引流淋巴结在肿瘤记忆CD8+T细胞形成中的作用

Role of tumor draining lymph nodes in generation of memory CD8+T cells in a murine model of colon cancer

目的探究引流淋巴结(tumor-draining lymph nodes,TDLN)在肿瘤记忆CD8^(+)T细胞形成中的作用,明确新辅助PD-L1抗体治疗对抗肿瘤免疫记忆的影响。方法建立小鼠CT26/MC38-OVA结直肠癌皮下移植瘤模型,采用新辅助PD-L1抗体治疗,荷瘤15 d后手术切除肿瘤和TDLN。手术30 d后运用肿瘤再挑战模型评估TDLN在新辅助PD-L1抗体治疗条件下对免疫记忆形成的影响。利用过继回输实验探究新辅助PD-L1对肿瘤特异性CD8^(+)T细胞的影响,运用流式细胞术在术时(效应阶段)和术后30 d(记忆阶段)检测CD45.1^(+)CD8^(+)T细胞在各组织中的分布以及记忆表型变化。结果新辅助PD-L1抗体治疗显著缩小手术时肿瘤大小(P<0.05),肿瘤再挑战结果显示,接受新辅助PD-L抗体治疗,肿瘤生长显著减缓,而TDLN的切除导致肿瘤生长加剧(P<0.05)。但对肿瘤再挑战进行长期观察发现,无论是否接受新辅助PD-L1抗体治疗,肿瘤最终均消退。过继回输实验结果表明,TDLN及非引流淋巴结(no-draining lymph nodes,NLN)中贮存的CD45.1^(+)CD8^(+)T细胞数量最多(P<0.05),且在记忆阶段,中枢记忆T细胞(Tcm)和组织驻留记忆T细胞(Trm)主要分布于TDLN及NLN(P<0.05)。新辅助PD-L1抗体治疗后,引流淋巴结及非引流淋巴结等组织中CD45.1^(+)CD8^(+)T细胞的分布和记忆表型没有明显变化。结论TDLN是记忆CD8^(+)T细胞的主要驻留场所,但新辅助PD-L1抗体治疗条件下,TDLN切除并不影响其长期抗肿瘤免疫能力。

ObjectiveTo investigate the role of tumor draining lymph nodes(TDLN)in the generation of memory CD8^(+)T cells under the context of neoadjuvant anti-PD-L1 therapy.MethodsA mouse transplanted model of CT26/MC38-OVA colon cancer was established to recapitulate clinic neoadjuvant anti-PD-L1 therapy,and then the tumor masses and TDLN were removed in 15 d after transplantation.On day 30 after surgery,the effect of TDLN on the formation of immune memory was assessed under the condition of neoadjuvant PD-L1 antibody therapy by tumor re-challenge.The effect of neoadjuvant PD-L1 on tumor-specific CD8^(+)T cells was investigated by adoptive transfer CD45.1^(+)OT-Ⅰcells.The distribution and phenotype of CD45+CD8^(+)T cells in various tissues were determined by flow cytometry at the time of surgery(effector phase)and 30 d after surgery(memory phase).ResultsThe tumor mass was reduced significantly after receiving neoadjuvant anti-PD-L1 therapy(P<0.05).After tumor re-challenge,tumor growth was significantly slowed in the mice treated with neoadjuvant PD-L antibody,whereas TDLN resection accelerated the tumor growth(P<0.05).However,long-term survival of tumor re-challenge showed that all tumors were eventually subsided,whether or not the mice treated with neoadjuvant PD-L antibody.The results of adoptive reinjection indicated that the number of CD45.1^(+)CD8^(+)T cells being stored in TDLN and no-draining lymph nodes(NLN)was the highest(P<0.05).Tcm(T central memory)cells and Trm(tissue-resident memory)cells were mainly distributed in TDLN and NLN in memory phase(P<0.05).The distribution and memory phenotype of CD45.1^(+)CD8^(+)T cells in TDLN and NLN had no obvous changes after neoadjuvant anti-PD-L1 therapy.ConclusionTDLN is the primary site for the storage of memory CD8^(+)T cells,but under the condition of neoadjuvant PD-L1 antibody treatment,TDLN resection does not affect their long-term anti-tumor immunity.