Table_3_Characterization of age-related immune features after autologous NK cell infusion: Protocol for an open-label and randomized controlled trial.docx

BackgroundAging is usually accompanied by functional declines of the immune system, especially in T-cell responses. However, little is known about ways to alleviate this.
MethodsHere, 37 middle-aged healthy participants were recruited, among which 32 were intravenously administrated with expanded NK cells and 5 with normal saline. Then, we monitored changes of peripheral senescent and exhausted T cells within 4 weeks after infusion by flow cytometry, as well as serum levels of senescence-associated secretory phenotype (SASP)-related factors. In vitro co-culture assays were performed to study NK-mediated cytotoxic activity against senescent or exhausted T cells. Functional and phenotypic alteration of NK cells before and after expansion was finally characterized.
ResultsAfter NK cell infusion, senescent CD28-, CD57+, CD28-CD57+, and CD28-KLRG1+ CD4+ and CD8+ T-cell populations decreased significantly, so did PD-1+ and TIM-3+ T cells. These changes were continuously observed for 4 weeks. Nevertheless, no significant changes were observed in the normal saline group. Moreover, SASP-related factors including IL-6, IL-8, IL-1α, IL-17, MIP-1α, MIP-1β, and MMP1 were significantly decreased after NK cell infusion. Further co-culture assays showed that expanded NK cells specifically and dramatically eliminated senescent CD4+ T cells other than CD28+CD4+ T cells. They also showed improved cytotoxic activity, with different expression patterns of activating and inhibitory receptors including NKG2C, NKG2A, KLRG1, LAG3, CD57, and TIM3.
ConclusionOur findings imply that T-cell senescence and exhaustion is a reversible process in healthy individuals, and autologous NK cell administration can be introduced to alleviate the aging.
Clinical Trial RegistrationClinicalTrials.gov, ChiCTR-OOh-17011878.

背景 衰老通常伴随免疫系统功能衰退，尤以T细胞应答异常为显著特征，但目前针对该类衰老相关免疫衰退的缓解手段仍知之甚少。
方法 本研究招募37名中年健康受试者，其中32例接受扩增NK细胞（natural killer cells）静脉输注，剩余5例给予生理盐水作为对照。于输注后4周内，通过流式细胞术监测外周血中衰老及耗竭T细胞的动态变化，并检测血清中衰老相关分泌表型（senescence-associated secretory phenotype, SASP）相关因子的水平。此外，开展体外共培养实验，以探究NK细胞对衰老或耗竭T细胞的细胞毒活性。最终对扩增前后NK细胞的功能与表型改变进行系统表征。
结果 NK细胞输注后，衰老表型的CD28⁻、CD57⁺、CD28⁻CD57⁺以及CD28⁻KLRG1⁺的CD4⁺和CD8⁺ T细胞亚群均显著减少，PD-1阳性与TIM-3阳性T细胞亦呈现类似的显著下降。上述动态变化可持续至输注后4周。而生理盐水对照组未观察到类似显著变化。此外，NK细胞输注后，血清中IL-6、IL-8、IL-1α、IL-17、MIP-1α、MIP-1β及MMP1等SASP相关因子的水平均显著降低。进一步共培养实验显示，扩增后的NK细胞可特异性高效清除衰老CD4⁺ T细胞，而对CD28⁺CD4⁺ T细胞无明显清除作用。同时，扩增后NK细胞的细胞毒活性显著提升，其活化性与抑制性受体（包括NKG2C、NKG2A、KLRG1、LAG3、CD57及TIM3）的表达模式亦发生显著改变。
结论 本研究结果提示，健康个体的T细胞衰老与耗竭过程具有可逆性，自体NK细胞输注可作为缓解机体衰老的潜在干预手段。
临床试验注册 本研究已在ClinicalTrials.gov平台完成注册，注册号为ChiCTR-OOh-17011878。