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Melanoma Cell Expression of Fas(Apo-1/CD95) Ligand: Implications for Tumor Immune Escape

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Science  22 Nov 1996:
Vol. 274, Issue 5291, pp. 1363-1366
DOI: 10.1126/science.274.5291.1363

Figures

  • Fig. 1.

    (A) Detection of soluble FasL (sFasL) in serum samples from patients (252, 256, and so forth) with melanoma. Serum aliquots were subjected to SDS-PAGE (10%) under nonreducing conditions and immunoblot analysis using an anti-FasL antibody (32). Serum-derived sFasL migrates as a oligomeric complex under nonreducing conditions (∼70 kD). The control lanes correspond to serum samples from a normal donor with no detectable sFasL (Control) and from a patient with alcoholic hepatitis with very high levels of FasL (Hepatitis), respectively (12). Immunodetection of sFasL was inhibited in the presence of peptide (100 μg/ml) corresponding to the epitope recognized by the antibody (33). (B) FasL expression in melanoma cell lines derived from patients with malignant melanoma (32). Triton extracts were analyzed as in (A). The transmembrane form of FasL is 40 kD; some cells show the proteolytically cleaved sFasL product (27 kD). (C) In vitro killing of Fas-sensitive cells by melanoma cells. Fas-bearing A20 B lymphoma cells were cultured with melanoma cell lines derived from various patients (190, 248, and so forth). Cell death was Fas-dependent, since only the FasL sensitive A20 lymphoma cell line but not the FasL-resistant variant cell line A20R was killed (34). Controls: Melanoma effector cells are replaced by A20 cells and FasL, respectively. (D) Melanoma cell lines resist killing by FasL. Various cell lines were incubated with recombinant FasL. Controls: A20 cells and mutant A20R cells were used as target cells.

  • Fig. 2.

    Expression of FasL in human malignant melanoma but not in melanocytes (A) Melanomas are positive for the MAGE antigen (35). (B) Expression of FasL in malignant melanoma (M) from patients 190 (B) and 225 (D), respectively. Higher magnification (F) suggests that FasL can be retained in the cytoplasm, in agreement with its subcellular localization in activated T cells (36). (C) Cells infiltrating the tumor mass (marked with M). A substantial portion of the infiltrating cells are Fas-positive (arrow). (E) Control: The antibody to FasL, A11, was replaced by rat Ig. Normal human skin was stained for FasL in (G) and for the melanocyte-specific antigen HMB45 (arrow) in (H). Keratinocytes (K) express low amounts of FasL and melanocytes expressed no detectable FasL. Scale bars are each 20 μm: bar in (E) applies to (A) through (E); bar in (H) also applies to (G).

  • Fig. 3.

    (A) In situ detection of T cells infiltrating a melanoma lesion (M). T cells extravasating a blood vessel (L, lumen) were stained using an antibody-CD3 specific antibody (closed arrow). (B) In situ detection of apoptotic cells (open arrow) in the tumor. Apoptotic cells were detected through the staining of DNA fragments with the TUNEL assay (37). Scale bar, 20 μm.

  • Fig. 4.

    Consequences of Fas-deficiency on tumor growth (38). (A) Expression of FasL in melanoma B16-F10 cell lines as detected by immunoblots and (B) by RT-PCR. Controls: In (A) inclusion of peptide (100 μg/ml) corresponding to the epitope detected by the antibody to FasL (+peptide), and in (B) amplification of FasL cDNA (543 bp) in Neuro-2a-FasL but not in Neuro-2a-mock transfectants, respectively, and amplification of actin cDNA (in B). (C) Tumor progression in C57BL/6 (WT), C57BL/6-lpr, and C57BL/6-gld mice. The number of mice (in %) with palpable tumors are indicated. Data are representative of three to five experiments with 9 to 13 mice per group. Tumor size was measured daily. (D) Tumor progression in MRL and MRL-lpr mice. Data are representative of three experiments with 9 to 12 mice per group. (E) Lytic activity of B16-F10 cells on Fas-sensitive A20 B lymphoma target cells. Melanoma cells were isolated from the tumor of C57BL/6 mice 5 days after injection. Controls: Melanoma effector cells were replaced by A20 cells and FasL, respectively. Melanoma cells do not kill the variant, FasL-resistant A20R cells.