Fig. 6

Tumor-infiltrating T cells display reduced cytotoxicity after culture in a high-density collagen matrix. Tumor-infiltrating T cells from melanoma MM33 were cultured for 3 days on plastic (2D) or in collagen matrices of high- or low density, after which the cells were assayed for their ability to lyze autologous MM3 melanoma cells using a 51Cr-release assay. T cells were transiently PMA/ionomycin stimulated before the culture period (a-c) or cultured without any stimulus (d-f). (a and d) Representative example of MM33 melanoma cell lysis after 4 h incubation with different numbers of T cells, which had been transiently PMA/ionomycin stimulated (a) or directly embedded in collagen (d) and pre-cultured as indicated. b Percentage of melanoma cell lysis at the highest T cell: melanoma cell ratio in 3 different experiments. The T cells had been transiently PMA/ionomycin stimulated and cultured for 3 days in a low-density or high-density collagen matrix before extraction and incubation with 51Cr-labeled MM33 melanoma cells. c and f IFNγ levels in conditioned media of MM33 T cells, which had been transiently PMA/ionomycin stimulated (c) or directly embedded in collagen (f) and cultured for 3 days in a low-density or high-density collagen matrix. e Percentage of melanoma cell lysis at the highest T cell: melanoma cell ratio in 5 different experiments. The T cells had been cultured for 3 days in a low-density or high-density collagen matrix before extraction and incubation with 51Cr-labeled MM33 melanoma cells