CD8⁺T-APC induce secondary trogocytosis by tumor specific CTL.

(A, B) CD8⁺T-APCs and non-T-APCs were generated by co-incubation with cognate or irrelevant melanoma, respectively. They were then sorted, labeled with DiIC18 (see Methods) and co-cultured with surface biotinylated 2C7 or 2E2 clones (effector CTL). The culture was then stained with anti-CD8 antibodies and streptavidin- allophycocyanin, and subjected to flow cytometry. (A) Histograms indicate secondary trogocytosis by the presence of DiIC18 on 2C7 or 2E2 CTLs, gated on CD8⁺streptavidin⁺ populations, following co-culture with CD8⁺T-APC (blue) or non-T-APC (grey). (B) Secondary trogocytosis was measured by presence of DiIC18 on the CD8⁺streptavidin⁺ population (effector CTL) and streptavidin-allophycocyanin on the CD8⁺DiIC18⁺ population (left column, non-T-APC, right column, T-APC). Numbers in upper right quadrants indicate the percentage of DiIC18- stained effector CTL (performing secondary trogocytosis). (C) PKH67-labeled CD8⁺T-APCs (red) were co-cultured with PKH26-labeled effector CTLs (blue). The lymphocytes were co-incubated in a chambered cover-glass and subjected to confocal microscopy. A snapshot series of 8 min is presented. Arrows, transfer of membrane fragments (secondary trogocytosis) from CD8⁺T-APC to CTL. Scale bars, 15 μm. Data are representative of at least five independent experiments (A, B) or of three experiments (C).