Maintenance Of Plasma Cells Research Articles

Long-term in vitro maintenance of plasma cells in a hydrogel-enclosed human bone marrow microphysiological 3D model system

Plasma cells (PCs) in bone marrow (BM) play an important role in both protective and pathogenic humoral immune responses, e.g. in various malignant and non-malignant diseases such as multiple myeloma, primary and secondary immunodeficiencies and autoimmune diseases. Dedicated microenvironmental niches in the BM provide PCs with biomechanical and soluble factors that support their long-term survival. There is a high need for appropriate and robust model systems to better understand PCs biology, to develop new therapeutic strategies for PCs-related diseases and perform targeted preclinical studies with high predictive value. Most preclinical data have been derived from in vivo studies in mice, as in vitro studies of human PCs are limited due to restricted survival and functionality in conventional 2D cultures that do not reflect the unique niche architecture of the BM. We have developed a microphysiological, dynamic 3D BM culture system (BM-MPS) based on human primary tissue (femoral biopsies), mechanically supported by a hydrogel scaffold casing. While a bioinert agarose casing did not support PCs survival, a photo-crosslinked collagen-hyaluronic acid (Col-HA) hydrogel preserved the native BM niche architecture and allowed PCs survival in vitro for up to 2 weeks. Further, the Col-HA hydrogel was permissive to lymphocyte migration into the microphysiological system´s circulation. Long-term PCs survival was related to the stable presence in the culture of soluble factors, as APRIL, BAFF, and IL-6. Increasing immunoglobulins concentrations in the medium confirm their functionality over culture time. To the best of our knowledge, this study is the first report of successful long-term maintenance of primary-derived non-malignant PCs in vitro. Our innovative model system is suitable for in-depth in vitro studies of human PCs regulation and exploration of targeted therapeutic approaches such as CAR-T cell therapy or biologics.

Unique and redundant roles of mouse BCMA, TACI, BAFF, APRIL, and IL-6 in supporting antibody-producing cells in different tissues

Antibody-producing plasma cells fuel humoral immune responses. They also contribute to autoimmune diseases such as systemic lupus erythematosus or IgA nephropathy. Interleukin-6 and the tumor necrosis factor (TNF) family ligands BAFF (B cell-activating factor) and APRIL (a proliferation-inducing ligand) participate in plasma cell survival. BAFF binds to three receptors, BAFFR (BAFF receptor), TACI (transmembrane activator and CAML interactor), and BCMA (B cell maturation antigen), while APRIL binds to TACI, BCMA, and proteoglycans. However, which ligand-receptor pair(s) are required to maintain plasma cells in different body locations remains unknown. Here, by combining mouse genetic and pharmacological approaches, we found that plasma cells required BCMA and/or TACI but not BAFFR. BCMA responded exclusively to APRIL, while TACI responded to both BAFF and APRIL, identifying three self-sufficient ligand-receptor pairs for plasma cell maintenance: BAFF-TACI, APRIL-TACI, and APRIL-BCMA. Together, these actors accounted for 90% of circulating antibodies. In BAFF-ko mice, the reduction of plasma cells upon APRIL inhibition indicated that APRIL could function in the absence of BAFF-APRIL heteromers. No evidence was found that in the absence of BCMA and TACI, binding of APRIL to proteoglycans would help maintain plasma cells. IL-6, alone or together with BAFF and APRIL, supported mainly splenic plasmablasts and plasma cells and contributed to circulating IgG but not IgA levels. In conclusion, survival factors for plasma cells can vary with body location and with the antibody isotype that plasma cells produce. To efficiently target plasma cells, in particular IgA-producing ones, dual inhibition of BAFF and APRIL is required.

Large-Scale Characterization and Functional Assessment of Adaptive Immunity in Patients with MGUS and SMM Reveals Significant Defects

Introduction Patients with Monoclonal Gammopathy of Undetermined Significance (MGUS) or Smoldering Multiple Myeloma (SMM) exhibit signs of immune dysregulation despite their early stage of oncogenesis. The SARS-CoV-2 pandemic and subsequent broad vaccination of the population presented an opportunity to compare the real-life immune response of patients and healthy individuals following exposure to a common antigen. Here, we have used SARS-CoV-2 vaccination as a model to study the adaptive immune response and have thus comprehensively assessed the adaptive immune response of patients with MGUS/SMM to SARS-CoV-2 vaccination compared to healthy donors (HD). Methods We performed (i) paired 5' single-cell RNA and T cell receptor (TCR) sequencing on 224 peripheral blood mononuclear cell (PBMC) samples drawn serially before and after two doses of SARS-CoV-2 vaccination (HD, n=26; MGUS, n=20; SMM, n=48; Multiple Myeloma (MM), n=24), (ii) ELISA for anti-Spike IgG antibodies post-vaccination (post-vx), (HD, n=237; MGUS, n=550; SMM, n=947) and MMR antibodies at baseline (HD, n=20; SMM, n=20), (iii) bulk TCR sequencing pre- and post-vx (HD, n=2; SMM, n=2) (Adaptive Biotechnologies), and (iv) IFN gamma ELISpot assay following in vitro stimulation with Spike peptide (HD, n=20; SMM, n=20). All post-vx samples were drawn after 2 doses of vaccination. Results Patients and HD showed comparable anti-Spike antibody titers within 14-50 days post-vx, however, we observed faster waning in patients with MGUS/SMM, who had significantly lower titers 50-100 days post-vx (p=0.014 & p=0.007, respectively), suggesting a potential defect in long-lived plasma cell maintenance. Indeed, patients with SMM showed significantly lower titers of antibodies against Mumps and Rubella, antigens encountered in childhood, compared to age-matched HD (p=0.002 & p=0.03, respectively), showing that this defect may also lead to loss of plasma cell memory. Next, we performed single-cell RNA/TCR-seq to assess the patients' T cell response to vaccination. Patients with SMM showed a significant increase in TCR repertoire diversity post-vx (paired t-test, p=0.046), suggesting at least partial preservation of T cell responses with recruitment of rare clones. Indeed, using bulk TCR-seq, we observed Spike-specific T cells in patients with MGUS/SMM. Interestingly, however, a significant enrichment in Spike-specific clones by single-cell TCR-seq was only observed in HD post-vx (paired t-test, p=0.01), suggesting patient T cell responses may be suboptimal. Functional assessment of T cell activation following stimulation with the Spike peptide showed significantly lower activation in patients with SMM compared to HD (t-test, p=0.002), confirming the suspected defect. Notably, a larger proportion of Spike-specific clones in patients was of the Treg phenotype compared to HD (empirical p=0.002), which may partly explain the observed defect. Conclusions This study demonstrates that despite the absence of symptoms, patients with MGUS/SMM may demonstrate defective humoral and cellular immune responses, including loss of plasma cell memory. Specific interventions, such as changes in the vaccination schedule, may be warranted for this population, and the morbidity associated with these defects should be studied further.

Sec22b is a critical and nonredundant regulator of plasma cell maintenance

Despite the essential role of plasma cells in health and disease, the cellular mechanisms controlling their survival and secretory capacity are still poorly understood. Here, we identified the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) Sec22b as a unique and critical regulator of plasma cell maintenance and function. In the absence of Sec22b, plasma cells were hardly detectable and serum antibody titers were dramatically reduced. Accordingly,Sec22b-deficient mice fail to mount a protective immune response. At the mechanistic level, we demonstrated that Sec22b contributes to efficient antibody secretion and is a central regulator of plasma cell maintenance through the regulation of their transcriptional identity and of the morphology of the endoplasmic reticulum and mitochondria. Altogether, our results unveil an essential and nonredundant role for Sec22b as a regulator of plasma cell fitness and of the humoral immune response.

A comparative study of adjuvants effects on neonatal plasma cell survival niche in bone marrow and persistence of humoral immune responses.

The neonatal immune system is distinct from the immune system of older individuals rendering neonates vulnerable to infections and poor responders to vaccination. Adjuvants can be used as tools to enhance immune responses to co-administered antigens. Antibody (Ab) persistence is mediated by long-lived plasma cells that reside in specialized survival niches in the bone marrow, and transient Ab responses in early life have been associated with decreased survival of plasma cells, possibly due to lack of survival factors. Various cells can secrete these factors and which cells are the main producers is still up for debate, especially in early life where this has not been fully addressed. The receptor BCMA and its ligand APRIL have been shown to be important in the maintenance of plasma cells and Abs. Herein, we assessed age-dependent maturation of a broad range of bone marrow accessory cells and their expression of the survival factors APRIL and IL-6. Furthermore, we performed a comparative analysis of the potential of 5 different adjuvants; LT-K63, mmCT, MF59, IC31 and alum, to enhance expression of survival factors and BCMA following immunization of neonatal mice with tetanus toxoid (TT) vaccine. We found that APRIL expression was reduced in the bone marrow of young mice whereas IL-6 expression was higher. Eosinophils, macrophages, megakaryocytes, monocytes and lymphocytes were important secretors of survival factors in early life but undefined cells also constituted a large fraction of secretors. Immunization and adjuvants enhanced APRIL expression but decreased IL-6 expression in bone marrow cells early after immunization. Furthermore, neonatal immunization with adjuvants enhanced the proportion of plasmablasts and plasma cells that expressed BCMA both in spleen and bone marrow. Enhanced BCMA expression correlated with enhanced vaccine-specific humoral responses, even though the effect of alum on BCMA was less pronounced than those of the other adjuvants at later time points. We propose that low APRIL expression in bone marrow as well as low BCMA expression of plasmablasts/plasma cells in early life together cause transient Ab responses and could represent targets to be triggered by vaccine adjuvants to induce persistent humoral immune responses in this age group.

Inflammation depletes humoral immunity by limiting plasma cell access to the bone marrow survival niche

Abstract The survival of plasma cells (PCs) is directly correlated to the longevity of prophylactic titers against pathogens. The bone marrow contains hematopoietic and non-hematopoietic cells that contribute to the maintenance of PCs. Studies have shown that pathogens contain a variety of mechanisms that result in the depletion of bone marrow PCs and antibody titers. Most, if not all, forms of infection induce inflammation, however, the role of inflammation in bone marrow PC depletion is unclear. Here, we show that acute Tumor Necrosis Factor Alpha (TNFa) treatment can deplete pre-existing antibody titers in a variety of models. We also found that recombinant TNFa treatment limits PC retention in the bone marrow via adoptive transfer experiments. To determine if PC retention in the bone marrow was mediated in a cell-intrinsic or − extrinsic manner, we generated bone marrow chimeras with wild-type and TNFa receptor knockout mice. Results showed that TNFa signaling through TNFa receptor 1 in non-hematopoietic cells regulates PC retention in the bone marrow, indicating that this mechanism is cell-extrinsic. Another cell-extrinsic mechanism that we identified is TNFa regulation of syndecan-1, a cell surface proteoglycan found to regulate PC survival and motility. These findings demonstrate that inflammation limits PC access to the bone marrow survival niche, ultimately leading to the reduction of antibody titers and long-term humoral protection. Supported by grants from the NIH (R01 HLI141491) and the Eric Heyer M.D. Ph.D Translational Research Grant

Prdi-BF-1 Strongly Detected in Active Multiple Myeloma By Droplet Digital PCR: A Comparison with Real-Time PCR

Human positive regulatory domain I binding factor 1 (PRDI-BF1 or BLIMP-1) is a transcription factor that has been demonstrated to act as a master regulator required and sufficient for the generation and for the prolonged maintenance of plasma cells (PC). PRDI-BF1 essentially functions as a repressor, causing exit from cell cycling and the extinction of the expression of several genes critical for B-cell development at earlier stages. The PRDM1 gene, which codifies for PRDI-BF1, contains an alternative promoter capable of generating a PRDI-BF1 deleted protein (called PRDI-BF1β), which lacks 101 amino acids comprising most of the regulatory domain. The potential role of Blimp-1/PRDM1 in multiple myeloma, may help in understanding the progression of the disease and the potential specific function of a new drug as elotuzumab that selectively blocks CS1. To the best of our knowledge this is the first report that uses two new molecular techniques to identify and perform a quantitative levels of PRD-BF1. We measured, using real time polymerase chain reaction (RT- PCR) and Droplet digital PCR (ddPCR), the levels of PRDI-BF1 in Myeloma patients'(n=15) and normal human bone marrow (n=12). qPCR represents the most widely used method for molecular analysis. However, it has a major limitation from being a relative quantification approach. This results in the need of a reference standard curve usually built by dilutions of the tumor-specific target obtained from diagnostic DNA, plasmids, or cell lines that contain the rearrangement of interest. ddPCR is based on sample compartmentalization in single oil droplets that represent independent PCR reactions and on end point amplification and Poisson statistics. ddPCR has several theoretical advantages compared with qPCR. most notably allowing for absolute quantification of target DNA molecules and avoiding the need for a reference standard curve. dd-PCR was performed using the QX100 platform (BioRad®, Hercules, CA, USA), consisting of two instruments: the droplet generator and the droplet reader. The droplet generator divides the sample by creating about 20,000 partitions (droplets). The droplets are then transferred into PCR plates and, at the end of the amplification cycles, placed into the droplet reader, where each droplet is read as mutated or wild-type by issuing specific fluorescence signals (FAM for BLIMP and Hex for GAPDH). These signals, after being counted, are redistributed according to the Poisson's algorithm. In our samples, total RNA was extracted and reverse transcribed from bone marrow blood using RNA isolation and transcriptor kits from Roche (Barcelona, Spain). Primer mixes and TaqMan probes, assay references Hs00153357_m1 for PRDIBF1 and s99999903_m1 for β-actin (used as an internal refer-ence), were obtained from Applied Biosystems (Foster City, CA, USA). The relative quantitative values were calculated using the 2-∆∆CT method. In all MM patients the mean expression of PRDI-BF1 was 5.92 (range 1.00-21.75) fold higher than in healthy controls with RT PCR. Using ddPCR, PRDI-BF1 was also expressed at considerable levels in all samples tested and the mean expression of PRDI-BF1 was 10.10 (range 0.63-52.52) fold higher than in healthy controls.In particular in 4 patients with very aggressive disease the PRDI-BF had a marked higher transcript level (mean 28.52 range 15.12-52.52), Similar results were obtained with RT-PCR higher transcript level (mean 13.5 range 7.41-21.75) Our data suggest that the total level of PRDI-BF is clearly detectable in MM patients . The expression of these gene could be a feature for identifying active disease of MM with particular interest in very aggressive disease. DisclosuresGalimberti:Pfizer: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau; Incyte: Speakers Bureau.

Humoral Immunity vs. Salmonella.

In primary infection with Salmonella, it has been reported—without consideration of Salmonella's functions—that humoral immunity plays no role in the clearance of bacteria. In fact, Salmonella targets and suppresses several aspects of humoral immunity, including B cell lymphopoiesis, B cell activation, and IgG production. In particular, the suppression of IgG-secreting plasma cell maintenance allows the persistence of Salmonella in tissues. Therefore, the critical role(s) of humoral immunity in the response to Salmonella infection, especially at the late phase, should be re-investigated. The suppression of IgG plasma cell memory strongly hinders vaccine development against non-typhoidal Salmonella (NTS) because Salmonella can also reduce humoral immune memory against other bacteria and viruses, obtained from previous vaccination or infection. We propose a new vaccine against Salmonella that would not impair humoral immunity, and which could also be used as a treatment for antibody-dependent autoimmune diseases to deplete pathogenic long-lived plasma cells, by utilizing the Salmonella's own suppression mechanism of humoral immunity.

A screening platform for human plasma cells (PCs) reveals a selective response to SYK inhibition

Abstract Therapeutic targeting of PC populations has potential value in various auto-immune, allergic and neoplastic cell states, but at present few options exist for selective targeting. However, the unique physiology and niche dependence of PCs suggests that such targeting may be achievable. To this end we have developed a sensitive screening platform capable of detecting changes in PC biology. Starting from our published methods for in vitro PC generation we optimised conditions to allow maintenance of PCs in polarised niche environments reflective of homeostatic and inflammatory conditions in microtitre plates. Using this assay format we probed PC biology via small molecule inhibitors (SMIs) targeting BCL2 family members, proteasome, and kinase signalling pathways. We were able to identify SMIs with broad and selective toxicity, as well as SMIs with no effect on PCs. Under all conditions analysed we found consistent divergence in the impact of targeting aspects of B cell receptor (BCR)-associated signalling pathways. Targeting SYK via two distinct SMIs consistently resulted in selective killing of subsets of PCs. By contrast BTK inhibition had no significant effect on PC viability. Whilst the importance of SYK signalling has been well documented at the B cell stage its importance to PCs has to our knowledge not been extensively documented. Retention of functional BCRs has been recently described in BM IgA+ and IgM+ PCs, and examination of expression data indicates selective retention of components of the BCR signalling complex in our assay system. The data presented here demonstrate the potential utility of an in vitro screening platform to evaluate the selective dependency of subsets of PCs on therapeutically targetable pathways.

Neutrophil-Derived MRP14 Supports Plasma Cell Commitment and Protects Myeloma Cells from Apoptosis.

Neutrophils have recently been proposed as cells with high functional plasticity and are involved in the pathogenesis of infections, malignancy, and autoimmune diseases. However, less is known about the role of neutrophil in humoral response. In this study, we examined the importance of neutrophils and the neutrophil-derived DAMP protein, MRP14, in antibody production. Splenic neutrophils and MRP14 that are present in the splenic peri-MZ region have a close contact with MZ B cells and promote their differentiation into plasma cells. Using neutrophil-depleting mice and an MRP14-blocking compound, we showed that the presence of neutrophil and MRP14 is required for class switch, plasma cell maintenance, and antibody production in the spleen. We found that MRP14 could also be produced by neutrophils in the bone marrow and support the maintenance of bone marrow plasma cells. MRP14 binding could enhance the effect of the BAFF signal and protect primary multiple myeloma cells from doxorubicin-induced apoptosis. Our data demonstrate the effects of neutrophils on neighboring B cells and plasma cells, which provides new insights into the connection between neutrophil and humoral responses.

A Study of Tbo-Filgrastim (Granix) to Disrupt the Bone Marrow Microenvironment in Patients with Multiple Myeloma Undergoing Autologous Stem Cell Transplantation

Multiple myeloma is the second most common hematologic malignancy in the US. The current standard of care for transplant eligible patients is therapy with high-dose melphalan (HDM) followed by autologous stem cell transplant (ASCT). Although ASCT improves progression-free (PFS) and overall survival (OS), it is not curative and virtually all patients will relapse. Attempts to improve upon HDM by adding other agents to transplant protocols have largely resulted in unacceptable increases in toxicity.Preclinical studies performed by our group suggest that granulocyte-stimulating factor (G-CSF) disrupts the bone marrow microenvironment, resulting in a striking loss of plasmablasts, plasma cells, and decreased expression of chemokine/cytokines contributing to plasma cell maintenance. Tbo-filgrastim (Granix, Teva Pharmaceuticals) is a recombinant methionyl human granulocyte colony-stimulating factor. We hypothesized that tbo-filgrastim treatment may provide a potent and well-tolerated method to disrupt the ‘myeloma cell niche’, rendering patients more sensitive to HDM.Methods: Here, we report results from an open label, single center, phase II randomized study to test the efficacy and safety of tbo-filgrastim plus HDM (tbo-filgrastim arm) versus HDM alone (SOC arm) prior to ASCT (NCT02112045). Patients were randomized 1:1 to tbo-filgrastim on Day -7 through Day -2 (480 or 960 mcg/day, based on weight) and melphalan on Day -2 prior to ASCT (140 or 200 mg/m2, based on age) or melphalan alone. The primary objective was to compare CR rate at day 100. Secondary objectives included comparison of the toxicity, overall response rate, PFS, OS, and rate of neutrophil and platelet engraftment between the two arms. Eligible patients were ≥18 years with symptomatic multiple myeloma enrolled within 12 months of receiving at least 2 cycles of any systemic therapy, were undergoing their first ASCT, and had an adequate ASCT collection product (at least 2 million CD34+ cells/kg). Target enrollment was 176 patients, with an interim analysis of efficacy and futility planned after 88 patients reached Day 100 post-ASCT. Early stopping rules for unacceptable toxicity were in place. Responses were evaluated by IMWG criteria.Results: Ninety patients were enrolled (median age 59.5, range 33 to 77) and 89 were evaluable for response. The early stopping rules for toxicity were not met. The planned interim analysis showed that the proportion of patients in CR at Day 100 was similar between the arms and the study was halted for futility (39.5% on the tbo-filgrastim arm vs. 37.8% on SOC arm). The overall response rate (CR + VGPR + PR) between the tbo-filgrastim and the SOC arm was 95% vs 93%, respectively. At the interim analysis, with median follow-up time for the study of 21.7 months, (range 8.8 to 25.8), the median PFS and OS had not been reached for either arm. There was no difference in PFS between the tbo-filgrastim and the SOC arm (84% vs 80%, respectively, p=0.60). There was no difference in OS between the tbo-filgrastim and the SOC arm (90.9% vs 95.6%, respectively, p=0.43). All patients in the study achieved neutrophil (ANC > 0.5 K/cumm) and platelet (> 20 K/cumm) engraftment. The median time to neutrophil engraftment for the tbo-filgrastim arm was 5 days (range, 3-9) vs 4 days (range, 3-7) in the SOC arm, p<0.001. There was no difference in the median time to platelet engraftment between the arms (11 days in the tbo-filgrastim arm, range 2-23, and 10 days in the SOC arm, range 1-24, p=0.67). Adverse events for both arms were typical of those observed in the ASCT population.Conclusions: The administration of tbo-filgrastim in the setting of HDM prior to ASCT is feasible, without excess toxicity or loss of engraftment. There was no difference in Day +100 CR or ORR rates, PFS, or OS in patients treated with tbo-filgrastim plus HDM versus HDM alone with a median follow-up of 21.7 months. The lack of efficacy may be secondary to the high pre-ASCT response rates seen with modern agents. DisclosuresJacoby:Celgene: Speakers Bureau; NovoNordisk: Consultancy. Schroeder:Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Vij:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jansson: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Eosinophils are not essential for maintenance of murine plasma cells in the bone marrow.

Eosinophils were reported to serve as an essential component of the plasma cell niche within the bone marrow. As the potential contribution of eosinophils to humoral immunity has remained incompletely understood, we aimed to further characterize their role during antibody responses and to additionally investigate their role in autoimmune disease. Contrary to our expectations and the currently prevailing paradigm, we found that eosinophils are fully dispensable for the survival of murine bone marrow plasma cells and accordingly do not contribute to antibody production and autoantibody-mediated disease. Littermate wild type and eosinophil-deficient ΔdblGATA-1 animals showed similar numbers and frequencies of plasma cells and did not differ in steady state levels of immunoglobulins or their ability to raise antigen-specific antibody responses. Eosinophils were likewise dispensable for autoantibody production or autoantibody-induced disease in a mouse model of systemic lupus erythematosus. Our findings thus argue against a role of eosinophils during the maintenance of the plasma cell pool and challenge the hitherto postulated concept of an eosinophil-sustained bone marrow niche.

Protein Homeostasis: A Key Target in Myeloma

Multiple myeloma is a disease of long-lived plasma cells that remains incurable in the majority of patients. However advances in the treatment have been made in the last 20 years resulting in significant improvement in the survival of patients with this disease. Interestingly almost all of these advances have been made through the targeting of pathways or molecules that are shared between normal and malignant plasma cells and are not due to the precision targeting of myeloma cell dependencies associated with the transformation process. Most notably targeting both specific and bulk protein homeostasis through the action of IMiDs and proteasome inhibitors have become the backbone for the treatment of newly diagnosed myeloma in the United States. We have focused on proteasome function and regulation to better understand why a macromolecular structure that is present and functions in every cell in the body is such an effective target for myeloma therapy. Our initial findings indicated that proteasome inhibition activated an endoplasmic reticulum stress response known as the unfolded protein response (UPR). The UPR is required for normal differentiation and maintenance of plasma cells, although our initial findings suggesting that the PERK arm of the response, which is not required for normal plasma cell maintenance was activated by proteasome inhibitors. We determined that the likely source of unfolded protein was immunoglobulin produced by myeloma cells and that the fate of immunoglobulin was a key determinant of sensitivity to proteasome inhibition. We have now focused on additional factors that determine the response to proteasome inhibition and consistent with previous findings have demonstrated that activation of heat shock response is a early protective event following proteasome inhibition. However we were unable to demonstrate that any single heat shock protein was sufficient to alter the response to proteasome inhibition. Rather it was the activation of the heat shock response itself and that protection was only efficiently blocked by inhibition of the transcriptional regulator of the response, HSF1. HSF1 is activated through post-translational modifications and proteasome inhibition results in phosphorylation of HSF1 at several sites, most notably S326. This can be blocked with the multi-kinase inhibitor TG02 that effectively blocks the proteasome inhibitor-induced heat shock response and sensitizes myeloma cells to proteasome inhibitor-induced cell death. Based on these findings, the combination of TG02 and carfilzomib are being tested clinically. The cellular response to proteasome inhibitors is also controlled through the regulation of proteasome assembly. We have recently demonstrated that 14-3-3ζ an anti-apoptotic phospho-serine binding protein, can limit proteasome assembly and activity through its binding to the PA28α subunit of the 11S regulator. This significantly lowers proteasome capacity and sensitizes cells to proteasome inhibition but not to other therapeutic agents. Finally in addition to regulation of bulk protein homeostasis, targeting of specific protein turnover has proven effective in myeloma treatment. IMiDs can alter the ability of the E3 ligase cereblon to recognize substrates for ubiquitination, resulting in the degradation of key B cell transcription factors IKZF1/3. These can regulate the plasma cell maintenance factor IRF4 as well as play a direct role in the regulation plasma cell superenhancers that control the expression of oncogenes translocated in myeloma. Another key mediator of myeloma cell survival, MCL1 is regulated via protein stability and can targeted in this manner. Direct and indirect targeting of MCL1 will be discussed. Disclosures Boise: Abbvie: Consultancy; Eli Lilly and Company: Research Funding.

Study of oxidative stress biomarkers to predict ischemia reperfusion injury and its effects in renal allograft transplant recipients

There are no known specific markers for memory B cells in mice, although studies suggest that the CD38low and CD38high phenotypes are indicative of isotype-switched germinal center (GC) and memory B cells in the mouse, respectively [1]. Further analysis suggested that IgG1+CD38high B cells, but not IgG1+CD38low B cells, are capable of inducing a significant IgG1 secondary response in the adoptive hosts [2], demonstrating that the CD38low and CD38high phenotype distinction can be used to monitor the development of the antigen-specific memory B cells in the T cell-dependent (TD) response. In humans, approximately 30–50% of the peripheral blood B cells are CD27+, and CD27 has been identified as a good surface marker for human memory B cells [3–5]. IgD−CD27+ cells in the peripheral blood have already undergone class switch recombination (CSR) and have accumulated somatic hypermutations (SHMs) in their VH genes in comparison to CD27− B cells, which have not. Polyclonal stimulation of B cells from anthrax vaccine adsorbed (AVA) vaccinated individuals generated AVA-specific IgG+ antibody-secreting cells (ASCs) in vitro, but the deletion of CD27+ B cells abrogated the response [6], indicating that human memory B cells are present in the CD27+ B cell compartment.The immune system memorizes the characteristics of pathogens to provide effective immune protection. Memory B cells and long-lived plasma cells (PCs) account for the long-term humoral immunity elicited by infections and many vaccines. Once generated, memory B cells enter a resting state and persist over long periods of time in the lymphoid organs in the apparent absence of immunizing antigen. T cell-dependent (TD) B cell memory is generated along two fundamentally distinct differentiation pathways. One of these is the classical generation pathway through antibody affinity maturation in the germinal center (GC) reaction with the help of T follicular helper (Tfh) cells. In the other pathway, memory B cells develop in response to a TD antigen before the onset and independently of the GC reaction with the help of T cells other than Tfh. The maintenance of these cells over time may depend on interactions with cells in their environment, perhaps in specialized “niches” akin to those postulated for the maintenance of PCs. Memory B cells provide the quick anamnestic antibody response that follows after antigen reexposure. This activity is critical for eliminating pathogens and toxins that are not efficiently eliminated by preexisting circulating antibodies.Plasma cells are terminally differentiated cells of the B lymphocyte lineage, the cells uniquely able to secrete antibody and thus the cell responsible for antibody-mediated immunity. In addition, because plasma cells can be maintained for extended periods, providing potentially life-long immunity to pathogens and their toxic products, they constitute a crucial component of immune memory. As such, plasma cell biology is fundamental in health in terms of immunity resulting from infection and vaccines. Furthermore, information regarding plasma cell development, differentiation, and survival and the molecular mediators of these processes provides potentially unprecedented insights into plasma cells participating in disease processes such as antibody-mediated autoimmune diseases such as systemic lupus erythematosus and the development of plasma cell cancers such as multiple myeloma.Memory plasma cells residing as mature long-lived plasma cells in bone marrow and inflamed tissues secrete antibodies independently of antigen contact, T cell help and memory B cells and are therefore crucial for maintaining antibody levels. They are refractory to irradiation, immunosuppression and therapies targeting B cells. Consequently, memory plasma cells secreting pathogenic antibodies substantially contribute to the chronicity and therapy resistance of antibody-mediated diseases. Their therapeutic targeting is a promising challenge.

TACI deficiency leads to alternatively activated macrophage phenotype and susceptibility to Leishmania infection

The TNF family member, transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), is a key molecule for plasma cell maintenance and is required in infections where protection depends on antibody response. Here, we report that compared with WT mouse, TACI KO Μϕs expressed lower levels of Toll-like receptors (TLRs), CD14, myeloid differentiation primary response protein 88, and adaptor protein Toll/IL-1 receptor domain-containing adapter-inducing IFN-β and responded poorly to TLR agonists. Analysis of Μϕ phenotype revealed that, in the absence of TACI, Μϕs adapt the alternatively activated (M2) phenotype. Steady-state expression levels for M2 markers IL-4Rα, CD206, CCL22, IL-10, Arg1, IL1RN, and FIZZ1 were significantly higher in TACI KO Μϕ than in WT cells. Confirming their M2 phenotype, TACI-KO Mϕs were unable to control Leishmania major infection in vitro, and intradermal inoculation of Leishmania resulted in a more severe manifestation of disease than in the resistant C57BL/6 strain. Transfer of WT Μϕs to TACI KO mice was sufficient to significantly reduce disease severity. TACI is likely to influence Mϕ phenotype by mediating B cell-activating factor belonging to the TNF family (BAFF) and a proliferation inducing ligand (APRIL) signals because both these ligands down-regulated M2 markers in WT but not in TACI-deficient Μϕs. Moreover, treatment of Μϕs with BAFF or APRIL enhanced the clearance of Leishmania from cells only when TACI is expressed. These findings may have implications for understanding the shortcomings of host response in newborns where TACI expression is reduced and in combined variable immunodeficiency patients where TACI signaling is ablated.

Quantitative image analysis of cell colocalization in murine bone marrow

Long-term antibody production is a key property of humoral immunity and is accomplished by long-lived plasma cells. They mainly reside in the bone marrow, whose importance as an organ hosting immunological memory is becoming increasingly evident. Signals provided by stromal cells and eosinophils may play an important role for plasma cell maintenance, constituting a survival microenvironment. In this joint study of experiment and theory, we investigated the spatial colocalization of plasma cells, eosinophils and B cells by applying an image-based systems biology approach. To this end, we generated confocal fluorescence microscopy images of histological sections from murine bone marrow that were subsequently analyzed in an automated fashion. This quantitative analysis was combined with computer simulations of the experimental system for hypothesis testing. In particular, we tested the observed spatial colocalization of cells in the bone marrow against the hypothesis that cells are found within available areas at positions that were drawn from a uniform random number distribution. We find that B cells and plasma cells highly colocalize with stromal cells, to an extent larger than in the simulated random situation. While B cells are preferentially in contact with each other, i.e., form clusters among themselves, plasma cells seem to be solitary or organized in aggregates, i.e., loosely defined groups of cells that are not necessarily in direct contact. Our data suggest that the plasma cell bone marrow survival niche facilitates colocalization of plasma cells with stromal cells and eosinophils, respectively, promoting plasma cell longevity.

The Plasma Cell Fountain of Youth?

Plasma cell longevity requires the antiapoptotic molecule Mcl-1.

Mcl-1 is essential for the survival of plasma cells

The long-term survival of plasma cells is entirely dependent on signals derived from their environment. These extrinsic factors presumably induce and sustain the expression of antiapoptotic proteins of the Bcl-2 family. It is uncertain whether there is specificity among Bcl-2 family members in the survival of plasma cells and whether their expression is linked to specific extrinsic factors. We found here that deletion of the gene encoding the antiapoptotic protein Mcl-1 in plasma cells resulted in rapid depletion of this population in vivo. Furthermore, we found that the receptor BCMA was needed to establish high expression of Mcl-1 in bone marrow but not spleen plasma cells and that establishing this survival pathway preceded the component of plasma cell differentiation that depends on the transcriptional repressor Blimp-1. Our results identify a critical role for Mcl-1 in the maintenance of plasma cells.

Immunization induces activation of bone marrow eosinophils required for plasma cell survival

Eosinophils not only have multiple functions as effector cells of the innate immune system but also as modulators of immune responses. As producers of cytokines required for plasma cell survival, they are essential for the long-term maintenance of plasma cells in the BM. Here we show that the activation of eosinophils both in vitro and in vivo enhances the expression of the plasma cell survival factors APRIL, IL-6, IL-4, IL-10 and TNF-α. The in vivo activation of eosinophils was independent of the type of adjuvant used for primary immunization. Although eosinophils were activated by adjuvant itself, a stable activation and a constant increase in BM eosinophils were observed only in the presence of antigen. Thus, the numbers and the quality of eosinophils were dependent on priming the adaptive immune system. With secondary immunization and re-activation of antigen-dependent memory cells, the ability of eosinophils to promote plasma cell survival was further increased. These findings suggest that in T-cell-dependent immune responses eosinophils are conditioned to support the long-term survival of plasma cells in the BM, and furthermore imply that through accelerated numbers of eosinophils, stable plasma cell survival niches are established and the long-term survival of plasma cells is ensured.

Sustained Antibody Responses Depend on CD28 Function in Bone Marrow Resident Plasma Cells

Abstract 182Protective immunity against infection requires sustained antibody production by long-lived plasma cells (LLPC) that survive for years/decades within specialized niches. What regulates/supports this survival remains largely unknown. However, it has been shown that normal and transformed (human multiple myeloma) LLPC are critically dependent on the bone marrow microenvironment, including cell-to-cell interactions. This lead us to hypothesize that modulating these interactions could either enhance antibody production for vaccine development or, conversely, compromise the survival of transformed/normal LLPC in the bone marrow microenvironment. We have shown that the T cell costimulatory receptor CD28 expressed on both normal and transformed LLPC, plays an essential role in survival. While LLPC and short-lived plasma cells (SLPC) both express CD28, its activation in vitro only significantly increases survival and IgG production in LLPC. Consistent with these findings, we show in vivo, vaccinated bone marrow CD28−/−:μMT chimeras had significantly reduced long-term antibody titers and decreased LLPC (but not SLPC) t1/2 from 426 to 63 days. These findings demonstrate the existence of a distinct bone marrow (BM) LLPC subset necessary to sustain antibody titers, and establish a central role for CD28 function in the maintenance of plasma cells and humoral immunity.While CD28 signaling has been shown to play an important role in maintaining long-term humoral immune responses, the mechanism by which CD28 signaling affects PC function has not yet been determined. To further elucidate CD28 signaling in BM PC, we utilized CD28 conditional knock-in mice. In these mice, the CD28 cytoplasmic tail is mutated at either the YMNM or proline-rich motifs, resulting in an inhibition of PI3K or vav signaling, respectively. We found that CD28-vav signaling deficient BM PC were selectively depleted in vivo and could not be rescued by CD28 activation in in vitro serum starvation conditions. Furthermore, anti-CD28 mAb drove a 1.5 fold increase in Blimp-1 expression in BM PC, compared to control. This increase was regulated through the CD28-vav signaling pathway, as CD28 activation in CD28-vav signaling deficient BM PC did not increase Blimp-1 expression. To further determine if CD28 is acting directly on the Blimp-1 promoter, we examined in silico for a CD28RE composite element, previously reported to transcriptionally regulate IL-2 production in T cells and IL-8 production in myeloma cells. To our surprise, we found a CD28RE “like” site 4712bp upstream of the Blimp-1 start site. To confirm CD28 transcriptionally regulates Blimp-1 promoter activity, we transfected the CD28+ plasmacytoma cell line J558 with full-length or truncated Blimp-1 promoter constructs (i.e. 7000bp, 4500bp, 1500bp). We found CD28 activation enhances Blimp-1 activity in J558 cells transfected with full-length-Blimp-1, and this activity was lost when the promoter was truncated. Using site-directed mutagenesis, we confirmed the CD28RE is required for induction of Blimp-1 in PC. Furthermore, we show CD28 activation of Blimp-1 increases the BCMA receptor in BM PC. Taken together, our data suggests the CD28-vav signaling pathway in PC induces a CD28RE composite element, which is necessary for the induction of the key PC transcriptional regulator Blimp-1, required to maintain LLPC and humoral immunity. Disclosures:No relevant conflicts of interest to declare.