Our findings suggest a novel mechanism by which PD-L2 distinctly regulates airway inflammation and AHR, in which the engagement of PD-L2 by its receptor on iNKT cells downregulates iNKT cell function and cytokine production. The analysis of PD-L2−/− mice shows that the loss of PD-L2 expression results in increased AHR and increased lung inflammation, and indicates that PD-L2 expression in the lung protects against the initiation and progression of iNKT cell-dependent airway inflammation. Our findings suggest that the severity of asthma is greatly enhanced in PD-L2−/− mice because of higher production of IL-4 by iNKT cells as PD-L2 engagement inhibited IL-4 production by iNKT cells. In contrast, PD-L1−/− mice showed reduced levels of AHR, minimal inflammation, and mucous secretion compared with WT mice, and enhanced production of IFN-γ by iNKT cells.
Several studies show the importance of iNKT cells in the induction of AHR in mice and humans,2,3,8,26
but the role of the co-inhibitory molecules PD-L1 and PD-L2 in the activation and modulation of iNKT cell-mediated AHR is still not understood. In this study we used ligand-deficient strains as an approach for understanding the functions of this immunoregulatory pathway. To focus on the role of iNKT cells, we used an α-GalCer model of AHR, as conventional CD4+
T cells do not respond to α-GalCer, but it is a potent activator of iNKT cells. We observed that upon challenge with α-GalCer, PD-L2−/−
mice developed severe AHR compared with WT mice, whereas PD-L1−/−
mice showed a reduced level of AHR and minimal airway inflammation. These disparate phenotypes were also observed after sensitization and challenge of mice with OVA in a conventional model of antigen-induced AHR. Deficiency of PD-L1 or PD-L2 expression did not affect the development of iNKT cells, as iNKT cell numbers in the thymus, liver, and spleen of PD-L1−/−
, and PDL1−/−
were similar to those of WT mice. This is in contrast to the loss of ICOS expression, which we have shown affects NKT cell homeostasis.22
Moreover, surface expression of PD-1 on resting or activated splenic iNKT cells from PD-L1−/−
was similar to that of WT mice. These results suggest that PD-L2 preferentially modulates the effector phase of the asthmatic response, which includes the activation of iNKT cells in the lungs, through interaction with a receptor on iNKT cells.
To further examine the role of iNKT cells in mediating the phenotype of the PD-L1−/−
mice, we performed adoptive transfer studies. We and others have shown that the development of AHR in iNKT-cell-deficient Jα18−/−
mice is restored by the transfer of iNKT cells from WT mice.2,27
mice reconstituted with splenic iNKT cells from PD-L2−/−
-deficient mice developed high levels of AHR. In contrast, Jα18−/−
mice reconstituted with iNKT from PD-L1−/−
-deficient mice developed only low levels of AHR. These results are consistent with the results observed in our in vitro
experiments with iNKT cells purified from PD-L1−/−
Although iNKT cells have an important role in the development of AHR, the development of asthma, which is characterized by airway inflammation and mucus production, also requires the presence of allergen-specific CD4 +
Th2 cells that respond to allergen. Th2 cells produce IL-4, IL-5, and IL-13 and interact with mast cells, basophils, and eosinophils to mediate airway inflammation.28
In this study, we showed that iNKT cell function and the development of AHR is regulated by PD-L1 and PD-L2. However, PD-L1 and/or PD-L2 engagement also regulates the responses of antigen-specific CD4 +
T cells that are involved in airway inflammation. For example, during the OVA sensitization phase, immune deviation away from Th2 responses may reduce AHR and airway inflammation, as observed in the PD-L1−/−
mouse, because CD4 +
T cells from an OVA-sensitized PD-L1−/−
mouse produce much higher levels of IFN-γ and less IL-4 than do CD4 +
T cells from sensitized WT mouse upon in vitro
re-stimulation with OVA (data not shown).
Although PD-L1 is expressed broadly on various hematopoietic and non-hematopoietic cell types, PD-L2 exhibits markedly restricted expression, being inducibly expressed only on DCs, macrophages, B1 B cells, and mast cells.29–31
PD-L2 expression was previously shown to be elevated on the lung and draining lymph node DCs after antigen challenge, 32
and upregulated on splenic DCs after in vivo
activation of iNKT cells by the administration of α-GalCer IP.24
In this study, we observed that upon OVA challenge, all lung DCs showed increased expression of PD-L2. We also found that expression of PD-L2 was upregulated on lung DCs from naive mice upon culture with IL-4, whereas treatment with IFN-γ plus LPS inhibited PD-L2 expression, suggesting that the local cytokine microenvironment in the lungs defines the expression pattern of PD-L2. As PD-L2 inhibits IL-4 production and iNKT-cell-mediated AHR, these results suggest that PD-L2 in the lung during the course of a Th2 cytokine-driven lung inflammatory response constitutes a feedback loop that is upregulated by IL-4 and then acts to reduce continued IL-4 production, thereby modulating the severity of asthma. Loss of PD-L2 in this PD-L2/IL-4 circuit results in increased IL-4 and AHR. In contrast, the enhanced PD-L1 expression observed on lung DCs, macrophages, and B cells after OVA challenge 32
(and this study) inhibits IFN-γ production, which could mitigate AHR. Loss of PD-L1 in this PD-L1/IFN-γ circuit results in increased IFN-γ and reduced AHR. Previous work in cutaneous Leishmania
infection has also shown differential effects of PD-L1 and PD-L2 on cytokine production and Th differentiation.33
Several other studies probed the role of PD-L2 in regulating airway inflammation using PD-L2 mAbs. Treatment with the blocking anti-PD-L2 mAb Ty25 during the challenge (effector), but not the sensitization phase of the response, augmented AHR and BAL eosinophilia, 32
which is consistent with our findings that mice lacking PD-L2 manifested increased AHR, and blocking iNKT cell function in vitro
with PD-L2 mAb increased IL-4 production. Other studies found that administration of sHIgM1, a human IgM autoantibody that recognizes human and murine PD-L2, prevented lung inflammation and AHR by activating mouse DCs that induced deviation from Th2 to Th1 cytokine production.34,35
However, the in vivo
effects of the sHIgM12 Ab may differ from other PD-L2 mAbs and the PD-L2/PD-1 interaction because this antibody regulates DC function by simultaneously recruiting and activating CD40 and TREM-2 on DCs,36
indicating that pathways other than PD-L2 are also involved.
PD-L1 and PD-L2 are ligands for PD-1; however, additional receptors for these B7 family members have been proposed 37–39
and a second ligand for PD-L1 has been molecularly identified.40
PD-L2 was reported to co-stimulate CD4 +
T-cell proliferation and cytokine production and this co-stimulation was independent of PD-1.37,41
PD-L1 interacts not only with PD-1 but also with a second receptor, B7-1, on activated T cells, and this interaction negatively regulates T-cell expansion.40
We found that the addition of a mAb that blocks the PD-L1 interaction with both PD-1 and B7-1 (9G2) resulted in greatly enhanced levels of IFN-γ production by splenic iNKT cells stimulated with α-GalCer, but did not affect IL-4 production. However, culture in the presence of PD-L1 mAb that blocks only the PD-L1/B7-1 interaction (2H11) had no effect on the production of IFN-γ or IL-4, suggesting that the PD-1/PD-L1 interaction is primarily responsible for inhibiting iNKT cell production of IFN-γ. In contrast, iNKT cells activated in the presence of PD-L2 blocking mAb produced higher amounts of IL-4. Our observation that when activated in vitro
with α-GalCer, iNKT cells from PD-L2−/−
mice produced more IL-4, whereas those from PD-L1−/−
mice produced more IFN-γ than WT controls is consistent with these mAb blocking studies. Together, these findings strongly suggest that PD-L2 specifically inhibits IL-4 production by iNKT cells, perhaps by binding to an additional receptor on iNKT cells other than PD-1. Alternatively, PD-L2 has a three-fold higher affinity for PD-1 than does PD-L140,42
and consequently may engage PD-1 in a different manner.
Studies of OVA-induced airway inflammation showed that PD-1- and BTLA-deficient mice displayed only a slight enhancement of acute airway inflammation, but showed a persistent inflammation, indicating an important role for these receptors in terminating the response.1
We found that the development of AHR in PDL1−/−
mice measured at the acute phase of airway inflammation was similar to that of WT mice, which is consistent with the reported studies of the PD-1−/−
mice are not directly comparable with PDL1−/−
mice, as PD1−/−
mice still have the interaction of PD-L1 with B7-1.
In summary, we show that PD-L1 and PD-L2 have important but distinct roles in modulating and polarizing iNKT cell function in AHR and airway inflammation. Understanding the complex mechanisms that regulate the development and effector function of Th2 and iNKT cells is crucial for developing new strategies for asthma therapies. Our finding gives impetus to the development of therapeutic approaches that exploit the PD ligands for controlling airway hyperresponsiveness and asthma.