Self-renewing cell populations such as hematopoietic stem cells and memory B and T lymphocytes might be regulated by shared signaling pathways1. Wnt/β-catenin is an evolutionarily conserved pathway that promotes hematopoietic stem cell self-renewal and multipotency by limiting stem cell proliferation and differentiation2,3, but its role in the generation and maintenance of memory T cells is unknown. We found that the induction of Wnt/β-catenin signaling using inhibitors of glycogen-sythase-kinase-3β or the Wnt protein family member, Wnt3a, arrested CD8+ T cell development into effector cells. By blocking T-cell differentiation, Wnt signaling enabled the generation of CD44low, CD62Lhigh, Sca-1high, CD122high, Bcl-2high self-renewing, multipotent CD8+ memory stem cells with proliferative and anti-tumor capacities exceeding those of central and effector memory T cell subsets. These findings reveal a key role for Wnt signaling in the maintenance of stemness in mature memory CD8+ T cells and have important implications for the design of novel vaccination strategies and adoptive immunotherapies.

Many tumor-associated antigens are derived from nonmutated “self” proteins. T cells infiltrating tumor deposits recognize self-antigens presented by tumor cells and can be expanded in vivo with vaccination. These T cells exist in a functionally tolerant state, as they rarely result in tumor eradication. We found that tumor growth and lethality were unchanged in mice even after adoptive transfer of large numbers of T cells specific for an MHC class I–restricted epitope of the self/tumor antigen gp100. We sought to develop new strategies that would reverse the functionally tolerant state of self/tumor antigen-reactive T cells and enable the destruction of large (with products of perpendicular diameters of >50 mm2), subcutaneous, unmanipulated, poorly immunogenic B16 tumors that were established for up to 14 d before the start of treatment. We have defined three elements that are all strictly necessary to induce tumor regression in this model: (a) adoptive transfer of tumor-specific T cells; (b) T cell stimulation through antigen-specific vaccination with an altered peptide ligand, rather than the native self-peptide; and (c) coadministration of a T cell growth and activation factor. Cells, vaccination, or cyto-kine given alone or any two in combination were insufficient to induce tumor destruction. Autoimmune vitiligo was observed in mice cured of their disease. These findings illustrate that adoptive transfer of T cells and IL-2 can augment the function of a cancer vaccine. Furthermore, these data represent the first demonstration of complete cures of large, established, poorly immunogenic, unmanipulated solid tumors using T cells specific for a true self/tumor antigen and form the basis for a new approach to the treatment of patients with cancer.

T cell differentiation is a progressive process characterized by phenotypic and functional changes. By transferring tumor-specific CD8 + T cells into tumor-bearing mice at various stages of differentiation, we evaluated their efficacy for adoptive immunotherapy. We found that administration of naive and early effector T cells, in combination with active immunization and IL-2, resulted in the eradication of large, established tumors. Despite enhanced in vitro antitumor properties, more-differentiated effector T cells were less effective for in vivo tumor treatment. Several events may underlie this paradoxical phenomenon: (a) downregulation of lymphoid-homing and costimulatory molecules; (b) inability to produce IL-2 and access homeostatic cytokines; and (c) entry into a proapoptotic and replicative senescent state. While the progressive acquisition of terminal effector properties is characterized by pronounced in vitro tumor killing, in vivo T cell activation, proliferation, and survival are progressively impaired. These findings suggest that the current methodology for selecting T cells for transfer is inadequate and provide new criteria for the generation and the screening of optimal lymphocyte populations for adoptive immunotherapy. IntroductionAdoptive cell transfer therapy (ACT), the administration of ex vivoactivated and -expanded autologous tumor-reactive T cells, is currently one of the few immunotherapies that can induce objective clinical responses in significant numbers of patients with metastatic solid tumors (1-3). In a previous study, ACT after lymphodepleting conditioning caused objective responses in 46% of patients with metastatic melanoma refractory to other therapeutic modalities (4). We have now tripled the size of the original study (4), and the objective response rate exceeds 50%, and 11% of all patients treated are complete responders (5).There are several theoretical advantages to the use of ACT in treatment of cancer. Tumor-specific T cells can be activated and expanded to large numbers ex vivo, independently of the immunogenic properties of the tumor. Perhaps most important, the functional and phenotypic qualities of T cells can be selected prior to their adoptive transfer.Much progress has been made in the understanding of the properties of T cell subpopulations associated with states of T cell differentiation in mice and humans, especially those states that are related to the generation of memory T cells capable of protecting against viral challenge (6-9). However, little progress has been made in identifying the characteristics of cell states that are associated with the successful treatment of large, established tumors in mice or in humans.