Abstract There is growing evidence that CD8 + cytotoxic T lymphocyte (CTL) responses can contribute to long-term remission of many malignancies. The etiological agent of adult T-cell leukemia/lymphoma (ATL), human T lymphotropic virus type-1 (HTLV-1), contains highly immunogenic CTL epitopes, but ATL patients typically have low frequencies of cytokine-producing HTLV-1-specific CD8 + cells in the circulation. It remains unclear whether patients with ATL possess CTLs that can kill the malignant HTLV-1 infected clone. Here we used flow cytometric staining of TCRVβ and cell adhesion molecule-1 (CADM1) to identify monoclonal populations of HTLV-1-infected T cells in the peripheral blood of patients with ATL.

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Thus, we quantified the rate of CD8 +-mediated killing of the putative malignant clone in ex vivo blood samples. We observed that CD8 + cells from ATL patients were unable to lyse autologous ATL clones when tested directly ex vivo. However, short in vitro culture restored the ability of CD8 + cells to kill ex vivo ATL clones in some donors.

The capacity of CD8 + cells to lyse HTLV-1 infected cells which expressed the viral sense strand gene products was significantly enhanced after in vitro culture, and donors with an ATL clone that expressed the HTLV-1 Tax gene were most likely to make a detectable lytic CD8 + response to the ATL cells. We conclude that some patients with ATL possess functional tumour-specific CTLs which could be exploited to contribute to control of the disease. Author Summary Human T lymphotropic virus-1 infects T cells, causing them to multiply. In some people, cellular replication is unchecked, resulting in an aggressive blood cancer called adult T-cell leukemia/lymphoma. The virus proteins are efficiently recognised as ‘foreign’ by the immune system in most infected individuals. People with cancer have weak immune responses to certain viral proteins, however it was not known whether the immune system can attack the malignant cells in this disease.

In this paper, we developed a method which allows us to directly monitor malignant cells, and used it to test whether malignant and non-malignant infected cells are killed by immune cells from people with the cancer. We found that some people had immune cells which could kill the cancer cells. These observations are both new and important because they raise the possibility of boosting the immune response to malignant cells as a novel therapeutic strategy for this aggressive and hard-to-treat disease. Citation: Rowan AG, Witkover A, Melamed A, Tanaka Y, Cook LBM, Fields P, et al. (2016) T Cell Receptor Vβ Staining Identifies the Malignant Clone in Adult T cell Leukemia and Reveals Killing of Leukemia Cells by Autologous CD8 + T cells.

PLoS Pathog 12(11): e1006030. Editor: Ronald C. Desrosiers, Miller School of Medicine, UNITED STATES Received: June 8, 2016; Accepted: October 28, 2016; Published: November 28, 2016 Copyright: © 2016 Rowan et al.

This is an open access article distributed under the terms of the, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by Bloodwise (formerly Leukaemia and Lymphoma Research UK, ) (grant ref. 12038), the Wellcome Trust (CRMB Senior Investigator Award WT100291MA), the Medical Research Council (MRC, ) (MR/K019090/1), and the Imperial National Institute for Health Research Biomedical Research Centre. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist. Introduction Adult T cell leukemia/lymphoma is a mature T cell malignancy caused by the retrovirus human T lymphotropic virus-1 (HTLV-1). Four clinical subtypes exist: acute, lymphoma, chronic and smouldering, which range from highly aggressive to indolent in their clinical course ,. Advances in chemotherapy protocols have contributed only a modest increase in overall survival of aggressive subtypes, and few patients receive potentially curative allogeneic hematopoietic stem cell transplantation (HSCT). Antiviral drugs (zidovudine and interferon alpha, AZT/IFN)– and molecular targeted therapy (anti-CCR4, Mogamulizumab)– have shown promising results, especially in chronic ATL, but their efficacy in the lymphoma and acute subtypes is limited. There is an urgent need for new therapies and strategies to consolidate existing treatments. HTLV-1 establishes persistent infection by integration of the provirus into the genomic DNA of T lymphocytes, and propagates in the host by both clonal proliferation and cell-to-cell transmission,.

Expression of structural genes on the sense strand of the 9kb genome is induced by the viral transcriptional transactivator protein Tax, triggering production of viral particles, cellular activation and proliferation. The antisense strand encodes HTLV-1 b-zip protein (HBZ), which opposes many of the actions of Tax. HTLV-1 + individuals carry thousands of long-lived infected CD4 + clones in their peripheral blood, each of which has arisen from a single infection event,.

Malignant cells in ATL are HTLV-1-infected clones: in 91% of ATL cases a single dominant proviral integration site makes up over 35% of the proviral load, circulating alongside subdominant populations of polyclonal infected and uninfected T cells. Although the genomic integration site influences clonal proliferation and proviral gene expression, it does not appear to explain clonal dominance in most cases of ATL. Spontaneous mutations in the T cell receptor (TCR)/NF-kB, CCR4, p53 and, Notch-1 signalling pathways are frequently observed in malignant clones. Several lines of evidence indicate that the outcome of HTLV-1 infection is determined by the equilibrium set between proliferation of infected cells and the activity of abundant, chronically activated, HTLV-1-specific cytotoxic T lymphocytes ,.

Major histocompatibility complex (MHC) class 1 alleles HLA-A.0201 and C.08 are associated with a low proviral load in southern Japan. Tax protein is highly immunodominant in the HTLV-1-specific CD8 + response, and tax is silenced or deleted in the dominant clone in over 50% of patients with ATL, implying the presence of strong CTL selection pressure. Paradoxically, ectopic expression of Tax can be oncogenic in vivo. The region of the viral genome which encodes HBZ is highly conserved in ATL, suggesting HBZ also has a role in oncogenesis. The ability of an individual to present peptides from HBZ to CD8 + cells is associated with a low proviral load, however, HBZ evades immune detection by means of low-level protein expression and weak immunogenicity. In addition, biological actions are exerted by untranslated HBZ mRNA,.

ATL patients are commonly immunosuppressed, and frequently present with opportunistic infections. Previous studies on samples from ATL patients have reported that the frequency and diversity of HTLV-1-specific CD8 + T cells is significantly lower in ATL patients than in non-malignant HTLV-1 infection,. In addition to the silencing of Tax expression, several mechanisms by which ATL cells might escape CTL have been proposed. The malignant clone in 5%-6% of ATL patients carries mutations in HLA-A or -B genes, and the MHC class 1-encoding region in ATL is frequently subject to hypermethylation and copy-number variation.

ATL cells frequently express the regulatory T-cell-associated transcription factor FoxP3 and the coinhibitory ligand PD-L1, but it remains unclear whether primary ATL clones directly suppress CD8 + responses. Indeed, the susceptibility of primary ATL clones to CD8 +-mediated lysis is not known, though rare occurrences of spontaneous disease remission, and successful allogeneic HSCT, have been reported to involve induction and maintenance of HTLV-1-specific CTLs.

Measuring the rate at which ATL clones are killed by CD8 + cells requires a reliable method to distinguish ATL clones from both non-malignant HTLV-1 infected cells and uninfected T cells. We recently published that CADM1 expression identifies 60–70% of infected cells in HTLV-1 carriers. ATL patients have high frequencies of CADM1 +,CCR4 +, CD25 + and CD7 − cells in their peripheral blood. These cells often express FoxP3 and low levels of CD3 epsilon. However, this combination of markers is also expressed by a subset of CD4 + T cells in uninfected donors and asymptomatic HTLV-1 carriers (ACs), particularly those with a high proviral load , thus may not be used to directly identify the ATL clone. Here, we used TCRVβ subunit staining, immunophenotyping and high-throughput sequencing to identify clonally expanded populations in a well-characterised cohort of ATL patients.

We show that in some individuals with ATL, the malignant clone is susceptible to lysis by cultured autologous CD8 + cells. Autologous CD8 + cells from ATL patients preferentially killed targets that expressed the viral sense strand: both Tax +ATL clones and Tax +non-malignant cells were killed. In all donors, cells which did not express Tax escaped killing by CD8 + cells. Flow cytometric quantification of TCRVβ subunits reveals expanded clones of HTLV-1-infected cells in ATL patients Peripheral blood mononuclear cells from ATL patients, asymptomatic HTLV-1 carriers and patients with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) were stained with a panel of antibodies specific for 24 TCRVβ subunits ( and Tables) and CADM1.

The frequency distributions of TCRVβ subunits in CADM1 + (which typically carry one proviral copy per cell) and CADM1 − (low proviral load) T cells (both CD4 + and CD8 +) were ascertained by dividing live CD3 + cells into 50 possible groups on the basis of TCRVβ staining (see ). Linker-mediated PCR (LM-PCR) followed by high-throughput sequencing (HTS) were performed to corroborate the observed frequency distributions. Flow cytometric staining of TCRVβ subunits reveals clonal expansions in ATL patients. Cryopreserved PBMCs from 52 individuals (28 ATL; 11 AC; 13 HAM) were thawed and stained with a viability stain followed by antibodies specific for 24 TCRVβ subunits, CD3, CD4, CD8, CADM1, CD7, CD127, CD25 and CCR4. Proviral genomic integration sites were mapped by LM-PCR and HTS.

OCI was calculated using the Gini index, as previously described. (A) Representative data from one individual with chronic ATL, and one high PVL AC. Pie charts show the relative frequency distribution of unique integration sites (green), and CD3 + cells (TCRVβ identified: CD4 +, red; CD8 +, blue; TCRVβ ‘off panel’: CD4 +, light grey; CD8 +, dark grey). (B) OCI-flow of CADM1 +CD3 + cells versus OCI-flow of CADM1 −CD3 + cells. Statistical analysis: Kruskal-Wallis test with Dunn post-test, 95% confidence interval (CI).

denotes p0.7 is associated with ATL (see below). In ATL patients, the OCI-flow for CADM1 +CD3 + T cells measured by flow cytometry was significantly correlated with the OCI-UIS measured by HTS.

In addition, the absolute frequency of the most abundant UIS detected by HTS was significantly correlated with the frequency of the most abundant population of T cells which shared a single Vβ subunit. We therefore refer henceforth to the dominant TCRVβ-expressing population of CD4 + cells, in individuals with an OCI-flow (CADM1 +CD3 +) 0.7, as the ‘ATL clone’. We detected putatively malignant expansions in patients with chronic (n = 12) or acute (n = 6) leukemia (, and Figs); in 16 cases by direct identification of the TCRVβ and two cases in which the TCRVβ subunit was not represented in the TCRVβ antibody panel. Each case had a population of T cells which shared a Vβ subunit comprising 35% of CADM1 + cells, and an OCI-flow of CADM1 +CD3 + cells 0.7.

There was no evidence of preferential transformation of cells expressing particular TCRVβ subunits. Two out of five lymphoma patients also had CADM1 +CD3 + PBMC with an OCI-flow 0.7.

Patients with leukemic type ATL who had an OCI-flow (CADM1 +CD3 +) 35% of the PVL) detectable by HTS. Comparison with other ATL cell markers Direct flow-cytometric identification of clonal HTLV-1-infected populations in ATL permitted detailed assessment of the sensitivity and specificity of other established immunophenotypic markers of ATL. Using multicolour flow cytometry we evaluated co-expression of phenotypic ATL markers (CD3, CD25, CD7, CCR4 and CADM1) on cells which carried the respective dominant TCRVβ (designated TCRVβX +) with those which did not (TCRVβX −). As described in the literature, CD3 epsilon was significantly downregulated on expanded clones in ATL , compared with cells from the same individual which expressed other TCRVβ subunits.

CCR4 was expressed by a median of 98% cells within the malignant clone; CADM1 by 93%; and CD7 was downregulated on 96%. CD25 had the poorest sensitivity of all the markers: a median of 66% of malignant cells were CD25 +.

Expression of candidate ATL cell surface markers by the dominant TCRVβ-expressing population. Staining was performed as described in. (A) Representative flow cytometry plots of total live CD3 +CD4 + cells from an ATL patient (LHN) and an AC (HHD). Plots display the most frequently expressed TCRVβ subunit in the respective donor.

(B) Expanded clones are CCR4 +CD7 −CADM1 +. Live CD3 +CD4 + T cells from ATL patients (n = 21) with an OCI-flow (CADM1 +CD3 +) 0.7 were gated on the basis of expression of the dominant TCRVβ (designated TCRVβX + or TCRVβX –). Total live CD4 + T cells from PBMC of n = 24 individuals without malignancy (patients with HAM or ACs) were included as controls.

Whiskers represent maximum and minimum values. Statistical analysis: Kruskal-Wallis test with Dunn post-test, 95% confidence interval (CI). OCI-flow(CADM1 +CD3 +) 0.7 indicates a high probability of clinically evident ATL Within our cohort of 24 age-matched HTLV-1-infected individuals without malignancy (in whom the PVL ranged from undetectable to 79 copies per 100 PBMC, ), the OCI-flow of CADM1 +CD3 + cells did not exceed 0.7. We plotted receiver operator curves (ROC) to evaluate the sensitivity and specificity by which the OCI-flow of CADM1 +CD3 + cells could identify individuals with clinically evident ATL , compared with the common diagnostic investigations: enumerating CD7 −CD4 + cells and CD25 +CD4 + cells. Five ATL patients within the original cohort who were in clinical remission were excluded from this analysis on the basis of clinical observations (not on the basis of oligoclonality). Area under the curve (AUC) analysis rated the diagnostic power of the OCI-flow (CADM1 +CD3 +)and CD7 −CD4 + frequency as ‘excellent’ (AUC 0.9–1), and CD25 +CD4 + frequency as ‘good’ (AUC 0.8–0.9), and both tests had significantly higher diagnostic power than the frequency of CD25 +CD4 + T cells (, p = 0.001, OCI-flow (CADM1 +CD3 +) vs. CD25; p = 0.03, CD7 vs.

CD25, one-tailed test ). OCI-flow of CADM1 +CD3 + cells is an excellent diagnostic test for monoclonal integration. Receiver operator curves illustrating the specificity and sensitivity by which the OCI-flow of CADM1 +CD3 + cells, the frequency of CD7 −CD4 + or the frequency of CD25 +CD4 + cells discriminate individuals with clinically evident ATL (n = 23) from individuals with non-malignant HTLV-1 infection (n = 24). Individuals previously diagnosed with ATL which were in clinical remission were excluded from this analysis. ATL clones express MHC class 1 and high levels of CADM1 In 16 individuals with a known dominant TCRVβ, all T cells (including ATL clones) expressed MHC class 1 at a similar intensity ( and ). CADM1 expression was significantly higher on ATL clones than on non-malignant infected cells within the same individual, or CADM1 + cells from ACs ( and ).

In polyclonal infected populations (CD4 +CADM1 + cells in ACs, CADM1 +VβX −cells in ATL patients), a median of 11–15% of CADM1 + cells expressed Tax after overnight culture (; and ). By contrast, ATL clones fell into two distinct groups: those in which 5% expressed Tax (Tax highATL). FoxP3 and PD-L1 were highly expressed in some cases, by both Tax high and Tax low ATL clones. Expression HLA-ABC, CADM1 and Tax by CADM1 +CD4 + T cells. CD8 + cells were depleted from PBMCs of 15 ATL patients with a dominant ATL clone detectable by TCRVβ staining, and 10 ACs. Cells were cultured for 18h, after which they were surface stained with a viability stain followed by antibodies specific for the most frequently utilised TCRVβ (VβX), CD3, CD4, CD8, CADM1, PD-L1, FoxP3 and HLA-ABC (; panels 3, 4, 6 and 7). Cells were then permeabilised and stained with antibodies specific for Tax and FoxP3 and analysed by flow cytometry.

Cells from ATL patients and ACs were gated on live CD3 +CD4 +CADM1 + cells which were positive or negative for the dominant TCRVβX as indicated. Intensity of expression of (A) MHC class 1 and (B) CADM1. (C) Frequency of Tax expression by ATL clones. Tax high ATL clones are plotted in red, and Tax low ATL clones are plotted in blue.

Statistical analysis: Kruskal-Wallis test with Dunn post-test, 95% CI. Patient-derived CD8 + cells do not efficiently lyse ATL clones ex vivo We tested the ability of autologous CD8 + cells to kill malignant clones using an ex vivo cell survival assay. In order to mimic in vivo CD8 + cell:target cell frequencies, we incubated CD4 + PBMCs from ATL patients for 18h with a range of ratios of autologous CD8 + T cells and quantified the absolute number of surviving cells in the following populations: CADM1 −CD4 + cells (which have a low proviral load ), malignant HTLV-1 infected CADM1 +VβX +CD4 + cells, and non-malignant CADM1 +VβX −CD4 + cells , which typically carry a single proviral copy per cell. This strategy permitted us to estimate the efficacy by which each subset was targeted by CD8 + cells in vivo, in the presence of other potential CTL targets.

Because previous reports indicated that ex vivo CD8 + cells from ATL patients had negligible lytic function , purified CD8 + cells were expanded in culture for 2 weeks, both to increase the effector: target ratio, and to allow potential reactivation of lytic function. Experimental design of cell survival assay.

PBMCs from each of 9 ATL patients with a dominant ATL clone detectable by TCRVβ staining were depleted of CD8 + T cells. The CD8 − PBMCs (CADM1 +CD4 +, purple; CADM1 −CD4 +, yellow) were cultured overnight either alone or in the presence of autologous CD8 + cells at a range of ratios, after which cells were stained with a viability stain and antibodies specific for CD3, CD4, CD8, CADM1 and the TCRVβ subunit which was most frequently used in that individual (‘TCRVβX’). Cells were then permeabilised, stained intracellularly with anti-Tax antibody, and analysed by flow cytometry. Absolute cell counts of CD3 +, CD4 + and CD8 + cells were performed in parallel. At the effector: target ratios tested, no significant lysis of the ATL clone by autologous ex vivo CD8 + T cells was detected. When compared with ACs, CD8 + from ATL patients also had a markedly reduced ability to lyse non-malignant Tax-expressing CADM1 +CD4 + cells. Tax −CADM1 + and CADM1 − CD4 + cells were not killed by ex-vivo CD8 + cells in either cohort.

By contrast, after expansion in vitro, cultured CD8 + cells from 3 of 9 donors with ATL killed a proportion of their respective ATL clone. Addition of 20nM concanamycin A blocked killing of ATL cells, indicating that the observed effect is perforin-dependent , as previously reported. Cultured CD8 + cells can kill autologous malignant cells in some donors. CD8 depleted PBMCs from ATL patients (n = 9) with a known dominant TCRVβ were incubated in the presence of ex vivo (A) or cultured (B) autologous CD8 + cells at the indicated E:T ratios.

After 18h, the absolute number of viable ATL cells (live CD3 +CD4 +CADM1 +TCRVβ +) was quantified by flow cytometry and used to calculate the proportion of the ATL clone which had been specifically killed in the presence of CD8 + cells. The proportion of Tax + and Tax −CADM1 + TCRVβ − cells which were killed was calculated in the same manner.

(C) Ex vivo CD8 + cell killing of Tax +CD3 +CD4 +CADM1 + cells, Tax −CD3 +CD4 +CADM1 + cells and CD3 +CD4 +CADM1 − cells in ACs (n = 10). Subsets of cells which expressed Tax (in both ATL and ACs) are plotted in red. Efficiency of CTL selection: preferential lysis of Tax high clones We observed that the ATL clone was not completely eliminated at any CD4 +:CD8 + ratio, even supraphysiological ratios. All donors (3/3) whose CD8 + cells regained the ability to lyse the malignant cells had an ATL clone which strongly expressed the proviral sense strand genes, as detected by intracellular expression of Tax protein (Tax high). In contrast, the malignant clones of all other donors in the cohort (6/6) were Tax low. Within the ATL clone, we observed a strong preferential lysis of Tax-expressing malignant cells; only one donor lysed Tax-negative malignant cells. Between 20–60% of malignant Tax expressing cells were cleared in each donor.

To quantify the preferential CD8 + targeting of cells that express the viral plus strand, we calculated for each donor the rate at which Tax-expressing and non-expressing cells were killed after in vitro culture. Cultured CD8 + cells killed Tax-expressing ATL clones at a higher rate than ex vivo CD8 + cells in 3 of 3 cases. In addition, cultured CD8 + cells from patients with Tax low clones also had enhanced ability to kill non-malignant HTLV-1 infected cells which expressed the viral plus strand. Tax-expressing cells are preferentially killed by cultured autologous CD8 + cells. (A) Selective loss of live Tax-expressing ATL cells after incubation with cultured CD8 + cells. Extended analysis of data from.

ATL clones from three Tax high ATL patients (LGZ, LGC and LGB) were gated on the basis of Tax expression as shown. For each donor, graphs show the percentage of Tax + or Tax −cells which were killed in the presence of cultured autologous CD8 + cells. Flow plots show Tax and CADM1 expression by live CD3 +CD4 +CADM1 +TCRVβX + cells from each individual after culture alone or in the presence of CD8 + cells at the highest E:T ratio tested. (B) Comparison of the rate at which ex vivo and cultured CD8 + cells supress survival of the populations indicated in ATL patients (n = 9) and ACs (n = 10). Subsets of cells which expressed Tax (in both ATL and ACs) are plotted in red. Data from was analysed by nonlinear regression to estimate the% change observed in each population with each 1% increase in CD8 + cells present in the co-culture.

A negative rate indicates that number of viable target cells recovered from the co-culture was greater in the presence of CD8 + cells versus in the absence of CD8 + cells. Statistical analysis (CADM1 +TCRVβ − groups only): Wilcoxon matched pairs test, two tailed, 95% confidence interval. Discussion An array of novel anti-cancer immune therapies are currently in clinical trials, which potentiate existing immune responses, and induce tumour-specific immunity by vaccination, or infusion of engineered tumour-specific T cells. Might these approaches be effective in ATL? We demonstrate that abnormal clonal expansions of HTLV-1-infected T cells are readily detectable in individuals with ATL by TCRVβ flow cytometry, which is faster, cheaper and less labour-intensive than the current gold-standard technique of high-throughput sequencing of proviral integration sites. In this cohort, an oligoclonality index of 0.7 within CADM1 +CD3 +cells reliably identified individuals who had a dominant ATL clone as validated by high-throughput sequencing.

Whilst CD25 expression is frequently high in ATL, we show that in most individuals, 40% of cells in the ATL clone are CD25 negative. Over 94% of ATL clones were CCR4 +CADM1 +CD7 −; the exceptions were one CD7 dim ATL clone and one CADM1 − clone. Analysis of TCRVβ expression within the heavily infected CADM1 +CD3 + population allows direct flow cytometric analysis of the clonal structure of HTLV-1 infected cells, and can distinguish ATL patients from age-matched HTLV-1 carriers with high specificity and sensitivity.

Whilst we did not have sufficient cases to independently test the diagnostic power of OCI-flow of CADM1 +CD3 + cells in an unrelated cohort of cases and controls, our observations indicate that this measure could be useful in the diagnosis of ATL: particularly in detecting the presence of monoclonal/oligoclonal populations of HTLV-1 infected cells. We exploited this technique to measure the rate at which ATL clones are lysed by ex vivo, autologous CTLs. Ex vivo CD8 + cells were unable to kill autologous malignant ATL cells, even at supraphysiological E:T ratios. In addition, CD8 + mediated killing of non-malignant cells which express viral proteins was less efficient in ATL patients than in asymptomatic carriers. In certain patients, in vitro culture of CD8 + cells revealed a population of CD8 + cells which could kill the ATL clone.

This ability was associated with expression of the viral plus-strand genes by the ATL clone: Tax highATL clones and Tax expressing non-malignant infected cells were preferentially targeted by cultured autologous CD8 + cells. In most subjects in the present study, Tax was expressed on.

Proviral load estimation and mapping of proviral integration sites Genomic DNA was extracted using a DNeasy kit (Qiagen, Manchester), according to the manufacturer’s instructions, and proviral load was estimated as described in Manivannan et al, 2016. Genomic DNA (20 ng, 6.7 ng or 2.2 ng in 4 μl H 2O) was subjected to thermal cycling in the presence of FastSYBR (Life Technologies) master mix and the following primer pairs: SK43/SK44- 5'CGGATACCCAGTCTACGTGT3' /5'GAGCCGATAACGCGTCCATCG3' ( tax gene) or GAPDHF/GAPDHR- 5’AACAGCGACACCCATCCTC3’/5’ CATACCAGGAAATGAGCTTGACAA3’ ( gapdh gene). DNA amplification was monitored in real time with a QuantStudio7 thermal cycler (Life technologies). DNA from a naturally-infected primary T cell clone which contained a single-copy of tax and two copies of gapdh as used as a standard. The proportion of PBMC which carry the provirus was estimated as follows: (copies of tax)/(2.copy number of gapdh).100. Where 1 copy of Tax is detected per 2 copies of GAPDH, the value exceeds 100%. Linker-mediated (LM)-PCR, high-throughput sequencing, data extraction and analysis of viral integration sites were carried out as described in Gillet et al.

Random fragments of genomic DNA (1 μg) generated by sonication were ligated to a partially double-stranded DNA adaptor. Nested PCR (two rounds) was used to amplify the region between the HTLV-1 LTR and the adaptor. Amplicons generated from adaptors with unique 6bp barcodes were combined into libraries; following which, sequence data from paired-end 50 bp reads and a 6 bp index (barcode) read were acquired on an Illumina HiSeq/MiSeq platform. Paired reads were then aligned to a human genome reference (Hg18). The number of individual cells which were sequenced within a given HTLV-1 infected clone were estimated by quantifying the number of distinct genomic shear sites generated by sonication (read2) for each paired unique integration site (junction between the provirus and human genome- read 1), and correcting to a calibration curve. The absolute abundance of unique integration sites per 100 PBMC was estimated by combining the proviral load and relative abundance of each clone. The oligoclonality index (OCI) was used as a metric to compare the clone frequency distribution between samples.

This was based directly on the Gini index , which calculates the relative inequality within a given distribution. The OCI was computed using the reldist package in R. Values range between 0 and 1, with 0 indicating that all clones make up an equal proportion of the load, and 1 indicating that a single clone dominates completely. Flow cytometric analysis Flow cytometric staining was performed as previously described using panels of antibodies and stains outlined in. Cells (3x 10 5-2x10 6) were stained for 5 min with 1 μl/ml fixable Live/Dead blue viability stain (Life technologies).

After incubation cells were washed once with FACS buffer (PBS containing 7% normal goat serum). Surface molecules were stained for 20 min at room temperature (RT) with the antibodies listed in. In order to quantify the frequency of T cells utilising each TCRVβ subunit, eight PBMC samples were stained with three anti-TCRVβ antibodies in parallel using the Beckman Coulter IOTest Beta mark kit. For the CD8 + killing experiments, PBMC were stained with an anti-TCRVβ antibody specific for the subunit most frequently utilised in that donor. Biotinylated antibodies were detected by staining with streptavidin-PeCy7 or -BV421 (Biolegend), 10 min at RT in FACS buffer. To stain intracellular antigens, cells were fixed and permeabilised using FoxP3 staining buffers (eBioscience, SanDiego), and stained with anti-Tax AF488 or anti-FoxP3 for 25 min at RT.

Cells which were surface stained only were fixed with 2% paraformaldehyde in PBS for 20 min at RT. Data was acquired using a BD LSRFortessa, and analysed using Kaluza software. Gating strategy is outlined in and Figs.

Analysis of TCRVβ oligoclonality The frequency of live CADM1 +CD4 +CD3 + cells which bound each anti-TCRVβ antibody was expressed as a percentage of total live CD3 + T cells. In order to estimate the frequency of T cells expressing Vβ subunits which were not recognised by antibodies in the panel, the sum of all positively identified TCRVβ subunits was subtracted from the total frequency of live CADM1 +CD4 + within CD3 + cells.

The frequencies of TCRVβ-expressing live CADM1 +CD8 +CD3 + cells were also calculated in the same manner, as CADM1 +CD8 + cells are also heavily infected with HTLV-1. The resulting 50 frequencies (including instances where a particular population was undetectable within CD3 + cells), were used to compute the oligoclonality index as described for the proviral integration site data. To avoid introducing sampling error in the case of low PVL (and thus low frequencies of CADM1 + cells) flow cytometric data from donors for which. Cell survival assay CD8 + cells were isolated from cryopreserved PBMC by positive selection using magnetic beads (Miltenyi Biotech) following the manufacturer’s protocol. The CD8 + fraction was placed in culture at 5 x10 5 cells/ml for 13 days in the presence of 1 μg/ml phytohemagluttinin-L (Sigma Aldrich) and 100 IU/ml IL-2 (Promocell).

At three day intervals, 50% of the culture medium was replaced and supplemented with 100 IU/ml IL-2. Cells were split as required. Flow cytometric analysis indicated that the mean frequency of live CD8 + cells in each culture was 99.6%; with a mean residual contamination of on average 0.28% of the ATL clone from that donor. On day 13, CD8 + cells were depleted from a second vial of cryopreserved PBMC. Cultured or freshly isolated ex-vivo CD8 + cells were added to between 3x10 5 and 5x10 5 CD8 − PBMCs at a range of effector: target (E:T) ratios in duplicate: CD8-depleted), the natural CD8 +:CD4 + ratio (median 1:23), 1:4 and 1:2 as permitted by the number of CD8 + cells recovered.

As significantly greater numbers of cultured CD8 + cells were recovered (versus ex vivo CD8 + cells) ratios of 1:1, 2:1 and 4:1 were tested where possible. Cells were co-cultured 1ml RPMI containing 10% FCS, 2 mM L-glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin (Gibco) and 20 μg/ml DNAse (Sigma).

After 18h, a 100 μl sample of each culture was harvested in order to count the absolute numbers of CD3 +, CD4 + and CD8 + cells present. This was performed by adding 50 μl of an antibody master mix containing 1 μl anti CD8 AF700, 0.5 μl anti-CD3 BV510 and 0.25 μl anti-CD4 BV605 to each sample. Samples were incubated at RT for 30 min, after which 150 μl 2% paraformaldehyde in PBS was added, without any centrifugation/washing steps. Prior to flow cytometric analysis, 10 μl of CountBright absolute counting beads (Life Technologies) were added to each tube.

The number of cells surviving was calculated as follows: # cells in tube = (# cells collected / # beads collected) × total # beads added to the tube. The remaining portion of each sample was analysed by flow cytometry as described above using the panel of antibodies outlined in. Estimation of the rate of lysis of target populations The rate at which cells in a given subpopulation of cells were cleared (% target cells killed/%CD8/day) was estimated in each subject as described in Asquith et al using the following equation: dy/dt = c- εyz; where y is the percentage of targets within total CD4 + cells, c is the rate of antigen presentation (assumed to be constant during the short-term culture), ε is the CD8 + cell-mediated lytic efficiency, and z is the proportion of CD3 + cells that are CD8 +.

This model was solved analytically and fitted to the data using nonlinear least-squares regression (SPSS v22). Acknowledgments The authors thank the blood donors who contributed to this study and the clinical and research staff at the HTLV-1 clinic in St Mary’s Hospital. We thank the St Mary’s flow cytometry core facility, Yanping Guo and Malte Paulsen; Laurence Game and Marian Dore of the Genomics Laboratory, MRC Clinical Sciences Centre, Hammersmith Hospital, London; and the High Performance Computing service staff at Imperial College. Many thanks also to Maria-Antonietta Demontis, Huseini Kagdi, Silva Hilburn, Kiruthika Manivannan, Becca Asquith, Masao Matsuoka, Yorifumi Satou and Kenji Sugata for advice and helpful discussions. References. 1. Diagnostic criteria and classification of clinical subtypes of adult T-cell leukaemia-lymphoma.

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For example, Japanese unexamined Patent Publication No. 11-212934 discloses a technique for having a creature that is raised in a virtual space perform a predetermined action by inputting a command via an input device such as a mouse or a keyboard.

According to the technique, a user takes care of a virtual pet using a computer. Specifically, the user feeds the pet, lays the pet down, praises the pet, reproves the pet or plays with the pet in a similar way to taking care of a real pet by using a computer. The pet is raised by the user as described above and the user can experience how to raise pet with confirming growth of the virtual pet via images and voices output from a display or a speaker. It is also possible to remotely raise the pet via a network. As a method for matching an output timing of voices of life with an output timing of images thereof, there is proposed a method disclosed in European Patent No.

0860811 in which the voices are synchronized with the images for output and a method disclosed in Japanese Unexamined Patent Publication No. 10-293860 in which the images are synchronized with the voices for output.

Above method enables production of animation and output of the voices at the same time with the animation; therefore, the user can realistically recognize the output images and voices. As a method for producing animation based on actual film images, there is proposed an animation synthesis technique by way of recognition of actual film images (P.98-106, December 1998, NTT Technical Journal). According to the technique, a portrait is automatically made by a picture and expressions of different opening states of eyes and a mouth and expressions of various emotions are automatically made based on the portrait. Then, the portrait is synchronized with a voice so that portrait animation can be synthesized. In the above-described technique disclosed in Japanese Unexamined Patent Publication No. 11-212934, the user can remotely communicate with the virtual pet via the network. In the conventional technique, however, the virtual pet is controlled by commands from the user that are input via the input device so as to be displayed on the display; therefore, the user can communicate with the virtual pet only in limited patterns.

For example, the technique does not allow conversation between the user and the virtual pet; therefore, realistic communication cannot be achieved by the technique. However, since the images have a large amount of data, a communication line having large capacity for communication is required in order to transmit and receive the images. In the case of transmission and reception of images via a general telephone line, it is impossible to send and receive more than a few frames as an image per second and, therefore, it is impossible to display a satisfactorily animated image. In turn, the usage of a high-speed private line enables display of animated images wherein a motion appears substantially natural, however, it has not been widely prevalent yet due to high communication cost.

In order to reduce communications traffic, there has been proposed a method in which images of a part of and whole parts of a face are previously produced at low resolution for registration in a database, and then the whole facial image is displayed on a screen of a receiver's terminal device at the start of a conversation and only a part of the facial image corresponding to a part in which expressions have changed is downloaded from the database to the terminal device so as to be displayed in Japanese Unexamined Patent Publication No. As shown in FIG. 2, the magnetic disk unit 0058 27 in the client 2 stores an OS 2 s as a basic program of the client 2, a client conversation program 2 p as an application program of the client in the communication system 1, data 2 d required therefor and the like.

The client conversation program 2 p serves to carry out a basic operation process 2 bs and other processes. The basic operation process 2 bs is a process for performing linkage with the OS 2 s, operations relative to a selection of a person HMN and input of the first message MG 1. The programs and data are loaded into the RAM 21 b as required so as to be executed by the CPU 21 a. The animation engine EG 0063 2 carries out a motion control process 3 ds and an animation generating process 3 an. Motion control data DSD are generated by the motion control process 3 ds. The motion control data DSD are control information for controlling facial image data FGD of the person HMN in such a manner that the facial image data FGD of the person HMN move in accordance with a timing of output of the second message MG 2 from the speaker 22 b or the display 22 a.

The animation generating process 3 an is a process for generating the facial animation data FAD based on the motion control data DSD and the facial image data FGD. The facial image data FGD are data represented by a structured three-dimensional model of a head of a person HMN wherein components such as a mouth, eyes, a nose and ears, skin, muscle and skeleton can move (See FIG. The persons HMN may be various actual or fictional humans, for example, celebrities such as actors, singers, other artists or stars, sport-players and politicians, ancestors of the user and historical figures. It is also possible to use animals, dolls or characters of cartoons. Generally, a skin model is used as a three-dimensional model. Muscle and skeleton may be added to the skin model to generate a three-dimensional model.

In the three-dimensional model with the muscle and the skeleton, motion of a person can be expressed more realistically by manipulating the construction points or the control points in the muscle or the skeleton. The data of the three-dimensional model mentioned above are the facial image data FGD. A list LST described below is prepared with respect to the facial image data FGD accumulated in the facial image database FDB. The each facial image data FGD can be specified by a person number NUM or the like in the list LST. As shown in FIG.

5, the list LST is a database for storing information of a plurality of persons HMN who can be persons with whom the user converses. The list LST includes a plurality of fields, for example, the person number NUM for discriminating each of the persons HMN, a person name NAM as a name of the person corresponding to the person number NUM and a sample image SMP indicating an example of a facial image. The list LST stores data concerning the persons HMN such as a person HMN 0077 1 and a person HMN 2. Turning to FIG. 7, the received first message MG 0093 1 is recognized in the server 3 (# 31).

In the case where the first message MG 1 comprises character data TXT 1, it is unnecessary to perform a language recognition process 3 gn. If the first message MG 1 comprises voice data SND 1, the language recognition process 3 gn is performed by using a language recognition conversation engine EGI so as to generate character data TXTa. If, however, the first message MG 1 is not received yet as shown in the step # 13, or if the conversation is interrupted for a predetermined period of time, the step # 31 is omitted. The character data TXT 0095 2 are generated with reference to the character data TXTa or TXT 1, sentence information BNJ and word information TNJ.

In the case where the character data TXTa or TXT 1 are ‘How are you?’, for example, sentence information BNJ having possibilities that the person HMN responds to the question is extracted from a conversation database KDB with reference to the person information HMJ so as to apply the word information TNJ to the sentence information BNJ. Thus, the character data TXT 2 such as ‘Fine, thank you.

How about yourself?’ or ‘OK, but I am a little bit tired. Are you all right?’ are generated.

Conversion from the character data TXT 0096 2 to the voice data SND 2 is performed by using known techniques. However, if the first message MG 1 is not received yet as shown in the step # 13, or if the conversation is interrupted for a prejudged period of time, character data TXT 2 having possibilities that the person HMN talks to the user are generated with reference to the person information HMJ, the sentence information BNJ, and the word information TNJ in the step # 32. Such character data TXT 2 include ‘Hello.’ or ‘Is everything OK with you?’. As described above, according to the first embodiment, facial animation data FAD are generated by a server 0101 3 so as to be transmitted to a client 2. Since the client 2 have only to receive and display the generated data, burden accompanying the data processing is relatively small. Accordingly, even if the client 2 has difficulties with production of animation due to low performance or low specifications thereof, it is possible to perform a conversation with a person HMN by using the client 2. Specifically, in the first embodiment, facial image data FGD extracted from a facial image database FDB in the server 0104 3B are temporarily stored in a RAM 31B or the magnetic disk unit 37 in the server 3B.

In turn, in the second embodiment, the facial image data FGD are transmitted to the client 2B so as to be temporarily stored in a RAM 21 b or the magnetic disk unit 27 in the client 2B. Then, facial animation data FAD are generated in the client 2B based on motion control data DSD transmitted from the server 3B. As shown in FIG. 11, in the client 0111 2B, a person HMN with whom a user converses is selected from a list LST (# 41). A person number NUM of the selected person HMN is sent to the server 3B at this point.

After reception of the person number NUM, the server 3B reads facial image data FGD corresponding to the person number NUM from the facial image database FDB so as to transmit the facial image data FGD to the client 2B (# 42). Such preprocesses for performing a conversation are automatically carried out as background processes. A whole structure of a communication system 0121 1C according to a third embodiment is the same as in the second embodiment. Accordingly, FIG. 1 is also applied to the third embodiment.

Contents of programs memorized in magnetic disk units 27 and 37 are substantially the same as those of the second embodiment shown in FIGS. However, since data stored in the magnetic disk units 27 and 37 that are provided in a client 2C and a server 3C are different from those of the second embodiment, contents processed by the client 2C and the server 3C are somewhat different. Other two embodiments of a communication system will be described. In communication systems 0138 1D and 1E according to the two embodiments, a conversation is performed with watching facial animation that is a partner's avatar (substitute) instead of an actual facial image of the partner of conversation.

Although a personal computer is used as a terminal device in the communication systems 1D and 1E, other communication equipment such as a telephone, a portable phone, mobile devices can be used as the terminal device. The communication controller 0146 24 controls transmission and reception of facial image data FGD as three-dimensional shape data of a face, motion control data DSD used for controlling the facial image data FGD in such a manner that the facial image data FGD move in accordance with a timing of the output of the voice, voice data SND obtained by digital conversion of voice and other data.

The CD-ROM drive 25, the floppy disk drive 26 and the magnetic disk unit 27 all stores data and a program. The host communication program 0155 3Dp includes programs such as a basic process program 3 pk, a motion control program 3 pd and a language translating program 3 py or a module.

The basic process program 3 pk performs linkage with the OS 3 s, supervises and controls an animation engine EM 2 and a language translation engine EM 3. The motion control program 3 pd generates the motion control data DSD based on the voice data SND. The motion control data DSD are control information used for controlling the facial image data FGD in such a manner that the facial image data FGD move in accordance with a timing of the output of the voice based on the voice data SND. The language translating program 3 py is used for translation from voice data SND of a natural language to voice data SND of another natural language. First, communication between the terminal devices 0163 5D and 6D is established (# 110).

In order to establish the communication, for example, a request for connection with the terminal device 6D is sent from the terminal device 5D to the host computer 3D. The host computer 3D notifies the terminal device 6D that a connection request is sent from the terminal device 5D. In the case where the connection is permitted, the terminal device 6D performs notification indicating the permission. Various known protocols can also be used for the communication. After establishment of the communication, the host computer 0164 3D transmits partner's facial image data FGD to the terminal devices 5D and 6D as shown in FIG. Specifically, facial image data FGD selected by the user of the terminal device 6D are sent to the terminal device 5D, and facial image data FGD selected by the user of the terminal device 5D are transmitted to the terminal device 6D. Each of the users selects facial image data FGD according to the user's preference from the facial image database FDB or a database wherein facial image data FGD for each user are previously registered.

In the selection, a list of selectable facial image data FGD may be displayed on a display of the each user, or the user may designate facial image data FGD the user like by specifying number or the like. Alternatively, one facial image data FGD previously designated by the each user may be transmitted.

The host computer 0167 3D judges whether translation is required in the conversation in accordance with designation of languages sent from the terminal devices 5D and 6D (# 113). When a language used by one user for speaking is different from a language used by the other user for listening, the host computer 3D judges that translation is required. In the case where there is no designation of languages, the host computer 3D judges that a specific language, for example, Japanese is used in the conversation. When the user of the terminal device 0174 5D says ‘Good morning’, the host computer 3D generates motion control data DSD for giving motion of ‘Good morning’ to a mouth of the facial image data FGD, and the generated motion control data DSD are transmitted to the terminal device 6D. In the terminal device 6D, a voice of ‘Good morning’ that is given by the user of the terminal device 5D is output from the speaker 22 b. The display 22 a displays the facial image data FGD of the user in the terminal device 5D and the mouth thereof opens and closes in connection with a voice of ‘Good morning’.

Additionally, the host computer 0175 3D analyzes emotions of the user in the terminal device 5D based on a tone of ‘Good morning’. For example, in the case where the host computer 3D analyzes that the user in the terminal device 5D has a congenial atmosphere, the host computer 3D generates motion control data DSD for moving eyes and a whole face of the facial image data FGD to cause the eyes and the whole face of the facial image data FGD to smile and then transmits the generated motion control data DSD to the terminal device 6D. Thus, the display 22 a in the terminal device 6D displays animation wherein the user in the terminal device 5D says ‘Good morning’ with smiling. As shown in FIG. 19, the host computer 0179 3D sends the facial image data FGD of the user as the receiver to the respective terminal devices 5D and 6D (# 131). In the case where the voice data SND are received from the terminal devices 5d and 6D (# 132), the host computer 3D carries out translation if required (# 133 and # 134) and generates the motion control data DSD are generated (# 135) followed by transmitting the motion control data DSD and the voice data SND to the respective user's terminal devices (# 136).

Further, communication can be performed among three or more terminal devices. In this case, facial image data FGD of all other users are transmitted to respective users.

Voice of each of the users is transmitted to the terminal devices of the all other users along with motion control data DSD based on the voice. In each of the terminal devices, only animation corresponding to the talking user may be selected from animation based on the received plural facial image data FGD to be displayed. Alternatively, animation of the all users may be simultaneously displayed or may be switched to be displayed one by one. Specifically, in the fourth embodiment, the facial image database FDB, the motion control program 0194 3 pd and the animation engine EM 2 are provided in the host computer 3D.

In the fifth embodiment, however, a facial image database FDB, a motion control program 5 pd and an animation engine EM 2 are provided in each of the terminal devices 5E and 6E, as shown in FIG. Therefore, the facial image database FDB, the motion control program 5 pd and the animation engine EM 2 are not provided in the host computer 3E, as shown in FIG. In order to start a conversation, a judgment is made as to whether translation is required (# 0197 142). The judgment is performed by the host computer 3D in the fourth embodiment, while the judgment is made by the terminal devices 5E and 6E in the fifth embodiment. For example, in the step # 141, each of the users of the terminal devices 5E and 6E sends information of language he/she uses to the other user together with the facial image data FGD. Each of the terminal devices of the users as receivers judges whether translation is required or not based on the received information. In the fourth and fifth embodiments described above, facial image data FGD are previously obtained in the each terminal device in order to start a conversation, and animation is generated based on the motion control data DSD during the conversation.

Thus, communications traffic can be reduced and animation expressing a natural motion can be displayed. Further, even if users uses different languages, it is possible to perform a conversation with watching each partner's animation wherein a motion is smooth and substantially natural. Transmitting the generated voice signal to another terminal device.