CD122+CD8+ Treg suppress vaccine-induced antitumor immune responses in lymphodepleted mice.

Lymphodeleption prior to adoptive transfer of tumor-specific T cells greatly improves the clinical efficacy of adoptive T-cell therapy for patients with advanced melanoma, and increases the therapeutic efficacy of cancer vaccines in animal models. Lymphodepletion reduces competition between lymphocytes, and thus creates "space" for enhanced expansion and survival of tumor-specific T cells. Within the lymphodepleted host, Ag-specific T cells still need to compete with other lymphocytes that undergo lymphopenia-driven proliferation. Herein, we describe the relative capacity of naïve T cells, Treg, and NK cells to undergo lymphopenia-driven proliferation. We found that the major population that underwent lymphopenia-driven proliferation was the CD122+ memory-like T-cell population (CD122+CD8+ Treg), and these cells competed with Ag-driven proliferation of melanoma-specific T cells. Removal of CD122+CD8+ Treg resulted in a greater expansion of tumor-specific T cells and tumor infiltration of functional effector/memory T cells. Our results demonstrate the lymphopenia-driven proliferation of CD122+CD8+ Treg in reconstituted lymphodepleted mice limited the antitumor efficacy of DC vaccination in conjunction with adoptive transfer of tumor-specific T cells.

Combined IL-21 and low-dose IL-2 therapy induces anti-tumor immunity and long-term curative effects in a murine melanoma tumor model.

BACKGROUND: In vivo studies have recently demonstrated that interleukin 21 (IL-21) enhances the anti-tumor function of T-cells and NK cells in murine tumor models, and the combined use of IL-21 and IL-15 has resulted in prolonged tumor regression and survival in mice with previously established tumors. However, the combined anti-tumor effects of IL-21 and low dose IL-2 have not been studied even though IL-2 has been approved for human use, and, at low dose administration, stimulates the proliferation of memory T cells, and does not significantly increase antigen-induced apoptosis or regulatory T cell (Treg) expansion. This study examined whether recombinant IL-21 alone or in combination with low-dose IL-2 could improve the in vivo anti-tumor function of naïve, tumor-antigen specific CD8+ T cells in a gp100(25-33) T cell receptor transgenic pmel murine melanoma model. METHODS: Congenic C57BL/6 (Ly5.2) mice bearing subcutaneous B16F10 melanoma tumors were sublethally irradiated to induce lymphopenia. After irradiation naive pmel splenocytes were adoptively transferred, and mice were immunized with bone marrow-derived dendritic cells pulsed with human gp100(25-33) (hgp100(25-33)). Seven days after vaccination groups of mice received 5 consecutive days of intraperitoneal administration of IL-2 alone (20 x 10(3) IU), IL-21 alone (20 microg) or IL-21 and IL-2. Control animals received no cytokine therapy. RESULTS: IL-21 alone and IL-2 alone both delayed tumor progression, but only IL-21 significantly augmented long-term survival (20%) compared to the control group. However, combination therapy with IL-21 and IL-2 resulted in the highest long-term (>150 days) tumor-free survival frequency of 46%. Animals that were tumor-free for > 150 days demonstrated tumor-specific protection after rechallenge with B16F10 melanoma cells. At peak expansion (21 days post vaccination), the combination of IL-21 plus IL-2 resulted in a 2- to 3-fold higher absolute number of circulating tumor antigen-specific pmel CD8+ T cells than was stimulated by IL-2 or IL-21 alone. Pmel CD8+ T cells were predominantly partitioned into central memory (CD62L+/CD127+) or effector-memory (CD62L-/CD127+) phenotypes by day 28-post vaccination in IL-21 + IL-2 treated mice. CONCLUSION: These observations support the potential use of IL-21 and low-dose IL-2 therapy in combination with a tumor-antigen vaccine and lymphopenic conditioning in future cancer clinical trials to maintain high numbers of anti-tumor memory CD8+ T cells with the potential to sustain long term tumor regression and survival.

Increased susceptibility to immune destruction of B16BL6 tumor cells engineered to express a novel pro-Smac fusion protein.

Recent developments in immunology have provided new strategies to induce and augment the immune response to cancer. Nonetheless, objective clinical responses after vaccination are rare and even when high frequencies of tumor-specific T cells are achieved after adoptive immunotherapy, tumor cells continue to evade the immune response. We hypothesize that 1 mechanism of resistance of tumor cells to destruction by T cells is an elevated threshold for the induction of apoptosis. Inhibitor of apoptosis proteins (IAPs) are overexpressed in various tumors and have been associated with treatment failure and poor prognosis. As the mitochondrial peptide second mitochondria-derived activator of caspase (Smac) can antagonize IAPs, we designed a GFP-Smac fusion protein with a granzyme B (GrB) cleavage site. This fusion protein should be cleaved when tumor-specific cytolytic T cells recognize the tumor and, using the pore-forming protein perforin, insert GrB into the target. Here we report that transfer of a construct encoding a novel eGFP-Smac fusion protein (pro-Smac) containing a specific cleavage site for GrB, into the poorly immunogenic mouse melanoma cell line, B16BL6-D5 (D5), sensitizes tumor cells for killing by tumor-specific wild type, but not perforin-deficient (perforin-knockout), effector T cells in vitro and in vivo. These results describe the first example of a tumor-specific, T-cell-mediated approach to amplify the GrB-mediated cytotoxicity pathway with a pro-Smac fusion protein and provide an innovative approach to overcome IAPs and improve the efficacy of immunotherapy.