IFN-gamma protects short-term ovarian carcinoma cell lines from CTL lysis via a CD94/NKG2A-dependent mechanism

Abstract

IFN-gamma regulates the immunogenicity of target cells by increasing their expression of HLA class I molecules. This facilitates the T cell receptor-mediated recognition by CD8(+) T cells but decreases target cell sensitivity to lysis by NK cells due to engagement of inhibitory NK receptors. In this study, short-term tumor cell lines from patients with advanced ovarian carcinomas were established. We demonstrate the paradoxical finding that IFN-gamma treatment of these short-term ovarian carcinoma cell lines (OVACs) resulted in resistance of tumor cells to lysis by peptide- and allospecific CD8(+) T cells. Blocking experiments revealed that this phenomenon was dependent on enhanced inhibitory signalling via CD94/NKG2A receptors expressed on the effector cells. This was associated with increased expression of HLA-E mRNA and HLA-G at the protein level in IFN-gamma-treated OVACs. Furthermore, pulsing of untreated OVACs with the leader sequence peptide of HLA-G protected these cells from lysis by CTLs, thus mimicking the inhibitory effect of IFN-gamma. This study provides evidence that CD94/NKG2A receptors play an important role in regulating T cell activity against tumors and shows that IFN-gamma modulation of target cells may shift the balance of triggering and inhibitory signals to T cells, turning off their cytolytic activity.

Figure 1

IFN-γ protects short-term ovarian carcinomas from CTL lysis. The effect of IFN-γ on CTL recognition of OVACs was evaluated in 4-hour ⁵¹Cr-release assays. (a) K562 and untreated or IFN-γ–treated OVAC 16 (HLA-A11⁺) were used as targets for the HLA-A11–specific, CD8⁺ CTL clone KV109. (b) OVAC 16 was untreated or treated with IFN-γ and then pulsed or not pulsed with the HLA-A11–restricted, EBNA4-derived epitope IVT at a concentration of 1 μg/ml and used as a target for the IVT-specific, CD8⁺ T cell clone BK289. (c) C1R/A11 was untreated or treated with IFN-γ and then pulsed or not pulsed with the IVT peptide (1 μg/ml) and used as a target for BK289. (d) Untreated or IFN-γ–treated OVAC 16 was prepulsed with IVT peptide at concentrations ranging from 10^–12 to 10^–6 g/ml and used as a target for BK289 at an effector-to-target ratio of 3:1. Recognition of non-pulsed targets was below 7%. Results from one of at least three experiments are shown. (e) K562 and untreated or IFN-γ–treated OVAC 16 was used as a target for autologous TALs.

Figure 2

Intact regulatory function of IFN-γ on HLA class I expression in OVACs. OVAC 16 was untreated or treated with IFN-γ for 48 hours and then stained for the expression of total HLA class I, HLA-A2, and HLA-A11. Stainings of untreated cells (dashed line) and IFN-γ–treated cells (dotted line) are shown. Isotype control stainings are depicted as a solid line. Histograms from one representative staining out of three of this particular tumor is shown.

Figure 3

Blockade of CD94/NKG2A inhibitory receptors restores killing of IFN-γ–treated OVACs. The effect of anti-CD56 (MOC-1) or anti-CD94 (HP3D9) mAb’s on the IFN-γ–mediated protection of OVACs was evaluated in the following situations: The HLA-A11⁺ OVAC 16 was used as target for the HLA-A11–specific T cell clone KV109 (a) and for the IVT-specific T cell clone BK289 (b). The peptide concentration in b was 1 μg/ml. Recognition of unpulsed targets by BK289 was below 5% and is not shown. (c) The HLA class I–restricted, tumor-reactive CTL line RF1 was used as an effector against untreated and IFN-γ–treated OVAC 19. (d) Reactivity of TAL 31 against autologous OVAC 31. Error bars represent SD of triplicate cultures. *P < 0.05, **P < 0.01 as determined by two-tailed Student t test.

Figure 4

Expression of nonclassical HLA molecules. (a) The expression of HLA-E mRNA was evaluated in indicated OVACs by real-time quantitative PCR assay. Results are presented as fold increase in IFN-γ–treated cells (IFN) compared with untreated cells (NT). Results from one of two experiments are shown. (b) The expression of HLA-G by freshly isolated ovarian tumor cells was monitored by flow cytometry analysis. Indicated cells were stained with isotype control (mouse IgG2a) (solid line) or anti–HLA-G1 (87G) (dotted line). Results from one representative staining of at least ten are shown. (c) Western blot analysis of HLA-G expression in indicated OVACs and Caov-4 was performed using the HLA-G–specific mAb MEM-G/1 at a concentration of 1 μg/ml. The LCLs 721.221 and 721.221 transfected with HLA-G1 (721.221/G1) were used as positive and negative controls. These were diluted 1:10 because of the excess amount of HLA-G expressed by the 721.221/G1 transfectant. Cell extracts of untreated and IFN-γ–treated samples were loaded in equal amounts as determined by Western blots for β-actin (data not shown).

Figure 5

Role of the HLA-G leader sequence peptide. OVAC 19 was incubated with different concentrations of Gsp as indicated in the figure. HLA-A2–binding HIV peptide (HIV-RT 476-484) was used as negative control at 100 μg/ml. HIV- and Gsp-pulsed OVAC 19 was then used as target in a 4-hour ⁵¹Cr-release assay. The HLA class I–restricted, tumor-specific CTL line RF1 was used as effector. Results from one of two experiments are shown.