1. 19.01.2009 Evgeniya Solodova

2. 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'

3. 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:

4. 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:

5. 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

6. RAG is induced in antigen-reactive early memory/plasma B cells activation following DWEYS-MAP immunization PNA – peanut agglutinin, stains germinal centers B220 – marker for B cell follicles 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

7. RAG is induced in antigen-reactive early memory/plasma B cells activation following DWEYS-MAP immunization Tet + cells Tet + B220 hi Tet + B220 lo Which cell population is expressing RAG? Tet + B220 lo cells express both RAG1 and RAG2

8. Characterization of antigen-reactive and nonreactive B cells qPCR analysis of expression of IgM and IgG1 heavy chain Tet + B220 lo subset represents a further stage in differentiation

9. 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 maturation? Evidence of somatic mutation, a characteristic of GC-matured B cells sequencing of Ig V genes of Tet + B220 lo and Tet + B220 hi cells Tet + B220 hi cells were primarily unmutated Tet + B220 lo cells – both IgH and IgL V genes were mutated Tet + B220 lo cells, but not Tet + B220 hi cells, have undergone GC differentiation Tet + B220 lo subset represents a further stage in differentiation

10. 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

11. 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 + B220 lo subset has progressed further through a differentiation pathway than the Tet + B220 hi subset

12. Characterization of antigen-reactive and nonreactive B cells If the Tet + B220 lo subset is a memory cell population? CD80 and CD95 surface markers are expressed at higher levels in memory than naive B cells Tet + B220 lo Tet + B220 hi Tet - B220 hi

13. Characterization of antigen-reactive and nonreactive B cells If the Tet + B220 lo subset is a memory cell population? CD80 and CD95 surface markers are expressed at higher levels in memory than naive B cells Tet + B220 lo Tet + B220 hi Tet - B220 hi Tet + B220 lo B cells identified on day 16 after immunization are early memory/preplasma B cells that are isotype switched, hypermutated and exiting the GC reaction

14. 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 DWEYS- MAP-immunized mice? IL-7R is highly upregulated in Tet + B220 lo cells IL-7R signalling is crucial for RAG2 expression

15. 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 + B220 lo ) cells and coexpression of Igλ and Ig κ light chains Inhibition of receptor revision inhibit λ light chain expression Receptor revision is mediated by RAG

16. 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

17. 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 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

18. 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 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

19. 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 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

20. A. Immunization with DWEYS-MAP B-D. Immunization 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 phosphorylcholin KLH – keyhole limpet hemocyanin BSA – bovine serum albumin dsDNA outside the GC environment (as an antigen) is essential for RAG induction in Tet + B220 lo cells protein carriers

21. 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

22. 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

23. 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

24. 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

25. Conclusions: Tet + B220 lo B cells are newly generated early memory/plasma B cells, that have matured in GC environment RAG is specifically induced in Tet + B220 lo 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

26. 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

27. Thank you for your attention!

28. 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

29. 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.