Journal: Biomolecules

Article Title: A Novel T Cell-Engaging Bispecific Antibody for Treating Mesothelin-Positive Solid Tumors

doi: 10.3390/biom10030399

Figure Lengend Snippet: In vivo efficacy of bispecific antibodies in the xenograft model. ( a ) Schematic depiction of the antibody treatment protocol. Human T cells were injected intraperitoneally (effector:target ratio, 2:1) into NOG mice at 5 days after the injection of AsPC-1 or H226 tumor cells. Mice bearing xenograft tumors were treated with MG1122-A or MG1122-B at a dose of 3 mg/kg twice per week, starting at day 7 after tumor cell injection. ( b ) Tumor growth was measured twice per week for the indicated time periods. Data points represent the mean ± standard deviation (SD) of five animals. * p < 0.01 vs. PBS, # p < 0.01 vs. MG1122-A. ( c ) Representative histologic images of tumor sections stained for human CD3 and CD8 (all brown). Scale bars are shown on each panel. Representative images of at least three mice were analyzed in each group.

Article Snippet: Following deparaffinization, the sections underwent heat antigen retrieval and were then stained with human CD3 (anti-CD3, Abcam, Cambridge, UK) or CD8 (anti-CD8, Abcam) using VECTASTAIN Elite ABC kits (VECTOR Lab, Burlingame, CA, USA).

Techniques: In Vivo, Injection, Standard Deviation, Staining

Journal: PLoS ONE

Article Title: Mouse SPNS2 Functions as a Sphingosine-1-Phosphate Transporter in Vascular Endothelial Cells

doi: 10.1371/journal.pone.0038941

Figure Lengend Snippet: (A–G) Blood from wild-type (WT, n = 3) and SPNS2-deficient mice (Δ, n = 3) was collected, and leukocytes, leukocyte subpopulations, erythrocytes and platelets were counted using flow cytometry. Bar graphs show the average values from three experiments. (H–J) Flow cytometric analysis of blood from wild-type (WT, red, n = 13) and SPNS2-deficient (Δ, blue, n = 7) mice. CD8 (H), CD4 (I) and B220 (J) were used to detect each cell type. Graphs show the average values from multiple experiments, with error bars representing the standard error. The P -value of comparisons between WT and Δ samples is indicated.

Article Snippet: CD4 or CD8 single positive cells were purified from the thymus of mice by negative and positive selection using specific MACS microbeads conjugated to anti-mouse CD4 or CD8 antibodies (Miltenyi Biotech).

Techniques: Flow Cytometry

Journal: PLoS ONE

Article Title: Mouse SPNS2 Functions as a Sphingosine-1-Phosphate Transporter in Vascular Endothelial Cells

doi: 10.1371/journal.pone.0038941

Figure Lengend Snippet: (A and B) Expression profiles for CD4 and CD8. Thymus-derived CD4 + and CD8 + cells from wild-type (A, WT, n = 5) and SPNS2-deficient (B, Δ, n = 7) mice were analyzed by flow cytometry. Each plot is representative of multiple experiments. Numbers show the percent of total lymphocytes, identified by their size. (C) The percentage corresponding to CD4 + CD8 + (DP), CD4 − CD8 + (CD8SP) or CD4 + CD8 − (CD4SP) populations. (D) Quantitative analysis of S1p1 mRNA in mature thymocytes. CD4 or CD8 single positive cells were purified from the thymus of wild-type (WT, n = 3) or SPNS2-deficient (Δ, n = 5) mice with MACS. The amount of S1p1 mRNA is normalized to that of Hprt . The primers and probes used for PCR are indicated in . The P -values from comparisons between WT and Δ samples are indicated. (E, F) Chemotaxis assays of mature thymocytes of wild-type (WT, n = 10) or SPNS2-deficient (Δ, n = 5) mice. The percentage of input cells of the CD62L hi and CD4 (E) or CD8 (F) single positive phenotype that migrated toward S1P is shown. open square, wild-type CD4 single positive; closed square, SPNS2-deficient CD4 single positive; open circle, wild-type CD8 single positive; closed circle, SPNS2-deficient CD8 single positive. The graphs show the average values from multiple experiments, with error bars representing the standard error.

Article Snippet: CD4 or CD8 single positive cells were purified from the thymus of mice by negative and positive selection using specific MACS microbeads conjugated to anti-mouse CD4 or CD8 antibodies (Miltenyi Biotech).

Techniques: Expressing, Derivative Assay, Flow Cytometry, Purification, Chemotaxis Assay

Journal: Journal of Translational Medicine

Article Title: Decidual CD8 + T cells exhibit both residency and tolerance signatures modulated by decidual stromal cells

doi: 10.1186/s12967-020-02371-3

Figure Lengend Snippet: Transcriptional analysis of CD8 + T cells from peripheral blood and decidua. a Gating strategy of CD8 + T cells from maternal peripheral blood and decidua, CD8 + T cells were gated from live CD3 + , CD56 − , and CD4 − cells. Fluorescence-activated cell sorting was performed to purify the CD8 + T cells by this gating strategy; b Left: Percentage of CD4 + T cells and CD8 + T cells in CD3 + T cells from paired PBMC and decidua. n = 4, paired t − test , * p = <0.05 ** p = <0.01; Right: Ratio of frequencies of CD4 versus CD8 from PBMC and decidua. c Volcano plot representation of differential expression analysis of genes; Red and blue points mark the genes with significantly increased or decreased expression, respectively, in samples of CD8 + dT cells compared with samples of CD8 + pT cells (FDR < 0.01). The x-axis shows fold-changes (log2) in expression, and the y-axis shows the log p value of a gene being expressed differentially. In both data sets, Smchd1 is the top-ranked gene. Blue dots are downregulated genes, and red dots are upregulated genes, of CD8 + dT cells compared with CD8 + pT cells; d Heatmap result of an unsupervised hierarchical clustering of genes that is significantly different ( p < 0.01) in CD8 + dT cell samples compared with CD8 + pT cell samples. Each column represents a patient (blue: CD8 + pT, red: CD8 + dT), and each row represents a gene. The heatmap indicates the level of row normalized gene expression. Red = high expression; Green = low expression

Article Snippet: For coculturing with either DSCs or trophoblast cells, CD8 + T cells from the decidua and PBMC via magnetic affinity cell were sorted using CD8 MicroBeads (MiltenyiBiotec, Germany), according to the manufacturer’s instructions.

Techniques: Fluorescence, FACS, Quantitative Proteomics, Expressing, Gene Expression

Journal: Journal of Translational Medicine

Article Title: Decidual CD8 + T cells exhibit both residency and tolerance signatures modulated by decidual stromal cells

doi: 10.1186/s12967-020-02371-3

Figure Lengend Snippet: Comparison of differentiation status of CD8 + dT cells and CD8 + pT cells. The gating strategy of CD8 + T cells from maternal peripheral blood ( a ) and decidua ( b ). The memory phenotype was evaluated by expression of CD45RA and CCR7, and each of the CCR7 versus CD45RA dot plot quadrants were further analyzed for CD27 versus CD28 staining. c Cell surface markers CD45RA and CCR7 were used to classify naive (CD45RA + CCR7 + ), terminally differentiated effector memory (EFF or TEMRA; CD45RA + /CCR7 − ), effector-memory (EM; CD45RA − CCR7 − ), and central-memory (CM; CD45RA − CCR7 + ) CD8 + T cells. d The proportion of each subset of CD8 + dT cells and CD8 + pT cells. n = 15, paired t − test , * p = < 0.05, ** p = < 0.01, *** p = <0.005, **** p = <0.0001

Article Snippet: For coculturing with either DSCs or trophoblast cells, CD8 + T cells from the decidua and PBMC via magnetic affinity cell were sorted using CD8 MicroBeads (MiltenyiBiotec, Germany), according to the manufacturer’s instructions.

Techniques: Comparison, Expressing, Staining

Journal: Journal of Translational Medicine

Article Title: Decidual CD8 + T cells exhibit both residency and tolerance signatures modulated by decidual stromal cells

doi: 10.1186/s12967-020-02371-3

Figure Lengend Snippet: GSEA analysis of upregulated genes in decidua CD8 + T cells

Article Snippet: For coculturing with either DSCs or trophoblast cells, CD8 + T cells from the decidua and PBMC via magnetic affinity cell were sorted using CD8 MicroBeads (MiltenyiBiotec, Germany), according to the manufacturer’s instructions.

Techniques:

Journal: Journal of Translational Medicine

Article Title: Decidual CD8 + T cells exhibit both residency and tolerance signatures modulated by decidual stromal cells

doi: 10.1186/s12967-020-02371-3

Figure Lengend Snippet: Combined residency and exhaustion signatures of CD8 + dT cells. a GSEA analysis with gene sets of core T RM signature, tumor-infiltrating monocytes and LCMV-specific exhausted CD8 + T cells, respectively. NES, normalized enrichment score; FDR, false discovery rate; Nom, nominal. b Heat map of selected genes that are expressed differentially between CD8 + dT cells and CD8 + pT cells; gene expression was row normalized. c Representative FACS plots (Left) and percentages (Right) of the activation markers CD69, HLA-DR, tissue residency associated marker, CD103, CXCR3, and the coinhibitory molecule PD-1, as well as the CD39 molecule expressed on freshly isolated CD8 + pT cells and CD8 + dT cells. Graphs depict data of six to ten samples in each group; paired t − test , ** p = < 0.01, *** p = < 0.005

Article Snippet: For coculturing with either DSCs or trophoblast cells, CD8 + T cells from the decidua and PBMC via magnetic affinity cell were sorted using CD8 MicroBeads (MiltenyiBiotec, Germany), according to the manufacturer’s instructions.

Techniques: Gene Expression, Activation Assay, Marker, Isolation

Journal: Journal of Translational Medicine

Article Title: Decidual CD8 + T cells exhibit both residency and tolerance signatures modulated by decidual stromal cells

doi: 10.1186/s12967-020-02371-3

Figure Lengend Snippet: Detection of Intracellular effector molecules in CD8 + dT cells and their activation after coculturing with autologous trophoblasts. a Representative FACS plots (Left) and percentages (Right) of the expression of intracellular granzyme B and perforin in total CD8 + pT cells and CD8 + dT cells; b IFN-γ, IL-4 expression in total CD8 + pT cells and CD8 + dT cells stimulated for 6 h with PMA/Ionomycin (1 μg/mL); c Lymphocytes from the decidua and PBMC were cultured separately with autologous trophoblasts for up to 24 h and were then tested for expression of CD69 and IFN-γ. Graphs show the data of four to ten samples in each group; a paired t - test was used to calculate the p-values, * p = < 0.05, *** p = < 0.005, **** p = < 0.0001

Article Snippet: For coculturing with either DSCs or trophoblast cells, CD8 + T cells from the decidua and PBMC via magnetic affinity cell were sorted using CD8 MicroBeads (MiltenyiBiotec, Germany), according to the manufacturer’s instructions.

Techniques: Activation Assay, Expressing, Cell Culture

Journal: Journal of Translational Medicine

Article Title: Decidual CD8 + T cells exhibit both residency and tolerance signatures modulated by decidual stromal cells

doi: 10.1186/s12967-020-02371-3

Figure Lengend Snippet: Upregulation of CD103 by decidual stromal cells and effector molecule expression in CD103 + dT cells. a Representative FACS plots and percentages of expression of CD69 + CD103 + cells in CD8 + pT cells and CD8 + dT cells. Percentages of CD69 + CD103 + cells are depicted within four subpopulations of CD8 + Tcell; b CD103 expression of CD8 + pT cells cocultured with DSCs and trophoblasts, respectively, for three days and analyzed by flow cytometry; c Representative FACS plots (Left) and percentages (Right) of the expression of intracellular granzyme B and IFN-γ in CD103 + and CD103 − CD8 + dT cells stimulated for 6 h with PMA/Ionomycin (1 μg/mL). Graphs show the data of six to ten samples in each group; a paired t-test was used to calculate the p-values, *p = < 0.05, ***p = < 0.005, ****p = < 0.0001. CD103 expression of CD8 + pT cells cocultured with DSCs and trophoblasts, respectively, for three days and analyzed by flow cytometry. Graphs show the data of six samples in each group; a paired t-test was used to calculate the p-values, *p = < 0.05, **p = < 0.01

Article Snippet: For coculturing with either DSCs or trophoblast cells, CD8 + T cells from the decidua and PBMC via magnetic affinity cell were sorted using CD8 MicroBeads (MiltenyiBiotec, Germany), according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry

Journal: BMC Cancer

Article Title: Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer

doi: 10.1186/1471-2407-9-463

Figure Lengend Snippet: Representative immunohistochemical stainings for B7-H3 (A, C) and CD8 (B, D) in pancreatic cancer tissues . (A) Pancreatic cancer tissue section with strong B7-H3 immunoreactivity. (B) Consecutive section with immunostaining for CD8 shows the infiltration of numerous CD8+ T cells (arrows). (C) Pancreatic cancer tissue section with weak tumor B7-H3 immunoreactivity. (D) Consecutive section with immunostaining for CD8 shows no CD8+ tumor-infiltrating T cells.

Article Snippet: Immunohistochemical analyses were carried out with mouse anti-human B7-H3 antibody (MAB 1027, R&D Systems, Minneapolis, MN), mouse anti-human CD4 antibody (1F6, Monosan, Uden, Netherlands), and mouse anti-human CD8 antibody (DAKO Diagnostics AG, Zürich, Switzerland).

Techniques: Immunohistochemical staining, Immunostaining

Journal: BMC Cancer

Article Title: Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer

doi: 10.1186/1471-2407-9-463

Figure Lengend Snippet: Semi-quantitative analysis of CD8+ T cells in pancreatic cancer . In areas with high tumor B7-H3 expression, the prevalence of CD8+ T cells was significantly increased compared to areas with low tumor B7-H3 expression (p = 0.018).

Article Snippet: Immunohistochemical analyses were carried out with mouse anti-human B7-H3 antibody (MAB 1027, R&D Systems, Minneapolis, MN), mouse anti-human CD4 antibody (1F6, Monosan, Uden, Netherlands), and mouse anti-human CD8 antibody (DAKO Diagnostics AG, Zürich, Switzerland).

Techniques: Expressing