Based on the profile of genetic alterations occurring in tumor samples from selected diffuse large B-cell lymphoma (DLBCL) patients, 2 recent whole-exome sequencing studies proposed partially ...overlapping classification systems. Using clustering techniques applied to targeted sequencing data derived from a large unselected population-based patient cohort with full clinical follow-up (n = 928), we investigated whether molecular subtypes can be robustly identified using methods potentially applicable in routine clinical practice. DNA extracted from DLBCL tumors diagnosed in patients residing in a catchment population of ∼4 million (14 centers) were sequenced with a targeted 293-gene hematological-malignancy panel. Bernoulli mixture-model clustering was applied and the resulting subtypes analyzed in relation to their clinical characteristics and outcomes. Five molecular subtypes were resolved, termed MYD88, BCL2, SOCS1/SGK1, TET2/SGK1, and NOTCH2, along with an unclassified group. The subtypes characterized by genetic alterations of BCL2, NOTCH2, and MYD88 recapitulated recent studies showing good, intermediate, and poor prognosis, respectively. The SOCS1/SGK1 subtype showed biological overlap with primary mediastinal B-cell lymphoma and conferred excellent prognosis. Although not identified as a distinct cluster, NOTCH1 mutation was associated with poor prognosis. The impact of TP53 mutation varied with genomic subtypes, conferring no effect in the NOTCH2 subtype and poor prognosis in the MYD88 subtype. Our findings confirm the existence of molecular subtypes of DLBCL, providing evidence that genomic tests have prognostic significance in non-selected DLBCL patients. The identification of both good and poor risk subtypes in patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) clearly show the clinical value of the approach, confirming the need for a consensus classification.
•Robust subtypes of DLBCL are identified by model-based clustering of genetic mutations in a large (n = 928) population-based cohort.•With full follow-up data available for all sequenced patients, the prognostic significance of these subtypes is identified.
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Ab-secreting cells survive in niche microenvironments, but cellular responses driven by particular niche signals are incompletely defined. The TNF superfamily member a proliferation-inducing ligand ...(APRIL) can support the maturation of transitory plasmablasts into long-lived plasma cells. In this study, we explore the biological programs established by APRIL in human plasmablasts. Under conditions allowing the maturation of ex vivo- or in vitro-generated plasmablasts, we find that APRIL drives activation of ERK, p38, and JNK, accompanied by a classical NF-κB response and activation of the AKT/FOXO1 pathway. Time-course gene expression data resolve coordinated transcriptional responses propagated via immediate early genes and NF-κB targets and converging onto modules of genes enriched for MYC targets and metabolism/cell growth-related pathways. This response is shared between APRIL and an alternate TNF superfamily member CD40L but is not a feature of alternative niche signals delivered by IFN-α or SDF1. However, APRIL and CD40L responses also diverge. CD40L drives expression of genes related to the activated B cell state whereas APRIL does not. Thus, APRIL establishes a broad foundation for plasma cell longevity with features of cellular refueling while being uncoupled from support of the B cell state.
Cell of origin classification of diffuse large B-cell lymphoma (DLBCL) identifies subsets with biological and clinical significance. Despite the established nature of the classification existing ...studies display variability in classifier implementation, and a comparative analysis across multiple data sets is lacking. Here we describe the validation of a cell of origin classifier for DLBCL, based on balanced voting between 4 machine-learning tools: the DLBCL automatic classifier (DAC). This shows superior survival separation for assigned Activated B-cell (ABC) and Germinal Center B-cell (GCB) DLBCL classes relative to a range of other classifiers. DAC is effective on data derived from multiple microarray platforms and formalin fixed paraffin embedded samples and is parsimonious, using 20 classifier genes. We use DAC to perform a comparative analysis of gene expression in 10 data sets (2030 cases). We generate ranked meta-profiles of genes showing consistent class-association using ≥6 data sets as a cut-off: ABC (414 genes) and GCB (415 genes). The transcription factor ZBTB32 emerges as the most consistent and differentially expressed gene in ABC-DLBCL while other transcription factors such as ARID3A, BATF, and TCF4 are also amongst the 24 genes associated with this class in all datasets. Analysis of enrichment of 12323 gene signatures against meta-profiles and all data sets individually confirms consistent associations with signatures of molecular pathways, chromosomal cytobands, and transcription factor binding sites. We provide DAC as an open access Windows application, and the accompanying meta-analyses as a resource.
Plasma cells are heterogenous in terms of their origins, secretory products, and lifespan. A current paradigm is that cell cycle exit in plasma cell differentiation is irreversible, following a ...pattern familiar in short-lived effector populations in other hemopoietic lineages. This paradigm no doubt holds true for many plasma cells whose lifespan can be measured in days following the completion of differentiation. Whether this holds true for long-lived bone marrow plasma cells that are potentially maintained for the lifespan of the organism is less apparent. Added to this the mechanisms that establish and maintain cell cycle quiescence in plasma cells are incompletely defined. Gene expression profiling indicates that in the transition of human plasmablasts to long-lived plasma cells a range of cell cycle regulators are induced in a pattern that suggests a quiescence program with potential for cell cycle re-entry. Here a model of relative quiescence with the potential for replicative self-renewal amongst long-lived plasma cells is explored. The implications of such a mechanism would be diverse, and the argument is made here that current evidence is not sufficiently strong that the possibility should be disregarded.
Long-lived human plasma cells (PCs) play central roles in immunity and autoimmunity and are enriched among the subpopulation of CD19
human PCs. However, whether human CD19
PCs are necessarily aged ...cells that have gradually lost CD19 expression is not known. Assessing peripheral blood samples at steady-state and during the acute response to influenza vaccination in healthy donors, we identify the presence of phenotypic CD19
plasmablasts, the proliferative precursor state to mature PCs, and demonstrate by ELISPOT that these are Ab-secreting cells (ASCs). During the acute response to influenza vaccination, CD19
, CD19
, and CD19
ASCs secrete vaccine-specific Abs and show linked
repertoires. To address precursor/product relationships, we use in vitro models that mimic T-dependent and T-independent differentiation, finding that the CD19
state can be established at the plasmablast to PC transition, that CD19
PCs increase as a percentage of surviving PCs in vitro, and that CD19
and CD19
PCs can be maintained independently. These data provide proof-of-principle for the view that newly generated ASCs can acquire a mature PC phenotype that is accompanied by loss of CD19 expression at an early stage of differentiation and that aging is not an obligate requirement for a CD19
state to be established.
Using a Burkitt lymphoma-like gene expression signature, we recently defined a high-risk molecular high-grade (MHG) group mainly within germinal centre B-cell like diffuse large B-cell lymphomas ...(GCB-DLBCL), which was enriched for MYC/BCL2 double-hit (MYC/BCL2-DH). The genetic basis underlying MHG-DLBCL and their aggressive clinical behaviour remain unknown. We investigated 697 cases of DLBCL, particularly those with MYC/BCL2-DH (n = 62) by targeted sequencing and gene expression profiling. We showed that DLBCL with MYC/BCL2-DH, and those with BCL2 translocation, harbour the characteristic mutation signatures that are associated with follicular lymphoma and its high-grade transformation. We identified frequent MYC hotspot mutations that affect the phosphorylation site (T58) and its adjacent amino acids, which are important for MYC protein degradation. These MYC mutations were seen in a subset of cases with MYC translocation, but predominantly in those of MHG. The mutations were more frequent in double-hit lymphomas with IG as the MYC translocation partner, and were associated with higher MYC protein expression and poor patient survival. DLBCL with MYC/BCL2-DH and those with BCL2 translocation alone are most likely derived from follicular lymphoma or its precursor lesion, and acquisition of MYC pathogenic mutations may augment MYC function, resulting in aggressive clinical behaviour.
Upon encounter with Ag, B cells undergo a sequential process of differentiation to become Ab-secreting plasma cells. Although the key drivers of differentiation have been identified, research has ...been limited by the lack of in vitro models recapitulating the full process for murine B cells. In this study, we describe methodology using BCR or TLR ligation to obtain plasma cells that are phenotypically mature, have exited cell cycle and express a gene signature concordant with long-lived plasma cells. Dependent on the initial stimuli, the transcriptomes also show variation including the enhanced expression of matrisome components after BCR stimulation, suggestive of unique functional properties for the resultant plasma cells. Moreover, using the new culture conditions we demonstrate that alternative promoter choice regulating the expression of the master transcription factor Blimp-1/Prdm1 can be observed; when the canonical B cell promoter for
is deleted, differentiating B cells exhibit flexibility in the choice of promoter, dictated by the initiating stimulus, with preferential maintenance of expression following exposure to TLR ligation. Thus our system provides a readily tractable model for furthering our understanding of plasma cell biology.
In vitro generation of long-lived human plasma cells Cocco, Mario; Stephenson, Sophie; Care, Matthew A ...
The Journal of immunology (1950),
2012-Dec-15, 2012-12-15, 20121215, Letnik:
189, Številka:
12
Journal Article
Recenzirano
Plasma cells (PCs), the terminal effectors of humoral immunity, are short-lived unless supported by niche environments in which they may persist for years. No model system has linked B cell ...activation with niche function to allow the in vitro generation of long-lived PCs. Thus, the full trajectory of B cell terminal differentiation has yet to be investigated in vitro. In this article, we describe a robust model for the generation of polyclonal long-lived human PCs from peripheral blood B cells. After a proliferative plasmablast phase, PCs persist in the absence of cell division, with viability limited only by elective culture termination. Conservative predictions for PC life expectancy are 300 d, but with the potential for significantly longer life spans for some cells. These long-lived PCs are preferentially derived from memory B cells, and acquire a CD138(high) phenotype analogous to that of human bone marrow PCs. Analysis of gene expression across the system defines clusters of genes with related dynamics and linked functional characteristics. Importantly, genes in these differentiation clusters demonstrate a similar overall pattern of expression for in vitro and ex vivo PCs. In vitro PCs are fully reprogrammed to a secretory state and are adapted to their secretory load, maintaining IgG secretion of 120 pg/cell/day in the absence of XBP1 mRNA splicing. By establishing a set of conditions sufficient to allow the development and persistence of mature human PCs in vitro, to our knowledge, we provide the first platform with which to sequentially explore and manipulate each stage of human PC differentiation.
Plasma cells (PCs) as effectors of humoral immunity produce Igs to match pathogenic insult. Emerging data suggest more diverse roles exist for PCs as regulators of immune and inflammatory responses ...via secretion of factors other than Igs. The extent to which such responses are preprogrammed in B-lineage cells or can be induced in PCs by the microenvironment is unknown. In this study, we dissect the impact of IFNs on the regulatory networks of human PCs. We show that core PC programs are unaffected, whereas PCs respond to IFNs with distinctive transcriptional responses. The IFN-stimulated gene 15 (ISG15) system emerges as a major transcriptional output induced in a sustained fashion by IFN-α in PCs and linked both to intracellular conjugation and ISG15 secretion. This leads to the identification of ISG15-secreting plasmablasts/PCs in patients with active systemic lupus erythematosus. Thus, ISG15-secreting PCs represent a distinct proinflammatory PC subset providing an Ig-independent mechanism of PC action in human autoimmunity.
Epstein-Barr virus (EBV)-driven B cell neoplasms arise from the reactivation of latently infected B cells. In a subset of patients, EBV was seen to drive a polymorphous lymphoproliferative disorder ...(LPD) in which B cell differentiation was retained. In this work, spontaneous EBV reactivation following B cell mitogen stimulation was shown to provide a potential model of polymorphic EBV-driven LPD. Here, we developed an in vitro model of plasma cell (PC) differentiation from peripheral blood memory B cells. To assess the frequency and phenotypes of EBV-associated populations derived during differentiation, we analysed eight differentiations during the PC stage with a targeted single-cell gene expression panel. We identified subpopulations of EBV-gene expressing cells with PC and/or B cell expression features in differentiations from all tested donors. EBV-associated cells varied in frequency, ranging from 3-28% of cells. Most EBV-associated cells expressed PC genes such as
or
and in all samples these included a quiescent PC fraction that lacked cell a cycle gene expression. With increasing EBV-associated cells, populations with B cell features became prominent, co-expressing a germinal centre (GC) and activating B cell gene patterns. The presence of highly proliferative EBV-associated cells was linked to retained
expression and
and
co-expression, while
class-switched cells were enriched in quiescent PC fractions. Thus, patterns of gene expression in primary EBV reactivation were shown to include features related to GC B cells, which was also observed in EBV-transformed lymphoblastoid cell lines. This suggests a particular association between spontaneously developing EBV-expansions and IgM+ IgD+ non-switched B cells.
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