Predicting combinations of immunomodulators to enhance dendritic cell-based vaccination based on a hybrid experimental and computational platform.

Dendritic cell (DC)-based vaccines have been largely used in the adjuvant setting for the treatment of cancer, however, despite their proven safety, clinical outcomes still remain modest. In order to improve their efficacy, DC-based vaccines are often combined with one or multiple immunomodulatory agents. However, the selection of the most promising combinations is hampered by the plethora of agents available and the unknown interplay between these different agents. To address this point, we developed a hybrid experimental and computational platform to predict the effects and immunogenicity of dual combinations of stimuli once combined with DC vaccination, based on the experimental data of a variety of assays to monitor different aspects of the immune response after a single stimulus. To assess the stimuli behavior when used as single agents, we first developed an in vitro co-culture system of T cell priming using monocyte-derived DCs loaded with whole tumor lysate to prime autologous peripheral blood mononuclear cells in the presence of the chosen stimuli, as single adjuvants, and characterized the elicited response assessing 18 different phenotypic and functional traits important for an efficient anti-cancer response. We then developed and applied a prediction algorithm, generating a ranking for all possible dual combinations of the different single stimuli considered here. The ranking generated by the prediction tool was then validated with experimental data showing a strong correlation with the predicted scores, confirming that the top ranked conditions globally significantly outperformed the worst conditions. Thus, the method developed here constitutes an innovative tool for the selection of the best immunomodulatory agents to implement in future DC-based vaccines.

Electroporation as a method of choice to generate genetically modified dendritic cell cancer vaccines.

In the last few decades, immunotherapy has emerged as an alternative therapeutic approach to treat cancer. Immunotherapy offers a plethora of different treatment possibilities. Among these, dendritic cell (DC)-based cancer vaccines constitute one of the most promising and valuable therapeutic options. DC-vaccines have been introduced into the clinics more than 15 years ago, and preclinical studies showed their general safety and low toxic effects on patients. However, their treatment efficacy is still rather limited, demanding for novel avenues to improve vaccine efficacy. One way to potentially achieve this is to focus on improving the DC-T cell interaction to further increase T cell priming and downstream activity. A successful DC-T cell interaction requires three different signals (Figure 1): (1) Major Histocompatibility Complex (MHC) and antigen complex interaction with T cell receptor (TCR) (2) interaction between co-stimulatory molecules and their cognate ligands at the cell surface and (3) secretion of cytokines to polarize the immune response toward a Type 1 helper (Th1) phenotype. In recent years, many studies attempted to improve the DC-T cell interaction and overall cancer vaccine therapeutic outcomes by increasing the expression of mediators of signal 1, 2 and/or 3, through genetic modifications of DCs. Transfection of genes of interest can be achieved through many different methods such as passive pulsing, lipofection, viral transfection, or electroporation (EP). However, EP is currently emerging as the method of choice thanks to its safety, versatility, and relatively easy clinical translation. In this review we will highlight the potential benefits of EP over other transfection methods as well as giving an overview of the available studies employing EP to gene-modify DCs in cancer vaccines. Crucial aspects such as safety, feasibility, and gene(s) of choice will be also discussed, together with future perspectives and opportunities for DC genetic engineering.

Adenosine mediates functional and metabolic suppression of peripheral and tumor-infiltrating CD8+ T cells.

BACKGROUND: Several mechanisms are present in the tumor microenvironment (TME) to impair cytotoxic T cell responses potentially able to control tumor growth. Among these, the accumulation of adenosine (Ado) contributes to tumor progression and represents a promising immunotherapeutic target. Ado has been shown to impair T cell effector function, but the role and mechanisms employed by Ado/Ado receptors (AdoRs) in modulating human peripheral and tumor-infiltrating lymphocyte (TIL) function are still puzzling. METHODS: CD8+ T cell cytokine production following stimulation was quantified by intracellular staining and flow cytometry. The cytotoxic capacity of tumor infiltrating lymphocytes (TILs) was quantified by the chromium release assay following co-culture with autologous or anti-CD3-loaded tumor cell lines. The CD8+ T cell metabolic fitness was evaluated by the seahorse assay and by the quantification of 2-NBDG uptake and CD71/CD98 upregulation upon stimulation. The expression of AdoRs was assessed by RNA flow cytometry, a recently developed technology that we validated by semiquantitative RT-PCR (qRT-PCR), while the impact on T cell function was evaluated by the use of selective antagonists and agonists. The influence of Ado/AdoR on the PKA and mTOR pathways was evaluated by phosphoflow staining of p-CREB and p-S6, respectively, and validated by western blot. RESULTS: Here, we demonstrate that Ado signaling through the A2A receptor (A2AR) in human peripheral CD8+ T cells and TILs is responsible for the higher sensitivity to Ado-mediated suppression of T central memory cells. We confirmed that Ado is able to impair peripheral and tumor-expanded T cell effector functions, and we show for the first time its impact on metabolic fitness. The Ado-mediated immunosuppressive effects are mediated by increased PKA activation that results in impairment of the mTORC1 pathway. CONCLUSIONS: Our findings unveil A2AR/PKA/mTORC1 as the main Ado signaling pathway impairing the immune competence of peripheral T cells and TILs. Thus, p-CREB and p-S6 may represent useful pharmacodynamic and efficacy biomarkers of immunotherapies targeting Ado. The effect of Ado on T cell metabolic fitness reinforces the importance of the adenosinergic pathway as a target for next-generation immunotherapy.