Induce Plasma Cell Research Articles

Fibroblast-like synoviocytes preferentially induce terminal differentiation of IgD+ memory B cells instead of naïve B cells.

Rheumatoid arthritis (RA) is a systemic autoimmune disease driven by highly active autoantibody-producing B cells. Activation of B cells is maintained within ectopic germinal centres found in affected joints. Fibroblast-like synoviocytes (FLS) present in inflamed joints support B-cell survival, activation, and differentiation. CD27+ memory B cells and naive B cells show very different responses to activation, particularly by CD40 ligand (CD40L). We show that FLS-dependent activation of human B cells is dependent on interleukin-6 (IL-6) and CD40L. FLS have been shown to activate both naive and memory B cells. Whether the activating potential of FLS is different for naive and memory B cells has not been investigated. Our results suggest that FLS-induced activation of B cells is dependent on IL-6 and CD40L. While FLS are able to induce plasma cell differentiation, isotype switching, and antibody production in memory B cells, the ability of FLS to activate naive B cells is significantly lower.

The genomic landscape of Vk*MYC myeloma highlights shared pathways of transformation between mice and humans

Multiple myeloma (MM) is a heterogeneous disease characterized by frequent MYC translocations. Sporadic MYC activation in the germinal center of genetically engineered Vk*MYC mice is sufficient to induce plasma cell tumors in which a variety of secondary mutations are spontaneously acquired and selected over time. Analysis of 119 Vk*MYC myeloma reveals recurrent copy number alterations, structural variations, chromothripsis, driver mutations, apolipoprotein B mRNA-editing enzyme, catalytic polypeptide (APOBEC) mutational activity, and a progressive decrease in immunoglobulin transcription that inversely correlates with proliferation. Moreover, we identify frequent insertional mutagenesis by endogenous retro-elements as a murine specific mechanism to activate NF-kB and IL6 signaling pathways shared with human MM. Despite the increased genomic complexity associated with progression, advanced tumors remain dependent on MYC. In summary, here we credential the Vk*MYC mouse as a unique resource to explore MM genomic evolution and describe a fully annotated collection of diverse and immortalized murine MM tumors.

OA-33 Aberrant B cell differentiation induce plasma cell malignancy

OA-33 Aberrant B cell differentiation induce plasma cell malignancy

Inherited CD19 Deficiency Does Not Impair Plasma Cell Formation or Response to CXCL12

BackgroundThe human CD19 antigen is expressed throughout B cell ontogeny with the exception of neoplastic plasma cells and a subset of normal plasma cells. CD19 plays a role in propagating signals from the B cell receptor and other receptors such as CXCR4 in mature B cells. Studies of CD19-deficient patients have confirmed its function during the initial stages of B cell activation and the production of memory B cells; however, its role in the later stages of B cell differentiation is unclear.ObjectiveUsing B cells from a newly identified CD19-deficient individual, we investigated the role of CD19 in the generation and function of plasma cells using an in vitro differentiation model.MethodsFlow cytometry and long-read nanopore sequencing using locus-specific long-range amplification products were used to screen a patient with suspected primary immunodeficiency. Purified B cells from the patient and healthy controls were activated with CD40L, IL-21, IL-2, and anti-Ig, then transferred to different cytokine conditions to induce plasma cell differentiation. Subsequently, the cells were stimulated with CXCL12 to induce signalling through CXCR4. Phosphorylation of key downstream proteins including ERK and AKT was assessed by Western blotting. RNA-seq was also performed on in vitro differentiating cells.ResultsLong-read nanopore sequencing identified the homozygous pathogenic mutation c.622del (p.Ser208Profs*19) which was corroborated by the lack of CD19 cell surface staining. CD19-deficient B cells that are predominantly naïve generate phenotypically normal plasma cells with expected patterns of differentiation-associated genes and normal levels of CXCR4. Differentiated CD19-deficient cells were capable of responding to CXCL12; however, plasma cells derived from naïve B cells, both CD19-deficient and sufficient, had relatively diminished signaling compared to those generated from total B cells. Additionally, CD19 ligation on normal plasma cells results in AKT phosphorylation.ConclusionCD19 is not required for generation of antibody-secreting cells or the responses of these populations to CXCL12, but may alter the response other ligands that require CD19 potentially affecting localization, proliferation, or survival. The observed hypogammaglobulinemia in CD19-deficient individuals is therefore likely attributable to the lack of memory B cells.

Innovative Anti-CD38 and Anti-BCMA Targeted Therapies in Multiple Myeloma: Mechanisms of Action and Resistance.

CD38 and B-cell maturation antigens (BCMAs) are prevalently expressed on neoplastic plasma cells in multiple myeloma (MM), making them ideal therapeutic targets. Anti-CD38 monoclonal antibodies, such as approved daratumumab and isatuximab, are currently the milestone in MM treatment because they induce plasma cell apoptosis and kill through several mechanisms, including antibody-dependent cellular cytotoxicity or phagocytosis. BCMA is considered an excellent target in MM, and three different therapeutic strategies are either already available in clinical practice or under investigation: antibody-drug conjugates, such as belantamab-mafodotin; bispecific T cell engagers; and chimeric antigen receptor-modified T cell therapies. Despite the impressive clinical efficacy of these new strategies in the treatment of newly diagnosed or multi-refractory MM patients, several mechanisms of resistance have already been described, including antigen downregulation, the impairment of antibody-dependent cell cytotoxicity and phagocytosis, T- and natural killer cell senescence, and exhaustion. In this review, we summarize the current knowledge on the mechanisms of action and resistance of anti-CD38 and anti-BCMA agents and their clinical efficacy and safety.

Structural Elements of the BCR Determine Permissiveness to Oncogenic RAS-Driven B-Cell Transformation

Background: RAS-pathway activating mutations (e.g. NRAS, KRAS, BRAF) invariably promote constitutive ERK-activation and are oncogenic drivers in B-cell acute lymphoblastic leukemia (B-ALL; Irving et al., 2014). Conversely, persistent ERK-activation can induce plasma cell differentiation (Yasuda et al., 2011), negative B-cell selection and cell death (Limnander et al., 2011) during B-cell development. In RAS-associated autoimmune leukoproliferative disorder (RALD), RAS-activating germline mutations cause lymphoproliferative disorder but not leukemia. The molecular determinants driving these divergent outcomes of RAS-activation have yet to be characterized. For these reasons, we here examined the mechanisms that enable permissiveness to RAS-driven transformation of B-cells. Results: Studying RAS-pathway activating mutations, we found that these genetic lesions are very common in B-cell malignancies derived from the earliest (e.g. B-ALL) and the latest (e.g. multiple myeloma) stages of B-cell development. In contrast, RAS-pathway lesions are exceedingly rare in B-cell receptor (BCR)-expressing B-cell malignancies, including mantle cell lymphoma, chronic lymphocytic leukemia, follicular lymphoma, Burkitt's lymphoma and diffuse large B-cell lymphoma (Figure 1). We also studied the consequence of oncogenic RAS-activation in mature B-cells, which express BCR. Consistent with previous findings that ERK-activation results in negative B-cell selection and cell death of B-cells (Limnander al., 2011), we found that expression of NRASG12D in mature B-cells resulted in upregulation of Prdm1, a tumor suppressor and mediator of negative B-cell selection and cell death (Hug et al., 2014; Setz et al., 2018). Altogether, our results suggest that mature B-cells are not permissive to RAS-driven transformation and prompted us to investigate whether expression of BCR counteracts constitutive ERK-activation resulting from oncogenic RAS signaling in B-cells. Reflecting incompatibility between oncogenic RAS-ERK activation and BCR-expressing malignant B-cells, expression of NRASG12D in BCR-positive mantle cell lymphoma cells transiently decreased surface expression of BCR but eventually led to depletion of cells from culture in growth competition assays. The non-receptor protein tyrosine kinase SYK is a key mediator of BCR signaling. Notably, treatment with small molecule inhibitor of SYK (entospletinib) largely reversed cell death caused by oncogenic RAS-signaling. Taken together, our results suggest that expression of an active BCR prevents permissiveness to constitutive ERK-activation. BCR consists of µ heavy chain (µHC) and conventional light chains (LCs; either kappa LC or lambda LC). Given that expression of a functional BCR opposes constitutive ERK-activation, this raised the question of whether structural elements of BCR are involved in determining outcome of RAS-activation. To this end, we performed CRISPR/Cas9-based gene editing to disrupt the constant region of immunoglobulin heavy chain (IGHM) or the constant region of the immunoglobulin kappa LC (IGKC) in kappa LC-expressing mantle cell lymphoma cells. While CRISPR-mediated deletion of IGHM reduced surface expression of IgM (μHC), this did not rescue cells from call death caused by oncogenic RAS-ERK activation. On the contrary, genetic ablation of IGKC enabled permissiveness to oncogenic RAS-signaling and prevented cell death resulting from constitutive ERK-activation. Hence, these findings showed that expression of kappa LC opposes constitutive RAS-ERK signaling in B-cell lymphoma cells. Conclusions: Surface expression of BCR is conserved in most mature B-cell lymphomas. Recent studies suggest that constitutive RAS-ERK signaling can override BCR requirement in B-cell lymphoma and that B-cell lymphoma cells acquiring RAS mutations may lose selective pressure to express a functional BCR (Varano et al., 2017). Here, our results demonstrated that conventional LCs of the BCR determine permissiveness to RAS-driven malignant transformation of B-cells. Conventional LCs were previously shown to inhibit the autonomous signaling capacity of BCR (Meixlsperger et al., 2007). We here showed that conventional LCs of BCR counteract oncogenic RAS signaling, providing a mechanistic basis of the low frequency of RAS-pathway activating mutations observed in BCR-expressing B-cell malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

First Report of Daratumumab in Clinical Lung Transplantation

<h3>Purpose</h3> Antibody mediated rejection (AMR) after lung transplantation is difficult to treat and often results in death or graft loss. Therapies targeting antibodies or B cells are in many cases inadequate for decreasing donor specific antibodies (DSA), particularly when they are directed against MHC class II. Daratumumab (DAR) is a humanized anti-CD38 monoclonal antibody, which induces plasma cell death through multiple mechanisms including complement- dependent cytotoxicity, antibody-dependent phagocytosis, and apoptosis. Based on these properties it may have the potential to reduce the amount of DSAs and therefore improve outcome after AMR. <h3>Methods</h3> Retrospective analysis of all patients who received daratumumab as an add on rescue therapy for AMR or for desensitization pre/post-transplant at our center. <h3>Results</h3> Six patients received daratumumab due to the following reasons: 5 patients with de novo DSAs and AMR, 1 patient with pre-transplant DSAs and post-transplant immunadsorption without clinical AMR. Daratumumab was safely administered with just mild infusion reactions and no severe adverse event. Of not, none of the patients developed and infectious complication. Five of the 6 patients showed a significant decrease of their DSAs with a reduction of MFI values after 6-8 weeks to <50% of the baseline . Despite this early success, four patients developed CLAD (,two of which required retransplantation. The remaining two patients stabilized with their lung function and did not develop CLAD. <h3>Conclusion</h3> Treatment for AMR remains challenging, especially in the presence of class II HLA antibodies. Daratumumab might be a promising addition to the AMR treatment panel, prospective clinical studies are needed.

PD-1+CXCR5−CD4+ Th-CXCL13 cell subset drives B cells into tertiary lymphoid structures of nasopharyngeal carcinoma

BackgroundA major current challenge is to exploit tertiary lymphoid structures (TLSs) to promote the lymphocyte infiltration, activation and differentiation by tumor antigens to increase antitumor immune responses. The mechanisms that...

Cdc37 suppression induces plasma cell immaturation and bortezomib resistance in multiple myeloma via Xbp1s

Multiple myeloma (MM) is the second most prevalent hematologic malignancy. Although the use of bortezomib (BTZ) significantly improves MM therapy, intrinsic and acquired drug resistance to BTZ remains a major clinical problem. In this study, we find that Cdc37, a key co-chaperone of Hsp90, is downregulated in relapsed MM patients, especially after BTZ treatment, suggesting a link between Cdc37 and BTZ resistance. Suppression of Cdc37 or inhibition of Cdc37/Hsp90 association induces plasma cell dedifferentiation, quiescence of MM cells, and BTZ resistance in MM. Furthermore, we discover that Cdc37 expression correlates positively with Xbp1s, a critical transcription factor for plasma cell differentiation in MM samples. Depletion/inhibition of Cdc37 downregulates Xbp1s, while overexpression of Xbp1s in MM cell lines partially rescues plasma immaturation and BTZ resistance. It is suggested that Xbp1s may act as a key downstream effector of Cdc37. Experiments with a mouse model also demonstrate that Cdc37 inhibition promotes plasma cell immaturation, confers BTZ resistance, and increases MM progression in vivo. Together, we identify a critical factor and a new signaling mechanism that regulate plasma cell immaturation and BTZ resistance in MM cells. Our findings may constitute a novel strategy that overcomes BTZ resistance in MM therapy.

Hyperactive Follicular Helper T Cells Contribute to Dysregulated Humoral Immunity in Patients With Liver Cirrhosis.

Objectives: Liver cirrhosis (LC) is usually accompanied by cirrhosis associated immune dysfunction (CAID), including reduced naïve T cells and memory B cells. However, little is known regarding on follicular helper T (Tfh) cell compartments in cirrhotic patients, especially in the secondary lymphoid organs such as spleen. This study characterizes splenic Tfh cells and explores its association with humoral immunity and disease progression in cirrhotic patients.Methods: Using flow cytometry and histological staining, we analyzed the frequency and cytokine production of splenic Tfh cells from LC patients and healthy controls (HCs). Co-culture experiments of sorted Tfh and B cells were performed for functional analysis in vitro. The correlations between Tfh cells and disease progression markers as well as B cell subset perturbations were also examined.Results: PD-1highICOS+CXCR5+ Tfh cells were preferentially enriched in the spleen of cirrhotic patients, where they expressed higher levels of CXCR3 and produced more interleukin (IL)-21. Histologically, more splenic Tfh cells occupied the B cell follicular structure in LC patients where they shaped more active germinal centers (GCs) than those in HC spleens. In vitro, splenic Tfh cells in cirrhotic patients robustly induce plasma cell differentiation through IL-21 dependent manner. Finally, increased Tfh cell frequency is positively correlated with the plasma cells and disease severity in LC patients.Conclusions: We conclude that hyperactive Tfh cells contribute to dysregulated humoral immunity in patients with liver cirrhosis.

The effects of altered TGFβ signaling on human T follicular helper cell development and function

Abstract T follicular helper (Tfh) cells are a subset of CD4+ T cells that play a crucial role in the development of memory B cells and plasma cells. T follicular regulatory (Tfr) cells oppose Tfh cells and suppress humoral immune responses. Patients with Loeys-Dietz Syndrome (LDS) type 1 and 2 have an autosomal dominant mutation in the genes encoding TGFβ Receptor 1 or 2, respectively, and have a strong predisposition to develop allergic diseases. We hypothesized that altered development and function of Tfh cells could contribute to increased IgE-mediated allergic phenotypes in LDS. Comparing a cohort of pediatric LDS patients with age-matched healthy volunteers, we found that LDS patients have significantly higher levels of serum IgE and IgG and lower levels of IgM. Congruently, LDS patients have an increased frequency of memory T cells, Th2 cells, and Tfh cells and a significantly lower frequency of Tfr cells. The increase in Tfh cells in LDS was restricted to the CCR4+ CCR6− subset, which has been associated with Th2 immune responses. In vitro, Tfh cells from LDS patients co-cultured with autologous naïve B cells demonstrated a greater ability to induce plasma cell differentiation compared to age-matched healthy volunteers. Additionally, naïve CD4+ T cells from LDS patients cultured in the presence of recombinant TGFβ1 and IL-12 or IL-4 were more likely to differentiate into Tfh cells compared to healthy age-matched controls, suggesting a cell-intrinsic propensity for naïve CD4+ T cells in LDS patients to acquire a Tfh fate. Collectively, our data suggest that LDS mutations alter the development and function of Tfh cells in a manner that promotes IgE-mediated disease.

Immunoproteasome inhibition induces plasma cell apoptosis and preserves kidney allografts by activating the unfolded protein response and suppressing plasma cell survival factors

Chronic antibody-mediated rejection is the leading cause of allograft dysfunction and loss after kidney transplantation, and current immunosuppressive regimens fail to target the plasma cells that produce alloantibodies. We previously showed that treatment with the immunoproteasome inhibitor ONX 0914 prevented the expansion of plasma cells and prevented chronic allograft nephropathy and organ failure after kidney transplantation in rats, but the mechanism has remained elusive. In the current study, we confirmed a long-term reduction in alloantibody production and improvements in allograft histology in rats treated with ONX 0914 or with the broad-spectrum proteasome inhibitor bortezomib. Plasma cells from allotransplanted rats expressed immunoproteasomes at high levels. Immunoproteasome inhibition with ONX 0914 led to ubiquitin-conjugate accumulation, activation of the unfolded protein response, and induction of apoptosis in plasma cells. In addition, ONX 0914 suppressed the expression of adhesion molecules (VLA-4 and LFA-1), plasma cell survival factors (APRIL and IL-6), and IFN-γ-inducible chemokines in bone marrow, while the APRIL receptor BCMA, the IL-6 receptor, and the chemokine receptors CXCR4 and CXCR3 were down-regulated on plasma cells. Taken together, immunoproteasome inhibition blocked alloantibody production by inducing apoptosis of plasma cells through activating the unfolded protein response and suppressing plasma cell survival factors in the bone marrow.

Activation of the IL-6/JAK2/STAT3 pathway induces plasma cell mastitis in mice

Plasma cell mastitis (PCM) is a chronic mastitis with limited treatment options and common recurrence. A histopathological hallmark of PCM is the infiltration of numerous plasma cells surrounding the mammary duct. Our previous study showed that the activity of the IL-6/STAT3 signaling pathway was elevated in patients with PCM. However, the etiology of PCM remains largely unclear. In this study, we sought to explore the effects of IL-6/JAK2/STAT3 signaling pathway in the pathogenesis of PCM. Histological analysis showed that the mammary glands of mice that received human breast tissue homogenates, followed by an injection of IL-6, exhibited features of PCM similar to human PCM. The IL-6/JAK2/STAT3 signaling activity was significantly elevated and Bcl-2 was highly expressed in CD138 + plasma cells in the mammary glands of mice with PCM. Furthermore, treatment with AG-490, an inhibitor of JAK family kinases, suppressed activation of the IL-6/JAK2/STAT3 signaling cascade, in turn resulting in a decreased number of plasma cells in the mammary gland and reversing the pathogenesis of PCM. Taken together, our study indicated that a PCM mouse model was successfully established through activation of the IL-6/JAK2/STAT3 pathway by injecting IL-6 into the mammary gland of mouse that had received homogenates of human breast tissue. Thus, the IL-6/JAK2/STAT3 signaling pathway plays a critical role in orchestrating the pathogenesis of PCM.

Neuroblastoma chemotherapy can be augmented by immunotargeting O-acetyl-GD2 tumor-associated ganglioside

ABSTRACTDespite recent advances in high-risk neuroblastoma therapy, the prognosis for patients remains poor. In addition, many patients suffer from complications related to available therapies that are highly detrimental to their quality of life. New treatment modalities are, thus, urgently needed to further improve the efficacy and reduce the toxicity of existing therapies. Since antibodies specific for O-acetyl GD2 ganglioside display pro-apoptotic activity against neuroblastoma cells, we hypothesized that combination of immunotherapy could enhance tumor efficacy of neuroblastoma chemotherapy.We demonstrate here that combination of anti-O-acetyl GD2 monoclonal antibody 8B6 with topotecan synergistically inhibited neuroblastoma cell proliferation, as shown by the combination index values. Mechanistically, we evidence that mAb 8B6 induced plasma cell membrane lesions, consistent with oncosis. Neuroblastoma tumour cells treated with mAb 8B6 indeed showed an increased uptake of topotecan by the tumor cells and a more profound tumor cell death evidenced by increased caspase-3 activation. We also found that the combination with topotecan plus monoclonal antibody 8B6 showed a more potent anti-tumor efficacy in vivo than either agent alone. Importantly, we used low-doses of topotecan with no noticeable side effect. Our data suggest that chemo-immunotherapy combinations may improve the clinical efficacy and safety profile of current chemotherapeutic modalities of neuroblastoma.

Effect of plasma-activated medium on the decrease of tumorigenic population in lymphoma

Nonequilibrium atmospheric pressure plasma (NEAPP) is a novel approach for blood coagulation, wound healing, and tumor elimination. NEAPP not only directly but also indirectly affects living cells via the medium exposed to NEAPP-yielding devises, called plasma-activated medium (PAM). The conservable and portable PAM serves as an alternative and advantageous approach over direct NEAPP. Here we examined the effect of PAM on lymphoplasmacytic lymphoma (LPL) cell lines. We found that PAM induced plasma cell differentiation and reduced tumorigenic population. PAM increased the expression level of PRDM1α, which is a transcription factor promoting plasma cell differentiation, suggesting that plasma cell differentiation of LPL might be mediated by PRDM1α. We previously reported that plasma cell component of LPL is vulnerable to apoptosis and less tumorigenic. These findings suggested that PAM treatment might become a novel therapy against LPL by inducing the transition from tumorigenic to non-tumorigenic population.

Pathologically expanded peripheral T helper cell subset drives B cells in rheumatoid arthritis.

CD4+ T cells are central mediators of autoimmune pathology; however, defining their key effector functions in specific autoimmune diseases remains challenging. Pathogenic CD4+ T cells within affected tissues may be identified by expression of markers of recent activation. Here we use mass cytometry to analyse activated T cells in joint tissue from patients with rheumatoid arthritis, a chronic immune-mediated arthritis that affects up to 1% of the population. This approach revealed a markedly expanded population of PD-1hiCXCR5-CD4+ T cells in synovium of patients with rheumatoid arthritis. However, these cells are not exhausted, despite high PD-1 expression. Rather, using multidimensional cytometry, transcriptomics, and functional assays, we define a population of PD-1hiCXCR5- 'peripheral helper' T (TPH) cells that express factors enabling B-cell help, including IL-21, CXCL13, ICOS, and MAF. Like PD-1hiCXCR5+ T follicular helper cells, TPH cells induce plasma cell differentiation in vitro through IL-21 secretion and SLAMF5 interaction (refs 3, 4). However, global transcriptomics highlight differences between TPH cells and T follicular helper cells, including altered expression of BCL6 and BLIMP1 and unique expression of chemokine receptors that direct migration to inflamed sites, such as CCR2, CX3CR1, and CCR5, in TPH cells. TPH cells appear to be uniquely poised to promote B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues.

Increased Abundance of Plasmacytoid Dendritic Cells and Interferon-Alpha Induces Plasma Cell Differentiation in Patients of IgA Nephropathy.

The roles of pDC and IFN-α have not been well defined in IgA nephropathy (IgAN). In this study, we investigated the abundance of pDCs and IFN-α in IgAN patients and the response of peripheral blood mononuclear cells (PBMCs) after stimulation of the pDC-preferred TLR9 ligand CpG2216. The effects of IFN-α on plasma cell differentiation and leukocyte migration were also investigated. Here, we found that the percentages of pDCs were increased in PBMCs of IgAN patients, than in those of healthy controls. Plasma levels of IFN-α proteins and abundance of plasma cells were higher in IgAN patients than in healthy donors. Plasma IFN-α levels were positively associated with proteinuria, renal IgM deposition, and renal tubular atrophy/interstitial fibrosis grade in IgAN patients. Ex vivo activation of TLR9 on pDCs resulted in increased IFN-α production and enhanced plasma cell differentiation in IgAN patients as compared with healthy donors. IFN-α treatment led to increased plasma cell differentiation in vitro. IFN-α also significantly promoted expression of chemokines IP-10 and MCP-1 in human mesangial cells, which subsequently facilitated the transendothelial migration of human CD4+ and CD14+ cells. In conclusion, pDC and its secreted cytokine IFN-α may play important roles in pathological changes of IgA nephropathy.

Characterization of an In Vitro model of MGUS Progression to Multiple Myeloma

Introduction: Multiple Myeloma (MM) is an incurable cancer characterized by a pre-malignant clonal phase of disease called monoclonal gammopathy of undetermined significance (MGUS). Most patients with MGUS do not develop overt MM and the biology underlying this potential transformation is unclear. Investigations to prevent the development of MM from MGUS are limited by the relative infrequency of MGUS progression. Unfortunately, MGUS cells have historically proven difficult to grow in vitro because of slow rates of proliferation and difficulty in sustaining cell cultures. We present evidence of an in vitro model that generates MM-like plasma cells from patients diagnosed with only MGUS. We further present gene expression patterns of primary patient cells versus the induced MM cells to provide guidance as to important initiating events within our model.Methods: We collected a CD38+ cell fraction and a mononuclear (CD38-) fraction from 4 patients with MGUS using a Miltenyl Biotec column Separator. The CD38- fraction was grown in RPMI with 10% FBS and 1% sodium pyruvate with or without a polyglycolic acid/ poly L-lactic acid 90/10 (PLGA) copolymer scaffold to create 3D culture conditions. The mononuclear layer from healthy donors and the MM cell line, U266, were grown as controls. We then analyzed the initial CD38+ fraction, the initial CD38- fraction, and the CD38- fraction grown in media or 3D co-culture by flow cytometry for expression of kappa, lambda, CD38, CD138, CD45, CD19, and CD56. Gene expression analysis was performed using RNA-sequencing data from the CD38+, CD38-, cultured CD38-, and control cells. Expression of the top 100 ranked differentially expressed genes, which demonstrated the largest variation, were further analyzed using the nCounter® Analysis System (NanoString Technologies).Results: The CD38- fraction from MGUS patients grew into an adherent layer of elongated cells, consistent with bone marrow stromal cells. After several months, the stromal cells were noted to change shape and new, round cells were observed budding off from the stromal layer. Over time, the stromal layer disappeared and the round plasmacytoid cells remained. Characterization of these round cells revealed them to be plasma cells by IHC and flow cytometry. When comparing these in vitro generated cells to the initial CD38+ fraction removed from patients, the new cells showed the re-emergence of CD38 and CD138, increased expression of CD56 and CD19, and decreased expression of CD45. Gene expression analysis revealed 3 distinct populations of cells. The initial CD38- fraction separated with the healthy mononuclear layer. The initial CD38+ fraction clustered independently while the grown plasma cells clustered with the U266 cells. Analysis of the differential gene expression patterns revealed differences in the expression of immunoglobulin genes, as well as alterations in expression of extracellular matrix and cell adhesion markers including PAI-1, MMP2, COL1A2, and GREM1; and alterations in expression of mitochondrial genes.Conclusion: To our knowledge, this is the first in vitro simulation of disease progression from MGUS to MM. Our model induced the growth of plasma cells with an aggressive phenotype as assayed by flow cytometry. The gene expression profile further demonstrates gene expression patterns from our induced plasma cells consistent with MM versus MGUS. The alterations in extracellular matrix proteins as seen in our induced plasma cells are consistent with an epithelial to mesenchymal type transition implicated in disease progression, metastasis, and bone lesions. Additionally, the alterations in mitochondrial gene expression have been implicated in early disease progression in colon cancer and MM. These findings provide further evidence that our model simulates disease transformation and the expression data suggest possible pathways that may be important in myeloma disease progression that can be further evaluated in vivo. DisclosuresNo relevant conflicts of interest to declare.

B-cell activation with CD40L or CpG measures the function of B-cell subsets and identifies specific defects in immunodeficient patients.

Around 65% of primary immunodeficiencies are antibody deficiencies. Functional tests are useful tools to study B-cell functions in vitro. However, no accepted guidelines for performing and evaluating functional tests have been issued yet. Here, we report our experience on the study of B-cell functions in infancy and throughout childhood. We show that T-independent stimulation with CpG measures proliferation and differentiation potential of memory B cells. Switched memory B cells respond better than IgM memory B cells. On the other hand, CD40L, a T-dependent stimulus, does not induce plasma cell differentiation, but causes proliferation of naïve and memory B cells. During childhood, the production of plasmablasts in response to CpG increases with age mirroring the development of memory B cells. The response to CD40L does not change with age. In patients with selective IgA deficiency (SIgAD), we observed that switched memory B cells are reduced due to the absence of IgA memory B cells. In agreement, IgA plasma cells are not generated in response to CpG. Unexpectedly, B cells from SIgAD patients show a reduced proliferative response to CD40L. Our results demonstrate that functional tests are an important tool to assess the functions of the humoral immune system.

Polarization of Tissue-Resident TFH-Like Cells in Human Hepatoma Bridges Innate Monocyte Inflammation and M2b Macrophage Polarization.

We identified a novel protumorigenic IL21+ TFH-like cell subset with a CXCR5-PD-1- BTLA-CD69hi tissue-resident phenotype in hepatoma. TLR4-mediated monocyte inflammation and subsequent T-cell STAT1 and STAT3 activation are critical for TFH-like cell induction. TFH-like cells operate via IL21-IFNγ pathways to induce plasma cells and create conditions for M2b macrophage polarization. Cancer Discov; 6(10); 1182-95. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1069.