OBJECTIVETo investigate the effects of interleukin 7 (IL-7) on B7 molecules expression and immunogenicity of acute leukemia (AL) cells.

METHODSThe B7 molecules expression on fresh acute leukemia cells and on the IL-7 exposed leukemia cells was detected by FACScan cytometer. B7-1 and B7-2 mRNA in IL-7 treated HL-60 cells were detected by reverse transcription-PCR (RT-PCR). The stimulation of proliferation of allogeneic peripheral blood mononuclear cells (PBMNC) by IL-7 treated leukemia cells was detected by MTT method. The level of interferon-gamma (IFN-gamma) secreted by the stimulated PBMNC was determined using enzyme-linked immunosorbent assays (ELISA). The blocking experiments were performed using monoclonal antibodies against B7-1, B7-2 and W6/32.

RESULTSB7-1 was weakly expressed in three, whereas B7-2 did in only one of eleven AL patients. IL-7 significantly enhanced B7 molecules expression on AL cells in a time-dependent manner. Furthermore, IL-7 could induce higher expression of B7-1 and B7-2 mRNAs on HL-60 cells. IL-7 treated leukemia cells could stimulate PBMNC proliferation and promote their IFN-gamma production. Anti-B7-1 and anti W6/32 but not anti-B7-2 monoclonal antibodies significantly inhibited the stimulated PBMNC proliferation and IFN-gamma secretion.

CONCLUSIONFresh AL cells express low level of B7-1 and B7-2 molecules. IL-7 enhances the B7 molecules expression on AL cells. The IL-7-treated leukemia cells can significantly stimulate the proliferation of allogeneic PBMNC and induce their IFN gamma secretion.

B7-1 Antigen ; genetics ; metabolism ; B7-2 Antigen ; genetics ; metabolism ; Humans ; Interleukin-7 ; pharmacology ; Leukemia ; immunology ; metabolism ; RNA, Messenger ; genetics ; Tumor Cells, Cultured ; Up-Regulation ; drug effects

AIMTo investigate the expression of interleukin-2 receptors (IL-2Rs) on MCF-7 cells, estradiol's regulation of IL-2Rs expression and the influence of IL-2 on the proliferation of MCF-7 cells.

METHODSImmunocytochemistry and flow cytometric analysis were used to investigate the expression of interleukin-2 receptors (IL-2Rs) by using of specific IL-2R polyclonal antibody; MTT method and 3H-TdR incorporation method were used to examine the changes of proliferation of MCF-7 cells.

RESULTSIL-2Ralpha, beta, gamma like immunoreactive substances can be found on MCF-7 cells and the IL-2Rgamma immunostaining was more strong than the other two. Estradiol of 10(-6) mol/L can increase the percentage of immunoreactive cells of IL-2Ralpha, beta and the expression of IL-2Rgamma. Exogenous addition of recombinant IL-2 of 100 U/ml to 1 000 U/ml can significantly increase the proliferation of MCF-7 cells.

CONCLUSIONMCF-7 cell can express IL-2R and estradiol can regulate their expression, IL-2 can influence the proliferation of MCF-7 cells.

Breast Neoplasms ; genetics ; metabolism ; Cell Division ; Estradiol ; pharmacology ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; MCF-7 Cells ; Receptors, Interleukin-2 ; metabolism

Adoptive immunotherapy based on chimeric antigen receptor-modified T cells (CAR-T) is one of the most promising strategies to treat malignant tumors, but its application in solid tumors is still limited. Glypican-3 (GPC3) is a meaningful diagnostic, therapeutic, and prognostic biomarker for hepatocellular carcinoma (HCC). The second/third generation GPC3-targeted CAR-T cells are generated to treat HCC. In order to improve the therapeutic effect, we constructed a fourth-generation lentiviral vector to express GPC3 CAR, human interleukin-7 (IL-7) and CCL19. Then the lentiviral vector and packaging plasmids were co-transfected into HEK293T cells to generate CAR lentiviral particles. Human T lymphocyte cells were transduced with CAR lentiviral to develop the fourth-generation GPC3-targeted CAR-T cells (GPC3-BBZ-7×19). In vitro, we used cell counting, transwell assay, luciferase bioluminescence assay and flow cytometry to compare the proliferation, chemotaxis, cytotoxicity and subtype distribution between GPC3-BBZ-7×19 CAR-T cells and the second generation GPC3-targeted CAR-T cells (GPC3-BBZ). In vivo, we established GPC3-positive HCC xenograft model in immunodeficient mice, then untransduced T cells (non-CAR-T) or GPC3-BBZ-7×19 CAR-T cells were injected. Tumor growth in mice was observed by bioluminescence imaging. Results showed that compared with GPC3-BBZ CAR-T, GPC3-BBZ-7×19 CAR-T cells had stronger proliferation, chemotactic ability, and higher composition of memory stem T cells (Tscm) (P values<0.05). However, there were no significant difference in cytotoxicity and cytokine secretion between them. In addition, GPC3-BBZ-7×19 CAR-T cells could significantly eliminate GPC3-positive HCC xenografts established in immunodeficient mice. Therefore, the fourth-generation GPC3-targeted CAR-T cells (secreting IL-7 and CCL19) are expected to be more durable and effective against HCC and produce tumor-specific memory, to provide a preclinical research basis for future clinical trials.
Animals ; Carcinoma, Hepatocellular ; Cell Line, Tumor ; Chemokine CCL19 ; metabolism ; Glypicans ; metabolism ; HEK293 Cells ; Humans ; Interleukin-7 ; metabolism ; Lentivirus ; genetics ; Liver Neoplasms ; Mice ; Receptors, Chimeric Antigen ; metabolism ; T-Lymphocytes ; metabolism ; Xenograft Model Antitumor Assays

BACKGROUNDImmunosuppressive regulatory T cells (Tregs) participate in tumor immune evasion and the number and suppressive function of Tregs change with the aging process, but it is not clear whether such change leads to a higher incidence of tumors in the elderly. To this end, we designed experiments to explore the changes of Tregs and the functional gene Forkhead box P3 (FoxP3) in the aging process and its relationship with lung tumors in humans and mice.

METHODSThe percentage of CD4(+)CD25(+)CD127(low) Tregs and expression of FoxP3 mRNA were analyzed using flow cytometry (FCM) and real-time fluorescence-based quantitative polymerase chain reaction (FQ-PCR). Markers were analyzed in the peripheral blood (PB) of 65 elderly patients (age ≥ 65 years) with primary non-small cell lung cancer (NSCLC), 20 younger patients (aged < 55 years) with NSCLC, 30 elderly healthy individuals and 30 young healthy individuals. Furthermore, we set up the Lewis lung cancer model with C57BL/6 female mice. Thirty-six mice were divided into a young healthy group, a middle-aged healthy group, an elderly healthy group, a young tumor group, a middle-aged tumor group, and an elderly tumor group. The percentage of CD4(+)CD25(+)FoxP3(+) Tregs and the expression level of FoxP3 mRNA in splenocytes were determined in the six groups.

RESULTSThe percentage of peripheral CD4(+)CD25(+)CD127(low) Tregs and the expression of FoxP3 mRNA were significantly increased in elderly patients with NSCLC comparing with the other groups and in elderly healthy individuals compared with young healthy individuals. Further analysis showed that the percentage of CD4(+)CD25(+)CD127(low) Tregs and the expression of FoxP3 mRNA were closely associated with tumor node metastasis (TNM) staging in elderly patients with NSCLC. In the mouse model, the percentage of CD4(+)CD25(+)FoxP3(+) Tregs and the expression of FoxP3 mRNA in splenocytes of the tumor groups were significantly higher than in the healthy groups, with the highest expression in the elderly tumor group. In the healthy groups, the elderly healthy mice had the highest percentage of Tregs and expression of FoxP3 mRNA. The elderly mice had larger and heavier tumors than did the young and middle aged mice.

CONCLUSIONSThe up-regulation of Tregs and the FoxP3 gene with aging may play an essential role in oncogenesis and development of lung tumors in an elderly population.

Aging ; genetics ; metabolism ; Animals ; CD4 Antigens ; metabolism ; Female ; Flow Cytometry ; Forkhead Transcription Factors ; genetics ; metabolism ; Humans ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Interleukin-7 Receptor alpha Subunit ; metabolism ; Lung Neoplasms ; immunology ; metabolism ; Mice ; Mice, Inbred C57BL ; Real-Time Polymerase Chain Reaction ; T-Lymphocytes, Regulatory ; immunology ; metabolism

Dendritic cells (DCs) comprise two functionally distinct subsets: plasmacytoid DCs (pDCs) and myeloid DCs (mDCs). pDCs are specialized in rapid and massive secretion of type I interferon (IFN-I) in response to nucleic acids through Toll like receptor (TLR)-7 or TLR-9. In this report, we characterized a CD56(+) DC population that express typical pDC markers including CD123 and BDCA2 but produce much less IFN-I comparing with pDCs. In addition, CD56(+) DCs cluster together with mDCs but not pDCs by genome-wide transcriptional profiling. Accordingly, CD56(+) DCs functionally resemble mDCs by producing IL-12 upon TLR4 stimulation and priming naïve T cells without prior activation. These data suggest that the CD56(+) DCs represent a novel mDC subset mixed with some pDC features. A CD4(+)CD56(+) hematological malignancy was classified as blastic plasmacytoid dendritic cell neoplasm (BPDCN) due to its expression of characteristic molecules of pDCs. However, we demonstrated that BPDCN is closer to CD56(+) DCs than pDCs by global gene-expression profiling. Thus, we propose that the CD4(+)CD56(+) neoplasm may be a tumor counterpart of CD56(+) mDCs but not pDCs.
Biomarkers ; metabolism ; CD56 Antigen ; genetics ; immunology ; Cell Lineage ; genetics ; immunology ; Dendritic Cells ; immunology ; metabolism ; pathology ; Gene Expression ; Hematologic Neoplasms ; genetics ; immunology ; pathology ; Humans ; Immunophenotyping ; Interferon Type I ; biosynthesis ; metabolism ; Interleukin-12 ; biosynthesis ; metabolism ; Interleukin-3 Receptor alpha Subunit ; genetics ; immunology ; Lectins, C-Type ; genetics ; immunology ; Membrane Glycoproteins ; genetics ; immunology ; Myeloid Cells ; immunology ; metabolism ; pathology ; Receptors, Immunologic ; genetics ; immunology ; Terminology as Topic ; Toll-Like Receptor 4 ; genetics ; immunology ; Toll-Like Receptor 7 ; genetics ; immunology ; Toll-Like Receptor 9 ; genetics ; immunology

OBJECTIVETo explore the roles of NF-κB in factor VIIa-induced proliferation and migration of a colon cancer cell line (SW620) in vitro and its possible mechanism.

METHODSThe expression levels of NF-κB (p65), inhibitory protein of NF-κB (IκB-α), caspase-7, interleukin 8 (IL-8) and tissue factor (TF) in SW620 cells treated with factor VIIa, PDTC (an inhibitor of NF-κB) and other factors were measured by Western-blotting and real-time PCR. Proliferation and migration of the cells were analyzed by flow cytometry and Transwell assay, respectively.

RESULTSFactor VIIa down-regulated the IκB-α level in SW620 cells and increased the intranuclear level of NF-κB. Those effects of factor VIIa were blocked by anti-TF or anti-PAR2 antibodies. The effects of factor VIIa on proliferation and migration of SW620 cells, expression of IL-8, TF as well as caspase-7, were interfered by PDTC (the inhibitor of NF-κB).

CONCLUSIONSTF/VIIa complex activates NF-κB pathway via PAR2, thereby up-regulates IL-8 and down-regulates caspase-7 expression in SW620 cells, finally promotes proliferation and migration of colon cancer cells. In addition, TF/VIIa/PAR2/NF-κB pathway also upregulates TF expression, thus to create a positive feedback loop of TF/VIIa/PAR2/NF-κB/TF.

Antineoplastic Agents ; pharmacology ; Caspase 7 ; genetics ; metabolism ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Colonic Neoplasms ; metabolism ; pathology ; Factor VIIa ; pharmacology ; Humans ; I-kappa B Proteins ; metabolism ; Interleukin-8 ; genetics ; metabolism ; NF-KappaB Inhibitor alpha ; Proline ; analogs & derivatives ; pharmacology ; RNA, Messenger ; metabolism ; Receptor, PAR-2 ; metabolism ; Thiocarbamates ; pharmacology ; Thromboplastin ; genetics ; metabolism ; Transcription Factor RelA ; antagonists & inhibitors ; metabolism

OBJECTIVETo quantify the CD4+ CD25+ CD127(low) regulatory T cell (Treg), the expression levels of forkhead/winged helix transcription factor FOXP3 and Notch1 mRNA in aplastic anemia (AA) patients before and after treatment, and explore the significance of Treg in pathogenesis of AA.

METHODCD4+ CD25+ and CD4+ CD25+ CD127(low) T cells in peripheral blood were examined with FACS in 29 AA patients at active phase, 14 at recovery phase, 11 at unrecovery phase, and 15 normal controls. The levels of FOXP3 mRNA and Notch1 mRNA expression were detected with RT-PCR, and the correlations between Treg, FOXP3 mRNA and Notchl mRNA were analyzed.

RESULTSThe percentages of peripheral activated CD4+ CD25+ T cells in AA patients at active phase (4.3 +/- 0.7)% and unrecovery phase (4.2 +/- 0.6)% were significantly higher than those in normal controls (2.4 +/- 0.8)% (P < 0.05). The proportion of these cells in AA patients at recovery phase was reduced to (2.6 +/- 0.7)% (P < 0.05), being no difference from that in control group. The number of CD4+ CD25+ CD127(low) T cells in AA patients at active phase (2.4 +/- 1.2)% and unrecovery phase (2.5 +/- 1.1)% was decreased significantly compared with those in normal controls (7.1 +/- 2.7)% (P < 0.01) and in AA patients at recovery phase (5.3 +/- 1.0)% (P < 0.01), there was no difference between the latter two groups. In active phase AA patients, the levels of FOXP3 mRNA and Notchl mRNA (0.260 +/- 0.011 and 0.018 +/- 0.005, respectively) were lower than that in control group (1.307 +/- 0.011 and 0.308 +/- 0.028, respectively) (P < 0.01 and P < 0.01). After treatment, the levels significantly increased to 1.287 +/- 0.012 and 0.281 +/- 0.013 (P < 0.01 and P < 0.01), but there was no difference with that of normal controls (P > 0.05). CD4+ CD25+ CD2(low) T cells and FOXP3 were positively related with Notchl (P < 0.01) in AA patients.

CONCLUSIONThe decreased number and suppressive activity of CD4 CD25+ CD127(low) Treg cells in the peripheral blood of AA patients cause over-activation of autoreactive T cells and suppression of haematopoiesis. One of the mechanisms maybe the reduced expression of Notch1 in the target cells.

Adolescent ; Adult ; Aged ; Anemia, Aplastic ; immunology ; metabolism ; CD4 Antigens ; Case-Control Studies ; Female ; Forkhead Transcription Factors ; genetics ; metabolism ; Humans ; Interleukin-2 Receptor alpha Subunit ; Interleukin-7 Receptor alpha Subunit ; Male ; Middle Aged ; RNA, Messenger ; genetics ; Receptor, Notch1 ; genetics ; metabolism ; T-Lymphocytes, Regulatory ; immunology ; Young Adult

BACKGROUNDBone morphogenetic protein (BMP) is a member of the superfamily of transforming growth factor-beta. Recent studies show that it is an indispensable factor in hematopoiesis. To better characterize the effect of recombinant human BMP (rhBMP)-2 in hematopoiesis, we set out to determine whether rhBMP-2 could promote the proliferation of mesenchymal stem cells (MSCs) and increase the levels of hematopoietic cytokines in MSCs.

METHODS2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino) carbonyl)-2H-tetrazolium hydroxide (XTT), real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the effects of rhBMP-2 on the proliferation and hematopoietic cytokine levels of MSCs. In addition, MSCs marked with Hoechst33342 were transplanted into BALB/c mice by the intravenous route or intra-bone marrow transplantation, and cluster numbers were counted.

RESULTSThe XTT test revealed that rhBMP-2 significantly induced proliferation of MSCs in doses ranging from 10 ng/ml to 0.1 mg/ml in a dose-dependent manner. The experiments in vivo showed that there were more clusters of donor cells in bone marrow, spleen, liver and lung of the BMP group than those in the control group after both intra-bone marrow transplantation (P < 0.001, P < 0.001, P < 0.001, and P = 0.001, respectively) and intravenous transplantation (P < 0.001, P < 0.001, and P < 0.001 respectively). The results of real-time PCR and ELISA revealed that rhBMP-2 significantly increased mRNA expressions and protein levels of IL-6, IL-7, IL-11, G-CSF, M-CSF and SCF.

CONCLUSIONSThe treatment with rhBMP-2 promotes the proliferation of MSCs in vivo and in vitro and increases the levels of hematopoietic cytokines in MSCs, which may contribute to the improvement of hematopoietic function.

Animals ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Enzyme-Linked Immunosorbent Assay ; Granulocyte Colony-Stimulating Factor ; genetics ; Humans ; Interleukin-11 ; genetics ; Interleukin-6 ; genetics ; Interleukin-7 ; genetics ; Macrophage Colony-Stimulating Factor ; genetics ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Mice ; Mice, Inbred BALB C ; Polymerase Chain Reaction ; Recombinant Proteins ; pharmacology ; Stem Cell Factor ; genetics ; Transforming Growth Factor beta ; pharmacology

OBJECTIVETo investigate the mechanisms that coagulation factor VIIa promotes proliferation and migration of a colon cancer cell line (SW620 cells) in vitro.

METHODSThe expression of interleukin 8 (IL-8), tissue factor (TF), caspase-7 and p-p38 MAPK in SW620 cells treated with factor VIIa or protease activated receptor 2 agonist (PAR2-AP) was measured by ELISA, Western-blotting and QT-PCR.

RESULTSFactor VIIa and PAR2-AP induced IL-8 expression at both mRNA and protein levels, upregulated TF mRNA expression and TF activity, but down-regulated caspase-7 mRNA and p-p38 MAPK levels in SW620 cells. The effects of factor VIIa were not only blocked by anti-TF but also by anti-PAR2 antibodies.

CONCLUSIONFactor VIIa binds to TF on cell surface, forming a complex which activates PAR2, then provoking IL-8 and TF expression, and suppresses caspase-7 expression, thus promotes the tumor cell proliferation and migration. p38 MAPK may negatively regulate this process.

Caspase 7 ; biosynthesis ; genetics ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Colonic Neoplasms ; metabolism ; pathology ; Factor VIIa ; pharmacology ; Humans ; Interleukin-8 ; biosynthesis ; genetics ; Oligopeptides ; pharmacology ; RNA, Messenger ; metabolism ; Receptor, PAR-2 ; agonists ; Thromboplastin ; biosynthesis ; genetics ; p38 Mitogen-Activated Protein Kinases ; metabolism

Dendritic Cells ; cytology ; metabolism ; Enzyme-Linked Immunosorbent Assay ; HEK293 Cells ; Humans ; Interferon Regulatory Factor-7 ; metabolism ; Interferon Type I ; metabolism ; Interferon-gamma ; analysis ; Interleukin-6 ; analysis ; Oligonucleotides ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Real-Time Polymerase Chain Reaction ; Receptors, Tumor Necrosis Factor ; antagonists & inhibitors ; genetics ; metabolism