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

Background: Aging is usually accompanied by functional declines of the immune system, especially in T-cell responses. However, little is known about ways to alleviate this. Methods: Here, 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. Results: After 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-1ß, 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. Conclusion: Our 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 Registration: ClinicalTrials.gov, ChiCTR-OOh-17011878.

Effect of Warming Process on the Survival of Cryopreserved Human Peripheral Blood Mononuclear Cells.

It is well known that the warming process is a critical step in cell cryopreservation, affecting the survival rate of the cryopreserved cells. However, there is a lack of understanding and optimization of the warming process for the cryopreserved human peripheral blood mononuclear cells (PBMCs) that are greatly needed for the cellular/immune therapies worldwide. In this study, the effect of the warming process on cryosurvival of the PBMCs was investigated, resulting in a recommendation of an optimal warming method. In the experiments, all PBMC samples were cooled by a fixed slow cooling process and stored in a liquid nitrogen tank. The frozen samples were then warmed in water baths with stirring at various temperatures, 37°C, 42°C, and 65°C, respectively. After thawing, PBMC's viability as well as phenotypic and functional analyses were performed and evaluated. It was shown that a relatively rapid warming process at 65°C in a water bath with stirring generated a significant improvement of cell viability, recovery, and functionality of the cryopreserved PBMCs. In addition, interferon-γ and interleukin-2 secretion were much higher in PBMCs thawed at 65°C than that in 42°C and 37°C, respectively. This study suggests that a rapid warming process at 65°C in a water bath should be used to replace the current conventional warming approach at 37°C.