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Abstract

Metastatic melanoma is a severe disease with a high rate of lethality. It is known to be resistant to current therapies. Since melanoma is immunogenic the development of an immunotherapy can be a promising possibility to enhance an antitumor effect in vivo. Memory T cells (MTC) have abilities to respond quicker to antigens and to release a broader spectrum of cytokines than naïve T cells. The ret transgenic mouse melanoma model was used in this study since it resembles the pathological situation of human melanoma in contrast to transplantation models. It has been previously shown that the bone marrow (BM) is a major site for the persistence of tumor-specific MTC in cancer patients. In addition, melanoma-specific MTC were also found in the BM of ret transgenic mice without macroscopic tumors. Therefore, we isolated CD3+ cells from the BM of ret transgenic mice with and without tumors. Therefore, we isolated CD3+ T cells from the BM of ret transgenic mice with and without tumors. After a 40 h ex vivo stimulation of bone marrow-derived T cells with melanoma antigen-loaded DC, which were treated with anti-PD-L1 antibody overnight, T cells revealed a higher IFN-γ production and an increased T cell activation in vitro. Moreover, activated CD8+ T cells displayed mainly a central memory phenotype and an increased level of CD69 expression after 40 h of co-culture with DC. Labeled melanoma-specific, stimulated memory T cells from ret transgenic mice migrated to skin tumor lesions, metastatic lymph nodes (LN), BM and spleen after adoptive transfer into ret transgenic tumor-bearing mice. A similar migration pattern was detected using stimulated TRP-2 TCR transgenic effector T cells. Furthermore, migrated CD8+ T cells showed an increase in effector memory (TEM) and effector phenotype at day 7 post injection and a decrease of central memory and naive CD8+ T cells within tumor lesions, whereas at day 3, central memory, effector memory, naive and effector CD8+ T cells were equally distributed. To investigate the anti-tumor activity of melanoma-specific memory T cells in vivo we adoptively transferred MTC, which were prior activated with DC, into tumor-bearing mice by i.c. injections. Mice receiving memory T cells showed a significantly longer survival compared to the control group. Mice receiving the phosphodiesterase-5 inhibitor sildenafil and adoptive transfer of MTCs displayed a significantly higher survival rate than mice treated with sildenafil or PBS only. We suggest that an adoptive transfer of melanoma-specific memory T cells activated with antigen-loaded DC, which were pre-treated with anti-PD-L1 antibodies, can enhance an anti-tumor response and therapeutic efficacy in vivo.