Figure 3. Importance of IFNγ-pretreatment of tumor cells for efficient detection of T-cell receptor (TCR)-mediated T-cell activation.

(A) Impact of IFNγ-pretreatment on the cell surface HLA class I and II expression, as detected by pan-HLA class I and class II monoclonal antibodies (APC-conjugated W6/32 and PE-conjugated LN3, respectively) for a representative human pancreatic ductal adenocarcinoma (PDAC) tumor cell line. (B) Flow cytometry data showing the outcome of functional testing in a 5 h co-cultivation experiment of three different CD8⁺ T-cell-derived TCRs, isolated from the same human PDAC tumor sample, upon transfection into human CD8⁺ T222 cells. Left to right: mock-transfected T-cells, followed by T-cells transfected with a non-tumor-reactive (NTR) TCR and two tumor reactive (TR) TCRs. The dot plots depict the expression of CD107a and TNFα in CD3⁺mTCRβ⁺ T-cells. The data illustrate that detection of TCR reactivity is strongly promoted by IFNγ-pretreatment of the tumor cells, especially for weaker TCRs as exemplified by TCR1. The same applies, even to greater extent, for the detection of reactivity by TCRs derived from CD4⁺ T-cell clones (see Procedure C).