Response: Stimulatory or tolerogenic role of CD40-activated B cells depends on the strength of the activation to T cells

Response

We thank Shimabukuro-Vornhagen et al for their comments on our recent study where we developed a simple and low-cost protocol using allogeneic CD40-activated B cells to induce and expand highly efficient human alloantigen-specific CD4^highCD25⁺Foxp3⁺ regulatory T cells (Tregs) from naive CD4⁺CD25⁻ T cells in large scale.¹  First, we have to clarify that we did not use autologous CD40-activated B cells but we used allogeneic cells to induce and expand Tregs instead. Second, the CD40-activated B cells used in our system are live cells but not the irradiated peptide-pulsed cells as others used.^2,3  Third, the ratio of B to T cells for induction and expansion of CD4^highCD25⁺ Tregs is 1:10 in our system but not 1:4 as others used to induce antigen-specific T and cytotoxic T cells.^2,3  Indeed, several reports have demonstrated that weekly stimulation with antigen-presenting cells (APCs) such as dendritic cells (DCs) is an effective way to induce and expand Tregs in vitro and in vivo.^4-9  Similar to our report,¹  Jonuleit et al demonstrated that allogeneic immature DCs induced Tregs from naive CD4 T cells at a 1:10 ratio of DC:T cells.⁶  Therefore, it is not surprising that CD40-activated B cells, as one of the APCs,^2,3  can induce and expand Tregs, in particularly under weekly stimulation.

Although the process of cryopreservation and thawing would undoubtedly affect the absolute number of live CD40-activated B cells (the recovery rate of live B cells preserved in liquid nitrogen for 6 months is approximately 80% in our system), it does not affect their function. As shown in Table 1, there are no significant differences in the induction, expansion, and suppressive ability of CD4^highCD25⁺ Tregs induced by frozen versus fresh CD40-activated B cells.

Another question raised by von Bergwelt-Baildon et al was the phenotype of CD40-activated B cells. We think the difference of the major histocompatibility complex (MHC-II) expression in B cells resulted from the different antibodies used. In our study, we determined MHC–II expression in these B cells with fluorescein isothiocyanate (FITC)–anti-human MHC-II antibody, which reacts with all MHC class II molecular HLA-DR, DP, and most DQ antigens (BD Biosciences, San Jose, CA). In contrast, von Bergwelt-Baildon et al only examined the HLA-DR expression in these B cells. We further checked the MHC-II and HLA-DR expressions in CD40-activated B cells (Figure 1). Consistent with our previous report,¹  more than 1 peak of MHC-II expressions were observed in these B cells, whereas only 1 peak of HLA-DR expression in the B cells was found (Figure 1).

More evidence of CD40-activated B cells as the tolerogenic cells was also found in our recent study (Zheng J. and Tu W. manuscript submitted). In this study, we demonstrated that allogeneic CD40-activated B cells induced novel CD8^highCD25⁺ cells from naive CD8⁺ T cells. These CD8^highCD25⁺ T cells were alloantigen-specific Tregs with relatively poor alloantigen-specific cytotoxicity.

Taken together, the process of cryopreservation and thawing does not affect the function of CD40-activated B cells to induce and expand alloantigen-specific Tregs, and the strength of the activation to T cells by CD40-activated B cells is critical for determining whether B cells act as the stimulatory or tolerogenic cells.