Cell purification and induction of CTL : Human PBMC were separated from the peripheral blood of tumor-bearing patients by the conventional Ficoll/Hypaque method. For preparation of a CD56-negative population of PBMC, CD56+ cells were removed from PBMC by standard immunomagnetic separation method (MACS). The CTL population generated was maintained in RHAMa medium containing 5% autologous plasma or 5% plasma protein fraction and an IL-cocktail consisting of IL-1 (167 U/ml), IL-2 (67 U/ml), IL-4 (67 U/ml), and IL-6 (134 U/ml). CTL were restimulated every two weeks with x-ray irradiated autologus tumor cells. IL-18 and IL-12 were used at 10 ng/ml and 50 pg/ml, respectively. CD8+ T cells were purified from T lymphocyte populations by MACS.

Tumor cells : Tumor cell lines TKB9 from glioblastoma multiforme (patient A), GT3TKB from ascites of a gastric adenocarcinoma patient, and TUHR13TKB (patient B) from renal carcinoma were established in our laboratory. These lines were maintained in RHAMa medium containing 10% feral bovine serum (FBS).

Cytotoxic activity : Cytotoxic activity of CTL was determined by coculturing lymphocytes and the target tumor cells for 24 hr. The number of target cells adhering to the bottom of wells was measured as the number of surviving cells by the crystal violet staining (CV) assay.

Fig. 1. Killing activity of the CTL induced from PBMC.

�@PBMC from patient A were stimulated with autologous tumor cells (TKB9) in medium containing IL-2, or an IL-cocktail consisting of IL-1, -2, -4, and -6, with or without IL-18, for 28 days. Cytotoxic activity of the CTL against TKB9 cells was determined by the crystal violet staining assay. Each point shown is the mean + SD of triplicate determinations.

The T cells cultured in medium containing the IL-cocktail together with IL-18 exhibited target killing activity in a dose responsive manner. The addition of IL-18 resulted in remarkable enhancement of the cytotoxic activity of the CTL at low E/T ratios of 2-8.

Fig. 2. Inhibition of the cytotoxic activity of CTL from patient A by anti-CD8 mAb.

Effector cells were pretreated with anti-CD8 mAb at 4 �C for 1 h. Then these cells were incubated with autologous target tumor cells (TKB9) at an E/T ratio of 4 at 37 �C for 24 h. Each bar shown is the mean + SD of triplicate determinations.

The cytotoxic activity of the CTL was blocked by anti-CD8 monoclonal antibody, suggesting that the cytotoxic activity was dependent on CD8+ CTL in the T cell population.

Table 1. Proportion of CD8+ T cells induced from PBMC

By IL-18 , the proportion of CD8+ T cells reached 90% after 35 days in the induction culture, while 54% of the cells induced with the IL-cocktail in the absence of IL-18 were CD8+ T cells.

Fig. 3. Direct activation of purified CD8+ CTL by IL-18

�@CD8+ T cells were purified from the IL-cocktail-induced CTL population derived from patient A, and then cultured in medium containing IL-2 alone or IL-2 in combination with IL-18 and IL-12 for 7 days. The killing assay was performed at an E/T ratio of 4 with TKB9 autologous target cells. Each bar shown is the mean + SD of triplicate determinations.

IL-18, like IL-12, augmented the cytotoxic activity of CD8+ CTL against the autologous tumor cells, TKB9 by culturing with IL-18 in the presence of IL-2 for 7 days.

Fig. 4. Induction of cytotoxic activity in inert CD8+ T cells against TUHR13TKB autologous tumor cells.

�@CD8+ T cells derived from patient B were purified from an inert T cell population which had been cultured in induction medium containing the IL-cocktail. The CD8+ T cells were cultured with IL-2 alone or IL-2 in combination with IL-18 and IL-12 for 7 days. Each point shown is the mean + SD of triplicate determinations.

The CD8+ T cells which showed no killing activity gained cytotoxicity against TUHR13TKB cells by culturing with IL-18 in the presence of IL-2 for 7 days. The cytotoxic activity induced by IL-18 was comparable to that obtained with IL-12.

Direct activation of human cytotoxic T lymphocytes (CTL) by IL-18 was observed.

1.IL-18 increased the activity of the CTL and the proportion of autologous CD8+ T cells present after 28 days in the induction culture (Figure 1, 2, Table 1).

2.The CTL showed enhanced cytotoxic activity against autologous tumor cells (Figure 3).

3. A purified CD8+ T cell population, which did not exhibit any apparent cytotoxic activity against autologous tumor cells, displayed cytotoxic activity after 7-day incubation with IL-18 (Figure 4).

The present results will have important implications for the induction and activation of CD8+ CTL for cancer immunotherapy.

ACKNOWLEDGMENTS

This work was partly supported by the special Coordination Fund from the Science and Technology Agency of Japan.