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Workshop A: Development and Differentiation of the Immune System, Abstract A1 bis A15
Immunobiology (2001) 204, pp. 1 – 354 © 2001 Urban & Fischer Verlag http://www.urbanfischer.de/journals/immunobiol
32nd Annual Meeting of the German Society of Immunology* Dresden September 26 – 29, 2001
Abstracts Workshop A Development and Differentiation of the Immune System Division of Molecular Immunology, Department of Medicine III, University of Erlangen, Erlangen, Germany
A. 1 Specific interaction of a soluble pre-B cell receptor with adherent cell lines H. BRADL, D. MILIUS, and H.-M. JÄCK Signals initiated by the pre-B cell receptor (pre-BCR) are critical for B cell progenitors (pro-B) to mature into precursor (pre) B cells. The pre-BCR consists of a homodimer of m heavy chains, the covalently associated surrogate light (SL) chain composed of VpreB and l5, and the transmembrane signal molecules Iga and Igb. One way to explain how maturation signals are initiated in late pro-B cells is that the pre-BCR is transported to the cell surface and from there interacts with a ligand on stroma cells. To address this hypothesis, we first produced soluble Fab-like pre-BCR and B cell receptor (BCR) fragments, as well as SL-chain, in baculovirus-infected insect cells. Flow cytometry revealed that, in contrast to Fab-like BCR fragments, the soluble pre-BCR binds to the surface of stroma and several other adherent cell lines, but not to suspension cells representing various stages of B cell development. The specific binding of the soluble pre-BCR to stroma cells is saturable, sensitive to trypsin digestion and not dependent on bivalent cations. The * We are obliged to PD Dr. Martin R. Hadam, Hannover, for guiding the online abstract submission and for editing this abstract volume. 0171-2985/01/204/01-02-001 $ 15.00/0
2 · 32nd Annual Meeting of the German Society of Immunology binding of pre-BCR seems to be independent of the m heavy chain, because SL-chain alone was able to interact with stroma cells. These findings not only demonstrate for the first time the capacity of a pre-BCR to bind to a structure on the surface of adherent cells, but also suggest that the pre-BCR interacts via its SL-chain with a putative ligand on stroma cells.
Institutes of 1Medical Microbiology and 2Biochemistry, Justus-Liebig-University, Giessen, Germany
A. 2 Detection of a therapeutic protein, human Ciliary NeuroTropic Factor, in the blood of nude mice implanted with transgenic mouse embryoid bodies U. CHEN1, C. ELMSHÄUSER1, D. ANHLAN2, and E. BECK2 Objectives: To test the possibility of using stem cells as the carrier to deliver a therapeutic protein conditionally in treating human diseases. Methods: Double transgenic ES clones were established, containing a plasmid expressing hCNTF gene under the control of the early promoter of polyomavirus JC (pJCV-hCNTF) as well as a plasmid coding for rtTA-ts A58 genes (tet-onsA58) which consists of the doxycyclineinducible reverse transactivator and the temperature sensitive SV40T antigen (Tag-ts). The tsA58 gene is transcribed in the presence of doxycycline, and the Tag-ts is active at 33°C only. Transcription of the JCV promoter depends on transactivation by either the transcription factor Oct6, or by JCV-Tag. The role of JCV-Tag can be substituted by SV40Tag. The former transactivator (Oct6) is expressed only in cells committed to the glial lineage, the later (SV40Tag-ts) requires the presence of doxycycline and is active at 33°C only. These double transgenic ES cell clones were released to differentiate in culture into embryoid bodies (EBs), and EBs were implanted subcutaneously into nude mice for 3 weeks, and blood was collected to test the level of hCNTF. Results: Culture supernates of ES and EBs under permissive condition and blood of such implanted nude mice were shown to contain hCNTF. Conclusions: The results suggest that pJCV-hCNTF is actively transcribed in glial (precursor) cells, leading to secretion of hCNTF in the blood of nude mice. Such ES-derived glial (precursor) cells must have developed in tissues derived from EB’s in this combined in vitro and in vivo ES differentiation system. It offers the possibility to have a tet-on delivery of therapeutic protein under the control of a tissue-specific promoter besides the tet-O promoter. It also provides a quick cellular readout to evaluate the biological action of SV40 Tag, and to obtain a large quantity of lineage-committed stem cell clones.
32nd Annual Meeting of the German Society of Immunology · 3 Hans-Spemann-Laboratorien, Max-Planck-Institut für Immunbiologie, Freiburg, Germany
A. 3 RFX1 and MIBP1 regulate the CD4 gene silencer M. EHLERS, K. LAULE-KILIAN, C.J. ALDRIAN, J. HUNN, and V. STEIMLE During thymic T cell development CD4+CD8+ precursor T cells develop either into CD4+CD8– helper T cells or into CD4–CD8+ cytotoxic T cells, which interact with cells presenting MHC class II/antigen or MHC class I/antigen complexes, respectively. The molecular mechanisms governing the complex selection and differentiation steps during thymic T cell development are not well understood. A silencer element in the first intron of the CD4 gene has been shown to mediate the downregulation of CD4 gene expression during the differentiation of CD4–CD8+ cytotoxic T cells. Here we show by band shift and antibody supershift experiments that the regulatory proteins RFX1 and MIBP1 bind to a newly identified region of the CD4 silencer element. Mutagenesis of this RFX1/MIBP1 binding site strongly reduces silencing in a new stable reporter assay with insulator elements between the reporter and the resistance cassettes, which faithfully reproduces silencing in CD4–CD8+ T cell lines. The functional importance of RFX1 in CD4 gene silencing was shown by antisense oligonucleotide-mediated inhibition of RFX1 that abolished silencing in CD4–CD8+ T cells.
1
Clinical Research Unit for Rheumatology and 2Department of Molecular Immunology, Institute of Biology, Albert-Ludwigs University, Freiburg, Germany
A. 4 Egr-1 activity partially rescues a defect in slp65 in B cells H. EIBEL1, P. RICHTER1, P. NIELSEN2, M. RETH2, and H. JUMAA2 Introduction: The transcription factor Egr-1 is a member of the Egr-transcription factor family which includes also Egr-2, Egr-3 and Egr-4. All factors share a highly homologous DNA binding domain composed from 3 zinc fingers and bind to a common DNA sequence motif. In B cells, Egr-1 expression is induced upon B cell receptor crosslinking. To study the role of Egr-1 in B cells we generated transgenic mice that overexpress Egr-1 from the immunoglobulin heavy chain promoter-enhancer. Starting at the pro-B cell stage, transgenic Egr-1 expression (tgEgr-1) is maintained throughout the maturation process. It enhances B cell maturation at defined stages of development. To study the signaling components in vivo that normally regulate Egr-1 expression in B cells, we backcrossed tgEgr-1 to mice deficient in the gene encoding for the BCR adapter protein SLP65. In these mice, B cell development is arrested at the transition from immature to mature B cells and in the generation of Ly1 positive (B1) B cells. Methods: B cells of SLP65–/– and tgEgr-1¥SLP65–/– were analyzed by flow cytometry with antibodies specific for IgM; IgD, pre-BCR, CD21, CD23 and CD45R (B220) and by in vitro cultivation on ST-2 feeder cells. Results: Our experiments revealed that Egr-1 overexpression partially complements the SLP65–/– defect. In contrast to SLP65–/– mice, tgEgr-1¥SLP65–/– littermates had less immature and more mature B cells in the spleen. However, the defect in the development of B1 B cells was not rescued.
4 · 32nd Annual Meeting of the German Society of Immunology Conclusions: Therefore, we conclude that Egr-1 activity promotes the transition of immature to mature B cells in the spleen but not the generation of B 1 cells.
Laboratory of Molecular Biology, Children’s Hospital, Charité, Humboldt-University, Berlin, Germany
A. 5 Model for a superantigen-like activation of B cells originating from VH germ-line genes 3-23 and 3-30/3-30.5 by IVIG and normal immunoglobulins P. FISCHER Intravenous immunoglobulins (IVIG) represent the IgG repertoire of several thousand healthy donors. The beneficial use of IVIG in certain immunodeficiencies and autoimmune diseases has been proven, but it is not clear how IVIG functions at the molecular level. Previously we sequenced 92 IgG and IgM clones specifically selected with IVIG from phage display libraries of patients with autoimmune thrombocytopenia (Eur. J. Immunol. 1998; 28:4236), SLE (Arthritis Rheum. 2000, 43:2722), Kawasaki disease (KD) (Clin. Immunol. 2001, 99:18), and a healthy individual (Clin. Exp. Immunol. 2000; 121:37). The most frequently used VH germline gene segments of all IVIG-selected Fabs (62/92, 67%) were 3–23 and 3–30/3–30.5, in contrast to only 3 out of 29 (10%) unselected controls. The combined results suggest a favoured interaction of a subset of IVIG or normal immunoglobulin repertoires with BCRs derived from those two genes. As light chains, antigen-specificity and the high variation in CDR3 showed only little influence on the selection by IVIG, this type of interaction is characteristic for a B cell superantigen. It may significantly shape the B cell repertoire, because 3–23 and 3–30/3–30.5 are the most frequently rearranged VH germ-line genes among humans. Further experiments revealed that despite possible homologies, at least some of the contact residues on Fabs for IVIG must be different from those for the known superantigens Staphylococcal protein A and HIV gp120. To investigate how IVIG influences this restricted interaction, we cloned Fabs from a patient with KD before and after IVIG therapy. Again, a favoured selection of IgG Fabs originating from the 3–23 or 3–30/3–30.5 genes was observed. Importantly, the reactivity with IVIG was significantly higher for clones from the library prepared after the IVIG treatment, providing the first in vivo functional evidence that a subset of IVIG may selectively activate B cells of this germ-line origin.
32nd Annual Meeting of the German Society of Immunology · 5 1
Institute of Medical Microbiology, Immunology and Hygiene, Technische Universität, Institute of Molecular Animal Breeding, Gene Center, and 5Institut für Molekulare Immunologie, GSF-Forschungszentrum für Umwelt and Gesundheit, München, 3Ingenium Pharmaceuticals AG, Martinsried, 4Institute of Experimental Genetics, GSF Research Center, Neuherberg, and 6German Research Center for Biotechnology (GBF), Braunschweig, Germany 2
A. 6 Phenotypic analysis and chromosomal mapping of mouse mutants with immunological defects generated by ENU mutagenesis H. FLASWINKEL1, B. RATHKOLB2, C. FÄRBER3, A. SERVATIUS1, D. SOEWARTO4, E. KREMMER5, N. SANDHOLZER1, M. HRABE DE ANGELIS4, R. BALLING6, E. WOLF2, and K. PFEFFER1 Analysis of gene function in vivo is currently mostly performed by transgenic insertion, inactivation of the respective gene by homologous recombination in embryonic stem cells and gene trapping. We have complemented this approach by isolation of mouse lines with phenotypic immunological alterations which were produced within the Munich ENU-mouse mutagenesis project. In this phenotypic approach C3HeB/FeJ are randomly mutagenized using ethylnitrosourea (ENU). ENU is most potent during spermatogenesis and therefore mutagenesis is performed on male mice which are subsequently crossed to female WT mice. Progeny thereof are analyzed for inherited immunological alterations using a panel of immunological parameters. Out of more than 50 mutant lines isolated with this approach we have analyzed two in more detail and mapped the corresponding mutation. Data from this analysis will be presented.
1
Institute of Medical Microbiology, Immunology and Hygiene, Technische Universität, Institute of Molecular Animal Breeding, Gene Center, and 4Institut für Molekulare Immunologie, GSF-Forschungszentrum für Umwelt und Gesundheit, München, 3Institute of Experimental Genetics, GSF Research Center, Neuherberg, and 5German Research Center for Biotechnology (GBF), Braunschweig, Germany 2
A. 7 Identification of recessive mutants with immunological phenotypes among offspring of ENU-treated C3HeB/FeJ mice H. FLASWINKEL1, B. RATHKOLB2, A. SERVATIUS1, D. SOEWARTO3, E. KREMMER4, N. SANDHOLZER1, M. HRABE DE ANGELIS3, R. BALLING5, E. WOLF2, and K. PFEFFER1 The Munich ENU-Mouse Mutagenesis Project has entered its second phase in which recessive mutants (G3) are being generated. Our group focuses on the identification of immunological phenotypes among those mice employing ELISAs for basal immunoglobulin levels, anti-DNA antibodies and rheumatoid factor as well as flow cytometry for the detection of cell surface molecules. In addition, we are currently measuring cytokine levels in established mutants of the dominant screen and plan to extend this analysis to a limited number of animals from the recessive screen. Nine recessive mutant mouse lines have been established. Additionally, 25 recessive variants are currently being crossed for genetic confirmation. The confirmed mutants encompass phenotypes with alterations in the Ig level of single or multiple isotypes, decreased percentages of
6 · 32nd Annual Meeting of the German Society of Immunology lymphocyte subpopulations and unusual expression patterns of lymphocyte markers. Chromosomal mapping of these recessive lines will start soon. At present we are selecting mutants that will be analyzed in more detail. We are continuing the ENU-program analysing increasing numbers of G3 animals with a broader panel of parameters. Future directions include functional assays i.e. vaccination experiments and the analysis of selected activation markers. Information can be obtained from the following hyperlinks: http:/www.mikrobio.med.tu-muenchen.de/forschung/enu.html http:/www.gsf.de/ieg/groups/enu/mutants/
Department of Cellular Immunology, Max-Planck-Institute of Immunobiology, Freiburg, Germany
A. 8 Characterization of an ADAM family member expressed in thymic epithelial cells G. HUBER, I.D. HAIDL, and K. EICHMANN T cell development in the thymus is dependent on thymic epithelial cells (TEC). However, the molecular explanation for the unique functions of TEC is still incomplete. Following subtractive enrichment for TEC cDNAs, a cDNA was isolated that encodes a protein containing all of the ADAM family member characteristics including a disintegrin and metalloproteinase domain. The novel ADAM family member was designated ADAM28. Expression was detected in adult and embryonic murine TEC and in developmentally related tissues including the trachea, thyroid, lung and stomach. The metalloproteinase active site contains all conserved residues required for activity, thus indicating a potential role in ectodomain shedding and extracellular matrix modeling. The disintegrin-like domain may function in cell-cell adhesion events such as TECthymocyte and/or TEC-TEC interactions. Given the crucial role of ADAM family members in ectodomain processing and cellular adhesion, ADAM28 may play an important role in organogenesis and in organ specific functions such as supporting thymic T cell development. In order to address the specific functions of ADAM28 we are generating recombinant molecules containing the entire extracellular domain or individual domains of the protein. Furthermore, in situ hybridization is being used to clarify the ADAM28 expression pattern in fetal development and in adult tissues.
32nd Annual Meeting of the German Society of Immunology · 7 Molecular Immunology, German Research Centre for Biotechnology (GBF), Braunschweig, Germany
A. 9 Strong antigenic selection acts on the immunoglobulin heavy chain repertoire of B1a lymphocytes in l2 MOPC315 transgenic mice K. KRETSCHMER, J. STOPKOWICZ, H. ENGEL, and S. WEISS The mouse line L2 – transgenic for the l light chain of the plasmacytoma MOPC315 – exhibits nearly complete exclusion of endogenous light chain isotypes and generates predominantly B lymphocytes of the CD5+ B1 type. The exclusive expression of the l2 315 light chain results in an oligoclonal immunoglobulin H chain repertoire. Both, clonally expanded and clonally independent B1a cells contribute to this very restricted IgH chain diversity. Many IgH chains identical in amino acid sequence were isolated repeatedly and in a very high frequency from peritoneal B1a cells of adult L2 mice. We could demonstrate that a significant proportion of these identical IgH chains were due to independent VDJ rearrangements. Comparison of the IgH chain repertoire of peritoneal cavity derived and splenic CD5+ B cells from an individual adult L2 mouse revealed that about 10% of sequences derived for each anatomical location were identical in amino acid sequence suggesting that the same BCR specificities are selected into both pools of mature B1a cells. In contrast, phosphatidylcholine specific CD5+ B1 cells were present only in the peritoneal cavity and like in controls could not be detected in the spleen of L2 mice. Similarly, the IgH chain repertoire of B cells from fetal and neonatal sources show compelling evidence for a high frequency of independent identical VDJ rearrangements resulting in identical amino acid sequences. This observation is contrasted by the clear increase of CDR3 diversity especially in CDR3 of fetal liver Pro/PreB-1 cells from L2 embryos differentiated in vitro when compared to B cells that had developed in vivo. These data suggest that the antibody repertoire of B1a cells is strongly selected by antigen during the fetal as well as during the adult phase of development and clearly indicate selective differences in the antibody repertoire of peritoneal and splenic B1a cells.
Institutes of 1Virology and Immunobiology and 2Pathology, University of Würzburg, Würzburg, Germany and 3National Institute of Medical Research, MI, London, UK
A. 10 Expression of constitutively active Akt/PKB induces lymphomas S.Y. NA1, Y. SCHEURING1, A. MARX2, A. PATRA1, M. TOLAINI3, D. KIOUSSIS3, T. HÜNIG1, and U. BOMMHARDT1 Akt, also known as Protein Kinase B (PKB), has been shown in many different cell systems to regulate cell survival, growth and differentiation. To study the function of Akt in T cell development and activation/differentiation of peripheral T cells we established transgenic mouse lines expressing constitutively active Akt (myristoylated Akt) in T cells using the human CD2 minicassette. Analyses of the Akt transgenic mice show: (1) Thymocytes from Akt heterozygous mice are hyper-reactive to anti-CD3 or PMA/ionomycin stimulation and proliferate even in the presence of the calcineurin inhibitors CsA or FK506. (2) The CD4+ T cell compartment is increased
8 · 32nd Annual Meeting of the German Society of Immunology compared to wildtype littermates whereas the numbers of B cells and CD8+ T cells are reduced. (3) CD4+ T cells have the phenotype of activated T cells, being Mel14lo, CD45RBlo and CD44hi. (4) In RNAse protection analyses (RPA) we did not find significant differences in the mRNA expression of several anti-apoptotic genes (e.g. A1, bcl2, bclx), however, in RPA for cytokine expression Akt heterozygous CD4+ T cells clearly showed enhanced mRNA production for certain cytokines, e.g. IFN-g, depending on the type of stimulation. (5) We will also present data on the effect of Akt on TCR transgenic T cells concerning positive and negative selection of thymocytes and peripheral T cell homeostasis. (6) The cellularity of thymi from Akt+/+ mice is reduced, mainly due to the loss of DP thymocytes which display aberrant expression of several selection markers. (7) Finally, Akt+/+ mice develop monoclonal CD4+ T cell lymphomas (or DP lymphomas, CD8+ T cell lymphomas were not detected) and immunohistological stainings reveal a profound destruction of splenic and thymic architecture as well as infiltration of the liver with CD4+ T cells. Thus, we provide evidence that dysregulation of Akt can be sufficient for the induction of T cell tumorigenesis.
Institute of Medical Immunology, Martin-Luther-University, Halle, Germany
A. 11 The two conserved C-terminal cysteines are necessary for proper folding and membrane localization of the raft-associated peptidase neprilysin/CD10 A. NAVARRETE SANTOS, J. WULFÄNGER, G. HELBING, J. LANGNER, and D. RIEMANN The membrane peptidase neprilysin/CD10, located e.g. on early B and T cells, is raft-associated and consists of 749 amino acids with two conserved cysteines (C734, C746) and a CAAX-resembling box at the C-terminus (aa 746–749). The molecule has been implicated in signal transduction. We investigated the role of the two C-terminal cysteines for the molecular sorting of neprilysin. Using PCR mutagenesis technique, three different mutants of neprilysin (C734S, C746S, and double mutant) were created and cloned into a green fluorescent protein vector. Substitution of C734 as well as C746 by serine disturbed the plasma membrane localization of the endopeptidase as detected by laser scanning microscopy and flow cytometry analysis of transfected CHO and Hek 293 cells. However, Western blot analysis revealed similar molecular weights of neprilysin wild type and mutants. After raft isolation (1% Triton X-100, 4°C, sucrose gradient centrifugation) and Western blot for neprilysin, we detected two bands (a high m.w. band in all fractions and a low m.w. only in the non-raft fractions). Endo-H treatment of protein lysate showed that in wild type and mutants a part of neprilysin is resistant to the glycosidase. When transfected cells were analysed by native PAGE we observed for wild type and mutant C746S three bands of approximately 170, 100 and 90 kDa, whereas C734S and double mutants lost the low m.w. band. Enzymatic endopeptidase activity was only slightly diminished for the C746S mutant, but was not detectable for C734S and the double mutant. Triton X-114 temperature-induced phase separation showed that all mutants partition into the aqueous phase whereas the wild type partitions both into the hydrophobic and the hydrophilic phase. We suggest that C746 (CAAX-box) could be function as transport signal to the plasma membrane whereas cysteine 734 could be important in folding (disulfide bond formation?).
32nd Annual Meeting of the German Society of Immunology · 9 Institutes of 1Immunology, 2Medical Biology and Genetics, and 3Department of Cardiology, University Hospital Motol, Prague, Czech Republic
A. 12 Age-dependent changes of immune parameters in children with DiGeorge syndrome A. SˇEDIVÁ1, J. BARTU˚ Nˇ KOVÁ1, R. ZACHOVÁ1, E. KOCˇ ÁREK2, D. NOVOTNÁ2, and T. KLEIN3 Hypo- or aplasia of thymus connected with syndrome DiGeorge should theoretically cause the profound immunodeficiency. We then followed, on a prospective basis, the cohort of children with syndrome DiGeorge and confirmed 22q11 deletion. One up to six repeated investigations was performed in 11 boys and 17 girls, in the age range from 1 month to 17 years. Immune investigation focused on T lymphocytes and their function. Three children had transient and 3 permanent total lymphopenia. Lower numbers of CD3 positive T cells, compared to normal age related values, were found in 10 children. Both spontaneous and PHA induced proliferation was unexpectedly increased in comparison with normal samples in all tested children less then 15 months old, however, increased T lymphocyte proliferation disappears between 18 months and 2 years. Repeated investigations of proliferative responses in children older than 2 years were comparable with controls. Due to the proposed role of thymus in T lymphocyte selection we followed the presence of double positive CD4+CD8+ and g/d T lymphocytes in the periphery. Double positive T cells were detected in one boy, together with transient positivity of antinuclear antibodies. Gamma/delta T cells were higher than 5% in 9 out of 28 children. Any of the described changes was not connected with particular clinical problems. Patients with lower CD3 positive T cells usually presented with repeated respiratory infections, which were successfully controlled by antibiotic therapy. We did not observe serious clinical signs of immunodeficiency or autoimmunity in any of the patients, all of them classified as partial syndrome DiGeorge. The prospective study is ongoing with specific focus on the development of autoimmunity on the genetic basis of inadequate T cell differentiation on the background of impaired thymic function. This work was supported by grants IGA 6247–3 and 00000064203 of Ministry of Health of Czech Republic.
1 2
Department of Internal Medicine III, Friedrich-Alexander-University, Erlangen and Department of Pediatrics, University of Mainz, Mainz, Germany
A. 13 Analysis of T cell effector functions in a patient with GATA3 haploinsufficiency A. SKAPENKO1, R. BEETZ2, J.R. KALDEN1, and H. SCHULZE-KOOPS1 The transcription factor GATA3 has been implicated in regulating Th2 cell differentiation in the mouse. Moreover, expression of GATA3 was shown to be critical for T cell development and thus, GATA3 knockout mice lack peripheral CD4 T cells. We investigated T cell effector functions in a patient with a dominant negative GATA3 haploinsufficiency related to a deletion of exon 2 in the dominant allele. At the time of investigation, the patient was 16 years old and had no histo-
10 · 32nd Annual Meeting of the German Society of Immunology ry of severe or opportunistic infections. He suffered from renal insufficiency, deafness and hypoparathyroidism, a trias that has been found in other patients with GATA3 haploinsufficiency. Vaccinations had been performed successfully throughout childhood. The patient had normal peripheral T cell counts and normal frequencies and absolute numbers of CD4 helper and CD8 cytotoxic T cells, CD19 B cells, CD56 NK cells and CD14 monocytes. Markers of in vivo Th2 mediated immunity, such as serum IgE, serum IgG2 and IgG4 levels were all within normal limits. T cell proliferation to mitogens and a panel of recall antigens was also without abnormalities. Cytometric analysis of cytoplasmic cytokines revealed a normal distribution of Th1 and Th2 cells within the purified CD4 memory T cell population. Moreover, after in vitro priming of CD4 memory T cells under Th1 or Th2 differentiation inducing conditions employing a previously described ex vivo cell culture system that permits the differentiation of Th effector cells after short term priming, no differences in the extent of Th1 and Th2 cell generation was detected. The data suggest, that GATA3 is not critical for T cell development in humans and that T cell function is completely normal in GATA3 haploinsufficient individuals. Importantly, Th2 generation and Th2 mediated effector functions are not altered in the absence of functional GATA3 in humans.
Department of Animal Physiology, Institute of Zoology, University of Leipzig, Leipzig, Germany
A. 14 Ability for monocytic differentiation in HL-60 cells from different sources U. WAGNER, I. LACHMANN, and K. DRÖSSLER Introduction: The differentiation along the monocytic or granulocytic pathway in response to various compounds was described for the human promyelocytic leukemia cell line HL-60. Nevertheless, the present study demonstrated the setup of a useful model of HL-60 monocytic differentiation related to phenotypical and physiological features. For this purpose, we have examined the sensitivity of HL-60 cells from two different sources to 1,25-dihydroxyvitamin D3 (VD3) and PMA. Methods: Proliferation and viability was estimated by MTT-assay and by flow cytometry of PI-stained cells, respectively. Expression of surface antigens CD11b, CD14, CD54, CD71 was analyzed by FACS using FITC-labeled monoclonal antibodies. Reactive oxygen species (ROS) were quantified using chemiluminescence measurements. The production of cytokines and prostaglandin E2 was analyzed using ELISAs whereas NO-metabolites were detected by the Griess reaction. Results: We demonstrated the monocytic differentiation of HL-60 cells from the source B after VD3-treatment which caused adherence, inhibition of growth, and changes of antigen expression in a concentration- and time-dependent manner. The monocytic cells exhibited a significant increase of CD11b, CD14 and CD54 expression whereas the expression of CD71 was down-regulated. Here we show that differentiated HL-60 B cells were able to produce PGE2, IL-1b and IL-6 after LPS stimulation whereas NO-production was not observed. Additionally, ROS-production was induced by different stimulators. In contrast, HL-60 from a different source showed no sensitivity to VD3-treatment. Surprisingly, the ability for production of PGE2, IL-and ROS was missing in the HL-60 cells from the two different sources after PMA-treatment and phenotypical changes.
32nd Annual Meeting of the German Society of Immunology · 11 Conclusions: Our data demonstrate that VD3 is a powerful promoter for the monocytic development of HL-60 cells (B) and give a useful model to study the mechanisms of the monocytic differentiation process. Furthermore, we found that PMA can obstruct signal pathways of HL-60 populations and origin-dependent differentiation pathways of HL-60 cells.
Department of Immunology, Institute of Genetics, University of Cologne, Cologne, Germany
A. 15 B cell development driven by g1 rather than m chain expression A. WAISMAN and K. RAJEWSKY During B cell development the m constant region gene is used to express the pre-B cell receptor and subsequently the B cell receptor. In order to study whether the g1 heavy chain can replace the m chain during B cell development, we generated a mutant mouse which could only express g1 heavy chains from its IgH locus. In mice expressing the inserted g1 constant region gene in a heterozygous manner, only 15% of the peripheral B cells express IgG1, showing that either the g1 heavy chain can not form an efficient pre-B receptor, or that IgM expressing cells are selected over IgG1 expressing cells in the periphery. In mice with only the IgG1 constant region insertion all B cells express IgG1 as their B cell receptor. However, when B cell development was analyzed in the bone marrow from these animals, we found an increased proportion of pro-B cells in comparison to wild type mice, indicative of a block at the pro- to pre-B cell transition.
32nd Annual Meeting of the German Society of Immunology* Dresden September 26 – 29, 2001
Abstracts Workshop A Development and Differentiation of the Immune System Division of Molecular Immunology, Department of Medicine III, University of Erlangen, Erlangen, Germany
A. 1 Specific interaction of a soluble pre-B cell receptor with adherent cell lines H. BRADL, D. MILIUS, and H.-M. JÄCK Signals initiated by the pre-B cell receptor (pre-BCR) are critical for B cell progenitors (pro-B) to mature into precursor (pre) B cells. The pre-BCR consists of a homodimer of m heavy chains, the covalently associated surrogate light (SL) chain composed of VpreB and l5, and the transmembrane signal molecules Iga and Igb. One way to explain how maturation signals are initiated in late pro-B cells is that the pre-BCR is transported to the cell surface and from there interacts with a ligand on stroma cells. To address this hypothesis, we first produced soluble Fab-like pre-BCR and B cell receptor (BCR) fragments, as well as SL-chain, in baculovirus-infected insect cells. Flow cytometry revealed that, in contrast to Fab-like BCR fragments, the soluble pre-BCR binds to the surface of stroma and several other adherent cell lines, but not to suspension cells representing various stages of B cell development. The specific binding of the soluble pre-BCR to stroma cells is saturable, sensitive to trypsin digestion and not dependent on bivalent cations. The * We are obliged to PD Dr. Martin R. Hadam, Hannover, for guiding the online abstract submission and for editing this abstract volume. 0171-2985/01/204/01-02-001 $ 15.00/0
2 · 32nd Annual Meeting of the German Society of Immunology binding of pre-BCR seems to be independent of the m heavy chain, because SL-chain alone was able to interact with stroma cells. These findings not only demonstrate for the first time the capacity of a pre-BCR to bind to a structure on the surface of adherent cells, but also suggest that the pre-BCR interacts via its SL-chain with a putative ligand on stroma cells.
Institutes of 1Medical Microbiology and 2Biochemistry, Justus-Liebig-University, Giessen, Germany
A. 2 Detection of a therapeutic protein, human Ciliary NeuroTropic Factor, in the blood of nude mice implanted with transgenic mouse embryoid bodies U. CHEN1, C. ELMSHÄUSER1, D. ANHLAN2, and E. BECK2 Objectives: To test the possibility of using stem cells as the carrier to deliver a therapeutic protein conditionally in treating human diseases. Methods: Double transgenic ES clones were established, containing a plasmid expressing hCNTF gene under the control of the early promoter of polyomavirus JC (pJCV-hCNTF) as well as a plasmid coding for rtTA-ts A58 genes (tet-onsA58) which consists of the doxycyclineinducible reverse transactivator and the temperature sensitive SV40T antigen (Tag-ts). The tsA58 gene is transcribed in the presence of doxycycline, and the Tag-ts is active at 33°C only. Transcription of the JCV promoter depends on transactivation by either the transcription factor Oct6, or by JCV-Tag. The role of JCV-Tag can be substituted by SV40Tag. The former transactivator (Oct6) is expressed only in cells committed to the glial lineage, the later (SV40Tag-ts) requires the presence of doxycycline and is active at 33°C only. These double transgenic ES cell clones were released to differentiate in culture into embryoid bodies (EBs), and EBs were implanted subcutaneously into nude mice for 3 weeks, and blood was collected to test the level of hCNTF. Results: Culture supernates of ES and EBs under permissive condition and blood of such implanted nude mice were shown to contain hCNTF. Conclusions: The results suggest that pJCV-hCNTF is actively transcribed in glial (precursor) cells, leading to secretion of hCNTF in the blood of nude mice. Such ES-derived glial (precursor) cells must have developed in tissues derived from EB’s in this combined in vitro and in vivo ES differentiation system. It offers the possibility to have a tet-on delivery of therapeutic protein under the control of a tissue-specific promoter besides the tet-O promoter. It also provides a quick cellular readout to evaluate the biological action of SV40 Tag, and to obtain a large quantity of lineage-committed stem cell clones.
32nd Annual Meeting of the German Society of Immunology · 3 Hans-Spemann-Laboratorien, Max-Planck-Institut für Immunbiologie, Freiburg, Germany
A. 3 RFX1 and MIBP1 regulate the CD4 gene silencer M. EHLERS, K. LAULE-KILIAN, C.J. ALDRIAN, J. HUNN, and V. STEIMLE During thymic T cell development CD4+CD8+ precursor T cells develop either into CD4+CD8– helper T cells or into CD4–CD8+ cytotoxic T cells, which interact with cells presenting MHC class II/antigen or MHC class I/antigen complexes, respectively. The molecular mechanisms governing the complex selection and differentiation steps during thymic T cell development are not well understood. A silencer element in the first intron of the CD4 gene has been shown to mediate the downregulation of CD4 gene expression during the differentiation of CD4–CD8+ cytotoxic T cells. Here we show by band shift and antibody supershift experiments that the regulatory proteins RFX1 and MIBP1 bind to a newly identified region of the CD4 silencer element. Mutagenesis of this RFX1/MIBP1 binding site strongly reduces silencing in a new stable reporter assay with insulator elements between the reporter and the resistance cassettes, which faithfully reproduces silencing in CD4–CD8+ T cell lines. The functional importance of RFX1 in CD4 gene silencing was shown by antisense oligonucleotide-mediated inhibition of RFX1 that abolished silencing in CD4–CD8+ T cells.
1
Clinical Research Unit for Rheumatology and 2Department of Molecular Immunology, Institute of Biology, Albert-Ludwigs University, Freiburg, Germany
A. 4 Egr-1 activity partially rescues a defect in slp65 in B cells H. EIBEL1, P. RICHTER1, P. NIELSEN2, M. RETH2, and H. JUMAA2 Introduction: The transcription factor Egr-1 is a member of the Egr-transcription factor family which includes also Egr-2, Egr-3 and Egr-4. All factors share a highly homologous DNA binding domain composed from 3 zinc fingers and bind to a common DNA sequence motif. In B cells, Egr-1 expression is induced upon B cell receptor crosslinking. To study the role of Egr-1 in B cells we generated transgenic mice that overexpress Egr-1 from the immunoglobulin heavy chain promoter-enhancer. Starting at the pro-B cell stage, transgenic Egr-1 expression (tgEgr-1) is maintained throughout the maturation process. It enhances B cell maturation at defined stages of development. To study the signaling components in vivo that normally regulate Egr-1 expression in B cells, we backcrossed tgEgr-1 to mice deficient in the gene encoding for the BCR adapter protein SLP65. In these mice, B cell development is arrested at the transition from immature to mature B cells and in the generation of Ly1 positive (B1) B cells. Methods: B cells of SLP65–/– and tgEgr-1¥SLP65–/– were analyzed by flow cytometry with antibodies specific for IgM; IgD, pre-BCR, CD21, CD23 and CD45R (B220) and by in vitro cultivation on ST-2 feeder cells. Results: Our experiments revealed that Egr-1 overexpression partially complements the SLP65–/– defect. In contrast to SLP65–/– mice, tgEgr-1¥SLP65–/– littermates had less immature and more mature B cells in the spleen. However, the defect in the development of B1 B cells was not rescued.
4 · 32nd Annual Meeting of the German Society of Immunology Conclusions: Therefore, we conclude that Egr-1 activity promotes the transition of immature to mature B cells in the spleen but not the generation of B 1 cells.
Laboratory of Molecular Biology, Children’s Hospital, Charité, Humboldt-University, Berlin, Germany
A. 5 Model for a superantigen-like activation of B cells originating from VH germ-line genes 3-23 and 3-30/3-30.5 by IVIG and normal immunoglobulins P. FISCHER Intravenous immunoglobulins (IVIG) represent the IgG repertoire of several thousand healthy donors. The beneficial use of IVIG in certain immunodeficiencies and autoimmune diseases has been proven, but it is not clear how IVIG functions at the molecular level. Previously we sequenced 92 IgG and IgM clones specifically selected with IVIG from phage display libraries of patients with autoimmune thrombocytopenia (Eur. J. Immunol. 1998; 28:4236), SLE (Arthritis Rheum. 2000, 43:2722), Kawasaki disease (KD) (Clin. Immunol. 2001, 99:18), and a healthy individual (Clin. Exp. Immunol. 2000; 121:37). The most frequently used VH germline gene segments of all IVIG-selected Fabs (62/92, 67%) were 3–23 and 3–30/3–30.5, in contrast to only 3 out of 29 (10%) unselected controls. The combined results suggest a favoured interaction of a subset of IVIG or normal immunoglobulin repertoires with BCRs derived from those two genes. As light chains, antigen-specificity and the high variation in CDR3 showed only little influence on the selection by IVIG, this type of interaction is characteristic for a B cell superantigen. It may significantly shape the B cell repertoire, because 3–23 and 3–30/3–30.5 are the most frequently rearranged VH germ-line genes among humans. Further experiments revealed that despite possible homologies, at least some of the contact residues on Fabs for IVIG must be different from those for the known superantigens Staphylococcal protein A and HIV gp120. To investigate how IVIG influences this restricted interaction, we cloned Fabs from a patient with KD before and after IVIG therapy. Again, a favoured selection of IgG Fabs originating from the 3–23 or 3–30/3–30.5 genes was observed. Importantly, the reactivity with IVIG was significantly higher for clones from the library prepared after the IVIG treatment, providing the first in vivo functional evidence that a subset of IVIG may selectively activate B cells of this germ-line origin.
32nd Annual Meeting of the German Society of Immunology · 5 1
Institute of Medical Microbiology, Immunology and Hygiene, Technische Universität, Institute of Molecular Animal Breeding, Gene Center, and 5Institut für Molekulare Immunologie, GSF-Forschungszentrum für Umwelt and Gesundheit, München, 3Ingenium Pharmaceuticals AG, Martinsried, 4Institute of Experimental Genetics, GSF Research Center, Neuherberg, and 6German Research Center for Biotechnology (GBF), Braunschweig, Germany 2
A. 6 Phenotypic analysis and chromosomal mapping of mouse mutants with immunological defects generated by ENU mutagenesis H. FLASWINKEL1, B. RATHKOLB2, C. FÄRBER3, A. SERVATIUS1, D. SOEWARTO4, E. KREMMER5, N. SANDHOLZER1, M. HRABE DE ANGELIS4, R. BALLING6, E. WOLF2, and K. PFEFFER1 Analysis of gene function in vivo is currently mostly performed by transgenic insertion, inactivation of the respective gene by homologous recombination in embryonic stem cells and gene trapping. We have complemented this approach by isolation of mouse lines with phenotypic immunological alterations which were produced within the Munich ENU-mouse mutagenesis project. In this phenotypic approach C3HeB/FeJ are randomly mutagenized using ethylnitrosourea (ENU). ENU is most potent during spermatogenesis and therefore mutagenesis is performed on male mice which are subsequently crossed to female WT mice. Progeny thereof are analyzed for inherited immunological alterations using a panel of immunological parameters. Out of more than 50 mutant lines isolated with this approach we have analyzed two in more detail and mapped the corresponding mutation. Data from this analysis will be presented.
1
Institute of Medical Microbiology, Immunology and Hygiene, Technische Universität, Institute of Molecular Animal Breeding, Gene Center, and 4Institut für Molekulare Immunologie, GSF-Forschungszentrum für Umwelt und Gesundheit, München, 3Institute of Experimental Genetics, GSF Research Center, Neuherberg, and 5German Research Center for Biotechnology (GBF), Braunschweig, Germany 2
A. 7 Identification of recessive mutants with immunological phenotypes among offspring of ENU-treated C3HeB/FeJ mice H. FLASWINKEL1, B. RATHKOLB2, A. SERVATIUS1, D. SOEWARTO3, E. KREMMER4, N. SANDHOLZER1, M. HRABE DE ANGELIS3, R. BALLING5, E. WOLF2, and K. PFEFFER1 The Munich ENU-Mouse Mutagenesis Project has entered its second phase in which recessive mutants (G3) are being generated. Our group focuses on the identification of immunological phenotypes among those mice employing ELISAs for basal immunoglobulin levels, anti-DNA antibodies and rheumatoid factor as well as flow cytometry for the detection of cell surface molecules. In addition, we are currently measuring cytokine levels in established mutants of the dominant screen and plan to extend this analysis to a limited number of animals from the recessive screen. Nine recessive mutant mouse lines have been established. Additionally, 25 recessive variants are currently being crossed for genetic confirmation. The confirmed mutants encompass phenotypes with alterations in the Ig level of single or multiple isotypes, decreased percentages of
6 · 32nd Annual Meeting of the German Society of Immunology lymphocyte subpopulations and unusual expression patterns of lymphocyte markers. Chromosomal mapping of these recessive lines will start soon. At present we are selecting mutants that will be analyzed in more detail. We are continuing the ENU-program analysing increasing numbers of G3 animals with a broader panel of parameters. Future directions include functional assays i.e. vaccination experiments and the analysis of selected activation markers. Information can be obtained from the following hyperlinks: http:/www.mikrobio.med.tu-muenchen.de/forschung/enu.html http:/www.gsf.de/ieg/groups/enu/mutants/
Department of Cellular Immunology, Max-Planck-Institute of Immunobiology, Freiburg, Germany
A. 8 Characterization of an ADAM family member expressed in thymic epithelial cells G. HUBER, I.D. HAIDL, and K. EICHMANN T cell development in the thymus is dependent on thymic epithelial cells (TEC). However, the molecular explanation for the unique functions of TEC is still incomplete. Following subtractive enrichment for TEC cDNAs, a cDNA was isolated that encodes a protein containing all of the ADAM family member characteristics including a disintegrin and metalloproteinase domain. The novel ADAM family member was designated ADAM28. Expression was detected in adult and embryonic murine TEC and in developmentally related tissues including the trachea, thyroid, lung and stomach. The metalloproteinase active site contains all conserved residues required for activity, thus indicating a potential role in ectodomain shedding and extracellular matrix modeling. The disintegrin-like domain may function in cell-cell adhesion events such as TECthymocyte and/or TEC-TEC interactions. Given the crucial role of ADAM family members in ectodomain processing and cellular adhesion, ADAM28 may play an important role in organogenesis and in organ specific functions such as supporting thymic T cell development. In order to address the specific functions of ADAM28 we are generating recombinant molecules containing the entire extracellular domain or individual domains of the protein. Furthermore, in situ hybridization is being used to clarify the ADAM28 expression pattern in fetal development and in adult tissues.
32nd Annual Meeting of the German Society of Immunology · 7 Molecular Immunology, German Research Centre for Biotechnology (GBF), Braunschweig, Germany
A. 9 Strong antigenic selection acts on the immunoglobulin heavy chain repertoire of B1a lymphocytes in l2 MOPC315 transgenic mice K. KRETSCHMER, J. STOPKOWICZ, H. ENGEL, and S. WEISS The mouse line L2 – transgenic for the l light chain of the plasmacytoma MOPC315 – exhibits nearly complete exclusion of endogenous light chain isotypes and generates predominantly B lymphocytes of the CD5+ B1 type. The exclusive expression of the l2 315 light chain results in an oligoclonal immunoglobulin H chain repertoire. Both, clonally expanded and clonally independent B1a cells contribute to this very restricted IgH chain diversity. Many IgH chains identical in amino acid sequence were isolated repeatedly and in a very high frequency from peritoneal B1a cells of adult L2 mice. We could demonstrate that a significant proportion of these identical IgH chains were due to independent VDJ rearrangements. Comparison of the IgH chain repertoire of peritoneal cavity derived and splenic CD5+ B cells from an individual adult L2 mouse revealed that about 10% of sequences derived for each anatomical location were identical in amino acid sequence suggesting that the same BCR specificities are selected into both pools of mature B1a cells. In contrast, phosphatidylcholine specific CD5+ B1 cells were present only in the peritoneal cavity and like in controls could not be detected in the spleen of L2 mice. Similarly, the IgH chain repertoire of B cells from fetal and neonatal sources show compelling evidence for a high frequency of independent identical VDJ rearrangements resulting in identical amino acid sequences. This observation is contrasted by the clear increase of CDR3 diversity especially in CDR3 of fetal liver Pro/PreB-1 cells from L2 embryos differentiated in vitro when compared to B cells that had developed in vivo. These data suggest that the antibody repertoire of B1a cells is strongly selected by antigen during the fetal as well as during the adult phase of development and clearly indicate selective differences in the antibody repertoire of peritoneal and splenic B1a cells.
Institutes of 1Virology and Immunobiology and 2Pathology, University of Würzburg, Würzburg, Germany and 3National Institute of Medical Research, MI, London, UK
A. 10 Expression of constitutively active Akt/PKB induces lymphomas S.Y. NA1, Y. SCHEURING1, A. MARX2, A. PATRA1, M. TOLAINI3, D. KIOUSSIS3, T. HÜNIG1, and U. BOMMHARDT1 Akt, also known as Protein Kinase B (PKB), has been shown in many different cell systems to regulate cell survival, growth and differentiation. To study the function of Akt in T cell development and activation/differentiation of peripheral T cells we established transgenic mouse lines expressing constitutively active Akt (myristoylated Akt) in T cells using the human CD2 minicassette. Analyses of the Akt transgenic mice show: (1) Thymocytes from Akt heterozygous mice are hyper-reactive to anti-CD3 or PMA/ionomycin stimulation and proliferate even in the presence of the calcineurin inhibitors CsA or FK506. (2) The CD4+ T cell compartment is increased
8 · 32nd Annual Meeting of the German Society of Immunology compared to wildtype littermates whereas the numbers of B cells and CD8+ T cells are reduced. (3) CD4+ T cells have the phenotype of activated T cells, being Mel14lo, CD45RBlo and CD44hi. (4) In RNAse protection analyses (RPA) we did not find significant differences in the mRNA expression of several anti-apoptotic genes (e.g. A1, bcl2, bclx), however, in RPA for cytokine expression Akt heterozygous CD4+ T cells clearly showed enhanced mRNA production for certain cytokines, e.g. IFN-g, depending on the type of stimulation. (5) We will also present data on the effect of Akt on TCR transgenic T cells concerning positive and negative selection of thymocytes and peripheral T cell homeostasis. (6) The cellularity of thymi from Akt+/+ mice is reduced, mainly due to the loss of DP thymocytes which display aberrant expression of several selection markers. (7) Finally, Akt+/+ mice develop monoclonal CD4+ T cell lymphomas (or DP lymphomas, CD8+ T cell lymphomas were not detected) and immunohistological stainings reveal a profound destruction of splenic and thymic architecture as well as infiltration of the liver with CD4+ T cells. Thus, we provide evidence that dysregulation of Akt can be sufficient for the induction of T cell tumorigenesis.
Institute of Medical Immunology, Martin-Luther-University, Halle, Germany
A. 11 The two conserved C-terminal cysteines are necessary for proper folding and membrane localization of the raft-associated peptidase neprilysin/CD10 A. NAVARRETE SANTOS, J. WULFÄNGER, G. HELBING, J. LANGNER, and D. RIEMANN The membrane peptidase neprilysin/CD10, located e.g. on early B and T cells, is raft-associated and consists of 749 amino acids with two conserved cysteines (C734, C746) and a CAAX-resembling box at the C-terminus (aa 746–749). The molecule has been implicated in signal transduction. We investigated the role of the two C-terminal cysteines for the molecular sorting of neprilysin. Using PCR mutagenesis technique, three different mutants of neprilysin (C734S, C746S, and double mutant) were created and cloned into a green fluorescent protein vector. Substitution of C734 as well as C746 by serine disturbed the plasma membrane localization of the endopeptidase as detected by laser scanning microscopy and flow cytometry analysis of transfected CHO and Hek 293 cells. However, Western blot analysis revealed similar molecular weights of neprilysin wild type and mutants. After raft isolation (1% Triton X-100, 4°C, sucrose gradient centrifugation) and Western blot for neprilysin, we detected two bands (a high m.w. band in all fractions and a low m.w. only in the non-raft fractions). Endo-H treatment of protein lysate showed that in wild type and mutants a part of neprilysin is resistant to the glycosidase. When transfected cells were analysed by native PAGE we observed for wild type and mutant C746S three bands of approximately 170, 100 and 90 kDa, whereas C734S and double mutants lost the low m.w. band. Enzymatic endopeptidase activity was only slightly diminished for the C746S mutant, but was not detectable for C734S and the double mutant. Triton X-114 temperature-induced phase separation showed that all mutants partition into the aqueous phase whereas the wild type partitions both into the hydrophobic and the hydrophilic phase. We suggest that C746 (CAAX-box) could be function as transport signal to the plasma membrane whereas cysteine 734 could be important in folding (disulfide bond formation?).
32nd Annual Meeting of the German Society of Immunology · 9 Institutes of 1Immunology, 2Medical Biology and Genetics, and 3Department of Cardiology, University Hospital Motol, Prague, Czech Republic
A. 12 Age-dependent changes of immune parameters in children with DiGeorge syndrome A. SˇEDIVÁ1, J. BARTU˚ Nˇ KOVÁ1, R. ZACHOVÁ1, E. KOCˇ ÁREK2, D. NOVOTNÁ2, and T. KLEIN3 Hypo- or aplasia of thymus connected with syndrome DiGeorge should theoretically cause the profound immunodeficiency. We then followed, on a prospective basis, the cohort of children with syndrome DiGeorge and confirmed 22q11 deletion. One up to six repeated investigations was performed in 11 boys and 17 girls, in the age range from 1 month to 17 years. Immune investigation focused on T lymphocytes and their function. Three children had transient and 3 permanent total lymphopenia. Lower numbers of CD3 positive T cells, compared to normal age related values, were found in 10 children. Both spontaneous and PHA induced proliferation was unexpectedly increased in comparison with normal samples in all tested children less then 15 months old, however, increased T lymphocyte proliferation disappears between 18 months and 2 years. Repeated investigations of proliferative responses in children older than 2 years were comparable with controls. Due to the proposed role of thymus in T lymphocyte selection we followed the presence of double positive CD4+CD8+ and g/d T lymphocytes in the periphery. Double positive T cells were detected in one boy, together with transient positivity of antinuclear antibodies. Gamma/delta T cells were higher than 5% in 9 out of 28 children. Any of the described changes was not connected with particular clinical problems. Patients with lower CD3 positive T cells usually presented with repeated respiratory infections, which were successfully controlled by antibiotic therapy. We did not observe serious clinical signs of immunodeficiency or autoimmunity in any of the patients, all of them classified as partial syndrome DiGeorge. The prospective study is ongoing with specific focus on the development of autoimmunity on the genetic basis of inadequate T cell differentiation on the background of impaired thymic function. This work was supported by grants IGA 6247–3 and 00000064203 of Ministry of Health of Czech Republic.
1 2
Department of Internal Medicine III, Friedrich-Alexander-University, Erlangen and Department of Pediatrics, University of Mainz, Mainz, Germany
A. 13 Analysis of T cell effector functions in a patient with GATA3 haploinsufficiency A. SKAPENKO1, R. BEETZ2, J.R. KALDEN1, and H. SCHULZE-KOOPS1 The transcription factor GATA3 has been implicated in regulating Th2 cell differentiation in the mouse. Moreover, expression of GATA3 was shown to be critical for T cell development and thus, GATA3 knockout mice lack peripheral CD4 T cells. We investigated T cell effector functions in a patient with a dominant negative GATA3 haploinsufficiency related to a deletion of exon 2 in the dominant allele. At the time of investigation, the patient was 16 years old and had no histo-
10 · 32nd Annual Meeting of the German Society of Immunology ry of severe or opportunistic infections. He suffered from renal insufficiency, deafness and hypoparathyroidism, a trias that has been found in other patients with GATA3 haploinsufficiency. Vaccinations had been performed successfully throughout childhood. The patient had normal peripheral T cell counts and normal frequencies and absolute numbers of CD4 helper and CD8 cytotoxic T cells, CD19 B cells, CD56 NK cells and CD14 monocytes. Markers of in vivo Th2 mediated immunity, such as serum IgE, serum IgG2 and IgG4 levels were all within normal limits. T cell proliferation to mitogens and a panel of recall antigens was also without abnormalities. Cytometric analysis of cytoplasmic cytokines revealed a normal distribution of Th1 and Th2 cells within the purified CD4 memory T cell population. Moreover, after in vitro priming of CD4 memory T cells under Th1 or Th2 differentiation inducing conditions employing a previously described ex vivo cell culture system that permits the differentiation of Th effector cells after short term priming, no differences in the extent of Th1 and Th2 cell generation was detected. The data suggest, that GATA3 is not critical for T cell development in humans and that T cell function is completely normal in GATA3 haploinsufficient individuals. Importantly, Th2 generation and Th2 mediated effector functions are not altered in the absence of functional GATA3 in humans.
Department of Animal Physiology, Institute of Zoology, University of Leipzig, Leipzig, Germany
A. 14 Ability for monocytic differentiation in HL-60 cells from different sources U. WAGNER, I. LACHMANN, and K. DRÖSSLER Introduction: The differentiation along the monocytic or granulocytic pathway in response to various compounds was described for the human promyelocytic leukemia cell line HL-60. Nevertheless, the present study demonstrated the setup of a useful model of HL-60 monocytic differentiation related to phenotypical and physiological features. For this purpose, we have examined the sensitivity of HL-60 cells from two different sources to 1,25-dihydroxyvitamin D3 (VD3) and PMA. Methods: Proliferation and viability was estimated by MTT-assay and by flow cytometry of PI-stained cells, respectively. Expression of surface antigens CD11b, CD14, CD54, CD71 was analyzed by FACS using FITC-labeled monoclonal antibodies. Reactive oxygen species (ROS) were quantified using chemiluminescence measurements. The production of cytokines and prostaglandin E2 was analyzed using ELISAs whereas NO-metabolites were detected by the Griess reaction. Results: We demonstrated the monocytic differentiation of HL-60 cells from the source B after VD3-treatment which caused adherence, inhibition of growth, and changes of antigen expression in a concentration- and time-dependent manner. The monocytic cells exhibited a significant increase of CD11b, CD14 and CD54 expression whereas the expression of CD71 was down-regulated. Here we show that differentiated HL-60 B cells were able to produce PGE2, IL-1b and IL-6 after LPS stimulation whereas NO-production was not observed. Additionally, ROS-production was induced by different stimulators. In contrast, HL-60 from a different source showed no sensitivity to VD3-treatment. Surprisingly, the ability for production of PGE2, IL-and ROS was missing in the HL-60 cells from the two different sources after PMA-treatment and phenotypical changes.
32nd Annual Meeting of the German Society of Immunology · 11 Conclusions: Our data demonstrate that VD3 is a powerful promoter for the monocytic development of HL-60 cells (B) and give a useful model to study the mechanisms of the monocytic differentiation process. Furthermore, we found that PMA can obstruct signal pathways of HL-60 populations and origin-dependent differentiation pathways of HL-60 cells.
Department of Immunology, Institute of Genetics, University of Cologne, Cologne, Germany
A. 15 B cell development driven by g1 rather than m chain expression A. WAISMAN and K. RAJEWSKY During B cell development the m constant region gene is used to express the pre-B cell receptor and subsequently the B cell receptor. In order to study whether the g1 heavy chain can replace the m chain during B cell development, we generated a mutant mouse which could only express g1 heavy chains from its IgH locus. In mice expressing the inserted g1 constant region gene in a heterozygous manner, only 15% of the peripheral B cells express IgG1, showing that either the g1 heavy chain can not form an efficient pre-B receptor, or that IgM expressing cells are selected over IgG1 expressing cells in the periphery. In mice with only the IgG1 constant region insertion all B cells express IgG1 as their B cell receptor. However, when B cell development was analyzed in the bone marrow from these animals, we found an increased proportion of pro-B cells in comparison to wild type mice, indicative of a block at the pro- to pre-B cell transition.