High expression of CD244 and SAP regulated CD8 T cell responses of patients with HTLV-I associated neurologic disease

Abstract

HTLV-I-specific CD8(+) T cells have been characterized with high frequencies in peripheral blood and cerebrospinal fluid and production of proinflammatory cytokines, which contribute to central nervous system inflammation in HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). However, little is known about the differences in CD8(+) T cell activation status between asymptomatic carrier (ACs) and patients with HAM/TSP. The expression of CD244, a signaling lymphocyte activation molecule (SLAM) family receptor, was significantly higher on CD8(+) T cells in HTLV-I-infected patients, both ACs and patients with HAM/TSP, than those on healthy normal donors (NDs). Blockade of CD244 inhibited degranulation and IFN-gamma production in CD8(+) T cells of patients with HAM/TSP, suggesting that CD244 is associated with effector functions of CD8(+) T cells in patients with HAM/TSP. Moreover, SLAM-associated protein (SAP) was overexpressed in patients with HAM/TSP compared to ACs and NDs. SAP expression in Tax-specific CTLs was correlated in the HTLV-I proviral DNA loads and the frequency of the cells in HTLV-I-infected patients. SAP knockdown by siRNA also inhibited IFN-gamma production in CD8(+) T cells of patients with HAM/TSP. Thus, the CD244/SAP pathway was involved in the active regulation of CD8(+) T cells of patients with HAM/TSP, and may play roles in promoting inflammatory neurological disease.

Figure 1. Expression of CD244 on CD8⁺ T cells of HTLV-I-infected patients.

(A) Representative histograms of CD244 expression on CD8⁺ T cells of a ND, an AC and a patient with HAM/TSP. Staining in ND (opened histogram), AC (grayed histogram) and patient with HAM/TSP (closed histogram) were shown. (B) Comparison of CD244 expression in CD8⁺ T cells of NDs and HTLV-I-infected patients. The data were obtained from 14 NDs, 8 ACs and 13 patients with HAM/TSP. The horizontal line represents the median. (C) CD244 expression on antigen-specific CTLs. CD244 expressions were shown in Tax11-19- specific and CMVpp65-specific CD8⁺ T cells of a patient with HAM/TSP.

Figure 2. Involvement of CD244 in CD8⁺ T cell degranulation and IFN-γ expression of patients with HAM/TSP.

(A) Representative dot plot of spontaneous degranulation and IFN-γ expression in CD8⁺ T cells of a ND and a patient with HAM/TSP after culture for 24 hours. The PBMCs were cultured for 24 hours without any exogenous stimulators. (B) Dose-dependent inhibition of spontaneous degranulation and IFN-γ expression in CD8⁺ T cells of a patient with HAM/TSP by anti-CD244 (closed circle) and anti-CD48 (opened circle). The PBMCs were cultured with each antibody for 24 hours. (C) Inhibitory effects of anti-CD244 and anti-CD48 on degranulation and IFN-γ production in CD8⁺ T cells of patients with HAM/TSP. The PBMCs were cultured with 1 µg/ml of control IgG, anti-CD244 or anti-CD48 for 24 hours. The amount of CD107a/IFN-γ expressions of CD8⁺ T cells cultured with control IgG were normalized to 100%, and then, those in PBMCs cultured with each antibody were calculated. The graph was prepared from data obtained from 7 patients with HAM/TSP. Error bars represent SD. (D) CD48 and Tax expression in HTLV-I-infected cells of a patient with HAM/TSP. The top panels show CD48 and Tax expression in CD4⁺ T cells before (left) and after (right) the culture for 20 hours. The bottom panels show CD48 and Tax expression in CD14⁺ cells before (left) and after (right) the culture for 20 hours.

Figure 3. Distribution of CD244 in PBMCs of patients with HAM/TSP.

After the culture for 8 hours, PBMCs were stained with antibodies against perforin, CD244 and CD48, and visualized through microscope. Two representative 3D images were shown in A and B. The image shows DAPI (blue), perforin (green), CD244 (orange) and CD48 (purple). In addition to DAPI and perforin (left), CD244 (middle) and CD48 (right) are merged. The white arrows indicate CD244 clustering at the cell contact area.

Figure 4. Expression of SLAM-associated proteins in CD8⁺ T cells of HTLV-I-infected patients.

(A) Representative histograms of SAP expression in CD8⁺ T cells of a ND (opened histogram), an AC (grayed histogram), and a patient with HAM/TSP (closed histogram). (B) Comparison of SAP expression in CD8⁺ T cells of NDs (n = 11), ACs (n = 8) and patients with HAM/TSP (n = 10). The horizontal line represents the median. (C) Comparison of EAT-2 expression in CD8⁺ T cells of NDs (n = 10), ACs (n = 5) and patients with HAM/TSP (n = 10). (D) SAP expression in Tax11-19-specific CTLs of HTLV-I-infected patients. SAP expressions were shown in Tax11-19-specific CD8⁺ T cells of AC and patient with HAM/TSP. (E) Correlation of the HTLV-I-proviral DNA loads (circles; R² = 0.4746, P = 0.0401) and the frequency of Tax11-19-specific CD8⁺ T cells (squares R² = 0.6811, P = 0.0062) with SAP expression in the cells of AC (n = 4, opened circles and squares) and patients with HAM/TSP (n = 5, closed circles and squares).

Figure 5. SAP and CD244 expressions in CD8⁺ T cells of NDs after stimulation with IL-2 or IL-15.

(A) SAP expressions in CD8⁺ T cells isolated from ND PBMCs were compared after the culture with rhIL-2 (opened circle) or rhIL-15 (closed circle) for 7 days. The graphs were prepared from data obtained from two NDs (left and right). (B) CD244 expressions in CD8⁺ T cells isolated from ND PBMCs were compared after the culture with rhIL-2 (opened circle) or rhIL-15 (closed circle) for 7 days. The graphs were prepared from data obtained from two NDs (left and right).

Figure 6. Inhibition of degranulation and IFN-γ expression in CD8⁺ T cells of patients with HAM/TSP by SAP siRNA.

(A) SAP and EAT-2 expression in transfected CD8⁺ T cells with either control, SAP or EAT-2 siRNA was determined after the culture for 6 hours. The cell lysates were prepared from transfected CD8⁺ T cells, and each 10µg of the cell lysates was loaded on the gel. (B) Inhibitory effects of SAP or EAT-2 siRNA on degranulation and IFN-γ expression in transfected CD8⁺ T cells. Isolated CD8⁺ T cells from patients with HAM/TSP were transfected with control, SAP or EAT-2 siRNA, and then cocultured with autologous CD14⁺ cells. The amounts of CD107a/IFN-γ expressions in CD8⁺ T cells transfected with control siRNA were normalized to 100%, and then, those in CD8⁺ T cells transfected with SAP or EAT-2 siRNA were calculated. The graph was prepared from data obtained from three patients with HAM/TSP. Error bars represent SD.