When engineered to express and present T1D antigens to antigen-reactive T cells, FRCs seeded on 3D collagen scaffold can engage T1D antigen-specific T cells more efficiently than 2D systems

When engineered to express and present T1D antigens to antigen-reactive T cells, FRCs seeded on 3D collagen scaffold can engage T1D antigen-specific T cells more efficiently than 2D systems. cells. Traditional systems for studying FRCs do not allow modulation of T1D-relevant antigen expression levels in FRCs and quantification of engagement of specific T cells more efficiently than in 2D systems. Research Design and Methods Mice All animal procedures were approved by the Institutional Animal Care and Use Committee of University or college of Miami. NOD/ShiLtJ, NOR/LtJ, NOD.Cg-Tg(TcraTcrbNY8.3)1Pesa/DvsJ (NY8.3) mice were purchased from your Jackson Laboratory (Bar Harbor, Maine). Tissue Harvest Pancreatic LNs (PLNs) and/or skin draining LNs (SDLNs, including brachial, axillary and inguinal LNs) were harvested from mice at either 4 or 12 weeks of age (low and high T1D risk, respectively). For LN volume quantification, LNs were photographed after harvesting, LN area was quantified using ImageJ, and the volume calculated using the ellipsoid formula (4/3Tracking of T1D antigen-specific T cells (CellTrace violet labeled: blue) in the GFP-expressing FRC reticula (green) was performed 24 h after co-culture by confocal imaging through time-lapse was quantified by circulation cytometry 3 days after FRC/T cell co-culture. Scaffolds were mechanically processed to isolate T cells, while 2D Klf2 controls were pipetted vigorously in order to obtain the T cells resting on top of the FRC layer. Isolated cells were stained with antibodies (gp38-PE, CD31-APC, CD45-PE/Cy7 or CD45-AF700, CD8-PerCP/Cy5.5, CD44-PE/CF594, CD25-PE/Cy7) and processed for flow cytometry. CD8+ T cell proliferation (CellTrace dilution) and CD25 upregulation were calculated using Kaluza (Beckman Coulter). Details of antibodies utilized for circulation cytometry are indicated in Supplementary Table?1. Statistical Analysis GraphPad Prism 6 was utilized for data analysis. Every set of data is usually offered as mean standard deviation. Paired test for comparison across different time points and Bonferroni for comparison across different experimental groups (NOD vs. NOR) were performed in analysis of data units with more than two populations. 95% confidence level was considered significant. The investigator carrying out the acquisition and analysis was blinded to the treatment groups. Results Reticular Properties of FRC Networks in Lymph Nodes are Altered in T1D Murine Models FRC network integrity in LNs plays a critical role for the activation of adaptive immune responses and may impact FRC-mediated peripheral tolerance mechanisms. We hypothesize that FRC reticular properties in LNs, in addition to the previously reported decreased frequency and T1D antigen expression levels, are altered in T1D. We characterized the reticular properties of FRC networks in PLNs (Fig.?1) and SDLNs (Fig. S1) of 4-week aged female NOD mice as low diabetes risk group (Figs.?1a, S1A) and of 12-week old mice (Figs.?1b, S1B) as high diabetes risk group since diabetes onset in female NOD mice occurs after 12 weeks of age. We euthanized the mice before they switched diabetic, harvested PLNs and SDLNs, measured their volume, and processed them for immunofluorescence to identify FRC reticula in LN sections as gp38+ Lyve-1? networks (to differentiate FRCs from gp38+ Lyve-1+ lymphatic endothelial cells) in T cell areas (recognized by staining with antibodies that label T cells and B cells). As controls, we harvested and processed PLNs (Figs.?1c and d) and SDLNs (Fig. S1C, D) from Cinobufagin age-matched diabetic-resistant NOR mice. Reticular properties were characterized through quantification of reticular porosity of FRC networks as Feret diameter of the irregularly shaped pores using ImageJ. We found that NOD PLN networks displayed larger reticular pores than NOR PLN controls at both 4 weeks (Feret NOD: 18 6 not significant, **not significant, *not significant, *in a physiomimetic environment. FRCs in Designed Reticula Expressing T1D Antigens Engaged T1D Autoreactive T Cells More Effectively than in 2D Culture Systems Finally, we aimed at validating the applicability of our designed FRC reticula to quantify engagement of T1D antigen-specific T cells using T1D antigen-expressing FRCs compared to traditional 2D culture systems. We transduced FRCs from 12-week aged NOD PLNs to express multiple T1D-relevant epitopes,7 including a natural peptide from IGRP Cinobufagin recognized Cinobufagin by NY8.3 CD8+ T cells in the context of H2-Kd MHC class I and GFP to allow T cell tracking on FRC reticula through confocal microscopy. To determine if FRC reticula designed.