The Roles of Type 2 Cytotoxic T Cells in Inflammation, Tissue Remodeling, and Prostaglandin (PG) D2 Production Are Attenuated by PGD2 Receptor 2 Antagonism

Key Points Multiple proinflammatory effects of Tc2 cells are inhibited by DP2 antagonism. Tissue-remodeling functions of Tc2 cells are attenuated by DP2 antagonism. Autocrine/paracrine PGD2 production in Tc2 cells is reduced by DP2 antagonism.

T ype 2 immunity plays critical roles in the pathogenesis of asthma, particularly in type 2high asthma, as type 2 cytokines, such as IL-4, IL-5, and IL-13, are important drivers of many features of airway inflammation in the disease, including bronchoconstriction, airway eosinophilia, and IgE upregulation. Increased local concentrations of type 2 cytokines are detected in both airways and bronchoalveolar lavage fluid of patients with asthma (1). It is well accepted that type 2 cells, including Th2 and group 2 innate lymphoid cells (ILC2) are important players in type 2 immunity (2). We have reported recently that type 2 cytotoxic T cells (Tc2), another group of type 2 cells, are significantly enriched in the peripheral blood and airways of patients with severe eosinophilic asthma, which can contribute to eosinophilia directly or indirectly, suggesting a crucial role of this group of cells in the disease (3). However, the biology of Tc2 cells has not been studied as well as that of Th2 and ILC2 cells, and many potential roles of Tc2 cells in the pathogenesis of inflammatory diseases remain unclear.
All these type 2 cells highly express PGD 2 receptor 2 (DP2), also known as chemoattractant receptor homologous molecule expressed on Th2 cells (CRTH2), a receptor for PGD 2 , which can be used as a marker of these type 2 cells, as depletion of DP2 1 cells from CD4 or CD8 populations can almost completely remove all type 2 cytokine-producing cells in the populations (24). PGD 2 is a major lipid mediator that is released from mast cells during an allergic response and is upregulated in asthma, according to disease severity (57). Two G proteincoupled receptors have been identified as PGD 2 receptors, PGD 2 receptor 1 (DP1), formerly known as DP, and DP2 (8,9). However, DP2 is the dominant receptor mediating the effect of PGD 2 in the above type 2 cells, as DP1 agonists or antagonists do not exhibit any obvious effect in these cells in vitro (10). Activation of DP2 elicits proinflammatory reactions in these cells, including cell migration, proinflammatory cytokine production, and suppression of apoptosis (3,1012). Therefore, inhibition of DP2 is considered as a potential approach to control type 2 immunity-mediated inflammatory diseases, and DP2 antagonists remain under clinical investigation (13,14). Fevipiprant is a potent and selective DP2 antagonist that has shown therapeutic benefit in certain subsets of asthma patients in phase 2 clinical trials (1517). In a mechanistic phase 2 clinical trial study in patients with persistent eosinophilic asthma, fevipiprant not only reduced airway inflammation but also improved epithelial integrity and reduced airway smooth muscle (ASM) mass (16,17). In two recently published *Respiratory Medicine Unit and National Institute for Health Research Oxford Biomedical Research Centre, University of Oxford, Oxford, United Kingdom; † Novartis Institutes for BioMedical Research, Cambridge MA; ‡ Novartis Pharma, AG, Basel, Switzerland; and x Translational Gastroenterology Unit and Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, United Kingdom phase 3 studies, although neither trial showed a statistically significant reduction in asthma exacerbations, consistent and modest reductions in exacerbation rates were observed with a high dose of fevipiprant in both studies (18). However, the underlying anti-inflammatory mechanism of fevipiprant is still only known to a limited extent. An x-ray crystallographic study illustrated how fevipiprant competitively occupies a semioccluded ligand-binding pocket in DP2 to block the function of the receptor (19). Previous in vitro studies demonstrated the anti-inflammatory effect of fevipiprant in DP2-mediated reactions in Th2 and ILC2 cells (17,20,21). The effects of DP2 antagonism in Tc2 cells are not fully understood.
In this study, we explored some novel proinflammatory and profibrotic functions of Tc2 cells, focusing on protissue remodeling effects and PGD 2 autocrine/paracrine production. Fevipiprant was used as a potent tool to dissect the role of the PGD 2 /DP2 axis and DP2 antagonism on these functions. Our observations provide further evidence of the important and pleiotropic roles of Tc2 cells in eosinophilic asthma and potential use of DP2 antagonism in type 2 inflammation.

Human clinical samples
Patients meeting the American Thoracic Society/European Respiratory Society definition of severe asthma with a sputum eosinophil count of >3% (eosinophilic) and healthy control subjects were recruited from John Radcliffe Hospital, Oxford, U.K. (22). The studies were approved by South CentralOxford B Research Ethics Committee, Oxford, U.K. (18/SC/0361), and written informed consent was obtained from each donor before sample collection.
Human CD8 1 DP2 1 Tc2 cell preparation and treatment Human Tc2 cells were isolated from fresh clinical blood samples collected in heparin-coated tubes or CD leukocyte cones (National Blood Service, Oxford, U.K.). PBMCs were prepared by gradient with Lymphoprep (STEM-CELL Technologies), then CD3 1 CD8 1 CD4 À DP2 1 cells were sorted into 96-well plates using a BD FACSAria III sorter. Cells were amplified in culture for about a month with RPMI 1640 supplemented with 10% human serum, 250 IU/ml IL-2, 50ng/ml anti-CD3 Ab, and irradiated feeder PBMCs. The purity (CD3 1 CD8 1 CD4 À DP2 1 cells >90%) of the cells after expansion and their response to PGD 2 was confirmed with flow cytometry and chemotaxis before use.
For cytokine or other protein production assays, Tc2 cells were incubated with 100200 nM PGD 2 in presence or absence of various concentrations of fevipiprant (Novartis Pharma), BW245C, BW868C, or TM30089 (Cayman Chemical), as indicated in the Results, for 4 h at 37 C. The cell supernatants were harvested for ELISA or Luminex assays, and the cell pellets were stored at À80 C for quantitative RT-PCR (qRT-PCR) and microarray.

Fibroblast and Tc2 coculture
Human fibroblast cell line MRC-5 cells (American Type Culture Collection) were seeded in a 96-well plate at 1 Â 10 3 cells per well in MEM (Life Technologies) supplemented with 10% FBS and cultured overnight. After a gentle wash with PBS, 1 Â 10 4 Tc2 cells were loaded on the top of fibroblasts in a final volume of 200 ml of MEM containing 2% FBS in the presence or absence of 200 nM PGD 2 or/and 1 mM fevipiprant. As a negative control, 200 ml of the same media were added into fibroblast cultures without Tc2 cells.
In some experiments, Tc2 cells were loaded into a 0.5-mm filter insert on the top of the wells containing MRC-5. Otherwise, Tc2 conditioned media, after treatment with 200 nM PGD 2 in the presence or absence of 1mM fevipiprant, or MEM, containing various concentrations of IL-4/13, were loaded instead of Tc2 cells. Images of the cell cultures were captured at different time points by using the IncuCyte ZOOM (Essen Bioscience). Confluency of fibroblasts was quantified as area of fibroblasts from images using the Trainable Weka Segmentation, a plugin in Fiji for ImageJ (21). The supernatants of the MRC-5 cultures were harvested for ELISA, and the cell pellets were collected for qRT-PCR analysis.

Chemotaxis
Tc2 cells were resuspended in RPMI 1640 containing 10% human serum and then incubated with various concentrations of fevipiprant, BW245C, BW868C, TM830089, or medium for 1 h at 37 C in the upper chamber (5mm pore size) of HTS Transwell-96 permeable supports (Corning). The upper chamber was then placed into the lower chamber of the supports containing prewarmed media supplemented with 100 nM PGD 2 , 1 mM BW245C, or medium for a further 1-h incubation. The cell migration to the lower chamber was quantified using an IncuCyte ZOOM.

qRT-PCR
Total RNA from the Tc2 cell pellets or MRC-5 cells was extracted using RNeasy Mini Kit (QIAGEN). cDNA was prepared using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was conducted using Master Mix and Probes (Roche) or Fast SYBR Green Master Mix (Applied Biosystems) in a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories) (Supplemental Table I). GAPDH was used as a reference gene.

Microarray
RNAs from cultured Tc2 cells treated with or without PGD 2 for 4 h were extracted with an RNeasy Mini Kit, and microarrays were conducted using an Illumina HumanHT-12 v4.0 Gene Expression BeadChip at the Transcriptomics Core Facility, The Jenner Institute, University of Oxford, Oxford, U.K. Preprocessing data analysis was performed using R Bioconductor packages. Genes involved in tissue-remodeling functions in Gene Ontology Terms 0060429, 0030198, 0048771, and 0042060 that reached an absolute log 2 fold change >1 and were significant at p < 0.05 were selected using the Limma package (23). The heatmap was generated using pheatmap (https://CRAN. R-project.org/package=pheatmap) package.

Apoptosis
Tc2 cells were incubated in RPMI 1640 in the presence or absence of 1 mM PGD 2 or/and 1 mM fevipiprant for 12 h at 37 C. Cells treated with normal culture medium containing human serum were used as control. The cells were harvested and stained with annexin V (BioLegend) in an Annexin V Binding Buffer (BioLegend) following the BioLegend instructions. Results were acquired with a LSR II flow cytometer (BD Biosciences).

Cell aggregation analysis
Cell aggregation was photographed using a Nikon Eclipse TS100 microscope. Images were analyzed with the Trainable Weka Segmentation. Briefly, cell clumps were identified using the Otsu method of thresholding. The intensity and size of identified objects were measured. Results are reported as integrated intensity.

Flow cytometry
For the ex vivo cytokine production assays, PBMCs isolated from fresh blood of the patients with severe eosinophilic asthma were incubated with 1 mM PGD 2 , 1 mM fevipiprant, or in combination for 6 h at 37 C. Brefeldin A (5 mg/ml, BioLegend) was added 30 min after starting stimulation. The cells were then stained with an Ab panel against CD3, CD4, and CD8 with Zombie Aqua dye (BioLegend) followed by fixation with IC Fixation Buffer (Invitrogen) overnight at 4 C. The cells were treated with permeabilization buffer (Invitrogen) followed by intracellular staining with anti-human IL-13 and IL-5 (BioLegend) Abs. CD3 1 CD4 -CD8 1 IL-5/13 1 cells were detected with flow cytometer.
For hPGDS staining, fresh blood was labeled with the Ab panel against CD3, CD4, and CD8 with Zombie Aqua dye followed by RBC lysis with a FACS lysing solution (BD Biosciences) and cell permeabilization with permeabilization buffer and then intracellularly stained with Ab to hPGDS (a kind gift from Advanced Technology and Development, BML, Saitama, Japan).
For cyclooxygenase (COX) staining in cultured Tc2 cells, the cells were treated with 400 nM DK-PGD 2 or 5 mg/ml antiCD3/CD28 Abs in the absence or presence of 1 mM fevipiprant for 4 h, then fixed and permeabilized using a FOXP3 Fix/Perm Buffer Set (BioLegend) followed by intracellular staining with anti-human COX-1 and COX-2 Abs (BD Biosciences).
For DP2 endocytosis analysis, cultured Tc2 cells with or without preincubation with 10 mM flurbiprofen or 1 mM hPGDS inhibitor 1 for 1 h were stimulated with 200 nM PGD 2 or 5 mg/ml antiCD3/CD28 Abs in the absence or presence of 1 mM fevipiprant for 4 h and then stained with anti-DP2 Ab together with Zombie Aqua.
Results of the above staining were acquired with BD LSRFortessa or LSR II flow cytometers.

Statistics
Data were analyzed by using one-way ANOVA, followed by a Tukey test.
All p values <0.05 were considered statistically significant.

DP2-mediated Tc2 cell migration, aggregation, and survival were inhibited by fevipiprant
To study the proinflammatory roles of human Tc2 cells in vitro, CD3 1 CD8 1 DP2 1 Tc2 cells were isolated from the blood of healthy or severe eosinophilic asthma donors and expanded. Because Tc2 cells are enriched in the airways of eosinophilic asthma patients (3), we first examined the cell reactions that potentially contribute to cell recruitment and enrichment, including cell migration, adhesion, and survival. PGD 2 (100 nM) but not DP1 agonist BW245C induced Tc2 cell migration strongly in a chemotaxis assay, which was inhibited by DP2 antagonist TM30089 but not DP1 antagonist BW868C, suggesting that the migration was mediated specifically by DP2 (Fig.  1A). Fevipiprant inhibited PGD 2 -induced (100 nM) Tc2 migration in a dose-dependent manner with an IC 50 = 3.5 ± 3.6 nM.
We next examined the effect of DP2 on Tc2 cell survival. Cell apoptosis was increased after serum deprivation in the cell culture (Fig. 1B). PGD 2 (1 mM) suppressed apoptosis, which was reversed by fevipiprant (1 mM, Fig. 1B).
Cell aggregation is a marker of cell adhesion in in vitro culture. Aggregation of Tc2 cells rapidly occurred within 1 h after PGD 2 (200 nM) treatment, which persisted for 24 h (Fig. 1C), and was inhibited by fevipiprant in a dose-dependent manner (Fig. 1C, 1D). This aggregation was dominantly mediated by the adhesion molecules ICAM and PECAM, as the expression of these molecules was upregulated by PGD 2 stimulation and inhibited by fevipiprant (Fig.  1E), and neutralization Abs to ICAM and PECAM significantly reduced the cell aggregation (Fig. 1F, 1G). Interestingly, inhibition of Tc2 aggregation with these neutralization Abs also reduced IL-5 and IL-13 production from the cells (Fig. 1H), suggesting that type 2 cytokine production in Tc2 is partly dependent on ICAM and PECAM expression.
We also compared type 2 cytokine production from cultured Tc2 cells between healthy donors and patients with severe eosinophilic asthma (Table I). The cells derived from asthmatics released slightly more IL-13 in response to PGD 2 than those from healthy donors (Fig. 2C, left panel). The maximum responses achieved by Tc2 from healthy blood were only 72% of that achieved by Tc2 from asthmatic donors. However, no difference of the potency of fevipiprant was detected between healthy and asthmatic Tc2 (Fig. 2C,  right panel).
To confirm the effects of PGD 2 and fevipiprant on type 2 cytokine production in Tc2 cells under physiological conditions, we tested the responses in fresh blood from patients with severe asthma using intracellular staining for type 2 cytokines ex vivo (Fig. 2D). CD3 1 CD4 À CD8 1 IL-5 1 /IL-13 1 T cells were increased in the blood after PGD 2 (1 mM) treatment. Coincubation with fevipiprant (1 mM) significantly blocked the increase. In contrast, it was difficult to detect type 2 cytokinepositive CD8 1 T cells in the blood from healthy donors in response to PGD 2 . PGD 2 also upregulated production of many other proinflammatory cytokines, including IL-3, GM-CSF, M-CSF, and TNF-a at both mRNA and protein levels in Tc2 cells (Fig. 3A, 3B). The production of these cytokines was ablated by fevipiprant in a dose-dependent manner, with an IC 50 = 6.08 nM (mRNA) and 8.2 nM (protein) for IL-3, an IC 50 = 4 nM (mRNA) and 8.1 nM (protein) for GM-CSF, an IC 50 = 14.3 nM (mRNA) and 2.8 nM (protein) for M-CSF, and an IC 50 = 11.1 nM (mRNA) and 2 nM (protein) for TNF-a. Such an inhibitory effect of fevipiprant was also observed in some other proteins, such as IL-8, CCL4, FasL, and Galectin-3 (Fig. 3C).
To further confirm the potential physiological effect of the above genes in tissue remodeling, we conducted a fibroblastTc2 coculture assay (Fig. 5A, 5B). MRC-5 cells, a fibroblast cell line, were cultured with Tc2 cells in the presence or absence of PGD 2 (200 nM) with or without fevipiprant (1 mM). The images of the cell cultures were recorded at different time points (Fig. 5A), then the confluence of MRC-5 cells was calculated (Fig. 5B). After a 24-h culture, Tc2 cells with PGD 2 significantly enhanced the MRC-5 confluence compared with the culture without PGD 2 . Addition of fevipiprant together with PGD 2 in the MRC-5-Tc2 coculture removed the enhancement, indicating the key role of PGD 2 /DP2 axis in this reaction. This enhancement was not due to the direct interaction between PGD 2 and MRC-5 cells, as no such enhancement was observed in the same cultures without Tc2 cells (Supplemental Fig.  1B). Both soluble tissue-remodeling factors in the Tc2 medium and direct cell contact of MRC-5Tc2 seem to contribute to the enhancement, as either using a filter insert to separate two types of cells in the coculture (Fig. 5C, Supplemental Fig. 1C) or culturing MRC-5 in a conditioned medium from activated Tc2 cells (Fig. 5D) still enhances MRC-5 confluence, but the level of the enhancement was slightly reduced compared with the coculture without insert (Fig. 5C, Supplemental Fig. 1C). Furthermore, in addition to the aforementioned tissue-remodeling proteins, type 2 cytokines could also enhance MRC-5 proliferation, as addition of IL-4 and IL-13 in MEM increased MRC-5 confluence in a dose-dependent manner (Fig. 5E), although the concentration of IL-13 (0.010.1 ng/ml) in the supernatants of cocultures or Tc2 conditioned medium (IL-4 was undetectable) was not enough to trigger the full enhancement (Supplemental Fig. 1D).
Increased levels of collagen I a1 and TGF-b1 were detected in the supernatant of MRC-5 cocultures with Tc2 in the presence of PGD 2 (Fig. 5F), and upregulations of gene transcription for collagen I a1 (COL1A1), actin a2 (ACTA2), and vimentin (VIM) were also detected in the MRC-5 cells cocultured with Tc2 and PGD 2 (Fig.  5G) that were inhibited by fevipiprant. These upregulations were not due to the direct interaction between PGD 2 and MRC-5 cells, as  such reactions were not detected in the same cultures without Tc2 cells (Supplemental Fig. 1E, 1F). These genes were not regulated by PGD 2 in Tc2 cells, based on the microarray results.

Production of PGD 2 in Tc2 cells was attenuated by fevipiprant
The level of PGD 2 is increased in the airways of asthmatics (3,7). To investigate whether Tc2 cells are capable of contributing to PGD 2 upregulation in the disease, we examined PGD 2 production in Tc2 cells activated through DP2 or TCR. To enable differentiation of stimulator and product PGD 2 in the PGD 2 ELISA, we used DK-PGD 2 , a specific DP2 agonist that was undetectable in the assay (Supplemental Fig. 2A), or antiCD3/CD28 to stimulate the cells (Fig. 6A). Both DK-PGD 2 and antiCD3/CD28 strongly induced PGD 2 production, which peaked after 4-h treatment. The capacity of PGD 2 production in Tc2 cells (22.3 ± 43.2 ng PGD 2 /1 Â 10 6 cells) was similar to that in Th2 cells (29.3 ± 57.1 ng PGD 2 /1 Â 10 6 cells) (Supplemental Fig. 2B) but was less than half of the capacity of mast cells (51.7 ng PGD 2 /1 Â 10 6 cells) (5). Blockade of DP2 with fevipiprant significantly inhibited the PGD 2 production triggered by DK-PGD 2 , confirming autocrine or paracrine generation of PGD 2 in Tc2 cells (Fig. 6B). Interestingly, fevipiprant also significantly reduced the PGD 2 production induced by TCR.
To confirm the biosynthetic pathway of PGD 2 in Tc2 cells, we examined the expression of hPGDS, an enzyme required for PGD 2 synthesis, in Tc2 cells with flow cytometry (Fig. 6C). The frequency of hPGDS-positive Tc2 cells was correlated with blood eosinophil counts in the asthma cohort (Fig. 6D). DK-PGD 2 but not antiCD3/CD28 upregulated the expression of hPGDS, which was reversed by fevipiprant (Fig. 6E). The expression of COX-1/2, another group of enzymes required for PGD 2 synthesis, in Tc2 cells was also examined (Fig. 6F, 6G; Supplemental  Fig. 2C, 2D). DK-PGD 2 slightly upregulated both COX-1 and COX-2 in Tc2 cells at mRNA (PTGS1 for COX-1 and PTGS2 for COX-2) and protein levels, which were inhibited by fevipiprant, although the change of COX-1 was difficult to determine with flow cytometry, as all the cells showed COX-1positive staining. AntiCD3/CD28 only upregulated COX-2, but this was not affected by fevipiprant. Inhibition of hPGDS with hPGDS inhibitor 1 or COXs with diclofenac or flurbiprofen completely blocked the PGD 2 production in Tc2 cells induced by DK-PGD 2 (Fig. 6H). The inhibition of COXs also blocked the PGD 2 production induced by antiCD3/CD28. However, the inhibition of hPGDS significantly but only partially inhibited the effect of antiCD3/CD28 (Fig. 6H). We also examined cytosolic phospholipase A2 (cPLA2) and phospholipase C (PLC), the potential enzymes upstream of COXs required for PGD 2 synthesis. Both enzymes, but dominantly cPLA2, were expressed in Tc2 cells (Supplemental Fig. 2E) and were not significantly regulated by the DK-PGD 2 activation of Tc2 cells, although PLA2 was weakly upregulated by TCR activation (Supplemental Fig. 2F). The PLA2 inhibitor ATK strongly inhibited PGD 2 production, the PLC inhibitor U-73122 only partially reduced PGD 2 production, and the combination of ATK and U-73122 further enhanced the inhibition (Supplemental Fig. 2G).
To further confirm the autocrine/paracrine production of PGD 2 in Tc2 cells, the levels of DP2 endocytosis were examined. Both PGD 2 and antiCD3/CD28 induced the loss of DP2 level on the cell surface because of endocytosis (Fig. 6I, 6J). Blockade of DP2 with fevipiprant completely blocked the loss mediated by PGD 2 and significantly reduced the loss caused by antiCD3/CD28 (Fig. 6I). Inhibition of COXs and hPGDS reduced the DP2 endocytosis induced by antiCD3/CD28 but not that by DK-PGD 2 (Fig. 6J). This was expected, as DP2 on the surface of DK-PGD 2 treated cells had already been occupied, and inhibition of PGD 2 synthesis could not reverse this. Furthermore, the inhibition of COXs and hPGDS also mitigated IL-13 production induced by antiCD3/CD28 (Fig. 6K), indicating the role of PGD 2 autocrine production in the TCR-mediated Tc2 activation (Fig. 7).
Although the synthesis of cysteinyl leukotrienes (cysLTs) shares the same upstream source of arachidonic acid with PGD 2 , LTE 4 levels detected in Tc2 cultures were low and not changed by the stimulation of DK-PGD 2 or antiCD3/CD28 (Supplemental Fig. 3A). The expression levels of 5-lipoxygenase (5-LO) and 5-LOactivating protein (FLAP), critical proteins required for leukotriene synthesis, in Tc2 cells were also low and not significantly affected by the stimulations (Supplemental Fig. 3B).

Discussion
Tc2 cells are enriched in both peripheral blood and airways in severe eosinophilic asthma (3). Our previous study has demonstrated that the activation of Tc2 cells could contribute to airway eosinophilia through producing proinflammatory cytokines IL-4/5/13 and GM-CSF. In this study, we revealed some previously, to our knowledge, unrecognized functions of Tc2 cells, including promoting tissue remodeling and IgE-independent PGD 2 autocrine production, which could play critical roles in the pathogenesis of asthma (Fig.  7). The PGD 2 /DP2 axis enables multi-proinflammatory functions in Tc2 cells, promoting cell recruitment and activation, particularly type 2 cytokine production (3). Using fevipiprant, we demonstrated in this study that competitive inhibition of DP2 not only completely blocked the cell migration, adhesion, proinflammatory cytokine production, and survival functions in Tc2 triggered by PGD 2 but also attenuated tissue-remodeling effects and PGD 2 autocrine production in Tc2. These findings will enable a better understanding of the potential role of Tc2 cells in the pathogenesis of asthma.
Airway remodeling, particularly airway wall thickening, is a characteristic feature of asthma (24) that involves structural changes in the airways, including epithelial hyperplasia and metaplasia, subepithelial fibrosis, smooth muscle cell hyperplasia, and angiogenesis, leading to deleterious consequences on lung function. The mechanisms regulating airway remodeling remain poorly understood. It has been suggested that airway remodeling could be regulated by the interaction of immune cells with tissue-forming cells (25). In this study, we demonstrated that PGD 2 upregulated many proteins produced in Tc2 cells that could play important roles in tissue remodeling. IL-1a, TNF-a, syndecan-4, TrkA, NRP1, and TWEAK/ Fn14 are capable of stimulating fibroblast and smooth muscle cell proliferation (2631). IL-1a, TNF-a and NRP1 promote fibrosis (3234). NRP1 can play versatile roles in angiogenesis (35). TWEAK/Fn14, glia-derived nexin, and TrkA are able to induce extracellular matrix generation and deposition (3638). The type 2 cytokines released by activated Tc2 cells can also contribute to tissue remodeling, as it has been demonstrated that tissue injury promotes type 2 cytokine production by the tissue-resident CD8 1 T cells that promote wound repair in mouse (39). Our data further confirmed this in human cells. The protissue remodeling role of Tc2 cells mediated by the PGD 2 /DP2 axis was also evidenced by our fibro-blastTc2 coculture assay, in which PGD 2 -activated Tc2 cells enhanced fibroblast proliferation that was mediated by both soluble tissue-remodeling factors in the Tc2 medium and direct cell contact. Activated Tc2 cells also promoted the synthesis of collagen I a1, actin a2, vimentin, and TGF-b in fibroblasts. Collagen I a1 is a member of type 1 collagen, which is the most abundant component of extracellular matrix and can contribute to myofibroblast differentiation (40); actin a2, also named as a smooth muscle actin, is a biomarker of myofibroblasts and smooth muscle differentiation (41); vimentin plays important roles on fibroblast proliferation during wound healing (42), whereas TGF-b signaling controls expression of type 1 collagen and actin a2 (43,44). In a recent publication, it was reported that fevipiprant reduced ASM mass in asthma patients (45). Although ASM cells were shown to express DP2, DK-PGD 2 did not directly induce migration of ASM cells in vitro. It was also demonstrated that there was a correlation between ASM mass reduction and the numbers of myofibroblasts or fibrocytes in the lamina propria, which may explain reduced airway remodeling with DP2 antagonism. Our results support these observations and provide a novel, to our knowledge, mechanism for how PGD 2 /DP2 could regulate airway remodeling via activation of Tc2 cells, suggesting a potential strategy to control airway remodeling in asthma (Fig. 7). Of course, further investigation is required to confirm the role of the PGD 2 /DP2/Tc2 axis in human airway remodeling. PGD 2 is a major arachidonic acid metabolite detected in high concentrations at sites of allergic inflammation and plays an important role in inflammatory reactions (6,7). It was traditionally considered that PGD 2 is predominantly released from activated mast cells during an allergic response initiated by IgE cross-linkage of its high-affinity receptor FceRI (5). However, high levels of PGD 2 are not always correlated with the levels of IgE in asthma patients. Increasing evidence suggests that PGD 2 can also be produced by some FceRI-low or -negative cells, such as dendritic cells, macrophages (46), eosinophils (47), Th2 cells (8), and ILC2s (48). In this study, we demonstrated for the first time, to our knowledge, that Tc2 cells are capable of producing PGD 2 via an IgE-independent pathway. The capacity of PGD 2 production in Tc2 cells is similar to that in Th2 cells and about half of that in mast cells. Tc2 cells possess and use the routine molecular machinery for PGD 2 synthesis, as inhibition of cPLA2/PLC, COX-1/2, or hPGDS blocked PGD 2 production in Tc2 cells. PLA2 and PLC are enzymes that cleave membrane phospholipids to arachidonic acid upstream of PGD 2 synthesis. Our data suggested that, in Tc2 cells, arachidonic acid synthesis was predominantly mediated by cPLA2. COX-1 and COX-2, also known as PG-endoperoxide synthases, convert arachidonic acid to prostanoids consisting of PGs, thromboxanes, and prostacyclins (49). The levels of COX-1/2 in Tc2 cells are upregulated during endogenous synthesis of PGD 2 . hPGDS is a cytosolic enzyme downstream of COXs in the PGD 2 synthesis pathway that isomerizes PGH 2 , a common precursor for all PGs and thromboxanes, to PGD 2 in a glutathione-dependent manner (50). Tc2 cells express hPGDS, and hPGDS-positive Tc2 cells are increased in eosinophilic asthma and correlated with blood eosinophil counts in asthma patients. These observations suggest that Tc2 cells could contribute to IgE-independent DP2-mediated airway inflammation in asthma, which could be supported by clinical evidence that $20% of severe eosinophilic asthma patients are nonatopic with low IgE levels (51). Stimulation of TCR also strongly promotes PGD 2 production in Tc2 cells, confirming a TCR-dependent mechanism of PGD 2 production (8). This response in Tc2 cells is likely involved in secondary autocrine or paracrine production of PGD 2 , as the inhibition of DP2 with fevipiprant markedly attenuated PGD 2 production induced by TCR stimulation and reversed TCR-mediated DP2 endocytosis, and the inhibition PGD 2 synthesis also mitigated TCRmediated cytokine production. In addition to the secondary autocrine synthesis of PGD 2 , TCR activation could also trigger some unknown pathways to regulate PGD 2 production in Tc2 cells because of the following factors: 1) TCR activation did not upregulate COX-1 or hPGDS, 2) COX-2 upregulation by TCR was not reduced by fevipiprant, and 3) inhibition of DP2 and hPGDS could not completely block the PGD 2 production induced by TCR activation. Therefore, further investigation is required to understand the complete mechanism involved in TCR-dependent PGD 2 production. Both PGD 2 and cysLTs are major arachidonic acid metabolites released from mast cells, playing critical roles in the pathogenesis of allergic disorders. They are synthesized by separate metabolic pathways downstream of arachidonic acid. In mast cells activated by IgE, PGD 2 and cysLTs are produced simultaneously, and inhibition of one of their pathways could potentiate the other one (52). However, in activated Tc2 cells, only PGD 2 but not cysLT production is detected. Considering that very low expression levels of 5-LO and FLAP were detected in Tc2 cells, they might be unable to produce cysLTs because of a lack of enzymes for cysLT synthesis.
DP2 inhibition with fevipiprant has been reported to abolish the proinflammatory effect of PGD 2 in Th2 and ILC2 cells (20,21). In this study, we demonstrated that fevipiprant is a potent and specific inhibitor of the DP2 pathway in Tc2 cells. It not only inhibited established Tc2 proinflammatory activation, including migration, adhesion, cytokine production and survival (3), but also suppressed the newly discovered tissue-remodeling effect and autocrine/paracrine PGD 2 production in Tc2 cells. These findings may be relevant to the design of clinical studies with ongoing DP2 antagonists (13,14).
Overall, these data expand the array of potential effector functions of type 2 cells, specifically of CD8 1 T cell populations, that are often overlooked. Additionally, the blockade of these cells and pathways remains of potential clinical value in asthma and other type 2driven inflammatory diseases.