靶向髓源性抑制细胞的叶酸循环增强肿瘤免疫治疗效果研究

doi:10.3969/j.issn.1674-8115.2024.08.010

摘要/Abstract

摘要：

目的·探究叶酸循环代谢对髓源性抑制细胞（myeloid-derived suppressor cells，MDSCs）免疫抑制作用的调控机制。方法·在C57BL/6小鼠骨髓细胞的培养体系中加入细胞因子粒细胞集落刺激因子（granulocyte colony-stimulating factor，G-CSF）、粒细胞巨噬细胞集落刺激因子（granulocyte-macrophage colony-stimulating factor，GM-CSF）和白细胞介素-6（interleukin-6，IL-6），体外诱导MDSCs。利用流式细胞仪检测诱导MDSCs的程序性死亡受体配体1（programmed death-ligand 1，PD-L1）表达水平和活性氧（reactive oxygen species，ROS）的产生水平。采用磁珠分选出小鼠脾脏CD8⁺ T细胞并用Celltrace violet或CFSE标记，再与MDSCs共培养，72 h后用流式细胞术检测CD8⁺ T细胞增殖情况。通过实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction，qPCR）检测MDSCs中叶酸循环相关代谢酶的表达水平。采用叶酸循环代谢酶亚甲基四氢叶酸脱氢酶2（methylenetetrahydrofolate dehydrogenase 2，MTHFD2）抑制剂DS18561882（DS18）和叶酸拮抗剂培美曲塞（Pemetrexed）处理MDSCs，并用流式分析检测MDSCs产生ROS和线粒体ROS的水平。将DS18或培美曲塞处理后的MDSCs与磁珠分选出来并用Celltrace violet或CFSE标记的CD8⁺ T细胞共培养，72 h后用流式细胞术检测CD8⁺ T细胞增殖情况。利用RNA测序（RNA-seq）检测DS18和培美曲塞处理后MDSCs在转录组水平的变化。建立小鼠结肠癌（mouse colon cancer cells，MC38）和Lewis 肺癌（Lewis lung carcinoma，LLC）皮下瘤模型。造模后第10天开始，采用Isotype抗体、抗CD8单抗（1 mg/kg，清除CD8⁺ T细胞）、培美曲塞以及抗CD8单抗联合培美曲塞处理MC38荷瘤小鼠；利用Isotype抗体、抗Gr1单抗（1.25 mg/kg，清除MDSCs）、培美曲塞以及抗Gr1单抗联合培美曲塞处理MC38荷瘤小鼠；分别给予MC38和LLC荷瘤小鼠培美曲塞（50 mg/kg）、抗PD-1单克隆抗体（250 μg/kg）以及培美曲塞联用抗PD-1单克隆抗体治疗；第14天收集小鼠肿瘤组织，记录肿瘤大小，绘制肿瘤生长曲线。结果·流式结果显示诱导后的骨髓细胞PD-L1的表达升高，ROS的产生也增加，且明显抑制CD8⁺ T细胞的增殖。qPCR结果显示MDSCs中叶酸循环相关代谢酶MTHFD2等表达升高。给予DS18和培美曲塞处理MDSCs会影响MDSCs的累积，抑制MDSCs的ROS产生，并解除对CD8 T细胞的免疫抑制。RNA-seq结果表明2种叶酸循环抑制剂处理后，与MDSCs分化相关基因S100钙结合蛋白a8（S100 calcium binding protein a8，S100a8）等下调，与MDSCs抑制功能相关基因如与ROS产生有关的基因细胞色素b-245β链（cytochrome b-245 beta chain，Cybb）等也有所下调。与对照组相比，培美曲塞处理组的小鼠肿瘤生长明显受到抑制。与培美曲塞处理组相比，抗CD8单抗联合培美曲塞处理组肿瘤进展加剧；与抗CD8单抗处理组相比，抗CD8单抗联合培美曲塞处理组肿瘤进展受到限制。清除MDSCs能显著抑制肿瘤生长，然而在清除MDSCs的荷瘤小鼠中，培美曲塞的抗肿瘤作用显著低于培美曲塞单独处理的小鼠。与抗PD-1抗体单独治疗相比，培美曲塞联用抗PD-1抗体治疗组的肿瘤生长限制更为显著。结论·培美曲塞依赖CD8⁺ T细胞发挥抗肿瘤作用，并通过重编程MDSCs为抗肿瘤表型，进一步阻碍肿瘤生长。通过调控MDSCs叶酸循环阻断其免疫抑制能力，可增强免疫检查点阻断剂治疗肿瘤效果。

关键词: 髓源性抑制细胞, 叶酸循环, 亚甲基四氢叶酸脱氢酶2, 培美曲塞, 肿瘤免疫治疗

Abstract:

Objective ·To explore the regulatory mechanism of folate cycle metabolism in the immunosuppressive effect of myeloid derived suppressor cells (MDSCs). Methods ·Bone marrow cells were isolated from C57BL/6 mice and cultured in RPMI 1640 medium supplemented with GM-CSF, G-CSF, and IL-6 to induce MDSCs in vitro. PD-L1 expression level and ROS production level of induced MDSCs were detected by flow cytometry. CD8⁺ T cells were enriched from the spleen by MACS with anti-CD8a-conjugated microbeads, labeled with Celltrace violet, and then co-cultured with MDSCs. After 72 h, proliferation was assessed by flow cytometry. Folate cycle-related metabolic enzymes in MDSCs were detected by real-time quantitative PCR. MDSCs were treated with folate cycle metabolic enzyme MTHFD2 inhibitor DS18561882 (DS18) and folic acid antagonist Pemetrexed. ROS and mitoROS production in MDSCs were assessed by flow cytometry. CD8⁺ T cells were enriched from the spleen by MACS with anti-CD8a-conjugated microbeads, labeled with Celltrace violet, and then co-cultured with Pemetrexed or DS18-treated MDSCs. After 72 h, proliferation was assessed by flow cytometry. Transcript levels of folate cycle-related metabolic enzymes in pemetrexed or DS18-treated MDSCs were detected by RNAseq. A subcutaneous tumor mouse model of colon cancer was established. From the tenth day post-implantation, tumor-bearing mice were intraperitoneally injected with Pemetrexed (200 mg/kg) and tumor size was recorded for tumor growth curve. On the fourteenth day, mice were sacrificed, and tumors were harvested. MC38 tumor-bearing mice were treated with isotype antibody, anti-CD8 monoclonal antibody (1 mg/kg, deplete CD8⁺ T cells), Pemetrexed (200 mg/kg), and combination of Pemetrexed with anti-CD8 antibody. MC38 tumor-bearing mice were treated with isotype antibody, anti-Gr1 monoclonal antibody (1.25 mg/kg, clearing MDSCs), combination of Pemetrexed with anti-Gr1 antibody. On the tenth day after implantation, tumor-bearing mice were treated with Pemetrexed (50 mg/kg), anti-PD-1 monoclonal antibody (250 μg/kg), Pemetrexed, and combination of Pemetrexed with anti-PD-1 antibody. Results ·Flow cytometry data showed that PD-L1 level and ROS production were increased in induced MDSCs, and CD8⁺ T cell proliferation was also suppressed significantly. qPCR data revealed the expression of folate cycle-related metabolic enzymes MTHFD2 and others was increased in MDSCs. The accumulation of MDSCs was affected by DS18 or Pemetrexed, ROS production in MDSCs was reduced, and the immunosuppression of CD8⁺ T cells was relieved. RNA-seq results showed that genes related to MDSCs differentiation, such as S100 calc-binding protein A8, and genes related to MDSCs inhibition, such as cytochrome b-245β chain, which is related to ROS production, were also down-regulated after treatment with two folic acid cycling inhibitors. Tumor growth was suppressed by Pemetrexed. Tumor progression was promoted by combination of Pemetrexed with anti-CD8 antibody, compared with Pemetrexed monotherapy. However, tumor growth delay was inhibited by combination of Pemetrexed and anti-CD8, compared with anti-CD8 monotherapy. Tumor growth delay was caused by MDSCs depletion. But tumor growth was promoted by combination of pemetrexed and anti-Gr1, compared with pemetrexed monotherapy. Tumor growth was restricted by combination of pemetrexed and anti-PD-1 antibody, compared with anti-PD-1 monotherapy. Conclusion ·Pemetrexed relies on CD8⁺ T cells for anti-tumor effects and further retards tumor growth by reprogramming MDSCs to an anti-tumor phenotype. Modulating MDSCs by targeting folate cycle could impair their immunosuppressive ability and enhance the efficacy of immune checkpoint blockade in cancer treatment.

Key words: myeloid-derived suppressor cells (MDSCs), folate cycle, methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), pemetrexed, cancer immunotherapy

中图分类号:

R73-3

何蕊, 颜克鹏, 王静. 靶向髓源性抑制细胞的叶酸循环增强肿瘤免疫治疗效果研究[J]. 上海交通大学学报（医学版）, 2024, 44(8): 1011-1022.

HE Rui, YAN Kepeng, WANG Jing. Targeting folate cycle enhances effects of cancer immunotherapy by modulating myeloid-derived suppressor cells[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(8): 1011-1022.

图/表 6

表1 实时定量PCR引物信息

Tab1 Primers information of qPCR

Primer	Forward sequence (5'→3')	Reverse sequence (5'→3')
Aldh1l1	CAGGAGGTTTACTGCCAGCTA	CACGTTGAGTTCTGCACCCA
Dhfr	CGCTCAGGAACGAGTTCAAGT	TGCCAATTCCGGTTGTTCAATA
Gapdh	AGGTCGGTGTGAACGGATTTG	TGTAGACCATGTAGTTGAGGTCA
Mthfd1	GGGAATCCTGAACGGGAAACT	TGAGTGGCTTTGATCCCAATC
Mthfd2	AGTGCGAAATGAAGCCGTTG	GACTGGCGGGATTGTCACC
Shmt1	CAGGGCTCTGTCTGATGCAC	CGTAACGCGCTCTTGTCAC
Shmt2	TGGCAAGAGATACTACGGAGG	GCAGGTCCAACCCCATGAT

图1 体外诱导MDSCs高表达叶酸循环通路相关代谢酶Note： A. Expression of PD-L1 in in vitro induced MDSCs detected by flow cytometry. B. ROS levels of in vitro induced MDSCs detected by flow cytometry. C. CD8 T cells proliferation was assessed by flow cytometry. D. One-carbon unit metabolic pathway. E?J. RNA levels of folate cycle-related metabolic enzymes [Dhfr (E), Mthfd2 (F), Shmt2 (G), Shmt1 (H), Mthfd1 (I), and Aldh1l1 (J)] in induced MDSCs detected by qRT-PCR. ①P=0.000, ②P=0.001, compared with the control group.

Fig 1 Expression of folate cycle-related metabolic enzymes in induced MDSCs in vitro

图2 靶向叶酸循环抑制MDSCs的分化Note： A/E. Cell viability of bone marrow cells treated with DS18 (A) or Pemetrexed (E) detected by flow cytometry. B/F. Percentages of CD11b+ Gr1+ cells treated with DS18 (B) or Pemetrexed (F). C/G. Absolute count of live cells treated with DS18 (C) or Pemetrexed (G). D/H. Absolute count of MDSCs treated with DS18 (D) or Pemetrexed(H). ①P= 0.008, ②P= 0.013, ③P= 0.005, compared with the control group.

Fig 2 Targeting folate cycle inhibits differentiation of MDSCs

图3 靶向叶酸循环解除MDSCs的免疫抑制功能Note： A. Proliferation of CD8+ T cells cocultured with NAC treated-MDSCs. B/C. Proliferation of CD8+ T cells cocultured with DS18-treated MDSCs (B) or Pemetrexed-treated MDSCs (C). D/H. ROS levels of DS18-treated MDSCs (D) or Pemetrexed-treated MDSCs (H) detected by flow cytometry. E/G. Quantitation of ROS Geo MFI. F/J. MitoROS levels of DS18-treated MDSCs (F) or Pemetrexed-treated MDSCs (J) detected by flow cytometry. I/K. Quantitation of MitoROS Geo MFI. ①P= 0.001, ②P= 0.024, ③P= 0.000, ④P= 0.035, compared with the control group.

Fig 3 Targeting folate cycle relieves immunosuppressive function of MDSCs

图4 叶酸循环抑制剂处理后MDSCs的转录组分析Note： A. Differentially expressed genes between DS18 and Pemetrexed-treated MDSCs, compared with the control group. B/C. Cluster analysis of differential gene groups in DS18 (B)- or Pemetrexed (C)-treated MDSCs, compared with the control group. D/E. GO enrichment analysis of differentially upregulated (D)or downregulated (E) genes in DS18-treated MDSCs (biological process). F/G. GO enrichment analysis of differentially upregulated (F) or downregulated (G) genes in Pemetrexed-treated MDSCs (biological process).

Fig 4 Transcript analysis of MDSCs after treatment with folate cycle inhibitors

图5 叶酸循环抑制剂的抗肿瘤作用Note： A. MC38 tumor burden assessed by gross visualization. B. Tumor weight of MC38 tumor-bearing mice in pemetrexed-treated group. C. Tumor growth curve of MC38 tumor-bearing mice in pemetrexed-treated group. D/G. After combination of anti-CD8 monoclonal antibody and Pemetrexed treatment, MC38 tumor burden(D) was assessed by gross visualization, and tumor growth curve (G) was drawn. E/H. After combination of anti-Gr1 monoclonal antibody and Pemetrexed treatment, MC38 tumor burden(E) was assessed by gross visualization, and tumor growth curve (H) was drawn. F/I. After combination of anti-PD-1 monoclonal antibody and Pemetrexed treatment, MC38 tumor burden(F) was assessed by gross visualization, and tumor growth curve (I) was drawn. J/K.After combination of anti-PD-1 monoclonal antibody and Pemetrexed treatment, LLC tumor burden (J) was assessed by gross visualization, and tumor growth curve(K) was drawn.①P=0.009, ②P=0.029, ③P=0.001, compared with the control group. ④P=0.000, compared with the control group, combination of anti-CD8 monoclonal antibody and Pemetrexed group, combination of anti-Gr1 monoclonal antibody and Pemetrexed group, or combination of anti-PD-1 monoclonal antibody and Pemetrexed group.

Fig 5 Antitumor effects of folate cycle inhibitor

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