*Purpose: Regulatory T cells (Tregs) are promising tool for induction and maintenance of transplantation tolerance. We have done a feasibility trial in kidney transplant recipients and are currently conducting a withdrawal trial in liver transplant recipients using CD4⁺CD25⁺CD127^lo purified alloantigen-reactive Tregs (arTregs), which are produced by 3 days mixed lymphocyte reaction (MLR) in the presence of CTLA4-IgG fusion protein as costimulatory blockade (CSB). Recently, Hokkaido University group in Japan published successful withdrawal results from a trial using unfractionated products with similar approach. Although regulatory potential of B cells is an emerging topic as direct suppressor or in the context of interaction with Treg, the role of B cell in this manufacturing product remains unclear. Here, we show that CSB product with B cell depleted responder (CSB-Bdep) increased the proportion of arTreg at day3 with a comparable phenotype with regard to stability and function.

*Methods: We compared the day 3 products from MLR without CSB, CSB, and CSB-Bdep using random allo-pairs of PBMC from healthy human donors by flow cytometry and DNA methylation analyses.

*Results: The cell yield was not different among three conditionings at day 3. With HLA discriminating markers to avoid stimulator contamination, only CD4⁺ T cell fraction was increased in comparison between CSB and CSB-Bdep, but CD8⁺ T, NK and NKT cells were not. Among the CD4⁺ T cells, we observed significant increase of CD4⁺C25⁺CD127^lo population in CSB-Bdep compared to CSB (mean 4.7% vs 2.9% in CD4⁺ cells, P=0.0001) with modestly higher expression of FOXP3, CTLA4 and ICOS. CSB-Bdep induced arTreg also showed comparable demethylation status in Treg specific demethylation region (TSDR) of FOXP3 gene (% demethylation in TSDR, 77.6±6.0 vs 79.2±5.9%), consistent with the expression of stability related markers, Helios, CD45RA and CD161.

*Conclusions: Collectively, responder B cell depletion increased number of arTreg in 3 day culture without loss of stability and functionality. Generating sufficient number of functionally stable Tregs remains still challenging. These findings suggest that responder B cell depletion improves the optimal Treg generation in allo-anergization. Further studies are necessary to evaluate the in vivo fate of these cells and understanding the interaction between Tregs and B cells may enhance the potential of Treg cell therapy.

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