Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Increased intratumoral mast cells foster immune suppression and gastric cancer progression through TNF-α-PD-L1 pathway

Increased intratumoral mast cells foster immune suppression and gastric cancer progression... Background: Mast cells are prominent components of solid tumors and exhibit distinct phenotypes in different tumor microenvironments. However, the nature, regulation, function, and clinical relevance of mast cells in human gastric cancer (GC) are presently unknown. Methods: Flow cytometry analyses were performed to examine level and phenotype of mast cells in samples from 114 patients with GC. Multivariate analysis of prognostic factors for overall survival was performed using the Cox proportional hazards model. Kaplan-Meier plots for patient survival were performed using the log-rank test. Mast cells, T cells and tumor cells were isolated or generated, stimulated and/or cultured for in vitro and in vivo function assays. Results: Patients with GC showed a significantly higher mast cell infiltration in tumors. Mast cell levels increased with tumor progression and independently predicted reduced overall survival. These tumor-infiltrating mast cells accumulated in tumors by CXCL12-CXCR4 chemotaxis. Intratumoral mast cells expressed higher immunosuppressive molecule programmed death-ligand 1 (PD-L1), and mast cells induced by tumors strongly express PD-L1 proteins in both time-dependent and dose-dependent manners. Significant correlations were found between the levels of PD-L1 mast cells and pro-inflammatory cytokine TNF-α in GC tumors, and tumor-derived TNF-α activated NF-κB signaling pathway to induce mast cell expression of PD-L1. The tumor-infiltrating and tumor-conditioned mast cells effectively suppressed normal T-cell immunity through PD-L1 in vitro, and tumor-conditioned mast cells contributed to the suppression of T-cell immunity and the growth of human GC tumors in vivo; the effect could be reversed by blocking PD-L1 on these mast cells. Conclusion: Thus, our results illuminate novel immunosuppressive and protumorigenic roles of mast cells in GC, and also present a novel mechanism in which PD-L1 expressing mast cells link the proinflammatory response to immune tolerance in the GC tumor milieu. Keywords: Gastric cancer, Tumor microenvironment, Mast cells, TNF-α,PD-L1, Immunotherapy * Correspondence: cj_doc@sina.com; yuanzhuang1983@yahoo.com Department of General Surgery and Centre of Minimal Invasive Gastrointestinal Surgery, Southwest Hospital, Third Military Medical University, No.30 Gaotanyan Street, Chongqing 400038, China National Engineering Research Center of Immunological Products, Department of Microbiology and Biochemical Pharmacy, College of Pharmacy, Third Military Medical University, No.30 Gaotanyan Street, Chongqing 400038, China Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 2 of 15 Background peripheral blood were obtained from patients with GC who Gastric cancer (GC), as a severe health problem, has been underwent surgical resection at the Southwest Hospital of the fourth most common malignancies and the second Third Military Medical University. None of the patients leading cause of cancer death worldwide [1]. In some low/ had received chemotherapy or radiation before the sample middle income countries with poor sanitation or high risk was taken. Patients with infectious diseases, autoimmune of helicobacter pylori infection, it has been one of the major diseases or multiple primary cancers were excluded. The causes of cancer death [2, 3]. Despite significant progress clinical stage of tumors was determined according to the made in prevention, diagnose, and therapeutic options in TNM classification system of the International Union recent years [4, 5], many questions remain unanswered, Against Cancer (7th edition). The study was approved by especially the pathogenesis of GC. Nowadays, it is generally the Ethics Committee of the Southwest Hospital of Third believed that the development and prognosis of GC are Military Medical University. Each subject provided written influenced by the cross-talk between tumors and host im- informed consent. Additional file 1: Table S1 lists antibodies mune system [6, 7]. Previous studies have focused on the and other reagents. crucial role for adaptive immunity in determining the clin- ical outcomes of GC patients [8]. However, little is known Isolation of single cells from GC tissues about the role of innate immunity and innate immune cells Fresh tissues were washed 3 times with Hank’ssolution during GC development and progression. containing 1% FCS and cut into small pieces. Specimens Mast cells are a group of innate immune cells, which were collected in RPMI 1640 medium containing collage- have profound immunomodulatory effects on tumor pro- nase IV (1 mg/ml) and deoxyribonuclease I (10 mg/ml) and gression [9, 10], such as angiogenesis [11], tumor micro- mechanically separated using the gentle MACS Dissociator environment reconstruction [12] and interaction with other (Miltenyi Biotec). Dissociated cell suspensions were further immune cells [13]. At present, limited studies on mast cells incubated for 1 h under continuous rotation at 37 °C. The in GC mainly focus on the correlation between the survival cell suspensions were then filtered by a 70 μmcellfilter rate of GC patients and their GC mast cell infiltration by (BD Labware). Cell viability, as measured by trypan blue ex- immunohistochemistry [14], and a few on the relationship clusion staining, was typically > 90%. between infiltrated mast cell density and local angiogenesis [15]. Overall, these studies suggest that mast cells may be a Isolation of mast cells and T cells therapeutic target for GC. However, the phenotype, func- As mentioned above, tumor and non-tumor tissues were tional regulation and clinical correlation of mast cells in hu- treated as single cell suspension. Then the single cell sus- man GC microenvironment remain unclear. pension was stained with anti-human CD45, anti-human Herein, we investigate the interplays among mast cells, CD117 and anti-human FcεRI antibodies, and mast cells T cells and tumor cells in the GC microenvironment. We from autologous tumor and non-tumor tissues were sorted show that mast cells could be recruited to tumor micro- by fluorescence activating cell sorter (FACS) (FACSAria II; environment through CXCL12-CXCR4 chemotaxis axis. BD Biosciences). Density gradient centrifugation was used Moreover, tumor-derived TNF-α efficiently induces pro- to isolate peripheral blood mononuclear cells (PBMCs) grammed death-ligand 1 (PD-L1) expression on mast cells from autologous GC patients and healthy donors by using by activating nuclear factor kappa-light-chain-enhancer of Ficoll-Paque Plus. CD3 T cells from PBMCs were purified activated B cells (NF-κB) signaling pathways. In turn, with CD3 microbeads. The sorted cells were used only these mast cells inhibit the normal function of T cells in a when their viability was determined > 90% and their purity PD-L1-dependent manner, which could suppress antitu- was determined > 95%. mor immunity in GC. Our data suggest a protumorigenic role of mast cells with an immunosuppressive phenotype in GC. These Preparation of TTCS and NTCS and supernatant- tumor-infiltrating mast cells increase with tumor pro- conditioned mast cells gression and are negatively correlated with patient sur- Tumor tissue culture supernatants (TTCS) or non-tumor vival after surgery, suggesting that these mast cells may tissue culture supernatants (NTCS) were prepared by plat- be a novel target in novel GC therapy. ing autologous tumor or non-tumor gastric tissues in 1 ml RPMI 1640 medium for 24 h. The supernatant was then Methods harvested by centrifugation. To generate supernatant-con- Patients and specimens ditioned mast cells, primary human umbilical cord Fresh gastric tumor (homogeneous cellularity, without foci blood-derived cultured mast cells (hCBMCs) or LAD2 of necrosis, including intratumoral and marginal tissues), cells were first harvested and cultured with autologous peritumoral and non-tumor (non-tumor tissues, at least 5 50% TTCS or NTCS for 24 h, and then washed with cm distant from the tumor site) tissues and autologous RPMI-1640 medium for 3 times. Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 3 of 15 Chemotaxis assay as described above, in the presence or absence of a neutral- Fluorescence-activated cell sorter sorted tumor-infil- izing antibody against human PD-L1 (20 μg/ml). After trating mast cells (1 × 10 ) from fresh human tumor 5-day incubation, the supernatants were harvested for tissues were transferred into the upper chambers of ELISA and the cells were harvested for intracellular cyto- 8-μm pore size Transwells (Corning). Autologous 50% kine staining. TTCS or NTCS as the sources of chemoattractants were placed in the lower chambers. After 24-h culture In vivo tumor inhibition assay at 37 °C, migration was quantified by counting cells in All animal experiments were undertaken with the ap- the lower chamber and cells adhering to the bottom of proval from the Animal Ethical and Experimental the membrane. In some cases, blocking antibody for Committee of Third Military Medical University. 10 CXCR4 (20 μg/ml, IgG2b) or control IgG2b was added GC cells (SGC-7901 cells) in 100 μl of buffered saline into mast cell suspensions and incubated for 2 h before were subcutaneously injected into the axillary tissues of chemotaxis assay. Furthermore, CXCL12 neutralizing female nonobese diabetic/severe combined immunodefi- antibody (20 μg/ml, IgG1) or control IgG1 was added ciency (NOD/SCID) mice (5–7 week, one tumor per into TTCS in some assays. RPMI-1640 medium and mouse). The hCBMCs (referred to as mast cells) were chemokine CXCL12 (100 ng/ml) were placed in the stimulated with 50% TTCS for 24 h. Then, 5 × 10 lower chambers as blank and positive controls anti-CD3- and anti-CD28-stimulated (2 μg/ml anti-CD3 respectively. and 1 μg/ml anti-CD28) polyclonal T cells were co-cul- tured with mast cells, or TTCS-conditioned mast cells Mast cell stimulation (TCM) at a 2:1 ratio in the presence or absence of a neu- The hCBMCs, LAD2 cells, HMC-1 cells were stimulated tralizing antibody against human PD-L1 (20 μg/ml) or a with 50% TTCS or 50% autologous NTCS, or 50% TTCS control IgG (20 μg/ml) for 5 days, and were subsequently for 3, 6 or 12 h, or with different concentrations TTCS injected into the peritoneum in 200 μl of buffered saline (20, 40, 80%) for 24 h, or with 50% TTCS together with on day 7 after inoculation. Tumor size was measured a neutralizing antibody against human TNF-α (20 μg/ml) every 2 days by two independent observers using calipers for 24 h, or with human recombinant (hr) cytokines fitted with a vernier scale. Tumor volumes (V) were calcu- (100 ng/ml) for 24 h. After stimulation, the cells were lated with the formula: V = A × B /2 (A = axial diameter; harvested for flow cytometric analysis and western blot. B = rotational diameter). Once the mice were sacrificed, For the signaling pathway inhibition experiments, the tumors were weighed and photographed, and were further cells were pretreated with 5 μl (10 μM) BAY 11–7082 fixed for immunohistochemical staining, ELISA or real- (an IκBα inhibitor), U0126 (a MEK1/2 inhibitor), time PCR, and the spleens were dissociated into single SP600125 (a c-Jun N-terminal kinase (JNK) inhibitor), cells for flow cytometry. SB203580 (a mitogen-activated protein kinase (MAPK) inhibitor) or Wortmannin (a PI3K inhibitor) for 1 h, then were stimulated with 50% TTCS or hr. TNF-α (100 ng/ml) for 24 h and harvested as above. As the in- Statistical analysis hibitors were dissolved in dimethyl sulfoxide (DMSO), Results are expressed as mean ± SEM. Student t test was parallel cell groups were treated with DMSO (5 μl) or generally used to analyze the differences between two culture media as controls. groups, but when the variances differed, the Mann- Whitney U test was used. Correlations between parame- In vitro mast cell-T cell co-culture system ters were assessed using the Pearson correlation analysis In a 5-day incubation, magnetic bead-purified peripheral and linear regression analysis as appropriate. Overall/ + 5 CD3 T cells (2 × 10 cells/well in 96-well plates) were disease-free survival was defined as the interval between labeled with carboxyfluorescein succinimidyl ester surgery and death/recurrence or between surgery and (CFSE) and co-cultured with autologous mast cells iso- the last observation for surviving/disease-free patients. lated from tumor or non-tumor tissues at a 2:1 (T cell: The known tumor-unrelated deaths (eg, accidental mast cell) ratio in 200 μl RPMI-1640 medium containing death) were excluded from the death record for this 10% fatal bovine serum (FBS), rh IL-2 (20 IU/ml), study. Cumulative survival time was calculated by the anti-CD3 (2 μg/ml), and anti-CD28 (1 μg/ml) antibodies, Kaplan-Meier method, and survival was measured in with or without a human PD-L1 neutralizing antibody month; the log-rank test was applied to compare (20 μg/ml). In another co-culture system, CFSE-labeled between 2 groups. Multivariate analysis of prognostic + 5 magnetic bead-purified peripheral CD3 T cells (2 × 10 factors for patient overall survival was performed using cells/well in 96-well plates) were co-cultured with TTCS- the Cox proportional hazards model. SPSS statistical or NTCS-conditioned hCBMCs or LAD2 cells at a 2:1 ratio software (version 13.0) was used for all statistical Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 4 of 15 analysis. All data were analyzed using 2-tailed tests, and these cells in GC were not actively proliferative (Additional P < 0.05 was considered statistically significant. file 7:FigureS2a andb). Next, wehypothesizedthatGC microenvironment might induce mast cell migration into Results the tumors by chemotaxis. We therefore examined the che- Mast cells are enriched in GC as tumor progress and mokine receptors that are involved in myeloid cell migra- independently predict poor patient survival tion. The majority of tumor-infiltrating mast cells expressed To evaluate the potential role of mast cells in human CXCR4 but not CCR2, CCR4, CCR5, CCR7, CXCR1, GC, we analyzed mast cell percentage within the total CXCR2 or CXCR7 (Fig. 2a; Additional file 7:FigureS2c), CD45 leukocytes from intratumoral, marginal, peritu- while, mast cells in peritumoral or non-tumor tissues moral, and non-tumor tissues of GC patients at various showed lower CXCR4 expression (Fig. 2a). In support of stages. Notably, patients with GC showed a higher mast this, dual immunofluorescence staining showed that cell percentage in intratumoral tissues than marginal, tryptase mast cells expressed CXCR4 in GC tumors peritumoral, and non-tumor tissues (Fig. 1a and b). (Fig. 2a). Taken together, these results suggest that mast Moreover, as the cancer progressed, the percentage of cells may be induced to migrate into the tumor micro- intratumoral mast cells increased significantly (Fig. 1a), environment through CXCR4-mediated chemotaxis. and such intratumoral mast cell accumulation was most Interestingly, we further found that the frequency of mast notable from stage II onwards (Fig. 1e). Similar obser- cells was positively correlated with CXCL12 production vations were made when analyzing the total number of (Fig. 2b), which most likely derived from EpCam tumor mast cells per million total cells in each tissue (Fig. 1c cells (Additional file 7: Figure S2d) in the GC microenvir- and f). Furthermore, immunohistochemical staining onment. Meanwhile, we found that the concentrations of also showed that mast cells were accumulated in tu- CXCL12 in tumor tissues or tumor tissue culture superna- mors (Fig. 1d), indicating a potential role for mast cells tants (TTCS) were significantly increased when compared in the GC microenvironment. In keeping with this find- to that in non-tumor tissues or non-tumor tissue culture ing, increased mast cell percentage and mast cell num- supernatants (NTCS) (Fig. 2c). To substantiate the func- ber were correlated with increased advanced lymphatic tional significance of CXCL12-CXCR4 in the recruitment invasion, tumor size and tumor stage (Additional file 2: of mast cells, mast cell chemotaxis assay was performed Figure S1). and showed that TTCS induced significantly more Next, we evaluated the clinical relevance of intratumoral tumor-infiltrating mast cell migrationthanNTCS fromthe mast cells in GC. Comparing patients with high (≥9.315% same GC patients, and this effect was lost upon pre-treat- median level) versus low (< 9.315%) mast cell percentage ment with neutralizing antibodies against CXCL12 and/or level, the 62-month overall survival rates were significantly CXCR4 (Fig. 2d). Taken together, these data support a lower for those within the higher mast cell percentage model wherein GC tumors secrete chemokine CXCL12 group (Fig. 1e). Similar results were obtained when the pa- which in turn recruits mast cells into the tumor micro- tient cohort was stratified based on intratumoral mast cell environment by CXCL12-CXCR4 interaction. number (Fig. 1f). Importantly, this finding that intratu- moral mast cell percentage and/or number independently Tumor-derived factor TNF-α induces mast cells to express predicted survival was verified by univariate and multivari- PD-L1 ate analyses using a Cox proportional hazard model To better understand these intratumoral mast cells’ (Additional file 3: Table S3). The clinical characteristics of likely function, we performed a detailed immune- all the GC patients are described in Additional file 4: Table phenotype. Notably, we found that intratumoral mast S2, and correlations between intratumoral mast cell per- cells expressed significantly higher level of immuno- centage or mast cell number and clinical characteristics of suppressive molecule PD-L1 (Fig. 3a) but not other GC patients are listed in Additional file 5: Table S4 and molecules with immunosuppressive potential such as Additional file 6: Table S5. Taken together, these findings 2B4, glactin-3, CTLA-4, or ICOSL (Additional file 8: suggest that increased intratumoral mast cells are associ- Figure S3a) than that expressed on peritumoral and ated with tumor progression and poor survival of GC non-tumor mast cells, indicating a potential role for patients. PD-L1 on mast cells in the GC microenvironment. Meanwhile, we hypothesized that GC environments Increased mast cell accumulation in GC is promoted by contribute to the immunosuppressive phenotype of CXCL12-CXCR4-mediated chemotaxis mast cells. Consistent with our hypothesis, compared The results described above suggested that GC microenvir- to NTCS-conditioned mast cells, TTCS-conditioned onment triggers mast cell accumulation, so we wondered mast cells significantly up-regulated PD-L1 expression the cause of such accumulation. We first found that intra- in both time-dependent and dose-dependent manners tumoral mast cells expressed little Ki-67, suggesting that (Fig. 3b). Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 5 of 15 Fig. 1 Mast cells accumulate in GC tumors with disease progression and predict poor patient survival. a Mast cell percentage in CD45 cells or (c)the total + + + number of mast cells per million total cells among TNM stages (I + II vs III + IV) in each tissue of patients with GC by gating on CD45 CD117 FcεRI cells or counting. Cumulative results from 114 GC patients were shown. b Dot plots of surface molecule staining for mast cells gating on CD45 cells. d Representative analysis of tryptase (brown) mast cell distributions in tumor tissues of GC patients by immunohistochemical staining. Scale bars: 100 μm. e and f Intratumoral mast cell percentage (e)ormastcellnumber(f) among TNM stages was compared. Kaplan-Meier plots for overall survival by median mast cell percentage (9.315%) (e) or median mast cell number (4749 per million) (f). The horizontal bars in panels a, c, e and f represent mean values. Each ring in panels a, c, e and f represents 1 patient. **, P < 0.01; n.s., P > 0.05 for groups connected by horizontal lines. MC (%), mast cell percentage; MC (NO.), mast cell number Tumor microenvironment can possess various soluble cytokines including IL-1β, IL-6, IL-10, IL-17, IL-22, IL-23, factors [16], including pro-inflammatory cytokines. To see M-CSF, G-CSF, TNF-α,IFN-γ,TGF-β,etc. We found that which cytokines might induce PD-L1 expression on mast only TNF-α remarkably up-regulated the expression of cells, we first screened pro-inflammatory cytokines in PD-L1 on mast cells (Fig. 3d; Additional file 8:Figure S3b). human GC environments by microarray (Fig. 3c), and Next, we found that the concentrations of TNF-α in tumor stimulated normal mast cells with highly-expressed tissues or TTSC were significantly increased when Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 6 of 15 Fig. 2 CXCL12-CXCR4 chemotaxis mediates mast cell migration and accumulation in GC tumors. a Statistics analysis of CXCR4 mast cell percentage in total mast cells in each samples of patients with GC (n = 26). Expression of molecule CXCR4 on mast cells by gating on + + + + + CD45 CD117 FcεRI cells. Color histograms represent staining of CXCR4; black, isotype control. Tumor-infiltrating CXCR4 tryptase mast cells were defined by immunofluorescence staining. Green, Tryptase; red, CXCR4; and blue, DAPI-stained nuclei. Scale bars: 10 μm. b The correlations between mast cells and CXCL12 production in GC tumors were analyzed. Results were expressed as percentage of mast cells in CD45 cells and CXCL12 concentration in tumor tissues of patients with GC. c CXCL12 concentration between autologous tumor and non-tumor tissues (n = 27) or between autologous TTCS and NTCS (n = 8) was analyzed. d Migration of tumor-infiltrating mast cells was assessed by Transwell assay as described in Materials and methods and statistically analyzed (n = 3). The horizontal bars in panels a and c represent mean values. Each ring or dot in panels a, b or c represents 1 patient. *, P < 0.05; **, P < 0.01 for groups connected by horizontal lines compared to that in non-tumor tissues or NTCS (Fig. 3e). Tumor-derived TNF-α activates NF-κB pathway to induce There was clearly a positive correlation between PD-L1 expression on mast cells PD-L1 mast cell infiltration and TNF-α production To further see which signaling pathways might operate (Fig. 3f) and a high expression of TNF-α receptor II in the mast cell PD-L1 induction, we first pre-treated (TNFRII) on mast cells (Additional file 8:FigureS3c) mast cells with corresponding inhibitors and then ex- within tumors. Next, to evaluate the potential role of posed them to the indicated TTCS. The results showed GC tumor-derived TNF-α in PD-L1 induction on mast that only blocking the signal transduction of NF-κB with cells, we added neutralizing antibody against TNF-α inhibitor BAY 11–7082 effectively suppressed PD-L1 ex- into TTCS/mast cell co-culture. Interestingly, antibody pression on TTCS-conditioned mast cells or blockade of TNF-α efficiently inhibited the induction of TNF-α-stimulated mast cells (Fig. 4a; Additional file 9: PD-L1 on mast cells (Fig. 3g). These findings show that Figure S4a). Furthermore, p65, a direct NF-κBpathway tumor-derived TNF-α plays an essential role in mast downstream substrate (Fig. 4b), but not other pathway cell PD-L1 induction. downstream substrates (Additional file 9:FigureS4b), was Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 7 of 15 Fig. 3 Tumor-derived factor TNF-α induces mast cells to express PD-L1. a Statistics analysis of PD-L1 mast cell percentage in total mast cells in + + + each samples of patients with GC (n = 26). Expression of molecule PD-L1 on mast cells by gating on CD45 CD117 FcεRI cells. Color histograms + + represent staining of PD-L1; black, isotype control. Tumor-infiltrating PD-L1 tryptase mast cells were defined by immunofluorescence staining. Green, Tryptase; red, PD-L1; and blue, DAPI-stained nuclei. Scale bars: 50 μm. b Expression of PD-L1 on hCBMCs exposed to 50% autologous TTCS and NTCS for 24 h, or exposed to 50% TTCS for 6, 12, 24 h, or exposed to 20, 40, 80% TTCS for 24 h. black, isotype control. c Clustering of microarray data for the expression of 40 pro-inflammatory cytokine genes in human tumor tissues from 8 GC patients. d Expression of PD-L1 on hCBMCs exposed to TNF-α for 24 h. black, isotype control. e TNF-α concentration between autologous tumor and non-tumor tissues (n = 24) or between autologous TTCS and NTCS (n = 8) was analyzed. f The correlations between TNF-α and PD-L1 mast cells in human tumors were analyzed. Results are expressed as the number of PD-L1 mast cells per million total cells and TNF-α concentration in tumor tissues. g Expression of PD-L1 on hCBMCs exposed to TTCS with anti-TNF-α antibody for 24 h. Each ring or dot in panels a and f represents 1 patient. *, P < 0.05, **, P < 0.01 for groups connected by horizontal lines predominantly more phosphorylated in mast cells after TTCS or TNF-α. Taken together, these data suggest that treatment with TTCS than that treatment with NTCS, and tumor-derived TNF-α activates NF-κB pathway to induce this phosphorylation was abolished when blocking TNF-α, PD-L1expressiononmastcells. implying that activation of NF-κB signaling pathway is cru- cial for mast cell PD-L1 induction by TNF-α in the GC en- Tumor-infiltrating mast cells suppress T cell immunity vironments. Similar results were obtained when analyzing through PD-L1 PD-L1 on mast cell (Fig. 4b). Furthermore, immunofluores- The co-localization of mast cells and T cells (Fig. 5a) in cence staining (Fig. 4c) and western blot (Fig. 4d) showed the tumoral area of GC tissues, and the significant nega- that PD-L1 expression or p65 phosphorylation was abol- tive correlations between the levels of mast cells and T ished when blocking the signal transduction of NF-κBwith cells in human GC tumors analyzed (Fig. 5b) suggest inhibitor BAY 11–7082 on mast cells either stimulated with that these tumor-infiltrating mast cells may promote Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 8 of 15 Fig. 4 Tumor-derived TNF-α activates NF-κB pathway to induce PD-L1 expression on mast cells. a Expression of PD-L1 on hCBMCs exposed to TTCS or TNF-α with or without BAY 11–7082 (an IκBα inhibitor) for 24 h. black, isotype control. b PD-L1, p65 and p-p65 on/in LAD2 cells exposed to autologous TTCS, NTCS, or TTCS with anti-TNF-α antibody for 24 h (detecting for PD-L1) or 3 h (detecting for p65 and p-p65) were analyzed by western blot. c PD-L1 on HMC-1 cells exposed to TTCS with or without BAY 11–7082 (an IκBα inhibitor) for 24 h was analyzed by immunofluorescence staining. Green, PD-L1; blue, DAPI-stained nuclei. Scale bars: 20 μm. d PD-L1, p65 and p-p65 on/in LAD2 cells exposed to TTCS with or without BAY 11–7082 (an IκBα inhibitor) were analyzed by western blot tumor progression by impairing T cell immunity. Mast human umbilical cord blood-derived cultured mast cells cells from tumor and non-tumor tissues of autologous GC (hCBMCs) for 5 days. Consistent with our hypothesis, patients were therefore isolated and cultured with purified TTCS-conditioned hCBMCs showed significantly more autologous peripheral CD3 T cells for 5 days. Mast cell/ suppression of T cell proliferation and IFN-γ production T-cell co-cultures showed that tumor-infiltrating mast (Fig. 5d). To see whether PD-L1 operates in this T cell sup- cells were superior to non-tumor-derived mast cells in pression, we added PD-L1 neutralizing antibodies in the T inhibiting T cell proliferation and IFN-γ production (Fig. cell/TTCS-conditioned hCBMCs co-culture system. As ex- 5c), suggesting an immunosuppressive function of tumor- pected, PD-L1 blocking by antibody efficiently attenuated infiltrating mast cells in tumor immunity. such T cell suppression mediated by TTCS-conditioned Our next objective was to determine the role of PD-L1 hCBMCs (Fig. 5d). Similar observations were made when on tumor-infiltrating mast cells in T cell suppression. using human mast cell line LAD2 cells (Additional file 10: Therefore, we added neutralizing antibodies against PD-L1 Figure S5a). Besides, tumor-associated mast cells could into our tumoral mast cell/peripheral T-cell co-culture sys- modulate cytotoxic function of T cells as perforin and gran- tem. Interestingly, blockade of PD-L1 significantly attenu- zyme B production from the T cells cocultured with ated such T cell suppression mediated by tumor-infiltrating PD-L1-expressing mast cells decreased significantly mast cells (Fig. 5c). Collectively, these findings show that (Additional file 10: Figure S5d and e). These results above PD-L1 contributes to tumor-infiltrating mast cell-mediated indicate that, in the GC microenvironment, mast cells ac- T cell suppression in vitro. quire ability to suppress T cell function through PD-L1. Given the tumor-infiltrating mast cells suppressed T-cell proliferation and IFN-γ production more than non-tumor Blockade of mast cell-associated PD-L1 on T cell mast cells, we hypothesized that the tumor microenviron- immunity inhibits tumor growth and GC progression ment might have played an important role in this process. To test the suppressive effect of PD-L1 mast cells on T To test this, purified peripheral CD3 Tcells were cell immunity in vivo, we treated TTCS-conditioned co-cultured with TTCS- or NTCS-conditioned primary mast cells (TCM) with PD-L1 blocking or control IgG and Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 9 of 15 Fig. 5 Tumor-infiltrating and tumor-conditioned mast cells suppress T cell immunity through PD-L1. a Representative analysis of tryptase mast cell (green) and CD3 T cell (red) interactions in tumor tissues of GC patients by immunofluorescence. Scale bars: 20 μm. b The correlations between mast cells and T cells in human GC tumors were analyzed. Results were expressed as percentage of mast cells and T cells in CD45 cells in tumor tissues of GC patients. c CFSE (carboxyfluorescein succinimidyl amino ester)-labeled peripheral CD3 T cells of patients with GC were co-cultured for 5 days with autologous mast cells from non-tumor or tumor tissues with or without anti-PD-L1 antibody. Representative data and statistical analysis of T cell proliferation and IFN-γ production were shown (n = 5). d CFSE-labeled peripheral CD3 T cells of donors were co-cultured for 5 days with autologous TTCS-, or NTCS-conditioned hCBMCs with or without anti-PD-L1 antibody. Representative data and statistical analysis of T cell proliferation and IFN-γ production were shown (n = 5). Each dot in panel b represents 1 patient. **, P < 0.01 for groups connected by horizontal lines. hCBMCs, human umbilical cord blood-derived cultured mast cells then injected them together with T cells into our estab- measuring time point from day 19 (Fig. 6a and b). lished human NOD/SCID mice bearing SGC-7901-de- Moreover, mice treated with T cells plus PD-L1 block- rived GC. As expected, mice without T cell transfusions, ing antibody-treated TCM, also showed decreased mice treated with T cells plus TCM or control IgG-treated tumor cell proliferation, and increased CD3 T cell TCM, showed tumor growth and disease progression infiltration and IFN-γ production (Fig. 6cand d; (Fig. 6a and b). Consistent with a vital role in assisting Additional file 11: Figure S6a), as well as an increased tumors of PD-L1 mast cells in vivo, mice treated with T production of cytolytic molecules perforin and gran- cells plus PD-L1 blocking antibody-treated TCM showed zyme B (Fig. 6e; Additional file 11:FigureS6b andc), reduced tumor volumes and disease progression at each compared with the mice treated with T cells plus TCM Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 10 of 15 Fig. 6 Blockade of mast cell-associated PD-L1 on T cell immunity inhibits tumor growth and GC progression in vivo. a Mice were injected with human SGC-7901 cells, as described in Materials and methods. The control animals ( ) received no further injections. The experimental treatments entailed injections with T cells ( ) or T cells in combination with TTCS-conditioned mast cells (TCM) ( ), or T cells in combination with TCM pre-treated with an anti-PD-L1 antibody ( ) or a control IgG ( ). The illustrated data represent tumor volumes (5 mice in each group). The day of tumor cell injection was counted as day 0. *P < 0.05, for groups injections with T cells in combination with TCM pre-treated with an anti-PD-L1 antibody ( ), compared with groups injections with T cells in combination with TCM pre-treated with a control IgG ( ). The tumors were excised and photographed 21 day after injecting the tumor cells. b The weights of tumors were compared. c-e Proliferating cell nuclear antigen (PCNA) (brown) expression, CD3 Tcell infiltration (brown) (c), or IFN-γ (d), perforin and granzyme B (e) production in tumors and IFN-γ-producing T cell response (d) in spleens of mice were compared (n = 5). Scale bars: 100 μm. The horizontal bars represent mean values. **, P < 0.01; n.s., P > 0.05 for groups connected by horizontal lines or control IgG-treated TCM. These findings suggest cells in GC [18], the roles of innate immune cells re- that tumor-associated mast cells suppress T cell main less well understood. Mast cells are a group of immunity in vivo depending on PD-L1 and thereby innate immune cells, which have been reported in GC contribute to tumor growth and GC progression. [14, 19], but the phenotype, functional regulation and clinical relevance of mast cells in human GC micro- Discussion environment remain largely unclear. In this study, we Illuminating the roles of host innate and adaptive im- have shown that within GC mast cells could play a mune cells within the tumor milieu is crucial for un- positive role on promoting tumor progression. We derstanding the development and progression of have found that the percentage of mast cells in tumors human tumors [17]. Although previous studies have was significantly increased at advanced stages of GC, been delineating the functions of adaptive immune with a high percentage of mast cells positively Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 11 of 15 correlating with poor overall survival of GC patients. but little CCR2, CCR4, CCR5, CCR7, CXCR1, CXCR2 Furthermore, tumor-infiltrating mast cells colocalized or CXCR7. Therefore, we are the first to define the vital with T cells, and tumor-derived TNF-α could induce role of CXCL12-CXCR4 chemotaxis in recruiting mast immunosuppressive mast cells (PD-L1 mast cells) cells in GC. Certainly, some other cells can also express (Fig. 3g). Although less efficiently than professional CXCR4 in GC, such as gastric cancer cells [27], mye- antigen-presenting cells (APCs), mast cells can upreg- loid cells [28], myeloid-derived suppressor cells ulate CD69 expression, proliferation and cytokine pro- (MDSCs) [29] and so on. Some studies have shown duction in effector T cells [20, 21] and others have CXCR4 pathway can promote epithelial-mesenchymal been clearly shown to be able to present antigens to T transition in gastric cancer cells [30]. In tumor micro- cells [22]. It is therefore easy to envisage, when they environment, CXCR4 MDSCsand neutrophilscan be express the immune inhibitory ligand PD-L1 they may recruited by CXCL12 and they impair the anti-tumor inhibit T-cell immunity via PD-L1 as shown in in vitro functions of CD8 cytotoxic T cells leading to increased co-culture as well as in vivo in the absence of PD-L1 tumor metastasis [29, 31]. neutralizing antibody in this study. These results are Mast cells have been identified in tumors recently. similar to those from our previous studies on neutro- However, very little is currently known about the phils, which are also not considered as professional phenotype and function of these tumor-infiltrating APCs [23]. The pathological role of mast cells within mast cells. Immunosuppression has been widely ac- the tumor microenvironment suggests a novel tumor knowledged as a hallmark of cancer [17]. Interestingly, immune escaping mechanism during GC progression. we screened the expression of immunosuppressive mol- In humans, mast cell infiltration in tumors influences ecules on tumor-infiltrating mast cells, and we found disease progression and patient survival [24]. Hence, the that mast cells within GC expressed high level im- analysis of mast cell infiltration in GC is a crucial area of munosuppressive molecule PD-L1, suggesting that they clinical investigation. Our data shed some light on the mayplaya role to directlymodulate effector function. clinical relevance of mast cells in GC. We observed a Crosstalk between PD-L1 and PD-1 is one of the main significant positive association between the percentage/ mechanisms leading to immunosuppression of T cells number of mast cells and advanced clinical features of [32]. In our present study, we uncovered that GC GC, such as lymphatic invasion, tumor size, and tumor tumor-associated mast cells effectively inhibited T-cell’s stage (Additional file 2: Figure S1). In addition, we immune function in a PD-L1–dependent manner in uncovered that an increased frequency/number of intra- vitro and in vivo. Furthermore, immunofluorescence tumoral mast cells predicted a lower rate of disease-free staining showed that most tumor–infiltrating mast cells survival, which was oppositely correlated with intratu- localized in close proximity to CD3 T cells in GC, in- moral mast cell levels (Additional file 12: Figure S7), dicating that immunosuppression could be mediated by suggesting that tumor-infiltrating mast cells may become the interaction of mast cells and T cells in a PD- a helpful clinical prognostic marker in the future. L1-dependent manner. We are the first to report that Regardless of patient outcome, the percentage/num- the induced PD-L1 expression on tumor-infiltrating ber of mast cells was notably increased in tumors com- mast cells enabled them to suppress T-cell proliferation pared with non-tumor tissues. This may be a and IFN-γ production. Consistent with our findings in consequence of either enhanced proliferation of intra- GC, blocking PD-L1/ PD-1 pathway could also achieve tumoral mast cells or an increased migration of mast a good prognosis in testicular germ cell tumors [33]. cells to tumor tissues. As for the little expression of We previously reported that degranulation of mast cells Ki-67 in mast cells in GC, we exclude the possibility of prompted GC progression [34], which offered add- the enhanced proliferation of intratumoral mast cells itional targets to consider for mast cell directed therap- and predicted that the tumors possibly induced migra- ies besides target for mast cell-associated PD-L1. In tion of mast cells by chemotaxis. Recently, mast cells addition, tumor-associated mast cells can also be acti- had been reported to be attracted into the tumor vated to secrete sufficient amount of biological mole- microenvironment in a SCF-dependent manner [25]. In cules to mediate tumor progression [35]. Previous our case, we identified a new mechanism for mast cell studies found that mast cell released histamine via chemotaxis in GC. In comparison with non-tumor tis- c-Kit/SCF, which increased cholangiocarcinoma growth, sue, gastric tumors could secrete more CXCL12, which and angiogenesis [36]. Moreover, mast-cell-derived me- attracted migration of mast cells. Additionally, some in- diators: histamine, CXCL1 and CXCL10 could increase vestigations showed that mast cells also expressed che- thyroid cancer cell survival and DNA synthesis in vitro mokine receptors such as CXCR1 and CXCR2 in [37]. In pancreatic cancer, activated mast cells promote different cancers [26]. In our study, we found that tumor progression by IL-13 and tryptase [38]. Import- intratumoral mast cells expressed high level CXCR4 antly, in vivo GC model, we found TTCS-conditioned Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 12 of 15 Fig. 7 A proposed model of cross-talks among mast cells, T cells, and tumor cells leading to mast cell-mediated immunosuppressive and protumorigenic effects in the GC microenvironment. CXCL12-CXCR4 chemotaxis mediates the recruitment and accumulation of mast cells into the GC microenvironment, which could be up-regulated PD-L1 expression via NF-κB signaling pathway activation by tumor-derived TNF-α. Mast cells inhibit T-cell proliferation and function in a PD-L1-dependent manner in GC, which promotes GC progression mast cells effectively inhibited T cell immunity and promoter [46]. In this study, we verify TNF-α as a cru- promoted GC progression, and such T cell suppression cial pro-inflammatory factor within GC microenviron- was through PD-L1 on the mast cells as blocking ment, which derived from GC tumor and effectively PD-L1 reversed it. Certainly, some other cell types are induces the expression of PD-L1 on mast cells via activa- also known to mediate T-cell suppression in tumor micro- tion of NF-κB signaling pathway. environment. For instance, a recent study showed that The recent success of checkpoint inhibitor monother- LC3-associated phagocytosis (LAP) in myeloid cells regu- apy opens up a new era of cancer treatment, however, lated macrophages in the tumor microenvironment, which it’s not suited for everyone. How to achieve precision as a result suppressed T cell function and promoted tumor immunotherapy further, select the preponderant benefi- tolerance [39]; and the number of tumor-infiltrating mye- ciaries, and improve the efficacy of pharmacoeconomics loid cells was correlated with early metastatic relapse [40]. are important topics. Tumor mutational burden (TMB) Furthermore, it has been recognized that tumors often re- is a promising new biomarker for predicting the efficacy cruit MDSCs that inhibits T cell infiltration and activation of cancer immunotherapy, which is measured by whole- [41]. Therefore tumor resistance to single-agent immune exome sequencing and associated with clinical benefit checkpoint inhibitor therapy could be a result of lack infil- from multiple checkpoint inhibitors. TMB exhibited trating T cells or too many suppressive immune cells [42, joint predictive utility in identifying responders and non- 43]. It will be very important to further explore the role of responders to the PD-1 antibody pembrolizumab, the the suppressive, PD-L1-expressing mast cells reported in number and type of alterations may prove to be valuable this study and their relation with other suppressive immune for judging the potential usefulness of immune check- cell types. Here, our results indicate that the important point inhibitors [47]. Besides, high TMB may be a re- PD-1-PD-L1 immunosuppression pathway operates in GC sponse biomarker for PD-1/PD-L1 blockade in tumors via mast cells. such as melanoma and non-small cell lung cancer PD-L1 is expressed in a wide range of cell types and (NSCLC), and results show a linear correlation between tissues and shown to be overexpressed with immune ac- higher TMB and favorable outcome parameters with tivation, such as inflammation or tumor [23, 44]. Previ- anti-PD-1/PD-L1 monotherapy [48]. In addition, PD-1/ ous studies have shown that PD-L1 up-regulation can be PD-L1 also plays an important role in some inflamma- induced mainly by activated CD8 cytotoxic T cells-de- tory disease. Asthma is a chronic airway inflammation rived interferon-γ in HCC milieu [45]. Meanwhile, our associated largely with CD4 T cells, eosinophils, and team identified that tumor-derived GM-CSF effectively mast cells. PD-L1 expressed on mast cells was shown to induces PD-L1 expression on neutrophils via activation regulate T-cell activation and tolerance. And PD-L1 of JAK-STAT3 pathway [23]. Besides, research has re- mast cell is a negative regulator of conventional CD4 T vealed that tumor-derived hypoxia inducible factor 1a cells, and understanding of the role will give propulsion (HIF-1a) upregulated PD-L1 on MDSCs by binding to a to the development of novel therapeutic approaches in hypoxia-response element (HRE) in the PD-L1 proximal allergic asthma [49]. Besides, alopecia areata (AA) is a Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 13 of 15 CD8 T-cell dependent autoimmune disease and mast + + tryptase mast cells and Ki-67 cells were defined by immunofluorescence cells are crucial immunomodulatory cells implicated in staining. Green, Tryptase; red, Ki-67; and blue, DAPI-stained nuclei. Scale bars: 50 μm. (c) Expression of CCR2, CCR4, CCR5, CCR7, CXCR1, CXCR2 and CXCR7 the regulation of T cell-dependent immunity in AA. A + + + on tumor-infiltrating mast cells by gating on CD45 CD117 FcεRI cells. Color report showed that a number of PD-L1 mast cells ap- histograms represent staining of chemokine receptors; black, isotype control. peared to be further reduced in lesional AA skin com- (d) Representative analysis of CXCL12-expressing (red) EpCam tumor cells (green) in tumor tissues of GC patients by immunofluorescence. Scale bars: pared to healthy skin, particularly during their 20 μm. (e) Expression of CD80 and CD86 in tumor-infiltrating mast cells by interactions with CD8 T-cells. Mast cells in AA are + + + gating on CD45 CD117 FcεRI cells. Color histograms represent staining of skewed towards pro-inflammatory activities and that CD80 and CD86; black, isotype control. (TIF 5879 kb) MC-CD8 T-cell interactions in AA are predominantly Additional file 8 Figure S3. Tumor-derived factor TNF-α induces mast cells to express PD-L1. (a) Expression of 2B4, glactin-3, CTLA-4, and ICOSL pro-inflammatory. This finding suggests that treatment + + + on mast cells by gating on CD45 CD117 FcεRI cells. Color histograms regimens which promote an immune-inhibitory pheno- represent staining of 2B4, glactin-3, CTLA-4, and ICOSL; black, isotype type and/or suppress the switch towards a pro-inflam- control. (b) Expression of PD-L1 on hCBMCs exposed to IL-1β, IL-6, IL-10, IL-17, IL-22, IL-23, M-CSF, G-CSF, IFN-γ, TGF-β (100 ng/ml) for 24 h. black, matory mast cells phenotype, should down-regulate isotype control. (c) Expression of TNF-α receptor II (TNFRII) on tumor- undesired CD8 T-cell responses in AA [50]. infiltrating mast cells. Black, isotype control. (TIF 1497 kb) Additional file 9 Figure S4. Tumor-derived TNF-α activates NF-κB pathway to induce PD-L1 expression on mast cells. (a) Expression of Conclusion PD-L1 on hCBMCs exposed to 50% TTCS with or without U0126 (an ERK In brief, based on our in vitro and in vivo data, we inhibitor), Wortmannin (a PI3K inhibitor), SB203580 (a MAPK inhibitor), or propose a model involving complex interactions between SP600125 (a JNK inhibitor) for 24 h. black, isotype control. (b) p44/42 and p-p44/42, Akt and p-Akt, p38 and p-p38, JNK and p-JNK in LAD2 cells mast cells, T cells and tumor cells within GC (Fig. 7). exposed to TTCS with or without anti-TNF-α antibody were analyzed by First, CXCL12-CXCR4 chemotaxis mediates the recruit- western blot. (TIF 1181 kb) ment of mast cells into GC microenvironment. And Additional file 10 Figure S5. Tumor-infiltrating and tumor-conditioned then, tumor-derived TNF-α induces the overexpression mast cells suppress T cell immunity through PD-L1. (a) CFSE-labeled peripheral CD3 T cells of donors were co-cultured for 5 days with TTCS-, of PD-L1 on mast cells via NF-κB signaling pathway ac- + or NTCS-conditioned LAD2 cells with or without anti-PD-L1 antibody. tivation. Finally, the PD-L1 mast cells inhibit T-cell Representative data and statistical analysis of T cell proliferation and IFN-γ proliferation and function in a PD-L1-dependent man- production were shown (n = 5). *, P < 0.05; **, P < 0.01 for groups con- nected by horizontal lines. (b) Surface staining of FcεRI and CD117 of ner. In conclusion, our study has highlighted a notable generated human umbilical cord blood-derived cultured mast cells role for mast cells in human GC and identified new (hCBMCs) was shown. Results were expressed as percentage of + + + mechanisms of GC-associated mast cells mediating CD117 FcεRI cells by gating on CD45 cells. Iso, Isotype control antibody. (c) Toluidine blue staining of sorted human tumor-infiltrating tumor progression. Overall, blocking these pathological mast cells was shown. Scale bars: 10 μm. (d) Granzyme B and mast cells and the TNF-α-PD-L1 immunosuppressive perforin production were compared in the supernatants (n = 5), which pathway may be a useful therapeutic strategy for pre- CD3 T cells of patients with GC were co-cultured with autologous mast cells from non-tumor or tumor tissues with or without anti-PD-L1 venting GC progress. antibody. (e) Granzyme B and perforin production were compared in the supernatants (n = 5), which peripheral CD3 T cells of donors were co- Additional files cultured with autologous TTCS-, or NTCS-conditioned hCBMCs with or without anti-PD-L1 antibody. **, P < 0.01 for groups connected by horizontal lines. hCBMCs, human umbilical cord blood-derived cultured Additional file 1 Table S1. Antibodies and other reagents. (DOCX 29 mast cells. (TIF 1676 kb) kb) Additional file 11 Figure S6. Blockade of mast cell-associated PD-L1 Additional file 2 Figure S1. Mast cell percentage (a) or mast cell on T cell immunity inhibits tumor growth and GC progression in vivo. number (b) and its potential correlations with clinical parameters. Mast (a-c) Mice were injected with human SGC-7901 cells, as described in cell percentage in CD45 leukocytes and mast cell number per million Materials and methods. The control animals () received no further total cells were analyzed for correlations with clinical pathological injections. The experimental treatments entailed injections with T parameters. **, P < 0.01; n.s., P > 0.05 for groups connected by horizontal cells () or T cells in combination with TTCS-conditioned mast cells lines. Each dot represents one patient. CEA, carcinoembryonic antigen; (TCM) (), or T cells in combination with TCM pre-treated with an anti- H.pylori Ab, Helicobacter pylori antibody. (TIF 1496 kb) PD-L1 antibody () or a control IgG (). The illustrated data represent Additional file 3 Table S3. Univariate and multivariate analyses of tumor volumes (5 mice in each group). The day of tumor cell factors associated with survival. (DOCX 20 kb) injection was counted as day 0. The expression of IFN-γ (a), anti- tumor molecules perforin (b)and granzyme B(c)in tumorsof mice Additional file 4 Table S2. Clinical characteristics of 114 patients with on day 21 after tumor cell injection were compared (n =5). The gastric cancer. (DOCX 19 kb) horizontal bars represent mean values. *, P < 0.05; **, P <0.01 for Additional file 5 Table S4. Correlations between mast cell percentage groups connected by horizontal lines. (TIF 841 kb) and clinic pathological features of patients with gastric cancer. (DOCX 20 Additional file 12 Figure S7. Kaplan-Meier plots for disease-free survival kb) by median mast cell percentage (9.315%) or median mast cell number Additional file 6 Table S5. Correlations between mast cell number and (4749 per million). MC (%), mast cell percentage; MC (NO.), mast cell clinic pathological features of patients with gastric cancer. (DOCX 21 kb) number. (TIF 108 kb) Additional file 7 Figure S2. CXCL12-CXCR4 chemotaxis mediates mast cell Additional file 13 Table S6. Primer and probe sequences for real-time migration and accumulation in GC tumors. (a) Expression of Ki-67 in tumor- PCR analysis. (DOCX 20 kb) + + + infiltrating mast cells by gating on CD45 CD117 FcεRI cells. Color histograms Additional file 14 Supplementary Materials and Methods. (DOCX 63 kb) represent staining of Ki-67; black, isotype control. (b) Tumor-infiltrating Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 14 of 15 Abbreviations 3. Wang F, Meng W, Wang B, Qiao L. Helicobacter pylori-induced gastric APCs: Antigen-presenting cells; GC: Gastric cancer; hCBMCs: Human umbilical inflammation and gastric cancer. Cancer Lett. 2014;345:196–202. cord blood-derived cultured mast cells; IFN: Interferon; IL: Interleukin; 4. Choi IJ, Kook MC, Kim YI, Cho SJ, Lee JY, Kim CG, et al. Helicobacter pylori MDSCs: Myeloid-derived suppressor cells; PBMCs: Peripheral blood therapy for the prevention of Metachronous gastric Cancer. N Engl J Med. mononuclear cells; PD-L1: Programmed death-ligand 1; TNF-α: Tumor 2018;378:1085–95. necrosis factor-α 5. Wen T, Wang Z, Li Y, Li Z, Che X, Fan Y, et al. A four-factor Immunoscore system that predicts clinical outcome for stage II/III gastric Cancer. Cancer Immunol Res. 2017;5:524–34. Acknowledgments The authors are grateful to Prof. Wei Zhang for donating the human mast 6. Ferrone C, Dranoff G. Dual roles for immunity in gastrointestinal cancers. J cell line LAD2 cells, and Xiao Luo for collecting samples from gastric cancer Clin Oncol. 2010;28:4045–51. patients. 7. Ying L, Yan F, Meng Q, Yu L, Yuan X, Gantier MP, et al. PD-L1 expression is a prognostic factor in subgroups of gastric cancer patients stratified according to their levels of CD8 and FOXP3 immune markers. Funding Oncoimmunology. 2018;7:e1433520. This work was supported by grant of National Natural Science Foundation of 8. Lee HE, Chae SW, Lee YJ, Kim MA, Lee HS, Lee BL, et al. Prognostic China (81670510 and 81872016), Founding by Southwest Hospital implications of type and density of tumour-infiltrating lymphocytes in (SWH2017YBXM-07) and National Key Research and Development Program gastric cancer. Br J Cancer. 2008;99:1704–11. of China (2016YFC1302200). 9. Kalesnikoff J, Galli SJ. New developments in mast cell biology. Nat Immunol. 2008;9:1215–23. Availability of data and materials 10. Maciel TT, Moura IC, Hermine O. The role of mast cells in cancers, All data generated or analyzed during this study are included in this F1000Prime Rep. 2015;7:9. published article [and its additional files]. Primer and probe sequences for 11. Sammarco G, Gadaleta CD, Zuccala V, Albayrak E, Patruno R, Milella P, et al. real-time PCR analysis and Supplementary Materials and Methods are shown Tumor-associated macrophages and mast cells positive to Tryptase are as Additional files 13 and 14. correlated with angiogenesis in surgically-treated gastric Cancer patients. Int J Mol Sci. 2018;19:1176-89. Authors’ contributions 12. Liu J, Zhang Y, Zhao J, Yang Z, Li D, Katirai F, et al. Mast cell: insight into Study concept and design and drafting of the manuscript: YZ. Acquisition of remodeling a tumor microenvironment. Cancer Metastasis Rev. 2011;30: data and analysis and interpretation of data: YZ, YL. Critical revision of the 177–84. manuscript for important intellectual content: YZ, and WC. Statistical analysis: 13. Danelli L, Frossi B, Gri G, Mion F, Guarnotta C, Bongiovanni L, et al. Mast YZ, YL. Obtained funding: YZ, YZ, and JC. Technical, or material support: YZ, cells boost myeloid-derived suppressor cell activity and contribute to the XW, NC, YT, TW, FM, LP, JZ, PC, YL, HK, CH, BH, QM, QZ, and JC. Final development of tumor-favoring microenvironment. Cancer Immunol Res. approval of the version to be published: YZ. All authors read and approved 2015;3:85–95. the final manuscript. 14. Lin C, Liu H, Zhang H, Cao Y, Li R, Wu S, et al. Tryptase expression as a prognostic marker in patients with resected gastric cancer. Br J Surg. 2017; Ethics approval and consent to participate 104:1037–44. The study was approved by the Ethics Committee of the Southwest Hospital 15. Guidolin D, Ruggieri S, Annese T, Tortorella C, Marzullo A, Ribatti D. Spatial of Third Military Medical University. Written informed consent was obtained distribution of mast cells around vessels and glands in human gastric from each subject. carcinoma. Clin Exp Med. 2017;17:531–9. 16. Mushtaq MU, Papadas A, Pagenkopf A, Flietner E, Morrow Z, Chaudhary SG, Consent for publication et al. Tumor matrix remodeling and novel immunotherapies: the promise of Informed consent for publication of data was obtained from all patients. matrix-derived immune biomarkers. J Immunother Cancer. 2018;6:65. 17. Schreiber RD, Old LJ, Smyth MJ. Cancer immunoediting: integrating Competing interests immunity's roles in cancer suppression and promotion. Science. 2011;331: The authors declare that they have no competing interests. 1565–70. 18. Stromnes IM, Hulbert A, Pierce RH, Greenberg PD, Hingorani SR. T-cell localization, activation, and clonal expansion in human pancreatic ductal Publisher’sNote adenocarcinoma. Cancer Immunol Res. 2017;5:978–91. Springer Nature remains neutral with regard to jurisdictional claims in 19. Ammendola M, Sacco R, Donato G, Zuccala V, Russo E, Luposella M, et al. published maps and institutional affiliations. Mast cell positivity to tryptase correlates with metastatic lymph nodes in gastrointestinal cancer patients treated surgically. Oncology-Basel. 2013; Author details 85:111–6. National Engineering Research Center of Immunological Products, 20. Gaudenzio N, Espagnolle N, Mars LT, Liblau R, Valitutti S, Espinosa E. Cell-cell Department of Microbiology and Biochemical Pharmacy, College of cooperation at the T helper cell/mast cell immunological synapse. Blood. Pharmacy, Third Military Medical University, No.30 Gaotanyan Street, 2009;114:4979–88. Chongqing 400038, China. Department of General Surgery and Centre of Minimal Invasive Gastrointestinal Surgery, Southwest Hospital, Third Military 21. Kambayashi T, Allenspach EJ, Chang JT, Zou T, Shoag JE, Reiner SL, et al. Medical University, No.30 Gaotanyan Street, Chongqing 400038, China. Inducible MHC class II expression by mast cells supports effector and Department of Obstetrics and Gynecology, Research Institute of Surgery, regulatory T cell activation. J Immunol. 2009;182:4686–95. Daping Hospital, Third Military Medical University, Chongqing, China. La 22. Stelekati E, Bahri R, D'Orlando O, Orinska Z, Mittrucker HW, Langenhaun R, Trobe Institute of Molecular Science, School of Molecular Science, La Trobe et al. Mast cell-mediated antigen presentation regulates CD8+ T cell University, Bundoora, Vic 3085, Australia. Affiliated Hospital of North Sichuan effector functions. Immunity. 2009;31:665–76. Medical College, Nanchong, Sichuan Province, China. 23. Wang TT, Zhao YL, Peng LS, Chen N, Chen W, Lv YP, et al. Tumour-activated neutrophils in gastric cancer foster immune suppression and disease Received: 19 July 2018 Accepted: 11 February 2019 progression through GM-CSF-PD-L1 pathway. Gut. 2017;66:1900–11. 24. Giannou AD, Marazioti A, Spella M, Kanellakis NI, Apostolopoulou H, Psallidas I, et al. Mast cells mediate malignant pleural effusion formation. J Clin Invest. 2015;125:2317–34. References 1. Van Cutsem E, Sagaert X, Topal B, Haustermans K, Prenen H. Gastric cancer. 25. Huang B, Lei Z, Zhang GM, Li D, Song C, Li B, et al. SCF-mediated mast cell Lancet. 2016;388:2654–64. infiltration and activation exacerbate the inflammation and 2. Ndegwa N, Ploner A, Liu Z, Roosaar A, Axell T, Ye W. Association between immunosuppression in tumor microenvironment. Blood. 2008;112:1269–79. poor oral health and gastric cancer: a prospective cohort study. Int J Cancer. 26. Varricchi G, Galdiero MR, Loffredo S, Marone G, Iannone R, Marone G, et al. 2018;143:2281-8. Are mast cells MASTers in Cancer? Front Immunol. 2017;8:424. Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 15 of 15 27. Xiang Z, Zhou ZJ, Xia GK, Zhang XH, Wei ZW, Zhu JT, et al. A positive 48. Goodman AM, Kato S, Bazhenova L, Patel SP, Frampton GM, Miller V, et al. crosstalk between CXCR4 and CXCR2 promotes gastric cancer metastasis. Tumor mutational burden as an independent predictor of response to Oncogene. 2017;36:5122–33. immunotherapy in diverse cancers. Mol Cancer Ther. 2017;16:2598–608. 28. Yang J, Kumar A, Vilgelm AE, Chen SC, Ayers GD, Novitskiy SV, et al. Loss of 49. Singh AK, Stock P, Akbari O. Role of PD-L1 and PD-L2 in allergic diseases CXCR4 in myeloid cells enhances antitumor immunity and reduces and asthma. Allergy. 2011;66:155–62. melanoma growth through NK cell and FASL mechanisms. Cancer Immunol 50. Bertolini M, Zilio F, Rossi A, Kleditzsch P, Emelianov VE, Gilhar A, et al. Res. 2018;6:1186–98. Abnormal interactions between perifollicular mast cells and CD8+ T- cells may contribute to the pathogenesis of alopecia areata. PLoS One. 29. Zhuang Y, Peng LS, Zhao YL, Shi Y, Mao XH, Chen W, et al. CD8(+) T cells 2014;9:e94260. that produce interleukin-17 regulate myeloid-derived suppressor cells and are associated with survival time of patients with gastric cancer. Gastroenterology. 2012;143:951–62. 30. Cheng Y, Song Y, Qu J, Che X, Song N, Fan Y, et al. The chemokine receptor CXCR4 and c-MET cooperatively promote epithelial-mesenchymal transition in gastric Cancer cells. Transl Oncol. 2018;11:487–97. 31. Seubert B, Grunwald B, Kobuch J, Cui H, Schelter F, Schaten S, et al. Tissue inhibitor of metalloproteinases (TIMP)-1 creates a premetastatic niche in the liver through SDF-1/CXCR4-dependent neutrophil recruitment in mice. Hepatology. 2015;61:238–48. 32. Kuang DM, Zhao Q, Peng C, Xu J, Zhang JP, Wu C, et al. Activated monocytes in peritumoral stroma of hepatocellular carcinoma foster immune privilege and disease progression through PD-L1. J Exp Med. 2009; 206:1327–37. 33. Siska PJ, Johnpulle R, Zhou A, Bordeaux J, Kim JY, Dabbas B, et al. Deep exploration of the immune infiltrate and outcome prediction in testicular cancer by quantitative multiplexed immunohistochemistry and gene expression profiling. Oncoimmunology. 2017;6:e1305535. 34. Lv YP, Peng LS, Wang QH, Chen N, Teng YS, Wang TT, et al. Degranulation of mast cells induced by gastric cancer-derived adrenomedullin prompts gastric cancer progression. Cell Death Dis. 2018;9:1034. 35. Marichal T, Tsai M, Galli SJ. Mast cells: potential positive and negative roles in tumor biology. Cancer Immunol Res. 2013;1:269–79. 36. Johnson C, Huynh V, Hargrove L, Kennedy L, Graf-Eaton A, Owens J, et al. Inhibition of mast cell-derived histamine decreases human cholangiocarcinoma growth and differentiation via c-kit/stem cell factor- dependent signaling. Am J Pathol. 2016;186:123–33. 37. Melillo RM, Guarino V, Avilla E, Galdiero MR, Liotti F, Prevete N, et al. Mast cells have a protumorigenic role in human thyroid cancer. Oncogene. 2010; 29:6203–15. 38. Ma Y, Hwang RF, Logsdon CD, Ullrich SE. Dynamic mast cell-stromal cell interactions promote growth of pancreatic cancer. Cancer Res. 2013;73: 3927–37. 39. Cunha LD, Yang M, Carter R, Guy C, Harris L, Crawford JC, et al. LC3- associated phagocytosis in myeloid cells promotes tumor immune tolerance. Cell. 2018;175:429–41. 40. Kim K, Skora AD, Li Z, Liu Q, Tam AJ, Blosser RL, et al. Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells. Proc Natl Acad Sci U S A. 2014;111: 11774–9. 41. Marvel D, Gabrilovich DI. Myeloid-derived suppressor cells in the tumor microenvironment: expect the unexpected. J Clin Invest. 2015; 125:3356–64. 42. Kamran N, Kadiyala P, Saxena M, Candolfi M, Li Y, Moreno-Ayala MA, et al. Immunosuppressive myeloid Cells' blockade in the glioma microenvironment enhances the efficacy of immune-stimulatory gene therapy. Mol Ther. 2017;25:232–48. 43. Kusmartsev S, Nagaraj S, Gabrilovich DI. Tumor-associated CD8+ T cell tolerance induced by bone marrow-derived immature myeloid cells. J Immunol. 2005;175:4583–92. 44. Tsukamoto H, Fujieda K, Miyashita A, Fukushima S, Ikeda T, Kubo Y, et al. Combined blockade of IL-6 and PD-1/PD-L1 signaling abrogates mutual regulation of their immunosuppressive effects in the tumor microenvironment. Cancer Res. 2018;78(17):5011-22. 45. XieQK, ZhaoYJ, PanT, Lyu N, Mu LW,LiSL, et al. Programmed death ligand 1 as an indicator of pre-existing adaptive immune responses in human hepatocellular carcinoma. Oncoimmunology. 2016;5:e1181252. 46. Noman MZ, Chouaib S. Targeting hypoxia at the forefront of anticancer immune responses. Oncoimmunology. 2014;3:e954463. 47. Cristescu R, Mogg R, Ayers M, Albright A, Murphy E, Yearley J, et al. Pan- tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy. Science. 2018;362:eaar3593. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal for ImmunoTherapy of Cancer Springer Journals

Increased intratumoral mast cells foster immune suppression and gastric cancer progression through TNF-α-PD-L1 pathway

Loading next page...
 
/lp/springer-journals/increased-intratumoral-mast-cells-foster-immune-suppression-and-O2WBfe5fML

References (54)

Publisher
Springer Journals
Copyright
Copyright © 2019 by The Author(s).
Subject
Medicine & Public Health; Oncology; Immunology
eISSN
2051-1426
DOI
10.1186/s40425-019-0530-3
Publisher site
See Article on Publisher Site

Abstract

Background: Mast cells are prominent components of solid tumors and exhibit distinct phenotypes in different tumor microenvironments. However, the nature, regulation, function, and clinical relevance of mast cells in human gastric cancer (GC) are presently unknown. Methods: Flow cytometry analyses were performed to examine level and phenotype of mast cells in samples from 114 patients with GC. Multivariate analysis of prognostic factors for overall survival was performed using the Cox proportional hazards model. Kaplan-Meier plots for patient survival were performed using the log-rank test. Mast cells, T cells and tumor cells were isolated or generated, stimulated and/or cultured for in vitro and in vivo function assays. Results: Patients with GC showed a significantly higher mast cell infiltration in tumors. Mast cell levels increased with tumor progression and independently predicted reduced overall survival. These tumor-infiltrating mast cells accumulated in tumors by CXCL12-CXCR4 chemotaxis. Intratumoral mast cells expressed higher immunosuppressive molecule programmed death-ligand 1 (PD-L1), and mast cells induced by tumors strongly express PD-L1 proteins in both time-dependent and dose-dependent manners. Significant correlations were found between the levels of PD-L1 mast cells and pro-inflammatory cytokine TNF-α in GC tumors, and tumor-derived TNF-α activated NF-κB signaling pathway to induce mast cell expression of PD-L1. The tumor-infiltrating and tumor-conditioned mast cells effectively suppressed normal T-cell immunity through PD-L1 in vitro, and tumor-conditioned mast cells contributed to the suppression of T-cell immunity and the growth of human GC tumors in vivo; the effect could be reversed by blocking PD-L1 on these mast cells. Conclusion: Thus, our results illuminate novel immunosuppressive and protumorigenic roles of mast cells in GC, and also present a novel mechanism in which PD-L1 expressing mast cells link the proinflammatory response to immune tolerance in the GC tumor milieu. Keywords: Gastric cancer, Tumor microenvironment, Mast cells, TNF-α,PD-L1, Immunotherapy * Correspondence: cj_doc@sina.com; yuanzhuang1983@yahoo.com Department of General Surgery and Centre of Minimal Invasive Gastrointestinal Surgery, Southwest Hospital, Third Military Medical University, No.30 Gaotanyan Street, Chongqing 400038, China National Engineering Research Center of Immunological Products, Department of Microbiology and Biochemical Pharmacy, College of Pharmacy, Third Military Medical University, No.30 Gaotanyan Street, Chongqing 400038, China Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 2 of 15 Background peripheral blood were obtained from patients with GC who Gastric cancer (GC), as a severe health problem, has been underwent surgical resection at the Southwest Hospital of the fourth most common malignancies and the second Third Military Medical University. None of the patients leading cause of cancer death worldwide [1]. In some low/ had received chemotherapy or radiation before the sample middle income countries with poor sanitation or high risk was taken. Patients with infectious diseases, autoimmune of helicobacter pylori infection, it has been one of the major diseases or multiple primary cancers were excluded. The causes of cancer death [2, 3]. Despite significant progress clinical stage of tumors was determined according to the made in prevention, diagnose, and therapeutic options in TNM classification system of the International Union recent years [4, 5], many questions remain unanswered, Against Cancer (7th edition). The study was approved by especially the pathogenesis of GC. Nowadays, it is generally the Ethics Committee of the Southwest Hospital of Third believed that the development and prognosis of GC are Military Medical University. Each subject provided written influenced by the cross-talk between tumors and host im- informed consent. Additional file 1: Table S1 lists antibodies mune system [6, 7]. Previous studies have focused on the and other reagents. crucial role for adaptive immunity in determining the clin- ical outcomes of GC patients [8]. However, little is known Isolation of single cells from GC tissues about the role of innate immunity and innate immune cells Fresh tissues were washed 3 times with Hank’ssolution during GC development and progression. containing 1% FCS and cut into small pieces. Specimens Mast cells are a group of innate immune cells, which were collected in RPMI 1640 medium containing collage- have profound immunomodulatory effects on tumor pro- nase IV (1 mg/ml) and deoxyribonuclease I (10 mg/ml) and gression [9, 10], such as angiogenesis [11], tumor micro- mechanically separated using the gentle MACS Dissociator environment reconstruction [12] and interaction with other (Miltenyi Biotec). Dissociated cell suspensions were further immune cells [13]. At present, limited studies on mast cells incubated for 1 h under continuous rotation at 37 °C. The in GC mainly focus on the correlation between the survival cell suspensions were then filtered by a 70 μmcellfilter rate of GC patients and their GC mast cell infiltration by (BD Labware). Cell viability, as measured by trypan blue ex- immunohistochemistry [14], and a few on the relationship clusion staining, was typically > 90%. between infiltrated mast cell density and local angiogenesis [15]. Overall, these studies suggest that mast cells may be a Isolation of mast cells and T cells therapeutic target for GC. However, the phenotype, func- As mentioned above, tumor and non-tumor tissues were tional regulation and clinical correlation of mast cells in hu- treated as single cell suspension. Then the single cell sus- man GC microenvironment remain unclear. pension was stained with anti-human CD45, anti-human Herein, we investigate the interplays among mast cells, CD117 and anti-human FcεRI antibodies, and mast cells T cells and tumor cells in the GC microenvironment. We from autologous tumor and non-tumor tissues were sorted show that mast cells could be recruited to tumor micro- by fluorescence activating cell sorter (FACS) (FACSAria II; environment through CXCL12-CXCR4 chemotaxis axis. BD Biosciences). Density gradient centrifugation was used Moreover, tumor-derived TNF-α efficiently induces pro- to isolate peripheral blood mononuclear cells (PBMCs) grammed death-ligand 1 (PD-L1) expression on mast cells from autologous GC patients and healthy donors by using by activating nuclear factor kappa-light-chain-enhancer of Ficoll-Paque Plus. CD3 T cells from PBMCs were purified activated B cells (NF-κB) signaling pathways. In turn, with CD3 microbeads. The sorted cells were used only these mast cells inhibit the normal function of T cells in a when their viability was determined > 90% and their purity PD-L1-dependent manner, which could suppress antitu- was determined > 95%. mor immunity in GC. Our data suggest a protumorigenic role of mast cells with an immunosuppressive phenotype in GC. These Preparation of TTCS and NTCS and supernatant- tumor-infiltrating mast cells increase with tumor pro- conditioned mast cells gression and are negatively correlated with patient sur- Tumor tissue culture supernatants (TTCS) or non-tumor vival after surgery, suggesting that these mast cells may tissue culture supernatants (NTCS) were prepared by plat- be a novel target in novel GC therapy. ing autologous tumor or non-tumor gastric tissues in 1 ml RPMI 1640 medium for 24 h. The supernatant was then Methods harvested by centrifugation. To generate supernatant-con- Patients and specimens ditioned mast cells, primary human umbilical cord Fresh gastric tumor (homogeneous cellularity, without foci blood-derived cultured mast cells (hCBMCs) or LAD2 of necrosis, including intratumoral and marginal tissues), cells were first harvested and cultured with autologous peritumoral and non-tumor (non-tumor tissues, at least 5 50% TTCS or NTCS for 24 h, and then washed with cm distant from the tumor site) tissues and autologous RPMI-1640 medium for 3 times. Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 3 of 15 Chemotaxis assay as described above, in the presence or absence of a neutral- Fluorescence-activated cell sorter sorted tumor-infil- izing antibody against human PD-L1 (20 μg/ml). After trating mast cells (1 × 10 ) from fresh human tumor 5-day incubation, the supernatants were harvested for tissues were transferred into the upper chambers of ELISA and the cells were harvested for intracellular cyto- 8-μm pore size Transwells (Corning). Autologous 50% kine staining. TTCS or NTCS as the sources of chemoattractants were placed in the lower chambers. After 24-h culture In vivo tumor inhibition assay at 37 °C, migration was quantified by counting cells in All animal experiments were undertaken with the ap- the lower chamber and cells adhering to the bottom of proval from the Animal Ethical and Experimental the membrane. In some cases, blocking antibody for Committee of Third Military Medical University. 10 CXCR4 (20 μg/ml, IgG2b) or control IgG2b was added GC cells (SGC-7901 cells) in 100 μl of buffered saline into mast cell suspensions and incubated for 2 h before were subcutaneously injected into the axillary tissues of chemotaxis assay. Furthermore, CXCL12 neutralizing female nonobese diabetic/severe combined immunodefi- antibody (20 μg/ml, IgG1) or control IgG1 was added ciency (NOD/SCID) mice (5–7 week, one tumor per into TTCS in some assays. RPMI-1640 medium and mouse). The hCBMCs (referred to as mast cells) were chemokine CXCL12 (100 ng/ml) were placed in the stimulated with 50% TTCS for 24 h. Then, 5 × 10 lower chambers as blank and positive controls anti-CD3- and anti-CD28-stimulated (2 μg/ml anti-CD3 respectively. and 1 μg/ml anti-CD28) polyclonal T cells were co-cul- tured with mast cells, or TTCS-conditioned mast cells Mast cell stimulation (TCM) at a 2:1 ratio in the presence or absence of a neu- The hCBMCs, LAD2 cells, HMC-1 cells were stimulated tralizing antibody against human PD-L1 (20 μg/ml) or a with 50% TTCS or 50% autologous NTCS, or 50% TTCS control IgG (20 μg/ml) for 5 days, and were subsequently for 3, 6 or 12 h, or with different concentrations TTCS injected into the peritoneum in 200 μl of buffered saline (20, 40, 80%) for 24 h, or with 50% TTCS together with on day 7 after inoculation. Tumor size was measured a neutralizing antibody against human TNF-α (20 μg/ml) every 2 days by two independent observers using calipers for 24 h, or with human recombinant (hr) cytokines fitted with a vernier scale. Tumor volumes (V) were calcu- (100 ng/ml) for 24 h. After stimulation, the cells were lated with the formula: V = A × B /2 (A = axial diameter; harvested for flow cytometric analysis and western blot. B = rotational diameter). Once the mice were sacrificed, For the signaling pathway inhibition experiments, the tumors were weighed and photographed, and were further cells were pretreated with 5 μl (10 μM) BAY 11–7082 fixed for immunohistochemical staining, ELISA or real- (an IκBα inhibitor), U0126 (a MEK1/2 inhibitor), time PCR, and the spleens were dissociated into single SP600125 (a c-Jun N-terminal kinase (JNK) inhibitor), cells for flow cytometry. SB203580 (a mitogen-activated protein kinase (MAPK) inhibitor) or Wortmannin (a PI3K inhibitor) for 1 h, then were stimulated with 50% TTCS or hr. TNF-α (100 ng/ml) for 24 h and harvested as above. As the in- Statistical analysis hibitors were dissolved in dimethyl sulfoxide (DMSO), Results are expressed as mean ± SEM. Student t test was parallel cell groups were treated with DMSO (5 μl) or generally used to analyze the differences between two culture media as controls. groups, but when the variances differed, the Mann- Whitney U test was used. Correlations between parame- In vitro mast cell-T cell co-culture system ters were assessed using the Pearson correlation analysis In a 5-day incubation, magnetic bead-purified peripheral and linear regression analysis as appropriate. Overall/ + 5 CD3 T cells (2 × 10 cells/well in 96-well plates) were disease-free survival was defined as the interval between labeled with carboxyfluorescein succinimidyl ester surgery and death/recurrence or between surgery and (CFSE) and co-cultured with autologous mast cells iso- the last observation for surviving/disease-free patients. lated from tumor or non-tumor tissues at a 2:1 (T cell: The known tumor-unrelated deaths (eg, accidental mast cell) ratio in 200 μl RPMI-1640 medium containing death) were excluded from the death record for this 10% fatal bovine serum (FBS), rh IL-2 (20 IU/ml), study. Cumulative survival time was calculated by the anti-CD3 (2 μg/ml), and anti-CD28 (1 μg/ml) antibodies, Kaplan-Meier method, and survival was measured in with or without a human PD-L1 neutralizing antibody month; the log-rank test was applied to compare (20 μg/ml). In another co-culture system, CFSE-labeled between 2 groups. Multivariate analysis of prognostic + 5 magnetic bead-purified peripheral CD3 T cells (2 × 10 factors for patient overall survival was performed using cells/well in 96-well plates) were co-cultured with TTCS- the Cox proportional hazards model. SPSS statistical or NTCS-conditioned hCBMCs or LAD2 cells at a 2:1 ratio software (version 13.0) was used for all statistical Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 4 of 15 analysis. All data were analyzed using 2-tailed tests, and these cells in GC were not actively proliferative (Additional P < 0.05 was considered statistically significant. file 7:FigureS2a andb). Next, wehypothesizedthatGC microenvironment might induce mast cell migration into Results the tumors by chemotaxis. We therefore examined the che- Mast cells are enriched in GC as tumor progress and mokine receptors that are involved in myeloid cell migra- independently predict poor patient survival tion. The majority of tumor-infiltrating mast cells expressed To evaluate the potential role of mast cells in human CXCR4 but not CCR2, CCR4, CCR5, CCR7, CXCR1, GC, we analyzed mast cell percentage within the total CXCR2 or CXCR7 (Fig. 2a; Additional file 7:FigureS2c), CD45 leukocytes from intratumoral, marginal, peritu- while, mast cells in peritumoral or non-tumor tissues moral, and non-tumor tissues of GC patients at various showed lower CXCR4 expression (Fig. 2a). In support of stages. Notably, patients with GC showed a higher mast this, dual immunofluorescence staining showed that cell percentage in intratumoral tissues than marginal, tryptase mast cells expressed CXCR4 in GC tumors peritumoral, and non-tumor tissues (Fig. 1a and b). (Fig. 2a). Taken together, these results suggest that mast Moreover, as the cancer progressed, the percentage of cells may be induced to migrate into the tumor micro- intratumoral mast cells increased significantly (Fig. 1a), environment through CXCR4-mediated chemotaxis. and such intratumoral mast cell accumulation was most Interestingly, we further found that the frequency of mast notable from stage II onwards (Fig. 1e). Similar obser- cells was positively correlated with CXCL12 production vations were made when analyzing the total number of (Fig. 2b), which most likely derived from EpCam tumor mast cells per million total cells in each tissue (Fig. 1c cells (Additional file 7: Figure S2d) in the GC microenvir- and f). Furthermore, immunohistochemical staining onment. Meanwhile, we found that the concentrations of also showed that mast cells were accumulated in tu- CXCL12 in tumor tissues or tumor tissue culture superna- mors (Fig. 1d), indicating a potential role for mast cells tants (TTCS) were significantly increased when compared in the GC microenvironment. In keeping with this find- to that in non-tumor tissues or non-tumor tissue culture ing, increased mast cell percentage and mast cell num- supernatants (NTCS) (Fig. 2c). To substantiate the func- ber were correlated with increased advanced lymphatic tional significance of CXCL12-CXCR4 in the recruitment invasion, tumor size and tumor stage (Additional file 2: of mast cells, mast cell chemotaxis assay was performed Figure S1). and showed that TTCS induced significantly more Next, we evaluated the clinical relevance of intratumoral tumor-infiltrating mast cell migrationthanNTCS fromthe mast cells in GC. Comparing patients with high (≥9.315% same GC patients, and this effect was lost upon pre-treat- median level) versus low (< 9.315%) mast cell percentage ment with neutralizing antibodies against CXCL12 and/or level, the 62-month overall survival rates were significantly CXCR4 (Fig. 2d). Taken together, these data support a lower for those within the higher mast cell percentage model wherein GC tumors secrete chemokine CXCL12 group (Fig. 1e). Similar results were obtained when the pa- which in turn recruits mast cells into the tumor micro- tient cohort was stratified based on intratumoral mast cell environment by CXCL12-CXCR4 interaction. number (Fig. 1f). Importantly, this finding that intratu- moral mast cell percentage and/or number independently Tumor-derived factor TNF-α induces mast cells to express predicted survival was verified by univariate and multivari- PD-L1 ate analyses using a Cox proportional hazard model To better understand these intratumoral mast cells’ (Additional file 3: Table S3). The clinical characteristics of likely function, we performed a detailed immune- all the GC patients are described in Additional file 4: Table phenotype. Notably, we found that intratumoral mast S2, and correlations between intratumoral mast cell per- cells expressed significantly higher level of immuno- centage or mast cell number and clinical characteristics of suppressive molecule PD-L1 (Fig. 3a) but not other GC patients are listed in Additional file 5: Table S4 and molecules with immunosuppressive potential such as Additional file 6: Table S5. Taken together, these findings 2B4, glactin-3, CTLA-4, or ICOSL (Additional file 8: suggest that increased intratumoral mast cells are associ- Figure S3a) than that expressed on peritumoral and ated with tumor progression and poor survival of GC non-tumor mast cells, indicating a potential role for patients. PD-L1 on mast cells in the GC microenvironment. Meanwhile, we hypothesized that GC environments Increased mast cell accumulation in GC is promoted by contribute to the immunosuppressive phenotype of CXCL12-CXCR4-mediated chemotaxis mast cells. Consistent with our hypothesis, compared The results described above suggested that GC microenvir- to NTCS-conditioned mast cells, TTCS-conditioned onment triggers mast cell accumulation, so we wondered mast cells significantly up-regulated PD-L1 expression the cause of such accumulation. We first found that intra- in both time-dependent and dose-dependent manners tumoral mast cells expressed little Ki-67, suggesting that (Fig. 3b). Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 5 of 15 Fig. 1 Mast cells accumulate in GC tumors with disease progression and predict poor patient survival. a Mast cell percentage in CD45 cells or (c)the total + + + number of mast cells per million total cells among TNM stages (I + II vs III + IV) in each tissue of patients with GC by gating on CD45 CD117 FcεRI cells or counting. Cumulative results from 114 GC patients were shown. b Dot plots of surface molecule staining for mast cells gating on CD45 cells. d Representative analysis of tryptase (brown) mast cell distributions in tumor tissues of GC patients by immunohistochemical staining. Scale bars: 100 μm. e and f Intratumoral mast cell percentage (e)ormastcellnumber(f) among TNM stages was compared. Kaplan-Meier plots for overall survival by median mast cell percentage (9.315%) (e) or median mast cell number (4749 per million) (f). The horizontal bars in panels a, c, e and f represent mean values. Each ring in panels a, c, e and f represents 1 patient. **, P < 0.01; n.s., P > 0.05 for groups connected by horizontal lines. MC (%), mast cell percentage; MC (NO.), mast cell number Tumor microenvironment can possess various soluble cytokines including IL-1β, IL-6, IL-10, IL-17, IL-22, IL-23, factors [16], including pro-inflammatory cytokines. To see M-CSF, G-CSF, TNF-α,IFN-γ,TGF-β,etc. We found that which cytokines might induce PD-L1 expression on mast only TNF-α remarkably up-regulated the expression of cells, we first screened pro-inflammatory cytokines in PD-L1 on mast cells (Fig. 3d; Additional file 8:Figure S3b). human GC environments by microarray (Fig. 3c), and Next, we found that the concentrations of TNF-α in tumor stimulated normal mast cells with highly-expressed tissues or TTSC were significantly increased when Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 6 of 15 Fig. 2 CXCL12-CXCR4 chemotaxis mediates mast cell migration and accumulation in GC tumors. a Statistics analysis of CXCR4 mast cell percentage in total mast cells in each samples of patients with GC (n = 26). Expression of molecule CXCR4 on mast cells by gating on + + + + + CD45 CD117 FcεRI cells. Color histograms represent staining of CXCR4; black, isotype control. Tumor-infiltrating CXCR4 tryptase mast cells were defined by immunofluorescence staining. Green, Tryptase; red, CXCR4; and blue, DAPI-stained nuclei. Scale bars: 10 μm. b The correlations between mast cells and CXCL12 production in GC tumors were analyzed. Results were expressed as percentage of mast cells in CD45 cells and CXCL12 concentration in tumor tissues of patients with GC. c CXCL12 concentration between autologous tumor and non-tumor tissues (n = 27) or between autologous TTCS and NTCS (n = 8) was analyzed. d Migration of tumor-infiltrating mast cells was assessed by Transwell assay as described in Materials and methods and statistically analyzed (n = 3). The horizontal bars in panels a and c represent mean values. Each ring or dot in panels a, b or c represents 1 patient. *, P < 0.05; **, P < 0.01 for groups connected by horizontal lines compared to that in non-tumor tissues or NTCS (Fig. 3e). Tumor-derived TNF-α activates NF-κB pathway to induce There was clearly a positive correlation between PD-L1 expression on mast cells PD-L1 mast cell infiltration and TNF-α production To further see which signaling pathways might operate (Fig. 3f) and a high expression of TNF-α receptor II in the mast cell PD-L1 induction, we first pre-treated (TNFRII) on mast cells (Additional file 8:FigureS3c) mast cells with corresponding inhibitors and then ex- within tumors. Next, to evaluate the potential role of posed them to the indicated TTCS. The results showed GC tumor-derived TNF-α in PD-L1 induction on mast that only blocking the signal transduction of NF-κB with cells, we added neutralizing antibody against TNF-α inhibitor BAY 11–7082 effectively suppressed PD-L1 ex- into TTCS/mast cell co-culture. Interestingly, antibody pression on TTCS-conditioned mast cells or blockade of TNF-α efficiently inhibited the induction of TNF-α-stimulated mast cells (Fig. 4a; Additional file 9: PD-L1 on mast cells (Fig. 3g). These findings show that Figure S4a). Furthermore, p65, a direct NF-κBpathway tumor-derived TNF-α plays an essential role in mast downstream substrate (Fig. 4b), but not other pathway cell PD-L1 induction. downstream substrates (Additional file 9:FigureS4b), was Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 7 of 15 Fig. 3 Tumor-derived factor TNF-α induces mast cells to express PD-L1. a Statistics analysis of PD-L1 mast cell percentage in total mast cells in + + + each samples of patients with GC (n = 26). Expression of molecule PD-L1 on mast cells by gating on CD45 CD117 FcεRI cells. Color histograms + + represent staining of PD-L1; black, isotype control. Tumor-infiltrating PD-L1 tryptase mast cells were defined by immunofluorescence staining. Green, Tryptase; red, PD-L1; and blue, DAPI-stained nuclei. Scale bars: 50 μm. b Expression of PD-L1 on hCBMCs exposed to 50% autologous TTCS and NTCS for 24 h, or exposed to 50% TTCS for 6, 12, 24 h, or exposed to 20, 40, 80% TTCS for 24 h. black, isotype control. c Clustering of microarray data for the expression of 40 pro-inflammatory cytokine genes in human tumor tissues from 8 GC patients. d Expression of PD-L1 on hCBMCs exposed to TNF-α for 24 h. black, isotype control. e TNF-α concentration between autologous tumor and non-tumor tissues (n = 24) or between autologous TTCS and NTCS (n = 8) was analyzed. f The correlations between TNF-α and PD-L1 mast cells in human tumors were analyzed. Results are expressed as the number of PD-L1 mast cells per million total cells and TNF-α concentration in tumor tissues. g Expression of PD-L1 on hCBMCs exposed to TTCS with anti-TNF-α antibody for 24 h. Each ring or dot in panels a and f represents 1 patient. *, P < 0.05, **, P < 0.01 for groups connected by horizontal lines predominantly more phosphorylated in mast cells after TTCS or TNF-α. Taken together, these data suggest that treatment with TTCS than that treatment with NTCS, and tumor-derived TNF-α activates NF-κB pathway to induce this phosphorylation was abolished when blocking TNF-α, PD-L1expressiononmastcells. implying that activation of NF-κB signaling pathway is cru- cial for mast cell PD-L1 induction by TNF-α in the GC en- Tumor-infiltrating mast cells suppress T cell immunity vironments. Similar results were obtained when analyzing through PD-L1 PD-L1 on mast cell (Fig. 4b). Furthermore, immunofluores- The co-localization of mast cells and T cells (Fig. 5a) in cence staining (Fig. 4c) and western blot (Fig. 4d) showed the tumoral area of GC tissues, and the significant nega- that PD-L1 expression or p65 phosphorylation was abol- tive correlations between the levels of mast cells and T ished when blocking the signal transduction of NF-κBwith cells in human GC tumors analyzed (Fig. 5b) suggest inhibitor BAY 11–7082 on mast cells either stimulated with that these tumor-infiltrating mast cells may promote Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 8 of 15 Fig. 4 Tumor-derived TNF-α activates NF-κB pathway to induce PD-L1 expression on mast cells. a Expression of PD-L1 on hCBMCs exposed to TTCS or TNF-α with or without BAY 11–7082 (an IκBα inhibitor) for 24 h. black, isotype control. b PD-L1, p65 and p-p65 on/in LAD2 cells exposed to autologous TTCS, NTCS, or TTCS with anti-TNF-α antibody for 24 h (detecting for PD-L1) or 3 h (detecting for p65 and p-p65) were analyzed by western blot. c PD-L1 on HMC-1 cells exposed to TTCS with or without BAY 11–7082 (an IκBα inhibitor) for 24 h was analyzed by immunofluorescence staining. Green, PD-L1; blue, DAPI-stained nuclei. Scale bars: 20 μm. d PD-L1, p65 and p-p65 on/in LAD2 cells exposed to TTCS with or without BAY 11–7082 (an IκBα inhibitor) were analyzed by western blot tumor progression by impairing T cell immunity. Mast human umbilical cord blood-derived cultured mast cells cells from tumor and non-tumor tissues of autologous GC (hCBMCs) for 5 days. Consistent with our hypothesis, patients were therefore isolated and cultured with purified TTCS-conditioned hCBMCs showed significantly more autologous peripheral CD3 T cells for 5 days. Mast cell/ suppression of T cell proliferation and IFN-γ production T-cell co-cultures showed that tumor-infiltrating mast (Fig. 5d). To see whether PD-L1 operates in this T cell sup- cells were superior to non-tumor-derived mast cells in pression, we added PD-L1 neutralizing antibodies in the T inhibiting T cell proliferation and IFN-γ production (Fig. cell/TTCS-conditioned hCBMCs co-culture system. As ex- 5c), suggesting an immunosuppressive function of tumor- pected, PD-L1 blocking by antibody efficiently attenuated infiltrating mast cells in tumor immunity. such T cell suppression mediated by TTCS-conditioned Our next objective was to determine the role of PD-L1 hCBMCs (Fig. 5d). Similar observations were made when on tumor-infiltrating mast cells in T cell suppression. using human mast cell line LAD2 cells (Additional file 10: Therefore, we added neutralizing antibodies against PD-L1 Figure S5a). Besides, tumor-associated mast cells could into our tumoral mast cell/peripheral T-cell co-culture sys- modulate cytotoxic function of T cells as perforin and gran- tem. Interestingly, blockade of PD-L1 significantly attenu- zyme B production from the T cells cocultured with ated such T cell suppression mediated by tumor-infiltrating PD-L1-expressing mast cells decreased significantly mast cells (Fig. 5c). Collectively, these findings show that (Additional file 10: Figure S5d and e). These results above PD-L1 contributes to tumor-infiltrating mast cell-mediated indicate that, in the GC microenvironment, mast cells ac- T cell suppression in vitro. quire ability to suppress T cell function through PD-L1. Given the tumor-infiltrating mast cells suppressed T-cell proliferation and IFN-γ production more than non-tumor Blockade of mast cell-associated PD-L1 on T cell mast cells, we hypothesized that the tumor microenviron- immunity inhibits tumor growth and GC progression ment might have played an important role in this process. To test the suppressive effect of PD-L1 mast cells on T To test this, purified peripheral CD3 Tcells were cell immunity in vivo, we treated TTCS-conditioned co-cultured with TTCS- or NTCS-conditioned primary mast cells (TCM) with PD-L1 blocking or control IgG and Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 9 of 15 Fig. 5 Tumor-infiltrating and tumor-conditioned mast cells suppress T cell immunity through PD-L1. a Representative analysis of tryptase mast cell (green) and CD3 T cell (red) interactions in tumor tissues of GC patients by immunofluorescence. Scale bars: 20 μm. b The correlations between mast cells and T cells in human GC tumors were analyzed. Results were expressed as percentage of mast cells and T cells in CD45 cells in tumor tissues of GC patients. c CFSE (carboxyfluorescein succinimidyl amino ester)-labeled peripheral CD3 T cells of patients with GC were co-cultured for 5 days with autologous mast cells from non-tumor or tumor tissues with or without anti-PD-L1 antibody. Representative data and statistical analysis of T cell proliferation and IFN-γ production were shown (n = 5). d CFSE-labeled peripheral CD3 T cells of donors were co-cultured for 5 days with autologous TTCS-, or NTCS-conditioned hCBMCs with or without anti-PD-L1 antibody. Representative data and statistical analysis of T cell proliferation and IFN-γ production were shown (n = 5). Each dot in panel b represents 1 patient. **, P < 0.01 for groups connected by horizontal lines. hCBMCs, human umbilical cord blood-derived cultured mast cells then injected them together with T cells into our estab- measuring time point from day 19 (Fig. 6a and b). lished human NOD/SCID mice bearing SGC-7901-de- Moreover, mice treated with T cells plus PD-L1 block- rived GC. As expected, mice without T cell transfusions, ing antibody-treated TCM, also showed decreased mice treated with T cells plus TCM or control IgG-treated tumor cell proliferation, and increased CD3 T cell TCM, showed tumor growth and disease progression infiltration and IFN-γ production (Fig. 6cand d; (Fig. 6a and b). Consistent with a vital role in assisting Additional file 11: Figure S6a), as well as an increased tumors of PD-L1 mast cells in vivo, mice treated with T production of cytolytic molecules perforin and gran- cells plus PD-L1 blocking antibody-treated TCM showed zyme B (Fig. 6e; Additional file 11:FigureS6b andc), reduced tumor volumes and disease progression at each compared with the mice treated with T cells plus TCM Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 10 of 15 Fig. 6 Blockade of mast cell-associated PD-L1 on T cell immunity inhibits tumor growth and GC progression in vivo. a Mice were injected with human SGC-7901 cells, as described in Materials and methods. The control animals ( ) received no further injections. The experimental treatments entailed injections with T cells ( ) or T cells in combination with TTCS-conditioned mast cells (TCM) ( ), or T cells in combination with TCM pre-treated with an anti-PD-L1 antibody ( ) or a control IgG ( ). The illustrated data represent tumor volumes (5 mice in each group). The day of tumor cell injection was counted as day 0. *P < 0.05, for groups injections with T cells in combination with TCM pre-treated with an anti-PD-L1 antibody ( ), compared with groups injections with T cells in combination with TCM pre-treated with a control IgG ( ). The tumors were excised and photographed 21 day after injecting the tumor cells. b The weights of tumors were compared. c-e Proliferating cell nuclear antigen (PCNA) (brown) expression, CD3 Tcell infiltration (brown) (c), or IFN-γ (d), perforin and granzyme B (e) production in tumors and IFN-γ-producing T cell response (d) in spleens of mice were compared (n = 5). Scale bars: 100 μm. The horizontal bars represent mean values. **, P < 0.01; n.s., P > 0.05 for groups connected by horizontal lines or control IgG-treated TCM. These findings suggest cells in GC [18], the roles of innate immune cells re- that tumor-associated mast cells suppress T cell main less well understood. Mast cells are a group of immunity in vivo depending on PD-L1 and thereby innate immune cells, which have been reported in GC contribute to tumor growth and GC progression. [14, 19], but the phenotype, functional regulation and clinical relevance of mast cells in human GC micro- Discussion environment remain largely unclear. In this study, we Illuminating the roles of host innate and adaptive im- have shown that within GC mast cells could play a mune cells within the tumor milieu is crucial for un- positive role on promoting tumor progression. We derstanding the development and progression of have found that the percentage of mast cells in tumors human tumors [17]. Although previous studies have was significantly increased at advanced stages of GC, been delineating the functions of adaptive immune with a high percentage of mast cells positively Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 11 of 15 correlating with poor overall survival of GC patients. but little CCR2, CCR4, CCR5, CCR7, CXCR1, CXCR2 Furthermore, tumor-infiltrating mast cells colocalized or CXCR7. Therefore, we are the first to define the vital with T cells, and tumor-derived TNF-α could induce role of CXCL12-CXCR4 chemotaxis in recruiting mast immunosuppressive mast cells (PD-L1 mast cells) cells in GC. Certainly, some other cells can also express (Fig. 3g). Although less efficiently than professional CXCR4 in GC, such as gastric cancer cells [27], mye- antigen-presenting cells (APCs), mast cells can upreg- loid cells [28], myeloid-derived suppressor cells ulate CD69 expression, proliferation and cytokine pro- (MDSCs) [29] and so on. Some studies have shown duction in effector T cells [20, 21] and others have CXCR4 pathway can promote epithelial-mesenchymal been clearly shown to be able to present antigens to T transition in gastric cancer cells [30]. In tumor micro- cells [22]. It is therefore easy to envisage, when they environment, CXCR4 MDSCsand neutrophilscan be express the immune inhibitory ligand PD-L1 they may recruited by CXCL12 and they impair the anti-tumor inhibit T-cell immunity via PD-L1 as shown in in vitro functions of CD8 cytotoxic T cells leading to increased co-culture as well as in vivo in the absence of PD-L1 tumor metastasis [29, 31]. neutralizing antibody in this study. These results are Mast cells have been identified in tumors recently. similar to those from our previous studies on neutro- However, very little is currently known about the phils, which are also not considered as professional phenotype and function of these tumor-infiltrating APCs [23]. The pathological role of mast cells within mast cells. Immunosuppression has been widely ac- the tumor microenvironment suggests a novel tumor knowledged as a hallmark of cancer [17]. Interestingly, immune escaping mechanism during GC progression. we screened the expression of immunosuppressive mol- In humans, mast cell infiltration in tumors influences ecules on tumor-infiltrating mast cells, and we found disease progression and patient survival [24]. Hence, the that mast cells within GC expressed high level im- analysis of mast cell infiltration in GC is a crucial area of munosuppressive molecule PD-L1, suggesting that they clinical investigation. Our data shed some light on the mayplaya role to directlymodulate effector function. clinical relevance of mast cells in GC. We observed a Crosstalk between PD-L1 and PD-1 is one of the main significant positive association between the percentage/ mechanisms leading to immunosuppression of T cells number of mast cells and advanced clinical features of [32]. In our present study, we uncovered that GC GC, such as lymphatic invasion, tumor size, and tumor tumor-associated mast cells effectively inhibited T-cell’s stage (Additional file 2: Figure S1). In addition, we immune function in a PD-L1–dependent manner in uncovered that an increased frequency/number of intra- vitro and in vivo. Furthermore, immunofluorescence tumoral mast cells predicted a lower rate of disease-free staining showed that most tumor–infiltrating mast cells survival, which was oppositely correlated with intratu- localized in close proximity to CD3 T cells in GC, in- moral mast cell levels (Additional file 12: Figure S7), dicating that immunosuppression could be mediated by suggesting that tumor-infiltrating mast cells may become the interaction of mast cells and T cells in a PD- a helpful clinical prognostic marker in the future. L1-dependent manner. We are the first to report that Regardless of patient outcome, the percentage/num- the induced PD-L1 expression on tumor-infiltrating ber of mast cells was notably increased in tumors com- mast cells enabled them to suppress T-cell proliferation pared with non-tumor tissues. This may be a and IFN-γ production. Consistent with our findings in consequence of either enhanced proliferation of intra- GC, blocking PD-L1/ PD-1 pathway could also achieve tumoral mast cells or an increased migration of mast a good prognosis in testicular germ cell tumors [33]. cells to tumor tissues. As for the little expression of We previously reported that degranulation of mast cells Ki-67 in mast cells in GC, we exclude the possibility of prompted GC progression [34], which offered add- the enhanced proliferation of intratumoral mast cells itional targets to consider for mast cell directed therap- and predicted that the tumors possibly induced migra- ies besides target for mast cell-associated PD-L1. In tion of mast cells by chemotaxis. Recently, mast cells addition, tumor-associated mast cells can also be acti- had been reported to be attracted into the tumor vated to secrete sufficient amount of biological mole- microenvironment in a SCF-dependent manner [25]. In cules to mediate tumor progression [35]. Previous our case, we identified a new mechanism for mast cell studies found that mast cell released histamine via chemotaxis in GC. In comparison with non-tumor tis- c-Kit/SCF, which increased cholangiocarcinoma growth, sue, gastric tumors could secrete more CXCL12, which and angiogenesis [36]. Moreover, mast-cell-derived me- attracted migration of mast cells. Additionally, some in- diators: histamine, CXCL1 and CXCL10 could increase vestigations showed that mast cells also expressed che- thyroid cancer cell survival and DNA synthesis in vitro mokine receptors such as CXCR1 and CXCR2 in [37]. In pancreatic cancer, activated mast cells promote different cancers [26]. In our study, we found that tumor progression by IL-13 and tryptase [38]. Import- intratumoral mast cells expressed high level CXCR4 antly, in vivo GC model, we found TTCS-conditioned Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 12 of 15 Fig. 7 A proposed model of cross-talks among mast cells, T cells, and tumor cells leading to mast cell-mediated immunosuppressive and protumorigenic effects in the GC microenvironment. CXCL12-CXCR4 chemotaxis mediates the recruitment and accumulation of mast cells into the GC microenvironment, which could be up-regulated PD-L1 expression via NF-κB signaling pathway activation by tumor-derived TNF-α. Mast cells inhibit T-cell proliferation and function in a PD-L1-dependent manner in GC, which promotes GC progression mast cells effectively inhibited T cell immunity and promoter [46]. In this study, we verify TNF-α as a cru- promoted GC progression, and such T cell suppression cial pro-inflammatory factor within GC microenviron- was through PD-L1 on the mast cells as blocking ment, which derived from GC tumor and effectively PD-L1 reversed it. Certainly, some other cell types are induces the expression of PD-L1 on mast cells via activa- also known to mediate T-cell suppression in tumor micro- tion of NF-κB signaling pathway. environment. For instance, a recent study showed that The recent success of checkpoint inhibitor monother- LC3-associated phagocytosis (LAP) in myeloid cells regu- apy opens up a new era of cancer treatment, however, lated macrophages in the tumor microenvironment, which it’s not suited for everyone. How to achieve precision as a result suppressed T cell function and promoted tumor immunotherapy further, select the preponderant benefi- tolerance [39]; and the number of tumor-infiltrating mye- ciaries, and improve the efficacy of pharmacoeconomics loid cells was correlated with early metastatic relapse [40]. are important topics. Tumor mutational burden (TMB) Furthermore, it has been recognized that tumors often re- is a promising new biomarker for predicting the efficacy cruit MDSCs that inhibits T cell infiltration and activation of cancer immunotherapy, which is measured by whole- [41]. Therefore tumor resistance to single-agent immune exome sequencing and associated with clinical benefit checkpoint inhibitor therapy could be a result of lack infil- from multiple checkpoint inhibitors. TMB exhibited trating T cells or too many suppressive immune cells [42, joint predictive utility in identifying responders and non- 43]. It will be very important to further explore the role of responders to the PD-1 antibody pembrolizumab, the the suppressive, PD-L1-expressing mast cells reported in number and type of alterations may prove to be valuable this study and their relation with other suppressive immune for judging the potential usefulness of immune check- cell types. Here, our results indicate that the important point inhibitors [47]. Besides, high TMB may be a re- PD-1-PD-L1 immunosuppression pathway operates in GC sponse biomarker for PD-1/PD-L1 blockade in tumors via mast cells. such as melanoma and non-small cell lung cancer PD-L1 is expressed in a wide range of cell types and (NSCLC), and results show a linear correlation between tissues and shown to be overexpressed with immune ac- higher TMB and favorable outcome parameters with tivation, such as inflammation or tumor [23, 44]. Previ- anti-PD-1/PD-L1 monotherapy [48]. In addition, PD-1/ ous studies have shown that PD-L1 up-regulation can be PD-L1 also plays an important role in some inflamma- induced mainly by activated CD8 cytotoxic T cells-de- tory disease. Asthma is a chronic airway inflammation rived interferon-γ in HCC milieu [45]. Meanwhile, our associated largely with CD4 T cells, eosinophils, and team identified that tumor-derived GM-CSF effectively mast cells. PD-L1 expressed on mast cells was shown to induces PD-L1 expression on neutrophils via activation regulate T-cell activation and tolerance. And PD-L1 of JAK-STAT3 pathway [23]. Besides, research has re- mast cell is a negative regulator of conventional CD4 T vealed that tumor-derived hypoxia inducible factor 1a cells, and understanding of the role will give propulsion (HIF-1a) upregulated PD-L1 on MDSCs by binding to a to the development of novel therapeutic approaches in hypoxia-response element (HRE) in the PD-L1 proximal allergic asthma [49]. Besides, alopecia areata (AA) is a Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 13 of 15 CD8 T-cell dependent autoimmune disease and mast + + tryptase mast cells and Ki-67 cells were defined by immunofluorescence cells are crucial immunomodulatory cells implicated in staining. Green, Tryptase; red, Ki-67; and blue, DAPI-stained nuclei. Scale bars: 50 μm. (c) Expression of CCR2, CCR4, CCR5, CCR7, CXCR1, CXCR2 and CXCR7 the regulation of T cell-dependent immunity in AA. A + + + on tumor-infiltrating mast cells by gating on CD45 CD117 FcεRI cells. Color report showed that a number of PD-L1 mast cells ap- histograms represent staining of chemokine receptors; black, isotype control. peared to be further reduced in lesional AA skin com- (d) Representative analysis of CXCL12-expressing (red) EpCam tumor cells (green) in tumor tissues of GC patients by immunofluorescence. Scale bars: pared to healthy skin, particularly during their 20 μm. (e) Expression of CD80 and CD86 in tumor-infiltrating mast cells by interactions with CD8 T-cells. Mast cells in AA are + + + gating on CD45 CD117 FcεRI cells. Color histograms represent staining of skewed towards pro-inflammatory activities and that CD80 and CD86; black, isotype control. (TIF 5879 kb) MC-CD8 T-cell interactions in AA are predominantly Additional file 8 Figure S3. Tumor-derived factor TNF-α induces mast cells to express PD-L1. (a) Expression of 2B4, glactin-3, CTLA-4, and ICOSL pro-inflammatory. This finding suggests that treatment + + + on mast cells by gating on CD45 CD117 FcεRI cells. Color histograms regimens which promote an immune-inhibitory pheno- represent staining of 2B4, glactin-3, CTLA-4, and ICOSL; black, isotype type and/or suppress the switch towards a pro-inflam- control. (b) Expression of PD-L1 on hCBMCs exposed to IL-1β, IL-6, IL-10, IL-17, IL-22, IL-23, M-CSF, G-CSF, IFN-γ, TGF-β (100 ng/ml) for 24 h. black, matory mast cells phenotype, should down-regulate isotype control. (c) Expression of TNF-α receptor II (TNFRII) on tumor- undesired CD8 T-cell responses in AA [50]. infiltrating mast cells. Black, isotype control. (TIF 1497 kb) Additional file 9 Figure S4. Tumor-derived TNF-α activates NF-κB pathway to induce PD-L1 expression on mast cells. (a) Expression of Conclusion PD-L1 on hCBMCs exposed to 50% TTCS with or without U0126 (an ERK In brief, based on our in vitro and in vivo data, we inhibitor), Wortmannin (a PI3K inhibitor), SB203580 (a MAPK inhibitor), or propose a model involving complex interactions between SP600125 (a JNK inhibitor) for 24 h. black, isotype control. (b) p44/42 and p-p44/42, Akt and p-Akt, p38 and p-p38, JNK and p-JNK in LAD2 cells mast cells, T cells and tumor cells within GC (Fig. 7). exposed to TTCS with or without anti-TNF-α antibody were analyzed by First, CXCL12-CXCR4 chemotaxis mediates the recruit- western blot. (TIF 1181 kb) ment of mast cells into GC microenvironment. And Additional file 10 Figure S5. Tumor-infiltrating and tumor-conditioned then, tumor-derived TNF-α induces the overexpression mast cells suppress T cell immunity through PD-L1. (a) CFSE-labeled peripheral CD3 T cells of donors were co-cultured for 5 days with TTCS-, of PD-L1 on mast cells via NF-κB signaling pathway ac- + or NTCS-conditioned LAD2 cells with or without anti-PD-L1 antibody. tivation. Finally, the PD-L1 mast cells inhibit T-cell Representative data and statistical analysis of T cell proliferation and IFN-γ proliferation and function in a PD-L1-dependent man- production were shown (n = 5). *, P < 0.05; **, P < 0.01 for groups con- nected by horizontal lines. (b) Surface staining of FcεRI and CD117 of ner. In conclusion, our study has highlighted a notable generated human umbilical cord blood-derived cultured mast cells role for mast cells in human GC and identified new (hCBMCs) was shown. Results were expressed as percentage of + + + mechanisms of GC-associated mast cells mediating CD117 FcεRI cells by gating on CD45 cells. Iso, Isotype control antibody. (c) Toluidine blue staining of sorted human tumor-infiltrating tumor progression. Overall, blocking these pathological mast cells was shown. Scale bars: 10 μm. (d) Granzyme B and mast cells and the TNF-α-PD-L1 immunosuppressive perforin production were compared in the supernatants (n = 5), which pathway may be a useful therapeutic strategy for pre- CD3 T cells of patients with GC were co-cultured with autologous mast cells from non-tumor or tumor tissues with or without anti-PD-L1 venting GC progress. antibody. (e) Granzyme B and perforin production were compared in the supernatants (n = 5), which peripheral CD3 T cells of donors were co- Additional files cultured with autologous TTCS-, or NTCS-conditioned hCBMCs with or without anti-PD-L1 antibody. **, P < 0.01 for groups connected by horizontal lines. hCBMCs, human umbilical cord blood-derived cultured Additional file 1 Table S1. Antibodies and other reagents. (DOCX 29 mast cells. (TIF 1676 kb) kb) Additional file 11 Figure S6. Blockade of mast cell-associated PD-L1 Additional file 2 Figure S1. Mast cell percentage (a) or mast cell on T cell immunity inhibits tumor growth and GC progression in vivo. number (b) and its potential correlations with clinical parameters. Mast (a-c) Mice were injected with human SGC-7901 cells, as described in cell percentage in CD45 leukocytes and mast cell number per million Materials and methods. The control animals () received no further total cells were analyzed for correlations with clinical pathological injections. The experimental treatments entailed injections with T parameters. **, P < 0.01; n.s., P > 0.05 for groups connected by horizontal cells () or T cells in combination with TTCS-conditioned mast cells lines. Each dot represents one patient. CEA, carcinoembryonic antigen; (TCM) (), or T cells in combination with TCM pre-treated with an anti- H.pylori Ab, Helicobacter pylori antibody. (TIF 1496 kb) PD-L1 antibody () or a control IgG (). The illustrated data represent Additional file 3 Table S3. Univariate and multivariate analyses of tumor volumes (5 mice in each group). The day of tumor cell factors associated with survival. (DOCX 20 kb) injection was counted as day 0. The expression of IFN-γ (a), anti- tumor molecules perforin (b)and granzyme B(c)in tumorsof mice Additional file 4 Table S2. Clinical characteristics of 114 patients with on day 21 after tumor cell injection were compared (n =5). The gastric cancer. (DOCX 19 kb) horizontal bars represent mean values. *, P < 0.05; **, P <0.01 for Additional file 5 Table S4. Correlations between mast cell percentage groups connected by horizontal lines. (TIF 841 kb) and clinic pathological features of patients with gastric cancer. (DOCX 20 Additional file 12 Figure S7. Kaplan-Meier plots for disease-free survival kb) by median mast cell percentage (9.315%) or median mast cell number Additional file 6 Table S5. Correlations between mast cell number and (4749 per million). MC (%), mast cell percentage; MC (NO.), mast cell clinic pathological features of patients with gastric cancer. (DOCX 21 kb) number. (TIF 108 kb) Additional file 7 Figure S2. CXCL12-CXCR4 chemotaxis mediates mast cell Additional file 13 Table S6. Primer and probe sequences for real-time migration and accumulation in GC tumors. (a) Expression of Ki-67 in tumor- PCR analysis. (DOCX 20 kb) + + + infiltrating mast cells by gating on CD45 CD117 FcεRI cells. Color histograms Additional file 14 Supplementary Materials and Methods. (DOCX 63 kb) represent staining of Ki-67; black, isotype control. (b) Tumor-infiltrating Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 14 of 15 Abbreviations 3. Wang F, Meng W, Wang B, Qiao L. Helicobacter pylori-induced gastric APCs: Antigen-presenting cells; GC: Gastric cancer; hCBMCs: Human umbilical inflammation and gastric cancer. Cancer Lett. 2014;345:196–202. cord blood-derived cultured mast cells; IFN: Interferon; IL: Interleukin; 4. Choi IJ, Kook MC, Kim YI, Cho SJ, Lee JY, Kim CG, et al. Helicobacter pylori MDSCs: Myeloid-derived suppressor cells; PBMCs: Peripheral blood therapy for the prevention of Metachronous gastric Cancer. N Engl J Med. mononuclear cells; PD-L1: Programmed death-ligand 1; TNF-α: Tumor 2018;378:1085–95. necrosis factor-α 5. Wen T, Wang Z, Li Y, Li Z, Che X, Fan Y, et al. A four-factor Immunoscore system that predicts clinical outcome for stage II/III gastric Cancer. Cancer Immunol Res. 2017;5:524–34. Acknowledgments The authors are grateful to Prof. Wei Zhang for donating the human mast 6. Ferrone C, Dranoff G. Dual roles for immunity in gastrointestinal cancers. J cell line LAD2 cells, and Xiao Luo for collecting samples from gastric cancer Clin Oncol. 2010;28:4045–51. patients. 7. Ying L, Yan F, Meng Q, Yu L, Yuan X, Gantier MP, et al. PD-L1 expression is a prognostic factor in subgroups of gastric cancer patients stratified according to their levels of CD8 and FOXP3 immune markers. Funding Oncoimmunology. 2018;7:e1433520. This work was supported by grant of National Natural Science Foundation of 8. Lee HE, Chae SW, Lee YJ, Kim MA, Lee HS, Lee BL, et al. Prognostic China (81670510 and 81872016), Founding by Southwest Hospital implications of type and density of tumour-infiltrating lymphocytes in (SWH2017YBXM-07) and National Key Research and Development Program gastric cancer. Br J Cancer. 2008;99:1704–11. of China (2016YFC1302200). 9. Kalesnikoff J, Galli SJ. New developments in mast cell biology. Nat Immunol. 2008;9:1215–23. Availability of data and materials 10. Maciel TT, Moura IC, Hermine O. The role of mast cells in cancers, All data generated or analyzed during this study are included in this F1000Prime Rep. 2015;7:9. published article [and its additional files]. Primer and probe sequences for 11. Sammarco G, Gadaleta CD, Zuccala V, Albayrak E, Patruno R, Milella P, et al. real-time PCR analysis and Supplementary Materials and Methods are shown Tumor-associated macrophages and mast cells positive to Tryptase are as Additional files 13 and 14. correlated with angiogenesis in surgically-treated gastric Cancer patients. Int J Mol Sci. 2018;19:1176-89. Authors’ contributions 12. Liu J, Zhang Y, Zhao J, Yang Z, Li D, Katirai F, et al. Mast cell: insight into Study concept and design and drafting of the manuscript: YZ. Acquisition of remodeling a tumor microenvironment. Cancer Metastasis Rev. 2011;30: data and analysis and interpretation of data: YZ, YL. Critical revision of the 177–84. manuscript for important intellectual content: YZ, and WC. Statistical analysis: 13. Danelli L, Frossi B, Gri G, Mion F, Guarnotta C, Bongiovanni L, et al. Mast YZ, YL. Obtained funding: YZ, YZ, and JC. Technical, or material support: YZ, cells boost myeloid-derived suppressor cell activity and contribute to the XW, NC, YT, TW, FM, LP, JZ, PC, YL, HK, CH, BH, QM, QZ, and JC. Final development of tumor-favoring microenvironment. Cancer Immunol Res. approval of the version to be published: YZ. All authors read and approved 2015;3:85–95. the final manuscript. 14. Lin C, Liu H, Zhang H, Cao Y, Li R, Wu S, et al. Tryptase expression as a prognostic marker in patients with resected gastric cancer. Br J Surg. 2017; Ethics approval and consent to participate 104:1037–44. The study was approved by the Ethics Committee of the Southwest Hospital 15. Guidolin D, Ruggieri S, Annese T, Tortorella C, Marzullo A, Ribatti D. Spatial of Third Military Medical University. Written informed consent was obtained distribution of mast cells around vessels and glands in human gastric from each subject. carcinoma. Clin Exp Med. 2017;17:531–9. 16. Mushtaq MU, Papadas A, Pagenkopf A, Flietner E, Morrow Z, Chaudhary SG, Consent for publication et al. Tumor matrix remodeling and novel immunotherapies: the promise of Informed consent for publication of data was obtained from all patients. matrix-derived immune biomarkers. J Immunother Cancer. 2018;6:65. 17. Schreiber RD, Old LJ, Smyth MJ. Cancer immunoediting: integrating Competing interests immunity's roles in cancer suppression and promotion. Science. 2011;331: The authors declare that they have no competing interests. 1565–70. 18. Stromnes IM, Hulbert A, Pierce RH, Greenberg PD, Hingorani SR. T-cell localization, activation, and clonal expansion in human pancreatic ductal Publisher’sNote adenocarcinoma. Cancer Immunol Res. 2017;5:978–91. Springer Nature remains neutral with regard to jurisdictional claims in 19. Ammendola M, Sacco R, Donato G, Zuccala V, Russo E, Luposella M, et al. published maps and institutional affiliations. Mast cell positivity to tryptase correlates with metastatic lymph nodes in gastrointestinal cancer patients treated surgically. Oncology-Basel. 2013; Author details 85:111–6. National Engineering Research Center of Immunological Products, 20. Gaudenzio N, Espagnolle N, Mars LT, Liblau R, Valitutti S, Espinosa E. Cell-cell Department of Microbiology and Biochemical Pharmacy, College of cooperation at the T helper cell/mast cell immunological synapse. Blood. Pharmacy, Third Military Medical University, No.30 Gaotanyan Street, 2009;114:4979–88. Chongqing 400038, China. Department of General Surgery and Centre of Minimal Invasive Gastrointestinal Surgery, Southwest Hospital, Third Military 21. Kambayashi T, Allenspach EJ, Chang JT, Zou T, Shoag JE, Reiner SL, et al. Medical University, No.30 Gaotanyan Street, Chongqing 400038, China. Inducible MHC class II expression by mast cells supports effector and Department of Obstetrics and Gynecology, Research Institute of Surgery, regulatory T cell activation. J Immunol. 2009;182:4686–95. Daping Hospital, Third Military Medical University, Chongqing, China. La 22. Stelekati E, Bahri R, D'Orlando O, Orinska Z, Mittrucker HW, Langenhaun R, Trobe Institute of Molecular Science, School of Molecular Science, La Trobe et al. Mast cell-mediated antigen presentation regulates CD8+ T cell University, Bundoora, Vic 3085, Australia. Affiliated Hospital of North Sichuan effector functions. Immunity. 2009;31:665–76. Medical College, Nanchong, Sichuan Province, China. 23. Wang TT, Zhao YL, Peng LS, Chen N, Chen W, Lv YP, et al. Tumour-activated neutrophils in gastric cancer foster immune suppression and disease Received: 19 July 2018 Accepted: 11 February 2019 progression through GM-CSF-PD-L1 pathway. Gut. 2017;66:1900–11. 24. Giannou AD, Marazioti A, Spella M, Kanellakis NI, Apostolopoulou H, Psallidas I, et al. Mast cells mediate malignant pleural effusion formation. J Clin Invest. 2015;125:2317–34. References 1. Van Cutsem E, Sagaert X, Topal B, Haustermans K, Prenen H. Gastric cancer. 25. Huang B, Lei Z, Zhang GM, Li D, Song C, Li B, et al. SCF-mediated mast cell Lancet. 2016;388:2654–64. infiltration and activation exacerbate the inflammation and 2. Ndegwa N, Ploner A, Liu Z, Roosaar A, Axell T, Ye W. Association between immunosuppression in tumor microenvironment. Blood. 2008;112:1269–79. poor oral health and gastric cancer: a prospective cohort study. Int J Cancer. 26. Varricchi G, Galdiero MR, Loffredo S, Marone G, Iannone R, Marone G, et al. 2018;143:2281-8. Are mast cells MASTers in Cancer? Front Immunol. 2017;8:424. Lv et al. Journal for ImmunoTherapy of Cancer (2019) 7:54 Page 15 of 15 27. Xiang Z, Zhou ZJ, Xia GK, Zhang XH, Wei ZW, Zhu JT, et al. A positive 48. Goodman AM, Kato S, Bazhenova L, Patel SP, Frampton GM, Miller V, et al. crosstalk between CXCR4 and CXCR2 promotes gastric cancer metastasis. Tumor mutational burden as an independent predictor of response to Oncogene. 2017;36:5122–33. immunotherapy in diverse cancers. Mol Cancer Ther. 2017;16:2598–608. 28. Yang J, Kumar A, Vilgelm AE, Chen SC, Ayers GD, Novitskiy SV, et al. Loss of 49. Singh AK, Stock P, Akbari O. Role of PD-L1 and PD-L2 in allergic diseases CXCR4 in myeloid cells enhances antitumor immunity and reduces and asthma. Allergy. 2011;66:155–62. melanoma growth through NK cell and FASL mechanisms. Cancer Immunol 50. Bertolini M, Zilio F, Rossi A, Kleditzsch P, Emelianov VE, Gilhar A, et al. Res. 2018;6:1186–98. Abnormal interactions between perifollicular mast cells and CD8+ T- cells may contribute to the pathogenesis of alopecia areata. PLoS One. 29. Zhuang Y, Peng LS, Zhao YL, Shi Y, Mao XH, Chen W, et al. CD8(+) T cells 2014;9:e94260. that produce interleukin-17 regulate myeloid-derived suppressor cells and are associated with survival time of patients with gastric cancer. Gastroenterology. 2012;143:951–62. 30. Cheng Y, Song Y, Qu J, Che X, Song N, Fan Y, et al. The chemokine receptor CXCR4 and c-MET cooperatively promote epithelial-mesenchymal transition in gastric Cancer cells. Transl Oncol. 2018;11:487–97. 31. Seubert B, Grunwald B, Kobuch J, Cui H, Schelter F, Schaten S, et al. Tissue inhibitor of metalloproteinases (TIMP)-1 creates a premetastatic niche in the liver through SDF-1/CXCR4-dependent neutrophil recruitment in mice. Hepatology. 2015;61:238–48. 32. Kuang DM, Zhao Q, Peng C, Xu J, Zhang JP, Wu C, et al. Activated monocytes in peritumoral stroma of hepatocellular carcinoma foster immune privilege and disease progression through PD-L1. J Exp Med. 2009; 206:1327–37. 33. Siska PJ, Johnpulle R, Zhou A, Bordeaux J, Kim JY, Dabbas B, et al. Deep exploration of the immune infiltrate and outcome prediction in testicular cancer by quantitative multiplexed immunohistochemistry and gene expression profiling. Oncoimmunology. 2017;6:e1305535. 34. Lv YP, Peng LS, Wang QH, Chen N, Teng YS, Wang TT, et al. Degranulation of mast cells induced by gastric cancer-derived adrenomedullin prompts gastric cancer progression. Cell Death Dis. 2018;9:1034. 35. Marichal T, Tsai M, Galli SJ. Mast cells: potential positive and negative roles in tumor biology. Cancer Immunol Res. 2013;1:269–79. 36. Johnson C, Huynh V, Hargrove L, Kennedy L, Graf-Eaton A, Owens J, et al. Inhibition of mast cell-derived histamine decreases human cholangiocarcinoma growth and differentiation via c-kit/stem cell factor- dependent signaling. Am J Pathol. 2016;186:123–33. 37. Melillo RM, Guarino V, Avilla E, Galdiero MR, Liotti F, Prevete N, et al. Mast cells have a protumorigenic role in human thyroid cancer. Oncogene. 2010; 29:6203–15. 38. Ma Y, Hwang RF, Logsdon CD, Ullrich SE. Dynamic mast cell-stromal cell interactions promote growth of pancreatic cancer. Cancer Res. 2013;73: 3927–37. 39. Cunha LD, Yang M, Carter R, Guy C, Harris L, Crawford JC, et al. LC3- associated phagocytosis in myeloid cells promotes tumor immune tolerance. Cell. 2018;175:429–41. 40. Kim K, Skora AD, Li Z, Liu Q, Tam AJ, Blosser RL, et al. Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells. Proc Natl Acad Sci U S A. 2014;111: 11774–9. 41. Marvel D, Gabrilovich DI. Myeloid-derived suppressor cells in the tumor microenvironment: expect the unexpected. J Clin Invest. 2015; 125:3356–64. 42. Kamran N, Kadiyala P, Saxena M, Candolfi M, Li Y, Moreno-Ayala MA, et al. Immunosuppressive myeloid Cells' blockade in the glioma microenvironment enhances the efficacy of immune-stimulatory gene therapy. Mol Ther. 2017;25:232–48. 43. Kusmartsev S, Nagaraj S, Gabrilovich DI. Tumor-associated CD8+ T cell tolerance induced by bone marrow-derived immature myeloid cells. J Immunol. 2005;175:4583–92. 44. Tsukamoto H, Fujieda K, Miyashita A, Fukushima S, Ikeda T, Kubo Y, et al. Combined blockade of IL-6 and PD-1/PD-L1 signaling abrogates mutual regulation of their immunosuppressive effects in the tumor microenvironment. Cancer Res. 2018;78(17):5011-22. 45. XieQK, ZhaoYJ, PanT, Lyu N, Mu LW,LiSL, et al. Programmed death ligand 1 as an indicator of pre-existing adaptive immune responses in human hepatocellular carcinoma. Oncoimmunology. 2016;5:e1181252. 46. Noman MZ, Chouaib S. Targeting hypoxia at the forefront of anticancer immune responses. Oncoimmunology. 2014;3:e954463. 47. Cristescu R, Mogg R, Ayers M, Albright A, Murphy E, Yearley J, et al. Pan- tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy. Science. 2018;362:eaar3593.

Journal

Journal for ImmunoTherapy of CancerSpringer Journals

Published: Feb 26, 2019

There are no references for this article.