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T Hamaoka

Publications and source records attributed to T Hamaoka.

At least 73 records · Page 4Linked to original sources
PubMed1998

B cell-mediated down-regulation of IFN-gamma and IL-12 production induced during anti-tumor immune responses in the tumor-bearing state.

Unfractionated spleen cells taken from tumor-bearing mice contained tumor-primed T cells which produced lymphokines such as IFN-gamma and IL-2 through collaboration with antigen-presenting cells (APC) binding tumor antigens when cultured in vitro. Here, we investigated the regulatory mechanisms underlying IFN-gamma production by T-APC interactions. Elimination of B cells from a splenic population of tumor-bearing mice resulted in enhanced IFN-gamma production. Adding B cells back into cultures down-regulated IFN-gamma production to almost the same levels as those induced by unfractionated spleen cells. IL-2 production was not enhanced by B cell depletion, but rather was significantly suppressed. IFN-gamma-selective up-regulation was due to an enhancement of IL-12 production because IL-12 was detected in B cell-depleted cultures and enhanced IFN-gamma production was prevented by addition of anti-IL-12 mAb or anti-CD40 ligand (CD40L) mAb capable of inhibiting CD40L-induced IL-12 production. These results indicate that B cells interfere with IFN-gamma production induced through interactions between anti-tumor T cells and APC, and this suppressive effect is based on the capacity of CD40+ B cells to down-regulate the CD40L-induced IL-12 production by APC.

Animals↗
PubMed1998

Ultrastructure of murine tumour cell lines defective in MHC class I expression before and after interferon-gamma treatment.

Two tumour cell clones, 6D1 and 4C2 cells, which are defective both in the major histocompatibility gene complex (MHC) class I expression and in the endogenous antigen presentation, are recovered with interferon (IFN)-gamma treatment. The present study describes the ultrastructure of these cells by using scanning and transmission electron microscopy in relation to the effect of IFN-gamma treatment. The general morphology of these cells was found to be similar to each other and comparable to that of a tumour cell clone, 4A1 cells, of the same origin, normal in MHC class I expression; they exhibited a fibroblast-like appearance and had many blebs on all the cell surfaces, with desmosome-like junctions between cells. On IFN-gamma treatment, surface fine blebs appeared less, and mitochondria became more densely stained. Expression of MHC class I molecules on the cell surface was much higher in the IFN-gamma treated 6D1 and 4C2 cells than in untreated cells, when estimated by immunoelectron microscopy. The addition of an epitope peptide to these cells did not enhance the class I expression, which differed from other antigen presentation-defective cells such as RMA-S cells, nor change the cell surface morphology.

Animals↗
PubMed1997

Positively charged liposome functions as an efficient immunoadjuvant in inducing immune responses to soluble proteins.

To design an optimum liposome immunoadjuvant for soluble protein antigens, we investigated the relationship between liposomal surface charge and adjuvant action. Positively charged multilamellar vesicles (MLV) were taken up efficiently by macrophages, while negatively charged and neutral MLVs were hardly picked up. Consistent with this, positively charged MLVs containing soluble chicken egg albumin (OVA) functioned as a more potent inducer of antigen-specific cytotoxic T lymphocyte (CTL) responses and antibody production than negatively charged and neutral MLVs containing the same concentrations of antigens. These results indicate that the positive charge on the surface of liposomes represents an important factor for enhancing their immunoadjuvancy in the induction of antigen-specific immune responses.

Adjuvants, Immunologic↗
PubMed1997

CD9-mediated costimulation of TCR-triggered naive T cells leads to activation followed by apoptosis.

The induction of full activation or death in TCR-triggered T cells depends largely on whether appropriate costimulatory signals are provided. In this study, we show that the costimulation of CD9 on naive T cells during TCR stimulation results in transient, albeit potent, activation followed by apoptosis, rather than full activation. Anti-CD9 mAb synergized with suboptimal doses of anti-CD3 mAb in inducing T cell activation. [3H]TdR incorporation determined 2 days after CD9 costimulation was as potent as that induced by CD28 costimulation. In contrast to progressive T cell proliferation induced by CD28 costimulation, CD9 costimulation led to the induction of apoptosis of once-activated T cells. Although IL-2R expression was induced significantly earlier and to a greater degree after CD9 costimulation than after CD28 costimulation, CD9 costimulation only transiently produced a small amount of IL-2 and induced apparently low levels of bcl-xL compared with those observed in CD28 costimulation. Addition of rIL-2 to cultures of CD9 costimulation induced strikingly enhanced expression of bcl proteins, especially of bcl-xL, and protected TCR-stimulated T cells from apoptosis. These data indicate that CD9-mediated costimulation of TCR-triggered naive T cells leads to activation followed by apoptosis as the result of failure to generate a positive signal for sufficient levels of IL-2 production.

Adjuvants, Immunologic↗
PubMed1997

Administration of IL-12 induces a CD3+ CD4- CD8- B220+ lymphoid population capable of eliciting cytolysis against Fas-positive tumor cells.

The present study investigates the effect of IL-12 administration on the generation of lymphoid cells that exhibit cytotoxicity against tumor cells expressing Fas Ag. Systemic injection of rIL-12 into BALB/c or (B6C3)F1 mice bearing syngeneic CSA1M or OV-HM tumor induced complete tumor regression. CSA1M tumor cells expressed Fas Ag, and exposure of these cells to IFN-gamma enhanced Fas expression. In contrast, Fas Ag was hardly detected on OV-HM cells even after IFN-gamma exposure. Only CSA1M cells were lysed by anti-Fas mAb or cells expressing Fas ligand (FasL), indicating that Fas on CSA1M cells is functional in mediating cell death. An increase in the frequency of lymphoid cells characterized as CD3+ CD4- CD8- B220+ was observed in spleens from both CSA1M and OV-HM tumor-bearing mice after IL-12 treatment. A splenic population enriched in cells with these unique phenotypes exhibited considerable degrees of cytotoxicity against Fas+ CSA1M, but not against Fas- OV-HM tumor cells. The lysis of CSA1M cells was almost completely blocked by addition of Fas-Fc, a fusion protein between the extracellular domain of mouse Fas and the Cgamma1 domain of human Ig. Regressing CSA1M and OV-HM tumor masses after IL-12 treatment exhibited a massive lymphoid cell infiltration and expressed significant levels of FasL mRNA, suggesting the infiltration of FasL-expressing cells to tumor sites. These results indicate that IL-12 induces the expansion of lymphoid cells that exhibit FasL-mediated cytolytic activity and accumulate into regressing tumor masses.

Animals↗
PubMed1997

Enhanced induction of antitumor T-cell responses by cytotoxic T lymphocyte-associated molecule-4 blockade: the effect is manifested only at the restricted tumor-bearing stages.

Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), a second counterreceptor for the B7 family of costimulatory molecules, functions as a negative regulator of T-cell activation. Here, we investigated whether the blockade of the CTLA-4 function leads to enhancement of antitumor T-cell responses at various stages of tumor growth. Unfractionated spleen cells taken from CSAIM fibrosarcoma-bearing mice 1-2 weeks after CSA1M cell implantation (early tumor-bearing mice) contained tumor-primed T cells that produced interleukin 2 and IFN-gamma through collaboration with antigen-presenting cell-binding tumor antigens when cultured in vitro. However, this initial lymphokine-producing capacity decreased at later stages of tumor growth (7-10 weeks after tumor cell implantation). Anti-CTLA-4 monoclonal antibody (mAb) was added to whole-spleen cell cultures from early or late tumor-bearing mice. Spleen cells from early tumor-bearing mice exhibited enhanced production of interleukin 2 and IFN-gamma upon in vitro culture in the presence of anti-CTLA-4 mAb. However, addition of anti-CTLA-4 mAb to whole-spleen cell cultures from late tumor-bearing mice failed to display such an enhancement. Consistent with these in vitro results, the in vivo antitumor effect of anti-CTLA-4 administration was observed in a tumor-bearing stage-restricted manner; in vivo administration of anti-CTLA-4 (1 mg/mouse, three times at 1-week intervals) into early tumor-bearing mice resulted in regression of growing tumors, whereas the same treatment did not affect tumor growth when performed for late tumor-bearing mice. Similar anti-CTLA-4 effect was observed in another tumor (OV-HM ovarian carcinoma) model. These in vitro and in vivo results indicate that CTLA-4 blockade in tumor-bearing individuals enhances the capacity to generate antitumor T-cell responses, but the expression of such an enhancing effect is restricted to early stages of tumor growth.

Abatacept↗
PubMed1997

A mechanism underlying synergy between IL-12 and IFN-gamma-inducing factor in enhanced production of IFN-gamma.

IL-12 and IFN-gamma-inducing factor (IGIF) have the capacity to stimulate IFN-gamma production by T cells. Using an IL-12-responsive T cell clone, 2D6, we investigated how these two cytokines collaborate for IFN-gamma production. 2D6 obtained from cultures containing rIL-12 produced IFN-gamma in response to rIGIF. 2D6 from cultures deprived of IL-12 for 24 h produced only marginal levels of IFN-gamma production following stimulation with either rIL-12 or rIGIF alone. However, simultaneous stimulation of these 2D6 cells with both cytokines resulted in strikingly enhanced levels of IFN-gamma production. 2D6 could also be maintained in the presence of rIL-2 instead of rIL-12. 2D6 lines maintained with rIL-12 (2D6(IL-12)) or rIL-2 (2D6(IL-2)) exhibited differential IGIF responsiveness: both lines responded similarly to rIL-2 or rIL-12, whereas the 2D6(IL-12) or 2D6(IL-2) exhibited high or marginal IGIF responsiveness, respectively. The 2D6(IL-12) line expressed IGIF receptor (IGIFR), whereas the 2D6(IL-2) did not. Overnight exposure of the 2D6(IL-12) to rIL-2 reduced IGIFR expression and conversely, exposure of the 2D6(IL-2) to rIL-12 restored IGIFR expression. IGIFR expression by these 2D6 lines correlated with the capacity to produce IFN-gamma in response to rIGIF. Purified naive T cells stimulated with anti-CD3 plus anti-CD28 mAb and subsequently cultured with rIL-12 were also found to express IGIFR and induce enhanced IFN-gamma production following IGIF stimulation. These results indicate that the induction of IGIFR by IL-12 represents one of the mechanisms underlying the synergy between IL-12 and IGIF in IFN-gamma production.

Animals↗
PubMed1997

Enhanced induction of very late antigen 4/lymphocyte function-associated antigen 1-dependent T-cell migration to tumor sites following administration of interleukin 12.

Administration of interleukin 12 (IL-12) into mice bearing CSA1M, OV-HM, Meth A, or MCH-1-A1 tumor induced complete regression of CSA1M and OV-HM tumors but induced only a slight growth inhibition of Meth A and MCH-1-A1 tumors. These effects of IL-12 were associated with high and only marginal levels of T-cell infiltration into CSA1M/OV-HM and Meth A/MCH-1-A1 tumor masses, respectively. Here, we investigated the role of IL-12 in the induction of T-cell migration. Spleen cells from untreated or IL-12-treated CSA1M-bearing mice were stained in vitro with a fluorescein chemical and transferred i.v. into IL-12-untreated CSA1M-bearing mice. Migration of donor cells was quantitated by counting the number of fluorescent cells on cryostat sections of tumor masses. Although only a slight migration was detected for spleen cells from IL-12-untreated CSA1M-bearing as well as IL-12-treated or untreated normal mice, enhanced migration was observed for cells from IL-12-treated CSA1M-bearing mice. A similar enhanced migration was observed for the OV-HM model. In contrast, such an enhancement was only marginal in the Meth A and MCH-1-A1 models. Immunohistochemical studies of tumors from IL-12-treated mice revealed that the predominant T-cell subset was CD4+ in CSA1M and CD8+ in OV-HM tumor masses. Consistent with this observation, the dominant subset of migrating T cells was found to be CD4+ in the CSA1M and CD8+ in the OV-HM models. T-cell migration was inhibited by pretreatment of recipients with either combination of anti-very late antigen 4 + anti-vascular cell adhesion molecule 1 or anti-lymphocyte function-associated antigen 1 + anti-intercellular adhesion molecule 1 monoclonal antibody. These results indicate that IL-12 can confer T cells with a capacity to migrate to tumor sites through very late antigen 4/lymphocyte function-associated antigen 1 adhesion pathways and that the in vivo acquisition of such a capacity following IL-12 treatment correlates with the induction of tumor regression.

Animals↗
PubMed1997

Expression of an inducible type of nitric oxide (NO) synthase in the thymus and involvement of NO in deletion of TCR-stimulated double-positive thymocytes.

The present study investigates the role of nitric oxide (NO) in the deletion of TCR-stimulated double-positive (DP) thymocytes. Fetal thymi expressed mRNA for an inducible type of NO synthase (iNOS). The levels of iNOS mRNA became maximal around gestation day 18 with a decline after birth. Administration of anti-CD3 mAb to fetal thymus organ culture (FTOC) or young mice resulted in enhanced expression of mRNAs for iNOS as well as IFN-gamma. Immunohistochemical analyses revealed that iNOS was produced in the corticomedullary junction and medulla. The effects of iNOS-induced NO on anti-CD3-unstimulated or anti-CD3-stimulated thymocytes were examined by culturing them in the presence or absence of a NO-generating compound. Stimulation of DP thymocytes with anti-CD3 alone induced the generation of CD4(low)CD8(low) thymocytes. The subsequent exposure of these anti-CD3-stimulated thymocytes to NO promoted down-regulation of CD4 and CD8 expression. The recovery of viable DP cells was considerably reduced compared with stimulation with anti-CD3 or NO alone. Even in a viable DP population, high incidences of DNA strand breaks were detected in the CD4(low)CD8(low) compartment. In contrast to DP cells, the recovery of viable single-positive cells was not decreased but rather slightly enhanced by treatment with anti-CD3 and/or NO. The recovery of anti-CD3-stimulated thymocytes were also reduced when cultured on the thymic stromal monolayer with the capacity to produce NO upon IFN-gamma stimulation. These results indicate that NO, which is generated in association with TCR stimulation in the thymus, functions to induce deletion of DP thymocytes, especially when their TCR is stimulated.

Animals↗
PubMed1997

Direct recognition of rat MHC antigens on rat antigen-presenting cells by mouse CD4+ and CD8+ T cells and establishment of T cell clones exhibiting a direct recognition pathway.

Alloantigens are recognized by T cells either through a direct pathway, which involves recognition of alloantigens expressed on allogeneic antigen-presenting cells (APC), or through an indirect pathway, which involves recognition of processed alloantigens presented by self APC. We investigated whether rat xenoantigens are also recognized by direct (xenogeneic APC-restricted) and/or indirect (self APC-restricted) pathways. C57BL/6 (B6) mouse anti-F344 or WKAH rat mixed lymphocyte reactions (MLRs) were partially inhibited by addition of either anti-mouse CD4 or CD8 monoclonal antibody (mAb) and almost completely blocked in the presence of both mAbs. These xenogeneic MLRs were almost completely inhibited by simultaneous depletion of both self and xeno APCs and only partially suppressed by the elimination of either type of APC, indicating that freshly prepared splenic mouse T cells can recognize rat xenoantigens through both direct and indirect pathways. Anti-F334 T cell lines were generated from B6 anti-F344 MLR cultures, and four CD4+ and four CD8+ T cell clones were isolated from these parental lines. The parental lines and those derived T cell clones were tested for their ability to proliferate depending on the presence of F344 APC. Proliferation of CD8 clones by stimulation with F344 APC was inhibited by the addition of anti-rat class I MHC mAb but not of anti-class II MHC mAbs. Conversely, proliferation of CD4 clones was reduced by addition of anti-class II MHC mAbs. Thus, these results indicate that xeno (rat)-reactive mouse T cells recognize xenoantigens via both indirect (self APC-restricted) and direct (xeno APC-restricted) pathways and that both CD4 and CD8 subsets of T cells participate in a direct pathway of xenoantigen recognition.

Animals↗
PubMed1997

B cells regulate CD40 ligand-induced IL-12 production in antigen-presenting cells (APC) during T cell/APC interactions.

Although stimulation of freshly isolated murine spleen cells with anti-CD3 mAb or Con A failed to generate IL-12 production, the same cell preparations depleted of B cells produced IL-12. Addition of normal B cells inhibited IL-12 production in a cell number-dependent manner. IL-12 production was dependent on the presence of CD4+, but not of CD8+, T cells, and inhibited by addition of anti-CD40 ligand (CD40L) mAb. Anti-CD3 or Con A stimulation induced CD40L expression only on CD4+ T cells, which was inhibited in the presence of B cells. IL-12 production was also induced by interactions between CD40L-transfected Chinese hamster ovary cells and splenocytes depleted of T and B cells, but not of APC, indicating CD40L-induced IL-12 production by APC. The involvement of CD40 molecules was examined by comparing the ability of cells from CD40-deficient (CD40 -/-) and wild-type mice (CD40 +/+) to produce IL-12. Spleen cells from CD40 -/- and CD40 +/+ mice produced comparable amounts of IL-12 in response to bacterial stimuli. However, the B cell-depleted fraction from CD40 -/- mice failed to produce IL-12 when stimulated with anti-CD3 or Con A or when cocultured with CD40L-expressing Chinese hamster ovary cells. These results indicate that CD40L expressed on activated T cells induces APC to produce IL-12 through CD40/CD40L interaction, but this pathway is competitively inhibited by CD40+ B cells incapable of producing IL-12 upon stimulation with CD40L. Thus, this might represent a novel mechanism underlying the regulation of cell-mediated and humoral immunity.

Animals↗
PubMed1997

Regulation of T cell-dependent and -independent IL-12 production by the three Th2-type cytokines IL-10, IL-6, and IL-4.

The production of IL-12 by macrophages/dendritic cells (Mphi/DC) is mediated either by a T cell-dependent pathway that is induced primarily by the interaction of CD40 ligand (CD40L) on activated T cells with CD40 on IL-12-producing cells or by a T cell-independent pathway that is induced by bacteria or bacterial products and enhanced by interferon-gamma (IFN-gamma). In this study we investigated the ability of the Th2-type cytokines interleukin (IL)-10, IL-6, and IL-4 to modulate IL-12 production in Mphi/DC induced through the two pathways. IL-12 production was induced in Mphi/DC from normal mice by stimulation with the combination of IFN-gamma plus lipopolysaccharide (LPS) or Staphylococcus aureus Cowan I (a model for the T cell-independent pathway) or by co-culture with Chinese hamster ovary (CHO) cells transfected with the CD40L (a model for the T cell-dependent pathway). The effects of three Th2-type cytokines on IL-12 production by Mphi/DC through the two pathways were examined. IL-10 inhibited IL-12 production induced through both pathways, although the inhibitory effect was more potent on the (IFN-gamma + LPS)-induced pathway. IL-6 inhibited only (LPS + IFN-gamma)-induced IL-12 production. The effect of IL-4 was particularly noteworthy: this cytokine inhibited (LPS + IFN-gamma)-induced IL-12 production, whereas it potentiated the production of IL-12 induced by CD40L. Regulation of IL-12 protein production by IL-10 and IL-4 was found to correspond to the levels of mRNA accumulation for the p40 and p35 IL-12 genes, whereas the presence of IL-6 during stimulation decreased IL-12 protein production without affecting steady-state mRNA levels. These results indicate that IL-12 production in Mphi/DC induced through a T cell-dependent or -independent pathway is positively or negatively regulated by particular cytokines at various control levels.

Animals↗
PubMed1997

Establishment of an IL-12-responsive T cell clone: its characterization and utilization in the quantitation of IL-12 activity.

We previously demonstrated that proliferation of terminally differentiated Th1 clones depends primarily on an interleukin-12 (IL-12)-paracrine mechanism mediated by their interactions with antigen-presenting cells (APC) rather than on an IL-2-autocrine mechanism. Such a Th1 clone (4-86, C57BL/6 origin) was cultured with recombinant IL-12 (rIL-12) in the absence of either antigen or APC. Some cells survived for several passages of culture with only rIL-12, and by limiting dilution, several clones highly reactive to rIL-12 alone were obtained. One of these clones, designated 2D6, was found to proliferate strongly in response to less than 1 pg/mL of rIL-12. This clone exhibited the following surface phenotypes: CD3+, T cell receptor (TCR) alpha beta+, Vbeta11+, NK-1.1-; CD4-CD8-; LFA-1+, ICAM-1+; and CD28+, CD80+, CD86+, CTLA-4-. In accordance with high responsiveness to IL-12, 2D6 cells were also found to express IL-12 receptor (IL-12R) as detected by incubation with rIL-12 and then staining with anti-IL-12 monoclonal antibody (mAb). Stimulation of 2D6 with rIL-12 resulted in the expression of interferon-gamma (IFN-gamma) and IL-10 mRNAs and production of these cytokines. The 2D6 clone responded to IL-2 (vigorously), IL-7 (moderately), and IL-4 (mildly) in addition to IL-12. However, the Ab capture assay using anti-IL-12 mAb enabled us to quantify IL-12-specific activity contained in a given sample. Thus, this study describes the unique features of the IL-12-responsive T cell clone and demonstrates the utilization of this clone in the quantitation of a specific IL-12 activity.

Animals↗
PubMed1997

IL-12-induced tumor regression correlates with in situ activity of IFN-gamma produced by tumor-infiltrating cells and its secondary induction of anti-tumor pathways.

Administration of recombinant interleukin-12 (rIL-12) into CSA1M fibrosarcoma-bearing mice results in complete regression of growing tumors. This tumor regression is associated with massive lymphoid cell infiltration to tumor sites and is completely blocked by injection of anti-interferon-gamma (IFN-gamma) monoclonal antibody (mAb). We investigated whether anti-IFN-gamma mAb exerts its suppressive effect on tumor regression by blocking the IL-12-induced lymphoid cell migration to tumor sites or by inhibiting the secondary effects of IFN-gamma produced by infiltrating cells. Injection of anti-IFN-gamma mAb to CSA1M-bearing mice before IL-12 treatment prevented the induction of tumor regression, whereas this treatment affected only marginally the infiltration of lymphoid cells to tumor masses. In accordance with this, IFN-gamma mRNA was expressed inside tumor masses by infiltrating cells after IL-12 therapy irrespective of whether anti-IFN-gamma mAb was injected. However, anti-IFN-gamma mAb treatment almost completely abrogated the in situ expression of inducible nitric oxide synthase (iNOS) as well as IFN-inducible protein-10 (IP-10) genes as examples of IFN-gamma-inducible genes. Immunohistochemical analyses also revealed that the expression of iNOS protein was completely inhibited by anti-IFN-gamma injection. These results suggest that the implementation of in situ IFN-gamma activity and its secondary induction of anti-tumor pathways such as iNOS and IP-10 expression are important processes in the IL-12-induced tumor regression.

Animals↗
PubMed1997

Synergy between CD28 and CD9 costimulation for naive T-cell activation.

Our previous study demonstrated that CD9 is expressed on most mature naive T-cells and delivers a potent costimulatory signal that functions independently of CD28. Here, we investigated whether this CD9-mediated signal is different from the CD28-mediated signal in the mode of costimulation and whether both signals function synergistically for T-cell activation. Anti-CD9 or anti-CD28 monoclonal antibody (mAb) increased [3H]TdR incorporation of naive T-cells in the absence of antigen-presenting cells (APC) when coimmobilized with submitogenic doses of anti-CD3 mAb. The levels of costimulation induced by ligation of CD9 and CD28 were comparable. However, the costimulatory effect differed between soluble anti-CD9 and CD28 mAb. A soluble form of anti-CD28 mAb could costimulate anti-CD3-triggered T-cells, whereas soluble anti-CD9 mAb failed to costimulate. Although anti-CD28 costimulated naive T-cells treated with phorbol myristate acetate (PMA) instead of anti-CD3 mAb, a combination of PMA plus anti-CD9 mAb could not induce T-cell activation. The combined costimulation of anti-CD3-triggered T-cells with anti-CD9 and anti-CD28 mAbs resulted in strikingly enhanced [3H]TdR uptake and lymphokine (IL-2 and IFN-gamma) production when compared to those induced by each costimulation. These results suggest that CD9 and CD28 induce T-cell costimulation using different signaling pathways, thereby inducing synergy in T-cell activation.

Animals↗
PubMed1997

Subunit composition of the pre-T-cell receptor complex analysed by monoclonal antibody against the pre-T-cell receptor alpha chain.

The pre-T-cell receptor (TCR) complex, which consists of a heterodimer of the TCR beta-chain and the pre-TCR alpha-chain, is known to regulate early thymocyte development. The pre-TCR complex contains CD3 subunits as a signal-transducing molecule, but the exact subunit composition of the fully assembled pre-TCR complex remains to be elucidated. In particular, the association of the CD3 zeta-chain with the pre-TCR is controversial. In the present study, we have generated a monoclonal antibody against the cytoplasmic portion of the pre-TCR alpha-chain, and analysed a subunit composition of the pre-TCR complex. We demonstrated that the CD3 zeta-chain is physically associated with the pre-TCR in immature T cells. Thus, the result strongly supports the previous findings that CD3 zeta contributes to signalling mediated through the pre-TCR complex.

Animals↗
PubMed1997

A defect in cell-to-cell adhesion via integrin-fibronectin interactions in a highly metastatic tumor cell line.

We investigated the role of integrin-fibronectin (FN) interactions in tumor cell adhesion. Two cloned tumor cell lines designated OV-LM (low-metastatic) and OV-HM (high-metastatic) were isolated from a murine ovarian carcinoma, OV2944. OV-LM and OV-HM cells exhibited high and low RGDS-sequence-dependent adhesiveness to FN, respectively. Both lines expressed comparable levels of alpha5 and alpha v integrins, which are capable of reacting with RGDS on FN. To compare the functions of these integrins between the two tumor lines, the signaling mechanism following FN stimulation was examined. Significant levels of phosphorylation of focal adhesion kinase (FAK) were detected in both OV-LM and OV-HM cells before FN stimulation. Whereas the level of FAK phosphorylation was appreciably enhanced in OV-LM cells stimulated with FN, stimulation of OV-HM cells with FN induced a reduction in the FAK phosphorylation in association with a significant decrease in the amount of FAK protein in the soluble compartment of cell lysates. A difference in the deposition of FN on the cell surface was also observed between the two types of tumor lines; OV-HM cells had an appreciably smaller amount of FN than OV-LM. Consistent with the functional abnormality of the integrin-FAK system and the smaller amount of FN on OV-HM, this clone exhibited a reduced cell-cell adhesion in the in vitro cell aggregation assay. Namely, OV-LM cells displayed a time-dependent increase in the formation of cell aggregates, whereas most OV-HM cells remained single. The formation of aggregates by OV-LM cells was inhibited by addition of RGDS peptide. These results indicate that the highly metastatic clone, OV-HM, exhibits a decreased capacity of cell-cell adhesion mediated by integrin-FN interactions and suggest that this defect is mainly due to the dysfunction of integrins/FAK rather than a decrease in the amount of integrins expressed on tumor cells.

Animals↗