Trained Immunity of IL-12-, IL-15-, and IL-18-Induced CD3+CD56+ NKT-Like Cells

CD3+CD56+ natural killer T (NKT)-like cells have an immune function of T cells and NK cells, which play an important role in antitumor and antiviral immune responses. This study aims to establish a CD3+CD56+ NKT-like cell model by simulating the memory NK effect induced by cytokines IL-12, IL-15, and IL-18 (IL-12/15/18) and explore the formation mechanism. Our study found that the IL-12/15/18 preactivated CD3+CD56+ NKT-like cells exhibited enhanced IFN-γ production in response to restimulation with IL-12/15/18 for 6h on day 7. The intrinsic potential of these trained cells was significantly improved, showing an increase in IFN-γ, TNF-α, and cell proliferation potential. The IFN-γ release, granzyme B level, and proliferation ability significantly increased when stimulated by NK-cell-sensitive K562 tumor cells. Among these cytokines, the combination of IL-12/15/18 was particularly effective. After the preactivation of IL-12/15/18, some cell surface proteins related to function and differentiation, such as CD11b, CD62 L, NKp46, NKG2A, and CD127, showed an evident and consistent change trend. The CDK4/6 inhibitor can effectively weaken this effect, and the expression of cyclin D1, Rb protein phosphorylation, and E2F-1 decreased significantly. Our work revealed that cytokine IL-12/15/18 can induce CD3+CD56+ NKT-like cells to obtain enhanced training immunity, which was a memory-like phenomenon.


Introduction
Sharing characteristics with natural killer (NK) and T cells, CD 3 +CD 56 + natural killer T (NKT)-like cells possess innate and acquire immune functions [1]. ey regulate different immune responses, including cancer immunity and autoimmunity development [1,2]. NK cells, iNKT cells, and CD 3 +CD 56 + NKT-like cells serve as the first line of defense of the natural immune system [3]. ese cells play an important role in inhibiting viral infection and controlling tumor by secreting interleukin, interferon (IFN), and chemokine [4,5]. CD 3 +CD 56 + NKT-like cells are large granular lymphocytes, which are CD1 d unrestricted, and they have polygenic TCR recombination [1,6]. Moreover, they can be activated to secrete cytokines and effectively kill cancer cells in a nonmajor histocompatibility complex (MHC)-restricted lysis in the absence of T-cell antigen receptor (TCR) activation [1][2][3][4]. Numerous studies have confirmed that exogenous IFN-c and cytokines such as IL-2 and IL-15 can activate immune cells [5]. As far as we know, the nature, function, and clinical relevance of cytokinetrained CD 3 +CD 56 + NKT-like cells remain unexplored.
Immunotherapy is a potentially valuable treatment of the 21st century [6]. Relevant immunotherapies in recent clinical trials engage either the cellular stage of adaptive immunity or effector molecules, such as cytokines. Trained immunity was first proposed by Netea et al. in recent years [7,8]. As an immunological memory of the innate immune system, trained immunity involves the epigenetic programming of myeloid lineage cells, leading to changes in their metabolic and phenotypical behavior [6,9]. e abovementioned process may strengthen the immune response to secondary stimuli [9][10][11]. Cooper MA first proposed cytokine-induced memory-like NK cells in 2009, representing a typical example of trained immunity of NK cells [12]. Since then, an increasing number of studies have confirmed that human NK cells display functional memory-like properties after cytokine activation, which may provide a novel rationale for NK cell immunotherapy strategies [4,13]. erefore, we investigate whether CD 3 +CD 56 + NKT-like cells can establish a similar cytokine-induced memory NK cell experimental model when they play a role in NK cellkilling activity.
is study aimed to establish a CD 3 +CD 56 + NKT-like cell model by simulating the memory NK effect induced by cytokines. e levels and functional activities of CD 3 +CD 56 + NKT-like cells in human peripheral blood mononuclear cells (PBMC) stimulated by IL-12, IL-15, and IL-18 (IL-12/15/18) were detected to observe their response to NK-cell-sensitive K562 tumor cells. e biological activity and function of IL-12/15/18 preactivated CD 3 +CD 56 + NKT-like cells were analyzed when the same cytokines were restimulated. Moreover, the mechanism of this memory-like NK effect produced by CD 3 +CD 56 + NKT-like cells was explored.

Blood Samples.
Fresh human blood specimens for flow cytometry experiments were obtained from Tianjin Medical University Cancer Hospital. All samples were provided anonymously after informed consent was obtained. e collection, distribution, and usage of all deidentified human peripheral blood were approved by the Institutional Review Committee on Human Research of the Tianjin Medical University Cancer Institute and Hospital.

Cell Lines and Culturing
Conditions. Human leukemia cell lines (K562) were obtained from the American Type Culture Collection. All cancer cell lines were maintained in RPMI 1640 media (Gibco, Grand Island, New York, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum ( ermo Fisher Scientific) and contained 100 U/mL of penicillin and 100 µg/mL of streptomycin (Gibco).

Flow Cytometry and
Antibodies. Cells were harvested and transferred into a 96-well plate at approximately 1 × 10 6 / well for staining, incubated with human Fc receptor blocking solution (422302; BioLegend), and then stained in sequence with cell viability dyes (423106; BioLegend), surface antibodies, and intracellular antibodies. e release of IFN-c, TNF-α, and granzyme B, proliferation, and degranulation were detected by flow cytometry.

Western Blot.
All harvested cells were lysed in ice-cold RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.). All protein concentrations were determined using the BCA protein assay reagent and measured with a NanoDrop2000 spectrophotometer ( ermo Fisher Scientific, Inc.). Equal amounts of protein samples (25 µg) were separated by 10% SDS-PAGE gels and then transferred onto PVDF membranes. After blocking with nonfat milk for 2 h at room temperature, the membranes were probed with the corresponding primary antibodies incubated at 4°C overnight. After incubating with horseradish peroxidase (HRP)conjugated secondary antibodies at room temperature for 2 h, protein bands were visualized using a chemiluminescence detection system (Bio-Rad Laboratories, Inc.). en, the density was evaluated by ImageJ 1.48v (National Institutes of Health 2.6. Statistical Analysis. Data are shown as the meansSEM in all graphs, and significant differences were calculated with Student's t-test or as indicated. P < 0.05 was considered significant. All statistical analyses and plots were produced in GraphPad Prism. 3 +CD 56 + NKT-Like Cells Increase IFN-c Release. Human CD 3 +CD 56 + NKT-like cells were preactivated with low-dose IL-15 as control or with IL-12/15/18 for 16 h, and then the control and experimental samples were cocultured with IL-15 for 7 days to induce training immunity. e cells were analyzed by flow cytometry at different time points (Figure 1(a)). During culture, the proportion of cells did not change (P � 0.334, Figure 1(b)). Consequently, trained CD3+CD56+ NKT-like cells exhibited enhanced IFN-c production in response to restimulation with IL-12/15/18 for 6 h on day 7 (P � 0.002, Figure 1(b)), which was similar to the memory effect, but TNF-α release and cell proliferation were not improved (P � 0.126; P � 0.432, Figure 1(c)). e preactivated CD 3 +CD 56 + NKT-like cells showed an increase in IFN-c, TNF-α, and cell proliferation potential after PMA + iono stimulation on day 7 (P < 0.001; P � 0.020;P � 0.008, Figure 2(a)). IFN-c release, granzyme B level, and proliferation ability significantly increased when IL-12/15/18 preactivated CD 3 +CD 56 + NKT-like cells were stimulated by K562 (P � 0.013; P < 0.001; P < 0.001, Figure 2(b)). When the proportion of tumor cells was adjusted, IL-12/15/18 preactivated CD 3 +CD 56 + NKT-like cells encountered different proportions of K562 tumor cells. e results showed that the effect of different proportions of K562 tumor cells was the same (P � 0.184; P � 0.245; P � 0.245, Figure 2(c)).
We selected several small-molecule drugs on the basis of epigenetics to study the mechanism of this memory effect in CD 3 +CD 56 + NKT-like cells, including the CDK4/6 inhibitor, DNA-demethylating agent, Myc inhibitor, and EZH2. We added these small-molecule drugs to the IL-12/15/18 preactivated CD 3 +CD 56 + NKT-like cells on day 1 and trained CD 3 +CD 56 + NKT-like cells using the above-mentioned method ( Figure 5(a)). e IFN-c release significantly reduced in the experimental group with the CDK4/6 inhibitor, whereas the IFN-c release in the other three drug experimental groups did not significantly decrease (P < 0.001; P � 0.963; P � 0.297; P � 0.158, Figure 5(b)). We detected the expression of CDK4/6 and its downstream pathway proteins by the Western blot to verify whether CDK4/6 and its downstream pathway proteins are involved in the biological effects of CD 3 +CD 56 + NKT-like cells. e experimental results showed that after adding the CDK4/6 inhibitor, the expression level of CDK4/6 in the drug group was significantly lower than that in the preactivated group (P � 0.008, Figure 5(c)). e expression of cyclin D1, the phosphorylation level of Rb, and the expression of E2F-1 significantly decreased (P < 0.001, Figures 5(c) and 5(d)).

Discussion
CD 3 +CD 56 + NKT-like cells are a large group of unique cell subsets that express TCR and NK cell markers, accounting for approximately 5%-15% of the peripheral T cell pool and up to 50% of T cells within the liver [14]. Different from the traditional T cells, CD 3 +CD 56 + NKT-like cells have innate and adaptive immune functions, T cells, and NK cells [1,14]. erefore, they can perform MHC-restricted cytotoxicity or MHC-unrestricted cytotoxicity and secrete a variety of cytokines [1,2,5]. CD 3 +CD 56 + NKT-like cells play an important role in antitumor and antiviral immune response [4]. e activation of CD 3 +CD 56 + NKT-like cells may be crucial because they can serve as an early source of regulatory cytokines and degranulation-related killing functions [4,5]. IFN-c is the key factor in activating the acquired immune response and resisting various infections, autoimmune diseases, and tumor infections [1][2][3]5]. It primarily exerts antiviral activity and specific cytotoxicity and promotes 1 transformation through overexpression of MHC class I and II molecules, antigen processing, and immunoglobulin conversion [1,2,14]. erefore, IFN-c may be an important indicator for CD 3 +CD 56 + NKT-like cells to exert immune function, and it is important to study the relationship and changes among these cells.
Recent studies have found that cytokine signal transduction is important for the differentiation, survival, maintenance, and activation of NK cells [12,13,15]. e cytokines IL-2, IL-12, IL-15, IL-18, and IL-21 and type I IFNs have been used to expand and activate NK cells in vitro before adoptive transfer, and this process efficiently boosts the quantity and function of NK cells [15][16][17]. In the study of memory NK cells, IL-12/15/18 preactivated NK cells showed an increase in IFN-c production in response to restimulation with IL-12/15/18 or K562 tumor cell lines. Compared with traditional NK cells, cytokine-induced memory NK cells had stronger recognizing and killing ability [5,12,17,18]. As an important natural immune cell, CD 3 +CD 56 + NKT-like cells  Journal of Oncology have many similar characteristics with NK cells in killing activity, such as cytotoxicity of sensitive tumor cells and expression of NK cell receptors to regulate their own functions [1,14]. erefore, we deeply explore whether cytokine-induced CD 3 +CD 56 + NKT-like cells, which are similar to memory NK cells, have biological characteristics to improve their role in resisting infection and tumor. Our study found that IL-12/15/18 preactivated CD 3 +CD 56 + NKTlike cells exhibited enhanced IFN-c production in response to restimulation with IL-12/15/18 for 6 h on day 7, which was similar to the memory effect, but the TNF-α release and cell proliferation were not improved. Among these cytokines, the combination of IL-12/15/18 was effective, whereas other cytokines, such as IL-2/IL-15, IL-12/IL-18, IL-15/IL-18, and IL-12/IL-15, could not stimulate a similar biological effect. erefore, our study found that CD 3 +CD 56 + NKT-like cells showed similar memory characteristics after cytokine activation, which may provide a new theoretical basis for CD 3 +CD 56 + NKT-like cell immunotherapy.
Tumor immunotherapy is a potential therapeutic method [7,19], which can be used to remove cancerous cells by activating immune cells in the body, and the advantages of tumor immunotherapy include high specificity, long action period, and small side effects [6][7][8]19]. Trained immunity is orchestrated by epigenetic reprogramming, broadly defined as sustained changes in gene expression and cell physiology that do not involve permanent genetic changes, such as mutations and recombination, which are essential for adaptive immunity [7,20]. erefore, the discovery of trained immunity may lead to new therapeutic strategies for the treatment of many immune diseases and could be a potential strategy for tumor immunotherapy. Some studies have focused on CD 3 +CD 56 + NKT-like cells in tumor. Jiang YongJun found that the function of CD 3 +CD 56 + NKT-like cells in long-term nonprogressors may be a protective mechanism to slow down the progression of HIV disease [14]. Peng Liu-Sheng indicated that the frequencies of CD 3 +CD 56 + NKT-like cells in gastric cancer tumors significantly decrease and that low levels of tumor-infiltrating CD 3 +CD 56 + NKT-like cells are positively correlated with poor survival and disease progression [1]. In patients with chronic myelogenous leukemia, Jani-Sofia Almeida observed significant alterations on the expression of tumor recognition (NCRsandNKp80) and immune regulatory receptors (LAG-3, TIM-3, and CD137) by NKT-like cells [2]. ese studies suggest that CD3+CD56+ NKT-like cells play an important role in the occurrence and development of cancer. In the present study, the preactivated CD3+CD56+ NKT-like cells showed an increase in IFN-c, TNF-α, and cell proliferation potential after PMA + iono stimulation on day 7, indicating that training immunization can directly activate the intracellular signaling pathways without being affected by cell surface receptors. When we stimulated these cytokine-induced CD 3 +CD 56 + NK-T-like cells with K562 tumor cells, we found that the IFN-c release, granzyme B level, and proliferation ability were significantly increased. K562 tumor cells can directly stimulate these trained CD 3 +CD 56 + NKT-like cells to release IFN-c and enhance their killing ability, which also indicates the potential of cytokine training immunity for clinical tumor therapy. e memory effect of CD 3 +CD 56 + NKT-like cells induced by cytokines is primarily manifested in the significantly increased release of IFN-c after encountering the same cytokine stimulation. is finding also suggests that after cytokine immune training, CD 3 +CD 56 + NKT-like cells can mobilize their internal potential and play a stronger immunological function when they encounter appropriate stimulation. erefore, optimizing the cytokine scheme, inducing CD 3 +CD 56 + NKT-like cells sensitive to certain tumor cells, and making this immune cell safe, stable, and effective in killing tumor cells in patients with cancer is necessary.   e specific mechanism of the memory effect in cytokine-trained CD 3 +CD 56 + NKT-like cells remains unclear, and we should consider the research process, causes, and results. In the present study, we compared the CD 3 +CD 56 + NKT-like cells preactivated with cytokines IL-12/15/18 on the first day (about 16 h) with those maintained for 7 days. When the same dose of IL-12/15/18 was restimulated, the IFN-c release was significantly increased on the 7th day but not on the 1st day, suggesting that the cultured cells must remain stable for a period of time to induce a memorylike effect, which also indicates that their formation exists during cell division and differentiation. In addition, we detected the expression of indicators on the surface of the cell membrane, and the results showed that after preactivation with IL-12/15/18, some cell surface proteins related to function and differentiation, such as CD11b, CD62 L, NKp46, NKG2A, and CD127, showed evident and consistent change trends, suggesting that this memory-like effect manifested in different differentiation states. On the basis of epigenetics, we selected several small-molecule drugs and observed their effects on the experimental results. Cyclindependent kinases (CDKs), first discovered by Tim Hunt, Paul Nurse, and Leland H. Hartwell, play an important role in the initiation of cell cycle and the regulation of cell cycle transition [21,22]. Human cells have a complete division and proliferation cycle, from G1 phase to S phase and G2 phase to M phase [23][24][25], and CDK4/6 in CDKs are key condition proteins of human cell division and proliferation cycle [23]. CDK4/6 inhibitors selectively inhibit CDK4/6, effectively preventing tumor cells from progressing from G1 phase to S phase, restoring cell cycle control, and blocking tumor cell proliferation [21][22][23]. Our results showed that when some small-molecule drugs were added to the culture induced by cytokines on day1, the Myc inhibitor, DNAdemethylating agent, and EZH2 could not change the IFN-c release, whereas CDK4/6 inhibitors can effectively weaken    this effect, suggesting that this memory-like effect may be related to cell cycle and cell proliferation. Moreover, after adding the CDK4/6 inhibitor, this biological effect has not completely disappeared, suggesting that its production mechanism results from multiple effects, which is still closely related to the cell microenvironment or other unknown proteins and receptors, and more research must be further explored. e Western blot results showed that CDK4/6 and cyclin D could phosphorylate the retinoblastoma gene (Rb) and then release the transcription factor E2F, which could promote the transcription of cell cycle-related genes, thereby regulating the occurrence of this memory-like phenomenon. is phenomenon provides insights into the mechanism study of such memory-like CD 3 +CD 56 + NKT-like cells.
e present study proposed that CD 3 +CD 56 + NKT-like cells in PBMCs preactivated by IL-12/15/18 could gain trained immunity with enhanced effector functions, a memory-like phenomenon, analyzing the possible response of these prestimulated cells to K562 tumor cells. In addition, this study found that IFN-c played a key role in the proliferation and cytotoxicity of CD 3 +CD 56 + NKT-like cells.
e important role of CDK4/6 in this memory response was also proposed, which provided experimental reference for other factors of its formation mechanism and paved the way for later research and clinical research. Tumor immunotherapy is a potential therapeutic method in the 21st century [7,8], which can be used to clear cancer cells by activating immune cells in vivo. Tumor training immunotherapy is modified on the basis of epigenetics. Epigenetics regulates the chromatin status and gene expression through DNA methylation and demethylation, histone modification, and chromatin remodeling, but it does not involve permanent genetic changes, which is important for adaptive immunity [7,26,27]. erefore, our study may provide a new theoretical basis for CD 3 +CD 56 + NKT-like cells in immunotherapy strategies, but further study on their differentiation, survival, maintenance and activation of microbial homeostasis, and clinical transformation of the idealized model of specific killing tumor cells is necessary.

Conclusions
Our work revealed that cytokine IL-12/15/18 can induce CD 3 +CD 56 + NKT-like cells to obtain enhanced training immunity, which was a memory-like phenomenon [28]. Data Availability e data and materials used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest.

Authors' Contributions
Siyu Zhu and Chen Zhang have contributed equally to this work. Siyu Zhu, Chen Zhang, and Xiubao Ren designed the project and wrote the paper. Siyu Zhu, Chen Zhang, Qian Sun, Yang Wang, Wenwen Yu, and Feng Wei conducted the experiments. All authors reviewed and approved the final manuscript.