Nature of antigens and antibodies in immune complexes isolated by staphylococcal protein A from plasma of melanoma patients

Summary Immune complexes (IC) were isolated from plasma of melanoma patients by absorption to staphylococcal protein A and subsequent elution with MgCl₂. The isolated ICs were purified by precipitation with polyethylene glycol and sucrose density gradient ultracentrifugation after radioiodination with ¹²⁵I. The purified ICs were dissociated and radiolabeled antigen/antibody components were separated by ultracentrifugation at low pH (2.6). Under these conditions, about 72% radioactivity of the purified IC remained in the light-density region as a wide band. After neutralization, 26%–60% radioactivity in the region of 5S sedimentation bound to immobilized autologous immunoglobulins, as opposed to a maximum of 23% to immobilized immunoglobulins from human normal serum. Significant levels (73%–77%) of radioactivity in 7S region bound to rabbit anti-human IgG immunobeads. Immunoprecipitation of the antigen fraction by allogeneic anti-melanoma and rabbit anti-melanoma antibodies followed by SDS-polyacrylamide gel electrophoresis revealed the presence of a fetal antigen (FA) and a melanoma tumor-associated antigen (TAA). In addition, the presence of auto-antigen(s) was indicated by using autologous antibody in immunoprecipitation. Immunoglobulins (IgG) isolated from purified IC bound to cultured melanoma, sarcoma, and normal fibroblasts, although the binding to sarcoma and normal fibroblasts could be inhibited by preincubation of isolated IgG with soluble FA but not with soluble melanoma TAA. Thus, results of this investigation provide evidence that circulating IC in melanoma patients are composed of at least IgG and different antigens, and some of these antigens are produced by their tumor.     

Complement-dependent lysis of tumor cells by a baboon IgM antibody to a tumor-associated antigen

Summary We developed a high-titer polyclonal antiserum to a glycoprotein tumor-associated antigen (TAA) by immunization of a baboon with the purified glycoprotein antigen. The baboon serum was fractionated into IgG and IgM components by DEAE Affi-Gel blue chromatography. The ability of the baboon IgM anti-TAA antibody to effect tumor cell lysis in the presence of complement was tested using a chromium-release assay. The baboon antibody was able to lyse melanoma target cells (20.8%–71.4% cytolysis), breast carcinoma cells (36.5%–38.9% cytolysis), and a neuroblastoma cell line (35.5% cytolysis) in the presence of complement but did not effect significant lysis of autologous lymphoblastoid cell lines (4.9% cytolysis) or peripheral blood lymphocytes from healthy volunteers (12.6% cytolysis). Cytolysis of melanoma target cells was completely inhibited by preabsorption of the IgM anti-TAA antibody with UCLA-SO-M14 (M14) cells and partially inhibited by preabsorption with several other melanoma cell lines. There was no significant inhibition of tumor cell lysis after preabsorption of the antibody with lymphoblastoid cell lines. Complement-dependent lysis of M14 targets could be blocked by addition of the purified antigen to the antibody prior to incubation with the tumor cells. Our results suggest that the glycoprotein TAA resides on the tumor cell surface and that the baboon IgM anti-TAA antibody recognizes the antigen on the cell surface and is able to fix complement and effect the lysis of the tumor cells.     

Characterization of the antigenic determinants and host components in immune complexes from patients with secondary syphilis

Sera from patients with secondary syphilis were evaluated for abnormal levels of circulating immune complexes (IC), immunoglobulins (Ig), and complement components. Clq-solid-phase assays (Clq-SPA) that made use of monoclonal and polyclonal antibodies directed against IgG subclasses indicated that human IC were composed primarily of IgG3 and IgG1; these findings appeared consistent with subclass profile responses of electrophoretically transferred blots (Western blots) of Treponema pallidum reacted with syphilitic sera. Complexes were isolated from reactive sera by polyethylene glycol precipitation followed by either anti-Clq column chromatography or protein A-Sepharose chromatography. Although qualitative and quantitative differences were noted, all purified materials contained a treponemal polypeptide antigen with a m.w. of approximately 87,000. Subsequent analysis of this polypeptide, which was also present in purified IC from rabbits with experimental syphilis, suggests that it may represent the fibronectin receptor of the organism. The 76,000 and 66,000 materials, earlier identified in purified rabbit IC, appeared to represent C-terminal degradation products of fibronectin presumably of host origin, rather than treponemal antigens. Although fibronectin binds avidly to Clq and could represent a co-precipitable contaminant throughout the isolation procedure, anti-fibronectin antibodies in the sera of patients detectable by radioimmunoassay and the present of antibodies to 76,000 and 66,000 dalton fibronectin fragments in the globulin fractions of disassociated complexes argues against such a conclusion.     

Structural studies of Fc receptors. III. Isolation and molecular weight analysis of the component chain of Fc gamma receptor of macrophage

Surface receptors of guinea pig peritoneal macrophages specific for the Fc region of IgG (Fc gamma receptor) were isolated and identified as a surface-radioiodinated component with a molecular weight of 44,000 that bound in an Fc-specific manner to IgG2 of guinea pig immunoglobulin immobilized in any of the following three different ways: IgG2 antibody in insoluble immune complex, IgG2 antibody bound to antigen-coupled Sepharose, and IgG2 covalently coupled to Sepharose. In order to obtain the Fc gamma receptor retaining the binding activity, the Fc-binding component was isolated by IgG2 affinity chromatography in which mild acidic buffer (pH 5.0-4.0) was chosen to elute the component bound to the affinity column. Forty-five to sixty-two percent of the eluted radioactivity was shown to rebind to the IgG2-affinity column. The bound fraction showed a single radioactive peak of 44,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Fc-binding component isolated by the affinity chromatography behaved similarly in gel filtration in the presence of a detergent, as did the detergent-solubilized Fc gamma receptor before isolation by affinity chromatography. These results suggested that the Fc gamma receptor was isolated in a native form. Furthermore, it was confirmed that the isolated Fc gamma receptor is distinct from actin or the actin-like protein (DNase I-binding protein) which had been reported to bind to IgG-affinity column.     

Effect of H chain V region on complement activation by immobilized immune complexes.

Activation of C by immune complexes (IC) in tissues and the inflammatory consequences are major determinants in the pathogenesis of many autoimmune disorders. To assess the factors involved in C activation by such IC, we examined the binding of C components by chimeric IgG1 antibodies bound to immobilized Ag. We previously reported that alterations in the H chain V regions can affect the binding of first component of C (C1q) and a major breakdown product of the third C component (C3b) when otherwise identical antibodies were bound to immobilized (Tyr, Glu)-Ala-Lys. To evaluate C activation of these antibodies in well defined IC, we utilized a 9-amino acid peptide conjugated to BSA as Ag. The peptide:BSA conjugate was bound similarly by the two IgG1 antibodies which differed mainly in the CDR3 regions, but also in 9 other amino acids in the H chain V region. When soluble IC were prepared with the two antibodies, they activated C similarly. However, C activation by solid phase Ag:antibody complexes differed; we found that antibody 10B bound more C1q and C3b than antibody B11 did, unless the Ag was present at high density on the plates. These data suggest that the variable region differences affect C activation by these antibody when they are bound to immobilized Ag. Furthermore, these results underscore the differences in C activation by the same antibody depending upon whether the IC are free in solution or immobilized.     

Non-fastidious,melanoma-specific CD8+ cytotoxic T lymphocytes from choroidal melanoma patients

To characterize the anti-melanoma reactivity of CD8⁺ cytotoxic T lymphocytes (CTL) from choroidal melanoma patients, CTL clones were isolated from the peripheral blood of three patients after mixed lymphocyte/tumor cell culture (MLTC). Clones were derived from lymphocytes stimulated by allogeneic (OCM-1, A24, A28) or autologous (OCM-3, Al, A30) melanoma cells. Their reactivity against a panel of HLA-typed melanoma and nonmelanoma cells was assessed, to determine whether a single CTL clone could recognize and lyse a variety of allogeneic melanoma cell lines. While proportionately more clones derived from autologous MLTC were melanoma-specific than allogeneic MLTC (42% versus 14%), melanoma-specific CTL were recovered from both. Notably, a novel melanoma specificity was identified. These CTL clones were termed non-fastidious because they were capable of lysing melanoma cells with which they had no HLA class I alleles in common. Nonetheless, lysis was mediated by the HLA class I molecule. Since lysis was specific for melanoma cells, these CTL appeared to recognize a shared melanoma peptide(s). Because of their prevalence, we propose that non-fastidious CTL are integral to human anti-melanoma T cell immunity. This reinforces clinical findings that allogeneic melanomas can substitute for autologous tumors in active specific immunotherapy. By circumventing the need for autologous melanoma, it is possible to treat patients after removal of the primary choroidal melanoma in an attempt to prevent metastasis.Supported by USPHS grants EY-09031 and EY-09427, and the Lucy Adams Choroidal Melanoma Research Fund to J. K.-M.     

Antibodies against p-aminophenyl-beta-D-galactopyranoside-containing proteins

The antibodies were prepared from antisera of rabbits immunized with bovine serum albumin containing covalently bound p-aminophenyl-beta-D-galactopyranoside (APG) and purified by affinity chromatography on APG-containing ovalbumin immobilized by BrCN-activated Sepharose 4B. The antibodies possessed a selective specificity for APG and interacted with different APG-containing proteins, including APG-containing lysosomal alpha-glucosidase. The purified antibodies are immunoglobulins of G type as was determined from the molecular weights of native and dissociated antibodies and from the immunochemical assays with antibodies against rabbit IgG.     

Recovery of a cell surface fetal antigen from circulating immune complexes of melanoma patients

Summary A well-characterized 69.5×10³ dalton glycoprotein fetal antigen (FA), isolated from the spent culture medium of a melanoma cell line, UCLA-SO-14 (M14), was utilized to characterize the antigen component of circulating immune complexes (CIC) from melanoma patients. Ten serum samples from five patients with stage II melanoma at 1 and 4 months prior to the clinical detection of recurrent disease were selected for study. The CIC were dissociated with low pH and ultrafiltered through a 100¢10³ dalton exclusion limit membrane. The low pH treatment resulted in an increase in antibody titer in eight of ten serum samples. The antibody activity in membrane immunofluorescence was quantitatively inhibited by the filtered antigen fraction and purified FA, suggesting the presence of anti-FA antibodies in the treated serum, which possibly were complexed with FA in the untreated sample. As determined by competitive inhibition in an enzyme-linked immunosorbent assay, the filtrate (antigen fraction) contained an antigen that was immunologically similar to FA. These results clearly demonstrate that FA, expressed on the cell surface of melanoma cells, is present in CIC of selected melanoma patients.Supported in part by NIH grants CA 30019, CA 12582, CA 09010, CA 29605 awarded by DHSS     

Delayed-type cutaneous hypersensitivity to melanoma antigens and its implication in active specific immunotherapy in cancer

Summary In this study, we explored whether soluble tumor-cell surface-associated antigens (TAA) might be derived from autochthonous as well as allogeneic sources as immunogens for active specific immunotherapy. Using two popular cell membrane-bound antigen extraction techniques (3 M KCl and isotonic-hypotonic NaCl), we examined the immunogenic potential of such TAA and the specificity of immunologic host reactivity through a delayed-type cutaneous hypersensitivity reaction (DTH) as a guideline for their immunogenic potential in a human malignant melanoma model system. We found that either extraction technique could provide soluble TAA from both autochthonous and allogeneic sources capable of eliciting DTH. While evidence of positive DTH with autochthonous TAA reaffirms the immunogenicity of such TAA, the specificity of host reactivity against TAA derived from allogeneic sources is extremely difficult to establish, even with TAA partially purified by column chromatography in Sephadex G-200. Patients exhibited reactivity to other TAA derived from tumors of different histologies and often to more than one component isolated by column chromatography. Furthermore, when a group of melanoma patients was tested against a panel of melanoma antigens in any random combination, DTH to allogeneic TAA was seen in an unpredictable order and with inconsistent frequency. We conclude, therefore, that while autochthonous antigen immunizations may be justified, more careful studies will be necessary to define the antigenic profile of a given tumor (individual specificity vs shared specificity), establish specificity of alloantigens, and devise suitable methods for testing immunologic specificity for alloantigens, before rational immunotherapy with allogeneic tumor antigens will be feasible.     

Investigations on the aminoacid content of tumor associated antigens of rat sarcoma cells induced by virus

In our previous work (Alexandrov et al., 1996) was reported that the rat sarcoma cells induced by SR-RSV express two tumor associated antigens (TAA). The one TAA has a molecular weight of 52 kD and is detected by the help of a monoclonal antibody 2C2 only on the outer side of the plasma membrane of the sarcoma cells. The other antigen, with molecular weight of 28 kD, is expressed on the outher and inner side of the membrane. The antigens were isolated as a pure fraction by polyacrylamide electrophoresis and prepared for aminoacid analysis after that. The consisting 16 bound aminoacids were in different amounts. Both antigens are rich in glycine and poor in aromatic and sulphur-containing aminoacids. The presence of glucosamine and galactosamine in the antigens proves their glycoprotein nature. The received data show that the both TAA-s differ not only in molecular weights, place of expression and functional activity, but also in the amount of the bound aminoacids which constitute their proteins.     

Radiolabeling of immunoglobulin without loss of antibody activity--use of 14C-phenylisothiocyanate with human cytotoxic antibody.

Radioiodination of immunoglobulins, particularly human cytotoxic IgG, induced a marked loss of antibody activity. Phenylisothiocyanate (PITC) readily reacts with alpha-amino groups and the epsilon-amino groups of lysines to form phenylthiocarbamyl derivatives. Since PITC is commercially available with 14C, 3H, and 35S substitutions, it provided an alternative means for labeling immunoglobulin which preserved antibody activity. There were approximately 80 PITC binding sites per IgG molecule, and 14C-PITC was bound with an efficiency in excess of 80%. With as many as 40 PITC molecules bound per IgG molecule, full cytotoxic activity was retained by both anti-HLA isoantisera and human anti-melanoma autoantisera. Even with 70 to 80 molecules of PITC bound per IgG molecule, over 80% of the antibody activity remained. Radiolabeling of antibody with 3H, 14C, or 35S-substituted PITC will greatly facilitate studies of antibody binding, particularly to cell surface antigens.     

Association between allo-immunoreactive and xeno-immunoreactive subunits of a glycoprotein tumor-associated antigen

Summary Using allogeneic antibody, we previously described a tumor-associated antigen (TAA) in the urine of 68% of melanoma patients. The TAA was purified from urine of a melanoma patient and used as immunogen to develop a murine monoclonal antibody (AD1-40F4) and xenopolyclonal antibodies in a baboon. Sera from melanoma patients treated with whole melanoma cell vaccine were used as the source of human antibody to the glycoprotein antigen. Treatment with 2-mercaptoethanol and separation by sodium dodecyl sulfate/polyacrylamide gel electrophoresis resolved the high-molecular-mass glycoprotein TAA into smaller subunits. Immunoblot analysis indicates that the murine monoclonal antibody (AD1-40F4) recognized a 90–100-kDa subunit of the antigen while human anti-TAA antibodies primarily recognized a 65-kDa subunit in addition to the 90–100-kDa subunit. Baboon polyclonal antibodies recognized the same subunits plus a 120-kDa subunit. Blocking studies indicated that the murine monoclonal and baboon polyclonal antibodies recognized the closely related epitopes on the 90–100-kDa subunit, while human antibodies recognized an epitope entirely distinct from that recognized by the mouse antibody. These results demonstrate the epitope complexity associated with the high-molecular-mass glycoprotein TAA.     

Immunopurification, characterization, and nature of membrane association of human melanoma-associated oncofetal antigen gp87 defined by monoclonal antibody 140.240

A melanoma-associated oncofetal antigen, gp87 (a p97-like molecule), defined by the monoclonal antibody (MoAb) 140.240 has been purified to homogeneity from the spent medium of cultured melanoma cells by a two-step immunoadsorbent procedure. The first immunoadsorbent step using glutaraldehyde-insolubilized MoAb 140.240 (ascites fluid) resulted in a 13-fold enrichment with 93% recovery in the bound material. In the second immunoadsorbent step constructed by the purified IgG2a of MoAb 140.240 (culture fluid) coupled to CNBr-activated Sepharose 4B the bound material from the first step was further purified resulting in a 330-fold purification with 90% recovery. SDS-polyacrylamide gel electrophoretic analysis of the final purified material revealed a single band migrating as a polypeptide with an approximate molecular weight of 87 Kd, consistent with the size of the molecule immunoprecipitated by MoAb 140.240 from lysates of radiolabelled melanoma cells. Preliminary amino acid analysis indicates a particularly high proportion of phenylalanine in gp87. We have also compared gp87 with two well defined antigens, HLA-A,B,C (integral membrane protein) and "94K" melanoma/carcinoma-associated antigen (peripheral membrane protein) with respect to antigen extractability from melanoma cells using phosphate-buffered saline, 0.1 M urea, 3 M NaCl, or nonionic detergent (NP-40). The results showed that whereas 94K antigen was extractable by each of the four different solutions, gp87, similar to HLA-A,B,C antigens, could only be extracted with NP-40, strongly suggesting that gp87 is an integral melanoma cell component.     

Tumor-associated antigen 90K/Mac-2-binding protein: possible role in colon cancer

The tumor-associated antigen 90K (TAA90K)/Mac-2-binding protein implicated in cancer progression and metastasis is modified by beta1-6 branched N-linked oligosaccharides in colon cancer cells, glycans shown to contribute to cancer metastasis. To elucidate the role of TAA90K in colon cancer, we examined its expression and function in human colon tumors and colon carcinoma cell lines. Immunohistochemical analyses of colon tumors revealed elevated expression of TAA90K in all samples analyzed compared to normal colon. To examine the function of TAA90K in colon cancer, we carried out protein and cell binding assays using TAA90K-His purified from HT-29 cells colon carcinoma cells infected with recombinant vaccinia virus expressing TAA90K containing a C-terminal poly-histidine tag. TAA90K-His bound to fibronectin, collagen IV, laminins-1, -5, and -10 and galectin-3 (Mac-2) but poorly to collagen I and galectin-1. As expected, binding of TAA90K to galectin-3 was dependent on carbohydrate since it was inhibitable by lactose and asiolofetuin, and a TAA90K-His glycoform purified from HT-29 cells treated with the glycosylation inhibitor 1-deoxymannojirimycin bound poorly to galectin-3. Unlike TAA90K isolated from other cell types, TAA90K-His isolated from colon cancer cells failed to mediate adhesion of colon cancer and normal cell lines, possibly due to cell-type specific glycosylation of TAA90K-His and/or its putative cellular receptor. However, at low concentrations, TAA90K-His enhanced galectin-3-mediated HT-29 cell adhesion while at high concentrations, it inhibited cell adhesion. Thus, a possible mechanism by which TAA90K may contribute to colon cancer progression is by modulating tumor cell adhesion to extracellular proteins, including galectin-3.     

Binding of anti-band 3 autoantibody to oxidatively damaged erythrocytes. Formation of senescent antigen on erythrocyte surface by an oxidative mechanism

Incubation of human erythrocytes oxidized by iron catalysts, ADP/Fe3+ or xanthine/xanthine oxidase/Fe3+, with autologous IgG resulted in IgG binding as detected by enzyme immunoassay using protein A-beta-galactosidase conjugate. The binding of autologous IgG to ADP/Fe3(+)-treated erythrocytes maximized when the cells were treated with 1.8:0.1 mM ADP/Fe3+, and declined when treated above this concentration, suggesting that autologous IgG binds to moderately but not to excessively oxidized erythrocytes. The antibody involved in the binding was anti-Band 3, the autoantibody known to bind to aged erythrocytes, because isolated anti-Band 3 bound to the oxidized cells, but anti-Band 3-depleted autologous IgG did not. In addition, purified Band 3 inhibited the autologous IgG binding. Anti-alpha-galactosyl IgG, another natural antibody which has been reported to bind to aged erythrocytes, did not bind to the oxidized cells. Oxidation of membrane lipids, SH-groups of membrane proteins, and Hb of these cells was slight, but the cells contained an increased amount of membrane-bound native Hb, indicating that the oxidized cell membrane has an altered property. alpha-Tocopherol prevented the lipid oxidation and the subsequent IgG binding. Reduction of the oxidized erythrocytes with dithiothreitol resulted in a loss of the IgG binding. These results suggest that anti-Band 3 binding sites (Band 3 senescent antigen) are formed on moderately oxidized erythrocytes as a result of oxidation of membrane protein SH-groups which can be mediated by the membrane lipid oxidation and that formation of the anti-Band 3 binding sites on the oxidized cells is an essentially reversible membrane event which is linked to oxidation and restoration of the protein SH-groups.     

Identification of immune complex antigens in sera of Indian kala-azar patients

Level of circulating immune complex (IC) in visceral leishmaniasis is much higher than that in control sera. In immunoblot experiment, treatment of kala-azar IC with patient sera showed at least 6 bands of which the band around 55 kDa region was most prominent. The band at 55 kDa is primarily due to the presence of an antigen recognized by its corresponding antibody present in the patient sera. This was confirmed by using radiolabelled antibody from kala-azar patient serum and antipromastigote serum. The heavy chain of IgG originating from IC is also present in the same region which was detected by its recognition with antihuman IgG. The IC gave a band at 55 kDa region with sea-urchin antitubulin. Kala-azar sera also reacted with purified rat brain tubulin. The present results suggest that a tubulin like protein is present at 55 kDa region along with the heavy chain of IgG.     

Recombinant, truncated CD4 molecule (rT4) binds IgG

CD4 is a cell surface glycoprotein that identifies the subset of human T lymphocytes that induces sIg+ B lymphocytes to differentiate and secrete Ig after intimate T-B cell contact. In the course of studying a recombinant, truncated form of CD4 (rT4) we noticed that goat antibodies of apparently irrelevant specificities bound to immobilized rT4. To directly study whether rT4 interacts with Ig molecules, purified human IgG was added to rT4-coated wells and a dose-dependent interaction between IgG and rT4 was observed by ELISA. Purified myeloma IgG proteins bound to immobilized rT4 with the same avidity as polyclonal IgG that suggests that rT4-IgG binding was not due to the presence of anti-rT4 antibodies in the IgG fraction. IgG from 6 sera bound to rT4 in concentration dependent manner similar to purified IgG. Immobilized rT4 specifically bound IgG, and not IgM, IgA, IgD, or beta 2-microglobulin. The specific interaction of rT4 and IgG was also observed when IgG or IgM were immobilized, demonstrating that IgG binding was not a unique property of immobilized rT4. As with low affinity receptors for IgG, rT4 bound heat aggregated IgG with increased avidity. Neither anti-CD4 mAb nor dextran sulfate inhibited rT4-IgG binding. rT4 bound Fc but not F(ab)2 fragments. Each of the purified IgG subclasses; IgG1, 2, 3, and 4 bound to rT4 with similar avidity. Taken together, these data suggest that rT4 specifically interacts with a public structure on IgG Fc.     

Isolation and characterization of root nodule proteins from lupin

A group of root nodule-specific plant proteins (nodulins) has been isolated from yellow lupin (Lupinus luteus) by immunoaffinity chromatography. The cytoplasmic nodule protein extract was initially enriched in nodulins on a column with immobilized IgG fraction. It was then purified by chromatography on Sepharose 4B - bound IgG against uninfected root proteins and finally on Sepharose 4B - bound IgG against Rhizobium lupini proteins. Rocket immunoelectrophoresis showed that the nodulin preparation did not react with antibodies against root or bacterial proteins. SDS gel electrophoresis of lupin nodulins revealed at least 23 polypeptides ranging in Mr, from 7,000 to 70,000, probably representing protein subunits.     

One-step purification of murine IgM and human alpha 2-macroglobulin by affinity chromatography on immobilized snowdrop bulb lectin

A new mannose-specific plant lectin (GNA) isolated from the snowdrop bulb was immobilized on Sepharose 4B and employed for the purification of certain glycoproteins with high-mannose type glycan chains. Murine IgM bound tightly to this column and was eluted with 0.1 M methyl alpha-D-mannoside whereas bovine and murine IgG were not bound. When a murine hybridoma serum containing IgM monoclonal antibody was applied to this column, highly purified IgM antibody was obtained after elution with methyl alpha-D-mannoside. On the contrary, human IgM was not bound by this column despite reports that it contains high-mannose type glycan chains. alpha 2-Macroglobulin was the sole glycoprotein present in human serum which was bound by the immobilized snowdrop lectin column. It appears that only glycoproteins containing multiple Man(alpha 1,3)Man units are bound to the immobilized lectin.     

MELOE-1 Antigen Contains Multiple HLA Class II T Cell Epitopes Recognized by Th1 CD4+ T Cells from Melanoma Patients

MELOE-1 is an overexpressed melanoma antigen containing a HLA-A2 restricted epitope, involved in melanoma immunosurveillance of patients adoptively transferred with tumour infiltrating lymphocytes (TIL). The use of the full-length antigen (46 aa) for anti-melanoma vaccination could be considered, subject to the presence of Th epitopes all along MELOE-1 sequence. Thus, in this study we evaluated in vitro the immunoprevalence of the different regions of MELOE-1 (i.e. their ability to induce CD4 T cell responses in vitro from PBMC). Stimulation of PBMC from healthy subjects with MELOE-1 induced the amplification of CD4 T cells specific for various regions of the protein in multiple HLA contexts, for each tested donor. We confirmed these results in a panel of melanoma patients, and documented that MELOE-1 specific CD4 T cells, were mainly Th1 cells, presumably favourable to the amplification of CD8 specific T cells. Using autologous DC, we further showed that these class II epitopes could be naturally processed from MELOE-1 whole protein and identified minimal epitopes derived from each region of MELOE-1, and presented in four distinct HLA contexts. In conclusion, vaccination with MELOE-1 whole polypeptide should induce specific Th1 CD4 responses in a majority of melanoma patients, stimulating the amplification of CD8 effector cells, reactive against melanoma cells.