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

Abstract

Tolerizing mice polygenically predisposed to lupus-like disease (NZB/NZW F1 females) with a peptide mimicking anti-DNA IgG sequences containing MHC class I and class II T cell determinants (pConsensus, pCons) results in protection from full-blown disease attributable in part to the induction of CD4 þ CD25 þ Foxp3 þ and CD8 þ Foxp3 þ regulatory T cells. We compared 45 000 murine genes in total white blood cells (WBC), CD4 þ T cells, and CD8 þ T cells from splenocytes of (NZBxNZW) F1 lupus-prone mice tolerized with pCons vs untreated naïve mice and found two-fold or greater differential expression for 448 WBC, 174 CD4, and 60 CD8 genes. We identified differentially expressed genes that played roles in the immune response and apoptosis. Using real-time PCR, we validated differential expression of selected genes (IFI202B, Bcl2, Foxp3, Trp-53, CCR7 and IFNar1) in the CD8 þ T cell microarray and determined expression of selected highly upregulated genes in different immune cell subsets. We also determined Smads expression in different immune cell subsets, including CD4 þ T cells and CD8 þ T cells, to detect the effects of TGF-b, known to be the major cytokine that accounts for the suppressive capacity of CD8 þ Treg in this system. Silencing of anti-apoptotic gene Bcl2 or interferon genes (IFI202b and IFNar1 in combination) in CD8 þ T cells from tolerized mice did not affect the expression of the other selected genes. However, silencing of Foxp3 reduced expression of Foxp3, Ifi202b and PD1-all of which are involved in the suppressive capacity of CD8 þ Treg in this model.

Genes, tolerance and systemic autoimmunity

“…Semplicini et al reported that reduction of RGS2 signaling increases Ca 2+ mobilization and ERK1/2 activation in response to GPCR stimulation [84]. We found that multiple RGS proteins are downregulated in tolerized CD8 + Ti cells [58]. …”

“…A recent study identifies the major role of p53 in the increased formation of exosomes, a non-classical protein secretion pathway, by upregulated expression of the target gene TSAP6 [65, 69]. We detected increased expression of p53 in CD8 + Ti [58]. Conceivably, p53 may play a novel role in support of CD8+Ti-mediated tolerance by promoting the exosome-mediated delivery of TGFβ or other key growth factors.…”

“…We previously demonstrated that apoptosis was significantly decreased in tolerized CD8 + Ti cells [10]. We have found increased expression of Bcl-2 in tolerized CD8 + Ti cells in BWF1 mice [58]. Similarly, others have found increased expression of Bcl-2 and Bcl-XL in a model of immune tolerance in SLE [59, 60].…”

“…Recently, we have shown that pCons-induced CD8 + Ti suppress autoimmunity in a murine model of SLE in a manner dependent on Foxp3 expression [10, 16, 17]. Following pCons administration, CD8 + Ti display a unique genetic profile, with upregulated genes including Foxp3, Trp53, Bcl2, CCR7, IFNAR1, and IFI202b and downregulated genes including regulator of G protein signaling proteins (RGS2, RGS16, and RGS17), glutamic pyruvate transaminase (GPT2), BAX, programmed cell death-1 (PD1), growth arrest and DNA damage inducible 45 beta (GADD45β), and phosphodiesterase 3b (PDE3b) [47]. CD8 + Ti in our tolerance model expressed low levels of PD1, CTLA4 and CD122, and a partial characterization of the genetic basis of their suppressive capacity indicates some dependence on the expression of FoxP3, PD1, and IFI202b [10, 16, 17, 19].…”

“…Our recent data highlight multiple genes whose products appear to be advantageous in these cells. We will next discuss further the salient genes we have identified in microarray analyses [47], offering insights into their possible roles in immune tolerance and autoimmunity. Validation of our results by RT-PCR and protein expression studies has substantiated the likelihood that these genes play roles in autoimmunity.…”