Establishment and characterization of the tumors of chronic lymphocytic leukemia cell line in nude and SCID mice.

A new cell line, designated MO1043, was established from the peripheral blood (PB) of a patient with B-cell chronic lymphocytic leukemia (CLL). Both the PB leukemia cells and MO1043 were found to have an abnormal cytogenetic marker of trisomy 12, the most common cytogenetic abnormality in CLL. In addition, both the PB cells and MO1043 expressed a cell surface phenotype of typical B-CLLs. The MO1043 was efficiently transplanted into X-irradiated athymic nude mice by i.p. inoculation after it was subjected to serial passages in new born (1 week old) and irradiated adult nude mice. The tumor of a CLL cell line (termed CLL tumor) was also generated in the nude mice by s.c. inoculation of the cells. The MO1043 was inoculated i.p. into mice with severe combined immunodeficiency (SCID) which had not been subject to any preconditionings. The CLL tumor in the non-conditioned SCID mice was disseminated to various tissues in a manner more analogous to CLL tumors in patients as compared with nude mice, where the CLL tumors were not as widely disseminated. At each of four different tumor doses, i.e. 2 x 10(6), 6 x 10(6), 1.8 x 10(7) and 5.4 +/- 10(7) cells of MO1043, the transplantability was 100%. Titration experiments revealed a reciprocal relationship between survival and the number of tumor cells inoculated. FACS analysis showed that several cell surface markers of the parental MO1043 were maintained in CLL tumors from nude and SCID mice. Fluorescence in situ hybridization with novel DNA probes demonstrated that CLL tumors of both nude and SCID mice maintained trisomy 12. The CLL tumor models developed here, particularly the SCID mouse model, may be very useful for therapeutic studies of CLL.

Adoptive immunotherapy for head and neck cancer with killer cells induced by stimulation with autologous or allogeneic tumour cells and recombinant interleukin-2.

Peripheral blood lymphocytes drawn by leukapheresis using Haemonetics V50 were mixed and cultured with autologous or allogeneic tumour cell line to activate killer cells by tumour antigenic stimulation, and further with recombinant interleukin-2 (rIL-2). Killer cells were intra-arterially infused, as a primary therapy, in 5 patients with maxillary and one with lingual cancer (squamous cell carcinoma). Effects on reduction of primary tumour size were significantly high without any severe side effects. The effects were interpreted mainly by direct day-by-day observation of the site, findings of CT and histology. Histological findings of the tissue obtained by surgical operation performed after adoptive immunotherapy were remarkable changes, such as infiltration of lymphoid cells around the cancer nets, degeneration of cancer cells, infiltration of scavenger macrophages (giant cells) and so on. The results suggested that adoptive immunotherapy by the killer cells can be a powerful treatment to bring the cancer under control, in with combination of other therapies.

Establishment of a human cell line from maxillary squamous cell carcinoma and its biological features as a stimulator for induction of cytotoxic T lymphocytes.

A cancer cell line named FS-1 was established from maxillary cancer of which histological diagnosis was well differentiated squamous cell carcinoma (SCC). The HLA class I typing showed that FS-1 expressed the same HLA class I antigens as those of the host lymphocytes. The chromosome analysis and nuclear DNA contents suggested that FS-1 was not a normal human cell. FS-1 is characteristic of SCC in that it grows adhesively on the surface of a culture flask. The structure of tumor tissue obtained from FS-1-transplanted nude mice is very much like the original tissue of SCC. The SCC-antigen was determined in the culture supernatant of FS-1. Autologous tumor killing activity was induced from peripheral blood lymphocytes of some patients suffering from head and neck SCC after mixed lymphocyte tumor culture with FS-1. Thus, FS-1 can serve as a useful allogeneic stimulator of SCC for induction of autologous tumor killing activity.

Electron microscopic observation of killer cells induced by mixed culture of lymphocytes with autologous cancer cells and further culture with recombinant interleukin-2.

Peripheral blood lymphocytes obtained from 2 patients with hypopharyngeal cancer were cultured with mitomycin C treated autologous tumor cells (autologous MLTC) for 10 days and further cultured with recombinant interleukin 2 (rIL-2). In one case 10-day MLTC induced increase of CD25-positive lymphocyte count, indicating that IL-2 receptors were expressed dominantly by the autologous tumor stimulation, and further culture with rIL-2 differentiated killing activity against autologous tumor cells. In the other case, however, MLTC alone induced killing activity against autologous tumor cells, indicating that the tumor cells from this patient might possess stimulatory activity sufficient to induce mature killer cells. Electron microscopic observation of the morphological features of lymphocytes cultured for 10 days revealed mostly small lymphocytes with low incidence of cytoplasmic granules. Further culture with rIL-2, however, induced slightly larger lymphocytes with well-developed microvilli, and cytoplasmic granules were found in many of the cells. Lymphokine activated killer (LAK) cells induced by culture of lymphocytes with rIL-2 alone were much larger and had long microvilli and abundant cytoplasmic granules, and were apparently morphologically different from the killer cells initiated by MLTC. The small lymphocytes induced by autologous MLTC alone might be autologous tumor specific cytotoxic T lymphocytes (CTL) and/or CTL precursors. Further culture with rIL-2 induced maturation of the CTL. However, the nature of the cytoplasmic granules remains obscure.

[Adoptive immunotherapy by intra-arterial infusion of ATLAK or Allo-TLAK cells in patients with head and neck cancer].

For clinical application of adoptive immunotherapy, it is necessary to prepare a sufficient number of autologous tumor specific effector cells. A large amount of peripheral blood lymphocytes was obtained by leukapheresis using a Heamonetics V50 pheresis system. Autologous tumor- and lymphokine-activated killer (ATLAK) cells were induced by autologous mixed lymphocyte tumor cell culture (autologous MLTC) and further activation with recombinant interleukin-2 (rIL-2). Another problem was the difficulty of obtaining a sufficient number of highly activated effector cells to reach the target tumor tissue. Direct infusion of effector cells into a feeding artery was effective for cell accumulation in the target. ATLAK cells were infused into maxillary artery in 4 patients with maxillary squamous cell carcinoma. The results indicated that the therapy was effective for reduction of the tumor mass. After immunotherapy, surgery was performed and the tissues were histologically examined. Degenerated tumor cells and intensive infiltration by mononuclear cells and macrophages were seen in the surrounding fibrous tissue. However, the quantity of fresh autologous tumor cells available from open biopsy was limited. Allogeneic cultured tumor cell line was used as stimulator of lymphocytes instead of autologous tumor cells. The killing activity of the allogeneic tumor and lymphokine activated killer (Allo-TLAK) cells was significantly induced against the autologous tumor cells. Antitumor effect was observed in 5 out of 9 patients. Side effects were minor, such as slight fever and blood eosinophilia, which may be due to the rIL-2 function. These results indicate that this method of therapy is an effective form of adoptive immunotherapy.

Killer cells induced by stimulation with allogeneic tumor cells and subsequent culture with recombinant interleukin-2.

Peripheral blood lymphocytes were cultured for 5 days with allogeneic tumor cells (allogeneic mixed lymphocyte/tumor cell culture), and subsequently cultured with recombinant interleukin-2 for 12 days. These cultured cells were found to be cytotoxic to autologous tumor cells. Results of two-color analysis using monoclonal antibodies to cell markers showed that more than 80% of their cultured cells were CD3+ cells, and CD4+ cells showed a higher distribution than CD8+ cells. However, CD8+ cells had a much higher killing activity with autologous tumor than did CD4+ cells, when estimated by an elimination study using monoclonal antibodies to T cell phenotypes and complement. The "cold-target" inhibition test showed that the cytotoxicity of these cells for autologous tumor cells was inhibited by unlabeled autologous tumor cells but not by unlabeled stimulator cells. Furthermore, about 40% of the cytotoxicity was suppressed by blocking of HLA class I antigen with a monoclonal antibody on autologous tumor cells. Thus, cytotoxic activity of lymphocytes to autologous tumor restricted by target cell HLA class I antigen is possibly induced by allogeneic tumor-stimulation.

The role of major histocompatibility complex expression on head and neck cancer cells in the induction of autologous cytotoxic T lymphocytes.

Using head and neck tumors, we studied the role of HLA class I and DR antigens on tumor cells in cytotoxic T lymphocyte (CTL) induction. Expression of major histocompatibility complex (MHC) antigens was investigated by two-color flow cytometry analysis and for this study we used the tumor cells, over 50% of which expressed both HLA class I and DR antigens on their surface. In seven cases, tumor cells were divided into three groups according to the specificity of monoclonal antibodies (mAb) to MHC to study the role of MHC antigens on tumor cells in CTL induction: one was not blocked (MHC double-positive tumor), a second was blocked by anti-class I mAb (class-I-negative DR-positive tumor) and third was blocked by anti-DR mAb (class-I-positive DR-negative tumor). Subsequently, these tumors were used to stimulate an autologous mixed lymphocyte/tumor cell culture for 5 days (MLTC) followed by further cultivation with interleukin-2 for 12 days. The induced autologous tumor killer cells were most cytotoxic when non-treated tumors, which consist mainly of cells that are both HLA-class I and DR-positive, were used as stimulator cells. When the tumor cells blocked by anti-DR mAb were used as stimulators, autologous tumor killer activity was lower than that induced by tumor cells blocked by anti-class-I mAb. Moreover, cytolysis by autologous tumor killer cells induced by stimulation of non-treated tumor cells was blocked during the effector phase, 26.6%-42.3% and 32.7%-53.8% by anti-class-I and anti-DR mAb respectively, suggesting that majority of the autologous tumor killer cells are MHC-restricted CD8+ or CD4+ CTL. These results suggest that both MHC class I and class II antigens on head and neck tumor cells play a critical role in inducing CTL.