Expert of Antibody Production and Genetic Modification

Scientific Publications

Cell-specific STORM superresolution imaging reveals nanoscale organization of cannabinoid signaling

Barna Dudok, László Barna, Marco Ledri, Szilárd I. Szabó, Eszter Szabadits, Balázs Pintér, Stephen G. Woodhams, Christopher M. Henstridge, Gyula Y. Balla, Rita Nyilas, Csaba Varga, Sang-Hun Lee, Máté Matolcsi, Judit Cervenak, Imre Kacskovics, Masahiko Watanabe, Claudia Sagheddu, Miriam Melis, Marco Pistis, Ivan Soltesz, and István Katona



A major challenge in neuroscience is to determine the nanoscale position and quantity of signaling molecules in a cell-type-, and subcellular compartment-specific manner. We therefore developed a novel approach combining cell-specific physiological and anatomical characterization with superresolution imaging, and studied the molecular and structural parameters shaping the physiological properties of synaptic endocannabinoid signaling in the mouse hippocampus. We found that axon terminals of perisomatically-projecting GABAergic interneurons possess increased CB1 receptor number, active-zone complexity, and receptor/effector ratio compared to dendritically-projecting interneurons, in agreement with higher efficiency of cannabinoid signaling at somatic versus dendritic synapses. Furthermore, chronic Δ9-tetrahydrocannabinol administration, which reduces cannabinoid efficacy on GABA release, evoked dramatic CB1- downregulation in a dose-dependent manner. Full receptor recovery required several weeks after  cessation of Δ9-tetrahydrocannabinol treatment. These findings demonstrate that cell-type-specific nanoscale analysis of endogenous protein distribution is possible in brain circuits, and identify novel molecular properties controlling endocannabinoid signaling and cannabis-induced cognitive dysfunction.

ORAI2 modulates  store-operated calcium entry and T cell-mediated immunity

Martin Vaeth, Jun Yang, Megumi Yamashita, Isabelle Zee, Miriam Eckstein, Camille Knosp, Ulrike Kaufmann, Peter Karoly Jani, Rodrigo S. Lacruz, Veit Flockerzi, Imre Kacskovics, Murali Prakriya & Stefan Feske



Store-operated  Ca2 þ entry (SOCE) through Ca2 þ release-activated Ca2 þ (CRAC) channels is critical for lymphocyte function and  immune  responses.  CRAC channels  are  hexamers of ORAI proteins  that  form  the  channel  pore,  but  the  contributions  of individual ORAI homologues to CRAC channel function are not well understood. Here we show that deletion of Orai1 reduces, whereas deletion of Orai2 increases, SOCE in mouse T cells. These distinct effects  are  due  to the  ability of ORAI2 to form heteromeric  channels  with ORAI1 and to attenuate CRAC channel function. The combined deletion of Orai1 and Orai2 abolishes SOCE and strongly impairs T cell function. In vivo, Orai1/Orai2  double-deficient mice have impaired T cell-dependent  antiviral immune responses,  and are  protected  from T cell-mediated  auto- immunity and alloimmunity in models  of colitis and graft-versus-host disease.  Our study demonstrates that  ORAI1  and  ORAI2 form heteromeric  CRAC channels,  in which ORAI2 fine-tunes  the magnitude of SOCE to modulate immune responses.

Overexpression of bovine Fcrn in mice enhances T-dependent immune responses by amplifying T helper cell frequency and germinal center enlargement in the spleen

Zita Schneider† ‡ , Péter Károly Jani ‡, Bence Szikora , Attila Végh † , Dorottya Kövesdi, Attila Iliás , Judit Cervenak , Péter Balogh, István Kurucz †  and Imre Kacskovics



The neonatal Fc receptor (FcRn) plays key roles in IgG and albumin homeostasis, maternal IgG transport, and antigen presentation of IgG-opsonized antigens. Previously, we reported that transgenic  (Tg) mice that overexpress the bovine FcRn (bFcRn) have augmented T-dependent humoral immune response with increased IgG protection, higher level of antigen-specific antibodies, greater number of antigen-specific B cells, and effective immune response even against weakly immunogenic epitopes. In the current study, we analyzed the localization of the bFcRn in secondary lymphoid organs, and focused to demonstrate the in vivo impact of its overexpression in the spleen on the course of antibody production. bFcRn was highly expressed by red pulp macrophages and marginal zone macrophages in the spleen and by subcapsular sinus macrophages and macrophage-like cells in the interfollicular areas in the lymph node cortex. We also demonstrated that splenic dendritic cells of Tg mice express bFcRn and intraperitoneal immunization of these mice with T-dependent antigens led to more than threefold increase in the number of antigen-specific activated T helper cells with increased size and numbers of germinal centers compared to wild-type controls. bFcRn expression in splenic B cells was also detected and that may also contribute to the enhanced B cell activation. Finally, we demonstrated that these Tg mice developed efficient immune response against very low dose of antigen, reflecting another important practical benefit of these Tg mice.


FcRn Overexpression in Transgenic Mice Results in Augmented APC Activity and Robust Immune Response with  Increased  Diversity  of  Induced Antibodies

Attila Végh, Anita Farkas, Dorottya Kövesdi, Krisztián Papp, Judit Cervenak, Zita Schneider, Balázs Bender, László Hiripi, Glória László, József Prechl, János Matkó, Imre Kacskovics



Our previous studies have shown that overexpression of bovine FcRn (bFcRn) in transgenic (Tg) mice leads to an increase in the humoral immune response, characterized by larger numbers of Ag-specific B cells and other immune cells in secondary lymphoid organs and higher levels of circulating Ag-specific antibodies (Abs). To gain additional insights into the mechanisms underlying this increase in humoral immune response, we further characterized the bFcRn Tg mice. Our Western blot analysis showed strong expression of the bFcRn transgene in peritoneal macrophages and bone marrow derived dendritic cells; and a quantitative PCR analysis demonstrated that the expression ratios of the bFcRn to mFcRn were 2.6- and 10-fold in these cells, respectively. We also found that overexpression of bFcRn enhances the phagocytosis of Ag- IgG immune complexes (ICs) by both macrophages and dendritic cells and significantly improves Ag presentation by dendritic cells. Finally, we determined that immunized bFcRn mice produce a much greater diversity of Ag-specific IgM, whereas only the levels, but not the diversity, of IgG is increased by overexpression of bFcRn. We suggest that the increase in diversity of IgG in Tg mice is prevented by a selective bias towards immunodominant epitopes of ovalbumin, which was used in this study as a model antigen. These results are also in line with our previous reports describing a substantial increase in the levels of Ag-specific IgG in FcRn Tg mice immunized with Ags that are weakly immunogenic and, therefore, not affected by immunodominance.


Accelerating antibody discovery using transgenic animals overexpressing the neonatal Fc receptor as a result of augmented humoral immunity.

Cervenak J, Kurrle R, Kacskovics I.



In recent years, there has been an increasing demand for the development of faster and more efficient technologies for the generation of monoclonal antibodies against challenging targets that are weakly immunogenic or available only in limited amounts. Typical classes of such targets are cell surface antigens such as G-protein related receptors (GPCRs) or ion channels. We have developed transgenic (Tg) mice and rabbits that overexpress the neonatal Fc receptor (FcRn), resulting in an augmented humoral immune response even if challenging antigens are used for immunization. The impressively enhanced FcRn-mediated immune reactions are characterized by improved IgG protection and enhanced antigen presentation leading to greater number of antigen-specific T-helper and B-cell activation in lymphoid organs. Notably, these animals do not show any sign of autoimmunity and can be efficiently bred. FcRn overexpression thus leads to a number of practical benefits for improved generation of monoclonal and polyclonal antibodies against multiple antigens, including weakly immunogenic epitopes or tiny amounts of proteins. This review summarizes our current understanding about the mechanisms by which FcRn overexpression leads to such a significantly enhanced humoral immune response.

NFκB induces overexpression of bovine FcRn A novel mechanism that further contributes to the enhanced immune response in genetically modified animals carrying extra copies of FcRn

Judit Cervenak†, Márton Doleschall†, Balázs Bender, Balázs Mayer, Zita Schneider, Zoltán Doleschall, Yaofeng Zhao, Zsuzsanna Bősze, Lennart Hammarström, Wolfgang Oster, and Imre Kacskovics



Among the many functions of the neonatal  Fc receptor (FcRn) for IgG, it binds to IgG-opsonized antigen complexes and propagates their traffic into lysosomes where antigen  processing  occurs. We previously reported that  transgenic  (Tg) mice and rabbits that carry multiple copies and overexpress FcRn have augmented humoral immune responses. Nuclear factor-kappa B (NFκB) is a critical molecule in the signaling cascade in the immune response. NFκB induces human FcRn expression and our previous in silico analysis suggested NFκB binding sites in the promoter region of the bovine (b) FcRn α-chain gene (FCGRT). Here, we report the identification of three NFκB transcription factor binding sites in the promoter region  of this gene  using  luciferase reporter  gene  technology, electromobility  shift assay and  supershift  analysis. Stimulation  of primary bovine  endothelial  cells with the  Toll-like receptor-4  ligand  lipopolysaccharide  (LPS), which mediates its effect via NFκB, resulted in rapid upregulation of the bFcRn expression and a control gene, bovine E-selectin. This rapid bFcRn gene induction was also observed in the spleen of bFcRn Tg mice treated with intraperitoneally injected LPS, analyzed by northern blot analysis. Finally, NFκB-mediated bFcRn upregulation was confirmed at the protein level in macrophages isolated from the bFcRn Tg mice using flow cytometry with a newly developed FcRn specific monoclonal antibody  that does not cross-react with the mouse FcRn. We conclude  that NFκB regulates  bFcRn expression and thus optimizes  its functions, e.g., in the  professional  antigen  presenting cells, and  contributes to the  much  augmented humoral immune response in the bFcRn Tg mice.


Transgenic  Rabbits  That Overexpress the  Neonatal Fc Receptor  (FcRn) Generate Higher  Quantities and Improved  Qualities  of Anti-Thymocyte  Globulin (ATG)

ria Baranyi, Judit Cervenak, Balázs Bender, Imre  Kacskovics



Immune suppression  with rabbit  anti-thymocyte globulin (rATG) is a well-established  therapeutic concept  for preventing host rejection of transplanted organs and graft versus host disease. Increasing the efficiency of rATG production by reducing the  number  of animals would  be  highly beneficial to lower cost and  to improve  quality standards.  We have developed transgenic  (Tg) mice and rabbits that overexpress the neonatal  Fc receptor  (FcRn) and have shown an augmented humoral immune  response  in these  animals. To test  whether  our FcRn Tg rabbits  produced rATG more efficiently, we immunized them  and  their New Zealand  White controls  with live Jurkat cells. By day 21 after immunization,  Tg animals produced significantly, 1.5 times higher amount  of total IgG compared  to their wt littermates. Also, the binding efficiency of Tg sera to Jurkat cells and their complement-mediated cytotoxicity was significantly higher. The purified Tg IgG preparation contained 2.6 the  amount  of Jurkat specific IgG as the  wt preparation analyzed by complement-mediated lysis, suggesting greater antigen-specific  B cell activation  in the  Tg rabbits. To test  this hypothesis,  immunization  with ovalbumin  and human  a1- antitrypsin  was performed,  resulting  in significantly greater  numbers  of antigen-specific  B-cells in the  FcRn Tg rabbits  as compared  with wt controls. The shift towards significantly larger populations of antigen-specific B cells relative to the non- specific B cell pool is further corroborated by our previous findings in FcRn Tg mice. Consequently, our FcRn Tg rabbits have the potential to offer substantial qualitative and quantitative  improvements for the production of rATG and other polyclonal or monoclonal  antibodies.


Characterization of the  Rabbit  Neonatal Fc Receptor (FcRn) and  Analyzing the  Immunophenotype of the Transgenic  Rabbits  That Overexpresses FcRn

Ana  Paula Catunda Lemos, Judit Cervenak, Balázs Bender, Orsolya Ivett Hoffmann, Mária  Baranyi, Andrea Kerekes, Anita Farkas, Zsuzsanna Bősze, László  Hiripi, Imre  Kacskovics



The neonatal  Fc receptor  (FcRn) regulates  IgG and albumin homeostasis,  mediates  maternal  IgG transport,  takes an active role in phagocytosis,  and  delivers antigen  for presentation. We have  previously shown  that  overexpression  of FcRn in transgenic   mice  significantly improves  the  humoral  immune  response.   Because  rabbits  are  an  important   source  of polyclonal and  monoclonal  antibodies,  adaptation of our  FcRn overexpression  technology  in this  species  would  bring significant advantages.  We cloned  the  full length  cDNA of the  rabbit  FcRn alpha-chain  and found  that  it is similar to its orthologous analyzed so far. The rabbit FcRn - IgG contact  residues are highly conserved, and based  on this we predicted pH dependent interaction, which we confirmed by analyzing the pH dependent binding of FcRn to rabbit IgG using yolk sac lysates  of  rabbit   fetuses  by  Western  blot.  Using  immunohistochemistry,  we  detected  strong   FcRn staining  in  the endodermal cells of the  rabbit  yolk sac membrane, while the  placental  trophoblast cells and  amnion  showed  no  FcRn staining.  Then, using  BAC  transgenesis   we  generated transgenic  rabbits  carrying  and  overexpressing  a  110 kb  rabbit genomic fragment encoding  the FcRn. These transgenic  rabbits – having one extra copy of the FcRn when hemizygous and two extra copies  when  homozygous - showed  improved  IgG protection and  an augmented humoral  immune  response when  immunized  with a variety of different  antigens.  Our results  in these  transgenic  rabbits  demonstrate an increased immune response,  similar to what we described  in mice, indicating that FcRn overexpression  brings significant advantages for the production of polyclonal and monoclonal  antibodies.


Neonatal FcR Overexpression Boosts Humoral Immune Response in Transgenic Mice

Judit Cervenak, Balázs Bender, Zita Schneider, Melinda Magna, Bogdan Valer Carstea, Károly Liliom, Anna Erdei, Zsuzsanna Bosze and Imre Kacskovics



The neonatal  FcR (FcRn) regulates  IgG and albumin  homeostasis, mediates  maternal IgG transport, takes active part  in phago- cytosis, and delivers Ag for presentation. We have previously shown that overexpression of FcRn in transgenic (Tg) mice extends the half-life of mouse IgG by reducing  its clearance.  In this paper,  we demonstrate that  immunization of these mice with OVA and trinitrophenyl-conjugated human  IgG results  in a 3- to 10-fold increase  of Ag-specific IgM and IgG in serum.  The IgM increase was unexpected because FcRn does not bind IgM. Our results showed that the affinity of the Ag-specific IgG was at least as good in Tg mice as in the wild-type (wt) controls, implying appropriate affinity maturation in both groups. Influenza vaccination produced a 2-fold increase in the amount of virus-specific Ab in Tg animals, which proved twice as efficient in a hemagglutination inhibition assay as was the case in wt controls. After immunization, Tg mice displayed significantly larger  spleens containing a higher number of Ag-specific B cells and plasma  cells, as well as many  more  granulocytes and dendritic cells, analyzed  by ELISPOT  and flow cytometric  studies. The neutrophils from these Tg mice expressed  the Tg FcRn and phagocytosed  IgG immune  complexes more efficiently than  did those from wt mice. These results  show that  FcRn overexpression  not only extends the IgG half-life but also enhances  the  expansion  of Ag-specific B cells and  plasma  cells. Although  both  effects  increase  the  level of Ag-specific IgG, the  increase  in  immune  response  and  IgG  production seems to  be more  prominent compared with  the  reduced  IgG  clear- ance.