Soluble factors released by activated cytotoxic T lymphocytes interfere with death receptor pathways in neuroblastoma

Abstract

Neuroblastoma (NB) is often described as an unfavorable target for both HLA-restricted and death receptor-mediated elimination by cytotoxic T lymphocytes (CTLs) due to low or absent HLA class I and caspase-8 expression. We investigated the effects of soluble factors released by CTLs activated by TCR triggering (named as activated supernatant; AS) on the levels and composition of cell surface molecules involved in HLA-restricted and HLA-independent NB cell recognition (surface immune phenotype). Using a panel of long-term propagated NB cell lines and freshly isolated primary human NB cells, we analyzed surface expression of the (1) cognate receptors for TNFalpha, Fas and TRAIL; (2) HLA class I and II heterodimers; (3) adhesion molecules; (4) the intracellular expression and activation of caspase-8, as well as (5) the susceptibility of NB cells to death receptor-mediated killing prior to and after exposure to AS. The exposure of NB cells to soluble factors released by activated CTLs skewed the surface immune phenotype of both long term cultured and primary NB cells, induced the expression and activation of caspase-8 and increased the susceptibility of tumor cells to lysis by TRAIL and Fas-agonistic antibody. Blocking experiments identified IFNgamma and TNFalpha as main factors responsible for modulating the surface antigens of NB cells by AS. Our data suggest that recruitment of CTLs activated on third party targets into the vicinity of the NB tumor mass, may override the "silent" immune phenotype of NB cells via the action of soluble factors.

Fig. 1

a Cell surface immune phenotype of the neuroblastoma cell line CHP-212. The expression of the molecules indicated in the figure was assessed by flow cytometry, following incubation with specific antibodies (open histograms) or relevant isotype controls (filled histograms) as described in Materials and methods. b, c Cell surface immune phenotype of tumor cells derived from neuroblastoma patients. b Cell surface phenotype of tumor cells derived from neuroblastoma sample NB-KI-3. Tumor cells were analyzed by flow cytometry at the day of tumor excision for the surface expression of the molecules indicated in the figure. Immunostainings were performed using specific antibodies (open histograms) or relevant isotype controls (filled histograms). c Statistical analysis of the tumor cell surface phenotype in low stage (I/II) versus high stage (III/IV) NB patients. The surface expression levels of the molecules indicated in the figure were calculated as specific geometric mean fluorescence intensity (sgMFI = Ab_{geo mean} − Iso_{geo mean}) as determined by flow cytometry. The boxes represent the interquartile range with the lower and upper quartiles surrounding a thicker line representing the median. The vertical lines connect the nearest observations within 1.5 IQRs (inter-quartile ranges) of the lower and upper quartiles. Squares (filled square) and circles (filled circle) indicate possible outliers more than 1.5 IQRs (near outliers) and 3.0 IQRs (far outliers) from the quartiles, respectively. P values for differences between independent groups were calculated using the non-parametric Mann–Whitney U test (*P < 0.05)

Fig. 2

The effect of activated supernatant (AS) on the surface immune phenotype of NB cells. a NB cell lines were cultured overnight in either medium alone (gray bars) or in the presence of 10% v/v of supernatant from activated CTLs (black bars) and subsequently analyzed for the surface expression of the indicated molecules using specific antibodies followed by flow cytometry. Specific geometric MFI was calculated as described in the legend to Fig. 1. Means ± SD from three independent experiments are shown in the figure. (MC, MC-IXC; CHP, CHP-212; SH, SK-N-SH; 5Y, SH-SY5Y; L5, LAN-5). b Tumor cells from patient NB-KI-6 were treated for 84 h with either medium alone (filled histogram) or in the presence of 10% v/v of supernatant from activated CTLs (thick open histogram), and then stained for the indicated surface markers using specific antibodies and analyzed by flow cytometry. The histograms representing fluorescence obtained with the isotype control antibodies on untreated and AS-treated NB cells overlapped and had similar geo mean values, therefore, only one histogram is shown as reference. c Specific geometric MFI values obtained in the experiment shown in b. d CHP-212 cells were cultured overnight in either medium alone (M) or AS. The AS was either non-manipulated or pre-incubated with IFNγ-neutralizing antibodies (α-IFNγ) and/or soluble TNFα receptor (α-TNFα) for 1 h at room temperature. The numbers in the ICAM-1 panel indicates the average geo MFI values

Fig. 3

Supernatant from activated CTLs both induce caspase-8 expression and modulate its activity in NB cells. a NB cell lines were treated for 48 h with either medium alone, or in the presence of 10% v/v of control supernatant (CS) or activated supernatant (AS). The expression of caspase-8 in total cell lystaes was monitored by Western blot using a specific antibody recognizing the 55/50 kD pro-form of caspase-8 as well as the 40/36 and 23 kD cleavage products obtained upon activation. Expression of actin was used as a control of sample loading. b The activity of caspase-8 in NB cells propagated for 24, 48 and 72 h in complete medium alone (thin open histograms), or in the presence of either CS (filled histograms) or AS (bold open histograms) was measured by flow cytometry using fluorescent caspase-8 specific substrates as described in Materials and Methods. Numbers show percentage of AS-treated cells positive for active caspase-8

Fig. 4

Pre-treatment of NB cells with AS renders them more sensitive to death receptor mediated killing. NB cell lines propagated in complete medium alone or in the presence of 10% v/v of activated supernatant (AS) were exposed to death-inducing signals for the periods of time described in Materials and Methods, and then stained with FITC-conjugated Annexin-V and analyzed by flow cytometry. Data from one representative experiment are shown as histograms for each cell line. The bar graphs show percentages of Annexin-V positive cells induced by the respective death inducer where gray bars represent medium-pretreated cells and black bars represent AS-pretreated cells. Data is presented as the difference between the numbers of Annexin V-positive cells in control cultures (kept in complete medium alone for TNFα and killerTRAIL or in the presence of IgM for CH-11) and cultures exposed to the relevant death inducer. Statistical significance (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001) was determined using the Student’s paired t test