Background Inhibiting DNA methylation in human or mouse CD4+ T cells causes overexpression of methylation sensitive genes including CD11a, CD70, CD40L and the killer-cell immunoglobulin like receptor (KIR) gene family, and the epigenetically altered murine cells are sufficient to cause lupus-like autoimmunity in syngeneic mice. CD4+ T cells from patients with active lupus also have hypomethylated DNA and overexpress the same genes. Whether the methylation sensitive genes are expressed on different T cells or co-expressed on the same cell is unknown but important to determine, because antibodies to one of the proteins would deplete the subset if all are expressed on the same cell, providing a safer and more effective treatment than currently used medications. We have now used multicolor flow cytometry to test if all these genes are co-expressed on the same or different T cells, using CD4+ T cells experimentally demethylated in vitro and CD4+ T cells from patients with active lupus.
Materials and methods Peripheral blood mononuclear cells (PBMC) were isolated from healthy women and women with lupus by density gradient centrifugation. PBMC from healthy women were stimulated with PHA then treated for 72 hours with the Dnmt1 inhibitor 5-azacytidine (5-azaC). Lupus disease activity was determined using the SLEDAI. T cells stained with fluorchrome conjugated antibodies biotin-CD40L/ PECY7-avidin, APC-CD11a, Pacific Blue-CD3, PECy5-CD28, FITC-CD70, APCCy7CD4, and a “cocktail” of anti-KIR antibodies including PE-anti- Kir2DL4/CD158D, PE-anti- CD158b, PE-anti- CD158i, PE-anti-CD158b1/b2,j, and PE-anti- CD158a,h were analyzed using a FACS ARIA IIIU flow cytometer and FACSDiva software or an iCyte Synergy flow cytometer and WINLIST software.
Results The 5-azaC treated cells, but not untreated T cells, contained a CD3 + CD4 + CD28 + CD11a hi KIR + CD70 + CD40L hi subset representing a range of 3–6% of the 5 AzaC-treated total CD4+ T cells. Similarly, PBMC from patients with active but not inactive lupus also contained a CD3 + CD4 + CD28 + CD11a hi KIR + CD70 + CD40L hi subset, and the size of the subset was directly proportional to disease activity (Figure 1).
Conclusion These results demonstrate that CD4+ T cells experimentally demethylated in vitro, and CD4+ T cells from women with active but not inactive lupus, contain a novel epigenetically altered subset that co-expresses the methylation sensitive genes. This subset may be a novel marker for lupus disease activity. Co-expression of the genes on the same cell also suggests that antibodies to a gene expressed on demethylated but not normal T cells may treat lupus flares.
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