Bone Marrow Derived Mesenchymal Stem Cells from Splenic Marginal Zone Lymphoma Patients Exhibit Altered Proliferative Potential and B Lymphocyte Immunomodulatory Properties

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2396-2396
Author(s):  
Athanasia Kalyva ◽  
Charalampos Pontikoglou ◽  
Christina Kalpadakis ◽  
Athina Trakaki ◽  
Nikitas Zorzos ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Marginal Zone ◽  
Ex Vivo ◽  
Proliferative Potential ◽  
Splenic Marginal Zone Lymphoma ◽  
Healthy Individuals ◽  
Healthy Donors ◽  
Normal Controls

Abstract Splenic marginal zone lymphoma (SMZL) originates from the neoplastic transformation of mature B-lymphocytes. However, there is a concurrent high prevalence of bone marrow (BM) infiltration, suggesting that BM microenvironment dynamics could have a potential involvement in disease pathology. In this regard, we aim to characterise BM derived mesenchymal stem cells (MSCs), since they comprise key components of the BM hematopoietic stroma, in order to investigate if MSCs show altered properties in SMZL patients compared to healthy controls. BM MSCs were isolated from 8 SMZL patients and 10 age- and sex-matched healthy controls. MSCs were in vitro expanded and re-seeded for a total of 5 passages (P). The colony forming unit-fibroblast (CFU-F) assay was used for the estimation of MSC frequency within the BM mononuclear cell (BMMC) fraction. Ex-vivo expanded MSCs were phenotypically characterized by flow cytometry (FC) using appropriate markers. In vitro differentiation to adipocytes and osteoblasts was assessed by cytochemical stains. The proliferative potential of ex vivo expanded MSCs was evaluated by Methyl Triazolyl Tetrazolium (MTT)-based assay and survival characteristics were studied using FC and 7-Aminoactinomycin D (7-AAD) staining. To assess the effect of patient MSCs on B cell growth, B cells were immunomagnetically isolated (Miltenyi Biotec GmbH, Germany) from peripheral blood (PB) of normal individuals, labeled with carboxy fluorescein succinimidyl ester (CFSE; Gibco Invitrogen, Paisley, Scotland) and subsequently cultured in the absence or presence of confluent layers of allogeneic BM-MSCs from SMZL patients or normal controls in the presence of CpG oligonucleotide 2006 (Invivogen, France) and IL-2 (R&D Systems, Minneapolis, MN). In a separate set of experiments, B cell survival was evaluated via FC and 7-AAD staining, after co-culturing with BM-MSCs from patients or healthy donors. Finally, to study BM-MSC capacity to chemotactically attract B-cells, transwell migration assays were set. In the bottom chambers MSCs from patients or healthy individuals were grown until confluency and then isolated B cells from PB of either patients or controls were added into the upper chamber. Twelve hours later migrated cells were enumerated. Grouped data are expressed as means± 1 standard error of the mean (SEM). MSCs were successfully expanded from all participants in the study. Adherent cells from both study groups displayed the typical spindle-shape morphology and immunophenotypic analysis at the end of P2-P3-P4 demonstrated that cultures constituted of a homogeneous cell population, typically expressing CD29, CD44, CD73, CD90 and CD105 while being negative for CD14, CD34 and CD45. SMZL-derived MSCs were similar to their normal counterparts in the capacity to differentiate towards adipocytes and osteocytes as evidenced by Oil Red O and Alizarin Red staining, respectively. The frequency of MSCs within the BMMC compartment was significantly lower in patients as compared to healthy individuals (2.5±0.68/105 ΒΜΜCs and 7.23±0.6/105 ΒΜΜCs, respectively; P=0.0032) apparently due to the predominance of the lymphoma cells within patient BMMCs. SMZL MSCs displayed defective proliferative potential as compared to their normal counterparts at P2, as evidenced by the MTT assay (P<0.0001). To explore the influence of SMZL BM-MSCs in B cells survival we compared the viability of B cells isolated from the PB of healthy individuals cultured in medium alone to that of such cells co-cultured with either BM-MSCs derived from patients or normal controls. 43.85±1.46% of B cells cultured alone were apoptotic, while only 20.8±2.63% and 12±0.77% of B cells co-cultured with either normal MSCs or SMZL MSCs were apoptotic (P<0.0001 and P<0.0001, respectively). Notably patient MSCs confer a survival advantage in B cell viability over their normal counterparts (P=0.0374). Finally SMZL MSCs had a more potent chemotactic activity on B cells from healthy donors, as compared to MSCs from normal controls ( P<0.05). In conclusion we have shown for the first time that SMZL lymphoma MSCs are intrinsically defective in terms of proliferative potential and exert an altered modulation of B cell apoptosis and B cell chemotaxis. These preliminary results concerning the properties of SMZL MSCs merit further investigation and provide the theoretical background for exploring their potential implication in lymphomagenesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals NR4A Expression by Human Marginal Zone B-Cells

Antibodies ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 50
Author(s):  
Kim Doyon-Laliberté ◽  
Josiane Chagnon-Choquet ◽  
Michelle Byrns ◽  
Matheus Aranguren ◽  
Meriam Memmi ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Population ◽  
Human Blood ◽  
Marginal Zone ◽  
Ex Vivo ◽  
Transcriptome Profiling ◽  
Marginal Zone B Cells ◽  
Healthy Donors ◽  
And Function

We have previously characterized a human blood CD19+CD1c+IgM+CD27+CD21loCD10+ innate-like B-cell population, which presents features shared by both transitional immature and marginal zone (MZ) B-cells, named herein “precursor-like” MZ B-cells. B-cells with similar attributes have been associated with regulatory potential (Breg). In order to clarify this issue and better characterize this population, we have proceeded to RNA-Seq transcriptome profiling of mature MZ and precursor-like MZ B-cells taken from the blood of healthy donors. We report that ex vivo mature MZ and precursor-like MZ B-cells express transcripts for the immunoregulatory marker CD83 and nuclear receptors NR4A1, 2, and 3, known to be associated with T-cell regulatory (Treg) maintenance and function. Breg associated markers such as CD39 and CD73 were also expressed by both populations. We also show that human blood and tonsillar precursor-like MZ B-cells were the main B-cell population to express elevated levels of CD83 and NR4A1-3 proteins ex vivo and without stimulation. Sorted tonsillar precursor-like MZ B-cells exerted regulatory activity on autologous activated CD4+ T-cells, and this was affected by a CD83 blocking reagent. We believe these observations shed light on the Breg potential of MZ populations, and identify NR4A1-3 as potential Breg markers, which as for Tregs, may be involved in stabilization of a regulatory status. Since expression and activity of these molecules can be modulated therapeutically, our findings may be useful in strategies aiming at modulation of Breg responses.

Ligation of tumour-produced mucins to CD22 dramatically impairs splenic marginal zone B-cells

2009 ◽  
Vol 417 (3) ◽  
pp. 673-683 ◽  
Author(s):  
Munetoyo Toda ◽  
Risa Hisano ◽  
Hajime Yurugi ◽  
Kaoru Akita ◽  
Kouji Maruyama ◽  
...  
Keyword(s):  
Sialic Acid ◽  
B Cells ◽  
B Cell ◽  
Marginal Zone ◽  
Cell Signalling ◽  
Negative Regulator ◽  
Mammary Adenocarcinoma ◽  
Marginal Zone B Cells ◽  
Tumour Bearing

CD22 [Siglec-2 (sialic acid-binding, immunoglobulin-like lectin-2)], a negative regulator of B-cell signalling, binds to α2,6- sialic acid-linked glycoconjugates, including a sialyl-Tn antigen that is one of the typical tumour-associated carbohydrate antigens expressed on various mucins. Many epithelial tumours secrete mucins into tissues and/or the bloodstream. Mouse mammary adenocarcinoma cells, TA3-Ha, produce a mucin named epiglycanin, but a subline of them, TA3-St, does not. Epiglycanin binds to CD22 and inhibits B-cell signalling in vitro. The in vivo effect of mucins in the tumour-bearing state was investigated using these cell lines. It should be noted that splenic MZ (marginal zone) B-cells were dramatically reduced in the mice bearing TA3-Ha cells but not in those bearing TA3-St cells, this being consistent with the finding that the thymus-independent response was reduced in these mice. When the mucins were administered to normal mice, a portion of them was detected in the splenic MZ associated with the MZ B-cells. Furthermore, administration of mucins to normal mice clearly reduced the splenic MZ B-cells, similar to tumour-bearing mice. These results indicate that mucins in the bloodstream interacted with CD22, which led to impairment of the splenic MZ B-cells in the tumour-bearing state.

scholarly journals Dual T cell– and B cell–intrinsic deficiency in humans with biallelic RLTPR mutations

2016 ◽  
Vol 213 (11) ◽  
pp. 2413-2435 ◽  
Author(s):  
Yi Wang ◽  
Cindy S. Ma ◽  
Yun Ling ◽  
Aziz Bousfiha ◽  
Yildiz Camcioglu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Cell Phenotype ◽  
Loss Of Function ◽  
Inborn Errors

Combined immunodeficiency (CID) refers to inborn errors of human T cells that also affect B cells because of the T cell deficit or an additional B cell–intrinsic deficit. In this study, we report six patients from three unrelated families with biallelic loss-of-function mutations in RLTPR, the mouse orthologue of which is essential for CD28 signaling. The patients have cutaneous and pulmonary allergy, as well as a variety of bacterial and fungal infectious diseases, including invasive tuberculosis and mucocutaneous candidiasis. Proportions of circulating regulatory T cells and memory CD4+ T cells are reduced. Their CD4+ T cells do not respond to CD28 stimulation. Their CD4+ T cells exhibit a "Th2" cell bias ex vivo and when cultured in vitro, contrasting with the paucity of "Th1," "Th17," and T follicular helper cells. The patients also display few memory B cells and poor antibody responses. This B cell phenotype does not result solely from the T cell deficiency, as the patients’ B cells fail to activate NF-κB upon B cell receptor (BCR) stimulation. Human RLTPR deficiency is a CID affecting at least the CD28-responsive pathway in T cells and the BCR-responsive pathway in B cells.

Interleukin-7 Over-Expression Regulates T Cell Versus B Cell Lineage Development in the Thymus.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3238-3238
Author(s):  
Nahed El Kassar ◽  
Philip J. Lucas ◽  
Frank Flomerfelt ◽  
Melinda Merchant ◽  
Catherine V. Bare ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Dose Effect ◽  
Proliferative Potential ◽  
Thymic Tissue ◽  
Over Expression ◽  
Normal Controls

Abstract We have shown that high IL-7 transgene (Tg) over-expression (39-fold at day 1 in thymic tissue) under the T cell specific, proximal lck promoter had a dose effect on TCRαβ that was accompanied by active B cell development in the thymus. To further characterize these affects in the thymus of IL-7 transgenic mice, we analyzed thymi from day 18 embryos and newborn Tg mice, as well as fetal thymic organ culture (FTOC) derived from using day 16 embryos. We show that arrested T-cell and increased B-cell thymic development is initiated during fetal development. Using mixed bone marrow chimeras and anti-IL-7 monoclonal antibody injection, we further demonstrate that abnormalities in thymic T and B cell development are non-cell autonomous and are due to IL-7 over-expression. Recently, it was shown that only the early thymocyte progenitor (ETP, c-kit+IL-7R−/lo) fraction within the DN1 subpopulation had a T-cell proliferative potential in contrast to the c-kit−IL-7R+ DN1 subset. Here we show that in Tg mice the ETPs were decreased, while the c-kit−IL-7R+ cells are increased in both percentage and absolute count when compared to normal controls. In order to explore the T vs. B ETP potential, we seeded re-aggregate thymic organ cultures with sorted lin−CD44+CD25−c-kit+IL-7R+ cells. While ETPs derived from normal controls were able to proliferate and produce 83% of DP thymocytes, ETPs sorted from Tg mice developed poorly (10-fold less) into DP cells (30%) and produced 14% of B220+ cells vs. 6% in controls. Moreover, sorted Pro/Pre B derived thymic B cells from Tg mice, but not BM-derived Pro/Pre B cells had the TCRβD-J rearrangement, suggesting a T-specific origin. Since the B-cell differentiation pathway in normal mice is selectively inhibited by thymic presentation of Notch ligands, we hypothesized that IL-7 down-regulates Notch signaling. To test this hypothesis, we analyzed thymocyte progenitors (DN1-DN4) in normal and Tg mice for the intra-cytoplasmic part of Notch, that is cleaved upon Notch/Notch-ligand activation. Notch staining was decreased in the lin−CD44+CD25inter representing the only DN2 population present in these Tg mice. These data favour a decrease of Notch signalling in mice with high IL-7 Tg over-expression, inducing a block in TCRαβ development, and skewing of thymic B cell development by T vs. B lineage subversion. These conclusions may have implications for IL-7 in the clinical setting.

Expression of a Dysfunctional Activation Induced Cytidine Deaminase Is Correlated with Disease Progression in Splenic Marginal Zone Lymphoma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2397-2397
Author(s):  
Gabriel Brisou ◽  
Laurent Jallades ◽  
Alexandra Traverse-Glehen ◽  
Francoise Berger ◽  
Aurélie Verney ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Marginal Zone ◽  
Cytidine Deaminase ◽  
Marginal Zone Lymphoma ◽  
Splenic Marginal Zone Lymphoma ◽  
Marginal Zone B Cells ◽  
Activation Induced Cytidine Deaminase ◽  
Splicing Variants

Abstract Abstract 2397 B cells can undergo at least two differentiation pathways, dependent of T cells or not, starting from follicular or marginal zone B cells respectively. The T-independent response, less understood than the germinal center reaction, is triggered by specific antigens and arises from marginal zone B cells. During this development, some B cells undergo somatic hypermutation (SHM) and class switch recombination (CSR), triggered by the same DNA editing enzyme called Activation Induced Cytidine Deaminase (AID). The splenic marginal zone lymphoma (SMZL) is a rare lymphoproliferative disorder characterized by a clonal expansion of B cells in the marginal zone of the spleen. These B-cells underwent SHM in roughly 60% of the cases but nearly none underwent CSR. These observations suggest that tumor clones originate from a particular activated B cell subset not transiting through the germinal center. In order to confirm this hypothesis, we focused our work on the status and impact of AID in this disease and worked on purified B cells extracted from spleen of well-characterized SMZL cases. We determined AID status by quantitative RT-PCR analysis on 27 SMZL samples and compared it with 5 controls. In the SMZL group the relative level of expression of AID is heterogeneous but two subgroups could be distinguished: one considered as expressing AID (14 cases out of the 27 analyzed), the remaining considered as not expressing AID. When we compared AID expression rate with occurrence of SHM and CSR, no clear correlation between AID expression and presence of SHM or CSR could be observed suggesting that AID, when expressed, is dysfunctional. To address this hypothesis, we first analyzed AID protein by immunohistochemistry and a good correlation between IHC signal and AID mRNA expression level has been observed. As AID gene was not mutated, we next focused our work on AID mRNA splicing variants as these variants exhibit different functions according to the domain of the protein they contain in a murine model. We found that SMZL B cells express various splicing variants of AID mRNA, some of those variants corresponding to the full length isoform (n = 6/17), and other variants corresponding to AID-ΔE4a (n = 2/17) or AID-ΔE4 (n = 7/17) isoforms known to be expressed in normal germinal center B cells as well as in Chronic Lymphocytic and Acute Lymphoblastic Leukemia. These findings indicate that although expressed at the mRNA and protein levels, AID may not be fully functional in SMZL cases. Finally we addressed the potential clinical significance of AID expression. We identified for that purpose a group of “progressive SMZL” patients that had received immuno-chemotherapy after splenectomy because of a significant risk of progression or transformation into aggressive large B cell lymphoma (n = 8/27) pre-empting outcome differences. We found a higher proportion of AID expressing patients in the defined “progressive SMZL” group (n = 7/8) as compared to the proportion found in the “indolent SMZL” group (n = 5/14, p = 0,03). Altogether, this data suggest that the B cell clone leading to SMZL originate from the marginal zone and support the hypothesis of a lymphoproliferative disorder affecting the T-independent response. AID expression in SMZL may reflect an advanced stage of the disease and could be correlated with the evolution of the lymphoma into a more clinically or pathologically aggressive form. Disclosures: No relevant conflicts of interest to declare.

In Vitro Co-Culture of CLL-B Cells Reveals Long-Term Survival, Proliferation, and Maintenance of Telomere Length

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 350-350
Author(s):  
Ceri H Jones ◽  
Thet Thet Lin ◽  
Elisabeth Jane Walsby ◽  
Guy E Pratt ◽  
Christopher Fegan ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Telomere Length ◽  
Erosion Rate ◽  
Ex Vivo ◽  
Effector Memory ◽  
Long Term Culture

Abstract Telomere length is a prognostic factor in Chronic Lymphocytic Leukemia (CLL) with short telomere length a powerful predictor of early time to first treatment and reduced overall survival. However, little is known about telomere dynamics through the course of an individual patient's disease. Our recent longitudinal analysis of CLL B-cell telomere length revealed very little dynamic change within individual patients with a mean erosion rate of -52bp/year (p=0.05). In marked contrast, T-cells derived from the same patients showed a significantly higher mean erosion rate of -119bp/year (p=0.02) with a median follow up time of 69 months. Here we present data derived from long-term in-vitro co-culture of peripheral blood from CLL patients coupled with temporal analysis of their telomere length dynamics. We utilized a multi-cellular co-culture system, comprised of autologous T-cells and CD40L-expressing mouse fibroblasts, to maintain CLL cells in long-term culture. Patient-derived peripheral blood mononuclear cells (n=16) were maintained for a median of 70 days (range 54-154); samples were analyzed every two weeks for tumor cell telomere length and evidence of proliferation. We used fluorescence-activated cell sorting (FACS) to sort populations of CD19+CD5+ CLL B-cells and CD3+ T-cells from each of the cultures. We then performed high-resolution single telomere length analysis (STELA) on these sorted subsets of cells and analyzed their telomere dynamics over this extended time course. Analysis of CLL B-cells from these cultures revealed significantly increased Ki-67+ at day 14 when compared to day 0 (p<0.001) and this was evident for the duration of the cultures. Despite sustained tumor cell proliferation, we observed no significant difference in the CLL B-cell telomere length with a mean TL at the start of 4.5kb vs 4.3kb at the end (p=0.14). The presence of T-cells was shown to be critical for the maintenance of the long-term cultures in two ways. Firstly, cultures that were treated with 4μM fludarabine showed a catastrophic reduction in T-cells (p=0.01), which was associated with a significantly shorter duration of survival of CLL B-cells when compared to untreated controls (median 17.5 days (range 7-70); p<0.001). Secondly, it proved impossible to maintain T-cell depleted, purified CLL B-cells, in long-term culture. T-cells isolated from the long-term cultures showed evidence of proliferation with Ki-67+ again being increased at day 14 in comparison to baseline (p=0.003). Furthermore, T-cells derived from these cultures showed a significant alteration in subset composition over time with a decrease in the numbers of naive CD4+ (p=0.05) and CD8+ (p=0.02) T-cells and a corresponding increase in effector memory (p=0.2) and terminally differentiated effector memory (EMRA) subsets (p=0.07). In conclusion, this study demonstrates that we have developed a robust, long-term culture method for the maintenance of CLL cells. Despite evidence of sustained CLL proliferation, CLL B-cells showed little telomere length erosion during long-term co-culture and this is compatible with our recent ex-vivo analysis, which showed that the telomere length of CLL B-cells are remarkably stable with a mean erosion rate of only -52bp/year. In both ex-vivo and in-vitro analysis, telomere erosion correlated with starting telomere length (r2=0.14, p=0.04 and r2=0.3 p=0.03 respectively). Taken together, our in-vitro and ex-vivo data imply that the radically short telomeres observed in some CLL patients are not the result of increased proliferation of the malignant B-cell, but rather the mutagenic event occurs in a B-cell which already has short telomeres. Furthermore, our novel long-term culture model has reinforced the vital role of T-cells in sustaining CLL B-cells viability and proliferation in-vitro. Given the consistent skewing of the T-cell pool towards a memory phenotype it seems unlikely that this is driven in-vitro by cognate TCR antigen recognition but rather a cytokine-mediated response. Disclosures Fegan: Gilead Sciences: Honoraria; Roche: Honoraria; AbbVie: Honoraria.

scholarly journals Autoreactive marginal zone B cells are spontaneously activated but lymph node B cells require T cell help

2006 ◽  
Vol 203 (8) ◽  
pp. 1985-1998 ◽  
Author(s):  
Laura Mandik-Nayak ◽  
Jennifer Racz ◽  
Barry P. Sleckman ◽  
Paul M. Allen
Keyword(s):  
Lymph Node ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Marginal Zone ◽  
B Cell Tolerance ◽  
Marginal Zone B Cells ◽  
T Cell Help

In K/BxN mice, arthritis is induced by autoantibodies against glucose-6-phosphate-isomerase (GPI). To investigate B cell tolerance to GPI in nonautoimmune mice, we increased the GPI-reactive B cell frequency using a low affinity anti-GPI H chain transgene. Surprisingly, anti-GPI B cells were not tolerant to this ubiquitously expressed and circulating autoantigen. Instead, they were found in two functionally distinct compartments: an activated population in the splenic marginal zone (MZ) and an antigenically ignorant one in the recirculating follicular/lymph node (LN) pool. This difference in activation was due to increased autoantigen availability in the MZ. Importantly, the LN anti-GPI B cells remained functionally competent and could be induced to secrete autoantibodies in response to cognate T cell help in vitro and in vivo. Therefore, our study of low affinity autoreactive B cells reveals two distinct but potentially concurrent mechanisms for their activation, of which one is T cell dependent and the other is T cell independent.

scholarly journals Circulating Exosomes Inhibit B Cell Proliferation and Activity

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2110
Author(s):  
Jan C. Schroeder ◽  
Lisa Puntigam ◽  
Linda Hofmann ◽  
Sandra S. Jeske ◽  
Inga J. Beccard ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
B Cells ◽  
B Cell ◽  
Surface Expression ◽  
Receptor Expression ◽  
Healthy Individuals ◽  
Inhibitory Effects ◽  
Healthy Donors ◽  
Checkpoint Receptors

(1) Background: Head and neck squamous cell carcinoma (HNSCC) is characterized by a distinctive suppression of the anti-tumor immunity, both locally in the tumor microenvironment (TME) and the periphery. Tumor-derived exosomes mediate this immune suppression by directly suppressing T effector function and by inducing differentiation of regulatory T cells. However, little is known about the effects of exosomes on B cells. (2) Methods: Peripheral B cells from healthy donors and HNSCC patients were isolated and checkpoint receptor expression was analyzed by flow cytometry. Circulating exosomes were isolated from the plasma of HNSCC patients (n = 21) and healthy individuals (n = 10) by mini size-exclusion chromatography. B cells from healthy individuals were co-cultured with isolated exosomes for up to 4 days. Proliferation, viability, surface expression of checkpoint receptors, and intracellular signaling were analyzed in B cells by flow cytometry. (3) Results: Expression of the checkpoint receptors PD-1 and LAG3 was increased on B cells from HNSCC patients. The protein concentration of circulating exosomes was increased in HNSCC patients as compared to healthy donors. Both exosomes from healthy individuals and HNSCC patients inhibited B cell proliferation and survival, in vitro. Surface expression of inhibitory and stimulatory checkpoint receptors on B cells was modulated in co-culture with exosomes. In addition, an inhibitory effect of exosomes on B cell receptor (BCR) signaling was demonstrated in B cells. (4) Conclusions: Plasma-derived exosomes show inhibitory effects on the function of healthy B cells. Interestingly, these inhibitory effects are similar between exosomes from healthy individuals and HNSCC patients, suggesting a physiological B cell inhibitory role of circulating exosomes.

Assessing CD137 (4-1BB) As a Therapeutic Target in B-Cell Neoplasms,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3735-3735
Author(s):  
Adam D Cohen ◽  
Indira D Joshi ◽  
Valentin Robu ◽  
Hossein Borghaei ◽  
Tahseen I. Al-Saleem ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
Tumor Cell ◽  
B Cell ◽  
Marginal Zone ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Tumor Cell Proliferation

Abstract Abstract 3735 Agonist monoclonal antibodies (mAbs) to CD137, a co-stimulatory TNF receptor family member expressed on activated T and NK cells, can induce immune-mediated rejection of multiple murine tumor types, and a fully human anti-CD137 mAb, BMS-663513, is in early-phase clinical trials in solid tumors. Significant activity has been seen in murine lymphoma models, both alone and in combination with anti-CD20 mAbs, providing rationale for clinical studies in lymphoma patients. Recently, however, CD137 up-regulation on activated human B cells has been reported, with CD137 ligation causing enhanced B cell proliferation and survival. This raises the concern that mAb binding to CD137, if present, on B cell neoplasms may promote tumor cell proliferation and/or resistance to apoptosis that may counteract the beneficial effects on T and NK cells. We therefore sought to assess the expression of CD137 on a series of human cell lines and primary tumor samples from patients with B-cell neoplasms, and if expressed, to explore the consequences of ligation with the anti-CD137 agonist BMS-66513. First, archived paraffin-embedded lymph node specimens from patients with low-grade B-cell lymphoma (n=11: 5 follicular, 4 marginal zone, 2 small lymphocytic) and diffuse large B-cell lymphoma (n=15) were stained for CD137 by immunohistochemistry. Reactive tonsillar tissue served as a positive control. No CD137 expression was observed within any tumor cells. Next, fresh samples from 14 additional patients with known tumor involvement of peripheral blood or bone marrow (8 chronic lymphocytic leukemia, 1 mantle cell lymphoma, 3 myeloma, 2 marginal zone lymphoma) were analyzed by multi-color flow cytometry. Again, no CD137 expression was observed on the gated neoplastic cells. Baseline surface expression of CD137 was similarly absent in all B cell-derived lines tested (Raji, FCTxFL2, FSCCL, DoHH2, Jeko-1, RPMI8226). However, activation with PMA/Ionomycin could reproducibly induce CD137 expression (% positive: 0.17% → 91%) after 24 hours in 1 of the lines: the follicular lymphoma FSCCL. Interestingly, this was the only line tested that lacked constitutive expression of CD137 ligand (CD137L), suggesting some reciprocal regulation of ligand and receptor expression. Despite this up-regulation of CD137, in vitro ligation of PMA/Ionomycin-activated FSCCL cells with BMS-66513 did not further increase tumor cell proliferation, nor protect the cells from activation-induced cell death, in contrast to effects of CD137 ligation reported in normal B cells (Zhang et al, J Immunol 2010; 184:787). Similarly, BMS-663513 treatment of activated, CD137+ FSCCL cells did not diminish the apoptosis induced by doxorubicin or bortezomib treatment. In addition, FSCCL cells recovered from ascites 7 and 14 days following intraperitoneal injection in SCID mice did not express CD137, implying that CD137 up-regulation is not occurring in vivo during tumor growth. Finally, treatment of FSCCL cells with rituximab, either in vitro or in vivo, did not induce CD137 expression. In conclusion, we demonstrate a lack of steady-state CD137 expression on malignant B cells, confirming the prior study by Houot et al (Blood 2009; 114:3431) and extending these findings to include CLL/SLL for the first time. While CD137 could be induced in a single cell line upon non-specific activation, CD137 expression on FSCCL cells was not seen under physiologic conditions likely to be encountered in the clinical setting, consistent with the primary patient data. Furthermore, even when CD137 was expressed, ligation with the agonist anti-CD137 mAb BMS-663513 did not provide a pro-proliferative or anti-apoptotic signal. These studies provide reassurance and further rationale for exploring agonist anti-CD137 antibodies as therapies for B cell neoplasms. Disclosures: Borghaei: Lilly, Genentech, Amgen, Pfizer: Honoraria, Research Funding. Jure-Kunkel:Bristol Meyers Squibb: Employment.

scholarly journals Ex vivo characterization and isolation of rare memory B cells with antigen tetramers

Blood ◽  
2011 ◽  
Vol 118 (2) ◽  
pp. 348-357 ◽  
Author(s):  
Bettina Franz ◽  
Kenneth F. May ◽  
Glenn Dranoff ◽  
Kai Wucherpfennig
Keyword(s):  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Ex Vivo ◽  
Cell Receptor ◽  
Memory B Cells ◽  
Cell Repertoire ◽  
Low Frequencies

Abstract Studying human antigen-specific memory B cells has been challenging because of low frequencies in peripheral blood, slow proliferation, and lack of antibody secretion. Therefore, most studies have relied on conversion of memory B cells into antibody-secreting cells by in vitro culture. To facilitate direct ex vivo isolation, we generated fluorescent antigen tetramers for characterization of memory B cells by using tetanus toxoid as a model antigen. Brightly labeled memory B cells were identified even 4 years after last immunization, despite low frequencies ranging from 0.01% to 0.11% of class-switched memory B cells. A direct comparison of monomeric to tetrameric antigen labeling demonstrated that a substantial fraction of the B-cell repertoire can be missed when monomeric antigens are used. The specificity of the method was confirmed by antibody reconstruction from single-cell sorted tetramer+ B cells with single-cell RT-PCR of the B-cell receptor. All antibodies bound to tetanus antigen with high affinity, ranging from 0.23 to 2.2 nM. Furthermore, sequence analysis identified related memory B cell and plasmablast clones isolated more than a year apart. Therefore, antigen tetramers enable specific and sensitive ex vivo characterization of rare memory B cells as well as the production of fully human antibodies.

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