Latest Articles Include:
- Support for peer review
- Nat Immunol 11(12):1063 (2010)
Does peer review help or hinder science? - Multistoried roles for B lymphocytes in autoimmunity
- Nat Immunol 11(12):1065-1068 (2010)
Nature Immunology | Meeting Report Multistoried roles for B lymphocytes in autoimmunity * Masaki Hikada1 Search for this author in: * NPG journals * PubMed * Google Scholar * Moncef Zouali2moncef.zouali@wanadoo.fr Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature ImmunologyVolume: 11,Pages:1065–1068Year published:(2010)DOI:doi:10.1038/ni1210-1065Published online16 November 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 In autoimmune disease, multifaceted approaches are being explored to tailor a particular treatment that targets a specific cell at the appropriate disease stage. The key roles of B lymphocytes have enabled them to enter the clinical arena. In turn, clinical trials have suggested several leads for future research. View full text Author information * Abstract * Author information Affiliations * Masaki Hikada is with the Center for Innovation in Immunoregulative Technology and Therapeutics, Graduate School of Medicine, Kyoto University, Kyoto, Japan. * Moncef Zouali is with Institut National de la Santé et de la Recherche Médicale U606, and Université Paris 7, Unité Mixte de Recherche-S 606, Paris, France. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Moncef Zouali (moncef.zouali@wanadoo.fr) Additional data - Ten years of the Global Alliance for Vaccines and Immunization: challenges and progress
- Nat Immunol 11(12):1069-1072 (2010)
Diseases preventable by underused vaccines cause the death of approximately 3 million children per year. The Global Alliance for Vaccines and Immunization (GAVI) was launched 10 years ago to tackle this appalling situation. - Lymphocytes, Jim Gowans and in vivo veritas
- Nat Immunol 11(12):1073-1075 (2010)
Jim Gowans opened a new era of immunology by combining physiological in vivo studies with prospective enrichment of small lymphocytes, validating the clonal selection theory, discovering lymphocyte homing and recirculation, and training others to follow. - Regulatory ripples
- Nat Immunol 11(12):1077-1078 (2010)
Regulatory T cells come in many different forms depending on their mode of action or developmental origin. Data now show that interleukin 35, an immunomodulatory cytokine secreted by regulatory T cells, and interleukin 10 induce so-called 'iTR35 cells', which may have an important role in the phenomenon of infectious tolerance. - Skin function for human CD1a-reactive T cells
- Nat Immunol 11(12):1079-1080 (2010)
Human CD4+ T cells that produce interleukin 22 are an essential component of skin defense and repair. New evidence shows that these T cells recognize CD1a-lipid complexes on Langerhans cells. - More things in heaven and earth: defining innate and adaptive immunity
- Nat Immunol 11(12):1080-1082 (2010)
Natural killer cells have emerged as key components of innate immunity with critical antimicrobial functions. New work showing that they can also be accessed by vaccination to deliver antigen-specific memory responses and protect against subsequent viral infections challenges the traditional distinctions made between innate and adaptive immunity. - Research Highlights
- Nat Immunol 11(12):1083 (2010)
- A genetically selective inhibitor demonstrates a function for the kinase Zap70 in regulatory T cells independent of its catalytic activity
- Nat Immunol 11(12):1085-1092 (2010)
Nature Immunology | Article A genetically selective inhibitor demonstrates a function for the kinase Zap70 in regulatory T cells independent of its catalytic activity * Byron B Au-Yeung1 Search for this author in: * NPG journals * PubMed * Google Scholar * Susan E Levin1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Chao Zhang2 Search for this author in: * NPG journals * PubMed * Google Scholar * Lih-Yun Hsu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Debra A Cheng1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nigel Killeen3 Search for this author in: * NPG journals * PubMed * Google Scholar * Kevan M Shokat2 Search for this author in: * NPG journals * PubMed * Google Scholar * Arthur Weiss1aweiss@medicine.ucsf.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 11,Pages:1085–1092Year published:(2010)DOI:doi:10.1038/ni.1955Received06 May 2010Accepted29 September 2010Published online31 October 2010Corrected online04 November 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 To investigate the role of the kinase Zap70 in T cells, we generated mice expressing a Zap70 mutant whose catalytic activity can be selectively blocked by a small-molecule inhibitor. We found that conventional naive, effector and memory T cells were dependent on the kinase activity of Zap70 for their activation, which demonstrated a nonredundant role for Zap70 in signals induced by the T cell antigen receptor (TCR). In contrast, the catalytic activity of Zap70 was not required for activation of the GTPase Rap1 and inside-out signals that promote integrin adhesion. This Zap70 kinase–independent pathway was sufficient for the suppressive activity of regulatory T cells (Treg cells), which was unperturbed by inhibition of the catalytic activity of Zap70. Our results indicate Zap70 is a likely therapeutic target. View full text Figures at a glance * Figure 1: T cell activation is dependent on the catalytic activity of Zap70. () Flow cytometry of Zap70+/− and Zap70(AS) CD4+ cells loaded with the fluorescent Ca2+ indicator Fluo-3. For T cells, arrows indicate the simultaneous addition of 3-MB-PP1 and mAb to CD3ε (left) and the addition of crosslinking antibodies (right); for B cells, arrow indicates the addition of anti-IgM plus 3-MB-PP1 (dose, key). () Flow cytometry analysis of staining for phosphorylated Erk (p-Erk) in Zap70+/− and Zap70(AS) CD4+ T cells left unstimulated (Unstim) or stimulated with biotinylated anti-CD3ε and anti-CD4 plus streptavidin in the presence of vehicle only or 5 μM or 10 μM 3MB-PP1 alone or 10 μM 3MB-PP1 plus PMA. () Frequency of CD69+Zap70+/− and Zap70(AS) T cells after stimulation for 16 h with plate-bound anti-CD3ε and anti-CD28 in the presence of 3-MB-PP1 (concentration, horizontal axis); results are presented to those of vehicle-treated cells, set as 100%. () Immunoblot analysis of whole-cell lysates of purified Zap70+/− and Zap70(AS) CD4+ T cells l! eft unstimulated or stimulated for 2 min with anti-CD3ε and crosslinking antibodies in the presence of vehicle or 5 μM or 10 μM 3-MB-PP1; blots probed with antibodies specific for epitopes along left margin (phosphorylated (p-) residue in parentheses; Y, tyrosine). Data are representative of at least three independent experiments. * Figure 2: Proliferation of CD4+ T cells requires the catalytic activity of Zap70. () [3H]thymidine uptake by Zap70+/− and Zap70(AS) purified polyclonal CD4+ T cells stimulated for 72 h with plate-bound anti-CD3ε and anti-CD28 in the presence of vehicle or 3-MB-PP1 (concentration, horizontal axis). () CFSE dilution in CFSE-loaded Zap70+/− OT-II T cells (gray filled histograms) and Zap70(AS) OT-II T cells (black lines) stimulated for 72 h with irradiated APCs and 1 μM ovalbumin peptide (amino acids 323–339) in the presence of 3-MB-PP1 (concentration, above plots). Data are representative of three experiments (mean and s.d. of triplicate cultures in ). * Figure 3: Execution of effector T cell functions requires the catalytic activity of Zap70. () Flow cytometry analysis of staining for IFN-γ and IL-4 in effector TH1 and TH2 Zap70+/− and Zap70(AS) OT-II cells, respectively, cultured with vehicle or with 3-MB-PP1 (concentration, above plots). Far right, separate sample of cells cultured with 3-MB-PP1 and stimulated with PMA and ionomycin (iono) as a control to bypass requirements for proximal TCR pathways. Numbers in quadrants indicate percent cells in each. () Lysis of allogeneic 51Cr-labeled P815 target cells by Zap70+/− and Zap70(AS) CTLs in cultures containing effector and target cells (ratio, horizontal axes) supplemented with vehicle alone (dimethyl sulfoxide (DMSO)), CsA or 10 μM 3-MB-PP1. () Frequency of TNF+ cells among alloreactive Zap70+/− and Zap70(AS) CTLs generated as in and stimulated by P815 cells in the presence of 3-MB-PP1 or CsA (final concentration, horizontal axes). Data are representative of three experiments (error bars (), s.d.). * Figure 4: CD8+ memory responses are dependent on the catalytic activity of Zap70. (,) Flow cytometry analysis of the production of IFN-γ and TNF by Zap70+/− and Zap70(AS) memory CD8+ cells after stimulation with the LCMV peptides glycoprotein, amino acids 33–41 (GP(33–41); ), or nucleoprotein, amino acids 396–404 (NP(396–404); ), in the presence of vehicle alone or 10 μM 3-MB-PP1. Plots are gated on CD8+CD44hi memory cells; numbers in quadrants indicate percent cells in each. (,) Frequency of IFN-γ+TNF+ cells at various concentrations of 3-MB-PP1 (horizontal axes) among CD8+CD44hi memory Zap70+/− and Zap70(AS) cells (n = 3 mice per genotype). Data are representative of three experiments (mean and s.d. in ,). * Figure 5: The catalytic activity of Zap70 is dispensable for the suppressive activity of Treg cells in vitro. () In vitro suppression assays with CD4+CD25− Tconv cells from Zap70+/− and Zap70(AS) mice, together with irradiated APCs, mAb to CD3ε and 'titrated' numbers of Zap70+/− or Zap70(AS) CD4+CD25+ Treg cells, cultured in the presence of vehicle alone or 5 μM or 10 μM 3-MB-PP1, presented as [3H]thymidine uptake. () [3H]thymidine uptake by Tconv cells or Treg cells alone in the presence of vehicle alone or 3-MB-PP1. () Immunoblot analysis of whole-cell lysates of purified Zap70+/− and Zap70(AS) Tconv and Treg cells, probed for expression of Syk, Zap70 and Erk1-Erk2 (Erk1/2). Data are representative of at least three experiments (mean and s.d. of triplicate cultures in ,). * Figure 6: TCR-induced activation of Rap1 and adhesion to ICAM-1 are independent of Zap70 kinase activity. () Flow cytometry analysis of intracellular calcium in CD4+CD25− Tconv and CD4+CD25+ Treg cells loaded with the fluorescent calcium indicators Fluo-3 and Fura red and stimulated in the presence of vehicle alone (red) or 3-MB-PP1 (blue); arrows indicate the addition of biotinylated anti-CD3ε and anti-CD4 (1), streptavidin (2) and ionomycin (3). Tconv cells were >99% Foxp3−; Treg cells were >98% Foxp3+. () Flow cytometry analysis of phosphorylated Erk in splenocytes stimulated ex vivo by crosslinking mAb to CD3 and left unstimulated or stimulated in the presence of vehicle alone, 5 μM or 10 μM 3-MB-PP1 alone, or 10 μM 3-MB-PP1 plus PMA; plots are gated on Foxp3− or Foxp3+ CD4+ cells. () Immunoassay of lysates of Zap70(AS) thymocyte stimulated for 2 min with crosslinking mAb to CD3; samples immunoprecipitated (IP) with anti-CrkII and whole-cell lysates (WCL) were analyzed by immunoblot (IB) for Zap70 and CrkII. () Precipitation of Rap1-GTP from Zap70+/− and Zap70(AS! ) thymocytes left unstimulated or stimulated for 2 min with crosslinking mAb to CD3 and treated with vehicle alone or 10 μM 3-MB-PP1. Below, total Rap1 in whole-cell lysates. () Flow cytometry of Zap70+/− and Zap70(AS) CD4+ T cells stimulated for 10 min with anti-CD3ε in wells coated with recombinant ICAM-1, then washed and counted. Data are representative of three independent experiments (error bars (), s.d.). * Figure 7: TCR-induced activation of Rap1 and adhesion to ICAM-1 are dependent on the adaptor function of Zap70. () Precipitation of Rap1-GTP from Zap70+/− and Zap70(YYAA) thymocytes stimulated for 2 min with the various doses of anti-CD3ε (above lanes) in the absence of 3-MB-PP1. () ICAM-1 adhesion by peripheral Zap70+/− and Zap70(YYAA) CD4+ T cells left unstimulated or stimulated (as in Fig. 6d) with 5 μg/ml or 10 μg/ml of anti-CD3ε. Data are representative of three independent experiments (error bars (), s.d.). Change history * Abstract * Change history * Author information * Supplementary informationCorrigendum 04 November 2010In the version of this article initially published online, the key above Figure 5a was mislabeled. The correct label is Treg:Tconv. The error has been corrected for the print, PDF and HTML versions of this article. Author information * Abstract * Change history * Author information * Supplementary information Affiliations * Howard Hughes Medical Institute, Rosalind Russell Medical Research Center for Arthritis, Department of Medicine, Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California, USA. * Byron B Au-Yeung, * Susan E Levin, * Lih-Yun Hsu, * Debra A Cheng & * Arthur Weiss * Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA. * Chao Zhang & * Kevan M Shokat * Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California, USA. * Nigel Killeen * Present address: Department of Biology, Tufts University, Medford, Massachusetts. * Susan E Levin Contributions B.B.A.-Y. did most of the experiments and wrote the paper; S.E.L. did the initial characterization of Zap70(AS) mice; N.K. designed the strategy for generating Zap70(AS) mice; D.A.C. assisted with the ICAM-1 adhesion assays and calcium-flux assays; L.-Y.H. helped with the Zap70(YYAA) Treg cell–suppression assay; K.M.S. and C.Z. provided advice and synthesized 3-MB-PP1; and A.W. directed the project. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Arthur Weiss (aweiss@medicine.ucsf.edu) Supplementary information * Abstract * Change history * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–7 Additional data - IL-35-mediated induction of a potent regulatory T cell population
- Nat Immunol 11(12):1093-1101 (2010)
Nature Immunology | Article IL-35-mediated induction of a potent regulatory T cell population * Lauren W Collison1 Search for this author in: * NPG journals * PubMed * Google Scholar * Vandana Chaturvedi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Abigail L Henderson1, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Paul R Giacomin2 Search for this author in: * NPG journals * PubMed * Google Scholar * Cliff Guy1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jaishree Bankoti1 Search for this author in: * NPG journals * PubMed * Google Scholar * David Finkelstein3 Search for this author in: * NPG journals * PubMed * Google Scholar * Karen Forbes1 Search for this author in: * NPG journals * PubMed * Google Scholar * Creg J Workman1 Search for this author in: * NPG journals * PubMed * Google Scholar * Scott A Brown1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jerold E Rehg4 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael L Jones5 Search for this author in: * NPG journals * PubMed * Google Scholar * Hsiao-Tzu Ni6 Search for this author in: * NPG journals * PubMed * Google Scholar * David Artis2 Search for this author in: * NPG journals * PubMed * Google Scholar * Mary Jo Turk7 Search for this author in: * NPG journals * PubMed * Google Scholar * Dario A A Vignali1vignali.lab@stjude.org Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 11,Pages:1093–1101Year published:(2010)DOI:doi:10.1038/ni.1952Received02 September 2010Accepted27 September 2010Published online17 October 2010 Abstract * Abstract * Accession codes * 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 Regulatory T cells (Treg cells) have a critical role in the maintenance of immunological self-tolerance. Here we show that treatment of naive human or mouse T cells with IL-35 induced a regulatory population, which we call 'iTR35 cells', that mediated suppression via IL-35 but not via the inhibitory cytokines IL-10 or transforming growth factor-β (TGF-β). We found that iTR35 cells did not express or require the transcription factor Foxp3, and were strongly suppressive and stable in vivo. Treg cells induced the generation of iTR35 cells in an IL-35- and IL-10-dependent manner in vitro and induced their generation in vivo under inflammatory conditions in intestines infected with Trichuris muris and within the tumor microenvironment (B16 melanoma and MC38 colorectal adenocarcinoma), where they contributed to the regulatory milieu. Thus, iTR35 cells constitute a key mediator of infectious tolerance and contribute to Treg cell–mediated tumor progression. Furthermore, iTR35 ce! lls generated ex vivo might have therapeutic utility. View full text Figures at a glance * Figure 1: Treatment of Tconv cells with human IL-35 confers a regulatory phenotype. () Quantitative PCR analysis of the expression of EBI3 mRNA (left) and IL12A mRNA (right) generated from RNA extracted from Tconv cells purified from cord blood and treated for 9 d with supernatants of HEK293T human embryonic kidney cells transfected with IL-35-expressing vector or control (empty) vector during activation with human IL-2 plus beads coated with anti-CD3 and anti-CD28; results are presented relative to those of control-treated Tconv cells. () Flow cytometry quantification of IL-35 in cells repurified from those in and activated for 4 h with the phorbol ester PMA and ionomycin, then stained with anti-p35 or isotype-matched control antibody (isotype). () Microscopy of p35 expression, assessed as in : yellow, anti-p35 or isotype-matched control antibody; grey, phalloidin; blue, DNA-intercalating dye (DAPI). Original magnification, ×63. () Proliferation of the cells in , assessed by incorporation of [3H]thymidine. () Suppression of freshly purified responder Tcon! v cells by control or IL-35-treated Tconv cells, mixed at various ratios (Tconv/treated Tconv) and cultured together for 9 d with activation as in . () Suppressive effect of control-treated or IL-35-treated Tconv cells (in the top chamber of Transwell plate) toward responder Tconv cells (in the bottom chamber), both activated as in , with [3H]thymidine added directly to the responder cells for the final 8 h of the 9-day assay. () Suppression of responder Tconv cells by IL-35-treated Tconv cells, as in , supplemented with neutralizing (NT Ab) anti-IL-10, anti-TGF-β or anti-IL-35. NS, not significant; *P < 0.05, **P < 0.005 and ***P < 0.001 (unpaired t-test). Data represent ten (,–), four () or three () independent experiments (mean and s.e.m.). * Figure 2: Treatment of Tconv cells with mouse IL-35 converts them into an IL-35-producing suppressive population. () Expression of Ebi3 and Il12a mRNA in control-treated or IL-35-treated Tconv cells activated for 72 h with beads coated with anti-CD3 and anti-CD28; results are presented relative to those of control-treated Tconv cells. () Immunoblot analysis of supernatants of wild-type (WT) or Ebi3−/− Tconv cells activated as in in the presence of supernatants of HEK293T cells transfected with IL-35-expressing vector (IL-35 SN) or control vector (Con SN), then repurified and cultured for an additional 24 h, followed by immunoprecipitation with monoclonal anti-p35; blots were probed with monoclonal anti-Ebi3. Far left two lanes, untreated Tconv cells and Treg cells (controls). () Proliferative capacity of control-treated or IL-35-treated Tconv cells, prepared as in , stimulated with beads coated with anti-CD3- and anti-CD28. () Proliferation of responder Tconv cells mixed at various ratios with control-treated or IL-35-treated Tconv cells (Tconv/treated Tconv) and cultured for 72 h i! n the presence of beads coated with anti-CD3 and anti-CD28. () Proliferation of responder Tconv cells (activated with beads coated with anti-CD3 and anti-CD28) in the bottom chamber of a Transwell plate, in the presence of control- or IL-35-treated Tconv cells in the top chamber stimulated with beads coated with anti-CD3- and anti-CD28. Results are presented relative to those of responder Tconv cells stimulated in the absence of cells in the top chamber. () Suppression of the proliferation of responder Tconv cells by wild-type, Ebi3−/− or Il10−/− IL-35-treated Tconv cells, prepared as in , at a ratio of 4:1 (responder Tconv cells/treated Tconv cells). () Suppression of the proliferation of responder Tconv cells as in , in the presence of neutralizing anti-IL-10, anti-TGF-β or anti-IL-35. () Suppressive capacity of IL-35-treated Tconv cells (Tconv + IL-35), assessed as in in the presense of neutralizating anti-IL-35 (NT IL-35) or isotype-matched control antibody (is! otype). *P < 0.05, **P < 0.005 and ***P < 0.001 (unpaired t-te! st). Data represent four to eight independent experiments (mean and s.e.m.). * Figure 3: Suppressive effects of iTR35 cells in vivo. (–) Prevention of disease in 3-week-old Foxp3−/− mice given intraperitoneal injection (at 2 or 3 d of age) of nTreg cells, iTRcon cells or wild-type, Ebi3−/− or Il12a−/− iTR35 cells or TGF-β–iTR cells, monitored by clinical score (), splenic CD4+ T cell numbers () and histology (). Far left (WT −), untreated, age-matched, wild-type littermates (control). () Histopathology of ear pinna of Foxp3−/− mice treated as in – or their untreated wild-type littermates. Original magnification, ×10. (,) Homeostatic population expansion of Thy-1.1+ Tconv cells (as target cells; ) injected intravenously into Rag1−/− mice alone or with Thy-1.2+ iTRcon cells, wild-type iTR35 cells or Ebi3−/− iTR35 cells (as regulatory cells; ). () EAE scores of C57BL/6 mice injected intravenously with wild-type nTreg, iTRcon, wild-type or Ebi3−/− iTR35 cells 12–18 h before disease induction via immunization with a peptide of amino acids 35–55 of myelin oligodendrocyt! e glycoprotein in complete Freund's adjuvant and pertussis toxin. P = 0.002, PBS versus iTR35 (Wilcoxon matched-pairs test). () Tumor diameter in Rag1−/− mice injected intravenously with various combinations of cells (key) and then injected intradermally with B16 cells. P = 0.0025, CD4 + CD8 versus CD4 + CD8 + nTreg, and P = 0.0048, CD4 + CD8 versus CD4 + CD8 + iTR35 (Wilcoxon matched-pairs test). (,) Weight () and colonic histology scores () of Rag1−/− mice injected intravenously with Tconv cells; after 3–4 weeks, mice that developed signs of IBD were given iTRcon cells or iTR35 cells. *P < 0.05, **P < 0.005 and ***P < 0.001 (unpaired t-test). Data represent at least two independent experiments with eight to twelve mice per group (mean and s.e.m.). * Figure 4: Stability of iTR35 cells and TGF-β–iTR cells in vivo. () Recovery of splenic iTR cells 25 d after adoptive transfer of iTR35 or TGF-β–iTR cells (generated in vitro from CD45.2+ Tconv cells) into CD45.1+ C57BL/6 mice, assessed by flow cytometry of CD45.2+ cells and presented as percentage of total cells injected. () Supression of Tconv cell proliferation by either freshly generated iTR cells (Before transfer) or iTR cells recovered after a period of in vivo 'resting' (After transfer), mixed at various ratios (Tconv/Treg) and stimulated for 72 h with beads coated with anti-CD3 and anti-CD28, assessed by [3H]thymidine incorporation. () Disease development in Foxp3−/− mice injected with natural Treg cells or iTR cells at 2–3 d of age; mice with a clinical score of 4 were considered moribund. *P < 0.05 and **P < 0.001 (unpaired t-test). Data represent at least two independent experiments with five to twelve mice per group (mean and s.e.m.). * Figure 5: Treg cells generate iTR35 cells in an IL-35- and IL-10-dependent manner. (,) Expression of Ebi3 () and Il12a () by Tconv cells activated for 72 h with Treg cells at a ratio of 4:1 in the presence (+) or absence (–) of beads coated with anti-CD3 and anti-CD28. () Immunoprecipitation (IP) and immunoblot (IB) analysis of the secretion of IL-35 into supernatants of Tconv cells suppressed by coculture as in , then repurified and cultured for an additional 24 h. () Proliferation of suppressed Tconv cells cocultured as in , then repurified and activated with anti-CD3 and anti-CD28. () Suppression of responder Tconv cell proliferation by suppressed Tconv cells generated as in . () Suppression of responder Tconv cell proliferation by suppressed Tconv cells generated as in , in the presence of neutralizing anti-IL-10, anti-TGF-β or anti-IL-35 during either culture with nTreg cells (Conversion; top) or suppression of responder Tconv cells (Function; bottom). () Splenic T cell numbers in Rag1−/− mice injected with Tconv cells (target cell) alone or to! gether with suppressed Tconv cells (as regulatory cells), assessed 7 d after transfer. () EAE scores of C57BL/6 mice injected intravenously with suppressed Tconv cells, nTreg cells or saline control 12–18 h before disease induction (via immunization as in Fig. 3g). *P < 0.05, **P < 0.005 and ***P < 0.001 (–; unpaired t-test). Data represent at least two independent experiments with eight to twelve mice per group (mean and s.e.m.). * Figure 6: IL-35-producing Foxp3− iTR35 cells develop in vivo. (,) Expression of Ebi3 () and Il12a () in CD4+Foxp3− and CD4+Foxp3+ cells purified from Foxp3gfp mice infected with T. muris. (,) Expression of Ebi3 () and Il12a () in CD4+Foxp3− and CD4+Foxp3+ cells purified from tumors and spleens excised from Foxp3gfp mice 15–17 d after intradermal injection of B16 cells (1.2 × 105). (,) Expression of Ebi3 () and Il12a () in CD4+Foxp3− and CD4+Foxp3+ cells purified from tumors and spleens excised from Foxp3gfp mice 12 d after subcutaneous injection of MC38 cells (2 × 106). Results in – are presented relative to those of CD4+Foxp3− cells purified from the spleen. () Immunoprecipitation (with monoclonal anti-p35) and immunoblot analysis (with monoclonal anti-Ebi3) of the secretion of IL-35 into supernatants of CD4+Foxp3− and CD4+Foxp3+ cells purified from Foxp3gfp or Ebi3−/−Foxp3gfp mice as in , and cultured for 24 h. () Proliferation of cells purified as in and mixed for 72 h at a ratio of 4:1 with fresh responder Tconv! cells, assessed by [3H]thymidine incorporation. *P < 0.05, **P < 0.005 and ***P < 0.001 (unpaired t-test). Data represent two to three independent experiments (B16) or one experiment (MC38) with eight to ten mice per group (mean and s.e.m.). * Figure 7: The suppressive T cell milieu in the tumor microenvironment is largely due to iTR35 cells. Tumor diameter of Rag1−/− mice reconstituted with wild-type C57BL/6 CD8+ T cells and wild-type or Ebi3−/− CD4+ Tconv cells with or without wild-type Treg cells, then injected intradermally the next day on the right flank with B16 cells (1.2 × 105), assessed on day 15 after inoculation. *P < 0.05 and **P < 0.001 (unpaired t-test). Data represent at least two independent experiments with six to twelve mice per group (mean and s.e.m.). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE24210 * GSM595497 * GSM595512 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. * Lauren W Collison, * Vandana Chaturvedi, * Abigail L Henderson, * Cliff Guy, * Jaishree Bankoti, * Karen Forbes, * Creg J Workman, * Scott A Brown & * Dario A A Vignali * Department of Microbiology and Department of Pathobiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA. * Paul R Giacomin & * David Artis * Department of Bioinformatics, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. * David Finkelstein * Department of Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. * Jerold E Rehg * Shenandoah Biotechnology, Monroe, Ohio, USA. * Michael L Jones * Department of Antibody Applications, R&D Systems, Minneapolis, Minnesota, USA. * Hsiao-Tzu Ni * Department of Microbiology and Immunology, Dartmouth Medical School, Lebanon, New Hampshire, USA. * Mary Jo Turk * Present address: Department of Research, Des Moines University, Des Moines, Iowa, USA. * Abigail L Henderson Contributions L.W.C. designed (with help from D.A.A.V.) and did all mouse experiments, analyzed data and wrote the manuscript; V.C. did human experiments; A.L.H. (with L.W.C.) did the B16 tumor experiments; J.B. did the MC38 tumor experiments; P.R.G. infected mice with T. muris; C.G. did confocal microscopy; D.F. analyzed Affymetrix data; K.F. and S.A.B (with C.J.W.) generated and screened monoclonal antibodies to IL-35; C.J.W. coordinated the development of monoclonal anti-IL-35 and aided in figure preparation; M.L.J. generated and purified mouse Ebi3 protein for immunization and the development of monoclonal antibodies; H.-T.N. provided reagents and information; J.E.R. created and did histological analyses of Foxp3−/− mice; D.A. designed T. muris experiments and provided input on their interpretation; M.J.T. provided training for the B16 tumor model and provided input to research design and interpretation; and D.A.A.V. conceived of the research, directed the study and edited the man! uscript. Competing financial interests D.A.A.V. and L.W.C. have submitted a patent based on this work that is now pending. Also, D.A.A.V., L.W.C. and C.J.W. have also submitted a patent on IL-35 and are entitled to a share in net income generated from licensing of these patent rights for commercial development. M.L.J. is employed by Shenandoah Biotechnology and H.-T.N. is employed by R&D Systems. Corresponding author Correspondence to: * Dario A A Vignali (vignali.lab@stjude.org) Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (844K) Supplementary Figures 1–18 Additional data - CD1a-autoreactive T cells are a normal component of the human αβ T cell repertoire
- Nat Immunol 11(12):1102-1109 (2010)
Nature Immunology | Article CD1a-autoreactive T cells are a normal component of the human αβ T cell repertoire * Annemieke de Jong1 Search for this author in: * NPG journals * PubMed * Google Scholar * Victor Peña-Cruz2 Search for this author in: * NPG journals * PubMed * Google Scholar * Tan-Yun Cheng1 Search for this author in: * NPG journals * PubMed * Google Scholar * Rachael A Clark3 Search for this author in: * NPG journals * PubMed * Google Scholar * Ildiko Van Rhijn1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * D Branch Moody1bmoody@rics.bwh.harvard.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 11,Pages:1102–1109Year published:(2010)DOI:doi:10.1038/ni.1956Received26 March 2010Accepted30 September 2010Published online31 October 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 CD1 activates T cells, but the function and size of the possible human T cell repertoires that recognize each of the CD1 antigen-presenting molecules remain unknown. Using an experimental system that bypasses major histocompatibility complex (MHC) restriction and the requirement for defined antigens, we show that polyclonal T cells responded at higher rates to cells expressing CD1a than to those expressing CD1b, CD1c or CD1d. Unlike the repertoire of invariant natural killer T (NKT) cells, the CD1a-autoreactive repertoire contained diverse T cell antigen receptors (TCRs). Functionally, many CD1a-autoreactive T cells homed to skin, where they produced interleukin 22 (IL-22) in response to CD1a on Langerhans cells. The strong and frequent responses among genetically diverse donors define CD1a-autoreactive cells as a normal part of the human T cell repertoire and CD1a as a target of the TH22 subset of helper T cells. View full text Figures at a glance * Figure 1: Population study of CD1-autoreactive cells in blood of human donors. () Surface expression of MHC and CD1 on mock-transfected K562 cells (K562-mock) or K562 cells transfected with plasmids containing human genes encoding various CD1 proteins (above plots). Open histograms, MHC or CD1 staining; filled histograms, isotype-matched control antibody staining. Data are representative of three or more experiments. () Bioassay of IL-2 in supernatant of K562 cells incubated for 24 h with T cell lines recognizing CD1a and dideoxymycobactin (DDM); CD1b and glucose monomycolate (GMM); CD1c and mannosyl phosphomycoketide (MPM); or CD1d and α-galactosylceramide. Data are representative of three or more experiments (mean ± s.d.). () ELISPOT analysis of IFN-γ release by polyclonal cell cultures stimulated in vitro with autologous DCs and analyzed for CD1 reactivity, with K562-CD1 cells as APCs; results presented after subtraction of background spots formed in response to mock-transfected K562 cells. Each symbol represents an individual donor (mean of trip! licate measurements; n = 14 donors total); small horizontal lines indicate the mean for the group. *P < 0.01 (Dunnett's multiple-comparison test after one-way ANOVA). * Figure 2: Autoreactive T cells in the blood recognize CD1a. () Bioassay of IL-2 release from polyclonal T cell cultures incubated for 24 h with mock-transfected K562 cells (K562-mock) or with K562-CD1a cells alone (No Antibody) or K562-CD1a cells pretreated with CD1a-blocking mAb (Anti-CD1a) or isotype-matched control antibody (Control IgG). Data are representative of three or more experiments (mean and s.d.). () Bioassay of IL-2 release by T cell clones incubated for 24 h with mock-transfected K562 cells or K562-CD1a cells. Data are representative of two or more experiments with each clone. () CD1a-autoreactive T cell clones in a panel of 1,291 autoreactive T cell clones generated from 14 donors by ex vivo limiting dilution, each assessed in triplicate for IL-2 release as in or by IFN-γ ELISPOT (when the number of cells was limited). Clones were considered CD1a autoreactive if cytokine secretion increased more than threefold in the presence of unblocked CD1a. * Figure 3: Quantitative detection of CD1a-autoreactive memory T cells. ELISPOT assay of IFN-γ-secreting cells among CD45RO+ T cells incubated with mock-transfected K562 cells (m) or with K562-CD1a cells preincubated for 1 h with mAb to CD1a or control IgG. Precursor frequency (bottom row): mean number of spots in response to K562-CD1a cells plus control IgG minus mean number of spots in response to K562-CD1a cells plus mAb to CD1a/total cells per well. *P < 0.05 (two-tailed Student's t-test). Data are presented as mean and s.d. of triplicate measurements. * Figure 4: CD1a-autoreactive T cells in the blood express skin-homing markers. RT-PCR analysis (right) of RNA extracted from CD45RO−CLA− (gate 1) T cells, CD45RO+CLA− (gate 2) T cells or CD45RO+CLA+ (gate 3) T cells (sorting, left and middle), with primers confirmed to be specific for the CDR3 regions of the TCR α- and β-chains of a CD1a-autoreactive T cell clone. TRAC, TCRα constant region primers (control for input cDNA). Data are representative of three experiments (TCR sequences) or two experiments (sorted T cells). * Figure 5: CD1a-dependent IL-22 production. () Real-time PCR analysis of the upregulation of cytokine-encoding genes in polyclonal CD1a-autoreactive T cell cultures incubated for 6 h together with K562-CD1a cells pretreated with mAb to CD1a or control IgG at a K562 cell/T cell ratio of 1:10; results were normalized to β-actin, and results for incubation with K562-CD1a cells plus control IgG are presented relative to those for incubation with K562-CD1a cells plus mAb to CD1a. () Enzyme-linked immunosorbent assay of IL-22 in supernatants of T cell lines showing IL-22 upregulation that were incubated for 24 h alone (No APC) or with increasing numbers of mock-transfected K562 cells or K562-CD1a cells. () Intracellular staining of IL-17 and IL-22 in T cell lines from the donors in analyzed without stimulation (Unstim) or in response to stimulation with PMA and ionomycin. Data for IL-22 protein are representative of two or more experiments with two donors (mean ± s.d. of triplicate measurements). * Figure 6: The TH22 subset contains CD1a-autoreactive T cells. () Real-time PCR analysis of the cytokine profiles of memory CD4+ T cells sorted into CCR6+CXCR3+CCR4−CCR10−, CCR6+CXCR3−CCR4+CCR10− and CCR6+CXCR3−CCR4+CCR10+ fractions (far left) and stimulated for 6 h with anti-CD3. Data are representative of experiments with three donors. () Real-time PCR analysis of the upregulation of cytokine-encoding genes in sorted TH1 (top) or TH22 (bottom) T cell fractions that underwent a single in vitro expansion with DCs and were cultured for 6 h with K562-CD1a cells preincubated with mAb to CD1a or control IgG, at a K562 cell/T cell ratio of 1:10; results were normalized to β-actin, and results for K562-CD1a cells plus control IgG are presented relative to those for incubation with K562-CD1a cells plus mAb to CD1a. * Figure 7: IL-22 producing CD1a-autoreactive T cells in human skin. () Enzyme-linked immunosorbent assay of IL-22 release by lymphocytes obtained from normal human dermis samples, followed by population expansion in vitro with DCs and incubation for 24 h with CD1-transfected K562 cells Data are representative of one experiment with three donors (mean and s.d.). () Bioassay of IL-2 in supernatants of freshly isolated LCs (left), monocyte-derived DCs and K562-CD1a cells incubated for 24 h with a CD1a-autoreactive T cell line. Right, flow cytometry of CD1a on APCs; numbers in plots indicate mean fluorescence intensity (open histograms, CD1a staining; filled histograms, isotype-matched control antibody staining). Data are representative of three experiments (mean and s.d.). () Real-time PCR analysis of the upregulation of cytokine-encoding genes in a CD1a-autoreactive T cell line incubated for 18 h together with freshly isolated LCs pretreated with control IgG or mAb to CD1a; results were normalized to β-actin, and results for incubation with L! Cs plus control IgG are presented relative to those for incubation with LCs plus mAb to CD1a. Data are presented as mean ± s.d. of triplicate measurements. Author information * Abstract * Author information * Supplementary information Affiliations * Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Annemieke de Jong, * Tan-Yun Cheng, * Ildiko Van Rhijn & * D Branch Moody * Dana-Farber Cancer Institute, Boston, Massachusetts, USA. * Victor Peña-Cruz * Department of Dermatology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Rachael A Clark * Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. * Ildiko Van Rhijn Contributions A.d.J. designed and did the experiments; A.d.J. and D.B.M. prepared the manuscript; D.B.M. supervised the experiments; V.P.-C. isolated LCs from human epidermis and lymphocytes from the dermis; T.-Y.C. did T cell culture and immunoblot analysis; I.V.R. assisted in experiments; and R.A.C. provided T cells isolated from human skin biopsies. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * D Branch Moody (bmoody@rics.bwh.harvard.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–5 and Supplementary Table 1 Additional data - Plasma cells negatively regulate the follicular helper T cell program
- Nat Immunol 11(12):1110-1118 (2010)
Nature Immunology | Article Plasma cells negatively regulate the follicular helper T cell program * Nadége Pelletier1 Search for this author in: * NPG journals * PubMed * Google Scholar * Louise J McHeyzer-Williams1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kurt A Wong1 Search for this author in: * NPG journals * PubMed * Google Scholar * Eduard Urich1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Nicolas Fazilleau1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael G McHeyzer-Williams1mcheyzer@scripps.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 11,Pages:1110–1118Year published:(2010)DOI:doi:10.1038/ni.1954Received02 July 2010Accepted29 September 2010Published online31 October 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 B lymphocytes differentiate into antibody-secreting cells under the antigen-specific control of follicular helper T cells (TFH cells). Here we demonstrate that isotype-switched plasma cells expressed major histocompatibility complex (MHC) class II, the costimulatory molecules CD80 and CD86, and the intracellular machinery required for antigen presentation. Antigen-specific plasma cells accessed, processed and presented sufficient antigen in vivo to induce multiple helper T cell functions. Notably, antigen-primed plasma cells failed to induce interleukin 21 (IL-21) or the transcriptional repressor Bcl-6 in naive helper T cells and actively decreased these key molecules in antigen-activated TFH cells. Mice lacking plasma cells showed altered TFH cell activity, which provided evidence of this negative feedback loop. Hence, antigen presentation by plasma cells defines a previously unknown layer of cognate regulation that limits the antigen-specific TFH cell program that controls! ongoing B cell immunity. View full text Figures at a glance * Figure 1: Isotype-switched plasma cells retain expression of MHCII and costimulatory molecules. () NP-specific plasma cell (PC) numbers over time (negative for propidium iodide (PI−), the dump channel (Dump−) and IgD−, and NP+CD138+) in the spleens of C57BL/6 mice after primary and secondary immunization with NP-KLH (open square, adjuvant-only for memory at day 5). () Expression of CD138 versus IgM on NP-specific cells (gated as PI−Dump−IgD− and NP+) at day 5 after secondary challenge (left); BrdU expression on NP-specific IgM−CD138+ plasma cells on day 5 after rechallenge (middle); and frequency of BrdU+ NP-specific IgM−CD138+ plasma cells on days 3 and 5 after rechallenge (Memory), relative to that of the total IgM−CD138+ plasma cell compartment (right), 20 h after a single intraperitoneal injection of BrdU. Number in outlined area (left) indicates percent CD138+IgM− cells. (,) Expression of I-Ab (MHCII; ) and CD80 and CD86 () on NP-specific IgM−CD138+ plasma cells, CD4+ helper T cells (T), CD11c+ DCs and naive B cells (B), presented as summary ! mean fluorescence intensity (MFI). *P < 0.05 and **P < 0.01 (Student's t-test). Data are representative of four or more () or three or more (–) experiments with one mouse per experiment (mean and s.e.m.). * Figure 2: Plasma cells express the intracellular machinery for MHCII antigen processing and presentation. (,) Intracellular expression of CD74 (Ii) and I-Ab–CLIP () and of H-2M and H-2O () on NP-specific IgM−CD138+ plasma cells in C57BL/6 spleens on day 5 after secondary immunization with NP-KLH, and in naive B cells and CD4+ helper T cells, presented as summary MFI. () Quantitative PCR analysis of the expression of mRNA for various proteases (vertical axes) in CD4+ helper T cells, CD11c+ DCs, naive B cells and IgM−CD138+ plasma cells, presented in arbitrary units (AU) relative to expression in naive T cells, set as 1. *P < 0.01 and **P < 0.001 (Student's t-test). Data are representative of three experiments (,) or three or more experiments () with one mouse each (mean and s.e.m.). * Figure 3: Antigen-specific plasma cells process and present antigen in vivo. () Isotype-switched B cell compartment (Dump−IgD−IgM− and CD19+CD138+; far left), HEL binding versus CD138 expression (middle) and total number of switched HEL-specific plasma cells (far right) in B10.BR mice given primary immunization with HEL or PCC (above plots or below graph), assessed on day 5 after secondary challenge with soluble HEL. Numbers adjacent to outlined areas indicate percent CD138+CD19+ cells (far left) or HEL+CD138+ cells (middle). () Isotype-switched B cells (Dump−IgD−IgM−; far left), HEL binding versus pMHCII expression (middle) and summary MFI for pMHCII (far right), for cells from mice treated as in . () Expression of CD44 and CFSE on CFSE-labeled 5C.C7 αβTCR–transgenic helper T cells cultured for 4 d with isotype-switched plasma cells isolated 10 d after vaccination of mice with adjuvant only (far left) or with PCC protein (middle), either alone (PCC) or with the addition of MCC peptide (+ peptide); numbers in outlined areas indicate f! requency of responders. Far right, total CD44hiCFSElo helper T cells. () Expression of CD44 and CFSE (left and middle) on CFSE-labeled 3A9 αβTCR–transgenic HEL-specific helper T cells 24 h after transfer into B10.BR mice immunized with peptide alone (HEL) amino acids 48–61 (HEL(48–61) only), given transfer of IgM−CD138+ plasma cells pulsed with 10 μM HEL peptide (Peptide-pulsed PCs), or immunized with HEL in adjuvant (HEL + adjuvant; n = 1 mouse); numbers in outlined areas indicate frequency of responders. Far right, total CD44hiCFSElo helper T cells. *P < 0.05 and **P < 0.01 (Student's t-test). Data are representative of three experiments with one mouse each (mean and s.e.m.). * Figure 4: Plasma cells induce proliferation, many helper T cell functions and Blimp-1 expression but not expression of Bcl-6 or IL-21. () Expression of CD44 and CFSE (left and middle) on CFSE-labeled 5C.C7 αβTCR–transgenic helper T cells activated for 4 d in vitro with IgM−CD138+ plasma cells or CD11c+ DCs alone (0), MCC (Peptide (MCC)) or PCC (Protein (PCC)); numbers in outlined areas indicate frequency of responder. Far right, total activated CD44hiCFSElo helper T cells. (,) Quantitative RT-PCR analysis of the expression of mRNA for various molecules (horizontal axes) in CD4+ helper T cells left unactivated (Naive) or activated in cultures with CD11c+ DCs (DC-activated) or IgM−CD138+ plasma cells (PC-activated) as APCs in the presence of PCC protein; results are normalized to β2-microglobulin and are presented relative to those of naive CD4+ helper T cells assessed ex vivo immediately after isolation, set as 1. TNF, tumor necrosis factor; IFN, interferon. *P < 0.05 (Mann-Whitney test). Data are representative of ten or more () or three or more (,) experiments with one mouse each (mean and s.e.m.). * Figure 5: Switched plasma cells selectively inhibit the expression of IL-21 and Bcl-6 in TFH cells. () Total CD44hiCD62Llo helper T cells after secondary culture of in vitro–activated CFSE-labeled 5C.C7 αβTCR–transgenic helper T cells in medium alone (0) or with CD11c+ DCs or IgM−CD138+ plasma cells as APCs with (Ag) or without (No) PCC antigen. () Quantitative RT-PCR analysis of the expression of IL-21 mRNA and Bcl-6 mRNA by in vitro–activated helper T cells reactivated in various secondary culture conditions (horizontal axes); results are normalized to β2-microglobulin and presented relative to those of naive helper T cells, set as 1. () Quantitative RT-PCR analysis of the expression of mRNA for various molecules (vertical axes) in 5C.C7 αβTCR–transgenic helper T cells activated in vivo, then sorted and reactivated in various culture conditions (horizontal axes); results presented as in . Open bars, in vivo–activated helper T cells before culture. () Quantitative RT-PCR analysis of the expression of mRNA for various molecules (vertical axes) in nontransg! enic, in vivo antigen–activated Vα11+Vβ3+ helper T cells on day 7 after immunization of mice (n = 4) with PCC and reactivated in various culture conditions (horizontal axes); results presented as in . *P < 0.05, **P < 0.01 and ***P < 0.001, compared with medium alone or compared according to brackets (Student's t-test). Data are representative of three or more experiments (–) or four experiments () with one mouse each (mean and s.e.m.). * Figure 6: More accumulation of TFH cells in the absence of plasma cells in vivo. () Expression of CXCR5 versus PD-1 (left) on activated CD4 helper T cells (gated as CD16-CD32−B220−CD8−CD4+CD62LloCD44hi) from lymph nodes (LN) and spleens (SP) of Blimp-1-cKO mice (cKO; right) and littermates (wild-type (WT); left) 11 d after vaccination with NP-KLH in adjuvant. () Expression of CXCR5 versus PD-1 on activated spleen CD4+ helper T cells from Blimp-1-cKO mice (right) and littermates (left) 11 d after vaccination with NP-KLH in adjuvant; 2 days before analysis, mice received mixtures of 5.0 × 107 unfractionated lymph node and spleen cells from C57BL/6 mice intraperitoneally and at the base of the tail from day 4 after secondary NP-KLH immunization. () Expression of CXCR5 versus PD-1 on activated splenic CD4 helper T cells (gated as in ) from Blimp-1-cKO mice (right) and littermates (left) 7 d after vaccination with NP-KLH in adjuvant. Numbers adjacent to outlined areas indicate percent CXCR5+PD-1+ cells (left; outer outlined areas) or CXCR5++PD-1++ cell! s (right; inset outlined areas). Right, total PD-1+CXCR5+ or PD-1++CXCR5++ activated helper T cells. NS, not significant; *P < 0.05 (Student's t-test). Data are representative of three experiments with one mouse each (mean and s.e.m.). * Figure 7: Plasma cells localize together with CD4+ T cells and negatively affect the antigen-specific TFH program in vivo. (–) Confocal microscopy of draining lymph nodes from PCC-immunized B10.BR mice at day 10 after priming. Top left, approximate location of medulla (M) relative to positions of images in –; below, colors of labeled antibodies for antigens. B, B cell follicle; T, T cell area. Areas outlined in and in , are enlarged at right in and in , respectively. White arrowheads () indicate contact of CD138+ plasma cells with Vβ3+ T cells. Scale bars, 100 μm. () Quantitative RT-PCR analysis of the expression of mRNA for various molecules (vertical axes) in TFH cells sorted as Vα11+Vβ3+CD44hiCD62Llo and CXCR5+ from lymph nodes of B10.BR mice on day 7 after PCC vaccination; mice received transfer of no cells (0) or plasma cells (PC) 2 d before analysis. Results are normalized to β2-microglobulin and presented relative to those of naive helper T cells, set as 1. *P < 0.05 and **P < 0.01 (Student's t-test). Data are representative of three experiments with one mouse each (mean and s.e.! m.). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, California, USA. * Nadége Pelletier, * Louise J McHeyzer-Williams, * Kurt A Wong, * Eduard Urich, * Nicolas Fazilleau & * Michael G McHeyzer-Williams * Present address: Roche, Basel, Switzerland (E.U.), and Institut National de la Santé et de la Recherche Médicale U563, Toulouse, France (N.F.). * Eduard Urich & * Nicolas Fazilleau Contributions N.P., L.J.M.-W. and M.G.M.-W. conceived of and designed the project; N.P. provided and analyzed the data for all experiments except Figures 3a,b and 6; K.A.W. designed and completed the experiments and analyzed the data for pMHCII HEL-specific plasma cells (Fig. 3a,b); E.U. laid the experimental foundation for staining HEL-specific B cells; N.F. prepared the activated helper T cell samples for quantitative PCR analysis (Fig. 4b) and provided expertise in setting up the T cell in vitro experiments; L.J.M.-W. and M.G.M.-W. did the experiments for and analyzed the data from the Blimp-1-cKO mice (Fig. 6); K.A.W. and N.F. contributed ideas and participated in the manuscript preparation; and N.P., L.J.M.-W. and M.G.M.-W. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Michael G McHeyzer-Williams (mcheyzer@scripps.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (16M) Supplementary Figures 1–15 and Supplementary Table 1 Additional data - A role for IL-27p28 as an antagonist of gp130-mediated signaling
- Nat Immunol 11(12):1119-1126 (2010)
Nature Immunology | Article A role for IL-27p28 as an antagonist of gp130-mediated signaling * Jason S Stumhofer1, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Elia D Tait1, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * William J Quinn III2, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Nancy Hosken3 Search for this author in: * NPG journals * PubMed * Google Scholar * Björn Spudy4 Search for this author in: * NPG journals * PubMed * Google Scholar * Radhika Goenka2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ceri A Fielding5 Search for this author in: * NPG journals * PubMed * Google Scholar * Aisling C O'Hara1 Search for this author in: * NPG journals * PubMed * Google Scholar * Yi Chen6 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael L Jones7 Search for this author in: * NPG journals * PubMed * Google Scholar * Christiaan J M Saris8 Search for this author in: * NPG journals * PubMed * Google Scholar * Stefan Rose-John4 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel J Cua6 Search for this author in: * NPG journals * PubMed * Google Scholar * Simon A Jones5 Search for this author in: * NPG journals * PubMed * Google Scholar * Merle M Elloso9 Search for this author in: * NPG journals * PubMed * Google Scholar * Joachim Grötzinger4 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael P Cancro2 Search for this author in: * NPG journals * PubMed * Google Scholar * Steven D Levin3 Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher A Hunter1chunter@vet.upenn.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 11,Pages:1119–1126Year published:(2010)DOI:doi:10.1038/ni.1957Received09 September 2010Accepted12 October 2010Published online07 November 2010 Abstract * Abstract * Accession codes * 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 The heterodimeric cytokine interleukin 27 (IL-27) signals through the IL-27Rα subunit of its receptor, combined with gp130, a common receptor chain used by several cytokines, including IL-6. Notably, the IL-27 subunits p28 (IL-27p28) and EBI3 are not always expressed together, which suggests that they may have unique functions. Here we show that IL-27p28, independently of EBI3, antagonized cytokine signaling through gp130 and IL-6-mediated production of IL-17 and IL-10. Similarly, the ability to generate antibody responses was dependent on the activity of gp130-signaling cytokines. Mice transgenic for expression of IL-27p28 showed a substantial defect in the formation of germinal centers and antibody production. Thus, IL-27p28, as a natural antagonist of gp130-mediated signaling, may be useful as a therapeutic for managing inflammation mediated by cytokines that signal through gp130. View full text Figures at a glance * Figure 1: IL-27p28 has biological activity in the absence of EBI3. () Enzyme-linked immunosorbent assay (ELISA) of IL-27p28 production by C57BL/6 wild-type (WT) or Ebi3−/− bone marrow–derived dendritic cells (DC) and macrophages (MΦ) left unstimulated (Unstim) or stimulated for 24 h with IFN-γ, LPS or a combination of LPS and IFN-γ. Data are representative of three independent experiments with similar results. () ELISA of IL-27p28 in the serum of wild-type and Ebi3−/− mice isolated before and on days 4 and 8 after infection with Toxoplasma gondii. ND, not detected. Data are representative of three independent experiments with groups of three to four mice (error bars, s.d.). (,) Flow cytometry of intracellular IL-17 (, left) or IL-10 (, left) and ELISA of the production of IL-17 (, right) or IL-10 (, right) in CD4+ T cells isolated from the spleens and lymph nodes of wild-type or Ebi3−/− mice and activated for 4 d with anti-CD3 and anti-CD28 in TH17-polarizing conditions in the presence or absence of IL-27 or IL-27p28, then ! stimulated for 4 h with PMA and ionomycin in the presence of brefeldin A; ELISAs were done after 72 h of stimulation. Numbers in outlined areas indicate percent IL-17+ cells () or IL-10+ cells (); numbers adjacent to outlined areas indicate the mean fluorescent intensity (MFI) of IL-17+ cells () or IL-10+ cells (). α-, anti-. Data are representative of three independent experiments with similar results with groups of two to three mice (error bars, s.d.). * Figure 2: IL-27p28 antagonizes gp130-mediated STAT phosphorylation. (,) Flow cytometry of intracellular phosphorylated STAT1 (p-STAT1) or STAT3 (p-STAT3) in CD4+ T cells purified from wild-type mice and stimulated for 15 min with IL-27p28, IL-6 or IL-27 alone () or hyper-IL-6 alone () or with IL-6 or IL-27 () or hyper-IL-6 () plus IL-27p28 preincubated with T cells for 2 h at 37 °C (+ IL-27p28). Numbers in outlined areas indicate percent CD4+ T cells positive for phosphorylated STAT1 or STAT3; numbers adjacent to outlined areas indicate the MFI of phosphorylated STAT1 or STAT3. Data are representative of four independent experiments with similar results. () Three-dimensional model of interaction of IL-27p28 with gp130 indicating amino acid residues key to this interaction that differ between IL-27p28 and IL-6. () Flow cytometry of intracellular phosphorylated STAT3 in mouse embryonic fibroblasts left unstimulated (gray shaded histograms) or stimulated with OSM or hyper-IL-6 for 15 min at 37 °C (blue lines) or incubated for 2 h at 37 °C wi! th IL-27p28 and then stimulated with OSM or hyper-IL-6 (red lines). Below, change in MFI of phosphorylated STAT3 in mouse embryonic fibroblasts preincubated with IL-27p28 before stimulation with OSM or hyper-IL-6. *P = 0.0059 (unpaired t-test). Data are representative of three individual experiments with similar results (flow cytometry) or five independent experiments (bottom; error bars, s.d.). * Figure 3: Phenotypic analysis of p28-transgenic mice. () IL-27p28 transgene construct. Functional elements include the juxtaposed Lck proximal promoter (Prom) and immunoglobulin intronic heavy-chain enhancer (Enh); the insertion site for IL-27p28; and a mutated (untranslatable) version of the gene encoding human growth hormone (hGX: filled boxes, exons; open boxes, introns). () Flow cytometry of intracellular IL-27p28 in wild-type (blue lines) and p28-transgenic (red lines) CD19+ B cells, CD4+ T cells and CD8+ T cells after stimulation for 48 h with LPS and anti-IgM or activation with anti-CD3 and anti-CD28; cells were incubated for 4 h with brefeldin A before staining. Shaded histogram, fluorescence-minus-phycoerythrin channel. () ELISA of IL-27p28 in serum of naive p28-transgenic (p28-TG) mice and their wild-type littermates. () Total CD19+B220+ B cells in the spleens of naive p28-transgenic mice and their wild-type littermates, calculated from percentages determined by flow cytometry. () Flow cytometry of splenocytes from na! ive p28-transgenic mice and their wild-type littermates, stained for CD4 and CD8. Numbers adjacent to outlined areas indicate percent CD4+CD8− cells (left) or CD8+CD4− cells (right). () Total CD4+ T cells (left) and CD8+ T cells (right) in spleens of the mice in , calculated from percentages determined by flow cytometry. *P = 0.0024 and **P = 0.0148 (unpaired t-test). () Total CD4+CD44hiCD62Llo T cells (left) and CD8+CD44hiCD62Llo T cells (right) in the spleens of p28-transgenic mice and their naive wild-type littermates, calculated from percentages determined by flow cytometry. *P = 0.0062 and **P = 0.0036 (unpaired t-test). Data are representative of two independent experiments (,) or three independent experiments with groups of two to four mice (,,) or three to four mice (; error bars, s.d.). * Figure 4: Transgenic overexpression of IL-27p28 antagonizes the activity of IL-6 and IL-27 on CD4+ T cells. (,) Flow cytometry (left) of intracellular IL-17 () or IL-10 () in CD4+ T cells isolated from the spleens and lymph nodes of wild-type and p28-transgenic mice, activated for 4 d with anti-CD3 and anti-CD28 in nonpolarizing conditions in the presence of TGF-β plus IL-6 (,) or IL-27 () and stimulated for 4 h with PMA and ionomycin in the presence of brefeldin A. Numbers in outlined areas indicate percent IL-17+ cells () or IL-10+ cells (); numbers adjacent to outlined areas indicate MFI of IL-17+ cells () or IL-10+ cells (). Right, frequency of IL-17+ cells () or IL-10+ cells () among the CD4+ T cells described above. *P = 0.0002, **P = 0.0009 and ***P = 0.0158 (unpaired t-test). Data are representative of four individual experiments with similar results (error bars, s.d.). () Flow cytometry of intracellular phosphorylated STAT1 or STAT3 in CD4+ T cells purified from p28-transgenic mice and then preincubated for 2 h at 37 °C, then left unstimulated or stimulated with IL-6 fo! r 15 min. Numbers in outlined areas indicate percent CD4+ T cells positive for phosphorylated STAT1 or STAT3; numbers adjacent to outlined areas indicate MFI of phosphorylated STAT1 or STAT3. Data are representative of three independent experiments with similar results. * Figure 5: Failure of p28-transgenic mice to generate an antigen-specific IgG response after immunization with a thymus-dependent antigen. () Enzyme-linked immunospot assay of IgM+ antibody-secreting cells (ASC) able to bind to NP33-BSA in the spleens of naive (untreated (NT)) p28-transgenic mice and their wild-type littermates (control) or 5 d after immunization with NP-Ficoll in saline. (–) Enzyme-linked immunospot assay of IgM+ () or IgG1+ () antibody-secreting cells able to bind NP33-BSA (,), or IgG1+ antibody-secreting cells able to bind NP4-BSA (), in the spleens of naive p28-transgenic mice and their wild-type littermates (control) or 7 d (D7) and 14 d (D14) after immunization with NP-CGG in alum. *P < 0.001 (nonparametric Mann-Whitney U-test). Each symbol represents an individual mouse; small horizontal lines indicate the mean. Data are representative of two independent experiments with groups of two to three mice () or two (day 7) or three (day 14) independent experiments with groups of three mice (–). * Figure 6: Transgenic expression of IL-27p28 blocks the formation of GC reactions after immunization with a thymus-dependent antigen. () Sections of spleens from p28-transgenic mice and their wild-type littermates left unimmunized (top) or immunized with NP-CGG in alum and assessed 14 d after immunization (bottom), stained with fluorescein isothiocyanate–conjugated antibody to the T cell antigen receptor β-chain (TCRβ; T cells); PNA conjugated to rhodamine (GC B cells); and Alexa Fluor 647–conjugated anti-IgD (B cell follicles). Original magnification, ×10. () Flow cytometry of PNA+ B cells (right) with NP bound to the λ-light chain (left) in spleens of naive p28-transgenic mice and their wild-type littermates (control) or 14 d after immunization with NP-CGG. Numbers in outlined areas indicate percent λ+NP+ B cells (left) or NP+PNA+ GC B cells (right). () Total NP+PNA+ B cells in the spleens in , calculated from percentages determined by flow cytometry. *P = 0.0049 (unpaired t-test). Data are representative of two () or three (,) independent experiments with groups of three mice (error bars (), s.! d.). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions GenBank * AY099297 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Jason S Stumhofer, * Elia D Tait & * William J Quinn III Affiliations * University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania, USA. * Jason S Stumhofer, * Elia D Tait, * Aisling C O'Hara & * Christopher A Hunter * University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA. * William J Quinn III, * Radhika Goenka & * Michael P Cancro * Department of Immunology, ZymoGenetics, Seattle, Washington, USA. * Nancy Hosken & * Steven D Levin * Institute for Biochemistry, Christian-Albrechts-University of Kiel, Kiel, Germany. * Björn Spudy, * Stefan Rose-John & * Joachim Grötzinger * Cardiff University, School of Medicine, Cardiff, UK. * Ceri A Fielding & * Simon A Jones * Merck Research Laboratories, DNAX Discovery Research, Palo Alto, California, USA. * Yi Chen & * Daniel J Cua * Shenandoah Biotechnology, Warwick, Pennsylvania, USA. * Michael L Jones * Department of Inflammation Research, Amgen, Thousand Oaks, California, USA. * Christiaan J M Saris * Centocor Research and Development, Radnor, Pennsylvania, USA. * Merle M Elloso Contributions J.S.S. and C.A.H. contributed to all studies and wrote the manuscript; E.D.T., W.J.Q. III, N.H., M.P.C. and S.D.L. were involved in analyzing p28-transgenic mice; R.G. contributed to studies of GC formation; C.J.M.S. contributed to the studies of Il27ra−/− mice; M.M.E. contributed to studies with Ebi3−/− mice; A.C.O. contributed to studies of intracellular staining for IL-27p28; B.S., S.R.-J. and J.G. did the p28-gp130 modeling and contributed to its analysis; C.A.F. and S.A.J. did the biacore assays and contributed to their analysis; M.L.J. provided the recombinant IL-27p28 protein; and Y.C. and D.J.C. did hydrodynamics-based transfection experiments with minicircle DNA and contributed to their analysis. Competing financial interests C.A.H. and J.S.S. have a patent application on the use of p28 to limit gp130 signaling. N.H. and S.D.L. are employees of ZymoGenetics; Y.C. and D.J.C. are employees of DNAX Discovery Research; M.L.J. is an employee of Shenandoah Biotechnology; C.J.M.S. is an employee of Amgen; and M.M.E. is an employee of Centocor Research and Development. Corresponding author Correspondence to: * Christopher A Hunter (chunter@vet.upenn.edu) Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–9 Additional data - Critical role for the chemokine receptor CXCR6 in NK cell–mediated antigen-specific memory of haptens and viruses
- Nat Immunol 11(12):1127-1135 (2010)
Nature Immunology | Article Critical role for the chemokine receptor CXCR6 in NK cell–mediated antigen-specific memory of haptens and viruses * Silke Paust1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Harvinder S Gill3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Bao-Zhong Wang3 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael P Flynn1 Search for this author in: * NPG journals * PubMed * Google Scholar * E Ashley Moseman1 Search for this author in: * NPG journals * PubMed * Google Scholar * Balimkiz Senman1 Search for this author in: * NPG journals * PubMed * Google Scholar * Marian Szczepanik5, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Amalio Telenti2, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Philip W Askenase7 Search for this author in: * NPG journals * PubMed * Google Scholar * Richard W Compans3 Search for this author in: * NPG journals * PubMed * Google Scholar * Ulrich H von Andrian1, 8uva@hms.harvard.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature ImmunologyVolume: 11,Pages:1127–1135Year published:(2010)DOI:doi:10.1038/ni.1953Received26 May 2010Accepted28 September 2010Published online24 October 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 Hepatic natural killer (NK) cells mediate antigen-specific contact hypersensitivity (CHS) in mice deficient in T cells and B cells. We report here that hepatic NK cells, but not splenic or naive NK cells, also developed specific memory of vaccines containing antigens from influenza, vesicular stomatitis virus (VSV) or human immunodeficiency virus type 1 (HIV-1). Adoptive transfer of virus-sensitized NK cells into naive recipient mice enhanced the survival of the mice after lethal challenge with the sensitizing virus but not after lethal challenge with a different virus. NK cell memory of haptens and viruses depended on CXCR6, a chemokine receptor on hepatic NK cells that was required for the persistence of memory NK cells but not for antigen recognition. Thus, hepatic NK cells can develop adaptive immunity to structurally diverse antigens, an activity that requires NK cell–expressed CXCR6. View full text Figures at a glance * Figure 1: Liver NK cells develop specific memory of haptens. () Hapten-specific CHS responses in naive Rag2−/−Il2rg−/− mice (n = 10–15 per group) that received hepatic CD45+NK1.1+Thy-1+ NK cells (1 × 105) from naive (vehicle-exposed; Acetone) or DNFB- or OXA-sensitized Rag1−/− donors (Sensitization) and were challenged 24 h or 4 months after transfer; ear swelling was calculated after 24 h by subtraction of background swelling in naive mice from that in recipients of NK cells. *P < 0.01 and **P < 0.001 (analysis of variance (ANOVA)). () Flow cytometry analysis of the survival and population expansion of adoptively transferred NK cells 2 weeks after the 4-month challenge in , presented as the number of liver-resident CD45+ NK1.1+ cells. Results were similar for mice challenged with DNFB or OXA, so data were pooled according to donor sensitization. No NK1.1+ cells were detected in mock (PBS)-injected control recipient Rag2−/−Il2rg−/− mice (data not shown). () Hapten-specific CHS responses in naive C57BL/6 mice (n ! = 10–15 per group) that received sorted CD45+NK1.1+CD3−Thy-1+ NK cells (1 × 105) from donors transgenic for expression of GFP-tagged actin, then were challenged 6 weeks later with OXA (left) or DNFB (right) and analyzed as in . *P < 0.001 and **P < 0.0001 (ANOVA). () Recruitment of memory NK cells to challenge sites in naive Rag2−/−Il2rg−/− mice (n = 6–7 per group) that received adoptive transfer of mixtures of hepatic CD45+NK1.1+Thy-1+ NK cells from naive CD45.1+ wild-type donors (C57BL/6) and from CD45.2+ DNFB- or OXA-sensitized wild-type or donors transgenic for expression of GFP-tagged actin (1 × 105 each) and, 1 month later, were challenged in the ears with either DNFB or OXA; livers and ears collected at 24, 48 and 72 h were analyzed by flow cytometry for NK cells whose origin was distinguished by congenic or fluorescent markers (results presented as mean of all mice at 24, 48 and 72 h). No NK cells were found in acetone-challenged control ears (data n! ot shown). *P < 10−11 (ANOVA). Data are representative of th! ree to five independent experiments (pooled results; error bars, s.d.). * Figure 2: Liver NK cells develop specific memory of viral antigens. () Virus-specific DTH in naive Rag2−/−Il2rg−/− mice (n = 8–10 per group) that received adoptive transfer of splenic or hepatic CD45+ NK1.1+ NK cells (8 × 104) 1 month after immunization of Rag1−/− donor mice, followed by challenge of ears of recipient mice by subcutaneous injection 2 months later and analysis after 24 h (as in Fig. 1a). P values, Student's t-test. () Survival of Rag2−/−Il2rg−/− mice (n = 15–19 per group) treated by the sensitization and transfer protocol in , then infected intranasally with influenza A strain A/PR/8/34 (500 PFU) 3 months after NK cell transfer. P values, log-rank Mantel-Cox test. () Survival of the recipients in (n = 19 per group) infected with influenza A strain A/PR/8/34 (500 PFU) 2 months after assessment of DTH; results are correlated to DTH. *P = 0.0001 (Spearman correlation). () Survival of Rag1−/− mice (n = 8–12 per group) immunized with PR8-VLPs, M1-VLPs or UV-VSV, then challenged 1 month later with liv! e virus (2,500 PFU influenza strain A/PR/8/34 intranasally or 500 PFU VSV intravenously). P values, log-rank Mantel-Cox test. () Survival of Rag2−/− mice (n = 15–22 per group) immunized with VLPs containing influenza (PR8-VLP) or HIV-1 (HIV-VLP) or with UV-VSV, then challenged 1 month later by intramuscular injection of VSV at the median lethal dose (250 PFU). *P = 0.0116 (log-rank Mantel-Cox test). () Virus-specific DTH in Rag1−/− mice (n = 10–15 per group) immunized with HA-containing VLPs (PR8) or HA-free VLPs (M1), then challenged 1 month later and analyzed as in . P values, unpaired Student's t-test. Data are representative of three to five independent experiments (pooled results; error bars, s.d.). * Figure 3: Mouse liver NK cells recognize and discriminate between HIV-1 and influenza A. () Ear swelling in naive Rag2−/−Il2rg−/− mice (n = 12–15 per group) that received adoptively transferred hepatic (left) or splenic (right) CD45+NK1.1+ NK cells (8 × 104 cells per mouse) from Rag1−/− donor mice immunized with VLPs containing influenza (PR8) or HIV-1 (HIV) 1 month before transfer; recipients were challenged by subcutaneous injection of VLPs into one ear and PBS in the other ear and were assessed 2 months after transfer. NS, not significant; *P < 0.01 and **P < 0.001 (unpaired Student's t-test). () Ear swelling in C57BL/6 Rag1−/− mice (left) and BALB/c Rag2−/− mice (right) immunized with VLPs and challenged 1 month later (n = 10–15 mice per group). P values, unpaired Student's t-test. Background ear swelling in nonimmunized mice was subtracted from ear swelling in the experimental groups. Data are representative of three to five independent experiments (pooled results; error bars, s.d.). * Figure 4: NK cell–expressed CXCR6 is required for NK cell–mediated adaptive immunity to haptens. () Frequency of CXCR6-expressing CD45+ NK1.1+ NK cells from Cxcr6+/− mice on a Rag1-sufficient (C57BL/6) or Rag1−/− background in different tissues, assessed by flow cytometry. LN, lymph node; BM, bone marrow. () Ear swelling in naive Rag2−/−Il2rg−/− mice (n = 10–12 per group) that received 1 × 105 NK cells from DNFB-sensitized Rag1−/−Cxcr6+/− donor spleen or liver, sorted for expression of NK1.1 and GFP; recipient mice were challenged 1 month later with DNFB on one ear and solvent on the other. () DNFB-induced CHS in lymphocompetent C57BL/6 mice (left) and Rag1−/− C57BL/6 mice (right; n = 10–12 mice per group). () DNFB-induced CHS in C57BL/6 mice (left) and Rag1−/− C57BL/6 mice (right) sensitized with hapten and given mAb to CXCR6 (100 μg per mouse) or isotype-matched control antibody intravenously 24 h before DNFB challenge (n = 10–15 mice per group). *P < 0.01, **P < 0.001 and ***P < 0.0001 (unpaired Student's t-test (,,) or ANOVA ()). ! Data are representative of three to five independent experiments (pooled results; error bars, s.d.). * Figure 5: NK cell–expressed CXCR6 is required for NK cell–mediated adaptive immunity to viruses. () Antiviral DTH responses in Rag1−/− C57BL/6 mice (left) or Rag2−/− BALB/c mice (right) immunized and challenged with various combinations of VLPs and UV-VSV (below graphs) and given mAb to CXCR6 (100 μg per mouse) or isotype-matched control antibody 24 h before challenge. P values, unpaired Student's t-test. () Survival of Rag1−/− and Rag2−/− mice (n = 8–12 per group) immunized with PR8-VLP or M1-VLP, challenged 1 month later by lethal infection with influenza A strain A/PR/8/34 (2,500 PFU for Rag1−/− (left) and 10,000 PFU for Rag2−/− (right)) and injected with mAb to CXCR6 (100 μg per mouse) or isotype-matched control antibody on days 1 and 5. P values, log-rank Mantel-Cox test. Data are representative of three to five independent experiments (pooled results; error bars, s.d.). * Figure 6: CXCR6 regulates hepatic NK cell homeostasis. () Frequency of GFP+ and GFP− NK cell subsets (identified as CD45+NK1.1+ cells) in various organs of Cxcr6+/− and Cxcr6−/− mice. *P < 0.01 and **P < 0.001 (unpaired Student's t-test). () Ratio of NK cell subsets (CXCR6+/CXCR6−) in liver and spleen of wild-type (Cxcr6+/+), Cxcr6+/− and Cxcr6−/− mice (all C57BL/6; n = 12–15 per group). *P < 0.00001 (unpaired Student's t-test). () Distribution of NK cells recovered from liver or spleen 1 month after adoptive transfer of sorted subsets (1 × 105 cells) into Rag2−/−Il2rg−/− recipients (n = 10–15 per group). *P < 0.001 and **P < 0.0001 (unpaired Student's t-test). () Flow cytometry of NK cells in livers and spleens of Rag2−/−Il2rg−/− recipients (n = 8–10 per group) 2 weeks after transfer of mixtures of 1 × 105 GFP+ or GFP− CD45+NK1.1+ NK cells sorted from Cxcr6+/− or Cxcr6−/− donors. *P < 0.001 and **P < 0.0001 (unpaired Student's t-test). () Ear swelling in Rag2−/−Il2rg−/− mi! ce (n = 8 per group) that received 8 × 104 DNFB-primed CD45+ NK1.1+ GFP+ NK cells from Cxcr6+/− or Cxcr6−/− donors and were challenged 24 h or 1 month later. *P < 0.01 and **P < 0.001 (unpaired Student's t-test). Data are representative of three to five independent experiments (pooled results; error bars, s.d.). * Figure 7: Hepatic memory NK cells mediate hapten-specific killing in vitro. () Hapten-specific killing of target B cells by naive and hapten-sensitized CD45+ NK1.1+ NK cells cultured for 12 h at various target cell/effector cell ratios (horizontal axis) with a mixture of two populations of B cells labeled with a large (CFSEhi) or small (CFSElo) amount of the cytosolic dye CFSE (n = 10–20 donor mice per group); alternatively, target and control B cells were distinguished by use of the congenic markers CD45.1 and CD45.2. CFSElo or CD45.1+ B cells served as a control; CFSEhi or CD45.2+ B cells were from wild-type donors and were haptenated with DNBS (left and middle) or were from MHC class I–deficient (MHC-KO) donors (right). Hapten-specific killing was assessed as the ratio of CFSElo to CFSEhi cells, corrected for input. *P < 10−8 and **P < 10−12, DNFB- or OXA-sensitized versus acetone (left and middle) or MHC-KO versus DNBS-labeled (right; unpaired Student's t-test). () Killing capacity of DNFB-primed hepatic CD45+ NK1.1+ NK cells from Cxcr6+! /− or Cxcr6−/− donor mice (n = 12 donor mice per group), assessed as in in the presence of mAb to CXCR6 or isotype-matched control mAb. *P < 0.01, **P < 0.001 and ***P < 0.00001, compared with Cxcr6−/− (unpaired Student's t-test). () Killing capacity of acetone- or DNFB-primed hepatic CD45+ NK1.1+ NK cells from Rag1−/− donors (n = 15 per group) at a target cell/effector cell ratio of 1:25, assessed in the presence of mAb to CXCR6 (10 μg/ml), mAb to CXCL16 (10 μg/ml) or CXCL16 (500 ng/ml); results are presented relative to those of cultures treated with isotype-matched control antibody (10 μg/ml). *P < 0.01 and **P < 0.001, compared with isotype-matched control antibody (unpaired Students t-test). () Flow cytometry analysis of the incorporation of anti-LAMP-1 by NK1.1+ NK cells sorted from the livers or spleens of Rag1−/− donor mice sensitized with acetone, DNFB or OXA on days 0 and 1 and injected with 100 μg mAb to CXCR6 or isotype-matched control mAb ! 12 h before NK cell isolation; NK cells were cultured together! with DNBS-labeled B cells in the presence of fluorescein isothiocyanate–conjugated anti-LAMP-1 (10 μg/ml) with or without mAb to CXCR6 or isotype-matched control mAb (10 μg/ml) and assessed after 3 h (n = 10–18 donor mice total with 12–20 wells per group). () Frequency of LAMP-1+ NK cells among the cells in . *P < 10−9 (unpaired Student's t-test). Data are representative of three to five independent experiments (pooled results; error bars, s.d.). Author information * Abstract * Author information * Supplementary information Affiliations * Harvard Medical School, Department of Pathology, Boston, Massachusetts, USA. * Silke Paust, * Michael P Flynn, * E Ashley Moseman, * Balimkiz Senman & * Ulrich H von Andrian * The Ragon Institute of MIT, MGH and Harvard, Charlestown, Massachusetts, USA. * Silke Paust & * Amalio Telenti * Department of Microbiology & Immunology and Emory Vaccine Center, Emory University, Atlanta, Georgia, USA. * Harvinder S Gill, * Bao-Zhong Wang & * Richard W Compans * Texas Tech University, Department of Chemical Engineering, Lubbock, Texas, USA. * Harvinder S Gill * Department of Human Developmental Biology, Jagiellonian University College of Medicine, Kraków, Poland. * Marian Szczepanik * Institute of Microbiology, University Hospital, University of Lausanne, Lausanne, Switzerland. * Amalio Telenti * Yale Medical School, Department of Medicine, New Haven, Connecticut, USA. * Marian Szczepanik & * Philip W Askenase * Immune Disease Institute, Boston, Massachusetts, USA. * Ulrich H von Andrian Contributions S.P. and U.H.v.A. designed the study; S.P., H.S.G., B.Z.W. and M.F. did experiments; S.P., A.T. and B.S. collected and analyzed data; E.A.M., H.S.G., B.Z.W. and R.H.C. provided reagents; E.A.M., M.S. and P.W.A. provided technical support and conceptual advice; and S.P. and U.H.v.A. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ulrich H von Andrian (uva@hms.harvard.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–6 Additional data - Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria
- Nat Immunol 11(12):1136-1142 (2010)
Nature Immunology | Article Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria * Edward A Miao1emiao@systemsbiology.org Search for this author in: * NPG journals * PubMed * Google Scholar * Irina A Leaf1 Search for this author in: * NPG journals * PubMed * Google Scholar * Piper M Treuting2 Search for this author in: * NPG journals * PubMed * Google Scholar * Dat P Mao1 Search for this author in: * NPG journals * PubMed * Google Scholar * Monica Dors1 Search for this author in: * NPG journals * PubMed * Google Scholar * Anasuya Sarkar3 Search for this author in: * NPG journals * PubMed * Google Scholar * Sarah E Warren1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Mark D Wewers3 Search for this author in: * NPG journals * PubMed * Google Scholar * Alan Aderem1aderem@systemsbiology.org Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature ImmunologyVolume: 11,Pages:1136–1142Year published:(2010)DOI:doi:10.1038/ni.1960Received21 April 2010Accepted15 October 2010Published online07 November 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 Macrophages mediate crucial innate immune responses via caspase-1-dependent processing and secretion of interleukin 1β (IL-1β) and IL-18. Although infection with wild-type Salmonella typhimurium is lethal to mice, we show here that a strain that persistently expresses flagellin was cleared by the cytosolic flagellin-detection pathway through the activation of caspase-1 by the NLRC4 inflammasome; however, this clearance was independent of IL-1β and IL-18. Instead, caspase-1-induced pyroptotic cell death released bacteria from macrophages and exposed the bacteria to uptake and killing by reactive oxygen species in neutrophils. Similarly, activation of caspase-1 cleared unmanipulated Legionella pneumophila and Burkholderia thailandensis by cytokine-independent mechanisms. This demonstrates that activation of caspase-1 clears intracellular bacteria in vivo independently of IL-1β and IL-18 and establishes pyroptosis as an efficient mechanism of bacterial clearance by the inna! te immune system. View full text Figures at a glance * Figure 1: Characterization of flagellin-expressing S. typhimurium. () Enzyme-linked immunosorbent assay of IL-1β in wild-type BMDMs infected for 4, 6 or 8 h with ST-WT or ST-FliCON (multiplicity of infection (MOI), key) under conditions in which SPI2 T3SS is expressed and SPI1 T3SS is not expressed, followed by treatment with gentamicin. () Immunoblot analysis of caspase-1 processing in wild-type (WT) and Nlrc4−/− BMDMs infected for 1 h with ST-WT or ST-FliCON as in at an MOI of 20. Pro-casp1, precursor form of caspase-1; p10, mature form of caspase-1. () IL-1β concentration in wild-type BMDMs infected for 4, 6 or 8 h with ST-FliCON or SPI2-mutant (ssaT) ST-FliCON at an MOI of 12. () IL-1β concentration in wild-type and Nlrc4−/− BMDMs infected for 2, 4, 6 or 8 h with vector only or vector containing a plasmid expressing ST-FliCON, at an MOI of 10. NS, not significant (P > 0.05); *P < 0.05, versus the relevant control (t-test). Data are representative of at least three experiments (error bars, s.d.). * Figure 2: Flagellin expression attenuates S. typhimurium in vivo. () Survival of wild-type and Nlrc4−/− mice infected with ST-WT or ST-FliCON (100 CFU). () CFU in the spleens of wild-type mice infected for 48 h with equal numbers of various combinations (above graph) of ST-WT, ST-FliCON, SPI2-mutant ST-FliCON (ssaT ST-FliCON) or SPI2-mutant (ssaT) bacteria marked with ampicillin resistance (AmpR) or kanamycin resistance (KanR). Results presented as CI (log of the ratio of ampicillin resistance to kanamycin resistance); a CI of −2 corresponds to 1 CFU of ampicillin-resistant bacteria for every 100 CFU of kanamycin-resistant bacteria. () CI of ST-FliCON verusus ST-WT in the spleens, livers and mediastinal lymph nodes (Med LN) of mice at various times (above graph) after infection. (–) CI of ST-FliCON versus ST-WT in wild-type and knockout mice at 48 h after infection. () CFU in the draining lymph nodes of wild-type mice (n = 4), Casp1−/− mice (n = 6) and Il1b−/−Il18−/− mice (n = 6) infected intraperitoneally for 24 h with! L. pneumophila. () CFU in the draining lymph nodes of wild-type mice (n = 6), Casp1−/− mice (n = 4) and Il1b−/−Il18−/− mice (n = 6) infected intraperitoneally for 24 h with B. thailandensis. *P < 0.05 (t-test). Data are representative of three (,,), more than three (), five (), two (,,) or four (,) experiments (error bars, s.d.; mouse numbers in –, Supplementary Table 1). * Figure 3: Different roles for ASC in NLRC4 signaling. () CI of ST-FliCON versus ST-WT (assessed as in Fig. 2) in wild-type mice (n = 6) and ASC-deficient mice (Pycard−/− (called 'Asc−/−' here); n = 4). (,) Enzyme-linked immunosorbent assay of IL-1β secretion, with (Ctx correx) or without (No correx) correction for the release of pro-IL-1β based on cytotoxicity results (), and cytotoxicity, assessed as release of lactate dehydrogenase (), of wild-type, ASC-deficient and Nlrc4−/− BMDMs infected for 6 h with ST-FliCON (as described in Fig. 1). () Pyroptosis of wild-type BMDMs infected for 8 h with ST-WT or ST-FliCON, assessed as release of lactate dehydrogenase. () Cytotoxicity in cells infected for 7 h with ST-FliCON with (Glycine) or without (Control) the addition of 10 mM glycine to the medium. P > 0.05, versus the relevant control (t-test). Data are representative of two experiments () or at least three experiments (–; error bars, s.d.). * Figure 4: Evidence of pyroptosis in vivo. () CI of ST-FliCIND versus ST-WT in wild-type mice infected for 48 h with GFP-expressing ST-WT or flagellin-inducible ST-FliCIND, followed by injection of doxycycline for synchronized induction of flagellin expression (n = 2 mice per time point, except at 0 h, n = 3). (,) Membrane integrity of GFP-containing cells from the peritoneal wash of wild-type mice (,) and Nlrc4−/− mice () infected for 48 h with 1 × 105 CFU GFP-expressing ST-WT or ST-FliCON, followed by injection of doxycycline and analysis of propidium iodide (PI) staining 3 h later by flow cytometry: numbers in outlined areas () indicate percent PI+GFP+ cells; each symbol () represents an individual mouse and small horizontal lines indicate the average. Mice per group: wild-type, n = 9 (ST-WT) and n = 11 (ST-FliCIND); Nlrc4−/−, n = 3 (ST-WT) and n = 4 (ST-FliCIND). *P < 0.001 (t-test). Data are representative of two experiments (; error bars, s.d.) or three experiments (,). * Figure 5: ST-FliCON bacteria are released from macrophages and cleared by ROS. () CI of ST-FliCON versus ST-WT in wild-type mice (n = 2) or Ncf1−/− mice (n = 2 (104 CFU) or n = 3 (103 CFU)) infected for 24 h with 1 × 103 or 1 × 104 CFU. (,) Analysis of CI () and CFU () in Ncf1−/− mice coinfected for 24 h with ST-FliCIND and ST-WT on a flgB-mutant background (with ablation of endogenous flagellin expression during growth in Luria-Bertani broth), followed by injection with doxycycline (dox) alone (n = 8 mice) or doxycycline plus gentamicin (gent; n = 10 mice) and CFU analysis 24 h later. *P < 0.05 (t-test). Data are representative of three experiments (error bars, s.d.). * Figure 6: ST-FliCON bacteria accumulate in neutrophils. (–) Flow cytometry analysis of markers for macrophages (F4/80) or neutrophils (Ly6G) in wild-type and Ncf1−/− mice infected for 48 h with GFP-expressing ST-WT or ST-FliCON, presented as the frequency of marker-positive cells (,) or as the ratio of GFP+ Ly6G+ cells to GFP+ F4/80+ cells (). ND, not detectable. *P < 0.05 (t-test). Data are representative of three experiments with four mice each (error bars, s.d.). (–) Quantitative PCR analysis of the expression of genes encoding NLRC4 or markers specific for macrophages (CD68) or neutrophils (S100a8) in BMDMs or neutrophils (PMN), presented relative to the expression of Hprt1 (encoding hypoxanthine guanine phosphoribosyl transferase). *P < 0.05 (t-test). Data are representative of three experiments (error bars, s.d.). * Figure 7: Sustained NLRC4 activation causes tissue damage. (–) Histopathology of spleens from wild-type or Ncf1−/− mice infected for 48 h with ST-WT or ST-FliCON (CFU, 1 × 105 (wild-type mice) and 1 × 103 (Ncf1−/− mice)). Scale bars, 100 μm. () Scores of pathological changes in spleens from Ncf1−/− mice (n = 3 per inoculum) left uninfected or infected for 48 h with ST-WT or ST-FliCON (CFU, horizontal axes; two doses of ST-WT used for comparison with ST-FliCON-infected mice normalized for either initial dose or bacterial load at 48 h). () CFU in spleens of the mice in (n = 3 per inoculum). *P < 0.05 (t-test). Data are representative of three experiments (–) (error bars, s.d.). Author information * Abstract * Author information * Supplementary information Affiliations * Institute for Systems Biology, Seattle, Washington, USA. * Edward A Miao, * Irina A Leaf, * Dat P Mao, * Monica Dors, * Sarah E Warren & * Alan Aderem * Department of Comparative Medicine, University of Washington, Seattle, Washington, USA. * Piper M Treuting * Pulmonary, Critical Care, Allergy and Sleep Medicine, The Dorothy M. Davis Heart and Lung Research Institute, Ohio State University, Columbus, Ohio, USA. * Anasuya Sarkar & * Mark D Wewers * Department of Immunology, University of Washington, Seattle, Washington, USA. * Sarah E Warren Contributions E.A.M., I.A.L. and A.A. conceived of the research plan and wrote the manuscript; E.A.M., I.A.L., D.P.M., M.D., A.S. and M.D.W. planned and did experiments; and P.M.T. planned and did histological analyses. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Edward A Miao (emiao@systemsbiology.org) or * Alan Aderem (aderem@systemsbiology.org) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (492K) Supplementary Figures 1–2 and Tables 1–3 Additional data
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