Prostaglandin D2 axis impairs immunity against melanoma via dendritic cells and γδ T cells in middle-aged mice

Abstract

The risk of developing melanoma increases with age. Although immune checkpoint blockade (ICB) therapy has shown considerable success, a significant portion of melanoma patients either fail to respond to ICB or eventually develop resistance. This leads to the urgent need for exploring novel treatments. Phospholipase A2 group IID (PLA₂G2D) is an inducible enzyme found in myeloid cells, especially in aging dendritic cells (DCs), that exert an immunosuppressive effect by producing anti- or proinflammatory small lipid molecules, including prostaglandin D2 (PGD₂). An aging-related increase of PLA₂G2D-PGD₂ expression makes this signaling a promising target for treating aging-associated diseases. The overexpression of hematopoietic PGD₂ synthase identified in both human and mouse melanoma tissue further highlights the potential of PLA₂G2D-PGD₂–targeting therapy. In this study, we show that the absence of PLA₂G2D or the PGD₂ receptor, PTGDR, restricts primary tumor growth and lung metastasis of subcutaneously implanted melanoma, as demonstrated using middle-aged Pla2g2d^−/− and Ptgdr^−/− mice. These therapeutic benefits are linked to increased tumor infiltration of activated γδ T cells, which can be amplified in B16F10-bearing wild-type mice through the adoptive transfer of Ptgdr^−/− DCs. These tumor-restraining effects were also confirmed in DC-specific PTGDR-deficient (zDC^crePtgdr^floxp) mice. Mechanistically, the enhanced production of IL-1β by Ptgdr^−/− DCs contributes to the activation and accumulation of γδ T cells in tumor tissue. In summary, our findings highlight the effectiveness of targeting the PLA₂G2D-PGD₂/PTGDR axis to reprogram aging dendritic cells, thereby inhibiting melanoma progression and presenting a promising therapeutic target, particularly for elderly patients.

Introduction

Melanoma accounts for 1.7% of global cancer diagnoses and is the fifth most common cancer in the United States, while its incidence keeps rising in developed countries. Although the overall 5-year survival has risen to 93.3% in the United States, survival for stage IV disease remains only 29.8% due to diverse metastasis.¹ According to a statistical analysis by the American Cancer Society, age represents a major risk for the incidence of melanoma. The rate of new melanoma patients increased by about 1% to 3% per year in people aged 50 years and older, while the average age of melanoma patients when it is diagnosed is 66 years (https://www.cancer.org/cancer/types/melanoma-skin-cancer/about/key-statistics.html). As with many other types of cancer, the lungs are by far the most common visceral site for melanoma. The modified lung environment in aged hosts, such as angiogenesis, extracellular matrix remodeling, dysregulated inflammatory response, and changes caused by chemotherapeutic agents, favors tumor metastasis.^2,3 Therefore, aging-related signaling represents potential targets for restricting the occurrence and lung metastasis of melanoma. Moreover, although cancer survival has significantly increased in the past decades, partly because of the advances in early screening and detection^4-7 and the development of immunotherapies,^5,8-10 the efficacy of current immunotherapeutic interventions, including immune checkpoint blockade (ICB), is far from satisfactory,^11-13 so there is a continuous need to develop novel antitumor strategies.

In addition to protumor characteristics of the lung environment mentioned above, the oxidative stress and chronic low-grade inflammation (inflammaging)^14,15 that occur in lung and other organs during aging are associated with immune system dysfunction^16,17 and result in defective immunoprotection.^18,19 Inflammaging in aged organs, especially lung and brain, is partially countered by age-dependent increased expression of a single inducible phospholipase, phospholipase A2 group IID (PLA₂G2D).^20,21 PLA₂G2D is increased in myeloid cells, especially dendritic cells (DCs), as mice age²¹ and contributes to the production of several pro- and anti-inflammatory lipid mediators, including prostaglandin D2 (PGD₂), prostaglandin E2 (PGE₂), prostaglandin F2-alpha (PGF_2α), and thromboxane B2 (TXB₂).^20-22 The immune regulation mediated by PLA₂G2D signaling may impede immune responses and delay pathogen clearance.²³ For example, age-related decreased survival rates of respiratory virus–infected mice were largely reversed in Pla2g2d^−/− mice because of enhanced migration of respiratory tract–resident DCs (rDCs) from the lungs to the draining lymph nodes (DLNs) and subsequently augmented virus-specific T-cell responses.^21,24 These benefits were also confirmed in mice lacking expression of D-prostanoid receptor 1 (PTGDR), a PGD₂ receptor,²⁴ suggesting that PLA₂G2D-PGD₂/PTGDR signaling can be targeted to enhance immune responses mediated by the DC–T-cell axis.

Compared to well-studied PGE₂, the biological function of PGD₂ is less understood due to its context-related anti- or proinflammatory roles. PGD₂ is considered an anti-inflammatory molecule when it signals through PTGDR expressed on myeloid cells, but it can also play a proinflammatory role when it binds to the CRTH2 receptor expressed by Th2 CD4 T cells.^25,26 PGD₂/PTGDR signaling in myeloid cells triggers G protein activation and cAMP production. Genome-wide expression analyses indicated that this signaling is required for the upregulation of a putative inflammasome inhibitor, pyrin domain only–containing protein 3 (PYDC3), which subsequently regulates inflammasome assembly and IL-1β/IL-18 production.^27-29 PTGDR signaling in human macrophages also results in the upregulation of a putative functional analogue of PYDC3, POP3, supporting the potential of PTGDR-targeting therapy in treating human inflammation-related diseases.³⁰ Notably, mRNA expression of PLA₂G2D and production of PGD₂ also increased in human PBMC-derived DCs during aging, making them potential targets for the development of novel strategies to modify age-dependent inflammation-related diseases. A recent study demonstrated that hematopoietic PGD₂ synthase (HPGDS) overexpression in tumor-associated macrophages impeded the responsiveness of human melanoma patients and a mouse model to anti-PD-1 therapy.³¹ However, the role of DC-specific PLA₂G2D-PGD₂/PTGDR signaling in primary tumor growth and lung metastasis of melanoma has never been investigated.

In this study, by comparing the outcomes of melanoma B16F10-implanted middle-aged wild-type (WT), Pla2g2d^−/−, and Ptgdr^−/− mice, we found that the deficiency of PLA₂G2D-PGD₂/PTGDR signaling, especially PTGDR, potently restricted primary tumor growth and lung metastasis. This benefit correlates with an increased accumulation of activated γδ T cells in tumor tissue, while the antitumor activity of γδ T cells was further confirmed by the adoptive transfer model. Mechanistically, the increased accumulation and activation of γδ T cells in tumor tissue are largely dependent on IL-1β produced by Ptgdr^−/− DCs. Taken together, our data demonstrate the potential of DC-specific PLA₂G2D-PGD₂/PTGDR signaling–targeting therapy in treating primary melanoma and lung metastasis by reprogramming aging DCs. The efficacy of this targeting therapy in patients lacking responsiveness to ICB treatment also merits further investigation.

Materials and methods

Mice

Eight- to 10-week-old or 8- to 10-month-old or 18-month-old specific pathogen-free (SPF) C57BL/6 and B6.129S-Il1rn^tm1Dih/J mice were purchased from The Jackson Laboratory. Pla2g2d^−/− mice were obtained from the Tokyo Metropolitan Institute of Medical Science (Japan) Ptgdr^−/− mice were generated as described previously³² and were obtained from Kyoto University (Japan). Pla2g2d^−/− and Ptgdr^−/− mice were kept in an SPF environment at the University of Iowa and the University of Louisville. Ptgdr^flox mice were obtained from Dr Richard Breyer (Vanderbilt University) and were cross-mated with B6.Cg-Zbtb46^{tm3.1(cre)Mnz}/J, B6·129P2-Lyz2^tm1(cre)Ifo/J, and B6·129P2-Cx3cr1^{tm2.1(cre/ERT2)Litt}/WganJ mice (Jackson Laboratory; all are C57BL/6J background) to generate cell-specific PTGDR knockout (KO) mice (zDC^crePtgdr^floxp, LyzM^crePtgdr^floxp, and CX3CR1^crePtgdr^floxp, respectively). Data shown in this manuscript were obtained from both male and female mice. No sex-related difference was found in the animal models used in this study. All animal studies were approved by the University of Iowa and the University of Louisville Animal Care and Use Committee and meet stipulations of the Guide for the Care and Use of Laboratory Animals.

Cell culture and tumor implantation

Melanoma cell line B16F10 (CRL-6475) and Lewis lung carcinoma LL/2 (CRL-1642) were purchased from American Type Culture Collection and grown in DMEM supplemented with 10% FBS, 100 U/mL penicillin/100 μg/mL streptomycin (15140-122), 0.7 mM sodium pyruvate (11360-070), 1× nonessential amino acid (11140-050), 0.15% sodium bicarbonate (25080-094), and 4 mM l-glutamine (25030-081) (all from Gibco).

To establish the tumor model, 1 × 10⁶ B16F10 were implanted on recipient mice via s.c. or i.v. injection in 0.2 mL 1× PBS under anesthesia induced by isoflurane. The general condition of tumor-implanted mice and tumor growth were monitored daily. As for s.c. injection, solid tumors typically form in the injection site after ~10 days, while lung metastasis usually occurs after 3 weeks. As for i.v. injection, lung metastasis usually occurs after 3 to 4 weeks.

Antibodies and flow cytometry

The following monoclonal antibodies were used: PE- or PerCP-Cy5.5–conjugated rat anti-mouse CD4 (RM4-5), FITC- or PE-Cy7–conjugated rat anti-mouse CD8α (53–6.7), FITC- or e450-conjugated hamster anti-mouse CD11c (HL3), APC-Cy7–conjugated mouse anti-mouse Ly-6C (HK1.4), APC-conjugated rat anti-mouse F4/80 (BM8), FITC-conjugated rat anti-mouse IL-1β (NJTEN3), PerCP-Cy5.5–conjugated mouse anti-mouse Foxp3 (FJK-16s), and rat anti-mouse CD16/32 (2.4G2) (eBioscience); APC-conjugated hamster anti-mouse γδ TCR (GL3), PE-conjugated hamster anti-mouse Vγ1.1 (2.11), FITC-conjugated mouse anti-mouse Vγ4 (49.2), PerCP-Cy5.5–conjugated hamster anti-mouse CD3 (145-2C11), PE-Cy7–conjugated mouse anti-mouse NK1.1 (PK-136), PerCP-Cy5.5– or PE-conjugated rat anti-mouse CD45 (30-F11), APC-Cy7-conjugated mouse anti-mouse CD69 (H1.2F3), FITC-conjugated mouse anti-mouse CD107a (LAMP-1), APC-conjugated mouse anti-mouse 4-1BBL (4B4-1), PE-conjugated mouse anti-mouse 4-1BBL (TKS-1), APC-conjugated mouse anti-mouse PD-L1 (10F.9G2), APC-Cy7–conjugated mouse anti-mouse MHC-I (28-8-6), Brilliant Violet 510–conjugated mouse anti-mouse MHC-II (M5/114.15.2), PE-conjugated mouse anti-mouse CD80 (2D10), PE-Cy7–conjugated mouse anti-mouse PD-1 (RPM1-30), PerCP-Cy5.5–conjugated mouse anti-mouse CTLA-4 (UC10-4B9), PE-Cy7–conjugated rat anti-mouse CD11b (M1/70), APC-conjugated rat anti-mouse BST-2 (927), Pacific Blue–conjugated hamster anti-mouse CD103 (2E7), Alexa Fluor 647– or APC-conjugated rat anti-mouse IFN-γ (XMG1.2), PE-conjugated mouse anti-mouse IL-17F (9D3.1C8), APC-conjugated rat anti-mouse TNF (MP6-XT22), Pacific Blue–conjugated rat anti-mouse perforin (S16009B), and FITC-conjugated mouse anti-human/mouse granzyme B (QA16A02) (BioLegend); and APC-conjugated mouse anti-mouse Vγ6 (1C10-1F7), FITC-conjugated mouse anti-mouse B220 (RA3-6B2), Brilliant Violet 510–conjugated rat anti-mouse Ly-6G (1A8), and FITC-conjugated rat anti-mouse CD86 (GL1) (BD Biosciences).

For surface staining, 10⁶ cells were blocked with 1 mg of anti-CD16/32 antibody and stained with the indicated antibodies at 4 °C for 30 minutes. For intracellular staining, cells were fixed using Cytofix Solution (BD) and stained for Foxp3 or intracellular cytokines. To determine the absolute number of cells, CountBright absolute counting beads (Invitrogen) were added at the beginning of sample preparation. A Live/Dead cell dye kit (Invitrogen) or FITC–Annexin V (AV)–propidium iodide (PI) kit (Thermo Fisher) was used to gate live/apoptotic cells. Flow cytometric data were acquired using an FACSVerse (BD) and analyzed using FlowJo software (TreeStar).

CFSE staining

In some experiments, B16F10 cells were resuspended with 1× PBS at the concentration of 1 × 10⁷/mL and stained with 5 mM CFSE at 37 °C for 10 minutes. After washing with 1× PBS for 3 times, CFSE-labeled B16F10 cells were used for implantation. The in vivo proliferation of B16F10 was determined by the intensity of CFSE via flow cytometry.

BrdU assay

Tumor-bearing mice were injected i.p. with 2 mg BrdU (BD) at 24 hours before sample collection. BrdU incorporation by B16F10 tumor cells was examined by flow cytometry according to the manufacturer’s protocol (APC BrdU flow kit; BD).

Cytotoxicity assay

Mouse alveolar macrophages (AMs) were purified from lung single-cell suspension of WT C57BL/6 mice by anti-Siglec-F microbeads (Miltenyi) and used as target cells, as well as B16F10 and LL/2, of cytotoxicity assay. Tumor-infiltrating γδ T cells or CD8 T cells were purified from tumor-bearing Ptgdr^−/− mice at day 20 postimplantation by anti-mouse γδ TCR or anti-mouse CD8 microbeads with an autoMACS system (Miltenyi) following the manufacturer’s protocols, respectively. Purified γδ T cells or CD8 T cells were then used as effector cells and cultured with target cells at indicated E:T ratios for 6 hours in a 37 °C incubator. FITC-AV-PI was added at the final 15 minutes of co-culture. The dying target cells were identified by the expression of AV or PI in γδ TCR⁻ or CD8⁻ population, respectively, by flow cytometry.

In some experiments, effector cells (γδ T cells) and target cells (B16F10, LL/2, or AMs) were cultured in a Transwell system (Abcam). In brief, effector cells were placed in the inserts while target cells were cultured in the bottom wells. After 6 hours of culture, the dying target cells were determined by AV-PI staining as described above.

Blocking/stimulating assay

In cytotoxicity assays, neutralizing antibodies against PD-1 (BE0146) and CTLA-4 (BE0131), agonist antibody against 4-1BB (BE0239), or their isotype control Ig (final concentration: 10mg/mL) were purchased from Bio X Cell and added at the start of coculture.

In the in vivo neutralizing assay, hamster anti-mouse IL-1β antibody or its isotype control Ig (Bio X Cell) was injected intravenously (10mg/kg weight) at the indicated timepoints.

Adoptive transfer of γδ T lymphocytes

A total of 2 × 10⁶ tumor-infiltrating γδ T cells were purified from tumor-bearing Ptgdr^−/− mice as described above and were adoptively transferred via intratumor or i.v. injection into tumor-bearing WT mice at day 14 postimplantation. Seven days later, recipient mice were euthanized for measuring tumor progression.

In the co-transfer model, peripheral γδ T cells isolated from the dermis and DLNs of different donors were stained with CFSE (5 mM or 0.1 mM) before the adoptive transfer.

CD11c⁺ DC separation using magnetic beads and adoptive transfer

CD11c⁺ DCs were either isolated from lung single-cell suspensions using anti-mouse CD11c microbeads or were derived from bone marrow (10% FBS-DMEM supplemented with 100 ng/mL GM-CSF and 50 ng/mL IL-4 [PeproTech]) of WT, Pla2g2d^−/−, or Ptgdr^−/− mice.

A total of 1 × 10⁵ CD11c⁺ rDCs or bone marrow–derived DCs (BMDCs) were adoptively transferred by i.v. injection into tumor-bearing WT mice at the indicated time points.

Histology

Animals were anesthetized and transcardially perfused with PBS followed by zinc formalin. Subcutaneous tumor tissue and lung were removed, fixed in zinc formalin, and paraffin embedded. Sections were stained with hematoxylin and eosin. Images were acquired using a BX61 light microscope (Olympus) and CellSens software (Olympus).

Fluorescence microscopy

Tissues were harvested as described above and fixed with 4% PFA at 4 °C for 4 hours, followed by immersion in 10%, 20%, and 30% sucrose in PBS for 12 hours each. Sections 7 mm thick were then prepared from OCT-embedded samples and fixed in acetone for 10 minutes at 4 °C. For staining, sections were blocked with goat serum for 2 hours in humidity chambers at room temperature. Sections were then treated with primary antibodies (hamster anti-mouse γδ TCR, GL3; rat anti-mouse CD11c, OX42; Abcam) at 4 °C overnight. After washing with PBS, samples were treated with secondary antibodies (Alexa 488–conjugated goat anti-hamster IgG, Alexa 647–conjugated goat anti-rat IgG, Abcam) for 30 minutes at room temperature. Finally, slides were overlaid with antifade mounting medium containing DAPI (Vector) and examined using a confocal microscope (Zeiss 710).

Imaging mass cytometry

Tumor tissue sections from WT, Pla2g2d^−/−, or Ptgdr^−/− mice were stained with metal-labeled antibodies: anti-Ki67-150Nd, 1:150; anti-CD3 (UCHT1)-170Er, 1:150; and anti-DNA-191Ir, 1:300 (Standard BioTools), respectively. All antibodies were first validated to confirm optimal staining intensity, specificity, and signal-to-noise ratio. Stained tissue sections were ablated by using the Hyperion mass cytometry imaging system (Standard BioTools). The Hyperion was autotuned using a 3-element tuning slide (Standard BioTools) as described in the Hyperion imaging system user guide. An extra minimum threshold of 1,000 mean duals of 175Lu were used. At least three 2500 × 1500-μm regions of interest per sample were selected and ablated at 200 Hz. Data were visualized with MCD viewer software (Standard BioTools). For downstream analysis, image data were exported as tiff. ome file from the MCD viewer, followed by cell segmentation using CellProfiler (developed by Broad Institute of Massachusetts Institute of Technology and Harvard, v4.2.1). The .fcs files or .cvs files were exported using histoCAT software. The .fcs files were further analyzed using FlowJo software (BD).

Statistical analysis

A Student t-test or one-way ANOVA with Turkey post hoc correction was used to analyze differences in mean values between groups. All results are expressed as mean ± SEM. P values of <0.05 were considered statistically significant.

Results

PLA₂G2D and PTGDR deficiency restrict the primary growth and lung metastasis of melanoma

The implantation of the melanoma B16F10 cell line represents a verified model to investigate tumor growth and lung metastasis in mice.^33,34 To identify the role of PLA₂G2D and PTGDR in primary tumor growth and lung metastasis, B16F10 cells were implanted in the lateral region of the abdomen of age-matched WT, Pla2g2d^−/−, and Ptgdr^−/− mice via s.c. injection (Fig. 1A). Since the expression of PLA₂G2D and PGD₂ increases with age,²¹ 8- to 10-month-old mice were used in this study. We did not use older mice because of the aging-related decline of global immunity and increase of comorbidities in older Pla2g2d^−/− and Ptgdr^−/− mice (such as dermatitis starting at 12 to 15 months of age, which may affect the immune cell composition and status in DLNs of skin). On the other hand, no sex-related differences have been reported in the expression of PLA₂G2D and PTGDR in humans or mice or were observed in our preliminary experiments. Therefore, data shown in this manuscript were obtained from both male and female mice.

Figure 1.

PLA₂G2D and PTGDR deficiency inhibit the in situ growth and metastasis of melanoma B16F10. (A) Eight- to 10-month-old (middle-aged) WT, Pla2g2d^−/−, and Ptgdr^−/− mice (all from C57BL/6 background and age-matched) were implanted with 1 × 10⁶ melanoma cell line B16F10 via s.c. injection. (B–D) Four weeks later, the primary tumor and lungs were harvested and examined for primary tumor growth and lung metastasis (circled areas) (B). The volume of primary tumor (C) and metastasis lesions (D) in different mice strains are summarized. n = 5. Data are mean ± SEM and are representative of 3 independent experiments. ****P < 0.0001. (E) Ki67 expression and CD3 T-cell infiltration in s.c. tumor tissue of WT, pla2g2d^−/−, and Ptgdr^−/− mice determined by imaging mass cytometry at 28 days postimplantation. The images represent data harvested from 8 mice. ****P < 0.0001. (F) Eight- to 10-month-old WT, pla2g2d^−/−, and Ptgdr^−/− mice were s.c. injected with 1 × 10⁶ CFSE-labeled B16F10 as described previously. The tumor tissues were harvested at indicated time points and examined for the proliferation of B16F10 by flow cytometry. n = 4. Data are representative of 3 independent experiments. n.s., not significant. **P < 0.01. (G, H) Eight- to 10-month-old WT, pla2g2d^−/−, and Ptgdr^−/− mice were s.c. injected with 1 × 10⁶ B16F10 as described previously and fed with BrdU-containing water starting from the implantation of B16F10. On day 20 postimplantation, tumor tissues were harvested while the percentage of BrdU⁺ and AV⁺/PI⁺ B16F10 cells were determined by flow cytometry. n = 4. Data are mean ± SEM and are representative of 2 independent experiments. n.s., not significant. **P < 0.01.

As shown in Fig. 1B-D, Pla2g2d^−/− and Ptgdr^−/− mice developed smaller primary tumors, as well as a significantly lower incidence of lung metastasis, compared to WT mice. Consistent with that, fewer Ki67⁺ dividing tumor cells and increased CD3⁺ T cells were identified in primary tumor tissue of Pla2g2d^−/− and Ptgdr^−/− mice (Fig. 1E). Nevertheless, CFSE and BrdU staining assays showed a comparable percentage of labeled B16F10 cells isolated from WT, Pla2g2d^−/−, and Ptgdr^−/− mice (Fig. 1F, G), indicating that PLA₂G2D and PTGDR deficiency did not directly affect the proliferative potential of tumor cells. In contrast, AV/PI staining assay showed a higher percentage of apoptotic B16F10 cells in primary tumor tissue of Pla2g2d^−/− and Ptgdr^−/− mice compared with WT mice (Fig. 1H). Taken together, these data suggest that PLA₂G2D and PTGDR deficiency–correlated tumor reduction was mainly caused by increased tumor cell apoptosis, which may result from the enhanced T-cell infiltration in tumor tissue.

In addition, this tumor resistance was replicated in older (18-month-old) Pla2g2d^−/− and Ptgdr^−/− mice (Fig. S1A, B) but not in young (8- to 10-week-old) mice (Fig. S1C, D), which is likely due to the low expression of PLA₂G2D-PGD₂ axis and its minor or no impact on antitumor activity in younger WT mice.

Increased γδ T-cell accumulation in tumor tissue inversely correlates with tumor growth

Immune cells, especially CD3 T cells and natural killer (NK) cells, represent the major effector cells in antitumor responses.^35-37 To determine the cellular mechanisms contributing to tumor-restraining effects in Pla2g2d^−/− and Ptgdr^−/− mice, tumor-infiltrating immune cells were determined by flow cytometry at indicated time points. As shown in Fig. 2A, tumor-infiltrating CD4, CD8, and NK cells increased in WT, Pla2g2d^−/−, and Ptgdr^−/− mice after melanoma implantation. However, only Pla2g2d^−/− and Ptgdr^−/− tumor-bearing mice showed a robust accumulation of γδ T cells in tumor tissue. More importantly, the accumulation of γδ T cells are inversely correlated with tumor growth (Fig. 2B, measured by the quantity of B16F10 cells present in tumor tissue), whereas the accumulation of CD4, CD8, and NK cells did not show a similar correlation with tumor growth. These data suggest a critical role of γδ T cells in restraining melanoma in Pla2g2d^−/− and Ptgdr^−/− mice.

Figure 2.

Increased tumor-infiltrating γδ T cells contribute to tumor reduction in Ptgdr^−/− mice. (A) The accumulation of CD4 T cells, CD8 T cells, γδ T cells, and NK cells in primary tumor tissue of WT, Pla2g2d^−/−, and Ptgdr^−/− mice was determined by flow cytometry at indicated time points. n = 4/group for each time point. Data are mean ± SEM and are representative of 2 independent experiments, *P < 0.05, **P < 0.01. (B) The correlation between the quantity of tumor-infiltrating immune cells and tumor cells (B16F10) was analyzed at day 20 postimplantation. n = 12/group. Data are representative of 2 independent experiments. (C) The immunophenotypes of tumor-infiltrating γδ T cells and ab-T cells in tumor-bearing Ptgdr^−/− mice were determined by flow cytometry at day 20 postimplantation. Data are mean ± SEM and are representative of 2 independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001. (D, E) Cytotoxicity assay. Tumor-infiltrating γδ T cells or CD8 T cells were purified from tumor-bearing Ptgdr^−/− mice by magnetic beads and cultured with B16F10, LL/2, or mouse alveolar macrophages (AMs) at indicated E:T ratios (effectors: γδ T cells, or CD8 T cells; targets: B16F10, LL/2, or AMs) for 6 hours. γδ T-cell or CD8 T-cell–mediated cytotoxicity in co-culture (D) or Transwell system, or with corresponding neutralizing (α-PD-1, α-CTLA-4) or agonist antibodies (α-4-1BB) (E) were determined by the percentages of PI⁺ target cells via flow cytometry. n = 4. IC, isotype control Ig. Data are mean ± SEM and are representative of 2 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (compared to IC-treated group). (F, G) Adoptive transfer assay: 2 × 10⁶ tumor-infiltrating γδ T cells purified from tumor tissue or dermis/DLNs, as well as tumor-infiltrating CD8 T cells of Ptgdr^−/− mice at day 21 postimplantation, were adoptively transferred into tumor-bearing WT mice (at day 14 postimplantation) by in situ or i.v. injection. The primary tumor growths were measured at day 7 post–adoptive transfer. n = 5. Data are mean ± SEM and are representative of 3 independent experiments. ****P < 0.0001.

Compared to Pla2g2d^−/− mice, Ptgdr^−/− mice showed greater tumor resistance (Fig. 1B-D), which may be due to the protumor activity of other lipid products produced by active PLA₂G2D.^20-22 Therefore, Ptgdr^−/− mice were mainly used to investigate γδ T-cell–mediated antitumor immunity in the following study.

Antitumor activity of γδ T cells

To further verify the antitumor activity of infiltrating γδ T cells (gating strategy shown in Fig. S2), the phenotypes of γδ T cells isolated from tumor-bearing Ptgdr^−/− mice were determined and compared with those of CD8 T cells isolated from the same mice. Although both effector cells showed similar expression of granzyme B and perforin, γδ T cells showed higher expression of CD69, IFN-γ, IL-17, and 4-1BB compared to CD8 T cells, indicating a more activated status (Fig. 2C). In addition, PD-1 expression levels on γδ T cells were lower than those of CD8 T cells, which may favor the resistance of γδ T cells to tumor microenvironment (TME)–associated immunosuppression.^38,39 Despite their comparable expression of CD107a, tumor-infiltrating γδ T cells showed more potent cytotoxicity against B16F10 and LL/2 (Lewis lung carcinoma) than CD8 T cells when examined in vitro (Fig. 2D). It is also noteworthy that γδ T cells in the dermis and DLNs (brachial and inguinal) of tumor-bearing Ptgdr^−/− mice exhibited lower cytolytic capacity than their tumor-infiltrating analogues despite their comparable composition of γδ T-cell subsets (Fig. S3), indicating that infiltrating γδ T cells were further activated in tumor tissue. In addition, γδ T-cell–mediated cytotoxicity was dependent upon cell–cell contact and could be blocked by physical isolation from target cells using the Transwell system, while blocking antibodies targeting PD-1 and CTLA-4, as well as agonist antibody targeting 4-1BB, improved γδ T-cell–mediated cytotoxicity on melanoma (Fig. 2E).

To further evaluate their in vivo function, γδ T cells isolated from tumor tissue or dermis/DLNs, as well as tumor-infiltrating CD8 T cells of Ptgdr^−/− mice, were adoptively transferred into B16F10-bearing WT mice (Fig. 2F) via intratumor (in situ) or i.v. injection. As shown in Fig. 2G, both tumor-infiltrating or dermis-related γδ T cells treatment led to significant tumor reduction irrespective of the manner of administration, while tumor-infiltrating γδ T cells showed stronger antitumor activity. On the contrary, the therapeutic efficacy of tumor-infiltrating CD8 T cells’ adoptive transfer was less impressive. Taken together, these data suggest that the accumulation and activation of tumor-infiltrating γδ T cells contribute to PTGDR deficiency-related tumor reduction.

Ptgdr^−/− DCs contribute to tumor reduction and γδ T-cell accumulation

Since PLA₂G2D and PTGDR are highly expressed in myeloid cells (monocytes, DCs, and macrophages) in an age-dependent manner,^26,40 we aimed to elucidate the mechanisms bridging PLA₂G2D and PTGDR deficiency to increased γδ T-cell infiltration and activation. Owing to their professional capacity in antigen presentation, DCs play a critical role in initiating and regulating T-cell responses, representing a potential tool for antitumor immunotherapy.^41-43 Compared to that of WT mice, melanoma implanted in Ptgdr^−/− mice was infiltrated with more DCs at day 20 postimplantation (Fig. 3A), which co-localized with γδ T cells (Fig. 3B). Despite comparable expressions of MHC-I/II, CD80, and CD86, enriched DCs (gating strategy shown in Fig. S4A) in Ptgdr^−/− (Fig. 3C) and Pla2g2d^−/− mice (Fig. S4B) exhibited higher expression levels of 4-1BB-L, TNF, and IL-1β, as well as lower PD-L1, compared to those of WT mice. These proinflammatory characteristics of DCs are expected to subsequently modify antitumor activity of T cells. We then adoptively transferred BMDCs from WT or Ptgdr^−/− mice⁴⁴ into B16F10-bearing WT mice at days 7 and 14 after melanoma implantation. As shown in Fig. 3D-G, reductions in primary tumor growth (Fig. 3E) and lung metastatic lesions (Fig. 3F) were observed in mice receiving BMDCs from Ptgdr^−/− mice. Consistent with increased γδ T-cell infiltration in tumor tissue of Pla2g2d^−/− and Ptgdr^−/− mice (Fig. 2), γδ T-cell accumulation in tumor tissue of WT recipients was increased with the adoptive transfer of Ptgdr^−/− (Fig. 3G) or Pla2g2d^−/− BMDCs (Fig. S5). These data support our hypothesis that PLA₂G2D and PTGDR deficiency–driving restraining of tumor growth and metastasis is mediated by DCs through recruiting and activating γδ T cells in both WT and Ptgdr^−/− tumor-bearing mice.

Figure 3.

Inflammatory DCs contribute to γδ T-cell accumulation and tumor reduction in Ptgdr^−/− mice. Eight- to 10-month-old age-matched WT and Ptgdr^−/− mice were s.c. implanted with 1 × 10⁶ B16F10 as described previously. (A) The quantity of DCs in tumor tissue was determined by flow cytometry at indicated time points. n = 5/group for each time point. Data are mean ± SEM and are representative of 2 independent experiments. *P < 0.05. (B) The distribution and co-localization of tumor-infiltrating DCs and γδ T cells in tumor tissue. Data are representative of 10 mice. (C) The immunophenotypes of tumor-infiltrating DCs in tumor-bearing WT or Ptgdr^−/− mice were determined by flow cytometry at day 20 postimplantation. Data are representative of 2 independent experiments (n = 5/group for each experiment). *P < 0.05, **P < 0.01, ****P < 0.0001. (D–G) Eight- to 10-month-old WT mice were implanted with 1 × 10⁶ melanoma cell line B16F10 as described above. On day 7 and 14 postimplantation, 1 × 10⁵ BMDCs from WT or Ptgdr^−/− mice were adoptively transferred into tumor-bearing WT mice via i.v. injection (D). On day 28 postimplantation, the volume of primary tumor (E) and lesions of lung metastasis (F) in tumor-bearing WT mice were measured, while the quantity of tumor-infiltrating γδ T cells was determined by flow cytometry (G). Data are mean ± SEM and represent 2 independent experiments. n = 8. **P < 0.01, ***P < 0.001. (H, J) Eight- to 10-month-old WT, Ptgdr^−/−, and zDC^crePtgdr^floxp mice were implanted with 1 × 10⁶ melanoma cell line B16F10 as described above. At day 20 postimplantation, the volume of primary tumors (H and I) and lung metastasis lesions (H and J) in different mice strains were examined. n = 5. Data are mean ± SEM and are representative of 3 independent experiments. ****P < 0.0001. (K–M) Eight- to 10-month-old WT, Ptgdr^−/−, and zDC^crePtgdr^floxp mice were implanted with 1 × 10⁶ melanoma cell line B16F10 via i.v. injection (K). At day 28 postimplantation, the lesions of lung metastasis of different mice strains were examined (L) and summarized (M). n = 5. Data are mean ± SEM and are representative of 2 independent experiments. ****P < 0.0001.

zDC^crePtgdr^floxp mice show resistance to the progression of s.c. and i.v. implanted melanoma

To verify the central role of DCs in PTGDR deficiency–related melanoma reduction, DC-specific PTGDR-KO (zDC^crePtgdr^floxp) mice were used. As shown in Fig. 3H-J, the primary tumor growth and lung metastasis of s.c. implanted B16F10 cells were largely restrained in zDC^crePtgdr^floxp mice, which was consistent with the outcome of Ptgdr^−/− mice and was accompanied by an increased accumulation of γδ T cells in primary tumor tissue (Fig. S6A). On the contrary, tumor restraining was not found in other myeloid cell–conditional KO mice (LysM^crePtgdr^floxp and CX3CR1^crePtgdr^floxp) (Fig. S6B), which further confirmed the indispensable role of DC-derived PTGDR signaling. Despite the equal reduction of primary tumor growth and lung metastasis in Ptgdr^−/− and zDC^crePtgdr^floxp mice, it remains unclear whether the reduction in lung lesions is a result of decreased primary tumor burden or if it can be independently influenced by PTGDR signaling within the affected lung. To exclude the potential impact of primary tumor burden on the incidence of lung metastasis, a lung melanoma metastasis model was established by i.v. injection of B16F10 (Fig. 3K).⁴⁵ As shown in Fig. 3L and M, lung lesions induced by i.v. injection of B16F10 were diminished in both Ptgdr^−/− and zDC^crePtgdr^floxp mice, suggesting direct anti-melanoma metastasis effects mediated by DCs in lung of implanted mice.

DCs-mediated accumulation of γδ T cells is dependent on IL-1β signaling

PGD₂/PTGDR signaling deficiency promotes the inflammasome assembly and IL-1β production^27-29 (Fig. 3C), while IL-1β has been found to contribute to the activity of γδ T cells.^46,47 Consistent with the protumor role of IL-1β by enhancing tumor invasiveness and angiogenesis,^48,49 the universal KO of IL-1β signaling in B6.129S-Il1rn^tm1Dih/J mice (Il1r^−/−) increased the hosts’ resistance to melanoma progression, characterized by a lower primary tumor volume and fewer lung metastases (Fig. 4A, B), making it unfit for studying the role of the PGD₂ axis. Nevertheless, the adoptive transfer of Ptgdr^−/− DCs failed to increase the accumulation of γδ T cells in Il1r^−/− mice (Fig. 4C), pushing us to further compare the accumulation of γδ T cells isolated from WT or Il1r^−/− mice using a co-transfer model (Fig. 4D). As expected, more γδ T cells isolated from dermis and DLNs of WT mice were recruited to tumor tissue compared to those from Il1r^−/− mice (Fig. 4E). Consistent with that, anti-mouse IL-1β blocking antibody partially reversed the Ptgdr^−/− DC-mediated inhibition on primary tumor growth in WT mice, accompanied by impaired accumulation of γδ T cells in tumor tissue (Fig. 4F-H). It is noteworthy that Ptgdr^−/− DC-mediated inhibition on lung metastasis was not affected by anti-IL-1β antibody, which may result from IL-1β blockade–mediated suppression on tumor invasiveness and angiogenesis.^48,49

Figure 4.

IL-1β contributes to the accumulation of γδ T cells. (A–C) Eight- to 10-month-old Il1r^−/− mice were implanted with 1 × 10⁶ melanoma cell line B16F10 as described above. On day 7 and 14 postimplantation, 1 × 10⁵ BMDCs from WT or Ptgdr^−/− mice were adoptively transferred into tumor-bearing mice via i.v. injection. On day 28 postimplantation, the volume of primary tumor (A) and lesions of lung metastasis (B) in tumor-bearing mice were measured, while the quantity of tumor-infiltrating γδ T cells was determined by flow cytometry (C). Data are mean ± SEM and are representative of 2 independent experiments. n = 5. n.s., not significant. (D) Protocol for co-transfer (0.2 × 10⁶ CFSE^hi γδ T cells from WT mice and 0.2 × 10⁶ CFSE^low γδ T cells from Il1r^−/− mice were mixed at 1:1 before being injected into each recipient mouse). (E) The ratio of tumor-infiltrating and peripheral blood γδ T cells was determined 1 day after the transfer by flow cytometry. Data are mean ± SEM and represent 2 independent experiments. n = 5. **P < 0.01. (F–H) Eight- to 10-month-old WT mice were implanted with 1 × 10⁶ melanoma cell line B16F10 as described above. On day 7 and 14 postimplantation, 1 × 10⁵ rDCs from Ptgdr^−/− mice were adoptively transferred into tumor-bearing mice via i.v. injection. On day 9, 12, 15, and 18 postimplantation, tumor-bearing mice were treated with 10 mg/kg weight hamster anti-mouse IL-1β antibody via i.v. injection. On day 28 postimplantation, the volume of tumor (F) and lung metastasis lesions (G) in tumor-bearing WT mice were measured, while the quantity of tumor-infiltrating γδ T cells was determined by flow cytometry (H). Data are mean ± SEM and represent 2 independent experiments. n = 5. *P < 0.05, **P < 0.01.

Discussion

Aging-associated microbiota dysbiosis and oxidative stress lead to altered soluble mediator networks, resulting in chronic inflammation and weakened immune defense and surveillance.^14-17 To counteract these pathogenic processes, the aging hosts develop diverse regulatory mechanisms, such as the PLA₂G2D-PGD₂/PTGDR signaling pathway, for “self-rescue.” Myeloid cell–expressing PLA₂G2D hydrolyzes phospholipids to release long-chain fatty acids that are further metabolized sequentially by COX1/2 and synthases to yield various lipid mediators, including PGD₂.^20-22 PGD₂, by binding its receptor PTGDR expressed on DCs and other myeloid cells, triggers G protein activation and cAMP production, which subsequently stimulates inflammasome-inhibiting IFN-related genes. PLA₂G2D or PTGDR deficiency or blockade relieve inflammasome suppression and thus may contribute to tumor reduction via recruiting or activating T cells and/or other immune effector cells.

As a member of the esterase family expressed in both rodent and human epithelial cells,^50,51 PLA₂ includes secretory and cytoplasmic forms, both of which are elevated in cancer, demonstrating significant specificity and sensitivity for cancer detection^52-57 and potential roles in regulating tumor cell invasion.^58-61 While direct inhibition of PLA2 has been shown to affect cancer cell growth and survival in vitro,^50,62 its in vivo efficacy and the underlying mechanisms have not been thoroughly investigated. In this study, we focused on the bioactivity of a myeloid cell–expressing PLA₂ enzyme, PLA₂G2D, which is correlated with the tissue accumulation of T cells, DCs, and macrophages.⁴⁰ PLA₂G2D exhibits immunosuppressive function, exerting beneficial or detrimental impact depending on pathophysiological contexts in inflammation and cancer.^20,22 Consistent with that, PGD₂, one of the downstream products of PLA₂G2D, was observed in multiple human cancers and associated with negative outcomes.⁶³ However, its contribution to tumor progression and corresponding mechanisms remain to be elucidated. In this study, we provided direct evidence for the detrimental role of PLA₂G2D-PGD₂/PTGDR signaling in primary melanoma growth and lung metastasis using B16F10-implanted Pla2g2d^−/− and Ptgdr^−/− mouse models. As shown in Fig. 1B-D, both KO mice showed striking tumor reduction compared to WT mice. Since Ptgdr^−/− mice exhibited even better outcomes than Pla2g2d^−/− mice, it is intriguing to identify the roles of other PLA₂G2D-derived lipid mediators in melanoma progression in future study. Due to the lack of PLA₂G2D expression in young WT mice,^20,21 it is not surprising that no advantages in tumor resistance were found in young Pla2g2d^−/− or Ptgdr^−/− mice (Fig. S1C, D). Identifying the signaling responsible for regulating antitumor immunity in young hosts deserves further investigation.

Owing to their unique capacity to initiate and regulate T-cell responses, DCs have been extensively explored as a potential candidate for immunotherapy to treat many types of cancer.^41,42 Although no differences in the composition of immune cells, including T cells and DCs, were identified in skin DLNs of middle-aged WT and Ptgdr^−/− mice (Fig. S7),⁶⁴ the immune profile of DCs and their precursors can be modified by aging or TME, thus affecting their potency in clinic therapy.⁶⁵ Tumor-conditioned DCs were found to downregulate the trafficking of endosomes⁶⁶ and the expression of cytokine/co-stimulatory molecule expression,⁶⁷ which are critical for tumor antigen presentation and tumor rejection. These “tolerant” DCs may also induce regulatory T cells (Tregs), which will further suppress antitumor activity mediated by immune effector cells.⁶⁷ However, no differences in Treg accumulation in tumor tissues were identified in our models (Fig. S8). On the contrary, Toll-like receptor activation⁶⁸ and Bruton tyrosine kinase and tryptophan-degrading enzyme indoleamine 2,3-dioxygenase inhibition or deletion⁶⁹ allowed robust differentiation of inflammatory DCs during chemotherapy, promoting antitumor T-cell responses and inhibiting tumor growth. In this study, we demonstrated the proinflammatory characteristics of DCs isolated from Ptgdr^−/− and Pla2g2d^−/− mice (Fig. 3C; Fig. S4B), as well as the potent tumor-restraining efficacy mediated by adoptive transfer of Ptgdr^−/− and Pla2g2d^−/− DCs (Fig. 3E-G; Fig. S5). These data support PLA₂G2D-PGD₂/PTGDR signaling to be a potential target in restoring DC quantity and function for treating cancer diseases. Moreover, PLA₂G2D-related treatments, such as COX-2 inhibition or PGE₂ blockade, were found to improve the expansion of tumor-infiltrating T cells,^70,71 as well as enhancing the efficacy of ICB^60,72 by regulating lung Th17 cell prevalence⁷³ or modifying PD-1 expression on T-cells.^61,74 Since PLA₂G2D and PTGDR are highly enriched in aged lung, the function of lung DCs (rDCs) may be further modified by the deficiency of PLA₂G2D-PGD₂/PTGDR signaling, which is confirmed by their superior tumor-restraining effects compared to Pla2g2d^−/− and Ptgdr^−/− BMDCs (Fig. S9). Nevertheless, the mechanisms underlying the superiority of rDCs remain to be clarified by single-cell sequencing or proteomics assays. In addition, the potential contribution of plasmacytoid DCs and the individual role of DC subsets (such as cDC1 and cDC2) need to be further identified in the following study, although no differences of these cell percentages were identified between WT and Ptgdr^−/− mice (Fig. S10). Further clarification of their separate contributions is expected to benefit the efficacy of cell-based therapy in future clinical practice.

Several small-molecule PLA₂ inhibitors have been evaluated in clinical trials to treat inflammatory diseases. However, none of them have reached the market yet, due to either failure to exhibit the expected in vivo efficacy or the presence of undesired side effects.⁷⁵ On the other hand, celecoxib, a COX-2 inhibitor, alone or combined with a stimulator of interferon genes (STING) agonist 2′3′-cyclic GMP-AMP (cGAMP), showed benefits on a variety of cancers, including lung metastasis of B16F10 melanoma.^5,74 Other potential drugs targeting PLA₂ include methyl arachidonylfluorophosphonate (a dual inhibitor of cPLA₂ and iPLA₂), bromoenol lactone (a selective inhibitor for iPLA₂),⁵⁵ and manoalide (a proven natural inhibitor of PLA₂).¹³ Besides PLA₂ inhibitors, a PTGDR inhibitor, asapiprant, has been proved to be safe in a phase 1 clinical trial and been tested in mouse models for multiple inflammatory disease. In our previous study, asapiprant improved antiviral immunity in mouse SARS-CoV-2 infection models without causing apparent side effects.²⁴ By optimizing the dose and route of treatment, asapiprant is expected to show benefits on tumor restriction. Additionally, CRISPR-Cas9–edited human DCs and macrophages (targeting at PLA₂G2D) exhibited proinflammatory phenotypes as discovered in mouse systems,⁷⁶ endorsing the application of this RNA editing system in modifying human primary cells for the treatment of human cancers.

In the past decade, γδ T cells have been increasingly explored as a potential immunotherapeutic approach for cancer control.^77-82 γδ T cells are unique among other T lymphocyte subsets in that their activity are independent of neoantigen and conventional MHC-mediated antigen presentation, which make them appealing effectors in treating cancer resistant to antigen-specific T-cell therapies. Nevertheless, the heterogeneity of γδ T cells, as well as the species differences between human and murine γδ T cells,^77,83-85 need to be further addressed. In this study, IL-1β signaling was identified to enhance the accumulation of γδ T cells by DCs (Fig. 4E, H). Nevertheless, the contribution of IL-1β from other resources and other inflammasome-related factors, such as IL-18 and type-I IFN, to the accumulation of γδ T cells cannot be excluded and is worth further study. In support of our findings, a recent report confirmed the beneficial role of IL-1β in correcting age-associated immune defects of DCs and subsequent T-cell–mediated antitumor activity.⁸⁶ In this study, both γδ T-cell and DC transfer exhibited potent antitumor activity; it is intriguing to identify whether the combined transfer of 2 cell types can lead to enhanced therapeutic effects, especially in aggressive tumor models.

In summary, this study identifies the detrimental role of PLA₂G2D-PGD₂/PTGDR signaling in the primary tumor growth and lung metastasis of melanoma. Based on its conservation among species- and age-related increased expression in human and mouse lungs, this signaling represents a potential target in future antitumor medication. Consistent with a recent report showing that PLA₂G2D expressed by macrophages and DCs facilitated immune escape in non-small-cell lung cancer (NSCLC),⁸⁷ we also identified increased PLA₂G2D expression in human NSCLC tumor tissues (Fig. S11), which further supports the potential involvement of PLA₂G2D-PGD₂/PTGDR signaling in the progression of human lung cancer. The expression of PLA₂G2D and PTGDR in nonmalignant lung tissue and specific cell populations (especially rDCs and AMs) deserves further investigation by histological examination or spatial transcriptomics.

Supplementary Material

Supplementary material is available at The Journal of Immunology online.

Data availability

The data underlying this article are available in the article and in its online supplementary material.