Epigenetic modification of the FoxP3 TSDR in HAM/TSP decreases the functional suppression of Tregs

Abstract

HTLV-1 is a human retrovirus that is associated with the neuroinflammatory disorder HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). In these patients, HTLV-1 is primarily found in the CD4(+)CD25(+) T cell subset (Regulatory T cells:Tregs), which is responsible for peripheral immune tolerance and is known to be dysfunctional in HAM/TSP. Recent evidence suggests that FoxP3 expression and function is determined epigenetically through DNA demethylation in the Treg-specific demethylated region (TSDR). We analyzed the methylation of the TSDR in PBMCs, CD4(+) T cells, and CD4(+)CD25(+) T cells from normal healthy donors (NDs) and HAM/TSP patients. We demonstrated that there is decreased demethylation in analyzed PBMCs and CD4(+)CD25(+) T cells from HAM/TSP patients as compared to NDs. Furthermore, decreased TSDR demethylation was associated with decreased functional suppression by Tregs. Additionally, increased HTLV-1 Tax expression in HAM/TSP PBMC culture correlated with a concomitant decline in FoxP3 TSDR demethylation. Overall, we suggest that HTLV-1 infection decreases Treg functional suppressive capacity in HAM/TSP through modification of FoxP3 TSDR demethylation and that dysregulated Treg function may contribute to HAM/TSP disease pathogenesis.

Fig. 1

(A) % FoxP3 TSDR demethylation in ND PBMC (n=10), isolated CD4⁺ T cells, and isolated CD4⁺CD25⁺ T cells. (B) % FoxP3 TSDR demethylation in HAM/TSP PBMC (n=9), isolated CD4⁺ T cells, and isolated CD4⁺CD25⁺ T cells. The long horizontal bars represent the mean for each group while the shorter bars represent the standard deviation. (C) A comparison of the %CD4⁺CD25⁺ in CD3⁺ T cells of ND and HAM/TSP PBMC. (D) Normalized % FoxP3 TSDR demethylation in isolated CD4⁺CD25⁺ T cells of ND and HAM/TSP patients. % FoxP3 TSDR demethylation was normalized to the % CD4⁺CD25⁺. (E) Normalized % FoxP3 TSDR demethylation in PBMC of ND and HAM/TSP patients. % FoxP3 TSDR demethylation was normalized to the % CD4⁺CD25⁺.

Fig. 2

(A) % FoxP3⁺ cells in CD4⁺CD25⁺ T cells of ND and HAM/TSP patients. The long horizontal bars represent the mean for each group while the shorter bars represent the standard deviation. (B) No correlation of the %FoxP3 TSDR demethylation in PBMCs with the % FoxP3⁺ cells in CD4⁺CD25⁺ T cells of ND (opened circles) and HAM/TSP patients (closed circles).

Fig. 3

Lymphoproliferation suppression assays were set up and ND (n=3) or HAM/TSP (n=2) CD4⁺CD25⁺ T cells were added at different ratios of Treg:Teff = 0.25:1 (A), 0.5:1 (B) and 1:1 (C). The % suppressions of Treg were normalized to Teff proliferation without Treg. (D) Correlation between CD4⁺CD25⁺ TSDR % demethylation and suppressive capacity as determined in lymphoproliferation suppression assays at Treg:Teff ratio of 1:1.

Fig.4

HTLV-1-infected and HTLV-1-uninfected cell line profiles. (A) Flow cytometry profiles of HTLV-1-infected and HTLV-1-uninfected cell lines. The x-axis denotes CD25 expression and the y-axis denotes FoxP3 expression. (B) FoxP3 TSDR demethylation analysis of HTLV-1-infected and HTLV-1-uninfected cell lines. (C) Tax mRNA expression in the cell lines by RTPCR. The data were calculated as relative amount to MT2 (1.00). (D) HBZ mRNA expression in the cell lines by RT-PCR. The data were calculated as relative amount to MT2 (1.00).

Fig. 5

Increased Tax expression correlates with reduced FoxP3 TSDR demethylation. (A) Tax protein expression in CD4⁺CD25⁺ T cells of HAM/TSP patients before and after culture. PBMCs of HAM/TSP patients (n=6) were cultured for 24 hours. (B) Correlation of the Tax protein expression in CD4⁺CD25⁺ T cells of HAM/TSP patients with the change in FoxP3 TSDR demethylation before and after culture for 24 hours. ND samples are circled in red. Coefficient of determination r²= 0. 7736 (C) %FoxP3 TSDR demethylation and tax mRNA expression in isolated HAM/TSP CD4⁺ T cells ex vivo.