Generation of novel killer hybridomas derived from proliferation-suppressed somatic cell hybrids between YACUT T cell lymphoma and normal lymphocytes activated in secondary mixed lymphocyte cultures.

Somatic cell hybridization between the YACUT T cell lymphoma cell line with normal lymphocytes activated in secondary mixed lymphocyte cultures (MLCs) consistently yielded IL-2-dependent CD4- CD8 alpha+ beta- Fc gamma RIII+ hybrids with cytotoxic function. The hybrids expressed T cell receptors other than that of YACUT origin, and fusion of the YACUT with a CD8 alpha+ beta+ Fc gamma RIII- T cell line also yielded hybrids with an unexpected CD8 alpha+ beta- Fc gamma RIII+ phenotype, which two observations strongly suggested that CD8+ T cells became the parental cell of the hybrids. Prolonged growth of the hybrids with IL-2 resulted in the generation of autonomously growing hybrids (hybridomas) without abrogating the cytotoxic function. The hybridomas exhibited MHC-unrestricted cytotoxicity in a Ca(2+)-dependent manner without prior stimulation and also mediated antibody-dependent cellular cytotoxicity. These results indicate that novel killer hybridomas can be produced following cell transformation of proliferation-suppressed cytotoxic YACUT x MLC cell hybrids. The killer hybridomas may be of value for analyzing recognition mechanisms and molecules involved in MHC-unrestricted cell-mediated cytotoxicity.

Three-dimensional collagen matrices as cell culture substrata affect the generation of alloreactive cytotoxic T lymphocytes.

Primary one-way mixed lymphocyte cultures (MLC) of C3H/He responder and DBA/2 stimulator were performed in three-dimensional (3-D) collagen matrices and the generation of alloreactive cytotoxic T cell (CTL) responses was compared to those in MLC which were done on usual plastic surfaces or on collagen-coated plastic surfaces. MLC in the 3-D collagen matrices were found to generate strong CTL responses. Flow cytometric analysis of Lyt-2 and L3T4 antigen expressions on the effector cells showed that the Lyt-2/L3T4 ratios were substantially higher in the 3-D collagen matrices, and that a larger proportion of the cells in the 3-D collagen matrices were Lyt-2+ lymphoblasts. These results indicate that the milieu of the 3-D collagen matrices favors the proliferation of Lyt-2+ lymphocytes, and suggests that cell-to-matrix interactions in 3-D collagen matrices may play a regulatory role in the maturation process of alloreactive CTLs.

CD4+CD8+ cells lacking self-Mls reactive T cells are induced in mesenteric lymph nodes of Salmonella enteritidis-infected mice.

The percentage of mesenteric lymph node (MLN) cells that co-expressed both CD4 and CD8 was found to be from 5 to 7% in BALB/c and AKR/N mice bred under conventional conditions. In mice maintained under specific pathogen-free (SPF) conditions, the percentage fell below 2%. When mice were infected with an attenuated strain of Salmonella enteritidis (SER), the percentage of CD4+CD8+ cells in MLN rose to 20-30% transiently. In these mice, the total cell number and the percentage of CD8+ cells were not changed, but the CD4+ cell percentage was decreased. The expression intensity of TCR-alpha beta on CD4+CD8+ cells in the infected mice was higher in the MLN than in the thymus, but was similar to that of mature peripheral T cells. Among the CD4+CD8+ cell population in MLN, TCR-V beta 3+ cells were deleted but V beta 6+ cells were present in BALB/c mice which possess endogenous superantigen Mls-2a, but lack Mls-la. In AKR mice with the inverse of the occurrence of the superantigens, TCR-V beta 3+ cells were present and V beta 6+ cells were absent. These data suggest that CD4+CD8+ cells in the MLN of SER-infected mice may belong to thymus-derived mature T cells undergoing negative selection and that they may appear following exogenous stimulation.

Dissociation of signal transduction via Thy-1 and CD3 antigens in murine T cells.

To understand the proliferation/differentiation of immature thymocytes which have not express T cell antigen receptor (TCR), we studied whether Thy-1 has signal-transducing capacity. Thy-1+ CD3-TCR- cells including thymocytes from BALB/c embryos and SCID mice and nude mouse splenic cells did not show proliferative responses in the culture with anti-Thy-1 (G7) plus phorbol myristate acetate (PMA), whereas Thy-1+ CD3+ cells from normal thymus or spleen did show a response to them. Since Thy-1-mediated activation is suggested to require co-expression of the CD3-TCR complex, we compared the T cell proliferative response in mature T cells stimulated with anti-Thy-1 (G7) and anti-CD3-epsilon (2C11). Under the presence of PMA or IL-2, accessory cell-depleted splenic T cells were cultured with G7 or 2C11. PMA augmented the proliferative response of splenic T cells cultured with G7 much more than that with 2C11. IL-2, however, showed reciprocal effect on the proliferation of G7 and 2C11-treated splenic T cells. These data suggest that signals triggered via Thy-1 and CD3-epsilon may provide a distinct intracellular pathway for T cell activation.

Deregulated c-myc expression is insufficient for emergence of the tumorigenic phenotype in malignancy-suppressed intratypic T-cell lymphoma hybrids: an in vitro model for multistep lymphomagenesis.

The T-cell lymphoma cell line YACUT was fused with the Mls-1a antigen-responsive non-tumorigenic T-cell line G4 to construct growth-arrested hybrids which could be induced to proliferate in the presence of Mls-1a antigen. Prolonged growth of the hybrids by repeated antigenic stimulation resulted in the emergence of cells with transformed phenotype, which was accompanied by a reversion of c-myc expression to the levels of the YACUT lymphoma parent and an increase in the number of YACUT-derived chromosome 15 carrying the rearranged pvt-1 gene. Despite these two changes, early passage transformed hybrids as well as proliferation-suppressed hybrids were non-tumorigenic in vivo. The fact that only late passage transformed hybrids produced tumors in vivo indicated that additional genetic and/or epigenetic alterations are required for the emergence of hybrid lines with tumorigenic phenotype. Thus, this experimental system offers tangible possibilities for delineation of the three distinct phenotypes which could be exploited for the investigation of the multistep process of lymphomagenesis.

CD40 ligation inhibits trichilemmoma cell proliferation and induces IL-6 production.

CD40 is a member of the tumor necrosis factor receptor superfamily expressed by B cells, monocytes, dendritic cells, epithelial cells, and hematopoietic progenitor cells. Recently, CD40 has been reported to also be expressed on human epidermal cells. We have elucidated the function of CD40 on epidermal tumor cells and have found that trichilemmoma (KTL-1) cells constitutively express CD40 and respond to CD40 ligation by anti-CD40 mAb (EA-5) with a significant decrease in proliferation. We were also able to demonstrate that KTL-1 cells respond to CD40 ligation by EA-5 with the up-regulation of interleukin-6 (IL-6) mRNA expression. Together, the results suggest that CD40 on KTL-1 cells may function to regulate their proliferation associated with the induction of IL-6 production.

Treatment of murine angiosarcoma with etoposide, TNP-470 and prednisolone.

To develop effective therapies for angiosarcoma, we investigated the anti-tumor effects of etoposide (ETO), TNP-470 and prednisolone (PSL) using an established murine angiosarcoma cell line (ISOS-1). We examined the direct anti-tumor and anti-angiogenic effects of these drugs on ISOS-1 cells and normal murine microvascular endothelial cells (mECs) in vitro. Cell growth of ISOS-1 was inhibited significantly by ETO, moderately by TNP-470, and not at all by PSL (IC(50): 0.25 microg/ml, 10 microg/ml, >8000 microg/ml, respectively). One the other hand, cell growth of mECs was inhibited significantly by TNP-470, slightly by PSL, and negligibly by ETO (IC(50): 0.85 ng/ml, 0.7 microg/ml, 10 microg/ml, respectively). In an in vivo assay, tumor growth of ISOS-1 was significantly inhibited by more than 2.5 mg/kg of ETO dose-dependently, and by more than 30 mg/kg of TNP-470, and 100 mg/kg of PSL individually. Combination treatments of ETO+TNP-470 and TNP-470+PSL showed synergistic enhancement of inhibition (% control inhibition: ETO vs. TNP-470 vs. ETO+TNP-470: 55 versus 55 vs. 16%) (% control inhibition: TNP-470 vs. PSL vs. TNP-470+PSL: 41 vs. 86 vs. 21%). ETO+PSL combination treatment, however, failed to show significant enhancement of anti-tumor effects. In conclusion, our results indicated that TNP-470 may be a very effective drug for angiosarcoma treatment, especially in combination with ETO or PSL. We eagerly anticipate the use of TNP-470 in clinical treatment of angiosarcoma.

Evaluation of recombinant interleukin-2 immunotherapy for human hemangiosarcoma in a SCID mice model (WB-SCID).

We treated the patients with cutaneous hemangiosarcoma with recombinant interleukin-2 (rIL-2) immunotherapy that showed clear therapeutic effects. This immunotherapy is popular for the treatment of hemangiosarcoma in Japan. The purpose of this study is to clarify the clinical effects in an animal experiment. After establishing a SCID mouse model of human hemangiosarcoma WB-SCID, we used this model to investigate anti-tumor effects of rIL-2 and LAK cells. We demonstrated that hemangiosarcoma cells are LAK-sensitive, and LAK cells induced by rIL-2 suppress the growth of hemangiosarcoma. Our results may assure the clinical effects of rIL-2 immunotherapy on hemangiosarcoma.

The in vivo role of murine natural killer cells in the development of B cell lineage in bone marrow.

Physiological functions of natural killer (NK) cells in the development of bone marrow cells into the cells expressing B cell characteristics were examined in recipient mice expressing different allotype immunoglobulins (Ig) or in mutants defective in lipopolysaccharide (LPS) response. When the irradiated BALB/c nude mice (Igha) were injected with bone marrow cells of C57BL/6N (B6,Ighb), the level of donor-type serum Ig (Ighb) was about 10 fold higher in the mice with NK activity depleted by injecting anti-asialo GM1 compared to that of mice with the normal NK activity on day 21 after bone marrow transplantation. In the irradiated C3H/HeJ (low responder) which received bone marrow cells of C3H/HeN (high responder), augmentation of polyclonal antibody response and of the cell proliferation with LPS was demonstrated in the NK depleted mice. However, the difference in the level of donor-type Ig or LPS response between the NK-depleted and intact mice disappeared in 3 to 4 weeks. In normal mice without irradiation and marrow cell transplantation, NK cell elimination from spleen cells did not give rise to distinctly enhanced responses to in vitro LPS stimulation, whereas mice with augmented NK activity by poly I:C demonstrated a suppressed response to LPS when the mice were immunized with LPS. Collectively, these observations suggested that NK cells are involved in the suppressive regulation of developing B cell lineage as well as activated B cells in vivo.

Rhabdomyosarcoma of the prostate--report of a case with an immunohistochemical study of the neoplastic rhabdomyoblast.

A case of rhabdomyosarcoma of the embryonal alveolar type arising from the prostate in a 6-year-old boy was reported with a immunohistochemical survey. Tumor cells in the biopsied inguinal lymph node were weakly positive for myoglobin and CPK-mm and showed positive staining for neuron specific enolase (NSE). At autopsy, an immunohistochemical examination demonstrated that the tumor cells, especially differentiated elongated cells, were strongly positive for myoglobin, CPK-mm and NSE. An enzyme immunoassay for three distinct subunits of NSE revealed a 320 fold increase in the beta subunit of enolase which is specific for heart and skeletal muscle. The diagnostic significance of immunohistochemical methods for rhabdomyoblasts was discussed.

[Establishment of monoclonal antibody mNI-11 which induces homotypic cell aggregation of LPS-treated U937 cells].

A mouse monoclonal antibody (mAb), designated as mNI-11, was produced. The reactivity of mNI-11 was assessed by immunofluorescence assay with various cells. Myelomonocytic cell line, U937, and lipopolysaccharide (LPS)-treated U937 (LPS-U937 cells) were found to bind strongly to the mAb. EBV-B cell line and peripheral blood mononuclear cells (PBMCs) were found to bind moderately to it. This mAb strongly induced homotypic cell aggregation of LPS-U937 cells. The mAbs to CD18 and CD54 completely inhibited the LPS-U937 cell aggregation induced by mNI-11. The surface antigens of the U937 and LPS-U937 cells recognized by mNI-11 had a molecular size of 95-97 kDa as determined by immunoblotting analysis.

[Establishment of monoclonal antibody NI-58 which inhibits homotypic cell adhesion of LPS-stimulated U937 cells].

A mouse monoclonal IgG1 antibody, referred to as NI-58, has been produced. In immunofluorescence assay, this antibody reacted with myelomonocytes, EBV-B cells, Burkitt's lymphoma cells, T cell leukemia cells and peripheral blood mononuclear cells, but not with erythroid cells. The surface antigen on U937 cells recognized by NI-58 had a molecular size of 65 kDa as determined by immunoblotting analysis. As a biological function, NI-58 strongly inhibited the homotypic cell adhesion of LPS-stimulated U937 cells. It was found that the antigen defined by NI-58 was distinct from CD54 (intercellular adhesion molecule-1) in it's pattern of cellular expression and molecular weight, suggesting that NI-58 recognizes a new adhesion molecule and inhibits the homotypic cell adhesion of LPS-stimulated U937 cells.

CD4+CD8+ thymocytes develop into CD4 or CD8 single-positive cells in athymic nude mice.

A differentiation pathway from CD4+CD8+ cells to CD4+CD8- or CD4-CD8+ cells was investigated in athymic nude mice. Using fluorescence-activated cell sorter, CD4+CD8+ cells were sorted out from AKR thymocytes (H-2k, Thy-1.1) stained with two monoclonal antibodies against CD4 and CD8 (anti-L3T4 and anti-Ly-2). These CD4+CD8+ AKR thymocytes were injected i.v. into CBA or C3H nude mice (H-2k, Thy-1.2) which had received 650 rads and had been reconstituted with syngeneic nude bone marrow cells. The lymph node cells of the nude recipients at 4 wks post-thymocyte transfer were shown to contain 50% AKR-derived Thy-1.1+ cells. The majority of the Thy-1.1+ cells were found to express either CD4 or CD8 alone but not to express both CD4 and CD8. These findings indicate that CD4+CD8+ thymocytes can develop into CD4+CD8- and CD4-CD8+ single-positive cells in extrathymic tissues.

Modulation of cell surface antigens and regulation of phagocytic activity mediated by CD11b in the monocyte-like cell line U937 in response to lipopolysaccharide.

Modulation of the cellular antigens and regulation of the phagocytic activity of the monocyte-like cell line U937 after culture with lipopolysaccharide (LPS) were investigated. CD14 expression was induced on the surface of the U937 cells after 48 h of culture with LPS and then they became adhesive with numerous filamentous filopodia extruded on the cell surface, exhibiting the enhanced expression of CD16 and CD23, the activation cell surface markers for differentiation into macrophage. However, no induction or enhancement of the cell surface expression was observed with respect to CD11b, CD18, HLA-A, B, C, HLA-DR, DQ, DP or CD57. These U937 cells also acquired the ability to produce superoxide anions and to phagocytose the Salmonella enteritidis strain, 116-54. This phagocytosis was inhibited by the anti-CD11b monoclonal antibodies, but not by the anti-CD14, anti-CD16, anti-CD18, anti-CD23, anti-HLA-A, B, C or anti-HLA-DR monoclonal antibodies. These findings indicate that the phagocytic activity against Salmonella enteritidis 116-54 induced by LPS is mediated mainly via the CD11b molecule, but is not associated with the increased expression of CD11b. Puromycin and cycloheximide, inhibitors of protein synthesis, or a divalent cation-chelating agent, EDTA completely inhibited this phagocytic activity. Interestingly, EDTA was found to suppress specifically the CD11b expression on the U937 cells cultured with LPS. No phagocytic activity was induced when the U937 cells cultured with LPS were incubated at 4 degrees C, but restored to the control level when shifted up to 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of CD4+ and CD8+ T cells in protective immunity to the murine nematode parasite Trichuris muris.

The role of CD4+ and CD8+ T cells in protective immunity to Trichuris muris was studied in CD4+ or CD8+ or both CD4+ and CD8+ T cell-depleted BALB/c mice. To assess in vivo depletion of T-cell subsets, CD4+ and CD8+ T cells in the Peyer's patches, the mesenteric lymph nodes (MLN), and the spleens of mice treated with T cell-specific monoclonal antibodies (MoAbs) were analysed by FACS. CD4+ T cells were selectively depleted in mice injected with anti-CD4 MoAb i.p. and injection of anti-CD8 MoAb resulted in selective depletion of CD8+ T cells. The expulsion of T. muris was inhibited in CD4+ T cell-depleted mice and numerous worms were detected in the large intestine on days 14 and 21 after infection, although no suppression of protective immunity to T. muris was observed in CD8+ T cell-depleted mice. Moreover, there was no difference in suppression of protective immunity to T. muris between CD4+ T cell-depleted and both CD4+ and CD8+ T cells play a central role in protective immunity to T. muris infection.

Unique properties of a cytotoxic CD4+CD8+ intraepithelial T-cell line established from the mouse intestinal epithelium.

Growth factor-dependent gut intraepithelial lymphocyte (IEL) cell lines were established from a long-term in vitro culture of BALB/c IEL with syngeneic irradiated spleen cells in the presence of concanavalin A-stimulated spleen supernatant fluids. The cell lines were preferentially consisted of very limited thymoindependent subsets of IEL; i.e., Thy-1+CD5-TCR alpha beta+CD4+CD8 alpha+beta- (double-positive; DP) IEL and Thy-1+CD5-TCR alpha beta+CD4-CD8 alpha+beta- (CD8 single-positive; CD8 SP) IEL. The CD8 SP IEL cell line had cytotoxic activities and was triggered to proliferate by T-cell receptor (TCR)-directed stimuli. The DP IEL cell line expressed high levels of the CD3-TCR alpha beta, exhibited cytotoxic activity in redirected lysis assays, and had perforin in the cytoplasm, indicating the functional maturity of this cell line. However, the DP IEL cell line did not proliferate in response to TCR alpha beta-directed stimuli, which indicated that TCR alpha beta-mediated signalling was able to initiate cytotoxic function but not to induce proliferation of the DP IEL cell line. Although both cell lines were shown to have functional competence, they expressed J11d antigen which marks immaturity in thymocyte differentiation pathways. These results indicate that the established thymoindependent DP and CD8 SP IEL cell lines have unique properties distinct from DP thymocytes and CD8 SP peripheral T cells. Together with a recent report on freshly isolated DP IEL (10), the unique properties of the DP IEL cell line seems to support the notion that DP IEL may undergo a unique maturation process in the gut microenvironment.

In the absence of Epstein-Barr virus infection, phorbol ester modulates apoptosis in cycloheximide-treated Burkitt's lymphoma (BJA-B) cells.

We have shown previously that cycloheximide (CHX), a potent protein synthesis inhibitor, induces high levels of apoptosis in Epstein-Barr virus free (EBV(-)) Burkitt's lymphoma (BJA-B) cells, with comparably reduced levels of apoptosis in the EBV positive (EBV(+)) cells. Modulation of CHX-induced apoptosis in EBV(-) and (+) B cells is reported here using concurrent treatment with phorbol ester (phorbol 12, 13-dibutyrate, PdBu). Cells were collected at 0, 3, 6, 12, 24 and 48 hours after treatment with (i) 1 microgram/ml CHX, (ii) 0.1 microgram/ml PdBu (1 hour pretreatment before 0 h), or (iii) CHX + PdBu (CHX added at 0 h, 1 hour after PdBu). Control cultures were untreated. Apoptotic, necrotic or viable cells were quantified using histological, ultrastructural and biochemical parameters. Protein synthesis was assessed using 35S-methionine incorporation. Intracellular calcium concentrations were measured using flow cytometry. PdBu alone had little effect on cell death: High levels of CHX-induced apoptosis in EBV(-) cells were significantly reduced by concurrent addition of PdBu (P < 0.005). In contrast, low levels of CHX-induced apoptosis in EBV(+) cells were not significantly altered by PdBu treatment. In EBV(-) cells, a negative relationship was observed between levels of apoptosis and calcium concentrations, whereas in EBV(+) cells, there was negligible correlation between these parameters. Thus high levels of CHX-induced apoptosis in EBV(-) cells occur via a PKC-dependent pathway, whereas CHX treatment of EBV(+) cells induces comparatively low levels of apoptosis that occur via a PKC-independent mechanism. The results application in the therapeutic intervention for cancers developing in association with EBV infection.

Oxygen-independent antimicrobial activity against Salmonella enteritidis of specially activated macrophage with living vaccine.

Characteristics of peritoneal macrophages recovered from mice infected with two attenuated strains SER and Jena of Salmonella enteritidis were compared. Strong resistance against lethal infection with a virulent strain 116-54 of S. enteritidis was seen in a group of mice immunized with strain SER, but not in a group of mice immunized with strain Jena as well as in a control group. Peritoneal macrophages from mice immunized with strain SER showed an enhanced Salmonella-killing activity, an increased generation of O2- and an increased expression of Ia antigen on 7 to 14 days after infection when compared with those from mice immunized with strain Jena and thioglycollate(TG)-elicited macrophages as a control. The bacterial number of strain Jena in organs decreased more rapidly than that of strain SER after day 4 of infection. These observations suggest that the survival of an attenuated Salmonella bacilli at reticulo-endothelium is essential to increase of their activities of macrophages. Macrophages from mice injected with recombinant interferon(IFN)-gamma for 3 days induced the activated stage of the same characteristics as noted in activated macrophages from mice immunized with strain SER. Effect of oxygen intermediates (OI) scavengers such as superoxide dismutase and catalase on Salmonella-killing activity of activated macrophages was not seen at all. These results suggest that an increased generation of OI may be not primarily responsible for the ability to inhibit the intracellular growth of a virulent strain of S. enteritidis in macrophages activated by immunization with live, attenuated strains and injection with rIFN-gamma.

A microfilament formation inhibitor, cytochalasin strongly enhances the low-affinity Fc epsilon receptor II (CD23) expression on the human monocyte-like cell line, U937.

Enhancement of the low-affinity Fc epsilon receptor (CD23) expression by cytochalasin was analyzed on the human monocytelike cell line, U937. The CD23 expression on the U937 cells was enhanced at 24 hr after culture with cytochalasin B, D, or E, especially cytochalasin E having the most remarkable effect on it at the low concentration. This enhanced expression was found to be associated with a concomitant increase of a CD23 (about 45-kDa) protein on the U937 cells as assessed by Western blotting analysis. On the other hand, CD11a, CD18, CD31, CD49d, or CD54 was not markedly enhanced on the U937 cells by culture with cytochalasin E, although the mean fluorescence intensities (MFIs) of CD11a, CD18, and CD54 on U937 was partially up-regulated. Cell growth of U937 cultured with cytochalasin E was completely suppressed for 72 hr, but cell viability was sufficiently maintained (more than 95%). Soluble-formed CD23 (sCD23) also was released from the U937 cells at 24 to 72 hr after culture with cytochalasin E. In addition, the protein tyrosine kinase activity was detected in the U937 cells cultured with cytochalasin E for 24 hr using the enzyme immunoassay. Enhancement of the CD23 expression on the U937 cells at 24 to 72 hr cultured with cytochalasin E was sufficiently blocked by protein tyrosine kinase inhibitors herbimycin A and genistein, and a protein synthesis inhibitor, cychloheximide. On the other hand, protein kinase C inhibitors such as H-7 and H-8 had no effect on this CD23 expression. These results suggest that a mechanism underlying enhancement of the CD23 expression on the U937 cells cultured with cytochalasin E is mediated through tyrosine phosphorylation and protein synthesis.

Production and characterization of a human monoclonal antibody recognizing a new antigen expressed on some lymphoid cells.

A cell line secreting a human monoclonal antibody was established by Epstein-Barr virus transforming B cells derived from an enlarged cervical lymph node excised from a patient bearing a carotid body tumor. The reactivity of the monoclonal antibody, designated as mNISP, was tested on various cells and cell lines. An antigen defined by the mNISP was expressed on some Burkitt's lymphoma cell lines and on a non-T non-B acute lymphoblastic leukemia cell line. Furthermore, this antigen was expressed on leukemic cells from 2 of 8 patients with chronic myelocytic leukemia, 2 of 10 patients with acute myeloblastic leukemia, one of 13 patients with acute lymphoblastic leukemia, and two patients with adult T cell leukemia, but it was not expressed on normal T, B and adherent (macrophage) cells. In addition, mNISP reacted with T cells obtained from human T-cell leukemia virus type I carriers. We found that the antigen defined by mNISP was distinct from any previously reported antigen in terms of its pattern of cellular expression and molecular weight, suggesting that mNISP recognizes a new antigen expressed on some lymphoid cells.