Saponin-based adjuvants (SBAs) are being used in animal and human (cancer) vaccines, as they induce protective cellular immunity. Their adjuvant potency is a factor of inflammasome activation and enhanced antigen cross-presentation by dendritic cells (DCs), but how antigen cross-presentation is induced is not clear. Here we show that SBAs uniquely induce intracellular lipid bodies (LBs) in the CD11b+ DC subset in vitro and in vivo. Using genetic and pharmacological interference in models for vaccination and in situ tumour ablation, we demonstrate that LB induction is causally related to the saponin-dependent increase in cross-presentation and T-cell activation. These findings link adjuvant activity to LB formation, aid the application of SBAs as a cancer vaccine component, and will stimulate development of new adjuvants enhancing T-cell-mediated immunity.
Multidrug resistance (MDR) is often related to expression of P‐glycoprotein (Pgp) or Multidrug Resistance Protein (MRP). Pgp‐mediated MDR can be evaluated by determining cellular retention of fluorescent substrates by flow cytometry. This study determined if agents used to evaluate Pgp function also can be used to evaluate MRP function. Cellular retention of doxorubicin (Dox), Rhodamine‐123 (Rh‐123), and 3,3′‐diethyloxacarbocyanine iodide (DiOC2(3)) were studied in MRP‐expressing cell lines (HL60/Adr and HT1080/DR4), whereas a Pgp expressing cell line (A2780/Dx5) served as a positive control. Overexpression of Pgp correlated inversely with retention of Dox, Rh‐123, and DiOC2(3); however, under identical experimental conditions (1 h reincubation in drug‐free medium), no retention difference of the three agents was detected between parental and MRP‐expressing resistant cells. Upon extending the reincubation time to 4 h, an efflux of Rh‐123 and Dox in the resistant lines became apparent and even more pronounced after 24 h; however, still no efflux was detectable for DiOC2(3). Incubation of the cells with a modulator of MDR, PAK‐104P, negated the observed drug efflux in Pgp and MRP expressing cells, which correlated with increased sensitivity of the MDR lines to doxorubicin. Thus both Dox and Rh‐123 can be used to evaluate MRP‐function, but DiOC2(3) can not. © 1996 Wiley‐Liss, Inc.
Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin lymphoma subtype arising from naïve B cells. Although novel therapeutics have improved patient prognosis, drug resistance remains a key problem. Here, we show that the SRC-family tyrosine kinase hematopoietic cell kinase (HCK), which is primarily expressed in the hematopoietic lineage but not in mature B cells, is aberrantly expressed in MCL, and that high expression of HCK is associated with inferior prognosis of MCL patients. HCK expression is controlled by the toll-like receptor (TLR) adaptor protein MYD88 and can be enhanced by TLR agonists in MCL cell lines and primary MCL. In line with this, primary MCL with high HCK expression are enriched for a TLR-signaling pathway gene set. Silencing of HCK expression results in cell cycle arrest and apoptosis. Furthermore, HCK controls integrin-mediated adhesion of MCL cells to extracellular matrix and stromal cells. Taken together, our data indicate that TLR/MYD88-controlled aberrant expression of HCK plays a critical role in MCL proliferation and survival as well as in retention of the malignant cells in the growth- and survival-supporting lymphoid organ microenvironment, thereby contributing to lymphomagenesis. These novel insights provide a strong rationale for therapeutic targeting of HCK in MCL.
AimsTo (1) reclassify ocular adnexal large B-cell lymphomas (OA-LBCLs) per 2016 WHO lymphoma classification and (2) determine the prevalence of MYD88 and CD79B mutations and their association with clinical parameters among OA-LBCLs.MethodsThis study is a retrospective analysis of all OA-LBCLs diagnosed in Denmark between 1980 and 2018. Medical records and tissue samples were retrieved. Thirty-four OA-LBCLs were included. Fluorescence in situ hybridisation and Epstein-Barr-encoded RNA in situ hybridisation were used for the reclassification. Mutational status was established by allele-specific PCR and confirmed by Sanger sequencing. Primary endpoints were overall survival, disease-specific survival (DSS) and progression-free survival (PFS).ResultsTwo LBCL subtypes were identified: diffuse large B-cell lymphoma (DLBCL) (27 of 32; 84%) and high-grade B-cell lymphoma (HGBL) with MYC and BCL2 and/or BCL6 rearrangements (5 of 32; 16%). cMYC/BCL2 double-expressor DLBCLs had a poorer DSS than non-double-expressor DLBCLs (5-year DSS, 25% vs 78%) (HR 0.23; 95% CI 0.06 to 0.85; p=0.014). MYD88 mutations were present in 10 (29%) of 34 lymphomas and carried a poorer PFS than wild-type cases (5-year PFS, 0% vs 43%) (HR 0.78; 95% CI 0.61 to 0.98; p=0.039). CD79B mutations were present in 3 (9%) of 34 cases.ConclusionOA-LBCL consists mainly of two subtypes: DLBCL and HGBL with MYC and BCL2 and/or BCL6 rearrangements. MYD88 mutations are important drivers of OA-LBCL. MYD88 mutations, as well as cMYC/BCL2 double-expressor DLBCL, appear to be associated with a poor prognosis. Implementing MYD88 mutational analysis in routine diagnostics may improve OA-LBCL prognostication.
The paracaspase mucosa-associated lymphoid tissue 1 (MALT1) is a protease and scaffold protein essential in propagating B-cell receptor (BCR) signaling to NF-κB. The deubiquitinating enzyme cylindromatosis (CYLD) is a recently discovered MALT1 target that can negatively regulate NF-κB activation. Here, we show that low expression of CYLD is associated with inferior prognosis of diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) patients, and that chronic BCR signaling propagates MALT1-mediated cleavage and, consequently, inactivation and rapid proteasomal degradation of CYLD. Ectopic overexpression of WT CYLD or a MALT1-cleavage resistant mutant of CYLD reduced phosphorylation of IκBα, repressed transcription of canonical NF-κB target genes and impaired growth of BCR-dependent lymphoma cell lines. Furthermore, silencing of CYLD expression rendered BCR-dependent lymphoma cell lines less sensitive to inhibition of NF-κΒ signaling and cell proliferation by BCR pathway inhibitors, e.g., the BTK inhibitor ibrutinib, indicating that these effects are partially mediated by CYLD. Taken together, our findings identify an important role for MALT1-mediated CYLD cleavage in BCR signaling, NF-κB activation and cell proliferation, which provides novel insights into the underlying molecular mechanisms and clinical potential of inhibitors of MALT1 and ubiquitination enzymes as promising therapeutics for DLBCL, MCL and potentially other B-cell malignancies.
A new cell line of nonhematopoietic and nonvascular endothelial origin that expresses CD34 in association with selection for MDR was cloned. A study of MDR mechanisms in this cell line revealed that reduced type II alpha topoisomerase levels were likely responsible for the MDR observed. A study of the causal relation between the selection of drug resistance and the expression of CD34 may provide insight into why CD34 correlates with poor clinical response in patients with acute myeloid leukemia.
T he development of precision medicine for diffuse large B-cell lymphoma (DLBCL) is complicated by its great clinical and molecular heterogeneity. Twenty years ago, two distinct cell-of-origin (COO) subtypes were identified based on distinct gene expression profiles that reflect different stages of B-cell development, i.e., germinal center B-cell-like (GCB) and activated B-celllike (ABC) DLBCL. 1 The ongoing revolution in genomics has since shed light on the genetic landscape of these subtypes. Whereas ABC DLBCL is characterized by frequent mutations in nuclear factor-κB (NF-κB) pathway drivers and intermediates, including TNFAIP3, MYD88, CARD11 and CD79B, as well as loss of cell cycle regulators CDKN2A and CDKN2B, GCB DLBCL carry frequent mutations in epigenetic modifiers, such as CREBBP, KMT2D and EZH2. 2,3 GCB DLBCL patients have a more favorable prognosis and a better clinical response to standard R-CHOP immunochemotherapy than those with ABC DLBCL. Nevertheless, the clinical outcome is het-lymphoma
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