Generation of affinity ranged antigen-expressing tumor cell lines.

The interaction strength between CD8 <sup>+</sup> T cells' TCR and cognate peptide-MHC (pMHC) impacts on the CD8 <sup>+</sup> T cell response against pathogens and tumors (Martinez-Usatorre, Donda, Zehn, & Romero, 2018; Zehn, Lee, & Bevan, 2009). CD8 <sup>+</sup> T cell responses against tumors are characterized by the presence of low affinity CD8 <sup>+</sup> T cells specific for nonmutated tumor associated self-antigens (TAA) and potentially high affinity tumor specific CD8 <sup>+</sup> T cells recognizing mutated self-antigens (Gros et al., 2016; Kvistborg et al., 2012; McMahan & Slansky, 2007). High affinity T cells display enhanced survival, expansion capacity and tumor control (Martinez-Usatorre et al., 2018; Schmid et al., 2010). In fact, recent clinical trials using neoantigen tumor vaccines showed prolonged progression free survival in melanoma patients (Ott et al., 2017; Sahin et al., 2017), while only modest clinical efficacy was obtained with TAA vaccines (Romero et al., 2016). However, the highly individual nature of neoantigens constitutes a major technical and economical hurdle for routine clinical application. Thus, the characterization of TAA-specific CD8 <sup>+</sup> T cell responses may reveal new strategies to enhance their anti-tumor properties. In parallel, the identification of high affinity antigens and CD8 <sup>+</sup> T cells may be essential to design effective tumor vaccines and adoptive cell transfer therapies. Therefore, in this chapter, we describe how to generate tumor cell lines with stable expression of affinity-ranged antigens and methods to assess T-cell affinity