Aviva Witkover

The Influence of HLA Class I Alleles and Heterozygosity on the Outcome of Human T Cell Lymphotropic Virus Type I Infection

The inflammatory disease human T cell lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM/TSP) occurs in only 1–2% of HTLV-I-infected individuals and is associated with a high provirus load of HTLV-I. We hypothesize that a person’s risk of developing HAM/TSP depends upon the efficiency of their immune response to the virus, which differs between individuals because of polymorphism in genes that influence this response. Previously we showed that the possession of HLA-A*02 was associated with a lower risk of HAM/TSP, and with a lower provirus load in healthy carriers of HTLV-I. However, HLA-A*02 did not account for all the observed difference in the risk of HAM/TSP. Here we present evidence, in the same study population in Japan, that HLA-Cw*08 was also associated with disease protection (probability value, two-tailed test = 0.002) and with a lower proviral load in healthy carriers. Possession of the A*02 and/or Cw*08 genes prevented 36% of potential HAM/TSP cases. In contrast, HLA-B*5401 was associated with a higher susceptibility to HAM/TSP (probability value, two-tailed test = 0.0003) and with a higher provirus load in HAM/TSP patients. At a given provirus load, B*5401 appeared to increase the risk of disease. The fraction of HAM/TSP cases attributable to B*5401 was 17%. Furthermore, individuals who were heterozygous at all three HLA class I loci have a lower HTLV-I provirus load than those who were homozygous at one or more loci. These results are consistent with the proposal that a strong class I-restricted CTL response to HTLV-I reduces the proviral load and hence the risk of disease.

The Role of CTLs in Persistent Viral Infection: Cytolytic Gene Expression in CD8 Lymphocytes Distinguishes between Individuals with a High or Low Proviral Load of Human T Cell Lymphotropic Virus Type 1

The proviral load in human T cell lymphotropic virus type 1 (HTLV-1) infection is typically constant in each infected host, but varies by >1000-fold between hosts and is strongly correlated with the risk of HTLV-1-associated inflammatory disease. However, the factors that determine an individual’s HTLV-1 proviral load remain uncertain. Experimental evidence from studies of host genetics, viral genetics, and lymphocyte function and theoretical considerations suggest that a major determinant of the equilibrium proviral load is the CD8+ T cell response to HTLV-1. In this study, we tested the hypothesis that the gene expression profile in circulating CD8+ and CD4+ lymphocytes distinguishes between individuals with a low proviral load of HTLV-1 and those with a high proviral load. We show that circulating CD8+ lymphocytes from individuals with a low HTLV-1 proviral load overexpressed a core group of nine genes with strong functional coherence: eight of the nine genes encode granzymes or other proteins involved in cell-mediated lysis or Ag recognition. We conclude that successful suppression of the HTLV-1 proviral load is associated with strong cytotoxic CD8+ lymphocyte activity in the peripheral blood.

Clonality of HTLV-2 in Natural Infection

Human T-lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) both cause lifelong persistent infections, but differ in their clinical outcomes. HTLV-1 infection causes a chronic or acute T-lymphocytic malignancy in up to 5% of infected individuals whereas HTLV-2 has not been unequivocally linked to a T-cell malignancy. Virus-driven clonal proliferation of infected cells both in vitro and in vivo has been demonstrated in HTLV-1 infection. However, T-cell clonality in HTLV-2 infection has not been rigorously characterized. In this study we used a high-throughput approach in conjunction with flow cytometric sorting to identify and quantify HTLV-2-infected T-cell clones in 28 individuals with natural infection. We show that while genome-wide integration site preferences in vivo were similar to those found in HTLV-1 infection, expansion of HTLV-2-infected clones did not demonstrate the same significant association with the genomic environment of the integrated provirus. The proviral load in HTLV-2 is almost confined to CD8+ T-cells and is composed of a small number of often highly expanded clones. The HTLV-2 load correlated significantly with the degree of dispersion of the clone frequency distribution, which was highly stable over ∼8 years. These results suggest that there are significant differences in the selection forces that control the clonal expansion of virus-infected cells in HTLV-1 and HTLV-2 infection. In addition, our data demonstrate that strong virus-driven proliferation per se does not predispose to malignant transformation in oncoretroviral infections.

Plasma proteome analysis in HTLV-1-associated myelopathy/tropical spastic paraparesis

BackgroundHuman T lymphotropic virus Type 1 (HTLV-1) causes a chronic inflammatory disease of the central nervous system known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM) which resembles chronic spinal forms of multiple sclerosis (MS). The pathogenesis of HAM remains uncertain. To aid in the differential diagnosis of HAM and to identify pathogenetic mechanisms, we analysed the plasma proteome in asymptomatic HTLV-1 carriers (ACs), patients with HAM, uninfected controls, and patients with MS. We used surface-enhanced laser desorption-ionization (SELDI) mass spectrometry to analyse the plasma proteome in 68 HTLV-1-infected individuals (in two non-overlapping sets, each comprising 17 patients with HAM and 17 ACs), 16 uninfected controls, and 11 patients with secondary progressive MS. Candidate biomarkers were identified by tandem Q-TOF mass spectrometry.ResultsThe concentrations of three plasma proteins - high [β2-microglobulin], high [Calgranulin B], and low [apolipoprotein A2] - were specifically associated with HAM, independently of proviral load. The plasma [β2-microglobulin] was positively correlated with disease severity.ConclusionsThe results indicate that monocytes are activated by contact with activated endothelium in HAM. Using β2-microglobulin and Calgranulin B alone we derive a diagnostic algorithm that correctly classified the disease status (presence or absence of HAM) in 81% of HTLV-1-infected subjects in the cohort.

Balancing the Robustness and Predictive Performance of Biomarkers

Recent studies have highlighted the importance of assessing the robustness of putative biomarkers identified from experimental data. This has given rise to the concept of stable biomarkers, which are ones that are consistently identified regardless of small perturbations to the data. Since stability is not by itself a useful objective, we present a number of strategies that combine assessments of stability and predictive performance in order to identify biomarkers that are both robust and diagnostically useful. Moreover, by wrapping these strategies around logistic regression classifiers regularized by the elastic net penalty, we are able to assess the effects of correlations between biomarkers upon their perceived stability. We use a synthetic example to illustrate the properties of our proposed strategies. In this example, we find that: (i) assessments of stability can help to reduce the number of false-positive biomarkers, although potentially at the cost of missing some true positives; (ii) combining assessments of stability with assessments of predictive performance can improve the true positive rate; and (iii) correlations between biomarkers can have adverse effects on their stability and hence must be carefully taken into account when undertaking biomarker discovery. We then apply our strategies in a proteomics context to identify a number of robust candidate biomarkers for the human disease HTLV1-associated myelopathy/tropical spastic paraparesis (HAM/TSP).

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.

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. 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.

Free serum haemoglobin is associated with brain atrophy in secondary progressive multiple sclerosis

Background: A major cause of disability in secondary progressive multiple sclerosis (SPMS) is progressive brain atrophy, whose pathogenesis is not fully understood. The objective of this study was to identify protein biomarkers of brain atrophy in SPMS. Methods: We used surface-enhanced laser desorption-ionization time-of-flight mass spectrometry to carry out an unbiased search for serum proteins whose concentration correlated with the rate of brain atrophy, measured by serial MRI scans over a 2-year period in a well-characterized cohort of 140 patients with SPMS. Protein species were identified by liquid chromatography-electrospray ionization tandem mass spectrometry. Results: There was a significant (p<0.004) correlation between the rate of brain atrophy and a rise in the concentration of proteins at 15.1 kDa and 15.9 kDa in the serum. Tandem mass spectrometry identified these proteins as alpha-haemoglobin and beta-haemoglobin, respectively. The abnormal concentration of free serum haemoglobin was confirmed by ELISA (p<0.001). The serum lactate dehydrogenase activity was also highly significantly raised (p<10 -12) in patients with secondary progressive multiple sclerosis. Conclusions: The results are consistent with the following hypothesis. In progressive multiple sclerosis, low-grade chronic intravascular haemolysis releases haemoglobin into the serum; the haemoglobin is subsequently translocated into the central nervous system (CNS) across the damaged blood-brain barrier. In the CNS, the haemoglobin and its breakdown products, including haem and iron, contribute to the neurodegeneration and consequent brain atrophy seen in progressive disease. We postulate that haemoglobin is a source of the iron whose deposition along blood vessels in multiple sclerosis plaques is associated with neurodegeneration. If so, then chelators of haemoglobin, rather than chelators of free serum iron, may be effective in preventing this neurodegeneration.

The human leukemia virus HTLV-1 alters the structure and transcription of host chromatin in cis

Chromatin looping controls gene expression by regulating promoter-enhancer contacts, the spread of epigenetic modifications, and the segregation of the genome into transcriptionally active and inactive compartments. We studied the impact on the structure and expression of host chromatin by the human retrovirus HTLV-1. We show that HTLV-1 disrupts host chromatin structure by forming loops between the provirus and the host genome; certain loops depend on the critical chromatin architectural protein CTCF, which we recently discovered binds to the HTLV-1 provirus. We show that the provirus causes two distinct patterns of abnormal transcription of the host genome in cis: bidirectional transcription in the host genome immediately flanking the provirus, and clone-specific transcription in cis at non-contiguous loci up to >300 kb from the integration site. We conclude that HTLV-1 causes insertional mutagenesis up to the megabase range in the host genome in >104 persistently-maintained HTLV-1+ T-cell clones in vivo.

Clonality of HTLV-2 in natural infection

We recently developed a high-throughput sequencing method for analysis and quantification of HTLV-1 integration sites in the host genome (Gillet et al, 2011, Blood). Using this method we investigated the effect of the genomic environment on integration targeting, clonal expansion and spontaneous HTLV-1 proviral expression (Gillet et al, 2011, Blood, Melamed et al, 2013, PLoS Pathogens). HTLV-2 preferentially infects CD8+ T cells, with a minority of the proviral load in CD4+ T cells. Here we describe the use of our high-throughput technique to investigate the distribution of HTLV-2 proviral integration sites in the host genome, in peripheral blood mononuclear cell (PBMC) DNA of HTLV-2 infected individuals (n=28). We also mapped and quantified proviral integration sites separately in flow-sorted CD4+CD8- and CD4-CD8+ populations. We quantified the clone frequency distribution and clonal survival over time in 10 individuals, using samples from 2 time points separated by a median of 10 years. The results show that the clone frequency distribution of HTLV-2 in PBMCs is distinct from that of HTLV-1 and resembles that of HTLV-1-infected CD8+ T cells. These results suggest that in both HTLV-1 and HTLV-2 infections, there is a greater degree of selective oligoclonal clonal expansion in infected CD8+ T cells than in CD4+ T cells. We are now investigating the selection forces that underlie this dichotomy between T cell lineages.

Impact of Hepatitis B Virus Coinfection on Human T-Lymphotropic Virus Type 1 Clonality in an Indigenous Population of Central Australia

Human T-cell lymphotropic virus type 1 (HTLV-1) and hepatitis B virus coinfection is frequent in certain Indigenous Australian populations, but its outcome remains unknown. We report a higher degree of HTLV-1 clonal expansion than in those with HTLV-1 alone.