Silva Hilburn

HLA Class I Binding of HBZ Determines Outcome in HTLV-1 Infection

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.

Susceptibility of Primary HTLV-1 Isolates from Patients with HTLV-1-Associated Myelopathy to Reverse Transcriptase Inhibitors

Since human T-lymphotropic virus type 1 (HTLV-1)-associated diseases are associated with a high HTLV-1 load, reducing this load may treat or prevent disease. However, despite in vitro evidence that certain nucleoside/nucleotide analogue reverse transcriptase inhibitors (NRTIs) are active against HTLV-1, in vivo results have been disappointing. We therefore assayed the sensitivity of HTLV-1 primary isolates to a panel of RT inhibitors. HTLV-1 primary isolates were obtained, pre- and post- NRTI treatment, from patients with HTLV-1-associated myelopathy. Sensitivity to azidothymidine (AZT), lamivudine (3TC), tenofovir (TDF) and three phosphonated carbocyclic 2’-oxa-3’aza nucleosides (PCOANs) was assessed in a RT inhibitor assay. With the exception of 3TC, HTLV RT from primary isolates was less sensitive to all tested inhibitors than HTLV-1 RT from MT-2 cells. HTLV-1 RT from primary isolates and from chronically infected, transformed MT-2 cells was insensitive to 3TC. Sensitivity of primary isolates to RT inhibitors was not reduced following up to 12 months of patient treatment with AZT plus 3TC. The sensitivity of HTLV-1 primary isolates to NRTIs differs from that of cell lines and may vary among patients. Failure of NRTIs to reduce HTLV-1 viral load in vivo was not due to the development of phenotypic NRTI resistance. AZT and the three PCOANs assayed all consistently inhibited primary isolate HTLV-1 RT.

Treatment of patients with HTLV-1-associated myelopathy with methotrexate

The lifetime risk of developing HTLV-1 associated myelopathy (HAM) is 0.25-3%. The main pathological feature is an immune-mediated response leading to chronic inflammation of the spinal cord. The optimal long term treatment has yet to be determined although clinical improvement with ciclosporin has been shown in a pilot study. Methotrexate, commonly used for autoimmune diseases, was introduced for the treatment of HAM at the National Centre for Human Retrovirology, London, UK as an alternative to ciclosporin.

The clinical utility of HTLV-1 viral load measurement

High levels of HTLV-1 proviral load in peripheral blood mononuclear cells (PBMCs) have been observed in patients with Adult T-cell Leukaemia Lymphoma (ATLL) and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). The proviral load has been reported to fluctuate in both asymptomatic carriers and in symptomatic patients. We aimed to quantify intra-patient variability, to identify “outliers”, and to characterise the range of viral loads in asymptomatic carriers and in patients with HTLV-1-associated diseases.

Long Terminal Repeat Circular DNA as Markers of Active Viral Replication of Human T Lymphotropic Virus-1 in Vivo

Clonal expansion of human T-lymphotropic virus type-1 (HTLV-1) infected cells in vivo is well documented. Unlike human immunodeficiency virus type 1 (HIV-1), HTLV-1 plasma RNA is sparse. The contribution of the “mitotic” spread of HTLV-1 compared with infectious spread of the virus to HTLV-1 viral burden in established infection is uncertain. Since extrachromosomal long terminal repeat (LTR) DNA circles are indicators of viral replication in HIV-1 carriers with undetectable plasma HIV RNA, we hypothesised that HTLV-1 LTR circles could indicate reverse transcriptase (RT) usage and infectious activity. 1LTR and 2LTR DNA circles were measured in HTLV-1 cell lines and peripheral blood mononuclear cells (PBMC) of asymptomatic carriers (ACs) and patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T cell leukaemia/lymphoma (ATLL). 1LTR DNA circles were detected in 14/20 patients at a mean of 1.38/100 PBMC but did not differentiate disease status nor correlate with HTLV-1 DNA copies. 2LTR DNA circles were detected in 30/31 patients and at higher concentrations in patients with HTLV-1-associated diseases, independent of HTLV-1 DNA load. In an incident case the 2LTR DNA circle concentration increased 2.1 fold at the onset of HAM/TSP compared to baseline. Detectable and fluctuating levels of HTLV-1 DNA circles in patients indicate viral RT usage and virus replication. Our results indicate HTLV-1 viral replication capacity is maintained in chronic infection and may be associated with disease onset.

CD25+CCR4+ cells as a marker of HTLV-1-infected cells in peripheral blood

The diagnosis of Human T cell Lymphotropic Virus type 1 (HTLV-1) infection is based on serology and PCR. The burden of HTLV-1 viral infection is monitored by measuring the proviral load [PVL] in peripheral blood. This assay is not useful for isolation of HTLV-1-infected cells. HTLV-1 Tax upregulates CD25 expression on infected CD4+ T cells. However, CD4+CD25+ is also the phenotype of activated T cells. CCR4 is a chemokine receptor expressed on subsets of activated T cells. HTLV-1-infected cells secrete the CCR4 ligand CCL22, and preferentially infect CCR4+ Cells. We studied the expression of CD25 and CCR4 in lymphocytes and its relation to PVL. We performed 11-colour immunophenotyping and HTLV-1 PVL quantification on 53 samples obtained from 36 HTLV-1 infected patients, [10 asymptomatic carriers (AC); 11 patients with HTLV-1-associated myelopathy (HAM); 4 co-infected with HIV; 11 with chronic/smouldering adult T-cell leukaemia/lymphoma (ATL)] and 3 uninfected individuals. Increased frequency of CD25+CCR4+ T cells [median: 14.7%, range: 1.95-91.3%] was observed in all HTLV-1 infected patients; the frequency correlated with PVL [Spearman r=0.89, P <0.001, Linear R2 = 0.61]. CD4+ and CD8+ cells from 12 patients [6ACs and 6 HAMs] were separated for PVL estimation. CD25+ CCR4+ cell counts correlated closely with PVL in CD4+ cells [Spearman r=0.816, P <0.001, linear R2=0.886] but not in CD8+ cells. Frequency of CD25+CCR4+ T cells correlated with PVL change in treated ATL patients. We conclude that CD25+CCR4+ cells can be used as a specific marker for monitoring of HTLV-1 infection and isolation of HTLV-1 infected cells.

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

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

The potential of CD127 as a prognostic and residual disease marker in chronic adult T cell leukaemia/lymphoma

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. FourATL 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<0.005) and disease remission status. CD127 expression appears to be useful to identify patients needing treatment and for monitoring the treatment of chronic ATL.