HTLV-1 modulates the frequency and phenotype of FoxP3+CD4+ T cells in virus-infected individuals

Abstract

Background: HTLV-1 utilizes CD4 T cells as the main host cell and maintains the proviral load via clonal proliferation of infected CD4+ T cells. Infection of CD4+ T cells by HTLV-1 is therefore thought to play a pivotal role in HTLV-1-related pathogenicity, including leukemia/lymphoma of CD4+ T cells and chronic inflammatory diseases. Recently, it has been reported that a proportion of HTLV-1 infected CD4+ T cells express FoxP3, a master molecule of regulatory T cells. However, crucial questions remain unanswered on the relationship between HTLV-1 infection and FoxP3 expression.

Results: To investigate the effect of HTLV-1 infection on CD4+ T-cell subsets, we used flow cytometry to analyze the T-cell phenotype and HTLV-1 infection in peripheral mononuclear cells (PBMCs) of four groups of subjects, including 23 HTLV-1-infected asymptomatic carriers (AC), 10 patients with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), 10 patients with adult T-cell leukemia (ATL), and 10 healthy donors. The frequency of FoxP3+ cells in CD4+ T cells in AC with high proviral load and patients with HAM/TSP or ATL was higher than that in uninfected individuals. The proviral load was positively correlated with the percentage of CD4+ T cells that were FoxP3+. The CD4+FoxP3+ T cells, themselves, were frequently infected with HTLV-1. We conclude that FoxP3+ T- cells are disproportionately infected with HTLV-1 during chronic infection. We next focused on PBMCs of HAM/TSP patients. The expression levels of the Treg associated molecules CTLA-4 and GITR were decreased in CD4+FoxP3+ T cells. Further we characterized FoxP3+CD4+ T-cell subsets by staining CD45RA and FoxP3, which revealed an increase in CD45RA-FoxP3low non-suppressive T-cells. These findings can reconcile the inflammatory phenotype of HAM/TSP with the observed increase in frequency of FoxP3+ cells. Finally, we analyzed ATL cells and observed not only a high frequency of FoxP3 expression but also wide variation in FoxP3 expression level among individual cases.

Conclusions: HTLV-1 infection induces an abnormal frequency and phenotype of FoxP3+CD4+ T cells.

Figure 1

CD4⁺T-cell subset in HTLV-1 infected individuals. (A) Percentages of CD4⁺ T cells in 4 distinct subjects. Data shown are gated on lymphocyte fraction based on the dot plot pattern of SSC and FSC. (B and C) Proportion of FoxP3⁻CD45RA⁺ naïve CD4⁺ T cells (B) or FoxP3⁻CD45RA⁻ effector/memory CD4⁺ T cells (C). (D) Percentages of FoxP3⁺ cells in CD4⁺ T cells. (E) Frequency of FoxP3⁺CD4⁺ cells in ACs or HAM/TSP patients showed significant correlation with HTLV-1 proviral load by Spearman’s rank correlation (P = 0.0051 or P = 0.0268, r = 0.60 or r = 0.7012, respectively).

Figure 2

Characterization of Tax expression afterex vivocultivation. (A) A flowchart of the experiment to detect Tax by flow cytometry. (B) The percentages of Tax expression in CD4⁺ T cells after ex vivo culture are shown from 3 distinct ACs. (C–E) Correlation between Tax positivity in CD4⁺ T cells and PVL in ATL patients (C), ACs (D) or HAM/TSP (E).

Figure 3

Frequency of HTLV-1-infection in each CD4⁺T-cell subset of asymptomatic HTLV-1 carriers. PBMCs from HTLV-1 asymptomatic carriers (n = 23) were cultivated for 18 hours, stained with anti-CD4, anti-CD8, anti-FoxP3, and anti-Tax antibodies, and analyzed by flow cytometry. (A) Representative dot plots of CD4 and CD8 and histograms of Tax in CD4⁺ or CD8⁺ T cells (Left panel). Right, cumulative results from 23 AC individuals are shown in graph (Right panel). (B) Representative histograms of Tax expression in FoxP3⁺ or FoxP3⁻ cell (Left panel). Right, cumulative results from 23 AC individuals are shown in graph (Right panel). (C) Tax positivity in CD4⁺ T cells showed significant correlation with FoxP3 positivity in CD4⁺ T cells by Spearman’s rank correlation (P = 0.0257, r = 0.48)

Figure 4

Characterization of FoxP3⁺CD4⁺T-cell subset in AC samples. (A) A result of flow cytometric dot plots of CD45RA and FoxP3 in CD4⁺ T cells of an AC sample is shown as an example. FoxP3⁺CD4⁺ T cells were classified into three subsets. a, CD45RA⁺FoxP3^low resting T_reg cells (rT_reg cells); b, CD45RA⁻FoxP3^low non-regulatory T cells (FoxP3^low non-T_reg cells), c, CD45RA⁻FoxP3^high activated T_reg cells (aT_reg cells). (B–D) Frequencies of the FoxP3⁺CD4⁺ T-cell subset in HD, AC^low, and AC^high. Percentages of resting T_reg cells (B), activated T_reg cells (C), and FoxP3^low non-T_reg cells (D) in CD4⁺ T cells are shown. (E) Representative flow cytometric dot plots of CD45RA and FoxP3 and histograms of Tax expression in each CD4⁺ T-cell subset in an AC sample (Left panel). Cumulative results from 13 AC^low and 10 AC^high individuals are shown in the graph (Middle and right panels).

Figure 5

Detailed characterizations of CD4⁺T-cell subsets in HAM/TSP patients. Freshly isolated PBMCs from HAM/TSP patients (n = 10) were stained with anti-CD4, anti-CD45RA, anti-FoxP3 antibodies and analyzed by flow cytometry. To detect Tax expression, PBMCs were cultivated for 18 hours before antibody staining. (A) Percentages of CD4⁺ or CD8⁺ T cells in HAM/TSP patients. (B) Tax positivity of CD4⁺, CD8⁺, non-T, FoxP3⁻, or FoxP3⁺ cell populations in HAM/TSP patients. (C) Expression levels of T_reg associated molecules in FoxP3⁺ cells of HD or HAM/TSP patients. (D–F) Frequencies of the FoxP3⁺CD4⁺ T-cell subset in HD and HAM/TSP patients. Percentages of rT_reg cells (D), aT_reg cells (E), and FoxP3^low non-T_reg cells (F) in CD4⁺ T cells are shown. (G) Tax positivity of each CD4⁺ T-cell subset in HAM/TSP patients

Figure 6

Characterization of CD4⁺T-cell subsets in ATL cells. PBMCs from ATL patients (n = 10) were stained with anti-CD4, anti-CD45RA, anti-CD25, and anti-FoxP3 antibodies and analyzed by flow cytometry. (A) The expression pattern of CD45RA and FoxP3 of CD4⁺ T cells in ATL patients. (B) The expression pattern of CD25 and FoxP3 of CD4⁺ T cells in ATL patients.