Analysis of CD20 and PD-L1 levels on small extracellular vesicles (sEV) produced by DLBCL cells and EBV-transformed B cells, and potential role in T cell inhibition.

Increasing evidence supports a role for small extracellular vesicles (sEV, including exosomes) in Diffuse Large B-cell lymphoma (DLBCL) progression and resistance to treatment. CD20 and PD-L1 are found on DLBCL-derived sEV, but little is known about their patient-level heterogeneity. Moreover, the capacity of PD-L1+ sEV to modulate T cells needs to be clarified. Herein we analyzed sEV produced by human DLBCL cell lines and EBV-transformed B cell-lymphoblastoid cell lines (LCLs), a model allowing autologous T cell co-cultures. We determined CD20 and PD-L1 levels on plasma sEV from patient samples vs healthy volunteers (HV). sEV functional relevance was also investigated on CD4+ and CD8+ T cells. sEV derived from all cell lines showed an enrichment of CD20 and a high glycosylated PD-L1 expression when compared to cell lysates. High PD-L1 expression on LCL-derived sEV was associated with higher CD4+ and CD8+ T cell apoptosis. In patients, plasma sEV concentration was higher vs HV. Compared to sEV-CD20 level that seemed higher in patients, PD-L1 level in sEV was not different from those of HV. A high glycosylated PD-L1 level was shown in sEV from both patients and HV plasma samples, that was associated with the same inhibiting effect on activated T cells. We conclude that sEV derived from EBV-transformed B cells realize an immunosuppressive role that involved cell-cell interaction and probably at least PD-L1. Furthermore, our findings suggest the potential of circulating sEV as a source of biomarkers in DLBCL, notably to have information on immunotherapeutic target levels of parental tumor cells.

CD20 expression, TrkB activation and functional activity of diffuse large B cell lymphoma-derived small extracellular vesicles.

BACKGROUND: Small extracellular vesicles (sEVs) including exosomes, carrying the CD20, could be involved in immunotherapy resistance in diffuse large B cell lymphoma (DLBCL). We have reported endogenous brain-derived neurotrophic factor/TrkB (tropomyosin-related kinase B) survival axis in DLBCL. Here, we performed a comparative study of sEV production by germinal centre B cell (GCB) and activated B cell (ABC)-DLBCL cell lines, and analysed TrkB activation on this process. METHODS: GCB (SUDHL4 and SUDHL6) and ABC (OCI-LY3, OCI-LY10 and U2932) cell lines were used. sEVs were characterised using nanoparticle tracking analysis technology and western blot. CD20 content was also analysed by enzyme-linked immunoassay, and complement-dependent cytotoxicity of rituximab was investigated. 7,8-Dihydroxyflavone (7,8-DHF) was used as a TrkB agonist. In vivo role of sEVs was evaluated in a xenograft model. RESULTS: sEVs production varied significantly between DLBCL cells, independently of subtype. CD20 level was consistent with that of parental cells. Higher CD20 expression was found in sEVs after TrkB activation, with a trend in increasing their concentration. sEVs determined in vitro and in vivo protection from rituximab, which seemed CD20 level-dependent; the protection was enhanced when sEVs were produced by 7,8-DHF-treated cells. CONCLUSIONS: DLBCL-derived sEVs have the differential capacity to interfere with immunotherapy, which could be enhanced by growth factors like neurotrophins. Evaluating the sEV CD20 level could be useful for disease monitoring.

Prohibitin (PHB) expression is associated with aggressiveness in DLBCL and flavagline-mediated inhibition of cytoplasmic PHB functions induces anti-tumor effects.

BACKGROUND: Diffuse large B-cell lymphomas (DLBCLs) are aggressive lymphomas accounting for approximately a third of non-Hodgkin lymphomas. Prohibitin 1 (PHB1) and prohibitin 2 (PHB2) are scaffold proteins that promote mitochondria homeostasis and consequently cell survival, but biological functions of cytoplasmic PHBs remain largely unknown in DLBCL. METHODS: PHB expression was analyzed in 82 DLBCL biopsies and five DLBCL cell lines by immunohistochemistry (IHC) and Western blotting. Pharmacological inhibition of PHB using the synthetic flavagline FL3 was realized in vitro to gain insight PHB cellular functions. Effects of FL3 on DLBCL cell line viability, apoptosis, C-Raf-ERK-MNK-eIF4E signaling pathway and eIF4F complex formation and activity were evaluated by XTT assay, annexin V-FITC/PI dual staining and Western blotting respectively. Subcutaneous DLBCL xenograft model in SCID mice was also performed to determine in vivo FL3 effect. RESULTS: As in DLBCL cell lines, PHB1 and PHB2 were expressed in germinal center B-cell-like (GCB) and activated B-cell-like (ABC) subtypes. In patient samples, high PHB levels were associated with higher serum LDH (PHB1 and PHB2), IPIaa (PHB2), and Ki-67 (PHB2) expression. Higher PHB1 expression tends to be associated with shorter event-free survival (EFS) in patients, especially in male patients. FL3 induced apoptosis of DLBCL cell lines that was associated with inhibition of the ERK-MNK-eIF4E signaling pathway, including aggressive double/triple-hit DLBCL cell lines. This resulted in altered eIF4F complex formation and activity leading to a reduction of Bcl-2 and c-Myc expression levels. Moreover, FL3 strongly downregulated DLBCL cellular levels of Akt protein and AKT mRNA. FL3 antitumor activity was also confirmed in vivo in a murine xenograft model. CONCLUSION: Our data indicate that PHB overexpression is associated with markers of tumor aggressiveness in DLBCL, and that targeting PHBs may be a therapeutic option, notably in aggressive subtypes.