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M. Montesinos-Rongen et al.
Table 1. Autoreactivity of the recombinant antibodies tested by HEp-2 immunofluorescence, ANAcombi and ANCAcombi ELISA.
PCNSL IGHV ANA-HEp-2
ANAcombi ANCAcombi tBCR nBCR tBCR
Patient n.
#01
#02
#03
#04
#05
#06
#07
#08
#09
#10
a: RNP-70 (small nuclear ribonucleoprotein U1 subunit 70); b: lysozyme; c: lysozyme, elastase, myeloperoxidase, BPI (bactericidal permeability-increasing protein); d: lysozyme, elastase, proteinase 3. PCNSL: primary central nervous system lymphoma; nBCR: naïve B-cell receptor; tBCR: tumor cell B-cell receptor.
nBCR
tBCR nBCR
IGHV3-23 - + - - - -
IGHV3-30 + +
- -
- - - - - - - - - - - - -+b - -
IGHV3-48 - -
- -
IGHV3-7 - -
- -
IGHV3-74 - +
- -
IGHV4-34 - -
- -
IGHV4-34 - +
- -
IGHV4-34 - +
- +a
IGHV4-34 - -
- -
IGHV4-34 - -
- - +c +d
five distinct members of the IGHV3 subgroup and five IGHV4-34+ PCNSL (Online Supplementary Table S2).10 All PCNSL had introduced somatic mutations into their rearranged Ig heavy and light chain variable genes. All Ig heavy and light chain sequences harbored mutations vary- ing from 2-15% with mean mutation frequencies of 9.9% and 8.3% for the heavy and the light chain V-genes, respectively. Mutations corresponded to point mutations in the heavy and light chain of all ten PCNSL. In addition, case #01 harbored an insertion of six base pairs which extended the heavy chain CDR2 from eight to ten amino acids (Online Supplementary Table S3). As we attributed this insertion to SHM, it was eliminated in the revertant sequence (Online Supplementary Table S3). Deletions and stop codons were absent. Thus, all tumors harbored rearranged and somatically mutated Ig heavy and light chain variable genes encoding a potentially functional BCR, in accordance with previous data.2-4
Recombinant antibodies of naïve and tumor B-cell receptor show self-reactivity with common autoantigens
Complete IgG recAb of nBCR and tBCR were compared for reactivity with common self-antigens using permeabi- lized human HEp-2 cells as target. In five cases, a positive reaction indicated autoreactivity (Table 1 and Online Supplementary Figure S4). In four of them, the SHM- induced modification of the nBCR yielded tBCR HEp-2 reactivity not noticed for the nBCR (Table 1). In the remaining case, both nBCR and tBCR were HEp-2 reactive (Table 1). None of the nBCR exhibited reactivity that was lost upon SHM. Remarkably, HEp-2 reactivity was much below the standard (Online Supplementary Figure S4), detectable at high, but not low recAb concentration (250 mg/mL vs. 10 mg/mL), indicating low affinity of the recAB.
ANA/ANCA ELISA detected reactivity in two IGHV4- 34+ PCNSL (Table 1). In one case (PCNSL patient #10), both nBCR and tBCR reacted with autoantigens, while in the other case (PCNSL patient #08) only the tBCR, but not the nBCR was reactive (Table 1). Again, BCR binding was much below the standard of the ELISA requiring a high antibody concentration (200 mg/mL). At a concentration recommended for the detection of high-affinity antibodies in patients with clinically active autoimmune disease (10 mg/mL), in this regard differing from PCNSL-derived
nBCR and tBCR, reactivity was completely lost, further suggesting low affinity. Together, these data show that the recAB of 6 of 10 (60%) PCNSL exhibit weak reactivity with self-antigens, mostly in the tBCR, indicating that SHM fosters self-reactivity.
Naïve and tumor B-cell receptor differ in their protein recognition pattern on the ProtoArray
All recAb specifically recognized proteins on the ProtoArray. A total of 821 proteins was specifically recog- nized by at least one recAb (mean: 165; median: 140; range: 106-300). Regarding all proteins recognized, 341 and 725 proteins were recognized by at least one of the nBCR and tBCR, respectively. The nBCR exclusively rec- ognized 96 proteins including several ribosomal proteins, B-cell linker, and ligase III (Online Supplementary Table S5), whereas the tBCR exclusively recognized 480 proteins including histones, myelin associated proteins (i.e., myelin basic protein, MBP; myelin-associated oligodendrocyte basic protein, MOBP; myelin protein zero-like protein 1, MPZL1), ribosomal proteins, and splicing factors (SNRP70, SNRPA, SNRPB) (Online Supplementary Table S5). A total of 245 proteins were recognized by both the nBCR and the tBCR including galectins, S100A1 protein, and SNRPC (Online Supplementary Table S4). With respect to the proteins recognized by the nBCR, 107 and 58 proteins were recognized exclusively by the nBCR derived from IGHV3+ and IGHV4-34+ PCNSL, respectively; 176 proteins were recognized in common. Regarding the proteins rec- ognized by the tBCR, 248 and 179 proteins were recog- nized exclusively by the tBCR derived from IGHV3+ and IGHV4-34+ PCNSL, respectively; 298 proteins were recog- nized in common (Figure 1A and Online Supplementary Table S5).
According to the Human Protein Atlas Project (www.pro- teinatlas.org), 482 proteins recognized by the recAb are expressed in the CNS, albeit mostly not exclusively (mean: 96; median: 82; range: 63-184). Of those CNS pro- teins recognized, 202 and 425 CNS proteins were recog- nized by at least one of the nBCR and the tBCR, respec- tively (Figure 1B). NBCR exclusively recognized 57 pro- teins including BLNK and LIG23 (Online Supplementary Table S5), whereas tBCR exclusively recognized 280 pro- teins including MBP, MOBP, MPZL1, SNRPA, SNRPB2, and S100A13 (Online Supplementary Table S5); 145 proteins were recognized in common including endoglin, galectins,
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