Static and dynamic components synergize to form a stable survival niche for bone marrow plasma cells

In the bone marrow (BM), memory plasma cells (PCs) survive for long time periods in dedicated microenvironmental survival niches, resting in terms of proliferation. Several cell types, such as eosinophils and reticular stromal cells, have been reported to contribute to the survival niche of memory PCs. However, until now it has not been demonstrated whether the niche is formed by a fixed cellular microenvironment. By intravital microscopy, we provide for the first time evidence that the direct contacts formed between PCs and reticular stromal cells are stable in vivo, and thus the PCs are sessile in their niches. The majority (～80%) of PCs directly contact reticular stromal cells in a non‐random fashion. The mesenchymal reticular stromal cells in contact with memory PCs are not proliferating. On the other hand, we show here that eosinophils in the vicinity of long‐lived PCs are vigorously proliferating cells and represent a dynamic component of the survival niche. In contrast, if eosinophils are depleted by irradiation, newly generated eosinophils localize in the vicinity of radiation‐resistant PCs and the stromal cells. These results suggest that memory PC niches may provide attraction for eosinophils to maintain stability with fluctuating yet essential accessory cells.     

CXCR4, but not CXCR3, drives CD8+ T‐cell entry into and migration through the murine bone marrow

The BM serves as a blood‐forming organ, but also supports the maintenance and immune surveillance function of many T cells. Yet, in contrast to other organs, little is known about the molecular mechanisms that drive T‐cell migration to and localization inside the BM. As BM accumulates many CXCR3‐expressing memory CD8⁺ T cells, we tested the involvement of this chemokine receptor, but found that CXCR3 is not required for BM entry. In contrast, we could demonstrate that CXCR4, which is highly expressed on both naive and memory CD8⁺ T cells in BM, is critically important for homing of all CD8⁺ T‐cell subsets to the BM in mice. Upon entry into the BM parenchyma, both naïve and memory CD8⁺ T cells locate close to sinusoidal vessels. Intravital imaging experiments revealed that CD8 T cells are surprisingly immobile and we found that they interact with ICAM‐1+VCAM‐1+BP‐1+ perivascular stromal cells. These cells are the major source of CXCL12, but also express key survival factors and maintenance cytokines IL‐7 and IL‐15. We therefore conclude that CXCR4 is not only crucial for entry of CD8⁺ T cells into the BM, but also controls their subsequent localization toward BM niches that support their survival.     

Stromal and hematopoietic cells in secondary lymphoid organs: partners in immunity

Secondary lymphoid organs (SLOs), including lymph nodes, Peyer's patches, and the spleen, have evolved to bring cells of the immune system together. In these collaborative environments, lymphocytes scan the surfaces of antigen-presenting cells for cognate antigens, while moving along stromal networks. The cell-cell interactions between stromal and hematopoietic cells in SLOs are therefore integral to the normal functioning of these tissues. Not only do stromal cells physically construct SLO architecture but they are essential for regulating hematopoietic populations within these domains. Stromal cells interact closely with lymphocytes and dendritic cells, providing scaffolds on which these cells migrate, and recruiting them into niches by secreting chemokines. Within lymph nodes, stromal cell-ensheathed conduit networks transport small antigens deep into the SLO parenchyma. More recently, stromal cells have been found to induce peripheral CD8⁺ T-cell tolerance and control the extent to which newly activated T cells proliferate within lymph nodes. Thus, stromal-hematopoietic crosstalk has important consequences for regulating immune cell function within SLOs. In addition, stromal cell interactions with hematopoietic cells, other stroma, and the inflammatory milieu have profound effects on key stromal functions. Here, we examine ways in which these interactions within the lymph node environment influence the adaptive immune response.     

Finding a home for plasma cells — A niche to survive

Long‐term survival of plasma cells (PCs) and sustained antibody secretion require a specific microenvironment that provides the appropriate prosurvival signals. This plasma cell niche involves both mesenchymal and hematopoietic components. Although a consensus exists about the essential contribution of CXCL12⁺ stromal cells in this environment, the identity of the hematopoietic participants remains a matter of debate. In this issue of the European Journal of Immunology, Zehentmeier et al. [Eur. J. Immunol. 2014. 44: 2306‐2317] aim to identify the components of the bone marrow plasma cell niche in C57BL/6 mice in an unbiased manner by using a streamlined analysis of histological colocalization data. Apart from stromal cells, the authors showed that eosinophils are the only population specifically localized in the vicinity of PCs. In addition, the authors performed intravital imaging demonstrating that PCs are sessile and form stable contacts with reticular stromal cells. This work opens the door to a more rational approach to characterize the plasma cell niche.     

Extracellular Vesicles and Cell–Cell Communication in the Cornea

One question that has intrigued cell biologists for many years is, “How do cells interact to influence one another's activity?” The discovery of extracellular vesicles (EVs) and the fact that they carry cargo, which directs cells to undergo changes in morphology and gene expression, has revolutionized this field of research. Little is known regarding the role of EVs in the cornea; however, we have demonstrated that EVs isolated from corneal epithelial cells direct corneal keratocytes to initiate fibrosis. Intriguingly, our data suggest that EVs do not penetrate epithelial basement membrane (BM), perhaps providing a mechanism explaining the importance of BM in the lack of scarring in scrape wounds. Since over 100-million people worldwide suffer from visual impairment as a result of corneal scarring, the role of EVs may be vital to understanding the mechanisms of wound repair. Therefore, we investigated EVs in ex vivo and in vivo-like three-dimensional cultures of human corneal cells using transmission electron microscopy. Some of the major findings were all three major cell types (epithelial, fibroblast, and endothelial cells) appear to release EVs, EVs can be identified using TEM, and EVs appeared to be involved in cell–cell communication. Interestingly, while our previous publication suggests that EVs do not penetrate the epithelial BM, it appears that EVs penetrate the much thicker endothelial BM (Descemet's membrane). These findings indicate the huge potential of EV research in the cornea and wound healing, and suggest that during homeostasis the endothelium and stromal cells are in communication. Anat Rec, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.     

Characterization of splenic CD21hi T2 B cells

B cell development is a highly regulated process that initiates in the bone marrow (BM) of adult mice. After reaching the IgM⁺ immature stage in the BM, these B cells migrate to the spleen to complete maturation and incorporation into the long-lived peripheral lymphocyte pool. Studies have identified these splenic immature B cells, and have further attempted to delineate the sequence whereby they transition into mature B cells. As such, these immature splenic populations are termed transitional B cells and have been the focus of intense study. The review summarizes the phenotype and currently known functions of the four putative transitional B cell subsets identified to date. Although most appear to represent short-lived transitional B cells, the CD21^hi T2 B cell population exhibits a number of qualities that question its label as a transitional B cell subset.     

Bone marrow stromal cells as targets for gene therapy

The bone marrow (BM) is composed of the non-adherent hematopoietic and adherent stromal cell compartment. This adherent BM stromal cell fraction contains pluripotent mesenchymal stem cells (MSCs) and differentiated mesenchymal BM stromal cells. The MSCs self-renew by proliferation while maintaining their stem-cell phenotype and give rise to the differentiated stromal cells which belong to the osteogenic, chondrogenic, adipogenic, myogenic and fibroblastic lineages. A more primitive adherent stem cell was recently identified, the multipotent adult progenitor cell (MAPC) or mesodermal progenitor cell, which co-purifies with MSCs. These MAPCs differentiate into MSCs, endothelial, epithelial and even hematopoietic cells. BM stroma cells, including the primitive pluripotent MSCs and MAPCs, are attractive targets for cell and gene therapy. The BM stromal cell population and its multipotent stem cells can be engineered to secrete a series of different proteins in vitro and in vivo that could potentially treat a variety of serum protein deficiencies and other genetic or acquired diseases, including bone, cartilage and BM stromal disorders or even cancer.     

Distinct phenotypes of plasma cells in spleen and bone marrow of autoimmune NOD.B10.H2b mice

Long-lived plasma cells (PCs) residing in the bone marrow (BM) are important producers of protective antibodies. However, when reacting against self-antigens, these PCs produce autoantibodies that contribute to progression of autoimmune diseases such as Sjögren's syndrome (SS). By using a murine model of primary SS, the NOD.B10.H2b mice, we characterized phenotype and generation of PCs at different stages of the pSS disease progression. In general, the PC population found in the NOD.B10.H2b mice expressed high amounts of MHCII, IgG, and BrdU. We further demonstrate the presence of both short- and long-lived PCs in autoimmune spleen and in autoimmune BM. A long-lived PC subset was also found in the spleen and BM of non-autoimmune BALB/c mice, which have not been treated with any immunological agent. Quantitative investigation of splenic and BM PCs revealed that in the NOD.B10.H2 mice, splenic PCs migrate not only to the BM but possibly also to the sites of inflammation. Finally, BM in the aged NOD.B10.H2b mice (40-week-old) presented significantly higher quantities of long-lived PCs than BM of BALB/c mice.     

GL7 defines the cycling stage of pre-B cells in murine bone marrow.

We have identified a novel subset of early B lineage cells in the mouse bone marrow (BM) by GL7 expression on cell surface. GL7(+)B220(low) BM cells have a large cell size and are CD43(-to low), CD95(-), Sca-1(-), I-A(low), IgM(-) and IgD(-), suggesting that they are large pre-B cells. These BM cells express lambda5 and VpreB but not terminal deoxytransferase (TdT) and Bcl-2, and approximately 50 % of them are in cell cycle. This fraction was not detected in BM cells of Rag-1-deficient and Scid mice, supporting that GL7(+)B220(low) BM cells belong to fraction C' and D according to Hardy's criteria or to an early large pre-B-II fraction according to Melchers-Rolink's criteria. Furthermore, GL7(+)B220(low) BM cells can differentiate into IgM(+) immature B cells in co-culture with stromal cells. These results suggest that B lymphocytes pass through the GL7(+) pre-B cell stage during differentiation in the BM. Thus, GL7 is the critical marker to define the proliferation stage of large pre-B cells.     

Splenic stromal cells mediate IL‐7 independent adult lymphoid tissue inducer cell survival

Lymphoid tissue inducer cells (LTi) play an important role in the development of lymphoid tissue in embryos. Adult CD4⁺CD3^? LTi‐like cells present a similar phenotype and gene expression to their embryonic counterpart and have important roles in CD4⁺ T‐cell memory and lymphoid tissue recovery following viral infection. However, adult LTi‐like cells are heterogeneous populations and the factors that regulate their survival and accumulation within secondary lymphoid organs remain unclear, in particular whether the T‐zone stroma is involved. Here we report the identification and characterization of a distinct subset of podoplanin⁺ murine splenic stromal cells that support adult LTi‐like cell survival. We have identified and isolated CD45^?podoplanin⁺ stromal cell populations which have a similar but distinct phenotype to T‐zone reticular cells in LN. CD45^?podoplanin⁺ fibroblast‐like cells mediate LTi‐like cell survival in vitro; surprisingly this was not dependent upon IL‐7 as revealed through blocking Ab experiments and studies using LTi‐like cells unable to respond to γ chain cytokines. Our findings show that adult LTi‐like cells require extrinsic signals from podoplanin⁺ splenic stromal cells to survive and suggest that IL‐7 is not necessary to mediate their survival in the adult spleen.     

The effects of microenvironment and internal programming on plasma cell survival

Two populations of plasma cells (PCs) are formed after immunization. A short-lived population in the spleen and lymph nodes provides rapid protection. A long-lived population, mainly in the bone marrow, provides lasting immunity. The mechanisms responsible for the differences in PCs life span remain largely unknown. The goal of the current study was to compare the intrinsic survival capacity of isolated short-lived (spleen) versus long-lived (bone marrow) PCs. We approached this question by using a previously established in vitro model that measures PC survival in a supportive stromal environment. Regardless of the tissue source or isolation time point after immunization, the two PC populations showed similar intrinsic ability to survive in vitro. To test differences in the stromal microenvironments, stromal cells from marrow, spleen or lymph nodes were evaluated for ability to support PCs survival. Survival of isolated PC was always greater when co-cultured with marrow stromal cells compared with those from spleen (or lymph node) despite the finding that IL-6, necessary for PC survival in culture, was secreted by all three stromal cell sources. Additionally, low expression of B-cell-activating factor belonging to the tumor necrosis factor-family was detected in all three stromal isolates. In contrast, marrow stromal cells were distinguished by cell-surface phenotype and CXC chemokine ligand (CXCL)12, IL-7 and stem cell factor expression. Although CXCL12 has been suggested as a possible survival factor for PC, addition or neutralization of CXCL12 had minimal effect on PC survival. We conclude the mechanisms regulating PC longevity appear extrinsically driven and marrow favored, but the factors that give marrow stromal cells a unique advantage remain unknown.     

Enrichment of IL-12–Producing Plasmacytoid Dendritic Cells in Donor Bone Marrow Grafts Enhances Graft-versus-Leukemia Activity in Allogeneic Hematopoietic Stem Cell Transplantation

A critical question in the field of allogeneic hematopoietic stem cell transplantation (HSCT) is how to enhance graft-versus-leukemia (GVL) activity while limiting graft-versus-host-disease (GVHD). We have previously reported that donor bone marrow (BM) precursors of plasmacytoid dendritic cells (pre-pDCs) can polarize donor T cells toward Th1 immunity and augment the GVL activity of donor T cells while attenuating their GVHD activity in a murine model of allogeneic HSCT. Clinical data on the role of donor pre-pDCs and conventional DCs (cDCs) on transplantation outcomes has been conflicting. To test the effect of increasing the proportion of pre-pDCs versus cDCs in a BM graft, we enriched CD11b⁻ pDCs by selectively depleting the CD11b⁺ myeloid DC (mDC) population from BM using FACS sorting in a murine model of allogeneic BM transplantation. Donor T cell expansion and GVL activity were greater in mice that received BM depleted of mDCs compared with mice that received undepleted BM. GVHD was not increased by depleting mDCs. To examine the mechanism through which mDC depletion enhances the GVL activity of donor T cells, we used BM and pDCs from IL-12p40KO mice, and found that the increased GVL activity of mDC-depleted BM was IL-12–dependent. This study indicates that a clinically translatable strategy of engineering the DC content of grafts can improve clinical outcomes in allogeneic HSCT through the regulation of donor T cell activation and GVL activity.     

The distribution of CD10 (NEP 24.11, CALLA) in humans and mice is similar in non-lymphoid organs but differs within the hematopoietic system: absence on murine T and B lymphoid progenitors

Prior studies in the human have implied an important function for CD10 (CALLA, neutral endopeptidase 24.11) in early lymphoid development. To examine the role of this ectoenzyme in an experimental system, a rat mAb specific for mouse CD10, termed R103, was generated. Immunohistological and flow cytometric analyses indicate that the distribution of CD10 in non-lymphoid anatomical compartments is virtually identical in human and mouse. However, CD10 expression within the hematopoietic system is strikingly different. In contrast to human spleen, lymph node and thymus, the corresponding mouse organs contain no detectable CD10⁺ cells. Mouse granulocytes, unlike human granulocytes, also lack CD10 expression. Five-color flow cytometric studies of adult bone marrow (BM) from C57BL/6 and BALB/c mice with mAb specific for CD43, B220, HSA, BP-1 and immunoglobulin M fail to detect any significant number of CD10⁺ cells at pro-B, pre-B or B cell stages. In addition, lymphoid cells in both (rIL-7) independent and rIL-7-dependent in vitro pro-B cell cultures lack CD10 expression. Consistent with this result, CD10 mRNA is not detected. Unlike the AA4.1⁺ population from day 13 and 14 fetal liver, the CD10⁺ subset is unable to reconstitute T and B lymphoid compartments in RAG-2^?/? mice. Nevertheless, mouse CD10 is readily found on BM stromal elements known to support early B lineage lymphoid development. Given the common expression of CD10 on human and mouse BM stromal elements, this enzyme may have an important function in the stromal cell-dependent phase of hematopoiesis.     

Distinct phenotypes of plasma cells in spleen and bone marrow of autoimmune NOD.B10.H2b mice

Long-lived plasma cells (PCs) residing in the bone marrow (BM) are important producers of protective antibodies. However, when reacting against self-antigens, these PCs produce autoantibodies that contribute to progression of autoimmune diseases such as Sj?gren's syndrome (SS). By using a murine model of primary SS, the NOD.B10.H2b mice, we characterized phenotype and generation of PCs at different stages of the pSS disease progression. In general, the PC population found in the NOD.B10.H2b mice expressed high amounts of MHCII, IgG, and BrdU. We further demonstrate the presence of both short- and long-lived PCs in autoimmune spleen and in autoimmune BM. A long-lived PC subset was also found in the spleen and BM of non-autoimmune BALB/c mice, which have not been treated with any immunological agent. Quantitative investigation of splenic and BM PCs revealed that in the NOD.B10.H2 mice, splenic PCs migrate not only to the BM but possibly also to the sites of inflammation. Finally, BM in the aged NOD.B10.H2b mice (40-week-old) presented significantly higher quantities of long-lived PCs than BM of BALB/c mice.     

T-bet-expressing B cells contribute to the autoreactive plasma cell pool in Lyn-/- mice

Systemic Lupus Erythematosus (SLE) is characterized by pathogenic autoantibodies against nucleic acid-containing antigens. Understanding which B-cell subsets give rise to these autoantibodies may reveal therapeutic approaches for SLE that spare protective responses. Mice lacking the tyrosine kinase Lyn, which limits B and myeloid cell activation, develop lupus-like autoimmune diseases characterized by increased autoreactive plasma cells (PCs). We used a fate-mapping strategy to determine the contribution of T-bet⁺ B cells, a subset thought to be pathogenic in lupus, to the accumulation of PCs and autoantibodies in Lyn^-/- mice. Approximately, 50% of splenic PCs in Lyn^-/- mice originated from T-bet⁺ cells, a significant increase compared to WT mice. In vitro, splenic PCs derived from T-bet⁺ B cells secreted both IgM and IgG anti-dsDNA antibodies. To determine the role of these cells in autoantibody production in vivo, we prevented T-bet⁺ B cells from differentiating into PCs or class switching in Lyn^-/- mice. This resulted in a partial reduction in splenic PCs and anti-dsDNA IgM and complete abrogation of anti-dsDNA IgG. Thus, T-bet⁺ B cells make an important contribution to the autoreactive PC pool in Lyn^-/- mice.