Antibody Diversity Immunoglobulin antibody Antibody response B cells

Antibody Diversity

Immunoglobulin: antibody • Antibody response: B cells, with the help of T cells, produce antibody to antigen, preserve the ability to produce antibody to recognize the same antigen in “memory” and can differentiate to professional secretory plasma cells to produce antibody • Antigen (antibody generator): any substance that can elicit an adaptive immune response • Lymphocytes: white blood cells responsible for adaptives immune response. Two classes: B cells, T cells

Primary immune response and secondary response • When an animal is injected with antigen, Ig. M is first produced against that antigen, then Ig. G will appear. The antibody titer then subsides. • Upon the second injection of antigen, the secondary response is at a higher level of titer, typically dominated b Ig. G even though there is still some Ig. M (possibly from newly immunized B cells). • The process thus involves a antibody class switch. The secondary response is from the memory cells which have been exposed to antigen in the primary response.

Primary immune response and secondary response

Antigen determinants • Antigen determinants (called epitopes) are the partsof the antigen molecule that bind to the antibodymolecule or the receptor on the surface of thelymphocytes. • Most large molecules have many determinants • When an animal responds to an antigen, pool antibodies are produced, recognizing different determinants of the antigen. Each subpool of antibodies recognizing the same antigen is produced by a B cell clone

Antibody Diversity • Human preimmune repertoire needs a capacity of ~ 1015 to cope with antigens encountered in life time. • Human genome has ~ 105 genes, not enough to code for all in preimmune repertoire.

Clonal selection • – Each B cell makes only one antibody and is a clone (each cell has ~105 immunoglobulin molecules on the surface). • The binding of antigen to the receptor on the surface of naïve cell (when the cell is in the peripheral lymphoid organ)activates the B cell clone to develop into effector cells and to proliferate

Clonal selection

Clonal selection • After the first exposure, some become memory cells. • Both memory and naïve B cells are activated by antigen binding. • Memory cells respond differently than naïve cells, resulting in a higher antibody titer

Clonal selection

Immunoglobulins or Antibodies • Classes of antibodies (differ in their heavy chain) • 5 classes of heavy chain Ig. A, Ig. D, Ig. E, Ig. G, Ig. M with α, δ, ε, γ, μ heavy chain respectively Ig. G, Ig. A have subclasses (Ig. G 1, Ig. G 3, Ig. G 4 with γ 1, γ 2, γ 3, γ 4 respectively). • 2 light chain κ or λ • Constant region and variable region • Both heavy chain and light chain have a constant region and a variable region. • Three hypervariable section are the loci which are really variable in the variable region of heavy and light chains, the rest of “framework” in the variable region is relatively invariant.

Immunoglobulins or Antibodies Three hypervariable regions

Immunoglobulins or Antibodies

• Ig. M has no hinge region • Major class in early primary antibody response • Also on naïve B cell and Pre-B cell surface

Ig. A: major class in secretion, saliva, tears, milk, intestines, lungs

Ig. E and histamine secretion by Mast cells

Ig. E and histamine secretion by Mast cells • Ig. E secreted by B cells is bound to Fc receptors on Mast cells • Each Mast Cell has many different Ig. Es recognizing different antigens • Antigen binding to Ig. E and cross linking causes Mast cells to secrete histamine by exocytosis

B cells initially expression Ig. M, later most undergo a class switch

L and H chain antibody molecules are each made of repeating immunoglobulin domains

These domains form repeating units on chromosomes (Constant region of a heavy chain)

Antibody diversity is generated by a number of mechanisms Combinational recombination Mammal recombine immunoglobulin gene segments before transcription. • Each chain (λ, κ, and heavy chains) has a pool of gene segments. • • Each pool is on a different chromosome. i. e. on one chromosome. • There a large pool of segments for variable region and a smaller pool of constantregion. On of those segments in each pool is selected to be included in the final make upof immunoglobulin. • During B cell development, a coding sequence is assembled by site specificrecombination to increase the diversity. An antibody Ig. D with a К light chain thus has 7 x 106 possible combination.

Each variable region of a light chain is made of a V and a J gene segment

Human heavy chain gene segment pool • Each variable region of a heavy chain is madeof a, V, J, D segment

Imprecise V(D)J joining • Only appropriate segments get joined (V always joins to J in a light chain, not V to V). It is site specific. • Catalysed by RAG-1 and RAG-2 (recombination activating genes) • During recombination, nucleotides can be lost or inserted (may shift reading frame) • Can have a second round of V(D)J rearrangement (receptor editing)

Affinity maturation by somatic hypermutation • During the course of immunization, with the passage of time, there is an increase in the affinity of antibody to antigen. This is caused by somatic hypermutation (only in V region in heavy and light chain). Point mutation rateincreases, about – 1 mutation/v region/cell generation • The DNA rearrangement has occurred in bone marrow in early B cell development. Those B cells with a high affinity to Ig molecules will be selected for, others will die by apoptosis. This happens when B cells encounter antigen and T-helper cells.

Antibody diversification

Allelic exclusion ensuring specificity • Each B cell clone produces only antibody against one antigen. The dimmeric antibody doesn’t carry two different antigen recognition sites on two different monomers – Ensuring cross-linking of antigens for aggregation and elimination – Ensuring no “co-stimulation” of non-specific antigen binding due to the stimulation of the specific antigen binding on the other monomer • However, there are two sets of chromosomes in each B cells – Will use only one each of the light chain and heavy chain genes

Alternative splicing and switching from membrane bound to secreted antibodies • For clonal expansion, the surface bound Ig. M plays a crucial role, but later B cells must be switched to produce other classes and/or secreted antibodies • Change from membrane bound Ig. M to secreted Ig. M involves changes in C-Terminus in CH region. Alternative splicing generates a • hydrophobic C-terminus of heavy chain for membrane bound form, but a hydrophilic C terminus for secreted form.

Class switching via class switch recombination • The mature naïve B cells express Ig. M and Ig. D on their surface • On primary response, responding to antigen and T helper cells, some activated to secrete Ig. M, the dominating antibody in primay response. They generate both. • Later, in the immuno response, many B cells switch to produce Ig. G, Ig. A, and Ig. E. They generate both memory cells (with surface bound Ig. G, Ig. E or Ig. A) and effector cells, which secrete antibodies • The secondary response is dominated by Ig. G, Ig. E, Ig. A secreting cells • The class switch recombination eliminates other C regions of heavy chain, retains only the right class • It does not affect coding sequences for VH • It allows more types of antibodies to be produced with the same antigen recognition.

Class switching via class switch recombination