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Background CD137 (4-1BB) is widely considered a comprehensive marker of T cell activation, and it is largely used in cancer immunology to determine the proportion of tumor-reactive T cells. However, CD137 detection does not provide specific information regarding the functions of activated T cells. Hence, in this study we tried to determine whether the simultaneous detection of CD137 plus common antitumor immune functions (type 1 functions such as production of cytokines TNFα and IFN-γ, or upregulation of CD107a) was feasible and provided advantages over using both methods separately. Methods The influence of common protein transport inhibitors used to allow cytokine detection, such as Brefeldin A (BFA) or Monensin (MN), on surface and intracellular expression of CD137 was tested in vitro. Activation of tumor infiltrating lymphocytes (TILs) was achieved with unspecific stimuli (PMA and Ionomycin) or naturally presented tumor-antigens (autologous tumor cells). TIL activation was defined as the proportion of CD4+ or CD8+ TILs staining for either CD137 (CD137+ TILs) and/or at least one of TNFα, IFNγ and CD107a (Type 1 function+ TILs) via flow cytometry. Results Our results showed that MN had minimal impact on the ability to detect surface CD137, whereas BFA had a much larger effect regardless of the stimulus employed. Nonetheless, robust results could be obtained when type 1 functions detection was combined with intracellular staining of CD137 in the presence of both BFA and MN. Using this method, we identified three tumor-antigen specific T cell subpopulations, characterized by combined expression of CD137 and type 1 functions or CD137 without type 1 functions and vice-versa. The combined method allowed the identification of a much larger proportion of tumor-reactive T cells, over either of the two methods used alone. Indeed, CD137 alone identified 46% of the total tumor-reactive CD8+ TILs, while 86% of the total tumor-reactive CD8+ TILs were positive for type 1 functions. Conclusion Combined detection of CD137 and common type 1 functions represents a more comprehensive method to identify and characterize the total repertoire of tumor-antigen specific, activated T lymphocytes, regardless of the antigen recognized. Legal entity responsible for the study Herlev and Gentofte Hospital. Funding Research grant A11806 and A12535, The Danish Cancer Society; Research grant R233-2016-3728 and R286-2018-991 Lundbeck Foundation; Clinician-Scientist Grant from the Herlev and Gentofte Hospital Research Council. Disclosure All authors have declared no conflicts of interest.

e13012 Background: Adoptive cell transfer (ACT) with tumor infiltrating lymphocytes (TIL) is an effective treatment for advanced melanoma, but to our knowledge generation of clinical scale TILs has...

e13084 Background: Several clinical trials have evaluated the effect of dendritic cell (DC) vaccination in the treatment of melanoma patients but so far the clinical results have been disappointing...

e13071 Background: T-cell based immune therapy in melanoma has shown markedly objective clinical responses in a single-center study at the NIH. In order to extend the treatment to other centers and...

16144 Background: CD4+CD25highFoxp3+ regulatory T cells (Treg) play an important role in the maintenance of immune tolerance and may be one of the obstacles of successful tumour immunotherapy. Meth...

2575 Background: Autologous dendritic cells (DC) pulsed with tumor-associated antigens as peptides or tumor lysate derived proteins can induce generation of cytotoxic T-cells in cancer patients. Survivin and telomerase are tumor-associated antigens overexpressed in renal cell carcinoma, and telomerase and survivin derived HLA-A2 binding peptides are able to induce effector T-cells cytotoxic to tumor cells. Tumor lysate can be generated from allogeneic renal carcinoma cell lines and has a natural high amount of antigens potentially enabling the induction of a polyclonal immune response against multiple targets on tumor cells. Methods: Twenty-two patients with progressive metastatic renal cell carcinoma were included and toxicity and efficacy of DC-based immunotherapy were evaluated. HLA-A2 positive patients were treated with mature autologous DCs pulsed with a broad panel of HLA binding telomerase and survivin peptides and PADRE; HLA-A2 negative patients received DCs pulsed with allogeneic tumor lysate and KLH. The vaccines were administered intradermally or intranodally weekly/biweekly ten times and repeated monthly until tumor progression. Patients received low dose IL-2 as an adjuvant. Immune response was monitored using ELISPOT assay. IL-6 and the biomarker YKL-40 were measured in serum using ELISA assay. Results: Vaccinations were well tolerated. 17/22 patients were evaluable and 10 patients had stable disease (SD) for up to 11+ months (range 2–11+ months). Interestingly, serum IL-6 increased in patients with progressive disease compared with a decrease in patients with SD. After vaccination mean values of YKL-40 were 91ng/ml in patients with SD and 208ng/ml in patients with PD. Immune monitoring is ongoing and preliminary data demonstrate that peptide specific CTLs are induced by the treatment. Conclusions: This pilot study demonstrates, that vaccination with autologous DCs pulsed with tumour antigens is safe and without severe toxicity. Disease stabilization was observed in half of the treated patients. Serum IL-6 and YKL-40 values might be useful parameters to predict clinical response during vaccination therapy. No significant financial relationships to disclose.