Figure 1.
α-GalCer significantly suppressed the severity of GPI peptide-induced arthritis.
DBA1 mice were immunized with GPI peptide and then treated with either DMSO (as a vehicle control) or α-GalCer followed by clinical assessment of arthritis. (n = 5, data shown are representative results of three experiments). (A) Severe swelling of ankle joints of control mice and markedly improved swelling of the ankle joints of α-GalCer-treated mice. (B) Clinical score, and (C) Ankle thickness. The latter represented the severity of arthritis. Arrow indicates α-GalCer administration. Data are mean±SD. *p<0.01, by Man-Whitney test.
Figure 2.
Expansion of iNKT cells in draining lymph nodes of α-GalCer-treated mice.
Mice were immunized with GPI peptide and then treated with either DMSO or α-GalCer and euthanized on day 6 or 10. The dLNs were obtained and examined for the presence of iNKT cells by FCM (Data are mean±SD. n = 4–5. Data are representative results of two experiments). (A) iNKT cells were detected as TCRβ and α-GalCer-loaded CD1d-tetramer double positive cells. (B) Proportion of iNKT cells among TCRβ+ αβT cells, and (C) absolute number of iNKT cells in dLNs of naive or immunized mice. *p<0.05 by Man-Whitney analysis.
Figure 3.
α-GalCer suppressed the production of autoantibodies against GPI, but not serum inflammatory cytokines.
Mice were immunized with GPI peptide and treated with either DMSO or α-GalCer. Serum samples were obtained on days 1, 3, 6, and 28 after the immunization. (A) Levels of anti-recombinant human GPI antibody (total IgG, IgG1, IgG2a, IgG2b, IgG3) in sera obtained on day 28 were measured by ELISA (n = 11). Levels of (B) TNF-α and (C) IL-6 in the sera were measured by CBA (n = 3–5). Data are mean±SD. *p<0.05, by Man-Whitney analysis.
Figure 4.
Suppression of antigen-specific CD4+ T cells by α-GalCer.
(A)(B) Mice were immunized with GPI peptide and treated with either DMSO or α-GalCer. On day 6 (A) or day 10 (B), mice were euthanized and dLNs and spleens were harvested. CD4+ T cells were isolated from dLNs using MACS and cultured with mitomycin-treated splenocytes as antigen-presenting cells (APC). After 24-h culture, the levels of IL-17, IFN-γ, IL-2, TNF-α, IL-4 and IL-10 in the supernatant were measured by ELISA (n = 4). (C) Ten days after immunization, the mice were euthanized and dLNs and spleens were harvested. CD4+ T cells isolated from control or α-GalCer-treated mice were cultured with splenocytes (described as APC in the figure) from control or α-GalCer-treated mice. Cytokine levels in the culture supernatants were measured by ELISA (n = 4). Data are mean±SD. *p<0.05, by Man-Whitney analysis.
Figure 5.
iNKT cells do not directly suppress antigen-specific CD4+ T cells in vitro.
Mice were immunized with GPI peptide and treated with either DMSO or α-GalCer. Mice were euthanized on day 10 and dLNs were harvested. (A) Total CD4+T cells (CD3+ CD4+ cells) and NKT-depleted CD4+T cells (CD3+ CD4+ α-GalCer loaded CD1d-tetramer negative cells) were sorted by flow cytometry. These cells were cultured with sorted MMC-treated CD3-negative cells as APC in the presence of 10 µM of GPI peptide for 24 h. (B) Supernatants were collected and subjects to quantitative analysis of IL-17 and IFN-γ levels by ELISA. Data are mean±SD. (n = 4–5). *p<0.05, **p<0.01, by Man-Whitney analysis.
Figure 6.
α-GalCer does not induce expansion of CD25+foxp3+ regulatory T (Treg) cells.
Mice were immunized with GPI peptide and treated with either DMSO or α-GalCer, then euthanized on day 6 or 10 and dLN cells were harvested. (A) Foxp3+ T reg cells were detected as TCRβ, foxp3- and CD25-positive cells by flow cytometry. (B) Proportion of foxp3+ CD25+ cells among TCRβ+ αβ T cells. Data are mean±SD. *p<0.05, by Man-Whitney analysis.