SUPPLEMENTAL MATERIALS

Supplementary Methods SUPPLEMENTAL MATERIALS Supplementary References Supplementary Video Legends Supplementary Figures and Legends SUPPLEMENTARY METHODS Additional animals and cell lines used for the Supplementary Figures. C3H/HeN mice were purchased from the National Cancer Institute (Frederick, MD), and AKR, FVB/NJ, and Cd247 -/- mice were purchased from Jackson Labs (Bar Harbor, ME). The BALB/c Thy1.1 mice were obtained from the Scripps Research Institute (La Jolla, CA). The OT-1/Rag1 -/- 1, 2, germ-free and conventional-housed Swiss Webster mice were purchased from Taconic Farms (Hudson, NY). Most experiments were performed on mice 8-12 weeks old, although mice up to 6 months old were also tested with similar results. To reconstitute the Tcrd -/- mice with γδ DETC precursors, unfractionated day 14, 15, or 16 fetal thymocytes from wild-type IL2p8-GFP mice were injected intraperitoneally into newborn Tcrd -/- pups (1.0-1.5 10 7 cells/mouse) and the mice were analyzed after 6 weeks. The 7-17 DETC cells 3 were provided by W. Havran (The Scripps Research Institute, CA). Antibodies and reagents. The following antibodies and reagents were used for immunofluorescence microscopy and/or flow cytometry: V γ 5-FITC (nomenclature according to Tonegawa, clone 536), γδ TCR-biotin (GL3), CD3ε-biotin (145-2C11), CD3-AF647 (17A2), CD103-FITC (M290) and py 142 -CD3ζ- AF647 (K25-407.69) from BD Pharmingen (San Diego, CA); γδ TCR-AF647 (UC7-13D5), LFA- 1/CD11a-AF647 (M17/4) and CD45.2-AF647 (104) from BioLegend (San Diego, CA); LAMP-1-AF647 (1D4B) from ebioscience (San Diego, CA); 4G10-biotin and FcεR1γ from Millipore-Upstate (Billerica, MA); py 418 -Src from GenScript (Piscataway, NJ); py 493 -ZAP70 from Signalway Antibody (Pearland, TX); NKG2D (H-15) from Santa Cruz Biotechnology (Santa Cruz, CA); ZO-1 (Z-R1), tricellulin (48-8300), anti-rabbit IgG, and anti-goat IgG (labeled with AF555 or AF647) from Invitrogen (Carlsbad, CA). FM4-64 and fluorescently labeled cholera toxin subunit B, phalloidin and streptavidin were from Invitrogen. DETC activation in vitro and in vivo. The epidermal cell suspensions were cultured in the Iscove s modified Dulbecco s medium (HyClone, Logan UT) with 10% FBS, 25 mm HEPES, 6 mm L-glutamine, 1

50 µm 2-ME, penicillin (100 U/ml), streptomycin (100 µg/ml), and IL-2 (50 U/ml). After 24 h, the cells were transferred into plates coated with streptavidin and biotinylated anti-cd3ε antibody (2C11, 5 µg/ml). After 3 days, the cells were detached using 2 mm EDTA in PBS and analyzed by flow cytometry. For the analysis of γδ TCR and CD45 distribution the 7-17 DETC line was cultured on glass cover slips in the medium described above. For the kinase inhibition studies, ears were harvested from the mice and split into dorsal and ventral sides. Sections of skin (40-50 mm 2 ) were floated on the Hank s balanced salt solution with PP2, PP3 or 428205 (an Lck-specific inhibitor) (all at 20 µm; EMD Chemicals, Inc., Gibbstown, NJ) for 3 h at 37 C. The skin sections were then fixed, stained, and imaged by confocal microscopy. The activity of each compound was analyzed in comparison with untreated skin sample from the same mouse. The vehicle control (DMSO) gave similar results as the not treated condition. To induce TCR cross-linking in DETC in vivo, 40 µl of biotinylated anti-cd3ε antibody (2C11, 2.5 µg/ml) and fluorescently labeled streptavidin (5 µg/ml) were injected into the dorsal ear dermis in anesthetized mice. The ears were harvested after 0.5-4 h or after 7 days and analyzed by immunofluorescence microscopy. Full-thickness skin wounding was performed by 1 mm diameter ear punch. Topical PMA was applied at 20-100 µg/ml in acetone. The skin was either imaged in vivo or processed for immunofluorescent staining at different time points. TLR9 agonist CpG-A 2216 (40-100 µm; sequence: 5' G*G*G GGA CGA TCG TCG* G*G*G* G*G, where * indicates a phosphothioate linkage; Trilink Biotechnologies, Inc., San Diego, CA) was injected intradermally. The same fields of view in the injected ear were imaged in anesthetized mice for up to 9 days after the injection. Spatiotemporal analysis. The whole-cell masks (WCM) were generated by thresholding the GFP or CD3ε channels, and the mid-body region was obtained by erosion followed by dilation. The dendrite regions were obtained by subtracting the mid-body regions from the WCM. The local background was read out from DETC-adjacent regions obtained by dilating and subtracting the WCM. Alternatively, for the PALP number analysis, local background channel was created by median filtering the image with a radius of 8.9 µm. The dendrite regions of interest were set by circles of a radius (1.5-1.75 µm) at the end or beginning of the dendrite shaft. The dendricity index 4 (= Perimeter 2 / Area 4Π) was determined in maximum intensity projection images based on the WCM statistics in the Slidebook software; the 3D dendricity was calculated based on the reciprocal of the sphericity parameter in the Imaris software version 6 and 7 (Bitplane AG, Saint Paul, MN). The PALPs were identified using an automated macro 2

procedure based on a three-fold increase in py signal over the local background followed by size filtering. The CD3ε polarization was calculated as the ratio of the mean CD3ε fluorescence intensity at the distal halves of dendrites to the mean CD3ε intensity at the mid-body. Alternatively, the polarization index (CD3ε, py 142 -CD3ζ, NKG2D, and CD103) was calculated within the dendrite region as the ratio of the mean fluorescence intensity of a molecule of interest at the dendrite end region to that of at the beginning of the dendrite. Three-dimensional visualization and cell tracking were performed using Imaris. For short-term motility and shape-retention analysis (1-2 hours), six frames, equally spaced in time, were binarized and added (time projection). Therefore, pixels with the intensity value of 6 (red in pseudocolor scale) indicated that the structure was immobilized throughout the recording time. The DETC shape retention index was calculated as a ratio of the immobilized area (pixels with the intensity value of 6) to the total cell area during the recorded time (pixels with the intensities 1). Quantification of dendrite motility. Based on 2 h video recordings of DETC motility (spaced 2 min apart) kymographs were created using the reslice function in the ImageJ software. After thresholding coordinates of dendrite membrane position (x, t) were read out using the ImageJ software and the instantaneous velocity was calculated according to the equation: v = (x n+1 x n )/(t n+1 t n ). Next, the average and maximum velocity were determined for each analyzed dendrite. SUPPLEMENTAL REFERENCES 1. Hogquist, K.A. et al. T cell receptor antagonist peptides induce positive selection. Cell 76, 17-27 (1994). 2. Mombaerts, P. et al. RAG-1-deficient mice have no mature B and T lymphocytes. Cell 68, 869-877 (1992). 3. Kuziel, W.A. et al. Regulation of T-cell receptor gamma-chain RNA expression in murine Thy-1+ dendritic epidermal cells. Nature 328, 263-266 (1987). 4. Baxter, C.S., Andringa, A., Chalfin, K. & Miller, M.L. Effect of tumor-promoting agents on density and morphometric parameters of mouse epidermal Langerhans and Thy-1+ cells. Carcinogenesis 12, 1017-1021 (1991). 3

Supplementary Fig. 1 a Stratum corneum Stratum granulosum Stratum spinosum Basal lamina XZ view - skin Epidermis Dermis Subcutaneous fat Stratum corneum Squamous/stratum granulosum Tip of dendrite XY views z = 0 µm z = 3 µm Cuboidal/stratum spinosum z = 6 µm z = 7.5 µm z = 9 µm z = 11 µm z = 13.5 µm z = 15 µm XZ view - epidermis GFP FM4-64 b Intravital GFP-depth z = 0 bulbous swellings -11 µm 4

Supplementary Figure 1. Morphological polarity of DETCs in epidermis. (a) Position of DETCs relative to the skin and epidermal keratinocyte layers revealed by confocal microscopy of wild-type IL2p8-GFP mouse skin stained with FM4-64. The perpendicular projection was reconstructed from a stack of confocal images (z step = 0.2 µm). The inset and selected XY planes show the localization of a GFP + DETC in the epidermis (Supplementary Video 2). (b) Intravital images, represented as a color-coded depth projection (based on GFP signal), show apical dendrites ending with bulbous swellings (arrows). Scale bars, 10 µm. 5

Supplementary Fig. 2 Immediate fixation γδ TCR py Phosphatase pre-treated γδ TCR py 6

Supplementary Figure 2. Specificity control for py staining. Left: Fixed C57BL/6 mouse skin was stained for γδ TCR (red) and py (green). Fluorescence overlap results in the yellow color. The arrowheads indicate py clusters (PALPs). Right: A similar specimen was pre-treated with alkaline phosphatase before identical staining. Scale bar, 10 µm. 7

Supplementary Fig. 3 Tcrd / CD3ε # CD3ε (FAU) * 100 80 60 40 20 0 20 40 60 Distance (µm) * CD3ε (FAU) a 100 80 60 40 20 0 20 40 60 Distance (µm) WT & # CD3ε (FAU) & CD3ε WT b Tcrd / CD3ε py(142)-cd3ζ 100 80 60 40 20 0 20 40 60 Distance (µm) Rare WT-like e Typical Tcrd / CD3ε py c WT d CD3ε py(142)-cd3ζ Tcrd / WT-like 1 Tcrd / WT-like WT 2 1 1 GFP py(142)-cd3ζ 1 2 2 CD3ε 2 py(142)-cd3ζ Overlay Overlay MIP z=6 µm 0 255 8 Time projection 0 2h

Supplementary Figure 3. The epidermis of Tcrd -/- mice contains rare V γ 5-like αβ DETC clusters. (a) Confocal microscopy of non-treated skin of Tcrd -/- mice stained for CD3ε showing a rare wild-typelike clone of αβ TCR DETCs. The graphs represent profiles of CD3ε fluorescence intensity along the dashed lines in the indicated cells. An area of similarly stained wild-type skin and one magnified wildtype DETC is shown for comparison. (b) Rare V γ 5-like αβ DETCs in Tcrd -/- mice form PALPs including CD3ζ tyrosine phosphorylation. Note that the Tcrd -/- cells in the upper left marked with the symbol resemble wild-type cells, magnified in panel c. (c) The cells indicated in panel b, two wild-type and two Tcrd -/- wild-type-like, are shown in higher magnification. Bottom panel represents py(142)-cd3ζ levels of the DETCs in fire color scale. (d) Close-ups of highlighted PALPs from the panel c. Images in c and d are maximum intensity projections (MIP) from the confocal stacks with indicated z-span. (e) Using idisc approach in IL2p8-GFP Tcrd -/- mice we identified rare αβ DETCs with wild-type-like TCR and py polarization and immobilized dendrites during a 2-h time lapse (white arrowheads). White scale bars, 10 µm; yellow scale bar, 1 µm. Data show one representative of at least two independent experiments. 9

Supplementary Fig. 4 a 28 day tracking #8 #4 #14.5 #8 11.5 #8 #16 #20 #20 #2 2 14 * #25 12.5 3 #2.5 b Older than 48h Settlement Migration A Newly divided B Days after cell division: A - 26, 16 B - 14 C - 8 D - 17 #5 2 nd 18 19.5 11 B D C #23.5 C D #23 A e cell division (day) # cell disappearance (day) c Displacement (µm) 100 80 60 40 20 *** 0 0 Older Newly than 48 h divided 28 d Live Day 21 Day 27 Day 28 d Dendrite label * 1 week 2 weeks 3 weeks 4 weeks Time point 1 7 14 21 28 35 42 49 56 a b c d e f g h i j k l m n o p Fixed z = 0-11 µm o * j i GFP-depth o * j o i * j i γδ TCR py * Overlay 10

Supplementary Figure 4. Long-term in vivo anchoring and post-cytokinesis migration of DETCs at steady state. (a) Color-coded time projection of DETC tracks in an IL2p8-GFP reporter mouse visualized every 12 h over the course of 28 days. The arrows represent net displacements and identify cell divisions. The asterisk indicates an example cell analyzed in detail in d and e. (b) DETCs migrate for several days after dividing. All cell tracks are divided into two groups: cells that did not recently divide (older than 48 h), and cells that divided during the observation (newly divided). The tracks are translated to a common origin. The letters identify daughter cell pairs. (c) Comparison of the 28-day displacements of nondividing versus dividing cells. (d) 28-day history of dendrite anchoring in the cell marked with the asterisk in panel a. Dendrite anchoring was identified based on 30-min recordings every 12 h; the letters represent individual sites of dendrite anchoring in the order of appearance. (e) The three frames from Supplementary Video 6 showing time points on days 21, 27 and 28, represented as depth projections, and the same area imaged with confocal microscopy after staining for γδ TCR and py at the conclusion of 28 day-long intravital monitoring. The arrowheads and arrows indicate the dendrites that remained anchored for the last 7 days or 24 h, respectively. The inset shows a close-up of a dendrite end containing a central TCR cluster (arrow) in a dendrite that persisted 7 days. Scale bars, 20 µm; ***P < 0.001. 11

Supplementary Fig. 5 V a γ 5 NKG2D Overlay * V γ 5-depth b 100 80 V γ 5 NKG2D 60 z = 0 NFI 40 20 * - 8 µm 2 4 6 8 10 12 Distance (µm) 12

Supplementary Figure 5. Unlike Vγ5 TCR, NKG2D is not polarized toward the ends of dendrites at steady state. (a) Confocal microscopy of a wild-type mouse skin stained for Vγ5 TCR (red) and NKG2D (green). The arrowheads indicate the apical dendrites. (b) Fluorescence intensity profiles for Vγ5 TCR and NKG2D staining normalized to the local maximum (NFI) along the dashed line drawn along the dendrite marked with an asterisk in a. Scale bar, 5 µm. Data show one representative of at least two independent experiments. 13

Supplementary Fig. 6 a Cd247 / WT CD3ε py(142)-cd3ζ py(142)-cd3ζ CD3ε py(142)-cd3ζ py(142)-cd3ζ b C57BL/6 AKR C3H/HeN Vγ5 py(142)-cd3ζ BALB/c FVB/NJ py(142)-cd3ζ c XY view apical dendrite XZ view YZ view γδ TCR-depth Vγ5 py(142)-cd3ζ F-actin downward/horizontal dendrite T-shaped ends Vγ5 py(142)-cd3ζ elongated PALPs d z=0-14 µm Overlay py-free TCR cluster Conventional Vγ5 py(142)-cd3ζ Germ-free py(142)-cd3ζ Vγ5 py(142)-cd3ζ 14 py(142)-cd3ζ

Supplementary Figure 6. TCR/CD3ζ tyrosine phosphorylation at the ends of dendrites is common in different mouse strains and does not depend on microbiota. (a) Specificity control for py(142)-cd3ζ staining by comparing signals upon staining the skin from wildtype or Cd247 -/- mice. (b) Confocal microscopy of untreated skin biopsies from different strains of mice showing steady-state apical phosphorylation of CD3ζ. (c) Confocal microscopy of skin biopsies from AKR mice. Left top panel: Color-coded depth projections of a DETC (based on γδ TCR staining). Left bottom panel: AKR PALPs are elongated and some contain non-phosphorylated TCR clusters (arrows). Right panel: AKR PALPs align with squamous keratinocyte junctions (arrows). (d) Tyrosine phosphorylation of CD3ζ is unaffected in the skin in germ-free Swiss Webster mice. Scale bars, 10 µm. 15

Supplementary Fig. 7 a FcεR1γ CD3ε py(142)-cd3ζ Overlay b WT Tcrd / chimera CD3ε py(142)-cd3ζ Vγ5 NT 1 Overlay CD3 XL WT NT 1 XZ view of cell 1 Sc py(142)-cd3ζ ( 104) (FAU) CD3 XL Tcrd / 6 **** 4 2 0 Vγ5+ 16 Vγ5

Supplementary Figure 7. TCR functionality in DETCs in wild-type, Tcrd -/- and wild-type Tcrd -/- chimeric mice. (a) Intradermal injection of biotinylated anti-cd3ε cross-linking antibody induces extensive phosphorylation of CD3ζ both in wild-type and Tcrd -/- DETCs. The skin was fixed after 4 h and stained with py(142)-cd3ζ. In addition, fluorescent-labeled streptavidin confirms intravital CD3ε binding. The cell body is visualized based on for FcεR1γ. Note that FcεR1γ, which is known to incorporate in γδ TCR complexes, is recruited to PALPs as expected. Since py(142)-cd3ζ is induced across the cell membrane, including outside PALPs in wild-type and in Tcrd -/- DETCs, all TCRs are accessible for ligand binding and functional. Data show one representative of at least two independent experiments. (b) Putative apical ligand for V γ 5 TCR is present in Tcrd -/- epidermis and the microenvironment supports PALP formation. Wild-type fetal thymocytes were transferred into newborn Tcrd -/- recipient, and six weeks later the ear skin of the recipient was stained for V γ 5, CD3ε, and py(142)-cd3ζ. Note that unlike the host-derived V γ 5 - CD3 + DETCs, the donor-derived V γ 5 + cell has an apically directed dendrite that ends with a PALP including py(142)-cd3ζ. The graph quantifies the results in terms of py(142)-cd3ζ levels in whole cells. n=13 (V γ 5 + ) and 47 (V γ 5 - ) cells, ****P < 0.0001. Scale bars, 10 µm. 17

Supplementary Fig. 8 a V γ 5 LFA-1 Overlay V γ 5-depth z = 0-7µm b NFI 100 80 60 40 V γ 5 LFA-1 c LFA-1 (FAU) 400 300 200 NS d WT Tcrd / 20 100 0 5 10 15 Distance (µm) 20 0 WT Tcrd / 10 0 10 1 10 2 10 3 10 4 LFA-1 18

Supplementary Figure 8. Co-clustering of LFA-1 with V γ 5 TCR at the ends of apical dendrites. (a) Whole-mount fixed skin from a C57BL/6 mouse was stained for V γ 5 TCR and LFA-1. As in all other confocal imaging, fluorescence was excited sequentially and no bleed-through signals were observed in singly stained specimens (not shown). The arrowheads show apical co-clustering. Scale bar, 10 µm. (b) V γ 5 TCR and LFA-1 image intensity profiles in a typical DETC along the dashed line in a. (c, d) LFA-1 levels do not change in the absence of γδ TCR as quantified by flow cytometry in epidermal cell suspensions from wild-type and Tcrd -/- mice, gated on CD3 + cells. Each symbol represents the mean from one mouse. The bars are the overall means. The graph in d shows representative histograms. Data show one representative of at least two independent experiments. 19

Supplementary Fig. 9 a γδ TCR CD45 Overlay γδ TCR-depth C57BL/6 z = 0-11µm AKR z = 0-14µm b γδ TCR CD45 Overlay C57BL/6 V γ 5 CD45 Overlay AKR NFI NFI 100 γδ TCR 80 CD45 60 40 20 0 1 2 3 Distance (µm) 100 80 60 40 20 V γ 5 CD45 0 1 2 3 4 5 Distance (µm) c In epidermis In vitro TCR/CD45 ratio 3.0 2.0 1.0 0.0 A A B C D *** B C D γδ TCR CD45 3.0 2.0 1.0 0.0 A B C D A B C D 20

Supplementary Figure 9. CD45 patterning in C57BL/6 and AKR mice. (a) Confocal microscopy of skin biopsies from C57BL/6 and AKR mice stained for γδ TCR (red) and CD45 (green) showing CD45 separation from γδ TCR in apical dendrites in DETCs visible as orange rather than yellow tint in the overlay. The depth is color-coded based on the TCR signal, and the arrowheads indicate the apical dendrites. (b) Maximum intensity projections of three to five confocal sections (z-step, 0.2 µm) of the stained skin as in a. The fluorescence intensity profiles were drawn along the indicated lines. (c) The differential distribution of CD45 and γδ TCR is evident in skin-resident but not in vitro-cultured DETCs. The TCR to CD45 ratios were quantified in four regions along dendrites as marked with the capital letters. The in vitro DETC is the 7-17 DETC line. The data was analyzed with the Friedman test, followed by Page s trend test. ***P < 0.001, n=41 (epidermis) and 12 (in vitro cultured) cells. The white scale bars, 5 µm; the yellow scale bars, 1 µm. Data show one representative of at least two independent experiments. 21

LEGENDS FOR SUPPLEMENTARY VIDEOS Supplementary Video 1 Apical dendrites are stably anchored. A 3.5-h time lapse shows the motility of DETCs in an IL2p8- GFP mouse. The GFP signal is represented as a color-coded depth projection. Note the dichotomy of dendrite anchoring depending on the vertical position of the dendrites. The DETC mid-bodies are immobilized. The fast moving cell is a GFP low dermal T cell. Supplementary Video 2 Three-dimensional confocal visualization of DETCs forming apical PALPs in healthy epidermis. Part 1: Travel through a confocal z-stack from the apical stratum corneum down through the epidermis and into the dermis in the skin from an IL2p8-GFP transgenic mouse. Cell boundaries were visualized by staining with FM4-64. The color-coded projection is based on the GFP signal. Note that DETCs extend projections toward squamous keratinocytes, ending with bulbous swellings. Asterisks indicate apical dendrites (blue tint). See also Supplementary Fig. 1b. Part 2: Three-dimensional visualization of DETC morphology in an AKR mouse using iso-surface rendering. The cell surface of the DETCs (red) was reconstructed from the γδ TCR signal. The green signal represents CD45 fluorescence of Langerhans cells. Part 3: Three-dimensional visualization of a group of DETCs in an IL2p8-GFP mouse stained for py. Note that the massive clusters of tyrosine-phosphorylated proteins are only present at the ends of apical dendrites. Part 4: A single DETC stained for γδ TCR and py. Supplementary Video 3 Intravital dynamics-immunosignal correlative microscopy shows that DETC dendrites are anchored in the apical epidermis through PALPs depending on γδ TCR. In a side-by-side representation, the first part shows the in vivo dynamics of several DETCs in wild-type (left) or Tcrd -/- mouse (right) over the course of 60 min. This dynamics is also represented in color-coded scale whereby the red represents persistence for at least 50 min, and the orange, yellow, green, and cyan colors represent areas of decreasing persistence in 10 min steps. Immediately after the recording, the skin was fixed, analyzed by immunofluorescence for CD3ε and phosphoproteins, and aligned with the intravital video. Note that the sites of dendrite anchoring that are apically located and enriched in TCR(CD3ε) and py are numerous in wild-type and infrequent in Tcrd -/- mice. Corresponds to Fig. 1a. 22

Supplementary Video 4 Anchoring of apical dendrites depends on γδ TCR. Comparison of DETC motility in wild-type- and Tcrd -/- - IL2p8-GFP reporter mice. Two color-coded time lapses are shown in a sequence. The arrows indicate anchored points that persisted during the 2-h recording. Supplementary Video 5 Anchoring of apical dendrites depends on V γ 5 TCR. Using the intravital dynamics-immunosignal correlative microscopy, the dynamics of DETCs in a wild-type IL2p8-GFP mouse was recorded followed by skin fixation and staining for V γ 5 γδ TCR. The cells that were immobilized throughout the 2-h recording (labeled with asterisks) express V γ 5 TCR, as shown in the last frame. See also Fig 1i, j. Supplementary Video 6 DETCs remain anchored for days. The movie shows the 28-day DETC persistence experiment in which the same group of cells was tracked over time every 12 h in a repeatedly anesthetized IL2p8- GFP mouse. The red asterisk in the first frame indicates the cell that is represented in more detail in Supplementary Fig. 3d, e. The white arrowheads indicate DETCs about to undergo division and the red arrowheads indicate the resulting daughter cells. Immediately after the last intravital recording, the skin was fixed and analyzed for py levels (shown is a composite of several images). In the tracking analysis image, green spheres correspond to the DETCs; the track lines are time-coded, and arrows show the track displacements. Cells that disappeared or underwent a division are labeled with symbols, with a number indicating a time point when the event occurred. Supplementary Video 7 DETC dynamics in response to stress stimuli. The analysis of DETC motility in stressed tissue. The movie contains a series of 2-h (or longer) intravital recordings of DETC motility at steady state, after punch biopsy and after applying two different doses of PMA in anesthetized IL2p8-GFP mice. The stress-affected areas were imaged 6 min, 24 h or 72 h after the stress stimulus was applied. The 2-h recordings are followed by a time-projection image based on six movie frames (every 24 min) showing DETC immobilization (immobilized area in red). The asterisk indicates a wound border-proximal DETC, with bulbous swellings at the dendrite ends (characteristic of PALPs) that detaches the dendrites after punch biopsy. The second steady-state movie and PMA (20 µg/ml) present in the same field of view 23

before and 24 h after the treatment. Arrowheads indicate the anchored dendrites at steady state that persisted despite PMA application and the detachment of the majority of dendrites. Supplementary Video 8 Skin inflammation after TLR9 stimulation induces redistribution of TCR signaling. Untreated or CpG-A 2219 injected skin from an IL2p8-GFP mouse (9 days after treatment) was stained for py(142)- CD3ζ (green) and F-actin (white); the GFP signal is in red. In addition, collagen fibers in the dermis were visualized by second harmonic generation using 2-photon microscopy. The three-dimensional rendering is based on XY stacks of images using the Imaris software. Supplementary Video 9 DETC-Langerhans cell synapse. This movie shows a 3D rotation of a DETC-LC conjugate that formed 9 days after intradermal injection of CpG-A 2219 into a CD11c-YFP mouse. The skin was harvested and stained for py(142)-cd3ζ (green) and V γ 5 TCR (red). The YFP signal from a LC is in blue. 24