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Ars2 in the pathogenesis of DLBCL
miRNA processing.23 Ars2 expression was shown to be linked to proliferative states24 and different roles were described in malignant disesases.25,26
All three target antigens of DLBCL-BCR identified in this study share the characteristic of being atypically post-translationally modified, which represents the most likely reason for their immunogenicity. For ubiquitinated FamH83 and sumoylated JmJD4, the lymphoma BCR were specific for the secondary modified isoforms. In contrast, Ars2-reactive BCR of DLBCL bound both the hypophosphorylated and the normally phosphorylated isoforms of Ars2. However, the hypophosphorylated iso- form of Ars2 was only observed in cell lines or cryospec- imens of DLBCL with BCR reactivity for Ars2 (P<0.0001). Similarly, regarding peripheral blood, the hypophospho- rylated isoform of Ars2 was only observed in lysates of peripheral blood cells of patients seropositive for Ars2 autoantibodies (P<0.0001). These statistically significant associations between Ars2-reactivity and the presence of hypophosphorylated Ars2 indicate that this post-transla- tional modification is involved in the immunogenicity.
Bringing this into the context of other B-cell neoplasias, in plasma cell dyscrasia SLP2-reactive paraprotein was also not specific for the differentially phosphorylated SLP2 isoform.27 As for CD4+ T-helper cells specific for hyperphosphorylated SLP2 in plasma cell dyscrasia,28 one might speculate about a possible role of hypothetical CD4+ T-helper cells specific for hypophosphorylated Ars2 epitopes, which might stimulate Ars2-reactive B cells, which by themselves do not differentiate between nor- mally phosphorylated and hypophosphorylated Ars2. Generally, atypical post-translational modifications repre- sent an accepted mechanism for the breakdown of self- tolerance, with numerous examples in clinical immunolo- gy, such as modified wheat gliadin in celiac disease,29 N- terminally acetylated myelin basic protein in multiple sclerosis,30 citrullinated fibrin/vimentin in rheumatoid arthritis,31,32 phosphorylated SR proteins during stress- induced apoptosis in systemic lupus erythematosus,33,34 immunogenic pSer81 progranulin isoform and progran- ulin autoantibodies,35,36 phosphorylated enolase in pancre- atic carcinoma,37-39 and the involvement of atypically modified BCR target antigens in lymphomagenesis including hyperphosphorylated SLP2, ATG13, and sumoylated HSP90 in plasma cell dyscrasia and hyper-N- glycosylated SAMD14/neurabin-I in primary central nervous system lymphoma.11,15,27,40,41
Of interest, Ars2 hypophosphorylation and reactivity of DLBCL BCR against Ars2 was nearly exclusively detected in DLBCL of the ABC type. All the cell lines with anti-Ars2 reactivity were of this cell of origin, and in a validation cohort of DLBCL with cell of origin charac- terized by GEP, hypophosphorylated Ars2 was detected in eight of 31 cases (26%) of the ABC type of DLBCL, but in only one of 20 cases (5%) of GCB-type DLBCL. In a combined analysis of GEP-typed cryospecimens and ana- lyzed cell lines, the hypophosphorylated Ars2 isoform was statistically significantly associated with ABC type (P=0.0188).
Considering possible functional effects of Ars2, we observed that its addition stimulated growth of DLBCL lines with Ars2-specific BCR (Figure 3), indicating that these lines still depend to some extent on BCR stimulation
by their cognate antigen Ars2. Regarding the mutational background of these cell lines with Ars2-reactive BCR and expression of hypophosphorylated Ars2, OCI-Ly10 has mutated MYD88 (L265P)42 and a truncating mutation of CD79A,7,43 U2932 has a mutated NFkB-pathway by TAK1 mutation,44 but wild-type CARD11, and wild-type MYD88,7,45 and OCI-Ly3 has a mutated CARD11 and mutated MYD88 (L265P).7,42 This demonstrates that, despite pathway-activating mutations, the proliferation of these cell lines might still benefit from an upstream BCR pathway stimulation by a cognate antigen.
From a therapeutic point of view, two hypothetical approaches arise from these data. Firstly, BCR antigens might be used as baits to target lymphoma cells in a spe- cific way (i.e., targeting of a cell-bound antibody by an antigen), similarly to anti-idiotype antibodies or pepti- bodies,46 with the advantage of not having to be selected and synthesized individually for each patient, since all Ars2-reactive DLBCL BCR bind the same epitope. Physiologically it is the major task of a surface immunoglobulin to bind its cognate antigen and then internalize it, enabling processing and antigen presenta- tion via MHC class II molecules. Targeting Ars2-reactive BCR of DLBCL cell lines resulted in a specific and effica- cious killing. Beside this, the Ars2 epitope could be used for bispecific constructs for T- or NK-cell engaging (e.g., CD3/Ars2 or CD16/Ars2),47 or as an additional ectodomain for chimeric antigen receptor T cells.48 Secondly, regarding the high relative risk of carriers of atypically hypophosphorylated Ars2, investigating ways of modulating this post-translational modification might be worthwhile in the future.
Disclosures
No conflicts of interest to disclose. MP died during the prepa- ration of the manuscript. Saarland University has applied for a relevant patent.
Contributions
LTh, SH, KDP, BK and MP designed the study. SH and MLH performed microdissection of DLBCL cases and interpret- ed data. WK, AW YJK, RMB, BK, FvB, LTr, MZ, NM, DKM VP and GH were of great help in the acquisition of DLBCL samples and clinical data. NF performed the protein array, phosphorylation and proteomic experiments. ER did site-directed point mutagenesis of Ars2. MK and ER performed sequencing studies. LTh, MK, ER and TB performed seminested IgV gene PCR and BCR expression cloning. LTh, MB, MK, ER and NF performed expression of Ars2-immunotoxins. LTh and MP were responsible for data analysis, interpretation of results and writ- ing the manuscript. This article is dedicated to the memory of MP, who died during its preparation.
Acknowledgments
We are grateful to the entire team of the José-Carreras-Center for Immuno- and Gene Therapy, the DSHNHL and GLA, the Department of Internal Medicine I of Saarland University Medical School for continuous logistic and intellectual support. We also thank Hans Drexler from DSMZ for constant support.
Funding
This work was supported by a grant from Wilhelm-Sander- Stiftung.
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