Quantitative correlation between HLA class I allele expression and recognition of melanoma cells by antigen-specific cytotoxic T lymphocytes

Abstract

MHC class I antigen expression is necessary for CD8+ T-cell-mediated recognition of tumors. Recently, several mechanisms leading to loss or decreased expression of MHC antigens on the tumor cell surface have been described that may account for tumor escape from immune recognition. It is yet unknown whether tumor recognition by CTL occurs at a threshold amount of MHC molecules or correlates with the level of HLA-allele expression. In this study, a model was developed in which clones derived from the 624-MEL melanoma cell line and expressing varying amounts of HLA-A2 molecules were lysed in a standard 51Cr release assay by an HLA-A2-restricted CTL clone (A42) or a bulk culture of tumor-infiltrating lymphocytes. The A42 clone and the tumor-infiltrating lymphocyte culture were characterized previously as specifically recognizing the melanoma antigen MART-1(27-35) peptide. A marked heterogeneity in the susceptibility to lysis by A42 was observed in tumor clones and was not due to heterogeneous expression of MART-1 by the clones or loss of accessory molecules involved in the lymphocyte-target interaction. Lysis by A42 and by the tumor-infiltrating lymphocyte culture significantly correlated with the level of HLA-A2 expression, evaluated as mean channel number of fluorescence by flow cytometry (P < 0.001). Transfection of an HLA-A2-negative clone (624.28) with the HLA-A2.1 gene produced a panel of clones expressing different levels of HLA-A2, the lysis of which was highly correlated with the expression of HLA-A2 (P < 0.001). The addition of exogenous MART-1(27-35) peptide enhanced lysis of clones expressing intermediate amounts of HLA-A2 but did not affect clones with high expression. These data suggest that the number of HLA molecules present on the surface of tumor cells can quantitatively affect their lysis by CTL in situations with borderline amounts of peptide and/or MHC.

Fig. 1

Flow cytometric analysis of allele-specific surface expression of HLA antigens in representative clones of 624-MEL. Data are presented as MCN of fluorescence. The following mAbs were used: MA2.1, anti-HLA-A2 and B17 (37); MB40.2, anti-HLA-B7, B40; 277-HA1, anti-HLA-B14; GAP A-3. anti-HLA-A3 (38). The original bulk culture (624-MEL) was HLA-A2, A3, -B7, B14 positive (first row) expressing the A loci in standard culture conditions (first two columns) and the B alleles only under stimulation with 500 units/ml of rIFN-γ for 48 h (last two columns). Clone 626.28 totally lost expression of HLA-A2 (second row), clone 624.23 has intermediate expression of HLA-A2 (third row), and clone 624.38 expresses the highest amount of HLA-A2 molecules on its cell surface (fourth row).

Fig. 2

Analysis of HLA-A genomic DNA in 624.28 and 624.38 clones of 624-MEL (HLA-A2, 3). Allele-specific amplification was done as described in the text (17, 18), using genomic DNA from the two clones and two relevant controls: DU-145 (HLA-A3, 33) and LN-CAP (HLA-A1, 2) prostatic cancer lines (data not shown). Included in each PCR reaction was an internal control primer that gave rise to a 256-bp fragment (42).

Fig. 3

Correlation between the level of expression of HLA-A2 surface antigens in clones of 624-MEL and lysis by an HLA-A2-restricted CTL clone A42 recognizing the melanoma MART-1_27–35 peptide. Cytotoxicity was measured in a standard 4-h ⁵¹Cr release assay as described previously (40). The percent lysis was calculated (see “Materials and Methods”). Data are presented at 20:1 (○) and 5:1 (●) E:T ratio. To simplify the analysis, a simple linear correlation was assumed for this range of lytic activity and HLA-A2 allele expression. The level of expression is described in a semiquantitative fashion by analyzing the MCN of fluorescence within the same experiment.

Fig. 4

Expression of MART-1 mRNA in representative clones of 624-MEL as analyzed by Northern blot. The level of expression of the melanoma antigen MART-1 is semiquantitatively compared (10 to 20 μg of total RNA per lane) with a Sal/I-digested fragment containing the full-length cDNA from the MART-1 and the β-actin cDNA probe (Clontech). Although variable among the different clones, the level of expression does not correlate with their sensitivity to lysis by A42 CTL. Melanoma line A 375 represents a MART-1-negative control as already described (29).

Fig. 5

Enhancement of lysis of 624.28 clones by exogenous addition of the MART-1_27–35 peptide. 624.28 clones were incubated with the MART 1_27–35 peptide (▲) before testing. As controls unpulsed, clones were used as targets in the ⁵¹Cr release assay (■). In these experimental conditions, lysis is enhanced in the clones expressing an intermediate amount of HLA-A2 molecules to a plateau corresponding to the maximum lysis for the clones with the highest expression in the absence or presence of pulsing with peptide. Lysis of subclone 28A.22, which does not express HLA-A2, is not affected by pulsing with peptide. For all data points, lysis is expressed at 20:1 (left) and 5:1 (right) E:T ratio. Numbers in parentheses, MCN of HLA-A2 expression.