A total of 65 individuals were studied: 17 patients with HAM/TSP, 23 asymptomatic HTLV-1 carriers (HAC), and 25 uninfected controls (CT), with fully informed consent. Leukocyte global counts and CD4/CD8T cell ratio were performed in all individuals. Samples of HTLV-1-infected individuals were recruited from the Neurology Department of Clinical Hospital of the Medical School at the University of São Paulo, Ribeirão Preto, São Paulo, Brazil. Inclusion criteria for this study were HTLV-1 positivity by enzyme immunoassay (rp21e-enhanced EIA; Cambridge Biotech) and molecular test (conventional LTR and TAX PCR). All HTLV-1-infected individuals were evaluated for clinical status according to the previously described criteria for ATL and HAM/TSP.11 Individuals from CT group were recruited from the Regional Blood Center of Ribeirão Preto, São Paulo, Brazil. Both HTLV-1-infected and CT individuals were serologically negative for Hepatitis B virus, Hepatitis C virus, Human Immunodeficiency virus, Chagas disease, and syphilis. This study was approved by the Institutional Ethic Committee (process number 3083/2007).

The DNA was extracted from the buffy coat from 17 HAC individuals and 17 HAM/TSP patients using the Super Quick Gene DNA isolation kit (Analytical Genetic Testing Center – AGTC, Denver, CO, USA) following the manufacturer's instructions. The in house PCR (LTR and TAX regions) was performed as previously described.12 The proviral load was quantified using TaqMan®Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The primers sequences were formerly reported.9 Real-time PCR was performed in duplicate for all DNA standards and samples using the ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The amplification program consisted of 50°C for 2min; 95°C for 10min; followed by 40 cycles at 95°C for 15s/60°C for 1min. The HTLV-1 proviral load value was calculated using the following formula: (copy number of TAX)/(copy number of β-actin)×2×100.000.

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using a Ficoll Hypaque™ PLUS (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). CD4+ cells were positively selected on a MACS column (Miltenyi Biotec, Germany) using magnetic microbeads according to the manufacturer's instructions. CD3+CD4+ T cells purity was confirmed by flow cytometry using the anti-CD4-FITC and anti-CD3-PE (FACSCalibur) (Becton & Dickinson, San Jose, CA, USA) surface markers. The total RNA was isolated with TRIzol®Reagent (Invitrogen, Carlsbad, CA, USA). The RNA was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), following the manufacturer's instructions.

The gene expression was analyzed by real-time PCR technique using TaqMan®Gene Expression Assays (Applied Biosystems, Foster City, CA, USA). The PCR amplification and fluorescence data collection were performed with ABI 7500 Sequence Detection (Applied Biosystems, Foster City, CA, USA).

The following genes were studied: CD3e molecule, epsilon (CD3-TCR complex) (CD3¿) (Hs99999153_m1), vav 1 guanine nucleotide exchange factor (VAV1) (Hs00232108_m1), zeta-chain (TCR) associated protein kinase 70kDa (ZAP70) (Hs_00896347), and lymphocyte-specific protein tyrosine kinase (LCK) (Hs00178427_m1). The geometric average of four housekeeping genes human β-actin (ACTB) (4326315E), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (4310884-E), beta-2-microglobulin (B2M) (4333766-0710013), and ribosomal protein L13a (RPL13A) (185720330-7)13 was used to normalize the mRNA amount for each sample (Applied Biosystems). The 2−ΔΔCT method was chosen to calculate the relative expression levels.14 All these experiments were performed in duplicates.

The Tax protein expression was evaluated in 11 HAC individuals and 9 HAM/TSP patients. To detect Tax protein expression in HTLV-1-infected cells, the whole PBMCs were cultured in RPMI 1640 (Sigma-Aldrich, Saint Louis, MO, USA) containing 10% fetal bovine serum (HyClone, Logan, UT, USA) and 20nM concanamycin A (Sigma-Aldrich, Saint Louis, MO, USA), a potent cytolysis inhibitor of CD8+ T cell that allows the CD4+ T cell survival. The cells were incubated for 12h under 5% CO2 at 37°C. Then, the cells were stained for the surface markers anti-CD4-PE, anti-CD8-PerCP, and anti-CD3-APC (Becton & Dickinson, San Jose, CA, USA). For intracellular analysis, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20min. After this time, the fixed cells were washed with PBS containing 4% of normal goat serum (NGS) (Sigma-Aldrich, Saint Louis, MO, USA) and then washed with PBS containing 0.1% Triton X-100 (Sigma-Aldrich, Saint Louis, MO, USA) for 10min at room temperature. Permeabilized cells were washed and resuspended in PBS/4% NGS containing isotype control mAb (Southern Biotechnology Associates, Birmingham, AL) or anti-HTLV-I Tax mAb (Lt-4; IgG3), provided by Dr. Yuetsu Tanaka, University of the Ryukyus, Okinawa. Finally, the cells were incubated for 30min at room temperature and were stained intracellularly with Alexa Fluor 488-labeled goat anti-mouse IgG3 (Invitrogen, Carlsbad, CA, USA) for 30min at room temperature. All samples were analyzed on a FACSCalibur flow cytometer (Becton & Dickinson, San Jose, CA, USA) and 10,000 total events were acquired.

Comparisons among groups were performed using analysis of variance (ANOVA) followed by Tukey test. Correlation among different parameters was evaluated using the Spearman's Rank Correlation. Data regarding gender and age were analyzed using the Fisher's exact test. Critical p-values were considered statistically significant if below 0.05. Data sets were compiled and analyzed in GraphPad PRISM, version 5.01 (GraphPad software, CA, USA).

The clinical features of the studied population are shown in Table 1. The HAM/TSP group consisted of 76.5% women and 23.5% men, mean age 55.5 years, age range 37–74 years. In the HAC group there were 56.5% of women and 43.5% of men, mean age 43.1 years, age range 22–72 years. No significant gender difference was observed between HAC and HAM/TSP groups, whereas age was significantly different (p=0.0053).

The CT group was composed of 72% women and 28% men, mean age 47.3 years, age range 22–76 years. HAM/TSP group had higher CD4/CD8T cell ratio than CT and HAC groups, however, this difference was not significant; no difference was observed in leukocyte global counts among groups.

Both Tax expression in CD4+ T cells and proviral load were increased in the HAM/TSP group (p=0.0007 and p=0.0061, respectively) when compared to the HAC group (Fig. 1A and B). Moreover, proviral load positively correlated to Tax expression (r=0.7208; p=0.0002) (Fig. 1C).

Fig. 1.

Proviral load (PVL) and Tax protein expression (percentage of Tax+CD3+CD4+ T cells) measurement in HTLV-1-infected individuals. (A) Proviral load was measured in HAC (n=17) and HAM/TSP (n=17) individuals. (B) Tax protein expression was quantified in HAC (n=11) and HAM/TSP (n=9) individuals. The Mann–Whitney U test was used to evaluate differences among groups (**p≤0.01; ***p≤0.001). (C) Correlation between PVL and Tax expression in HTLV-1-infected individuals (n=20). Correlation was calculated by the Spearman method. HAC: Asymptomatic HTLV-1 carrier; HAM/TSP: HTLV-1 associated mielopathy/tropical spastic paraparesis.

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The levels of CD3¿, LCK, VAV1, and ZAP70 gene expression were evaluated by quantitative real-time PCR (Fig. 2A–D). We observed that all these genes were significantly overexpressed in HAM/TSP group compared to HAC group. Additionally, VAV1 and ZAP70 genes were increased in HAM/TSP group compared to CT group (p=0.0364 and p=0.0120, respectively).

Fig. 2.

CD3¿, LCK, VAV1, and ZAP70 genes expression by real-time PCR and correlation among them. Gene expression levels comparison of CD3¿ (A), LCK (B), VAV1 (C), and ZAP70 (D) among CT (n=25), HAC (n=23), and HAM/TSP (n=17) groups. The Mann–Whitney U test was used to evaluate differences among groups (*p≤0.05; **p≤0.01). A positive correlation between LCK and CD3¿ (E), VAV1 and CD3¿ (F), ZAP70 and CD3¿ (G), VAV1 and LCK (H), ZAP70 and LCK (I), ZAP70 and VAV1 (J), CD3¿ and FOXP3 (K), LCK and FOXP3 (L), VAV1 and FOXP3 (M), and ZAP70 and FOXP3 (N) was observed in the samples from HTLV-1-infected individuals. Correlation was calculated by the Spearman method. CT: Healthy control; HAC: Asymptomatic HTLV-1 carrier; HAM/TSP: HTLV-1 associated mielopathy/tropical spastic paraparesis.

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In order to determine the activation of T cell receptor signaling pathway, we studied the correlation among CD3¿, LCK, VAV1, and ZAP70 genes in HTLV-1-infected individuals. We observed a positive correlation among all these genes (Fig. 2E–J). Correlations among genes were also performed in HAC and HAM/TSP groups individually. Both groups revealed a positive correlation between most of the genes, except for the correlation between ZAP70 and CD3¿ genes in HAC group, and ZAP70 and VAV1 genes in both groups (data not shown). In this individual analysis of genes correlation, we observed that HAM/TSP group showed a more significant genes correlation compared to HAC group (data not shown).

Since the regulatory T-cells (Treg) are related to CD4+ T-cells in HTLV-1-infected population,15 we believe that FOXP3, one of the main Treg cell markers,16 might be associated to the genes of T cell receptor signaling pathway. In a recent study9 we reported that FOXP3 gene expression was upregulated in HAM/TSP group compared to the CT and HAC groups. Moreover, the percentage of CD4+FOXP3+ cells was increased in HTLV-1-infected individuals compared to CT group. Therefore, we performed the correlation between FOXP3 gene expression and the CD3¿, LCK, VAV1, and ZAP70 genes in HTLV-1-infected individual. This correlation was based on the FOXP3 results obtained in our previously study.9 A positive correlation was observed between FOXP3 and all the analyzed genes (Fig. 2K–N).

Since CD3¿, LCK, VAV1, and ZAP70 genes were upregulated in HAM/TSP compared to HAC group; we suggested that these genes could be correlated to the PVL and Tax expression. We performed Spearman's correlation test and verified that CD3¿, LCK, and VAV1 genes were positively correlated to plasma viral load and Tax expression in HTLV-infected samples, while ZAP70 gene did correlate neither with plasma viral load nor with Tax expression (Fig. 3A–H).

Fig. 3.

Correlation between genes expression of CD3¿, LCK, VAV1, and ZAP70, and percentage of Tax expression or proviral load (PVL) in HTLV-1-infected individuals. A positive correlation between PVL and CD3¿ (A), LCK (B), and VAV1 (C) was observed. (D) Correlation between PVL and ZAP70. The Tax expression positively correlated to CD3¿ (E), LCK (F), and VAV1 (G). H) Correlation between Tax expression and ZAP70. Correlation was calculated by the Spearman method.

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