Identification of a PD-L1+Tim-1+ iNKT subset that protects against fine particulate matter–induced airway inflammation

Although air pollutants such as fine particulate matter (PM2.5) are associated with acute and chronic lung inflammation, the etiology of PM2.5-induced airway inflammation remains poorly understood. Here we report that PM2.5 triggered airway hyperreactivity (AHR) and neutrophilic inflammation with concomitant increases in Th1 and Th17 responses and epithelial cell apoptosis. We found that γδ T cells promoted neutrophilic inflammation and AHR through IL-17A. Unexpectedly, we found that invariant natural killer T (iNKT) cells played a protective role in PM2.5-induced pulmonary inflammation. Specifically, PM2.5 activated a suppressive CD4– iNKT cell subset that coexpressed Tim-1 and programmed cell death ligand 1 (PD-L1). Activation of this suppressive subset was mediated by Tim-1 recognition of phosphatidylserine on apoptotic cells. The suppressive iNKT subset inhibited γδ T cell expansion and intrinsic IL-17A production, and the inhibitory effects of iNKT cells on the cytokine-producing capacity of γδ T cells were mediated in part by PD-1/PD-L1 signaling. Taken together, our findings underscore a pathogenic role for IL-17A–producing γδ T cells in PM2.5-elicited inflammation and identify PD-L1+Tim-1+CD4– iNKT cells as a protective subset that prevents PM2.5-induced AHR and neutrophilia by inhibiting γδ T cell function.


Introduction
Air pollution is a factor associated with hospitalization for respiratory diseases and, therefore, is a cause of increased medical care burden worldwide (1)(2)(3). Toxicities and biological effects of ambient particulate matter (PM) depend on the particle diameter and source. PM with a diameter of 10 μm, known as PM 10 , is mostly restricted to the upper airway and is removed through breathing. Particles smaller than 10 μm, especially those with diameters of 2.5 μm or less, known as fine particulate matter (PM 2.5 ), can reach distal airways and can accumulate (4). Clinically, short-term exposure to PM 2.5 results in increased risk of wheezing in children with asthma (5), and long-term exposure results in the development of poorly controlled asthma and reduced lung function in both children and adults (6). Nevertheless, the pathophysiology of PM 2.5 -induced airway inflammation and asthma remains unclear.
Invariant natural killer T (iNKT) cells are a subset of T cells with a restricted TCRα chain (Vα14-Jα18 in mouse and Vα24-Jα18 in human) (7). This T cell receptor (TCR) rearrangement enables iNKT cells to recognize lipids and glycolipids presented by CD1d on antigen-presenting cells. iNKT cells are a heterogeneous population with 3 major subsets, NKT1, NKT2, and NKT17, which produce IFN-γ, IL-4, and IL-17A, respectively (8). iNKT cells can either inhibit or exacerbate allergic responses. For instance, iNKT cells activated by α-galactosylceramide (9) exert suppressive function, whereas iNKT cells activated by house dust extracts promote ovalbumin-induced sensitization (10,11). iNKT cells play protective roles in autoimmune diseases, including multiple sclerosis and rheumatoid arthritis (12).
γδ T cells are a specialized T cell population that bear the γδ TCR instead of the conventional αβ TCR; these cells are found in various mucosal sites such as the skin and lungs (13). These T cells exhibit "innate-like" properties: they do not engage MHC antigen but can be activated directly by TLR stimulation to produce immunomodulatory regulators as the first line of defense (14). This is especially true Although air pollutants such as fine particulate matter (PM 2.5 ) are associated with acute and chronic lung inflammation, the etiology of PM 2.5 -induced airway inflammation remains poorly understood. Here we report that PM 2.5 triggered airway hyperreactivity (AHR) and neutrophilic inflammation with concomitant increases in Th1 and Th17 responses and epithelial cell apoptosis. We found that γδ T cells promoted neutrophilic inflammation and AHR through IL-17A. Unexpectedly, we found that invariant natural killer T (iNKT) cells played a protective role in PM 2.5 -induced pulmonary inflammation. Specifically, PM 2.5 activated a suppressive CD4 -iNKT cell subset that coexpressed Tim-1 and programmed cell death ligand 1 (PD-L1). Activation of this suppressive subset was mediated by Tim-1 recognition of phosphatidylserine on apoptotic cells. The suppressive iNKT subset inhibited γδ T cell expansion and intrinsic IL-17A production, and the inhibitory effects of iNKT cells on the cytokine-producing capacity of γδ T cells were mediated in part by PD-1/PD-L1 signaling. Taken together, our findings underscore a pathogenic role for IL-17A-producing γδ T cells in PM 2.5 -elicited inflammation and identify PD-L1 + Tim-1 + CD4 -iNKT cells as a protective subset that prevents PM 2.5 -induced AHR and neutrophilia by inhibiting γδ T cell function. for IL-17A-producing γδ T cells, which, unlike Th17 cells, can differentiate into Th17 lineage in response to IL-1β and IL-23 without TCR stimulation (15). IL-17A-producing γδ T cells are critical in the early defense against bacterial infection (16). Their role in asthma remains controversial, with studies showing both protective and pathogenic roles in allergen-induced asthma (17,18).
In this study, we found that PM 2.5 induces acute neutrophilic inflammation and airway hyperreactivity (AHR) accompanied by mixed Th1 and Th17 responses. Pathological analysis revealed that PM 2.5 exposure induces pulmonary cell apoptosis, alveolar leakage, and epithelial cell hypertrophy. Pulmonary γδ T cells and iNKT cells are activated after PM 2.5 exposure. Whereas γδ T cell-derived IL-17A contributes to PM 2.5 -induced lung pathogenesis, iNKT cells confer protection by suppressing γδ T cell function. Detailed analysis revealed that activation of a suppressive CD4 -iNKT cell subset that coexpresses Tim-1 and programmed cell death ligand 1 (PD-L1) blocks γδ T cell function in part through PD-1/PD-L1 signaling. This suppressive subset is activated by apoptotic cells through recognition of phosphatidylserine (PtdSer) by Tim-1, which is upregulated specifically on the CD4 -iNKT subset upon PM 2.5 exposure. In sum, this study demonstrates the pathogenic function of γδ T cells in PM 2.5 -mediated airway inflammation and AHR and underscores the protective function of the Tim-1 and PD-L1 coexpressing CD4 -iNKT cell subset in air pollutant-induced lung pathogenesis.

PM 2.5 induces acute AHR and airway inflammation characterized by neutrophilic inflammation.
We first investigated the kinetics of PM 2.5 -induced AHR and airway inflammation in mice. Mice were exposed to 200 μg of PM 2.5 once daily for 3 days and were sacrificed 1, 3, or 5 days after the last exposure. AHR was quantified by measuring lung resistance in response to methacholine. We observed that PM 2.5 increased airway resistance on days 1 and 3 after exposure to a similar degree, whereas resistance was markedly lower 5 days after the last exposure ( Figure 1A). Neutrophil numbers in bronchoalveolar lavage fluid (BALF) were profoundly increased upon exposure to PM 2.5 , peaking on day 1 after exposure ( Figure 1B). Notably, no eosinophils were detected throughout the duration of the experiment. Consistent with an acute response, H&E staining of lung tissue sections at 1 day after the last exposure revealed bronchial epithelium thickening ( Figure 1C). Likewise at this time point, total protein concentration in BALF was increased 2.5-fold, indicating alveolar leakage ( Figure 1D). Furthermore, pulmonary cell apoptosis increased after exposure to PM 2.5 , as detected by TUNEL assay ( Figure 1E).
Next, we examined the levels of inflammatory cytokines. The levels of Th17-associated cytokines (namely, IL-17A, IL-1β, and IL-23) were elevated as early as day 1 after exposure ( Figure 1F). IL-17A kinetics mirrored that of neutrophils in the BALF ( Figure 1B), whereas IL-1β and IL-23 levels in the BALF increased continuously throughout the observation period. Consistent with the protein levels, mRNA levels of all 3 cytokines were increased in PM 2.5 -exposed mice ( Figure 1G). The Th1-related cytokines IFN-γ and IL-18 were also induced by PM 2.5 at both the protein ( Figure 1H) and mRNA ( Figure 1I) levels. Of note, PM 2.5 did not trigger a Th2 response (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.164157DS1). Further confirming the lack of a Th2 response was the observation that IL-13 deficiency did not affect the levels of BALF neutrophils, IL-17A, or neutrophil chemoattractant-encoding cxcl1 and cxcl2 (Supplemental Figure 1, B-D). Overall, these data indicate that PM 2.5 induces acute airway obstruction, neutrophilic inflammation, and pulmonary cell apoptosis with concomitant Th1/Th17-biased cytokine production.
IL-17A derived from γδ T cells contributes to PM 2.5 -mediated lung inflammation. IL-17A is associated with AHR and neutrophilic inflammation (19,20), and some studies have reported that IFN-γ is detrimental in asthma pathogenesis (21,22). We investigated the individual roles of these cytokines using ifng -/mice and IL-17A Cre homozygous mice that are deficient in IL-17A (hereafter referred to as il17a -/mice). Lack of IFN-γ did not alter neutrophil numbers in BALF, whereas IL-17A deficiency partially suppressed neutrophilia ( Figure 2A) without affecting IFN-γ levels ( Figure 2B). IL-17A production is regulated by the transcription factor RORγt in various cell types (23,24). Consistent with this, rorc -/mice did not produce IL-17A when exposed to PM 2.5 ( Figure 2C). These mice also developed lower neutrophilia upon exposure to PM 2.5 ( Figure 2D), further confirming the role of IL-17A in neutrophil inflammation.
Next, we sought to identify the cellular source of IL-17A. Flow cytometry analysis revealed that IL-17A was produced by various lymphocytes, including innate lymphoid cells and Th cells, as well as neutrophils in mice exposed to PM 2.5 ( Figure 2E). γδ T cells were the major producers under these JCI Insight 2022;7(23):e164157 https://doi.org/10.1172/jci.insight.164157 conditions, accounting for more than 40% of the total IL-17A production. We also observed a marked increase in the frequencies of γδ T cells and their intrinsic IL-17A-producing capacity in mice exposed to PM 2.5 , compared with controls ( Figure 2F). Accordingly, total numbers of γδ T cells and IL-17A-producing γδ T cells were higher in PM 2.5 -exposed mice than unexposed mice ( Figure 2G). Although γδ T cells can be induced to produce IFN-γ (25), we did not observe any production of IFN-γ by these cells in the PM 2.5 model (Supplemental Figure 2A). We next assessed the impact of γδ T cell depletion using Tcrd -/mice. Mice lacking γδ T cells had lower levels of IL-17A and impaired AHR relative to WT mice upon exposure to PM 2.5 ( Figure 2, H and I). Notably, neutrophilia was less severe in Tcrd -/mice than in WT counterparts ( Figure 2J). Taken together, these data indicate that γδ T cells contribute to the pathogenesis of PM 2.5 through IL-17A.

PM 2.5 activates iNKT cells through induction of apoptotic epithelial cells.
In addition to increases in γδ T cell numbers upon treatment with PM 2.5 , we also observed an increase in the numbers of lung iNKT cells, particularly the CD4subset in PM 2.5 -exposed mice ( Figure 3, A-C). There was also greater expression of the activation marker CD69 in the overall iNKT cell population in mice exposed to PM 2.5 , compared with controls, and the increase was most significant in the CD4subset in terms of frequency ( Figure 3, D-F). Although iNKT cells can be stimulated to produce IL-17A and IFN-γ (26,27), neither cytokine was induced by PM 2.5 (Supplemental Figure 2, B and C). PM 2.5 exposure increased pulmonary cell apoptosis in mice ( Figure 1E), and PM 2.5 directly induced apoptosis of epithelial MLE-12 cells in culture ( Figure 3G). Apoptotic cells can activate iNKT cells through binding of PtdSer to Tim-1 expressed on the iNKT cell surface (28). Our results show that PM 2.5 To confirm the role of PtdSer/Tim-1 signaling, we cocultured PM 2.5 -exposed MLE-12 cells and iNKT cells in the absence or presence of annexin V, which binds to PtdSer and blocks its recognition (29). As expected, PM 2.5 -exposed MLE-12 cells increased the percentage and number of CD69 + iNKT cells in the coculture. Importantly, addition of annexin V impaired iNKT cell activation ( Figure 3, K and L). These results suggest that PM 2.5 -induced iNKT cell activation is mediated by the PtdSer-Tim-1 axis.
iNKT cells protect against PM 2.5 -induced AHR and airway inflammation through suppression of γδ T cells. The role of iNKT cells in asthma is controversial, with some studies showing protective function and others reporting detrimental roles of these innate-like lymphocytes (30,31). To determine the role of iNKT cells in PM 2.5 -induced AHR and neutrophilic inflammation, we used CD1d -/mice that lack all NKT cells (both type I and II). Interestingly, NKT cell-deficiency exacerbated AHR and neutrophilia detected in BALF ( Figure 4, A and B). Flow cytometry analysis revealed higher frequencies of γδ T cells with enhanced intrinsic potential to produce IL-17A in PM 2.5 -exposed CD1d -/mice compared with WT mice ( Figure 4C). In support of this finding, total numbers of lung γδ T cells and IL-17A + γδ T cells were higher in PM 2.5 -exposed mice lacking iNKT cells than in PM 2.5 -exposed WT mice (Figure 4, D and E). Of note, naive γδ T cells from iNKT cell-deficient mice also had enhanced capacity to produce IL-17A, compared with their WT counterparts (Supplemental Figure 3). We also observed substantially higher levels of IL-17A in BALF in PM 2.5 -exposed CD1d -/mice than in PM 2.5 -exposed WT mice ( Figure 4F).
To confirm the role of iNKT cells, we examined the effects of PM 2.5 on Jα18 -/mice, which lack only iNKT cells. The findings in PM 2.5 -treated Jα18 -/mice recapitulated those in CD1d -/mice in terms of augmented neutrophilic inflammation and increased IL-17A + γδ T cells (Figure 4, G and H). Similarly, IL-17A levels were higher in Jα18 -/mice than in WT mice after exposure to PM 2.5 at both protein and mRNA levels ( Figure 4, I and J). Furthermore, bronchial epithelial hypertrophy and cellular infiltration were more severe in mice lacking iNKT cells ( Figure 4K). Notably, Th1 and Th2 responses were similar upon PM 2.5 treatment in Jα18 -/and WT mice (Supplemental Figure 4). The stronger Th17 response and more intense pulmonary pathology in iNKT cell-deficient mice upon PM 2.5 exposure suggest that iNKT cells moderate PM 2.5 -induced pathology.
Reconstitution of CD4 -iNKT cell subset alleviates PM 2.5 -induced airway inflammation. To further confirm the protective role of iNKT cells, we performed adoptive transfer of splenic iNKT cells into Jα18 -/mice. Efficiency of lung iNKT cell reconstitution was validated by flow cytometry. Although the population size of reconstituted iNKT cells in the lung was equivalent to only a quarter of the endogenous pulmonary iNKT cells in WT mice (~0.25% and ~1% of the total CD45 + population, respectively), these cells had a response to PM 2.5 that was similar to the endogenous iNKT cells in WT mice with PM 2.5 exposure resulting in accumulation of CD4 -iNKT cells in the lungs ( Figure 5, A and B). Importantly, reconstitution of iNKT cells attenuated neutrophilia and IL-17A production ( Figure 5, C and D). Likewise, Vα14 Tg mice, a mouse strain with greatly increased iNKT cell numbers compared with WT mice (32), developed less severe neutrophilia and produced less IL-17A than did WT mice ( Figure 5, E and F). These data reinforce the notion that iNKT cells protect against PM 2.5 -induced lung pathogenesis.
A recent study reported a suppressive CD4 -iNKT cell subset with high CD38 expression (31). Our flow cytometry analysis revealed a significant increase in the CD38 hi CD4 -iNKT cell subset upon exposure to PM 2.5 ( Figure 5, G and H). Therefore, we hypothesized that the CD38 hi CD4 -iNKT cell subset is responsible for protection against the effects of PM 2.5 . To test this, we sorted CD38 hi CD4and CD38 lo CD4 -iNKT cell subsets and adoptively transferred these cells into Jα18 -/mice. In contrast to our hypothesis, we found that both subsets suppressed PM 2.5 -induced neutrophilia and reduced γδ T cell frequency and total numbers, although the CD38 hi CD4 -iNKT cell subset showed slightly stronger suppression ( Figure 5, I and J). Taken together, these data imply that both CD38 hi CD4and CD38 lo CD4 -iNKT cell subsets suppress airway neutrophilia and γδ T cell accumulation in the lungs of PM 2.5 -treated mice.
iNKT cells directly suppress γδ T cell function through PD-1/PD-L1 interaction. Upon activation, γδ T cells transiently upregulate various inhibitory receptors, such as PD-1 and cytotoxic T lymphocyte associated protein 4 (33). Mass cytometry by TOF (CyTOF) analysis revealed substantial increases in PD-1 expression in group 2 innate lymphoid cells, group 3 innate lymphoid cells, and γδ T cells after exposure to PM 2.5 ( Figure 6A). Likewise, flow cytometry analysis showed that there were significant increases in both frequencies and total numbers of PD-1 + γδ T cells in PM 2.5 -exposed mice compared with control mice (Figure 6, B-D). Notably, other T cell types (e.g., CD3 + TCRγδcells) did not show any significant increases in PD-1 expression after exposure to PM 2.5 ( Figure 6B).
Both PD-1 and PD-L1 are expressed on splenic iNKT cells (34,35). Consistent with this, a small fraction of CD4 + and CD4 -iNKT cell subsets expressed PD-1 at steady state; however, PD-1 expression decreased after PM 2.5 exposure (Supplemental Figure 5, A-C). In contrast, exposure to PM 2.5 markedly increased PD-L1 expression on iNKT cells, particularly the CD4subset ( Figure 6, E and F). Notably, the frequencies of PD-L1-expressing CD38 hi and CD38 lo subsets did not show significant differences (Supplemental Figure 5, D and E), suggesting that the inherent suppressive capacity of the CD38 hi subset likely mediates the stronger inhibitory effect of this subset, as seen in Figure 5, I and J. Consistent with the frequency, numbers of PD-L1 + CD4 -iNKT cells were also increased ( Figure 6G). To examine whether the observed increment was due to local cell proliferation or recruitment of circulating CD4 -iNKT cells, we injected CFSE i.v. into PM 2.5 -exposed mice, which labeled up to 85% of iNKT cells in the circulation (Supplemental Figure 6A). Cell proliferation was determined by Ki-67 labeling. Circulating CD4 -iNKT cells (CFSE + cells) constituted approximately 30% of total PD-L1 + CD4 -iNKT cells but were nonproliferative (Ki-67 -). Proliferating tissue-resident iNKT cells (CFSE -Ki-67 + cells), on the other hand, accounted for approximately 10% of the total PD-L1-expressing CD4 -iNKT cells (Supplemental Figure 6B). These data indicate that the increase in PD-L1 + CD4 -iNKT cell numbers is attributed to both recruitment of circulating CD4 -iNKT cells and, to a lesser extent, proliferation of tissue-resident CD4 -iNKT cells.
In addition to the aforementioned findings, we also noted that the PD-L1-expressing subset coexpressed Tim-1 ( Figure 6H), and numbers of these cells were substantially elevated in PM 2.5 -exposed mice ( Figure 6I). On these bases, we postulated that PD-1/PD-L1 signaling is involved in iNKT cell-mediated suppression of γδ T cell function. To determine whether iNKT cells modulate γδ T cell function through the PD-1/PD-L1 interaction, we administered anti-PD-l Ab to mice to block PD-1/PD-L1 signaling. Treatment with anti-PD-1 Ab boosted the intrinsic production of IL-17A by γδ T cells but did not increase the total number of these cells; this effect was not seen in Jα18 -/mice ( Figure 6, J-M). Together, these results indicate that iNKT cells are required for PD-1-mediated inhibition of γδ T cell function.
To examine whether this inhibition is direct, we cocultured CD4 -iNKT cells and γδ T cells in the presence or absence of anti-PD-1 or anti-PD-L1 neutralizing Abs. Consistent with the suppressive role of iNKT cells, addition of the CD4 -iNKT cell subset reduced IL-17A levels in the culture supernatant, and treatment with either anti-PD-1 or anti-PD-L1 reversed this inhibition ( Figure 6N). Moreover, iNKT cell-mediated suppression was abolished in a Transwell culture system (Figure 6O), indicating a requirement for cell-to-cell contact. Taken together, our results show that PD-1/PD-L1 signaling plays a crucial role in iNKT cell-mediated suppression of IL-17A production by γδ T cells.

Discussion
Exposure to PM 2.5 is associated with increased frequency of exacerbations of and hospitalizations for asthma. Although it is known that PM 2.5 stimulates a wide range of immune effector responses, the exact immunological factors that contribute to pulmonary inflammation and development of asthma remain incompletely understood. Here, we showed that PM 2.5 elicits AHR and airway inflammation characterized by acute neutrophilic inflammation, epithelial cell hypertrophy, and mixed Th1/Th17 responses. We demonstrated that these pathological features are mediated by IL-17A, which is produced mainly by γδ T cells. Concomitantly, PM 2.5 -induced epithelial cell apoptosis exposes PtdSer on apoptotic cell surfaces and activates the CD4 -iNKT cell subset through interaction with Tim-1 expressed on iNKT cells. Importantly, we showed that this CD4 -iNKT cell subset upregulates PD-L1 expression upon activation and plays a protective role by counter-regulating IL-17A production by γδ T cells through PD-1/PD-L1 signaling. PM 2.5 aggravates asthma-like symptoms in various allergic asthma models induced by cockroach extract and house dust mites through Th17 cell-driven inflammation (36,37). The effects of PM 2.5 alone on AHR development and lung inflammation had not been thoroughly studied, however. Our results indicated that PM 2.5 alone rapidly induced both Th1 and Th17 cytokines with concomitant increases in neutrophilic inflammation and lung resistance as early as day 1 after exposure. As also shown in previous studies (36,37), we found that IL-17A is the effector cytokine that drove lung pathology and AHR upon exposure to PM 2.5 . However, we found that γδ T cells are the dominant early source of this cytokine rather than Th17 cells, indicating that γδ T cells are the first responders during short-term PM 2.5 exposure. This is consistent with their role as initiators of immune responses, which result from their rapid, innate-like properties (38). We also detected substantial increases in the mRNA and protein levels of IL-1β and IL-23, which have been shown to stimulate IL-17A production by γδ T cells (15). Because γδ T activation does not require antigen presentation by MHC molecules (39), it is likely that these cells are activated by these proinflammatory cytokines.
T cell coinhibitory molecules such as PD-1 help keep T cell responses in check to avoid self-damage (40). In this study, we found that CD4 -iNKT cells suppressed IL-17A production by γδ T cells through PD-1/PD-L1 signaling. To our knowledge, this is the first study to report suppression of γδ T cell function by iNKT cells through coinhibitory molecule signaling. Although it was reported that iNKT cells can suppress IL-17A production by γδ T cells in Salmonella enterocolitis-induced reactive arthritis (41), the mechanism involved was not elucidated. Moreover, several studies have also shown that PD-1 signaling attenuates IL-17A production by γδ T cells under various disease conditions (42,43); however, the cellular source of the PD-1 ligand was not defined. iNKT cells express PD-L1 but not PD-L2 ligand (34). We found that PM 2.5 upregulated PD-L1 expression on the CD4 -iNKT cell subset and caused increased PD-1 expression on γδ T cells and that PD-1/PD-L1 signaling regulated the intrinsic IL-17A-producing ability of γδ T cells. Nonetheless, anti-PD-1 did not completely restore the IL-17A production potential of γδ T cells to the levels seen in Jα18 -/mice, indicating the involvement of other mechanisms that remains to be defined. PD-L1 is expressed on various immune cells, including macrophages, DCs, and activated B and T cells (44). Moreover, PD-1 can also interact with PD-L2, which is expressed primarily on macrophages and DCs (45). Because blockade of PD-1 signaling in mice lacking iNKT cells did not further enhance intrinsic IL-17A production by γδ T cells, the expression of PD-1 ligands on other cells does not affect γδ T cell function.
Our analyses also revealed that CD4 -iNKT cells suppressed γδ T cell expansion upon exposure to PM 2.5 . This effect was independent of PD-1 signaling, given that anti-PD-1 treatment did not enhance the frequency or total number of lung γδ T cells. A CD38 hi CD4 -iNKT cell subset was recently shown to suppress CD4 + T cell proliferation and may serve as a marker to distinguish suppressive iNKT cells (31). Although PM 2.5 induces this particular subset, the suppressive effect of CD4 -iNKT cells is not limited to the CD38 hi population, because CD38 lo iNKT cells also suppress lung γδ T cell expansion, although to a lesser degree. Exactly how PM 2.5 -activated CD4 -iNKT cells inhibit γδ T cell expansion remains to be determined. Of note, we also observed a marked increase in IFN-γ production in mice exposed to PM 2.5 ; IFN-γ suppresses Th17 differentiation and IL-17A production in experimental models of arthritis and autoimmune encephalomyelitis (46,47). Moreover, IFN-γ-producing iNKT cells are associated with protection from airway inflammation (48). We did not detect IFN-γ production by iNKT cells in response to PM 2.5 , and, importantly, IFN-γ deficiency did not alter the pathological outcome of PM 2.5 , indicating that this cytokine is not necessary for the function of iNKT cells in the response to PM 2.5 .
JCI Insight 2022;7(23):e164157 https://doi.org/10.1172/jci.insight.164157 We found that PM 2.5 upregulated Tim-1 expression on the suppressive PD-L1 + CD4 -iNKT cell subset. Tim-1 is a member of the family of T cell immunoglobulin and mucin domain family of proteins and is expressed on mast cells, macrophages, activated Th2 cells, and iNKT cells (49)(50)(51)(52). Previous studies have shown that Tim-1 mediates iNKT cell activation through recognition of PtdSer on apoptotic cells (28,53). Consistent with these findings, we found that PM 2.5 induced bronchial epithelial cell apoptosis and that blockade of PtdSer exposed on PM 2.5 -treated epithelial cells with annexin V prevents iNKT cell activation. Despite functioning as a T cell costimulatory molecule, Tim-1 signaling alone can stimulate iNKT cell proliferation and cytokine production (52). It is unclear whether this was the case in our study or whether PM 2.5 also triggers CD1d-mediated activation, because the lipid antigens produced upon PM 2.5 exposure have yet to be identified. A recent study showed that ozone exposure can lead to iNKT cell activation, likely through recognition of oxidized lipids (54). Chronic exposure to PM 2.5 can also induce formation of oxidized lipids (55), but whether the same mechanism occurs in our acute model remains to be determined.
In conclusion, we demonstrate that PM 2.5 exposure activated a protective subset of iNKT cells that suppressed airway inflammation and AHR by inhibiting γδ T cell expansion and function. Mechanistically, PM 2.5 triggers epithelial cell apoptosis, which, in turn, activates the CD4 -iNKT cell subset through Ptd-Ser-mediated Tim-1 signaling. Activated Tim-1 + CD4 -iNKT cells upregulated PD-L1 expression on their surface and suppressed IL-17A production by γδ T cell through PD-1/PD-L1 interaction. Therefore, CD4 -iNKT cells could serve as a potential target for immunotherapy, and strategies to exploit their function, such as the use of Tim-1-activating monoclonal Abs, should be explored as a possible therapeutic option for management of nonallergic asthma.

Methods
Mice. BALB/c and C57BL/6 mice were purchased from Taiwan National Laboratory Animal Center. Vα14Tg mice were purchased from Jackson Laboratory. IL-17A Cre and Rorc eGFP mice were provided by Jr-Wen Shui (Academia Sinica). Homozygous IL-17A Cre and Rorc eGFP mice were used as IL-17A-and RORγt-deficient mice, respectively. Jα18 -/and CD1d -/mice were provided by Masaru Taniguchi (RIKEN Center for Integrative Medical Sciences, Yokohama, Japan); Tcrd -/mice were obtained from Leo Yung-Ling Lee (Academia Sinica); Il13 -/mice were obtained from Andrew J. McKenzie (Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom); and Ifng -/mice were provided by Nan-Shih Liao (Academia Sinica). Experiments were performed with age-and sex-matched mice.
In vivo administration of PM 2.5 or neutralizing Ab. Mice received 200 μg of PM 2.5 (SRM2786, Sigma-Aldrich) i.n. once a day for 3 days. Mice were sacrificed 1 day after the last exposure unless specified otherwise. To block the PD-1/PD-L1 interaction, anti-PD-1 (RMP1-14) was administered at 100 μg/mouse 1 day before and 1 day after the first exposure to PM 2.5 . Rat IgG2b.κ (100 μg/mouse) was given as the isotype control. Both Abs were purchased from Bio X Cell.
In vivo CFSE labeling. To track migration of iNKT cells into the lung, blood cells were labeled with CFSE (Cayman Chemical) through i.v. injection, as previously described (56). Briefly, mice were exposed to PM 2.5 daily through the i.n. route for 3 days. Mice were injected with CFSE 2 μg/g mouse weight after the second exposure to PM 2.5 and sacrificed 1 day after the last exposure to PM 2.5 .
Measurement of airway AHR. Mice were anesthetized with pentobarbital (Sigma-Aldrich) at 100 mg/ kg body weight. AHR was determined by measuring airway resistance in response to increasing doses of methacholine (Sigma-Aldrich), using the FinePointe RC system (Buxco Research Systems).
BALF collection for differential cell counting and ELISA. Mouse trachea was exposed and lungs were lavaged twice with 2% FCS in PBS using a 20-gauge i.v. catheter (Terumo). Cells were obtained from BALF by centrifugation at 400g for 5 minutes at 4°C. RBCs were lysed with RBC lysis buffer (Omics Bio), and cells were spun onto slides and stained with Diff-Quick solution (Polysciences, Inc.). The BALF cellular profile was assessed by differential cell counting. For ELISA measurement, lavage was performed with PBS supplemented with protease inhibitor III (Merck), phosphatase inhibitor II (Merck), and phosphatase inhibitor III (Merck).
Isolation of mononuclear cells from the blood. Blood was drawn from the heart using a 27-gauge needle and immediately mixed with PBS solution containing EDTA (final concentration, 4 mM). The mixed blood solution was overlaid on an equal volume of Histopaque-1077 (Sigma-Aldrich) and centrifuged at 400g for 30 minutes without a brake at room temperature. The opaque interface containing mononuclear cells was collected and washed twice with 2% FCS and PBS. Lung processing for flow cytometry and CyTOF. Whole lungs were minced and digested in DMEM containing 0.1% (vol/vol) DNase I (Worthington Biochemicals) and 1.6 mg/mL collagenase IV (Worthington Biochemicals) at 37°C. After 30 minutes of digestion, tissue aggregates were dissociated with an 18-gauge needle and lung tissues were further incubated at 37°C for 15 minutes. Tissues were filtered through a 70 μm mesh to obtain single-cell suspensions. RBCs were removed from the cell suspensions using ACK lysing buffer (Gibco Laboratories).
Sample processing for CyTOF analysis. After lung digestion, single-cell suspensions were washed once with cell-staining medium (CSM; PBS with 0.5% BSA and 0.02% sodium azide). Fc receptors were blocked with anti-mouse CD16/32, and cells were stained with a surface Ab cocktail for 1 hour. Cells were then washed with CSM and stained with cisplatin (Sigma-Aldrich) at a final concentration of 25 μM for 1 minute at room temperature to label dead cells. After quenching by adding an equal volume of complete medium, cells were fixed and permeabilized using the Foxp3 transcription factor staining kit (Thermo Fisher Scientific) and then stained with an intracellular Ab cocktail. Cells were washed twice with CSM and stained for DNA with Cell-ID Intercalator-Ir ( 191 Ir and 193 Ir; Fluidigm). Samples were resuspended in MilliQ water containing EQ Four Element Calibration Beads (Fluidigm) for normalization.
CyTOF analysis. Sample acquisition was performed on a CyTOF2 instrument (Fluidigm). Raw flow cytometry standardfiles acquired from CyTOF2 machine were normalized using the Fluidigm Helios software (Fluidigm). Normalized data were analyzed and visualized using viSNE and Flow-SOM (Cytobank). The Abs and gating strategies used for CyTOF analysis are listed in Supplemental  Tables 1 and 2, respectively. iNKT and γδ T cell sorting. Total iNKT cells, CD38 hi CD4 -iNKT cells, and CD38 lo CD4 -iNKT cells were sorted from the spleens of Vα14Tg mice. Spleens were harvested, minced, and dispersed into single cells in 2% FCS in PBS. RBCs were lysed and B cells were removed by incubating splenocytes in AffiniPure goat anti-mouse IgG plus IgM (heavy and light chains) for 15 minutes at room temperature. Cells were then stained with surface Abs for 30 minutes at 4°C. Total iNKT cells were sorted as CD45 + TCRβ + CD1d-tetramer + cells, whereas CD38 hi CD4and CD38 lo CD4 -iNKT cell subsets were sorted as CD45 + TCRβ + CD1d-tetramer + CD38 hi CD4cells and CD45 + TCRβ + CD1d-tetramer + CD38 lo CD4cells, respectively.
To obtain a sufficient number of γδ T cells, mice were pretreated with murine recombinant IL-1β and IL-23 (both at 0.1 μg/mouse) daily for 3 days. Mice were sacrificed 4 days after the last treatment. Lungs were digested as described above, and mononuclear cells were obtained using a 1-step density gradient centrifugation in 33% Percoll (GE Healthcare, now Cytiva). γδ T cells were sorted as CD45 + CD3 + TCRγδ + cells. Cell sorting was performed with a FACS Aria cell sorter (BD Biosciences) with a sorting purity of greater than 95%.
Adoptive transfer. Total iNKT cells (5 × 10 6 cells/mouse), CD38 hi CD4 -iNKT cells (2 × 10 5 cells/mouse), and CD38 lo CD4 -iNKT cells (2 × 10 5 cells/mouse) were sorted from the spleens of Vα14Tg mice and injected i.v. into Jα18 -/mice 30 minutes before the first exposure to PM 2.5 . Mice were given PM 2.5 i.n. daily for 3 days and were sacrificed 1 day after the last exposure. Reconstitution of cells in the lungs was confirmed by flow cytometry analysis.