PSGL-1/selectin and ICAM-1/CD18 interactions are involved in macrophage-induced drug resistance in myeloma.

Chemoresistance is the major obstacle in multiple myeloma (MM) management. We previously showed that macrophages protect myeloma cells, on a cell contact basis, from melphalan or dexamethasone-induced apoptosis in vitro. In this study, we found that macrophage-mediated myeloma drug resistance was also seen with purified macrophages from myeloma patients' bone marrow (BM) in vitro and was confirmed in vivo using the human myeloma-SCID (severe combined immunodeficient) mouse model. By profiling differentially regulated and paired plasma membrane protein genes, we showed that PSGL-1 (P-selectin glycoprotein ligand-1)/selectins and ICAM-1/CD18 played an important role in macrophage-mediated myeloma cell drug resistance, as blocking antibodies against these molecules or genetic knockdown of PSGL-1 or ICAM-1 in myeloma cells repressed macrophages' ability to protect myeloma cells. Interaction of macrophages and myeloma cells via these molecules activated Src and Erk1/2 kinases and c-myc pathways and suppressed caspase activation induced by chemotherapy drugs. Thus, our study sheds new light on the mechanism of drug resistance in MM and provides novel targets for improving the efficacy of chemotherapy in patients.

Regression of human T-cell leukemia virus type I (HTLV-I)-associated lymphomas in a rat model: peptide-induced T-cell immunity.

BACKGROUND: Human T-cell leukemia virus type I (HTLV-I) is etiologically linked to adult T-cell leukemia (ATL). The disease has a high mortality rate and is resistant to chemotherapy; therefore, immunologic approaches to treatment could be of interest. We have previously shown that athymic rats inoculated with a syngeneic (i.e., with the same genetic background) HTLV-I-infected T-cell line (FPM1-V1AX) develop ATL-like disease and that the transfer of T cells from normal syngeneic rats immunized with FPM1-V1AX cells prevents disease development. In this study, we further characterized the host antitumor immunity to explore the possibility of peptide-based vaccination against the ATL-like disease. METHODS: Immune T cells from rats immunized with FPM1-V1AX cells were analyzed for their phenotypes and cytotoxic properties. The epitope recognized by the T cells was analyzed by fine mapping. To evaluate the antitumor effects of a peptide-based vaccine, normal rats were immunized with synthetic oligopeptides corresponding to the epitope, the T cells were transferred to athymic rats inoculated with HTLV-I infected cells, and tumor size was monitored. RESULTS: Both CD4+ and CD8+ T-cell populations from rats immunized with FPM1-V1AX cells inhibited the growth of FPM1-V1AX cell-induced lymphomas in vivo. Long-term culture of splenic T cells from the immunized rats repeatedly resulted in establishment of CD8+ HTLV-I-specific cytotoxic T lymphocyte (CTL) lines restricted to the rat major histocompatibility complex class I molecule, RT1.A(l). The cytotoxicity of these lines was directed against the HTLV-I regulatory protein Tax and, specifically, against the epitope, amino acids 180-188 (GAFLTNVPY). Adoptive transfer of the Tax 180-188-specific CTL line or freshly prepared T cells from rats vaccinated with the Tax 180-188 oligopeptide prevented the development of FPM1-V1AX-cell induced lymphomas in athymic rats in comparison with control groups (two rats in each group). CONCLUSIONS: This study indicated a potential therapeutic effect of peptide-based vaccination against HTLV-I-induced lymphoproliferative disease.

Immunological aspects of rat models of HTLV type 1-infected T lymphoproliferative disease.

The level of host immune responses against human T cell leukemia virus type 1 (HTLV-1) varies among HTLV-1-infected individuals. In the present study, we investigate the role of host immunity on HTLV-1 leukemogenesis in vivo by using animal models. At first, we examined the effect of the routes of HTLV-1 transmission on the host anti-HTLV-1 immune responses. When immune competent adult rats were inoculated with HTLV-1-infected cells, the orally infected rats were persistently infected with HTLV-1 without humoral and cellular immune responses against HTLV-1, whereas all intravenously or intraperitoneally inoculated rats showed significant levels of immune responses. Next, we examined in vivo tumorigenicity of HTLV-1-immortalized cells in the absence of T cell immunity, by using athymic F344/N Jcl-rnu/rnu (nu/nu) rats. When inoculated into nu/nu rats, not all but some HTLV-1-immortalized rat cell lines including syngeneic FPM1-V1AX could grow and form T cell lymphoma in vivo. This syngeneic lymphoma formation was inhibited by adoptively transferred immune T cells. Furthermore, immunocompetent rats allowed in vivo growth of HTLV-1-infected lymphoma, when treated with antibodies that block costimulatory signals for T cell activation. These observations indicated that (1) host anti-HTLV-1 immunity can be affected by the conditions of the primary infection, (2) under the low pressure of anti-HTLV-1 immunity, some HTLV-1-infected cell clones grow in vivo, and (3) T cell immunity is required for in vivo surveillance against these HTLV-1-infected cell clones.

Prevention of adult T-cell leukemia-like lymphoproliferative disease in rats by adoptively transferred T cells from a donor immunized with human T-cell leukemia virus type 1 Tax-coding DNA vaccine.

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) in infected individuals after a long incubation period. To dissect the mechanisms of the development of the disease, we have previously established a rat model of ATL-like disease which allows examination of the growth and spread of HTLV-1 infected tumor cells, as well assessment of the effects of immune T cells on the development of the disease. In the present study, we induced HTLV-1 Tax-specific cytotoxic T lymphocyte (CTL) immunity by vaccination with Tax-coding DNA and examined the effects of the DNA vaccine in our rat ATL-like disease model. Our results demonstrated that DNA vaccine with Tax effectively induced Tax-specific CTL activity in F344/N Jcl-rnu/+ (nu/+) rats and that these CTLs were able to lyse HTLV-1 infected syngeneic T cells in vitro. Adoptive transfer of these immune T cells effectively inhibited the in vivo growth of HTLV-1-transformed tumor in F344/N Jcl-rnu/rnu (nu/nu) rats inoculated with a rat HTLV-1 infected T cell line. Vaccination with mutant Tax DNA lacking transforming ability also induced efficient anti-tumor immunity in this model. Our results indicated a promising effect for DNA vaccine with HTLV-1 Tax against HTLV-1 tumor development in vivo.

Development of human T-cell leukemia virus type 1-transformed tumors in rats following suppression of T-cell immunity by CD80 and CD86 blockade.

Host immunity influences clinical manifestations of human T-cell leukemia virus type 1 (HTLV-1) infection. In this study, we demonstrated that HTLV-1-transformed tumors could develop in immunocompetent rats by blocking a costimulatory signal for T-cell immune responses. Four-week-old WKA/HKm rats were treated with monoclonal antibodies (MAbs) to CD80 and CD86 and subcutaneously inoculated with syngeneic HTLV-1-infected TARS-1 cells. During MAb treatment for 14 days, TARS-1 inoculation resulted in the development of solid tumors at the site of inoculation, which metastasized to the lungs. In contrast, rats not treated with MAbs promptly rejected tumor cells. Splenic T cells from MAb-treated rats indicated impairment of proliferative and cytotoxic T-lymphocyte responses against TARS-1 in vitro compared to untreated rats. However, tumors grown in MAb-treated rats regressed following withdrawal of MAb therapy. Recovery of TARS-1-specific T-cell immune responses was associated with tumor regression in these rats. Our results suggest that HTLV-1-specific cell-mediated immunity plays a critical role in immunosurveillance against HTLV-1-transformed tumor development in vivo.

Establishment of a seronegative human T-cell leukemia virus type 1 (HTLV-1) carrier state in rats inoculated with a syngeneic HTLV-1-immortalized T-cell line preferentially expressing Tax.

Human T-cell leukemia virus type 1 (HTLV-1) causes T-cell malignancies in a small percentage of the population infected with the virus after a long carrier state. In the present study, we established a seronegative HTLV-1 carrier state in rats inoculated with a newly established HTLV-1-infected rat T cell line, FPM1. FPM1 originated from rat thymocytes cocultured with a human HTLV-1 producer, MT-2 cells, and expressed rat CD4, CD5, CD25, and HTLV-1 Tax. However, FPM1 scarcely expressed other major HTLV-1 structural proteins and failed to induce typical antibody responses against HTLV-1 in inoculated rats. In contrast, control rats inoculated with MT-2 cells generated significant levels of anti-HTLV-1 antibodies. HTLV-1 proviruses were detected in peripheral blood cells of syngeneic rats inoculated with FPM1 for more than 1 year. Analysis of the flanking region of HTLV-1 provirus integrated into host cells suggested that FPM1 cells remained in these animals over a relatively long period of time. However, a similar seronegative HTLV-1 carrier state was induced in the rats inoculated with mitomycin C-treated FPM1 cells and also in FPM1-inoculated allogeneic rats, suggesting that FPM1 could also transmit HTLV-1 into host cells in vivo. Our findings indicated that (i) HTLV-1-immortalized T cells which preferentially express HTLV-1 Tax persisted in vivo but failed to induce any diseases in immunocompetent syngeneic rats and that (ii) suboptimal levels of HTLV-1 for antibody responses allowed the establishment of persistent HTLV-1 infection.

Induction of adult T-cell leukemia-like lymphoproliferative disease and its inhibition by adoptive immunotherapy in T-cell-deficient nude rats inoculated with syngeneic human T-cell leukemia virus type 1-immortalized cells.

Human T-cell leukemia virus type 1 (HTLV-1) has been shown to be the etiologic agent of adult T-cell leukemia (ATL), but the in vivo mechanism by which the virus causes the malignant transformation is largely unknown. In order to investigate the mechanisms of HTLV-1 leukemogenesis, we developed a rat model system in which ATL-like disease was reproducibly observed, following inoculation of various rat HTLV-1-immortalized cell lines. When previously established cell lines, F344-S1 and TARS-1, but not TART-1 or W7TM-1, were inoculated, systemic multiple tumor development was observed in adult nude (nu/nu) rats. FPM1 cells, newly established from a heterozygous (nu/+) rat syngeneic to nu/nu rats, caused transient tumors only at the injection site in adult nu/nu rats, but could progressively grow in newborn nu/nu rats and metastasize in lymph nodes. The derivative cell line (FPM1-V1AX) serially passed through newborn nu/nu rats acquired the potency to grow in adult nu/nu rats. These results indicated that only some with additional changes but not all of the in vitro HTLV-1-immortalized cell lines possessed in vivo tumorigenicity. Using the syngeneic system, we further showed the inhibition of tumor development by transferring splenic T cells from immunized rats, suggesting the involvement of T cells in the regression of tumors. This novel and reproducible nude rat model of human ATL would be useful for investigation of leukemogenesis and antitumor immune responses in HTLV-1 infection.

Oral administration of human T-cell leukemia virus type 1 induces immune unresponsiveness with persistent infection in adult rats.

The major route of human T-cell leukemia virus type 1 (HTLV-1) infection is mother-to-child transmission caused by breast-feeding. We investigated the host immune responses to orally established persistent HTLV-1 infection in adult rats. HTLV-1-producing MT-2 cells were inoculated into immunocompetent adult rats either orally, intravenously, or intraperitoneally. HTLV-1 proviruses were detected in the peripheral blood and several organs for at least 12 weeks. Transmission of HTLV-1 to these animals was confirmed by analysis of HTLV-1 flanking regions. Despite persistent HTLV-1 presence, none of the orally inoculated rats produced detectable levels of anti-HTLV-1 antibodies, whereas all intravenously or intraperitoneally inoculated rats showed significant anti-HTLV-1 antibody responses. T-cell proliferative responses against HTLV-1 were also absent in orally inoculated rats. Our findings suggest that gastrointestinal exposure of adult rats to HTLV-1-infected cells induces persistent HTLV-1 infection in the absence of both humoral and cellular immune responses against HTLV-1. This immune unresponsiveness at primary infection may subsequently affect the host defense ability against HTLV-1.

Fas and its ligand in a general mechanism of T-cell-mediated cytotoxicity.

To investigate the mechanisms of T-cell-mediated cytotoxicity, we estimated the involvement of apoptosis-inducing Fas molecule on the target cells and its ligand on the effector cells. When redirected by ConA or anti-CD3 monoclonal antibody, a CD4+ T-cell clone, BK1, could lyse the target cells expressing wild-type Fas molecule but not those expressing death signaling-deficient mutants. This indicates the involvement of Fas-mediated signal transduction in the target cell lysis by BK1. Anti-CD3-activated but not resting BK1 expressed Fas ligand as detected by binding of a soluble Fas-Ig fusion protein, and the BK1-mediated cytotoxicity was blocked by the addition of Fas-Ig, implicating the inducible Fas ligand in the BK1 cytotoxicity. Ability to exert the Fas-mediated cytotoxicity was not confined to BK1, but splenic CD4+ T cells and, to a lesser extent, CD8+ T cells could also exert the Fas-dependent target cell lysis. This indicates that the Fas-mediated target cell lytic pathway can be generally involved in the T-cell-mediated cytotoxicity. Interestingly, CD4+ T cells prepared from gld/gld mice did not mediate the Fas-mediated cytotoxicity, indicating defective expression of functional Fas ligand in gld mice.