Tsutomu Ikawa

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.

Suppressor cells in the effector phase of autologous cytotoxic reactions in cancer patients.

SummaryCytotoxicity was induced in lymphocytes (CL) from 10 out of 15 patients by autologous mixed lymphocyte tumor cell culture and further cultivation with recombinant interleukin-2. In cells from 3 of the 10 patients, cytotoxicity was suppressed by more than 50% when autologous peripheral blood mononuclear cells (PBMC) from the patients with large tumors were added to the autologous killing system. The cells responsible for suppressing the cytotoxicity in the effector phase were adherent or nonadherent to plastic depending on the patient examined. The T cell fraction from 1 patient significantly suppressed the cytotoxic activity, and this suppression was seen only in the autologous system. On the other hand, plastic adherent cells but not T cells from PBMC of 2 subjects suppressed the cytotoxic activity of CL. The reason why the main cell population suppressing the CL activity differed among the patients is unclear. However, the findings that the suppression was mostly abrogated following resection of the tumor mass suggested that suppressor cells, either of macrophage lineage or T cells, are induced in patients with a large tumor mass. This speculation is supported by the finding that the PBMC from a patient with tumor recurrence regained the suppressive activity.

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

SummaryPeripheral 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.

Differences of Sensitivity to Autologous Cytotoxic Lymphocytes Between Primary Tumor and its Cervical Lymph Node Metastases

Antigenic differences between primary tumors and their cervical lymph node metastases of 12 patients with head and neck cancers were examined by measuring their sensitivity to cytotoxic lymphocytes (CL). Cytotoxicity was induced by autologous mixed lymphocyte (CL). Cytotoxicity was induced by autologous mixed lymphocyte tumor cell culture (MLTC), and further cultivation with recombinant interleukin-2 (rIL-2). The effector cells which were used in this study consisted of OKT3+8+ and OKT3+4+ subpopulations. Their cytotoxic nature was different from lymphokine activated killer cell (LAK cell) activity. Cytotoxicity of CLs stimulated by autologous primary tumor cells (CLP) was observed in 7 out of 12 patients (58.3%). In contrast, cytotoxicity of CLs stimulated by metastatic tumor cells (CLM) was observed in 4 out of 12 patients (33.3%). In the cases in which both CLP and CLM were successfully induced, cross-reactivity tests and cold target inhibition tests were performed. These results suggested that a reduction in immunogenicity had occurred at the metastatic site, and sensitivity against autologous CL was different between primary and metastatic tumor cells.

Immunological Study of Pustulosis Palmaris Et Plantaris: Blastoid Transformation of Tonsil and Peripheral Blood Lymphocytes by Stimulation with Human Skin Extract

The experiment was performed in order to approach the question whether denatured epithelial debris in the tonsillar crypts could be an antigenic substance which might be responsible for inducing autoallergic reactions in the skin. Identical antigenicity of the tonsillar epithelium and of the skin was determined by immunofluorescence study. Then heat-denatured skin extract was used as a model system for studying the antigenicity of denatured epithelial debris. A blastoid transformation study of tonsil and peripheral blood lymphocytes obtained from patients with pustulosis palmaris et plantaris and control individuals was carried out by stimulating with heat-denatured skin extract. Tonsil lymphocytes responded well to skin extract, but peripheral blood lymphocytes scarcely responded at all. Skin extract induced transformation of tonsil lymphocytes occurred in the T-cell fraction, but not in the B-cell fraction. The results indicate that denatured tonsillar epithelium can induce an immune response of tonsil lymphocytes as autoantigen, and that the blastogenesis observed was dependent upon T cells.

Mucoepidermoid Carcinoma of the Hard Palate

A case of mucoepidermoid carcinoma originating in the hard palate was presented. Histological diagnosis of the removed mass at the first operation was pleomorphic adenoma. Tumor recurred at the same site three years later. Subtotal maxillectomy was performed and recurrent tumor was histologically diagnosed as low grade malignant mucoepidermoid carcinoma. A two-year follow-up showed no evidence of recurrence. The low grade malignant mucoepidermoid carcinoma strongly resembles pleomorphic adenoma. The differential diagnosis is important for the better treatment and prognosis.

Autologous Immune Response of Tonsillar Lymphocytes

Tonsillar (TL) and peripheral blood lymphocytes (PBL) from patients with pustulosis palmaris et plantaris (PPP) or recurrent tonsillitis (RT) were analysed for their proliferative response of T-cells upon stimulation with non-T-cells in the autologous mixed lymphocyte reaction (AMLR). TL and PBL from the same donor were cultured in the AMLR for up to 7 days. Maximum proliferation was observed after a 6-day culture in PBL, whereas that of TL was observed on the 3rd day of the culture. It was also observed that the AMLR of TL in patients with PPP was significantly (p less than 0.01) lower than in patients with RT. On the other hand, it has previously been proved by an immunofluorescence study that there is an identical antigenicity between tonsillar epithelium and skin. From this result, a blastoid transformation study of TL and PBL from patients with PPP was performed by using homologous or autologous skin extracts. Both homologous and autologous skin extract induced blastoid transformation of TL but PBL scarcely responded. These results may support the opinion that an auto-immunological mechanism may take part in the onset of PPP.

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.

Autologous Mixed Lymphocyte Reaction in Head and Neck Cancer

The proliferative response of T cells cultured with autologous non-T cells is known as the autologous mixed lymphocyte reaction (AMLR). The AMLR in patients with head and neck tumors was found to be significantly decreased in comparison with that in normal donors and patients without evidence of recurrent disease more than 3 years after treatment. Responder T cells in AMLR were CD4+ T cells but not CD8+ T cells. In one patient, AMLR were performed between lymphocytes in condition with tumor burden and those in condition with tumor free by using cryopreserved lymphocytes. T cells in tumor burden state poorly responded to both of these non-T cells. On the other hand, T cell in tumor free state vigorously responded to non-T cells in the same state, but not to non-T cells in tumor burden state. These data indicated that impairment of AMLR in patients with cancer was due to deficit of both responder T cells and autologous stimulator non-T cells.

Two Cases of Nasopharyngeal Angiofibroma —Effect of Preoperative Transarterial Embolization—

Recently, two cases of nasopharyngeal angiofibroma were experienced. We emphasized in this paper that polytomography, computed tomography scan and angiography are necessary for preoperative evaluation of locating the lesion and delineating the vascular supply. An important problem in surgical treatment is massive bleeding, and the transarterial embolization is helpful to diminish it.