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

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 CD307bhi, CD258hiCD72hi, and CD21loPD-1hi 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.

Microbiota maintain colonic homeostasis by activating TLR2/MyD88/PI3K signaling in IL-10-producing regulatory B cells.

Resident microbiota activate regulatory cells that modulate intestinal inflammation and promote and maintain intestinal homeostasis. IL-10 is a key mediator of immune regulatory function. Our studies described the functional importance and mechanisms by which gut microbiota and specific microbial components influenced the development of intestinal IL-10-producing B cells. We used fecal transplant to germ-free (GF) Il10+/EGFP reporter and Il10-/- mice to demonstrate that microbiota from specific pathogen-free mice primarily stimulated IL-10-producing colon-specific B cells and T regulatory-1 cells in ex-GF mice. IL-10 in turn down-regulated microbiota-activated mucosal inflammatory cytokines. TLR2/9 ligands and enteric bacterial lysates preferentially induced IL-10 production and regulatory capacity of intestinal B cells. Analysis of Il10+/EGFP mice crossed with additional gene-deficient strains and B cell co-transfer studies demonstrated that microbiota-induced IL-10-producing intestinal B cells ameliorated chronic T cell-mediated colitis in a TLR2, MyD88 and PI3K-dependent fashion. In vitro studies implicated PI3Kp110δ and AKT downstream signaling. These studies demonstrated that resident enteric bacteria activated intestinal IL-10-producing B cells through TLR2, MyD88 and PI3K pathways. These B cells reduced colonic T cell activation and maintained mucosal homeostasis in response to intestinal microbiota.

Killer (FASL regulatory) B cells are present during latent TB and are induced by BCG stimulation in participants with and without latent tuberculosis.

Regulatory B cells (Bregs) have been shown to be present during several disease states. The phenotype of the cells is not completely defined and the function of these cells differ between disease. The presence of FASL expressing (killer) B cells during latent and successfully treated TB disease have been shown but whether these cells are similar to regulatory B cells remain unclear. We assessed the receptor expression of FASL/IL5 (killer B cells), CD24/CD38 (regulatory B cells) on whole peripheral blood of participants with untreated active TB and healthy controls. We then isolated B cells from a second cohort of M.tb exposed (Quantiferon (QFN) positive) and unexposed (Quantiferon negative) HIV negative participants, and evaluated the frequency of killer B cells induced following stimulation with BCG and/or CD40 and IL5. Our data reveal no difference in the expression on CD24 and CD38 between participants with active TB and the controls. There was also no difference in the frequency of regulatory B cells measured in the peripheral blood mononuclear cells (PBMC) fraction between latent TB and uninfected controls. We did however notice that regulatory B cells (CD24hiCD38hi) population express the FASL receptor. The expression of killer B cell phenotype (CD178+IL5RA+) was significantly higher in controls compared to those with active TB disease (1,06% vs 0,455%). Furthermore, we found that BCG restimulation significantly induced the FASL/IL5RA B cells but this was only evident in the QFN positive group. Our data suggest that both regulatory and killer B cells are present during latent and active TB disease but that the frequency of these populations are increased during latent disease. We also show that the FASL+IL5RA+ B killer B cells are induced in latent TB infection following BCG restimulation but whether these cells are indicative of protection remains unclear.