We demonstrate here a novel function for PD-L1 in maintaining peripheral immune tolerance by downregulating the expression of chemokine gene transcripts. In addition, we demonstrate that in DED, one of the most common ophthalmic conditions, PD-L1 expression is suppressed. We also examine the functional significance of PD-L1 expression in DED by studying DED pathogenesis using a blocking anti–PD-L1 mAb and PD-L1
−/− mice showing that blockade or elimination of PD-L1 is associated with amplified dry eye–associated inflammation and overexpression of chemokines relevant for the recruitment of T cells (
Fig. 5).
We have previously shown that corneal epithelial cells constitutively express high levels of PD-L1, which is capable of directly suppressing the local T-effector response, contributing to the immunoprivileged status of the cornea.
17 Recently, it was reported that an extremely low number of T cells may be present in the cornea of the normal mouse.
26 Interestingly, in normal PD-L1
−/− mice, we found a significantly increased number of T cells (compared with normal WT mice), comparable to the numbers of T cells present in the dermis of the normal skin of WT mice, which, unlike the cornea, has a significant constitutive population of resident T cells and which, unlike the cornea, has no detectable PD-L1 expression in the uninflamed state.
28,30–32
The increased infiltration of T cells into the corneas of PD-L1
−/− mice suggested to us a potential role for PD-L1 in regulating the expression of chemokines in the cornea. Accordingly, we determined the transcript levels of chemokines and relevant receptors expressed by the cornea by real-time PCR in normal PD-L1
−/− mice and normal WT mice. Interestingly, we found a remarkable increase, primarily in the expression of CXCL9, CCL5, and their corresponding receptors in PD-L1
−/− corneas. In the cornea, chemokines can be expressed by epithelial cells, keratocytes, immune cells, and endothelial cells.
33–35 In the PD-L1
−/− mice, most chemokines are expressed by the epithelial cells because chemokine expression in the stroma/endothelium (which contains keratocytes, immune cells, and endothelial cells) was below the detectable level detected by real-time PCR (data not shown).
CXCL9 is an essential chemokine for corneal T-cell infiltration, as demonstrated in a murine model of HSV keratitis in which CXCL9
−/− corneas are extremely resistant to T-cell infiltration.
36
Our data suggest that corneal PD-L1 expression is tightly inversely correlated with the expression of major T-cell chemokine gene transcripts such as CXCL9. The mechanism by which PD-L1 exerts its inhibitory function on chemokine expression requires further investigation. It is known that there are two ligands for PD-L1, CD80 and PD-1. PD-1 is not expressed by the cornea, but CD80 is expressed at low levels under normal conditions.
17,37,38 CD80 expression is increased in corneal antigen-presenting cells (Langerhans cells in the corneal epithelium and dendritic cells in the corneal stroma) under inflammatory conditions.
37,38 Therefore, it is possible that PD-L1 may exert its function by binding to CD80 in the cornea or to an unidentified ligand on the corneal epithelium. On the other hand, there are myriad questions to be determined, not the least of which is the precise receptor-ligand interactions in the PD-L1 response system. Additionally, future studies are clearly needed to determine the downstream PD-L1–mediated signaling mechanisms that regulate chemokine gene expression. In many chronic inflammatory and autoimmune diseases, IFN-γ leads to increased expression of PD-L1 by parenchymal cells, which is believed to be a protective mechanism against T cell–mediated inflammation.
39 Interestingly, however, in DED, corneal expression of PD-L1 is suppressed, thus disinhibiting chemokine expression normally afforded by high constitutive expression of PD-L1. PD-L1/PD-1 or PD-L1/CD80 interaction decreases the production of IFN-γ by T cells.
40 Hence, we propose that the decreased expression of PD-L1 in DED corneas facilitates T-cell infiltration and IFN-γ production in the cornea. In addition, in DED, the ocular surface epithelial expression of molecules that inhibit T-cell function is also suppressed.
12 This suggests that the decreased expression of the corneal immune privilege properties, such as PD-L1, is a precedent step that promotes corneal T-cell infiltration and IFN-γ secretion in DED.
To further substantiate the role of PD-L1 in the pathogenesis of DED in vivo, we induced DED in PD-L1
−/− mice and WT mice treated with anti–PD-L1 blocking mAb and found a significant increase in the expression levels of CXCL9 and CCL5 and relevant receptors. This was associated with a further significant increase in T-cell infiltration into the corneas of these mice and a significant increase in corneal fluorescein staining score as a result of dry eye. The systemic administration of anti–PD-L1 antibody does not allow precise localization of where this antagonism is effecting the immune response. However, the data derived from both (normal and challenged) PD-L1
−/− mice strongly suggest that PD-L1 does in fact regulate corneal immune response locally. In the aggregate, these data suggest that the downregulation of corneal epithelial PD-L1 amplifies dry eye–associated corneal inflammation and epitheliopathy by modulating the expression of chemokine gene transcripts, thus facilitating T-cell infiltration into the cornea in DED (
Fig. 5).
Data from several laboratories have strongly suggested that tissue-specific expression of PD-L1 protects against autoimmune diseases, improves the success of tissue grafts, and promotes immune quiescence.
16–20 In addition, it has been shown that the gene transfer or treatment with molecules that stimulates PD-L1 signaling prolongs allogeneic and xenogeneic organ transplant survival.
41,42 However, the potential therapeutic usefulness of overexpressing PD-L1, or PD-L1–mediated signaling, in ocular disease remains unknown, although topical blockade of corneal chemokine expression has been shown to suppress inflammation in microbial keratitis and to promote the survival of corneal transplants.
43–45 Hence, our data not only shed light on important immunoregulatory mechanisms in dry eye disease, they suggest potentially novel therapeutic strategies based on enhancing PD-L1 signaling (e.g., through PD-L1 agonistic fusion protein).
Supported in part by a research grant from Allergan Inc.