Virus-induced dysfunction of CD4+CD25+ T cells in patients with HTLV-I-associated neuroimmunological disease

Abstract

CD4(+)CD25(+) Tregs are important in the maintenance of immunological self tolerance and in the prevention of autoimmune diseases. As the CD4(+)CD25(+) T cell population in patients with human T cell lymphotropic virus type I-associated (HTLV-I-associated) myelopathy/tropical spastic paraparesis (HAM/TSP) has been shown to be a major reservoir for this virus, it was of interest to determine whether the frequency and function of CD4(+)CD25(+) Tregs in HAM/TSP patients might be affected. In these cells, both mRNA and protein expression of the forkhead transcription factor Foxp3, a specific marker of Tregs, were lower than those in CD4(+)CD25(+) T cells from healthy individuals. The virus-encoded transactivating HTLV-I tax gene was demonstrated to have a direct inhibitory effect on Foxp3 expression and function of CD4(+)CD25(+) T cells. This is the first report to our knowledge demonstrating the role of a specific viral gene product (HTLV-I Tax) on the expression of genes associated with Tregs (in particular, foxp3) resulting in inhibition of Treg function. These results suggest that direct human retroviral infection of CD4(+)CD25(+) T cells may be associated with the pathogenesis of HTLV-I-associated neurologic disease.

Figure 1

Decreased Foxp3 expression in CD4⁺CD25⁺ T cells from HAM/TSP patients. (A) Quantitative expression of foxp3 mRNA was determined by real-time RT-PCR. The level of foxp3 mRNA expression was calculated as the relative quantity of foxp3 mRNA expression divided by the relative quantity of endogenous control HPRT mRNA expression, as described in Methods. The data represent isolated cell subsets (CD4⁺CD25⁺ or CD4⁺CD25^–) from 13 uninfected HDs and 13 HAM/TSP patients (HAM). Foxp3 mRNA expression was significantly reduced in the CD4⁺CD25⁺ T cell subset from HDs compared with that from HAM/TSP patients. (B) A representative histogram of intracellular expression of Foxp3 protein showing results from flow cytometric analysis of PBMC samples from HAM/TSP patients and HDs. Foxp3 protein expression was detected in the CD4⁺CD25⁺ T cell subset from HDs but not in CD4⁺CD25^– or in total CD4^– T cell subsets. In contrast, the number of Foxp3-positive cells in CD4⁺CD25⁺ T cells from HAM/TSP patients was clearly reduced. (C) Data represent averaged percentage of Foxp3-positive cells in each T cell subset. The percentage (mean ± SD) of Foxp3-positive cells in CD4⁺CD25⁺ T cells of 8 HAM/TSP patients (3.09% ± 1.04%) was significantly lower than that of 8 HDs (25.9% ± 8.23%; P = 0.0014). No difference in the protein expression levels of Foxp3 was observed in CD4⁺CD25^– or CD4^– cells between HAM/TSP patients and HDs.

Figure 2

Lack of regulatory function in CD4⁺CD25⁺ T cells from HAM/TSP patients. A total of 1 × 10⁵ CD4⁺CD25^– T cells/well from HDs were labeled with CFSE. They were cultured for 6 days in the culture medium in the absence or presence of 2.5 μg/ml anti-CD3 antibody (top 2 panels). They were also cultured for 6 days in 2.5 μg/ml anti-CD3 antibody added to culture medium with 1 × 10⁵ irradiated allogeneic CD4⁺CD25⁺ T cells from HDs or with 1 × 10⁵ irradiated CD4⁺CD25⁺ T cells from HAM/TSP patients (bottom 2 panels). The data indicate that regulatory function in CD4⁺CD25⁺ T cells from HAM/TSP patients is reduced in comparison with that in CD4⁺CD25⁺ T cells from HDs. Failure of CD4⁺CD25⁺ T cells to suppress lymphoproliferation of activated HD cells was observed in separate experiments with cells from 4 HAM/TSP patients, while suppression of activated HD cell proliferation by allogeneic HD CD4⁺CD25⁺ T cells from 2 HDs was demonstrated.

Figure 3

HTLV-I Tax suppresses Foxp3 expression. Purified CD4⁺CD25⁺ T cells and CD4⁺CD25^– T cells from HDs were transfected with the HTLV-I tax gene (n = 7) or HTLV-I env gene (n = 4). The foxp3 mRNA expression in these T cell populations before and after transfection was measured by real-time RT-PCR. (A) The foxp3 mRNA expression level in CD4⁺CD25⁺ T cells was significantly decreased by transfection with HTLV-I tax gene (P = 0.018). By contrast, there was no significant decrease in foxp3 mRNA expression in CD4⁺CD25^– T cells. (B) foxp3 mRNA expression was significantly decreased in HTLV-I tax–transfected CD4⁺CD25⁺ T cells compared with HTLV-I env–transfected CD4⁺CD25⁺ T cells (P = 0.020). There was no significant difference between the foxp3 mRNA expression in HTLV-I tax transfected CD4⁺CD25^– T cells and that in HTLV-I env–transfected CD4⁺CD25^– T cells. env, HTLV-I env gene; tax, HTLV-I tax gene.

Figure 4

Loss of regulatory function in HTLV-I tax–transfected HD CD4⁺CD25⁺ T cells. CD4⁺CD25⁺ or CD4⁺CD25^– T cells from uninfected HDs were stimulated with 2.5 μg/ml anti-CD3 antibody and irradiated PBMCs and cultured for 4 days (HD CD25⁺ and HD CD25^–). Furthermore, to compare the suppressive activity of HD CD4⁺CD25⁺ T cells before and after HTLV-I tax gene transfection, CD4⁺CD25^– T cells from HDs were stimulated with 2.5 μg/ml anti-CD3 antibody and irradiated PBMCs and cultured for 4 days in the presence of equal numbers of HD CD4⁺CD25⁺ T cells or HTLV-I tax–transfected HD CD4⁺CD25⁺ T cells (Tax⁺ HD CD25⁺). After culture, [³H]thymidine was added for additional 16 hours. The suppressive activity of CD4⁺CD25⁺ T cells from HDs was inhibited by transfection with the HTLV-I tax gene. Data represent the mean of experiments with cells from 3 HDs.