Subcapsular Sinus Macrophage Fragmentation and CD169+ Bleb Acquisition by Closely Associated IL-17-Committed Innate-Like Lymphocytes

Subcapsular sinus macrophages (SSMs) in lymph nodes are rapidly exposed to antigens arriving in afferent lymph and have a role in their capture and display to B cells. In tissue sections SSMs exhibit long cellular processes and express high amounts of CD169. Here, we show that many of the cells present in lymph node cell suspensions that stain for CD169 are not macrophages but lymphocytes that have acquired SSM-derived membrane blebs. The CD169 bleb+ lymphocytes are enriched for IL-17 committed IL-7RαhiCCR6+ T cells and NK cells. In addition, the CD169 staining detected on small numbers of CD11chi dendritic cells is frequently associated with membrane blebs. Counter intuitively the CD169 bleb+ lymphocytes are mostly CD4 and CD8 negative whereas many SSMs express CD4. In situ, many IL-7Rαhi cells are present at the subcapsular sinus and interfollicular regions and migrate in close association with CD169+ macrophages. These findings suggest SSMs undergo fragmentation during tissue preparation and release blebs that are acquired by closely associated cells. They also suggest an intimate crosstalk between SSMs and IL-17 committed innate-like lymphocytes that may help provide early protection of the lymph node against lymph-borne invaders.


Introduction
Subcapsular sinus macrophages (SSMs) are a unique subset of lymph node macrophages that form a dense layer overlapping with the lymphatic lining that separates the lymphatic sinus and B cell follicle. In situ staining has shown that SSMs express high amounts of the sialic acid binding Ig-like lectin 1 (Siglec1 or CD169) and the integrin CD11b (Mac1), and, in contrast to their counterparts in the medulla, lack expression of the macrophage marker F4/80 [1,2,3,4]. Many SSMs straddle the lymphatic lining cells at the base of the subcapsular sinus, extending a ''head'' into the sinus and long cellular processes (or ''tails'') into the adjacent B cell follicle [1]. In contrast to the dynamic behavior of dendritic cell processes [5,6,7], real time imaging studies have revealed that the long cellular processes of SSMs are relatively static, potentially indicating tight adhesion to adjacent stromal cells or extracellular matrix [1,2,3,4].
Due to this unique localization, SSMs are poised to rapidly encounter pathogens and antigens that reach the lymph node via the lymph. Indeed, a number of studies have revealed that SSMs have the capacity to capture a range of antigens, including viral particles, immune complexes, antigen-loaded beads, and other opsonized antigens [8]. In contrast to classical macrophages, which typically internalize and degrade antigen, SSMs are thought to be poorly phagocytic [9,10], a property that may contribute to their capacity to function as antigen-presenting cells for B cells. Antigen captured by SSMs is displayed on macrophage ''tails'' that extend into the B cell follicle, where B cells can directly acquire antigen via complement or B cell receptors [1,2,3,4]. SSMs have also been shown to activate iNKT cells. Subcutaneously injected a-GalCer-coated microspheres were captured by SSMs, processed, and presented via CD1d to iNKT cells [11]. In addition to these antigen presentation functions, a number of recent studies have shown that SSMs are an early site of replication for a number of viruses [12][13] as well as the parasite Toxoplasma gondii [14]. SSMs have been suggested to be unusually permissive for pathogen infection, a property that might foster local production of cytokines that protect other cell types from infection [15].
As part of a continued effort to understand the biology of SSMs, attempts to isolate and study these CD169 hi cells have been made by us [2] and others [3,15,16,17,18]. Here we report that many of the cells in lymph node cell suspensions that stain for CD169 are lymphocytes that have acquired SSM-derived membrane blebs and thus masquerade as CD169 hi SSMs during flow cytometric analysis. This acquisition does not appear to be a random process as CD169 bleb + lymphocytes are enriched for IL-17 committed IL-7Ra hi CCR6 + T cells and NK cells. Moreover, in situ and realtime imaging analysis reveals that IL-7Ra hi CXCR6 hi cells are present at the subcapsular sinus and in interfollicular regions, often migrating over the membrane processes of CD169 + macrophages. These observations raise the possibility of crosstalk between SSMs and innate-like lymphocyte populations that may have important roles in the early phases of lymph node immune responses.

Results
Detection of CD169 + staining on IL-7Ra hi CCR6 + lymphocytes In situ staining has revealed that SSMs are CD169 hi CD11b + CD11c lo cells that, in contrast to the medullary macrophages, are F4/80 2 [1,2,3,4]. Previously, we identified a population of CD169 hi CD11b + CD11c lo F4/80 2 cells by flow cytometry that appeared to correspond to SSMs [2]. In further efforts to characterize CD169 hi CD11c lo cells by flow cytometry, we observed that many of the cells with a CD169 hi CD11c lo phenotype also expressed IL-7Ra ( Figure 1A). However, subsequent analysis of IL-7Ra expression in tissue sections revealed that SSMs were IL-7Ra 2 . Instead, we observed a population of IL-7Ra hi cells, with a rounded, lymphocyte-like morphology, which were present at the subcapsular sinus and interfollicular regions of the lymph node ( [19] and Figure 1B and Supplemental Figure S1). These cells expressed higher amounts of IL-7Ra than the bulk population of T zone T cells and were often closely associated with the macrophages ( Figure 1B). In some cases, CD169 + SSM ''arms'' appeared to be wrapped tightly around the IL-7Ra hi cells ( Figure 1B, far right bottom panel), although the resolution of the imaging analysis could not exclude the possibility that some IL-7Ra hi cells were expressing CD169.
These results suggested that IL-7Ra hi lymphocytes that stained positively for CD169 in flow cytometric analysis might have been contaminating the SSM gate. Further analysis of the IL-7Ra hi CD169 hi CD11c lo cells by flow cytometry revealed that the majority were CCR6 + ( Figure 1C). When we gated on IL-7Ra hi CCR6 + cells from total lymph node cells, we observed that this gate included both CD3e + T lymphocytes and CD3e 2 non-T cells. Moreover, there was a ''smear'' of CD169 staining spanning almost a 2-log range on a fraction of both the IL-7Ra hi CCR6 + CD3e + T lymphocytes and CD3e 2 non-T cells ( Figure 1D, top panels). CD3e + IL-7Ra hi CCR6 + cells were absent in a TCRbddeficient mouse, confirming that a subset of the IL-7Ra hi CCR6 + cells were T lymphocytes ( Figure 1D).
To exclude the possibility that the CD169 antibody Ser4 was cross-reacting with a non-CD169 epitope on IL-7Ra hi CCR6 + cells, we stained lymph node cell suspensions with 3D6, an antibody that recognizes a distinct epitope on CD169 [20]. IL-7Ra hi CCR6 + lymphocytes that bound Ser4 also stained with 3D6 ( Figure 2A), suggesting that CD169 was present on the lymphocytes. As a further test that these cells were specifically staining for CD169, we utilized CD169-DTR mice, in which the diptheria toxin receptor (DTR) is knocked into the Siglec1 locus. Intraperitoneal administration of diphtheria toxin (DT) causes ablation of CD169-expressing cells, including the SSMs in these mice [16,21]. Following DT treatment, there was a loss of CD169 + cells by flow cytometry, including CCR6 + CD169 + cells ( Figure 2B), indicating that CD169 staining on IL-7Ra hi CCR6 + lymphocytes is specific.
However, when we measured CD169 transcripts on sorted CD169 + and CD169 2 IL-7Ra hi CCR6 + lymphocytes, we detected low levels of Siglec1 mRNA in both the CD169 + and CD169 2 fraction ( Figure 2C). In contrast, Siglec1 mRNA was abundant in sorted CD169 + CD11c lo F4/80 + cells (i.e. cells that stain positive for medullary sinus macrophage markers) ( Figure 2C). These data do not exclude the possibility that IL-7Ra hi CCR6 + cells do intrinsically express low levels of Siglec1 mRNA. However given the low abundance of mRNA detected in both CD169 + and CD169 2 IL-7Ra hi CCR6 + lymphocytes, despite more than a 10-fold difference in CD169 staining by flow cytometry, we wondered whether these cells were acquiring CD169 in trans from other cells.
These combined data led us to hypothesize that IL-7Ra hi CCR6 + lymphocytes acquire CD169 + macrophage-derived membrane fragments or ''blebs''. Consistent with this possibility, we observed that CD169 staining intensity on IL-7Ra hi lymphocytes correlated with other markers expressed by SSMs based on in situ staining, such as CD11b ( Figure 2F). To explore the possibility that these cells acquire CD169 + blebs, CD169 + IL-7Ra hi CCR6 + lymphocytes were sorted and fixed to slides to visualize CD169 staining. CD169-staining membrane blebs were observed attached to the surface of IL-7Ra hi CCR6 + lymphocytes ( Figure 2G). Consistent with the range in CD169 staining intensity observed by FACS, we noted significant variation in the number and size of blebs attached to each cell. In some cases the amount of CD169 surface staining was extensive. However, even in many of these cases, the marker did not appear uniformly distributed across the plasma membrane, suggesting that the staining was associated with a macrophage-derived membrane process that encompassed a large part of the lymphocyte surface ( Figure 2G, far right panel).
To assess CD169 + bleb acquisition by IL-7Ra hi CCR6 + lymphocytes more quantitatively, experiments were carried out using ImageStreamX imaging flow cytometry (Amnis Corp). Cells with the CD169 + IL-7Ra hi CCR6 + marker profile were gated and CD169 surface distribution was analyzed. Similar to our observations with sorted cells, we observed CD169 + ''blebs'' of various sizes on the surface of the cells ( Figure 3A, B). To quantify the fraction of cells with small compared to large CD169 + ''blebs'', we calculated the area of CD169 staining on the gated cells and separated the images into small, medium, and large area gates ( Figure 3A, B). Most CD169 + IL-7Ra hi CCR6 + cells had a small area of CD169 staining, while approximately 15% had a large area of staining ( Figure 3B). CD169 + IL-7Ra hi CCR6 + cells in the small area gate (R11) tended to have small, punctate CD169 staining, while those in the large area gate (R13) tended to have CD169 staining covering the majority of the cell ( Figure 3B). Whether these rare cells with a large area of CD169 staining have acquired very large CD169 + blebs or actually express CD169 will require future study.
High CD169 staining was recently reported on a subset of CD11c hi tumor-antigen-presenting lymph node cells [16]. Given the above findings, we wondered whether CD169 hi CD11c hi cells all express CD169, or whether some might be CD11c hi dendritic cells that have acquired CD169 + blebs. Consistent with the latter possibility, analysis of mixed bone marrow chimeras revealed no difference in CD169 staining on Siglec1 2/2 and Siglec1 +/+ CD11c hi cells (Supplemental Figure S2B). Thus, like IL-7Ra hi CCR6 + lymphocytes, CD11c hi cells can acquire CD169 in trans. We then asked whether we could visualize CD169 + blebs on CD11c hi cells using imaging flow cytometry. Indeed, analysis of gated CD169 + CD11c + cells showed bleb-like CD169 staining on many of these cells ( Figure 3D). Quantification of the CD169 staining area revealed that the majority of CD169 + CD11c hi cells had a small area of CD169 staining, corresponding to CD11c hi cells with small blebs of CD169 staining ( Figure 3C, D). Less than 15% of CD169 + CD11c hi cells had a large area of CD169 staining, which sometimes appeared uniform and covered much of the cell ( Figure 3C, D). These cells may correspond to rare CD11c hi cells that express CD169 or that have acquired large CD169 + blebs. Together, these data suggest that the majority of CD169 + CD11c hi cells are dendritic cells that have acquired CD169+ blebs.

CD169 bleb + cells are enriched for IL-7Ra hi CCR6 + cells and NK cells
These results suggested that IL-7Ra hi CCR6 + cells do not express CD169, but rather that they acquire CD169 + macrophage-derived blebs. Bleb acquisition does not appear to be a random process, as CD169 + staining was enriched on IL-7Ra hi CCR6 + lymphocytes compared to total T and B cells ( Figure 4A). Furthermore, we observed enrichment for CD169 staining on NK1.1 + DX5 + CD3e 2 NK cells, suggesting that NK cells also acquire blebs from CD169 hi macrophages ( Figure 4A). This may suggest that these cell types have a unique capacity to capture blebs that are shed by the SSMs either in vivo or during tissue preparation. Alternatively, given that IL-7Ra hi cells are located near CD169 + SSMs in situ ( Figure 1B and Supplemental Figure S1), bleb acquisition may be dependent on localization  adjacent to SSMs. Notably, tissue digestion was not required for CD169 + bleb acquisition as the fraction of IL-7Ra hi CCR6 + lymphocytes that were CD169 + by flow cytometry was similar in digested compared to undigested cell suspensions ( Figure 4B). The total number of IL-7Ra hi CCR6 + lymphocytes was greater in digested samples, suggesting that while digestion improved the recovery of these lymphocytes, it was not required for bleb acquisition per se.
IL-7Ra hi CCR6 + lymphocytes are IL-17 committed cells that interact with CD169 + macrophages IL-7Ra hi CCR6 + cells express high levels of CXCR6 ( [19] and Figure 5A), a chemokine receptor enriched on effector and memory T cells [22]. Consistent with an effector phenotype, flow cytometric analysis revealed that IL-7Ra hi CCR6 + cells were CD44 hi and CD62L lo ( Figure 5A). Based on their expression of CCR6, a marker of IL-17 committed cells [23], we hypothesized that these cells were programmed to make IL-17A. Indeed, the IL-7Ra hi CCR6 + lymphocytes rapidly produced IL-17A following phorbol ester plus ionomycin stimulation ex vivo, although there was no difference in the capacity of CD169 bleb + cells to make IL-17A compared to the bleb 2 fraction ( Figure 5B). The ability of the IL-7Ra hi CCR6 + lymphocytes to rapidly produce cytokines ex vivo in addition to their effector phenotype suggested that these cells correspond to the IL-17A committed cells recently described by several groups [24].
The IL-7Ra hi CCR6 + gate included cdT, abT, and non-T cells ( Figure 5C) all of which showed a smear of CD169 staining. The IL-7Ra hi CCR6 + cdT cells correspond to innate IL-17 producing cdT that have been described in the dermis and peripheral lymph nodes [19,25,26]. We observed that approximately 15-20% of IL-7Ra hi CCR6 + cells were CD1d-tetramer + CD3e int iNKT cells, which showed a smear of CD169 staining ( Figure 5D). These CCR6 + iNKT cells likely correspond to the IL-17 producing iNKT cells recently described in skin and peripheral lymph nodes [27].
Analysis of CD4 expression by the IL-7Ra hi CCR6 + T cells showed a smear of CD4 signal, much of which correlated with CD169 staining ( Figure 5E, top panel, upper quadrants) as well as a population of T cells with a conventional CD4 hi CD169 2 phenotype ( Figure 5E, top panel, lower right quadrant). In contrast, the IL-7Ra hi CCR6 + T cells were CD8 2 ( Figure 5E, bottom panel). The smear of CD4 staining suggested, counter intuitively, that the SSMs express CD4 and that the CD4 2 IL-7Ra hi CCR6 + T cells were acquiring CD4 + CD169 + blebs from the macrophages. Indeed, analysis of CD4 expression in situ revealed CD4 staining on the majority of CD169 + SSMs in control but not CD4-deficient mice ( Figure 5F).
Finally, we took advantage of the high expression of CXCR6 by IL-7Ra hi CCR6 + cells to gain a more precise assessment of their localization in lymph node tissue sections than can be achieved by IL-7Ra-staining alone. Approximately 60-80% of IL-7Ra hi CXCR6 hi lymphocytes identified by flow cytometry are CCR6 + [19] (and data not shown), suggesting that the majority of IL-7Ra hi CXCR6 hi cells on sections correspond to IL-7Ra hi CCR6 + IL-17-committed lymphocytes. Using Cxcr6 GFP/+ reporter mice [22], we found that the IL-7Ra hi CXCR6 hi cells were abundant in subcapsular and interfollicular regions, often adjacent to CD169 + macrophages (Supplementary Figure S3). Moreover, two photon laser scanning microscopy of intact lymph nodes revealed CXCR6 hi (GFP hi ) cells migrating in close association with CD169+ macrophages in subcapsular sinus and interfollicular regions (Supplementary Movies S1 and S2).

Discussion
We report here that many of the cells in lymph node cell suspensions that stain positively for SSM-markers are not macrophages, but rather IL-7Ra hi CCR6 + lymphocytes and NK cells that have acquired CD169 + SSM-derived membrane blebs. This conclusion is established by: (i) the incomplete concordance between surface marker expression on CD169 + cells detected in tissue sections and by flow cytometry; (ii) the low abundance of CD169 transcripts in CD169 + IL-7Ra hi CCR6 + cells by quantitative PCR as well as in microarray analysis of FACS sorted CD169 + CD11b + CD11c lo F4/80 2 cells [2,28]; (iii) the positive CD169 staining on Siglec1 2/2 IL-7Ra hi CCR6 + cells in Siglec1 +/+ hosts, and; (iv) the visualization of CD169 + blebs on the surface of sorted cells and cells examined using ImageStreamX flow cytometry. The tendency of IL-7Ra hi CCR6 + lymph node cells to acquire CD169 + macrophage-derived blebs suggests there may be a propensity for these two cell types to interact in vivo. Future studies should explore whether a specific interaction between IL-7Ra hi CCR6 + lymphocytes and CD169 + macrophages occurs in vivo, and, if so, define the functional consequences.
An important question that arises from these observations is whether bleb acquisition by IL-7Ra hi CCR6 + lymphocytes occurs in vivo or during cell preparation. Arguing against acquisition being a prominent process in vivo, during real-time imaging studies of intact lymph nodes in mice intravitally labeled with CD169 antibodies, we have so far not observed CD169 marker acquisition by migrating lymphocytes (Supplementary Movies S1 and S2, and data not shown). Therefore, we favor the view that bleb acquisition predominantly occurs during tissue preparation. The finding that CD169 acquisition by IL-7Ra hi CCR6 + cells was similar whether the lymph nodes were gently teased apart and enzyme digested or simply mechanically separated suggests that SSMs may be highly prone to fragmentation, perhaps due to strong attachments to the surrounding cells or extracellular matrix. Alternatively, the cells may be programmed to undergoing blebbing during apoptosis. In this regard it is notable that dying germinal center B cells in intact lymph nodes can release blebs that are acquired by neighboring lymphocytes [29] and active ROCKdependent membrane blebbing during apoptosis has been reported in a number of in vitro studies [30]. In addition, one physiological mechanism of axon pruning involves active membrane blebbing [31]. Whatever the mechanism for the SSM blebbing, the present findings highlight the challenges associated with isolating pure populations of SSMs, challenges that likely extend to other cell populations with long membrane processes, such as the closely related CD169 + marginal metallophillic macrophages (MMMs) in the spleen and the non-hematopoietic follicular dendritic cells in all secondary lymphoid tissues. MMM isolation and analysis by flow cytometry has been reported in a number of studies but few of these studies have examined the isolated cells by microscopy. In a report where the low density (macrophage-enriched) fraction of cells from digested spleen and lymph nodes was examined by microscopy, less than 0.1% of the cells stained for markers corresponding to MMM and SSM; these cells were noted to have a granular cytoplasm and in some cases appeared tightly associated with lymphocyte-sized cells [32].
Given that CD169 bleb + cells are enriched for IL-7Ra hi CCR6 + cells and NK cells, in future efforts to isolate SSMs it will be important to include IL-7Ra, CCR6, and NK1.1 in addition to CD3e and B220 in a lineage 'dump' gate, while keeping in mind that SSMs are CD4 + . In our initial efforts to perform such an analysis we have found that there are very few lineage 'dump' negative cells (unpubl. obs.). However, it must also be considered likely that SSMs themselves will be strongly associated with IL-7Ra hi CCR6 + cells, causing them to be lost during doublet or 'dump' gating. In future studies, it will be important to perform microscopy on CD169 hi cells of large size, including cells that might be classified as doublets on flow cytometry, to more definitively test for the amount of SSM recovery that can be achieved with current tissue digestion procedures. Macrophage reporter mice, such as the MacGreen mice [33] or Lysozyme Mcre [34] x Rosa-stop flox YFP mice may be of utility in identifying intact SSMs, given that the cytoplasmic reporter molecules may be restricted to intact macrophages and absent from macrophagederived blebs. Until improved isolation and cell tracing procedures are developed it will be important to confirm any unique properties of the cells suggested based on gene expression analysis of sorted cells [2,15,17] through assessments of gene or protein expression by the cells in situ or after isolation from snap-frozen tissue by laser capture microscopy [35].
Our finding that mouse SSMs express CD4 is consistent with an earlier study in rats showing CD4 expression by these cells [36]. Most other mouse macrophage populations, including peritoneal macrophages, Kupffer cells, and red pulp macrophages do not express CD4, in contrast to rats as well as humans, in which CD4 expression by monocytes and macrophages is more widespread [37]. Thus, CD4 expression may be a special feature of these lymph node macrophages. CD4 expression by human lymph node sinus macrophages, which may correspond to mouse subcapsular sinus macrophages, has been reported [38]. SSMs are thought to be uniquely permissive for viral replication [12,15,35] raising the possibility that expression of the HIV-coreceptor CD4 by SSMs may play an important role during HIV infection and perhaps also in the capture and display of HIV-1 virions for recognition by B cells.
We describe a population of IL-7Ra hi CCR6 + CXCR6 hi lymphocytes that are abundant at the subcapsular sinus and in interfollicular regions. This is a diverse population of cells, including cdT, abT, and non-T cells, all of which rapidly produce IL-17A when stimulated with PMA and ionomycin ex vivo. The IL-7Ra hi CCR6 + TCRcd + cells correspond to innate IL-17 producing cdT that have been described in the dermis and peripheral lymph nodes [19,25,26]. 15-20% of IL-7Ra hi CCR6 + cells were iNKT cells, which likely correspond to the IL-17 producing iNKT cells recently described in skin and peripheral lymph nodes [27]. In another study, approximately 10% of adoptively transferred iNKT cells localized to the interfollicular or subcapsular sinus regions of the lymph node [17], consistent with the notion that at least a subset of iNKT localize near SSMs in the steady state. What types of effector cells the remaining TCRb + CD4 and CD8 double negative cells and CD3e 2 cells in the CD169 + IL-7Ra hi CCR6 + gate correspond to is unclear, although they may include IL-17 producing LTi-like cells [24,39].
The presence of IL-17 committed lymphocytes in or near the subcapsular sinus and their migration in close association with CD169 + macrophages raises the possibility that these cell types functionally interact. The spontaneous clustering or 'swarming' of CXCR6 hi (GFP hi ) cells in close proximity with SSMs observed in some of our imaging experiments (Supplementary Movie S2) also provides support for crosstalk between these cells though the type(s) of stimuli that provoke this behavior are not yet defined. IL-17 plays an important role in immunity at barrier surfaces, such as the skin [24]. One might consider the subcapsular sinus lining cells as a second barrier, given the dense network of macrophages and size-exclusion properties of this site as well as the constant exposure to lymph fluid that delivers antigens to the sinus within seconds to minutes of inoculation [1,3,40,41]. CD169 + macrophages, including cells at the subcapsular sinus and in interfollicular regions, constantly sample the lymph draining the skin for antigen and likely also for inflammatory cytokines. Thus, one intriguing possibility is that SSMs sense lymph-derived signals and activate adjacent lymphocytes by producing IL-17-promoting cytokines, such as IL-1b and IL-23 [42,43] or upregulating presentation of their cognate ligands, such as the recently described endogenous iNKT ligand, b-GlcCer [44]. Early production of IL-17 and possibly additional cytokines may enhance pro-inflammatory cytokine production and microbicidal activity of the macrophages [45,46] and help maintain the barrier function of the subcapsular sinus while influencing the initial induction of effector T [42] and B cells [47] within the lymph node parenchyma. Future studies should explore whether crosstalk between IL-17 committed lymphocytes and SSMs plays a role in protecting the lymph node from invading pathogens and in guiding early phases of lymph node immune responses.

Ethics Statement
All experiments conformed to ethical principles and guidelines approved by the UCSF Institutional Animal Care and Use Committee under protocol authorization number AN087331-01.

Tissue preparation
Unless otherwise indicated, lymph nodes were digested as described [2]. Briefly, lymph nodes were teased apart in DMEM containing penicillin/streptomycin and HEPES buffer and digested with 67 mg/ml Liberase TM (Roche) and 20 mg/ml DNAse I (Sigma) for 20 minutes while rotating. The digestion was then quenched by the addition of 10% fetal bovine serum (FBS) and 5 mM EDTA and lymph nodes were disaggregated by mashing through 100 mm nylon sieve (BD Bioscience).
To detect IL-17A, cells were stimulated for 2 h with 50 ng/ml PMA (Sigma) and 1 mg/ml Ionomycin (I, EMD Biosciences) in Brefeldin A (BD Biosciences), stained for surface antigens, treated with BD Cytofix Buffer and Perm/Wash reagent (BD Biosciences), and stained with anti-IL-17A.
RNA was extracted and Siglec1 mRNA was quantified as previously described [2]. For imaging, a fraction of sorted cells were washed with PBS containing 0.1% BSA and 0.1% sodium azide and applied to a chambered coverslip (CultureWell, Molecular Probes). After allowing cells to settle for 20 minutes at room temperature, supernatent was carefully aspirated from the coverslips. The cells were then fixed to the coverslip for 10 minutes at room temperature with 40 ml of 4% paraformaldehyde. Coverslips were washed with PBS, stained with DAPI, and then mounted onto a glass slide. Images were acquired with a Zeiss AxioObserver Z1 inverted microscope using equivalent exposure times. Optimal exposure times were determine based on the CD169 staining intensity of the majority of the sorted cells.

Two-photon microscopy
CFP + B cells were transferred intravenously and anti-CD169biotin/streptavidin-PE was injected s.c. to label SSMs in Cxcr6 gfp/+ mice 20-24 hours prior to imaging. Lymph node explants were prepared for imaging as previously described [29,54] and imaged with a Zeiss LSM 7MP equipped with a Chameleon laser (Coherent). Fluorophores were excited at 870 nm and detected with 450-490 nm (CFP), 500-550 nm (GFP) and 570-640 nm (PE) emission filters. Images were acquired with Zen (Zeiss), and time-lapse images generated with Imaris 7.4.0 (Bitplane). Videos were processed with a Gaussian noise filter. Annotation and final compilation of videos were with After Effects 7.0 software (Adobe Systems). Video files were converted to MPEG format with AVI-MPEG Converter for Windows 1.5 (FlyDragon Software). In some early experiments (imaging data not shown), images were acquired and videos generated as previously described [55]. Figure S1 IL-7Ra hi lymphocytes are located adjacent to CD169+ macrophages. Three additional examples of lymph node sections stained as in Figure 1B to detect CD169 (green) and IL-7Ra (red). FO, follicle; T, T zone. Scale bar = 50 mm. (TIF) Figure S2 CD169 staining on Siglec1 -/-(CD169-deficient) IL-7Ra hi CCR6 + lymphocytes and CD11c hi dendritic cells in mixed bone marrow chimeras. Flow cytometric detection of CD169 staining on CD45.2 + Siglec1 -/or Siglec1 +/+ (red) compared to CD45.1 + Siglec1 +/+ (blue) IL-7Ra hi CCR6 + cells (A) and CD11c hi cells (B) in mixed bone marrow chimeric mice. Data are representative of two experiments. (TIF) Figure S3 IL-7Ra hi CXCR6 hi lymphocytes at the subcapsular sinus and interfollicular regions. Immunofluorescence microscopy of lymph node sections from Cxcr6 GFP/+ mice stained with anti-CD169 (blue) and anti-IL-7Ra (red) monoclonal antibodies. Two examples are shown and are representative of sections from four lymph nodes from two mice. FO, follicle; T, T zone. Scale bar = 50 mm.

(TIF)
Movie S1 Two photon imaging of CXCR6 hi cells migrating in association with CD169 + macrophages. Intravital TPSLM showing CXCR6 hi cells migrate in close association with CD169 + macrophages (labeled with anti-CD169-PE) at the subcapsular sinus and interfollicular regions of an explanted Cxcr6 GFP/+ lymph node. Movie shows 12 mm maximum intensity z projection. Time is shown as hr:min:sec. Data in Movies S1 and S2 are representative of one experiment in which a total of five follicles in two lymph nodes were analyzed. Similar findings were observed in a second experiment of this type, in which macrophages were labeled with PE-ICs as described [2]. FO, follicle; IF, interfollicular region; SCS, subcapsular sinus.

(MPG)
Movie S2 Two photon imaging of CXCR6 hi cells migrating in association with CD169 + macrophages. Intravital TPSLM showing CXCR6 hi cells migrate in close association with CD169 + macrophages (labeled with anti-CD169-PE) at the subcapsular sinus and interfollicular regions of an explanted Cxcr6 GFP/+ lymph node. The clustering of CXCR6 hi cells seen in Movie S2 was observed in 4 of 10 movies. Movie shows 12 mm maximum intensity z projection. Time is shown as hr:min:sec. FO, follicle; IF, interfollicular region; SCS, subcapsular sinus. (MPG)