Immunoglobulin Superfamily of Genes and Diversity of Antigen Recognition

1
Immunoglobulin Superfamily of Genes and Diversity
of Antigen Recognition
2
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

3
Antigen Recognition Members of the Ig Superfamily

• Cellular Adhesion
• Cell-Cell Interactions
• Antigen Recognition
• undergo DNA recombination

4
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!!

5
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

6
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

7
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
8
The BIG PICTURE
B-cell Development
Light Chain Generated
9
Immense Diversity in Antigen Recognition
Typically, most individuals recognize 107 109
different antigens
10
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

11
Path of the Lymphocyte
12
When and Where does Somatic Recombination Occur?
13
Development of Lymphocytes

• Steps of Lymphocyte Development
• Development (T or BM)
• Functional Receptor or DIE
• Positive Selection (T or BM)
• Interact w/ appropriate Receptor (MHC)
• or DIE
• Negative Selection (T or BM)
• React to self and DIE
• IV. Functional Competence (SLT)

Somatic Recombination occurs during development-
formation of functional receptor
14
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

15
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

16
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

17
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

18
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

19
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
20
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

21
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.
22
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

23
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)

24
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

25
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

26
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.

27
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

28
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

29
The BIG PICTURE
B-cell Development
Light Chain Generated
30
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

31
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

32
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

33
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

34
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.

35
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
36
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

37
Questions? Comments?