cd4 t cell isolation kit  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

doi: 10.64898/2026.01.28.701378

Figure Lengend Snippet: A. Expression of CAR, CD4, and CD8 in WT and 218F CAR-T cells 7-10 days after transduction. Each point represents an individual healthy donor (n = 11). Statistical significance was determined by one-way ANOVA. B. The lysis of HPAC WT cells when treated with WT or 218F CAR-T cells was evaluated using a real-time cytotoxicity assay (xCELLigence). Left Panel: Representative graph showing % of cytolysis over a 72 hour period after the addition of the effector cells. Right Panel: % of cytolysis of WT and 218F CAR-T cells at 12 hours after the addition of the effector cells. Significance was determined by paired t-test. Each symbol represents an independent experiment using one of five different healthy donors. C. Proliferation capacity of WT vs 218F CAR-T cells, analyzed by CellTrace Violet dilution. WT and 218F CAR-T cells were co-cultured at a 2:1 E:T ratio and incubated at 37°C for 4 days. Percentage of Divided cells (left) and percentage of cells in the last division (right) of 218F CAR-T cells is shown normalized to WT CAR-T cells. Significance was determined by t-test with Welch’s correction. * = P<0.05. Each symbol represents an independent experiment from 4 different healthy donors. Data is represented as the mean ± standard deviation (SD). D. CD4 and CD8 CAR-T cells were sorted using magnetic MicroBeads. WT and 218F CAR-T cells were cocultured with HPAC WT cells at different CD4:CD8 ratios. Supernatant was collected after 24 hours and cytokine production was measured by ELISA. Left Panel. Representative graph showing the IL-2 production at different CD4:CD8 ratio. Dotted lines indicate IL-2 production by unsorted CAR-T cells. Center and left panel show production of IL-2 production of 218F CAR-T cells with respect to WT CAR-T cells. Significance was determined by t-test with Welch’s correction. ** = P<0.01. Each symbol represents an independent experiment from 3 different healthy donors.

Article Snippet: To independently assess the contribution of CD4+ and CD8+ cells, these two populations were isolated using human CD4 MicroBeads (Miltenyi, 130-045-101) and human CD8 MicroBeads (Miltenyi, 130-045-201) according to the manufacturer’s instructions.

Techniques: Expressing, Transduction, Lysis, Cytotoxicity Assay, Cell Culture, Incubation, Standard Deviation, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: C-terminus CD28 phosphorylation (Y218) modulates IL-2 secretion and antitumor effect of CAR-T cells

doi: 10.64898/2026.01.28.701378

Figure Lengend Snippet: A-B . PSCA WT and 218F CAR-T cells were cocultured with HPAC WT cells for 0, 1, 10, 30 or, 60 min. A. After protein extraction, CAR-associated proteins were co-immunoprecipitated using protein-L coated beads. PLCγ abundance in the immunoprecipitate was analyzed by Western blot. Left panel: Representative Western blot. Right panel: Normalized CAR-bound PLCγ intensity quantified by densitometry (representative plot), and CAR-bound PLCγ 60 min post-stimulation, showing 3 biological replicates corresponding to different donors, analyzed in independent experiments. Densitometry values were normalized to total CAR (CD3ζ) B. SLP76 pY376 was analyzed using phospho-specific antibodies. Left panel: Representative Western blot of SLP76 pY376 in CAR-T cells after stimulation with HPAC WT cells. Right panel: Normalized SLP76 pY376 intensity quantified by densitometry (representative plot) and SLP76 pY376 at 60 min post-stimulation. Each symbol represents an independent experiment from 3 different healthy donors. Untransduced cells (UT) were included as a negative control of immunoprecipitation. Densitometry results were normalized relative to total SLP76. Significance was determined by paired t-test. * = P<0.05. C and E . Transcriptomic profile of WT vs 218F CAR-T cells. CAR-T cells were cocultured with HPAC WT cells for 24 hours and subsequently sorted into CD4⁺ and CD8⁺ subsets using the MACSQuant® Tyto® system prior to RNA extraction. C. Volcano plot representing the genes that are differentially expressed in 218F CAR-T cells vs WT CAR-T. CD4+ cell is the left, CD8+ cell in the right. D. CAR-T cells were cocultured with HPAC WT target cells and supernatant was collected 24hours hours after. IL-17A production was analyzed by ELLA. Cytokine production of 218F and UT was normalized to WT CAR. Significance was determined by t-test with Welch’s correction. * = P<0.05. Each symbol represents an independent experiment from 3 different healthy donors. Data is represented as the mean ± standard deviation (SD). E. Gene expression of cytokine-related genes associated with Th17 cells. Bar plot shows log₂ fold change in gene expression of 218F CAR-T cells relative to WT CAR-T cells. CD4⁺ cells are shown on the left and CD8⁺ cells on the right.

Article Snippet: To independently assess the contribution of CD4+ and CD8+ cells, these two populations were isolated using human CD4 MicroBeads (Miltenyi, 130-045-101) and human CD8 MicroBeads (Miltenyi, 130-045-201) according to the manufacturer’s instructions.

Techniques: Protein Extraction, Immunoprecipitation, Western Blot, Negative Control, RNA Extraction, Standard Deviation, Gene Expression