The impact of CXCL13 and IL-9 expression on CAR T cell functionality

CXCL13 expression is highly elevated in T cells from patients who respond to PD-1 blockade, suggesting that CXCL13 may play a role in boosting T cell antitumor responses. Compared to conventional Th1/Tc1 cells, Th9/Tc9 cells exhibit stronger antitumor functions, and IL-9 plays a critical role in mediating their antitumor efficacy. Therefore, we investigate the respective effects of CXCL13 and IL-9 on CAR T cells. CD3+ T cells were extracted from C57BL/6J mouse splenocytes and cultured on plates coated with anti-CD3 (5 mg/mL) in the presence of soluble anti-CD28 (1 mg/mL) and IL-2 (100 U/mL) to induce differentiation. CD3+ T cells were then cultured with different viruses (control CAR, CXCL13 CAR, IL-9 CAR) and polybrene (10 µg/mL), then incubated for 24 hours. After three days, CAR T cells were carefully removed from the plates, centrifuged to remove the supernatant, and grown in fresh media with IL-2 (100 U/mL). RNAex Pro RNA Reagent was used to obtain total RNA of CAR T cells on day 5 for RNAseq.