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Plasma Cell Homeostasis: the Effects of Chronic Antigen Stimulation and Inflammation

Plasma Cell Homeostasis: the Effects of Chronic Antigen Stimulation and Inflammation

Plasma Homeostasis: The Effects of Chronic Stimulation and

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

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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. 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 to previously encountered Ags occupy a compartment in the (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 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 (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 is key to waned. Therefore, the GC-derived plasmablasts do not settle in the F providing protection from reinfection. The main source of red , 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 . 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 –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 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 . If the (20), megakaryocytes (21), and myeloid cells (22) pos- GC were initiated by classical , 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 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 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 –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. TLR4-, and TRIF-deficient mice were generously supplied by Dr. Shizuo Akira (Osaka University, Osaka, Japan). K/BxN mice were generated ELISA by crossing NOD mice with heterozygous TCR-transgenic KRN mice (27); the latter were originally kindly supplied by Drs. Diane Mathis and Chris- Ninety-six–well Nunc MaxiSorp plates (Fischer Scientific) were coated in Downloaded from tophe Benoist (Harvard Medical School, Boston, MA). Development of ar- NP-BSA or FITC-OVA (at 10 mg/ml) or /chemokine capture Abs thritis was assessed by scoring joint swelling. Nonarthritic littermates were (at the manufacturer’s recommended concentration) and incubated overnight used as controls in all experiments. Experiments were covered by a Project at 4˚C. Plates were washed in PBS + 0.1% Tween and blocked with PBS + License granted by the Home Office under the Animal (Scientific Proce- 1% BSA. Sera (2-fold dilutions) were incubated for 2 h, and bound Abs were dures) Act 1986. This license was approved locally by the University of detected with alkaline phosphatase–labeled anti-IgM, -IgG1, -IgG2b, -IgG2c, Edinburgh Ethical Review Committee. -IgG3, or total IgG (all from Southern Biotech). Titers are the dilution at which samples reached half of the maximal OD of the standard for each http://www.jimmunol.org/ and plate. For primary immunizations, mice received 100 mg alum (Sigma)-precipitated Statistics FITC-OVA (Biosearch), nitrophenyl (NP)–keyhole limpet hemocyanin The Student t test, one-way ANOVA, or two-way ANOVA was used to 3 8 Bordetella per- (KLH), or both i.p. injections together with 2 10 killed determine statistical significance. The Dunnett posttest or Bonferroni posttest tussis m (Lee Labs). For secondary immunizations, mice received 100 gNP- was used, where required, for one-way ANOVA; the Bonferroni posttest was KLH or FITC-OVA, either in soluble form or alum precipitated, with or used for two-way ANOVA. Tests were performed using GraphPad Prism without B. pertussis, also i.p., unless otherwise stated. Recombinant mouse software (GraphPad, San Diego, CA). TNF-a (R&D Systems) was supplied to mice i.v. in PBS. Mice received 0.5 mg/d for eight consecutive days. Mice were infected i.v. with ∼106

CFU the Salmonella enterica serovar Typhimurium strain SL3261. Results by guest on September 26, 2021 To assess cell division in vivo, BrdU (Sigma) was given to the mice in the Dynamics of accumulation of Ag-specific PC populations in drinking water at 0.8 mg/ml (protected from light and changed every 3 d). spleen and BM following primary and secondary immunization For short-term labeling, mice were injected i.p. with 1 mg BrdU. For recom- binant cytokine administration, three 1-mg doses of recombinant mouse with NP-KLH TNF-a or 10 mgIFN-g (both from BioLegend), diluted in PBS, were injected In these studies, PCs were identified by expression of both CD138 i.v. at 24-h intervals. Mice were sacrificed 48 h after the final injection. and intracellular Ig (IgG, k L chain) or Ag binding. Mature PCs FACS staining were distinguished from less mature plasmablasts by their lack of MHC class II expression. Five days after primary immunization using Cells were stained with an Aqua Live/Dead kit (Invitrogen) before surface staining with anti–CD19-PE-Cy7 (6D5; BioLegend), anti–MHC class II– alum-precipitated NP-KLH with killed B. pertussis, large numbers Pacific blue (M5/114.15.2; BioLegend), anti–CD138-allophycocyanin or ofPCswereformedinthespleen(increasingfrom0.03to1%during biotin (281-2; BD), anti–CD11b-allophycocyanin-Cy7 (M1/70; BD), anti– this time) (Fig. 1A). Few of these splenic PCs were NP specific at Gr1-AF488 (RB6-8C5; ABD Serotec), anti–F4/80–Pacific Blue (BM8; day 5 (Fig. 1B) and likely were secreting Ab against B. pertussis Ags BioLegend), anti–Siglec-F–PE (E50-2440; BD), anti–CXCR3–Brilliant Vi- or were activated in a polyclonal manner through TLR stimulation. olet 421 (CXCR3-173; BioLegend), or anti–CXCR4-allophycocyanin (2B11/ CXCR4; BD) and FcR block (2.4G2; in-house) diluted in FACS buffer for By day 45 after primary immunization, a significant frequency (5.48 30 min at 4˚C. Cells were washed in FACS buffer and suspended in Foxp3 6 0.16%) of NP-specific PCs had accumulated in the spleen (Fig. Fixation/Permeabilization kit (eBioscience) overnight at 4˚C prior to in- 1B, 1C). In the BM, throughout the primary response, the frequency tracellular staining. For this, cells were stained with goat anti-mouse IgG- of PCs (Fig. 1A) or the total number of PCs (data not shown) changed PerCP (Jackson ImmunoResearch), goat anti-mouse k L chain (FITC or PE), goat anti-mouse IgM (FITC or PE) (both from Southern Biotech), or little. However, Ag-specific PCs accumulated in the BM over time, anti–APRIL-PE (A3D8; BioLegend) in permeabilization buffer (eBioscience) reaching 6.17 6 0.52% at 45 d (Fig. 1C). for 30 min at 4˚C. For Ag-specific intracellular staining, 1 mgNP-PEor Following secondary immunization (boosting) with soluble NP- 6 FITC-OVA (both from Biosearch) was used per 1 3 10 cells in conjunction KLH, very large numbers of PCs were generated in the spleen with IgG or IgM Abs. For BrdU staining, cells were treated with 10 mg/ml (7.51 6 1.15%; Fig. 1C), and, at day 5 postboost, 21.25 6 1.97% DNase I (Sigma) and diluted in permeabilization buffer for 45 min at 37˚C before washing and staining with anti–BrdU-FITC (B44; BD) for 30 min at of the PCs were NP specific. The frequency of NP-specific PCs room temperature. Samples were acquired on an LSR II or FACSCanto (BD) increased rapidly to .50% at day 9 and, surprisingly, was maintained and analyzed using FlowJo software (TreeStar). at ∼60% of PCs at day 45 (Fig. 1B, 1C), although the total number Migration assays of PCs in the spleen had waned by this point (Fig. 1C, lower left panel). In the BM, secondary immunization caused very little in- Splenic leukocytes were prepared using Lympholyte (Cedarlane). Washed crease in total PC numbers (Fig. 1A, 1C); however, there was a rapid cells were resuspended at 1 3 107/ml (in 0.5% BSA) in IMDM (both from Sigma). Transwell plates (0.5-mm pore size; Corning) were coated with appearance of large numbers of NP-specific PCs (with similar ki- mouse fibronectin (10 mg/ml in sterile water; Sigma) for 60 min at 37˚C, netics to the spleen), and these were maintained stably until the end after which fibronectin was removed, and plates were dried for 2 h at 37˚C. of the study at day 45 (Fig. 1B, 1C). Interestingly, despite the BM The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/

FIGURE 1. Secondary immunization with soluble NP-KLH generates large numbers of NP-specific LLPCs that persist in the BM but not in the spleen. (A) PCs expressing intracellular IgG in spleen and BM after primary immunization with alum-precipitated NP-KLH with killed B. pertussis and boosting with soluble NP-KLH. FACS plots show individual mice that are representative of 24 mice used in three experiments. (B) Frequency (%) of NP-specific PCs by guest on September 26, 2021 in the spleen and BM at various time points following primary or secondary immunization with NP-KLH (plots are representative of data from 15 mice in three separate experiments). (C) Summary graphs showing frequencies and numbers of NP-specific IgG PCs in the spleen and BM following primary (dashed) and secondary (solid) immunization with NP-KLH. Each point on the graphs is the mean and SE of data from five mice. The experiment was repeated twice with similar results. (D) BrdU incorporation in NP-specific IgG PCs in the spleen and BM between days 15 and 24 following secondary immunization with NP-KLH (bars show the mean and SE of data from five mice/group that are representative of two experiments). (E) titers of NP- specific Ab (of indicated class) at various time points following primary or secondary immunization with NP-KLH (points on graph represent four or five mice from one of at least six experiments). being a site where PCs of many specificities persist as a form of hu- from CD20+ B cell precursors is required to sustain an SLPC moral memory, after boosting with soluble Ag, approximately half population). In mice, in a model of systemic erythematosus, of IgG PCs in the BM were specific for the single immunizing Ag some (e.g., anti-dsDNA) are derived from SLPCs, (NP) and formed a stable population for $45 d (Fig. 1C). A BrdU whereas others (against RNA-containing complexes) are the prod- pulse between 15 and 24 d following NP-KLH boost showed that uct of LLPCs (32). Also, in the K/BxN model of arthritis, the PCs very few of the PCs became labeled (Fig. 1D); therefore, the majority making anti–glucose 6-phosphate isomerase Abs seem to be short- of NP-specific PCs in both spleen and BM had already become non- lived (33). To investigate whether a persistent supply of Ag affects dividing LLPCs. Although the numbers of NP-specific PCs declined PC lifespan, we primed mice with NP-KLH and boosted them 5 wk rapidlyinthespleen,theyweremaintainedintheBMfor$45dafter later with either a single 100-mg dose of soluble NP-KLH (acute) or boosting(Fig.1C).ThiswasreflectedinNP-specificAblevelsinthe 20 5-mg doses of NP-KLH at 3-d intervals (chronic) (Fig. 2A). serum, which peaked by day 9 and declined slightly by day 24, but Providing a single 100-mg dose of NP-KLH generated far greater subsequently were maintained until at least day 45 (Fig. 1E). numbers of NP-specific PCs in the spleen at day 10 and, as before, a proportion of these became established in the BM where they Persistent antigenic stimulation causes SLPC production at the declined only marginally over the course of the 120-d experiment expense of LLPCs (Fig. 2B). Few new NP-specific PCs were generated after the first We wanted to determine how PC turnover and lifespan were af- 10 d, as revealed by 10-d BrdU pulses at days 1–10, 50–60, or 110– fected in situations in which persist (autoimmune dis- 120 of the experiment (Fig. 2C). Conversely, continuous supply of 5 ease or chronic infection). In patients with mg NP-KLH generated lower numbers of NP-specific PCs in the treated with to deplete B cells, the titers of autoantibodies spleen (Fig. 2B), but in contrast to the acute immunization, these decrease much more rapidly after B cell depletion than do Abs to cells were maintained until day 60. This was due to continued gen- vaccine Ags (31). This has been interpreted to indicate that the au- eration of new PCs, as shown by BrdU incorporation (Fig. 2C). In toantibody response is derived from SLPCs (i.e., continued input contrast to the spleen, few of these PCs became established in the 4 INFLAMMATION AND PLASMA CELL HOMEOSTASIS Downloaded from http://www.jimmunol.org/

FIGURE 2. Chronic delivery of Ag generates SLPCs without establishment of LLPCs in the BM. (A) Schematic diagram of experimental design. All mice were primed with alum-precipitated NP-KLH and B. pertussis and were pulsed with BrdU for 10 d prior to culling at time points 1, 2, or 3. (B)

Numbers of NP-specific IgG PCs in the spleen (left panel)orBM(right panel) of mice from the groups described above (acute: solid line; chronic: dashed by guest on September 26, 2021 line; no boost: dotted line). Period of chronic Ag administration is indicated below the graph. (C) Numbers of BrdU+ NP-specific IgG PCs in the spleen and BM of groups of mice described above. Numbers of BM cells were adjusted to number/100 mg femur, because the bones increase in size with the age of the mice over the course of the experiment. Points on graphs represent the mean and SE of data from five or six mice. (D)EnumerationofGCBcells(PNA+/IgD2) by flow cytometry at points during or following acute or chronic Ag delivery. Two-way ANOVAwith Bonferroni posttest was used to evaluate the significance of observed differences. **p = 0.001–0.01, ***p , 0.001. ns, Not significant.

BM (i.e., only a third of the numbers seen following acute NP- nonarthritic control mice (Fig. 3C). This suggested a failure to KLH administration) (Fig. 2B). BrdU incorporation data suggest enter or survive in the BM compartment. To investigate whether that, during the first 10-d, PCs generated in the periphery entered the this phenomenon was also a feature of chronic infection, we gave BM; however, at later time points, these PCs either failed to migrate 4-d BrdU pulses to mice at various time points following infection to the BM or were unable to survive (Fig. 2C). The failure to es- with Salmonella. This revealed an increase in the number of di- tablish LLPC populations in the BM after persistent stimulation was viding PCs in the BM at day 10, which declined by day 18 (Fig. not due to any failure to generate GCs; Fig. 2D shows that the num- 3D) but increased again at day 55, when anti-Salmonella Ab titers bers of GC B cells in the of the chronic Ag group remained are maximal (34). To see whether these dividing PCs (in BM or high until 60 d, when Ag administration was stopped. Also, there spleen) were able to survive as LLPCs in the BM, we performed was no discernable difference in the balance of IgM versus IgG a BrdU pulse-chase experiment: mice were given 4-d BrdU pulses isotypes in the anti-NP Ab responses between chronic and acute at days 5–8 or 25–28 postinfection, and the survival of labeled PCs immunizations (data not shown). was determined 7 and 28 d after BrdU. Few BM PCs retained We used K/BxN mice, which develop arthritis as a result of the BrdU for .7 d when pulsed at either time point (Fig. 3E), sug- production of circulating Ab (IgG) to anti–glucose 6-phosphate gesting that PCs entering the BM at early time points in Salmo- isomerase, to test whether other chronic inflammatory conditions led nella infection do not have the capacity to become long lived. to reduced establishment of PCs in the BM (27). At 3 wk after birth, The lack of LLPCs in the BM in these models could be due to arthritic mice had increased levels of splenic PCs compared with failure to migrate there (K/BxN arthritis) or the failure to incor- nonarthritic littermates (Fig. 3A, 3B). BrdU pulses revealed that porate into the LLPC pool (Salmonella). The chemokine CXCL12 these PCs had a consistent turnover (∼50% in 3 d) (Fig. 3A), and the has a principal role in effecting entry of PCs into the BM; therefore, percentage (data not shown) and, especially, the number of BrdU+ expression of its receptor CXCR4 might indicate reasons for the PCs were greater in arthritic mice compared with nonarthritic mice failure to incorporate in the BM PC compartment. We assessed the (Fig. 3C). Despite the uptake of BrdU by many PCs in the spleen expression of CXCR4 on the surface of PCs from the spleens of during a 10-d pulse (50.79 6 3.06% in arthritic mice), very few mice 5 d after secondary immunization with soluble NP-KLH, which BrdU+ PCs could be detected in the BM, indeed fewer than in generates many PCs that travel to the BM, and 16 d postinfection The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 3. PCs generated in the spleens of arthritic K/BxN mice do not become established as LLPCs in the BM. (A) Representative plots showing CD138+ PCs in the spleen of nonarthritic or arthritic littermates (upper panels) and BrdU incorporation in PCs following a 3-d pulse (lower panels). Data are representative of five mice from one of five similar experiments. (B) Summary data of total PC numbers in the spleen (upper panels) and BM (per femur) by guest on September 26, 2021 (bottom panel) of nonarthritic and arthritic mice (horizontal line indicates median value). (C) Numbers of PCs labeled with BrdU in the spleen and BM of nonarthritic or arthritic mice following a 10-d BrdU pulse at day 1, 7, 14, or 28 after BrdU. (D) BrdU-labeled PCs in the spleen (solid line) or BM (dashed line) following a 4-d BrdU pulse prior to each indicated time point after Salmonella infection. (E) Survival of BrdU-labeled PCs in the BM of Salmonella- infected mice 7 or 28 d after receiving 4-d BrdU pulses at days 5–8 or 25–28 postinfection. Mean and SE of data from five mice/group (data are rep- resentative of three experiments). (F) Mean fluorescence intensity (MFI) of CXCR4 on PCs in the spleen of mice boosted with soluble NP-KLH or infected with Salmonella (at day 5). Statistical significance was evaluated using the Student t test (B, F) or two-way ANOVAwith Bonferroni posttest (C, E). ***p , 0.001. ns, Not significant. with Salmonella. In the spleen, expression of CXCR4 was similar specific PCs declined over time postinfection (by day 75 postin- (Fig. 3F) between the two models, indicating that this did not fection they were 10-fold lower than in uninfected mice), whereas underlie the failure of PC entry. they were maintained at stable levels in uninfected controls (Fig. 4D, panel 3). Total PC numbers in the BM recovered to normal Salmonella infection causes depletion of previously established between days 25 and 60 postinfection (Fig. 4B); however, the NP- LLPC populations in the BM specific PCs remained lower than in uninfected controls until the Salmonella-infected mice generated large numbers of PCs in the end of the analysis at 75 d (Fig. 4D, panels 2, 3). This indicates that spleen, but these failed to persist in the BM (Fig. 3D). Moreover, there was no significant re-entry to the BM by PCs that were lost intriguingly, we observed a considerable contraction of the BM PC during infection (Fig. 4D). Finally, we observed an overall neg- compartment following Salmonella infection (Fig. 4A, 4B). There- ative effect of Salmonella infection on the NP-specific Ab levels in fore, we investigated the effects of Salmonella infection on previ- the serum (Fig. 4E). These data indicate that Salmonella infection ously established BM PC populations. To induce a readily detectable causes a disruption of the steady-state equilibrium of BM PC pop- NP-specific PC population, mice were primed and boosted with NP- ulations that affects circulating Ab levels. KLH and then rested for 5 wk before infection with Salmonella. Bacterial loads in the spleen peaked between days 7 and 14 (Fig. 4C). Inflammation, not influx of new PCs, causes the loss of By day 16 postinfection, a dramatic depletion in NP-specific PCs pre-existing LLPCs in the BM was seen in the BM (Fig. 4D, panels 1, 2). Interestingly, at the same In humans, following booster vaccination with tetanus toxoid (TT), time, increased numbers of NP-specific PCs were detected in the TT-specific PCs can be observed entering the BM from approxi- spleen (Fig. 4D, panel 4). This is not the result of the generation of mately day 5, and an increased frequency of mature non–TT-specific new NP-specific PCs (no BrdU was incorporated into NP-specific PCs is detected briefly in the (26). The PCs in the blood are PCs after 48-h pulses on days 6–8 or 14–16); however, it could be thought to be LLPCs of other specificities that have been “pushed due to an influx of NP-binding PCs from the BM. Splenic NP- out” of their survival niches by the new influx of TT-specific PCs 6 INFLAMMATION AND PLASMA CELL HOMEOSTASIS Downloaded from http://www.jimmunol.org/

FIGURE 4. Salmonella infection causes loss of LLPCs from BM. (A) Plots showing PC induction in the spleen 16 d postinfection with Salmonella and reduced frequencies of PCs in BM (plots are representative of groups of five mice in experiments performed six times). (B) Numbers of PCs in the spleen and BM at various time points following infection with Salmonella. Data points are mean and SE of five mice/group. (C) CFU in the spleens of mice primed by guest on September 26, 2021 and boosted with NP-KLH 5 wk before infection with Salmonella (dashed line) versus uninfected controls (solid line). (D) Total IgG (left panel) or NP- specific IgG PCs (left middle panel) per 100 mg femur, BM NP-specific PCs as a percentage of total IgG PCs (right middle panel), and number of NP- specific IgG PCs per spleen (right panel) of mice shown in (C). Numbers of BM cells were adjusted to the number per 100 mg femur, because the bones increase in size with the age of the mice over the course of the experiment. (E) Titers of NP-specific Abs of indicated class in serum of mice shown in (C). Points on graph are mean and SE of data from five mice/group from one of two experiments. Statistical significance was evaluated using one-way ANOVA (B) or two-way ANOVA with Bonferroni posttest (D, E). *p = 0.01–0.05, **p = 0.001–0.01, ***p , 0.001. ns, Not significant.

(35). The mechanism of this competition between new and old NP plots); however, the numbers of NP-specific PCs in the BM BM PCs is unknown. The loss of BM PC populations that we saw decreased quite dramatically following the Ag + adjuvant formu- during Salmonella infection was unlikely to be the result of newly lation, being reduced to ,20% of preboost numbers (Fig. 5C, arrived PCs pushing out old ones, because there was no early influx bottom panel;meanof5.63 103/femur down to 1 3 103 at day 10), of PCs, and the decrease in numbers was also very pronounced. We a much greater proportional depletion than seen in total PC numbers hypothesized that the change in BM PC numbers was more likely in these mice (mean of 3.2 3 104/femur down to 2.1 3 104 at day the result of circulating inflammatory mediators. To test whether 10; data not shown). Soluble FITC-OVA also accelerated the decay simple competition for niches (35) or inflammatory signals were of NP-specific PCs in the BM (compared with nonboosted controls), important, we developed a model to observe the competition be- but this was not as striking as the effect of FITC-OVA + adjuvant tween PCs of two Ab specificities (NP and FITC) delivered in soluble (Fig. 5C). In the spleen we saw a transient increase in NP-specific or inflammatory formulations (Fig. 5A). Mice were primed with both PCs after the Ag + adjuvant boost, which might arise from an influx NP-KLH and FITC-OVA. Five weeks later, the mice were boosted of PCs from the BM or from a nonspecific activation/differentiation with soluble NP-KLH to establish NP-specific BM LLPCs (Fig. of NP-specific memory B cells; the study of Benson et al. (36) 1B). An additional 5 wk later, mice were boosted with either soluble suggests that the latter is less likely. Importantly, with regard to FITC-OVA or alum-precipitated FITC-OVA with killed B. pertussis serum Ab responses, the Ag + adjuvant caused a noticeable low- (Fig. 5A). As with Salmonella infection, FITC-OVA delivered with ering of anti-NP levels over a period of 75 d (Fig. 5C, bottom adjuvant caused a significant loss of total PCs (∼50%) from the BM panel). The adjuvant effect that we describe in this study is medi- (Fig. 5B, right panel, CD138 versus intracellular IgG stain). In the ated by the killed B. pertussis component, because alum alone has spleen the opposite was true, in that Ag + adjuvant caused a large no effect on BM PC numbers (Supplemental Fig. 1). expansion of total PC numbers (Fig. 5B, left panel). Focusing first Turning to the generation of new FITC-specific PCs following on the fate of the pre-existing NP-specific PCs, the frequency of the boost with FITC-OVA, both Ag formulations caused a con- NP-specific cells within the BM PC population remained the same siderable increase in the spleen (Fig. 5D), although the frequency following all forms of boosting (Fig. 5B, right panel, FITC versus was much greater after the soluble boost (Fig. 5B). The adjuvant The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 5. The effect of antigenic competition and adjuvant on the exchange of new for existing PCs in the BM. (A) Schematic diagram of experimental design. All mice were primed with alum-precipitated NP-KLH and FITC-OVA with killed B. pertussis and boosted 5 wk later with soluble NP-KLH. Five weeks later, mice were boosted with FITC-OVA in different formulations. (B) Representative FACS plots showing total IgG PC staining in spleen and BM by guest on September 26, 2021 at day 5 following boost with FITC-OVA (upper panels) and NP or FITC specificity of IgG PCs (lower panels). Data shown are representative of five mice/ group in this experiment, which was repeated four times. (C) Summary graphs showing numbers of NP-specific PCs in the spleen and BM of mice im- munized as described in (A). (D) Summary graphs showing numbers of FITC-specific PCs in the spleen (left panel) and BM (right panel) of mice described in (A). (E) FITC-specific IgG titers in the serum of mice described in (A). caused a splenomegaly that explains the differences in frequency. CXCL12 is also the main chemokine to mediate entry of PCs into In the BM, only soluble FITC-OVA caused a major (.20-fold) the BM (7). We confirmed that both Salmonella infection (Fig. increase in the number of FITC-specific PCs. Thus, although the 6A) and alum-precipitated NP-KLH + B. pertussis (data not shown) adjuvant formulation made space in the BM compartment, it did caused a decrease in B lineage cells in the BM and an increase in not allow entry of newly generated PCs. The anti-FITC titers were . In addition, we noted that, at the peak of Salmonella similar in both groups at 30 d postboost, but by 60 d after boost the infection, Siglec-F+ eosinophils were much reduced in the BM (Fig. Ag + adjuvant group exhibited reduced titers compared with the mice 6B). Eosinophils have been identified as providing a survival niche receiving soluble FITC-OVA, reflecting the failure to establish a for PCs in the BM (20). Furthermore, we found that the BM cells population of FITC-specific long-lived BM PCs (Fig. 5E). Although flushed from the bone of infected mice secreted little, if any, these data show that the arrival of new PCs into the BM has some CXCL12 compared with those from noninfected mice (Fig. 6C). effect on resident PCs (soluble FITC-OVA caused some loss of NP- We also examined CXCL12 production in the BM from mice re- specific PCs; Fig. 5C), adjuvant- or infection-induced inflammation ceiving boost injections of soluble versus alum-precipitated Ag + B. has a much more profound influence. Intriguingly, this infection/ pertussis. At day 5 postboost with the adjuvant formulation, we inflammation-driven loss of BM PCs does not involve TLRs, because could not detect CXCL12 production compared with normal levels MyD88-, TRIF-, and TLR4-knockout mice all show equivalent or in the BM from the mice receiving the soluble boost (Fig. 6D). The greater degrees of PC depletion after Salmonella infection (Sup- production of CXCL12 in the BM returned to normal by 12 d plemental Fig. 2) postboost (Fig. 6D). In the case of Salmonella infection, the de- crease in CXCL12 correlated with the increase in the circulating The effect of inflammation on the BM microenvironment levels of a number of inflammatory mediators (Supplemental Fig. The BM microenvironment is sensitive to systemic inflammation 3), including TNF-a.IFN-g (prevalent in the response to Salmo- (37): for instance, TNF-a (induced by adjuvant) caused a signifi- nella) has been linked to the induction of CXCR3-expressing PCs cant decrease in BM (25), accompanied by an ex- that migrate toward CXCL9 (13), leading to their preferential mi- pansion of granulocytes, and MCP1 caused the mobilization of gration to inflammatory sites rather than the BM. However, the from the BM (38). The former was related to a decrease splenic PCs generated following immunization increased both in CXCL12 production by resident VCAM-1+ stromal cells (25). CXCR3 and CXCR4 expression, especially when B. pertussis was 8 INFLAMMATION AND PLASMA CELL HOMEOSTASIS Downloaded from http://www.jimmunol.org/

FIGURE 6. Changes to the BM cellular compartments following Salmonella infection or bacterial adjuvant injection. (A) Frequencies of CD19+ (B cells) and Gr1+ cells (granulocytes) in the BM of naive or Salmonella-infected mice (at day 16). (B) Frequency of eosinophils (Siglec-Fhi/CD11bint) in the BM of A B C naive or Salmonella-infected mice (at day 16). Data in ( ) and ( ) are representative of five mice from one of five experiments. ( ) CXCL12 levels in the by guest on September 26, 2021 supernatant of BM cultures from Salmonella-infected mice (at day 16). (D) CXCL12 levels in the supernatant of BM cultures from mice receiving no boost, soluble FITC-OVA boost, or FITC-OVA + adjuvant boost at days 5, 12, and 60 (all mice were primed with alum FITC-OVA + B. pertussis). Each symbol represents a single mouse, and horizontal lines represent the means; data are representative of three other experiments. (E) Migration across Transwells toward medium or medium containing CXCL12 (400 nM) or CXCL9 (400 nM), expressed as the percentage of FITC-OVA IgG PCs that migrated across the Transwell. Data are mean and SE of ELISA titers from five mice/group (experiment was run three times). Statistical significance was evaluated using the Student t test (C) or one-way ANOVA with Bonferroni posttest (D). ***p , 0.001. ns, Not significant. included in the adjuvant (Supplemental Fig. 3). Importantly, and mobilization from the BM. We also noted that TNF-a and Salmo- despite the upregulation of CXCR3 and CXCR4, migration of PCs nella infection caused a transient loss of cells within the BM that across Transwell membranes toward both CXCL9 and CXCL12 was were making the PC survival factor APRIL (Fig. 7C). impaired in mice immunized with B. pertussis as adjuvant (Fig. 6E). It seems likely that the loss of CXCL12 production by BM Discussion cells and the reactivity of PCs to this chemokine play important Following acute immunization, the generation of PCs in the local roles in the homeostatic control of LLPC populations postinfection. lymphoid organ is transient; even following secondary immuni- zation the number of Ag-specific PCs wanes significantly beyond 2 a TNF- causes the loss of PCs from the BM wk (e.g., in the spleen; Fig. 1). If Ag is supplied over a longer period, Ueda et al. (25) reported that TNF-a reduced levels of CXCL12 the generation of PCs is also extended, presumably evoked by from the BM. To test whether TNF-a was responsible for the mo- continued stimulation of Ag-specific B cells. Although the persistent bilization of PCs from the BM, we injected mice three times at 48-h antigenic stimulus causes cell turnover (BrdU incorporation) in the intervals with 1 mg TNF-a. Analysis of the BM PCs 2 d after the last spleen throughout the period of Ag administration, newly divided injection showed that the number and frequency of PCs were sig- PCs only enter the BM during the first few days following immu- nificantly reduced (Fig. 7A, 7B). There was no change in splenic PC nization (boosting). There could be several reasons for this. First, the populations. Given the role of IFN-g and downstream mediators in source of new LLPC precursors, the GC, might be impaired in some the mobilization of myeloid populations from the BM (38) and the way; however, this is probably not the case because GCs are en- largeincreaseinIFN-g in the circulation of infected mice (Sup- hanced in mice given Ag chronically (Fig. 2D). There are two other plemental Fig. 3), we also injected this cytokine; however, no change possibilities that we cannot differentiate: either the BM is receptive in BM or spleen PC numbers was observed (Fig. 7), despite changes to new PC entrants only for a defined period following immuni- to MHC class II levels on monocytes/ (data not shown). zation (both acute and chronic groups exhibit increased numbers of On this basis we conclude that TNF-a induced by Salmonella in- BrdU+ PCs in the BM when labeled from days 1 to 10, but not after fection or B. pertussis–containing adjuvant is a major cause of PC this) or the environment of the spleen during chronic Ag admin- The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/

FIGURE 7. LLPCs and APRIL-producing cells in BM are mobilized by TNF-a but not IFN-g.(A) Representative FACS plots of PCs in the spleen and BM following i.v. injection of TNF-a or IFN-g (three times over 72 h). (B) Summary graphs of PC frequencies (left panel) or numbers (right panels)in spleen and BM of TNF-a/IFN-g–injected mice. (C) Number and frequency of APRIL-producing cells, identified by intracellular flow cytometric staining, in BM of mice injected with TNF-a or IFN-g. Means and SE are shown; this experiment was performed twice. Statistical significance was evaluated using by guest on September 26, 2021 one-way ANOVA with Bonferroni posttest. *p = 0.01–0.05, **p = 0.001–0.01, ***p , 0.001. ns, Not significant. istration retains the new PCs locally. The latter may be a reflection demonstration of this hypothesis remains to be provided. The of stromal cells providing chemotactic and survival factors over observation that chronic Ag supply favors SLPC production at the a prolonged period as a result of the inflammatory signals. We expense of LLPCs may have profound consequences for certain observed these same phenomena in two other models of chronic vaccination protocols, such as DNA vaccination, in which small stimulation: arthritis and prolonged infection. In the K/BxN ar- amounts of Ag are administered over a prolonged period. thritis model, we found that many SLPCs were generated in the We noted the dramatic loss of total PC numbers from the BM in spleen, but they did not appear in the BM (Fig. 3). Following the first week following Salmonella infection and supposed that Salmonella infection, SLPCs were generated early, and these en- this may underlie the exchange of newly formed PCs with pre- tered the BM but again did not persist as long-lived cells. Some vious occupants. We provide ample evidence that infection- and insight may be provided by our experiments comparing the BM adjuvant-induced inflammatory signals cause the mobilization of incorporation of Ag-specific PCs after soluble or adjuvant- PCs from the BM and that this efflux reduces the size of the associated boosting. After soluble Ag there is no barrier to entry existing LLPC populations, with concomitant reduction in circu- of new PCs into the BM compartment; however, inclusion of lating Abs derived from these PC populations (Figs. 4, 5). This is adjuvant (especially bacterial) inhibits this process. So in the case associated with a dramatic decrease in the amount of CXCL12 of the soluble Ag boost the plasmablasts are allowed to enter the produced by BM cells isolated from infected or adjuvant (B. BM, whereas bacterial adjuvant causes retention of these cells in pertussis)-immunized mice (Fig. 6), as well as with a loss of cells the spleen. More generally, the window for entry of new PCs into that was previously shown to provide survival signals for LLPCs the BM compartment is controlled by inflammatory signals arising in the BM (i.e., eosinophils) (Fig 6) (20). Because a generalized from infection or bacterial adjuvant and as a result of chronic loss of B lineage cells from the BM occurs following infection stimulation/inflammation. We (in this work) and other investigators (39) and is a feature of TNF-a administration (25), we wondered (33) observed that chronic stimulation drives the formation of whether the mobilization of PCs would also be TNF-a dependent. SLPCs; these cells seem incapable of converting into stable LLPCs Indeed, this was the case and, as in previous studies (25), is likely (in the BM) and so must be sustained locally. This is possible be- to be the result of decreased CXCL12 production (Fig. 6). Thus, cause inflamed tissues contain a number of cell types that can injection of TNF-a caused a rapid loss of PCs from the BM; in- provide survival signals (a “niche”) but only for a short period, terestingly, this also provoked a noticeable decrease in the num- because the size of this niche is limited, and the input of new PCs bers of cells in the BM that produce APRIL (Fig. 7). It seems from the chronic activation of B cells is likely to overwhelm the unlikely that TNF-a acts directly on PCs to cause their exit from niche capacity (hence, cells are pushed out and die). A formal the BM. Interestingly a number of mediators that affect PC survival 10 INFLAMMATION AND PLASMA CELL HOMEOSTASIS

(e.g., IL-6, stem cell factor, VCAM-1) are increased in the circu- each subsequent (unrelated) infection. Attrition of CD8 memory lation during Salmonella infection (Supplemental Fig. 3). In sum- populations following unrelated viral infections was noted mary, systemic inflammation, specifically that mediated by TNF-a, previously (42, 43). We predict that a similar impairment of these has quite profound effects on the PC compartment. Our competitive Ab titers may be apparent in patients with chronic autoimmune immunization experiments (Fig. 5) show that these effects have conditions, when severe flares occur. Estimating LLPC lifespan significant long-term consequences for the Ab response; pre-exist- experimentally is generally done with mice in pathogen-free con- ing anti- Ab responses decrease rapidly when adjuvant-de- ditions; based on these findings, we believe that, in a more normal, livered immunization or an infection is introduced. pathogen-challenged environment, the decay of LLPC populations Contrary to our expectations, the loss of existing BM PCs fol- may be accelerated. lowing immunization in association with a bacterial adjuvant did not lead to enhanced entry of new PCs into this compartment. Indeed, when B. pertussis was used as adjuvant the incorporation Disclosures of new FITC-specific PCs in the BM was much reduced compared The authors have no financial conflicts of interest. with a boost with soluble or alum-precipitated Ag. 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