Tax-specific Cytotoxic T Lymphocytes Research Articles

Live attenuated VZV vaccination induces antitumor immunity in ATLL patients

Adult T cell leukemia/lymphoma (ATLL) is a CD4-positive peripheral T cell lymphoma caused by human T cell lymphotropic virus type 1 (HTLV-1). Although ATLL is quite difficult to be cured, up-regulation of cellular immunity such as HTLV-1 Tax-specific cytotoxic T lymphocytes (CTLs) has been proved to be important to obtain long-term survival. At present, no efficacious method to activate ATLL-specific cellular immunity is available. This study aimed to investigate whether live attenuated varicella-zoster virus (VZV) vaccination to ATLL can activate HTLV-1 Tax-specific cellular immune response. A total of 3 indolent- and 3 aggressive-type ATLL patients were enrolled. All aggressive-type patients had the VZV vaccination after completing anti-ATLL treatment including mogamulizumab, which is a monoclonal antibody for C–C chemokine receptor 4 antigen, plus combination chemotherapy, whereas all indolent-type patients had the VZV vaccination without any antitumor treatment. Cellular immune responses including Tax-specific CTLs were analyzed at several time points of pre- and post-VZV vaccination. After the VZV vaccination, a moderate increase in 1 of 3 indolent-type patients and obvious increase in all 3 aggressive-type patients in Tax-specific CTLs percentage were observed. The increase in the cell-mediated immunity against VZV was observed in all indolent- and aggressive-type patients after VZV vaccination. To conclude, VZV vaccination to aggressive-type ATLL patients after mogamulizumab plus chemotherapy led to the up-regulation of HTLV-1 Tax-specific CTLs without any adverse event. Suppression of regulatory T lymphocytes by mogamulizumab may have contributed to increase tumor immunity in aggressive-type ATLL patients. Japan Registry of Clinical Trials number, jRCTs051180107.

Improved Overall Survival Among Newly Diagnosed Aggressive Adult T-Cell Leukemia/Lymphoma Patients Treated with Mogamulizumab Plus EPOCH Compared to Mogamulizumab Plus Vcap-AMP-Vecp

Introduction: Adult T-cell leukemia/lymphoma (ATLL) is a rare and quite refractory peripheral T-cell lymphoma. The human T-lymphotropic virus type 1 (HTLV-1) retrovirus causes ATLL. Tax protein produced by HTLV-1 has oncogenic activities, and its peptides can be recognized as tumor antigens by the host immune system. Therefore, some immunological approaches to treat ATLL are effective. Mogamulizumab (Moga), an anti-CCR4 monoclonal antibody, and lenalidomide, an immunomodulator, have been recently approved for newly diagnosed and/or relapsed/refractory aggressive ATLL in Japan. However, to date, satisfactory treatment results have not been obtained with these drugs. Before approval of Moga and lenalidomide, VCAP-AMP-VECP (mLSG15) therapy was one of the standard combination chemotherapies for aggressive ATLL. A randomized phase II study comparing Moga plus mLSG15 and mLSG15 alone showed a higher %CR in Moga plus mLSG15 arm but no significant difference in overall survival (OS) between the two arms. In this study, we compared the outcomes of Moga plus EPOCH with those of Moga plus mLSG15. Methods: Aggressive ATLL patients, who were ineligible for hematopoietic stem cell transplantation (HSCT) and were newly diagnosed after June 2000 in our hospital, were retrospectively analyzed. Results: Table 1 summarizes the patient characteristics. Twenty-two patients were treated with Moga plus EPOCH, and 16 were treated with Moga plus mLSG15. There was no significant difference in gender and disease subtype between the groups. Moga-induced skin disorders, such as rash and erythema, were observed in 14 (64%) patients treated with Moga plus EPOCH group and 9 (56%) patients treated within Moga plus mLSG15; the difference was not significant. Figure 1 shows OS of both groups. There was no significant difference (P=0.3092), but a 30% risk reduction was observed in the Moga plus EPOCH group (hazard ratio [HR]: 0.6859, 95% confidence interval [CI]: 0.3165−1.430), though patients treated with Moga plus EPOCH were significantly older than those treated with Moga plus mLSG15 (Table 1). Figure 2 shows OS of patients with and without Moga-induced skin disorders in both groups. The OS of patients with skin disorders was significantly greater than that of those without skin disorders (P<0.0001, HR: 0.2653, 95% CI: 0.05879−0.3628), as previously reported. Elevation of the Tax-specific cytotoxic T-lymphocytes (CTL) percentage was observed after occurrences of skin disorders in some patients. Representative data are shown in Figure 3. These data suggest that the combination chemotherapy regimen might be irrelevant for patients with Moga-induced skin disorders. Next, the OS of the Moga plus EPOCH group was compared with that of those treated with chemotherapy alone, such as mLSG15 and the CHOP-like regimen, as the historical control (Table 2). There was no statistical difference between the groups, but a 16% risk reduction was observed in the Moga plus EPOCH group (P=0.5885, HR: 0.8401, 95% CI: 0.4491−1.568) (Figure 4), though those treated with Moga plus EPOCH were significantly older than those treated with chemotherapy alone (Table 2). A 36% risk reduction in OS was observed in patients with Moga-induced skin disorders compared to those treated with chemotherapy alone (Figure 5). Conclusion: Improved OS was observed more frequently in the Moga plus EPOCH group than in the Moga plus mLSG15 group. However, to date, Moga plus chemotherapy has not shown a significant impact on OS for newly diagnosed aggressive ATLL patients in non-HSCT settings, except for patients with Moga-induced skin disorders. Elevation of the Tax-specific CTL percentage was observed in some patients with Moga-induced skin disorders. We previously reported in the 2014 ASH annual meeting that several aggressive ATLL patients, who obtained long-time survival using chemotherapy alone, had maintained Tax-specific CTLs for long periods. Therefore, it might be necessary to induce, up-regulate, and maintain ATLL-specific CTLs to obtain long-time survival. Moga is useful, but insufficient, for inducing such CTLs by suppressing regulatory T-cells. No effective method is available to activate an ATLL-specific cellular immune response. Further studies are urgently needed to establish effective methods for attaining ATLL-specific CTLs. Disclosures No relevant conflicts of interest to declare.

Detection of Tax-specific CTLs in lymph nodes of adult T-cell leukemia/lymphoma patients and its association with Foxp3 positivity of regulatory T-cell function.

Human T-cell lymphotropic virus type (HTLV)-1 Tax is a viral protein that has been reported to be important in the proliferation of adult T-cell leukemia/lymphoma (ATLL) cells and to be a target of HTLV-1-specific cytotoxic T lymphocytes (CTLs). However, it is not clear how Tax-specific CTLs behave in lymph nodes of ATLL patients. The present study analyzed the immunostaining of Tax-specific CTLs. Furthermore, ATLL tumor cells are known to be positive for forkhead box P3 (Foxp3)and to have a regulatory T (Treg)-cell-like function. The association between T-reg function and number and activity of Tax-specific CTLs was also investigated. A total of 15 ATLL lymphoma cases with human leukocyte antigen (HLA)-A24, for which Tax has a high affinity, were selected from the files of the Department of Pathology, School of Medicine, Kurume University (Kurume, Japan) using a polymerase chain reaction (PCR) method. Immunostaining was performed for cluster of differentiation (CD) 20, CD3, CD4, CD8, T-cell intracellular antigen-1 and Foxp3 in paraffin sections, and for Tax, interferon γ and HLA-A24 in frozen sections. In addition, the staining of Tax-specific CTLs (HLA-A24-restricted) was analyzed by MHC Dextramer® assay in frozen sections. In addition, the messenger RNA expression of Tax and HTLV-1 basic leucine zipper factor were also evaluated by reverse transcription-PCR. Immunohistochemical staining of Tax protein in lymphoma tissue revealed the presence of positive lymphoma cells ranging from 5 to 80%, and immunohistochemical staining of HLA-A24 revealed the presence of positive lymphoma cells ranging from 1 to 95%. The expression of Tax and HLA-A24 was downregulated by viral function. Foxp3, a marker for Treg cells, was expressed in 0–90% of cells. Several cases exhibited Tax-specific CTL (HLA-A24-restricted)-positive cells, and there was an inverse correlation between Tax-specific CTLs and Foxp3. However, neither Tax nor HLA-A24 expression was associated with CTL or Foxp3. Our study indicated the possibility that ATLL cells, which expressed Tax, target of CTL, evade the CTL-mediated immune control by expression of Foxp3 as a Treg function.

Molecular Pathology of Adult T-Cell Leukemia/Lymphoma

Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell neoplasm of highly pleomorphic lymphoid cells. ATLL is usually widely disseminated, and it is caused by human T-cell leukemia virus type 1 (HTLV-1). It is a disease with a long latency, and affected individuals are usually exposed to the virus very early in life. The cumulative incidence of ATLL is estimated to be 2.5% among HTLV-1 carriers. ATLL cells express CD2, CD3, CD5, CD4, and CD25, as well as CCR4 and FoxP3 of the regulatory T-cell marker. HTLV-1 is causally linked to ATLL, but infection alone is not sufficient to result in neoplastic transformation. A significant finding in this connection is that the Tax viral protein leads to transcriptional activation of many genes, while the HTLV-1 basic leucine zipper factor is thought to be important for T-cell proliferation and oncogenesis. Half of ATLL cases retain the ability to express HTLV-1 Tax, which is a target of HTLV-1-specific cytotoxic T lymphocytes (CTL). An increase in HTLV-1-specific CTL responses is observed in some asymptomatic HTLV-1 carriers. Although HTLV-1-specific CTL are also present in the peripheral blood of ATLL patients, they do not expand sufficiently. We investigated the clinicopathological features and analyzed the staining of Tax-specific CTL and FoxP3. Tax-specific CTL correlated inversely with FoxP3, an increase in the ratio of CD163+ tumor-associated macrophages was associated with worse clinical prognosis, and ATLL cell lines proliferated significantly following direct co-culture with M2 macrophages. Several clinical variants of ATLL have been identified: acute, lymphomatous, chronic, and smoldering. Oligo-array comparative genomic hybridization revealed that genomic loss of 9p21.3 was a significant characteristic of acute-type, but not of chronic-type ATLL. Furthermore, we found that genomic alteration of CD58, which is implicated in immune escape, is more frequently observed in acute than in chronic ATLL. Interestingly, the chronic cases with cell cycle deregulation and disruption of immunosurveillance mechanism were associated with faster progression to acute ATLL. Immune evasion, microenvironment, and genetic alteration are therefore important in the multi-step progression of ATLL lymphomagenesis.

Clinical outcomes of a novel therapeutic vaccine with Tax peptide‐pulsed dendritic cells for adult T cell leukaemia/lymphoma in a pilot study

Adult T cell leukaemia/lymphoma (ATL) is a human T cell leukaemia virus type-I (HTLV-I)-infected T cell malignancy with poor prognosis. We herein developed a novel therapeutic vaccine designed to augment an HTLV-I Tax-specific cytotoxic T lymphocyte (CTL) response that has been implicated in anti-ATL effects, and conducted a pilot study to investigate its safety and efficacy. Three previously treated ATL patients, classified as intermediate- to high-risk, were subcutaneously administered with the vaccine, consisting of autologous dendritic cells (DCs) pulsed with Tax peptides corresponding to the CTL epitopes. In all patients, the performance status improved after vaccination without severe adverse events, and Tax-specific CTL responses were observed with peaks at 16-20weeks. Two patients achieved partial remission in the first 8weeks, one of whom later achieved complete remission, maintaining their remission status without any additional chemotherapy 24 and 19months after vaccination, respectively. The third patient, whose tumour cells lacked the ability to express Tax at biopsy, obtained stable disease in the first 8weeks and later developed slowly progressive disease although additional therapy was not required for 14months. The clinical outcomes of this pilot study indicate that the Tax peptide-pulsed DC vaccine is a safe and promising immunotherapy for ATL.

Allogeneic Peripheral Blood Stem Cell Transplantation Using Reduced-Intensity Conditioning Regimen with Fludarabine and Busulfan from HLA-Matched Related Donor for Elderly Adult T-Cell Leukemia/Lymphoma: Results of Multicenter Phase II Study (ATL-NST-3)

Background: Adult T-cell leukemia-lymphoma (ATL) is a peripheral T-cell malignancy caused by human T-lymphotropic virus type 1 (HTLV-1) infection and commonly affects individuals at an average age of 67 years. Since the prognosis of aggressive ATL patients (pts) treated with conventional chemotherapy has been dismal, we had so far conducted two clinical trials to evaluate the feasibility of allogeneic peripheral blood stem cell transplantation (allo-PBSCT) from an HLA-identical sibling donor using reduced-intensity conditioning regimen (RIC) consisting of fludarabine and busulfan. We reported that allo-PBSCT using RIC provided a long-term survival in about one third of ATL pts and that it exerted not only graft-versus-ATL effects but also graft-versus-HTLV-1 effects (Okamura J, et al. Blood 2005; Tanosaki R, et al. Biol Blood Marrow Transplant 2008; Choi IL, Bone Marrow Transplant 2011). To confirm these promising results in a larger number of ATL pts, we conducted a phase II study (UMIN C000000409).Patients and Methods: Between September 2006 and July 2011, 20 pts were registered in this study. Because ATL is a rare disease affecting elderly people, their siblings were also old and were not appropriate for donors, and the enrollment of pts needed much more time than we had expected. Therefore, though we had originally planned to enroll 35 pts, we had to close this study incompletely. The conditioning regimen consisted of fludarabine 30 mg/m2/day intravenously days -8 to -3, and busulfan 3.2 mg/kg/day intravenously or 4 mg/kg/day orally days -6 and -5. GVHD prophylaxis was CsA alone from day -1 and PBSCs (CD34+ cells >2x106/kg) were infused on day 0. Primary endpoint was 2-year overall survival (OS). Engraftment, adverse events such as graft-versus-host disease (GVHD) and infectious events, and progression free survival (PFS) were also evaluated as well as donor chimerism by STR-PCR analysis, HTLV-1 proviral load, and Tax-specific cytotoxic T lymphocytes (CTL) by tetramers. The eligibility criteria at registration included acute- or lymphoma-type ATL, pts with an HLA-matched related donor, age between 50 and 70, disease status of CR, PR or NC with stable disease, no uncontrollable active infections, no major organ dysfunction, and no active CNS involvement.Results: Median age was 54 years (range 50-68), genders were 10 males and 10 females, ATL types were 13 acute and 7 lymphoma, and disease status at transplantation was 9 CR, 8 PR, and 3 NC. 13 donors were HTLV-1 positive. Engraftment as confirmed by donor chimerism analysis was achieved in all pts. OS and PFS were 95% (95%CI, 86-100%) and 60% (95%CI, 39-82%) at day 100, 74% (95%CI, 55-94%) and 45% (95%CI, 23-67%) at 1 year, and 53% (95%CI, 31-76%) and 40% (95%CI, 19-62%) at 2 years. Acute GVHD occurred in 13 pts (grade I in 2, II in 5, III in 6 pts), and chronic GVHD developed in 12 pts (63%) out of 19 evaluable pts. Eleven pts died at the time of analysis; ATL 6, GVHD 3, IP 1, PCP 1. There was no significant relationship between OS and grades of acute GVHD. While HTLV-1 proviral load decreased as early as 1 month after transplantation and became undetectable level in 14 pts (70%) out of 20, the increase of A2- or A24-restricted Tax-specific CTLs in HLA-A2 or A24-positive pts, respectively, were observed in some pts after 3 months or later, and there was no clear relationship between them. With a median follow-up of 36 months (range 24-86), 9 pts (5 CR and 4 PR at transplantation) were alive without disease.Conclusions: It was confirmed that allo-PBSCT using RIC consisting of fludarabine and busulfan was relatively safe and effective, and provided a long-term survival at least in chemo-sensitive aggressive ATL pts. DisclosuresNakamae:Novartis: Honoraria, Research Funding, Speakers Bureau, Travel/accomidations/meeting expenses Other.

Cytotoxic T lymphocyte lysis of HTLV-1 infected cells is limited by weak HBZ protein expression, but non-specifically enhanced on induction of Tax expression.

BackgroundImmunogenetic evidence indicates that cytotoxic T lymphocytes (CTLs) specific for the weak CTL antigen HBZ limit HTLV-1 proviral load in vivo, whereas there is no clear relationship between the proviral load and the frequency of CTLs specific for the immunodominant antigen Tax. In vivo, circulating HTLV-1-infected cells express HBZ mRNA in contrast, Tax expression is typically low or undetectable. To elucidate the virus-suppressing potential of CTLs targeting HBZ, we compared the ability of HBZ- and Tax-specific CTLs to lyse naturally-infected cells, by co-incubating HBZ- and Tax-specific CTL clones with primary CD4+ T cells from HLA-matched HTLV-1-infected donors. We quantified lysis of infected cells, and tested whether specific virus-induced host cell surface molecules determine the susceptibility of infected cells to CTL-mediated lysis.ResultsPrimary infected cells upregulated HLA-A*02, ICAM-1, Fas and TRAIL-R1/2 in concert with Tax expression, forming efficient targets for both HTLV-1-specific CTLs and CTLs specific for an unrelated virus. We detected expression of HBZ mRNA (spliced isoform) in both Tax-expressing and non-expressing infected cells, and the HBZ26–34 epitope was processed and presented by cells transfected with an HBZ expression plasmid. However, when coincubated with primary cells, a high-avidity HBZ-specific CTL clone killed significantly fewer infected cells than were killed by a Tax-specific CTL clone. Finally, incubation with Tax- or HBZ-specific CTLs resulted in a significant decrease in the frequency of cells expressing high levels of HLA-A*02.ConclusionsHTLV-1 gene expression in primary CD4+ T cells non-specifically increases susceptibility to CTL lysis. Despite the presence of HBZ spliced-isoform mRNA, HBZ epitope presentation by primary cells is significantly less efficient than that of Tax.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0116-6) contains supplementary material, which is available to authorized users.

WS3-2 - Pathological Features and Microenvironment of Atll

Histopathology of ATLL usually shows a pleomorphic type. Some cases show a pleomorphic small cell, or anaplastic large cell type. In addition, some patients with pre-overt ATLL show a Hodgkin's disease-like and lymph nodes in non-neoplastic carriers with features of lymphadenitis. Lymphoma cells express T-cell-associated antigens (CD2, CD3, CD5, CD4), and CD25. CCR4 of the chemokine receptor is frequently expressed, and FoxP3 of the regulatory T-cell marker also. Half of ATLL cases express HTLV-1 Tax which is a target of HTLV-1-specific cytotoxic T lymphocyte (CTL). And there was inverse correlation between Tax-specific CTL and FoxP3. In addition, the increased CD163+ tumor associated macrophages were associated with worse prognosis. Peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) with genomic aberrations has been shown to resemble ATLL in terms of its genomic aberration patterns, histopathology, phenotype and prognosis.

Excellent Quality of Lives for Previously Treated Adult T-Cell Leukemia/Lymphoma Patients After Autologous Dendritic Cell Vaccine Therapy Targeting Htlv-1 Tax Antigens

ABSTRACT Aim: Adult T-cell leukemia/lymphoma (ATL) is a human T-cell leukemia virus type-1 (HTLV-1)-infected T-cell malignancy with extremely poor prognosis due to frequent relapse. Among HTLV-1 antigens, the viral regulatory protein Tax is widely recognized as a target of cytotoxic T lymphocytes (CTLs). We investigated the safety and feasibility of Tax peptide-pulsed dendritic cell (Tax-DC) vaccine therapy administered to augment Tax-specific CTL responses in ATL patients. Methods: Three previously treated acute-type ATL patients possessing HLA-A*0201, A*2402 and/or A*1101 in stable condition were enrolled in the study. Autologous monocyte-derived dendritic cells (DCs) that exhibited the mature phenotype were pulsed with Tax peptides corresponding to the major epitopes of Tax-specific CTL previously identified from ATL patients who received stem cell transplantation. The vaccine protocol consists of three subcutaneous injections of Tax peptide-pulsed DC at 2-week intervals in a total 8-week observation period to assess adverse events. The primary endpoint of the study was to evaluate safety and feasibility of Tax-DC vaccine. Results: DC vaccine-related toxicities were grade 1 or 2 and were considered relatively mild, and non-hematological toxicity was acceptable. Two patients achieved partial remission and one obtained stable disease. The performance status improved after vaccination in all patients and two of them are currently alive without any additional chemotherapy for 17 and 12 months respectively, while the third patient developed slowly progressive disease, although additional therapy was not required until 14 months after vaccination. In all three patients, the level of HTLV-1 proviral load in the peripheral blood mostly revealed below 100 copies per 1,000 PBMCs during the observation period, and Tax-specific CTL responses were observed with a peak at 16-20 weeks. Conclusions: The good quality of lives and long-term treatment-free survival after vaccination observed in this first pilot study indicates that the Tax-DC vaccine is a safe and promising long-lasting maintenance therapy for ATL. Disclosure: All authors have declared no conflicts of interest.

HTLV-1 gene expression by individual infected clones determines susceptibility to lysis by cytotoxic T lymphocytes specific for Tax and HBZ

Host cytotoxic T-lymphocyte (CTL) responses are critical in limiting expansion of HTLV-1-infected CD4+T-cells in vivo, and most individuals generate abundant Tax-specific CTL. Although Tax is immunodominant, the ability to efficiently present peptides from HBZ on MHC class 1 is associated with a lower proviral load and a reduced frequency of HAM/TSP. HBZ mRNA is expressed in vivo, directing proliferation of infected cells. However, HBZ-specific CTL were detectable in fresh PBMCs in only 25% of chronically infected individuals. We hypothesised that HBZ has evolved to evade the generation of effective HBZ-specific CTLs. To evaluate the selective capacity of a potent HBZ-specific CTL response, we assayed the ability of equally efficient Tax-and HBZ-specific CTL clones to kill unstimulated, naturally infected cells from 16 HLA-A*02+HTLV-1+ individuals. Infected cells which expressed Tax during the course of the assay upregulated surface expression of HLA-A*02, and were eliminated efficiently by Tax-specific CTL. HBZ-specific CTL killed Tax+ cells less efficiently, preferentially killing cells with high levels of HLA-A*02. As a proportion of infected cells do not express Tax owing to silencing, mutation or viral integration site (IS) location, we tested whether HBZ-specific CTLs could kill Tax- infected cells, using high-throughput sequencing to monitor survival of infected clones after CTL selection. We are now validating our findings using patient-derived HBZ-specific CTL, and mapping HBZ epitopes recognised in vivo. In conclusion, the efficacy of HBZ-specific CTLs appears to be limited by the level of antigen presentation, but may confer the ability to target infected cells which escape Tax-specific CTL.

Combined Cytolytic Effects of a Vaccinia Virus Encoding a Single Chain Trimer of MHC-I with a Tax-Epitope and Tax-Specific CTLs on HTLV-I-Infected Cells in a Rat Model

Adult T cell leukemia (ATL) is a malignant lymphoproliferative disease caused by human T cell leukemia virus type I (HTLV-I). To develop an effective therapy against the disease, we have examined the oncolytic ability of an attenuated vaccinia virus (VV), LC16m8Δ (m8Δ), and an HTLV-I Tax-specific cytotoxic T lymphocyte (CTL) line, 4O1/C8, against an HTLV-I-infected rat T cell line, FPM1. Our results demonstrated that m8Δ was able to replicate in and lyse tumorigenic FPM1 cells but was incompetent to injure 4O1/C8 cells, suggesting the preferential cytolytic activity toward tumor cells. To further enhance the cytolysis of HTLV-I-infected cells, we modified m8Δ and obtained m8Δ/RT1AlSCTax180L, which can express a single chain trimer (SCT) of rat major histocompatibility complex class I with a Tax-epitope. Combined treatment with m8Δ/RT1AlSCTax180L and 4O1/C8 increased the cytolysis of FPM1V.EFGFP/8R cells, a CTL-resistant subclone of FPM1, compared with that using 4O1/C8 and m8Δ presenting an unrelated peptide, suggesting that the activation of 4O1/C8 by m8Δ/RT1AlSCTax180L further enhanced the killing of the tumorigenic HTLV-I-infected cells. Our results indicate that combined therapy of oncolytic VVs with SCTs and HTLV-I-specific CTLs may be effective for eradication of HTLV-I-infected cells, which evade from CTL lysis and potentially develop ATL.

The phase-I study of a therapeutic vaccine to ATL patients with autologous dendritic cells pulsed with peptides corresponding to Tax-specific CTL epitopes

In the last decade, various kinds of clinical trials of hematopoietic stem cell transplantation (HSCT) have been performed for ATL patients, and showed therapeutic effects with long-term remission in a part of recipients. Our previous finding of activation of CD8+ Tax-specific cytotoxic T-lymphocytes (CTL) in post-HSCT ATL patients indicated the presence of Tax expression in vivo and potential contributions of CTL to GVL effects. These findings let us attempt developing an anti-ATL therapeutic vaccine consisting of autologous dendritic cells (DC) pulsed with Tax peptides corresponding to the major epitopes of Tax-specific CTL previously identified from HLA-A2, A24 or A11-possessing post-HSCT ATL patients. In preliminary experiments, the DC induced with a conventional method showed matured phenotype and produced IL-12 in 2 of 3 ATL patients tested. Under the official approval by the institutional ethical committees, we conducted the phase-I clinical study of anti-ATL immunotherapy for ATL patients at stable conditions after other therapy. The peptide-pulsed DC was subcutaneously injected for three times with 2 weeks intervals. The first patient showed a significant reduction in the proviral loads in 1 week after administration of DC, which gradually increase during the treatment, then decreased again at 8 weeks after the initiation of the therapy. Reduction in the size of surface lymph nodes by CT confirmed the therapeutic effects. So far two patients completed the course of the study without severe side effects except for fever and skin rash, and their clinical outcomes were partial remission and stable disease respectively.

Augmentation of donor-derived Tax-specific CTL responses by a novel Tax epitope-specific CD4+ helper T-cells in ATL patients after allogeneic hematopoietic stem cell transplantation

Adult T-cell leukemia/ lymphoma (ATL) is an aggressive T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1) and characterized by extremely poor prognosis because of intrinsic drug resistance to cytotoxic agents. Recently, allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been shown to be an effective treatment for ATL due to graft-versus-leukemia effects. However, donor-derived HTLV-1-specific T-cell responses in ATL patients after allo-HSCT are not well understood. In this study, blood samples from 15 ATL patients at 180 days following allo-HSCT from seronegative donors were screened for IFN-g production of donor-derived T-cells against Tax protein. Among 15 patients, Tax-specific T-cell responses were observed in 11 patients. Direct detection with Tax/MHC class I tetramers revealed that donor-derived Tax-specific CD8+ cytotoxic T-lymphocytes (CTLs) were present in 12 of 15 patients. We then identified a novel Tax epitope (Tax155-167) presented by HLA-DR1 (HLA-DRB1*0101) and examined the presence of Tax-specific CD4+ T-cells in 3 HLA-DR1+ patients after allo-HSCT using a newly generated Tax155-167/HLA-DR1 tetramer. Tax155-167-specific CD4+ T-cells were found to be present in all HLA-DR1+ patients tested. Furthermore, in vitro stimulation of PBMCs from HLA-DR1+ HLA-A*2402+ patients with both Tax155-167 and HLA-A*2402-restricted CTL epitope (Tax301-309) led to robust expansion of Tax301-309-specific CTLs. Our results suggest that donor-derived HTLV-1-specific both CD4+ and CD8+ T-cells could be induced in ATL patients after allo-HSCT from seronegative donors, probably due to exposure to HTLV-1 antigens from recipient-derived ATL cells, and the HTLV-1-specific CD4+ T-cells may contribute to efficient induction of donor-derived HTLV-1-specific CTL responses.

A Therapeutic Vaccine In Adult T-Cell Leukemia Patients With Autologous Dendritic Cells Pulsed With Peptides Corresponding To Tax-Specific Cytotoxic T Lymphocyte Epitopes

Adult T-cell leukemia (ATL) is an aggressive peripheral T-cell neoplasm with extremely poor prognosis. It typically develops due to long-term infection with human T-cell leukemia virus type 1 (HTLV-1). Among HTLV-1 antigens, the viral regulatory protein Tax is widely recognized as a target of cytotoxic T lymphocytes (CTLs), and Tax-specific CTLs are often activated in ATL patients following hematopoietic stem cell transplantation (HSCT). This strongly suggests the presence of Tax expression in vivo and potential contributions of CTL to GVL effects. Based on these findings, we developed an anti-ATL therapeutic vaccine consisting of autologous dendritic cells (DC) pulsed with Tax peptides corresponding to the major epitopes of Tax-specific CTL that were previously identified from human leukocyte antigen (HLA)-A2, A24 or A11-possessing ATL patients post-HSCT (Cancer Res. 2004, J Virol. 2005). In preliminary experiments, the DC induced with a conventional method showed matured phenotype and produced interleukin (IL)-12 in two of three ATL patients tested.After approval by the institutional ethics committees, we conducted a phase I clinical trial of anti-ATL immunotherapy for pretreated ATL patients in stable condition in order to evaluate whether HTLV-1 Tax targeting immunotherapy is safe and feasible. The vaccine protocol consists of three subcutaneous injections of peptide-pulsed DC at 2-week intervals in a total 8-week observation period to assess adverse events. Three acute-type patients enrolled and completed the course of the study. Modest clinical symptoms, such as injection site reaction, fever, and skin damage, were observed in all cases. DC vaccine-related toxicities were grade 1 or 2 and were considered relatively mild, and non-hematological toxicity was acceptable. In addition, significant reduction in sIL-2R levels were observed at least in one patient, and the HTLV-1 proviral loads were maintained below 60 copies /1000 PBMCs during the monitored period after administration of DC in the all patients. Enhanced proliferative responses of Tax-specific CTLs were also observed in vitro after therapy. Clinical effects, as evaluated by RECIST, were partial remission in two patients and stable disease in one patient. Durable remissions and improvements in performance status were observed in all three patients without the requirement for new treatment for 4-10 months after treatment with Tax-targeted DC vaccination. These results indicate that Tax-specific DC vaccination is safe and well tolerated, and may be promising as a new therapeutic strategy for ATL patients. Disclosures:No relevant conflicts of interest to declare.

Functional impairment of Tax-specific but not CMV-specific CTLs in a minor population of asymptomatic HTLV-1-carriers

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a small percentage of infected individuals. ATL is often associated with general immune suppression and an impaired HTLV-1-specific T cell response. We previously found that a small fraction of asymptomatic HTLV-1-carriers (AC) also showed impaired T cell responses against the major target antigen Tax. However, it is unclear whether the impaired HTLV-1-specific T cell response in these individuals is an HTLV-1-specific phenomenon, or merely reflects general immune suppression. In this study, we investigated the function of Tax-specific cytotoxic T lymphocytes (CTLs) using tetramers consisting of major CTL epitopes and HLA-A0201, A2402, or A1101 in various HTLV-1-infected individuals possessing these HLAs. Tax-specific CTLs were detected in 33.3% (n=7/21), 100% (n=18/18), and 86.4% (n=19/22) of chronic ATL (cATL) patients, HAM/TSP patients, and AC, respectively. Tax-specific CTLs of all HAM/TSP patients tested proliferated well in culture and produced IFN-γ when stimulated with Tax peptides, while Tax-specific CTLs detected in cATL patients did not. In ACs, the Tax-specific CTL responses were retained in most cases. However, Tax-specific CTLs in one AC hardly produced IFN-γ and failed to proliferate or express a degranulation (CD107a) marker upon Tax peptide stimulation. In contrast, cytomegalovirus (CMV) pp65-specific CTLs in the same donor could be fully activated by pp65 peptide stimulation. These findings indicated that HTLV-1-specific T cell function is impaired in a limited population in an HTLV-1-specific manner during an asymptomatic stage.

Reduced Tim-3 expression on HTLV-I Tax-specific cytotoxic T lymphocytes in HTLV-I infection

T-cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3) and programmed cell death-1 (PD-1) are T-cell exhaustion molecules. We investigated the expression of Tim-3 and PD-1 in HTLV-I infection. Tim-3 expression, but not PD-1 expression, was reduced on CD4+ and CD8+ T cells of HAM/TSP patients and HTLV-I carriers as compared to healthy controls. Tim-3 expression was also reduced in HTLV-I Tax-specific cytotoxic T lymphocytes (CTLs) as compared to cytomegalovirus-specific CTLs. Tim-3+, but not PD-1+, Tax-specific CTLs produced less interferon-γ and exhibited low cytolytic activity. However, we observed no difference in the expression of Tim-3 or cytolytic activity between Tax-specific CTLs of HAM/TSP patients or carriers. Moreover, HTLV-I-infected CD4+ T cells showed decreased Tim-3 expression. The decreased expression of Tim-3 in HTLV-I infection is a marked contrast to other chronic viral infections such as HIV and HCV infection, where Tim-3 expression is increased in T cells, including the virus-specific CTLs. In HTLV-I infection, CTL response may not be negatively regulated by Tim-3. Rather, immune cells such as HTLV-I-specific CTLs may be resistant to cell death through the Tim-3/galectin-9 pathway. In summary, our data suggest that Tim-3 expression is reduced in HTLV-I infection and that a high number of Tim-3- HTLV-I-specific CTLs preserves their cytolytic activity, thereby controlling viral replication.

The roles of innate and acquired immune responses on HTLV-I infection

The level of HTLV-I expression is low in vivo. We found that recombinant interferon (IFN)-α and β suppressed viral expression in HTLV-I-infected cells. Non-lymphoid stromal cells also suppressed HTLV-I expression through type-I IFNs. The suppression was reversible after isolation of infected cells from the source of IFNs, mimicking the status of viral expression in freshly isolated ATL cells which is rapidly induced in culture. Nevertheless, HTLV-I-infected individuals maintain acquired immune responses against HTLV-I such as antibodies and cytotoxic T lymphocytes (CTLs), indicating the presence of HTLV-I proteins in vivo. Analysis on Tax-specific CTLs revealed that they were activated in HAM/TSP but unresponsive in ATL patients. We found that a subpopulation of HTLV-I carriers at asymptomatic stage exhibited impaired Tax-specific T-cell response and elevated HTLV-I proviral load. This combination is a feature of ATL and likely to be an underlying risk of ATL. Collectively, HTLV-I is doubly controlled by acquired and innate immunity; HTLV-I-specific CTLs eliminate infected cells, and IFNs suppress viral expression. Both would contribute the reduction of viral pathogenesis, while the efficiency of CTLs could be partial because of limited viral expression. An increase in viral expression would activate CTLs but also accelerate inflammation. When the viral expression is well-controlled, viral pathogenesis may not be apparent until infected cell clones with a malignant phenotype finally emerge, which may occur earlier without proper CTL responses. Diversity in innate and acquired immune responses among individuals might be important determinants of disease manifestation in HTLV-I infection.

Modulation of dendritic cell functions by HTLV-1 with DCs able to direct the presentation of Tax through infected T cells, live virus, and the Tax protein

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of a debilitating neurologic disorder, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease features a robust immune response including the oligoclonal expansion of CD8+ cytotoxic T lymphocytes (CTLs) specific for the viral oncoprotein Tax. The key pathogenic process resulting in the proliferation of CTLs and the presentation of Tax peptide remains uncharacterized. We have investigated the role of APCs, particularly dendritic cells (DCs), in priming of the anti-Tax CTL response under both in vitro and in vivo conditions. We investigated 2 routes (direct versus indirect) of Tax presentation using live virus, infected primary CD4+/CD25+ T cells, and the CD4+ T-cell line (C8166, an HTLV-1-mutated line that only expresses Tax). Our results indicated that DCs are capable of priming a pronounced Tax-specific CTL response in cell cultures consisting of naive PBLs as well as in HLA-A*0201 transgenic mice (line HHD II). DCs were able to successfully direct the presentation of Tax through infected T cells, live virus, and cell-free Tax. These observations were comparable to those made with a known stimulant of DC maturation - a combination of CD40L and IFN-γ. Our studies clearly establish a role for this important immune cell component in HTLV-1 immuno/neuropathogenesis and suggest that modulation of DC functions could be an important tool for therapeutic interventions.

Dynamics of dendritic cells and T cells in HTLV-1-associated neuroinflammatory disease: implications in immunomodulatory therapies and diagnostic tools

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), is a debilitating neurodegenerative disease characterized by a robust immune response including the oligoclonal expansion of cytotoxic T lymphocytes (CTLs) specific for the viral oncoprotein Tax. However, the underlying mechanism resulting in the disease process is currently unknown. The CTL response is affected by many factors including the efficiency of epitope processing and presentation. In this respect, dendritic cells (DCs), the most potent antigen presenting cells, have long been recognized as key regulators of the immune system. We have previously demonstrated that DCs are capable of priming a pronounced Tax-specific CTL response in naive PBLs and in HLA-A2 transgenic mice. Since DCs are such crucial cells of the immune system, an extensive assessment of their function and interaction with T cells in HAM/TSP is critical. Therefore, utilizing a newly standardized DC and pre-standardized T cell polychromatic antibody cocktails, we have investigated the immune activation of these cells in HTLV-1 infected samples from the Jamaican region including the seronegative controls, asymptomatic carriers (ACs), and HAM/TSP patients. The extensive immune cell profiling was compared to the matched proviral loads and Tax mRNA levels leading to the identification of unique signatures distinguishing ACs from HAM/TSP patients. Collectively, these studies possess great potential to enable immune cell monitoring and development of diagnostic and therapeutic strategies for the HTLV-associated neuroinflammatory disease.

Reduced Tim-3 Expression on Human T-lymphotropic Virus Type I (HTLV-I) Tax-specific Cytotoxic T Lymphocytes in HTLV-I Infection

T cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3) and programmed cell death-1 (PD-1) are T cell exhaustion molecules. We investigated the expression of Tim-3 and PD-1 in human T-lymphotropic virus type I (HTLV-I) infection. Tim-3 expression, but not PD-1 expression, was reduced on CD4(+) and CD8(+) T cells of HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients and HTLV-I carriers as compared with healthy controls. Tim-3 expression was also reduced in HTLV-I Tax-specific cytotoxic T lymphocytes (CTLs) as compared with cytomegalovirus-specific CTLs. Tim-3(+), but not PD-1(+), Tax-specific CTLs produced less interferon-γ and exhibited low cytolytic activity. However, we observed no difference in the expression of Tim-3 or cytolytic activity between Tax-specific CTLs of HAM/TSP patients or carriers. Moreover, HTLV-I-infected CD4(+) T cells showed decreased Tim-3 expression. These data suggest that Tim-3 expression is reduced in HTLV-I infection and that a high number of Tim-3(-) HTLV-I-specific CTLs preserves their cytolytic activity, thereby controlling viral replication.