B Cell Tolerance

The mechanisms of B cell tolerance were studied in an attempt to learn whether B cells rendered tolerant are present in the immune system in a potentially responsive form. The author tested the in vitro anti-trinitrophenyl (TNP) antibody-forming cell (anti-TNP AFC) response to TNP-immunogens and polyclonal B cell activators (PBA) of spleen cells taken from mice injected with a tolerogen, TNP-carboxymethylcellulose (TNP-CMC). Spleen cells from mice injected 5 days previously with 10 μg of TNP-CMC did not respond to TNP-sheep red blood cells (TNP-SRBC), T-dependent (TD) antigen or TNP-Ficoll, T-independent (TI) antigen. However, the same spleen cells responded to PBA, lipopolysaccharide (LPS) of Salmonella enteritidis and purified protein derivative (PPD) of BCG. The results indicate that B cells specific for TNP are present in a potentially responsive form. Spleen cells from mice injected with 500 μg of TNP-CMC did not respond to either TNP-immunogens or PBA. The state of unresponsiveness to PBA lasted for 12 days after the tolerogen injection. Responsiveness to PBA reappeared within the short period of 2 days, whereas unresponsiveness to TNP-immunogens lasted much longer. Unresponsiveness to PBA was relieved considerably by treating tolerant spleen cells with the proteolytic enzyme trypsin before in vitro stimulation. These results indicate that B cells rendered refractory are present in the immune system in a potentially responsive form.     

B细胞不对称分裂

细胞的不对称分裂对于细胞多样性产生的重要性已经被大部分人所认识。B细胞的不对称分裂首先是在抗体类别转换的研究中发现的。最近,美国5科学家对B细胞在免疫发生中心中不对称分裂的原因进行了探索。结果发表在2012年1月20日出版的《Science》中。B细胞的不对称分裂参与体液免疫的抗体类别转换和抗体亲和力成熟过程。对于其机制仍不清楚,但目前研究初步提示细胞内分子的不对称分布是其发生的上游因素。并且B细胞的不对称分裂可能与不对称抗原分离可能在抗体亲和力成熟过程中具有独立协同作用。     

B细胞稳态的信号调节

在人类,65%的骨髓产生的B细胞是自身反应性的,它们大部分在骨髓中被克隆删除了。但有些B细胞通过免疫无力的方式逃脱了这种克隆删除到达外周,产生抗自身的抗体。研究表明,在鼠和人类中,B细胞存活时间过长是引发自身性免疫病的原因之一。B细胞的过度活化将导致自身反应性B细胞的产生和破坏自身免疫耐受,引起自身免疫性疾病或肿瘤;但B细胞的活化不足将使B细胞数量大大减少,抗原应答能力降低,从而使适应性免疫应答失衡。细胞因子和其他信号分子对B细胞稳态的调节是十分严密的,它们或调节B细胞的发育、成熟和分化,或调节B细胞向外周的迁移,或通过调节B细胞周期而使B细胞停留在特定时期,从而使B细胞避免凋亡,或通过调节抗凋亡蛋白或凋亡蛋白而决定B细胞的生存或死亡。本文就细胞因子、转录因子、蛋白激酶等信号分子对B细胞稳态的调节做一综述。     

Alterations in Peripheral Blood B Cell Subsets and Dynamics of B Cell Responses during Human Schistosomiasis

Antibody responses are thought to play an important role in control of Schistosoma infections, yet little is known about the phenotype and function of B cells in human schistosomiasis. We set out to characterize B cell subsets and B cell responses to B cell receptor and Toll-like receptor 9 stimulation in Gabonese schoolchildren with Schistosoma haematobium infection. Frequencies of memory B cell (MBC) subsets were increased, whereas naive B cell frequencies were reduced in the schistosome-infected group. At the functional level, isolated B cells from schistosome-infected children showed higher expression of the activation marker CD23 upon stimulation, but lower proliferation and TNF-α production. Importantly, 6-months after 3 rounds of praziquantel treatment, frequencies of naive B cells were increased, MBC frequencies were decreased and with the exception of TNF-α production, B cell responsiveness was restored to what was seen in uninfected children. These data show that S. haematobium infection leads to significant changes in the B cell compartment, both at the phenotypic and functional level.     

Polyclonal B Cell Activation by Cell Wall Preparations of Gram-Positive Bacteria

The effects of polyclonal B cell activation (PBA) of cell walls and their cell wall fractions obtained from several kinds of gram-positive bacteria were studied using the anti-sheep red blood cell (SRBC) or anti-trinitrophenylated (TNP) SRBC plaque forming cell (PFC) responses of cultured spleen cells from Balb/c, athymic nu/nu, their littermates (nu/+), C3H/He (LPS-responder), C3H/HeJ (LPS-non-responder), (CBA/N × Balb/c) F1 male with an X-linked defect in B cell function and the F1 female mice. The cell walls of Staphylococcus epidermidis (ATCC 155), Lactobacillus plantarum (ATCC 8014), Micrococcus lysodeikticus (NCTC 2665), Mycobacterium rhodochrous (ATCC 184), Streptomyces gardneri (ATCC 23911) and Nocardia corynebacteriodes (ATCC 14898) had the ability to induce polyclonal B cell responses in the spleen cells of Balb/c, nu/nu, nu/+, C3H/He and C3H/HeJ mice. The cell wall fractions prepared by enzymatic digestion from the cell walls of S. epidermidis, S. gardneri or N. corynebacteriodes were also capable of inducing polyclonal B cell responses. The responses of spleen cells from (CBA/N × Balb/c) F1 male mice to these active preparations, except the cell walls of M. rhodochrous, were much lower than those of the F1 female mice. These findings indicate that the majority of the cell wall preparations lacks PBA ability for spleen cells with the CBA/N defect, except for the cell walls of M. rhodochrous which possess this ability. The PBA-ability of synthetic peptidoglycan, muramyl dipeptide (N-acetylmuramyl-L -alanyl-D -isoglutamine, MDP), was also examined, and a similar activity was observed in MDP.     

Cell cycle-dependent methyl esterification of lamin B

Previous work from this laboratory has shown that approximately 24 proteins are reversibly modified by methyl esterification in a mouse lymphoma cell line. Here, we analyze several mouse tissues as well as other mouse, hamster, and human cell lines and find that many protein-methyl esters are ubiquitous while others show apparent tissue specificity. One of the modified proteins is identified by cellular localization and immunological detection as lamin B, a nuclear envelope structural protein which undergoes depolymerization during mitosis. The average stoichiometry of methylation is at least 0.5 methyl groups per lamin B molecule as determined by radioactive incorporation. By immunoblotting, however, demethylation appears to result in a gain of two negative charges suggesting the loss of two neutral methyl esters producing two carboxylic acid groups per molecule. By comparing mitotic and interphase cells, lamin B is found to be demethylated in mitosis while most other methyl esterified proteins show no appreciable cell cycle dependence. In addition to the correlation with cell cycle, it is shown that lamin B does not incorporate radioactive methyl esters in intact mouse brain tissue yet can do so if the cells are lysed. Analysis of lamin B charge by immunoblotting after isoelectric focusing indicates that this protein is fully methylated in brain suggesting that turnover of methyl groups in intact brain tissue is inhibited. We propose that methylation of lamin B may be involved in the control of disassembly and reassembly of the nuclear envelope during mitosis. If this were the case, the apparent lack of methyl group turnover in brain would be consistent with the inability of those cells to divide.     

Cell Cycle-Related Cyclin B1 Quantification

Background

To obtain non-relative measures of cell proteins, purified preparations of the same proteins are used as standards in Western blots. We have previously quantified SV40 large T antigen expressed over a several fold range in different cell lines and correlated the average number of molecules to average fluorescence obtained by cytometry and determined cell cycle phase related expression by calculation from multi-parametric cytometry data. Using a modified approach, we report quantification of endogenous cyclin B1 and generation of the cell cycle time related expression profile.

Methodology

Recombinant cyclin B1 was purified from a baculovirus lysate using an antibody affinity column and concentrated. We created fixed cell preparations from nocodazole-treated (high cyclin B1) and serum starved (low cyclin B1) PC3 cells that were either lyophilized (for preservation) or solubilized. The lysates and purified cyclin B1 were subjected to Western blotting; the cell preparations were subjected to cytometry, and fluorescence was correlated to molecules. Three untreated cell lines (K562, HeLa, and RKO) were prepared for cytometry without lyophilization and also prepared for Western blotting. These were quantified by Western blotting and by cytometry using the standard cell preparations.

Results

The standard cell preparations had 1.5×10⁵ to 2.5×10⁶ molecules of cyclin B1 per cell on average (i.e., 16-fold range). The average coefficient of variation was 24%. Fluorescence varied 12-fold. The relationship between molecules/cell (Western blot) and immunofluorescence (cytometry) was linear (r² = 0.87). Average cyclin B1 levels for the three untreated cell lines determined by Western blotting and cytometry agreed within a factor of 2. The non-linear rise in cyclin B1 in S phase was quantified from correlated plots of cyclin B1 and DNA content. The peak levels achieved in G2 were similar despite differences in lineage, growth conditions, and rates of increase through the cell cycle (range: 1.6–2.2×10⁶ molecules per cell).

Conclusions

Net cyclin B1 expression begins in G1 in human somatic cells lines; increases non-linearly with variation in rates of accumulation, but peaks at similar peak values in different cell lines growing under different conditions. This suggests tight quantitative end point control.     

B 细胞抗原表位的研究进展及其应用

抗表位是抗分子中决定抗特异性的特殊化学基团,其对研究特异性免疫应答有着重要意义.简要综述了蛋白质抗表位的种类及特性,回顾了近几年来理论预测和实验确定 B 细胞抗表位的常用方法及 B 细胞抗表位分析的研究方法,以及抗表位在流感流行预测及疫苗安全性方面的应用.     

Genomic Uracil Homeostasis during Normal B Cell Maturation and Loss of This Balance during B Cell Cancer Development

Activation-induced deaminase (AID) converts DNA cytosines to uracils in immunoglobulin genes, creating antibody diversification. It also causes mutations and translocations that promote cancer. We examined the interplay between uracil creation by AID and its removal by UNG2 glycosylase in splenocytes undergoing maturation and in B cell cancers. The genomic uracil levels remain unchanged in normal stimulated B cells, demonstrating a balance between uracil generation and removal. In stimulated UNG^−/− cells, uracil levels increase by 11- to 60-fold during the first 3 days. In wild-type B cells, UNG2 gene expression and enzymatic activity rise and fall with AID levels, suggesting that UNG2 expression is coordinated with uracil creation by AID. Remarkably, a murine lymphoma cell line, several human B cell cancer lines, and human B cell tumors expressing AID at high levels have genomic uracils comparable to those seen with stimulated UNG^−/−splenocytes. However, cancer cells express UNG2 gene at levels similar to or higher than those seen with peripheral B cells and have nuclear uracil excision activity comparable to that seen with stimulated wild-type B cells. We propose that more uracils are created during B cell cancer development than are removed from the genome but that the uracil creation/excision balance is restored during establishment of cell lines, fixing the genomic uracil load at high levels.     

前B细胞受体及转录因子在B细胞发育中的作用

B细胞在骨髓中,由造血干细胞发育、成熟是一复杂、分阶段的过程。这一过程是受到高度调控的。PU．1、E2A、EBF和Pax5等转录因子分级控制B细胞系基因的表达,决定了B细胞的定型和进入B细胞发育途径。而重链重排完成后形成的前B细胞受体,传递分化信号,促进B细胞进一步的发育。未完成重链重排的前B细胞不能组成前B细胞受体,通过凋亡被清除,保证了成熟B细胞的功能。     

The B Cell Antigen Receptor and Overexpression of MYC Can Cooperate in the Genesis of B Cell Lymphomas

A variety of circumstantial evidence from humans has implicated the B cell antigen receptor (BCR) in the genesis of B cell lymphomas. We generated mouse models designed to test this possibility directly, and we found that both the constitutive and antigen-stimulated state of a clonal BCR affected the rate and outcome of lymphomagenesis initiated by the proto-oncogene MYC. The tumors that arose in the presence of constitutive BCR differed from those initiated by MYC alone and resembled chronic B cell lymphocytic leukemia/lymphoma (B-CLL), whereas those that arose in response to antigen stimulation resembled large B-cell lymphomas, particularly Burkitt lymphoma (BL). We linked the genesis of the BL-like tumors to antigen stimulus in three ways. First, in reconstruction experiments, stimulation of B cells by an autoantigen in the presence of overexpressed MYC gave rise to BL-like tumors that were, in turn, dependent on both MYC and the antigen for survival and proliferation. Second, genetic disruption of the pathway that mediates signaling from the BCR promptly killed cells of the BL-like tumors as well as the tumors resembling B-CLL. And third, growth of the murine BL could be inhibited by any of three distinctive immunosuppressants, in accord with the dependence of the tumors on antigen-induced signaling. Together, our results provide direct evidence that antigenic stimulation can participate in lymphomagenesis, point to a potential role for the constitutive BCR as well, and sustain the view that the constitutive BCR gives rise to signals different from those elicited by antigen. The mouse models described here should be useful in exploring further the pathogenesis of lymphomas, and in preclinical testing of new therapeutics.     

Cell intrinsic TGF-beta 1 regulation of B cells

TGF-beta family cytokines play multiple roles in immune responses. TGF-beta1-null mice suffer from multi-organ infiltration that leads to their premature death. T cells play a central role in the TGF-beta1 phenotype, as deficiency of TGF-beta1 only in T cells reproduces the lethal phenotype. Although it is known that TGF-beta1 controls B cells isotype switch and homeostasis, the source responsible for this control has not been characterized. Because of the major role that T cells play in regulating B cell responses, we addressed the T cell dependency of the TGF-beta1 control of B cells. The analysis of T cell-deficient, TGF-beta1 knockout mice and the production of chimeras in which B but not T cells lacked TGF-beta1 allowed us to show that B cells are controlled in part by cell autonomous production of TGF-beta1.     

Automated Detection of B Cell and T Cell Acute Lymphoblastic Leukaemia Using Deep Learning

PurposeLeukaemia is diagnosed conventionally by observing the peripheral blood and bone marrow smear using a microscope and with the help of advanced laboratory tests. Image processing-based methods, which are simple, fast, and cheap, can be used to detect and classify leukemic cells by processing and analysing images of microscopic smear. The proposed study aims to classify Acute Lymphoblastic Leukaemia (ALL) by Deep Learning (DL) based techniques.ProceduresThe study used Deep Convolutional Neural Networks (DNNs) to classify ALL according to WHO classification scheme without using any image segmentation and feature extraction that involves intense computations. Images from an online image bank of American Society of Haematology (ASH) were used for the classification.FindingsA classification accuracy of 94.12% is achieved by the study in isolating the B-cell and T-cell ALL images using a pretrained CNN AlexNet as well as LeukNet, a custom-made deep learning network designed by the proposed work. The study also compared the classification performances using three different training algorithms.ConclusionsThe paper detailed the use of DNNs to classify ALL, without using any image segmentation and feature extraction techniques. Classification of ALL into subtypes according to the WHO classification scheme using image processing techniques is not available in literature to the best of the knowledge of the authors. The present study considered the classification of ALL only, and detection of other types of leukemic images can be attempted in future research.     

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability ¹. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease ^2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle¹. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/C^Cdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths¹.Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects ⁵.To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B ⁶.To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety ⁶ (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) ^7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium ⁷ (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation ⁶ (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable ^9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium ⁶.     

Interleukin-21 Enhances Rituximab Activity in a Cynomolgus Monkey Model of B Cell Depletion and in Mouse B Cell Lymphoma Models

Rituximab, a monoclonal antibody targeting CD20 on B cells, is currently used to treat many subtypes of B cell lymphomas. However, treatment is not curative and response rates are variable. Recombinant interleukin-21 (rIL-21) is a cytokine that enhances immune effector function and affects both primary and transformed B cell differentiation. We hypothesized that the combination of rIL-21 plus rituximab would be a more efficacious treatment for B cell malignancies than rituximab alone. We cultured human and cynomolgus monkey NK cells with rIL-21 and found that their activity was increased and proteins associated with antibody dependent cytotoxicity were up-regulated. Studies in cynomolgus monkeys modeled the effects of rIL-21 on rituximab activity against CD20 B cells. In these studies, rIL-21 activated innate immune effectors, increased ADCC and mobilized B cells into peripheral blood. When rIL-21 was combined with rituximab, deeper and more durable B cell depletion was observed. In another series of experiments, IL-21 was shown to have direct antiproliferative activity against a subset of human lymphoma cell lines, and combination of murine IL-21 with rituximab yielded significant survival benefits over either agent alone in xenogeneic mouse tumor models of disseminated lymphoma. Therefore, our results do suggest that the therapeutic efficacy of rituximab may be improved when used in combination with rIL-21.