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Background: \ud The SARS-CoV-2 delta (B.1.617.2) variant was first detected in England in March, 2021. It has since rapidly become the predominant lineage, owing to high transmissibility. It is suspected that the delta variant is associated with more severe disease than the previously dominant alpha (B.1.1.7) variant. We aimed to characterise the severity of the delta variant compared with the alpha variant by determining the relative risk of hospital attendance outcomes.\ud \ud Methods: \ud This cohort study was done among all patients with COVID-19 in England between March 29 and May 23, 2021, who were identified as being infected with either the alpha or delta SARS-CoV-2 variant through whole-genome sequencing. Individual-level data on these patients were linked to routine health-care datasets on vaccination, emergency care attendance, hospital admission, and mortality (data from Public Health England's Second Generation Surveillance System and COVID-19-associated deaths dataset; the National Immunisation Management System; and NHS Digital Secondary Uses Services and Emergency Care Data Set). The risk for hospital admission and emergency care attendance were compared between patients with sequencing-confirmed delta and alpha variants for the whole cohort and by vaccination status subgroups. Stratified Cox regression was used to adjust for age, sex, ethnicity, deprivation, recent international travel, area of residence, calendar week, and vaccination status.\ud \ud Findings: \ud Individual-level data on 43 338 COVID-19-positive patients (8682 with the delta variant, 34 656 with the alpha variant; median age 31 years [IQR 17–43]) were included in our analysis. 196 (2·3%) patients with the delta variant versus 764 (2·2%) patients with the alpha variant were admitted to hospital within 14 days after the specimen was taken (adjusted hazard ratio [HR] 2·26 [95% CI 1·32–3·89]). 498 (5·7%) patients with the delta variant versus 1448 (4·2%) patients with the alpha variant were admitted to hospital or attended emergency care within 14 days (adjusted HR 1·45 [1·08–1·95]). Most patients were unvaccinated (32 078 [74·0%] across both groups). The HRs for vaccinated patients with the delta variant versus the alpha variant (adjusted HR for hospital admission 1·94 [95% CI 0·47–8·05] and for hospital admission or emergency care attendance 1·58 [0·69–3·61]) were similar to the HRs for unvaccinated patients (2·32 [1·29–4·16] and 1·43 [1·04–1·97]; p=0·82 for both) but the precision for the vaccinated subgroup was low.\ud \ud Interpretation: \ud This large national study found a higher hospital admission or emergency care attendance risk for patients with COVID-19 infected with the delta variant compared with the alpha variant. Results suggest that outbreaks of the delta variant in unvaccinated populations might lead to a greater burden on health-care services than the alpha variant.\ud \ud Funding: \ud Medical Research Council; UK Research and Innovation; Department of Health and Social Care; and National Institute for Health Research.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections were rising during early summer 2021 in many countries as a result of the Delta variant. We assessed reverse transcription polymerase chain reaction swab positivity in the Real-time Assessment of Community Transmission–1 (REACT-1) study in England. During June and July 2021, we observed sustained exponential growth with an average doubling time of 25 days, driven by complete replacement of the Alpha variant by Delta and by high prevalence at younger, less-vaccinated ages. Prevalence among unvaccinated people [1.21% (95% credible interval 1.03%, 1.41%)] was three times that among double-vaccinated people [0.40% (95% credible interval 0.34%, 0.48%)]. However, after adjusting for age and other variables, vaccine effectiveness for double-vaccinated people was estimated at between ~50% and ~60% during this period in England. Increased social mixing in the presence of Delta had the potential to generate sustained growth in infections, even at high levels of vaccination.

Background Coinfection with human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1) diminishes the value of the CD4+ T-cell count in diagnosing AIDS, and increases the rate of HTLV-1–associated myelopathy. It remains elusive how HIV-1/HTLV-1 coinfection is related to such characteristics. We investigated the mutual effect of HIV-1/HTLV-1 coinfection on their integration sites (ISs) and clonal expansion. Methods We extracted DNA from longitudinal peripheral blood samples from 7 HIV-1/HTLV-1 coinfected, and 12 HIV-1 and 13 HTLV-1 monoinfected individuals. Proviral loads (PVL) were quantified using real-time polymerase chain reaction (PCR). Viral ISs and clonality were quantified by ligation-mediated PCR followed by high-throughput sequencing. Results PVL of both HIV-1 and HTLV-1 in coinfected individuals was significantly higher than that of the respective virus in monoinfected individuals. The degree of oligoclonality of both HIV-1– and HTLV-1–infected cells in coinfected individuals was also greater than in monoinfected subjects. ISs of HIV-1 in cases of coinfection were more frequently located in intergenic regions and transcriptionally silent regions, compared with HIV-1 monoinfected individuals. Conclusions HIV-1/HTLV-1 coinfection makes an impact on the distribution of viral ISs and clonality of virus-infected cells and thus may alter the risks of both HTLV-1– and HIV-1–associated disease.

Objectives The wide spectrum of clinical outcomes to SARSCoV-2 exposure suggests that early immune responses play a pivotal role 1 We aim to describe early, longitudinal, local (nasal mucosal lining fluid) and systemic (peripheral blood) cytokine and cellular immune responses to SARS-CoV-2 in a symptomatic index case and their household contacts with detailed clinical and virological phenotyping We hypothesise that immune responses at symptom onset would correlate with outcomes Methods Participants from the London area are referred to INSTINCT study by general practitioners as suspected, or Public Health England as laboratory-confirmed, cases (ethical review details: IRAS 282820, approved 24 04 2020) Households are visited the day after identification and again on days 7, 15 and 28 Clinical and exposure questionnaires, samples of environment (surface swabs and air);oropharynx (swabs);nasal mucosa (synthetic absorptive matrix) and blood, and daily symptom diaries are collected Samples are analysed by PCR, serology, 20-plex cytokine assay and flow cytometry in institutional laboratories Results The index case was the first SARS-CoV-2 PCR-positive recruit of INSTINCT, confirmed on oropharyngeal swab 5 days after symptom onset Contacts 1 and 2, the spouse and daughter, became symptomatic 2 days after the index case and were confirmed PCR-positive 3 days after symptom onset The three PCR-positive individuals seroconverted during follow-up Contact 3, the son, remained asymptomatic, PCRand serology-negative throughout (figure 1a-b) None required hospitalisation Swabs of the kettle and fridge handles were positive for virus, while other household surfaces and air samples were negative Induction, peak and decline of interferonl-1 and IP-10 levels were captured in nasal mucosa, with lower serum levels (figures 1c-f) Conclusion These data demonstrate the ability of the INSTINCT household contact study to capture early immune responses in mild SARS-CoV-2 infection, not captured by COVID-19 hospital cohort studies Early nasal mucosal cytokine responses to SARS-CoV-2 infection are not reflected in serum The correlations observed provide cogent hypotheses that will be tested in the larger INSTINCT cohort, with implications for COVID-19 risk stratification, therapeutics, prophylaxis and vaccinology

There is growing evidence that measurement of SARS-CoV-2 viral copy number can inform clinical and public health management of SARS-CoV-2 carriers and COVID-19 patients. Here we show that quantification of SARS-CoV-2 is feasible in a clinical setting, using a duplex RT-qPCR assay which targets both the E gene (Charite assay) and a human RNA transcript, RNase P (CDC assay) as an internal sample sufficiency control. Samples in which RNase P is not amplified indicate that sample degradation has occurred, PCR inhibitors are present, RNA extraction has failed or swabbing technique was insufficient. This important internal control reveals that 2.4 % of nasopharyngeal swabs (15/618 samples) are inadequate for SARS-CoV-2 testing which, if not identified, could result in false negative results. We show that our assay is linear across at least 7 logs and is highly reproducible, enabling the conversion of Cq values to viral copy numbers using a standard curve. Furthermore, the SARS-CoV-2 copy number was independent of the RNase P copy number indicating that the per-swab viral copy number is not dependent on sampling- further allowing comparisons between samples. The ability to quantify SARS-CoV-2 viral copy number will provide an important opportunity for viral burden-guided public health and clinical decision making.

Adult T cell leukaemia/lymphoma (ATL) arises from clonally expanded T cells that are infected with human T cell leukaemia virus type-1 (HTLV-1). Here, we show that ATL can be detected early in HTLV-1-carriers through quantification of T-cell receptor (TCR)Vβ subunit diversity on T-cells infected with HTLV-1 (CD3+ CCR4+ CD26− T-cells) using an ‘oligoclonality index’ (OCI-flow). We established a reference range for OCI-flow by analysing peripheral blood mononuclear cells (PBMCs) from HTLV-1-carriers who had not developed ATL in a median of 10.5 years follow up (n = 38) and patients with ATL (n = 30). In the third cohort of HTLV-1-carriers with no history or clinical evidence of ATL (n = 106), 19% of high proviral load (PVL, ≥4 copies of HTLV-1/100 PBMCs) carriers had an OCI-flow in the ATL range, >0.770. Carriers with an OCI-flow >0.770 (n = 14) had higher lymphocyte counts and PVLs and were more likely to have a family history of ATL than carriers with OCI-flow ≤0.770. ATL subsequently developed in two of these 14 carriers but no carriers with OCI-flow ≤0.770 (p = 0.03, cumulative follow-up 129 person-years). This method can be used to identify a subset of high-PVL HTLV-1-carriers at increased risk of developing ATL who may benefit from intervention therapy, prior to the detection of disease.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive hematological malignancy caused by human T-cell leukemia virus type-1 (HTLV-1). ATL is preceded by decades of chronic HTLV-1 infection, and the tumors carry both somatic mutations and proviral DNA integrated into the tumor genome. In order to gain insight into the oncogenic process, we used targeted sequencing to track the evolution of the malignant clone in 6 individuals, 2 to 10 years before the diagnosis of ATL. Clones of premalignant HTLV-1–infected cells bearing known driver mutations were detected in the blood up to 10 years before individuals developed acute and lymphoma subtype ATL. Six months before diagnosis, the total number and variant allele fraction of mutations increased in the blood. Peripheral blood mononuclear cells from premalignant cases (1 year prediagnosis) had significantly higher mutational burden in genes frequently mutated in ATL than did high-risk, age-matched HTLV-1 carriers who remained ATL-free after a median of 10 years of follow-up. These data show that HTLV-1–infected T-cell clones carrying key oncogenic driver mutations can be detected in cases of ATL years before the onset of symptoms. Early detection of such mutations may enable earlier and more effective intervention to prevent the development of ATL.

Human T-cell leukemia virus type 1 (HTLV-1) has infected approximately 20 million people worldwide. While 90% are asymptomatic, 5% develop severe diseases including adult T-cell leukemia/lymphoka (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). No vaccine against HTLV-1 exists, and screening programs are not universal. However, patients with chronic HTLV-1 infection have high frequencies of HTLV-1-activated CD8+ T cells, and the two main HLA alleles (A2, A24) are present in 88% of infected individuals. We thus utilized an immunoproteomics approach to characterize MHC-I restricted epitopes presented by HLA-A2+, A24+ MT-2 and SLB-1 cell lines. Unlike traditional motif prediction algorithms, this approach identifies epitopes associated with cytotoxic T-cell responses in their naturally processed forms, minimizing differences in antigen processing and protein expression levels. Out of nine identified peptides, we confirmed six novel MHC-I restricted epitopes that were capable of binding HLA-A2 and HLA-A24 alleles and used in vitro and in vivo methods to generate CD8+ T cells specific for each of these peptides. MagPix MILLIPLEX data showed that in vitro generated epitope-specific CD8+ T cells secreted IFN-γ, granzyme B, MIP-1α, TNF-α, perforin and IL-10 when cultured in the presence of MT-2 cell line. Degranulation assay confirmed cytotoxic response through surface expression of CD107 on CD8+ T cells when cultured with MT-2 cells. A CD8+ T-cell killing assay indicated significant antiviral activity of CD8+ T cells specific against all identified peptides. In vivo generated CD8+ T cells similarly demonstrated immunogenicity on ELISpot, CD107 degranulation assay, and MagPix MILLIPLEX analysis. These epitopes are thus candidates for a therapeutic peptide-based vaccine against HTLV-1, and our results provide preclinical data for the advancement of such a vaccine.

Adult T-cell leukemia/lymphoma (ATL) is a highly aggressive malignancy that arises in 2-5% of carriers of human T-cell lymphotropic virus type 1 (HTLV-1). The median overall survival of acute and lymphoma subtypes remains approximately 9–13 months and depressingly, with chemotherapy based approaches survival is largely unchanged in the ~40 years since it was first described. There is a clear and urgent need to conduct clinical trials of novel therapies in this disease. A high proviral load (PVL) (>4%, percentage of HTLV-1 infected mononuclear cells), male gender and smoking were previously the only major known risk factors for developing ATL, and so it has been difficult to advise patients about their individual risk of future ATL. Here, we describe the recent evidence that malignant disease does not occur randomly amongst all asymptomatic carriers but is more likely to arise in a subset of high PVL individuals with abnormally abundant clonal expansions of circulating HTLV-1 infected T-cells which typically express CD3dim+ CD4+ CD5-CD7- CD25+ CCR4+ with monoclonal TCRVβ. These clones also typically harbour known ATL driver mutations such as PLCG1, PRKCB, CARD11, STAT3, VAV1, NOTCH1, IRF4, CCR4, CCR7, TP53 and CDKN2, and may be detectable 10 years prior to disease presentation providing an opportunity to identify at risk individuals prior to clinical ATL. We describe the current classification and clinical features of ATL, and the exciting work of the last few years that underpins our new understanding of the genetic and epigenetic landscape with implications for future therapy. Whilst current therapy for aggressive ATL remain largely ineffective, recent advances may allow for early identification of at-risk individuals, and for pre-emptive therapies, and hope for a new era of effective targeted biological agents.

© 2017 The Japanese Society of Hematology Globally, > 5–10 million people are estimated to be infected with Human T-lymphotropic virus type 1 (HTLV-1), of whom ~ 5% develop adult T-cell leukemia/lymphoma (ATL). Despite advances in chemotherapy, overall survival (OS) has not improved in the 35 years since HTLV-1 was first described. In Europe/USA, combination treatment with zidovudine and interferon-α (ZDV/IFN-α) has substantially changed the management of patients with the leukemic subtypes of ATL (acute or unfavorable chronic ATL) and is under clinical trial evaluation in Japan. However, there is only a single published report of long-term clinical remission on discontinuing ZDV/IFN-α therapy and the optimal duration of treatment is unknown. Anecdotal cases where therapy is discontinued due to side effects or compliance have been associated with rapid disease relapse, and it has been widely accepted that the majority of patients will require life-long therapy. The development of molecular methods to quantify minimal residual disease is essential to potentially guide therapy for individual patients. Here, for the first time, we report molecular evidence that supports long-term clinical remission in a patient who was previously treated with ZDV/IFN-α for 5 years, and who has now been off all therapy for over 6 years.

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Background Human T-lymphotropic virus type 1 (HTLV-1)–associated myelopathy (HAM) is an inflammatory condition characterized by severe disability and high levels of infected white blood cells. The circulating cellular inflammatory changes that distinguish this condition from asymptomatic infection are not well understood. Methods To investigate the immune characteristics of individuals with low or high HTLV-1 proviral load (pVL), symptomatic disease, and the impact of immunosuppressive therapy, 38 women living with HTLV-1 infection, at a median age of 59 (52–68) years, were studied. Nineteen were asymptomatic carriers with low or high pVL; 19 were diagnosed with HAM, with 10 receiving anti-inflammatory therapy. Peripheral blood mononuclear cells were stained and analyzed for frequency distribution and activation of innate and adaptive immune cell subsets using multiparameter flow cytometry. Results Inflation of the CD4:CD8 ratio (>2) was observed among all groups irrespective of pVL. The frequency of naive CD4+ T cells correlated inversely with HTLV-1 pVL (rs = –0.344, P = .026). Mature T effector memory TEM CD4+ T cells were expanded in patients with untreated HAM compared with asymptomatic carriers (P < .001) but less so in those on therapy. High levels of exhausted (PD-1+) and senescent (CD28null) CD4+ and CD8+ T cells were observed in all individuals, particularly in those with HAM, while monocytes showed increased aggregation and CD14+CD56- monocytes were less frequent. Conclusions CD4:CD8 ratio inflation is a feature of HTLV-1 infection, whereas enhanced CD4+ T cell maturation and monocyte aggregation are features of HAM, reflecting widespread inflammatory change, which may be detectable presymptomatically and be amenable to anti-inflammatory treatment.

The prognosis of adult T-cell leukemia-lymphoma (ATL) remains very poor, and there is an urgent clinical need to investigate novel therapies for ATL. The expression of phosphatidylinositol 3-kinase-δ (PI3k-δ) is normally restricted to hematopoietic cells and is known as a key determinant of cell survival in certain cancers. The inhibitor of PI3k-δ, idelalisib, has been shown to be effective in the treatment of chronic lymphocytic leukemia. Here, we report the expression of PI3k-δ and the ability of idelalisib to promote apoptosis in ex vivo ATL samples. The activity of PI3K was quantified by a PI3-Kinase Activity ELISA kit. Although there was no significant difference in mean PI3K activity between healthy donors and patients with ATL, certain cases of ATL showed extremely high PI3K activities. The expression of PI3k-δ protein was detectable in most ATL cases. The freshly isolated cells from ATL patients were cultured with or without idelalisib for 0–10 days, and cell survival was then quantified. Idelalisib induced apoptosis in ATL cells in a time-dependent manner, and significantly reduced the frequency of viable ATL cells at 10 days. No time-dependent effects of idelalisib were observed in non-malignant T cells from the same patients. CCL22 has been reported to promote survival of ATL cells in part through the PI3K-AKT pathway. Idelalisib blocked this CCL22-induced phosphorylation of AKT and significantly inhibited the proliferation of ATL cells. These results validate the PI3K-AKT pathway as a potential therapeutic target in ATL. Electronic supplementary material The online version of this article (10.1186/s40364-018-0138-7) contains supplementary material, which is available to authorized users.

ContentsIntroduction. . 131HTLV-1 pathogenesis 132HTLV-1 and disease. . . 132ATL 132HAM/TSP . . 132Determinants of proviral load 132Integration and clonal expansion . . 132CD8þ T cell lysis 133The immune response to HTLV-1. . . 133Determinants of protective immunity 133Immunodominance 134‘‘Innate’’ receptors enhance the HLA class I-restricted response 135Persistence in the face of a strong immune response 136Immunogenicity of HTLV-I-infected cells 136Slow clearance of Tax-expressing cells in vivo 137Concluding remarks 137Is HBZ a viable target for HTLV-I vaccine design? 137Why is CTL killing low in vivo? 137How do KIRs affect the outcome of HTLV-1 infection? . . 137References . . . 137

Human T lymphotropic virus 1 (HTLV-1) was discovered in 1980, when Robert Gallo and his colleagues observed production of retroviral particles by a cell line established from a patient with a T-cell lymphoma (Poiesz et al. 1980). Concurrently, two groups in Jamaica and Japan detected HTLV-1-specific antibodies in the cerebrospinal fluid (CSF) and serum of patients with a progressive myelopathy that was previously known as tropical spastic paraparesis (TSP) and named by the Japanese group HTLV-1-associated myelopathy (HAM) (Gessain et al. 1985; Osame et al. 1986). TSP and HAM were subsequently identified as the same condition, and the disease is now designated HAM/TSP. HAM/TSP is characterised by lesions in the spinal cord, resulting in a loss of control of motor functions below the waist, constipation, incontinence and neuropathic pain.

ATLL is an aggressive malignancy of T cells that affects about 5% of individuals infected with HTLV-1. The precise mechanism of oncogenesis is not known, but there is evidence that two regulatory viral proteins, Tax and HBZ, are involved. A high set point proviral load is associated with development of ATLL or a chronic inflammatory condition, HAM/TSP. Several lines of evidence, including HLA class 1 association studies and in vitro killing assays, indicate that cytotoxic T lymphocytes are instrumental in determining this proviral load set point. Prior studies have focused chiefly on the CTL response to the immunodominant Tax protein: efficient lysis of Tax-expressing cells inversely correlates with proviral load in nonmalignant infection. However, a recent study showed that strong binding of peptides from HBZ, but not Tax, to HLA class 1 molecules was associated with a low proviral load and a reduced risk of developing HAM/TSP, indicating an important role for HBZ-specific CTL in determining infection outcome. In comparison with nonmalignant infection, HTLV-1-specific CTLs in ATLL patients are reduced in frequency and functionally deficient. Here we discuss the nature of protective CTL responses in nonmalignant HTLV-1 infection and explore the potential of CTLs to protect against ATLL.

Oncogenic transformation of CD4+ T cells by human T-cell lymphotropic virus type 1 (HTLV-1) is understood as the initial step to adult T-cell leukemia/lymphoma, a process that is mainly initiated by perturbation of cellular signaling by the viral Tax oncoprotein, a potent transcriptional regulator. In search of novel biomarkers with relevance to oncogenesis, we identified the tumor marker and actin-bundling protein Fascin (FSCN1) to be specifically and strongly up-regulated in both HTLV-1–transformed and adult T-cell leukemia/lymphoma patient-derived CD4+ T cells. Fascin is important for migration and metastasis in various types of cancer. Here we report that a direct link can exist between a single viral oncoprotein and Fascin expression, as the viral oncoprotein Tax was sufficient to induce high levels of Fascin. Nuclear factor-κB signals were important for Tax-mediated transcriptional regulation of Fascin in T cells. This suggests that Fascin up-regulation by Tax contributes to the development of HTLV-1–associated pathogenesis.

Human T-lymphotropic virus type 1 (HTLV-1), the first discovered human retrovirus, is prevalent in Japan, the Caribbean, parts of sub-Saharan Africa, South America, Melanesia, and the Middle East, with worldwide prevalence once estimated to be 10–20 million [1]. Infection with HTLV-1 is lifelong. Most subjects remain asymptomatic carriers (ACs), but a significant minority, 7%, will develop adult T cell leukemia/lymphoma (ATLL) [2] or HTLV-1-associated myelopathy (HAM) [3]. The overall risk of disease may reach 10% if HTLV-1–associated uveitis [4], HTLV-1-associated infective dermatitis [5], and other, apparently associated, manifestations such as polymyositis [6], a broad spectrum of lung disease [7], and thyroiditis [8] are included. In addition to these overt diseases, a reduction in life expectancy, of uncertain cause, has been described [5, 9, 10]. HTLV-1, like other retroviruses, contains the structural and enzymatic genes gag, pro, pol, and env flanked by 2 long terminal repeats (LTR). In addition, HTLV-1 encodes accessory and regulatory proteins in 4 open reading frames (ORFs) I–IV located at the 3′ end of the genome. ORFs III and IV encode 2 well-characterized genes, tax and rex. Tax appears to play an important role in HTLV-1 pathogenesis; it is the first viral protein detected in HTLV-1-expressing cells [11] and has been shown to be the immunodominant antigen recognized by cytotoxic T-lymphocytes (CTL) of patients with HAM and of ACs [12–16]. Recently a new viral protein, HTLV-1 basic leucine zipper (HBZ), has been identified; it is encoded by an ORF located between the env and tax/rex genes on the minus, or antisense, strand of the HTLV-1 provirus [17]. Transcription of HBZ is controlled by a promoter in the 3′ LTR. HBZ contains a transcription activation domain at the N-terminus and a leucine zipper at the C-terminus and the transcribed RNA has been shown to exist in 3 forms, unspliced HBZ (unHBZ), HBZ spliced-1 (HBZ-SP1), and HBZ spliced-2 (HBZ-SP2) [18, 19]. Unspliced HBZ and HBZ-SP1 have been studied the most, both forms have been detected in multiple HTLV-1 cell lines [18–21] and in peripheral blood mononuclear cells (PBMCs) isolated from patients with ATLL or HAM and from ACs [19–21]. HBZ appears to have biologically important activities at both the RNA and protein levels. HBZ protein inhibits Tax-mediated transactivation of viral transcription from the 5′ LTR through an interaction with JUN and ATF/CREB families [22–25]. HBZ RNA reportedly promotes T cell proliferation [19]. In transgenic mice HBZ promotes CD4+ T cell proliferation, while in inoculated rabbits HBZ enhances infectivity and persistence of HTLV-1 [26]. Similar genes encoded on the antisense strand of HTLV-2, HTLV-3 and HTLV-4 have been described [27–29]. The antisense transcript of HTLV-2 encodes the antisense protein APH-2 that remains able to bind to CREB and to down-regulate Tax-2 dependent expression although APH-2 lacks a leucine zipper domain [28]. We describe the use of an immunological assay to detect evidence of HBZ protein expression in vivo in patients with HAM and in ACs, and report a significant association between the detection of HBZ-specific CD8+ T cell responses with low viral load (LVL) and asymptomatic carriage of HTLV-1. These results are consistent with the conclusion of our recent study of class I binding of HTLV-1 epitopes [30], that the HLA class 1–restricted T cell response plays an important part in limiting HTLV-1 viral load and the risk of HAM.

Adult T cell leukaemia/lymphoma (ATL) is a predominantly CD4+ T cell neoplasia caused by human T cell lymphotropic Virus 1 (HTLV-1) infection. A few cases of CD8+ ATL have been reported but its existence has been debated. An Afrocaribbean female presented to the dermatology clinic with widespread papulo-nodular rash. HTLV-1 infection was detected by serology. Skin biopsy showed dense dermal infiltration by mainly CD8+ T cells which also expressed CD2, CD3, CD5, CD7 and occasionally CD25. Further investigations showed a mild lymphocytosis (4.4 x 106/L), circulating flower cells, absolute CD4+ count 1247 x 106/L (28.3% of lymphocytes), absolute CD8+ count x 2617 x 106/L (59.8% of lymphocytes), LDH 241 U/L (NR 135 – 214), Calcium 2.7 mmol/L (NR 2.2 – 2.6) but with increased serum PTH level. HTLV-1 proviral load (PVL) in peripheral blood mononuclear cells was 46%. PET/CT scan demonstrated FDG avid lymph nodes above and below the diaphragm. Axillary lymph node core biopsy showed CD20+ B cells nodules admixed with mainly small-to-medium sized T cells and a small population of large T cells. The small lymphocytes were mainly CD8+ T cells (also expressed CD2, CD3, CD5, and CD7 whereas the large cells expressed CD8, CD25 and CD30. Fewer than 5% expressed Ki67 (MIB-1). TCR clonality studies by PCR revealed expansion of an identical clone in all three compartments (skin, lymph node and blood). Detailed immunophenotyping on peripheral blood demonstrated a single dominant TCR vβ1+ clone (>90% of total CD8 T cells) which was CD3+, CD7+, CCR4+, CD25 low and, TSCL-1-. In summary we report a malignant CD8+ clone with atypical immunophenotype in a patient with high PVL. To confirm a working diagnosis of CD8+ ATL high throughput sequencing of each proviral integration sites is underway.

Adult T cell leukemia (ATL) is typically characterised by expansion of a single dominant malignantly transformed HTLV-1-infected clone. Clinically, the abundance of this clone can be estimated by flow cytometric assessment of CD4, CD25 and CD7 expression, and, more recently, by quantitative LM-PCR analysis of viral integration site. In addition to the unique proviral integration site, malignant clones arising from a single infection event also share an identical T cell receptor (TCR) sequence, utilising one of a potential 47 TCR Vbeta subunit genes. Using multiparametric flow cytometry, we quantified the frequency of peripheral T cells bearing each of 24 TCR Vbeta subunits in a cohort of individuals with adult T cell leukemia or lymphoma (n=26) at various stages of disease progression/remission. To establish an appropriate baseline, we also analysed T cells from asymptomatic carriers (n=11) and donors with HAM/TSP (n=13). Vbeta-specific antibodies stained 75% of CD3+ cells in donors without malignancy, and the median size of the largest single subset (CD4+Vbeta2+) was 7.6 % of CD3+ cells. We detected putative T cell clones which exceeded 20% of CD3+ T cells only in individuals with active chronic or acute leukemia (n=17), reaching frequencies of up to 96% of CD3+ T cells. With this method, we quantified the expression on clonally expanded populations of the putative ATL cell markers CD25, CCR4, TSLC1, CD7 and CD127. Almost 1% of expanded clonal populations in ATL patients were CCR4+TSLC1+CD7–. Surprisingly, in most donors only 60% of the dominant clonal population expressed high levels of CD25. This flow cytometric protocol is a potentially powerful method to both monitor disease progression and response to therapy, providing fast and accurate quantification of clonally expanded populations. We are currently employing this technique to assay lysis of expanded clones by autologous cytotoxic T lymphocytes in patients with ATL.

Adult T cell leukaemia/lymphoma (ATL) is a highly heterogeneous neoplasm caused by human T cell lymphotropic virus type 1 (HTLV-1) infection. Shimoyama et al first proposed a prognostic classification based on clinical features. However subsequent studies have highlighted even greater heterogeneity and progression from indolent to aggressive subtypes. The molecular basis for this heterogeneity and progression remains unknown. Our aims were to identify molecular markers that characterize this heterogeneity and progression. We studied expression of T-cell markers and clonality (Ligation Mediated PCR) on peripheral blood mononuclear cells in samples collected from HTLV-1 infected patients (16 asymptomatic carrier [AC], 18 with HTLV-1-associated myelopathy [HAM], 18 with indolent ATL [five smouldering & 13 chronic] and seven with acute ATL) attending the National Centre for Human Retrovirology, London. 11/18 indolent ATL required no systemic treatment and one progressed to acute ATL. The remainder required systemic therapy and all except one achieved complete remission with zidovudine/interferon. 6/7 with acute ATL had primary refractory disease. Clonality testing showed no dominant clones in the AC, patient with HAM and 6 of the 11 patients with indolent ATL who didn't require systemic therapy. We confirmed CD4+CCR4+ as the main reservoir of HTLV-1 infection. These CD4+CCR4+ T cells had CD7+CD25+CD26+CD127+CCR7-immunophenotype in AC, HAM and indolent ATL with no dominant clones. CD7 down-regulation within CD4+CCR4+ T cells identified ATL patients with dominant clone(s). CD127 down-regulation within CD4+CCR4+ T cells was present in all indolent ATL patients which required systemic therapy. Nine patients with ATL (all acute and two indolent) had CCR7 up- regulation of which all but two had primary refractory disease. Longitudinal follow up in six patients showing changing immunophenotype which correlated with clinical features and prognosis. In conclusion CD7, CD127 and CCR7 expression within CD4+CCR4+ infected cells further characterises the heterogeneity within ATL with therapeutic implications.

HTLV-1 Tax protein is usually undetectable in freshly isolated peripheral blood mononuclear cells (PBMCs). After in vitro culture, the provirus is reactivated and Tax protein is detectable only in a proportion of infected CD4+ T cells: the percentage of Tax+ cells is always lower than the proviral load (PVL). To identify and further analyse the latently infected cells we have measured the expression of tumour suppressor of lung cancer 1 (TSLC1). TSLC1 is a member of the immunoglobulin super family that is expressed on all cells with the exception of PBMCs. It mediates cell-to-cell adhesion by either homophilic or heterophilic interactions with other members of the immunoglobulin family and also signals to the actin cytoskeleton. TSLC1 has been shown to be expressed on primary adult T cell leukaemia (ATL) cells and ATL cell lines. We assayed the expression of TSLC1 in the PBMCs of 13 asymptomatic carriers (AC) and 13 HAM/TSP patients by flow cytometry. We found that the percentage of TSLC1+ CD4+ T cells was positively correlated with the PVL (P < 0.0001). To test whether TSLC1+ cells themselves were infected, we flow sorted TSLC1+ CD4+ T cells of 3 ACs and 3 HAM/TSP patients. A median of 95% of TSLC1+ CD4+ T cells carried the provirus. Whilst only 20% of infected CD4+ T cells expressed Tax, a further 47% were identified on the basis of TSLC1 expression. As the host cytotoxic T cell (CTL) response is an important protective factor, we are now testing the hypothesis that the presence of TSLC1 on the surface of infected cells affects its recognition and subsequent killing by CTLs.

Background Human T-lymphotropic Virus Type I (HTLV-1) is a retrovirus that persistently infects 5–10 million individuals worldwide and causes disabling or fatal inflammatory and malignant diseases. The majority of the HTLV-1 proviral load is found in CD4+ T cells, and the phenotype of adult T cell leukemia (ATL) is typically CD4+. HTLV-1 also infects CD8+ cells in vivo, but the relative abundance and clonal composition of the two infected subpopulations have not been studied. We used a high-throughput DNA sequencing protocol to map and quantify HTLV-1 proviral integration sites in separated populations of CD4+ cells, CD8+ cells and unsorted peripheral blood mononuclear cells from 12 HTLV-1-infected individuals. Results We show that the infected CD8+ cells constitute a median of 5 % of the HTLV-1 proviral load. However, HTLV-1-infected CD8+ clones undergo much greater oligoclonal proliferation than the infected CD4+ clones in infected individuals, regardless of disease manifestation. The CD8+ clones are over-represented among the most abundant clones in the blood and are redetected even after several years. Conclusions We conclude that although they make up only 5 % of the proviral load, the HTLV-1-infected CD8+ T-cells make a major impact on the clonal composition of HTLV-1-infected cells in the blood. The greater degree of oligoclonal expansion observed in the infected CD8+ T cells, contrasts with the CD4+ phenotype of ATL; cases of CD8+ adult T-cell leukaemia/lymphoma are rare. This work is consistent with growing evidence that oligoclonal expansion of HTLV-1-infected cells is not sufficient for malignant transformation. Electronic supplementary material The online version of this article (doi:10.1186/s12977-015-0221-1) contains supplementary material, which is available to authorized users.

The majority of hepatitis C virus (HCV) infections become chronic, despite the presence of HCV-specific cellular and humoral immune responses. We have previously suggested that IL-10-secreting antigen-specific regulatory T cells may contribute to viral persistence, and demonstrate here that peripheral blood mononuclear cells (PBMC) from chronically HCV-infected patients secrete IL-10, but not IFN-gamma, in response to HCV nonstructural protein 4 (NS4). A neutralizing anti-IL-10 antibody restored this defective antigen-specific IFN-gamma production in vitro. Furthermore, PBMC from normal individuals secreted IL-10 in response to NS4, suggesting that cells of the innate immune system, in addition to T cells, produced IL-10 in the HCV-infected patients. Cell separation experiments revealed that the innate IL-10 was produced by blood monocytes, but not dendritic cells (DC). In addition, NS4 inhibited IL-12 production by PBMC in response to LPS and IFN-gamma, and Th1 responses to recall antigens in normal individuals. Furthermore, supernatants from NS4-stimulated monocytes inhibited LPS-induced maturation of DC and suppressed their capacity to stimulate proliferation and IFN-gamma production by allospecific T cells. Our data suggest that HCV subverts cellular immunity by inducing IL-10 and inhibiting IL-12 production by monocytes, which in turn inhibits the activation of DC that drive the differentiation of Th1 cells.

Human T lymphotropic virus type 1 (HTLV-1) appears to persist in the chronic phase of infection by driving oligoclonal proliferation of infected T cells. Our recent high-throughput sequencing study revealed a large number (often > 104) of distinct proviral integration sites of HTLV-1 in each host that is greatly in excess of previous estimates. Here we use the highly sensitive, quantitative high-throughput sequencing protocol to show that circulating HTLV-1+ clones in natural infection each contain a single integrated proviral copy. We conclude that a typical host possesses a large number of distinct HTLV-1–infected T-cell clones.

In human T-lymphotropic virus type 1 (HTLV-1) infection, a high frequency of HTLV-1-specific CTLs can co-exist stably with a high proviral load and the proviral load is strongly correlated with the risk of HTLV-1-associated inflammatory diseases. These observations led to the hypothesis that HTLV-1 specific CTLs are ineffective in controlling HTLV-1 replication but contribute to the pathogenesis of the inflammatory diseases. But evidence from host and viral immunogenetics and gene expression microarrays suggests that a strong CTL response is associated with a low proviral load and a low risk of HAM/TSP. Here, we quantified the frequency, lytic activity and functional avidity of HTLV-1-specific CD8+ cells in fresh, unstimulated PBMCs from individuals with natural HTLV-1 infection. The lytic efficiency of the CD8+ T cell response—the fraction of autologous HTLV-1-expressing cells eliminated per CD8+ cell per day—was inversely correlated with both the proviral load and the rate of spontaneous proviral expression. The functional avidity of HTLV-1-specific CD8+ cells was strongly correlated with their lytic efficiency. We conclude that efficient control of HTLV-1 in vivo depends on the CTL lytic efficiency, which depends in turn on CTL avidity of Ag recognition. CTL quality determines the position of virus-host equilibrium in persistent HTLV-1 infection.

IL-17-secreting T (Th17) cells play a protective role in certain bacterial infections, but they are major mediators of inflammation and are pathogenic in organ-specific autoimmune diseases. However, human Th17 cells appear to be resistant to suppression by CD4+CD25+FoxP3+ regulatory T cells, suggesting that they may be regulated by alternative mechanisms. Herein we show that IL-10 and TGF-β suppressed IL-17 production by anti-CD3-stimulated PBMC from normal individuals. TGF-β also suppressed IL-17 production by purified CD4+ T cells, whereas the inhibitory effect of IL-10 on IL-17 production appears to be mediated predominantly by its effect on APC. An examination of patients infected with hepatitis C virus (HCV) demonstrated that Ag-specific Th17 cells are induced during infection and that these cells are regulated by IL-10 and TGF-β. PBMC from HCV Ab-positive donors secreted IL-17, IFN-γ, IL-10, and TGF-β in response to stimulation with the HCV nonstructural protein 4 (NS4). Furthermore, NS4 induced innate TGF-β and IL-10 expression by monocytes from normal donors and at higher levels from HCV-infected patients. Neutralization of TGF-β, and to a lesser extent IL-10, significantly enhanced NS4-specific IL-17 and IFN-γ production by T cells from HCV-infected donors. Our findings suggest that both HCV-specific Th1 and Th17 cells are suppressed by NS4-induced production of the innate anti-inflammatory cytokines IL-10 and TGF-β. This may represent a novel immune subversion mechanism by the virus to evade host-protective immune responses. Our findings also suggest that TGF-β and IL-10 play important roles in constraining the function of Th17 cells in general.

Protective immunity against viruses, bacteria, parasites and fungi is mediated by innate and adaptive immune responses of the hosts. However, pathogens have developed strategies for subverting these responses and thereby establishing persistent or chronic infections. Furthermore, anti-pathogen effector immune responses must be tightly regulated to prevent collateral damage to host tissues. Regulatory T (Treg) cells that secrete the immunosuppressive cytokines IL-10 and /or TGF-β have been implicated in both processes, assisting the host to prevent immunopathology and helping the pathogen to evade protective immunity. This chapter discusses the induction of antigen-specific IL-10 and TGF-β-producing regulatory T cells during infection and gives some examples of their different roles in immunity to parasites, bacteria, viruses and fungi.

The integration of Human T-lymphotropic virus type I (HTLV-1) is known to be diverse but biased towards genomically active regions. Each infected clone is identified by a unique integration site position. The integration site position is known to be associated with the expression pattern of the provirus and its propensity to expand in vivo but it is not known how it would impact the gene expression of the host genes. The viral transactivator Tax has been shown (mostly in vitro) to be capable of altering the expression of many host genes however it is not known what role the genomic integration site may have in determining the alteration of host gene expression or how the gene expression pattern would vary from one infected T-cell clone to another. We have developed a panel of HTLV-1 infected T-cell clones. CD4+ T-cells were isolated from HTLV-1 infected individuals and cloned by limiting dilution. Each clone was cultured in the presence of integration inhibitor to prevent super infection and defined by a single unique integration site. Uninfected T cell clones were also established in the same way. In order to address this question and to analyze gene expression pattern, total RNAs were isolated from each clone for RNA sequencing (RNA-seq). We will present the results of high-throughput sequencing of the human and viral mRNA, highlighting the similar and variable between the different clones. # Poster award winnder - 2nd place

HTLV-1 genes are encoded on both strands of the provirus, such as tax in the plus and HBZ in the minus strand. The HBZ gene is constitutively expressed from the negative strand of the integrated provirus, whereas plus-strand expression, required for viral propagation to uninfected cells, is expressed only intermittently in vivo, perhaps to escape from host immune surveillance. However, it remains unknown what regulates this pattern of proviral transcription in vivo. We have found that CTCF binds to the HTLV-1 provirus. CTCF is a DNA-binding protein that plays a fundamental role in controlling higher-order chromatin structure and gene expression in vertebrates. We identified several candidate regions for CTCF binding in the HTLV-1 genome. Chromatin immunoprecipitation assays showed that CTCF bound selectively to the pX region of HTLV-1. Furthermore, electromobility shift assays revealed that CTCF bound directly to the pX DNA sequence. Consistent with the CTCF binding, there was a sharp border of histone modification patterns at the pX region, consistent with CTCF's role as a chromatin insulator. Finally, the CTCF-binding region (1bp) showed enhancer-blocking activity. The CTCF binding and epigenetic border were detectable not only in HTLV-1 cell lines and ATL cell lines but also in fresh PBMCs of ATL patients. These observations suggest that CTCF plays a central role in the regulation of HTLV-1 transcription. #Poster award winnder - 1st place

Human T-lymphotropic virus type I (HTLV-1) integration is known to be weakly biased towards genomically active regions but can occur throughout the genome. We have shown that there are of the order of 10^4 HTLV-1-infected T-cell clones in each individual, each clone defined by a unique genomic integration site of the single-copy HTLV-1 provirus. HTLV-1 dysregulates many host genes by expressing a transcriptional transactivator, Tax, but it remains unknown whether HTLV-1 also alters host gene expression by insertional mutagenesis. In recent work (Satou et al, submitted) we have shown that HTLV-1 encodes a functional binding site for CCCTC binding factor (CTCF), a known mediator of chromatin looping. We hypothesize that the HTLV-1 provirus forms CTCF-mediated chromatin loops with the flanking host genome and dysregulates the expression of host genes. To test this hypothesis, we performed a circular chromatin conformation capture assay followed by high-throughput sequencing (4C-seq) which enables high resolution screening of physical long range interactions between chromosomes. Using the proviral CTCF site as bait, we have detected long-range interactions between the proviral and the host genome which suggest the formation of novel chromatin looping in clones of HTLV-1-infected T cells from both cases of ATL and from non-malignant cases of HTLV-1 infection. Chromosome conformation capture using specific primers (3C) confirmed clone-specific interactions. We are now testing whether these long-range interactions are associated with clone-specific alterations in the expression of host genes.

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.

Host cytotoxic lymphocyte (CTL) responses play an essential role in limiting clonal expansion of infected CD4+ T cells in HTLV-1+ individuals. We have reported that individuals who possess HLA class 1 alleles that strongly bind peptides from HTLV-1 basic leucine zipper protein (HBZ) have a lower proviral load and are less likely to develop HAM/TSP. In general, peptides from HBZ bind weakly to MHCI, and we detected low frequencies of HBZ-specific CD8+ T cells in ≈25% of infected individuals. Despite its poor immunogenicity, high avidity CTL lines can be generated in vitro which efficiently lyse HBZ-presenting cells [1]. HBZ mRNA, encoded on the antisense strand of the viral genome, can induce proliferation of T cells, and once translated it can inhibit expression of Tax. HBZ mRNA is expressed in vivo in the majority of infected individuals, however, HBZ protein expression levels are extremely low in PBMCs ex vivo. To investigate the relationship between Tax and HBZ expression in naturally infected CD4+ T cells at the single cell level, we cloned T cells from individuals with HAM/TSP. We detected HBZ mRNA in all clones tested. In contrast, a subset of infected clones stably expressed Tax protein. Thus, individual clones may have a unique viral gene expression profile, with differing susceptibility to lysis by HTLV-1 specific CTL. We are testing the hypothesis that targeting of Tax– clones by HBZ-specific CTL eliminates clones which otherwise escape lysis by abundant Tax-specific CTL.

In search of novel biomarkers of HTLV-1- transformed cells with relevance to oncogenesis, we identified the tumour marker and actin-bundling protein Fascin (FSCN1) to be specifically and strongly upregulated in both HTLV-1-transformed and ATLL-patient-derived CD4+ T cells. Fascin is important for migration and metastasis in various types of cancer. Here we report that a direct link can exist between a single viral oncoprotein and Fascin expression, as the viral oncoprotein Tax of HTLV-1 was sufficient to induce high levels of Fascin. In contrast, Tax-2 of HTLV-2 could not induce Fascin. Expression of Fascin was also detectable in primary CD4+ T cells of ATLL-patients after they expressed Tax spontaneously. Immunofluorescence analysis revealed that Fascin could colocalize with Actin in the cytoplasm and at the membrane of HTLV-1-transformed cells. We found a novel mode of transcriptional regulation of Fascin by showing the importance of NF-κB signals for Tax-mediated induction of Fascin in T cells. Chemical and dominant-negative inhibition of the NF-κB pathway as well as a NF-κB-deficient Tax-mutant led to a strong decrease of Fascin mRNA and protein in Tax-transfected Jurkat cells. Additionally, in HTLV-1-/Tax-transformed cells Fascin transcripts were strongly reduced after chemical inhibition of IκB-kinase. Knockdown of Fascin by lentiviral transduction of shRNA decreased the invasive capacity of ATLL-derived cells into extracellular matrix. Thus, we have clearly shown that the tumour marker Fascin can be induced by the viral Tax oncoprotein through NF-κB signals. Our data suggest that Fascin up-regulation by Tax contributes to the development of HTLV-1-associated pathogenesis.

CD8+ T cells can exert both protective and harmful effects on the virus-infected host. However, there is no systematic method to identify the attributes of a protective CD8+ T cell response. Here, we combine theory and experiment to identify and quantify the contribution of all HLA class I alleles to host protection against infection with a given pathogen. In 432 HTLV-1-infected individuals we show that individuals with HLA class I alleles that strongly bind the HTLV-1 protein HBZ had a lower proviral load and were more likely to be asymptomatic. We also show that in general, across all HTLV-1 proteins, CD8+ T cell effectiveness is strongly determined by protein specificity and produce a ranked list of the proteins targeted by the most effective CD8+ T cell response through to the least effective CD8+ T cell response. We conclude that CD8+ T cells play an important role in the control of HTLV-1 and that CD8+ cells specific to HBZ, not the immunodominant protein Tax, are the most effective. We suggest that HBZ plays a central role in HTLV-1 persistence. This approach is applicable to all pathogens, even where data are sparse, to identify simultaneously the HLA Class I alleles and the epitopes responsible for a protective CD8+ T cell response., Author Summary A large immune response to the retrovirus HTLV-1 does not always prevent HTLV-1-associated diseases. Indeed, it has been shown that CD8+ T cells may contribute towards the inflammatory disease associated with HTLV-1 infection. This observation has led to the hypothesis that it is the ‘quality’ of the immune response towards HTLV-1 that is important, and not simply a response in itself. Using a combination of computational and experimental methods we have investigated T cell ‘quality’. We have found that specificity is an important determinant of CD8+ T cell quality with recognition of the viral protein HBZ enabling the host to make a more effective immune response. This approach can be used for other pathogens to identify what HLA class 1 alleles and the parts of the pathogen they bind to are responsible for a protective CD8+ T cell immune response. This work informs basic immunology: “what constitutes a protective CD8+ T cell response?”; vaccine design: “which antigens elicit the most effective response” and virology: “which viral proteins are key players in the strategy of persistence?”.

Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes malignant and inflammatory diseases in 10% of infected people. A typical host has between 10 4 and 10 5 clones of HTLV-1- infected T lymphocytes, each clone distinguished by the genomic integration site of the single-copy HTLV-1 provirus. The HBZ gene is constitutively expressed from the minus strand of the provirus, whereas plus-strand expression, required for viral propagation to uninfected cells, is suppressed or intermittent in vivo , allowing escape from host immune surveillance. It remains unknown what regulates this pattern of proviral transcription and latency. Here we show that CTCF, a key regulator of chromatin structure and function, binds to the provirus at a sharp border in epigenetic modifications in the pX region of the HTLV-1 provirus, in T cells naturally infected with HTLV-1. CTCF is a zinc-finger protein that binds to an insulator region in genomic DNA and plays a funda- mental role in controlling higher-order chromatin structure and gene expression in vertebrate cells. We show that CTCF bound to HTLV-1 acts as an enhancer blocker, regulates HTLV-1 mRNA splicing, and forms long-distance interactions with flanking host chromatin. CTCF binding sites have been propagated through- out the genome by transposons in certain primate lineages, but CTCF binding has not previously been described in present-day exogenous retroviruses. The presence of an ectopic CTCF binding site introduced by the retrovirus in tens of thousands of genomic locations has the potential to cause widespread abnormalities in host cell chromatin structure and gene expression.

Adult T cell Leukaemia Lymphoma [ATL], a mature T-cell neoplasm has been classified into 4 subtypes: smouldering; chronic leukaemia; lymphoma and acute leukaemia. The diagnosis depends on clinical features, the immunophenotype, and demonstration of HTLV-1 infection & ideally of monoclonal proviral integration. The typical immunophenotype of ATL is not specific. The methods used to detect monoclonality are labour-intensive and/or expensive and are not widely available. We developed a flow cytometry assay for diagnosis and monitoring of ATL. We performed 11-colour immunophenotyping , HTLV-1 proviral quantification and proviral integration site [IS] analysis on 53 samples from 36 patients [25 non ATL HTLV-1-infected,11 chronic/smouldering ATL], 3 uninfected individuals and 2 HTLV-1-immortalized cell lines. The non-ATL patients had CD127+ & CCR7-lo expression in CD4+CD25+CCR4+ cells, and a polyclonal distribution on IS analysis. Four ATL patients had CD127+ & CCR7-lo expression in CD4+CD25+CCR4+ cells and polyclonal distribution on IS analysis. These patients had an excellent outcome achieving remission with either PUVA or no therapy. Eight ATL patients had CD127-lo expression on CD4+ CD25+ CCR4+ cells with mono/oligoclonal distribution on IS analysis. One of nine patients with chronic ATLL had high CCR7 expression. Foxp3 expression was variable. All 8 patients required systemic ATL treatment and longitudinal study of 5 patients found the change in frequency of CD4+CD25+CCR4+ to correlate with PVL whilst CD127 expression correlated with IS analysis (p

HTLV-1 propagates in the host by two parallel mechanisms: infectious spread, i.e. direct transfer of virions from cell to cell via the virological synapse; and mitotic spread, i.e. proliferation of HTLV-1-infected T cell clones. During chronic infection, mitotic spread predominates, generating very large clones in some individuals. Yet the reasons for the remarkable variation within and between hosts in the abundance of HTLV-1-infected clones remain unknown. We aim to identify and quantify the selection forces that determine the size of HTLV-1-infected T cell clones in vivo and hence the risk of the associated inflammatory and malignant diseases. We shall summarize evidence from host and viral genetics, cellular immunology and high-throughput proviral integration site analysis that: 1. The ‘quality’ or efficiency of a person’s genetically-determined cytotoxic T lymphocyte (CTL) response to the HBZ protein is a major determinant of that person’s proviral load and the risk of inflammatory diseases. 2. The chief determinant of CTL quality is the host genotype in HLA Class 1 and Killer cell Immunoglobulin-like Receptor (KIR) loci. 3. The CTL response to the Tax protein is immunodominant but does not protect against HAM/TSP. 4. The site and orientation of the HTLV-1 provirus integrated in the T cell genome determine the size of each T cell clone in the host. 5. Negative selection on the virus dominates during chronic infection: we postulate that this selection is exerted by CTLs.