhans cells, eosinophils, and tissue mast cells by means of specific surface proteins, termed high-affinity FC ε RI receptors. Plasma cells that produce IgE are located primarily in the lungs and in the skin

25. IgE IgE functions against parasites. In the absence of parasites, IgE responds to allergens. They react with neutrophils and eosinophils during acute inflammation to help destroy invading antigens that have penetrated IgA defenses.

26. Theories of Antibody Diversity Ehrlich’s Side-Chain Theory : Ehrlich postulated that certain cells had specific surface receptors for antigen that were present before contact with antigen occurred. Once antigen was introduced, it would select the cell with the proper receptors, combination would take place, and then receptors would break off and enter the circulation as antibody molecules. New receptors would form in place of those broken off, after which this process could be repeated. Clonal Selection Hypothesis: states that individual lymphocytes are genetically preprogrammed to produce one type of immunoglobulin and that a specific antigen finds or selects those particular cells capable of responding to it, causing them to proliferate.

27. Terminologies Class switching , process whereby daughter plasma cells can produce antibody of another type. Monoclonal antibodies are Ab derived from a single parent antibody-producing cell that has reproduced many times, thus forming a clone. The five different types of heavy chains are called isotypes . Minor variations in a particular type of heavy chain are called allotypes. The variable portion of the light and heavy chains unique to a particular immunoglobulin molecule is known as the idiotype

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