Genetics in the Generation of Antibody Diversity

Folder Title: AbGenesNoTP

Updated: October 14, 2015

Chapter Seven, 7th Edition: “Organization and Expression of Lymphocyte Receptor Genes”

Includes Immunoglobulin and T-Cell Receptor Genetics

Questions About B-Cell Responses

Biochemical Questions:1.How can Immunoglobulins recognize so many different

epitopes?

2.What does an antibody protein look like?

3.How do specific antibodies work as proteins binding so many different specific antigenic determinants?

Representation of Sequence Comparisons Among Light Chains from Antibodies with Three Different Antigen Specificities

H3N-Ser-Val-Ile-Thr-Gly-Gly-Tyr-Ala... Thr-Glu-Ala-Val-Tyr-Ser-Met-COO-

H3N-Ser-Ile-Met-Thr-Arg-Leu-Tyr-Gly..Thr-Glu-Ala-Val-Tyr-Ser-Met-COO-

H3N-Thr-Gly-Gly-Thr-Lys-Leu-Tyr-Ile..Thr-Glu-Ala-Val-Tyr-Ser-Met-COO-

Variable Amino Terminal Half Conserved Carboxyl Terminal Half

(Positions 1 to 107) (Positions 108 - 214)

LiteComp

See Figure 4-6,Kuby 6th Editionp. 85

See Figure 4-6,Kuby 6th Editionp. 85

Heavy ChainIso-forms

Variable RegionsIncluding hypervariable CDR’s (Complementarity determining regions)

κ or λ forms

The Heavy and light chains are labeled incorrectly in the Kuby Immunology Powerpoint slides. The figure is labeled correctly in the book.

Questions About B-Cell Responses

Biochemical Questions:1.How can Immunoglobulins recognize so many different epitopes?

2.What does an antibody protein look like?

3.How do specific antibodies work as proteins binding so many different specific antigenic determinants?

Genetic Question:

How can this diversity of structure leading to enormous diversity of function be coded and controlled by a very limited host genome?

How can we have “one gene - one polypeptide” and make a virtually limitless selection of polypeptides?

Problems for Genetics in Generating Antibody Sequence Diversity

Vast Sequence Diversity-

Encoded by Very Limit Genome

Heavy and Light Chain Sequence Variations-

Almost Exclusively in only one region

Exactly the same V-region sequences-

End up on different C-region Isotypes.

How is all of this possible?????

Myeloma Cell DNADifferentiated Neoplastic Plasma Cell making a single antibody.

Myeloma Cell Conclusions

Variable and conserved regions of light chain are linked in the differentiated end-product cell line

DNA coding for the mRNA for the light chain is all in oneContinuous sequence as for any gene for any protein.

Embryonic Mouse Cell DNA

Variable and conserved regions of light chain are not linked originally in the stem cell lineage!

Immunology and Phone Numbers315-443-1870212-345-1775478-367-8903537-503-2078409-159-6309610-970-3970934-620-8122909-603-7023800-620-6021704-590-5307703-725-0153207-502-6671435-431-0890412-830-0048740-592-1954307-620-4450490-501-5672601-909-7002554-891-7712335-592-0944

20 Phone Numbers

Immunology and Phone Numbers-443-1870-345-1775-367-8903-503-2078-159-6309-970-3970-620-8122-603-7023-620-6021-590-5307-725-0153-502-6671-431-0890-830-0048-592-1954-620-4450-501-5672-909-7002-891-7712-592-0944

315-212-478-537-409-610-934-909-800-704-703-207-435-412-740-307-490-601-554-335-

400 Phone Numbers

Immunology and Phone Numbers-1870-1775-8903-2078-6309-3970-8122-7023-6021-5307-0153-6671-0890-0048-1954-4450-5672-7002-7712-0944

315-212-478-537-409-610-934-909-800-704-703-207-435-412-740-307-490-601-554-335-

-443--345--367--503--159--970--620--603--620--590--725--502--431--830--592--620--501--909--891--592-

8000 Phone Numbers

Kappa Chain DNA: Vk’s

This and the next two slides deal with Kappa Light Chain DNA. Human Kappa DNA was 40 different “V” region genes to work with, and 5 “J” region genes. (200 Possibilities)

Lambda light chain DNA works the same way except that the Human Lambda light chain works with 30 “V” regions and 4 “J” region genes. (120 Possibilities)

Kappa Chain: K-J- C

Complete Kappa Chain

Heavy Chain Dna: VH’s, C-Genes

This and the next two slides deal with Heavy chain DNA. Human heavy chain DNA has 51 “V” region genes to work with, 27 “D” (diversification) region genes, and 6 “J” (joining) region genes.

8262 Possibilities even with no additional variations

Coupling a VH option with DH option and a JH option to a Cu gene sequence would give a Mu Heavy chain isotype with a specific antibody recognizing specificity.

Coupling that exact same VH-DH-JH genetic information to Cd or Cg or Ce or Ca Heavy chain isotype gene would give the exact same antigen specificity on a different isotype.

We would have Isotype Switching

Chain Structures of the five immunoglobulin classes in humans

(adapted from Kuby, 2nd edition)

Class Heavy Light Sub-Classes Subunit Formula

Chain Chain

IgG γ κ or λ γ1, γ2, γ3, γ4 γ2κ2 γ2λ2

IgA α κ or λ α1, α2 (α2κ2)n

(α2λ2)n

n =1,2,3,4

IgM μ κ or λ None (μ2κ2)n

(μ2λ2)n

n = 1 or 5

IgD δ κ or λ None δ2κ2 δ2λ2

IgE ε κ or λ None ε2κ2 ε2λ2

Complete Heavy Chain

The puzzle of immunoglobulin gene structure

• B cells use groups of PARTS of genes to create different possible antibodies using recombination

– Like shuffling a deck of cards, dealing out different hands

– Tightly regulated machinery controls the recombination processes

The puzzle of immunoglobulin gene structure

• B cells use groups of PARTS of genes to create different possible antibodies using recombination– There are variable (V), diversity (D), joining (J), and constant

(C) region gene segments• D segments are used in antibody heavy chains only

Multigene organization of Ig genes• Recall that Ig proteins consist of:

– two identical heavy chains– two identical light chains

• Light chains can be either kappa or lambda

– Each set of gene families are encoded on separate chromosomes

The mechanism of V(D)J recombination• Five mechanisms generate antibody diversity in naïve B

cells– Multiple gene segments―which gene segments are put together

– P nucleotide addition―templated nucleotide addition between joints, resulting from assymetrical cleaving of hairpin structures

– Exonuclease trimming―sometimes occurs at junctions, losing nucleotides and changing reading frames

– Non-templated N nucleotide addition―mediated by TdT activity, adding in random nucleotides between joints

Combinatorial diversity―which heavy chains pair with which light chains

The mechanism of V(D)J recombination• Five mechanisms generate antibody diversity in

naïve B cells (See previous slide)

Additional sequence diversity estimated at several orders of magnitude (i.e. perhaps 1000-fold) is generated by junctional flexibility, nucleotide addition, and somatic hypermutation. This allows for an estimated 100 million to a billion different possible antibody seuquences in humans.

B-Cell Differenatiation

B-cell receptor expression• Allelic exclusion ensures that each B cell synthesizes only one heavy

and one light chain.Recombination is a very ordered process.Nonproductive arrangements lead to programmed cell death (apoptosis) during development.

Detailed genetic mechanisms for generating antibody diversity are shown in Chapter 7 of Edition 7.

These specifics are beyond the scope of this introductory course but are important for those interested in advanced work in immunobiology or medical genetics.

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