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leukemic clones, and the activation of BcR-triggered signalling pathways drives CLL ontogeny. Strikingly, CLL-derived BcRs are unique in being able to mediate antigen-independent signalling, raising the possibility that BcRs recognise intrinsic motifs.

To understand the structural basis of cell-autonomous signalling in CLL, the crystal structures of different CLL-derived BcR Fabs were determined at resolutions ranging from 2.1 to 3.4 Å, by collecting single-crystal diffraction data at beamlines ID30A-1, ID23-1 and ID29 (Figure 23). The BcR Fabs belonged to three different CLL subsets: two from clinically indolent subset #4, and one each from clinically aggressive subsets #2 and #169. In subset #4 BcRs, the stereotyped HCDR3 mediates intermolecular interactions with residues in a second molecule s heavy chain (Figure 24a). On the other hand, the light chain dominates the homotypic interactions in the structures of subsets #2 and #169, and the LCDR1 and 2 of the light chain play a crucial role (Figure 24a).

Analysis of the residues involved in these intermolecular contacts through mutagenesis, analytical

ultracentrifugation (AUC) and Ca2+ influx assays demonstrated that the molecular arrangements in the crystals recapitulate the physiological BcR self-recognition leading to CLL cell proliferation [2]. This study enabled a structural understanding of the sculpting and stronger complementarity of the observed epitopes-paratopes given by specific gene usage, mutations and class-switch recombination phenomena prevalent within CLL subsets, hence explaining functional selection. Finally, affinity and half-life measurements in solution showed that BcR IGs derived from indolent clinical cases are stably associated, whereas in the aggressive ones, the interactions are weak and short-lived (Figure 24b).

This work sheds light on the atomic structure and interaction between BcRs, crucial for the classification of CLL patients into homogenous groups and for rationalisation of the diverse clinical outcomes and functional behaviours. The structures will inform the rational identification of antagonist molecules towards personalised treatments that target specific BcR self-recognition and activation patterns, thus taking into consideration the diversity of the pathology in different patients.

Fig. 24: Analysis of the interactions between CLL-derived Fab from subsets #4, #2 and

#169. a) Close-up of the interacting residues (sticks) for Fabs of the indicated subsets,

coloured and labelled as in Figure 23. b) AUC analysis shows stable association

between the clinically indolent subset #4 BcR Fabs (left), but not the aggressive subsets #2

and #169 (right).