Identification Of CD40~+MDSC Subpopulation And Preliminary Analysis Of Its Biology In Gastric Tumor Model

Objective Gr-1+CD11b+myeloid-derived suppressor cells (MDSC) are considered asthe most critical cells participating in tumor immune escape mechanism in the tumorenvironment. The heterogeneity and biological complexity of these cells limit a comprehensiveunderstanding of MDSC. Thus identifying their subpopulations is the prerequisite for MDSCbiological investigations. Here we intended to identify subpopulations of MDSC andexplore their biology, provide new strategies for the gastric cancer immunotherapy.Methods C57BL/6mices were injected subcutaneously with tumor cell lines to buildtumor-bearing mice models. The levels of splenic and tumor-infiltrating MDSC and CD40on MDSC were detected dynamically by flow cytometry. In vitro, MDSC were collectedafter stimulation with agonist anti-CD40and stained with anti-Gr-1mAb, anti-CD11bmAb and Annexin-V (invitrogen). The proportions of Gr-1+CD11b+Annexin-V+cells weredetermined by flow cytometry to analyze the effects of CD40signal on MDSC apoptosis.CD40+MDSC, CD40-MDSC were sorted from splenocytes of gastric-tumor bearing miceusing the BD FACSAria cell sorting system. Sorted CD40+MDSC and CD40-MDSC wererespectively centrifuged onto microscope slides, stained with Wright to observe theirmorphology. Different gene expressions between two subsets were further analyzed bygenome-wide microarray experiments. Real-time quantitative polymerase chain reaction(RT-PCR) was performed to confirm the results of gene chip.Results (1) CD40is expressed on splenic and tumor-infiltrating MDSC fromtumor-bearing mice, the percentages of CD40expression were obviously higher than thatof tumor-free mice. However in gastric tumor model, a significantly decreased level ofCD40expression was observed along with an increased number of MDSC during tumorprogression. Pearson’s correlation analysis revealed that MDSC levels positively correlatedwith tumor progression (spleen: r=0.9318, p<0.0001; tumor: r=0.9169, p<0.0001), CD40expression levels inversely correlated with MDSC accumulation (spleen: r=-0.8690, p=0.0002; tumor: r=-0.8673, p=0.0003). Apoptosis detectation indicated CD40activationcan induce MDSC apoptosis. Compared with PBS treatment, the apoptosis oftumor-infiltrating MDSC significantly increased from71.37±2.68to79.09±3.29(mean±SEM,%; p=0.0352). The splenic MDSC apoptosis significantly increased from25.43±10.45to32.30±10.27(mean±SEM,%; p=0.0167).(2) CD40, as a specific marker,can divide MDSC to CD40+MDSC, CD40-MDSC. Morphological observation showed thatCD40+MDSC displayed mononuclear, whereas CD40-MDSC displayed ring-shaped nucleor polymorphonuclear. The results of RT-PCR indicated that expression of TNF-α, IL-6,IL-12, prostaglandin E2(PGE2), IL-10, Arginase (Arg), IL-1β from CD40+MDSC werehigher compared to CD40-MDSC, especially PGE2(Foldchange=7.29) and Arginase(Foldchange=3.12) were upregulated significantly (Foldchange≥2.0). In our whole genomeexpression analysis, gene expression of chemokines and suppressive phenotype onCD40+MDSC, such as chemokine (C-X-C motif) ligand9(CXCL9), chemokine (C-X-Cmotif) ligand10(CXCL10), chemokine (C-X-C motif) receptor5(CXCR5), CD40, CD83,fms-like tyrosine kinase3(FLT3), were significantly upregulated than that of CD40-MDSC(Foldchange≥2.0).Conclusion (1) CD40was expressed on MDSC from tumor-bearing mice and itsexpression level was obviously higher than tumor-free mice. However a significantlydecreased level of CD40expression on MDSC was observed and correlated with MDSCaccumulation during gastric-tumor progression. CD40activation can induce MDSCapoptosis. Down-regulation of CD40expression may contribute to MDSC accumulationby facilitating MDSC resistance to apoptosis. All that indicated CD40on MDSC canpromote inflammatory responses at the early phase of tumor, create an inflammatoryenvironment suitable for the growth of gastric cancer; with CD40down-regulation intumor progression, CD40-mediated MDSC apoptosis gradually weakened, leading to thegradual accumulation of MDSC, creating immune escape environment for tumor.(2) CD40is another specific marker which can divide MDSC to CD40+MDSC and CD40-MDSC.There was great difference in phenotype and functional factors between CD40+MDSC andCD40-MDSC, suggesting that two subsets represented different biological functions andmay exert absolutely different effects on promoting tumor progression.