CD8 T cells induce T-bet-dependent migration toward CXCR3 ligands by differentiated B cells produced during responses to alum-protein vaccines.

Antibody-forming cells (AFCs) expressing the chemokine receptor CXCR3 are recruited to sites of inflammation where they help clear pathogens but may participate in autoimmune diseases. Here we identify a mechanism that induces CXCR3 expression by AFC and germinal center (GC) B cells. This happens when CD8 T cells are recruited into CD4 T cell-dependent B-cell responses. Ovalbumin-specific CD4 T cells (OTII) were transferred alone or with ovalbumin-specific CD8 T cells (OTI) and the response to subcutaneous alum-precipitated ovalbumin was followed in the draining lymph nodes. OTII cells alone induce T helper 2-associated class switching to IgG1, but few AFC or GC B cells express CXCR3. By contrast, OTI-derived IFN-γ induces most responding GC B cells and AFCs to express high levels of CXCR3, and diverse switching to IgG2a, IgG2b, with some IgG1. Up-regulation of CXCR3 by GC B cells and AFCs and their migration toward its ligand CXCL10 are shown to depend on B cells' intrinsic T-bet, a transcription factor downstream of the IFN-γR signaling. This model clarifies how precursors of long-lived AFCs and memory B cells acquire CXCR3 that causes their migration to inflammatory foci.

CXCR3 promotes the production of IgG1 autoantibodies but is not essential for the development of lupus nephritis in NZB/NZW mice.

OBJECTIVE: Autoantibody immune complexes and cellular infiltrates drive nephritis in patients with systemic lupus erythematosus (SLE) and in murine lupus. The chemokine receptor CXCR3 is assumed to promote cellular infiltration of inflamed tissues. Moreover, CXCR3 deficiency ameliorates lupus nephritis in the MRL/MpJ-Fas(lpr) (MRL/lpr) mouse model of SLE. Hence, CXCR3 blockade has been suggested as a novel therapeutic strategy for the treatment of lupus nephritis. We undertook this study to test the effect of CXCR3 in the (NZB × NZW)F(1) (NZB/NZW) mouse model of SLE. METHODS: CXCR3(-/-) NZB/NZW mice were generated and monitored for survival, proteinuria, and kidney infiltration. Anti-double-stranded DNA (anti-dsDNA) and total IgG1, IgG2a, and IgG2b antibody levels were determined by enzyme-linked immunosorbent assay. T cell and plasma cell infiltrates in the kidneys and interferon-γ production were determined by flow cytometry. Plasma cell infiltrates were measured using enzyme-linked immunospot assay. Kidney tissue was evaluated for pathologic changes. RESULTS: CXCR3(-/-) NZB/NZW mice exhibited reduced production of total and anti-dsDNA antibodies of the IgG1 subclass, but had normal titers of IgG2a and IgG2b antibodies compared to CXCR3(+/+) NZB/NZW mice. Cellular infiltrates and glomerulonephritis were not reduced in CXCR3(-/-) mice. CONCLUSION: CXCR3 has an effect on (auto)antibody production but is not essential for lupus pathogenesis in NZB/NZW mice, indicating that the effect of CXCR3 on the development of kidney disease varies between MRL/lpr and NZB/NZW mice. These results suggest that CXCR3-dependent and -independent mechanisms can mediate lupus nephritis. Hence, therapeutic CXCR3 blockade could be beneficial for only a subgroup of patients with SLE.

Bone marrow of NZB/W mice is the major site for plasma cells resistant to dexamethasone and cyclophosphamide: implications for the treatment of autoimmunity.

Antibodies contribute to the pathogenesis of many chronic inflammatory diseases, including autoimmune disorders and allergies. They are secreted by proliferating plasmablasts, short-lived plasma cells and non-proliferating, long-lived memory plasma cells. Memory plasma cells refractory to immunosuppression are critical for the maintenance of both protective and pathogenic antibody titers. Here, we studied the response of plasma cells in spleen, bone marrow and inflamed kidneys of lupus-prone NZB/W mice to high-dose dexamethasone and/or cyclophosphamide. BrdU+, dividing plasmablasts and short-lived plasma cells in the spleen were depleted while BrdU- memory plasma cells survived. In contrast, all bone marrow plasma cells including anti-DNA secreting cells were refractory to both drugs. Unlike bone marrow and spleen, which showed a predominance of IgM-secreting plasma cells, inflamed kidneys mainly accommodated IgG-secreting plasma cells, including anti-DNA secreting cells, some of which survived the treatments. These results indicate that the bone marrow is the major site of memory plasma cells resistant to treatment with glucocorticoids and anti-proliferative drugs, and that inflamed tissues and secondary lymphoid organs can contribute to the autoreactive plasma cell memory. Therefore, new strategies targeting autoreactive plasma cell memory should be considered. This could be the key to finding a curative approach to the treatment of chronic inflammatory autoantibody-mediated diseases.

Megakaryocytes constitute a functional component of a plasma cell niche in the bone marrow.

Long-lived plasma cells in the bone marrow produce memory antibodies that provide immune protection persisting for decades after infection or vaccination but can also contribute to autoimmune and allergic diseases. However, the composition of the microenvironmental niches that are important for the generation and maintenance of these cells is only poorly understood. Here, we demonstrate that, within the bone marrow, plasma cells interact with the platelet precursors (megakaryocytes), which produce the prominent plasma cell survival factors APRIL (a proliferation-inducing ligand) and IL-6 (interleukin-6). Accordingly, reduced numbers of immature and mature plasma cells are found in the bone marrow of mice deficient for the thrombopoietin receptor (c-mpl) that show impaired megakaryopoiesis. After immunization, accumulation of antigen-specific plasma cells in the bone marrow is disturbed in these mice. Vice versa, injection of thrombopoietin allows the accumulation and persistence of a larger number of plasma cells generated in the course of a specific immune response in wild-type mice. These results demonstrate that megakaryocytes constitute an important component of the niche for long-lived plasma cells in the bone marrow.

HAX1 deficiency: impact on lymphopoiesis and B-cell development.

HAX1 was originally described as HS1-associated protein with a suggested function in receptor-mediated apoptotic and proliferative responses of lymphoid cells. Recent publications refer to a complex and multifunctional role of this protein. To investigate the in vivo function of HAX1 (HS1-associated protein X1) in B cells, we generated a Hax1-deficient mouse strain. Targeted deletion of Hax1 resulted in premature death around the age of 12 wk accompanied by a severe reduction of lymphocytes in spleen, thymus and bone marrow. In the bone marrow, all B-cell populations were lost comparably. In the spleen, B220(+) cells were reduced by almost 70%. However, as investigated by adoptive transfer experiments, this impairment is not exclusively B-cell intrinsic and we hypothesize that a HAX1-deficient environment cannot sufficiently provide the essential factors for proper lymphocyte development, trafficking and survival. Hax1(-/-) B cells show a significantly reduced expression of CXCR4, which might have an influence on the observed defects in B-cell development.

SHIP is required for a functional hematopoietic stem cell niche.

SH2-domain-containing inositol 5'-phosphatase-1 (SHIP) deficiency significantly increases the number of hematopoietic stem cells (HSCs) present in the bone marrow (BM). However, the reconstitution capacity of these HSCs is severely impaired, suggesting that SHIP expression might be an intrinsic requirement for HSC function. To further examine this question, we developed a model in which SHIP expression is ablated in HSCs while they are resident in a SHIP-competent milieu. In this setting, we find that long-term repopulation by SHIP-deficient HSCs is not compromised. Moreover, SHIP-deficient HSCs from this model repopulate at levels comparable with wild-type HSCs upon serial transfer. However, when HSCs from mice with systemic ablation of SHIP are transplanted, they are functionally compromised for repopulation. These findings demonstrate that SHIP is not an intrinsic requirement for HSC function, but rather that SHIP is required for the BM milieu to support functionally competent HSCs. Consistent with these findings, cells that comprise the BM niche express SHIP and SHIP deficiency profoundly alters their function.

Short-lived plasmablasts and long-lived plasma cells contribute to chronic humoral autoimmunity in NZB/W mice.

The current view holds that chronic autoimmune diseases are driven by the continuous activation of autoreactive B and T lymphocytes. However, despite the use of potent immunosuppressive drugs designed to interfere with this activation the production of autoantibodies often persists and contributes to progression of the immunopathology. In the present study, we analyzed the life span of (auto)antibody-secreting cells in the spleens of NZB x NZW F1 (NZB/W) mice, a murine model of systemic lupus erythematosus. The number of splenic ASCs increased in mice aged 1-5 mo and became stable thereafter. Less than 60% of the splenic (auto)antibody-secreting cells were short-lived plasmablasts, whereas 40% were nondividing, long-lived plasma cells with a half-life of >6 mo. In NZB/W mice and D42 Ig heavy chain knock-in mice, a fraction of DNA-specific plasma cells were also long-lived. Although antiproliferative immunosuppressive therapy depleted short-lived plasmablasts, long-lived plasma cells survived and continued to produce (auto)antibodies. Thus, long-lived, autoreactive plasma cells are a relevant target for researchers aiming to develop curative therapies for autoimmune diseases.

Stromal niches, plasma cell differentiation and survival.

Contacts made with other cells and stroma have a major impact on proliferation, differentiation, survival, migration and immunoglobulin class switching of plasma cell precursors as well as on the lifespan of the antibody-secreting cells. Induction of tissue-specific chemokine receptors and adhesion molecules directs migratory plasma cell precursors to tissues close to those in which the original immune stimulation occurred. This mechanism focuses the production of specific antibodies within a particular type of tissue, thus providing a means for the most efficient protection against tissue-specific pathogens. Relocation does not apply to long-lived plasma cells responsible for sustained titers of high-affinity systemic antibody. These are formed in germinal centers and migrate to specific niches in the bone marrow that support their further differentiation and long-term survival.

Long-lived plasma cells in immunity and immunopathology.

Following tetanus vaccination, a wave of antibody-secreting cells appear in the peripheral blood composed of vaccine-specific, migratory plasmablasts and plasma cells secreting antibodies specific for other antigens. The latter probably were tissue resident plasma cells formed in earlier immune responses that are mobilized due to competition with the newly formed tetanus-specific plasmablasts. Newly formed plasma cells secreting antibodies specific for a particular antigen/vaccine are accommodated in the bone marrow likely at the global expense of the pre-existing long-lived plasma cell population providing humoral memory for other antigens. Plasmablasts but not mature plasma cells are attracted by the ligands for the chemokine receptors CXCR4 and CXCR3. While CXCR4 and its cognate ligand is important for plasma cell homing to the bone marrow, CXCR3 and its ligand IP10 are likely to be involved in attracting them to inflamed tissue. In NZB/W mice, a model for systemic lupus, long-lived autoreactive plasma cells are present not only in bone marrow, but also in inflamed tissues and spleen. Autoreactive plasma cells in the spleen are present long before the onset of the disease, suggesting that these cells contribute to induction of immunopathology.

Long-lived plasma cells are early and constantly generated in New Zealand Black/New Zealand White F1 mice and their therapeutic depletion requires a combined targeting of autoreactive plasma cells and their precursors.

INTRODUCTION: Autoantibodies contribute significantly to the pathogenesis of systemic lupus erythematosus (SLE). Unfortunately, the long-lived plasma cells (LLPCs) secreting such autoantibodies are refractory to conventional immunosuppressive treatments. Although generated long before the disease becomes clinically apparent, it remains rather unclear whether LLPC generation continues in the established disease. Here, we analyzed the generation of LLPCs, including autoreactive LLPCs, in SLE-prone New Zealand Black/New Zealand White F1 (NZB/W F1) mice over their lifetime, and their regeneration after depletion. METHODS: Bromodeoxyuridine pulse-chase experiments in mice of different ages were performed in order to analyze the generation of LLPCs during the development of SLE. LLPCs were enumerated by flow cytometry and autoreactive anti-double-stranded DNA (anti-dsDNA) plasma cells by enzyme-linked immunospot (ELISPOT). For analyzing the regeneration of LLPCs after depletion, mice were treated with bortezomib alone or in combination with cyclophosphamide and plasma cells were enumerated 12 hours, 3, 7, 11 and 15 days after the end of the bortezomib cycle. RESULTS: Autoreactive LLPCs are established in the spleen and bone marrow of SLE-prone mice very early in ontogeny, before week 4 and before the onset of symptoms. The generation of LLPCs then continues throughout life. LLPC counts in the spleen plateau by week 10, but continue to increase in the bone marrow and inflamed kidney. When LLPCs are depleted by the proteasome inhibitor bortezomib, their numbers regenerate within two weeks. Persistent depletion of LLPCs was achieved only by combining a cycle of bortezomib with maintenance therapy, for example cyclophosphamide, depleting the precursors of LLPCs or preventing their differentiation into LLPCs. CONCLUSIONS: In SLE-prone NZB/W F1 mice, autoreactive LLPCs are generated throughout life. Their sustained therapeutic elimination requires both the depletion of LLPCs and the inhibition of their regeneration.

The proteasome inhibitor bortezomib depletes plasma cells and protects mice with lupus-like disease from nephritis.

Autoantibody-mediated diseases like myasthenia gravis, autoimmune hemolytic anemia and systemic lupus erythematosus represent a therapeutic challenge. In particular, long-lived plasma cells producing autoantibodies resist current therapeutic and experimental approaches. Recently, we showed that the sensitivity of myeloma cells toward proteasome inhibitors directly correlates with their immunoglobulin synthesis rates. Therefore, we hypothesized that normal plasma cells are also hypersensitive to proteasome inhibition owing to their extremely high amount of protein biosynthesis. Here we show that the proteasome inhibitor bortezomib, which is approved for the treatment of multiple myeloma, eliminates both short- and long-lived plasma cells by activation of the terminal unfolded protein response. Treatment with bortezomib depleted plasma cells producing antibodies to double-stranded DNA, eliminated autoantibody production, ameliorated glomerulonephritis and prolonged survival of two mouse strains with lupus-like disease, NZB/W F1 and MRL/lpr mice. Hence, the elimination of autoreactive plasma cells by proteasome inhibitors might represent a new treatment strategy for antibody-mediated diseases.

Phosphoproteomic analysis of rat liver by high capacity IMAC and LC-MS/MS.

Proper liver function is crucial for metabolism control and to clear toxic substances from the bloodstream. Many small-molecule therapeutics accumulate in the liver, negatively impacting liver function and often resulting in hepatotoxicity and cell death. Several analytical methods are currently utilized to evaluate hepatotoxicity and monitor liver function. To date, none of these methods have specifically targeted protein phosphorylation-mediated signal transduction pathways which should be altered in response to toxic effects of small molecule therapeutics. To develop novel assays to probe specific signaling pathways in the liver, identification and quantification of specific protein phosphorylation sites in this complex organ is necessary. Here, we have utilized an optimized immobilized metal affinity chromatography (IMAC) protocol to enrich phosphorylated peptides from a tryptic digest of proteins isolated from whole liver lysate. LC-MS/MS analysis of IMAC-enriched peptides resulted in the identification of more than 300 phosphorylation sites from over 200 proteins in rat liver, a significant advance over previously published analyses of the liver phosphoproteome. Previously characterized phosphorylation sites and potentially novel sites were identified in the current study, including sites on proteins implicated in metabolism regulation, transcription, translation, and canonical signaling pathways. Moreover, protein phosphorylation analysis was performed without prior fractionation of the sample, enabling analysis of small sample amounts while minimizing analysis time, potentially allowing for high-throughput assays to be performed with this methodology. From these data, it appears that this methodology can be used to identify new phosphorylation sites and, in combination with a stable isotope-labeling step, to investigate the effects of liver diseases, cancer and evaluate potential toxicology of new drug substances.

Adaptation of humoral memory.

Immunological memory, as provided by antibodies, depends on the continued presence of antibody-secreting cells, such as long-lived plasma cells of the bone marrow. Survival niches for these memory plasma cells are limited in number. In an established immune system, acquisition of new plasma cells, generated in response to recent pathogenic challenges, requires elimination of old memory plasma cells. Here, we review the adaptation of plasma cell memory to new pathogens. This adaptation is dependent upon the influx of plasmablasts, generated in a secondary systemic immune reaction, into the pool of memory plasma cells, the efficiency of competition of new plasmablasts with old plasma cells, and the frequency of infection with novel pathogens. To maintain old plasma cells at frequencies high enough to provide protection and to accommodate as many specificities as possible, an optimal influx rate per infection exists. This optimal rate is approximately three times higher than the minimal number of plasma cells providing protection. Influx rates of plasmablasts generated by vaccination approximately match this optimum level. Furthermore, the observed stability of serum concentrations of vaccine-specific antibodies implies that the influxing plasmablasts mobilize a similar number of plasma cells and that competitive infectious challenges are not more frequent than once per month.