CD8+ T Cell-Mediated Suppression of Autoimmunity in a Murine Lupus Model of Peptide-Induced Immune Tolerance Depends on Foxp3 Expression

Abstract

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.

Distinct gene signature revealed in white blood cells, CD4+ and CD8+ T cells in (NZBx NZW) F1 lupus mice after tolerization with anti-DNA Ig peptide

“…Table shows CD8 genes differentially expressed in naive vs pCons-treated mice We have long been interested in the role of CD8 þ T cells in murine lupus and in their specific role in the system of pCons-induced tolerance. 5,7,[24][25][26] The analysis of CD8 þ T cells from naive vs pCons-treated mice indicated that 60 genes were differentially expressed in this cell subset ( Figure 1a). Gene and condition clustering of CD8 þ T cells showed that large numbers of genes were downregulated in naive cells with higher expression in tolerized cells ( Figure 1a bottom two lines).…”

“…We have shown previously that interference with the expression of this gene via siRNA abrogated 60-70% of the suppressive capacity of the CD8 þ T cells from tolerized mice. 7,24,25 Silencing of one gene (Bcl2) in CD8 þ T cells from tolerized mice does not effect expression of other selected genes known to be involved (or not) in the suppressive capacity of CD8 þ Treg in our system To test whether silencing of bcl2 in tolerized CD8 þ T cells affects expression of other genes, we silenced tolerized CD8 þ T cells with different siRNA as described in the Materials and methods section and in the figure legends. Results shown in Figure 5 illustrate that silencing of bcl2 did not significantly affect the expression of other genes studied.…”

“…Silencing of Foxp3 affects expression of other genes in addition to silenced gene/genes in tolerized CD8 þ T cells To address whether silencing of selected genes and combinations of these genes affects other genes in tolerized CD8 þ T cells, we did siRNA-silencing experiments for single genes, known to be involved in immune tolerance in our system 7,24,25 and for the combination of Ifi202b and IFNar1. As shown in Figure 6, we found that silencing of the These data suggest that there is a correlation between Figure 3 Reproducibility of CD8 arrays.…”

“…[8][9][10][11][12][13][14][15][16] Similarly, self-antigen-derived peptides have also been used to induce immune tolerance and ameliorate disease in murine lupus models. 15,[17][18][19][20][21][22][23][24][25] Our group has developed a novel peptide pConsensus (pCons) that is based on MHC Class I and Class II T-cell determinants in the heavy chain region of murine anti-DNA IgG. 26 Intravenous administration of the peptide in BWF1 mice has been shown to reduce anti-DNA antibodies, reduce CD4 þ Th secreted Interferon-g, and prolong survival through a mechanism at least partially dependent on the generation of CD4 þ CD25 þ FoxP3 þ Tregs (CD4 þ Tregs) and CD8 þ T inhibitory cells (CD8 þ Ti).…”

“…We identified a subset of differentially expressed genes in WBC, CD4 þ T cells and CD8 þ T cells from tolerized mice 1 week after a single dose of pCons-at which time tolerance (that is, suppression of anti-DNA synthesis by CD8 þ Treg or CD4 þ CD25 þ Treg) is well established. 6,7,24,25 We examine here the differential expression of several immune response, defense response, signal transduction and apoptosis genes. We assessed the reproducibility of CD8 þ T cells arrays and validated the gene expression patterns of some selected genes in WBC and CD8 þ T cells using real-time PCR.…”