Transcript Slide 1

Immunoglobulin Superfamily of
Genes and Diversity of Antigen
Recognition
Outline
• Immunoglobulin Superfamily
• Antigen Recognition Members:
Antibodies
T-Cell Receptor
• Immense Diversity:
The Problem: Package all that Information
The 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 Recognition
undergo DNA recombination
The Problem: All That Information
•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!!
Discovery of Immunoglobulin Gene Organization
•Dreyer and Bennett
Amino acid sequencing of Ig
Same isotype of protein had highly variable V-region
Must come from different genes that are joined at DNA/RNA level
•Tonegawa
Ig 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
Antigen Recognition Receptor Gene Loci
•Gene Segments are located in different genes on different CHROMOSOMES!
•Each locus:
Sets of gene segments
Gene segments (exons) separated by non-coding regions (introns)
Immunoglobulin Gene Segments
TCR Gene Segments
The BIG PICTURE
B-cell Development
Light Chain Generated
Immense Diversity in Antigen Recognition
Typically, most individuals recognize 107 – 109 different antigens
Outline
• Immunoglobulin Superfamily
• Antigen Recognition Members:
Antibodies
T-Cell Receptor
• Immense Diversity:
The Problem: Package all that Information
The Solution: Somatic Recombination
• Development of a Lymphocyte: A Time for
Diversity
• Somatic Recombination
• VDJ: The Making of the Variable Region
• Isotype Switching
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
Outline
• Immunoglobulin Superfamily
• Antigen Recognition Members:
Antibodies
T-Cell Receptor
• Immense Diversity:
The Problem: Package all that Information
The Solution: Somatic Recombination
• Development of a Lymphocyte: A Time for
Diversity
• Somatic Recombination
• VDJ: The Making of the Variable Region
• Isotype Switching
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:
Combinatorial
Junctional
Mechanisms of Somatic Recombination
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
Combinatorial Diversity
•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
Mechanisms of Somatic Recombination
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 Recombination
Junctional Diversity
•Addition or removal of nucleotides between V/D, D/J or V/J segments at the
time of joining
Mechanisms of Somatic Recombination
Junctional Diversity
•RAG mediated cleavage
•Endonuclease may remove Nu
•P-nucleotides = make blunt ends
from “over hangs” made by RAG
enzymes
•N-nucleotides = new nucleotides
randomly added by TdT (Terminal
Deoxyribonucleotidyl Transferase)
Outline
• Immunoglobulin Superfamily
• Antigen Recognition Members:
Antibodies
T-Cell Receptor
• Immense Diversity:
The Problem: Package all that Information
The Solution: Somatic Recombination
• Development of a Lymphocyte: A Time for
Diversity
• Somatic Recombination
• VDJ: The Making of the Variable Region:
B-Cells/T-Cells
• Isotype Switching
B-Cell Development
PRE-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
B-Cell Development
IMMATURE B-CELL
•The V joins with the J gene segment of chromosome 2 for k or 22 for l.
•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.
Heavy and Light Chain Gene Recombination
•Heavy chain 1st
D to J
V to DJ
•Light chain 2nd
V to J
•Primary RNA transcript is
spliced to remove introns
•Final heavy and light chains
are assembled into functional
protein
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 or
IgD
•Other isotypes available only by isotype
switching
The BIG PICTURE
B-cell Development
Light Chain Generated
T-Cell Development
PRE-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 Development
IMMATURE 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 a and b Chain Gene Recombination and Expression
•Beta chain 1st
D to J
V to DJ
•Alpha chain 2nd
V to J
•Primary RNA transcript is
spliced to remove introns
•Final alpha and beta chains
are assembled into functional
protein
Outline
• Immunoglobulin Superfamily
• Antigen Recognition Members:
Antibodies
T-Cell Receptor
• Immense Diversity:
The Problem: Package all that Information
The Solution: Somatic Recombination
• Development of a Lymphocyte: A Time for
Diversity
• Somatic Recombination
• VDJ: The Making of the Variable Region:
B-Cells/T-Cells
• Isotype Switching
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.
Isotype Switching
•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 known
as 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
IL-4
Outline/Summary
• Immunoglobulin Superfamily
• Antigen Recognition Members:
Antibodies
T-Cell Receptor
• Immense Diversity:
The Problem: Package all that Information
The Solution: Somatic Recombination
• Development of a Lymphocyte: A Time for
Diversity
• Somatic Recombination
• VDJ: The Making of the Variable Region
T-cell/B-cell
• Isotype Switching