Screening of adjuvant for PD-L1 vaccine based on nitrated T cell epitope
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摘要:
为了充分发挥本课题组前期设计的基于硝基化T表位的靶向PD-L1的肿瘤疫苗(PD-L1-NitraTh)的抑瘤活性,选择了作用机制不同的几种佐剂进行比较,以期筛选出最适用于此类疫苗的佐剂。研究结果显示,Poly(I:C)、CPG1018、肿节风多糖SGP2及GM-CSF等佐剂均可以提高PD-L1-NitraTh疫苗的免疫原性,其中,Poly(I:C)组诱导产生的抗体滴度最高。对T细胞分化相关转录因子的qPCR检测结果显示,Poly(I:C)减少了GATA3和FoxP3的表达,提示对CD4+T细胞分化有较强的影响。同时,相比其他佐剂,Poly(I:C)可以辅助PD-L1-NitraTh增加肿瘤内T淋巴细胞以及CD11b+细胞浸润,提示 Poly(I:C)佐剂可能适用于以硝基化T表位为基础的肿瘤疫苗。
Abstract:To enhance the anti-tumor activity of tumor vaccine targeting PD-L1 based on the nitrated T-epitope (PD-L1-NitraTh), this research compared several adjuvants with different mechanisms to screen out the adjuvant most suitable for PD-L1-NitraTh. The results showed that Poly(I:C), CPG1018, swollen knotted polysaccharide SGP2 and GM-CSF could enhance the immunogenicity of PD-L1-NitraTh when used as adjuvants, with the Poly(I:C) group inducing the highest antibody titer. The results of qPCR for T cell differentiation-related cytokines showed that Poly(I:C) reduced the expression of GATA3 and FoxP3, indicating a strong effect on CD4+ T cell differentiation. Besides, compared with other adjuvants, Poly(I:C) could assist PD-L1-NitraTh to increase the infiltration of T cells as well as CD11b+ cells within tumor, suggesting that Poly(I:C) may be the suitable adjuvant for tumor vaccines based on the nitrated T epitopes.
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Keywords:
- tumor vaccine /
- adjuvant /
- Toll-like receptor /
- Poly(I:C)
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Figure 1. Immunogenicity of PD-L1-NitraTh with different adjuvants ($ \bar{x}\pm s $, n =5)
A: Determination of anti-PD-L1 antibody titer in mouse serum; B: Number of IFN-γ ELISPOT spots of mouse spleen cells; C: IFN-γ ELISPOT speckle pattern of mouse spleen cells **P < 0.01,***P < 0.001, ****P < 0.0001
Figure 2. Antitumor activity of PD-L1-NitraTh with different adjuvants ($ \bar{x}\pm s $, n =5)
A:Tumor volume measured from the beginning of PD-L1-NatriTh treatment; B: Volume of the tumors excised after the mice were sacrificed (circle indicates tumor regression; triangle represents the death of the mouse); C: Tumor volume of mice at the end of the experiment**P < 0.01
Table 1 Grouping situation and immune dose of mice
Group Dose of
PD-L1-NitraTh/μgAdjuvant Dose of
adjuvant/μgBlank - - - PD-L1 50 - - CPG1018 50 CPG1018 35 Poly(I:C) 50 Poly(I:C) 50 GM-CSF 50 GM-CSF 0.2 SGP2 50 SGP2 200 Blank group was inoculated with saline, PD-L1 group was inoculated with PD-L1-NitraTh only, and the other groups were inoculated with PD-L1-NitraTh mixed with the corresponding adjuvant respectively
GM-CSF:Granulocyte-macrophage colony-stimulating factor ;SGP2:
An acidic polysaccharide from Sarcandra glabraTable 2 Primer sequence for PCR
Gene Forward primer sequence(5′→3′) Reverse primer sequence(5′→3′) TNF-α CCCTCACACTCAGATCATCTTCT GCTACGACGTGGGCTACAG INF-γ ATGAACGCTACACACTGCATC CCATCCTTTTGCCAGTTCCTC GATA3 CTCGGCCATTCGTACATGGAA GGATACCTCTGCACCGTAGC CD11c CTGGATAGCCTTTCTTCTGCTG GCACACTGTGTCCGAACTCA CD206 CTCTGTTCAGCTATTGGACGC CGGAATTTCTGGGATTCAGCTTC Foxp3 CCCATCCCCAGGAGTCTTG ACCATGACTAGGGGCACTGTA β-actin ACTCCTATGTGGGTGACGAG CATCTTTTCACGGTTGGCCTTAG -
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