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• W2023143826 abstract "Article13 April 2012Open Access The location of splenic NKT cells favours their rapid activation by blood-borne antigen Patricia Barral Corresponding Author Patricia Barral Lymphocyte Interaction Laboratory, Cancer Research UK, London Research Institute, London, UK Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK Search for more papers by this author María Dolores Sánchez-Niño María Dolores Sánchez-Niño Lymphocyte Interaction Laboratory, Cancer Research UK, London Research Institute, London, UK Search for more papers by this author Nico van Rooijen Nico van Rooijen Department of Molecular Cell Biology, Faculty of Medicine, Vrije Universiteit, VUMC, Amsterdam, The Netherlands Search for more papers by this author Vincenzo Cerundolo Vincenzo Cerundolo Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK Search for more papers by this author Facundo D Batista Corresponding Author Facundo D Batista Lymphocyte Interaction Laboratory, Cancer Research UK, London Research Institute, London, UK Search for more papers by this author Patricia Barral Corresponding Author Patricia Barral Lymphocyte Interaction Laboratory, Cancer Research UK, London Research Institute, London, UK Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK Search for more papers by this author María Dolores Sánchez-Niño María Dolores Sánchez-Niño Lymphocyte Interaction Laboratory, Cancer Research UK, London Research Institute, London, UK Search for more papers by this author Nico van Rooijen Nico van Rooijen Department of Molecular Cell Biology, Faculty of Medicine, Vrije Universiteit, VUMC, Amsterdam, The Netherlands Search for more papers by this author Vincenzo Cerundolo Vincenzo Cerundolo Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK Search for more papers by this author Facundo D Batista Corresponding Author Facundo D Batista Lymphocyte Interaction Laboratory, Cancer Research UK, London Research Institute, London, UK Search for more papers by this author Author Information Patricia Barral 1,2, María Dolores Sánchez-Niño1, Nico van Rooijen3, Vincenzo Cerundolo2 and Facundo D Batista 1 1Lymphocyte Interaction Laboratory, Cancer Research UK, London Research Institute, London, UK 2Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK 3Department of Molecular Cell Biology, Faculty of Medicine, Vrije Universiteit, VUMC, Amsterdam, The Netherlands *Corresponding authors. Lymphocyte Interaction Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln's Inn Fields, London, WC2A 3LY, UK. Tel.:+44 (0)20 7269 3593; Fax:+44 (0)20 7269 3479; E-mail: [email protected] or Tel.:+44 (0)20 7269 2059; Fax:+44 (0)20 7269 3479; E-mail: [email protected] The EMBO Journal (2012)31:2378-2390https://doi.org/10.1038/emboj.2012.87 Correction(s) for this article The location of splenic NKT cells favours their rapid activation by blood-borne antigen29 June 2012 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Natural killer T (NKT) cells play an important role in mounting protective responses to blood-borne infections. However, though the spleen is the largest blood filter in the body, the distribution and dynamics of NKT cells within this organ are not well characterized. Here we show that the majority of NKT cells patrol around the marginal zone (MZ) and red pulp (RP) of the spleen. In response to lipid antigen, these NKT cells become arrested and rapidly produce cytokines, while the small proportion of NKT cells located in the white pulp (WP) exhibit limited activation. Importantly, disruption of the splenic MZ by chemical or genetic approaches results in a severe reduction in NKT cell activation indicating the need of cooperation between both MZ macrophages and dendritic cells for efficient NKT cell responses. Thus, the location of splenic NKT cells in the MZ and RP facilitates their access to blood-borne antigen and enables the rapid initiation of protective immune responses. Introduction NKT cells are a heterogeneous subset of classical αβ T lymphocytes that share the common characteristic of being restricted by the MHC class I-like molecule CD1, which can present self and foreign lipids (Barral and Brenner, 2007; Bendelac et al, 2007). As such, NKT cells can be directly activated in response to lipids from several bacteria (like Shingomonas, Erlichia, Borrelia or Streptococcus; Kinjo et al, 2005, 2006, 2011; Mattner et al, 2005) or in response to endogenous lipid antigens upregulated in antigen presenting cells (APCs) after TLR stimulation (Brigl et al, 2003; Paget et al, 2007; Salio et al, 2007). The activation of NKT cells by these two mechanisms explains their involvement in a broad range of immune responses against viruses, bacteria, fungus, anti-tumour immunity and autoimmune processes (Bendelac et al, 2007; Tupin et al, 2007; Cerundolo et al, 2009). Upon activation, NKT cells rapidly secrete large amounts of cytokines and induce downstream activation of different cell types, including dendritic cells (DCs), NK cells, B cells and conventional T cells. On this basis, NKT cells have been described as a bridge between innate and adaptive immune responses (Bendelac et al, 2007). In recent years, various studies have highlighted the particular importance of NKT cells in responding to blood-borne pathogens and these cells have been shown to accumulate in the vasculature of both lung and liver (Geissmann et al, 2005; Velázquez et al, 2008; Lee et al, 2010; Scanlon et al, 2011). However, in spite of the fact that the spleen is the major site where immune responses to blood-borne antigens are initiated (Mebius and Kraal, 2005), little is currently understood about where NKT cells are located and their dynamic features in this organ. Indeed, due to the technical difficulties associated with unambiguous identification of these cells using immunohistochemistry (Berzins et al, 2005), the distribution of endogenous NKT cells in the spleen has remained an open question in the field. The spleen is highly structured and composed of the red pulp (RP) that acts mainly as a blood filter, and the white pulp (WP) where the majority of B and T lymphocytes reside, separated by the marginal zone (MZ). The MZ contains a specialized subset of MZ B cells, DCs and two subsets of macrophages: MZ macrophages characterized by the expression of the surface receptors SIGN-R1 and/or MARCO (Elomaa et al, 1995; Geijtenbeek et al, 2002), and metallophilic macrophages expressing sialoadhesin (CD169, Siglec-1 (Kraal and Janse, 1986)). Arterial branches in the spleen terminate in the marginal sinus, allowing blood to transit through the MZ and into the RP (Mebius and Kraal, 2005). This organization allows cells in the MZ to be in continuous contact with blood-borne antigens and indeed, MZ B cells and macrophages have been described as main players in the initiation of immune responses against blood-borne pathogens (Martin et al, 2001; Aichele et al, 2003; Kang et al, 2004). The MZ is a very complex structure in which the various intercellular interactions appear to be critical for the organization and function of the individual cell types. However, the precise role of the MZ populations, particularly in antigen presentation during the initiation of immune responses, remains unclear (Ciavarra et al, 1997; Aichele et al, 2003; Backer et al, 2010; You et al, 2011). Here, we have used a combination of pulse-labelling, immunofluorescence and multi-photon microscopy to show that the majority of splenic NKT cells localize to and patrol around the MZ and RP. In response to lipid antigen, these NKT cells arrest in close proximity to antigen-containing cells in the MZ and become activated to secrete cytokines. In contrast, NKT cells located in the periarteriolar lymphoid sheath (PALS) showed limited activation. Moreover, the disruption of macrophages and dendritic cells in the MZ dramatically reduces NKT cell activation, suggesting that the cooperation of cells in the MZ is necessary for efficient NKT cell responses. Results NKT cells are highly accessible to the blood entering the spleen Since the role of NKT cells in response to blood-borne antigen has been well established, we sought to examine the accessibility of NKT cells to the blood entering the spleen as an indication of their location. To do this, we adapted an in vivo pulse-labelling procedure that allows the selective labelling of cells according to their exposure to the blood (Figure 1; Cinamon et al, 2008; Pereira et al, 2009; Muppidi et al, 2011). Mice were intravenously (i.v.) injected with phycoerythrin-labelled anti-CD45 antibody (CD45-PE) and spleen sections were imaged by confocal microscopy (Figures 1A and B). As expected, the MZ region became highly labelled after a brief (3 min) exposure to CD45-PE, while no staining was detected in the WP that was protected from antibody arrival (Figure 1A). In line with this, flow cytometric analysis of the extent of CD45-PE labelling in total splenocytes revealed that a large proportion of MZ B cells (B220+CD21hiCD23lo) were highly labelled with CD45-PE compared with follicular B (B220+CD21loCD23hi) and T cells (Figure 1A). Using this approach, we observed that the majority of splenic NKT cells, identified as either TCR-β+αGalCer-CD1d tetramer+B220− cells (Figure 1A) or TCR-β+NK1.1+B220− cells (Figure 1D), were highly labelled with CD45-PE (72±7% and 75±5%, respectively), indicating their proximity to the blood supplied to the spleen. Unlike MZ B cells, the proportion of NKT cells labelled after longer (20 min) antibody treatments remains stable (Figures 1B and C), although the mean fluorescence intensity (MFI) of labelling in the NKT population increased over time (Figure 1C). Interestingly, we did not observe striking phenotypical differences between highly and poorly in vivo labelled NKT cells in terms of the expression of CD4, CD8, DX5, CD44, CD122, NK1.1 and CD62L, although CD69 expression seemed to be higher in CD45-PE+ NKT cells (Supplementary Figure S1). Figure 1.Splenic NKT cells are accessible to the blood entering the spleen. (A–D) Mice were injected with CD45-PE antibody 3 min (A, C, D) or 20 min (B, C) before analyses. (A, B) Immunofluorescence (left) from spleens of mice injected with CD45-PE (red) stained with CD169 (blue). Bars, 50 μm. Flow cytometry for CD45-PE binding by splenic MZ B cells, T cells and NKT cells (black line; grey solid profile, un-injected control) (C) MFI (left) and percentage of cells (right) binding to CD45-PE in the referred splenic populations at 3 and 20 min after injection. Each dot represents an individual animal. (D) Flow cytometry of B220− splenocytes showing TCR-β and NK1.1 (left), and binding of CD45-PE by TCR−β+NK1.1+B220− cells (right). Data represent 5 independent experiments with 2 mice per experiment. Download figure Download PowerPoint Therefore, our results indicate that the majority of NKT cells are readily accessible to blood entering the spleen, suggesting that they reside outside the splenic WP. NKT cells are preferentially located in the splenic MZ and RP We moved on to directly visualize the distribution of endogenous NKT cells in the spleen and initially adopted an approach using CD1d tetramer staining of splenic frozen sections. However, consistent with previous reports, this proved technically challenging (Berzins et al, 2005; Thomas et al, 2011) and as a result of high levels of background staining we were unable to unambiguously identify endogenous NKT cells. To overcome this, we have used two alternative strategies to elucidate the distribution of splenic NKT cells. First, endogenous NKT cells were identified in situ in flash-frozen cryostat sections of spleens of mice previously perfused with neutral buffered formalin (Figures 2A and B; Supplementary Figure S2; Andrews et al, 2001). This method allows discrimination of TCR-β+NK1.1+ NKT cells from NK cells (NK1.1+TCR-β−) and conventional T cells (TCR-β+NK1.1−). However, as both TCR and NK1.1 can be down-regulated in activated NKT cells, we have used a second, complementary strategy involving the adoptive transfer of highly purified NKT cells into congenic recipients (Figures 2C and D; Supplementary Figure S2; Barral et al, 2010). Figure 2.Splenic NKT cells are predominantly located in the MZ and RP. (A–D) Immunofluorescence from spleen sections stained with B220 (cyan), CD169 (green), TCR-β (red) and NK1.1 (blue, A) or CD45.2 (blue, C). White dots depict NKT cells. Bars, 200 μm. (B, D) Percentages (left) and proportion of cells per area (right) of NKT cells in the RP, MZ, B cell follicles (B) and PALS (T) for endogenous (B) and adoptively transferred (D) NKT cells. (E) 2 × 106 sorted NKT cells were labelled with CellTrace Violet (CTV) and injected into WT mice. 16 h later recipients were injected with CD45-PE 3 min before analysis. Transferred cells were detected in the recipient spleen as CTV+ cells (left) and showed high CD45-PE labelling (right). (F, top) Multi-photon microscopy image of the spleen showing collagen capsule (blue) and NKT cells (red). Tracks for individual cells are depicted in pink. Bar, 50 μm. (F, bottom) Snapshot images showing NKT cells (red) and T cells (blue) at the indicated time points. Individual cell tracks are coloured in pink (NKT cells) and blue (T cells). Stamp, min:sec. Bars, 20 μm. (G–J) Average speed (G), speed distribution (H), arrest coefficient (I) and migratory tracks (J) for NKT cells and T cells. Each dot represents an individual cell. Data were pooled from 2 independent experiments with 2 mice each. p, unpaired two-tailed t-test. Download figure Download PowerPoint Importantly, both endogenous (n=247) and adoptively transferred (n=81) NKT cells occupied a similar distribution within the splenic sections. We observed that splenic NKT cells were predominantly located in the RP, MZ and T cell areas (47%, 25 and 23%, respectively) but virtually excluded from B cell follicles. In order to compare the relative distribution of NKT cells amongst splenic regions, the proportion of NKT cells was related to the cross-sectional area of each region (Supplementary Figures S2C and D, previously reported (Aoshi et al, 2008)). This type of analysis revealed the preferential accumulation of NKT cells in the MZ (Figures 2B and D). Notably, the total proportion of NKT cells present in the RP and MZ (∼70%), and therefore readily accessible to the blood entering the spleen, is equivalent to the percentage of endogenous NKT cells labelled by in vivo antibody injection (Figure 1A). Similarly, the majority of adoptively transferred NKT cells were highly stained after pulse-labelling with CD45-PE (∼72%), confirming that they occupy a similar distribution in the spleen than that of endogenous cells (Figure 2E). To characterize the spatiotemporal dynamics of NKT cells in the spleen we have used time-lapse multi-photon microscopy. This method presents important technical difficulties since the maximum imaging depth achievable in the spleen is severely hampered by the absorption and scattering of light by blood. However, as the majority of NKT cells are excluded from the PALS, we imaged the spleen directly through the collagen capsule (to a maximum depth of 70 μm), which allowed us to visualize NKT cells outside the WP (Figures 2F–J, Supplementary Movies S1 and S2). Under these conditions, we observed that splenic NKT cells migrate with an average speed of 6.2±2.7 μm/min (Figure 2G). The distribution of instantaneous speeds for NKT cells showed maximum values of around 20 μm/min and low arrest coefficients (13%) similar to those measured for conventional T cells (10%; Figures 2H and I). To visualize NKT cells located in deeper regions of the spleen (MZ and PALS) we performed an alternative imaging approach by cutting open the spleen across the transverse axis and placing the tissue in warm oxygenated medium for imaging (as previously reported (Aoshi et al, 2008; Bajénoff et al, 2008)). Under these imaging conditions NKT cells showed comparable dynamics to those of NKT cells visualized in intact spleen (average speed, 5.9±0.4 μm/min; arrest coefficient, 13%; Supplementary Figure S3, Supplementary Movie S3). Therefore, splenic NKT cells predominantly reside in the MZ and RP and patrol the resting spleen with similar dynamic features as those observed for conventional T cells. NKT cells are activated by blood-borne lipid antigen outside the WP The observed location of splenic NKT cells renders them in an ideal position to sense circulating antigen in its arrival to the spleen (Mebius and Kraal, 2005). Therefore we have used different approaches in order to investigate the behaviour of splenic NKT cells in response to antigen administration. To mimic conditions occurring during infection with bacteria encoding NKT cell agonists we injected mice i.v. with particles (200 nm diameter) coated with either α-linked galacturonic glycosphingolipid (GalA-GSL) from Sphingomonas yanoikuyae or αGalCer (Kinjo et al, 2005; Mattner et al, 2005; Barral et al, 2010). We observed that the majority of splenic NKT cells were activated to produce IFN-γ and IL-4, within 2 h of i.v. injection with bacterial lipids (Figure 3A). A similar activation was observed following administration of particulate αGalCer while no cytokine production was detected after injection of control lipids (Figure 3A). Next we examined the distribution of both endogenous and adoptively transferred NKT cells 2 h after i.v. administration of lipid antigen (Figures 3B–D, Supplementary Figure S4A and B). At this time, we observed that the proportion of NKT cells in the splenic MZ increased (25±4% to 38±4%, n=187, Figures 3C and D). However, the total proportion of NKT cells outside the splenic WP did not significantly change following antigen administration (∼70%, Figures 3C and D), suggesting that cells relocate from the RP into the splenic MZ. Figure 3.NKT cell activation in response to lipid antigen (A) Intracellular IFN-γ (top) and IL-4 (bottom) staining for splenic NKT cells (TCR-β+αGalCer-CD1d tetramer+B220−) 2 h after injection of particulate control lipids (left), GalA-GSL (middle) or αGalCer (right). (B–D) Confocal microscopy identification of endogenous NKT cells 2 h after antigen injection. (B) Spleen sections were stained with B220 (cyan), CD169 (green), TCR-β (red) and NK1.1 (blue). Bars, 200 μm. (C, D) Percentages (C) and proportion of cells per area (D) for NKT cells in the RP, MZ, B cell follicles (B) and PALS (T). Data represent 2 independent experiments. p, unpaired two-tailed t-test. Download figure Download PowerPoint Although the majority of NKT cells localize in the MZ after antigen administration still a number of cells remained in the WP. Therefore we sought to investigate a potential relationship between NKT cell location and their activation status by extending the in vivo pulse-labelling approach. Two hours after i.v. injection of particulate lipid antigen, mice received a 3 min pulse of CD45-PE and the extent of labelling was correlated with the activatory status of NKT cells (Figure 4). Notably, in response to either bacterial-derived GalA-GSL or αGalCer, the majority of NKT cells that were activated to secrete IL-4 and IFN-γ were highly labelled in vivo with CD45-PE. In contrast, those NKT cells located in the WP that were protected from CD45-PE labelling, exhibited very limited activation. Comparably, i.v. administration of soluble αGalCer induced the same kind of differential activation in NKT cells located inside or outside the splenic WP (Supplementary Figures S4C–F). Figure 4.NKT cell activation occurs preferentially outside the WP. (A–H) Mice were injected with particulate GalA-GSL (A–D) or αGalCer (E–H) and 2 h later they received CD45-PE 3 min before analyses. Flow cytometry profiles and quantification of intracellular IFN-γ (A, B and E, F) and IL-4 (C, D and G, H) for total (left) and highly (middle, CD45-PE+) or poorly (right, CD45-PE−) CD45-PE labelled NKT cells (TCR-β+αGalCer-CD1d tetramer+B220−). p, paired t-test. Data represent 3 independent experiments with at least 3 mice each. Download figure Download PowerPoint Thus, the localization of NKT cells outside the splenic WP promotes their early activation in response to blood-borne antigen arrival. Splenic NKT cells arrest in the MZ Since NKT cell activation takes place within hours of antigen administration we asked the question of where blood-borne particulate lipids localized on their arrival to the spleen. The location of fluorescently labelled particulate lipids in the spleen was tracked using confocal microscopy of frozen sections (Figure 5A). Two hours after i.v. administration, lipid antigen was predominantly located in the MZ and co-localized with MARCO+ and SIGN-R1+ MZ macrophages and CD11c+ DCs (Figure 5A). Importantly, we observed that endogenous NKT cells were also situated in the MZ and appeared in close proximity to cells containing particulate GalA-GSL and αGalCer (Figures 5B and C). Figure 5.NKT cells arrest in the MZ in response to lipid antigen. (A–C) Mice received lipid antigen 2 h before analyses. (A) Immunofluorescence of spleen sections showing particulate αGalCer (green) stained with CD169, SIGN-R1 or MARCO (red) and B220, CD11c or F4/80 (blue). (B, C) Immunofluorescence of spleen sections showing particulate GalA-GSL (B, green) and αGalCer (C, green) and endogenous NKT cells stained with TCR-β (red) and NK1.1 (blue). Bars, 50 μm. (D–I) NKT cell dynamics after antigen administration. Mice were injected with particulate αGalCer (green) 2 h before imaging. (D) Multi-photon microscopy image of the spleen showing particulate lipids (green) and NKT cells (red). Average speed (E), migratory tracks (F), snapshot images (G), arrest coefficient (H) and speed distribution (I) for splenic NKT cells (red) and T cells (blue). Individual cell tracks are coloured in pink (NKT cells) and blue (T cells). Bars, 20 μm. p, unpaired two-tailed t-test. Data were pooled from 2 independent experiments with 2 mice each. Download figure Download PowerPoint In order to understand the effect of antigen arrival in NKT cell dynamics we imaged the spleen by multi-photon microscopy 2 h after antigen administration. At this time point we observed that NKT cells stopped their migration and became confined in antigen-rich regions (Figures 5D–I, Supplementary Movies S4 and S5). Consequently we measured a decrease in the average speed of NKT cells (3.7±2.5 μm/min versus 6.2±2.7 μm/min in the absence of antigen; Figure 5E) that was accompanied by an increase in arrest coefficient (56% versus 13%; Figure 5H). In contrast, the average speed and arrest coefficient observed for conventional T cells was not changed following lipid antigen administration (6.2±1.9 μm/min and 13.4%, respectively; Figures 5D–I). The alteration of the dynamic behaviour of splenic NKT cells in response to lipid antigen is similar to that reported for NKT cells both in lymph nodes and liver, suggesting that their migration provides a means of immune surveillance in tissues (Geissmann et al, 2005; Barral et al, 2010; Lee et al, 2010). Role of different APCs in activation of splenic NKT cells The identity of the APCs that participate in the initiation of NKT cell activation in the spleen is largely unknown. To assess the ability of primary splenic DCs and MZ macrophages to retain lipid antigens arriving to the spleen we injected mice with fluorescent particulate lipids and 2 h later we analysed antigen retention by splenic cell populations by flow cytometry. At this time point around 50% of DCs and SIGN-R1+ MZ macrophages were found to retain particulate lipids after in vivo injection (Figures 6A and B). Importantly, both of these cell types showed high expression of CD1d (Figures 6C and D). Figure 6.Role of different APCs in NKT cell activation (A) Flow cytometry analyses of splenic single cell suspensions showing CD11chigh DC and SIGN-R1+ macrophage populations. (B) Flow cytometry analyses of lipid uptake by CD11chigh DCs (left) and SIGN-R1+ macrophages (right) in the spleen 2 h after injection of αGalCer particles (un-injected control, grey filled histogram). (C, D) CD1d expression in splenic DCs (C) and SIGN-R1+ macrophages (D). (E) DCs (left) and SIGN-R1+ macrophages (right) were purified by sorting and stained with CD11c and SIGN-R1 antibodies. (F) Lipid presentation by sorted DCs (O) and SIGN-R1+ MZ macrophages () incubated with αGalCer particles previous to co-culture with DN32.D3 NKT cells. Secretion of IL-2 into the culture medium by DN32.D3 cells was measured as a read-out for lipid presentation. Download figure Download PowerPoint Subsequently, we went on analysing the ability of MZ macrophages and DCs to stimulate NKT cell activation in vitro. To this end, SIGN-R1+ macrophages and CD11chi DCs were enriched from splenic single cell suspensions by depletion of B and T cells and further purified by flow cytometry sorting (Figure 6E). After pre-incubation for 2 h with particulate lipids, sorted cells were co-cultured with NKT cell hybridoma DN32.D3 cells. As shown in Figure 6F αGalCer was efficiently presented by both DCs and SIGN-R1+ cells, as measured by IL-2 production by DN32.D3 cells, with DCs showing a stronger presentation capacity than macrophages. Thus, DCs and MZ macrophages retain lipid antigen on its arrival to the spleen and they can efficiently present lipids to mediate NKT cell activation. NKT cell activation depends on the integrity of the MZ To investigate the role of DCs and macrophages in mediating NKT cell activation in vivo we have used two different approaches that allowed us to disrupt particular cell populations present in the spleen. Firstly, mice were treated with clodronate liposomes (CLL) to selectively deplete macrophages in the MZ and RP. However, in line with previous reports (Aoshi et al, 2008), two days after CLL treatment we observed a depletion not only of MARCO+, SIGN-R1+, F4/80+ and CD169+ cells but also a reduction in CD11chi cells (Figure 7A, Supplementary Figure S5A–E). Concomitant with the loss of these cells, antigen was not efficiently retained in the splenic MZ and was instead dispersed throughout the RP. Moreover, at this time we detected a dramatic decrease in the percentage of NKT cells that were activated to secrete IFN-γ (23±5% versus 69±3%; Figure 7B, Supplementary Figure S5E), accompanied by a decrease in MFI for intracellular IFN-γ secreted by NKT cells (46±13% MFI reduction after CLL treatment versus untreated mice). Figure 7.Disruption of the MZ reduces NKT cell activation. (A–J) Mice were injected with particulate αGalCer 2 h before analyses. Confocal microscopy images (A, C, E, G, I) and flow cytometry profiles (B, D, F, H, J) of spleens from WT mice treated with CLL for 2 days (A, B), 6 days (C, D), 16 days (E, F) or 22 days (G, H) and CD11c-DOG mice treated with DT (I, J). Immunofluorescence images show particulate αGalCer (green), MARCO (red) and B220 (blue); Bars, 50 μm. Flow-cytometry plots show NKT cells (left), intracellular IFN-γ staining for NKT cells (middle) and quantification of IFN-γ production (right) for NKT cells in treated (+) and untreated (−) mice. p, unpaired two-tailed t-test. Data represent 2 independent experiments with at least 3 mice per experiment. Download figure Download PowerPoint As the DC population recovers more rapidly than macrophages after CCL administration, we went on to assess the contribution of these populations in NKT cell activation six days after CCL treatment which allows recovering of DCs while macrophages remain absent (Figures 7C and D, Supplementary Figure S5F–H). At this time, antigen was not efficiently retained at the MZ however we detected that a substantial proportion of NKT cells recovered their ability to secrete cytokines after antigen injection (46±9% IFN-γ+ NKT cells; 28±18% MFI reduction for IFN-γ after CLL treatment versus untreated mice; Figure 7D, Supplementary Figure S5H), suggesting that DCs play a significant role in mediating NKT cell activation. At later times after CLL treatment (16 days) we found that DC numbers were equivalent to those of WT mice and the populations of F4/80+ and MARCO+ macrophages started to recover, with cells appearing scattered through the RP and partially colocalizing with injected particulate lipids (Figures 7E and F, Supplementary Figure S5I–K). However CD169+ and SIGN-R1+ cells remained absent (Supplementary Figure S5I). Despite the reappearance of some macrophage populations, the extent of NKT cell activation after antigen injection (41±4% IFN-γ NKT cells, Figure 7F) was still comparable to the one measured at 6 days after CLL treatment suggesting that the positioning of the different macrophage populations in the MZ is important for efficient antigen retention and subsequent NKT cell activation. Twenty two days after CLL treatment we observed a complete recovery of all macrophage populations, accompanied by an efficient antigen retention in the" @default.
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• W2023143826 date "2012-04-13" @default.
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• W2023143826 title "The location of splenic NKT cells favours their rapid activation by blood-borne antigen" @default.
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