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

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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 signaling 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- Français 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 León 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. Detections and BLIMP1. This study highlights early events that could take were performed with HRP-conjugated secondary Ab (Bio-Rad) and en- place when cognate B and T cells meet and interact before seeding hanced chemiluminescent (ECL Plus) reagent (Amersham). GAPDH or follicle, where crucial inputs may condition final GC B cell destiny. b-actin on the same membrane served as loading control. Quantitative RT-PCR analysis Downloaded from Materials and Methods RNA was extracted using RNeasy microkit (Qiagen, Courtaboeuf, France) Primary B cell purification and reversed transcribed into cDNA with Superscript II (Invitrogen). Quantitative RT-PCR (qRT-PCR) was performed using the TaqMan Uni- PBMCs from healthy volunteers were obtained from the Etablissement versal Master Mix and assays-on-demand from Applied Biosystems (Foster Français du Sang (Rennes, France) after Ficoll density centrifugation City, CA). Gene expression levels were quantified using HPRT1 as en- (Sigma-Aldrich, St. Louis, MO). NBCs were purified by negative selection dogenous control. The 2 exp(2DDCt) method was used to determine the using magnetic cell separation (Naive B Cell Isolation Kit II; Miltenyi relative expression of each gene.

Biotech, Bergisch Gladbach, Germany), following the manufacturer’s http://www.jimmunol.org/ instructions, using the AutoMACS deplete-sensitive program. Purity of Microarray hybridization isolated CD19+CD272 NBCs was routinely .99%, and in some cases IgG+ CD272 memory B cells were removed by FACS sorting. Tonsil-derived paired CFSE-labeled NBCs from three donors were ﬁrst stimulated at day 0 [CD40L, memory B cells and centroblasts were isolated as previously described (29). CpG, and F(ab9)2 anti-human Ig]. Then, IL-2 was added or not at day 0 plus 16 h leading for each donor to two parallel cell culture conditions: IL-2+ 2 Cell culture, immunophenotyping, and cell sorting and IL-2 cells. Cells were removed at different time points for microarray analysis (day 0, day 0 + 16 h, day 0 + 22 h). At day 4, IL-2+ and IL-22 All cultures were performed in complete medium consisting of RPMI 1640 cells were separated into CSFEhi and CSFElo fractions (Fig. 4A). Extracted (Invitrogen, Carlsbad, CA) supplemented with 10% FCS (Biowest, Nuaille´, RNAs (RNeasy microkit; Qiagen) were hybridized onto Illumina Human France) and antibiotics (Invitrogen). To evaluate cell division, puriﬁed HT-12 v4 Whole-Genome Gene Expression BeadChips according to

NBCs were labeled with 1 mM CFSE (Invitrogen) at 37˚C for 10 min and standard Illumina protocols (Illumina, San Diego, CA). The 18 samples by guest on September 26, 2021 washed in complete medium. were randomly distributed on two beadchips. Data extraction and quality Purified human NBCs were cultured at 7.5 3 105 cells/ml in 24-well control were performed using BeadStudio. plates and stimulated during 4 d with 2 mg/ml F(ab9)2 fragment goat anti- human IgA+IgG+IgM (H+L) (Jackson ImmunoResearch Laboratories, Statistical analyses and functional interpretation West Grove, PA), 50 ng/ml recombinant human soluble CD40L associated Data are available at the Gene Expression Omnibus database (accession no. m m with 5 g/ml cross-linking Ab (R&D Systems, Abingdon, U.K.), 2.5 g/ml GSE36975; http://www.ncbi.nlm.nih.gov/geo/). Microarray statistical anal- CpG oligodeoxynucleotide 2006 (Cayla Invivogen, Toulouse, France), and yses were performed using Partek Genomics Suite software, version 6.5 50 U/ml recombinant IL-2 (SARL Pharmaxie, Aigueperse, France). When (Partek, St. Louis, MO) and the R language (www.R-project.org). First, indicated, neutralizing Abs for IL-2, IL-2Ra, IL-2Rb, and IL-2Rg and prefiltering of the raw data was performed by filtering out all probes for their isotype controls (all from R&D Systems) were used at 10 mg/ml. which detection above background p value was .5% for all samples as MEK1/2 and STAT5 pathways were inhibited with specific chemical well as all uncharacterized probes (associated to gene symbols starting by inhibitors, U0126, PD184161, and STAT5 inhibitor (all from Calbiochem, Cxorf, FLJ, KIAA, HS., LOC and MGC). Hence, by reducing the number San Diego, CA), respectively. Neutralizing Abs and inhibitors where added of probes to 21,788, the power of statistical tests was improved. Data were at least 1 h prior to stimulation with IL-2. 3 5 log2 transformed without normalization. Any batch effect highlighted with Day 4-activated B cells were washed and cultured at 4 10 cells/ml for PCA was corrected using the Partek batch-removal ANOVA-based method. up to 4 d with 50 U/ml IL-2, 10 ng/ml IL-10, and 10 ng/ml IL-4 (R&D Statistical analysis focused on the effect of stimulation 1 at the different Systems). time points. Hierarchical clustering analysis (HCA) was used to study overall B and T cell coculture. At day 0, CFSE-labeled NBCs were stimulated by dynamics of the genes throughout the time course. Probesets differentially + CpG plus F(ab9)2 anti-human Ig (BCR). At day 1, autologous CD4 T cells expressed between different time points were identified using paired t test were purified by magnetic cell separation using CD4+ microbeads (Miltenyi with p value ,5%, restricting the analysis to probes with absolute log2 fold Biotech) and added to B cells (ratio 1T:2B) together with 0.5 mg/ml anti- change .1.2 and mean of the linear-scale gene expression difference .150. CD3 plus 0.5 mg/ml anti-CD28 stimulation (Sanquin, Amsterdam, The HCA was used to highlight dynamics of these differentially expressed genes Netherlands) in the presence of a blocking anti–IL-2 or a control isotype. throughout the time course. Functional interpretation of these differentially IL-2 plus CD40L stimulated B cells were used as control. At day 4, T cells expressed genes was performed using IPA (Ingenuity Systems; www. were removed from the coculture by cell sorting based on the CD2 ex- ingenuity.com), more specifically the canonical pathway analysis tool that + pression. In parallel, CD4 T cells were activated or not with anti-CD3 and helped visualization of gene expressions on pathways of interest. anti-CD28 stimulation for 1 d, and their supernatants were collected and Statistical analyses for different experiments were performed using added to B cells at day 2, in the presence of CD40L and blocking Abs to GraphPad Prism software (GraphPad Software), and p values were cal- IL-2 or IL-2R(a+b+g). At day 4, B cells were subsequently cultured in the culated by two-tailed Student t test. presence of IL-2, IL-10, and IL-4 for two additional days. Abs used in this study are summarized in Table I. For quantitative RT-PCR analyses and ELISA assays, B cells were isolated at day 6 by FACS sorting Results after DAPI (Sigma-Aldrich) staining to exclude dead cells. Sorts were per- Experimental design to differentiate human unswitched NBC formed on a BD FACSAria cell sorter (BD Biosciences, San Jose, CA). precursors into PCs Differentiated and nondifferentiated B cells were sorted as CD20loCD38hi and CD20+CD38+ cells, respectively. After surface staining, the Cytofix/Cytoperm To explore T-dependent B cell terminal differentiation, we de- kit (BD Biosciences) was used for intracellular staining of IgM and IgG. veloped a two-step B cell culture model (Fig. 1A). All Abs used The Journal of Immunology 163 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

+ 2 FIGURE 1. Human B cell differentiation in vitro model: from NBCs to PCs. Purified CD19 CD27 NBCs were stimulated at day 0 with anti-F(ab9)2, CD40L, CpG, and IL-2. After 4 d activation, cells were washed and stimulated with IL-2, IL-4, and IL-10. (A) Representation of the B cell differentiation model. (B) The B cell differentiation was monitored by flow cytometry based on CD20 and CD38 membrane expression. Events were gated on DAPIneg population. Upper panels, Percentages represent the proportion of differentiated B cells (CD20loCD38hi) among viable cells. Lower panels, Percentages represent the proportion of CD138+ cells among the CD20loCD38hi B cells. Dot plot represents the expression of CD38 and CD138 among viable cells at day 7. Histogram plots are representative of 20 independent experiments. (C) Proliferation, survival, and differentiation were characterized among different generated B cell subpopulations. Proliferation (i) and apoptosis (ii) was determined respectively by BrdU incorporation and active caspase-3 staining by flow cytometry at days 2, 3, and 4. At day 6, analyses were done on differentiated (CD38hi) and nondifferentiated (CD38+) B cells. The relationship between proliferation and differentiation was assessed by CSFE and CD38 staining (iii). Events were gated on the DAPIneg population. Data are representative of three independent experiments. (D) Cumulative data of six independent experiments based on DAPI staining evaluate the viability among the CD20+CD38+ and CD20loCD38hi B cell subpopulations at day 6 and day 8, respectively. (E) Assessment of Ig class switching and secretion in CD38hi CD20lo and CD38+CD20+ B cell subpopulations. Left and middle panels, At day 6, after CD38 surface staining, the percentage (Figure legend continues) 164 IL-2–ENHANCED ERK1/2 SIGNALING AND PC DIFFERENTIATION

Table I. List of Abs used for ﬂow cytometry analysis, cell sorting, and Western blot

Anti-human Abs Clone Isotype Source CD19–FITC J3-119 Mouse IgG1 Beckman Coulter CD20–FITC B9E9 Mouse IgG2a Beckman Coulter CD27–PE 1A4CD27 Mouse IgG1 Beckman Coulter CD138–PE B-A38 Mouse IgG1 Beckman Coulter CD38–PC5 LS198-4-3 Mouse IgG1 Beckman Coulter IgM–FITC G20-127 Mouse IgG1 BD Biosciences IgG–PE G18-145 Mouse IgG1 BD Biosciences CD25–PE M-A251 Mouse IgG1 BD Biosciences CD122–PE Mik-b3 Mouse IgG1 BD Biosciences CD132–PE AG184 Mouse IgG1 BD Biosciences STAT5 89 Mouse IgG2b BD Biosciences Phospho-STAT5 (PY694) 47 Mouse IgG1 BD Biosciences BLIMP1 3H2-E8 Mouse IgG1 Novus Biologica PAX5 3852-1 Rabbit IgG Abcam BACH2 Polyclonal Rabbit Abcam b-Actin AC-15 Mouse IgG1 Sigma GAPDH 14C10 Rabbit IgG Cell Signaling P44/42 MAPK 137F5 Rabbit IgG Cell Signaling Phospho-p44/42 MAPK (T202/Y204) D13.14.4E Rabbit IgG Cell Signaling Downloaded from

for this study are listed in Table I. CD19+CD272 NBCs were first whether proliferation and differentiation were connected to- lo cultured with F(ab9)2 anti-Ig(A+G+M), CD40L, CpG, and IL-2. gether, we labeled B cells with CFSE at day 0. The CD20 hi At day 4, activated B cells were washed and then reseeded with CD38 subset was exclusively constituted by cells that had di- http://www.jimmunol.org/ IL-2, IL-4, and IL-10 in the absence of CD40L. luted the CSFE (Fig. 1Ciii). By sorting both CSFElo and CSFEhi CD38 and CD20 expression was monitored by flow cytometry subsets at day 4 before further culture, we demonstrated that only (Fig. 1B). During the activation phase, cells remained phenotypically cells that underwent three or more divisions gave rise to CD38hi cells homogeneous as a CD20+CD38+ population. In the second step, a (data not shown). CD20loCD38hi subpopulation appeared as soon as day 5 and in- To test generated B cell subsets for Ab secretion, intracytoplasmic creased progressively reaching 22 6 13% (n = 19) of the total Ig production was assessed by flow cytometry. As shown in Fig. 1E viable B cells at day 6. Unlike CD20+CD38+ cells, CD20loCD38hi (left and middle panel), CD20+CD38+ cells were mainly IgM cells concomitantly increased the expression of the CD138 PC positive (80.18 6 12.28%, n = 6), whereas CD20loCD38hi cells marker (Fig. 1B). Although recently described IgG+CD272 expressed preferentially IgG (52.36 6 7.71%, n = 6) reflecting a by guest on September 26, 2021 memory B cells (30) were found at an extremely low percentage in class-switching process among those cells. The upregulation in our purified NBC starting populations (1.13 6 1.04%, n = 3), we mean fluorescence intensity of intracytoplasmic IgM staining in decided to apply our differentiation model on CD272 NBCs cleared CD20loCD38hi compared with CD20+CD38+ cells suggested that for IgG+ cells. Both CD20loCD38hi and CD20+CD38+ subsets were IgM expression was mainly cytoplasmic in CD20loCD38hi cells. generated without any significant difference in PC generation Moreover, we detected only low levels of surface IgM or IgG compared with the standard procedure ruling out the possibility on CD20loCD38hi cells (data not shown). These results were in that the CD20loCD38hi cells resulted from the differentiation of agreement with the increase of IgM, IgA, and IgG secretion by CD272IgG+ memory B cells (data not shown). Therefore, in all CD20loCD38hi cells detected by ELISA (Fig. 1E, right panel) and further experiments, we used CD272 NBCs. ELISPOT assay (data not shown). A 3-fold increase of viable B cell number was observed during the activation phase, whereas the differentiation phase correlated Recapitulation at the transcriptional level of B cell with a decrease of cell viability, as evaluated by trypan blue exclusion differentiation hallmarks initially described in mouse models (data not shown). A dual BrdU and active caspase-3 staining was Cells were monitored from day 0 to day 6 by mRNA expression thus performed to assess apoptosis and proliferation in the course profiling for various molecules that are crucial in the transcriptional of PC generation (Fig. 1C). During the activation phase, a prolif- cascade that converts B cells into PCs (9). Gene expression levels eration burst was distinguished with more than 40% of the cells in of BACH2, PAX5, BCL6, PRDM1, IRF4, BIP, SPIB, SPI1, ETS1, S-phase at day 3 while only 4% of cells showed positive staining MITF, POU2AF1, AICDA, and the spliced variant of XBP1 (XBP1s) for caspase-3 (Fig. 1Ci–ii). At day 6 within the differentiation phase, at day 4 and day 6 were studied relative to their gene expression at cell proliferation was almost restricted to the CD20loCD38hi day 0, arbitrary defined as 1. At day 6, CD20+CD38+ and CD20lo subset with 34.4 6 7.9% of BrdU+ cells compared with 4.4 6 2.8% CD38hi subsets were sorted and tested separately. The two pop- for CD20+CD38+ cells that instead increased their susceptibility ulations presented a downregulation of MITF, a suppressor of PC for apoptosis (Fig. 1Ci–ii). Beyond day 6, cell viability decreased differentiation (31). Unlike CD20loCD38hi cells, CD20+CD38+ dramatically as cells arrest to proliferate (Fig. 1D). To ascertain cells did not modify the expression of PAX5, IRF4, and BACH2

of positive B cells for intracellular staining of IgM and IgG was evaluated by flow cytometry. Dot plots (left panel) are representative from five experiments, and bars (middle panel) represent mean values 6 SEM, n =5.Right panel, IgM, IgA, and IgG secretion was assessed in CD20+CD38+ and CD20loCD38hi B cells previously sorted by FACS at day 6 using CD20 and CD38 expression. Cells were reseeded at 1 3 106 cells/ml and subsequently cultivated for 18 h with IL-2, IL-4, and IL-10. IgG, IgA, and IgM secretion was assessed by ELISA. Bars represent mean values 6 SEM from four independent experiments. *p , 0.05, ***p , 0.001. D, Day. The Journal of Immunology 165 and maintained BCL6 and AICDA expression compared with day activated autologous CD4+ T cells promoted plasmablast gen- 4 cells consistent with a blockage in the differentiation process of eration. This effect strikingly decreased in the presence of anti– these cells (Fig. 2A, 2B). Their comparison with freshly isolated IL-2 neutralizing Ab (Fig. 3Ciii). Furthermore, supernatant of human centroblasts and memory B cells for BCL6 and AICDA activated CD4+ T cells was sufficient to trigger differentiation expression supported the notion of uncommitted activated B cell and was again blocked with anti–IL-2 or anti–IL-2R Abs (Fig. 3Civ). state (Fig. 2C). In contrast, CD20loCD38hi cells upregulated PRDM1, Finally, IL-2 was substitutable by IL-15 but not by IL-21 (data not IRF4, POU2AF1, BIP, and XBP1s and downregulated drastically shown). AICDA. These cells completed their differentiation program as seen by the downregulation of the Ets family genes SPI1, ETS1, IL-2 sustains the initial transcriptional burst and initiates PC and SPIB that are repressors of the B cell differentiation program, commitment as early as day 4 along with a strike increase of CD27 expression (Fig. 2A, 2B) To decipher signaling that sustains IL-2 effect, a microarray gene confirmed at the protein level (data not shown). Altogether, we expression profiling (GEP) was applied on highly purified cell conclude that our human B cell differentiation model mimics the fractions obtained at different time points of the cell culture. To molecular dynamic that takes place during in vivo GC B cell disconnect IL-2 signal effects from other stimuli and because IL-2R maturation. Thus, it is a potentially valuable tool for the analy- chains expression was induced only after initial activation of NBCs sis of molecular and temporal aspects of human NBCs differ- (Supplemental Fig. 1B), IL-2 was added 16 h after starting cell entiation into PCs. culture. Therefore, at point day 0 plus 16 h, cells were cultivated either in presence or absence of IL-2 (Fig. 4A). We generated lists

NBCs primed for PC commitment require IL-2 at early time point of genes differentially expressed between cell subsets (gene list; Downloaded from As previously described (10, 32), we confirmed that only highly GL). GL1 contained 2456 differentially expressed probes and proliferative B cells are primed for PC differentiation. We first characterized the initial transcriptional burst signature that takes compared the ability of several combinations of factors to promote place between day 0 and day 0 plus 16 h (Fig. 4A). The outcome proliferation during the first phase of our model. Both CpG and of this initial burst at day 0 plus 22 h and day 4 was explored by BCR cross-linking were required to induce the highest cell pro- unsupervised analysis that showed no clear and distinguishable

liferation (Fig. 3A). Omitting either CD40L or IL-2 did not sig- effect of IL-2 (Fig. 4B). Nevertheless, a global enhancement of the http://www.jimmunol.org/ nificantly affect cell division. However, at day 6 we found that mean intensity signal of the 2456 probesets was observed at CpG plus BCR alone or completed with CD40L did not allow day 0 plus 22 h for IL-2+ cells (Fig. 4C, left panel). Later on, at plasmablast generation under the second stimulation condition, day 4 the comparison of the mean intensity values between the two which included IL-2, IL-4, and IL-10. In fact, IL-2 was required in conditions showed a restricted number of 13 distinct genes with the activation phase to trigger PC differentiation (Fig. 3B). a fold change (FC) $ 2, all of them belonging to the IL-2+ con- In agreement with the demonstration that BCR triggering and dition (Fig. 4C, right panel). Among these genes, seven (CCND2, CD40 signaling could synergize with TLR9 to induce B cell acti- PHGDH, MAPKAPK3, ATF5, DUSP5, CCL3, SLC7A5) were vation (33–35), CD40L plus CpG stimulation also triggered B cell described elsewhere as preferentially expressed in plasmablasts or proliferation, but to a lower extent than CpG plus BCR (Fig. 3A). mature PCs compared with memory or activated B cells (38), by guest on September 26, 2021 Consequently, the number of cell divisions obtained with CD40L whereas some of these seven plus two more genes (LTA, IL-2RA) plus CpG was probably not sufficient to promote significantly were known as regulated by IL-2 (36, 39). plasmablast generation, even in the presence of IL-2 (Fig. 3B) (10). Kinetic analysis revealed that IL-2 needed to be added in the first IL-2 enhances the expression of genes involved in the 48 h of the culture to trigger maximum PC generation (Fig. 3Ci). MAPK–ERK signaling pathway Dose-response experiment showed that 0.5 U/ml IL-2 was suffi- GEP analysis at day 0 plus 22 h between IL-2+ and IL-22 cells cient to trigger differentiation (Supplemental Fig. 1A). Whereas (GL2) revealed 113 probes corresponding to 112 genes, all of IL-2 receptor expression was undetectable on NBCs, the three them overexpressed in the IL-2+ condition (Supplemental Table I, chains of the IL-2R, IL-2Ra (CD25), b (CD122), and g (CD132), left panel). By using Ingenuity software to identify molecular were upregulated upon stimulation with CpG, CD40L, and BCR pathways specific to GL2, only MAPK–ERK and integrin sig- triggering at the mRNA and protein levels (Supplemental Fig. 1B, naling were significantly represented (p value ,5%) (Fig. 5A). 1C). CD25 expression was further upregulated in IL-2–treated Both pathways are tightly connected, and 24 GL2 genes belonged cells at day 4, whereas CD122 and CD132 expression was only directly or indirectly to the MAPK–ERK pathway (Fig. 5B). weakly modulated by IL-2. Thus, IL-2 drives a positive feedback Notably, this pathway was further enriched by several molecules loop in B cells as IL-2 signaling increased the expression of high- related to protein and metabolism, cellular development and affinity IL-2R, an effect previously described in T cells (36, 37). movement including ERK itself after we built a specific IL-2 We next tested the role of IL-2 in the second step of the culture. In signature constituted of 334 probesets obtained after addition of absence of IL-2 between day 4 and day 6, the absolute number GL2toGL4andsubtractionofGL6probesets (Supplemental Fig. 2). of generated CD38hi cells only decreased by 20% on average We then investigated GEP at day 4 with GL3 and found 86 genes: (Supplemental Fig. 1D). Thus, IL-2 in the second step of dif- 76 overexpressed by IL-2+ cells and 10 by IL-22 cells (Supplemental ferentiation is not as crucial as IL-2 in the first step of culture to Table I, right panel). Functionally, the 76 former genes were trigger PC differentiation. IL-4 removal from the second culture connected mainly to cell growth and proliferation and cell-to-cell condition did not modify the number of generated plasmablasts at interaction (Ingenuity, data not shown). Several of these genes day 6 (Supplemental Fig. 1D). were described in previous GEP as related to preplasmablast, IL-2–mediated B cell differentiation was inhibited when we plasmablast, or even fully mature bone marrow PC compared with added in parallel anti–IL-2 or when IL-2R was blocked with anti– memory or activated B cells (http://amazonia.transcriptome.eu). IL-2Ra alone and even more efficiently with the combination of For instance, the two genes with the highest FC ratio, 5.99 and blocking Abs against the three IL-2R chains (Fig. 3Cii). Because 4.43 for GZMH and CCND2, respectively, were found upregulated T cells are the major source of IL-2, we tested whether CD4+ cells in plasmablast. These findings are in accordance with the fact that sustained PC production in our model. Indeed, CD3- and CD28- B cells committed to PC differentiation are intensively cycling, 166 IL-2–ENHANCED ERK1/2 SIGNALING AND PC DIFFERENTIATION

FIGURE 2. Transcriptional analysis of factors Downloaded from involved in B cell terminal differentiation. CD20+ CD38+ and CD20loCD38hi B cells were isolated at day 6 by FACS sorting after DAPI staining to exclude dead cells. (A) Expression of transcriptional factors in sorted CD20+CD38+ (dotted line) and CD20loCD38hi (full line) B cell subsets. Differen- tially regulated transcripts (BCL6, BACH2, PAX5, http://www.jimmunol.org/ PRDM1, MITF, POU2AF, SPI1, ETS1, IRF4, XBP1s, and SPIB) in B cells are shown relative to the day of experiment. Results of qRT-PCR analyses are expressed relative to gene expression in B cells at day 0. Bars represent mean values 6 SEM from five independent experiments. (B) Expression of AICDA, BIP, and CD27 in sorted cell fractions. Results of qRT-PCR analysis are expressed relative to gene expression in B cells at day 0. Bars repre- by guest on September 26, 2021 sent mean values 6 SEM from five independent experiments. (C) Relative expression of BCL6 and AICDA in CD20+CD38+ B cells compared with GC CXCR4+ centroblasts and tonsil memory B cells. Results are expressed using the relative DCt method. Bars represent mean values 6 SEM from five independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. D, Day.

what we demonstrated for the CD20loCD38hi cells generated be- IL-2 is required at the beginning of the cell culture to produce PCs yond day 4 (see earlier). Furthermore, at least 8 of those 76 genes suggest that IL-2 primes cells to PC differentiation by enhancing were described as upregulated by IL-2 (Supplemental Table I, MAPK–ERK signaling, which induces as soon as day 4 the ex- right panel), Altogether, these results associated with the fact that pression of genes involved in plasmablast generation. The Journal of Immunology 167

Pharmacological inhibition of MEK–ERK signaling blocks PC differentiation Considering that stimulation by IL-2 at day 2 is sufficient to trigger maximum PC differentiation and to disconnect IL-2 signal effects from other stimuli, IL-2 was added in the culture medium at day 2 for the rest of the experiments. We first compared signaling events at the protein level triggered by IL-2, IL-15, and IL-21 keeping in mind our earlier results where only the two first cytokines triggered PC differentiation. All three factors induced STAT5 phosphorylation, but only IL-2 and to a lesser extent IL-15 but not IL-21 activated ERK1/2 (Fig. 6A). Similar effects of IL-2 and IL-15 but not IL-21 on PC generation suggested a major role of the ERK1/2 pathway in cytokine-mediated PC differentiation. There- fore, we decided to use pharmacological inhibitors of ERK and STAT5 pathways at concentration and timing (day 2) where CFSE proliferation profiles were unaffected (data not shown). At day 4 and day 6, no difference in terms of cell viability was observed between all conditions. MEK inhibitors U0126 and PD184161 blocked the IL-2 property in a dose-dependent manner, whereas Downloaded from vehicle (DMSO) showed no effect on IL-2–mediated PC differentiation. In contrast, STAT5 inhibitor did not affect IL-2 effect (Fig. 6B). By Western blot we found that B cells primed by IL-2 for 24 h had an increased IL-2 phospho-ERK response compared with untreated cells. The MEK inhibitor U0126 at 0.5 mM blocked this phospho-ERK IL-2–induced response (Fig. 6C). In our model, http://www.jimmunol.org/ inhibition of IL-2–induced PC generation is only partial in the presence of MEK pharmacological inhibitors, whereas the complete absence of IL-2 abolished B cell differentiation. However, differentiation inhibition was superior when higher dose of drugs were used, but proliferation was also affected, reflecting probably drug toxicity. We could thus not exclude an additional ERK- independent role for IL-2.

IL-2–induced ERK signaling leads primarily to by guest on September 26, 2021 a downregulation of BACH2, a transcriptional repressor of PRDM1 Generation of PCs is associated with the downregulation of B cell transcription factors, PAX5, BCL6, IRF8, BACH2, and SPIB, and the upregulation of PC transcription factors, IRF4, BLIMP1, and XBP1. To understand mechanisms by which IL-2–induced ERK1/2 activation triggers PC differentiation, we sought for genes whose expression was modiﬁed in the presence of IL-2 and counteracted by PD184161 MEK–ERK inhibitor. To this end, we separated the CSFElo and CFSEhi cell fractions at day 4 from three different culture conditions: without cytokine, IL-2 added at day 2, and IL-2 in the presence of PD184161 at a concentration capable of inhibiting differentiation by 50% (Supplemental Fig. 3). Relative gene expression analyses by qRT-PCR showed a signiﬁcant de-

in presence of CpG, BCR cross-linking, and CD40L; results are expressed as absolute number of generated CD38hi cells; (ii) the presence of anti–IL-2 neutralizing Ab (“antiIL2”) or anti–IL-2 receptor a-chain Ab (“anti- IL2Ra") or anti–IL-2 receptor a, b, and g Abs (“antiIL2R”) for cells cultivated in presence of IL-2 during the activation phase; results are expressed in percentage relative to the condition with IL-2 alone, “none” FIGURE 3. Cell proliferation and IL-2 are required for PC differentia- indicating condition without IL-2; (iii) the coculture of NBCs with anti- tion. (A) Proliferation assessment comparing several combinations of CD3 and anti-CD28–activated autologous CD4+ T cells in presence of factors used for the activation phase during day 0 to day 4 on cells pre- CpG and BCR cross-linking, with anti–IL-2 neutralizing Ab (“antiIL2”) or viously treated with CSFE. Representative images of one experiment its control isotype (control Ig); (iv) the use of anti-CD3 and anti-CD28 among three realized. (B) Generation of CD20loCD38hi plasmablasts at activated autologous CD4+ T cells supernatant added to NBCs in presence day 6 according to factors used for the activation phase during day 0 to day of CpG, CD40L, and BCR cross-linking, anti–IL-2 neutralizing Ab 4. Cells of interest are in the circle (dotted line). (C) Generation of CD20lo (“antiIL2”) or anti–IL-2 receptor a, b, and g Abs (“antiIL2R”), or control CD38hi plasmablasts at day 6 according to: (i) the presence of IL-2 added isotype (control Ig). *p , 0.05, **p , 0.01, ***p , 0.001. ns, not sig- or not during the activation phase at the indicated day for B cells cultured niﬁcant. D, Day. 168 IL-2–ENHANCED ERK1/2 SIGNALING AND PC DIFFERENTIATION

FIGURE 4. IL-2–inducible GEP, impact of initial transcriptional burst, and initiation of PC differentiation at day 4. (A) Representation of the ﬂowchart for GEP analysis. For each experiment, cells were collected before RNA extraction as indicated by the red arrows at the corresponding time point. Generated GLs were obtained as described in Materials and Methods. The table on the right gives the total number of probes and the number of upregulated/downregulated probes for

each data set. (B) Unsupervised HCA clustering of the Downloaded from 2456 probes from GL1, corresponding to genes differentially expressed between day 0 and day 0 plus 16 h for NBCs in culture in presence of CpG, CD40L, and BCR cross-linking and without IL-2, leading to two main distinct clusters (upper cluster includes day 4 cells, lower cluster corresponds to cells collected at day

0 plus 22 h). (C) Scatterplot of the average expression http://www.jimmunol.org/ values per subject and per condition on the 2456 probesets, the condition being the presence or absence of IL-2 at day 0 plus 22 h; for all subjects. ***p , 0.001 (left panel). Scatterplot of pairwise global gene expression comparisons between NBCs at day 4 grown with or without IL-2; gene expression values are plotted on a log scale, and genes differentially expressed between the two groups (determined using a 5% p value and an FC $ 2) are highlighted in green (right panel). by guest on September 26, 2021 D, Day.

crease of BACH2, IRF8, PAX5, and SPIB in IL-2–primed CFSElo different between no-cytokine and IL-2–primed cells in CFSElo cells compared with the condition without IL-2 (Fig. 7A). This IL- fractions. Furthermore, BACH2 and PAX5 were less abundant in 2 effect was efficiently counteracted by PD184161. In contrast, in CFSElo compared with CFSEhi fractions suggesting posttranscrip- CFSEhi fractions—those cells are unable to differentiate further— tional BACH2 and PAX5 regulatory mechanisms linked to cell IL-2 did not modify gene expression (Fig. 7B). We finally took the division. PAX5 is a transcriptional repressor of PRDM1 and XBP1. CSFEhi gene expression level as reference to compare gene ex- This could explain the upregulation at the mRNA level of PRDM1 pression levels between cell treatments in CFSElo fractions. This and XBP1 specifically in CFSElo fractions whatever the culture analysis revealed that IL-2 effect goes through a significant conditions (Fig. 7C). downregulation of BACH2 and IRF8. Unexpectedly, PRDM1, BLIMP1 expression was not detected at day 4 (data not shown). BCL6,andXBP1s expression was significantly modulated in the However, at day 5 BLIMP1 protein was specifically detected in the CSFElo fractions whatever the considered culture conditions CFSElo fractions and was more abundant in IL-2–primed cells (Fig. 7C). than in cells activated without IL-2 (Fig. 8B). In contrast, BACH2 We then determined BACH2 protein levels at day 4 and day 5 in was no more detectable in CFSElo IL-2–primed cells consistent highly purified CFSEhi and CFSElo cell fractions. We first con- with the results obtained at day 4 regarding BACH2 mRNA ex- firmed an increase of IL-2–mediated ERK activation specifically pression levels. PAX5 was further repressed in this CFSElo IL-2– in the CFSElo fraction of cells pretreated with IL-2 at day 4 primed fraction suggesting a possible BLIMP1-dependent re- (Fig. 8A). At this time point, BACH2 protein expression was not pression of PAX5. The Journal of Immunology 169 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. IL-2 enhancement of genes within MAPK–ERK signaling after 6 h of treatment by IL-2. (A) Ingenuity’s canonical pathways analysis identified the pathways that were most significantly represented in the GL2 data set. The significance of this association was measured using Fisher’s exact test. Threshold indicates pathways that are significantly represented in the data set (p value ,5%). (B) Differentially expressed genes issued from the comparison between cells collected at day 0 plus 22 h generating the GL2 data set (112 distinct genes) that are directly or indirectly connected to the MAPK–ERK signaling pathway. 170 IL-2–ENHANCED ERK1/2 SIGNALING AND PC DIFFERENTIATION

Discussion Since the 1990s, major contributions have been made concerning GC biology and T cell-dependent B cell maturation. Several mouse models, eventually associated with intravital imaging, allowed identification of key factors and specific cell dynamics that govern normal GC reaction and GC B cell fate. At the cellular level, parsing the signals that direct cellular differentiation versus cell division remains largely uncovered. Identical remarks can be made concerning mechanisms guiding GC B cells to memory B cell differentiation or PC commitment. Although rodent models represent crucial tools to approach fundamental biological questions, they present some limitations in cell signaling, and translation of the findings to human is sometimes difficult. To address these questions, we decided to develop an in vitro model of B cell differentiation using peripheral human NBCs collected from healthy blood donors. This differentiation process includes two successive steps of culture: expansion of activated B cells in a first phase followed by PC maturation. This second step hosts the emergence of cycling CD38hi plasmablasts that give rise to PCs identified by Downloaded from CD138 expression on the cell surface; meanwhile cells undergo Ig class-switch recombination. We confirmed the successful differentiation of NBCs toward PCs by microarray GEP and immunophenotyping along the differentiation process. These analyses showed that this model recapitulated functionally cellular tran-

sitions and pinpoint crucial molecular events characteristic of http://www.jimmunol.org/ GC B cell differentiation. For instance, the BLIMP1 and BCL6 double-negative feedback loop is clearly involved in the emergence of CD38hi cells and associated with the extinction of the transcriptional program that establishes the B cell phenotype (8). The transcriptional cascade that sustains PC differentiation is highly dependent on soluble factors issued largely from T cells present in the GC microenvironment. CD40 signaling for instance may steer toward memory B cell pathway rather than PC pathway, although its effect might be dependent on the timing and the level by guest on September 26, 2021 of B cell activation (19, 40). We demonstrated that CD38hi cells appeared after CD40L withdrawal and exposure to IL-2, IL-4, and IL-10. Meanwhile, these cells continued to cycle whereas the undifferentiated CD20+CD38+ cells underwent apoptosis. This finding suggests that B cells steering toward the PC pathway result from precursors present in the human IgG-negative NBCs. The number of those precursors is highly variable from blood donor to blood donor; we noticed an unpredictable variability of PC generation throughout all the experiments although culture conditions are fixed and proliferation properties unaffected (data not shown). Finally, our data showed also that cells initiated for PC differentiation respond dramatically to mitogenic signals. Indeed, plasmablasts emerged only from cells with the lowest CSFE staining at day 4, which is consistent with previous data obtained with memory B cells (32). Interestingly, our study is, to our knowledge, the first to show that XBP1, BCL6, and PRDM1 expression levels are driven by the B cell proliferation, but that proliferation is not sufficient per se to trigger PC differentiation. The intermingled relationships between differentiation and proliferation during GC reaction underline the presence of intense FIGURE 6. IL-2 priming of B cells for PC commitment is mediated by intracellular signaling systems that remain poorly defined. In this the MEK–ERK pathway. (A) Activated day 2 NBCs were starved and stimulated for 10 min with cytokines (100 U/ml IL-2; 200 ng/ml IL-21; 50 ng/ml IL-15) or medium (Ctrl), lysed, and analyzed by Western blotting. STAT5 pathway is activated by IL-2, IL-15, and IL-21, whereas IL-2 and Differentiation was assessed by flow cytometry at day 6 based on CD38hi IL-15 but not IL-21 increased ERK1/2 phosphorylation. (B) NBCs were cells. The percentage of CD38hi cells was arbitrary fixed to 100 in the cultured in presence of IL-2 and of different concentrations of the MEK condition IL-2. n =4.*p , 0.05, **p , 0.01, ***p , 0.001. (C) Activated inhibitors PD184161 (PD184) and U0126 or STAT5 inhibitor (STAT5i) day 2 NBCs were stimulated or not by IL-2 for 24 h, starved, and stim- between day 2 and day 4 of the in vitro differentiation protocol. A control ulated for 10 min with IL-2 or medium (Ctrl), lysed, and analyzed by using the maximal DMSO concentration used as solvent for inhibitors was Western blotting. The MEK inhibitor U0126 (0.5 mM) blocks IL-2–me- included (“IL2”) as well as a culture condition without IL-2 (“none”). diated phosphorylation of ERK1/2. The Journal of Immunology 171 Downloaded from http://www.jimmunol.org/

FIGURE 8. Cell division and IL-2 priming effects on protein expression involved in PC differentiation. Immunoblot analysis of proteins from cell- by guest on September 26, 2021 sorted CFSEhi and CFSElo subsets at day 4 (A) and day 5 (B), primed with (“IL-2”) or without IL-2 (“No cytokine”). (A) Immunoblot analysis of p- ERK1/2, BACH2, and PAX5 in shortly starved cells stimulated with IL-2 for 10 min at day 4. b-Actin serves as a loading control throughout. (B) Immunoblot analysis of BLIMP1, BACH2, and PAX5 at day 5. Data are representative of two independent experiments.

study, investigations of the initial mitogenic stimulus revealed that the combination of CpG and BCR activation was sufficient and indispensable to get optimal cell proliferation but not to steer cells to the PC pathway. We demonstrated the need of an early IL-2 signal to initiate the differentiation. Along with IL-4, IL-7, IL-9, IL-15, and IL-21, IL-2 shares the common cytokine receptor g-chain. Using activated autologous CD4+ T cells or supernatant of those cells, we showed that both conditions were able to replace FIGURE 7. Effect of IL-2 on transcription factor expression in cell-sorted CD40L and IL-2 in the first mitogenic phase of our model to get CFSEhi and CFSElo subsets. IL-2 and the MEK inhibitor PD184161 (0.5mM) PC differentiation. This differentiation was suppressed when we were added at day 2. At day 4, CFSEhi and CFSElo cells of each condition blocked IL-2 or its receptor. We found also that IL-2 was not were cell-sorted and assessed for transcriptional analysis. (A) Expression of substitutable by IL-21, in contrast to IL-15, suggesting that the transcription factors in sorted CFSElo cells cultured in the presence of IL-2 IL-21 positive effect on PC differentiation may intervene later in (black bars) or IL-2 plus PD184161 (gray bars). Results of qRT-PCR anal- this process and especially in the establishment of long-lived lo yses are expressed relative to gene expression in sorted CFSE cells in the PCs (21, 41). B hi absence of IL-2. ( ) Expression of transcription factors in sorted CFSE cells Our investigations concerning the impact of IL-2 on the initial cultured in the presence of IL-2 (black bars) or IL-2 plus PD184161 (gray transcriptional burst (Fig. 4) allowed us to underline a broad en- bars). Results of qRT-PCR analyses are expressed relative to gene expression hancement of gene expression in the presence of IL-2, in agreement in sorted CFSEhi cells in the absence of IL-2. (C) Expression of transcription factors in sorted CFSElo cells cultured in the absence (white bars) or the with the fact that IL-2 is a pleiotropic cytokine with a broad array presence of IL-2 (black bars) or IL-2 plus PD184161 (gray bars). Results of of actions. However, despite this large and unspecified transcrip- qRT-PCR analyses are expressed relative to gene expression in sorted CFSEhi tional effect, we sought whether a signature of genes connected to cells in absence of IL-2. Bars represent mean values 6 SEM from five in- PC differentiation may be detectable during the mitogenic phase, dependent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. which could be consistent with descriptions made for T cells 172 IL-2–ENHANCED ERK1/2 SIGNALING AND PC DIFFERENTIATION where IL-2 helps for specific differentiated states and their ducing the expression of BCL6 and AICDA in centroblasts (54). maintenance (42, 43). After 4 d of culture, we highlighted 13 genes Taking into account all these findings, we propose to complete the issued from the initial transcriptional burst that were overexpressed previous scheme of PRDM1/BLIMP1 controlling PC differentia- in cells cultured with IL-2, among which 7 genes were already tion (55) by adding the T cell-delivered IL-2 effect, which sustains described in the plasmablastic differentiation. We confirmed those indirectly the mitogenic-induced BLIMP1 expression by down- data by the analysis at day 4 of genes differentially expressed regulating IRF8 and BACH2 via an ERK signal. This cytokine- between cells cultivated with or without IL-2 (GL3; Fig. 4A). mediated signaling is necessary early in B cell activation to allow Taken together, our data demonstrated, besides the broad impact the emergence of cells primed for PC differentiation among a of IL-2 at the transcriptional level, the emergence of genes spe- highly proliferative cell population. cifically related to the initiation of PC differentiation within highly cycling B precursors. The translation of this finding within in vivo Acknowledgments GC reaction could correspond to the step when B cells encounter We thank Joelle Dulong and the Etablissement Français du Sang of Bre- cognate T cells and start to proliferate while migrating to the tagne for providing the peripheral blood mononuclear cells, the “Institut follicle interior (3, 7). Concerning the small magnitude of the gene Fe´de´ratif de Recherche-140” of Rennes University for the cell sorting core expression differences, this might reflect heterogeneity within the facility, and the Biogenouest Genomic Platform Rennes for bioinformatics cell population and could fit with previous studies indicating that advice. a minority of GC B cells exhibits PC properties (5, 44). IL-2 is known to activate several signaling pathways including Disclosures RAS–MAPK, JAK–STAT, and PI3K/Akt/p70 S6 kinase pathways The authors have no financial conflicts of interest. Downloaded from (43). The GEP analysis between cells collected at time point day 0 plus 22 h with or without IL-2 revealed a specific and significant representation of the MAPK–ERK signaling pathway References + 1. MacLennan, I. C. 1994. Germinal centers. Annu. Rev. Immunol. 12: 117–139. in IL-2 cells. By using pharmacological inhibition with selective 2. Harwood, N. E., and F. D. Batista. 2009. 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25000 * * * 20000

15000 * 10000 ns 5000

Nb CD38hi cells /ml cells Nb CD38hi 0 none 0.05 0.5 5 50 500 IL-2 (U/ml)

B D0 D1 D2 D3 D4

CD25

CD122

CD132

C IL2RA IL2RB IL2RG / Day Day 0 / mRNA expression mRNA

** Relative number Relative cells of CD38hi

Supplemental Figure 1: IL-2 cytokine and receptor analysis

A) Dose-response assay of IL-2 cytokine and generation of absolute number of CD38hi plasmablasts per mL. (*; p<0.05). B) Specific IL-2 receptor chain expression onto NBCs get in our differentiation model at different time points; one representative experiment among 3 distinct. Grey histogram, isotype Ig; red histogram, without IL-2; blue histogram, with IL-2. C) mRNA expression of IL2 receptor chains on B cells cultured with (bold line) or without (dotted line) IL2. D) IL-4 and IL-2 effect during the differentiation phase (step 2) on plasmablast generation (N=4). Supplemental Table 1 Genelist GL2: Genelist GL3 (see also footnotes): 112 single genes (113 probes) differentially 86 single genes differentially expressed with expressed with a FC>= 1.2, at D0+22h a FC>= 1.2, at D4 between CSFElo IL2+ & CSFElo IL2- cells between IL2+ & IL2- FC: D0+22h,CFSElo, IL2+ / FC: D4-CFSElo, IL2+ / D0+22h,CFSElo, IL2- Column ID TargetID p-value eanFC (Day0+22h IL2+/Day0+22h IL Probe ID TargetID p-value CSFEloD4-CFSElo, IL2+/D4 IL2- 4480288 0.0294072 ISG20L1 1.49017 2370010 GZMH 0.0204272 5.98886 1440564 0.0475587 RUNX3 1.43492 1570348 0.0468738 4.43831 4730181 FTHL12 0.0308831 1.40699 CCND2 450468 MRPL44 0.0243757 1.40195 130021 IL2RA 0.0226678 3.3176 2970431 FTHL7 0.0252716 1.38331 1440440 MAPKAPK3 0.0202046 3.25386 4040360 ZNF483 0.0342887 1.3385 6770673 SOCS2 0.0356731 3.23171 5820681 0.0183986 PBX2 1.334 7320288 0.0279072 3.12019 4200450 G6PD 0.00534329 1.33282 CXCR4 2850433 SPSB3 0.0259232 1.33237 4850301 PTRF 0.00639325 3.11387 4760538 ZC3H3 0.0334508 1.32721 5220093 HAVCR2 0.0216012 2.91303 990358 LIMS1 0.0492346 1.32021 1030743 LTA 0.0126373 2.78672 6940202 R3HCC1 0.00288754 1.3199 270152 SLC7A5 0.048731 2.63811 2350209 YY1AP1 0.0125286 1.3135 4540328 RNF220 0.0025394 1.31002 1570484 ATF5 0.0101804 2.39129 5080482 TNPO1 0.0199022 1.30242 2490537 TNFRSF1B 0.0335157 2.20538 840544 RBM4 0.0151596 1.29834 830735 CENTA1 0.0202725 2.14856 1850047 FAM189B 0.00259803 1.289 240086 PHGDH 0.000875239 2.08106 4730739 RPS6KA1 0.0202923 1.28839 540681 S100A10 0.0179661 1.28722 1780273 ALOX5 0.0133416 1.85219 2320356 VASP 0.00140197 1.28672 2480326 HSP90B1 0.0499817 1.81744 770524 EEF1D 0.0164436 1.28622 2350538 RPS6KB2 0.00566355 1.80156 430669 0.04192 SPTLC1 1.28544 6660382 CDKN1A 0.0256676 1.7336 5890528 PPIAL4A 0.00544786 1.28255 2690561 RPLP1 0.0393095 1.2822 2230288 TST 0.0356684 1.59279 1980594 FTHL8 0.0378127 1.28027 610148 GSDMD 0.00314109 1.58116 4640435 CHD4 0.0230079 1.27476 4590494 YIF1A 0.0453352 1.57629 2810669 0.00929455 LCMT1 1.27365 5670180 TUBG1 0.0416095 1.55224 6270037 RAPGEF1 0.0130052 1.27149 6180465 0.0350301 1.53675 3800487 ZNFX1 0.00547642 1.27073 SLC2A6 3180273 PDXP 0.0315421 1.26625 1770717 HIRIP3 0.0351694 1.52434 840358 EXOSC10 0.0481548 1.26238 5130577 SLC37A4 0.0251123 1.51691 6110609 0.036946 FAM39DP 1.26222 1230187 CSNK1E 0.0109496 1.50001 5670037 MUTYH 0.0113059 1.26067 6220070 0.0179373 1.49735 3370300 CCBE1 0.0419224 1.26066 NT5C 6980156 CIAO1 0.042332 1.25733 6380370 CCND3 0.0130323 1.49312 840551 CEP27 0.0370393 1.25726 6110605 KRTCAP2 0.0277168 1.49269 60148 0.0102241 BOLA2 1.25643 2630433 CDT1 0.0187117 1.48358 3390092 0.0131578 PLEKHM2 1.2556 3890681 0.040688 1.47306 2630102 CCDC28B 0.0298375 1.25383 SLC41A3 730647 ANAPC1 0.022605 1.25373 6660270 MRPL17 0.00927692 1.4673 5310768 PAF1 0.0284634 1.2529 1500010 CDC20 0.00629866 1.46224 6380128 0.0332148 CLTA 1.25235 1990630 TRIB3 0.0410713 1.45917 3310463 0.0414889 ARHGAP15 1.25121 270736 0.040309 1.45886 2970563 RPRC1 0.00529062 1.25098 TRABD 2490730 SF4 0.0294099 1.24975 5560131 ATOX1 0.0285416 1.44303 360445 ITPA 0.0108141 1.24959 1570523 ASCC2 0.0202116 1.44225 1980301 FKTN 0.0281427 1.2488 7570711 PRR14 0.0177223 1.43536 1820722 0.0199047 APBB1IP 1.2485 1010364 0.0343938 1.42862 2470259 FBXO18 0.0246597 1.24829 DGCR6 830047 GSTP1 0.017282 1.24804 7650315 JMJD8 0.0219739 1.40776 6110035 DDX42 0.00630556 1.24799 290603 AARS 0.0227463 1.39028 6940255 PLSCR3 0.0358544 1.24544 150692 SLC26A6 0.0302638 1.38309 2100576 0.0107691 ARAF 1.24488 1850047 0.0141194 1.37806 4290196 SCRIB 0.00135044 1.24423 FAM189B 4860224 WARS 0.0129734 1.24315 4150670 CDC37 0.0256873 1.37475 3830273 NIT2 0.0243849 1.24278 6280360 SPG7 0.0110889 1.36593 6960037 EDC4 0.0469187 1.24245 6770343 ETFB 0.023446 1.36212 7210634 ZBED5 0.0405135 1.24222 60437 PLOD1 0.00983202 1.35372 4670487 SIVA 0.036272 1.24114 3990215 RPL3 0.0307856 1.24095 2030148 WDR54 0.0458358 1.35356 4290014 FLAD1 0.0237271 1.24047 2470367 INPPL1 0.0201836 1.35167 4220180 WDR68 0.0457381 1.24013 7400554 WDR18 0.0101739 1.34564 6590201 ATP6AP1 0.00889757 1.23976 1110072 PEX16 0.0262879 1.3348 3360681 TNFAIP3 0.0223769 1.23917 1300315 MRPL21 0.0299255 1.23773 3190274 LLPH 0.0125215 1.33399 380369 TRRAP 0.00784854 1.23392 5220347 BCKDK 0.0426319 1.32986 7200452 BCL2L13 0.0420196 1.23372 2350504 ECGF1 0.0462949 1.32689 3830538 0.013753 CCDC130 1.23144 3130349 CPSF1 0.0294645 1.32429 1090646 FAM119A 0.0378484 1.23114 4250372 KLHDC3 0.0426002 1.23006 2100576 ARAF 0.0225422 1.32083 6420180 TJAP1 0.0381346 1.22969 3140438 GRAMD1A 0.0390934 1.31935 360747 COASY 0.030792 1.22909 6370068 GNL1 0.0482471 1.31229 5090068 0.0331492 STX5 1.22901 2070170 UBE2L6 0.00609296 1.31117 1340689 RPN1 0.0158027 1.22896 5910128 0.015784 1.31064 650564 CCDC125 0.011742 1.22828 VAMP8 6620356 ARPP19 0.00216779 1.22806 5690228 CDK5RAP2 0.00900298 1.31049 2940451 RAB9A 0.0344138 1.2275 6860131 HNRNPUL2 0.0386839 1.30206 4540475 0.0223216 FABP5L2 1.22713 1230747 SF3A3 0.0423418 1.30188 3370274 ANKRD11 0.0339847 1.22594 1470315 0.0446092 1.28611 2650477 TRAF4 0.0189341 1.2247 PIAS4 2900594 PGD 0.0075781 1.22379 1340047 PHRF1 0.0384334 1.27307 4480162 SND1 0.0231838 1.22371 6380431 RXRB 0.0221392 1.26538 4560110 0.0137226 ARMET 1.22359 4900079 UIMC1 0.0209351 1.26418 4250753 0.00742646 TSPAN33 1.22327 3190288 0.0243864 1.26004 1170164 RPL35 0.00694207 1.22259 LYL1 20491 UBE2F 0.016096 1.22146 6110477 NUDC 0.0464155 1.25128 1300491 POLE3 0.00963564 1.21955 2510411 SLC44A4 0.0326771 1.24338 2140630 0.0236988 GLE1 1.21828 6040609 PNPT1 0.017573 1.24304 6100768 0.0221077 CYFIP2 1.21639 4730086 0.014989 1.23727 2680100 TNIP1 0.00318681 1.21623 SHCBP1 1570523 ASCC2 0.0132371 1.21608 5870452 TMEM14C 0.0260779 1.23318 6110392 GNS 0.0137419 1.21593 6900424 TYK2 0.0283992 1.23263 1410079 CDC26 0.0128702 1.21591 2450093 VCP 0.0230563 1.22859 3890300 0.0206457 PRR13 1.21453 6590661 0.025803 1.22613 5310068 NUP93 0.0318226 1.21434 MED22 4150142 SUCLG1 0.0209793 1.21322 510079 HLA-DRB4 0.0197213 -1.20783 6770242 MARCKSL1 0.00461462 1.21131 2260349 MIR1974 0.025226 -1.25214 5860162 ATP5J 0.0267964 1.21046 3710647 MXD4 0.045236 -1.26273 6660382 0.0233915 CDKN1A 1.20948 5050577 0.0255899 -1.39602 1440440 MAPKAPK3 0.0297581 1.20836 PNPLA7 4390288 TH1L 0.0327688 1.20821 5820768 DEF8 0.0499232 -1.48543 6250280 PRDX1 0.0229894 1.208 4200546 CD72 0.0410273 -1.60731 6270241 TPD52L2 0.0332761 1.20753 1820681 DPEP2 0.0271145 -1.69384 620240 ABR 0.00251076 1.20708 6620026 CD83 0.029319 -1.80214 7210224 DYNLRB1 0.0490815 1.20593 2030730 UBE2I 0.0295814 1.2054 7210192 ADA 0.0411682 -1.82966 1240593 CFLAR 0.0226573 1.20537 6180228 FCRL3 0.0477405 -1.84431 620376 PPP1R11 0.0299983 1.20421 2060082 MRPS10 0.00843868 1.20364 3850053 ANP32B 0.0203647 1.20231 6250017 TH1L 0.0134835 1.20132 5420592 SRC 0.0236376 1.20127 4570136 NHP2 0.0393187 1.20083 Supplemental Table 1 (Footnotes Right Panel)

Upregulated genes by IL-2: GZMH 1, MAPKAPK3 2, SOCS2 3, TYK2 4, ATF5 5 ,IL-2RA and LTA, plus TNFRSF1B regulated by both, IL-2 & LTA 3,6

1. Boyman O, Purton JF, Surh CD, Sprent J. Cytokines and T-cell homeostasis. Curr Opin Immunol. 2007;19:320-326. 2. Kovanen PE, Rosenwald A, Fu J, et al. Analysis of gamma c-family cytokine target genes. Identification of dual-specificity phosphatase 5 (DUSP5) as a regulator of mitogen-activated protein kinase activity in interleukin-2 signaling. J Biol Chem. 2003;278:5205-5213. 3. Marzec M, Halasa K, Kasprzycka M, et al. Differential effects of interleukin-2 and interleukin-15 versus interleukin-21 on CD4+ cutaneous T-cell lymphoma cells. Cancer Res. 2008;68:1083-1091. 4. Eckenberg R, Rose T, Moreau JL, et al. The first alpha helix of interleukin (IL)-2 folds as a homotetramer, acts as an agonist of the IL-2 receptor beta chain, and induces lymphokine- activated killer cells. J Exp Med. 2000;191:529-540. 5. Devireddy LR, Green MR. Transcriptional program of apoptosis induction following interleukin 2 deprivation: identification of RC3, a calcium/calmodulin binding protein, as a novel proapoptotic factor. Mol Cell Biol. 2003;23:4532-4541. 6. Zheng L, Trageser CL, Willerford DM, Lenardo MJ. T cell growth cytokines cause the superinduction of molecules mediating antigen-induced T lymphocyte death. J Immunol. 1998;160:763-769. GL4 A 230 334 IL-2-specific probes

161 23 2 81 152 7 GL6 GL2

Supplemental Figure 2: Extended gene list specific of the IL-2 condition obtained by microarray analysis

A) Venn diagram between the three data sets GL2, GL4 and GL6 that represent different ways how to explore gene differentially expressed after 6 hours post- treatment by IL-2 or vehicule as control for naïve B cells activated at D0 by CD40L, CpG and BCR crosslinking. The 334 probes specific for GL4 and/or GL2 and absent in GL6 may be considered as IL-2-induced.

B) Molecules involved directly or indirectly within the ERK/MAPK signaling pathway based on the 334 probes specifically induced by IL-2 after 6 hours of IL-2 treatment. Day 4 cell sorting CFSE Lo CFSE Hi Day 6 19% 43%

No IL-2 ***

23% 39% *** IL-2

21% 39% IL-2+ PD184161

Supplemental Figure 3: Experiment set up to assess the impact of IL-2-induced ERK1/2 activation on the transcriptional network involved in the PC differentiation release

Human naïve B cells from 5 distinct blood donors were used in our differentiation model using as stimulation 1 cocktail CpG, anti-IgM and CD40L following by the standard stimulation 2 conditions beyond D4. The number of generated plasmablasts (CD38hi cells) was assessed at D6 (Right Panel). Three different culture conditions were tested in parallel following the addition or not of factors at D2: no cytokine adjunction, IL-2 addition, and IL-2 addition plus MEK inhibitor PD184161 (0.5μM). At D4, CFSEhi and CFSElo cell fractions were cell-sorted before total RNA extraction.