Novel GMP-Compatible Protocol Employing an Allogeneic B Cell Bank for Clonal Expansion of Allospecific Natural Regulatory T Cells

The adoptive transfer of natural regulatory T cells (nTreg) is a new option to reshape undesired immune reactivity in autoimmunity and transplantation toward tolerance. The first clinical trials using adoptive transfer of polyclonal nTreg demonstrated safety and hints of efficacy. However, the low frequencies of antigen-specific cells among the pool of polyclonal nTreg and their broad antigen nonspecific suppression are limitations of this approach regarding efficacy and safety. Recently, the isolation and expansion of (allo)antigen-specific nTreg have successfully been achieved by using Treg-specific activation markers but the yield is relatively low. Here, we describe a novel good manufacturing practice (GMP)-compatible expansion protocol of alloantigen-specific nTreg based on the stimulation of nTreg by allogeneic activated B cells. Their functionality and specificity are superior compared to polyclonal nTreg both in vitro and in vivo. Employing an allogeneic B cell bank, designed to cover the majority of HLA types, allows fast GMP-compliant manufacturing for donor-specific nTreg for clinical application in organ and stem cell transplantation. TCR repertoire analyses by next generation sequencing revealed impressive expansion by several log-steps of even very low-abundance alloantigen-specific nTreg clones. This novel method offers a simple approach for expanding antigen-specific nTreg and is characterized by high replicability and easy transferability to full GMP standards. An allogeneic B cell bank allows fast and GMP-compatible expansion of even low-abundance allospecific nTreg clones, which are more potent suppressors of specific alloresponses than their polyclonal counterparts, both in vitro and in vivo.