Graft-Versus-leukemia Tissue-restricted T cell alloresponses across HLA barriers: selection and identification of leukemia-restricted CTL in HLA-mismatched stimulator–responder pairs H Fujiwara, G Sconocchia, J Melenhorst, R Eniafe, R Nakamura, N Hensel and AJ Barrett Allogeneic Stem Cell Transplant Section, National Heart, Lung and Blood Institute, Bethesda, MD, USA Summary: Exploiting the graft-versus-leukemia (GVL) effect in mismatched transplants requires its separation from graft- versus-host disease (GVHD). We generated leukemia- specific cytotoxic T lymphocytes (CTL) in three haplo- type-mismatched, two class I-mismatched and two single HLA-A locus-matched stimulator–responder pairs. Six patients with chronic myelogenous leukemia and one patient with acute myeloid leukemia transformed from MDS were studied. CTL generated after 10 days stimulation with unselected leukemic peripheral blood mononuclear cells inhibited leukemic CFU-GM colony growth (485% at 10:1 effector:target ratio) with no third-party colony inhibition. In five pairs, responders were cultured separately with leukemia cells, PHA-B or LCL from the stimulator. After 2–4 restimulations, the T cell repertoire was examined by flow analysis using Vb-specific antibodies. Test cultures (but not controls) showed preferential expansion of 1–4 Vb families either common to two or more stimulators or unique to a particular stimulator. Notably, we elicited leukemia-specific TCR Vb expansions on four out of five occasions. In two pairs, responder cells selected for the appropriate leukemia-specific Vb family were shown to have leukemia-specific cytotoxicity. These leukemia-restricted T-cells were CD8+ or CD4+ and CD25+ or CD57+. The results support the development of strategies to selectively deplete GVHD and conserve GVL reactivity in mismatched transplants. Bone Marrow Transplantation (2003) 32, 371–378. doi:10.1038/sj.bmt.1704142 Keywords: leukemia-restriction; cytotoxic T-lymphocytes; HLA-mismatched; stem cell transplant The curative effect of allogeneic bone marrow transplanta- tion (allo-BMT) from an HLA-identical donor is well established for a variety of hematological malignancies. 1 Nevertheless, despite the increasing availability of unre- lated donors, a suitable HLA-identical donor cannot be identified in a timely fashion for at least 50% of patients. 2,3 Animal transplantation models indicate that powerful antitumor cytotoxic T lymphocyte (CTL) can be elicited in an MHC-mismatched setting. 4,5 These observations support limited clinical experience in HLA-mismatched transplants indicating a strong graft-versus-leukemia (GVL) effect in high-risk patients with advanced leukemia and in leukemia relapsing after BMT treated with donor lymphocyte infusions. 6–9 However, it is also clear that HLA disparity increases the risk of severe graft-versus-host disease (GVHD). 10–12 As a result of the risk from GVHD, current HLA-mismatched transplant protocols use T cell depletion and potent immunosuppression to prevent GVHD, which in turn compromises the GVL effect. Separation of GVL alloresponses from the GVHD T cell alloresponse would permit the full exploitation of the therapeutic effect of the haplotype-mismatched transplant. Previous studies in HLA-matched transplants have defined an immunological basis for separating T cell clones with GVHD or GVL alloresponsiveness; while many alloanti- gens are shared between hematopoietic and nonhemato- poietic tissue, there are also opportunities for specific clonal T cell responses to antigens presented only by leukemia cells or hematopoietic cells, which make possible GVL (or graft-versus-marrow)-specific alloresponses. These tissue-restricted antigens have been identified either as minor histocompatibility antigens or nonallelic leukemia- restricted antigens. 13–16 The question remains whether such tissue-restricted responses can also be elicited in HLA-mismatched donor– recipient pairs. To study this possibility, we examined alloresponses between mismatched unrelated stimulator– responder pairs. Here we report preliminary observations indicating that tissue-restricted (including leukemia- restricted) alloresponses can regularly be identified in an HLA class I or II-mismatched context. Materials and methods Patients and responders Patients. Peripheral blood mononuclear cells (PBMC) were obtained from a cryopreserved leukapheresis Received 25 November 2002; accepted 26 February 2003 Correspondence: Professor AJ Barrett, Allogeneic Stem Cell Transplant Section, Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bldg.10, Rm. 7C103, 9000 Rockville Pike, Bethesda, MD 20892, USA Bone Marrow Transplantation (2003) 32, 371–378 & 2003 Nature Publishing Group All rights reserved 0268-3369/03 $25.00 www.nature.com/bmt