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- Nature Immunology 11(4):273 (2010)
As the fallout from 'Climategate' rumbles on, the scientific community needs to stick to what it is best at: the gathering, interpretation and dissemination of accurate data. - The biological century: coming to terms with risk in the life sciences
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Although the life sciences promise huge benefits, the possibility of doing harm from deliberate misuse of knowledge is an increasingly worrisome issue. Discussion and mitigation of these risks by life scientists must be encouraged. - Do NK cells always need a license to kill?
- Nature Immunology 11(4):279-280 (2010)
The ability of natural killer cells to eliminate abnormal cells has been shown to be enhanced by triggering of certain inhibitory receptors during their maturation. New data show that sometimes the opposite can happen. - Quieting T cells with Slfn2
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- Nature Immunology 11(4):282-284 (2010)
Neutrophils and other cells secrete the pentraxin PTX3, which promotes innate immunity by binding to pathogens and activating complement. PTX3 can also limit neutrophil recruitment by inhibiting rolling on P-selectin in inflamed venules. - Signal 0 for guided priming of CTLs: NKT cells do it too
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The cross-priming of antigen-specific CD8+ T cells requires help. The mechanism by which natural killer T cells provide such help is now characterized. - Research Highlights
- Nature Immunology 11(4):287 (2010)
- Sensing the outside world: TSLP regulates barrier immunity
- Nature Immunology 11(4):289-293 (2010)
Nature Immunology | Review Sensing the outside world: TSLP regulates barrier immunity * Steven F Ziegler1 Search for this author in: * NPG journals * PubMed * Google Scholar * David Artis2 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorsJournal name:Nature ImmunologyVolume:11,Pages:289–293Year published:(2010)DOI:doi:10.1038/ni.1852Published online19 March 2010 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Thymic stromal lymphopoietin (TSLP) is an interleukin 7 (IL-7)-like cytokine originally characterized by its ability to promote the activation of B cells and dendritic cells (DCs). Subsequent studies have shown that TSLP promotes T helper type 2 (TH2) cell responses associated with immunity to some helminth parasites and the pathogenesis of many inflammatory diseases, including atopic dermatitis and asthma. This review will focus on recent findings indicating that in addition to influencing B cell and DC function, TSLP can promote TH2 cytokine–associated inflammation by directly promoting the effector functions of CD4+ TH2 cells, basophils and other granulocyte populations while simultaneously limiting the expression of DC-derived proinflammatory cytokines and promoting regulatory T cell responses in peripheral tissues. View full text Figures at a glance * Figure 1: Pantheon of TSLP-responsive cells. NKT, natural killer T. * Figure 2: TSLP regulates TH2 cytokine responses after helminth infection and exposure to allergens. () After exposure to helminth parasites, infection and/or disruption of colonic epithelium elicits responses to Toll-like receptor (TLR) and Nod-like receptor agonists that are able to induce IL-12p40 expression and subsequent TH1 cytokine responses. TSLP, which is also induced during infection, acts to suppress p40 expression by DCs, which inhibits the development of TH1 responses, while also inducing OX40L to promote TH2 responses. TSLP may also act to recruit IL-4-producing basophils to draining lymph nodes (LN) that act cooperatively with DCs to prime TH2 cytokine responses. () After allergen exposure, proteases present in the allergen complex activate PAR-2, which in turn induces TSLP expression. TSLP induces resident DCs to upregulate OX40L and to produce chemokines (CCL17 and CCL22) to promote TH2 responses. TSLP acts on resident mast cells and natural killer T cells to increase cytokine production, which further promotes the TH2 inflammatory cascade. MHCII, major his! tocompatibility complex class II. Author information * Abstract * Author information Affiliations * Immunology Program, Benaroya Research Institute, Seattle, Washington, USA. * Steven F Ziegler * Department of Pathobiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA. * David Artis Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Steven F Ziegler (sziegler@benaroyaresearch.org) or * David Artis (dartis@vet.upenn.edu) Additional data - An intravascular immune response to Borrelia burgdorferi involves Kupffer cells and iNKT cells
Lee WY Moriarty TJ Wong CH Zhou H Strieter RM van Rooijen N Chaconas G Kubes P - Nature Immunology 11(4):295-302 (2010)
Nature Immunology | Article An intravascular immune response to Borrelia burgdorferi involves Kupffer cells and iNKT cells * Woo-Yong Lee1 Search for this author in: * NPG journals * PubMed * Google Scholar * Tara J Moriarty2 Search for this author in: * NPG journals * PubMed * Google Scholar * Connie H Y Wong1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hong Zhou1 Search for this author in: * NPG journals * PubMed * Google Scholar * Robert M Strieter4 Search for this author in: * NPG journals * PubMed * Google Scholar * Nico van Rooijen5 Search for this author in: * NPG journals * PubMed * Google Scholar * George Chaconas2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Paul Kubes1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume:11,Pages:295–302Year published:(2010)DOI:doi:10.1038/ni.1855Received05 November 2009Accepted12 February 2010Published online14 March 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Here we investigate the dynamics of the hepatic intravascular immune response to a pathogen relevant to invariant natural killer T cells (iNKT cells). Immobilized Kupffer cells with highly ramified extended processes into multiple sinusoids could effectively capture blood-borne, disseminating Borrelia burgdorferi, creating a highly efficient surveillance and filtering system. After ingesting B. burgdorferi, Kupffer cells induced chemokine receptor CXCR3–dependent clustering of iNKT cells. Kupffer cells and iNKT cells formed stable contacts via the antigen-presenting molecule CD1d, which led to iNKT cell activation. An absence of iNKT cells caused B. burgdorferi to leave the blood and enter the joints more effectively. B. burgdorferi that escaped Kupffer cells entered the liver parenchyma and survived despite Ito cell responses. Kupffer cell–iNKT cell interactions induced a key intravascular immune response that diminished the dissemination of B. burgdorferi. View full text Figures at a glance * Figure 1: Distribution of iNKT cells and Kupffer cells in the hepatic microvasculature. Spinning-disk confocal intravital microscopy of the vasculature of Cxcr6gfp/+ and BALB/c mouse livers. (,) CXCR6+ cells in the liver of Cxcr6gfp/+ mice. () Liver-specific Kupffer cells (red) labeled with phycoerythrin-conjugated anti-F4/80 in the hepatic sinusoids of a BALB/c mouse. () GFP+ cells and F4/80+ cells in the sinusoids, post-sinusoidal venules and pre-sinusoidal venules of Cxcr6gfp/+ mouse livers (n = 7 mice) in 21 FOV (iNKT cells) or 10 FOV (Kupffer cells). Numbers in graph indicate percent iNKT cells per FOV for bars not visible; ND, not detected. () Dextran conjugated to tetramethylrhodamine (2 megadalton; red) and polychromatic microspheres (green and yellow) bound to Kupffer cells. Original magnification, ×4 ( and iNKT cells in ), ×10 (, and Kupffer cells in ) or ×20 (). Data are representative of more than three independent experiments (–,) or seven experiments (; error bars, s.e.m.). * Figure 2: Binding capacity of Kupffer cells, iNKT cells and SECs for beads and bacteria. (,) Microscopy of red Kupffer cells (arrowheads) before () and after () intravenous injection of polychromatic microspheres (arrows, ) into BALB/c mice. (,) Quantification of the binding of beads or E. coli () or B. burgdorferi () to Kupffer cells (KC), iNKT cells or SECs in Cxcr6gfp/+ mice given intravenous injection of polychromatic microspheres, or E. coli () or B. burgdorferi () expressing GFP or labeled with the red fluorescent nucleic acid stain Syto 60. () Binding of B. burgdorferi to the endothelium (arrow) as well as to Kupffer cells (arrowheads) in the hepatic sinusoids of a mouse treated as described in (differences in activity, Supplementary Video 5). () Microcopy of B. burgdorferi in the liver of a mouse treated as described in ; yellow arrowhead indicates a GFP+ spirochete in the process of emigrating, and yellow arrow indicates a spirochete that has left the vasculature. Original magnification, ×20 (,,,). P values (,), Bonferroni's multiple-comparison test. D! ata are representative of more than three independent experiments (–) or two experiments (). * Figure 3: Ingestion of B. burgdorferi by Kupffer cells and Ito cells. () Visualization of the hepatic vasculature of a Cxcr6gfp/+ mouse at 2 h after the administration of GFP-expressing B. burgdorferi (yellow dots, shown interacting with red-labeled Kupffer cells). () Association of GFP-expressing or Tomato-expressing B. burgdorferi with Kupffer cells and Ito or dendritic cells (Ito-DC), measured in intravital videos of Cxcr6gfp/+ and Cx3cr1gfp/+ mice, respectively. () A z-stack reconstruction of two-dimensional microscopy, showing attachment and ingestion of spirochetes by Kupffer cells (rotation, Supplementary Fig. 1). () Total ingested B. burgdorferi confirmed after 360° rotation of z-stack-reconstructed images. (–) CD11c−, GFP+ big stellate (Ito) cells (arrows) and relatively small CD11c+GFP+ (dendritic) cells (arrowheads) in Cx3cr1gfp/+ mouse liver. () Tomato-expressing B. burgdorferi bound by GFP+ Ito cells in Cx3cr1gfp/+ mouse liver. Arrows indicate associated spirochetes; arrowheads indicate spirochetes out of Ito cells. Vessels w! ere stained with anti-PECAM-1 (blue). (,) Ingestion of Tomato-expressing B. burgdorferi by Ito cells, assessed after z-stack reconstruction. Original magnification, ×20 (,,–). P values (,), Bonferroni's multiple-comparison test. Data are representative of two independent experiments per group with more than four FOV each (–,–; error bars (,), s.e.m.) or two experiments (–). * Figure 4: Antigen presentation by Kupffer cells and Ito cells. () Release of IFN-γ from liver-derived mixed-lymphocyte populations stimulated for 4 d with Kupffer or Ito cells isolated from BALB/c mice infected by injection of B. burgdorferi. (,) Expression of CD69 in mixed-lymphocyte populations treated as described in () and a pure iNKT cell population isolated from Cxcr6gfp/+ mice (Supplementary Methods) and cultured alone or together with Kupffer cells or Ito cells (as described in ) with or without anti-CD1d (), evaluated by flow cytometry. P values (,), Bonferroni's multiple-comparison test. Data are representative of one experiment (mean and s.e.m. of three to four wells () or pooled cells from three to four wells ()) or three independent experiments (). * Figure 5: Changes in iNKT cell activity after B. burgdorferi infection. (,) GFP+ cell tracks in vehicle-treated mice () and at 24 h after injection of B. burgdorferi into Cxcr6gfp/+ mice (). (,) Distribution of GFP+iNKT cells and red-labeled Kupffer cells at 8 h () and 24 h () after injection of GFP-expressing B. burgdorferi. Arrows indicate surviving spirochetes. () A z-stack reconstruction showing Kupffer cells (red) and iNKT cells (green) at 24 h after infection. Original magnification, ×20 (,) or ×10 (). () Enzyme-linked immunosorbent assay of IFN-γ and IL-4 in serum samples obtained from wild-type (WT) and Cd1d−/− mice before or 5, 8 or 24 h after B. burgdorferi injection. P values, Bonferroni's multiple-comparison test. Data are representative of three independent experiments with more than ten FOV () or more than two independent experiments (–) or one experiment with four to six mice per group (; error bars, s.e.m.). * Figure 6: Inhibition of iNKT cell cluster formation, average crawling velocity and stationary adhesion by pertussis toxin, anti-CXCR3 and anti-CD1d. Analysis of the hepatic microvasculature (visualized by spinning-disk confocal intravital microscopy) and formation of iNKT cell clusters (counted by intravital video) in Cxcr6gfp/+ mice pretreated with pertussis toxin (PTX), anti-CXCR3, anti-CD1d and/or α-GalCer (α-GC) before injection of B. burgdorferi. () Distribution of iNKT cells after PTX treatment, assessed 24 h after infection. () Cluster formation by B. burgdorferi at 24 h after infection. Original magnification, ×4 (,). (,) Effect of pretreatment on the average crawling velocity () and stationary arrest () of iNKT cells at 24 h after infection. () Liver iNKT cells during the first 24 h of B. burgdorferi infection. () Effect of pretreatment on the number of iNKT cells in the liver sinusoids (n = 4–6 mice per group). Original magnification, ×10 (,). P values (–), Bonferroni's multiple-comparison test. Data are representative of three experiments () or more than two independent experiments per group (–; erro! r bars, s.e.m.). * Figure 7: Role of Kupffer cells in B. burgdorferi infection. () Kupffer cells labeled with anti-F4/80 before (−CLL) and 24 h after (+CLL) intravenous injection of CLLs (administered 24 h before B. burgdorferi infection to deplete mice of Kupffer cells). Original magnification, ×10. () Spirochetes remaining in the liver 12 h and 24 h after B. burgdorferi injection in untreated mice (−CLL) and CLL-treated mice (+CLL). () Spirochetes remaining in the blood 3 d after B. burgdorferi injection in untreated, CLL-treated and splenectomized (–Spl) wild-type mice and in Cd1d−/− mice, assessed by quantitative PCR analysis of B. burgdorferi flaB. () Distribution of iNKT cell velocities at 5 h after spirochete challenge; nonfluorescent B. burgdorferi were administered to avoid possible confusion of GFP-expressing B. burgdorferi with GFP+iNKT cells in CLL-treated mice. (,) Formation of iNKT cell clusters at 24 h after B. burgdorferi infection. () Mortality of untreated, CLL-treated and splenectomized (−Spl) wild-type mice and of Cd1d�! �/− mice after B. burgdorferi infection. () Migration of GFP-expressing spirochetes (arrows) out of microvasculature (space of Dissé) in a CLL-treated mouse. Original magnification, ×20. P values Bonferroni's multiple-comparison test () or Student's t-test (,). Data are representative of three experiments (), more than two independent experiments per group (,,,), one experiment with duplicate pooled results of three mice () or one experiment with five mice per group (; error bars (,,), s.e.m.). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Physiology & Pharmacology, University of Calgary, Alberta, Canada. * Woo-Yong Lee, * Connie H Y Wong, * Hong Zhou & * Paul Kubes * Department of Biochemistry & Molecular Biology, University of Calgary, Alberta, Canada. * Tara J Moriarty & * George Chaconas * Department of Microbiology and Infectious Diseases, University of Calgary, Alberta, Canada. * George Chaconas * Department of Medicine, University of Virginia, Charlottesville, Virginia, USA. * Robert M Strieter * Department of Molecular Cell Biology, Free University, Amsterdam, The Netherlands. * Nico van Rooijen Contributions W.-Y.L. and T.J.M. designed and did most of the experiments and prepared the manuscript; C.H.Y.W. and H.Z. did some intravital and cell culture experiments; R.M.S. provided anti-CXCR3 serum and helped design anti-CXCR3 experiments; N.v.R. provided CLLs and intellectual input; G.C. provided supervision for the preparation of fluorescent B. burgdorferi and prepared the manuscript; and P.K. provided overall supervision, helped design all the experiments and prepared the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Paul Kubes (pkubes@ucalgary.ca) Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Video 1 (924K) Distribution and movement of iNKT cells in the hepatic sinusoids of Cxcr6gfp/+ mouse. Low magnification (×4) video shows the general crawling pattern of iNKT cells. Experimental conditions were as described in the legend and in more detailed Supplementary Methods. Elapsed time is shown at the top right. The time lapse was recorded at 0.3 fps and exported to video at 30 fps. * Supplementary Video 2 (940K) iNKT cell movement in the hepatic sinusoids of Cxcr6gfp/+ mouse. Intermediate magnification (×10) video shows the general crawling pattern of iNKT cells. Experimental conditions were as described in the legend and in more detailed Supplementary Methods. Elapsed time is shown at the top right. The time lapse was recorded at 0.3 fps and exported to video at 30 fps. * Supplementary Video 3 (2M) Differential behavior of iNKT cells and Kupffer cells in the hepatic sinusoids of Cxcr6gfp/+ mouse. Intermediate magnification (×10) video shows the procedure of Kupffer cell labelling and behavioral patterns of iNKT cells/Kupffer cells. Experimental conditions were as described in the Supplementary Methods. Elapsed time is shown at the top right. The time lapse was recorded at 0.3 fps and exported to video at 30 fps. * Supplementary Video 4 (2M) Distribution and behavior of iNKT cells. Intermediate magnification (×10) video shows that iNKT cells are localized widely within sinusoids, but rarely in venules. iNKT cells that attempted to move into post-sinusoidal venule were often swept away (indicated by arrow). Experimental conditions were as described in the Supplementary Methods. Elapsed time is shown at the top right. The time lapse was recorded at 0.3 fps and exported to video at 30 fps. * Supplementary Video 5 (1M) Filtering function of Kupffer cells in the hepatic sinusoids I. High magnification (×20) video shows the typical binding pattern of foreign molecules between Kupffer cells and beads. Experimental conditions were as described in the and legends and in more detailed Supplementary Methods. Elapsed time is shown at the top right. The time lapse was recorded at 0.3 fps and exported to video at 30 fps. * Supplementary Video 6 (600K) Strikingly different behavior for B. burgdorferi attached to Kupffer cells and endothelium. High magnification (×20) video shows that B. burgdorferi bound to Kupffer cells were relatively immobilized (indicated by arrow) when compared to endothelium (indicated by arrowhead; moved back and forth over 10-20 μm). Experimental conditions were as described in the Supplementary Methods. Elapsed time is shown at the top right. The time lapse was recorded at 0.3 fps and exported to video at 30 fps. * Supplementary Video 7 (568K) The cluster formed by iNKT cells 24 h after B. burgdorferi treatment I. Intermediate magnification (×10) video shows a big iNKT cluster at 24 h after B. burgdorferi treatment. Experimental conditions were as described in the legend and in more detailed Supplementary Methods. Elapsed time is shown at the top right. The time lapse was recorded at 0.3 fps and exported to video at 30 fps. * Supplementary Video 8 (772K) The cluster formed by iNKT cells 24 h after B. burgdorferi treatment II. High magnification (×20) video shows a big iNKT cluster formed on Kupffer cells 24 h after B. burgdorferi treatment and few spirochetes in the liver. Experimental conditions were as described in the and legend and in more detailed Supplementary Methods. Elapsed time is shown at the top right. The time lapse was recorded at 0.3 fps and exported to video at 30 fps. * Supplementary Video 9 (692K) Filtering function of Kupffer cells in the hepatic sinusoids II. At 12 h after B. burgdorferi treatment, high magnification (×20) video shows that Kupffer cell-depleted liver could not clear the pathogens. Experimental conditions were as described in the Supplementary Methods. Elapsed time is shown at the top right. The time lapse was recorded at 0.3 fps and exported to video at 30 fps. * Supplementary Video 10 (516K) iNKT crawling in anti-CD1d antibody treated mouse liver at 24 h after B. burgdorferi treatment. Intermediate magnification (×10) video shows that anti-CD1d did not reduce the speed with which the iNKT cells crawl in the sinusoids, while it inhibited iNKT cluster formation. A small cluster of iNKT cells is seen in the video, but iNKT cells are still crawling on Kupffer cells. Experimental conditions were as described in the . Elapsed time is shown at the top right. The time lapse was recorded at 0.2 fps and exported to video at 30 fps. PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–8, Supplementary Table 1 and Supplementary Methods Additional data - CD169+ macrophages present lipid antigens to mediate early activation of iNKT cells in lymph nodes
Barral P Polzella P Bruckbauer A van Rooijen N Besra GS Cerundolo V Batista FD - Nature Immunology 11(4):303-312 (2010)
Nature Immunology | Article CD169+ macrophages present lipid antigens to mediate early activation of iNKT cells in lymph nodes * Patricia Barral1 Search for this author in: * NPG journals * PubMed * Google Scholar * Paolo Polzella2 Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas Bruckbauer1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nico van Rooijen3 Search for this author in: * NPG journals * PubMed * Google Scholar * Gurdyal S Besra4 Search for this author in: * NPG journals * PubMed * Google Scholar * Vincenzo Cerundolo2 Search for this author in: * NPG journals * PubMed * Google Scholar * Facundo D Batista1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature ImmunologyVolume:11,Pages:303–312Year published:(2010)DOI:doi:10.1038/ni.1853Received14 December 2009Accepted12 February 2010Published online14 March 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Invariant natural killer T cells (iNKT cells) are involved in the host defense against microbial infection. Although it is known that iNKT cells recognize glycolipids presented by CD1d, how and where they encounter antigen in vivo remains unclear. Here we used multiphoton microscopy to visualize the dynamics and activation of iNKT cells in lymph nodes. After antigen administration, iNKT cells became confined in a CD1d-dependent manner in close proximity to subcapsular sinus CD169+ macrophages. These macrophages retained, internalized and presented lipid antigen and were required for iNKT cell activation, cytokine production and population expansion. Thus, CD169+ macrophages can act as true antigen-presenting cells controlling early iNKT cell activation and favoring the fast initiation of immune responses. View full text Figures at a glance * Figure 1: Distribution and dynamics of iNKT cells in lymph nodes. () Flow cytometry of iNKT cells in mediastinal lymph nodes from wild-type mice, identified as TCRβ+CD1d-tet+ cells (outlined area). () Frequency of iNKT cells among TCRβ+B220− cells in the mediastinal (Md), inguinal (In), mesenteric (Ms) and popliteal (Po) lymph nodes. () CD1d-tet staining of iNKT-S2 cells. max, maximum. () Detection of CFSE-labeled iNKT-S2 cells after transfer into CD45.1+ congenic mice, detected in lymph nodes as CFSE+CD45.2+B220− (outlined area). () Three-dimensional reconstruction of multiphoton microscopy of iNKT-S2 cells (green), CD4+ T cells (red) and B cells (blue) after adoptive transfer into a wild-type recipient, in a section of a popliteal lymph node 200 μm in thickness showing the distribution of iNKT cells (white arrowheads). Long hash marks, 50 μm. () Frequency of iNKT cells in various locations in the lymph node in . (–) Average speed (), instantaneous speed distribution (), arrest coefficient () and confinement index () of iNKT-S1 ! cells (pale red) and iNKT-S2 cells (red) adoptively transferred together with CD4+ T cells (blue) and/or B cells (gray) and imaged in intact lymph nodes by multiphoton microscopy. Each symbol represents an individual cell; small horizontal lines indicate the mean. P values, two-tailed unpaired Mann-Whitney test; NS, not significant. () Displacement of iNKT cells and CD4+ T cells plotted against the square root of time. The mean motility coefficient (M) is calculated as M = x2 / 6t, where 'x' is the slope of the linear part of the graph and 't' is time. Data are representative of six (,), three (–) or four (–) experiments (mean ± s.e.m.). * Figure 2: Early activation of lymph node iNKT cells. () Popliteal lymph nodes of wild-type mice 3 d after injection of control particles (top) or α-GalCer particles (bottom). (,) Total cell number () and cells in various populations () in lymph nodes of wild-type mice (WT; ,) and Jα18-deficient mice (NKT-KO, ) 3 d after injection of α-GalCer particles, presented relative to that of mice injected with control particles. *P < 0.05 (two-tailed t-test). () Flow cytometry of iNKT cells in mediastinal lymph nodes 3 d after intraperitoneal injection of control particles (left) or α-GalCer particles (right). Numbers adjacent to outlined areas indicate percent TCRβ+CD1d-tet+ cells. (,) Frequency of iNKT cells in lymph nodes 3 d after intraperitoneal injection () or subcutaneous footpad injection () of α-GalCer particles. Each symbol represents one lymph node; small horizontal lines indicate the mean. +, draining lymph nodes; −, nondraining lymph nodes. *P < 0.005 and **P = 0.0007 (two-tailed t-test). () Detection of CFSE-labele! d adoptively transferred iNKT cells as CFSE+CD1d-tet+TCRβ+B220−DAPI− cells 2 d after injection of α-GalCer particles (black line) or control particles (gray filled histogram). (–) Activation of iNKT cells, assessed as expression of CD25 (–) and CD69 () after intraperitoneal injection of α-GalCer particles (black lines, ,) or control particles (gray filled histograms, ,). In ,, mean fluorescent intensity (MFI) for mice injected with α-GalCer particles (gray bars) is presented relative to that of mice injected with control particles. () NK1.1 expression in lymph node (LN) iNKT cells (left), and expression of CD25 (middle) and CD69 (right) in NK1.1+ (black) and NK1.1− (red) iNKT cells in mediastinal lymph nodes 12 h after injection with α-GalCer (open profiles) or control particles (filled profiles). Numbers below outlined areas (left) indicate percent CD1d-tet+NK1.1− cells (left) or CD1d-tet+NK1.1+ cells (right). () Intracellular IFN-γ staining of mediastina! l lymph node iNKT cells 12 h after injection of α-GalCer part! icles (black line) or control particles (gray filled histogram). Data are representative of three (–), four (–,–) or two (,,) experiments. * Figure 3: Arrest of iNKT cells in the lymph nodes in response to specific antigen. Analysis of recipient mice given intravenous injection of labeled iNKT-S2 cells (red) together with CD4+ T cells (blue), which were then allowed to home for 14–16 h. (–) Average speed (), instantaneous speed distribution () and arrest coefficient () of iNKT cells, determined by multiphoton microscopy of draining mediastinal lymph nodes from wild-type recipient mice injected intraperitoneally with particles coated with α-GalCer (+ α-GalCer) or control lipids (− α-GalCer). (,) Migratory tracks () and confinement index () for iNKT cells at 16 h after injection of particulate lipids as described in –. () Displacement of iNKT cells in mice injected with particles coated with α-GalCer (red) or control lipids (black), plotted against the square root of time. (–) Average speed (), confinement index () and migratory tracks () of iNKT and CD4+ T cells from CD1d-deficient recipient mice injected intraperitoneally with particles coated with α-GalCer; mediastinal lymph nod! es were imaged at 16 h after antigen administration. In ,,,, blue indicates the dynamic parameters of CD4+ T cells at 16 h after injection of particulate α-GalCer. In ,,,,, each symbol represents one cell; small horizontal lines indicate the mean. *P < 0.01 and **P < 0.0005 (two-tailed unpaired Mann-Whitney test). Data were obtained in at least two independent experiments per condition (mean ± s.e.m. in ). * Figure 4: Arrest of iNKT cells on SCS CD169+ macrophages. (,) Multiphoton microscopy of mediastinal lymph nodes iNKT-S2 (red) and B cells (blue) transferred into wild-type recipients before intraperitoneal injection of α-GalCer particles (green). () Time-lapse images from a movie acquired 15–65 μm below the lymph node surface, showing an iNKT cell arrested in the SCS (white circle) 2 h after injection of particulate α-GalCer. Scale bar, 20 μm. () Three-dimensional reconstruction of the SCS 16 h after injection of particulate α-GalCer, including tracks of B cells and iNKT cells (from a 20-minute movie). Long hash marks, 50 μm. () Confocal microscopy of mediastinal lymph nodes 2 h after intraperitoneal injection of α-GalCer particles (green), stained with CD169 (blue, left and middle) or CD11c (blue, right). Scale bars, 300 μm (left), 5 μm (middle) and 15 μm (right). () Flow cytometry analysis of lipid uptake by CD169+ macrophages (left) or CD11chi DCs (right) in mediastinal lymph nodes 4 h after no injection (gray filled! histograms) or injection of α-GalCer particles (black lines). () Three-dimensional reconstruction of a lymph node (section 100 μm in thickness) after injection of α-GalCer particles (top) or control particles (bottom), showing iNKT cells (blue), particulate lipids (red) and CD169 cells (green). Long hash marks, 50 μm. () Frequency of iNKT cells in direct contact with CD169+ macrophages after injection of α-GalCer or control particles. () Time-lapse images from two different movies (top and bottom) showing iNKT cells (arrowheads) in contact with CD169+ macrophages (green) 6 h after injection of α-GalCer (red). Dashed curved lines indicate SCS. Scale bars, 20 μm (top) and 15 μm (bottom). Time (bottom right in ,), minutes:seconds. Data are representative of three (–) or six (–) experiments. * Figure 5: Macrophages are required for early iNKT cell activation in the lymph nodes. () Confocal microscopy of draining lymph nodes from wild-type mice 6 d after injection of CLLs (+CLL) or no CLL injection (−CLL), stained for B220 (blue) and CD169 (red). () Frequency of CD169+ macrophages (Mφ; left) and iNKT cells (right) among total mononuclear cells in lymph nodes from mice 6 d after injection of CLLs or no CLL injection. *P < 0.0001 (two-tailed t-test). () CD25 expression in lymph node iNKT cells 12 h after injection of α-GalCer particles (lines) or control particles (gray filled histogram) into wild-type mice injected with CLLs or not 6 d before removal and analysis of draining lymph nodes. MFI (right) is presented relative to that of mice injected with control particles. *P = 0.0023 (two-tailed t-test). (–) Flow cytometry () and the frequency () and number () of iNKT cells in draining lymph nodes 3 d after injection of particles into mice injected with CLLs or not as described in . Numbers adjacent to outlined areas () indicate percent TCRβ+CD1d! -tet+ cells. *P = 0.0025 () or 0.0408 (), and **P = 0.0002 () or 0.0015 (; two-tailed t-test). Each symbol (,,) represents one lymph node in an individual mice; small horizontal lines indicate the mean values. Data are pooled from two independent experiments (error bars (), s.e.m.). * Figure 6: Lymph node CD169+ macrophages present lipids to iNKT cells. () CD1d expression (black line) in CD169+CD11b+ macrophages. Gray filled histogram, isotype-matched control antibody. () CD11b+ cells before sorting (left and middle) or after sorting (right), stained with anti-CD169 (middle and right) or isotype-matched control antibody (left). Numbers above outlined areas indicate percent CD11b+ isotype-positive cells (left) or CD11b+CD169+ cells (middle and right). (,) Lipid presentation by purified CD169+ cells from incubated with α-GalCer particles (+, ), Gal(α1&→;2)α-GalCer particles (+, ) or control particles (−) before culture together with DN32.D3 cells, assessed as IL-2 production by the DN32.D3 cells. () Lipid presentation by lymph node CD169+ macrophages, CD11chi DCs and B cells incubated with α-GalCer particles (1 × 104 to 5 × 104 cells) before culture together with 5 × 104 DN32.D3 cells, assessed as in ,. () Flow cytometry of CD169+CD11b+CD11cint cells stained with anti-CD11b and anti-F4/80 before (left) and after (r! ight) purification of SCS macrophages. Numbers below outlined areas indicate percent CD11b+F4/80+ cells (left) or CD11b+F4/80− cells (right). () Lipid presentation by SCS macrophages incubated with α-GalCer particles (+) or control particles (−) before culture together with DN32.D3 cells (assessed as in ,). (,) Lipid presentation by CD169+ cells from mice injected with α-GalCer particles (+, ), Gal(α1&→;2)α-GalCer particles (+, ) or control particles (−), purified from draining lymph nodes 2 h later and cultured together with DN32.D3 cells (assessed as in ,). () Lipid presentation by CD169+ macrophages, CD11chi DCs and B cells purified from mice injected with α-GalCer particles, then cultured together with DN32.D3 cells (assessed as in ,). (,) Lipid presentation (assessed as IFN-γ production; ) and iNKT cell proliferation (detected as CFSE dilution; ) of CD169+ cells pulsed with α-GalCer particles (+) or control particles (−) before culture for 2 or 3 d tog! ether with CFSE-labeled primary iNKT cells purified from lymph! nodes. No Mφ, iNKT cells cultured without macrophages (control). *P < 0.0001 (two-tailed t-test). Data represent one of at least two independent experiments (error bars (,,–), s.e.m.). * Figure 7: Bacterial glycolipids stimulate macrophage-dependent activation of iNKT cells. (,) Total cell number () or cells of various populations () in lymph nodes of wild-type mice (,) and Jα18-deficient mice () after injection with GSL-1′ particles, presented relative to that of mice injected with control particles, *P < 0.05 (two-tailed t-test). Data are representative of four experiments (error bars, s.e.m.). (,) CD25 expression in mediastinal lymph node iNKT cells at various times after intraperitoneal injection of GSL-1′ (black lines, ) or control particles (gray filled histograms, ). MFI for mice injected with GSL-1′ (gray bars, ) is presented relative to that of mice injected with control particles. Data are representative of three experiments. (,) Expression of CD69 () and intracellular IFN-γ () in iNKT cells from mediastinal lymph nodes 12 h after intraperitoneal injection of GSL-1′ (black lines) or control particles (gray filled histograms). Data are representative of three independent experiments. () CD25 expression in lymph node iNKT cells! 12 h after injection of GSL-1′ particles into CLL-treated mice (red) or untreated mice (black). Mean fluorescent intensity (right) is presented relative to that of mice injected with particles containing control lipids; each symbol represents one mediastinal lymph node from individual mice, and small horizontal black lines indicate the mean. *P = 0.0006 (two-tailed t-test). Data are representative of two independent experiments with at least two mice per experiment. (–) Average speed (), instantaneous speed distribution (), arrest coefficient () and mean displacement () of iNKT-S1 cells (red) and CD4+ T cells (blue) transferred into wild-type recipients injected intraperitoneally with GSL-1′ particles, assessed 16 h after antigen administration. Each symbol (,) represents one cell; small horizontal lack lines indicate the mean. *P < 0.005 (two-tailed unpaired Mann-Whitney test). Data were obtained in at least two independent experiments (error bars (), s.e.m.). Author information * Abstract * Author information * Supplementary information Affiliations * Lymphocyte Interaction Laboratory, Cancer Research UK, London Research Institute, London, UK. * Patricia Barral, * Andreas Bruckbauer & * Facundo D Batista * Tumor Immunology Group, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, UK. * Paolo Polzella & * Vincenzo Cerundolo * Department of Molecular Cell Biology, Faculty of Medicine, Vrije Universiteit, VUMC, Amsterdam, The Netherlands. * Nico van Rooijen * School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK. * Gurdyal S Besra Contributions P.B. and F.D.B. design and conceived of the research in consultation with V.C. and P.P.; P.B. did all experiments; P.P. made initial observations that led to the study development and provided reagents; A.B. assisted with the multiphoton microscopy; N.v.R. provided clodronate liposomes; G.S.B. provided Gal(α1→2)α-GalCer; and P.B. and F.D.B. prepared the manuscript (in consultation with V.C. and P.P.). Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Vincenzo Cerundolo (vincenzo.cerundolo@imm.ox.ac.uk) or * Facundo D Batista (facundo.batista@cancer.org.uk) Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (3M) Dynamics of iNKT-S1 cells in resting LNs. iNKT-S1 cells (red) and CD4+T cells (blue) were adoptively transferred into WT recipients and mediastinal LNs were imaged by multi-photon microscopy. Representative tracks of cell movement are traced and coloured according to cell type. Long ticks represent 20 μm. * Supplementary Movie 2 (2M) Dynamics of iNKT-S2 cells in resting LNs. iNKT-S2 cells (red) and CD4+T cells (blue) were adoptively transferred into WT recipients and mediastinal LNs were imaged by multi-photon microscopy. Representative tracks of cell movement are traced and coloured according to cell type. Long ticks represent 20 μm. * Supplementary Movie 3 (2M) iNKT-S2 cells are arrested following stimulation with specific antigen. iNKT-S2 cells (red) were adoptively transferred into WT recipients, prior to i.p. injection with particles coated with α-GalCer (right panel). Mediastinal LNs were imaged by multi-photon microscopy 16 h after particle administration. Representative tracks of iNKT movement are traced in pale red. Long ticks represent 20 μm. * Supplementary Movie 4 (736K) iNKT-S1 cells are arrested following stimulation with specific antigen. iNKT-S1 cells (red) were adoptively transferred into WT recipients, prior to i.p. injection with particles coated with α-GalCer. Mediastinal LNs were imaged by multi-photon microscopy 16 h after particle administration. Representative tracks of iNKT movement are traced in pale red. Long ticks represent 20 μm. * Supplementary Movie 5 (2M) iNKT-S2 cells arrest in response to specific antigen is dependent on CD1d expression. iNKT-S2 cells (red) and CD4+ T cells (blue) were adoptively transferred into CD1d-KO recipients, prior to i.p. injection of particles coated with α-GalCer. Mediastinal LNs were imaged by multi-photon microscopy 16 h after particle administration. Representative tracks of cell movement are traced and coloured according to cell type. Long ticks represent 20 μm. * Supplementary Movie 6 (6M) iNKT-S2 cells are retained at the SCS in response to specific antigen. iNKT-S2 cells (red) and B cells (blue) were adoptively transferred into WT recipients, prior to injection of particles containing α-GalCer (green). Draining LNs were imaged by multi-photon microscopy 2 h after particle administration. Two different examples are shown (left panel and right panels) with XY (upper panels) and XZ (bottom panels) projections of the imaged volumes. Representative tracks of cell movement are traced and coloured according to cell type. Long ticks represent 20 μm. * Supplementary Movie 7 (11M) iNKT-S2 cells exhibit long-lasting arrests at the SCS in response to specific antigen. iNKT-S2 cells (red) and B cells (blue) were adoptively transferred into WT recipients, prior to injection of particles coated with α-GalCer (green). Draining LNs were imaged by multi-photon microscopy 16 h after particle administration. A movie of the XY projection (left) is shown together with three-dimensional representations of the imaged volume (right and middle). Representative tracks of cell movement corresponding to the full length of the movie are traced and coloured according to cell type. Long ticks represent 20 μm. * Supplementary Movie 8 (5M) iNKT-S1 cells exhibit long-lasting arrests at the SCS in response to specific antigen. iNKT-S1 cells (red) and CD4+T cells (blue) were adoptively transferred into WT recipients, prior to injection of particles coated with α-GalCer (green). Draining LNs were imaged by multi-photon microscopy 16 h after particle administration. A movie of the XY projection (left) is shown together with a three-dimensional representation (right) of the imaged volume. Representative tracks of cell movement are traced and coloured according to cell type. Long ticks represent 20 μm. * Supplementary Movie 9 (9M) iNKT cells exhibit long-lasting contacts with CD169+ SCS macrophages in response to specific antigen administration. iNKT-S2 cells (blue) were adoptively transferred into WT recipients, prior to injection of particles coated with αGalCer (red). Animals received anti-mouse CD169 antibody (green) 15 min before imaging. Draining LNs were imaged by multi-photon microscopy 6 h after particle administration. Movies with XY (left panels) and XZ (right lower panel) projections are shown together with a three-dimensional representation of the imaged volume (right upper panel). Representative tracks of cell movement are traced. Long ticks represent 20 μm. * Supplementary Movie 10 (3M) iNKT cells are retained at CD169+ SCS macrophages in response to specific antigen. iNKT-S2 cells (blue) were adoptively transferred into WT recipients, prior to injection of particles containing αGalCer (red). Animals received anti-mouse CD169 antibody (green) 15 min before imaging. Draining LNs were imaged by multi-photon microscopy 6 h after injection with particles. Long ticks represent 20 μm. * Supplementary Movie 11 (2M) iNKT-S1 cells are arrested following stimulation with particulate GSL-1'. iNKT-S1 cells (red) together with CD4+ T cells (blue) were adoptively transferred into WT recipients, prior to i.p. injection with particles coated with GSL-1'. Mediastinal LNs were imaged by multi-photon microscopy 16 h after particle administration. Representative tracks of cell movement are traced and coloured according to cell type. Long ticks represent 20 μm. PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–7 Additional data - Alternative cross-priming through CCL17-CCR4-mediated attraction of CTLs toward NKT cell–licensed DCs
Semmling V Lukacs-Kornek V Thaiss CA Quast T Hochheiser K Panzer U Rossjohn J Perlmutter P Cao J Godfrey DI Savage PB Knolle PA Kolanus W Förster I Kurts C - Nature Immunology 11(4):313-320 (2010)
Nature Immunology | Article Alternative cross-priming through CCL17-CCR4-mediated attraction of CTLs toward NKT cell–licensed DCs * Verena Semmling1, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Veronika Lukacs-Kornek1, 9, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Christoph A Thaiss1 Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Quast2 Search for this author in: * NPG journals * PubMed * Google Scholar * Katharina Hochheiser1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ulf Panzer3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jamie Rossjohn4 Search for this author in: * NPG journals * PubMed * Google Scholar * Patrick Perlmutter5 Search for this author in: * NPG journals * PubMed * Google Scholar * Jia Cao5 Search for this author in: * NPG journals * PubMed * Google Scholar * Dale I Godfrey6 Search for this author in: * NPG journals * PubMed * Google Scholar * Paul B Savage7 Search for this author in: * NPG journals * PubMed * Google Scholar * Percy A Knolle1 Search for this author in: * NPG journals * PubMed * Google Scholar * Waldemar Kolanus2 Search for this author in: * NPG journals * PubMed * Google Scholar * Irmgard Förster8 Search for this author in: * NPG journals * PubMed * Google Scholar * Christian Kurts1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature ImmunologyVolume:11,Pages:313–320Year published:(2010)DOI:doi:10.1038/ni.1848Received22 October 2009Accepted02 February 2010Published online28 February 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Cross-priming allows dendritic cells (DCs) to induce cytotoxic T cell (CTL) responses to extracellular antigens. DCs require cognate 'licensing' for cross-priming, classically by helper T cells. Here we demonstrate an alternative mechanism for cognate licensing by natural killer T (NKT) cells recognizing microbial or synthetic glycolipid antigens. Such licensing caused cross-priming CD8α+ DCs to produce the chemokine CCL17, which attracted naive CTLs expressing the chemokine receptor CCR4. In contrast, DCs licensed by helper T cells recruited CTLs using CCR5 ligands. Thus, depending on the type of antigen they encounter, DCs can be licensed for cross-priming by NKT cells or helper T cells and use at least two independent chemokine pathways to attract naive CTLs. Because these chemokines acted synergistically, this can potentially be exploited to improve vaccinations. View full text Figures at a glance * Figure 1: Cognate NKT cell licensing of splenic DCs for cross-priming. (–) In vivo OVA-specific cytotoxic response in the spleen (,) or the blood () 5 d after priming of wild-type (WT) mice with soluble OVA with or without α-GC (), 5 d after priming of wild-type mice with OVA plus α-GC at 1 d or 3 d after sham operation or splenectomy (splX) () or after priming of MHC class II–deficient mice (MHCII-KO) or CD1d-deficient mice (Cd1d−/−) with OVA plus α-GC (). () Flow cytometry analysis of OT-I cells among splenic CD8+ cells from bm1 mice reconstituted with 50% bm1 bone marrow and 50% CD1d-deficient or wild-type bone marrow 8 weeks before, analyzed 3 d after adoptive transfer of 1 × 106 OT-I cells and priming with OVA plus α-GC. Tet+, OT-I cells identified by staining with specific tetramers. Data are representative of two individual experiments with four to five mice per group in each (mean and s.d.). * Figure 2: NKT cell–licensed cross-priming requires CCL17 and CCR4. (,) OVA-specific cytotoxicity 5 d after priming of wild-type mice, CCR5-deficient mice (Ccr5−/−) or CCR4-deficient mice (Ccr4−/−) with soluble OVA plus α-GC () or wild-type mice, CCL17-deficient mice (Ccl17−/−) or CCR4-deficient mice with soluble OVA with or without α-GC (). () Division indices of endogenous CTLs, labeled with the cytosolic dye CFSE, obtained from wild-type, CCL17-deficient or CCR4-deficient mice and left untreated or stimulated with anti-CD3 plus anti-CD28. Data are representative of two individual experiments with four mice (,) or four individual samples () per group in each (mean and s.d.). * Figure 3: NKT cells induce CCL17 in splenic DCs. () RT-PCR analysis of CCL17 RNA in splenic CD11c+ cells from mice at 1, 3 or 5 h after injection of α-GC or vehicle (Veh). β-actin, loading control. () CCL17 mRNA expression in wild-type or CD1d-deficient mice 5 h after injection of α-GC or vehicle alone, presented relative to 18S RNA. () Immunofluorescence staining of B cell zones (blue; B220) in heterozygous CCL17-eGFP reporter mice (Ccl17+/−) and CCL17-deficient mice injected with α-GC, presented with the CCL17-eGFP signal (green). Scale bars, 200 μm. (,) Flow cytometry of spleen cells from CCL17-eGFP reporter mice 5 h after injection of vehicle or α-GC, with gating of CD11c+CCL17+ cells (red box, ) followed by analysis of the expression of CD8 and CCL17 (). Numbers adjacent to outlined areas () indicate percent CD8+CCL17+ cells (top) or CD8−CCL17+ cells (bottom). () Flow cytometry analysis of CCL17-eGFP in CD11c+ splenocytes 20 h after injection of NKT cell ligands or vehicle (right margin). Numbers adjacent to! outlined areas indicate percent CCL17+ cells. SSC, side scatter. () Flow cytometry analysis of the proportion of OT-I cells in splenic CD8+ cells of bm1 mice reconstituted with 50% bm1 bone marrow and 50% CCL17-deficient or wild-type bone marrow 8 weeks before, analyzed on day 3 after adoptive transfer of 1 × 106 OT-I cells and priming with OVA plus α-GC. Data are representative of two experiments (mean and s.d. of three to four mice per group in each). * Figure 4: CCL17 enhances cross-priming neither by activating DCs nor by recruiting NKT cells. () Flow cytometry analysis of CD86 expression on splenic DCs from wild-type, CCL17-deficient and CCR4-deficient mice injected 14 h before with α-GC or vehicle. MFI, mean fluorescent intensity. () Division index (left) and IFN-γ concentration in supernatants (right) of 1.5 × 105 OT-I cells cultured for 2 d together with 0.5 × 105 wild-type or CCR4-deficient splenic DCs coated with OVA peptide (SIINFEKL). () CCL17 mRNA expression in wild-type and CCR4-deficient DCs 5 h after injection of α-GC, presented relative to 18S RNA expression. () Division indices of splenic CD8+ T cells obtained from α-GC-injected wild-type mice, then stimulated with anti-CD3 plus anti-CD28 and cultured in the presence or absence of splenic NKT cells from mice injected with α-GC 5 h before. () CCL22 mRNA expression in wild-type and CCR4-deficient NKT cells (left) and DCs (right) 5 h after injection of α-GC, presented relative to 18S RNA expression. Data are representative of two experiments (me! an and s.d. of three to four mice per group in each). * Figure 5: Splenic DC–derived CCL17 acts directly on CTLs. (,) In vivo cytotoxicity on day 4 in wild-type mice, CCR4-deficient mice () and MHC class II–deficient mice () given 5 × 103 CCR4-sufficient or CCR4-deficient OT-I cells and primed with OVA plus α-GC 1 d later. Horizontal lines, average background cytotoxicity in wild-type mice on day 4 due to endogenous CTLs. (,) Division index (left) and IFN-γ content in supernatants (right) of 2 × 105 OT-I cells stimulated for 2 d with anti-CD3 plus anti-CD28 () or stimulated with anti-CD3 plus anti-CD28 with or without recombinant CCL17 (600 ng/ml; ). () Flow cytometry analysis of endogenous OVA-specific CTLs among splenic CD8+ cells of wild-type mice reconstituted with 50% CCR4-deficient bone marrow and 50% wild-type, CCR4-deficient or CD8-deficient bone marrow, assessed 2 d after priming with OVA plus α-GC or OVA alone. Data are representative of two experiments (mean and s.d. of four to five mice per group in each). * Figure 6: DC-derived CCL17 recruits CTLs into the splenic T cell–DC zone. () Immunofluorescence staining of spleen cryosections from CCL17-eGFP reporter mice or CCL17-deficient mice injected with 2.5 × 106 far red fluorochrome–labeled CCR4-sufficient OT-I cells on day −1 and then injected with OVA with or without α-GC on day 0. Blue staining indicates B220+ cells (defines B cell zones). Scale bars, 200 μm. (,) Absolute number of CCR4-sufficent or CCR4-deficient OT-I cells in CCL17-deficient or CCL17-eGFP reporter mice 10 h after injection with α-GC, presented as cells per mm2 of the T cell–DC zone enclosed by the (blue) B cell zone. () Proportion of OT-I cells in positioned adjacent to GFP+ DCs. () Absolute number of CCR4-sufficent or CCR4-deficient OT-I cells in CCL17-deficient or CCL17-sufficient mice 10 h after injection of iGb3, presented as described in ,. () Absolute number of far red fluorochrome–labeled polyclonal wild-type or CCR4-deficient CTLs transferred into α-GC-injected CCL17-deficient mice or CCL17-eGFP reporter mice a! s described in –. NS, not significant; *P < 0.0005 (Kruskal-Wallis and Dunn's post-test). Data are representative of two individual experiments with at least 25 T cell–DC zones per group derived from three to four nonconsecutive sections from three mice each (mean and s.d.). * Figure 7: CCL17 improves the directional migration of CTLs toward CCL17-producing DCs and increases their contact time. () Transwell assay of the migration of polyclonal CTLs toward CCL17 (800 ng/ml); cells were from mice injected with α-GC 3, 6, 8 or 12 h before analysis. (,) Flow cytometry of the binding of fluorescein isothiocyanate–labeled CCL17 to CTLs from spleens of vehicle- or α-GC-injected wild-type mice after 4, 8 and 14 h () or CD1d-deficient mice after 14 h (), presented as mean fluorescence intensity. Dashed lines, background fluorescence of control-stained CTLs. (,) In vitro migration of CTLs with or without CCR4 expression toward DCs with or without CCL17 production, recorded by time-lapse videomicroscopy over 2–3 h and presented as CTL directionality before physical contact with DCs () or subsequent duration of CTL-DC contact (). Below graphs: α-GC indicates DCs or CTLs from donor mice injected with α-GC 14 h before (+) or not (–). Numbers adjacent to vertical brackets () indicate percent contacts lasting longer than 40 min. (,) CTL directionality () and contact dura! tion () of mixed populations of DCs with or without CCL17 production, recorded by time-lapse videomicroscopy over 2–3 h. (,) CTL directionality () and contact duration () of mixed populations of CTLs with or without CCR4 expression, recorded by time-lapse videomicroscopy over 2–3 h. In –, each symbol represents an individual cell (n = 30–40 cells (directionality) or n = 100–300 cells (contact duration)); small horizontal lines indicate the mean. *P < 0.005 and **P < 0.0005 (Kruskal-Wallis and Dunn's post-test (,) or Mann-Whitney (–)). Data are representative of three experiments with three to four mice per group each (mean and s.d.). * Figure 8: Helper T cell– and NKT cell–licensed cross-priming are synergistically regulated by distinct chemokines. (,) Absolute number of adoptively transferred CCR4-deficient T cells () or CCR5-deficient T cells () in OVA-primed wild-type mice injected with α-GC or CpG, assessed by histology as described in Figure 6 (presented as cells per mm2 of the T cell–DC zone). () In vivo cytotoxicity on day 5 in OVA-primed wild-type mice injected with α-GC, CpG or both agents. () Absolute number of adoptively transferred polyclonal T cells, assessed as described in ,. *P < 0.05 and **P < 0.0005 (Kruskal-Wallis and Dunn's selected columns). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Verena Semmling & * Veronika Lukacs-Kornek Affiliations * Institutes of Molecular Medicine and Experimental Immunology, Friedrich-Wilhelms-Universität, Bonn, Germany. * Verena Semmling, * Veronika Lukacs-Kornek, * Christoph A Thaiss, * Katharina Hochheiser, * Percy A Knolle & * Christian Kurts * Life and Medical Sciences Institute, Friedrich-Wilhelms-Universität, Bonn, Germany. * Thomas Quast & * Waldemar Kolanus * III Medizinische Klinik, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany. * Ulf Panzer * Department of Biochemistry and Molecular Biology, Clayton, Australia. * Jamie Rossjohn * Department of Chemistry, Monash University, Clayton, Australia. * Patrick Perlmutter & * Jia Cao * Department of Microbiology and Immunology, University of Melbourne, Parkville, Australia. * Dale I Godfrey * Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah, USA. * Paul B Savage * Molecular Immunology, Institut für Umweltmedizinische Forschung an der Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany. * Irmgard Förster * Present address: Dana-Farber Cancer Institute, Boston, Massachusetts, USA. * Veronika Lukacs-Kornek Contributions V.S. and V.L-K. designed and did most experiments, analyzed and interpreted data and contributed to the writing of the manuscript; C.A.T., T.Q. and K.H. designed, did and analyzed individual experiments; U.P., J.R., P.P., J.C., D.I.G., P.B.S., P.A.K., W.K. and I.F. contributed tools, discussed and interpreted results and edited the manuscript; and C.K. conceived the project, designed and interpreted experiments and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Irmgard Förster (irmgard.foerster@uni-duesseldorf.de) or * Christian Kurts (ckurts@web.de) Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (964K) Short DC contact duration when CTL had not been exposed to α-GC. * Supplementary Movie 2 (836K) Long DC contact duration when CTL had been exposed to α-GC. * Supplementary Movie 3 (1M) Impaired migration of CCR4-competent CTL towards DCs that cannot produce CCL17 * Supplementary Movie 4 (1M) Impaired migration of CCR4-deficient CTL towards CCL17-producing DCs. * Supplementary Movie 5 (1M) Direct comparison of migration of CCR4-competent and –deficient CTL towards CCL17-producing DCs. PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–16 Additional data - 'Unlicensed' natural killer cells dominate the response to cytomegalovirus infection
Orr MT Murphy WJ Lanier LL - Nature Immunology 11(4):321-327 (2010)
Nature Immunology | Article 'Unlicensed' natural killer cells dominate the response to cytomegalovirus infection * Mark T Orr1 Search for this author in: * NPG journals * PubMed * Google Scholar * William J Murphy2 Search for this author in: * NPG journals * PubMed * Google Scholar * Lewis L Lanier1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume:11,Pages:321–327Year published:(2010)DOI:doi:10.1038/ni.1849Received30 November 2009Accepted03 February 2010Published online28 February 2010 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Natural killer (NK) cells expressing inhibitory receptors that bind to self major histocompatibility complex (MHC) class I are 'licensed', or rendered functionally more responsive to stimulation, whereas 'unlicensed' NK cells lacking receptors for self MHC class I are hyporesponsive. Here we show that contrary to the licensing hypothesis, unlicensed NK cells were the main mediators of NK cell–mediated control of mouse cytomegalovirus infection in vivo. Depletion of unlicensed NK cells impaired control of viral titers, but depletion of licensed NK cells did not. The transfer of unlicensed NK cells was more protective than was the transfer of licensed NK cells. Signaling by the tyrosine phosphatase SHP-1 limited the proliferation of licensed NK cells but not that of unlicensed NK cells during infection. Thus, unlicensed NK cells are critical for protection against viral infection. View full text Figures at a glance * Figure 1: MHC class I inhibition overrides NK cell licensing. () IFN-γ production and degranulation by naive Ly49H+ B6 NK cells stimulated ex vivo with RMA, RMA-s, RMA-s–m157 or RMA-m157 target cells in the presence or absence of blocking antibody to H-2Kb (α-H-2Kb). Plots are gated on Ly49H+ NK1.1+ cells. Numbers in quadrants indicate percent cells in each. () IFN-γ production and degranulation by Ly49H+ B6 NK cells stimulated with RMA-s–m157 or RMA-m157 target cells in the presence or absence of blocking antibody to H-2Kb. () IFN-γ production and degranulation by Ly49D+ B6 NK cells stimulated with RMA target cells transduced with Hm1-C4 (RMA–Hm1-C4) in the presence or absence of blocking antibody to H-2Kb. NS, not significant; P values, paired two-tailed Student's t-test. Data are representative of five (,) or three () experiments with three to four mice per experiment (error bars, s.e.m.). * Figure 2: Licensed NK cells become under-represented during MCMV infection. () CD69 upregulation and ex vivo expression of IFN-γ and granzyme B by Ly49C/I− (unlicensed) and Ly49C/I+ (licensed) Ly49H+ NK cells from naive B6 mice or B6 mice infected for 36 h with MCMV. Max, maximum. Data are representative of three experiments with three to four mice per group. (,) Ly49C/I expression on Ly49H+ NK cells from wild-type (WT) B6 mice () or MHC class I (H-2Kb and H-2Db) -deficient mice () before infection (Naive) and 5 d after MCMV infection (MCMV). Each symbol represents an individual mouse; small horizontal lines indicate the mean. P values, paired two-tailed Student's t-test. Data are representative of three experiments with three to four mice per group. * Figure 3: Ly49C/I is stably expressed and limits the proliferation of NK cells during MCMV infection. () CFSE dilution by Ly49C/I+ and Ly49C/I− Ly49H+ NK cells transferred into wild-type recipients left uninfected or infected for 5 d after with MCMV. Data are representative of five experiments with three to four mice each. () Ly49C/I expression on NK cells sorted (left) as Ly49G2+Ly49C/I− (top left outlined area) or Ly49G2−Ly49C/I+ (bottom right outlined area), transferred into naive recipients and analyzed for Ly49C/I expression 6 d after MCMV infection (right). Data are representative of two experiments. * Figure 4: Ly49C/I-mediated inhibition requires functional SHP-1. (,) IFN-γ production and degranulation by Ly49H+ wild-type NK cells () and Ly49H+ Me-v NK cells () obtained from mixed–bone marrow chimeras and stimulated with RMA-s–m157 or RMA–m157 target cells in the presence or absence of blocking antibody to H-2Kb. () Ly49C/I expression on Ly49H+ wild-type and Me-v NK cells from mixed–bone marrow chimeras, assessed before infection and 5 d after MCMV infection. Each symbol represents an individual mouse; small horizontal lines indicate the mean. () CFSE dilution by Ly49C/I+ and Ly49C/I− Ly49H+ wild-type and Me-v NK cells from mixed–bone marrow chimeras, assessed 5 d after MCMV infection. P values, paired two-tailed Student's t-test. Data are representative of three experiments with three to four (–) or two to three () mice per experiment. * Figure 5: Unlicensed NK cells control MCMV infection. (,) MCMV titers in the salivary glands () and liver () 1 week after infection of B6 wild-type mice left untreated (PBS) or depleted of Ly49C/I+ NK cells (α-Ly49C/I), Ly49G2+ NK cells (α-Ly49G2) or all NK cells (α-NK1.1). (,) MCMV titers in the salivary glands () and liver () 1 week after infection of B6 wild-type and B2m−/− mice depleted of CD8+ T cells. Each symbol represents an individual mouse; small horizontal lines indicate the mean. P values, unpaired two-tailed Mann-Whitney test. Data are representative of two experiments with five mice per group. * Figure 6: Licensed NK cells do not protect neonates from MCMV infection. (,) Survival of Ly49H-deficient neonates given PBS or 1 × 105 Ly49H+ NK cells sorted as Ly49G2+Ly49C/I+ or Ly49G2+Ly49C/I− () or given PBS or 7.5 × 104 Ly49H+ NK cells from donors predepleted of Ly49C/I+ or Ly49G2+ cells in vivo () and challenged with 2 × 103 plaque-forming units of MCMV. P values, Mantel-Cox test. Data are representative of two experiments. Author information * Abstract * Author information Affiliations * Department of Microbiology and Immunology and the Cancer Research Institute, University of California, San Francisco, California, USA. * Mark T Orr & * Lewis L Lanier * Department of Dermatology, University of California, Davis Health System, Sacramento, California, USA. * William J Murphy Contributions M.T.O. planned and did experiments and wrote the manuscript; W.J.M. contributed to experimental design and provide essential reagents; and L.L.L. contributed to experimental design, data evaluation and writing of the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Lewis L Lanier (lewis.lanier@ucsf.edu) Additional data - Regulation of leukocyte recruitment by the long pentraxin PTX3
Deban L Russo RC Sironi M Moalli F Scanziani M Zambelli V Cuccovillo I Bastone A Gobbi M Valentino S Doni A Garlanda C Danese S Salvatori G Sassano M Evangelista V Rossi B Zenaro E Constantin G Laudanna C Bottazzi B Mantovani A - Nature Immunology 11(4):328-334 (2010)
Nature Immunology | Article Regulation of leukocyte recruitment by the long pentraxin PTX3 * Livija Deban1 Search for this author in: * NPG journals * PubMed * Google Scholar * Remo Castro Russo1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Marina Sironi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Federica Moalli1 Search for this author in: * NPG journals * PubMed * Google Scholar * Margherita Scanziani4 Search for this author in: * NPG journals * PubMed * Google Scholar * Vanessa Zambelli3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Ivan Cuccovillo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Antonio Bastone3 Search for this author in: * NPG journals * PubMed * Google Scholar * Marco Gobbi3 Search for this author in: * NPG journals * PubMed * Google Scholar * Sonia Valentino1 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrea Doni1 Search for this author in: * NPG journals * PubMed * Google Scholar * Cecilia Garlanda1 Search for this author in: * NPG journals * PubMed * Google Scholar * Silvio Danese1 Search for this author in: * NPG journals * PubMed * Google Scholar * Giovanni Salvatori5 Search for this author in: * NPG journals * PubMed * Google Scholar * Marica Sassano6 Search for this author in: * NPG journals * PubMed * Google Scholar * Virgilio Evangelista7 Search for this author in: * NPG journals * PubMed * Google Scholar * Barbara Rossi8 Search for this author in: * NPG journals * PubMed * Google Scholar * Elena Zenaro8 Search for this author in: * NPG journals * PubMed * Google Scholar * Gabriela Constantin8 Search for this author in: * NPG journals * PubMed * Google Scholar * Carlo Laudanna8 Search for this author in: * NPG journals * PubMed * Google Scholar * Barbara Bottazzi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Alberto Mantovani1, 9 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume:11,Pages:328–334Year published:(2010)DOI:doi:10.1038/ni.1854Received11 January 2010Accepted12 February 2010Published online07 March 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Pentraxins are a superfamily of conserved proteins involved in the acute-phase response and innate immunity. Pentraxin 3 (PTX3), a prototypical member of the long pentraxin subfamily, is a key component of the humoral arm of innate immunity that is essential for resistance to certain pathogens. A regulatory role for pentraxins in inflammation has long been recognized, but the underlying mechanisms remain unclear. Here we report that PTX3 bound P-selectin and attenuated neutrophil recruitment at sites of inflammation. PTX3 released from activated leukocytes functioned locally to dampen neutrophil recruitment and regulate inflammation. Antibodies have glycosylation-dependent regulatory effect on inflammation. Therefore, PTX3, which is an essential component of humoral innate immunity, and immunoglobulins share functional outputs, including complement activation, opsonization and, as shown here, glycosylation-dependent regulation of inflammation. View full text Figures at a glance * Figure 1: Interaction of PTX3 with P-selectin. (,) Microtiter plate assay of the binding of PTX3 (220 nM; ) or CRP (470 nM; ) to plates coated with P-selectin (P), E-selectin (E) or L-selectin (L), presented as absorbance at 450 nm (A450). C1q serves as a positive control. Results are from three independent experiments (mean and s.d. of duplicate or triplicate wells). () Affinity of the interaction between P-selectin immobilized on microtiter wells and various amounts of biotinylated PTX3 (bPTX3); specific binding was measured in accordance with a standard curve of bPTX3, with nonlinear fitting analysis. Inset, Scatchard plot of binding data. Data are representative of four experiments (error bars, s.d.). () SPR analysis of the binding of 100, 300 and 900 nM P-selectin injected onto immobilized PTX3. Sensorgrams were fitted by the 1:1 interaction model (white lines) to obtain the corresponding association and dissociation rate constants (kon and koff) and are presented as time course of the SPR signal in resonance units ! (RU). Data are representative of two experiments. * Figure 2: Binding site for P-selectin on PTX3. () Microtiter plate assay of the binding of recombinant PTX3 (R; 220 nM), its recombinant C- and N-terminal domains (C (220 nM) and N (220 nM), respectively) and PMN-derived PTX3 (PMN; 5 nM) to P-selectin. Results are from three independent experiments (mean and s.d. of duplicate or triplicate wells). () SPR analysis of the binding of 165, 325, 650 and 1300 nM P-selectin injected onto the immobilized C-terminal domain of PTX3, presented as the time course of the SPR signal. Sensorgrams were fitted by a model that included two different Kd values (white lines). Data are representative of two experiments. * Figure 3: The role of the PTX3 glycosidic moiety in the PTX3–P-selectin interaction. () Binding of untreated PTX3 (PTX3; 220 nM), enzymatically deglycosylated PTX3 (DG; 220 nM) or PTX3 deglycosylated by site-directed mutagenesis (MUT; 220 nM) to P-selectin-coated wells. Results are from four independent experiments (mean and s.d. of duplicate wells). () Binding of PTX3 (110 nM) to P-selectin-coated wells in the presence of increasing doses of recombinant PSGL-1, presented as percentage of binding in the absence of PSGL-1. Results are from four independent experiments (mean ± s.d. of duplicate wells). * Figure 4: PTX3 binds P-selectin in a cell-based context. () Binding of PTX3 to P-selectin-transfected CHO cells, assessed by flow cytometry. Results are presented as the percentage of maximum (% of max) to allow the presentation of normalized data and represent the number of events in each bin divided by the number of events in the bin with the largest number of cells. PE-A (horizontal axis), area under the curve for the intensity of phycoerythrin fluorescence. Mean fluorescence intensity (MFI): streptavidin-phycoerythrin (Strep-PE), 119.62; 220 nM PTX3, 996.02; 440 nM PTX3, 2309.71; 2,200 nM PTX3, 5768.92. Data are representative of two independent experiments. () Binding of PTX3 (1 μM) to resting or thrombin-activated platelets in the presence or absence of a monoclonal P-selectin-blocking antibody (mAb to P-sel (CLB-Thromb/6); 20 μg/ml), assessed by flow cytometry. *P < 0.001 (one-way analysis of variance (ANOVA)). Data are representative of three experiments (mean and s.d.). () Analysis of leukocyte rolling on P-selectin- an! d E-selectin-coated glass capillary tubes in the presence of increasing doses of PTX3; results were recorded for single areas of 0.2 mm2 for at least 30 s. Rolling interactions (corresponding to interactions lasting less than 0.5 s) were considered important and were given scores. Data are from five experiments with five to seven areas analyzed in each (mean and s.d.). * Figure 5: PTX3 inhibits rolling interactions in vivo. () Frequency of rolling interactions of PMNs in the mesenteric venules of wild-type mice (WT) and Ptx3−/− mice (PTX3-KO) and in wild-type mice with (PTX3) or without (Ctrl) PTX3 pretreatment (right), presented as rolling fractions (venules and mice per group, Supplementary Tables 1 and 2). *P < 0.01 (paired or unpaired two-tailed Student's t-test). Data are representative of five experiments (mean and s.e.m.). () Rolling PMNs (arrowheads) after thrombin stimulation in mesenteric venules before PTX3 administration (Control) and after PTX3 treatment (PTX3). Scale bar, 50 μm. Data are representative of five experiments. * Figure 6: Exogenous PTX3 dampens early leukocyte recruitment in vivo. () Recruitment of PMNs into the pleural cavity of wild-type mice treated intravenously with vehicle (0.9% saline solution (NaCl); n = 15), PTX3 (100 μg/mouse; n = 15), deglycosylated PTX3 (PTX3(DG); 100 μg/mouse; n = 8), heat-inactivated PTX3 (HI PTX3; 100 μg/mouse; n = 6), P-selectin-blocking antibody (α-P-sel; 30 μg/mouse; n = 8) or the C-terminal domain of PTX3 (PTX3(C-term); 100 μg/mouse; n = 8) 10 min before intrapleural injection of KC. () Recruitment of PMNs into the pleural cavity of wild-type and P-selectin-deficient (P-sel-KO) mice pretreated intravenously with vehicle (0.9% saline solution; wild type, n = 14; P-selectin-deficient, n = 8), PTX3 (100 μg/mouse; wild type, n = 15; P-selectin-deficient, n = 7), P-selectin-blocking antibody (30 μg/mouse; n = 15) or PTX3 and the P-selectin-blocking antibody together (n = 8). () Recruitment of PMNs into the pleural cavity of wild-type mice pretreated intravenously with vehicle (0.9% saline solution; n = 15) or PTX! 3 (100 μg/mouse; n = 15), assessed 2 h after injection of lipopolysaccharide (400 ng/mouse). *P < 0.01 and **P < 0.001 (one-way ANOVA). Data are representative two to six different experiments (,) or two experiments (; mean and s.e.m.). * Figure 7: Role of endogenous PTX3 in leukocyte recruitment. () Recruitment of PMNs into the pleural cavity of wild-type and PTX3-deficient mice pretreated intravenously with vehicle (0.9% saline solution; wild type, n = 16; PTX3-deficient, n = 16) or PTX3 (PTX3-KO + PTX3; 100 μg/mouse; n = 13). () Recruitment of PMNs into the pleural cavity of lethally irradiated wild-type (WT) and PTX3-deficient (KO) mice that received wild-type or PTX3-deficient bone marrow (wild type→wild type, n = 13; PTX3-deficient→wild type, n = 20; wild type→PTX3-deficient, n = 12; PTX3-deficient→PTX3-deficient, n = 11). *P < 0.05, **P < 0.01 and ***P < 0.001 (one-way ANOVA). Data are representative of two experiments () or one experiment (; mean and s.e.m.). * Figure 8: PTX3 dampens PMN recruitment in a model of acid-induced acute lung injury. () PMN infiltration of lung interstitium at 3 h after acid-induced acute lung injury (HCl) in mice pretreated intravenously with vehicle (0.9% saline solution; n = 13), PTX3 (100 μg/mouse; n = 16) or P-selectin-blocking antibody (30 μg/mouse; n = 6). () Migration of neutrophils into the bronchoalveolar space at 3 h after acid-induced acute lung injury in mice pretreated intravenously with vehicle (0.9% saline solution; n = 7), PTX3 (100 μg/mouse; n = 8) or P-selectin-blocking antibody (30 μg/mouse; n = 6). *P < 0.05 and **P < 0.01 (one-way ANOVA). Data are representative of two experiments (mean and s.e.m.). Author information * Abstract * Author information * Supplementary information Affiliations * Laboratory for Immunology and Inflammation, Instituto di Ricovero e Cura a Carattere Scientifico–Istituto Clinico Humanitas, Rozzano, Italy. * Livija Deban, * Remo Castro Russo, * Marina Sironi, * Federica Moalli, * Ivan Cuccovillo, * Sonia Valentino, * Andrea Doni, * Cecilia Garlanda, * Silvio Danese, * Barbara Bottazzi & * Alberto Mantovani * Laboratório de Imunofarmacologia, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. * Remo Castro Russo * Mario Negri Institute, Milan, Italy. * Vanessa Zambelli, * Antonio Bastone & * Marco Gobbi * Department of Experimental Medicine, University of Milano-Bicocca, Milan, Italy. * Margherita Scanziani & * Vanessa Zambelli * Sigma-Tau Research and Development, Pomezia, Italy. * Giovanni Salvatori * Tecnogen, Ricerca e Sviluppo, Piana di Monte Verna, Italy. * Marica Sassano * Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy. * Virgilio Evangelista * Department of Pathology, University of Verona, Verona, Italy. * Barbara Rossi, * Elena Zenaro, * Gabriela Constantin & * Carlo Laudanna * Dipartimento di Medicina Traslazionale, University of Milan, Milan, Italy. * Alberto Mantovani Contributions L.D., B.B. and A.M. planned the research, analyzed and interpreted data and wrote the manuscript; L.D., R.C.R., M.Sironi., F.M., M.Scanziani and V.Z. did the animal experiments; A.B. and M.G. did the SPR experiments; C.L. did and analyzed leukocyte rolling experiments; S.V. and I.C. participated in purification of recombinant proteins and helped with mouse breeding and genotyping; L.D. and A.D. did and analyzed flow cytometry and microtiter plate binding assays; G.S. generated deglycosylated PTX3 by enzymatic digestion; M.Sassano. generated deglycosylated PTX3 by site-directed mutagenesis; G.C., B.R. and E.Z. did and analyzed the intravital microscopy experiments; and C.G., V.E. and S.D. contributed to data analysis and interpretation. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Alberto Mantovani (alberto.mantovani@humanitasresearch.it) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (616K) Supplementary Figures 1–10, Tables 1–2 and Supplementary Methods Additional data - An Slfn2 mutation causes lymphoid and myeloid immunodeficiency due to loss of immune cell quiescence
Berger M Krebs P Crozat K Li X Croker BA Siggs OM Popkin D Du X Lawson BR Theofilopoulos AN Xia Y Khovananth K Moresco EM Satoh T Takeuchi O Akira S Beutler B - Nature Immunology 11(4):335-343 (2010)
Nature Immunology | Article An Slfn2 mutation causes lymphoid and myeloid immunodeficiency due to loss of immune cell quiescence * Michael Berger1 Search for this author in: * NPG journals * PubMed * Google Scholar * Philippe Krebs1 Search for this author in: * NPG journals * PubMed * Google Scholar * Karine Crozat1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaohong Li1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ben A Croker1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Owen M Siggs1 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel Popkin2 Search for this author in: * NPG journals * PubMed * Google Scholar * Xin Du1 Search for this author in: * NPG journals * PubMed * Google Scholar * Brian R Lawson2 Search for this author in: * NPG journals * PubMed * Google Scholar * Argyrios N Theofilopoulos2 Search for this author in: * NPG journals * PubMed * Google Scholar * Yu Xia1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kevin Khovananth1 Search for this author in: * NPG journals * PubMed * Google Scholar * Eva Marie Y Moresco1 Search for this author in: * NPG journals * PubMed * Google Scholar * Takashi Satoh3 Search for this author in: * NPG journals * PubMed * Google Scholar * Osamu Takeuchi3 Search for this author in: * NPG journals * PubMed * Google Scholar * Shizuo Akira3 Search for this author in: * NPG journals * PubMed * Google Scholar * Bruce Beutler1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume:11,Pages:335–343Year published:(2010)DOI:doi:10.1038/ni.1847Received12 November 2009Accepted25 January 2010Published online28 February 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Here we describe a previously unknown form of inherited immunodeficiency revealed by an N-ethyl-N-nitrosourea–induced mutation called elektra. Mice homozygous for this mutation showed enhanced susceptibility to bacterial and viral infection and diminished numbers of T cells and inflammatory monocytes that failed to proliferate after infection and died via the intrinsic apoptotic pathway in response to diverse proliferative stimuli. They also had a greater proportion of T cells poised to replicate DNA, and their T cells expressed a subset of activation markers, suggestive of a semi-activated state. We positionally ascribe the elektra phenotype to a mutation in the gene encoding Schlafen-2 (Slfn2). Our findings identify a physiological role for Slfn2 in the defense against pathogens through the regulation of quiescence in T cells and monocytes. View full text Figures at a glance * Figure 1: Mice homozygous for the elektra mutation are highly susceptible to infection with MCMV, LCMV and L. monocytogenes. () Survival curves for wild-type mice (n = 8) and elektra homozygotes (n = 8) after challenge with 2 × 105 PFU of MCMV. () Survival curves after infection with 2 × 105 PFU of MCMV, assessed in recipient mice reconstituted with 5 × 106 bone marrow cells (reciprocal bone marrow transplantation) 1 d after a 10-Gy dose of irradiation (C57BL/6J→C57BL/6J, n = 3; C57BL/6J→elektra, n = 6; elektra→C57BL/6J, n = 6; elektra→elektra, n = 3; congenic C57BL/6.SJL (PtprcaPep3b; Ly5.1+), C57BL/6J (PtprcbPep3a; Ly5.2+) wild-type and elektra mutant mice serve as both recipients and donors). () Viral load in spleens of wild-type and elektra homozygotes (n = 3 mice per group) 7 d after intravenous injection of 2 × 102 or 2 × 106 PFU of LCMV (Armstrong strain). Each symbol represents an individual mouse; small horizontal lines indicate the mean. *P = 0.028 and **P < 0.0001 (two-tailed Student's t-test). () Survival curves for wild-type mice (n = 5), elektra homozygotes (n = 7) and m! ice deficient in Toll-like receptor signaling due to mutation of Myd88 (Myd88poc/poc; n = 2) challenged with 2 × 105 colony-forming units of L. monocytogenes. Results are representative of five (), two (,) or four () independent experiments. * Figure 2: Defect in peripheral T cells in elektra homozygotes. (,) Flow cytometry analysis of the expression of CD4 and CD8 by cells from the spleen, lymph node (LN) and blood () or by thymocytes () from wild-type or elektra-homozygous mice. Numbers in quadrants indicate percent cells in each. () Total CD8+ splenocytes among splenocytes isolated from wild-type and elektra-homozygous mice 7 d after intravenous injection of 2 × 102 or 2 × 106 PFU of LCMV, Armstrong strain (top), and frequency of cells with intracellular IFN-γ expression among CD8+ splenocytes then restimulated ex vivo with gp33 or NP396 (peptides derived from LCMV) in the presence of brefeldin A (bottom). *P < 0.01 and **P < 0.001 (two-tailed Student's t-test). Results are representative of three experiments with ten mice per genotype (,) or two independent experiments with three mice per genotype per condition (; error bars, s.d.). * Figure 3: Apoptosis of elektra-homozygous T cells in response to activation signals. () CFSE intensity of CD8+ lymph node cells left untreated (None) or stimulated with anti-CD3ϵ plus anti-CD28, with IL-2, or with phorbol 12-myristate 13-acetate (PMA) plus ionomycin. () BrdU incorporation by CD8+ T cells obtained from wild-type or elektra mice (n = 3 each) and left untreated or stimulated with anti-CD3ϵ and anti-CD28 (left) and BrdU incorporation of CD8+ T cells from wild-type or elektra mice (n = 4 each) 4 h after injection with BrdU (right). () Propidium iodide (PI) and annexin V staining of CD8+ T cells obtained from wild-type or elektra mice (n = 3 each) and stimulated for 48 h ex vivo with anti-CD3ϵ and anti-CD28. () Induction of apoptosis (annexin V–positive cells; gated on the CD8+ propidium iodide–negative population) by γ-irradiation. () Immunoblot analysis (top) of pooled CD8+ T cells from wild-type mice (n = 2) or elektra mice (n = 4) after treatment with anti-CD3ϵ and anti-CD28, assessed with antibody to phosphorylated p38 (p-p38) or ant! i-p38 and flow cytometry analysis (bottom) of the staining of phosphorylated p38 (p-p38) or phosphorylated Jnk (p-Jnk) in wild-type (n = 3) or elektra (n = 3) CD8+ T cells. *P < 0.05, **P < 0.01 and ***P < 0.001 (two-tailed Student's t-test). Results are representative of four experiments with three mice per genotype (), two experiments (,), two experiments with three mice per genotype () or three experiments (). Error bars (,), s.d. * Figure 4: Apoptosis of elektra-homozygous T cells in response to homeostatic expansion signals. () Annexin V staining of adoptively transferred wild-type or elektra CD8+CD45.1− cells from spleens of wild-type (Ly5.1+) recipient mice (n = 4). Numbers in top right quadrants indicate percent CD8+ apoptotic cells. () CD8+ T cells (left) and CD4+ T cells (right) in 7-week-old wild-type or elektra mice (n = 5 or 6, respectively) at various times after thymectomy, presented as relative percent of T cells ((% T cells in blood after thymectomy) / (% T cells in blood before thymectomy) × 100). () Number of CD8+ T cells, CD4+ T cells and B cells and frequency of annexin V–positive (apoptotic) CD8+ T cells, in spleens from wild-type or elektra mice (n = 4 each) injected with PBS (white) or with IL-7 plus anti-IL-7 (M25) on days 1 and 3, with cells collected on day 6 (black), or on day 1, with cells collected on day 3 (gray). () Annexin V staining of splenic CD8+CD44hi or CD8+CD44lo cells from wild-type or elektra mice (n = 4 each). Numbers above bracketed lines indicate perce! nt apoptotic cells. NS, not significant; *P < 0.01 and **P < 0.001 (two-tailed Student's t-test). Results are representative of three (,) or two (,) experiments. Error bars (,), s.d. * Figure 5: T cells homozygous for the elektra mutation die via the intrinsic apoptotic pathway. () Staining of CD4 and CD8 in blood cells from 6-week-old littermate offspring of crosses of mice homozygous for the elektra mutation (eka) with mice homozygous for the lymphoproliferation mutation (lpr) of Fas to generate double-mutant mice (eka/eka;Faslpr/lpr). Numbers in quadrants indicate percent CD8+CD4− cells (top left) or CD8−CD4+ cells (bottom right). () Flow cytometry of Bcl-2 expression in splenic CD8+CD44hi or CD8+CD44lo cells from wild-type or elektra-homozygous mice. () Staining of CD4 and CD8 on splenic cells from 4-week-old littermate offspring from crosses of eka/eka and eka/+ mice expressing a BCL2 transgene (BCL2(Tg)) to generate elektra homozygotes overexpressing Bcl-2 in T cells. Numbers in quadrants indicate percent CD4+CD8− cells (top left) or CD4−CD8+ cells (bottom right). () Flow cytometry of the CFSE intensity of CD3+CD8+ cells among CFSE-labeled spleen cells from the mice in , stimulated in vitro with a combination of anti-CD3ϵ and anti-CD2! 8 and collected after 72 h. Results are representative of two independent experiments with three to four mice per genotype in each. * Figure 6: T cells homozygous for the elektra mutation exist in a semiactivated state. () Flow cytometry analysis of the staining of CD44 and CD122 (IL-2Rβ) in splenic CD8+ cells from wild-type or elektra mice (n = 3 per genotype) injected twice (one dose every 3 d) with PBS or with IL-7 plus anti-IL-7. Numbers in quadrants indicate percent cells in each. () Flow cytometry of CD44loCD8+ or CD44hiCD8+ cells stained for IL-7Rα, CD62L, CD5, CD69 or PD-1 (n = 3 mice per genotype). ISM, isotype-matched control antibody. () Staining of CD44 and CD122 in splenic CD8+ cells from 4-week-old littermates of various genotypes (above plots; n = 3 mice per genotype). Numbers in quadrants indicate percent CD44hiCD122− cells (top left) or CD44hiCD122+ cells (top right). () CD62L staining of spleen CD44loCD8+ or CD44hiCD8+ cells from elektra-heterozygous mice (eka/+) or elektra-homozygous mice (eka/eka) expressing a BCL2 transgene (n = 3 per genotype). (,) Flow cytometry of wild-type (CD45.1+) and elektra (CD45.2+) donor CD8+ T cells in CD3-deficient mixed–bone marrow ch! imeras. () CD44 versus CD122 staining. () CD62L staining of CD44loCD8+ or CD44hiCD8+ cells (top). Below, mean fluorescence intensity (MFI) of CD62L staining in the CD44lo population (left) and frequency of CD62Lhi and CD62Llo cells in the CD44hi population (right). *P < 0.001 (two-tailed Student's t-test). Results are representative of two (,,) or three () experiments or two experiments with six recipient mice (,). Error bars, s.d. * Figure 7: Apoptosis of elektra-homozygous monocytes in response to activation signals. () Ly6C and CD11b staining (left) of splenocytes from uninfected or L. monocytogenes–infected wild-type or elektra mice (n = 5 per genotype). Numbers adjacent to outlined areas indicate percent inflammatory monocytes (R1 gate; top left) or neutrophils (R2 gate; bottom right). Right, frequency of inflammatory monocytes from spleen, blood and bone marrow of wild-type or elektra mice (n = 5 each) without infection or at 48 h after infection. () Flow cytometry of wild-type (CD45.2−) and elektra (CD45.2+) donor CD4+ T cells, CD8+ T cells, B cells (B220+) and inflammatory monocytes (gated on CD11b+ population) in the blood of CD3-deficient mixed–bone marrow chimeras (n = 3). Numbers in quadrants indicate percent cells in each. (–) Analysis of isolated bone marrow inflammatory monocytes from wild-type or elektra mice (n = 2 each) cultured for 3 d in medium alone (Untreated) or in medium supplemented with IFN-γ and heat-killed L. monocytogenes (Treated). () MHC class II sta! ining (left) and nitric oxide concentration (right). () Forward-scatter (FSC) and side-scatter (SSC) profiles. Outlined areas (top left) indicate populations of live and dead cells. () Annexin V staining of the gated high-forward-scatter population. *P < 0.05, **P < 0.01 and ***P < 0.001 (two-tailed Student's t-test). Results are representative of three (,–) or two () experiments. Error bars (,,), s.d. * Figure 8: 'Rescue' of the elektra phenotype by BAC transgenesis. () DNA sequence of the BAC clone and genomic DNA from an Slfn2eka/eka mouse in the region of the Slfn2eka mutation. The BAC clone contains the wild-type Slfn2 sequence. Gray highlighting indicates that the nucleotide of interest is in heterozygous form in the transgenic mouse (wild-type nucleotide from the BAC transgene; mutated nucleotide from the genomic DNA); red highlighting indicates nucleotides of interest; lower case indicates heterozygosity; curved lines indicate sequencing peaks (colors match nucleotides above). () Flow cytometry analysis of the staining of CD8 and CD4 in blood from a wild-type mouse, an Slfn2eka/eka mouse carrying the Slfn2 transgene (elektra–BAC-Tg) and a littermate lacking the transgene (elektra). Results are representative of three experiments with four mice per genotype. () Survival curves for BAC-transgenic mice (n = 7), littermates without the BAC transgene (n = 4) and wild-type mice (n = 5) after challenge with 2 × 105 PFU of MCMV. Result! s are representative of two experiments. () Survival curves for BAC-transgenic mice (n = 7), littermates without the BAC transgene (n = 5) and wild-type mice (n = 7) after challenge with 5 × 105 colony-forming units of L. monocytogenes. Results are representative of two experiments. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Genetics, The Scripps Research Institute, La Jolla, California, USA. * Michael Berger, * Philippe Krebs, * Karine Crozat, * Xiaohong Li, * Ben A Croker, * Owen M Siggs, * Xin Du, * Yu Xia, * Kevin Khovananth, * Eva Marie Y Moresco & * Bruce Beutler * Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, California, USA. * Daniel Popkin, * Brian R Lawson & * Argyrios N Theofilopoulos * Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan. * Takashi Satoh, * Osamu Takeuchi & * Shizuo Akira * Present addresses: Centre d'Immunologie de Marseille-Luminy, Marseille, France (K.C.) and Cancer and Haematology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia (B.A.C.). * Karine Crozat & * Ben A Croker Contributions M.B. and B.B. designed the research with critical suggestions from P.K., K.C. and A.N.T.; M.B., P.K., K.C., X.L., B.A.C., O.M.S., D.P. and B.R.L. did experiments; X.L. and X.D. generated the BAC-transgenic mice; M.B., Y.X. and K.K. did all genome mapping; T.S., O.T. and S.A. generated Slfn3−/− and Slfn1−/− mice; and M.B., E.M.Y.M. and B.B. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Bruce Beutler (bruce@scripps.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–7 Additional data - Vitamin D controls T cell antigen receptor signaling and activation of human T cells
von Essen MR Kongsbak M Schjerling P Olgaard K Odum N Geisler C - Nature Immunology 11(4):344-349 (2010)
Nature Immunology | Article Vitamin D controls T cell antigen receptor signaling and activation of human T cells * Marina Rode von Essen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Kongsbak1 Search for this author in: * NPG journals * PubMed * Google Scholar * Peter Schjerling2, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Klaus Olgaard5 Search for this author in: * NPG journals * PubMed * Google Scholar * Niels Ødum1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Carsten Geisler1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume:11,Pages:344–349Year published:(2010)DOI:doi:10.1038/ni.1851Received30 November 2009Accepted08 February 2010Published online07 March 2010 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Phospholipase C (PLC) isozymes are key signaling proteins downstream of many extracellular stimuli. Here we show that naive human T cells had very low expression of PLC-γ1 and that this correlated with low T cell antigen receptor (TCR) responsiveness in naive T cells. However, TCR triggering led to an upregulation of ~75-fold in PLC-γ1 expression, which correlated with greater TCR responsiveness. Induction of PLC-γ1 was dependent on vitamin D and expression of the vitamin D receptor (VDR). Naive T cells did not express VDR, but VDR expression was induced by TCR signaling via the alternative mitogen-activated protein kinase p38 pathway. Thus, initial TCR signaling via p38 leads to successive induction of VDR and PLC-γ1, which are required for subsequent classical TCR signaling and T cell activation. View full text Figures at a glance * Figure 1: Lower sensitivity of naive T cells to TCR triggering. () TCR expression in naive and primed T cells after stimulation with beads coated with anti-CD3 and anti-CD28 (cell/bead ratio, above graphs), presented relative to that of untreated cells. Data are from five independent experiments (error bars, s.d.). () Immunoblot analysis of tyrosine phosphorylation (pY) and expression of Zap70, Lat, Erk and TCRζ (loading control) in naive and primed T cells stimulated for 0–10 min with beads coated with anti-CD3 and anti-CD28. Each vertically aligned lane represents the same cell sample. Data are representative of three independent experiments. () Calcium flux in naive and primed T cells left unstimulated (Medium) or stimulated at 30 s with a mixture of antibody to CD3 (anti-CD3) and anti-CD28, with ionomycin added at 465 s. Results are for gated CD8+ T cells; CD4+ T cells produced the same results. Data are representative of three independent experiments. * Figure 2: Phosphorylation and expression of PLC-γ1 in naive and primed T cells. () Immunoblot analysis of tyrosine phosphorylation and expression of PLC-γ1 and TCRζ (loading control) in naive and primed T cells stimulated for 0–10 min with beads coated with anti-CD3 and anti-CD28 (cell/bead ratio, 1:3). () RNA-hybridization analysis of the expression of PLC-γ1 mRNA and 28S rRNA (loading control) in T cells stimulated for 0–9 d. () Quantification of PLC-γ1 mRNA expression during differentiation, normalized to the loading control and presented (per T cell) relative to basal expression in naive T cells. () Immunoblot analysis of the expression of PLC-γ1, TCRζ (loading control) and Glut1 (activation marker22) in T cells stimulated for 0–9 d. () Quantification of PLC-γ1 expression during differentiation, normalized to the loading control and presented (per T cell) relative to basal expression in naive T cells. Data are representative of three separate experiments (,,) or are from three experiments (,; error bars, s.d.). * Figure 3: VDR induction precedes PLC-γ1 upregulation. () Immunoblot analysis of VDR, PLC-γ1 and TCRζ (loading control) in the cytoplasmic and nuclear fractions of T cells stimulated for 0–3 d with beads coated with anti-CD3 and anti-CD28 (cell/bead ratio, 1:3). () Immunoblot analysis of the expression of VDR, PLC-γ1 and TCRζ in purified naive and primed CD4+ and CD8+ T cells. () Immunoblot analysis of the expression of VDR, PLC-γ1 and TCRζ in T cells stimulated for 3 d in the presence of the VDR antagonists ZK191784 and ZK203278 (concentration, above lanes). () Flow cytometry of naive T cells (far left, top row) and T cells stimulated for 3 d in the presence of various concentrations (above plots) of ZK191784 (other plots, top row) or ZK203278 (bottom row). Numbers adjacent to outlined areas indicate percent cells in each. SSC, side scatter; FSC, forward scatter. () CFSE profiles of naive T cells (top row; gray filled histogram) and primed T cells stimulated for 3 d without further additions (black lines) or in the pres! ence of vehicle (middle; gray filled histogram) or ZK191784 (bottom row; gray filled histogram). () Immunoblot analysis of the expression of VDR, PLC-γ1 and TCRζ in T cells stimulated for 3 d in the presence of ketoconazole (0 or 1 μg/ml) and either 25(OH)D3 (25; 0.5 μM) or 1,25(OH)2D3 (1,25; 0.5 μM). () Proliferation index of T cells from patients with chronic deficiency of 25(OH)D3 and 1,25(OH)2D3 and controls with normal serum concentration of 25(OH)D3 and 1,25(OH)2D3, stimulated for 3 d. () Concentration of 25(OH)D3 (left vertical axis; open circles) and 1,25(OH)2D3 (right vertical axis; filled circles) in patient and control sera. Each symbol represents an individual serum sample; small horizontal lines indicate the mean. Data are representative of three separate experiments. * Figure 4: The alternative TCR signaling pathway induces VDR expression. () Immunoblot analysis of Thr180 phosphorylation (pT) and expression of p38 and TCRζ (loading control) in naive and primed T cells stimulated for 0–10 min with beads coated with anti-CD3 and anti-CD28 (cell/bead ratio, 1:3). () Immunoblot analysis of the expression of VDR, PLC-γ1 and GAPDH (glyceraldehyde phosphate dehydrogenase; loading control) in E6-1 (E6) and J.gamma1 (Jγ1) cells stimulated for 24 h with anti-CD3 and anti-CD28. () Immunoblot analysis of Thr180 phosphorylation and expression of p38 in unstimulated naive T cells and in naive T cells stimulated for 30 min with magnetic beads coated with anti-CD3 and anti-CD28 (cell/bead ratio, 1:3) in the presence or absence of the p38 inhibitor SB203580 (concentration, above lanes). () Immunoblot analysis of the expression of VDR, PLC-γ1 and TCRζ in T cells stimulated for 3 d in the presence or absence of SB203580 (concentration, above lanes). () Flow cytometry of T cells stimulated for 3 d in the presence or absenc! e of SB203580 (concentration, above plots). Numbers adjacent to outlined areas indicate percent cells in each. () CFSE profiles of primed T cells stimulated for 3 d with beads coated with anti-CD3 and anti-CD28 (black line) or beads plus 10 μM SB203580 (gray filled histogram). Author information * Abstract * Author information Affiliations * Department of International Health, Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark. * Marina Rode von Essen, * Martin Kongsbak, * Niels Ødum & * Carsten Geisler * Center for Healthy Aging, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. * Peter Schjerling * Department of Biology, Faculty of Science, University of Copenhagen, Copenhagen, Denmark. * Niels Ødum * Institute of Sports Medicine, Bispebjerg Hospital, Rigshospitalet and Faculty of Health Sciences, University of Copenhagen, Denmark. * Peter Schjerling * Department of Nephrology, Rigshospitalet and Faculty of Health Sciences, University of Copenhagen, Denmark. * Klaus Olgaard Contributions M.R.v.E. did most of the experiments, analyzed data and contributed to the writing of the manuscript; M.K. and P.S. contributed to the ketoconazole and mRNA experiments; K.O. contributed to the planning and analyses of studies involving patients; N.Ø. contributed to the design and analysis of some of the experiments; and C.G. conceptualized the research, directed the study, analyzed data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Carsten Geisler (cge@sund.ku.dk) Additional data - Unique autoreactive T cells recognize insulin peptides generated within the islets of Langerhans in autoimmune diabetes
Mohan JF Levisetti MG Calderon B Herzog JW Petzold SJ Unanue ER - Nature Immunology 11(4):350-354 (2010)
Nature Immunology | Article Unique autoreactive T cells recognize insulin peptides generated within the islets of Langerhans in autoimmune diabetes * James F Mohan1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Matteo G Levisetti1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Boris Calderon1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jeremy W Herzog1 Search for this author in: * NPG journals * PubMed * Google Scholar * Shirley J Petzold1 Search for this author in: * NPG journals * PubMed * Google Scholar * Emil R Unanue1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume:11,Pages:350–354Year published:(2010)DOI:doi:10.1038/ni.1850Received03 December 2009Accepted04 February 2010Published online28 February 2010Corrected online05 March 2010 Abstract * Abstract * Change history * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In addition to the genetic framework, there are two other critical requirements for the development of tissue-specific autoimmune disease. First, autoreactive T cells need to escape thymic negative selection. Second, they need to find suitable conditions for autoantigen presentation and activation in the target tissue. We show here that these two conditions are fulfilled in diabetic mice of the nonobese diabetic (NOD) strain. A set of autoreactive CD4+ T cells specific for an insulin peptide, with the noteworthy feature of not recognizing the insulin protein when processed by antigen-presenting cells (APCs), escaped thymic control, participated in diabetes and caused disease. Moreover, APCs in close contact with beta cells in the islets of Langerhans bore vesicles with the antigenic insulin peptides and activated peptide-specific T cells. Our findings may be relevant for other cases of endocrine autoimmunity. View full text Figures at a glance * Figure 1: Insulin-reactive T cells in NOD mice. (–) Interleukin 2 (IL-2) production by three representative CD4+ T cell hybridomas, type B T cell 2D10 (), type A T cell 4F7 () and type A T cell IIT-9 (), in response to stimulation with insulin (INS) or B:9-23, assessed by standard bioassay of the IL-2-dependent mouse cell line CTLL-2 (CTLL proliferation bioassay). () Enzyme-linked immunospot (ELISPOT) assay of IL-2 production by NOD mice immunized with insulin and restimulated without antigen (No Ag) or with insulin or B:9-23, presented as IL-2-secreting cells (spot-forming cells (SFC)) per 1 × 106 draining lymph node cells. () ELISPOT assay of IL-2 secretion by NOD mice immunized with B:9-23 and restimulated with B:9-23 or insulin. () ELISPOT assay of IL-2 secretion by NOD mice immunized with B:9-23 and restimulated with B:9-23 in the presence or absence of a specific blocking antibody to I-Ag7 or an irrelevant antibody to I-Ak. () ELISPOT assay of IL-2 secretion by B16:A-dKO mice immunized with insulin and restimulat! ed with B:9-23 or insulin in the presence or absence of anti-I-Ag7. () ELISPOT of IL-2 secretion by B16:A-dKO mice immunized with B:9-23 and restimulated with B:9-23 or insulin. () IL-2 production by two T cell hybridomas (118 and 22) derived from B16:A-dKO mice and two hybridomas (4F7 and 4E4-62) derived from NOD mice, in response to insulin presented by the C3.G7 mouse B cell lymphoma line. () IL-2 production by type A (4F7) and type B (2D10 and 10F9) hybridomas incubated with the M12.C3 cell line bearing a covalent B:9-23–I-Ag7 complex (M12.C3.G7β9-23). P values, one-tailed unpaired Student's t-test. Data are representative of two independent experiments (–) with three to five mice per group (–; error bars, s.e.m.). * Figure 2: Type B T cells are diabetogenic. () Diabetes incidence of type B T cell lines (n = 3 lines representative of 11) transferred into NOD.SCID recipients (n = 3 mice each); mice were considered diabetic after two consecutive blood glucose readings of ≥ 250 mg/dl. () Cytokine profile of CD4+ T cell lines 48 h after stimulation with plate-bound anti-CD3 and anti-CD28. TNF, tumor necrosis factor; IFN-γ, interferon-γ. (,) Hematoxylin- and eosin-stained pancreatic sections of a NOD.SCID recipient of 6D8 T cells that developed insulitis but not diabetes () and a NOD.SCID recipient of 3E6 T cells that developed overt diabetes (). Original magnification, ×200. Data are representative of a single experiment with 11 individual T cell lines. * Figure 3: Intra-islet DCs pulsed with secretory granules present insulin peptides. () B:9-23 presentation by cells from various tissues (horizontal axis) to a type B hybridoma (1.7). LN, lymph node; Flt3L, ligand for the cytokine receptor Flt3; tx, treated; Con A, concanavalin A; PECs, peritoneal exudate cells. () IL-2 production by type A (4E4-62) and type B (2D10) hybridomas stimulated with dispersed islets from NOD Rag1−/− mice. () IL-2 production by type A (4F7) and type B (2D10 and 1.7) hybridomas stimulated with CD11c+ cells isolated from the islets of 8-week-old male NOD mice. () Electron microscopy of Flt-3L CD11c+ cell incubated for 24 h together with NIT-1 insulinoma cells (arrows indicate granules in DC). Scale bar, 500 nm. (,) IL-2 production by type B (2D10; ) and type A (4F7; ) hybridomas stimulated with NIT-1 cells incubated together with purified splenic CD11c+ DCs. () IL-2 production by type A (4F7) and type B (2D10 and 1.7) hybridomas stimulated with splenic CD11c+ cells incubated together with purified insulin granules from primary d! ispersed islet cells. Data are representative of two independent experiments (error bars, s.e.m.). * Figure 4: Secretory granules contain proteolytic fragments of the insulin β-chain in NOD Rag1−/− islets. () Immunofluorescence microscopy of an islet stained for CD11c (red) and intracellular B:9-23 (green). Original magnification, ×200. () Immunofluorescence microscopy of an islet stained for CD11c (red) and B:9-23 (green) in the presence of competing exogenous B:9-23 peptide. Scale bars, 20 μm. () Confocal microscopy of an isolated beta cell stained for B:9-23 (green) and insulin (red). Reconstruction is from a single stack (1.5 μm in thickness). Scale bar, 15 μm. (,) Reconstruction from a stack of 60 compiled optical sections acquired in 0.5-μm increments, stained for CD11c (red) and intracellular B:9-23 (green) in the absence () or presence () of competing free B:9-23 peptide. Scale bars, 20 μm. () Three-dimensional reconstruction by confocal microscopy of an islet stained for CD11c (red) and intracellular B:9-23 (green). Bottom left, projection along the z-axis (top view) from a stack of 30 optical sections acquired in 0.5-μm increments. Top and right, zx and zy rec! onstructions (side view) of the same image stack (white lines). Yellow merge indicates B:9-23 staining in the islet DC. Scale bar, 20 μm. () Reconstruction of an isolated islet DC costained for B:9-23, MHC class II and LAMP-1. Images represent a single stack with thickness of 1.5 μm. Scale bars, 10 μm. Data are representative of three (–) or two () independent experiments. Change history * Abstract * Change history * Author information * Supplementary informationCorrected online 05 March 2010In the version of this article initially published online, some hybridomas in Figure 3 were misidentified. The correct text for Figure 3c is "type A (4F7) and type B (2D10 and 1.7) hybridomas" and the correct text for Figure 3e,f is "type B (2D10; e) and type A (4F7; f) hybridomas." The error has been corrected for the print, PDF and HTML versions of this article. Author information * Abstract * Change history * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * James F Mohan & * Matteo G Levisetti Affiliations * Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, Missouri, USA. * James F Mohan, * Matteo G Levisetti, * Boris Calderon, * Jeremy W Herzog, * Shirley J Petzold & * Emil R Unanue * Present address: Merck, Rahway, New Jersey, USA. * Matteo G Levisetti Contributions J.F.M., M.G.L. and E.R.U. designed and evaluated the experimental work; J.F.M., M.G.L. and J.W.H. did most of the cellular experiments; B.C. did immunofluorescence and confocal microscopy; S.J.P. isolated insulin granules for mass spectrometry and quantified insulin content in granules; and J.F.M. and E.R.U. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Emil R Unanue (unanue@pathbox.wustl.edu) Supplementary information * Abstract * Change history * Author information * Supplementary information PDF files * Supplementary Text and Figures (400K) Supplementary Figures 1–6 and Supplementary Table 1 Additional data - RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci
- Nature Immunology 11(4):355-356 (2010)
Nature Immunology | Addendum RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci * Susannah L Hewitt Search for this author in: * NPG journals * PubMed * Google Scholar * Bu Yin Search for this author in: * NPG journals * PubMed * Google Scholar * Yanhong Ji Search for this author in: * NPG journals * PubMed * Google Scholar * Julie Chaumeil Search for this author in: * NPG journals * PubMed * Google Scholar * Katarzyna Marszalek Search for this author in: * NPG journals * PubMed * Google Scholar * Jeannette Tenthorey Search for this author in: * NPG journals * PubMed * Google Scholar * Giorgia Salvagiotto Search for this author in: * NPG journals * PubMed * Google Scholar * Natalie Steinel Search for this author in: * NPG journals * PubMed * Google Scholar * Laura B Ramsey Search for this author in: * NPG journals * PubMed * Google Scholar * Jacques Ghysdael Search for this author in: * NPG journals * PubMed * Google Scholar * Michael A Farrar Search for this author in: * NPG journals * PubMed * Google Scholar * Barry P Sleckman Search for this author in: * NPG journals * PubMed * Google Scholar * David G Schatz Search for this author in: * NPG journals * PubMed * Google Scholar * Meinrad Busslinger Search for this author in: * NPG journals * PubMed * Google Scholar * Craig H Bassing Search for this author in: * NPG journals * PubMed * Google Scholar * Jane A Skok Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature ImmunologyVolume:11,Pages:355–356Year published:(2010)DOI:doi:10.1038/ni0410-355 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Immunol.10, 655–664 (2009); published online 17 May 2009; corrected after print 18 May 2009, 19 August 2009 and 12 March 2010; addendum published online 12 March 2010. In the version of this article initially published online, some hybridomas in Figure 3 are misidentified. The correct text for Figure 3c is "type A (4F7) and type B (2D10 and 1.7) hybridomas" and the correct text for Figure 3e,f is "type B (2D10; ) and type A (4F7; ) hybridomas." The error has been corrected for the print, PDF and HTML versions of this article. In our published article, we found that RAG-dependent γ-H2AX foci showed monoallelic colocalization with Igh and Igk alleles in developing B lymphocytes. We sought to determine whether this was due to a stochastic cleavage mechanism or a regulated cleavage process. In and and initially published, we made a calculation we thought would yield the predicted number of cells in which we would expect to find biallelic colocalization of γ-H2AX foci if recombinase targeting were stochastic and compared that with the actual number obtained. The percentage of cells with biallelic colocalization of γ-H2AX foci in wild-type cells undergoing recombination was significantly lower than the predicted frequency. We believed this calculation supported the idea that monoallelic V(D)J recombination occurs as a result of the restriction of RAG-mediated cleavage to one allele and is not simply a result of stochastic recombination. It was called to our attention, however, that our calculation of the predicted frequencies of biallelic colocalization of γ-H2AX foci was incorrect and should have been done on an allele basis, not on a per-cell basis. After consulting with biostatisticians and after further reflection, we realized that it was not possible to accurately calculate a predicted frequency on either basis, because such a calculation of predicted frequencies rests on the assumption that pre-pro-B cells, pro-B cells and pre-B cells are homogeneous populations in which all alleles are equally available for cleavage. This assumption is clearly not true for the following reasons. Some cells have two alleles available for rearrangement, whereas others have already undergone a nonfunctional rearrangement on one allele and therefore only have one allele left to rearrange. Also, the calculation does not take account of cells that have functionally rearranged one allele, as these cells move to the next de! velopmental stage. Consequently, the data presented in and in and as initially published do not allow us to make any statements regarding whether monoallelic recombination in wild-type cells is due to a stochastic process or a regulated process. Thus, the discussion of on page 659 is now obsolete. However, we feel that the remainder of the text is still supported. We feel it is important to correct the article and to alert the community to the caveats raised above regarding attempts to make this sort of calculation. We therefore reexamined the data through a more straightforward lens, the χ2 test, and present here the results. We assessed the number of wild-type and Atm−/− cells at various stages of development showing monoallelic or biallelic association of γ-H2AX with immunoglobulin loci (Addendum Fig. 1). These data represent the percentage of cells with monoallelic or biallelic association of γ-H2AX with Igh or Igk; the actual number of cells analyzed (n) and statistical P values comparing wild-type and Atm−/− cells are in Addendum Tables 1 and 2. The numbers of cells analyzed differ from those originally published, as we have added new experiments to make this statistical calculation more robust. The frequency with which γ-H2AX is associated with immunoglobulin alleles varies between experiments because the vagaries o! f both FISH and mice. Because of this, we always assessed wild-type controls alongside the mutants, and the results consistently showed differences between wild-type and Atm−/− mice. ATM deficiency caused a statistically significant increase in biallelic cleavage, as shown by biallelic γ-H2AX foci in both pre-pro-B cells and pre-B cells, with the greatest difference occurring in pre-B cells. Our analysis of monoallelic and biallelic cleavage in pro-B cells in the absence of ATM is complicated because DH-JH recombination has already occurred, creating many breaks in chromosomes that are not repaired rapidly in the absence of ATM, which decreases the number of alleles available for possible VH-DJH rearrangements in the next developmental stage. Pro-B cells thus contain many broken and missing alleles, and as we assigned scores only to the association of γ-H2AX with immunoglobulin alleles in cells with two alleles, the numbers we could analyze were lower. We believe this! leads to a misleadingly small set of pro-B cells with biallel! ic cleavage from ATM-deficient mice. These results, together with the molecular data presented in of the original article, provide support for the conclusion that RAG cleavage activates ATM-mediated signals that inhibit further V(D)J recombination to suppress biallelic recombination and diminish the risk of chromosome translocations. Figure 1: Wild-type and Atm−/− cells at various stages of development with monoallelic or biallelic association of γ-H2AX with Ig loci. Results are presented as the percentage of pre-pro-B, pro-B and pre-B cells assigned scored as described in Addendum Tables 1 and 2. Data are a composite of three independent experiments. * Full size image (44 KB) * Figures/tables index Table 1: Statistical analysis of γ-H2AX foci co-localization with lg alleles Full table * Figures/tables index * Next table Table 2: Addendum Table 2 Statistical analysis of data in Addendum Table 1 using Fisher's exact test and 3 × 2 contingency: Full table * Previous table * Figures/tables index Additional data - RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci
- Nature Immunology 11(4):356 (2010)
Nature Immunology | Corrigendum RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci * Susannah L Hewitt Search for this author in: * NPG journals * PubMed * Google Scholar * Bu Yin Search for this author in: * NPG journals * PubMed * Google Scholar * Yanhong Ji Search for this author in: * NPG journals * PubMed * Google Scholar * Julie Chaumeil Search for this author in: * NPG journals * PubMed * Google Scholar * Katarzyna Marszalek Search for this author in: * NPG journals * PubMed * Google Scholar * Jeannette Tenthorey Search for this author in: * NPG journals * PubMed * Google Scholar * Giorgia Salvagiotto Search for this author in: * NPG journals * PubMed * Google Scholar * Natalie Steinel Search for this author in: * NPG journals * PubMed * Google Scholar * Laura B Ramsey Search for this author in: * NPG journals * PubMed * Google Scholar * Jacques Ghysdael Search for this author in: * NPG journals * PubMed * Google Scholar * Michael A Farrar Search for this author in: * NPG journals * PubMed * Google Scholar * Barry P Sleckman Search for this author in: * NPG journals * PubMed * Google Scholar * David G Schatz Search for this author in: * NPG journals * PubMed * Google Scholar * Meinrad Busslinger Search for this author in: * NPG journals * PubMed * Google Scholar * Craig H Bassing Search for this author in: * NPG journals * PubMed * Google Scholar * Jane A Skok Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature ImmunologyVolume:11,Page:356Year published:(2010)DOI:doi:10.1038/ni0410-356 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Immunol.10, 655–664 (2009); published online 17 May 2009; corrected after print 18 May 2009, 19 August 2009 and 12 March 2010; addendum published online 12 March 2010. In the version of this article initially published, two values on page 657, the title to Figure 3 and several Supplementary Table citations are incorrect. Sentence 2 of paragraph 2 on page 657, column 2, should end "than those in similar wild-type populations (55% and 61%, respectively)." The title for Figure 3 should begin "RAG-1 cleavage marks paired immunoglobulin alleles differently...." The Supplementary Table citations should be as follows: Figure 3 legend, "(complete statistical results, )"; Figure 5 legend, "(complete statistical results, , , , , and )"; Figure 8 legend, "(complete statistical results, , and )"; and page 662, column 2, sentence 2 " and ." The errors have been corrected in the HTML and PDF versions of the article. Additional data
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