Efficient Expansion of Human Granzyme B-Expressing B Cells with Potent Regulatory Properties

Abstract

Granzyme B-expressing B cells have been shown to be an important regulatory B cell subset in humans. However, it is unclear which subpopulations of B cells express GZMB under normal conditions and which protocols effectively induce ex vivo expansion of GZMB⁺ B cells. We found that in the peripheral blood of normal individuals, plasmablasts were the major B cell subpopulation that expressed GZMB. However, when using an in vitro plasmablast differentiation protocol, we obtained only 2% GZMB⁺ B cells. Nevertheless, using an expansion mixture containing IL-21, anti-BCR, CpG oligodeoxynucleotide, CD40L, and IL-2, we were able to obtain more than 90% GZMB⁺ B cells after 3 d culture. GZMB⁺ B cells obtained through this protocol suppressed the proliferation of autologous and allogenic CD4⁺CD25^- effector T cells. The suppressive effect of GZMB⁺ B cells was partially GZMB dependent and totally contact dependent but was not associated with an increase in effector T cell apoptosis or uptake of GZMB by effector T cells. Interestingly, we showed that GZMB produced by B cells promoted GZMB⁺ B cell proliferation in ERK1/2-dependent manner, facilitating GZMB⁺ B cell expansion. However, GZMB⁺ B cells tended to undergo apoptosis after prolonged stimulation, which may be considered a negative feedback mechanism to limit their uncontrolled expansion. Finally, we found that expanded GZMB⁺ B cells exhibited a regulatory phenotype and were enriched in CD307b^hi, CD258^hiCD72^hi, and CD21loPD-1^hi B cell subpopulations. Our study, to our knowledge, provides new insight into biology of GZMB⁺ B cells and an efficient method to expand GZMB⁺ B cells for future cell therapy applications.