In this paper, we demonstrate a novel function for PD-L1 in promoting the development and sustaining the function of iT reg cells. There was a profound defect in conversion of naive CD4 T cells into Foxp3+
iT reg cells in the absence of PD-L1. Consistent with this observation, PD-L1 presented on beads (along with anti-CD3 and anti-CD28) could induce the development of functional Foxp3+
iT reg cells, demonstrating that PD-L1 is responsible for promoting iT reg cell development. Although TGF-β signaling is important for the conversion of naive CD4+
T cells toward Foxp3-expressing cells with suppressive capacity (Chen et al., 2003
; Fantini et al., 2004
), PD-L1 could induce T reg cells in the absence of exogenous TGF-β, suggesting that PD-L1 signaling alone can serve to promote iT reg cell development. Our signaling studies support the conclusion that PD-L1 reduces signaling of the Akt–mTOR pathway in naive T cells, which is critical for their conversion into iT reg cells. Furthermore, we show that PD-L1 has a novel role in sustaining expression of Foxp3 in iT reg cells and in enhancing iT reg cell suppressive function. Thus, these studies reveal a new mechanism by which PD-L1 mediates T cell tolerance. PD-L1 can inhibit self-reactive T cell responses by promoting iT reg cell development and maintaining iT reg cell function.
Where does PD-L1 exert its critical effects on iT reg cell development and function? PD-L1 is widely expressed on hematopoietic and nonhematopoietic cells (Keir et al., 2008
). There may be a critical interaction between PD-L1 expressing DC and T cells during the induction of T reg cell development from naive T cells (Brown et al., 2003
). PD-L1 is also expressed on T cells; however, WT T cells transferred into PD-L1−/−
recipients could not convert to iT reg cells, suggesting that T cell–T cell interaction via PD-L1 is not sufficient to drive naive T cell conversion. Instead an interaction with the host environment is crucial for T reg cell conversion.
Our bone marrow chimera studies have demonstrated an important role for PD-L1 on nonhematopoietic cells in mediating tissue tolerance (Keir et al., 2006
). Our findings of critical roles for PD-L1 in the development and maintenance of iT reg cell function suggest that PD-L1 may protect tissues from the potentially pathogenic self-reactive T eff cells not only by inhibiting the function of T eff cells but also by increasing the frequency and function of T reg cells in the target tissue. By promoting de novo generation of iT reg cells in situ, PD-L1 may play a role in mediating immune privilege, especially in environments where TGF-β is present (e.g., placenta and eye).
PD-L1 may also exert its inhibitory effects on anti-tumor and anti-microbial immunity, at least in part by inducing T reg cell development and sustaining iT reg cell function. PD-L1 is expressed on a wide variety of tumors, and high levels of PD-L1 expression strongly correlate with unfavorable prognosis in several cancers (Dong et al., 2002
; Iwai et al., 2002
; Strome et al., 2003
; Konishi et al., 2004
; Thompson et al., 2004
; Blank et al., 2005
; Hirano et al., 2005
; Ohigashi et al., 2005
; Dorfman et al., 2006
; Wu et al., 2006
; Inman et al., 2007
; Nakanishi et al., 2007
; Nomi et al., 2007
; Zhang et al., 2008
). The numbers of Foxp3+
T cells within tumors also correlates with a poor prognosis. Our work provides a mechanism by which high PD-L1 expression can lead to increased numbers of Foxp3+
T reg cells and thus, poor prognosis. Increased PD-L1 expression by tumor cells may induce and maintain iT reg cells in the periphery, thereby augmenting the suppression of anti-tumor T cell responses and allowing tumor progression.
Increased T reg cells are also seen during chronic infections (Belkaid, 2008
), correlating with a lack of sterilizing immunity. During persistent infection by Helicobacter pylori
, PD-L1 is up-regulated on gastric epithelial cells (Das et al., 2006
). Blocking PD-L1 on gastric epithelial cells enhances CD4 T eff cell function and prevents the generation of CD4+
T reg cells in vitro (Beswick et al., 2007
). During chronic viral infections, PD-L1 has a key role in limiting the function of exhausted CD8 T cells (Barber et al., 2006
; Sharpe et al., 2007
). PD-L1 blockade reinvigorates the function of these T cells and enhances viral clearance. Thus, PD-L1 may exert inhibitory effects in chronic infections by promoting iT reg cell development and maintaining iT reg cell function, as well as by inhibiting anti-microbial T eff cell function.
recipients of Foxp3−
naive T cells resemble scurfy mice, which have a deficit in Foxp3 and die by 3 wk of age as a result of multiorgan infiltration of CD4+
T cells (Clark et al., 1999
; Brunkow et al., 2001
; Schubert et al., 2001
recipients had a marked deficit in Foxp3+
T reg cell development, altering the T reg/T eff cell ratio. These findings illustrate the important role of PD-L1 in regulating the dynamic balance between T eff and T reg cells in vivo. PD-L1 may do the following: (a) control T reg cell development in lymphoid organs, which is important for immune homeostasis; (b) promote T reg cell development at target tissues, protecting against immune-mediated tissue damage, and (c) sustain and enhance T reg cell function within an inflammatory microenvironment, effectively counterbalancing the pathogenic T eff cells.
Foxp3 is a transcription factor only expressed in the T reg cell lineage (Fontenot et al., 2003
; Hori et al., 2003
; Vignali et al., 2008
). Along with contributing a distinct genetic signature to T reg cells, Foxp3 conveys regulatory activity to nT reg cells, iT reg cells, and, upon ectopic expression, in conventional T cells (Schubert et al., 2001
; Fontenot et al. 2003
; Hori et al., 2003
; Gavin et al., 2007
; Hill et al., 2007
). PD-L1 can induce and maintain the expression of Foxp3 in iT reg cells, suggesting that PD-L1 may function to stabilize and sustain T reg cell function in the periphery, similar to effects reported for TGF-β (Marie et al., 2005
; Pyzik and Piccirillo, 2007
). This maintenance of Foxp3 expression may explain both the increased efficiency of suppression seen for T reg cells cultured with PD-L1 and the lack of T reg cell conversion in the PD-L1−/−
adoptive transfer recipients. It has been suggested that although Foxp3 expression is a salient feature of the regulatory cell signature, it is not the master regulator of T reg cell development (Ramsdell, 2003
; Collison et al., 2007
; Hill et al., 2007
). In fact, it is increasingly clear that other factors (such as TGF-β and IL-2; Ramsdell, 2003
; Setoguchi et al., 2005
) induce key changes within the regulatory cell transcriptome that contribute to the identity of T reg cells separate from, and in addition to, the effects attributed to Foxp3 (Lin et al., 2007
). In vivo, TGF-β production is maintained at a basal level and up-regulated with inflammation. Interestingly, endogenous TGF-β was not sufficient to induce and/or maintain Foxp3+
iT reg cells in PD-L1−/−
recipients. In complementary studies, PD-L1 blockade similarly impaired iT reg cell development in vivo, showing that loss of PD-L1 can diminish the effect of TGF-β, which is critical for iT reg cell identity and function. This indicates that PD-L1 contributes unique and essential signals that drive iT reg cell development and function.
We find that PD-L1 attenuates the Akt signaling pathway during the conversion of naive T cells to T reg cells by reducing the phosphorylation of Akt and its downstream substrates mTOR and S6 while simultaneously augmenting PTEN. Previous work has shown that truncation of TCR signaling and inhibition of the Akt–mTOR signaling axis is critical for T reg cell development (Qu et al., 2007
; Strauss et al., 2007
; Haxhinasto et al., 2008
; Long and Buckner, 2008
; Sauer et al., 2008
). In these studies, drug inhibitors (FK506, rapamycin) or retrovirally modified Akt were used to demonstrate the role of Akt and mTOR in T reg cell development. We provide the first demonstration of a naturally occurring protein that can inhibit the Akt–mTOR cascade and regulate the development of T reg cells.
Similar to the Akt–mTOR pathway, recent data indicate an important role for the MAP kinase cascade in iT reg cell development (Adler et al., 2007
; Huber et al., 2008
; Luo et al., 2008
). We found that PD-L1 attenuated the phosphorylation of p42/ERK2, suggesting that PD-L1 may mediate the induction of T reg cells by modulating ERK2 activity and, hence, the MAP kinase signaling cascade. No discernable effects of PD-L1–Ig on p38 were detected (unpublished data).
There is great interest in generating T reg cells ex vivo as a therapy for autoimmune diseases and transplant rejection (Roncarolo and Battaglia, 2007
). However, recent studies indicate that T reg cells exhibit functional plasticity and can produce proinflammatory cytokines at the site of inflammation (Joetham et al., 2008
; Yang et al., 2008
). Thus, in order for T reg cell therapy to be a viable approach, it is critical to find ways to maintain and enhance the suppressive function of T reg cells. Our work suggests that administration of PD-L1–Ig or PD-1 agonists may harness the therapeutic potential of iT reg cells by providing a novel means of sustaining and enhancing their function in vivo while concomitantly suppressing the expansion and functions of activated T eff cells.