Journal: Frontiers in Immunology

Article Title: Blocking CD30 on CD19 CAR T cells augments their functional capacities against B-cell leukemia/lymphoma

doi: 10.3389/fimmu.2026.1725641

Figure Lengend Snippet: T cells with CD30/CD19 CAR induce superior killing of CD19 + target cells. (A) Modular composition of the chimeric antigen receptors (CARs). The anti-CD30 single-chain variable fragment (scFv) is derived from the HRS3 antibody, the anti-CD19 scFv from the FMC63 antibody, and the anti-CEA scFv from the BW431/26 antibody which was used as irrelevant specificity control. Li, (Gly 4 -Ser) 8 linker. (B) T cells were engineered by retroviral gene transfer with the respective CAR and recorded by flow cytometry for CD3 and CAR surface expression by the PE-conjugated anti-IgG1 antibody that detects the common IgG1 CH2CH3 spacer within the CAR exodomain. Representative dot plots from one out of three blood donors are shown. (C) CAR T cells (5 × 10 4 CAR + T cells/well) and non-engineered T cells (w/o CAR) were co-incubated with HEK293 cells expressing CD19, CD30, or both at a effector-to-target cell (E:T) ratio of 0.3:1 and monitored for IFN-γ spot formation after 20 h. Data represent mean values of two technical replicates + SEM. (D) CAR T cells (2 × 10 4 CAR + T cells/well) or non-modified T cells (2 × 10 4 T cells/well) were co-incubated with CD19 + CD30 - or CD19 - CD30 - HEK293 cells (2 × 10 4 cells/well) for 48 h. Cytotoxicity was determined by an XTT-based viability assay. Data represent mean values pooled from three individual T-cell donors + SEM. Student’s t test: p = 0.0395, Cohen’s D: 2.46, effect size r: 0.776 (E–G) Enhanced green fluorescent protein (eGFP)-marked Nalm6 cells (2 × 10 4 cells/well) were co-incubated with the indicated CAR T cells (2 × 10 4 cells/well) and cytolysis of Nalm6 leukemia cells was recorded by IncuCyte real-time imaging; cell numbers were normalized to the starting cell number (100%). Data represent mean values pooled from three individual T-cell donors ± SEM. Statistical analysis was performed using the Student’s t test for the 48-h timepoint: (E) p = 0.0003; (F) p = 0.0055; (G) p = 0.0055. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Article Snippet: Nalm6 (CRL-3273, ATCC) is a human acute lymphoblastic leukemia cell line.

Techniques: Derivative Assay, Control, Retroviral, Flow Cytometry, Expressing, Incubation, Modification, Viability Assay, Imaging

Journal: Frontiers in Immunology

Article Title: Blocking CD30 on CD19 CAR T cells augments their functional capacities against B-cell leukemia/lymphoma

doi: 10.3389/fimmu.2026.1725641

Figure Lengend Snippet: Distal CD30 scFv positioning is essential for enhancing CAR T cell cytolytic activity. (A) T cells engineered with the CD30/CD19 CAR and CD19/CD30 were recorded by flow cytometry using the PE-conjugated anti-IgG antibody that detects the common IgG1 CH2CH3 spacer within the exodomain. Representative dot plots from one out of three blood donors are shown. (B) T cells engineered with the indicated CAR or non-modified T cells (w/o CAR) (2 × 10 4 CAR + T cells/well) were co-incubated with CD19 + CD30 - or CD19 - CD30 - HEK293 cells (2 × 10 4 cells/well) for 48 h. Cytotoxicity was determined by an XTT-based viability assay. Data represent mean values pooled from five individual T-cell donors + SEM. Statistical analysis was performed by one-way ANOVA with Dunnett’s post-hoc test: CD19/CD30 CAR vs. CD30/19 CAR: p = 0.042; CD30/19 CAR vs. CD19 CAR: p = 0.0173. (C) EGFP + Nalm6 cells (2 × 10 4 cells) were co-incubated with CAR + or non-modified T cells (2 × 10 4 cells), and cytolysis of Nalm6 leukemia cells was recorded by IncuCyte real-time imaging. Cell numbers were normalized to the starting cell number (100%). Data represent mean values pooled from two individual T-cell donors ± SEM. Statistical analysis was performed using the Student’s t test for the 48-h timepoint: p = 0.0031. (D) Prediction of protein–protein interactions reveals favored synapse formation by the CD30/CD19 CAR. The protein folding of the extracellular moiety of the CD19 CAR, CD30/CD19 CAR, and CD19/CD30 CAR and the most likely mode of interaction with the respective cognate targets CD30 on the T cell and CD19 on the target cell were predicted using AlphaFold2. The predicted synaptic gap between T and target cells was 14–27 nm in the case of the CD30/CD19 CAR and 14–45 nm for the CD19/CD30 CAR. *p ≤ 0.05; **p ≤ 0.01.

Article Snippet: Nalm6 (CRL-3273, ATCC) is a human acute lymphoblastic leukemia cell line.

Techniques: Activity Assay, Flow Cytometry, Modification, Incubation, Viability Assay, Imaging, Protein-Protein interactions

Journal: Frontiers in Immunology

Article Title: Blocking CD30 on CD19 CAR T cells augments their functional capacities against B-cell leukemia/lymphoma

doi: 10.3389/fimmu.2026.1725641

Figure Lengend Snippet: CD30/CD19 CAR T cells downregulate CD30 on T cells without fratricide. (A) T cells engineered with the CD19 CAR or the CD30/CD19 CAR (2.5 × 10 4 cells/well each) were co-incubated together with T cells (w/o CAR) (1 × 10 5 cells/well) and stimulated with anti-CD3 (OKT3) and anti-CD28 (15E8) antibodies in the presence of IL-2 (1,000 U/mL) for 48 h. CD30 surface expression before (dark-filled histograms) and after stimulation (light-filled histograms) was recorded by flow cytometry. Representative histograms from one out of three T-cell donors are shown. The graph represents mean values of CD30 + cells pooled from three individual donors + SEM. Statistical analysis was performed by the Student’s t test: p = 0.0132. (B) Non-activated and anti-CD3/anti-CD28-activated T cells (5 × 10 4 cells/well) were stained with the membrane dye eF450 and co-incubated with CAR T cells (2 × 10 4 cells/well each) for 6 h. Annexin V-PE staining of membrane-labeled T cells was recorded by flow cytometry. Data represent the mean proportion (%) of Annexin V + cells from three T-cell donors + SEM. Statistical analysis was performed by one-way ANOVA with Dunnett’s post-hoc test. p < 0.05 was considered significant (ns, not significant). Nalm6 cells (5 × 10 4 cells/well) were co-incubated with CD30/CD19 CAR T cells (2 × 10 4 cells/well) for control demonstrating the killing capacity of the used CAR T cells. Data represent the proportion (%) of Annexin V + Nalm6 cells. Statistical analysis was performed by the Student’s t test: p = 0.0056. (C) CD30/CD19 CAR T cells were stimulated through anti-CD3 (OKT3) and anti-CD28 (15E8) antibodies in the presence of IL-2 (1,000 U/mL) for 24 h. CD30 surface expression and PI-9 intracellular expression were monitored by flow cytometry before and after activation. Representative dot plots from one out of three blood donors are shown. Graph represents PI-9 MFI mean values + SEM of sorted CAR T cells pooled from three T-cell donors. Statistical analysis was performed by the Student’s t test: p = 0.0018. *p ≤ 0.05; **p ≤ 0.01.

Article Snippet: Nalm6 (CRL-3273, ATCC) is a human acute lymphoblastic leukemia cell line.

Techniques: Incubation, Expressing, Flow Cytometry, Staining, Membrane, Labeling, Control, Activation Assay

Journal: bioRxiv

Article Title: Feeder cell – the key component in producing scalable and fit NK cells for therapeutic use

doi: 10.64898/2026.04.21.718880

Figure Lengend Snippet: NK cell cytotoxicity against K562 target cells was assessed in a 24-hour co-culture assay at varying effector-to-target (E:T) ratios. NK cells were collected for the assay at multiple timepoints: starting immediately after thawing (A), and at weeks 1 (B) and 4 (C), as well as during each sample’s the week precedin the last week of survival lon -term culture (D). (E) Responsiveness to IL-2 stimulation was evaluated in thawed NK cells using a 24-hour co-culture assay with K562 cells immediately after thawing. 500 IU/ml IL-2 was either added (+) or omitted (–) from the co-culture to assess its effect on cytotoxic activity. (F) Cytotoxicity against NALM-6 cells and (G) SH-Sy5y cells were measured one week after thawing using 24-hour co-culture assays. Each dot represents an individual donor; bars indicate mean values. Data are presented as mean ± SD from (A) n = 3, (B) n = 6, (C) n = 3, (D) n = 3, (E) n = 3, (F) n = 6 and (G) n = 6 individual donors. Data (E) points represent individual donors: donor A (circles), B (triangles) and C (squares). Statistical analysis comparisons were made against the Day 1, non-expanded NK cell condition and in (E) by comparing IL-2 added and omitted condition, using two-tailed paired t-test. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

Article Snippet: Briefly, NALM-6 cells (ATCC, cat# CRL-3273) were transduced with the IVISbrite Red F-luc-GFP lentiviral vector (RediFect, PerkinElmer) at an MOI of 10.

Techniques: Co-culture Assay, Co-Culture Assay, Activity Assay, Two Tailed Test