Postranslationally Modified Proteins in the Central Nervous System (CNS) Are the Dominant Antigenic Target/Stimulus of the B-Cell Receptor (BCR) in Primary CNS Lymphomas (PCNSL) Providing Strong Evidence for the Role of Chronic Autoantigenic Stimulation As an Early Step in the Pathogenesis of Aggressive B-Cell Lymphomas
Authors: Thurner Lorenz , Kemele Maria , Fadle Natalie , Regitz Evi , Roth Patrick , Weller Michael , Szczepanowski Monika , Klapper Wolfram , Monoranu Camelia-Maria , Rosenwald Andreas , Buslei Rolf , Bohle Rainer Maria , Kim Yoo-Jin , Preuss Klaus-Dieter , Pfreundschuh Michael 
Published: October 2019

Abstract
Background: Sequence analyses of variable immunoglobulin gene fragments of PCNSL from immunocompetent patients have shown a VH4-34 restriction raising speculations on a selection/stimulation of a functional BCR by an autoantigen in the central nervous system. The present study focused on the search for these hypothetic autoantigens presumably driving the malignant transformation of B-cells into PCNSL cells by chronic BCR stimulation in immunocompetent patients.
Methods: BCRs were expressed as recombinant Fabs based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA of snap-frozen lymphoma specimens and checked for binding to proteins expressed on macroarrays of human cDNA expression libraries.
Results: Recombinant BCR expression was attempted in 21 and successful in twelve PCNSL cases. The VH4-34 family was overrepresented, but was found in less than a quarter of the PCNSL patients (4/21). Screening of the recombinant BCRs on protein arrays revealed that 8 of 12 recombinant PCNSL-BCRs reacted with SAMD14 and the SAM domain of neurabin-1, two proteins with high homology and preferential expression in the CNS. Subsequent proteomic analysis of cryoconserved lymphoma specimens showed that SAMD14 and the SAM domain of neurabin-1 were alternatively N-glycosylated in patients with a PCNSL-BCR specific for SAMD14 and neurabin-1, but not in the remaining PCNSL patients with BCR specificities other than for SAMD14/neurabin-1. Compared to SAMD14 and neurabin-1 from healthy controls, Asn 339 of SAMD14 and Asn 1277 of neurabin-1 were shown to carry additionally glycosylated Asn residues. Of interest, both additional glycosylation sites belonged to atypical, non-canonical Asn-Leu-Glu-Gln (N-L-E-Q) sites instead of the Asn-X-Ser/Thre consensus sequence (N-X-S/T; where X can be any amino acid except proline), which is reported to constitute 97% of N-glycosylation sites under physiologic circumstances. These atypical N-hyperglycosylations were shown for every case with sufficient biopsy material for this proteomic analysis and a PCNSL-BCR specific for SAMD14 and neurabin-1 in their PCNSL and CNS, and to a lesser degree in their peripheral blood mononuclear cells and patient-derived EBV-transformed lymphoblastoid cell lines (LCLs). Of the recombinant BCRs of all cases with sufficient material to test for hyperglycosylation, only the BCRs of the 6 cases with hyperglycosylated SAMD14/neurabin-1 reacted against SAMD14/neurabin-1. Of note, glycosylation status of 2/8 cases with recombinant SAMD14/neurabin-1 reactive BCRs could not be analyzed due to insufficient cryomaterial left after variable region gene PCRs. No hyperglycosylation of SAMD14 and neurabin-1 was found in the peripheral blood of 400 healthy controls, 100 newborns and 50 nursery residents. Moreover, antibodies against SAMD14 and neurabin-1 were detected in the sera and cerebrospinal fluids of an independent second cohort of patients with PCNSL (8/22), but not in sera of patients with secondary CNS manifestations of systemic DLBCL (0/17) or of healthy controls (0/92).
Conclusion: Our results strongly suggest that the atypical (NLEQ) glycosylation of the highly homologous SAMD14 and the SAM domain of neurabin-1 maintains a chronic autoimmunogenic stimulation in the CNS, ultimately leading to the malignant transformation of B-cells with a BCR specific for these atypically N-hyperglycosylated proteins into an aggressive B-cell lymphoma in the CNS in a majority of patients with PCNSL. The fact that the VH4-34 family represents only a minority of VH families recognizing SAMD14/neurabin-1 underlines the extraordinary autoimmunogenicity of these posttranslationally modified proteins in a broad range of individuals. Our results provide the first and strongest experimental evidence for the role of chronic autoantigenic stimulation as a first step in the pathogenesis of aggressive B-cell lymphomas.
Supported by Deutsche Forschungsgemeinschaft DFG, Deutsche José Carreras Leukämie Stiftung, Wilhelm-Sander-Stiftung, Deutsche Krebshilfe e.V.

Disclosures
No relevant conflicts of interest to declare.