Background In systemic lupus erythematosus (SLE), dysregulated production of autoantibodies is a consequence of disrupted T cell homeostasis. We have published that blockade of Programmed death-1 (PD-1), a negative regulator in T cells, limits helper T cell (Th) activation, restores regulatory T cell (Treg) suppression, and reinstates immune cell function. We have also shown that attenuated PD-1 expression in Treg down-regulates OX40L, which helps restore the suppressive capacity of Treg. However, concurrent administration of different immunotherapies may negate positive outcomes. We hypothesize that the sequence of blocking PD-1 and OX40L influences the induction and sustainability of Treg suppressivity.

Methods We treated 8-week-old BWF1 mice with a neutralizing Ab against PD-1 or OX40L intraperitoneally. At age 25 weeks when anti-dsDNA began to rise, mice were treated with either anti-PD1 or anti-OX40L that they had not received at age 8 weeks. OX40L, Foxp3 and PD-1 expression on CD4 +CD25+Treg from spleens, apoptosis of Treg and CD4 +CD25 Th were measured by flow cytometry. Seum production of IFN (Th1), IL4 (Th2), IL17a (Th17) and TGF- (Treg), and anti-dsDNA (B cells) were measured by ELISA. The survival of these mice were compared to those treated with anti-PD1 alone at 8 weeks, which we previously demonstrated prolonged survival with delayed onset of proteinuria.

Results Anti-OX40L suppressed Th function and proliferation independent of Foxp3 expression in Treg with decreased anti-dsDNA production. Subsequent blockade of PD-1 in anti-OX40L-treated mice generated more PD1loTreg with increased TGF- production; it sustained Treg suppressivity and delayed onset of proteinuria when compared to mice treated with anti-PD1 alone. Conversely, sequential blockade of anti-OX40L in anti-PD1-treated mice did not promote Treg survival and their disease inconsistently progressed: these mice had predominantly PD1hi or PD1-Treg, and antagonistic OX40L could not restore their suppressivity.

Conclusions Effective induction of Treg is associated with low expression of PD-1 and OX40L, which permits Treg to survive and perform cell suppressive function. Combination of Abs targeting OX40L and PD-1 can improve Treg function and survival outcomes, but it is determined by the timing and sequence of Ab administration: blocking OX40L followed by PD-1 has an additive effect which is not observed when the order of Abs given was reversed. OX40L and PD-1 signaling communicate sequentially with Treg to regulate its suppressive capacity and survival to achieve peripheral tolerance in SLE, suggesting that treatment with one immunotherapy could change the biology of T-cell signaling such that another immunotherapy may lose its efficacy or has unexpected negative outcome.

Funding Source(s): None