Recognition and internalisation of conventional and cytotoxic tetramers.

<p><b>(A-F)</b> Representative dot plots of PBMC staining in an HIV-infected HLA-B*27:05-positive subject with conventional fluorescently labeled tetramer HLA-B*27:05-Gag-KK10-PE <b>(A)</b> or SAP-coupled HLA-B*27:05-Gag-KK10-SAP tetramer, detected with a secondary Alexa Fluor 488 anti-SAP antibody in permeabilised <b>(B)</b> or not permeabilised <b>(C)</b> cells. Absence of non-specific binding of HLA-mismatched tet-PE <b>(D)</b> and tet-SAP <b>(E)</b> and free unconjugated SAP <b>(F)</b> is shown. Gated on live CD3+ cells around CD8+tet+ cells; numbers indicate % tet+ cells (of CD8+). <b>(G)</b> Kinetics of tetramer binding and internalisation, determined by measurements of total and internal tetramer fluorescence in cells at internalisation-promoting (37°C) or internalisation-inhibiting (4°C) conditions. No further increase in internal fluorescence is observed after 90–120 minutes of internalisation time, suggesting that nearly all cognate metabolically active (37°C) CD8+ T-cells have internalised the tetramer. PBMC from an HIV-negative healthy donor with an EBV-HLA-A*02:01-GL9 response were used here. ‘Total tet-PE’ = surface-bound and internal tetramer fluorescence measured in cells not stripped of any surface-bound tetramer; ‘internal tet-PE’ = internal tetramer fluorescence measured in acid-stripped cells. A-F, representative of 8 independent experiments with PBMC from different donors. G, representative of 2 independent experiments with cells from two individuals.