Ig-producing Cells Research Articles

Abstract 101: B1b Cell Homeostasis Is Maintained By Cd40 In Atherosclerosis

Background: The co-stimulatory CD40-CD40L dyad is an important driver of atherosclerosis. CD40 exerts divergent, cell-specific roles, which differentially impacts atherogenesis. Objectives: We here investigate the role of the most prominent CD40-expressing cell type, the CD40 + B cell, in atherosclerosis. Methods: B cell subset specific CD40-expression of patients with mild and severe coronary artery disease (CAD) was determined by mass-cytometry and correlated to CAD severity. Underlying mechanisms were revealed using CD19-CD40 flfl -ApoE -/- (CD40 BKO ) and CD40 flfl -ApoE -/- (CD40 BWT ) mice. Results: Patients with severe CAD had similar CD40 expression levels on most B cell subsets but showed a profound decrease in CD40 on B1 cells. This was associated with increased atherosclerotic plaque burden and decreased plaque fibrosis. Likewise, CD40 BKO mice exhibited an increased plaque area and a more advanced stage of atherosclerosis. Absence of B cell CD40 caused a decrease in immunoglobulin (Ig) producing cells, including germinal center-, plasma-, but especially B1 cells, thereby reducing levels of (anti-ox/MDA-LDL) IgM and (anti-ox/MDA-LDL) IgG. Interestingly, transcriptomics analysis revealed that the absence of CD40 on B1b cells in particular, caused altered gene expression pathways related to lipid uptake ( Cd36, Ldlr ), cellular stress and metabolism ( Nr4a1, Hif1a ), and cell death ( Naip5/6, Top1 ). Indeed, in vitro analysis showed that B1b CD40KO cells took up excessive amounts of ac/oxLDL, exhibited defective BCR signaling, and were prone to apoptosis, especially in hyperlipidemic conditions. Finally, transfer of wild-type B1b cells into CD40 BKO mice prevented the increase in atherosclerosis. Conclusions: We here show that CD40 is a critical modulator of B1 cell protective function in hypercholesterolemia and atherosclerosis.

Increased Activated Circulating T Follicular Helper Cells and Changes in B Cell Subsets in Immune TTP (iTTP) and in Response to Rituximab Treatment

Background: T follicular helper cells (Tfh), characterised by surface expression of CXCR5, PD1 and ICOS, regulate development of antigen-specific B cell immunity through germinal centre (GC) formation, generation of long-lived memory B cells and high-affinity plasma cells. Methods: In this prospective study of peripheral blood B and circulating Tfh (cTfh) cell subsets, iTTP patients at 32 acute presentations, 23 elective rituximab (ER) episodes and 27 age & sex-matched healthy controls (HC) were studied using flow cytometry. All acute cases received PEX, steroids and RTX. All ER patients previously received RTX as acute therapy or previous ER at a median of 22 months (range 12-191 months). Serial samples were taken post-rituximab (RTX). B cell return was defined by laboratory CD19 count (<5 x 106/L). Statistical analysis was performed using GraphPad Prism 8. Results: 11/32 (34%) acute cases received potentially immunomodulatory therapy prior to blood sampling and were excluded. Median ADAMTS13 activity was <5 IU/dL (<5-10.4 IU/dL) and anti-ADAMTS13 IgG 46% (2-127%). In 23 ER cases, median ADAMTS13 activity was 9 IU/dL (<5-24IU/dL) and antibody 8% (2-89%). At acute presentation, CD4+CXCR5+ and CD4+CXCR5+PD1+ cTfh were decreased compared to HC (6.1% vs 9.4%; [p=0.003] and 1.2% vs 1.8%; [p=0.003] respectively), whereas activated cTfh (CD4+CXCR5+PD1+ICOS+) were increased (0.95% vs 0.55%; [p=0.01]) (Table 1). B cell subsets in acute iTTP showed decreased pre-switch and switched memory subsets compared to HC: IgD+/CD27+ [p=0.003]; IgD-/CD27+ [p=0.02] and IgD-/CD38+ [p=0.008]. Plasmablasts (IgD-CD38++) were increased [p=0.03] (Figure 1). ER patients pre-RTX had increased transitional and naïve B cells compared to HC [p=0.001; p<0.0001 respectively] and increased percentages of plasmablasts [p<0.0001]. Memory subsets defined by IgD/CD38 were all significantly decreased [p<0.0001] (Figure 1). Activated cTfh were increased in ER pre-RTX compared to HC [p<0.0001], whereas CD4+CXCR5+ICOS+ cells were reduced. There was no difference in CD4+CXCR5+ cells (Table 1). Memory subsets (defined by IgD/CD38) in ER were all significantly reduced compared with acute iTTP cases (Figure 1) likely to be due to previously described (often long term) changes in B cell subsets following B cell return after RTX. Longitudinal analysis: B cell return post-RTX in acute cases occurred mainly with transitional/naïve cells at a median of 8 months (0.5-14) but was not associated with iTTP relapse. Memory B cell subsets (defined by IgD/CD38) were significantly reduced at B cell return (Table 2). In ER patients, B cell subsets at repopulation were generally similar to levels seen prior to re-treatment with RTX. Frequency of cTfh was not significantly altered by RTX therapy in either acute TTP or ER. Two ER patients were followed longitudinally from RTX therapy, through ADAMTS13 normalisation & subsequent fall requiring further RTX re-treatment. Asymptomatic ADAMTS13 relapse (activity <15 IU/dL) was temporally related with an apparent maturation to memory phenotype and increase in % plasmablasts. Conclusions: At acute iTTP presentation and prior to elective re-treatment with RTX, activated (CD4+CXCR5+PD1+ICOS+) cTfh cells are increased, suggesting a role of T cell help in development of anti-ADAMTS13 IgG antibodies. Prior to RTX, B cell phenotype is also altered in acute TTP, with decreased frequency of memory subsets and a trend to increased naïve cells and plasmablasts. Persistent changes in B cell subsets were seen in ER patients who had received previous RTX with naive cells predominating and reduced memory cells. Interestingly, no patient relapsed/ required re-treatment related to B cell return, with relapse occurring at least 4 months after detection of B cells. Resumption of the autoimmune response therefore appeared limited by the rate of maturation of autoantigen(ADAMTS13)-specific B cells, either by selection/differentiation of ADAMTS13-naive B cells and/or expansion of ADAMTS13-specific memory B cells to Ig producing cells. This process, presumably driven by interaction with Tfh cells, suggests a role of T cell help in development of anti-ADAMTS13 IgG antibodies. Longitudinal analysis of the evolution of B and cTfh cells may help in predicting relapse in iTTP. Disclosures Scully: Alexion: Consultancy, Speakers Bureau; Ablynx/Sanofi: Consultancy, Other: Advisory Board, Speakers Bureau; Novartis: Other: Advisory Board, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau; Takeda: Speakers Bureau; Shire/Takeda: Other: Advisory Board, Research Funding, Speakers Bureau. Thomas:Ablynx: Honoraria, Other: Advisory Board; Sanofi: Honoraria, Other: Advisory Board; Bayer: Honoraria, Speakers Bureau.

Differential Effects of Reactive Oxygen Species on IgG versus IgM Levels in TLR-Stimulated B Cells.

It is becoming increasingly evident that reactive oxygen species (ROS) have critical roles as "second messengers" in cell signaling. In B cells, ROS can be generated either as a byproduct of mitochondrial respiration, as a result of the endoplasmic reticulum stress response induced by high production of Igs, or by the activation of NADPH oxidase (NOX) complexes. Having previously shown that costimulation of B cells via TLR 9 and the TLR-related receptor RP105 drives maturation of human peripheral blood B cells into Ig-producing cells, we aimed to study the role of ROS generated during this vital process. To this end, the ROS levels were either reduced by the NOX inhibitor VAS2870 or by the ROS scavenger N-acetyl cysteine (NAC). We revealed that TLR9/RP105-mediated stimulation of human B cells involved a rapid activation of NOX. Moreover, VAS2870 blocked the TLR9/RP105-induced B cell activation and thereby all Ig production. Importantly, we showed that ROS targeted by NAC was selectively required for IgG but not for IgM production. The endoplasmic reticulum stress response in the TLR9/RP105-stimulated cells was higher in IgG+ than in IgG- cells and was reduced by NAC in IgG+ cells only. Of note, we revealed that substantially higher levels of IgG than IgM were produced per cell and that IgG+ cells produced significantly higher ROS levels than IgG- cells. Taken together, our results imply that NAC-targeted ROS may be particularly important for sustaining the high Ig production in IgG+ B cells.

Increase of follicular helper T cells skewed toward a Th1 profile in CVID patients with non-infectious clinical complications

Common variable immunodeficiency (CVID) is characterized by low levels of circulating immunoglobulins and defects in B cell maturation leading to an increased susceptibility to infections. Some patients develop complications such as autoimmune diseases, enteropathy, and lymphoproliferation, resulting in higher morbidity and mortality. Follicular helper T (Tfh) cells are specialized in helping B cell differentiation into Ig-producing cells. Three subsets have been described, namely non B-cell helper Tfh1 and the two B-helper cell subsets Tfh2 and Tfh17. We determined that circulating Tfh cells were elevated in CVID patients and skewed toward a Tfh1 profile. Interestingly, elevated levels of Tfh1 cells were significant only in patients harboring non-infectious complications regardless of the type of complication and inversely correlated with switched memory B cells. Moreover, CXCR3+ cells are increased in splenic CVID germinal centers. Our observations suggest that the altered balance in Tfh subsets in CVID is linked to a more severe disease.

Overexpression of Mcl-1 exacerbates lymphocyte accumulation and autoimmune kidney disease in lpr mice

Cell death by apoptosis has a critical role during embryonic development and in maintaining tissue homeostasis. In mammals, there are two converging apoptosis pathways: the ‘extrinsic’ pathway, which is triggered by engagement of cell surface ‘death receptors’ such as Fas/APO-1; and the ‘intrinsic’ pathway, which is triggered by diverse cellular stresses, and is regulated by pro-survival and pro-apoptotic members of the Bcl-2 family of proteins. Pro-survival Mcl-1, which can block activation of the pro-apoptotic proteins, Bax and Bak, appears critical for the survival and maintenance of multiple haemopoietic cell types. To investigate the impact on haemopoiesis of simultaneously inhibiting both apoptosis pathways, we introduced the vavP-Mcl-1 transgene, which causes overexpression of Mcl-1 protein in all haemopoietic lineages, into Faslpr/lpr mice, which lack functional Fas and are prone to autoimmunity. The combined mutations had a modest impact on myelopoiesis, primarily an increase in the macrophage/monocyte population in Mcl-1tg/lpr mice compared with lpr or Mcl-1tg mice. The impact on lymphopoiesis was striking, with a marked elevation in all major lymphoid subsets, including the non-conventional double-negative (DN) T cells (TCRβ+CD4–CD8–B220+) characteristic of Faslpr/lpr mice. Of note, the onset of autoimmunity was markedly accelerated in Mcl-1tg/lpr mice compared with lpr mice, and this was preceded by an increase in immunoglobulin (Ig)-producing cells and circulating autoantibodies. This degree of impact was surprising, given the relatively mild phenotype conferred by the vavP-Mcl-1 transgene by itself: a two- to threefold elevation of peripheral B and T cells, no significant increase in the non-conventional DN T-cell population and no autoimmune disease. Comparison of the phenotype with that of other susceptible mice suggests that the development of autoimmune disease in Mcl-1tg/lpr mice may be influenced not only by Ig-producing cells but also other haemopoietic cell types.

Human IgGs induce synthesis and secretion of IgGs and neonatal Fc receptor in human umbilical vein endothelial cells

Human IgGs are increasingly used in the therapy of many different immune and inflammatory diseases, however their mechanism of action still remains unclear in most diseases. To gain insight into the mechanism by which IgGs might also exert their effects on endothelial cells, we tested human IgGs on human umbilical vein endothelial cells (HUVECs). IgGs induced a time-dependent increase in the synthesis and secretion of IgGs, together with a marked angiogenic-like transformation of HUVECs that was maximal after a 20-h incubation. IgGs stimulated IG gene transcription without affecting the process of gene rearrangement, already present in control HUVECs. The mechanism involved the activation of transcription factors with the increased expression of HSP90, HSP70 and inactive MMP-9 responsible for the phenotypic differentiation associated with the most intense IgG synthesis and secretion. However, even a short incubation with IgGs followed by recovery of cells was sufficient to trigger and sustain in time the synthesis and secretion of new IgGs, independently of the angiogenic-like transformation visible only when cells were continuously exposed to IgGs. Under the stimulus of IgGs, specific secretory pathways were also activated in HUVECs together with the expression of FcRn, which was always associated with IgGs of new synthesis, forming complexes that were also secreted. Our results disclose a so far unknown and unexpected mechanism of IgGs on HUVECs that behave as Ig-producing immune cells. Results might have relevance for the effects that IgGs also exert in vivo in physiological conditions.

Spotlight on tocilizumab and its potential in the treatment of systemic sclerosis

Systemic sclerosis (SSc) is a multisystem disease characterized by extensive collagen deposition in skin and internal organs, fibrointimal microvasculopathy, and activation of the immune system. T cells and B cells can promote fibrosis in SSc. Interleukin (IL)-6 is implicated in the pathogenesis of SSc. IL-6 is increased in the peripheral blood and lesional skin from patients with SSc, and induces fibroblast collagen production directly and indirectly by inducing profibrotic M2 macrophages. IL-6 also induces Th17 differentiation and promotes B cell differentiation toward Ig-producing plasma cells. IL-6 is also implicated in the pathogenesis of SSc in animal models as it is increased in mice with bleomycin-induced fibrosis, whereas neutralization of IL-6 in these mice prevents skin fibrosis. IL-6 acts on cells by binding to IL-6 receptor (IL-6R) which is transmembrane or soluble, and then recruits the signal-transducing glycoprotein 130 which is ubiquitously expressed. Tocilizumab is an anti-IL-6R humanized monoclonal antibody that blocks IL-6-mediated signaling. Tocilizumab has been approved for the treatment of moderate-to-severe rheumatoid arthritis, for polyarticular and systemic juvenile idiopathic arthritis, and for Castleman’s disease, and is well tolerated. Case reports and a Phase II, randomized trial in SSc have shown some improvement of skin tightness and delayed deterioration of lung function. A Phase III randomized trial in SSc is anticipated.

B Cell Depletion Increases Sphingosine-1-Phosphate–Dependent Airway Inflammation in Mice

Sphingosine-1-phosphate (S1P) has been widely associated with inflammation-based lung pathologies. Because B cells play a critical role as antigen-presenting and/or Ig-producing cells during asthmatic conditions, we wanted to dissect the role of these cells in S1P-dependent airway hyperreactivity and inflammation. Mice were sensitized to ovalbumin or exposed to S1P. Ovalbumin sensitization caused airway hyperreactivity coupled to an increased lung infiltration of B cells, which was significantly reduced after the inhibition of sphingosine kinases I/II. Similarly, the sole administration of S1P increased bronchial reactivity compared with vehicle and was accompanied by a higher influx of B cells in a time-dependent manner. This effect was associated with higher levels of IL-13, transforming growth factor-β, IL-10, and T regulatory cells. In addition, isolated S1P-derived lung B cells increased CD4(+) and CD8(+) T cell proliferation in vitro, and their suppressive nature at Day 14 was associated with the higher release of transforming growth factor-β and IL-10 when they were cocultured. Therefore, to prove the role of B cells in S1P-mediated airway inflammation, and because CD20 expression, contrary to major hystocompatibility complex I and major hystocompatibility complex II, was up-regulated at Day 14, CD20(+) B cells were depleted by means of a specific monoclonal antibody. The absence of CD20(+) B cells increased airway reactivity and inflammation in S1P-treated mice compared with control mice. These data imply that sphingosine kinase/S1P-mediated airway inflammation is countered by B cells via the induction of an immune-suppressive environment to reduce asthma-like outcomes in mice.

The comparative profile of lymphoid cells and the T and B cell spectratype of germ-free piglets infected with viruses SIV, PRRSV or PCV2.

Lymphocyte subsets isolated from germ-free piglets experimentally infected with swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV) or porcine circovirus type 2 (PCV2) were studied and the profile of these subsets among these three infections was monitored. Germ-free piglets were used since their response could be directly correlated to the viral infection. Because SIV infections are resolved even by colostrum-deprived neonates whereas PRRSV and PCV2 infections are not, SIV was used as a benchmark for an effectively resolved viral infection. PRRSV caused a large increase in the proportion of lymphocytes at the site of infection and rapid differentiation of B cells leading to a high level of Ig-producing cells but a severe reduction in CD2—CD21+ primed B cells. Unlike SIV and PCV2, PRRSV also caused an increase in terminally differentiated subset of CD2+CD8α+ γδ cells and polyclonal expansion of major Vβ families suggesting that non-specific helper T cells drive swift B cell activation. Distinct from infections with SIV and PRRSV, PCV2 infection led to the: (a) prevalence of MHC-II+ T cytotoxic cells, (b) restriction of the T helper compartment in the respiratory tract, (c) generation of a high proportion of FoxP3+ T cells in the blood and (d) selective expansion of IgA and IgE suggesting this virus elicits a mucosal immune response. Our findings suggest that PRRSV and PCV2 may negatively modulate the host immune system by different mechanisms which may explain their persistence.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-014-0091-x) contains supplementary material, which is available to authorized users.

Polymeric immunoglobulin receptor expression and local immunoglobulin A production in bovine sublingual, submandibular and parotid salivary glands

The submandibular and parotid glands are the main sources of immunoglobulins A (IgAs) in human and rat saliva. These glands express the polymeric immunoglobulin receptor (pIgR), which transports IgAs into saliva. The main source of IgAs in saliva and pIgR expression in salivary glands has not been well documented in cattle. Expressions of pIgR were determined in the major bovine salivary glands (sublingual, submandibular, and parotid) by RT-PCR for mRNA and by Western blot analysis and immunohistochemistry (IHC) using an anti-human pIgR antibody for protein. The protein detected with the antibody was identified by nano-liquid chromatography-quadrupole time of flight mass spectrometry. Additionally, the distribution of Ig-producing plasma cells was analyzed by IHC.RT-PCR showed that pIgR was expressed in the sublingual and submandibular glands, but not in the parotid gland. Higher protein levels were observed in sublingual glands than in submandibular glands by Western blot. By IHC, pIgR was mainly located on the apical side of the cytoplasmic membrane in the sublingual gland, whereas it was observed only on the basal side in the submandibular gland. The highest density of plasma cells expressing IgAs was observed in the sublingual gland. These results suggest that the sublingual gland plays an important role in first-line defence of the oral cavity in cattle in contrast to humans and rats.

Immunologic Studies in Humans.

Immunologic Studies in Humans.

Human tonsil B - cell lymphoma 6 ( BCL6 )-expressing CD4 + T-cell subset specialized for B-cell help outside germinal centers

T follicular helper (Tfh) cells represent a Th subset engaged in the help of B-cell responses in germinal centers (GCs). Tfh cells abundantly express the transcription repressor B-cell lymphoma 6 (Bcl6), a factor that is necessary and sufficient for their development in vivo. Whether Tfh or Tfh-committed cells are involved in the help of B cells outside GCs remains unclear, particularly in humans. In this study, we identified a previously undefined BCL6-expressing CD4(+) T-cell subset in human tonsils. This subset expressed IL-7 receptor and chemokine receptor 5 (CXCR5) and inducible costimulator (ICOS) at low levels (CXCR5(lo)ICOS(lo)), and it was found exclusively outside GCs. CXCR5(lo)ICOS(lo) CD4(+) T cells secreted larger amounts of IL-21 and IL-10 than CXCR5(hi)ICOS(hi) GC-Tfh cells upon activation, and they induced proliferation and differentiation of naïve B cells into Ig-producing cells more efficiently than GC-Tfh cells. However, this subset lacked the capacity to help GC-B cells because of the induction of apoptosis of GC-B cells through the FAS/FAS-ligand interaction. CXCR5(lo)ICOS(lo) CD4(+) T cells expressed equivalent amounts of BCL6 transcript with CXCR5(hi)ICOS(hi) GC-Tfh cells, but they expressed less Bcl6 protein. Collectively, our study indicates that CXCR5(lo)ICOS(lo) CD4(+) T cells in human tonsils represent Tfh lineage-committed cells that provide help to naïve and memory B cells outside GCs.

Epstein–Barr virus in bone marrow of rheumatoid arthritis patients predicts response to rituximab treatment

Objectives. Viruses may contribute to RA. This prompted us to monitor viral load and response to anti-CD20 therapy in RA patients.Methods. Blood and bone marrow from 35 RA patients were analysed for CMV, EBV, HSV-1, HSV-2, parvovirus B19 and polyomavirus using real-time PCR before and 3 months after rituximab (RTX) treatment and related to the levels of autoantibodies and B-cell depletion. Clinical response to RTX was defined as decrease in the 28-joint disease activity score (DAS-28) >1.3 at 6 months.Results. Before RTX treatment, EBV was identified in 15 out of 35 patients (EBV-positive group), of which 4 expressed parvovirus. Parvovirus was further detected in eight patients (parvo-positive group). Twelve patients were negative for the analysed viruses. Following RTX, EBV was cleared, whereas parvovirus was unaffected. Eighteen patients were responders, of which 12 were EBV positive. The decrease in the DAS-28 was significantly higher in EBV-positive group compared with parvo-positive group (P = 0.002) and virus-negative patients (P = 0.04). Most of EBV-negative patients that responded to RTX (75%) required retreatment within the following 11 months compared with only 8% of responding EBV-positive patients. A decrease of RF, Ig-producing cells and CD19+ B cells was observed following RTX but did not distinguish between viral infections. However, EBV-infected patients had significantly higher levels of Fas-expressing B cells at baseline as compared with EBV-negative groups.Conclusions. EBV and parvovirus genomes are frequently found in bone marrow of RA patients. The presence of EBV genome was associated with a better clinical response to RTX. Thus, presence of EBV genome may predict clinical response to RTX.

IL-21: An Executor of B Cell Fate

IL-21 is a type I cytokine that shares the common receptor gamma-chain with IL-2, IL-4, IL-7, IL-9, and IL-15. B cells are one of the lymphoid cell types whose development and function are regulated by IL-21. Depending on the interplay with costimulatory signals and on the developmental stage of a B cell, IL-21 can induce proliferation, differentiation into Ig-producing plasma cells, or apoptosis in both mice and humans. Alone and in combination with Th cell-derived cytokines IL-21 can regulate class switch recombination to IgG, IgA, or IgE isotypes, indicating its important role in shaping the effector function of B cells. This review highlights the role of IL-21 in B cell development, function, and disease and provides some perspectives on the future studies in this area.

Distribution of IgM, IgD and IgZ in mandarin fish, Siniperca chuatsi lymphoid tissues and their transcriptional changes after Flavobacterium columnare stimulation

The gene sequences of three different immunoglobulin (Ig) heavy chains, namely IgM, IgD and IgZ, were cloned from mandarin fish ( Siniperca chuatsi) recently. In this study the distribution of these three kinds of Ig-producing cells in lymphoid-related tissues as head kidney, spleen, gill and intestine were investigated by using in situ hybridization, and their transcriptional changes were also analyzed by quantitative real-time PCR during 8 weeks after immunization. IgM-producing cells could be detected obviously and abundantly in all the tissues examined. A few numbers of IgD and IgZ positive cells were both detected in head kidney and spleen. IgZ positive cells could be detected in gill moderately while IgD showed negative results, otherwise no IgD or IgZ positive cells could be detected in intestine. After stimulated with bacterial pathogen Flavobacterium columnare G 4, the transcripts of these three Ig genes exhibited quite different kinetics. Significantly increased transcription of IgM gene was observed in almost all the tissues examined especially in boosted group. In contrast with IgM, seldom strong increase was examined for IgD and IgZ genes. For IgD, it seemed that the first injection could stimulate the immune response easier, since in almost all the tissues significant increase was detected at 1 or 2 weeks after injection. For IgZ, boosted injection could not enlarge the up-regulation of gene expression of first injection. This is the first case to report the transcriptional kinetics of three Ig genes in teleost after bacterin immunization.

Distinct regulation of IgE, IgG4 and IgA by T regulatory cells and toll‐like receptors

Allergic diseases are characterized by the activation of the immune system and formation of immunoglobulin (Ig)E antibodies against normally innocuous environmental antigens, whereas IgG4 and IgA represent noninflammatory and blocking antibody isotypes. The T helper 2 (Th2) cells induce and T regulatory (Treg) cells suppress several features of allergic inflammation. Our aim was to investigate the role of allergen-specific T regulatory type 1 (Tr1) cells and CD4(+)CD25(+) Treg cells and toll-like receptors (TLRs) on IgE, IgG4 and IgA production. Germline or productive Ig-transcripts are determined by real-time reverse transcriptase-polymerase chain reaction, secreted Igs are measured by enzyme-linked immunosorbent assay and the frequency of Ig-producing plasma cells is investigated by enzyme-linked immunosorbent spot. Circulating CD4(+)CD25(+) Treg cells and allergen-specific Tr1 cells are used. Both allergen-specific, interleukin-10-secreting Tr1 cells and CD4(+)CD25(+) Treg cells from healthy individuals induced IgG4 and suppressed IgE production in peripheral blood mononuclear cells and purified B-cell cultures. In contrast, induction of IgA production is independent of T-cell help and the role of Tr1 or Treg cells is very limited, whereas it was highly induced by direct B-cell activation via TLR7 and 9. These data suggest that T regulatory cells may contribute to the suppression of allergic diseases by suppression of IgE and induction of IgG4, whereas IgA production is enhanced by B-cell activation via TLR7 and TLR9.

Distribution of immunoglobulin-producing cells in immunized mouse spleens revealed with “in vivo cryotechnique”

To identify immunoglobulin (Ig)-producing cells with immunohistochemistry, conventional methods of preparation using chemical fixatives have problems such as the artificial diffusion of components and antigen masking. The “diffusion artifact” is caused by the translocation of soluble proteins like Ig from the serum to cytoplasm or vice versa. We have examined the immunolocalization of serum proteins, such as Ig kappa light chain (Igκ), IgG1 heavy chain (IgG1), and albumin, in immunized mouse spleens after a peritoneal injection of human hemoglobin. Better preservation of morphology and immunoreactivity was obtained with the “in vivo cryotechnique” (IVCT) followed by freeze-substitution, than with conventional preparative methods. Although Ig-producing cells were not clearly detected in red pulp of 2-day-immunized spleens with the conventional methods, Igκ-immunopositive cells with rich cytoplasm were detected in the red pulp with IVCT, especially in the subcapsular and peritrabecular areas, where IgG1-immunopositive cells were rarely observed. In 7-day-immunized spleens prepared with IVCT, Igκ- or IgG1-immunopositive cells were mostly located in peritrabeculae. The development of Ig-producing cells was clarified in the specimens prepared with IVCT, which proved to be useful for analyzing the native morphology and distribution of Ig-producing cells.

Silver-stained nucleolar organizer regions and immunoglobulins in cutaneous squamocellular tumors

This study was conducted to elucidate the biological activity of epidermal cells in cutaneous squamocellular tumors by counting the number of silver-stained nucleolar organizer regions (AgNORs), to estimate the quantity of Ig-producing cells and the inflammatory cellular infiltrate (ICI), and to achieve a comparative evaluation. Twenty cases of actinic keratosis (AK), 20 in situ squamous cell carcinomas (ISC), and 20 invasive squamous cell carcinomas (SCCs) were assessed using a silver colloid technique. Ig-producing or binding cells and ICI were also investigated immunohistochemically. In all samples, AgNORs, Ig-producing cells, and ICI increased in proportion to the degree of malignancy. With regard to AgNORs values, a statistically significant difference was confirmed between AK and ISC ( p<0.01), AK and SCC ( p<0.001), and ISC and SCC ( p<0.05). IgG-producing cells predominated in each case. Furthermore, a linear correlation was detected between ICI and AgNORs in AK and ISC. The significant difference in the number of AgNORs among the 3 stages of involution of SCC reinforces the value of AgNORs as a marker for malignant potential. Despite the absence of a correlation between AgNORs and the proportion of Ig-producing cells, the association between ICI and AgNORs in AK and ISC was obvious.

Silver-stained organizer regions and immunoglobulins in cutaneous keratoacanthomas and squamous cell carcinomas

The aim of this study was to investigate the biologic activity of epidermal cells in keratoacanthomas (KAs) and squamous cell carcinomas (SCCs) by counting the number of silver-stained nucleolar organizer regions (AgNORs), to estimate the quantity of Ig-producing cells and the inflammatory cellular infiltrate (ICI), and to make a comparative evaluation. Thirty KAs (10 at growth stage, 10 at mature stage, and 10 at involution stage) and 28 SCCs (nine well differentiated-Grade 1 (G1), seven moderately differentiated-Grade 2 (G2), five poorly differentiated-Grade 3 (G3), and seven pseudoadenoid) were investigated. The KAs examined had a mean number of 1.727 AgNORs (S.D. 0.232), and IgG predominated in most cases. IgG and IgE increase at the involution, IgA remains at almost the same level, and IgM decreases during the maturity stage. The SCCs examined had a mean number of 2.105 AgNORs (S.D. 0.446). IgG predominated and gradually increased in proportion to the degree of malignancy. There is a significant difference in the number of AgNORs and the proportion of Ig subclasses in contrast to the cellular infiltrate among the three stages of KA. In SCCs, the number of AgNORs and the percentage of Igs and ICI increased gradually in proportion to the degree of malignancy.

The Human CD77− B Cell Population Represents a Heterogeneous Subset of Cells Comprising Centroblasts, Centrocytes, and Plasmablasts, Prompting Phenotypical Revision

The process of becoming an Ig-producing plasma cell takes the mature B cell through the germinal center, where Ig genes are diversified through somatic hypermutation and class switch recombination. To more clearly define functional characteristics of the germinal center dark zone centroblasts and the light zone centrocytes, we have performed expression analysis of the CD77(+) and CD77(-) populations, because CD77 has been accepted as a discriminator of centroblasts and centrocytes. Our results demonstrated that the CD77(+) and the CD77(-) populations lack functional associated expression programs discriminating the two populations. Both populations are shown to be actively cycling and to share common features associated with cell cycle regulation and DNA maintenance. They are also shown to have an equally active DNA repair program, as well as components involved in somatic hypermutation and class switch recombination. Moreover, the data also demonstrated that the CD77(-) population comprises cells with an already initiated plasma cell differentiation program. Together this demonstrates that CD77 does not discriminate centroblasts and centrocytes and that the CD77(-) population represents a heterogeneous subset of cells, comprising centroblasts, centrocytes, and plasmablast.