Individually distinct tumor-specific cell surface antigen identified by monoclonal antibody on a Rous sarcoma virus-induced mouse tumor.

Hybrid cell lines were prepared by the fusion of BALB/c myeloma P3U-1 cells with the lymphocytes of BALB/c mice that were immunized with syngeneic Rous sarcoma virus (RSV)-induced tumor CSA1M cells. Three clones of the hybrid progeny (3.4B2, 3.4C6, and 3.5C11) produced cytotoxic IgM antibodies against CSA1M cells. One of the clones, 3.5C11, was chosen for analysis of the detailed specificity. Both direct cytotoxicity assays and absorption tests revealed that monoclonal antibody from 3.5C11 was positive only with CSA1M cells and that it failed to react with other tumors, including 20 RSV-induced mouse tumors, and normal cells. The 3.5C11 monoclonal antibody alone, with or without exogenous complement, was suppressive in the therapy of ip injected CSA1M tumor in syngeneic hosts, and significant prolongation in survival was seen in the treated mice. These results clearly showed presence of an individually distinct tumor-specific cell surface antigen on an RSV-induced mouse tumor.

Effect of staphylococcal enterotoxin B-induced anergy on cytokine gene expression: anergy-sensitive and resistant mRNA expression.

We examined the effect of staphylococccal enterotoxin B (SEB)-induced anergy on expression of six different cytokine genes in T cells restimulated with SEB in vitro. We found that although IL-2, IL-3, and IL-4 mRNA levels are substantially reduced in anergic T cells, mRNAs for IL-6, IL-10, IFN-gamma, and TNF-alpha are expressed normally. Thus, there appeared both anergy-sensitive and resistant cytokine mRNA expression in restimulated anergic T cells. The same pattern of cytokine mRNA responses was observed in anergic CD4+ T cells, indicating that the preferential induction of anergy in Th1-like cells is not evident in this in vivo model. Employing TCR V beta 8.2 transgenic mice in which almost all T cells become anergic, we found that the TCR/CD3 complex can transduce both anergy-sensitive and resistant signals. Furthermore, a series of experiments using FK506, A23187, and PMA suggests that signals between TCR and activation of calcineurin and protein kinase C may be blocked in anergic T cells. This is supported by our gel mobility shift assays indicating that calcineurin and/or PMA-inducible NF-ATp, OAP40, and AP-1, but not calcineurin-independent Oct-2, are repressed in anergic spleen T cells upon restimulation with SEB. Taken together, these results suggest that, among signals elicited by stimulation of TCR with SEB, a Ca2+/calcineurin-NF-ATp pathway and other signals, including protein kinase C, are repressed in anergic T cells upstream of their activation, which are essential for the cytokine mRNA expression of the anergy-sensitive type but are dispensible for those of the anergy-resistant type.

Restriction molecules involved in the interaction of human T cell clones with antigen-presenting cells.

Two of three distinct human Ia molecules detected by murine monoclonal antibodies (MoAb) have been suggested to be involved in antigen presentation for T cell responses to purified protein derivatives (PPD) and herpes simplex virus (HSV). This observation was first suggested from studies on the inhibition of proliferative responses of whole T cell populations with MoAb against human Ia molecules. To determine whether a single T cell recognizes the antigen in the context of both Ia molecules or in the context of each one of two Ia molecules, we isolated and propagated PPD-reactive T cell clones from an HLA-DR heterozygous individual. They showed four different restriction patterns: type I and type II clones each appeared to be restricted to one of two HLA-DR antigens, type III clone gave anomalous patterns of response and seemed to be restricted to non-DR antigens, and type IV clone recognized antigen when both DR antigens were presented on the same antigen-presenting cells (APC) surface. Blocking study with monoclonal anti-Ia antibodies suggested that type I, II and IV clones are restricted to DR molecules and type III clones are restricted to 1B4 molecules distinct from DR or MB1 molecules. These data imply that human T cell clones recognizing PPD in the context of each one of two Ia molecules are clonally distributed.

Clonal distribution of human T cells recognizing PPD in the context of each of two distinct Ia molecules.

Participation of two of three distinct human Ia molecules, HLA-DR and the Ia molecule detected by monoclonal antibody (MoAb) 1B4 (1B4 molecules), in antigen presentation for T cell responses to purified protein derivative (PPD) and herpes simplex virus (HSV) was first suggested from studies on the inhibition of proliferative responses of whole T cell populations with MoAb against human Ia molecules. To determine whether a single T cell recognizes the antigen in the context of both Ia molecules or in the context of each one of two Ia molecules, we isolated and propagated PPD-reactive T cell clones from an HLA-DR heterozygous individual. The clones showed four different restriction patterns: type I and type II clones appeared to be restricted to one of two HLA-DR antigens, type III clones gave anomalous patterns of response and seemed to be restricted to non-DR antigens, and type IV clone recognized antigen when both DR antigens were presented on the same APC surface. Blocking study with MoAb to Ia molecules suggested that type I and type II clones are restricted to DR molecules and type III clones are restricted to 1B4 molecules distinct from DR or MB1 molecules. Furthermore, it is most likely that type IV clone was restricted to the interaction molecule associated with DR antigens. These data imply that human T cell clones recognizing PPD in the context of each one of two Ia molecules are clonally distinct.

Both HLA-DR restricted and nonrestricted functions of adherent cells are required for induction of T lymphocyte proliferative responses.

There appeared to be two distinct functions of adherent cells in human T lymphocyte proliferative responses to purified protein derivative (PPD). The first is antigen-presenting ability which is mediated by antigen-presenting cells (APC) among adherent cells. By employing antiserum blocking pretreatment of APC, it was revealed that HLA-DR antigens are involved in this function and that identity or partial identity of HLA-DR antigens between APC and T lymphocytes is required for T lymphocyte antigen recognition. The second function is mediated by the soluble factor produced by adherent cells and is HLA-DR nonrestricted. Although it remains unclear whether APC and adherent cells producing the soluble factor belong to the same cell population, this second function might lead T lymphocytes to proliferative as long as T lymphocytes recognize antigen (PPD) via APC.

A rapid and sensitive method for HLA-DRB1 typing by acridinium-ester-labeled DNA probes.

A rapid and accurate detection of HLA class II antigen is essential for transplantation and for the understanding of disease susceptibility. Recent molecular genetic studies have revealed that the number of class II loci and the number of alleles at these loci are greater than had been previously detected. It is, therefore, of great importance to detect these extensive polymorphisms. A great deal of effort has been made on identification of individual HLA class II specificities at the DNA level, called "DNA typing." What seems to be lacking, however, is handling simplicity. Here we accomplished a simple method for HLA-DRB1 typing based on hybridization of acridinium-ester (AE)-labeled DNA probes to amplified DNA. This method is called hybridization protection assay (HPA). By using 13 AE-labeled probes, 20 homozygous B-cell lines and leukocytes from 80 healthy individuals were typed by HPA. The results were completely consistent with those obtained by polymerase chain reaction--restriction fragment length polymorphism. This method is suitable for mass screening because of its procedural simplicity and swiftness.

Analysis of pathogenesis of autoimmune insulitis in NOD mice: adoptive transfer experiments of insulitis in ILI and NOD nude mice.

In an effort to study the pathophysiological events in the development of insulitis in NOD mice, we have developed ILI- and NOD-nu/nu mice. ILI mice are a nondiabetic inbred strain but are derived from the same JcI:ICR mouse as NOD mice and share the same H-2 allotype with NOD mice. Splenocytes and CD4+ cells from diabetic NOD mice appeared to transfer insulitis to ILI-nu/nu mice, suggesting that ILI mice already express autoantigen(s) responsible for insulitis. But reciprocal thymic grafts from NOD mice into ILI-nu/ nu mice and those from ILI mice into NOD-nu/nu mice failed to allow the development of insulitis, implying that ILI mice possess neither precursor T cells nor the thymic environment responsible for the development of insulitis. In addition, splenocytes from ILI mice appeared to contain regulatory cells which suppress the development of diabetes but not that of insulitis in NOD mice. The use of these nude mice should provide more information on the products of insulitis-susceptibility genes of NOD mice.

[Studies on the antitumor activity of human recombinant interferon alpha-2b in vitro].

Correlation between antiproliferative and binding activities of interferon (IFN) alpha-2b to various human cell lines was examined using human recombinant IFN alpha-2b. Burkitt's lymphoma Daudi cells and human renal cell carcinoma OS-RC-2 cells were sensitive to IFN alpha-2b, whereas two EB-virus-transformed B cell lines, FS and L-KT3, and human A375 melanoma cells showed low or no sensitivity. 125I-IFN alpha-2b binding assay revealed that the difference in IFN alpha-2b sensitivity was related to the number and the affinity of IFN alpha-2b receptors per cell. Experiments were then performed to investigate the influence of recombinant IFN alpha-2b on the cytostatic activity of monocytes against A375 cells in vitro. IFN alpha-2b enhanced the cytostatic activity of monocytes against A375 cells which showed low sensitivity to the direct growth inhibitory effect of IFN alpha-2b. Depletion of NK cells from the monocyte preparations by anti-Leu-11b monoclonal antibody and complement did not affect the monocyte activation by IFN alpha-2b, indicating that NK cells were not involved in this system.

[A new anticancer drug sensitivity test using the microplate culture and surviving tumor cell staining method].

We have developed a new anticancer drug sensitivity test using the short-term microplate culture and viable cell staining method, and the details of this method are reported. The tumor cells are cultured with the anticancer drugs for 2 days and 4 days. After culture, the microplate is centrifuged. The surviving tumor cells are fixed with ethanol and stained with crystal violet. After washing out the remaining crystal violet, 200 microliters of sodium lauryl sulfate is put into each microwell and the absorbance of each is measured. When we examined this method using leukemic cell lines, we found that the numbers of cells were in proportion to the absorbance, and that the surviving cells could be counted by the absorbance within the range of 0.4 to 1.7. On the assumption that the effective range is more than 60% of the cytotoxicity index (CI), tables of CI classified into drugs, concentrations and duration of culture for each leukemic cell line were made. With these tables, comparison of the anticancer drugs and subsequent selection of the effective ones became easier. This method is simple, rapid and convenient for handling a large volume of material. Its application to further clinical practice is therefore expected.

[Anticancer drug sensitivity test using the short-term microplate culture and MTT dye reduction assay].

We have applied the MTT dye reduction assay to the anticancer drugs sensitivity test using short-term microplate cultures. The tumor cells were cultured with the anticancer drugs for 2 and 4 days. After culture, MTT dye was placed in each microwell and culture was carried out again for 4 more hours. The formazans generated by living cells were dissolved in acidified isopropyl alcohol and the absorbances of each well were measured at a wavelength of 540 nm. When tables of cytotoxicity indices classified into anticancer drugs, concentrations and durations of culture for each type of leukemic cell were made, it became possible to compare each drug and to select the effective ones. This assay is simple, precise, rapid, has no washing steps and is convenient for handling a large volume of material. We apply this assay in clinical practice.

[Effective mechanisms of ATX-S10, a new photosensitizer, on HeLa tumors in nude mice].

Photodynamic therapy (PDT) is a treatment modality that utilizes a photosensitizing drug activated by laser generated light. PDT is effective for oncologic applications. Many cancer patients have undergone a hematoporphyrin derivative (HpD)-mediated PDT. The HpD showed a side effect causing prolonged cutaneous photosensitivity. But ATX-S10, a new photosensitizer, provides rapid plasma and tissue clearance, comparable photoactivation efficiency, and superior light absorption of visible red. In this study, the tumor rejection mechanisms of PDT using ATX-S10 on HeLa tumors in nude mice were investigated with morphological and fluorometric methods. The mice were intracutaneously inoculated with HeLa cells, 5 x 10(5) or 1 x 10(7) cells. When tumors grew to about 10-12 mm in diameter, mice were intraperitoneally administered ATX-S10, 30 mg/kg, and 2 hours later the ATX-S10 in tumors was indirectly measured by a fluorometric machine, PMA-10 (Hamamatsu Photonics K. K.) and the tumors were irradiated by Optical Parametric Oscillator (Hamamatsu Photonics K. K.) tuned to a wave length at 670 nm, 5 mJ/pulse, 100 J/tumor. Before and after the irradiation, the effective mechanisms of PDT with ATX-S10 were studied by histological and ultrastructural approaches. The results showed occlusive thrombi in the microvasculature of the tumors and tumor cell death. These occlusive thrombi were observed within one hour after PDT at both light and electron microscopy levels, and were more remarkable as time passed after PDT. Therefore, the morphological studies of PDT with ATX-S10 suggested that the rejection mechanisms occurred mainly as a result of the destructive changes of the microvasculature in the tumors first, and secondly or simultaneously, tumor cells were destroyed through necrosis, and finally the tumors were rejected.

[Analysis of T cell receptor V beta gene expression in bronchoalveolar lavage fluid lymphocytes from patients with idiopathic interstitial pneumonia].

T lymphocytes in bronchoalveolar lavage (BAL) from idiopathic interstitial pneumonia (IIP) patients are considered to recognize unknown antigens, such as dust, fume, virus or degenerated autoantigens. To analyse the nature of these T lymphocytes, we investigated T cell receptor (TCR) V beta gene usage (22 kinds) in BAL Lymphocytes and Peripheral blood lymphocytes from 10 11P patients and 9 normal controls, using reverse transcriptase-polymerase chain reaction method. In BAL lymphocytes, predominant usage of V beta genes (> 15% of the sum of all V beta transcripts) was recognized in 7 of 10 IIP patients, which appeared to vary in individuals (Case 1: V beta 14, case 2: V beta 3, V beta 5.1, case 5: V beta 2, case 6: V beta 6, case 7: V beta 8, case 8: V beta 7, V beta 20, case 9: V beta 6), whereas no predominant V beta usage was demonstrated in normal controls. It remains to be elucidated whether the BAL lymphocytes expressing predominant V beta genes are involved in the activation of alveolar macrophages, fibroblasts, thereby inducing the production of autoantibodies.

[Antibody-producing ability of mononuclear cells from human tonsils: a consideration for immunological pathogenesis and operative indication of recurrent tonsillitis].

Antibody-production of mononuclear cells from human tonsils was examined by enzyme-linked immunosorbent assays in the culture system following polyclonal stimulations with pokeweed mitogen. The results were summarized and discussed as follows; 1) Age-related changes were not observed. 2) Antibody-producing ability in the intermittent phase of recurrent tonsillitis was significantly impaired. 3) Antibody-production in the recrudescent phase of recurrent tonsillitis was increased and might be reactivated probably by stimuli of the recent infection in vivo. 4) It was suggested that the recurrent tonsillitis was due to the low antibody-production ability of tonsillar mononuclear cells. 5) These results gave an immunological basis on the operative indication of recurrent tonsillitis.

[The signal transduction mechanism responsible for interferon-gamma-inducible indoleamine 2,3-dioxygenase (IDO) gene expression in T98G cells].

The interferon (IFN)-gamma-induced indoleamine 2,3-dioxygenase (IDO) is implicated in the inhibition of intracellular pathogens, e.g. Chlamydia psittaci and Toxoplasma gondii. The intracellular signaling molecules responsible for the induction of IDO gene expression were investigated by the quantitative polymerase chain reaction. The gene expression was inhibited by a tyrosine kinase inhibitor, genistein. Being consistent with this, IFN-gamma induced increased tyrosine phosphorylation and this was inhibited by genistein. The transcription of IDO gene was not inhibited by protein kinase C (PKC) inhibitors, H-7 and staurosporine, or a calmodulin inhibitor, W-7. Irrelevance of PKC in IDO gene expression was supported by the failure of PMA or PMA + A23187 to induce IDO gene expression. These results all suggest that the tyrosine phosphorylation is a critical event in IFN-gamma-inducible IDO gene expression and PKC is not involved in the gene expression.