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 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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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 Investigacio´n, Hospital Universitario Puerta del Mar, 11009 Ca´diz, of hyper-IgE syndrome, a disease characterized by impaired IL-21 † Spain; and Servicio de Inmunologı´a, 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 Andalucı´a 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 Inmunologı´a, 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).