- Email: [email protected]
Workshop C Antigen Processing and Presentation
DKFZ-Department of Molecular Immunology, Heidelberg, Germany
c.
1 A role for HLA-DO in peptide loading of HLA·DR molecules
E. A. ARMANDOLA, H. KROPSHOFER, A. B. VOGT, G. MOLDENHAUER, B. LI and G.]. HAMMERLING
The non classical human leukocyte antigen (HLA) DO is expressed in antigen presenting cells and has been shown to interact and co-localize with DM in lysosomal-like vesicles. The function of DO is not known, but its interaction and its co-localization with DM suggest that it may have a function in antigen presentation and act as a regulator of DM. We have produced recombinant DO and investigated its influence on DM-catalysed peptide loading of DR molecules in vitro. The following observations were made: 1. DO does not bind peptides 2. by itself, DO does not support peptide loading onto DR molecules 3. however, in the presence of DM, DO strongly increases the efficiency of DR loading by acceleration of CLIP release. These effects appear to be mediated by a more efficient stabilization of intermediate empty DR molecules by DM-DO complexes in comparison with DM alone. Thus DO improves the function of DM in peptide loading.
'Division of Cellbiology and Immunology, GBF, Braunschweig, and 2Institute for Medical Microbiology, University Giessen, Giessen, Germany
C. 2 Impaired signalling and induction of Tcell anergy by antigen presenting cells treated with Ilemolysin of Listeria monocytogenes A. DARJI',]. WEHLAND 1, T. CHAKRABORTY2 and S. WEISS' Influence of listeriolysin 0 (LLO), the hemolysin secreted by pathogenic Listeria monocytogenes, on MHC class II-dependent T cell activation was investigated. Presentation of native
protein antigens but not the peptides was inhibited by antigen presenting cells (APC) treated with active LLO. Coculture experiments revealed that LLO treated APC inactivate the T cells irreversibly since T cells separated from primary cultures stimulated with LLO treated APC and antigen do not respond to fresh antigen in secondary cultures and also exclude the possibility of T cell inactivation via non specific factors released by the treatment of APC with LLO. The inhibition of T cells mediated by LLO treated APC and antigen was specific and could be observed even in presence of synthetic peptides normally leading to T cell stimulation. Thus it
resembles very much the results obtained using altered peptide ligands that can act antagonistically in the presence of stimulatory peptides. The impaired T cell response can not be restored by addition of IL-2 or PMA. However these T cells do respond to Ca++-ionophore ionomycin.
28th Annual Meeting 1997 . 157 Analysis of early events associated with T cell activation revealed defective T cell signals delivered by LLO treated APC resulted in impaired phosphoinositol turn over and T cell receptor down regulation. Our results represents a novel type of immune interference exerted by this pathogen.
Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany
C. 3 Antigen presentation of heat shock protein IHSP)-chaperoned peptides M. BRELOER, B. FLEISCHER, and A. VON BONIN Immunisation with HSP preparations derived from tumor cell lines have been shown to elicit tumor-specific immunity in the mouse model. The immunisation is mediated by CD8 positive cytotoxic T-lymphocytes (CTL) and directed against tissue-specific peptides associated with the HSP. This priming of CD8+ CTL requires antigen presentation in the context of major histocompatibility complex class-I molecules (MHC-I). While exogenous antigens are usually introduced into the MHC-II pathway. HSP-chaperoned peptides seem to follow the class-I pathway of professional antigen presenting cells (R. SUTO and P. K. SRIVASTAVA, 1995. Science 269: 1585). Here we show that the cytosolic HSP70 and the ER-resident GP96 proteins, purified from the ovalbumin (OVA) transfected cell line EG7 are associated with OVA-derived peptides which can be detected by the SIINFEKLlLKb specific clone 4G3. Macrophages pulsed with HSP preparations from OVA-positive tissue induced TNF-a production but no IFN-y release or specific killing in 4G3 cells, whereas macrophages pulsed with synthetic SIINFEKL induced TNF-a, IFN-y release and specific target cell killing in a chromium release assay. HSP preparations from OVA-negative tissue induced a lower, but significant, amount of TNF-a, suggesting that HSP-chaperoned peptides may stimulate also non-specifically. The incubation of macrophages with HSP/peptide-complexes, thus, may generate a qualitatively different stimulus for the responding CTL.
Division of Immunobiology, University of Bonn, Bonn, Germany
C. 4 Antigenic sequences fused to invariant chain bind to the MHC class II cleft C. CARSTENS, 1. FREISEWINKEL, and N. KOCH The invariant chain (Ii) has a basic function in class II restricted presentation of exogenous antigens. Ii associates with newly synthesised class II polypeptides and directs them to intracellular compartments where peptides are loaded. We generated recombinant polypeptides by replacing the class II groove binding segment of Ii with the sequence of DR1 or DR4 restricted peptides from influenza matrix protein (MP19-31). These fusion proteins were coexpressed with human class II molecules in transfected simian COS-7 cells. Immunoisolation of class II molecules showed that the recombinant II was coprecipitated. Different to wild type Ii which binds to all class II allotypes, the recombinant Ii binds specifically to the DR1 or DR4 dimers. Studies are in progress to determine whether the antigenic Ii fusion proteins are
presented to CD4 T cells.
158 . 28th Annual Meeting 1997 Institut fur Immunologie, Universitat Mainz, Mainz
C. 5 In vitro production of RT1.DM~ proteins by an eukaryotic expression system D. KURTZ, B. HESSER, J. NEUMANN and K. RESKE Endosomalllysosomal peptide loading of MHC class II molecules is facilitated by the nonclassical MHC class II heterodimer DM. For mechanistic studies on antigen processing and peptide loading of rat class II molecules in a variety of accessory cells we produced RT1.DM~ proteins by a yeast expression system and established a number of RTl.FM transfectants. Full length eDNA of RTl.DMb was cloned into expression vector pPICZaB to yield the recombinant vector pPICZaB/RT1.DMb. Pichia pastoris GSl15 cells were transformed with this construct. The vector encodes the secretion signal sequence of Saccharomyces cerevisiae a factor prepro peptide that was introduced to allow secretion of the recombinant protein. Transformed clone pPICZaB/RT1.DMb ~4-6 was isolated and shown to secrete the RT1.DM~ protein at rather low levels with the majority of recombinant protein being retained intracellulady. ~4-6 cells were disrupted and the RT1.DM~ protein was purified from the lysate by immunoaffinity chromatography using the RT1.DM~-specific mA 6D4 coupled to Protein GSepharose. Following SDS-PAGE analysis of the column effluent with subsequent Western blotting a number of proteins were detected that exhibited the mAb 6D4 epitope.
IDepartment of Dermatology, University of Freiburg, Germany, 2Department of Medical Microbiology, University of Edinburgh, United Kingdom
C. 6 Urocanic acid does not alter the antigen presenting activity or the development of murine dendritic cells M. B. LAPPIN!,J. M. WEISS!, E. SCHOPF I, M. NORVAL 2,J. C. SIMONI Exposure to UVB results in the isomerization of trans-urocanic acid (UCA), localized in the stratum corneum, to cis-UCA. Cis-UCA can mediate at least some of the immunosuppressive effects of UVB, though the mechanism of cis-UCA action remains incompletely defined. Alterations in Langerhans cells, and other dendritic antigen presenting cell populations in the skin, may contribute to the loss of skin immune function following UVB exposure. Hence, this study was designed to investigate whether cis-UCA directly, can induce changes in the immunostimulatory capacity of dendritic cells (DC) and the development of DC from precursor cells. Murine DC were generated from C57BL/6 bone marrow (BM) using granulocyte-macrophage colonystimulating factor (GM-CSF), and were used as stimulator cells in mixed lymphocyte reactions (MLR) using BALB/c lymph node cells (LNS) as responders. The addition of cis and transUCA at concentrations ranging from 0.1-500 mg/ml to the MLR, did not affect proliferative responses. Cis or trans-UCA (100 mg/ml) were added to GM-CSF stimulated mouse BM cells on day 0, day 3 or day 5 of culture and the phenotype and allo-stimulatory function of the DC were analysed on day 7. Treatment with cis or trans-UCA did not affect the numbers or the viability of cells in the BM cultures. In addition the expression on DC of lab, CD11c or the costimulatory molecules ICAM-1, B7-1, B7-2 and CD40, was not altered by the addition of cisUCA
to
BM cultures. The inability of cis-UCA to alter the development of DC in vitro was
confirmed by analysing the functional capacity of DC in MLR. DC generated in the presence of cis-UCA were equally efficient in the induction of allo-stimulation, when compared with control DC. These results suggest that cis-UCA does not exert its immunosuppressive activity through direct effects on DC. Such activity may be independent of DC, or alternatively, cisUCA may influence DC function indirectly, through the induction of secondary mediators.
28th Annual Meeting 1997 . 159 Institute of Clinical Immunology and Transfusion Medicine, University of Leipzig, Germany
e. 7 Phenotypical and functional analysis of the Tcell stimulatory potential of human monocyte-derived denaritic cells (moDe) R. LAUB, Y. SCHWITALLE, K. WENK, F. EMMRICH Culturing human monocytes in the presence of IL-4 and GM-CSF is a suitable approach to generate dendritic cells (moDC) at sufficient numbers for immunization or diagnostic purposes. Here we phenotypically and functionally characterize resulting cells of that in vitro differentiation process. FACS analysis of moDC at days 3 and 7 revealed dramatic upregulation of MHC class II and CD86 (B7-2), while the monocyte marker CD14 is downregulated. Only minor increases of CD4 and CD80 (B7-1) surface expression were detected. In cocultures (MLC) with allogeneic PBMC depleted of adherent cells, 7-day moDC stimulated strong proliferative responses comparable in magnitude to those obtained with dendritic cells isolated from peripheral blood. Treatment of moDC with cross-linking agents resulted in metabolic inactivation and a marked decrease of their T cell stimulatory potential, indicating the loss of activation signals initiated during MLC. Since cytokines are known to possess costimulatory activity, we sought to detect candidate cytokines in untreated moDC by intracellular immunostaining and subsequent FACS analysis. Preliminary results confirm the production of IL-1~, IL-6, IL-10 and TNF-a.. We suggest that moDC-derived cytokines contribute substantially to the T cell stimulating potential.
IDepart~ent of Immunobiology, University of Bonn, Germany, and 2Department of Pathology, Washington University, St. Louis, MO, USA
e. 8 Associa~on of peptide and protein antigen with MHe class II on distinct processmg pathways R. LINDNER I and E. UNANUE 2 The binding site of MHC class II molecules is open at both ends and does therefore not restrict the length of the bound ligand. Here we show that a partially folded protein antigen (':-HEL) spontaneously formed SDS-unstable complexes with the purified MHC class II molecule I-Ak (= Ak). These complexes also stimulated T cells when generated on pre-fixed antigen presenting cells (APCs). ':-HEL peptides and, unexpectedly, full-length ':-HEL. In contrast to "HEL peptides, the SDS-stable association of full-length ':-HEL occurred in a fast pathway that required mature class II molecules and did not involve HLA-DM or proteolytic processing. SDS-stable ':-HEL-Ak complexes were not formed by direct conversion of SDSunstable complexes, but by a reaction of mature Ak with free, endocytosed ':·HEL. Our work establishes a fundamental difference between the two MHC class II processing pathways and for the first time demonstrates a MHC class II-bound full-length protein as a cellular processing product.
160 . 28th Annual Meeting 1997 Institute of Hygiene and Microbiology, University of Wurzburg, Wurzburg, Germany
C. 9 Interference of Toxoplasma goncJii with the MHC class " antigen presentation pathway - a putative immune escape mechanism? C. G. K. LODER, and U. GROSS CD4+ T lymphocytes are thought to play a central role in regulating protective immunity against the intracellular parasite Toxoplasma gondii. Since macrophages may activate specific T cells by presenting pathogen-derived peptides bound to molecules of the major histocompatibility complex (MHC), we investigated the in vitro expression of host cell molecules involved in antigen-processing and presentation after infection of murine bone marrow-derived macrophages (BMM) with T gondii. IFN-y-induced upregulation of total as well as cell surfacelocated I-A/I-E antigens was dose-dependently inhibited by T gondii, while H-2D molecules were similarly detected in infected and uninfected BMM cultures. Furthermore, expression of the Ii invariant chain was also reduced by the parasite. Infection of IFN-y-preactivated macrophages revealed that T gondii is able to actively down-regulate an already established expression of I-A/I-E molecules beginning 20 hours p.i. While I-A/I-E molecules were most obviously reduced in parasite-positive host cells, culture supernatant from infected BMM cultures also significantly inhibited expression of I-A/I-E antigens in uninfected macrophages. Our d<1ta indicate that T gondii interferes with the MHC class II antigen presentation pathway of murine macrophages. This may be an important strategy for evasion of the host's immune response during acute infection and for intracellular survival of the parasite.
I Abteilung Molekulare Immunologie, Deutsches Krebsforschungszentrum, Heidelberg, und 2Max von Pettenkofer-Institut fur Virologie, Ludwig Maximilians Universitat, Miinchen
C. 10 The human cytomegalovirus-encoded US6 glycoprotein inhibits the transporter associated with antigen processing F. MOMBURG J, J.-O. KOOPMANN J, T. FLOHR 2, W. MURANYI 2, G. J. HAMMERLING J, U. H. KOSZINOWSKI 2, and H. HENGEL 2 Human cytomegalovirus (HCMV) inhibits peptide import into the endoplasmic reticulum (ER) by the MHC-encoded TAP peptide transporter. We identified the open reading frame US6 to mediate this effect. Expression of the 21 kDa US6 glycoprotein in HCMV-infected cells correlates with the inhibition of peptide transport during infection. The subcellular localization of US6 is restricted to the ER and identical with TAP. US6 protein translated in vitro into ER membrane inhibits peptide translocation indicating that it can interact with preformed TAP1/TAP2 dimers. By coimmunoprecipitation, US6 is detected in complexes with TAPlITAP2, MHC class I heavy chain, ~2-microglobulin, tapasin, calreticulin, and calnexin. Inhibition of TAP-mediated peptide transport is, however, independent of the presence of class I heavy chain and tapasin as shown with respective mutant cell lines. The results provide evidence for a novel mechanism for viral immune escape and a potential role for ER resident proteins to regulate TAP via its luminal face.
28th Annual Meeting 1997 . 161 Institut fur Immunobiologie, Johannes-Gutenberg Universitat Mainz, Mainz, and Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA
C. 11 Expression of rat nonconventional MHC class II molecules Rn.OM in Clendritic cells as revealed by the OMp chain-specific mAb 604 J. NEUMANN, D. KURTH, D. SUN and K. RESKE To investigate the expression of RT1.DM in different intracellular compartments of dendritic cells, mAb 6D4 directed against the light chain of the DM a,p heterodimer was established. The luminal segment of RT1.DMb was amplified by PCR using primers containing an enzyme cleavage site at their 5' end, along with a cDNA pool from IFN-y-stimulated LEW.AVN bone marrow macrophages. TA-cloning was performed to introduce the PCR product into cloning vector pCR 2.1. Recloning of the pCR 2.1 associated insert into the expression vectors pMALC2 and pGEX-3x with consecutive bacterial expression resulted in fusion proteins mBPRT1.DMP and GST-RT1.DMp. The two fusion proteins were purified by affinity chromatography using an amylose resin and a glutathione matrix, respectively. Following several injections of BALB/c mice with MBP-RT1.DMP, the spleen cells were collected, fused with mouse myeloma cells and a few days later culture supernatants were assayed by ELISA using GSTRT1.DMp-coated plates. The strategy to employ fusion protein MBP-RT1.DMP for immunization and GST-RT1.DMP for antibody testing ensured that interference with antibodies directed against the fusion partner MBP was excluded. Monoclonal antibody 6D4 was established by repetitive single cell cloning of positively scoring hybridoma cells.
Institut fur Virologie und Immunbiologie, Wurzburg, Germany
C. 12 Defective recognition of measles virus by cytotoxic T cells C. NEUMEISTER, V. TER MEULEN and S. NIEWIESK H2-d mice are resistent against measles virus (MV) induced encephalitis (MVE) and generate a good cytotoxic T cell (CTL) response. H2-k mice generate a poor CTL response and priming of CTL does not protect C3H (H2-k) mice against MVE. The lack of protection correlates with an impaired MV-specific CTL response. Target cells infected with a vaccinia viruses recombinant (VVR) expressing the nucleocapsid protein (N) are lysed whereas MV are recognized intermediately. The difference is not due to the amount of protein present in target cells nor to binding of the peptide to H2-Kk, retention of the Kk-molecule nor transport of peptide. The block in recognition was also seen in transfected cell lines and not overcome by IFN-y. Using VVR expressing either the full length N, the peptide epitope or the epitope with a leader sequence no difference in recognition could be detected. So far it seems that MV exerts an inhibitory effect on the recognition of certain MV peptide epitopes. As the vaccinia system is not useful in resolving the issue we are in the process of designing recombinant MV to discern the mechanism.
162 . 28th Annual Meeting 1997 Institut fur Immunologie der Universitat Heidelberg, Heidelberg, Germany
c. 13
Polym~rphonuclear neutrophils (PMN) as accessory cells for T cell
activation
M. RADSAK, R. RICHTER, B. REIS, K. ANDRASSY and G. M. Hansch PMN are considered to be terminally differentiated cells with a short half-life time and a limited capacity to synthesize proteins. However, under appropriate culture conditions, PMN are able to de novo synthesize proteins including CD64 and MHC class II antigens. Depending on the individual donor, 24 h to 72 h after adding either gamma interferon or GM-CSF 15-35% of the PMN expressed CD64 and MHC class II. To test whether class II positive PMN were able to activate T cells, they were cocultured with highly purified autologus peripheral T lymphocytes and staphylococcal enterotoxin E (SEE). After four to six days T cell proliferation was seen. When compared to T cell proliferation induced by antigen presenting cells (B cells or monocytes) PMN were equally efficient. Essentially similar data were obtained when cloned T cells were used in place of the peripheral T cells. Expression of MHC class II was not only seen after in vitro manipulation but also on peripheral PMN of patients with acute infections. Particularly during bacterial infections, including infections of the upper respiratory tract, the urinary tract or meningitis, about 10-15% of the peripheral PMN were positive for MHC class II antigens as measured by flow cytometry in whole blood. The biological significance of MHC class II expression on PMN is not yet known; a participation in the regulation of the local T lymphocyte activity appears possible.
Humboldt-Universitat Berlin, Charite, Institut fur Immunologie, Berlin, Germany
c. 14
Transforming growth factor ~ reduces monocytic antigen presenting capacity by Climinishing accessory molecules
F. RANDOW, R. ECKERT, H.-D. VOLK
TGF-p is abundant in the eye's aqueous humor and contributes to its immune privilege. The anterior chamber associated immune deviation (ACAID) is mediated by TGF-p primed F4/80+ APC, which after uptaking locally administered antigens migrate to the spleen to initiate the systemic immune deviation. Because the influence of TGF-p on the antigen presenting capacity of monocytic cells resulting in immune deviation has not been studied in detail, we investigated the changes induced by TGF-pl and its antagonist IFN-y on monocytic expression of HLA-DR and accessory molecules. After contact with TGF-pl human monocytes reduced their expression of CD40, CD54, CD80, and CD86 and of the HLA-DR antigen. IFN-y increased their expression and antagonized TGF-Pl. Reduced expression or induction of HLA-DR and costimulatory antigens resulted in a diminished or increased stimulation of allogeneic T cells by monocytes during MLC, respectively. Moreover, cytokine pretreated human monocytes injected i.v. into mice in order to mimic the ACAID-inducing antigen loaden F4/80+ cells that migrate from the eye to the spleen, influenced a subsequently initiated DTH reaction. While injection of medium treated monocytes did not significantly modulate the DTH reaction priming of the cells with TGF-pl led to a significantly lowered swelling reaction. In contrast, after IFN-y treatment monocytes induced a stronger DTH. These results suggest that TGF-pl's major mode of action on the antigen presenting capacity of monocytes results in downregulation of T cell stimulatory antigens which in turn may prime T cells for the inhibition of subsequently initiated DTH reactions and thus, may explain TGF-p's ACAID-inducing features.
28th Annual Meeting 1997 . 163 Medizinische Klinik I, Unikliniken des Saarlandes, Homburg/Saar, Germany
C. 15 Restin, which is s~ifically e~ressed in H&RS cells mediates macropinocytosis of human dendritic cells U. SAHIN, b. TORECI and M. PFREUNDSCHUH Restin has been described as a novel intermediate filament associated protein specifically expressed in Hodgkin&Reed Sternberg (H&RS) cells. Significant expression of the protein has not been detected in normal cells and its functional role in H&RS cells is unknown, so far. A highly homologous variant of Restin, CLIP170, was recently reported to mediate the binding of endosomes to microtubules. Here we report that Restin/CLIP170 are highly expressed in human dendritic cells and are involved in the extensive formation and transport of macropinosomes, a characteristical feature of professional antigen presenting cells, enabling the high level uptake of exogenous antigens for presentation. Whereas Restin expression is not detectable in freshly isolated peripheral blood mononuclear cells by immunoblot, expression is induced after 48 h culture of monocytes in GM-CSF and IL-4 supplemented medium. Highest expression levels, comparable to the amount of antigen found in H&RS cells, are detected in immature dendritic cells cultured for 5-6 days. Confocal microscopy analysis demonstrated that the protein is located in patches under the dendritic cell plasma membrane. The nature of those patches was identified by using the macropinosome marker fluorescein-dextran for colocalisation studies, demonstrating that Restin is strongly associated with early macropinocytotic vesicles. Flowcytometric analysis revealed that macropinocytotic activity of dendritic cells is positively correlated with the expression levels for Restin/CLIP-170. This disruption of the microtubule system, essential for the functional activity of Restin/CLIP, by nocodazole resulted in a complete loss of macropinocytosis in dendritic cells demonstrating the functional significance of Restin/CLIP170 for this process.
Division of Immunobiology, University of Bonn, Bonn, Germany
C. 16 The importance of N-glycosylation for MHC class II/invariant chain assembly N. SCHACH, H. BROSTERHUS, G. REUTER, R. LINDNER, and N. KOCH MHC class II IX and ~ chains form heterodimers after synthesis and translocation into the endoplasmic reticulum (ER). They associate with a third component, a pre-assembled trimer of invariant chain (Ii). Ii promotes correct folding of MHC class II and prevents premature binding of newly synthesized polypeptides to the binding groove. The assembly of nonameric class lI(1i complexes is facilitated by Calnexin, an ER-resident molecular chaperone that retains unfolded, incomplete complexes. To investigate the consequences of this interaction, we mutated consensus sequences for N-glycosylation in the 31 and 41 kDa splice forms of Ii. Murine fibroblasts (L cells) were transfected with these mutants. We could demonstrate that mutated unglycosylated 31 kDa Ii is not associated with Calnexin and cells transfected with this mutant can not efficiently present antigen. Since trimeric Ii is a prerequisite for efficient assembly and transport of MHC class II, a failure of unglycosylated Ii to oligomerize may explain these observations. In crosslinking experiments with transfected L cells oligomeric complexes of mutated 31 kDa Ii were not resolved.
164 . 28th Annual Meeting 1997 Department of Immunobiology, University Bonn, Germany
C. 17 Alternative splicing of the primary RNA transcript from the invariant chain gene E. SIEVERS and N. KOCH The invariant chain (Ii) has an important function in antigen processing and presentation. The Ii gene codes for two proteins, Ii31 and Ii41. Ii31 is the dominant isoform, which is expressed in all antigen presenting cells (APCs). The Ii41 isoform in contrast, is found in high amounts in professional APCs like dendritic cells, Langerhans cells and macrophages, whereas B cells express only minor amounts of Ii41. Both Ii isoforms promote antigen processing, but the role of the additional segment in Ii41 encoded by the exon 6b has not been identified yet. We studied the structural requirements for expression of exon 6b. DNA constructs containing parts of the Ii genomic DNA and parts of the Ii eDNA were generated. Successive deletion of the introns showed that only sequences flanking exon 6b were necessary for the production of Ii41. Exchange of the Ii promotor by an SV 40 promotor showed that the endogenous promotor is not required for the generation of Ii41. Thus we conclude that Ii41 and Ii31 are produced by alternative splicing of the primary RNA transcript. Since cytokines that regulate the expression of the Ii gene do not alter the ratio of Ii41 to Ii31 we conclude that other components, possibly tissue-specific nuclear factors, regulate the expression of Ii41.
Department of Immunobiology, University of Bonn, Bonn, Germany
C. 18 Presentation of a single CD4 TCell epitope using Invariant Chain (Ii) fusion proteins A.-M. SPONAAS and N. KOCH The aim of this study was to produce APCs (antigen presenting cells) which predominantly present a single peptide. The IAk restricted minimal epitope 51-61 of HEL (Hen Egg Lysosyme) was cloned into Ii 31 eDNA and super transfected into Ii negative class II Ak transfected L cell fibroblasts. The HEL epitope 52-61 was efficiently presented to the T cell hybridoma 3A9 by the transfectant. This endogenous HEL epitope could not be competed out by other HEL epitopes generated when the transfectant was pulsed with native HEL. In contrast, the presentation of the Ii independent antigen RNAse A was not affected by the presence of the endogenously produced HEL peptide. We hypothesize that the recombinant Ii delivers the HEL sequence to intracellular processing compartments and that after Ii is degraded, the HEL peptide binds to class II when CLIP is removed. Experiments are in progress to determine where the endogenous HEL peptide is loaded.
28th Annual Meeting 1997 . 165 tpediatric Cardiology, Herzzentrum GmbH, University Leipzig, Germany, 2Department of Immunology, RPMS and 3Department of Transplantation Biology, CSC, MRC, Hammersmith Hospital, London, UK
C. 19 Altered calcium signaling in MHC class II restricted antigen presentation by Tcells A. TARNOKt, G. LOMBARDI 2, R. HARGREAVES 2, S. SIDHU 2, N. IMAMI 2, L. LIGHTSTONE l, SH. T. FULLER-EsPIE2, and M. RITTERl , P. ROBINSON 3, R. LECHLERl Activated human T cells express MHC class II molecules and thus acquire the capacity to present antigens to other T cells. As we could show, T cell antigen presentation of influenza hemagglutinin (HA) induces in HA specific T cell clones anergy and a Th2 phenotype Immunol. 156: 2769,1996). As detected by flow cytometry T cell antigen presentation induces a transient [Ca2+1 elevation in the recipient cells that has a lower amplitude (150 mM) than following B cell antigen presentation (850 nM). A more detailed analysis of the T cell signaling indicated that a subpopulation of recipient T cells increased the [Cal+l. The [Cal +]; level in the responding cells was up to 1 mM. [Cal +]; increase was completely abolished in the absence of extracellular [Cal +]; and blocked up to 80% by Ni l + or COl+. These data indicated an influx of extracellular Cal + via voltage gated channels. Inhibition of B-cell induced signaling by Ni l + or 2 COl+ was <30%. The [Ca +]jchanges were further investigated by confocal microscopy. Immobilized recipient T cells stained with the Cal +sensitive fluorescent dye Flu03 were stimulated with HA antigen preincubated FuraRed stained T lymphocytes. Single cell Ca l+imaging showed that all recipient T cells increased after contact with HA presenting T cells their [Cal +]; level. However, in contrast to HA presentation by B cells the residual [Cal +]; kinetics were heterogeneous with regard to shape, amplitude and lag time following stimulation. As seen by changes in the FuraRed fluorescence there was also a retrograde stimulation of AG presenting cells that slightly preceded that in the recipient cells. Taken together the findings suggest that T cell antigen presentation induces an altered TCR:CD3 transduced signal. As antigen presenting T cells express high levels of B7, LFA-3 and ICAM-l the failure of these cells is clearly not due to a lack of conventional costimulation. Potential mechanisms could be the bi-directional stimulation via the same costimulatory signals or lack of TCR crosslinking and are currently under investigation.
a.
German Cancer Research Center, Department of Molecular Immunology, Heidelberg, Germany
C. 20 HLA-DM stabilizes empty HLA-DR molecules in chaperone-like fashion A. B. VOGT, H. KROPSHOFER, E. A. ARMANDOLA, G. MOLDENHAUER, S. O. ARNDT and G. HAMMERLING
J.
HLA-DM (DM) is a non-classical major histocompatibility complex (MHC) class II molecule that interacts with classical MHC II molecules in acidic compartments. During this interaction DM is supposed to catalyze the release of invariant chain (li)-derived CLIP peptides thereby rendering the peptide binding groove accessible for antigenic peptide loading. At lysosomal pH in the absence of peptide DR molecules undergo functional inactivation and aggregation. In the presence of DM, however, DR molecules are stabilised and kept receptive for peptide loading. In lysosomal compartments a considerable fraction of DM is found to be stably associated with empty DR a~ dimers to keep them function: Upon encounter with antigenic peptide the DMassociated DR molecules can be rapidly loaded, whereupon they do no longer bind to DM.
166 . 28th Annual Meeting 1997 Recently another non-classical class II molecules, HLA-DO (DO) has been described to be tightly associated with DM. In lysosomal compartments DO is engaged in heterotypic DR/DM/DO complexes. In vitro binding studies have shown that DP is able to increase the activity of DM during peptide loading. Thus, the DM/DO complex seems to act as a class IIspecific chaperone/co-chaperone system that rescues uncharged ap dimers. Empty class II molecules that are kept receptive for loading by DM/DO may enable the antigen processing system to respond promptly to the challenge be newly entering antigens.
Abteilung Immungenetik, Universitat Gottingen, Gottingen, Germany
C. 21 Identification of a rat class I-related gene which is unlinked to the MHC and is homologous to the human MR 1gene L. WALTER and E. GUNTHER A considerable number of genes distantly related to MHC class I genes have been identified, for example CDI, FCRGT, MIC, HFE, MR1. The FCRGT molecules is involved in binding and uptake of maternal immunoglobulin in the neonatal gut, whereas CDI gene products could be shown to present peptides or fatty acids of bacterial origin to cytotoxic T cells. For the MIC, HFE and MRl gene products no antigen-presenting function has been described so far. In order to identify novel class I-related genes in the rat we applied the PCR strategy originally described by HASHIMOTO et al. (1990). The deduced amino acid sequence of one clone shows 76% identity with the human MRl gene product, which is a MHC class I-related gene located on human chromosome Iq25. Thus, the gene isolated appears to be the rat homolog of the human MRl gene. The amino acid sequence of rat MR1 contains the conserved cysteine residues, the beta-2 microglobulin contact sites and the binding sites for the CD8 molecule as in classical class I genes. According to Southern blot data, Mrl is a single copy gene. The sequences of alpha 1 and alpha 2 domains were determined in 4 different inbred rat strains and no nucleotide polymorphism could be detected. Among 8 inbred rat strains only a single restriction fragment length polymorphism (RFLP) could be detected between strains DA and LEW. Therefore, rat Mrl does not appear to be conspicuously polymorphic. This RFLP was used to map Mrl in backcross hybrids. Mrl cosegregates with the Renin (Ren) gene located on rat chromosome (RNO) 13. Thus, also rat Mrl is not linked to the MHC which maps to RN020p12. Mrl is broadly expressed in lymphoid and non-lymphoid tissues, although at a low level. Analysis of lung RNA by RT-PCR revealed the expected and a further amplificate that represents an alternatively spliced Mrl transcript lacking exon 3. We are currently producing recombinant MR1 protein for production of monoclonal antibodies.
c.
1 A role for HLA-DO in peptide loading of HLA·DR molecules
E. A. ARMANDOLA, H. KROPSHOFER, A. B. VOGT, G. MOLDENHAUER, B. LI and G.]. HAMMERLING
The non classical human leukocyte antigen (HLA) DO is expressed in antigen presenting cells and has been shown to interact and co-localize with DM in lysosomal-like vesicles. The function of DO is not known, but its interaction and its co-localization with DM suggest that it may have a function in antigen presentation and act as a regulator of DM. We have produced recombinant DO and investigated its influence on DM-catalysed peptide loading of DR molecules in vitro. The following observations were made: 1. DO does not bind peptides 2. by itself, DO does not support peptide loading onto DR molecules 3. however, in the presence of DM, DO strongly increases the efficiency of DR loading by acceleration of CLIP release. These effects appear to be mediated by a more efficient stabilization of intermediate empty DR molecules by DM-DO complexes in comparison with DM alone. Thus DO improves the function of DM in peptide loading.
'Division of Cellbiology and Immunology, GBF, Braunschweig, and 2Institute for Medical Microbiology, University Giessen, Giessen, Germany
C. 2 Impaired signalling and induction of Tcell anergy by antigen presenting cells treated with Ilemolysin of Listeria monocytogenes A. DARJI',]. WEHLAND 1, T. CHAKRABORTY2 and S. WEISS' Influence of listeriolysin 0 (LLO), the hemolysin secreted by pathogenic Listeria monocytogenes, on MHC class II-dependent T cell activation was investigated. Presentation of native
protein antigens but not the peptides was inhibited by antigen presenting cells (APC) treated with active LLO. Coculture experiments revealed that LLO treated APC inactivate the T cells irreversibly since T cells separated from primary cultures stimulated with LLO treated APC and antigen do not respond to fresh antigen in secondary cultures and also exclude the possibility of T cell inactivation via non specific factors released by the treatment of APC with LLO. The inhibition of T cells mediated by LLO treated APC and antigen was specific and could be observed even in presence of synthetic peptides normally leading to T cell stimulation. Thus it
resembles very much the results obtained using altered peptide ligands that can act antagonistically in the presence of stimulatory peptides. The impaired T cell response can not be restored by addition of IL-2 or PMA. However these T cells do respond to Ca++-ionophore ionomycin.
28th Annual Meeting 1997 . 157 Analysis of early events associated with T cell activation revealed defective T cell signals delivered by LLO treated APC resulted in impaired phosphoinositol turn over and T cell receptor down regulation. Our results represents a novel type of immune interference exerted by this pathogen.
Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany
C. 3 Antigen presentation of heat shock protein IHSP)-chaperoned peptides M. BRELOER, B. FLEISCHER, and A. VON BONIN Immunisation with HSP preparations derived from tumor cell lines have been shown to elicit tumor-specific immunity in the mouse model. The immunisation is mediated by CD8 positive cytotoxic T-lymphocytes (CTL) and directed against tissue-specific peptides associated with the HSP. This priming of CD8+ CTL requires antigen presentation in the context of major histocompatibility complex class-I molecules (MHC-I). While exogenous antigens are usually introduced into the MHC-II pathway. HSP-chaperoned peptides seem to follow the class-I pathway of professional antigen presenting cells (R. SUTO and P. K. SRIVASTAVA, 1995. Science 269: 1585). Here we show that the cytosolic HSP70 and the ER-resident GP96 proteins, purified from the ovalbumin (OVA) transfected cell line EG7 are associated with OVA-derived peptides which can be detected by the SIINFEKLlLKb specific clone 4G3. Macrophages pulsed with HSP preparations from OVA-positive tissue induced TNF-a production but no IFN-y release or specific killing in 4G3 cells, whereas macrophages pulsed with synthetic SIINFEKL induced TNF-a, IFN-y release and specific target cell killing in a chromium release assay. HSP preparations from OVA-negative tissue induced a lower, but significant, amount of TNF-a, suggesting that HSP-chaperoned peptides may stimulate also non-specifically. The incubation of macrophages with HSP/peptide-complexes, thus, may generate a qualitatively different stimulus for the responding CTL.
Division of Immunobiology, University of Bonn, Bonn, Germany
C. 4 Antigenic sequences fused to invariant chain bind to the MHC class II cleft C. CARSTENS, 1. FREISEWINKEL, and N. KOCH The invariant chain (Ii) has a basic function in class II restricted presentation of exogenous antigens. Ii associates with newly synthesised class II polypeptides and directs them to intracellular compartments where peptides are loaded. We generated recombinant polypeptides by replacing the class II groove binding segment of Ii with the sequence of DR1 or DR4 restricted peptides from influenza matrix protein (MP19-31). These fusion proteins were coexpressed with human class II molecules in transfected simian COS-7 cells. Immunoisolation of class II molecules showed that the recombinant II was coprecipitated. Different to wild type Ii which binds to all class II allotypes, the recombinant Ii binds specifically to the DR1 or DR4 dimers. Studies are in progress to determine whether the antigenic Ii fusion proteins are
presented to CD4 T cells.
158 . 28th Annual Meeting 1997 Institut fur Immunologie, Universitat Mainz, Mainz
C. 5 In vitro production of RT1.DM~ proteins by an eukaryotic expression system D. KURTZ, B. HESSER, J. NEUMANN and K. RESKE Endosomalllysosomal peptide loading of MHC class II molecules is facilitated by the nonclassical MHC class II heterodimer DM. For mechanistic studies on antigen processing and peptide loading of rat class II molecules in a variety of accessory cells we produced RT1.DM~ proteins by a yeast expression system and established a number of RTl.FM transfectants. Full length eDNA of RTl.DMb was cloned into expression vector pPICZaB to yield the recombinant vector pPICZaB/RT1.DMb. Pichia pastoris GSl15 cells were transformed with this construct. The vector encodes the secretion signal sequence of Saccharomyces cerevisiae a factor prepro peptide that was introduced to allow secretion of the recombinant protein. Transformed clone pPICZaB/RT1.DMb ~4-6 was isolated and shown to secrete the RT1.DM~ protein at rather low levels with the majority of recombinant protein being retained intracellulady. ~4-6 cells were disrupted and the RT1.DM~ protein was purified from the lysate by immunoaffinity chromatography using the RT1.DM~-specific mA 6D4 coupled to Protein GSepharose. Following SDS-PAGE analysis of the column effluent with subsequent Western blotting a number of proteins were detected that exhibited the mAb 6D4 epitope.
IDepartment of Dermatology, University of Freiburg, Germany, 2Department of Medical Microbiology, University of Edinburgh, United Kingdom
C. 6 Urocanic acid does not alter the antigen presenting activity or the development of murine dendritic cells M. B. LAPPIN!,J. M. WEISS!, E. SCHOPF I, M. NORVAL 2,J. C. SIMONI Exposure to UVB results in the isomerization of trans-urocanic acid (UCA), localized in the stratum corneum, to cis-UCA. Cis-UCA can mediate at least some of the immunosuppressive effects of UVB, though the mechanism of cis-UCA action remains incompletely defined. Alterations in Langerhans cells, and other dendritic antigen presenting cell populations in the skin, may contribute to the loss of skin immune function following UVB exposure. Hence, this study was designed to investigate whether cis-UCA directly, can induce changes in the immunostimulatory capacity of dendritic cells (DC) and the development of DC from precursor cells. Murine DC were generated from C57BL/6 bone marrow (BM) using granulocyte-macrophage colonystimulating factor (GM-CSF), and were used as stimulator cells in mixed lymphocyte reactions (MLR) using BALB/c lymph node cells (LNS) as responders. The addition of cis and transUCA at concentrations ranging from 0.1-500 mg/ml to the MLR, did not affect proliferative responses. Cis or trans-UCA (100 mg/ml) were added to GM-CSF stimulated mouse BM cells on day 0, day 3 or day 5 of culture and the phenotype and allo-stimulatory function of the DC were analysed on day 7. Treatment with cis or trans-UCA did not affect the numbers or the viability of cells in the BM cultures. In addition the expression on DC of lab, CD11c or the costimulatory molecules ICAM-1, B7-1, B7-2 and CD40, was not altered by the addition of cisUCA
to
BM cultures. The inability of cis-UCA to alter the development of DC in vitro was
confirmed by analysing the functional capacity of DC in MLR. DC generated in the presence of cis-UCA were equally efficient in the induction of allo-stimulation, when compared with control DC. These results suggest that cis-UCA does not exert its immunosuppressive activity through direct effects on DC. Such activity may be independent of DC, or alternatively, cisUCA may influence DC function indirectly, through the induction of secondary mediators.
28th Annual Meeting 1997 . 159 Institute of Clinical Immunology and Transfusion Medicine, University of Leipzig, Germany
e. 7 Phenotypical and functional analysis of the Tcell stimulatory potential of human monocyte-derived denaritic cells (moDe) R. LAUB, Y. SCHWITALLE, K. WENK, F. EMMRICH Culturing human monocytes in the presence of IL-4 and GM-CSF is a suitable approach to generate dendritic cells (moDC) at sufficient numbers for immunization or diagnostic purposes. Here we phenotypically and functionally characterize resulting cells of that in vitro differentiation process. FACS analysis of moDC at days 3 and 7 revealed dramatic upregulation of MHC class II and CD86 (B7-2), while the monocyte marker CD14 is downregulated. Only minor increases of CD4 and CD80 (B7-1) surface expression were detected. In cocultures (MLC) with allogeneic PBMC depleted of adherent cells, 7-day moDC stimulated strong proliferative responses comparable in magnitude to those obtained with dendritic cells isolated from peripheral blood. Treatment of moDC with cross-linking agents resulted in metabolic inactivation and a marked decrease of their T cell stimulatory potential, indicating the loss of activation signals initiated during MLC. Since cytokines are known to possess costimulatory activity, we sought to detect candidate cytokines in untreated moDC by intracellular immunostaining and subsequent FACS analysis. Preliminary results confirm the production of IL-1~, IL-6, IL-10 and TNF-a.. We suggest that moDC-derived cytokines contribute substantially to the T cell stimulating potential.
IDepart~ent of Immunobiology, University of Bonn, Germany, and 2Department of Pathology, Washington University, St. Louis, MO, USA
e. 8 Associa~on of peptide and protein antigen with MHe class II on distinct processmg pathways R. LINDNER I and E. UNANUE 2 The binding site of MHC class II molecules is open at both ends and does therefore not restrict the length of the bound ligand. Here we show that a partially folded protein antigen (':-HEL) spontaneously formed SDS-unstable complexes with the purified MHC class II molecule I-Ak (= Ak). These complexes also stimulated T cells when generated on pre-fixed antigen presenting cells (APCs). ':-HEL peptides and, unexpectedly, full-length ':-HEL. In contrast to "HEL peptides, the SDS-stable association of full-length ':-HEL occurred in a fast pathway that required mature class II molecules and did not involve HLA-DM or proteolytic processing. SDS-stable ':-HEL-Ak complexes were not formed by direct conversion of SDSunstable complexes, but by a reaction of mature Ak with free, endocytosed ':·HEL. Our work establishes a fundamental difference between the two MHC class II processing pathways and for the first time demonstrates a MHC class II-bound full-length protein as a cellular processing product.
160 . 28th Annual Meeting 1997 Institute of Hygiene and Microbiology, University of Wurzburg, Wurzburg, Germany
C. 9 Interference of Toxoplasma goncJii with the MHC class " antigen presentation pathway - a putative immune escape mechanism? C. G. K. LODER, and U. GROSS CD4+ T lymphocytes are thought to play a central role in regulating protective immunity against the intracellular parasite Toxoplasma gondii. Since macrophages may activate specific T cells by presenting pathogen-derived peptides bound to molecules of the major histocompatibility complex (MHC), we investigated the in vitro expression of host cell molecules involved in antigen-processing and presentation after infection of murine bone marrow-derived macrophages (BMM) with T gondii. IFN-y-induced upregulation of total as well as cell surfacelocated I-A/I-E antigens was dose-dependently inhibited by T gondii, while H-2D molecules were similarly detected in infected and uninfected BMM cultures. Furthermore, expression of the Ii invariant chain was also reduced by the parasite. Infection of IFN-y-preactivated macrophages revealed that T gondii is able to actively down-regulate an already established expression of I-A/I-E molecules beginning 20 hours p.i. While I-A/I-E molecules were most obviously reduced in parasite-positive host cells, culture supernatant from infected BMM cultures also significantly inhibited expression of I-A/I-E antigens in uninfected macrophages. Our d<1ta indicate that T gondii interferes with the MHC class II antigen presentation pathway of murine macrophages. This may be an important strategy for evasion of the host's immune response during acute infection and for intracellular survival of the parasite.
I Abteilung Molekulare Immunologie, Deutsches Krebsforschungszentrum, Heidelberg, und 2Max von Pettenkofer-Institut fur Virologie, Ludwig Maximilians Universitat, Miinchen
C. 10 The human cytomegalovirus-encoded US6 glycoprotein inhibits the transporter associated with antigen processing F. MOMBURG J, J.-O. KOOPMANN J, T. FLOHR 2, W. MURANYI 2, G. J. HAMMERLING J, U. H. KOSZINOWSKI 2, and H. HENGEL 2 Human cytomegalovirus (HCMV) inhibits peptide import into the endoplasmic reticulum (ER) by the MHC-encoded TAP peptide transporter. We identified the open reading frame US6 to mediate this effect. Expression of the 21 kDa US6 glycoprotein in HCMV-infected cells correlates with the inhibition of peptide transport during infection. The subcellular localization of US6 is restricted to the ER and identical with TAP. US6 protein translated in vitro into ER membrane inhibits peptide translocation indicating that it can interact with preformed TAP1/TAP2 dimers. By coimmunoprecipitation, US6 is detected in complexes with TAPlITAP2, MHC class I heavy chain, ~2-microglobulin, tapasin, calreticulin, and calnexin. Inhibition of TAP-mediated peptide transport is, however, independent of the presence of class I heavy chain and tapasin as shown with respective mutant cell lines. The results provide evidence for a novel mechanism for viral immune escape and a potential role for ER resident proteins to regulate TAP via its luminal face.
28th Annual Meeting 1997 . 161 Institut fur Immunobiologie, Johannes-Gutenberg Universitat Mainz, Mainz, and Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA
C. 11 Expression of rat nonconventional MHC class II molecules Rn.OM in Clendritic cells as revealed by the OMp chain-specific mAb 604 J. NEUMANN, D. KURTH, D. SUN and K. RESKE To investigate the expression of RT1.DM in different intracellular compartments of dendritic cells, mAb 6D4 directed against the light chain of the DM a,p heterodimer was established. The luminal segment of RT1.DMb was amplified by PCR using primers containing an enzyme cleavage site at their 5' end, along with a cDNA pool from IFN-y-stimulated LEW.AVN bone marrow macrophages. TA-cloning was performed to introduce the PCR product into cloning vector pCR 2.1. Recloning of the pCR 2.1 associated insert into the expression vectors pMALC2 and pGEX-3x with consecutive bacterial expression resulted in fusion proteins mBPRT1.DMP and GST-RT1.DMp. The two fusion proteins were purified by affinity chromatography using an amylose resin and a glutathione matrix, respectively. Following several injections of BALB/c mice with MBP-RT1.DMP, the spleen cells were collected, fused with mouse myeloma cells and a few days later culture supernatants were assayed by ELISA using GSTRT1.DMp-coated plates. The strategy to employ fusion protein MBP-RT1.DMP for immunization and GST-RT1.DMP for antibody testing ensured that interference with antibodies directed against the fusion partner MBP was excluded. Monoclonal antibody 6D4 was established by repetitive single cell cloning of positively scoring hybridoma cells.
Institut fur Virologie und Immunbiologie, Wurzburg, Germany
C. 12 Defective recognition of measles virus by cytotoxic T cells C. NEUMEISTER, V. TER MEULEN and S. NIEWIESK H2-d mice are resistent against measles virus (MV) induced encephalitis (MVE) and generate a good cytotoxic T cell (CTL) response. H2-k mice generate a poor CTL response and priming of CTL does not protect C3H (H2-k) mice against MVE. The lack of protection correlates with an impaired MV-specific CTL response. Target cells infected with a vaccinia viruses recombinant (VVR) expressing the nucleocapsid protein (N) are lysed whereas MV are recognized intermediately. The difference is not due to the amount of protein present in target cells nor to binding of the peptide to H2-Kk, retention of the Kk-molecule nor transport of peptide. The block in recognition was also seen in transfected cell lines and not overcome by IFN-y. Using VVR expressing either the full length N, the peptide epitope or the epitope with a leader sequence no difference in recognition could be detected. So far it seems that MV exerts an inhibitory effect on the recognition of certain MV peptide epitopes. As the vaccinia system is not useful in resolving the issue we are in the process of designing recombinant MV to discern the mechanism.
162 . 28th Annual Meeting 1997 Institut fur Immunologie der Universitat Heidelberg, Heidelberg, Germany
c. 13
Polym~rphonuclear neutrophils (PMN) as accessory cells for T cell
activation
M. RADSAK, R. RICHTER, B. REIS, K. ANDRASSY and G. M. Hansch PMN are considered to be terminally differentiated cells with a short half-life time and a limited capacity to synthesize proteins. However, under appropriate culture conditions, PMN are able to de novo synthesize proteins including CD64 and MHC class II antigens. Depending on the individual donor, 24 h to 72 h after adding either gamma interferon or GM-CSF 15-35% of the PMN expressed CD64 and MHC class II. To test whether class II positive PMN were able to activate T cells, they were cocultured with highly purified autologus peripheral T lymphocytes and staphylococcal enterotoxin E (SEE). After four to six days T cell proliferation was seen. When compared to T cell proliferation induced by antigen presenting cells (B cells or monocytes) PMN were equally efficient. Essentially similar data were obtained when cloned T cells were used in place of the peripheral T cells. Expression of MHC class II was not only seen after in vitro manipulation but also on peripheral PMN of patients with acute infections. Particularly during bacterial infections, including infections of the upper respiratory tract, the urinary tract or meningitis, about 10-15% of the peripheral PMN were positive for MHC class II antigens as measured by flow cytometry in whole blood. The biological significance of MHC class II expression on PMN is not yet known; a participation in the regulation of the local T lymphocyte activity appears possible.
Humboldt-Universitat Berlin, Charite, Institut fur Immunologie, Berlin, Germany
c. 14
Transforming growth factor ~ reduces monocytic antigen presenting capacity by Climinishing accessory molecules
F. RANDOW, R. ECKERT, H.-D. VOLK
TGF-p is abundant in the eye's aqueous humor and contributes to its immune privilege. The anterior chamber associated immune deviation (ACAID) is mediated by TGF-p primed F4/80+ APC, which after uptaking locally administered antigens migrate to the spleen to initiate the systemic immune deviation. Because the influence of TGF-p on the antigen presenting capacity of monocytic cells resulting in immune deviation has not been studied in detail, we investigated the changes induced by TGF-pl and its antagonist IFN-y on monocytic expression of HLA-DR and accessory molecules. After contact with TGF-pl human monocytes reduced their expression of CD40, CD54, CD80, and CD86 and of the HLA-DR antigen. IFN-y increased their expression and antagonized TGF-Pl. Reduced expression or induction of HLA-DR and costimulatory antigens resulted in a diminished or increased stimulation of allogeneic T cells by monocytes during MLC, respectively. Moreover, cytokine pretreated human monocytes injected i.v. into mice in order to mimic the ACAID-inducing antigen loaden F4/80+ cells that migrate from the eye to the spleen, influenced a subsequently initiated DTH reaction. While injection of medium treated monocytes did not significantly modulate the DTH reaction priming of the cells with TGF-pl led to a significantly lowered swelling reaction. In contrast, after IFN-y treatment monocytes induced a stronger DTH. These results suggest that TGF-pl's major mode of action on the antigen presenting capacity of monocytes results in downregulation of T cell stimulatory antigens which in turn may prime T cells for the inhibition of subsequently initiated DTH reactions and thus, may explain TGF-p's ACAID-inducing features.
28th Annual Meeting 1997 . 163 Medizinische Klinik I, Unikliniken des Saarlandes, Homburg/Saar, Germany
C. 15 Restin, which is s~ifically e~ressed in H&RS cells mediates macropinocytosis of human dendritic cells U. SAHIN, b. TORECI and M. PFREUNDSCHUH Restin has been described as a novel intermediate filament associated protein specifically expressed in Hodgkin&Reed Sternberg (H&RS) cells. Significant expression of the protein has not been detected in normal cells and its functional role in H&RS cells is unknown, so far. A highly homologous variant of Restin, CLIP170, was recently reported to mediate the binding of endosomes to microtubules. Here we report that Restin/CLIP170 are highly expressed in human dendritic cells and are involved in the extensive formation and transport of macropinosomes, a characteristical feature of professional antigen presenting cells, enabling the high level uptake of exogenous antigens for presentation. Whereas Restin expression is not detectable in freshly isolated peripheral blood mononuclear cells by immunoblot, expression is induced after 48 h culture of monocytes in GM-CSF and IL-4 supplemented medium. Highest expression levels, comparable to the amount of antigen found in H&RS cells, are detected in immature dendritic cells cultured for 5-6 days. Confocal microscopy analysis demonstrated that the protein is located in patches under the dendritic cell plasma membrane. The nature of those patches was identified by using the macropinosome marker fluorescein-dextran for colocalisation studies, demonstrating that Restin is strongly associated with early macropinocytotic vesicles. Flowcytometric analysis revealed that macropinocytotic activity of dendritic cells is positively correlated with the expression levels for Restin/CLIP-170. This disruption of the microtubule system, essential for the functional activity of Restin/CLIP, by nocodazole resulted in a complete loss of macropinocytosis in dendritic cells demonstrating the functional significance of Restin/CLIP170 for this process.
Division of Immunobiology, University of Bonn, Bonn, Germany
C. 16 The importance of N-glycosylation for MHC class II/invariant chain assembly N. SCHACH, H. BROSTERHUS, G. REUTER, R. LINDNER, and N. KOCH MHC class II IX and ~ chains form heterodimers after synthesis and translocation into the endoplasmic reticulum (ER). They associate with a third component, a pre-assembled trimer of invariant chain (Ii). Ii promotes correct folding of MHC class II and prevents premature binding of newly synthesized polypeptides to the binding groove. The assembly of nonameric class lI(1i complexes is facilitated by Calnexin, an ER-resident molecular chaperone that retains unfolded, incomplete complexes. To investigate the consequences of this interaction, we mutated consensus sequences for N-glycosylation in the 31 and 41 kDa splice forms of Ii. Murine fibroblasts (L cells) were transfected with these mutants. We could demonstrate that mutated unglycosylated 31 kDa Ii is not associated with Calnexin and cells transfected with this mutant can not efficiently present antigen. Since trimeric Ii is a prerequisite for efficient assembly and transport of MHC class II, a failure of unglycosylated Ii to oligomerize may explain these observations. In crosslinking experiments with transfected L cells oligomeric complexes of mutated 31 kDa Ii were not resolved.
164 . 28th Annual Meeting 1997 Department of Immunobiology, University Bonn, Germany
C. 17 Alternative splicing of the primary RNA transcript from the invariant chain gene E. SIEVERS and N. KOCH The invariant chain (Ii) has an important function in antigen processing and presentation. The Ii gene codes for two proteins, Ii31 and Ii41. Ii31 is the dominant isoform, which is expressed in all antigen presenting cells (APCs). The Ii41 isoform in contrast, is found in high amounts in professional APCs like dendritic cells, Langerhans cells and macrophages, whereas B cells express only minor amounts of Ii41. Both Ii isoforms promote antigen processing, but the role of the additional segment in Ii41 encoded by the exon 6b has not been identified yet. We studied the structural requirements for expression of exon 6b. DNA constructs containing parts of the Ii genomic DNA and parts of the Ii eDNA were generated. Successive deletion of the introns showed that only sequences flanking exon 6b were necessary for the production of Ii41. Exchange of the Ii promotor by an SV 40 promotor showed that the endogenous promotor is not required for the generation of Ii41. Thus we conclude that Ii41 and Ii31 are produced by alternative splicing of the primary RNA transcript. Since cytokines that regulate the expression of the Ii gene do not alter the ratio of Ii41 to Ii31 we conclude that other components, possibly tissue-specific nuclear factors, regulate the expression of Ii41.
Department of Immunobiology, University of Bonn, Bonn, Germany
C. 18 Presentation of a single CD4 TCell epitope using Invariant Chain (Ii) fusion proteins A.-M. SPONAAS and N. KOCH The aim of this study was to produce APCs (antigen presenting cells) which predominantly present a single peptide. The IAk restricted minimal epitope 51-61 of HEL (Hen Egg Lysosyme) was cloned into Ii 31 eDNA and super transfected into Ii negative class II Ak transfected L cell fibroblasts. The HEL epitope 52-61 was efficiently presented to the T cell hybridoma 3A9 by the transfectant. This endogenous HEL epitope could not be competed out by other HEL epitopes generated when the transfectant was pulsed with native HEL. In contrast, the presentation of the Ii independent antigen RNAse A was not affected by the presence of the endogenously produced HEL peptide. We hypothesize that the recombinant Ii delivers the HEL sequence to intracellular processing compartments and that after Ii is degraded, the HEL peptide binds to class II when CLIP is removed. Experiments are in progress to determine where the endogenous HEL peptide is loaded.
28th Annual Meeting 1997 . 165 tpediatric Cardiology, Herzzentrum GmbH, University Leipzig, Germany, 2Department of Immunology, RPMS and 3Department of Transplantation Biology, CSC, MRC, Hammersmith Hospital, London, UK
C. 19 Altered calcium signaling in MHC class II restricted antigen presentation by Tcells A. TARNOKt, G. LOMBARDI 2, R. HARGREAVES 2, S. SIDHU 2, N. IMAMI 2, L. LIGHTSTONE l, SH. T. FULLER-EsPIE2, and M. RITTERl , P. ROBINSON 3, R. LECHLERl Activated human T cells express MHC class II molecules and thus acquire the capacity to present antigens to other T cells. As we could show, T cell antigen presentation of influenza hemagglutinin (HA) induces in HA specific T cell clones anergy and a Th2 phenotype Immunol. 156: 2769,1996). As detected by flow cytometry T cell antigen presentation induces a transient [Ca2+1 elevation in the recipient cells that has a lower amplitude (150 mM) than following B cell antigen presentation (850 nM). A more detailed analysis of the T cell signaling indicated that a subpopulation of recipient T cells increased the [Cal+l. The [Cal +]; level in the responding cells was up to 1 mM. [Cal +]; increase was completely abolished in the absence of extracellular [Cal +]; and blocked up to 80% by Ni l + or COl+. These data indicated an influx of extracellular Cal + via voltage gated channels. Inhibition of B-cell induced signaling by Ni l + or 2 COl+ was <30%. The [Ca +]jchanges were further investigated by confocal microscopy. Immobilized recipient T cells stained with the Cal +sensitive fluorescent dye Flu03 were stimulated with HA antigen preincubated FuraRed stained T lymphocytes. Single cell Ca l+imaging showed that all recipient T cells increased after contact with HA presenting T cells their [Cal +]; level. However, in contrast to HA presentation by B cells the residual [Cal +]; kinetics were heterogeneous with regard to shape, amplitude and lag time following stimulation. As seen by changes in the FuraRed fluorescence there was also a retrograde stimulation of AG presenting cells that slightly preceded that in the recipient cells. Taken together the findings suggest that T cell antigen presentation induces an altered TCR:CD3 transduced signal. As antigen presenting T cells express high levels of B7, LFA-3 and ICAM-l the failure of these cells is clearly not due to a lack of conventional costimulation. Potential mechanisms could be the bi-directional stimulation via the same costimulatory signals or lack of TCR crosslinking and are currently under investigation.
a.
German Cancer Research Center, Department of Molecular Immunology, Heidelberg, Germany
C. 20 HLA-DM stabilizes empty HLA-DR molecules in chaperone-like fashion A. B. VOGT, H. KROPSHOFER, E. A. ARMANDOLA, G. MOLDENHAUER, S. O. ARNDT and G. HAMMERLING
J.
HLA-DM (DM) is a non-classical major histocompatibility complex (MHC) class II molecule that interacts with classical MHC II molecules in acidic compartments. During this interaction DM is supposed to catalyze the release of invariant chain (li)-derived CLIP peptides thereby rendering the peptide binding groove accessible for antigenic peptide loading. At lysosomal pH in the absence of peptide DR molecules undergo functional inactivation and aggregation. In the presence of DM, however, DR molecules are stabilised and kept receptive for peptide loading. In lysosomal compartments a considerable fraction of DM is found to be stably associated with empty DR a~ dimers to keep them function: Upon encounter with antigenic peptide the DMassociated DR molecules can be rapidly loaded, whereupon they do no longer bind to DM.
166 . 28th Annual Meeting 1997 Recently another non-classical class II molecules, HLA-DO (DO) has been described to be tightly associated with DM. In lysosomal compartments DO is engaged in heterotypic DR/DM/DO complexes. In vitro binding studies have shown that DP is able to increase the activity of DM during peptide loading. Thus, the DM/DO complex seems to act as a class IIspecific chaperone/co-chaperone system that rescues uncharged ap dimers. Empty class II molecules that are kept receptive for loading by DM/DO may enable the antigen processing system to respond promptly to the challenge be newly entering antigens.
Abteilung Immungenetik, Universitat Gottingen, Gottingen, Germany
C. 21 Identification of a rat class I-related gene which is unlinked to the MHC and is homologous to the human MR 1gene L. WALTER and E. GUNTHER A considerable number of genes distantly related to MHC class I genes have been identified, for example CDI, FCRGT, MIC, HFE, MR1. The FCRGT molecules is involved in binding and uptake of maternal immunoglobulin in the neonatal gut, whereas CDI gene products could be shown to present peptides or fatty acids of bacterial origin to cytotoxic T cells. For the MIC, HFE and MRl gene products no antigen-presenting function has been described so far. In order to identify novel class I-related genes in the rat we applied the PCR strategy originally described by HASHIMOTO et al. (1990). The deduced amino acid sequence of one clone shows 76% identity with the human MRl gene product, which is a MHC class I-related gene located on human chromosome Iq25. Thus, the gene isolated appears to be the rat homolog of the human MRl gene. The amino acid sequence of rat MR1 contains the conserved cysteine residues, the beta-2 microglobulin contact sites and the binding sites for the CD8 molecule as in classical class I genes. According to Southern blot data, Mrl is a single copy gene. The sequences of alpha 1 and alpha 2 domains were determined in 4 different inbred rat strains and no nucleotide polymorphism could be detected. Among 8 inbred rat strains only a single restriction fragment length polymorphism (RFLP) could be detected between strains DA and LEW. Therefore, rat Mrl does not appear to be conspicuously polymorphic. This RFLP was used to map Mrl in backcross hybrids. Mrl cosegregates with the Renin (Ren) gene located on rat chromosome (RNO) 13. Thus, also rat Mrl is not linked to the MHC which maps to RN020p12. Mrl is broadly expressed in lymphoid and non-lymphoid tissues, although at a low level. Analysis of lung RNA by RT-PCR revealed the expected and a further amplificate that represents an alternatively spliced Mrl transcript lacking exon 3. We are currently producing recombinant MR1 protein for production of monoclonal antibodies.