Identification of T-cell epitopes on U1A protein in MRL/lpr mice: double-negative T cells are the major responsive cells

Abstract

Systemic lupus erythematosus (SLE) is characterized by the existence of a heterogeneous group of autoantibodies such as anti-DNA, chromatin, histone, and ribonucleoprotein antibodies (Abs). Although the B-cell antigenic determinants have been well characterized, very limited data about the T-cell epitopes of self-antigen (Ag) have been reported. In the present study, we analysed auto-T-cell epitopes using bone marrow-derived dendritic cells (BM-DCs) pulsed with murine U1A (mU1A) protein capable of activating autoreactive T cells from unprimed MRL/lpr mice in vitro. The data suggested that there are at least four T-cell epitopes on the U1A protein, U1A31-50, U1A61-80, U1A201-220 and U1A271-287, and U1A31-50 had the most significant T-cell proliferative response. In addition, the main responsive T cells are the CD4- CD8- double-negative subgroup of T cells. Furthermore, we also demonstrated that the activation of double-negative T cells is major histocompatibility complex class II restricted. The study here provides information on T-cell epitope analysis of the U1A antigen using BM-DCs as the effective antigen-presenting cells.

Figure 1

The level of autoantibodies in MRL/lpr mice over time with age. Sera obtained from five BALB/c and five MRL/lpr mice at different time-points were tested for anti-dsDNA IgG (a) and anti- U1A IgG (b) by ELISA. Sera were diluted 1 ≕ 100 for detecting these two kinds of autoantibodies. Values that were greater than the mean + 3SD (horizontal dash line) from 4-month-old BALB/c mice (n = 5) were regarded as positive. *Indicates P < 0·01 when compared to age-matched BALB/c mice.

Figure 2

The T-cell proliferation against U1A protein by syngeneic bone marrow-derived dendritic cells (BMDCs). T cells from C3H (a) or MRL/lpr mice (b) at 3–4 months of age were cocultured with OVA (10 μg/ml) or U1A (10 μg/ml) -pulsed syngeneic BMDCs for 5 days. The data are presented as mean ± SD, n = 3 in C3H mice, n = 5 in MRL/lpr mice. These results were obtained from two independent experiments. When SI (stimulation index) was > 2 (horizontal line), we referred to it as positive. *Indicates P < 0·01 when compared to mU1A on C3H mice group.

Figure 3

Identification of auto-T-cell epitopes in the U1A protein by using bone marrow-derived dendritic cells (BMDCs) as antigen-presenting cells. T cells from C3H (a) or MRL/lpr mice (b) at 3–4 months of age were cocultured with overlapping peptides of U1A-pulsed syngeneic BMDCs (100 μg/ml). Results are expressed as mean ± SD, n = 3 in C3H mice, n = 4 in MRL/lpr mice. These results were obtained from two independent experiments. Dotted horizontal line indicates an SI of 2·0; *indicates P < 0·01 when compared to each peptide on C3H mice group.

Figure 4

Identification of auto-T cell epitopes in the U1A protein recognized by CD4T cells or DNT cells by using bone marrow-derived dendritic cells (BMDCs) as antigen-presenting cells. CD4T cells (a) or DNT cells (b) were acquired from MRL/lpr mice at 3–4 months of age and cocultured with some peptides of U1A-pulsed syngeneic BMDCs. The data are shown as mean ± SD, n = 5 in CD4T cells group, n = 4 in DNT cells group. These results were obtained from two independent experiments. The dotted horizontal line indicates an SI of 2·0; *represents P < 0·01 when compared to each peptide on CD4⁺T-cell group.

Figure 5

The per cent of DNT cell proliferation in response to coculture with U1A₃₁₋₅₀-pulsed bone marrow-derived dendritic cells (BMDCs) together with MHC class II blocker, I-A^d (act as negative control) and I-A^k. The concentrations of MHC class II blocker were 6·25 and 12·5 μg/ml. The data were shown as the c.p.m. of T-cell proliferation in groups with MHC class II blocker added or not. (The value of per cent of proliferation was defined as 100% in DNT cells cocultured with U1A₃₁₋₅₀-pulsed BMDCs alone). Results from three mice were presented as mean ± SD. *Indicates P < 0·01 when compared to that of I-A^d control.