A8.2 An unconventional anti-inflammatory role for TNF recpetor (TNFR)1 in tregs: TNFR1 and 2 differetnialy modulate FOXP3+ TREG function

Background and Objectives Regulatory T cells (Tregs) can protect against rheumatoid arthritis (RA), but their function is compromised in RA. Anti-TNF therapy restores Treg function. However, despite its clinical significance, TNF responses in Tregs are incompletely understood. TNF exists in two forms, the soluble (sTNF) and membrane-bound (mTNF) form, which differentially activate two distinct TNF receptors (TNFR). Notably, only mTNF, but not sTNF, efficiently activates TNFR2 (CD120b), whereas TNFR1 (CD120a) is activated equally well by both TNF forms. Activation of TNFR1 and TNFR2 often result in opposing TNF responses, including pro-and anti-inflammatory actions. The potential importance of TNFR1 in the regulation of natural regulatory T cells (nTreg) is widely underscored by the apparent lack of TNFR1 expression and the high level expression of TNFR2 on these cells. However, since even very low TNFR1 levels are sufficient for profound TNF-mediated cell responses, previous negative results to detect TNFR1 do not rule out a significant role of TNFR1 in the TNF-mediated modulation of nTreg functions. This study sought to assess the expression, function and crosstalk of TNFR1 and TNFR2 in modulating nTreg function. Material and Methods nTreg (CD4 + CD25 ++ FoxP3 + cells) were isolated from healthy donors and expanded for 2-3 weeks in vitro . TNFR function was assessed by stimulating nTregs or monocyte-depleted PBMCs with unique TNF variants selectively stimulating TNFR1, TNFR2 or both receptors. nTreg phenotype was analysed using flow cytometry and cytokine production of the expanded Tregs was determined by ELISA. The modulation of nTreg function was assessed in T cell proliferation suppression assays. Results nTregs express TNFR1 comparable to effector T cells (CD4 + CD25 - FoxP3 - cells) at low but significant levels, whereas the cell surface expression of TNFR2 is higher on nTregs. Similar TNFR expression levels were observed for freshly isolated nTregs and expanded Tregs. Furthermore, using TNFR-selective ligands, we have demonstrated signalling competence for both TNFR1 and TNFR2 on expanded nTregs. Surprisingly, our data indicate that TNFR1-mediated signalling enhances the suppressive activity of nTregs in proliferation suppression assays, whereas TNFR2-signalling does not affect nTreg function in vitro. Conclusion Our data indicate that TNFR1 and TNFR2 differentially control nTreg function and challenge the current model of TNF-mediated modulation of Tregs. A more detailed understanding of TNF-mediated modulation of Treg function will facilitate the development of new strategies to improve anti-TNF therapies.