Abstract SY03-02: Wnt signaling generates “stem-like” self-renewing T cells that can eradicate tumors

The infusion of tumor-specific T lymphocytes, also known as adoptive cell transfer (ACT)-based immunotherapy, can have significant anti-tumor activities in mice and in humans. The most successful T cells being given in the clinic now are derived from tumor-infiltrating lymphocytes (TIL) in patients with malignant melanoma. Objective response rates to these therapies are approximately 50% in patients that receive treatment, and complete and durable long-term responses are observed in 10-15% of the patients treated. Thus, results are encouraging but these therapies must be improved upon. In its current form, successful ACT involves the adoptive transfer of large numbers of tumor-specific T cells. TIL that are being given in current clinical immunotherapy trials are predominantly highly differentiated CD45RO+ lymphocytes that are negative for the expression of CD62L and CCR7 and have low expression of CD27, CD28, and CD127. Functionally, the TIL that are currently being used have cytolytic properties and release large amounts of interferon-γ. Work using pmel-1 T cell receptor transgenic mice has revealed the functional and phenotypic qualities of adoptively transferred T cells that are associated with tumor rejection. We have observed that the state of differentiation of anti-tumor T cells prior to their adoptive transfer is critically important for their effectiveness. Until recently, it was thought that the optimal anti-tumor CD8+ T cells would be those IFN-gamma producing cells that were highly cytolytic upon encounter with tumor cell targets when tested prior to adoptive transfer. However, it seems clear now that cytotoxic T cells represent a terminal, pro-apoptotic differentiation state of CD8+ T cells. Paradoxically, the acquisition of full effector function in vitro impairs the in vivo antitumor efficacy of adoptively transferred CD8+ T cells.1 Central memory CD8+ T cells (e.g., those grown in IL-15) confer superior antitumor immunity compared with effector memory T cells expanded using IL-2.2,3 Furthermore, naive-derived cells or CD8+ T memory stem cells activated in the presence of IL-21, which are even less mature, are still more effective than central memory cells.4,5 Thus, the state of maturation of CD8+ T cells before adoptive transfer is inversely correlated with their effectiveness in vivo: “Younger,” less-differentiated T cells are more efficacious at triggering tumor eradication. It is important to note that undifferentiated T cells must be capable of maturing into fully functional T cells after transfer. This in vivo activation and differentiation of early CD8+ T cell subsets can be enhanced by homeostatic cytokines and encounter with the cognate antigen.6 Most surprisingly, we have found recently that the activities of anti-tumor T lymphocytes can be greatly enhanced by Wnt signaling.7 The Wnt-beta-catenin pathway is an evolutionarily conserved pathway that promotes hematopoietic stem cell self-renewal and multipotency by limiting stem cell proliferation and differentiation, but its role in the generation and maintenance of memory T cells was unknown. We found that induction of Wnt-beta-catenin signaling by inhibitors of glycogen synthase kinase-3beta or the Wnt protein family member Wnt3a arrested CD8+ T cell development into effector cells. By blocking T cell differentiation, Wnt signaling promoted the generation of CD44lowCD62LhighSca-1highCD122highBcl-2high self-renewing multipotent CD8+ memory stem cells with proliferative and antitumor capacities exceeding those of central and effector memory T cell subsets. These findings indicate that Wnt signaling has a key role in the maintenance of 9stemness9 in mature memory CD8+ T cells. T cell differentiation is also critically important for anti-tumor CD4+ T cells, which can be robustly polarized in vitro prior to transfer. This polarization can skew their expression of transcription factors, cytokines, chemokines and cell surface markers. Using the Trp-1 T cell receptor transgenic mouse system, we found that the adoptive transfer of tumor-specific, CD4+ T cells polarized to produce IL-17 (Th17 cells) have increased effectiveness in anti-tumor models when compared with other T cell subsets tested.8,9 Importantly, these Th17 cells evolve after transfer into an immunoablated host into cells that are capable of producing IFN-gamma. IL-17-secreting CD8+ T cells have been described, and their potential roles in cancer immunotherapy have been explored in two recent publications.10,11. IL-17-producing CD8+ T cells have greatly enhanced expression of RORgamma and reduced expression of Eomes and canonical cytolytic activity is diminished. Like their CD4+ counterparts, these cells can acquire the ability to produce IFN-gamma and mediate regression of large, established tumors. This enhanced anti-tumor activity is associated with increased in vivo expansion and persistence of the transferred cells. Type 17-skewed CD8+ cells display reduced expression of killer cell lectin-like receptor G1 (KLRG1), a marker of CD8+ T cell senescence, and increased expression IL-7Ralpha, a cytokine receptor associated in memory T cell formation. Thus, the polarization and differentiation states of anti-tumor CD8+ and CD4+ T cells are critical determinants of their anti-tumor effectiveness in vivo. Taken together, work in experimental mice using naturally-occurring T cell epitopes targeted by the pmel-1 CD8 and the Trp-1 CD4 systems indicates that indicate that “young”/undifferentiated T cells have increased effectiveness in the adoptive immunotherapy of melanoma and point the way towards the use of appropriately polarized CD4+ T cells in patients with cancer. References 1. Gattinoni, L., et al. Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+ T cells. The Journal of Clinical Investigation 115, 1616-1626 (2005). 2. Klebanoff, C.A., et al. IL-15 enhances the in vivo antitumor activity of tumor-reactive CD8+ T cells. Proceedings of the National Academy of Sciences of the United States of America 101, 1969-1974 (2004). 3. Klebanoff, C.A., et al. Central memory self/tumor-reactive CD8+ T cells confer superior antitumor immunity compared with effector memory T cells. Proceedings of the National Academy of Sciences of the United States of America 102, 9571-9576 (2005). 4. Hinrichs, C.S., et al. Adoptively transferred effector cells derived from naive rather than central memory CD8+ T cells mediate superior antitumor immunity. Proceedings of the National Academy of Sciences of the United States of America 106, 17469-17474 (2009). 5. Hinrichs, C.S., et al. IL-2 and IL-21 confer opposing differentiation programs to CD8+ T cells for adoptive immunotherapy. Blood 111, 5326-5333 (2008). 6. Gattinoni, L., Powell, D.J., Jr., Rosenberg, S.A. & Restifo, N.P. Adoptive immunotherapy for cancer: building on success. Nature Reviews 6, 383-393 (2006). 7. Gattinoni, L., et al. Wnt signaling arrests effector T cell differentiation and generates CD8+ memory stem cells. Nature Medicine 15, 808-813 (2009). 8. Muranski, P., et al. Tumor-specific Th17-polarized cells eradicate large established melanoma. Blood 112, 362-373 (2008). 9. Muranski, P. & Restifo, N.P. Adoptive immunotherapy of cancer using CD4(+) T cells. Current Opinion in Immunology 21, 200-208 (2009). 10. Hinrichs, C.S., et al. Type 17 CD8+ T cells display enhanced antitumor immunity. Blood 114, 596-599 (2009). 11. Yen, H.R., et al. Tc17 CD8 T cells: functional plasticity and subset diversity. J Immunol 183, 7161-7168 (2009). Citation Format: Nicholas P. Restifo, Luca Gattinoni. Wnt signaling generates “stem-like” self-renewing T cells that can eradicate tumors [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY03-02