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The increasingly recognized role of different types of B cells and plasma cells in protective and pathogenic immune responses combined with technological advances have generated a plethora of information regarding the heterogeneity of this human immune compartment. Unfortunately, the lack of a consistent classification of human B cells also creates significant imprecision on the adjudication of different phenotypes to well-defined populations. Additional confusion in the field stems from: the use of non-discriminatory, overlapping markers to define some populations, the extrapolation of mouse concepts to humans, and the assignation of functional significance to populations often defined by insufficient surface markers. In this review, we shall discuss the current understanding of human B cell heterogeneity and define major parental populations and associated subsets while discussing their functional significance. We shall also identify current challenges and opportunities. It stands to reason that a unified approach will not only permit comparison of separate studies but also improve our ability to define deviations from normative values and to create a clean framework for the identification, functional significance, and disease association with new populations., (Copyright © 2019 Sanz, Wei, Jenks, Cashman, Tipton, Woodruff, Hom and Lee.)

Plasma cell segmentation is the first stage of a computer assisted automated diagnostic tool for multiple myeloma (MM). Owing to large variability in biological cell types, a method for one cell type cannot be applied directly on the other cell types. In this paper, we present PCSeg Tool for plasma cell segmentation from microscopic medical images. These images were captured from bone marrow aspirate slides of patients with MM. PCSeg has a robust pipeline consisting of a pre-processing step, the proposed modified multiphase level set method followed by post-processing steps including the watershed and circular Hough transform to segment clusters of cells of interest and to remove unwanted cells. Our modified level set method utilizes prior information about the probability densities of regions of interest (ROIs) in the color spaces and provides a solution to the minimal-partition problem to segment ROIs in one of the level sets of a two-phase level set formulation. PCSeg tool is tested on a number of microscopic images and provides good segmentation results on single cells as well as efficient segmentation of plasma cell clusters., Competing Interests: The authors have declared that no competing interests exist.

Plasma cells are rare cells that have been notoriously difficult to detect by flow cytometry. New advances have described B220+ CD138+ plasma cells in the bone marrow that are particularly difficult to distinguish between CD138 intermediate B220+ developing B cells. Herein we describe a novel method for detecting plasma cells in the bone marrow using a combination of CD138 and Sca-1 staining., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We provide a robust four-color fluorescence-based flow cytometry protocol that distinguishes viable dividing plasmablasts from nondividing plasma cells and, based on CD19 surface abundance, identifies two mature plasma cell populations in the spleen and the bone marrow of mice., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: To study the clinical, pathologic, immunophenotype, molecular characteristics and prognosis of HIV-negative plasmablastic lymphoma (PBL)., Methods: Twelve cases of HIV-negative PBLs diagnosed between 2005 and 2014 in Guangdong General Hospital were identified according to WHO classification of tumors of haematopoietic and lymphoid tissues (2008). The clinicopathologic features and outcome were analyzed and the relevant literatures were reviewed., Results: The patients were predominantly male (11/12) with a median age of 55.5 years. The tumor cells showed the characteristic combination of immunoblastic/plasmablastic morphology, plasma cell phenotype and high proliferation, no expression of mature B cell markers. 7/10 of the cases were EBER positive. Two cases were positive for C-myc translocation. Four of twelve patients were died., Conclusions: PBL is a rare, aggressive B-cell lymphoma. HIV-negative PBL has lower rate of oral involvement and EBER expression than HIV-positive patients, the differential diagnosis is very challenging, and the prognosis is worse.

Normal and reactive plasma cells (PC) are easy to ascertain on human bone marrow films, due to their small mature-appearing nucleus and large cytoplasm, the latter usually deep blue after Giemsa staining. Cytoplasm is filled with long strands of rough endoplasmic reticulum and one large Golgi apparatus (paranuclear hof), demonstrating that PC are dedicated mainly to protein synthesis and excretion (immunoglobulin). Deregulation of the genome may induce clonal expansion of one PC that will lead to immunoglobulin overproduction and eventually to one among the so-called PC neoplasms. In multiple myeloma (MM), the number of PC is over 10% in most patients studied. Changes in the morphology of myeloma PC may be inconspicuous as compared to normal PC (30-50% patients). In other instances PC show one or several morphological changes. One is related to low amount of cytoplasm, defining lymphoplasmacytoid myeloma (10-15% patients). In other cases (40-50% patients), named immature myeloma cases, nuclear-cytoplasmic asynchrony is observed: presence of one nucleolus, finely dispersed chromatin and/or irregular nuclear contour contrast with a still large and blue (mature) cytoplasm. A peculiar morphological change, corresponding to the presence of very immature PC named plasmablasts, is observed in 10-15% cases. Several prognostic morphological classifications have been published, as mature myeloma is related to favorable outcome and immature myeloma, peculiarly plasmablastic myeloma, is related to dismal prognosis. However, such classifications are no longer included in current prognostic schemes. Changes related to the nucleus are very rare in monoclonal gammopathy of unknown significance (MGUS). In contrast, anomalies related to the cytoplasm of PC, including color (flaming cells), round inclusions (Mott cells, Russell bodies), Auer rod-like or crystalline inclusions, are reported in myeloma cases as well as in MGUS and at times in reactive disorders. They do not correspond to malignant changes of PC but are related to abnormal synthesis, trafficking, or excretion of the immunoglobulin that is stored in excess within the cytoplasm. Occurrence of crystalline inclusions within PC may be the first anomaly leading to the diagnosis of adult Fanconi syndrome. After a historical perspective, the authors report on the various morphological aspects of PC that may occur in multiple myeloma and related disorders, and discuss about their clinical and pathophysiological significance. Today, morphological identification and accurate determination of % PC within bone marrow remain ancillary criteria for the diagnosis of MM and help for the diagnosis of rare renal disorders., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Specific serum antibodies mediating humoral immunity and autoimmunity are provided by mature plasma cells (PC) residing in the bone marrow (BM), yet their dynamics and composition are largely unclear. We here characterize distinct subsets of human PC differing by CD19 expression. Unlike CD19(+) PC, CD19(-) PC were restricted to BM, expressed predominantly IgG, and they carried a prosurvival, distinctly mature phenotype, that is, HLA-DR(low)Ki-67(-)CD95(low)CD28(+)CD56(+/-), with increased BCL2 and they resisted their mobilization from the BM after systemic vaccination. Fewer mutations within immunoglobulin VH rearrangements of CD19(-) BMPC may indicate their differentiation in early life. Their resistance to in vivo B-cell depletion, that is, their independency from supply with new plasmablasts, is consistent with long-term stability of this PC subset in the BM. Moreover, CD19(-) PC were detectable in chronically inflamed tissues and secreted autoantibodies. We propose a multilayer model of PC memory in which CD19(+) and CD19(-) PC represent dynamic and static components, respectively, permitting both adaptation and stability of humoral immune protection., (© 2015 by The American Society of Hematology.)

Circulating myeloma tumor cells (CTCs) as defined by the presence of peripheral blood (PB) clonal plasma cells (PCs) are a powerful prognostic marker in multiple myeloma (MM). However, the biological features of CTCs and their pathophysiological role in MM remains unexplored. Here, we investigate the phenotypic, cytogenetic, and functional characteristics as well as the circadian distribution of CTCs vs paired bone marrow (BM) clonal PCs from MM patients. Our results show that CTCs typically represent a unique subpopulation of all BM clonal PCs, characterized by downregulation (P < .05) of integrins (CD11a/CD11c/CD29/CD49d/CD49e), adhesion (CD33/CD56/CD117/CD138), and activation molecules (CD28/CD38/CD81). Fluorescence in situ hybridization analysis of fluorescence-activated cell sorter-sorted CTCs also unraveled different cytogenetic profiles vs paired BM clonal PCs. Moreover, CTCs were mostly quiescent and associated with higher clonogenic potential when cocultured with BM stromal cells. Most interestingly, CTCs showed a circadian distribution which fluctuates in a similar pattern to that of CD34(+) cells, and opposite to stromal cell-derived factor 1 plasma levels and corresponding surface expression of CXC chemokine receptor 4 on clonal PCs, suggesting that in MM, CTCs may egress to PB to colonize/metastasize other sites in the BM during the patients' resting period.

Bone marrow flow cytometric analysis is a powerful and rapid tool for evaluating aberrant plasma cell. In this study, we have examined the utility of multiparameter flow cytometry (MFC) in 52 patients with multiple myeloma (MM) and in 45 patients with monoclonal gammopathy with unknown significance (MGUS) into routine evaluation for the management of patients with plasma cell-related disorders. The plasma cells (PC) were identified by their light scatter distribution and reactivity patterns to CD138, CD38, and CD45. The combination of these parameters was helpful for identifying distinct subpopulations of PCs. Moderate to bright expression of CD56, CD20, CD24, CD28, and CD117 was detected in 67%, 26%, 13%, 27%, and 57% of MM cases and in 58%, 20%, 11%, 43% and 44% of MGUS cases, respectively. In MGUS group, the median percentage abnormal PCs/total PCs was 88% with 37 patients out of 45 (82%) with ratio <95%. The median ratio of the MM group was 98.9% and a ratio ≥ 95% was observed in 37 samples out of 44 (84%). In conclusion, MFC immunophenotyping of PCs has obvious clinical relevance in differential diagnosis between MM and others monoclonal gammopathies, identification of high-risk MGUS and smouldering MM, and minimal residual disease monitoring of MM. Our results showed that this tool can be easily applied in haematology laboratories.

While endometrial neutrophils and plasma cells are criteria used to diagnose histologic endometritis in epidemiologic pelvic inflammatory disease (PID) research, plasma cell misidentification and nonspecificity may limit the accuracy of these criteria. Herein, we examined: (1) the identification of endometrial plasma cells with conventional methyl green pyronin-based methodology versus plasma cell-specific (CD138) immunostaining, (2) the prevalence of endometrial plasma cells among women at low risk for PID, and (3) endometrial leukocyte subpopulations among women diagnosed with acute or chronic histologic endometritis by conventional criteria. We observed an absence of CD138+ cells in 25% of endometrial biopsies in which plasma cells had been identified by conventional methodology, while additional immunohistochemical analyses revealed indistinguishable inflammatory infiltrates among women diagnosed with acute or chronic endometritis by conventional criteria. Among women considered at lower risk for PID development, flow cytometric analyses detected plasma cells in 30% of endometrial biopsy specimens, suggesting that these cells, even when accurately identified, only nonspecifically identify upper genital tract inflammatory processes. Combined, our findings underscore the limitations of the criteria used to diagnose histologic endometritis in PID-related research and suggest that satisfactory understanding of PID pathogenesis, treatment, and prevention is hindered by continued use of these criteria., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Sustained long-term antibody levels are the cornerstone of protective immunity, yet it remains unclear how they are durably maintained. A predominant theory implicates antigen-independent antibody production by a subset of long-lived plasma cells (LLPCs) that survive within bone marrow (BM). Central tenets of this model--that BM LLPCs constitute a subset defined by intrinsic biology distinct from PCs in other tissues and contribute to long-term antibody titers--have not been definitively demonstrated. We now report that long-term humoral immunity depends on the PC-intrinsic function of CD28, which selectively supports the survival of BM LLPC but not splenic short-lived PC (SLPC). LLPC and SLPC both express CD28, but CD28-driven enhanced survival occurred only in the LLPC. In vivo, even in the presence of sufficient T cell help, loss of CD28 or its ligands CD80 and CD86 caused significant loss of the LLPC population, reduction of LLPC half-life from 426 to 63 d, and inability to maintain long-term antibody titers, but there was no effect on SLPC populations. These findings establish the existence of the distinct BM LLPC subset necessary to sustain antibody titers and uncover a central role for CD28 function in the longevity of PCs and humoral immunity.

Conventional measurement of antibody responses to vaccines largely relies on serum antibodies, which are primarily produced by bone marrow plasma cells and may not represent the entire vaccine-induced B cell repertoire, including important functional components such as those targeted to mucosal sites. After immunization or infection, activated B cells differentiate into plasmablasts in local lymphoid organs, then traffic through circulation to the target sites where they further develop into plasma cells. On day 7 after influenza vaccination, a burst of plasmablasts, highly enriched for vaccine-specific antibody secreting cells, appears in the peripheral blood. This provides a unique window to the overall B cell response to the vaccine, without interference of pre-existing cross-reactive serum antibody. In this study we isolated B cells from volunteers on day 7 after immunization with the inactivated influenza vaccine and cultured them ex vivo to collect plasmablast-derived polyclonal antibodies (PPAb). The PPAb contained secreted IgG and IgA, which was approximately 0.2ng per antibody secreting cell. Influenza-specific IgG and IgA binding activity was detected in PPAb at dilutions up to 10(5) by ELISA. The ratio of the titers of influenza-specific IgA to IgG by ELISA was 4-fold higher in PPAb than in day 28 post-vaccination sera, suggesting that vaccine-induced IgA is enriched in PPAb compared to sera. Functional activity was also detected in PPAb as determined by microneutralization and hemagglutination inhibition assays. In addition to bulk B cell cultures, we also cultured plasmablast subsets sorted by cell surface markers to generate PPAb. These results suggest that PPAb better reflects the mucosal IgA response than serum samples. Since PPAb are exclusively produced by recently activated B cells, it allows assessing vaccine-induced antibody response without interference from pre-existing cross-reactive serum antibodies and permits an assessment of antibody avidity based on antigen specific binding and antibody quantity. Therefore this assay is particularly useful for studying vaccine/infection-induced antibodies against antigens that might have previously circulated, such as antibody responses to rotavirus, dengue or influenza viruses in which cross-reactive antibodies against different virus serotypes/subtypes play a critical role in immunity and/or pathogenesis., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Multiple myeloma (MM) is a hematological malignancy caused by clonal proliferation of malignant plasma cells (PC). The aim of the work is to determine prognostic significance of morphological subtypes of PC in relation to overall treatment response, long-term survival and other conventional prognostic parameters. One hundred and thirty-nine newly diagnosed MM patients who underwent autologous transplantation in clinical trials conducted in one center were included. Percentual representation of subtypes of plasma cells in bone marrow was measured based on progressive analysis of nucleolus, nuclear chromatin and ratio of nuclei to the volume of cytoplasm (N/C ratio) creating 8 subtypes P000-P111 and four subclassifications of cells. Mature plasma cells (P000, P001) were found in 42.4% of patients; proplasmocytes I (P010, P011, P100) in 38.1% of patients, and proplasmocytes II (P101, P110) in 19.4% of patients. Patients who reached treatment response after autologous transplantation had statistically significant lower frequency of mature plasma cells than patients with no treatment response (median 24.0% vs. 36.0 %; p=0.032). Patients with mature plasma cells of subtype P000 an patients with value P000 ≥ 37% (median 46.8 months vs. 77.8 months; p = 0.020). Patients with proplasmocytes II subtype P110 rings valuable prognostic information and correlation with other prognostic factors as well as total treatment response and survival in MM patients who underwent autologous transplantation.

In this paper, initial processing of biological material, cell separation algorithms and other procedures are discussed. For samples with initial infiltration of plasma cells > 5%, CD138 MicroBeads and Auto-Magnetic-Activated Cell Sorting program are used. Fluorescence-Activated Cell Sorting is used exclusively for cell populations with low-abundance; these samples are detected using fluorescently labeled antibodies only. Isolated plasma cells are further processed for molecular biological studies, for cytogenetics and protein analyses. Furthermore, this work examines the pitfalls of research related to multiple myeloma; some of them we have overcome, while the others are still problematic.

The technological development of flow cytometry (FC) together with new findings reveal the need for immunophenotyping in research of monoclonal gammopathy (MG) because of its diagnostic, prognostic and predictive significance. The aim of the European Myeloma Network (EMN) is to standardize this analytical method and implement it into routine clinical examination. Since the overall significance and application of FC are still analysed, standardisation could help obtain more clinical relevant information in terms of MG pathophysiology.

Objectives: Castleman's disease (CD) is a group of rare atypical lymphoproliferative disorders classified as hyaline vascular (HV) and plasma cell (PC) types. CD may be closely mimicked by IgG4-related sclerosing disease (IgG4-SD) involving the lymph nodes. We retrospectively analyzed findings in patients with CD to elucidate the relationship between CD and IgG4-SD., Methods: Clinicopathological and immunophenotypical characteristics, including IgG+ and IgG4 expression by plasma cells, were analyzed in 87 consecutive patients diagnosed with CD from 1999 to 2010 at two major Korean hospitals., Results: The numbers of IgG+ (p < 0.001) and IgG4+ (p < 0.001) cells and the IgG4:IgG ratio (p = 0.003) were significantly higher in the PC than in the HV group. The mean IgG4+:IgG+ plasma cell ratio in the PC group was 25.1%, with 10 patients having a ratio >40%, the threshold IgG4:IgG ratio in patients with IgG4-SD., Conclusions: Patients with the PC form of CD and IgG4-related lymphadenopathy share some features, including the pattern of plasma cell distribution and high-level infiltration of IgG4+ cells. Some patients thought to have the PC form of CD could be reclassified as showing IgG4-related lymphadenopathy., (Copyright © 2011 S. Karger AG, Basel.)

Although integration of laboratory data and radiological findings are required, quantity and qualitative evaluation of plasma cells in bone marrow is still useful for the diagnosis of multiple myeloma. However, there are considerable variations in morphology of myeloma and proliferation pattern among the individual patients. Therefore, it is important to recognize cytopathological features of myeloma cells for better understanding of patients' status. For example, the finding of immature plasmablastic myeloma cells predicts the poor prognosis of the patients. In the present review, we described the cytopathological features, which is useful for diagnosis and estimation of the patients' prognosis and discrimination from reactive non-tumorous plasma cells.

Increases in free light chain (FLC) production are associated with disease progression in multiple myeloma (MM). Using a double immunofluorescence staining method to produce a differential count of plasma cells in bone marrow, single populations were demonstrated, containing intact monoclonal immunoglobulins (M-Igs) in 74% and FLCs only in 8% of cases. However, 18% contained a mixture of both cell populations. Progression from cells making intact M-Ig to cells restricted to FLC only production occurred in individual cases during the course of their disease. The presence of FLC only cells was associated with shortened survival.

Although there is evidence for distinct roles of myeloid dendritic cells (DCs [mDCs]) and plasmacytoid pre-DCs (pDCs) in regulating T cell-mediated adaptive immunity, the concept of functional DC subsets has been questioned because of the lack of a molecular mechanism to explain these differences. In this study, we provide direct evidence that maturing mDCs and pDCs express different sets of molecules for T cell priming. Although both maturing mDCs and pDCs upregulate the expression of CD80 and CD86, only pDCs upregulate the expression of inducible costimulator ligand (ICOS-L) and maintain high expression levels upon differentiation into mature DCs. High ICOS-L expression endows maturing pDCs with the ability to induce the differentiation of naive CD4 T cells to produce interleukin-10 (IL-10) but not the T helper (Th)2 cytokines IL-4, -5, and -13. These IL-10-producing T cells are T regulatory cells, and their generation by ICOS-L is independent of pDC-driven Th1 and Th2 differentiation, although, in the later condition, some contribution from endogenous IL-4 cannot be completely ruled out. Thus, in contrast to mDCs, pDCs are poised to express ICOS-L upon maturation, which leads to the generation of IL-10-producing T regulatory cells. Our findings demonstrate that mDC and pDCs are intrinsically different in the expression of costimulatory molecules that drive distinct types of T cell responses.

The St. Vincent's Comprehensive Cancer Center (SVCCC) has a large multiple myeloma program in downtown New York City. The laboratory at SVCCC is an integral part of the diagnosing and monitoring of its myeloma patients. Circulating plasma cells are not a common finding in multiple myeloma. Being able to detect plasma cells in peripheral blood is important because they are a prognostic indicator that correlates with disease progression. Furthermore, the peripheral blood plasma cell population can demonstrate morphologic variability. Immature plasma cells, both plasmablasts and proplasmacytes are associated with more aggressive disease and shortened survival. We encountered 3 multiple myeloma patients with circulating immature plasma cells that appeared as distinct populations on our hematology analyzer's automated white blood cell (WBC) differential. The immature plasma cells, given their unique cellular characteristics, appeared in a common place within the WBC differential scatterplot in each patient. In our laboratory, we have utilized this common graphic pattern to screen for immature plasma cells. This pattern has proven to be a useful tool in our large population of multiple myeloma patients. We have also used examination of the scatterplots in other hematologic malignancies such as chronic lymphocytic leukemia. Using this review policy, the laboratory has been able to achieve a smear review of 25% in our highly abnormal patient population.

We have recently identified two groups of plasmacytoid dendritic cells (pDCs) isolated from murine liver based on the expression of CD4 and other cell surface markers uniquely expressed by pDCs. Herein, we describe the identification of both CD4+ and CD4- pDCs that clearly exist in lymph nodes (LNs), spleen, liver, thymus, bone marrow, and lung. Normally, CD4+ pDCs are enriched in LNs. However, after in vivo systemic injection with bacterial CpG, a larger number of CD4- pDCs are recruited to the LNs and local inoculation by CpG drives CD4- pDCs migrating into local sentinel LNs, suggesting that CD4- pDCs are the main subpopulation migrating to the peripheral LNs. Furthermore, although both freshly isolated CD4+ pDCs and CD4- pDCs appear as an immature plasmacytoid cell and develop into a DC morphology following activation, the two subsets have strikingly different immune features, including differences in the production pattern of cytokines stimulated with CpG and in T cell activation.

Plasmacytoid dendritic cells (pDCs) have been associated with several names and functions over time, reflecting the limited availability of specific markers and their ability to produce large amounts of type I interferons, present antigens, as well as prime disparate T-cell helper responses. Yet, there is increasing evidence that pDCs are a distinct cell type in the innate immune system. This review highlights aspects in which pDCs are unique in comparison to other antigen-presenting cells in regulating innate and adaptive immune responses.

We propose a model where human dendritic cell (DC) subsets control, in a coordinated fashion, B cell growth and differentiation. Plasmacytoid DCs drive memory B cell differentiation into effector plasma cell via type I interferon and IL-6. Type I interferon activates myeloid DCs that regulate B cell priming and acquisition of memory phenotype via IL-12, IL-6 and Blys/Baff. This model also integrates the role of antigen-specific T cells activated by myeloid DCs. Thus, protective humoral immunity results from a highly coordinated interplay of human DC subsets. This interplay may explain the spreading of immune response to deal with antigenic drift and to maintain an active immunity against mutating microbe. It may also provide an explanation for spreading of the autoimmune repertoire as autoimmunity develops.

Recent studies have shown that two recurrent translocations, t(4;14)(p16;q32) and t(11;14)(q13;q32), define distinct entities with different prognosis in multiple myeloma (MM). We addressed the issue of whether these illegitimate IGH rearrangements could contribute to the morphological heterogeneity of the malignant plasma cells (PC). Bone marrow aspirates of 178 untreated MM cases with successful molecular cytogenetics analysis using fluorescence in situ hybridization were reviewed. PC of 25/48 (52%) patients with t(11;14) exhibited a lymphoplasmacytoid morphology. Moreover, 25/27 (93%) of the cases with this morphological profile bore the t(11;14). In addition, both cytogenetics and morphological subtypes shared higher incidence of nonsecretory MM. In contrast, 17 out of 28 cases (61%) with t(4;14) exhibited PC with diffuse chromatin pattern. Interestingly, both t(4;14) translocation and immature morphology correlated with higher incidence of high tumor mass and chromosome 13 abnormality. In conclusion, our results suggest that a particular morphology can be the signature of chromosomal abnormalities in MM.

Background: Cyclosporin A (CsA) is an immunosuppressive agent that is known to induce gingival overgrowth (GO). Pharmacological, genetic, immunologic, and inflammatory factors seem to be involved in the complex pathogenesis of drug-induced GO. Lymphocyte subpopulations in human gingival connective tissue have been implicated in the pathogenesis of inflammatory periodontal diseases. One purpose of this study was to quantify CD4, CD8-, CD57-, and epithelial membrane antigen (EMA)-positive cells in the gingiva of renal transplant recipients treated with CsA, and compare them to findings in healthy controls. A second aim was to correlate cell numbers with clinical findings., Methods: The study included 19 kidney recipients who were taking CsA and had significant GO (CsAGO+), 13 recipients who were taking CsA but showed no GO (CsAGO-), and 14 systemically healthy individuals with gingivitis (C). Sections from gingival biopsies were incubated with monoclonal antibodies for CD4, CD8, EMA, and CD57, and then analyzed using the avidin-biotin complex method. In each specimen, the mononuclear cell types were quantified and their distribution was evaluated in 3 separate tissue zones: S = subepithelial connective tissue beneath the sulcular epithelium; O = subepithelial connective tissue beneath the oral epithelium; and M = middle connective tissue., Results: There were no significant differences among the groups with respect to the numbers of CD4+ and CD8+ cells in each of the 3 zones (P >0.05). In zone S, the CsAGO+ group had significantly more EMA-positive cells than either the C or CsAGO- groups (P <0.05). There were significant differences among the groups regarding numbers of CD57+ (natural killer) cells in zone M, with the lowest cell numbers in the CsAGO+ patients (P<0.05)., Conclusions: The results showed that low numbers of natural killer cells are important in the expression of plaque-induced inflammatory changes in CsA-associated GO. It appears that these cells may influence the drug's ability to induce proliferative activity.

Human plasmacytoid dendritic cells (pDCs) are major producers of IFNalpha, are activated by CpG motifs, and are believed to enter lymph nodes (LNs) via L-selectin dependent extravasation across high endothelial venules. To identify a similar murine DC type, CD11c(+) cells in the LNs of L-selectin-deficient and control BALB/c mice were compared, revealing a population of CD11c(+)CD11b(-) cells that is reduced 85% in the LNs of L-selectin-deficient mice. These cells are Gr-1(+)B220(+)CD19(-), either CD4(+) or CD8(+), and localize within T cell zones of LNs. Freshly isolated CD11c(+)Gr-1(+) cells express major histocompatibility complex class II at low levels, display a plasmacytoid morphology, and survive poorly in culture. Their survival is increased and they develop a DC-like morphology in interleukin 3 and CpG. Like human pDCs, CD11c(+)Gr-1(+) cells stimulate T cell proliferation after activation with CpG and produce IFNalpha after stimulation with influenza virus. These cells also display a strain-specific variation in frequency, being fivefold increased in the LNs of BALB/c relative to C57BL/6 mice. These CD11c(+)CD11b(-)B220(+)Gr-1(+) cells appear to be the murine equivalent of human pDCs.

This paper describes the histopathological features and the cellular distribution of T lymphocytes (CD3), B cells (CD79a), immunoglobulin (IgG, IgA, IgM)-bearing plasma cells, macrophages (Mac387 and alpha-1-antitrypsin), MHC class II antigen and S-100 protein in hepatic white spots associated with naturally occurring Ascaris suum parasitism in 35 pigs. Hepatic granulomas were observed in 10 pigs, whereas lymphoid proliferation with a diffuse or lymphonodular pattern was the main histopathological lesion in 14 other pigs, and portal fibrosis in a further 11 animals. In lymphonodular lesions, the distribution of immunoreactive cells with all antibodies tested was closely similar to that found in the cortex of lymph nodes. Thus, lymphoid follicles were composed mainly of CD79a(+)B cells and interfollicular tissue was composed mainly of CD3(+)T lymphocytes. The presence of follicular dendritic and interdigitating cells expressing S-100 protein and MHC class II antigen in lymphonodular lesions suggested that these are highly organized structures developed to enhance antigen presentation to B and T cells, and consequently the local immune response against the parasite. The humoral local response was represented mainly by IgG-secreting plasma cells., (Copyright Harcourt Publishers Ltd.)

The relationship between plasmablastic cells and outcome in multiple myeloma (MM) has been established for nearly 15 years. But the assessment of these cells is not easy to perform and it allows the identification of only a small proportion of patients. We investigated the plasma cell morphology using a progressive evaluation of consecutive criteria: nucleolus, chromatin and nuclear-cellular ratio (N/C). The combination of these three items produces a subclassification where four cellular subtypes identify 93% of the plasma cells, and these subtypes are related to the outcome. The interest of this methodology is to be based on the mature plasma cells that are easier to identify than the plasmablastic cells. These new cell subtypes introduce a new classification for patients: Group 1 includes patients with at least 66% mature plasma cells (P000). Both Group 2 and 3 have less than 66% P000 and are separated by their degree of maturation (Proplasma I > or = Proplasma II + plasmablastic). The distinction of these three groups of patients is highly related to the prognosis (P < 10(-4)). These results have been confirmed on a second group of patients coming from a different institution. In conclusion, we propose a new methodology for the plasma cell evaluation in MM, that is based on the morphological criteria and that has the advantage of identifying an intermediate (30%) subgroup of patients with a prognostic significance.

We have identified two cell subsets in human blood based on the lack of lineage markers (lin-) and the differential expression of immunoglobulin-like transcript receptor 1 (ILT1) and ILT3. One subset (lin-/ILT3+/ILT1+) is related to myeloid dendritic cells. The other subset (lin-/ILT3+/ILT1+) corresponds to 'plasmacytoid monocytes'. These cells are found in inflamed lymph nodes in and around the high endothelial venules. They express CD62L and CXCR3, and produce extremely large amounts of type I interferon after stimulation with influenza virus or CD40L. These results, with the distinct cell phenotype, indicate that plasmacytoid monocytes represent a specialized cell lineage that enters inflamed lymph nodes at high endothelial venules, where it produces type I interferon. Plasmacytoid monocytes may protect other cells from viral infections and promote survival of antigen-activated T cells.

The B cell-specific activator protein (BSAP) is a DNA-binding transcription factor expressed in pro-B, pre-B, and mature B cells but not in plasma cells. We explored the role of BSAP in B cell function by creating clones in a late B cell and a plasma cell line transfected with a BSAP expression plasmid. We found that the plasma cell line MPC11, which does not produce BSAP, is still permissive to BSAP production driven by heterologous promoter. Overexpression of BSAP in a late B cell line (CH12.LX.A2) and a plasma cell line augmented cell proliferation and led to greater suppression of Ig synthesis in a late B cell line than in the plasma cell line. The reduction was seen mostly in synthesis of a secretory form of Ig. Overexpression of BSAP reduced Blimp-1 expression in CH12.LX.A2 clones but not in MPC11 clones. In addition, overexpression of BSAP in CH12.LX.A2 cells suppressed spontaneous appearance of cells with high Syndecan-1 expression and high amounts of intracytosolic as well as secreted Ig synthesis. To corroborate the above findings, we cloned nontransfected CH12.LX.A2 cells and found reduced BSAP mRNA expression in the high Ig-secreting clones, which produced more Blimp-1 mRNA with greater Syndecan-1 expression than the low Ig-secreting clones. Taken together, these results indicate that BSAP expression is sufficient to reduce Ig production in late B cells; this effect is mediated in part by suppression of differentiation to cells of plasma-cell phenotype.

We investigated the ability of blood B cells, bone marrow (BM) plasma cells, and terminal leukemic plasma cells (T-PCL) from patients with multiple myeloma (MM) to migrate on extracellular matrix proteins. Hyaluronan (HA), but not collagen type I, collagen type IV, or laminin, promoted migration of MM blood B cells, as determined by time-lapse video microscopy. Between 13% and 20% of MM blood B cells migrated on HA with an average velocity of 19 micron/min, and greater than 75% of MM blood B cells exhibited vigorous cell movement and plasma membrane deformation, as did circulating T-PCL and extraskeletal plasma cells from patients with MM. In contrast, plasma cells obtained from BM of patients with MM lacked motility on all substrates tested and did not exhibit cell membrane protrusions or cellular deformation. MM blood B cells and MM plasma cells from all sources examined expressed the HA-binding receptors receptor for HA-mediated motility (RHAMM) and CD44. On circulating MM B cells, both RHAMM and CD44 participated in HA-binding, indicating their expression ex vivo in an activated conformation. In contrast, for the majority of BM plasma cells in the majority of patients with MM, expression of RHAMM or CD44 was not accompanied by HA binding. A minority of patients did have HA-binding BM plasma cells, involving both RHAMM and CD44, as evidenced by partial blocking with monoclonal antibodies (MoAbs) to RHAMM or to CD44. Despite HA binding by both RHAMM and CD44, migration of MM blood B cells on HA was inhibited by anti-RHAMM but not by anti-CD44 MoAbs, indicating that RHAMM but not CD44 mediates motility on HA. Thus, circulating B and plasma cells in MM exhibit RHAMM- and HA-dependent motile behavior indicative of migratory potential, while BM plasma cells are sessile. We speculate that a subset(s) of circulating B or plasma cells mediates malignant spread in myeloma.

Objective: To describe the immunophenotype of normal and myelomatous plasma cells (PCs) and the changes in immunoregulatory nonmyelomatous cells in multiple myeloma (MM)., Design: The cell surface markers (antigens) associated with this common cancer were reviewed., Material and Methods: Immunophenotypic characterization of both normal PCs and their counterpart malignant hematopoietic cells can be achieved by using monoclonal antibodies and either flow cytometry or immunocytochemical techniques., Results: Normal PCs are heterogeneous and express, in addition to cytoplasmic immunoglobulins, the antigens CD9, CD10, CD13, CD19, CD20, CD33, CD38, and D-related human leukocyte antigen (HLA-DR). This heterogeneity also occurs in malignant PCs. Myelomatous PCs may express, in addition to CD38 (the most typical PC marker), the antigens CD9, CD10, HLA-DR, and CD20. Other non-B-cell lineage markers such as myeloid (CD13, CD14, CD15, CD33, CD41, and glycophorin A), T-cell (CD2 and CD4), and natural killer-associated (CD56) antigens, as well as CD23, CD24, CD25, CD37, CD39, CDw40, CD45R, CD71, and certain unclustered antigens (R1-3, PCA-1, PCA-2, PC1, 62B1, 8A, 8F6, and MM4), have been noted in myelomatous PCs. The presence of these antigens in the myeloma cells may have a prognostic value--for example, the expression of CD20 and of myelomonocytic antigens (CD11b, CD13, CD14, CD15, and CD33) may be related to a poor prognosis. The adverse prognostic implication of the expression of CD10 initially described in MM has not been subsequently confirmed. Patients with MM may have mononuclear cells in their peripheral blood that express the same antigens as those expressed by the myeloma cells in their bone marrow. The presence of such cells or their therapy-associated decrease or disappearance may be related to the prognosis of patients with MM., Conclusion: The presence of cell surface markers on PCs and their prognostic significance in patients with MM warrant further investigation.

Multiple myeloma is recognized as a neoplasm of phenotypically mature plasma cells which produces a variety of clinical symptoms related both to tumour infiltration of the bone marrow and cytokine production. The latter results in bone disease and a complex interactive network between plasma cells, marrow stromal cells and other hematopoietic cells. This serves to sustain the myeloma proliferative pool and promote maturation and secretion of monoclonal immunoglobulin. Whereas the recognizable tumour cells in myeloma are the most mature B cells, early lymphoid cells are involved in the disease and probably represent the proliferative pre-plasma cell compartment. The definition of the myeloma 'stem cell' remains controversial, but our understanding of early pre-plasma cell differentiation in multiple myeloma has been aided by studies on normal non-neoplastic equivalents. Techniques like high resolution flow cytometry, flow cytometric DNA analysis and improvements in our ability to obtain karyotypic data in multiple myeloma will improve our understanding of myeloma cell biology, hopefully yielding new prognostic information. Finally, improvements in assessing prognosis will help identify patients whose survival with standard therapy is limited and who may require innovative or aggressive treatment protocols. These individuals must be separated from patients who either require no initial therapy or who are likely to have good outcomes with standard approaches.

A mouse monoclonal antibody (R1-3) raised against a human plasmacytoma xenograft and reactive with human plasma cells is described. The antibody reacts with a subset of plasma cells and lymphocytes, but is nonreactive with granulocytes, normoblasts, thymus, spleen or T lymphocytes. Flow cytometer double labelling experiments showed that approximately 50% of R1-3 positive bone marrow cells expressed surface immunoglobulin, but no R1-3 positive cells also expressed B1 or Leu 4. In a myeloma patient the R1-3 reactive cells were found to have a higher rate of DNA proliferation and were aneuploid as determined by flow cytometry. The R1-3 antigen is expressed on late B lymphocytes and early plasma cells.

We classified 100 cases of myeloma before chemotherapy as mature (28), intermediate (38), immature (19), or plasmablastic (15). The plasmablastic group had an estimated median survival (Kaplan-Meier method) of ten months, compared to 35 months for the other types (P less than .05). Decreased survival in the plasmablastic group was due to more frequent deaths in the first six months. There were no significant differences in survival among the mature, intermediate, and immature groups or among patients with different morphological grade or asynchrony scores. The plasmablastic myeloma group had more frequent renal insufficiency and higher plasma cell labeling indices, which may have contributed to the shorter survival.

Plasma cells are currently held to represent the end-stage differentiation of B-lymphocytes and hence do not undergo division. However, binucleated and occasionally multi-nucleated forms of plasma cells have often been recorded in histological materials containing plasma cell infiltrates. It was decided that a study be conducted to provide more information on the microscopic features of these binucleated cells. Of the fifty surgical specimens from different oral lesions examined by light microscopy, binucleated plasma cells were found in thirty-three specimens. The mean maximum diameter, surface area and volume were 26.87 microns, 534.61 microns 2 and 9466.28 microns 3 respectively. Morphologically, they resembled the mononucleated or single nucleated plasma cells. Their cytoplasm contain abundant basophilic cytoplasm and a prominent perinuclear golgi image. The two nuclei exhibit a clock-face or cartwheel appearance.

As they differentiate, precursor cells from the gut-associated lymphoid tissue are known to travel via the lymphatic system to the blood and then preferentially to home to various mucosal and exocrine sites such as the lamina propria of the gut and the lactating mammary gland, where they give rise to IgA-secreting plasma cells. The present study, directed at the mechanism by which the circulating precursors of mucosal IgA plasma cells selectively lodge in characteristic locations, explored the hypothesis that such homing is due to a locally produced chemotactic factor and that milk might be a source of such a factor. Subsets of lymphocytes bearing particular surface markers and purified by panning from lymph nodes of mice were examined in a micropore chemotaxis assay to search for the presence of chemotactic activity in mouse milk. The globulin fraction of whey was shown to contain a nondialyzable factor that is chemotactic for IgA (and also IgG)-positive lymphocytes when these are obtained from mesenteric lymph nodes as a source of mucosal-associated lymphoid tissue. Lymphocytes from peripheral lymph nodes, nonmucosal associated, were unaffected as were surface IgM-positive lymphocytes and T lymphocytes obtained from mesenteric nodes. Chemotactic activity for IgA lymphocytes was undetectable in mouse serum. The data are consistent with the idea that precursors of mucosal IgA plasma cells home to mucosal and exocrine sites in response to a specific chemotactic factor elaborated by local differentiated epithelial cells.

Inflammatory cell infiltration was characterized in five classical seminomatous testicular tumours using immunohistochemical techniques. These cells of immunological lineage were sited mostly around vessels. Lymphocytes were the most numerous, T cells outnumbering B cells in all sections studied. The helper/inducer subgroup predominated in the T cell family with suppressor cells more common than cytotoxic cells. Plasma cells constituted only a small percentage of this population as did natural killer cells. No Langerhans cells were identified. Phagocytic macrophages were twice as common as antigen-presenting macrophages yet T lymphocytes were 15 times more common than these antigen-presenting macrophages.

In vitro regulation of IgA subclass synthesis was investigated in pokeweed mitogen (PWM)-stimulated cultures of peripheral blood lymphocytes. In past experiments we have demonstrated that 50% of the IgA plasma cells derived from PWM-stimulated cultures are positive for IgA1 and 50% are positive for IgA2. This observation is surprising because approximately 80% of the IgA B cells in the peripheral circulation bear IgA1 and 20% bear IgA2. To determine if the shift toward IgA2 predominance in PWM-stimulated cultures might be due to an enriched source for IgA2 plasma cells from a precursor pool of immature B cells, we used panning techniques to separate immature precursors that express surface IgM (sIgM+) from mature precursors that no longer express IgM (sIgM-). These separated B cells were cultured with equal numbers of T cells and PWM for 7 days. In all 10 experiments there was an enrichment for IgA2 in the sIgM+ cultures; 55 +/- 9.6% of the total IgA plasma cells were positive for IgA2 in the sIgM+ cultures vs 38 +/- 6.3% in the sIgM- cultures (p less than 0.001). These results indicate that both sIgM+ and sIgM- cells can give rise to IgA plasma cells in PWM-stimulated cultures and that there is an enrichment for IgA2 precursors in the sIgM+ population. Other possible regulatory mechanisms were also investigated. To determine if there was isotype switching from IgA1 to IgA2, monoclonal anti-IgA1 antibodies were added to PWM cultures. These antibodies resulted in a mean suppression of IgA1 plasma cell production of 82% with a concomitant 45% suppression of total IgA but only 4.6% suppression of IgA2. These results make it unlikely that IgA2 plasma cells in PWM-stimulated cultures are derived from cells that initially produced IgA1. To investigate the possibility that one IgA subclass might be more T cell dependent than the other, T and B cells were separated and B cells were reconstituted with T cells in ratios that varied from 1:10 to 10:1 or with irradiated T cells. These procedures did not alter the proportion of IgA plasma cells positive for IgA1 or IgA2, indicating that the two subclasses do not differ in their response to T cell signals in PWM-stimulated cultures.

Plasma cells synthesizing rheumatoid factors (RF) were identified by fluorescent staining of sections of synovium and macrophage-depleted cells from dispersed synovial tissue. The latter avoided problems related to sampling errors in studying tissue sections and in the uncertainty raised by the staining of macrophages with intracellular complexes. Plasma cells producing IgG predominated, and seropositive patients had a higher proportion of IgM producers than seronegative subjects. None the less, in both groups of patients more than 90% of the IgM plasma cells were synthesizing RF, whereas the corresponding figure for IgG was between 50% and 60%. Only around 10% of IgA plasma cells were positive for RF. The high percentage of IgM plasma cells making RF would tend to argue for an IgG-specific response and against direct polyclonal activation as the stimulus. The percentage of IgG-producing cells positive for RF is also consistent with a dominant response to IgG. Accepting the difference in the relative proportion of total IgM- to IgG-producing plasma cells in seropositive as against seronegative patients, the close similarity between the two groups in the fraction of cells making RF favours the view that the two groups have a comparable underlying immunopathology dependent on IgG autosensitization. From the technical standpoint, the dispersed cell method gives results in line with those obtained with sections but which are easier to read, whereas the fluorescent techniques described give clear and reproducible results for the detection of RF of different heavy-chain isotype.

To investigate the in vitro regulation of IgA subclass synthesis, peripheral blood lymphocytes from healthy adults were cultured with the polyclonal B cell activator, pokeweed mitogen. Although 50% of the IgA plasma cells from a 7-d culture were positive for cytoplasmic IgA1 and 50% were positive for IgA2, less than 10% of the IgA released into the culture supernatant was IgA2. This discrepancy could not be explained by failure of the assay to detect in vitro synthesized IgA2, selective loss or destruction of IgA2 in culture media, delayed release of IgA2, or failure of IgA2 plasma cells to produce J chain. The results suggest that additional signals may be required for the differentiation of plasma cells into immunoglobulin-secreting cells.

Immunofluorescent study of paraffin sections of 80 lesions of the buccal cavity, for the most part malignant or pre-malignant revealed marked plasmocytosis in premalignant or early malignant cases. This plasmocytosis, easily seen by the use of anti-immunoglobulin sera, is of polyclonal type, with marked preponderance of IgG secretors. In infiltrating carcinomas and in recurrences, the number of plasmocytes decreases and the percentage of IgG secretors falls markedly. By contrast, that of IgA immunocytes remains the same. The significance of these findings is considered and their practical value in the surveillance of cases of leukoplasia is stressed.

In AIDS, although there is a lack of humoral responsiveness in vitro and in vivo, many patients persistently have an increased number of B cells which continue to produce increased amounts of immunoglobulin. An objective, reproducible morphologic classification scheme for B cells was devised. Comparison of cell kinetic parameters in various disease states will require such a classification. Although not immunologically responsive to new stimuli, the marrow B cells in the AIDS patients were shown to be replicating and turning over. The latter may be due to either death in situ or to migration. Plasmacytic lymphocytes and lymphocytic plasma cells, morphologic transitions between lymphocytes and mature plasma cells, had the largest fractions in DNA synthesis. Because of their relative cell numbers, the lymphocytic plasma cells contained most of the cells in DNA synthesis. The position of plasmablasts in the sequential compartments is unclear. Only small numbers are dividing. Within a given morphologic category, large cells were more likely to be in DNA synthesis than smaller cells. These studies can serve as a basis for comparison with marrow B-cell proliferation in other disease states.

Cytoplasmic immunoglobulin of lamina propria plasma cells was analyzed by immunofluorescence on the flow cytometer. Lymphoid cells were made permeable to immunofluorescent reagents by treatment with Triton X-100. These cells were then reacted with FITC (green) anti-light chain and with phycoerythrin (red) anti-heavy chain antibodies. Flow cytometric analysis of these cells revealed that plasma cells expressing cytoplasmic immunoglobulin light and heavy chains were specifically stained by the immunofluorescent reagents. These plasma cells were also shown to have membrane Ig as detected by cell surface immunofluorescence. Light scatter analysis indicated that these plasma cells could be distinguished from lymphocytes by 90 degrees light scatter. These studies provide a method by which several parameters of gut plasma cells can be analyzed by flow cytometry.