Naturally-occurring anti-G-CSF antibodies produced by human cord blood B-cell lines infected with Epstein-Barr virus Roberto P Revoltella* ,1 , Leopoldo Laricchia Robbio 1 , Stefania Moscato 1 , Fabrizio Vinante 2 , Alessandro Fasciani 3 , Anna Maria Liberati 4 , Gigliola Reato 5 and Robin Foa 5 1 Institute of Mutagenesis and Dierentiation, C.N.R., Pisa, Italy; 2 Department of Clinical and Experimental Medicine, Section of Hematology, University of Verona, Verona, Italy; 3 Division of Gynecology and Obstetrics, University of Pisa, Pisa, Italy; 4 Institute of Internal Medicine and Oncological Sciences, Policlinico Monteluce, Perugia, Italy; 5 Department of Cellular Biotechnologies and Hematology, University ‘La Sapienza’, Rome, Italy Introduction: Naturally occurring antibodies (auto-Abs) recognizing human granulocyte- colony stimulating factor were detected with high frequency in serum samples obtained from umbilical cord blood of newborns (12 of 65 samples screened) and maternal peripheral blood serum samples from women at the end of gestation (seven of 56 cases tested). The aim of this paper was to demonstrate that auto-Abs anti-G-CSF revealed in the blood of newborns were produced during foetal life. Materials and methods: Mononuclear cells from cord blood samples of dierent newborns containing high titer anti-G-CSF Abs were infected with Epstein-Barr virus in vitro, and EBV-immortalized B-cell lines were isolated and characterized for specific anti-G-CSF Ab production. Results: Six dierent, unrelated cell lines of male origin which showed the presence of EBNA-2 antigen in the nucleus, displayed a B-cell phenotype (CD30+, CD57, CD107, HLA-DR+, CD19+, CD20+, CD23+, CD38+, CD25+), coexpressed low intensity sIgM and sIgD, and produced only IgM with prevailing l clonal restriction and anti-rhG-CSF Ab reactivity. The Ab specificity was proven against either glycosylated or unglycosylated G-CSF by saturable binding in direct enzyme-linked immunosorbent assays, by competition binding and Western immunoblotting assays. Conclusion: The secreted Abs did not aect the in vitro generation of granulocyte colonies by human normal adult haemopoietic progenitor cells in soft agar clonogenic assays, suggesting that these Abs were not neutralizing. The Hematology Journal (2001) 2, 161 – 171 Keywords: G-CSF; auto-antibodies; cord-blood cultures; EBV-infection Introduction Granulocyte-colony stimulating factor (G-CSF) is a glycoprotein that specifically stimulates the prolifera- tion, dierentiation and maturation of haemopoietic myeloid progenitors, prolongs the survival of mature neutrophils and enhances their functional properties. 1– 3 Administration of G-CSF in vivo stimulates peripheral blood granulocytosis and induces the mobilization of stem cells and progenitor cells from the bone marrow to the peripheral blood. 4 Biological responses induced by G-CSF occur following its binding to a specific cell membrane receptor whose encoding gene has been cloned. 5 Recombinant human (rh) G-CSF is being extensively used in patients with dierent diseases (reviewed in 6 ). High levels of specific Abs anti-rhG-CSF are observed in lymphoma patients who received multiple rhG-CSF injections; however, circulating anti-G-CSF Abs do not aect haematolo- gic recovery. 7,8 Moreover, low levels of high avidity, non-neutralizing anti-G-CSF auto-Abs (both IgM and IgG) are detected in about 10% of sera from healthy adult individuals. 7,8 These findings stimulated us to investigate whether anti-G-CSF Abs are produced during foetal life and to analyse the potential functional role of these natural foetal Abs. In the present study, we demonstrate the presence of natural anti-G-CSF Abs in cord blood *Correspondence: RP Revoltella, Institute of Mutagenesis and Dierentiation, C.N.R., Area della Ricerca di San Cataldo, Via G. Moruzzi, 1. Ghezzano – 56100 Pisa, Italy; Tel: +39 050 3152772; Fax: +39 050 3153367; E-mail: r.revoltella@imd.pi.cnr.it Received 9 August 2000; accepted 29 November 2000 The Hematology Journal (2001) 2, 161 – 171 ª 2001 The European Haematology Association All rights reserved 1466 – 4680/01 $15.00 www.nature.com/thj