Journal: Molecular Therapy Oncology

Article Title: Intrinsic immunosuppressive features of monocytes suppress CAR-T19 through IL-1 pathway modulation in mantle cell lymphoma

doi: 10.1016/j.omton.2025.200985

Figure Lengend Snippet: Neutralization of recombinant human IL-1ra mitigates CAR-T19 proliferation inhibition and promotes CAR-T19 antitumor activities in an MCL xenograft mouse model (A) Recombinant human (rh)IL-1ra inhibits IL-1β-dependent CAR-T19 proliferation, which could be restored by IL-1ra neutralizing antibody. CAR-T19 was cocultured with JeKo-1 cells for 3 days with the addition of IL-1β, IL-1ra, or human IL-1ra neutralizing antibody (clone 10309) as indicated. PBS was used as vehicle control. Three biological replicates were analyzed. One-way ANOVA and paired t test were used. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; data are represented as mean ± SD. (B) Confirmation of M2-like phenotype of engrafted macrophages and production of IL-1ra 7 days after tumor injections by immunofluorescence staining. Ex vivo polarized M0 macrophages and luciferase + JeKo-1 were subcutaneously engrafted in NSG mice. Tumor tissues were harvested and prepared for immunofluorescence staining. Luciferase + JeKo-1 generated in the lab expressed GFP as well (green); human CD206 or IL-1ra was stained with rabbit-anti-human monoclonal antibody followed by goat-anti-rabbit secondary antibody Cyanine5 (magenta); DAPI is presented in blue. (C) Schematic outline of in vivo experimental design. NSG mice were engrafted with ex vivo polarized M0 macrophages (Mϕ) and luciferase + JeKo-1 cells delivered with Matrigel as indicated. After tumor establishment, mice were treated with CAR-T19 intravenously in combination with IL-1ra neutralizing antibody or control IgG (C1.18.4, Bio X Cell) or UTD in combination with IL-1ra neutralizing antibody or control IgG. Tumor burden was measured by bioluminescence imaging twice per week until the survival endpoint was reached. (D and E) Tumor burden changes over time. Two-way ANOVA was used; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001; data are represented as mean ± SEM. (F) Kaplan-Meier survival curve of the treatment groups. Log rank (Mantel-Cox) test was used; ∗ p < 0.05. UTD, untransduced T cell. Median overall survival of each treatment group is listed as follows: JeKo-1+Mϕ_CAR-T19+IL-1ra mAb: not reached; JeKo-1+Mϕ_CAR-T19+IgG control: 52 days; JeKo-1_CAR-T19+IgG control: not reached; JeKo-1+Mϕ_UTD+IL-1ra mAb: 40 days; JeKo-1+Mϕ_UTD+IgG control: 43 days; JeKo-1_UTD+IL-1ra mAb: 43 days; JeKo-1_UTD+IgG control: 43 days.

Article Snippet: 100 pg/mL rhIL-1β (R&D Systems, catalog no. 201-LB-010), 100 ng/mL rhIL-1ra (R&D Systems, catalog no. 280-RA-010), or 20 μg/mL IL-1ra neutralizing monoclonal antibody (clone 10309, Invitrogen, Carlsbad, CA; catalog no. MA5-23802) were added at the beginning of the coculture according to the indicated experimental conditions.

Techniques: Neutralization, Recombinant, Inhibition, Control, Immunofluorescence, Staining, Ex Vivo, Luciferase, Generated, In Vivo, Imaging

Journal: Molecular Therapy Oncology

Article Title: Intrinsic immunosuppressive features of monocytes suppress CAR-T19 through IL-1 pathway modulation in mantle cell lymphoma

doi: 10.1016/j.omton.2025.200985

Figure Lengend Snippet: IL-1ra inhibition of IL-1β-mediated CAR-T19 proliferation is dependent on interactions with IL-1RI on stimulated CAR-T19 (A) IL-1RI expression on both CD4 and CD8 CAR-T19 cells under indicated stimulation conditions. CAR-T19 was cocultured with JeKo-1 cells for up to 3 days. Expression of IL-1RI was measured on both CD4 and CD8 T cells by flow cytometry. Three biological replicates were analyzed in total. Two-way ANOVA was used; ∗∗ p < 0.01; ns, not significant; data are represented as mean ± SD. (B) IL-1RI expression on CD4 CAR-T after being cocultured with ex vivo polarized M2-like macrophages. CAR-T19, JeKo-1, and ex vivo polarized M2-like macrophages were cocultured for 3 days as described in Figure 1 A. Expression of IL-1RI was measured on CD4 CAR-T19 daily by flow cytometry. Three biological replicates were analyzed in total. Two-way ANOVA was used; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, not significant; data are represented as mean ± SD. (C) Confirmation of IL-1RI KO CAR-T19 at the protein level. IL-1RI KO CAR-T19 and CTsgRNA CAR-T19 were cocultured with JeKo-1 for 24 h. Expression of IL-1RI on CD4 T cells was measured by flow cytometry. Paired t test was used; ∗ p < 0.05. Three biological replicates were analyzed. Data are represented as mean ± SD. The graphic was created in BioRender ( https://BioRender.com/h3w3mb7 ). (D) Functional confirmation of IL-1RI KO CAR-T19 according to its response to IL-1β stimulation. IL-1RI KO CAR-T19 was cocultured with JeKo-1 and supplemented with vehicle control, IL-1β, or IL-1β+IL-1ra for 3 days. CAR-T19 antigen-specific proliferation was measured on day 3 by flow cytometry. Three biological replicates were analyzed in total. Multiple t test was used. Data are represented as mean ± SD.

Article Snippet: 100 pg/mL rhIL-1β (R&D Systems, catalog no. 201-LB-010), 100 ng/mL rhIL-1ra (R&D Systems, catalog no. 280-RA-010), or 20 μg/mL IL-1ra neutralizing monoclonal antibody (clone 10309, Invitrogen, Carlsbad, CA; catalog no. MA5-23802) were added at the beginning of the coculture according to the indicated experimental conditions.

Techniques: Inhibition, Expressing, Flow Cytometry, Ex Vivo, Functional Assay, Control