Journal: Nature Immunology

Article Title: Lymph node homeostasis and adaptation to immune challenge resolved by fibroblast network mechanics

doi: 10.1038/s41590-022-01272-5

Figure Lengend Snippet: a-b) Flow cytometric gating strategy and representative dot plots and percentages of a) CD45 − stroma cells and b) subpopulations. Fibroblastic reticular cells (FRC, PDPN + CD31 − ), blood endothelial cells (BEC, PDPN − CD31 + ), lymphatic endothelial cells (LEC, PDPN + CD31 + ). Red arrow shows GFP + FRCs c) Representative gating of GFP + Fibroblasts. d) A membrane-targeted GFP molecule is driven under the PDGFR α promoter. e) LN nodes were embedded in low melt agarose and sliced at 200 μm thickness before being secured by cover glass for imaging. f) IFA/OVA is used as model immunization with inguinal draining (dr) and non-draining (nDr) lymph nodes (LNs) harvested day 3 or day 5 post immunization (right). This figure supports Figs. – .

Article Snippet: Mice were immunized via subcutaneous injection into the right flank, proximal to the hip, with 100 μl of an emulsion of OVA with IFA or complete Freunds adjuvant (CFA) where stated (100 μg OVA per mouse) (Hooke Laboratories).

Techniques: Membrane, Imaging

Journal: Nature Immunology

Article Title: Lymph node homeostasis and adaptation to immune challenge resolved by fibroblast network mechanics

doi: 10.1038/s41590-022-01272-5

Figure Lengend Snippet: a , LN mass change after IFA/OVA immunization. One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons, **** P = 1.00 × 10 −6 . Each point represents one LN at day 0 ( n = 10), day 3 ( n = 11), and day 5 ( n = 12). b , T cell packing in the LN during inflammation for PDPN (FRCs, yellow), CD3 (T cells, blue), and DAPI (nuclei, gray). Scale bars, 25 µm. c , Quantification of CD3 + T cell nuclei per 100 µm 2 . Box plot indicates median, interquartile range, and minimum/maximum. Dotted line represents median of day 0. One-way ANOVA with Tukey’s multiple comparisons, * P = 0.0362, **** P = 0.0003. n indicates individual image ROI on day 0 ( n = 17), day 3 ( n = 15), and day 5 ( n = 20) from three independent LNs. d , FRC gap analysis during inflammation. PDPN (FRCs), binary, and circle overlay. Scale bars, 50 µm. e , Quantification of circle radius. Box plot indicates median, interquartile range, and minimum/maximum. Dotted line represents median of day 0. One-way ANOVA with Tukey’s multiple comparisons, **** P = 2.00 × 10 −6 . Each point represents a circle radius. n indicates individual image ROI on day 0 ( n = 12), day 3 ( n = 15), and day 5 ( n = 15) from three independent LNs. f , Laser ablation of the FRC network throughout inflammation. PDGFR α + mGFP + (FRCs) ablation ROI (white box) and cut site (red dotted line). Scale bars, 50 µm. Recoil displacement (arrowheads) with pre- (green) and postcut (magenta) overlay. Scale bars, 10 µm. g , Recoil curves of network displacement (μm) (mean ± standard error of the mean (s.e.m.)). h , Initial recoil velocity (μm s −1 ) after IFA/OVA immunization. Box plot indicates median, interquartile range, and minimum/maximum. Kruskal–Wallis test with Dunnett’s test, * P < 0.05. g , h , n indicates an ablation on day 0 ( n = 30), day 3 ( n = 37), and day 5 ( n = 32) for three independent experiments.

Article Snippet: Mice were immunized via subcutaneous injection into the right flank, proximal to the hip, with 100 μl of an emulsion of OVA with IFA or complete Freunds adjuvant (CFA) where stated (100 μg OVA per mouse) (Hooke Laboratories).

Techniques:

Journal: Nature Immunology

Article Title: Lymph node homeostasis and adaptation to immune challenge resolved by fibroblast network mechanics

doi: 10.1038/s41590-022-01272-5

Figure Lengend Snippet: a , b , Actomyosin structure in FRCs day 3 (a) and day 5 (b) after IFA/OVA immunization for perlecan (matrix, magenta), PDPN (FRC, yellow), phalloidin (F-actin, cyan), and pMLC (red). Asterisks and arrowheads indicate F-actin cables with or without perlecan alignment. Orthogonal views (yellow dotted line, yz axis, 10 μm depth). Representative image of three independent experiments. Scale bars, 10 µm. c , Percentage of Fibers. Stacked bar plots with mean ± standard deviation (s.d.) error bars. d , Confetti-labeled FRCs (yellow) spanning space without (white asterisk) ECM (magenta). Scale bar, 5 µm. e , Percentage of Fibers. Box plot indicates median, interquartile range, and minimum/maximum. One-way ANOVA with Tukey’s multiple comparisons, ** P < 0.01. c , e , n indicates image ROI on day 0 ( n = 14), day 3 ( n = 13), and day 5 ( n = 12) from three mice. f , Initial recoil velocity (µm s −1 ) after IFA/OVA immunization, ± ROCK inhibition ( Y27632 ). Resistive myosin forces (blue text indicates the ratio between PBS/Y27). Box plot indicates median, interquartile range, and minimum/maximum. Two-way ANOVA with Tukey’s multiple comparisons, **** P < 0.0001, ** P = 0.001973. g , Recoil curves of network displacement (µm) (mean ± s.e.m.). f , g , n indicates an ablation on day 0 ( n = 54), day 0 + Y27 ( n = 43), day 3 ( n = 18), day 3 + Y27 ( n = 20), day 5 ( n = 19), and day 5 + Y27 ( n = 15) over three independent experiments . h , Actomyosin structure in FRCs day 5 after IFA/OVA immunization with or without ROCK inhibition ( Y27632 ). Perlecan (matrix, magenta), PDPN (FRC, yellow), phalloidin (F-actin, cyan), and pMLC (red). Asterisks and arrowheads indicate F-actin cables with or without perlecan alignment. Representative image of three independent experiments. Scale bars, 10 µm. i , Percentage of Fibers. Stacked bar plots with mean ± s.d. error bars. Two-way ANOVA with Tukey’s multiple comparisons, **** P = 0.000577, * P = 0.03824. j , Percentage of Fibers. Box plot indicates median, interquartile range, and minimum/maximum. One-way ANOVA with Tukey’s multiple comparisons, **** P < 0.0001. i , j , n indicates image ROI on day 0 ( n = 12), day 0 + Y27 ( n = 12), day 5 ( n = 18), and day 5 + Y27 ( n = 18) from three mice. NS, not significant.

Article Snippet: Mice were immunized via subcutaneous injection into the right flank, proximal to the hip, with 100 μl of an emulsion of OVA with IFA or complete Freunds adjuvant (CFA) where stated (100 μg OVA per mouse) (Hooke Laboratories).

Techniques: Standard Deviation, Labeling, Inhibition

Journal: Nature Immunology

Article Title: Lymph node homeostasis and adaptation to immune challenge resolved by fibroblast network mechanics

doi: 10.1038/s41590-022-01272-5

Figure Lengend Snippet: a , LN tile scan of PDPN + (yellow) and Ki67 + (magenta) cells. Scale bars, 500 µm (zoom-in T cell area (white box), 50 µm). Arrowheads mark PDPN + Ki67 + FRCs. b , Flow cytometric analysis of Ki67 + percentage of FRCs after IFA/OVA immunization. Two-way ANOVA with Dunnett’s test, **** P = 1.00 × 10 −6 . n indicates LNs on day 0 ( n = 5), day 3 ( n = 5), and day 5 ( n = 4). c , FRC GFP nuclei are tracked over 72 h on different polyacrylamide gel stiffnesses. Scale bar, 50 µm . d – f , Average number of divisions per cell in 72 h and over time on different substrate rigidities. d , Control FRCs. e , FRCs treated with ROCK inhibition ( Y27632 ). f , PDPN KO FRCs. Box plot indicates median, interquartile range, and minimum/maximum. One-way ANOVA with Tukey’s multiple comparisons, * P = 0.0298. n indicates image ROI for 2 kPa ( n = 8), 12 kPa ( n ≥ 4), 30 kPa ( n ≥ 4), and glass ( n ≥ 5) over three independent experiments. Division curves compare glass and 2 kPa (mean ± s.e.m.). g , Flow gating and PDPN histograms geometric mean fluorescent intensity (gMFI) of isolated primary FRCs after ROCK inhibition ( Y27632 ). h , Fold change in gMFI of PDPN on FRCs following ROCK inhibition ( Y27632 ). Mann–Whitney test (two tailed), **** P < 0.0001. n = 3 LNs.

Article Snippet: Mice were immunized via subcutaneous injection into the right flank, proximal to the hip, with 100 μl of an emulsion of OVA with IFA or complete Freunds adjuvant (CFA) where stated (100 μg OVA per mouse) (Hooke Laboratories).

Techniques: Control, Inhibition, Isolation, MANN-WHITNEY, Two Tailed Test

Journal: Nature Immunology

Article Title: Lymph node homeostasis and adaptation to immune challenge resolved by fibroblast network mechanics

doi: 10.1038/s41590-022-01272-5

Figure Lengend Snippet: a , Tamoxifen and immunization strategy for Pdgfra mGFPΔPDPN mice. Draining LNs (Dr) and non-draining LNs (nDr). b , LN paracortex maximum z-projection. GFP ( Pdgfra mGF P , green), PDPN (FRCs, magenta). Scale bars, 50 µm (zoom, 30 µm). c , Representative histograms of surface protein expression for PDPN in control and Pdgfra mGFPΔPDPN steady-state LNs. d , LN mass after CFA/OVA immunization. Box plot indicates median, interquartile range, and minimum/maximum. Two-way ANOVA with Dunnett’s test, * P = 0.030. n indicates LNs on day 0 ( n = 10), day 0 ΔPDPN ( n = 9), day 3 ( n = 6), day 3 ΔPDPN ( n = 5), day 5 ( n = 4), and day 5 ΔPDPN ( n = 4). e , Representative image of GFP + (green), PDPN − (magenta) FRC cell body in day 5 after immunization in Pdgfra mGFPΔPDPN LNs from two independent experiments. Scale bar, 25 µm. f , Representative images of actomyosin and ECM structures within control and Pdgfra mGFPΔPDPN FRCs 5 days after immunization. Arrowheads and asterisks mark PDPN − and PDPN + FRCs respectively; perlecan (magenta), PDPN (yellow), F-actin (cyan), and pMLC (red). Scale bars, 10 µm. g , Recoil curves of network displacement (µm) (mean ± SEM) for control and Pdgfra mGFPΔPDPN mice. h , Initial recoil velocity (µm s −1 ) after CFA/OVA immunization in control and Pdgfra mGFPΔPDPN mice. Box plot indicates median, interquartile range, and minimum/maximum. Two-way ANOVA with Sidak’s multiple comparisons, * P < 0.05, **** P = 1.00E ^−6 . Each point represents an ablation. i , Laser ablation of the FRC network throughout inflammation in control and Pdgfra mGFPΔPDPN mice. PDGFR α + mGFP + (FRCs) ablation ROI (white box) and cut site (red dotted line). Scale bars, 50 µm. Recoil displacement (arrowheads) with pre- (green) and postcut (magenta) overlay. Scale bars, 10 µm. g – i , n indicates ablation at day 0 ( n = 48), day 0 ΔPDPN ( n = 44), day 3 ( n = 18), day 3 ΔPDPN ( n = 18), day 5 ( n = 28), and day 5 ΔPDPN ( n = 21) over three independent experiments.

Article Snippet: Mice were immunized via subcutaneous injection into the right flank, proximal to the hip, with 100 μl of an emulsion of OVA with IFA or complete Freunds adjuvant (CFA) where stated (100 μg OVA per mouse) (Hooke Laboratories).

Techniques: Expressing, Control

Journal: Nature Immunology

Article Title: Lymph node homeostasis and adaptation to immune challenge resolved by fibroblast network mechanics

doi: 10.1038/s41590-022-01272-5

Figure Lengend Snippet: a) Example PDPN contour plots for GFP + FRCs from control and Pdgfra mGFPΔPDPN LNs. b) Representative histograms of surface protein expression for PDPN in control and Pdgfra mGFPΔPDPN LNs 5 Days post CFA/OVA. c) Fold change in PDPN expression (gMFI) in FRCs (GFP + CD31 − , CD140 a + CD31 − ). Box plot indicates median, interquartile range and minimum/maximum. One-way ANOVA with Tukey’s multiple comparisons. ****p = 1.00E ^−6 , ***p = 0.000143, 0.0004, **p = 0.0084, *p = 0.0145. d) Fold change in PDPN expression (gMFI) for LECs (PDPN + CD31 + ). Box plot indicates median, interquartile range and minimum/maximum. One-way ANOVA with Tukey’s multiple comparisons. **p = 0.0033. c, d) n = individual LNs where (n = 12), Day 0 ΔPDPN (n = 12), Day 5 (n = 6), Day 5 ΔPDPN (n = 8) over 2 independent experiments. This figure supports Figs. , .

Article Snippet: Mice were immunized via subcutaneous injection into the right flank, proximal to the hip, with 100 μl of an emulsion of OVA with IFA or complete Freunds adjuvant (CFA) where stated (100 μg OVA per mouse) (Hooke Laboratories).

Techniques: Control, Expressing

Journal: Nature Immunology

Article Title: Lymph node homeostasis and adaptation to immune challenge resolved by fibroblast network mechanics

doi: 10.1038/s41590-022-01272-5

Figure Lengend Snippet: a , Schematic of immunization and Y27 treatment timeline. Draining LNs (Dr) and non-draining LNs (nDr). b , LN mass and cellularity. Box plots indicate median, interquartile range, and minimum/maximum. One-way ANOVA with Tukey’s multiple comparisons, **** P = 1.00 × 10 −6 . n indicates LNs on day 0 ( n ≥ 28), day 0 Y27 ( n ≥ 28), day 5 ( n ≥ 7), and day 5 Y27 ( n ≥ 9) over two independent experiments. c , CD45 + and CD45 − cell numbers. Box plots indicate median, interquartile range, and minimum/maximum. One-way ANOVA with Tukey’s multiple comparisons, **** P = 1.00 × 10 −6 . d , e , CD19 + and CD3 + cell numbers and percentages of live. One-way ANOVA with Tukey’s multiple comparisons, **** P = 1.00 × 10 −6 . f , Ratio of the percentage of CD19 + and CD3 + compared to day 0 steady state. Box plots indicate median, interquartile range, and minimum/maximum. Dotted line marks control. Mann–Whitney test (two tailed), ** P = 0.0033. g , Total number of LECs (left), BECs (middle), and FRCs (right) after IFA/OVA immunization, ± ROCK inhibition ( Y27632 ). Box plot indicates median, interquartile range, and minimum/maximum. One-way ANOVA with Tukey’s multiple comparisons, **** P = 1.00 × 10 −6 , ** P = 0.0016, * P < 0.05. h , Scatter plots of FRC numbers against initial recoil velocity. Each dot represents the mean ± s.d. for both Y27 (Figs. 8g and ) and ΔPDPN (Figs. and ) cell number and initial recoil velocity.

Article Snippet: Mice were immunized via subcutaneous injection into the right flank, proximal to the hip, with 100 μl of an emulsion of OVA with IFA or complete Freunds adjuvant (CFA) where stated (100 μg OVA per mouse) (Hooke Laboratories).

Techniques: Control, MANN-WHITNEY, Two Tailed Test, Inhibition