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Role of autonomous B cell receptor signalling and external antigen in the pathogenesis of chronic lymphocytic leukaemia (CLL)

Periodic Reporting for period 2 - Autonomous CLL-BCRs (Role of autonomous B cell receptor signalling and external antigen in the pathogenesis of chronic lymphocytic leukaemia (CLL))

Reporting period: 2018-05-01 to 2019-10-31

The ERC project ‘autonomous CLL-BCR’ aims at investigating the molecular mechanisms that activate B cell antigen receptor signalling in chronic lymphocytic leukaemia (CLL). The major goals of the project are: 1. Characterization of the interplay between antigen-driven and autonomous BCR signalling in CLL 2. The impact of BCR isotype on autonomous vs. antigen-induced signalling in CLL 3. Animal models for investigating the role of BCR isotype in signalling processes and CLL pathogenesis in vivo and 4. Characterization of the signalling fingerprints induced by failed differentiation in CLL.
In the first objective, our aim is to determine the fingerprints of autonomous signalling as compared to antigen-mediated signalling for the CLL-BCRs. We wish to express different CLL derived BCRs in TKO expression system and measure their autonomous signalling capacity as well as binding with several internal antigens such as double stranded (ds) DNA (as a measure of autoreactivity), oxidised LDL etc. We also measure the alteration of autonomous signalling of these BCRs upon antigen binding. Once the antigen binding profile is set up for different CLL-BCRs, we will treat primary CLL samples with those selected antigens. Using CyTOF technology, we will then determine the crosstalk between BCR signalling pathway and pathways such as TLR, BAFF/TACI or integrin signalling pathways.
In the second objective, we would like to investigate the role of BCR isotypes in CLL-BCR signalling. It has been observed that certain CLL subsets express only a certain isotype of BCR (for e.g. subset4 always express IgG) and autonomous signalling is only observed with this isotype. This triggers the idea that BCR isotype plays an essential role in mediating CLL pathogenesis. We therefore, clone and express different CLL derived BCRs as IgM and IgG in TKO expression system and measure their autonomous signalling as well as antigen driven signalling properties. Additionally, in order to understand which subclass(es) of IgG is necessary for autonomous signalling, we clone the BCRs as IgG1to IgG4 and determine their signalling properties.
Given the important role of BCR isotypes in CLL pathogenesis, in our third objective, we generate a mouse model for CLL that enables inducible switching from IgM to IgG1 isotype in vivo. For this we use the IgHµγ1flip/flop cassette from IgHµγ1flip/flop mice with a mutated loxP site that allows only a single switch from Cµ to Cγ1. The variable regions of subset 4 and subset 8 CLL cases can be additionally inserted upstream of the cassette leading to expression of CLL BCRs initially as IgM. These mice are then crossed with mb1-creERT2 mouse line. When cre is activated by tamoxifen, the BCR with same variable region will switch to IgG1. We then measure the in vivo signalling properties of these BCRs before and after switching using CyTOF and other conventional techniques.
Since transformation of B cells is often associated with a block in terminal differentiation and deregulation of genes controlling cell cycle and/or apoptosis, our fourth objective is to study the interplay of defective terminal differentiation and other survival and proliferation associated aberrations with the autonomous BCR signalling in developing CLL. For this purpose, we cross the mice generated in objective 3 with mice carrying floxed alleles for B cell terminal differentiation factor Blimp1 and also TP53 and ATM floxed mice. Analysis of CLL pathogenesis in the resulting mice will allow us to understand the role of Blimp1, TP53 or ATM in the development of CLL.
The work done so far in this project enables us to elucidate the structural basis of autonomous activation of CLL B cells. We observed that homotypic BCR-BCR interaction between epitopes leads to the initiation of intracellular signaling in CLL and the strength of this homotypic interaction determines the severity of the disease. Stronger interaction with longer half-lives lead to indolent cases while weaker, short-lived contacts mediate the aggressive ones. The amino acid residues that are important for these contacts are all acquired during class switch recombination or somatic hypermutation (SHM). Our findings provide a molecular and a structural basis for autonomous activation of BCR in CLL. These observations have already led to a publication in 2017 in Nature Communication entitled: Distinct homotypic B-cell receptor interactions shape the outcome of chronic lymphocytic leukaemia (Minici et al., 2017).
In this project, by employing a novel cell based system for the analysis of BCR autoreactivity, isotype specificity and autonomous signaling we could clearly show that autonomous signaling of certain CLL derived BCRs are isotype specific. Similarly, binding of these BCRs to internal antigen such as dsDNA which is also a measure of BCR autoreactivity is also dependent upon BCR isotype. For e.g. a certain CLL derived BCR shows autonomous signaling only when expressed as IgM but not as IgG1. In the same way, autoreactivity of this BCR is only manifested only as IgM BCR but not as IgG1 BCR. It is important to mention here that our cell based approach for determining the BCR autoreactivity is more accurate than the conventional ELISA based method which uses secreted soluble antibodies. We have tested many different BCRs that were already known to be autoreactive in our TKO system. Surprisingly, only a few of the tested BCRs are found to be actually autoreactive. This clearly suggests that autoreactivity of soluble antibodies do not necessarily reflect the autoreactivity of corresponding membrane bound BCRs. These findings are also published as a paper in 2019 in Journal of Immunology entitled: Differences in Self-Recognition between Secreted Antibody and Membrane-Bound B Cell Antigen Receptor (Iype et al., 2019).
The characterization of BCR-intrinsic motifs that are involved in mutual BCR-BCR interactions in crystallography and the subsequent validation in cellular experimental systems will establish autonomous BCR signaling as a central and distinctive mechanism in CLL pathogenesis. Thus, the conventional scenarios suggesting that the BCR on CLL cells is activated by unknown external antigen(s) need to be revisited.
By end of the project, we hope to establish the central and unique role of the BCR in CLL pathogenesis. In addition, we hope that our knowledge of the BCR-intrinsic motifs and the development of respective antibodies recognizing such crucial motifs will not only improve our understanding of CLL pathogenesis but will also result in advanced CLL diagnosis and treatment.