Development of a novel, highly sensitive assay for quantification of minimal residual B cells in autoimmune disease and comparison to traditional methods across B-cell-depleting agents.

Targeted B-cell depletion is a useful therapy for many diseases, including autoimmune disorders and certain cancers. We developed a sensitive blood B-cell depletion assay, MRB 1.1, compared its performance with the T-cell/B-cell/NK-cell (TBNK) assay, and assessed B-cell depletion with different therapies. The empirically defined lower limit of quantification (LLOQ) for CD19+ cells in the TBNK assay was 10 cells/µL, and 0.441 cells/µL for the MRB 1.1 assay. The TBNK LLOQ was used to compare differences between B-cell depletion in similar lupus nephritis patient populations who received rituximab (LUNAR), ocrelizumab (BELONG), or obinutuzumab (NOBILITY). After 4 weeks, 10% of patients treated with rituximab retained detectable B cells vs 1.8% with ocrelizumab and 1.7% for obinutuzumab; at 24 weeks 93% of patients who received obinutuzumab remained below LLOQ vs 63% for rituximab. More-sensitive measurements of B cells may reveal differences in potency among anti-CD20 agents, which may associate with clinical outcomes.

All hematopoietic cells develop from hematopoietic stem cells through Flk2/Flt3-positive progenitor cells.

While it is clear that a single hematopoietic stem cell (HSC) is capable of giving rise to all other hematopoietic cell types, the differentiation paths beyond HSC remain controversial. Contradictory reports on the lineage potential of progenitor populations have questioned their physiological contribution of progenitor populations to multilineage differentiation. Here, we established a lineage tracing mouse model that enabled direct assessment of differentiation pathways in vivo. We provide definitive evidence that differentiation into all hematopoietic lineages, including megakaryocyte/erythroid cell types, involves Flk2-expressing non-self-renewing progenitors. A Flk2+ stage was used during steady-state hematopoiesis, after irradiation-induced stress and upon HSC transplantation. In contrast, HSC origin and maintenance do not include a Flk2+ stage. These data demonstrate that HSC specification and maintenance are Flk2 independent, and that hematopoietic lineage separation occurs downstream of Flk2 upregulation.