Transcript No Slide Title

B cell effector functions II
•Overview of antibody response and changes in antibodies
•Antibody class switching
-brief review of effector functions
•Class switching mechanism- part 1 overview and targeting
-distinction from V(D)J recombination
-targeting of switch recombination by cis acting elements
-relationship of targeting to sterile transcription
•Somatic mutation
-the striking restriction of mutation
-mutation or lack of repair?
-models of mutation with relation to DNA repair
-paradoxical effect of repair mutants
•AID and a unified model of somatic mutation and class switch
-initial lesion
-repair and resolution
The purpose of the antibody response is to tag specifically the
microbe, marking it for appropriate disposal.
•The variable portion of the antibody confers affinity/specificity
•The constant portion regulates the effector function
•A high affinity and appropriate effector class must be established
Antibody protein sequence and bioactivity changes during the immune response
Low affinity
IgM class
High affinity
IgG class
IgM
Antibody protein sequence is altered in several independent ways
•(Pre-immune) membrane IgM/IgD coexpression RNA splicing
•Regulation of membrane vs secreted IgM RNA splicing
•Switch to IgG, IgA, IgE antibody H-chain C exons
DNA class switch recombination
•Affinity maturation
Somatic hypermutation
(in some species, gene conversion)
VDJ joining here creates heavy chain variable region domain
a
VD
Note: there are exons encoding the membrane and secreted
forms of each of the antibody heavy chains.
Antibody heavy chain locus
VDJ recombination
Class switch is a DNA
recombination, distinct from
VDJ recombination, that occurs
in immune responses
RNA splicing controls expression of
membrane vs secreted IgM
Immunoglobulin classes
Information/
Specificity
Effector/ triage
function
Antibodies come in different classes
2
3
4
2
V(D)J recombination
•Short, conserved recognition sequence
•RAG1/2 cleaves DNA
•Resolved by DNA repair proteins that
specialize in non-homologous end joining.
•“Calculated imprecision” introduced to
create diversity.
Sm
VDJ
Sa
Cm
Ca
•Joining can occur anywhere within the large, repetitive
S-regions. There is no obvious conserved sequence motif.
•Unlike V(D)J recombination, this gene splicing occurs
between, rather than within, coding sequences.
•Like V(D)J recombination, targeting of elements for
rearrangement is correlated with prior “sterile transcription”
Like V(D)J recombination, class switch recombination is regulated by
1) expression of a recombination machine
2) targeted “accessibility” mediated by nearby enhancers and promoters
Promoter upstream of human IgE I-region
Accumulation of Vregion point mutations
during the antibody
response.
Somatic mutations are focussed on the variable region exon
Promoter
Enhancer elements
From Gearhart and Bogenhagen PNAS 80:3439, 1983
Patricia J. Gearhart and Richard D. Wood
One proposed way that
a mutator could have
localized action
Clues from the pattern of somatic mutation
Strand bias, transitions>transversions,
bias vs pyrimidines (as assessed on coding strand).
To
From
A
G
C
T
A
68
20
14
G
46
0
14
% of observed
C
29
24
72
T
25
8
80
-
mutations
33
35
21
10
From Betz et al, PNAS 90:2385.
Is antibody mutation induced?
•Natural mutation rate in the absence of repair is high
~10-5/bp/generation
•With repair, spontaneous mutation rate is ~10-9 or less.
•Repair pathways
-DNA polymerase 3’-->5’ exonucleolytic proofreading
improves fidelity ~100X
-mismatch repair system
improves fidelity ~100X
•Initial estimates found values of 10-3-10-5/bp/generation
in clonally related B cells carrying mutations.
•Suggested that repair is either turned off or mutation is
induced.
Characteristics of Somatic Mutation
1. Occurs at high rates: 10 -4 -10 -3 /bp/generation.
2. Occurs by untemplated single base substitutions.
3. Restricted to a brief period of B cell differentiation.
4. Restricted to the rearranged V region and its immediate flanking sequences.
5. Occurs in germinal centers with T cell help.
6. Occurs throughout the V region but more frequently in RGYW (A/G G C/T A/T) motifs.
7. Mutations in kappa light chain transgenes require intronic and 3’ enhancers
but not in the V region promoter or V coding region.
BioEssays 20:227–234, 1998
-If mutations are routinely removed from replicating
DNA, a process that prevents repair locally would target
mutation. If so, knockouts of DNA repair genes would
have little or no effect.
-If mutations are introduced by massive local DNA
damage, possibly needed to overwhelm the normal
repair mechanisms, then repair mutants would have
increased mutation rates in the targeted regions (near
assembled VDJs).
-Alternatively, DNA repair enzymes may be needed to
generate mutations.
Mutation models involving error-prone DNA polymerases
Mismatch repair in bacteria
Mut mutants in bacteria have “mutator” phenotype
Marti et al. J. Cell. Physiol. 191:28 (2002)
Mismatch repair in eukaryotes
Msh2,6 +/- linked to hereditary non-polyposis colon cancer (HNPCC)
Martin and Scharff Nat. Rev. 2:605 (2002)
MSH2-/- mice
have reduced
antibody gene
mutation
The authors suggested that the
mutator might target the “wrong”
strand for repair,
in effect co-opting the mismatch
repair process to introduce
mutations.
Cascalho et al, Science (1998) 279:1207
Table 2. Distribution of Mutations in Hot Spots vs Background Mutations
Position
39
56
62
253
TGT
AGC
GCA
GCT
Msh2+/-
Msh-/-
2.1% (11)
2.7% (14)
1.8% (9)
1.6% (8)
8.2% (42)
4.6% (7)
9.3% (14)
4.6% (7)
6.6% (10)
25.1% (38)
Rada et al. (1998) Immunity, 9:135.
,i
… but what would cause the initial mismatch?
A surprising finding: these three processes are
dependent on a single gene, activation induced
deaminase (AID). AID-deficient mice do not switch to
IgG, IgA, IgE or show much antibody hypermutation.
The closest homologue to activation induced deaminase (AID)
is APOBEC-1, an RNA editase involved in lipid metabolism.
Figure 2. Occurrence of somatic mutation in one DNA strand in the G1 phase of the cell cycle. Somatic mutation
was induced in BL2 cells in the G1 phase of the cell cycle. Single cells were either analyzed for mutations in the
V4-39 gene after 90 min of stimulation or isolated in single wells and left for 24 or 48 h (one or two divisions)
before analysis. (a) Three representative mutations in the V4-39 gene, which show a mixed sequence. (b )
Visualization of one, two and four cells. Note the streptavidin beads that cross-link the biotinylated anti-IgM
bound at the cell surface. (c ) Three patterns were observed when two BL2 cells that differed at a single position
in their V gene (nucleotide 57) were amplified. In addition to the expected configuration of amplification of a
mixed sequence (left), cases of biased amplification were observed, which resulted in amplification of either of the
two "alleles" (middle and right). (d) Schematic representation of mutation occurrence on a single strand of DNA,
segregated in the same 50:50 proportion after cell division.
Faili, A. et al. Nature Immunology 3, 815 - 821 (2002)
,i
Point mutations
in the Sµ region can
occur under switching
culture conditions, but
prior to switching,
consistent with a
common mechanism for
the two
types of DNA
modification.
Deletions,
mutations and
short
duplications
are associated
with switch
recombination
DNA repair
system involved
Mismatch repair
Homologous
recombination
Non-homologous
end joining
Relationship to disease
•The hypermutation/ class switch mechanism could be
disastrous if not correctly targeted, leading to
translocations, point mutations, in appropriate gene
conversions. It is not know if the mutator can be
activated in non-B cell tumors.
•Surprisingly, the gene of the germinal center B cell
specific transcription factor Bcl6 is often mutated in
normal B cells, but many other tested genes are not
mutated at higher rates.
•Many lymphoid tumors involve breakpoints between Ig
genes and oncogenes. Some of these are associated
with V(D)J type recombination, others with class switch,
others with functional V regions.
Modern drug research and development (duration ~10 years):
•random screening of millions of compounds in bioassay
to find initial candidates
•refining candidates based on minor substitutions and selection
for improved affinity and specificity
•toxicity and efficacy trials
The antibody response (duration ~14 days):
•select a few thousand (or fewer) cells among millions of B cells
•point mutate and select to develop highest affinities*
•class switch to appropriate effector class
*Important for recognition of microbes that mutate rapidly
Harnessing the power of the immune system
•Rapid generation of antibody reagents.
•AID can mutate many genes when transiently
overexpressed in cell lines.Recruiting the
mutator system to selected non-Ig genes may be
useful for protein engineering.
•Understanding selection and how it is
optimized.