DISTINCT HOMOTYPIC B-CELL RECEPTOR INTERACTIONS SHAPE THE OUTCOME OF CHRONIC LYMPHOCYTIC LEUKEMIA
(Abstract release date: 05/19/16)
EHA Library. Ghia P. 06/10/16; 135149; S116
Dr. Paolo Ghia
Contributions
Contributions
Abstract
Abstract: S116
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 11:45 - 12:00
Location: Auditorium 2
Background
Signaling initiated by antigen binding to the B-cell receptor immunoglobulin (BcR IG) is paramount for CLL development and evolution. Indeed, BcR signaling pathways are constitutively active in all CLL cases and recently, a cell-autonomous model of CLL cell activation has been proposed, whereby all CLL-derived BcR IGs could promote Ca2+ influx and NF-κB target gene transcription through the binding of a common internal epitope on the BcR IG.
Aims
We investigated the relevant molecular interactions underlying cell-autonomous signaling in CLL cases with distinct and opposite clinical outcome in order to understand the molecular basis of the clinical disease heterogeneity. We focused on CLL stereotyped subset #4 (IGHV4-34/IGKV2-30), a particularly indolent clinical subgroup with a characteristic anergic functional phenotype of the malignant cells, and stereotyped subset #2 (IGHV3-21/IGLV3-21), noted for a dismal prognosis, largely independent of p53 dysfunction.
Methods
We produced recombinant IG molecules from CLL leukemic cells. The BcR IGs were analyzed for self-association using analytical ultracentrifugation, and their structure was determined by x-ray crystallography. Recombinant BcRs were transfected in the RAG2/λ5/SLP65 triple knockout murine pre B-cell line (TKO cells) engineered to include a tamoxifen-inducible ERT2-SLP65 fusion protein. Addition of 4-hydroxy tamoxifen in the absence of exogenous antigen induces an increase in Ca2+ influx in case of autonomous signaling of the tested BcR.
Results
The BcR IGs of both subsets interact in the crystals in a geometry highly indicative of specific homotypic recognition, albeit with distinct features. In subset #4, the VH CDR3 binds to a conformational epitope spanning the Heavy chain (VH and CH1 domains), while in subset #2 the VL CDR1 and CDR2 loops contact a surface composed of residues from the Light chain (VL FR1 and the linker between the VL and CL domains). Using analytical ultracentrifugation, we confirmed that the contacts observed in the experimental crystal structures for both subsets were also occurring in solution. Interestingly, the association in solution of the receptors was markedly different for the two subsets with persistent binding in subset #4 cases with indolent disease, versus weaker interactions in subset #2 aggressive cases. For both subsets, using the TKO tamoxifen-inducible system and Ca2+ influx as a readout, we found that the observed interactions activate cell-autonomous intracellular signaling in the absence of exogenous antigen. Conversely, cells expressing epitope or paratope mutants of either receptor did not demonstrate significant variations in Ca2+ influx, confirming the relevance of the homotypic interactions revealed by the crystal structures.
Conclusion
We provide the first description to high resolution of a homotypic association process in BcRs that resembles antibody-antigen recognition and leads to intracellular signaling in CLL cells. BcR IGs from CLL cases with different prognosis bind homotypically via their combining sites to specific, diverse epitopes to initiate intracellular signalling. Our results suggest that the avidity of BcR self-recognition may directly underlie the clinical course of CLL, since tight, persistent binding was noted in cases with indolent disease whereas weaker interactions characterized the aggressive progressive cases.
Session topic: CLL natural history and progression
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 11:45 - 12:00
Location: Auditorium 2
Background
Signaling initiated by antigen binding to the B-cell receptor immunoglobulin (BcR IG) is paramount for CLL development and evolution. Indeed, BcR signaling pathways are constitutively active in all CLL cases and recently, a cell-autonomous model of CLL cell activation has been proposed, whereby all CLL-derived BcR IGs could promote Ca2+ influx and NF-κB target gene transcription through the binding of a common internal epitope on the BcR IG.
Aims
We investigated the relevant molecular interactions underlying cell-autonomous signaling in CLL cases with distinct and opposite clinical outcome in order to understand the molecular basis of the clinical disease heterogeneity. We focused on CLL stereotyped subset #4 (IGHV4-34/IGKV2-30), a particularly indolent clinical subgroup with a characteristic anergic functional phenotype of the malignant cells, and stereotyped subset #2 (IGHV3-21/IGLV3-21), noted for a dismal prognosis, largely independent of p53 dysfunction.
Methods
We produced recombinant IG molecules from CLL leukemic cells. The BcR IGs were analyzed for self-association using analytical ultracentrifugation, and their structure was determined by x-ray crystallography. Recombinant BcRs were transfected in the RAG2/λ5/SLP65 triple knockout murine pre B-cell line (TKO cells) engineered to include a tamoxifen-inducible ERT2-SLP65 fusion protein. Addition of 4-hydroxy tamoxifen in the absence of exogenous antigen induces an increase in Ca2+ influx in case of autonomous signaling of the tested BcR.
Results
The BcR IGs of both subsets interact in the crystals in a geometry highly indicative of specific homotypic recognition, albeit with distinct features. In subset #4, the VH CDR3 binds to a conformational epitope spanning the Heavy chain (VH and CH1 domains), while in subset #2 the VL CDR1 and CDR2 loops contact a surface composed of residues from the Light chain (VL FR1 and the linker between the VL and CL domains). Using analytical ultracentrifugation, we confirmed that the contacts observed in the experimental crystal structures for both subsets were also occurring in solution. Interestingly, the association in solution of the receptors was markedly different for the two subsets with persistent binding in subset #4 cases with indolent disease, versus weaker interactions in subset #2 aggressive cases. For both subsets, using the TKO tamoxifen-inducible system and Ca2+ influx as a readout, we found that the observed interactions activate cell-autonomous intracellular signaling in the absence of exogenous antigen. Conversely, cells expressing epitope or paratope mutants of either receptor did not demonstrate significant variations in Ca2+ influx, confirming the relevance of the homotypic interactions revealed by the crystal structures.
Conclusion
We provide the first description to high resolution of a homotypic association process in BcRs that resembles antibody-antigen recognition and leads to intracellular signaling in CLL cells. BcR IGs from CLL cases with different prognosis bind homotypically via their combining sites to specific, diverse epitopes to initiate intracellular signalling. Our results suggest that the avidity of BcR self-recognition may directly underlie the clinical course of CLL, since tight, persistent binding was noted in cases with indolent disease whereas weaker interactions characterized the aggressive progressive cases.
Session topic: CLL natural history and progression
Abstract: S116
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 11:45 - 12:00
Location: Auditorium 2
Background
Signaling initiated by antigen binding to the B-cell receptor immunoglobulin (BcR IG) is paramount for CLL development and evolution. Indeed, BcR signaling pathways are constitutively active in all CLL cases and recently, a cell-autonomous model of CLL cell activation has been proposed, whereby all CLL-derived BcR IGs could promote Ca2+ influx and NF-κB target gene transcription through the binding of a common internal epitope on the BcR IG.
Aims
We investigated the relevant molecular interactions underlying cell-autonomous signaling in CLL cases with distinct and opposite clinical outcome in order to understand the molecular basis of the clinical disease heterogeneity. We focused on CLL stereotyped subset #4 (IGHV4-34/IGKV2-30), a particularly indolent clinical subgroup with a characteristic anergic functional phenotype of the malignant cells, and stereotyped subset #2 (IGHV3-21/IGLV3-21), noted for a dismal prognosis, largely independent of p53 dysfunction.
Methods
We produced recombinant IG molecules from CLL leukemic cells. The BcR IGs were analyzed for self-association using analytical ultracentrifugation, and their structure was determined by x-ray crystallography. Recombinant BcRs were transfected in the RAG2/λ5/SLP65 triple knockout murine pre B-cell line (TKO cells) engineered to include a tamoxifen-inducible ERT2-SLP65 fusion protein. Addition of 4-hydroxy tamoxifen in the absence of exogenous antigen induces an increase in Ca2+ influx in case of autonomous signaling of the tested BcR.
Results
The BcR IGs of both subsets interact in the crystals in a geometry highly indicative of specific homotypic recognition, albeit with distinct features. In subset #4, the VH CDR3 binds to a conformational epitope spanning the Heavy chain (VH and CH1 domains), while in subset #2 the VL CDR1 and CDR2 loops contact a surface composed of residues from the Light chain (VL FR1 and the linker between the VL and CL domains). Using analytical ultracentrifugation, we confirmed that the contacts observed in the experimental crystal structures for both subsets were also occurring in solution. Interestingly, the association in solution of the receptors was markedly different for the two subsets with persistent binding in subset #4 cases with indolent disease, versus weaker interactions in subset #2 aggressive cases. For both subsets, using the TKO tamoxifen-inducible system and Ca2+ influx as a readout, we found that the observed interactions activate cell-autonomous intracellular signaling in the absence of exogenous antigen. Conversely, cells expressing epitope or paratope mutants of either receptor did not demonstrate significant variations in Ca2+ influx, confirming the relevance of the homotypic interactions revealed by the crystal structures.
Conclusion
We provide the first description to high resolution of a homotypic association process in BcRs that resembles antibody-antigen recognition and leads to intracellular signaling in CLL cells. BcR IGs from CLL cases with different prognosis bind homotypically via their combining sites to specific, diverse epitopes to initiate intracellular signalling. Our results suggest that the avidity of BcR self-recognition may directly underlie the clinical course of CLL, since tight, persistent binding was noted in cases with indolent disease whereas weaker interactions characterized the aggressive progressive cases.
Session topic: CLL natural history and progression
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 11:45 - 12:00
Location: Auditorium 2
Background
Signaling initiated by antigen binding to the B-cell receptor immunoglobulin (BcR IG) is paramount for CLL development and evolution. Indeed, BcR signaling pathways are constitutively active in all CLL cases and recently, a cell-autonomous model of CLL cell activation has been proposed, whereby all CLL-derived BcR IGs could promote Ca2+ influx and NF-κB target gene transcription through the binding of a common internal epitope on the BcR IG.
Aims
We investigated the relevant molecular interactions underlying cell-autonomous signaling in CLL cases with distinct and opposite clinical outcome in order to understand the molecular basis of the clinical disease heterogeneity. We focused on CLL stereotyped subset #4 (IGHV4-34/IGKV2-30), a particularly indolent clinical subgroup with a characteristic anergic functional phenotype of the malignant cells, and stereotyped subset #2 (IGHV3-21/IGLV3-21), noted for a dismal prognosis, largely independent of p53 dysfunction.
Methods
We produced recombinant IG molecules from CLL leukemic cells. The BcR IGs were analyzed for self-association using analytical ultracentrifugation, and their structure was determined by x-ray crystallography. Recombinant BcRs were transfected in the RAG2/λ5/SLP65 triple knockout murine pre B-cell line (TKO cells) engineered to include a tamoxifen-inducible ERT2-SLP65 fusion protein. Addition of 4-hydroxy tamoxifen in the absence of exogenous antigen induces an increase in Ca2+ influx in case of autonomous signaling of the tested BcR.
Results
The BcR IGs of both subsets interact in the crystals in a geometry highly indicative of specific homotypic recognition, albeit with distinct features. In subset #4, the VH CDR3 binds to a conformational epitope spanning the Heavy chain (VH and CH1 domains), while in subset #2 the VL CDR1 and CDR2 loops contact a surface composed of residues from the Light chain (VL FR1 and the linker between the VL and CL domains). Using analytical ultracentrifugation, we confirmed that the contacts observed in the experimental crystal structures for both subsets were also occurring in solution. Interestingly, the association in solution of the receptors was markedly different for the two subsets with persistent binding in subset #4 cases with indolent disease, versus weaker interactions in subset #2 aggressive cases. For both subsets, using the TKO tamoxifen-inducible system and Ca2+ influx as a readout, we found that the observed interactions activate cell-autonomous intracellular signaling in the absence of exogenous antigen. Conversely, cells expressing epitope or paratope mutants of either receptor did not demonstrate significant variations in Ca2+ influx, confirming the relevance of the homotypic interactions revealed by the crystal structures.
Conclusion
We provide the first description to high resolution of a homotypic association process in BcRs that resembles antibody-antigen recognition and leads to intracellular signaling in CLL cells. BcR IGs from CLL cases with different prognosis bind homotypically via their combining sites to specific, diverse epitopes to initiate intracellular signalling. Our results suggest that the avidity of BcR self-recognition may directly underlie the clinical course of CLL, since tight, persistent binding was noted in cases with indolent disease whereas weaker interactions characterized the aggressive progressive cases.
Session topic: CLL natural history and progression
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