Journal: Communications Biology

Article Title: A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism

doi: 10.1038/s42003-024-06297-0

Figure Lengend Snippet: a Experimental scheme for characterizing the effect of BBM on primary CD3+ T cells. b Fold changes in cell number and c viability determined using the standard trypan blue method. Error bars indicate SD from six donors. d Representative FACS plots illustrating the expression of CCR7 and CD45RA in CD4+ cells (top) and CD8β+ cells (bottom) on day 14. e Estimated numbers of CD4+CCR7+CD45RA+ cells (left) and CD8β+CCR7+CD45RA+ cells (right) in cultured CD3+ T cells calculated from the total cell number and frequencies of the populations that are PI- on day 14. f , g Results of the CFSE assay in CD4+ ( f ) and CD8β+ ( g ) fractions within CD3+ cells on day 3. Error bars indicate SD from six ( b , c ) or four donors ( e – g ). Statistical significance is denoted as follows: * p < 0.05; ** p < 0.01; and *** p < 0.001 (paired t -test).

Article Snippet: Cultured CD3+ T cells (as described in “Primary T cell culture”) were stained with anti-CD3-APC (3 uL/sample), anti-CD4-FITC (2 uL/sample), and anti-CD8β-PE (4 uL/sample) antibodies and sorted using a SONY MA900 cell sorter at 9 d after stimulation.

Techniques: Expressing, Cell Culture, CFSE Assay

Journal: Communications Biology

Article Title: A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism

doi: 10.1038/s42003-024-06297-0

Figure Lengend Snippet: a Experimental scheme designed to assess the in vivo persistence of BBM-treated primary CD8+ T cells. b Gating strategy employed to determine the expression of CCR7 and CD45RA in the CD45+CD8β+ fractions. c Frequencies of injected CD45+CD8β+ cells in the spleen (left), peripheral blood (center), and bone marrow (right) on day 14. Each data point represents one injected mouse and its corresponding donors: circle; donor A, triangle; donor B, square; donor C. All error bars indicate SD. Statistical significance is denoted as follows: * p < 0.05; ** p < 0.01; and *** p < 0.001 (unpaired t -test). d Representative flow cytometry plots of CCR7 and CD45RA in CD45+CD8β+ cells in the spleen samples.

Article Snippet: Cultured CD3+ T cells (as described in “Primary T cell culture”) were stained with anti-CD3-APC (3 uL/sample), anti-CD4-FITC (2 uL/sample), and anti-CD8β-PE (4 uL/sample) antibodies and sorted using a SONY MA900 cell sorter at 9 d after stimulation.

Techniques: In Vivo, Expressing, Injection, Flow Cytometry

Journal: Communications Biology

Article Title: A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism

doi: 10.1038/s42003-024-06297-0

Figure Lengend Snippet: a Gating strategy for FACS to purify the naïve and memory T cell fractions from healthy donor-derived CD8+ cells. b , c Gene ontology analysis of DEGs in BBM-treated T SCM cells. Upregulated ( b ) or downregulated ( c ) genes from RNA-sequencing results on day 12. Genes were queried in the DAVID functional annotation database. d Transcripts per million (TPM) values of cell cycle-related and effector function-related genes. Statistical significance is denoted as follows: * p < 0.05; ** p < 0.01; and *** p < 0.001 ( n = 4, paired t -test). T SCM stem cell memory T, T CM central memory T, T EM effector memory T.

Article Snippet: Cultured CD3+ T cells (as described in “Primary T cell culture”) were stained with anti-CD3-APC (3 uL/sample), anti-CD4-FITC (2 uL/sample), and anti-CD8β-PE (4 uL/sample) antibodies and sorted using a SONY MA900 cell sorter at 9 d after stimulation.

Techniques: Derivative Assay, RNA Sequencing, Functional Assay

Journal: Communications Biology

Article Title: A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism

doi: 10.1038/s42003-024-06297-0

Figure Lengend Snippet: a Metabolomics of BBM-treated T SCM cells from healthy donor-derived CD8+ cells. Lipids commonly upregulated or downregulated in all three donors are shown. b Representative flow cytometry plots of Phosflow assay in BBM-treated CD3+ cells on day 10. Phosphorylated proteins in CD8+ T cells are shown. c Mean fluorescence intensity (MFI) of phosphorylated p38 in CD8+ T cells. Error bars indicate SD from four donors. d Oxygen consumption rate (OCR) across time for BBM-treated CD3+ T cells. e Spare respiratory capacity (SRC) calculated by OCRmax-OCRbasal. f Extracellular acidification rates (ECAR) across time for BBM-treated CD3+ T cells. Error bars indicate the standard error of the mean from six donors ( d – f ). Statistical significance is denoted as follows: * p < 0.05; ** p < 0.01; and *** p < 0.001 (paired t -test) ( c , e ).

Article Snippet: Cultured CD3+ T cells (as described in “Primary T cell culture”) were stained with anti-CD3-APC (3 uL/sample), anti-CD4-FITC (2 uL/sample), and anti-CD8β-PE (4 uL/sample) antibodies and sorted using a SONY MA900 cell sorter at 9 d after stimulation.

Techniques: Derivative Assay, Flow Cytometry, Fluorescence