Transcript Characterization of antigen-reactive and nonreactive B cells

19.01.2009
Evgeniya Solodova
Introduction:
Autoimmunity is the failure of an organism to recognize its own constituent parts as
self, which results in an immune response against its own cells and tissues
Examples:
- systemic lupus erythematosus (SLE)
- rheumatoid arthritis
- diabetes mellitus type 1
- others
In terms of antibody-producing B lymphocytes, diseases such as rheumatoid arthritis
and thyrotoxicosis are associated with of loss of immunological tolerance, which is
the ability of an individual to ignore 'self', while reacting to 'non-self'
Introduction:
Autoreactive B cells are regulated in the BM
during development by:
- Clonal deletion, which purges B cells reactive to
self antigens from the repertoire
- Secondary
variable
joining
[V(D)J]
rearrangement mediated by recombination
activating genes (RAG)1 and RAG2, which is
termed receptor revision and occurs at Ig light
chain loci, helps maintain tolerance by modifying
the specificity of the BCR
- Induction of clonal anergy, which renders
autoreactive B cells nonresponsive to BCR
stimulation
Hypothesis:
Receptor revision could be possible mechanism for regulating
self-reacting B cells during an ongoing immune response through
modifying the specificity of the BCR
What was shown before:
• Immunization with a peptide mimetope of dsDNA (DWEYS-MAP) can induce a SLElike serology in the non-autoimmune BALB/c strain
• Using a fluorochrome-tagged tetrameric peptide it was possible to identify the
antigen-specific B cells in the spleens of mice immunized with DWEYS-MAP
• The tetramer binding subset (Tet+) is enriched for B cells reactive to dsDNA
• RAG and λ light chain expression occurs in antigen-activated autoreactive B cells
• No detected RAG expression in antigen-specific B cells in response to a control
peptide that does not generate autoreactivity
RAG is induced in antigen-reactive early memory/plasma B cells
activation following DWEYS-MAP immunization
Where are the RAG-expressing B
cells located?
•Tet+ cells, but not RAG2, are
located in GCs
• RAG2 is coexpressed in
extrafollicular tetramer-binding cells,
but not in the follicular or GC cells
PNA – peanut agglutinin, stains germinal centers
B220 – marker for B cell follicles
RAG is induced in antigen-reactive early memory/plasma B cells
activation following DWEYS-MAP immunization
Which cell population is expressing RAG?
Tet+ cells
Tet+B220hi Tet+B220lo
 Tet+B220lo cells express both RAG1 and RAG2
Characterization of antigen-reactive and nonreactive B cells
qPCR analysis of expression of IgM and IgG1
heavy chain
• Tet+B220lo subset represents a further
stage in differentiation
Characterization of antigen-reactive and nonreactive B cells
qPCR analysis of expression of IgM and IgG1
heavy chain
• Tet+B220lo subset represents a further
stage in differentiation
Do these antigen reactive B cells have experienced GC
maturation?
Evidence of somatic mutation, a characteristic of GC-matured B
cells
sequencing of Ig V genes of Tet+B220lo and Tet+B220hi
cells
Tet+B220hi cells were primarily unmutated
Tet+B220lo cells – both IgH
and IgL V genes were
mutated
Tet+B220lo cells, but not Tet+B220hi cells, have undergone GC differentiation
Characterization of antigen-reactive and nonreactive B cells
AID – activation-induced deaminase – is highly expressed in GC B cells and required for both somatic
hypermutation and class switch recombination
Blimp1, transcriptional repressor, and Xbp1, transcriptional activator, are required for plasma cell differentiation
Characterization of antigen-reactive and nonreactive B cells
AID – activation-induced deaminase – is highly expressed in GC B cells and required for both somatic
hypermutation and class switch recombination
Blimp1, transcriptional repressor, and Xbp1, transcriptional activator, are required for plasma cell differentiation
Tet+B220lo subset has progressed further through a differentiation pathway than
the Tet+B220hi subset
Characterization of antigen-reactive and nonreactive B cells
If the Tet+B220lo subset is a memory cell population?
CD80 and CD95 surface markers are expressed at higher levels in memory than naive B cells
Tet-B220hi
Tet+B220lo
Tet+B220hi
Characterization of antigen-reactive and nonreactive B cells
If the Tet+B220lo subset is a memory cell population?
CD80 and CD95 surface markers are expressed at higher levels in memory than naive B cells
Tet-B220hi
Tet+B220lo
Tet+B220hi
Tet+B220lo B cells identified on day 16 after immunization are early
memory/preplasma B cells that are isotype switched, hypermutated and exiting the
GC reaction
DWEYS-MAP induced RAG expression requires IL-7R signalling
Expression of RAG proteins, or their functionality, in
developing B cells is dependent on IL-7 receptor
(IL-7R) signalling
If IL-7R is involved in the regulation of
RAG expression in the spleen of DWEYSMAP-immunized mice?
• IL-7R is highly upregulated in Tet+B220lo cells
• IL-7R signalling is crucial for RAG2 expression
Inhibition of RAG leads to decrease of λ chain expression
• Variable joining rearrangements lead to exhaustion of the recombinant potential at the κ chain V gene locus and
expression of a λ light chain
• Sign for receptor revision
chains
an increase in Igλ+ (Tet+B220lo) cells and coexpression of Igλ and Igκ light
• Receptor revision is mediated by RAG
Inhibition of receptor revision inhibit λ light chain expression
Inhibition of RAG leads to decrease of λ chain expression
If receptor revision is occurring
selectively in antigen-activated
B cells?
- serum levels of λ chain associated
with IgM or IgG
• Total serum level of IgG was not altered by the treatment with anti-IL-7R antibody
Inhibition of RAG leads to decrease of λ chain expression
If receptor revision is occurring
selectively in antigen-activated
B cells?
- serum levels of λ chain associated
with IgM or IgG
• Total serum level of IgG was not altered by the treatment with anti-IL-7R antibody
Decreased expression of λ light chain is due to inhibition of receptor revision in antigenspecific B cells that are undergoing or have undergone isotype switching
Receptor revision is a mechanism to regulate an antibody response
If receptor revision plays a role in the regulation
of autoreactivity induced by antigen challenge?
primary and memory responses
immunisation with DWEYS-MAP
after
• no significant influence of anti-IL-7R treatment
in primary response on titers of both antipeptide
and anti-DNA IgM and IgG
Receptor revision is a mechanism to regulate an antibody response
If receptor revision plays a role in the regulation
of autoreactivity induced by antigen challenge?
primary and memory responses
immunisation with DWEYS-MAP
after
• no significant influence of anti-IL-7R treatment
in primary response on titers of both antipeptide
and anti-DNA IgM and IgG
- Receptor revision attenuates the autoantibody production arising in the course of a response to
foreign antigen
- Inhibition of receptor revision leads to increased expression of autoreactivity
Soluble antigen induces RAG expression and receptor revision
in the spleens of immunized mice
 RAG is expressed in autoreactive early
memory/plasma B cells, but not in the
equivalent
compartment
in
mice
immunized with non-self peptide (i.e. 10-2)
A. Immunization with DWEYS-MAP
• dsDNA outside the GC environment
(as an antigen) is essential for RAG
induction in Tet+B220lo cells
B-D. Immunization with 10-2-KLH
10-2 peptide – mimetope of phosphorylcholin
KLH – keyhole limpet hemocyanin
protein carriers
BSA – bovine serum albumin
Soluble antigen induces RAG expression and receptor revision
in the spleens of immunized mice
- Increase in Igλ transcript in antigen-reactive B cells
- Increase in IgG-assosiated λ light chain
Soluble antigen induces RAG expression and receptor revision
in the spleens of immunized mice
- Increase in Igλ transcript in antigen-reactive B cells
- Increase in IgG-assosiated λ light chain
Soluble antigen induces receptor revision in antigen-actived B cells
Soluble antigen reduces the humoral response
by induction of receptor revision
If induction of receptor revision alters the
production of 10-2 specific antibody?
• Administration of anti-IL-7R or isotype
control to mice treated with soluble antigen
and measuring serum antibody production
Soluble antigen reduces the humoral response
by induction of receptor revision
If induction of receptor revision alters the
production of 10-2 specific antibody?
• Administration of anti-IL-7R or isotype
control to mice treated with soluble antigen
and measuring serum antibody production
Soluble antigen induces tolerance in antigen-actived B cells during the ongoing
immune response through the induction of receptor revision
Conclusions:
• Tet+B220lo B cells are newly generated early memory/plasma B cells, that have
matured in GC environment
• RAG is specifically induced in Tet+B220lo B cells and its expression requires
signalling through IL-7R
• Soluble antigens induce receptor revision in antigen-reactive cells and diminish
non-autoreactive antibody response
Take home message:
Reinduction of RAG in antigen-activated autoreactive early
memory B cells requires IL-7R signalling, and the resulting receptor
revision contributes to the regulation of autoreactivity
Thank you for your attention!
Adaptive immunity is based on
clonally distributed antigen receptors
that arise from random
recombination of V, D and J
segments encoded in H and L chain
and TCR loci.
Variable (diversity) joining [V(D)J]
rearrangement mediated by RAG1
and RAG2, which is termed receptor
editing, occurs particulary in L chain
of Ig, modifies specificity of the BCR
Genetic recombination within the immunoglobulin locus is the major source of BCR
diversity, as different immunoglobulin variable domain sequences confer different
antigen binding specificities to the receptor. The Rag 1 and Rag 2 proteins, which
mediate recombination, are up-regulated under conditions when rearrangement of the
heavy and light chain sequences is required and down-regulated at other times. Once a
B cell produces an antigen receptor, it is normally prevented from further rearrangement
of the heavy and light chain sequences (allelic exclusion). In the process of receptor
editing, however, a B cell re-expresses the Rag proteins and then can produce alternate
light chain sequences. Replacement light chains are paired with the existing heavy
chain and the modified BCR is once again subjected to antigen selection. If receptor
editing results in a BCR unresponsive to self antigen, the B cell continues along the
development pathway. If receptor editing results in a different BCR that is still
autoreactive, rearrangement of the light chain locus will continue. Autoreactive B cells
which cannot re-express their Rag proteins will be deleted by apoptosis.
Individual fate of autorreactive B cell is determined by various factors:
Strength of signalling through BCR
The developmental stage at which BCR engagement by self antigen occurs
Whether the antigen is soluble or membrane bound
Presence of costimulatory factors, cytokines or TLR ligands that can rescue B cells
triggered for tolerance induction
Perioheral B cell tolerance is also important for protection from autoimmune damage,
although the mechanisms are less defined.