Induction of Potent Anti-B-Cell Lymphoma CTL Responses by the Same Immunoglobulin Variable Region Subfamily Framework-Derived Peptides.

The immunoglobulin variable region (IgHV) which also be called the idiotype (Id) is the best-characterized tumor antigen expressed in B-cell lymphoma. It can function as a target for T-cell-mediated immune responses. However, the usefulness of this approach is limited because vaccines must be patient-specific. IgHV gene can be divided into seven subfamilies, which share the same gene sequence within their framework regions (FR). Here, we choice some peptides derived from the different gene subfamily FRs, and tried to identify if these peptides were capable of inducing cytotoxic T-lymphocyte (CTL) responses, and if the special CTL had possibility to kill B-cell lymphoma cells that belonging to the same subfamily. Meantime we explore if the limitations could be overcome by using the FR-derived peptides. In our study, we amplified seven respective IgHV gene subfamilies by PCR and directly sequenced for 108 cases of acute lymphoblastic leukemias. Complete VHDJH rearrangements were identified in 71(66%)out of 108 cases with 34 (47.89%) monoallelic rearrangements, 30 (42.25%) biallelic rearrangements and 7 (9.86%) oligoclonal rearrangements. SYFPEITHI and BIMAS software databases were used to predict T-cell epitopes in the seven different IgHV subfamily’s FR, and to find that these peptides have the same sequence as their germ line gene. Using an antigen-specific T-cell expansion system in vitro, we stimulated the PBMC from the health HLA-A0201 donor by the IgHV1 peptide(QLVQSGAEV) loading APC every week, and demonstrated that the predicted peptides could generate peptide-specific CTL successfully in healthy donors. The frequency of CD8+ and IgHV1 peptide(QLVQSGAEV)/tetramer+ double positive cells in the gated lymphocyte population was 0.38% before stimulation and increased to 49.38 % after forth stimulation . The cytokine release assay and the cytotoxicity assay indicate that these CTLs are capable of killing the lymphoma cell line belonging to the same family in a peptide-specific, and MHC-restricted way. Furthermore, the CTL stimulated by the IgHV1 peptide cannot kill the target cell pulsing a different subfamily peptide, indicating that the cytotoxicity is family-specific. Using the RT-PCR method, TCRBV expression of the CTLs were analyzed and the PCR products were sequenced directly. We found after four stimulations, the TCRBV expression were skewed in several families, and all the clones were found to have identical nucleotide sequences. In conclusion, our results demonstrate that the family-specific peptides derived from the IgHV FR can successfully lead to the generation of peptide-specific CTLs in vitro. Such defined antigenic epitopes may not only serve as candidates for novel lymphoma immunotherapy strategies, but also may help in discovering the interaction between the B-cell and T-cell clones.