Radiol Oncol 2002; 36(2): 185-8. Characterization of monoclonal antibodies against MHC class II-associated p41 invariant chain fragment Valentina Zavašnik Bergant1, Nataša Kopitar-Jerala1, Tadeja Bevec1 and Janko Kos1,2 1Department of Biochemistry and Molecular Biology, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia; 2Krka, d.d., R & D Division, Department of Biochemical Research and Drug Design, 1000 Ljubljana, Slovenia Mouse monoclonal antibodies directed against human MHC class II-associated p41 invariant chain fragment have been generated. Mice were immunized with human recombinant Ii-isoform p26. For hybridoma production mouse splenocytes and myeloma cells were fused. Hybridoma cells were screened using ELISA and immunoblotting. Three cell lines (42B10, 42G11 and 43C8) were used for production of specific antibodies, which reacted with p41 fragment and did not bind to cathepsins L or S or their proenyzmes. As primary antibody for immunofluorescence staining of lymph node tissue sections clone 2C12 MAb was selected. Specific localization of p41 fragment in certain cells in lymph nodes was observed. Introduction MHC class II molecules display antigenic peptides on cell surface of APC (dendritic cells, B cells, macrophages, thymic epithelial cells) for recognition by CD4+ T lymphocytes.1,2,3 The MHC class II-associated Ii is a transmembrane protein that complexes with newly synthesized MHC class II heterodimers and directs their trafficking through the endosomal compartments of APC. The luminal domain of Ii organizes MHC class II dimers into nonomeric complexes and prevents premature association of MHC class II molecules with endogenous polypeptides. Within endosomal/lysosomal compartments, Ii undergoes stepwise proteolytic degradation to yield progressively smaller fragments that remain associated with the peptide-binding groove of MHC class II dimmers.4 Dissociation of CLIP (a set of 3 kDa peptides) from the peptide-binding groove allows loading and subsequent surface expres sion of MHC class II molecules with antigenic peptides generated from endocytosed or phagocytosed protein. The key enzymes that degrade Ii are the cysteine proteases (cathepsins S5, L6 and/or V7). In human Ii exists in two alternatively spliced forms, p31 in p413, the latter containing an additional 64-amino acid sequence at the C-terminal end (hereafter called the p41 fragment). The discovery of p41 fragment- cathepsin L complex isolated from human liver8 and its known crystal structure9, led to the suggestion that the p41 fragment (Ii respectively) may enhance antigen presentation by providing a mechanism to inhibit otherwise destructive cathepsin L but not cathepsin S activity.10 The aim of the present study was to generate specific mouse monoclonal antibodies directed against MHC class II-associated p41 invariant chain. Furthermore these antibodies were used in immunohistochemical studies of p41 fragment in lymph-node tissue. Zavašnik Bergant V et al. / Characterization of monoclonal antibodies Materials and methods Preparing and purification of antibodies Human recombinant Ii-isoform p26, comprising only the luminal domain11 (together with p41 fragment) was purified by Ni-chelate chromatography (a gift from Dr. Klaus Dornmair, Max Planck Institute, Martinsried) and was subsequently used as an antigen for immunization of mice. BALB/c mice were injected subcutaneously with p26 (50 µg/mouse) emulsified in complete Freund’s adjuvant, followed by intraperitoneal injections of the same amount of antigen in incomplete Freund’s adjuvant. Test bleeds were taken and titer of specific antibodies determined using antigen immobilized ELISA in which recombinant p26 and p41 fragment-cathepsin L complex, respectively, were used as antigens. The mouse with the highest titer was boosted intraperitoneally with p26 (50 µg/mouse). For hybridoma production splenocytes and myeloma cells (NS1/1-Ag4-1) were fused by a modification of the method of Kohler and Milstein.12 The screening of the positive wells was performed by antigen immobilised ELISA as described above for test bleeds. Hybridomas producing antibodies against p41 fragment were cloned twice by means of the limiting dilution method13 and expanded into large volumes. The cell culture supernatants were concentrated by ultrafiltration and MAbs purified by affinity chromatography on Protein A-Sepharose (Pharmacia, Sweden). Antibody containing fractions were pooled and dialysed against PBS, pH 7.2. Small aliquots of purified antibodies were stored at – 20 °C. Immunoblotting Samples were first separated by SDS-PAGE on 8 – 25 % polyacrylamide gels using Phast- System (Pharmacia, Sweden). After the electrophoresis the proteins were transferred on to PVDF membrane (Millipore, USA) by passive diffusion accelerated with higher temperature. Non-specific binding sites were blocked with 0.4 % Tween 20 in PBS, pH 7.2. After this and all subsequent steps the membrane was washed with PBS, pH 7.2 containing 0.5 % Tween. Primary anti-p41 fragment antibodies were incubated with the membrane, followed by secondary goat anti-mouse IgG conjugated to HRP (Dianova, Germany). Detection was performed using 0.05 % DAB (Sigma, USA) and 0.01 % H2O2 in 50 mM Tris- HCl buffer, pH 7.5. Immunofluorescence tissue staining Sections from formalin fixed, paraffin embedded lymph nodes were used for IHA. Tissue sections on micro cover glasses were deparaffinised in xylene and rehydrated through ethanol series. They were placed in 10 mM sodium citrate buffer, pH 6.0 and put into a microwave oven (5 min, 400 W) for antigen retrieval. Non-specific binding sites were blocked with 3 % BSA in PBS, pH 7.4. After this and all subsequent steps tissue sections were rinsed in PBS, pH 7.4. Primary anti-p41 fragment antibody was added (clone 2C12, 25 µg/ml, for 2 h at 37 °C), followed by Alexa FluorTM 488-labeled goat anti-mouse IgG secondary antibody. Molecular Probes, USA). Tissue sections were mounted on slides with ProLongTM AntiFade Kit (Molecular Probes, USA). Fluorescence microscopy and optical slicing were performed by confocal laser scanning microscope LSM 510 (Carl Zeiss Inc., USA). Results Purified monoclonal antibodies were tested for specificity using immunoblotting. As shown in Figure 1, 2C12 MAb (derived from clone 42B10) reacted with p41 fragment in complex with cathepsin L (32 kDa, lane 5), Radiol Oncol 2002; 36(2): 185-8. Zavašnik Bergant V et al. / Characterization of monoclonal antibodies with p41 fragment detached from the complex (14 kDa, lane 1 and 2), as well as with recombinant p26 (lane 3). There was no cross- reactivity observed with recombinant procathepsin L (lane 4), cathepsin L (31 kDa, lane 1) nor its heavy chain (25 kDa, lane 1) and light chain (below 14 kDa, lane 1), respectively. Also, antibody specificity and cross-reactvity towards different cathepsins was tested by ELISA. Recombinant (pro)cathepsins L and S were added to the wells instead of p26 Figure 1. Demonstration of the specificity of anti-p41 fragment Mab. (A) SDS-PAGE silver staining. (B) Immunoblot of the equivalent gel stained with 2C12 MAb. Samples: (lane 1) p41 fragment-cathepsin L complex reduced (with 5 % 2-mercaptoethanol) and exposed to 100 °C in the presence of SDS for 5 minutes; (lane 2) p41 fragment detached from the native complex by HPLC; (lane 3) p26 reduced (with 5 % 2mercaptoethanol) and exposed to 100 °C in the presence of SDS for 5 minutes; (lane 4) nonreduced recombinant procathepsin L; (lane 5) nonreduced p41 fragment-cathepsin L complex; (lane 6) LMW standards. or p41 fragment-cathepsin L complex. With all three selected cell lines negligible reactivity was observed. For immunohistochemical localization of p41 fragment in lymph node tissue sections 2C12 MAb was selected. Results are shown in Figure 2. Figure 2. Immunohistochemical staining of lymph-node tissue sections for p41 fragment. Conclusions Specific monoclonal antibodies recognizing MHC class II-associated p41 fragment were successfully produced. They do not cross react with cathepsins L nor S or their proenzymes. We have shown specific localization of Ii fragments in certain cells in lymph nodes. These antibodies provide new tools for investigating subcellular colocalization of Ii together with cathepsins S and L. Acknowledgements This work was supported by the Ministry of Science and Technology of the Republic of Slovenia. The authors thank Professor Robert Zorec and Dr. Marko Kreft for their assistance with confocal laser scanning microscope. Radiol Oncol 2002; 36(2): 185-8. Zavašnik Bergant V et al. / Characterization of monoclonal antibodies References 1. Nakagawa TY, Brissette WH, Lira PD, Griffiths RJ, Petrushova N, Stock J, McNeish J D, Eastman S, Howard ED, Clarke SRM, Rosloniec EF, Elliot EA, Rudensky AY. Impaired invariant chain degradation and antigen presentation and diminished collagen- induced arthritis in cathepsin S null mice. Immunity 1999; 10: 207-17. 2. Riese RJ, Mitchell RN, Villadangos JA, Shi, G-P, Palmer JT, Karp ER, De Sanctis GT, Ploegh HL, Chapman HA. Cathepsin S activity regulates antigen presentation and immunity. J Clin Invest 1998; 101: 2351-63. 3. Chapman HA. Endosomal proteolysis and MHC class II. function. Curr Opin Immunol 1998; 10: 93102. 4. Shi G-P, Villadangos JA, Dranoff G, Small C, Gu L, Haley KJ, Riese R, Ploegh HL, Chapman HA. Cathepsin S required for normal MHC Class II peptide loading and center development. Immunity 1999; 10: 197-206. 5. Cresswell, P. Proteases, processing and thymic selection. Science 1998; 280: 394-5. 6. Nakagawa T, Roth W, Wong P, Nelson A, Farr A, Deussing J, Villadangos JA, Ploegh H, PetersC, Rudensky AY. Cathepsin L: Critical role in Ii degradation and CD4 T cell selection in the thymus. Science 1998; 280: 450-3. 7. Brömme D, Li Z, Barnes M, Mehler E. Human cathepsin V functional expression, tissue distribution, electrostatic surface potential, enzymatic characterization and chromosomal localization. Biochemistry 1999; 38: 2377- 85. 8. Bevec T, Stoka V, Pungerčič G, Dolenc I, Turk V. Major histocompatibility complex class II-associated p41 invariant chain fragment is a strong inhibitor of lysosomal cathepsin L. J Exp Med 1996; 183: 1331-8. 9. Gunčar G, Pungerčič G, Klemenčič I, Turk V, Turk D. Crystal structure of MHC class II-associated p41 Ii fragment bound to cathepsin L reveals the structural basis for differentiation between cathepsins L and S. EMBO J 1999; 18: 793-803. 10. Turk D, Gunčar G, Turk V. The p41 fragment story. IUBMB Life 1999; 48: 7-12. 11. Strubin M, Mach B, Long E O, The complete sequence of the mRNA for the-HLA-DR-associated invariant chain reveals a polypeptide with an unusual transembrane polarity. EMBO J 1984; 3: 869-72. 12. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 265: 495-7. 13. Robb A. J. Microcloning and replica plating of mammalian cells. Science 1970; 170: 857-8. Radiol Oncol 2002; 36(2): 185-8.