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Signaling This Information Is Current As of September 26, 2021 IL-2 Requirement for Human Plasma Cell Generation: Coupling Differentiation and Proliferation by Enhancing MAPK−ERK Signaling This information is current as of September 26, 2021. Simon Le Gallou, Gersende Caron, Céline Delaloy, Delphine Rossille, Karin Tarte and Thierry Fest J Immunol 2012; 189:161-173; Prepublished online 25 May 2012; doi: 10.4049/jimmunol.1200301 Downloaded from http://www.jimmunol.org/content/189/1/161 Supplementary http://www.jimmunol.org/content/suppl/2012/05/25/jimmunol.120030 http://www.jimmunol.org/ Material 1.DC1 References This article cites 55 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/189/1/161.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 26, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology IL-2 Requirement for Human Plasma Cell Generation: Coupling Differentiation and Proliferation by Enhancing MAPK–ERK Signaling Simon Le Gallou,*,†,‡,1,2 Gersende Caron,*,†,‡,x,1 Ce´line Delaloy,*,†,‡,x,1 Delphine Rossille,x,{ Karin Tarte,*,†,‡,x and Thierry Fest*,†,‡,x Mature B cell differentiation involves a well-established transcription factor cascade. However, the temporal dynamics of cell sig- naling pathways regulating transcription factor network and coordinating cell proliferation and differentiation remain poorly de- fined. To gain insight into the molecular processes and extrinsic cues required for B cell differentiation, we set up a controlled primary culture system to differentiate human naive B cells into plasma cells (PCs). We identified T cell-produced IL-2 to be crit- ically involved in ERK1/2-triggered PC differentiation. IL-2 drove activated B cell differentiation toward PC independently of its Downloaded from proliferation and survival functions. Indeed, IL-2 potentiated ERK activation and subsequent BACH2 and IRF8 downregulation, sustaining BLIMP1 expression, the master regulator for PC differentiation. Inhibition of the MAPK–ERK pathway, unlike STAT5 signaling, impaired IL-2–induced PC differentiation and rescued the expression profile of BACH2 and IRF8. These results identify IL-2 as a crucial early input in mature B cell fate commitment. The Journal of Immunology, 2012, 189: 161–173. cell-dependent immune response is initiated in germinal the follicle (3–7). In GCs, B cells undergo terminal differentiation http://www.jimmunol.org/ centers (GCs) after seeding by a small number of rapidly and selection, which depend tightly on the light zone microenvi- T dividing Ag-responding B cells. These cells undergo a se- ronment (3, 4). The gene regulatory network that governs transi- ries of proliferation/selection steps to give rise to memory B cells tion between GC B cells and plasmablasts is well understood and or long-lived plasma cells (PCs) (1). The GC response initiates in the heavily controlled by cytokines, particularly those that reduce outer follicle where naive B cells (NBCs) encounter their specific BCL6 expression and induce BLIMP1, two mutually exclusive Ags (2). Subsequently, activated B cells relocate to the B zone–T transcriptional regulators. BLIMP1 orchestrates PC differentiation zone boundary where Ag-specific B and T cells interact with each by extinguishing the mature B cell gene expression program in- other and form long-lived pairs. In this pairing, B and T cells up- cluding BCL6, freeing factors like IRF4, and XBP1 (8, 9). Notably, regulate BCL6, proliferate (3, 4), and acquire a centroblastic or a in response to stimulation, B cells stochastically pursue various by guest on September 26, 2021 follicular Th (TFH) cell identity respectively before migrating inside distinct fates. Some cells undergo differentiation and isotype switching depending on cell division and cytokine environment (10–12). *INSERM, Unite´ Mixte de Recherche 917, Rennes F-35043, France; †Universite´ de Rennes 1, Unite´ Mixte de Recherche 917, Rennes F-35043, France; ‡Etablissement The T cell help provides distinct signals that are critical in de- Franc¸ais du Sang de Bretagne, Unite´ Mixte de Recherche 917, Rennes F-35043, termining B cell behavior. During initial T–B cognate interaction, France; xCentre Hospitalier Universitaire de Rennes, Poˆle Cellules & Tissus, Rennes { T cell potentially induces a large variety of inputs including F-35033, France; and INSERM, Unite´ Mixte de Recherche 936, Rennes F-35043, France CD40 and CD80/CD86 engagement (13, 14). However, T cell- 1S.L.G., G.C., and C.D. contributed equally to this work. derived signals seem not to be mandatory, as T cell-independent B cell clonal expansion, GC formation, and acquisition of GC 2Current address: INSERM U783, Universite´ Descartes, Paris, France. B cell phenotype may be observed in the absence of T cells (15). Received for publication January 24, 2012. Accepted for publication April 25, 2012. However, stable contacts of TFH cells with B cells are absolutely This work was supported by an internal grant from the Hematology and Immunology Laboratory, Poˆle Cellules & Tissus, Centre Hospitalier Universitaire de Rennes. S.L.G. required to sustain GC maturation, centrocyte selection, and PC was supported by a research grant from La Ligue Contre le Cancer/Re´gion Bretagne, generation (5, 16). TFH cells are not statically providing a single and C.D. was supported by an internal grant from the Poˆle Cellules & Tissus, Centre stimulus to B cells, but instead at some point they promote high- Hospitalier Universitaire de Rennes. affinity GC B cells to become either PCs or memory B cells. This S.L.G. performed research, analyzed data, and established the B cell differentiation model; G.C. designed experiments and performed research; C.D. performed research could be an early or late input that might function with a con- and analyzed cell signaling; D.R. performed microarray analyses; K.T. contributed to comitant CD40 signal. First, T cells drive NBC proliferation and study design; and T.F. designed and supervised research, raised funds, and wrote the inhibit PC differentiation (17). At a later stage, T cells promote paper. FH a reinforcement of BCL6 repression signals in high-affinity B cells Microarray data presented in this article have been submitted to the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under accession no. GSE36975. (18, 19). Besides CD40L, TFH cells produce IL-21, which acts directly on GC B cells maximizing BCL6 expression leading to Address correspondence and reprint requests to Prof. Thierry Fest, INSERM U917, Faculte´ de Me´decine, 2 Avenue du Professeur Le´on Bernard, CS 34317, 35043 cell survival and proliferation (20–23). In contrast, IL-21 induces Rennes Cedex, France. E-mail address: [email protected] PC differentiation through a STAT3-dependent BLIMP1 induction The online version of this article contains supplemental material. (21, 24, 25). Numerous other T cell-derived cytokines enhance PC + Abbreviations used in this article: FC, fold change; GC, germinal center; GEP, gene differentiation such as IL-2 produced by memory CD4 T cells expression profiling; GL, gene list; HCA, hierarchical clustering analysis; NBC, (26). Recently in mice, ERKs have been shown to be necessary to naive B cell; PC, plasma cell; qRT-PCR, quantitative RT-PCR; T , follicular Th. FH trigger PC generation, mediating the cytokine-induced production Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 of BLIMP1 (27). Altogether, these findings reinforce the well-known www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200301 162 IL-2–ENHANCED ERK1/2 SIGNALING AND PC DIFFERENTIATION assumption that a combination of a BCR signal and T cell help is Apoptosis and proliferation assays required to initiate PC differentiation (28). Apoptosis and proliferation were analyzed using a PE-conjugated anti-active To explore events that govern human NBC differentiation, we caspase-3 apoptosis kit (BD Biosciences) and an FITC-conjugated anti-BrdU designed an in vitro two-step culture model combining BCR signal, kit (BD Biosciences), respectively, according to the manufacturer’s instruc- TLR activation, and T cell help in the form of CD40L and tions. For BrdU staining, B cells were incubated with BrdU (10 mM) during cytokines. Unswitched human naive precursors differentiated into 45 min followed by CD38 staining. Cells were then fixed, permeabilized, and DNAse treated before subsequent staining and FACS analysis. CD20+CD38+ and CD20loCD38hi cells characterized by distinct cell fates. We explored one by one factors used in our model and Ig secretion and Western blotting found that T cell-produced IL-2 was critical for human NBC For Ig secretion assay, differentiated and nondifferentiated B cells were commitment to PCs. IL-2 activated the ERK pathway at a threshold isolated and reseeded with IL-2, IL-4, and IL-10 at 1 3 106 cells/ml for 18 h. level that triggered, beyond the induction of cell cycle progression, IgG, IgA, and IgM secretion was assessed by ELISA using a goat anti- PC generation. BACH2 and IRF8 expression was downregulated human Ig for coating and secondary alkaline phosphatase-coupled Abs specific for g, a,orm chain, respectively (all from Jackson Immuno- through the MEK–ERK signaling pathway in IL-2–primed B cells. Research Laboratories). SDS-PAGE and Western blotting were performed Therefore, IL-2 reinforced the mutual repression between BCL6 according to standard procedures and Abs listed in Table I.
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