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The transcriptional profiling of human in vivo-generated plasma cells identifies selective imbalances in monoclonal gammopathies.

Plasma cells (PC) represent the heterogeneous final stage of the B cells (BC) differentiation process. To characterize the transition of BC into PC, transcriptomes from human naïve BC were compared to those of three functionally-different subsets of human in vivo-generated PC: i) tonsil PC, mainly consisting of early PC; ii) PC released to the blood after a potent booster-immunization (mostly cycling plasmablasts); and, iii) bone marrow CD138+ PC that represent highly mature PC and include the long-lived PC compartment. This transcriptional transition involves subsets of genes related to key processes for PC maturation: the already known protein processing, apoptosis and homeostasis, and of new discovery including histones, macromolecule assembly, zinc-finger transcription factors and neuromodulation. This human PC signature is partially reproduced in vitro and is conserved in mouse. Moreover, the present study identifies genes that define PC subtypes (e.g., proliferation-associated genes for circulating PC and transcriptional-related genes for tonsil and bone marrow PC) and proposes some putative transcriptional regulators of the human PC signatures (e.g., OCT/POU, XBP1/CREB, E2F, among others). Finally, we also identified a restricted imbalance of the present PC transcriptional program in monoclonal gammopathies that correlated with PC malignancy.

Clinical relevance of circulating anti-ENA and anti-dsDNA secreting cells from SLE patients and their dependence on STAT-3 activation.

Disturbances of plasma cell homeostasis and auto-antibody production are hallmarks of systemic lupus erythematosus. The aim of this study was to explore the presence of circulating anti-ENA and anti-dsDNA antibody-secreting cells, to determine their dependence on plasma cell-niche cytokines and to analyze their clinical value. The study was performed in SLE patients with serum anti-ENA and/or anti-dsDNA antibodies (n = 57). Enriched B-cell fractions and sorted antibody-secreting cells (CD19low CD38high ) were obtained from blood. dsDNA- and ENA-specific antibody-secreting cells were identified as cells capable of active auto-antibody production in culture. The addition of a combination of IL-6, IL-21, BAFF, APRIL, and CXCL12 to the cultures significantly augmented auto-antibody production and antibody-secreting cell proliferation, whereas it diminished apoptosis. The effect on auto-antibody production was dependent on STAT-3 activation as it was abrogated in the presence of the JAK/STAT-3 pathway inhibitors ruxolitinib and stattic. Among patients with serum anti-dsDNA antibodies, the detection of circulating anti-dsDNA-antibody-secreting cells was associated with higher disease activity markers. In conclusion, auto-antibody production in response to plasma cell-niche cytokines that are usually at high levels in SLE patients is dependent on JAK/STAT-3 activation. Thus, patients with circulating anti-dsDNA antibody-secreting cells and active disease could potentially benefit from therapies targeting the JAK/STAT3 pathway.

Survival of human circulating antigen-induced plasma cells is supported by plasma cell-niche cytokines and T follicular helper lymphocytes.

Human circulating Ag-induced plasma cells (PCs) contain a high proportion of cycling cells. This study reveals that these PCs spontaneously proliferate in culture during 72 h, as determined by BrdU-uptake detection. Transcriptome analysis indicates that, in comparison with tonsil and bone marrow (BM) PCs, these PCs distinctively upregulate genes involved in cell division. Blood PC proliferation occurs simultaneously with increasing apoptosis rates, and is associated with PC survival. In addition, the proliferating activity of these PCs is enhanced by the addition of cytokines present in PC survival niches. Moreover, blood Ag-induced, but not BM, PCs exhibit the expression of molecules involved in the interaction between memory B cells and T follicular helper (Tfh) cells. In fact, purified circulating and tonsil Tfh cells increased IgG secretion by blood Ag-induced, but not by BM, PCs. This effect is exerted by augmenting blood PC survival through a mechanism partly dependent on cell contact. These results strongly suggest that the proliferating capacity of circulating Ag-induced PCs contributes to their competitive migration to survival niches, either to long-living PC niches or to temporal niches present in reactive lymphoid organs and inflamed tissues, structures where Tfh cells appear to participate.

Syntaxin-4 is implicated in the secretion of antibodies by human plasma cells.

PCs are responsible for the production and secretion of antibodies, the effector molecules of the humoral immune response. The molecular mechanisms responsible for vesicle docking and secretion implicated in the antibody-secretion process are not well-known, as they have not been studied, but it is known that SNARE proteins are responsible for many membrane-fusion processes in the cell. We show here that freshly isolated human colon LP-PCs and T-PCs from MM-PC patients and the U266 cell line, as a model for PC secretion, contain a set of these proteins. SNAP23, STX3, and STX4 were localized mainly in the plasma membrane of PCs, and interactions of SNAP23 with STX3 and with STX4 were proven by IP. Interaction between SNAP23 and STX4 was also confirmed in situ. With the use of siRNA, as well as shRNA, the functional role of SNAP23, STX3, and STX4 in antibody secretion was also examined. The findings demonstrate that in addition to SNAP23, STX4 is implicated in the antibody secretion by a myeloma cell line and by normal human colon LP-PCs.

STAT-3 activation by differential cytokines is critical for human in vivo-generated plasma cell survival and Ig secretion.

Maturation and survival of plasma cells (PCs) depends on extrinsic factors provided in specialized niches. In addition, B lymphocyte differentiation into PCs requires the activation of the JAK-STAT-3 pathway. However, whether STAT-3 is needed only during the transition of B lymphocytes to PC, or it is also involved in the survival and function of PCs at different stages of maturation, has not been unequivocally clarified. This study analyzes the effect of IL-10, IL-21, and IL-6 on human in vivo-generated PCs isolated from secondary lymphoid organs, blood (circulating, recently Ag-induced PCs), and bone marrow. PCs from these different organs show specific profiles of receptors for, and responsiveness to, these cytokines required for their survival and sustained Ab secretion. However, IL-10, IL-21, and IL-6 commonly induce STAT-3 phosphorylation in the three PC subsets, and all of their effects are exerted strictly through the STAT-3 activation. The inhibition or nonactivation of this pathway in the three PC populations impairs not only the effect of STAT-3-activating cytokines, but also the action of other cytokines important at the PC level, including a proliferation-induced ligand, BAFF, insulin-like growth factor 1, vascular endothelial growth factor, and stromal cell-derived factor-1α. These results indicate that STAT-3 activation is critical for human PCs throughout their maturation.

Cutting edge: IL-21 derived from human follicular helper T cells acts as a survival factor for secondary lymphoid organ, but not for bone marrow, plasma cells.

IL-21 induces the differentiation of activated B lymphocytes into plasma cells (PC), but its direct effect on PC remains uncertain. This study analyzes the role of IL-21 on human in vivo-generated PC. IL-21R was clearly expressed on PC from the human tonsil, the lymph node, and the spleen (secondary lymphoid organs [SLO]) but barely on terminally mature bone marrow PC. IL-21 enhanced Ig secretion by isolated SLO PC but not bone marrow PC. Tonsillar T follicular helper (Tfh) lymphocytes are known to secrete IL-21. Purified Tfh cells induced a marked increase of Ig production by tonsillar PC, and this effect was impaired when endogenous IL-21 production was blocked. IL-21 provoked a rapid and transient phosphorylation of STAT3 in tonsillar PC. Tfh cells or exogenous IL-21 reduce tonsillar PC apoptosis and increases PC recovery but does not modify their nonproliferating status. These results suggest that IL-21 derived from Tfh cells acts as a survival factor for SLO PC in vivo.

Differential expression of SLAMS and other modulatory molecules by human plasma cells during normal maturation.

Plasma cells (PCs) are specialized in antibody (Ab) production and they are, therefore, responsible for maintaining humoral immune responses. The human PC compartment is heterogeneous. PCs from inductive secondary lymphoid organs and from peripheral blood (PB) show less capability for prolonged survival and Ab production than bone marrow (BM) PCs, a pool consisting of fully mature cells. The HLDA9 workshop has allowed the use of labeled-monoclonal Abs (moAbs) recognizing a variety of recently identified lymphocyte modulatory surface receptors. In this study, flow cytometry analysis has been used to define the presence of these receptors on human PCs obtained from human tonsil (as an example of inductive organ), from PB and from BM. It was found that human PCs commonly expressed SLAMF1 (CD150), SLAMF2 (CD48), SLAMF3 (CD229), SLAMF6 (CD352) and SLAMF7 (CD319), but not SLAMF4 (CD244). In addition, PCs distinctively showed a low level of SLAMF5 (CD84) and a very high level of SLAMF7 expression in comparison with earlier stages of B cell maturation. All PC subsets exhibited a similar pattern of expression of SLAMF receptors suggesting a stage-dependent role for these proteins. In addition, most circulating PCs clearly expressed TNFRSF14 (CD270), BTLA (CD272), B7-1 (CD80) and B7-2 (CD86), and a substantial fraction of them were also positive for TNFRSF18 (CD357), FCRL1 (CD307a) and LAIR-1 (CD305). In contrast, tonsil and BM PCs only exhibited partial expression of TNFRSF14 and B7-2, a pattern of molecular expression similar to that detected on germinal center (GC) B cells. Present results indicate that human PCs exhibit a common pattern of SLAMF proteins, but differ in the rest of the receptors examined; this difference might be associated with their distinctive homing and functional requirements.

Decreased frequency and activated phenotype of blood CD27 IgD IgM B lymphocytes is a permanent abnormality in systemic lupus erythematosus patients.

INTRODUCTION: Systemic lupus erythematosus (SLE) is characterized by B cell hyper-activation and auto-reactivity resulting in pathogenic auto-antibody generation. The phenotypic analysis of blood B cell subsets can be used to understand these alterations. METHODS: The combined detection of CD19, CD27 and IgD (or IgM) by flow cytometry (FC) analysis delineates five well-defined blood B cell-subsets: naive, switched (S) memory, double negative (DN) memory and CD27 IgD IgM (non-switched memory) B lymphocytes, and plasma cells (PCs). This phenotypic study was performed in 69 consecutive SLE patients and 31 healthy controls. RESULTS: SLE patients exhibited several abnormalities in the distribution of these B cell subsets, including elevated levels of DN memory B cells and PCs, and decreased CD27 IgD IgM B cells. Active SLE patients also showed decreased presence of S memory B cells and increased proportions of naive B lymphocytes. Nevertheless, when the patients in remission who did not require treatment were studied separately, the only remaining abnormality was a reduction of the CD27 IgD IgM B cell-subset detectable in most of these patients. The level of reduction of CD27 IgD IgM B cells was associated with elevated values of serum SLE auto-antibodies. Further analysis of this latter B cell-subset specifically showed increased expression of CD80, CD86, CD95, 9G4 idiotype and functional CXCR3 and CXCR4. CONCLUSIONS: The presence of a reduced blood CD27 IgD IgM B cell-subset, exhibiting an activated state and enriched for auto-reactivity, is a consistent B cell abnormality in SLE. These findings suggest that CD27 IgD IgM B lymphocytes play a role in the pathogenesis of this disease.

Modulated selection of IGHV gene somatic hypermutation during systemic maturation of human plasma cells.

Systemic antigen-induced PCs are generated in inductive lymphoid tissues. Some of them are selected to travel through the circulation and finally, to home onto BM niches. BM PCs show prolonged survival and secrete high-affinity antibodies. In this study, human PCs were isolated from tonsil, blood, and BM, their IGHV3 and IGHV6 genes were sequenced, and their SHM were evaluated. The SHM analysis reveals the existence of a maturational gradient in these genes, as demonstrated by a progressive increase in the frequency of total and R mutations and total and NC aa changes following the direction: tonsil --> blood --> BM. The ratio of R to S mutations in the CDR1 and -2, but not in the FRs, increases from tonsil to blood and BM; this parameter reaches a maximum threshold when more than 10 mutations/sequence occur. Further analyses indicate that CDR1 and CDR2 SHM followed different strategies to provide appropriate amino acid changes, but both exhibited maximal resistance to incorporating drastic molecular alterations in the BM PCs. Finally, all of the findings are similar in IGHV3 and IGHV6 sequences, indicating that they reflect general rules imposed by in vivo antigen selection.

Transcription of PRDM1, the master regulator for plasma cell differentiation, depends on an SP1/SP3/EGR-1 GC-box.

The positive regulatory domain containing 1, encoded by the PRDM1 gene, is a transcriptional repressor considered as a master regulator that is required and sufficient for plasma cell differentiation. In the present study we have performed sequence analysis of the upstream region of the human PRDM1 gene to detect the minimal promoter region necessary for PRDM1 gene transcription. This region comprises the region upstream of the initiation site, as well as the first exon. Collectively, deletion and mutation analysis in conjunction with luciferase reporter assays, EMSA and supershift assays identified a phylogenetically conserved GC-box as an essential element for PRDM1 expression. This GC-box element matches to a binding site for multiple transcription factors such as SP1 and SP3 isoforms as well as early growth response 1. Chromatin immunoprecipitation assays confirmed the in vivo binding capability of these factors to the human PRDM1 promoter. These studies together characterize for the first time the basal activity of the human PRDM1 promoter, through which several factors, including SP1, SP3 and early growth response 1, modulate its expression through a conserved GC-box.

Chemokine receptor expression on infiltrating lymphocytes from abdominal aortic aneurysms: role of CXCR4-CXCL12 in lymphoid recruitment.

This is a study to define the profile of chemokine receptors expressed on isolated infiltrating lymphocytes in human abdominal aortic aneurysms (AAAs), and to examine their role in lymphoid recruitment. AAA T-lymphocytes were CXCR4-positive, CCR7-negative and partially CXCR3 and CCR5-positive. Functionally, AAA T-cells were proinflammatory effector cells, as they produced IFN-gamma and granzyme A. AAA B-lymphocytes were CXCR4-positive and exhibited low CXCR5 expression. A relevant feature of infiltrating T- and B-lymphocytes was the high intensity of CXCR4 expression and the capability to migrate to CXCL12. CXCL12-producing cells were found in the adventitia of AAA. These cells were CD45-negative and partially VCAM-1 and DR-positive. In summary, the present results suggest that CXCR4, expressed on infiltrating lymphocytes, and CXCL12, expressed on stromal cells, is involved in the recruitment of lymphocytes within the arterial wall in AAA.

Increased survival is a selective feature of human circulating antigen-induced plasma cells synthesizing high-affinity antibodies.

The present study shows that tetanus toxoid (tet) booster releases to the human circulation 2 subsets of specific plasma cells (PCs), as defined by phenotype and morphology, which clearly differed in the staining capacity of their cytoplasmic antibodies (Abs) with fluorescein isothiocyanate (FITC)-labeled tet-fragment C (tetC). These cells, called tetCHIGH and tetCINT PCs according to their either high or intermediate FITC-tetC staining capacity, exhibit similar rapid temporary kinetics in the blood (5-8 days after boost), contain many cycling cells, express equivalent amounts of BLIMP-1 mRNA, and produce similar quantities of IgG. However, Abs synthesized by tetCHIGH PCs show a tetC affinity more than 10 times higher than that exhibited by tetCINT PC Abs, and indicated by IGVH sequence analysis. Chemotaxis to CXCL12, a requisite for bone marrow (BM) PC homing, is similar for both cell types. Circulating nonspecific and tetCINT PCs, but not tetCHIGH PCs, tend to undergo spontaneous apoptosis, as demonstrated by APO2.7 and activated caspase-3 expression, and cell recovery. These results indicate that tet booster generates 2 discrete subsets of specific PCs exhibiting different ranges of Ab affinity for the immunogen, and that only those synthesizing high-affinity Abs show enhanced survival. This inherent property may be essential for determining the BM fate of PCs secreting high-affinity Ab.

Higher maturity and connective tissue association distinguish resident from recently generated human tonsil plasma cells.

Human plasma cells (PC) are present in cell suspensions obtained from the tonsil by mechanical disaggregation (PC(MECH)). The present study shows that a collagenase treatment of tonsillar debris remaining after mechanical disaggregation yielded similar proportions of PC (PC(COLL)). Moreover, PC(MECH) were present in suspensions highly enriched in germinal center cells whereas PC(COLL) contained most of the IgA-secreting cells, suggesting their predominant location in follicular and parafollicular areas and connective tissue-rich zones such as tonsil subepithelium, respectively. Tonsil PC(MECH) and PC(COLL) shared the phenotype CD38(high) CD19(+) CD20(low) CD45(high), expressed equivalent amounts of PRDI BF1/Blimp-1 transcription factor, and carried similarly mutated IgVH6 genes. However, they differed in several features. 1) PC(MECH) still expressed the early B cell transcription factor BSAP and were HLA-DR(high); in contrast, PC(COLL) were BSAP(-)and HLA-DR(low). 2) PC(MECH) were CD95(+) and Bcl-2(+/-) whereas PC(COLL) showed CD95(+/-) and Bcl-2(+) expression; in addition, PC(MECH) exhibited increased spontaneous apoptosis. 3) The two PC subsets exhibited distinctive adhesion molecule profiles, since PC(COLL) expressed higher levels of CD31, CD44, and CD49d, but a lower level of CD11a than PC(MECH). These results suggest that PC(MECH) are recently generated, short-living PC, and PC(COLL) constitutes a subset with higher maturity and survival, which resides in connective tissue-rich areas.

Human BSAP and BLIMP1 conform an autoregulatory feedback loop.

B-lymphocyte-induced maturation protein-1 (BLIMP1), encoded by the PRDM1 gene, is a transcriptional repressor considered a master regulator that is required and sufficient for plasma cell (PC) differentiation. BLIMP1 represses the PAX5 gene, coding for the B-cell lineage-specific activator protein (BSAP), which is required for B-cell identity and survival. Mutations in PAX5 gene as well as in PRDM1 gene have been recently implicated in lymphomas. In the present study, sequence analysis of PRDM1 gene revealed a binding site for BSAP transcription factor. By analyzing different human cell lines, we have found that a specific nuclear factor for B-cell lines binds to a site on the PRDM1 promoter. Electrophoretic mobility shift assays identified this factor as BSAP, and chromatin immunoprecipitation assays confirmed its binding in vivo to the human PRDM1 promoter. Moreover, by ectopically expressing BSAP, and using a PRDM1 promoter with the BSAP-binding site mutated, we demonstrated that this factor represses the expression of BLIMP1. Therefore, repression of PRDM1 by BSAP reveals an autoregulatory negative-feedback loop that could play a relevant role in controlling human PC differentiation.

The prognostic role of CXCR3 expression by chronic lymphocytic leukemia B cells.

BACKGROUND AND OBJECTIVES: Chemokine receptors are involved in tumor progression and several of these receptors, including CXCR3, are expressed by chronic lymphocytic leukemia (CLL) B cells. This study was aimed to examine a possible relationship between CXCR3 expression in CLL and the clinical evolution of the disease. DESIGN AND METHODS: Using flow activated cell sorting (FACS), we analyzed the level of expression of CXCR3 on blood CLL B cells from 76 consecutive patients. The results were correlated with CD38 expression, IgVH gene status and clinical outcome. RESULTS: CXCR3, measured as mean fluorescence intensity (MFI), was unimodally expressed by blood tumor cells at various levels (range, 3.5 to 232.3) but levels within individual patients were remarkably stable over time. Low CXCR3 expression by CLL B cells was strongly associated with Rai disease stages III and IV (p<0.0001) and a pattern of diffuse tumor infiltration of the bone marrow (p<0.0001). In the 28 cases available for genetic studies, low CXCR3 expression also showed good concordance with tumor unmutated IgVH gene status (p<0.04), and tended to correlate with high CD38 expression (p<0.06). Patients with low CXCR3 expression (MFI < or =15) had a shorter survival (p<0.0001) and, in multivariate analysis, low CXCR3 expression (MFI pound15) was an independent predictor of poor outcome (hazard ratio 24.5; p<0.01). INTERPRETATION AND CONCLUSIONSL: CXCR3 expression by CLL B cells appears to be stable within individual patients. Tests to assay this chemokine receptor are cheap and easy to perform and the results could be of prognostic value in CLL.

CD95-Mediated control of anti-citrullinated protein/peptides antibodies (ACPA)-producing plasma cells occurring in rheumatoid arthritis inflamed joints.

OBJECTIVE: Serum anti-citrullinated protein/peptides antibodies (ACPA) are a valuable diagnostic parameter that might be involved in rheumatoid arthritis (RA) pathogenesis. CD95-dependent apoptosis is defective in RA synovium. The present study explores the occurrence of ACPA IgG, and the CD95-mediated control of ACPA IgG-secreting plasma cells (PC) in RA patients. METHODS: Mononuclear cells (MC) were purified from synovial fluid (SF) and peripheral blood (PB) of 15 RA patients. PC capable of secreting ACPA IgG were detected in MC cultures. ACPA IgG present in serum and SF, and PB and SF MC culture supernatant was measured by ELISA. CD95, CD27 and CD138 expression was examined on RA PC identified as CD19(low) CD38(high) cells by flow cytometry. CD95-ligation was obtained by treatment of cultured MC with the anti-CD95 Ab CH11. Apoptotic PC were identified as Annexin-V+. RESULTS: ACPA IgG level was found higher in patients' SF than in their serum. PC were detectable in SF and PB, and exhibited high CD95 and CD27 expression. In contrast, SF, but not PB, PC expressed elevated levels of CD138. SF, but not PB, PC actively secreted ACPA IgG in cultures, in a linear fashion for at least 14 days, and CD95-ligation markedly reduced this activity and provoked PC apoptosis. CONCLUSIONS: The results suggest that RA synovium is a prominent site for ACPA IgG formation and for the accumulation of ACPA IgG-secreting PC exhibiting prolonged survival, probably due to RA defective CD95-mediated control.

The expression of PRDI-BF1 beta isoform in multiple myeloma plasma cells.

The PRDM1 gene, a master regulator of plasma cells (PC), can generate two transcription factor isoforms: PRDI-BF1alpha and PRDI-BF1beta. The present study shows that purified human normal PC have a significantly lower levels of PRDI-BF1beta expression than that in tumoral PC isolated from multiple myeloma (MM) (0.06+/-0.01 and 0.25+/-0.05, respectively; p<0.001). The role of this finding in MM is discussed.

Inorganic polyphosphate and specific induction of apoptosis in human plasma cells.

BACKGROUND AND OBJECTIVES: Inorganic polyphosphate (polyP), a ubiquitous phosphate polymer with ATP-like bonds, has recently been related to a variety of functions including blood coagulation and cell proliferation. We investigated the effects of polyP in the biology of human plasma cells (PC), responsible for the production and maintenance of antibodies in response to antigens. DESIGN AND METHODS: The U266 myeloma cell line was used to study whether polyP affects immunoglobulin (Ig) secretion and survival. Different human cell lines were used to test the specificity of polyP on viability. We analyzed Ig secretion of PC from bone marrow and peripheral blood after polyP addition. A conventional tetanus toxoid booster immunization was used to increase the proportion of PC in order to examine the ex vivo effects of polyP. We also tested the effects of polyP on primary myeloma cells. Ig secretion and apoptosis were determined by ELISA and FACS respectively. RESULTS: Addition of polyP to human PC produced an unexpected inhibition of Ig secretion and stimulation of apoptosis. PolyP generated apoptosis specifically in PC, myeloma (malignant PC) cell lines, primary myeloma cells, and B lymphoid cell lines. Normal B cells, T cells, total blood mononuclear cells, and non-lymphoid cell lines were not affected by polyP. In the U266 myeloma cell line, polyP induced externalization of phosphatidylserine, activation of caspase-3, and arrest of the cell cycle. The protective effects of interleukin-6 did not overcome the polyP-induced apoptosis Interpretation and CONCLUSIONS: Taken together, our results suggest for the first time the relevance of the use of polyP to the humoral immune response and open prospects for polyP as a novel therapy for myeloma.