3.1 Baseline characteristics
During July 2017 to September 2018, 42 out of total 49 recruited subjects were with ages from 45 to 55 years old, for which these middle-age populations shown a significantly decreased percentage and activity of NK cells. During screening, 8 volunteers were excluded, among which 3 had incomplete detection index, 2 had two or more abnormal results in live function tests, 1 had abnormal immune index, 1 had positive infectious index and 1 had abnormal blood biochemical. Therefore, 34 volunteers were enrolled in the study, and received leukapheresis and subsequent NK cell administration. However, 2 subjects missed their sample collection after NK cell administration and had to exit from the study. Finally, 32 subjects successfully completed the study, among which 15 males and 17 females were included (Figure 1). Importantly, all the volunteers consulted and signed the informed consent form before participation. Meanwhile, 14 of 32 volunteers were re-injected cryopreserved NK cell,18 of 32 volunteers were re-injected fresh NK cell. The baseline characteristics of the subjects and information of NK cells used were listed in Supplementary Table 1.
3.2 Safety of autologous NK cells
After cell administration, all subjects had normal body temperature and blood pressure. And no one developed skin rashes, local infection and bleeding, fever, chills, difficult breathing, nausea and vomiting. Besides, one subject developed agrypnia in one week after cell infusion and recovered thereafter. One developed dizziness in one week after cell infusion and this phenomenon lasted for two weeks before recovering. 2 subjects developed fatigue, among which one developed mild fatigue and the other developed media fatigue, and both recovered in two weeks ( Supplementary Table 2). Furthermore, we conducted routine blood test, hematological examination, urinary and virological examination at one month later after cell infusion. No hepatotoxicity and nephrotoxicity were observed according to normal serum levels of ALT, AST, Urea, creatinine. Additionally, no abnormal C response protein (CRP), anti-thyroglobulin antibody (TGAb) and anti-thyroid peroxidase autoantibody (TPOAb) occurred, indicating no immune response and autoimmune effects were induced. Furthermore, no increased plasma levels of alpha fetoprotein (AFP) and carcinoembryonic antigen (CEA) were observed one month later, strongly confirming that autologous NK cell infusion is safe in terms of tumorigenicity.
3.3 Senescent CD4+ T cells decreased after NK cell infusion
Previous researches have proved that senescent cell accumulation accelerates aging-associated disorders and clearance of p16 positive cell delays this phenomenon 29. NK cells play important roles in innate immunity for clearing senescent cells and defending against cancer 30, 31. Thus, flow cytometry was carried out to detect populations of CD4+CD28-, CD4+CD57+, CD4+KLRG1+, CD4+CD28-CD57+, CD4+CD28-KLRG1+ as senescent CD4+ T cells at baseline, one- and four-weeks after infusion. Results showed no significant changes in CD4+ and CD8+ populations at two time-points after cell infusion (Figure 2A-B). However, senescent CD4+ T cells significantly decreased after NK cell infusion at one week and four weeks after infusion (Figure 2C). In addition, gender does not influence reducing extent caused by NK cell infusion (Figure 2D).
3.4 Senescent CD8+ T cells decreased after NK cell infusion
As we know, CD8+ T cells are the main tumor killing cell group. CD8+ cytotoxic T cell can attenuate tumor growth by expressing FasL and secreting granzyme B and IFN-γ. However, senescent T cell accumulation impaired T cell-mediated responses. So we check CD8+CD28-, CD8+CD57+, CD4+KLRG1+, CD8+CD28-CD57+, CD4+CD28-KLRG1+ percentage at baseline, one- and four-weeks after infusion. We found that senescent CD8+ T cells significantly decreased after NK cell infusion at both one week and four weeks after NK cell infusion (Figure 2E). also, The effects induced by NK cell infusion are independent with gender (Figure 2F).
3.5 Exhausted T cells decreased after NK cell infusion
During chronic infections and cancer, which include persistent antigen exposure and inflammation, memory T cell differentiation exists 14. It has been reported in human that T cell exhaustion happens during infections such as HIV and hepatitis C virus (HCV), as well as in cancer 17, 32, 33. Importantly, exhausted T cells were characterized by high expression of PD-1, TIM-3, CTLA-4 and activation of their involved signaling pathways. The successful applications of anti-PD-1/PD-L1 antibodies in cancer immunotherapy recently have proved the significance and efficacy of treatments targeting T cell exhaustion 17. Then we checked whether NK cell infusion affected the percentage of exhausted CD4+ and CD8+ T cells. Results showed that CD4+PD-1+T cell, CD8+ PD-1+ T cell, CD4+TIM-3+T cell and CD8+TIM-3+T cell were all significantly decreased after NK cell infusion at both one week and four weeks (Figure 3A). These results suggested that NK cell infusion might improve the function of T cells by alleviating exhausted status of T cells.
Moreover, The percentages of PD-1+ and TIM-3+ T cell decreased at one week and four weeks after NK cell infusion, which are independent of gender (Figure 3B).
3.6 Cell types influenced the effects of induced by NK cell infusion
In this study, we finally recruited 32 volunteers in all experiments. It should be pointed out that 14 of 32 volunteers were re-injected cryopreserved NK cell, and 18 of 32 volunteers were re-injected fresh NK cell. So we wanted to explore whether immune system effects influenced by NK cell infusion was dependent on infused cell types influence or not. We analyzed this two groups by two-tailed paired Student t test. The results suggested that fresh NK cell infusion group is better than frozen NK cell infusion group in promote immune system in sub-health population (Figure 4).
3.7 Key SASP-related factors reduced after NK cell infusion
Senescent cells accumulate with aging and lead to SASP-related factors including pro-inflammatory cytokines (IL-1α, IL-1β, IL-6), chemokines (IL-8, CXCL1), proteases (MMP-1,MMP-3,MMP-13). These SASP-related factors play critical roles to aging-related inflammation, diseases and morbidity 34-36. Therefore, to check whether NK cell infusion decrease systematic levels of SASP-related factors, we measured cytokine levels in plasma collected before and after NK cell infusion. We found lower levels of key SASP components in plasma by NK cell infusion including IL-6, IL-8, IL- 1α, IL-17, MIP-1α, ΜΙP-1β, MMP1, whereas a non-SASP-related factor, IFN-γ was not continuously significantly altered (Figure 5). These results indicated that NK cell infusion could attenuate SASP accumulation and improve CD4+ and CD8+ T cell activity.