Adoptive Cell Therapy Using In Vitro Generated Human CD4+CD25+ Regulatory T Cells With Indirect Allospecificity to Promote Donor-Specific Transplantation Tolerance

The holy grail in clinical transplantation is the establishment of long-term donor-specific transplantation tolerance with the minimum use of immunosuppressive agents. CD4+ CD25+ regulatory T cells (Tregs) play a crucial role in the prevention of autoimmunity, and appear to mediate transplantation tolerance. Harnessing Tregs for potential adoptive cell therapy to promote donor-specific transplantation tolerance is promising. Here we show that human CD4+ CD25+ Tregs with indirect allospecificity for an HLA-A2 (103-120) peptide can be generated from purified peripheral blood CD4+ CD25+ by priming with HLA-DR0101+ A2- autologous dendritic cells (DCs) pulsed with the A2 peptide in vitro. The antigen specificity for the A2 peptide was demonstrated in functional assays and flow cytometric analysis using a fluorescent tetramer composed of HLA-DR0101 and the A2 peptide. The CD4+ CD25+ Tregs with indirect allospecificity for the A2 peptide showed potent suppression of an indirect alloresponse by effector CD4+ CD25- T-helper cells. Importantly, the selected CD4+ CD25+ Tregs can be expanded substantially to meet a therapeutic end after T-cell receptor (TCR) stimulation by CD3/CD28 antibody-coated beads in the presence of high doses of interleukin-2 (IL-2). The expanded CD4+ CD25+ Tregs highly expressed Foxp3, and retained their suppressive properties and maintained expression of lymphoid homing receptor CD62L. Taken together, these data pave the way for clinical studies using in vitro generated and expanded human CD4+ CD25+ Tregs with indirect allospecificity as therapeutic reagents to promote donor-specific transplantation tolerance.