Tumor derived cell-free nucleic acid upregulates programmed death-ligand 1 expression in neutrophil via intracellular Toll-like receptor signaling

IF 20.1 1区 医学 Q1 ONCOLOGY
Suguru Saito, Duo-Yao Cao, Tomohiro Shibata, Yan Liu, Aoi Otagiri-Hoshi, Xiaojiang Cui, Kenneth E. Bernstein
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Although some stimuli have been identified, it is still unclear whether the nucleic acid sensing system (NAS) participates in PD-L1 upregulation in neutrophils [<span>6</span>]. Here, we report that increased cell-free nucleic acid (CFNA) upregulates PD-L1 expression via intracellular Toll-like receptor (TLR) activation in neutrophils following tumor expansion.</p><p>Flow cytometry analysis showed that the expression of PD-L1 was gradually increased in peripheral blood (PB) neutrophil after inoculating B16-F10 melanoma cells or EO771 breast cancer cells into wildtype (WT) mice (Figure 1A, protocol is shown in the Supplementary Materials and gating strategy of flow cytometry is shown in Supplementary Figure S1). Notably, the expression of PD-L1 was significantly increased in PB neutrophils of B16-F10-inoculated mice as early as day 3 post-injection compared to those of naïve mice. Although EO771-inoculated mice did not show significantly increased PD-L1 expression in PB neutrophil at days 3 and 7 of post tumor inoculation, there was a significant, pronounced upregulation at day 14 (Figure 1A). Intratumor (IT) neutrophils showed the largest increase of PD-L1 expression compared to neutrophils in PB, spleen and bone marrow (BM) 14 days post inoculation in both types of tumors. The PD-L1 expression level in BM neutrophils was lower than that of PB and spleen neutrophils in B16-F10 inoculated mice. In EO771-inoculated mice, the PD-L1 expression levels in BM and spleen neutrophils were similar, but slightly lower than that in PB (Supplementary Figure S2A and B). Interestingly, similar to the observation in PB, spleen and BM neutrophils also showed significant increases in PD-L1 levels in tumor-bearing mice compared to those of naïve mice, implying that neutrophil PD-L1 upregulation occurs systematically in these murine tumor models (Supplementary Figure S2C and D). Given these data, we decided to investigate circulating factors that may induce changes in PD-L1 levels in neutrophils of tumor-bearing mice, and found that the plasma CFNA levels were significantly increased in the tumor-bearing mice compared to the mice before tumor inoculation (Figure 1B). Linear regression analyses showed strong positive correlations between the plasma CFNA and PB neutrophil-associated PD-L1 expression levels in tumor-bearing mice (Figure 1C). Of note, both the plasma CFNA (Figure 1D) and neutrophil PD-L1 expression levels (Supplementary Figure S3) were positively correlated with the tumor volumes in these mice. In vitro experiments revealed that medium supplemented with plasma of tumor-bearing mice (14 days post tumor inoculation) significantly increased PD-L1 expression in naïve mouse BM-derived neutrophils compared to naïve mouse plasma-supplemented medium (Figure 1E). DNase or RNase treatment abolished this effect suggesting that CFNA components are capable of increasing PD-L1 expression in neutrophils, an observation that has important clinical implications given that cancer patients often have increased plasma cell-free DNA (cfDNA) levels compared to healthy individuals [<span>7</span>].</p><p>Next, we investigated whether the activation of intracellular NAS, particularly TLR signaling, triggers PD-L1 upregulation in neutrophils [<span>8</span>]. For this purpose, in vitro stimulation assays were performed using intracellular TLR ligands, such as polyinosinic: polycytidylic acid (Poly (I:C)) for TLR3 stimulation, R837 (Imiquimod) for TLR7/8 stimulation, R848 (Resiquimod) for TLR7/8 stimulation, and ODN1826 (Class B CpG oligonucleotide) for TLR9 stimulation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte-colony-stimulating factor (G-CSF) were used as positive controls to increase PD-L1 expression in the neutrophils [<span>4, 5, 9</span>]. Except for Poly (I:C), R837, R848, and ODN1826 significantly increased PD-L1 expressions in BM-derived neutrophils compared to the controls (Figure 1F and G). The conditioned media (CM) of B16-F10 or EO771 cell culture also exhibited similar effects in neutrophil PD-L1 upregulation. The responsibility of intracellular TLR in PD-L1 upregulation was proven by an inhibition assay using E6446 (TLR7/9 inhibitor) in the neutrophils upon R848 or ODN1826 stimulation. PD-L1 expression was suppressed in neutrophils by E6446 treatment (Figure 1H). Intracellular TLR inhibition also suppressed PD-L1 upregulation in neutrophils cultured in tumor-bearing mice plasma supplemented medium or cancer cell CM (Figure 1I). These results further support the essential role of intracellular TLR signaling in neutrophil PD-L1 upregulation. The upregulation of <i>Pdl1</i> mRNA expression was confirmed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in GM-CSF or R848 treated BM-derived neutrophils (Supplementary Figure S4). In addition, flow cytometry analysis showed that R837 or R848 stimulation increased PD-L1 expression in dimethyl sulfoxide (DMSO)-differentiated HL-60 (dHL-60) cells, a human neutrophil model, suggesting that this intracellular TLR activation-mediated PD-L1 upregulation was mechanistically conserved in both mouse and human cells (Supplementary Figure S5). Western blot (WB) showed that GM-CSF, R837, R848, and ODN1826 treatments increased phosphorylated-signal transducer and activator of transcription 3 (pSTAT3) levels in BM-derived neutrophils, respectively (Figure 1J). PD-L1 upregulation in these treatments was suppressed by inhibition of STAT3 phosphorylation using Stattic in the stimulated neutrophils (Figure 1K). The STAT3 inhibition also suppressed PD-L1 upregulation in neutrophils cultured in cancer cell CM (Supplementary Figure S6). Additionally, STAT3 inhibition significantly suppressed <i>Pdl1</i> mRNA upregulation in neutrophils upon GM-CSF or R848 stimulation as compared to control treatment (Supplementary Figure S7). As STAT3 has been known as one of the regulators in PD-L1 expression [<span>6</span>], our results suggest that STAT3 activation may be involved in intracellular TLR-dependent PD-L1 upregulation in neutrophils.</p><p>Since PD-L1<sup>+</sup> myeloid cells are characterized as possessing immunosuppressive activity against T cells [<span>4, 5</span>], we investigated whether intracellular TLR-stimulated or CFNA-exposed neutrophils also exhibits a suppressive effect by employing a co-culture system with T cell (Figure 1L). BM-derived neutrophils were first pre-cultured in the medium supplemented with or without R837, R848, ODN1826, or plasma originating from B16-F10 tumor-bearing mice to increase PD-L1 expression as verified by flow cytometry analysis (Supplementary Figure S8). GM-CSF was added to all the cultures to sustain neutrophil survival. The pre-treated neutrophils were washed and then co-cultured with splenic CD3<sup>+</sup> T cells in the presence of anti-CD3 and anti-CD28 monoclonal antibodies (mAbs), and the suppressive effect of neutrophils on T cells was assessed by examining the levels of interferon-gamma (IFN-γ) production and activation marker CD69 expression in the T cells. GM-CSF-stimulated neutrophils suppressed IFN-γ production as well as CD69 expression in CD8<sup>+</sup> T cells (Figure 1M and N, Supplementary Figure S9). Notably, the TLR ligand or tumor-bearing mouse plasma-exposed neutrophils further decreased IFN-γ production and CD69 expression in CD8<sup>+</sup> T cells compared to the neutrophils treated with GM-CSF alone. These pre-cultured neutrophils could also suppress CD4<sup>+</sup> T cell function in the co-culture system (Supplementary Figure S10). Nuclease treatments for tumor-bearing mouse plasma supplemented medium suppressed PD-L1 upregulation in the pre-cultured neutrophils (Supplementary Figure S11), and these neutrophils reduced their suppressive effects against CD8<sup>+</sup> T cells which resulted in equivalent IFN-γ production to GM-CSF-pre-cultured neutrophils in the co-culture systems (Supplementary Figure S12). Finally, we investigated whether PD-1/PD-L1 blockade can restore CD8<sup>+</sup> T cell function in the presence of PD-L1-upregulated neutrophils. The reduced IFN-γ production in CD8<sup>+</sup> T cells cocultured with immunosuppressive neutrophils was significantly restored by anti-PD-L1 mAb treatment compared with isotype Ab treatment (Figure 1O).</p><p>In summary, this study found that intracellular TLR stimulation upregulated PD-L1 expression in neutrophils. Moreover, we posit that tumor-released CFNA participates in PD-L1 upregulation of neutrophils via intracellular NAS represented by TLR7, 8, and 9. The TLR-mediated PD-L1 upregulation results in neutrophils gaining immunosuppressive activity which dampens T cell function, and thus implicates a potential new target for anti-cancer immunotherapy (Figure 1P). An important remaining question is the mechanism of CFNA uptake in neutrophils, which may be via endocytosis or micropinocytosis. Other NASs, such as cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) and retinoic acid-inducible gene-I (RIG-I)-like receptors, which recognize cytosolic DNA and double-stranded RNA (dsRNA), respectively, may also play roles in sensing tumor-derived CFNA and regulating PD-L1 expression in neutrophils [<span>10</span>]. In addition, specific CFNA sequences triggering intracellular TLR-mediated PD-L1 upregulation in neutrophils also remain to be identified. Our results suggest the need to investigate PD-L1-upregulated immunosuppressive neutrophils in cancer patients and determine whether they may serve as a predictable marker for effectiveness of PD-1/PD-L1 blockade therapy.</p><p>Suguru Saito, Duo-Yao Cao, Tomohiro Shibata, Yan Liu, and Aoi Otagiri-Hoshi performed all experiments. Suguru Saito performed data analysis and finalization. Suguru Saito, Xiaojiang Cui and Kenneth E. Bernstein established methodology. Suguru Saito wrote original manuscript. Suguru Saito, Xiaojiang Cui and Kenneth E. Bernstein finalized manuscript. Xiaojiang Cui and Kenneth E. Bernstein supervised this study. All authors read and approved the final manuscript.</p><p>The authors have no conflict of interests.</p><p>This study was supported by the National Institutes of Health grants (R01AI164519, 2R01CA151610, R21CA280458),  and American Heart Association's Career Development award (23CDA1052548), U.S. Department of Defense (W81XWH-18-1-0067) and the Glazer Foundation.</p><p>All animal experimental protocols were reviewed and approved by the Animal Welfare Committee of Cedars-Sinai Medical Center (#8780, #8109).</p>","PeriodicalId":9495,"journal":{"name":"Cancer Communications","volume":"45 1","pages":"4-8"},"PeriodicalIF":20.1000,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11758147/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cancer Communications","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cac2.12615","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"ONCOLOGY","Score":null,"Total":0}
引用次数: 0

Abstract

Neutrophils are innate immune cells that function predominantly against pathogens, while recent studies have revealed additional crucial roles in various diseases, including cancers [1-3]. For instance, neutrophils expressing the co-inhibitory molecule programmed death-ligand 1 (PD-L1) were identified as novel immunosuppressive myeloid cells that impair cytotoxic T cell (CTL) activity via programmed cell death protein 1 (PD-1)/PD-L1 interaction [4, 5]. Although some stimuli have been identified, it is still unclear whether the nucleic acid sensing system (NAS) participates in PD-L1 upregulation in neutrophils [6]. Here, we report that increased cell-free nucleic acid (CFNA) upregulates PD-L1 expression via intracellular Toll-like receptor (TLR) activation in neutrophils following tumor expansion.

Flow cytometry analysis showed that the expression of PD-L1 was gradually increased in peripheral blood (PB) neutrophil after inoculating B16-F10 melanoma cells or EO771 breast cancer cells into wildtype (WT) mice (Figure 1A, protocol is shown in the Supplementary Materials and gating strategy of flow cytometry is shown in Supplementary Figure S1). Notably, the expression of PD-L1 was significantly increased in PB neutrophils of B16-F10-inoculated mice as early as day 3 post-injection compared to those of naïve mice. Although EO771-inoculated mice did not show significantly increased PD-L1 expression in PB neutrophil at days 3 and 7 of post tumor inoculation, there was a significant, pronounced upregulation at day 14 (Figure 1A). Intratumor (IT) neutrophils showed the largest increase of PD-L1 expression compared to neutrophils in PB, spleen and bone marrow (BM) 14 days post inoculation in both types of tumors. The PD-L1 expression level in BM neutrophils was lower than that of PB and spleen neutrophils in B16-F10 inoculated mice. In EO771-inoculated mice, the PD-L1 expression levels in BM and spleen neutrophils were similar, but slightly lower than that in PB (Supplementary Figure S2A and B). Interestingly, similar to the observation in PB, spleen and BM neutrophils also showed significant increases in PD-L1 levels in tumor-bearing mice compared to those of naïve mice, implying that neutrophil PD-L1 upregulation occurs systematically in these murine tumor models (Supplementary Figure S2C and D). Given these data, we decided to investigate circulating factors that may induce changes in PD-L1 levels in neutrophils of tumor-bearing mice, and found that the plasma CFNA levels were significantly increased in the tumor-bearing mice compared to the mice before tumor inoculation (Figure 1B). Linear regression analyses showed strong positive correlations between the plasma CFNA and PB neutrophil-associated PD-L1 expression levels in tumor-bearing mice (Figure 1C). Of note, both the plasma CFNA (Figure 1D) and neutrophil PD-L1 expression levels (Supplementary Figure S3) were positively correlated with the tumor volumes in these mice. In vitro experiments revealed that medium supplemented with plasma of tumor-bearing mice (14 days post tumor inoculation) significantly increased PD-L1 expression in naïve mouse BM-derived neutrophils compared to naïve mouse plasma-supplemented medium (Figure 1E). DNase or RNase treatment abolished this effect suggesting that CFNA components are capable of increasing PD-L1 expression in neutrophils, an observation that has important clinical implications given that cancer patients often have increased plasma cell-free DNA (cfDNA) levels compared to healthy individuals [7].

Next, we investigated whether the activation of intracellular NAS, particularly TLR signaling, triggers PD-L1 upregulation in neutrophils [8]. For this purpose, in vitro stimulation assays were performed using intracellular TLR ligands, such as polyinosinic: polycytidylic acid (Poly (I:C)) for TLR3 stimulation, R837 (Imiquimod) for TLR7/8 stimulation, R848 (Resiquimod) for TLR7/8 stimulation, and ODN1826 (Class B CpG oligonucleotide) for TLR9 stimulation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte-colony-stimulating factor (G-CSF) were used as positive controls to increase PD-L1 expression in the neutrophils [4, 5, 9]. Except for Poly (I:C), R837, R848, and ODN1826 significantly increased PD-L1 expressions in BM-derived neutrophils compared to the controls (Figure 1F and G). The conditioned media (CM) of B16-F10 or EO771 cell culture also exhibited similar effects in neutrophil PD-L1 upregulation. The responsibility of intracellular TLR in PD-L1 upregulation was proven by an inhibition assay using E6446 (TLR7/9 inhibitor) in the neutrophils upon R848 or ODN1826 stimulation. PD-L1 expression was suppressed in neutrophils by E6446 treatment (Figure 1H). Intracellular TLR inhibition also suppressed PD-L1 upregulation in neutrophils cultured in tumor-bearing mice plasma supplemented medium or cancer cell CM (Figure 1I). These results further support the essential role of intracellular TLR signaling in neutrophil PD-L1 upregulation. The upregulation of Pdl1 mRNA expression was confirmed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in GM-CSF or R848 treated BM-derived neutrophils (Supplementary Figure S4). In addition, flow cytometry analysis showed that R837 or R848 stimulation increased PD-L1 expression in dimethyl sulfoxide (DMSO)-differentiated HL-60 (dHL-60) cells, a human neutrophil model, suggesting that this intracellular TLR activation-mediated PD-L1 upregulation was mechanistically conserved in both mouse and human cells (Supplementary Figure S5). Western blot (WB) showed that GM-CSF, R837, R848, and ODN1826 treatments increased phosphorylated-signal transducer and activator of transcription 3 (pSTAT3) levels in BM-derived neutrophils, respectively (Figure 1J). PD-L1 upregulation in these treatments was suppressed by inhibition of STAT3 phosphorylation using Stattic in the stimulated neutrophils (Figure 1K). The STAT3 inhibition also suppressed PD-L1 upregulation in neutrophils cultured in cancer cell CM (Supplementary Figure S6). Additionally, STAT3 inhibition significantly suppressed Pdl1 mRNA upregulation in neutrophils upon GM-CSF or R848 stimulation as compared to control treatment (Supplementary Figure S7). As STAT3 has been known as one of the regulators in PD-L1 expression [6], our results suggest that STAT3 activation may be involved in intracellular TLR-dependent PD-L1 upregulation in neutrophils.

Since PD-L1+ myeloid cells are characterized as possessing immunosuppressive activity against T cells [4, 5], we investigated whether intracellular TLR-stimulated or CFNA-exposed neutrophils also exhibits a suppressive effect by employing a co-culture system with T cell (Figure 1L). BM-derived neutrophils were first pre-cultured in the medium supplemented with or without R837, R848, ODN1826, or plasma originating from B16-F10 tumor-bearing mice to increase PD-L1 expression as verified by flow cytometry analysis (Supplementary Figure S8). GM-CSF was added to all the cultures to sustain neutrophil survival. The pre-treated neutrophils were washed and then co-cultured with splenic CD3+ T cells in the presence of anti-CD3 and anti-CD28 monoclonal antibodies (mAbs), and the suppressive effect of neutrophils on T cells was assessed by examining the levels of interferon-gamma (IFN-γ) production and activation marker CD69 expression in the T cells. GM-CSF-stimulated neutrophils suppressed IFN-γ production as well as CD69 expression in CD8+ T cells (Figure 1M and N, Supplementary Figure S9). Notably, the TLR ligand or tumor-bearing mouse plasma-exposed neutrophils further decreased IFN-γ production and CD69 expression in CD8+ T cells compared to the neutrophils treated with GM-CSF alone. These pre-cultured neutrophils could also suppress CD4+ T cell function in the co-culture system (Supplementary Figure S10). Nuclease treatments for tumor-bearing mouse plasma supplemented medium suppressed PD-L1 upregulation in the pre-cultured neutrophils (Supplementary Figure S11), and these neutrophils reduced their suppressive effects against CD8+ T cells which resulted in equivalent IFN-γ production to GM-CSF-pre-cultured neutrophils in the co-culture systems (Supplementary Figure S12). Finally, we investigated whether PD-1/PD-L1 blockade can restore CD8+ T cell function in the presence of PD-L1-upregulated neutrophils. The reduced IFN-γ production in CD8+ T cells cocultured with immunosuppressive neutrophils was significantly restored by anti-PD-L1 mAb treatment compared with isotype Ab treatment (Figure 1O).

In summary, this study found that intracellular TLR stimulation upregulated PD-L1 expression in neutrophils. Moreover, we posit that tumor-released CFNA participates in PD-L1 upregulation of neutrophils via intracellular NAS represented by TLR7, 8, and 9. The TLR-mediated PD-L1 upregulation results in neutrophils gaining immunosuppressive activity which dampens T cell function, and thus implicates a potential new target for anti-cancer immunotherapy (Figure 1P). An important remaining question is the mechanism of CFNA uptake in neutrophils, which may be via endocytosis or micropinocytosis. Other NASs, such as cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) and retinoic acid-inducible gene-I (RIG-I)-like receptors, which recognize cytosolic DNA and double-stranded RNA (dsRNA), respectively, may also play roles in sensing tumor-derived CFNA and regulating PD-L1 expression in neutrophils [10]. In addition, specific CFNA sequences triggering intracellular TLR-mediated PD-L1 upregulation in neutrophils also remain to be identified. Our results suggest the need to investigate PD-L1-upregulated immunosuppressive neutrophils in cancer patients and determine whether they may serve as a predictable marker for effectiveness of PD-1/PD-L1 blockade therapy.

Suguru Saito, Duo-Yao Cao, Tomohiro Shibata, Yan Liu, and Aoi Otagiri-Hoshi performed all experiments. Suguru Saito performed data analysis and finalization. Suguru Saito, Xiaojiang Cui and Kenneth E. Bernstein established methodology. Suguru Saito wrote original manuscript. Suguru Saito, Xiaojiang Cui and Kenneth E. Bernstein finalized manuscript. Xiaojiang Cui and Kenneth E. Bernstein supervised this study. All authors read and approved the final manuscript.

The authors have no conflict of interests.

This study was supported by the National Institutes of Health grants (R01AI164519, 2R01CA151610, R21CA280458),  and American Heart Association's Career Development award (23CDA1052548), U.S. Department of Defense (W81XWH-18-1-0067) and the Glazer Foundation.

All animal experimental protocols were reviewed and approved by the Animal Welfare Committee of Cedars-Sinai Medical Center (#8780, #8109).

Abstract Image

肿瘤衍生的无细胞核酸通过细胞内 Toll 样受体信号传导,上调中性粒细胞中程序性死亡配体 1 的表达。
中性粒细胞是先天免疫细胞,其主要功能是对抗病原体,而最近的研究揭示了在包括癌症在内的各种疾病中具有重要作用[1-3]。例如,表达共抑制分子程序性死亡配体1 (PD-L1)的中性粒细胞被鉴定为通过程序性细胞死亡蛋白1 (PD-1)/PD-L1相互作用损害细胞毒性T细胞(CTL)活性的新型免疫抑制骨髓细胞[4,5]。虽然已经发现了一些刺激,但目前尚不清楚核酸传感系统(NAS)是否参与中性粒细胞[6]中PD-L1的上调。在这里,我们报道了在肿瘤扩张后,增加的无细胞核酸(CFNA)通过在中性粒细胞中激活toll样受体(TLR)来上调PD-L1的表达。流式细胞术分析显示,将B16-F10黑色素瘤细胞或EO771乳腺癌细胞接种野生型(WT)小鼠后,外周血(PB)中性粒细胞中PD-L1的表达逐渐升高(图1A,方案见补充资料,流式细胞术的门控策略见补充图S1)。值得注意的是,与naïve小鼠相比,早在注射后第3天,接种b16 - f10的小鼠PB中性粒细胞中PD-L1的表达就显著增加。虽然接种了eo771的小鼠在肿瘤接种后的第3天和第7天PB中性粒细胞中PD-L1的表达没有明显增加,但在第14天出现了显著的上调(图1A)。两种肿瘤接种后14天,肿瘤内(IT)中性粒细胞与PB、脾脏和骨髓(BM)中性粒细胞相比,PD-L1表达增加最多。B16-F10接种小鼠BM中性粒细胞中PD-L1的表达水平低于PB和脾脏中性粒细胞。在接种eo771的小鼠中,BM和脾脏中性粒细胞中PD-L1的表达水平相似,但略低于PB(补充图S2A和B)。有趣的是,与PB中观察到的结果相似,荷瘤小鼠中脾脏和BM中性粒细胞中PD-L1的表达水平也比naïve小鼠显著升高,这表明中性粒细胞PD-L1的上调在这些小鼠肿瘤模型中系统性地发生(补充图S2C和D)。我们决定研究可能诱导荷瘤小鼠中性粒细胞中PD-L1水平变化的循环因子,发现荷瘤小鼠的血浆CFNA水平与接种肿瘤前相比显著升高(图1B)。线性回归分析显示,荷瘤小鼠血浆CFNA与PB中性粒细胞相关的PD-L1表达水平呈正相关(图1C)。值得注意的是,血浆CFNA(图1D)和中性粒细胞PD-L1表达水平(补充图S3)与这些小鼠的肿瘤体积呈正相关。体外实验显示,与naïve小鼠血浆补充培养基相比,添加荷瘤小鼠血浆的培养基(肿瘤接种后14天)显著增加了naïve小鼠bm源性中性粒细胞中PD-L1的表达(图1E)。DNase或RNase治疗消除了这种作用,表明CFNA成分能够增加中性粒细胞中PD-L1的表达,这一观察结果具有重要的临床意义,因为与健康个体相比,癌症患者通常具有更高的血浆游离DNA (cfDNA)水平。接下来,我们研究了细胞内NAS的激活,特别是TLR信号的激活,是否会触发中性粒细胞[8]中PD-L1的上调。为此,使用细胞内TLR配体进行体外刺激试验,如TLR3刺激的多肌苷:多胞苷酸(Poly (I:C)), TLR7/8刺激的R837(咪喹莫特),TLR7/8刺激的R848(雷西喹莫特)和TLR9刺激的ODN1826 (B类CpG寡核苷酸)。粒细胞-巨噬细胞集落刺激因子(GM-CSF)和粒细胞-集落刺激因子(G-CSF)作为阳性对照,增加中性粒细胞中PD-L1的表达[4,5,9]。除Poly (I:C)外,与对照组相比,R837、R848和ODN1826显著增加了bm源性中性粒细胞中PD-L1的表达(图1F和G)。B16-F10或EO771细胞培养的条件培养基(CM)在中性粒细胞PD-L1上调方面也表现出类似的效果。在R848或ODN1826刺激下,在中性粒细胞中使用E6446 (TLR7/9抑制剂)进行抑制实验,证实了细胞内TLR在PD-L1上调中的作用。E6446处理可抑制中性粒细胞中PD-L1的表达(图1H)。细胞内TLR抑制也抑制了荷瘤小鼠血浆补充培养基或癌细胞CM中培养的中性粒细胞中PD-L1的上调(图1I)。 这些结果进一步支持了细胞内TLR信号在中性粒细胞PD-L1上调中的重要作用。通过逆转录定量聚合酶链反应(RT-qPCR)在GM-CSF或R848处理的脑源性中性粒细胞中证实了Pdl1 mRNA表达的上调(补充图S4)。此外,流式细胞术分析显示,R837或R848刺激增加了二甲亚砜(DMSO)分化的HL-60 (dHL-60)细胞(人中性粒细胞模型)中PD-L1的表达,表明这种细胞内TLR激活介导的PD-L1上调在小鼠和人细胞中都具有机制保守性(Supplementary Figure S5)。Western blot (WB)结果显示,GM-CSF、R837、R848和ODN1826处理分别增加了脑源性中性粒细胞中磷酸化信号转导因子和转录激活因子3 (pSTAT3)的水平(图1J)。在这些处理中,PD-L1的上调通过在受刺激的中性粒细胞中使用STAT3磷酸化抑制而被抑制(图1K)。STAT3抑制也抑制了CM癌细胞培养的中性粒细胞中PD-L1的上调(补充图S6)。此外,与对照治疗相比,在GM-CSF或R848刺激下,STAT3抑制显著抑制中性粒细胞中Pdl1 mRNA的上调(补充图S7)。由于STAT3被认为是PD-L1表达的调节因子之一,我们的研究结果表明,STAT3的激活可能参与了中性粒细胞细胞内tlr依赖性PD-L1的上调。由于PD-L1+骨髓细胞具有对T细胞的免疫抑制活性[4,5],我们通过与T细胞共培养系统研究了细胞内tlr刺激或cfna暴露的中性粒细胞是否也表现出抑制作用(图1L)。首先在添加或不添加R837、R848、ODN1826或B16-F10荷瘤小鼠血浆的培养基中预培养bm来源的中性粒细胞,流式细胞术分析证实PD-L1表达增加(补充图S8)。在所有培养物中加入GM-CSF以维持中性粒细胞存活。将预处理后的中性粒细胞清洗后,在抗CD3和抗cd28单克隆抗体(mab)存在下与脾CD3+ T细胞共培养,通过检测T细胞中干扰素γ (IFN-γ)的产生水平和活化标志物CD69的表达水平来评估中性粒细胞对T细胞的抑制作用。gm - csf刺激的中性粒细胞抑制了IFN-γ的产生以及CD69在CD8+ T细胞中的表达(图1M和N,补充图S9)。值得注意的是,与GM-CSF单独处理的中性粒细胞相比,TLR配体或肿瘤小鼠血浆暴露的中性粒细胞进一步降低了CD8+ T细胞中IFN-γ的产生和CD69的表达。这些预培养的中性粒细胞也可以抑制共培养系统中CD4+ T细胞的功能(补充图S10)。对载瘤小鼠血浆补充培养基进行核酸酶处理可抑制预培养中性粒细胞中PD-L1的上调(补充图S11),这些中性粒细胞降低了它们对CD8+ T细胞的抑制作用,从而导致共培养系统中gm - csf预培养的中性粒细胞产生相同的IFN-γ(补充图S12)。最后,我们研究了PD-1/PD-L1阻断是否可以在PD-L1上调的中性粒细胞存在下恢复CD8+ T细胞的功能。与同型Ab处理相比,抗pd - l1单抗处理显著恢复了与免疫抑制性中性粒细胞共培养的CD8+ T细胞中IFN-γ产生的减少(图10)。综上所述,本研究发现细胞内TLR刺激上调中性粒细胞中PD-L1的表达。此外,我们假设肿瘤释放的CFNA通过以TLR7、8和9为代表的细胞内NAS参与中性粒细胞PD-L1上调。tlr介导的PD-L1上调导致中性粒细胞获得免疫抑制活性,从而抑制T细胞功能,从而暗示了抗癌免疫治疗的潜在新靶点(图1P)。一个重要的问题是中性粒细胞摄取CFNA的机制,这可能是通过内吞作用或微胞饮作用。其他NASs,如干扰素基因环GMP-AMP合成酶刺激因子(cGAS-STING)和视黄酸诱导基因-i (RIG-I)样受体,分别识别胞质DNA和双链RNA (dsRNA),也可能在检测肿瘤来源的CFNA和调节中性粒细胞[10]中PD-L1的表达中发挥作用。此外,在中性粒细胞中触发细胞内tlr介导的PD-L1上调的特异性CFNA序列也有待鉴定。我们的研究结果表明,有必要研究癌症患者中PD-L1上调的免疫抑制中性粒细胞,并确定它们是否可以作为PD-1/PD-L1阻断治疗有效性的可预测标记物。 Saito Suguru, Cao uo- yao, shibattomhiro, Liu Yan和Aoi Otagiri-Hoshi进行了所有实验。Suguru Saito负责数据分析和最终定稿。斋藤苏汝、崔晓江和伯恩斯坦建立了方法论。原稿由斋藤Suguru Saito撰写。斋藤Suguru,崔小江和Kenneth E. Bernstein定稿。崔晓江和Kenneth E. Bernstein指导了这项研究。所有作者都阅读并批准了最终的手稿。作者没有利益冲突。本研究得到了美国国立卫生研究院拨款(R01AI164519, 2R01CA151610, R21CA280458),美国心脏协会职业发展奖(23CDA1052548),美国国防部(W81XWH-18-1-0067)和Glazer基金会的支持。所有动物实验方案均由雪松-西奈医学中心动物福利委员会(#8780,#8109)审查和批准。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Cancer Communications
Cancer Communications Biochemistry, Genetics and Molecular Biology-Cancer Research
CiteScore
25.50
自引率
4.30%
发文量
153
审稿时长
4 weeks
期刊介绍: Cancer Communications is an open access, peer-reviewed online journal that encompasses basic, clinical, and translational cancer research. The journal welcomes submissions concerning clinical trials, epidemiology, molecular and cellular biology, and genetics.
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