Induction of neoantigen-reactive T cells from healthy donors

The identification of immunogenic neoantigens and their cognate T cells represents the most crucial and rate-limiting steps in the development of personalized cancer immunotherapies that are based on vaccination or on infusion of T cell receptor (TCR)-engineered T cells. Recent advances in deep-sequencing technologies and in silico prediction algorithms have allowed rapid identification of candidate neoepitopes. However, large-scale validation of putative neoepitopes and the isolation of reactive T cells are challenging because of the limited availablity of patient material and the low frequencies of neoepitope-specific T cells. Here we describe a standardized protocol for the induction of neoepitope-reactive T cells from healthy donor T cell repertoires, unaffected by the potentially immunosuppressive environment of the tumor-bearing host. Monocyte-derived dendritic cells (DCs) transfected with mRNA encoding candidate neoepitopes are used to prime autologous naive CD8+ T cells. Antigen-specific T cells that recognize endogenously processed and presented epitopes are detected using peptide-MHC (pMHC) multimers. Single multimer-positive T cells are sorted for the identification of TCR sequences, after an optional step that includes clonal expansion and functional characterization. The time required to identify neoepitope-specific T cells is 15 d, with an additional 2-4 weeks required for clonal expansion and downstream functional characterization. Identified neoepitopes and corresponding TCRs provide candidates for use in vaccination and TCR-based cancer immunotherapies, and datasets generated by this technology should be useful for improving algorithms to predict immunogenic neoantigens

Scientific Nonknowledge and Its Political Dynamics: The Cases of Agri-Biotechnology and Mobile Phoning

While in the beginning of the environmental debate, conflicts over environmental and technological issues had primarily been understood in terms of "risk", over the past two decades the relevance of ignorance, or nonknowledge, was emphasized. Referring to this shift of attention to nonknowledge the article presents two main findings: first, that in debates on what is not known and how to appraise it different and partly conflicting epistemic cultures of nonknowledge can be discerned and, second, that drawing attention to nonknowledge in technology conflicts results in significant institutional effects and new constellations of actors in public debates. To illustrate and substantiate this political dynamics of nonknowledge we draw upon examples from the areas of agri-biotechnology and mobile phoning. In a first step, we develop in greater detail the concept of scientific cultures of nonknowledge and identify three such cultures involved in the social conflicts within the two areas. Subsequently, we analyze the specific dynamics of the politicisation of nonknowledge looking at the variety of actors involved and the pluralisation of perceptions and evaluations of what is not known. Then, we point out some of the institutional reactions to the political and cultural dynamics of scientific nonknowledge. We argue that the equal recognition of the diverse cultures of nonknowledge is a key prerequisite for socially legitimate and "robust" decision-making under conditions of politicised scientific nonknowledge. © The Author(s) 2010