Ph.D thesis Functional analysis of the effet of T cells and antigen presenting cells on humoral immune response of FcRn transgenic animals Anita Farkas Supervisor: Imre Kacskovics DVM, Ph.D János Matkó Ph.D, DSc. Ph.D School of Biology, Eötvös Loránd University Ph.D School Leader: Prof. Anna Erdei, D.Sc Immunology Program Program leader: Prof. Anna Erdei, D.Sc Department of Immunology, Institute of Biology Eötvös Loránd University, Budapest, Hungary 2013
Introduction The role of the neonatal Fc receptor (FcRn) is already known in the IgG homeostasis. Its basic functions are involved in the maternal IgG transport and in preventing IgG and albumin destruction in the endothelial cells, increasing thus the halflife of these two important plasma proteins. Lately it was proposed that the FcRn may have an important tole in the phagocytosis of immunecomplexes (IC) by antigen presenting cells (APC) and it enhances the antigen presentation by directing the antigen to the lysosomal pathway. Kacskovics et al. cloned and characterized the bovine FcRn (bfcrn) and for its functional analysis they created the bfcrn overexpressing transgenic mouse by BAC transgenesis. The transgene is expressed tissue- specifically in FcRn expressing tissues (endothelial cells, liver, kidney, spleen, lymph nodes, uterus, placenta, APCs). According to the results of our work group the breakdown of IgG is decreased due to the overexpression of bfcrn, the humoral immune response is elevated, shown by the increased IgG and IgM titers. In the spleen the number of the antigen specific cells is increased and in addition the diversity of the antibody producing clones also increased. These transgenic mice show an effective 1
immune response even to weakly immonogenic antigens. The effective humoral immune responsiveness of the FcRn overexpression and all these properties together may be highly beneficial also in the therapeutic antibody production of animals (including rabbits). Aims of the study Checking expression of rabbit FcRn (rfcrn) in immune cells Rabbits may be efficient sources in producing monoclonal and polyclonal antibodies, therefore creating a rabbit line, overexpressing FcRn would also be of central importance in pharmaceutical industry. Our aim was to check whether rfcrn is expressed in isolated peritoneal rabbit macrophages and in the blood neutrophil granulocytes. bfcrn expession in mouse immune cells The expression level of bfcrn on mrna level from the immune cells (T, B, Mf, DC, Gr) sorted (by FACS) from the spleen and peritoneal macrophages of bfcrn tg mouse was the subject of our analysis. The effectiveness of phagocytosis in peritoneal macrophages and bone marrow derived dendritic cells The comparative anaylsis of phagocytotic function of the antigen presenting cells from bfcrn tg and wild type mice was 2
the subject of our investigations, using ovalbumin containing (OVA-IgG) immunecomplex. We were interested whether FcRn overexpression is beneficial for the APCs in the antigen uptake mechanism. We wanted to test whether the expression levels of membrane proteins involved directly in antigen presentation (MHC-II, CD80 and CD86 costimulatory molecules, Fc receptors) are altered in peritoneal macrophages and bone marrow derived dendritic cells of bfcrn tg animals. We studied the presence of Fcγ receptors on the cell surface taking part in antigen uptake, the expression of costimulator molecules (CD80, CD86) needed for effective T cell co-activation presentation and the expression of cell surface MHC II in macrophages and dendritic cells from wild type and bfcrn tg mice after IC treatment. We wanted to test whether bfcrn overexpression influences the efficacy of T cells in proliferation Our aim was to compare the T cell proliferation capacity of APCs from wild-type and bfcrn tg mice with IC-loaded peritoneal macrophages and dendritic cells. Methods: PCR Reverse- transcription PCR Flow-cytometry (FCM) 3
Fluorescent cell sorting (FACS) Confocal laser-scanning microscopy Bone marrow stem cell differentiation into dendritic cells Isolation of peritoneal macrophages T cell proliferation assay by detecting incorporation of radioactive thymidine Results: We could detect rfcrn expression from peritoneal macrophages and neutrophil granulocytes isolated from blood of rabbits. We found significant bfcrn expression in macrophages, dendritic cells, neutrophil granulocytes isolated from spleen bfcrn tg mice. The FcRn expression of B and T cells still remained controversial, to be elucidated. We could show elevated phagocytotic function from peritoneal macrophages and dendritic cells, if we added the antigen in the form of an immunocomplex. We could show that the dendritic cells from the tg mice were more effective in triggering antigen specific T cell proliferation after the uptake of the immunocomplex, than DCs of the control animals. Using soluble antigen, no alterations could be shown in the antigen specific T cell 4
response, which strongly supports the contribution of the FcRn. Antigen presentation by macrophages was only slightly but not significantly more effective in the bfcrn overexpressing mice. Cell surface proteins involved in antigen presentation, such as the costimulatory molecules CD80 and CD86, the MHCII and Fcγ receptors on dendritic cells did not show altered expression in bfcrn overexpressing mice. Thus, we can say that it is not their altered cell surface expression which is responsible for the elevated antigen-specific T cell activation. Discussion: We could detect FcRn expression in imune cells of rabbit and mouse, alike. In these cells the FcRn is mainly responsible for regulation of immune processes, classically through its IgGprotecting mechanism. In addition the FcRn overexpression efficiently increses the phagocytosis efficiency of peritoneal macrophages and bone marrow derived dendritic cells in the bfcrn tg mice, if the antigen appears in the form of an immunocomplex. We could not observe increased expression of the cell surface Fcγ receptor. The antigen presentation 5
efficacy of peritoneal macrophages did not increase significantly, either. However, the increased T cell activation by APCs through the enhanced antigen-presentation is not due to enhanced MHC-II, CD80 or CD86 expression, but to the presence of overexpressed FcRn. Publications related to the Ph.D thesis Vegh A, Farkas A, Kovesdi D, Papp K, Cervenak J, Schneider Z, Bender B, Hiripi L,Laszlo G, Prechl J, Matko J, Kacskovics I (2012) FcRn Overexpression in Transgenic Mice Results in Augmented APC Activity and Robust Immune Response with Increased Diversity of Induced Antibodies. PLoS One 7 (4):e36286.doi:10.1371/ journal.pone.0036286 (IF: 4,411) Contributed equally. Catunda Lemos AP, Cervenak J, Bender B, Hoffmann OI, Baranyi M, Kerekes A, Farkas A, Bosze Z, Hiripi L, Kacskovics I. (2012) Characterization of the rabbit neonatal Fc receptor (FcRn) and analyzing the immunophenotype of the transgenic rabbits that overexpresses FcRn. PLoS One. 2012;7(1):e28869. doi: 10.1371/journal.pone.0028869. (IF: 4,411) 6
Other publications Izsepi E, Balogh A, Farkas A, Molnar A, Solymos E, Toth EA, Csepanyi-Komi R, Matko J. (2012) The AC8 IgG3 monoclonal anti-cholesterol antibody modulates uptake and presentation of antigens for T cell activation. Immunol Lett.2012 Mar 30;143(1):106-15. doi: 10.1016/j.imlet. 2012.01.009. (IF: 2,511) Farkas A, Horváth É, Paul M, Varankáné László K, Fekete M, (2003) A B12-vitaminhiány korai diagnosztizálásának laboratóriumi lehetőségei, Klinikai és kísérletes laboratóriumi medicina 30:(3) pp. 106-118. (2003) Conference abstracts Anita Farkas, Dorottya Kövesdi, Glória László, János Matkó, Imre Kacskovics: Enhanced IgG-immune complex phagocytosis and antigen presentation inmacrophages and dendritic cells isolated from tg mice that overexpress FcRn Immune-related Pathologies: Understanding Leukocyte Signaling and Emerging therapies, Visegrad, Hungary, 2011 Farkas Anita, Kövesdi Dorottya, Magna Melinda, Bender Balázs, László Glória, Matkó János, Kacskovics Imre: Az FcRn fokozott kifejeződése növeli az immunkomplexek fagocitózisának hatékonyságát makrofágokban és neutrofil granulocitákban, The Youth Conference of the Hungarian Immunological Society, 2009, Harkány Farkas Anita, Kövesdi Dorottya, László Glória, Matkó János, Kacskovics Imre: Az FcRn fokozott kifejeződése dendritikus 7
sejteken és makrofágokban növeli az immunkomplexek fagocitózisának hatékonyságát és az antigén prezentációt, The 40 th Conference of the Hungarian Immunological Society, 2011, Kecskemét Cervenak Judit, Schneider Zita, Vegh Attila, Baranyi Mária, Farkas Anita, Bender Balázs, Papp Krisztián, Prechl József, Bősze Zsuzsanna, Erdei Anna és Kacskovics Imre: Az FcRn transzgénikus állatok kimagasló immunválasza jelentősen fokozza a mono- és poliklonális ellenanyagok előállításának hatékonyságát, The 40 th Conference of the Hungarian Immunological Society, 2011, Kecskemét Lemos Ana Paula Catunda, Cervenak Judit, Bender Balázs, Hoffmann Ivett Orsolya, Baranyi Mária, Kerekes Andrea, Farkas Anita, Bõsze Zsuzsanna, Hiripi László,Kacskovics Imre: A nyúl FcRn karakterizálása; az FcRn transzgénikus nyulak immunfenotípusának jellemzése, The 41 th Conference of the Hungarian Immunological Society, 2012, Debrecen 8