2. Genetics of Antigen Receptor Diversity – I & II

3. Antibodies and T Cell Receptor Genetics Learning Objectives
To understand mechanisms for creating antigen receptor (BCR/TCR) diversity
Be able to identify changes at the DNA level required to produce a functional immunoglobulin gene
To understand isotype switching at the molecular level
To recognize basic differences between antigen receptors (structure and function) on B and T cells

4. VARIABILITY Variability in the antibody variable region
Variability in the antibody heavy chain constant region – isotype switching
Variability in the T cell antigen receptor variable region
No T cell isotypes

5. Immunoglobulins Have Two Roles
As an Antigen Receptor
To selectively recognize and bind antigens
These include toxins, viruses, and exposed molecules on the surface of pathogenic organisms
V domain function (VH and VL)
As an Effector Molecule
To eliminate or inactivate the foreign antigen or the cell that bears that antigen
C domain function (CH)

7. Light chain gene in a B cell CDR CL VL L CDR
Sequence myeloma light chain proteins. Bence-Jones. With more sequences, C and V, then V hypervariable or CDR 1-3. CDR3 most variable. Why? Mechanisms restricted to CDR3. Another mechanism effects entire V region.
CDR

8. 1920’s Karl Landsteiner
Analysis of antibody specificity in “Specificity of Serological Reactions”
Antibodies can distinguish between chemically identical molecules
that differ only in the position of the COOH group – stereoisomers
Immunologists struggled for many years to figure out how it is possible to make antibodies to any substance
Landsteiner was a busy guy. He also discovered ABO blood groups (Nobel Prize) and poliovirus

9. Generation of Antigen Receptor Diversity “The Repertoire”
Survival requires B and T cell receptor diversity to respond to the diversity of pathogens
The immune system must “Be Prepared” to respond to antigens it has never encountered

10. Generation of Diversity
One to 100 million different antibodies with different specificities can be produced
Nine antibody isotypes IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE
Similar numbers of T cell receptors for antigen
10 x 106 genes X 103 base pairs DNA/gene = 10 x 109 bp DNA
No isotypes with TCR

11. Generation of Diversity Cellular Solutions
Diversity operates at the level of the lymphocyte. Each lymphocyte has a single receptor specificity
Clonal selection:
1. Generate one million different lymphocytes per day
2. Antigen selects cells by binding to a complementary receptor and stimulating cell division and differentiation
Cell that don’t meet antigen die.

12. Clonal selection Plus CD4 T cell help IgM, IgG, IgA or IgE
Unselected cells die. T cells in thymus, >95% die.
Clonal selection

13. Generation of Diversity Genetic Solutions
Genes for antigen receptors do not exist until they are generated during the development of lymphocytes
Light chain gene CL VL L
Variable regions exons are formed by splicing together segments of genes inherited through the germline

14. Generation of Diversity Genetic Solutions
Functional genes for antigen receptors do not exist until they are generated during the development of lymphocytes
Variable regions exons are formed by splicing together segments of genes inherited through the germline
The process is called Ig or TCR gene rearrangement and generates tremendous diversity without monopolizing the genome

15. Anatomy of Immunoglobulin Genes in B Lymphocytes
Intron
V Region
Exon
V Region
Exon
C Region
Exon
C Region Exons
DNA
mRNA
Protein
Light chain
Heavy chain

16. Variable region genes are constructed from gene segments
Germline
DNA
Stem cell
B Cell
DNA
mRNA
Protein
J here is not J in J chain of IgM and IgA.

17. Germline Ig Genes

18. V-region Gene Segments are Joined by Somatic Recombination
VL Exon
Deletion Circle
Light chain gene CL VL L

19. Visualization of deletion circles by electron microscopy

20. VL VH VH
IgM B Cell
IgM B Cell VH VL

21. Somatic recombination at the Ig heavy chain locus
VH gene segments
DH segments
JH segments
Germline DNA *
D  JH rearrangement
No Ig produced at this point *
V  DJH rearrangement
Primary RNA transcript
Splicing and polyA  mRNA
Nascent protein
Mature μ heavy chain protein
Same thing as with light chain where we have juxtaposition of the segments and looping out of intervening DNA.

22. VL VH
IgM B Cell VH VL

23. Recombination Activating Genes
Critical protein complex for VDJ and VJ recombination
Two closely-linked genes RAG-1 and RAG-2 required for activity
“Knock-out” of either gene leads to total absence of B and T cells
How do RAG1 and RAG2 know where to cut the DNA?
Baltimore. Found in all species with Gene rearrangement, not in others. The recombination activating gene 1 component is thought to contain most of the catalytic activity, while the N-terminal of the recombination activating gene 2 component is thought to form a six-bladed propeller in the active core that serves as a binding scaffold for the tight association of the complex with DNA.

24. RAGs Recognize Recombination Signal Sequences
DNA sequences that flank rearranging gene segments of all immunoglobulin and T cell receptor loci
Highly conserved palindromic heptamer and A/T-rich nonamer separated by a 12 or 23 bp spacer

25. Recombination Signal Sequences

26. RAGs Recognize Recombination Signal Sequences
DNA sequences that flank rearranging gene segments of all immunoglobulin and T cell receptor loci
Highly conserved palindromic heptamer and A/T-rich nonamer separated by a 12 or 23 bp spacer
12 and 23 bp correspond to one and two turns of DNA helix
12/23 bp rule:
only RSS with 12 and 23 bp spacers can recombine with each other

27. Benefits of Antigen Receptor Gene Rearrangement
Combinatorial joining The random somatic recombination of
gene segments (VJ and VDJ) to create a variable region exon
Vκ: 40 Vκ X 5 Jκ = 200 κ V region exons
Vλ: 32 Vλ X 4 Jλ = 128 λ V region exons
Light chain genes
VH: 39 VH X 27 DH X 6 JH = 6,318 V region exons
Heavy
chain genes
How many antibodies can be made?
Combinatorial Association
(6,318 HC) x (200 κ LC) = 1.3 x 106 IgM κ antibodies
(6,318 HC) x (128 λ LC) = 0.8 x 106 IgM λ antibodies
>2 million total

29. Benefits of Antigen Receptor Gene Rearrangement
Combinatorial joining The random somatic recombination of
gene segments (VJ and VDJ) to create a variable region exon
Vκ: 40 Vκ X 5 Jκ = 200 κ V region exons
Vλ: 32 Vλ X 4 Jλ = 128 λ V region exons
Light chain genes
VH: 39 VH X 27 DH X 6 JH = 6,318 V region exons
Heavy
chain genes
How many antibodies can be made?
Combinatorial Association
(6,318 HC) x (200 κ LC) = 1.3 x 106 IgM κ antibodies
(6,318 HC) x (128 λ LC) = 0.8 x 106 IgM λ antibodies
>2 million total
Is this enough diversity?
Apparently not!

30. Strategies to Increase Diversity at the Site of V(D)J Joining (CDR3)
Junctional flexibility – imprecise ligation of V, D, J segments
Exonuclease nibbling
P-nucleotide addition
N-nucleotide addition
P-nucleotide (palindromic) addition
Non DNA template-encoded nucleotides generated during
resolution of the hairpin at the coding end
N-nucleotide addition
Non DNA template-encoded nucleotides added to the free
3’ ends of the cleaved DNA of the coding joints
More common in heavy chain VDJ rearrangements than in
light chain VJ rearrangements
Catalyzed by the enzyme
terminal deoxynucleotidyl transferase (TdT)

31. Strategies to Increase Diversity at the Site of V(D)J Joining (CDR3)
V(D)J Joining is imprecise
Junctional flexibility – imprecise ligation of V, D, J segments
Exonuclease nibbling

32. Frame shift more common. Genetic code is read 3 nucleotides at a time

33. Addition of N and P nucleotides to rearranging gene segments
Occurs in CDR3 of H and L, but that is often the main determinant of Ag binding DJ

34. Benefits of Junctional Flexibility
Increased diversity
- only in CDR3
- CDR3 major contributor to antigen binding

35. CDR3 often directly involved in binding antigen
CDR3 focus of diversity. Usually H more than L
HWS

36. Benefits of Junctional Flexibility
Increased diversity
- only in CDR3
- CDR3 major contributor to antigen binding
Cost
Most V(D)J rearrangements are non-functional
Solutions
Rearrange on second chromosome if first fails
Generate lots of cells

37. Allelic and Isotypic Exclusion of Ig Genes

38. Ig gene rearrangement during B cell development

39. Generation of Antibody Variable Region Diversity
Antigen Independent
V(D)J rearrangement (Combinatorial joining)
Junctional flexibility
P nucelotides
N nucleotides
Exonuclease nibbling
Combinatorial association
Antigen and T Cell Dependent
Somatic Hypermutation
Changes the DNA sequence of Ig Vh and Vl exons

40. Primary and Secondary Serum Antibody Responses
Higher titer
Higher affinity
Tetanus toxoid. Somatic mutation has produced antibodies that can bind better to the antigen. CSR has produced IgG memory B cells.

41. Immune response to the hapten 2-phenyl-5-oxazolone (phOx)
Demonstration of somatic mutation of Ig Vh and Vl exons during an immune response
Immune response to the hapten 2-phenyl-5-oxazolone (phOx)
Immunize mice with phOx-protein carrier
At various times isolate spleen cells and make hybridomas secreting anti-phOx antibodies
Sequence Ig Vh and Vl exons and compare it to the germline gene sequence to look for any mutations
Most antibodies to phOx use a particular Vh and Vl gene – VhOx1 and VkOx1
Hapten alone, no response.

42. Somatic Mutation of Variable Regions during an Immune Response
Isolate hybridomas making anti-phOX monoclonal antibodies
VDJ-Cm
VJ-Ck
2-phenyl-5-oxazolone
(phOx)
Claudia Berek and Ceaser Milstein. Day 7 mostly IgM.
Berek, et al., Nature 316:412, 1985

44. Antibody
Affinity
Actual data. 100 fold increase in affinity.

45. Somatic Hypermutation
Introduction of random mutations in the variable regions (VH and VL) of B cells responding to antigen
T cell help is essential
CD40 ligand deficiency → hyper IgM immunodeficiency
Rate
~ 10-3/bp/cell division
~ 10-8 – 10-9 for conventional genes
Mutations random
Silent
Replacement
Selection of B cells bearing mutated receptors with increased affinity for antigen
Competition for help from follicular helper T cells (TFH) in the GC

46. Somatic Hypermuatation Is Targeted to Ig V genes

47. Somatic Hypermutation II
Cell no longer able to bind antigen or get insufficient T cell help die by apoptosis
SHM occurs in germinal centers of secondary lymphoid organs
Exact mechanism unknown – requires transcription of targeted gene
Requires the activation induced deaminase (AID) gene

48. Somatic hypermutation in the germinal center
Summarizes a lot of events that occur during the generation of a T cell dependent antibody response. Microevolution, survival of fittest in this GC. Other GC, completion may be different.

50. IgG
IgA or
IgE
Plus T cell Help
Isotype switching

51. Switch Recombination Switch region Switch region VDJ C C C C VDJ
DNA deletion
Simplified Ig locus.

52. Switch Recombination Advantage No requirement for separate VDJH
recombination for each isotype
Only cells that switch will be those
responding to antigen
Features Irreversible
Individual plasma cell produces
one isotype
one specificity
Since VDJ recombination is so wasteful, don’t want to have to do it for each isotype.

53. Ig Class (Isotype) Switching
Mouse

54. Human has duplicated part of the locus. One Ce is psuedogene
Human has duplicated part of the locus. One Ce is psuedogene. Anthropomorphically, maybe not good to have two Ce.

55. Regulation of Isotype Switching
IgM/IgD
Dr. Chaplin mentioned. Different in humans. Directed by sterile transcription of target C gene

56. Switch Recombination II
Not site specific recombination like V(D)J
Recombination is in S-region in introns
Targeted constant region genes must be transcribed
Similar to somatic hypermutation and V(D)J rearrangement
Activation-Induced Cytidine Deaminase (AID)
required for both SHM and isotype switching
Although many cytidine deaminases are RNA
editing enzymes, AID edits DNA
AID introduces single stranded nicks in DNA. Error
prone repair get SHM
AID introduces double stranded breaks in DNA
switch regions. Ligation of two S-regions leads
to isotype switching
AID also required for gene conversion. Used in chickens and sheep, cows to diversify antibodies.

57. Sterile transcripts
Need to activate the B cell to get this process rolling. Here using LPS to polyclonally activate. Even though this is kind of TI response, in vitro can get switching to other isotypes by adding cytokines normally provided by T cell in a TD response

58. Ig Gene Diversification
RAG 1/2
Summary of the action at the DNA level in IgH
Teng and Papavasiliou Annu. Rev. Genet. 41:107 (2007)

59. Summary of the cellular level.

60. T CELL RECEPTOR DIVERSITY
ANTIBODY DIVERSITY
Variable region
Antigen independent – bone marrow
Ig gene segment rearrangements
Antigen dependent – 20 lymphoid organs
Somatic hypermutation
Constant region
Antigen dependent – 20 lymphoid organs
IgM  IgG or IgA or IgE
T CELL RECEPTOR DIVERSITY
Finished with B, lets look at T. Only antigen independent VDJ recombination.
Variable region
Antigen independent – thymus
TCR gene segment rearrangements

61. The T Cell Receptor for Antigen
TCR

62. Comparison of the TCR and the BCR (Immunoglobulin)
V regions
encoded by
rearranging
genes
Rearranging parts of two receptors

63. Structure of the T-cell Receptor
Both chains TM proteins, C and V regions

64. The T Cell Receptor
Heterodimer that only exists as a transmembrane antigen receptor
It is not secreted since T cells function by direct cell contact
The variable regions of the TCR are generated by somatic gene recombination as the T cells develop in the Thymus
The process is identical to Ig gene rearrangement, but different genes are used
Kill or help, cant do at a distance.

65. B cells recognize intact protein antigens
Heavy Chain
Lysozyme
Ab recognize epitopes on intact molecules.
Antigenic Determinant
Epitope
Light Chain
Ab recognize epitopes on intact molecules.

66. T cells recognize processed (degraded) protein antigens Peptide-MHC
T cell recognize peptide MHC.

67. Differences in antigen recognition by B and T lymphocytes
B cell antigen receptor
Immunoglobulin (Ig)
B cells
Transmembrane protein
Secreted by effector cells (Plasma cell)
Humoral Immunity
T cell antigen receptor
T cell receptor (TCR)
T cells
Transmembrane protein
Transmembrane protein on effector cells
CD4 Helper T Cells
CD8 Cytotoxic T Cells
Cell Mediated Immunity

68. Differences in T and B Cells
Recognize native protein antigens in solution or on cell surfaces
Secreted antibody is effector molecule
Antibodies can operate at a distance
T cells
Recognize peptides from degraded antigens
Peptides are displayed on cell surfaces in association with specialized proteins (MHC)
Antigen-specific T cell functions require direct cellcell interactions

69. Here are the two receptors again.

70. T-cell Receptor - and -chain V Regions are Generated by Gene Segment Rearrangement
B1 and B2 not isotypes.

71. T-cell Receptor - and -chain V Regions are Generated by Gene Segment Rearrangement
How locus really looks. Delta locus embedded in alpha locus Va>Ja delete delta. Again B1 and 2 not isotypes.

72. The  TCR Basic structure similar to  TCR
Encoded in separate gene loci
Only a minor subpopulation of T cells in the blood are  T cells
The  T cells may be enriched in tissues such as skin and intestinal epithelium
A more primitive T cell? The first line of defense?

73. The T Cell Receptor Gene Loci
Gamma a bit like Ig lamda of Ig. Multiple C with upstream V and J. No evidence that they are isotypes in either case.

74. Diversity Variable region Antigen independent [V(D)J]
ANTIBODY DIVERSITY
Variable region
Antigen independent [V(D)J]
Antigen dependent (SHM)
Constant region
Antigen dependent
IgM  IgG or IgA or IgE
T CELL RECEPTOR DIVERSITY
Variable region
Antigen independent

75. Dangers in Diversity Mechanism is essentially a random draw
By chance, some Ig and TCR will react with self antigens
Autoreactive B and T cells must be eliminated or silenced to prevent autoimmune diseases
Chromosomal translocations arising during VDJ recombination or isotype switching may lead to lymphoid malignancies