Evaluation of Immune Profile in Patients with Multiple Myeloma Using Cytof Technology

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3404-3404
Author(s):  
Jana Jakubikova ◽  
Efstathios Kastritis ◽  
Danka Cholujova ◽  
Teru Hideshima ◽  
Ludmila M Flores ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Natural Killer ◽  
Plasma Cells ◽  
Memory B Cells ◽  
High Dimensional ◽  
Newly Diagnosed ◽  
D Cells

Abstract Introduction: CyTOF (time-of-flight mass cytometry) is a novel high-dimensional technology which permits immunophenotyping and analysis of signaling in single cells. This approach enables simultaneous evaluation of up to 40 parameters using antibodies tagged with distinct elemental isotopes, by combining flow cytometry with atomic mass spectrometry. Since multiple myeloma (MM) is characterized by immune dysfunction, we used CyTOF technology to define the complex immune profile in MM patient bone marrow (BM) samples. Methods: We used 40 different markers to define various B, T, natural killer (NK) subsets, as well as cells of monocytic, granulocytic, erythroid and platelet lineages. Our preliminary data are results from 10 patients with MGUS/ smoldering MM (SMM); 10 newly diagnosed MM; 20 relapsed/refractory MM; and 15 WM patients (5 newly diagnosed and 10 receiving treatment) in comparison with age-matched healthy donors’ BM (HD). A significantly larger cohort of MM (N=150) and WM (N=50) patients is being similarly analyzed and will be presented. To evaluate phenotypic abnormalities in various B cell subsets, we used B lineage markers CD10, CD19, CD20, CD22, CD27, CD34, CD38, CD45, IgA, IgD, IgG and IgM to define B cells maturation stages from hematopoietic stem cells (HSC) to naïve to mature B lymphocytes (pro-B; pre-B-I; pre-B-II; immature B; and mature (naïve) B cells), as well as memory non-switched and memory switched B cells, plasmablasts, normal (CD138+CD38+CD19+CD45+) and clonal plasma cells (CD138+CD38+CD19-CD45-/low), which reside in the specific BM niche. Furthermore, natural killer (NK) subsets (such as NK and NKT cells) and T cells (such as memory CD4T, naive CD4T, memory CD8T, naive CD8T, T regulatory cells and Tg/d cells) were examined. High-dimensional data was obtained using CyTOF technology and analyzed by SPADE and viSNE software. Results: Our data showed significantly decreased HSC in patients with newly diagnosed and relapsed/refractory MM compared to HD (P<0.025). A significant increase in pre-B-I cells was detected in relapsed/refractory MM vs. MGUS/SMM (P<0.028), but the opposite trend was observed in the pre-B-II subpopulation (P<0.005). No differences in immature B cell populations were observed in different stages of MM. However, a significantly higher percentage of immature B cells was present in relapsed/refractory MM compared to HD (P=0.008), and transitional B cells were significantly decreased in newly diagnosed MM compared to HD (P<0.001). Moreover, memory B cells were significant decreased in all MM stages compared to HD (P<0.003). Non-switched memory B cells were significantly increased in MGUS and SMM compared to newly diagnosed MM, while a significant increase of switched memory B cells was present in newly diagnosed MM compared to relapsed/refractory MM. A significant increase in plasmablasts was seen in relapsed/refractory MM in comparison with other MM stages (P<0.011) by CyTOF analyses. Malignant plasma cells (PC) were defined as CD19-, CD38++, CD45-/dim, CD138+ and either cyk or cyl positive. Importantly, a significant increase in clonal PC was found in all MM stages vs. HD, as well as in newly diagnosed MM compared to relapsed/refractory MM (P<0.01). The percentage of PC from CyTOF analyses correlated with % of PC obtained using flow cytometry by Bland-Altman method comparison. We also observed significant differences in T cell subsets including naïve, central memory, effector, and effector memory CD4+ and CD8+T populations between MGUS and newly diagnosed MM, but no significant changes in T regulatory and Tg/d cells. Furthermore, plasmacytoid dendritic (pDC) cells were significantly increased in newly diagnosed MM, and PD-1 expressed on pDC was significantly decreased in newly diagnosed MM compared to MGUS (P=0.007). Interestingly, PD-1 and its ligand PD-L1 were variably expressed on B cells (2-9% and 3-27%) and PC (0.5-46% and 3-41%) from MM BM samples. Other surface molecules including CD269 (4-32%), CD289 (1-8%), CD362 (0.5-1%) and CD329 (1-4%), were variably expressed in PC. Conclusion: A better understanding of the neoplastic BM milieu will provide the framework for identifying and validating novel targeted therapies directed against MM. CyTOF technology represents a novel diagnostic tool to assess the status not only of MM, but also of host immunity, and may allow for the development of rational personalized therapies. Disclosures No relevant conflicts of interest to declare.

MO251HIGHLY SENSITIVE FLOW CYTOMETRIC DETECTION OF RESIDUAL B-CELLS AFTER RITUXIMAB IN ANTI-NEUTROPHIL CYTOPLASMIC ANTIBODY-ASSOCIATED VASCULITIS PATIENTS*

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Y K O Teng ◽  
L Van Dam ◽  
Jelle Oskam ◽  
S W A Kamerling ◽  
E J Arends ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
Memory B Cells ◽  
Cell Depletion ◽  
B Cell Depletion ◽  
Peripheral Blood Mononuclear

Abstract Background and Aims B-cell depletion with rituximab (RTX) is an effective treatment for anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) patients. Nevertheless, relapses are frequent after RTX, often preceded by B-cell repopulation suggesting that residual autoreactive B-cells persist despite therapy. Therefore, this study aimed to identify minimal residual autoimmunity (MRA) in the B-cell compartment of AAV patients treated with RTX. Method EuroFlow-based highly-sensitive flow cytometry (HSFC) was employed to study B-cell and plasma cell (PC) subsets in-depth in AAV patients before and after RTX treatment. Additionally, peripheral blood mononuclear cells (PBMCs) of these RTX-treated AAV patients were cultured and in vitro stimulated with CpG, IL-2, and IL-21 to induce antibody-secreting cells (ASC). (ANCA)-IgG was measured in these supernatants by ELISA. Results By employing EuroFlow-based HSFC, we detected circulating CD19+ B-cells at all timepoints after RTX treatment, in contrast to conventional low-sensitive flow cytometry. Pre-germinal center (Pre-GC) B-cells, memory B-cells and CD20+CD138− plasmablasts (PBs) were rapidly and strongly reduced, while CD20−CD138− PrePC and CD20-CD138+ mature (m)PCs were reduced slower and remained detectable. Both memory B-cells and CD20− PCs remained detectable after RTX. Serum ANCA-IgG decreased significantly upon RTX. Changes in ANCA levels strongly correlated with changes in naive, switched CD27+ and CD27− (double-negative) memory B-cells, but not with plasma cells. Lastly, we demonstrated in vitro ANCA production by AAV PBMCs, 24 and 48 weeks after RTX treatment reflecting MRA in the memory compartment of AAV patients. Conclusion We demonstrated that RTX induced strong reductions in circulating B-cells, but never resulted in complete B-cell depletion. Despite strongly reduced B-cell numbers after RTX, ANCA-specific memory B-cells were still detectable in AAV patients. Thus, MRA is identifiable in AAV and can provide a potential novel approach in personalizing RTX treatment in AAV patients.

scholarly journals Potential anti-EBV effects associated with elevated interleukin-21 levels: a case report

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Kristian Assing ◽  
Christian Nielsen ◽  
Marianne Jakobsen ◽  
Charlotte B. Andersen ◽  
Kristin Skogstrand ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Germinal Center ◽  
Plasma Cells ◽  
Cell Formation ◽  
Memory B Cells ◽  
Memory B Cell

Abstract Background Germinal center derived memory B cells and plasma cells constitute, in health and during EBV reactivation, the largest functional EBV reservoir. Hence, by reducing germinal center derived formation of memory B cells and plasma cells, EBV loads may be reduced. Animal and in-vitro models have shown that IL-21 can support memory B and plasma cell formation and thereby potentially contribute to EBV persistence. However, IL-21 also displays anti-viral effects, as mice models have shown that CD4+ T cell produced IL-21 is critical for the differentiation, function and survival of anti-viral CD8+ T cells able to contain chronic virus infections. Case presentation We present immunological work-up (flow-cytometry, ELISA and genetics) related to a patient suffering from a condition resembling B cell chronic active EBV infection, albeit with moderately elevated EBV copy numbers. No mutations in genes associated with EBV disease, common variable immunodeficiency or pertaining to the IL-21 signaling pathway (including hypermorphic IL-21 mutations) were found. Increased (> 5-fold increase 7 days post-vaccination) CD4+ T cell produced (p < 0.01) and extracellular IL-21 levels characterized our patient and coexisted with: CD8+ lymphopenia, B lymphopenia, hypogammaglobulinemia, compromised memory B cell differentiation, absent induction of B-cell lymphoma 6 protein (Bcl-6) dependent peripheral follicular helper T cells (pTFH, p = 0.01), reduced frequencies of peripheral CD4+ Bcl-6+ T cells (p = 0.05), compromised plasmablast differentiation (reduced protein vaccine responses (p < 0.001) as well as reduced Treg frequencies. Supporting IL-21 mediated suppression of pTFH formation, pTFH and CD4+ IL-21+ frequencies were strongly inversely correlated, prior to and after vaccination, in the patient and in controls, Spearman’s rho: − 0.86, p < 0.001. Conclusions To the best of our knowledge, this is the first report of elevated CD4+ IL-21+ T cell frequencies in human EBV disease. IL-21 overproduction may, apart from driving T cell mediated anti-EBV responses, disrupt germinal center derived memory B cell and plasma cell formation, and thereby contribute to EBV disease control.

A reservoir of rituximab-resistant splenic memory B cells contributes to relapses after B-cell depletion therapy

2019 ◽  
Author(s):  
Etienne Crickx ◽  
Pascal Chappert ◽  
Sandra Weller ◽  
Aurélien Sokal ◽  
Imane Azzaoui ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Complete Response ◽  
Memory B Cells ◽  
Chain Gene ◽  
Gene Repertoire ◽  
Heavy Chain Gene ◽  
Immunoglobulin Heavy Chain Gene ◽  
Pathogenic Antibodies

AbstractImmune thrombocytopenia (ITP) is an autoimmune disease mediated by pathogenic antibodies directed against platelet antigens, including GPIIbIIIa. Taking advantage of spleen samples obtained from ITP patients, we characterized by multiples approaches the onset of disease relapses occurring after an initial complete response to rituximab. Analysis of splenic B cell immunoglobulin heavy chain gene repertoire at bulk level and from single anti-GPIIbIIIa B cells revealed that germinal centers were fueled by B cells originating from the ongoing lymphopoiesis, but also by rituximab-resistant memory B cells, both giving rise to anti-GPIIbIIIa plasma cells. We identified a population of splenic memory B cells that resisted rituximab through acquisition of a unique phenotype and contributed to relapses, providing a new target in B cell mediated autoimmune diseases.

scholarly journals Hyperproliferation of B Cells Specific for a Weakly Immunogenic PorA in a Meningococcal Vaccine Model

2008 ◽  
Vol 15 (10) ◽  
pp. 1598-1605 ◽  
Author(s):  
Thomas A. Luijkx ◽  
Jacqueline A. M. van Gaans-van den Brink ◽  
Harry H. van Dijken ◽  
Germie P. J. M. van den Dobbelsteen ◽  
Cécile A. C. M. van Els
Keyword(s):  
B Cells ◽  
B Cell ◽  
Bactericidal Activity ◽  
Plasma Cells ◽  
Affinity Maturation ◽  
Memory B Cells ◽  
Vaccine Antigen ◽  
Cell Subpopulation ◽  
Cell Subpopulations ◽  
Group B

ABSTRACT Highly homologous meningococcal porin A (PorA) proteins induce protective humoral immunity against Neisseria meningitidis group B infection but with large and consistent differences in the levels of serum bactericidal activity achieved. We investigated whether a poor PorA-specific serological outcome is associated with a limited size of the specific B-cell subpopulation involved. The numbers of PorA-specific splenic plasma cells, bone marrow (BM) plasma cells, and splenic memory B cells were compared between mice that received priming and boosting with the weakly immunogenic PorA (P1.7-2,4) protein and those that received priming and boosting with the highly immunogenic PorA (P1.5-1,2-2) protein. Immunoglobulin G (IgG) titers (except at day 42), bactericidal activity, and the avidity of IgG produced against P1.7-2,4 were significantly lower at all time points after priming and boosting than against P1.5-1,2-2. These differences, however, were not associated with a lack of P1.7-2,4-specific plasma cells. Instead, priming with both of the PorAs resulted in the initial expansion of comparable numbers of splenic and BM plasma cells. Moreover, P1.7-2,4-specific BM plasma cells, but not P1.5-1,2-2-specific plasma cells, expanded significantly further after boosting. Likewise, after a relative delay during the priming phase, the splenic P1.7-2,4-specific memory B cells largely outnumbered those specific for P1.5-1,2-2, upon boosting. These trends were observed with different vaccine formulations of the porins. Our results show for the first time that B-cell subpopulations involved in a successfully maturated antibody response against a clinically relevant vaccine antigen are maintained at smaller population sizes than those associated with poor affinity maturation. This bears consequences for the interpretation of immunological memory data in clinical vaccine trials.

The Loss of Kdm6a in B-Cell Development Causes Germinal Center Hyperplasia and Impedes the B-Cell Immune Response in a Specific Manner

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 5-5
Author(s):  
Ling Tian ◽  
Monique Chavez ◽  
Lukas D Wartman
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Plasma Cells ◽  
Cell Development ◽  
Young Female ◽  
B Cell Development ◽  
Cell Immune Response ◽  
Germinal Center Hyperplasia

Putative loss-of-function mutations in KDM6A, an X-linked H3K27 demethylase, occur recurrently in B-cell malignancies, including B-cell non-Hodgkin lymphoma. How the KDM6A in normal B cell development and function, as well as the mechanism(s) by which its loss contributes lymphomagenesis has not been defined. To address this issue, we generated a conditional knockout mouse of the Kdm6a gene (with LoxP sites flanking the 3rd exon) and crossed these mice with Vav1-Cre transgenic mice to selectively inactivate Kdm6a in hematopoietic stem/progenitor cells. Our previous data have shown young Kdm6a-null mice have a myeloid skewing in the bone marrow, spleen and peripheral blood. These changes became more pronounced with age and were specific to the female, homozygous Kdm6a knockout mice. Early B-cell development is also altered in female Kdm6a-null mice. Flow cytometry showed a decrease in multipotent progenitor cells (MPPs) with a decrease in both common lymphoid progenitors (CLPs) and B cell-biased lymphoid progenitors (BLPs) in young, female Kdm6a-null mice bone marrow. B-cell progenitor analysis (Hardy profiles) showed an increase in Fraction A with a concomitant decrease in Fraction B/C and Fraction D. The GC B-cells are thought to be the cell-of-origin of diffuse large B-cell lymphoma (DLBCL). To determine if the loss of Kmd6a could impact the mature B cells undergo germinal center (GC) reaction, we immunized the young, female Kdm6a-null mcie and wildtype littermates with T cell-dependent antigen sheep red blood cell (SRBC). Mice were scrificed 14 days after immunization, spleen cells were examined by flow cytometry. As expected, we observed a significant increase in the percentage of GC B cells (B220+GL7+CD95+) from female Kdm6a-null mice compared to control mice. We also observed differences in the percentage of other B-cell subsets between these mice, including an increase in plasma cells (B220-CD138+) and memory B cells (B220+CD19+CD27+), concomitant with an increase trend towards the elevated marginal zone B cells (B220+CD23loCD21+) and transitional B cells (B220+CD23-CD21-). In contrast, there was a decrease in the follicular zone B cells (B220+CD23-CD21-) and plasmablast (B220+CD138+). To analyze the levels of SRBC-specific Abs from immunized mice, serum was collected from blood at day 14. A flow cytometry-based assay was performed to detect the fluorescent-labeled SRBC-specfic Abs for immunoglobulin. Results showed that the abundance of non-class-switched anti-SRBC IgM level was significantly increased in female Kdm6a-null mice serum compared with control mice. In contrast, these mice had significantly decreased anti-SRBC IgA, IgG, IgG1, IgG3 and IgE levels indicating a isotype class switch defect. The aberrant GC phenotype induced by SRBC indeicated that kdm6a loss results in expansion of GC B cells, which subsequently enhances the plasma cell generation. This finding prompted us to investigate if the Kdm6a impairs the immunoglobulin affinity maturation. Therefore, we analyzed the ability of female Kdm6a-null mice and wildtype littermates to generate specific Abs against another T cell-dependent antigen NP-Chicken Gamma Globulin (NP-CGG). Mice were immunized with NP-CGG (29) and serum were collected weekly up to 8 weeks total. ELISA analysis of serum revealed that NP-specfic total Ig level were similar for both groups of mice over time. However, consistent with the SRBC immunization results, we did observed a sinificant reduction in the titers of NP-specific IgA and IgG1 Abs in female Kdm6a-null mice compared with control mice at each time point, while these mice had a sinificant increase in NP-specific IgM Abs, which indicating the loss of Kdm6a disrupts the balance between non-class-switched and class-switched NP-specific Abs isotypes (Figure 1A-D). Likewise, we also observed an increase in the percentage of GC B cells and plasma cells 8 weeks after NP-CGG immunization by flow cytometry. Again, our findings indicate the loss of Kdm6a causes germinal center hyperplasia, enhances plasma cell differentiation, and likely impairs class switch recombination (CSR). Taken together, our data shows that Kdm6a plays an important, but complex, role in B-cell transiting in the GC reaction and that loss of Kdm6a causes germinal center hyperplasia and impedes the B-cell immune response in a specific manner that may contribute to infection and B-cell malignancies. Disclosures Wartman: Novartis: Consultancy; Incyte: Consultancy.

The Role of B Cell-Activating Factor (BAFF) in the Biology of Multiple Myeloma (MM).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3380-3380 ◽  
Author(s):  
Noopur Raje ◽  
Shaji Kumar ◽  
Teru Hideshima ◽  
Kenji Ishitsuka ◽  
Hiroshi Yasui ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Cell Development ◽  
Newly Diagnosed ◽  
Growth And Survival ◽  
Affymetrix U133a ◽  
Apoptotic Proteins

Abstract BAFF is a member of the tumor necrosis factor (TNF) family and is critical for the maintenance and homeostasis of normal B-cell development. Importantly, BAFF promotes the generation of rapidly dividing immunoglobulin secreting plasmablasts from activated memory B cells by enhancing their survival. Given that MM is a cancer of plasma cells and that the signaling cascades implicated in receptor ligand interactions of BAFF are crucial in MM cell biology, we hypothesized that this cytokine may play a critical role in MM cell development, survival, and proliferation. We performed gene expression profiling (GEP) on CD 138+ plasma cells isolated from 90 MM patients (45 newly diagnosed and 45 relapsed) and 11 healthy controls using the Affymetrix U133A arrays. Our data demonstrates increased expression of transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA), 2 receptors used by BAFF to exert its effects. Our data also shows an increased expression of a proliferation-inducing ligand (APRIL), another member of the TNF family with homology to BAFF. Expression levels of BAFF and BAFF-R could not be determined because of lack of these probe sets on the Affymetrix U133A arrays. GEP analysis shows increased BCMA expression (p&lt;0.0001, student T test) on newly diagnosed and relapsed MM versus normal plasma cells. Flow cytometry on MM cell lines demonstrated a differential expression of the three receptors of BAFF, with BCMA present on most cell lines but BAFF-R expressed at low levels only on LR5 cells and DOX40 MM cells. In contrast, flow cytometry performed on MM patient cells demonstrated the presence of all 3 receptors on CD 138+ cells. ELISA assays performed on 30 MM sera demonstrated a mean BAFF level of 618 pg/ml (range: 128–2126pg/ml) versus 235pg/ml (range: 158–326pg/ml) in 7 normal donor sera. Fifty six% (17/30) of MM patients had BAFF levels in excess of the highest value noted in normals. To understand the role BAFF might play in the biology of MM, we studied the effects of recombinant BAFF (rh-BAFF) on MM cells directly and in the context of its bone marrow microenvironment. (abstract # 554746) rh-BAFF conferred a survival advantage to MM cells and protected them against dexamethasone-induced cytotoxicity. Importantly, anti-apoptotic proteins Bcl2 and Mcl-1 were upregulated, as were growth and survival signals belonging to the JAK/STAT and MAPKinase pathways. Conversely, neutralizing antibody to BAFF blocked, at least in part, blocked the upregulation of anti-apoptotic proteins with associated growth and survival, confirming that these effects were due to BAFF. Importantly, all of these signals were downregulated even in the presence of bone marrow stromal cells (BMSCs). These data therefore show a role for BAFF mediating MM cell survival and provide the framework for inhibiting BAFF, either alone or in combination with dexamethasone, to improve patient outcome in MM.

CS1: A Potential New Therapeutic Target for the Treatment of Multiple Myeloma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3457-3457 ◽  
Author(s):  
Eric D. Hsi ◽  
Roxanne Steinle ◽  
Balaji Balasa ◽  
Aparna Draksharapu ◽  
Benny Shum ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cell ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
Target Cells ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Effector Cells

Abstract Background: To identify genes upregulated in human memory B and plasma cells, naïve B cell cDNA was subtracted from plasma cell and memory B cell cDNA. One gene that was highly expressed in plasma cells encodes CS1 (CD2 subset 1, CRACC, SLAMF7), a cell surface glycoprotein of the CD2 family. CS1 was originally identified as a natural killer (NK) cell marker. Monoclonal antibodies (mAbs) specific for CS1 were used to validate CS1 as a potential target for the treatment of multiple myeloma (MM). Methods: Anti-CS1 mAbs were generated by immunizing mice with a protein comprising of the extracellular domain of CS1. Two clones, MuLuc63 and MuLuc90, were selected to characterize CS1 protein expression in normal and diseased tissues and blood. Fresh frozen tissue analysis was performed by immunohistochemistry (IHC). Blood and bone marrow analysis was performed using flow cytometry with directly conjugated antibodies. HuLuc63, a novel humanized anti-CS1 mAb (derived from MuLuc63) was used for functional characterization in non-isotopic LDH-based antibody-dependent cellular cytotoxicity (ADCC) assays. Results: IHC analysis showed that anti-CS1 staining occurred only on mononuclear cells within tissues. The majority of the mononuclear cells were identified as tissue plasma cells by co-staining with anti-CD138 antibodies. No anti-CS1 staining was detected on the epithelia, smooth muscle cells or vessels of any normal tissues tested. Strong anti-CS1 staining was also observed on myeloma cells in 9 of 9 plasmacytomas tested. Flow cytometry analysis of whole blood from both normal healthy donors and MM patients showed specific anti-CS1 staining in a subset of leukocytes, consisting primarily of CD3−CD(16+56)+ NK cells, CD3+CD(16+56)+ NKT cells, and CD3+CD8+ T cells. Flow cytometry of MM bone marrow showed a similar leukocyte subset staining pattern, except that strong staining was also observed on the majority of CD138+CD45−/dim to + myeloma cells. No anti-CS1 binding was detected to hematopoietic CD34+CD45+ stem cells. To test if antibodies towards CS1 may have anti-tumor cell activity in vitro, ADCC studies using effector cells (peripheral blood mononuclear cells) from 23 MM patients and L363 MM target cells were performed. The results showed that HuLuc63, a humanized form of MuLuc63, induced significant ADCC in a dose dependent manner. Conclusions: Our study identifies CS1 as an antigen that is uniformly expressed on normal and neoplastic plasma cells at high levels. The novel humanized anti-CS1 mAb, HuLuc63, exhibits significant ADCC using MM patient effector cells. These results demonstrate that HuLuc63 could be a potential new treatment for multiple myeloma. HuLuc63 will be entering a phase I clinical study for multiple myeloma.

Induction Of Proliferation and Survival Of Multiple Myeloma Cells By CD137 Ligand Reverse Signaling

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2537-2537
Author(s):  
Chengcheng Fu ◽  
Hui Liu ◽  
Juan Wang ◽  
Ling Ma ◽  
Songguang Ju ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Cell Surface ◽  
B Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Cell Counting ◽  
Myeloma Cells ◽  
Reverse Signaling

Abstract CD137 and its ligand are members of the Tumor Necrosis Factor (TNF) receptor and TNF superfamilies, respectively, regulate cell activation and proliferation of immune system. CD137L, in addition to its ability to costimulate T cells by triggering CD137 receptor, also signals back into antigen presenting cells inducing proliferation, prolonging survival and enhancing secretion of proinflammatory cytokines. The expression of CD137L and its function on multiple myeloma cells is unknown. We identified the constitutive expression of CD137L by flow cytometry on U266, RPMI 8226, LP1, MY5 and KMS-11 of Multiple myeloma (MM) cell lines as high as 96%, 97.5%, 89%, 93% and 94%.But, CD137 expressed on the cell surface was low as 4%, 5%, 1%, 2%, 5% respectively. Now that, CD137L was expressed very strongly on MM cell lines, next, we investigated CD137L expression of MM cells from 85 BM samples of patients seen in the hematological Dept of the First Affiliated Hosp. of Soochow University between January 2012 and June 2013 and diagnosed of active multiple MM, including the patients of newly-diagnosed (n=35), relapsed (n=5) and after 2- 4 prior therapies (n=45). The BM samples were examined using antibodies against CD45RO PE-Cy7, CD138 APC-H7, CD38 FITC and CD137L PE, according to standard protocols for surface staining. Indeed, CD137L protein was expressed by a select group of CD45-CD38++CD138+cells as higher than 95%, the same, CD38 and CD138 are expressed more than 90% of the cells of CD45-CD137L+.There were 22 samples from 11 cases collected before and after treatment and this was further evidence that CD137L molecule was consistently expressed on the MM cell surface. However, CD137L expression was not or hardly detectable on normal plasma cells confirmed by CD45+CD38++CD138+ CD56- CD19+, indicating that CD137L was ectopically expressed by MM cells and probably a specific marker of MM cells. The ectopic CD137L expression was not a mere epiphenomenon but was selected for, what function of it? We hypothesized that it would also act as a growth stimulus for B cell cancers. Then we selected U266-a MM cell line to explore the biological effect of CD137L reverse signaling and its underlying mechanism. As a result, in vitro study, U266 cells(2X105/ml))were cultured plate pre-coated with mAb 1F1 or irrelevant mouse IgG at l ug/ml in PBS and at 400 ul per well of 24-well plate or 80 ul per well of 96-well plate and washed twice after overnight incubation at 4°C. The proliferation and survival of U266 was enhanced by stimulating- CD137L mAb (1F1) than those induced by control mouse IgG by cell counting (4.2 X105/ml VS 3.3 X105/ml), WST-8(1.15 VS 0.81) and CFSE assay (930 VS 991) at incubation for 48h. In addition, the cell cycle analysis showed that CD137L induces proliferation and increases the number of cells in the S phase from 36.1% to 42.5% after 72h incubation. The percentage of apoptosis cells (Annexin V+ and PI+) was 19.6% VS 21.2% with no statistical significance. In order to explore the mechanism of the function of CD137L on MM cells, we surveyed the cytokine profiles during the incubation of U266 cells cultured for 2 days with different stimuli with mAb 1F1 compared with the control group. Intracellular cytokine staining showed that treatment of cells with 1F1 increased the production of IL-6 from 3.8% to 63.9% by Flow cytometry. When neutralizing anti-IL-6 mAb (5 ug/ml) was added to the culture medium, the cells(2X105/ml))were cultured for 48 h in pure medium or plus 10 ng/ml Fc or CD137–Fc protein and the cell proliferation measured by WST-8 was 0.79 VS 0.80 VS 0.72.1F1-induced cell proliferation was effectively inhibited. IL-6 can promote cell proliferation and survival of MM. An increase of these cytokines might explain why CD137L expression could stimulate the proliferation of U266. Finally, the U266 cells were treated with bortezomib and the growth of cells was analyzed by WST-8 assay. It demonstrated that bortezomib could inhibit the function of 1F1 and the inhibition ratio of bortezomib was 22%, 51% and 58% at 24h, 48h and 72h. MM is a B-cell malignancy characterized by the clonal expansion and accumulation of malignant plasma cells in the bone marrow. In our study, CD137L is not only a novel ectopic constitutive marker of MM, but also a promoting proliferation factor. This suggests the possibility that its expression on MM cells may be directly target for immunomodulatory therapy for MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Inter and Intra-Clonal Heterogeneity in Multiple Myeloma and Waldenstrom Macroglobulinemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2070-2070
Author(s):  
Jana Jakubikova ◽  
Danka Cholujova ◽  
Teru Hideshima ◽  
Jacob P. Laubach ◽  
Nikhil C. Munshi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
High Dimensional Data ◽  
Signaling Molecules ◽  
High Dimensional ◽  
Disease Pathogenesis ◽  
Clonal Heterogeneity ◽  
Cytogenetic Abnormalities

Abstract Introduction: Intra-clonal heterogeneity in malignant plasma (PC) cells and B-cells has recently been reported in both multiple myeloma (MM) and Waldenstrom macroglobulinemia (WM). Further phenotypic and molecular characterization of inter- and intra-clonal genetic complexity will enhance our understanding of disease pathogenesis and identify novel therapeutic strategies. Methods: In this study, we compared normal and malignant PC maturation-associated B-cell subsets using bone marrow samples from individuals with monoclonal gammopathy of undetermined significance (MGUS), smoldering MM (SMM), newly diagnosed MM, and relapsed/refractory MM versus age-matched healthy donors (HD). We also similarly analyzed WM. In addition to corrupted B-cell lineage, we examined phenotypic and molecular features of intra-clonal architecture (complexity) of malignant PC in MM and clonal B-cells in WM on a single cell level using time-of-flight mass cytometry (CyTOF) technology. CyTOF technology is based on rare stable earth elemental isotopes-bound to antibodies to target epitopes on and within cells: up to 40 different markers on a single cell can simultaneously assess including phenotype, transcription factors, regulatory signaling molecules and enzymes, as well as activation of signaling molecules. The resulting high-dimensional data were analyzed by SPADE, viSNE and Wanderlust software. Results: Our high-dimensional data of clustered analyses showed significantly decreased CD19+CD27- patient cells in MM with cytogenetic abnormalities (cytog+) including del(13q), t(4;14), t(14;16), t(3;14), +1q or t(11;14) versus patient cells in MM without any cytogenetic abnormalities (cytog-; P=0.013). In contrast, there was a significant increase of transitional B cells (CD19+CD27-IgM+CD10-IgDlow) in patients with MM cytog+ vs. MM cytog- (P=0.028). A significant increase of mature (naïve) B cells (CD19+CD27-IgM+CD10-IgD+) was also detected in MM cytog+ versus MM cytog- patients (P=0.013), but not in WM cytog- vs. WM cytog+ (46XY, -Y, +18q, +6p, 14q). Clonal PC (CD19-CD38++CD138+CD45-/dim; either cyk or cyl +) were significantly upregulated in MM cytog+ compared to MM cytog- (P=0.021) by CyTOF analyses. To investigate phenotypic profiles and molecular signature of intra-clonal heterogeneity of PC in MM, high-dimensional analyses by SPADE and viSNE revealed that clonal PC clustered separately from B cells by, virtue of high CD319 and CD47 expression; variable expression of CD52, CD56, CD81, CD44, CD200; and low expression of CD28, CD117, CD338, CD325, and CD243. For example, adhesion CD56 and anti-adhesion CD52 molecules were significantly increased in MM cytog+ compared to MM cytog-. Clonal PC highly expressed IFR4 and Notch1; variously expressed FGFR3, sXBP-1, KLF4 and c-Myc; and only minimally expressed Bcl-6, WHSC-1 (MMSET) and RARa2. sXBP-1 was significantly upregulated in all MM stages compared to HD. Furthermore, expression of stem cell markers including Sox-2, Oct3/4 and Nestin was detected only at low level in clonal PC, except for higher expression of Nanog. In WM, clonal B cells expressed Bcl-6 (4-36%) and MYD88 (2-27%) by CyTOF analyses. Finally, cluster analyses by SPADE and viSNE allows for detection of phenotypic and molecular changes not only in clonal populations but also at distinct B-lineage maturation stages, such as expression of Pax-5 and Bcl-2 on early B cell progenitors. This data represents a cohort of MM (N=35) and WM (N=15) patients; a significantly larger data set of MM (N=100) and WM (N=50) will be presented. Conclusion: This study characterizes the molecular and phenotypic profile associated with inter- and intra-clonal heterogeneity in MM and WM. It not only enhances our understanding of disease pathogenesis, but may allow for individualized targeted therapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals Multiscale Modeling of Germinal Center Recapitulates the Temporal Transition From Memory B Cells to Plasma Cells Differentiation as Regulated by Antigen Affinity-Based Tfh Cell Help

2021 ◽  
Vol 11 ◽  
Author(s):  
Elena Merino Tejero ◽  
Danial Lashgari ◽  
Rodrigo García-Valiente ◽  
Xuefeng Gao ◽  
Fabien Crauste ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Fate ◽  
Plasma Cell ◽  
Germinal Center ◽  
Plasma Cells ◽  
Asymmetric Division ◽  
Memory B Cells ◽  
Memory B Cell ◽  
Germinal Center Reaction

Germinal centers play a key role in the adaptive immune system since they are able to produce memory B cells and plasma cells that produce high affinity antibodies for an effective immune protection. The mechanisms underlying cell-fate decisions are not well understood but asymmetric division of antigen, B-cell receptor affinity, interactions between B-cells and T follicular helper cells (triggering CD40 signaling), and regulatory interactions of transcription factors have all been proposed to play a role. In addition, a temporal switch from memory B-cell to plasma cell differentiation during the germinal center reaction has been shown. To investigate if antigen affinity-based Tfh cell help recapitulates the temporal switch we implemented a multiscale model that integrates cellular interactions with a core gene regulatory network comprising BCL6, IRF4, and BLIMP1. Using this model we show that affinity-based CD40 signaling in combination with asymmetric division of B-cells result in switch from memory B-cell to plasma cell generation during the course of the germinal center reaction. We also show that cell fate division is unlikely to be (solely) based on asymmetric division of Ag but that BLIMP1 is a more important factor. Altogether, our model enables to test the influence of molecular modulations of the CD40 signaling pathway on the production of germinal center output cells.

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