Anti cd4 or anti cd8 microbeads

Ex vivo human T cells were isolated from IMR5-bearing mice spleens using Miltenyi Biotec tumor dissociation kit. Viable CAR-T cells were enriched using Ficoll. CD4⁺/CD8⁺ T cell subsets were separated using anti-CD4 or anti-CD8 microbeads (Miltenyi Biotec) and stimulated with B7-H3-positive IMR32 or IMR32 B7-H3 KO cells at a ratio of 1:2 for 20 h. Both CD4⁺ and CD8⁺ T populations were then recovered from the tumor cell-incubation systems. A single cell functional profile was determined as described previously^{32 (link),54 (link)}. Briefly, a total of 30,000 single cells were loaded onto an IsoCode chip (IsoPlexis) which contains ~12,000 microchambers. A 32-plex cytokine/chemokine antibody array was designed and embedded in each microchamber, including Effector: Granzyme B, IFN-γ, MIP-1α, Perforin, TNFα, TNFβ; Stimulatory: GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15, IL-21; Chemoattractive: CCL11, IP-10, MIP-1β, RANTES; Regulatory: IL-4, IL-10, IL-13, IL-22, sCD137, sCD40L, TGFβ1; Inflammatory: IL-1β, IL-6, IL-17A, IL-17F, MCP-1, MCP-4. The single-cell functional subsets within the different CD4⁺ CAR-T cells groups were shown in a heatmap, and the combination of multiple cytokine/chemokine proteins in each sample was quantified as the percentage of polyfunctional cells.

Peptide-specific T cells were expanded using an aAPC as described previously23 (link)24 (link). PBMCs were isolated from healthy volunteers and stimulated with 50 ng/ml anti-CD3 mAb (clone OKT3) in the presence of 100 IU/ml human IL-2 (Novartis) 3 days before transduction. Activated T cells were retrovirally transduced with TCR genes by centrifuging 1 hour at 1,000 g at 32 °C. Following transduction, CD4⁺ or CD8⁺ T cells were purified using anti-CD4 or anti-CD8 Microbeads (Miltenyi Biotec) and plated at 2 × 10⁶ cells/well in RPMI 1640 supplemented with 10% human AB serum. The stimulator aAPC was pulsed with 10 μg/ml A2-restricted wild-type MART1_27–35 for 6 hours at room temperature. The aAPC was then irradiated at 200 Gy, washed, and added to the responder T cells at a responder to stimulator ratio of 20:1. Starting the next day, 10 IU/ml IL-2 (Novartis) and 10 ng/ml IL-15 (Peprotech) were added to the cultures every 3 days. T cells were harvested, counted, and restimulated every week. T cell analysis was performed one day prior to or on the day of restimulation.

For flow cytometry assay, samples were suspended and adjusted to standard concentrations according to specifications. All fluochrome-conjugated Abs, unless specifically stated, were purchased from eBiosicience. The PE anti-human CD69, APC-anti-human FoxP3, and human Foxp3 staining buffer sets were purchased from BD (USA). For magnetic cell separation, PBMCs were separated from whole blood using Lymphoreo (Fresenius Kabi Norge AS, Norway). Then the proper volume (10 μl per 10⁶ cells) of anti-CD4 or anti-CD8 microbeads (Miltenyi, De) was added to the cell suspension and allowed to react on ice for 15 min before the cells went through a magnetic separator. The magnetic separator was washed three times to thoroughly separate CD4⁻ or CD8⁻ cells from CD4⁺ or CD8⁺ cells. The positive cells were then collected from the magnetic separator.