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Notice that the CH regions are arranged in a sequential order-this is noaccident-it is related to the developmental appearance of the Ig classes inthe course of an immune response.

Variable region gene rearrangements occur in an ordered sequence duringB cell maturation in the bone marrow. The heavy chain variable regiongenes rearrange first then the light chain variable region genes. At the endof the process, each B cell contains a single functional variable regionDNA sequence for its H chain and a single functional variable regionDNA sequence for its light chain.

Although variable region gene rearrangements occur in an orderedsequence, they are random events that result in the random determinationof B cell specificity.

Once the H chain gene rearrangements take place, RNA polymerase canbind and transcribe the entire H chain gene.

Other Properties

B cells like all somatic cells are diploid and contain both maternal andpaternal chromosomes. Even so, it expresses the rearranged H chain genesfrom only one chromosome and the rearranged light chain genes from onechromosome. This process called allelic exclusion ensures that functionalB cells never contain more than one VDJand one VJ unit. (If it expressedboth the B cell would be multispecific). Same is true for the T cell receptor.

DNA sequencing revealed the presence of unique DNA recombinationsignal sequences (RSSs) flanking each germ line V,(D), and J segment.These signals function as signals for the recombination process. Due totheir structure, the RSSs only allow the proper joining of segments- ie. itprevents 2 J segments from joining. The RSSs vary in sequence but itslength is conserved and corresponds to one or two turns of the DNAdouble helix. This would bring the sequences to one side of the DNA helixwhere they can be bound by the protein complex that catalyzesrecombination.

Generation of Ab Diversity

As the organization of the Ig genes was deciphered, the sources of the vastdiversity in the variable region began to come clear.

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The germ line theory argues that the entire variable region repertoire is encodedin the germ line of the organism and is transmitted from parent to offspring viathe germ cells (egg and sperm).

The somatic variation theory held that the germ line contains a limited numberof variable genes which are diversified in the somatic cells by mutational orrecombinational events during development of the immune system.

Upon cloning and sequencing of Ig genes it become clear that diversity was duepartially to both theories.

Virtually any substance can elicit an Ab response and the response to a simpleAg is diverse initiating many different Ab molecules each with a unique affinityand specificity.

Multiple Germ Line V,D,J SegmentsAlthough the numbers of germ line genes are far fewer than originally predicted,multiple germ line V,D,J genes clearly do contribute to diversity of the Agbinding sites in Abs.

Combinatorial Joining

The contribution of multiple germ line gene segments to Ab diversity ismagnified by the random rearrangement of these segments in somatic cells.

The total number of possible combinations is conservatively estimated to be ~10 8 different Ab specificities generated by the mammalian immune system.(Remember in humans the and loci contain roughly equal numbers of Vgenes.

Combinatorial V-J and V-D-J joining: (mouse) 300(V) X 4 (J)= 1.2 x10 3

x 300-1000(V) x13(D)x 4(J)=1.6 x 104 (minimum combos)=~2 x 10 7

Each different combination yields an Ab with a different specificity.

Recombination only occurs between gene segments located on the samechromosome and it follows the special linking rule imposed by the RSSs.

Only one joining event is needed for light chain genes whereas two are neededfor H chains.

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The most common mode of rearrangement involves the looping out and deletionof the DNA intervening between the two gene segments. The DNA is broken andre-ligated.

Junctional Flexibility

The enormous diversity generated by means of V,D, and J combinations isfurther augmented by a phenomenon known as junctional flexibility. This bringsthe potential repertoire up to 10 9 to 1011 diversity.

The signal sequences join precisely to form a signal joint in a circular piece ofDNA (where it has looped out) which is then lost from the genome when the celldivides. The joining of the V and J segments (ect) form what is called the coding joint and it is an imprecise joining and consequently generates more Abvariability.

Inaccurate or imprecise DNA rearrangement occurs because thenucleotide sequences at the 3' end of a V gene and the 5' end of a Jsegment in a light chain , or the ends of V,D, J gene segments in a H chaincan each recombine at any of several nucleotides in the germlinesequence. As long as the recombination does not generate nonfunctionalDNA, different nucleotide and thus different aa sequences arise.

The amino acid sequence variation generated by junctional flexibility has beenshown to fall within the CDR3 (3rd HV region) in both the H and L chain. SinceCDR3s are a major contributor to the Ag binding site, junctional flexibility canhave a major impact in generating Ab diversity.

P Nucleotide Addition

P nucleotides are so called because they comprise palindromic sequences addedto the ends of the gene segments.

Nucleotides at the end of coding sequences are thought to be sealed to form ahairpin structure. This hairpin is later cleaved by an endonuclease. Cleavagesometimes occurs at a position that leaves a short single stranded region at the

end of the coding sequences. Then subsequent addition of complementarynucleotides by repair enzymes occurs. Variation in the position at which thehairpin is cut thus leads to variation in the sequence of coding joint.

N region nucleotide addition

Nucleotides called N sequences because they are non-template encoded in otherwords they are not present in the germline. They can be added to the junctions of

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rearranged VDJ genes after the hairpin has been cleaved by (only occurs in Hchain DNA). This addition of new nucleotides is a random process with thepossible addition of up to 20 nucleotides and is mediated by an enzyme calledterminal deoxyribonucleotidyl transferase (TdT).

*N nucleotides are absent from light chains because the enz TdT is expressedonly for a short period of time during the assembly of the H chain genes whichoccurs before the L chain gene are assembled.

Addition of nucleotides often disrupts the reading frame with roughly two inthree rearrangements being nonproductive-with many B cell never succeeding toproduce a fxnl Ab so diversity is achieved only with considerable waste.

(Junctional diversity is even more important in the T cell receptor).

Somatic HypermutationAll Ab diversity discussed to this point has been due to mechanisms that operateduring formation of specific variable regions by gene rearrangement. Once thefunctional variable region gene unit is formed a process called somatichypermutation can take place.

In somatic hypermutation individual nucleotides in VJ or VDJ units arereplaced with alternative bases thus most likely altering the specificity ofthe encoded Ig.

The rate of somatic mutation is a million fold higher than the spontaneousmutation rate in other genes and an average of one mutation will beintroduced in every one to two cell divisions.

The mechanism of somatic hypermutation has not yet been determined. Ithas been suggested that mutations are introduced by an error prone DNApolymerase.

Source of variation CDR1 CDR2 CDR3

sequence coded by: V segment V segment VL-JL junction

VH-DH-JH junctions

junctional flexibility - - +

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P-nucleotide addition - - +

N-Nucleotide addition * - - +

somatic hypermutation

+

+

+

*H chain DNA only

Combinatorial association of heavy and light chains

Because the specificity of an Abs Ag binding site is determined by the variableregions in both its heavy and light chains , combinatorial association can alsogenerate diversity.

In the mouse 1.6 x 104 H chain genes can associate with 1.2 x 103 possible lightchain genes= ~1.9 x 107 possible combinations

SUMMARY-

All these sources of diversity creates a vast repertoire of Ab specificities from areleatively limited number of genes!

Class Switching

Following Antigenic stimulation of a B cell, the heavy chain DNA can undergo afurther rearrangement in which the VDJ unit can combine with any CH genesegment.

The exact mechanism is unclear, but evidence suggests that DNA flankingsequences (termed switch sites ) are located 2-3 kb upstream (the 5'side) fromeach CH segment.

These switch sites are composed of multiple copies of short repeated sequences.One hypothesis is that a series of class specific recombinase proteins bind tothese switch sites and facilitate DNA recombination. The particular Ig class that

is expressed thus may depend onthe specificity of the recombinase protein expressed and

on the cytokine environment. Cytokines are thought to induce the accessibility ofthe switch sites.

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Circular excision products are produced during class switching so switching canonly progess downstream there is no switching from IgG back to IgM in otherwords there is orderly deletion of H chain genes.

Switch recombination is unlike variable gene segment recombination in severalways

All isotype switch recombinations are productive

It uses different signal switches and enzymes

It happens after Ag stimulation and not during B cell development

The process is not random but is regulated by T cells and their products-lymphokines!