Journal: The Journal of Immunology Author Choice
Article Title: An Isolated TCR αβ Restricted by HLA-A*02:01/CT37 Peptide Redirecting CD8+ T Cells To Kill and Secrete IFN-γ in Response to Lung Adenocarcinoma Cell Lines
doi: 10.4049/jimmunol.1701054
Figure Lengend Snippet: Features of lung ADC cell lines used ( A ) and cytotoxic capacity of CD8 + T cells transduced with the three different constructs ( B ). In (A), total RNA was isolated from cell lines and negative control (A549 cell line) as indicated, and RT-PCR results obtained. The mean of triplicates is shown (A i ). Protein lysates from cell lines indicted and negative control A549 were immunoprecipitated with rabbit polyclonal anti-human CT37 Ab HPA011284, proteins were separated by SDS-PAGE, transferred to a membrane, and CT37 blotted using rabbit polyclonal AP51690PU-N Ab and HRP-conjugated goat anti-rabbit IgG. Blots were detected by chemiluminescence using autoradiography films [Kodak BioMax Light; (Aii)]. In (B), retroviral transduction of effector (E) allogeneic CD8 + . For results presented in (Bi)–(Biii), six [ 51 Cr] release assays, with each variable in triplicate, were performed. After 2 cycles of 10 d stimulation in vitro, E cells were harvested, washed, counted, and plated to be tested at 16:1 E:T ratios. Target (T) lung ADC lines HLA-A*A-02:01– positive HCC2935 (Bi), and H1993 (Bii), and HLA-A*02:01– negative line H1299, were labeled with 200 μCi of Na 2 51 CrO 4 for 1 h and washed. [ 51 Cr]-released cytotoxic assay proceed for 4 h at a 16:1 E:T ratio. Cultures proceeded without or with an anti–HLA-A2 Ab to block the HLA-A*02:01 molecule, and at the indicated concentrations. The HCC2935 cell line carries wild type (wt) TP53 and KRAS genes, and an EGFR 2237_2254del10 (deletion E746-S752). The H1993 line carries a TP53 726C > G (C242W) substitution, and wt EGFR and KRAS genes. The H1299 carries a TP53 gene deletion, and wt EGFR and KRAS genes. Data on TP53 . We genotyped mutations in the EGFR and KRAS . The p values from Mann–Whitney comparison of two means are presented.
Article Snippet: To determine the identity of CD3 chains, immunoprecipitated proteins obtained as indicated above from cells transfected with construct 3 were blotted using rabbit anti-CD3δ EP4426, anti-CD3γ 134684, anti-CD3ε 133628, and Armenian hamster anti-CD3ζ H136-968 Abs (Abcam), 1:2000 HRP-conjugated goat anti-rabbit IgG or 1:000 HPR-conjugated rabbit anti-Armenian hamster IgG (Abcam), and the Immun-Star HRP kit, and blots were revealed by chemiluminescence using autoradiography Kodak BioMax Light film.
Techniques: Transduction, Construct, Isolation, Negative Control, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, SDS Page, Autoradiography, In Vitro, Labeling, Blocking Assay, MANN-WHITNEY
![Features of lung ADC cell lines used ( A ) and cytotoxic capacity of CD8 + T cells transduced with the three different constructs ( B ). In (A), total RNA was isolated from cell lines and negative control (A549 cell line) as indicated, and RT-PCR results obtained. The mean of triplicates is shown (A i ). Protein lysates from cell lines indicted and negative control A549 were immunoprecipitated with rabbit polyclonal anti-human CT37 Ab HPA011284, proteins were separated by SDS-PAGE, transferred to a membrane, and CT37 blotted using rabbit polyclonal AP51690PU-N Ab and HRP-conjugated goat anti-rabbit IgG. Blots were detected by chemiluminescence using autoradiography films [Kodak BioMax Light; (Aii)]. In (B), retroviral transduction of effector (E) allogeneic CD8 + . For results presented in (Bi)–(Biii), six [ 51 Cr] release assays, with each variable in triplicate, were performed. After 2 cycles of 10 d stimulation in vitro, E cells were harvested, washed, counted, and plated to be tested at 16:1 E:T ratios. Target (T) lung ADC lines HLA-A*A-02:01– positive HCC2935 (Bi), and H1993 (Bii), and HLA-A*02:01– negative line H1299, were labeled with 200 μCi of Na 2 51 CrO 4 for 1 h and washed. [ 51 Cr]-released cytotoxic assay proceed for 4 h at a 16:1 E:T ratio. Cultures proceeded without or with an anti–HLA-A2 Ab to block the HLA-A*02:01 molecule, and at the indicated concentrations. The HCC2935 cell line carries wild type (wt) TP53 and KRAS genes, and an EGFR 2237_2254del10 (deletion E746-S752). The H1993 line carries a TP53 726C > G (C242W) substitution, and wt EGFR and KRAS genes. The H1299 carries a TP53 gene deletion, and wt EGFR and KRAS genes. Data on TP53 . We genotyped mutations in the EGFR and KRAS . The p values from Mann–Whitney comparison of two means are presented.](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5890535/bin/ji1701054f5.jpg)