Abstract
Background. The roles of the human T-lymphotropic virus type 1 (HTLV-1) basic leucine zipper (HBZ) gene are not clearly understood. We examined CD8+ and CD4+ T cell responses to HBZ and compared these with Tax responses.
Method. Interferon (IFN)-γ 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 CD4+ T cells and 12 had HBZ-specific CD8+ T cell responses. Detection of Tax-specific CD4+ T cells (IL-2- or IFN-γ-secreting) did not differ by disease status, but Tax-specific CD8+ T cell responses were more commonly detected in patients with HAM. HBZ-specific CD4+ or CD8+ T cells were less likely to be detected than Tax-specific T cells. IL-2-secreting Tax-specific CD8+ T cells, and IFN-γ–secreting Tax-specific CD4+ T cells were associated with HAM. Low viral load, asymptomatic HTLV-1 carriage was associated with IL-2–secreting CD8+ 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, 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.
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 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 CD8+ or CD4+ 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 CD4+ and CD8+ 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 = 57) and HBZ peptides (n = 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.
Table 1.
Peptide Sequences for Tax and HTLV-1 Basic Leucine Zipper Proteins
| HBZ Peptides | |
| Tax Peptides | Accession No. DQ2732132 (Peptides 1–33) |
| 1 MAHFPGFGQSLLFGYPVYVF | 1 MAASGLFRCLPVSCPEDLLV |
| 2 FGQSLLFGYPVYVFGDCVQG | 2 FRCLPVSCPEDLLVEELVDG |
| 3 FGYPVYVFGDCVQGDWCPIS | 3 SCPEDLLVEELVDGLLSLEE |
| 4 VFGDCVQGDWCPISGGLCSA | 4 LVEELVDGLLSLEEELKDKE |
| 5 QGDWCPISGGLCSARLHRHA | 5 DGLLSLEEELKDKEEEKAVL |
| 6 ISGGLCSARLHRHALLATCP | 6 EEELKDKEEEKAVLDGLLSL |
| 7 SARLHRHALLATCPEHQITW | 7 KEEEKAVLDGLLSLEEESRG |
| 8 HALLATCPEHQITWDPIDGR | 8 VLDGLLSLEEESRGRLRRGP |
| 9 CPEHQITWDPIDGRVIGSAL | 9 SLEEESRGRLRRGPPGEKAP |
| 10 TWDPIDGRVIGSALQFLIPR | 10 RGRLRRGPPGEKAPPRGETH |
| 11 GRVIGSALQFLIPRLPSFPT | 11 GPPGEKAPPRGETHRDRQRR |
| 12 ALQFLIPRLPSFPTQRTSKT | 12 APPRGETHRDRQRRAEEKRK |
| 13 PRLPSFPTQRTSKTLKVLTP | 13 THRDRQRRAEEKRKRKKERE |
| 14 PTQRTSKTLKVLTPPITHTT | 14 RRAEEKRKRKKEREKEEEKQ |
| 15 KTLKVLTPPITHTTPNIPPS | 15 RKRKKEREKEEEKQIAEYLK |
| 16 TPPITHTTPNIPPSFLQAMR | 16 REKEEEKQIAEYLKRKEEEK |
| 17 TTPNIPPSFLQAMRKYSPFR | 17 KQIAEYLKRKEEEKARRRRR |
| 18 PSFLQAMRKYSPFRNGYMEP | 18 LKRKEEEKARRRRRAEKKAA |
| 19 MRKYSPFRNGYMEPTLGQHL | 19 EKARRRRRAEKKAADVARRK |
| 20 FRNGYMEPTLGQHLPTLSFP | 20 RRAEKKAADVARRKQEEQER |
| 21 EPTLGQHLPTLSFPDPGLRP | 21 AADVARRKQEEQERRERKWR |
| 22 HLPTLSFPDPGLRPQNLYTL | 22 RKQEEQERRERKWRQGAEKA |
| 23 FPDPGLRPQNLYTLWGGSVV | 23 ERRERKWRQGAEKAKQHSAR |
| 24 RPQNLYTLWGGSVVCMYLYQ | 24 WRQGAEKAKQHSARKEKMQE |
| 25 TLWGGSVVCMYLYQLSPPIT | 25 KAKQHSARKEKMQELGIDGY |
| 26 VVCMYLYQLSPPITWPLLPH | 26 ARKEKMQELGIDGYTRQLEG |
| 27 YQLSPPITWPLLPHVIFCHP | 27 QELGIDGYTRQLEGEVESLE |
| 28 ITWPLLPHVIFCHPGQLGAF | 28 GYTRQLEGEVESLEAERRKL |
| 29 PHVIFCHPGQLGAFLTNVPY | 29 EGEVESLEAERRKLLQEKED |
| 30 HPGQLGAFLTNVPYKRIEEL | 30 LEAERRKLLQEKEDLMGEVN |
| 31 AFLTNVPYKRIEELLYKISL | 31 KLLQEKEDLMGEVNYWQGRL |
| 32 PYKRIEELLYKISLTTGALI | 32 EDLMGEVNYWQGRLEAMWLQ |
| 33 ELLYKISLTTGALIILPEDC | 33 TSRVRQSVESRLSLGLFRCLa |
| 34 SLTTGALIILPEDCLPTTLF | 34 SVESRLSLGLFRCLPVSCPE |
| 35 LIILPEDCLPTTLFQPARAP | 35 SLGLFRCLPVSCPEDLLVEE |
| 36 DCLPTTLFQPARAPVTLTAW | 36 MVNFVSVGLFRCLPVSCPEDb |
| 37 LFQPARAPVTLTAWQNGLLP | 37 VGLFRCLPVSCPEDLLVEEL |
| 38 APVTLTAWQNGLLPFHSTLT | 38 DGLLSLEEELKDKEEEEAVLc |
| 39 AWQNGLLPFHSTLTTPGLIW | 39 EEELKDKEEEEAVLDGLLSL |
| 40 LPFHSTLTTPGLIWTFTDGT | 40 KEEEEAVLDGLLSLEEESRG |
| 41 LTTPGLIWTFTDGTPMISGP | 41 RKRKKEREKEEEKQTAEYLKd |
| 42 IWTFTDGTPMISGPCPKDGQ | 42 REKEEEKQTAEYLKRKEEEK |
| 43 GTPMISGPCPKDGQPSLVLQ | 43 KQTAEYLKRKEEEKARRRRR |
| 44 GPCPKDGQPSLVLQSSSFIF | |
| 45 GQPSLVLQSSSFIFHKFQTK | |
| 46 LQSSSFIFHKFQTKAYHPSF | |
| 47 IFHKFQTKAYHPSFLLSHGL | |
| 48 TKAYHPSFLLSHGLIQYSSF | |
| 49 SFLLSHGLIQYSSFHSLHLL | |
| 50 GLIQYSSFHSLHLLFEEYTN | |
| 51 SFHSLHLLFEEYTNIPISLL | |
| 52 LLFEEYTNIPISLLFNEKEA | |
| 53 TNIPISLLFNEKEADDNDHE | |
| 54 LLFNEKEADDNDHEPQISPG | |
| 55 EADDNDHEPQISPGGLEPPS | |
| 56 HEPQISPGGLEPPSEKHFRE | |
| 57 QISPGGLEPPSEKHFRETEV |
NOTE. HTLV-1, human T-lymphotropic virus type 1; HBZ, HTLV-1 basic leucine zipper; VL, viral load; IFN, interferon; IL, interleukin; AC, asymptomatic HTLV-1 carriers.
Peptides 33–35 HBZ-SP2 head domain (Cavanagh et al [18]).
Peptides 36–37 unspliced HBZ head domain (Cavanagh et al [18]).
Peptides 38–40 amino acid change (in bold) from peptides 5–7 (accession no. AB219938).
Peptides 41–43 amino acid change (in bold) from peptides 15–17 (accession no. AB219938).
Enzyme-linked Immunosorbent Spot Assays for Interferon-γ and Interleukin-2
Flat-bottomed 96-well polyvinylidine difluoride (PVDF) membrane-backed plates (MAIPS4510, Millipore) were used. Each well was activated with 15 μL 35% ethanol and washed with sterile PBS. The wells were coated with 100 μL of the primary capture antibody, anti-IFN-γ (mAb clone 1-D1K) or anti-IL-2 (mAb clone IL2-I) (all mAbs from MabTech), each at a concentration of 10 μg/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 to each well. In CD4+ T cell assays, to duplicate wells stimulatory monoclonal antibodies were added, anti-CD49d (clone HP2/1) and anti-CD28 (clone 28.2) (both from Becton Dickinson), each at a final concentration of 0.5 μg/mL, and peptide pools (either the Tax pool or the HBZ pool) containing each peptide at a final concentration of 2 μmol/L, in a total volume of 100 μL CM. CD8+ T cell assays were prepared in the same way except for the omission of anti-CD49d mAb [13]. To induce nonspecific cytokine production by PBMCs depleted of CD8+ T cells, we added 0.1 ng/mL phorbol myristate acetate (PMA) and 0.5μg/mL calcium ionophore A23187 (both from Sigma-Aldrich) to the positive control wells. The plates were incubated at 37oC in 5% CO2 for 6 h.
After incubation, the cells were discarded and the plate washed. A second-layer biotinylated antibody specific to either IFN-γ (clone 7-B6-1) or IL-2 (clone IL2-II) at 1μg/mL in .5% FCS in 100 μL PBS was added for 2 h at RT. Excess antibody was discarded and the plate washed. Streptavidin alkaline phosphatase conjugate (1:1000 dilution in 100 μL PBS/0.5% FCS) was added for 1 h at RT. The solution was discarded and the plate washed. Chromogenic alkaline phosphatase substrate (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate [NBT/BCIP], Europa Bioproducts) was added and placed the plates for 10–15 min at RT in the dark after which the reaction was terminated by washing the plates with tap water. Plates were allowed to dry and the number of spots per well were counted using AID 3.5 software (AID ELIspot reader, Germany). A response was designated positive if the number of spots exceeded the mean + 2 standard deviations (SDs) of the spot count in the negative wells (no peptide added). The frequencies of cytokine-producing CD8+ T cells or CD4+ T cells are presented as the number of spots per 105 CD8+ or CD4+ T cells [34].
Human T-Lymphotropic Virus Type 1 Viral Load Quantification
Genomic DNA was extracted from PBMCs using QIAamp DNA mini kit (Qiagen). DNA extracted from MT-2 cell lines was serially diluted 10-fold to generate standard curves ranging from 2 to 20,000 β-globin copies and 7 to 70,000 HTLV-1 tax copies. Real time polymerase chain reaction (PCR) was performed using LightCycler FastStart DNA MasterPLUS SYBR Green 1 (Roche), 0.6U LightCycler Uracil DNA Glycosylase (Roche, Germany), 5 pmol of each forward (F) and reverse (R) primer for Tax (F: 5′-CGGATACCCAGTCTACGTGT-3′, R: 5′-GAGCCGATAACGCGTCCATCG-3′) and β-globin (F: 5′-GCAAGGTGAACGTGGATG-3′, R: 5′-TAAGGGTGGAAAATTGACC-3′), 750 ng - 7.5 ng DNA per reaction, in a Roche LightCycler 1.5. Thermal cycler conditions were: denaturation at 40oC for 8 min and 95oC for 10 min, followed by 45 amplification cycles of 95oC for 10 s, 58oC for 5 s, 72oC for 8 s, and a single 10 s acquisition at 85oC for Tax and 81oC for β-globin. A final melting curve was performed with continuous acquisition between 60 and 95oC. HTLV-1 DNA copy number was calculated from the standard curves and standardized to the number of β-globin copies divided by 2 and reported as HTLV-1 DNA copies/100 PBMCs (%).
Statistical Analysis
Data were analysed in GraphPad PRISM, version 5.01 (GraphPad software) using nonparametric statistical tests to limit the assumptions made about the distribution of the data. Categorical comparisons of Tax and HBZ responses were made using the χ2 test, and Fisher exact test was used for comparison between clinical states. The Mann–Whitney U test was used to compare continuous variables.
RESULTS
Human T-Lymphotropic Virus Type 1 Viral Load (Table 2)
Table 2.
Human T-Lymphotropic Virus Type 1 (HTLV-1) Tax and HTLV-1 Basic Leucine Zipper–Specific CD8+ and CD4+ T Cell Responses in HTLV-1–Infected Subjects
| CD8+ T cell IFN-γ responsesb | CD8+ T cell IL-2 responsesb | CD4+ T cell IFN-γ responsesb | CD4+ T cell IL-2 responsesb | ||||||
| Subjects | VLa | Tax | HBZ | Tax | HBZ | Tax | HBZ | Tax | HBZ |
| HAM | |||||||||
| TCG | 7.69 | 45 | 14 | 0 | 0 | 232 | 0 | 210 | 0 |
| TAL | 46.15 | 46 | 0 | 44 | 0 | 19 | 4 | 16 | 9 |
| TCL | 6.7 | 295 | 0 | 39 | 0 | 5 | 0 | 0 | 0 |
| TCO | 4.91 | 0 | 0 | 2 | 0 | 31 | 0 | 17 | 0 |
| TBU | 3.12 | 111 | 0 | 2 | 0 | 21 | 12 | 12 | 0 |
| TCQ | 13.79 | 0 | 0 | 2 | 9 | 23 | 5 | 17 | 0 |
| TBO | 13.87 | 763 | 0 | 30 | 0 | 5 | 0 | 0 | 0 |
| TAA | 6.87 | 814 | 0 | 7 | 0 | 0 | 0 | 0 | 0 |
| TCF | 7.17 | 24 | 0 | 4 | 0 | 0 | 0 | 0 | 0 |
| TBW | 15.83 | 7 | 3 | 10 | 3 | 15 | 0 | 46 | 14 |
| Median | 7.17 | 79 | -c | 7 | -c | 20 | 5 | 17 | -c |
| AC high | |||||||||
| HCH | 9.5 | 0 | 0 | 0 | 0 | 63 | 0 | 0 | 0 |
| HEZ | 5.1 | 80 | 0 | 0 | 0 | 5 | 4 | 5 | 4 |
| HES | 11.4 | 44 | 81 | 0 | 0 | 18 | 0 | 7 | 7 |
| HEI | 3.4 | 0 | 20 | 0 | 0 | 4 | 0 | 17 | 6 |
| HBX | 11.25 | 0 | 0 | 0 | 0 | 64 | 27 | 15 | 5 |
| HDR | 1.92 | 77 | 17 | 0 | 0 | 0 | 3 | 9 | 0 |
| HCP | 11.72 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| HAY | 8.58 | 0 | 0 | 0 | 0 | 0 | 0 | 6 | 0 |
| HBT | 2.44 | 292 | 0 | 13 | 0 | 17 | 0 | 2 | 0 |
| HBE | 7.88 | 5 | 0 | 1 | 0 | 0 | 0 | 0 | 0 |
| Median | 8.23 | 77 | 20 | -c | NDd | 5 | 4 | 6 | 6 |
| AC low | |||||||||
| HBA | 0.001 | 0 | 19 | 0 | 6 | 0 | 0 | 0 | 0 |
| HDH | 0.13 | 97 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| HDN | 0.72 | 177 | 0 | 5 | 5 | 18 | 7 | 12 | 33 |
| HBV | 0.01 | 0 | 0 | 78 | 0 | 8 | 0 | 43 | 8 |
| HAE | 0.03 | 17 | 23 | 0 | 0 | 26 | 2 | 0 | 0 |
| HCL | 0.17 | 246 | 3 | 53 | 5 | 0 | 0 | 4 | 0 |
| HFL | 0.1 | 2 | 2 | 0 | 0 | 0 | 0 | 0 | 2 |
| HX | 0.09 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
| HAD | 0.47 | 277 | 70 | 87 | 7 | 22 | 21 | 5 | 0 |
| HDT | 0.23 | 0 | 0 | 0 | 0 | 7 | 0 | 7 | 8 |
| Median | 0.115 | 137 | 19 | 66 | 6 | 18 | 7 | 7 | 8 |
NOTE. IFN, interferon; IL, interleukin; HAM, HTLV-1-associated myelopathy; AC, asymptomatic HTLV-1 carriers; HBZ, HTLV-1 basic leucine zipper.
Data presented as spot-forming cells per 1E5 CD8 or CD4 T cells for each cytokine measured.
Viral load is presented as HTLV-1 Tax DNA copies per 100 PBMCs.
No median value calculated.
Not detected.
As per the study design, there was no statistical significance in the median viral load between patients with HAM (7.17%, range 3.12%–46.14%) and AC high (8.23%, range 1.92%–11.72%), whereas AC low (.115%, range .001%–.72%) had significantly lower viral loads than AC high or patients with HAM (P = .002 in each case; Mann–Whitney U test).
Detection of Any Tax or HBZ Immune Responses (Table 2, 3A and 3B)
Table 3.
Number of Subjects with Detectable Interferon-γ or Interleukin-2 Response to Tax and HBZ
| A) Tax | ||||||||
| CD8+ | CD4+ | |||||||
| IFN- γ | IL-2 | Dual responsea | Any responseb | IFN- γ | IL-2 | Dual responsea | Any responseb | |
| HAM (n = 10) | 8 | 9 | 7 | 10 | 8 | 6 | 6 | 8 |
| AC high (n = 10) | 5 | 2 (P = .003)c | 2 (P = .03)c | 5 (P = .02)c | 6 | 7 | 6 | 8 |
| AC low (n = 10) | 6 | 4 (P = .03)c | 3 | 7 | 5 | 5 | 3 | 6 |
| HAM (n = 10) | 8 | 9 | 7 | 10 | 8 | 6 | 6 | 8 |
| AC (n = 20) | 11 | 6 (P = .003)c | 5 (P = .02) c | 12 (P = .02) c | 11 | 12 | 9 | 14 |
| HVLd patients (n = 20) | 13 | 11 | 9 | 15 | 14 | 13 | 12 | 16 |
| LVLe patients (n = 10) | 6 | 4 | 3 | 7 | 5 | 5 | 3 | 6 |
| B) HBZ | ||||||||
| CD8+ | CD4+ | |||||||
| IFN-γ | IL-2 | Dual responsea | Any responseb | IFN-γ | IL-2 | Dual responsea | Any responseb | |
| HAM (n = 10) | 2 | 2 | 1 | 3 | 3 | 2 | 1 | 4 |
| AC high (n = 10) | 3 | 0 | 0 | 3 | 3 | 4 | 2 | 5 |
| AC low (n = 10) | 5 | 4 (P = .04)c | 3 | 6 | 3 | 5 | 1 | 7 |
| HAM (n = 10) | 2 | 2 | 1 | 3 | 3 | 2 | 1 | 4 |
| AC (n = 20) | 8 | 4 | 3 | 9 | 6 | 9 | 3 | 12 |
| HVLd patients (n = 20) | 5 | 2 | 1 | 6 | 6 | 6 | 3 | 11 |
| LVLe patients (n = 10) | 5 | 4 (P = .04)c | 3 | 6 | 3 | 5 | 1 | 7 |
NOTE. HBZ, HTLV-1 basic leucine zipper; IFN, interferon; IL, interleukin; HAM, HTLV-1-associated myelopathy; AC, asymptomatic HTLV-1 carriers; HVL, high viral load; LVL, low viral load.
Dual response refers to patients with BOTH IFN-γ and IL-2 responses and not to cells that express both cytokines.
Any response refers to patients with EITHER IFN-γ or IL-2 response.
Fisher exact test 1 tail P values quotes when significant and they refer to the values above the parenthesis.
HVL: High viral load patients are patients with HAM and AC with viral load >1%.
LVL: Low viral load patients refers to asymptomatic carriers with viral load <1%.
Tax-specific T cell responses were detected in all patients with HAM. Although 2 out of 10 patients lacked a detectable CD4+ T cell response to Tax, usually both IFN-γ and IL-2 responses were detected in the same patient. Tax-specific CD8+ T cells were also detected in most HTLV-1 asymptomatic carriers, but CD8+ T cell IL-2 responses, and hence dual responses, were significantly less common in symptomatic carriers. However, this was driven by ACs with high viral load in whom Tax-specific IL-2 CD8+ T cell responses were least common. Furthermore, CD8+ T cell Tax responses differed by clinical condition and not by viral load. Tax-specific CD4+ T cell responses significantly differed between AC with low viral load and patients with HAM or ACs with high viral load regardless of whether IFN-γ or IL-2 responses were considered (Table 3A).
HBZ-specific CD4+ T cell responses were detected in a minority of patients with no significant difference between ACs high, ACs low or patients with HAM regardless of cytokine (IFN-γ or IL-2). HBZ responses were much less likely to be detected than Tax-specific CD8+ T cell responses. However, HBZ-specific CD8+ T cell IL-2 responses were significantly more commonly detected in AC with low viral load than patients with high viral load (P = .04).
We next examined the size of the response, where detected, by quantifying the number of spot-forming cells specific for HBZ or Tax (Table 4). CD8+ T cell Tax responses detected in patients with HAM and in ACs with high viral load were predominantly IFN-γ secreting. However, when detected the absolute number of Tax-specific CD8+ IL-2–secreting SFC was higher in AC low than in subjects (patients with HAM and AC high) with high viral load, although they remained fewer than IFN-γ–producing Tax-specific CD8+ T cells.
Table 4.
Median Frequency and Range of Tax- or HBZ-Specific CD8+ and CD4+ T Cells
| Tax |
HBZ |
|||||||
| CD8 |
CD4 |
CD8 |
CD4 |
|||||
| IFN-γ | IL-2 | IFN-γ | IL-2 | IFN-γ | IL-2 | IFN-γ | IL-2 | |
| HAM (n = 10) | 79 (7–814) | 7 (2 - 44) | 20 (5–232) | 17 (12–210) | -a (3 and 14) | -a (3 and 9) | 5 (4–12) | -a (9 and 14) |
| AC high (n = 10) | 77 (5–295) | -a (1 and 13) | 5 (4–46) | 6 (2–17) | 20 (17–81) | NDb | 4 (3–27) | 6 (4–7) |
| AC low (n = 10) | 137 (2–277) | 66 (5–87) | 18 (7–26) | 7 (4–43) | 19 (2–70) | 6 (5–7) | 7 (2–21) | 8 (1–33) |
| All participants (n = 30) | 80 (2–814) | 10 (1–87) | 18 (4–232) | 12 (2- 210) | 18 (2–81) | 6 (3–9) | 7 (2–27) | 7 (1–33) |
| HAM (n = 10) | 79 (7–814) | 7 (2 - 44) | 20 (5–232) | 17 (12–210) | -a (3 and 14) | -a (3 and 9) | 5 (4–12) | -a (9 and 14) |
| AC (n = 20) | 80 (2 -295) | 33 (1–87) | 28 (4–46) | 7 (2–43) | 20 (2–81) | 6 (5–7) | 6 (2–27) | 6 (1–33) |
| HVLc patients (n = 20) | 77 (5–814) | 7 (1–44) | 19 (4–232) | 15 (2–210) | 19 (3–81) | 6 (3–9) | 5 (3–27) | 7 (4–14) |
| LVLd patients (n = 10) | 137 (2–277) | 66 (5–87) | 18 (7–26) | 7 (4–43) | 19 (2–70) | 6 (5–7) | 7 (2–21) | 8 (1–33) |
NOTE. HBZ, HTLV-1 basic leucine zipper; IFN, interferon; IL, interleukin; HAM, HTLV-1-associated myelopathy; AC, asymptomatic HTLV-1 carriers; HVL, high viral load; LVL, low viral load.
Median frequency and in parenthesis the range of frequencies
No median value calculated.
Not detected.
HVL: High viral load patients with viral load more than 1%, which includes patients with HAM and AC.
LVL: Low viral load patients with viral load less than 1% and only includes asymptomatic carriers.
CD4+ T cell Tax responses were numerically less than CD8+ T cell Tax responses with no significant difference between patient categories.
The size of the HBZ responses was generally smaller than the Tax responses (comparing the number of spot forming cells [SFC]) for both CD4+ T cells and CD8+ T cells and did not differ by cytokine secreted or disease state.
DISCUSSION
HBZ is Expressed In Vivo; T Cell Response to HBZ is Associated with Low Viral Load and Asymptomatic Carriage
Detection of HBZ-specific CD4+ and CD8+ T cell responses ex vivo in these short-term ELISpot assays implies expression of this protein in vivo in asymptomatic carriers as well as in patients with HAM. Importantly, CD8+ T cell responses to HBZ were found significantly more frequently in patients with low HTLV-1 viral load. This accords with our recent immunogenetic study [30], in which HLA Class 1 genotypes that predicted efficient T cell recognition of HBZ were associated with low viral load. In our study, 4 of 10 ACs with a low viral load (median, .1%) had a detectable CD8+ T cell IL-2 response to HBZ compared with none of the 10 ACs with a high viral load (median, 8.2%; Fisher exact test P = .08). Suemori et al reported that although HBZ can be presented by HLA-A*0201, HBZ-specific CTLs were not able to discriminate between HTLV-1-infected and uninfected cells because HBZ is expressed at low levels. In their study, HBZ-specific CTLs were scarcely detectable using HLA-A*0201/HBZ 26-34 tetramer analysis on PBMCs from 6 HLA-A*0201-positive patients, 5 with ATLL and 1 AC [35].
Previously, the Tax protein has been shown to be immunodominant in the CD8+ T cell response to HTLV-1 [13]. Our finding that Tax-specific responses were detected in all patients with HAM and in most asymptomatic carriers is consistent with previous reports [13, 34]. Furthermore, Tax-specific responses (both IFN-γ and IL-2) were found in both CD4+ and CD8+ T cell populations, and, when tax responses were present, the frequency of responding cells was high compared with HBZ-specific T cells. There is considerable interest in whether it is the frequency (ie, the numerical size of the response) or the efficacy of the response (as measured by lysis of infected cells) that is important in controlling viral replication [36]. Like Suemori et al [35], we find, where detected at all, a very low frequency of HBZ specific CD8+ T cells. However, our comparison of patient categories both by disease and by viral load leads us to conclude that even a very low frequency response to a critical target antigen may contribute significantly to the control of viral replication.
IL-2 Responses May Be More Protective than IFN-γ Responses
A previous study has suggested that IL-2 does not contribute to initial antigen-stimulated T cell activation but is necessary for the survival of the activated T cells and the generation of memory T cells [37]. We found that the presence of any detectable CD8+ T cell response to Tax (ie, IL-2 or IFN-γ) was associated with HAM, independently of viral load, whereas an IL-2 response to HBZ was associated with low viral load. In some ACs, IL-2 responses in the absence of an IFN-γ response could be detected, whereas in patients with HAM IL-2 responses were detected only when an IFN-γ response was present. This suggests that following HBZ recognition re-exposure of a lymphocyte or its progeny leads to activation of the IL-2 secreting T cell and better infection control, ie, a protective response. We hypothesise that a low level of HTLV-1 HBZ antigen expression is associated with an efficient immune response, re-exposure of HBZ-specific CD8+ T cells or their progeny is treated as a secondary infection with IL-2 production and this results in asymptomatic carriage. This would be similar to the situation in Mycobacterium tuberculosis infection wherein IL-2–secreting cells were more common in latent infection than in active M. tuberculosis [38]. Conversely, we speculate that in the absence of this response HTLV-1 antigen exposure is repeatedly seen as a new infection leading to high levels of the IFN-γ and thus is found in the patients with inflammatory disease. Therefore, HBZ-specific CD8+ T cells secreting IL-2 would be associated with asymptomatic carriage, whereas Tax-specific CD8+ T cell secreting IFN-γ would be associated with an increased risk of disease.
Funding
This work was funded by the Medical Research Council G041616, UK and supported by the NIHR Biomedical Research Centre.
Acknowledgments
We wish to thank the patients and staff at the National Centre for Human Retrovirology at St Mary's Hospital, London.
References
- 1.de Thé G, Bomford R. An HTLV-I vaccine: Why, how, for whom? AIDS Res Hum Retroviruses. 1993;9:381–6. doi: 10.1089/aid.1993.9.381. [DOI] [PubMed] [Google Scholar]
- 2.Arisawa K, Soda M, Endo S, et al. Evaluation of adult T-cell leukemia/lymphoma incidence and its impact on non-Hodgkin lymphoma incidence in southwestern Japan. Int J Cancer. 2000;85:319–24. doi: 10.1002/(sici)1097-0215(20000201)85:3<319::aid-ijc4>3.0.co;2-b. [DOI] [PubMed] [Google Scholar]
- 3.Tosswill JH, Taylor GP, Tedder RS, Mortimer PP. HTLV-I/II associated disease in England and Wales, 1993–7: Retrospective review of serology requests. BMJ. 2000;320:611–2. doi: 10.1136/bmj.320.7235.611. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Mochizuki M, Watanabe T, Yamaguchi K, et al. Uveitis associated with human T-cell lymphotropic virus type I. Am J Ophthalmol. 1992;114:123–9. doi: 10.1016/s0002-9394(14)73974-1. [DOI] [PubMed] [Google Scholar]
- 5.LaGrenade L, Hanchard B, Fletcher V, Cranston B, Blattner W. Infective dermatitis of Jamaican children: A marker for HTLV-I infection. Lancet. 1990;336:1345–7. doi: 10.1016/0140-6736(90)92896-p. [DOI] [PubMed] [Google Scholar]
- 6.Morgan OS, Rodgers-Johnson P, Mora C, Char G. HTLV-1 and polymyositis in Jamaica. Lancet. 1989;2:1184–7. doi: 10.1016/s0140-6736(89)91793-5. [DOI] [PubMed] [Google Scholar]
- 7.Okada F, Ando Y, Yoshitake S, et al. Pulmonary CT findings in 320 carriers of human T-lymphotropic virus type 1. Radiology. 2006;240:559–64. doi: 10.1148/radiol.2402050886. [DOI] [PubMed] [Google Scholar]
- 8.Kawai H, Inui T, Kashiwagi S, et al. HTLV-I infection in patients with autoimmune thyroiditis (Hashimoto's thyroiditis) J Med Virol. 1992;38:138–41. doi: 10.1002/jmv.1890380212. [DOI] [PubMed] [Google Scholar]
- 9.Arisawa K, Sobue T, Yoshimi I, et al. Human T-lymphotropic virus type-I infection, survival and cancer risk in southwestern Japan: A prospective cohort study. Cancer Causes Control. 2003;14:889–96. doi: 10.1023/b:caco.0000003853.82298.96. [DOI] [PubMed] [Google Scholar]
- 10.Devita DA, Murphy EL. Living with HTLV: Insights from a prospective study [abstract] AIDS Res Hum Retroviruses. 2009;25:A13. [Google Scholar]
- 11.Li M, Kesic M, Yin H, Yu L, Green PL. Kinetic analysis of human T-cell leukemia virus type 1 gene expression in cell culture and infected animals. J Virol. 2009;83:3788–97. doi: 10.1128/JVI.02315-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Daenke S, Kermode AG, Hall SE, et al. High activated and memory cytotoxic T-cell responses to HTLV-1 in healthy carriers and patients with tropical spastic paraparesis. Virology. 1996;217:139–46. doi: 10.1006/viro.1996.0101. [DOI] [PubMed] [Google Scholar]
- 13.Goon PK, Biancardi A, Fast N, et al. Human T cell lymphotropic virus (HTLV) type-1-specific CD8+ T cells: Frequency and immunodominance hierarchy. J Infect Dis. 2004;189:2294–8. doi: 10.1086/420832. [DOI] [PubMed] [Google Scholar]
- 14.Hanon E, Asquith RE, Taylor GP, Tanaka Y, Weber JN, Bangham CR. High frequency of viral protein expression in human T cell lymphotropic virus type 1-infected peripheral blood mononuclear cells. AIDS Res Hum Retroviruses. 2000;16:1711–5. doi: 10.1089/08892220050193191. [DOI] [PubMed] [Google Scholar]
- 15.Hanon E, Hall S, Taylor GP, et al. Abundant tax protein expression in CD4+ T cells infected with human T-cell lymphotropic virus type I (HTLV-I) is prevented by cytotoxic T lymphocytes. Blood. 2000;95:1386–92. [PubMed] [Google Scholar]
- 16.Parker CE, Daenke S, Nightingale S, Bangham CR. Activated, HTLV-1-specific cytotoxic T-lymphocytes are found in healthy seropositives as well as in patients with tropical spastic paraparesis. Virology. 1992;188:628–36. doi: 10.1016/0042-6822(92)90517-s. [DOI] [PubMed] [Google Scholar]
- 17.Gaudray G, Gachon F, Basbous J, Biard-Piechaczyk M, Devaux C, Mesnard JM. The complementary strand of the human T-cell leukemia virus type 1 RNA genome encodes a bZIP transcription factor that down-regulates viral transcription. J Virol. 2002;76:12813–22. doi: 10.1128/JVI.76.24.12813-12822.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Cavanagh MH, Landry S, Audet B, et al. HTLV-I antisense transcripts initiating in the 3'LTR are alternatively spliced polyadenylated. Retrovirology. 2006;3:15. doi: 10.1186/1742-4690-3-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Satou Y, Yasunaga J, Yoshida M, Matsuoka M. HTLV-I basic leucine zipper factor gene mRNA supports proliferation of adult T cell leukemia cells. Proc Natl Acad Sci U S A. 2006;103:720–5. doi: 10.1073/pnas.0507631103. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Saito M, Matsuzaki T, Satou Y, et al. In vivo expression of the HBZ gene of HTLV-1 correlates with proviral load, inflammatory markers and disease severity in HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) Retrovirology. 2009;6:19. doi: 10.1186/1742-4690-6-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Usui T, Yanagihara K, Tsukasaki K, et al. Characteristic expression of HTLV-1 basic zipper factor (HBZ) transcripts in HTLV-1 provirus-positive cells. Retrovirology. 2008;5:34. doi: 10.1186/1742-4690-5-34. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Basbous J, Arpin C, Gaudray G, Piechaczyk M, Devaux C, Mesnard JM. The HBZ factor of human T-cell leukemia virus type I dimerizes with transcription factors JunB and c-Jun and modulates their transcriptional activity. J Biol Chem. 2003;278:43620–7. doi: 10.1074/jbc.M307275200. [DOI] [PubMed] [Google Scholar]
- 23.Isono O, Ohshima T, Saeki Y, et al. Human T-cell leukemia virus type 1 HBZ protein bypasses the targeting function of ubiquitination. J Biol Chem. 2008;283:34273–82. doi: 10.1074/jbc.M802527200. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Lemasson I, Lewis MR, Polakowski N, et al. Human T-cell leukemia virus type 1 (HTLV-1) bZIP protein interacts with the cellular transcription factor CREB to inhibit HTLV-1 transcription. J Virol. 2007;81:1543–53. doi: 10.1128/JVI.00480-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Matsumoto J, Ohshima T, Isono O, Shimotohno K. HTLV-1 HBZ suppresses AP-1 activity by impairing both the DNA-binding ability and the stability of c-Jun protein. Oncogene. 2005;24:1001–10. doi: 10.1038/sj.onc.1208297. [DOI] [PubMed] [Google Scholar]
- 26.Arnold J, Yamamoto B, Li M, et al. Enhancement of infectivity and persistence in vivo by HBZ, a natural antisense coded protein of HTLV-1. Blood. 2006;107:3976–82. doi: 10.1182/blood-2005-11-4551. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Chevalier SA, Ko NL, Calattini S, et al. Construction and characterization of a human T-cell lymphotropic virus type 3 infectious molecular clone. J Virol. 2008;82:6747–52. doi: 10.1128/JVI.00247-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Halin M, Douceron E, Clerc I, et al. Human T-cell leukemia virus type 2 produces a spliced antisense transcript encoding a protein that lacks a classic bZIP domain but still inhibits Tax2-mediated transcription. Blood. 2009;114:2427–38. doi: 10.1182/blood-2008-09-179879. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Switzer WM, Salemi M, Qari SH, et al. Ancient, independent evolution and distinct molecular features of the novel human T-lymphotropic virus type 4. Retrovirology. 2009;6:9. doi: 10.1186/1742-4690-6-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.MacNamara A, Rowan A, Hilburn S, et al. HLA Class I binding of HBZ determines outcome in HTLV-1 infection. PLoS Pathog. 2010;6 doi: 10.1371/journal.ppat.1001117. e1001117. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.World Health Organization (WHO) Virus diseases: Human T lymphotropic virus type I, HTLV-I. Wkly Epidemiol Rec. 1989;64:382–3. [Google Scholar]
- 32.Seiki M, Hattori S, Hirayama Y, Yoshida M. Human adult T-cell leukemia virus: Complete nucleotide sequence of the provirus genome integrated in leukemia cell DNA. Proc Natl Acad Sci U S A. 1983;80:3618–22. doi: 10.1073/pnas.80.12.3618. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Murata K, Hayashibara T, Sugahara K, et al. A novel alternative splicing isoform of human T-cell leukemia virus type 1 bZIP factor (HBZ-SI) targets distinct subnuclear localization. J Virol. 2006;80:2495–505. doi: 10.1128/JVI.80.5.2495-2505.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Goon PK, Hanon E, Igakura T, et al. High frequencies of Th1-type CD4(+) T cells specific to HTLV-1 Env and Tax proteins in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis. Blood. 2002;99:3335–41. doi: 10.1182/blood.v99.9.3335. [DOI] [PubMed] [Google Scholar]
- 35.Suemori K, Fujiwara H, Ochi T, et al. HBZ is an immunogenic protein, but not a target antigen for human T-cell leukemia virus type 1-specific cytotoxic T lymphocytes. J Gen Virol. 2009;90:1806–11. doi: 10.1099/vir.0.010199-0. [DOI] [PubMed] [Google Scholar]
- 36.Bangham CR. CTL quality and the control of human retroviral infections. Eur J Immunol. 2009;39:1700–12. doi: 10.1002/eji.200939451. [DOI] [PubMed] [Google Scholar]
- 37.Hoyer KK, Dooms H, Barron L, Abbas AK. Interleukin-2 in the development and control of inflammatory disease. Immunol Rev. 2008;226:19–28. doi: 10.1111/j.1600-065X.2008.00697.x. [DOI] [PubMed] [Google Scholar]
- 38.Millington KA, Innes JA, Hackforth S, et al. Dynamic relationship between IFN-γ and IL-2 profile of Mycobacterium tuberculosis-specific T cells and antigen load. J Immunol. 2007;178:5217–26. doi: 10.4049/jimmunol.178.8.5217. [DOI] [PMC free article] [PubMed] [Google Scholar]