pConsensus Peptide Induces Tolerogenic CD8+ T Cells in Lupus-Prone (NZB × NZW)F1 Mice by Differentially Regulating Foxp3 and PD1 Molecules

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disease caused primarily by autoantibodies (including IgG anti‐DNA) and immune complexes. Tolerization with an artificial peptide (pCONSENSUS, pCons) based on murine anti‐DNA IgG sequences containing MHC class I and class II T cell determinants, lupus‐prone NZB/NZW F1 female (BWF1) mice develop regulatory CD4+CD25+T cells and inhibitory CD8+T cells, both of which suppress anti‐DNA Ig production and immune glomerulonephritis. In the present work, we show that splenocytes from BWF1 mice treated with pCons had significant expansion of primarily CD8+T cells. CD4+ T cells and B cells were each directly suppressed by CD8+T cells from tolerized mice in a contact‐independent manner. Both pCons‐ induced CD8+CD28+ and CD8+CD28− T cells suppressed production of anti‐DNA in vitro. Silencing with siRNA of Foxp3 abrogated the suppression mediated by both CD8+ T cell subsets. In addition, CD8+ T cells from tolerized mice were weakly cytotoxic against syngeneic B cells from old anti‐DNA‐producing mice, but not from young mice. Importantly, CD8+ suppressor T cells from tolerized mice, increases the intracellular expression of Foxp3 while decreases the surface expression of PD1 molecules. Blocking PD1/PDL1 interactions in the CD8+ T cells from tolerized mice reduced their expression of Foxp3 and their ability to suppress CD4+CD25− proliferation.

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.…”