MATERIAL AND METHODS RESULTS Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 DISCUSSION ACKNOWLEDGEMENTS REFERENCESHIGH LEVEL ANTIBODY RESPONSE TO RETROVIRUS-ASSOCIATED BUT NOTTO MELANOCYTE LINEAGE-SPECIFIC ANTIGENS IN MICE PROTECTEDAGAINST B16 MELANOMALucia SFONDRINI1, Daniele MORELLI1, Alessandra BODINI1, Maria I. COLNAGHI1, Sylvie ME´NARD1and Andrea BALSARI2*1Molecular Targeting Unit, Department of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy2Institute of Pathology, University of Milan, Milan, ItalyMice vaccinated with Mycobacterium tuberculosis Ag38 gene-transduced B16 melanoma cells showed significant protec-tion from intravenous challenge with parental B16 melanomacells. No cytotoxic T-cell activity was found against mela-noma cells, although the endogenous presence of the myco-bacterial gene induced a preferential Th1 response. Afterimmunization, a low serological response against melanomacells was detected, while a high titer of antibodies directed toparental B16 cells, mainly of IgG2aisotype, was found inprotected mice after challenge. These antibodies exhibitedcomplement-dependent cytotoxicity against melanoma cellsin vitro, while in vivo, used in passive immunization, theyinduced a decrease in a number of experimental B16 lungmetastases. Most of the antibodies were directed againstendogenous murine leukemia viruses. No reactivity againstmelanocyte lineage-specific antigens was observed. In particu-lar, no reactivity was found in sera from protected miceagainst tyrosinase-related protein 2 (TRP-2), either stablyexpressed in a non-melanoma cell line or obtained by in vitrotranscription-translation, or against tyrosinase, TRP-1 andgp100 antigens immunoprecipitated from B16 cells. Thus, inthe B16 murine model, the presence of dominant viralantigens induces a very strong humoral response that mightbe protective and may inhibit or mask the presence of minorclonotypes. Int. J. Cancer 83:107–112, 1999.r1999 Wiley-Liss, Inc.To date, studies on tumor immunity have focused mainly on Tcells, in particular on CD8⫹HLA class I-restricted cytotoxic Tlymphocyte (CTL) responses (Boon et al., 1994). Indeed, CD8⫹CTLs present in tumor-infiltrating lymphocytes (TIL) and periph-eral blood lymphocytes (PBLs) of melanoma patients recognizeseveral melanoma-associated antigens. Among the epitopes recog-nized by CD8⫹CTLs, most are derived from melanocyte lineage-specific proteins, including tyrosinase and its related proteins, aswell as TRP-1 and TRP-2, Pmel 17/gp 100 and MART-1/melan-A(Coulie et al., 1994; Kawakami et al., 1994, 1995; Wo¨lfel et al.,1994; Kang et al., 1995). Genetic engineering techniques havebeen used to introduce different genes into melanoma tumor cells inorder to raise an effective cellular immune response againstmelanoma-associated antigens (Tuting et al., 1997). By contrast,induction of an anti-cancer humoral response by active immuniza-tion has received less attention, perhaps because the clinicalsignificance of anti-tumor antibodies in patients is largely unknown(Apostolopoulos et al., 1998). Indeed, natural or vaccine-inducedantibodies to tumor antigens might be associated with clinicallyrelevant features and/or might represent useful serodiagnostic orprognostic markers (Old and Cher, 1998).Common features shared by human and murine tumor antigenssuggest the usefulness of murine cancer antigens as tools for theevaluation of antigen-based vaccines for cancer treatment. Forexample, the non-mutated product of a normal differentiationantigen, tyrosinase-related protein 2 (TRP-2) (Wang et al., 1996), isrecognized by CTL clones derived from TIL of melanoma patients,while the corresponding murine TRP-2 antigen is recognized bymurine T-cell clones reactive with B16 melanoma cells (Bloom etal., 1997). The highly consistent expression of the TRP-2 proteinby all types of melanoma tumors makes it an excellent targetmolecule for immunologically based diagnostic and/or therapeuticstrategies. In the present study, we examined the humoral responseof mice protected against a challenge with B16 melanoma cells.These mice, after immunization with B16 melanoma cells trans-duced with the Mycobacterium tuberculosis gene encoding a 38kDa antigen (Sfondrini et al., 1998), evoked a protective immuneresponse against untransduced B16 melanoma cells.MATERIAL AND METHODSCells and antibodiesB16 murine melanoma cells and Chinese hamster ovary (CHO)cells were routinely cultured in Dulbecco’s modified Eagle’smedium supplemented with 10% FCS, glutamine and antibiotics.Ag38-transduced B16 cells were obtained as described by Sfon-drini et al. (1998) and were maintained in G418 selection medium(0.8 mg/ml). The murine NIH/3T3 transformed tumor cell line pTand its variant pT-N, derived from in vivo passage of pT cells intoSwiss nude mice and subsequently reestablished in culture (Traver-sari et al., 1989), were kindly supplied by Dr. M.L. Sensi (Milan,Italy).Rabbit polyclonal antiserum ␣PEP8 (Tsukamoto et al., 1992),generated against a synthetic peptide that corresponds to theC-terminus of murine TRP-2, was a gift of Dr. V. Hearing(Bethesda, MD). Goat anti-gp70 and anti-total virus antisera weregenerated against purified MuLV gp70 and total proteins (Traver-sari et al., 1989). The anti-TRP1 and anti-tyrosinase mousemonoclonal antibodies (MAbs) TA99 (Thomson et al., 1985) andT311 (Chen et al., 1995), respectively, were a gift of Dr. L.J. Old(New York, NY). The gp100-specific polyclonal rabbit serumAZN-LAM (Schreurs et al., 1997) was a gift of Dr. M.W.J.Schreurs (Nijmegen, The Netherlands).In vivo experimentsC57BL/6 female mice, 6 to 8 weeks old, were purchased fromCharles River (Calco, Italy). All mice were handled and treated inaccordance with institutional guidelines. Mice were immunized s.c.(twice with a 4-week interval) with 106irradiated (20,000 rad)Ag38-transduced B16 cells and challenged i.v. with 5 ⫻ 105viableparental B16 cells. Control mice only received the i.v. inoculation.Differences between groups were analyzed using the log rank test.Interferon (IFN)-␥ and interleukin (IL)-4 released by lympho-cytes obtained from popliteal lymph nodes of mice immunized intothe right hind footpad with untransduced or transduced irradiatedB16 cells were determined as previously described (Sfondrini etal., 1998). Values represent the IFN-␥:IL-4 ratio expressed asmean ⫾ SE obtained from 3 independent experiments performed induplicate.Grant