Differential responses to Smith D autoantigen by mice with HLA-DR and HLA-DQ transgenes: dominant responses by HLA-DR3 transgenic mice with diversification of autoantibodies to small nuclear ribonucleoprotein, double-stranded DNA, and nuclear antigens

Abstract

Anti-Smith (Sm) D autoantibodies are specific for systemic lupus erythematosus. In this investigation, the influence of HLA-D genes on immune responses to SmD was investigated. Mice with HLA-DR3, HLA-DR4, HLA-DQ0601, HLA-DQ0604, or HLA-DQ8 transgenes were immunized with recombinant SmD1, and their Ab responses were analyzed. Analysis by ELISA showed that all strains responded well to SmD. However, when synthetic SmD peptides were used as substrate, DR3 mice had the highest Ab response followed by DQ8, DQ0604, DQ0601, and DR4. A similar trend was observed in Western blot analysis using WEHI 7.1 cell lysate as the substrate, with the exception that DR4 mice did not generate detectable amounts of Abs. Only sera from DR3 and DQ0604 mice immunoprecipitated A-ribonucleoprotein (RNP), SmB, and SmD. Intermolecular epitope spreading to A-RNP and SmB was evident in DR3 and DQ0604 mice, as sera depleted of anti-SmD Abs were reactive with these proteins. DR3 mice also generated an immune response to C-RNP. Anti-nuclear Abs were detected in the majority of the DR3 mice, whereas moderate reactivities were seen in DQ0604 and DQ8 mice. Interestingly, only DR3 mice mounted an anti-dsDNA Ab response. Approximately half of the anti-dsDNA Abs were cross-reactive with SmD. Ab responses correlated with the strength of the T cell responses. Thus, HLA-DR3 appears to be the dominant HLA-D gene that determines the magnitude and quality of the anti-SmD immune response. In addition, our findings provide insights into the origin of the anti-dsDNA Abs often detected in patients with systemic lupus erythematosus.

Figure 1. HLA-DR and HLA–DQ transgenic mice immunized with SmD protein generate a robust antibody response against the immunogen

Reactivity of sera with SmD was analyzed in ELISA. Filled symbols represent individual serum samples from transgenic mice immunized with SmD. Open symbols represent sera from mice immunized only with adjuvants. Data is shown for sera obtained 90 days after immunization, at 1:300 dilution and is represented as mean duplicate O.D. at 490nm. To determine statistical significance, Student’s t-test was performed and a p<0.05 considered significant. Note mean O.D values in all strains of mice are significantly higher than the HLA-DQ0601 mice.

Figure 2. SmD B cell epitope specificities are different between the HLA-DR3 and HLA-DQ transgenic mice

Sera from mice immunized with SmD were pooled and their reactivity with overlapping peptides of SmD was analyzed in ELISA. Data is shown for sera obtained 90 days post immunizations and were used at 1:100 dilution. Sera from HLA-DR3 transgenic mice recognized more SmD peptides than any other group of mice.

Figure 3. Diversification of autoantibody responses in HLA-D transgenic mice immunized with SmD

Reactivity of sera with different cellular proteins was analyzed by Western blotting using WEHI 7.1 cell extract. All sera were tested at 1:100 dilution. The figure shows reactivity of representative serum samples from different transgenic mice. As control, only reactivity of sera from CFA/IFA immunized DR3 mice are shown. Note that sera from HLA-DR3 and HLA-DQ8 mice immunized with SmD recognize the most number of cellular proteins.

Figure 4. Intermolecular epitope spreading to other proteins within the snRNP complex occurs in HLA-D transgenic mice immunized with SmD

Pooled sera from HLA-DQ0604 and HLA-DR3 mice immunized with SmD were absorbed with either SmD-coupled sepharose beads or equivalent amount of control beads. Reactivity of unabsorbed and absorbed sera with different proteins was analyzed by Western blotting. Absorption with SmD-beads completely depleted antibodies reactive with SmD and some additional proteins. However, in DQ0604 mice, reactivity to A-RNP and SmB; and in DR3 mice reactivity to A-RNP, SmB and C-RNP persisted, which is indicative of epitope spreading.

Figure 5. Sera from HLA-DR3 and HLA-DQ0604 transgenic mice immunoprecipitate A-RNP, SmB and SmD

To confirm the reactivity of sera with proteins within the snRNP complex, sera were used to immunoprecipitate ³⁵S labeled-A-RNP, -SmB and SmD. Sera (50µl) obtained 3 months post immunization were used to immunoprecipitate the in vitro transcribed and translated proteins. The figure shows a representative autoradiograph.

Figure 6. Anti-nuclear antibodies are generated in HLA-DR3 transgenic mice immunized with SmD

Reactivity of representative serum samples from HLA-DR3 transgenic mice, either immunized with SmD (A and B) or only with adjuvants (C and D) is shown. Panels A and C, show nuclei stained with DAPI. Panels B shows the presence of ANA and panel D the absence of ANA using indirect immunofluorescence for antibody detection. The sera were used at 1:50 dilution.

Figure 7. Only HLA-DR3 transgenic mice immunized with SmD generate anti-dsDNA reactive antibodies

A) Sera from different HLA-D transgenic mice, obtained 90 days post immunization were tested for reactivity to dsDNA. Filled symbols represent sera from SmD immunized mice and open symbols represent sera from adjuvant immunized mice. B) To determine whether anti-dsDNA antibodies were cross-reactive with SmD, pooled sera from DR3 transgenic mice were absorbed with SmD-coupled beads. Absorbed sera were used to immunoprecipitate ³⁵S-labeled SmD. The precipitated radioactivity was measured by scintillation counting and data are represented as CPM. Note all antibodies reactive with SmD were depleted following absorption. C) SmD absorbed sera were tested for their reactivity to dsDNA in ELISA. Data are represented as amount of anti-dsDNA antibody ng/ml and was calculated using a purified anti-dsDNA monoclonal antibody as standard. Note almost 50% of anti-dsDNA antibody reactivity is reduced following absorption with SmD. Control absorption (abs) was carried out with untreated Sepharose beads.

Figure 8. HLA-DR3 mice recognize more T cell epitopes on SmD protein

Different HLA-D transgenic mice (3–5 per group) were immunized with SmD in the foot pad and the base of the tail. On day 12, draining lymph node cells were used to set up a proliferation assay. Synthetic peptides were used at a final concentration of 10µm and reactivity to SmD is shown at a concentration of 10µg/ml. Data are represented as stimulation index, which is ratio of mean triplicate CPM with peptide to mean triplicate CPM without peptide. A S.I.>2.0 was considered positive and is indicated by a solid line. Similar results were obtained in an additional cohort of mice.