Adoptive immunotherapy was carried out using tumor infiltrating lymphocytes (TIL)cultured with rlL-2 after isolation from transplantable spontaneous squamous cell carcino-ma from inbred WHT/Ht lnice. Effector cells involved in antitumor reaction were investigated. TIL were collected at l×10⁵ cells/1gof tumor tissue. The viabnity amounted to more than 85% and the ratio of mixed tumor cells was less than 8%. By being cultured with rlL-2, TIL could be proliferated and maintained without tumor cells. A lymphocyte subsets were analyzed by FACScan. It was found that, fresh TIL were dominated by L3T4⁺ cells but a significant increase in Lyt-2⁺ cells was observed when TIL were cultured with rlL-2. The cytolytic activities of both cell lines were determined by means of ⁵¹ Cr release assay. Fresh TIL had no activity against syngeneic tumor cells, but cultured TIL had higher activity. These findings showed that the cytolytic activity was induced by rIL-2. Adoptive immunotherapy performed with cultured TIL produced no therapeutic effect in the group of TILalone, but had an inhibitory effect against tumor growth and a significant life-prolongation effect in the groups of TIL十rlL2 and CY十TIL十rlL-2. In conclusion, Lyt-2⁺ cells were presumed to play a main role in the cytolytic activity in TIL proliferated with rIL-2. TIL was suggested to be useful as a source of adoptive immunotherapy.

The cytotoxic activity of subcellular fractions in LAK cells derived from normal spleen cells of inbred WHT/Ht mice was investigated. The cytotoxic activity of LAK cells,determined by ⁵¹Cr-release assay with syngeneic tunlor cells as a target, was found to reach the maximum level of 44.2% at the 5 th day of cultivaton with 8,000 units of rlL-2/ml culture. LAK cells lysed by sonication under hypotonic conditions were fractionated into 4 subcellular fractions. The mitochondrial fraction contained a component which, even at low concentration, had an immediate cytotoxic effect. The microsomal and cytosol fractions also contained a component which acted with a larger quantity of proteins and a longer period of incubation time than the factor of the mitochondrial fraction. These observations suggested that at least two types of cytotoxic sub-stances, which have different subcellular localizations and mechanisms of action, are present in LAK cells.

The following experiment was undertaken to investigate the distribution and structure of lymphatic capillaries in connective tissue underlying an early DMBA-induced cancer of the tongue using golden hamsters. The left lingual margin was rubbed with a dental cleanser in the control group. In an experilnental group, the left lingual margin was treated likewise and was further treated with a 0.5% 9,10-dimethyl 1, 2-benzanthracene acetate solution for induction of an early tongue cancer. Lymphatic vessels in the tongues of both groups were identified by intravascular injection of Indian ink and impregnation with a silver nitrate solution and by double staining with 5’-nucleotidase-alkaline phosphatase.

In the control group, lymphatic capillaries formed a plexus directly above the muscle layer in submucosal connective tissue. A few lymphatic capillaries were found to extend as cecal lymphatic capillaries from the plexus into connective tissue papillae. In the experimental group, the lymphatic capillaries formed another layer of lymphatic network on the epithelial side in addition to a network directly above the muscle layer of connective tissue underlying the early cancer. Furthermore, the cecal lymphatic capillaries extending from the newly formed lymphatic network appeared more frequently than they did in the control group. These cecal lymphatic capillaries were thick and meandered. This pattern of the distribution and structure of lymphatic capillaries was considered to suggest hypermetabolism at the local site of early cancer formation and cancer proliferation.

The 5’-Nase-ALPase double staining is a simple method for the identification of lymphatic capillaries. This study suggested the feasibility of its application to clinicopathological research.

A single intradermal injection of bacterial lipopolysaccharide (LPS) caused hemorrhagic necrosis in mouse skin (skin reaction). A tissue factor, membrane glycoprotein, is known to activate the extrinsic blood coagulation cascade. In this study, an attempt was made to compare the structural requirements of LPS to induce skin reaction in vivo, and to produce tissue factor by peritoneal macrophages in vitro. The skin reaction was induced by injection of a smooth type (S)-LPS, rough type (R: Ra-Re)-LPS, and lipid A derived from Salmonella typhimurium, Salmonella minnesota and Escherichia coli in a dose-dependent manner, but not by the polysaccha-ride portion of S-LPS. Re-LPS induced the strongest dermal inflammation (activation) in ddY mice. This was followed by Rc-LPS, lipid A (the activity of Rc-LPS was similar to lipid A), Ra-LPS and S-LPS in the order mentioned. In the C3H/HeN mice, Re-LPS and lipid A induced the skin reaction to almost the same levels as in the ddY mice. The activity of synthetic E.coκtype lipid A (#506) which has a double acyl structure was similar to or sHghtly weaker than that by natural lipid A, whereas the activity of a synthetic counterpart of a Iipid A precursor (#406) was considerably weak. In the C3H/HeJ mice, Re-LPS and lipid A did not induce any hemorrhagic response. The macrophage suspension of C3H/HeN mice was stimulated with Re-LPS at 37℃ for 6 hours, and the sonic lysate of the cells was used to estimate the tissue factor activity by the clotting method using a fibrometer. The tissue factor was produced by Re-LPS in ddY and C3H/HeN mouse macrophages. The activity of #406 was the same as that of #506. C3H/HeJ macrophages did not respond to even a high dose of Re-LPS. These results indicate that the lipid A portion of LPS is very important to cause a reaction both in viro. As to the lipid A structure, the double acyl structure is not required for the production of tissue factor by macrophages, but is required for the induction of skin reaction.