Abstract Background Epstein-Barr virus (EBV) is a double-stranded DNA oncogenic virus. Several types of solid tumors, such as nasopharyngeal carcinoma, EBV-associated gastric carcinoma, and lymphoepithelioma-like carcinoma of the lung, have been linked to EBV infection. Currently, several TCR-T-cell therapies for EBV-associated tumors are in clinical trials, but due to the suppressive immune microenvironment of solid tumors, the clinical application of TCR-T-cell therapy for EBV-associated solid tumors is limited. Figuring out the mechanism by which EBV participates in the formation of the tumor immunosuppressive microenvironment will help T cells or TCR-T cells break through the limitation and exert stronger antitumor potential. Methods Flow cytometry was used for analyzing macrophage differentiation phenotypes induced by EBV-infected and EBV-uninfected tumors, as well as the function of T cells co-cultured with these macrophages. Xenograft model in mice was used to explore the effects of M2 macrophages, TCR-T cells, and matrix metalloprotein 9 (MMP9) inhibitors on the growth of EBV-infected tumors. Results EBV-positive tumors exhibited an exhaustion profile of T cells, despite the presence of a large T-cell infiltration. EBV-infected tumors recruited a large number of mononuclear macrophages with CCL5 and induced CD163+M2 macrophages polarization through the secretion of CSF1 and the promotion of autocrine IL10 production by mononuclear macrophages. Massive secretion of MMP9 by this group of CD163+M2 macrophages induced by EBV infection was an important factor contributing to T-cell exhaustion and TCR-T-cell therapy resistance in EBV-positive tumors, and the use of MMP9 inhibitors improved the function of T cells cocultured with M2 macrophages. Finally, the combination of an MMP9 inhibitor with TCR-T cells targeting EBV-positive tumors significantly inhibited the growth of xenografts in mice. Conclusions MMP9 inhibitors improve TCR-T cell function suppressed by EBV-induced M2 macrophages. TCR-T-cell therapy combined with MMP9 inhibitors was an effective therapeutic strategy for EBV-positive solid tumors. Keywords: Head and Neck Neoplasms, Immune Evation, Immunotherapy, Macrophages, Tumor Microenvironment __________________________________________________________________ WHAT IS ALREADY KNOWN ON THIS TOPIC. * Epstein-Barr virus (EBV)-infected tumors are infiltrated with large numbers of lymphocytes, but T cells infiltrating to EBV-infected solid tumors do not exhibit their full potential in killing tumor cells. TCR-T cell therapy in EBV-infected tumors is also hampered by an immunosuppressive microenvironment. WHAT THIS STUDY ADDS * EBV induced CD163+M2 macrophage polarization through the synergistic action of CSF1 and IL10, and this cluster of M2 macrophages secreted a large amount of matrix metalloprotein 9 (MMP9), resulting in resistance to TCR-T cell therapy. Combined use of MMP9 inhibitors and TCR-T cell therapy can effectively inhibit the growth of EBV-infected xenografts. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY * Our study proposes a new and effective therapeutic approach for EBV-infected tumors—the combination of TCR-T-cell therapy and MMP9 inhibitors, and provides a theoretical basis for its clinical application. Background Epstein-Barr virus (EBV), a type of human herpesvirus, possesses a double-stranded DNA genome with an estimated length of 172 KB. EBV predominantly infects B lymphocytes and epithelial cells during its normal life cycle. Since its discovery in a patient with Burkitt’s lymphoma in 1964, EBV has been implicated in the development of a variety of tumors, primarily lymphomas and epithelial malignancies such as nasopharyngeal and gastric cancers (GCs), which may also be associated with the cellular susceptibility to EBV infection.[64]1–3 The carcinogenic mechanism of EBV infection varies in different types of cancer. While it can promote the malignant conversion of B lymphocytes into persistently proliferating lymphoblastoid cells,[65]4 it does not directly induce the malignant transformation of epithelial cells. However, in nasopharyngeal carcinoma, EBV infection promotes metastasis and angiogenesis by modulating the expression of non-coding RNAs.[66]5 6 It is common to observe a large amount of lymphocyte infiltration in nasopharyngeal carcinomas. However, these cells do not appear to fully function as tumor killers and instead seem to play a role in tumor maintenance and immune evasion.[67]3 The exact impact of EBV infection on the formation of an immunosuppressive microenvironment in tumors is still not well defined. T-cell exhaustion is a prevalent phenomenon in cancer. Exhausted T cells exhibit inhibited effector functions, high expression of suppressor molecules (eg, PD-1, LAG-3 and Tim-3) and metabolic and epigenetic alterations.[68]7 Although the development of PD-1/PD-L1 immunotherapy has made it possible to reverse T-cell exhaustion to some extent, its clinical efficacy remains limited.[69]8 T-cell exhaustion is influenced by the tumor microenvironment, with the presence of suppressor molecules on tumor cells and the infiltration of suppressor cells such as Tregs, myeloid-derived suppressor cells (MDSCs), and M2 macrophages within the tumor. Persistent infection also contributes to T-cell exhaustion,[70]7 although there is no direct evidence establishing the relationship between EBV infection and T-cell exhaustion in malignant tumors. The existence of this intricate regulatory network may be responsible for the difficulty in reversing T-cell exhaustion. It is necessary to further investigate the mechanisms underlying T-cell exhaustion in tumors and identify novel drug targets to reverse T-cell exhaustion. Mononuclear macrophages originate from the bone marrow or yolk sac. In the process of tumor formation, mononuclear macrophages are recruited into the tumor via blood circulation to differentiate into macrophages, the most abundant type of immune cells in the tumor microenvironment.[71]9 10 Tumor-associated macrophages (TAMs) are plastic, with anticancer M1 and procancer M2 types coexisting in the tumor immune microenvironment, and they can switch from one state to another in response to different signals.[72]11 Although the specific markers for distinguishing M1 and M2 macrophages are not yet fully defined, some surface markers have been identified for M1 (CD16, CD64, CD86) and M2 (CD163, CD206, CD226) macrophages.[73]12 In progressive tumors, procancer M2 macrophages tend to predominate in the microenvironment and play a detrimental role in tumor progression by promoting tumor invasion and metastasis through the secretion of extracellular matrix-degrading enzymes such as metalloproteinases and serine proteases.[74]13 Additionally, M2 macrophages can also inhibit the tumor-killing abilities of T cells or natural killer (NK) cells by downregulating the expression of tumor antigens.[75]14 M2 macrophages are an important component of the immunosuppressive tumor microenvironment, but the precise mechanisms of the interactions between M2 macrophages and other immune cells, such as T cells, are still not completely understood. Matrix metalloprotein 9 (MMP9) is a zinc-dependent matrix metalloproteinase that can remodel the extracellular matrix by degrading gelatin and collagen. This property makes it an important factor in cancer progression.[76]15 Apart from facilitating tumor metastasis and angiogenesis by degrading the extracellular matrix,[77]16 17 MMP9 can also have a negative impact on immune responses by cleaving the activating receptors on immune cells or antigen-presenting complex molecules on tumor cells.[78]18 Despite the demonstrated involvement of MMP9 in promoting cancer, there are no specific inhibitors of MMP9 that have been approved in clinical trials to date.[79]19 Therefore, it is crucial to gain a deeper understanding of the mechanisms behind MMP9 upregulation in tumors and to explore new combination therapies to address the low effectiveness of MMP9 inhibitors. In this study, we found that EBV-positive tumors recruited large numbers of CD8+T cells and mononuclear macrophages into the tumor by secreting CCL5. Subsequently, in the presence of CSF1 and IL10, mononuclear macrophages were polarized into CD163+M2 macrophages and produced high levels of MMP9, which significantly inhibited the tumor-killing ability of T cells. Altogether, these results revealed the mechanism through which EBV infection induced T-cell exhaustion. Moreover, the combination of MMP9 inhibitors and TCR-T-cell therapy presented a promising novel approach for the treatment of EBV-positive tumors. Methods Cell culture The tumor cell lines HK1, HK1-EBV, AGS and AGS-EBV were obtained from Tong Xiang, Sun Yat-sen University Cancer Center and cultured in RPMI 1640 (GIBCO) supplemented with 10% fetal bovine serum (NEZERUM). C666-1-A11-LMP2A cells and compatible TCR-T cells were provided by TCRCure Biological Technology Company; the former were cultured in DMEM (GIBCO) supplemented with 10% FBS, and the latter were cultured in X-vivo medium (LONZA)+1000 IU/mL IL2 (Beijing Four Rings Bio-Pharmaceutical). CD14+mononuclear macrophages and CD8+T cells were isolated from peripheral blood mononuclear cells (PBMCs) by magnetic bead sorting. Mononuclear macrophages were cultured with RPMI 1640 supplemented with 10% Australian fetal bovine serum (ExCell Bio), and CD8+T cells were cultured with X-vivo supplemented with 1000 IU/mL IL2 similar to TCR-T cells. All cells were cultured in a constant temperature incubator containing 5% CO2 at 37 ℃. Multiplex immunohistochemistry staining We conducted multiplex immunohistochemical staining using a Panovue multiplex fluorescent immunohistochemistry (IHC) kit (Panovue, 0004100100) according to the manufacturer’s guidelines. Briefly, paraffin sections were first dewaxed, hydrated, fixed and subjected to antigen repair and block. Subsequently, the primary antibody for the first marker was applied, followed by a horseradish peroxidase-conjugated secondary antibody. The appropriate tyramide signal amplification was then selected for labeling. This process was then repeated for each marker, and finally, the nuclei were labeled with DAPI. To obtain multispectral images, we used Olympus vs200 and Olympus UPLXAPO 20× objective lenses for whole-slide fluorescence image scanning. The whole-slide multispectral images were analyzed with Qupath software. The following primary antibodies were used: CD8 (CST, 1:400), Granzyme B (CST, 1:200), CD163 (CST, 1:400), and Pan-CK (CST, 1:400). PBMC isolation and magnetic absorption cell sorting PBMCs were isolated using density gradient centrifugation with Ficoll (TBD, LTS1077-1). Briefly, anticoagulated peripheral blood from healthy donors was diluted three times with saline after plasma removal and subsequently added to centrifuge tubes containing Ficoll for density gradient centrifugation at 800×g for 25 min. Then, the intermediate layer containing PBMCs was collected and washed with PBS for subsequent experiments. Specific immune cell populations were isolated from PBMCs using Miltenyi magnetic absorption cell sorting (MACS). PBMCs were resuspended in 80 µL auto MACS Running Buffer (Miltenyi, 130-091-221-1) at a concentration of 1×10^7 cells. Then, a 20 µL of magnetic beads was added and incubated for 15 min at 4ºC in the dark. The cells were then diluted with MACS buffer and added to a separation column (Miltenyi, 130-042-401) placed in a strong magnetic field. The labeled cells were adsorbed in the separation column and washed off with buffer after removing the strong magnetic field to obtain a specific immune cell population with a purity of over 90%. CD14 magnetic beads (Miltenyi, 130-050-201) were used to sort CD14+mononuclear macrophages, and CD8 magnetic beads (Miltenyi, 130-045-201) were used to sort CD8+T cells. Chemotaxis assay We used 3 µm Transwell chambers (Corning, 353096, JET Biofil, TCS012024) to evaluate the migratory capacity of CD14+mononuclear macrophages and CD8+T cells. For chemotaxis experiments using tumor supernatant, 1×10^6 CD14+mononuclear macrophages or CD8+T cells were resuspended in 200 µL serum-free medium and then added to the upper chamber. The lower chamber contained 500 µL of tumor supernatant. The plates were then incubated at 37 ℃ with 5% CO2 for 4 hours. Afterward, the upper chamber was carefully removed, and 1–3 randomly selected fields of the lower chamber were photographed with a phase contrast microscope to visualize the cells that had migrated to the lower chamber. For chemotaxis experiments involving chemokines, mononuclear macrophages or T cells were treated with tumor supernatant overnight, centrifuged, resuspended in 200 µL of serum-free medium and added to the upper chamber. The lower chamber contained 200 ng/mL CCL5 (PeproTech, 300-06-20), 200 ng/mL CXCL10 (PeproTech, 300-12-5) or 500 µL of serum-free medium. After 4 hours of chemotaxis in a 37°C incubator with 5% CO2, cells that had migrated to the lower chamber were photographed. For chemotaxis experiments involving CCR5-blocking, cells were first treated with 200 nM of CCR5 antibody (ab65850) overnight to block CCR5, whereas controls treated without CCR5 antibody. Both types of cells were centrifuged, resuspended in serum-free medium, and then chemotaxis with 200 ng/mL of CCL5, serum-free medium, or tumor supernatant. Immunohistochemical staining Paraffin sections of pathological tissues from patients with nasopharyngeal or gastric cancer were first dewaxed and hydrated and then subjected to antigen repair under high temperature and pressure conditions. Afterward, endogenous peroxidase was blocked by treatment with 3% H[2]O[2]. The sections were blocked with sheep serum for 1 hour (ZS bio.co., ZLI-9056) and incubated with primary antibody for 1 hour and secondary antibody (Dako, GK500710) for half an hour at 37 ℃. After the antibody-binding site was labeled with DAB (ZS bio.co., ZLI-9017), the nuclei were stained with hematoxylin (HUAYUN, HYR800). The following primary antibodies were used: CCL5 (Abcam, ab52562, 1:1000), CXCL10 (Abcam, ab214668, 1:1000), CD163 (Abcam, ab182422, 1:200), CD3 (ZS, ZA-0503-6.0), Granzyme B (CST, 46 890S, 1:200), and MMP9 (Abcam, ab76003, 1:1000). ELISA We used a CCL5 ELISA kit (Proteintech, KE00093), CXCL10 ELISA kit (Neobioscience, EHC157.96), CSF1 ELISA kit (Neobioscience, EHC028.96), IL10 ELISA kit (Neobioscience, EHC157.96) and MMP9 ELISA kit (Neobioscience, EHC115.96) to measure the levels of CCL5, CXCL10, CSF1, IL10 and MMP9 in the cell supernatant according to the manufacturer’s protocol. Flow cytometry For surface marker staining, CD14+mononuclear macrophages, THP-1 cells or CD8+T cells were resuspended in PBS (Bio-Channel, BC-BPBS-01) at a concentration of 1×10^6/100 µL. Fixable Viability Stain 780 (BD, 564996) was then added at a 1:1000 dilution and incubated for 10 min. Afterward, APC-CD163 (Biolegend, 333610), PE-CD206 (Biolegend, 321106), PE-Tim3 (BD, 563422) or BV421-LAG3 (BD, 565720) was added to the cell suspension at a 1:100 dilution and incubated for 25 min at room temperature. The cells were then centrifuged, resuspended in 300 µL of PBS and analyzed by flow cytometry. For intracellular marker staining, CD8+T cells or TCR-T cells were first activated with Cell Activation Cocktail (Biolegend, 423304) for 5 hours. The cells were then resuspended in PBS and stained with Fixable Viability Stain 780 for 10 min, followed by PE/Cyanine7-CD8 (Biolegend, 100722) or PE/Cyanine7-CD3 (Biolegend, 300316) staining at a 1:100 dilution for 20 min. After washing with PBS, the Fixation/Permeabilization Kit (BD, 554714) was then used for cell permeabilization, fixation, and intracellular molecule staining. Briefly, cells were treated with Cytofix/Cytoperm solution for 25 min to fix and permeabilize the cells, followed by washing and resuspension in Perm/Wash solution. APC-IFN γ (Biolegend, 502512), PE-TNF α (Biolegend, 502909), FITC-perforin (Biolegend, 308104) and BV421-Granzyme B (BD, 563389) were added at a 1:100 dilution for intracellular staining and incubated for 25 min. Finally, the cells were resuspended in 300 µL of PBS and analyzed by flow cytometry. CD14+mononuclear macrophage differentiation CD14+mononuclear macrophages isolated from PBMCs by MACS were resuspended in culture medium (CM) at a concentration of 2×10^6 cells/mL. Subsequently, a 500 µL of the cell suspension was added to each well of a 24-well plate. To induce differentiation of the CD14+mononuclear macrophages or THP-1 cells, EBV−tumor/EBV+tumor cell supernatant was added at a volume of 500 µL/well and incubated for 3 days. Alternatively, for differentiation induction with cytokines, 50 ng/mL CSF1 (PeproTech, 300-25-10), 80 ng/mL IL10 (PeproTech, 200-10-10) or both were added to 24-well plates and incubated for 3 days. To block differentiation, 200 nM pexidartinib (CSF1Rin) (Selleck, S7818), 5 µg/mL anti-IL10R antibody (Biolegend, 308817) or both were added simultaneously with the induction of EBV+tumor cell supernatant for 3 days. Coculture experiments and treatments CD14+mononuclear macrophages were treated with tumor supernatant or cytokines for 3 days to induce CD163+M2 macrophages. Afterward, the cells were centrifuged, and the medium was replaced. Subsequently, CD163+M2 macrophages or mononuclear macrophages without polarization were added to 24-well plates at a density of 1×10^6 cells per well. After that, 1×10^6/well CD8+T cells or TCR-T cells were added to the same 24-well plate and cocultured with CD163+M2 macrophages or mononuclear macrophages without polarization for another 3 days with or without 20 µM MMP9 inhibitor (MCE, HY-135232). Then, the cells were either analyzed using flow cytometry or used directly for killing assays or apoptosis assays after replacing with fresh CM. Additionally, pure TCR-T cells (>95% purity) were isolated using the MACS technique after coculture and subsequently employed in the killing experiments or apoptosis assays. RNA sequencing and analysis CD14+mononuclear macrophages were treated with TRIzol after induction of differentiation with tumor supernatant or cytokines. The extracted RNA samples were then sent to Gene Denovo for purification, sequencing and analysis. Differential gene expression between the two groups was analyzed using DESeq2 software, followed by Gene Ontology (GO) pathway enrichment analysis. Additionally, gene set enrichment analysis (GSEA) was performed using software such as GSEA and MSigDB. Lactate dehydrogenase release C666-1-A11-LMP2A cells were seeded in 96-well plates overnight at a concentration of 1×10^4 cells per well and allowed to adhere to the well surface. Afterward, 5×10^4 TCR-T cells, 5×10^4 mock-T cells, 5×10^4 mononuclear macrophages+5×10^4 TCR-T cells or 5×10^4 CD163+M2 macrophages+5×10^4 TCR-T cells were added to each well to kill the tumor cells for 16 hours. Subsequently, the culture supernatant was collected. Lactate dehydrogenase (LDH) is originally present in the cytoplasm and can be released into the CM on cell death.[80]20 To assess the level of cytotoxicity, we used a CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, G1780) according to the manufacturer’s protocol to measure the LDH level in the collected supernatant. Apoptosis assay C666-1-A11-LMP2A cells (2×10^5/well) were added to 24-well plates overnight for adherence. Subsequently, 1×10^6 TCR-T cells or 1×10^6 mononuclear macrophages/CD163+M2 macrophages+1×10^6 TCR-T cells were added to induce tumor cell apoptosis for 16 hours. Afterward, the cells were collected and labeled with an Annexin V-AF647/PI apoptosis kit (ESscience, AP006), and then the apoptosis rate of C666-1-A11-LMP2A cells was determined by flow cytometry. Animal studies Five-week-old female NCG mice were obtained from Gempharmatech Company. A total of 5×10^6 C666-1-A11-LMP2A cells were subcutaneously inoculated in the right flank of the mice. The injection of immune cells via the tail vein was started in the second week after subcutaneous inoculation of the tumor cells, when the tumor maximum diameter reached 5 mm. CD14+mononuclear macrophages were induced to differentiate into CD163+M2 macrophages (>90% purity) by treatment with CSF1 (50 ng/mL)+IL10 (80 ng/mL). A total of 1×10^7 CD163+M2 macrophages were then injected into the tail vein of mice once a week. Additionally, 1×10^7 TCR-T cells, prepared by TCRCure Biological Technology Company, were injected either alone or together with CD163+M2 cells via the tail vein. For mice treated with TCR-T cells, 10,000 IU IL2 was intraperitoneally injected every other day to maintain TCR-T-cell activity. Furthermore, MMP9 inhibitors (MCE, HY-135232) were intraperitoneally administered at a dose of 20 mg/kg twice a week from the time of immune cell injection. The length (L) and width (W) of the tumors were measured every 2 days with Vernier calipers, and the tumor volume was calculated based on the following formula: tumor volume=(L×W^2)/2. The xenograft tumors were harvested on day 26 for tumor weight evaluation, IHC staining, and other analyses. Statistical analysis GraphPad Prism V.8 was used for statistical analysis. The statistical methods used are described in the figure legends. Significance was determined as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Student’s t-test was used to make comparisons between two groups. One-way analysis of variance (ANOVA) was employed to assess differences between multiple groups while two-way ANOVA was used to compare tumor growth curves. Pearson’s correlation coefficient was used to assess the correlations between CD163+cells and Granzyme B/CD8. Results A higher abundance of exhausted CD8+T cells in EBV-positive tumors than EBV-negative tumors is associated with an increased population of CD163+ M2 macrophages EBV infection is a risk factor for the progression of several malignancies.[81]3 T-cell exhaustion is a state manifested by a decrease in T-cell effector function, accompanied by a possible upregulation of the expression of suppressor molecules, which is an important mechanism of immune evasion in tumors.[82]7 To investigate whether there is a potential link between EBV infection and T-cell exhaustion, we collected paraffin-embedded tumor sections from three types of EBV-associated tumor patients: nasopharyngeal cancer (NPC), gastric cancer (GC), and lymphoepithelioma-like carcinoma of the lung (LELC), who were initially diagnosed at the Sun Yat-sen University Cancer Center and underwent surgical procedures to obtain gross specimens. First, we performed EBER in situ hybridization to determine whether the tumors were infected by EBV. EBER-positive ones were defined as EBV-positive tumors, and vice versa as EBV-negative, and ultimately, three pairs of roughly matched EBV± tumor paraffin sections for each type of carcinoma were used to perform multiplex immunohistochemical staining. Interestingly, in all three tumor types, we observed that EBV-positive tumors recruited more CD8+T cells than EBV-negative tumors ([83]figure 1A,B). However, the expression of Granzyme B, a major effector molecule of intratumoral CD8+T cells, did not show a significant upregulation in the EBV-positive tumors except in the LELC sections ([84]figure 1A,C). We further investigated the ratio of Granzyme B/CD8 and found that this ratio was significantly lower in EBV-positive tumors ([85]figure 1D), indicating a loss of T-cell effector function and a higher state of exhaustion. Many factors contribute to T-cell exhaustion, and an increase in suppressor immune cells is one of them. Previous studies have suggested that EBV can activate ATR and subsequently promote M2 polarization.[86]21 Although there is a lack of specific markers that distinguish between M1 and M2 macrophages, CD163 is a widely used surface marker for M2 macrophages.[87]12 Accordingly, we included the marker CD163 for M2 macrophages in our multiplex IHC staining and found a significant increase in the number of suppressor immune cells (CD163+M2 macrophages) in EBV-positive tumors ([88]figure 1E,F). Furthermore, we observed a negative correlation between the number of CD163+M2 macrophages and the Granzyme B/CD8 ratio ([89]figure 1G), suggesting a potential connection between T-cell exhaustion and the increase in CD163+M2 macrophages in EBV-positive tumors. Overall, our findings indicate an elevated numbers of exhausted T cells in EBV-positive tumors, which may be associated with the increased number of CD163+M2 cells. Figure 1. [90]Figure 1 [91]Open in a new tab Increased numbers of exhausted CD8+T cells and CD163+M2 macrophages within EBV-positive tumors. (A) Representative single-stained and merged images of panels (CD8, Granzyme B, DAPI) used for multiplex immunohistochemistry in EBV-positive and EBV-negative tumors. Magnification: ×400 (single-stained images and big merged images), ×200 (small merged images in the bottom right corner). (B–D) Statistical analysis of the density of CD8 (B) or Granzyme B (C) and the ratio of Granzyme B/CD8 (D) in multiplex immunohistochemistry. Mean±SD, n=3, two-tailed t-test. (E) Representative merged images of panels (CD8, Granzyme B, CD163, Pan-CK, DAPI) used in multiplex immunohistochemistry. Magnification: ×400 (small merged images in the top left corner), ×100 (big merged images). (F) Statistical analysis of the density of CD163 in EBV-positive and EBV-negative tumors by multiplex immunohistochemistry. Mean±SD, n=3, two-tailed t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (G) Correlation analysis between CD163/mm^2 and the Granzyme B/CD8 ratio in NPC, GC and LELC. EBV, Epstein-Barr virus; GC, gastric cancer; LELC, lymphoepithelioma-like carcinoma of the lung; NPC, nasopharyngeal cancer. EBV infection in tumor promotes CCL5 secretion to recruit CD8+T cells and mononuclear macrophages The numbers of both CD8+T cells and CD163+M2 macrophages were found to be increased in EBV-positive tumors. To further investigate the underlying mechanism, we conducted immune cell chemotaxis assays. We used two pairs of cell lines, one infected with EBV (HK1-EBV and AGS-EBV) and the other not infected (HK1 and AGS), as previously described.[92]22 We collected the supernatants from both EBV-infected and uninfected tumor cell lines. Then, both EBV-infected and EBV-uninfected tumor supernatants were used to recruit CD8+T cells or CD14+mononuclear macrophages, which were isolated from healthy donor PBMCs using MACS with a purity of over 90% ([93]online supplemental figure 1A). The results showed that the supernatant from EBV-infected tumors had a significantly stronger recruitment ability for both CD8+T cells and CD14+mononuclear macrophages than the supernatant from EBV-uninfected tumors ([94]figure 2A,B). Then, to investigate the chemokines involved in the recruitment of CD8+T cells and CD14+monocytes, we performed RNA sequencing of EBV-infected and uninfected tumor cell lines. Pathway enrichment analysis revealed that the chemokines CCL5 and CXCL10 were upregulated in pathways related to T-cell chemotaxis and mononuclear macrophage chemotaxis ([95]online supplemental figure 1B,C). Immunohistochemical staining and ELISA measurements confirmed that EBV-infected tumors secreted increased levels of CCL5 and CXCL10, consistent with the sequencing results ([96]figure 2C,D). Next, we used 200 ng/mL CCL5, 200 ng/mL CXCL10 or serum-free RPMI 1640 (control) to recruit CD8+T cells and CD14+mononuclear macrophages treated with different EBV-infected tumor supernatants overnight and found that CCL5 had a significantly higher recruitment capacity than CXCL10 or serum-free RPMI 1640 for both cell types ([97]figure 2E,F and [98]online supplemental figure 1D,E), suggesting that EBV-infected tumors may recruit these immune cells by secreting more CCL5. CCL5 interacts with three known receptors: CCR1, CCR3, and CCR5. CCL5 has a stronger affinity for CCR5 than CCR1 and CCR3.[99]23 To verify whether CCL5 secreted by EBV-infected tumors binds predominantly to CCR5 with the highest affinity, we used a 200 nM CCR5 antibody overnight to block CCR5 on CD8+T cells and CD14+mononuclear macrophages. The recruitment of CCL5 to immune cells treated with both EBV-infected tumor supernatant and blockade of CCR5 was markedly attenuated in comparison to cells treated with tumor supernatant only overnight ([100]figure 2G and I and [101]online supplemental figure 1F and H). Similarly, EBV-infected tumor supernatants had a diminished ability to recruit CD8+T cells and CD14+mononuclear macrophages when their CCR5 was blocked ([102]figure 2H and J and [103]online supplemental figure 1G and I). In summary, EBV-infected tumors secrete CCL5, which binds to CCR5 on immune cells and subsequently recruits CD8+T cells and CD14+monocyte macrophages. Figure 2. [104]Figure 2 [105]Open in a new tab EBV-infected tumors secrete CCL5 to recruit T cells and monocytes. (A, B) Representative images (left) and quantification (right) of CD8+T cells (A) or CD14+monocytes (B) recruited to the lower chamber by EBV-infected or EBV-uninfected tumor supernatants. Magnification: ×400. Mean±SD, n=3, two-tailed t-test. (C, D) Left: Representative images of EBV-uninfected or EBV-infected nasopharyngeal and gastric cancer pathology sections stained with antibodies targeting CCL5 (C) or CXCL10 (D). Magnification: ×200. Right: Measurement of CCL5 (C) and CXCL10 (D) levels in the supernatant of EBV-uninfected or EBV-infected nasopharyngeal and gastric cancer cell lines by ELISA. Mean±SD, n=4, two-tailed t-test. (E, F) Quantification of CD8+T cells (E) or CD14+ monocytes (F) recruited to the lower chamber by 200 nM CCL5, 200 nM CXCL10 or serum-free RPMI 1640. CD8+T cells and CD14+monocytes were pretreated overnight with AGS-EBV or HK1-EBV cell supernatants. Mean±SD, n=3, one-way ANOVA. (G, I) Quantification of CD8+T cells (G) and CD14+monocytes (I) recruited to the lower chamber. CD8+T cells and CD14+monocytes were pretreated overnight with AGS-EBV or HK1-EBV cell supernatant, and anti-CCR5 or CCL5+anti-CCR5 groups were simultaneously treated with 200 nM CCR5 antibody overnight to block CCR5. CD8+T cells or CD14+ monocytes were then recruited with 200 nM CCL5 or serum-free RPMI 1640. Mean±SD, n=3, one-way ANOVA. (H, J) CD8+T cells or CD14+ monocytes were treated with or without CCR5 antibody overnight to block CCR5. The above cells were recruited with EBV-infected or EBV-uninfected tumor supernatants. Quantification of CD8+T cells (H) and CD14+ monocytes (J) recruited to the lower chamber. Mean±SD, n=3, one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; EBV, Epstein-Barr virus. Supplementary data [106]jitc-2023-008375supp001.pdf^ (1.2MB, pdf) EBV infection in tumors induces CD163+M2 macrophages polarization by secreting CSF1 and enhancing autocrine IL10 production in mononuclear macrophages EBV-infected tumors recruit large numbers of mononuclear macrophages, but their direction of polarization within the tumor remains unclear, and the mechanisms underlying the increase in CD163+M2 macrophages within EBV-positive tumors still need to be further investigated. We first induced THP-1 cell or CD14+mononuclear macrophage differentiation with EBV-infected or EBV-uninfected tumor supernatant and found that EBV-infected tumor supernatant significantly promoted the polarization of both toward CD163+M2 macrophages than EBV-uninfected tumor supernatant ([107]figure 3A), suggesting that the increase in CD163+M2 macrophages within EBV-positive tumors may be related to the polarization of CD163+M2 macrophages promoted by EBV infection. In the RNA sequencing results of the EBV-infected and EBV-uninfected tumor cell line, CSF1 enrichment scores were the highest in pathways associated with mononuclear macrophage differentiation ([108]online supplemental figure 1C). Further analysis using ELISA revealed elevated levels of CSF1 in EBV-infected tumor supernatants compared with EBV-uninfected tumor supernatants ([109]figure 3B). Monocytes can differentiate into mature macrophages in response to CSF1, but macrophage polarization to CD163+M2 still requires the involvement of other inhibitory molecules such as IL10 or TGF β.[110]24 25 When treated with recombinant CSF1 alone, CD14+monocytes showed a tendency to differentiate more toward CD163+CD206+ M2 cells. However, when treated with IL10 alone or with both CSF1 and IL10, they tended to differentiate toward CD163+CD206- M2 cells, similar to the effect of treatment with EBV-infected tumor supernatant ([111]figure 3C). We thus hypothesized that IL10 plays an important role in the EBV-induced differentiation of CD163+M2 macrophages. Although there was a slight increase in the IL10 level in the EBV+tumor supernatant, the absolute level was relatively low ([112]figure 3D). It has been reported that macrophages themselves can secrete IL10 in addition to tumor cells.[113]26 Therefore, we examined the level of IL10 secreted by mononuclear macrophages treated with EBV-infected or EBV-uninfected tumor supernatant and found that mononuclear macrophages treated with EBV-infected tumor supernatant exhibited higher autocrine production of IL10 ([114]figure 3E). These results revealed that IL10 was more frequently produced by macrophages than tumor cells in EBV-infected tumor. Next, we further explored the role of CSF1 in IL10 autocrine secretion. We found that blocking the receptor of CSF1 impaired the ability of EBV-infected tumor supernatant to promote IL10 autocrine secretion while treatment of macrophages with CSF1 alone increased IL10 secretion ([115]figure 3F), suggesting that CSF1 was involved in the promotion of EBV-induced IL10 autocrine secretion by mononuclear macrophages. Next, to further confirm these findings, we performed blocking experiments. Blocking the CSF1 receptor reduced the differentiation of CD163+M2 macrophages when CD14+mononuclear macrophages were treated with EBV-infected tumor CM (EBV+CM), while blocking IL10 receptor alone or blocking both the CSF1 and IL10 receptor resulted in minimal differentiation toward CD163+M2 macrophages ([116]figure 3G). These findings indicate that EBV-infected tumors secrete higher levels of CSF1, which in turn promotes IL10 autocrine secretion by mononuclear macrophages recruited into the tumor. The combined effect of CSF1 and IL10 then drives the differentiation of monocytes toward CD163+M2 macrophages. Figure 3. [117]Figure 3 [118]Open in a new tab EBV-infected tumors promote CD163+M2 macrophages polarization. (A) Flow cytometry analysis of the CD163+M2 phenotype in THP-1 or CD14+ monocytes treated with EBV-infected or EBV-uninfected tumor supernatants. Mean±SD, n=4 (HK1), n=3 (AGS), two-tailed t-test. (B) Level of CSF1 in EBV-infected and EBV-uninfected tumor supernatants as measured by ELISA. Mean±SD, n=4, two-tailed t-test. (C) Flow cytometry analysis of the CD163+M2 phenotype of CD14+monocytes treated with 50 ng/mL CSF1, 80 ng/mL IL10, or both together. Mean±SD, n=3, one-way ANOVA. (D) Level of IL10 in EBV-infected and EBV-uninfected tumor supernatants as measured by ELISA. Mean±SD, n=4, two-tailed t-test. (E) Levels of IL10 in supernatants of mononuclear macrophages treated with EBV-infected and EBV-uninfected tumor supernatants for 3 days as measured by ELISA. Mean±SD, n=3, two-tailed t-test. (F) Levels of IL10 in mononuclear macrophage supernatants treated for 3 days with EBV-infected tumor supernatant, EBV-infected tumor supernatant and CSF1R inhibitor, 50 ng/mL CSF1, or no treatment as measured by ELISA. Mean±SD, n=4, two-tailed t-test. (G) Flow cytometry analysis of the CD163+M2 phenotype of CD14+monocytes treated with EBV-infected tumor supernatant (control), EBV-infected tumor supernatant and CSF1R inhibitor, EBV-infected tumor supernatant and anti-IL10R antibody, or EBV-infected tumor supernatant and CSF1R inhibitor and anti-IL10R antibody. Mean±SD, n=3, one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; EBV, Epstein-Barr virus. EBV-induced CD163+M2 polarization contributes to T-cell exhaustion To further investigate whether there is a direct correlation between increased CD163+M2 macrophage numbers and T-cell exhaustion in EBV-positive tumors, we first conducted RNA sequencing on mononuclear macrophages induced by EBV-infected or EBV-uninfected tumor supernatant. We identified a total of 94 upregulated genes and 327 downregulated genes in mononuclear macrophages treated with EBV-infected tumor supernatant compared with those treated with EBV-uninfected tumor supernatant ([119]figure 4A). GO pathway enrichment of these differentially expressed genes revealed several pathways associated with T-cell activation in the top 20 pathways ([120]figure 4B). Next, GSEA also suggested that mononuclear macrophages induced by EBV-infected tumor supernatants downregulated pathways associated with lymphocyte activation or T-cell activation ([121]figure 4C,D). We examined the expression of inhibitory molecules on mononuclear macrophages induced by EBV-infected or EBV-uninfected tumor supernatant and found that EBV-infected tumor supernatant upregulated the expression of TGFB1, Galectin9, Galectin3 and LSECtin ([122]online supplemental figure 2A) on mononuclear macrophage. Subsequently, we cocultured mononuclear macrophages induced by EBV-infected or EBV-uninfected tumor supernatant with CD8+T cells and found that mononuclear macrophages induced by EBV-infected tumor supernatant significantly inhibited T-cell function ([123]figure 4E) and upregulated the expression of inhibitory receptors (Tim3 and LAG3) on the surface of T cells ([124]online supplemental figure 2B), leading to the manifestation of T-cell exhaustion characteristics. These evidences suggested a direct link between macrophage polarization induced by EBV infection and T-cell exhaustion, but whether it was EBV-induced CD163+M2 macrophages that led to T-cell exhaustion still needed to be further explored. We then generated CD163+M2 macrophages (>90% purity) by treated CD14+monocytes with CSF1 and IL10 and then cocultured them with CD8+T cells, and found that these CD8+T cells similarly exhibited reduced function compared with those co-cultured with untreated monocyte macrophages (control) ([125]figure 4F,G). In contrast, reduced CD163+M2 macrophage differentiation by blocking either CSF1 or IL10 receptors alone or together during the treatment of mononuclear macrophages with EBV-infected tumor CM (EBV+CM) improved T-cell function ([126]figure 4H,I). These results confirmed that EBV-induced generation of CD163+M2 macrophages contributed to T-cell exhaustion. Overall, we find that there is a direct link between T-cell exhaustion and the induction of CD163+M2 macrophage polarization by EBV infection via CSF1 and IL10 within EBV-positive tumors. Figure 4. [127]Figure 4 [128]Open in a new tab CD163+M2 macrophages polarized by EBV suppresses T-cell function. (A) Volcano plot of differentially expressed genes between mononuclear macrophages induced with EBV-uninfected tumor supernatant and those induced with EBV-infected tumor supernatant for 3 days. (B) GO pathway enrichment analysis of differentially expressed genes between mononuclear macrophages treated with EBV-infected and EBV-uninfected tumor supernatant. (C, D) GSEA of differentially expressed genes between mononuclear macrophages treated with EBV-infected and EBV-uninfected tumor supernatant. (E) Flow cytometry analysis of T-cell functional molecules (Granzyme B, IFN γ, TNF α, Perforin) after 3 days of coculture with mononuclear macrophages polarized with EBV-uninfected or EBV-infected tumor supernatants. Mean±SD, n=3, two-tailed t-test. (F) Flow cytometry analysis of T-cell functional molecules after 3 days of coculture with mononuclear macrophages untreated (control) or polarized with 50 ng/mL CSF1+80 ng/mL IL10 (M2). (G) Statistical analysis of T-cell functional molecules in (F). Mean±SD, n=3, two-tailed t-test. (H) Flow cytometry analysis of T-cell functional molecules after 3 days of coculture with mononuclear macrophages induced by EBV-infected tumor culture medium (control), EBV-infected tumor culture medium and CSF1R inhibitor (CSF1Rin), EBV-infected tumor culture medium and anti-IL10R antibody (anti-IL10) or EBV-infected tumor culture medium and CSF1R inhibitor and anti-IL10R antibody (CSF1Rin+anti-IL10). (I) Statistical analysis of T-cell functional molecules in (H). Mean±SD, n=3, one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; EBV, Epstein-Barr virus; GO, Gene Ontology; GSEA, gene set enrichment analysis. CD163+M2 macrophages impair the cytotoxic potential of TCR-T cells in vitro and in vivo To research the effect of CD163+M2 polarized by EBV-infected tumors on the cytotoxicity of T cells, we constructed C666-1-A11-LPM2A cells as well as TCR-T cells that have TCRs (AU011) capable of directly recognizing and targeting C666-1-A11-LPM2A cell antigen (A11-LMP2A) ([129]online supplemental figure 3A). When cocultured with C666-1-A11-LMP2A cells, TCR-T cells secreted more effector molecules, such as perforin, TNF-α, IFN-γ and granzyme B, than mock-T cells ([130]online supplemental figure 3B). Furthermore, TCR-T cells effectively killed C666-1-A11-LPM2A tumor cells, leading to a higher release of LDH than mock-T cells ([131]online supplemental figure 3C). These results confirmed that our TCR-T cells can directly recognize, target and kill C666-1-A11-LPM2A cells. However, in the presence of CD163+M2 macrophages, the release of effector molecules by TCR-T cells was significantly reduced ([132]figure 5A). Isolation of TCR-T cells cocultured with CD163+M2 cells using MACS revealed their decreased cytotoxic capacity against C666-1-A11-LMP2A cells by LDH release and apoptosis assay ([133]figure 5B,C). Similar results were observed when TCR-T cells were not isolated ([134]online supplemental figure 3D,E). We then subcutaneously implanted NCG mice with C666-1-A11-LPM2A cells and injected CD163+M2 macrophages and TCR-T cells through the tail vein on day 13 and day 21 separately or together to investigate the effect of M2 macrophages on the cytotoxicity of TCR-T cells in vivo ([135]figure 5D). Compared with the control group (without treatment), the treatment group injected with TCR-T cells alone or M2+TCR T cells exhibited decreased tumor volume and weight (although the difference between the M2+TCR T and control groups was not statistically significant) while M2 macrophages injection alone had little effect on tumor growth. However, tumor growth was accelerated in the M2+TCR T group compared with the TCR-T group, indicating that M2 macrophages inhibited the tumor-killing capability of TCR-T cells in vivo ([136]figure 5E and [137]online supplemental figure 4A–D). Hematoxylin-eosin (HE) and immunohistochemical staining of the tumors revealed infiltration of injected M2 macrophages and TCR-T cells into the tumor, but coinjection of M2 with TCR-T cells resulted in a significant reduction in the functional molecule granzyme B compared with TCR-T cells alone. Interestingly, although the difference of the number of CD163+cells between M2 and M2+TCR T was not statistically significant, injection of TCR-T cells appeared to promote infiltration of CD163+M2 macrophages, which we speculated that apoptotic tumor cells resulting from TCR-T cell activity released certain chemokines that further recruited CD163+M2 macrophages ([138]figure 5F). In conclusion, CD163+M2 macrophages inhibited the targeted killing of C666-1-A11-LMP2A cells by TCR-T cells both in vitro and in vivo. Figure 5. [139]Figure 5 [140]Open in a new tab EBV-polarized CD163+M2 macrophages inhibit the cytotoxic capacity of TCR-T cells against tumors. (A) Flow cytometry analysis of functional molecules in TCR-T cells cocultured with CSF1+IL10-polarized M2 (M2) or untreated mononuclear macrophages (control) for 3 days. Mean±SD, n=4, two-tailed t-test. (B) Left: Images of C666-1-A11-LMP2A cells killed by TCR-T cells after coculture with untreated mononuclear macrophages (control treated TCR-T) or CSF1+IL10-polarized M2 macrophages (M2 treated TCR-T) for 3 days and then isolated via MACS at 0 hour and 16 hours. Magnification: ×100. Right: Statistical analysis of LDH released from C666-1-A11-LMP2A cells after 16 hours of killing by TCR-T cells. Mean±SD, n=3, two-tailed t-test. (C) Apoptosis rate analysis of C666-1-A11-LMP2A cells killed by TCR-T cells with different treatment for 16 hours by flow cytometry. Mean±SD, n=6, two-tailed t-test. (D) Experimental scheme diagram for the subcutaneous xenograft tumor model in NCG mice. (E) Tumor volume (left) and tumor weight (right) at day 26 after implantation. Mean±SD, n=7 (M2, M2+TCR T), n=8 (TCR-T, control), two-tailed t-test. (F) Left: Serial sections of mouse xenografts were stained with HE and antibodies targeting CD163, CD3, and Granzyme B. Magnification: ×200. Right: Statistical analysis of the number of CD163+cells, CD3+cells, and Granzyme B. Mean±SD, n=3, two-tailed t-test or Mann-Whitney U test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. EBV, Epstein-Barr virus; LDH, lactate dehydrogenase; HE, hematoxylin-eosin. CD163+M2 macrophages induced by EBV infection secrete MMP9, leading to T-cell exhaustion To investigate the mechanism through which EBV-induced CD163+M2 macrophages promote T-cell exhaustion, we performed spatial transcriptome analysis on EBV-positive and EBV-negative tumor tissue sections ([141]online supplemental figure 5A). We observed a significant upregulation of MMP9 expression in CD163+M2 macrophages within EBV-positive tumors compared with CD163+M2 macrophages within EBV-negative tumors ([142]online supplemental figure 5B). We first measured MMP9 levels in mononuclear macrophages treated with EBV-infected or EBV-uninfected tumor supernatant, as well as CSF1+IL10 or untreated (control), and in tumor supernatant by ELISA. The results showed that macrophages induced by EBV-infected tumor supernatant or CSF1+IL10 secreted higher levels of MMP9 than those induced by EBV-uninfected tumor supernatant or untreated, whereas the tumor supernatant itself only contained low levels of MMP9 ([143]figure 6A). Immunohistochemical staining was performed on both EBV-positive and negative gastric and NPC tissues, and the expression level of MMP9 was found to be significantly higher in EBV-positive tumors ([144]figure 6B). To confirm whether MMP9 secreted by macrophages is a factor contributing to T-cell exhaustion, we introduced an MMP9 inhibitor during the coculture of TCR-T cells with macrophages induced by CSF1+IL10 or tumor supernatant (without C666-1-A11-LMP2A). The results demonstrated that the inhibition of TCR-T-cell function by either EBV-infected tumor supernatant-induced or CSF1+IL10-induced CD163+M2 macrophages was reduced on the addition of an MMP9 inhibitor. However, MMP9 inhibitor did not improve the function of TCR-T cells co-cultured with untreated monocyte macrophages (control) or macrophages induced by EBV-uninfected tumor supernatants ([145]figure 6C and [146]online supplemental figure 5C). These results indicated that MMP9 plays a crucial role in the exhaustion of T cells caused by CD163+M2 macrophages induced by EBV-infected tumor. Next, TCR-T cells were first co-cultured with M2 macrophages, M2 macrophages+MMP9 inhibitor, untreated monocytes or untreated monocytes+MMP9 inhibitor for 3 days, after which they were isolated by MACS or not isolated but used as a co-culture system for subsequent killing experiments in vitro. The results showed that the tumor killing ability of M2+MMP9 inhibitor-treated TCR-T cells was close to that of control+MMP9 inhibitor-treated TCR-T cells, slightly lower than that of control-treated TCR-T cells, but significantly higher than that of M2-treated TCR-T cells, as indicated by the increased release of LDH from tumor cells and the increased apoptosis rate of tumor cells ([147]figure 6D–F), demonstrating that the MMP9 inhibitor could restore the killing function of M2-inhibited TCR-T cells to the level of lacking CD163+M2 macrophages. Similar results were presented when killing experiments were performed using the co-culture system ([148]online supplemental figure 6A–C). Collectively, MMP9 secreted by CD163+M2 macrophages was an important factor contributing to the diminished function and killing capacity of TCR-T cells. Figure 6. [149]Figure 6 [150]Open in a new tab MMP9 secreted by EBV-induced CD163+M2 macrophages suppresses TCR-T-cell function in vitro. (A) Levels of MMP9 in the supernatants of mononuclear macrophages treated with different conditions (left) as well as in EBV-infected or EBV-uninfected tumor supernatants (right) measured by ELISA. Mean±SD, n=4, two-tailed t-test. (B) Left: Representative images of EBV-positive or EBV-negative nasopharyngeal and gastric cancer tissues stained with MMP9 antibody. Magnification: ×200. Right: IHC scores of MMP9. Mean±SD, n=5 (GC and NPC+), n=4 (NPC−), two-tailed t-test. (C) Flow cytometry analysis of functional molecules in TCR-T cells after 3 days of coculture with M2 macrophages induced by CSF1+IL10, M2 macrophages and MMP9 inhibitors, untreated monocytes (control), or untreated monocytes and MMP9 inhibitors (without C666-1-A11-LMP2A). Mean±SD, n=4, one-way ANOVA. (D) Images of C666-1-A11-LMP2A cells killed by TCR-T cells at 0 hour and 16 hours which were cocultured with M2 macrophages, M2 macrophages+MMP9 inhibitor, untreated monocytes or untreated monocytes+MMP9 inhibitor for 3 days and then isolated via MACS. Magnification: ×100. (E) Statistical analysis of LDH released from C666-1-A11-LMP2A cells after 16 hours of killing by TCR-T cells with different treatment. Mean±SD, n=5, two-tailed t-test. (F) Apoptosis analysis of C666-1-A11-LMP2A cells killed by TCR-T cells for 16 hours which were cocultured with M2 macrophages, M2 macrophages+MMP9 inhibitor, untreated monocytes or untreated monocytes+MMP9 inhibitor for 3 days and then isolated via MACS Mean±SD, n=4, two-tailed t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; EBV, Epstein-Barr virus; IHC, immunohistochemistry. TCR-T cells and MMP9 inhibitors have a synergistic effect on inhibiting EBV-infected xenograft growth in vivo Knowing that MMP9 inhibitors could improve the function of TCR-T cells that were resisted by CD163+M2 macrophages, we devoted our efforts to exploring whether a synergistic effect existed between MMP9 inhibitors and TCR-T cells in combating EBV-infected tumors. To evaluate this, C666-1-A11-LMP2A cells were subcutaneously implanted in NCG mice. On days 13 and 21, TCR-T cells and M2 macrophages induced with CSF1+IL10 were administered through the tail vein separately or together. To assess the impact of MMP9 inhibitors, some groups of mice were intraperitoneally injected twice a week with 20 mg/kg MMP9 inhibitors, and all mice were sacrificed on day 26 ([151]figure 7A). Our findings revealed a deceleration in tumor growth within the M2+TCR-T+MMP9in group compared with the M2+TCR T group, indicating an antitumor effect of MMP9 inhibitor within the tumor microenvironment where both M2 macrophages and T cells coexist. Additionally, tumor growth was attenuated in the M2+TCR-T+MMP9in group compared with the M2+MMP9 in group, suggesting the antitumor efficacy of the engineered TCR-T cells; however, there was no significant difference in tumor growth between the M2+MMP9in group and the M2 group, the TCR-T+MMP9in group and the TCR-T group, or the MMP9in group and the control group, suggesting that the MMP9 inhibitor exerts its antitumor effect by reinstating the cytotoxic function of TCR-T cells that had been suppressed by M2 macrophages ([152]figure 7B–D). Immunohistochemical staining of xenograft sections showed TCR-T-cell infiltration, but the injection of M2 impeded the secretion of granzyme B, a functional molecule of TCR-T cells, while the application of an MMP9 inhibitor along with M2 injection enhanced the secretion of granzyme B by TCR-T cells. Importantly, the use of an MMP9 inhibitor did not alter the expression of MMP9, suggesting that its improvement of TCR-T killing capacity may be achieved through its catalytic function ([153]figure 7E). EBV-infected tumors are characterized by a significant infiltration of M2 macrophages, which impede the efficacy of TCR-T cell therapy. The application of an MMP9 inhibitor can potentiate the TCR-T killing function that is inhibited by M2 macrophages, suggesting that there is a synergistic effect of MMP9 and TCR-T cells in the treatment of EBV-infected tumors. Overall, these results validated that the combination of TCR-T cells and MMP9 inhibitors is an effective treatment strategy for EBV+tumors in the presence of M2 macrophages. Figure 7. [154]Figure 7 [155]Open in a new tab The combination of TCR-T cells and MMP9 inhibitors synergistically induces the regression of EBV-positive xenograft tumors. (A) Experimental scheme diagram for the subcutaneous xenograft tumor model in NCG mice. (B) Tumor growth curves of subcutaneous xenograft tumors. Mean±SEM, n=3, two-way ANOVA. (C) Images of C666-1-A11-LMP2A xenograft tumors in mice treated with 1×10^7 TCR-T cells (once a week, twice in total, i.v.), 1×10^7 M2 macrophages (once a week, twice in total, i.v.), or an MMP9 inhibitor (20 mg/kg, twice a week, five times in total, i.p.) separately or together or untreated (control). (D) Statistical analysis of C666-1-A11-LMP2A xenograft weight in (C). Mean±SD, n=3, two-tailed t-test.(E) Left: Serial sections of mouse xenografts were stained with HE and antibodies targeting CD3, Granzyme B and MMP9. Magnification: ×200. Right: Statistical analysis of the number of CD3+cells, Granzyme B and IHC score of MMP9. Mean±SD, n=3, one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; IHC, immunohistochemistry; i.v., intravenous; i.p.,intraperitoneal. Discussion EBV is the first oncogenic DNA virus discovered in humans. Persistent EBV infection leads to a decrease in tumor immunogenicity. In nasopharyngeal carcinoma, both EBV-encoded miRNAs and LMP2A have been reported to downregulate the expression of HLA molecules on the tumor cell surface, limiting antigen presentation and allowing tumor cells to evade immune attack.[156]27–29 In addition, EBV infection can also affect tumor lethality of effector T-cell by upregulating PD-L1 expression in nasopharyngeal carcinoma through related genes such as EBER1,[157]6 LMP1,[158]30 and miR-BART6-3p.[159]31 However, due to the complexity of the tumor immune microenvironment, which includes multiple suppressive immune cell types, such as Tregs, MDSCs and M2 macrophages,[160]32 the processes and mechanisms by which EBV infection reshapes the tumor microenvironment are still poorly understood. In this study, we observed an increased number of exhausted T cells within EBV-associated tumors, which was closely associated with an increase in suppressor immune cells, M2 macrophages. Furthermore, EBV infection promoted tumor cells to secrete large amounts of CCL5, attracting T cells while also inducing massive infiltration of mononuclear macrophages that can be polarized to M2 macrophages. These recruited and polarized suppressor immune cells (M2 macrophages) promoted the exhaustion of T cells, enhanced immune evasion and drove tumor progression. Blocking the recruitment of suppressor immune cells by EBV-associated tumors represents a potential therapeutic direction, and our study identified CCL5 as a key molecule that could serve as a target for the treatment of EBV-associated solid tumors. Nevertheless, it is worth noting that CCL5 also plays an important role in T cell recruitment. Its dual role in recruiting both tumor-killing T cells and cancer-promoting monocytes that can polarize into M2 macrophages prevents it from being an ideal target for the treatment of EBV-associated tumors. Interestingly, in our experimental investigations, we observed that blocking the CXCR3 receptor on T cells resulted in reduced T cell recruitment by EBV-infected tumor supernatants, suggesting the involvement of CXCL10-CXCR3 axis in the T cell recruitment by EBV-infected tumors (data not shown). However, the chemotactic effect of CXCL10 alone on T cells treated overnight with EBV-infected tumor supernatants was extremely weak ([161]figure 2E). We speculate that there may be some factor in the EBV-infected tumor supernatant that is necessary for CXCL10 to exert T-cell chemotaxis, whereas CXCL10 alone has a weak chemotactic effect on T cells due to the lack of synergistic factors. Besides, the low concentration of CXCL10 and short chemotaxis time may also contribute to its weak T cell chemotaxis effect. Macrophages are the most abundant immune cells within tumors, and tumor-associated macrophages include both the anticancer M1 phenotype and the procancer M2 phenotype.[162]26 EBV-infected nasopharyngeal carcinoma cells can promote the polarization of tumor-associated macrophages toward an M2 phenotype by secreting ISG15[163]33 or activating ATR.[164]21 Our transcriptome sequencing analysis of EBV-negative and EBV-positive tumor cells identified CSF1 as a key molecule involved in EBV-mediated macrophage polarization. CSF1 not only acted synergistically with IL10 to promote EBV-mediated CD163+M2 macrophages polarization but also played a role in promoting the autocrine secretion of IL10 by macrophages. Interestingly, the ability of monocytes treated with CSF1 alone to secrete IL10 was inferior to that of monocytes treated with the supernatant from EBV-infected tumors, suggesting that there may be other factors present in the supernatant of EBV-infected tumors that promoted the autocrine secretion of IL10 by TAMs, which needed further in-depth investigations. Furthermore, since that fully differentiated mature CD163+M2 macrophages serve as the primary source of autocrine IL10 while monocytes treated with CSF1 alone differentiate into immature CD163+M2 macrophages or an intermediate state, the ability of such cells to autocrine IL10 is somewhat restricted. Consequently, while CSF1 may exert a promotional effect on IL10 autocrine secretion, its impact appears to be constrained. Nonetheless, our study identified, for the first time, a novel mechanism by which EBV-infected tumors promoted TAM differentiation toward CD163+M2 phenotype through the synergistic effect of CSF1 and IL10. This study enriches our understanding of how EBV remodels the tumor microenvironment. Tumor-associated macrophages have recently emerged as potential therapeutic targets in tumor therapy.[165]34 Studies in mice have shown that deletion of CD163+TAMs through genetic engineering promotes T-cell-mediated melanoma regression.[166]35 Our study found that the use of CSF1R inhibitors suppressed CD163+M2 differentiation induced by EBV-infected tumors. When CSF1R inhibitors were used in combination with IL10R antibodies, TAMs almost completely avoided polarization towards CD163+M2 macrophages. Several CSF1R inhibitors or antibodies are currently being studied in clinical trials, but no drugs have been officially approved yet.[167]34 Our study suggests that the combination of CSF1R inhibitors and IL10R antibodies may enhance the effect of targeted clearance of CD163+M2 cells, offering a new approach to address the current challenge of poor clinical efficacy of CSF1R inhibitor. MMP9 is a 92 kDa multidomain protein belonging to the zinc metalloendopeptidase family, which plays a dual role in tumor progression.[168]36 On the one hand, it facilitates cancer metastasis by degrading extracellular matrix components, such as E-cadherin.[169]37 38 Additionally, it suppresses immune effects by proteolyzing receptors associated with the immune system, such as TNFR1.[170]39 On the other hand, studies showed that MMP9-deficient pancreatic neuroendocrine tumors exhibited increased invasiveness.[171]40 In breast cancer, silencing the MMP9 gene with an adenoviral vector promoted neutrophil infiltration and thus inhibited tumor growth.[172]41 We discovered that, in addition to the tumor cells themselves, polarized M2 macrophages in EBV-infected tumors also secreted a substantial amount of MMP9. These M2 cell-derived MMP9 molecules inhibited T-cell function and were closely related to T-cell exhaustion, confirming a pro-carcinogenic role of MMP9 in EBV-infected tumors. Our study highlighted the importance of M2 cells in EBV-infected tumors as a critical source of MMP9. Furthermore, this study demonstrated that the use of MMP9 inhibitors can improve the function of T cells, which was suppressed by M2 macrophages, providing a rationale for the application of MMP9 inhibitors in EBV-infected tumors. TCR-T cells, which are engineered by transferring a TCR gene sequence that recognizes tumor antigens, constitute a group of cells that can selectively eliminate tumor cells.[173]42 43 TCR-T-cell therapy has emerged as a promising approach in the treatment of solid tumors.[174]44 However, the clinical application of TCR-T-cell therapy is hindered by challenges such as the lack of appropriate tumor antigens, the presence of suppressive factors in the tumor microenvironment, and the occurrence of side effects. Hence, identifying suitable tumor antigens for constructing TCRs is crucial for the success of TCR-T-cell therapy.[175]45 Most EBV-infected tumor cells express LMP2A,[176]3 and thus, developing TCR-T cells that specifically recognize LMP2A may be effective in treating EBV-infected tumors. In our study, we generated TCR-T cells that specifically targeted the C666-1-A11-LMP2A cell antigen and conducted both in vitro and in vivo experiments to demonstrate their ability to effectively target and eliminate tumor cells. However, the presence of suppressive immune cells or molecules in the tumor microenvironment, such as M2 macrophages and MMP9, diminished the killing function of TCR-T cells. Although some TCR-T cells targeting LMP1 or LMP2 for the treatment of EBV-infected tumors are in clinical trials, none have been approved for clinical use yet.[177]46 It is worth noting that tumor growth inhibition is the result of the combined effect of multiple factors. Although TCR-T cells can directly target and kill tumor, their cytotoxic potential is weakened by the immunosuppressive microenvironment. Moreover, various protumor elements present within the tumor, such as M2 macrophages that stimulate angiogenesis[178]47 and vasculogenic mimicry,[179]22 contribute to tumor progression through alternative pathways. In addition, apoptotic tumor cells killed by TCR-T cells can release specific chemokines to recruit suppressor immune cells,[180]48 also limiting the antitumor capacity of TCR-T cells. Consequently, the ability to impede tumor growth following TCR-T cell therapy hinges not only on the intrinsic activity and cytotoxicity of TCR-T cells but also on the intricate immune landscape within the tumor microenvironment. Overcoming the limitations imposed by the tumor suppressive microenvironment remains a major challenge in the field of TCR-T-cell therapy.[181]49 At present, researchers have designed TCR-T cells that secrete PD1 antibodies or IL-12 to break through the limitations of the tumor immunosuppressive microenvironment, thus targeting and killing EBV-infected tumors, but its efficacy still needs further verification. In our research, by either clearing M2 macrophages (without M2 injection) or inhibiting MMP9, a suppressor molecule secreted by M2 cells, we significantly enhanced the killing potential of TCR-T cells. Besides, we combined the application of MMP9 inhibitors with TCR-T cells and observed remarkable regression of C666-1-A11-LMP2A xenograft tumors in mice even in the presence of M2 macrophages. This study highlighted the potential of TCR-T-cell combined with MMP9 inhibitor therapy as a means to overcome the tumor suppressive microenvironment, offering a new and effective treatment approach for EBV-infected solid tumors. Our research has discovered that EBV-infected tumors can induce CD163+M2 macrophages polarization through the synergistic effect of CSF1 and IL10. These M2 macrophages secrete a large amount of MMP9, leading to T cell exhaustion. Simultaneous blockade of CSF1R and IL10R prevents the differentiation of TAMs into CD163+M2 phenotype, providing a new approach to target and eliminate M2 macrophages within EBV-infected tumors. Additionally, by combining MMP9 inhibitors with TCR-T cells, we effectively suppress EBV-infected tumor growth in mice, providing preclinical evidence for the application of this therapy in the treatment of EBV-positive tumors and theoretical guidance for the future development of TCR-T or CAR-T cells secreting MMP9 antibody. Acknowledgments