Toll-like receptor stimulation in splenic marginal zone lymphoma can modulate cell signaling, activation and proliferation

Abstract

© F e r r a t a S t o r t i F o u n d a t i o nadaptor molecule MyD88 connects distinct TLRs to proximal IRAK kinases which then trigger a cascade of phosphorylation events, eventually leading to MAPK and IKK activation and the induction of specific transcriptional programs including that of the NF-κB complex. 16 With all the above in mind, we herein aimed at characterizing TLR expression and function in SMZL, in order to assess the potential impact of TLR signaling on the biology of this disease. Methods Cell purificationBlood and tissue samples were obtained from SMZL patients after informed consent as part of a study (ViVi-NHL) approved by the institutional ethics committee. SMZL cells were negatively selected and purified using a B-cell enrichment kit (RosetteSep; StemCell Technologies) following the manufacturer's instructions. Normal B cells were purified by negative selection (EasySep; StemCell Technologies) from buffy coats. Preparations were virtually devoid of NK cells, T-lymphocytes and monocytes.In 19 SMZL cases, total peripheral blood mononuclear cells (PBMC) were isolated by Ficoll gradient centrifugation without further purification (the percentage of CD19 + cells in these cases is reported in Online Supplementary Table S1). In all cases, subsequent flow cytometric analyses were performed after gating on CD19+ cells. TLR expression analysisSMZL cells were analyzed either at the time of blood withdrawal or after thawing. The analysis was performed by flow cytometry using the following antibodies: anti-TLR1 and anti-TLR7 (AbCam); anti-TLR2 (Invitrogen); anti-TLR6 and anti-TLR10 (IMGENEX); anti-TLR8 (DENDRITICS), and anti-TLR9 (eBiosciences). The BD Cytofix/Cytoperm reagent was used for the intracellular staining of TLR7, TLR8 and TLR9. Antibodies against IgD, IgM, CD38 and CD19 were used to detect normal MZ B cells in spleen samples, as previously described. 7-AminoActinomycin D (7-AAD, BD Pharmingen™) was used for the exclusion of nonviable cells in flow cytometric assays. All flow cytometric analyses were performed on a FC-500 (Beckman Coulter) or on a FACSAria II (Becton Dickinson). Cell culture and functional studiesSMZL cells were either unstimulated or stimulated with specific TLR ligands (Online Supplementary Methods). Collection was performed either after 24 hours for CD86 (BD Pharmingen) and CD25 (Beckman Coulter) flow cytometry analysis, or after 48 hours to assess cell proliferation (Ki67 staining, BD Biosciences), apoptosis (Annexin V and Propidium Iodide, Bender Med Systems) and viability (CellTiter-Glo Luminescent Cell Viability Assay, Promega). The IRAK1/4 inhibitor I (Sigma) was used at the concentration of 3 μM 30 minutes before TLR stimulation. The MyD88 inhibitory peptide and control peptide (Novus Biologicals) were used at the concentration of 100 μM. Molecular studiesi. Immunogenetic analysis. PCR amplification and sequence analysis of IGHV-IGHD-IGHJ gene rearrangements was performed as previously described. 20 ii. Qualitative assessment of TLR1-10 and TIR8 expression b...

The splenic marginal zone shapes the phenotype of leukemia B cells and facilitates their niche-specific retention and survival

“…In human SMZL, a B cell lymphoma located in the MZ of SLOs, lymphoma cells express functional toll-like receptors (TLRs) and their stimulation by microbial antigens contributes to disease pathobiology. 7 Despite a denied access to the follicle, we observed expansion of Cxcr5 ¡/¡ Em-Tcl1 leukemic cells within the MZ. 5 We now asked if these tumor cells have the flexibility to adapt to their microenvironment and what factors facilitate this phenotypic diversity.…”

“…Antigenic stimulation through immune receptors such as BCR and TLR, has been postulated to be involved in the development of CLL and SMZL, [40][41][42] in addition expression of functional TLRs and signaling molecules were described in CLL, 28,29,43 and SMZL. 7 Mutations in genes that are activated through BCR signaling or toll-like and interleukin signaling were associated with constitutive activation of TLR-signaling and a higher histological score in SMZL. 44,45 Antibody-mediated BCR stimulation, a stimulus provided in B cell follicles, induced upregulation of ALCAM in a probably BTK-and PKC-dependent manner, whereas a PKC-dependent downregulation of CD49d occurred.…”

“…47 Human B-CLL and SMZL cells also express a wide range of TLRs and their stimulation leads to activation and proliferation of tumor cells. 7,28,29 An IRAK1/4 kinase-specific inhibitor blocked TLR-mediated pro-survival effects and it was suggested that this pathway represents a putative therapeutic target. 7 In the Em-Tcl1 mouse model, unabated TLR-mediated stimulation caused accelerated leukemia progression.…”

“…25,27 Human B-CLL and SMZL cells express numerous TLRs, i.e., TLR1, TLR2, TLR6, TLR7, TLR9, and TLR10. 7,28,29 Engagement of these receptors with their respective ligands leads to activation or proliferation of CLL, 28,29 and SMLZ 7 cells. CLL cells in LNs showed upregulation of gene sets, indicating TLR signaling, compared with CLL cells from blood and BM.…”

“…Next, we introduced an inhibitor for IRAK1/4 that inhibits the activation of IRAK kinases by TLR1/2, TLR6/2, and TLR9 stimulation. 7 Three hours later, cells were stimulated with FSL-1. TLR6/2-ligand induced changes in ALCAM and CD49d surface expression could be effectively reversed by IRAK1/4 inhibition, whereas upregulation of NOTCH2 was only partially inhibited (Fig.…”