Lecture 14: Antibody class swapping and Memory

Detailed Antibody Structure

• Light and heavy chains are composed of repeating Ig domains.
• The N-terminal domains of both chains are called ‘variable’ (V) domains.
• The remaining domains are ‘constant’ (C) domains.
  • LC: VL-CL
  • HC: VH-CH1-hinge-CH2-CH3-(CH4) Typical IgG1

IgM Structure

• IgM: $(VH-CH1-h-CH2-CH3-CH4)5+J$
• Contains multiple disulfide bonds and is heavily N-glycosylated.
• N-glycans are complex carbohydrates added to asparagine residues during folding before secretion.
• N-glycans are large and hold domains apart, exposing functional motifs (e.g., complement binding sites).

IgG Structure

• IgG: $VH-CH1-h-CH2-CH3$
• Different domains have different functions:
  • Cγ1, Cγ2: bind complement components
  • Cγ2, Cγ3: bind Fc receptors on neutrophils
  • Cγ3: binds Fc receptor on macrophages and NK cells

IgE Structure

• IgE: $VH-CH1-h-CH2-CH3-CH4$
• Multiple N-glycans make it a stiff, rigid molecule.
• Good for targeting large pathogens but cannot cross-link small targets.

IgA Structure

• IgA: $(VH-CH1-h-CH2-CH3)2+J+S$
• Flexible, good cross-linker, valency = 4.

Class Switching

• Pre-B cells in the bone marrow express membrane-bound IgM.
• During maturation, they express both IgM and IgD (membrane-bound) in lymphoid tissue.
• IgM+ IgD+ B cells are selected by antigen and undergo clonal selection.
• Mature B cells can switch classes from IgM to other Ig classes while maintaining the same specificity for the antigen.
• Requires the same VH domain on a different heavy chain.

H Chain Gene Arrangement

• The Ig H chain gene encodes a variable (VH) domain and all the H chain constant regions, separated by non-coding introns.
  • $VH Cμ Cδ Cγ Cε Cα$

Somatic Recombination

• Class switching occurs through somatic recombination of DNA.
• Intervening DNA is excised to allow expression of VH with Cγ, Cε, or Cα.
• Genomic DNA is looped, and recombination occurs between switch regions.
• Requires specialized sets of proteins.

DNA Cutting and Rejoining

• Cutting and rejoining of DNA results in excision of the loop and class switching.
• For example, a class switch from IgM to IgA.
  • $Cμ Cδ Cγ Cε$

Expression after Class Switching

• Expression relies on removal of the intron from the mRNA.
• The rearranged gene is transcribed to generate a primary transcript.
• The segments encoding VH and Cα are fused in frame at the RNA level by excision of the intron to generate the mRNA.
• In this case, this mRNA is transcribed to make an IgA heavy chain with the same specificity as the original IgM.

Primary Response and Class Switching

• During the primary response, antigen stimulates clonal expansion of B and T cells that have receptors that already recognize the antigen.
• Mature B cells produce secreted IgM.
• They can switch antibody heavy chain classes by somatic recombination, maintaining their VARIABLE domains and original specificity.
• The same mechanism is used for:
  • IgM → IgG
  • IgM → IgE
  • IgM → IgA

Pre-Existing Diversity

• The clonal selection theory: An antigen activates only those lymphocytes already committed to respond.
• Lymphocytes committed to an antigen display cell surface receptors that specifically recognize the antigen, even if that antigen has never been encountered before.
• Receptors: TCR and BCR (membrane-bound antibodies).
• Millions of different clones of lymphocytes in the human immune system.
• Upon encountering antigen, lymphocytes undergo clonal expansion and differentiation.

Clonal Selection and Expansion

• Clonal selection: Individual clones are selected by antigen based on how well the antigen and the receptor fit together.
• Clonal expansion: The selected clones undergo mitosis, proliferate, and differentiate into effector cells.
• Clonal deletion: Lymphocytes that react inappropriately with ‘self’ antigens are destroyed.

Somatic Gene Recombination

• Antigen-specific receptors (TCR and membrane-bound antibodies) are encoded by unusual segmented genes.
• These genes are assembled from a series of gene segments by somatic gene recombination.

Antibody Genes

• There are only 3 antibody genes.
• There are two classes of light chains, which increases diversity.
  • $VH Cμ Cδ Cγ Cε Cα$
  • $Vλ C$
  • $Vκ C$

Two LC Classes

• The same VH domain can be partnered with variable domains from two classes of light chain, increasing the repertoire of possible binding sites.

Multiple Gene Segments

• There are multiple gene segments encoding V domains that can be combined with C domains by somatic recombination.
  • $V1 V2 V3 V~65$
  • $V1 V2 V3 V~30$
  • $V1 V2 V3 V~40$

V(D)J Recombination

• Somatic recombination (V(D)J recombination) involves selection of small pieces of ‘diversity’ and ‘joining’ DNA.
• 3 genes (1H, 2L) can generate > $10^{14}$ proteins with unique potential antigen binding sites.
• There are only $10^{12}$ B cells in a human, so the repertoire is greater than can be carried by the known number of B cells.

Affinity Maturation

• Antibodies made by B cells improve in affinity and become more specific over time.
• This process is called affinity maturation; the cause is the accumulation of point mutations in the V domains.
• Occurs in the lymph nodes.
• Mutation rate in germinal centers is about 1 million times greater than the spontaneous mutation rate in other genes.
• Confined to the gene segments that encode V domains, often referred to as somatic hypermutation.

Affinity Maturation Steps

1. Antigen stimulation causes activation and clonal expansion of B cells.
2. Some B cells proliferate in germinal centers and undergo somatic hypermutation.
3. Most hypermutated clones are worse than the original and will die, but rare B cells with higher affinity for the original antigen will proliferate.

• Darwinian process: survival by selective advantage.

Memory

• Exposure to antigen results in a primary response that appears after a few days, rises rapidly, and then declines gradually.
• Second exposure results in a greater and more efficient secondary response with a short lag period.
• The secondary response is greater and more specific because it is dominated by class-switched antibodies that have undergone somatic hypermutation.

Immunological Memory

• Memory is generated by the primary response.
• Naïve T and B cells differentiate into effector cells.
• Some antigen-stimulated cells multiply and differentiate into memory cells (both B and T lineages do this).
• Memory cells do not perform immunological functions but can be induced to become effectors by subsequent antigenic stimulation.
• Most effectors die after an immune response; memory cells do not.

Memory T Cells

• Multiple classes of memory T cells exist.
• Some carry cell-surface markers characteristic of TH cells; others carry cell-surface markers characteristic of TC cells.
• Memory T cells migrate to tissues.

Memory B Cells

• B cells that can respond to antigen increase in frequency after priming (10- to 100-fold) and produce antibody of higher average affinity.
• The secondary antibody response antibodies are produced by memory B cells that have already switched from IgM to more mature isotypes.
• Memory B cells circulate through the same secondary lymphoid compartments that contain naive B cells.

Immunological Memory Failures

• Some pathogens manage to avoid being remembered by our immune system.
• Neisseria gonorrhoeae takes host-derived sialic acid and adds it to its LOS, masquerading as us and avoiding recognition.
• N. gonorrhoeae secretes a protease that specifically cleaves IgA, the adaptive response that protects our moist mucosal surfaces.