Sanne Hoeks

PD-1 signalling and PKB in augmented CB Treg induction 29 2 Inhibition of downstream signaling pathways of TCR and CD28 on T-cell activation is able to promote FOXP3 induction as well. The phosphoinositide 3-kinase/protein kinase B (PKB) pathway is central in T-cell differentiation. 8 Prevention of activation of this pathway by limited co-stimulation or signaling through inhibitory molecules, such as programmed death 1 (PD-1), promotes FOXP3 upregulation. 7, 9 We hypothesized that differential downstream signaling on activation might be pivotal to the high levels of FOXP3 found on day 6 of activation and onward (Fig 1, D ). We therefore investigated the role of PD-1 and PKB in the induction of FOXP3 in CB-naive T cells. On activation, CB CD4 + T cells have an increased expression of the PD-1 molecule compared with APB (Fig 2, B ). When we blocked the interaction between PD-1 and its ligand PD-L1 by including a blocking mAb to PD-L1 in the culture, a significant reduction in the percentage of FOXP3 + T cells was observed (Fig 2, C ), isotype mAb had no effect (not shown). A PD-1–blocking mAb showed comparable results. As a consequence, the phosphorylation status of PKB on activation by different concentrations of anti-CD3 was lower for CB than APB cells (Fig 2, D ). Additionally, CB T cells required approximately 10-fold more TCR triggering than APB T cells before an increase in PKB phosphorylation was observed. Fig 2 Journal of Allergy and Clinical Immunology 2011 1281369-1371DOI: (10.1016/j.jaci.2011.08.006) FIGURE 2. PD-1/PD-L1 interaction stimulates Treg cell induction. A, Treg cell percentage after 6-day stimulation of naive T cells in the presence of anti-CD3/viable APCs with blocking mAb as indicated (n= 6). B, PD-1 expression on 6-day anti-CD3–activated T cells in the presence of viable monocytes. C, Treg cell percentages of anti-CD3–activated naive T cells with viable monocytes in the presence of blocking mAb (CB, n= 11; APB, n= 3). D, Relative phosphorylated PKB levels in CD4 + T cells cultured with a graded amount of plate-bound anti-CD3 (n= 3). *P ≤.05. Shortly after birth, the immune system of the newborn encounters all kinds of neoantigens. The low percentages of Treg cells found ex vivo in CB are not likely to play a major role in maintaining tolerance to these neoantigens. However, we here show that PD-1 signaling in CB T cells facilitates their differentiation into induced functional FOXP3 + Treg cells through a mechanism involving reduced PKB signaling. This phenomenon might well represent a mechanism that is developed to ensure active tolerance in the neonatal immune system.