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Rheumatoid arthritis (RA) is a T cell mediated autoimmune disease and associated with HLA-DR4 or HLA-DR1 subtypes.1,2 Type II collagen (CII) has been implicated as an autoantigen of RA, and CD4+ T cell responses to CII or CII derived peptides are mainly presented by HLA-DR4/1 molecules.3,4 Inhibition of antigen presentation by HLA-DR4/1 molecules can interfere with T cell mediated autoimmune responses in RA.
Our previous studies have suggested that altered CII263–272 peptides inhibited CII263–272-induced T cell activation by blocking antigen presentation.5,6 In this study we examine the role of the altered influenza virus haemagglutinin (HA) 308–317 peptides (altered peptide ligands (APLs)) with single or multiple substitutions of T cell receptor (TCR) contact residues in T cell responses of peripheral blood mononuclear cells (PBMC) and inhibitory effects of APLs on CII263–272-induced T cell activation in RA.
Twenty seven HLA-DR4/1 positive patients with RA (21 female, 6 male; mean (SD) age 53.6 (13.3) years; mean (SD) disease duration 10.4 (8.4) years) were included in the study. All patients fulfilled the American College of Rheumatology revised criteria for the classification of RA. Of 27 patients with RA, 24 (89%) were positive for DR4 and 3 (11%) for DR1.
Sequences of three APLs and CII263–272 were YVAQNTLKLA (APL1), YAKQATLKLA (APL2), YAKQATLALA (APL3), and FKGEQGPKGE, respectively. T cell proliferation experiments were performed by [3H]thymidine incorporation assay. PBMC (2.0×105/well) were incubated with CII263–272 or APLs at 10 μg/ml for 5 days. In competitive studies, PBMC were preincubated with various concentrations of APLs as indicated for 2 hours before addition of CII263–272. Cultures were pulsed with [3H]thymidine (0.25 μCi/well) before the last 12 hours. The data are presented as the stimulation index (SI). Enzyme linked immunosorbent assay (ELISA) kits were used to detect the levels of interferon γ (IFNγ) or interleukin (IL) 4 in the supernatants.
T cell proliferative responses to APLs in PBMC from RA were 7.4% for APL1, 3.7% for APL2 or APL3, which were lower than for CII263–272 (62.2%). The mean (SD) SI values for T cell responses to APLs were 1.2 (0.4) for APL1, 1.3 (0.4) for APL2, and 1.1 (0.4) for APL3, which were significantly lower than for CII263–272 (2.0 (0.8)). In addition, it was shown that T cell proliferative responses to CII263–272 were suppressed by APLs in a dose dependent manner in a range from 2.0 μg/ml to 50 μg/ml (fig 1).
The levels of IFNγ were significantly increased when stimulated with CII263–272 (77.3 (60.8) pg/ml), compared with the levels of 37.8 (14.1) pg/ml for APL1, 37.1 (23.5) pg/ml for APL2, and 34.8 (19.0) pg/ml for APL3, which were similar to the level for unstimulated control (32.1 (15.8) pg/ml). No significant differences were found in IL4 production when PBMC were stimulated with APLs or CII263–272 (table 1).
In this study we assessed T cell responses to APLs in patients with RA and demonstrated that T cell responses to CII263–272 could be inhibited by the APLs with substitutions of the TCR contact residues. HA306–318 peptide can bind to HLA-DR4/1 molecules with a much higher affinity than CII263–272.7 Altered HA306–318 peptides with the substitutions of TCR contact residues are not recognised by HA-specific T cell clones, although these peptide analogues still bind to DR4/1.8 Therefore, altered HA peptides might be more efficient antagonist peptides in the inhibition of immune responses induced by HLA-DR4/1-specific peptides.
The mechanism by which APLs antagonise T cell responses cannot be based on HLA-DR blockade only. Alternatively, APLs may alter the cytokine production profile of T cells.9,10 In this study we showed that APLs down regulated the production of IFNγ compared with CII263–272, which promoted IFNγ secretion. These results suggest that APLs are not Th1 stimulators. It is not clear whether they regulate Th2 cells because no effects on IL4 production were found in the present study. Further studies are necessary to investigate whether APLs effectively inhibit T cell activation in vivo, such as in the HLA-DR4 transgenic animal model.
This work was supported by National Science Foundation of China (30271223) and National Outstanding Youth Grant of China (30025040).
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