BLOCK 3: IMM: (3.3) ANTIBODIES (PT. 2)

Immunoglobulin Diversity in B Cells Before Antigen Encounter

• Immunoglobulin is first made in a membrane-bound form that's present on the B-cell surface.
• Igα and Igβ form a disulfide-linked complex that interacts with the immunoglobulin molecule.
• Igα and Igβ have long cytoplasmic tails that interact with intracellular signaling proteins.
• Igα and Igβ have binding sites for immunoglobulin C region on their extracellular portions.
• The complex of immunoglobulin with Igα and Igβ serves as the functional B-cell receptor.
• All isotypes can serve as B-cell receptors.
• Igα and Igβ are also called CD79a and CD79b.

Diversification of Antibodies After Antigen Encounter

• Secreted antibodies are produced by an alternative pattern of heavy-chain RNA processing.
• Each heavy-chain C gene has two exons (membrane-coding, MC & secretion-coding, SC) encoding the carboxy terminus.
• Membrane-coding (MC) encodes the transmembrane region & cytoplasmic tail of the surface form.
• Secretion-coding (SC) sequence encodes the carboxy terminus of the secreted form.
• Alternative RNA processing determines whether a heavy-chain RNA will result in a secreted or transmembrane immunoglobulin.
• Each heavy-chain C gene has two potential polyadenylation sites (pAμs & pAμm).
• To generate the transmembrane form of the heavy chain, the transcript is cleaved and polyadenylated at the second polyadenylation site (pAμm).
• Splicing between a site located between the fourth Cμ exon & SC sequence, & a second site at the 5ʹ end of MC exons, removes the SC sequence and joins MC exons to the fourth Cμ exon.

The Structural Basis of Antibody Diversity

• The DNA sequence encoding a V region is assembled from two or three gene segments.
• The λ light-chain locus has ~30 functional Vλ gene segments, 4 pairs of functional Jλ, and a Cλ gene segment.
• The κ light-chain locus has ~35 functional Vκ gene segments & 5 Jκ gene segments with ONE Cκ gene segment.
• The heavy-chain locus has ~40 functional VH gene segments, a cluster of ~23 D segments, & 6 JH gene segments.
• A monoclonal antibody is produced by a clone of antibody-producing cells.
• To make a monoclonal antibody, lymphocytes from a mouse are immunized with an antigen of choice and fused with myeloma cells.
• Fused cells are grown with a drug that kills myeloma cells but permits growth of hybrid cells.
• Cultures of hybrid cells (hybridomas) are tested to determine whether they make the desired antibody.
• Cultures containing a hybridoma making the desired antibody are cloned to produce a homogeneous culture of cells making the monoclonal antibody.
• Myelomas are tumors of plasma cells; those used to make hybridomas were selected not to express antibody heavy and light chains.

Overview

• Epitope specificity of immunoglobulins is determined before antigen encounter.
• The number of possible epitope-binding specificities is greater than the number of genes within the genome.
• Each heavy-chain locus has multiple V, D, and J gene segments that are rearranged to produce a diverse array of antibodies.
• This process is known as V(D)J recombination.
• The rearrangement of these gene segments is mediated by specific enzymes, including RAG1 and RAG2.
• Junctional diversity is generated during gene rearrangement, increasing the diversity of antibodies.
• The process of V(D)J recombination contributes significantly to the diversity of the immune system.

Immunoglobulin Heavy Chains

• Immunoglobulin molecules are arranged in a specific order with IgM appearing first, followed by IgG, IgA and then IgE.
• The order reflects the location of the heavy chain genes on the genome.
• IgM and IgD genes are closely adjacent on the genome leading to their co-expression on the surface of B cells.
• B cells express both IgM and IgD during development, but only express IgD upon maturation.
• There are four subtypes of IgG (IgG1, IgG2, IgG3, and IgG4) and two subtypes of IgA (IgA1 and IgA2).
• Each immunoglobulin domain is coded by a separate exon.

Immunoglobulin Heavy Chain Assembly

• During the assembly of heavy chains, the D and J segments recombine first, followed by the V segment.
• The recombined VDJ region then joins with a constant region gene, determining the immunoglobulin class.
• The constant region gene determines the class of the immunoglobulin molecule, such as IgM, IgG, IgA, or IgE.
• Two membrane coding exons (MC) specify a hydrophobic sequence that anchors the immunoglobulin to the cell membrane.
• This hydrophobic sequence will later be cleaved off during the maturation process.

Immunoglobulin Light Chains

• The light chains are assembled by recombining a variable segment (V) with a joining segment (J).
• The VDJ region then joins with a constant region gene to produce a complete light chain.