Masao Hanaoka

Induction of human glioma-specific cytotoxic T-lymphocyte lines by autologous tumor stimulation and interleukin 2

SummaryTwo human glioma-specific cytotoxic T-lymphocyte (G-S-CTL) lines were established by autologous tumor stimulation (ATS) with the aid of lectin free interleukin 2 (IL 2). Coculture of patients peripheral blood lymphocytes and autologous irradiated glioma cells and subsequent addition of partially purified IL 2 enhanced the tumoricidal activity of the lymphocytes. These CTL lines possessed cross-cytotoxic activity against autologous and allogeneic glioma cells and exhibited low cytotoxic activity against non-glial tumor cells. They did not lyse autologous lymphoblasts. This phenomenon suggested the existence of a common gliomaspecific antigen recognized by the CTL lines.T-cell subset depletion test revealed that the major surface phenotype of G-S-CTL line, responsible for cytotoxic activity was OKT 3 positive, OKT 4 negative and OKT 8 positive.G-S-CTL lines were composed of a low proportion of OKT 8 positive subpopulation after primary ATS and successive propagation with IL 2. The proportion of OKT 8 positive subpopulation was increased by secondary ATS, which enhanced the cytotoxic activity to glioma cells more effectively.

Immunocytology of cultured IgM-forming cells of mouse. II. Purification of phagocytic cell factor and its role in antibody formation.

Abstract A factor required for the proliferation of IgM-forming tumor cells of mouse was purified approximately 1500-fold from culture supernatant of phagocytic cells through conventional protein fractionation procedures. The isoelectric point of the factor was pH 6.1 ± 0.1 as measured by isoelectric focusing. Its molecular weight was estimated to be approximately 5 × 10 4 daltons. These characteristics of the factor were not identical with those of the stimulating factor for granulocyte and macrophage colony formation. Antiserum against the purified factor inhibited the growth-promoting activity of the factor. The effect of the antiserum on the normal antibody response of mouse to sheep red blood cells was examined in the tissue culture system. The antiserum inhibited only the late stage in the formation of direct plaque-forming cells.

Regulatory factors of lymphocyte-lymphocyte interaction. I. Con A-induced mitogenic factor acts on the late G1 stage of T-cell proliferation.

DNA synthesis in murine lymphocytes was augmented by a soluble factor in the supernatant of serum‐free cultures of syngeneic spleen cells activated with concanavalin A (Con A). This so‐called mitogenic factor (MF), which is probably identical with interleukin II, partially purified by DEAE‐cellulose and Sephadex G‐75 chromatography, is a fairly homogeneous molecule of 17–25 × 103 daltons. By using partially purified MF, the role of MF in lymphocyte proliferation was investigated.

Letterer-Siwe disease: Immunopathologic study with a new monoclonal antibody

Three cases of Letterer-Siwe disease were studied with the monoclonal antibody Lag, which reacts to the antigen on the membranes of Birbeck granules and related structures of human Langerhans cells. Both lymph nodes and lesional skin contained abundant Lag-positive cells. By two-dimensional gel electrophoresis, antigenic substances in the lymph nodes of patients with Letterer-Siwe disease were found to have the same molecular weight of 40,000 dalton and isoelectric points extending from 4.7 to 6.5 as those in normal human skin and lymph nodes. Our results support the contention that Letterer-Siwe disease is a proliferative disorder of Langerhans cells. A double-staining method with Lag and anti-T6 antibody revealed that Lag reacted to 70% of T6-positive cells in the lymph nodes but to almost all such cells in skin lesions of patients with Letterer-Siwe disease, suggesting that the proliferating cells consist of at least Lag+, T6+, and Lag-, T6+ subpopulations.

Interleukin 2 induces rapid phosphorylation of cellular proteins in murine T-lymphocytes

When quiescent murine T‐lymphocyte cells were stimulated by the addition of interleukin 2 (IL‐2), they reinitiated DNA synthesis after a lag period of 5 h. Under these conditions, rapid but transient phosphorylation of two cellular proteins with M r, values of 27000 and 26000 was detected; maximal phosphorylation occurred within 10–15 min after the addition of IL‐2. The protein of M r, 27 000 contained phosphoserine, while the protein of M r 26000 contained phosphothreonine.

Synthesis of Sterols and 5‐Lipoxygenase Products are Required for the G1‐S Phase Transition of Interleukin‐2‐Dependent Lymphocyte Proliferation

A murine killer T cell line, G‐CTLL 1, whose proliferation depends on the presence of interleukin 2 (IL‐2), was used to analyze the mechanism of IL‐2 action with respect to sterol synthesis and arachidonate metabolism. De novo sterol synthesis was substantially enhanced much earlier than DNA synthesis, and the rate reached a maximum at 13 hr after the addition of IL‐2. Compactin, which is a potent competitive inhibitor of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMG CoA reductase, the enzyme in the rate‐limiting step of the sterol synthesis), inhibited the IL‐2‐induced DNA synthesis. The addition of mevalonate, the product of HMG CoA reductase, prevented the inhibition of DNA synthesis by compactin, suggesting that the supply of a sufficient amount of sterol is an essential prerequisite for IL‐2 action. The IL‐2‐induced DNA synthesis was also inhibited by AA861, a specific inhibitor of arachidonate 5‐lipoxygenase, and by other lipoxygenase inhibitors such as nordihydroguaiaretic acid and esculetin. In contrast, indomethacin, an inhibitor of arachidonate cyclooxygenase, had no effect. These findings suggest that synthesis of 5‐lipoxygenase products is also a prerequisite. The inhibition of DNA synthesis was effectively inhibited only when compactin or lipoxygenase inhibitors were added early enough to block the synthesis of sterols or 5‐lipoxygenase products; addition of the reagents after 3 hr decreased the inhibition with time. Therefore, about 3 hr after the addition of IL‐2, several drastic intracellular changes are assumed to begin and to lead to DNA synthesis.

Expression of proto-oncogene products during drug-induced differentiation of a neuroblastoma cell line SK-N-DZ.

SummaryFour human neuroblastoma cell lines exhibited differences in their ability to differentiate into neuron-like cells in response to three different treatments, serum deprivation, or additions of dibutyryl cyclic-AMP or retinoic acid. Expression of N-myc gene product was reduced in neuroblastoma cell line SK-N-DZ differentiated by retinoic acid as compared with untreated cells. On the contrary, expression of c-src gene product, pp60c-src, was considerably enhanced in differentiated SK-N-DZ cells. Tyrosine phosphorylation of several cellular proteins was found to be enhanced in differentiated cells. Alteration in expression of these proto-oncogene products might be important in the differentiation of neuroblastoma cells into neuron-like cells.

Studies on the Discrimination of Lymphocytes and Plasma Cells SUPPLEMENTS ON ADVOCATION OF THE “LYMPHOGONIA”-THEORY*

In the lymphatic gland of well sensitized rabbits, there locate three groups of pyroninophile cells stained with the Unna‐Pappenheims method. Namely, they are (1) germ centers, (2) sinus, especially the intermedially sinus and (3) lymphatic pulp near the medullary sinus.

Lethal graft-versus-host disease in nude mice. I. Establishment of model systems

We examined whether nude mice, which are deficient in T cell function, could be used as a model for induction of lethal graft-versus-host disease. Nude mice injected with MHC-disparate spleen cells exhibited only transient GVH reaction such as splenomegaly. Inoculation of B6 spleen cells into BALB/c nude mice produced high titers of alloantibodies to the donor cells. These alloantibodies eliminated host-MHC-reactive donor T cells from the host. After abolition by 400 rads irradiation of the capacity of nude mice to produce antibody, lethal GVHD could be induced by allogeneic spleen cell transfer and was mediated by donor T cells. This lethal GVHD was prevented by prior administration of antidonor alloantibody to the irradiated recipients at least 24 hr before donor-cell grafting. The role of alloantibody was substantiated in 2 other combinations in which little or no alloantibodies to donor spleen cells were produced. Engraftment of either MHC-identical but non-MHC disparate donor spleen cells into BALB/c nude mice or of parental spleen cells into F1 nude mice resulted in death mediated by T cells. In addition, irradiated BALB/c nude mice inoculated with non-MHC-incompatible B10.D2 spleen cells were much more sensitive to alloaggression by the donor cells than were nonirradiated hosts, indicating the presence of some radiation-sensitive component(s) acting in nude mice against GVHD induction by donor T cells. Thus the nude mouse is considered to be a useful recipient for clarifying the basic mechanisms involved in lethal GVHD.

A new approach to the detection of autoantibodies against insulin receptors that inhibit the internalization of insulin into human cells

It has been shown that the conjugate of the fragment A of diphtheria toxin to insulin is cytotoxic to cultured cells bearing insulin receptors, apparently through the endocytosis of fragment A. We examined the effect of autoantibodies against insulin receptors on the cyto-toxicity of the conjugate. The conjugate was cytotoxic to a rat fibroblast cell line that was resistant to the intact toxin, and the cytotoxicity was inhibited by exogenous insulin, indicating that the fragment A underwent endocytosis through insulin receptors. Immunoglobulins from three patients with type B syndrome of insulin-resistant diabetes blocked the cytotoxicity of the conjugate to Changs liver cells in a dose-dependent manner. When the cells were pretreated with the immunoglobulins, cytotoxicity of the conjugate was also blocked. These results suggest that autoantibodies against insulin receptors interfere with the binding of the conjugate to insulin receptors or with the endocytosis of fragment A after binding. This assay system seems useful for detecting autoantibodies against the determinants that are involved in the internalization of the ligand-receptor complex.