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1
Lin, Ling, Andrea J. Gerth, and Stanford L. Peng. "Active Inhibition of Plasma Cell Development in Resting B Cells by Microphthalmia-associated Transcription Factor." Journal of Experimental Medicine 200, no. 1 (June 28, 2004): 115–22. http://dx.doi.org/10.1084/jem.20040612.
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B cell terminal differentiation involves development into an antibody-secreting plasma cell, reflecting the concerted activation of proplasma cell transcriptional regulators, such as Blimp-1, IRF-4, and Xbp-1. Here, we show that the microphthalmia-associated transcription factor (Mitf) is highly expressed in naive B cells, where it antagonizes the process of terminal differentiation through the repression of IRF-4. Defective Mitf activity results in spontaneous B cell activation, antibody secretion, and autoantibody production. Conversely, ectopic Mitf expression suppresses the expression of IRF-4, the plasma cell marker CD138, and antibody secretion. Thus, Mitf regulates B cell homeostasis by suppressing the antibody-secreting fate.
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Yu, Tian, Jonathan Hull, Andrea Ruiz, Ashwini Bhat, and Amar Basu. "Expediting antibody discovery using Bioelectronica’s HypercellTM platform." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 86.36. http://dx.doi.org/10.4049/jimmunol.204.supp.86.36.
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Abstract Antibody-based drugs have been successful in a range of therapeutic categories. However, generating monoclonal antibodies is time-consuming and expensive. A common approach is Hybridoma technology, which overcomes the short life-span of IgG-secreting plasma B cells in vitro. However, many plasma B cells are lost due to the low efficiency of hybridoma cell fusion (typically <10%). Direct single B cell screening strategies have emerged to bypass hybridoma fusion and recombinatorial display, coupled with the generation of recombinant monoclonal antibodies through mammalian expression systems. Obtaining expression systems with the required productivity, specificity and stability for clinical or commercial use requires screening millions of cells and thousands of clones. Bioelectronica’s HypercellTM platform is an emerging technology used throughout antibody discovery and cell-line development to identify and isolate single, high-antibody secreting cells from large pools (~10,000,000 cells) in short periods (ca. 48 hrs). This scalable “electrofluidic” sorting system reduces time and cost by integrating antigen-detection reagents and real-time computer vision analysis to expedite single cell sorting. In this paper antigen-specific IgG-secreting hybridoma cells are identified and sorted by their secretion rate. The cells and reagents are encapsulated in a Polydisperse Oblate Dispersion system (PODs), incubated for 1–4 hours for signal gain, and loaded into the HypercellTM device for cell sorting. Alternatively, the mixture can be analyzed without sorting to produce single cell secretion “finger print” signatures that can help identify unique expression patterns and monitor cell line stability over culturing time.
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Andreani, Virginia, Senthilkumar Ramamoorthy, Abhinav Pandey, Ekaterina Lupar, Stephen L. Nutt, Tim Lämmermann, and Rudolf Grosschedl. "Cochaperone Mzb1 is a key effector of Blimp1 in plasma cell differentiation and β1-integrin function." Proceedings of the National Academy of Sciences 115, no. 41 (September 26, 2018): E9630—E9639. http://dx.doi.org/10.1073/pnas.1809739115.
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Plasma cell differentiation involves coordinated changes in gene expression and functional properties of B cells. Here, we study the role of Mzb1, a Grp94 cochaperone that is expressed in marginal zone (MZ) B cells and during the terminal differentiation of B cells to antibody-secreting cells. By analyzing Mzb1−/−Prdm1+/gfp mice, we find that Mzb1 is specifically required for the differentiation and function of antibody-secreting cells in a T cell-independent immune response. We find that Mzb1-deficiency mimics, in part, the phenotype of Blimp1 deficiency, including the impaired secretion of IgM and the deregulation of Blimp1 target genes. In addition, we find that Mzb1−/− plasmablasts show a reduced activation of β1-integrin, which contributes to the impaired plasmablast differentiation and migration of antibody-secreting cells to the bone marrow. Thus, Mzb1 function is required for multiple aspects of plasma cell differentiation.
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Golding, B., S. R. Pillemer, P. Roussou, E. A. Peters, G. C. Tsokos, J. E. Ballow, and T. Hoffman. "Inverse relationship between proliferation and differentiation in a human TNP-specific B cell line. Cell cycle dependence of antibody secretion." Journal of Immunology 141, no. 8 (October 15, 1988): 2564–68. http://dx.doi.org/10.4049/jimmunol.141.8.2564.
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Abstract Studies of an EBV-transformed and TNP-specific human B cell line revealed that, unlike myeloma or hybridoma cell lines that consist mainly of fully differentiated cells, most of the cloned EBV-transformed cells were not fully differentiated, as judged by inability to bind TNP-SRBC and to secrete anti-TNP antibody. The minority of more differentiated cells were selected by TNP-SRBC rosetting. They were found to proliferate to a lesser extent than nonrosetting cells and to contain increased numbers of antibody-secreting cells. This inverse relationship between proliferation and differentiation was also shown to be cell cycle related in that the TNP-SRBC rosetting cells resided, to a greater extent than the nonrosetting cells, in the G1 phase of the cell cycle. The finding that the G1 phase of the cell cycle was associated with differentiation into anti-TNP secreting cells was confirmed by demonstrating that treatment with hydroxyurea, which arrests the cells in G1, resulted in decreased proliferation and an increased proportion of antibody-secreting cells. Similarly, addition of phorbol ester resulted in increased antibody secretion and decreased proliferation, suggesting a role for protein kinase C in this differentiation pathway. The strategy of increasing the number of antibody-producing cells in this human EBV line, by promoting differentiation of the cells in G1, may be relevant to the large scale production of specific human mAb for the treatment and diagnosis of human diseases.
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Keane, John T., and Avery D. Posey. "Abstract 4089: PanCAR-specific antibody-cytokine fusion proteins." Cancer Research 83, no. 7_Supplement (April 4, 2023): 4089. http://dx.doi.org/10.1158/1538-7445.am2023-4089.
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Abstract CAR-T cells are a potent immunotherapy that redirect T cell specificity towards cell surface tumor-associated antigens. While this therapy has been effective in hematological malignancies, therapies against solid tumors not achieved the same efficacy due to a multitude of additional challenges, including a lack of tumor specific antigens and an immunosuppressive tumor microenvironment. One strategy to increase T cell potency is to use fourth generation CAR-T cells that also secrete an immunostimulatory factor, such as a cytokine. We have created CAR-T cells targeting Tn-MUC1 utilizing a scFv from the 5E5 antibody that also secrete interleukin-12 (IL-12), interleukin-18 (IL-18), and interleukin-23 (IL-23). These cytokine-secreting CAR-T cells have shown an increase in efficacy against multiple epithelial based tumors in impedance-based killing assays. In a xenograft model of human breast cancer, CAR-T cells secreting IL-12 and IL-23 were able to significantly delay tumor growth compared to non-transduced T cells, CAR-T cells alone, and CAR-T cells secreting IL-18. While these cells showed good efficacy in vivo, clinical translation is difficult due to concerns over cytokine storm due to the constitutive secretion of cytokines. To overcome this, we created a fusion protein utilizing an antibody specific to an invariant region found in majority of CAR molecules, including those utilized commercially available therapies and under pre-clinical development, fused to the three immunostimulatory cytokines. In impedance-based killing assays, antibody-cytokine fusions stimulated non-cytokine secreting CAR-T cells to completely clear the tumor at an E:T ratio in which the CAR-T cells incubated with only the antibody alone were unable to control the tumor growth. Interestingly, combination therapy of CAR-T cells and antibody-cytokine fusions outperformed CAR-T cells constitutively secreting the same cytokines in in vitro assays. This work highlights the use of a single reagent capable of increasing the efficacy of the majority of CAR-T cell therapies and may be an important contribution towards treating solid tumor malignancies with CAR-T cell therapies. Citation Format: John T. Keane, Avery D. Posey. PanCAR-specific antibody-cytokine fusion proteins. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4089.
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Hoogenboom, H. R., J. C. Raus, and G. Volckaert. "Cloning and expression of a chimeric antibody directed against the human transferrin receptor." Journal of Immunology 144, no. 8 (April 15, 1990): 3211–17. http://dx.doi.org/10.4049/jimmunol.144.8.3211.
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Abstract The cloning, construction and expression of chimeric Ig genes, encoding a mAb directed against the human transferrin receptor, is described. From a mouse hybridoma cell line, secreting an antitransferrin receptor antibody, mRNA was prepared and converted into cDNA using Ig-specific oligonucleotides. H and L chain encoding cDNA fragments were isolated and sequenced. Chimeric genes were constructed by linking the murine V region cDNA fragments to human C region exons. After sequential transfection of nonproducing mouse hybridoma cells with the expression vectors containing the chimeric H and L chain genes, antibody secreting transfectomas were obtained. ELISA and immunoblot analysis clearly demonstrate the secretion of human kappa- and gamma-1 chain. Flow microfluorimetry analysis of the chimeric antibody shows that the Ag-binding capacity has been retained. The chimeric antibody most likely will be less immunogenic then the original mouse antibody when used in human cancer therapy.
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McHeyzer-Williams, M. G., and G. J. Nossal. "Clonal analysis of autoantibody-producing cell precursors in the preimmune B cell repertoire." Journal of Immunology 141, no. 12 (December 15, 1988): 4118–23. http://dx.doi.org/10.4049/jimmunol.141.12.4118.
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Abstract Evidence for anti-self-reactivity in the preimmune B cell repertoire has been well documented. The present study aimed to determine the frequency of antibody-forming cell precursors in this repertoire whose Ig V regions impart reactivity to "self" or autologous Ag. Clones were activated in vitro with LPS and their secreted IgM antibody was assayed for reactivity by direct binding to cell surface or intracellular Ag. An IL-4-containing lymphokine mixture was added to the clonal cultures to induce the secretion of IgG1. The reactivity of secreted IgG1 with Ag would more closely resemble the binding required to activate B cells through their monomeric surface IgM and/or IgD. The results indicate a high frequency of precursors secreting IgM with reactivity to intracellular Ag, namely 1 in 37 +/- 6 B cells, with a marked paucity of response to cell surface molecules. The repertoire was markedly deficient in precursors secreting IgG1 able to bind to intracellular Ag, with only one clone detected by the screening of 3.0 x 10(6) spleen cells. No positives were detected for cell surface Ag. This suggested that the frequency of clones in the preimmune repertoire that express IgR with sufficient affinity to bind "self" molecules must be very low.
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Marquez, C., M. L. Toribio, M. A. Marcos, A. de la Hera, A. Barcena, L. Pezzi, and C. Martinez. "Expression of alkaline phosphatase in murine B lymphocytes. Correlation with B cell differentiation into Ig secretion." Journal of Immunology 142, no. 9 (May 1, 1989): 3187–92. http://dx.doi.org/10.4049/jimmunol.142.9.3187.
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Abstract Alkaline phosphatases (ALPase) (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.1) are implicated in many biologic phenomena including ossification and differentiation of human neutrophils and choriocarcinoma cells. Another trait, demonstrated by microinjection into Xenopus oocytes, is their ability to block the first mitotic division. Previous work in our laboratory has established that ALPase is also present on murine B lymphocytes activated by either polyclonal mitogens or Th cells. We have now characterized the ALPase present on murine B cells as belonging to the liver-bone-kidney isoenzyme and found it to be implicated in B cell differentiation into antibody secretion. Thus, B cell proliferative responses, elicited either by high concentrations of rabbit anti-IgM antibodies or by LPS in the presence of PMA, are characterized by the lack of both antibody secretion and expression of ALPase activity. In contrast, B cells stimulated to differentiate into Ig-secreting cells by B cell differentiation factors, nearly in the absence of a proliferative response, express high levels of ALPase activity, as did those that were LPS-stimulated. These data showing the association of the ALPase expression with the process of B cell differentiation into antibody-secreting cells are discussed in the context of the possible role that phosphorylation-dephosphorylation mechanism may play in controlling the growth/differentiation rate in the B cell lineage.
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Sankari, Hanna, Minna Hietikko, Kalle Kurppa, Katri Kaukinen, Eriika Mansikka, Heini Huhtala, Kaija Laurila, et al. "Intestinal TG3- and TG2-Specific Plasma Cell Responses in Dermatitis Herpetiformis Patients Undergoing a Gluten Challenge." Nutrients 12, no. 2 (February 13, 2020): 467. http://dx.doi.org/10.3390/nu12020467.
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Dermatitis herpetiformis (DH), a cutaneous manifestation of coeliac disease, is characterized by transglutaminase (TG) 3-targeted dermal immunoglobulin A (IgA) deposits. The treatment for DH is the same as for coeliac disease, namely a life-long gluten-free diet. DH patients typically have gluten-dependent circulating autoantibodies targeting TG3 and TG2, and plasma cells secreting such autoantibodies have been detected in the small intestinal mucosa. This study investigates the gluten-responsiveness of intestinal TG3 and TG2 antibody-secreting plasma cells in 16 treated DH patients undergoing a gluten challenge. The frequency of both plasma cell populations increased significantly during the challenge, and their frequency correlated with the corresponding serum autoantibody levels at post-challenge. TG3-specific plasma cells were absent in all 18 untreated coeliac disease patients and seven non-coeliac control subjects on gluten-containing diets. These findings indicate that, in DH, both intestinal TG3- and TG2-antibody secreting plasma cells are gluten-dependent, and that TG3-antibody secreting plasma cells are DH-specific.
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Kirchenbaum, Greg A., Giuseppe A. Sautto, Rodrigo B. Abreu, Paul V. Lehmann, and Ted M. Ross. "Assessment of Antibody Functional Affinity using FluoroSpot." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 86.11. http://dx.doi.org/10.4049/jimmunol.204.supp.86.11.
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Abstract Studies of B cell immunity often rely upon serologic methodologies that primarily assess antibody specificity. However, functional affinity of the antigen-specific antibody repertoire is a key determinant of protective efficacy. Presently, detailed assessment of single B cell/antibody functional affinity is labor-intensive and low-throughput. Therefore, we sought to evaluate whether B cell FluoroSpot assays would enable distinction between B cells with differential functional affinity since fluorescent intensity is directly proportional to antibody abundance in this assay. To test our prediction, murine B cell hybridomas (P65C6-2, 36–65 and 36–71) secreting monoclonal antibodies (mAb) with differential affinity for the p-azophenylarsonate (Ars) hapten were utilized as model antibody-secreting cells (ASC). Additionally, usage of an anti-idiotypic mAb (17–63) specific for the high-affinity anti-Ars mAb (36–71) confirmed unambiguous segregation of Ars-specific ASC. Moreover, we also evaluated the capacity of a multiplexed FluoroSpot assay to simultaneously distinguish antibody functional affinity among influenza hemagglutinin-reactive murine B cell hybridomas, or in vivo differentiated ASC from immunized mice, secreting various IgG subclasses (IgG1/IgG2a/IgG2b) in parallel. Collectively, our data support the notion that FluoroSpot assays can be used to assess the functional affinity of antigen-specific B cells, and that this approach is well-suited for detailed assessment of humoral immunity.
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Savage, Hannah P., Vanessa M. Yenson, Sanjam S. Sawhney, Betty J. Mousseau, Frances E. Lund, and Nicole Baumgarth. "Blimp-1–dependent and –independent natural antibody production by B-1 and B-1–derived plasma cells." Journal of Experimental Medicine 214, no. 9 (July 11, 2017): 2777–94. http://dx.doi.org/10.1084/jem.20161122.
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Natural antibodies contribute to tissue homeostasis and protect against infections. They are secreted constitutively without external antigenic stimulation. The differentiation state and regulatory pathways that enable continuous natural antibody production by B-1 cells, the main cellular source in mice, remain incompletely understood. Here we demonstrate that natural IgM-secreting B-1 cells in the spleen and bone marrow are heterogeneous, consisting of (a) terminally differentiated B-1–derived plasma cells expressing the transcriptional regulator of differentiation, Blimp-1, (b) Blimp-1+, and (c) Blimp-1neg phenotypic B-1 cells. Blimp-1neg IgM-secreting B-1 cells are not simply intermediates of cellular differentiation. Instead, they secrete similar amounts of IgM in wild-type and Blimp-1–deficient (PRDM-1ΔEx1A) mice. Blimp-1neg B-1 cells are also a major source of IgG3. Consequently, deletion of Blimp-1 changes neither serum IgG3 levels nor the amount of IgG3 secreted per cell. Thus, the pool of natural antibody-secreting B-1 cells is heterogeneous and contains a distinct subset of cells that do not use Blimp-1 for initiation or maximal antibody secretion.
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Landi, D. B., J. T. Rahkola, and E. N. Janoff. "MEASURING ANTIBODY-SECRETING B-CELL RESPONSES TO IMMUNOLOGICAL STIMULI." Journal of Investigative Medicine 55, no. 1 (January 2007): S121. http://dx.doi.org/10.1097/00042871-200701010-00279.
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Carrell, Jeffrey, and Christopher J. Groves. "OMIP-043: Identification of human antibody secreting cell subsets." Cytometry Part A 93, no. 2 (December 29, 2017): 190–93. http://dx.doi.org/10.1002/cyto.a.23305.
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Amadori, A., R. Zamarchi, V. Ciminale, A. Del Mistro, S. Siervo, A. Alberti, M. Colombatti, and L. Chieco-Bianchi. "HIV-1-specific B cell activation. A major constituent of spontaneous B cell activation during HIV-1 infection." Journal of Immunology 143, no. 7 (October 1, 1989): 2146–52. http://dx.doi.org/10.4049/jimmunol.143.7.2146.
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Abstract B cell activation is a well known consequence of HIV-1 infection, and seropositive subjects show high numbers of spontaneously activated Ig-secreting cells in circulation. To better define the importance of the HIV-1-specific response in this phenomenon, we first studied whether in vitro spontaneous anti-HIV-1 antibody production was accompanied by reactivation of memory B lymphocytes. Unstimulated PBL from HIV-1-infected individuals with prior history of hepatitis B and/or EBV infection did not consistently show spontaneous in vitro synthesis of anti-hepatitis B core Ag or anti-EBV antibodies; in addition, PWM-induced synthesis of anti-hepatitis B virus and anti-EBV antibodies was decreased compared to HIV-1-seronegative subjects. Moreover, in comparing the frequencies of activated HIV-1-specific B cell precursors and activated Ig-secreting precursors in limiting dilution experiments, a sizable fraction (20 to 40%) of circulating cells spontaneously secreting Ig produced antibody against HIV-1 determinants. The ratio between the two frequencies fitted in very well with the amount of Ig removed from unstimulated culture supernatants after HIV-1-specific antibody absorption with solid-phase HIV-1. These findings indicate that B cell activation during HIV-1 infection is mainly oriented toward a specific response to HIV-1 determinants; the possible relevance of this phenomenon to lymphomagenesis in AIDS patients is discussed.
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Pioli, KimAnh T., and Peter D. Pioli. "Retro-orbital CD45 antibody labeling to evaluate antibody-secreting cell trafficking in mice." STAR Protocols 4, no. 2 (June 2023): 102308. http://dx.doi.org/10.1016/j.xpro.2023.102308.
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Basu, Uttiya, Jianbo Sun, and Jiguang Wang. "B cell development and IL-10 secretion (P1476)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 174.23. http://dx.doi.org/10.4049/jimmunol.190.supp.174.23.
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Abstract Accumulating evidence confirms the existence of regulatory B cells (Bregs). Bregs are hypothesized to negatively regulate immune responses in autoimmunity and inflammation through the production of regulatory cytokines such as IL-10 and TGF-β. The precise cellular origin and function of Bregs remains unknown. IL-10 is known to exert broad anti-inflammatory effects which may negatively influence the activation of T cells, macrophages, and dendritic cells (DCs) and thereby influence both cellular and humoral immunity. Many questions exist regarding the function and biogenesis of Bregs. Do IL-10-generating Bregs only transiently express IL-10 during particular developmental stages? And what is the functional consequence of B cell mediated IL-10 secretion in vivo? We observe that in a population of naïve splenic B cells, a significant fraction is competent to secrete IL10. Even in B cells stimulated for class switch recombination (CSR), the unswitched population shows a high proportion of IL-10 secreting Bregs. Finally, we discuss the expansion of IL-10 secreting Bregs and their ex vivo matured progenitors in activation-induced cytidine deaminase (AID) deficient mice which are incompetent to undergo CSR and somatic hypermutation, two processes that generate antibody diversity. Put together, these observations point to a correlation between Breg biogenesis and antibody diversification processes.
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Guo, Muyao, Madeline J. Price, Dillon G. Patterson, Benjamin G. Barwick, Robert R. Haines, Anna K. Kania, John E. Bradley, Troy D. Randall, Jeremy M. Boss, and Christopher D. Scharer. "EZH2 Represses the B Cell Transcriptional Program and Regulates Antibody-Secreting Cell Metabolism and Antibody Production." Journal of Immunology 200, no. 3 (December 29, 2017): 1039–52. http://dx.doi.org/10.4049/jimmunol.1701470.
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Wilson, Eric, and Eugene C. Butcher. "CCL28 Controls Immunoglobulin (Ig)A Plasma Cell Accumulation in the Lactating Mammary Gland and IgA Antibody Transfer to the Neonate." Journal of Experimental Medicine 200, no. 6 (September 20, 2004): 805–9. http://dx.doi.org/10.1084/jem.20041069.
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The accumulation of immunoglobulin (Ig)A antibody-secreting cells (ASCs) in the lactating mammary gland leads to secretion of antibodies into milk and their passive transfer to the suckling newborn. This transfer of IgA from mother to infant provides transient immune protection against a variety of gastrointestinal pathogens. Here we show that the mucosal epithelial chemokine CCL28 is up-regulated in the mammary gland during lactation and that IgA ASCs from this tissue express CCR10 and migrate to CCL28. In vivo treatment with anti-CCL28 antibody blocks IgA ASC accumulation in the mammary gland, inhibiting IgA antibody secretion into milk and the subsequent appearance of antibody in the gastrointestinal tract of nursing neonates. We propose that CCL28 is a key regulator of IgA ASC accumulation in the mammary gland and thus controls the passive transfer of IgA antibodies from mother to infant.
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Petersen, Jøgen, Ulla Feldt-Rasmussen, Flemming Larsen, and Kai Siersbæk-Nielsen. "Autoreactive lymphocytes in thyroid disorders. Quantitation of anti-thyroglobulin antibody formation by a specific haemolytic plaque forming cell (PFC) assay." Acta Endocrinologica 113, no. 1 (September 1986): 50–55. http://dx.doi.org/10.1530/acta.0.1130050.
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Abstract. Blood mononuclear cells (MNC) from 21 patients with autoimmune thyroiditis were assayed for secretion of immunoglobulins in vitro by a reverse haemolytic plaque forming cell (PFC) assay. An anti-gen-specific assay was employed to quantify anti-thyroglobulin antibody (TgAb) secreting cells. The sensitivities of the two PFC assays were similar. The antigen specificity of the Tg-PFC assay was demonstrated by the ability of free Tg to inhibit PFC formation. The number of spontaneous TgAb-secreting cells was low (median 3 IgG-Tg-PFC/106, range 0–35/106); TgAb activity was found in 3% (range 0–11%) of total IgG-PFC. The number of spontaneous IgG-TgAb-secreting cells correlated positively to TgAb titres in serum. MNC from most patients secreted IgG-TgAb upon polyclonal stimulation in vitro for six days with pokeweed mitogen (52 IgG-Tg-PFC/106, range 0–478/106); TgAb activity was found among 2% (range 0–8%) of total IgG-PFC. Again, pokeweed mitogen-induced TgAb secretion correlated positively to TgAb titres in serum. Finally, MNC from most patients secreted TgAb after culture with Tg. The Tg-induced response was about 1/3 of the pokeweed mitogen-induced TgAb response. Tg did not increase the production of total IgG indicating that Tg is not a polyclonal stimulus. Few TgAb-secreting MNC were discovered in euthyroid sex and age-matched control patients.
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Rizzo, L. V., R. H. DeKruyff, and D. T. Umetsu. "Generation of B cell memory and affinity maturation. Induction with Th1 and Th2 T cell clones." Journal of Immunology 148, no. 12 (June 15, 1992): 3733–39. http://dx.doi.org/10.4049/jimmunol.148.12.3733.
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Abstract We used an adoptive transfer system and CD4+ T cell clones with defined lymphokine profiles to examine the role of CD4+ T cells and the types of lymphokines involved in the development of B cell memory and affinity maturation. Keyhole limpet hemocyanin (KLH)-specific CD4+ Th2 clones (which produce IL-4 and IL-5 but not IL-2 or IFN-gamma) were capable of inducing B cell memory and affinity maturation, after transfer into nude mice or after transfer with unprimed B cells into irradiated recipients and immunization with TNP-KLH. In addition, KLH-specific Th1 clones, which produce IL-2 and IFN-gamma but not IL-4 or IL-5, were also effective in inducing B cell memory and high affinity anti-TNP-specific antibody. The induction of affinity maturation by Th1 clones occurred in the absence of IL-4, as anti-IL-4 mAb had no effect on the affinity of the response whereas anti-IFN-gamma mAb completely blocked the response. Th1 clones induced predominantly IgG2a and IgG3 antibody, although Th2 clones induced predominantly IgG1 and IgE antibody. We thus demonstrated that some Th1 as well as some Th2 clones can function in vivo to induce Ig synthesis. These results also suggest that a single type of T cell with a restricted lymphokine profile can induce both the terminal differentiation of B cells into antibody secreting cells as well as induce B cell memory and affinity maturation. Moreover, these results suggest that B cell memory and affinity maturation can occur either in the presence of Th2 clones secreting IL-4 but not IFN-gamma, or alternatively in the presence of Th1 clones secreting IFN-gamma but not IL-4.
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McHeyzer-Williams, M. G., M. J. McLean, P. A. Lalor, and G. J. Nossal. "Antigen-driven B cell differentiation in vivo." Journal of Experimental Medicine 178, no. 1 (July 1, 1993): 295–307. http://dx.doi.org/10.1084/jem.178.1.295.
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The secretion of specific antibodies and the development of somatically mutated memory B cells in germinal centers are consequences of T cell-dependent challenge with the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP). Using six-parameter flow cytometry and single cell molecular analysis we can directly monitor the extent of somatic hypermutation in individual responsive (isotype switched) antigen-specific B cells. The current study provides a direct quantitative assessment of recruitment into the antibody-secreting compartment on the one hand, and the germinal center pathway to memory on the other. Cellular expansion in both compartments is exponential and independent during the first week after challenge. The first evidence of somatic mutation, towards the end of the first week, was restricted to the germinal center pathway. Furthermore, germinal center cells express a significantly shorter third hypervariable region (CDR3), even when unmutated, than their antibody-secreting counterparts, suggesting a secondary selection event may occur at the bifurcation of these two pathways in vivo. By the end of the second week, the majority of mutated clones express a shorter CDR3 and affinity-increasing mutations as evidence of further selection after somatic mutation. These data provide evidence for substantial proliferation within germinal centers before the initiation of somatic mutation and the subsequent selection of a significant frequency of mutated clonotypes into the memory compartment.
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Chan, Wing Fuk, Timothy M. Johanson, and Rhys S. Allan. "Three-dimensional genome rewiring during the development of antibody-secreting cells." Biochemical Society Transactions 48, no. 3 (May 26, 2020): 1109–19. http://dx.doi.org/10.1042/bst20191104.
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The development of B lymphocytes into antibody-secreting plasma cells is central to the adaptive immune system in that it confers protective and specific antibody response against invading pathogen. This developmental process involves extensive morphological and functional alterations that begin early after antigenic stimulation. These include chromatin restructuring that is critical in regulating gene expression, DNA rearrangement and other cellular processes. Here we outline the recent understanding of the three-dimensional architecture of the genome, specifically focused on its contribution to the process of B cell activation and terminal differentiation into antibody-secreting cells.
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Bauer, Natalie, Christina Hausl, Rafi U. Ahmad, Bernhard Baumgartner, Hans Peter Schwarz, and Birgit M. Reipert. "Comparable Long-Term Persistence of Anti-FVIII Antibodies in Hemophilic E17 Mice on Different Genetic Backgrounds Despite Significant Differences in the Amplitude of the Immune Responses." Blood 108, no. 11 (November 16, 2006): 1027. http://dx.doi.org/10.1182/blood.v108.11.1027.1027.
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Abstract About 30% of patients with severe hemophilia A develop neutralizing antibodies against FVIII (FVIII inhibitors) following replacement therapy. The type of FVIII gene mutation as well as other predisposing genetic factors contribute to the inhibitor phenotype. Based on these findings, we asked if the genetic background modulates the long-term persistence of anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells in the E17 murine hemophilia model. Furthermore, we asked if the recently described inhibition of memory-B-cell re-stimulation by high doses of FVIII is influenced by the genetic background of the murine model. E17 mice on two different genetic backgrounds (C57Bl/6J and Balb/c) were treated with four doses of 200 ng human FVIII at weekly intervals. Anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells were followed up to 12 months after the last dose of FVIII. Antibody titers and subclasses of antibodies (IgM, IgG1, IgG2a, IgG2b, IgG3) were measured by ELISA. Antibody secreting plasma cells in spleen and bone marrow were detected by ELISPOT as described (Hausl et al., Thromb Haemost 2002). The re-stimulation of FVIII-specific memory B cells was studied as described recently (Hausl et al., Blood 2005). Anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells were first detectable in E17 Balb/c mice. IgM antibodies in the circulation and IgM secreting plasma cells in the spleen were observed after the first dose of FVIII, IgG antibodies and IgG secreting plasma cells after the second dose. No anti-FVIII antibodies after the first dose of FVIII were observed in E17 C57BL/6J mice but both IgM and IgG antibodies as well as IgM and IgG producing plasma cells were detectable after the second dose of FVIII. The antibody response involved all IgG subclasses in both mouse strains. However, IgG1 was dominant in E17 Balb/c mice whereas IgG2a was dominant in E17 C57BL/6J mice. When the in vitro restimulation of FVIII-specific memory B cells was examined, similar patterns were observed for both mouse strains. Low concentrations of FVIII between 10 and 100 ng/ml FVIII restimulated memory B cells and induced their differentiation into antibody secreting plasma cells whereas high concentrations of FVIII between 1,000 and 20,000 ng/ml FVIII inhibited memory-B-cell-restimulation. These results indicate that the dose-dependent effect of FVIII on the restimulation of FVIII-specific memory B cells does not depend on the genetic background. The major difference between both hemophilic mouse strains was the amplitude of the anti-FVIII immune response. Peak titers of anti-FVIII antibodies and peak concentrations of anti-FVIII antibody secreting plasma cells in spleen and bone marrow were significantly higher in E17 C57BL/6J mice than in E17 Balb/c mice. Whether or not higher ELISA titers correlate with higher Bethesda titers of neutralizing antibodies is currently being investigated. Despite the substantial differences in the amplitude of the immune response, anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells persisted for the whole observation period of 12 months after the last dose of FVIII in both mouse strains. We conclude that the amplitude of the anti-FVIII immune response in hemophilic mice is significantly different between E17 C57BL/6J and E17 Balb/c mice. However, the persistence of the immune response is comparable.
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Corcoran, Lynn M., Jhagvaral Hasbold, Wendy Dietrich, Edwin Hawkins, Axel Kallies, Stephen L. Nutt, David M. Tarlinton, Patrick Matthias, and Philip D. Hodgkin. "Differential requirement for OBF-1 during antibody-secreting cell differentiation." Journal of Experimental Medicine 201, no. 9 (May 2, 2005): 1385–96. http://dx.doi.org/10.1084/jem.20042325.
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Resting B cells can be cultured to induce antibody-secreting cell (ASC) differentiation in vitro. A quantitative analysis of cell behavior during such a culture allows the influences of different stimuli and gene products to be measured. The application of this analytical system revealed that the OBF-1 transcriptional coactivator, whose loss impairs antibody production in vivo, has two effects on ASC development. Although OBF-1 represses early T cell–dependent (TD) differentiation, it is also critical for the completion of the final stages of ASC development. Under these conditions, the loss of OBF-1 blocks the genetic program of ASC differentiation so that Blimp-1/prdm1 induction fails, and bcl-6, Pax5, and AID are not repressed as in control ASC. Retroviral complementation confirmed that OBF-1 was the critical entity. Surprisingly, when cells were cultured in lipopolysaccharide to mimic T cell–independent conditions, OBF-1–null B cells differentiated normally to ASC. In the OBF-1−/− ASC generated under either culture regimen, antibody production was normal or only modestly reduced, revealing that Ig genes are not directly dependent on OBF-1 for their expression. The differential requirement for OBF-1 in TD ASC generation was confirmed in vivo. These studies define a new regulatory role for OBF-1 in determining the cell-autonomous capacity of B cells to undergo terminal differentiation in response to different immunological signals.
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Meyer Sauteur, Patrick M., Johannes Trück, Annemarie M. C. van Rossum, and Christoph Berger. "Circulating Antibody-Secreting Cell Response During Mycoplasma pneumoniae Childhood Pneumonia." Journal of Infectious Diseases 222, no. 1 (February 8, 2020): 136–47. http://dx.doi.org/10.1093/infdis/jiaa062.
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Abstract Background We recently demonstrated that the measurement of Mycoplasma pneumoniae (Mp)-specific immunoglobulin (Ig)M antibody-secreting cells (ASCs) improved diagnosis of Mp infection. Here, we aimed to describe Mp ASC kinetics and duration in comparison to conventional measures such as pharyngeal Mp deoxyribonucleic acid (DNA) and serum antibodies. Methods This is a prospective longitudinal study of 63 community-acquired pneumonia (CAP) patients and 21 healthy controls (HCs), 3–18 years of age, from 2016 to 2017. Mycoplasma pneumoniae ASCs measured by enzyme-linked immunospot assay were assessed alongside Mp DNA and antibodies during 6-month follow-up. Results Mycoplasma pneumoniae ASCs of the isotype IgM were found in 29 (46%), IgG were found in 27 (43%), and IgA were found in 27 (43%) CAP patients. Mycoplasma pneumoniae ASCs were detected from 2 days to a maximum of 6 weeks after symptom onset, whereas Mp DNA and antibodies persisted until 4 months (P = .03) and 6 months (P < .01). Mycoplasma pneumoniae ASCs were undetectable in HCs, in contrast to detection of Mp DNA in 10 (48%) or antibodies in 6 (29%) controls for a prolonged time. The Mp ASC response correlated with clinical disease, but it did not differ between patients treated with or without antibiotics against Mp. Conclusions Mycoplasma pneumoniae-specific ASCs are short-lived and associated with clinical disease, making it an optimal resource for determining Mp pneumonia etiology.
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Kelso, A. "Frequency analysis of lymphokine-secreting CD4+ and CD8+ T cells activated in a graft-versus-host reaction." Journal of Immunology 145, no. 7 (October 1, 1990): 2167–76. http://dx.doi.org/10.4049/jimmunol.145.7.2167.
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Abstract Lymphokine secretion by in vivo-activated T cells was analyzed at the population and single-cell levels in lymphocytes from mice undergoing an acute allogeneic graft-vs-host reaction (GVHR). Three observations were made. First, constitutive lymphokine production by these cells was very low but could be dramatically up-regulated by TCR ligation. Thus, even when harvested at the peak of the GVHR, fewer than 0.1% of lymphocytes secreted detectable granulocyte-macrophage (GM)-CSF, IFN-gamma, or IL-3 in the first 24 h in vitro, and average production of these lymphokines in bulk cultures was less than 10(-5) U/cell. However, when cultured for 24 h with anti-CD3 antibody under conditions which activated less than 0.1% of normal cells, about 30% of GVHR T cells secreted GM-CSF, IFN-gamma, and/or IL-3, and average production levels were increased by 10(3)- to 10(4)-fold. Together with evidence that host alloantigen-induced lymphokine secretion was 10 to 100 times lower than the anti-CD3 response, these data suggest that physiologic lymphokine synthesis by most T cells is low (less than 10(-18) mol of IL-3 per cell) but can be raised above the threshold of detection by TCR cross-linking. Second, individual GVHR lymphocytes varied markedly in their total and relative production of different lymphokines in response to anti-CD3 stimulation, with some cells secreting IL-3 alone, some secreting IL-3 accompanied by other lymphokines (GM-CSF and/or IFN-gamma), and some secreting other lymphokines without detectable IL-3. Finally, both CD4+ and CD8+ T cells from GVHR mice responded to anti-CD3 antibody by secreting IL-3 and other lymphokines: purified CD4+ cells contained an average of 16% and CD8+ cells an average of 10% anti-CD3-inducible lymphokine-secreting cells. By contrast, only 2 to 3% of cells of either subset formed clones in cultures with host allogeneic cells and IL-2, suggesting that clonogenic alloreactive cells were a minority of the T cells activated in the GVHR.
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Mei, Henrik, Daniela Frölich, Claudia Giesecke, Christoph Loddenkemper, Karin Reiter, Stefanie Schmidt, Eugen Feist, Capucine Daridon, Hans-Peter Tony, and Thomas Dörner. "Chronic differentiation of human mucosal IgA-secreting cells during B cell depletion therapy with rituximab. (161.3)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 161.3. http://dx.doi.org/10.4049/jimmunol.186.supp.161.3.
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Abstract During mucosal immune responses, IgA-secreting plasmablasts are generated from mucosal B cells. We previously demonstrated that at steady-state, most human peripheral blood antibody-secreting cells produce IgA and express mucosal homing receptors, and suggested their generation in mucosal immune responses. In the current study, we analyzed the blood of patients who received B cell depletion therapy with rituximab (anti-CD20) for treatment of rheumatoid arthritis before and at 2-9months after rituximab infusion by flow cytometry. While CD20+ B cells were reduced to <0.02% of their intial numbers before therapy, CD19+/CD27hi/CD20- antibody-secreting cells remained detectable in the blood at 26-119% of their initial numbers. These circulating antibody-secreting cells expressed IgA that bound to bacterial antigens, beta7 integrin and CCR10 before and during B cell depletion, suggesting their mucosal origin. High expression of HLA-DR and Ki-67 and in vitro migration towards CCL28 and CXCL12 qualified these cells as recently activated plasmablasts, reflecting the survival of functional B cells during rituximab treatment. Consistently, IgA+ plasmablasts and plasma cells were identified in the lamina propria during B cell depletion. Our data implicate that a population of mucosal B cells is resistant to rituximab and self-sufficient in adult humans and not replenished by CD20+ B cells immigrating from blood, lymphoid tissue, or bone marrow, that is, B cells depleted by rituximab.
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Bole, D. G., L. M. Hendershot, and J. F. Kearney. "Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas." Journal of Cell Biology 102, no. 5 (May 1, 1986): 1558–66. http://dx.doi.org/10.1083/jcb.102.5.1558.
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A rat monoclonal antibody specific for immunoglobulin (Ig) heavy chain binding protein (BiP) has allowed the examination of the association of BiP with assembling Ig precursors in mouse B lymphocyte-derived cell lines. The anti-BiP monoclonal antibody immunoprecipitates BiP along with noncovalently associated Ig heavy chains. BiP is a component of the endoplasmic reticulum and binds free intracellular heavy chains in nonsecreting pre-B (mu+, L-) cell lines or incompletely assembled Ig precursors in (H+, L+) secreting hybridomas and myelomas. In the absence of light chain synthesis, heavy chains remain associated with BiP and are not secreted. The association of BiP with assembling Ig molecules in secreting hybridomas is transient and is restricted to the incompletely assembled molecules which are found in the endoplasmic reticulum. BiP loses affinity and disassociates with Ig molecules when polymerization with light chain is complete. We propose that the association of BiP with Ig heavy chain precursors is a novel posttranslational processing event occurring in the endoplasmic reticulum. The Ig heavy chains associated with BiP are not efficiently transported from the endoplasmic reticulum to the Golgi apparatus. Therefore, BiP may prevent the premature escape and eventual secretion of incompletely assembled Ig molecules.
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Hinterleitner, Martina, Clemens Hinterleitner, Elke Malenke, Birgit Federmann, Ursula Holzer, Martin Müller, Wolfgang A. Bethge, and Stefan Wirths. "Early Reconstitution of Antibody Secreting Cells after Allogeneic Stem Cell Transplantation." Journal of Clinical Medicine 11, no. 1 (January 5, 2022): 270. http://dx.doi.org/10.3390/jcm11010270.
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Immune cell reconstitution after stem cell transplantation is allocated over several stages. Whereas cells mediating innate immunity recover rapidly, adaptive immune cells, including T and B cells, recover slowly over several months. In this study we investigated kinetics and reconstitution of de novo B cell formation in patients receiving CD3 and CD19 depleted haploidentical stem cell transplantation with additional in vivo T cell depletion with monoclonal anti-CD3 antibody. This model enables a detailed in vivo evaluation of hierarchy and attribution of defined lymphocyte populations without skewing by mTOR- or NFAT-inhibitors. As expected CD3+ T cells and their subsets had delayed reconstitution (<100 cells/μL at day +90). Well defined CD19+ B lymphocytes of naïve and memory phenotype were detected at day +60. Remarkably, we observed a very early reconstitution of antibody-secreting cells (ASC) at day +14. These ASC carried the HLA-haplotype of the donor and secreted the isotypes IgM and IgA more prevalent than IgG. They correlated with a population of CD19− CD27− CD38low/+ CD138− cells. Of note, reconstitution of this ASC occurred without detectable circulating T cells and before increase of BAFF or other B cell stimulating factors. In summary, we describe a rapid reconstitution of peripheral blood ASC after CD3 and CD19 depleted haploidentical stem cell transplantation, far preceding detection of naïve and memory type B cells. Incidence before T cell reconstitution and spontaneous secretion of immunoglobulins allocate these early ASC to innate immunity, eventually maintaining natural antibody levels.
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Werwitzke, Sonja, Marcus von Hornung, Katy Kalippke, Arne Trummer, Arnold Ganser, and Andreas Tiede. "Deletion or Inhibition of Fc Gamma Receptor IIb (CD32) Prevents the Memory B Cell Response to Factor VIII in a Hemophilia A Mouse Model." Blood 118, no. 21 (November 18, 2011): 204. http://dx.doi.org/10.1182/blood.v118.21.204.204.
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Abstract Abstract 204 The formation of inhibitory antibodies to factor VIII (FVIII) is the foremost complication of replacement therapy in hemophilia A. Patients with inhibitors are treated with very high doses of FVIII, over prolonged periods of time, to induce immune tolerance. Studies in a hemophilia A mouse model demonstrated that very high doses of FVIII can induce apoptosis in FVIII-specific memory B cells and prevent their differentiation into antibody-secreting cells. The Fc gamma receptor IIb (FcgRIIb) is expressed on B cells and mediates inhibitory signals after crosslinking with the B cell receptor. Here, we studied the potential role of this receptor in the regulation of memory B cell response to FVIII. FVIII knockout mice (B6;129S4-F8tm2Kaz/J) were crossed with FcgRIIb knockout mice (B6;129S4-Fcgr2btm1Ttk/J). Comparing F8−/− mice and F8−/−/FcgR2b−/− double knockout mice, the initial anti-FVIII antibody formation was similar after intravenous exposure to 4 weekly doses of 80 or 400 IU/kg. Similar numbers of FVIII-specific antibody-secreting cells were detected in the spleen and bone marrow by ELISPOT. As previously shown, in vitro re-stimulation of memory B cells from spleens of immunized F8−/− mice at doses of 1 to 200 ng/ml induced their differentiation into antibody-secreting cells. Higher doses of 400 to 800 ng/ml prevented differentiation. In F8−/−/FcgR2b−/− double knockout mice, however, formation of antibody-secreting cells was completely inhibited across all FVIII doses tested. Addition of B220-depleted splenocytes from F8−/− mice did not restore memory B cell function in F8−/−/FcgR2b−/− double knockout mice, indicating that the observed effect was not due to dysfunction of follicular dendritic cells or other antigen-presenting cells. Inhibition of FcgRIIb using a monoclonal antibody prevented the FVIII-specific memory B cell response in splenocytes from immunized F8−/− mice. Staining with propidium iodide, annexin V, or anti-caspase 3 indicated increased rates of apoptosis when FcgRIIb was blocked during re-stimulation. In summary, FcgRIIb plays a crucial role for the differentiation of FVIII-specific splenic memory B cells into antibody-secreting cells. Inhibition of FcgRIIb appears to sensitize B cells for apoptosis during re-stimulation with FVIII. This mechanism could potentially facilitate the eradication of FVIII-specific memory B cells during ITI. Disclosures: No relevant conflicts of interest to declare.
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Effros, R. B., C. M. Hulette, R. Ettenger, L. C. Dillard, E. Zeller, R. Duong, and R. L. Walford. "A human-human hybridoma secreting anti-HLA class II antibody." Journal of Immunology 137, no. 5 (September 1, 1986): 1599–603. http://dx.doi.org/10.4049/jimmunol.137.5.1599.
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Abstract In this report, we describe the production and characterization of the first human-human hybridoma secreting antibody to HLA Class II determinants. The hybridoma (GMEC101), which has been stable in tissue culture for greater than 20 mo, secretes 10 to 50 micrograms/ml of IgM-kappa antibody. This antibody binds to a wide range of human cell lines, but not to the HLA-A,B,C, and DR-negative K562 cell line. Functionally, GMEC101 strongly inhibits a unidirectional mixed lymphocyte reaction (MLR) at the level of the stimulator cell. Neither the cellular ELISA binding nor the MLR inhibition is lost after a triple platelet absorption (which removes Class I but not Class II activity). Because the binding and MLR blocking show no correlation with the known DR or DQ specificities, we suggest that GMEC101 may be detecting a novel HLA Class II determinant.
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Groves, C. J., J. Carrell, R. Grady, B. Rajan, C. A. Morehouse, R. Halpin, J. Wang, et al. "CD19-positive antibody-secreting cells provide immune memory." Blood Advances 2, no. 22 (November 26, 2018): 3163–76. http://dx.doi.org/10.1182/bloodadvances.2017015172.
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Abstract Long-lived antibody-secreting cells (ASCs) are critical for the maintenance of humoral immunity through the continued production of antibodies specific for previously encountered pathogen or vaccine antigens. Recent reports describing humoral immune memory have suggested the importance of long-lived CD19− bone marrow (BM) ASCs, which secrete antibodies recognizing previously encountered vaccine antigens. However, these reports do not agree upon the unique contribution of the CD19+ BM ASC subset toward humoral immunity. Here, we found both CD19+ and negative ASCs from human BM were similar in functional capacity to react to a number of vaccine antigens via ELISpot assays. The CD19+ cells were the predominant ASC population found in lymphoid tissues, and unlike the CD19− ASCs, which were found only in spleen and BM, the CD19+ ASCs were found in tonsil and blood. CD19+ ASCs from the BM, spleen, and tonsil were capable of recognizing polio vaccine antigens, indicating the CD19+ ASC cells play a novel role in long-lasting immune defense. Comparative gene expression analysis indicated CD19+ and negative BM ASCs differed significantly by only 14 distinct messenger RNAs and exhibited similar gene expression for cell cycle, autophagy, and apoptosis control necessary for long life. In addition, we show identical CDR-H3 sequences found on both BM ASC subsets, indicating a shared developmental path. Together, these results provide novel insight for the distribution, function, genetic regulation, and development of long-lived ASCs and may not only impact improved cell therapies but also enhance strategies for vaccine development.
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Tang, Jun, Daniel Ramis-Cabrer, Víctor Curull, Xuejie Wang, Liyun Qin, Mercé Mateu-Jiménez, Xavier Duran, et al. "Immune Cell Subtypes and Cytokines in Lung Tumor Microenvironment: Influence of COPD." Cancers 12, no. 5 (May 13, 2020): 1217. http://dx.doi.org/10.3390/cancers12051217.
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Background: The immune microenvironment plays a role in tumorigenesis. Chronic Obstructive Pulmonary Disease (COPD) is an independent risk factor for lung cancer (LC). We hypothesized that immune profile characterized by T regulatory (Treg), natural killer (NK), and plasma cells, as well as interleukin (IL)-10 and interferon-gamma, may differ within tumors of LC patients with/without COPD. Methods: Treg (anti-CD3 and anti-forkhead boxP3 antibodies), NK (anti-NCR1 antibody), IgG (anti-CD138-IgG antibody), IgA (anti-CD138-IgA antibody) using immunohistochemistry, and both IL-10 and interferon-gamma (ELISA) were quantified in tumor and non-tumor specimens (thoracotomy for lung tumor resection) from 33 LC–COPD patients and 20 LC-only patients. Results: Immune profile in tumor versus non-tumor specimens: Treg cell counts significantly increased in tumors of both LC and LC–COPD patients, while in tumors of the latter group, IgG-secreting plasma cells significantly decreased and IL-10 increased. No significant differences were seen in levels of NK cells, IgA-secreting cells, IgA/IgG, or interferon-gamma. Immune profile in tumors of LC–COPD versus LC: No significant differences were observed in tumors between LC–COPD and LC patients for any study marker. Conclusions: Immune cell subtypes and cytokines are differentially expressed in lung tumors, and the presence of COPD elicited a decline in IgG-secreting plasma cell levels but not in other cell types.
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Habibzadeh, Parham, and Mohammad Sajadi. "450 Developing Methods for High-Resolution Characterization of Plasma Cells." Journal of Clinical and Translational Science 8, s1 (April 2024): 133. http://dx.doi.org/10.1017/cts.2024.385.
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OBJECTIVES/GOALS: Antibodies play an important role in the pathogenesis of a wide range of diseases, including cancer, autoimmune diseases, and infections. There are currently no reliable methods to isolate and study specific plasma cell subpopulations as antibody production sources. We aim to develop methods to study plasma cells in high resolution. METHODS/STUDY POPULATION: We will use molecular cloning to engineer fusion proteins that would bind plasma cell proteins to study these cells based on their surface features. The first phase of our study consists of assessing the efficacy of this plasma cell isolation method in established cell lines (e.g., RPMI 8226) and also antibody-secreting cell lines that we are establishing as a part of this study. In the second phase of the study, we will assess the efficacy of this method by studying antigen-specific plasma cell populations in the bone marrow aspiration samples of 20 healthy volunteers using various assays, including ELISPOT, flow cytometry, and fluorescent microscopy. RESULTS/ANTICIPATED RESULTS: We have designed the constructs and have completed the cloning. The final plasmids have been verified using various restriction enzymes and Sanger sequencing. Following the transfection of Freestyle HEK 293F cells and isolation of respective proteins, we expect to be able to utilize these engineered proteins to differentiate various antibody-secreting plasma cells. We will use cell lines for proof-of-concept experiments and will subsequently move this method to human bone marrow samples. We expect to be able to visualize multiple specific antibody-secreting plasma cell populations using fluorescent microscopy and utilize this method to isolate them by cell sorting via flow cytometry. DISCUSSION/SIGNIFICANCE: We expect to be able to use this method to target specific plasma cell clones in the advancement of precision medicine regarding the treatment of plasma cell disorders (e.g., multiple myeloma) and also expand its use in other areas, such as antibody discovery and the assessment of the humoral immune responses in infectious diseases.
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Price, Madeline J., Dillon Patterson, Christopher D. Scharer, and Jeremy M. Boss. "Progressive upregulation of oxidative metabolism facilitates plasmablast differentiation to a T-independent antigen." Journal of Immunology 200, no. 1_Supplement (May 1, 2018): 171.5. http://dx.doi.org/10.4049/jimmunol.200.supp.171.5.
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Abstract Transitioning from a metabolically quiescent naïve B cell to an antibody-secreting plasmablast requires division-dependent cellular differentiation. Though cellular processes such as cell division and antibody secretion demand significant ATP and metabolites, the metabolic processes used to generate ATP during plasmablast formation are not well described. Here, the metabolic machinery requirements for plasmablast formation were determined. Following T-independent stimulation with lipopolysaccharide, B cells increased expression of the oxidative phosphorylation machinery in a step-wise manner. Such activated B cells have increased capacity to perform oxidative phosphorylation, but showed additional dependency on glycolytic metabolism. Plasmablasts displayed higher oxidative metabolism to support antibody secretion, as inhibiting oxidative ATP production with oligomycin resulted in decreased antibody titres. Differentiation orchestrated by Blimp1 was required for this increase in oxidative metabolism as Blimp1-deficient cells proliferate normally, but neither differentiate to plasmablasts nor upregulate oxidative phosphorylation. Therefore, Blimp1 expression is required for full metabolic activity, and proliferation alone is insufficient to induce full oxidative phosphorylation. Together, these findings identify a shift in metabolic pathways as B cells differentiate, as well as the requirement for increased metabolic potential to support antibody production.
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Trung, Nguyễn Thị, and Trương Nam Hải. "Study on some properties of the hybrid cell B4D10C9 producing the anti B monoclonal antibody which was agglutinated the b antigens on the surface of red blood cells." Vietnam Journal of Biotechnology 15, no. 2 (April 20, 2018): 243–49. http://dx.doi.org/10.15625/1811-4989/15/2/12340.
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The B4D10C9 hybridoma cell line producing the monoclonal antibody which was specifically agglutinated red blood cells B group, belongs to the IgM class and kappa light chains that have been created in our previous study. This study presents some biological charateristics of B4D10C9 such as the capability of growth, anti B monoclonal antibody producing, the stability of growth and cell line and antibody secreting of this cell line. The hybridoma cells grew well in the DMEM medium added with fetal bovine serum (FBS) at the final concentration of 1% or 10%. However, the maximum density of cells reached 9,9 x 105 cells per ml after 50 hours post incubation only when it was cultured in the medium containing 10% of FBS. The maximum antibody titers in the supernatant that collected after 150 hours of culture reached 1/512. In contrast to that, the maximum density of cells only reached 3,4 x 105 cells/ml after 72 hours post incubation when was cultured in DMEM medium containing 1% FBS. The maximum antibody titer was 1/64 after 50 hours post incubation. These cells were cultured through 3 different generations and the ability of antibody production in each generation was evaluated. As a result, this cell line had continuous growth stability and antibody secretion through 3 generations. The quantity of monoclonal antibody obtained from the supenantant of the culture medium at 150 hours of incubation was about 100 micrograms per ml. Thus, the growth of the cell line B4D10C9 and its ability of secretion of anti B monoclonal antibody were better when was cultured in the DMEM medium containing 10% fetal bovine serum. Preservation, storage and recovery does not affect to the growth and antibody producing of this cell line.
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Wimperis, J. Z., D. Gottlieb, A. S. Duncombe, H. E. Heslop, H. G. Prentice, and M. K. Brenner. "Requirements for the adoptive transfer of antibody responses to a priming antigen in man." Journal of Immunology 144, no. 2 (January 15, 1990): 541–47. http://dx.doi.org/10.4049/jimmunol.144.2.541.
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Abstract Antibody responses to recall vaccines can be adoptively transferred after marrow transplantation in man. Transfer of responses to priming Ag has not been successful, although this would broaden the range of organisms to which recipients could be protected. To investigate the importance of T cells and Ag in such transfer we primed marrow donors with keyhole limpet hemocyanin (KLH) 1 or 3 wk before marrow harvesting. B cells secreting IgM and IgG anti-KLH antibody were present in donor marrow at both 1 and 3 wk after immunization. After T cell depletion, donor marrow was infused into chemo-irradiated recipients, half of whom were immunized pretransplant with KLH. We found no evidence for the transfer of the IgM component of the response. Clonal expansion of the transferred IgG antibody-secreting cells with a corresponding rise in recipient serum IgG antibody levels was seen only when donors were primed 3 wk before marrow harvest and when the recipients were also immunized. IEF and immunoblotting demonstrated that successful transfer coincided with maturation of the IgG primary response from a polyclonal to an oligoclonal pattern and confirmed that donor oligoclonal bands appeared in the recipient serum. We conclude that the immunization protocols required for the transfer of antibody responses to priming Ag reflect the initial dependence of unprimed B cells on T cell help and on prolonged Ag stimulation. Ag-stimulated primary B cells in T cell-depleted marrow respond only to the noncognate growth and differentiation signals available in the chemo-irradiated recipient after an initial period of clonal selection and expansion in the donor which is both T cell and Ag dependent. Even after this initial selection, continued expansion of antibody-secreting clones in recipients retains an absolute dependence on Ag stimulation. Immunization techniques to protect transplant recipients against organisms such as Pseudomonas and CMV may need to be modified accordingly.
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Heilmann, C., T. Barington, and T. Sigsgaard. "Subclass of individual IgA-secreting human lymphocytes. Investigation of in vivo pneumococcal polysaccharide-induced and in vitro mitogen-induced blood B cells by monolayer plaque-forming cell assays." Journal of Immunology 140, no. 5 (March 1, 1988): 1496–99. http://dx.doi.org/10.4049/jimmunol.140.5.1496.
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Abstract The subclass of individual human IgA B cells was investigated by means of monolayer plaque-forming cell assays permitting analysis of all IgA-secreting cells as well as of cells secreting IgA anti-pneumococcal polysaccharide antibody. Center cells were examined by indirect immunofluorescence staining with mouse mAb against either of the two IgA subclasses as primary antibodies and FITC-conjugated rabbit anti-mouse Ig as the second antibody. Blood lymphocytes spontaneously secreting IgA (mean 399/10(6) mononuclear cells) produced mainly IgA1 (73%). A similar distribution of subclasses was recorded among IgA-secreting blood cells in PWM- and EBV-stimulated cultures. In contrast, a predominance of IgA2 (54%) was found among IgA-secreting cells (2531/10(6)) isolated from the blood 7 days after in vivo stimulation with pneumococcal polysaccharides, and a similar proportion (51%) of IgA2 producing cells was found among IgA anti-pneumococcal polysaccharide-secreting cells. It was thus confirmed that IgA1 is the predominant subclass of blood IgA-secreting cells in general. However, the high percentage of IgA2-secreting cells found after vaccination with pneumococcal polysaccharides suggests that these Ag have an unusually high ability to activate IgA2 B cells, or that the B cells stimulated originate from lymphatic tissues with a high frequency of IgA2 committed cells.
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Watanabe, Norihiko, Koichi Ikuta, Sidonia Fagarasan, Shujiro Yazumi, Tsutomu Chiba, and Tasuku Honjo. "Migration and Differentiation of Autoreactive B-1 Cells Induced by Activated γ/δ T Cells in Antierythrocyte Immunoglobulin Transgenic Mice." Journal of Experimental Medicine 192, no. 11 (November 27, 2000): 1577–86. http://dx.doi.org/10.1084/jem.192.11.1577.
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Using normal and transgenic (Tg) mice, we have shown that peritoneal B-1 cells are activated by administration of cytokines or lipopolysaccharide and migrate to other lymphoid organs where they differentiate into antibody-secreting cells. However, little is known about the process of B-1 cell migration and differentiation in vivo. We developed a mouse line by crossing the antierythrocyte antibody Tg mice (HL mice) with TCR-γ/δ Tg mice specific for a self-thymus leukemia (TL) antigen in the recombination activating gene (RAG)2−/− background. In the presence of the self-antigen, Tg γ/δ T cells increased in number and manifested activated phenotypes. Peritoneal B-1 cells in these mice migrated into mesenteric lymph nodes and differentiated into autoantibody-secreting cells, resulting in strong autoimmune hemolytic anemia. Furthermore, transfer of RAG2−/− × HL bone marrow or peritoneal cells into the peritoneal cavity of RAG2−/− × TCR-γ/δ Tg mice gave rise to donor-derived B-1 cells in mesenteric lymph nodes, and these cells produced the autoantibody. Thus, this study demonstrates that the migration of B-1 cells and differentiation into the antibody-secreting cells can be induced by noncognate T cell help and implies the possibility that γ/δ T cells may induce B-1 cell differentiation in vivo.
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Shearman, C. W., E. J. Kanzy, D. K. Lawrie, Y. W. Li, P. Thammana, G. P. Moore, and R. Kurrle. "Construction, expression, and biologic activity of murine/human chimeric antibodies with specificity for the human alpha/beta T cell receptor." Journal of Immunology 146, no. 3 (February 1, 1991): 928–35. http://dx.doi.org/10.4049/jimmunol.146.3.928.
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Abstract Murine/human chimeric antibodies with specificity for the human TCR-alpha/beta have been produced by genetic engineering. The L and H chain V region exons encoding the murine mAb BMA 031 were isolated and inserted into mammalian expression vectors containing the human kappa and gamma 1 or gamma 4 C region exons. The chimeric genes were transfected into murine Sp2/O hybridoma cells by electroporation and transfectomas secreting chimeric antibody were isolated. Secretion levels ranged from 1 to 7 pg/cell/24 h. The chimeric antibodies bound specifically to T cells and competed effectively with the parental murine mAb for binding to these sites. The ability to promote antibody-dependent cell-mediated cytolysis was significantly enhanced in the chimeric antibodies as compared with murine BMA 031. C-dependent cytolysis, however, was not detectable with any of the antibodies. Chimeric BMA 031 is a clinically relevant, genetically engineered antibody with potential uses in transplantation, graft-vs-host disease, autoimmune diseases and other T cell-related disorders.
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Chace, J. H., and D. W. Scott. "Activation events in hapten-specific B cells from tolerant mice." Journal of Immunology 141, no. 10 (November 15, 1988): 3258–62. http://dx.doi.org/10.4049/jimmunol.141.10.3258.
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Abstract We have studied hapten-binding cells from the spleens of normal and tolerant adult mice in terms of their ability to enlarge, proliferate, and differentiate into antibody-secreting cells. Tolerant B cells showed clear defects in intermediate activation events in addition to a deficit in antibody-secreting cells. In these studies, isolated B cells were stimulated by T cell independent Ag with lymphokines, or with mitogens, in the absence of filler cells. The number of antibody-secreting cells generated from the tolerant population was consistently reduced by 70 to 80% of the control response to the specific Ag, fluoresceinated Brucella abortus (FL-BrA) +/- lymphokines. We found that similar numbers of normal and tolerant cells enlarged (entered cell cycle) when stimulated by FL-BrA, LPS, IL-4, or alpha-Ig coupled to dextran (alpha-Ig-dex). Ia induction stimulated by IL-4, LPS, FL-BrA, or alpha-Ig-dex was the same in normal and tolerant cells. However, DNA synthesis stimulated by FL-BrA, FL-BrA + IL-5, or suboptimal concentrations of LPS was reduced by 70% in the tolerant cell population. Proliferation in response to 50 micrograms/ml LPS or to low doses of alpha-Ig-dex was similar in normal and tolerant B cells. These data suggest that the primary defect in adult B cell tolerance is the ability to proliferate in response to Ag.
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Huang, Kuan-Ying Arthur, Chris Ka-Fai Li, Elizabeth Clutterbuck, Cecilia Chui, Tom Wilkinson, Anthony Gilbert, John Oxford, et al. "Virus-Specific Antibody Secreting Cell, Memory B-cell, and Sero-Antibody Responses in the Human Influenza Challenge Model." Journal of Infectious Diseases 209, no. 9 (January 10, 2014): 1354–61. http://dx.doi.org/10.1093/infdis/jit650.
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Mountz, J. D., J. Smith, C. M. Snapper, J. F. Mushinski, and F. D. Finkelman. "Polyclonal activation of the murine immune system by an antibody to IgD. VIII. Stimulation of IgD secretion." Journal of Immunology 139, no. 7 (October 1, 1987): 2172–78. http://dx.doi.org/10.4049/jimmunol.139.7.2172.
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Abstract The low levels of serum IgD found in mice and the lack of a typical DNA switch sequence between C delta and C mu raise the possibility that the generation of murine IgD-secreting cells results from a chance "mistake" rather than a controlled process. The recent observation that injection of mice with purified IgD upregulates IgD receptor expression on helper T cells and enhances the ability of these T cells to induce B cells to differentiate into antibody secreting cells led us to look for evidence of controlled differentiation of B cells into IgD-secreting cells. To do this, we injected mice with a goat antibody to IgD (GaM delta), because this antibody stimulates large increases in IgM, IgG1, IgG2a, and IgE secretion. Mice injected with GaM delta demonstrated a large increase in splenic content of mRNA specific for the secreted form of delta-chain, as well as a greater than 100-fold increase in the percentage of splenic IgD-containing plasmablasts. The secretory IgD response was totally T-dependent. Production of the secretory form of IgD was not seen until 7 days after GaM delta injection, and peaked sharply on day 8, whereas by day 6 IgM secretion had already peaked and IgG1 and IgG2 secretion had attained substantial levels. This observation suggests that: 1) either cells that synthesize large quantities of the secretory form of delta-chain, unlike cells that synthesize large quantities of the secretory forms of gamma-, epsilon-, or alpha-chains, do this without deleting C mu, or, despite the absence of a typical DNA switch sequence between C mu and C delta, controls must exist to effect the C mu deletion and VDJ-C delta joining; and 2) if secreted IgD has a role in the regulation of a humoral immune response it most likely is involved in later processes, such as memory cell generation or response termination, rather than in relatively early processes, such as helper T cell activation.
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Nguyen, Doan C., Matthew C. Woodruff, Ignacio Sanz, and F. Eun-Hyung Lee. "Modulating Immunoglobulin Secretion of a Single Human Plasma Cell by Glycolysis Inhibition." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 112.27. http://dx.doi.org/10.4049/jimmunol.208.supp.112.27.
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Abstract Early-minted antibody-secreting cells (ASC) undergo further maturation to become long-lived plasma cells (LLPC) in the bone marrow (BM) microniche to afford life-long humoral protection. As terminally-differentiated cells, ASC are thought to be antibody factories that secrete immunoglobulins (Ig) constitutively. This model suggests that Ig secretion is linked with survival and loss of Ig secretion is associated with apoptosis. Here, we show that the Ig secretory function of human ASC is inhibited after treatment with an GAPDH inhibitor as measured by IgG Elispot assays and by direct visualization of IgG secretion from a single ASC. Although Ig secretion ceases quickly with the initial treatment, ASC promptly recover secretory function upon removal of the inhibitor. The modulation of Ig secretory function was directly visualized in both human early-minted blood ASC and BM LLPC by single-cell analysis using an optofluidic technology. Thus, we conclude that ASC Ig secretion is not constitutive but a dynamic process which can be modulated by metabolic processes such as glycolysis. Supported by NIH/NIAID 1R01AI121252, 1P01AI125180, U01AI141993, U54CA260563, U19AI110483, T32AI070081 and the Bill & Melinda Gates Foundation Grant INV-002351.
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Pond, L., D. L. Wassom, and C. E. Hayes. "Influence of resistant and susceptible genotype, IL-1, and lymphoid organ on Trichinella spiralis-induced cytokine secretion." Journal of Immunology 149, no. 3 (August 1, 1992): 957–65. http://dx.doi.org/10.4049/jimmunol.149.3.957.
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Abstract The relative importance of cell-mediated inflammatory responses and antibody-mediated responses in controlling parasitic helminth infection is debated. To study the relationship between these responses and resistance or susceptibility to primary Trichinella spiralis infection, we infected resistant AKR mice and susceptible B10.BR mice and analyzed the lymphokines IL-2, IFN-gamma, and IL-5 produced by their T cells as a function of time and lymphoid organ. IL-2-secretors occurred maximally between days 3 and 6 postinfection, whereas IL-5-secretors peaked between days 6 and 9. Previously, we found that IFN-gamma producers peaked before day 6, whereas IL-4 producers peaked between days 6 and 9. Most cytokine secretors were CD4+. The simultaneous development of IL-2- and IFN-gamma-secreting cells, and IL-4- and IL-5-secreting cells, suggests that the infection may be stimulating T cells to differentiate into cells capable of secreting specific cytokine sets, analogous to the postulated Th1 and Th2 subsets. In the spleen and mesenteric lymph nodes, cells from B10.BR mice secreted more IL-5 than cells from AKR mice, as we found previously for IL-4. For both strains, mesenteric lymph node cells produced more IL-5 than splenocytes. The AKR mesenteric lymph node cells produced more IL-2 than the B10.BR cells, but the reverse occurred in splenocytes. The AKR peripheral lymph node cells also secreted more IFN-gamma than the B10.BR cells, but the strains were equivalent for peritoneal exudate cell IFN-gamma production. Thus, the lymphoid organ microenvironment plays an important role in regulating cytokine-secreting cell development in this system. We also tested the possible regulatory role of IL-1. Exogenous rIL-1 alpha increased IFN-gamma secretion early but not late in mesenteric lymph node cells from both strains; this reflected an increased IFN-gamma-secreting cell frequency, not a change in IFN-gamma mRNA transcript level. Exogenous rIL-1 alpha did not consistently affect IL-2, IL-4, or IL-5 secretion. These data suggest that IL-1 alpha availability in vivo may regulate IFN-gamma-secreting cell development. In sum, early activation of IFN-gamma-secreting T cells in lymph nodes, with little subsequent activation of IL-4- and IL-5-secreting cells, distinguished the resistant from susceptible strain responses to T. spiralis infection, and IL-1 alpha and lymphoid organ environment influence IFN-gamma-secreting cell activation.
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Reinhardt, Richard Lee, Hong-Erh Liang, and Richard M. Locksley. "Cytokine-secreting follicular T cells shape the antibody repertoire (34.19)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 34.19. http://dx.doi.org/10.4049/jimmunol.182.supp.34.19.
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Abstract High-affinity antibodies are critical for host protection against pathogens and toxins, and underlie successful vaccines. Generation of such antibodies requires T cell-dependent help, which mediates germinal center (GC) reactions where mutation and selection of B cells expressing receptors with high-affinity for antigen occurs. Follicular CD4 T (TFH) cells, which comprise only 5-10% of GC cells, have been imaged in stable conjugates with GC B cells, but no functional analysis of B/TFH cell interactions in GC has been performed. Using an IL-4 reporter system, we show that TFH cells comprise essentially all of the cytokine-secreting T cells in lymph nodes and are functionally distinct from T cells secreting the same cytokine in peripheral tissues. TFH cells with different cytokine profiles could be visualized in the same GC and could be isolated as conjugates with B cells undergoing cytokine-specific immunoglobulin isotype class switching, expressing high levels of AID, and evidence of somatic hypermutation. These findings support a model wherein GC B cells compete for cytokines produced by rare antigen-specific GC TFH cells that mediate highly localized effects and regulate the size, affinity and isotype of the B cell antibody response.
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Martel, F., R. Bazin, S. Verrette, and R. Lemieux. "Characterization of higher avidity monoclonal antibodies produced by murine B-cell hybridoma variants selected for increased antigen binding of membrane Ig." Journal of Immunology 141, no. 5 (September 1, 1988): 1624–29. http://dx.doi.org/10.4049/jimmunol.141.5.1624.
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Abstract Somatic mutation in the Ig genes plays a major role in the increase of antibody affinity observed in secondary immunologic responses. It has been shown that the mechanism responsible for the high rate of somatic mutation in the Ig genes was active not only in normal B lymphocytes but also in B-cell hybridomas secreting mAb. Also, it has been reported that B-cell hybridomas were positive for membrane Ig of the same specificity as the secreted mAb. The presence of membrane Ig suggested that somatic variants secreting mAb of higher affinity could be selected by the increased capacity of these hybridoma cells to bind immobilized Ag. This hypothesis was tested with hybridoma cells secreting an IgM mAb reacting with the A Ag of the ABO blood group system. In two selection experiments, we have isolated several variant cell lines secreting mAb of increased avidity for the A Ag under similar IgM concentrations. Biochemical characterization of one of the variant mAb indicated that the mutation responsible for the increased avidity has occurred in the heavy chain gene. The method developed may have profound implications for the diagnostic and therapeutic use of mAb and will permit the study, in an in vitro system, of the role of somatic mutations in antibody diversity.
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Denkers, E. Y., C. C. Badger, J. A. Ledbetter, and I. D. Bernstein. "Influence of antibody isotype on passive serotherapy of lymphoma." Journal of Immunology 135, no. 3 (September 1, 1985): 2183–86. http://dx.doi.org/10.4049/jimmunol.135.3.2183.
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Abstract We assessed the in vivo anti-tumor effectiveness of monoclonal antibodies of different isotypes. Starting with a hybridoma cell secreting an IgG3 anti-Thy-1.1 antibody, we isolated three variant hybridoma cell lines secreting anti-Thy-1.1 antibody of the IgG1, IgG2a, and IgG2b isotypes. Each antibody displayed identical antigen binding properties, but differed in their ability to mediate in vitro lysis of Thy-1.1+ AKR/J SL2 lymphoma cells. In assays of complement dependent cytotoxicity, the relative activity of each antibody isotype was IgG2a = IgG2b greater than IgG3 greater than IgG1. In assays of antibody-dependent cell-mediated cytotoxicity when using non-immune spleen cells as effectors, the relative activities were IgG2a greater than or equal to IgG2b greater than IgG1 greater than IgG3. Infusion of equivalent amounts of each antibody (1.5 mg) in AKR/Cum (Thy-1.2+) mice inoculated subcutaneously with 3 X 10(5) AKR/J SL2 lymphoma cells resulted in significant inhibition of tumor growth only in mice treated with IgG2a antibody. However, the antibodies were cleared at different rates, with the IgG2a antibody having the slowest clearance. When antibody doses were adjusted to achieve equivalent serum levels 24 hr after infusion, all of the antibody isotypes exhibited at least some anti-tumor activity, although IgG2a antibody was again the most effective. These studies demonstrate that the difference in anti-tumor activity between antibodies of different isotypes may result from differences both in their serum clearance rate and their ability to interact with host effector mechanisms.
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Tumang, Joseph R., Ruben Frances, and Thomas L. Rothstein. "A Unique Transcriptional Profile for Spontaneously Immunoglobulin Secreting B-1 Cells." Blood 104, no. 11 (November 16, 2004): 3236. http://dx.doi.org/10.1182/blood.v104.11.3236.3236.
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Abstract Natural antibodies are spontaneously arising polyreactive Ig, usually IgM, that are encoded by V genes in germline configuration and are produced by B-1 cells. Because of their ability to promiscuously bind to a variety of antigens, especially those on bacteria and viruses, natural antibodies act as a primary line of defense against infection. A key shortfall in our understanding of these key effector molecules centers on the molecular mechanisms regulating natural antibody secretion by B-1 cells. Recent advances in our understanding of plasmacytic differentiation now provide us with a conceptual framework by which we can begin to explore this issue. In the studies described herein, we demonstrate that secreting B-1 cells utilize some aspects of the plasmacytic differentiation program and deviate away from it in others. Specifically, we show that key repressors of the plasmacytic program, BCL-6 and PAX-5a, are indeed reduced in secreting B-1 B cells. Surprisingly, we found that key promoters of the plasmacytic program, BLIMP-1 and XBP-1, are not upregulated in secreting B-1 B cells. These data demonstrate that B-1 B cells operate under a differentiation program unique from prototypic secreting B-2 cells and cast BCL-6 and PAX-5a but not BLIMP-1 and XBP-1 as central regulators of natural immunoglobulin secretion.
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McHeyzer-Williams, Louise J., Melinda Cool, and Michael G. McHeyzer-Williams. "Antigen-Specific B Cell Memory." Journal of Experimental Medicine 191, no. 7 (April 3, 2000): 1149–66. http://dx.doi.org/10.1084/jem.191.7.1149.
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The mechanisms that regulate B cell memory and the rapid recall response to antigen remain poorly defined. This study focuses on the rapid expression of B cell memory upon antigen recall in vivo, and the replenishment of quiescent B cell memory that follows. Based on expression of CD138 and B220, we reveal a unique and major subtype of antigen-specific memory B cells (B220−CD138−) that are distinct from antibody-secreting B cells (B220+/−CD138+) and B220+CD138− memory B cells. These nonsecreting somatically mutated B220− memory responders rapidly dominate the splenic response and comprise >95% of antigen-specific memory B cells that migrate to the bone marrow. By day 42 after recall, the predominant quiescent memory B cell population in the spleen (75–85%) and the bone marrow (>95%) expresses the B220− phenotype. Upon adoptive transfer, B220− memory B cells proliferate to a lesser degree but produce greater amounts of antibody than their B220+ counterparts. The pattern of cellular differentiation after transfer indicates that B220− memory B cells act as stable self-replenishing intermediates that arise from B220+ memory B cells and produce antibody-secreting cells on rechallenge with antigen. Cell surface phenotype and Ig isotype expression divide the B220− compartment into two main subsets with distinct patterns of integrin and coreceptor expression. Thus, we identify new cellular components of B cell memory and propose a model for long-term protective immunity that is regulated by a complex balance of committed memory B cells with subspecialized immune function.