The programmed death 1 (PD-1) pathway is a way of inhibiting T-cell proliferation and cytokine production. This pathway is important for maintaining peripheral tolerance. Different mechanisms and cytokines are involved in this pathway. Herein, we show the contribution of endogenous TGF-b to increasing PD-1 expression after T cell receptor (TCR) activation. The receptor for PD-1, also called CD279, is a regulatory protein of the CD28 family and is expressed at low levels on the surface of resting T and B lymphocytes. 1 PD-1 is induced by the expression of TCR or B-cell receptor signaling. 2 Its induction on the surface of activated T cells can prevent a runaway immune response. Indeed, the interaction between PD-1 and its ligand (PD-L1 or PD-L2) inhibits proliferation and effector functions of T cells and induces apoptosis. 1, 3 The regulatory pathway PD-1 thus constitutes a safeguard against autoimmunity and excessive tissue destruction by T cells. 3 Conversely, it can be involved in tumor escape if it is hyperactivated in tumor-infiltrating CD81 T cells. 4 The PD-1 pathway plays a key role in the loss of tolerance in systemic lupus erythematosus (SLE). Consistent with this role, it has been demonstrated that mice lacking PD-1 expression develop a disease similar to SLE. 5 Blockade of PD-1 has been shown to affect disease severity in a mouse model of lupus. 6 Moreover, lower PD-1 receptor expression on CD4 T cells was observed in SLE patients, and several polymorphisms of the PD-1.3 allele are associated with this disease. 7–9 We have recently demonstrated an impaired response of peripheral cells to TGF-b1 in patients with active SLE. 10 Such a defect may contribute to the pathogenesis of the disease. 8 Herein, we hypothesized that PD-1 expression is increased in TCR-stimulated T cells through activation of TGF-b signaling and that resistance to TGF-b could explain the loss of tolerance because the PD-1 pathway would be affected. However, the effect of TGF-b1 on PD-1 expression has not been fully deciphered.

To explore the involvement of TGF-b in the PD-1 pathway, venous peripheral blood was collected in heparin tubes from healthy donors. All experiments were approved by the local ethics committee, and informed consent was obtained from all donors. Peripheral blood mononuclear cells were isolated using a Ficoll-Hypaque density gradient and cultured for 96 h under different conditions: unstimulated, stimulated with exogenous recombinant TGF-b or stimulated with anti-CD3/CD28 antibodies in the presence or absence of an anti-TGF-b blocking antibody or isotype control. Flow cytometry analysis was performed after surface staining with CD279-PE and CD3-PE-Cy5 conjugated antibodies. The percentage and the median fluorescence of CD2791 cells among CD31 lymphocytes were compared between stimulated and unstimulated conditions. Data obtained from patients and healthy controls were compared by the non-parametric Mann–Whitney U test. Statistical significance was assigned to a value of P, 0.05. Our results showed that TGF-b plays a key role in activation of the PD-1 pathway because exogenous TGF-b significantly increased the membrane expression of PD-1 (P50. 0065) after 48 h of stimulation (Figure 1a). Moreover, PD-1 expression is significantly enhanced after TCR activation by anti-CD3/CD28 antibodies compared with unstimulated cells (P50. 0039)(Figure 1b). This increase in PD-1 is partially but significantly reduced after blocking endogenous TGF-b (P50. 0104), while it is not affected when an isotype control is added (P. 0.05)(Figure 1b). These results are formal proof that PD-1 induction through TCR …