The Study On The Mechanism Of NLRP3 Inflammasome And ??T17/MDSCs Mediated Immune Imbalance In Lymphoma

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The role of NLRP3 inflammasome-mediated immunosuppression in DLBCL pathogenesisBackground:Diffuse large B-cell lymphoma(DLBCL)is the most common form of non-Hodgkin lymphoma(NHL)and accounts for 30-35%of all NHLs worldwide.Although rituximab-based chemotherapy as a first-line treatment significantly improved the efficacy,still one-third of patients relapsed or had a refractory disease,the clinical prognosis of which was very poor.90%of relapsed or refractory cases cannot be relieved by conventional salvage immunochemotherapy and hematopoietic stem cell transplantation.Therefore,there is an urgent need to further clarify the pathogenesis of DLBCL and identify high-risk factors,thereby finding novel therapeutic targets.Inflammation plays a critical role in tumorigenesis,and the inflammatory response is an important part of tumor microenvironment.Inflammasomes are important factors that lead to inflammatory cytokines secretion and the subsequent inflammatory cascade.NLRP3 is the most widely studied inflammasome.Pathogen invasion or cells damage will recruit and assemble the NLRP3 inflammasome,which activates caspase-1,cleaves IL-1? and IL-18 precursors,and produces active IL-1? and IL-18.On one hand,the NLRP3 inflammasome contributes to restricting pathogen invasion and clearing endogenous damage.On the other hand,the inflammatory response as a result of NLRP3 inflammasome activation can form a pro-tumorigenic environment,thereby promoting tumor growth and metastasis.We previously reported that the serum IL-18 levels were elevated in newly-diagnosed NHL patients and decreased after remission.Furthermore,NLRP3 inflammasome activation in lymphoma cells by adenosine triphosphate and lipopolysaccharides(LPS)is accompanied by increased expression of NLRP3-related genes.However,the precise role of NLRP3 inflammasome in the pathogenesis of DLBCL remains unclear.The malignant cells can escape and suppress immune surveillance in various ways.The major components of the tumor immune microenvironment include effector T cells,myeloid-derived suppressor cells(MDSCs),tumor-associated macrophages(TAMs),and regulatory T cells(Tregs).MDSCs,TAMs,and Trees are characterized by their ability to potently impair anti-tumor immune responses.In addition,immune checkpoints,expressed on immune cells and tumor cells,can regulate immune homeostasis and lead to tumor immune escape by inhibiting the anti-tumor immune responses of T cells and NK cells.Some malignant B cells escape immune surveillance by overexpressing programmed cell death ligand 1(PD-L1),which interacts with programmed cell death protein 1(PD-1)on activated T cells to relay an inhibitory signal that blocks T-cell proliferation and cytokine production.Furthermore,a mechanistic study has reported MDSCs-dependent T-cell inhibition in DLBCL,which is associated with the release of IL-10 and S100A12 and expression of PD-L1.A previous study has reported a close relationship among the NLRP3 inflammasome,immunosuppressive cells,and immune checkpoints in head and neck squamous cell carcinoma(HNSCC).Given the crucial roles of the NLRP3 inflammasome in tumorigenesis and immunosuppression,we hypothesized that it contributes to lymphomagenesis by regulating anti-tumor immunity.At present,the mechanism of immunosuppression in DLBCL is still unclear.Therefore,a better understanding of the mechanisms underlying immune suppression in DLBCL would help identify predictive biomarkers,reverse the immunosuppressive status,and improve immunotherapeutic outcomes.Objective:This study aims to elucidate the function of NLRP3 inflammasome in the pathogenesis of DLBCL and their critical role in mediating immunosuppression in lymphoma.We intend to confirm the pro-tumorigenic role of NLRP3 inflammasome in a murine lymphoma model,especially to explore the effect of NLRP3 inflammasome on T cell phenotype and function,further to explore the effect of NLRP3 inhibition on the efficacy of immune checkpoint blockade therapy.Materials and Methods:1.Tissue microarray:35 DLBCL tissues and 35 normal lymphoid tissues were contained in tissue microarray and assessed with immunohistochemistry.We compared the expression of IL-1?,IL-18 and PD-L1 between DLBCL tissues and normal lymphoid tissues,and analyzed the correlation between their expression and clinical features of patients with DLBCL.2.Flow cytometry:peripheral blood samples from DLBCL patients with initial diagnosis,remission,or refractory/relapse stage were obtained.Peripheral blood samples from healthy donors and other types of NHL were also obtained.Peripheral blood mononuclear cells(PBMCs)were collected for further investigation.The anti-tumor immune responses in DLBCL patients were analyzed by flowcytometry.The frequencies of CD3+T cells,CD4+T cells,CD8+T cells,and NK cells in peripheral blood were detected,as well as the expression of immune checkpoints on T cells and NK cells,cytokines production by T cells and NK cells,and the proportion of MDSCs and Tregs.The relationship between the levels of TIM-3 and PD-1 expressed on T cells and the clinical features of DLBCL patients was analyzed.3.RT-qPCR:total RNA of PBMCs was extracted from DLBCL patients and healthy donors.RT-qPCR were conducted to analyze the NLRP3 inflammasome-related genes including IL1B,IL18,NLRP3,ASC,CASP1,NFKB1.4.Cell culture and inflammasome activation assay:human DLBCL cell lines(DB,OCI-LY3,and RC)were cultured as per the manufacturer's instructions.LPS and adenosine 5'-triphosphate(ATP)disodium salt hydrate were used to activate NLRP3 inflammasomes in cells,and MCC950 was used to effectively inhibit the activation of NLRP3 inflammasomes.Total proteins were extracted from the cells of different treatment groups,and Western Blot was used to verify whether NLRP3 inflammasome was activated or not.5.Co-cultures of lymphoma cells and PBMCs:OCI-LY3 and RC cells were pretreated with LPS and ATP,with or without MCC950 inhibition,and PBMCs were isolated from peripheral blood of healthy donors.Both OCI-LY3 and RC cells were separately co-cultured with freshly isolated PBMCs.On days 3,7,and 10,T cells were harvested,stained with antibodies,and then analyzed using flow cytometry.6.Murine lymphoma model:A murine lymphoma model was established by subcutaneously injection with murine lymphoma cells,and randomly allocated to different groups.For NLRP3 inflammasome blockade,mice were intraperitoneally injected with 15mg/kg MCC950.For in vivo IL-18 and IL-1? neutralization,anti-mouse IL-18 and anti-mouse IL-1? antibodies were respectively administrated intraperitoneally at a dose of 200 ?g per mouse,three times a week for two weeks.For the PD-L1 blockade,the tumor-bearing mice were intraperitoneally injected with 200 ?g anti-mouse PD-L1 antibody on alternate days.The control group was treated with the same dose of PBS or IgG.Tumor growth was monitored every other day.The levels of IL-1? and IL-18 in tumor and plasma were analyzed by ELISA.Western Blot was used to assess the expression levels of NLRP3 inflammasome-related proteins and PD-L1.The expression of PD-1 and TIM-3 on the T cells,the proportion of T cells and their ability to secrete IFN-??TNF-? and Granzyme B,and the frequencies of immunosuppressive MDSCs,TAMs and Tregs were assessed by flowcytometry.7.Statistical analysis:All data in this study was analyzed using SPSS 21 and GraphPad Prism 7 system.Data are presented as the mean ± SD of three independent experiments.The P values were assessed with 2-tailed unpaired Student t tests or Mann-Whitney U,and values less than 0.05 were considered statistically significant.Results:1.Higher levels of IL-18 were detected in DLBCL tissues and correlated with PD-L1 levels:tissue microarray analysis showed that IL-18 levels were significantly elevated in DLBCL tissues,and DLBCL patients with the non-GCB phenotype expressed higher IL-18 than patients with GCB phenotype.Meanwhile,We also found that IL-18 and IL-1? expression is associated with CD 10 and MUM1,which have been identified as prognostic biomarkers in DLBCL.By analyzing the immunohistochemical data of patients,we observed higher IL-18 and IL-1?expression in the CD 10(-)or MUM1(+)patients.Furthermore,PD-L1 in DLBCL tissue was significantly up-regulated compared with normal lymphoid tissue,and the level of PD-L1 was correlated with Ki-67 expression-Further analysis showed that the PD-L1 expression in the TME was positively correlated with IL-18 levels.In addition,the IL1B and IL18 expression in PBMCs of DLBCL patients was significantly elevated compared with that in healthy donors.2.The anti-tumor immune responses were inhibited in DLBCL patients:flow cytometric analysis showed that TNF-? and IFN-y secretion by T cells was significantly decreased,but there was no significant change in the proportion of T cells.Moreover,the proportion of NK cells in DLBCL patients and their ability to secrete perforin and granzyme B was significantly reduced.The PD-1/TIM-3-expressing T cells and TIM-3/BTLA-expressing INK cells in DLBCL patients were significantly increased.By analyzing clinical data of DLBCL patients,we found that the PD-1 expression on T cells was positively correlated with levels of serum lactate dehydrogenase(LDH)in patients,but there was no significant correlation between TIM-3 expression and LDH levels.In addition,patients with advanced-stage DLBCL(Ann Arbor ? or ?)and international prognostic index(IPI)>3 harbors high levels of PD-1+and TEM-3+T cells,suggesting that PD-1 and TIM-3 is related to poor prognosis.Moreover,compared with healthy donors,the proportion of MDSCs and Tregs in peripheral blood of DLBCL patients was significantly increased,indicating an immunosuppressive state in DLBCL patients.The proportion of Tregs in patients with remission was significantly lower than that in newly-diagnosed patients,whereas the proportion of Tregs in refractory or relapsed patients was significantly higher than that in patients with remission.3.NLRP3 inflammasome in DLBCL cell lines can be activated and affect PD-L1 expression:after stimulation with LPS and ATP,the cleaved IL-1?(p17)and PD-L1 levels were significantly elevated.In contrast,the NLRP3 inhibitor MCC950 reduced cleaved IL-1? levels and suppressed PD-L1 expression in OCI-LY3,RC,and DB cell lines.DLBCL cell lines(RC or OCI-LY3 cells)pretreated with LPS and ATP,or MCC950 were co-cultured with PBMCs.We found that stimulation of the NLRP3 inflammasome in DLBCL cell lines significantly decreased the proportion of CD8+T cells in the co-culture system,and this effect was abrogated by the NLRP3 inflammasome inhibitor MCC950.4.NLRP3 inflammasome blockade inhibited lymphoma growth and reduced PD-L1 expression:high levels of IL-18 and IL-1? were observed in tumor homogenates of the lymphoma-bearing mice,which pointed to an inflamed local tumor microenvironment.To directly assess the involvement of NLRP3 inflammasome in lymphomagenesis,we subsequently treated the lymphoma-bearing mice with MCC950 or placebo.Importantly,NLRP3 inflammasome blockade by MCC950 significantly reduced lymphoma growth and delayed tumor progression,thus confirming its tumorigenic role in lymphoma.The western blot analysis supported that MCC950 markedly decreased NLRP3 inflammasome activation.Of note,MCC950 markedly decreased PD-L1 and pSTAT3 expression in lymphoma tissues.5.Blocking NLRP3 inflammasome in vivo enhanced anti-tumor immune responses and reduced immunosuppressive cell populations:MCC950 treatment significantly increased the proportion of tumor-infiltrating CD8+T cells and splenic T cells,as well as TNF-? producing CD3+and CD4+T cells,while IFN-y production by splenic CD3+and CD4+T cells was significantly reduced in the mice treated with MCC950 compared with that in control group.Additionally,NLRP3 inhibition significantly decreased the expression of PD-1 on CD3+ and CD4+ T cells in the spleen and lymph nodes,and reduced the expression of TIM-3 on splenic T cells and lymphatic CD4+T cells.Furthermore,blocking NLRP3 inflammasome markedly decreased the proportion of MDSCs and TAMs in the tumor tissues,spleen,and peripheral blood,along with that of Tregs in the tumor tissues and spleen,indicating a strong association between NLRP3 inflammasome activation and immunosuppression.6.Blocking IL-18 in vivo reduced immunosuppressive cell populations:we injected lymphoma-bearing mice with IL-1? or IL-18 neutralizing antibody,respectively,and found that blocking IL-1? or IL-18 inhibited lymphoma growth in mice.Flow cytometric analysis showed that the percentages of tumor-infiltrating CD8+T cells as well as lymphatic and circulating CD8+T cells in mice treated with anti-IL-18 antibody were significantly increased compared with those in the control group.Anti-IL-1? treatment increased the percentage of CD8+T cells in lymph nodes,whereas decreased the proportion of CD8+ T cells in peripheral blood.Furthermore,IL-18 inhibition in lymphoma-bearing mice resulted in lower expression of PD-1 on CD8+cells in lymph nodes and peripheral blood,as well as reduced levels of PD-1 on CD4+cells in peripheral blood,spleen,and tumor,whereas IL-1?inhibition did not affect PD-1 expression on T cells.Administration of anti-IL-18 antibody also downregulated TIM-3 expression on tumor-infiltrating T cells,whereas anti-IL-1? increased TIM-3-expressing T cells in tumors.In addition,the frequencies of MDSCs and TAMs were significantly decreased in the peripheral blood of lymphoma-bearing mice treated by anti-IL-18 and anti-IL-1? antibodies.The proportion of Tregs in tumor and spleen decreased in response to IL-18 neutralization.Consistent with the data from MCC950-treated mice,the lymphoma-bearing mice undergoing anti-IL-1? and anti-IL-18 treatments exhibited downregulated PD-L1 levels,along with reduced pSTAT3 levels in the tumor tissues.7.NLRP3 inflammasome blockade combined with anti-PD-L1 treatment has antagonistic effects:in order to detect the role of NLRP3 inflammasome in the efficacy of anti-PD-L1 treatment,the lymphoma-bearing mice were treated with MCC950,anti-PD-L1 antibody,or combination therapy.We found that both MCC950 and anti-PD-L1 antibody markedly reduced the tumor burden of lymphoma mice.However,the combination treatment diminished the tumor-suppressive effect.Flow cytometry revealed an elevation of the tumor-infiltrating CD3+and CD8+T cells upon PD-L1 blockade,and a significant increase of IFN-y and TNF-? production by splenic T cells.However,the proportion of tumor-infiltrating CD8+T cells in the combination treatment group was significantly lower than that in the anti-PD-L1 monotherapy group.Similarly,the levels of IFN-? and TNF-? produced by splenic T cells were also decreased in the combination treatment group compared with those in the anti-PD-L1 blockade alone group.On the other hand,PD-1 levels on splenic and lymphatic T cells were significantly elevated after the PD-L1 blockade compared with those in the control or MCC950-treated group.Moreover,TIM-3 levels on splenic and lymphatic T cells were also higher in the anti-PD-L1-treated group than those in the MCC950-treated group.In addition,PD-1 expression on splenic and lymphatic T cells,as well as TIM-3 expression on lymphatic T cells,were significantly higher in the combination treatment group than that in the MCC950 monotherapy group.Furthermore,compared with MCC950 monotherapy,both PD-L1 blockade and the combination treatment significantly increased the frequencies of tumor-infiltrating MDSCs and TAMs,but did not affect the proportion of tumor-infiltrating Tregs.Conclusion:1.The NLRP3 inflammasome was activated in DLBCL patients,and the expression of effector cytokine IL-18 was up-regulated in DLBCL tissues,which was positively correlated with the PD-L1 expression.2.The anti-tumor immune responses in DLBCL patients were suppressed.The PD-L1 expression was significantly elevated in the TMEs.The immunosuppressive cell populations such as MDSCs,TAMs and Tregs were significantly expanded,and the expression of immune checkpoints PD-1 and TIM-3 increased,which was closely related to the poor prognosis of DLBCL.3.Blocking NLRP3 inflammasome significantly inhibited the lymphoma progression in mice,and enhanced the anti-tumor immune responses,reduce the proportion of immunosuppressive cells.IL-18 served as the main effector cytokine involved in the negative regulation of anti-lymphoma immunity.4.The NLRP3 inflammasome inhibitor and anti-PD-L1 antibody had antagonistic effect in the treatment of lymphoma in mice.Section ? The role of ?8T17 and myeloid-derived suppressor cells in the development of gastric MALT lymphomaBackground:Gastric mucosa-associated lymphoid tissue(MALT)lymphoma is closely related to H.Pylori(HP)infection,which could regulate immune homeostasis in the stomach.Gastric MALT lymphoma is a typical model of chronic inflammation-induced gastric tumor development.During this process,the infiltration of a large number of immune cells and the production of cytokines causes immune homeostasis imbalance in the local microenvironment of the gastric mucosa,which plays an important role in the development of gastric MALT lymphoma.Therefore,exploring the role of immune regulatory factors in gastric MALT lymphoma is of great significance to clarify the mechanism of inflammation-tumor transformation.Myeloid-derived suppressor cells(MDSCs)are composed of myeloid cells at an early differentiation stage and mediate tumor immune tolerance mainly by suppressing T and NK cell functions.The highly active arginase 1(Arg-1)in MDSCs consumes the L-arginine,which is essential for T cell receptor(TCR)synthesis,and inhibits the expression of Cyclin D3 and CDK4,resulting in the blockade of T cells proliferation.MDSCs also produce nitric oxide through inducible nitric oxide synthase(iNOS)to induce T cell apoptosis.In addition to the above inhibitory pathways,MDSCs could exert immunosuppressive effects through regulatory T cells(Tregs).In conclusion,MDSCs play an important role in tumor immune tolerance.??T17,as an important component of mucosal immunity,is highly enriched in the gastrointestinal mucosa.??T17 plays an important role in maintaining homeostasis of the gastric mucosa microenvironment,resisting the invasion of microorganisms and regulating the adaptive immune response.Studies have found that ??T cells are activated in gastric mucosa by H.Pylori associated chronic inflammation,suggesting that ??T cells exert important functions in the development of gastric MALT lymphoma.??T17 is a subset of ??T cells which secretes interleukin 17(IL-17).Although ??T17 could inhibit tumorigenesis in some studies,other studies have shown that ??T17 would promotes tumor growth in multiple malignancies.It has been found that ??T17 infiltrates the tumor microenvironment and produces IL-17 in ovarian cancer,fibrosarcoma,skin cancer and colon cancer,which plays pro-tumorigenic roles.However,the role of ??T17 in gastric MALT lymphoma is unclear.In studies on colorectal cancer and hepatocellular carcinoma,it was found that??T17 can mediate immune tolerance in the tumor microenvironment by recruiting and activating MDSCs.Moreover,activated MDSCs can secrete IL-23 and IL-1?,which would promote the polarization of ??T17 cells,forming a positive feedback loop.In view of these findings,we speculate that ??T17 may contribute to the development of gastric MALT lymphoma by regulating MDSCs.Chronic inflammation caused by Helicobacter pylori infection leads to imbalance of the microenvironmental immune homeostasis.The activated ??T17 secretes IL-17 and other cytokines,recruits MDSCs and mediates tumor immune escape,participating in the development of gastric MALT lymphoma.Objective:This study focused on the regional immune microenvironment of gastric MALT lymphoma and intends to clarify the regulatory role of ??T17,MDSCs and related cytokines in the process of gastric MALT lymphomagenesis,and explore the mechanism underlying immune regulation of gastric MALT lymphoma from inflammation to tumor.Materials and Methods:1.The mice were divided into the H.felis-infected and control groups,and accordingly inoculated with the bacterial suspension or PBS three times every other day via the intragastric route.Bacterial colonization was assessed by rapid urease test.Hematoxylin and eosin staining was performed to record the presence of lymphoid follicles and lymphoepithelial lesions.The features of lymphoid aggregates were assessed through immunohistochemistry.2.Peripheral blood,spleen and stomach tissues of mice at different time points after H.felis infection were isolated,and MDSCs proportions in different groups were detected by flow cytometry.Immunofluorescence and immunohistochemistry were performed to detect the level and distribution of MDSCs(Gr-1+CD11b+),Arg-1 and iNOS in the murine stomach.3.ELISA and RT-qPCR were performed to detect the IL-17 levels in the gastric tissues of control and H.felis-infected mice.The frequencies of ??T17(TCR??+IL-17+),Th 17(CD4+IL-17+)and Tc 17(CD8+IL-17+)from the two groups of mice at different time points after infection were analyzed by flow cytometry.4.The comparison of different expression of cytokines and chemokines in the two group of mice:RT-qPCR was performed to detect the Toll-like receptor(TLR),cytokines and chemokines.IL-1? and IL-23 levels were detected by ELISA.NLRP3 inflammasome activation in murine stomach was evaluated by Western Blot.5.Peripheral blood and tissues samples of gastric MALT lymphoma and healthy donors were collected and detected by flow cytometry to compare the difference of MDSCs(CD45+Lin-CD33+HLA-DR-CD11b+),Tregs(CD4+CD25+Foxp3+),??T17(TCR??+IL-17+),Th17(CD4+IL-17+)and Tc17(CD8+IL-17+)proportions in patients and controls.Immunofluorescence was performed to detect the levels of MDSCs(Gr-1+CD11b+).Immunohistochemistry was performed to detect Arg-1,iNOS,IL-1? and IL-23 in gastric MALT lymphoma tissues.6.Data were expressed as mean ± standard deviation(SD)and evaluated by the Fisher's exact test,Student's t-test,and Mann-Whitney test as appropriate.All tests were performed using SPSS 17.0 and GraphPad Prism 6.0 system.Two-tailed P-values<0.05 were considered statistically significant.Results:1.Long-term infection of H.felis lead to gastric MALT lymphomagenesis,the process of which is highly consistent with the development of human gastric MALT lymphoma.From 8 months after infection,we observed lymphoid follicles formation in the gastric mucosa of H.felis infected mice.And the severity of gastric pathological change in H.felis-infected mice gradually increased in a time-dependent manner.Lymphoepithelial lesions(LEL)appeared in the gastric mucosa 14 months after infection.These changes simulated the histopathological characteristics of MALT lymphomagenesis.Furthermore,the lymphoid aggregates consisted predominantly of B-cells.2.Increased infiltration of MDSCs was observed in the immune microenvironment of gastric mucosa in H.felis infected mice.As shown by flowcytometric analysis,the number of MDSCs in peripheral blood was unaffected at the early-stage of infection(8-to 11-months).However,at the later stages(14 to 19 months)of infection,the circulating MDSCs increased significantly,accompanied by gastric accumulation of MDSCs and severe LELs.Moreover,Arg-1 was significantly upregulated in the stomach of H.felis-infected mice.3.Large amounts of ??T17 cells were accumulated in the gastric mucosa of H.felis-infected mice.Eight months after H.felis infection,the mRNA and protein levels of IL-17 in the infected mice were significantly higher than those in control mice.Flow cytometry data confirmed that among the IL-17-producing T cells,the proportion of ??T17 in the spleen of H.felis-infected mice was higher than that in control mice,but there was no significant change in the percentage of Th17 and Tc17,suggesting that ??T17 is possibly the main immune regulatory cell population in H.felis-induced pathologies.Moreover,the proportion of splenic??T17 cells increased in a time-dependent manner during chronic H.felis infection,suggesting its association with disease progression.4.The expression of TLR2,TLR7 and TLR9 increased in gastric tissue after H.felis infection.Western Blot analysis showed that NLRP3 inflammasome was activated in murine stomach after H.felis infection.Importantly,IL-1? and IL-23 were significantly elevated in the gastric homogenates of H.felis-infected mice at 8-months post-infection,which may participate in the activation of ??T17 cells.On the other hand,we found that the expression of CCR6 and CCL20 in the gastric tissue of H.felis-infected mice was significantly higher than that of control mice,suggesting that ??T17 is recruited to the gastric tissues by these chemokines.5.Flow cytometry showed that MDSCs and Tregs in peripheral blood of patients with gastric MALT lymphoma were significantly increased.Furthermore,MDSCs were also enriched in the gastric MALT lymphoma tissues,which was accompanied by a significant upregulation of Arg-1 and iNOS.The expression of IL-1? and IL-23 was also significantly up-regulated in gastric MALT lymphoma tissues compared to the paired normal tissues.These results indicate an imbalance of immune homeostasis in patients with gastric MALT lymphoma,which may be involved in lymphomagenesis.Conclusion:1.Persistent H.felis infection induces lymphoepithelial lesions in murine stomach,which is consistent with the development of human gastric MALT lymphoma.2.MDSCs are accumulated in the tumor microenvironment of gastric MALT lymphoma,both in patients and H.felis-infected murine model.The upregulation of Arg-1 or iNOS is involved for MDSCs to mediate immunosuppression in gastric MALT lymphomagenesis.3.Accumulation of ??T17 cells was both validated in gastric MALT lymphoma patients and murine model.The elevated cytokines IL-23 and IL-1?,as well as chemokines CCL20/CCR6,may be involved in the accumulation of ??T17 cells and the subsequent immunosuppression.And ??T17 increases in a time-dependent manner,which may participate in the progression of gastric MALT lymphoma by recruiting MDSCs.