Background/Purpose: Systemic lupus erythematosus (SLE) is a disease where autoreactive antibodies are produced as a result of abnormal B cell activation and broken self-tolerance. This activation is perhaps most prominently observed in elevated levels of circulating activated cells within the naïve compartment of SLE patients. The origin, diversity, and direct developmental consequences of this elevated activation are largely unclear though. In this study, we use a variety of multi-color flow cytometry panels to isolate and sort specific bone marrow (BM) and circulating B cell populations for immunoglobulin heavy chain (IGH) deep sequencing analysis. By developing a novel program to trace their inter-population relationships through mutational analysis, we aim to further characterize these cells and their contribution to the pathogenesis of SLE.

Methods: Peripheral blood and BM aspirates from healthy donors and volunteers with SLE were collected and sorted using several multi-color flow cytometry panels. The IGH gene was amplified from these cells using multiple sets of VH family-specific and isotype-specific primers. Deep sequencing was conducted using Illumina MiSeq technology, and results were analyzed using an internally developed analysis program that performs quality filtering, detailed mutational analysis, and identification and matching of clonal lineages.

Results: We found that a population of circulating mitotracker green (MTG) positive, CD23- activated naïve cells were highly elevated in flaring SLE patients, slightly elevated in non-flaring patients, and largely absent in healthy controls. This population was also identified in BM of SLE patients, and it was found to be highly elevated in all SLE samples. Deep sequencing analysis showed that unlike most other populations in the circulation and BM, this population was highly oligoclonal, with some clones accounting for greater than 5% of the total population. Sequences from these activated naïve cells were somatically mutated and typically found to be of lower VH mutation rates than memory and plasmablasts (PB), but higher than resting naïve cells. We found clonal relations between several circulating and BM B cell subsets and tracked the lineage maturation using mutation analysis. Surprisingly, the activated naïve fraction was highly related to PB and through mutational tree analysis, they were found to inhabit the more proximal portion of the same lineage branches as the more distal PB.

Conclusion: Our data suggest that in SLE, there is an expansion of activated MTG+, CD23- naive cells which may be an early implication of abnormal B cell activation as a result of damaged self-tolerance. We show that these cells are highly related to circulating and BM antibody-producing PB, which are typically also expanded in SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/bone-marrow-in-systemic-lupus-erythematosus-patients-contain-a-highly-elevated-proportion-of-somatically-mutated-activated-naive-cells-comprised-of-substantial-clonal-expansions/