Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Screening and Identification of an H-2K b -Restricted CTL Epitope within the Glycoprotein of Hantaan Virus

doi: 10.3389/fcimb.2016.00151

Figure Lengend Snippet: Cytotoxic activity of splenocytes derived from HTNV-infected mice in vitro . The primary antiviral CTL responses were first detected. Splenocytes from C57BL/6 mice infected with HTNV (1 × 10 5 pfu/head) were used as effector cells. As targets, EL-4 cells were pulsed with GP6 (A) and NP1 (B) at a concentration of 10 μg/mL. Splenocytes that were restimulated with GP6 (C) and NP1 (D) were used as effector cells in the second round of the CTL assay. Macrophages infected with HTNV (■) and peptide-pulsed EL-4 cells (•) were used as target cells. P815 cells (▴) were used as negative controls. (E) Evaluation of the mean lysis percentage of naïve CD8+T cells to kill no peptide-pulsed target cells. Macrophages (■), EL-4 cells (•), and P815 cells (▴) were served as target cells, respectively. Data are expressed as the mean ± SEM ( n = 6). ** P < 0.001, *** P < 0.0001.

Article Snippet: EL-4 and P815 cells, which express H-2 b and H-2 d as MHC class I molecules, were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Activity Assay, Derivative Assay, Infection, In Vitro, Concentration Assay, CTL Assay, Lysis

Journal:

Article Title: Antigen-pulsed dendritic cells expressing macrophage-derived chemokine elicit Th2 responses and promote specific humoral immunity

doi:

Figure Lengend Snippet: Chemotaxis of human and murine T lymphocytes to conditioned media of genetically modified DCs. (a) CEM cells. Human T lymphocyte CEM cells placed in the upper chamber of a transwell chamber were assayed for chemotaxis in response to serial dilution of supernatant from DCs transduced with AdMDC, AdNull, or PBS alone (naive control) in the lower chamber. (b) EL4 cells. The study was similar to that in a, but the murine T lymphocyte EL4 cell line was used. (c) Suppression of AdMDC-mediated chemotaxis. CEM and EL4 cells were assayed for chemotaxis in response to 1:2 dilution of supernatant from AdMDC-modified DCs. Where indicated, anti–MDC neutralizing Ab or mouse IgG control Ab was added to the supernatant in the lower chambers at 10 μg/ml at the initiation of assay or cells were placed to the upper chamber in the presence of recombinant (r) MDC or TARC at 100 ng/ml. For all parts, the number of cells migrating to the lower chamber at 37°C for 4 hours was counted by FACS analysis. Migration index was calculated as the number of cells migrating to the conditioned media over the number of cells migrating to control medium. Results represent the mean ± SE (n = 3 per data point).

Article Snippet: To demonstrate the function of the MDC protein expressed by the AdMDC-modified DCs, human T lymphocyte CEM cells or murine T lymphocyte EL4 cells (no. CCL-119 and TIB-39, respectively; American Type Culture Collection, Manassas, Virginia, USA) were suspended in 100 μl of RPMI-1640 media containing 1 mg/ml BSA at 10 7 cells/ml and loaded to the upper wells of 5-μm pore, polycarbonate, transwell chambers in 24-well plates (Corning Costar Inc., Corning, New York, USA).

Techniques: Chemotaxis Assay, Genetically Modified, Serial Dilution, Transduction, Control, Modification, Recombinant, Migration

Journal: Scientific Reports

Article Title: Allogeneic dendritic cells induce potent antitumor immunity by activating KLRG1 + CD8 T cells

doi: 10.1038/s41598-019-52151-3

Figure Lengend Snippet: Allogeneic dendritic cells could elicit efficient antitumor effects. ( a ) Schematic diagram of the investigation of antitumor effects by allogeneic immunocytes. ( b , c ) B6 mice were pre-immunized with 10 8 or 10 7 of splenocytes from DBA/2 mice (▲) or B6 mice (●), respectively. After immunization for two times, they were inoculated subcutaneously with 2 × 10 6 EL4 cells. EL4 tumor sizes were detected at indicated time points. (d-f) 5 × 10 7 TCRβ − CD19 + B cells ( d ), 1 × 10 6 TCRβ − CD19 − CD11c + Ia + dendritic cells ( e ) and 3 × 10 7 TCRβ + CD19 − T cells ( f ) were isolated from splenocytes of DBA/2 (▲) or B6 (●) mice by flow cytometry and injected intraperitoneally into recipient B6 mice for two times. Recipient mice were inoculated subcutaneously with 2 × 10 6 EL4 cells. EL4 tumor sizes were detected at indicated time points. ( g – i ) B6 mice were pre-immunized intraperitoneally by 1 × 10 6 DBA DC (▲) or B6 DC (●) for two times. Then mice were inoculated subcutaneously with 1 × 10 6 S180 cells ( g ) or 1 × 10 6 H22 cells ( h ), or injected intravenously with 5 × 10 5 B16 cells ( i ), and tumor growth were monitored. ( j – l ) DCs from FVB mice (▲) or B6 mice (●) were immunized into B6 mice for two times. Then recipient mice were inoculated subcutaneously with 2 × 10 6 EL4 cells ( j ), 1 × 10 6 H22 cells ( k ) or 1 × 10 6 S180 cells. ( l ) Tumor sizes were recorded at indicated time points. Recipient mice injected with PBS (■) were used as the blank control. These experiments were repeated for 3 times. n = 5 for each repeat. P values indicated the statistical significance when comparing with blank control group (ip PBS group).

Article Snippet: B16-F10 and EL4 cell lines were purchased from ATCC.

Techniques: Isolation, Flow Cytometry, Injection, Control

Journal: Scientific Reports

Article Title: Allogeneic dendritic cells induce potent antitumor immunity by activating KLRG1 + CD8 T cells

doi: 10.1038/s41598-019-52151-3

Figure Lengend Snippet: CD8 T cells were involved in alloDC-elicited antitumor effects. ( a – e ) Peripheral blood was collected from DBA DC-, B6 DC- or PBS-injected mice and main lymphocyte subsets involved in antitumor immunity were examined. Proportions of indicated subsets were statistically compared among the three groups. ( f – h ) B6 mice were pre-injected with 1 × 10 7 CD8 T cells from DBA DC-immunized mice (▲, labeled as DBA DC-CD8 T), B6 DC-immunized mice (●, labeled as B6 DC-CD8 T) or PBS (■). Then recipient mice were inoculated with 2 × 10 6 EL4 cells ( f ), or 1 × 10 6 H22 cells ( g ) or 1 × 10 6 S180 cells ( h ). Tumor growth curves were depicted and compared among the three groups. These experiments were repeated thrice, where n = 5 for each repeat. P values indicated the statistical significance when comparing with blank control group (PBS group). ( i , j ) RNAseq on CD8 T cells from autoDC- and alloDC-vaccinated mice was performed and their DEGs were analyzed using R language and Cytoscape software. KEGG pathway enrichment of DEGs ( i ) and protein-protein interaction network analysis focusing on genes in the three most significantly enriched pathways ( j ) was demonstrated. Significantly enriched antitumor-associated pathways and molecules were highlighted.

Article Snippet: B16-F10 and EL4 cell lines were purchased from ATCC.

Techniques: Injection, Labeling, Control, Software

Journal: Scientific Reports

Article Title: Allogeneic dendritic cells induce potent antitumor immunity by activating KLRG1 + CD8 T cells

doi: 10.1038/s41598-019-52151-3

Figure Lengend Snippet: KLRG1 + CD8 T cells were involved in alloDC-elicited antitumor effects. ( a , b ) B6 mice were immunized with B6 DC, FVB DC or DBA DC. Then percentages of KLRG1 + CD8 T cells in peripheral blood lymphocytes were detected at indicated time points using flow cytometry. Statistical data were shown in ( b ). ( c ) DBA/2 mice were immunized with either β 2 m −/− DC or B6 DC. Then percentages of KLRG1 + CD8 T cells were detected on day 7. ( d ) B6 mice were pre-vaccinated by different doses of DBA DC and then inoculated with EL4 subcutaneously or B16 intravenously. Peripheral KLRG1 + CD8 T cells were analyzed by flow cytometry and their tumor growth was shown. ( e ) Frozen 8-μm sections were processed from B6 DC- or DBA DC-immunized spleens and were stained with DAPI (blue), PE labeled anti-CD8 (green) and APC labeled anti-KLRG1 (red). In the highlighted images, green balls represent cells only expressing CD8, while red balls represent cells only expressing KLRG1. When both CD8 and KLRG1 were expressed on the same cells, they were highlighted by a larger yellow ball. The length of the scale bar is 100 μm. ( f ) Frozen 8-μm sections from tumor-bearing lungs were stained with DAPI (blue), PE labeled anti-CD8 (green) and APC labeled anti-KLRG1 (red). The dotted white line shows the tumor border. The length of the scale bar is 100 μm. ( g ) Metastatic lung tissue was harvested and the proportions and absolute numbers of KLRG1 + CD8 T cells were compared among the mice pre-immunized by PBS, B6 DC and DBA DC. (h) 1 × 10 6 KLRG1 + CD8 T cells, KLRG1 − CD8 T cells as well as naïve CD8 T cells were sorted from DBA DC-immunize B6 mice and adoptive transferred intravenously into B6 mice injected with 1 × 10 4 B16 cells. Survival rates of each group were recorded and shown. These experiments were repeated for 3 times. n = 6 for each repeat. Error bars show standard deviation. P values indicated the statistical significance when comparing with blank control group (PBS group or naive CD8 T group).

Article Snippet: B16-F10 and EL4 cell lines were purchased from ATCC.

Techniques: Flow Cytometry, Staining, Labeling, Expressing, Injection, Standard Deviation, Control

Journal: Scientific Reports

Article Title: Allogeneic dendritic cells induce potent antitumor immunity by activating KLRG1 + CD8 T cells

doi: 10.1038/s41598-019-52151-3

Figure Lengend Snippet: Mechanisms for KLRG1 + CD8 T cells suppressing tumors. ( a ) KLRG1 + CD8 T cells or KLRG1 − CD8 T cells were co-cultured with B16-GFP cells (green) at the E:T ratio of 5:1, and the killing process was captured by PE spinning disk live cell confocal microscope with a 60 × oil immersion lens. ( b ) KLRG1 + CD8 T cells or KLRG1 − CD8 T cells were co-cultured with B16-GFP cells at the E:T ratio of 5:1 for 24 hours. Then target cells were collected and stained with 7-AAD. Percentages of 7-AAD positive populations indicated the killing rates. ( c ) KLRG1 + CD8 T cells or KLRG1 − CD8 T cells were co-cultured with EL4 cells at the E:T ratio of 20:1 for 12 hours. Then target cells were collected and stained with 7-AAD. Percentages of 7-AAD positive populations indicated the killing rates. ( d ) KLRG1 + CD8 T cells and KLRG1 − CD8 T cells were co-cultured with EL4 cells at the E:T ratio of 5:1 and 20:1 for 24 h with or without 50ug/mL anti-FasL, 50ug/mL anti-TRAIL, and 50 μM Granzyme B inhibitor Z-AAD-CMK. Cytotoxicity against target cells was evaluated and shown. ( e ) In an in vitro matrigel invasion experiment, KLRG1 + CD8 T cells or KLRG1 − CD8 T cells were sorted and inoculated on the upper layer. After 24 hours, penetrated cells on the lower layer were collected and calculated. ( f – h ) Real-time PCR ( f , g ) was carried out to examine the gene expression of heparanase and p53, which were also confirmed by RNA-seq analysis. ( h ) In vitro experiments were performed in triplicates for three times.

Article Snippet: B16-F10 and EL4 cell lines were purchased from ATCC.

Techniques: Cell Culture, Microscopy, Staining, In Vitro, Real-time Polymerase Chain Reaction, Gene Expression, RNA Sequencing