Supporting Information

Iftakhar-E-Khuda et al. 10.1073/pnas.1602357113 SI Methods P (Roche) and 0.8 mg/mL neutral protease (Worthington Mice. C57BL/6J (stock 000664) WT, and MSR KO (B6.Cg- Biochemical) for 45 min at 37 °C. The isolated cells were cul- tm1Csk tured in gelatin-coated flasks in complete mouse endothelial cell Msr1 /J (stock 006096) mice were purchased from The − − medium/w (Cell Biologics) for 7 d. Thereafter, CD45 /CD11b / Jackson Laboratory. All animal experiments were approved by + + + the Ethical Committee for Animal Experimentation in Finland. podoplanin /CD31 /Lyve-1 cells were low-pressure sorted using FACSAria IIu fitted with a 100-μm tip. Sorted LECs were seeded Antibodies. Detailed information of the antibodies used is given in at a concentration of 5 × 105 cells/cm2 andculturedfor7d. Table S3. Microarray Analysis. RNA amplification, labeling, and cDNA LCMP. Inguinal, axillary, brachial lymph nodes (LNs) from C57BL/ microarray hybridization and expression analyses were 6J mice aged 8 wk were used for the isolation of LECs from the SS performed at the Miltenyi Biotec Genomic Services (Miltenyi and LS. LNs were collected in Rnase-free phosphate buffer saline Biotec). The sorted cells in SuperAmp Lysis Buffer were sub- containing 1 unit/μL SUPERase-In (Applied Biosystem) and jected to SuperAmp RNA amplification based on a global PCR thereafter, placed onto a layer of OCT containing 20 μg/mL protocol using mRNA-derived cDNA according to Miltenyi vanadyl ribonucleoside complex (Sigma-Aldrich) solution. More Biotec’s undisclosed procedure. The integrity of the cDNA was solution was added over the LNs until they were completely checked with the Agilent 2100 Bioanalyzer platform (Agilent covered. The filled cryomolds were immediately frozen in dry-ice Technologies). The average length of the highly amplified cDNA chilled isopentane and stored in a −70 °C freezer until use. products ranged between 200 and 1,000 bp. The cDNAs (250 ng) Alexa Fluor 488 anti-mouse CD31 (Biolegend) and PE-Texas were used as templates for Cy3 labeling using Miltenyi Biotec’s red anti-mouse F4/80 (Invitrogen) antibodies were applied onto undisclosed protocol. The Cy3-labeled cDNAs were hybridized the frozen sections of the LNs for 3 min at 4 °C. After washing, overnight (17 h, 65 °C) to Agilent Whole Mouse Genome Oligo the sections were dehydrated through ascending concentrations Microarrays 8× 60 K using Agilent’s recommended hybridization of ice cold [70%, 95%, and 100% (vol/vol)] for 10 s each chamber and oven. Fluorescence signals of the hybridized Agi- and finally in xylene for 10 s. lent Microarrays were detected using Agilent’s Microarray Scanner Microscopic observations of the cells were performed using a System (Agilent Technologies). The Agilent Feature Extraction Zeiss microscope with filter set 25 (Zeiss) and LCMP was con- Software was used to read out and process the microarray image ducted on a PALM microbeam instrument including Palm-Robo files. The software determines feature intensities (including back- version 3 software. To maximize the collection of the right cells, in ground subtraction), rejects outliers, and calculates statistical + the SS only discrete CD31 cells were collected. In the LS area, confidences. The microarray data produced in this study has been the blood vessels stain much brighter for CD31 than do LECs. deposited to GEO archive (GSE 68371). Moreover, LS lymphatics can be morphologically discriminated In bioinformatics analyses, the intensity data were background from the blood vessels. SS and LS endothelial cells were col- corrected and quantile normalization was conducted between the lected based on these characteristics using a 40× objective and arrays. The normalized intensities were log2 transformed and the fluorescence filter. LCMP of the labeled cells was performed used as a basis for further analysis. Interexperiment correlation under bright-field optics. The following settings were used for analysis of normalized log2 intensities was performed using Eu- the microdissection procedure: dissection method, Robo LPC; clidean distance metric. The differential be- UV energy, 50–65; UV focus, 45–50; and cutting speed, 20. tween the SS and the LS of the LCM samples was tested with a Microdissected cells were collected to adhesive caps (Zeiss); one-way ANOVA and an adjusted P value was calculated by the before their use, the caps were irradiated with UV (254 nm) for method of Benjamini and Hochberg (24). All samples of the SS 30 min. Five cells were isolated per slide to minimize the time and LS populations regardless of the isolation method were (15–20 min) and maximize the quality of RNA. Altogether 50 further used as a test and reference group, respectively, by per- cells were collected per experiment. The cells were collected forming Student’s t test (two tailed). The differentially expressed into the superamp lysis buffer (Miltenyi Biotec), incubated at 45 °C (DE) were selected requiring an absolute fold-change >2 for 10 min, and thereafter stored at −40 °C. and a P < 0.05. For stringent selection, the magnitude of the expression difference was at least eightfold. A detection P value Isolation and Sorting of LECs. Peripheral LNs and MLNs from 12 (Rosetta resolver error model) (25) was also calculated for each mice were pooled and digested in a freshly prepared mix reporter to indicate whether the signal is reliable or not. This [PBS containing 25 μg/mL liberase TM research grade (Roche) P value was used in further filtering of the data to allow only and 100 μg/mL DNase I (Roche)]. For cell sorting, the following records with the majority of samples fulfilling the detection cri- antibodies were used: APC-conjugated lineage (CD3e, CD11b, teria (detection P < 0.01) in the group with higher expression. CD45R/B220, erythroid cells, Ly-6G, and Ly-6C) mixture, with Functional grouping analyses were performed using the dif- an isotype control (BD Pharmingen), PE anti-CD73 (BD Phar- ferentially expressed genes as input gene populations. The an- mingen), and PE/Cy7 anti-podoplanin (Biolegend). Seven-AAD notations used were derived from databases (Calbiochem) was used to exclude dead cells. The 106 cells/100 μL and various other pathway resources curated by Miltenyi Biotec. were incubated with 1 μL of each antibody for 15 min at 4 °C The differentially expressed reporters were tested for significant before acquisition on a FACSAria IIu (BD Bioscience). A total of enrichments of annotations using the TreeRanker software 1,200 cells were sorted into FCS-coated FACS tubes con- (Miltenyi Biotec). The frequency of the association of a category taining RPMI-1640 and 20% (vol/vol) FCS; cell purity was with the input reporter set was compared with that of a back- more than 98%. The gating was always done based on the ground set (Agilent 8× 60 K array genes). P values were com- stainings with negative control antibodies. puted by Fisher’s exact test with Benjamini–Hochberg correction For primary cell cultures, mouse peripheral and mesenteric for multiple testing. Values ≤0.05 indicate a significant enrichment LNs were digested with PBS containing 0.2 mg/mL collagenase of the respective category relative to the background (all reporters

Iftakhar-E-Khuda et al. www.pnas.org/cgi/content/short/1602357113 1of10 with a gene ID () of the Agilent Whole Mouse Genome Flow Cytometric Analyses. Enzyme digestions and antibody staining Oligo Microarray 8× 60 K). The data were further analyzed using of LNs were performed as described in Isolation and Sorting of GENE-E analysis platform (https://www.broadinstitute.org/cancer/ LECs. The skin was digested for 45 min in a freshly prepared software/GENE-E/). enzyme mix (PBS containing 8 mg/mL collagenase IV from Sigma and 100 μg/mL DNase from Roche). The following pri- Immunohistochemical Analyses. Frozen sections were stained with mary, secondary, and isotype-control antibodies were used for primary antibodies: rat anti-mouse MSR1/CD204 (Lifespan stainings: APC-Cy7 rat anti-mouse CD45 (BD Pharmingen), Bioscience), rat anti-mouse Siglec-1/CD169 (AbD Serotec), rat APC-Cy7 rat anti-mouse CD11b (BD Pharmingen), PE/Cy7 Sy- anti-mouse EMCN (EMD Millipore), rabbit anti-mouse Lyve-1 rian hamster anti-mouse podoplanin (Biolegend), APC rat anti- (ReliaTech), Syrian hamster anti-mouse podoplanin/gp36 (Abcam), mouse CD31 (Biolegend), PE-conjugated rat anti-mouse Lyve-1 and rat anti-mouse PLVAP-1 (Meca 32, gift from Eugene Butcher, (R&D Systems), PE rat IgG2A κ-isotype control antibody (BD Stanford University, Stanford, CA). The second stage reagents Pharmingen), rat anti-mouse CD204 (Lifespan Bioscience), rat were: Alexa Fluor 546 goat anti-rat IgG (H+L) (Invitrogen), Alexa anti-mouse CD169 (AbD Serotec), rat anti-mouse EMCN (EMD Fluor 488 goat anti-rabbit IgG (H+L) (Invitrogen), Alexa Fluor Millipore), eFluor 660 anti-mouse EMCN (eBioscience), FITC- 546 goat anti-rabbit IgG (H+L) (Invitrogen), and Alexa Fluor 488 conjugated rat IgG1 λ-isotype control (Biolegend), FITC-conjugated goat anti-hamster IgG (H+L) (Invitrogen). The following direct rat IgG2A κ-isotype control (BD Pharmingen), FITC-conjugated rat conjugates were used: Alexa Fluor 488-conjugated rat anti-mouse IgG2B κ-isotype control (BD Pharmingen), and FITC-conjugated CD204 (BIO-RAD) and Alexa Fluor 488-conjugated rat anti- goat anti-rat IgG (whole molecule) (Sigma). Cells were analyzed with mouse Lyve-1 (R&D Systems), Alexa Fluor 647 rat anti-mouse FACSAria IIu and LSR Fortessa (both BD Biosciences) using CD4 (BD), Pacific Blue rat anti-mouse CD45R (BD), and Alexa FlowJo software (Tree Star). Fluor 488 rat anti-mouse CD8 (BD) or Alexa Fluor 488 rat anti- mouse CD31 (Biolegend). Finally, the sections were mounted in Adhesion Assays. Ex vivo. ProlongGold anti-fade reagent (Molecular Probes) and images In principle the adhesion assays were performed as de- were acquired using an Olympus BX60 microscope or an LSM 780 scribed earlier (26). Briefly, human were isolated confocal microscope (Carl Zeiss) and Intelligent Imaging Inno- from peripheral blood by Ficoll purification and mouse lym- vations (3i) Marianas Spinning disk confocal microscope (Intelligent phocytes from mesenteric and peripheral LNs. Isolated lym- Imaging Innovations). Slice thickness was determined with a pin- phocytes were incubated on freshly cut frozen sections of human hole adjustment. Background subtraction and adjustment of bright- LNs or mouse peripheral LNs pretreated with the following antibodies for 30 min: for human, anti-human MSR1 (Abcam), ness and contrast were performed using ImageJ software. anti-human CD169 (Abcam) and the negative isotype control Alternatively, the LNs were fixed in 4% (wt/vol) parafor- antibody AK-1 (In Vivo Biotech) and normal rabbit serum; for maldehyde followed by embedding in paraffin. Sections were mouse, a pool of anti-mouse MSR1 antibodies (monoclonal from stained with primary antibodies: rabbit anti-mouse Lyve-1 anti- Lifespan Bioscience and rabbit polyclonal from GeneTex rec- body (ReliaTech) and Syrian hamster anti-mouse podoplanin/ ognizing both mouse and human MSR1) and a pool of normal gp36 antibody (Abcam). The secondary antibodies were: Alexa rabbit serum and rat serum as a negative control. Thereafter the Fluor 546 goat anti-rabbit IgG (H+L) (Invitrogen) and Alexa + sections were washed twice. In another set of assays, the LNs were Fluor 488 goat anti-hamster IgG (H L) (Invitrogen). taken from MSR1 KO and WT mice. Lymphocytes were allowed Primary MLECs were stained with directly conjugated primary to bind to the sinuses for 5 min in rotatory conditions, then in antibodies: Alexa Fluor 647 rat anti-mouse CD204 antibody steady state conditions for 15 min, followed by 5 min in rotatory (BIO-RAD) and Alexa Fluor 488 anti-mouse CD31 (Biolegend). conditions and thereafter, 15 min without rotation at +7 °C. The For intracellular staining, the cells were fixed with 4% parafor- nonbound cells were gently decanted off from the sections and maldehyde, blocked with 0.1 M glycine and incubated with 0.2% adherent cells were fixed in 1% glutaraldehyde. The number of saponin. Goat anti-human PROX1 antibody (R&D Systems) + lymphocytes bound to the SS and LS was counted under dark- followed by Alexa Fluor 546 rabbit anti-goat IgG (H L) field microscopy. To be able to standardize day-to-day variations (Invitrogen) was used for staining. Images were acquired with between the experiments, the binding to the control sections was an LSM 780 confocal microscope (Carl Zeiss) and analyzed set to 100% by definition. Macroscopically and microscopically with Zen 2010 software (Carl Zeiss). normal LNs from three anonymous human donors (obtained For whole mount staining, the ears collected from WT and from surplus tissue from surgical operations) and blood samples MSR1 KO mice were mechanically separated into the dorsal and for purification from healthy volunteers (informed ventral halves. The dorsal sheets were fixed in 2% PFA for 20 min consent obtained) with the permission of the Ethical Committee and dehydrated with 50% methanol followed by 100% methanol of Turku University Hospital and three different KO and WT − for 10 min in each phase. The ears were stored in 20 °C in 100% mice were used. methanol. Before staining, the ears were rehydrated in 50% In vitro 1. A MSR1-Fc fusion expressing the extracellular methanol followed by two 10-min washes with PBS. The ears domain of mouse MSR1 fused to the human IgG2 Fc-tail was were blocked with 1% NMS and 0.5% FCS in PBS-MT [PBS generated. The extracellular domain (amino acids 79–458) was with 1% skim milk and 0.4% Triton X-100 (Sigma)] for 1 h on PCR cloned from a full-length cDNA clone for mouse MSR1 ice. The samples were incubated with antibodies in PBS-MT (cat. no. MG222674; Origene) using primers containing BamHI at +4 °C overnight. The primary antibodies used were rabbit anti- and NheI restriction enzyme sites. The PCR was carried out mouse Lyve-1 (ReliaTech) and rat anti-mouse MSR1 (Lifespan using Phusion High-Fidelity DNA Polymerase (ThermoFisher Biosciences). The secondary antibodies were Alexa Fluor 546 Scientific). The amplified fragment was purified and annealed to goat anti-rabbit IgG (H+L) (Invitrogen) and Alexa Fluor 647 BamHI and NheI digested pFUSEN-hG2Fc vector (from Invi- Goat anti-rat IgG (H+L) (Invitrogen), which were incubated in voGen). The resulting clone was sequenced for verification. the presence of 5% NMS. Samples were imaged with the In- MSR1-Ig chimera and control human Ig (50 μg/mL) were telligent Imaging Innovations (3i) Marianas Spinning disk confo- incubated with mouse lymphocytes isolated from WT PLNs and cal microscope (Intelligent Imaging Innovations) using the 20×/0.8 MLNs for 30 min at room temperature. The binding was de- N.A. objective and the Hamamatsu ORCA-Flash 4.0 v2 sCMOS tected using biotinylated anti-human Ig Fc (Abcam) followed by camera (Hamamatsu Photonics). SlideBook 6 (Intelligent Imaging detection with Alexa Fluor 488 streptavidin (Invitrogen). Lym- Innovations) software was used for acquiring the images. phocytes were stained with the following lymphocyte markers:

Iftakhar-E-Khuda et al. www.pnas.org/cgi/content/short/1602357113 2of10 PE-conjugated CD45R (BD), APC Cy7-conjugated CD4 (BD), with ProlongGold antifade reagent (Molecular Probes). Tile scan BV650-conjugated CD8a (BD), and APC-conjugated CD11b images from LN sections were acquired with an LSM 780 confocal (BD). The samples were ran with LSR Fortessa (BD Biosci- microscope (Carl Zeiss) with a c-Apochromat Korr M27 40× ences) and analyzed with FlowJo (Tree Star). Statistics were objective with a N.A. of 1.20 and Zen software (Carl Zeiss). With obtained by using the Mann-Whitney u test. the confocal microscope the slice thickness was determined with + In vitro 2. Lymphocyte binding to primary MLECs was tested as a pinhole adjustment. The distance of CFSE cells in the LN previously described (27). MLECs were seeded and cultured parenchyma (excluding the cells in the medullary area) from the overnight, followed by treatment with anti-MSR1 or control SS was measured manually using Zen2012 (Carl Zeiss). Roundish antibody for 30 min. Antibody solutions were removed and particles with diameter >5 μm were measured. The shortest dis- carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled tance from the cell center to the SS was measured. Numerical lymphocytes were added and incubated for 30 min at 7 °C with values obtained from WT and KO samples were compared with 60 rpm. Adherent lymphocytes were fixed with 1% glutaralde- Mann–Whitney u test. The P < 0.05 were considered significant. hyde. The number of lymphocytes bound to MLECs in presence of anti-MSR1 and the control antibody was counted manually in × ’ Depletion of . SS macrophages were depleted by 60 microscopic fields using a 40 objective. Student s t test was subcutaneous injections of 0.1 mg clodronate or control liposomes used for the statistical analyses of both types of adhesion assays. (28) to the footpads. Lymphocyte Migration In Vivo. Lymphocytes isolated from the μ RT-PCR. To detect the MSR1 transcript variants Buffy coats from spleens and PLNs of WT donor mice were labeled with 0.5 M healthy donors were obtained from the Finnish Red Cross, and CFSE and injected subcutaneously into the hind paws of WT or monocytes were purified from peripheral blood mononuclear MSR1 KO mice in the concentration 10 × 106 cells/25 μLor1× 106 cells using negative or positive selection kits (Miltenyi Biotec). cells/25 μL of PBS. Cells were allowed to migrate via lymphatic Monocytes were cultured with M-CSF 10 ng/mL (Biolegend) for vessels for 12 h (Fig. 5G)or4.5h(Fig.5H) before the mice γ were killed, and draining popliteal LNs were collected for six days and polarized for 1 d with IFN- 20 ng/mL (R&D Sys- further analyses. tems) or IL-4 20 ng/mL (PeproTech) toward the M1 or M2 Single cell suspensions of the LNs for flow cytometry were phenotypes. M0 cells were cultured with M-CSF blocked with mouse BD Fc block (BD) followed by antibody 10 ng/mL for 7 d. Human lymphatic endothelial cells (HLECs) staining with lymphocyte markers Pacific Blue-conjugated anti- and human dermal lymphatic endothelial cells (HDLECs) were mouse CD45R (BD), Alexa Fluor 647-conjugated anti-mouse purchased from ScienCell Research Laboratories and PromoCell, CD4 (BD), and PerCP Cy5.5-conjugated anti-mouse CD8a (BD). respectively. HLECs were cultured in endothelial cell medium and Flow cytometry was performed using LSRII or LSR Fortessa (BD HDLECs in endothelial cell growth medium. The cells were Biosciences) and data were analyzed using Flowing Software 2 used for RNA isolation up to passage seven. Total RNA was (Perttu Terho, Center for Biotechnology, www.uskonaskel.fi/ isolated using the NucleoSpin RNA extraction kit (Macherey- flowingsoftware/) or FlowJo software (Tree Star). Gating strat- Nagel) and PCR reactions were carried out with Kapa Robust egies of lymphocyte subpopulations are shown in Fig. S6 A–C. (Kapa Biosystems). LN samples for microscopy were collected under stereomi- croscope (Leica) and all samples were embedded in a similar Statistical Analyses. Statistical analyses have been indicated in each orientation. Unfixed frozen sections were stained with un- methodological description and if not, a two-tailed Student’s t test conjugated rabbit anti-mouse Lyve-1 (Reliatech) followed by was performed. The P values have been presented in figures/ Alexa Fluor 546 goat anti-rabbit IgG (Invitrogen) and mounted figure legends.

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Fig. S1. Gating startegy for the identification and isolation of fresh SS and LS LECs by triple immunolabeling and FACS sorting. The starting population (P1 in + − A) was further gated to exclude aggregated (B and C), dead (D), lineage and podoplanin cells (E and F). LECs were identified by their lineage negativity and positive podoplanin expression. Within the vital podoplanin+ LEC population, the nonoverlapping subpopulations of SS and LS were discriminated by anti- CD73 immunolabeling (F and the histogram in G), the sorted CD73 negative and positive populations are indicated (G).

Iftakhar-E-Khuda et al. www.pnas.org/cgi/content/short/1602357113 4of10 Fig. S2. Heat map of the 50 most up- or down-regulated genes in LECs of the SS in comparison with the LS. The expression values are centered to the median log2 intensity value of all samples and the colors represent the relative expression of a given gene in comparison with the median of all samples. The heat map color scheme with the corresponding log2 values is given. Only the genes with a Refseq record are shown. (Left) The genes are up-regulated in LS; (Right)the genes are down-regulated in LS.

Iftakhar-E-Khuda et al. www.pnas.org/cgi/content/short/1602357113 5of10 Chemokines IL-6 family receptors TLR Transcription factors Ccl1 Il11ra1 Tlr1 Ar Ccl2 Il27ra Tlr2 Dach1 Ccl3 Il6ra Tlr3 Eqr2 Ccl4 Il6st Tlr4 Esrrq Ccl5 Lifr Trl5 Ets2 Osmr Foxp4 Ccl6 Gata2 Ccl7 Heparan sulfate Heyl Ccl8 IL-10, interferon proteoglycans Hlx Ccl9 family and Hoxd8 Ccl11 Id1 Ccl12 Ifna5 Sdc4 Id3 Ccl17 Ifnar1 Cd44 Junb Ccl19 Ifnar2 Col8a1 Klf12 Ifnqr1 Gpc1 Ccl20 Gpc4 Lhx2 Ccl21a Ifnqr2 Gpc6 Meis2 Ccl21b Il10rb Hspq2 Meox2 Ccl22 Il20rb Sdc1 Msx1 Ccl24 Sdc3 Mycn Ccl25 IL-12 family and receptor Sdc4 Nfil3 Ccl26 Nkx2-3 Il12rb1 Nr2f2 Ccl27a Il23a Nr4a1 Ccl28 Nr4a2 Cx3cl1 Common β chain receptor Itqa1 Nr4a3 Cxcl1 Itqa2 Pparq Cxcl2 Csf2ra Itqa2t Rarq Cxcl3 Csf2rb Itqa3 Smad2 Cxcl5 Il3ra Itqa5 Smad6 Cxcl9 Itqa6 Smad7 Cxcl10 Itqa9 Sox17 Cxcl11 Common γ chain receptor Itqam Sox7 Cxcl12 and Itqav Tcf7l1 Cxcl13 Itqb1 Tsc22d1 Cxcl14 Il15 Itqb3 Cxcl15 Il15ra Itqb4 GPCR Cxcl16 Il4ra Itqb5 Cxcl17 Il2rq Adora2a Pf4 Ig CAM Adrb2 Rarres2 TNF family Adrb3 Cadm1 Bdkrb2 Chemokine receptors Ltb Esam Cd97 Tnf Icam1 Celsr1 Darc Tnfsf9 Icam2 Ednrb Ccbp2 Tnfsf10 Jam2 F2rl3 Ccr1 Tnfsf11 L1cam Fzd6 Ccr2 Tnfsf12 Madcam1 Gpr124 Ccr3 Tnfsf15 Pecam1 Gpr17 Ccr4 Pvrl1 Gpr182 Ccr5 TNF receptor Pvrl2 Gpr35 Ccr6 Pvrl3 Gpr4 Ccr7 Cd40 Vcam1 Gpr44 Ccr8 Eda2r Vsiq2 Gpr68 Ccr9 Fas Gpr81 Ccr10 Ltbr VEGF family and Gpr97 Ccr1l1 Nqfr receptor Gprc5a Ccrl1 Relt Gprc5b Ccrl2 Tnfrsf1a Lpar6 Cx3cr1 Tnfrsf1b Veqfa Npr2 Cxcr1 Tnfrsf9 Veqfb Npr3 Cxcr2 Tnfrsf10b Veqfc P2ry2 Cxcr3 Tnfrsf11a Flt1 Ptqer4 Cxcr4 Tnfrsf11b Flt4 Sstr4 Cxcr5 Tnfrsf12a Kdr Tbax2r Cxcr6 Tnfrsf13b Nrp1 Tshr Cxcr7 Tnfrsf13c Nrp2 Tbfrsf14 SS LS Tnfrsf21 TGF-β family and IL-6 family Tnfrsf22 Tnfrsf23 receptor Cntf Tnfrsf26 Tqfb1 Il27 Tqfb2 Il6 SS LS Tqfb3 SS LS Tqfbr1 Tqfbr2 Tqfbr3 1.1 18.9 SS LS

Fig. S3. Expression of selected groups of genes in LS vs. SS. The genes in the heat map are colored based on the average normalized log2 intensities. Red font: genes differentially expressed between SS and LS with a fold-change >8. Blue font: genes differentially expressed between SS and LS with a fold-change between 2 and 8.

Iftakhar-E-Khuda et al. www.pnas.org/cgi/content/short/1602357113 6of10 PLN MLN A MSR1 Podoplanin Merge B MSR1 Lyve-1 Merge SS SS LS SS

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Fig. S4. MSR1, Siglec-1, and EMCN expression in PLN and MLN. (A) Two color immunostaining of PLN with anti-MSR1 (magenta) and anti-podoplanin (cyan). (B) MLN staining with anti-MSR1 (magenta) and anti–Lyve-1 (cyan). (C) MLN staining with anti–Siglec-1 (magenta) and anti–Lyve-1 (cyan). (D) MLN staining with anti-EMCN (magenta) and anti–Lyve-1 (cyan). EMCN+ positive stromal cells (negative for Lyve-1) in SS are indicated by an open arrowhead. Insets of the first + − rows in A–D are shown in higher magnifications in the Middle rows (also in the Bottom row in A, SS area) and Bottom rows (LS area, B–D). EMCN , MSR1 ,and − Siglec-1 LS are marked with arrowheads, the arrows point to the floor of SS and the open arrows to the roof of SS. (E) Double stainings of Peyer’s patches with anti-MSR1, anti–Siglec-1, and anti-EMCN antibody (magenta) and anti–Lyve-1 (cyan). The arrow points to double positive . Images are repre- sentative of at least 10 samples per group. (Scale bars, 50 μm.)

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Fig. S5. Gating of skin LECs. (A) The gating of CD45−/CD11b−/CD31+/Lyve-1+/podoplanin+ population from abdominal and back skin of WT mice. (B) Whole mount of the split ear of a WT and MSR1 KO mouse. Weak MSR1 positivity (white) in Lyve-1+ (magenta) lymphatics is indicated by small arrows in WT whole mount. (Scale bar, 100 μm.)

Iftakhar-E-Khuda et al. www.pnas.org/cgi/content/short/1602357113 8of10 A WT MSR1 KO

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Fig. S6. Examples of ex vivo adhesion assays, MSR1-Fc chimera binding and gating and counting strategies. (A) Examples of lymphocyte binding to the SS of WT mice (Left) and KO mice (Right) in ex vivo adhesion assays. The SS-bound cells are pointed out by arrows; the focus is always a compromise with the bound cells and the tissue; dark field. As a comparison, lymphocyte binding to (HEVs) is also shown. The basement membranes of HEVs are outlined by dashed lines. Both in WT and KO mice, lymphocytes bind with comparable efficiency to HEVs, whereas fewer cells bind to SS of MSR1 KO mice than to SS of WT mice. (Magnification: 200×.) (B) Examples of two-color FACS blots illustrating binding of MSR1-Ig chimera (Left) and control Ig (Right) to CD4 positive and negative lymphocytes. (C–E) Gating of lymphocyte populations in 12-h lymphocyte migration experiments. (C) CFSE-labeled donor cells, which were + + + injected into the footpads of recipient mice were highly CFSE .(D) Practically no CFSE cells were detected in the control LNs (axillary) of the recipient mice. (E) MSR1 KO migration experiments: CFSE lymphocytes in the draining popliteal + − LNs of recipient mice (Left). Lymphocyte subpopulations from CFSE and CFSE (endogenous) were calculated from the blots showing CFSE against Pacific Blue CD45R/B220, Alexa Fluor 647 CD4, or PerCP Cy5.5 CD8a (Right). (F) The counting strategy used to measure the entrance of the injected lymphocytes into the lymph nodes. An example of measuring distances from the SS is shown. (Scale bars, 100 μm.)

Iftakhar-E-Khuda et al. www.pnas.org/cgi/content/short/1602357113 9of10 Fig. S7. The Msr1 transcript variants 1 and 2, but not a clearly detectable amount of the transcript variant 3, are expressed in cultured HLECs, M0, and polarized M1 and M2 macrophages. RT-PCR using specific primers for the Msr1 transcript variants was performed on RNA extracted from HLECs and HDLECs as well as from macrophages (M0, cultured with M-CSF) and in vitro polarized M1 (cultured with additional IFN-γ) and M2 (cultured with additional IL-4) mac- rophages as described in Methods.(Upper Left) PCR performed with the primer pair 1 (primers specific for the transcript variants 1 and 3, 500 bp and 300 bp, respectively). (Right) The primer pair 2 (primers specific for the transcript variant 2, 400 bp). (Lower) Controls (primers specific for β-actin).

Other Supporting Information Files

Table S1 (DOCX) Table S2 (DOCX) Table S3 (DOCX)

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