Target cells and culture condition. We selected three cell lines with the same MHC-class I subtype HLA-A2402, namely, a gastric adenocarcinoma cell line MKN45 that is poorly differentiating and well known to produce a high level of CEA and, as controls, a stomach adenocarcinoma cell line GT3TKB and kidney adenocarcinoma cell line Hpt.10. All the cell lines were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) in an atmosphere of humidified 5% CO2 in air.

Preparation of antigen presenting cells and pulsing CEA-beads. Human PBMC were prepared by the conventional Ficoll-Paque centrifugation method from heparinized peripheral blood of a healthy volunteer who has been identified as carrying HLA-A-2402 subtype. The cells were washed once with PBS, then once with RHAMa medium supplemented with 5% autologous plasma and centrifuged by centrifugation at 1400 rpm for 10 min at room temperature as previously described[8]. The adherent cells with monocytes/macrophages-like morphology were further cultured overnight in the 24-well plate with 2 ml of the medium containing CEA-beads (50ug). As controls, CEA protein alone (50ug/ml), none, or 50 ug of naked latex beads was used. After 24 hr incubation, the adherent cells were fixed with 10% (v/v) formalin in PBS for 1 hr at room temperature and washed thoroughly with the culture medium before the induction of CTLs.

Induction of CTLs from PBMC. On the fixed adherent cells, PBMC (1000000 cells) freshly prepared from the same volunteer were added with 2 ml of RHAMa medium supplemented with human interleukin(IL)-1beta (Otsuka Pharmaceutical Co., Ltd., 167 U/ml), IL-2 (Shionogi & Co., Ltd., 67 U/ml), IL-4 (Ono Pharmaceutical Co., Ltd, 67 U/ml), IL-6 (Ajinomoto, Co., Inc., 134 U/ml), and 5%(v/v) autologous plasma. The induction culture was continued for 4 weeks with half volume of medium change every other day. The effector cells were re-stimulated weekly to a total of 3 re-stimulation at an effector: APC ratio of 2. The fixed adherent cells were renewed by transferring the cultured PBMC once a week. In addition, CEA-peptide specific CTLs were generated by re-stimulating the PBMC 3 times with the fixed adherent cells previously pulsed with CEA652(9) peptide (TYALFVSNL, 50 ug/ml) for 1 hr at 37 ˇC.

Cytotoxicity assay. The target cells, 1000 cells/well in 200ul RPMI 1640 medium containing 5% FBS, were seeded in each well of 96-well plates and were precultured for 12 hrs. The cultured target cells were washed once with PBS(-), then, the cultured PBMC suspended in 200ul of RHAMa containing 5% autologous plasma were added as effector cells to each well at the indicated effector to target ratio (E/T ratio). The cells were cocultured for 24 hrs.

Percentage of surviving target cells was defined as follows:

Surviving target cell(%)=(A-C)/(B-C)x100

where A is the OD570 of the well containing the target cells and the CTLs, B is the OD570 of the 100% control well of the target cells, and C is OD570 of the well containing medium alone.

Inhibition of the cytotoxic activity of the cultured lymphocytes with monoclonal antibodies. Effector cells were pretreated with monoclonal antibodies against CD3, CD8, or CD4 as described previously[9]. These antibodies were used at a final concentration of 5 ug/ml. Target MKN45 cells were precultured overnight in 96-well plates at 10000 cells/well. These target cells were pretreated also with antibodies against human MHC-class I or MHC-class II at a final concentration of 10 ug/ml and then incubated with the effector cells at 37 ˇC for 24 hr.

The inhibition of cytotoxic activity was calculated according to following formula:

% inhibition = (A-B)/(T-B) x 100

where A is OD570 value of the target cells to which the effector cells were added, in which the target cells or the effector cells were pretreated with a monoclonal antibody; B is OD570 value of the target cells to which the effector cells were added (both of the cells were not pretreated with any monoclonal antibodies; and T is OD570 value of the target cells to which neither CTL nor antibodies were added (OD570 of 100% control).

The present results suggest that the cultured lymphocytes on the fixed adherent blood cells pre-loaded with CEA-beads contain MHC class I-restricted CD8+ CTLs (Fig. 5) specific to CEA-producing carcinoma cells (Fig. 2a), but not, or at very low extent, to CEA-non-producing HLA-A2402 carcinoma cells (Fig. 2b and c). MHC- class II molecules were not involved in the cytotoxic response of the CTLs (Fig. 5). The effector cells cultured on the fixed adherent cells pre-loaded with CEA protein alone showed no specific cytotoxicity on any target cells (Fig. 2). These observations provide evidence that the exogenous antigen on the latex beads was processed and presented by MHC-class I molecules after delivery into cells[10].

We also demonstrated here that the CTLs recognized efficiently the CEA epitope peptide TYACFVSNL (CEA652(9)) and less efficiently other peptides so far tested (Fig. 3). CEA652(9) contains two anchor-motif amino acid residues, Y and L with the space of 6 amino acid residues, but the binding affinity to the HLA-A2402 molecule was the third in the tested 11 peptides [K. Takesako and I. Nukaya, personal communication].

Since the present CTL induction was carried out strictly under autologous condition as the lymphocytes were cultured for weeks on the autologous adherent blood cells that previously phagocytized CEA-latex beads and then were fixed with formalin, the results strongly imply that the technique described here will be able to induce autologous CTLs of the tumor-bearing patients against the CEA producing autologous cancer cells in vitro.

We also demonstrated that loading of the CEA peptide to the adherent cells resulted in the generation of the CTLs in vitro (Fig. 4). By this approach, however, so many trials must be made to identify the real epitope peptide among the candidate peptides. Our present approach is simple since CEA whole protein was used as the antigen and previous selective culture of dendritic cells that are hard to proliferate was not required. Fixed antigens on the adherent PBMC will provide long lasting stimulating effect on the expansion of CTLs[11]. Consequently, however, the CTLs responded polyclonally to the CEA epitope peptides (Fig. 3).

We also consider that, if any specific tumor antigens are available and their epitope peptides are unknown, the technique described here will be useful for the induction of autologous CTLs specific to the target tumor antigen even if the target cancer cells are not available.

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

1. Babbit BP, Allen PM, Matsueda G, Habre E, Unanue ER (1985) Binding of immunogenic peptides to Ia histocompatibility molecules. Nature 317: 359
2. Falo Jr LD, Kovacsovics-Bankowski M, Thompson K, Rock KL (1995) Targeting antigen into the phagocytic pathway in vivo induces protective tumour immunity. Nature Med 1: 649
3. Rock KL, Clark K (1996) Analysis of the role of MHC class II presentation in the stimulation of cytotoxic T lymphocytes by antigens targeted into the exogenous antigen MHC class I presentation pathway. J Immunol 156: 3721
4. Nair S, Zhou F, Reddy R, Huang L, Rouse BT (1992) Soluble proteins delivered to dendritic cells via pH-sensitive liposomes induce primary cytotoxic T lymphocyte responses in vitro. J Exp Med 175: 609
5. De Bruijn MLH, Jackson MR, Peterson PA (1995) Phagocyte-induced antigen-specific activation of unprimed CD8+ T cells in vitro. Eur J Immunol 25: 1274
6. Kantor J, Irvine K, Abrams S, Kaufman H, Dipierto J, Schlom J (1992) Antitumor activity and immune responses induced by a recombinant carcinoembryonic antigen-vaccinia virus vaccine. J Natl Cancer Inst 84: 1084
7. Tsang KY, Zaremba S, Nieroda CA, Zhu MZ, Hamilton JM, Schlom J (1995) Generation of human cytotoxic T cells specific for human carcinoembryonic antigen epitopes from patients immunized with recombinant vaccinia-CEA vaccine. J Natl Cancer Inst 87: 982
8. Liu SQ, Shiba R, Kim BS, Saijo K, Ohno T (1994) Long-term serum/plasma free culture of human cytotoxic T lymphocytes induced from peripheral blood mononuclear cells. Cancer Immunol Immunother 39: 279
9. Liu SQ, Saijo K, Todoroki T, Ohno T (1995) Induction of human autologous cytotoxic T lymphocytes on formalin-fixed and paraffin-embedded tumour sections. Nature Med 1: 267
10. Harding CV, Song R (1994) Phagocytic processing of particulate antigen by macrophages for presentation by class I MHC molecules. J Immunol 153: 4925
11. Horiuchi K, Tsurushima H, Kim BS, Liu SQ, Saijo K, Saijo Y, Nukiwa T, Nomura N, Matsumura M, Ohno T (1998) Expansion of human autologous cytotoxic T lymphocytes on fixed target tumor cells. Cytotechnol in press