Fig 2: Increased expression of immune inhibitory checkpoints was shown in CD3+CD7− T cells in T-NHL patients with BM invasion. (A) CD3+CD7− T cells had a higher LAG-3 expression than CD3+CD7+ T cells. (B) CD3+CD7− T cells had a higher PD-1 expression than CD3+CD7+ T cells. (C) CD3+CD7− T cells had a higher ration of CD4/CD8 than CD3+CD7+ T cells. (D) CD3+CD7−CD4+ T cells had a higher PD-1 expression than CD3+CD7+CD4+ T cells. APC, allophycocyanin; BM, bone marrow; LAG-3, lymphocyte-activation-gene-3; PD-1, programmed cell death protein 1; PE, phycoerythrin; T-NHL, T-cell non-Hodgkin’s lymphoma.

Fig 3: SwR-STEAP1 CAR T cells show a higher polyfunctionality after repeated in vitro stimulation of tumor cells. ‘Parts of Whole’ graphs showing the co-expression of the activation maker CD25 with the checkpoint receptors PD1, LAG3, TIM3, and TIGIT on CD4+ (a) and CD8+ (b) JK11 and JK59 CAR T cells. Samples were collected before the start of long-term co-culture assay (day 0) and at days 6 and 13 of co-culture. T cells were co-cultured with 22Rv1 cells or with 22Rv1 target cells in the presence of rhTGFβ (22Rv1 + rhTGFβ). Co-cultures were performed at an E:T ratio of 1:1 (see also Figure 7 and Figure S8). Average expression values from the two healthy donors of the long-term co-culture assay were used. Each co-culture from each donor was performed in duplicate.

Fig 4: Analysis of STEAP1 and SwR-STEAP1 CAR T cell phenotyping after repeated in vitro stimulation of STEAP1+ tumor cells. Long-term co-culture was achieved by stimulating every 3 to 4 days the JK11, JK59, and CD19 CAR T cells and NT T cells, with irradiated 22Rv1 and 22Rv1-KO target cells at an E:T ratio of 1:1 in the absence or presence of 10 ng/mL rhTGFβ. Before the start of the co-culture (day 0), at day 6, and day 13, CAR T cell phenotyping was assessed with flow cytometry. (a,b) The CD4+ and CD8+ T cell populations co-cultured with 22Rv1 target cells (a) or with 22Rv1-KO target cells (b) were assessed for surface expression of the activation marker CD25 and the checkpoint receptors PD1, LAG3, TIM3, and TIGIT. Background staining for each marker was identified using Fluorescence Minus One controls (see Figure S7a–c for gating strategy). Data represent the mean values ± SEM from two healthy donors. Each co-culture was performed in duplicate, and these were kept separate throughout the assay.