A dichotomy of gene regulatory associations during the activated B-cell to plasmablast transition.

The activated B-cell (ABC) to plasmablast transition encompasses the cusp of antibody-secreting cell (ASC) differentiation. We explore this transition with integrated analysis in human cells, focusing on changes that follow removal from CD40-mediated signals. Within hours of input signal loss, cell growth programs shift toward enhanced proliferation, accompanied by ER-stress response, and up-regulation of ASC features. Clustering of genomic occupancy for IRF4, BLIMP1, XBP1, and CTCF with histone marks identifies a dichotomy: XBP1 and IRF4 link to induced but not repressed gene modules in plasmablasts, whereas BLIMP1 links to modules of ABC genes that are repressed, but not to activated genes. Between ABC and plasmablast states, IRF4 shifts away from AP1/IRF composite elements while maintaining occupancy at IRF and ETS/IRF elements. This parallels the loss of BATF expression, which is identified as a potential BLIMP1 target. In plasmablasts, IRF4 acquires an association with CTCF, a feature maintained in plasma cell myeloma lines. Thus, shifting occupancy links IRF4 to both ABC and ASC gene expression, whereas BLIMP1 occupancy links to repression of the activation state.

Site-1 protease function is essential for the generation of antibody secreting cells and reprogramming for secretory activity.

The unfolded protein response (UPR) and activation of XBP1 is necessary for high secretory efficiency and functional differentiation of antibody secreting cells (ASCs). The UPR additionally includes a branch in which membrane-bound transcription factors, exemplified by ATF6, undergo intramembrane-proteolysis by the sequential action of site-1 (MBTPS1/S1P) and site-2 proteases (MBTPS2/S2P) and release of the cytoplasmic domain as an active transcription factor. Such regulation is shared with a family of CREB3-related transcription factors and sterol regulatory element-binding proteins (SREBPs). Of these, we identify that the CREB3 family member CREB3L2 is strongly induced and activated during the transition from B-cell to plasma cell state. Inhibition of site-1 protease leads to a profound reduction in plasmablast number linked to induction of autophagy. Plasmablasts generated in the presence of site-1 protease inhibitor segregated into CD38high and CD38low populations, the latter characterized by a marked reduction in the capacity to secrete IgG. Site-1 protease inhibition is accompanied by a distinctive change in gene expression associated with amino acid, steroid and fatty acid synthesis pathways. These results demonstrate that transcriptional control of metabolic programs necessary for secretory activity can be targeted via site-1 protease inhibition during ASC differentiation.

SPIB and BATF provide alternate determinants of IRF4 occupancy in diffuse large B-cell lymphoma linked to disease heterogeneity.

Interferon regulatory factor 4 (IRF4) is central to the transcriptional network of activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL), an aggressive lymphoma subgroup defined by gene expression profiling. Since cofactor association modifies transcriptional regulatory input by IRF4, we assessed genome occupancy by IRF4 and endogenous cofactors in ABC-DLBCL cell lines. IRF4 partners with SPIB, PU.1 and BATF genome-wide, but SPIB provides the dominant IRF4 partner in this context. Upon SPIB knockdown IRF4 occupancy is depleted and neither PU.1 nor BATF acutely compensates. Integration with ENCODE data from lymphoblastoid cell line GM12878, demonstrates that IRF4 adopts either SPIB- or BATF-centric genome-wide distributions in related states of post-germinal centre B-cell transformation. In primary DLBCL high-SPIB and low-BATF or the reciprocal low-SPIB and high-BATF mRNA expression links to differential gene expression profiles across nine data sets, identifying distinct associations with SPIB occupancy, signatures of B-cell differentiation stage and potential pathogenetic mechanisms. In a population-based patient cohort, SPIBhigh/BATFlow-ABC-DLBCL is enriched for mutation of MYD88, and SPIBhigh/BATFlow-ABC-DLBCL with MYD88-L265P mutation identifies a small subgroup of patients among this otherwise aggressive disease subgroup with distinct favourable outcome. We conclude that differential expression of IRF4 cofactors SPIB and BATF identifies biologically and clinically significant heterogeneity among ABC-DLBCL.

In vitro generation of long-lived human plasma cells.

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.

BLIMP-1 and STAT3 counterregulate microRNA-21 during plasma cell differentiation.

During cellular differentiation, mRNA transcription and translation require precise coordination. The mechanisms controlling this are not well defined. IL-21 is an important regulator of plasma cell differentiation, and it controls the master regulator of plasma cell differentiation, B lymphocyte-induced maturation protein-1 (BLIMP-1), via STAT3 and IRF4. Among the other targets of STAT3 is microRNA-21 (miR-21). miR-21 is the most frequently deregulated microRNA in malignancy, including B cell lymphomas, and it has oncogenic potential downstream of STAT3. However, the regulation and function of miR-21 during plasma cell differentiation are not characterized. In contrast to the induction of miR-21 observed in response to STAT3 activation in other systems, we demonstrate that miR-21 is repressed during IL-21-driven plasma cell differentiation. We explored the molecular basis for this repression and identify primary miR-21 transcription as a direct target of BLIMP-1-dependent repression, despite continued STAT3 activation and phospho-STAT3 binding to the primary miR-21 promoter. Thus, STAT3 and BLIMP-1 constitute an incoherent feed-forward loop downstream of IL-21 that can coordinate microRNA with mRNA expression during plasma cell differentiation.

B-cell differentiation in EBV-positive Burkitt lymphoma is impaired at posttranscriptional level by miRNA-altered expression.

Endemic, sporadic and HIV-associated Burkitt lymphoma (BL) all have a B-cell phenotype and a MYC translocation, but a variable association with the Epstein-Barr virus (EBV). However, there is still no satisfactory explanation of how EBV participates in the pathogenesis of BL. A recent investigation suggested that EBV-positive and EBV-negative BL have different cells of origin. In particular, according to immunoglobulin gene mutation analysis, EBV-negative BLs may originate from early centroblasts, whereas EBV-positive BLs seem to arise from postgerminal center B cells or memory B cells. The appearance of a germinal center phenotype in EBV-positive cells might thus derive from a block in B-cell differentiation. The exit from the germinal center involves a complex series of events, which require the activation of BLIMP-1, and the consequent downregulation of several target genes. Here, we investigated the expression of specific miRNAs predicted to be involved in B-cell differentiation and found that hsa-miR-127 is differentially expressed between EBV-positive and EBV-negative BLs. In particular, it was strongly upregulated only in EBV-positive BL samples, whereas EBV-negative cases showed levels of expression similar to normal controls, including microdissected germinal centers (GC) cells. In addition, we found evidence that hsa-miR-127 is involved in B-cell differentiation process through posttranscriptional regulation of BLIMP1 and XBP1. The overexpression of this miRNA may thus represent a key event in the lymphomagenesis of EBV positive BL, by blocking the B-cell differentiation process.

CD34+ cord blood cell-transplanted Rag2-/- gamma(c)-/- mice as a model for Epstein-Barr virus infection.

Recent studies suggest that Epstein-Barr virus (EBV) can infect naïve B cells, driving them to differentiate into resting memory B cells via the germinal center reaction. This hypothesis has been inferred from parallels with the biology of normal B cells but has never been proven experimentally. Rag2(-/-) gamma(c)(-/-) mice that were transplanted with human CD34(+) cord blood cells as newborns were recently shown to develop human B, T, and dendritic cells, constituting lymphoid organs in situ. Here we used this model to better define the strategy of EBV infection of human B cells in vivo and to compare this model system with different conditions of EBV infection in humans. Our results support the model of EBV persistence in vivo in cases that were characterized by follicular hyperplasia and a relatively normal CD4(+) and CD8(+) T-cell distribution. Intriguingly, in cases that were characterized by nodular and diffuse proliferation with a preponderance of CD8(+) T cells, similar to infectious mononucleosis, EBV still infects naïve B cells but also induces clonal expansion and ongoing somatic mutations without germinal center reactions. Our results reveal different strategies of EBV infection in B cells that possibly result from variations in the host immune response. Future experiments might allow understanding of the mechanisms responsible for persistent EBV infection and provide targets for more highly tailored therapeutic interventions.

Cdk9/Cyclin T1 complex: a key player during the activation/differentiation process of normal lymphoid B cells.

Cdk9/Cyclin T1 complex is very important in controlling specific differentiative pathways of several cell types. Limited data are available regarding the expression of Cdk9/Cyclin T1 in hematopoietic and lymphoid tissues. Cdk9/Cyclin T1 expression seems to be related to particular stages of lymphoid differentiation/activation. In this study, we observed that the expression level of Cdk9/Cyclin T1 in vivo increases in memory B cells compared to naïve B cells, and in activated B cells, compared to non-activated ones. The expression level of the Cdk9/Cyclin T1 complex does not increase in cells induced to differentiate in vitro. In addition, we showed that Cdk9 interacts with E12 and E47, specifically activated during Germinal Center (GC) reaction. Taken together this data suggests an active role for the Cdk9/Cyclin T1 complex during lymphoid differentiation through germinal center reaction.

Translocation detection in lymphoma diagnosis by split-signal FISH: a standardised approach.

Lymphomas originating from the lymphatic system comprise about 30 entities classified according to the World Health Organization (WHO). The histopathological diagnosis is generally considered difficult and prone to mistakes. Since non-random chromosomal translocations are specifically involved in different lymphoma entities, their detection will be increasingly important. Hence, a split-signal fluorescence in situ hybridisation (FISH) procedure would be helpful in discriminating the most difficult classifications. The Euro-FISH programme, a concerted action of nine European laboratories, has validated a robust, standardised protocol to improve the diagnostic approach on lymphoma entities. Therefore, 16 fluorescent probes and 10 WHO entities, supplemented with reactive cases, were selected. The results of the Euro-FISH programme show that all probes were correctly cytogenetically located, that the standardised protocol is robust, resulting in reliable results in approximately 90% of cases, and that the procedure could be implemented in every laboratory, bringing the relatively easy interpretation of split-signal probes within the reach of many pathology laboratories.

The role of the Cdk9/Cyclin T1 complex in T cell differentiation.

The Cdk9/Cyclin T1 complex is very important in controlling specific differentiative pathways of several cell types, including muscle cells and neurons. We recently demonstrated the involvement of this complex in B cell activation/differentiation. To check whether the Cdk9/Cyclin T1 complex is also involved in the T cell activation/differentiation process, we isolated different T cell populations by magnetic separation, based on their surface antigens. We observed that the expression level of Cdk9/Cyclin T1 increases in effector T cells (CD27(+)), as well as in activated T cells (CD25(+)) and memory T cells (CD45RA(-)), thus suggesting a specific upregulation of the Cdk9/Cyclin T1 complex following antigen encounter. We have previously demonstrated that in B cells, Cdk9 interacts in vivo with the E2A gene products E12/E47 (members of the basic helix-loop-helix family) which are involved in differentiation. In this article, we show that this interaction also occurs in T cells. This suggests an active role for the Cdk9/Cyclin T1 complex during lymphoid differentiation, through physical binding with E12 and E47. These preliminary results suggest that the Cdk9/Cyclin T1 complex may be important in the activation and differentiation program of lymphoid cells and that its upregulation, which is due to still unknown mechanisms, may contribute to malignant transformation.

Overlapping morphologic and immunophenotypic profiles in small B-cell lymphoma. A report of two cases.

We present two cases of small B-cell lymphomas of particular diagnostic interest because the histological patterns were at variance with their immunophenotype. One of these lymphomas, involving the gallbladder and duodenum, showed a marginal zone lymphoma-like (MALT type) pattern of cellular infiltration with CD5 negativity but (unexpectedly) Cyclin D1 positivity. Fluorescence in situ hybridization analysis of this case was performed because of the aberrant expression of Cyclin D1, and was clearly positive for the Cyclin D1 gene translocation. The second case, occurring in a lymph node, showed the typical growth pattern of a follicular lymphoma but it had an atypical immunophenotype, namely, expression of Cyclin D1, CD10, and Bcl2 and focally Bcl6, accompanied by a lack of CD5 and CD23. The Cyclin D1 gene translocation was detected by fluorescence in situ hybridisation (FISH), whereas c-myc and Bcl2 genes translocation were absent. Numerical chromosomal changes, which were visualized for chromosomes 8, 11, and 18 could be correlated to the aberrant immunoprofile. In this context, we discuss the diagnostic value of Cyclin D1, CD5, CD23, CD10, Bcl6 markers revealed by immunohistochemistry, as well as the significance of detection by FISH of chromosomal translocations such as t(11;14) and t(14;18). The question still remains as to whether such cases should be designated as specific lymphoma entities or reported as unclassifiable and the chromosome aberration reported.

Routine assessment of hormonal receptor and her-2/neu status underscores the need for more therapeutic targets in Kenyan women with breast cancer.

OBJECTIVE: To report the expression of estrogen receptors, progesterone receptors and human epidermal growth factor receptor (Her-2/neu) in 158 Kenyan women with breast cancer and correlation with other prognostic indicators in this high-risk group. This study stressed the importance of routine assessment of the steroid receptors and Her-2/neu as a mode of therapeutic selection of patients for antihormonal or targeting monoclonal antibody (Herceptin) therapy, directed at the juxtamembrane domain of Her-2/neu protein in the developing countries such as Kenya. STUDY DESIGN: The study population consisted of 158 female patients with histologically confirmed breast carcinoma seen at the pathology department of The Nairobi Hospital. An immunohistochemical (IHC) study of ER, PR and Her-2/neu was conducted, followed by fluorescent in situ hybridization (FISH) validation for Her-2/neu gene amplification in cases initially scored as positive 2+ with IHC. Mastectomy samples registered at the pathology department of The Nairobi Hospital were used for this study. The study was approved by the institution's ethical review committee and informed consent obtainedfrom the concerned patients. RESULTS: In the studied cohort, positivity for both hormonal receptors and Her-2/neu was noted in 10 (6.33%) cases and negativity in 44 (27.85%) cases. Conversely, Her-2/neu negativity was noted in 32 (20.25%) cases with both steroid receptors positive and Her-2/neu positivity with both steroid receptors negative in 20 (12.66%) cases. Overall, no predictive factor was found in the Her-2/neu amplified 31/153 (20.26%) cases completely assessed with IHC and FISH. Grade III invasive ductal carcinomas, however, had a high prevalence of Her-2/neu overexpression. Association of both menopausal status (p = 0.044) and progesterone receptor status (p = 0.004) with high grade tumors were found to be statistically significant at 95% CI (p < 0.5). Consistent with other studies, Her-2/neu overexpression in this cohort was 20.26%. CONCLUSION: Her-2/neu positivity may activate ER expression through signaling kinases, and the combined target of mitogenic estrogen plus the monoclonal antibody therapy against Her-2/neu-overexpressing tumors expand chances of survival for patients in developing countries such as Kenya. The cost factor for these tests, selection for appropriate combined therapies and lack of awareness were noted as limiting factors for access to basic health care service and resulted in advanced tumor grade at time of patient presentation.