signature=e02a2bb00a81a3c3c773b778d49febc4,Distinct gene signature revealed in white blood cells, CD...

Differential gene expression in splenic WBC from naive vs pCons-treated mice

To examine the profile of differentially expressed genes induced by pCons treatment, total RNA was isolated from WBC, CD4+ T cells and CD8+ T cells of pCons-treated and naive untreated mice splenocytes. Subsequent analysis and comparison identified 448 genes in WBC, 174 genes in CD4+ T cells and 60 genes in CD8+ T cells that were differentially expressed after pCons treatment (Figure 1a). As expected, larger numbers of genes were differentially upregulated in WBC than in isolated CD4 or CD8 subsets (Figure 1b), suggesting that pCons tolerance has a significant effect on several peripheral blood cell subsets.

Figure 1

(a) Gene and condition cluster with all differentially expressed genes together. Heat map diagram comparing changes in gene expression in splenic WBC, CD4+ T cells and CD8+ T cells from naive BWF1 mice (1st, 3rd and 5th horizontal row) to splenic WBC, CD4+ CD8+ T cells from BWF1 mice treated 1 week earlier with pCons (2nd, 4th and 6th horizontal row). Cells were pooled from spleens of 4–5 mice in tolerized vs naive groups. Note the increased expression of multiple genes in the cells from tolerized mice. (b) Venn diagram of all differentially expressed genes together in various cell types. Overlap of differentially regulated gene expression in different cell types is relatively small, although for a small number of genes, expression is changed in more than one cell type. (c) Gene ontology (amiGO) and pathways graphs were generated using Gene Sifter Net Software (Seattle, WA, USA), Gene and condition clustering and Venn Diagram graphs were generated by Gene Spring 6.0 software.

Gene and condition cluster with all changed genes in WBC

Gene and condition clustering indicate that pCons treatment induces significant upregulation of many genes (Figures 1a and b). The Venn diagram of all changed gene expressions shows that some gene expression is changed in more than one cell type (Figure 1b). However, except for the sharing of 21 (5%) of the differentially expressed genes between total WBC and CD8+ T cells, there is remarkably little sharing of similarities between the three cell subsets. White blood cells (WBC) from tolerized mice showed upregulation of more than 48 genes (Table 1a) having roles in transcriptional regulation (Tal1, 5-fold, E2f8, 6.5-fold), signal transduction (Tetraspanin-8 (Tm4sf3), 19.7-fold; G protein-signaling modulator 2; 14.9-fold; Discoidin domain receptor family, member 1, 8-fold), cell division (S17 protein (Spag 5), 26-fold; 6720463M24Rik (9.2-fold)), DNA-protein binding (Ribonucleotide reductase M2, 5-fold; Tal1, 5-fold; Synuclein α (Snca), 5-fold; BC004690, 8-fold; Klf1, 5.7-fold) and coding for integral membrane proteins (Apol2, 4.6-fold; Tetraspanin 33 (1300010A20Rik), 4.6-fold). There were 11 downregulated genes showing more than two-fold changes in expression (Table 1b). Notable decreases in expression were seen in genes coding for rRNA processing (WD repeat domain 3, 42-fold), transaminase activity/lipid metabolism (glutamic pyruvate transaminase, 9-fold), signal transduction (platelet-activating factor receptor, 9-fold) and proteolysis (Elastase 3B, 5-fold).

Table 1 Differentially expressed genes in pCons-tolerized splenocytes vs naive splenocytes from BWF1 mice

Functional analysis of differentially expressed genes in total WBC revealed three major categories: (1) apoptotic genes, (2) signal transduction genes and (3) immune response genes. The 27 genes listed in Table 2 are considered to be involved in apoptosis and their fold changes and gene function/ontology have been depicted. Signal transduction genes and immune response genes have been described in Tables 1, 3 and 4.

Table 2 Apoptosis-related genes in pCons-tolerized BWF1 mice splenocytes vs naive mice splenocytes

Table 3 Differentially expressed genes in CD4+T cell subsets

Table 4 Differentially expressed genes in CD8+ T cell subsets

Affymetrix Gene chip analysis of naive WBC vs pCons-treated WBC showed 448 genes that were differentially expressed (Figure 1 and Table 1). The differentially expressed genes were at least two-fold increased or decreased in their normalized expression value. Gene ontology (amiGO), shown in Figure 1c, showed distribution of more than 50% of differentially regulated WBC genes in physiological processes, with more than one-third of the genes involved in cellular processes. The other categories of genes influenced significantly, as shown by hierarchical gene clustering were development (9.18%) and unknown biological processes (3.93%). In summary, the differences in gene expression associated with pCons-induced tolerance showed effects mainly on physiological and cellular processes.

Expression of selected highly upregulated genes in different cell subsets within WBC

As upregulation of gene expression was most dramatic in WBC, we determined whether a few of the highly upregulated genes (three-fold or more) were similar in different immune cell subsets, using real-time PCR on RNA from purified and isolated immune cell populations. The results are shown in Figure 2. In tolerized (t) mice compared with naive (n) mice, phosphatidylserine synthetase-2 (Figure 2a) was significantly decreased in CD4, CD8 and B cells and increased in granulocytes. Tnfaip2 tumor necrosis factor, α-induced protein 2 (Figure 2b) was decreased in tolerized CD8+ T cells. mRNA expression of GATA-binding protein-1 (Figure 2c) was decreased in CD4+ T cells, CD8+ T cells and B cells from tolerized mice compared with naive mice. Tolerized granulocytes have increased Guanylate-binding protein 1 expression. The mRNA gene expression of adenosine kinase and protein kinase are reduced in pCons-treated groups in tested CD8+ T cell, CD11b and CD11c cell subsets (Figure 2e). Similarly the adenosine deaminase, RNA-specific and GATA 4 expression was reduced in tolerized CD8, CD11c and Gr-1 subsets (Figure 2e). Finally, copropyrinogen oxidase (Figure 2d) was increased in tolerized B cells from pCons-treated mice. Cpox gene expression was significantly reduced in tolerized CD8+ T cells and in granulocytes. These data indicate that the upregulation of some genes in WBC shown in Figure 1 and Table 1 is primarily attributable to changes in single cell subsets, and that such changes may be unique to selected immune cell subpopulations and may even be downregulated in other subsets in the same tolerized mice.

Figure 2

(a–d) Gene expression pattern in splenocyte subsets before and after pCons treatment in BWF1 mice. Non-T cells show large differential expression of some genes after pCons treatment. Different cell subsets (CD4+ T cells, CD8+ T cells, B cells, NK cells, macrophages, granulocytes and dendritic cells) were isolated from saline-treated naive and pCons-treated BWF1 mice spleen by AutoMACS using specific microbeads. RNA from cell subsets, pooled from 4–5 mice in each group was isolated, measured and used for RT-PCR. 100 ng of RNA was used for real-time PCR. A separate standard curve for each gene was constructed and the relative input amount was calculated. Real-time PCR was performed with gene-specific primers and TaqMan probes. (e) RT-PCR was performed from the isolated RNA from different cell subsets (CD8, CD4, CD11b+ macrophages, CD11c+ DC, NK1.1+ NK cells, B 220+B cells and Gr-1+ granulocytes from naive and pCons-treated mice). cDNA was prepared from isolated RNA with reverse transcriptase enzyme and individual gene-specific primers were designed and used for subsequent PCR analysis. The amplified products were size-fractionated by 2% agarose gel electrophoresis. GAPDH was used as a housekeeping gene. *P<0.05, **P<0.005.

CD4 genes differentially expressed in naive vs pCons-treated mice

The differentially expressed genes in naive CD4+ T cells vs pCons-treated CD4+ T cells showed 174 genes that are differentially expressed (Figure 1a). pCons treatment upregulated some genes and downregulated others (Figure 1a— top panel —right 2nd row). There was little overlap in the regulation of genes between CD4+ and CD8+ T cells, with only 3 of the 174 differentially regulated in both types of T cells (Figure 1b). Comparisons between CD4+ T cells from tolerized and naive mice displayed a distinct gene expression pattern (Table 3a and 3b). Notable genes upregulated in CD4+ T cells from tolerized mice (Table 3a) were those coding for protein kinases (Vaccinia-related kinase 2, 18-fold), Rio kinase 3 (Rio kinase 3, 3-fold), DNA binding (Bbx, 4.6-fold), signal transduction (C-type lectin domain family 2, member d (Ocil), 8-fold; Tachykinin receptor 2, 3.5-fold)) and heat-shock proteins (Hsp105, 3-fold). Tolerization of CD4+ T cells seems to have had the effect of downregulating several genes (Table 3b), notably genes that have a role in the regulation of transcription (Zinc finger protein 146 (Zfp146), 24.3-fold; transcription factor 12, 2.8-fold; and Zinc finger homeobox 1b, 4-fold among others), proteolysis (Elastase 3B, pancreatic, 3.7-fold; 2210010C04Rik, 3.5 fold), ubiquitin protein ligase activity (Pellino 1 (Peli1), 4.3-fold; thyroid hormone receptor interactor 12, 3.7-fold), ATPase activity (DEAD box polypeptide 6 (Ddx6), 3.0-fold; DEAD box polypeptide 6 (Ddx50), 2.8-fold), and RNA binding/modification (RNA binding motif protein 5, 4.3-fold; DEAD box polypeptide 6 (Ddx6), 3-fold; and Williams–Beuren syndrome chromosome region 1 homolog (Wbscr1), 17-fold among others).

CD8 genes differentially expressed in naive vs pCons-treated mice

We have long been interested in the role of CD8+ T cells in murine lupus and in their specific role in the system of pCons-induced tolerance.+ 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+ T cells showed that large numbers of genes were downregulated in naive cells with higher expression in tolerized cells (Figure 1a bottom two lines).

CD8+ T cells from tolerized mice had a variety of genes that were either upregulated or downregulated relative to naive cells. Upregulated genes (Table 4a) included those that have a role in calcium ion binding and chemotaxis (S100 calcium-binding protein A8 (S100a8), 3.2-fold; S100 calcium-binding protein A9 (S100a9), 2.8-fold), the regulation of T-cell expansion (CD24a antigen, 2.5-fold), anti-apoptotic activity (Prdx2, 2.8-fold), and interferon-inducible viral activity (Viperin (Rsad2), 2.6-fold). Downregulated genes (Table 4b) include those having a role in ATPase and GTPase activity (Smc4l1, 3.7-fold; Igtp, 2.5-fold), cytoskeletal organization (LIM and SH3 protein 1 (Lasp1), 4.6 fold; Capping protein (actin filament) muscle Z-line, α-1 (Capza1), 3.0-fold; and Utrophin, 2.3-fold), and Phosphodiesterase activity (Phosphodiesterase 4B, 5.3-fold; Phosphodiesterase 7A (Pde7a), 5.3-fold).

Apoptosis-related genes

We have found earlier that both CD4+and CD8+ T cells from tolerized mice are protected from apoptosis.Table 2). These genes encoded proteins that had either indirect or direct roles in apoptosis and had been reported to have either pro-apoptotic or anti-apoptotic functions. Twenty-six genes in the apoptosis microarray were downregulated and one, Peroxiredoxin 2, was upregulated. Of the downregulated genes, 12 (44%) were downregulated more than 3-fold.

Signal transduction genes

Genes involved in signal transduction were differentially regulated among the CD4+, CD8+ and splenocyte subsets. In the splenocyte microarray (Tables 1a and b), these genes included Tetraspanin 8 (Tm4sf3), G protein-signaling modulator 2, Myosin Ixa, platelet-activating factor receptor, and Discoidin domain receptor family, member 1. In CD4+ T cells (Tables 3a and b), the differentially expressed genes in signal transduction were Tachykinin receptor 2, Thymoma viral proto-oncogene 1 (Akt1), Ocil and thyroid hormone receptor interactor 12. Genes that were differentially regulated in the CD8+ subset (Tables 4a and b) were LIM and SH3 protein 1 (Lasp1), phosphodiesterase 4B (Pde4b), and phosphodiesterase 7A (Pde7a).

Reproducibility

To determine the reproducibility of CD8+ T cells genes from naive and tolerized animals, microarray analyses were performed twice. As shown in Figure 3, excellent reproducibility of the CD8 arrays for differentially expressed genes was found.

Figure 3

Reproducibility of CD8 arrays. Reproducibility of CD8 arrays were performed by analyzing microarray analysis twice from isolated CD8+ T cells (pooled from five mice in each group). RNA was isolated and Affymetrix 430 2.0 chips were hybridized with RNA. The scatter plot of signal log ratios of up- and downregulated genes from the first CD8 experiments against the signal log ratios of the up- and downregulated genes from the second CD8 experiment are shown. Red, increased expression. Green, decreased expression. Both the upregulated and downregulated genes were found in both arrays indicating excellent reproducibility of the data.

Real-time PCR validation shows consistent upregulation of selected genes in CD8+ T cells

From the WBC arrays, we selected 10 upregulated genes that were also upregulated at least 2-fold in the CD8+ arrays of pCons-treated vs untreated naive mice, and that could be related to specific immune functions in autoimmunity and immune tolerance. Using real-time PCR, we confirmed the upregulation of 6 of those 10 genes tested. The 10 genes studied were: interferon inducible Ifi202b, Foxp3, bcl2, bcl-2 modifying factor, muscle intestine and stomach expression 1, tumor necrosis factor receptor super family member 11b (osteoprotegerin), orosomucoid-2, transformation-related protein 53 (Trp-53), interferon α receptor IFNar1 and CCR7. Regulator of G protein signaling was chosen as a negative control, as the microarray analyses suggested that it is downregulated in CD8+ T cells from tolerized mice. Statistically significant increased expression patterns with real-time PCR of six genes in CD8+ T cells from tolerized mice was repeatedly found, as shown in Figure 4. Bcl2, CCR7, Ifi202b, IFNar1, Trp53 and Foxp3 were upregulated two- to threefold as compared with cells from naive control mice. In contrast, regulator of G protein signaling was downregulated in pCons-treated cells compared with naive CD8+ T cells (Figure 4). The expression pattern of muscle intestine and stomach expression 1, OPG or orosomucoid-2 could not be confirmed as different from that in naive mice. Of significance was the upregulated expression of Foxp3 in tolerized CD8+ T cells. We have shown previously that interference with the expression of this gene via siRNA abrogated 60–70% of the suppressive capacity of the CD8+ T cells from tolerized mice.

Figure 4

Validation of microarray analysis by real-time PCR for selected genes differently regulated in CD8+ T cells from pCons-treated mice compared with saline-treated controls. mRNA-fold differences by real time PCR are shown on the y axis for tolerized CD8+Ti cells compared with saline-treated naive CD8+ T cells by real-time PCR. RNA was isolated from splenic CD8+ T cells from naive and pCons-tolerized mice. Splenocytes were pooled from 4–5 mice in each group. 100 ng of RNA was used with TaqMan Primer and probe from Applied Biosystems. Expression values are normalized to house keeping gene GAPDH. Data are means±s.e.m. of two to three independent experiments for each gene.

Silencing of one gene (Bcl2) in CD8+ 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+ Treg in our system

To test whether silencing of bcl2 in tolerized CD8+ T cells affects expression of other genes, we silenced tolerized CD8+ 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.

Figure 5

Silencing of one gene (Bcl2) in CD8+ T cells from tolerized mice does not effect expression of other selected genes known to be involved (or not) in suppressive capacity of CD8+ Treg in our system. CD8+ T cells were isolated from pCons-treated mice 1 week after pCons tolerization using antibody-specific microbeads from AUTOMACS. Cells were pooled from 3–4 mice in each group. The cells were transfected with siRNA of Foxp3, CCR7, IFI202b, IFNar1, bcl2 (50–100 nM) or with siRNA controls (GAPDH or scrambled siRNA, 50–100 nM). The negative control scrambled sequence has random sequences with no homology to mouse, rat or human genomes. Incubation with siPORT amine served as a control for the transfection reagent. Then the transfected CD8+ T cells (1 × 105) were cultured for 3 days and RNA was isolated. Real-time PCR for Bcl2 was performed with isolated RNA (100 ng). tolerized CD8=tCD8. Columns: a; tCD8; b; tCD8+ Bcl2 siRNA, c; tCD8+ Foxp3 siRNA, d; tCD8+ p53 siRNA, e; tCD8+ IFNar1 siRNA, f; tCD8+ ifi202b siRNA, g; tCD8+ CCR7 siRNA, h; tCD8+ GAPDH siRNA, I; tCD8+ scrambled siRNA, j; tCD8+ si PORT amine.

Silencing of Foxp3 affects expression of other genes in addition to silenced gene/genes in tolerized CD8+ T cells

To address whether silencing of selected genes and combinations of these genes affects other genes in tolerized CD8+ T cells, we did siRNA-silencing experiments for single genes, known to be involved in immune tolerance in our system Figure 6, we found that silencing of the Foxp3 gene or of the Ifi202b gene both downregulated Foxp3 expression (Figure 6 panel a, compare columns b and e with column a). Silencing of Foxp3 also downregulated expression of PD-1 mRNA (Figure 6 panel b, column b). In contrast, silencing of the p53 and CCR7 genes had no significant effect on either Foxp3 or PD-1 expression (Figure 6 panels a and b) These data suggest that there is a correlation between Foxp3 and PD1 gene expression in our model of tolerance.

Figure 6

Silencing of Foxp3 affects expression of other genes in addition to the silenced gene in tolerized CD8+ T cells; silencing of interferon genes (IFI202b and IFNar1 combination) has no effect on PD1, Foxp3 and bcl2 gene expression. CD8+ T cells were isolated from pCons-treated mice after 1 week of pCons tolerization using antibody-specific microbeads from AutoMACS. Cells were pooled from 3 to 4 mice in each group. Isolated CD8+ T cells from pCons-tolerized mice were transfected with siRNA of Foxp3, PD1, CCR7, IFI202b, IFNar1 and bcl2 (50–100 nM) alone and in combinations, and with controls (GAPDH-50–100 nM), as indicated on the x axis. Real-time PCR was performed with isolated RNA (100 ng). House keeping gene GAPDH-positive siRNA and scrambled-negative siRNA were used as controls. Another control, the transfectant siPORT amine by itself, was also used. The negative control scrambled sequence has random sequences with no homology to mouse, rat or human genomes. (a): Foxp3 mRNA gene expression (b): PD1 mRNA gene expression. Gene expression values were normalized to GAPDH. *P<0.05.

Silencing of interferon genes (IFI202b and IFNar1 combination) has no effect on PD1, Foxp3 and bcl2 gene expression

As interferon gene signatures have been found in SLE patients,Figures 6A and B, column i). These data suggest that PD1, Foxp3 and bcl2 expression and their regulation is independent of the interferon genes tested (IFI202b and IFNar1 combination). A future detailed study will be required to understand these interactions and their biological significance.

Smads expression is increased in tolerized CD8+ T cells

Earlier we found that TGFβ was significantly increased in tolerized CD8+ T cells and was at least in part responsible for the suppression of anti-DNA Ab in our tolerance model.+ T cells but these changes were not significant (Figures 7 a and b). However, we found significantly increased expression of Smad7 in tolerized CD8+ T cells (Figure 7c, P<0.0147). These data suggest TGFβ receptor signaling through Smad 7 has an important role in CD8+ inhibitory T cells in our tolerance model.

Figure 7

Expression of Smad2 (a), Smad3 (b), and Smad7 (c) increases in tolerized CD8+ T cells. CD8+ T cells were isolated from BWF1 mice splenocytes treated with negative control peptide (p58) or pCons. After 1 week, cells were isolated from splenocytes by AUTOMACS and RNA isolated. Real-time PCR was performed for Smad 2, Smad3 and Smad7 with specific primers and probes from Applied Biosystem. 100 ng of RNA was used. Data were normalized with GAPDH. *P<0.05 was considered significant.