Interleukin 2 (il 2)

Various CAR-T cells were co-cultured with target cells at 5:1 (E:T ratio) in 96-well plates (V bottom). Supernatants were collected after 16 h and cytokine release was analyzed using TNF-α- (Neobioscience) and IL-2- (MultiSciences) specific enzyme-linked immunosorbent assay kit according to the manufacturer’s instructions.

The population analysis and sorting of Tregs, naive CD4⁺ T cells, CD8⁺ T cells, and TAMs were performed by flow cytometry using the FACSAria III flow cytometer (BD Biosciences) or NovoCyte flow cytometer (ACEA Biosciences). The antibodies used in flow cytometry assay were as follows: PE-Cy7-conjugated CD45 antibody (25-0451-82, eBioscience), FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz Biotechnology), PE-conjugated CD206 antibody (141706, Biolegend), PE-conjugated CD4 antibody (100407, Biolegend), APC/Cyanine7-conjugated CD8a antibody (100714, Biolegend), PE-Cy7-conjugated CD25 antibody (25-0251-81, Invitrogen), APC-conjugated FOXP3 antibody (17–4777-42, eBioscience), and BV650-conjugated CD45RA antibody (564360, BD Biosciences). The sorted lymphocytes were re-suspended in RPMI-1640 complete culture medium and stimulated by 50 ng/ml PMA, 1 ug/ml ionomycin, and 10 ng/ml IL-2 (Multiscience, Hangzhou, China) in vitro for 24–48 h, and then continuously cultured in RPMI-1640 complete culture medium.

The FACSAria III flow cytometer (BD Biosciences, California, US) or the NovoCyte Quanteon Flow cytometer (Agilent Technologies, California, USA) was used to analyze and sort the population of CD8⁺ T cells. The antibodies used in the flow cytometry assay were APC - conjugated CD8a antibody (100711, Biolegend, California, US) and FITC-conjugated CD3 antibody (100204, Biolegend, California, US). The sorted CD8⁺ T cells were re-suspended in RPMI-1640 complete culture medium and stimulated by 50 ng/ml phorbol 12-myristate 13-acetate (PMA), 1 ug/ml ionomycin, and 10 ng/ml IL-2 (Multiscience, Hangzhou, China) in vitro for 48 h, and then continuously cultured in RPMI-1640 complete culture medium.
To assess the effects of HTR2A or HTR2B inhibitor (ketanserin or RS-127445 hydrochloride) on the proliferation of CD8⁺ T cells, 5 (6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE) staining assay was performed by CFSE (C1031, Beyotime, Shanghai, China) for 15 min. Subsequently, cells were re-suspended with RPMI-1640 medium and seeded in the 96 well plates. After treatment with 0.1 nM ketanserin (HTR2A inhibitor, 74050-98-9, MCE) or 0.1 nM RS-127445 hydrochloride (HTR2B inhibitor,199864-86-3, MCE) for 24 h, CD8⁺ T cells were collected and analyzed by the NovoCyte Quanteon Flow cytometer.