CIK cells in tumor immunotherapy, Treg cells and their correlation

Title: CIK cells in tumor immunotherapy, Treg cells and their correlation Author: Ren Pengtao Degree-granting units: Hebei Medical University Keywords: dendritic cells;; cytokine-induced killer cells;; CD4 ~ + CD25 ~ + T cells;; melanoma;; immune tolerance;; immunotherapy Abstract:
Objective: The infusion of immune cells adoptive cellular immunotherapy (adoptive cell immunotherapy ACI) is a biological therapy for cancer research focus. This treatment is not only conventional surgery, radiotherapy, chemotherapy and other anti-cancer treatment supplement, it is for the promotion of the reconstruction of the immune system of patients, to Strong magnets eliminate residual disease and others have good results. Not suitable for more advanced surgical treatment or can not afford to radiotherapy and chemotherapy in patients with side effects caused by opening a new therapeutic approach, a human anti-tumor therapy one of the most promising measures.
Cytokine-induced killer cells CIK (cytokine induced killer, CIK), as a new and efficient proliferation of immune cells because of their ability to kill tumor spectrum, and strong tumoricidal activity of other effector cells can not match the superior characteristics of in adoptive immunotherapy has been widely used. How to improve the number of CIK cells, to enhance its anti-tumor and anti-tumor-specific cytotoxic activity and so is the CIK cells for research on the key. Dendritic cells (DC) is by far the strongest effect found in antigen-presenting cells, with high uptake, processing, presenting tumor-associated antigens, and activation of the unique features of the initial immune response. In this study, DC and CIK cells co-cultured cells, and load-specific antigen, in order to obtain a greater number, higher killing activity, more specific anti-tumor effector cells.
However, in practical application process, the anti-tumor effector cells in the tumor micro-environment is often induced into the "immune incompetence" (immune anergy) status and can not effectively recognize and kill tumor cells. Removal of the body's immune suppressor cells, give full play to effector cell function, for improving the treatment of cancer is important. CD4 ~ + CD25 ~ + Treg cells are newly discovered inhibition of immune cell populations, now more commonly used is based on different ways to produce the CD4 ~ + CD25 ~ + T cells into naturally occurring regulatory T cells (natural regulatory T cells nTreg) and induced regulatory T cells (induced regulatory T cells iTreg) categories. Large number of studies found in the stomach, lung, breast and other http://www.chinamagnets.biz/Neodymium/Ball-Neodymium-Magnets.php cancer patients peripheral blood, tumor-infiltrating lymph nodes can detect the CD4 ~ + CD25 ~ + T cells, CD4 ~ + CD25 ~ + T cells may recognize tumor antigens, activated play its immune inhibitory anti-tumor immune responses generated, so that the body is low on tumor response or no response to a state. The study shows that CD4 ~ + CD25 ~ + Treg cells for tumor immunotherapy is an important obstacle. So look for both to enhance anti-tumor immune effector cell function and can inhibit or remove CD4 ~ + CD25 ~ + T cells in ways that will enhance and improve tumor immunotherapy offers a new way.
In this study, the mature DC-specific antigen co-cultured with CIK cells, as anti-tumor immune effector cells, morphology, cell proliferation and apoptosis and tumor cell killing activity on different aspects of the effects of different stages of the application magnetic activated cell sorting to remove CD4 ~ + CD25 ~ + Treg cell-specific antigen components of DC-CIK cells on B16 melanoma animal model for the role, to explore and analyze the CIK cells, Treg cells in anti-tumor immunity the role and relationship to anti-tumor immune therapy for the clinical application of ideas to provide more treatment and theoretical basis.
Method:
In this experiment, C57BL / 6 mice as animal models of melanoma study, preparation of animal models of melanoma, and the application of radioactive cobalt-60 mice were irradiated animal models of melanoma, the mice formed body of non-myeloid cells removed state, the C57BL / 6 mice in animal models of melanoma loss of the immune system.
Mice peripheral blood mononuclear cells (PBMC) preparation of dendritic cells and CIK cells, the two co-culture, and the load of tumor-specific antigen, and detecting the immune phenotype. By magnetic activated cell sorting methods were cultured in culture before and after removal of CD4 ~ + CD25 ~ + Treg cell components. Were obtained to remove CD4 ~ + CD25 ~ + Treg cell components and then by in vitro induced specific antigen load DC-CIK effector cells in vitro induced by tumor-specific antigen loaded DC-CIK effector cells, and then the beads sorting methods to remove CD4 ~ + CD25 ~ + Treg cell-specific antigen components of the load of DC-CIK effector cells and did not remove CD4 ~ + CD25 ~ + Treg cell-specific antigen components of the load of DC-CIK effector cells were three groups of anti-tumor immune effector cells.
Then a good preparation to remove non-myeloid cell state C57BL / 6 mice animal models of melanoma were randomly divided into four groups. Group I: the first transfusion by magnetic separation to remove CD4 ~ + CD25 ~ + Treg cell components and then by in vitro induced specific antigen load DC-CIK effector cells, Group II: in vitro induction of the first transfusion produce tumor-specific antigen loaded DC-CIK effector cells, and then magnetic separation to remove CD4 ~ + CD25 ~ + Treg cell-specific antigen components of the load of the DC-CIK effector cells. The third group: reinfusion not remove CD4 ~ + CD25 ~ + Treg cell components of DC-CIK cells group. Group IV: control group.
Each group by measuring tumor volume, weight inhibitory rate was calculated to compare their inhibitory effect and the application of TEM-specific antigen load DC-CIK cells kill the tumor cells and morphological features. Application's flow cell technology (FCM) to detect the proliferation of tumor cells in G0/G1 phase cycle, S phase, G2 / M phase fraction, proliferation index (proliferation Index, PI) and apoptosis index (apoptosis Index, AI) of the changes observed in antigen load of DC-CIK to tumor cell proliferation and apoptosis.
By MTT staining before culture to remove CD4 ~ + CD25 ~ + Treg cell-specific antigen components of the load of DC-CIK cells group, to develop after the removal of CD4 ~ + CD25 ~ + Treg cell-specific antigen components of DC -CIK cells group, did not remove CD4 ~ + CD25 ~ + Treg cell-specific antigen components of the load of the DC-CIK cells group three groups of anti-tumor immune effector cells of B16 melanoma tumor cell-killing effect.
Results:
The use of specific tumor antigen-pulsed DC cells co-cultured with CIK cells, the immune phenotype analysis, TEM-specific antigen loaded DC-CIK cells increase in size, there is notch nuclear, cytoplasmic organelles rich in rough endoplasmic reticulum, cell surface processes, in close contact with tumor cells, a large number of tumor cell apoptosis and necrosis.
Before culture to remove CD4 ~ + CD25 ~ + Treg cell-specific antigen components of the load of DC-CIK cells group mean tumor volume of 0.0377 ± 0.0128cm3, cultured remove CD4 ~ + CD25 ~ + Treg cell-specific antigen components of the load The DC-CIK cells group mean tumor volume of 0.0359 ± 0.0131cm3, did not remove CD4 ~ + CD25 ~ + Treg cell-specific antigen components of the load of the DC-CIK cells group mean tumor volume of 0.1278 ± 0.0362 cm3, while the control group mean tumor volume of 0.4052 ± 0.0429cm3. Three sets of experimental tumor volume was significantly smaller than the control group (P <0.05), different stages of the removal of CD4 ~ + CD25 ~ + Treg cell composition between the two groups, although the train before the removal of CD4 ~ + CD25 ~ + Treg cellular components of the antigen load of the DC-CIK cells group than in the culture after the removal of CD4 ~ + CD25 ~ + Treg cell components group size slightly smaller, but no significant difference (P> 0.05). But the two groups and did not remove CD4 ~ + CD25 ~ + Treg cell-specific antigen components of the load of the DC-CIK cells group tumor volume were significantly different (P <0.05).
Three group-specific antigen of DC-CIK effector cell inhibition rate was significantly higher group (P <0.05); remove CD4 ~ + CD25 ~ + Treg cell inhibition rate was significantly higher than the two components are not removed CD4 ~ + CD25 ~ + Treg cells in DC-CIK effector cells group (P <0.05), but no significant difference between the two groups (P> 0.05).
Tumor weight of control group 3.361 ± 1.07g, before culture to remove CD4 ~ + CD25 ~ + Treg cell-specific antigen components of the load of the DC-CIK cells group 0.958 ± 0.32g, cultured remove CD4 ~ + CD25 ~ + Treg cellular components of the antigen load of the DC-CIK cells group 0.939 ± 0.41g, did not remove CD4 ~ + CD25 ~ + Treg cell-specific antigen components of the load of the DC-CIK cells group 1.651 ± 0.57g. The same three experimental group tumor weight was significantly less than the control group (P <0.05). But at different stages to remove CD4 ~ + CD25 ~ + Treg cell components are less than the weight of the two groups of tumors did not remove CD4 ~ + CD25 ~ + Treg cell components group, and a statistically significant difference (P <0.05). Different stages of the removal of CD4 ~ + CD25 ~ + Treg cell composition between the two groups no significant difference (P> 0.05).
TEM-specific antigen load can be observed in the DC-CIK cells can promote tumor cell apoptosis ultrastructural changes. Flow cytometry (FCM) detection of cancer cell cycle changes; three experimental groups compared with the control group the ratio of G0/G1 cells, AI was significantly increased, S phase no significant changes, G2 / M phase fraction, PI significantly down; remove CD4 ~ + CD25 ~ + Treg cells in G0/G1 phase cell ratio of two components, AI was significantly higher than removal of CD4 ~ + CD25 ~ + Treg group, G2 / M phase fraction, PI was significantly lower than remove CD4 ~ + CD25 ~ + Treg group, S-phase no significant changes. Remove CD4 ~ + CD25 ~ + Treg cells in G0/G1 phase composition between the two groups, S phase, G2 / M phase cell ratio and AI, PI were not significantly different (P> 0.05).
MTT assay before culture to remove CD4 ~ + CD25 ~ + Treg cells in DC-CIK cells group, to develop after the removal of CD4 ~ + CD25 ~ + Treg cells in DC-CIK cells group and did not remove CD4 ~ + CD25 ~ + Treg cell DC-CIK cells group three effector cell for B16 melanoma cells in vitro results showed that three groups of effector cells to tumor cells have a strong killing effect. Before culture to remove CD4 ~ + CD25 ~ + Treg cells in DC-CIK cells group and cultured to remove CD4 ~ + CD25 ~ + Treg cells in DC-CIK cells group of B16 melanoma cells in vitro was significantly higher than (P <0.05) did not remove CD4 ~ + CD25 ~ + Treg cells in DC-CIK cells group. And comparison between different T ratio, effector-target ratio, the stronger the higher the cytotoxicity. Before culture to remove CD4 ~ + CD25 ~ + Treg cells in DC-CIK cells group and cultured to remove CD4 ~ + CD25 ~ + Treg cells in DC-CIK cells group comparison between the two groups of B16 melanoma cells in vitro there was no significant differences (P> 0.05).
Conclusion:
1, the specific antigen of DC-CIK cells on B16 melanoma has a significant anti-tumor effect, and on B16 melanoma tumor cells have strong killing effect. Specific antigen of DC-CIK cells can affect the cell cycle of tumor cells, and induce tumor cell apoptosis. Description CIK cells is a powerful tumor-killing ability of a new generation of anti-tumor adoptive immunotherapy.
2, removal of CD4 ~ + CD25 ~ + Treg cells after specific antigen components of DC-CIK cells the role of anti-tumor immunity is more efficient and specific, indicating that CD4 ~ + CD25 ~ + Treg cells capable of specific antigen-induced Min DC-CIK cells kill the tumor cells significantly inhibited the activity of the formation. Remove CD4 ~ + CD25 ~ + Treg cells, effector T cells, re-raising to enhance the body's anti-tumor effect, this will become a viable method of tumor immunotherapy.
3, at different times to remove CD4 ~ + CD25 ~ + Treg cells from the inhibitory effect, for the tumor cells and the cell cycle of tumor cells in vitro has certain differences, but not statistically significant, indicating CD4 ~ + CD25 ~ + Treg cells in various subsets of the source and mechanism of action needs further study. Degree Year: 2009