Studies of the mechanism of cytolysis by tumour-infiltrating lymphocytes

Cell lines

Five pairs of autologous tumour cell lines and TIL were derived: four melanoma cell lines (M-1, M-3, M-5 and Mel-EW), one glioma cell line (Gli-PI) and the corresponding TIL. M-1, M-3 and M-5 melanomas and TIL have been previously characterized [52,53]. Mel-EW was derived from a metastatic melanoma patient and Gli-PI from a glioma. Cultures supplemented with IL-2 demonstrated propagation of activated T cells in those specimens containing lymphocytic infiltrates. These cultures were kept in IL-2 and restimulated with phytohaemagglutinin (PHA) and irradiated feeder cells every 2–4 weeks to enhance proliferation, as previously described [54]. Other cell lines used included Jurkat (derived from a T cell leukaemia), U-937 (derived from a promonocytic leukaemia) and K562 (from an erythroleukaemia). These cell lines were obtained from ATCC (Rockville, MD).

Cloning of TIL cultures

T cell clones were obtained by limiting dilution as previously reported [27,55]. Limiting dilution of IL-2-propagated TIL was performed within 4 weeks from original establishment of the culture.

Antibodies

TIL (bulk cultures and clones) were studied with a panel of MoAbs which included anti-CD3, CD4, CD8 (Ortho Diagnostic System, Raritan, NJ). MoAbs were used to stain cells in an indirect immunofluorescence test followed by analysis using a Becton Dickinson FACScan (Palo Alto, CA). Fas expression was detected by anti-Fas CH-11 MoAb obtained from UBI (Lake Placid, NY). Fas expression was also studied after pre-incubation of tumour cells with interferon-gamma (IFN-γ) as previously described in detail [53]. FasL expression was detected on TIL activated for 3 h in culture with PHA, anti-CD3 (purified OKT3 from Ortho, used at appropriate dilutions) or phorbol myristate acetate (PMA) and Ca-ionophore (ionomycin, obtained from Sigma (St Louis, MO) and used at 3 μg/ml), with a polyclonal rabbit anti-FasL (N-20) serum (Santa Cruz Biotechnology, Santa Cruz, CA), in an indirect immunofluorescence test with an anti-rabbit immunoglobulin FITC serum.

Anti-Fas IgM CH-11 antibody was also used to test susceptibility to Fas-mediated lysis by tumour cell lines at the concentration of 0.5 μg/ml. Anti-Fas MoAb (IgG ZB4; Immunotech, Westbrook, ME) was used at a concentration of 1 μg/ml to inhibit Fas-mediated lysis.

Perforin accumulations were detected in the cytoplasm of cytocentrifuged smears by immunofluorescence with an anti-perforin antibody (obtained from Kamiya Biomedical Co., Thousand Oaks, CA), and used at a concentration of 150 μg/ml. The percentage of stained cells was calculated from a minimum of 200 cells counted, with cells scored as positive if they contained distinctly fluorescent granules.

Cytoplasmic staining with Bcl-2 was assessed using anti-Bcl-2 (Novacastra Labs, Newcastle upon Tyne, UK) using cells which were first fixed for 10 min in 1% paraformaldehyde, followed by membrane permeabilization with 0.1% saponin for 10 min prior to addition of the anti-Bcl-2 antibody for 45 min. Following two washes, FITC-labelled goat anti-mouse IgG was added for 30 min. All steps were performed at room temperature (22°C). Assessment of staining was made by FACScan flow cytometry (Becton Dickinson).

Cytotoxicity

Cytotoxic activity was assessed by the lysis of ⁵¹Cr-labelled target cells in a 4-h Cr-release assay, as previously reported [56]. Per cent specific lysis was calculated as: (experimental −spontaneous) × 100/(maximum −spontaneous), where all experimental points were calculated as the average of triplicate cultures, and the spontaneous and maximum release by the target cells was the average of six wells each. Cytotoxicity was determined at serial dilutions of effector cells at ratios of 50:1, 25:1, 12.5:1 and 6.25:1 to determine the level at which maximum lysis could be achieved for each target/effector pair. As maximum release was achieved using 25 effectors per target, the data shown are for this ratio only in order to simplify data presentation. In all cases the ‘spontaneous release’ (background) was < 15% of the maximum, including cultures in which antibodies and cyclohexamide were added. Target cells included autologous tumour cell lines, as well as the series of tumour lines described above. Effector cells were TIL maintained in IL-2 (100 U/ml) for between 8 and 20 days after the previous restimulation with PHA and irradiated feeder cells. Assays were performed at multiple dilutions resulting in effector:target ratios of 50, 25, 12 and 6:1. As maximal lysis was apparent at ratios of 25:1, results are reported as percentage of specific lysis at this ratio. Effects on cytotoxicity by a calcium chelant were measured after adding 2 mm EGTA (Sigma) to the cultures.

Detection of Fas-induced cell death

After treatment of the tumour cell lines with anti-Fas (CH-11, at 1 μg/ml), apoptosis was evaluated by flow cytometry as previously described [57] with minor modifications. Briefly, the cells were centrifuged and resuspended in 0.6 ml of propidium iodide (PI) hypotonic fluorochrome solution (PI 50 μg/ml in 0.1% sodium citrate plus 0.1% NP-40; Sigma). The tubes were incubated overnight at 4°C in the dark before the flow cytometric analysis. The PI fluorescence was measured using an Ortho Absolute flow cytometer.

Assessment of cell death was also evaluated by the reduction of tetrazolium salt, MTT, by living cells to form a blue formazan product as previously reported [58].

Fas-induced apoptosis was also evaluated after coincubation of tumour cells with CH11 and cyclohexamide (CHX; used at a concentration of 1 μg/ml) as previously reported [59].

Fas and Bcl-2 expression on tumour cell line

We evaluated Fas expression on eight tumour cell lines. These included Jurkat (derived from a T cell leukaemia), U-937 (derived from a promonocytic leukaemia), K562 (from erythroleukaemia), four melanoma cell lines (M-1, M-3, M-5 and Mel-EW) and one glioma cell line (Gli-PI). Our data confirmed Fas expression on the Jurkat and U937 cell lines, while K562 was negative. In addition, we observed Fas expression on three melanoma cell lines (M-3, M-5, Mel-EW) and the Gli-PI glioma cell line, while M-1 cells were negative. Pre-incubation of the tumour cells with IFN-γ had only insignificant effects on Fas expression in all the cell lines tested (not shown). All of the melanomas and the glioma cell lines showed detectable cytoplasmic Bcl-2, although levels were generally low.

Fas-mediated apoptosis in tumour cells lines

We then tested the ability of an anti-Fas MoAb (clone CH-11) to induce apoptosis of target cells. Tumour cell lines, incubated with the anti-Fas antibody CH-11, were evaluated for apoptosis using PI and flow cytometry and by MTT assays. As shown in Table 1, the MTT assay (data shown are representative of four separate assays) revealed that Fas ligation was able to induce cell death in Jurkat (80%) and, to a lesser extent, in U-937 (32%) cells. Of the other cell lines tested, only 19% of the M-3 cell line population was killed, while the other cells tested showed very little susceptibility to cytotoxicity (≤ 12%). PI staining confirmed that the cell death observed in these cultures was due to apoptosis (data not shown).

As it has been shown that Fas-induced apoptosis in certain tumour cells may be inhibited by short lived cytoplasmic proteins [59], we also evaluated the ability of CH-11 to kill tumour targets after pre-incubation with CHX (an inhibitor of protein synthesis). Data show that, under these conditions, Fas was able to trigger increased apoptotic death in some of the cells lines (Fig. 1). (Data shown are representative of three separate assays).

Mechanisms of killing involved in TIL-mediated cytotoxicity of tumours. Ca²⁺-dependent killing by CD8⁺ and CD4⁺ cells

We first investigated the mechanisms involved in cell-mediated cytotoxicity by CD8⁺ TIL. We studied a previously described CD8⁺ TIL cell line, derived from a melanoma patient, with strong cytotoxic capacity against autologous tumour cells (M-1 cell line). Some clones derived from this bulk culture were also studied (MU-63, MU-79, MU-115). These CD8⁺ clones kill the autologous tumours through the recognition of the Melan A/MART-1 presented on HLA-A2 (not shown). As expected, killing by the bulk culture and the three derived clones was dependent on the presence of Ca²⁺, as shown by the inhibition of cytotoxicity in the presence of the Ca²⁺ chelant EGTA, whereas it could not be blocked by the addition of the anti-Fas MoAb ZB-4 (Table 2). (Data shown are representative of three experiments.)

The mechanisms involved in killing by CD4⁺ cells were investigated in TIL bulk cultures with a CD4⁺ phenotype and in CD4⁺ clones derived from both a melanoma and a glioma patient. TIL derived from a glioma patient, for which an autologous tumour cell line (Gli-PI) was available, were shown to be predominantly CD4⁺ (63%). This TIL cell line showed only marginal lytic capacity against the autologous tumour (5%, 10% and 15% in three different experiments). Three CD4⁺ clones (PI-A, PI-D and PI-H) derived from the glioma TIL also showed little cytotoxicity (not shown).

The expression of MHC class II antigens on glioma tumour cells was undetectable by staining with an anti-DR antibody and class II expression could not be induced by pretreatment of the glioma cells with IFN-γ (data not shown). As HLA class II molecules were not expressed on tumour cells, this precluded efficient recognition of the target by CD4 effectors. Therefore, we cross-linked effectors and target cells with PHA before performing the cytotoxic assay. Under these conditions, both the bulk culture and the three clones showed killing of the autologous tumour. In clones PI-A, -D and -H killing was 57%, 35% and 87%, respectively. Killing was Ca²⁺-dependent, as shown by > 90% inhibition observed with EGTA, while marginal (< 10%) effects were seen with the addition of the ZB-4 anti-Fas antibody (representative of three separate assays).

Expression of perforin on CD4⁺ cells

Ca²⁺-dependent killing manifested by CD4⁺ cells in the experiments with PHA-cross-linked targets suggested that Fas–FasL-mediated killing of tumour cells may not have been involved in the mechanism of lysis by these cells. Instead, the Ca²⁺-dependent killing observed is more consistent with perforin-mediated lysis. Therefore, we evaluated the presence of perforin in the cytoplasm of six CD4⁺ clones (three from the glioma and three from melanoma patient Mel-EW), by indirect immunofluorescence. In three out of six CD4⁺ clones examined, we observed the presence of scattered perforin staining in the cytoplasm in a granular distribution (Fig. 2). The positive clones were glioma clones PI-A and PI-H, and melanoma clone EW-2E5. This clone showed 43% killing of the autologous tumour cells, but killing was not significantly inhibited by either EGTA or ZB-4. In the three positive CD4⁺ clones, perforin staining was strong in only 15–50% of the cells (scored + or stronger (out of + + + +) among 200 cells counted per sample). In contrast, in CD8⁺ cultures the number of strongly perforin-positive cells was > 80% (most + + + or + + + +). In addition, Giemsa staining revealed the presence of the characteristic azurophilic cytoplasmic granules in the CD8⁺ cells, while in the CD4⁺ clones azurophilic granules were not detected. Glioma clones PI-D and melanoma clones EW-2E10 and 3F8 did not show detectable perforin.

Fas-mediated killing by CD4 TIL

To determine if the CD4⁺ effectors derived from tumour lesions also had the potential to kill by Fas engagement, we first evaluated the ability of TIL cell lines to express FasL. Bulk cultures, maintained in IL-2 for 6 or more days after restimulation with PHA and feeder cells, showed little expression of FasL. However, when we tested the same cell lines 3 h after stimulation with either PMA and ionomycin, anti-CD3 or PHA (without feeder cells), we observed increased FasL expression in all the tested cell lines, suggesting transient expression after stimulation (Fig. 3).

To assess the ability of CD4⁺ TIL cells to kill via Fas–FasL interactions, we developed a model using a Fas-sensitive target. As shown in Table 1, among the tumour cell lines derived in our laboratory from fresh tumours, only the M-3 targets were susceptible to some Fas-mediated apoptosis (19%). We therefore tested the ability of the melanoma-derived TIL CD4⁺ cell line M-3 and some of the Mel-EW CD4⁺ clones to induce killing of the Fas-sensitive target cell lines Jurkat and M-3. The K562 cell line was used as a Fas-insensitive control. FasL expression on effector cells was enhanced by pre-incubation with anti-CD3 antibodies for 3 h before performing the cytotoxic assay (representative of duplicate experiments). As shown in Fig. 4, the M3 (Fig. 4a) and Jurkat targets (Fig. 4b) were lysed in the presence of anti-CD3 antibody with or without anti-Fas or EDTA (P < 0.05 versus IL-2 alone). In contrast, K562 cells (Fig. 4c) were not lysed significantly by the EW clones, even in the presence of anti-CD3 antibody and added anti-Fas and EGTA. The M-3 TIL bulk had anti-K562 activity even in IL-2, which was not enhanced significantly by anti-CD3 or anti-Fas antibody, indicating the Fas-independent lysis of this target.

Cells cultured in IL-2 alone (which expressed low levels of FasL, see Fig. 3) showed no killing of the M-3 target (Fig. 4a). With Jurkat (Fig. 4b) and K562 (Fig. 4c) targets, the M-3 TIL bulk cultured in IL-2 alone showed some cytotoxic activity. After activation with anti-CD3, M-3 cells and two EW clones (EW-2E5 and EW-2E10) could lyse the Jurkat (Fig. 4b) and (to a lesser degree), the M-3 targets (Fig. 4a). Killing was consistent with the differential Fas sensitivity of Jurkat and M3 cell lines (Table 1).

Killing of Jurkat by M-3 bulk and EW clones was inhibited by anti-Fas antibody, but EGTA induced little or no inhibition (Fig. 4b). On the other hand, lysis of the M-3 target by M-3 bulk and clone EW-2E5 appeared to be mediated by both Ca²⁺- and Fas-dependent mechanisms, since some inhibition could be observed with both EGTA and anti-Fas (Fig. 4a). Lysis of this target by clone 2E10 could not be blocked by either EGTA or anti-Fas, thus precluding a delineation of the mechanism involved in this interaction. Killing of the Fas-resistant K562 target by M-3 bulk could be inhibited only by EGTA, and not by anti-Fas, confirming that killing of this target occurred in a Ca²⁺-dependent, Fas-independent fashion.