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Impact of GC reaction on self-/polyreactivity in PCNSL
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B
Figure 1. Recognition pattern of recombinant antibodies (recAb) derived from naïve B-cell receptor (nBCR) and tumor B-cell receptor (tBCR) of primary lymphoma of the central nervous system (PCNSL) on the ProtoArray. (A) Quantitative Venn diagrams show numbers of proteins recognized by at least one recAb. The numbers of proteins recognized exclusively by nBCR or tBCR are shown in the middle panel. The bottom panel depicts numbers of proteins recognized exclusively by nBCR or tBCR derived from IGHV3+ or IGHV4-34+ PCNSL. (B) Quantitative Venn diagrams show numbers of CNS proteins recognized by at least one recAb (upper panel). The numbers of CNS proteins recognized exclusively by nBCR or tBCR are shown in the middle panel. The bottom panel depicts numbers of CNS proteins recognized exclusively by nBCR or tBCR derived from either IGHV3+ or IGHV4-34+ PCNSL.
and SNRPC (Online Supplementary Table S5). Regarding CNS proteins recognized by nBCR, 64 and 33 CNS pro- teins were exclusively recognized by nBCR derived from IGHV3+ and IGHV4-34+ PCNSL, respectively; 105 proteins were recognized in common (Figure 1B). Regarding CNS proteins recognized by tBCR, 143 and 112 CNS proteins were exclusively recognized by the tBCR derived from IGHV3+ and IGHV4-34+ PCNSL, respectively; 170 proteins were recognized in common (Online Supplementary Table S5).
Focusing on individual PCNSL, tBCR of both IGHV3+ and IGHV4-34+ PCNSL recognized significantly increased numbers of proteins compared to nBCR (Figure 2A-D). Proteins recognized exclusively by tBCR expressed in the CNS included histones, ribosomal proteins, CNS hor- mones (arginine vasopressin-induced 1, neuropeptide Y), proteins involved in glial cell metabolism (S100-A13, MPZL1, MBP, MOBP), and mitochondria (ATP/ATPases, cytochrome C) (Online Supplementary Table S5).
Thus, nBCR alter their protein recognition pattern with B-cell maturation. In addition to proteins recognized by both nBCR and tBCR and those exclusively bound by tBCR, we also detected proteins that were recognized by
the nBCR and lost reactivity with modification towards the tBCR. Importantly and of potentially functional rele- vance, the BCR increases its protein reactivity upon SHM enabling recognition of increased numbers of antigens expressed in the CNS.
Reactivity of naïve and tumor B-cell receptor with central nervous system proteins determined by immunoprecipitation
Normal brain biopsies were used for immunoprecipita- tion with all recAb. A total of 270 proteins (mean: 80.2, median: 82.5, range: 44-124) co-immunoprecipitated with at least one recAb (Figure 3A); 214 and 241 proteins co- immunoprecipitated with at least one nBCR and tBCR, respectively. NBCR and tBCR exclusively immunoprecip- itated 29 and 56 proteins, respectively; 185 proteins were recognized in common. Regarding nBCR corresponding to IGHV3+ and IGHV4-34+ PCNSL, 59 and 42 proteins were exclusively immunoprecipitated, respectively; 113 were recognized in common. With respect to the tBCR, 47 pro- teins each were immunoprecipitated exclusively by IGHV3+ and IGHV4-34+ PCNSL; 147 proteins were immunoprecipitated in common (Figure 3A-C).
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