Interferon-γ secretion by natural killer cells. The experimental approach is detailed in Supplementary Fig 1B online. (A,B) Natural killer (NK), cells alone; NK+CD4, NK cells co-cultured with CD4 T cells that did not contact tumour cells; NK+CD4/C, NK+CD4/1, NK+CD4/Ian, NK+CD4/M8 and NK+CD4/Mel-888, NK cells co-cultured with CD4 T cells previously co-cultured with tumour clone C or tumour clone 1, or IIB-MEL-IAN, M8 or Mel-888 cells, respectively. CD4, CD4/C, CD4/1, CD4/Ian, CD4/M8 and CD4/Mel-888, CD4⁺ T cells cultured in the absence or in the presence of tumour clone C or 1, or IIB-MEL-IAN, M8 or Mel-888 cells, respectively. For blocking experiments, NK cells were co-cultured with CD4⁺ T cells imprinted by tumour clone C (C) or tumour clone 1 (D), in the absence (−) or in the presence (+) of the indicated blocking monoclonal antibodies. Each dot or line in (A–D) represents the result obtained with cells from a different donor. To circumvent the heterogeneity commonly observed in IFN-γ secretion among different donors, the ratio of IFN-γ secretion observed in the presence to that obtained in the absence of the blocking monoclonal antibodies is shown (E). The mean±s.e.m. obtained in independent experiments carried out with cells from five to eight different donors is shown. ^*P<0.05; ^**P<0.01; ^***P<0.001. IFN-γ, interferon-γ.

Transfer of molecules between tumour and T cells. Acceptor unlabelled cells (peripheral blood mononuclear cells (PBMCs) stimulated with phytohaemagglutinin (PHA) and interleukin (IL)-2 in (A) and cells of tumour clone 1 in (B)) were co-cultured for 1 h with 3,3′-dioctadecyloxacarbocyanine perchlorate (DiOC₁₈)- or carboxyfluorescein succinimidyl ester (CFSE)-labelled donor cells (melanoma cells in (A) or PBMCs stimulated with PHA+ IL-2 in (B)), and fluorescence was assessed on (A) non-adherent CD3⁺ T cells or (B) tumour cells after removing PBMCs. Numbers represent the mean fluorescent intensity (MFI) of CFSE- or DiOC₁₈-labelled donor cells or acceptor cells that captured CFSE or DiOC₁₈. The extent of trogocytosis was calculated as the ratio of fluorescence between acceptor and donor cells for each fluorescent probe. In addition, tumour clone 1 (unlabelled or labelled with DiOC₁₈ or CFSE) was co-cultured for 1 h with PBMCs stimulated with PHA and IL-2, and cell surface expression of major histocompatibility complex class I chain-related protein A (MICA) was assessed by flow cytometry on non-adherent CD3⁺ cells (C). The percentages in quadrant 2 represent T cells that captured the fluorescent probe and MICA (quadrants were set with acceptor cells that did not contact tumour cells). Results are representative of three experiments carried out with cells from three different donors. The increase in green fluorescence on acceptor cells after co-culture with CFSE- and DiOC₁₈-labelled donor cells (A,B) or the increase in percentage of double-positive cells after co-culture with CFSE- and DiOC₁₈-labelled donor cells (C) was statistically significant, compared with acceptor cells cultured alone (P<0.05).

Cytotoxicity and degranulation of natural killer cells. The experimental approach is detailed in Supplementary Fig 1A online. (A) Degranulation of natural killer (NK) cells triggered by activated CD4⁺ T cells cultured in the absence (ii) or previously co-cultured for 1 h with tumour clone C (iii) or tumour clone 1 (iv); NK cells cultured alone are also shown (i). The graph shows results from three different donors. ^**P<0.01. (B) NK cells were used as effector cells of cytotoxicity against tumour clone 1, tumour clone C or activated CD4⁺ T cells cultured in the absence or in the presence of the tumour clones at two effector:target (E:T) ratios. Results correspond to one of four independent experiments carried out with cells from different donors (data are expressed as mean±s.e.m.).

Transfer of major histocompatibility complex class I chain-related protein A to T cells. (A) Peripheral blood mononuclear cells (PBMCs) stimulated with phorbol-12-myristate-13-acetate (PMA) and ionomycin (Iono) or (B) phytohaemagglutinin (PHA) and interleukin (IL)-2, (C) purified CD4⁺ T cells or (D) CD8⁺ T cells stimulated with PHA and IL-2, (E) resting PBMCs or (F) natural killer (NK) cells stimulated with IL-15 were cultured without (medium) or with tumour clone C or tumour clone 1 for 1 h and major histocompatibility complex class I chain-related protein A (MICA) was detected on non-adherent CD3⁺ T cells (A–E) or CD56⁺ cells (F). (H) Similar experiments were carried out with the indicated cell lines and MICA expression was assessed on CD4⁺ T cells. (I) Similar experiments as in (B) were carried out with fixed PBMCs (p-formaldehyde (pfa)). (J) PBMCs stimulated with PMA+Iono were co-cultured for 1 h with tumour clone 1 previously fixed with pfa, or with live cells of tumour clone 1 separated by transwells (clone 1/transwell), and MICA was assessed on CD3⁺ T cells. (K) Kinetic analysis of MICA on T cells stimulated with PMA+Iono on co-culture with tumour clone 1 for different periods of time (indicated at the top of each dot plot). 0 min: MICA on stimulated T cells not co-cultured with tumour clone 1. MICA expression was assessed as in (J). (L) PBMCs stimulated with PHA+IL-2 co-cultured for 1 h with tumour clone 1 were harvested, cultured alone for 3 or 24 h (in this case, in the absence or in the presence of chloroquine or cytochalasin B), and MICA was assessed as in (J). 0 h: MICA on activated T cells co-cultured with clone 1 for 1 h analysed immediately after co-culture. Numbers within dot-plots show the percentage of MICA⁺ cells. One of four representative experiments carried out with PBMCs from four different donors is shown. The mean±s.e.m. of these experiments is shown in (G). ^***P<0.001. The increase in cell-surface MICA on T and NK cells co-cultured with tumour clone 1 was statistically significant, compared with equivalent cells cultured alone (P<0.05; data not shown).

Confocal microscopy. (A) The tumour clone 1 was stained with the MICA (major histocompatibility complex class I chain-related protein A) monoclonal antibody (mAb; i) or isotype-matched control mAb (IC mAb; ii) and Cy3-labelled donkey anti-mouse IgG. (B) Cells were cultured as detailed in Supplementary Fig 9 online. Fluorescein isothiocyanate (FITC)-labelled stimulated CD4⁺ T cells after co-culture with red-labelled MICA⁺ tumour clone 1 cells are shown in i–iii. CD4⁺ T cells stained with an IC mAb co-cultured with tumour clone 1 labelled with an IC control mAb are also shown (iv). (C) Higher magnification of some CD4⁺ T cells from (B). (D,E) Unlabelled CD4⁺ T cells stimulated with phytohaemagglutinin (PHA) and interleukin (IL)-2 were co-cultured for 1 h with cells of tumour clone 1 labelled with (D) 3,3′-dioctadecyloxacarbocyanine perchlorate (DiOC₁₈; green) or (E) PKH26 (red), and with the anti-MICA mAb and Cy3-labelled anti-mouse IgG (for DiOC₁₈-labelled tumour cells) or FITC-labelled anti-mouse IgG (for PKH26-labelled tumour cells). Thereafter, cells were analysed by confocal microscopy. Insets show the synapse between T cells and tumour cells. Results are representative of three independent experiments carried out with cells from three different donors.