Five separate clones of different lengths were sequenced in both strands, and all overlapping regions were found to have identical sequence. malignancies and may be a target for antigen-specific immunotherapy. The therapeutic benefits of allogeneic bone marrow transplantation (BMT) derive in part from the antitumor effect of high-dose chemotherapy and radiation (1, 2). However, several clinical observations provide convincing evidence that donor immune elements also contribute to the elimination of residual leukemia after BMT. These observations include the reduced risk of relapse after BMT in patients who develop graft-versus-host disease and the increased risk of relapse in patients who receive AZD0156 T cell-depleted donor marrow (3, 4). It also has been demonstrated that relapse after BMT often can be successfully treated by donor lymphocyte infusion (DLI) without additional therapy (5C7). The demonstration that adoptive immunotherapy with donor T cells can provide long-lasting remissions provides compelling evidence that these cells play an important role in mediating a graft-versus-leukemia (GVL) response after allogeneic BMT (8, 9). Appreciation of the importance of GVL has led to the development of less intensive nonmyeloablative approaches for transplantation of allogeneic hematopoietic stem cells with subsequent infusion of donor AZD0156 T cells to enhance antitumor immunity (10C12). Initial reports using these approaches are encouraging and provide evidence that the therapeutic effects of DLI can be extended to provide effective immunity against solid tumors as well as hematopoietic malignancies (13). Although reconstitution with allogeneic stem cells can provide effective antitumor immunity, the mechanisms whereby donor T cells exert this activity are unknown and the target antigens of this response have not been well defined. To better characterize the antitumor effect LeptinR antibody of DLI we previously examined the reconstitution of T AZD0156 and B cell immunity in patients with chronic myelocytic leukemia (CML) who received infusions of CD4+ donor lymphocytes for treatment of relapse after allogeneic BMT (14). Patients with CML were selected for this analysis because the great majority demonstrate a complete cytogenetic and molecular response within a defined time period after DLI and without additional intervention (15). These patients thus represent a unique opportunity to examine a consistently effective antitumor response Hybridization (FISH) Chromosome Localization Analysis. Phage (1 106) from a lambda Dash II human genomic DNA library (Stratagene) were screened by using described methods (20). Genomic DNA from purified positive phage were prepared by using Qiagen Lambda Midi Kit (Qiagen, Valencia, CA). The insert size of positive genomic DNA clones was determined by gel electrophoresis. Exon sequences in the genomic DNA AZD0156 clones encoding CML66 cDNA were confirmed by DNA sequencing. Human FISH chromosome localization was performed by using a CML66 genomic clone with an insert of 23 kb labeled with digoxigenin dUTP by nick translation (Incyte Genomics, St. Louis). Labeled probe was combined with sheared human DNA and hybridized to metaphase chromosomes derived from phytohemagglutinin-stimulated peripheral blood lymphocytes in a solution containing 50% formamide, 10% dextran sulfate, and 2 SSC. Specific hybridization signals were detected by incubating the hybridized slides with fluorescein-conjugated antidigoxigenin antibodies followed by counterstaining with 4,6-diamidino-2-phenylindole. Reverse TranscriptaseCPCR, PCR Cloning, and 5 Rapid Amplification of cDNA Ends. Total RNA was prepared from AZD0156 cultured tumor cell lines, patient CML cells, and normal human PBMC by using RNAzole (Tel-Test, Friendswood, TX). Reverse transcriptaseCPCR and PCR cloning were performed as described (20). A sense primer (25k) specific for the 5 upstream CML66 (5-CGGAGAATTCGGCACGAGTCCCAGTCTCTGTGCGA-3) and a second antisense primer (25c) specific for the 3 downstream CML66 (5-CGGAGAATTCTCATTCTCTGTATTTACTTTTATTAA-3) were used for PCR cloning. All of the PCR cloning reactions were performed by using high-fidelity enzymes such as Pfu Turbo (Stratagene). The 5 rapid amplification of cDNA ends by PCR was performed by using human testis Marathon-Ready cDNAs as templates with a CML66-specific antisense primer 25H (5-CCCAGGTAGAAGATGAGAAATGGATA-3) and the primer AP1 or AP2 specific for the adapter sequence (CLONTECH). PCR-amplified products were subcloned into the pCRII-TOPO vector (Invitrogen), followed by DNA sequencing. Real-Time Quantitative PCR. Quantitative PCR was performed by using were subjected to 10C12% SDS/PAGE with Tris-glycine buffer and transferred onto nitrocellulose filters in 20% methanol in Tris-glycine buffer. Proteins on the blots were visualized as described (16). Detection of.
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