CIK (cytokine-induced killer, Chinese name: [autologous cell immunotherapy] multiple cytokine-induced killer cells) is a group of heterogeneous cells obtained by co-culturing human peripheral blood mononuclear cells with multiple cytokines (such as anti-CD3 monoclonal antibody, IL-2 and IFN-γ, etc.) in vitro for a period of time. Since these cells express both CD3+ and CD56+ membrane protein molecules, they are also known as NK cell-like T lymphocytes, which have both the powerful anti-tumor activity of T lymphocytes and the non-MHC-restricted tumor-killing advantage of NK cells. Therefore, the application of CIK cells is considered to be the first choice for the next generation of antitumor pericyte immunotherapy. The effector CD3+ and CD56+ cells in CIK cells are extremely rare in normal human peripheral blood, only 1%-5%.CIK characteristicsThe effector CD3+CD56+ cells in CIK cells are extremely rare in normal human peripheral blood, only 1%-5%, in vitro by multi-factor culture 28-30 days, CD3+CD56+ cells The CD3+CD56+ cells increased rapidly in 28-30 days after multifactor culture in vitro, and the rate of increase was more than 1000 times compared with that before culture. It was demonstrated that the expanded CD3+CD56+ cells were derived from CD3+CD56-T cells, but not CD3-CD56+ NK cells. It was also found that among the CD3+CD56- T cells, all three T cell subsets (CD4-CD8+, CD4-CD8-, CD4+CD8+) except CD4-CD8- T cells could obtain CD56 molecule expression by in vitro multi-factor culture, and since the levels of CD4+CD8+ cells and CD4-CD8- cells in normal human peripheral blood Indirectly, this CD3+CD56+ cells were derived from CD4-CD8+ T cells in peripheral blood due to the extremely low levels of CD4+CD8+ cells and CD4-CD8- cells in normal human peripheral blood. Since nearly 56% of CD4-CD8-T cells expressed both CD56 and CD3 after 1 month of culture, this suggests that they are also an important source of CIK cells. Comparing CD3+CD56+ CIK cells expressing CD8+ and CD8-,there was no significant difference in their tumoricidal activity, suggesting that the cytotoxicity of CIK cells tended to correlate with CD3CD56 expression, whereas it did not show a correlation with CD8 expression. Killing principle CIK cells are able to kill tumor cells and virus-infected cells through three pathways: ① Direct killing of tumor cells and virus-infected cells by CIK cells: CIK cells can recognize tumor cells through different mechanisms and release toxic particles such as granzyme/perforin, leading to tumor cell lysis. (ii) A large number of inflammatory cytokines released by CIK cells have tumor-inhibitory and tumor-killing activity: CIK cells cultured in vitro can secrete a variety of cytokines, such as IFN-γ, TNF-α, IL-2, etc., which not only have a direct inhibitory effect on tumor cells, but also can kill tumor cells indirectly by regulating the responsiveness of the body’s immune system. ③CIK cells can induce apoptosis of tumor cells: CIK cells express FasL (type II transmembrane glycoprotein) in culture induce apoptosis of tumor cells by binding to Fas (type I transmembrane glycoprotein) expressed in the tumor cell membrane. The three pathways by which CIK cells act to kill tumors Characteristic application of LAK cells is currently a more popular tumor over-the-top immunotherapy regimen, widely used in melanoma, renal cell carcinoma, non-Hodgkin’s lymphoma, lung cancer, and colon cancer. Because LAK cells have limited expansion and lower tumor-killing activity than TIL cells, they have a broad spectrum of tumor killing but limited effect. In contrast, TIL cells have more potent antitumor activity than LAK cells, but their clinical application is limited by their narrow tumor killing spectrum, the difficulty of preparation, and the functional changes that may occur during collection. Compared with the above two effector cells, CIK cells have unique advantages, which are listed below. 1, fast proliferation rate CIK cells in the culture process after the addition of IFN-γ, IL-1α, anti-CD3, McAb, IL-2 and other multi-factors, cell proliferation rate is rapidly accelerated, far more than LAK cells. On the 22nd day of culture, the proliferation curve peaked at about 100-fold increase, in which the absolute number of CD3+CD56+ cells not only increased by more than 1000-fold, and the percentage also increased significantly, reaching a plateau at 28-30 days of culture, and the cytotoxic activity also peaked, while the number of LAK cells did not increase significantly before and after culture. 2, high tumor killing activity CIK cells are a heterogeneous population of CD3+CD56+ T cells, a large number of in vitro and ex vivo experiments have confirmed that CIK cells have more powerful tumor killing activity than NK cells-based LAK cells, and their in vivo maintenance of tumor killing cytotoxicity does not have to rely on the continuous administration of large doses of exogenous IL-2. In in vitro experiments, Ren Huan and Lu et al. found that equal numbers of CIK cells had a slightly higher or similar killing capacity than LAK cells against tumor cell lines in vitro, but the total killing unit (TLU) of CIK cells was 73 times or more than that of LAK cells because of the rapid growth of CD3+CD56+ effector cells in culture, and the killing efficiency of CIK cells was significantly higher than that of LAK cells. The tumor clonogenic inhibition assay showed that the tumor cell inhibition Log index of CIK cells was 2,5-3,5, which was 2 Log higher than that of LAK cells. The effects of CIK cells were significantly better than those of LAK cells in clearing tumor foci, inhibiting metastasis and prolonging survival. Although CIK cells use CD3+CD56+ T cells as the main effector cells, they do not have the MHC restriction of T lymphocyte killing, so they are more effective against a variety of tumor cell lines (including NK-sensitive K562 and NK-insensitive Hela, HL60, human T-cell acute leukemia cell line OCRF-CEM, human lymphoma cell lines OCI-LY8 and LAM53, human colon cancer cell line OCRF-CEM, human lymphoma cell line OCI-LY8 and LAM53). LAM53, human colon cancer cell lines HT-29 and CR75, human kidney cancer cell line A704) and fresh tumor tissues all exhibited potent killing activity. 4, Equally sensitive to multi-drug resistant tumor cells Wolf induced multi-drug resistant cell lines K562/DOX and CCRF-CEM-VBL with adriamycin and vincristine and found that CIK cells showed potent killing activity against both parental cells sensitive to chemotherapeutic drugs and insensitive transformed cells, with no difference between the two comparisons. 5, Tumor killing activity was not affected by immunosuppressants such as CsA and FK506 Mehta observed that although immunosuppressants CsA and FK506 could inhibit the anti-CD3 monoclonal antibody-mediated degranulation process of CIK cells, they did not affect the target cell-induced degranulation of CIK cells, and the killing activity of CIK cells on target cells would not be reduced as a result. 6, the normal bone marrow hematopoietic precursor cell toxicity is very small Seheffold by CFU-GM formation assay to detect the effect of CIK cells on bone marrow hematopoietic precursor cells, found that CIK cells on K562 cells kill intensity up to level 3, but only less than 1 level of inhibition of GM-CFU. Holye also confirmed that CIK cells have little effect on normal myeloid clonogenesis, only the generation of red lineage showed mild inhibition, which may be related to the secretion of higher levels of IFN-γ by CIK cells themselves. 7, can resist tumor cell-triggered effector cell Fas-FasL apoptosis has been shown that an important reason for the failure of successive immunotherapy is the induction of apoptosis in the successive effector cells by certain proteins expressed on the surface of tumor cells (mainly FasL), whereas CIK cells, although they cause a small amount of apoptosis after Fas is occupied, have no significant effect on their tumor-killing cytotoxicity. verneris’ experiments suggested the expression of anti-apoptotic genes in CIK cells and detected upregulation of transcript levels of various protective genes, such as cFLIP, Bcl-2, Bcl-X1, DAD1 and survivin. CIK cells were also found to have the ability to synthesize FasL, and biologically active water-soluble FasL could be detected in CIK cell culture supernatants, indicating that CIK cells can counteract the decrease or even loss of effector cell activity triggered by FasL-positive tumors in vivo. Factors affecting killing activity 1, exogenous cytokine supplementation CIK cell expansion in vitro requires the assistance of exogenous cytokines, such as IL-2, IL-7, IL-12, etc. These factors control the expansion of various antigen-specific cells in the human immune system and their biological activity. Exogenous IL-2, IL-7, and IL-12 significantly promote the growth of lymphocytes, and the proliferation rate of CIK cells is particularly high in the presence of IL-2 and IL-7, whereas exogenous IL-2, IL-7, and IL-12 have no effect on the cytotoxic activity of CIK cells. Stimulation of exogenous IL-2 and IL-7 decreased the expression of the corresponding receptors on the surface of CIK cells, whereas CD28 molecules were more highly expressed in the presence of IL-7 than in the presence of IL-2. IL-12 decreased the expression of ICAM-1 on the surface of CIK cells, while IL-7 increased the expression of CD56. IL-7 significantly increased the proportion of CD4+ cells compared to IL-2. Although exogenous IL-2, IL-7 and IL-12 can all appear in small numbers of apoptotic cells during culture, some studies have shown that the addition of exogenous IL-12 increases the proportion of necrotic cells in CIK cells. Lefterov’s pre-incubation of anti-CD3McAb with target cells increased the killing sensitivity of CIK cells, and this enhancement was partially blocked by antibodies against FcR (e.g. anti-CD36, anti-CD32). anti-CD32) were partially blocked, indirectly demonstrating that the increased killing activity triggered by anti-CD3McAb was associated with FcR-mediated antibody binding. 2, transfection of multiple cytokine genes Since CIK cell expansion is dependent on exogenous cytokines, transferring relevant genes into CIK cells by gene transfer methods not only reduces the amount of exogenous cytokines used, but also improves the antitumor activity of CIK cells themselves. IL-7: Fitke transfected CIK cells with human IL-7 gene using a modified adenoviral transgenic system and found that the transfected cells could produce high concentrations of IL-7, up to 1,100 pg/106cell/24 h. The synthesized IL-7 had significant biological activity and could promote the proliferation of transfected CIK cells, which was significantly higher than that of untransfected cells. The expression of exogenous IL-7 gene also altered the secretion of other cytokines by CIK cells, especially TNF-α, which was not observed when IL-7 was added to untransfected CIK in vitro. Although various surface antigens associated with cell killing activity, such as ICAM-1, were not significantly changed on the surface of transfected CIK cells compared to untransfected CIK cells, the killing ability of transfected CIK cells was significantly enhanced compared to untransfected CIK cells for a variety of tumor cell lines, such as renal cancer, malignant melanoma, and colon cancer. IL-2: Lu et al. found that the expression of CD56 molecules during CIK cell culture was IL-2 dependent, but the presence of IL-2 alone reduced the magnitude of phenotypic changes in CIK cells after culture. Although some experiments have indicated that in vivo treatment of CIK cells does not require a continuous supply of IL-2 in vitro, the results of Zoll et al. showed that IL-2 in vitro culture promoted the proliferation and killing function of CIK cells. Schmidt-Wolf introduced recombinant plasmids containing human IL-2 gene fragments into CIK cells for the treatment of metastatic solid tumors by electroporation and found that after transfection The cells secreted higher levels of IL-2 (330-1,800 pg/106 cell/24h, with an average of 836 pg/106 cell/24h). Although there was no significant change in the expression of various membrane proteins on the surface of CIK cells before and after transfection, the proliferation rate and cell killing activity of transfected CIK cells were higher than those of untransfected cells in an in vitro assay. Preparation process CIK cells were prepared by drawing peripheral blood from patients, incubated at 37°C for 2h in a 5% CO2 incubator, separated using a high-speed centrifuge, collected in suspension, and cultured in vitro (simulating the human in vivo environment) with the addition of various cytokines such as CD3 monoclonal antibody, IFN-R, IL-2, etc. The culture medium was changed every 2-3 days and collected on days 7, 11, 13 and 15 CIK cells, CD3+ and CD56+ cells increased rapidly, up to 1000 times more than before culture. Development history In 1985 American surgeons first discovered that high doses of IL-2 in vitro could culture lymphocytes into cells with a strong tumor-killing effect, called LAK cells. Later, it was found that the addition of CD3 monoclonal antibody to the culture medium could increase the tumor-killing activity of these cells by more than tenfold, and the number of expanded cells was also greatly increased, which was called CD3AK. These cells are called CIK and have a higher proliferation rate and greater virulence than LAK cells. CIK cells were first reported by Schmidt Wolf et al. at Stanford University in 1991. Domestic clinical applications and related policies and regulations as early as 1996, there are CIK cell therapy technology clinical applications of research papers published in China, more than a hundred medical units have carried out the clinical application of this treatment technology and research. According to the progress of international and domestic clinical application of cell therapy, on May 1, 2009, the Ministry of Health issued and implemented the “clinical management of medical technology application” will be “autoimmune cell therapy technology” as three types of medical technology. Indications CIK cell therapy is a type of relay cell immunotherapy. Since CIK cell lysis is non-MHC-restricted, i.e. not restricted by tumor tissue type, it can kill any kind of tumor, but it is most effective in cancers with high antigen expression, such as myeloid leukemia, melanoma, renal cell carcinoma, metastatic kidney cancer, non-Hodgkin’s lymphoma, etc. Other cancers such as lung cancer, colon cancer, breast cancer, liver cancer, gastric cancer, colorectal cancer, etc. CIK cell therapy is also effective for patients with any stage of cancer, but it is effective for patients with early stage tumors or those with less tumor load after surgery and radiotherapy. It plays an important role in removing small residual lesions in patients after surgery, radiotherapy or hematopoietic stem cell transplantation, preventing the spread and recurrence of cancer cells, improving patients’ own immunity and reducing toxic reactions. CIK cell therapy can improve the quality of life and prolong the survival time with tumor for certain patients with intermediate and advanced tumors who are not suitable for surgery and cannot tolerate radiotherapy or chemotherapy. Features 1, CIK cells proliferate rapidly, anti-tumor active cells can proliferate in large numbers, and cell activity is also greatly enhanced. 2, CIK cells have the mechanism to recognize tumor, and have no toxic effect on normal cells. 3.Wide tumor killing spectrum, can be used for the treatment of leukemia, lymphoma, lung cancer, gastric cancer, intestinal cancer and other tumors, and is equally sensitive to multi-drug resistant tumor cells. 4. It is a typical personalized biological treatment mode. After infusion of these cells back into the body, they can also improve the immune ability of the body and produce specific antiviral effects, thus exerting a dual effect on tumor treatment. 5. Since CIK cells are activated autologous cells, they are very safe to use. Effective CIK cells directly inhibit the growth of tumor cells and induce apoptosis through direct killing and secretion of various cytokines to play a therapeutic role. Most of the cells that perform the killing function perform their function immediately after transfusion, with a half-life of about 2 weeks to a month. The returned cells include some memory cells, which can survive for several years to decades and are rapidly activated in vivo to kill target cells when they encounter the corresponding stimuli. Therefore, the interval between courses of CIK cell therapy is in principle one month, and it is recommended to do three courses of treatment consecutively at an interval of one month first, and then one course every six months thereafter.