19.01.2009 Evgeniya Solodova. Introduction: Autoimmunity is the failure of an organism to recognize its own constituent parts as self, which results in.

19.01.2009Evgeniya 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'

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

Introduction:

Receptor revision could be possible mechanism for regulating self-reacting B cells during an ongoing immune response through

modifying the specificity of the BCR

Hypothesis:

What was shown before:

• Immunization with a peptide mimetope of dsDNA (DWEYS-MAP) can induce a SLE-like 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

PNA – peanut agglutinin, stains germinal centersB220 – marker for B cell follicles

Where are the RAG-expressing B Where are the RAG-expressing B cells located?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

RAG is induced in antigen-reactive early memory/plasma B cells activation following DWEYS-MAP immunization

Tet+ cells

Tet+B220hi Tet+B220lo

Which cell population is expressing RAG?Which cell population is expressing RAG?

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

Do these antigen reactive B cells have experienced GC Do these antigen reactive B cells have experienced GC maturation?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 unmutatedTet+B220lo cells – both IgH and IgL V genes were mutated

Tet+B220lo cells, but not Tet+B220hi cells, have undergone GC differentiation

• Tet+B220lo subset represents a further stage in 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 TetIf the Tet++B220B220lolo subset is a memory cell population? subset is a memory cell population?

CD80 and CD95 surface markers are expressed at higher levels in memory than naive B cells

Tet+B220lo

Tet+B220hi

Tet-B220hi

Characterization of antigen-reactive and nonreactive B cells

If the TetIf the Tet++B220B220lolo subset is a memory cell population? subset is a memory cell population?

CD80 and CD95 surface markers are expressed at higher levels in memory than naive B cells

Tet+B220lo

Tet+B220hi

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 If IL-7R is involved in the regulation of RAG expression in the spleen of DWEYS-RAG expression in the spleen of DWEYS-MAP-immunizedMAP-immunized mice? 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 an increase in Igλ+ (Tet+B220lo) cells and coexpression of Igλ and Igκ light chains

Inhibition of receptor revision inhibit λ light chain expression

• Receptor revision is mediated by RAG

Inhibition of RAG leads to decrease of λ chain expression

If receptor revision is occurring If receptor revision is occurring selectively in antigen-activated selectively in antigen-activated B cells?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

Decreased expression of λ light chain is due to inhibition of receptor revision in antigen-specific B cells that are undergoing or have undergone isotype switching

If receptor revision is occurring If receptor revision is occurring selectively in antigen-activated selectively in antigen-activated B cells?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

Receptor revision is a mechanism to regulate an antibody response

If receptor revision plays a role in the regulation If receptor revision plays a role in the regulation of autoreactivity induced by antigen challenge?of autoreactivity induced by antigen challenge?

primary and memory responses after immunisation with DWEYS-MAP

• 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 If receptor revision plays a role in the regulation of autoreactivity induced by antigen challenge?of autoreactivity induced by antigen challenge?

primary and memory responses after immunisation with DWEYS-MAP

• 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

A. A. Immunization with DWEYS-MAPImmunization with DWEYS-MAP

B-D. B-D. Immunization with 10-2-KLHImmunization with 10-2-KLH

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)

10-2 peptide – mimetope of phosphorylcholinKLH – keyhole limpet hemocyaninBSA – bovine serum albumin

• dsDNA outside the GC environment (as an antigen) is essential for RAG induction in Tet+B220lo cells

protein carriers

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 If induction of receptor revision alters the production of 10-2 specific antibody?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 If induction of receptor revision alters the production of 10-2 specific antibody?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 BCRThe developmental stage at which BCR engagement by self antigen occursWhether the antigen is soluble or membrane boundPresence 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.