- Email: [email protected]
Workshop H: Development and Differentiation of the Immune System
Workshop H Development and Differentiation of the Immune System
Department of Cellular Immunology, Max-Planck-Institute of Immunobiology, Freiburg, Germany
bic+TCRd dic+ DN4 thymocyte population: H. 1 Analysis of the TCRb Possible gd lineage commitment C.E. BUSSE, G. NERZ, A. KROTKOVA, and K. EICHMANN Approximately 1–2% of all CD4–CD8–CD25–CD44– (DN4) thymocytes express both the TCR b and TCR d chains intracellularly (ic). Until now, the fate of this population, whose existence cannot be easily explained when assuming an inductive lineage commitment model, could not be determined. In order to determine the fate of TCRbic+/TCRdic+ DN4 cells we first studied the correlation between intracellular and surface (sf ) expression of the TCR d chain, which turned out to be greater than 97%. Due to the fact that TCR d chains can only get to the surface when paired with a TCR g chain, we conclude that nearly all TCRdic+ cells also express TCR g. Next, we analyzed the mRNA expression of TCR b and preTa in gd TCRsf+ DN4 cells by semiquantitative RT-PCR. While the expression of TCR b in this population was about 25% of that in CD4–CD8–CD25+CD44– (DN3) thymocytes, preTa expression is less than 1% of that in DN3. Since the ratio of TCRbic– to TCRbic+ cells within the gd TCRsf+ DN4 population is approximately 3:1, this implies that TCRbic+/TCRdic+ DN4 cells express TCR b at a level comparable to DN3 cells – which is confirmed by FACS analysis –, while expressing little or no preTa. To determine whether the TCR b chains expressed in the TCRbic+/TCRdic+ DN4 population possess unusual V(D)J joints, which might lead to an inability of the TCR b chain to pair efficiently with preTa, we sequenced the Vb8.2DbJb2.7 joints of sorted TCRbic+/TCRdic– and TCRbic+/TCRdic+ DN4 cells. The comparison of 20 TCRbic+/TCRdic– and 19 TCRbic+/ TCRdic+ sequences did not reveal any striking differences between the two populations. In summary, our results are consistent with the hypothesis that TCRbic+/TCRdic+ DN4 cells are committed to the gd lineage and might be the precursors of TCRbic+ gd T cells in the periphery.
Institute of Immunology, Ludwig-Maximilians-University, Munich, Germany
H. 2 Dendritic cells induce negative, but not positive selection of CD8 T cells and cannot educate NK cells in vivo M.A. CANNARILE and T. BROCKER During the selection process of T cells in the thymus, CD8 T cells are first positively selected by functional recognition of MHC class I molecules. Then autoreactive CD8 T cells are eliminated in the thymus through negative selection. We generated mice with MHC class I molecules selectively on dendritic cells (DC) by expressing b2m under the control of the CD11c-promoter
33rd Annual Meeting of the German Society of Immunology · 103 (CD11c-MHC I) in a b2m knockout (b2m KO) background. The influence of DC on the selection of CD8 T cells was investigated by comparing CD8 T cell numbers and the T cell receptor (TCR)-repertoires of C57BL/6, b2m KO and CD11c-MHC I mice. Flow cytometric analysis revealed that, compared to wildtype C57BL/6 mice, CD11c-MHC I transgenic and b2m KO mice have 7-fold fewer CD8 positive T cells in the thymus and a 15-fold reduction in the periphery. These data suggests MHC class I expression on thymic DC is not sufficient for positive selection of CD8 T cells. Analysis of TCR-Vb- domains also showed that the CD8 T cell repertoire of CD11c-MHC I mice is more similar to the repertoire in b2m KO mice than to wild type animals. Transfer of MHC class I splenocytes into wildtype, transgenic and b2m KO mice showed that CD11c-MHC I-mice tolerize MHC class I expressing cells, whereas they are rejected in b2m KO mice, indicating that DC are sufficient to negatively select CD8 T cells. Interestingly, adoptively transferred cells from b2m KO mice to the three described mouse strains were also accepted by CD11c-MHC I mice. These data indicate that despite the presence of MHC class I on DC in CD11c-MHC I mice, NK function towards cells missing MHC class I was not restored. Thus only negative, but no positive, selection events can be induced by DC in vivo. The exact influence of DC on NK cell development has to be further investigated.
Institute of Transplantation Diagnostics and Cell Therapeutics, Heinrich-Heine-University, Düsseldorf, Germany
H. 3 Induction of CD94:NKG2A expression on NK cells but not T cells by treatment with the histone deacetylase inhibitor trichostatin A B. EISERMANN, S. SANTOURLIDIS, G. KÖGLER, P. WERNET, and M. UHRBERG The target cell specificity of human NK cells is determined by the clonally-distributed expression of two MHC class I-specific NK receptor (NKR) families called KIR (Killer Immunoglobulin-like Receptors) and CD94:NKG2. However, it is largely unknown how the human NKR repertoire is formed and tolerance induction is achieved during NK cell development. As a simple in vitro model of NK cell differentiation, human cord blood (CB)-enriched CD34+ stem cells were cultured with Flt3-L in combination with either IL-2, IL-12, or IL-15. After four weeks of culture, the strongest induction of CD94:NKG2A was seen with IL-15/Flt3L (55,9% of CD56+ NK cells) followed by IL-12/Flt3-L (49,1% of NK cells). IL-2/Flt3-L led to the highest frequency of NK cells but a smaller percentage of CD94:NKG2A (33,3%). In contrast, no induction of KIR expression was detectable under the chosen culture conditions. We next investigated if the induction of CD94:NKG2A expression was dependent on epigenetic modification of DNA as shown recently for the expression of the KIR gene family. Treatment of the CD94:NKG2A-negative NK cell line YT with the demethylating agent 5Aza-dC led to a moderate induction of CD94:NKG2A surface expression (24,3%). Surprisingly, a much stronger induction of CD94:NKG2A expression (40,1%) was observed with the histone deacetylase inhibitor trichostatin A (TSA), a chemical agent which induces an open chromatin structure. A synergistic effect was seen upon combinatory treatment of YT with 5Aza-dC and TSA (71,9%). In contrast, treatment of polyclonally-stimulated PBMC-derived T cells with TSA and/or 5AzadC did not result in any CD94:NKG2A induction. This parallels the distribution of CD94:NKG2A in vivo, which is largely restricted to NK cells and only rarely found on T cells.
104 · 33rd Annual Meeting of the German Society of Immunology Our results indicate that the NK cell-specific expression of CD94:NKG2A is modulated by changes in chromatin structure and to a lesser extend by DNA methylation.
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Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Universität zu Köln, Köln, Germany and 2Department of Biological Sciences, University of Chicago, Chicago IL, USA
H. 4 Molecular portraits of B cell lineage commitment N. FELDHAHN1, F. KLEIN1, S. LEE2, V.S. BARATH1, H. WANG1, K. TANG1, M. KRÖNKE1, J.D. ROWLEY2, S.M. WANG2, and M. MÜSCHEN1 In an attempt to characterize early development of human B cells including the commitment of progenitor cells to the B cell lineage, we generated and compared genome-wide gene expression profiles of CD34+ human hematopoietic stem cells (HSC) and pre-B cells using the serial analysis of gene expression (SAGE) technique. Human CD34+ HSC and CD10+CD19+CD20– preB cells were purified by MACS from bone marrow and umbilical cord blood samples from four and 28 healthy donors, respectively. The identity of the purified populations was confirmed by flow cytometry and PCR amplification, cloning and sequencing of IgH germline fragments, DHJH- and VH-DHJH-rearrangements. Thereafter, both populations were subjected to SAGE-analysis, in which mRNA molecules are transformed into 14-bp cDNA sequences, the so-called SAGE-tags. For HSC and pre-B cells, more than 100,000 SAGE-tags were collected, which establish a quantitative and genome-wide transcriptome of these cells. While the transcriptome of CD34+ HSC encompasses 42,399 individual transcripts, only 16,786 were identified in the preB cell SAGE-profile, indicating that about 60% of genes expressed in HSC were silenced during or after commitment to the B cell lineage. On the other hand, mRNAs of pre-B cell receptorassociated genes are missing in HSC but account for more than 10% of the transcriptome of preB cells, which also show markedly increased expression of apoptosis-related genes. Both, concentration of the transcriptional repertoire on pre-B cell receptor-related genes together with upregulation of apoptosis-mediators in pre-B cells might reflect selection for the expression of a functional pre-B cell receptor within the bone marrow. Besides known regulator genes of early B cell development such as PAX5, E2A, and EBF, the most abundantly expressed genes in pre-B cells include ATM, PDGFRA, SIAH1, PIM2, C/EBPB, WNT16 and TCL1, which were, so far, not implicated in the regulation of early B cell development.
33rd Annual Meeting of the German Society of Immunology · 105 Institut für Virologie und Immunbiologie, Universität Würzburg, Würzburg, Germany
H. 5 Suppression of germ line g1 transcripts and arrest of isotype switching in cells expressing Blimp-1 E. FEOKTISTOVA, D.S. VALLABHAPURAPU, I. BERBERICH, and A. SCHIMPL Following stimulation, primary B cells either directly undergo terminal differentiation to IgM secreting plasma cells or enter the memory pathway, characterised by affinity maturation and isotype switching. High rate secretion correlates with endogenous Blimp-1 expression which in turn is stimulated by cytokines such as Il-2 and IL-5 but inhibited by ligation of the BCR, CD40 or by IL-4. Importantly, introduction of Blimp-1 by retroviral gene transfer is sufficient to overcome the block in Ig secretion caused by BCR-, CD40- or IL-4R ligation. At the same time, the drastic increase in membrane IgG1+ cells with time in cultures treated with IL-4 is greatly diminished in cells forced to express Blimp-1 while cells that have already switched are turned into IgG1 secreting plasma cells. We now show by RT-PCR analysis that arrest in isotype switching caused by Blimp-1 is accompanied by a loss of excision circle transcription accompanying the class switch process while expression of AID (activation induced cytidine deaminase) is less drastically affected. We conclude that suppression of Blimp-1 by antigen-BCR interaction and T helper cell dependent CD40 and IL-4 signaling is necessary to facilitate class switch recombination and prevent terminal differentiation.
Institutes of 1Molecular Animal Breeding, Gene Center, 2Medical Microbiology, Immunology and Hygiene, Technical University, and 4Molecular Immunology, GSF National Research Center for Environment and Health, Munich, 3Institute of Experimental Genetics, GSF Research Center, Neuherberg, and 5German Research Center for Biotechnology (GBF), Braunschweig, Germany
H. 6 A phenotype-driven approach for the identification of mutants with immunological alterations among offspring of ENU-treated C3HeB/FeJ mice H. FLASWINKEL1, B. RATHKOLB1, M. HOWALDT1, A. SERVATIUS2, D. SOEWARTO3, S. MARSCHALL3, E. KREMMER4, N. SANDHOLZER2, M. HRABE DE ANGELIS3, R. BALLING5, E. WOLF1, and K. PFEFFER2 Dominant as well as recessive mouse mutants have been generated in large scale and analyzed for a multitude of parameters by a consortium of screening groups. The project is a part of the German Human Genome Project and aims at the production of mouse models resembling human diseases. More information about the ENU-Mouse Mutagenesis Screen Project can be obtained via the world wide web. Employing ELISAs for basal immunoglobulin levels, anti-DNA antibodies and rheumatoid factor as well as flow cytometry for the detection of cell surface molecules on a semi automated basis we were able to analyze more than 6000 F1 mice in the dominant and more than 3000
106 · 33rd Annual Meeting of the German Society of Immunology Generation 3 (G3) mice in the recessive screen. Second samples have been taken from 1181 mice. In 286 cases phenotypes were tested for inheritance. While 28 confirmation crosses are still running, more than 70 mutant mouse lines with immunological alterations could be established thus far. The established mutant lines encompass phenotypes with complete loss or alterations in the Ig level of single or multiple isotypes as well as complete loss or skewed percentages of lymphocyte subpopulations. Comparison of the dominant and recessive phases of the screen revealed interesting differences. As expected the recessive screen delivered a higher percentage and more severe phenotypes compared to the dominant screen. At the same time fertility of the respective phenotypes decreased. However, despite the loss of phenotypes due to fertility problems, the yield of mutant mouse lines with immunological alterations was higher compared to the dominant screen. The mutant mouse lines with immunological alterations are available to non commercial users. For an updated list of mutants and their phenotypes contact the author. Website: http://www.gsf.de/ieg/groups/enu/mutants/index.html Email: [email protected]
Hematopoiesis Unit, Chair of Genetics, Nikolaus-Fiebiger-Center, Friedrich-Alexander University, Erlangen, Germany
H. 7 Downregulation of the V(D)J-recombinase machinery in mouse pre-B cells is mediated by mH-chain surface expression and is not dependent on surrogate light chain G.R. GALLER and T.H. WINKLER Early B cell development in mouse bone marrow is characterized by stepwise, ordered rearrangement of the immunoglobulin genes. Only one of the two potential immunoglobulin loci of the heavy and light chain becomes functionally rearranged, a phenomenon called allelic exclusion. The molecular basis of this regulation is poorly understood. It has been suggested, that exposure of the newly synthesized mH-chain chain in complex with surrogate light chain (pre-BCR) signals downregulation of the V(D)J-recombinase machinery (RAG-1, RAG-2 and TdT), thereby preventing further rearrangement on the second allele. In a mouse model we could show that expression of a transgenic mH-chain in RAG-2–/– pro-B cells induces downregulation of TdT, and a transgenic GFP-reporter reflecting endogenous RAG expression, on protein level. Surprisingly, similar effects were observed in surrogate light chain mutant mice (l5–/– or VpreB–/–) although a functional pre-BCR cannot be assembled and thus pre-B cells do not proliferate upon mH-chain induction. It is assumed that mH-chains are retained in the ER in the absence of l5 or VpreB, raising questions about the origin of the observed signals. Using enzymatic-amplification-staining we could detect surface m-expression in l5–/– cells, showing that surrogate light chain is obviously not required for m-heavy chain ER exit and transportation to the cell surface. These results are consistent with the fact that all knockout mice for surrogate light chain components still display allelic exclusion in the periphery and provide evidence for two distinct signaling pathways in pre-B cells. Whilst a functional pre-BCR is required to signal proliferation, m-heavy chain expression alone could be responsible for signaling allelic exclusion.
33rd Annual Meeting of the German Society of Immunology · 107 1
Department of Molecular Immunology, Institute of Biology, University of Freiburg, Freiburg, Germany and 2Department of Immunology, National Jewish Center for Medical Research, Denver CO, USA
H. 8 Inducible B cell development in MerCreMer knock-in mice E. HOBEIKA1, R. PELANDA2, and M. RETH1 We generated knock-in (k.i.) mice with a B cell-specific expression of the Tamoxifen-regulated Cre recombinase MerCreMer carrying at both termini a mutated murine estrogen receptor binding domain (Mer). The MerCreMer cDNA was inserted in the mb-1 gene encoding the Ig-a chain of the B cell receptor (BCR). In order to study B cell development we crossed the MerCreMer mouse to the GFP/mb-1 k.i. mouse (Genesis 2002 32:154–157). The GFP/mb-1 cassette can be inverted by Cre recombination and thus alternatively gives rise either to Ig-a or GFP expression. We show that administration of Tamoxifen in vitro as well, as in vivo, induces inversion of the GFP/mb-1 cassette. Furthermore, no background recombination was detected in the absence of Tamoxifen. In MerCreMer and GFP/mb-1 double k.i. mice, B cell development is blocked at the pro-B cell stage. Tamoxifen administration allows gene inversion and expression of Ig-a resulting in a wave of B cells emigration from the bone marrow to the periphery. These newly generated B lymphocytes could be isolated and characterized. Alternatively, in MerCreMer and mb-1/GFP double k.i. mice Ig-a expression can be terminated by Tamoxifen induced gene inversion resulting in loss of BCR and induction of GFP expression. Preliminary results show the presence of a long-lived population of GFP positive B cells. This double k.i. system will be used to test the requirement for the BCR and its signaling elements during B lymphocyte development. Specifically we will test how long mature B cells can survive without the BCR and what characteristics BCR-negative B cells have. The protocols for Tamoxifen-induced Cre activation are also being optimized.
Basel Institute of Immunology, Basel, Switzerland
H. 9 Rules for gene usage inferred from a comparison of large-scale gene expression profiles of T and B lymphocyte development R. HOFFMANN*, L. BRUNO, T. SEIDL, A. ROLINK, and F. MELCHERS RNA expression profiles of seven consecutive stages of mouse thymocyte development were generated on high-density oligonucleotide arrays. Previously known expression patterns of several genes were confirmed. Ten percent (1304 of over 13000) of the monitored genes were found with 99% confidence to be differentially expressed across all T cell developmental stages. When compared with 1204 genes differentially expressed in five consecutive B lineage developmental stages of bone marrow, more than 40% (546 genes) appeared to be shared by both lineages. However, when four pools of functionally distinct cell stages were compared between B and T cell development, DJ-rearranged precursor cells and resting immature precursor cells before and after sur-
108 · 33rd Annual Meeting of the German Society of Immunology face antigen receptor expression shared less than 10%, mature resting lymphocytes between 15% and 20%, and only cycling precursors responding to precursor lymphocyte receptor deposition share more than 50% of these differentially expressed genes. Three general rules emerge from these results: 1) Proliferation of cells at comparable stages is in majority executed by the same genes; 2) intracellular signaling and intercellular communication is effected largely by different genes, and 3) most genes are not used strictly at comparable, but rather at several, stages, possibly in different functional contexts. *present address: Max-von-Pettenkofer-Institut, Ludwg-Maximilians-Universität, München, Germany
Departments of 1Bacteriology, Max-von-Pettenkofer-Institut and 3Medical Informatics, Biometrics and Epidemiology, University of Munich, Munich, Germany, and 2Section of Gene Function and Regulation, The Institute of Cancer Research, London, UK
H. 10 A novel set of lineage- and stage-specific markers in lymphoid development identified by gene expression profiling and supervised machine learning R. HOFFMANN1, T. SEIDL2, L. BRUNO2, and M. DUGAS3 B and T lymphocytes develop through a series of cellular stages which are defined by recombination status of the immunoglobulin- and TCR loci and can be separated by analysis of cell surface marker proteins. We have generated high-density oligonucleotide array based gene expression profiles containing approx. 13,000 genes from five B cell and eight T cell precursor stages. Analysis of these datasets with supervised machine learning algorithms identified novel lineage and stage specific markers. Eight Genes are identified that correctly predict all cell samples as belonging to either the B- or the T cell lineage. Individual stages of the two lineages can optimally be predicted by models containing 29 (for B-lineage cells) or 39 (for T-lineage cells) genes. These models contain ESTs as well as known genes that have not previously been associated with lymphocyte development, and only very few of the established markers. When analyzed by Principal Component Analysis, a perfect separation based on the first two principal components can be made for the distinction between B- and T-lineage cells as well as for stages within the B cell lineage, while stages within the T cell lineage do not separate well. Thus, the markers currently widely used for separating B cell stages are more in accordance with the cellular gene expression profile as those used for separating T cell stages.
33rd Annual Meeting of the German Society of Immunology · 109 1
Division Environmental Dermatology and Allergology, GSF-TUM and 4Institute of Experimental Genetics, GSF Research Center, Neuherberg, Institutes of 2Molecular Animal Breeding, Gene Center, 3Medical Microbiology, Immunology and Hygiene, Technical University, Munich, and 5German Research Center for Biotechnology (GBF), Braunschweig, Germany
H. 11 Analysis and chromosomal mapping of selected mouse mutants with immunological alterations derived from the Munich ENU mouse mutagenesis project T. JAKOB1, B. RATHKOLB2, M. HOWALDT2, A. SERVATIUS3, D. SOEWARTO4, N. SANDHOLZER3, M. HRABE DE ANGELIS4, R. BALLING5, H. BEHRENDT1, E. WOLF2, K. PFEFFER3, and H. FLASWINKEL2 Out of more than 70 mouse mutant lines with immunological alterations established within the Munich ENU Mouse Mutagenesis Project we are currently analyzing selected lines in more detail. One of these mutant lines TUM079/IEHR01 recovered from the recessive screen is especially interesting in that T cells are almost absent in peripheral blood and IgE levels are increased more than 10–30 fold above normal range between 10–14 weeks of age. All other isotypes tested are deviating much more moderately from normal. First consumptive analysis revealed several important alterations. The thymus is reduced or not visible at all. Spleen size is increased more than twofold. Coat is present but rough and bare patches are visible at six month of age. Female mutants frequently die early and female as well as male mice are growth retarded. Further phenotypic analysis and mapping of the respective mutation are in progress. Two other mutant mouse lines are almost devoid of basal immunoglobulin. In one case all isotypes measured are reduced to a similar fraction. In the second line, IgM, IgG1, IgG3 and IgA are drastically reduced while IgG2a is about normal. Yet another recessive mutant line TUM038/Dia001 exhibits anti-DNA antibodies, a drastic increase in basal IgA in combination with chronic diarrhea. Mutants are fertile but viability is reduced. All mutant mouse lines are inbred C3Heb/FeJ. We will present recent data on selected mutant mouse lines recovered from the Munich ENU Mouse Mutagenesis Project.
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Molecular Immunology, German Research Center for Biotechnology (GBF), Braunschweig and 2Department of Bacteriology, Max-von-Pettenkofer-Institut, Munich, Germany
H. 12 Transgenic expression of a l2315 light chain results in a severe abrogation of B2 development but normal generation of marginal zone B lymphocytes A. JUNGEBLOUD1, J. STOPKOWICZ1, R. HOFFMANN2, S. WEISS1, and K. KRETSCHMER1 The mature preimmune splenic B cell compartment of normal adult mice consists of CD23+CD21int follicular B cells (FO or B2 cells), CD23lo/–CD21hi marginal zone (MZ) B cells and CD5+ B1 cells, each of which exhibit specific developmental and functional characteristics.
110 · 33rd Annual Meeting of the German Society of Immunology Here, we describe that the high expression of a transgenic l2315 light chain differentially affects these mature B lymphocyte subpopulations in the recombinant mouse line L2. A severe block in B cell development in adult bone marrow at the transition from large to small preB-II cells is observed. Consistently, B2 cells are completely lacking in such mice. CD5+ B1 cells, generated early in mouse development, predominate in both peritoneal cavity and spleen. Interestingly, CD5– B cells present in the spleen show the CD23lo/–CD21hi MZ B cell surface marker phenotype. Gene expression profiling was carried out with B cells from such mice. Using high-density oligonucleotide arrays representative of approximately 12.000 genes clearly demonstrated that CD23lo/–CD21hi B cells from L2 mice correspond to MZ B cells from non-transgenic control mice. Substantial differences in the gene expression pattern between splenic B1a and MZ B cells were detected and paralleled by a clear difference in the IgH chain repertoire. Whereas the light chain restriction results in high frequency of identical IgH chains with a high number of independent rearrangement events in the B1a compartment, we could not find any evidence for such an oligoclonal IgH chain repertoire in the MZ B cell compartment. IgH chains from these MZ B cells show a high frequency of N nucleotide insertions, indicating their generation in adult bone marrow in the presence of TdT activity. These data show that B1a and MZ B lymphocytes mediate specialized effector functions of the splenic B cell compartment.
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Clinical T Cell Immunology, Deutsches Rheuma-Forschungszentrum and 4Haematology und Oncology, Virchow Klinikum, Berlin, and 3Department of Haematology und Oncology, ErnstMoritz-Arndt-University, Greifswald, Germany, and 2Institute of Human Genetics, Poznan, Poland
H. 13 Two subsets of peripheral blood naïve T helper cells with distinct T cell receptor excision circle content indicate peripheral homeostatic expansion of recent thymic emigrants in adult humans S. KIMMIG1, G.K. PRZYBYLSKI2, C.A. SCHMIDT3, K. LAURISCH4, B. MÖWES1, B. OPPMANN1, A. RADBRUCH1, and A. THIEL1 Introduction: During ageing thymic function declines and is unable to meet the demand for peripheral Th cell replenishment. Therefore population maintenance of naïve Th cells must be at least partly peripherally based. Such peripheral postthymic expansion of recent thymic emigrants (RTEs) during ageing consequently should lead to loss or dilution of T cell receptor excision circles (TRECs) from a subset of naïve Th cells. Results: We have identified two subsets of naïve Th cells in human adult peripheral blood characterized by a striking unequal content of TRECs, indicating different peripheral proliferative histories. In nine analyzed healthy donors TRECs are highly enriched in peripheral naïve CD45RA+ Th cells coexpressing CD31 as compared to peripheral naïve CD45RA+ Th cells lacking CD31 expression, in which TRECs can hardly be detected. Furthermore we show that CD31–CD45RA+ Th cells increase in frequency during ageing but retain all phenotypic and functional features of naïve Th cells. Based on our results it is now possible to distinguish peripherally postthymically expanded centralnaïve Th cells, described by loss of CD31 expression and highly reduced TREC content, from thymicnaïve Th cells recently emigrated from the thymus.
33rd Annual Meeting of the German Society of Immunology · 111 Interestingly, more recent investigations have shown that CD31 triggering leads to inhibition of TCR signaling via immunoreceptor tyrosine-based inhibitory motifs (ITIM). Conclusion: Since TRECs are reduced up to 10fold in centralnaïve Th cells peripheral postthymic expansion implicates extensive cell proliferation of thymicnaïve Th cells. Our results enable direct access and thereby further functional and molecular analysis of these two human peripheral naïve Th cell subsets.
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Molecular Immunology, German Research Center for Biotechnology (GBF), Braunschweig and 2Department of Bacteriology, Max-von-Pettenkofer-Institut, Munich, Germany
H. 14 Antibody repertoire and gene expression profile of splenic and peritoneal B1a lymphocytes: Implications on different developmental and functional traits K. KRETSCHMER1, A. JUNGEBLOUD1, J. STOPKOWICZ1, R. HOFFMANN2, and S. WEISS1 High expression level of the transgenic l2315 light chain in L2 mice results in near complete exclusion of endogenous light chains and a predominance of B1a cells in both peritoneum and spleen, whereas B2 cells are completely absent. Availability of a single light chain has been shown to lead to an oligoclonal IgH chain repertoire of peritoneal B1a lymphocytes. Here, we show that splenic and peritoneal B1a cells of L2 mice differ considerably in their functional properties. The IgH chain repertoire of splenic, peritoneal and blood derived B1a cells was established using the clonotypic IgH chain sequences as potential diagnostic markers for migration and exchange. Splenic B1a cells exhibit much more IgH chain diversity under such L chain limitation due to high frequencies of N nucleotides. Despite few oligoclonal overlaps between the splenic and peritoneal B1a compartment, some B cell receptor specificities are clearly restricted to the peritoneum. In addition, reconstitution experiments using a congenic IgH allotype system suggest that peritoneal B1a cells from L2 mice transferred into the peritoneum of congenic L2 recipients have a very limited capacity to enter the splenic B1a compartment. Thus, identical IgH chains overlapping between spleen and peritoneum appear to be unlikely due to migratory exchange. Consistently, gene expression profiling revealed substantial differences in the pattern of expressed genes between the two B1a compartments. Some upregulated genes found in splenic B1a cells are potentially involved in mediating specialized «first line of defense» effector functions and interaction with T cells. Interestingly, splenic B1a and peritoneal B1b cells appear to be very similar concerning IgH chain repertoire and gene expression profile. The striking differences in antibody repertoire and pattern of expressed genes between splenic and peritoneal B1a cells indicate that both B1a subpopulations differ not only in their functional properties but also in their developmental program.
112 · 33rd Annual Meeting of the German Society of Immunology 1
Clinical Research Unit for Rheumatology, University Medical Center, 2Department of Molecular Immunology, Institute of Biology, University of Freiburg, and 3Max-Planck-Institute of Immunobiology, Freiburg, Germany; and 4Department of Immunology, University of Basel, Basel, Switzerland
H. 15 Egr-1 controlled B cell differentiation M. RAKHMANOV1, H. JUMAA2, P. NIELSEN2, C. BLEUL3, A. ROLINK4, M. RETH2, and H. EIBEL1 Expression of the transcription factor Egr-1 in B cells is rapidly induced upon antigen binding to the B cell receptor. The intracellular signal chain that initiates the transcription of the Egr-1 gene involves the sequential activation of phosphotyrosine protein kinases and the generation of second messengers. The adapter protein SLP65 is a key component of this process as it serves as a scaffold for the assembly of B cell receptor complex-associated enzymes. Deletion of the slp65 gene results in impaired B cell maturation and arrests development at the stage of immature B cells in the spleen. Since the induction of Egr-1 expression depends on signals transmitted through SLP65 we generated SLP65-deficient mice expressing an Egr-1 transgene (tgEgr-1) in B cells to study the cellular responses regulated by this transcription factor. Phenotypic comparison of slp–/– and of tgEgr-1¥slp–/– spleen B cells revealed that Egr-1 activity partially rescued the developmental block caused by the SLP65 defect and allowed the differentiation into B cells with a more mature phenotype. To identify Egr-1 target genes that promote Egr-1 induced B cell maturation, we purified total RNA from B cells isolated from spleens of slp65-deficient, of tgEgr1¥slp65–/– and of BALB/c control mice and prepared probe sets for hybridization experiments with DNA microarrays. Using Affymetrix U74 genechips, we then established gene expression profiles of these samples and analyzed the changes in the expression patterns between tgEgr1¥slp65–/–, slp65–/– and BALB/c mice. Our results show, that Egr-1 activity induces genes that are expressed under normal conditions in B cells of BALB/c control animals but not in slp65 knockout mice. Based on these data, we now are able to establish a signal chain that starts at the B cell receptor complex and uses SLP65 and Egr-1 to induce B maturation.
Molecular Immunology, German Research Center for Biotechnology (GBF), Braunschweig, Germany
H. 16 Differential effect of control elements on somatic hypermutation of a transgenic l2 immunoglobulin light chain I. RODE, M. NACKE, H. ENGEL, K. KRETSCHMER, and S. WEISS During an antibody response the affinity of the induced antibodies increases by the accumulation of single nucleotide exchanges in the variable regions of the heavy and light chains. This so called somatic hypermutation takes place in the dark zone of the germinal centers and is dependent on strong transcription of the Ig chains that is effected by juxtaposition of strong promoters and enhancers during the rearrangement process. Here we wanted to test which of the three characterized light chain enhancers is able to support somatic hypermutation of a transgenic l2
33rd Annual Meeting of the German Society of Immunology · 113 light chain. To this end we established four types of transgenic mice carrying a genomic fragment encoding the l2 chain of MOPC315 driven by the Í intron enhancer, the Í 3’enhancer, both Í enhancers and the enhancer found downstream of the l2–4 cluster. All lines showed similar expression patterns of the transgenes, i.e. endogenous L chain rearrangement was only partially inhibited and a significant population of B cell displayed Í and l at their cell surface. For analysis PNAhi and PNAlo B cells from Peyer’s patches of 9–18 months old mice were sorted, RNA was isolated and the transgenic transcript or transcripts of an endogenous Í chain was amplified by RT-PCR, cloned and sequenced. Alternative, genomic DNA from such cells was used for amplification. A clear difference was found between constructs that contained the intron enhancer of the Í chain and those that were driven by the 3’ or the l enhancer, respectively. Only the intron enhancer rendered the constructs a substrate for the somatic hypermutation mechanism independent of whether the 3’ enhancer was additionally present in the construct. The mutations accumulated in the expected hot spots. Some mutations were also found in the PNAlo population although such cells are usually not considered to be in the process of mutating.
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Department of Functional and Applied Anatomy, Medical School, Hannover and Department of Internal Medicine I, University Hospital, Regensburg, Germany, and 2 Department of Periodontology, Faculty of Dentistry, University of Oslo, Oslo, Norway 3
H. 17 Different postnatal experiences determine adult disease susceptibility: Stress protection as an option for the treatment of periodontal disease M. STEPHAN1, T. BREIVIK2, R.H. STRAUB3, R. PAPST1, and S. VON HÖRSTEN1 Objective: Stress affects wound healing in the oral cavity of humans. Individual differences in the stress responsiveness may therefore determine susceptibility to oral and dental diseases. Early experiences including maternal deprivation (as a paradigm for adverse early life events) or handling (as a paradigm of postnatal stimulation) alter the development of neural systems including the hypothalamic-pituitary-adrenal axis, which govern endocrine responses to stress. The present studies investigated whether acquired differences in the stress response interact with a given genetic background and whether interventional strategies, considered as a stress protection, are successful in preventing increased disease susceptibility in rats. Methods: Male F344 or LEW rats were daily handled (i.e. brief maternal separation and exposure to novelty), maternally deprived (i.e. maternal separation for h), or left undisturbed until weaning. Prophylactic treatment strategies (additional tactile stimulation directly after maternal deprivation) or treatment using antidepressants in adulthood (imipramine via drinking water) were applied to protect from the maternal deprivation induced effects. At 5 months of age, placing silk-ligatures around molar teeth induced periodontal disease. At 7 months, neuroendocrine stress response (corticosterone, NPY), cytokines, and clinical outcome of periodontal disease were determined. Results: Stress high responding F344 rats developed significantly more periodontal disease. Only in the stress low responding LEW rat strain, handling-induced reduction of adult stress responsiveness was protective while maternal deprivation aggravated experimental periodontal disease. Both, interventions by additional tactile stimulation in infancy as well as antidepressants in adulthood protected from the deprivation-induced aggravation of the disease.
114 · 33rd Annual Meeting of the German Society of Immunology Conclusions: Individual differences acquired in the postnatal period specifically alter neuroendocrine stress responsiveness, thereby shifting disease susceptibility and aggravating periodontal disease an the basic of the genetic background. It is suggested that in a subgroup of genetically stress low responding individuals stress protection is effective in the treatment of periodontal disease.
Institute of Virology and Immunobiology, University of Würzburg, Würzburg, Germany
H. 18 Notch signaling and rat thymocyte development J. VAN DEN BRANDT, S.H. KWON, M. SCHOTT, and H.M. REICHARDT Notch, a family of transmembrane receptors, is involved in directing the choice between alternative cell fates. In mouse thymopoiesis, Notch-1 has been implicated in bipolar lineage decisions between T and B cells, ab and gd T cells and CD4 and CD8 single-positive cells. However, the role of Notch in these processes is controversially discussed. Recent studies have shown that similar stimuli can cause opposite lineage decisions in mouse and rat thymocytes. This prompted us to study thymus development in the absence of Notch signaling taking a pharmacological approach in the rat. Generation of active Notch requires cleavage of the intracellular domain by g-secretase. Consequently, fetal thymus organ culture (FTOC) in the presence of inhibitor can be used to study the role of Notch in thymocyte lineage decisions. Thymuses were recovered at day e16.5 and cultured for six days. Subsequently, the cells were stained with Abs against CD2, CD4, CD8, CD45RC, CD45RA, abTCR and gdTCR and analysed by FACS. We found that interfering with Notch signaling arrests thymocyte development at an early double-negative stage. In particular, the development of the abTCR lineage but not of the gdTCR lineage was affected under these conditions. The observed phenotypic changes correlate with a reduced expression of several Notch target genes, such as Hes-1. Finally, in contrast to mice, lack of Notch signaling in the rat was found to promote the generation of CD8 at the dispense of CD4 singlepositive cells. Collectively, we believe that this pharmacological approach should help to further decipher the role of Notch in thymus development as well as to define differences between rats and mice in more detail.
Institute of Clinical Transfusion Medicine and Immunogenetics, DRK-Blutspendedienst Baden-Württemberg-Hessen, Ulm, Germany
H. 19 An enhanced green fluorescence protein based assay: Making V(D)J recombination visible Z. XIAO and K. SCHWARZ A single cell in vivo green fluorescence protein (GFP) based assay was successfully developed to monitor V(D)J recombination efficiency. This assay makes V(D)J recombination visible at a single cell level. Furthermore, efficiency of V(D)J recombination can be evaluated in real time.
33rd Annual Meeting of the German Society of Immunology · 115 Our results show that the in vivo V(D)J recombination efficiency is low (≈1%) in 293 cells. This in vivo V(D)J recombination efficiency correlated well with that achieved by a classical in vitro V(D)J recombination assay designed by Lieber and colleagues. The in vivo V(D)J recombination efficiency depended on the relative RAG-1 and RAG-2 expression vector concentrations used for co-transfection. The test differentiates RAG null mutants as seen in severe combined immunodeficiency (SCID) from Omenn syndrome hypomorphic RAG mutants. In addition, this novel assay is also used to screen novel factors involved in V(D)J recombination.
Department of Cellular Immunology, Max-Planck-Institute of Immunobiology, Freiburg, Germany
bic+TCRd dic+ DN4 thymocyte population: H. 1 Analysis of the TCRb Possible gd lineage commitment C.E. BUSSE, G. NERZ, A. KROTKOVA, and K. EICHMANN Approximately 1–2% of all CD4–CD8–CD25–CD44– (DN4) thymocytes express both the TCR b and TCR d chains intracellularly (ic). Until now, the fate of this population, whose existence cannot be easily explained when assuming an inductive lineage commitment model, could not be determined. In order to determine the fate of TCRbic+/TCRdic+ DN4 cells we first studied the correlation between intracellular and surface (sf ) expression of the TCR d chain, which turned out to be greater than 97%. Due to the fact that TCR d chains can only get to the surface when paired with a TCR g chain, we conclude that nearly all TCRdic+ cells also express TCR g. Next, we analyzed the mRNA expression of TCR b and preTa in gd TCRsf+ DN4 cells by semiquantitative RT-PCR. While the expression of TCR b in this population was about 25% of that in CD4–CD8–CD25+CD44– (DN3) thymocytes, preTa expression is less than 1% of that in DN3. Since the ratio of TCRbic– to TCRbic+ cells within the gd TCRsf+ DN4 population is approximately 3:1, this implies that TCRbic+/TCRdic+ DN4 cells express TCR b at a level comparable to DN3 cells – which is confirmed by FACS analysis –, while expressing little or no preTa. To determine whether the TCR b chains expressed in the TCRbic+/TCRdic+ DN4 population possess unusual V(D)J joints, which might lead to an inability of the TCR b chain to pair efficiently with preTa, we sequenced the Vb8.2DbJb2.7 joints of sorted TCRbic+/TCRdic– and TCRbic+/TCRdic+ DN4 cells. The comparison of 20 TCRbic+/TCRdic– and 19 TCRbic+/ TCRdic+ sequences did not reveal any striking differences between the two populations. In summary, our results are consistent with the hypothesis that TCRbic+/TCRdic+ DN4 cells are committed to the gd lineage and might be the precursors of TCRbic+ gd T cells in the periphery.
Institute of Immunology, Ludwig-Maximilians-University, Munich, Germany
H. 2 Dendritic cells induce negative, but not positive selection of CD8 T cells and cannot educate NK cells in vivo M.A. CANNARILE and T. BROCKER During the selection process of T cells in the thymus, CD8 T cells are first positively selected by functional recognition of MHC class I molecules. Then autoreactive CD8 T cells are eliminated in the thymus through negative selection. We generated mice with MHC class I molecules selectively on dendritic cells (DC) by expressing b2m under the control of the CD11c-promoter
33rd Annual Meeting of the German Society of Immunology · 103 (CD11c-MHC I) in a b2m knockout (b2m KO) background. The influence of DC on the selection of CD8 T cells was investigated by comparing CD8 T cell numbers and the T cell receptor (TCR)-repertoires of C57BL/6, b2m KO and CD11c-MHC I mice. Flow cytometric analysis revealed that, compared to wildtype C57BL/6 mice, CD11c-MHC I transgenic and b2m KO mice have 7-fold fewer CD8 positive T cells in the thymus and a 15-fold reduction in the periphery. These data suggests MHC class I expression on thymic DC is not sufficient for positive selection of CD8 T cells. Analysis of TCR-Vb- domains also showed that the CD8 T cell repertoire of CD11c-MHC I mice is more similar to the repertoire in b2m KO mice than to wild type animals. Transfer of MHC class I splenocytes into wildtype, transgenic and b2m KO mice showed that CD11c-MHC I-mice tolerize MHC class I expressing cells, whereas they are rejected in b2m KO mice, indicating that DC are sufficient to negatively select CD8 T cells. Interestingly, adoptively transferred cells from b2m KO mice to the three described mouse strains were also accepted by CD11c-MHC I mice. These data indicate that despite the presence of MHC class I on DC in CD11c-MHC I mice, NK function towards cells missing MHC class I was not restored. Thus only negative, but no positive, selection events can be induced by DC in vivo. The exact influence of DC on NK cell development has to be further investigated.
Institute of Transplantation Diagnostics and Cell Therapeutics, Heinrich-Heine-University, Düsseldorf, Germany
H. 3 Induction of CD94:NKG2A expression on NK cells but not T cells by treatment with the histone deacetylase inhibitor trichostatin A B. EISERMANN, S. SANTOURLIDIS, G. KÖGLER, P. WERNET, and M. UHRBERG The target cell specificity of human NK cells is determined by the clonally-distributed expression of two MHC class I-specific NK receptor (NKR) families called KIR (Killer Immunoglobulin-like Receptors) and CD94:NKG2. However, it is largely unknown how the human NKR repertoire is formed and tolerance induction is achieved during NK cell development. As a simple in vitro model of NK cell differentiation, human cord blood (CB)-enriched CD34+ stem cells were cultured with Flt3-L in combination with either IL-2, IL-12, or IL-15. After four weeks of culture, the strongest induction of CD94:NKG2A was seen with IL-15/Flt3L (55,9% of CD56+ NK cells) followed by IL-12/Flt3-L (49,1% of NK cells). IL-2/Flt3-L led to the highest frequency of NK cells but a smaller percentage of CD94:NKG2A (33,3%). In contrast, no induction of KIR expression was detectable under the chosen culture conditions. We next investigated if the induction of CD94:NKG2A expression was dependent on epigenetic modification of DNA as shown recently for the expression of the KIR gene family. Treatment of the CD94:NKG2A-negative NK cell line YT with the demethylating agent 5Aza-dC led to a moderate induction of CD94:NKG2A surface expression (24,3%). Surprisingly, a much stronger induction of CD94:NKG2A expression (40,1%) was observed with the histone deacetylase inhibitor trichostatin A (TSA), a chemical agent which induces an open chromatin structure. A synergistic effect was seen upon combinatory treatment of YT with 5Aza-dC and TSA (71,9%). In contrast, treatment of polyclonally-stimulated PBMC-derived T cells with TSA and/or 5AzadC did not result in any CD94:NKG2A induction. This parallels the distribution of CD94:NKG2A in vivo, which is largely restricted to NK cells and only rarely found on T cells.
104 · 33rd Annual Meeting of the German Society of Immunology Our results indicate that the NK cell-specific expression of CD94:NKG2A is modulated by changes in chromatin structure and to a lesser extend by DNA methylation.
1
Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Universität zu Köln, Köln, Germany and 2Department of Biological Sciences, University of Chicago, Chicago IL, USA
H. 4 Molecular portraits of B cell lineage commitment N. FELDHAHN1, F. KLEIN1, S. LEE2, V.S. BARATH1, H. WANG1, K. TANG1, M. KRÖNKE1, J.D. ROWLEY2, S.M. WANG2, and M. MÜSCHEN1 In an attempt to characterize early development of human B cells including the commitment of progenitor cells to the B cell lineage, we generated and compared genome-wide gene expression profiles of CD34+ human hematopoietic stem cells (HSC) and pre-B cells using the serial analysis of gene expression (SAGE) technique. Human CD34+ HSC and CD10+CD19+CD20– preB cells were purified by MACS from bone marrow and umbilical cord blood samples from four and 28 healthy donors, respectively. The identity of the purified populations was confirmed by flow cytometry and PCR amplification, cloning and sequencing of IgH germline fragments, DHJH- and VH-DHJH-rearrangements. Thereafter, both populations were subjected to SAGE-analysis, in which mRNA molecules are transformed into 14-bp cDNA sequences, the so-called SAGE-tags. For HSC and pre-B cells, more than 100,000 SAGE-tags were collected, which establish a quantitative and genome-wide transcriptome of these cells. While the transcriptome of CD34+ HSC encompasses 42,399 individual transcripts, only 16,786 were identified in the preB cell SAGE-profile, indicating that about 60% of genes expressed in HSC were silenced during or after commitment to the B cell lineage. On the other hand, mRNAs of pre-B cell receptorassociated genes are missing in HSC but account for more than 10% of the transcriptome of preB cells, which also show markedly increased expression of apoptosis-related genes. Both, concentration of the transcriptional repertoire on pre-B cell receptor-related genes together with upregulation of apoptosis-mediators in pre-B cells might reflect selection for the expression of a functional pre-B cell receptor within the bone marrow. Besides known regulator genes of early B cell development such as PAX5, E2A, and EBF, the most abundantly expressed genes in pre-B cells include ATM, PDGFRA, SIAH1, PIM2, C/EBPB, WNT16 and TCL1, which were, so far, not implicated in the regulation of early B cell development.
33rd Annual Meeting of the German Society of Immunology · 105 Institut für Virologie und Immunbiologie, Universität Würzburg, Würzburg, Germany
H. 5 Suppression of germ line g1 transcripts and arrest of isotype switching in cells expressing Blimp-1 E. FEOKTISTOVA, D.S. VALLABHAPURAPU, I. BERBERICH, and A. SCHIMPL Following stimulation, primary B cells either directly undergo terminal differentiation to IgM secreting plasma cells or enter the memory pathway, characterised by affinity maturation and isotype switching. High rate secretion correlates with endogenous Blimp-1 expression which in turn is stimulated by cytokines such as Il-2 and IL-5 but inhibited by ligation of the BCR, CD40 or by IL-4. Importantly, introduction of Blimp-1 by retroviral gene transfer is sufficient to overcome the block in Ig secretion caused by BCR-, CD40- or IL-4R ligation. At the same time, the drastic increase in membrane IgG1+ cells with time in cultures treated with IL-4 is greatly diminished in cells forced to express Blimp-1 while cells that have already switched are turned into IgG1 secreting plasma cells. We now show by RT-PCR analysis that arrest in isotype switching caused by Blimp-1 is accompanied by a loss of excision circle transcription accompanying the class switch process while expression of AID (activation induced cytidine deaminase) is less drastically affected. We conclude that suppression of Blimp-1 by antigen-BCR interaction and T helper cell dependent CD40 and IL-4 signaling is necessary to facilitate class switch recombination and prevent terminal differentiation.
Institutes of 1Molecular Animal Breeding, Gene Center, 2Medical Microbiology, Immunology and Hygiene, Technical University, and 4Molecular Immunology, GSF National Research Center for Environment and Health, Munich, 3Institute of Experimental Genetics, GSF Research Center, Neuherberg, and 5German Research Center for Biotechnology (GBF), Braunschweig, Germany
H. 6 A phenotype-driven approach for the identification of mutants with immunological alterations among offspring of ENU-treated C3HeB/FeJ mice H. FLASWINKEL1, B. RATHKOLB1, M. HOWALDT1, A. SERVATIUS2, D. SOEWARTO3, S. MARSCHALL3, E. KREMMER4, N. SANDHOLZER2, M. HRABE DE ANGELIS3, R. BALLING5, E. WOLF1, and K. PFEFFER2 Dominant as well as recessive mouse mutants have been generated in large scale and analyzed for a multitude of parameters by a consortium of screening groups. The project is a part of the German Human Genome Project and aims at the production of mouse models resembling human diseases. More information about the ENU-Mouse Mutagenesis Screen Project can be obtained via the world wide web. Employing ELISAs for basal immunoglobulin levels, anti-DNA antibodies and rheumatoid factor as well as flow cytometry for the detection of cell surface molecules on a semi automated basis we were able to analyze more than 6000 F1 mice in the dominant and more than 3000
106 · 33rd Annual Meeting of the German Society of Immunology Generation 3 (G3) mice in the recessive screen. Second samples have been taken from 1181 mice. In 286 cases phenotypes were tested for inheritance. While 28 confirmation crosses are still running, more than 70 mutant mouse lines with immunological alterations could be established thus far. The established mutant lines encompass phenotypes with complete loss or alterations in the Ig level of single or multiple isotypes as well as complete loss or skewed percentages of lymphocyte subpopulations. Comparison of the dominant and recessive phases of the screen revealed interesting differences. As expected the recessive screen delivered a higher percentage and more severe phenotypes compared to the dominant screen. At the same time fertility of the respective phenotypes decreased. However, despite the loss of phenotypes due to fertility problems, the yield of mutant mouse lines with immunological alterations was higher compared to the dominant screen. The mutant mouse lines with immunological alterations are available to non commercial users. For an updated list of mutants and their phenotypes contact the author. Website: http://www.gsf.de/ieg/groups/enu/mutants/index.html Email: [email protected]
Hematopoiesis Unit, Chair of Genetics, Nikolaus-Fiebiger-Center, Friedrich-Alexander University, Erlangen, Germany
H. 7 Downregulation of the V(D)J-recombinase machinery in mouse pre-B cells is mediated by mH-chain surface expression and is not dependent on surrogate light chain G.R. GALLER and T.H. WINKLER Early B cell development in mouse bone marrow is characterized by stepwise, ordered rearrangement of the immunoglobulin genes. Only one of the two potential immunoglobulin loci of the heavy and light chain becomes functionally rearranged, a phenomenon called allelic exclusion. The molecular basis of this regulation is poorly understood. It has been suggested, that exposure of the newly synthesized mH-chain chain in complex with surrogate light chain (pre-BCR) signals downregulation of the V(D)J-recombinase machinery (RAG-1, RAG-2 and TdT), thereby preventing further rearrangement on the second allele. In a mouse model we could show that expression of a transgenic mH-chain in RAG-2–/– pro-B cells induces downregulation of TdT, and a transgenic GFP-reporter reflecting endogenous RAG expression, on protein level. Surprisingly, similar effects were observed in surrogate light chain mutant mice (l5–/– or VpreB–/–) although a functional pre-BCR cannot be assembled and thus pre-B cells do not proliferate upon mH-chain induction. It is assumed that mH-chains are retained in the ER in the absence of l5 or VpreB, raising questions about the origin of the observed signals. Using enzymatic-amplification-staining we could detect surface m-expression in l5–/– cells, showing that surrogate light chain is obviously not required for m-heavy chain ER exit and transportation to the cell surface. These results are consistent with the fact that all knockout mice for surrogate light chain components still display allelic exclusion in the periphery and provide evidence for two distinct signaling pathways in pre-B cells. Whilst a functional pre-BCR is required to signal proliferation, m-heavy chain expression alone could be responsible for signaling allelic exclusion.
33rd Annual Meeting of the German Society of Immunology · 107 1
Department of Molecular Immunology, Institute of Biology, University of Freiburg, Freiburg, Germany and 2Department of Immunology, National Jewish Center for Medical Research, Denver CO, USA
H. 8 Inducible B cell development in MerCreMer knock-in mice E. HOBEIKA1, R. PELANDA2, and M. RETH1 We generated knock-in (k.i.) mice with a B cell-specific expression of the Tamoxifen-regulated Cre recombinase MerCreMer carrying at both termini a mutated murine estrogen receptor binding domain (Mer). The MerCreMer cDNA was inserted in the mb-1 gene encoding the Ig-a chain of the B cell receptor (BCR). In order to study B cell development we crossed the MerCreMer mouse to the GFP/mb-1 k.i. mouse (Genesis 2002 32:154–157). The GFP/mb-1 cassette can be inverted by Cre recombination and thus alternatively gives rise either to Ig-a or GFP expression. We show that administration of Tamoxifen in vitro as well, as in vivo, induces inversion of the GFP/mb-1 cassette. Furthermore, no background recombination was detected in the absence of Tamoxifen. In MerCreMer and GFP/mb-1 double k.i. mice, B cell development is blocked at the pro-B cell stage. Tamoxifen administration allows gene inversion and expression of Ig-a resulting in a wave of B cells emigration from the bone marrow to the periphery. These newly generated B lymphocytes could be isolated and characterized. Alternatively, in MerCreMer and mb-1/GFP double k.i. mice Ig-a expression can be terminated by Tamoxifen induced gene inversion resulting in loss of BCR and induction of GFP expression. Preliminary results show the presence of a long-lived population of GFP positive B cells. This double k.i. system will be used to test the requirement for the BCR and its signaling elements during B lymphocyte development. Specifically we will test how long mature B cells can survive without the BCR and what characteristics BCR-negative B cells have. The protocols for Tamoxifen-induced Cre activation are also being optimized.
Basel Institute of Immunology, Basel, Switzerland
H. 9 Rules for gene usage inferred from a comparison of large-scale gene expression profiles of T and B lymphocyte development R. HOFFMANN*, L. BRUNO, T. SEIDL, A. ROLINK, and F. MELCHERS RNA expression profiles of seven consecutive stages of mouse thymocyte development were generated on high-density oligonucleotide arrays. Previously known expression patterns of several genes were confirmed. Ten percent (1304 of over 13000) of the monitored genes were found with 99% confidence to be differentially expressed across all T cell developmental stages. When compared with 1204 genes differentially expressed in five consecutive B lineage developmental stages of bone marrow, more than 40% (546 genes) appeared to be shared by both lineages. However, when four pools of functionally distinct cell stages were compared between B and T cell development, DJ-rearranged precursor cells and resting immature precursor cells before and after sur-
108 · 33rd Annual Meeting of the German Society of Immunology face antigen receptor expression shared less than 10%, mature resting lymphocytes between 15% and 20%, and only cycling precursors responding to precursor lymphocyte receptor deposition share more than 50% of these differentially expressed genes. Three general rules emerge from these results: 1) Proliferation of cells at comparable stages is in majority executed by the same genes; 2) intracellular signaling and intercellular communication is effected largely by different genes, and 3) most genes are not used strictly at comparable, but rather at several, stages, possibly in different functional contexts. *present address: Max-von-Pettenkofer-Institut, Ludwg-Maximilians-Universität, München, Germany
Departments of 1Bacteriology, Max-von-Pettenkofer-Institut and 3Medical Informatics, Biometrics and Epidemiology, University of Munich, Munich, Germany, and 2Section of Gene Function and Regulation, The Institute of Cancer Research, London, UK
H. 10 A novel set of lineage- and stage-specific markers in lymphoid development identified by gene expression profiling and supervised machine learning R. HOFFMANN1, T. SEIDL2, L. BRUNO2, and M. DUGAS3 B and T lymphocytes develop through a series of cellular stages which are defined by recombination status of the immunoglobulin- and TCR loci and can be separated by analysis of cell surface marker proteins. We have generated high-density oligonucleotide array based gene expression profiles containing approx. 13,000 genes from five B cell and eight T cell precursor stages. Analysis of these datasets with supervised machine learning algorithms identified novel lineage and stage specific markers. Eight Genes are identified that correctly predict all cell samples as belonging to either the B- or the T cell lineage. Individual stages of the two lineages can optimally be predicted by models containing 29 (for B-lineage cells) or 39 (for T-lineage cells) genes. These models contain ESTs as well as known genes that have not previously been associated with lymphocyte development, and only very few of the established markers. When analyzed by Principal Component Analysis, a perfect separation based on the first two principal components can be made for the distinction between B- and T-lineage cells as well as for stages within the B cell lineage, while stages within the T cell lineage do not separate well. Thus, the markers currently widely used for separating B cell stages are more in accordance with the cellular gene expression profile as those used for separating T cell stages.
33rd Annual Meeting of the German Society of Immunology · 109 1
Division Environmental Dermatology and Allergology, GSF-TUM and 4Institute of Experimental Genetics, GSF Research Center, Neuherberg, Institutes of 2Molecular Animal Breeding, Gene Center, 3Medical Microbiology, Immunology and Hygiene, Technical University, Munich, and 5German Research Center for Biotechnology (GBF), Braunschweig, Germany
H. 11 Analysis and chromosomal mapping of selected mouse mutants with immunological alterations derived from the Munich ENU mouse mutagenesis project T. JAKOB1, B. RATHKOLB2, M. HOWALDT2, A. SERVATIUS3, D. SOEWARTO4, N. SANDHOLZER3, M. HRABE DE ANGELIS4, R. BALLING5, H. BEHRENDT1, E. WOLF2, K. PFEFFER3, and H. FLASWINKEL2 Out of more than 70 mouse mutant lines with immunological alterations established within the Munich ENU Mouse Mutagenesis Project we are currently analyzing selected lines in more detail. One of these mutant lines TUM079/IEHR01 recovered from the recessive screen is especially interesting in that T cells are almost absent in peripheral blood and IgE levels are increased more than 10–30 fold above normal range between 10–14 weeks of age. All other isotypes tested are deviating much more moderately from normal. First consumptive analysis revealed several important alterations. The thymus is reduced or not visible at all. Spleen size is increased more than twofold. Coat is present but rough and bare patches are visible at six month of age. Female mutants frequently die early and female as well as male mice are growth retarded. Further phenotypic analysis and mapping of the respective mutation are in progress. Two other mutant mouse lines are almost devoid of basal immunoglobulin. In one case all isotypes measured are reduced to a similar fraction. In the second line, IgM, IgG1, IgG3 and IgA are drastically reduced while IgG2a is about normal. Yet another recessive mutant line TUM038/Dia001 exhibits anti-DNA antibodies, a drastic increase in basal IgA in combination with chronic diarrhea. Mutants are fertile but viability is reduced. All mutant mouse lines are inbred C3Heb/FeJ. We will present recent data on selected mutant mouse lines recovered from the Munich ENU Mouse Mutagenesis Project.
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Molecular Immunology, German Research Center for Biotechnology (GBF), Braunschweig and 2Department of Bacteriology, Max-von-Pettenkofer-Institut, Munich, Germany
H. 12 Transgenic expression of a l2315 light chain results in a severe abrogation of B2 development but normal generation of marginal zone B lymphocytes A. JUNGEBLOUD1, J. STOPKOWICZ1, R. HOFFMANN2, S. WEISS1, and K. KRETSCHMER1 The mature preimmune splenic B cell compartment of normal adult mice consists of CD23+CD21int follicular B cells (FO or B2 cells), CD23lo/–CD21hi marginal zone (MZ) B cells and CD5+ B1 cells, each of which exhibit specific developmental and functional characteristics.
110 · 33rd Annual Meeting of the German Society of Immunology Here, we describe that the high expression of a transgenic l2315 light chain differentially affects these mature B lymphocyte subpopulations in the recombinant mouse line L2. A severe block in B cell development in adult bone marrow at the transition from large to small preB-II cells is observed. Consistently, B2 cells are completely lacking in such mice. CD5+ B1 cells, generated early in mouse development, predominate in both peritoneal cavity and spleen. Interestingly, CD5– B cells present in the spleen show the CD23lo/–CD21hi MZ B cell surface marker phenotype. Gene expression profiling was carried out with B cells from such mice. Using high-density oligonucleotide arrays representative of approximately 12.000 genes clearly demonstrated that CD23lo/–CD21hi B cells from L2 mice correspond to MZ B cells from non-transgenic control mice. Substantial differences in the gene expression pattern between splenic B1a and MZ B cells were detected and paralleled by a clear difference in the IgH chain repertoire. Whereas the light chain restriction results in high frequency of identical IgH chains with a high number of independent rearrangement events in the B1a compartment, we could not find any evidence for such an oligoclonal IgH chain repertoire in the MZ B cell compartment. IgH chains from these MZ B cells show a high frequency of N nucleotide insertions, indicating their generation in adult bone marrow in the presence of TdT activity. These data show that B1a and MZ B lymphocytes mediate specialized effector functions of the splenic B cell compartment.
1
Clinical T Cell Immunology, Deutsches Rheuma-Forschungszentrum and 4Haematology und Oncology, Virchow Klinikum, Berlin, and 3Department of Haematology und Oncology, ErnstMoritz-Arndt-University, Greifswald, Germany, and 2Institute of Human Genetics, Poznan, Poland
H. 13 Two subsets of peripheral blood naïve T helper cells with distinct T cell receptor excision circle content indicate peripheral homeostatic expansion of recent thymic emigrants in adult humans S. KIMMIG1, G.K. PRZYBYLSKI2, C.A. SCHMIDT3, K. LAURISCH4, B. MÖWES1, B. OPPMANN1, A. RADBRUCH1, and A. THIEL1 Introduction: During ageing thymic function declines and is unable to meet the demand for peripheral Th cell replenishment. Therefore population maintenance of naïve Th cells must be at least partly peripherally based. Such peripheral postthymic expansion of recent thymic emigrants (RTEs) during ageing consequently should lead to loss or dilution of T cell receptor excision circles (TRECs) from a subset of naïve Th cells. Results: We have identified two subsets of naïve Th cells in human adult peripheral blood characterized by a striking unequal content of TRECs, indicating different peripheral proliferative histories. In nine analyzed healthy donors TRECs are highly enriched in peripheral naïve CD45RA+ Th cells coexpressing CD31 as compared to peripheral naïve CD45RA+ Th cells lacking CD31 expression, in which TRECs can hardly be detected. Furthermore we show that CD31–CD45RA+ Th cells increase in frequency during ageing but retain all phenotypic and functional features of naïve Th cells. Based on our results it is now possible to distinguish peripherally postthymically expanded centralnaïve Th cells, described by loss of CD31 expression and highly reduced TREC content, from thymicnaïve Th cells recently emigrated from the thymus.
33rd Annual Meeting of the German Society of Immunology · 111 Interestingly, more recent investigations have shown that CD31 triggering leads to inhibition of TCR signaling via immunoreceptor tyrosine-based inhibitory motifs (ITIM). Conclusion: Since TRECs are reduced up to 10fold in centralnaïve Th cells peripheral postthymic expansion implicates extensive cell proliferation of thymicnaïve Th cells. Our results enable direct access and thereby further functional and molecular analysis of these two human peripheral naïve Th cell subsets.
1
Molecular Immunology, German Research Center for Biotechnology (GBF), Braunschweig and 2Department of Bacteriology, Max-von-Pettenkofer-Institut, Munich, Germany
H. 14 Antibody repertoire and gene expression profile of splenic and peritoneal B1a lymphocytes: Implications on different developmental and functional traits K. KRETSCHMER1, A. JUNGEBLOUD1, J. STOPKOWICZ1, R. HOFFMANN2, and S. WEISS1 High expression level of the transgenic l2315 light chain in L2 mice results in near complete exclusion of endogenous light chains and a predominance of B1a cells in both peritoneum and spleen, whereas B2 cells are completely absent. Availability of a single light chain has been shown to lead to an oligoclonal IgH chain repertoire of peritoneal B1a lymphocytes. Here, we show that splenic and peritoneal B1a cells of L2 mice differ considerably in their functional properties. The IgH chain repertoire of splenic, peritoneal and blood derived B1a cells was established using the clonotypic IgH chain sequences as potential diagnostic markers for migration and exchange. Splenic B1a cells exhibit much more IgH chain diversity under such L chain limitation due to high frequencies of N nucleotides. Despite few oligoclonal overlaps between the splenic and peritoneal B1a compartment, some B cell receptor specificities are clearly restricted to the peritoneum. In addition, reconstitution experiments using a congenic IgH allotype system suggest that peritoneal B1a cells from L2 mice transferred into the peritoneum of congenic L2 recipients have a very limited capacity to enter the splenic B1a compartment. Thus, identical IgH chains overlapping between spleen and peritoneum appear to be unlikely due to migratory exchange. Consistently, gene expression profiling revealed substantial differences in the pattern of expressed genes between the two B1a compartments. Some upregulated genes found in splenic B1a cells are potentially involved in mediating specialized «first line of defense» effector functions and interaction with T cells. Interestingly, splenic B1a and peritoneal B1b cells appear to be very similar concerning IgH chain repertoire and gene expression profile. The striking differences in antibody repertoire and pattern of expressed genes between splenic and peritoneal B1a cells indicate that both B1a subpopulations differ not only in their functional properties but also in their developmental program.
112 · 33rd Annual Meeting of the German Society of Immunology 1
Clinical Research Unit for Rheumatology, University Medical Center, 2Department of Molecular Immunology, Institute of Biology, University of Freiburg, and 3Max-Planck-Institute of Immunobiology, Freiburg, Germany; and 4Department of Immunology, University of Basel, Basel, Switzerland
H. 15 Egr-1 controlled B cell differentiation M. RAKHMANOV1, H. JUMAA2, P. NIELSEN2, C. BLEUL3, A. ROLINK4, M. RETH2, and H. EIBEL1 Expression of the transcription factor Egr-1 in B cells is rapidly induced upon antigen binding to the B cell receptor. The intracellular signal chain that initiates the transcription of the Egr-1 gene involves the sequential activation of phosphotyrosine protein kinases and the generation of second messengers. The adapter protein SLP65 is a key component of this process as it serves as a scaffold for the assembly of B cell receptor complex-associated enzymes. Deletion of the slp65 gene results in impaired B cell maturation and arrests development at the stage of immature B cells in the spleen. Since the induction of Egr-1 expression depends on signals transmitted through SLP65 we generated SLP65-deficient mice expressing an Egr-1 transgene (tgEgr-1) in B cells to study the cellular responses regulated by this transcription factor. Phenotypic comparison of slp–/– and of tgEgr-1¥slp–/– spleen B cells revealed that Egr-1 activity partially rescued the developmental block caused by the SLP65 defect and allowed the differentiation into B cells with a more mature phenotype. To identify Egr-1 target genes that promote Egr-1 induced B cell maturation, we purified total RNA from B cells isolated from spleens of slp65-deficient, of tgEgr1¥slp65–/– and of BALB/c control mice and prepared probe sets for hybridization experiments with DNA microarrays. Using Affymetrix U74 genechips, we then established gene expression profiles of these samples and analyzed the changes in the expression patterns between tgEgr1¥slp65–/–, slp65–/– and BALB/c mice. Our results show, that Egr-1 activity induces genes that are expressed under normal conditions in B cells of BALB/c control animals but not in slp65 knockout mice. Based on these data, we now are able to establish a signal chain that starts at the B cell receptor complex and uses SLP65 and Egr-1 to induce B maturation.
Molecular Immunology, German Research Center for Biotechnology (GBF), Braunschweig, Germany
H. 16 Differential effect of control elements on somatic hypermutation of a transgenic l2 immunoglobulin light chain I. RODE, M. NACKE, H. ENGEL, K. KRETSCHMER, and S. WEISS During an antibody response the affinity of the induced antibodies increases by the accumulation of single nucleotide exchanges in the variable regions of the heavy and light chains. This so called somatic hypermutation takes place in the dark zone of the germinal centers and is dependent on strong transcription of the Ig chains that is effected by juxtaposition of strong promoters and enhancers during the rearrangement process. Here we wanted to test which of the three characterized light chain enhancers is able to support somatic hypermutation of a transgenic l2
33rd Annual Meeting of the German Society of Immunology · 113 light chain. To this end we established four types of transgenic mice carrying a genomic fragment encoding the l2 chain of MOPC315 driven by the Í intron enhancer, the Í 3’enhancer, both Í enhancers and the enhancer found downstream of the l2–4 cluster. All lines showed similar expression patterns of the transgenes, i.e. endogenous L chain rearrangement was only partially inhibited and a significant population of B cell displayed Í and l at their cell surface. For analysis PNAhi and PNAlo B cells from Peyer’s patches of 9–18 months old mice were sorted, RNA was isolated and the transgenic transcript or transcripts of an endogenous Í chain was amplified by RT-PCR, cloned and sequenced. Alternative, genomic DNA from such cells was used for amplification. A clear difference was found between constructs that contained the intron enhancer of the Í chain and those that were driven by the 3’ or the l enhancer, respectively. Only the intron enhancer rendered the constructs a substrate for the somatic hypermutation mechanism independent of whether the 3’ enhancer was additionally present in the construct. The mutations accumulated in the expected hot spots. Some mutations were also found in the PNAlo population although such cells are usually not considered to be in the process of mutating.
1
Department of Functional and Applied Anatomy, Medical School, Hannover and Department of Internal Medicine I, University Hospital, Regensburg, Germany, and 2 Department of Periodontology, Faculty of Dentistry, University of Oslo, Oslo, Norway 3
H. 17 Different postnatal experiences determine adult disease susceptibility: Stress protection as an option for the treatment of periodontal disease M. STEPHAN1, T. BREIVIK2, R.H. STRAUB3, R. PAPST1, and S. VON HÖRSTEN1 Objective: Stress affects wound healing in the oral cavity of humans. Individual differences in the stress responsiveness may therefore determine susceptibility to oral and dental diseases. Early experiences including maternal deprivation (as a paradigm for adverse early life events) or handling (as a paradigm of postnatal stimulation) alter the development of neural systems including the hypothalamic-pituitary-adrenal axis, which govern endocrine responses to stress. The present studies investigated whether acquired differences in the stress response interact with a given genetic background and whether interventional strategies, considered as a stress protection, are successful in preventing increased disease susceptibility in rats. Methods: Male F344 or LEW rats were daily handled (i.e. brief maternal separation and exposure to novelty), maternally deprived (i.e. maternal separation for h), or left undisturbed until weaning. Prophylactic treatment strategies (additional tactile stimulation directly after maternal deprivation) or treatment using antidepressants in adulthood (imipramine via drinking water) were applied to protect from the maternal deprivation induced effects. At 5 months of age, placing silk-ligatures around molar teeth induced periodontal disease. At 7 months, neuroendocrine stress response (corticosterone, NPY), cytokines, and clinical outcome of periodontal disease were determined. Results: Stress high responding F344 rats developed significantly more periodontal disease. Only in the stress low responding LEW rat strain, handling-induced reduction of adult stress responsiveness was protective while maternal deprivation aggravated experimental periodontal disease. Both, interventions by additional tactile stimulation in infancy as well as antidepressants in adulthood protected from the deprivation-induced aggravation of the disease.
114 · 33rd Annual Meeting of the German Society of Immunology Conclusions: Individual differences acquired in the postnatal period specifically alter neuroendocrine stress responsiveness, thereby shifting disease susceptibility and aggravating periodontal disease an the basic of the genetic background. It is suggested that in a subgroup of genetically stress low responding individuals stress protection is effective in the treatment of periodontal disease.
Institute of Virology and Immunobiology, University of Würzburg, Würzburg, Germany
H. 18 Notch signaling and rat thymocyte development J. VAN DEN BRANDT, S.H. KWON, M. SCHOTT, and H.M. REICHARDT Notch, a family of transmembrane receptors, is involved in directing the choice between alternative cell fates. In mouse thymopoiesis, Notch-1 has been implicated in bipolar lineage decisions between T and B cells, ab and gd T cells and CD4 and CD8 single-positive cells. However, the role of Notch in these processes is controversially discussed. Recent studies have shown that similar stimuli can cause opposite lineage decisions in mouse and rat thymocytes. This prompted us to study thymus development in the absence of Notch signaling taking a pharmacological approach in the rat. Generation of active Notch requires cleavage of the intracellular domain by g-secretase. Consequently, fetal thymus organ culture (FTOC) in the presence of inhibitor can be used to study the role of Notch in thymocyte lineage decisions. Thymuses were recovered at day e16.5 and cultured for six days. Subsequently, the cells were stained with Abs against CD2, CD4, CD8, CD45RC, CD45RA, abTCR and gdTCR and analysed by FACS. We found that interfering with Notch signaling arrests thymocyte development at an early double-negative stage. In particular, the development of the abTCR lineage but not of the gdTCR lineage was affected under these conditions. The observed phenotypic changes correlate with a reduced expression of several Notch target genes, such as Hes-1. Finally, in contrast to mice, lack of Notch signaling in the rat was found to promote the generation of CD8 at the dispense of CD4 singlepositive cells. Collectively, we believe that this pharmacological approach should help to further decipher the role of Notch in thymus development as well as to define differences between rats and mice in more detail.
Institute of Clinical Transfusion Medicine and Immunogenetics, DRK-Blutspendedienst Baden-Württemberg-Hessen, Ulm, Germany
H. 19 An enhanced green fluorescence protein based assay: Making V(D)J recombination visible Z. XIAO and K. SCHWARZ A single cell in vivo green fluorescence protein (GFP) based assay was successfully developed to monitor V(D)J recombination efficiency. This assay makes V(D)J recombination visible at a single cell level. Furthermore, efficiency of V(D)J recombination can be evaluated in real time.
33rd Annual Meeting of the German Society of Immunology · 115 Our results show that the in vivo V(D)J recombination efficiency is low (≈1%) in 293 cells. This in vivo V(D)J recombination efficiency correlated well with that achieved by a classical in vitro V(D)J recombination assay designed by Lieber and colleagues. The in vivo V(D)J recombination efficiency depended on the relative RAG-1 and RAG-2 expression vector concentrations used for co-transfection. The test differentiates RAG null mutants as seen in severe combined immunodeficiency (SCID) from Omenn syndrome hypomorphic RAG mutants. In addition, this novel assay is also used to screen novel factors involved in V(D)J recombination.