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• W2987796490 abstract "Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Lymph nodes (LNs) contain innate-like lymphocytes that survey the subcapsular sinus (SCS) and associated macrophages for pathogen entry. The factors promoting this surveillance behavior have not been defined. Here, we report that IL7RhiCcr6+ lymphocytes in mouse LNs rapidly produce IL17 upon bacterial and fungal challenge. We show that these innate-like lymphocytes are mostly LN resident. Ccr6 is required for their accumulation near the SCS and for efficient IL17 induction. Migration into the SCS intrinsically requires S1pr1, whereas movement from the sinus into the parenchyma involves the integrin LFA1 and its ligand ICAM1. CD169, a sialic acid-binding lectin, helps retain the cells within the sinus, preventing their loss in lymph flow. These findings establish a role for Ccr6 in augmenting innate-like lymphocyte responses to lymph-borne pathogens, and they define requirements for cell movement between parenchyma and SCS in what we speculate is a program of immune surveillance that helps achieve LN barrier immunity. https://doi.org/10.7554/eLife.18156.001 eLife digest The lymphatic system is a network of vessels and a vital part of our immune system. Amongst other things, the lymphatic system carries microbes that have entered the body – for example via to a cut or mosquito bite – to small, oval-shaped organs called lymph nodes. The lymph nodes are packed with immune cells that can be activated to help fight off infections, however certain microbes actually replicate inside the lymph nodes themselves. Lymph nodes protect themselves from these infections by having some pre-armed immune cells that are ready to respond rapidly as soon as an invading microbe is detected. These cells, referred to as innate-like lymphocytes, position themselves at the exposed surfaces of the lymph node – the locations where microbes are most likely to enter the organ. However, it was not known which cues caused these immune cells to assemble and remain at these locations. Zhang et al. now reveal that a signaling molecule called CCL20 attracts the innate-like lymphocytes to the lymph node’s exposed surfaces, while a protein known as CD169 helps to securely attach the innate-like lymphocytes in place. Further experiments then confirmed that positioning the innate-like lymphocytes at this location made mice more able to fight off the disease-causing bacterium Staphyloccus aureus. Unexpectedly, Zhang et al. also found that innate-like lymphocytes can move from the surfaces of lymph node through to the underlying tissue. This unusual migratory behavior might allow the lymphocytes to search a larger area for the infectious microbes, though further studies are needed to test this hypothesis. Future studies are also likely to focus on elucidating how the innate-like lymphocytes recognize different types of invaders, and how their activity keeps the lymph nodes healthy. https://doi.org/10.7554/eLife.18156.002 Introduction Our ability to mount adaptive immune responses against skin-invading pathogens depends on the delivery of antigens to lymph nodes (LNs) for encounter by naive lymphocytes (Cyster, 2010; Qi et al., 2014). However, activation, clonal expansion and effector lymphocyte differentiation takes several days, whereas pathogens can undergo marked replication in a matter of hours. The skin contains immune effector cells that help keep pathogen replication in check, in a process referred to as ‘barrier immunity’ (Belkaid and Segre, 2014). Despite this, in many cases, intact pathogens travel within minutes via lymph fluid to draining LNs. Indeed some pathogens, such as Yersinia pestis, appear to have evolved to undergo marked expansion only after arrival in the LN (St John et al., 2014). Recently, there has been evidence indicating the existence of barrier immunity within LNs. The first LN cells exposed to lymph-borne antigens include the CD169+ macrophages that extend between the subcapsular sinus (SCS) or medullary sinuses and the underlying parenchyma (Barral et al., 2010; Iannacone et al., 2010; Phan et al., 2009). Crosstalk between sinus-associated macrophages and IFNγ precommitted CD8 T cells and NK cells is important for mounting rapid Th1-like and NK cell responses against acute infection by Pseudomonas aeroguinosa, Salmonella typhimurium and Toxoplasma gondii (Coombes et al., 2012; Kastenmüller et al., 2012). IL17 is a cytokine with roles in anti-bacterial and anti-fungal defense that is made abundantly by effector T cells at epithelial surfaces (Littman and Rudensky, 2010). Whether IL17 is produced rapidly during responses to subcapsular sinus-invaders in LNs is unclear. In recent work, our group and others identified populations of innate-like (pre-formed effector) lymphocytes that are enriched near the SCS in peripheral LNs and are pre-committed to produce IL17 (Do et al., 2010; Doisne et al., 2009; Gray et al., 2012; Roark et al., 2013). These cells express high amounts of the chemokine receptors Ccr6 and Cxcr6 as well as the cytokine receptor IL7R, and they include a majority of αβ T cells but also considerable numbers of γδ T cells as well as non-T cells (Gray et al., 2012). Within the IL17-committed γδ T cell population a major subset expresses a Vγ4-containing TCR (according to the nomenclature of [Heilig and Tonegawa, 1986]), and undergoes expansion in response to challenge with imiquimod or complete Freund’s adjuvant (Gray et al., 2013; Ramirez-Valle et al., 2015; Roark et al., 2013). In previous work, we found that innate-like lymphocytes isolated from peripheral LNs were heavily coated with CD169+ macrophage-derived membrane fragments (‘blebs’) (Gray et al., 2012). This observation suggested there may be strong adhesive interactions between these cells and the CD169+ macrophages. CD169 is the founding member of the Siglec family of sialic acid-binding lectins (Crocker et al., 2007; Macauley et al., 2014). Although CD169 is a defining feature of LN SCS macrophages and targeting antigens to CD169 can promote antibody responses (Macauley et al., 2014), the function of CD169 on these cells is not fully understood. Pre-enrichment of innate-like lymphocytes near LN sinuses is thought to be important for allowing very rapid responses against lymph-borne invaders (Gray et al., 2012; Kastenmüller et al., 2012). Despite this, it is not known whether IL17-committed innate-like lymphocytes in LNs respond rapidly upon pathogen challenge, and little is understood about how these cells localize to or move in the subcapsular region. In this study, we found IL7RαhiCcr6+ innate-like lymphocytes were mostly LN resident and they produced IL17 within hours of bacterial or fungal challenge. Their proximity to the SCS was mediated by Ccr6 and was important for the rapid induction of IL17 following bacterial challenge. Real time intravital two photon microscopy and in vivo labeling procedures revealed that innate-like lymphocytes exchanged between the LN parenchyma and the SCS. Movement into the SCS was S1pr1 dependent, whereas return to the parenchyma involved LFA1 and ICAM1. Within the SCS, CD169-mediated adhesive interactions that helped retain the cells, presumably against the shear stresses exerted by lymph flow. This requirement was most prominent for the Vγ4+γδ T cell subset of innate-like lymphocytes. These observations provide a model for understanding the mechanism by which innate-like lymphocytes survey the pathogen-exposed surface of the LN to protect the organ from infection. Results IL7RαhiCcr6+ innate-like lymphocytes near the SCS respond rapidly to pathogens IL7RαhiCcr6+ innate-like lymphocytes within peripheral LNs express high amounts of Cxcr6 and they include ~70% αβ T cells, ~20% γδ T cells and 5–10% non-T cells (Figure 1A,B) (Gray et al., 2012). Consistent with previous findings, the IL7RαhiCcr6+gd T cell subset produced IL17 rapidly upon treatment with phorbol 12-myristate 13-acetate (PMA) and ionomycin or with the cytokines IL1β and IL23 (Figure 1C, lower graph) (Cai et al., 2014; Gray et al., 2012; Gray et al., 2011). These treatments also triggered rapid IL17 production from the IL7RαhiCcr6+αβ T cells (Figure 1C). We therefore tested whether both the αβ and γδ subsets of IL7RαhiCcr6+ T cells produce IL17 in skin draining LNs following bacterial or fungal challenge. IL17 is known to play a role in host defense against cutaneous Candida albicans and Staphyloccus aureus infection (Cho et al., 2010; Conti and Gaffen, 2015) and to be induced in rats by Y. pestis (Comer et al., 2010). Three hours after heat-killed C. albicans, S. aureus bioparticle, or attenuated Y. pestis footpad challenge, IL17 production in draining popliteal LNs was observed (Figure 1D). IL7RαhiCcr6+ lymphocytes were the dominant IL17 producers at this early time point after challenge (Figure 1D). The induction of IL17 expression in IL7RαhiCcr6+ cells upon bacterial challenge was dependent on CD169+ SCS macrophages as the response was greatly blunted in CD169-DTR mice (Miyake et al., 2007) pretreated with DT to ablate these cells (Figure 1E). Since IL1β and IL23 induced IL17 production from IL7RαhiCcr6+ T cells in vitro, we looked for expression of these cytokines after S. aureus bioparticle challenge. Transcripts for both Il1b and Il23a were upregulated (Figure 1F). Induction of IL17 following S. aureus bioparticle challenge was compromised in mice lacking the ASC (Apoptosis-associated speck-like protein containing a CARD) inflammasome subunit (Figure 1G), consistent with a role for IL1β in activating IL7RαhiCcr6+ cells during the response to these bacteria. Figure 1 Download asset Open asset Rapid induction of IL17 expression by IL7RαhiCcr6+ innate-like lymphocytes in a CD169+ macrophage-dependent manner following bacterial and fungal challenge. (A) Representative FACS plot showing IL7RαhiCcr6+ staining of peripheral LN cells from a Cxcr6GFP/+ mouse, and Cxcr6-GFP intensity on the gated cells. (B) Representative FACS plots showing CD3ε, TCRβ and TCRγδ staining of the IL7RαhiCcr6+ population. Bar graph shows summary frequency data (mean ± sd) for more than 10 mice. (C) Intracellular FACS showing IL17 production among LN cells after 3 hr in vitro stimulation with IL1β and IL23 or phorbol 12-myristate 13-acetate and ionomycin. Graph shows summary data from 3 experiments. (D) Representative FACS plots showing IL17 production among popliteal LN cells 3 hr after footpad challenge with heat inactivated C. albicans, S. aureus coated bioparticles, and attenuated Y. pestis. Summary graph shows% IL17+ cells among IL7RαhiCcr6+ cells. (E) IL17 production by IL7RαhiCcr6+ cells in control and CD169-DTR macrophage ablated mice treated with S. aureus bioparticles as in D, Summary graph shows% IL17+ cells among IL7RαhiCcr6+ cells. (F) Il1b and Il23a mRNA level in popliteal LNs of S. aureus bioparticle challenged mice relative to controls, determined by qRT-PCR. (G) Summary graph to show% IL17+ cells among IL7RαhiCcr6+ cells between control and ASC-deficient mice after 3 hr S. aureus bioparticle challenge. ***p<0.001 by student’s t test. Data are representative of at least two experiments for panels A–C. Data are representative of two or more experiments with at least two mice per group for panels D–G. LN, Lymph node. https://doi.org/10.7554/eLife.18156.003 IL7RαhiCcr6+ innate-like lymphocytes are mostly LN resident IL7RαhiCcr6+ lymphocytes make up ~0.5% of peripheral LN cells yet they represent only ~0.1% of cells in blood (Figure 2A), suggesting that the cells are largely non-recirculatory under homeostatic conditions. To test this more directly, we ‘time stamped’ cells in inguinal LNs of KikGR photoconvertible protein-expressing transgenic mice by brief violet light exposure (Gray et al., 2013). At 24 hr after photoconversion almost three quarters of the IL7RαhiCcr6+ cells and a similar fraction of the Vγ4+Ccr6+ cells remained resident in the LN, whereas more than 80% of the conventional αβ T cells and Ccr6– Vγ4+ T cells had left the LN and been replaced by newly arriving cells. At 48 hr, the innate-like lymphocyte pool showed little further exchange, whereas naive αβ T cells were 90% replaced (Figure 2B). In a further approach, we examined the amount of cell exchange that occurred in parabiotic mice. Two weeks following surgery the naive αβ T cell compartment and the Ccr6– Vγ4+ T cells had achieved full chimerism, whereas the innate-like lymphocytes showed only limited exchange between the paired mice (Figure 2C). Taken together, these findings indicate that most IL7RαhiCcr6+ cells are resident in the LN for multiple days and are not extensively recirculating under homeostatic conditions. Figure 2 Download asset Open asset IL7RαhiCcr6+ innate-like lymphocytes are mostly LN resident. (A) Representative FACS plots showing frequency of IL7RαhiCcr6+ and Vγ4+Ccr6+ cells in LNs and blood. Graphs show summary data for more than 30 mice of each type. (B) FACS analysis of LN IL7RαhiCcr6+ cells and naïve αβ T cells in KikGR mice before, immediately after and 24 and 48 hr after photoconversion. Summary data are pooled from three experiments and each point indicates an individual mouse. (C) FACS analysis of LN IL7RαhiCcr6+ cells and naive αβ T cells in GFP-host and GFP+ host from parabiotic pairs. Summary data are pooled from two experiments and each point indicates an individual mouse. **p<0.01, ***p<0.001, by student’s t test. Data are representative for at least two experiments. LN, Lymph node. https://doi.org/10.7554/eLife.18156.004 Migration dynamics of innate-like lymphocytes at the SCS Cxcr6-GFP is highly expressed by all IL7RαhiCcr6+ cells (Figure 1A), and in previous work, we observed that Cxcr6GFP/+ cells migrate extensively in outer and inter-follicular regions, often in close association with CD169+ SCS macrophages (Gray et al., 2012). A closer examination of Cxcr6GFP/+ cell behavior in this region revealed that the cells frequently made contact with CD169+ macrophages, and occasionally, lymphocytes could be observed crossing the layer of macrophages to reach the SCS (Figure 3A and Videos 1 and 2). Reciprocally, cells that were initially detected within the SCS could be observed migrating across the thick CD169+ macrophage layer into the LN parenchyma (Figure 3A and Videos 1 and 2). To quantify the crossing events, cell tracks were generated automatically (Figure 3—figure supplement 1) and tracks crossing the SCS floor were manually enumerated. Among all the tracks of Cxcr6GFP/+ cells within 50 µm of the capsule, ~25% were in the SCS (Figure 3B). Cell tracking analysis showed that about 3% of the cells in the SCS region traveled from the parenchyma into the sinus, and 3% of the cells traveled in the reverse direction, in the 30 min imaging periods (Figure 3C). Figure 3 with 1 supplement see all Download asset Open asset Migration dynamics, sinus exposure and CD169+ macrophage interaction of LN innate-like lymphocytes. (A) Time series of Cxcr6GFP/+ cell movement with respect to CD169+ SCS macrophages. Upper panels: white arrow indicates a Cxcr6GFP/+ lymphocyte in the LN parenchyma that crosses into the SCS. 300 s time series was taken from a 46 μm z stack. Lower panel: white arrow indicates a Cxcr6GFP/+ lymphocyte that begins in the SCS and crosses into the LN parenchyma. 360 s time series was taken from a 34 μm z stack. Green, Cxcr6-GFP+ lymphocytes; Red, CD169+ macrophages; Blue, second harmonic. White dashed line indicates boundary between SCS and LN parenchyma. (B, C) Percent tracks in SCS compartment among the total tracks enumerated (B) and frequency of tracks crossing from the parenchyma into the SCS or out of the SCS into the parenchyma (C) in Cxcr6GFP/+ control mice. Each point represents data from a single movie (two independent experiments). (D) In vivo 5 min Thy1-PE labeling of IL7RαhiCcr6+ cells, Vγ4+Ccr6+ cells and αβ T cells, analyzed by flow cytometry. Data are representative of at least 10 mice. (E) In vivo Thy1-PE labeling of cells analyzed in tissue sections. Costaining was with CD3-A488 (green) and Lyve1-A647 (blue). White arrows point out Thy1 and CD3 costained cells. (F) Frequency of IL7RαhiCcr6+, Vγ4+Ccr6+ and naive αβ T cells positive for CD169. Data are representative of 10 mice. (G) In vivo Thy1-PE labeling and CD169 staining on IL7RαhiCcr6+, Vγ4+Ccr6+ and naive αβ T cells, analyzed by flow cytometry. Data are representative of at least two experiments in each panel. https://doi.org/10.7554/eLife.18156.005 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Cxcr6-GFP+ cell shuttling between parenchyma and the SCS. Representative intravital time-lapse imaging of the popliteal LNs from two Cxcr6GFP/+ mice. Overhead 3D video exemplifies the dynamic movement of Cxcr6-GFP+ cells (green) within the LN. Two-dimensional video of a 20 µm maximal intensity projection from an orthogonal plane demonstrates the anatomy of the SCS region. An afferent lymphatic vessel (rarely visualized) drains into the SCS, bounded by the collagenous LN capsule (blue, second harmonic signal) and CD11b+ SCS macrophages (red). The second example further reveals the motility of Cxcr6-GFP+ cells both within the SCS and the LN parenchyma. Cxcr6-GFP+ cells are observed to cross from within the LN parenchyma into the SCS, as well as from within the SCS into the LN parenchyma (examples highlighted by circles). LN, Lymph node; SCS, Subcapsular sinus. https://doi.org/10.7554/eLife.18156.007 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Representative examples of individual Cxcr6-GFP+ cells crossing into and out of SCS. Intravital time-lapse imaging of the popliteal LN from a Cxcr6GFP/+ control mouse, highlighting one cell crossing from the parenchyma into the SCS, and one crossing from the SCS into the parenchyma. Cells such as these that clearly crossed from one region were manually identified from automated tracking of all Cxcr6-GFP+ cells in an experiment. SCS, Subcapsular sinus. https://doi.org/10.7554/eLife.18156.008 To quantitate the proportion of cells in the LN lymphatic sinuses at a given moment in time, we optimized an in vivo procedure to label lymph-exposed cells based on the established method of antibody pulse-labeling of blood-exposed cells (Cinamon et al., 2008). We targeted Thy1 since this marker is expressed by all the IL7RαhiCcr6+ cells while not being present on SCS macrophages (not shown). PE-conjugated antibody was used as the large size of PE (250 kD) reduces the rate at which the antibody accesses the lymphoid tissue parenchyma (Pereira et al., 2009). Thy1-PE antibody (0.2 μg) was injected into the footpad, the draining popliteal LN was isolated 5 min later and the frequency of labeled cells determined by flow cytometry. Approximately 10% of the total IL7RαhiCcr6+ cells were brightly labeled with Thy1-PE antibody compared with about 0.3% of naive T cells (Figure 3D). Among the innate-like lymphocytes, γδ T cells (predominantly Vγ4+Ccr6+ cells) were preferentially labeled, with around 15–20% of these cells being antibody exposed. By immunofluorescence microscopy, Thy1-PE-labeled CD3ε+ cells were observed in the SCS and in nearby lymphatic sinuses, and few labeled cells were detected within the LN parenchyma, confirming that footpad injection of PE-conjugated antibody led to preferential labeling of lymph-exposed LN cells (Figure 3E). The broader labeling of the sinus by Thy-1 than by CD3 reflects the expression of Thy1 by lymphatic endothelial cells (Jurisic et al., 2010). We also obtained information about lymphocyte-SCS macrophage proximity by following up on our finding that isolated innate-like lymphocytes are heavily coated with CD169+ macrophage-derived membrane fragments (‘blebs’) (Gray et al., 2012). This coating is thought to occur at the time of LN cell dissociation, possibly because the SCS macrophages are tightly bound to the extracellular matrix and become fragmented during mechanical preparation of the tissue. Importantly, the blebs only become bound to cells that are associated with the macrophages at the time of isolation since co-preparation of LN cells from congenically distinct animals did not lead to cross acquisition of macrophage-derived blebs by innate-like lymphocytes from the different LNs (Gray et al., 2012). In accord with previous findings, 30–40% of the IL7RαhiCcr6+ innate-like lymphocytes isolated from control mice were CD169 macrophage-derived membrane bleb positive (Figure 3F). A higher frequency (40–55%) of the Vγ4+γδ T cells were CD169 bleb positive, suggesting these cells may be preferentially associated with SCS macrophages (Figure 3F). Combining this analysis with Thy1-PE labeling showed that lymph-exposed innate-like lymphocytes, but not total αβ T cells, were enriched for CD169-bleb-positive cells (Figure 3G). The Thy1-PE+ CD169– cells amongst total αβ T cells most likely correspond to recirculating cells that are exiting the LN via cortical and medullary sinuses. Ccr6 promotes innate-like lymphocyte positioning near the SCS Given the high Ccr6 expression on the innate-like lymphocyte population, we asked whether this CCL20 receptor had a role in guiding innate-like lymphocytes to the subcapsular region. Although CCL20 is not abundantly expressed in LNs, it is expressed in LN lymphatic endothelial cells (LECs) at levels more than 100-fold higher than other LN lymphoid stromal cells (Figure 4A). Immunofluorescence microscopy showed evidence of CCL20 protein in the subcapsular sinus region (overlying follicular and interfollicular regions) but not in the medullary sinus region (Figure 4B, Figure 4—figure supplement 1) consistent with findings in primate LNs (Choi et al., 2003; Pegu et al., 2007). Innate-like lymphocytes were responsive to CCL20 by in vitro migration assays (Figure 4C). When CCL20 was injected subcutaneously into Cxcr6GFP/+ mice, IL7RαhiCcr6+Cxcr6hi lymphocytes became clustered near and within the SCS in the draining LNs (Figure 4D, Figure 4—figure supplement 2). Cells from these LNs showed reduced surface Ccr6, and increased CD169 staining and Thy1 labeling, consistent with their having been exposed to increased amounts of CCL20 and localizing near and within the SCS (Figure 4E). Figure 4 with 4 supplements see all Download asset Open asset Ccr6 promotes innate-like lymphocyte positioning near the SCS. (A) Ccl20 mRNA abundance in sorted LN lymphatic endothelial cells (LEC), blood endothelial cells (BEC), fibroblastic reticular cells (FRC) and double negative stromal cells (DN) determined by qRT-PCR, shown relative to Hprt. (B) CCL20 staining of LN section (red). The control (no primary) section was stained with the secondary anti-goat-Cy3 antibody alone. B cells were detected in blue (B220). (C) Transwell migration of IL7RαhiCcr6+ cells to CCL20. (D) Distribution of Cxcr6GFP/+ cells in LNs 3 hr after saline or CCL20 s.c. injection. Sections were stained to detect Cxcr6-GFP (green) and Lyve1 (blue). (E) Representative FACS plots show Ccr6 surface level, in vivo Thy1-PE labeling and CD169 macrophage bleb level on IL7RαhiCcr6+ cells from control (con) or CCL20 injected mice. Summary graph shows comparison of Thy1-PE labeling and CD169+ staining frequency of IL7RαhiCcr6+ and Vγ4+Ccr6+ cells from control or CCL20 injected mice. (F) LN sections from Ccr6GFP/+ or Ccr6GFP/GFP mice stained for EGFP (green) and B220 (blue). White arrow indicates subcapsular sinus area; FO: B cell follicle; T: T zone. (G) Comparison of Thy1-PE labeling on IL7RαhiCcr6+ cells from WT and Ccr6GFP/+ or Ccr6GFP/GFP mice. Ccr6 in Het and KO mice was detected based on GFP reporter expression. (H) Comparison of CD169 staining on IL7RαhiCcr6+ cells from WT, Ccr6GFP/+ or Ccr6GFP/GFP mice. (I) Representative FACS plots showing Vγ4+Scart2+ cells amongst γδT cells and the fraction that are Ccr6+ (upper), and in vivo Scart2-BV605 labeling and CD169 staining (lower). Graph shows summary data. (J) LN sections from Ccr6 Het or KO mice stained for Scart2+ (green) and B220 (blue). White arrow indicates subcapsular sinus area; FO: B cell follicle; T: T zone. (K) Representative histogram plot and summary mean fluorescence intensity (MFI) data of IL17 intracellular staining in IL7RαhiCcr6+ LN cells from Ccr6 Het or KO mice 3 hr after S. aureus bioparticle challenge. *p<0.05, **p<0.01, ***p<0.001, by student’s t test. Data are representative of at least two experiments for panel A–D, F, J. Data are representative of two or more experiments with at least two mice per group for panels E, G–I, K. LN, Lymph node; SCS, Subcapsular sinus. https://doi.org/10.7554/eLife.18156.009 We next examined the distribution of Ccr6-deficient (KO) cells in Ccr6GFP/GFP mice and found that the cells were reduced in density near the SCS (Figure 4F, Figure 4—figure supplement 3) despite being slightly increased in total frequency in the LN (not shown). Instead, the cells were often distributed along the B-T zone interface (Figure 4F, Figure 4—figure supplement 3). In Thy1-PE labeling experiments, Ccr6 KO mice showed reduced frequencies of labeled cells, a result that was most significant for the Vγ4+ population (Figure 4G). Consistent with reduced proximity to SCS macrophages, Ccr6-GFP+ IL7Rαhi cells and the Vγ4+ subset from Ccr6 KO mice were associated with less CD169+ blebs compared with Ccr6-sufficient controls (Figure 4H). The CD169+ macrophage population in LN sections was unaffected by Ccr6-deficiency (not shown). Given that the Vγ4+Ccr6+ cell population was most dependent on Ccr6 for Thy1 labeling and macrophage bleb acquisition (Figure 4G,H), we further studied the properties of these cells. Vγ4+Ccr6+ T cells uniquely express the surface marker Scart2 (Figure 4I) (Gray et al., 2013; Kisielow et al., 2008). In a complementary approach to Thy1 labeling, Scart2 antibody treatment was found to label fewer Vγ4+ cells in LNs from Ccr6-deficient mice than from wild-type mice (Figure 4I). By immunofluorescence microscopy, Scart2+ γδ T cells were underrepresented in the SCS region in LN sections from Ccr6-deficient mice compared with those from control mice (Figure 4J, Figure 4—figure supplement 4). Together these data support the conclusion that Ccr6 plays a role in guiding innate-like lymphocytes toward the SCS region. Importantly, when Ccr6 KO mice were immunized with S. aureus bioparticles, the IL7RαhiCcr6+ cells mounted a diminished IL17 response (Figure 4K), whereas they responded normally to activation stimuli in vitro (not shown). These data provide further evidence that proximity to SCS macrophages is important for innate-like lymphocytes to mount rapid IL17 responses following pathogen exposure. S1pr1 is required for innate-like lymphocyte movement into the SCS S1pr1 is needed in naive lymphocyte for access to cortical and medullary lymphatic sinuses during LN egress (Cyster and Schwab, 2012). S1pr1 is also required for marginal zone (MZ) B cell shuttling between the S1P high MZ and the S1P low lymphoid follicle (Arnon et al., 2013). We therefore tested whether S1pr1 played a role in guiding innate-like lymphocytes into the SCS. The innate-like lymphocytes had detectable surface S1pr1 (Figure 5A) and they responded to S1P by in vitro migration in a Transwell assay (Figure 5B). Pretreatment of mice for 6 hr with FTY720, a functional antagonist of S1pr1, greatly diminished in vivo Thy1-PE labeling on IL7RαhiCcr6+ lymphocytes (Figure 5C). Similarly, in vivo Scart2 antibody labeling of Vγ4+γδ T cells was decreased after FTY720 treatment (Figure 5D). FTY720 also decreased the CD169 membrane bleb positive fraction to 20–25% in both the total IL7RαhiCcr6+ population and the γδ T cell subpopulation (Figure 5C). The reductions in Thy1-PE-labeled cells (from ~10 to ~1%) and in CD169-bleb+ cells (from ~35 to~25%) were similar, representing ~10% of the total IL7RαhiCcr6+ population in both cases (Figure 5C), suggesting that the reduced frequency of CD169-bleb+ cells was due to the loss of cells accessing the sinus. Comparable findings were made after treatment with the more selective S1pr1 functional antagonist, AUY954 (Figure 5E). These data provided evidence that S1pr1 was required for the cells to have normal access to the SCS. Examination of tissue sections by immunofluorescence microscopy showed a loss of Cxcr6GFP/+ cells from the SCS following FTY720 treatment (Figure 5F). FTY720 treatment also caused a depletion of Scart2+ cells from the SCS (Figure 5G, Figure 5—figure supplement 1). When FTY720-treated mice were challenged with S. aureus bioparticles, the IL7RαhiCcr6+ population mounted an IL17 response of normal magnitude (not shown). We speculate that SCS access is needed for other types of responses. Figure 5 with 2 supplements see all Download asset Open asset S1pr1 is required for innate-like lymphocyte movement into the SCS. (A) S1pr1 surface expression on IL7RαhiCcr6+ and Vγ4+Ccr6+ cells from control or FT" @default.
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• W2987796490 title "Author response: Migratory and adhesive cues controlling innate-like lymphocyte surveillance of the pathogen-exposed surface of the lymph node" @default.
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