Danish Society of Immunology Danish Society for Flow Cytometry

2 Mannan-binding lectin recognizes structures on ischemic reperfused mouse kidneys and is implicated in tissue injury Mette Møller-Kristensen, Weidong Wang, Marieta Ruseva, Steffen Thiel, Søren Nielsen, Kazue Takahashi, Lei Shi, Alan Ezekowitz, Jens Chr. Jensenius, Mihaela Gadjeva. Department of Medical Microbiology and Immunology, Fax: +45 86196128, Tel: +45 89421776, The Water and Salt Research Center, University of Aarhus, Denmark Developmental Immunology, Massachusetts General Hospital, Boston, USA Organ damage as a consequence of ischemia and reperfusion (I/R) is a major clinical problem in an acute renal failure and transplantation. Ligands on surfaces of endothelial cells that are exposed due to the ischemia may be recognized by pattern recognition molecules such as mannan-binding lectin (MBL), inducing complement activation. We examined the contribution of the MBL complement pathway in a bilateral renal I/R model (45 min of ischemia followed by 24 h of reperfusion), using transgenic mice deficient in MBL-A and MBL-C (MBL double knock out, MBL DKO) and in wild type (WT) mice. Kidney damages, which were evaluated by levels of blood urea nitrogen (BUN) and creatinine, showed that MBL DKO mice were significantly protected compared with WT mice. MBL DKO mice reconstituted with recombinant human MBL showed a dose-dependent severity of kidney injury increasing to a comparable level to WT mice. Acute tubular necrosis was evident in WT mice but not in MBL DKO mice after I/R, confirming renal damages in WT mice. MBL ligands in kidneys were observed to be present after I/R but not in sham-operated mice. C3a (desArg) levels in MBL DKO mice were decreased after I/R compared with that in WT mice, indicating less complement activation that was correlated with less C3 deposition in the kidneys of MBL DKO mice. Our data implicate a role of MBL in I/R induced kidney injury. Abstract 3 Hormonal regulation of mannan-binding lectin synthesis in cultured hepatocytes Christian Møller Sørensen , Troels Krarup Hansen , Rudi Steffensen, Jens Chr. Jensenius, Steffen Thiel 13 Hormonal regulation of mannan-binding lectin synthesis in cultured hepatocytes Christian Møller Sørensen , Troels Krarup Hansen , Rudi Steffensen, Jens Chr. Jensenius, Steffen Thiel 1 Danish Society of Immunology Annual meeting 2005 Abstracts Danish Society for Flow Cytometry www.immunologisk-selskab.dk www.flowcytometri.dk [email protected] Page 4 of 15 [email protected] Department of Medical Microbiology and Immunology, Aarhus University, Aarhus, phone 89421776. Immunoendocrine Research Unit, Medical Department M, Aarhus University Hospital, Aarhus, Regional Centre for Blood Transfusion and Clinical Immunology, Aalborg Hospital, Aalborg, Serum MBL levels are mainly genetically determined, but also influenced by growth hormone (GH) and insulin in vivo. Our aim was to study in more details the hormonal regulation of MBL synthesis using an in vitro model with cultured hepatocytes. Cells from the human hepatocyte line HuH-7 were seeded and challenged over a 3 day period with either GH, hydrocortisone, IGF-1, insulin, IL-6, T3 or T4. The concentration of MBL and human albumin in the culture supernatants was measured with immunoassays. mRNA was measured with quantitative real time reverse PCR using β2 microglobulin as household protein. T3 and T4 had the strongest influence on MBL production causing an 8-fold increase (p=0.003), while GH augmented the production of MBL 3-fold (p=0.028) and IL-6 caused a weak but significant dose dependent increase in MBL production. Hydrocortisone, IGF-1 and insulin had no effect on the MBL production. None of the hormones significantly affected production of albumin. MBL mRNA levels were stable during the first 24 hours of T3 stimulation, but increased significantly between 24 and 48 hours reflecting the increased synthesis of MBL. In conclusion, thyroid hormone and GH significantly increase MBL synthesis. Abstract 4 Surfactant protein D is proatherogenic in mice Grith L Sorensen,, Jens Madsen,, Karin Kejling, Ida Tornoe, Ole Nielsen, Paul Townsend, Francis Poulain, Claus H. Nielsen, Kenneth B M Reid, Samuel Hawgood, Erling Falk, Uffe Holmskov 1. Medical Biotechnological Centre, University of Southern Denmark, Odense, Denmark. 2. Department of Pediatrics and Cardiovascular Research Institute, University of California, San Francisco, USA 3. Department of Pathology, Odense University Hospital, Odense, Denmark 4. Department of Biochemistry, Medical Research Council Immunochemistry Unit, University of Oxford, Oxford, United Kingdom 5. Department of Pediatrics, University of California, Davis, USA 6. Copenhagen Blood Transfusion Centre, Copenhagen University Hospital, Denmark 7. Department of Cardiology, Aarhus University Hospital (Skejby), Aarhus N, Denmark4 Surfactant protein D is proatherogenic in mice Grith L Sorensen,, Jens Madsen,, Karin Kejling, Ida Tornoe, Ole Nielsen, Paul Townsend, Francis Poulain, Claus H. Nielsen, Kenneth B M Reid, Samuel Hawgood, Erling Falk, Uffe Holmskov 1. Medical Biotechnological Centre, University of Southern Denmark, Odense, Denmark. 2. Department of Pediatrics and Cardiovascular Research Institute, University of California, San Francisco, USA 3. Department of Pathology, Odense University Hospital, Odense, Denmark 4. Department of Biochemistry, Medical Research Council Immunochemistry Unit, University of Oxford, Oxford, United Kingdom 5. Department of Pediatrics, University of California, Davis, USA 6. Copenhagen Blood Transfusion Centre, Copenhagen University Hospital, Denmark 7. Department of Cardiology, Aarhus University Hospital (Skejby), Aarhus N, Denmark Background Atherogenesis involves arterial inflammation and lipid deposition. We investigated the role of surfactant protein D (SP-D) in disease development because SP-D is an endogenous modulator of inflammation. Methods and Results SP-D synthesis was localized to vascular endothelial cells. The use of a diet-induced model of atherosclerosis showed that the induced atherosclerotic lesion areas were 5.6 fold smaller in the aortic roots in SP-D deficient (Spd-/-) mice compared to wild-type mice. High-density lipoprotein cholesterol (HDL-C) was significantly elevated the Spd-/mice and HDL-C inversely correlated to lesion areas in the study. Treatment of the Spd-/mice, with a recombinant fragment of human SP-D resulted in decreases of HDL-C (21%) as well as total cholesterol (TC) (26%), and low-density lipoprotein (LDL-C) (28%). Plasma tumor necrosis factor-α (TNF-α) was significantly reduced in the Spd-/mice (45% difference). Conclusions SP-D is proatherogenic and retards development of atherosclerosis in the used mouse model. The effect is not likely to be due to the observed disturbances of plasma lipid metabolism. We suggest that altered progression of the inflammatory process underlies the reduced susceptibility to atherosclerosis in SP-D deficient mice. Abstract 5 Assessment of immunogenicity of pharmaceuticals in vitro L. Bærentzen, J. Glamann Cancer & ImmunoBiology, Novo Nordisk A/S, Måløv, Denmark [email protected], Phone 4442 36295 Assessment of immunogenicity of pharmaceuticals in vitro L. Bærentzen, J. Glamann Cancer & ImmunoBiology, Novo Nordisk A/S, Måløv, Denmark [email protected], Phone 4442 3629 Insulin mimetics are non-self synthetic proteins that react specifically with the insulin receptor which lead to a reduction in blood sugar levels. Due to the fact that insulin mimetics are foreign proteins to both mouse and man, it is interesting to utilize these proteins in the assessment of immunogenicity. Based on antibody Elisa assays testing antiserum from immunized Balb/c inbred and NMRI outbred mice, we showed a marked difference in immunogenicity, although the proteins included in the study only differed by one conservative substitution. In the Balb/c mice the antibody titer was high towards one of the proteins and low towards the other, whereas in the NMRI mice it was low at all times. Scrutinizing the T cell response in the immunized Balb/c mice, we performed a proliferation study where mice were Danish Society of Immunology Annual meeting 2005 Abstracts Danish Society for Flow Cytometry www.immunologisk-selskab.dk www.flowcytometri.dk [email protected] Page 5 of 15 [email protected] treated with the proteins in adjuvant every 2 weeks for 4 weeks total. In this immunization setting a difference in immunogenicity between the peptides was not detectable, but interestingly a marked difference was seen when we added 12-mer overlapping peptides to the wells, allowing us to discover the immunogenic epitopes of the proteins. In the Elispot assay we showed a ten-fold increase in sensitivity as compared to the proliferation assay. These methods and in particular the Elispot assay are a valuable supplement to the antibody Elisa in measuring immunogenicity in vitro. Abstract 6 Chaperones are not required for association and dissociation of MHC class I and tapasin Kajsa M. Paulsson, Catarina Betou, Ping Wang, Suling Li. Institute of Cell and Molecular Science, Barts and London School of Medicine, DDRC, Institute for Medical Microbiology and Immunology, Panum Institute, Department of Biological Sciences, Brunel University. Presenting author: Phone (45) 3532 7889, Fax (45) 3532 76966 Chaperones are not required for association and dissociation of MHC class I and tapasin Kajsa M. Paulsson, Catarina Betou, Ping Wang, Suling Li. Institute of Cell and Molecular Science, Barts and London School of Medicine, DDRC, Institute for Medical Microbiology and Immunology, Panum Institute, Department of Biological Sciences, Brunel University. Presenting author: Phone (45) 3532 7889, Fax (45) 3532 7696 Tapasin is important for the quality control of MHC class I assembly. It has been discovered that the chaperones calreticulin and/or ERp57, are also associated with tapasin. However, it is unknown whether the association of tapasin with chaperones and/or association of MHC class I with chaperones are compulsory for the process of quality control. In this study, we have characterised the interaction of chaperone-free tapasin with MHC class I or chaperone-free MHC class I with tapasin. In both conditions, the interaction of tapasin and MHC class I was detected and the interaction could be dissociated with class I binding peptides. Furthermore, the interaction of tapasin and MHC class I could be reconstituted in the absence of any other cellular proteins. Thus, the interaction of tapasin with MHC class I is independent on the association of calreticulin or ERp57 with tapasin, as well as on MHC class I association with these two chaperones. In conclusion the regulation of MHC class I assembly by tapasin relies on the de novo interaction between tapasin and MHC class I, but not on the interaction of chaperones with either tapasin or MHC class I. Abstract 7 Probiotic Bacteria Induce Regulatory Cytokine Production via Dendritic cells Christian Lodberg Hvas*, Jens Kelsen*, Jørgen Agnholt*, Per Höllsberg , Michael Tvede§, and Jens F. Dahlerup* from *Department of Medicine V, Aarhus University Hospital, Århus Sygehus Department of Medical Microbiology and Immunology, University of Aarhus §Department of Clinical Microbiology, Rigshospitalet, University of Copenhagen, Denmark. Tel 8949 3828; fax 8949 2740; e-mail: [email protected] Probiotic Bacteria Induce Regulatory Cytokine Production via Dendritic cells Christian Lodberg Hvas*, Jens Kelsen*, Jørgen Agnholt*, Per Höllsberg , Michael Tvede§, and Jens F. Dahlerup* from *Department of Medicine V, Aarhus University Hospital, Århus Sygehus Department of Medical Microbiology and Immunology, University of Aarhus §Department of Clinical Microbiology, Rigshospitalet, University of Copenhagen, Denmark. Tel 8949 3828; fax 8949 2740; e-mail: [email protected]. Probiotic bacteria, e.g. Lactobacillus spp., may improve inflammatory diseases by altering the signaling between dendritic cells and T cells. Here we present an in vitro model system where gut-derived CD4+ T cells and autologous monocyte-derived dendritic cells are used in co-culture to study the effects of various probiotic strains. T cell cultures were stimulated with autologous bacterial sonicates or strains of Lactobacillus spp., with and without addition of autologous dendritic cells. Cytokine levels (IFN-γ and IL-10) and phenotype (CD4, CD25, CD69) were measured on day 4. Lactobacillus spp. induced higher IL-10 production than autologous bacteria, and dendritic cells induced an increased production of all cytokines. However, the increase of IFN-γ was more pronounced in wells with autologous bacteria than in wells with Lactobacillus spp. The addition of dendritic cells upregulated CD25 expression without simultaneous upregulation of CD69. The upregulation was pronounced after stimation with Lactobacillus rhamnosus GG compared with autologous bacteria and other Lactobacilli. Future studies will address whether such IL-10 producing T cells with a CD25+ phenotype represent a differentiation into competent regulatory T cells. In a clinical context, such cells might be used for treatment of inflammatory diseases. Abstract 8 Induction of immune response using dendritic cells transfected with selected tumor antigens associated with human breast carcinomas Özcan Met, Jens Eriksen, Mogens H. Claesson, Inge Marie Svane Department of Oncology, Herlev University Hospital, Herlev, Denmark Department of Medical Anatomy, University of Copenhagen, the Panum Institute, Copenhagen, Denmark8 Induction of immune response using dendritic cells transfected with selected tumor antigens associated with human breast carcinomas Özcan Met, Jens Eriksen, Mogens H. Claesson, Inge Marie Svane Department of Oncology, Herlev University Hospital, Herlev, Denmark Department of Medical Anatomy, University of Copenhagen, the Panum Institute, Copenhagen, Denmark The development of protocols for the ex vivo generation of DC has provided a rationale to design and develop DCbased vaccination studies for the treatment of infectious and malignant diseases. The efficacy of antigen loading and delivery into DC is pivotal for the optimal induction of T-cell-mediated immune responses. The use of DC transfected Danish Society of Immunology Annual meeting 2005 Abstracts Danish Society for Flow Cytometry www.immunologisk-selskab.dk www.flowcytometri.dk [email protected] Page 6 of 15 [email protected] with RNA encoding tumor antigen offers the prospect of antigen specific immunization without requiring prior knowledge of the immunogenic epitope or restricting allele, since epitopes from the translated protein are processed by the endogenous antigen-presentation machinery. We have previously established an anti-tumor vaccine using autologous DC pulsed with p53 peptides for treatment of metastatic breast cancer, and the object now is to improve this treatment by transfecting DCs with mRNA encoding selected tumor antigens. We have developed a non-viral transfection method protocol that uses in vitro synthesized mRNA and square-wave electroporation for transient expression of antigens in DC. Using the mRNA encoding the green fluorescence protein (EGFP), the square-wave electroporation data show high yield and viability (>90 %), and high transfection efficiency (>90 %) of DC. The flow cytometry analysis also demonstrated that the expression of EGFP peaked 48-72 h after electroporation in DC transfected either before or after maturation. However, higher levels of expression and viability were obtained when DC were electroporated after maturation. Furthermore, cryopreservation of DC before and after electroporation did not alter the level of EGFP expression. Taken together, these preliminary results show that the use of square-wave electroporation as a transfection method seems to be a useful and effective technique to charge DC with tumor antigens. Next, in vitro analysis of anti-tumor T cell-reactivity from patients operated for primary breast cancer will be initiated using square-wave electroporation for transfection of DC with the tumor antigens p53, survivin and hTERT. Abstract 9 Dendritic cell based vaccination in combination with VEGF receptor 2 and CTLA-4 blockade. A. E. Pedersen*, S. Buus¤, and M. H. Claesson*. *Laboratory of Cellular Immunology, Department of Medical Anatomy A and ¤Institute of Medical Microbiology and Immunology, The Panum Institute, University of Copenhagen, Denmark. Telephone: +45 35327397; Fax: +45 35327269; email:[email protected] Dendritic cell based vaccination in combination with VEGF receptor 2 and CTLA-4 blockade. A. E. Pedersen*, S. Buus¤, and M. H. Claesson*. *Laboratory of Cellular Immunology, Department of Medical Anatomy A and ¤Institute of Medical Microbiology and Immunology, The Panum Institute, University of Copenhagen, Denmark. Telephone: +45 35327397; Fax: +45 35327269; email:[email protected] We investigated the anti CT26 tumour effect of dendritic cell based vaccination with the MuLV gp70 envelope proteinderived peptides AH1 and p320-333. Vaccination lead to generation of AH1 specific cytotoxic lymphocytes (CTL) and some decrease in tumour growth of simultaneously inoculated CT26 cells. After combination with an antibody against VEGF receptor 2 (DC101), a significant increase in survival of the tumour cell recipients was observed. Also, monotherapy with an antibody against CTLA-4 (9H10), lead to nearly 100% survival of tumour cell recipients. However, effective treatment of mice with already established tumours was only obtained after combination of vaccination, DC101 and 9H10 treatment in which setting 80% of the mice rejected their tumours. Abstract 10 Immunomodulatory dendritic cells require autologous serum to circumvent non-specific immunosuppressive activity in vivo Claus Haase, Mette Ejrnaes, Amy E. Juedes, Tom Wolfe, Helle Markholst and Matthias G. von Herrath Hagedorn Research Institute, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121, USA10 Immunomodulatory dendritic cells require autologous serum to circumvent non-specific immunosuppressive activity in vivo Claus Haase, Mette Ejrnaes, Amy E. Juedes, Tom Wolfe, Helle Markholst and Matthias G. von Herrath Hagedorn Research Institute, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121, USA In immunotherapy, dendritic cells (DCs) can be used as powerful antigen-presenting cells to enhance or suppress antigen-specific immunity upon in vivo transfer in mice or humans. However, to generate sufficient numbers of DCs most protocols include an ex vivo culture step, wherein the cells are exposed to heterologous serum and/or antigenic stimuli. In mouse models of virus infection and virus-induced autoimmunity, we tested how heterologous serum affects the immunomodulatory capacity of immature DCs generated in the presence of IL-10, comparing FBSor normal mouse serum (NMS)-supplemented DC-cultures. We show that immature DCs generated in FBS-supplemented cultures induced a non-antigen-specific systemic immune deviation characterized by reduction of virus-specific T cells, delayed viral clearance and enhanced systemic production of IL-4 and IL-10 to FBS-derived antigens, including BSA. By contrast, DCs generated in NMS-supplemented cultures modulated immunity and autoimmunity in an antigen-specific fashion, did not induce systemic IL-4 or IL-10 production and inhibited generation of virus-specific T-cells or autoimmunity only if pulsed with a viral antigen. These data underscore the importance of using autologous serumderived immature DCs in preclinical animal studies to accurately assess their immunomodulatory potential in future human therapeutic settings, where application of FBS will not be feasible. Abstract 11 Delayed Expansion of Dysfunctional Virus Specific CD8+ T-Cells Following Systemic Recombinant Adenovirus Infection in mice11 Delayed Expansion of Dysfunctional Virus Specific CD8+ T-Cells Following Systemic Recombinant Adenovirus Infection in mice Danish Society of Immunology Annual meeting 2005 Abstracts Danish Society for Flow Cytometry www.immunologisk-selskab.dk www.flowcytometri.dk [email protected] Page 7 of 15 [email protected] Peter J. Holst, Cathrine Orskov, Allan R. Thomsen, Jan P. Christensen 1: Institute of Medical Microbiology and Immunology, University of Copenhagen, The Panum Institute bldg.: 22.5, Blegdamsvej 3C, DK-2200 2: Institute of Medical Anatomy, University of Copenhagen, The Panum Institute bldg.: 18.2, Blegdamsvej 3C, DK2200 Infection with hepatotropic viruses are characterised by prolonged infection, delayed immune activation, immunopathology, and for some viruses the tendency to cause persistent infection. As systemically administered adenovirus has pronounced hepatotropism we compared the immune response and viral clearance following i.v. and peripheral infection with recombinant adenovirus. Our results demonstrate a marked qualitative and kinetic impairment of the immune response following i.v. administered adenovirus, remarkably similar to what can be seen in human viral hepatitis. These effects are demonstrated to be route and not virus specific. By infection of T-cell receptor transgenic mice targeting an adenovirus encoded epitope we demonstrate that naïve T-cells initially become activated, then dysfunctional after systemic infection with this hepatotropic model virus. Thus, systemic adenoviral infection recapitulates key features of human viral hepatitis and allows for dissection of mechanisms responsible for the induction of immunity and immune dysfunction following infection. Abstract 12 Opposing effects of CXCR3 and CCR5 deficiency on CD8 T cell response to intracerebral infection with LCMV *Carina de Lemos, Jeanette Erbo Christensen, Anneline Nansen, Torben Moos, Bao Lu, Craig Gerard, Jan Pravsgaard Christensen and *Allan Randrup Thomsen *Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark Phone: +45 35327871. Fax: +45 35327891.12 Opposing effects of CXCR3 and CCR5 deficiency on CD8 T cell response to intracerebral infection with LCMV *Carina de Lemos, Jeanette Erbo Christensen, Anneline Nansen, Torben Moos, Bao Lu, Craig Gerard, Jan Pravsgaard Christensen and *Allan Randrup Thomsen *Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark Phone: +45 35327871. Fax: +45 35327891. To study the interplay of the chemokine receptors CXCR3 and CCR5 in regulating virus-induced CD8 T-cell mediated inflammation in the brain, CXCR3/CCR5-deficient mice were generated and infected intracerebrally (i.c.) with lymphocytic choriomeningitis virus. Since these chemokine receptors were expressed by overlapping subsets of activated CD8 T cells, it was expected that absence of both receptors would additively impair effector T cell invasion, and therefore protect mice against the fatal meningitis. Contrary to expectations double deficient mice were more susceptible to i.c. infection than CXCR3-deficient mice. Analysis of effector T cell generation revealed an accelerated antiviral CD8 T cell response in CXCR3/CCR5-deficient mice. Furthermore, while the accumulation of CD8 T cells into the neural parenchyma was delayed in both CXCR3and CXCR3/CCR5-deficient mice the number of CD8 T cells is statistically significant higher in the parenchyma of the CXCR3/CCR5-deficient mice around the time were most mice succumbed the infection. Taken together these results indicate that while CXCR3 play an important role in controlling CNS invasion, other receptors but not CCR5 also contribute significantly. Furthermore, CCR5 primarily seems to play a role as a negative regulator of the antiviral T cell response. Abstract 13 Impaired virus control and severe CD8 T cell-mediated immunopathology in chimeric mice deficient in IFN-γ receptor expression on both parenchymal and hematopoietic cells Pernille Henrichsen, Christina Bartholdy, Jan Pravsgaard Christensen, and Allan Randrup Thomsen Phone: +45 35327878. Fax: +45 35327891. E-mail: [email protected] Impaired virus control and severe CD8 T cell-mediated immunopathology in chimeric mice deficient in IFN-γ receptor expression on both parenchymal and hematopoietic cells Pernille Henrichsen, Christina Bartholdy, Jan Pravsgaard Christensen, and Allan Randrup Thomsen Phone: +45 35327878. Fax: +45 35327891. E-mail: [email protected]. Bone marrow chimeras were used to determine the cellular target(s) for the antiviral activity of interferon-γ. By transfusing such mice with high numbers of naive virus-specific CD8 T cells a system was created, in which the majority of virus-specific CD8 T cells would be capable of responding to interferon-γ, but expression of the relevant receptor on non-T cells could be experimentally controlled. Only when the IFN-γ receptor is absent on both radioresistent parenchymal and bone marrow-derived cells, will chimeric mice challenged with a highly invasive, noncytolytic virus completely lack the ability to control the infection and develop severe wasting disease. Further, the study shows that IFN-γ receptor expression on parenchymal cells in the viscera is more important for virus control than IFN-γ receptor expression on bone marrow-derived cells. Danish Society of Immunology Annual meeting 2005 Abstracts Danish Society for Flow Cytometry www.immunologisk-selskab.dk www.flowcytometri.dk [email protected] Page 8 of 15 [email protected] Abstract 14 MEK kinase 1 is a negative regulator of virus specific CD8 T cells. Tord Labuda, Jan Pravsgaard Christensen, Barbara Bonnesen, Michael Karin, Allan Randrup Thomsen and Niels Ødum. Department of Medical Microbiology and Immunology, University of Copenhagen 2200 Copenhagen N, Denmark. Phone +45 35327754 fax +45 35327876 Mek kinase 1(MEKK1) is a potent JNK activating kinase, a regulator of T helper cell differentiation, cytokine production and proliferation in vitro. Using mice deficient for MEKK1 kinase activity (Mekk1) exclusively in their hematopoietic system, we show, that MEKK1 has a neagative regulatory role in the generation of a virus-specific immune response. Mekk1 mice challanged with vesicular stomatitis virus (VSV) showed a 4-fold increase in spleenic CD8 T cell numbers. In contrast, spleenic T cells in infected wt mice where only marginally increased. The CD8 T cell expansion in Mekk1 mice following VSV infection is virus-specific and the frequency of virus-specific T cells is significantly higher (> 3-fold) in Mekk1 as compared to wt animals. Moreover, the hyper expansion of T cells seen in Mekk1 mice after VSV infection is a result of increased proliferation, since a significantly higher percentage of virus-specific Mekk1 CD8 T cells incorporated BrdU as compared to virus-specific wt CD8 T cells. In contrast, similar levels of apoptosis were detected in Mekk1 and wt T cells following VSV infection. These results strongly suggest that MEKK1 plays a negative regulatory role in the expansion of virus specific CD8 T cells in vivo. Abstract 15 Response of allergic bakers to a food challenge (DBPCFC) with flour HJ Hoffmann , T Skjold , M Raithel , K Adolf , O Hilberg 1 and R Dahl . 1 Dept Pulmonary Medicine, AUH, Aarhus, DENMARK and 2 Dept of Medicine I, Uni Erlangen-Nürnberg, Erlangen, GERMANY.15 Response of allergic bakers to a food challenge (DBPCFC) with flour HJ Hoffmann , T Skjold , M Raithel , K Adolf , O Hilberg 1 and R Dahl . 1 Dept Pulmonary Medicine, AUH, Aarhus, DENMARK and 2 Dept of Medicine I, Uni Erlangen-Nürnberg, Erlangen, GERMANY. A number of occupational respiratory allergens are food related. Little is known about the responses these algs elicit in sensibilised persons that ingest them. 9 allergic exbakers were exposed in a double blind placebo controlled food challenge with relevant allergen. Significant increases in urinary methyl histamine (MH) and serum tryptase and decrease in blood basophils and nasal volume after ingestion of allergen compared with placebo suggest an allergic response to ingested allergen. There was no change in FEV1. The number of blood DC2 decreased after exposure to allergen (p=0,011) and placebo (p=0,036). Dendritic cell HLA DR was reduced after both exposures (p<0,001). Expression of CXCR4 on these cells was reduced after allergen exposure (p=0,033). CD4+ memory T cell expression of CD25 was upregulated after placebo exposure (p=0,021) but reduced after allergen. The reduction of CD25 expression after allergen compared to placebo was significant (p=0,024). CD152 was downregulated on these cells after allergen exposure (p=0,039), less so after placebo. Allergic bakers respond to relevant ingested allergen. The allergen challenge reduced plasmacytoid dendritic cell nrs and memory T cell expression of CD25 and CD152.