Figure 2. RT-PCR analysis of protocadherin 18 in tissues and immune cells.

(2a) Amino acid sequence comparison of the p56^lck Y505 motif in protocadherins. (2b) RT-PCR analysis of mouse tissues. The indicated tissues and organs were isolated from a control mouse, RNA was extracted and used to prepare cDNA, and PCR performed using control (pcdh8 and β-actin) or pch18 primers as described in ‘Materials and Methods’. (2c) RT-PCR analysis of spleen cells. Spleens were isolated from a 7 week old mouse and the indicated cells were purified by FACS (top panel). TIL were isolated from an MCA38 tumor. CTLL-2 is a CD8⁺ lytic cell line. RNA was isolated and used to program RT-PCR as described in ‘Materials and Methods’. Similarly spleen CD8⁺ T cells were purified and activated in vitro with anti-CD3 for the indicated times before analysis (bottom panel). (2d) TIL qRT-PCR analyses. Single cell suspensions of MCA38 tumors were prepared and CD4⁺ or CD8⁺ TIL were isolated by magnetic immunobeading. (Liver tissue was isolated and used for RNA isolation in certain control analyses. TIL were labeled with anti-CD4 or CD8 Ab and further purified by FACS (example of flow cytometry analysis shown in left panels) before RNA isolation and qRT-PCR analysis. TIL used to prepare RNA immediately after isolation are indicated in as ‘nonlytic’ or ‘TIL 0 hr’. As indicated some TIL samples were cultured in vitro for 8 or 24 h before RNA isolation during which time TIL recover both proximal TCR-mediated signaling and lytic function [5], [7]. PCR analyses of various target RNAs are shown and include several control reactions that demonstrate specificity of the expression patterns observed (e.g. pcdh18 and pcdh12 in CD8⁺ TIL, Dab1 and Dab2a in CD8⁺ TIL, granzyme B in CD8⁺ TIL and liver, as well as TNF, IFN, IL-2, PD-1, and PD-1L). Data show SD from three independent experiments. (2e) qRT-PCR analysis of purified spleen cells. Spleen immune cells were isolated by FACS from young (4 week) or old (>48 week) control mice and RNA was isolated and used to program pcdh18 qRT-PCR as described in ‘Materials and Methods’. The data shown are representative of multiple repetitions.