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(+)CD25(+)Foxp3+ and CD8(+)Foxp3+ regulatory T cells. We compared 45 000 murine genes in total white blood cells (WBC), CD4(+) T cells, and CD8(+) 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(+)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(+) T cells and CD8(+) T cells, to detect the effects of TGF-beta, known to be the major cytokine that accounts for the suppressive capacity of CD8(+) Treg in this system. Silencing of anti-apoptotic gene Bcl2 or interferon genes (IFI202b and IFNar1 in combination) in CD8(+) 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(+) Treg in this model.

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.

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.

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.

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.

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.

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.

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.