The frequency of CD127low expressing CD4+CD25high T regulatory cells is inversely correlated with human T lymphotrophic virus type-1 (HTLV-1) proviral load in HTLV-1-infection and HTLV-1-associated myelopathy/tropical spastic paraparesis

1471-2172-9-41 1471-2172 Research article The frequency of CD127low expressing CD4+CD25high T regulatory cells is inversely correlated with human T lymphotrophic virus type-1 (HTLV-1) proviral load in HTLV-1-infection and HTLV-1-associated myelopathy/tropical spastic paraparesis Michaëlsson Jakob Jakob.michaelsson@ki.se Barbosa Marcelo R Hugo hugomarcelo.barbosa@gmail.com Jordan A Kimberley kjordan@gmail.com Chapman M Joan joanmchapman@yahoo.com Brunialti KC Milena milena.brunialti@unifesp.br Neto Kleine Walter walterkleine@yahoo.com.br Nukui Youko ynukui@terra.com.br Sabino C Ester sabinoec@uol.com.br Chieia Antonio Marco abrela99@hotmail.com Oliveira Souza Bulle Acary abrela99@hotmail.com Nixon F Douglas douglas.nixon@ucsf.edu Kallas G Esper kallas.dmed@epm.br

Division of Experimental Medicine, Department of Medicine, University of California, San Francisco, San Francisco, CA, USA

Infectious Diseases Division, Federal University of São Paulo, São Paulo, Brazil

São Paulo Blood Bank, São Paulo, Brazil

Clinical Immunology and Allergy Division, University of São Paulo, Brazil

Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Stockholm, Sweden

BMC Immunology 1471-2172 2008 9 1 41 http://www.biomedcentral.com/1471-2172/9/41 18664281 10.1186/1471-2172-9-41 25 6 2008 29 7 2008 29 7 2008 2008 Michaëlsson et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

CD4⁺CD25^highregulatory T (T_Reg) cells modulate antigen-specific T cell responses, and can suppress anti-viral immunity. In HTLV-1 infection, a selective decrease in the function of T_Regcell mediated HTLV-1-tax inhibition of FOXP3 expression has been described. The purpose of this study was to assess the frequency and phenotype of T_Regcells in HTLV-1 asymptomatic carriers and in HTLV-1-associated neurological disease (HAM/TSP) patients, and to correlate with measures of T cell activation.

Results

We were able to confirm that HTLV-I drives activation, spontaneous IFNγ production, and proliferation of CD4+ T cells. We also observed a significantly lower proportion of CTLA-4⁺T_Regcells (CD4⁺CD25^highT cells) in subjects with HAM/TSP patients compared to healthy controls. Ki-67 expression was negatively correlated to the frequency of CTLA-4⁺T_Regcells in HAM/TSP only, although Ki-67 expression was inversely correlated with the percentage of CD127^lowT_Regcells in healthy control subjects. Finally, the proportion of CD127^lowT_Regcells correlated inversely with HTLV-1 proviral load.

Conclusion

Taken together, the results suggest that T_Regcells may be subverted in HAM/TSP patients, which could explain the marked cellular activation, spontaneous cytokine production, and proliferation of CD4⁺T cells, in particular those expressing the CD25^highCD127^lowphenotype. T_Regcells represent a potential target for therapeutic intervention for patients with HTLV-1-related neurological diseases.

Background

Between 10 and 20 million people are infected with HTLV-1 worldwide 1. Although most subjects are clinically asymptomatic during their lifetime, a proportion (5 to 10%) develop adult T cell leukemia/lymphoma (ATLL) or HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) 2. Epidemiological surveys have identified regions in the world where prevalence rates are considerably higher, including Japan, the Caribbean, South America, Africa, Melanesia and the Middle East 13. It has been estimated that the prevalence of HTLV-1 infection in South America ranges from 2 to 5% 4, with an estimated 1–2 million infected people in Brazil 5. The prevalence in blood donors ranges from 0.17 to 1.8% in different areas of the country 67, with a 0.3% seroprevalence in the city of Sao Paulo blood donors 8.

HTVL-1 is a retrovirus encoding the group specific antigen (gag), protease (pro), polymerase (pol), and envelope (env) genes. Six proteins are encoded by the pX region of the genome, including the Tax protein, which is critical to viral replication and induction of cellular activation and transformation, increasing the expression and production of cytokines and receptors involved in T cell growth and transformation, such as IL-15 910 and IL-2 111213. Tax also has the ability of interfering in the expression of several transcription factors and proto-oncogenes, as well as in the nucleic acid repair and apoptosis 14151617. These effects combined seem to play a key role in the potential of HTLV-1 to induce cellular transformation and, consequently, trigger the development of ATLL.

It has been previously demonstrated that HTLV-1 proviral load is one of the key factors in the pathogenesis of HAM/TSP 1819, although host genetic factors are also independently associated with the development of the diseases, e.g. certain HLA 2021 and non-HLA 2223 genes. These invoke the hypothesis that both viral and genetic host factors are implicated in the pathogenesis of HAM/TSP.

The CD8⁺T cell response to HTLV-1 can be readily detected 2425262728293031, commonly directed against the HTLV-1-tax protein. The contribution of the CD8⁺T cell response might be particularly important for viral control in HTLV-1 infection, since infected lymphocytes produce virtually no cell-free infectious HTLV-1 particles. However, it is noteworthy that the magnitude of the HTLV-1-specific T cell response is associated with higher proviral loads, highlighting the fact that T cells frequencies are determined by proviral load, as well as being a determinant of proviral load. CD4⁺T cells are the main target for HTLV-1 infection, which induces CD4⁺T cell activation, including proliferation and IFNγ production. The HTLV-1-specific CD4⁺T cell response is directed mainly against Env, the HTLV-1 envelope surface 32.

T_Regcells are crucial for the control of autoimmune disease and maintenance of peripheral T cell tolerance (reviewed in Sakagushi et al. 33). In addition, they can suppress pathogen-specific T cell responses, including response to viruses 34353637. The mechanisms whereby T_Regcells suppress T cell responses are not yet fully understood, but are likely to include both soluble factors, e.g. IL-10 and TGF-β, as well as cell-cell contact dependent mechanisms, e.g. through CTLA-4. CTLA-4 (CD152) is expressed by a large fraction of CD4⁺CD25⁺T cells, and by a majority of CD4⁺CD25^highT cells. CTLA-4 has also been shown to be one of mediators of T_Regfunction 3839, and is considered a marker for T_Regcells. In addition, it was recently demonstrated that T_Regcells are characterized by low levels of the IL-7Rα (CD127^low) 404142, which together with CD25 help to distinguish T_Regcells from activated normal CD4⁺T cells in healthy individuals. FOXP3 is a key regulator of T_Regcell function, but is not exclusive to T_Regcells; it has been identified in human nonregulatory activated CD4⁺FoxP3⁺T cells. Humans with mutations in FOXP3 present with a syndrome characterized by severe autoimmune and inflammatory disorders often early in life, denominated IPEX 33. Interestingly, it was recently shown that HTLV-1 tax can downregulate Foxp3 expression 4344.

We hypothesized that HTLV-1 compromises T_Regcell function, resulting in higher T cell activation, which contributes to HAM/TSP development. We found a significantly higher frequency of CD4⁺Ki-67⁺T cells and a lower proportion of CTLA-4⁺T_Regcells in subjects with HAM/TSP, compared to healthy controls. Moreover, we found an inverse correlation between HTLV-1 proviral load and frequency of CD127^low/CTLA-4⁺T_Regcells. Our data suggest a role for T_Regcells in the pathogenesis of HAM/TSP, and reveal a potential new therapeutic target for patients with HAM/TSP.

Results

Study subjects

Blood samples were collected at the Federal University of Sao Paulo outpatient clinics, after informed consent. PBMC were isolated by Ficoll-Paque PLUS density gradient centrifugation and cryopreserved. The demographics of the study subjects are shown in Table 1, including gender, age, proviral load, CD3, CD4 and CD8 absolute T cell counts. No statistically significant differences were observed in gender and age distribution among groups.

Table 1

Characteristics of study subjects.

Sample

Group

Gender

Age (years)

Proviral Load (copies/1,000 cells)

CD4+ T cells/μl

CD8+ T cells/μl

CD3+ T cells/μl

101

Control

Male

690

389

1220

102

Control

Male

1116

525

1649

103

Control

Male

104

Control

Male

105

Control

Female

106

Control

Female

107

Control

Male

917

233

1235

201

HTLV+

Female

1321

619

1970

202

HTLV+

Female

183

710

700

1402

203

HTLV+

Male

548

351

989

204

HTLV+

Female

314

749

494

1248

205

HTLV+

Female

120

705

227

934

206

HTLV+

Female

105

738

482

1341

209

HTLV+

Female

1274

622

1909

212

HTLV+

Male

160

904

527

1500

213

HTLV+

Female

1320

496

1831

214

HTLV+

Male

535

251

782

301

HAM/TSP

Female

723

735

354

1092

302

HAM/TSP

Female

1012

242

303

HAM/TSP

Male

304

HAM/TSP

Female

Undetectable

727

356

1076

305

HAM/TSP

Female

563

327

972

306

HAM/TSP

Female

151

881

334

1272

307

HAM/TSP

Female

Undetectable

899

500

1472

308

HAM/TSP

Female

433

1154

478

1694

309

HAM/TSP

Female

175

614

165

520

310

HAM/TSP

Female

565

734

286

1039

CD4+ T cell activation and IFNγ production in healthy donors, HTLV-1 seropositive asymptomatics, and HAM/TSP patients

We initially investigated the expression of Ki-67, HLA-DR and CD38 on CD4⁺T cells in PBMC from healthy donors (Control), HTLV-1 infected patients who were clinically asymptomatic (HTLV), or had associated neurological disease (HAM/TSP). HAM/TSP had significantly higher frequencies of CD4⁺Ki-67⁺T cells compared to HTLV or Control subjects (Fig. 1A, 1C). In addition, HAM/TSP patients had an increase in the frequency of CD4⁺HLA-DR⁺T cells compared to Controls (Fig. 1E), whereas no statistically significant difference in frequency of CD4⁺CD38⁺T cells was noted (data not shown). Furthermore, CD4⁺T cells from both HTLV and HAM/TSP groups had an increase in the spontaneous expression of IFNγ (Fig. 1B, 1D).

Figure 1

Expression of Ki-67, HLA-DR and spontaneous production of IFNγ by CD4⁺T cells from Control, HTLV and HAM/TSP subjects

Expression of Ki-67, HLA-DR and spontaneous production of IFNγ by CD4⁺T cells from Control, HTLV and HAM/TSP subjects. (A) Representative dot plots showing Ki-67 expression in CD4⁺T cells from a Control, a HTLV, and a HAM/TSP subject. (B) Representative dot plots showing spontaneous IFNγ production by CD4⁺T cells. (C) Percent CD4⁺Ki-67⁺T cells; and (D) percent CD4⁺IFNγ⁺T cells; or (E) percent CD4⁺HLA-DR⁺T cells from Control, HTLV, or HAM/TSP. The line shown represents the mean.

Decreased frequency of T_Regcells in HAM/TSP patients

In order to assess the frequency of T_Regcells in HTLV-1 infected subjects, we measured the expression of CD25, CTLA-4, CD127 and GITR on CD4⁺T cells by flow cytometry. Gating strategies are shown in Fig. 2A–C. The frequency of CD4⁺T cells expressing CD25 was very similar between the groups (Fig. 2D). As CD25 is upregulated on activated CD4⁺T cells, and thus is not a specific marker for T_Regcells, we sought to determine the frequency of T_Regcells using more specific phenotypes. First, we analyzed the frequency of CD4⁺CD25^highT cells, known to be composed mainly of T_Regcells 45, and no significant difference in the frequency of T_Regcells could be found between controls and HTLV-1 subjects (Fig. 2E). We determined the intracellular expression of CTLA-4 in CD4⁺CD25⁺, and CD4⁺CD25^highT cells and observed a decrease in frequency of CTLA-4⁺from HAM/TSP patients (Fig. 2F and 2G). Next, we assessed the expression of CD127. HAM/TSP patients had a statistically significant increase in the frequency of CD4⁺CD25⁺CD127^lowT cells compared to Controls (Fig. 2H). In contrast, there was a slight decrease in frequency of CD4⁺CD25^highCD127^lowT cells in HTLV-1 infected groups, although this did not reach statistical significance (Fig. 2I). There were no differences in percent expression of GITR between the groups (Fig. 2J, 2K).

Figure 2

Immunophenotypes of CD25⁺or CD25^highCD4⁺T cells in Control, HTLV, or HAM/TSP subjects

Immunophenotypes of CD25⁺or CD25^highCD4⁺T cells in Control, HTLV, or HAM/TSP subjects. Dot plots depict the gating strategies for CD25⁺and CD25^highcells (A); CD127^low(B); and CTLA-4 expression (C) are shown. The gates for CD25⁺and CD4⁺CD25^highT cells were set based on the expression level of CD25 on CD4^-T cells, where the pattern of CD25 expression is more distinct. Accordingly, CD4⁺CD25^highT cells were defined as those expressing CD25 at a level higher than the bulk CD4^-CD25⁺population. For D-K, individual results are represented in symbols, and the line represents the mean. (D) % CD4⁺CD25⁺T cells; (E) % CD4⁺CD25^highT cells; (F) % CD4⁺CD25⁺CTLA-4⁺T cells; (G) % of CD4⁺CD25^highCTLA-4⁺T cells; (H) % CD4⁺CD25⁺CD127^lowT cells; (I) % CD4⁺CD25^highCD127^lowT cells; (J) % CD4⁺CD25^highGITR⁺T cells; and (K) % CD4⁺CD25^highGITR⁺T cells.

Decreased CTLA-4+ T_Regcells correlate with increased CD4+ T cell proliferation in HAM/TSP patients

Our initial analysis indicated that the frequency of CD4⁺CD25⁺CD127^lowT cells was higher in HAM/TSP patients. However, there were a lower percentage of CTLA-4⁺T_Regcells, indicating that HAM/TSP subjects might have T_Regcells with a dysfunctional phenotype. Moreover, we showed that HAM/TSP patients had an increased proliferation and T cell activation, as evidenced by higher frequencies of HLA-DR, Ki-67 and INFγ-expressing CD4⁺T cells. To test whether the higher frequency of T_Regcells was associated with lower levels of activation or proliferation of CD4⁺T cells, we compared the frequency of CTLA-4⁺, CD127^lowor GITR⁺T_Regcells with the percentage of CD4⁺Ki-67⁺T cells. The frequency of CTLA-4⁺T_Regwas negatively correlated with the frequency of CD4⁺Ki-67⁺T cells in HAM/TSP patients only (Fig. 3A). In addition, there was a negative correlation between the frequency of CD127^lowT_Regcells and the percentage of CD4⁺Ki-67⁺T cells in controls, whereas no such correlation was found in HTLV-1 infected subjects (Fig. 3B). There was no association between GITR⁺T_Regcells with the percentage of CD4⁺Ki-67⁺T cells in any group (Fig. 3C).

Figure 3

Correlation between different immunophenotypes of T_regwith expression of Ki-67

Correlation between different immunophenotypes of T_regwith expression of Ki-67. Frequency of CTLA-4⁺, CD127^low, or GITR⁺T_regwere correlated with proportion of CD4⁺Ki67⁺T cells in Control (Blue diamond), HTLV (Green triangle) and HAM/TSP (Red circle). A. There was a negative correlation between CTLA-4⁺T_regwith percentage of CD4⁺Ki67⁺T cells in HAM/TSP only (r = -0.7833, p = 0.017). B. CD127^lowT_regwere inversely correlated with proportion of CD4⁺Ki67⁺T cells in Control group (r = -0.8108, p = 0.034). C. There was no association between GITR⁺T_regwith percentage of CD4⁺Ki67⁺T cells in any group.

The frequency of CD4⁺CD25⁺CTLA-4⁺and CD127^lowT_Regcells was negatively correlated to HTLV-1 proviral load

Finally, we wanted to determine whether the frequency of T_Regcells was related to HTLV-1 proviral load. High CD4⁺T cells activation and elevated HTLV-1 proviral load are observed in HAM/TSP. We hypothesized that this phenomenon would be related to a lower proportion of T_Regcells. We quantified HTLV-1 proviral load by real-time PCR and correlated it with the frequency of the different CD25-expressing CD4⁺T cell subsets. There was no correlation between the frequency of CD4⁺CD25⁺or CD4⁺CD25^highT cells with proviral load (Fig. 4A and 4B). In contrast, there was a negative correlation between the frequency of CD4⁺CD25⁺CTLA-4⁺T cells and proviral load in HTLV only (Fig. 4C). Moreover, there was an inverse correlation between CD127^lowT_Regcells and HTLV-1 proviral load (Fig. 4F).

Figure 4

Correlation between HTLV-1 proviral load of HTLV (Green triangle), and HAM/TSP (Red circle) with expression of CD127 or CTLA-4 in CD25⁺or CD25^highCD4⁺T cells subsets

Correlation between HTLV-1 proviral load of HTLV (Green triangle), and HAM/TSP (Red circle) with expression of CD127 or CTLA-4 in CD25⁺or CD25^highCD4⁺T cells subsets. HTLV-I proviral load is expressed in copies per 1,000 cells. There was no correlation with CD4⁺CD25⁺T cells (A). No correlation with CD4⁺CD25^highT cells (B). Inverse correlation with the percentage of CD4⁺CD25⁺CTLA-4⁺T cells in HTLV only; r = -0.7500, p = 0.0255 (C). No correlation with CD4⁺CD25^highCTLA-4⁺T cells (D). Lack of correlation with the percentage of CD4⁺CD25⁺CD127^lowT cells (E). (F) Negative correlation with CD4⁺CD25^highCD127^lowT cells in HTLV (r = -0.7333, p = 0.03) and HAM/TSP (r = -0.8108, p = 0.034).

Discussion

Regulatory T cells are important for the maintenance of peripheral T cell tolerance to self antigens, and can also suppress T cell responses to tumors, parasites, viruses and bacteria. In this study we addressed the relationship between T_Regcells, T cell activation, and HTLV-1 proviral load. Infection with HTLV-1 was associated with higher spontaneous IFNγ release by CD4⁺T cells, but only in HAM/TSP there was a marked increase in T cell proliferation.

The HTLV-1 derived tax protein can downregulate expression of the FOXP3, which presence is associated with T_Regcell function 4344. Increased expression of tax can be expected in patients with HAM/TSP, who have higher proviral loads compared to asymptomatic carries. We observed a higher proportion of CD4⁺IFNγ⁺T cells in HTLV-1 infected subjects, which could also be indicative of a decreased T_Regcell fraction. Interestingly, only the HAM/TSP patients presented with a higher cell proliferation, as measured by Ki-67 staining, which correlated markedly with HTLV-1 proviral load (data not shown). These observations suggest that HTLV-1 directly affects T_Regcell number, and as proviral load increases, not only is the control of IFNγ lost, but controls on cell proliferation as well. Our data, together with the recent findings that HTLV-1 tax downregulates FOXP3 expression, indicate that T_Regcell dysfunction can be a direct consequence of HTLV-1 infection.

In order to better understand the role of T_Regcells in HTLV-I infection and disease, we used CTLA-4 and CD127 staining in CD4⁺CD25^highcells as markers for T_Regsubsets. CD127 and CTLA-4 have been described as useful markers for T_Reg, and facilitate the identification of T_Regcells, even without staining for FOXP3 40. In this study, we found that an increased frequency of CD127^lowCD4⁺CD25⁺T_Regin controls correlated negatively with CD4⁺T cell proliferation (Ki-67), indicating that these cells indeed have a regulatory immunophenotype. In contrast, increased frequency of these cells correlated with increased CD4⁺T cell proliferation in HTLV-1 infected individuals, suggesting that these cells are not regulatory T cells in these individuals. In addiction, the elevated frequency of CTLA-4⁺T_Regcells was negatively correlated to CD4⁺T cell proliferation only in HAM/TSP patients, which suggest that it is a better immunophenotype of T_Regcells in HAM/TSP patients, but more studies are necessary to confirm this.

We could detect a negative association between the frequency of CTLA-4⁺or CD127^lowT_Regcells and proviral load, extending recent findings of an association between FOXP3 expression and HTLV-1 infection 44. We speculate that therapeutic manipulation of regulatory T cells could positively impact disease pathogenesis. Two mechanisms might be involved, the first by suppressing the exuberant anti-HTLV-1 CD8⁺T cell mediated immune response, and the second by suppression of CD4⁺T cell proliferation, which can result in lower proviral load. However, stimulating an expansion of T_Regcells could also provide additional targets for HTLV-1 replication, so such studies should proceed with great caution.

In this study, there are some limitations. The study was cross sectional, and with a limited number of patients in each group. We hope that future longitudinal studies can assess changes in T_Regcells over time in HTLV-1 infected patients. We, and others, working in the regulatory T cells field, are limited by the lack of definitive phenotypic markers of T_Reg, and CD4⁺CD25^+/highremains the standard identifiers. In this study we have added other markers, but at the time the study was conducted, the FOXP3 antibody, commonly used to detect a T_Regcell population was not commercially available. However, this may have been a fortuitous event, as recent reports suggest that FOXP3 is also expressed on non regulatory T cells in humans 4647. As we did not have access to tissue samples from these subjects, we cannot exclude redistribution of cells out of the peripheral blood into tissues, and the study of regulatory T cells at secondary lymphoid sites and within CSF will be of interest for a future study of HTLV-1 associated disease.

In conclusion, our data suggest a role of T_Regcells in the pathogenesis of HAM/TSP. Further studies should help delineate the ability of expanded T_Regcells to affect T cell proliferation in HTLV-1 patients and the potential development of therapeutic modulation of regulatory T cells in HTLV-1 patients.

Conclusion

In this study, we showed that HTLV-I drives activation, spontaneous IFNγ production, and proliferation of CD4+ T cells. HAM/TSP patients have a decreased frequency of T_Regcells in peripheral blood, compared to healthy subjects, markedly in the CD4⁺CD25^highCTLA⁺phenotype. The proportion of CD127^lowT_Regcells correlated inversely with HTLV-1 proviral load. These results suggest that T_Regcells may be subverted in HAM/TSP patients, and contributes to the identification of novel therapeutic targets for patients with HTLV-1-related disease.

Methods

Study subjects

Three groups of volunteers were enrolled. The first consisted of seven HTLV-1-negative control volunteers; the second consisted of ten HTLV-1 seropositive volunteers without clinical and laboratory evidence of HTLV-1-associated disease, and the last group was composed of nine patients with the diagnosis of HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). After approval by the Institutional Review Board, written informed consent was obtained from all the participants according to the guidelines of Brazilian Ministry of Health. Samples were collected in EDTA-treated vacuum tubes, and PBMC were frozen into liquid nitrogen after separation using a ficoll gradient.

DNA extraction and determination of HTLV-1 proviral load

HTLV-1 proviral DNA was extracted from PBMCs using a commercial kit (Qiagen GmbH, Hilden Germany) following the manufacturer's instructions. The extracted DNA was used as a template to amplify a fragment of 158 bp from the viral tax region using previously published primers 48. The SYBR green real-time PCR assay was carried out in 25 μl PCR mixture containing 10× Tris (pH 8.3; Invitrogen, Brazil), 1.5 mM MgCl₂, 0.2 μM of each primer, 0.2 mM of each dNTPs, SYBR Green (18.75 Units/r × n; Cambrex Bio Science, Rockland, ME) and 1 unit of platinum Taq polymerase (Invitrogen, Brazil). The amplification was performed in the Bio-Rad iCycler iQ system using an initial denaturation step at 95°C for 2 minutes, followed by 50 cycles of 95°C for 30 seconds, 57°C for 30 seconds and 72°C for 30 seconds. The human housekeeping β globin gene primers GH20 and PC04 49 were used as an internal control calibrator. For each run, standard curves for the value of HTLV-1 tax were generated from MT-2 cells of log₁₀dilutions (from 10⁵to 10⁰copy). The threshold cycle for each clinical sample was calculated by defining the point at which the fluorescence exceeded a threshold limit. Each sample was assayed in duplicate and the mean of the two values was considered as the copy number of the sample. The amount of HTLV-1 proviral load was calculated as follows: copy number of HTLV-1 (tax) per 1,000 cells = (copy number of HTLV-1 tax)/(copy number of β globin/2) × 1000 cells. The method could detect 1 copy per 10³PBMCs cells.

Flow cytometry

PBMCs were thawed and stained with directly conjugated antibodies. Three different panels of antibodies were used to evaluate the expression of proteins associated with T_Regcells and T cell activation. All antibodies were from BD Biosciences, unless otherwise noted. All panels contained PerCP-conjugated anti-CD4 and allophycocyanin-conjugated anti-CD25, and in addition contained (1) FITC-conjugated anti-GITR and PE-conjugated anti-CD127 (Beckman Coulter, Miami, FL), (2) FITC-conjugated anti-CD45RA and PE-conjugated anti-HLA-DR and (3) FITC-conjugated anti-Ki-67 and PE-conjugated anti-CD152 (CTLA-4). Cells stained with PerCP-conjugated anti-CD4 alone and allophycocyanin-conjugated CD25 alone were used to establish positive gates for FITC- and PE-conjugated antibodies. For panel 1 and 2, cells were stained with all antibodies in PBS supplemented with 0.5% bovine serum albumin (BSA) and 2 mM EDTA (FACS buffer), followed by two washes in FACS buffer and fixation in 1% paraformaldehyde (PFA). For panel 3, cells were first stained with PerCP-conjugated anti-CD4 and allophycocyanin-conjugated anti-CD25, followed by two washes in FACS buffer and fixation in 1% PFA. The cells were subsequently washed twice with PBS containing 0.1% saponin (perm buffer), prior to staining with PE-conjugated anti-CD152 and FITC-conjugated anti-Ki-67 diluted in perm buffer. All samples were analyzed on a FACSCalibur flow cytometer (Becton Dickinson) equipped with a 488 nm argon and a 633 nm red-diode lasers for four color detection. Acquisition and analyses were performed using CellQuest software (Becton Dickinson). Fluorescence voltages and compensation values were determined using unstained cells and cells single-stained with each of the fluorochrome-conjugated antibodies, respectively. The gating strategy used was to gate on lymphocytes using a forward scatter versus side scatter gate, followed by gating on CD4⁺cells. The gate for CD4⁺CD25⁺cells was set using cells cells stained with the PerCP-conjugated anti-CD4 antibody alone. Positive gates for the FITC- and PE-conjugated antibodies were set using cells stained with only PerCP-conjugated anti-CD4 and APC-conjugated anti-CD25 antibodies.

Cytokine flow cytometry

PBMCs were thawed and cultured for 24 hours in 96-well U-bottom plates at a concentration of 4 × 10⁵cells/well. Brefeldin A (BFA) was added at a concentration of 5 μg/ml for the last 5 hours of the culture. After culture, cells were harvested, stained with PE-conjugated anti-CD4, fixed in 4% PFA for 20 min, prior to being washed twice with perm buffer. The cells were subsequently stained with PerCP-conjugated anti-CD3 and allophycocyanin-conjugated anti-IFNγ, washed twice in perm buffer and resuspended in FACS buffer, prior to being analyzed on a FACSCalibur. All antibodies were from BD Biosciences.

Statistical analyses

Data sets were compiled and analyzed in Statistica, release 6.0 (Statsoft, Tulsa, OK) and Prism, release 4.0 (GraphPad Software, San Diego, CA). Groups comparisons were performed using non-parametric Kruskal Wallis ANOVA by ranks test; associations between variables were evaluated by Spearman rank order correlation's test. Critical p values were considered statistically significant if below 0.05.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

JM planed and conducted the experiments, performed the statistical analyses, and wrote the manuscript; HMRB wrote the original protocol, collected the samples, prepared the database, helped in the experiments, performed statistical analyses, and wrote the manuscript; KAJ conducted the experiments and helped in the flow cytometry analyses; JMC conducted the experiments and helped in the flow cytometry analyses; MKCB conducted the experiments and helped in the flow cytometry analyses; WKN performed the HTLV-I viral load; YN supported the protocol design and the conduction of HTLV-I viral load; ECS performed the HTLV-I serology, helped in the project design, the molecular biology experiments, and writing the manuscript; MAC selected subjects for the cohort and supervised the blood collection of samples from HAM/TSP patients; ASBO selected subjects for the cohort and supervised the blood collection of samples from HAM/TSP patients; DFN participated in the experimental design, obtained funds for the project, discussed the results, and wrote the manuscript; EGK wrote the original protocol, participated in the experimental design, obtained funds for the project, discussed the results, and wrote the manuscript.

Acknowledgements

We would like to thank Marli Campos, Rosemeire Gabriel, and Perpétua for the assistance in blood collection, and Dr. Lishomwa Ndhlovu for helpful scientific discussions. We are also grateful to Karina I. Carvalho, Helena Tomiyama, and Raphaella Goulart for the continuous laboratory support. This work was supported by the Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), grant #04/10918-6, NIH grants (AI41531, AI52731, AI060379, AI064520), and The Fogarty International Center, grant #D43 TW00003. Dr. Barbosa and Ms. Brunialti were supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazilian Ministry of Education.

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