Plasma Cell Homeostasis: The Effects of Chronic Antigen Stimulation and Inflammation This information is current as Tom Slocombe, Sheila Brown, Katherine Miles, Mohini of September 26, 2021. Gray, Tom A. Barr and David Gray J Immunol published online 9 August 2013 http://www.jimmunol.org/content/early/2013/08/09/jimmun ol.1301163 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2013/08/12/jimmunol.130116 Material 3.DC1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published August 9, 2013, doi:10.4049/jimmunol.1301163 The Journal of Immunology Plasma Cell Homeostasis: The Effects of Chronic Antigen Stimulation and Inflammation Tom Slocombe,*,† Sheila Brown,*,† Katherine Miles,‡ Mohini Gray,‡ Tom A. Barr,*,†,1 and David Gray*,† Long-lived plasma cells (LLPCs) that maintain humoral immunity to previously encountered Ags occupy a compartment in the bone marrow (BM). The rules and mechanisms by which cells enter (and leave) this compartment are poorly understood. We looked at what happens to the LLPC compartment and to plasma cell lifespan in general, in situations in which Ag stimulation and/or inflammation persist. We find that chronic Ag supply causes the generation of short-lived plasma cells in the local lymphoid organ, at the expense of any LLPC production. Furthermore, we find that inflammation caused by infection (mediated via TNF-a)causesa dramatic mobilization of LLPCs from the BM, with a concomitant reduction in circulating Ab levels against previously immunized Ags. These data are discussed in the context of the capacity of the BM LLPC compartment and competition for entry to it. The Downloaded from Journal of Immunology, 2013, 191: 000–000. ollowing vaccination or infection, Ab levels persist for mablasts emerge at a time when the extrafollicular PC response has many years (1, 2), and this humoral immunity is key to waned. Therefore, the GC-derived plasmablasts do not settle in the F providing protection from reinfection. The main source of red pulp, but rather respond to CXCL12 (7) in the circulation and these Abs is long-lived plasma cells (LLPCs) that are generated in migrate to the BM (10–12). Under other conditions, such as infec- http://www.jimmunol.org/ the secondary lymphoid organs but reside primarily in the bone tion, the local CXCL12 production may be increased and prolonged marrow (BM) (3–5). However, many short-lived PCs (SLPCs) are and so GC-derived plasmablasts may remain in the spleen. The GC- also generated during the early phases of T-dependent immune derived plasmablasts are also capable of migrating to sites of in- responses. These two types of plasma cells (PCs) are the result of flammation guided by CXCR3 (attracted by CXCL9, CXCL10, and two distinct waves of differentiation; a first wave occurs following CXCL11) (13). initial interactions between activated, Ag-specific CD4 T and B cells PCs are inherently short-lived, surviving for just 2–3 d unsupported, at the border of the T zone (6). Some B cells become plasmablasts and yet their lifespan can be extended, almost indefinitely, by the andupregulateCXCR4,migratingtotheredpulpinresponseto provision of survival factors (14). These include soluble factors (such CXCL12 gradients (7). Here they form the extrafollicular foci of as a proliferation-inducing ligand [APRIL], BAFF, IL-6, TNF-a,and by guest on September 26, 2021 PCs (8), which no longer divide and have lost many B cell–associated CXCL12) and membrane receptors (such as CD44, LFA-1:ICAM-1, markers (e.g., MHC class II, B220, CD19, CD20). Although mas- VLA-4:VCAM-1) (14) that act synergistically to provide a survival sive in scale, this extrafollicular PC response is transient, because niche. BCMA ligands (APRIL and BAFF) seem especially impor- most of these cells die within a few days (9). Several days later, a tant for long-term survival in the BM (15, 16), as is CD93 (17) and second wave of plasmablasts emerges from the germinal center possibly CD28 (18, 19). These factors are enriched only in the BM (GC) reaction. These cells are generally class switched and secrete under homeostatic conditions, provided by a complex cellular niche: high-affinity Ab molecules containing V-region mutations. If the eosinophils (20), megakaryocytes (21), and myeloid cells (22) pos- GC were initiated by classical immunization, generally the plas- sibly clustering around VCAM-1+ stromal cells (attracted by CXCL12) where they provide signals to plasmablasts and early PCs that also migrate toward CXCL12. Supported by a survival niche, *Institute of Immunology and Infection Research, School of Biological Sciences, PCs endure at this site for many months or even years without di- University of Edinburgh, Edinburgh EH9 3JT, United Kingdom; †Centre for Immu- vision or replenishment from the peripheral B cell pool. Survival nity, Infection, and Evolution, School of Biological Sciences, University of Edinburgh, niches are also induced in other organs during inflammation, al- Edinburgh EH9 3JT, United Kingdom; and ‡The Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, EH16 4TJ, United Kingdom though their characterization is incomplete (23). In the secondary + 2 1Current address: Institute of Infection, Immunity, and Inflammation, University of lymphoid organs, IL-6–secreting CD11c CD8a dendritic cells Glasgow, Glasgow, U.K. support plasmablast survival, whereas APRIL- and BAFF-secreting Received for publication May 1, 2013. Accepted for publication July 8, 2013. populations support PC survival in the red pulp and medullary cords This work was supported by a Medical Research Council studentship (to T.S.) and by (8, 24). These populations are expanded during inflammation, a Programme Grant from the Wellcome Trust (to D.G.). allowing for the temporary survival of large numbers of plasmablasts Address correspondence and reprint requests to Prof. David Gray, Institute of Immu- and PCs. However, under steady-state conditions, relatively few nology and Infection Research, University of Edinburgh, Ashworth Laboratories, LLPCs are supported in the secondary lymphoid organs (9). King’s Buildings, West Mains Road, Edinburgh EH9 3JT, U.K. E-mail address: [email protected] In this study, we wished to determine how chronic Ag supply and/ The online version of this article contains supplemental material. or inflammation affected the survival and turnover of PCs in local Abbreviations used in this article: APRIL, a proliferation-inducing ligand; BM, bone lymphoid tissues and in the LLPC compartment of the BM. Both marrow; GC, germinal center; KLH, keyhole limpet hemocyanin; LLPC, long-lived myeloid and B lymphocyte development are known to be influ- plasma cell; NP, nitrophenyl; PC, plasma cell; SLPC, short-lived plasma cell; TT, enced by systemic inflammatory mediators (25), and mobilization tetanus toxoid. of BM PCs upon Ag rechallenge was noted previously (26). This Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 suggests that inflammatory signals might also affect the LLPC www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301163 2 INFLAMMATION AND PLASMA CELL HOMEOSTASIS compartment. Therefore, we investigated how competing immune A total of 600 ml medium, with or without recombinant mouse chemokines responses under inflammatory or noninflammatory conditions affect at 400 nM (CXCL9 or CXCL12; R&D Systems), was added to the lower pre-existing BM PC populations and the laying down of new LLPCs well, and 100 ml spleen cells was added to the upper well. Cells were allowed to migrate for 3 h at 37˚C. The contents of the lower well were harvested and in the BM. Our findings suggest that, in addition to Ag chronicity and resuspended in 5% FCS IMDM before culturing on ELISPOT plates over- inflammation causing the generation of SLPCs at the expense of night. For assay of chemokines in spleen or BM ex vivo, cells were mashed LLPCs, inflammation (e.g., TNF-a) caused a general mobilization of or flushed into PBS and spun down, and supernatants were collected for existing LLPCs from the BM that may be important to facilitate entry assay. of newly formed LLPCs into the compartment. ELISPOT Ninety-six–well multiscreen plates (Millipore) were treated with 15% eth- Materials and Methods anol for 1 min, washed in PBS, and coated with NP-BSA or FITC-OVA, at Mice 10 mg/ml, in carbonate/bicarbonate buffer (Sigma) overnight at 4˚C. Plates were washed (PBS), blocked with 10% FCS-PBS for 2 h, and washed. Du- C57BL/6 mice, K/BxN mice (27), MyD88-deficient mice (28), TLR4- plicate 5-fold cell dilutions were incubated overnight at 37˚C before washing deficient mice (29), and TRIF-deficient mice (30) were bred and main- with PBS and PBS-0.1% Tween (nine washes). Finally, anti–IgG–alkaline tained in specific pathogen–free conditions at the School of Biological phosphatase (Southern Biotech) was added to each well for 4 h at room Sciences Animal Facility at the University of Edinburgh. Mice were aged temperature. Plates were developed using SIGMAFAST (Sigma), and spots 6–10 wk at the start of experiments, unless otherwise stated. The MyD88-, were scored under a microscope.