Journal: bioRxiv

Article Title: Organoid Polymer Functionality and Mode of Klebsiella Pneumoniae Membrane Antigen Presentation Regulates Ex Vivo Germinal Center Epigenetics in Young and Aged B Cells

doi: 10.1101/2020.02.16.947408

Figure Lengend Snippet: A) Chemical structure of the vinyl sulfone, acrylate, and maleimide functional groups at the end of four-arm PEG. B) Schematic of immune organoid formation with co-cultured CD40L- and BAFF-presenting stromal cells and primary, naive B cells. C) Effects of pH and degradability on 40LB cell viability in PEG-4VS and PEG-4MAL hydrogels. D) Representative brightfield images illustrating the effects of pH and degradability on stromal cell network formation in PEG-4VS and PEG-4MAL organoids. E) Quantitative flow cytometric analysis of CD19+ B cell in four-arm PEG hydrogels modified with maleimide, vinyl sulfone, or acrylate, indicating survival in hydrogels. F) Flow cytometric gating strategy for germinal center-like B cell (CD19+GL7) formation in four-arm PEG hydrogels modified with maleimide and vinyl sulfone. G) Effects of vinyl sulfone and acrylate functionalization on stromal cell network formation and survival of B cells in PEG-based organoids. H) Confocal imaging of germinal center-like B cell (GL7+) and its relative localization with 40LB stromal in PEG-4MAL organoids. Green: GL7, Yellow: Actin, Magenta: DAPI. I) Quantitative flow cytometric analysis of proliferative germinal center-like B cell (CD19+GL7+Fas+) formation in four-arm PEG hydrogels modified with maleimide, vinyl sulfone, or acrylate. J) Effect of four-arm PEG end-modification chemistry on Fas expression in CD19 +GL7+ B cells. All data represent mean ± S.E.M. and were analyzed via one-way ANOVA with Tukey’s post-hoc multiple comparisons test. ****p<0.0001, ns=not significant; N=5.

Article Snippet: Where indicated, organoid media was replenished on days 1 or 2 with 50 μg/mL mouse anti-CD40L (Clone MR-1, Bio X Cell #BE0017-1), 50 μM BCL6 inhibitor 79-6 (Sigma #197345), or 10 ng/mL IL-4 alone.

Techniques: Functional Assay, Cell Culture, Modification, Imaging, Expressing

Journal: bioRxiv

Article Title: Organoid Polymer Functionality and Mode of Klebsiella Pneumoniae Membrane Antigen Presentation Regulates Ex Vivo Germinal Center Epigenetics in Young and Aged B Cells

doi: 10.1101/2020.02.16.947408

Figure Lengend Snippet: A) EZH2 expression in naïve murine B cells, generation 1, ex vivo 3D organoids made of RGD-rich matrix, in vivo germinal centers in immunized mice, and ex vivo 2D co-cultures. B) Effect of integrin-binding peptides on percentage of B220+EZH2+ B cells over time in organoid grown cultures of naïve B cells with 40LB and IL-4. C) Change in B220+EZH2+ B cells between days 4 and 6. D-E) Effect of integrin-binding peptides on D) percentage and E) number of B220+ B cells with high EZH2 expression. Left side flow cytometry histogram in D) indicates expression of EZH2 on Day 6. F-G) Effect of integrin-binding peptides on histone methylation in terms on F) number of positive cells and G) median fluorescence intensity. Left side flow cytometry histogram in G) indicates expression of H3k27Me3 on Day 6. H) Percent change in H3k27 methylation over time in response to varying integrin ligand. I) Effect of blocking CD40L and BCL6 on days 1 or 2 of ex vivo organoid culture on H3k27 methylation. Left side flow cytometry scatter plot and histogram indicates EZH2 populations and expression of H3k27Me3. Data represent mean ± S.E.M. Significance determined via one- or two-way ANOVA with Tukey’s post-hoc multiple comparisons test; N=5. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Ψp<0.05, ΨΨp<0.01, ΨΨΨp<0.001, ΨΨΨΨp<0.0001 relative to day 4 for corresponding ligand.

Article Snippet: Where indicated, organoid media was replenished on days 1 or 2 with 50 μg/mL mouse anti-CD40L (Clone MR-1, Bio X Cell #BE0017-1), 50 μM BCL6 inhibitor 79-6 (Sigma #197345), or 10 ng/mL IL-4 alone.

Techniques: Expressing, Ex Vivo, In Vivo, Binding Assay, Flow Cytometry, Methylation, Fluorescence, Blocking Assay