anti-cd69-apc cy7 fn50  (Thermo Fisher)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Identification of genetic modifiers enhancing B7-H3-targeting CAR T cell therapy against glioblastoma through large-scale CRISPRi screening

doi: 10.1186/s13046-024-03027-6

Figure Lengend Snippet: B7-H3-specific CAR T cells recognize and eradicate GBM cells. A Retroviral vectors construct encoding the B7-H3.CAR and bicistronic mCherry reporter. B Schematics of transduction and expansion of CAR T cells used in this study. C Expression of the B7-H3.CAR in transduced human T cells. The transduction efficiency of CAR molecules was measured either by mCherry expression or by direct staining of B7-H3 specific scFv molecules. Data shown are a representative FACS plot and quantifications of 3 independent donors. Error bars denote SEM. D Phenotypic analysis of CAR + T cells at 12 days post transduction showing the frequency of CD4 + and CD8 + population in CAR + T cells (left) as well as percentage of stem cell memory T cells (T SCM , CD45RA + CCR7 + ), central memory T cells (T CM , CD45RA − CCR7 + ), effector memory T cells (T EM , CD45RA − CCR7 − ), and effector T cells (T E , CD45RA + CCR7 − ) in CD4 + T cells and CD8 + T cells ( n = 3). Error bars denote SEM. E Surface staining for CD69, CD25, and CD137 of CAR T cells after co-culture with the indicated cell lines for 24 h ( n = 3). Error bars denote SEM. F Expression of granzyme B and cytokine (TNF-α, IFN-γ and IL-2) secretion of CAR T cells after co-culture with the indicated cell lines for 24 h ( n = 3). Error bars denote SEM. G Counts of residual tumor cell lines after 5-day co-culture with CAR T cells or Ctrl T cells at 1:1 E: T ratio ( n = 3). Error bars denote SEM. H&I Counts of U87 MG tumor cells and T cells (CAR T or Ctrl T cells) on indicated days post co-culture at 1:1 E: T ratio ( n = 3). Error bars denote SEM. J Counts of U87 MG tumor cells on day two post co-culture with CAR T cells or Ctrl T cells at 1:1, 1:2, and 1:4 E: T ratio ( n = 3). Error bars denote SEM

Article Snippet: The following antibodies used for the flow cytometry analysis were obtained from BD Biosciences: FITC-conjugated anti-CCR7 (Clone 150,503, Cat. no. 561,271), APC-Cy7-conjugated anti-CD69 (Clone FN50, Cat. no. 557,756), V450-conjugated anti-Granzyme B (Clone GB11, Cat. no. 561,155).

Techniques: Construct, Transduction, Expressing, Staining, Co-Culture Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Identification of genetic modifiers enhancing B7-H3-targeting CAR T cell therapy against glioblastoma through large-scale CRISPRi screening

doi: 10.1186/s13046-024-03027-6

Figure Lengend Snippet: TNFSF15 is an immunostimulatory factor that enhances CAR T cell cytotoxicity. A Cell surface staining of CD69 and CD25 in CAR T cells stimulated by plate-bound recombinant B7-H3-Fc protein with or without recombinant trimeric TNFSF15 protein. ( n = 3 for 0, 100 ng/mL and = 2 for 400 ng/mL). Error bars denote SEM. B Tumor killing of U87 MG cells by B7-H3-targeting CAR T cells or control T cells at an E: T ratio of 1:2 after two-day co-culture with or without exogenous addition of recombinant TNFSF15 protein ( n = 4). Error bars denote SEM. C Gene expression correlation between TNFSF15 and the T cell activation signature ( GZMB 、 GZMK 、 GZMA 、 IFNG 、 TNF 、 IL2 、 IL2R 、 CD69 and CD137 ) in human GBM samples. Data were obtained from TCGA. D Schematic of a recent study that performed single-cell RNA sequencing on mouse skin-draining lymph nodes for probing cellular response to various cytokines, including TNFSF15 (TL1A). E Violin plots showing the expression levels of genes involved in T cell activation and NF-κB pathway in CD8 + T cells following PBS or TNFSF15 treatment in vivo. F-I Gene expression correlation between TNFSF15 and NFKB2 ( F ), NFKB1 ( G ), RELB ( H ) and ICAM1 ( I ) in human GBM samples. Data were obtained from TCGA. J Schematic of our proposed model. Inhibiting ARPC4 or NDUFV1 in GBM cells upregulates TNFSF15. TNFSF15 acts as an immunostimulatory factor that activates the NF-κB pathway in CAR T cells, leading to increased production and release of proinflammatory and cytotoxic factors, thus enhancing the anti-tumor activity of CAR T cells

Article Snippet: The following antibodies used for the flow cytometry analysis were obtained from BD Biosciences: FITC-conjugated anti-CCR7 (Clone 150,503, Cat. no. 561,271), APC-Cy7-conjugated anti-CD69 (Clone FN50, Cat. no. 557,756), V450-conjugated anti-Granzyme B (Clone GB11, Cat. no. 561,155).

Techniques: Staining, Recombinant, Co-Culture Assay, Expressing, Activation Assay, RNA Sequencing Assay, In Vivo, Activity Assay

Journal: PLoS ONE

Article Title: Interferon-α Improves Phosphoantigen-Induced Vγ9Vδ2 T-Cells Interferon-γ Production during Chronic HCV Infection

doi: 10.1371/journal.pone.0037014

Figure Lengend Snippet: Representative flow cytometry panels on Vγ9Vδ2 T-cells frequency and differentiation profile are shown for one healthy donor and one HCV-infected patient. Differentiation was analyzed by monitoring CD27 and CD45RA expression. Naïve: CD45RA+CD27+; Central Memory: CD45RA-CD27+; Effector Memory: CD45RA-CD27-; Effectors: CD45RA+CD27-. ( B ) Statistical analysis of Vγ9Vδ2 T-cell differentiation profile from HD (white boxes, n = 35) and HCV (grey boxes n = 24) was performed by Mann-Whitney test. *p<0.05. ( C ) Representative flow cytometry panels on CD25 and CD69 expression on Vγ9Vδ2 T-cells are shown for one healthy donor and one HCV-infected patient. ( D ) Statistical analysis of CD25 and CD69 expression on Vγ9Vδ2 T-cells from HD (white boxes, n = 35) and HCV (grey boxes n = 24) was performed by Mann-Whitney test. *p<0.05.

Article Snippet: Specifically, Vδ2 T-cell subsets were analyzed by using the following monoclonal antibodies: anti- Vδ2 FITC (clone IMMU389), anti-CD3 PerCP-PC5 (clone UCHT-1), from Beckman Coulter (Immunotech, France); anti-CD27 APC (clone L128), anti-CD45RA CY-Chrome (clone HI100), anti-CD69 APC-Cy7 (clone FN50), anti-CD25 APC (clone M-A251) from BD Biosciences (San Jose, CA, USA).

Techniques: Flow Cytometry, Infection, Expressing, Cell Differentiation, MANN-WHITNEY