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Molecular characterization of human skin response to diphencyprone at peak and resolution phases: therapeutic insights
research-article
Author(s):
Nicholas Gulati
1 ,
Mayte Suárez-Fariñas
1
,
2 ,
Judilyn Fuentes-Duculan
1 ,
Patricia Gilleaudeau
1 ,
Mary Sullivan-Whalen
1 ,
Joel Correa da Rosa
2 ,
Inna Cueto
1 ,
Hiroshi Mitsui
1 ,
James G. Krueger
1
Publication date (Electronic):
21 April 2014
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Abstract
Diphencyprone (DPCP) is a hapten that induces delayed-type hypersensitivity (DTH) reactions. It is used as an immune modulating therapeutic, but its molecular effects in human skin are largely unknown. We studied cellular and molecular characteristics of a recall response to 0.04% DPCP at 3 day (peak) and 14 day (resolution) timepoints using immune markers, RT-PCR and gene array approaches. A peak response showed modulation of ~7,500 mRNA transcripts, with high expression of cytokines that define all major effector T-cell subsets. Concomitant increases in T-cell and CD11c+ dendritic cell (DC) infiltrates were measured. The resolution reaction was characterized by unexpectedly high levels of T-cells and mature (DC-LAMP+) DCs, but with marked decreases in expression of IL-2, IFNγ, and other T-cell derived cytokines. However, negative immune regulators such as IDO1 that were high in peak reactions, continued to have high expression in resolution reactions. In the resolution reaction, ~1,500 mRNA transcripts were significantly different from placebo-treated skin. These data suggest the response to DPCP evolves from an inflammatory/effector peak at day 3 to a more regulated immune response after 14 days. This model system could be useful for further dissection of mechanisms of immune activation or negative immune regulation in human skin.
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Most cited references25
- Record: found
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Origin and development of dendritic cells.
Kang Liu, Michel Nussenzweig (2010)
Dendritic cells (DCs) are specialized antigen-presenting cells and essential mediators of immunity and tolerance. This group of cells is heterogeneous in terms of cell-surface markers, anatomic location, and function. Here, we review the development and function of DCs found in lymphoid and non-lymphoid tissues in the steady state. DC and monocyte lineages originate from a common progenitor, the monocyte and dendritic cell progenitor (MDP). The two cell types diverge when MDPs give rise to monocytes and committed DC progenitors (CDPs) in the bone marrow. CDPs give rise to pre-DCs, which migrate from the bone marrow to lymphoid and non-lymphoid tissues to produce the two major subpopulations of lymphoid tissue DCs and non-lymphoid tissue CD103(+) DCs. Within tissues and during development, DC division and homeostasis are regulated by the hormone Flt3L.
- Record: found
- Abstract: found
- Article: not found
L-arginine metabolism in myeloid cells controls T-lymphocyte functions.
Vincenzo Bronte, Paolo Serafini, Alessandra Mazzoni … (2003)
Although current attention has focused on regulatory T lymphocytes as suppressors of autoimmune responses, powerful immunosuppression is also mediated by a subset of myeloid cells that enter the lymphoid organs and peripheral tissues during times of immune stress. If these myeloid suppressor cells (MSCs) receive signals from activated T lymphocytes in the lymphoid organs, they block T-cell proliferation. MSCs use two enzymes involved in arginine metabolism to control T-cell responses: inducible nitric oxide synthase (NOS2), which generates nitric oxide (NO) and arginase 1 (Arg1), which depletes the milieu of arginine. Th1 cytokines induce NOS2, whereas Th2 cytokines upregulate Arg1. Induction of either enzyme alone results in a reversible block in T-cell proliferation. When both enzymes are induced together, peroxynitrites, generated by NOS2 under conditions of limiting arginine, cause activated T lymphocytes to undergo apoptosis. Thus, NOS2 and Arg1 might act separately or synergistically in vivo to control specific types of T-cell responses, and selective antagonists of these enzymes might prove beneficial in fighting diseases in which T-cell responses are inappropriately suppressed. This Opinion is the second in a series on the regulation of the immune system by metabolic pathways.
- Record: found
- Abstract: found
- Article: found
Aire-dependent production of XCL1 mediates medullary accumulation of thymic dendritic cells and contributes to regulatory T cell development
Yu Lei, Adiratna Mat Ripen, Naozumi Ishimaru … (2011)
The thymus provides a microenvironment that is central to the establishment of self-tolerance (Takahama, 2006; Anderson et al., 2007; Klein et al., 2009). In the medullary region of the thymus, medullary thymic epithelial cells (mTECs) and thymic DCs (tDCs) display systemic and tissue-restricted self-antigens and cooperate to induce the negative selection of self-reactive thymocytes (Liston et al., 2003; Gallegos and Bevan, 2004; Anderson et al., 2005). mTECs express a diverse set of self-antigens, including the promiscuously expressed tissue-restricted antigens, in part regulated by the nuclear protein Aire (Derbinski et al., 2001; Anderson et al., 2002). tDCs cross-present the mTEC-derived self-antigens (Gallegos and Bevan, 2004), whereas a fraction of tDCs are derived from the circulation importing various self-antigens into the thymus (Bonasio et al., 2006). The cooperation between mTECs and tDCs contributes to the negative selection of tissue-restricted antigen-reactive thymocytes (Gallegos and Bevan, 2004). In addition to the negative selection of self-reactive thymocytes, the thymus produces naturally occurring regulatory T cells (nT reg cells) that are essential for the establishment of self-tolerance (Sakaguchi et al., 2008). It has been suggested that mTECs (Aschenbrenner et al., 2007; Spence and Green, 2008), tDCs (Proietto et al., 2008; Hanabuchi et al., 2010), and their cooperation (Watanabe et al., 2005) contribute to the generation of nT reg cells in the thymus. However, how these cells contribute to and/or cooperate for the generation of nT reg cells is still elusive. tDCs are predominantly accumulated in the medullary region of the thymus and sparsely detectable in the cortex (Barclay and Mayrhofer, 1981; Flotte et al., 1983; Kurobe et al., 2006). The accumulation of tDCs in the medulla is presumed to contribute to their efficient cooperation with mTECs in the establishment of negative selection and nT reg cell generation. Nonetheless, how tDCs are accumulated in the thymic medulla and how this medullary accumulation of tDCs contributes to negative selection and nT reg cell generation are unknown. The present study identifies the chemokine XCL1, also known as lymphotactin, as being essential for the medullary accumulation of tDCs. Cells that produce XCL1 in the thymus include mTECs, whereas XCR1, the receptor for XCL1, is expressed by tDCs. We also find that Aire is essential for the mTEC production of XCL1. In mice deficient for XCL1 or Aire, most tDCs fail to accumulate in the medulla and are arrested at the cortico-medullary junction (CMJ). The generation of nT reg cells is impaired in both Xcl1-deficient mice and Aire-deficient mice. Like Aire-deficient mice (Anderson et al., 2002, 2005), nude mice transferred with thymocytes from Xcl1-deficient mice exhibit inflammatory lesions in lacrimal glands. These results reveal that the XCL1-mediated medullary accumulation of tDCs critically contributes to the development of nT reg cells, and they suggest a role for Aire in facilitating the XCL1-mediated medullary accumulation of tDCs. RESULTS Screening for chemokines involved in the localization of tDCs tDCs are predominantly accumulated in the medulla and sparsely detectable in the cortex (Barclay and Mayrhofer, 1981; Flotte et al., 1983; Kurobe et al., 2006). To identify chemokines that mediate the medullary accumulation of tDCs, we screened for the expression of chemokine receptors in isolated tDCs by RT-PCR analysis. Among the mouse chemokine receptors so far identified, Ccr2, Ccr4, Ccr5, Ccr6, Ccr7, Ccr8, Cxcr1, Cxcr3, Cxcr4, Xcr1, and Cx3cr1 were detected in CD11c+ cells isolated from the thymus (Fig. 1 A). We then examined chemokines that could bind to these transcript-detectable receptors for their ability to attract CD11c+ thymic cells. We found that CCL19 (CCR7 ligand), CCL21 (CCR7 ligand), CXCL12 (CXCR4 ligand), or XCL1 (XCR1 ligand) attracted CD11c+ thymic cells (Fig. 1 B). These results revealed the potential roles of CCR7, CXCR4, and XCR1 in the chemotactic regulation of tDCs. However, we detected no obvious defects in the medullary accumulation of CD11c+ DCs in the thymus of mice deficient for CCR7, CCR7 ligands, or CXCR4 (Fig. 1 C; also see Fig. 3 D), even though the medullary region in CCR7- or CCR7 ligand–deficient mice was smaller than that in control mice (Fig. 1 C; Kurobe et al., 2006; Nitta et al., 2009). Thus, instead of CCR7 and CXCR4, the XCL1–XCR1 chemokine axis may play a major role in regulating the medullary accumulation of tDCs. Figure 1. Screening for chemokines that regulate the localization of tDCs. (A) RT-PCR analysis of chemokine receptor expression in isolated tDCs. Shown are the results of ethidium bromide detection of electrophoretically separated PCR products. Hprt, hypoxanthine-guanine phosphoribosyltransferase. Shown are the representative data of three independent experiments. (B) Chemotactic indexes of tDCs to the ligands of expressed chemokine receptors CCL1 (CCR8 ligand), CCL3 (CCR5 ligand), CCL7 (CCR2 ligand), CCL8 (CCR5 ligand), CCL17 (CCR4 ligand), CCL19 (CCR7 ligand), CCL20 (CCR6 ligand), CCL21 (CCR7 ligand), CCL22 (CCR4 ligand), CXCL1 (CXCR1 ligand), CXCL9 (CXCR3 ligand), CXCL10 (CXCR3 ligand), CXCL11 (CXCR3 ligand), CXCL12 (CXCR4 ligand), XCL1 (XCR1 ligand), and CX3CL1 (CX3CR1 ligand) were determined. Bar graphs indicate means ± standard errors of the data from at least three independent measurements. Red underlines indicate the chemokines that attracted the tDCs (based on the statistical significance as shown in the graph). *, P 0.05) altered by the Xcl1 deficiency (Fig. 3, A and B). Similarly to Xcl1-deficient mice of BALB/c background, Xcl1-deficient mice of C57BL/6 background exhibited altered distribution of tDCs, namely sparseness in the M region and density in the CMJ-C region (Fig. S2 B). Nonetheless, the absolute numbers of tDCs, including lymphoid DC, myeloid DC, and plasmacytoid DC subsets, were not affected in Xcl1-deficient mice (Fig. 3 C). These results indicate that XCL1 is essential for the accumulation of tDCs in the inner medullary region and that the deficiency of XCL1 causes the aberrant arrest of tDCs in the CMJ-C region. Figure 3. Distribution of DCs and macrophages in the thymus of Xcl1-deficient mice. (A) Two-color immunofluorescence analysis of thymus sections stained for CD11c (green) and the mTEC-specific ER-TR5 determinant (red). Representative images from three independent experiments are shown. White lines indicate borders among the indicated regions of the thymus section identified as in Fig. S2 A. C, cortex; M, medulla. Representative images from three independent experiments are shown. (B) Number of CD11c+ cells per unit area (1 mm2) of the indicated regions of the thymus sections was measured. Means and standard errors of cell numbers from three independent measurements are shown. (C) Means and standard errors (n = 6) of the absolute numbers of lymphoid DCs (lyDCs), myeloid DCs (mDCs), and plasmacytoid DCs (pDCs) in the thymus of indicated mice are shown. (D) Relative density of CD11c+ cells in the indicated regions to the density in the medullary region of the thymus section (in percentile). Means and standard errors (n = 3) for WT, plt/plt, Mx-Cre × CXCR4f/f, Xcl1-deficient, and Xcl1-deficient plt/plt mice are plotted. (E and F) The thymus sections were analyzed for CD11b (green) and the ER-TR5 determinant (red). Representative images from three independent experiments are shown in E, and means and standard errors of cell numbers from three independent analyses are shown in F. ***, P 0.05). The localization of tDCs was specifically altered in Xcl1-deficient mice but not in CCR7 ligand–deficient plt/plt mice or CXCR4-deficient mice (Fig. 1 C and Fig. 3 D). The defective accumulation of tDCs in the thymic medulla caused by the XCL1 deficiency was not further compromised by an additional lack of CCR7 ligands (Fig. 3 D). Thus, among the chemokines that exert chemotactic activity on tDCs (Fig. 1 B), XCL1 appears to be the major regulator of the tDC medullary accumulation. Unlike tDCs, CD11b+ macrophages in the thymus were not enriched in the medulla of WT mice but were most densely distributed in the CMJ-C region (Fig. 3, E and F). This distribution was not affected in Xcl1-deficient mice (Fig. 3, E and F). The distribution and number of CD4+CD8+, CD4+CD8−, or CD4−CD8+ thymocytes were normal in Xcl1-deficient mice (unpublished data). These results indicate that in the thymus, XCL1 influences the distribution of tDCs specifically. Negative selection of self-reactive thymocytes in Xcl1-deficient mice It was previously shown that tDCs contribute to the negative selection of self-reactive thymocytes (Jenkinson et al., 1992; Gallegos and Bevan, 2004). We therefore examined whether negative selection might be affected in the thymus of Xcl1-deficient mice. As tDCs have been implicated for their roles in negative selection by mammary tumor virus (Mtv)–encoded superantigens (Moore et al., 1994), we first analyzed the negative selection of Vβ3+, Vβ5+, and Vβ11+ T cells in Mtv-expressing BALB/c mice. We found that the deletion of Vβ3+ and Vβ5+ TCRhigh CD4+CD8− thymocytes in the Mtv + BALB/c background, as compared with Mtv − C57BL/6 background, was slightly but significantly (P 0.05). Unlike the reduced number of nT reg cells in the thymus, the number of total Foxp3+ cells in the periphery was not reduced in Xcl1-deficient mice throughout ontogeny (Fig. S4, B-D). However, the number of thymic nT reg cell–derived spleen nT reg cells, which were identified by the expression of the nuclear factor Helios along with Foxp3 (Sugimoto et al., 2006; Thornton et al., 2010), was reduced in Xcl1-deficient mice (Fig. 5 H and Fig. S4 E), suggesting that XCL1 deficiency causes the reduction of nT reg cells in the periphery. No signs of tissue inflammation were detected in Xcl1-deficient mice at 6 wk old and 3 mo old (unpublished data). However, we detected inflammatory lesions and autoantibody deposits in several tissues, including heart, liver, stomach, salivary glands, and lacrimal glands, in Xcl1-deficient mice at 12–18 mo old (unpublished data). Importantly, the intravenous transfer of thymocytes from Xcl1-deficient BALB/c background mice into athymic BALB/c-nu/nu mice caused severe lymphocyte infiltration and tissue damage of lacrimal glands (Fig. 6, A and B). The tissue lesions were prominent in lacrimal glands, weakly detectable in salivary glands, and not detected in heart, liver, stomach, pancreas, or kidney. The administration of equal numbers of WT BALB/c thymocytes along with thymocytes from Xcl1-deficient mice significantly reduced the lesions in lacrimal glands (Fig. 6, A and B), suggesting that nT reg cells generated in the normal thymus are capable of suppressing the dacryoadenitis that nT reg cells generated in the thymus of Xcl1-deficient mice fail to control. These results indicate that nT reg cell development is impaired in the thymus of Xcl1-deficient mice and that the thymocytes from Xcl1-deficient mice are potent in causing, and fail to regulate, autoimmune dacryoadenitis. Figure 6. Thymocytes from Xcl1-deficient mice elicit inflammatory lesions in lacrimal glands in nude mice. 5 × 107 thymocytes isolated from WT or Xcl1-deficient (Xcl1-KO) mice of BALB/c background were intravenously transferred into BALB/c-nu/nu mice. Where indicated, equal numbers of thymocytes (5 × 107 + 5 × 107) were mixed before the transfer. Paraffin-embedded sections of lacrimal glands at 8 wk after the transfer were stained with hematoxylin and eosin. (A) Representative images of the sections at two different magnifications. (B) Histological scores of inflammatory lesions in the lacrimal glands (n = 5). Horizontal bars indicate the means. ***, P 0.05). The density of tDCs in the middle medullary M region was significantly (P 0.05) altered by the Aire deficiency (Fig. 7, B and C). The absolute numbers of tDCs and their subsets were not affected in Aire-deficient mice (Fig. 7 D). These results indicate that as in Xcl1-deficient mice, Aire-deficient mice exhibit defective accumulation of tDCs in the medullary region. The density of Foxp3+ cells in the thymic medulla (CMJ-M and M regions) was accordingly and significantly (P 0.05) different between Xcl1-deficient mice and Aire-deficient mice. Our results also indicate that Aire deficiency causes severe loss of XCL1 expression by mTECs, whereas Xcl1 deficiency does not reduce Aire expression by mTECs. We therefore think that Aire is essential for the mTEC expression of XCL1, which attracts XCR1-expressing tDCs to the medullary region and contributes to the optimal generation of T reg cells in the thymus. Proximal interactions between tDCs and mTECs in the thymic medulla may promote optimal generation of nT reg cells, possibly via the production of γc cytokines, including IL-2, IL-7, and thymic stromal lymphopoietin (Watanabe et al., 2005; Ziegler and Liu, 2006; Mazzucchelli et al., 2008; Vang et al., 2008). Among the tDC subpopulations, lymphoid DCs most highly express XCR1 and, thus, may play a major role in the interaction with mTECs and the generation of nT reg cells. Unlike tDCs, CD4+CD8−CD25+ thymic T reg cells do not express detectable Xcr1 transcripts (unpublished data), ruling out the possibility of a direct effect of XCL1 on T reg cells. Thus, our results suggest that XCL1-producing mTECs attract tDCs into the inner medullary region, thereby promoting the generation of nT reg cells, and that optimal nT reg cell development in the thymus requires a specialized medullary microenvironment that is formed by the XCL1-mediated attraction of tDCs by Aire-dependent mTECs. Whether XCL1-mediated tDC accumulation in the medulla affects TCR repertoire of nT reg cells remains unclear. A recent work using CD11c-Cre mice crossed with mice that express diphtheria toxin A under the control of a loxP-flanked neomycin resistance cassette from the ROSA26 locus has shown that these DC-depleted mice are not defective in the generation of nT reg cells in the thymus (Ohnmacht et al., 2009), potentially contradicting our results indicating the role of tDCs in nT reg cell generation. However, that work also described that the depletion of tDCs is incomplete in the DC-depleted mice (Ohnmacht et al., 2009) but did not describe the intrathymic localization of the remaining tDCs. Thus, the incompletely depleted tDCs in the DC-depleted mice may be enriched in the inner medullary region and be sufficient for the unreduced generation of nT reg cells. Our results show that the number of nT reg cells in the thymus is reduced in Aire-deficient mice. However, several studies have described that the generation of T reg cells is not defective in Aire-deficient mice (Anderson et al., 2002, 2005; Liston et al., 2003; Kuroda et al., 2005; Hubert et al., 2009), particularly in the spleen and the lymph nodes (Anderson et al., 2005; Kuroda et al., 2005; Hubert et al., 2009). Indeed, our results show that the number of T reg cells in Aire-deficient mice is reduced in the thymus to approximately half of that in WT mice but is not reduced in the spleen and the lymph nodes of Aire-deficient mice. It should be noted that previous studies have indicated that the number of T reg cells in the thymus is slightly reduced in Aire-deficient mice, although those studies concluded no loss of T reg cell generation in the thymus (Anderson et al., 2005; Kuroda et al., 2005; Hubert et al., 2009). We think that Aire indeed contributes to the optimal generation of nT reg cells in the thymus and that the generation of induced T reg cells, particularly in the periphery (Piccirillo and Shevach, 2004; Curotto de Lafaille and Lafaille, 2009), has veiled the reduced cellularity of nT reg cells in Aire-deficient mice. The present results show that thymocytes from Xcl1-deficient mice are potent in eliciting severe lymphocyte infiltration and tissue damage of lacrimal glands in athymic nu/nu mice, indicating that the thymocytes generated in Xcl1-deficient mice fail to establish self-tolerance. The mixture of thymocytes from normal mice reduces the dacryoadenitis caused by the thymocytes from Xcl1-deficient mice, supporting the possibility that the thymocytes from Xcl1-deficient mice are potent in triggering, and fail to regulate, the autoimmunity and that nT reg cell development is impaired in the thymus of Xcl1-deficient mice. In a similar manner, Aire-deficient mice tend to exhibit inflammatory failure of exocrine tissues, including lacrimal glands (Anderson et al., 2002, 2005; Kuroda et al., 2005; Hubert et al., 2009). The commonness of the target organs may reflect a similarity in the breakdown of self-tolerance in Xcl1-deficient mice and Aire-deficient mice. Nonetheless, our results do not rule out the possibility that the absence of XCL1 in peripheral T cells also contributes to the onset of the dacryoadenitis. Finally, our results reveal that the XCL1-mediated medullary accumulation of tDCs plays only a minor role in the negative selection of self-reactive thymocytes. In contrast, tDCs are known for their roles in negative selection, particularly in the intrathymic presentation of peripheral antigens, by cooperating with promiscuous gene expression in mTECs (Gallegos and Bevan, 2004; Koble and Kyewski, 2009; Nitta et al., 2009) and by transport from the circulation (Bonasio et al., 2006). The tDCs that are mislocalized in the CMJ regions in the absence of XCL1 may be sufficient for the cross-presentation of mTEC-expressed tissue-restricted antigens and the negative selection of developing thymocytes reactive to those self-antigens. In addition, recent studies indicated that DCs in the thymic cortex are capable of inducing the deletion of negatively selected thymocytes (McCaughtry et al., 2008) and that CCR2 is involved in the accumulation of CD8−Sirpα+ tDC subpopulation in the thymic cortex and the intrathymic negative selection against blood-borne antigens (Baba et al., 2009). Thus, the XCL1-mediated medullary accumulation is not needed for the deletion of the majority of negatively selected thymocytes. In conclusion, the present results indicate that the XCL1–XCR1 chemokine axis contributes to the medullary accumulation of tDCs and the thymic development of nT reg cells. The results also suggest that Aire expressed in mTECs regulates the XCL1-mediated medullary accumulation of tDCs and that XCL1-mediated proximal interaction between tDCs and mTECs in the thymic medulla contributes to the thymic development of nT reg cells. Our results imply a novel role of Aire in regulating autoimmunity via the XCL1-mediated medullary accumulation of tDCs and that the breakdown of self-tolerance in Aire deficiency may involve the failure to localize tDCs in the medulla in an XCL1-dependent manner. MATERIALS AND METHODS Mice. Xcl1-deficient mice were generated at Merck Research Laboratories (Fig. S1, A–C). Aire-deficient mice were generated at the University of Basel (Fig. S5). Ccr7-deficient mice (Förster et al., 1999), Mx-Cre x Cxcr4 flox/flox mice (Sugiyama et al., 2006), and plt/plt mice (Nakano et al., 1998), as well as OT-I and OT-II TCR-transgenic and RIP-mOVA–transgenic mice (Kurts et al., 1996; Barnden et al., 1998), were described previously. Mx-Cre x Cxcr4 flox/flox mice were injected with poly I poly C, and only mice that exhibited a nearly complete loss of CXCR4 genomic sequence and the undetectable expression of CXCR4 gene in hematopoietic cells were further analyzed (Sugiyama et al., 2006). Mice were maintained under specific pathogen-free conditions in our animal facility, and experiments were performed under the approval of the Institutional Animal Care Committee of the University of Tokushima. Bone marrow chimeras. Bone marrow cells were magnetically depleted of T cells using biotin-conjugated antibodies specific for CD4, CD8, and Thy1.2 and streptavidin-conjugated magnetic beads (Miltenyi Biotec). Recipient mice were injected with T cell–depleted bone marrow cells (4 × 107) 1 d after 9.25 Gy x-ray irradiation. The mice were analyzed 4–5 wk after the reconstitution. Thymocyte transfer into nude mice. 5 × 107 thymocytes isolated from WT or Xcl1-deficient (Xcl1-KO) mice of BALB/c background were intravenously transferred into BALB/c-nu/nu mice. Where indicated, equal numbers of thymocytes (5 × 107 + 5 × 107) were mixed before the transfer. Various organs at 8 wk after the transfer were fixed with 4% phosphate-buffered formaldehyde, pH 7.2. Paraffin-embedded sections were stained with hematoxylin and eosin. Two pathologists independently evaluated the histology without being informed of the conditions of individual mice. Inflammatory lesions of the tissues were scored as previously described (Ishimaru et al., 2008) and as follows: 0 = no inflammation, 1 = 1–5 foci composed of >20 mononuclear cells per focus, 2 = >5 such foci but without significant parenchymal destruction, 3 = degeneration of parenchymal tissue, and 4 = extensive infiltration with mononuclear cells and extensive parenchymal destruction. Flow cytometry analysis. Multicolor flow cytometry analysis and cell sorting were performed using FACSCalibur and FACSAria II (BD). Intracellular staining of Foxp3 and Helios was performed according to the manufacturer’s instructions (eBioscience). Thymic stromal cells were prepared by digesting thymic fragments with collagenase, dispase, and DNase I (Roche) and enriched by depleting CD45+ cells with a magnetic cell sorter (Miltenyi Biotec) before cell sorting, as described previously (Gray et al., 2002). For DC analysis, thymus cells were prepared by digesting thymic fragments with collagenase D and DNase I. Immunofluorescence analysis. Frozen thymus tissues embedded in OCT compound (Sakura) were sliced into 5-µm-thick sections, fixed with acetone, and stained with the following antibodies: mTEC-specific monoclonal antibody ER-TR5 (a gift from W. van Ewijk, Erasmus University, Rotterdam, Netherlands) followed by Alexa Fluor 633–conjugated anti–rat IgG antibody (Invitrogen); biotinylated UEA1 (Vector Laboratories) followed by Alexa Fluor 633–conjugated streptavidin (Invitrogen); FITC-conjugated anti-Foxp3 monoclonal antibody (eBioscience); biotinylated anti-CD11c or anti-CD11b monoclonal antibody (eBioscience) followed by Alexa Fluor 488–conjugated or Alexa Fluor 546–conjugated streptavidin (Invitrogen); and anti-Aire antibody (Santa Cruz Biotechnology, Inc.) followed by FITC-conjugated anti–rabbit IgG antibody (Invitrogen). Images were analyzed with a TSC SP2 confocal laser-scanning microscope and Confocal software version 2.6 (Leica). Chemotaxis assay. 106 collagenase-digested thymus cells were placed in a Transwell chamber (6.5-mm diameter, 5-µm pore; Corning) that was inserted into a 100-nM chemokine-containing culture well. Cells were incubated for 2 h, counted, stained for CD11c and I-Ab, and analyzed by flow cytometry. Chemotactic index is the ratio of the numbers of CD11c+I-Ab+ DCs that migrated to the bottom of culture wells in the presence and absence of chemokines. Measurement of T reg cell function. According to the methods previously reported (Tai et al., 2005), 5 × 104 CFSE-labeled CD4+CD25− lymph node T cells isolated from B6-Ly5.1 mice were cultured with 5 × 104 CD25+CD4+CD8− or CD25−CD4+CD8− thymocytes in the presence of 105 20 Gy-irradiated T cell–depleted spleen cells from B6 mice and 1 µg/ml anti-CD3 monoclonal antibody (clone 2C11). Cells were harvested at 72 h and analyzed by flow cytometry. RT-PCR analysis. Total cellular RNA was reverse transcribed with oligo-dT primer and Superscript III reverse transcription (Invitrogen). cDNA was PCR amplified, electrophoresed, and visualized with ethidium bromide. For quantitative analysis, real-time RT-PCR was performed using SYBR Premix Ex Taq (Takara Bio Inc.) and Light Cycler DX400 (Roche). Amplified products were confirmed to be single bands by gel electrophoresis and were normalized to the amount of HPRT products. Primer sequences are listed in Table S1. Statistical analysis. Statistical comparison was performed with the Student’s t test (two-tailed) using Excel software (Microsoft). Online supplemental material. Fig. S1 shows genomic structure of Xcl1-deficient mice. Fig. S2 shows analysis of CD11c+ cells in thymus sections. Fig. S3 shows negative selection of T cells in the periphery of Xcl1-deficient mice. Fig. S4 shows thymocytes, splenocytes, and Aire+ mTECs in Xcl1-deficient mice. Fig. S5 shows targeting strategy to generate Aire-Cre-GFP knockin mice. Table S1 shows primer sequences used for RT-PCR analysis. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20102327/DC1.
Author and article information
Journal
Journal ID (nlm-journal-id): 0426720
Journal ID (pubmed-jr-id): 4839
Journal ID (nlm-ta): J Invest Dermatol
Journal ID (iso-abbrev): J. Invest. Dermatol.
Title:
The Journal of investigative dermatology
ISSN
(Print):
0022-202X
ISSN
(Electronic):
1523-1747
Publication date Nihms-submitted: 20
May
2014
Publication date
(Electronic):
21
April
2014
Publication date
(Print):
October
2014
Publication date PMC-release: 01
April
2015
Volume: 134
Issue: 10
Pages: 2531-2540
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Author notes
CORRESPONDING AUTHOR: James G. Krueger, Laboratory for Investigative Dermatology, The Rockefeller University,
1230 York Avenue, Box 178, New York, NY, 10065-6399, Ph: 212-327-7730, Fax: 212-327-8232,
kruegej@
123456rockefeller.edu
Article
Manuscript ID: NIHMS587093DOI: 10.1038/jid.2014.196
PMC ID: 4165712
PubMed ID: 24751728
SO-VID: 8f4e4805-a2f2-4b72-b476-760b54eab212
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ScienceOpen disciplines: Dermatology
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ScienceOpen disciplines: Dermatology