Characterization of self-T-cell response and antigenic determinants of U1A protein with bone marrow-derived dendritic cells in NZB x NZW F1 mice

Abstract

Systemic lupus erythematosus (SLE) is characterized by the existence of a heterogeneous group of autoantibodies directed against nuclear intact structures, such as nucleosomes and small nuclear ribonucleoproteins (snRNPs). Autoantibodies against snRNPs are of special interest because they are detectable in the majority of SLE patients. Although the B-cell antigenic determinants have been well characterized, very limited data have been reported in regard to the T-cell epitopes of snRNPs. Furthermore, several studies have demonstrated that determination of the auto-T-cell epitopes recognized by freshly isolated T cells is difficult from unprimed lupus mice when self-antigen-pulsed B cells or macrophages are used as antigen-presenting cells (APCs) in vitro. In the present study, we showed a novel approach for determining the auto-T-cell epitopes, using bone marrow-derived dendritic cells (BMDCs) pulsed with the murine U1A protein - an immunodominant antigen of the U1 snRNPs - which is capable of activating freshly isolated T cells from unprimed (NZB x NZW) F1 (BWF1) mice in vitro. The T-cell epitope area was found to be located at the C-terminus of U1A, overlapping the T-cell epitope of human U1A that has been reported in human SLE. Identification of the autoreactive T-cell epitope(s) in snRNPs will help to elucidate how reciprocal T-B determinant spreading of snRNPs emerges in lupus. The results presented here also indicate that it is feasible to use this approach to further explore strategies to design immunotherapy for patients with lupus.

Figure 3

Bone-marrow-derived dendritic cells (BMDCs) pulsed with U1A protein elicited an autoreactive T-cell response from unprimed BWF₁ mice in vitro. (a) Purified T cells isolated from unmanipulated 12-week-old BWF₁ mice or DBA-2 × NZW F₁ mice were cocultured with syngeneic BMDCs pulsed with increasing concentrations of U1A. After 5 days, the proliferation assay was performed. Results are expressed as stimulation index (SI) (mean ± SD, n = 3–6). The mean background (T cells plus non-antigen-pulsed BMDCs) counts per minute (c.p.m.) was 1375. Results are representative of two independent experiments. An asterisk indicates a statistically significant difference (P < 0·01) in [³H]thymidine incorporation. (b) BMDCs were incubated with U1A (10 µg/ml), human gamma globulin (HGG) (100 µg/ml), hepatitis D virus (HDV) surface antigen (20 µg/ml) or histidine TAG control peptide (1 µg/ml). Experimental conditions were similar to those described in Figure 3(a). Results are shown as SI (mean ± SD, n = 3–6). Background (T cells plus non-antigen-pulsed BMDCs) c.p.m. varied from 733 to 2016. Splenocytes (SC) from 12-week-old BWF₁ mice (n = 4) were cocultured with U1A proteins (0·1, 1 or 10 µg/ml) for 5 days. The value of baseline proliferation ranged from 253 to 550. Dotted horizontal lines indicate an SI of 3·0. These data are representative of four separate experiments.

Figure 1

The level of anti-U1A immunoglobulin G (IgG) in BWF₁ mice over time with age. Sera obtained from 15 BWF₁ mice at each time-point shown were tested for anti-U1A IgG by enzyme-linked immunosorbent assay (ELISA), as described in the Materials and methods. Sera was diluted 1 : 100 for anti-U1A antibodies. Values that were greater than the mean ELISA units + 3 SD (horizontal line) from 4-month-old BALB/c mice (n = 6) were regarded as positive.

Figure 2

Bone marrow-derived dendritic cells (BMDCs) could present ovalbumin (OVA) to stimulate T cells from OVA-immunized DBA-2 × NZW F₁ mice. Purified T cells from phosphate-buffered saline (PBS) or OVA-immunized DBA-2 × NZW F₁ mice were cocultured with non-antigen- or OVA-pulsed syngeneic BMDCs for 5 days. [³H]Thymidine was added during the last 4–6 hr of culture. The T-cell proliferation was calculated as the stimulation index (SI) (mean ± SD, n = 3). Background (T cells plus non-antigen-pulsed BMDCs) counts per minute (c.p.m.) varied from 323 to 804. The dotted horizontal line indicates an SI of 3·0.

Figure 4

U1A-pulsed bone marrow-derived dendritic cells (BMDCs) stimulated the proliferative response of autoreactive CD4⁺ T cells in a major histocompatibility complex (MHC) class II-dependent manner in vitro. Purified T cells (a) or Thy1.2⁺ T cells (b) from 12-week-old BWF₁ mice were cocultured with U1A (10 µg/ml)-pulsed syngeneic BMDCs in the presence or absence of blocking monoclonal antibody (mAb), as indicated above. The concentrations of mAbs were as follows: anti-IA^d/E^d mAb at 25 µg/ml, anti-IA^b mAb at 10 µg/ml, and anti-CD4 and anti-CD8 mAbs at 5 µg/ml. Responses are presented as mean stimulation index (SI) (± SD) from one of three similar experiments. The mean background counts per minute (c.p.m.) was 807 (a) and 1007 (b). Dotted horizontal lines indicate an SI of 3·0.

Figure 5

The T-cell proliferation against U1A stimulated by allogeneic bone marrow-derived dendritic cells (BMDCs). Thy1.2⁺ T cells from BWF₁ (left) or DBA-2 × NZW F₁ (right) mice at 6 months of age were cocultured with ovalbumin (OVA) (10 µg/ml) or U1A (10 µg/ml)-pulsed allogeneic BMDCs from DBA-2 × NZW F₁ (left) or BWF₁ mice (right) for 5 days, respectively. The mean background counts per minute (c.p.m.) was 3313 (left) or 62 310 (right). The data shown (mean ± SD, n = 3 each strain of mice) represent results obtained in two independent experiments. SI, stimulation index.

Figure 6

The T-cell response against U1A in young and old BWF₁ mice. Purified T cells from either 12-week-old or 28-week-old BWF₁ mice were cocultured with ovalbumin (OVA) (10 µg/ml) or U1A (10 µg/ml)-pulsed syngeneic bone marrow-derived dendritic cells (BMDCs). Data are expressed as the stimulation index (SI) (mean±SEM, n = 7 each group). The mean background counts per minute (c.p.m.) (T cells plus non-antigen-pulsed BMDCs) of young or old mice was 771 or 470, respectively. Dotted horizontal lines indicate an SI of 3·0. Results are representative of two separate experiments.

Figure 7

Cytokine production of U1A-specific T cells from BWF₁ mice stimulated by bone marrow-derived dendritic cells (BMDCs). Purified T cells isolated from unmanipulated 12-week-old BWF₁ (n = 5, mean ± SD) mice were cocultured with irradiated syngeneic BMDCs pulsed with increasing concentrations of U1A, as indicated in the figure. The level of cytokines produced from ovalbumin (OVA)-pulsed BMDCs plus purified T cells was similar to that of non-antigen-pulsed BMDCs plus T cells. Culture supernatants were collected at 24 hr for interleukin (IL)-5, at 48 hr for IL-2 and interferon-γ (IFN-γ), and at 72 hr for IL-4. These data are representative of two independent experiments.

Figure 8

Identification of auto-T-cell epitopes in the U1A protein using bone marrow-derived dendritic cells (BMDCs) as antigen-presenting cells (APCs). Thy1.2⁺ T cells from unprimed BWF₁ (a) or DBA-2 × NZW F₁ mice (b) at 10 months of age were cocultured with overlapping peptides of U1A-pulsed syngeneic BMDCs. T cells from identical BWF₁ mice were cocultured with purified non-T cells (B cells + macrophages) (c) in the presence or absence of U1A peptides. Results are expressed as the mean stimulation index (SI) ± SD calculated from six mice tested individually in two separate experiments. The mean background counts per minute (c.p.m.) was 657 (a), 1401 (b) or 242 (c). The c.p.m. elicited by OVA_323−339 was similar to that induced by non-antigen-pulsed BMDCs. Dotted horizontal lines indicate an SI of 3·0.

Figure 9

Restimulation of peptide-specific T-cell lines with U1A protein-pulsed bone marrow-derived dendritic cells (BMDCs). T-cell lines were generated by the coculture of Thy1.2⁺ T cells from 7-month-old BWF₁ mice and U1A peptide-pulsed syngeneic BMDCs, as indicated on the x-axis. After 10 days, the T-cell lines were harvested and restimulated with ovalbumin (OVA) (10 µg/ml) or U1A protein (10 µg/ml)-pulsed syngeneic BMDCs. The mean background counts per minute (c.p.m.) ranged from 984 to 2053. Results are representative of two similar experiments and the values shown represent the mean stimulation index (SI) from triplicate wells.