Differences in the Reactivity of CD4⁺ T‐Cell Lines Generated against Free Versus Nucleosome‐Bound SV40 Large T Antigen

Abstract

Previous results have revealed a strong correlation between polyomavirus BK reactivation and disease activity and antinuclear auto-antibody production in the human autoimmune disease systemic lupus erythematosus. BK virus establishes a latent infection in most humans, and reactivation requires the production of the DNA-binding large T antigen. Experimentally induced expression of the polyomavirus SV40 large T antigen in mice induces both an immune response to large T antigen and autoimmune response to nuclear antigens and antinuclear antibody production. Previous results have indicated that human Tantigen-specific CD4 1 T-cell lines are stimulated equally by free, soluble and nucleosome-bound T antigen. This study was designed to determine how antigen processing of nucleosomes containing bound SV40 large T antigen may affect the specificity and response characteristics of experimentally induced T-antigenspecific CD4 1 T cells. The results indicated that CD4 1 T-cell lines generated from mice immunized with soluble, free T antigen responded very poorly in response to stimulation with T antigen bound to nucleosomes. CD4 1 T-cell lines generated from mice immunized with nucleosomes that had bound T antigen in situ responded to both free and nucleosome-bound T antigen. The T-antigen-specific, CD4 1 memory T cells induced by latent polyomavirus infections in humans may be uniquely suited to initiate autoimmunity to nuclear antigens upon virus reactivation.

Autoimmunity to DNA and Nucleosomes in Binary Tetracycline-Regulated Polyomavirus T-Ag Transgenic Mice

“…In a clinical study, a highly significant correlation between productive polyomavirus infection and Ab production against T-Ag and dsDNA was observed (12,13,17). These results (discussed in Ref.…”

“…Polyomavirus SV40 T-Ag was expressed in recombinant baculovirus, purified by affinity chromatography, and characterized by SDS-PAGE and Western blot using the anti-T-Ag Ab Ab-2 as previously described (14,15). T-Ag was virtually pure and had a molecular mass of ϳ90 kDa and some degradation products (14,17). Human and calf thymus (CT) dsDNA (Sigma-Aldrich) were S1 nuclease digested as described previously (13,27), while the circular cloning vector pUC18 (Amersham Pharmacia Biotech) was used without further structural modifications as described in detail elsewhere (27).…”

“…Preparation of spleen or lymph node cells and stimulation with selected Ags was performed largely as described previously (17). The cells were cultured in Iscove's DMEM (Life Technologies Laboratories, Grand Island, NY) supplied with 25 mM HEPES buffer, 1ϫ nonessential amino acids (Life Technologies), 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, and 10% FCS (Sigma-Aldrich), in round-bottom 96-well culture plates.…”

“…The cells were cultured in Iscove's DMEM (Life Technologies Laboratories, Grand Island, NY) supplied with 25 mM HEPES buffer, 1ϫ nonessential amino acids (Life Technologies), 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, and 10% FCS (Sigma-Aldrich), in round-bottom 96-well culture plates. The T cells (5 ϫ 10 5 cells as spleen or lymph node cells, in 200 l medium/well) were sham-stimulated (medium) or stimulated with 10 g/ml T-Ag, 15 g/ml (as DNA) nucleosomes or nucleosome-T-Ag complexes, or 20 g/ml histones (17). All experiments were performed in triplicate.…”

“…In some experiments, mAbs against CD4 and CD8 molecules (Santa Cruz Biotechnology, Santa Cruz, CA), both at final concentrations of 10 g/ml IgG, were added in parallel triplicate medium-, T-Ag-, or nucleosome-T-Ag-stimulated cultures. T cell proliferation was measured at day 3 after Ag stimulation in vitro, if not otherwise stated in the text, by [ 3 H]thymidine (Amersham Pharmacia Biotech) incorporation (1 Ci per well) as described previously (17).…”