Background/Purpose: CD4+CD28- T cells are enriched in chronic inflammatory diseases like rheumatoid arthritis (RA) and lupus.  They are cytotoxic and resistant to apoptosis.  Compared to CD28+ cells, CD28- CD4 T cells over-express killer immunoglobulin-like receptors (KIRs), perforin, and other molecules that are expected to contribute to their pro-inflammatory phenotype.  These genes are regulated by DNA methylation, so they are over-expressed by CD4 T cells that are demethylated in vitro.  This is a result of decreased signaling through the ERK and JNK pathways.  This consequently decreases activity of the DNA methyltransferase enzymes (DNMTs) responsible for DNA methylation.  Protein phosphatase 5 (PP5) is a stress induced regulator of gene expression in multiple signaling pathways, including ERK, JNK, and those involved in aging.  It is expressed in CD4+CD28-, but not CD4+CD28+ T cells.  We hypothesized that PP5 is over-expressed in CD4+ T cells in patients with RA and lupus, and that over-expressing PP5 in CD4 T cells from healthy donors will induce expression of methylation sensitive genes unique to CD4+CD28- T cells. 

Methods: CD4+ T cells were isolated from healthy controls and patients with RA and lupus, and PP5 mRNA expression was measured by RT-PCR.  To study the effects of PP5 on methylation sensitive genes, PBMCs from healthy donors were stimulated with phytohemagglutinin (PHA) and cultured for 3 days with IL-2.  CD4+ T cells were then isolated by negative selection, transfected (Amaxa Nucleofector) with constructs encoding GFP and PP5 or GFP alone, and cultured an additional 24-72 hours.  Expression of DNMT1, KIR, and perforin was assessed by RT-PCR in sorted CD4+GFP+ T cells.  DNMT1 expression was measured 24 hours after transfection, and KIR and perforin were analyzed 72 hours after transfection.  KIR protein expression was also measured by flow cytometry with unsorted cells 72 hours after transfection.   

Results: Compared to CD4+ T cells from healthy controls, PP5 mRNA is over-expressed in CD4+ T cells from patients with lupus (1.97 fold change ±0.18 SEM, p=0.03) and RA (1.6±0.2,  p=0.06).  When transfected into CD4+ T cells from healthy donors, PP5 increased KIR mRNA expression (2DL4 gene, 2.4±0.7, n=3, p=0.04) and CD70 mRNA expression (1.38±0.07, n=3, p=0.03).  PP5 transfection also increased the percentage of cells expressing KIR proteins on their surface (33±7% with control vs. 62±7% with PP5, n=7, p<0.01).  Finally, PP5 caused a corresponding 20±8% decrease (n=3, p=0.05) in DNMT1 mRNA expression.

Conclusion: CD4+CD28- T cells, which are enriched in RA and lupus, over-express methylation sensitive genes that contribute to their pro-inflammatory phenotype.  These data demonstrate, for the first time, that protein phosphatase 5 (PP5) contributes to the regulation of methylation sensitive genes in CD4+ T cells.  Specifically, PP5 increases expression of KIR and perforin, and it decreases expression of DNMT1.  Its effects on other methylation sensitive genes, including CD70 and IFN-g, are currently being studied.  PP5 has not been studied in T cells before, and it potentially links aging and DNA methylation with the pathogenesis of multiple rheumatologic disorders.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/protein-phosphatase-5-pp5-regulates-methylation-sensitive-gene-expression-in-cd4-t-cells/