Journal: Molecular Therapy Oncology

Article Title: Antigen-binding affinity is a key determinant of the durable antitumor activity of CD5 CAR-T cells

doi: 10.1016/j.omton.2026.201158

Figure Lengend Snippet: Low-affinity CD5 CAR-T cells exhibit reduced fratricide and exhaustion phenotypes (A) Viability of CAR-T cells measured on day 3 after cell seeding. Data are presented as mean ± SD from multiple donors ( n = 3). (B) Culture supernatants were collected on day 3 after cell seeding. IFN-γ and TNF-α levels secreted by CAR -T cells were measured by cytometric bead array. Data are presented as mean ± SD from multiple donors ( n = 3). (C) CD69 expression level of CAR-T cells on day 10 after seeding. Fluorescence intensity was quantified by flow cytometry. Data are presented as mean ± SD from multiple donors ( n = 3). (D) Total T cell number harvested on day 10 after cell seeding. Data are presented as mean ± SD from multiple donors ( n = 3). (E) Expression levels of PD-1 and LAG-3 (surface markers), and TOX (intracellular marker) in CAR-T cells on day 10 after seeding. Surface markers were analyzed by flow cytometry, and TOX expression was assessed via intracellular staining following fixation and permeabilization. Data are presented as mean ± SD from multiple donors ( n = 3). All statistical significance was assessed using a linear mixed-effects model with donor as a random effect and Tukey’s test.

Article Snippet: The following antibodies were used: BioLegend, CD5-PE (clone UCHT2), CD69-PE (clone FN50), LAG3-FITC (Clone 7H2C65), mIgG2a-AF647 (clone RMG2a-62), and streptavidin-AF647; BD Biosciences, CD4-V500 (clone RPA-T4), CD8-V450 (clone RPA-T8), αβ TCR-PE (clone IP26), and γδ TCR-BV421 (clone 11F2); Thermo Fisher Scientific, PD-1-PE (clone J105) and CD27-PE (clone O323); Miltenyi Biotec, TOX-APC (clone REA473), Vδ1-PE-Cy7 (clone REA173), and Vδ2-V500 (clone 123R3); Cell Signaling Technology, Myc-AF647 or PE (clone 9B11).

Techniques: Expressing, Fluorescence, Flow Cytometry, Marker, Staining

Journal: Molecular Therapy Oncology

Article Title: Antigen-binding affinity is a key determinant of the durable antitumor activity of CD5 CAR-T cells

doi: 10.1016/j.omton.2026.201158

Figure Lengend Snippet: Affinity-tuned A2 variants provide functional evidence that antigen-binding affinity plays a key role in regulating fratricide and T cell exhaustion (A) Binding level of recombinant anti-CD5 scFvs to CD5-positive and CD5-negative cell lines, as measured by flow cytometry. Data are presented as mean ± SD from technical replicates ( n = 3). (B) Affinity properties of anti-CD5 scFvs as determined by biolayer interferometry using Octet system. A2 scFv was used as controls. (For definitions of K D , K a , K dis , and curve fit parameters, refer to legend.) (C) Viability of CAR-T cells measured on day 3 after cell seeding. Data are presented as mean ± SD from multiple donors ( n = 3). (D) Culture supernatants were collected on day 3 to assess IFN-γ and TNF-α secretion, measured using cytometric bead array. Data are presented as mean ± SD from technical replicates ( n = 3). (E) Total T cell number harvested on day 10 after cell seeding. Data are presented as mean ± SD from multiple donors ( n = 3). (F) CD69 expression levels in CAR-T cells on day 10 after cell seeding, measured by surface staining and flow cytometry. Data are presented as mean ± SD from multiple donors ( n = 3). (G) Expression levels of PD-1 and LAG-3 (surface markers) and TOX (intracellular) in CAR-T cells on day 10 after seeding. Surface markers were analyzed by flow cytometry, and TOX expression was assessed via intracellular staining. Data are presented as mean ± SD from multiple donors ( n = 3). (H) 3 × 10 5 Myc + CAR-T cells were co-cultured with 1 × 10 5 Jurkat cells every 3–4 days for repeated antigen stimulation. Expansion of Myc + CAR-T cells was measured weekly by flow cytometry. Data are presented as mean ± SD from technical replicates ( n = 3). Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparisons test for (A, D, and H) and a linear mixed-effects model with donor as a random effect followed by Tukey’s test for (C–G).

Article Snippet: The following antibodies were used: BioLegend, CD5-PE (clone UCHT2), CD69-PE (clone FN50), LAG3-FITC (Clone 7H2C65), mIgG2a-AF647 (clone RMG2a-62), and streptavidin-AF647; BD Biosciences, CD4-V500 (clone RPA-T4), CD8-V450 (clone RPA-T8), αβ TCR-PE (clone IP26), and γδ TCR-BV421 (clone 11F2); Thermo Fisher Scientific, PD-1-PE (clone J105) and CD27-PE (clone O323); Miltenyi Biotec, TOX-APC (clone REA473), Vδ1-PE-Cy7 (clone REA173), and Vδ2-V500 (clone 123R3); Cell Signaling Technology, Myc-AF647 or PE (clone 9B11).

Techniques: Functional Assay, Binding Assay, Recombinant, Flow Cytometry, Expressing, Staining, Cell Culture

Journal: Molecular Therapy Oncology

Article Title: Antigen-binding affinity is a key determinant of the durable antitumor activity of CD5 CAR-T cells

doi: 10.1016/j.omton.2026.201158

Figure Lengend Snippet: High-affinity C7 variant-based CAR5 exhibited increased levels of both fratricide and T cell exhaustion compared to the C7 wild-type CAR5 (A) Binding level recombinant anti-CD5 scFvs to CD5-positive and CD5-negative cell lines, as measured by flow cytometry. Data are presented as pooled mean ± SD from technical replicates ( n = 3). (B) Affinity properties of anti-CD5 scFvs as determined by biolayer interferometry using Octet system. C7 scFv was used as controls. (For definitions of K D , K a , K dis , and curve fit parameters, refer to legend.) (C) Viability of CAR-T cells measured on day 3 after cell seeding. Data are presented as mean ± SD from multiple donors ( n = 3). (D) Culture supernatants were collected on day 3 to assess IFN-γ and TNF-α secretion, measured using cytometric bead array. Data are presented as mean ± SD from technical replicates ( n = 3). (E) Total T cell number harvested on day 10 after cell seeding. Data are presented as mean ± SD from multiple donors ( n = 3). (F) CD69 expression levels in CAR-T cells on day 10 after cell seeding, measured by surface staining and flow cytometry. Data are presented as mean ± SD from multiple donors ( n = 3). (G) Expression levels of PD-1 and LAG-3 (surface markers) and TOX (intracellular) in CAR-T cells on day 10 after seeding. Surface markers were analyzed by flow cytometry, and TOX expression was assessed via intracellular staining. Data are presented as mean ± SD from multiple donors ( n = 3). (H) 3 × 10 5 Myc + CAR-T cells were co-cultured with 1 × 10 5 Jurkat cells every 3–4 days for repeated antigen stimulation. Expansion of Myc + CAR-T cells was measured weekly by flow cytometry. Data are presented as mean ± SD from technical replicates ( n = 3). Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparisons test for (A, D, and H) and a linear mixed-effects model with donor as a random effect followed by Tukey’s test for (C–G).

Article Snippet: The following antibodies were used: BioLegend, CD5-PE (clone UCHT2), CD69-PE (clone FN50), LAG3-FITC (Clone 7H2C65), mIgG2a-AF647 (clone RMG2a-62), and streptavidin-AF647; BD Biosciences, CD4-V500 (clone RPA-T4), CD8-V450 (clone RPA-T8), αβ TCR-PE (clone IP26), and γδ TCR-BV421 (clone 11F2); Thermo Fisher Scientific, PD-1-PE (clone J105) and CD27-PE (clone O323); Miltenyi Biotec, TOX-APC (clone REA473), Vδ1-PE-Cy7 (clone REA173), and Vδ2-V500 (clone 123R3); Cell Signaling Technology, Myc-AF647 or PE (clone 9B11).

Techniques: Variant Assay, Binding Assay, Recombinant, Flow Cytometry, Expressing, Staining, Cell Culture

Journal: Cell Research

Article Title: Dynamic magneto-mechanical force in lysosomes induces durable macrophage repolarization for antitumor immunity

doi: 10.1038/s41422-025-01217-1

Figure Lengend Snippet: a RAW 264.7 cells were polarized into M0, M1 (induced by IFN-γ and LPS), or M2 (induced by IL-4 and IL-13) macrophages. M0 macrophages were incubated with MNMs and then treated with 1 Hz RMF for 15 min. Macrophages were stained with ActinRed (red), and nuclei were stained with Hoechst (blue). b Polarized M0 RAW 264.7 cells were incubated with MNMs and then treated with 1 Hz RMF for 15 min. Flow cytometry analysis of CD86 and CD206 expression was performed. c , d RNA-sequencing analyses of polarized M2 RAW 264.7 cells treated with or without 1 Hz RMF for 15 min. KEGG enrichment analysis and heat map analysis were performed ( n = 3 independent biological replicates). e , f RAW 264.7 cells or BMDMs were polarized into M0, M1, or M2 macrophages. M2 macrophages were incubated with MNMs and then treated with or without 1 Hz RMF for 15 min (M2-MNM represents M2 macrophages incubated with MNMs, and M2-RMF represents M2 macrophages incubated with MNMs and treated with RMF). Concentrations of M1-associated proteins (IL-1β) and M2-associated proteins (IL-10) were examined in the conditioned medium of these cells. Data are presented as means ± SD. Statistical significance was defined as P < 0.05 ( n = 3 independent biological replicates). g RAW 264.7 cells were polarized into M0, M1, or M2 macrophages. M0 macrophages were cultured in conditioned medium derived from LLC and incubated with or without MNMs, then treated with or without 1 Hz RMF for 15 min. mRNA levels of M1-associated genes ( Cd80 , Cd86 , Il1b , Il6 ) and M2-associated genes ( Cd163 , Cd206 , Il4 , Il10 ) were examined. Data are presented as means ± SD ( n = 4 independent biological replicates).

Article Snippet: To explore the proportions of different macrophage subtypes in tumor tissues after MagLMP treatment, macrophages in the sections were stained by immunofluorescence with anti-mouse CD206 (R&D Systems, AF2535), anti-mouse CD80 (Proteintech, 14292-1-AP), anti-mouse F4/80 (R&D Systems, MAB5580), and DAPI (Beyotime Biotech, C1002).

Techniques: Incubation, Staining, Flow Cytometry, Expressing, RNA Sequencing, Cell Culture, Derivative Assay