Journal: PLoS ONE

Article Title: Over-Expression of CD200 Protects Mice from Dextran Sodium Sulfate Induced Colitis

doi: 10.1371/journal.pone.0146681

Figure Lengend Snippet: Cytokines and chemokines were analyzed in colonic tissue from CD200 tg , WT, CD200R1KO and CD200KO mice with DSS-induced acute colitis. A and B, Real-time RT-PCR was performed using total RNA extracted from colonic tissue (5 samples/group) or LP cells (3 samples/group), with data normalized to the expression of GAPDH and HPRT in the same organ. Expression levels in CD200 tg , CD200R1KO and CD200KO mice (mean ± SD in 5 mice) are shown relative to that in WT at day 0 (designated as 1). * p < 0.05; ** p < 0.01; *** p < 0.001 compared to WT control (day 0). (C) Tissue explants from the four experimental groups were cultured and used for multi-analyte Elisarray. Again, relative expression levels of cytokine/chemokine (mean ± SD in 5 mice) in CD200 tg , CD200R1KO and CD200KO mice are shown compared to that in WT at day 0 (designated as 1), using the manufacturer’s software supplied. * p < 0.05; ** p < 0.01; *** p < 0.001, compared to this WT control (day 0). (D) Inflammatory cytokines measured by commercial ELISA (Biolegend: USA) in 36hr culture supernatants of colon-derived CD11c + , CD11b + , or CD4 + T cells (isolated by MACS columns) from WT, CD200 tg , CD200KO or CD200RKO mice 9 days post initiation of DSS treatment. Cells were obtained from a pool of 4 mice/group, and cultured without further stimulation for 36hrs in complete medium. In some cases, as shown, cells were cultured in the presence of CD200Fc (5mg/ml). Data show mean ( + SD) of triplicate measurements. *, indicates p<0.05 compared with WT control; **indicates p<0.05 compared to equivalent group with no CD200Fc. E, Comparison of weight loss in 4 groups of DSS treated mice receiving anti-CD4mAb (right hand panel) or isotype control Ig (left hand panel)-see also . Data are for 4 mice/group, given 50mg Ig at days 4/7 post initiation of DSS treatment. Comparison of equivalent groups in right/left hand panels (Mann Whitney U-test) revealed no statistically significant differences.

Article Snippet: Where CD4 + T cells were depleted from mice with acute DSS-induced colitis, mice received 50mg/mouse of anti-mouse CD4 mAb (GK1.5), purchased from Cedarlane Labs, Hornby, Ontario, iv at 4 and 7 days post DSS initiation, and were sacrificed at 9 days post DSS initiation.

Techniques: Quantitative RT-PCR, Expressing, Control, Cell Culture, Software, Enzyme-linked Immunosorbent Assay, Derivative Assay, Isolation, Comparison, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Over-Expression of CD200 Protects Mice from Dextran Sodium Sulfate Induced Colitis

doi: 10.1371/journal.pone.0146681

Figure Lengend Snippet: mRNAs for cytokines and chemokines were analyzed in colonic tissue harvested 8 days after initiation of the third cycle of DSS-induced chronic colitis from CD200 tg , WT, CD200R1KO and CD200KO mice. Real-time RT-PCR was performed using total RNA extracted from colonic tissue (panel A: 5 samples/group) or LP cells (panel B: 3 samples/group), with data normalized to the expression of GAPDH and HPRT in the same organ. Expression levels in CD200 tg , CD200R1KO and CD200KO mice (mean ± SD in 5 mice) are shown relative to that in WT at day 0 (designated as 1). * p < 0.05; ** p < 0.01; *** p < 0.001, compared with WT control (day 0). (C) Tissue explants from the four experimental groups were cultured and used for multi-analyte Elisarray. Again, relative expression levels of cytokine/chemokine (mean ± SD in 5 mice) in CD200 tg , CD200R1KO and CD200KO mice are shown compared to that in WT at day 0 (designated as 1), using manufacturer’s software. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to WT control (day 0). (D) Inflammatory cytokines measured in 36hr culture supernatants of colon-derived CD11c + , CD11b + , or CD4 + T cells (isolated by MACS columns) from WT, CD200 tg , CD200KO or CD200RKO mice 8 days post initiation of the third cycle of DSS treatment. Cells were obtained from a pool of 4 mice/group, and cultured without further stimulation for 36hrs in complete medium. Data show mean ( + SD) of triplicate measurements. *, indicates p<0.05 compared with WT control-see also .

Article Snippet: Where CD4 + T cells were depleted from mice with acute DSS-induced colitis, mice received 50mg/mouse of anti-mouse CD4 mAb (GK1.5), purchased from Cedarlane Labs, Hornby, Ontario, iv at 4 and 7 days post DSS initiation, and were sacrificed at 9 days post DSS initiation.

Techniques: Quantitative RT-PCR, Expressing, Control, Cell Culture, Software, Derivative Assay, Isolation

Journal: PLoS ONE

Article Title: Over-Expression of CD200 Protects Mice from Dextran Sodium Sulfate Induced Colitis

doi: 10.1371/journal.pone.0146681

Figure Lengend Snippet: Foxp3, CCR4, CCL-17 and CCL-22 mRNA expression was analyzed in (A) colons and (B) LP cells by real-time RT-PCR from CD200 tg , WT, CD200R1KO and CD200KO mice with DSS-induced chronic colitis. All values were normalized to the expression of GAPDH and HPRT in the same organ with expression levels in CD200 tg , CD200R1KO and CD200KO mice expressed relative to that of WT (designated as 1). Data show mean ± SD for 5 mice/group. * p < 0.05; ** p < 0.01. (C) FACS staining for CD3 + and CD4 + Foxp3 + (gated on CD3 + cells) in mesenteric lymph node (MLN) cells of mice in panels A and B. Data show typical staining patterns from pooled samples (3 mice/group). (D) Loss of protection from weight loss in DSS-induced chronic colitis in CD200 tg mice by treatment with anti-CD25mAb (Fig 7Da), and protection from weight loss in CD200RKO mice by treatment with anti-CD4 mAb (Fig 7Db). Data are shown for 4 mice/group (SD not shown to retain clarity in panels). Mice received 5mg antibody iv 1 day following completion of each DSS treatment. *p<0.05 (Mann-Whitney U-test) compared with equivalent groups not receiving mAb.

Article Snippet: Where CD4 + T cells were depleted from mice with acute DSS-induced colitis, mice received 50mg/mouse of anti-mouse CD4 mAb (GK1.5), purchased from Cedarlane Labs, Hornby, Ontario, iv at 4 and 7 days post DSS initiation, and were sacrificed at 9 days post DSS initiation.

Techniques: Expressing, Quantitative RT-PCR, Staining, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Co-Expression Profile of TNF Membrane-Bound Receptors Type 1 and 2 in Rheumatoid Arthritis on Immunocompetent Cells Subsets

doi: 10.3390/ijms21010288

Figure Lengend Snippet: Subpopulations of T helper cells. Proportion of cells expressing type 1 and 2 receptors for TNFα among T helper cells. Data have been normalized to the total median value.

Article Snippet: Evaluation of phenotypic characteristics was performed by flow cytometry (FACSVerse cytometer (BD, USA)) using monoclonal antibodies: anti-human CD3 APC/Cy7, anti-human CD19 PE/Cy7, anti-human CD8 APC/Cy7, anti-human CD14 PerCP, anti-human CD25 FITC, anti-human CD127 (IL-7Rα) APC/Cy7, anti-human CD45RO FITC, anti-human CD45RA Pacific Blue, anti-human CD45 PerCP, anti-human CD4 Pe/Cy7, anti-human TNFRI-PE, anti-human TNFRII-PE, anti-human TNFRI-APC, and anti-human TNFRII-APC (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Co-Expression Profile of TNF Membrane-Bound Receptors Type 1 and 2 in Rheumatoid Arthritis on Immunocompetent Cells Subsets

doi: 10.3390/ijms21010288

Figure Lengend Snippet: Number of type 1 and 2 receptors for TNFα on the surfaces of immune cells. Data are presented as median and interquartile range.

Article Snippet: Evaluation of phenotypic characteristics was performed by flow cytometry (FACSVerse cytometer (BD, USA)) using monoclonal antibodies: anti-human CD3 APC/Cy7, anti-human CD19 PE/Cy7, anti-human CD8 APC/Cy7, anti-human CD14 PerCP, anti-human CD25 FITC, anti-human CD127 (IL-7Rα) APC/Cy7, anti-human CD45RO FITC, anti-human CD45RA Pacific Blue, anti-human CD45 PerCP, anti-human CD4 Pe/Cy7, anti-human TNFRI-PE, anti-human TNFRII-PE, anti-human TNFRI-APC, and anti-human TNFRII-APC (R&D Systems, Minneapolis, MN, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Co-Expression Profile of TNF Membrane-Bound Receptors Type 1 and 2 in Rheumatoid Arthritis on Immunocompetent Cells Subsets

doi: 10.3390/ijms21010288

Figure Lengend Snippet: Correlation analysis ( p < 0.05).

Article Snippet: Evaluation of phenotypic characteristics was performed by flow cytometry (FACSVerse cytometer (BD, USA)) using monoclonal antibodies: anti-human CD3 APC/Cy7, anti-human CD19 PE/Cy7, anti-human CD8 APC/Cy7, anti-human CD14 PerCP, anti-human CD25 FITC, anti-human CD127 (IL-7Rα) APC/Cy7, anti-human CD45RO FITC, anti-human CD45RA Pacific Blue, anti-human CD45 PerCP, anti-human CD4 Pe/Cy7, anti-human TNFRI-PE, anti-human TNFRII-PE, anti-human TNFRI-APC, and anti-human TNFRII-APC (R&D Systems, Minneapolis, MN, USA).

Techniques:

Journal: Advanced Science

Article Title: CXCL13 Expression Promotes CAR T Cell Antitumor Activity and Potentiates Response to PD‐1 Blockade

doi: 10.1002/advs.202508095

Figure Lengend Snippet: CXCL13 is highly expressed in T cells from responders to ICB therapy. a). UMAP plot displaying 237231 CD3 + T cells across 8 cancer types: BCC (basal cell carcinoma, n = 11 patients; n = 22 samples), SCC (squamous cell carcinoma, n = 3 patients; n = 8 samples), BLCA (bladder cancer, n = 6 patients; n = 6 samples), TNBC (triple‐negative breast cancer, n = 8 patients; n = 14 samples), RCC (Renal cell carcinoma, n = 7 patients; n = 7 samples), HNSCC (head and neck cancer, n = 4 patients; n = 8 samples), NSCLC (non‐small‐cell lung cancer, n = 36 patients; n = 47 samples), and PRAD (prostate cancer, n = 10 patients; n = 12 samples), categorized into 13 distinct cell types. b). Comparison of differential genes in CD3 + , CD8 + , and CD4 + T cells in the response and non‐response groups. c,d). UMAP plots (c) and dot plots (d) showing the expression of CXCL13 in CD3 + , CD8 + , and CD4 + T cells of pre‐treatment non‐response, pre‐treatment response, post‐treatment non‐response, and post‐treatment response groups. e). CXCL13 expression levels in responders and non‐responders across multiple tumor cohorts treated with ICB: HNSC, melanoma cohort 1 (anti‐PD‐1 and anti‐CTLA‐4), melanoma cohort 2 (anti‐PD‐1), STAD (stomach adenocarcinoma), and BLCA. f). Comparison of CXCL13 expression in pre‐treatment and post‐treatment groups from melanoma cohort 1 and melanoma cohort 2 treated with ICB. g). Kaplan‐Meier survival curves stratified by high and low CXCL13 expression in ICB‐treated patients from BLCA, melanoma cohort 1, and melanoma cohort 2. h). Heatmap showing the correlation of CXCL13 expression with various cell types across tumor types in the TCGA dataset. Cell‐type deconvolution was performed using TIMER2.0, with significant correlations ( p < 0.05) marked by solid indicators.

Article Snippet: The following antibodies were used for staining: anti‐mouse CD45.1, BV450 (E‐AB‐F1184UQ), Elabscience, Cat # AF19855; anti‐mouse CD45.1, PE (A20), Biolegend, Cat # 110 708; anti‐mouse CD3ε, PE/Cyanine7 (145‐2C11), Biolegend, Cat # 100 320; anti‐mouse PD‐1, PE/Cyanine7 (RMP1‐30), Biolegend, Cat # 109 110; anti‐mouse CD4, APC/Cy7 (E‐AB‐F1353UJ), Elabscience, Cat # AF18028; anti‐mouse CD4, PerCP/Cyanine5.5 (RM4‐5), Biolegend, Cat # 100 540; anti‐mouse CD4, PE/Cyanine7 (RM4‐5), Biolegend, Cat # 100 527; anti‐mouse CD4, Brilliant Violet 785 (RM4‐5), Biolegend, Cat # 100 552; anti‐mouse CD8a, PE (53‐6.7), Biolegend, Cat # 100 708; anti‐mouse CD8a, APC (53‐6.7), Biolegend, Cat # 100 712; anti‐mouse CD8a, PE/Cyanine7 (53‐6.7), Biolegend, Cat # 100 721; anti‐mouse CD45, PerCP/Cyanine5.5 (30‐F11), Biolegend, Cat # 103 132; anti‐mouse Ly108, PE (330‐AJ), Biolegend, Cat # 134 605; anti‐mouse LAG‐3, PerCP/Cyanine5.5 (C9B7W), Biolegend, Cat # 125 211; anti‐mouse LAG‐3, APC (C9B7W), Biolegend, Cat # 125 210; anti‐mouse CD69, Brilliant Violet 605 (H1.2F3), Biolegend, Cat # 104 530; anti‐mouse/human CD44, Brilliant Violet 510 (IM7), Biolegend, Cat # 103 044; anti‐mouse CD62L, PerCP/Cyanine5.5 (MEL‐14), Biolegend, Cat # 104 432; anti‐mouse CTLA‐4, APC (UC10‐4B9), Biolegend, Cat # 106 309; anti‐human CD3, APC (OKT3), Biolegend, Cat # 317 318; anti‐human CD4, PerCP/Cyanine5.5 (RPA‐T4), Biolegend, Cat # 300 530; anti‐human CD8, APC/Cyanine7 (SK1), Biolegend, Cat # 344 713; anti‐human CD279(PD‐1), PE (A17188A), Biolegend, Cat # 379 209; anti‐human CD366(Tim3), PE/CY7 (F38‐2E2), Biolegend, Cat # 345 014.

Techniques: Comparison, Expressing

Journal: Advanced Science

Article Title: CXCL13 Expression Promotes CAR T Cell Antitumor Activity and Potentiates Response to PD‐1 Blockade

doi: 10.1002/advs.202508095

Figure Lengend Snippet: CXCL13 CAR T cells display enhanced antitumor characteristics. a). GSEA analysis of the mitochondrial membrane potential gene set in bulk RNA‐seq comparing CXCL13 CAR T cells with control CAR T cells. b–d). Flow cytometry analysis of mitochondrial membrane potential (TMRM) in control CAR T and CXCL13 CAR T cells. Representative TMRM peak plots are shown for GFP + (b) and GFP − (c) T cells cultured in vitro on day 10 and for cells isolated from the spleen (d). TMRM: Tetramethylrhodamine, methyl ester. Mean ± SEM, n = 3 (b, c); n = 5 (control CAR T), n = 4 (CXCL13 CAR T) (d). e). Intracellular ATP levels in control CAR T cells and CXCL13 CAR T cells. Mean ± SEM, n = 4. f,g). GSEA analysis of positive regulation of T cell migration gene set in CXCL13 CAR T cells versus control CAR T cells using bulk RNA‐seq data (f) and CXCL13 + cells versus CXCL13 − cells using scRNA‐seq data (g). h). Schematic overview of the transwell migration assay. i,j). The migration ability of CAR T cells was assessed by the transwell assay, showing the proportion (i) and number (j) of cells that migrated through the membrane. Mean ± SEM, n = 4. k–m). C57BL/6 mice injected s.c. with 5 × 10 5 B16‐CD19 cells were treated with 2×10⁶ CAR T cells on day 5 post‐tumor establishment. The number of CAR T cells was detected after 7 days of adoptive transfer in the spleen (k), draining lymph nodes (l), and tumor sites (m). Mean ± SEM, n = 3. n). Percentage of CD4 + T cells among CD3 + T cells from patients with GPC3‐positive solid tumors treated with CAR T cells (non‐responders [NR], n = 6; responders [R], n = 15). Mean ± SEM. o,p). Flow cytometry plots (o) and line graphs (p) showing the percentage of CD4⁺ CAR T cells in vitro from day 4 to day 10. Mean ± SEM, n = 4. q–s). Proportions of CD4 + and CD8 + CAR T cells in blood (q), spleen (r), and draining lymph nodes (s). Mean ± SEM, blood ( n = 6), spleen ( n = 6 for control CAR T; n = 4 for CXCL13 CAR T), draining lymph nodes ( n = 6 for control CAR T; n = 4 for CXCL13 CAR T). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; two‐tailed unpaired t‐test (b–e,i–n,p–s).

Article Snippet: The following antibodies were used for staining: anti‐mouse CD45.1, BV450 (E‐AB‐F1184UQ), Elabscience, Cat # AF19855; anti‐mouse CD45.1, PE (A20), Biolegend, Cat # 110 708; anti‐mouse CD3ε, PE/Cyanine7 (145‐2C11), Biolegend, Cat # 100 320; anti‐mouse PD‐1, PE/Cyanine7 (RMP1‐30), Biolegend, Cat # 109 110; anti‐mouse CD4, APC/Cy7 (E‐AB‐F1353UJ), Elabscience, Cat # AF18028; anti‐mouse CD4, PerCP/Cyanine5.5 (RM4‐5), Biolegend, Cat # 100 540; anti‐mouse CD4, PE/Cyanine7 (RM4‐5), Biolegend, Cat # 100 527; anti‐mouse CD4, Brilliant Violet 785 (RM4‐5), Biolegend, Cat # 100 552; anti‐mouse CD8a, PE (53‐6.7), Biolegend, Cat # 100 708; anti‐mouse CD8a, APC (53‐6.7), Biolegend, Cat # 100 712; anti‐mouse CD8a, PE/Cyanine7 (53‐6.7), Biolegend, Cat # 100 721; anti‐mouse CD45, PerCP/Cyanine5.5 (30‐F11), Biolegend, Cat # 103 132; anti‐mouse Ly108, PE (330‐AJ), Biolegend, Cat # 134 605; anti‐mouse LAG‐3, PerCP/Cyanine5.5 (C9B7W), Biolegend, Cat # 125 211; anti‐mouse LAG‐3, APC (C9B7W), Biolegend, Cat # 125 210; anti‐mouse CD69, Brilliant Violet 605 (H1.2F3), Biolegend, Cat # 104 530; anti‐mouse/human CD44, Brilliant Violet 510 (IM7), Biolegend, Cat # 103 044; anti‐mouse CD62L, PerCP/Cyanine5.5 (MEL‐14), Biolegend, Cat # 104 432; anti‐mouse CTLA‐4, APC (UC10‐4B9), Biolegend, Cat # 106 309; anti‐human CD3, APC (OKT3), Biolegend, Cat # 317 318; anti‐human CD4, PerCP/Cyanine5.5 (RPA‐T4), Biolegend, Cat # 300 530; anti‐human CD8, APC/Cyanine7 (SK1), Biolegend, Cat # 344 713; anti‐human CD279(PD‐1), PE (A17188A), Biolegend, Cat # 379 209; anti‐human CD366(Tim3), PE/CY7 (F38‐2E2), Biolegend, Cat # 345 014.

Techniques: Membrane, RNA Sequencing, Control, Flow Cytometry, Cell Culture, In Vitro, Isolation, Migration, Transwell Migration Assay, Transwell Assay, Injection, Adoptive Transfer Assay, Two Tailed Test

Journal: Journal of Translational Medicine

Article Title: Niraparib exhibits a synergistic anti-tumor effect with PD-L1 blockade by inducing an immune response in ovarian cancer

doi: 10.1186/s12967-021-03073-0

Figure Lengend Snippet: The information of antibodies used for western blot, immunohistochemistry, and flow cytometry

Article Snippet: CD4 and isotype , Human , 5 μl of antibody per test , Elabscience, E-AB-F1109I , Flow cytometry.

Techniques: Western Blot, Immunohistochemistry, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Low IL7R Expression at Diagnosis Predicted Relapse in Adult Acute Myeloid Leukemia Patients With t(8;21)

doi: 10.3389/fimmu.2022.909104

Figure Lengend Snippet: Comparison of IL7R tested by RQ-PCR and FCM. (A) The frequency of CD127 in different cell subsets, numbers on the figure represent median (range); (B) Correlation of IL7R transcript expression and percentage of CD8 + T in NCs (FCM) (bivariate Spearman correlation test); (C) Correlation of IL7R transcript expression and percentage of CD4 + T in NCs (FCM) (bivariate Spearman correlation test).

Article Snippet: The monoclonal antibodies included CD127(IL7R)-APC (Biolegend, Clone A019D5), CD45-V500 (BD Biosciences, Clone HI30), CD19-BV605 (Biolegend, Clone HIB19), CD34-FITC (BD Biosciences, Clone 8G12), CD56-APC-F750 (Biolegend, Clone 5.1H11), CD3-BV421 (Biolegend, Clone OKT3), CD4-PE (Caprico Biotechnologies, Clone OKT4), CD8-PE-Cy7 (BD Biosciences, Clone SK1).

Techniques: Comparison, Expressing