High frequency of antitumor T cells in the blood of melanoma patients before and after vaccination with tumor antigens

Abstract

After vaccination of melanoma patients with MAGE antigens, we observed that even in the few patients showing tumor regression, the frequency of anti-vaccine T cells in the blood was often either undetectable or <10(-5) of CD8 T cells. This frequency being arguably too low for these cells to be sole effectors of rejection, we reexamined the contribution of T cells recognizing other tumor antigens. The presence of such antitumor T cells in melanoma patients has been widely reported. To begin assessing their contribution to vaccine-induced rejection, we evaluated their blood frequency in five vaccinated patients. The antitumor cytotoxic T lymphocyte (CTL) precursors ranged from 10(-4) to 3 x 10(-3), which is 10-10,000 times higher than the anti-vaccine CTL in the same patient. High frequencies were also observed before vaccination. In a patient showing nearly complete regression after vaccination with a MAGE-3 antigen, we observed a remarkably focused antitumoral response. A majority of CTL precursors (CTLp's) recognized antigens encoded by MAGE-C2, another cancer-germline gene. Others recognized gp100 antigens. CTLp's recognizing MAGE-C2 and gp100 antigens were already present before vaccination, but new clonotypes appeared afterwards. These results suggest that a spontaneous antitumor T cell response, which has become ineffective, can be reawakened by vaccination and contribute to tumor rejection. This notion is reinforced by the frequencies of anti-vaccine and antitumor CTLs observed inside metastases, as presented by Lurquin et al. (Lurquin, C., B. Lethe, V. Corbiere, I. Theate, N. van Baren, P.G. Coulie, and T. Boon. 2004. J. Exp. Med. 201:249-257).

Figures

Figure 1.

Limiting dilution analysis of antitumor CTLp's in patient EB81. Lytic patterns obtained at days 20 and 27 with limiting dilution microcultures set up with 700 and 350 blood CD8 lymphocytes and stimulated with irradiated autologous tumor cells, IL-2, IL-4, and IL-7 on days 0, 7, 14, and 21. Microcultures deemed to contain specific antitumor T cells are boxed. The lytic activity of aliquots of the microcultures was measured against autologous tumor cells EB81-MEL, autologous EBV-transformed B cells (EB81-EBV), and K562. On day 20 only, effector cells and 51Chromium-labeled targets were incubated in the presence of a 50-fold excess of unlabeled K562 to quench the lytic activity of NK-like effectors. Microcultures were considered to contain antitumor CTLs if they satisfied on day 20 as well as on day 27 the following criteria: lysis of tumor >10% and lysis of EBV-B and K562 lower than one third of the lysis of tumor.

Figure 2.

Frequencies and target antigens of antitumor CTLs from patient EB81. The clinical evolution of the patient and the frequencies of antitumor and anti-vaccine CTLp's are indicated in the top. Frequencies of antitumor CTLp's were measured by MLTC with a tumor cell line derived from the invaded lymph node, whereas those of anti–MAGE-3.A1 and anti–MAGE-C2336–344 CTLp's were measured by clonotypic PCR. The bottom panels represent antitumor CTL clones with different numbers for each TCR sequence and the occurrence of repeated clones. Most of these CTL clones were derived from lymphocytes collected in September 1999 and March 2000 and were stimulated in autologous MLTC. Additional CTL clones were derived from lymphocytes collected in October 2000 by stimulation with MAGE-C2 peptides and identification of the positive microcultures with the appropriate tetramer. For peptide MAGE-C2336–344, 15 additional CTL clones were obtained. 14 turned out to be the CTL 16 clonotype, whereas one, CTL 40, had another TCR. A similar experiment performed with MAGE-C2191–200 revealed a new highly repeated clonotype, CTL 41.

Figure 3.

Lytic activity of antitumor CTL clones of patient EB81. Target cells included autologous melanoma cells EB81-MEL (•), autologous EBV-B cells (○), and K562 cells (▵).