In vivo Expression of Human T-lymphotropic Virus Type 1 Basic Leucine-Zipper Protein Generates Specific CD8+ and CD4+ T-Lymphocyte Responses that Correlate with Clinical Outcome

MAJOR ARTICLE In vivo Expression of Human T-lymphotropic Virus Type 1 Basic Leucine-Zipper Protein Generates Specific CD81 and CD41 T-Lymphocyte Responses that Correlate with Clinical Outcome Silva Hilburn,1 Aileen Rowan,2 Maria-Antonietta Demontis,1 Aidan MacNamara,2 Becca Asquith,2 Charles RM. Bangham,2 and Graham P. Taylor1 Diseases and 2Immunology, Faculty of Medicine, Imperial College, Norfolk Place, London, England 1Infectious Background. The roles of the human T-lymphotropic virus type 1 (HTLV-1) basic leucine zipper (HBZ) gene are not clearly understood. We examined CD81 and CD41 T cell responses to HBZ and compared these with Tax responses. Method. Interferon (IFN)-c and interleukin (IL)-2–secreting T cells were detected by enzyme-linked immunosorbent spot (ELISpot) assays of freshly isolated peripheral blood mononuclear cells (PBMCs) stimulated with synthetic HBZ or Tax peptides. Ten patients with HTLV-1–associated myelopathy (HAM) and 20 asymptomatic HTLV-1 carriers (ACs), (10 high, 10 low viral load). Results. Of 30 study participants, 17 had detectable HBZ-specific CD41 T cells and 12 had HBZ-specific CD81 T cell responses. Detection of Tax-specific CD41 T cells (IL-2- or IFN-c-secreting) did not differ by disease status, but Tax-specific CD81 T cell responses were more commonly detected in patients with HAM. HBZ-specific CD41 or CD81 T cells were less likely to be detected than Tax-specific T cells. IL-2-secreting Tax-specific CD81 T cells, and IFN-c–secreting Tax-specific CD41 T cells were associated with HAM. Low viral load, asymptomatic HTLV-1 carriage was associated with IL-2–secreting CD81 T cells specific for HBZ. Conclusion. HBZ protein is expressed in vivo in patients with HAM and in ACs. Our results are consistent with the idea that the T cell response to HBZ plays an important part in restricting HTLV-1 viral load. 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, Received 11 June 2010; accepted 7 October 2010; electronically published 5 January 2011. Potential conflicts of interest: none reported. Reprints or correspondence: Dr Graham P. Taylor, Reader in Communicable Diseases, Section of Infectious Diseases, Imperial College, Norfolk Place, London W2 1PG, England (). The Journal of Infectious Diseases 2011;203:529–536 Ó The Author 2011. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: 1537-6613/2011/2034-0001$15.00 DOI: 10.1093/infdis/jiq078 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 HTLV1–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 The Importance of HBZ in vivo d JID 2011:203 (15 February) d 529 MATERIALS AND METHODS Patients and Cells The patient cohort is based at the National Centre for Human Retrovirology at St Mary’s Hospital, London. HTLV-1 infection was confirmed by Western blot and the diagnosis of HAM was made according to World Health Organization criteria [31]. Of 30 patients who participated in this study, 10 had HAM, and 20 were ACs, of whom 10 had HTLV-1 viral loads similar to patients with HAM (ie, .1 HTLV-1 DNA copy/100 PBMCs [%] and referred to as AC high) and 10 had HTLV-1 viral loads ,1% (AC low). Uninfected controls were relatives of patients who had 530 d JID 2011:203 (15 February) d Hilburn et al. been investigated for HTLV-1/2 infection at the Centre and laboratory staff. All patients and controls donated blood for research purposes after giving written informed consent. Preparation of T Lymphocytes PBMCs were isolated from fresh whole blood by density gradient centrifugation on Histopaque-1077 (Sigma-Aldrich), washed in phosphate buffer saline (PBS, Sigma-Aldrich), cryopreserved in 10% dimethyl sulphoxide (Sigma-Aldrich) and 90% heat inactivated fetal calf serum (FCS) (Gibco), and stored in liquid nitrogen until use. Cryopreserved cells were thawed and washed with cold PBS/0.5% FCS 3 times. PBMCs were depleted of CD81 or CD41 T cells using anti-CD4 or anti-CD8 magnetically labeled beads according to the manufacturer’s protocol (Miltenyi Biotec). Depleted PBMCs were washed once with complete medium (CM, RPMI 1640, 10% FCS, 1% penicillin/streptomycin [all from Gibco]). A fraction (105) of depleted cells were stained with CD8 and CD4 (Beckman-Coulter) surface markers and analyzed by flow cytometry to determine the percentage of CD41 and CD81 T-cells present in each sample. Peptide Libraries Peptides spanning Tax (ATK strain, [32]) and HBZ [18, 19, 33] proteins of HTLV-1 were commercially synthesized by Mimotopes Pty Ltd Europe and reconstituted in 50% acetonitrial:molecular-grade water (both Sigma-Aldrich) according to their instructions. HBZ peptides were based on HBZ SP-1 and designed to include the head domain of unspliced HBZ and HBZ SP-2 [18] and the 2 published amino acid polymorphisms, a lysine to glutamic acid at position 41 and isoleucine to threonine at position 99. Peptide purity was determined by reverse phase HPLC and ion spray mass spectroscopy. Tax peptides (n 5 57) and HBZ peptides (n 5 43) were overlapping 20-mers offset by 6 amino acids (accession number J02029 for Tax and DQ273132 for HBZ). All peptides used are presented in Table 1. Enzyme-linked Immunosorbent Spot Assays for Interferon-c and Interleukin-2 Flat-bottomed 96-well polyvinylidine difluoride (PVDF) membrane-backed plates (MAIPS4510, Millipore) were used. Each well was activated with 15 lL 35% ethanol and washed with sterile PBS. The wells were coated with 100 lL of the primary capture antibody, anti-IFN-c (mAb clone 1-D1K) or anti-IL-2 (mAb clone IL2-I) (all mAbs from MabTech), each at a concentration of 10 lg/mL. The antibody was allowed to bind overnight at 4oC. Plates were washed (unless otherwise stated, 6 times with PBS) and blocked with CM for 1 h at room temperature (RT). The blocking solution was discarded and 105 cells were added t (...truncated)