Introduction: Several features of the B-cell receptor (BCR) complex have emerged as major markers for prognostic classification of B-cell chronic lymphocytic leukemia (B-CLL). Characterizing BCR signaling profiles may help to identify novel biomarkers for patient classification, disease monitoring and therapeutic targeting of B-CLL. In this study, we have used phage-display random peptide libraries (RPLs) for identifying peptide ligands of the Ig-BCR of human B-cell Chronic Lymphocytic Leukemia (CLL) in order to determine the epitope diversity recognized by the Ig-BCR. Materials and methods: The full-length Ig gene transcripts (VDJ) of the B-CLL patient #1 were amplified by two nested PCRs using a combination of forward primers specific for the respective VH, Vκ or Vλ leader regions and a reverse primer specific for the respective constant region. The PCR products were sequenced and analysed by IgBlast to identify the IgV mutation status. The IgVH and IgVL PCR products were cloned into eukaryotic expression vectors and co-expressed in 293T cells in order to produce the corresponding IgBCR. The quality of the recombinant IgBCR was assessed by SDS-PAGE under non-reducing and reducing conditions. Recombinant IgBCR was used as bait to screen a phage-display RPL to identify mimotope ligands, which were sequenced to obtain the primary structure of Id-peptides. The binding specificity of Id-peptides to the relative IgBCR on B-CLL #1 cells was determined by flow cytometry. Results and discussion: We identified Id-peptides composed by nine amminoacidsfour, which specifically bound to the idiotypic determinants of the CLL Ig-BCR patient #1. A pool of these Id- peptides was shared by other ten B-CLL patients, suggesting the possibility of common epitopes recognition. We plan to extend this analysis to a statistically significant number of mutated and unmutated CLL patients in order to determine differences in the antigenic reactivity between the two classes of CLL patients as well as to follow the epitope recognition in the clinical course of disease. Conclusion: The described strategy is able to provide us of (i) patient-specific and tumor-specific Ig-BCR for screening RPLs, (ii) monitoring the CLL clinical progression by recognizing the circulating B-CLL in lymph nodes and bone marrow, and (iii) prognostic information based on the IgVH genetic status of each patient.

Id-peptides as biomarkers of B-cell Chronic Lymphocytic Leukemia

Mimmi S;Vecchio E;Fiume G;Palmieri C;Quinto I;
2015-01-01

Abstract

Introduction: Several features of the B-cell receptor (BCR) complex have emerged as major markers for prognostic classification of B-cell chronic lymphocytic leukemia (B-CLL). Characterizing BCR signaling profiles may help to identify novel biomarkers for patient classification, disease monitoring and therapeutic targeting of B-CLL. In this study, we have used phage-display random peptide libraries (RPLs) for identifying peptide ligands of the Ig-BCR of human B-cell Chronic Lymphocytic Leukemia (CLL) in order to determine the epitope diversity recognized by the Ig-BCR. Materials and methods: The full-length Ig gene transcripts (VDJ) of the B-CLL patient #1 were amplified by two nested PCRs using a combination of forward primers specific for the respective VH, Vκ or Vλ leader regions and a reverse primer specific for the respective constant region. The PCR products were sequenced and analysed by IgBlast to identify the IgV mutation status. The IgVH and IgVL PCR products were cloned into eukaryotic expression vectors and co-expressed in 293T cells in order to produce the corresponding IgBCR. The quality of the recombinant IgBCR was assessed by SDS-PAGE under non-reducing and reducing conditions. Recombinant IgBCR was used as bait to screen a phage-display RPL to identify mimotope ligands, which were sequenced to obtain the primary structure of Id-peptides. The binding specificity of Id-peptides to the relative IgBCR on B-CLL #1 cells was determined by flow cytometry. Results and discussion: We identified Id-peptides composed by nine amminoacidsfour, which specifically bound to the idiotypic determinants of the CLL Ig-BCR patient #1. A pool of these Id- peptides was shared by other ten B-CLL patients, suggesting the possibility of common epitopes recognition. We plan to extend this analysis to a statistically significant number of mutated and unmutated CLL patients in order to determine differences in the antigenic reactivity between the two classes of CLL patients as well as to follow the epitope recognition in the clinical course of disease. Conclusion: The described strategy is able to provide us of (i) patient-specific and tumor-specific Ig-BCR for screening RPLs, (ii) monitoring the CLL clinical progression by recognizing the circulating B-CLL in lymph nodes and bone marrow, and (iii) prognostic information based on the IgVH genetic status of each patient.
2015
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.12317/58324
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