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• W1779160407 abstract "•Aspergillus-germinated morphotypes induce IL-2 in lung CD103+ dendritic cells•IL-2 production is dependent on the Ca2+-calcineurin-NFAT pathway•Deletion of dendritic-cell-derived IL-2 leads to higher mortality upon infection•Dendritic-cell-derived IL-2 regulates Th17 cell phenotype Th17 cells express diverse functional programs while retaining their Th17 identity, in some cases exhibiting a stem-cell-like phenotype. Whereas the importance of Th17 cell regulation in autoimmune and infectious diseases is firmly established, the signaling pathways controlling their plasticity are undefined. Using a mouse model of invasive pulmonary aspergillosis, we found that lung CD103+ dendritic cells (DCs) would produce IL-2, dependent on NFAT signaling, leading to an optimally protective Th17 response. The absence of IL-2 in DCs caused unrestrained production of IL-23 and fatal hyperinflammation, which was characterized by strong Th17 polarization and the emergence of a Th17 stem-cell-like population. Although several cell types may be affected by deficient IL-2 production in DCs, our findings identify the balance between IL-2 and IL-23 productions by lung DCs as an important regulator of the local inflammatory response to infection. Th17 cells express diverse functional programs while retaining their Th17 identity, in some cases exhibiting a stem-cell-like phenotype. Whereas the importance of Th17 cell regulation in autoimmune and infectious diseases is firmly established, the signaling pathways controlling their plasticity are undefined. Using a mouse model of invasive pulmonary aspergillosis, we found that lung CD103+ dendritic cells (DCs) would produce IL-2, dependent on NFAT signaling, leading to an optimally protective Th17 response. The absence of IL-2 in DCs caused unrestrained production of IL-23 and fatal hyperinflammation, which was characterized by strong Th17 polarization and the emergence of a Th17 stem-cell-like population. Although several cell types may be affected by deficient IL-2 production in DCs, our findings identify the balance between IL-2 and IL-23 productions by lung DCs as an important regulator of the local inflammatory response to infection. In recent years, the view of Th17 cells as a short-lived, transient population has been overturned by data showing that, under some circumstances, they exhibit marked functional plasticity, multipotency, and a stem-cell-like phenotype (Muranski et al., 2011Muranski P. Borman Z.A. Kerkar S.P. Klebanoff C.A. Ji Y. Sanchez-Perez L. Sukumar M. Reger R.N. Yu Z. Kern S.J. et al.Th17 cells are long lived and retain a stem cell-like molecular signature.Immunity. 2011; 35: 972-985Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). In parallel, the contribution of the Th17/IL-17 response to both physiological and pathological inflammation has become well established (Korn et al., 2009Korn T. Bettelli E. Oukka M. Kuchroo V.K. IL-17 and Th17 Cells.Annu. Rev. Immunol. 2009; 27: 485-517Crossref PubMed Scopus (3859) Google Scholar). What remains unknown is which cellular interactions, factors, and signaling pathways determine the functional programs that are expressed by Th17 cells and how Th17 functionality is regulated under different conditions. One possible candidate is IL-2 (Boyman and Sprent, 2012Boyman O. Sprent J. The role of interleukin-2 during homeostasis and activation of the immune system.Nat. Rev. Immunol. 2012; 12: 180-190PubMed Google Scholar). Because of the potency and pleiotropic roles of IL-2, the finding that myeloid dendritic cells (DCs) will produce the cytokine in response to microbial stimuli (Granucci et al., 2001Granucci F. Vizzardelli C. Pavelka N. Feau S. Persico M. Virzi E. Rescigno M. Moro G. Ricciardi-Castagnoli P. Inducible IL-2 production by dendritic cells revealed by global gene expression analysis.Nat. Immunol. 2001; 2: 882-888Crossref PubMed Scopus (412) Google Scholar, Wuest et al., 2011Wuest S.C. Edwan J.H. Martin J.F. Han S. Perry J.S. Cartagena C.M. Matsuura E. Maric D. Waldmann T.A. Bielekova B. A role for interleukin-2 trans-presentation in dendritic cell-mediated T cell activation in humans, as revealed by daclizumab therapy.Nat. Med. 2011; 17: 604-609Crossref PubMed Scopus (215) Google Scholar, Zelante et al., 2012Zelante T. Fric J. Wong A.Y. Ricciardi-Castagnoli P. Interleukin-2 production by dendritic cells and its immuno-regulatory functions.Front. Immunol. 2012; 3: 161Crossref PubMed Scopus (66) Google Scholar) has posed intriguing questions about IL-2’s role in the context of DC-T cell interactions. After exposure to fungal antigens in vitro, DCs undergo an influx of Ca2+ that causes calcineurin, a Ca2+-calmodulin-dependent phosphatase, to dephosphorylate nuclear factor of activated T cells (NFAT) in the cytoplasm, resulting in NFAT nuclear translocation and Il2 transcription (Fric et al., 2014Fric J. Zelante T. Ricciardi-Castagnoli P. Phagocytosis of particulate antigens - all roads lead to calcineurin/NFAT signaling pathway.Front. Immunol. 2014; 4: 513Crossref PubMed Scopus (25) Google Scholar). We therefore asked what impact the absence of IL-2 in DCs would have on immune polarization and outcome of fungal infection by using a murine model of invasive pulmonary aspergillosis. Mice selectively lacking IL-2 in DCs expressed higher levels of IL-17 in their lungs during Aspergillus fumigatus infection and frequently died as a result of a pathological Th17 response. DCs that were not competent for IL-2 production would instead secrete IL-23, which drove fatal Th17-mediated immune pathology. These data suggest a crucial role for Aspergillus-induced IL-2 production by lung DCs that shapes the Th17 response to infection and determines disease resolution versus an ultimately fatal condition of hyperinflammation. Fungal β-glucan particles trigger IL-2 release from DCs (Rogers et al., 2005Rogers N.C. Slack E.C. Edwards A.D. Nolte M.A. Schulz O. Schweighoffer E. Williams D.L. Gordon S. Tybulewicz V.L. Brown G.D. Reis e Sousa C. Syk-dependent cytokine induction by Dectin-1 reveals a novel pattern recognition pathway for C type lectins.Immunity. 2005; 22: 507-517Abstract Full Text Full Text PDF PubMed Scopus (734) Google Scholar, Zelante et al., 2012Zelante T. Fric J. Wong A.Y. Ricciardi-Castagnoli P. Interleukin-2 production by dendritic cells and its immuno-regulatory functions.Front. Immunol. 2012; 3: 161Crossref PubMed Scopus (66) Google Scholar). We first confirmed that whole fungal cells were also able to induce IL-2 in DCs in vitro. We exposed long-term growth-factor-dependent D1 cells—an immature murine myeloid CD103+ CD11b+ DC line (Winzler et al., 1997Winzler C. Rovere P. Rescigno M. Granucci F. Penna G. Adorini L. Zimmermann V.S. Davoust J. Ricciardi-Castagnoli P. Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures.J. Exp. Med. 1997; 185: 317-328Crossref PubMed Scopus (724) Google Scholar)—to different Aspergillus morphotypes, namely, conidia (A-con), partially germinated swollen conidia (A-sw), and fully germinated hyphae (A-hyp), as compared to whole-glucan particles (WGP). All morphotypes induced IL-2 release by D1 cells (Figure 1A). Although A-hyp was the strongest stimulus for IL-2 release, the physical properties of A-con and A-sw render them most suited to in vitro studies, and they were therefore used in the subsequent experiments. Exposure of D1 cells to A-con, and more so to A-sw, was marked by a cytosolic Ca2+ influx (Figure 1B, left panel). Pre-incubating D1 cells with EDTA to chelate extracellular Ca2+, or the cell-permeant Ca2+ chelator BAPTA, revealed a role for both extracellular and intracellular Ca2+ stores. Moreover, the addition of SKF-96365, which inhibits Ca2+-release-activated channels (CRACs) (Hsu et al., 2001Hsu Sf. O’Connell P.J. Klyachko V.A. Badminton M.N. Thomson A.W. Jackson M.B. Clapham D.E. Ahern G.P. Fundamental Ca2+ signaling mechanisms in mouse dendritic cells: CRAC is the major Ca2+ entry pathway.J. Immunol. 2001; 166: 6126-6133Crossref PubMed Scopus (75) Google Scholar), similarly abrogated Ca2+ influx in response to Aspergillus exposure in D1 cells (Figure 1B, right panel). Exposure to all morphotypes induced nuclear translocation of NFAT, measured by an NFAT-luciferase reporter assay, with the greatest luciferase activity recorded in response to germinated fungi (Figure 1C). In D1 cells exposed to A-sw, NFAT nuclear translocation was detected by immune-fluorescence microscopy at 0.5–3 hr (Figures 1D and S1A). IL-2 production by D1 cells exposed to A-sw depended on NFAT signaling, as treatment with the calcineurin B inhibitors cyclosporin A (CsA) or tacrolimus (FK506) significantly reduced the amount of IL-2 in culture supernatants (Figure 1E). Consistent with a need for fungal uptake, the inhibition of actin polymerization with cytochalasin D revealed the parallel importance of phagocytic function in D1 cells for IL-2 production (Figure S1B). Finally, IL-2 release did not require the MyD88-TRIF signaling complex but was instead contingent on dectin-1, which initiates Ca2+-calmodulin-dependent events in response to β-glucan (Figures S1C and S1D). The NFAT family of transcription factors has emerged as a key mediator of the initiation of immune responses, and specifically, NFATc2 is known to mediate the transcription of IL-2 released by DCs in response to LPS upon Ca2+ entry (Zanoni et al., 2009Zanoni I. Ostuni R. Capuano G. Collini M. Caccia M. Ronchi A.E. Rocchetti M. Mingozzi F. Foti M. Chirico G. et al.CD14 regulates the dendritic cell life cycle after LPS exposure through NFAT activation.Nature. 2009; 460: 264-268Crossref PubMed Scopus (247) Google Scholar). To establish whether NFATc2 was acting directly or indirectly in modulating IL-2 production, we performed a chromatin immunoprecipitation (ChIP) assay in D1 cells. In the absence of a commercially available NFATc2 antibody suitable for use in ChIP assays, we devised a V5-tagging approach (Yu et al., 2011Yu H.B. Johnson R. Kunarso G. Stanton L.W. Coassembly of REST and its cofactors at sites of gene repression in embryonic stem cells.Genome Res. 2011; 21: 1284-1293Crossref PubMed Scopus (42) Google Scholar) (Figure S1E). This confirmed that NFATc2 bound the promoter region of Il2 upon WGP stimulation of D1 cells (Figure S1E), which is consistent with our recent finding that NFATc2 occupancy is responsible for curdlan-mediated activation of multiple genes in DCs (Yu et al., 2015Yu H.B. Yurieva M. Balachander A. Foo I. Leong X. Zelante T. Zolezzi F. Poidinger M. Ricciardi-Castagnoli P. NFATc2 mediates epigenetic modification of dendritic cell cytokine and chemokine responses to dectin-1 stimulation.Nucleic Acids Res. 2015; 43: 836-847Crossref PubMed Scopus (21) Google Scholar). Thus, A. fumigatus fungal morphotypes trigger IL-2 release from murine myeloid DCs through the receptor dectin-1, phagocytosis, and the downstream Ca2+-calmodulin-dependent NFAT signaling pathway. Several subpopulations of murine lung DCs are present in the steady state: “classical” CD11c+ DCs (encompassing CD103+ [type 1] and CD11b+ [type 2] DC subsets) and plasmacytoid DCs (Guilliams et al., 2014Guilliams M. Ginhoux F. Jakubzick C. Naik S.H. Onai N. Schraml B.U. Segura E. Tussiwand R. Yona S. Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny.Nat. Rev. Immunol. 2014; 14: 571-578Crossref PubMed Scopus (1195) Google Scholar). Recent data suggest that CD11b+ DCs are essential for T-helper cell priming in aspergillosis (Schlitzer et al., 2013Schlitzer A. McGovern N. Teo P. Zelante T. Atarashi K. Low D. Ho A.W. See P. Shin A. Wasan P.S. et al.IRF4 transcription factor-dependent CD11b+ dendritic cells in human and mouse control mucosal IL-17 cytokine responses.Immunity. 2013; 38: 970-983Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar), and pulmonary CD103+ DCs have an established role in the response to particulate antigens (Greter et al., 2012Greter M. Helft J. Chow A. Hashimoto D. Mortha A. Agudo-Cantero J. Bogunovic M. Gautier E.L. Miller J. Leboeuf M. et al.GM-CSF controls nonlymphoid tissue dendritic cell homeostasis but is dispensable for the differentiation of inflammatory dendritic cells.Immunity. 2012; 36: 1031-1046Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar); however, it is unknown whether any of those subpopulations are able to release IL-2 and whether NFAT proteins are important in their signaling pathways. We generated mice conditionally lacking the calcineurin B1 subunit (CnB) in the CD11c+ DC population (Cd11cCreCnbfl/fl mice; see Experimental Procedures). CD11c+ DCs were isolated from the lungs of Cd11cCreCnbfl/fl mice and the CD11b+ and CD103+ subpopulations then exposed to A-sw ex vivo. A substantive Ca2+ flux was only observed in the CD103+ fraction (Figure 2A), and it was accompanied by a significant induction of IL-2 in response to A-sw (Figure 2B), which accumulated in culture medium over the following 18 hr (Figure 2C). Moreover, CD103+ DCs from the lungs of Cd11cCreCnbfl/fl mice had markedly diminished IL-2 responses to A-sw (Figure 2B). These results confirmed that, similar to our observations in D1 cells, CD103+ DCs from mouse lung transcribe Il2 in an NFAT pathway-dependent manner following exposure to A-sw ex vivo. We asked whether Il2 expression also occurred in lung DCs from wild-type (WT) mice during acute aspergillosis. Mice were exposed intranasally to A-sw, and after 1 day, DC subpopulations were sorted from dissociated lung tissue. We found that Il2 mRNA was selectively induced in CD103+ DCs (Figure 2D). Interestingly, whereas lung CD11b+ DCs also expressed the calcineurin (Ppr1) and Nfat1 genes (Figure S2A), they were evidently unable to produce IL-2. Further investigation of their transcriptional profile revealed significantly lower levels of Sept4 expression in response to Aspergillus relative to the CD103+ fraction (Figure S2B). Septin4 is essential for store-operated Ca2+ entry organization and therefore required for NFAT translocation (Sharma et al., 2013Sharma S. Quintana A. Findlay G.M. Mettlen M. Baust B. Jain M. Nilsson R. Rao A. Hogan P.G. An siRNA screen for NFAT activation identifies septins as coordinators of store-operated Ca2+ entry.Nature. 2013; 499: 238-242Crossref PubMed Scopus (183) Google Scholar), thereby likely explaining/contributing to the absence of Il2 induction in those cells. Because of the IL-2 production from CD103+ lung DCs ex vivo—which is associated with Il2 expression in vivo—we investigated the role of those events in aspergillosis. As IL-2 is crucial for Th cell differentiation (Malek, 2008Malek T.R. The biology of interleukin-2.Annu. Rev. Immunol. 2008; 26: 453-479Crossref PubMed Scopus (779) Google Scholar), we first asked whether T cell polarization differed in mice lacking DC-derived IL-2. Inguinal, popliteal, and para-aortic lymph node CD4+ T cells from mice immunized with A. fumigatus crude culture filtrate antigens (CCFA) (Grünig et al., 1997Grünig G. Corry D.B. Leach M.W. Seymour B.W. Kurup V.P. Rennick D.M. Interleukin-10 is a natural suppressor of cytokine production and inflammation in a murine model of allergic bronchopulmonary aspergillosis.J. Exp. Med. 1997; 185: 1089-1099Crossref PubMed Scopus (267) Google Scholar) were cultured with GM-CSF bone-marrow-derived DCs (GM-DCs), either from WT mice or from mice lacking IL-2 in all tissues (i.e., Il2−/− mice). In vitro proliferative responses of T cells isolated from immunized mice and stimulated with CCFA (Figure S2C) were significantly lower when GM-DCs lacked the ability to produce IL-2 (Figures S2D and S2E). Using pulmonary CD103+ DCs and CD11b+DCs, we investigated the effect of IL-2 deficiency on Th cell polarization. After overnight incubation of lung DC subsets with A-sw and CCFA to allow full induction of DC IL-2 production (as revealed by the kinetics in Figure 2C), DCs were co-cultured with T cells isolated from immunized mice (Figure 2E). The absence of IL-2 in pulmonary CD103+ DCs led to a significantly higher IL-17 production relative to culturing T cells with IL-2-competent DCs (Figure 2E). This effect was directly attributable to the absence of IL-2, as adding recombinant IL-2 to cultures containing Il2−/− CD103+ DCs significantly reduced IL-17 production from co-cultured T cells (Figure 2F). To confirm that DCs and not T cells were the critical source of IL-2, we added anti-IL-2 antibodies (or an isotype control) to cultures either before (Figure 2G, early) or after (Figure 2G, late) including T cells. Increased IL-17 production was selectively induced by adding anti-IL-2 at the time of DC priming with antigen, in the absence of T cells (Figure 2G). Thus, Il2—transcribed through a Ca2+-calcineurin-NFAT-signaling pathway in CD103+ DCs—modulated Th17 cell expansion in vitro in response to Aspergillus-germinated particles. Optimally balanced Th17 functionality in response to fungal antigens is required for resolution of invasive pulmonary aspergillosis (Romani et al., 2008Romani L. Fallarino F. De Luca A. Montagnoli C. D’Angelo C. Zelante T. Vacca C. Bistoni F. Fioretti M.C. Grohmann U. et al.Defective tryptophan catabolism underlies inflammation in mouse chronic granulomatous disease.Nature. 2008; 451: 211-215Crossref PubMed Scopus (454) Google Scholar). To assess the impact of DC-derived IL-2 deficiency in vivo, we also generated mice conditionally lacking Il2 in the CD11c+ DC population (Cd11cCreIl2fl/fl mice; see Figures S3A–S3C). We compared the Cd11cCreIl2fl/fl mice bearing a selective deficiency of IL-2 in CD11c+ DCs (DC-Il2−/−) with mice lacking IL-2 in all tissues (Il2−/−) or lacking IL-2 expression in CD4+ T cells (T-Il2−/−), as well as with WT mice. We selected Cd11cCreIl2fl/fl mice with specific targeted deletion in DCs, where T cells and DCs had comparable secretion of IL-2 in response to thapsigargin (60 nM) relative to WT T cells (Figure S3D). Mice of each genotype were infected intranasally with A. fumigatus, resulting in invasive pulmonary aspergillosis. Clinical signs of the disease were obvious in all groups at 7 days post-infection. However, significantly higher fungal loads and Aspergillus 18S expression in the lungs were observed in the three mutant strains than in WT animals (Figure 3A). Despite the fact that Il2−/− and T-Il2−/− mice had significantly higher fungal burdens in their lungs than DC-Il2−/− mice, they frequently survived longer than did animals selectively lacking IL-2 in DCs, 80% of which would succumb to challenge by day 6 of infection (Figure 3B). This indicated that fungal load was not, per se, the major determinant of disease outcome and that IL-2 production by lung DCs contributed substantially to overall protection. Whereas either generalized or T-cell-confined IL-2 deficiency increased local invasiveness by Aspergillus, selective lack of IL-2 in lung-resident DCs appeared to trigger an acute and potentially fatal effect not directly attributable to an increased fungal burden in the lungs. Kinetic analysis of fungal load during the first week post inoculation with Aspergillus revealed a consistent trend of exacerbated infection in DC-Il2−/− mice from day 3, which was maximal at 7 days (Figure 3C). Histopathology revealed multifocal bronchoalveolar histiocytic pneumonia and a greater multifocal perivascular neutrophil infiltration compared to WT (Figure 3C), T-Il2−/−, or Il2−/− mice (Figure S4A). Differences in cellular infiltrates among genotypes were not due to global changes in immune cell composition, as DC-Il2−/− mice had CD4+ and CD8+ T cell frequencies in their lungs and spleens similar to those in WT mice (Figure S4B). Considering innate immune cells, only the frequency of GR1+ CD11b+ neutrophils was significantly higher in the lungs of DC-Il2−/− mice (Figures 3D and S4C). IL-17 and IFN-γ are secreted in mice with pulmonary Aspergillus infections (Romani, 2011Romani L. Immunity to fungal infections.Nat. Rev. Immunol. 2011; 11: 275-288Crossref PubMed Scopus (900) Google Scholar), and they have a potential for contributing to resolution of infection as well as causing severe tissue inflammation. Mice were inoculated with A-con, and 7 days later, the cytokine content of their lung homogenates was measured by ELISA, which revealed that DC-Il2−/− mice produced significantly higher amounts of IL-17 than did their WT counterparts. Lung-associated IFN-γ was equally expressed across the four genotypes (Figure 3E). Therefore, lung DC-derived IL-2 seemed to control lung inflammation and susceptibility to invasive aspergillosis by regulating the IL-17 response. Because IL-2 is known to control T-reg cell survival (Fontenot et al., 2005Fontenot J.D. Rasmussen J.P. Gavin M.A. Rudensky A.Y. A function for interleukin 2 in Foxp3-expressing regulatory T cells.Nat. Immunol. 2005; 6: 1142-1151Crossref PubMed Scopus (1435) Google Scholar) and one function of T-reg cells is to restrain pathological inflammation, we investigated whether the abundance of Foxp3+ CD4+ T-reg cells varied in mice of the different genotypes. The relative frequencies of Foxp3+ CD4+ T cells in the lungs and thoracic lymph nodes (TLNs) of DC-Il2−/− and WT mice were similar, regardless of any exposure to intranasal Aspergillus (Figure 4A). mRNA expression of Foxp3 in T cells did not change during infection (Figure 4B). Although not statistically significant, T-Il2−/− and Il2−/− mice had lower T-reg-cell-associated Foxp3 expression in their lungs (Figure 4C) and splenic (Figure S4D) CD4+ T cell compartments than did WT and DC-Il2−/− animals. In agreement with the pattern of cytokine production seen in lung homogenates (Figure 3E), flow cytometry analysis of freshly harvested, inflammatory CD45+ cells from the lungs revealed a higher frequency of IL-17-producing cells in DC-Il2−/− mice infected with Aspergillus relative to WT counterparts (Figure 4D). The increased IL-17 expression in the lungs of DC-Il2−/− mice was entirely attributable to CD45+ CD90+ cells (Figure 4E), which were still present at 14 days of infection in the lungs from DC-Il2−/− mice (Figure 4F), but not in TLNs from the same animals (Figure 4E). CD90+ IL-17+ cells were not detected in the lungs of T-Il2−/− and Il2−/− mice or of WT animals (Figure 4G). Because both innate lymphoid cells (ILCs) and T cells in the lung express the cell surface marker CD90, CD90+ IL-17+ cells were further examined for their expression of other T cell markers. The analysis revealed that 70% of the population expressed CD3, with 60% expressing CD4 and 9% expressing CD8 (Figure 4H). Collectively, these data demonstrated that IL-2 produced by lung DCs was responsible for the increased frequency of IL-17+ CD45+ CD90+ T cells late in infection with Aspergillus. We investigated the nature of the pulmonary immune cells responsible for the shift toward a predominantly pathogenic IL-17 response under conditions of DC-derived IL-2 deficiency. We used a recently developed technique, single-cell mass spectrometric cytometry by time-of-flight (CyTOF mass cytometer), which is a unique means of characterizing phenotypically and functionally heterogeneous lymphocyte populations (Bandura et al., 2009Bandura D.R. Baranov V.I. Ornatsky O.I. Antonov A. Kinach R. Lou X. Pavlov S. Vorobiev S. Dick J.E. Tanner S.D. Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry.Anal. Chem. 2009; 81: 6813-6822Crossref PubMed Scopus (904) Google Scholar, Newell et al., 2012Newell E.W. Sigal N. Bendall S.C. Nolan G.P. Davis M.M. Cytometry by time-of-flight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of CD8+ T cell phenotypes.Immunity. 2012; 36: 142-152Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar). Using a panel of heavy metal isotope-labeled antibodies recognizing numerous surface and soluble molecules expressed by lung immune cells, we simultaneously analyzed the expression of 25 markers (Table S1). CD45+ CD90+ cells were also examined in detail (Figure 5A). Mass cytometry analysis revealed that pulmonary T cells naturally cluster into distinct subsets according to the presence of CD4 and CD8 on the cell surface (Figure 5B). We employed density-based clustering using support vector machine technology to improve the efficiency of image grouping so to result in grouped images being assigned to different clusters. We first reduced the dimensionality to 2D using t-SNE (Becher et al., 2014Becher B. Schlitzer A. Chen J. Mair F. Sumatoh H.R. Teng K.W. Low D. Ruedl C. Riccardi-Castagnoli P. Poidinger M. et al.High-dimensional analysis of the murine myeloid cell system.Nat. Immunol. 2014; 15: 1181-1189Crossref PubMed Scopus (275) Google Scholar). On the t-SNE map, we generated a density plot from which we identified density peaks. Cells located in the neighborhood of each density peak were classified to clusters represented by their respective peaks. Cells resulting as otherwise unclassified were assigned by using support vector machine. DC-Il2−/− mice expressed clusters 10, 6, 9, and 4 to a greater extent than did WT mice (Figure 5C). The frequency of T cell marker expression in the different clusters is graphically represented as a heatmap (Figure 5D). Interestingly, the T cell subsets were clustered similarly in the lungs of T-Il2−/− and Il2−/− mice but to a different extent as compared to DC-Il2−/− mice (Figures S5A–S5C). Because clusters upregulated in DC-Il2−/− mice were all Sca1+, we concluded that Aspergillus-infected DCs affect the T cell differentiation status in the lung by releasing IL-2 after infection. As both ILC2 and ILC3 subsets in the lung also express Sca1 and CD90, it is possible that IL-2 derived from DCs might additionally impact these populations. With this in mind, we explored the impact of DC-restricted IL-2 deficiency in Rag2−/− mice, which completely lack mature lymphocytes but possess ILCs. We compared Rag2−/− mice with DC-Il2−/−Rag2–/– mice and asked whether we could detect ILC2 or ILC3 expansion in the absence of T cells. We did not observe any increase in frequency of lung IL-17+ CD45+ CD90+ cells in either Rag2−/− or DC-Il2−/−Rag2−/− at 7 days of inoculation with Aspergillus (Figure 5E), indicating that the effects of DC-derived IL-2 on the IL-17+ CD45+ CD90+ CD3+ cell compartment are mostly confined to T cells and do not affect ILCs. Because DC-Il2−/−Rag2−/− mice did not show any expansion of IL-17+ CD45+ CD90+ CD3+ T cells in response to Aspergillus, we wanted to analyze the susceptibility of DC-Il2−/−Rag2−/− mice to fungal infection. At 7 days of infection, these mice showed neither higher colonization nor increased mortality relative to Rag2−/− counterparts (Figure 5F). Taken together, these data showed that the IL-17+ CD45+ CD90+ CD3+ T cells—which were expanded in T cell-sufficient DC-Il2−/− mice—directly contributed to the lung pathology in invasive aspergillosis. These results also confirmed previous data showing that, in acute phases post-infection, the role of IL-17+ CD3+ T cells in the lung mostly contributes to exacerbating disease (Zelante et al., 2007Zelante T. De Luca A. Bonifazi P. Montagnoli C. Bozza S. Moretti S. Belladonna M.L. Vacca C. Conte C. Mosci P. et al.IL-23 and the Th17 pathway promote inflammation and impair antifungal immune resistance.Eur. J. Immunol. 2007; 37: 2695-2706Crossref PubMed Scopus (458) Google Scholar). To further analyze the phenotype of IL-17+ CD45+ CD90+ CD3+ T cells in DC-Il2−/− mice, we used contour plots of mass cytometry data, with IL-17A+ cells being gated and superimposed on the t-SNE map of pulmonary CD45+ CD90+ cells from WT and DC-Il2−/− mice (Figure 6A). Contour plot cluster analysis indicated a greater abundance of cluster-9 IL-17-producing cells in DC-Il2−/− mice than in WT mice (Figure 6A). This cell population was characterized by an IL17+ CD4+ CD45+ CD90+ Sca1+ CD44hi CD27lo CD23Rhi phenotype (Figure 6B). When the analysis was extended to cells from T-Il2−/− or Il2−/− mice, there was no expansion of the Th17 subset, in marked contrast to DC-Il2−/− mice (Figure 6C). The phenotype of the expanded IL-17-producing population in DC-Il2−/− mice is reminiscent of IL-17+ T memory stem cells, which have self-renewal potential and a stem-cell-like signature characterized by β-catenin accumulation (Luckey and Weaver, 2012Luckey C.J. Weaver C.T. Stem-cell-like qualities of immune memory; CD4+ T cells join the party.Cell Stem Cell. 2012; 10: 107-108Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, Muranski et al., 2011Muranski P. Borman Z.A. Kerkar S.P. Klebanoff C.A. Ji Y. Sanchez-Perez L. Sukumar M. Reger R.N. Yu Z. Kern S.J. et al.Th17 cells are long lived and retain a stem cell-like molecular signature.Immunity. 2011; 35: 972-985Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar), and it is notably distinct from that of the CD4− IL-23+-responsive innate lymphoid population with colitogenic properties found in Rag−/− mice (Buonocore et al., 2010Buonocore S. Ahern P.P. Uhlig H.H. Ivanov I.I. Littman D.R. Maloy K.J. Powrie F. Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology.Nature. 2010; 464: 1371-1375Crossref PubMed Scopus (860) Google Scholar). Although IL-17+ T memory stem cells express markers of terminally differentiated T cells, they are long lived and maintain a molecular signature in line with their stem-cell-like nature (Muranski et al., 2011Muranski P. Borman Z.A. Kerkar S.P. Klebanoff C.A. Ji Y. Sanchez-Perez L. Sukumar M. Reger R.N. Yu Z. Kern S.J. et al.Th17 cells are long lived and retain a stem cell-like molecular signature.Immunity. 2011; 35: 972-985Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). This suggested th" @default.
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• W1779160407 title "CD103+ Dendritic Cells Control Th17 Cell Function in the Lung" @default.
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