Fig 2: The TLTs developing in murine models of chronic inflammation form a network of podoplanin+ stromal cells in the T zone, similar to LNs. Immunofluorescence analysis of frozen sections of pancreas from NOD and RIP-CXCL13 tg+ mice and kidney from RIP-LTa tg+ mice and peripheral LNs. A: B220+ B cells (green) and CD3+ T cells (red), the TRC marker podoplanin (podo, red) combined with the endothelial marker CD31 (green) in serial sections. Tg- pancreata did not show reticular podoplanin staining typical for TRCs (data not shown). Scale bar = 100 µm. i indicates islet. B: Infiltrate labeled with podoplanin (red), the lymphatic vessel marker Lyve-1 (green), and DAPI+ nuclei (blue). Arrows indicate lymphatic vessels; and arrowheads, the more abundant fibroblastic reticular cells. Scale bar = 100 µm. C: Higher-magnification images of the T-cell–rich zone of the infiltrate labeled with podoplanin (red) and desmin and the conduit marker ER-TR7 (green). Scale bar = 20 µm. D: The podoplanin (red) and CD35 (green) staining showing two distinct stromal cell networks in infiltrates of RIP-CXCL13+ pancreas. Scale bar = 100 µm. Data are representative of at least three independent experiments.

Fig 3: Co-expression of L-selectin and PECAM-1 in dHL-60 cells significantly increases TTT across TNF-activated HUVECs, and functionally blocking PECAM-1 in primary human neutrophils significantly reduces TTT across TNF-activated HUVEC. (A) Time series of frames (from Movie 3, which includes the time code) showing how TEM of HL-60 cells is quantified. Cells transitioning from phase bright (image 2) to completely phase dark (image 10). Scale bars: 5 µm. (B) Quantification of average TTT for dHL-60 cells transmigrating across TNF-activated HUVECs. The dHL-60 cells analysed are stably expressing GFP alone (green column scatter), PECAM-1–GFP alone (blue column scatter), L-selectin–RFP alone (red column scatter) or fluorescently tagged constructs of both PECAM-1 and L-selectin (orange column scatter). The TTT of at least 180 cells were quantified (mean±s.e.m.) per group across three independent experiments. ****P<0.0001 (one-way ANOVA followed by multiple comparisons corrected for by the Sidak method). (C) Quantification of mean±s.e.m. TTT for dHL-60 cells transmigrating across IL-1ß-activated HUVEC. The dHL-60 cells analysed are stably expressing GFP alone (green column scatter), PECAM-1–GFP alone (blue column scatter), L-selectin–RFP alone (red column scatter) or fluorescently tagged constructs of both PECAM-1 and L-selectin (orange column scatter). The TTT of at least 180 cells were quantified per group across three independent experiments. ****P<0.0001 (one-way ANOVA followed by multiple comparisons corrected for by the Sidak method). (D) Primary human neutrophils were pre-treated with either 1 µg/ml anti-PECAM-1 function-blocking antibody (HEC7) or isotype-matched control IgG2a (IgG Control) at 37°C for at least 20 min prior to perfusion over TNF-activated HUVECs. TTT were recorded using wide-field time-lapse microscopy. Column scatter graph represents the mean±s.e.m. TTT of 140 and 145 cells for IgG2a and HEC7 treatment groups, respectively. Data is collected over three independent experiments using three different donors. ****P<0.0001 (two-tailed unpaired Student's t-test with Welch's correction). (E) Primary human neutrophils were pre-treated with either 1 µg/ml anti-PECAM-1 function-blocking antibody (HEC7) or isotype-matched control IgG2a (IgG Control) at 37°C for at least 20 min prior to perfusion over IL-1ß-activated HUVECs. TTT were recorded using wide-field time-lapse microscopy. Column scatter graph represents the mean±s.e.m. TTT of 140 and 145 cells for IgG2a and HEC7 treatment groups, respectively. Data is collected over three independent experiments using three different donors.

Fig 4: L-selectin shedding is activated in neutrophils crossing TNF-, but not IL-1ß-, activated HUVECs. (A) Primary human neutrophils were perfused over TNF-activated HUVEC monolayers and after 6 min of perfusion were fixed in 4% PFA and prepared for immunostaining for PECAM-1 (blue, Alexa Fluor® 633), L-selectin (green, Alexa Fluor® 488) and F-actin (red, TRITC–phalloidin). Two primary human neutrophils are captured in different stages of the multi-step adhesion cascade – firm adhesion (right-hand side) and mid-TEM (left-hand side) under flow conditions. Two optical sections, Top and Base, reveal the subcellular organisation of endogenous L-selectin present within the transmigrated pseudopod (arrowhead) and the non-transmigrated cell body (or uropod). Distinct morphological characteristics of flat pseudopodial extensions in the subendothelial space and rounded non-transmigrated body afford easy interpretation of transmigrated and non-transmigrated regions. Anti-PECAM-1 staining of TNF-activated HUVEC reveals the well-documented discontinuity of the signal specifically at the region of paracellular TEM. Note that L-selectin expression is present in both the transmigrated pseudopod and non-transmigrated uropod, which has been observed previously in primary human (CD14-positive) monocytes (Rzeniewicz et al., 2015). Scale bar: 15 µm. (B) Analysis of HUVEC PECAM-1 protein expression through western blot analysis, following overnight stimulation with or without the cytokines IL-1ß and TNF (UNT, untreated). An anti-actin western blot is used as a loading control for PECAM-1. Bar graph represents the mean±s.d. band intensity values corresponding to PECAM-1, after normalisation against the actin western blot bands (n=3 independent experiments). (C) Schematic representation of how line intensity profiles were drawn and quantified for neutrophils within the subendothelial space. More details are provided in the Materials and Methods section. (D) Primary human neutrophils, pre-treated for 20 min with either DMSO or 10 µM TAPI-0, were perfused over TNF-activated HUVEC monolayers at a density of 106 cells per ml. Cells were perfused for 5 min, followed by a further 15 min with neat HL-60 medium. By the end of perfusion, over 95% of all neutrophils had successfully completed TEM (see Movie 2). Specimens were immediately fixed in 4% PFA prior to permeabilisation and immunostaining for L-selectin with LAM1-14 and TRITC-phalloidin. Bar graph (mean±s.e.m.) showing L-selectin surface expression, quantified as a percentage of the LAM1-14 signal that is relative to the maximal L-selectin signal assigned to the fluorescence intensity in the TAPI-0 treatment group (set at 100%). At least 420 cells were analysed per group across three independent experiments. *P<0.05 (two-tailed unpaired Student's t-test with Welch's correction). Scale bars: 10 µm. Images shown are (E) Primary human neutrophils were perfused over IL-1ß-activated HUVEC using the same method as in D. Fluorescence intensity of the LAM1-14 signal corresponding to L-selectin was assessed through analysing line intensity profiles using Volocity software (see C and Materials and Methods). Bar graph (mean±s.e.m.) showing L-selectin surface expression, quantified as a percentage of the LAM1-14 signal that is relative to the maximal L-selectin signal assigned to the fluorescence intensity in the TAPI-0 treatment group (set at 100%). At least 420 cells were analysed per group across three independent experiments. No significant difference was seen (two-tailed unpaired Student's t-test with Welch's correction). Scale bars: 10 µm.

Fig 5: In vitro cardiovascular tissue formation in the multiscale scaffold. (a) (left) Immunofluorescence images for CD31 in the fibrin hydrogel region of the multiscale scaffold at each time point. Yellow dashed rectangle insets show × 40 magnified images of each group. Scale bars = 200 µm and 100 µm in low magnification and high magnification images, respectively. (right) Quantitative analysis of CD31 expression by qRT-PCR at 3-week culture. (b) Quantitative expression of cardiac markers (MHC, ACTC1) from ADSCs in the multiscale scaffold by qRT-PCR at 3-week culture. The expression levels to relative to the housekeeping gene (GAPDH) were calculated for analysis of qRT-PCR results (n = 3). Data are mean ± s.d. One-way ANOVA with Tukey’s post-hoc test was used. ***P < 0.001, **P < 0.01.