Journal: International Journal of Molecular Sciences

Article Title: Artificial Tertiary Lymphoid Structures: Exploring Mesenchymal Stromal Cells as a Platform for Immune Niche Formation

doi: 10.3390/ijms252413286

Figure Lengend Snippet: MSC-Lymphocyte Organoid Formation and Analysis of Marker Expression. ( a ) Schematic Overview of Experimental Setup. Human or mouse MSCs and lymphocytes were mixed at a 1:3 ratio and initially cultured in hanging drops for one day to promote spheroid formation. Following this compaction phase, the spheroids were either transferred to agarose-coated wells, where they were maintained for an additional 15 days or implanted into mice; ( b ) Representative phase-contrast image at 100× magnification of a typical MSC-lymphocyte spheroid on agarose, showing its general morphology and structure; ( c ) An enlarged view of the spheroid (200×); ( d – g ) Representative immunofluorescence images of spheroids assembled with TNF-α-pretreated MSCs, illustrating marker localization within the spheroid ( d ) CD3 Staining (green) CD20 (red) identifying T and B lymphocytes, respectively, with nuclei counterstained with DAPI (blue) (200× magnification); ( e ) CD35 staining (red); ( f ) PDPN staining (green); ( g ) PAI2 staining (red); ( h – k ) Quantitative PCR Analysis of Gene Expression; ( h , i ) relative mRNA expression levels of selected genes in both 2D co-cultures and 3D spheroids in both 2D co-cultures and 3D spheroids from human MSCs pre-treated with TNF-α (5 ng/mL) or left untreated before co-culture. Data are shown as a ratio relative to control MSCs cultured without lymphocytes and normalized to both β-actin and CD45 to ensure equal lymphocyte representation. Statistically significant differences from the control 2D group (CTRL-2D) are marked with an asterisk (* p < 0.05); ( j,k ) Changes in relative mRNA expression levels of various genes in 2D co-cultures and 3D spheroids from mouse MSC that were either pre-treated with recombinant mouse TNF-α (5 ng/mL) or left untreated.

Article Snippet: Sections were incubated overnight at 4 °C with primary antibodies against CD21 (MA5-32227, Thermo Fisher Scientific Inc., Waltham, MA, USA), CD3 (NB600-1441SS, Novus Biologicals, Centennial, CO, USA), CD20 (PA5-16701, Thermo Fisher Scientific Inc., Waltham, MA, USA), and CD35 (A3661, Abclonal, Woburn, MA, USA).

Techniques: Marker, Expressing, Cell Culture, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Co-Culture Assay, Control, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: Artificial Tertiary Lymphoid Structures: Exploring Mesenchymal Stromal Cells as a Platform for Immune Niche Formation

doi: 10.3390/ijms252413286

Figure Lengend Snippet: Histological and immunohistochemical characterization of implanted MSC-Lymphocyte organoids ( a ) Low-magnification immunohistochemistry images of adipose tissue cross-sections containing an intact lymph node, an implanted MSC-derived organoid, and a sham-operated control. Sections are stained for CD21 using a peroxidase-based detection system with DAB chromogen (brown) and counterstained with hematoxylin (blue) to highlight cell nuclei. Scale bars = 1 mm; ( b ) Higher magnification images of a section stained for CD21 and ( c ) CD35. Scale bars = 100 μm; ( d ) immunohistochemistry images showing CD3 and ( e ) CD20 staining, indicating the presence of T cells (CD3-positive) and B cells (CD20-positive) within the implanted organoids; ( f ) calculation of T- and B-cells in organoids. MEAN ± SD. ( g ) representative phase-contrast image showing the overall structure and cellular density of the implanted organoid at 200× magnification; ( h ) representative immunofluorescence image of an entire organoid stained with CD3 (red) to highlight T-cell distribution with nuclei stained using DAPI (blue). Scale bars = 100 μm ( i ) immunofluorescence images of frozen sections from the implanted organoid showing CD31 (green) and alpha-SMA (green) expression.

Article Snippet: Sections were incubated overnight at 4 °C with primary antibodies against CD21 (MA5-32227, Thermo Fisher Scientific Inc., Waltham, MA, USA), CD3 (NB600-1441SS, Novus Biologicals, Centennial, CO, USA), CD20 (PA5-16701, Thermo Fisher Scientific Inc., Waltham, MA, USA), and CD35 (A3661, Abclonal, Woburn, MA, USA).

Techniques: Immunohistochemical staining, Immunohistochemistry, Derivative Assay, Control, Staining, Immunofluorescence, Expressing

Journal: Cell Death and Differentiation

Article Title: ARID1A mutations protect follicular lymphoma from FAS-dependent immune surveillance by reducing RUNX3/ETS1-driven FAS-expression

doi: 10.1038/s41418-025-01445-3

Figure Lengend Snippet: A Schematic overview of the GLSG2000 FL cohort and available data. B Lollipop plot of ARID1A mutations in the evaluable GLSG2000 FL cohort. C FAS RNA expression in primary FL biopsies ( ARID1A WT ( N = 39) vs ARID1A MUT ( N = 12)) by digital multiplex gene expression profiling (DMGEP). P-values from Mann-Whitney U-test . D FAS protein abundance in the CD20 + cells normalized to CD3 + cells in primary FL biopsies ( ARID1A WT ( N = 36) vs ARID1A MUT ( N = 7)) by quantitative multispectral imaging (QMI). P-values from Welch test . E Representative multispectral images. Scale bar is 20 µm (low magnification) or 400 µm (high magnification).

Article Snippet: We used the following antibodies: FAS-R (EP208; AC-0178RUO; Abcam, Cambridge, UK; 1:100), CD3 (SP7; RBK024-05; Zytomed System, Berlin, Germany; 1:150); CD20 (L26; 120M-85; Cell Marque, Rocklin, CA, USA; 1:200).

Techniques: RNA Expression, Multiplex Assay, Gene Expression, MANN-WHITNEY, Quantitative Proteomics, Imaging