OBJECTIVE: To investigate the biological activity and antitumor effect of pegylated recombinant human interleukin 2 (PEG-rhIL-2) obtained by site-specific conjugation of polyethylene glycol (PEG) with non-natural amino acids, and to explore its antitumor mechanism. METHODS: The binding activities of PEG-rhIL-2 at three different sites (T41, Y45, and V91) to human interleukin 2 receptors α (IL-2R_α) and β (IL-2R_β) and were detected by surface plasmon resonance (SPR) technology. Western blot was used to detect the levels of the Janus kinase-signal transducer and activator of transcription 5 (JAK-STAT5) signaling pathway activated by different doses of rhIL-2 and PEG-rhIL-2 in CTTL-2 and YT cells. Blood was collected after a single administration in mice to detect the drug concentration at different time points and evaluate the pharmacokinetic parameters of Y45-PEG-rhIL-2. Mouse hepatoma cell line Hepa1-6, pancreatic cancer cell line Pan-02, and colon cancer cell line MC-38 were selected. Tumor models were constructed in C57BL/6 mice. Different doses of Y45-PEG-rhIL-2 and excipient control were administrated respectively to evaluate the tumor suppression effect of the drug. In the MC-38 colon cancer model, the tumor suppression effect of Y45-PEG-rhIL-2 combined with anti-programmed death-1 (PD-1) monoclonal antibody was evaluated. Hepa1-6 mouse tumor models were constructed and rhIL-2, Y45-rhIL-2 and Y45-PEG-rhIL-2 were administrated respectively. The proportion of tumor-infiltrating lymphocytes was analyzed by flow cytometry. RESULTS: The SPR detection results showed that the binding activities of PEG-rhIL-2 to IL-2R_α/IL-2R_β were both reduced. The affinity of Y45-PEG-rhIL-2 to IL-2R_α was reduced to approximately 1/250, and its affinity to IL-2R_β was reduced to 1/3. Western blot results showed that the activity of Y45-PEG-rhIL-2 in stimulating JAK-STAT5 signaling in CTLL-2 cells expressing heterotrimeric IL-2 receptor complex IL-2R_αβγwas reduced to approximately 1/300, while its activity in YT cells expressing heterodimeric IL-2 receptor complex IL-2R_βγwas reduced to approximately 1/3. The pharmacokinetic evaluation after a single dose in the mice showed that the elimination half-life of Y45-PEG-rhIL-2 was 17.7 h. Y45-PEG-rhIL-2 has pharmacokinetic characteristics superior to those of rhIL-2. Y45-PEG-rhIL-2 showed dose-dependent tumor suppression activity, and the combination of Y45-PEG-rhIL-2 and anti-PD-1 antibody had a better tumor-inhibiting effect than the single use of Y45-PEG-rhIL-2 or anti-PD-1 antibody. Flow cytometry analysis demonstrated that 72 h after the administration of Y45-PEG-rhIL-2, the proportion of tumor-infiltrating cytotoxic T lymphocytes (CD8⁺T cells) increased by 86.84%. At 120 h after administration, the ratio of CD8⁺T cells to regulatory T cells (Treg) increased by 75.10%. CONCLUSION Y45-PEG-rhIL-2 obtained by site-specific conjugation via non-natural amino acids changed its receptor binding activity and inhibited tumor growth in dose-dependent manner in multiple tumor models by regulating CD8⁺T cells.
Interleukin-2/pharmacokinetics* ; Animals ; Mice ; Humans ; Recombinant Proteins/pharmacology* ; Polyethylene Glycols/chemistry* ; Cell Line, Tumor ; Antineoplastic Agents/pharmacokinetics* ; Signal Transduction/drug effects* ; STAT5 Transcription Factor/metabolism* ; Interleukin-2 Receptor alpha Subunit/metabolism* ; Interleukin-2 Receptor beta Subunit/metabolism*

The study aims to explore the active molecules of traditional Chinese medicine that specifically bind to interleukin-15 receptor α (IL-15Rα) using molecular docking and surface plasmon resonance (SPR) technology. AutoDock molecular docking software was used to perform simulated docking of more than 3 000 compounds from 48 traditional Chinese medicines at IL-15Rα and screen the specific binding compounds. Then Biocore T200 biomolecular interaction analysis system of SPR was used to confirm the binding specificity of the selected target compounds. Finally, the biological effects of the target compounds on IL-15Rα were verified by cell biological experiments. The results showed that neoprzewaquinone A (Neo) possessed the highest specific binding affinity among the active molecules from traditional Chinese medicine, and the dissociation constant (K_D) value was (0.62 ± 0.20) µmol/L. The results of cell experiment showed that Neo significantly inhibited the proliferation of Mo7e cells induced by IL-15, and the IC₅₀ was 1.075 µmol/L, approximately 1/120 of the IC₅₀ of Cefazolin (IL-15 specific antagonist). These results suggest that Neo is a specific inhibitor of IL-15Rα and may be a potential active drug for the treatment of diseases related to the dysfunction of the IL-15Rα signaling.
Molecular Docking Simulation ; Interleukin-15/pharmacology* ; Surface Plasmon Resonance ; Interleukin-15 Receptor alpha Subunit/metabolism* ; Protein Binding

The aim of this study was to investigate the functional characteristics and in vitro specific killing effect of EGFRvIII CAR-T cells co-expressing interleukin-15 and chemokine CCL19, in order to optimize the multiple functions of CAR-T cells and improve the therapeutic effect of CAR-T cells targeting EGFRvIII on glioblastoma (GBM). The recombinant lentivirus plasmid was obtained by genetic engineering, transfected into 293T cells to obtain lentivirus and infected T cells to obtain the fourth generation CAR-T cells targeting EGFRvIII (EGFRvIII-IL-15-CCL19 CAR-T). The expression rate of CAR molecules, proliferation, chemotactic ability, in vitro specific killing ability and anti-apoptotic ability of the fourth and second generation CAR-T cells (EGFRvIII CAR-T) were detected by flow cytometry, cell counter, chemotaxis chamber and apoptosis kit. The results showed that compared with EGFRvIII CAR-T cells, EGFRvIII-IL-15-CCL19 CAR-T cells successfully secreted IL-15 and CCL19, and had stronger proliferation, chemotactic ability and anti-apoptosis ability in vitro (all P < 0.05), while there was no significant difference in killing ability in vitro. Therefore, CAR-T cells targeting EGFRvIII and secreting IL-15 and CCL19 are expected to improve the therapeutic effect of glioblastoma and provide an experimental basis for clinical trials.
Humans ; Receptors, Chimeric Antigen/metabolism* ; Glioblastoma/metabolism* ; Interleukin-15/metabolism* ; Chemokine CCL19/metabolism* ; Cell Line, Tumor ; T-Lymphocytes/metabolism*

Objective This study aims to construct and identify the chimeric antigen receptor NK92 (CAR-NK92) cells targeting NKG2D ligand (NKG2DL) (secreting IL-15Ra-IL-15) and verify the killing activity of NKG2D CAR-NK92 cells against multiple myeloma cells. Methods The extracellular segment of NKG2D was employed to connect 4-1BB and CD3Z, as well as IL-15Ra-IL-15 sequence to obtain a CAR expression framework. The lentivirus was packaged and transduced into NK92 cells to obtain NKG2D CAR-NK92 cells. The proliferation of NKG2D CAR-NK92 cells was detected by CCK-8 assay, IL-15Ra secretion was detected by ELISA and killing efficiency was detected by lactate dehydrogenase (LDH) assay. The molecular markers of NKp30, NKp44, NKp46, the ratio of apoptotic cell population, CD107a, and the secretion level of granzyme B and perforin were detected using flow cytometry. In addition, the cytotoxic mechanism of NKG2D CAR-NK92 cells on the tumor was verified by measuring the degranulation ability. Moreover, after NKG2D antibody inhibited effector cells and histamine inhibited tumor cells, LDH assay was utilized to detect the effect on cell-killing efficiency. Finally, the multiple myeloma tumor xenograft model was constructed to verify its anti-tumor activity in vivo. Results Lentiviral transduction significantly increased NKG2D expression in NK92 cells. Compared with NK92 cells, the proliferation ability of NKG2D CAR-NK92 cells was weaker. The early apoptotic cell population of NKG2D CAR-NK92 cells was less, and NKG2D CAR-NK92 cells had stronger cytotoxicity to multiple myeloma cells. Additionally, IL-15Ra secretion could be detected in its culture supernatant. NKp44 protein expression in NKG2D CAR-NK92 cells was clearly increased, demonstrating an enhanced activation level. Inhibition test revealed that the cytotoxicity of CAR-NK92 cells to MHC-I chain-related protein A (MICA) and MICB-positive tumor cells was more dependent on the interaction between NKG2D CAR and NKG2DL. After stimulating NKG2D CAR-NK92 cells with tumor cells, granzyme B and perforin expression increased, and NK cells obviously upregulated CD107α. Furthermore, multiple myeloma tumor xenograft model revealed that the tumors of mice treated with NKG2D CAR-NK92 cells were significantly reduced, and the cell therapy did not sensibly affect the weight of the mice. Conclusion A type of CAR-NK92 cell targeting NKG2DL (secreting IL-15Ra-IL-15) is successfully constructed, indicating the effective killing of multiple myeloid cells.
Humans ; Mice ; Animals ; Receptors, Chimeric Antigen/genetics* ; Interleukin-15 ; NK Cell Lectin-Like Receptor Subfamily K/metabolism* ; Granzymes ; Cell Line, Tumor ; Multiple Myeloma/therapy* ; Perforin

OBJECTIVE: To establish the in vivo traceable acute myeloid leukemia mice model with Luciferase-Expressing KG1a Cells. METHODS: KG1a cells with stable luciferase gene expression (called as KG1a-Luc cells) were constructed by lentivirus transfection, then sifted out by puromycin. Eighteen male NOD-SCID-IL2rg RESULTS: KG1a cells expressing luciferase stably were successfully obtained. The tumor luminescence wildly spread at day 17 captured by in vivo imaging. The KG1a-Luc tumor cells could be detected in the peripheral blood of the mice, with the average percentage of (16.27±6.66)%. The morphology and pathology result showed that KG1a-Luc cells infiltrate was detected in bone marrow, spleens and livers. The survival time of the KG1a-Luc mice was notably shorter as compared with those in the control group, the median survival time was 30.5 days (95%CI: 0.008-0.260). CONCLUSION The acute myeloid leukemia NOD-SCID-IL2rg
Animals ; Disease Models, Animal ; Interleukin Receptor Common gamma Subunit ; Leukemia, Myeloid, Acute ; Luciferases/genetics* ; Male ; Mice ; Mice, Inbred NOD ; Mice, SCID

BACKGROUND: Small cell lung cancer (SCLC) is characterized by poor differentiation, high malignancy and rapid growth fast, short double time, early and extensive metastatic malignancy. In clinical, chemotherapy is the main treatment method, while resistance to multiple chemotherapy drugs in six to nine months has been a major clinical challenge in SCLC treatment. Therefore, It has important clinical value to building SCLC aninimal model which is similar to patients with SCLC. Animal model of xenotransplantation (PDX) from the patients with small cell lung cancer can well retain the characteristics of primary tumor and is an ideal preclinical animal model. The study is aimed to establish SCLC PDX animal model and induce the chemoresistance model to help to study the mechanism of chemoresistance and individual treatment. METHODS: Fresh surgical excision or puncture specimens from SCLC patients were transplanted into B-NSGTM mice subcutaneous tissues with severe immunodeficiency in one hour after operation the B-NSGTM mice subcutaneous in 1 hour, and inject chemotherapy drugs intraperitoneally after its tumor growed to 400 mm³ with EP which is cisplatin 8 mg/kg eight days and etoposide 5 mg/kg every two days until 8 cycles. Measure the tumor volum and mice weights regularly, then re-engrafted the largest tumor and continue chemotherapy. RESULTS: Nine cases were conducted for B-NSG mice modeling. Three of nine cases could be engrafted to new B-NSG mice at least two generation. The SCLC PDX animal models have been established successfully. After adopting chemotherapy drugs, the chemoresistance PDX models have been established. High homogeneity was found between xenograft tumor and patient's tumor in histopathology, immunohistochemical phenotype (Syn, CD56, Ki67). CONCLUSIONS The SCLC PDX animal model and the chemoresistance PDX animal model have been successfully constructed, the success rate is 33%, which provides a platform for the clinical research, seeking for biological markers and choosing individual treatment methods of SCLC.
Animals ; Antineoplastic Combined Chemotherapy Protocols ; pharmacology ; Cisplatin ; administration & dosage ; Disease Models, Animal ; Drug Resistance, Neoplasm ; Etoposide ; administration & dosage ; Female ; Humans ; Interleukin Receptor Common gamma Subunit ; deficiency ; genetics ; Lung Neoplasms ; drug therapy ; metabolism ; pathology ; Mice, Inbred BALB C ; Mice, Inbred NOD ; Mice, Knockout ; Mice, SCID ; Small Cell Lung Carcinoma ; drug therapy ; metabolism ; pathology ; Transplantation, Heterologous ; methods ; Xenograft Model Antitumor Assays

This research investigated the clinical features of immunodeficiency disease and the features of the mutation of its pathogenic genes. All 7 patients were boys aged 5 months to 4 years and 6 months and had a history of recurrent respiratory infection and pneumonia, low levels of IgM and IgG, and abnormal absolute values or percentages of lymphocyte subsets. High-throughput sequencing showed c.1684C>T mutations in the BTK gene in patient 1 and IVS8+2T>C splice site mutations in the BTK gene in patient 2. Both of these mutations came from their mothers. Patients 3, 4, and 5 had mutations in the IL2RG gene, i.e., c.298C>T, IVS3-2A>G, and c.164T>A, among which c.164T>A mutations had not been reported. Patient 6 had c.204C>G mutations in the RAG2 gene. Patient 7 had complex heterozygous mutations of c.913C>T and c.824G>A in the RAG2 gene, which came from his father and mother, respectively. Patients with immunodeficiency disease have abnormal immunological indices, and high-throughput sequencing helps to make a definite diagnosis.
Agammaglobulinaemia Tyrosine Kinase ; Agammaglobulinemia ; genetics ; Child, Preschool ; Computational Biology ; DNA-Binding Proteins ; genetics ; Genetic Diseases, X-Linked ; genetics ; High-Throughput Nucleotide Sequencing ; Humans ; Immunologic Deficiency Syndromes ; genetics ; therapy ; Infant ; Interleukin Receptor Common gamma Subunit ; genetics ; Male ; Mutation ; Nuclear Proteins ; genetics ; Protein-Tyrosine Kinases ; genetics

Immunosuppressive regulatory T lymphocytes (Treg) expressing the transcription factor Foxp3 play a vital role in the maintenance of tolerance of the immune-system to self and innocuous non-self. Most Treg that are critical for the maintenance of tolerance to self, develop as an independent T-cell lineage from common T cell precursors in the thymus. In this organ, their differentiation requires signals from the T cell receptor for antigen, from co-stimulatory molecules, as well as from cytokine-receptors. Here we focus on the cytokines implicated in thymic development of Treg, with a particular emphasis on the roles of interleukin-2 (IL-2) and IL-15. The more recently appreciated involvement of TGF-β in thymic Treg development is also briefly discussed. Finally, we discuss how cytokine-dependence of Treg development allows for temporal, quantitative, and potentially qualitative modulation of this process.
Animals ; Cell Differentiation ; genetics ; Cytokines ; immunology ; Forkhead Transcription Factors ; genetics ; immunology ; Gene Expression Regulation ; Immune Tolerance ; genetics ; Interleukin-15 ; genetics ; immunology ; Interleukin-2 ; genetics ; immunology ; Mice ; Receptors, Antigen, T-Cell ; genetics ; immunology ; T-Lymphocytes, Regulatory ; immunology ; Transforming Growth Factor beta ; genetics ; immunology

IL-15 is a cytokine of the common γ-chain family that is critical for natural killer (NK), invariant natural killer T (iNKT), and CD8 memory T cell development and homeostasis. The role of IL-15 in regulating effector T cell subsets, however, remains incompletely understood. IL-15 is mostly expressed by stromal cells, myeloid cells, and dendritic cells (DCs). Whether T cells themselves can express IL-15, and if so, whether such T cell-derived IL-15 could play an autocrine role in T cells are interesting questions that were previously addressed but answered with mixed results. Recently, three independent studies described the generation of IL-15 reporter mice which facilitated the identification of IL-15-producing cells and helped to clarify the role of IL-15 both in vitro and in vivo. Here, we review the findings of these studies and place them in context of recent reports that examined T cell-intrinsic IL-15 expression during CD4 effector T cell differentiation.
Animals ; Cell Differentiation ; Dendritic Cells ; Homeostasis ; Humans ; In Vitro Techniques ; Inflammation ; Interleukin-15 ; Memory ; Mice ; Myeloid Cells ; Receptors, Cytokine ; Stromal Cells ; T-Lymphocyte Subsets ; T-Lymphocytes ; Th17 Cells

Animals ; Cell Line ; Cytological Techniques ; methods ; Embryonic Stem Cells ; cytology ; Interleukin Receptor Common gamma Subunit ; genetics ; Male ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Mutation