Technical Advance: Amine-reactive OVA multimers for auto-vaccination against cytokines and other mediators: perspectives illustrated for GCP-2 in L. major infection Catherine Uyttenhove,* ,† Reece G. Marillier,* ,‡ Fabienne Tacchini-Cottier, § Me ´lanie Charmoy, § Rachel R. Caspi,  Jesse M. Damsker,  Stanislas Goriely, † Dan Su, ‡ Jo Van Damme, ¶ Sofie Struyf, ¶ Ghislain Opdenakker, # and Jacques Van Snick* , ‡, 1 *Ludwig Institute for Cancer Research, Brussels Branch, Brussels, Belgium; † Cellular Genetics Unit and ‡ Experimental Medicine Unit, de Duve Institute, Universite ´ Catholique de Louvain, Brussels, Belgium; § WHO Immunology Research and Training Center, Department of Biochemistry, University of Lausanne, Epalinges, Switzerland;  Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, Maryland, USA; and Laboratories of ¶ Molecular Immunology and # Immunobiology, Rega Institute for Medical Research, K.U. Leuven, Leuven, Belgium RECEIVED DECEMBER 21, 2010; REVISED JANUARY 25, 2011; ACCEPTED FEBRUARY 5, 2011. DOI: 10.1189/jlb.1210699 ABSTRACT Anticytokine auto-vaccination is a powerful tool for the study of cytokine functions in vivo but has remained rather esoteric as a result of numerous technical diffi- culties. We here describe a two-step procedure based on the use of OVA multimers purified by size exclusion chromatography after incubation with glutaraldehyde at pH 6. When such polymers are incubated with a target protein at pH 8.5 to deprotonate reactive amines, com- plexes are formed that confer immunogenicity to self- antigens. The chemokine GCP-2/CXCL6, the cytokines GM-CSF, IL-17F, IL-17E/IL-25, IL-27, and TGF-1, and the MMP-9/gelatinase B are discussed as examples. mAb, derived from such immunized mice, have obvious advantages for in vivo studies of the target proteins. Using a mAb against GCP-2, obtained by the method described here, we provide the first demonstration of the major role played by this chemokine in rapid neutro- phil mobilization after Leishmania major infection. Pre- activated OVA multimers reactive with amine residues thus provide an efficient carrier for auto-vaccination against 9 –90 kDa autologous proteins. J. Leukoc. Biol. 89: 1001–1007; 2011. Introduction Anticytokine auto-vaccination opens many perspectives for the study and modulation of cytokine function in vivo, as reviewed recently by Ratsimandresy et al. [1]. The procedure is based on the fact that self-cytokines, linked chemically to a nonself protein [2] or genetically associated to a defined foreign se- quence [3] or viral-like particles [4], become immunogenic. The proposed rationale underlying this reaction is that the self-reactive B cell, which has captured the complex or fusion protein, will present foreign peptides on its MHC class II membrane proteins and thus, attract help from T cells reactive with the nonself structure [5]. Examples of successful applica- tion of this approach include TNF- [3], IL-1 and IL-1 [6], IL-5 [7], IL-9 [8], IL-12 [9], IL-17A [10], VEGF [11], and IFN- [12]. We recently noted a positive correlation between immuno- genicity and immunogen size in a series of anti-IL-12–OVA vaccines that were fractionated according to size (unpublished results). This raises a dilemma, as the larger the complex, the greater the risk for structural alterations of the antigen. To circumvent this problem, we tried a two-step procedure. We first made large OVA multimers by treating OVA with glu- taraldehyde and after purifying the polymerized products by size exclusion chromatography, reacted these with the target cytokine before saturating remaining glutaraldehyde sites with PADRE [13] to maximize immunogenicity. Here, we show the efficacy of this procedure for mouse auto-vaccination against seven proteins involved in the control of immune and inflam- matory responses. Using this methodology, we obtained mouse anti-mouse GCP-2 mAb that demonstrate the crucial role of GCP-2 in early neutrophil mobilization following Leishmania major infection. 1. Correspondence: Ludwig Institute for Cancer Research, Brussels Branch, Avenue Hippocrate 74 B-1200, Bruxelles, Belgique. E-mail: jacques. vansnick@bru.licr.org Abbreviations: EBI3EBV-induced gene 3, FDC-P1factor-dependent cell progenitor 1, GCP-2granulocyte chemotactic protein 2, GCP-2(9- 78)granulocyte chemotactic protein 2 lacking 8 N terminal amino acids, MMP-9matrix metalloproteinase 9, NGCP-2Alexa Fluor 488-labeled GCP2 peptide, OVAgluOVA polymers covered with glutaraldehyde, PADREpan HLA-DR epitope peptide, TMLECTGF-R-transfected mink lung epithelial cell line f TECHNICAL ADVANCE 0741-5400/11/0089-1001 © Society for Leukocyte Biology Volume 89, June 2011 Journal of Leukocyte Biology 1001