Immunology, Kuby's fifth edition notes for strong background in the topic, General introduction, Types of Antibody and Structure, Experiments, Mechanisms

3. IgG
• Highest concentration in serum.
• Four subclasses: IgG1 - 4
• Activates complement
• Binds to Fcg -receptors on neutrophils,
macrophages and NK cells

4. • Membrane-bound monomer and secreted pentamer.
• First immunoglobulin to be synthesized during
ontogeny and in the immune response.
• Activates complement pathway; agglutination.
• Can be transported into mucosal secretions.
IgM

5. IgA
• Usually dimer
• Secretory IgA is a dimer with a secretory
component.
• Two subclasses: IgA1 and IgA2
• Major immunoglobulin in mucosal
secretions
• Neutralization; Prevents binding of micro-
organisms to receptors
• Not effective activator of complement

6. • Very low serum concentration in healthy individuals.
• Concentration is higher in patients with helminth
infections and often in patients with allergies.
• Lacks hinge region; extra CH domain
• Binds to Fce receptor on mast cells and basophils.
Cross-linking results in degranulation and release of
pro-inflammatory mediators.
IgE

7. IgD
• Very low concentration in
serum
• Primarily found with IgM on
naïve mature B cells
• Function is unknown

8. All Antibodies are coded by genes
of Germ line cells. Germ line
proponents proposed that the
genome contributed by the germ
cells contains a large repertoire of
Immunoglobulin genes. But they
were unable to explain the genetic
mechanism to account for
Antibody diversity.
Genome of Somatic cells contains a
relatively small number of
Immunoglobulin genes, from
which a large no of antibody
specificities are generated by
Mutation or Recombination.
SOMATIC VARIATION THEORY:
GERM LINE THEORY:

9. Dreyer – Bennett’s two- gene model
hypothesis
V
V
VV
V
V
VV
V
V
VV
V
A mechanism to rearrange V and
C genes in the genome exists so
that they can fuse to form a
complete Immunoglobulin gene
CV
C
A single C region gene is
encoded in the germ line
and separated from the
multiple V region genes
Find a way to show the existence of multiple V genes and
rearrangement to the C gene

10. Approach
Tools:
• A set of cDNA probes to specifically distinguish V regions from C regions
• DNA restriction enzymes to fragment DNA
• Examples of germ line (e.g. placenta) and mature B cell DNA (e.g. a
plasmacytoma/myeloma)
C
V
V
VV
V
V
VV
V
Germ line DNA
CV
V
VV
V
Rearranged DNA

12. * *
**
B-cellV C
V C
Embryonal cell
V-CmRNS probe
CmRNS probe
*
*
The experiment of Susumi Tonegawa 1976

13. There are many variable genes but only one constant gene
V CV V V
GERM LINE
V and C genes get close to each other in B-cells only
CV V V
B-CELL
CONCLUSION
Fehérje
Gén

14. Ig gene sequencing complicated the model
The structures of germ line VL genes were similar for Vk, and Vl,
However there was an ANOMALY between germ line and rearranged
DNA:
Where do the extra 13
amino acids come from?
CLVL
~ 95aa ~ 100aa
L CLVL
~ 95aa ~ 100aa
JL
Some of the extra amino
acids are provided by
one of a small set of J or
JOINING regions
L
CLVL
~ 208aa
L

15. The kappa ( and lambda light chains and the heavy chains are
encoded by separate gene families located on separate chromosomes.
In germ line DNA, each of these families contains several gene
sequences called the gene segments, which are separated by
noncoding regions.
During B-cell maturation, these gene segments are rearranged and
brought together to form functional immunoglobulin genes.
The light chain families consist of gene segments : V, J, and C
Rearranged VJ segments encodes- variable region of light chain
The heavy chain families consist of gene segments: V, D, J, and C
Rearranged VDJ segment encodes- variable region of heavy chain
 C gene encodes for constant region. D gene for Diversity.
V gene segment is preceded by 5’ end by a small exon that encodes a
short signal or leader (L) peptide which guides heavy and light chain
through ER
Signal peptide is cleaved from nascent light and heavy chains before
assembly of Ig.

16. l-Chain Multigene Family
V region: 2 Vl gene segments
3 Jl gene segments(13 aa)
C region: 3 Cl gene segments – l1, l2, l3 subtypes (mouse)
In humans: 30 Vl, 4 Jl and 4 Cl segments

17. k-Chain Multigene Family
V region: 85 Vk gene segments
4 Jk gene segments
C region: 1 Ck gene segments (mouse)
In humans: 40 Vk, 5 Jk and 1 Ck segments

18. H-Chain Multigene Family
V region: 134 VH gene segments
13 DH gene segments
4 JH gene segments
C region: 8 CH gene segments (mouse)
In humans: 51 VH, 27 DH, 6 JH and 9 CH segments

19. The H-chain V-region genes rearrange first,
then the L-chain V-region genes.
- The rearrangements are random events

20. 1st rearrangement
2nd rearrangement
A mature , immunocompetent
B cell expresses both IgM &
IgD with identical antigenic
specificity on its surface.

23. Two unique Recombination signal sequences (RSSs)
flanking each germ-line V, D, and J gene segment.
One-turn RSS: located at 3’ to each Vk, 5’ to each Jl, and
both sides of each DH gene segment
Two-turn RSS: located at 3’ to each Vl & VH and
5’ to each Jk & JH gene segment

24. Recombination Signal Sequences (RSS)

25. One turn/two-turn joining rule
-Signal sequences having a one-turn spacer (12 bp)
can join only with sequences having a two-turn
spacer (23 bp) (one-turn/two turn joining rule).
- This joining rule ensures that a VL segment joins
only to a JL segment and not to another VL segment.
- The rule likewise ensures that VH, DH, and JH
segments join in proper order and that segments
of the same type do not join each other.

26. Enzymatic Joining of Gene Segments
 Recombination-Activating Genes:
RAG-1, and RAG-2
(mediate V-(D)-J joining)

28. Deletional joining (coding joint):
-two gene segments are in the same
transcriptional orientation
Inversional joining (signal joint):
- two gene segments have opposite
orientation

33. Deletion of the signal joint and
intervening DNA as a circular
excision product
Retention of both the coding joint
and the signal joint (and inter-
vening DNA) on the chromosome

34. <15 nt

35. Imprecise Joining
- Productive and non-
productive
rearrangements
- Productive
rearrangement in one
allele is enough
- If rearrangement is not
produced, the B cell
dies by apoptosis.

36. Allelic Exclusion
A single B cell is only
specific for a single
epitope !!!
1- J&D rearrange
then V joins.

39. Generation of Antibody Diversity
Seven means of generation of Antibody diversity:
1. Multiple germ-line V, D, and J gene segments.
2. Combinatorial V-(D)-J joining.
3. Junctional flexibility.
4. P-region nucleotide addition (P-addition)
5. N-region nucleotide addition (N-addition))
6. Somatic hypermutation.
7. Combinatorial association of light and heavy
chains.

41. Junctional Flexibility

44. - Up to 15 N-nucleotides can be added to both
the DH-JH and VH-DHJH joints.
- Thus, a complete H-chain V region is encoded
by a VHNDHNJH unit.
- N regions appears to consist of wholly random
sequences

45. Somatic Hypermutation
- Somatic hypermutation occurs only within germinal
centers, structures that form in secondary lymphoid
organs within a week or so of immunization with an
Ag that activates a T-cell-dependent B-cell response.
- Somatic hypermutation occurs at a frequency approaching
10-3/bp/generation.
- This rate is at least 100,000s-fold higher than the
spontaneous mutation rate, about 10-8/bp/penetration, in
other genes.
- B cells with higher-affinity Ig receptors will be
preferentially selected for survival because of their greater
ability to bind to the Ag. ----- Affinity Maturation

46. A Lymph Node

47. The Spleen

48. Peyer’s Patch

49. Class (isotype) switching among C-region genes
-Class-specific recombinase proteins may
bind to switch regions and facilitate DNA
recombination.
- Cytokines secreted by activated TH cells
have been shown to induce B cells to class
switch to a particular isotype.
-IL-4 induces Cm to Cg1 or Ce

53. Secreated μ Membrane μ