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. Immunoglobulin Gene Segments
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?
Isotype Switching
VDJ

13. Steps of Lymphocyte Development
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
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?