Gatekeeper role of brain antigen‐presenting CD11c + cells in neuroinflammation
2015; Springer Nature; Volume: 35; Issue: 1 Linguagem: Inglês
10.15252/embj.201591488
ISSN1460-2075
AutoresMagdalena Paterka, Volker Siffrin, Jan Oliver Voß, Johannes Werr, Nicola Hoppmann, René Gollan, Patrick Belikan, Julia Bruttger, Jérôme Birkenstock, Steffen Jung, Enric Esplugues, Nir Yogev, Richard A. Flavell, Tobias Bopp, Frauke Zipp,
Tópico(s)Immune cells in cancer
ResumoArticle26 November 2015free access Gatekeeper role of brain antigen-presenting CD11c+ cells in neuroinflammation Magdalena Paterka Magdalena Paterka Department of Neurology, Focus Program Translational Neurosciences (FTN), Research Center for Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University, Mainz, Germany Search for more papers by this author Volker Siffrin Volker Siffrin Department of Neurology, Focus Program Translational Neurosciences (FTN), Research Center for Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University, Mainz, Germany Search for more papers by this author Jan O Voss Jan O Voss Molecular Neurology, Max Delbrück Center for Molecular Medicine Berlin-Buch, Berlin, Germany Search for more papers by this author Johannes Werr Johannes Werr Molecular Neurology, Max Delbrück Center for Molecular Medicine Berlin-Buch, Berlin, Germany Search for more papers by this author Nicola Hoppmann Nicola Hoppmann Department of Neurology, Focus Program Translational Neurosciences (FTN), Research Center for Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University, Mainz, Germany Search for more papers by this author René Gollan René Gollan Department of Neurology, Focus Program Translational Neurosciences (FTN), Research Center for Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University, Mainz, Germany Search for more papers by this author Patrick Belikan Patrick Belikan Department of Neurology, Focus Program Translational Neurosciences (FTN), Research Center for Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University, Mainz, Germany Search for more papers by this author Julia Bruttger Julia Bruttger Institute for Molecular Medicine, Research Center for Immunotherapy (FZI), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Search for more papers by this author Jérôme Birkenstock Jérôme Birkenstock Department of Neurology, Focus Program Translational Neurosciences (FTN), Research Center for Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University, Mainz, Germany Search for more papers by this author Steffen Jung Steffen Jung Department of Immunology, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Enric Esplugues Enric Esplugues Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Nir Yogev Nir Yogev Institute for Molecular Medicine, Research Center for Immunotherapy (FZI), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Search for more papers by this author Richard A Flavell Richard A Flavell Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Tobias Bopp Tobias Bopp Institute for Immunology, Research Center for Immunotherapy (FZI), University Medical Center of the Johannes Gutenberg University, Mainz, Germany Search for more papers by this author Frauke Zipp Corresponding Author Frauke Zipp Department of Neurology, Focus Program Translational Neurosciences (FTN), Research Center for Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University, Mainz, Germany Search for more papers by this author Magdalena Paterka Magdalena Paterka Department of Neurology, Focus Program Translational Neurosciences (FTN), Research Center for Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University, Mainz, Germany Search for more papers by this author Volker Siffrin Volker Siffrin Department of Neurology, Focus Program Translational Neurosciences (FTN), Research Center for Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University, Mainz, Germany Search for more papers by this author Jan O Voss Jan O Voss Molecular Neurology, Max Delbrück Center for Molecular Medicine Berlin-Buch, Berlin, Germany Search for more papers by this author Johannes Werr Johannes Werr Molecular Neurology, Max Delbrück Center for Molecular Medicine Berlin-Buch, Berlin, Germany Search for more papers by this author Nicola Hoppmann Nicola Hoppmann Department of Neurology, Focus Program Translational Neurosciences (FTN), Research Center for Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University, Mainz, Germany Search for more papers by this author René Gollan René Gollan Department of Neurology, Focus Program Translational Neurosciences (FTN), Research Center for Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University, Mainz, Germany Search for more papers by this author Patrick Belikan Patrick Belikan Department of Neurology, Focus Program Translational Neurosciences (FTN), Research Center for Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University, Mainz, Germany Search for more papers by this author Julia Bruttger Julia Bruttger Institute for Molecular Medicine, Research Center for Immunotherapy (FZI), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Search for more papers by this author Jérôme Birkenstock Jérôme Birkenstock Department of Neurology, Focus Program Translational Neurosciences (FTN), Research Center for Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University, Mainz, Germany Search for more papers by this author Steffen Jung Steffen Jung Department of Immunology, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Enric Esplugues Enric Esplugues Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Nir Yogev Nir Yogev Institute for Molecular Medicine, Research Center for Immunotherapy (FZI), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany Search for more papers by this author Richard A Flavell Richard A Flavell Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Tobias Bopp Tobias Bopp Institute for Immunology, Research Center for Immunotherapy (FZI), University Medical Center of the Johannes Gutenberg University, Mainz, Germany Search for more papers by this author Frauke Zipp Corresponding Author Frauke Zipp Department of Neurology, Focus Program Translational Neurosciences (FTN), Research Center for Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University, Mainz, Germany Search for more papers by this author Author Information Magdalena Paterka1,‡, Volker Siffrin1,7,‡, Jan O Voss2,8,‡, Johannes Werr2,9,‡, Nicola Hoppmann1, René Gollan1, Patrick Belikan1, Julia Bruttger3, Jérôme Birkenstock1, Steffen Jung4, Enric Esplugues5,10, Nir Yogev3, Richard A Flavell5, Tobias Bopp6 and Frauke Zipp 1 1Department of Neurology, Focus Program Translational Neurosciences (FTN), Research Center for Immunotherapy (FZI), Rhine-Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University, Mainz, Germany 2Molecular Neurology, Max Delbrück Center for Molecular Medicine Berlin-Buch, Berlin, Germany 3Institute for Molecular Medicine, Research Center for Immunotherapy (FZI), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany 4Department of Immunology, Weizmann Institute of Science, Rehovot, Israel 5Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA 6Institute for Immunology, Research Center for Immunotherapy (FZI), University Medical Center of the Johannes Gutenberg University, Mainz, Germany 7Present address: Charité - Universitätsmedizin Berlin, ECRC, Berlin, Germany 8Present address: Department of Craniomaxillofacial Surgery, Surgical Navigation, Charité - Universitätsmedizin Berlin, Campus Virchow-Klinikum, Berlin, Germany 9Present address: Department of Neurology, Klinikum rechts der Isar, Technische Universität München, München, Germany 10Present address: Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +49 6131 17 7156; Fax: +49 30 17 5697; E-mail: [email protected] The EMBO Journal (2016)35:89-101https://doi.org/10.15252/embj.201591488 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 Abstract Multiple sclerosis is the most frequent chronic inflammatory disease of the CNS. The entry and survival of pathogenic T cells in the CNS are crucial for the initiation and persistence of autoimmune neuroinflammation. In this respect, contradictory evidence exists on the role of the most potent type of antigen-presenting cells, dendritic cells. Applying intravital two-photon microscopy, we demonstrate the gatekeeper function of CNS professional antigen-presenting CD11c+ cells, which preferentially interact with Th17 cells. IL-17 expression correlates with expression of GM-CSF by T cells and with accumulation of CNS CD11c+ cells. These CD11c+ cells are organized in perivascular clusters, targeted by T cells, and strongly express the inflammatory chemokines Ccl5, Cxcl9, and Cxcl10. Our findings demonstrate a fundamental role of CNS CD11c+ cells in the attraction of pathogenic T cells into and their survival within the CNS. Depletion of CD11c+ cells markedly reduced disease severity due to impaired enrichment of pathogenic T cells within the CNS. Synopsis Brain antigen-presenting CD11c+ cells play a fundamental role in the attraction of pathogenic T cells into and their survival within the CNS, critically influencing the immune response in neuroinflammation. Investigation of role of CNS CD11c+ cells using experimental autoimmune encephalomyelitis in CD11c-DTR/GFP transgenic mouse model and two-photon laser-scanning microscopy. IL-17 expression correlates with GM-CSF expression by T cells and with accumulation of CD11c+ cells. Accumulated CD11c+ cells organize in perivascular clusters and strongly express the inflammatory chemokines Ccl5, Cxcl9, and Cxcl10. Depletion of CD11c+ cells reduced the encephalitogenicity of adoptively transferred T cells in CNS and thus markedly reduced EAE disease severity. Introduction Multiple sclerosis (MS) is a complex disease of the central nervous system (CNS) involving T cells in its pathology, as also recently evidenced in a genomewide association study (GWAS) (International Multiple Sclerosis Genetics Consortium (IMSGC) et al, 2011; 2013). It has been well established that the clinical severity of the animal disease model, experimental autoimmune encephalomyelitis (EAE), directly correlates with the number and differentiation of CD4+ T-helper cells inside the target organ (Hofstetter et al, 2007), among which Th17 cells strongly contribute to tissue destruction (Siffrin et al, 2010). Indeed, preliminary data from a phase II clinical study of a blocking antibody against IL-17 (AIN457) in patients with MS indicate that targeting Th17 cells might be a viable strategy in the treatment of multiple sclerosis (Fernández et al, 2013). Several recent reports indicate that another Th17 cytokine, the granulocyte-macrophage colony-stimulating factor (GM-CSF), represents a major effector molecule in these processes in EAE (Codarri et al, 2011; El-Behi et al, 2011) and MS (Hartmann et al, 2014; Noster et al, 2014). Since GM-CSF is crucial for the differentiation and survival of certain DC subsets (Markowicz & Engleman, 1990), the role of DCs in neuroinflammation might be more relevant for disease persistence than previously thought. DCs are professional antigen-presenting cells, which have a crucial role in the differentiation of T cells and integrate multiple stimuli, most importantly from the innate immune system and invading pathogens, in the decision of whether a pro-inflammatory or regulatory adaptive immune response is induced. However, DCs are very heterogenic, dependent on their anatomical location and phenotype (Schlitzer & Ginhoux, 2014). It has been reported that DCs are sufficient for promoting an autoreactive response inside the CNS (Greter et al, 2005) and are capable of inducing and amplifying EAE (McMahon et al, 2005; Karman et al, 2006). However, the priming of encephalitogenic T cells has been found to be unaffected or even exaggerated by the absence of DCs in active EAE induced by myelin peptides and strong adjuvants (Isaksson et al, 2012; Yogev et al, 2012). Therefore, a dual role of DCs seems possible, that is, a regulatory role in the context of T-cell differentiation in the secondary lymphoid organs and a rather pro-inflammatory role in the CNS. Focusing on CNS DCs, which are a very rare cell population under physiologic conditions, it has been unclear to what extent they contribute to local T-cell recruitment, activation, and lesion development. In this context, the role of inflammatory chemokines for recruitment and orchestration of tissue inflammation is of significant interest. T cells that express the chemokine receptors CCR2 and CCR5 are more abundant in patients with exacerbated MS (Misu et al, 2001). Furthermore, perivascular T cells in MS and EAE express the chemokine receptor CXCR3. However, the sources of the relevant chemokines remain unclear. Here, we investigate the role of CNS CD11c+ cells by exploiting a transgenic mouse model, CD11c-DTR/GFP (Jung et al, 2002), which uses the commonly accepted DC marker CD11c to specifically target DCs. This transgenic model allows the conditional depletion of classical DCs after diphtheria toxin (DTX) treatment (Jung et al, 2002). We performed conditional depletion experiments in the effector phase of adoptive transfer EAE, which targets primarily the processes within the CNS. Furthermore, we isolated CNS CD11c+ cells and characterized their chemokine expression profiles, which revealed a unique role of this cell subset for T-cell migration and recruitment. In addition, on the T-cell side, we made use of IL-17 reporter mice, which express the fluorescent protein EGFP, concomitantly to IL-17 (Esplugues et al, 2011). We utilized in vivo two-photon laser scanning microscopy (TPLSM) in order to observe the real behavior of DCs and IL-17-producing cells at the barrier of, and within, the CNS. Results Depletion of CD11c-GFP+ cells aborts EAE induction by adoptive transfer of encephalitogenic T cells To investigate the role of CD11c-GFP+ cells in the effector phase of EAE, we used the adoptive transfer EAE model of transgenic myelin-specific (MOG35-55-specific, 2d2) CD4+ T cells, which had been differentiated to Th17 cells in vitro (Siffrin et al, 2010). To analyze the role of CD11c+ cells in the adoptive transfer model, ablation of CD11c+ cells by subcutaneous DTX application was started in chimeric CD11c-DTR/GFP→C57BL/6 mice before transfer of 2d2.tdRFPxIl-17-EGFP Th17 cells and continued every other day during the whole observation period, resulting in reliable CD11c+ cell depletion. Experiments that required CD11c depletion by DTX were performed in chimera to bypass the reported problem of lethality after repetitive DTX injection in the CD11c-DTR/GFP mice (Probst & van den Broek, 2005; Zaft et al, 2005). Bone marrow chimeric mice were generated as described previously (Siffrin et al, 2009) and yielded a chimerism of > 85% when looking at CD11c-GFP+ cells of CD11c+ cells and > 93% for CD11c-DTR-depleted cells of CD11c+ cells (Appendix Fig S1A and B). Repetitive depletion, even after 11 and more DTX injections, did not result in a loss of depletion efficiency in the CNS or spleen and depleted all relevant subsets of CD11c+ cells (Appendix Fig S1C and D). In adoptive transfer EAE in CD11c+-depleted recipients, we found a dramatically reduced mean clinical disease score and lower disease incidence (Fig 1A, Appendix Table S1). FACS analysis of mononuclear cells, which were isolated from the CNS of these EAE animals, revealed that the frequencies of transferred Th17 cells were markedly higher in the presence of CD11c+ cells (16.2% in DC-depleted animals vs. 39.1% in control animals; pooled data for three animals; Fig 1B). In addition, the absence of CD11c+ cells led to the reduction of IL-17 producers (Fig 1C and D) instead of IFN-γ producers (Fig 1E). This is supported by similar results in an active EAE model of MOG35-55/CFA-immunized CD11c-DTR/GFP→C57BL/6 mice, which were CD11c+-depleted after onset of clinical signs to interfere with the effector phase (Appendix Fig S2). Here, a significant reduction of clinical deficits was achieved by CD11c+ cell depletion when started after onset of clinical signs in comparison with non-CD11c+-depleted control groups. Figure 1. Adoptive transfer EAE of encephalitogenic CD4+ T cells into CD11c-GFP+-depleted and control mice A. Mean clinical scores (±SEM) of C57BL/6→C57BL/6 controls treated with DTX and CD11c-DTR/GFP→C57BL/6 treated with DTX or PBS during the whole observation period starting from day −1. All animals received in vitro generated 2d2.tdRFP Th17 intravenously on day 0. DC depletion in CD11c-DTR/GFP→C57BL/6 reduces the encephalitogenicity of adoptively transferred 2d2.tdRFP Th17 cells. Mann–Whitney U-test was performed on mean clinical scores for DTX vs. PBS-treated CD11c-DTR/GFP→C57BL/6 (n = 11 PBS/13 DTX); *P < 0.05, **P < 0.01. See also Appendix Table S1. Pooled data from two independent experiments are shown. B. Mononuclear cells were isolated on day 30 of animals shown in (A) from the CNS of PBS- and DTX-treated CD11c-DTR/GFP→C57BL/6 that had been transferred with 2d2.tdRFP Th17 cells. Flow cytometry was performed on surface-stained cell samples. Cells were pre-gated on lymphocyte cells (FSC/SSC gate) and PI negative; pooled data of three animals are shown, representative of two independent experiments. C. Mononuclear cells were isolated from the CNS of PBS- and DTX-treated 2d2.tdRFP Th17-transferred CD11c-DTR/GFP→C57BL/6 mice 2–3 days after onset of the disease. Upper panels: Cells were stimulated with plate-bound anti-CD3/anti-CD28, stained for CD4 and cytokines IFN-γ, IL-17 (pre-gated in addition to FSC/SSC on CD45+CD4+). Lower panels: expression of transcription factor FoxP3. Pooled data from three animals are shown, representative for two independent experiments. D, E. Quantification of cytokine expression data as shown in (C) for pooled data from two independent experiments. Mann–Whitney U-test, *P < 0.05. F. 2d2 Th17 cells were transferred into lymphopenic Rag2−/−cγ−/−. Mononuclear cells were isolated from the spleen and brain at the peak of the disease and analyzed for IL-17 and GM-CSF production after stimulation with anti-CD3/anti-CD28. G. GM-CSF production of IL-17 producers versus IL-17 non-producers of CNS-isolated 2d2 Th17 cells (see also F). Each dot/square represents a single animal. One-way ANOVA and Dunn's multiple comparison test; *P < 0.05, **P < 0.01, ****P < 0.0001. Download figure Download PowerPoint To exclude a peripheral effect of CD11c+ cell depletion, which has been described in active EAE models where DC depletion led to exacerbation of the disease due to a lack of peripheral FoxP3+ regulatory T-cell (Treg) induction (Yogev et al, 2012), we also checked lymph nodes and the spleen in our adoptive transfer model. Here, we found similar numbers of transferred cells (Appendix Fig S3A and B), no differences in cytokine production capacity (Appendix Fig S3D and E), and FoxP3 expression by CD4+ T cells (Appendix Fig S3F) in secondary lymphoid organs. This argues against CD11c-GFP+ cells being relevant in inducing tolerance outside of the brain in this adoptive transfer EAE model. Next, we analyzed the co-expression of the CD11c+-relevant T-cell cytokine GM-CSF, which we found to be strongly upregulated in IL-17-producing Th17 cells in the CNS, but not in the spleen (Fig 1F), indicating that Th17 cells acquire the capacity to produce this cytokine after invasion of the CNS. Furthermore, IL-17hi 2d2 Th17 cells consistently co-expressed more frequently GM-CSF than IL-17 non-producers, which shows that these cytokines are selectively co-expressed within the CNS (Fig 1G). Pathogenic Th17 cells carry distinct chemokine receptor signatures To further determine the prerequisites for CD11c-GFP+ cells and encephalitogenic Th17 cell encounters, we analyzed the chemokine expression pattern of pathogenic T cells by microarray analysis. We compared (i) naïve CD4+CD62Lhi 2d2 T cells before differentiation (Tnaive) and (ii) after repetitive in vitro Th17 differentiation (Th17iv), and (iii) 2d2 Th17 cells, which were isolated from the CNS of these mice at the peak of EAE (Th17eae; see also (Hoppmann et al, 2015)). Additionally, we isolated (iv) CD4+ T cells from the CNS of C57BL/6 mice with active, MOG35-55-immunized EAE (CD4eae). Differential gene expression was analyzed using Agilent Whole Mouse Genome Oligo Microarrays 4x44K V2. We concentrated our analysis on chemokine receptors, which showed a distinct activation pattern during EAE development when compared by ratio (Fig 2A). We performed stringent statistical testing on these targets of the microarray, which identified Ccr2, Ccr3, Ccr5, and Cxcr1 to be upregulated in Th17iv cells in comparison with Tnaive, which might indicate their role for homing to the CNS (Fig 2B). For Cxcr1, the expression pattern was confined to the in vitro period, whereas Ccr2 and Ccr5 were strongly expressed in both EAE subtypes (Th17eae and CD4eae). In addition, we identified Ccr8, Cxcr3, and Cxcr4 to be upregulated not in vitro (Th17iv) but in EAE-derived Th17eae and/or CD4eae, which might indicate a role in their intraparenchymal distribution. Interestingly, the Th17-associated Ccr6 was not significantly regulated, as the other chemokine receptors involved in the array also did not show relevant regulation in the observed T cells (data not shown). Figure 2. Regulation of chemokine receptors in T cells at distinct points in and before EAE Expression of chemokine receptors was assessed by microarray analysis of different CD4+ T-cell populations. Statistical analysis revealed a strong upregulation of most of the chemokine receptors covered in the array for Th17iv/Tnaive (column 2), Th17eae/Tnaive (column 3), and MOG35-55-induced EAE-recovered CD4eae/Tnaive. Microarray signal intensities of most strongly regulated genes from (A) are shown in detailed statistical analysis for the different T-cell subgroups. Values are depicted as signal intensity mean ± SEM from three independent experiments. Statistical significance was determined using one-way ANOVA with post hoc Tukey test for multiple comparisons. P-values < 0.05 were regarded as statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Download figure Download PowerPoint CNS dendritic cells grant encephalitogenic T cells access to the CNS To visualize MOG35-55-specific T-cell receptor transgenic (2d2) Th17 cells, we used genetically encoded constitutively red fluorescent (Rosa26-tdRFP) and IL-17 reporter (Il17a-IRES-EGFP) IL-17hi 2d2 Th17 cells. These 2d2 Th17 cells were transferred into CD11c-DTR/GFP mice, and their interaction with CD11c-GFP+ cells was monitored at the onset and peak of clinical signs of EAE. Before disease onset, only very few CD11c-GFP+ cells in CD11c-DTR/GFP mice could be detected by TPLSM in the CNS. Some of these CD11c-GFP+ cells were found near the surface of larger venules on the parenchymal side (Appendix Fig S4) and have been described elsewhere as bipolar dendritic cells (Prodinger et al, 2011). At the onset of the clinical disease (days 1–2), vessel-associated CD11c-GFP+ cells (Fig 3A) closely interacted with intravascular, rolling IL-17hi 2d2 Th17 cells (Fig 3B and Video EV1). The elongated perivascular CD11c-GFP+ cells were closely associated with the vessel walls and made contact with the rolling Th17 cells via filopodium-like dendrites (Fig 3C). Interestingly, most of the long-lasting contacts were with Th17 cells actively producing IL-17. Within the perivascular area, the interaction of CD11c-GFP+ cells with 2d2 Th17 cells was associated with high EGFP expression as a sign of strong IL-17 production (Fig 3D), was long-lasting, and preceded tissue invasion (Fig 3E–G and Video EV2). This holds true not only for the perivascular DCs but also for the few intraparenchymal, round-shaped CD11c-GFP+ cells in early inflammatory CNS lesions. As an example, an IL-17hi 2d2 Th17 cell is shown that migrated directly toward a CD11c-GFP+ DC and engaged in long-lasting contact (Fig 3H, Videos EV3 and EV4), which is reminiscent of what has previously been described for the antigen recognition process in secondary lymphoid organs (SLO) (Cahalan & Parker, 2008). The quantitative analysis of the migration pattern of IL-17hi 2d2 Th17 cells as compared with all 2d2 Th17 cells revealed that the cytokine-expressing cells were slower in mean velocity but also had a larger number of stopping points (instantaneous velocity) compared to the whole 2d2 Th17 population—despite all of these T cells recognizing the same myelin antigen. This motility pattern was clearly CD11c+-GFP cell dependent as the depletion of CD11c-GFP+ cells during T-cell monitoring led to an increase in the mean velocity of the IL-17hi cells (Fig 3I) and lower static motility, as shown by reduced (< 2 μm/min) instantaneous velocity (Fig 3J). To exclude any bias by the adoptive transfer EAE model, we performed TPLSM in active EAE of single-transgenic Il17-EGFP mice. In active EAE lesions, Il17-EGFP+ cells showed a perivascular migration pattern with regionalized migration (Appendix Fig S5, Video EV5) in the same way as IL-17hi 2d2 Th17 cells. Recently, it has been shown ex vivo, based on functional and developmental criteria, that these CNS CD11c-GFP+ cells are classic DCs (Anandasabapathy et al, 2011) and function as potent antigen-presenting cells (APCs) (Greter et al, 2005; Bailey et al, 2007). Our in vivo observations by time-lapse imaging show that these CNS DCs have a crucial role in the interplay of CD11c-GFP+ cells with IL-17-producing Th17 cells. Figure 3. Preferential interaction of CNS CD11c-GFP+ cells with IL-17hi 2d2 Th17 cells at the onset of the diseaseTPLSM of EAE lesions in the brainstem of adoptive transfer EAE at the onset of the disease. EAE was induced by transfer of in vitro differentiated 2d2 Th17 cells (tdRFP, red; IL-17-EGFP, green) into CD11c-DTR/GFP mice (CD11c-GFP, green); imaging area 300 × 300 μm. A. Intravascular IL-17hi 2d2 Th17 cell (arrow; double positive, green: IL-17EGFP and red: 2d2.tdRFP) rolling cell toward a CD11c-GFP+ cell (arrowhead). White dotted lines mark a venous vessel. B. Time-lapse TPLSM (maximal intensity projections) of the boxed area in (A) shows that the perivascular elongated CD11c-GFP+ cell (arrowhead) enters into contact with IL-17-expressing (arrow; double positive, green: IL-17EGFP and red: 2d2.tdRFP) Th17 cells. Scale bar, 20 μm. C. XYZ-resolved TPLSM depiction reveals that the elongated perivascular CD11c-GFP+ cell makes contact with the intravascular 2d2 Th17 cells via a filopodium-like dendrite (xy-plane, 300 × 300 μm; z-depth, 70 μm). D. Strong interaction of a perivascular IL-17hi 2d2 Th17 cells with a perivascular CD11c-GFP+ cell (insert upper left contact). E. Time lapse of the insert in (D): Stopping motility of the round-shaped IL-17hi 2d2 Th17 cell (arrow) near to a Cd11c-GFP+ cell (arrowhead) is followed by entry into the CNS parenchyma. F, G. Magnified image of interaction of the (F) upper left and (G) lower right perivascular T-cell–DC contacts as shown in (D) and (E). H. Time-lapse imaging of parenchymal CD11c-GFP+ cells (arrowhead), targeted migration of an IL-17hi 2d2 Th17 cell (arrow, double positive; green, IL-17EGFP; red, 2d2.tdRFP) toward the CD11c-GFP+ cell (arrowhead). Scale bar, 20 μm. I. Quantification of the motility pattern of IL-17hi 2d2.tdRFP vs. all 2d2.tdRFP in CD11c-DTR/GFP brainstem lesions before and after (2–5 h) intraperitoneal DTX injection. Evaluation of mean track velocity of the single-cell tracking (each dot represents a track); pooled data from at least two independent experiments (> 3 imaging areas). For the statistical analysis, the Mann–Whitney U-test was performed (***P < 0.001; n.s., not significant). J. Percentage of stopping cells (red bar, instantaneous velocity < 2 μm/min) of the data set as shown in (I). Download figure Download PowerPoint CNS CD11c-GFP+ cells are a mixed population of conventional DCs and monocyte-derived CD11c+ cells with distinct proportions depending on disease stage In order to further characterize CNS CD11c+ cells, we isolated and phenotyped mononuclear cells from the CNS of EAE-affected mice before onset (day 8–9), at the peak (day 13–17) and in the chronic phase of the disease (day 22–27) by flow cytometry. We found that conve
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