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STAT-3 Activation by Differential Cytokines Is Critical for Human In Vivo−Generated Plasma Cell Survival and Ig Secretion

This information is current as Beatriz Rodríguez-Bayona, Ana Ramos-Amaya, Rubén of October 2, 2021. López-Blanco, Antonio Campos-Caro and José A. Brieva J Immunol 2013; 191:4996-5004; Prepublished online 7 October 2013; doi: 10.4049/jimmunol.1301559 http://www.jimmunol.org/content/191/10/4996 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2013/10/08/jimmunol.130155 Material 9.DC1 http://www.jimmunol.org/ References This article cites 51 articles, 30 of which you can access for free at: http://www.jimmunol.org/content/191/10/4996.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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

STAT-3 Activation by Differential Cytokines Is Critical for Human In Vivo–Generated Plasma Cell Survival and Ig Secretion

Beatriz Rodrı´guez-Bayona,* Ana Ramos-Amaya,* Rube´nLo´pez-Blanco,*,† Antonio Campos-Caro,* and Jose´ A. Brieva*,†

Maturation and survival of plasma cells (PCs) depends on extrinsic factors provided in specialized niches. In addition, B lymphocyte differentiation into PCs requires the activation of the JAK–STAT-3 pathway. However, whether STAT-3 is needed only during the transition of B lymphocytes to PC, or it is also involved in the survival and function of PCs at different stages of maturation, has not been unequivocally clarified. This study analyzes the effect of IL-10, IL-21, and IL-6 on human in vivo– generated PCs isolated from secondary lymphoid organs, blood (circulating, recently Ag-induced PCs), and bone marrow. PCs from these different organs show specific profiles of receptors for, and responsiveness to, these cytokines required for their survival Downloaded from and sustained Ab secretion. However, IL-10, IL-21, and IL-6 commonly induce STAT-3 phosphorylation in the three PC subsets, and all of their effects are exerted strictly through the STAT-3 activation. The inhibition or nonactivation of this pathway in the three PC populations impairs not only the effect of STAT-3–activating cytokines, but also the action of other cytokines important at the PC level, including a proliferation-induced ligand, BAFF, insulin-like growth factor 1, vascular endothelial growth factor, and stromal cell–derived factor-1a. These results indicate that STAT-3 activation is critical for human PCs throughout their maturation. The Journal of Immunology, 2013, 191: 4996–5004. http://www.jimmunol.org/

nimal models reveal that in vivo differentiation of B thermore, comparative studies of the phenotype, BLIMP1 ex- lymphocytes into plasma cells (PCs) in response to sys- pression, gene expression profiling, and IGVH gene somatic A temic Ag takes place in inductive areas (extrafollicular foci hypermutation support the view that human PCs obtained from and germinal centers) of the secondary lymphoid organs (SLOs). It tonsil (To) and lymph node (as examples of early PCs from in- is generally accepted that most of these SLO PCs have a limited life ductive SLOs), from the peripheral blood (PB) drawn at the peak span and only relatively few PCs, that is, those capable of secreting time of appearance of Ag-induced PCs (as a source of transitional high-affinity Ab, migrate through the circulation to be localized PCs), and from the BM (as terminally differentiated PCs) exhibit by guest on October 2, 2021 eventually in specialized survival niches of the bone marrow (BM), a gradient of increasing maturation in the following direction: where they become long-living PCs (1–7). SLOs→blood→BM (16–20). Besides this well-established mi- Human PCs seem to follow a progression similar to that de- gratory pathway, evidence in both humans and mice indicates that scribed in rodents. Thus, the human BM is also the main reservoir a non-negligible fraction of PCs survives in the SLOs, as well as in of mature PCs induced during systemic humoral responses, and inflamed tissues, where they continue to produce Abs for pro- these PCs do not appear to be generated in situ (8). In addition, BM longed periods (21–24). PCs exhibit greater capacity for prolonged survival and Ig secre- Differentiation of B lymphocytes into PCs in response to IL-21, tion than PCs from other organs (9–13). Moreover, 5–7 d after Ag CD40L, and other stimuli requires the activation of STAT-3; in booster immunization of healthy volunteers, most circulating PCs conjunction with IRF4, this leads to BCL-6 repression and BLIMP-1 secrete specific Abs, show features of intermediate maturity, and are transcription, two molecular events essential for this transition thought to contain the precursors of the BM PCs (14–16). Fur- (25, 26). The relevance of STAT-3 in this process is emphasized by the observation that mutations in STAT3 cause ∼60% of the cases *Unidad de Investigacioń, Hospital Universitario Puerta del Mar, 11009 Ca´diz, of hyper-IgE syndrome, a disease characterized by impaired IL-21 † Spain; and Servicio de Inmunologıá, Hospital Universitario Puerta del Mar, 11009 induction of PC differentiation and loss of PC formation in re- Ca´diz, Spain sponse to T cell–dependent Ag (27). This is consistent with the Received for publication June 12, 2013. Accepted for publication September 9, 2013. observation that Stat3 conditioned knockout in mice B cells results This study was supported by Fondo de Investigaciones Sanitarias Grants PI 08/1618 and PI 11/02193 and Junta de Andalucıá of Spain Grant CTS 02840. in a loss of T cell–dependent PCs (28). However, these studies do Address correspondence and reprints to Dr. Jose´ A. Brieva, Servicio de Inmunologıá, not clarify whether STAT-3 is required only during the transition Hospital Universitario Puerta del Mar, Avenida Ana de Viya 21, 11009 Ca´diz, Spain. of B lymphocytes into PCs, or it is also needed for PC maturation E-mail address: [email protected] and function. The online version of this article contains supplemental material. The fate of PCs is thought to depend on intrinsic Ag-recognition– Abbreviations used in this article: APRIL, a proliferation-induced ligand; BM, bone related signals (29, 30), as well as on extrinsic factors. These latter marrow; BMMC, bone marrow mononuclear cell; CT, threshold cycle; IGF, insulin- are generated in tissue niches, which provide the PCs with ap- like growth factor; MIX, mixture of IGF-1, SDF-1a, BAFF, VEGF121, and APRIL; PB, peripheral blood; PC, plasma cell; SDF, stromal cell–derived factor; sIL, soluble propriate survival factors. The different auxiliary cell types, and IL; SLO, secondary lymphoid organ; To, tonsil; VEGF, vascular endothelial growth the cytokines and other molecules reported as participants in PC factor. survival niches have been recently revised (24, 31). In a previous Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 study, we have demonstrated that human SLO, but not BM, PCs www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301559 The Journal of Immunology 4997 express IL-21R and respond to IL-21 by activating STAT-3 and containing pre-enriched populations of PCs were next obtained by im- increasing their survival (32). IL-10 induces the differentiation of munomagnetic cell selection. Finally, To, PB, and BM PCs were isolated B lymphocytes to PCs (33); nevertheless, its role in PCs has not by sorting in a FACSAria instrument (BD), based on the gating on CD38high cells (.96% purity by morphological and intracytoplasmic Ig- been clarified. IL-6 is a well-established inductive factor for hu- staining criteria). To B cells were sorted from the CD31+ fraction as man PCs (10, 16, 34, 35), although a comparison of the responsive CD19+CD38low/2 cells (32). capacity and the mechanism of action of this cytokine on PCs at different maturational stages have not been comprehensively ex- Cell culture and functional analysis amined. IL-10 and IL-6 can induce STAT-3 activation (25, 27, 36). Cell cultures were set up in a previously described medium (32), in 24- or This study shows that human PCs obtained from different organs 96-well flat-bottom culture plates (Nunc, Roskilde, Denmark), in a final m exhibit distinctive patterns of receptor expression not only for IL- volume of 1 ml and 250 l, respectively. The final concentrations of the cytokines, growth factors, neutralizing mAb, and inhibitors used in this 21, but also for IL-10 and IL-6, and that these cytokines differ- study were as follows: IL-10 (20 ng/ml); IL-6 (5 ng/ml); IL-21, sIL-6R, entially support PC survival and Ig secretion. Despite these dif- and VEGF (50 ng/ml); BAFF, SDF-1a,andIGF-1(100ng/ml);mega- ferences, all these effects are mediated through the activation of the APRIL (1 mg/ml); anti–IL-6 mAb (100 ng/ml); and stattic and rux- JAK–STAT-3 pathway. Moreover, the inhibition or nonactivation olitinib (1 mM). Indicated PC cultures were treated with a combination of a of this pathway prevents not only the effect of STAT-3–inducing PC-inducing factors consisting of a mixture of IGF-1, SDF-1 , BAFF, VEGF121, and APRIL (MIX). This cytokine combination and their cor- cytokines, but also the functioning of other relevant cytokines responding concentrations were found optimal in preliminary experiments operative at the PC level including a proliferation-induced ligand of PC Ig secretion. The elimination of either of these factors partially (APRIL), BAFF, insulin-like growth factor (IGF)-1, vascular en- reduced the effect of MIX on the three PC populations under study. Flow high dothelial growth factor (VEGF), and stromal cell–derived factor cytometry studies on CD38 cells (PCs), including detection of PC a proliferation, STATs phosphorylation, and apoptosis determined by la- Downloaded from (SDF)-1 . These results demonstrate unambiguously that STAT-3 beling active caspases with FAM-VAD-fmk (1 mM), and ELISA detection activation is essential for human PCs. of Ig secretion were performed as previously reported (16, 32). IL-6 and sIL-6R production were also determined in the culture supernatants of To and PB non-T cells and BMMCs by ELISA. The presence of IL-6 was Materials and Methods measured in the supernatants of PC cultures by a high-sensitivity ELISA. Human samples Ig and cytokine secretion data included in this study represented active cell production, because, in every experiment, the addition of cycloheximide

To were obtained from patients undergoing tonsillectomy for recurrent http://www.jimmunol.org/ (10 mg/ml) inhibited .90% of the secretion. For Ig-production experi- tonsillitis. PB samples were obtained from healthy donors 6 d after receiving ments, To and PB non-T cells and BMMCs were cultured at 1.25 3 105 a conventional booster immunization against tetanus-diphtheria toxoids and cells/250 ml for 7 d and 2.5 3 105 cells/250 ml for 14 d, respectively. acellular pertussis (Boostrix; GlaxoSmithKline Biologicals SA, Rixensart, Puriﬁed PCs were cultured at 104 cells/250 ml for 7 (To and PB) and Belgium). At this time point, most circulating CD38high cells (PCs) still 14 d (BM). Proliferation assays were carried out on the To CD31+ non- exhibit a high expression of HLA-DR (Supplemental Fig. 2A), a clear Tcells(106 cells/ml). For apoptosis experiments, PCs were cultured at indication of their recent entry in the PC maturational program (7, 15, 16). 5 3 104 cells/250 mlandBMMCsat106 cells/ml. For analysis of In addition, @60% of the IgG secreted in culture by these cells was anti- STAT3-phosphorylation, 2 3 106 cells/ml of To and PB non-T cells tetanus toxoid IgG. BM specimens showing no abnormalities were collected and BMMCs were used. from discarded BM aspirates obtained for diagnostic purposes. All studies were approved by the Institutional Review Board (Comite´ E´tico del Hospital

Analysis of mRNA expression by guest on October 2, 2021 Universitario Puerta del Mar), and informed consent was obtained according to the Declaration of Helsinki. mRNA for IL-10R1 present in purified To and BM PCs was quantified by real-time PCR. Total RNA was reverse-transcribed to cDNA and then Reagents and Abs amplified using the TaqMan probes assay (Hs00155485_m1; Applied The following mAbs were used: purified anti–IL-6, PerCP-Cy5.5– Biosystems, Foster City, CA). Threshold cycle (CT) for IL10R1 was nor- anti-CD38, PE–anti–IL-10R1 (CD210), –anti-CD126, –anti-CD130, –anti- malized to the CT of a control gene GAPDH (Hs99999905_m1; Applied TACI, –anti–IGF-1R, Alexa Fluor 647–anti–phospho-STAT1 (pY701), –anti– Biosystems). The relative quantitative values were calculated using the 2- DD phospho-STAT3 (pY705), and –anti–phospho-STAT5 (pY694), and CT method. Gene expression of TNFRSF17 (BCMA), TNFRSF13B isotype-matched controls (BD Pharmingen, San Diego, CA); PE–anti– (TACI), and TNFRSF13C (BAFFR) by To, PB, and BM PCs was deter- BAFFR (Biolegend, San Diego, CA); PE–anti-VEGF R1, –anti-VEGF R2 mined by microarrays assays. In brief, isolated PCs were pelleted and lysed and –anti-VEGF R3 and goat PE–anti-BCMA polyclonal Ab and isotype- with SuperAmp buffer (Miltenyi Biotec, Bergisch Gladbach, Germany) 2 matched controls (R&D Systems, Minneapolis, MN); FITC–anti-CD38, before being stored at 80˚C. RNA isolation and amplification were PE– and allophycocyanin–anti-CD19, allophycocyanin–anti-CD31, and performed according to Super Amp Services, and gene expression analysis magnetic microbeads bound to anti-allophycocyanin, anti-CD27, and –anti- was performed by One-color Agilent Whole Human Genome Oligo 3 CD138, and MS and LS columns for immunomagnetic selection were Microarrays 4 44K Microarrays Service (Miltenyi Biotec). Data are provided by Miltenyi Biotec (Auburn, CA). IL-6, IL-21, IL-10, BAFF, represented as relative quantitation normalized to average gene expression level in BM PCs (average BM = 1). SDF-1a,IGF-1,VEGF121, and soluble IL (sIL)-6R were purchased from PeproTech (London, U.K.). Mega-APRIL was provided by AdipoGen (Liestal, Switzerland). Goat unconjugated and peroxidase-conjugated anti- Statistical analysis human IgG, IgM, and IgA Abs used for ELISA were purchased from Statistical differences were established by the nonparametric Mann– Biosource (Camarillo, CA). IL-6, sIL-6R, and IL-6 high-sensitivity ELISAs Whitney U test (two-tailed) or Wilcoxon test for unpaired or paired samples, were provided by eBioscience (Vienna, Austria). STAT3 inhibitor V, Stattic, respectively. Results are generally expressed as the mean and SEM. and JAK2 inhibitor InSolution AG490 were provided by EMD Millipore (Darmstadt, Germany) and the JAK 1, 2 inhibitor ruxolitinib was purchased from SelleckBio (Houston, TX). FAM-VAD-fmk (carboxyfluorescein- Results Val-Ala-Asp OMe-fluoromethyl ketone) was provided by SM Biochemicals Effect of IL-10 on human in vivo–generated PCs (Anaheim, CA). The BrdU Flow Kit (BD Pharmingen) was used to detect proliferating cells. Previous studies have revealed that IL-10 stimulates Ig production by human spleen PCs (22). Accordingly, the possible role of IL-10 Cell preparation and cell sorting was examined in human in vivo–generated PCs obtained from To were mechanically disrupted and the resulting cell suspension was To (as an example of SLO PCs), from the blood 6 d after a po- + depleted of T lymphocytes (11) (To non-T cells). Subsequently, CD31 tent booster immunization (as transitional PCs), and from BM (as cells were purified from the To non-T cell fraction by immunomagnetic terminally differentiated PCs) (17). As can be seen in Fig. 1A (left cell selection to pre-enrich PC (17, 32). PB and BM mononuclear cells high (BMMCs) were isolated by density gradient centrifugation, and PBMCs panel), human PCs can be defined as CD38 cells (17, 32). Fig. were T cell–depleted. PB non-T CD27+ and BMMC CD138+ cell fractions 1A (right panel) and 1B show that human PCs differed in their 4998 PLASMA CELL MATURATION REQUIRES STAT-3 Downloaded from

FIGURE 1. Expression of IL-10R on and response to IL-10 by human To, PB, and BM PCs. (A) PCs were identified as CD19+CD38high cells, for To and PB, and as CD19+/2 CD38high cells for BM PCs (left panel). IL-10R expression was analyzed and representative histograms for To, PB, and BM PCs are depicted (right panel). Isotype-matched controls (shaded area) are included. (B) The bar graph summarizes the data of IL-10R expression in several experiments (mean fluorescence intensity [MFI]; n = 6, for each organ). (C) m-RNA for IL-10R1 was quantified by real-time PCR in To (n = 4) and BM (n = 4) PCs. (D) Effect of IL-10 on the IgG secretion by To, PB, and BM PCs cultured for 7 d. Values were calculated as the percentage of the IgG production in nonstimulated (CTRL) cultures of PCs isolated from To (180.14 6 27.73 ng/ml; n = 29), PB (153.8 6 28.81 ng/ml; n = 16), and BM (1787.91 6 488.71 ng/ http://www.jimmunol.org/ ml; n = 11). (E) Effect of IL-10 on the kinetics of IgG secretion by To PCs. Values were calculated as in (D). Control IgG production was 232.57 6 67.94 ng/ml (n = 7). (F) IL-10–induced proliferation on To PCs (after 48 h). Representative histograms of one of three independent experiments are depicted (upper panel). As a positive control (lower panel), blood B lymphocytes were polyclonally activated with 5 mM ODN 2216 (Invivogen, San Diego, CA) plus IL-21 during 6 d, and BrdU incorporation for the last 24 h was detected in CD38high plasmablasts. (G–I) Effect of IL-10 on the percentage of apoptotic To PCs (after 48 h). Histograms of one representative of six similar experiments are depicted in (G). Graphs in (H) and (I), respectively, show the results of apoptotic PCs and of the number of viable PCs recovered in all the experiments. In (B)–(E), data represent mean 6 SEM. *p , 0.05, **p , 0.001. surface IL-10R expression: To PCs clearly expressed this receptor, rations for endogenous IL-6 generation (37) (Supplemental Fig. whereas blood and BM PCs exhibited lower levels of expression. 2B). The effect of this endogenous IL-6 on the IgG secretion by by guest on October 2, 2021 This finding was confirmed at the mRNA level (Fig. 1C). The the PCs contained in To and PB non-T cells and in BMMCs is relevance of these observations was tested on the capacity for IgG revealed by the marked, but differential, inhibitory effect of adding secretion of purified PC cultures. As shown in Fig. 1D, IL-10 saturating quantities of neutralizing anti–IL-6 mAbs (Supplemental increased PC IgG-secretion, although the greatest effect was ob- Fig. 2C, 2D). These differences in IL-6–responsiveness (Supple- served in To PCs (a mean increase of 3- to 4-fold). The induction mental Fig. 2D) were then analyzed in purified PC preparations. of IL-10 on To PC Ig secretion was concentration dependent Fig. 2C shows that the blockade of endogenous IL-6 did not modify (Supplemental Fig. 1A), and this effect was similar for the three To PC IgG secretion, whereas it inhibited 50% of IgG production by main Ig isotypes (Supplemental Fig. 1B). IL-10 exerted its effect blood and BM PCs (p , 0.007). These observations were consistent during the first 4–5 d of culture (Fig. 1E). IL-10 treatment of with the occurrence of marginal, but detectable, IL-6 secretion in purified To B lymphocytes neither induced substantial IgG se- cultures of the majority of blood and BM PC samples, but not, or cretion nor triggered the appearance of CD38high cells (Supple- less often, in cultures of To PCs (Fig. 2D). PCs, or more probably, mental Fig. 1C and 1D, respectively). Therefore, the IL-10 induction a few stromal cells contaminating the purified PC preparations of IgG secretion was not due to the differentiation into PCs of could be the source of endogenous IL-6 secretion in these cultures. B lymphocytes eventually contaminating the PC preparation. Ad- This was not investigated further. The inhibitory effect of anti–IL-6 dition of IL-10 did not modify the nonproliferating status of To PCs mAb on the IgG production by purified PCs was specific, because after 24 (data not shown) and 48 h of culture (Fig. 1F). Similar the addition of IL-6 reversed this effect (Supplemental Fig. 2F). results were obtained with isolated To PCs (data not shown). How- Moreover, the addition of exogenous IL-6 (5 ng/ml) increased IgG ever, IL-10 addition reduced the tendency of To PCs to undergo secretion by all purified PCs, although the effect was greater in apoptosis as determined by FAM-VAD-fmk staining (Fig. 1G, 1H) blood and BM PCs (Fig. 2E). Addition of higher concentrations of and FITC–Annexin V staining (data not shown). The recovery of IL-6 did not provoke greater PC responses (data not shown). Ki- viable To PCs was clearly increased in the presence of IL-10, netics analysis revealed that IL-6–mediated IgG secretion occurred confirming this finding (Fig. 1I). The effect of IL-10 on lymph during 7 and 14 d, for PB and BM PCs, respectively (Supplemental node PCs was similar to that described for To PCs (data not shown). Fig. 2G). A graph was produced by combining the effect exerted by endogenous (Fig. 2C) and exogenous (Fig. 2E) IL-6 in this system. Effect of IL-6 on human in vivo–generated PCs As can be seen, the IL-6 responsiveness of blood and BM PCs was Fig. 2A and 2B show that the expression of CD126 and CD130, 2.5 to 3.5 times higher than that of To PCs (Fig. 2F). No difference the two IL6R components, was markedly higher in circulating Ag- was observed in the expression of CD126 and CD130 by BM PCs induced PCs. One difficulty faced in analyzing the effect of IL-6 cultured for 48 h in the presence and in the absence of IL-6. The on human PCs is the capacity of different lymphoid cell prepa- effect of IL-6 on lymph node PCs was similar to that observed on The Journal of Immunology 4999 Downloaded from http://www.jimmunol.org/

FIGURE 2. To, PB, and BM PCs exhibit distinctive patterns of response to IL-6. (A) Representative histograms showing CD126 and CD130 expression by To, PB, and BM PCs. Isotype-matched controls (shaded area) are included. (B) The graphs summarize all the experiments of CD126 and CD130 expression by PCs expressed as mean ﬂuorescence intensity (MFI; CD126: nTo = 12; nPB =6;nBM = 7; CD130: nTo =5;nPB =7;nBM = 7). (C) Effect of adding neutralizing anti–IL-6 mAb on IgG secretion by isolated PCs. (D) Endogenous IL-6 production by isolated PCs. (E) Effect of adding exogenous IL-6 on IgG secretion by To, PB, and BM PCs. In (C) and (E), IgG production was calculated as the percentage of the control (CTRL) cultures of puriﬁed PCs from To (251.2 6 52.81 ng/ml; n = 5), PB (83.67 6 20 ng/ml; n = 6), and BM (1474.00 6 416.98 ng/ml; n = 7). (F) An IL-6 response index was obtained by combining the data contained in (C) and (E) as follows: the IL-6–independent IgG production (non–IL-6) is that observed in the absence of endogenous IL-6 (PCs cultured with anti–IL-6 mAb); this value is considered as 1 (F, gray color). The other fractions of IgG production are proportionally adjusted to this by guest on October 2, 2021 unity for each PC population. The fraction of IgG production dependent on endogenous IL-6 (end–IL-6) represents the IgG fraction inhibited by neutralizing anti–IL-6 mAb (F, black color). Exo–IL-6 represents the IgG production in the presence of exogenous IL-6 (F, white color). In (B), (C), and (E), data represent mean 6 SEM. In (D), mean is represented by a horizontal bar. *p , 0.05, **p , 0.001.

To PCs (data not shown). A possible role of endogenous sIL-6R cordantly, a clear decrease of the PC recovery was simultaneously (38) in these experiments was not conﬁrmed in vitro (Supplemental observed in the same cultures (Fig. 3C). Moreover, the addition of Fig. 2H, 2I). IL-6 reduced the apoptosis observed in cultures of puriﬁed PB Fig. 3A and 3B show that caspase activation in cultures of blood PCs (see Fig. 4E). In contrast, the addition of anti–IL-6 mAb dur- PCs was clearly increased when endogenous IL-6 was neutralized ing a similar period did not provoke a consistent change in the by adding anti–IL-6 mAb after 2 d of culture. Similar results were viability of BM PCs (Fig. 3D–F). A similar lack of effect of IL-6 observed with FITC–Annexin V staining (data not shown). Con- neutralization was obtained when BM PCs were examined after

FIGURE 3. Cellular mechanism of IL-6 inductive effect on blood and BM PCs. The effect of endogenous IL-6 on PB and BM PCs survival was explored. PB and BM PCs were cultured for 48 h in the presence and in the absence of neutralizing anti–IL-6 mAb, and the percentage of apoptotic PCs was determined. (A) His- tograms of one representative experiment carried out on PB PCs are depicted. The results of apoptotic PB PCs (B) and the number of viable PB PCs (C) recovered in six experiments. (D) Histograms of one of four independent experiments on BM PCs are depicted. The results of apoptotic BM PCs (E) and the number of viable BM PCs (F) recovered in all the experiments. (G) BMMCs were cultured for 1 wk in the absence and in the presence of neutralizing anti–IL-6 mAb, and the percentage of apoptotic PCs and the number of recovered PCs (numbers inserted in square) were determined as in (B) and (C). One of two similar experiments is shown. *p , 0.05. 5000 PLASMA CELL MATURATION REQUIRES STAT-3 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. Cytokine-mediated distinctive induction of To, PB, and BM PCs show a common dependency on STAT-3 phosphorylation. (A) Effect on STAT-3 activation of IL-10 in To PCs and of IL-6 on PB and BM PCs. To and PB non-T cells and BMMCs were cultured with cytokines indicated and STAT-3 phosphorylation was assessed. Histograms show temporal kinetics of p-STAT-3 labeling after cytokine addition. Shaded areas represent the labeling of matched nonstimulated cultures. Results of one of three independent and similar experiments are shown for every PC subset. (B and C) Effect of stattic and ruxolitinib on STAT-3 phosphorylation in cytokine-induced PCs after 15 min. Histograms are representative of three independent and similar experiments for To, PB, and BM. Shaded areas represent labeling of cells cultured in medium alone. (D) Effect of stattic and ruxolitinib on IgG secretion by cytokine-induced cultures of isolated PCs after 7 d. Values are expressed as the percentage of the control (CTRL) culture production of PCs from To (251.0 6 50.5 ng/ml; n = 5), PB (390.4 6 210.1 ng/ml; n = 7), and BM (1372.2 6 341.7 ng/ml; n = 5). Data represent mean 6 SEM. (E) Effect of ruxolitinib and stattic on cytokine-induced survival of isolated PCs after 48 h of culture. Results are expressed as percentage of apoptotic PCs. Histograms contain data of one of three independent experiments showing similar results for each PC population. #p , 0.05 different from all the other sets of data, *p , 0.05 different from the control set of data.

7 d of culture (Fig. 2G), indicating that BM PCs did not require These data suggest that IL-21 and IL-10 have redundant effects IL-6 for initial survival. on To PCs. Phosphorylation of STAT-1 and -5 was not elicited in these PC cultures (data not shown). The kinetics of p-STAT-3 Appropriated induction of human PCs by IL-10, IL-21, and appearance after the addition of IL-6 to blood and BM PCs also IL-6 depends on STAT-3 activation followed a similar timeframe (Fig. 4A). STAT-3 activation in these Fig. 4A shows that IL-10 provoked a rapid (detectable in 15 m) PCs is specific, as it was prevented by anti–IL-6 mAb addition and transient appearance of p-STAT-3 in To PCs. This result was (Supplemental Fig. 3A). similar to that obtained with IL-21 (32), as well as with the com- The role of cytokine-induced STAT-3 activation on PCs was bination of IL-21 plus IL-10 (data not shown), a result equivalent to explored by using specific inhibitors of the STAT-3 phosphorylation that observed in To PC IgG production (Supplemental Fig. 1E). (stattic), and of the function of several JAK members (ruxolitinib), The Journal of Immunology 5001 which are required for upstream steps of this pathway. Fig. 4B shows that the inclusion of each of these inhibitors impaired IL-10 plus IL-21 induction of p-STAT-3 in To PCs. This inhibitory effect was specific, because the inclusion of AG490, an inhibitor of JAK2, which does not participate in the STAT-3 activation induced by IL-10 or IL-21, did not impair STAT-3 phosphorylation provoked by these cytokines (Supplemental Fig. 3B). Similarly to To PCs, the p-STAT-3 response induced by IL-6 in blood and BM PCs was also blocked by these inhibitors (Fig. 4C). The impairment of STAT-3 activation by these inhibitors also stopped the increasing effect on IgG secretion induced by IL-10 plus IL-21 in To PCs, and by IL-6 in blood and BM PCs (Fig. 4D). The inclusion of the inhibitors alone provoked a reduction of the control IgG secretion by PCs (Fig. 4D). This effect was probably due to the blockade of reduced quantities of STAT-3 activation caused by the residual presence in the cultures of endogenous STAT-3–activating cytokines. In this regard, in the absence of exogenous cytokine addition, a reduced but detectable level of STAT-3 phosphorylation (when compared with the isotypic negative control) could be observed in some samples, mainly of blood PCs. In fact, this inhibition Downloaded from of IgG secretion was similar to that obtained when endogenous IL-6 was neutralized in blood and BM PC cultures (Fig. 2C and Sup- plemental Fig. 2G). Finally, the blockade of the JAK–STAT-3 pathway by stattic or ruxolitinib in these cultures also impeded the viability improvement caused by IL-10 plus IL-21 in To PCs, andbyIL-6inbloodPCs(Fig.4E). http://www.jimmunol.org/ The inhibition of STAT-3 in human PCs prevents the inductive effect of other cytokines active at the PC level The expression of the receptors for APRIL, BAFF, IGF-1, VEGF, FIGURE 5. Expression of BCMA, TACI, and BAFFR on human PCs. and SDF-1a by human PCs was explored by flow cytometry. As The presence of these receptors was examined in PCs from To, PB, and A shown in Fig. 5A and 5B, BCMA and TACI, two common re- BM. ( ) Representative histograms showing BCMA, TACI, and BAFFR B ceptors for BAFF and APRIL, and BAFFR, a receptor for BAFF expression by human PCs. ( ) Graphs summarize the results obtained in several experiments (expressed as mean fluorescence intensity [MFI]; alone, were heterogeneously expressed by PCs from different BCMA: n =6,n =5,n = 5; TACI: n =8,n =4,n =5; by guest on October 2, 2021 organs. Thus, BCMA was clearly shown by To and PB PCs, but To PB BM To PB BM BAFFR: nTo =3,nPB =5,nBM = 6, for To, PB, and BM PCs, respectively). poorly expressed in BM PCs. In addition, To PCs did not express Data represent mean 6 SEM. (C) Quantification of mRNA for TNFRSF17 TACI, whereas PB and BM PCs did express this receptor. Finally, (BCMA), TNFRSF13B (TACI), and TNFRSF13C (BAFFR) by To (n = 7), BAFFR was highly expressed in To PCs, but its expression was PB (n = 6), and BM (n = 7) PCs is shown. Mean is represented by hori- lower in PB and BM PCs. These results were confirmed at the zontal bars. *p , 0.05, **p , 0.001. mRNA level (Fig. 5C). Low expression of IGF-1R and VEGF receptors were detectable only in BM PCs (Supplemental Fig. 4). The expression on human PCs of CXCR4, the receptor for SDF-1a,has this activity still remained in cultures of BM PCs after 21 d (Fig. been previously reported to be higher in BM PCs compared with 6A). Subsequently, the requirement for STAT-3 activation in the that observed in To and PB PCs (17). The incubation of blood and MIX-inducing effect was investigated. Fig. 6B shows that the BM PCs during 48 h with IL-6 did not modify their expression of addition of MIX alone induced a 2-fold increase of IgG secretion these receptors. Lymph node PCs show a profile of receptor ex- in To and blood PCs, and a 3-fold increase of IgG secretion in BM pression identical to that of To PCs (data not shown). PCs. In addition, as shown in Fig. 6A, the treatment of To PC The effect of the mixture of APRIL, BAFF, IGF-1, VEGF, and cultures with MIX in the presence of IL-21 plus IL-10 provoked SDF-1a (MIX) on PCs was explored. Initial experiments dem- an increase of the IgG secretion additional to that caused by the onstrated that the treatment with MIX neither induced STAT-3 latter cytokines. A similar finding was observed when MIX was phosphorylation by itself nor modified the STAT-3 phosphoryla- added to IL-6–stimulated cultures of blood and BM PCs. Finally, tion provoked by appropriated STAT-3–activating cytokines in the Fig. 6B also shows that STAT-3 inhibition by stattic completely three human PC subsets (Supplemental Fig. 3C). In addition, MIX abrogated the effect of STAT-3–activating cytokines, MIX, and the did not increase endogenous IL-6 production in blood (1.3 6 1.1 combination of both. A possible explanation for these observa- and 0.93 6 0.57 pg/ml, control and MIX, respectively; mean 6 tions could be that STAT-3 activation is a prerequisite for MIX SEM; n = 3) and BM (4.6 6 3.1 and 5.1 6 4.45 pg/ml, control and function. To confirm this possibility, we assessed the effect of elim- MIX, respectively; mean 6 SEM; n = 3) PC cultures. The effect of inating residual endogenous STAT-3–activating cytokines on MIX- MIX combined with appropriated STAT-3–activating cytokines on induced PC IgG secretion. Fig. 6C shows that the blockade of IgG secretion by human PCs was examined next. Kinetics studies endogenous IL-6 by anti–IL-6 mAb in PB and BM PC cultures revealed that the inclusion of MIX in cultures of the three PC prevented most of the inductive effect of MIX on PC IgG secretion. populations provoked a clear increase of IgG secretion additional to that induced by the corresponding STAT-3–activating cytokines Discussion (Fig. 6A). The MIX-dependent effect on PC IgG secretion ceased Present results demonstrate that IL-10 exerts a direct effect on by days 10 and 14 for To and blood PCs, respectively; however, human in vivo–generated PCs leading to increased IgG secretion. 5002 PLASMA CELL MATURATION REQUIRES STAT-3

FIGURE 6. Inhibition of the JAK/ STAT-3 pathway prevents the inductive effect of other cytokines active at the PC level. (A) Kinetics studies of IgG secretion by isolated To, PB, and BM PCs under the induction of STAT- 3–activating cytokines and a combination of additional PC-inducing factors (MIX) consisting of IGF-1, SDF- 1a, BAFF, VEGF, and APRIL. One experiment for each PC population is shown out of three independent experiments for every organ showing similar results. (B) Isolated PCs were culturedinmediumaloneandinthe presence and in the absence of the appropriate STAT-3–activating cyto-

kine (as in Fig. 4D), MIX, and stattic. Downloaded from Values are expressed as the percentage of the control (CTRL) IgG production by PCs from To (383.4 6 86.9 ng/ml; n = 8), PB (331.8 6 116.3 ng/ml; n = 6), and BM (1838.4 6 540.7 ng/ml; n =5).(C) PB and BM isolated PCs

were also cultured in medium alone http://www.jimmunol.org/ and in the presence and in the absence of neutralizing anti–IL-6 mAb and MIX. Values are expressed as the percentage of the control (CTRL) culture IgG production by PCs from PB (365.5 6 108.1 ng/ml; n =6)andBM (1293.0 6 540.7 ng/ml; n = 5). In (B) and (C), data represent mean 6 SEM. #p , 0.05 different from all the other set of data, *p , 0.05 different from by guest on October 2, 2021 the control set of data.

This effect is heterogeneous and apparently depends on the level The analysis of the precise role of IL-6 in human PCs is hampered of receptor expression. Thus, IL-10R expression and IL-10 re- by the common occurrence of endogenous IL-6 production in the sponsiveness by To PCs is clearly higher than that shown by blood majority of PC preparations. In this study, the combined assessment and BM PCs. The cellular mechanism underlying this effect is an of endogenous IL-6 blockade and exogenous IL-6 addition on the IL-10–mediated increase in To PC survival. The physiological rel- same PC preparations has allowed a more complete examination of evance of this observation is unknown. It is well-established that IL- its role. IL-6 effect in human PCs shows a distribution markedly 10 can be produced by several cell types present in normal tissues, different from that observed for IL-10 and IL-21. To and lymph as well as in inflammatory situations (39). It is known that PCs can node PCs show low levels of CD126 and CD130 expression, and migrate into inflamed tissues and survive there (24, 40). It could a modest response to this cytokine. In contrast, blood PCs exhibit be hypothesized that IL-10, combined with other cytokines, might levels of expression of both molecules and IL-6–mediated IgG- contribute to PC survival in inflamed territories. In fact, studies in secretion increase several times higher than those shown by SLO a mice model of liver inflammation reveal that the blockade of IL- PCs. IL-6 effect on blood PCs is also mediated through increased 10R leads to the development of uncontrolled disease because of survival. Interestingly, BM PCs show a low level of expression a lack of adequate PC recruitment within the liver (41). of the two IL-6R components but respond to IL-6 as strongly as The pattern of IL-10 responsiveness exhibited by human PCs is blood PCs do. The reason for this is uncertain. BMMCs distinc- similar to that previously demonstrated for IL-21 (32). Thus, To tively produce considerable quantities of sIL-6R (Supplemental PCs respond well to IL-21 and IL-10, whereas mature BM PCs Fig. 2G). Unfortunately, we were unable to demonstrate that BM have a poor response to both cytokines. The inductive effect of PCs have the capacity of responding to sIL-6R by increasing their these cytokines is redundantly exerted through increasing To PC IL-6–mediated IgG secretion (Supplemental Fig. 2I). Neverthe- survival. This pattern of response may extend to human lymph node less, these data do not totally exclude a trans-activating role for PCs (32 and data not shown). These similarities probably indicate sIL-6R on human BM PCs in vivo. IL-6 effect on BM PCs also that PCs from human SLOs, consisting mainly of early PCs (17), differs from that described for blood PCs, because that cytokine have reached an equivalent level of maturation and functional does not induce increased BM PC viability, but these cells do capacity. appear to require IL-6 to sustain IgG secretion. A similar effect of The Journal of Immunology 5003

IL-6 on IgG secretion has also been reported for mice BM PCs a cytokine milieu close to that occurring in PC survival niches (42), which suggests that IL-6 plays a general role in the main- in vivo. Present data reveal that the inductive effect of MIX on the tenance of IgG production by terminally differentiated BM PCs. three human PC populations is lost when the STAT-3 pathway is Collectively, these observations indicate that, during their phys- inhibited. Furthermore, the addition of all these factors was in- iological progression, human PCs exhibit distinctive patterns of capable of reversing the IgG-secretion blockade observed after receptor expression and preferential responsiveness to IL-10, IL-21, endogenous IL-6 neutralization in blood and BM PC cultures. and IL-6. This fact might reflect the development of specific ad- Therefore, STAT-3 activation appears to be necessary for enabling aptation of PCs to the functional requirements present in particular the functioning of other cytokines required for PC acquisition of organs and maturational stages. Thus, human SLO PCs predominantly the long-living status; this would indicate that STAT-3 activation is respond to IL-21 and IL-10 by increasing their survival; the two a central event throughout the PC maturation. In this context, it cytokines might be provided to these PCs in vivo by follicular Th has recently been demonstrated in several cell models that STAT cells (23, 32, 43–45) and by regulatory cells in inflammatory sit- components are responsible for connecting environmental cyto- uations (24, 39). In contrast, blood Ag-induced PCs preferentially kine signaling with specialized active gene enhancer profiles re- depend on IL-6 for survival, a mechanism that might give to these quired for particular programs of differentiating cells (50). Present PCs an advantage for homing onto BM survival niches, where IL-6 data suggest that STAT-3 might play this role in human PC dif- is a well-known component (10, 34–37). Finally, mature BM PCs ferentiation. show low expression of receptors for and limited response to IL-10 Finally, inhibitors of the JAK–STAT-3 pathway are under cur- and IL-21, but, to maintain prolonged Ig secretion, require IL-6 rent investigation for their possible use in the treatment of certain produced by auxiliary BM cells (10, 34–37, 46). diseases (51). Present results raise the possibility that these ther- The relevant role of STAT-3 in the differentiation of B lympho- apeutic tools might be beneficial in diseases involving long-living Downloaded from cytes into PCs is a well-established event (25–27). IL-21–induced PCs capable of producing pathogenic Ab. increased survival in human SLO PCs is associated with PC STAT-3 activation (32). Present results reveal that IL-10 effect Disclosures on To PCs and IL-6 effect on blood and BM PCs also activate The authors have no financial conflicts of interest. this signaling route. This supports the notion that, although these cytokines can elicit a variety of signaling pathways, STAT-3 acti- http://www.jimmunol.org/ vation seems to play a prominent role in the PC response to these References cytokines. Moreover, the inhibition either of STAT-3 or of required 1. Liu, Y. J., J. Zhang, P. J. Lane, E. Y. Chan, and I. C. MacLennan. 1991. Sites of specific B cell activation in primary and secondary responses to T cell-dependent upstream JAK components leads to a complete blockade of the and T cell-independent antigens. Eur. J. Immunol. 21: 2951–2962. effects of IL-10 and IL-21 on To PCs and of IL-6 on blood and BM 2. Smith, K. G., T. D. Hewitson, G. J. Nossal, and D. M. Tarlinton. 1996. The phenotype and fate of the antibody-forming cells of the splenic foci. Eur. J. PCs. These results indicate that, despite showing distinctive patterns Immunol. 26: 444–448. of cytokine responsiveness, STAT-3 activation is a common mech- 3. Manz, R. A., A. Thiel, and A. Radbruch. 1997. Lifetime of plasma cells in the anism present in human PCs during various different stages of bone marrow. Nature 388: 133–134. 4. Smith, K. G., A. Light, G. J. Nossal, and D. M. Tarlinton. 1997. The extent their physiological maturation. Therefore, this study unequivocally

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