Age-associated B cells expanded in autoimmune mice are memory cells sharing H-CDR3-selected repertoires.

Age-associated B cells (ABCs) represent a distinct cell population expressing low levels of CD21 (CD21-/low ). The Ig repertoire expressed by ABCs in aged mice is diverse and exhibits signs of somatic hypermutation (SHM). A CD21-/low B-cell population is expanded in autoimmune diseases, e.g. systemic lupus erythematosus, as well as in lupus-prone NZB/W mice and in mice lacking a pre-B cell receptor (SLC-/- ). However, the nature of the CD21-/low B cells (hereafter ABCs) in autoimmunity is not well understood. Here we show that in young SLC-/- mice, the vast majority of the ABCs express memory B-cell (MBC) markers in contrast to wild-type controls. A similar population is present in lupus-prone MRL mice before and at disease onset. In SLC-/- mice, a majority of the ABCs are IgM+ , their VH genes have undergone SHM, show clonal diversification and clonal restriction at the H-CDR3 level. ABC hybridomas, established from SLC-/- mice, secrete typical lupus autoantibodies, e.g. anti-Smith antigen, and some of those that bind to DNA comprise a H-CDR3 that is identical to previously described IgM anti-DNA antibodies from lupus-prone mice. Together, these results reveal that ABCs in autoimmune mice are comprised of autoreactive MBCs expressing highly restricted H-CDR3 repertoires.

Clonal relationships of memory B cell subsets in autoimmune mice.

Immunological memory protects our body from re-infection and it is composed of a cellular and a humoral arm. The B-cell branch with its memory B cells (MBCs), plasma cells and antibodies, formed either in a germinal centre (GC) -dependent or -independent manner, ensure that we can rapidly mount a recall immune response. Previous work in immunised wildtype (WT) mice have identified several subsets of MBCs whereas less is known under autoimmune conditions. Here, we have investigated the heterogeneity of the MBC compartment in autoimmune mouse models and examined the clonal relationships between MBC subsets and GC B cells in one of the models. We demonstrate the presence of at least four different MBC subsets based on their differential expression pattern of CD73, CD80 and PD-L2 in surrogate light chain-deficient (SLC-/-), MRL+/+ and MRLlpr/lpr mice, where most of the MBCs express IgM. Likewise, four MBC subsets could be identified in WT immunised mice. In SLC-/- mice, high-throughput sequencing of Ig heavy chains demonstrates that the two CD73-positive subsets are generally more mutated. Lineage tree analyses on expanded clones show overlaps between all MBC subsets and GC B cells primarily in the IgM sequences. Moreover, each of the three IgM MBC subsets could be found both as ancestor and progeny to GC B cells. This was also observed in the IgG sequences except for the CD73-negative subset. Thus, our findings demonstrate that several MBC subsets are present in autoimmune and WT mice. In SLC-/- mice, these MBC subsets are clonally related to each other and to GC B cells. Our results also indicate that different MBC subsets can seed the GC reaction.

Short-term assessment of BCR repertoires of SLE patients after high dose glucocorticoid therapy with high-throughput sequencing.

We analyze and assess BCR repertoires of SLE patients before and after high dose glucocorticoid therapy to address two fundamental questions: (1) After the treatment, how the BCR repertoire of SLE patient change on the clone level? (2) How to screen putative autoantibody clone set from BCR repertoire of SLE patients? The PBMCs of two SLE patients (P1 and P2) at different time points were collected, and DNA of these samples were extracted. High-throughput sequencing technology was applied in detection of BCR repertoire. Finally, we used bioinformatic methodology to analyse sequence data. We found that these two patients lost some IGHV3 family genes usage after treatment compared with before treatment. For pairing of IGHV-IGHJ gene, no significant change was shown for each patient. In addition, analyses of the composition of H-CDR3 showed overall AA compositions of H-CDR3 at three time points in each SLE patients were very similar, and the results of H-CDR3 AA usage that had the same length (14 AA) and the same position were similar. Antinuclear antibody tests of SLE patients showed that level of some antinuclear antibodies reduced after treatment; however, there was no sign that the percentage of autoantibody clones in BCR repertoires would reduce. High dose glucocorticoid treatment in short term will have little impact on composition of BCR repertoire of SLE patient. Treatment can reduce the amount of autoantibody in the protein level, but may not reduce the percentage of autoantibody clones in BCR repertoire in the clonal level.