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© 2006 Nature Publishing Group http://www.nature.com/natureimmunology
Alloantigen-presenting plasmacytoid dendritic cells mediate tolerance to vascularized grafts
Jordi C Ochando1, Chiho Homma1, Yu Yang1, Andres Hidalgo2, Alexandre Garin3, Frank Tacke1, Veronique Angeli1, Yansui Li4, Peter Boros4, Yaozhong Ding1, Rolf Jessberger1,5, Giorgio Trinchieri6, Sergio A Lira4, Gwendalyn J Randolph1 & Jonathan S Bromberg1,4,7
The induction of alloantigen-specific unresponsiveness remains an elusive goal in organ transplantation. Here we identify plasmacytoid dendritic cells (pDCs) as phagocytic antigen-presenting cells essential for tolerance to vascularized cardiac allografts. Tolerizing pDCs acquired alloantigen in the allograft and then moved through the blood to home to peripheral lymph nodes. In the lymph node, alloantigen-presenting pDCs induced the generation of CCR4+CD4+CD25+Foxp3+ regulatory T cells (Treg cells). Depletion of pDCs or prevention of pDC lymph node homing inhibited peripheral Treg cell development and tolerance induction, whereas adoptive transfer of tolerized pDCs induced Treg cell development and prolonged graft survival. Thus, alloantigen-presenting pDCs home to the lymph nodes in tolerogenic conditions, where they mediate alloantigen-specific Treg cell development and allograft tolerance. self-reactive T cells9. In several transplantation models, Treg cells have been shown to be important in the induction and maintenance of tolerance to foreign molecules10–13, although little is known about the identity of alloantigen-presenting cells that promote their development into antigen-specific suppressor cells or the location in which this process occurs. Plasmacytoid dendritic cells (pDCs) may be important in the in vivo generation of antigen-specific Treg cells, as their precursors have been reported to prolong graft survival14. Along with other published observations15,16, those results suggest that interactions between pDCs and CD4+ T cells in the lymph node may be required for Treg cell development and indefinite graft survival. Here we performed fully allogeneic vascularized cardiac transplants and subjected recipient mice to a well characterized tolerogenic protocol that is dependent on Treg cell development16,17. Using the YAe monoclonal antibody (mAb), which recognizes a donor-derived I-Ed peptide presented in the context of recipient MHC class II I-Ab molecules, we identified pDCs as alloantigen-presenting cells that mediate tolerance to vascularized allografts. Alloantigen-presenting pDCs acquired and processed cell-derived transplantation antigens and, after tolerogenic treatment, migrated 'preferentially' to the lymph node, where they induced antigen-specific CD4+CD25+Foxp3+ Treg cell development. These data are consistent with published results that have established that during tolerance, antigen-specific Treg cells must develop in the lymph node16 and provide direct evidence that pDCs can act as 'professional' tolerance-inducing alloantigenpresenting cells in vivo.
The main unsolved goal in clinical transplantation is the establishment of tolerance to transplant antigens. Although much has been learned over the past decade about the various cell populations involved in transplant rejection1–4, the precise cell subpopulations involved in the induction and maintenance of transplant tolerance remain unidentified. That is due in part to the difficulty in defining the precise interactions between diverse antigen-presenting cells (APCs) and T cell subsets with disparate trafficking patterns5. Antigen-specific immune responses are initiated after the acquisition of foreign molecules in the periphery by phagocytic APCs6. After alloantigen uptake, activated APCs undergo a shift in their expression of chemokine receptors and migrate to secondary lymphoid organs7. Entry into the spleen is considered nonspecific, whereas entry into the lymph node requires expression of the L-selectin CD62L and the chemokine receptor CCR7, which bind to peripheral node addressin and the chemokine CCL21, respectively, and allow extravasation across high endothelial venules (HEVs)8. Once in the secondary lymphoid organs, APCs present complexes of foreign peptide and major histocompatibility complex (MHC) molecules on their surfaces to alloantigen-specific CD4+ T cells, and signals arising from these interactions are key factors in the establishment of either immunity or tolerance. Central tolerance is established in the thymus by clonal deletion of potentially self-reactive T cells. In addition, CD4+CD25+Foxp3+ regulatory T cells (Treg cells) help to maintain peripheral tolerance by actively suppressing the activation and population expansion of
1Department of Gene and Cell Medicine, 2Hematology Center, 3Immunobiology Center and 4Recanati/Miller Transplantation Institute, Mount Sinai School of Medicine, New York, New York 10029-6574, USA. 5Department of Physiological Chemistry, Dresden University of Technology, Dresden, Germany. 6Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892-8003, USA. 7Department of Surgery, Mount Sinai School of Medicine, New York, New York 10029-6574, USA. Correspondence should be addressed to J.C.O. (firstname.lastname@example.com) or J.S.B. (email@example.com).
Received 19 December 2005; accepted 10 March 2006; published online 23 April 2006; doi:10.1038/ni1333
RESULTS pDCs acquire alloantigen in donor grafts To investigate whether a particular DC subtype is involved in alloantigen uptake and processing in allografts during tolerance, we characterized intracardiac alloantigen-presenting cells between 1 and 10 weeks after vascular cardiac transplantation. The YAe mAb recognizes donor-derived I-Ed peptide presented by recipient I-Ab (ref. 18). Two-color immunohistochemical analysis showed that intragraft YAe+ cells from tolerized recipient mice expressed CD11c, B220, the marker Gr-1 and pDC marker PDCA-1 but not CD19 (Fig. 1a). This surface phenotype is characteristic of mouse pDCs19. Using six-color flow cytometry, we confirmed that YAe+ cells in both tolerized grafts (Fig. 1b) and rejecting grafts (data not shown) were pDCs. These data indicate that pDCs acquire and process alloantigen in vivo. This result was unexpected, as no reports indicate that pDCs, which are poorly endocytic20, can internalize cells, a process that would probably involve phagocytosis. To assess the phagocytic capacity of pDCs, we cultured pDCs together with fluorescein isothiocyanate (FITC)–labeled latex particles (beads) and monitored engulfment. We used naive splenic B cells and peritoneal macrophages as negative and positive controls, respectively. Immunofluorescence images (Fig. 1c) showed that both pDCs and
© 2006 Nature Publishing Group http://www.nature.com/natureimmunology
5 97
2 96
Gr-1 Gr-1
3 98
1 6
B cell (B220)
104 103 102 101
104 103 102 101
B cell
101 102 103 104 105
101 102 103 104 105
% of max. % of max. % of max.
105 91
PE-Cy7 anti-FITC PE-Cy7 anti-FITC PE-Cy7 anti-FITC
100 80 60 40 20 0
101 102 103 104 105 100 80 60 40 20 0 100 80 60 40 20 0
pDC (PDCA-1)
104 103 102 101 105 15 104 103 102 101
104 103 102 101 105 104 103 102 101
101 102 103 104 105
101 102 103 104 105
101 102 103 104 105
101 102 103 104 105
101 102 103 104 105
101 102 103 104 105
PE-Cy7 anti-FITC
Figure 1 Phagocytic pDCs cells take up and process antigen in donor cardiac allografts. Fully mismatched vascularized BALB/c (H-2d) cardiac allografts were transplanted into C57BL/6 (H-2b) mice. Recipients were injected intravenously with 1 Â 107 donor splenocytes on day –7 and 250 mg mAb to CD40L on days –7, –4, 0 and +4 (times relative to transplantation). Untreated rejecting control mice received hamster immunoglobulin in PBS and rejected their grafts within 1 week (n ¼ 6 mice per group). (a,b) Two-color microscopy (a; staining of first column indicated along left margin) or six-color flow cytometry gated on YAe cells (b) of tolerized (1-, 5- and 10-week) and rejecting (1-week) cardiac allografts. Data are representative results from '10-week tolerized' mice. Original magnification, Â250. (c,d) In vitro pDC phagocytic assay of the uptake of 1-mm FITC-labeled latex beads (FITC-beads) over 12 h of culture. (c) May¨ Grunwald Giemsa staining and live fluorescent images of splenic B cells, splenic pDCs and peritoneal macrophages (Mf). Original magnification, Â1,000. Data are representative results of ten images. (d) Flow cytometry of phagocytic B cells, pDCs and macrophages. Gated FITC-labeled beads were stained with phycoerythrin-indotricarbocyanine-labeled (PE-Cy7) anti-FITC or the isotype control mAb. All experiments were done at least three times. Numbers in quadrants (b,d) indicate percentage of cells positive for markers along margins.
YAe+ pDC
4,000 3,000 2,000 1,000 0 4,000 101102103104105 4,000
4,000 3,000 2,000 1,000 0 4,000 101102103104105
3,000 2,000 1,000 0 4,000 101102103104105
Naive pDC
Naive mDC
SSC Lymph
3,000 2,000 1,000 0
3,000 2,000 1,000 0
3,000 2,000 1,000
Blood PDCA-1
104 0.5% 103 102 101
Tolerized 4.4%
105 104 0.8% 103 102
Naive 0.1%
© 2006 Nature Publishing Group http://www.nature.com/natureimmunology
101 102 103 104 105
% of max.
101 102 103 104 105
Expression (relative units × 103)
102 104 102 104 102 104 102 104 102 104 102 104
* * * * *
% of max.
102 104
102 104
102 104
102 104
102 104
102 104
% of max.
0 CCR2 Tolerized CCR7 Rejecting ChemR23 Naive
102 104
102 104
102 104 102 104 Tolerized Rejecting
102 104
102 104
Figure 2 Alloantigen-presenting pDCs circulate systemically through blood. (a) Percentage of total alloantigen-presenting YAe+ cells recovered from blood and lymph of tolerized mice (1-, 5- and 10-week), rejecting mice (1-week) and naive mice. Data are representative results of three independent experiments ¨ from '10-week tolerized' mice (n ¼ 3 mice per group). (b) May-Grunwald Giemsa staining (representative of two independent experiments) showing the morphology of freshly sorted blood naive pDCs (PDCA-1+CD11c+B220+), splenic naive mDCs (CD11c+CD11b+B220–) and '10-week tolerized' blood circulating YAe+PDCA-1+ pDCs. Original magnification, Â1,000; n ¼ 3 mice per group. (c) Flow cytometry of blood cells from tolerized mice (1- and 10-week). Representative results of three independent experiments from '10-week tolerized' mice show that more than 90% of YAe+ cells express PDCA-1 (n ¼ 3). (d) Flow cytometry of the expression of cell surface molecules on freshly isolated blood YAe+PDCA-1+ cells from tolerized mice (1- and 10-week) and rejecting mice (1-week) compared with that of PDCA-1+ cells from naive mice. MHC-II, MHC class II. Data are representative results of three independent experiments from '10-week tolerized' mice (n ¼ 3 mice per group). (e) Real-time PCR analysis of chemokine receptor expression on blood circulating YAe+PDCA-1+ cells from '10-week tolerized' and '1-week rejecting' mice compared with that of naive pDCs. *, P o 0.01 (one-way ANOVA). Data are from two independent experiments; n ¼ 3 mice per group.
macrophages acquired FITC-labeled beads, as demonstrated by fluorescent localization of FITC-coated beads in the cells. In contrast, FITC-labeled beads were bound only to the external cell membranes of B cells. We further confirmed the phagocytic capacity of pDCs by flow cytometry with a FITC-specific mAb (Fig. 1d), which showed that 88% of FITC+ cultured pDCs and 53% of FITC+ macrophages, but only 8% of FITC+ B cells, stained negative for cell surface FITC. Alloantigen-presenting pDCs circulate in blood To determine if alloantigen uptake and processing by pDCs results in subsequent maturation and egress from the allograft to lymphoid organs, we determined whether YAe+ cells were present in blood and/or lymph (Fig. 2a). YAe+ cells from tolerized recipient mice were present in blood but not in efferent lymph, suggesting that the main route for egress from the allograft is direct re-entry into blood. To further characterize blood-circulating YAe+ cells, we used WrightGiemsa staining of cytospin-sorted DC preparations. Freshly isolated blood pDCs (CD11c+PDCA-1+) were rounded cells with a smooth surface and large nuclei, whereas blood alloantigen-presenting plasmacytoid cells (YAe+PDCA-1+) had slightly indented nuclei and had pseudopods, and splenic myeloid DCs (mDCs) (CD11c+CD11b+B220–) contained more lobulated nuclei and demonstrated classical stellate morphology (Fig. 2b). More detailed
examination of the YAe+PDCA-1+ population showed that alloantigen-presenting cells had high expression of mRNA encoding interferon-a and Toll-like receptors 3, 7 and 9 (Supplementary Figure 1 online), consistent with a mature pDC lineage. We next defined the phenotype of the blood YAe+ cells by flow cytometry (Fig. 2c) and found that more than 90% of YAe+ blood cells were PDCA-1+, confirming their plasmacytoid phenotype. Detailed cell surface phenotypic analysis of YAe+ PDCA-1+ cells (Fig. 2d) showed that these pDCs had low expression of CD11c, high expression of Gr-1 and intermediate expression of B220 and lacked expression of CD19 and CD11b. More detailed examination of costimulatory molecule expression showed that YAe+PDCA-1+ cells had very low expression of CD80 and CD86 but high expression of MHC class II (Fig. 2d), indicative of a partially mature phenotype. Furthermore, YAe+PDCA-1+ cells had high expression of the phagocytic receptor CD206, consistent with the observation that pDCs acquired and processed allopeptides in the allograft. Further analysis of the phenotype of YAe+PDCA-1+ cells demonstrated large amounts of the adhesion molecules LFA-1, ICAM-1, VLA-4 and CD62L (Fig. 2d) and the lymph node–homing chemokine receptors CCR7, ChemR23 and CCR2 (Fig. 2e) in tolerized recipient mice. These findings collectively suggest that alloantigen-presenting pDCs migrate 'preferentially' to the lymph nodes in tolerized recipient mice but migrate to other lymphoid organs during rejection.
pDCs migrate to lymph node during tolerance We next used laser-capture microdissection of lymph node sections from tolerized mice to characterize the function of HEVs in YAe+ pDC lymph node homing. CCR2 and CCR7 ligands CCL2, CCL7 and CCL21 were upregulated in HEVs during tolerization (Fig. 3a), which may favor lymph node extravasation of CCR2+CCR7+YAe+ pDCs. As CCL21 produced by HEVs mediates rapid binding of integrin to CD62L ligands21, which can be induced during inflammation22, we also investigated whether CD62L ligands were upregulated on HEVs during tolerization. Using a CD62L fusion protein (Fig. 3b), we found upregulation of CD62L ligands on HEVs in tolerized mice, which favors the adhesion of CD62L+ cells to the endothelial barrier. MAdCAM1 is one of the CD62L ligands specifically synthesized by HEVs23 that was upregulated during tolerization (Supplementary Fig. 2 online). To further determine the functional importance of CD62L and CD62L ligand upregulation on circulating YAe+ pDCs and HEVs from tolerized mice, we did a modified in vitro HEVbinding assay24 (Fig. 3c). Blood-derived YAe+ pDCs from tolerized mice bound four to five times more effectively than did YAe+ pDCs from rejecting mice to the same HEVs, consistent with the upregulation of CD62L on YAe+ pDCs from tolerized but not rejecting mice. We next monitored the in vivo localization of alloantigen-presenting YAe+ cells in the lymph node and spleen. In tolerized mice, YAe+ cells were distributed systemically throughout the lymphoid compartment (inguinal, axillary, cervical, paraortic and hepatic lymph nodes) yet were almost completely absent from the spleen (Fig. 3d). In contrast, YAe+ cells were localized mainly in the spleens of rejecting mice and were almost completely absent from the lymph node. Further phenotypic analysis of YAe+ cells in secondary lymphoid organs (Fig. 3e) indicated that more than 90% were pDCs during both rejection and tolerization. Localization of pDCs and Treg cells together We next characterized the specific localization of YAe+ cells in the lymph node. During tolerization, YAe+ cells localized specifically in the vicinity of HEVs and T cell zones outside the follicles and germinal
© 2006 Nature Publishing Group http://www.nature.com/natureimmunology
Expression (relative units)
150 100 50 0
Tolerized HEV
Rejecting HEV
Naive HEV
EDTA control
10 8 6 4 2 0 10 8 6 4 2 0 10 8 6 4 2 0
Expression (relative units ×103)
Tolerized HEV Tolerized YAe pDC 9±2
20 15 10 5 0
YAe+ pDC bound/HEV
Rejecting YAe pDC
** *
Tolerized Rejecting Naive
Inguinal LN Axillary LN Cervical LN Inguinal LN Axillary LN Cervical LN
105 104 103 102 101
YAe+ pDC 5.0%
0.4% Tolerized
Paraortic LN
Hepatic LN
Paraortic LN
Hepatic LN
Spleen PDCA-1
105 104 103 102 101
0.3% YAe
101 102 103 104 105 101 102 103 104 105
Figure 3 cells home to lymph nodes during tolerance and to the spleen during rejection. (a) Right, real-time PCR analysis of chemokine expression in HEVs isolated from '10-week tolerized', '1-week rejecting' and naive mice by laser-capture microdissection. Left, hematoxylin and eosin staining before (top) and after (middle) excision of tissue by laser-capture microdissection. Cap (bottom), excised tissue. Original magnification, Â100. *, P o 0.01 (one-way ANOVA); n ¼ 3 mice per group. (b) Immunofluorescent analysis of CD62L ligands expressed on HEVs of tolerized mice (1-, 5- and 10-week), rejecting mice (1-week) and naive mice. Numbers in images indicate mean fluorescence intensity (determined over entire field). Data are representative results from '10-week tolerized' and '1-week rejecting' mice (n ¼ 3 mice per group). (c) Left, representative staining of Stamper-Woodruff assay of the binding of YAe+PDCA-1+ cells from '10-week tolerized' mice (red) and '1-week rejecting' mice (blue) to the same '10-week tolerized' lymph node tissue section. Numbers in images indicate number of cells bound to HEV. Original magnification, Â250. Right, quantitative analysis of the Stamper-Woodruff assay, determined from ten lymph node sections per slide. N, naive; T, tolerized; R, rejecting. **, P o 0.01, and *, P o 0.05 (one-way ANOVA). (d) Immunofluorescent analysis of YAe+ distribution in secondary lymphoid organs in tolerized mice (1-, 5- and 10-week) and rejecting mice (1-week). Data are representative results from '10-week tolerized' mice. Original magnification, Â100. (e) Flow cytometry of lymph node and splenic alloantigen-presenting cells from '10-week tolerized' and '1-week rejecting' mice. Numbers beside outlined areas indicate percentage of cells in each. Data are representative results from '10-week tolerized' mice. All experiments were done three times.
centers, consistent with a pDC-like pattern of distribution25 (Fig. 4a). This 'strategic' positioning near the HEVs favors the encounter of tissue-derived DCs with antigen-specific T cells and results in retention of those T cells that recognize the alloantigen, which optimizes T cell activation26. We analyzed chemokine expression in YAe+ and YAe– lymph node and spleen pDCs and found considerable CCL17 mRNA in lymph node YAe+PDCA-1+ DCs (Fig. 4b, left). Conversely, CCL17+ pDCs were absent from the spleen (Fig. 4b, left), consistent with published results27. That result was further corroborated by histological visualization of CCL17 in the YAe+ cells in the lymph node (Fig. 4b, right). The pDCs from rejecting mice did not express CCL17 (Supplementary Fig. 3 online). To determine whether CCL17+YAe+PDCA-1+ cells influence the migration or accumulation of T cells28, we isolated lymph node CD4+ T cells and analyzed them by PCR and multicolor flow cytometry. There was high expression of CCR4 mRNA and protein by CD4+CD25+Foxp3+ T cells in the lymph nodes but not the spleens of tolerized recipient mice (Fig. 4c). Further histological data demonstrated localization of Foxp3+ cells around the
© 2006 Nature Publishing Group http://www.nature.com/natureimmunology
B cell follicle HEV YAe HEV Merged
CCL17 (relative units)
300 250 200 150 100 50 0
p YA DC e– pD C YA e+ pD C YA e– pD C YA e
Merged HEV
B cell follicle YAe CCL17 Tolerized LN Tolerized SPL
100 Graft survival (%) 80 60 40 20 0 0 10 20 30 Time after transplantation (d)
105 94 WT Ccr4 –/–
Tolerized LN
Tolerized spleen
CCR4 (relative units) 300 250 200 CD25 150 100 50 0
+ + – –
Tolerized LN
105 104 103 102 101
105 104
103 102 101 105
Ccr4 –/– LN 15
101 102 103 104 105 105 104 1 3
104 103 102 101 105 104 24 103 102 101 105 104 3 103 102 101 105 14 104 103 0 101 102 103 104 105 6 0 0 101 102 103 104 105 96 1 0 101 102 103 104 105 4 0
CD25 Ccr4 –/– (DST + α-CD40L)
101 102 103 104 105
104 103 102 101 105
103 102
WT (DST + α-CD40L)
101 102 103 104 105
101 102 103 104 105
Tolerized LN
Tolerized spleen
Tolerized spleen
105 104 103
103 102 101 105
Ccr4 –/– SPL
105 104 103 23
101 102 103 104 105
102 101
21 Foxp3
101 102 103 104 105
103 102 101
102 101
101 102 103 104 105
101 102 103 104 105
102 101
1 101 102 103 104 105
Figure 4 'Strategic' localization of YAe+ cells in the lymph nodes during tolerance. (a) Immunohistochemical analysis of YAe+ staining around HEVs and B cell follicles (stained with MECA-79) in the lymph nodes of tolerized mice (1-, 5- and 10-week). Data are representative results from '10-week tolerized' mice. Original magnification, Â100; n ¼ 4 mice per group. (b) Left, CCL17 mRNA expression in '10-week tolerized' lymph node and splenic YAe+PDCA-1+ and YAe–PDCA-1+ cells (n ¼ 4 mice per group). Right, immunofluorescent analysis of YAe+ cells expressing CCL17. Original magnification, Â250; n ¼ 2. SPL, splenic. (c) Left, CCR4 expression in '10-week tolerized' lymph node and splenic CD4+CD25+ and CD4+CD25– T cells. Right, CCR4 and Foxp3 expression in CD4+CD25– and CD4+CD25+ T cell populations from tolerized lymph nodes and spleen (n ¼ 4 mice per group). WT, wild-type; a-, anti-. (d) Immunohistochemical analysis of Foxp3+ cell distribution around HEVs and B cell follicles (stained with MECA-79) in the lymph nodes of '10-week tolerized' mice. Original magnification, Â100; n ¼ 2 mice per group. (e) Graft survival in CCR4-deficient (Ccr4–/–) and wild-type (WT) recipient mice after treatment with DST and anti-CD40L (n ¼ 4 mice per group). (f) CCR4 and Foxp3 expression on day of rejection in CD4+CD25– and CD4+CD25+ T cell populations from CCR4-deficient lymph nodes and spleens after treatment with DST plus mAb to CD40L. Data are representative results of three independent experiments. Numbers in quadrants (c,f) indicate percentage of cells in each.
105 104
Splenic naive CD4+ TEa
4,000 3,000
Blood 25% 75%
105 104 103 102 101
104 103 102 101
2,000 1.3% 1,000 0 4,000 3,000 25% 75% 2,000 1,000 0 101 102 103 104 105 SPL 101 102 103 104 105 Graft
101 102 103 104 105 105 101 102 103 104 105 104 103 Graft 102 101 101 102 103 104 105 105 104 101 102 103 104 105 103 102 101 101 102 103 104 105 105 LN CD4+CD25+ 104 103 101 102 103 104 105 102 101 101 102 103 104 105 105 LN CD4+CD25– 104 103 101 102 103 104 105 SPL Graft
103 102 101
97% 101 102 103 104 105
105 104 103 102 101
CFSE TEa
© 2006 Nature Publishing Group http://www.nature.com/natureimmunology
CD4 Sorted CD4+CD25–
% of max.
80 60 40 20 0
Foxp3 Isotype
4,000 3,000
105 104 15% 85% 103 102 101
CFSE TEa
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Tolerized YAe
Blood 5.6% 72 h 105 Blood 104 103 102 101 101 102 103 104 105 Graft 2.9% 105 72 h 104 103 102 101 101 102 103 104 105 4,000 3,000 2,000
Cultured YAe+ + CD4+ TEa cells
100 6.7% 80 60 Blood
8 [3H]TdR uptake (SI)
Splenic naive CD4+ TEa
105 104 103 102 101 101 102 103 104 105 1.1%
40 20 101 102 103 104 105 Graft 2.9% 0 100 80 60 40 20 0 101 102 103 104 105 101 102 103 104 105 100 5.5% 80 60 40 20 0 101 102 103 104 105 101 102 103 104 105 100 12.1% 80 LN 100 80 30% 60 40 20 0 101 102 103 104 105 7% SPL 101 102 103 104 105 Graft
1,000 0 4,000 3,000
2,000 1,000 0
CD4 Sorted CD4+CD25– +
105 72 h 104 103 102 101
0 Stimulators C57BL/6 BALB/c BALB/c Responders (CD4+CD25–)
% of max.
80 60 40 20 0
Foxp3 Isotype
1,000 0 101 102 103 104 105 LN 7.2% 72 h
C57BL/6 C57BL/6 C57BL/6 + + Cultured Cultured CD4+CD25+ CD4+CD25+
4,000 101 102 103 104 105 3,000 2,000
105 104 103
LN α-TGF-β
% of max.
60 40 20 0
1,000 0 101 102 103 104 105
102 101 101 102 103 104 105
101 102 103 104 105
Figure 5 Lymph node YAe+ pDCs induce the generation of Foxp3+CD4+CD25+ cells from of Foxp3–CD4+CD25– T cells. (a) Flow cytometry of CFSElabeled TCR-transgenic CD4+CD25–Foxp3– TEa T cells isolated from various organs 72 h after adoptive transfer into '10-week tolerized' mice (n ¼ 3 mice per group). (b) Foxp3 (red) immunofluorescent analysis of adoptively transferred CFSE-labeled TCR-transgenic TEa CD4+ T cells (green) in proximity with YAe+ cells (blue). Original magnification, Â250. (c) Flow cytometry of CFSE-labeled TCR-transgenic TEa CD4+ T cells cultured for 72 h with YAe+PDCA-1+ cells from various organs of '10-week tolerized' mice with and without 10 mg/ml of anti-TGF-b1,2,3 (n ¼ 3 mice per group). (d) Suppressive function of CD4+CD25+ TCR-transgenic TEa cells cultured with lymph node YAe+PDCA-1+ cells. C57BL/6 responder CD4+CD25– T cells (4 Â 104) were cultured together with irradiated T cell–depleted splenocyte samples (mouse strains, below graphs). Results are expressed as stimulation index (SI) and represent the mean of triplicate determinations ± s.e.m. Numbers in quadrants (a,c) indicate percentage of cells in each. All experiments were done three times.
Swap70–/– LN Swap70–/– Spleen
Graft survival (%)
100 80 60 40 20 0 0 20 40 60 80 Time after transplantation (d)
α-CD62L DST + α-CD40L DST + α-CD40L + α-CD62L
α-CD62L LN
α-CD62L Spleen
b 100
Graft survival (%) 80 60 40 20 0 0 10 20 30 40
129 WT (DST + α-CD40L) Swap70 –/– (DST + α-CD40L)
WT Spleen
129 WT Spleen
Time after transplantation (d)
Untreated + naive pDC
% 0.5 11 86
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Graft survival (%)
100 80 60 40 20
Untreated DST + α-CD40L DST + α-CD40L + 120G8
DST + α-CD40L DST + α-CD40L + 120G8 1.08% 0.02%
100 Graft survival (%) 80
Untreated + tolerized YAe+ pDC Untreated + naive pDC
LN Spleen
60 40 20 0 0 15 5 10 20 25 Time after transplantation (d) 30
14 85
% of max.
% 0.1
15 5 10 20 25 Time after transplantation (d)
Untreated + tolerized YAe+ pDC
% 11 45%
LN Spleen
Figure 6 Alloantigen-presenting pDC lymph node homing is necessary for Treg cell development and tolerance induction. 77 (a,b) Graft survival (left) and immunofluorescent analysis of YAe+ cells on day of rejection (right) in mice treated with mAb to % 18% 6 CD62L (+ a-CD62L; a) or Swap70–/– mice (b). Original magnification, Â250; n ¼ 4 mice per group. 129, 129/Sv. (c) Graft 11 survival in mice receiving 500 mg of pDC-specific mAb 120G8 on days 0, 1, 3 and 4 after transplantation (downward arrows; 82 n ¼ 4 mice per group). (d) Flow cytometry of YAe+ pDCs in lymph nodes of recipient mice treated with mAb 120G8 on day 9 CD4 Foxp3 after transplantation. Numbers in quadrants and beside outlined areas indicate percent cells in each. SSC, side scatter. Data are 6 naive pDCs representative of two independent experiments. (e) Graft survival of untreated mice after adoptive transfer of 2 Â 10 or YAe+PDCA-1+ cells from the lymph nodes of '10-week tolerized' mice (n ¼ 3 mice per group). (f) Flow cytometry (two independent experiments) of the CD4+CD25+Foxp3+ cell population 72 h after adoptive transfer of 2 Â 106 naive pDCs or YAe+PDCA-1+ cells from '10-week tolerized' mice. Numbers beside outlined areas and bracketed lines indicate percent cells in each area.
HEVs, adjacent to the B cell follicles (Fig. 4d), similar to that of YAe+ cells (Fig. 4a), which suggested that interactions between CCL17+ YAe+ and CCR4+CD4+ T cells in the lymph nodes of tolerized mice might influence Treg cell development. To further investigate the importance of CCR4-dependent interactions during transplantation, we transplanted cardiac allografts into CCR4-deficient mice and found that Treg cells did not develop and rejection occurred despite administration of the tolerogenic regimen (Fig. 4e,f). pDCs induce Treg cell development in lymph nodes To determine whether alloantigen-presenting pDCs mediate tolerance by inducing Treg cell development in the lymph node, we monitored expression of the transcription factor Foxp3 in T cell receptor (TCR)– transgenic CD4+CD25–Foxp3– TEa T cells. We isolated TEa T cells, which recognize the same I-Ed–I-Ab complex of peptide and MHC as the YAe mAb, labeled the cells with carboxyfluoroscein succinimidyl ester (CFSE) and transferred them into tolerized mice. At 72 h after adoptive transfer, 98% of TEa cells in the lymph nodes had proliferated, in contrast to 15% in the spleen, 25% in the allograft and 25% in blood (Fig. 5a). Monitoring of CD25 expression showed that 20–30% of the lymph node TEa cells became CD25+ and FoxP3+, whereas spleen, graft and blood TEa T cells remained CD25–. Because Foxp3+ cells also expressed CCR4 (Fig. 4c), we monitored these markers and found that only the CD25+ TEa cells had high expression of both Foxp3 and CCR4 (Fig. 5a). Those results were further confirmed by fluorescent immunohistochemistry, which showed that CFSE-labeled TEa cells expressing Foxp3 in the lymph nodes of tolerized mice were localized together in proximity to YAe+ cells (Fig. 5b). To further determine the unique function of lymph node alloantigen-presenting pDCs during Treg cell development, we cultured CD4+CD25–Foxp3– naive TEa cells with YAe+PDCA-1+ cells from lymph node, spleen, blood or allograft. YAe+ cells from each anatomic compartment were able to induce CD25 expression in CD4+ T cells (Fig. 5c). However,
only the YAe+PDCA-1+ cells isolated from tolerized lymph nodes were able to induce Foxp3 expression in CD4+CD25+ T cells, and this Foxp3 induction occurred in a transforming growth factor-b (TGF-b)–dependent way (Fig. 5c). CD4+CD25+Foxp3+ Treg cells generated from antigen-specific CD4+CD25–Foxp3–TEa cells suppressed the in vitro population expansion of CD4+CD25– responder T cells in an alloantigen-specific way (Fig. 5d), demonstrating their specific regulatory nature. pDCs in the lymph node are necessary for tolerance Published reports have demonstrated that lymph node occupancy by T cells is critical for the establishment of tolerance15,16. Interfering with normal T cell homing by CD62L blockade or deficiency of CD62L, CCL19, CCL21 or CCR2 (refs. 15,16) or deficiency of CCR4 (Fig. 4) prevented tolerization. To further demonstrate the essential function of lymph node homing and occupancy of blood alloantigenpresenting pDCs in Treg cell development and tolerance induction, we did cardiac transplantation and monitored the localization of YAe+ cells in the lymphoid tissues after administration mAb to CD62L. Treatment with mAb to CD62L abrogated tolerance within 1 week (Fig. 6a, left) and caused an almost complete absence of YAe+ cells in the lymph node (Fig. 6a, right). Because treatment with mAb to CD62L blocked lymph node homing of both alloantigen-presenting pDCs and CD4+ T cells, we used the 129/Sv EMS (H-2b) Swap70–/– mouse to further determine whether alloantigen-presenting pDC lymph node occupancy was necessary for Treg cell development. Swap-70 is involved in actin rearrangement and cell migration29,30 and is expressed in pDCs but not in CD4+ T cells (Supplementary Fig. 4 online). Steady-state lymph node homing was unimpaired in Swap70–/– mice (Supplementary Fig. 5 online), but after transplantation there was a profound reduction in the number of YAe+ cells (Fig. 6b, right) but not CD4+ T cells (Supplementary Fig. 6 online) in the lymph node. Treg cells did not develop in the lymph
% of max.
node (Supplementary Fig. 7 online) and tolerance was not induced, despite administration of the tolerizing regimen (Fig. 6a, left). As CD62L and Swap-70 deficiency also alter B cell migration, we investigated the specific function of pDCs during the induction of tolerance to vascularized allografts with a pDC-specific mAb (120G8)31. Treatment with 120G8 prevented tolerance induction in recipient mice subject to tolerogenic treatment (Fig. 6c) and caused peripheral depletion of pDCs (Fig. 6d). This provides definitive evidence that pDCs are required for the induction of tolerance to vascularized allografts. We next investigated the potential therapeutic use of toleranceinducing alloantigen-presenting pDCs by adoptive transfer of YAe+PDCA-1+ cells isolated from the lymph nodes of tolerized mice into untreated recipient mice. When untreated recipient mice received YAe+PDCA-1+ cells from the lymph nodes of tolerized donor mice, graft survival was prolonged for up to 24 d (Fig. 6e). In contrast, graft survival was not prolonged when untreated recipient mice received YAe–PDCA-1+ cells from naive donor mice, and rejection occurred 8–10 d after transplantation. We also studied the population expansion of CD4+CD25+Foxp3+ cells to further investigate the involvement of YAe+PDCA-1+ cells in Treg cell development and prolonged graft survival. In mice receiving naive control pDCs, CD4+ T cells in the lymph node and the spleen had low expression of CD25 and no expression of Foxp3 (Fig. 6f). However, when we adoptively transferred tolerized YAe+PDCA-1+ cells, we found a distinct population of CD25hiCD4+ T cells (Fig. 6f). This population expressed Foxp3 in the lymph node (45%) and spleen (18%), suggesting that adoptively transferred YAe+PDCA-1+ cells induce Treg cell population expansion and prolong graft survival. DISCUSSION Here we have reported that tolerance to vascular alloantigens is mediated at least in part by a distinct pDC subpopulation that is capable of acquiring and processing MHC class II–derived donor allopeptide in allografts; migrating to peripheral lymph node across HEVs due to their expression of a repertoire of integrins, selectins and chemokine receptors; and inducing the peripheral development of CD4+CD25+Foxp3+ Treg cells. Our findings indicate several potential therapeutic targets for pharmacological manipulation, including pDC antigen uptake and presentation, pDC migration and Treg cell development. This and other tolerizing protocols rely not only on the induction of Treg cells but also on the engagement other tolerance mechanisms, such as clonal anergy or deletion17. The tolerogenic regimen of donor-specific transfusion (DST) plus mAb to CD40L used here may contribute to pDC specialization for tolerization by ensuring proper trafficking of pDCs into the lymph node or unique interactions between pDCs and naive T cells. Indeed, pDCs activated through CD40L have reduced CD62L expression32, which may explain the localization of blood pDCs to the lymph nodes but not the spleen after treatment with mAb to CD40L. The identification of pDCs as phagocytic cells that transport foreign molecules from a donor allograft to recipient secondary lymphoid organs bears relevance to transplantation and demonstrates previously unknown functional attributes of this DC subpopulation. Although pDCs have been identified in peripheral tissue33, published work has suggested that human DCs derived from CD11c–CD4+ pDCs have poor phagocytic abilities20, indicating that pDCs only present cellassociated endogenous antigen and prime CD8+ T cells34. However, our in vitro experiments have demonstrated that freshly isolated mouse pDCs are capable of phagocytosis. That finding raises the issue of whether mouse pDCs act like human pDCs and demonstrates that pDCs also present phagocytosed membrane-associated exogenous antigen and can prime CD4+ T cells. Supporting the idea of phagocytic functions for pDCs, and consistent with our observation that the C-type lectin mannose receptor CD206 was upregulated after capture of donor allograft MHC II molecules, a C-type lectin receptor on human pDCs that functions as an antigen-capturing molecule has been identified35. Defining the mechanisms of alloantigen-presenting pDC trafficking is critical for understanding tolerance to vascular transplants, as secondary lymphoid organs have different functions in the establishment of immunological recognition of or 'ignorance to' vascularized allografts36. In vascularized transplants, alloantigens are not presented in a localized way but are distributed systemically in many secondary lymphoid organs37,38, indicating that the designation of 'draining' is dependent on the type of allograft and its anatomic location and the surgical manipulation required for its placement. During tolerization, CD62L expression was upregulated on circulating alloantigen-presenting cells and CD62L ligand expression was upregulated on HEVs, and interactions between these two molecules facilitated binding of pDCs to HEVs and extravasation of pDCs into lymph nodes. We also found that LFA-1, VLA-4, ICAM-1 and chemokine receptors were upregulated on pDCs, whereas chemokines were upregulated in HEVs, during tolerization. Expression of a(1,3)-fucosyltransferase VII, a fucosyltransferase essential for the biosynthesis of selectin ligands, can be induced by TGF-b39. Therefore, it is possible that in tolerogenic conditions, TGF-b secreted by pDCs or other cell types upregulates L-selectin ligands on HEVs, which favor extravasation of alloantigenpresenting pDCs into the lymph node and subsequent Treg cell development40. Indeed, failure to visualize alloantigen-presenting cells in the lymph nodes after administration of mAb to CD40L and in Swap-70-deficient mice correlates with a failure of tolerance induction in these mice. Thus, tolerance may rely on an integrated signaling pathway orchestrated toward extravasation of alloantigenpresenting pDCs from the blood to the lymph node. These findings indicate that treatment with a new generation of immunomodulatory drugs directed against LFA-1 and ICAM-1, which prolong allograft survival41 by inhibiting the signals necessary for antigen-dependent T cell activation42 and trafficking into inflamed allografts, may also interfere with processes such as pDC homing, which are essential for tolerance43. It is unclear why the lymph node provides the anatomic location for the tolerogenic immune response to vascular alloantigen. It is noteworthy that the spleen is the main site where antigen-specific immune responses are initiated by systemic priming in mice and humans44,45 and, in contrast to lymph nodes, provides the anatomical site where B cells differentiate into immunoglobulin-secreting plasma cells46. It has been shown that splenic DCs 'preferentially' present alloantigen during the immune response against vascular antigens47. Tolerogenic DST treatment inhibits alloreactive proliferative responses in the spleen but not in the lymph nodes after vascular cardiac transplantation48 and induces a decrease in splenic alloantibody-secreting cells49. In addition, treatment with mAb to CD40L has been reported to disrupt germinal center structure50. We noted the most alloantigenpresenting pDCs in the spleens of rejecting mice and the lymph nodes of tolerized mice. Those findings suggest that the spleen may be a dominant location for immune priming and responsiveness, whereas the lymph node may be poised to provide regulatory signals depending on many factors, such as the presence of exogenous immunosuppression or whether splenic function has been disrupted. Important variables that determine priming versus tolerance include the DC subset that presents alloantigen. It has been proposed
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that the particular DC subset that presents antigen determines whether immunity (mDCs) or tolerance (pDCs) is induced. Specifically, T cell priming has been noted when mDCs present FITC-labeled ovalbumin (OVA) protein in an inflammatory airway disease model51. In contrast, suppression of the asthmatic response occurs when the antigen is transported by pDCs. Skin-derived DCs present alloantigen and migrate through lymphatics to local draining lymph nodes, where they prime antigen-specific T cells52. Here we have reported that for a vascularized transplant, alloantigen is presented systemically by pDCs that circulate through blood. Thus, anatomical differences may determine the type of APC that enters the grafted tissue. Although pDCs had the ability to generate Treg cells in our model and expand CD4+CD25+Foxp3+ T cell populations in vitro53, pDCs may not be an exclusive tolerogenic DC subset, so other DCs may have similar functions in other settings54. We did not further investigate other DC subpopulations in our study here of the APC subpopulations involved in alloantigen presentation and mediating tolerance to vascularized allografts. The reason for that is that when studied at different time points, the only DC subpopulation that expressed the alloantigen recognized by the YAe mAb was the pDC subpopulation. We noted a very small population of YAe+ cells ( o5%) that were not pDCs or mDCs; future experiments should be directed toward elucidating the phenotype of these cells. The observation that pDCs represent more than 95% of the alloantigen-presenting cells in the allograft challenges whether conventional mDC are able to migrate into the inflamed vascularized tissue and process alloantigen. Although homing of pDCs from blood into peripheral organs has been reported25, not much is known about the migration of blood circulating mDCs into inflamed vascularized tissue. It is possible that differences in graft size and type and site of engraftment may influence the type of APC that present alloantigen in different transplant settings. As for Treg cell induction, it has been shown that Foxp3 is not induced anew in non–Treg cells during pathogen-dependent immune responses, although the induction of Foxp3 in non–Treg CD4+ T cells using systemic tolerogenic protocols was not addressed55. In transplantation models, Treg cell development has been reported in the periphery of tolerant athymic mice12, suggesting that new CD25+CD4+ T cells induced in the periphery of transplanted mice mediate tolerance to transplant antigens after tolerogenic treatment13. Supporting that hypothesis, it has been reported that peripheral development of alloantigen-specific CD4+CD25+Foxp3+ Treg cells occurs in the lymph nodes, as blocking lymph node homing by treatment with mAb to CD40L abrogates Treg cell population expansion and tolerance16. The findings presented here suggest that pDCs mediate new Treg cell induction in the periphery in a transplantation setting. We have shown that an essential pathway for achieving tolerance to vascular allografts is provided by antigen-specific CD4+CD25+Foxp3+ regulatory cells that can be induced anew in the lymph nodes of transplanted mice by pDCs with systemic tolerogenic regimens. We have provided evidence that pDCs mediate Treg cell development in vivo. We suggest that the identification of pDCs as alloantigenpresenting cells that either activate antigen-specific effector T cells in the spleen or induce the population expansion of Treg cells in the lymph nodes may help in the design of specific protocols to accomplish indefinite graft survival. METHODS
Mice. BALB/c, C57BL/6 and CBA mice 8–10 weeks of age were purchased from The Jackson Laboratory. C57BL/6 TCR-transgenic TEa mice were provided by A.Y. Rudensky (University of Washington, Seattle, Washington). C57BL/6 CCR4-deficient mice were provided by C. Hogaboam (University of Michigan, Ann Arbor, Michigan), and Swap-70-deficient 129/Sv EMS and congenic 129/ Sv EMS mice have been described29,31. All mice were housed in a specific pathogen–free facility in microisolator cages. All experiments used age- and sex-matched mice in accordance with protocols approved by the Mount Sinai Institutional Animal Care and Utilization Committee. Reagents. The YAe hybridoma was a gift from A.Y. Rudensky. The MR1 hamster anti–mouse CD40L hybridoma was a gift from R.J. Noelle (Dartmouth University, Hanover, New Hampshire). The MEL-14 rat immunoglobulin G2a (IgG2a) anti–mouse CD62L hybridoma was purchased from the American Type Culture Collection. The 120G8 rat anti-mouse hybridoma has been described31. All hybridomas were grown in culture and supernatants were purified over protein G or protein A columns (Amersham Pharmacia Biotech). Vascularized cardiac transplantation. BALB/c hearts were transplanted as fully vascularized heterotopic grafts into C57BL/6 mice as described56. BALB/c cardiac grafts were transplanted by suturing of donor aorta and donor pulmonary artery end-to-side to the C57BL/6 recipient lower abdominal aorta and inferior vena cava, respectively. Recipient mice received intravenous injections in 0.5 ml PBS at various times. For tolerance, mice were treated with DST (1 Â 107 donor splenocytes intravenously) on day –7 and 250 mg mAb to CD40L on days –7, –4, 0 and +4 (times relative to transplantation). One group received 100 mg mAb to CD40L 30 d after tolerization and mice rejected at 37–40 d. Graft function was monitored every other day by abdominal palpation. Tolerizing mice were studied at 1, 5 and 10 weeks after transplantation. Mice that had graft survival 10 weeks or more were considered 'tolerized' (called '10-week tolerized' here). Untreated control mice received hamster IgG in PBS and rejection, defined as complete cessation of a palpable beat and confirmed by direct visualization at laparotomy, occurred 1 week after transplantation. Cell preparations. Single-cell suspensions were prepared as described16. Lymph (20 ml/mouse) was collected from the cisterna chyli and was resuspended in icecold Hank's balanced-salt solution medium until further use. Mixed-lymphocyte reaction and in vitro suppression assay. A total of 2 Â 104 sorted YAe+ cells from tolerized and rejecting lymph nodes or spleens were cultured in triplicate with 5 Â 104 with TCR-transgenic TEa CD4+ T cells. For testing of the antigen-specific suppressive properties of CD4+CD25+ T cells derived from those cultures, 4 Â 104 freshly isolated responder CD4+CD25– T cells from naive C57BL/6 mice were cultured together in triplicate with 5 Â 104 irradiated (1,500 rads) BALB/c splenocyte samples that had been depleted of T cells by negative selection with Mouse pan T Dynabeads according to the manufacturer's protocol (Dynal), along with 2 Â 104 CD4+CD25+ TEa T cells that had been previously cultured for 72 h with tolerized YAe+ cells. Cells were then cultured for 3 d in 96-well plates. At 18 h before termination of culture, wells were pulsed with 1 mCi [3H]thymidine and incorporation was quantified with a scintillation counter. Results are expressed as stimulation index, determined from mean of triplicate determinations ± s.e.m. Adoptive transfer and culture conditions of TCR-transgenic CD4+ TEa cells. CD4+ T cell subsets were isolated from the spleens of TCR-transgenic TEa mice using the Mouse T-cell CD4 Subset Column Kit (R&D Systems) according to the manufacturer's protocol and were purified to 85–90% purity. Enriched CD4+ cells were stained with CyChrome-labeled antibody to CD4 (anti-CD4) and phycoerythring-indotricarbocyanine–anti-CD25, and CD4+CD25– cells were isolated by sorting on a MoFlo cell sorter (DakoCytomation) to 97–99% purity. Cells were then stained with 5 mM CFSE (Molecular Probes), and 1 Â 107 cells were adoptively transferred into transplanted C57BL/6 mice. In vitro CFSElabeled TCR-transgenic TEa CD4+ T cells were cultured together for 72 h at a ratio of 10:1 with YAe+PDCA-1+ cells from the blood, lymph node, spleen or graft from '10-week tolerized' mice in 96 well-plates with 200 ml of complete RPMI medium with or without anti-TGF-b1,2,3 (10 mg/ml; R&D Systems). Laser-capture microdissection. Lymph node frozen sections 10 mm in thickness were fixed in acetone for 1 min and were washed with PBS containing
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RNA inhibitors (200 U/ml; GenHunter). Stained sections were microdissected with a PixCell II Laser Capture Microscope (Arcturus Engineering). Microdissected cells from at least ten tissue sections were placed together on the same cap until its entire surface was covered. The transfer film cap with the cells derived by laser-capture microdissection was then inserted into a microcentrifuge tube containing 50 ml of extraction buffer for RNA isolation (Epicentre) according to the manufacturer's instructions. Flow cytometry. For YAe staining, naive untreated cells from C57BL/6 mice that lack I-Ad molecules were used as negative controls, and cells from C57BL/6 Â BALB/c F1 mice were used as positive controls. Anti–mouse CD16-32 and biotinylated mouse IgG2b isotype control (PharMingen) were used to block Fcg III/II receptors and as a YAe isotype control antibody, respectively. Fluorochrome-conjugated antibodies specific for CD4, CD25, CD11c, B220, Gr-1, CD19, CD62L, CD11b, GR1, B220 and CD11b and purified rat antibody specific for mouse a4b7 were purchased from PharMingen. Purified goat anti-mouse CCR4 was from Capralogics and was visualized by secondary staining with a phycoerythrin-conjugated monoclonal anti-goat (Jackson ImmunoResearch). Purified anti-CD62L was from PharMingen and was visualized by secondary staining with Alexa Fluor 594– conjugated goat anti-rat (Molecular Probes). FITC-conjugated rat anti–mouse DEC-205 was from Serotec. FITC-conjugated rat anti–mouse MAdCAM1 was from Southern Biotech. FITC-conjugated rat antibodies specific for mouse CD80, CD86, CD40, MHC-II, CD206, LFA-1, ICAM, VLA-2, VLA-4 and CD2 were from eBioscience. Internalization of FITC-labeled beads was confirmed with biotinylated anti-FITC (Jackson Laboratory) that recognizes the FITClabeled beads on the cell surface of cultured cells or with biotinylated mouse IgG isotype control (PharMingen), plus streptavidin-allophycocyanin (Caltag) to recognize the membrane-bound FITC-labeled beads. For intracellular analysis of Foxp3 expression, cells were stained with rat anti-Foxp3 or rat IgG2a as an isotype control, according to the manufacturer's protocol (eBioscience). Anti-TGF-b1,2,3 was from R&D Systems. An LSR II (BD Biosciences) was used for flow cytometry, and data were analyzed with FlowJo software (Tree Star). Results are expressed as a percentage of cells with staining above background, and mAbs were 'titered' at regular intervals during the course of these studies to ensure that saturating concentrations were used. In vitro phagocytic assay. Sorted splenic B cells, splenic pDCs and peritoneal macrophages (495% purity) were cultured with 1-mm FITC-labeled beads (Polysciences) in 10% FBS RPMI medium for time periods ranging from 2 h to 12 h. After that time, cells were placed on glass slides and were examined with a fluorescence microscope. Images of cell suspensions were acquired with a Leica DMRA2 fluorescence microscope (Wetzlar) and a digital Hamamatsu chargecoupled device camera for monitoring internalization of FITC-labeled beads. In addition, two-color flow cytometry was done on the resuspended cell populations to demonstrate that the FITC-labeled beads were not bound to the external cell membranes of pDCs. Real-time PCR. Total RNA was extracted with the ArrayPure Nano-scale RNA purification Kit (Epicentre) according to the manufacturer's instructions. Reverse transcription used 1 mg of RNA and the Sensiscript RT Kit (Qiagen) and random primers (Invitrogen) according to the manufacturer's protocol. Quantitative real-time PCR was done in duplicate or triplicate using 25 mg cDNA with 0.4 mM of each primer in a final reaction volume of 30 ml of 1Â SYBR Green PCR Master Mix (Applied Biosystems). PCR cycling conditions were as follows: 50 1C for 2 min and 95 1C for 10 min, and then 40 cycles of 95 1C for 15 s followed by 60 1C for 1 min. Relative expression was calculated as 2(C T ubiquitin – C T gene) (where CT is cycling threshold; ABI PRISM 7700 User Bulletin 2) with ubiquitin RNA as the endogenous control. All samples were run in triplicate. Immunofluorescence microscopy. Hamster anti–mouse CD11c (HL3), purified rat anti–mouse B220, purified rat anti–mouse Ly6G and purified rat anti– mouse CD19 were purchased from PharMingen. Phycoerythrin-conjugated rat anti–mouse PDCA and purified rat anti–mouse PDCA were purchased from Miltenyi. Purified rat anti–mouse TARC-CCL17 was purchased from R&D Systems. Indocarbocyanine-conjugated goat anti-hamster IgG and indocarbocyanine-conjugated goat anti-rat IgG were purchased from Jackson ImmunoResearch. Phycoerythrin-conjugated rat ant–mouse Foxp3 was purchased from eBioscience. Biotin-labeled YAe antibody was developed with streptavidin–horseradish peroxidase, biotinyl tyramide (NEN; PerkinElmer) and streptavidin-carbocyanine (Jackson ImmunoResearch). All slides were mounted with Vectashield (Vector Laboratories) to preserve fluorescence. Images were acquired with a Leica DMRA2 fluorescence microscope (Wetzlar) and a digital Hamamatsu charge-coupled device camera. Separate green, red and blue images were collected and analyzed with Openlab software (Improvision). Final image processing was done with Volocity software (Improvision). For imaging of CD62L ligands on HEVs, chimeric CD62L fused to human m-chain (a gift from J.B. Lowe, Case Western Reserve University, Cleveland, Ohio) followed by FITC–anti-human IgM (Jackson ImmunoResearch) was used. The fluorescence intensity of HEVs was measured with Slidebook 4.1 Digital Software (Intelligent Imaging Innovations) with masking of at least four representative areas for each section to obtain the mean fluorescence intensity over 10 mm2. The z-projection images of 8-mm sections were analyzed at maximum fluorescence intensities every 0.5 mm. In vitro HEV-binding assay. A modified Stamper-Woodruff assay was done24. Frozen sections 8 mm in thickness were overlaid with tolerized and rejecting blood YAe+ pDCs (1 Â 104 in 100 ml) at a ratio of 1:1, labeled with PKH red (2.5 mM) and PKH green (2.5 mM), respectively (Sigma). Sections were incubated at 4 1C for 45 min with horizontal rotation at 60 r.p.m. Slides were then washed in cold PBS and then were fixed for 10 min in 3% paraformaldehyde on ice. Samples were stained with rat anti–mouse peripheral node addressin followed by FITC–goat anti-rat for HEV localization and were examined by fluorescent microscopy. The adhesion assay used ten lymph node sections per slide with triplicate slides. Results are expressed as number of cells bound to HEVs ± s.e.m. Statistics. For graft survival, one-way analysis of variance (ANOVA) was done. For chemokine receptor expression, one-way ANOVA and the Dunnett test were done to examine individual differences versus various controls. For binding assays, one-way ANOVA and the Dunnett test were done. For total cell percentages, one-way ANOVA was done.
Note: Supplementary information is available on the Nature Immunology website.
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ACKNOWLEDGMENTS We acknowledge the technical contributions of J. Llodra, H. Nikolayevskiy and S. Freeman, and discussions with M. Merad. Supported by National Institutes of Health (R01 AI41428, AI44929 and AI62765 to J.S.B.). COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests.
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12. Karim, M., Kingsley, C.I., Bushell, A.R., Sawitzki, B.S. & Wood, K.J. Alloantigeninduced CD25+CD4+ regulatory T cells can develop in vivo from CD25–CD4+ precursors in a thymus-independent process. J. Immunol. 172, 923–928 (2004). 13. Cobbold, S.P. et al. Induction of Foxp3+ regulatory T cells in the periphery of T cell receptor transgenic mice tolerized to transplants. J. Immunol. 172, 6003–6010 (2004). 14. Abe, M., Wang, Z., de Creus, A. & Thomson, A.W. Plasmacytoid dendritic cell precursors induce allogeneic T-cell hyporesponsiveness and prolong heart graft survival. Am. J. Transplant. 5, 1808–1819 (2005). 15. Bai, Y. et al. L-selectin-dependent lymphoid occupancy is required to induce alloantigen-specific tolerance. J. Immunol. 168, 1579–1589 (2002). 16. Ochando, J.C. et al. Lymph node occupancy is required for the peripheral development of alloantigen-specific Foxp3+ regulatory T cells. J. Immunol. 174, 6993–7005 (2005). 17. Quezada, S.A. et al. Analysis of the underlying cellular mechanisms of anti-CD154induced graft tolerance: the interplay of clonal anergy and immune regulation. J. Immunol. 175, 771–779 (2005). 18. Murphy, D.B. et al. A novel MHC class II epitope expressed in thymic medulla but not cortex. Nature 338, 765–768 (1989). 19. Nakano, H., Yanagita, M. & Gunn, M.D. CD11c+B220+Gr-1+ cells in mouse lymph nodes and spleen display characteristics of plasmacytoid dendritic cells. J. Exp. Med. 194, 1171–1178 (2001). 20. Grouard, G. et al. The enigmatic plasmacytoid T cells develop into dendritic cells with interleukin (IL)-3 and CD40-ligand. J. Exp. Med. 185, 1101–1111 (1997). 21. Pachynski, R.K., Wu, S.W., Gunn, M.D. & Erle, D.J. Secondary lymphoid-tissue chemokine (SLC) stimulates integrin a4b7-mediated adhesion of lymphocytes to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) under flow. J. Immunol. 161, 952–956 (1998). 22. Rosen, S.D. Endothelial ligands for L-selectin: from lymphocyte recirculation to allograft rejection. Am. J. Pathol. 155, 1013–1020 (1999). 23. Berg, E.L., McEvoy, L.M., Berlin, C., Bargatze, R.F. & Butcher, E.C. L-selectinmediated lymphocyte rolling on MAdCAM-1. Nature 366, 695–698 (1993). 24. Stamper, H.B., Jr & Woodruff, J.J. Lymphocyte homing into lymph nodes: in vitro demonstration of the selective affinity of recirculating lymphocytes for high-endothelial venules. J. Exp. Med. 144, 828–833 (1976). 25. Yoneyama, H. et al. Evidence for recruitment of plasmacytoid dendritic cell precursors to inflamed lymph nodes through high endothelial venules. Int. Immunol. 16, 915–928 (2004). 26. Bajenoff, M., Granjeaud, S. & Guerder, S. The strategy of T cell antigen-presenting cell encounter in antigen-draining lymph nodes revealed by imaging of initial T cell activation. J. Exp. Med. 198, 715–724 (2003). 27. Alferink, J. et al. Compartmentalized production of CCL17 in vivo: strong inducibility in peripheral dendritic cells contrasts selective absence from the spleen. J. Exp. Med. 197, 585–599 (2003). 28. Iellem, A. et al. Unique chemotactic response profile and specific expression of chemokine receptors CCR4 and CCR8 by CD4+CD25+ regulatory T cells. J. Exp. Med. 194, 847–853 (2001). 29. Shinohara, M. et al. SWAP-70 is a guanine-nucleotide-exchange factor that mediates signalling of membrane ruffling. Nature 416, 759–763 (2002). 30. Sivalenka, R.R. & Jessberger, R. SWAP-70 regulates c-kit-induced mast cell activation, cell-cell adhesion, and migration. Mol. Cell. Biol. 24, 10277–10288 (2004). 31. Asselin-Paturel, C., Brizard, G., Pin, J.J., Briere, F. & Trinchieri, G. Mouse strain differences in plasmacytoid dendritic cell frequency and function revealed by a novel monoclonal antibody. J. Immunol. 171, 6466–6477 (2003). 32. Cella, M., Facchetti, F., Lanzavecchia, A. & Colonna, M. Plasmacytoid dendritic cells activated by influenza virus and CD40L drive a potent TH1 polarization. Nat. Immunol. 1, 305–310 (2000). 33. Jahnsen, F.L. et al. Experimentally induced recruitment of plasmacytoid (CD123high) dendritic cells in human nasal allergy. J. Immunol. 165, 4062–4068 (2000). 34. Salio, M., Palmowski, M.J., Atzberger, A., Hermans, I.F. & Cerundolo, V. CpG-matured murine plasmacytoid dendritic cells are capable of in vivo priming of functional CD8 T cell responses to endogenous but not exogenous antigens. J. Exp. Med. 199, 567–579 (2004). 35. Dzionek, A. et al. BDCA-2, a novel plasmacytoid dendritic cell-specific type II C-type lectin, mediates antigen capture and is a potent inhibitor of interferon a/b induction. J. Exp. Med. 194, 1823–1834 (2001). 36. Lakkis, F.G., Arakelov, A., Konieczny, B.T. & Inoue, Y. Immunologic 'ignorance' of vascularized organ transplants in the absence of secondary lymphoid tissue. Nat. Med. 6, 686–688 (2000). 37. Chen, Y., Demir, Y., Valujskikh, A. & Heeger, P.S. The male minor transplantation antigen preferentially activates recipient CD4+ T cells through the indirect presentation pathway in vivo. J. Immunol. 171, 6510–6518 (2003). 38. Reed, A.J. et al. Alloreactive CD4 T cell activation in vivo: an autonomous function of the indirect pathway of alloantigen presentation. J. Immunol. 171, 6502–6509 (2003). 39. Wagers, A.J. & Kansas, G.S. Potent induction of a(1,3)-fucosyltransferase VII in activated CD4+ T cells by TGF-b1 through a p38 mitogen-activated protein kinasedependent pathway. J. Immunol. 165, 5011–5016 (2000). 40. Chen, W. et al. Conversion of peripheral CD4+CD25– naive T cells to CD4+CD25+ regulatory T cells by TGF-b induction of transcription factor Foxp3. J. Exp. Med. 198, 1875–1886 (2003). 41. Corbascio, M. et al. CTLA4Ig combined with anti-LFA-1 prolongs cardiac allograft survival indefinitely. Transpl. Immunol. 10, 55–61 (2002). 42. Kandula, S. & Abraham, C. LFA-1 on CD4+ T cells is required for optimal antigendependent activation in vivo. J. Immunol. 173, 4443–4451 (2004). 43. Brandt, M., Steinmann, J., Steinhoff, G. & Haverich, A. Treatment with monoclonal antibodies to ICAM-1 and LFA-1 in rat heart allograft rejection. Transpl. Int. 10, 141–144 (1997). 44. Liu, Y.J., Oldfield, S. & MacLennan, I.C. Memory B cells in T cell-dependent antibody responses colonize the splenic marginal zones. Eur. J. Immunol. 18, 355–362 (1988). 45. Ellyard, J.I. et al. Antigen-selected, immunoglobulin-secreting cells persist in human spleen and bone marrow. Blood 103, 3805–3812 (2004). 46. Shapiro-Shelef, M. & Calame, K. Regulation of plasma-cell development. Nat. Rev. Immunol. 5, 230–242 (2005). 47. Crowley, M., Inaba, K. & Steinman, R.M. Dendritic cells are the principal cells in mouse spleen bearing immunogenic fragments of foreign proteins. J. Exp. Med. 172, 383–386 (1990). 48. Josien, R., Heslan, M., Brouard, S., Soulillou, J.P. & Cuturi, M.C. Critical requirement for graft passenger leukocytes in allograft tolerance induced by donor blood transfusion. Blood 92, 4539–4544 (1998). 49. Baldwin, W.M., III, Rhoton, K. & Sanfilippo, F. IgM and IgG alloantibody production by splenocytes and deposition in rat renal allografts are decreased by donor-specific blood transfusion. Transplantation 51, 481–485 (1991). 50. Han, S. et al. Cellular interaction in germinal centers. Roles of CD40 ligand and B7–2 in established germinal centers. J. Immunol. 155, 556–567 (1995). 51. de Heer, H.J. et al. Essential role of lung plasmacytoid dendritic cells in preventing asthmatic reactions to harmless inhaled antigen. J. Exp. Med. 200, 89–98 (2004). 52. Itano, A.A. et al. Distinct dendritic cell populations sequentially present antigen to CD4 T cells and stimulate different aspects of cell-mediated immunity. Immunity 19, 47– 57 (2003). 53. Moseman, E.A. et al. Human plasmacytoid dendritic cells activated by CpG oligodeoxynucleotides induce the generation of CD4+CD25+ regulatory T cells. J. Immunol. 173, 4433–4442 (2004). 54. Kretschmer, K. et al. Inducing and expanding regulatory T cell populations by foreign antigen. Nat. Immunol. 6, 1219–1227 (2005). 55. Fontenot, J.D. et al. Regulatory T cell lineage specification by the forkhead transcription factor Foxp3. Immunity 22, 329–341 (2005). 56. Corry, R.J., Winn, H.J. & Russell, P.S. Primarily vascularized allografts of hearts in mice. The role of H-2D, H-2K, and non-H-2 antigens in rejection. Transplantation 16, 343–350 (1973).
© 2006 Nature Publishing Group http://www.nature.com/natureimmunology
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November 4, 2013 - Products
The Goodyear Tire & Rubber Co. has launched the SmartWay-verified Goodyear G572 1AD truck tire for single-axle drive and 6x2 applications.
Features of the G572 1AD include:
The G572 1AD is available in sizes 295/75R22.5 and 11R22.5, both Load Range G, and can use the popular G572 Goodyear UniCircle and precure retread products.
Go directly to this company's home page
We offer e-newsletters that deliver targeted news and information for the entire fleet industry.
The expert, Mark Betner from CITGO, will answer your questions
The expert, Brian Fletcher from Eaton, will answer your questions
Cotter Pin Designed to Prevent Brake Seizing
Mainstay Introduces Faster, Lighter, More Compact CNG System
Easy Operating Kin-Sider, Kin-Slider
Rand McNally Publishes 36th Edition of Truckers' Atlas

subset: commoncrawl


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Metro Transit on crash course with major layoffs, fare hikes
The looming government shutdown will almost certainly impact bus and light rail service across the Twin Cities. But the long-range impact of the budget mess carries even worse news for commuters.
Metro Transit will be forced to lay off 500 employees, eliminate 200 bus routes, and curtail service by 25 percent if a $109 million budget cut proposed by the GOP-controlled Legislature is enacted.
Because when it comes to commuting, the folks who got elected promising "jobs, jobs, jobs" actually meant cutting jobs, and making it harder for people with jobs to get to work.
And that's despite the fact that more people are using Metro Transit than ever.
Transit authority representatives presented the numbers to the Met Council yesterday. And they've scheduled a series of public hearings all over the Twin Cities and affected suburbs through August to help riders brace for the impact.
In April, business leaders told lawmakers in St. Paul the proposed budget cuts would cripple local economies. Gov. Mark Dayton vetoed the proposed transit cuts in May, but that doesn't mean they aren't in play now.
The full list of scheduled hearings on transit cuts is published here; the first is July 6 at Minneapolis Central Library.
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Tom White, the Nashville-based attorney representing McDonald's, told my colleague William Williams of the Nashville Post today that work should begin in 30 days on the new building. Construction should take about four months, White said.
The new McDonald's will be sited in a more pedestrian-friendly way with less asphalt separating the building from 12th Avenue South. White said there will also be more extensive landscaping than the codes require.
"Overall, it will be a great looking site when they are finshed," said Bill Herbert, zoning administrator for Metro Codes. "We are looking forward to it. It was a real process — a lot of give and take and a lot of meetings. McDonald's, quite frankly, has done a lot of work to make it comply with the code. They had to make some concessions."
Showing 1-3 of 3


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Company Overview of The Trump Hotel Collection
Company Overview
United States
Key Executives for The Trump Hotel Collection
The Trump Hotel Collection Key Developments
The Trump Hotel Collection Presents at The Americas Lodging Investment Summit 2016, Jan-26-2016 02:30 PM. Venue: JW Marriott Los Angeles, 900 W. Olympic Boulevard, Los Angeles, CA 90015, United States. Speakers: Dan Wakeling, SVP, Global Hotel Development & Acquisitions.
The Trump Hotel Collection Presents at Caribbean Hotel Investment Conference & Operations Summit (CHICOS), Nov-12-2015 04:00 PM. Venue: The Ritz-Carlton San Juan, Puerto Rico, United States. Speakers: Daniel R. Wakeling, SVP Global Hotel Development & Acquisitions.
TRUMP HOTEL COLLECTION has announced it will advance its vision for delivering exceptional resort offerings across the globe with a new ultra-luxury resort in Indonesia located in Lido in West Java. The development will be the second property in Asia for TRUMP HOTEL COLLECTION in partnership with globally-renowned MNC Group. The Lido news arrives hot on the heels of TRUMP HOTEL COLLECTION and MNC Group's official New York City announcement to develop an opulent resort and residential development in Bali, a project designed to reflect the authenticity of the local culture in a magnificent location overlooking the southwest coast. Both the Bali and the Lido developments, with the latest agreement officially signed on August 19, 2015, represent projects for MNC Group in partnership with key global players such as TRUMP HOTEL COLLECTION. TRUMP HOTEL COLLECTION will be involved with phase one of the 700-hectare Lido development including a six-star luxury resort, 18-hole signature Ernie Els championship golf course, elite Lifestyle Country Club, wellness destination Spa as well as a high-end residential offering including luxury villas and condominiums. Set within the lush tropical forests of the mountains of West Java, approximately 65 kilometers south of Jakarta, the ultra-luxury resort will offer residents a luxurious retreat within a short distance, yet a world away from the hustle and bustle of the city. TRUMP HOTEL COLLECTION plans to revive the allure of the fabled destination by offering a retreat with elevated services for luxury-minded guests, establishing Lido as the preferred getaway in West Java. The golf course in Lido will be Trump's first in Asia as well as Ernie Els' first in Indonesia, and will showcase the Trump brand's impeccable standard of excellence by offering a captivating golf experience that features facilities, 24/7 lifestyle concierge at the Lifestyle Country Club, uncompromising personal service and attention to detail. As the foremost golf course developer in the world, Trump delivers the finest portfolio of courses, set against the most spectacular landscapes, presenting new levels of challenge. Aligned with other Trump Golf clubs across the globe, the ultra-exclusive Lido destination will offer unprecedented lifetime membership opportunities to a select few, providing unmatched access to experiences only limited by one's imagination. The new Bogor-Sukabumi toll road, developed by MNC Group, will provide direct access to Lido Lakes, reducing the drive time from Jakarta to 60 minutes. Lido is located in Bogor city in West Java and is within 45 minutes driving distance to the famous protected forests of Bodogol and Cikaweni waterfalls. In addition to the Trump luxury components in the development's first phase, MNC Group will introduce Indonesia's first world-class theme park in partnership with a global leading design and production firm based in Los Angeles.
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text:
Gavin and Katie's TEN-HOUR bender: Rugby star Henson holds Price's hand during marathon all-night date
Katie Price mixed business with pleasure after going on a marathon drinking session with Gavin Henson, following a photo-shoot with him.
The glamour model went all out on a date with the rugby ace and the pair were apparently knocking back the drinks for 10 hours.
Katie, 34, and Gavin, 30, were seen heading into Balans, in Soho, central London, at 1am holding hands and being very flirty with one another.
Party starters: Katie Price and Gavin Henson seen hand in hand going in to Balans restaurant and bar at 1am, where they drank for 10 hours
The pair are both well known for their partying and they lived up to their reputations as they emerged from the Soho restaurant and bar at 11am.
Katie was dressed smart-casual in a pair of True Religion jeans with a beige jumper and eye catching leopard print shoes.
Hunky Gavin was also laid back in a blue V-neck jumper which he wore with jeans and shoes.
Walk of shame: Katie and Gavin left Balans via the back door after having a marathon drinking session
Walk this way: Gavin and Katie were very comfortable in each others company and were flirting all night
One onlooker said the pair didn't even bother to order any food but spent the time necking drinks and chatting.
A source told The People newspaper: 'They arrived at 1am and immediately ordered a round of cocktails. It became apparent that there was a budding romance happening.
'They both looked like they fancied one another and were not attempting to make any secret of it in front of other diners.
'Their date went on for hours. They were downing shots and getting very drunk indeed.'
Letting her hair down: Katie and Gavin ordered a lot of drinks including several cocktails
The tanning club: Gavin arrived looking as orange as ever as he prepared for his big date
Once they decided to call it a day the couple attempted to sneak out the back door of the restaurant where a worse for wear looking Katie was seen struggling to walk in her heels.
An eye-witness told Heat magazine: 'They didn't eat anything, just had drinks. They came in with one of his friends to start off with and he left about half way through, leaving them on their own.'
It is believed the pair met at Mahiki's Coconut party, where lover of fake tan Gavin had been taking part in a photo shoot with Katie.
Dressed to thrill: Katie wore a pair of True Religion jeans with her leopard print heels
A pal said Katie, who recently split from Argentine Leandro Penna, had always been a fan of rugby players because of their chunky thighs.
'She does like a rugby boy or footballer player, she loves muscular thighs', the friend admitted.
Gavin starred in last year's season of The Bachelor, picking Carrieann Barrow as his girlfriend, although their 'relationship' didn't last longer than two months.
Katie's ex Leandro recently said his ex would rather Google herself than be intimate and even went as far to brand his former wife-to-be crazy.
'She's on the prowl!': Katie Price is said to have spent the early hours of Wednesday with Gavin Henson
Earlier on: Gavin grins with a blonde, hours later he is thought to have met up with 'the Pricey'
Out and about: Katie was seen at the Clothes Show Live on Sunday after her marathon drinking session with Gavin
He added, in a glossy magazine interview, 'I would need a psychologist in the future if I stayed with her. I just want to live a peaceful, normal life,'
The young model, who met the 34-year-old at an Oscars party in LA in 2011, seemed smitten at first, but quickly came to see Katie's life as 'strange.'
New friend Rylan Clark told heat last month, 'We'll definitely go on the pull. Katie will rise to the occasion now! We'll be going out together in the clubs, going out on the prowl.'
Gavin has two children with Voice Of An Angel singer Charlotte, who he separated from in 2010 after five years.
On the prowl: According to pals Katie has her sights set on getting a new man... and quick
Happier times: Leandro met Katie in 2011 at a post-Oscar party in LA
Split: Gavin and Charlotte separated in 2010 after having two children together
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text:
EK P360 Performance Liquid Cooling KIT review
Hitman 2016: PC graphics performance benchmark review
Guru3D Rig of the Month - April 2016
AMD Radeon Pro Duo Preview and Details
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Guru3D.com » Files » HdTach
HdTach 3.0
Posted by: Hilbert Hagedoorn on: 07/28/2008 02:37 AM [ 0 comment(s) ]
HD Tach is a low level hardware benchmark for random access read/write storage devices such as hard drives, removable drives (ZIP/JAZZ), flash devices, and RAID arrays. HD Tach uses custom device drivers and other low level Windows interfaces to bypass as many layers of software as possible and get as close to the physical performance of the device possible.
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Top Definition
Top quality dank coverd with white crystals and magenta (purple) fibers
Incredibley good
Incredibley good
That magenta dank got me stonned in like 2 hits outta my bong
by kartoon January 16, 2005
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GOLDEN VALLEY, Minn. - Kat Perkins debuted her new single on iTunes on Tuesday.
"Fearless" is already climbing up the rock charts. It's received dozens of 5-star reviews.
Kat finished fourth on "The Voice" last season.
The rocking nanny is now back in Minneapolis and making music.
Read or Share this story: http://www.kare11.com/story/entertainment/music/2014/08/19/the-voice-star-kat-perkins-debuts-new-single/14316173/


text:
Recovering from Moral Injury after War
Excerpt from the book Soul Repair: Recovering from Moral Injury after War discusses how moral injury is not Post Traumatic Stress Disorder (PTSD), but often overlaps with it.
When such dialogues occur, they mine a deeper level of moral questioning in which language moves from being descriptive to being deeply transformative. Speaking about moral injury places morality, justice, and human dignity at the center of public attention and exposes a collective amnesia about war, its victims and its aftermath. To listen to the witnesses of veterans that struggle with moral injury shifts the conversation from the individual issues of some soldiers after the war to larger questions about war.
The veterans that speak about their moral injury and the cost of the latest wars on US soldiers do so with a deep concern for the people they fought against. They are not asking for public interest in U.S. veterans that would disregard the realities and the humanity of Iraqi and Afghani people.
The veterans in this book are very clear about the complexity of their moral positions and do not indulge in the easy and self-indulgent stance of slipping into a victim role. They remain fiercely committed to avoiding denial and forgetting. They seek to remember what they did personally to harm others and to take responsibility for how they violated their own moral conscience as their route to recovery.
Engaging in collective conversations about moral injury and war can help us all to strengthen the moral fabric of society and the connections that tie us to the rest of the world. Our collective engagement with moral injury will teach us more about the impact of our actions and choices on each other, enable us to see the world from other perspectives, and chart pathways for our future. If we achieve deeper and more open ways to grasp the complexities of human relationships, we will be able to understand power and the vast and complex ways we can misuse our power.
We cannot turn the clock back to pre-war times; we cannot bring back the dead, or undo atrocities and environmental destruction. We must resist offering hasty forgiveness to absolve ourselves and others. If we can take the time, instead, to listen to what veterans say to us, to befriend them as we journey together toward a different world, we can together, discover how deep transformation leads us toward the moral conscience that is the deepest most important dimension of our shared humanity. In doing so, we can come to understand the honor and integrity of military service and the importance of the moral criteria for war, which the military itself teaches, and what it would require of every one of us to send any one of us to war.
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Here's a quick introduction to five of the top 10 companies shortlisted as part of the Qualcomm® Design in India Challenge 2015-16.
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The idea of creating plug-and-play or DIY smart home automation systems came to Shatruddha Singh Kushwaha and Ravi Prakash Lakotia, says Kunal Bhatia one of the three Co-Founders of Switch. The trio got together late last year and early this year to create easy intelligent systems that can take homes off the energy grid. Graduates […]
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Technology advances are making amazing things possible: we finally have a chance to solve the problems that have long plagued humanity such as hunger, disease, energy, and education. The same technologies that make this abundance possible are however setting the stage for the elimination of the majority of human jobs. Within two decades, we will […]
22 Dec 2014
ASIMOV is the one-stop shop for all industry robotics and automation needs. They provide engineering product solutions and consultancy in areas like robotic simulation and control, machine-vision, training, virtual reality and navigation applications. ASIMOV Robotics private limited is a Kochi-based startup which was started in April 2012. It was started by Jayakrishnan T (Founder and […]
23 Oct 2014
Consumption of machine tool (CNC machines and robotic systems) has been growing significantly with its increasing application in electronic and automobile sectors, machining of metals and plastic parts among others. According to an industry research, India has spent Rs.11265 crore on machine tool with local production contributing merely 35% of the overall pie. Most of […]
06 Mar 2014


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Brown will be No. 1 candidate for several jobs
Originally Published: May 27, 2003By Marc Stein | ESPN.com
If the Nets can't do it in the Finals and give us some June suspense for the first time all century, don't worry.
There's always the coaches.
The forthcoming offseason is going to be a wild one, with a crush of marquee free agents bound for the open market and few dollars to go around, but the chaos didn't want to wait.
Larry Brown's resignation from the Philadelphia 76ers on Monday created the league's fifth coaching opening, except that there are really eight openings if you ...
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Jefferies & Co analyst Douglas Lane remains skeptical about the company's ability to grow topline."While the fourth quarter may re-establish some credibility, the topline remains under pressure across its businesses," Lane, who has an "underperform" rating on the stock, wrote in a note to clients.
guys give it up and stop making things upretails don't move this stock anyways
BTH priced target of $38 by Jefferies this morning.BTH closed friday at $47.Sell it!
you have a link?
the BTH CEO blatantly said the exact same thing as the analyst in the earnings press release:""We recognize that this strong performance is unlikely to be repeated at fiscal year 2010 levels in the near term."BTH will give up half its gains Monday


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"PRAIRIE CITY - GATEWAY TO THE STRAWBERRY MOUNTAINS" Prairie City by mtncorg
Prairie City Travel Guide: 4 reviews and 7 photos
Hidden away from the Portland and Willamette Valley metroplexes, the small, compact Strawberry Wilderness Area is located just south of Prairie City and east of John Day, both small communities accessible from US 26. The wilderness area is not very large, comprising a long narrow protective zone along the crest of the range. Forests, alpine lakes and far-reaching views greet the few visitors who do manage to venture out this far. Most of the Area's visitors will be found fishing in the Upper or Lower Strawberry Lakes - the Lower Lake being only 1.5 miles in from the road end just south of Prairie City. The town itself is oriented towards the ranches and logging industry of the region. There are a few hot springs in the area. Careful investigation and you can discover where they are for a nice soak after scurrying about on the peaks. Nearby John Day features an museum devoted to an early Chinese settler who ran his own 19th century pharmacy, among other occupations.
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This trail runs for 14 miles and traverse the wilderness area, which is itself about 18 miles long and 2-5 miles wide,... more travel advice
Trails continue from the lakes up to the highest peak in the range, Strawberry Peak. The top is 9044 feet high and you... more travel advice
The road from Prairie City runs due south for about 10 miles. The last couple of miles become a little bit rough, but... more travel advice
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Average Microbiologist Analyst Salaries in Edison, NJ
|In USD as of May 3, 2016
microbiologist analyst
Average Microbiologist Analyst Salaries
The average salary for microbiologist analyst jobs in Edison, NJ is $82,000. Average microbiologist analyst salaries can vary greatly due to company, location, industry, experience and benefits.
This salary was calculated using the average salary for all jobs with the term "microbiologist analyst" anywhere in the job listing.
Average Salary of Jobs with Related Titles
|In USD as of May 3, 2016
Microbiologist Analyst Salary Information
This free salary calculator uses salary data from millions of job listings indexed by Simply Hired's job search engine. The microbiologist analyst salary comparisons feature allows job seekers to get the information required to make important career decisions and negotiate salary ranges.
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'L'Homme' Eau de Toilette (1.3 oz.)
L'Homme Yves Saint Laurent is the force of attraction of a man with style and sensuality. He has the inner ability to attract and inspire people thanks to his charisma. His universe is the city, where he can express his creative energy and live by his own rules. From the ... Learn More


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The Grouped Parishes of Llanasa and Ffynnongroyw in Flintshire, North Wales.
A new year. Fresh beginnings, looking forward with hope.
Just before Christmas we finally received the good news we had all been waiting for. We will be getting a new vicar.
The Rev Dot Gosling will be joining us just after Easter. Dot has two daughters and enjoys singing and plays guitar and keyboard.
Dot was ordained in Chester in 2010, and has been working as a pioneer priest for the Dyffryn Clwyd Mission Area for the last year and a half, working across all the parishes in the Mission Area with a special responsibility for engagement with families and young people.
Dot's life experiences have been varied, she has been a caterer, night nurse, parish administrator, night shelter coordinator, youth worker, mentor, family support worker, Diocesan Youth Officer, and senior lecturer and chaplain at Chester University.
With her experience with mission areas, and her energy and passion for life, we are sure that Dot will ably lead us into this new era of the church.
We would like to thank everyone working to keep the Parishes running through this interregnum, and look forward to Dot taking up her Post in April.
Ann and Sharon (Wardens of Llanasa Parish) – David and John (Wardens of Ffynnongroyw Parish)
St Winifred's Church, Gronant
"For where two or three are gathered in my name, there am I among them" Matthew 18:20
We are sure that many of you will have already heard that the Parochial Church Council (PCC) have been holding discussions regarding St Winifred's Church building.
Gronant is in the parish of Llanasa, and in years gone by worshippers from the other villages of the parish would travel, probably on foot, to worship at the Parish Church in Llanasa. Worship in Gronant started in a small way, meeting at Anwyl Cottage, Lower Gronant. Alternative accommodation for the services was required as the congregation grew. The first service in the present Church building was held on 28th November 1948, and the building was dedicated to St Winifred on 28th January 1949. It was hoped that the building would accommodate worship for the next 20 years. This hope was surpassed, worship having been held in the building now for almost 70 years. Unfortunately, we have now reached a point where the fabric of the building is in need of a great deal of work at huge cost, and the difficult decision to close the building has been taken by the PCC.
However, St Winifred's Church will continue to worship in Gronant.
Looking to the future, the PCC has agreed that they apply to the County Council to look at Stage 1 – Expression of Interest, regarding Community Asset Transfer of Gronant Youth and Community Centre building in Pentre Lane. Stage 1 will enable the PCC to visit the building to assess its suitability.
For the present, St Winifred's services will be held at the Rural North Flintshire Family Centre, Gronant, on Sundays at 4 pm. The Midweek Services will return to Llanasa on Wednesdays, as in Spring/Summer of previous years.
The final service to be held at St Winifred's Church building will be a Service of Thanksgiving for the years of Worship at the Building, on 28th February at 4 pm.
Our usual joint service will be in Llanasa on Mothering Sunday, 6th March at 11 am.
The first service to be held at the Family Centre will be Sunday 13th March at 4 pm.
Sharon Ellmore and Ann Johnson, Church Wardens, Llanasa Parish
The First Sunday of each month is normally a Joint Service.
9.30 am – Joint Holy Eucharist Service at All Saints, Ffynnongroyw will be taken by Canon John Glover.
11:00 am – Holy Eucharist and Imposition of Ashes St Winifred's Gronant will be taken by Canon Clive Southerton.
4 pm – St Winifred's Gronant, Service of Thanksgiving for the years of Worship at the building.
11 am – Mothering Sunday. Joint Holy Eucharist Service at Llanasa.