immunoglobulin superfamily of genes and diversity of antigen recognition

Immunoglobulin Superfamily of Genes and Diversity of Antigen

Recognition

Outline• Immunoglobulin Superfamily • Antigen Recognition Members:

AntibodiesT-Cell Receptor

• Immense Diversity:The Problem: Package all that InformationThe Solution: Somatic Recombination

• Development of a Lymphocyte: A Time for Diversity

• Somatic Recombination• VDJ: The Making of the Variable Region• Isotype Switching

Antigen Recognition Members of the Ig Superfamily

•Cellular Adhesion

•Cell-Cell Interactions

•Antigen Recognitionundergo DNA recombination

•Typically, most individuals recognize 107 – 109 different antigens

•If each antigen receptor was a gene, then genes dedicated to this purpose alone would take a LARGE portion of the genome!!

The Problem: All That Information

Discovery of Immunoglobulin Gene Organization

•Dreyer and BennettAmino acid sequencing of IgSame isotype of protein had highly variable V-regionMust come from different genes that are joined at DNA/RNA level

•TonegawaIg genes are different in a cell committed to the synthesis of that protein(lymphoid-tumor vs. stem cell)Difference due to events that must occur during the development of that cell

The Solution: Packaging• Germline DNA: NO functional Ig/TCR protein can be made

without extensive processing

• Genes are packaged into loci each containing the pieces (gene segments) of a functional antibody or TCR

• The gene segments are put together in a step-wise fashion that results in a functional protein

• Recombination allows for the “piecing together” of genes

• Splicing finalizes the RNA transcripts

• END RESULT: Antibody or TCR

Immunoglobulin Gene Segments

Antigen Recognition Receptor Gene Loci

TCR Gene Segments

•Gene Segments are located in different genes on different CHROMOSOMES!

•Each locus: Sets of gene segmentsGene segments (exons) separated by non-coding regions (introns)

The BIG PICTURE

B-cell Development

Light Chain Generated

Immense Diversity in Antigen Recognition

Typically, most individuals recognize 107 – 109 different antigens

Path of the Lymphocyte

When and Where does Somatic Recombination Occur?

VDJ Isotype Switching

Development of Lymphocytes

Steps of Lymphocyte Development

I. Development (T or BM)Functional Receptor or DIE

II. Positive Selection (T or BM) Interact w/ appropriate Receptor (MHC)

or DIE

III. Negative Selection (T or BM) React to self and DIE

IV. Functional Competence (SLT)

Somatic Recombination occurs during development- formation of functional receptor

B-Cell Development•Occurs in the bone marrow•Roughly 109 cell are produced per day•Occurs in a highly controlled, stepwise fashion•Mistakes are not tolerated

T-Cell Development•Occurs mostly in the thymus – progenitors come from bone marrow•Occurs in a highly controlled, stepwise fashion•Mistakes are not tolerated•95% of the cells never make it to the state of naïve mature T-cell

Resident Cells of the Bone Marrow and Thymus Play An Important Role in Lymphocyte Development

•Stromal cells of both the bone marrow and thymus play an important role in development of lymphocytes

•Other cell types are also present (epithelial cells/dendritic cells/macrophage)

•Secrete cytokines to stimulate growth (IL-7 = lymphopoietic growth factor)

•Help with positive and negative selection

Somatic Recombination

•Somatic Recombination = process of DNA recombination by which functional genes encoding variable regions of Ag R are formed during lymphocyte development.

•Rearrangements occur in strict developmental sequences

•Two levels of diversity:CombinatorialJunctional

Mechanism:Each gene segment (V, D, and J) has an adjacent Recombination Signal Sequence (RSS)

at the 3' end of each V segment at both ends of each D segment at the 5' end of each J segment

These are recognized by two proteins encoded by two Recombination Activating Genes

RAG-1 and RAG-2

The RAG-1 and RAG-2 proteins cut through both strands of DNA at the RSS forming

Mechanisms of Somatic Recombination


•Structures to know: heptamer/nonamer: Conserved 12 or 23 nucleotide spacer: Not Conserved

•12/23 Rule: Recombination will occur only with segment that have a 12 or 23 at the 5’ region

Combinatorial Diversity


Combinatorial Diversity:

Brings regulatory elements (promoters/enhancers) closer together, thus strongly promoting expression of the gene elements in proximity of one another.

Mechanisms of Somatic RecombinationJunctional Diversity

•Addition or removal of nucleotides between V/D, D/J or V/J segments at the time of joining


Junctional Diversity•RAG mediated cleavage

•Endonuclease may remove Nu

•P-nucleotides = make blunt endsfrom “over hangs” made by RAGenzymes

•N-nucleotides = new nucleotidesrandomly added by TdT (TerminalDeoxyribonucleotidyl Transferase)





• Somatic Recombination• VDJ: The Making of the Variable Region:

B-Cells/T-Cells

• Isotype Switching

B-Cell DevelopmentPRE-B CELL

•The first rearrangement is DH and JH. Then, the DJ joins with the V gene segment of chromosome 14. •The heavy chain is coupled with a surrogate light chain.•Expressed on cell surface•Called pre-B-Cell Receptor (pre-BCR)•Pre-BCR important for survival signals and allelic exclusion

IMMATURE B-CELL

•The V joins with the J gene segment of chromosome 2 for or 22 for •Expressed on cell surface as a complete IgM•IgD/IgM is the final set of markers present on the surface of a mature B-cell.

B-Cell Development

Heavy and Light Chain Gene Recombination

•Heavy chain 1st D to JV to DJ

•Light chain 2nd

V to J

•Primary RNA transcript isspliced to remove introns

•Final heavy and light chains are assembled into functionalprotein

Coexpression of IgM and IgD Does Not Require Isotype Switching

•IgM and IgD C gene segments are part of the same open reading frame (ORF)

•Variations in splicing of primary RNA transcript will result in generation of IgM orIgD

•Other isotypes available only by isotypeswitching

The BIG PICTURE

B-cell Development

Light Chain Generated

T-Cell DevelopmentPRE-T CELL

•The first rearrangement is D and J. Then, the DJ joins with the V gene segment of chromosome 7. This makes the beta chain of the TCR. •The beta chain is coupled with an invariant protein.•Expressed on cell surface•Called pre-T-Cell Receptor (pre-TCR)•Pre-TCR important for survival signals and allelic exclusion

T-Cell DevelopmentIMMATURE T-CELL

•The V joins with the J gene segment of chromosome 14 (alpha or 7 for lambda). This makes the alpha chain of the TCR. •The beta chain assembled with alpha chain to complete TCR•Expressed on cell surface

TCR and Chain Gene Recombination and Expression

•Beta chain 1st D to JV to DJ

•Alpha chain 2nd

V to J

•Primary RNA transcript isspliced to remove introns

•Final alpha and beta chains are assembled into functionalprotein





• Somatic Recombination• VDJ: The Making of the Variable Region:

B-Cells/T-Cells


Isotype Switching

• During an antibody response to a T-dependent antigen a switch occurs in the class of Ig produced from IgM to some other class (except IgD).

• During class switching another DNA rearrangement occurs between a switch site (Sµ) in the intron between the rearranged VDJ regions and the Cµ gene and another switch site before one of the other heavy chain constant region genes.

• This recombination event brings the VDJ region close to one of the other constant region genes and allows expression of a new class of heavy chain.

IL-4

•Switch Recombination rearranged VDJ gene segment in a B-cell recombines with a downstream C region gene and the intervening DNA is deleted

•Involves nucleotide sequences knownas switch regions that are in the 5’ of each CH locus

•Cytokines and CD40 stimulate the read through of these regions and I exons which contain nonsense and many stop codons, these stimulate recombination downstream C regions

Isotype Switching

Outline/Summary• Immunoglobulin Superfamily • Antigen Recognition Members:




• Somatic Recombination• VDJ: The Making of the Variable Region

T-cell/B-cell


immunoglobulin superfamily of genes and diversity of antigen recognition

Documents

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lymphocyte slide

functional receptor

tcell development

tcr slide

information slide

dna recombination slide

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