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M. Montesinos-Rongen et al.
counterparts' encoded poly-/autoreactive antibodies, indi- cating that SHM plays an important role in disease devel- opment by altering original BCR autoreactivity and nega- tively selecting for auto-/polyreactivity.12 In this regard, SHM performs its physiological function in B-cell differen- tiation to eliminate self-reactive B cells.17 SHM also strong- ly impacts on BCR reactivity in PCNSL, however, with opposite results, at least in some instances. In PCNSL, a failure of the selection process in the GC reaction may be a crucial event broadening autoantigenic reactivity, there- by fostering survival upon antigen encounter.
Physiologically, low-affinity, autoreactive B cells with IgD+ IgM+ immunophenotype, a hallmark of PCNSL,24 are anergic, short-lived and have long been considered to be excluded from the GC.25 However, it was recently shown that in particular conditions autoreactive B cells may enter a GC reaction, but lose their autoreactivity as a result of SHM.26 Such a mechanism has also been reported for B cells using the IGHV4-34 gene that often encodes autore- active antibodies.2 PCNSL also preferentially rearrange the IGHV4-34 gene.2-4 On the other hand, B cells can acquire poly-/self-reactivity during the GC reaction, which may increase antibody affinity for a pathogen.28,29 Whether such a mechanism depicted for memory B cells28 may also underlie the increased poly-/self-reactivity of the tumor cells of PCNSL, and whether they have been selected for a foreign antigen, is unknown. In this regard, it is of note that the mutation pattern of the IG genes of PCNSL sug- gested triggering even by a (viral) superantigen.2,3
Similar to autoreactive VH4-34+ B cells redeemed from elimination in the GC, 72% of IGHV4-34+ PCNSL showed a mutational loss of the CDR2 sequence motif4 that pro- motes N-linked glycosylation of CDR2, an event increas- ing accessibility of the binding site to eliciting foreign anti- gen.26 Thus, one may hypothesize that in PCNSL, naïve precursor B cells may enter the GC where they undergo SHM as part of the rescue process.
However, if the B cells in the process of developing into lymphoma cells or already corresponding to tumor cells were rescued by redemption of autoreactive non-malig- nant B cells, this process, nevertheless, failed, because it did not yield B cells with reduced auto-/polyreactivity, but rather with increased polyreactivity, although some autospecificities were lost. Interestingly, with progression from the naïve to the mutated BCR, overall, the tBCR gained autoreactivity for proteins expressed in the CNS including S100 protein family members and constituents of myelin/oligodendrocytes, including MPLZ1, MBP, and MOBP, which are widely and prominently expressed in the CNS, particularly in the white matter and also, at low level, by neurons.30 This expression pattern may explain, at least in part, the preferential growth of PCNSL in deep brain structures of the cerebral hemispheres, along fiber tracts, and in the basal ganglia.31
Another antigen recognized by nBCR and/or tBCR was SNRPC, which is involved in pre-mRNA splicing.32 In con- ditions with chronic B-cell activation, such as systemic lupus erythematosus and other rheumatic diseases, autoantibodies against various ribonucleoproteins are fre- quent.33-36 SNRPC, expressed by the majority of tumor cells in 90% of PCNSL, may become accessible to tBCR upon necrosis, which is frequent in PCNSL.37 These data extend our observation on galectin-3, also expressed on PCNSL tumor cells,10 as a potential target for interactions with the BCR. Additionally, nBCR and tBCR may recog-
nize intrinsic BCR structures, ultimately inducing cell- autonomous signaling similar to CLL.38
Our data on polyreactive BCR in PCNSL are in line with reports from other mature B-cell lymphomas,16,39-41 suggest- ing a role for chronic antigenic stimulation in PCNSL pathogenesis. This hypothesis is supported by a recent study on PCNSL which extended the list of autoantigenic targets by abnormally hyperglycosylated SAMD14 and neurabin-I.42 SAMD14 reactivity was shown for 8 of 12 (67%) PCNSL with hyperglycosylation of SAMD14/neurabin-I in six (50%).42 We noticed a compa- rable neurabin-I reactivity in 14 of 20 (70%) PCNSL in an independent series (Online Supplementary Table S1); how- ever, western blots bands were mostly weak (Online Supplementary Figure S9). These data indicate neurabin-I, expressed by neurons and axons (Online Supplementary Figure S10), as potential antigen in a fraction, but not all, PCNSL. Our recAb, however, did not bind to proteins in PCNSL lysates (Online Supplementary Figure S6), because western blot experimental conditions were too stringent. A lack of further concordant data between our work and that of Thurner et al.42 may be attributed to technical dif- ferences. The ProtoArray and the Unipex differ in their proteins. Focussing on high-affinity antibodies, Thurner et al.42 pooled Fab fragments while we designed complete IgG antibodies which were studied individually, consider- ing all recAb-reactive proteins in subsequent analyses.
In conclusion, B cells with auto-/polyreactive nBCR may enter the GC where SHM may further increase BCR auto-/polyreactivity (Online Supplementary Figure S11). Together with mutation-induced sustained active BCR signaling and the inability to terminate SHM, the cells may become eligible for reaction with a plethora of anti- gens, particularly of CNS antigens. Thus, in the target organ, a microenvironment fertile for PCNSL prolifera- tion may result from stimulating CNS antigens, autoanti- gens liberated by dying tumor cells or expressed on the surface of tumor cells, resulting in a vicious cycle of uncontrolled proliferation sustained by the interaction of an active tBCR with (auto)antigens, as a consequence of a faulty GC reaction. While this concept does not fit into the 'classical' view that, physiologically, the GC reaction exclusively selects for high-affinitiy antibodies, it extends novel findings on low-affinity BCR that play a role in immunological memory by adding PCNSL as a lym- phoma resulting from a dysregulated GC reaction.
Disclosures
No conflicts of interest to disclose.
Contributions
MMR, MT, AB, MD and CM performed experiments; MMR, MT, AB and MD analyzed results and prepared the fig- ures; MMR, AB and MD designed the research; IB provided crucial material; IB and KM discussed the manuscript; MMR, RK and MD discussed the results and provided important intel- lectual input; MD wrote the paper; MH, MMR and MT per- formed statistical analysis.
Acknowledgments
The authors thank Mariana Carstov, Elena Fischer, and Diana Rudakova for their expert technical assistance.
Funding
This work was supported by a grant from the Wilhelm Sander- Stiftung (Grant 2011.092.2) (to MD and MMR) and the
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