anti cd69 apc cy7  (Miltenyi Biotec)

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: Frontiers in Immunology

Article Title: Ligand-based targeting of c-kit using engineered γδ T cells as a strategy for treating acute myeloid leukemia

doi: 10.3389/fimmu.2023.1294555

Figure Lengend Snippet: Design of novel ligand-based mSCF CAR and modification of αβ T cells. (A) Schematic of lentiviral GFP mSCF CAR DNA construct. (B) Transduction efficiency as depicted by %GFP + live Jurkat T cells. Error bars represent SD. n = 3 experimental replicates. (C) Activation of live mSCF CAR- or CD19 CAR-modified Jurkat T cells as depicted by %CD69 + when stained with 20 ng murine c-kit-Fc chimera. Error bars represent SD. n = 1-3 experimental replicates. (D) %CD69 activation of live mSCF CAR- or CD19 CAR-modified GFP + Jurkat T cells when co-cultured with 4 different leukemia cell lines at 4 different effector-to-target (E:T) ratios. Error bars represent SD. n = 2 experimental replicates. (E) %CD69 activation of live un-modified GFP- Jurkat T cells under the same conditions as (D) . Error bars represent SD. n = 2 experimental replicates. (F) c-kit MFI of 4 leukemia cell lines when co-cultured with mSCF CAR- or CD19 CAR-modified Jurkat T cells under the same conditions as (D) . Error bars represent SD. n = 2 experimental replicates. (G) Schematic of lentiviral mSCF CAR DNA construct without GFP marker. (H) Representative flow plots depicting mSCF CAR expression on primary T cells transduced at MOI 20 when stained with 20 ng of murine or human c-kit-Fc chimera. (I) Western blot depicting CAR expression from whole cell lysates of primary T cells transduced with mSCF CAR at MOI 20. Western blot antibody against human CD3ζ. (J) Four- and 24-hour flow cytometry cytotoxicity assays of mSCF CAR-modified primary T cells against c-kit expressing AML cell line CMK compared to mock T cell controls. Percent cytotoxicity is the sum of 7AAD + , Annexin V + , and 7AAD + Annexin V + cells when gated on VPD450-stained target cells only. Error bars represent SD. n = 2 experimental replicates with 1 donor.

Article Snippet: Co-cultures were then washed once with FACS buffer and stained with 3 μL anti-CD69-APC-Cy7 antibody (BD, Franklin Lakes, NJ, USA) for 20 minutes at 4°C.

Techniques: Modification, Construct, Transduction, Activation Assay, Staining, Cell Culture, Marker, Expressing, Western Blot, Flow Cytometry