Aim: T and B cells collaborate during alloresponses in transplantation. The transcription factor (TF) Bcl6 is critical in both T follicular helper cells (Tfh) and germinal center B cells. We developed a novel in vitro model of Tfh differentiation based on bi-directional activation of T and B cells to test the immunosuppressive effects of novel Bcl6 inhibitors (Bcl6i).
Method: T-B interactions are required for full differentiation of mature Tfh, which in turn are required for generation of affinity matured GC B cells. We therefore used naïve T cells (Tn) in coculture with B cells isolated from healthy donor blood to simulate Tfh differentiation. To provide antigenic stimulation, we used allogeneic B cells (allo BC)(n=6). To provide costimulatory signals, B cell activation with CPG was necessary prior to coculture. After 6 days of coculture, T cell proliferation, Tfh markers, T and B cell Bcl6 expression and B cell phenotype markers were measured by flow cytometry. In addition, Bcl6i (FX1 12.5, 3.125 and 0.78 ug/ml), Belatacept (Bela, 10 and 5 ug/ml), or Tacrolimus (Tac, 10 or 5 ng/ml) were added to cocultures (n=5). To gain insight into the mechanism of action of Bcl6i, gene expression was analyzed in CD3/CD28 stimulated isolated Tn by qPCR (n=8). To explore the role of Bcl6 in kidney transplantation (Ktx), Bcl6 expression was analyzed in Ktx biopsies (7 TCMR, 3 ABMR/TCMR, 10 non-rejection) using miFISH (multiplex immunofluorescence in situ hybridization).
Results: Coculture with activated allo BC potently stimulated T cell proliferation (0.6% vs 47%, p=0.0025) and expression of Tfh markers CXCR5 (p=0.014), PD-1 (p=0.015) and ICOS (p=0.12) compared to unstimulated Tn. CD4⁺CXCR5⁺PD-1⁺ Tfh-like cells increased from 0.2% to 30.8% (p=0.032). Bcl6 expression was upregulated in 70% of T cells and significantly enriched in CD4⁺CXCR5⁺PD-1⁺ cells (vs. CD4⁺CXCR5^-PD-1^- cells, p=0.031). Furthermore, cocultured allo BC upregulated Bcl6 expression (p=0.013) and differentiated into a memory phenotype (IgD⁺CD27⁺/IgD^-CD27⁺)(p=0.005, vs. allo BC alone). Using this model of B cell-dependent Tfh differentiation, we found that both FX1 and Tac significantly inhibited expression of T cell activation markers CD45RO (FX1 p=0.0003; Tac p<0.0001), PD-1 (FX1 p=0.051; Tac p<0.0001), and ICOS (FX1 p=0.028; Tac p=0.0003) in a dose-dependent manner, while Bela moderately inhibited expression of ICOS (p=0.034), but not other markers. Furthermore, mRNA expression of TCF-1 and ASCL2, two TF upstream of Bcl6 during Tfh differentiation, was significantly increased in the presence of Bcl6i, which we interpreted as an interruption of the Tfh differentiation pathway at a pre-Tfh stage. Meanwhile, expression of Bcl6-antagonizing TF T-bet and Blimp-1 was unchanged.  Within Ktx tissue, preliminary analyses showed significantly more Bcl6 expressing CD4+ cells in Ktx biopsies with mixed ABMR/TCMR compared to TCMR (p=0.027) or no rejection (p=0.011).
Conclusion: Our study highlights T-B collaboration as an important mechanism of T and B cell activation. Using our novel model of Tfh differentiation, we showed that Bcl6i can effectively inhibit Tfh differentiation and have potential as a novel strategy against humoral allosensitization.