OBJECTIVE: To detect the expression level of suppressors of cytokine signaling 3 （SOCS3） in acute lymphoblastic leukemia (ALL), and to observe the effect of over-expresson of SOCS3 in Jurkat cells on the cytotoxicity of NK cells. METHODS: The expression levels of SOCS3 mRNA in peripheral blood mononuclear cells of 20 children with ALL and 20 healthy children (normal control group) were detected by RT-PCR. The peripheral blood NK cells from healthy subjects were selected by immunomagnetic technique, and the purity was detected by flow cytometry. SOCS3 was overexpressed in Jurkat cells infected with lentivirus vector, and SOCS3 mRNA expression was detected by RT-PCR after lentivirus infection. The NK cells were co-cultured with the infected Jurkat, and LDH release method was used to detect the cytotoxicity of NK cells on the infected Jurkat cells. The concentrations of TNF-α and IFN-γ were determined by ELISA. The expression of NKG2D ligands MICA and MICB on the surface of Jurkat cells were detected by flow cytometry. Western blot was used to detect the effect of SOCS3 overexpression on STAT3 phosphorylation in Jurkat cells. RESULTS: Compared with the control group, the mRNA expression of SOCS3 in the peripheral blood mononucleated cells of ALL children was significantly decreased. The purity of NK cells isolated by flow cytometry could reach more than 70%. The expression of SOCS3 mRNA in Jurkat cells increased significantly after lentivirus infection. Overexpression of SOCS3 in Jurkat cells significantly promoted the killing ability of NK cells and up-regulated the secretion of TNF-α and IFN-γ from NK cells. The results of flow cytometry showed that the expression of NKG2D ligands MICA and MICB on Jurkat cells increased significantly after SOCS3 overexpression. Western blot results showed that overexpression of SOCS3 significantly reduced the phosphorylation level of STAT3 protein in Jurkat cells. CONCLUSION SOCS3 mRNA expression was significantly decreased in ALL patients, and overexpression of SOCS3 may up-regulate the expression of MICA and MICB of NKG2D ligands on Jurkat cell surface through negative regulation of JAK/STAT signaling pathway, thereby promoting the cytotoxic function of NK cells.
Child ; Histocompatibility Antigens Class I/metabolism* ; Humans ; Killer Cells, Natural/cytology* ; Leukocytes, Mononuclear/cytology* ; Ligands ; NK Cell Lectin-Like Receptor Subfamily K/metabolism* ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics* ; RNA, Messenger/genetics* ; Suppressor of Cytokine Signaling 3 Protein/metabolism* ; Tumor Necrosis Factor-alpha/metabolism*

Traumatic brain injury (TBI) contributes to the key causative elements of neurological deficits. However, no effective therapeutics have been developed yet. In our previous work, extracellular vesicles (EVs) secreted by stem cells from human exfoliated deciduous teeth (SHED) offered new insights as potential strategies for functional recovery of TBI. The current study aims to elucidate the mechanism of action, providing novel therapeutic targets for future clinical interventions. With the miRNA array performed and Real-time PCR validated, we revealed the crucial function of miR-330-5p transferred by SHED-derived EVs (SHED-EVs) in regulating microglia, the critical immune modulator in central nervous system. MiR-330-5p targeted Ehmt2 and mediated the transcription of CXCL14 to promote M2 microglia polarization and inhibit M1 polarization. Identified in our in vivo data, SHED-EVs and their effector miR-330-5p alleviated the secretion of inflammatory cytokines and resumed the motor functional recovery of TBI rats. In summary, by transferring miR-330-5p, SHED-EVs favored anti-inflammatory microglia polarization through Ehmt2 mediated CXCL14 transcription in treating traumatic brain injury.
Animals ; Brain Injuries, Traumatic/therapy* ; Chemokines, CXC/metabolism* ; Extracellular Vesicles/metabolism* ; Histocompatibility Antigens/metabolism* ; Histone-Lysine N-Methyltransferase/metabolism* ; Humans ; MicroRNAs/metabolism* ; Microglia/metabolism* ; Rats ; Stem Cells/metabolism*

To study the relationship between acute graft-versus-host disease (aGVHD) and the SIRT1 expression in peripheral blood CD4+T cells from patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT).
 Methods: We collected 40 patients who underwent allo-HSCT from human leukocyte antigen (HLA)-identical sibling donors. SIRT1 expression level in CD4+T cells was measured by real-time PCR and Western blot. Acetylation and phosphorylation of STAT3 in CD4+T cells were detected by Western blot. The binding level between SIRT1 and STAT3 in CD4+T cells was analyzed by co-immunoprecipitation and Western blot. Over-expression of SIRT1 in aGVHD CD4+T cells, as well as STAT3 acetylation and phosphorylation were measured by Western blot. The mRNA levels of RORγt, IL-17A, IL-17F related to Th17 were detected by real-time PCR.
 Results: SIRT1 expression was significantly down-regulated, while STAT3 expression, acetylation and phosphorylation levels were significantly up-regulated in patients with aGVHD compared with patients without aGVHD. The STAT3 acetylation was positively correlated with STAT3 phosphorylation (r=0.69, P<0.01). Less SIRT1-STAT3 complexes were found in CD4+T cells from patients with aGVHD compared with patients without aGVHD. After SIRT1 over-expression in aGVHD CD4+T cells, the STAT3 acetylation and phosphorylation, and the expression of RORγt, IL-17A, and IL-17F related to Th17 were significantly down-regulated (P<0.05).
 Conclusion: SIRT1 deficiency in CD4+T cells plays a crucial role in up-regulation of STAT3 acetylation and phosphorylation, the increase of Th17 related gene expression, and induction of aGVHD after allogeneic hematopoietic stem cell transplantation.
Acute Disease ; CD4-Positive T-Lymphocytes ; metabolism ; Down-Regulation ; Graft vs Host Disease ; etiology ; metabolism ; Hematopoietic Stem Cell Transplantation ; Histocompatibility Antigens Class I ; Humans ; Interleukin-17 ; metabolism ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; metabolism ; STAT3 Transcription Factor ; metabolism ; Sirtuin 1 ; deficiency ; metabolism ; Transplantation, Homologous ; Up-Regulation

Animals ; Antibodies, Monoclonal ; pharmacokinetics ; therapeutic use ; Antibodies, Viral ; therapeutic use ; Hepatitis B virus ; immunology ; Histocompatibility Antigens Class I ; metabolism ; Humans ; Macaca fascicularis ; Mice ; Protein Binding ; Protein Engineering ; Receptors, Fc ; metabolism

Killer cell immunoglobulin-like receptors (KIRs) are members of the immunoglobulin superfamily expressed on natural killer (NK) cells and a subset of T cells. Given the receptor-ligand relationship between certain KIR and human leukocyte antigen (HLA) classⅠmolecules, the KIRs are involved in the regulation of NK cell activation through conveying activating or inhibitory signals, which plays an important role in immunities involved in transplantation, tumor, infection as well as autoimmune diseases. This paper has provided a review for the research on KIR gene polymorphisms and summarized the characteristics of the sequence-based typing method for KIR genes.
HLA Antigens ; genetics ; Histocompatibility Antigens Class I ; genetics ; Humans ; Killer Cells, Natural ; metabolism ; Polymorphism, Genetic ; genetics ; Receptors, KIR ; genetics

OBJECTIVE: To investigate the mRNA and protein expression of MICA in laryngeal squamous cell carcinoma tissue and the Hep-2 cells. METHOD: Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western-blot were used to detect the expression of MICA mRNA and protein levels in the Hep-2 cells and laryngeal cancer tissues. RESULT: The MICA mRNA showed higher expression in Hep-2 cells by RT-PCR. Compared with the control, the mRNA expression of MICA was significantly enhanced in laryngeal cancer tissues (t = 11.878, P < 0.01). The intensity of MICA expression is not related to the clinical stage of cancer. MICA protein demonstrated higher level expression by Western blot. The intensity of MICA protein expression was decreased with increased clinical stage in laryngeal cancer tissues. CONCLUSION The MICA mRNA showed stronger expression in Hep-2 cells and laryngeal cancer tissues. The intensity of its expression is not related to clinical stage of cancer. The MICA protein expression was strong in Hep-2 cells. The intensity of MICA protein expression was decreased with increased clinical stage in laryngeal cancer tissues. MICA may play an important role in laryngeal carcinoma process.
Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Line, Tumor ; Head and Neck Neoplasms ; metabolism ; pathology ; Histocompatibility Antigens Class I ; metabolism ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Squamous Cell Carcinoma of Head and Neck

BACKGROUND: The impact of HLA and KIR ligand mismatching on the outcome of hematopoietic stem cell transplantation (HSCT) remains unclear. Previous reports have identified considerable ethnic differences in the impact of HLA and KIR ligand mismatches, as well as KIR ligand status, on HSCT; however, to date, no data has been acquired in Korean adult patients. METHODS: We investigated the association of high-resolution HLA matching on five loci (HLA-A, -B, -C, -DRB1, and -DQB1), KIR ligand mismatching, and KIR ligand status on the outcome of allogeneic HSCT from unrelated donors in 154 Korean adult patients treated at Seoul National University Hospital. RESULTS: In a multivariate analysis, less than 9/10 allelic matches in five HLA loci was an independent risk factor for acute graft-versus-host disease (GVHD) (grade II to IV) (P=0.019, odds ratio [OR]=2.7). In addition, HLA-A allele mismatching was increasingly prevalent in patients with acute GVHD compared to patients without (61.9% vs. 34.5%, P=0.06). For KIR ligand status, the patient and donor combination of both C1/C1 ligands showed better event-free and overall survival than combinations with C2 ligand patients or donors (P=0.048, P=0.034, respectively) by log-rank test. CONCLUSIONS: Korean adult transplant patients with less than 9 of 10 HLA allele matches in the HLA-A, -B, -C, -DRB1, and DQB1 loci have a higher likelihood of developing acute GVHD (grade II to IV). Impact of KIR ligand status on clinical outcome should be further studied in a larger patient population.
Adolescent ; Adult ; Alleles ; Female ; Genetic Loci ; Graft vs Host Disease/etiology ; HLA Antigens/*genetics/metabolism ; *Hematopoietic Stem Cell Transplantation/adverse effects/standards ; Histocompatibility Testing ; Humans ; Kaplan-Meier Estimate ; Leukemia/mortality/therapy ; Male ; Middle Aged ; Multivariate Analysis ; Receptors, KIR/*chemistry/metabolism ; Republic of Korea ; Risk Factors ; Transplantation, Homologous ; Young Adult

In allogeneic transplantation, including the B6 anti-BALB.B settings, H60 and H4 are two representative dominant minor histocompatibility antigens that induce strong CD8 T-cell responses. With different distribution patterns, H60 expression is restricted to hematopoietic cells, whereas H4 is ubiquitously expressed. H60-specific CD8 T-cell response has been known to be dominant in most cases of B6 anti-BALB.B allo-responses, except in the case of skin transplantation. To understand the mechanism underlying the subdominance of H60 during allogeneic skin transplantation, we investigated the dynamics of the H60-specific CD8 T cells in B6 mice transplanted with allogeneic BALB.B tail skin. Unexpectedly, longitudinal bioluminescence imaging and flow cytometric analyses revealed that H60-specific CD8 T cells were not always subdominant to H4-specific cells but instead showed a brief dominance before the H4 response became predominant. H60-specific CD8 T cells could expand in the draining lymph node and migrate to the BALB.B allografts, indicating their active participation in the anti-BALB.B allo-response. Enhancing the frequencies of H60-reactive CD8 T cells prior to skin transplantation reversed the immune hierarchy between H60 and H4. Additionally, H60 became predominant when antigen presentation was limited to the direct pathway. However, when antigen presentation was restricted to the indirect pathway, the expansion of H60-specific CD8 T cells was limited, whereas H4-specific CD8 T cells expanded significantly, suggesting that the temporary immunodominance and eventual subdominance of H60 could be due to their reliance on the direct antigen presentation pathway. These results enhance our understanding of the immunodominance phenomenon following allogeneic tissue transplantation.
Animals ; Antigen Presentation ; Antigen-Presenting Cells/immunology/metabolism ; CD8-Positive T-Lymphocytes/*immunology ; Epitopes, T-Lymphocyte/*immunology ; Female ; Graft Rejection/*immunology ; Interferon-gamma ; Lymphocyte Activation/immunology ; Lymphocyte Count ; Mice ; Minor Histocompatibility Antigens/*immunology/metabolism ; *Skin Transplantation ; Transplantation, Homologous

Analysis of the T-cell receptor (TCR) repertoire of innate CD4+ T cells selected by major histocompatibility complex (MHC) class II-dependent thymocyte-thymocyte (T-T) interaction (T-T CD4+ T cells) is essential for predicting the characteristics of the antigens that bind to these T cells and for distinguishing T-T CD4+ T cells from other types of innate T cells. Using the TCRmini Tg mouse model, we show that the repertoire of TCRalpha chains in T-T CD4+ T cells was extremely diverse, in contrast to the repertoires previously described for other types of innate T cells. The TCRalpha chain sequences significantly overlapped between T-T CD4+ T cells and conventional CD4+ T cells in the thymus and spleen. However, the diversity of the TCRalpha repertoire of T-T CD4+ T cells seemed to be restricted compared with that of conventional CD4+ T cells. Interestingly, the frequency of the parental OT-II TCRalpha chains was significantly reduced in the process of T-T interaction. This diverse and shifted repertoire in T-T CD4+ T cells has biological relevance in terms of defense against diverse pathogens and a possible regulatory role during peripheral T-T interaction.
Amino Acid Sequence ; Animals ; Antigens, Surface/metabolism ; CD4-Positive T-Lymphocytes/cytology/*immunology/*metabolism ; Cell Communication ; Cell Differentiation/genetics/immunology ; Clonal Evolution ; Histocompatibility Antigens Class II/*immunology ; *Immunity, Innate ; Immunophenotyping ; Lymphocyte Count ; Mice ; Mice, Knockout ; Mice, Transgenic ; Peptide Fragments/chemistry ; Phenotype ; Receptors, Antigen, T-Cell/chemistry/*genetics/metabolism ; Receptors, Antigen, T-Cell, alpha-beta/chemistry/genetics ; Spleen/cytology ; Thymocytes/cytology/immunology/metabolism

Our previous studies indicate that phosphatidylinositol 4-kinase IIα can promote the growth of multi-malignant tumors via HER-2/PI3K and MAPK pathways. However, the molecular mechanisms of this pathway and its potential for clinical application remain unknown. In this study, we found that PI4KIIα could be an ideal combinatorial target for EGFR treatment via regulating EGFR degradation. Results showed that PI4KIIα knockdown reduced EGFR protein level, and the expression of PI4KIIα shows a strong correlation with EGFR in human breast cancer tissues (r = 0.77, P < 0.01). PI4KIIα knockdown greatly prolonged the effects and decreased the effective dosage of AG-1478, a specific inhibitor of EGFR. In addition, it significantly enhanced AG1478-induced inhibition of tumor cell survival and strengthened the effect of the EGFR-targeting anti-cancer drug Iressa in xenograft tumor models. Mechanistically, we found that PI4KIIα suppression increased EGFR ligand-independent degradation. Quantitative proteomic analysis by stable isotope labeling with amino acids in cell culture (SILAC) and LC-MS/MS suggested that HSP90 mediated the effect of PI4KIIα on EGFR. Furthermore, we found that combined inhibition of PI4KIIα and EGFR suppressed both PI3K/AKT and MAPK/ERK pathways, and resulted in downregulation of multiple oncogenes like PRDX2, FASN, MTA2, ultimately leading to suppression of tumor growth. Therefore, we conclude that combined inhibition of PI4KIIα and EGFR exerts a multiple anti-tumor effect. Dual inhibition of EGFR at protein and activity level via combinatorial blocking of PI4KIIα presents a novel strategy to combat EGFR-dependent tumors.
Animals ; Antineoplastic Agents ; pharmacology ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Survival ; drug effects ; ErbB Receptors ; antagonists & inhibitors ; metabolism ; Female ; HSP90 Heat-Shock Proteins ; metabolism ; Humans ; MCF-7 Cells ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Minor Histocompatibility Antigens ; Mitogen-Activated Protein Kinases ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphotransferases (Alcohol Group Acceptor) ; antagonists & inhibitors ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Quinazolines ; pharmacology ; Transplantation, Heterologous ; Tyrphostins ; pharmacology