• adoptive cell therapy
• antitumor specificity
• bone marrow transplantation

A major issue in allogeneic hematopoietic stem cell transplantation (alloHSCT) is the difficulty of dealing with disease relapse after the transplant. The current standard approach is the use of donor lymphocyte infusions which have documented efficacy but are associated with significant mortality and relevant adverse events related to graft-versus-host disease (GvHD). Another recent approach is the development of adoptive T-cell therapies, but its major limitation is the limited availability of T-cells that are tumor-specific and display a broad antigenic repertoire. We show here that allogeneic donor-derived marrow-infiltrating lymphocytes (alloMILs) obtained from 9 patients who underwent alloHSCT with post-transplant cyclophosphamide and polyclonally stimulated with antiCD3/CD28 beads were reproducibly expanded and activated. Phenotypic characterization of activated alloMIL subpopulations revealed that the prevalent T-cell subtype exhibited a central memory phenotype in both CD4+ and CD8+ compartments. Activated alloMILs specifically recognized tumor cells and displayed in vitro antitumor activity. Selective expansion of small alloMIL subsets specific for different tumor antigens led to heightened tumor specificity and maintenance of a broad antigenic repertoire. Furthermore, activated alloMILs from all patients effectively targeted third-party allogeneic antigens, but did not show any reactivity towards autologous antigens presented in an HLA-dependent fashion, thereby suggesting a possible reduction in the risk of GvHD development. Collectively, these results underscore the intrinsic polyclonal tumor-specificity of activated alloMILs and describe a novel approach for the generation of tumor-specific T-cells that are suitable for adoptive immunotherapy of hematological malignancies relapsed after alloHSCT and that may significantly reduce the toxicities associated with current standard approaches.