Lecture 2: Antibody Diversity LIM 210: Immunology II Lecture 2: Antibody Diversity

Mechanisms contributing to generation of antibody diversity

Mechanisms contributing to generation of antibody diversity Genesis of antibody diversity Combinatorial diversity (somatic recombination) Junctional diversity Somatic hypermutations Random assortment of H and L chains Class switching in the C-region

Genesis of antibody diversity Presence of multiple V genes in the germ line contributes to Ig diversity

Immunogenetics Multi germ line gene segments: Antibody genes are composed of segments

B Cell: humoral or antibody mediated immunity Humoral Immunity: ANTIBODIES Matures in bone marrow Moves to lymph nodes (& other locations) Secretes antibodies “as plasma cells” Also creates “memory” B cells B cells secrete soluble antibodies: humoral immunity. T cells interact directly with their targets: cellular immunity B-lymphocyte

B cell Development: gene rearrangement and RNA processing

Genesis of antibody diversity A mature B-cell which has undergone V(D)J recombination is distinct from all others at 3 levels: The choice of V, D and J segments from germ line DNA The combination of re-arranged heavy and light chains The junctional insertion and deletion of nucleotides which occurs during rearrangement

CLONAL SELECTION

Mechanisms contributing to generation of antibody diversity How is Ig diversity specified genetically? Multiple germ line gene segments Combinatorial diversity (or V-J & V-D-J recombination) Junctional diversity Somatic hypermutations Random assortment of H and L chains Class switching in C region gene

Functional gene segments for the V regions of human heavy and light chains Presence of multiple V genes in the germ line contributes to Ig diversity

Mechanisms contributing to generation of antibody diversity Germ line genes H k λ V segments 65 40 30 J segments 6 5 4 D segments 27 Combinatorial joining V × J (×D) 11,000 200 120 H-L chain associations H × k 2.2 x 106 From 177 segments H × λ 1.3 x 106 Note: Junctional diversity is also estimated to add substantially to overall diversity

Immature B cell: gene rearrangement and RNA processing

Mature B cell: Antigen stimulation, Activation & differentiation, Class switching

Somatic recombination of gene segments

Light Chain Splicing There are two different L chain genes, called kappa and lambda. Immunoglobulins use one or the other. At the kappa L chain gene, there is a single region of DNA that codes for the C domain. Upstream from the C domain is a group of about 250 V domains and another group of 5 J (for "joining") regions. Each V region has a 5’UTR segment, a separate exon, attached to it: the leader. During the development of the B cell, a randomly chosen V domain joins with a randomly chosen J domain to form a VJ domain. This occurs by splicing out the DNA between them. Note that this is a very different mechanism from intron spicing that occurs in the RNA of most genes! Once the VJ domain is formed, it can be transcribed into RNA along with the C domain and any DNA that lies between the VJ and C. All the intervening RNA is spliced out as an intron, so the final messenger RNA has VJC all together; this is then translated into a L chain protein. Lambda light chain genes are slightly different: fewer V regions, and four different C regions each of which has its own J. Lambda chains use only a single DNA splice, to join a randomly selected V region to one of the 4 J-C regions.

Heavy Chains The process of producing a heavy chain is very similar to L chains, except that there is an additional group of 5 D regions between the V regions and the J regions. Two DNA splices occur for H chains: first a random J joins with a random D to form a DJ region, then a random V region is added to form a VDJ domain. Just as in the L chain, this VDJ is transcribed along with the C region, and any intervening RNA is spliced out as an intron. The final product has VDJC all joined together and gets translated into an H chain protein. All of the C regions are at the heavy chain locus. Initially, the CM region is transcribed. CD is also transcribed and expressed using an alternative RNA splicing mechanism.

Heavy Chain Rearrangement

DNA rearrangements bring together segments of a gene for expression. Figure 21.23 Fig. 22.24 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Mechanism of junctional diversity 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. Catalyzed 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)

Mechanism of junctional diversity

Somatic Hypermutation is a mechanism by which random base change mutations occurs in the V regions in B cells. This mechanism doesn't work in other cells and doesn't affect other genes: only a region of about 1.5 kb is affected. It only occurs as the B cell is maturing: after it has been stimulated to divide by an antigen, somatic hypermutation occurs to modify the antigen binding region. Those cells that bind the antigen most tightly survive and divide more than the others. This process is called “affinity maturation”. It is triggered by the enzyme “activation-induced cytidine deaminase” (AID), which deaminates cytidine to uracil. This base mismatch can be incorrectly repaired by several different mechanisms to generate mutations. Occurs randomly after antigenic stimulation and principally in CDR1, CDR2, CDR3 regions (more frequent in CDR3). Introduces point mutations in V genes at a higher rate than for normal mammalian genes. Mutation rate of V genes is 1 base pair change per 103 base pairs/cell division; it is 10-7 in other mammalian genes. Can give rise to Ig with different (new) antigen specificities leading to high or low affinity Abs. High affinity B cell clones are selectively expanded (Affinity Maturation)

Class Switching Heavy chains fall into 5 classes, based on their C regions. Each H gene has C regions for all 5 classes arranged on the chromosome, with the IgM C region nearest to the V regions. There are several different C regions for some of the classes. IgM is the initial Ab made by each B cell. However, after a while the B cell switches to a different class. This is done using a third DNA splice, in which the DNA between the VDJ and the constant region for the new class is spliced out.

Mature B cell: Antigen stimulation, Activation & differentiation, Class switching

Random Assortment of H and L chains

Changes in immunoglobulin and TCR genes that occur during B-cell and T-cell development and differentiation