Fig 1: Wild-type T cell clone PAPPA-2G6 specifically recognizes and kills HLA-A*02:01+/PAPPA+ ES cell lines. (A) PAPPA1434 and PAPPA601 bind to HLA-A2 and stabilize MHC I molecules in Tap deficient T2 cells. (B) PAPPA-2G6 T cells show peptide specificity against peptide loaded T2 cells. (C) Reactivity is dose dependent in IFNγ ELISpot T2 titration assays. IFNγ release diminishes at a threshold of < 1 nM. (D) HLA-A*02:01+/PAPPA+ ES cell lines are recognized specifically compared to the controls SK-N-MC and K562 in IFNγ ELISpot assays. (E) Killing/detachment of A673 ES cell line is shown in real time in xCELLigence assay. The control cell line SK-N-MC is not affected in its growth by the presence of the TCR transgenic T cells. Data are presented as mean and SEM. A673, EW7 and TC-71: HLA-A*02:01+ ES; SK-N-MC: HLA-A*02:01− ES; K562: MHC− NK cell control. Error bars represent standard deviation of triplicate experiments. Asterisks indicate significance levels. p values < 0.05 were considered statistically significant (*p < 0.05; **p < 0.005; ***p < 0.0005).
Fig 2: Identification of the PAPPA-2G6 TCR sequence. Full TCR PCR with specific primers for TRAV5 and TRBV4-4. PCR products (green boxes) of expected sizes were extracted and sequenced.
Fig 3: Detection of engrafted and tumor-infiltrating T cells in PAPPA-2G6 TCR transgenic T cells treated mice via FACS. FACS staining for CD8+ and specific multimer shows PAPPA-2G6 transgenic T cells circulating in blood (left). Further T cells were detected in bone marrow (middle) and infiltrating into the A673 tumors (right). An irrelevant multimer served as a control.
Fig 4: Isolation and ES specificity of PAPPA-2G6 TCR transgenic T cells. (A) Transduction efficiency for PAPPA-2G6 TCR transgenic T cells of 47.3% was determined via FACS multimer staining (middle). Multimer-PE stained transgenic T cells were isolated via magnetic beads (right) (B) PAPPA-2G6 TCR transgenic T cells show peptide specificity against PAPPA1434 peptide loaded T2 cells. (C) Reactivity is dose dependent in IFNγ ELISpot T2 titration assays. IFNγ release diminishes at a threshold of < 10 nM. (D) HLA-A*02:01+/PAPPA+ ES cell lines are recognized specifically compared to the controls in IFNγ ELISpot assays. (E) Killing of A673 ES cells is shown via detachment in xCELLigence assay. Addition of PAPPA-2G6 TCR transgenic T cells specifically kills HLA-A*02:01+ A673 tumor cells (top) whereas the negative SK-N-MC control is not affected. Data are presented as mean and SEM. A673, EW7 and TC-71: HLA-A*02:01+ ES; SK-N-MC and SB-KMS-KS1: HLA-A*02:01− ES; K562: MHC− NK cell control. Error bars represent standard deviation of triplicate experiments. Asterisks indicate significance levels. p values < 0.05 were considered statistically significant (*p < 0.05; **p < 0.005; ***p < 0.0005).
Fig 5: Immunohistochemistry staining confirms tumor infiltration by transgenic T cells and target gene expression. (A) Tumor slides were stained with a specific antibody against CD8+ in immunohistochemistry. Infiltration by T cells could be shown in PAPPA-2G6-treated mice (top). CD8 positivity upon mice treated with unspecific T cells was less frequent (bottom). (B) Immunohistochemistry further showed strong immunoreactivity in trophoblast layers of placental villi (left; positive control) and xenografted A673 (right).
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