Shu Qin Liu

Induction of human autologous cytotoxic T lymphocytes on formalin-fixed and paraffin-embedded tumour sections

Human autologous tumour-specific cytotoxic T lymphocytes (CTL) were generated from peripheral blood on small formalin-fixed paraffin-embedded sections of a gastric cancer. The CTL killed live target cells at an effector/target ratio of 1 within 24 hours and showed the same target specificity as those induced on live cancer cells. The killing activity of the CTL lasted for more than four months in culture and was inhibited by antibodies against CD8 and MHC-class I. These results suggest that adoptive immunotherapy of tumours will be possible with CTL induced on a stable source of tumour antigen.

Reduction of end-stage malignant glioma by injection with autologous cytotoxic T lymphocytes.

Autologous cytotoxic T lymphocytes (CTL) against primary‐cultured malignant gliomas were generated from peripheral blood mononuclear cells in vitro in 4 patients. Activities of the CTL were highly specific to the corresponding autologous glioma and were inhibited, in one patient, with antibodies against CD3, CD8 and MHC‐class I molecules. When the CTL were injected 3 times into the primary‐tumor‐resected cavity via an Ommaya tube, reduction of the recurrent tumors with magnetic resonance imaging (MRI)‐measured volumes exceeding 45 cm3 was observed in 3 patients. In a patient with glioblastoma multiforme (GBM), the tumor volume (estimated, 130 cm3) was rapidly reduced to 1/3, although re‐recurrence of the tumor followed 40 days later. A slight but distinct rapid reduction of the tumor volume was observed in another GBM patient and in an anaplastic astrocytoma patient; essentially no change was observed in a further GBM patient. These results suggest that adoptive immunotherapy with autologous CTL will be clinically effective against end‐stage malignant gliomas.

Study of blood compatible polymers: III. Copolymers of N-benzyl, N-(2-hydroxyethyl) acrylamide and 2-hydroxyethyl methacrylate

Copolymerization of 2-hydroxyethyl methacrylate (HEMA) and N-benzyl, N-(2-hydroxyethyl) acrylamide (BENAAm) was carried out at different mole ratios of the monomers to obtain copolymers of varying composition. BENAAm content of the copolymers varies between 13 and 70%. Investigation of the interaction of rabbit platelets with these polymer surfaces showed that copolymers with higher BENAAm content inhibit the platelet deformation. Human umbilical cord fibroblast cells proliferated very well on the copolymer surfaces. The cell growth rate on polyHEMA was relatively low. Maximum cell growth was observed on the copolymer having 87% HEMA.

High Rate of Induction of Human Autologous Cytotoxic T Lymphocytes against Renal Carcinoma Cells Cultured with an Interleukin Cocktail

A high rate of induction (9 of 10 cases) of human autologous cytotoxic T lymphocytes (CTL) was achieved in vitro from peripheral blood mononuclear cells of renal carcinoma patients by applying an interleukin (IL)‐cocktail consisting of IL‐1, ‐2, ‐4, and ‐6. The CTL specifically lysed their own target carcinoma cells within 24 h but did not kill neighboring autologous normal kidney cells or allogeneic renal cancer cell lines. In the case of TUHR4TKB, for which autologous CTL were not induced, no expression of MHC class‐I molecules was observed on the surface of these carcinoma cells, although they were sensitive to autologous natural killer cells. The results imply that adoptive immunotherapy for metastasized renal carcinoma will be feasible with autologous CTL in combination with natural killer cells.

Study of blood compatible polymers II. Poly(N, N-disubstituted) acrylamides

Poly(N,N-disubstituted) acrylamides with both hydrophilic and hydrophobic groups as substituents were synthesized. Different degrees of hydrophilicity were achieved by varying the bulk of the hydrophobic substituent. N-alkyl, N-(2-hydroxyethyl) acrylamides with alkyl substituents propyl (PROPAAm), octyl (OCTAAm) and benzyl (BENAAm) were synthesized. The swelling capacity of the polymers decreased with increase in bulk of the hydrophobic substituent. In vitro studies showed that the surfaces of these polymers did not induce platelet aggregation. Cell compatibility of these polymers was assessed by following the growth of human umbilical cord fibroblast cells. Pronounced cell growth and spreading was observed on the surfaces of polyOCTAAm and polyBENAAm. The relatively low cell growth on polyPROPAAm was ascribed to its high water content.

Expansion of human autologous cytotoxic T lymphocytes on fixed target tumor cells

Human tumor specific cytotoxic T lymphocytes (CTL) were expanded on formalin-fixed autologous target tumor cells derived from glioblastoma multiforme. Growth response of the CTL restimulated with the fixed target cells was larger than those with live target cells. The results suggest that formalin-fixed tumor cells will be stable sources of tumor antigen for efficient autologous CTL expansion and be useful for adoptive immunotherapy of tumors.

Assessment of the number of local cytotoxic T lymphocytes required for degradation of micrometer-size tumor spheroids

Adoptive immunotherapy with human cytotoxic T lymphocytes (CTL) is a promising cancer treatment. Previously we showed that human CTLs against various types of tumors can be efficiently produced by coculturing peripheral blood cells with target cells. The aims of this study were to simulate the interaction of CTLs and micrometer-size tumor tissues in vitro and to assess the required number of CTLs at local tumor sites for degradation of a tumor. Allogeneic CTLs against a human transitional cell carcinoma cell line and autologous CTLs against a renal cell carcinoma cell derived from a surgical specimen were generated. The cytotoxic activities of CTLs against tumor cells in monolayer culture and tumor spheroids formed in U-bottom 96-well culture plates were assessed. Both allogeneic and autologous CTLs showed greater destructive activity than lymphokine activated killer (LAK) cells against target tumor spheroids. CTLs inoculated at E/T ratios of 0.1 to 1 coexisted with the tumor spheroid for 5 to 6 days and then increased in number with apparently lethal activity against the tumor spheroid. In contrast to CTLs, the increase in LAK cell numbers was scarcely observed, and the proliferated LAK cells did not show cytotoxicity against the tumor spheroid. These observations suggest that, when a small number of CTLs reach a local tumor site, they can destroy micrometer-size tumors after considerable local proliferation.

Growth Stimulation of Tumor-Specific Cytotoxic T Lymphocytes on Concanavalin A-Immobilized Carrier Beads

Human tumor-specific CD4+ cytotoxic T lymphocytes (CTL) were generated against duodenum papilloma cell line TGBC18TKB from HLA type-matched peripheral blood mononuclear cells. Concanavalin A (Con A) immobilized on carrier beads stimulated growth of the CTL in a long-term culture without repeated antigen stimulation, while soluble Con A induced death of the CTL. The CTL exhibited the target-specific cytotoxicity in a more potent manner than those before the long-term culture in the presence of the immobilized Con A. Enhanced expression of the adhesion molecule, CD11b, was observed on the CTL. These results suggest that immobilized Con A will be useful for continuous growth stimulation and large scale expansion of CTL without tumor antigen.

Intelligent matrices for tumor-specific cytotoxic T lymphocytes

Abstract Specific matrices for the corresponding cytotoxic T lymphocytes were investigated with tumor cells and tumor cell membrane proteins that contain specific antigens. Human tumor-specific cytotoxic T lymphocytes were obtained by sensitising peripheral blood mononuclear cells on the confluent tumor cell layer. The induced cytotoxic T lymphocytes killed the corresponding tumor cells at cell-to-cell level. They proliferated in the presence of target tumor cells or solid-phase tumor cell membrane proteins. The lasting killing activities were observed over 3 months even in the serum-free culture system. Due to low antigenicity of human tumor cells, it is difficult to induce cytotoxic T lymphocytes even on some established tumor cell lines. These problems were resolved by a proper composite of immunoactivation stimulators such as interleukins and interferons. Generally, the lymphocytes grow only in the presence of tumor antigens of target tumor cells. In most clinical cases, no sufficient tumor cells are used to maintain tumor-specific cytotoxic T lymphocytes as the routine antigen-presenting feeder cells. The repeated use of antigen-fixed matrices makes possible the long-period culture of the antigen-specific lymphocytes.