tissuescan human major tissue qpcr panel  (OriGene)

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: MAGE-C2-Specific TCRs Combined with Epigenetic Drug-Enhanced Antigenicity Yield Robust and Tumor-Selective T Cell Responses.

doi: 10.4049/jimmunol.1502024

Figure Lengend Snippet: FIGURE 4. Treatment of melanoma, head and neck, bladder, and breast carcinoma cells with AZA and VPA enhances MC2 gene expression and T cell recognition. (A) Melanoma cell lines (n = 6), HaCaT keratinocytes, and fibroblasts (Fibrobl.) were pretreated or not with AZA (1 mM for 72 h), VPA (1 mM for 48 h), and IFN-g (50 pg/ml for 48 h). RNAwas isolated from 1 3 106 cells, followed by cDNA synthesis and RT-PCR with primers for detection of MC2, HLA-A2, and GAPDH transcripts. PCR products were subjected to gel electrophoresis, and intensities of MC2 and HLA-A2 bands were quantified by Quantity One (version 4.6.7), corrected for background noise and standardized for GAPDH levels, and displayed in relative intensity units/mm2. (B) MC2 TCR T cells were cocultured with pretreated melanoma cells from (A) at an E:T ratio of 3:1 for 24 h. IFN-g levels in culture supernatants were measured by ELISA and are displayed as mean 6 SEM (n = 5). IFN-g production by MC2 TCR T cells was corrected for IFN-g production by mock T cells; IFN-g levels produced by mock T cells ranged from 2 to 300 pg/ml. (C) Cell lines from HNSCC (n = 4), TNBC (n = 4), and bladder carcinoma (n = 3) were pretreated as described in (A). RNA was isolated, used for RT- PCR, and subjected to analysis of MC2 and HLA-A2 expression. (D) MC2 TCR T cells were cocultured with pretreated tumor cell lines from (C) and analyzed for IFN-g production, as described in (B). IFN-g production of mock T cells ranged from 9 to 180 pg/ml. Data are from one healthy donor out of two tested with similar results. *p , 0.05, **p , 0.005, responses of TCR T cells toward MC2+ versus MC22 target cells, Student t test.

Article Snippet: For quantification of MC2 mRNA in healthy human tissues, quantitative PCR (qPCR) was conducted with a normal human tissue cDNA panel (OriGene Technologies, Rockville, MD) and primers and TaqMan probes for MC2 (Gene ID: 51438) and GAPDH (Gene ID: 2597) (Life Technologies, Carlsbad, CA).

Techniques: Gene Expression, Isolation, cDNA Synthesis, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Enzyme-linked Immunosorbent Assay, Produced, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: MAGE-C2-Specific TCRs Combined with Epigenetic Drug-Enhanced Antigenicity Yield Robust and Tumor-Selective T Cell Responses.

doi: 10.4049/jimmunol.1502024

Figure Lengend Snippet: FIGURE 7. MC2 expression in healthy tissue is restricted to testis, and TCR6 shows no reactivity to other, including highly similar, epitopes. (A) MC2-specific qPCR was performed using a cDNA library of 48 healthy, human tissue samples. MC2 mRNA levels are expressed as fold increase compared with testis, the only healthy sample positive for MC2 mRNA. Fold increase was calculated based on the DDCt method and normalized for GAPDH expression. Patient-derived MC2+ melanoma cell line EB81-MEL served as positive control. (B) TCR6 T cells were cocultured with T2 cells loaded with 10 mM of 114 different HLA-A2–restricted peptides for 24 h. IFN-g levels in culture supernatants were measured by ELISA and are displayed as mean (n = 3). IFN-g response against the MC2 LLF peptide (positive control) is depicted as a triangle, and against no peptide (T2 cells only; negative control) is depicted as a hollow circle. (C) TCR6 T cells were cocultured with T2 cells loaded with 100 nM of highly similar peptides of MB4 (LVFGLALKEV) and MB10 (LIFGLDLKEV) (amino acids different from the MC2 LLF peptide being underlined). IFN-g levels in culture supernatants were measured by ELISA and are displayed as mean 6 SEM (n = 4). IFN-g response against the MC2 LLF peptide served as a positive control. (D) Again, TCR6 T cells were cocultured with T2 cells but now with titrated amounts of MB4 and MC2 LLF peptide for 24 h. IFN-g levels in culture supernatants were measured by ELISA and are displayed as mean 6 SEM (n = 4). Curves and EC50 values were analyzed using GraphPad Prism (EC50 for MC2: 3.1 3 1029 M; EC50 for MB4: 7.15 3 1029 M). (E) Finally, TCR6 T cells were cocultured with EB81-MEL (MC2+/MB42/A2+), OEC-19 (MC22/MB4+/A2+), or T2 (MC22/MB42/A2+) tumor cells. Expression of MC2, MB4, and HLA-A2 was verified or ruled out via RT-PCR. IFN-g levels in culture supernatants were measured after 24 h by ELISA and are displayed as mean 6 SEM (n = 4). IFN-g production by TCR6 T cells was corrected for IFN-g production by mock T cells; IFN-g levels produced by mock T cells ranged from 5 to 13 pg/ml.

Article Snippet: For quantification of MC2 mRNA in healthy human tissues, quantitative PCR (qPCR) was conducted with a normal human tissue cDNA panel (OriGene Technologies, Rockville, MD) and primers and TaqMan probes for MC2 (Gene ID: 51438) and GAPDH (Gene ID: 2597) (Life Technologies, Carlsbad, CA).

Techniques: Expressing, cDNA Library Assay, Derivative Assay, Positive Control, Enzyme-linked Immunosorbent Assay, Negative Control, Reverse Transcription Polymerase Chain Reaction, Produced