Contributions
Abstract: EP609
Type: E-Poster Presentation
Session title: Chronic lymphocytic leukemia and related disorders - Biology & Translational Research
Background
Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients carrying stereotyped i.e. quasi-identical B-cell receptor immunoglobulins (BcR IG), strongly suggesting restricted antigenic selection. Data showing autonomous BcR signaling through homotypic interactions between BcR IG molecules, distinctive for each subset, suggest that the selecting epitopes may lie within the clonotypic BcR IG itself. Antigenic drive appears relevant also for T cells in CLL, where published evidence supports that CLL T cells are selected by restricted, subset-specific tumor antigens. That said, whether the antigens selecting CLL progenitors and T cells are the same remains obscure.
Aims
We performed ad hoc prediction of putative T-cell class I neoepitopes contained within the clonotypic BcR IGs of CLL patients, with an intended bias towards major stereotyped subsets.
Methods
We selected 27 patients to represent the full spectrum of CLL: (i) with mutated IGHV genes (M-CLL, n=5), (ii) with unmutated IGHV genes (U-CLL, n=5), (iii) assigned to major stereotyped subsets (subset #1, n=7; subset #2, n=5; subset #4, n=5). RT-PCR was performed for the heavy (H) and the light (K/L) IG chain using subgroup-specific leader primers for the IGHV/IGK/LV gene and universal primers annealing to the constant region (IGHG, IGHM, IGKC, IGLC), in order to produce a full-length V-(D)-J gene rearrangement sequence, plus the start of the constant domain. PCR products were subjected to direct double-strand Sanger sequencing with a quality-optimized protocol. The amino acid sequences were subsequently parsed in peptides and subjected to NetMHCpan. The rank score was calculated, considering the 4-digit HLA-A and -B typing for each patient separately. Only high-binding peptides (rank score <.5%) were selected. Exact matches to germline and/or proteome databases were excluded.
Results
Overall, 202 predicted neoepitopes were identified in 22/27 patients. All 22 patients had predicted CD8+ T-cell epitopes within the heavy chain, and 19/22 also within the light chain. Half of the peptides (n=106, 52.5%) resulted from somatic hypermutations (SHMs) at the intersections of FR and CDR3 regions (FR1/CDR1, n=14; CDR1/FR2, n=28; FR2/CDR2, n=6; FR3/CDR3, n=17; CDR3/FR4, n=41). The remaining mostly involved the FR3 (n=52) and CDR3 (n=21). Overall, the majority of peptides involved (at least part of) the CDR3 (79/202, n=39.1%), which is particularly relevant given its small length (9-27 aa) within the full sequence (331-660 aa). There was no statistically significant difference in the rank score of peptides involving the CDR3 vs. all others. Peptide clustering revealed similar or identical predicted neoepitopes among different patients (17 clusters of two, 2 clusters of three, 1 cluster of four). Importantly, these clusters involved: (i) shared CDR3 patterns in patients assigned to the same subset, but also (ii) subset-specific recurrent SHMs in the IGHV gene, e.g. G to E SHM at position 28 in the CDR1 of subset #4, which was previously reported to be present in 114/265 (43%) subset #4 cases.
Conclusion
Ad hoc prediction with established bioinformatics algorithms identified a significant number of putative T-cell class I neoepitopes contained within the clonotypic BcR IG of CLL patients. Many of them culminate from subset-specific (‘stereotypic’) CDR3 patterns or recurrent SHMs, suggesting that the targeted SHM which shapes the CLL BcR IG repertoire may produce immunogenic CD8+ T-cell epitopes. Their actual immunogenicity has to be tested in ex vivo studies, currently underway by our group.
Keyword(s): Chronic lymphocytic leukemia, Immunoglobulin, T cell
Abstract: EP609
Type: E-Poster Presentation
Session title: Chronic lymphocytic leukemia and related disorders - Biology & Translational Research
Background
Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients carrying stereotyped i.e. quasi-identical B-cell receptor immunoglobulins (BcR IG), strongly suggesting restricted antigenic selection. Data showing autonomous BcR signaling through homotypic interactions between BcR IG molecules, distinctive for each subset, suggest that the selecting epitopes may lie within the clonotypic BcR IG itself. Antigenic drive appears relevant also for T cells in CLL, where published evidence supports that CLL T cells are selected by restricted, subset-specific tumor antigens. That said, whether the antigens selecting CLL progenitors and T cells are the same remains obscure.
Aims
We performed ad hoc prediction of putative T-cell class I neoepitopes contained within the clonotypic BcR IGs of CLL patients, with an intended bias towards major stereotyped subsets.
Methods
We selected 27 patients to represent the full spectrum of CLL: (i) with mutated IGHV genes (M-CLL, n=5), (ii) with unmutated IGHV genes (U-CLL, n=5), (iii) assigned to major stereotyped subsets (subset #1, n=7; subset #2, n=5; subset #4, n=5). RT-PCR was performed for the heavy (H) and the light (K/L) IG chain using subgroup-specific leader primers for the IGHV/IGK/LV gene and universal primers annealing to the constant region (IGHG, IGHM, IGKC, IGLC), in order to produce a full-length V-(D)-J gene rearrangement sequence, plus the start of the constant domain. PCR products were subjected to direct double-strand Sanger sequencing with a quality-optimized protocol. The amino acid sequences were subsequently parsed in peptides and subjected to NetMHCpan. The rank score was calculated, considering the 4-digit HLA-A and -B typing for each patient separately. Only high-binding peptides (rank score <.5%) were selected. Exact matches to germline and/or proteome databases were excluded.
Results
Overall, 202 predicted neoepitopes were identified in 22/27 patients. All 22 patients had predicted CD8+ T-cell epitopes within the heavy chain, and 19/22 also within the light chain. Half of the peptides (n=106, 52.5%) resulted from somatic hypermutations (SHMs) at the intersections of FR and CDR3 regions (FR1/CDR1, n=14; CDR1/FR2, n=28; FR2/CDR2, n=6; FR3/CDR3, n=17; CDR3/FR4, n=41). The remaining mostly involved the FR3 (n=52) and CDR3 (n=21). Overall, the majority of peptides involved (at least part of) the CDR3 (79/202, n=39.1%), which is particularly relevant given its small length (9-27 aa) within the full sequence (331-660 aa). There was no statistically significant difference in the rank score of peptides involving the CDR3 vs. all others. Peptide clustering revealed similar or identical predicted neoepitopes among different patients (17 clusters of two, 2 clusters of three, 1 cluster of four). Importantly, these clusters involved: (i) shared CDR3 patterns in patients assigned to the same subset, but also (ii) subset-specific recurrent SHMs in the IGHV gene, e.g. G to E SHM at position 28 in the CDR1 of subset #4, which was previously reported to be present in 114/265 (43%) subset #4 cases.
Conclusion
Ad hoc prediction with established bioinformatics algorithms identified a significant number of putative T-cell class I neoepitopes contained within the clonotypic BcR IG of CLL patients. Many of them culminate from subset-specific (‘stereotypic’) CDR3 patterns or recurrent SHMs, suggesting that the targeted SHM which shapes the CLL BcR IG repertoire may produce immunogenic CD8+ T-cell epitopes. Their actual immunogenicity has to be tested in ex vivo studies, currently underway by our group.
Keyword(s): Chronic lymphocytic leukemia, Immunoglobulin, T cell