The role of MHC class I glycoproteins in the regulation of induction of cell death in immunocytes by malignant melanoma cells

Abstract

A deranged expression of MHC class I glycoproteins, characteristic of a variety of malignancies, contributes to the ability of cancer to avoid destruction by T cell-mediated immunity. An abrogation of the metastatic capacity of B16 melanoma cells has been achieved by transfecting an MHC class I-encoding vector into class I-deficient B16 melanoma clones [Gorelik, E., Kim, M., Duty, L. & Galili, U. (1993) Clin. Exp. Metastasis 11, 439-452]. We report here that the deranged expression of class I molecules by B16 melanoma cells is more than a mere acquisition of the capacity to escape immune recognition. Namely, cells of the B16 melanoma prompted splenic lymphocytes to commit death after coculture. However, a class I-expressing and nonmetastatic CL8-2 clone was found to be less potent as an inducer of apoptosis than class I-deficient and metastatic BL9 and BL12 clones. Both Thy1.2(+) and Thy1.2(-) splenocytes underwent cell death when exposed to the class I-deficient BL9 clone. A proportion of CD4(+) and CD8(+) cells among splenocytes exposed to the BL9 clone was lower than that observed in a coculture with cells of the CL8-2 clone. Consistently, none of the melanoma clones studied produced a ligand to the FAS receptor (FAS-L). Thus, our results provide evidence that (i) the production of FAS-L may not be the sole mechanism by which malignant cells induce apoptosis in immunocytes, and (ii) absence of MHC class I glycoproteins plays an important role in preventing the elimination of potential effector immunocytes by tumor cells.

Figure 1

Cell death in spleen cells after coculture with MHC class I-deficient melanoma cells. (A) Splenocytes were cocultured with BL9, BL12, and CL8-2 clones for the indicated times and harvested, and their viability mean ± SEM by trypan blue is shown as the vertical axis. The results are one of 10 independent experiments. (B) Splenocytes were exposed to either BL8, BL12, or CL8-2 cells for 48 h, and propidium-iodide analysis by flow cytometry is shown. The results are one of 10 independent experiments. (C) Splenocytes were exposed to either BL9 or CL8-2 cells for 3 or 6 h and analyzed for DNA fragmentation. Lanes 1, 3, 4, 5, 9, 10, and 11 are splenocytes cocultured with BL9 cells; lanes 2, 6, 7, 8, 12, 13, and 14 are splenocytes cocultured with CL8-2 cells; lanes 1 and 2 are cocultured for 5 min; lanes 3, 4, 5, 6, 7, and 8 are cocultured for 6 h; and lanes 9, 10, 11, 12, 13, and 14 are cocultured for 3 h. Y-VAD has been used in the following concentrations: lanes 4, 7, 10, and 13, 100 μM; lanes 5, 6, 11, and 14, 50 μM. The results are one of three independent experiments. (D) Splenocytes were exposed to either BL9 or CL8-2 cells for 3 h and examined for membrane expression of phosphatidyl serine by staining with FITC-conjugated annexin V. Necrotic cells were excluded from the analysis by staining with propidium iodide and gating on propidium-iodide-negative cells. The results are one of three independent experiments.

Figure 2

The expression of FAS and FAS-L in splenocytes and melanoma cells. (A) Protein lysates prepared from either spleen (Left) or melanoma (Right) cells were subjected to immunoblotting with anti-FAS antibodies. The results are one representative of three independent experiments. (B) RNA samples isolated from BL9 and CL8-2 cells as well as from freshly isolated splenocytes in duplicates (1 and 2) were subjected to reverse transcription—PCR. The results are one of three independent experiments (Left). Protein lysates were prepared from melanoma clones and subjected to immunoblotting with anti-FAS-L antibodies. Splenocytes exposed to 600 units/ml of IFN-γ for 24 h were used as a positive control. The results are one of two independent experiments (Right).

Figure 3

H-2K-deficient melanoma cells deplete distinct subsets of splenocytes from a mixed spleen-cell population after coculture. (A) Splenocytes were cocultured for 72 h with either BL9 or CL8-2 melanoma cells. Lymphocytes then were harvested and fractionated to Thy1.2⁺ and Thy1.2⁻ cells. Each fraction was subjected to the analysis of cellular DNA content by flow cytometry. The results represent one of two independent experiments. (B) After coculture of splenocytes with melanoma cells for 72 h, splenocytes were harvested, and the expression of CD4 (thin curve), CD8 (bold curve), and NK1.1 (bold curve) was analyzed by flow cytometry by using appropriate antibodies. In the upper left, CD4 and CD8 curves are overlapping. The results are one representative of three independent experiments.