Immune normalization strategy against suboptimal health status: safe and efficacious therapy using mixed-natural killer cells

“Immune normalization” has emerged as a new paradigm in immunotherapy, which is proposed in cancer patients instead of conventional “immune-enhancement” therapy. Immune normalization may also be implemented in cancer prevention of “sub-healthy” individuals. We established in vitro cultured mixed-natural killer (NKM) cells to achieve immune normalization. The in vitro cytotoxicity of NKM cells was tenfold higher than that of peripheral blood mononuclear cells (PBMCs). The cytotoxicity of NKM cells was negatively correlated with the proportion of T-helper cells (cluster of differentiation: CD3+CD4+ T), and positively correlated with the proportion of NK cells (especially CD56brightCD16bright NK cells). Then, we defined “sub-healthy individuals” after measuring Programmed cell death protein-1 (PD-1) expression in PBMCs from 95 donors aged > 50 years. Furthermore, we evaluated the potential clinical application of NKM-cell therapy in 11 patients with malignant lymphoma, one patient with pancreatic cancer, and four sub-healthy individuals. NKM-cell therapy elicited good tolerance and side-effects were not found. In sub-healthy individuals, the proportion of CD3+PD-1+ T cells and CD3+CD8+PD-1+ T cells was reduced significantly after NKM-cell treatment. We demonstrated that a new method using NKM cells was safe and efficacious as adjuvant treatment for cancer patients as well as therapy for sub-healthy individuals. Normalization of the peripheral immune system through NKM-cell therapy could expand its scope of application in different disorders.


INTRODUCTION
The immune system is a crucial barrier for the human body to resist diseases. Its ability has rhythmic fluctuations [1,2], and is affected by several factors (e.g., genetic, age [3,4]). Reduction in the response capacity of immune cells can lead to diseases (e.g., cancer), whereas excessive activation of immune cells can cause autoimmune diseases. Thus, balance in the immune system is important for health [5].

AGING
In recent decades, epidemiological data for colorectal cancer and lung cancer have revealed that prevention and early detection of cancer are far more efficacious than their treatment for controlling cancer mortality [9,10]. Application of immune-cell therapy in disease prevention (especially cancer) can greatly reduce the occurrence or relapse of diseases [11,12]. For older individuals who may have long-term immunocompromise, immune-cell therapy could "rebalance" their immune system, which would eliminate mutant cells and pathogens (e.g., viruses, bacteria), thereby reducing the disease risk.
We established an in vitro cultured mixed-NK (NKM) cell system using PBMCs and then tested its potential anti-tumor activity in vivo and in vitro. Our data represent the first NKM-cell therapy, and could be used for healthcare and, possibly, cancer treatment.

Generation and characterization of NKM cells
To achieve safe and efficacious immunotherapy for patients, we established an in vitro culture system for the production of immune cells without separation or biological additives. The medium used in this culture system was designed specifically for the expansion of NK cells and T cells. Autologous plasma was prepared and inactivated, then used for cell culture. Autologous immune cells were manufactured as shown in Figure 1A and in the Methods and Materials section: these were NKM cells. After the culture was complete, we could obtain ~2.5 billion immune cells, the main components of which were NK cells and T cells.

In vitro cytotoxicity and its correlations with the subpopulation of NKM cells
We tested the in vitro cytotoxicity of NKM cells on a cancer cell line (K562), and compared its cytotoxicity with that of PBMCs and NK92 cells. In PBMCs (from healthy donors), the mean cytotoxicity was ~7.4% (effector/target (E/T) ratio = 10:1, 2-h incubation; n = 9) and 16.8% (E/T ratio = 10:1, 4-h incubation; n = 28). The mean cytotoxicity of NKM cells (n = 198) that we manufactured was ~65.6% (E/T ratio = 10:1, 2-h incubation), which was almost tenfold higher than that of PBMCs (Figure 2A and Supplementary Table 1). Approximately 84% of NKM cells showed >40% cytotoxicity. Even with more effector cells and longer incubation times, the cytotoxicity of PBMCs remained far inferior to that of NKM cells ( Figure 2B, 2C). Then, the cytotoxicity of NK92 cells was investigated with different E/T ratios (Supplementary Figure 2A). We discovered that the cytotoxicity of NKM cells (E/T ratio = 10:1) was comparable with that of NK92 cells, with an E/T ratio of 5:1 (2-h incubation) (Supplementary Figure 2B). Taken together, these results suggested that, although NKM cells had only half the cytotoxicity of that observed in "pure" NK cells (NK92), their cytotoxicity was much higher than that of PBMCs.
NKM cells are a mixed population of cells, so we wondered which subsets of these cells were responsible for in vitro cytotoxicity. We analyzed the subpopulations of NKM cells, and undertook correlation analyses of their in vitro cytotoxicity. We separated  AGING NKM cells into NK cells (CD3 − CD16 + or CD56 + ), NK T-like cells (CD3 + CD16 + or CD56 + ), CD4 T cells (T h cells, CD3 + CD4 + ) or CD8 T cells (cytotoxic T cells, CD3 + CD8 + ). A strong positive correlation was observed between the cytotoxicity of NKM cells and the proportion of NK cells (r = 0.58, p < 0.0001), but a negative correlation with the proportion of T h cells was noted (r = −0.32, p < 0.0001) ( Figure 3A, 3C). There was no correlation with the proportion of NK T-like cells and cytotoxic T cells ( Figure 3B, 3D).
Taken together, these data suggested that the in vitro cytotoxicity of NKM cells was caused mainly by the cytotoxicity of CD56 + CD16 + NK cells, and was suppressed by T h cells.

Potential function of NKM cells for adjuvant cancer treatment (in vivo cytotoxicity)
After the characterization of NKM cells, the potential in vivo anti-tumor activities of NKM cells were investigated in cancer patients for adjuvant cancer therapy. We recruited 11 patients with malignant lymphoma (diffuse large B-cell lymphoma [seven patients], peripheral T-cell lymphoma [two patients], anaplastic large-cell lymphoma [one patient], and Hodgkin's lymphoma [one patient]) who suffered primary relapse/were refractory to treatment (one patient), relapsed after salvage chemotherapy (eight patients), or relapsed after autologous stem-cell therapy (two patients). The treatment process is shown in Figure  4A. Each patient received an intravenous injection of 20-30 ×10 8 NKM cells each time (treatment once every 2 weeks). The response to NKM treatment was not robust (a complete response in one case and stable disease in one case) (Supplementary Table 2), but quality of life was improved greatly. In the patient who had a complete response (case #2), the number of AGING cancer foci was reduced significantly after treatment with NKM cells ( Figure 4B). During and after intravenous infusion of NKM cells, the body temperature, blood pressure, and heartbeat remained stable, and adverse reactions were not found. The patients tolerated the treatment and clinical side-effects were absent.
Systematic reviews of therapy with NK cells have indicated that immunotherapy with allogeneic NK cells AGING has better clinical efficacy than that using autologous cells [29][30][31]. Thus, we recruited another patient with pancreatic ductal carcinoma (T4N1M1c) whose cancer cells had metastasized, and he could not sustain chemotherapy. Then, we treated this patient with a human leukocyte antigen-matched infusion of haploidentical NKM cells (donor was the patient's son). The patient received an intravenous injection of 20-30×10 8 AGING NKM cells every week. After six treatments, the patient showed good toleration of treatment, and the number of metastatic foci in the renal pelvis and abdominal pelvic peritoneum was reduced significantly ( Figure 4C). The patient's vital signs and physical status were improved significantly.
Taken together, these results suggested that NKM cells displayed efficacious anti-tumor activity in cancer patients, and suggested that NKM cells could be used for adjuvant cancer therapy.

Identification and recharacterization of sub-healthy individuals on the basis of PD-1 expression in PBMCs
When analyzing the PBMCs of cancer patients, researchers found that PD-1 expression was significant-ly induced in peripheral immune cells [18,19,[32][33][34]. Previously, we found that among adolescents (10-20 years), the proportion of PD-1 + PBMCs (CD3 + CD4 + PD-1 + or CD3 + CD8 + PD-1 + ) was very low (<2%) (data not shown). However, the proportion of PD-1 + PBMCs in adults varies greatly, and the proportion in individuals is stable if external intervention is not applied. Here, to identify sub-healthy individuals, we measured expression of PD-1 and cytotoxic T lymphocyteassociated protein (CTLA)-4 in the NK cells and T cells of PBMCs in 95 individuals aged >50 years (Supplementary Table 3). PD-1 was expressed only on T cells, including T h cells and cytotoxic T cells, but not on NK cells, whereas CTLA-4 was not expressed in any PBMCs ( Figure 5A, 5B). High expression of PD-1 in T cells may indicate that a large number of T cells are exhausted [13,14] which, in turn implies an abnormal immune system.

NKM cells are a mixture of immune-cell types, including T h cells (which inhibit cytotoxicity) and NK cells (which elicit cytotoxicity). We wondered if these NKM cells may display more efficacious functions in the immune normalization of sub-healthy individuals.
We recruited four potential sub-healthy individuals and the treatment schedule is shown as Figure 7A. Approximately 20×10 8 autologous NKM cells were used for each treatment. Measurement of PD-1 expression in T cells was done before treatment, after the first injection, and after the third injection. In Patient-M and Patient-Z, upon a single injection of NKM cells, PD-1 expression in CD3 + , CD3 + CD8 + , and CD3 + CD4 + cells could be reduced significantly to a normal level (CD3 + PD-1 + cells/CD3 + cells was <4%; CD3 + CD4 + PD-1 + cells/CD3 + CD4 + cells was <10%; CD3 + CD8 + PD-1 + cells/CD3 + CD8 + cells was <10%) ( Figure 7B, 7C). In Patient-J, the effect of one injection of NKM cells was not obvious. After three injections of NKM cells, PD-1 expression in CD3 + , CD3 + CD4 + , and AGING CD3 + CD8 + cells decreased eventually to a normal level ( Figure 7D). In Patient-C, three injections of NKM cells reduced PD-1 expression in CD3 + cells and CD3 + CD8 + cells, but not in CD3 + CD4 + cells ( Figure 7E). All sub-healthy individuals showed good tolerance to treatment, and side-effects were not observed.
In summary, we found that with three therapies with autologous NKM cells, we could reduce PD-1 AGING expression in PBMCs, and achieve renormalization of the immune system in sub-healthy individuals.

DISCUSSION
For decades, researchers have focused efforts on "enhancing/improving" the immune system to eliminate the potential risks of serious diseases (e.g., cancer). The development of immune-cell therapies have made these concepts a reality.
A safe and efficacious method using NKM cells for the potential adjuvant treatment and prevention of cancer is presented here. We characterized the composition of NKM cells and investigated their cytotoxicity in vitro and in vivo. We showed that NKM cells were significantly cytotoxic to K562 cells or hepatocellular carcinoma cells [35]. However, compared with the number of cancer cells in cancer patients, the number of NKM cells that can reach a cancerous area is relatively small, so the anti-cancer effect is limited. Although the study cohort was and treatment effect was not robust, we showed the potential adjuvant anticancer function of NKM-cell therapy. The latter may not be as efficacious as therapy using pure NK cells. Nevertheless, due to its ease of operation and safety (fewer immune-related side-effects were found), we believe that NKM-cell therapy is potent adjuvant therapy for cancer patients, especially in combination with surgery, to improve the immune response and eliminate residual cancer cells.
Recently, scholars have proposed that cancer immunotherapy should focus on normalization of the immune system [6]. The proportion of PD-1 + immune cells (exhausted immune cells) is increased in the cancer tissues and PBMCs of cancer patients [13,20,23]. We postulate that this abnormality in PD-1 expression in PBMCs may be related to a high risk of cancer. A high proportion of PD-1 + T cells in PBMCs was found in some older adults (4.21%) in our cohort. Among the subhealthy individuals defined in our research, NKM-cell therapy could reduce PD-1 expression in PBMCs and quickly restore/rebalance the immune system. However, for some individuals (e.g., Patient C), more injections or larger doses of NKM-cell therapy may be required to reduce PD-1 expression in PBMCs.
We expanded NKM-cell therapy to not only treat cancer patients, but also prevent cancer in patients through normalization of the peripheral immune system. NKMcell therapy for sub-healthy individuals must be studied in randomized clinical trials. Our study could be used as reference to expand the number of applications of immune-cell therapy.
The COVID-19 pandemic is wreaking havoc on healthcare and economic systems worldwide. In patients with severe COVID-19, reduction in the number of immune cells and exhaustion of functional T cells in peripheral blood has been documented [28]. Just like our sub-healthy individuals, PD-1 expression is increased significantly in the CD4 + T cells and CD8 + T cells of PBMCs in COVID-19 patients. Our experiments in sub-healthy individuals normalized their peripheral immune system, so NKM-cell treatment for COVID-19 patients could be possible.

CONCLUSIONS
We demonstrated that a new method using NKM cells was safe and efficacious as adjuvant treatment for cancer patients as well as therapy for sub-healthy individuals. Normalization of the peripheral immune system through NKM-cell therapy could expand its scope of application in different disorders.

NKM cells were manufactured in an ISO Class 5 Good Manufacturing Practice Laboratory within the International Research Center for Regenerative
Medicine in BOAO International Hospital (Qionghai, China). Primary PBMCs were separated from whole blood (30-60 mL). A T75 flask was pre-coated with OKM25 medium (Fukoku, Ageo, Japan). PBMCs from individuals were cultured for 3-5 days in T75 and T225 flasks with OKM100 medium containing 1750 JRU/mL of IL-2 (Fukoku) and 10% autologous inactivated plasma. Then, NKM cells were transferred to a CO 2 Bag containing 1 L of OKM200 medium (containing 175 JRU/mL of IL-2) (Fukoku) and 1% autologous inactivated plasma, followed by culture for 3-5 days. NKM cells were incubated at 37° C in a humidified incubator in an atmosphere of and 5% CO 2 .

Ethical approval and consent to participate
The study protocol was approved by the Medical

Cytokine analyses with a cytometric bead array (CBA) kit
The cytokines secreted by NKM cells were analyzed by Human Th1/Th2 Cytokine Kit II (551809) from BD Biosciences. NKM cells (~2×10 9 cells) were harvested from 1.5 L of culture medium. The cytokines in these media were dyed and analyzed by flow cytometry according to the instructions of the CBA-kit supplier. Then, the collected data were analyzed with FCAP Array v3.0 (BD Biosciences).

Statistical analyses
Experiments were carried out independently in triplicate. Data are the mean ± SD. Graphs were plotted and analyzed with Prism 7.1 (GraphPad, La Jolla, CA, USA). P < 0.05 was considered significant.

Availability of data and material
All data have been added in the Supplementary Tables.