Journal: Immunotherapy Advances

Article Title: Targeting intratumoral regulatory T cells by CD137 aptamer-shRNA chimeras

doi: 10.1093/immadv/ltaf037

Figure Lengend Snippet: Selective, dose-dependent binding and uptake of murine CD137 aptamer by CD137-expressing cells. A total of 2 × 10 5 A20 or A20-CD137 cells prefixed with 1% paraformaldehyde were treated with fluorescein maleimide (FAM)-labeled murine CD137 aptamer at different concentrations (50, 100, and 500 nM). (a) The size of murine aptamer (222 nt) and the successful labeling of the aptamer with fluorescein maleimide (FAM) were detected using agarose gel electrophoresis. (b) CD137 expression on A20 and A20-CD137 cells. (c) Histograms showing the binding of CD137 aptamer to A20 (upper panel) or A20-CD137 (bottom panel) cells when given at different concentrations. (d) Comparisons of CD137 aptamer binding across different concentrations in A20 or A20-CD137 cells. (e) Comparisons of the % population between A20 and A20-CD137 cells that had bound to the CD137 aptamer at a given concentration. A total of 5 × 10 5 CD137-expressing (A20-CD137 or B16-CD137) or control (A20 or B16) cell lines were incubated with murine CD137 aptamer (0.5, 1, or 2 μg) for 2 h at 37°C. Comparisons between the amount of CD137 aptamer internalized by (f) A20 and A20-CD137 or (g) B16 and B16-CD137 cells. Comparisons of CD137 aptamer uptake when given at different amounts for (h) A20-CD137 and (i) B16-CD137 cells. The extent of CD137 aptamer internalization was expressed as fold change relative to control cells treated with 0.5 μg aptamer, or as absolute copy number per 1 μg total RNA. Dashed lines at a ratio of 1 represent the normalization reference. Data are shown as means ± SEM. Numbers above the brackets indicate P -values. * P < .05, ** P < .01, and *** P < .001 using unpaired Students’ t -test for (e), (f), and (g), while using one-way ANOVA with Bonferroni’s multiple comparison test for (d), (h), and (i).

Article Snippet: Murine B lymphoma cell line A20, melanoma cell line B16, and hepatoma cell line Hepa1-6 were purchased from American Type Cell Culture (ATCC; Manassas, VA, USA).

Techniques: Binding Assay, Expressing, Labeling, Agarose Gel Electrophoresis, Concentration Assay, Control, Incubation, Comparison

Journal: Immunotherapy Advances

Article Title: Targeting intratumoral regulatory T cells by CD137 aptamer-shRNA chimeras

doi: 10.1093/immadv/ltaf037

Figure Lengend Snippet: Murine CD137 aptamer blocks trogocytosis induced by intercellular CD137-CD137L interaction. CD137L-expressing RAW264.7 were incubated with A20 or A20-CD137 cells at a 1:1 ratio for 1 h at 37°C. During this process, anti-CD137 blocking antibody (clone: 17B5) or CD137 aptamer was added. Changes in expressions of CD137 and CD137L on (a) RAW264.7 and A20 cells or (b) RAW264.7 and A20-CD137 cells during coculture. The number in each histogram plot represents the percentage of positive cells. Each symbol represents an independent experiment. Data are presented as means ± SEM. *** P < .001 using one-way ANOVA with Bonferroni’s multiple comparison test. MFI, mean fluorescence intensity.

Article Snippet: Murine B lymphoma cell line A20, melanoma cell line B16, and hepatoma cell line Hepa1-6 were purchased from American Type Cell Culture (ATCC; Manassas, VA, USA).

Techniques: Expressing, Incubation, Blocking Assay, Comparison, Fluorescence

Journal: Frontiers in Immunology

Article Title: The tropomyosin 3.1/3.2 inhibitor ATM-3507 alters B-cell actin dynamics and impairs the growth and motility of diffuse large B-cell lymphoma cell lines

doi: 10.3389/fimmu.2025.1668379

Figure Lengend Snippet: ATM-3507 treatment alters BCR-induced actin remodeling and impairs B cell spreading. (A) A20 cells were pre-treated with either 3 µM ATM-3507, an equivalent volume of DMSO (0.03% final concentration), or 50 µM snBB for 1 h at 37°C. The cells were added to coverslips coated with 2.4 μg/cm 2 anti-IgG and allowed to spread for 30 min before being stained with rhodamine-phalloidin to visualize F-actin. For each condition, more than 105 cells from 3 independent experiments were imaged by confocal microscopy. Representative confocal microscopy images are shown (top panel; scale bar is 10 µm). Cell areas (middle panel) and cell circularity (lower panel) were quantified from the confocal microscopy images using ImageJ. The graphs on the left show one of three independent experiments (biological replicates) with similar results. Each dot represents one cell, and the medians and interquartile ranges are shown for >35 cells per condition. p-values were calculated using the Mann–Whitney U test. The graphs on the right show combined data from the 3 independent experiments. Each symbol is an individual experiment, and the data are presented as the mean ± SEM for the median values from the 3 experiments. p-values were calculated using two-tailed paired t -tests. (B) A20 cells were pre-treated with either 3 µM ATM-3507 or an equivalent volume of DMSO (0.03% final concentration) for 1 h at 37°C. The cells were added to coverslips coated with 2.4 μg/cm 2 anti-IgG and allowed to spread for 30 min before being stained with rhodamine-phalloidin. For each condition, 15–20 cells were imaged by STED super-resolution microscopy. Representative images are shown (Scale bar is 5 µm). (C) LPS-activated primary murine B cells were pre-treated with either 3 µM ATM-3507 or 0.03% DMSO for 1 h at 37°C before being added to coverslips coated with 2.4 μg/cm 2 anti-IgM for 30 min and then stained with rhodamine-phalloidin. For each condition, more than 105 cells from 3 independent experiments were imaged by confocal microscopy. Representative confocal microscopy images are shown (top panel; scale bar is 10 µm). Cell areas (middle panel) and cell circularity (lower panel) were quantified from the confocal microscopy images, and the data are presented as in panel (A) .

Article Snippet: The A20 IgG + murine B-lymphoma cell line (ATCC #TIB-208; https://www.cellosaurus.org/CVCL_1940 ) and the B16-F1 murine melanoma cell line (ATCC #CRL-6323; https://www.cellosaurus.org/CVCL_0158 ) were obtained from ATCC (Manassas, VA, USA).

Techniques: Concentration Assay, Staining, Confocal Microscopy, MANN-WHITNEY, Two Tailed Test, Super-Resolution Microscopy

Journal: Frontiers in Immunology

Article Title: The tropomyosin 3.1/3.2 inhibitor ATM-3507 alters B-cell actin dynamics and impairs the growth and motility of diffuse large B-cell lymphoma cell lines

doi: 10.3389/fimmu.2025.1668379

Figure Lengend Snippet: Tpm3 co-localizes with the peripheral F-actin ring during B cell spreading. A20 cells were pre-treated for 1 h with 0.03% DMSO or 3 µM ATM-3507 and then allowed to spread for 30 min on coverslips that had been coated with 2.4 µg/cm 2 anti-IgG. Cells were stained for F-actin (rhodamine-phalloidin) and Tpm3 and then imaged by confocal microscopy. (A) Representative images from one of 3 independent experiments. Scale bar: 10 µm. (B) The Manders’ coefficient represents the fraction of Tpm3 staining that co-localized with F-actin staining. The super-plot displays combined data for >25 cells per condition from two independent experiments. Each dot is one cell, and the different colors represent the two experiments. The large symbols represent the median values for each experiment. p-values for DMSO versus ATM-3507-treated cells were calculated using the Mann–Whitney U test.

Article Snippet: The A20 IgG + murine B-lymphoma cell line (ATCC #TIB-208; https://www.cellosaurus.org/CVCL_1940 ) and the B16-F1 murine melanoma cell line (ATCC #CRL-6323; https://www.cellosaurus.org/CVCL_0158 ) were obtained from ATCC (Manassas, VA, USA).

Techniques: Staining, Confocal Microscopy, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: The tropomyosin 3.1/3.2 inhibitor ATM-3507 alters B-cell actin dynamics and impairs the growth and motility of diffuse large B-cell lymphoma cell lines

doi: 10.3389/fimmu.2025.1668379

Figure Lengend Snippet: Tpm3 co-localizes with actin arcs, and Tpm3.1/3.2 function is important for their formation. (A) A20 cells were allowed to spread for 30 min on coverslips that had been coated with 0.625 µg/cm 2 anti-IgG plus 0.15 µg/cm 2 ICAM-1. Cells were stained for F-actin (rhodamine-phalloidin) and Tpm3 and then imaged by confocal microscopy. Representative images from one of 3 independent experiments are shown, in which yellow arrows indicate an actin arc. Scale bar: 10 µm. (B-D) A20 cells were pre-treated for 1 h with 0.03% DMSO or 3 µM ATM-3507 and then allowed to spread for 30 min on coverslips coated with 0.625 µg/cm 2 anti-IgG plus 0.15 µg/cm 2 ICAM-1. F-actin staining was used to define the cell periphery, quantify cell areas, and visualize actin arcs. In (B) , each symbol represents the median value from one of 3 independent experiments in which n >30 cells per condition were analyzed. The mean ± SEM for the median values from the 3 experiments is shown. Representative images of DMSO- and ATM-3507-treated cells are shown in (C) , with an actin arc indicated by a yellow arrow. The percent of DMSO- and ATM-3507-treated cells in which actin arcs were clearly visible is shown in (D) . Each symbol represents the median value from one of 3 independent experiments in which n >30 cells per condition were analyzed. The mean ± SEM for the median values from the 3 experiments is shown. p-values in panels (B) and (D) were calculated using two-tailed paired t -tests.

Article Snippet: The A20 IgG + murine B-lymphoma cell line (ATCC #TIB-208; https://www.cellosaurus.org/CVCL_1940 ) and the B16-F1 murine melanoma cell line (ATCC #CRL-6323; https://www.cellosaurus.org/CVCL_0158 ) were obtained from ATCC (Manassas, VA, USA).

Techniques: Staining, Confocal Microscopy, Two Tailed Test