Bosch NKT 92 User Manual

NKT cells between innate and acquired immunity: function and specificity D i s s e r t a t i o n zur Erlangung des akademischen Grades d o c

Introduction 2 1.1 Cells comprising the immune system Immune responses are mediated by a variety of specialized cells with diverse funtions and by s

Discussion 92 6.5 Towards an understanding of NKT cells A global picture on NKT cell properties and functions as well as regulation of NKT cell activ

Summary 93 7 Summary NKT cells are involved in the regulation of a wide range of immune responses. They play a role in autoimmunity, antitumour

Summary 94 cell activation by endogenous and exogenous antigens still remains to be determined. Regulation of NKT cell activation was examined b

Zusammenfassung 95 8 Zusammenfassung NKT-Zellen sind an der Regulation einer Vielzahl von Immunantworten beteiligt. Sie spielen eine Rolle in Autoim

Zusammenfassung 96 2) Mit Hilfe der Transkriptome Analyse konnte das genetische Profil von NKT-Zellen und das Expressionsmuster nach NKT-Zel

Zusammenfassung 97 Antigene befähigt zur effizienten Beobachtung des Immunsystems und zur Teilnahme an Immunantworten, entweder in einer un

Literature 98 9 Literature Adamson,E.D. and Mercola,D. (2002). Egr1 transcription factor: multiple roles in prostate tumor cell growth and survival

Literature 99 Berger,M.S., Taub,D.D., Orlofsky,A., Kleyman,T.R., Coupaye-Gerard,B., Eisner,D., and Cohen,S.A. (1996). The chemokine C10: immunologica

Literature 100 Chan,W.L., Pejnovic,N., Liew,T.V., Lee,C.A., Groves,R., and Hamilton,H. (2003). NKT cell subsets in infection and inflammation. Immuno

Literature 101 Dorner,B.G., Scheffold,A., Rolph,M.S., Huser,M.B., Kaufmann,S.H., Radbruch,A., Flesch,I.E., and Kroczek,R.A. (2002). MIP-1alpha, MIP-1

Introduction 3 spleen, lymph nodes, Peyer’s patches). Here, they develop further into effector cells. During infection, effector T cells are found no

Literature 102 Fournie,J.J., Mullins,R.J., and Basten,A. (1991). Isolation and structural characteristics of a monoclonal antibody-defined cross-reac

Literature 103 Gumperz,J.E. and Brenner,M.B. (2001). CD1-specific T cells in microbial immunity. Curr Opin Immunol 13, 471-8. Gumperz,J.E., Miyake,S.

Literature 104 Jayawardena-Wolf,J., Benlagha,K., Chiu,Y.H., Mehr,R., and Bendelac,A. (2001). CD1d endosomal trafficking is independently regulated by

Literature 105 Kim,C.H., Butcher,E.C., and Johnston,B. (2002). Distinct subsets of human Valpha24-invariant NKT cells: cytokine responses and chemoki

Literature 106 Ljutic,B., Carlyle,J.R., Filipp,D., Nakagawa,R., Julius,M., and Zuniga-Pflucker,J.C. (2005). Functional requirements for signaling thr

Literature 107 Mills,K.H. (2004). Regulatory T cells: friend or foe in immunity to infection? Nat Rev Immunol 4, 841-55. Mittrucker,H.W. and Kaufmann

Literature 108 Park,S.H., Benlagha,K., Lee,D., Balish,E., and Bendelac,A. (2000). Unaltered phenotype, tissue distribution and function of Valpha14(+

Literature 109 Ramachandra,L., Noss,E., Boom,W.H., and Harding,C.V. (1999). Phagocytic processing of antigens for presentation by class II major hist

Literature 110 Schofield,L., McConville,M.J., Hansen,D., Campbell,A.S., Fraser-Reid,B., Grusby,M.J., and Tachado,S.D. (1999). CD1d-restricted immunog

Literature 111 Spada,F.M., Borriello,F., Sugita,M., Watts,G.F., Koezuka,Y., and Porcelli,S.A. (2000). Low expression level but potent antigen present

Introduction 4 from NK cells, which promotes antigen processing and presentation to T cells and T helper type 1 (Th1) cell polarization. DCs are,

Literature 112 Wang,S., Miura,M., Jung,Y., Zhu,H., Gagliardini,V., Shi,L., Greenberg,A.H., and Yuan,J. (1996). Identification and characterization of

Literature 113 Zhou,D., Mattner,J., Cantu,C.3., Schrantz,N., Yin,N., Gao,Y., Sagiv,Y., Hudspeth,K., Wu,Y.P., Yamashita,T., Teneberg,S., Wang,D., Proi

Publications 114 Publications Kalka-Moll, W. M., Tzianabos, A. O., Bryant, P. W., Niemeyer, M., Ploegh, H. L., and Kasper, D. L. (

Erklärung 115 Erklärung Hiermit erkläre ich, dass ich die vorliegende Doktorarbeit selbständig und nur unter Zuhilfenahme der genannt

Danksagung 116 Danksagung Mein großer Dank gilt Prof. Dr. Stefan H. E. Kaufmann für die Überlassung dieses faszinierenden Themas und

Supplier 117 Suppliers Supplier Location URL Adobe SystemsGmbH Ohmstrasse 1 D-85716 Unterschleißheim www.adobe.de Agilent Technologies Deutschla

Supplier 118 Supplier Location URL GeneCraft Tresckowstrasse 10 D-48163 Münster www.genecraft.de Graph Pad Software, Inc. 11452 El Camino Real,

Supplier 119 Supplier Location URL Pfizer / Pharmacia 235 East 42nd Street New York, NY 10017, USA www.pfizer.com Promega High-Tech-Park, Schild

Introduction 5 al., 2004). TLRs 1, 2, 4, 5 and 6 seem to specialize in the recognition of mainly bacterial products that are not made by the h

Introduction 6 cells, the plasma cells. In a T-cell dependent process, a subpopulation of activated B cells undergoes differentiation to produce othe

Introduction 7 foreign peptides presented by major histocompatibility complex (MHC) class I molecules on the surface of virtually every cell of

Introduction 8 NKT cells posses both regulatory and effector functions, are self-reactive and express a semi-invariant T-cell receptor (T

Introduction 9 MHC class II molecules present peptide fragments derived from proteins taken up via phagocytosis and which are processed by th

Introduction 10 1.6 CD1 molecules During the recent years it became clear that not only peptides are presented to T cells, but that T cells also reco

Introduction 11 the CD1 heavy chain noncovalently paired with β2m (Figure 2). CD1 proteins are presented TAP-independently but association with β

Introduction 12 CD1 heavy chains are translocated into the ER where N-linked glycans are attached, interactions with ER chaperones occur

Introduction 13 tuberculosis in humans (Mycobacterium tuberculosis) as well as bovine tuberculosis (Mycobacterium bovis) belong to this

Introduction 14 1987). Additionally, inhibition of antigen-specific monocyte activation can be inhibited by PIMs. However, there are severa

Introduction 15 The identification of the first antigen presented by CD1 molecules to T cells demonstrated that CD1 presented antigens are lipids (Be

Introduction 16 1.9 NKT cells Unlike classical T cells reactive to peptides presented by MHC molecules, NKT cells are reactive to lipid

Introduction 17 enhance cell-mediated immunity and immunosuppressive cells, respectively (Smyth and Godfrey, 2000). Apart from cytokine produ

Introduction 18 Furthermore it was shown that proinflammatory cytokines secreted by APCs after exposure to microbial products augmented the bas

Aims of this thesis 19 2 Aims of this thesis NKT cells are a unique T lymphocyte lineage that has been implicated in the regulation of immune respo

Material 20 3 Material 3.1 Cells, Cell lines A20 Murine B-Cell lymphoma line (ATCC) A20-mCD1d A20 Cells transfected with DNA for the full-l

Material 21 Middlebrook medium: 4,7g Middlebrook 7H9 Broth, 2ml Bacto glycerol and 0,5g Tween 80 were dissolved in 900ml ddH2O and autoclaved. Sub

Content I Content I Abbreviations IV 1 Introduction 1 1.1 Cells comprising the immune system...

Material 22 Specificity Clone Application Source CD16/CD32 (anti-FcR) 24G2 FACS ATCC* CD25 PC61 FACS Pharmingen CXCR4 2B11 FACS ATCC* F4/

Material 23 3.8 Primers The table below shows the sequences of the synthetic oligonucleotides used for semi-quantitative RT-PCR. All primers were

Material 24 3.9 Plasmids pgD-hTLR2 Full length hTLR2 with NH2-terminal gD tag (Yang et al., 1998). The wild type signal sequence for hTLR2 was

Material 25 3.11 Buffers and solutions Solutions were made up in H2O prepared with a Millipore water purifier, unless stated otherwise.

Material 26 3.12 Enzymes T4 DNA ligase reverse transcriptase superscript II DNAse I RNAse H Gibco Pfu DNA polymerase DNA polymerase I, large frag

Material 27 Rotational Evaporator RotiVap (Büchi) Rocking Plate Biometra Vortex IKA-Labortechnik Stir- and Heatingplate IKA-Labortechnik

Material 28 Text Word (Microsoft) Microarray analysis Resolver (Rosetta Inpharmatics) ELISA Analysis Softmax Pro, Spf 3.0 (Molec

Methods 29 4 Methods 4.1 Immunological methods 4.1.1 Cell culture Cell lines and primary cells were cultured under sterile conditions at 37°C, 95% h

Methods 30 4.1.4 Infection of mice with M. bovis BCG C57BL/6 mice were infected i.v. with 2x106 M. bovis BCG. Bacteria were grown in Mi

Methods 31 4.1.7 Generation and restimulation of human NKT cell lines and clones Human NKT cell lines and clones were provided by Dr. M. B

Content II 3.12 Enzymes...26 3.13 Equipme

Methods 32 To measure intracellular cytokines, cells were first stained for surface expressed proteins, then washed in PBS and fixed for

Methods 33 cells were kept at 4°C at all times. The sorting regions were defined in the dot blot diagrams showing forward (FSC) versus 90° (SSC)

Methods 34 patients with Fabry disease. DCs and macrophages were pulsed with the respective lipids for 3-6 h, irradiated (5,000 rad) and subsequently

Methods 35 4.2 Biochemical methods 4.2.1 ELISA Immuno-Maxisorp ELISA plates (Nunc) were coated with the primary antibody in 50 µl/well coating

Methods 36 filter and the volume of the chloroform/methanol extract was reduced by using a rotational evaporator. Lipids were further purified by liq

Methods 37 which was replaced after 10 seconds with fresh solution. The reaction was stopped after 2 min by addition of citric acid (50%) in ddH2

Methods 38 5B1-4) cells (Invitrogen) using the Lipofectin Reagent® (Gibco BRL) by following the manufacturer's protocol. Recombinant virus wa

Methods 39 finally incubated for 15 min at 70°C in order to inactivate the reverse transcriptase and to stop the reaction. 4.3.3 Real-time RT-PCR

Methods 40 data analysis, a color swap was performed. The RNA samples were labeled vice versa with the two fluorescent dyes (fluorescence reversal).

Results 41 5 Results 5.1 Characterization of NKT cells NKT cells influence and regulate a wide range of immune responses. They play a role in aut

Content III 5 Results 41 5.1 Characterization of NKT cells...41 5.1.1

Results 42 cells with high efficacy, spleen cells were stained with biotin-conjugated antibodies against Mac-1, B220, CD11c and CD8. Magnetic beads,

Results 43 In all cell purifications, PI or DAPI was added shortly before sorting the cells to discriminate between viable and dead cells. Only PI /

Results 44 hybridized to Agilent custom made microarrays that contained oligonucleotide probes representing 8013 different gene sequence

Results 45 cells. Only genes with P values <0,05 and a minimum of 2 fold difference are considered to be significantly regulated. In the first mi

Results 46 The second microarray analysis detected 1,164 genes differentially expressed between the analyzed cell types with 135 genes up- or

Results 47 20 genes with lower expression levels in all other cells compared to NKT cells were identified in both arrays. These genes describe a grou

Results 48 Genes with decreased expression levels in NKT cells included caspase 1, caspase 11, integrin- α4β1, L-selectin, KLF2 and

Results 49 prominent genes up- or downregulated in NK cells, conventional CD4+ T cells and Treg cells compared with NKT cell expression levels. These

Results 50 Expression of IL-2 receptor, IL-4 receptor and IL-7 receptor has not been specifically demonstrated for NKT cells. However, IL

Results 51 eosinophils (Berger et al., 1996). The LPS-inducible CC chemokine receptor (L-CCR) is an orphan chemokine receptor that was recently

Abbreviations IV Abbreviations Ab antibody αGalCer alpha-galactosyl-ceramide αGalA alpha-galactosidase A Ag antigen APC antigen presenting cel

Results 52 Twenty-one % of NKT cells expressed LFA-1 on their surface and 1.47 % were positive for FasL expression. Moreover, 2.56 % NKT cells were s

Results 53 microarray analysis was performed to compare the 2 microarray analysis with each other. Only genes differentially regulated in both

Results 54 Genes with increased expression levels can be classified into two groups. One group consists of cytokines and chemokines

Results 55 production by NKT cells was detected by gating spleen cells for NK1.1 expression. NKT cells were identified by αGalCer-CD1d tetramer and a

Results 56 5.2 Endogenous NKT cell lipid ligands The best described antigen for NKT cells is αGalCer, a glycosphingolipid derived from the marine sp

Results 57 negative control. αGalCer and ConA were used as positive control antigens since they induce strong production of IFNγ and IL

Results 58 incubated for 24 h. Proliferation and cytokine production was measured by [3H] thymidine incorporation and ELISA, respectively. iGb3 p

Results 59 5.2.2 iGb3 and iGb3 analogues Professor Dr. Peng George Wang, University of Detroit, who initially provided us with iGb3, chemically synth

Results 60 DCs from healthy donors were incubated with iGb3, PW15, PW16, PW17 and PW18 for 6 h. APCs were additionally preincubated for 1 h ei

Results 61 Cresswell, 2004; Winau et al., 2004). Human NKT cells from a polyclonal NKT cell line were added and culture supernatant was removed after

Abbreviations V Lamp1 lysosomal-associated membrane protein 1 Li invariant chain LM lipomannan LPS lipopolysaccharid mAb monoclonal antibody M

Results 62 NKT cells were detected and 12.36 % NKT cells after iGb3 stimulation (Figure 20 c). Thus, incubation of PBMCs from Fabry patients with αGa

Results 63 5.3 Mycobacterial PIM is a natural NKT cell ligand A characteristic feature of NKT cells is the recognition of lipid antigens presente

Results 64 αGalCer-CD1d tetramers and ∼ 0.26% to PIM-CD1d tetramers (Figure 23). The tetramer-TCR interactions were blocked by preincubation of cel

Results 65 αGalCer-reactive NKT cells display a very limited TCR repertoire with constant Vα chain paired with the vβ chains Vβ8, V

Results 66 Relative results from FACS analysis of Vβ usage and analysis of NKT cell numbers during mycobacterial infections show no major differ

Results 67 5.4 TCR vs. TLR induced NKT cell activation NKT cell activation can be mediated directly by CD1d presented antigens recognized by the NKT

Results 68 In comparison to PIM, PI lacks the mannose sugar part and phospholipase A treatment of PIM results in the removal of one acyl chain (Fi

Results 69 solvent and characteristic bands for phosphatidylinositol-mono/di-mannoside (PIM1,2) was detected. Higher mannosylated PIMs, li

Results 70 Transfectants containing the empty vector lacking TLR2 were added as negative control for TLR2 independent NF-κB activation. Li

Results 71 Experiments for CD1d-mediated NKT cell activation and TLR2-mediated NF-κB activation revealed that lipid fraction 7 induced respon

Results 72 activate NKT cells as well as being recognized by TLR2. Peaks, indicating the masses of highly mannosylated PIMs were not detected in fra

Results 73 c) Fraction 16 d) Fraction 19 Figure 31 continued: MALDI-MS analysis of lipid fractions isolated from M. bovis BCG. Lipids were

Results 74 5.4.3 Toll-like receptor expression by NKT cells TLR2 and TLR4 recognize a wide range of bacterial cell wall products including lipopr

Results 75 However, the detection of minute percentages of TLR4 positive NKT cells cannot be considered as a TLR4+ NKT cell populati

Discussion 76 6 Discussion NKT cells possess very diverse capacities and are involved in a variety of immune responses (Godfrey and Kronenberg, 2004

Discussion 77 The lower purity of NKT cell sorts was surprising but can be attributed to two factors. Limitations of the FACS sorter,

Discussion 78 NKT cells display an activated, effector phenotype The recruitment of leukocytes into tissues depends on a series of adhesive and activ

Discussion 79 downregulated in NKT cells. In contrast to BLK, caspase-1, caspase-11 and KLF2 exhibited decreased expression levels in NKT cells (Tabl

Discussion 80 Genes with similar expression levels in NKT cells and conventional T cells suggest adaptive immunity-like qualities of NKT cells. S

Discussion 81 other cytokines. It is noteworthy that expression of some genes, e.g. IL-13 and MIP-1α, detected for activated NKT cells c

Introduction 1 1 Introduction The environment contains a huge variety of pathogenic microbes - viruses, bacteria, fungi, protozoa and multicellula

Discussion 82 6.2 NKT cell activity: induction by iGb3 is controlled through antigen availability Lysosomal storage disorders are a family of

Discussion 83 1996). Moreover, iGb3 stimulates NKT cells in a similar manner as αGalCer. Synthesis of iGb3 from lactosylceramide by lactosylceram

Discussion 84 sphingomonas lipids are directly recognized by the NKT cell receptor via CD1d. Moreover, Griffonia simplicifolia I-B4 (IB4), a lectin b

Discussion 85 Effects of IL-12 and IL-15 on NKT cell activation by iGb3 It has been shown that APC derived IL-12 can bias the self-reactiv

Discussion 86 healthy donors. This should increase the amount of iGb3 available for antigen presentation and thus lead to NKT cell stimulation. Jirim

Discussion 87 demonstrated that NKT cells are relatively resistant to activation induced cell death compared to other T cells and ra

Discussion 88 (Emoto et al., 1999a; Leite-de-Moraes et al., 2000; Osman et al., 2000). Recent data showed that NKT cells rather downregulate their TC

Discussion 89 6.4 Toll like receptor vs. T cell receptor mediated recognition of mycobacterial lipids CD1d-mediated NKT cell activation involv

Discussion 90 These results emphasize the importance of discrimination between conserved mammalian and bacterial structures to evoke a TLR mediate i

Discussion 91 surface and/or intracellular expression of TLR2 and TLR4 on NKT cells. Therefore, direct activation of NKT cell-expressed TLR2