Nanae Harashima

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.

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.

Tumor immunity against adult T‐cell leukemia

Human T‐cell leukemia virus type‐I (HTLV‐I) causes adult T‐cell leukemia (ATL) in a small population of infected individuals after a long incubation period. Although the process of clonal evolution of ATL cells may involve multiple steps, ATL cells from half of the ATL cases still retain the ability to express HTLV‐I Tax, a key molecule of HTLV‐I leukemogenesis. A recent finding of reactivation of Tax‐specific cytotoxic T lymphocytes (CTL) in ATL patients after hematopoietic stem cell transplantation suggests the presence of Tax expression in vivo and potential contribution of the CTL to antitumor immunity. This is consistent with the results of a series of animal experiments indicating that Tax‐specific CTL limit the growth of HTLV‐I‐infected cells in vivo, although the animal model mimics only an early phase of HTLV‐I infection and leukemogenesis. Establishment of an insufficient HTLV‐I‐specific T‐cell response and an increased viral load in orally HTLV‐I‐infected rats suggests that host HTLV‐I‐specific T‐cell response at a primary HTLV‐I infection can be a critical determinant of persistent HTLV‐I levels thereafter. These findings indicate that Tax‐targeted vaccines may be effective for prophylaxis of ATL in a high‐risk group, and also for therapy of ATL in at least half the cases. (Cancer Sci 2005; 96: 249 –255)

The HSP70 and Autophagy Inhibitor Pifithrin-μ Enhances the Antitumor Effects of TRAIL on Human Pancreatic Cancer

TRAIL and agonistic death receptor-specific antibodies can induce apoptosis in cancer cells with little cytotoxicity to normal cells. To improve TRAIL-induced antitumor effects, we tested its effectiveness in combination with pifithrin (PFT)-μ, which has the potential to inhibit HSP70 function and autophagy, both of which participate in TRAIL resistance in cancer cells. Among the four human pancreatic cancer cell lines tested, MiaPaca-2, Panc-1, and BxPC-3 cells showed varying sensitivities to TRAIL. In MiaPaca-2 and Panc-1 cells, knockdown of HSP70 or beclin-1, the latter an autophagy-related molecule, by RNA interference augmented TRAIL-induced antitumor effects, decreasing cell viability, and increasing apoptosis. On the basis of these findings, we next determined whether the TRAIL-induced antitumor effects could be augmented by its combination with PFT-μ. The combination of TRAIL plus PFT-μ significantly decreased the viability and colony-forming ability of MiaPaca-2 and Panc-1 cells compared with cells treated with either agent alone. When applied alone, PFT-μ increased Annexin V+ cells in both caspase-dependent and -independent manners. It also promoted TRAIL-induced apoptosis and arrested cancer cell growth. Furthermore, PFT-μ antagonized TRAIL-associated NF-κB activation in cancer cells. In a xenograft mouse model, combination therapy significantly inhibited MiaPaca-2 tumor growth compared with treatment with either agent alone. The results of this study suggest protective roles for HSP70 and autophagy in TRAIL resistance in pancreatic cancer cells and suggest that PFT-μ is a promising agent for use in therapies intended to enhance the antitumor effects of TRAIL. Mol Cancer Ther; 12(4); 341–51. ©2013 AACR.

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.

Identification of Two New HLA-A*1101-Restricted Tax Epitopes Recognized by Cytotoxic T Lymphocytes in an Adult T-Cell Leukemia Patient after Hematopoietic Stem Cell Transplantation

ABSTRACT We previously reported that Tax-specific CD8+ cytotoxic T lymphocytes (CTLs), directed to single epitopes restricted by HLA-A2 or A24, expanded in vitro and in vivo in peripheral blood mononuclear cells (PBMC) from some adult T-cell leukemia (ATL) patients after but not before allogeneic hematopoietic stem cell transplantation (HSCT). Here, we demonstrated similar Tax-specific CTL expansion in PBMC from another post-HSCT ATL patient without HLA-A2 or A24, whose CTLs equally recognized two newly identified epitopes, Tax88-96 and Tax272-280, restricted by HLA-A11, suggesting that these immunodominant Tax epitopes are present in the ATL patient in vivo.

Adult T-cell leukemia: future prophylaxis and immunotherapy.

A small population of human T-cell leukemia virus Type I (HTLV-I) carriers develop adult T-cell leukemia after a long incubation period. The results of a series of experiments using animal models suggest that insufficiency of HTLV-I-specific T-cell response induced by vertical HTLV-I infection allows enlargement of the HTLV-I-infected cell reservoir in vivo, a crucial risk factor of adult T-cell leukemia. In this review it is proposed that prophylactic Tax-targeted vaccines for the high-risk group of adult T-cell leukemia, which is characterized by low HTLV-I-specific T-cell response and high proviral load, can reduce the risk. Immunological studies on adult T-cell leukemia patients after hematopoietic stem cell transplantation also suggest that Tax-targeted immunotherapy may be effective against full-blown disease, although its indication may be limited.

Impaired Tax‐specific T‐cell responses with insufficient control of HTLV‐1 in a subgroup of individuals at asymptomatic and smoldering stages

Human T‐cell leukemia virus type‐1 (HTLV‐1)‐specific T‐cell immunity, a potential antitumor surveillance system in vivo, is impaired in adult T‐cell leukemia (ATL). In this study, we aimed to clarify whether the T‐cell insufficiency in ATL is present before the disease onset or occurs as a consequence of the disease. We investigated T‐cell responses against Tax protein in peripheral blood mononuclear cells (PBMCs) from individuals at earlier stages of HTLV‐1‐infection, including 21 asymptomatic HTLV‐1 carriers (ACs) and four patients with smoldering‐type ATL (sATL), whose peripheral lymphocyte count was in normal range. About 30% of samples tested showed clear Tax‐specific interferon (IFN)‐γ producing responses. Proviral loads in this group were significantly lower than those in the other less‐specific response group. The latter group was further divided to two subgroups with or without emergence of Tax‐specific responses following depletion of CC chemokine receptor 4 (CCR4)+ cells that contained HTLV‐1‐infected cells. In the PBMCs with Tax‐specific responses, CD8+ cells efficiently suppressed HTLV‐1 p19 production in culture. The remaining group without the emergence of Tax‐specific response after CCR4+ cell‐depletion included at least two sATL and one AC samples, which spontaneously produced HTLV‐1 p19 in culture, where tetramer‐binding, Tax‐specific cytotoxic T‐lymphocytes were either undetectable or unresponsive. Our results indicated that HTLV‐1‐specific T‐cell responsiveness widely differed among HTLV‐1 carriers, and that impairment of HTLV‐1‐specific T‐cell responses was observed not only in advanced ATL patients but also in a subpopulation at earlier stages, which was associated with insufficient control of HTLV‐1. (Cancer Sci 2009; 100: 481–489)

Abstract P5-01-13: Poly(I:C), an innate adjuvant receptor ligand, can induce the antitumor effects on human breast cancer cells

[Background] Innate adjuvant receptors are expressed in immune cells and some types of cancers. If antitumor therapies targeting adjuvant receptors in cancer cells are established, it could be therapeutically useful because antitumor effects and activation of the immune system can be induced simultaneously. [Objective]Poly(I:C) is one of innate adjuvant receptors ligands and triggers signaling through not only endosome Toll-like receptor (TLR) 3 but also cytoplasmic melanoma differentiation-associated gene (MDA) 5. In this study, we investigated antitumor effects of poly(I:C) on human breast cancer cell lines, and then elucidated the mechanisms. [Methods] Three breast cancer cell lines (MCF-7, MDA-MB-231, and BT-549) were used in this study. Poly(I:C) was transfected into these cancer cells, and cell viability and apoptosis were evaluated by WST8 assay and flow cytometry, respectively. The expression of adjuvant receptors and cell cycle-related molecules were examined by immunobotting. Autophagy was evaluated by immunoblotting and confocal imaging. In vivo antitumor effect was determined using a xenograft mouse model. [Results] All three cell lines were positive for both TLR3 and MDA5. Poly(I:C) transfection drastically reduced their cell viabilities in a partially MDA5-dependent manner. Poly(I:C)-transfected cancer cells showed caspase-dependent apoptosis and growth arrest as a result of suppression of c-Myc and cyclinD1. Interestingly, poly(I:C) transfection was accompanied by autophagy, which is known to work cytoprotectively under starvation and stress, and knockdown of beclin-1, an essential molecule for autophagy, by RNA interference promoted poly(I:C) transfection-induced apoptosis. In vivo poly(I:C) transfection exerted the antitumor effect on xenografted MDA-MB-231 in nude mice. [Conclusions] These results suggest that the innate adjuvant receptors are promising targets in human breast cancer and that autophagy plays a protective role in this treatment modality. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-01-13.