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Impact of GC reaction on self-/polyreactivity in PCNSL
rosis phases,5 formal proof of GC and a GC reaction in the brain is still lacking. In addition to a lack of classical, fully functional GC in the brain, the CNS microenvironment may also, at least in part, be responsible for the absence of Ig class switch in PCNSL due to a lack of signals in the GC required for class switching. The recent demonstration that SHM and class switch recombination occur inde- pendently and are topographically distinct is in line with this notion.6
So far, studies of PCNSL aiming at the identification of foreign antigens that might trigger the tumor B cells have been inconclusive; in particular, viruses able to persist in the CNS, e.g., HHV6, HHV8, and SV40 were excluded.7-9 Taking advantage of the fact that the tumor cells of PCNSL express a functional BCR, we previously reconstructed the tumor cell BCR (tBCR) as recombinant antibodies (recAb) in 23 PCNSL to identify their antigen recognition pattern on a large-scale protein microarray. This approach aimed at the identification of proteins that were expressed in the CNS and on the cell surface, thus enabling their recogni- tion as antigen by the BCR; it revealed that the tumor cells are polyreactive including reactivity with proteins physio- logically expressed in the CNS. Neuronally expressed GRINL1A, centaurin, and BAIAP2 were recognized by tBCR.10 The majority of tBCR recognized galectin-3, upregulated on microglia/macrophages, astrocytes, and endothelial cells upon CNS invasion by PCNSL.10 Thus, proteins differentially expressed by resident CNS cell pop- ulations may trigger the tBCR and support PCNSL survival in the CNS.
These data prompted us to revert somatic mutations of the tBCR to their preimmune sequence, yielding recAb with sequences similar to the BCR of the naïve B-cell receptor (nBCR) from which the tumor cells originated, to obtain further insight into the impact of SHM on BCR characteristics of PCNSL. To this end, we focused on IGHV4-34+ and IGHV3+ PCNSL, as the IGHV4-34 gene is preferentially rearranged in PCNSL, while genes of the large IGHV3 subgroup also occur frequently, but without biased usage.2-4 Comparison of nBCR with tBCR reactivity demonstrated that autoreactivity and polyreactivity increased upon SHM. Thus, in PCNSL, B cells exhibit fea- tures that, principally, qualify for elimination; however, they are rescued by paradoxical redemption in the GC reaction.
Methods
Patients
Stereotactic PCNSL biopsies (tumor load >80%) from 50 HIV- negative patients were included (Online Supplementary Table S1). PCNSL were diagnosed according to World Health Organization guidelines.1 The study was approved by the Ethics Committee of the University Hospital of Cologne (06-187, 07-109) and per- formed according to the Declaration of Helsinki.
Tumor B-cell receptor reversion
TBCR sequences (PCNSL patients #01 - #10) (Online Supplementary Tables S1 and S2)10 were reverted to their unmutated form using the ImMunoGeneTics (IMGT) information system database (www.imgt.org11) similar to the approach reported by Hervé et al.12 reverting V-derived sequences of the VH and VK/VL domain. Mutated nucleotides were replaced by those in the vari- ant region being most similar to the germline gene segment
according to IMGT, which was considered as naïve sequence. These sequences were synthetized by GeneArt (Thermo Fisher Scientific, Rockford, IL, USA); correctness was controlled by Sanger sequencing.
Generation of recombinant antibodies with binding specificity identical to tumor B-cell and naïve B-cell receptors
Monoclonal recAb with binding specificity identical to the tBCR and their corresponding nBCR were generated as described10 (Online Supplementary Figure S1). Ig V gene sequences are detailed in Online Supplementary Table S3.
As control, the recombinant anti-tetanus toxoid antibody recSA13 was generated from the SA13 cell line (HB8501, LCG Standard, Wesel, Germany) using the same strategy.10 For specifici- ty of recSA13 see Online Supplementary Table S4.
Protein microarray processing
Protein microarray slides (9374 human proteins, ProtoArray, Thermo Fisher Scientific) including internal controls were ana- lyzed as described previously.10,13,14
Immunohistochemistry
RecAb of PCNSL patients #01, #03, and #09 labeled with green fluorescent dye CF488A (Antibody Labeling Kit, Sigma, Deisenhofen, Germany), were applied to PCNSL sections, fol- lowed by incubation with monoclonal rabbit anti-SNRPC (clone EPR16034, Abcam, Cambridge, UK) and species-specific Cy3-cou- pled immunoglobulin (Dianova, Hamburg, Germany).
Double immunofluorescence was performed with rabbit anti- SNRPC and mouse anti-GFAP (clone GA-5, Biogenex, Fremont, CA, USA), mouse anti-Olig2 (clone OLIG2/2400, Abcam), mouse anti-NeuN (clone A60, Merck, Burlington, MA, USA), mouse anti- CD68 (clone KP1, DCS, Hamburg, Germany), and mouse anti- CD34 (clone Qbend/10, Biogenex, Palo Alto, CA, USA), respec- tively, including appropriate positive and negative controls (Online Supplementary Figures S2 and S3) was performed.
Analysis of gene expression profiling
Gene expression profiling (GEP) (10 normal CNS tissues, 21 PCNSL) obtained by the Affymetrix U95Av2 microarray (Santa Clara, CA, USA) published previously15 were analyzed for SNRPC mRNA expression.
Statistical analysis
To test for statistical significance between the number of pro- teins recognized by the recAb derived from nBCR and tBCR, the exact Wilcoxon signed rank test (calculated with R version 3.5.0, R Foundation for Statistical Computing, Vienna, Austria) was applied. Statistical significance between SNRPC mRNA expres- sion in normal CNS and PCNSL was assessed by Student t-test. P<0.05 was considered significant. All experiments were per- formed at least in duplicate.
Results
Immunoglobulin heavy and light chain variable gene rearrangements in primary lymphoma of the central nervous system used for generation of recombinant antibodies
All PCNSL harbored a monoclonally rearranged Ig heavy chain variable and a monoclonally rearranged Ig light chain variable gene. A gene of the IGHV3 and the IGHV4 subgroup was rearranged in five tumors each with
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