Osteopontin protects from autoimmunity-driven diffuse large B cell lymphoma development

Several studies have highlighted the association between certain autoimmune diseases and increased occurrence of hematological malignancies, such as Systemic lupus erythematosus (SLE). Patients affected by SLE have indeed a high risk of developing non-Hodgkin lymphoma (NHL), mostly diffuse large B cell lymphoma (DLBCL). Osteopontin (OPN) has been associated with SLE pathogenesis, as SLE patients have increased serum levels of OPN and often polymorphisms in SPP1, the gene encoding for OPN. To study the role of this molecule in autoimmunity-associated lymphomagenesis, the Faslpr/lpr mutation of the autoimmunity-prone mouse strain has been transferred onto a OPN-deficient background. Interestingly, OPN-/-Faslpr/lpr mice showed a significantly higher incidence of splenic B cell lymphomas in comparison to Faslpr/lpr mice. Indeed, in OPN-deficient autoimmune mice we observed an expansion of mature CD19+IgMCD23-CD21/35- cells, which showed the typical signature of the activated type of DLBCL (ABC-DLBCL), BCL6-, BCL2+, c-MYC+, IRF4+, Ki67hi, and a striking activation of the STAT3 pathway. Additionally, OPN-deficient autoimmune CD19+ B cells showed a baseline hyper-activation of Toll-like receptor 9 (TLR9)-dependent MYD88 signaling pathway, and were more activated when stimulated with the TLR9 agonist CpG 1826, which mimics double strand-DNA circulating in autoimmune conditions. Immunohistochemistry on splenic tissues and immunofluorescence staining on CD19+ cells showed that CD19+ B cells from Faslpr/lpr mice express the intracellular isoform of OPN at the endosomal surface, where TLR9 is also located. To gain mechanistic insights, I genetically modified ABC-DLBCL cell lines, derived from lymphomatous spleens of OPN-/- mice, to over-express either the soluble (sOPN) or the intracellular isoform of OPN (iOPN). Remarkably, in vitro experiments showed that iOPN was able to prevent the CpG-mediated activation of STAT3 and, partially, of NFkB pathway, suggesting a negative regulatory role for iOPN in this setting. Additionally, when injected in vivo, while the over-expression of sOPN conferred a growth advantage over control cells, iOPN-expressing cells were less tumorigenic. Interestingly, other than B cell-intrinsic, the molecular mechanisms at the basis of the aggressive lymphomagenesis observed in OPN-/-Faslpr/lpr mice were also likely stroma related. Indeed, mainly myeloid cell subsets of the splenic microenvironment of OPNdeficient autoimmune mice expressed more Tnfsf13B (encoding for BAFF) and Il6, both key factors for B cell survival and proliferation, in comparison with OPN-competent counterparts. Interestingly, in lymphomatous areas of the spleen from OPN-deficient mice, a high number of infiltrating CD8+ T cells with an activated morphology was notable. However, their analysis by flow cytometry showed markers of exhaustion (TIM3 and PD1), suggesting that, despite being recruited at the tumour site, they were likely dysfunctional and unable to contrast tumour expansion. The translational relevance of these findings comes from the analysis of ABC-DLBCL biopsies that expressed a lower SPP1/OPN level than GC-DLBCL counterparts, suggesting how a diminished expression of B-cell intrinsic OPN may be associated with the development of the most aggressive DLBCL. However, the existence of a specific intracellular form and/or localization of OPN in humans remains to be investigated. Altogether, my data suggest that B cell-intrinsic iOPN is contrasting autoimmunity driven ABC-DLBCL development, also in concert with microenvironment-related dynamics, which in an OPN-competent host seem to contribute in preventing lymphomagenesis