HTLV-1 induces a Th1-like state in CD4+CCR4+ T cells

Abstract

Human T-lymphotropic virus type 1 (HTLV-1) is linked to multiple diseases, including the neuroinflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T cell leukemia/lymphoma. Evidence suggests that HTLV-1, via the viral protein Tax, exploits CD4+ T cell plasticity and induces transcriptional changes in infected T cells that cause suppressive CD4+CD25+CCR4+ Tregs to lose expression of the transcription factor FOXP3 and produce IFN-γ, thus promoting inflammation. We hypothesized that transformation of HTLV-1-infected CCR4+ T cells into Th1-like cells plays a key role in the pathogenesis of HAM/TSP. Here, using patient cells and cell lines, we demonstrated that Tax, in cooperation with specificity protein 1 (Sp1), boosts expression of the Th1 master regulator T box transcription factor (T-bet) and consequently promotes production of IFN-γ. Evaluation of CSF and spinal cord lesions of HAM/TSP patients revealed the presence of abundant CD4+CCR4+ T cells that coexpressed the Th1 marker CXCR3 and produced T-bet and IFN-γ. Finally, treatment of isolated PBMCs and CNS cells from HAM/TSP patients with an antibody that targets CCR4+ T cells and induces cytotoxicity in these cells reduced both viral load and IFN-γ production, which suggests that targeting CCR4+ T cells may be a viable treatment option for HAM/TSP.

Figure 5. CCR4 shows potential as a molecular target for HAM/TSP immunotherapy.

(A–G) Cells isolated from HAM/TSP patients were sorted via FACS (A; n = 7) or cultured for 7 days under the following conditions: PBMCs were cultured with various concentrations of KM2760 or 1 μg/ml PSL (B–E; n = 9), and CSF cells were cultured with 1 μg/ml KM2760 (F and G; n = 8). (A, C, and F) HTLV-1 proviral DNA loads were measured using quantitative PCR. (D) Degree of spontaneous proliferation was assessed by measuring ³H-thymidine incorporation. (E and G) IFN-γ production in the culture media was evaluated using CBA assays. HTLV-1 resided in CD4⁺CCR4⁺ rather than CCR4^– cells among PBMCs (A), and KM2760 treatment effectively targeted these cells (B). Consequently, KM2760 treatment successfully reduced HTLV-1 proviral DNA load (C), suppressed spontaneous proliferation (D), and decreased IFN-γ production (E) in PBMC cultures as well as reducing HTLV-1 DNA load (F) and IFN-γ production (G) in CSF cell cultures derived from HAM/TSP patients. (A and C–E) Data are mean ± SD. (B, F, and G) Thick horizontal bars represent mean value for all patients; line segments represent individual patients. Statistical analyses were performed using Friedman test followed by Dunn test for multiple comparisons (C–E) or Wilcoxon test (A, B, F, and G). *P < 0.05, **P < 0.01, ***P < 0.001 vs. untreated control.

Figure 4. HTLV-1–infected Th1-like CCR4⁺ cells invade the CNS of HAM/TSP patients.

(A) Detection of CCR4⁺ cells expressing T-bet, IFN-γ, and CXCR3 infiltrating the spinal cord of a HAM/TSP patient. Representative images show immunofluorescent codetection of CCR4 with T-bet, IFN-γ, and CXCR3, as well as the merged images, in thoracic spinal cord sections. Rabbit and goat IgG antibody served as a negative control. Scale bars: 20 μm. (B) Presence of HTLV-1–infected CCR4⁺ cells in HAM/TSP patient CSF. Representative images show immunofluorescence-FISH codetection of CCR4 (green) and HTLV-1 provirus (red) in Jurkat cells (uninfected control), MT-2 cells (infected control), and CSF cells from the patients. Arrows denote red provirus signal in the CSF sample. Scale bars: 20 μm. (C) CD4⁺ T cells in HAM/TSP patient CSF were mostly CCR4⁺CXCR3⁺. A dot plot of CCR4 and CXCR3 expression in CD4⁺ gated cells isolated from the CSF of a representative HAM/TSP patient is shown. (D) CD4⁺CCR4⁺CXCR3⁺ cells were numerous in CSF, but not elevated in peripheral blood, of HAM/TSP patients. Graphs show the percentages of CCR4^–CXCR3^–, CCR4^–CXCR3⁺, CCR4⁺CXCR3^– and CCR4⁺CXCR3⁺ T cells among CD4⁺ PBMCs and CSF cells from HAM/TSP patients (n = 8) and PBMCs from HDs (n = 4). Analysis was performed using FACS. Data are mean ± SD. (E) Proliferation was not observed in CD4⁺CCR4⁺CXCR3⁺ cells from HAM/TSP patient CSF. The rate of Ki67 expression, a marker for cell proliferation, is shown for CD4⁺CCR4⁺CXCR3⁺ gated cells from the CSF of a representative HAM/TSP patient.

Figure 3. Tax and Sp1 cooperatively enhance TBX21 promoter activity.

(A) Co-IP of endogenous Tax and Sp1. Nuclear extracts from MT-2 cells were immunoprecipitated with anti-Tax or anti–Sp1 antibodies or with normal IgG as a control, then immunoblotted with anti-Tax or anti-Sp1 antibodies as indicated. (B) Tax bound to the TBX21 promoter in vivo. ChIP assay using anti-Tax antibody followed by primers encompassing the TBX21 promoter region (–179 to –59) was performed on genomic DNA isolated from MT-2 cells. DNA (input) and IP with anti-Sp1 served as positive controls, and normal IgG served as a negative control. (C) Coactivation of TBX21 promoter by Sp1 and Tax. HEK293 cells were transfected with 100 ng of TBX21-Luc reporter plasmid or Sp1 expression plasmid, as well as 0–100 ng of Tax expression plasmid as indicated. Values were normalized to β-galactosidase activity as an internal control. Data are mean ± SD.

Figure 2. Tax induces IFN-γ production via T-bet.

(A) Tax-dependent IFNG mRNA expression in JPX-9 cells. Experiments were performed in triplicate. (B) Elevated TBX21 mRNA expression in CD4⁺CD25⁺CCR4⁺ T cells from HAM/TSP patients relative to HDs (n = 4 per group). (C) Tax-dependent TBX21 mRNA expression in JPX-9 cells. Experiments were performed in triplicate. (D) Reduced TBX21 mRNA expression after silencing Tax in CD4⁺CD25⁺CCR4⁺ T cells from HAM/TSP patients. PBMCs from HAM/TSP patients (n = 5) were FACS sorted, transfected with either Luc or Tax siRNA, and incubated for 24 hours. (E and F) Tax expression correlated with T-bet expression and IFN-γ production in CD4⁺CCR4⁺ T cells from HAM/TSP patients. CD4⁺CCR4⁺ T cells isolated from HDs and HAM/TSP patients (n = 4 per group) were cultured before being stained for Tax and T-bet protein and analyzed using FACS. IFN-γ production in the culture medium was measured using a CBA assay. ND, not detectable. All data are mean ± SD. P values were calculated using (A and C) 1-way ANOVA followed by Dunnett test for multiple comparisons, (B) Mann-Whitney U test, (D) paired t test, or (E and F) Friedman test followed by Dunn test for multiple comparisons. *P < 0.05, ***P < 0.001 vs. time point 0.

Figure 1. HTLV-1 mainly infects Tregs and inhibits their regulatory function.

(A) Higher HTLV-1 proviral DNA load in CD4⁺FOXP3⁺ cells (Tregs) compared with CD4⁺GATA3⁺ cells (P = 0.0020, Wilcoxon test) from asymptomatic carriers (AC; n = 6) and HAM/TSP patients (n = 4). PBMCs were FACS sorted, and proviral load was measured using quantitative PCR. Horizontal bars represent the mean value for each set. (B) Loss of regulatory function in Tax-expressing CD4⁺CD25⁺CCR4⁺ cells (Tregs). CD4⁺CD25^– T cells from an HD were stimulated with CD2, CD3, and CD28 antibodies and cultured alone or in the presence of equal numbers of CD4⁺CD25⁺CCR4⁺ T cells, GFP lentivirus–infected HD CD4⁺CD25⁺CCR4⁺ T cells, or GFP-Tax lentivirus–infected HD CD4⁺CD25⁺CCR4⁺ T cells. As a control, CD4⁺CD25^– T cells alone were cultured without any stimulus. Proliferation of T cells was determined using ³H-thymidine incorporation by adding ³H-thymidine for 16 hours after 4 days of culture. All tests were performed in triplicate. Data are mean ± SD. **P < 0.01, ***P < 0.001, ANOVA followed by Tukey test for multiple comparisons.