HIGHER ORDER IMMUNOGLOBULIN REPERTOIRE RESTRICTIONS IN CLL: THE ILLUSTRATIVE CASE OF STEREOTYPED SUBSETS #2 AND #169

Chronic lymphocytic leukemia (CLL) major stereotyped subset #2 (IGHV3-21/IGLV3-21, ~2.5% of all CLL) is an aggressive disease variant, irrespective of the somatic hypermutation (SHM) status of the clonotypic IGHV gene. Minor stereotyped subset #169 (IGHV3-48/IGLV3-21, ~0.2% of all CLL) is related to subset #2 as it displays a highly similar variable antigen-binding site. We further explored this relationship through next-generation sequencing and crystallographic analysis of the clonotypic B cell receptor immunoglobulin (BcR IG). Branching evolution of the predominant clonotype through intraclonal diversification (ID) in the context of ongoing SHM was evident in both heavy and light chain genes of both subsets. Molecular similarities between the two subsets were highlighted by the finding of shared SHMs within both the heavy and the light chain genes in all analyzed cases at either clonal or subclonal level. Particularly noteworthy in this respect was a ubiquitous SHM at the linker region between the variable and the constant domain of the IGLV3-21 light chains, previously reported as critical for IG homotypic interactions underlying cell-autonomous signaling capacity. Notably, crystallographic analysis revealed that the IGLV3-21-bearing CLL subset #169 IG retains the same geometry and contact residues for the homotypic intermolecular interaction observed in subset #2, including the SHM at the linker region, and from a molecular standpoint belong to a common structural mode of autologous recognition. Collectively, our findings document that stereotyped subsets #2 and #169 are very closely related, displaying shared IG features that can be explained only in the context of shared functional selection.