Transfer of Specific T Cell Clones with Graft-Versus-Leukemia (GVL) Activity from Donor to Recipient in Stem Cell Transplantation: Identification, Quantification and Characterization.

Abstract

The self-antigens PR1 and WT1 that are aberrantly expressed on malignant cells may be important target antigens for GVL effects from donor-derived anti-leukemia T cells. It is now clear that T cells recognizing these antigens circulate in transplant recipients and can be detected in small numbers in healthy individuals. To determine whether the same T cell clones in the donor are transferred to the recipient and induce GVL effects we sought for presence of leukemia-reactive T cell clones in healthy donors and their transfer into the patient after transplant and following DLI. We identified CD8+ T cell clones specific for PR1 and WT1 from 2 healthy donors. The HLA-A2/PR1-binding and HLA-A2/WT1-binding CD8+ T cells were purified by flow cytometric cell sorting and analyzed for their T cell receptor (TCR) usage by template switch anchored RT-PCR. This showed an oligoclonal population of WT1-specific CD8+ T cells and a polyclonal population of PR1-specific CD8+ T cells. In addition, using a fluorescent peptide/MHC class I multimeric complex incorporating mutations in the a3 domain that abrogate binding to the CD8 coreceptor, we selectively isolated WT1-specific CD8+ T cells of high functional avidity and demonstrated that high avidity T cells comprise a single clonotype. One patient with CML received an alloSCT from the donor in whom PR1-specific CD8+ T cell clones were detected. Using quantitative real-time PCR for IFN-g production and HLA-A2/PR1 tetrameric complexes, we showed the emergence of PR1-specific CD8+ T cells in the blood of the recipient 10 weeks after SCT and again 8 weeks post-DLI given to treat a molecular relapse of CML. HLA-A2/PR1 tetramer-positive CD8+ T cells were sorted by flow cytometry post-alloSCT and again following DLI. By comparing TCRb CDR3 sequences, we confirmed direct transfer and expansion of PR1-specific CD8+ T cell clones from the donor into the recipient and the reemergence of the same PR1-specific clones following DLI. The appearance of these HLA-A2/PR1 tetramer-positive CD8+ T cells was followed by complete molecular remission of CML by sensitive PCR for BCR/ABL The PR1-specific CD8+ T cells in the donor were of memory phenotype and expanded in the recipient after both alloSCT and DLI. During the early phase post-transfer in the recipient, the majority of PR1-specific CD8+ T cells had an effector memory phenotype (CD45RO+ CD57+). There was a shift towards a central memory phenotype (CD45 RO+ CD57−) during the course of the GVL effect. This is the first direct demonstration of the transfer of leukaemia-reactive specific T cell clones from a healthy donor to a patient with leukemia. Further, the identification and monitoring of T cell clones that mediate the GVL effect as described here can be undertaken before stem cell transplantation and could aid donor selection.