Structural and Functional Analysis of the Clonotypic B Cell Receptor Immunoglobulin in Splenic Marginal Zone Lymphoma: Ontogenetic and Therapeutic Implications
Authors: Iatrou Anastasia , Patrone Marco , Sarrigeorgiou Ioannis , Pechlivanis Nikolaos , Oscier David Graham , Traverse-Glehen Alexandra , Sandaltzopoulos Raphael , Stavroyianni Niki , Agathangelidis Andreas , Petrakis Georgios , Koletsa Triantafyllia , Lymberi Peggy , Ghia Paolo , Chatzidimitriou Anastasia , Degano Massimo , Stamatopoulos Kostas 
Published: December 2024

Immunogenetic evidence implicates the B cell receptor immunoglobulin (BcR IG) in the natural history of splenic marginal zone lymphoma (SMZL). Indeed, SMZL carries distinct features of somatic hypermutation (SHM) amongst cases utilizing particular IGHV genes in their BcR IG. Moreover, the BcR IG gene repertoire in SMZL is restricted, whereby ~30% of cases utilize the IGHV1-2*04 gene and allele, notable for carrying a tryptophan (W) residue at position VH FR3-75 instead of the arginine (R) residue that is encoded by all remaining IGHV1-2 gene alleles and almost all other human IGHV genes and alleles. IGHV1-2*04 cases display heterogeneous CDR3 features and light chains, as well as pronounced intraclonal diversification within the heavy chain gene rearrangements. Overall, this constellation of features argues for heavy chain dominance in this major immunogenetic subgroup of SMZL. Against that, scant information exists regarding the functional and structural properties of the clonotypic BcR IG in SMZL, while the relevance of the IGHV1-2*04 overuse remains an enigma. In order to address this knowledge gap, we performed immunological, biochemical and crystallographic studies of the clonotypic BcR IG from SMZL cases that we expressed as recombinant monoclonal antibodies (rmAbs). ELISA against molecules that are common antigenic targets of naturally-occurring autoantibodies and disease-occurring/related pathological autoantibodies, namely DNA, actin, myosin, β-amyloid, TNP, thyroglobulin, IgG F(ab)'2 fragments, and trinitrophenyl (TNP), revealed that SMZL rmAbs (n=42, of which 14 utilized the IGHV1-2*04) were poly/autoreactive, regardless of the expressed IGHV gene and their SHM status. Of note, comparison with CLL rmAbs (n=35), known for their poly/autoreactivity, revealed significantly (p&lt;0.05) stronger binding of the SMZL rmAbs to all tested autoantigens on autoantigen protein microarrays. Autoreactivity was corroborated by flow cytometry showing that 38/42 SMZL rmAbs bound viable MEC1, HEK293 and HS-5 cells; and, by immunohistochemistry using the SMZL rmAbs as primary Abs, which documented binding to human tissues (appendix, tonsil, lymph nodes and kidney), albeit with distinct profiles depending on the expressed IGHV gene. Next, in order to assess the functional relevance of the W residue in IGHV1-2*04 BcR IG, we used PCR-based site-directed mutagenesis for modifying W75 to R (IGHV1-2*04 W75R, n=10), documenting significant (p&lt;0.05) differences in antigen reactivity profile between the authentic IGHV1-2*04 rmAbs vs the respective IGHV1-2*04 W75R rmAbs. Further, in order to test the hypothesis of heavy chain dominance, we coupled the IGHV1-2*04 heavy chains with random light chains and compared their antigen reactivity profile versus the authentic rmAbs, observing no differences. Finally, we crystallized two different IGHV1-2*04 SMZL BcR Fabs and determined their structures to high resolution. In all crystals obtained, regardless of pH and precipitating agent, pairs of Fab molecules were found to interact homotypically through a symmetrical stacking cation-π interaction mediated by the allele-specific W75 residue and side chain of R95 in the IGHV1-2*04 heavy chain, burying an extensive surface of ~650 Å2. In silico analysis with FOLDX highlighted the high energetic contribution of these residues in promoting the formation of the homodimers in the crystals. In conclusion, we document pronounced poly/autoreactivity in SMZL suggesting that SMZL likely derives from a progenitor B-cell population with highly restricted BcR IG structures and autoreactive potential. We also provide experimental support to the hypothesis of heavy chain dominance in the IGHV1-2*04 subgroup which raises the intriguing possibility that the IGHV1-2*04 heavy chain may represent a target for immunotherapeutic interventions in a sizeable fraction of SMZL.