WASHINGTON, DC—The effectiveness of tumor necrosis factor (TNF) inhibitors in rheumatoid arthritis (RA) appears to be partly due to a restoration of regulatory T-cells' ability to suppress damaging inflammation, Xavier Valencia, MD, of the US National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) reported at the American College of Rheumatology 2006 Meeting.¹

"T regulatory cells are essential for maintaining tolerance to self antigens," Dr. Valencia said.

Dr. Valencia described the regulatory T-cells (T-regs, formerly known as "T suppressor cells") as thymic derived and anergic upon polyclonal stimulation. Murine knock-out models lacking T-regs develop autoimmune diseases, and diminished T-reg function has been documented in human autoimmune diseases including RA and multiple sclerosis.

Synovial fluid in RA patients has increased numbers of CD4+CD25+ T-regs, but reduced T-reg functioning. "We have previously characterized a functional loss of suppressor function on CD4+CD25hi T-regulatory cells in clinically active RA and its correction after successful anti-TNF therapy," Dr. Valencia said. "Here we report evidence for an in vivo down modulatory effect on T-reg function by TNF."

The investigators harvested peripheral blood mononuclear cells from age-matched normal donors and active RA patients on methotrexate before treatment with infliximab and after completion of 6 monthly infliximab infusions. CD4+ T-cells were isolated, double-stained with anti-CD4 and anti-CD25, and sorted into CD4+ single positive (SP) and CD4+CD25hi T-regs (defined as the 2% brightest of CD25+).

To assess proliferation, CD4+ SP cells were incubated with plate bound anti-CD3 (clone 64.1). To measure  regulatory function, sorted CD4+ SP and CD4+CD25hi T-reg cells were cultured together at a 1:1 ratio with anti-CD3, and incorporation of [3H] thymidine and/or cytokine production were measured. Flow cytometry was used to perform surface and intracellular phenotyping of cells, and intracellular staining was used to assess the transcription factor FoxP3, which is lineage specific for CD4+CD25+ T-regs.

Dr. Valencia explained that normally TNF abolished T-reg functioning. TNF receptor crosslinking reverses T-reg suppression and also reverses T-reg interferon-gamma suppression.

Patients with active RA had normal percentages of CD4+CD25+ T-reg, but T-regs from active RA patients contained more cells expressing TNFRII than cells from normals (44±10%, mean±SD vs 18±3.7%). Expression of the FoxP3 transcription factor was significantly lower in freshly isolated T-regs from active RA than in T-regs from normal donors (35% vs 85%).

"More importantly, T-regs from patients with active RA pre-infliximab failed to suppress proliferation and cytokine secretion of CD4+SP cells from normals or RA patients, although normal CD4+CD25hi T-regs could suppress proliferation of RA CD4+SP cells," Dr. Valencia said.

Notably, after successful infliximab therapy, TNFRII expression on T-regs dropped into the normal range (down to 12± 5%) , and reacquisition of FoxP3 expression increased up to 85%. "T-regs from infliximab-treated RA patients also recovered their ability to suppress the proliferation and cytokine secretion of autologous CD4+ SP cells," Dr. Valencia said. "These data indicate that T-regs are abnormal in phenotype and function in patients with active RA and could participate in the pathogenesis of disease flares in these patients."


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Reference

1. Valencia X, Simone J, Goldbach-Mansky R, Wilson M, Lipsky P. Successful anti-TNF therapy improves the suppressor function of CD4+CD25hi T regulatory cells in RA. Presented at: American College of Rheumatology Meeting; November 13, 2006; Washington, DC. Abstract 653.