Lupus-prone (NZB × NZW)F1 mice spontaneously develop elevated titers of anti-DNA Abs that contain T cell determinants in their VH regions. We have previously shown that tolerization with an artificial peptide based on these T cell determinants (pConsensus (pCons)) can block production of anti-DNA Abs and prolong survival of the mice. In this study, we show that this protection depends in part on the generation of peripheral TGFβ- and Foxp3-expressing inhibitory CD8+ (Ti) cells. These CD8+ Ti cells suppress anti-DNA IgG production both in vitro and in vivo and require up-regulated expression of both Foxp3 and TGFβ to exert their suppressive function, as indicated by microarray analyses, small interfering RNA inhibition studies, and blocking experiments. Additionally, CD8+ Ti cells from pCons-tolerized mice were longer-lived suppressors that up-regulated expression of Bcl-2 and were more resistant to apoptosis than similar cells from naive mice. These data indicate that clinical suppression of autoimmunity after administration of pCons depends in part on the generation of CD8+ Ti cells that suppress secretion of anti-DNA Ig using mechanisms that include Foxp3, TGFβ, and resistance to apoptosis.

Systemic lupus erythematosus is an autoimmune disease caused by autoantibodies, including IgG anti-DNA. New Zealand Black/New Zealand White F1 female mice, a model of spontaneous polygenic systemic lupus erythematosus, tolerized with an artificial peptide (pConsensus) based on anti-DNA IgG sequences containing MHC class I and class II T cell determinants, develop regulatory CD4+CD25+ T cells and CD8+ inhibitory T cells (CD8+ Ti), both of which suppress autoantibody production. CD8+ Ti inhibit primarily via secretion of TGF-β. In the present study, we show that the inhibitory function of CD8+ T cells from tolerized mice is sustained for up to 8 wk and at all times depends on expression of Foxp3. Both CD28-positive and CD28-negative CD8+ T cells contain inhibitory cells, but the expression of mRNA for Foxp3 and for TGF-β is higher and lasts longer in the CD28− subset. In vitro addition of TGF-β (in the presence of IL-2) induces Foxp3 expression in a dose-response manner. Gene inhibition or blockade with small interfering RNA of Foxp3 abrogates the ability of the CD8+ Ti to inhibit anti-DNA production and the proliferation of CD4+ Th cells. Moreover, a significant correlation between expression of Foxp3 and ability of CD8+ Ti to secrete TGF-β is observed. Therefore, CD8+ Ti in this system of tolerance are similar to CD4+CD25+ regulatory T cells in their dependence on expression of Foxp3, and there may be a bidirectional Foxp3/TGF-β autocrine loop that determines the ability of the CD8+ T cells to control autoimmunity.

Summary(NZB x NZW) F1 (BWF1) mice develop spontaneous T cell autoimmunity to VH region determinants of syngeneic anti-DNA before the onset of clinical disease. In this study, we characterized the immunogenicity, MHC binding, and lymphokine secretion patterns induced by T cell determinants from the VH region of one such anti-DNA mAb (A6.1) and examined their role in the regulation of autoimmunity. Determinants were identified by proliferation of syngeneic splenic T cells from young, unprimed BWF1 mice in response to overlapping 12-mer peptides representing the entire Vu region sequence. Immunization of young BWF1 mice with any of three determinants (A6H 34-45 [p34], A6H 58-69 [p58], and A6H 84-95 [p84]) elicited proliferative responses upon in vitro recall. Upon immunization with the whole A6.1 molecule, however, proliferative responses could be recalled only to the p58 peptide, defining this as immunodominant. The other two peptides (p34 and p84) elicited minimal or no proliferation and could be termed cryptic. Proliferative responses elicited by the cryptic determinants were restricted by a single class II (I-E d for p34 and I-A u for p84), whereas the immunodominant p58 determinant was restricted by both bE d and I-E u. The cryptic p34 and p84 bound strongly to I-E a and I-A u, respectively, whereas the immunodominant p58 peptide bound poorly to I-E a. A6H p84 elicited T cells that secreted lymphokines in a pattern consistent with a Thl-like phenotype, whereas p58 induced a Th2-1ike cytokine pattern. Immunization with p34 or p84, or adoptive transfer of a p84-reactive T cell line to young BWF1 mice significantly increased IgG anti-DNA levels, accelerated nephritis, and decreased survival. In conclusion, in BWF1 mice, autoreactive T cells recognizing both cryptic and dominant self-determinants on anti-DNA autoantibodies escape deletion or anergy induction. Furthermore, since these cells are spontaneously activated before the onset of clinical disease, they may be involved in the development of the autoimmune process.

Three pathogenic and two non-pathogenic NZB/NZW F1 mAbs to DNA were compared. Pathogenicity was defined as the ability to induce nephritis in BALB/c mice. All mAbs were IgG2a or 2b, had high avidity for double-stranded DNA and fixed complement well. All three pathogens expressed idiotype IdGN2. Mice receiving pathogenic mAbs (compared with non-pathogenic) had more glomerular IgG deposits. The unique properties of two of the pathogens were: strong homogeneous staining of Hep-2 nuclei and the ability to bind (i) nucleosomes, (ii) histone (after mAb complexed with DNA), (iii) heparan sulfate in renal basement membranes (after complexing with DNA/histone) and (iv) nuclei in vivo. Comparison of nucleotide and amino acid sequences of the V regions of heavy and light Ig chains showed use of multiple VHDJH and V kappa J kappa gene families, with representation of several anti-DNA 'families' described by others. Arginine (R) occurred in the CDR2 or CDR3 of VH chains in all pathogens; R was absent in the CDRs of VH chains of non-pathogens. Positively and negatively charged AA were more frequent in VH CDR of pathogens than of non-pathogens. We hypothesize that the tertiary structure of mAbs determined by VH CDR regions permits stronger binding to negatively charged antigens (DNA and heparan sulfate) and to positively charged molecules (histone) in pathogens compared with non-pathogens.

Sera from MRL/1, BXSB, and NZB/NZW mice, which develop IgG antibodies to DNA and glomerular deposits of DNA-antiDNA immune complexes, were studied by isoelectric focusing. A large array of IgC antibodies with isoelectric points ranging from pH 5.5-9.0 were found to bind double-stranded DNA. Antibodies with isoelectric points from 8.0-8.5 were significantly more frequent than antibodies focusing in all other pH ranges. In contrast, glomerular eluates from MRL/1 and NZB/NZW mice contained a restricted number of DNA-binding bands, all of which focused at pH 8.0-9.0. Anti-DNA with isoelectric points from pH 8.0-9.0 may be more pathogenic for the kidney than other subpopulations.Circulating antibodies to DNA are characteristic of human and murine systemic lupus erythematosus (SLE). They serve not only as markers of the disease but also are implicated in pathogenesis (1-13). Circulating DNA-antiDNA immune complexes are detectable in sera of patients with active SLE (13), and DNA antigen and antibody are detectable in murine and human tissue lesions in glomeruli, skin, and choroid plexus (5-Many populations of DNA antibodies are pres-7,9-12). Submitted for publication August 6, 1979; accepted in revised form December 10, 1979. ent in sera from individuals with SLE. Some react with both double (ds) and single-stranded (ss) DNA; others react exclusively with dsDNA or ssDNA (2,14,15). Especially in murine lupus, DNA antibodies of the IgG subclasses which fix complement are more pathogenic than noncomplement fixing or IgM antibodies (16-1 8). Differences in pathogenic potential may also be related to size and configuration, which determine characteristics such as precipitability (19-21) or avidity (22)(23)(24)(25).With this large variety of antibodies to DNA in human and murine SLE, it would be desirable to study lupus sera with a technique that permits identification and separation of multiple DNA antibody subpopulations. We have successfully identified a large number of these subpopulations in individual murine sera by isoelectric focusing.Several inbred strains of mice spontaneously develop a disease similar to human SLE that is characterized by circulating IgG antibodies to DNA and lethal immune complex glomerulonephritis. Three of these strains were studied: NZB/NZW F, hybrids, MRL/l, and BXSB. The characteristics of each strain have been reviewed recently (26). As mice of each strain aged, an array of anti-DNA antibodies appeared in their sera that was characterized by as many as 20 IgG DNA-fixing bands with isoelectric points ranging from pH 5.5 to 9.0. The location of bands after isoelectric focusing was similar in all strains, with alkaline bands from 8.0-9.0 being most constant. IgG from glomerular eluates was less heterogeneous and formed 3 bands focusing from pH 8.0-9.0. These studies may provide a basis for studying specific subpopulations of antiDNA to determine their relative pathogenicity.