Shino Hanabuchi

Graft-versus-Tax Response in Adult T-Cell Leukemia Patients after Hematopoietic Stem Cell Transplantation

Adult T-cell leukemia (ATL) caused by human T-cell leukemia virus type I (HTLV-I) is characterized by poor prognosis after chemotherapy. Recent clinical trials have indicated, however, that allogeneic but not autologous hematopoietic stem cell transplantation (HSCT) for ATL can yield better clinical outcomes. In the present study, we investigated cellular immune responses of ATL patients who obtained complete remission after nonmyeloablative allogeneic peripheral blood HSCT from HLA-identical sibling donors. In the culture of peripheral blood mononuclear cells (PBMCs) from a post-HSCT but not pre-HSCT ATL patient, CD8+ CTLs proliferated vigorously in response to stimulation with autologous HTLV-I-infected T cells that had been established before HSCT in vitro. These CTLs contained a large number of monospecific CTL population directed to a HLA-A2-restricted HTLV-I Tax 11-19 epitope. The frequency of Tax 11-19-specific CD8+ CTLs in this patient markedly increased also in vivo after HSCT, as determined by staining with HLA-A2/Tax 11-19 tetramers. Similar clonal expansion of HTLV-I Tax-specific CTLs exclusively directed to a HLA-A24-restricted Tax 301-309 epitope was observed in the PBMCs from another ATL patient after HSCT from a HTLV-I-negative donor. Among four post-HSCT ATL patients tested, HTLV-I-specific CTLs were induced in the PBMC culture from three patients but not from the remaining one who had later recurrence of ATL. These observations suggested that reconstituted immunity against antigen presentation in ATL patients after HSCT resulted in strong and selective graft-versus-HTLV-I response, which might contribute to graft-versus-leukemia effects.

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

ABSTRACT 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.

Expansion of human T-cell leukemia virus type 1 (HTLV-1) reservoir in orally infected rats: inverse correlation with HTLV-1-specific cellular immune response.

ABSTRACT Adult T-cell leukemia (ATL) occurs in a small population of human T-cell leukemia virus type 1 (HTLV-1)-infected individuals. Although the critical risk factor for ATL development is not clear, it has been noted that ATL is incidentally associated with mother-to-child infection, elevated proviral loads, and weakness in HTLV-1-specific T-cell immune responses. In the present study, using a rat system, we investigated the relationships among the following conditions: primary HTLV-1 infection, a persistent HTLV-1 load, and host HTLV-1-specific immunity. We found that the persistent HTLV-1 load in orally infected rats was significantly greater than that in intraperitoneally infected rats. Even after inoculation with only 50 infected cells, a persistent viral load built up to considerable levels in some orally infected rats but not in intraperitoneally infected rats. In contrast, HTLV-1-specific cellular immune responses were markedly impaired in orally infected rats. As a result, a persistent viral load was inversely correlated with levels of virus-specific T-cell responses in these rats. Otherwise very weak HTLV-1-specific cellular immune responses in orally infected rats were markedly augmented after subcutaneous reimmunization with infected syngeneic rat cells. These findings suggest that HTLV-1-specific immune unresponsiveness associated with oral HTLV-1 infection may be a potential risk factor for development of ATL, allowing expansion of the infected cell reservoir in vivo, but could be overcome with immunological strategies.

Potential immunogenicity of adult T cell leukemia cells in vivo.

Experimental vaccines targeting human T cell leukemia virus type‐I (HTLV‐I) Tax have been demonstrated in a rat model of HTLV‐I‐induced lymphomas. However, the scarcity of HTLV‐I‐expression and the presence of defective HTLV‐I‐proviruses in adult T cell leukemia (ATL) cells have raised controversy about the therapeutic potential of HTLV‐I‐targeted immunotherapy in humans. We investigated the expression of HTLV‐I antigens in fresh ATL cells by using both in vitro and in vivo assays. In flow cytometric analysis, we found that 3 of 5 acute‐type and six of fifteen chronic‐type ATL patients tested showed significant induction of HTLV‐I Tax and Gag in their ATL cells in a 1‐day culture. Concomitantly with HTLV‐I‐expression, these ATL cells expressed co‐stimulatory molecules such as CD80, CD86 and OX40, and showed elevated levels of antigenicity against allogeneic T cells and HTLV‐I Tax‐specific cytotoxic T‐lymphocytes (CTL). Representative CTL epitopes restricted by HLA‐A2 or A24 were conserved in 4 of 5 acute‐type ATL patients tested. Furthermore, spleen T cells from rats, which had been subcutaneously inoculated with formalin‐fixed uncultured ATL cells, exhibited a strong interferon gamma‐producing helper T cell responses specific for HTLV‐I Tax‐expressing cells. Our study indicated that ATL cells from about half the patients tested readily express HTLV‐I antigens including Tax in vitro, and that ATL cells express sufficient amounts of Tax or Tax‐induced antigens to evoke specific T cell responses 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

ABSTRACT 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.

Repression of tax expression is associated both with resistance of human T-cell leukemia virus type 1-infected T cells to killing by tax-specific cytotoxic T lymphocytes and with impaired tumorigenicity in a rat model.

ABSTRACT Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). Although the viral transactivation factor, Tax, has been known to have apparent transforming ability, the exact function of Tax in ATL development is still not clear. To understand the role of Tax in ATL development, we introduced short-interfering RNAs (siRNAs) against Tax in a rat HTLV-1-infected T-cell line. Our results demonstrated that expression of siRNA targeting Tax successfully downregulated Tax expression. Repression of Tax expression was associated with resistance of the HTLV-1-infected T cells to Tax-specific cytotoxic-T-lymphocyte killing. This may be due to the direct effect of decreased Tax expression, because the Tax siRNA did not alter the expression of MHC-I, CD80, or CD86. Furthermore, T cells with Tax downregulation appeared to lose the ability to develop tumors in T-cell-deficient nude rats, in which the parental HTLV-1-infected cells induce ATL-like lymphoproliferative disease. These results indicated the importance of Tax both for activating host immune response against the virus and for maintaining the growth ability of infected cells in vivo. Our results provide insights into the mechanisms how the host immune system can survey and inhibit the growth of HTLV-1-infected cells during the long latent period before the onset of ATL.

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.

Correlation of Major Histocompatibility Complex Class I Downregulation with Resistance of Human T-Cell Leukemia Virus Type 1-Infected T Cells to Cytotoxic T-Lymphocyte Killing in a Rat Model

ABSTRACT Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) in infected individuals after a long incubation period. Despite the apparent transforming ability of HTLV-1 under experimental conditions, most HTLV-1 carriers are asymptomatic. These facts suggest that HTLV-1 is controlled by host immunity in most carriers. To understand the interplay between host immunity and HTLV-1-infected cells, in this study, we isolated several HTLV-1 Tax-specific cytotoxic T-lymphocyte (CTL) lines from rats inoculated with Tax-coding DNA and investigated the long-term effects of the CTL on syngeneic HTLV-1-infected T cells. Our results demonstrated that long-term mixed culture of these CTL and the virus-infected T cells led to the emergence of CTL-resistant HTLV-1-infected cells. Although the Tax expression level in these resistant cells was equivalent to that in the parental cells, expression of surface major histocompatibility complex class I (MHC-I) was significantly downregulated in the resistant cells. Downregulation of MHC-I was more apparent in RT1.Al, which presents a Tax epitope recognized by the CTL established in this study. Moreover, peptide pulsing resulted in killing of the resistant cells by CTL, indicating that resistance was caused by a decreased epitope density on the infected cell surface. This may be one of the mechanisms for persistence of HTLV-1-infected cells that evade CTL lysis and potentially develop ATL.