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Patients with hematological malignancies can be successfully treated with HLA-matched T cell-depleted allogeneic stem cell transplantation (alIoSCT) and subsequent donor lymphocyte infusions (DLIs). The efficacy of DLI is mediated by donor T cells recognizing minor histocompatibility antigens (mHags) on malignant recipient cells. Because HLA class II molecules are predominantly expressed on hematopoietic cells, mHag-specific [CD4.sup.+] T cells may selectively mediate graft versus leukemia (GvL) reactivity without graft versus host disease (GvHD). In this study, we used a recombinant bacteria cDNA library for the identification of the first autosomal HLA class II (HLA-DQBl*0603)-restricted mHag LBPI4K2B-1S encoded by the broadly expressed phosphatidylinositol 4-kinase type II [beta] gene. A polyclonal [CD4.sup.+] T cell response against LB-PI4K2B-1S was demonstrated in a patient with relapsed chronic myeloid leukemia (CML) who responded to DLI after HLA-matched alIoSCT. LB-PI4K2B-1S-specific [CD4.sup.+] T cells recognized and lysed the [CD34.sup.+] CML cells of the patient and other leukemic cells as well as high HLA-DQ-expressing normal hematopoietic cells. HLA-DQ expression on normal cells of nonhematopoietic origin was moderately up-regulated by IFN-[gamma], and not sufficient for T cell recognition. We hypothesize that LB-PI4K2B-1S-specific [CD4.sup.+] T cells contributed to the antitumor response by both directly eliminating malignant cells as effector cells and stimulating [CD8.sup.+] T cell immunity as helper cells. donor lymphocyte infusions | hematological malignancies | immunotherapy | stem cell transplantation | T lymphocytes

CD4(+) Th cells play an important role in the induction and maintenance of adequate CD8(+) T cell-mediated antitumor responses. Therefore, identification of MHC class II-restricted tumor antigenic epitopes is of major importance for the development of effective immunotherapies with synthetic peptides. CAMEL and NY-ESO-ORF2 are tumor Ags translated in an alternative open reading frame from the highly homologous LAGE-1 and NY-ESO-1 genes, respectively. In this study, we investigated whether CD4(+) T cell responses could be induced in vitro by autologous, mature dendritic cells pulsed with recombinant CAMEL protein. The data show efficient induction of CAMEL-specific CD4(+) T cells with mixed Th1/Th2 phenotype in two healthy donors. Isolation of CD4(+) T cell clones from the T cell cultures of both donors led to the identification of four naturally processed HLA-DR-binding CAMEL epitopes: CAMEL(1-20), CAMEL(14-33), CAMEL(46-65), and CAMEL(81-102). Two peptides (CAMEL(1-20) and CAMEL(14-33)) also contain previously identified HLA class I-binding CD8(+) T cell epitopes shared by CAMEL and NY-ESO-ORF2 and are therefore interesting tools to explore for immunotherapy. Furthermore, two CD4(+) T cell clones that recognized the CAMEL(14-33) peptide with similar affinities were shown to differ in recognition of tumor cells. These CD4(+) T cell clones recognized the same minimal epitope and expressed similar levels of adhesion, costimulatory, and inhibitory molecules. TCR analysis demonstrated that these clones expressed identical TCR beta-chains, but different complementarity-determining region 3 loops of the TCR alpha-chains. Introduction of the TCRs into proper recipient cells should reveal whether the different complementarity-determining region 3 alpha loops are important for tumor cell recognition.

Tumor Ag NY-ESO-1 is an attractive target for immunotherapy of cancer, since both CD8(+) CTL and CD4(+) Th cells against NY-ESO-1 have been described. Moreover, NY-ESO-1 as well as the highly homologous tumor Ag LAGE-1 are broadly expressed in various tumor types. Interestingly, the NY-ESO-1 and LAGE-1 genes also encode for proteins translated in an alternative open reading frame. These alternatively translated NY-ESO-ORF2 and CAMEL proteins, derived from the NY-ESO-1 and LAGE-1 genes, respectively, have been demonstrated to be immunogenic, since CTL specific for these proteins have been isolated from melanoma patients. In this study a panel of advanced melanoma patients was screened for the presence of Th cells specific for the alternatively translated tumor Ags NY-ESO-ORF2 and CAMEL. PBMC of melanoma patients were stimulated for 4 days with mixes of overlapping peptides covering the entire NY-ESO-ORF2 and CAMEL protein sequences and were tested for the release of type 1 (IFN-gamma) and type 2 (IL-13) cytokines in ELISPOT assays. In three of 15 patients, T cells specific for two CAMEL peptides (CAMEL(71-92) and CAMEL(81-102)) could be detected. From one of these patients, CD4(+) T cell clones specific for CAMEL(81-102) could be generated. These clones recognized a naturally processed epitope presented in both HLA-DR11 and HLA-DR12 and produced high levels of IL-4, IL-5, and IL-13. In conclusion, this study shows the presence of Th cells specific for the alternatively translated tumor Ag CAMEL in melanoma patients and is the first report that describes the isolation of tumor Ag-specific CD4(+) Th 2 clones.