143 General discussion and future perspectives higher ST-ST overlap than SF-ST overlap. This might indicate that there is more circulation of B-cells than T-cells from the synovium to the synovial fluid. Since intra-joint overlaps observed in the ST-SF and ST-ST comparisons are in the same range, our data suggest that B-cells derived from synovial fluid or alternatively from synovial tissue are equally informative regarding antigen specificity of B-cells in the synovium. However, more studies are required to further characterize these shared TCR and BCR clones. This might entail functional characterization, including antigen specificity, in order to develop antigen receptor directed therapies. Of note, it might also require additional genomic and proteomic analyses in order to identify more uniform cellular targets selectively present in the activated B- and/or T-cell clones. Isolation of B-cells from ST without modification of the expression of surface markers is challenging, but our studies suggest that the use of SF could be a good alternative. In 2018 Germar et al. performed a proof-of-concept study and were able to immortalize an antigen-specific subset of B-cells (i.e. anti-citrullinated protein antibody-producing B-cells), by genetic reprogramming using B cell lymphoma-6 (Bcl-6) and Bcl-xL proteins, isolated from both blood and SF to study their molecular and phenotypic properties [1,2]. However, this also had its challenges, as only a very limited number of ACPA-specific clones, i.e. three, were stably immortalized, even though many clones (> 300) were generated from 12 RA patients. So, this is a very challenging approach, but not impossible, and it revealed important information on auto-reactive B-cells in RA, more specifically increased CD40 expression and secretion of both pro- and anti-inflammatory cytokines. We believe it to be important to embark on these types of analyses in the near future, preferably in non-transformed cells analyzed directly ex vivo, in order to develop more selective immunotherapy. Aforesaid analyses combined with a selection of the most expanded and shared TCR/BCR clones could shine light on such potential new RA-specific targets. Furthermore, we noticed that the overlap observed in the top T-cell clones within and between synovial tissue samples taken in the same and in different joints is much more pronounced than the overlap in the B-cell compartment. This is even more clear in the comparison between synovial tissue and peripheral blood. This suggests that B-cell responses might be much more localized than T-cell responses. This might suggest localized proliferation and/or maturation accompanied by an increase in BCR expression, e.g. in the transformation of mature B-cells to plasma cells. Another explanation could be an antigen-driven influx of specific B-cell clones. Such a localized influx of B-cells into the joint on a stable TCR background might result in clinical arthritis. It would be very interesting to further investigate this into more detail, e.g. in a prospective study in seropositive individuals at increased risk of RA which involves sampling of paired ST, SF, and blood in all phases of the 7
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