Correlation between tumor regression and T cell responses in melanoma patients vaccinated with a MAGE antigen

Abstract

The cancer-germline gene MAGE-3 codes for tumor-specific antigens recognized on many tumors by T lymphocytes. A MAGE-3 antigen presented by HLA-A1 has been used in several vaccination trials on metastatic melanoma patients. Only a small minority of patients have shown evidence of tumor regression. Attempts to correlate the tumor rejections with the cytotoxic T lymphocyte (CTL) response against the vaccine have been hampered by the low level of these responses. In noncancerous individuals, the frequency of the T cell precursors against antigen MAGE-3.A1 is approximately 4 x 10(-7) CD8 T cells. The diversity of the T cell receptor repertoire of these anti-MAGE-3.A1 precursors was analyzed in one individual. The results indicate that it is very likely that the repertoire comprises >100 clonotypes. On this basis, it is possible to use not only the frequency of CTL precursors in the blood but also the presence of dominant clonotypes to ascertain in patients the existence of anti-MAGE-3.A1 responses as low as 10(-6) of CD8. With this approach, we observed a correlation between tumor regression and anti-MAGE-3.A1 CTL responses in patients vaccinated with a recombinant virus encoding the antigen and also in patients vaccinated with peptide-pulsed dendritic cells. In contrast, for patients showing tumor regression after vaccination with peptide alone, CTL responses were almost never observed. It is possible that even those CTL responses that are below our present detection level can trigger a sequence of events that leads to tumor regression.

Fig. 1.

Overview of the procedure to obtain anti-MAGE-3.A1 CTL clones in experiment I. The cells represented in the FACS panel are CD8 T cells. The control tetramer is an HLA-A1 tetramer containing an influenza peptide.

Fig. 2.

Tetramer analysis and anti-MAGE-3.A1 lytic activity of some tetramer-positive clones. (A) The clones were analyzed 7 days after the last stimulation after labeling with the two tetramers and anti-CD8 antibodies. (B) Lytic activity was tested on day 14 after the last stimulation. Targets were HLA-A1 EBV-B cells, MZ2-MEL.43 melanoma cells expressing HLA-A1 and MAGE-3, and NK target K562. When indicated, MAGE-3 peptide EVDPIGHLY was added at 0.5 μg/ml. CTLs were added at various effector-to-target ratios, and chromium release was measured after 4 h.

Fig. 3.

Probability of obtaining a series of 23 with one repeat by sampling from a repertoire of diversity d. The probabilities were calculated for each d with the formula

Fig. 4.

Overview of the procedure to evaluate CTL responses of vaccinated patients. Typically, 250,000 PBMC were distributed in each microwell.

Fig. 5.

Frequencies and diversity of anti-MAGE-3.A1 CTLs. Different clonotypes are indicated by circled numbers. We assume here that humans have a total of 4 × 10¹⁰ CD8 T cells (10⁶ PBMC per ml of blood containing 15% CD8 T cells, 5 liters of blood, and 2% of the total lymphocytes located in the blood) (17).