Journal: STAR Protocols

Article Title: Protocol for the preparation and analysis of patient-derived organotypic tumor spheroids

doi: 10.1016/j.xpro.2025.104286

Figure Lengend Snippet: PDOTS surface marker staining (A) Scheme showing the steps for surface marker staining of PDOTS “on chip”. (B) Surface markers staining of melanoma PDOTS. The left image is an overlay of the indicated markers. White arrows denote CD38 + CD8 + T cells inside a tumor spheroid.

Article Snippet: CD8 MicroBeads , Miltenyi , Cat#130-045-201.

Techniques: Marker, Staining

Journal: Journal of Clinical Apheresis

Article Title: Suppressive CD8 + T‐Cells Are Key Cellular Mediators of Extracorporeal Photopheresis

doi: 10.1002/jca.70094

Figure Lengend Snippet: The protective effect of ECP is dependent on CD8+ T cells. For all experiments 6–8 week old female mice of the indicated strain were used. EAE was induced with the indicated peptide on Day 0 and mice were monitored daily for signs of disease. (A) ECP was performed using spleen + LN cells obtained from PLP 139‐151 immunized mice on Day 12 post immunization. 10 million cells were transferred to recipient mice on Day 0. Control mice received no cells. N = 17 mice/group. (B) ECP was performed using spleen + LN cells obtained from PLP 178‐191 immunized mice on Day 12 post immunization. 10 million cells were transferred to recipient CD8 −/− mice on Day 0. Control mice received no cells. N = 11 mice/group. (C, D) ECP was performed using spleen + LN cells obtained from PLP 178‐191 immunized mice on Day 12 post immunization. 10 million cells were transferred to recipient mice on Day 0. Control mice received no cells. On Day 14 of EAE, mice were euthanized, spleens and LNs were harvested, and cells were incubated with IL‐2 and PLP 178‐191 for 72 h. CD8+ T‐cells were isolated, and five million cells were transferred to recipients at the time of EAE induction with PLP 178‐191 . Control mice received no cells (control), the remainder of the mice received CD8+ T cells obtained from ECP treated (cells from ECP‐treated) or untreated (cells from control) mice. N = 6 mice/group. Statistics were performed using Student's t ‐test with *** indicating p < 0.001.

Article Snippet: First, bulk CD8+ T cells were isolated by positive selection using the CD8+ T Cell Isolation Kit (Miltenyi Biotec, 130‐096‐495).

Techniques: Control, Incubation, Isolation

Journal: Journal of Clinical Apheresis

Article Title: Suppressive CD8 + T‐Cells Are Key Cellular Mediators of Extracorporeal Photopheresis

doi: 10.1002/jca.70094

Figure Lengend Snippet: Suppressive CD8+ T cells are increased in patients undergoing ECP. For all experiments PBMCs were isolated from whole blood obtained from healthy donors or patients undergoing ECP. (A) Flow cytometry was performed, and CD8+ suppressor cells were identified as indicated. (B–E) CD4+ CD25− cells were isolated from PBMCs, stained with CFSE, and incubated with CD8+ cells at the indicated ratios for 5–7 days. Flow cytometry was performed to identify CFSE dilute CD4+ cells (divided) and the % suppression was calculated by comparing the divided populations to CD4+ cells that were incubated in the absence of CD8+ cells (more details in the materials and methods). (B) CD8‐suppression assays in healthy donors versus ECP patients. (C) CD8‐suppression assays in paired samples obtained prior to the start of ECP (Baseline) or after 1 month of therapy. (D, E) Allo‐suppression assays. CD4+ CD25− cells were isolated from healthy donors or ECP patients. Each donor was paired with an ECP patient to allow for direct comparison between autologous and allogenic suppression results. (D) CD4+ CD25− cells from the healthy donors were incubated with CD8+ cells from the same healthy donor (auto) or a paired ECP patient (allo). (E) CD4+ CD25− cells from the ECP patients were incubated with CD8+ cells from a paired healthy donor (allo) or the same ECP patient (auto). Suppression of cell proliferation was evaluated as described above. Statistics were performed using Student's t ‐test with * indicating p < 0.05, ** indicating p < 0.01, and *** indicating p < 0.001.

Article Snippet: First, bulk CD8+ T cells were isolated by positive selection using the CD8+ T Cell Isolation Kit (Miltenyi Biotec, 130‐096‐495).

Techniques: Isolation, Flow Cytometry, Staining, Incubation, Comparison

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