Fig 1.
SRL effectively inhibited B-cell proliferation.
Purified CD19+ B cells were labeled with CFSE, stimulated with anti-IgM, anti-CD40 mAb and IL-21 (BCR method) in the absence (control; CTRL) or presence of TAC (6ng/ml) or SRL (2ng/ml or 6ng/ml) and flow cytometric analyses were performed after 6 days in culture. (A) A representative experiment: cells were gated on viable lymphocytes and analyzed for CFSE diluting proliferating cells. This scheme of analysis was used in all subsequent experiment, unless indicated otherwise. (B) The percentage of proliferating CD19+ B cells as obtained in A from 7 different experiments. (C) Absolute number of proliferating CD19+ B cells was calculated in each experiment by multiplying the recovered cell counts with the percentage of proliferating cells as in A (n = 7). Statistically significant (*p < 0.05) inhibition of B cell proliferation was observed with SRL at both subtherapeutic (2ng/ml) and therapeutic (6ng/ml) doses.
Fig 2.
B-cell stimulation in the presence of SRL resulted in a population shift toward an activated phenotype.
Purified CD19+ B cells were stimulated with anti-IgM, anti-CD40 mAb and IL-21 and multi-color flow cytometric analyses were performed on day 6 as described in Fig 1. The figures A, B and C show the expression of various surface markers on stimulated B cells (Percentage of positive cells/total proliferating CD19+ cells) p < 0.05, **p < 0.01. CTRL, control; TAC6, 6 ng/ml TAC; SRL2, 2 ng/ml SRL; SRL6, 6 ng/ml SRL.
Fig 3.
SRL but not TAC inhibited the proliferation of CD19+CD27− naïve and CD19+CD27+ memory B cells.
B cells were purified by depleting non-B cells resulting in >95% CD19+ cells which subsequently sorted into CD27− (naïve) and CD27+ (memory) B-cell fractions. These subsets were labeled with CFSE, stimulated with anti-IgM, anti-CD40 mAb and IL-21 in the absence (CTRL) or presence of TAC or SRL at 6ng/ml and were analyzed by multicolor flow cytometry after 6 days in culture. (A) A representative experiment: histogram plots show dilution of CFSE in the proliferating CD19+CD27− (upper panel) or CD19+CD27+ (lower panel) cells. (B) Data are from four different independent experiments are shown as mean ± SD percent proliferating naïve CD19+CD27− and memory CD19+CD27+ B cells. (C) B cell subsets showing indicated surface markers were analyzed and plotted as mean ± SD (n = 4) percent of proliferating cells in the cultures of naïve CD19+CD27− (upper panel) and memory CD19+CD27+ B cells (lower panel). The residual cells that proliferated in presence of SRL demonstrated an activated phenotype. *p < 0.05, **p < 0.01.
Fig 4.
SRL, but not TAC effectively inhibited the differentiation of B cells into plasma cells.
Purified CD19+ B cells were cultured as in Figs 1 and 2. (A) A representative experiment showing flow cytometric profile indicative of putative plasma cells (CD19low) in proliferated B cells (gated on viable lymphocytes; see Fig 1A). (B) The mean ± SD percentage of such CD19low B cells from 4 different independent experiments. (C) Mean ± SD (n = 4) percentage of CD19lowCD38++ plasmablasts, CD19lowCD138+ plasma cells, Blimp1+PAX5- cells and CD138+Blimp1+ cells in the proliferating CD19low cells. *p < 0.05, **p < 0.01. CTRL, control; TAC, 6 ng/ml TAC; SRL, 6 ng/ml SRL.
Fig 5.
SRL was more effective in inhibiting IL-6 and IL-10 production in B cells.
Cytokine levels were measured in the culture supernatants of stimulated B cells in the absence or presence of 6ng/ml SRL or TAC on day 6. SRL markedly inhibited production of IL-10 and IL-6 by B cells compared to control and TAC. No significant difference was found for IL-1beta, IL-4, IL-7, IL-8, TNF-alpha and GM-CSF. *p < 0.05, **p < 0.01.
Fig 6.
SRL-treated B cells enhances the proliferation and differentiation of CD4+ T cells.
Purified CD19+ B cells were pre-stimulated for 6 days with anti-IgM, anti-CD40 mAb and IL-21 in the absence (CTRL) or presence of 6ng/ml TAC or SRL. These cultured B cells were used as stimulators in 6-day MLRs of allogeneic CFSE-labelled CD4+CD25− T cell responders. (A) Level of proliferation differentially induced by pre-cultured B cells as detected by CFSE dilution in the allogeneic CD4 responder cells (representative experiment on the left, and compiled data from 8 independent experiments on the right). (B) Percentage of responding T cells positive for memory marker (CD45RO) and activation markers (CD62L, CD25, CD69, CD95) after co-culture with pre-stimulated B cells (n = 8); (C) Mean ± SD (n = 4) percentage of responding proliferating T cells expressing intracellular cytokines (top row) and transcription factors (bottom row). Taken together the data indicated that B cells that proliferated in presence of SRL on a per cell basis were capable of inducing alloreactive T cell proliferation towards a Th1 phenotype. *p < 0.05. ** p < 0.01.