Long-lived plasma cells (PCs) in the bone marrow (BM) are a critical source of antibodies after infection or vaccination, but questions remain about the factors that control PCs. We found that systemic infection alters the BM, greatly reducing PCs and regulatory T (Treg) cells, a population that contributes to immune privilege in the BM. The use of intravital imaging revealed that BM Treg cells display a distinct behavior characterized by sustained co-localization with PCs and CD11c-YFP + cells. Gene expression profiling indicated that BM Treg cells express high levels of Treg effector molecules, and CTLA-4 deletion in these cells resulted in elevated PCs. Furthermore, preservation of Treg cells during systemic infection prevents PC loss, while Treg cell depletion in uninfected mice reduced PC populations. These studies suggest a role for Treg cells in PC biology and provide a potential target for the modulation of PCs during vaccine-induced humoral responses or autoimmunity.

Many studies have demonstrated that systemic infection or inflammation results in marked changes in BM populations (). Here, challenge with Toxoplasma gondii, a clinically relevant parasite for which the mouse is a natural host (), demonstrated that infection-induced changes in the BM lead to a transient loss of PCs and Tregs. Multiphoton imaging revealed that at homeostasis, PCs interact with a CD11c-YFPpopulation and Treg cells in the BM. Further studies indicate that Treg cells have a complex interaction with BM PCs, including a CTLA-4-dependent mechanism of PC regulation, while supporting PC populations. Together, these studies suggest an unanticipated role for Treg cells in the maintenance and operation of the PC niche within the BM.

Inflammation and the reciprocal production of granulocytes and lymphocytes in bone marrow.

Diminished hematopoietic activity associated with alterations in innate and adaptive immunity in a mouse model of human monocytic ehrlichiosis.

Infection with Toxoplasma gondii alters lymphotoxin expression associated with changes in splenic architecture.

Long-lived PCs present in the BM constitutively produce high levels of antibodies that result in life long serum antibody titers against previously encountered pathogens or vaccines (). Consequently, there is interest in understanding the mechanisms that maintain these cells (). It is known that stromal cells provide survival signals to PCs through the production of CXCL13, BLyS, April, and IL-6 (). Furthermore, eosinophils, basophils, and megakaryocytes are implicated in the maintenance of PCs in the BM (), and there is evidence that perivascular clusters of DCs in the BM provide critical signals for B cells (). Although these factors promote PC survival, they are not sufficient, and the cellular composition of this niche and requirements for PC maintenance are major questions (). However, there is a paucity of intravital imaging studies to describe the behavior of PCs and their interactions with other cell populations. Thus, there remains a need to better define the composition of this niche to understand how PCs are maintained and whether there are regulatory networks that limit PC responses.

The establishment of the plasma cell survival niche in the bone marrow.

Eosinophils are required for the maintenance of plasma cells in the bone marrow.

The establishment of the plasma cell survival niche in the bone marrow.

A variety of immune cell precursors reside and develop in the bone marrow (BM), a site that is also home to several populations of mature lymphocytes. There are multiple mechanisms to allow pluripotent or long-lived cells, including hematopoietic and cancer stem cells, plasma cells (PCs), and memory T cells, to persist in the BM (). However, the spatial relationship and interactions between these disparate cellular populations are still being defined. For example, BM stromal cells provide growth and survival factors necessary for PC and hematopoietic stem cell (HSC) maintenance, but the relationship between these niches is unclear (). Moreover, in the BM, regulatory T cells (Treg) are enriched and may contribute to the maintenance of the BM as an immune privileged site, necessary for HSC survival (). However, the behavior of Treg cells in the BM and their interactions with other immune populations have not been visualized and it remains unclear whether their activity is relevant to other hematopoietic cell populations in the BM.

Second, to assess the impact of Treg cell loss on PC populations in the absence of infection, Treg cells were depleted in naive Foxp3-DTR mice using diphtheria toxin (DT) and the PC compartment was assessed. In these mice, DT treatment resulted in a >90% decrease in the percentage and number of Foxp3Treg cells in the BM ( Figures 6 A and 6B ) and a >50% decrease in PCs ( Figures 6 C and 6D). Of note, when Treg cells were eliminated and PCs were reduced, the numbers of CD11cMHCIIcells remained constant, although these cells did express elevated levels of CD80 (data not shown). Thus, despite recent findings that Treg cell depletion enhances PC formation in the periphery (), the depletion of Treg cells resulted in a reduction in the BM PC population, similar to the phenomenon observed in infected mice. Although Treg cell depletion using the Foxp3-DTR system results in widespread inflammation (), the studies described in Figure 5 are performed in the context of systemic infection-induced inflammation. Thus, the ability of IL-2C treatment to preserve Treg cells and mitigate the PC loss suggests that inflammation alone would not result in the PC reduction observed in the Foxp3-DTR experiments. In these mice, Treg cell depletion did not result in significant changes in the eosinophil population, further emphasizing that eosinophils are not sufficient for PC maintenance ( Figures 6 E and 6F). These independent approaches implicate Treg cells as part of a network that maintains and regulates PC populations in the BM.

(A–F) Populations of Treg cells (A and B), PCs (C and D), and eosinophils (E and F) were evaluated and enumerated in the BM.

Naive Foxp3-DTR Mice Were Treated with DT on Days 0, 1, 4, and 6 and Evaluated on Day 7.

Next, complementary approaches were used to understand whether Treg cells are directly involved in the maintenance of PCs. First, in gain-of-function experiments, we asked whether treatment with an interleukin-2 (IL-2)-anti-IL-2 antibody complex that promotes Treg cell survival () could prevent the loss of BM PCs during infection. Treatment of naive mice with IL-2 complex resulted in elevated Treg cell numbers by percentage, but not absolute number in the BM ( Figures 5 A and 5B ), and did not alter PC numbers ( Figures 5 C and 5D). IL-2 complex treatment of infected mice did result in a modest increase in parasite burden (data not shown), but mitigated the loss of the BM Treg cells and preserved the PCs in the BM ( Figures 5 A–5D). As PCs do not express the IL-2 receptor component CD25 () (data not shown), this rescue is unlikely due to direct effects of the IL-2-anti-IL-2 complex on PCs. Of note, because of the experimental variation we observed in the levels of PC reduction, the overall relationship between the frequency of Treg cells and PCs was assessed by comparing Treg cell numbers with the numbers of PCs in the BM from these experiments. When the number of Treg cells was plotted versus the number of PCs in the BM from these IL-2C treatment experiments, a highly significant correlation was observed between these populations ( Figure 5 E). Of note, it has been proposed that eosinophils promote PC survival () and these cells are transiently lost from the BM along with PCs during acute infection ( Figure 5 F). However, as previously reported (), treatment of naive mice with the IL-2 complex resulted in an eosinophilia, but when infected mice were treated with IL-2 complex, the eosinophil population was not rescued, although the PC population was maintained ( Figure 5 F). These data suggest that the role for Treg cells in supporting PC populations is distinct from that of the eosinophils present in the BM.

(A–F) WT mice were infected with T. gondii and treated with IL-2-anti-IL-2 complex every 3 days for 14 days. BM was evaluated for Treg cells (A and B), PCs (C and D), and representative flow plots are shown, with a concatenation of mice within each group for PC plots (D). The numbers of Treg cells and PCs were plotted to identify correlations between the cell numbers of these two populations (E). Eosinophils were evaluated and quantified by flow cytometry (F).

Eosinophils are required for the maintenance of plasma cells in the bone marrow.

Because PCs in the BM co-localize with Treg cells, it was relevant to determine whether the reduction in PCs during infection is a consequence of the loss of the BM Treg cell population or a secondary consequence of inflammation due to the loss of the suppressive niche. Based on the transcriptional profiling, we focused on CTLA-4 as being highly expressed by BM Treg cells and having a prominent role in Treg suppressive activity (). Analysis of CTLA-4 expression revealed that splenic Treg cells express negligible surface levels of CTLA-4, whereas BM Treg cells have elevated levels ( Figure 4 D). Next, we analyzed mice with a Treg cell-specific loss of CTLA-4 expression (Foxp3/CTLA4), which develop systemic inflammation (). Despite the presence of an ongoing inflammatory response, there was no decrease in the numbers of Foxp3T cells in the BM and these mice had a ∼3-fold increase in the percentage of Treg cells ( Figure 4 E). However, in contrast to the loss of PCs observed when Treg cells are reduced during infection-induced inflammation, compromising Treg cell function in this system without decreasing Treg cell number resulted in a 3-fold increase in the number of PCs ( Figure 4 E). However, CTLA-4 expression on Treg cells negatively regulates PC development in the periphery (), and PCs in the BM express high levels of CD28, an important regulator of PC function (). Thus, although CTLA4-deficient Treg cells are unable to prevent fatal inflammation (), these experiments suggest that Treg cell expression of CTLA-4 acts to limit the size of the PC pool in the BM.

Treg cells in non-lymphoid tissues have unique transcriptional profiles and functions associated with individual tissues (). However, despite the enrichment of Treg cells in the BM, these cells have not been transcriptionally characterized. Therefore, to determine whether BM Treg cells have a transcriptional signature distinct from that of peripheral Treg cells, gene expression profiles of BM or splenic Treg cells were compared ( Figure 4 A; Table S1 ). Differentially expressed transcripts are dominated by chemokine/cytokine receptors (Ccr2, Ccr3, Cxcr3, Il9r, Il1rl1) and effector molecules (Ctla4, Il10, Fgl2, Gzmb), suggestive of an activated population with increased Treg suppressive capabilities ( Figures 4 A and 4B). These transcripts are also overexpressed in other tissue-Treg cells relative to those isolated from lymphoid organs, with those from injured muscle being most similar to BM Treg cells ( Figure 4 B). Indeed, principal component analysis encompassing all differentially represented transcripts showed that BM Treg cells reside with tissue Treg cells, not lymphoid Treg cells ( Figure 4 C). These findings highlight the effector capacity of BM Treg cells, consistent with the idea that these cells have a role in the maintenance of an immune privileged niche.

(E) PC and Treg cell populations in the BM, Spl, and LN of WT or Foxp3 Cre /CTLA4 flox/flox (CKO) mice were assessed by flow cytometry.

(D) CTLA-4 expression was determined by flow cytometry on BM or splenic Treg cells, which was concatenated from five mice (left) and MFI was quantified (right).

(A–C) Treg cells sorted from spleen and BM of naive Foxp3-GFP + mice were evaluated by microarray. Cells were assessed for differentially regulated genes (A), which were then compared to Treg cells from other sites by specific gene expression (B) or as total populations by principle component analysis (C).

Treg Cells in the BM Exhibit an Effector Phenotype with Regulatory Effects on PCs

Few studies have used multi-photon imaging to visualize Treg cell behavior and interactions with DCs in situ and these were performed in LNs in the context of immunization and diabetes (). Therefore, we decided to compare how Treg cells in the BM or spleen interact with resident DC populations at homeostasis. Analysis of reporter mice revealed that splenic Treg cells were highly motile based on their velocity and displacement rate, and in the spleens of Foxp3-GFP/CD11c-YFP dual reporter mice, <10% of the Treg cells had sustained interactions with CD11c-YFPcells ( Figures 3 D, 3E, 3G, and 3H; Movie S9 ). In contrast, Treg cells in the BM had reduced velocity, low displacement, and >50% of Treg cells had sustained interactions with CD11c-YFPcells ( Figures 3 C, 3D, 3F–3H; Movie S10 ). Therefore, the Treg cell populations in the spleen and BM are characterized by fundamental differences in their behavior and those in the BM display unanticipated interactions with PCs, as well as with a CD11c-YFPpopulation.

Previous studies have described a population of mature recirculating B cells in the BM that are closely associated with DCs (), but it is unclear whether DCs are a component of the PC niche. Therefore, mice that express CD11c-YFP/BLIMP1-GFP or CD11c-YFP/Foxp3-GFP were generated to determine whether DCs are localized near to or interact with PCs or Treg cells in the BM. In the cavarial BM of naive CD11c-YFP/BLIMP1-GFP mice, there were numerous large stationary CD11c-YFPpopulations that displayed irregularly shaped dendrites ( Figures 3 A and 3B ), and ∼70% of PCs interacted with a population of CD11c-YFPcells, while 40%–50% of Treg cells interacted with CD11c-YFPcells ( Figure 3 C; Movie S8 ). The use of mixed BM chimeras demonstrated that the CD11c-YFPcells present in the BM are of hematopoietic origin, as wild-type (WT) recipients that were reconstituted with BM from CD11c-YFP mice contained an extensive network of YFPcells in the BM, while YFPcells were rare in irradiated reporter mice that were recipients of WT BM, associated with a 40-fold decrease in the YFP volume ( Figure S3 ). These data support a model in which CD11c-YFPcells, implicated in providing co-stimulation required for PC survival and antibody production (), are a component of the PC niche and form close associations with PCs and Treg cells.

(D–H) Imaging and quantification of Treg cell velocity (D) and CD11c + cell-Treg cell location (E and F) in the spleen and BM of Foxp3-GFP (53 BM Foxp3-GFP cells and 150 splenic Foxp3-GFP cells) (D) or CD11c-YFP/Foxp3-GFP dual (E–G) reporter mice, as well as interaction times between these cells (68 BM Foxp3-GFP cells and 94 splenic Foxp3-GFP cells) (G) and Treg cell movement shown by tracks (H).

(A–C) Intravital imaging of skull BM in BLIMP-GFP/CD11c-YFP dual reporter mice with quantum dot-labeled vasculature (red), with interacting cells highlighted in close up (B). Four mice were imaged, for a total of 83 BLIMP-GFP cells counted for quantification. (C) Quantification of the percentage of cells observed in interactions between CD11c-YFP + cells and BLIMP1-GFP + or Foxp3-GFP + cells in respective dual reporter mice.

The infection-induced loss of PCs in the BM raises questions about which of the regulatory or cellular elements that support the PC niche are disrupted. Because Foxp3-GFPcells are preferentially localized in the endosteal region of naive BM and contribute to the status of the BM as an immune privileged site (), we examined the impact of infection on this population. Intravital imaging of uninfected mice showed Foxp3-GFPTreg cells throughout the BM, predominantly in extravascular sites, but by day 14 post-infection, these cells were largely absent ( Figure 2 A). Flow cytometric analysis confirmed that infection resulted in an ∼80% decrease in Treg cells in the BM ( Figures 2 B and 2C) that mirrored the changes in Treg cell numbers at peripheral sites during acute toxoplasmosis (). The generation of mice that express both BLIMP1-YFP and Foxp3-GFP allowed for simultaneous imaging of PC and Treg cell populations in the BM. While Treg cells exhibited a greater motility than the PCs, these cells have a range of mobility with a large proportion of the Treg cell population displaying a more sessile phenotype ( Movie S2 ). Indeed, the majority of the Foxp3-GFPcells were found closely associated with BLIMP1-YFPPCs ( Figures 2 D–2F), as ∼10% of PCs interact with Treg cells for 4 min or less, while more than 60% of interactions between Foxp3-GFPand BLIMP1-YFPcells were >10 min, with 30% exceeding the 20-min imaging window ( Figure 2 F; Movies S3 and S4 ). In contrast, Foxp3-GFPcells in the spleen exhibited a higher velocity and shorter interaction times with BLIMP1-YFPcells ( Figure 2 F; Movie S5 ). It should be noted that the transfer of polyclonally activated CD8T cells into Foxp3-GFP reporter mice resulted in a small population of CD8T cells in the BM that could be imaged simultaneously with Treg cells. These effector T cells exhibited a higher velocity than that of the resident Treg cell population ( Figure S2 A; Movie S6 ). This internal control indicates that the sessile Treg cell phenotype in the BM was not an artifact associated with sample preparation. Further analysis of the cellular localization of PCs and Treg cells using fixed long bones showed that they were present in areas rich in HSCs (CD150), and PCs were intimately associated with the network of stromal cells that are a source of CXCL12 in the BM ( Figures S2 B and S2C; Movie S7 ). Thus, Treg cells in the BM display a behavior distinct from that observed in the spleen and many are spatially associated with PCs within local niches in the BM.

(D–F) BLIMP1-YFP/Foxp3-GFP dual reporter mice were imaged using intravital 2-photon microscopy of the skull BM. (D and E) BLIMP1-YFP (white), Foxp3-GFP (green), and vasculature (red) expression in BLIMP1-YFP/Foxp3-GFP dual reporter mice reveals short and long term interactions, highlighted in the close up (E). Three mice were imaged for these experiments, with 53 Foxp3-GFP cells recorded in the BM and 104 imaged in the spleen. (F) Quantification of the duration of PC-Treg cell interactions in the spleen and BM.

(B and C) BM from naive or infected WT mice was evaluated by flow cytometry for the presence of Treg cells.

(A) Naive or infected Foxp3-GFP reporter mice were imaged using intravital 2-photon microscopy of the skull BM. The Foxp3-GFP-expressing cells are green (demarcated by the ∗ ) and quantum dots were injected intravenously to label the vasculature red. At least three mice were imaged for each time point.

Microbial infection-induced expansion of effector T cells overcomes the suppressive effects of regulatory T cells via an IL-2 deprivation mechanism.

Changes in the BM, including increased hematopoiesis and reduced lymphopoiesis, are a hallmark of the immune response to numerous pathogens (). Indeed, following infection with the systemic pathogen T. gondii, histological analysis of the BM revealed extensive changes in the cellular composition of this compartment, including increased numbers of hematopoietic progenitors ( Figures 1 A and 1B ). Unexpectedly, resident PC populations, identified based on their oval shape, purple cytoplasm, and characteristic eccentric nucleus with clock-face chromatin and perinuclear clearing, were diminished (large arrows) ( Figures 1 A and 1B). Long-lived PC populations are an independent pool of terminally differentiated B cells maintained through slow homeostatic proliferation with a half-life of >120 days (). PCs express high levels of the transcription factor BLIMP-1 () and in uninfected BLIMP-YFP or BLIMP-GFP mice, 70%–85% of the CD138PCs were positive for the reporter, which indicated sufficient fidelity to visualize PC populations (). Therefore, intravital imaging of the BM in the skull of BLIMP1-YFP reporter mice was used to visualize the location and motility of PC populations in uninfected and infected mice ( Figure 1 C). In naive mice, PCs are localized in clusters adjacent to blood vessels in the endosteal region of the bone and were stationary over the imaging periods used in these studies ( Movie S1 ). However, by 14 days post infection, the PC population was markedly reduced ( Figure 1 C), and flow cytometry confirmed the infection-induced loss of PCs ( Figures 1 D and 1E). This analysis also revealed a decrease of >95% in the total number of pro, pre, immature, and mature recirculating B cells ( Figures S1 A–S1C). Although short-term blockade of B cell lymphopoiesis has minimal effects on mature B cell compartments (), it was possible that these alterations might impact the development of PC populations. To ascertain whether infection reduced an established PC population, mice were immunized with 4-hydroxy-3-nitrophenylacetyl (NP)-ovalbumin. This antigen results in a long-lived NP-specific PC population in the BM, detectable by intracellular staining with NP conjugated to allophycocyanin ( Figure 1 F). During acute T. gondii infection, the number of NP-specific B cells in the spleen was unchanged (data not shown), but there was a decrease in the number of NPPCs in the BM, accompanied by a significant drop in serum NP-specific IgG1 ( Figures 1 F and 1G). However, by the chronic phase of infection, the NPpopulation was restored to its original levels (data not shown) indicating that infection results in the transient loss of a pre-established PC compartment.

(F and G) WT mice were immunized with NP-OVA. Six weeks later, these mice were challenged with T. gondii and evaluated for NP-specific PCs (F) and NP-specific antibody titers (G).

(D and E) BM from naive or infected WT mice was evaluated by flow cytometry (using a “dump” gate to eliminate CD3 + , F4/80 + , and/or Gr1 + potential contaminating cells) for the presence of PCs.

(C) Naive or infected BLIMP1-YFP reporter mice were imaged using intravital 2-photon microscopy of the skull BM. The BLIMP1-YFP-expressing cells are yellow and quantum dots were injected intravenously to label the vasculature red. At least three mice were imaged for each time point.

(B) Day 14 T. gondii infected mouse. Medullary vascular sinuses (V) are surrounded by increased numbers of hematopoietic progenitors characterized by hyperchromatic nuclei. Few mature neutrophils (arrowheads) and immature band neutrophils (arrows) are observed. Mature PCs are not identified. Bone cortex (C).

(A) Naive mouse. The marrow cavity contains vascular sinuses (V) surrounded by mature neutrophils (arrowheads) admixed with predominantly myelopoietic precursors and few mature PCs (arrows). Bone cortex (C).

Homeostasis of peripheral B cells in the absence of B cell influx from the bone marrow.

Blimp-1, a novel zinc finger-containing protein that can drive the maturation of B lymphocytes into immunoglobulin-secreting cells.

Blimp-1 controls plasma cell function through the regulation of immunoglobulin secretion and the unfolded protein response.

Discussion