Search

BackgroundDysregulated interleukin (IL)-6 production can be characterised by the levels present, the kinetics of its rise and its inappropriate location. Rapid, excessive IL-6 production can exacerbate tissue damage in vital organs. In this situation, therapy with an anti-IL-6 or anti-IL-6 receptor (IL-6R) monoclonal antibody, if inappropriately dosed, may be insufficient to fully block IL-6 signalling and normalise the immune response.MethodsWe analysed inhibition of C-reactive protein (CRP) – a biomarker for IL-6 activity – in patients with COVID-19 or idiopathic multicentric Castleman disease (iMCD) treated with tocilizumab (anti-IL-6R) or siltuximab (anti-IL-6), respectively. We used mathematical modelling to analyse how to optimise anti-IL-6 or anti-IL-6R blockade for the high levels of IL-6 observed in these diseases.ResultsIL-6 signalling was insufficiently inhibited in patients with COVID-19 or iMCD treated with standard doses of anti-IL-6 therapy. Patients whose disease worsened throughout therapy had only partial inhibition of CRP production. Our model demonstrated that, in a scenario representative of iMCD with persistent high IL-6 production not controlled by a single dose of anti-IL-6 therapy, repeated administration more effectively inhibited IL-6 activity. In a situation with rapid, high, dysregulated IL-6 production, such as severe COVID-19 or a cytokine storm, repeated daily administration of an anti-IL-6/anti-IL-6R agent, or alternating daily doses of anti-IL-6 and anti-IL-6R therapies, could neutralise IL-6 activity.ConclusionIn clinical practice, IL-6 inhibition should be individualised based on pathophysiology to achieve full blockade of CRP production.FundingEUSA Pharma funded medical writing assistance and provided access to the phase II clinical data of siltuximab for analysis.

Abstract Background Mesenchymal stromal cells (MSCs) represent an interesting tool to improve pancreatic islet transplantation. They have immunomodulatory properties and secrete supportive proteins. However, the functional properties of MSCs vary according to many factors such as donor characteristics, tissue origin, or isolation methods. To counteract this heterogeneity, we aimed to immortalize and characterize adherent cells derived from human pancreatic islets (hISCs), using phenotypic, transcriptomic, and functional analysis. Methods Adherent cells derived from human islets in culture were infected with a hTERT retrovirus vector and then characterized by microarray hybridization, flow cytometry analysis, and immunofluorescence assays. Osteogenic, adipogenic, and chondrogenic differentiation as well as PBMC proliferation suppression assays were used to compare the functional abilities of hISCs and MSCs. Extracellular matrix (ECM) gene expression profile analysis was performed using the SAM (Significance Analysis of Microarrays) software, and protein expression was confirmed by western blotting. Results hISCs kept an unlimited proliferative potential. They exhibited several properties of MSCs such as CD73, CD90, and CD105 expression and differentiation capacity. From a functional point of view, hISCs inhibited the proliferation of activated peripheral blood mononuclear cells. The transcriptomic profile of hISCs highly clusterized with bone marrow (BM)-MSCs and revealed a differential enrichment of genes involved in the organization of the ECM. Indeed, the expression and secretion profiles of ECM proteins including collagens I, IV, and VI, fibronectin, and laminins, known to be expressed in abundance around and within the islets, were different between hISCs and BM-MSCs. Conclusion We generated a new human cell line from pancreatic islets, with MSCs properties and retaining some pancreatic specificities related to the production of ECM proteins. hISCs appear as a very promising tool in islet transplantation by their availability (as a source of inexhaustible source of cells) and ability to secrete a supportive “pancreatic” microenvironment.

An amendment to this paper has been published and can be accessed via the original article.

FCRL4 is an immunoregulatory receptor that belongs to the Fc receptor-like (FCRL) family. In healthy individuals, FCRL4 is specifically expressed by memory B cells (MBCs) localized in sub-epithelial regions of lymphoid tissues. Expansion of FCRL4+ B cells has been observed in blood and other tissues in various infectious and autoimmune disorders. Currently, the mechanisms involved in pathological FCRL4+ B cell generation are actively studied, but they remain elusive. As in vivo FCRL4+ cells are difficult to access and to isolate, here we developed a culture system to generate in vitro FCRL4+ B cells from purified MBCs upon stimulation with soluble CD40 ligand and/or CpG DNA to mimic T-cell dependent and/or T-cell independent activation, respectively. After 4 days of stimulation, FCRL4+ B cells represented 17% of all generated cells. Transcriptomic and phenotypic analyses of in vitro generated FCRL4+ cells demonstrated that they were closely related to FCRL4+ tonsillar MBCs. They strongly expressed inhibitory receptor genes, as observed in exhausted FCRL4+ MBCs from blood samples of HIV-infected individuals with high viremia. In agreement, cell cycle genes were significantly downregulated and the number of cell divisions was two-fold lower in in vitro generated FCRL4+ than FCRL4- cells. Finally, due to their reduced proliferation and differentiation potential, FCRL4+ cells were less prone to differentiate into plasma cells, differently from FCRL4- cells. Our in vitro model could be of major interest for studying the biology of normal and pathological FCRL4+ cells.

Background: Multiple myeloma (MM) is the second most common hematological cancer after lymphoma. It is characterized by the accumulation of clonal malignant plasma cells within the bone marrow. The development of drug resistance remains a major problem for effective treatment of MM. Understand the mechanisms underlying drug resistance in MM is a focal point to improve MM treatment. Methods: In the current study, we analyzed further the role of redox imbalance induction in melphalan-induced toxicity both in human myeloma cell lines (HMCLs) and primary myeloma cells from patients. Results: We developed an in-vitro model of short-term resistance to high-dose melphalan and identified that pretreatment with physiological concentration of GSH protects HMCLs from melphalan-induced cell cycle arrest and cytotoxicity. We validated these results using primary MM cells from patients co-cultured with their bone marrow microenvironment. GSH did not affect the ability of melphalan to induce DNA damages in MM cells. Interestingly, melphalan induced reactive oxygen species, a significant decrease in GSH concentration, protein and lipd oxydation together with NRF2 (NF-E2-related factor 2) pathway activation. Conclusions: Our data demonstrate that antioxidant defenses confers resistance to high dose melphalan in MM cells, supporting that redox status in MM cells could be determinant for patients’ response to melphalan.

DNA microarrays have considerably helped to improve the understanding of biological processes and diseases. Large amounts of publicly available microarray data are accumulating, but are poorly exploited due to a lack of easy-to-use bioinformatics resources. The aim of this study is to build a free and convenient data-mining web site (www.genomicscape.com). GenomicScape allows mining dataset from various microarray platforms, identifying genes differentially expressed between populations, clustering populations, visualizing expression profiles of large sets of genes, and exporting results and figures. We show how easily GenomicScape makes it possible to construct a molecular atlas of the B cell differentiation using publicly available transcriptome data of naïve B cells, centroblasts, centrocytes, memory B cells, preplasmablasts, plasmablasts, early plasma cells and bone marrow plasma cells. Genes overexpressed in each population and the pathways encoded by these genes are provided as well as how the populations cluster together. All the analyses, tables and figures can be easily done and exported using GenomicScape and this B cell to plasma cell atlas is freely available online. Beyond this B cell to plasma cell atlas, the molecular characteristics of any biological process can be easily and freely investigated by uploading the corresponding transcriptome files into GenomicScape.

High throughput DNA microarray has made it possible to outline genes whose expression in malignant plasma cells is associated with short overall survival of patients with Multiple Myeloma (MM). A further step is to elucidate the mechanisms encoded by these genes yielding to drug resistance and/or patients' short survival. We focus here on the biological role of the DEP (for Disheveled, EGL-10, Pleckstrin) domain contained protein 1A (DEPDC1A), a poorly known protein encoded by DEPDC1A gene, whose high expression in malignant plasma cells is associated with short survival of patients. Using conditional lentiviral vector delivery of DEPDC1A shRNA, we report that DEPDC1A knockdown delayed the growth of human myeloma cell lines (HMCLs), with a block in G2 phase of the cell cycle, p53 phosphorylation and stabilization, and p21(Cip1) accumulation. DEPDC1A knockdown also resulted in increased expression of mature plasma cell markers, including CXCR4, IL6-R and CD38. Thus DEPDC1A could contribute to the plasmablast features of MMCs found in some patients with adverse prognosis, blocking the differentiation of malignant plasma cells and promoting cell cycle.

The glycome, i.e. the cellular repertoire of glycan structures, contributes to important functions such as adhesion and intercellular communication. Enzymes regulating cellular glycosylation processes are related to the pathogenesis of cancer including multiple myeloma. Here we analyze the transcriptional differences in the glycome of normal (n = 10) and two cohorts of 332 and 345 malignant plasma-cell samples, association with known multiple myeloma subentities as defined by presence of chromosomal aberrations, potential therapeutic targets, and its prognostic impact. We found i) malignant vs. normal plasma cells to show a characteristic glycome-signature. They can ii) be delineated by a lasso-based predictor from normal plasma cells based on this signature. iii) Cytogenetic aberrations lead to distinct glycan-gene expression patterns for t(11;14), t(4;14), hyperdiploidy, 1q21-gain and deletion of 13q14. iv) A 38-gene glycome-signature significantly delineates patients with adverse survival in two independent cohorts of 545 patients treated with high-dose melphalan and autologous stem cell transplantation. v) As single gene, expression of the phosphatidyl-inositol-glycan protein M as part of the targetable glycosyl-phosphatidyl-inositol-anchor-biosynthesis pathway is associated with adverse survival. The prognostically relevant glycome deviation in malignant cells invites novel strategies of therapy for multiple myeloma.

Background Genetic abnormalities are common in patients with multiple myeloma, and may deregulate gene products involved in tumor survival, proliferation, metabolism and drug resistance. In particular, translocations may result in a high expression of targeted genes (termed spike expression) in tumor cells. We identified spike genes in multiple myeloma cells of patients with newly-diagnosed myeloma and investigated their prognostic value.Design and Methods Genes with a spike expression in multiple myeloma cells were picked up using box plot probe set signal distribution and two selection filters.Results In a cohort of 206 newly diagnosed patients with multiple myeloma, 2587 genes/expressed sequence tags with a spike expression were identified. Some spike genes were associated with some transcription factors such as MAF or MMSET and with known recurrent translocations as expected. Spike genes were not associated with increased DNA copy number and for a majority of them, involved unknown mechanisms. Of spiked genes, 36.7% clustered significantly in 149 out of 862 documented chromosome (sub)bands, of which 53 had prognostic value (35 bad, 18 good). Their prognostic value was summarized with a spike band score that delineated 23.8% of patients with a poor median overall survival (27.4 months versus not reached, P

Gene expression-based scores used to predict risk in cancer frequently include genes coding for DNA replication, repair or recombination. Using two independent cohorts of 206 and 345 previously-untreated patients with Multiple Myeloma (MM), we identified 50 cell cycle-unrelated genes overexpressed in multiple myeloma cells (MMCs) compared to normal human proliferating plasmablasts and non-proliferating bone marrow plasma cells and which have prognostic value for overall survival. Thirty-seven of these 50 myeloma genes (74%) were enriched in genes overexpressed in one of 3 normal human stem cell populations--pluripotent (18), hematopoietic (10) or mesenchymal stem cells (9)--and only three genes were enriched in one of 5 populations of differentiated cells (memory B lymphocytes, T lymphocytes, polymorphonuclear cells, monocytes, osteoclasts). These 37 genes shared by MMCs and adult or pluripotent stem cells were used to build a stem cell score ((SC)score), which proved to be strongly prognostic in the 2 independent cohorts of patients compared to other gene expression-based risk scores or usual clinical scores using multivariate Cox analysis. This finding highlights cell cycle-unrelated prognostic genes shared by myeloma cells and normal stem cells, whose products might be important for normal and malignant stem cell biology.

Background Multiple myeloma is a plasma-cell tumor with heterogeneity in molecular abnormalities and treatment response.Design and Methods We have assessed whether human myeloma cell lines have kept patients’ heterogeneity using Affymetrix gene expression profiling of 40 human myeloma cell lines obtained with or without IL6 addition and could provide a signature for stratification of patient risk.Results Human myeloma cell lines, especially those derived in the presence of IL6, displayed a heterogeneity that overlaps that of the patients with multiple myeloma. Human myeloma cell lines segregated into 6 groups marked by overexpression of MAF, MMSET, CCND1, FRZB with or without overexpression of cancer testis antigens (CTA). Cell lines of CTA/MAF and MAF groups have a translocation involving C-MAF or MAFB, cell lines of groups CCND1-1 and CCND1-2like have a t(11;14) and cell lines of group MMSET have a t(4;14). The CTA/FRZB group comprises cell lines that had no or no recurrent 14q32 translocation. Expression of 248 genes accounted for human myeloma cell line molecular heterogeneity. Human myeloma cell line heterogeneity genes comprise genes with prognostic value for survival of patients making it possible to build a powerful prognostic score involving a total of 13 genes.Conclusions Human myeloma cell lines derived in the presence of IL6 recapitulate the molecular diversity of multiple myeloma that made it possible to design, using human myeloma cell line heterogeneity genes, a high-risk signature for patients at diagnosis. We propose this classification to be used when addressing the physiopathology of multiple myeloma with human myeloma cell lines.

Background Anti-CD20 monoclonal antibodies are major therapeutic agents for patients with follicular lymphoma and work through complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity. Optimization of antibody-dependent cellular cytotoxicity, in particular by amplifying its effectors, could further increase the efficacy of anti-CD20 monoclonal antibodies.Design and Methods We investigated the cytotoxic activity of Vγ9Vδ2 T cells against follicular lymphoma cells and whether this killing could be increased by promoting antibody-dependent cellular cytotoxicity with anti-CD20 monoclonal antibodies, in particular a type-II glycoengineered anti-CD20. Vγ9Vδ2 T cells were expanded in vitro in the presence of bromohydrin pyrophosphate (Phosphostim) and interleukin-2 and their ability to kill follicular lymphoma primary cells or cell lines was evaluated by flow cytometry cytotoxic T-lymphocyte assays in the presence or absence of three anti-CD20 monoclonal antibodies: the afucosylated GA101, the chimeric rituximab or the humanized ofatumumab. The ability of these cells to release perforin/granzyme and secrete interferon-γ when co-cultured with follicular lymphoma primary cells or cell lines in the presence or not of the three anti-CD20 monoclonal antibodies was also evaluated by CD107a staining and Elispot assays.Results Phosphostim and interleukin-2 expanded Vγ9Vδ2 T cells were cytotoxic to primary follicular lymphoma cells and their cytotoxic potential was dramatically increased by GA101, a type II glycoengineered anti-CD20 monoclonal antibody, and to a lesser extent, by rituximab and ofatumumab. The increased cytotoxicity was associated with increased secretion of perforin/granzyme and interferon-γ.Conclusions In-vitro expanded Vγ9Vδ2 T cells efficiently kill primary follicular lymphoma cells and express CD16; anti-CD20 monoclonal antibodies, in particular GA101, dramatically increase the cytotoxic activity of expanded Vγ9Vδ2 T cells. These preclinical results prompt the development of clinical trials using this antibody dependent cellular cytotoxicity property of Vγ9Vδ2 T cells and anti-CD20 monoclonal antibodies.

Background Proliferation of malignant plasma cells is a strong adverse prognostic factor in multiple myeloma and simultaneously targetable by available (e.g. tubulin polymerase inhibitors) and upcoming (e.g. aurora kinase inhibitors) compounds.Design and Methods We assessed proliferation using gene expression-based indices in 757 samples including independent cohorts of 298 and 345 samples of CD138-purified myeloma cells from previously untreated patients undergoing high-dose chemotherapy, together with clinical prognostic factors, chromosomal aberrations, and gene expression-based high-risk scores.Results In the two cohorts, 43.3% and 39.4% of the myeloma cell samples showed a proliferation index above the median plus three standard deviations of normal bone marrow plasma cells. Malignant plasma cells of patients in advanced stages or those harboring disease progression-associated gain of 1q21 or deletion of 13q14.3 showed significantly higher proliferation indices; patients with gain of chromosome 9, 15 or 19 (hyperdiploid samples) had significantly lower proliferation indices. Proliferation correlated with the presence of chromosomal aberrations in metaphase cytogenetics. It was significantly predictive for event-free and overall survival in both cohorts, allowed highly predictive risk stratification (e.g. event-free survival 12.7 versus 26.2 versus 40.6 months, P

Generation of B and plasma cells involves several organs with a necessary cell trafficking between them. A detailed phenotypic characterization of four circulating B-cell subsets (immature-, naïve-, memory- B-lymphocytes and plasma cells) of 106 healthy adults was realized by multiparametric flow cytometry. We show that CD10, CD27 and CD38 is the minimal combination of subsetting markers allowing unequivocal identification of immature (CD10+CD27−CD38+, 6±6 cells/μL), naïve (CD10−CD27−CD38−, 125±90 cells/μL), memory B lymphocytes (CD10−CD27+CD38−, 58±42 cells/μL), and plasma cells (CD10−CD27++CD38++, 2.1±2.1 cells/μL) within circulating CD19+ cells. From these four subsets, only memory B lymphocytes and plasma cells decreased with age, both in relative and absolute counts. Circulating plasma cells split into CD138− (57±12%) and CD138+ (43±12%) cells, the latter displaying a more mature phenotypic profile: absence of surface immunoglobulin, lower CD45 positivity and higher amounts of cytoplasmic immunoglobulin, CD38 and CD27. Unlike B lymphocytes, both populations of plasma cells are KI-67+ and show weak CXCR4 expression.

BackgroundHematopoietic cells are endowed with very specific biological functions, including cell motility and immune response. These specific functions are dramatically altered during hematopoietic cell differentiation, whereby undifferentiated hematopoietic stem and progenitor cells (HSPC) residing in bone marrow differentiate into platelets, red blood cells and immune cells that exit into the blood stream and eventually move into lymphoid organs or inflamed tissues. The contribution of alternative splicing (AS) to these functions has long been minimized due to incomplete knowledge on AS events in hematopoietic cells.Principal findingsUsing Human Exon ST 1.0 microarrays, the entire exome expression profile of immature CD34+ HSPC and mature whole blood cells was mapped, compared to a collection of solid tissues and made freely available as an online exome expression atlas (Amazonia Exon! : http://amazonia.transcriptome.eu/exon.php). At a whole transcript level, HSPC strongly expressed EREG and the pluripotency marker DPPA4. Using a differential splicing index scheme (dsi), a list of 849 transcripts differentially expressed between hematopoietic cells and solid tissues was computed, that included NEDD9 and CD74. Some of these genes also underwent alternative splicing events during hematopoietic differentiation, such as INPP4B, PTPLA or COMMD6, with varied contribution of CD3+ T cells, CD19+ B cells, CD14+ or CD15+ myelomonocytic populations. Strikingly, these genes were significantly enriched for genes involved in cell motility, cell adhesion, response to wounding and immune processes.ConclusionThe relevance and the precision provided by this exon expression map highlights the contribution of alternative splicing to key feature of blood cells differentiation and function.

Thymosin β4 (Tβ4) is a polypeptide involved in cellular proliferation, differentiation, and migration, over-expressed in several tumor entities. We evaluated its expression and function in 298 newly diagnosed multiple myeloma patients and the murine 5TMM model. Mean Tβ4 expression was significantly lower in myeloma cells compared to normal plasma cells (P

Background and Objectives BAFF and APRIL stimulate the growth of multiple myeloma (MM) cells. BAFF and APRIL share two receptors – TACI and BCMA – and BAFF binds to a third receptor, BAFF-R. We previously reported that TACI gene expression is bimodal in 18 human MM cell lines (HMCL), being either present or absent, unlike BCMA that is expressed on all HMCL. BAFF-R is lacking. TACI expression is a good indicator of a BAFF-binding receptor in HMCL. In primary MM cells, the level of TACI expression correlates with a characteristic phenotypic pattern: TACIhigh MM cells resemble bone marrow plasma cells and TACIlow resemble plasmablasts. The aim of this study was to further characterize the role of TACI expression in MMDesign and Methods Using gene expression profiling, we investigated whether these patterns are kept in TACI+ or TACI− HMCL.Results Eighty genes/EST interrogated by Affymetrix microarrays were differentially expressed between TACI+ and TACI− HMCL, particularly c-maf, cyclin D2, and integrin β7. Triggered by the finding that TACI and c-maf expressions correlate in TACI+ HMCL, we demonstrated that TACI activation influences c-maf expression: (i) activation of TACI by BAFF or APRIL increases c-maf, cyclin D2, and integrin β7 gene expressions in TACI+ HMCL, (ii) blocking of autocrine BAFF/APRIL stimulation in some TACI+ HMCL by the TACI-Fc fusion protein reduces c-maf, cyclin D2, and integrin β7 gene expression, (iii) nucleofection of siRNA to c-maf decreases c-maf mRNA levels and reduces the expression of cyclin D2 and integrin β7 gene expressions, without affecting TACI expressionInterpretation and Conclusions We conclude that TACI activation can upregulate c-maf expression which, in turn, controls cyclin D2, and integrin β7 gene expression.