Volume 27, Issue 9, Pages 1314-1325 (May 2017) Dendritic Cells Interpret Haptotactic Chemokine Gradients in a Manner Governed by Signal-to-Noise Ratio and Dependent on GRK6  Jan Schwarz, Veronika Bierbaum, Kari Vaahtomeri, Robert Hauschild, Markus Brown, Ingrid de Vries, Alexander Leithner, Anne Reversat, Jack Merrin, Teresa Tarrant, Tobias Bollenbach, Michael Sixt  Current Biology  Volume 27, Issue 9, Pages 1314-1325 (May 2017) DOI: 10.1016/j.cub.2017.04.004 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Visualization and Quantification of the Interstitial CCL21 Gradient (A–C) CCL21 distribution in mouse ear dermal explants. Z projections of explanted ear dermis of membrane-labeled mice (mTmG) were stained for lymphatic vessels (LYVE-1; A) and CCL21 (B). An overlay is shown (merge; C). Lymphatic vessels (LVs) are indicated by red arrows and blood vessels by yellow arrows. Areas in the yellow boxes are enlarged and the borders of the lymphatic vessels indicated as dotted, yellow lines. The scale bar represents 100 μm. (D–F) Quantification of the interstitial CCL21 gradient. (D) Schematic of CCL21 quantification in vicinity of lymphatic vessels. CCL21 staining intensities were quantified starting from lymphatic vessels (yellow, dotted lines). (E) Normalized intensities of CCL21 stainings plotted against the distance from the lymphatic vessel (SEM; n = 4; c0 was normalized to 1). (F) Fit of an exponential decay to the experimentally acquired data (E). c0, background signal; decay length δ = 54.3 ± 2.8 μm. See also Figure S1. Current Biology 2017 27, 1314-1325DOI: (10.1016/j.cub.2017.04.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Haptotaxis Assay and In Vitro Reconstruction of the Interstitial CCL21 Gradient (A) 3D environment. Interstitial 3D migration conditions are mimicked by physical confinement of DCs between two glass surfaces. d, distance between glass surfaces. (B) Immobilized CCL21 gradient. Haptotactic, immobilized CCL21 gradient are mimicked by chemokine patterning. (A and B) Confining DCs on surface immobilized gradients of CCL21 (haptotaxis assay). The scale bar represents 50 μm. (C) Schematics of chemokine patterning. Biotin-4-fluorescein (B4F) is surface immobilized by laser-assisted photobleaching. CCL21 24-98 bio is surface immobilized on the B4F gradient via dye-labeled streptavidin (SA-Cy3). (D and E) In vitro reconstruction of the interstitial CCL21 gradient measured in vivo (Figure 1). (D) SA-Cy3 image of the patterned gradient. (E) Fit of an exponential decay to the printed gradient (D). Decay length δ = 55.1 ± 0.7 μm. The scale bar represents 100 μm. See also Figures S2 and S3 and Table S1. Current Biology 2017 27, 1314-1325DOI: (10.1016/j.cub.2017.04.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Analysis of DC Haptotaxis on Defined Gradients of Surface-Immobilized CCL21 (A) Gradient profiles (SA-Cy3 staining) of linear (green profiles) and in-vivo-like, exponential (blue profiles) gradients of CCL21 24-98 bio. I = 1 represents maximal printable concentration; I = 1/2 represents half-maximal printable concentration. n ≥ 5 profiles for each gradient type. The shades indicate the SD among different prints. (B) Average haptotactic index. <HI> = 1 represents migration in gradient direction, <HI> = −1 represents migration against the gradient direction, and <HI> = 0 represents random migration. (C) Schematic of cell trajectories (black) on a gradient of CCL21 (red). Cell trajectories in each concentration bin (c1–c8, gray bins) were pooled for each gradient or genotype condition. (D) Haptotactic indices, <HI>, of DCs migrating in the absence of chemokine (R10 cell culture medium), on areas of maximal chemokine deposition (cMAX), or next to areas with chemokine patterns (c0). Error bars represent bootstraps. (E and F) Haptotactic indices of DCs migrating on gradients reaching half-maximal CCL21 24-98 bio deposition (I = 1/2; E) and maximal CCL21 24-98 bio deposition (I = 1; F). Linear gradients are in green and exponential-like gradients in blue. Each bin (c1–c8) of the respective gradients is represented by its mean haptotactic index (n = 8–19) independent experiments for each gradient condition; error bars represent bootstraps. See also Figures S3–S5. Current Biology 2017 27, 1314-1325DOI: (10.1016/j.cub.2017.04.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 DC Haptotaxis Reflects Signal-to-Noise Ratio of Immobilized Chemokine Gradient (A) Schematic of differential CCL21 binding to CCR7, homogenously distributed over the cell surface. (B) Left: linear (green) and exponential-like (blue) gradients of CCL21. CCL21 concentration plotted against the location on the respective gradient. Middle: receptor occupancy of linear (green) and exponential-like (blue) gradients assuming equilibrium binding and a fixed number of receptors. Receptor occupancy is plotted against the location on the respective gradient. Right: SNR of signal recognition on receptor level of linear (green) and exponential-like (blue) gradients. SNR is plotted against the respective receptor occupancy. (C and D) SNR matched to the haptotactic response (<HI>) of DCs migrating on linear (green) or exponential-like (blue) gradients of CCL21 (I = 1, C; I = 1/2, D). SNRs (solid lines) are fitted using a single dissociation constant K and different scaling factors (αlin and αexp) for linear and exponential-like gradients. Shading indicates variations in the SNR from corresponding SDs in the gradient profiles (Figure 3A). Current Biology 2017 27, 1314-1325DOI: (10.1016/j.cub.2017.04.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Influence of Immobilized CCL21 Gradients on Cell Speed (A) Varying concentrations of CCL21 24-98 bio represented by SA-Cy3 fluorescence intensity and intensity of medium only (0%). N is the number of tracks evaluated for each CCL21 24-98 bio concentration. (B) DC velocities on patches of CCL21 24-98 bio with varying concentration (see A) increase linearly with homogeneous CCL21 concentration. Mean ± SEM. (C) DC velocities in cell culture medium with (CCL21 24-98 bio; 625 ng/mL) and without (R10 cell culture medium) CCL21 24-98 bio. (D) DC velocities at low concentrations (cMIN; bins c1–c4) and high concentrations (cMAX; bins c5–c8) of linear (left graphs) and exponential (right graphs) gradients of surface-immobilized CCL21 24-98 bio. (E) Average velocities of DCs migrating on gradients reaching maximal CCL21 24-98 bio deposition (I = 1), with linear gradients in green and exponential-like gradients in blue. Each bin (c1–c8) of the respective gradient is represented by its average velocity (n = 8–19 independent experiments for each gradient condition. Error bars represent bootstraps.). (F) Cell velocity as a function of haptotactic index for the data in (E), with correlation coefficients shown in the inset. Error bars represent bootstraps. See also Figures S3 and S6. Current Biology 2017 27, 1314-1325DOI: (10.1016/j.cub.2017.04.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 CCL21-Guided DC Haptotaxis Is GRK6 Dependent In Vitro (A) Sketch of chemotaxis and haptotaxis. In a collagen-gel-based chemotaxis assay, chemokine of given concentration is immersed into a collagen gel chamber, resulting in a diffusion-based gradient. (B) Collagen-gel-based chemotaxis assay. Average displacement of WT (different CCL21 24-98 bio concentrations in greys) and Grk6−/− (different CCL21 24-98 bio concentrations in red/yellow) DCs toward a diffusion-generated gradient of CCL21 24-98 bio (y displacement) plotted against the corresponding time (n = 9 independent experiments; frame rate 3 min; mean ± SEM). (C) Haptotactic indices (C) and average velocities (D) of WT (dots; blue and green) and Grk6−/− (diamonds; purple and pink) DCs migrating on linear (green and purple) and exponential-like (blue and pink) gradients (I = 1) of CCL21 24-98 bio. Each bin (c1–c8) of the respective gradients is represented by its average haptotactic index (C) or velocity (D). n = 8–19 independent experiments for each gradient condition. Error bars represent bootstraps. See also Figure S4. Current Biology 2017 27, 1314-1325DOI: (10.1016/j.cub.2017.04.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 7 DC Haptotaxis on In Vivo CCL21 Gradients Is GRK6 Dependent (A) Schematic of in vivo migration assay. Left: DC suspension is incubated on mouse ear dermal explants, including LV and CCL21 gradients, allowing CCL21-dependent invasion of the tissue. Right: non-invading DCs are washed away. Invading DCs migrate further toward the LV [2, 32]. (B) Z stack projections of WT DCs (red) and LYVE-1 immunostaining (green) after a 35 min co-incubation of DCs with mouse ear dermal explants. The scale bar represents 100 μm. (C) Schematic of experiment analysis. Lymphatic vessels (LVs) are represented by binary masks (vessel mask). DC numbers are determined by red and yellow pixel counts (total cell area). DC distances to the next vessel are measured as distances of each red pixel to the respective vessel mask. Cells aligned to vessels (yellow) are excluded. (D) Number of tissues invading WT and Grk6−/− DCs after 20 min of incubation with mouse ear dermal explants. Cell number is presented as total cell area. Mean ± SEM. (E) Mean distance of WT and Grk6−/− DCs to the next LV after 75 min of incubation with mouse ear dermal explants. Mean ± SEM. (F) Alignment of WT and Grk6−/− DCs with LVs after 75 min of incubation with mouse ear dermal explants. Alignment is calculated as ratio of cells inside LV compared to an image with the LV mask rotated by 90° with respect to cells (this value is ∼1 if the cell distribution is random. The value is high if the cells localize to the vessel.). Mean ± SEM. See also Figure S7. Current Biology 2017 27, 1314-1325DOI: (10.1016/j.cub.2017.04.004) Copyright © 2017 Elsevier Ltd Terms and Conditions