Fig. 3: Tumour-restricted o9R signalling improves the potency of CAR T cells.
From: Potentiating adoptive cell therapy using synthetic IL-9 receptors
a, Top, schematic of an adenoviral vector encoding oIL-2 (Ad-oIL-2) under the cytomegalovirus (CMV) promoter. LITR, left inverted terminal repeat; RITR, right inverted terminal repeat. Bottom left, in vitro expression of oIL-2 through Ad-oIL-2 in cell culture supernatants. mIL-2, mouse IL-2. Bottom right, quantification of oIL-2 in tumour homogenates and sera 72 h after intratumoral (IT) injection of 109 viral particles (VP) of Ad-oIL-2, or daily intraperitoneal (IP) injection of 2.5 × 104 units MSA-oIL-2 (n = 5 mice per group). b, Representative western blot analysis of pSTAT1, pSTAT3 and pSTAT5 expression in T cells 30 min after stimulation with MSA-IL-2 (100 nM) or MSA-oIL-2 (5 μM). For gel source data, see Supplementary Fig. 1. c, In vitro T cell killing of mesothelin-positive PDA7940b (2:1 E:T ratio) pre-incubated with MSA-oIL-2 (5 μM) (mean ± s.d., n = 4 per group). d–f, Representative surface expression of CD44 and CD62L (d), expression of Fas (CD95) (e), and secreted cytokines (f) in CAR-o2R or CAR-o9R T cell cultures after four days of stimulation with MSA-IL-2 (100 nM) or MSA-oIL-2 (5 μM). ****P < 0.0001 (ANOVA). g, Heat maps of genes associated with T cell stemness and dysfunction (left) and activation and effector function (right), and differentially expressed between o2R and o9R CAR T cells treated with MSA-oIL-2. h, Schematic of the syngeneic ACT model using PDA7940b tumours (created with Biorender.com). Ad-oIL-2 dose, 109 VP. CAR T cell dose, 5 × 106 cells. Cyclophosphamide (CTX) dose, 120 mg kg−1. IV, intravenous; precond., preconditioning; SC, subcutaneous. i,j, Individual growth curves of PDA7940b tumours (n = 12 mice per group), with (i) and without (j) conditioning CTX. Black lines indicate deaths due to ICANS. n = 12 mice per group. CR, complete response; Tox, deaths due to neurotoxicity. NS, not significant; ****P < 0.0001 (ANOVA). k, Quantification of tumour-infiltrating CAR T cells (top) and frequency of IFNγ-positive tumour-infiltrating CAR T cells (bottom) on day 9 in mice treated with CTX. *P < 0.05, ****P < 0.0001 (ANOVA). Data are mean ± s.e.m. with n = 3 biological replicates, unless stated otherwise.
