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. 2017 Oct 11;12(10):e0184496. doi: 10.1371/journal.pone.0184496

Fig 2. Tet-SAP effectively and specifically depletes cognate CD8+ T-cells.

Fig 2

(A) Representative dot plots of a time course, showing depletion of HIV HLA-B*27:05-Gag-KK10-specific CD8+ T-cells mediated by KK10-SAP. At time -2 hours, cells were left untreated or treated with KK10-SAP, mismatch-SAP or free SAP for 2 hours at 37°C, washed and left in media (time = 0 hours). Every 24 hours aliquots of cells from each condition were stained with the relevant fluorochrome-conjugated tetramer and analysed by flow cytometry. Gated on live CD3+ cells around CD8+tet+ cells; numbers indicate % tet+ cells (of CD8+) assessed at indicated times after treatment. (B) Graphical representation of 4 depletion time courses (performed as in A) with tet-SAP of different HIV, CMV and EBV specificities and restricted by different HLA class I molecules using cells from different individuals with the corresponding specificities. At time -2 hours cells were treated with tetramer-SAP of interest (red lines and symbols): HIV HLA-B*27:05 KK10-SAP (top left), CMV HLA-B*07:02 TM10-SAP (top right), HIV HLA-A*02:01 GL9-SAP (bottom left), or EBV HLA-A*02:01 GL9-SAP (bottom right). Controls included: untreated cells (black lines and symbols), treated with mismatch-SAP tetramer (blue lines and symbols; when sufficient cell numbers were present), or treated with free SAP (green lines and symbols, when sufficient cell numbers were present). % tet+ cells is normalised to baseline pre-treatment levels (time = -2h). Tx = treatment. (C,D) KK10-SAP-mediated depletion (assessed 48 hours post-treatment) of HLA-B*27:05-restricted KK10-specific CD8+ T-cells does not have an off-target effect on HLA-B*27:05-restricted KY9-specific CD8+ T-cells, while KY9-SAP-mediated elimination of KY9-specific cells does not affect KK10-specific cells. A,B, representative of at least 10 separate experiments with cells from different individuals with different HLA types and with tetramers of different specificities. C,D, representative of 3 independent experiments with cells from different HIV-positive HLA-B*27:05-positive donors.