Locus-specific analysis of human leukocyte antigen class I expression in melanoma cell lines

Abstract

Surface expression of human leukocyte antigen (HLA) class I antigens on melanoma lines was evaluated by locus-specific monoclonal antibodies (mAbs) with three different techniques: Fluorescence-activated cell sorting (FACS), immunohistochemistry with cytospin preparation (ICP), and complement-mediated cytotoxicity (CMC). Eleven HLA class I-expressing cell lines developed from metastases were used. Specific expression of HLA loci was examined under routine culture conditions and after 48-h incubation in interferon-gamma (IFN-gamma; 500 U/ml). Loss of allelic expression was seen in one line (586-MEL): Products of genes coding for HLA-A29 and -B44, in strong linkage disequilibrium, were not detectable. HLA-A antigens were consistently detected by all methodologies and minimally affected by pretreatment with IFN-gamma. HLA-B antigens were detectable in 8 of 11 lines by ICP and 3 of 11 lines by CMC. By FACS the supratypic specificity HLA-Bw6 was expressed at low levels in most lines (mean fluorescence 47.2 +/- 13.4 and rose to 259.8 +/- 45.9 after incubation with IFN-gamma; p < 0.001). HLA-Cw antigen detection by CMC correlated with HLA-B (p < 0.01), suggesting that down-regulation and sensitivity to IFN-gamma are shared by the two loci. This low expression of the HLA-B antigens may play a role in the evasion of the host immune response and its up-regulation may be useful in allowing tumor antigen recognition.

FIG. 1

Three examples of different patterns of expression of human leukocyte antigen (HLA) class I in three distinct melanoma cell lines as detected by fluorescence-activated cell sorting analysis. A: Line 938-MEL expressing both HLA-A and -B antigens in baseline conditions. After 48-h incubation in interferon-γ (IFN-γ; 500 U/ml), only the HLA-B specificities are up-regulated. B: Line 624-MEL (most common pattern) expressing HLA-A but only minimally HLA-B loci in baseline conditions. Also in this case treatment with IFN-γ up-regulates the expression of HLA-B. C: Line 586-MEL. Total loss of HLA-A and -B allelic expression not detectable even after IFN-γ. GAMF, fluorescein-conjugated goat anti-mouse IgG; GAMIgM, fluorescein-conjugated goat anti-mouse IgM; GARF, fluorescein-conjugated goat anti-rat IgG.

FIG. 2

A: Example of differential expression of human leukocyte antigen (HLA)-Bw4 supratypic specificity by different cell types including a tumor-infiltrating lymphocyte (TIL) line (1128 TIL), an Epstein-Barr virus (EBV) B-cell line (888-EBV), a fibroblast line (1154-FIB), and a melanoma line (1088 MEL) with and without pretreatment with interferon-γ (IFN-γ). B: Example of differential expression of HLA-Bw6 supratypic specificity by different cell types including a TIL line (1143 TIL), an EBV B-cell line (583-EBV), a fibroblast line (Malme-3), and a melanoma line (526-MEL) with and without pretreatment with IFN-γ. For other abbreviations see the legend to Fig. 1.

FIG. 3

Immunohistochemistry with cytospin preparation of line 537-MEL stained with anti-HLA-Bw6 antibody in baseline culture conditions (A) and after 48-h incubation with interferon-γ (IFN-γ) (B). Obviously increased expression of human leukocyte antigen (HLA)-B locus is detected after IFN-γ.