Journal: NPJ Precision Oncology

Article Title: RevCAR-mediated T-cell response against PD-L1-expressing cells turns suppression into activation

doi: 10.1038/s41698-025-00828-6

Figure Lengend Snippet: a Target cells (5 × 10 3 ) were co-cultured with RevCAR T-cells (E:T = 5:1) in the presence or absence of PD-L1 RevTM for 24 h. The supernatant was harvested, and the secretion of pro-inflammatory cytokines was determined. Statistical significance was assessed by Student’s independent t test. P ≤ 0.05 (*), P ≤ 0.01 (**). b RevCAR T-cells were seeded in the absence of target cells, in the presence of target cells alone, or in the presence of target cells and PD-L1 RevTM for 24 h. Then, the RevCAR T-cells were stained with CD69 antibody. Statistical significance was assessed by two-way ANOVA followed by Tukey’s multiple comparisons test; P ≤ 0.0001 (****). c Co-cultures were set as described in b , but in this case, after incubation, the T-cells were stained with CD62L and CD45RO antibodies, and according to the expression levels of these markers, RevCAR T-cells were divided into four groups of increasing differentiation: Naive (T N ): CD62L high and CD45RO low ; Central memory (T CM ): high expression of both markers. Effector memory (T EM ): CD62L low and CD45RO high ; and Terminal effector (T TE ): low expression of both markers. a – c Data for three individual T-cell donors are shown as mean ± SD.

Article Snippet: After the co-culture, the T-cells of each condition were collected and incubated for 20 min at 4 °C with either anti-CD69-APC mAb (Miltenyi Biotec GmbH) in the case of activation staining or anti-CD45RO-PE mAb (Miltenyi Biotec GmbH) and anti-CD62L-Pacific BlueTM mAb (Biolegend) in the case of memory staining.

Techniques: Cell Culture, Staining, Incubation, Expressing

Journal: NPJ Precision Oncology

Article Title: RevCAR-mediated T-cell response against PD-L1-expressing cells turns suppression into activation

doi: 10.1038/s41698-025-00828-6

Figure Lengend Snippet: a Dual RevCAR T-cells express two separate receptors: the signaling (SIG) RevCAR-E7B6 triggering the CD3z signal and the costimulatory (COS) RevCAR-E5B9 triggering the CD28 co-stimulatory signal. The SIG contains intracellular CD3z signaling domain (purple), transmembrane domain (gray), hinge domain (yellow), and peptide epitope E7B6 (dark yellow). The COS comprises intracellular CD28 domain (brown), transmembrane domain (gray), hinge domain (pink), and peptide epitope E5B9 (red). Dual RevCAR T-cells are redirected to PSCA + PD-L1 + cancer cells via PSCA RevTM and PD-L1 RevTM. The simultaneous recognition of PSCA and PD-L1 by SIG and COS RevCARs via the respective RevTMs triggers both CD3z and CD28 signals resulting in complete activation of Dual RevCAR T-cells. b Comparative expression of PD-L1 and PSCA on the surface of PC-3 PSCA/PD-L1. c , d 5 × 10 3 PC-3 PSCA/PD-L1 cells were co-cultured for 24 h with Dual RevCAR T-cells (E:T = 5:1) in the absence of RevTM, in the presence of only PD-L1 or PSCA RevTM, or in the presence of both RevTMs. c Specific lysis and d cytokine secretion were determined. e , f Dual RevCAR T-cells were seeded in the absence of target cells, in the presence of target cells alone, in the presence of target cells and either PD-L1 RevTM or PSCA RevTM, or in the presence of target cells and both RevTMs, for 24 h (E:T = 5:1). Then, the T-cells were stained with e anti-CD69 antibody or f anti-CD62L and anti-CD45RO antibodies. Dual RevCAR T-cells were divided into four groups of increasing differentiation: Naive (T N ): CD62L high and CD45RO low ; Central memory (T CM ): high expression of both markers; Effector memory (T EM ): CD62L low and CD45RO high ; and Terminal effector (T TE ): low expression of both markers. c – f Data for three individual T-cell donors are shown as mean ± SD. c – e , statistical significance was assessed by ordinary one-way ANOVA ( c , e ) or two-way ANOVA ( d ) followed by Tukey’s multiple comparisons test; P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.0001 (****).

Article Snippet: After the co-culture, the T-cells of each condition were collected and incubated for 20 min at 4 °C with either anti-CD69-APC mAb (Miltenyi Biotec GmbH) in the case of activation staining or anti-CD45RO-PE mAb (Miltenyi Biotec GmbH) and anti-CD62L-Pacific BlueTM mAb (Biolegend) in the case of memory staining.

Techniques: Activation Assay, Expressing, Cell Culture, Lysis, Staining