Immunophenotypic Analysis of Myeloma Precursors: Antigens for Therapeutic Targeting

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. SCI-5-SCI-5
Author(s):  
Martin Perez-Andres ◽  
Bruno Paiva ◽  
Leandro Thiago ◽  
Nico A Bos ◽  
Dirk Hose ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Tumor Cells ◽  
Research Funding ◽  
Plasma Cells ◽  
Somatic Hypermutation ◽  
Ex Vivo ◽  
Multidrug Resistant

Abstract Abstract SCI-5 Multiple myeloma (MM) is a malignant disorder characterized by the (mono)clonal expansion of terminally-differentiated plasma cells (M-PC) in the bone marrow (BM) that produce and secrete a monoclonal immunoglobulin (Ig), detectable in the serum and/or urine. Infiltration by the expanded M-PC is easily identified in the involved tissues, through conventional morphology and immunophenotyping. However, the possibility exists that rather than M-PC, a less differentiated B-cell that represents a minor fraction of all tumor cells and retains self-renewal properties, is responsible for the outgrowth of the more differentiated M-PC compartment. In B-cell disorders, the idiotypic Ig produced by tumor cells and defined by its CDR3 sequence, acts as a genetic fingerprint for clonally-related B-cells. In MM, the specificity of the idiotype is further enhanced through modification of V genes by somatic hypermutation (SHM) at the germinal center (GC), since M-PC display extensively mutated VH genes which are stable throughout the disease. This suggests that in MM, malignant transformation could occur in a post-GC B-cell. Based on the CDR3 sequences of the Ig genes of M-PC, preliminary studies have identified tumor-associated circulating peripheral blood (PB) CD19+ B-cells, whose malignant/clonogenic potential remained to be demonstrated. More recently, the Matsui group has reported that while in MM cell lines both CD138− and CD138+ cells retain clonogenic capacity after in vitro serial plating clonogenic assays, in primary MM samples, such (ex vivo and in vivo) ability would be restricted to the CD34−/CD138− compartment. Of note, the clonogenic growth of these later cells significantly decreased after depletion by CD19, CD22, CD20 and CD45 antibodies, and it was associated with an in vitro multidrug-resistant functional phenotype (restricted to CD19+/CD27+, CD138− cells but not CD138+ cells) and the Hedgehog (Hh) stem cell-associated signaling pathway. These results point out the potential existence of a CD19+, CD20+, CD138− pre-PC compartment responsible for the expansion of M-PC in MM. In turn, evidence also exists in both the SCID-Hu model and in Rituximab treated MM patients, which suggests that plasmablasts/PC -but not pre-plasmablasts-, could act as MM “stem” cells, the precise characteristics of such cells remaining to be precisely defined. Alternatively, it could also be possible that both cell cellular components coexist and are relevant to MM progression through appropriate interaction with the BM stroma. Independently of all the above, trafficking of such cells through PB to BM niches could also play a key role in the spread of the tumor and its malignant behavior. In this regard, we recently confirmed that a relatively high percentage of MM patients (and a substantial fraction of all MGUS cases) show circulating PB PC with i) tumor-related clonal VH gene rearrangements and ii) an aberrant immunophenotypic profile which largely overlaps with that of BM M-PC from the same subjects; the only minor differences consisted of a significantly lower expression of CD38 and CD138, smaller size and internal complexity, features that indicate a slightly more immature plasmablastic/PC profile. Noteworthy, this was the only PB B-cell compartment for which clonally-related B-cells were detected with a sensitivity of <1 cell/50μ L in all cases investigated. In summary, accumulating evidence suggest the existence of a clonal hierarchy in MM but uncertainties remain as regards the precise immunophenotypic features of those cells responsible for tumor growth in primary patient samples, that could be of help in developing new targeted therapies. Disclosures: Sonneveld: Millennium: Consultancy; Celgene: Consultancy. Orfao:Becton/Dickinson Biosciences Europe: Patents & Royalties, Research Funding; Cytognos SL: Patents & Royalties; Alexion: Membership on an entity's Board of Directors or advisory committees; Vivia Biotech: Research Funding; Mundipharma: Research Funding.

scholarly journals SIRPαFc, a CD47-Blocking Cancer Immunotherapeutic, Sensitizes Malignant B Cells to Macrophage-Mediated Destruction

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2191-2191
Author(s):  
Natasja Nielsen Viller ◽  
Saman Maleki Vareki ◽  
Karen Dodge ◽  
Hui Chen ◽  
Vivian Lee ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Research Funding ◽  
B Lymphoma ◽  
Established Cell Lines ◽  
Established Cell

Abstract Macrophages commonly infiltrate tumor microenvironments and can phagocytose and destroy malignant cells. Cancer cells, however, can inhibit the tumoricidal activity of macrophages by expressing CD47 on their surface. CD47 delivers an anti-phagocytic ("do not eat") signal by binding signal-regulatory protein α (SIRPα) on the surface of macrophages. There is strong evidence that many liquid and solid tumors exploit the CD47-SIRPα pathway to escape macrophage-mediated destruction. Blockade of this inhibitory axis using a soluble SIRPα-Fc fusion protein (SIRPαFc) has emerged as a promising strategy to neutralize the suppressive effects of CD47 and promote the eradication of tumor cells. Here we have examined the effect of SIRPαFc on malignant human B cells in vitro and in vivo. We first assessed the binding of SIRPαFc to a panel of established cell lines and primary cells from patients with diffuse large B cell lymphoma, Burkitt's lymphoma, multiple myeloma and acute lymphoblastic leukemia. SIRPαFc exhibited strong, dose-dependent binding to all tumor cells, with an average effective half-maximal concentration of approximately 150 nM. Next, the ability of SIRPαFc to promote macrophage-mediated phagocytosis of human tumor cells was examined using confocal microscopy. In cultures left untreated or treated with a control Fc fragment, macrophages exhibited a low level of phagocytosis, consistent with CD47-mediated suppression. Blockade of CD47 on the target cells using SIRPαFc dramatically increased macrophage phagocytosis of tumor cells. The majority of established cell lines and all primary human tumors were sensitized to macrophage-mediated destruction, including both peripheral blood- and bone marrow-derived primary tumor samples. Finally, we assessed the in vivo activity of SIRPαFc in CD20hi (Raji) and CD20low (Namalwa) B lymphoma xenograft models. SIRPαFc treatment significantly reduced Raji growth and increased host mouse survival (time to euthanasia), and completely ablated the growth of Namalwa tumors, the latter being insensitive to rituximab therapy. In conclusion, SIRPαFc demonstrated in vitro activity against a broad range of human B cell tumors and was highly effective at controlling the growth of aggressive B lymphoma xenografts in mice, including a CD20low tumor that was non-responsive to rituximab. These data support the evaluation of SIRPαFc in patients with B cell malignancies. Disclosures Nielsen Viller: Trillium Therapeutics Inc.: Employment. Vareki:Trillium Therapeutics Inc.: Research Funding. Dodge:Trillium Therapeutics Inc.: Employment. Chen:Trillium Therapeutics Inc.: Employment. Lee:Trillium Therapeutics Inc.: Employment. Chai:Trillium Therapeutics Inc.: Employment. Pang:Trillium Therapeutics Inc.: Employment. Wong:Trillium Therapeutics Inc.: Employment. Trudel:Novartis: Honoraria; Oncoethix: Research Funding; BMS: Honoraria; Trillium Therapeutics Inc.: Research Funding; Celgene: Equity Ownership, Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Figueredo:Trillium Therapeutics Inc.: Research Funding. Pampillo:Trillium Therapeutics Inc.: Research Funding. Koropatnick:Trillium Therapeutics Inc.: Research Funding. Petrova:Trillium Therapeutics Inc.: Employment. Uger:Trillium Therapeutics Inc.: Employment.

scholarly journals Differential Regulation of Mouse B Cell Development by Transforming Growth Factor β1

2002 ◽  
Vol 9 (2) ◽  
pp. 86-95 ◽  
Author(s):  
Denise A. Kaminski ◽  
John J. Letterio ◽  
Peter D. Burrows
Keyword(s):  
B Cells ◽  
Growth Factor ◽  
B Cell ◽  
Transforming Growth Factor ◽  
Plasma Cells ◽  
Population Analysis ◽  
Cell Development ◽  
B Cell Development

Transforming growth factor β (TGFβ) can inhibit thein vitroproliferation, survival and differentiation of B cell progenitors, mature B lymphocytes and plasma cells. Here we demonstrate unexpected, age-dependent reductions in the bone marrow (BM) B cell progenitors and immature B cells in TGFβ1-/-mice. To evaluate TGFβ responsiveness during normal B lineage development, cells were cultured in interleukin 7 (IL7)±TGFβ. Picomolar doses of TGFβ1 reduced pro-B cell recoveries at every timepoint. By contrast, the pre-B cells were initially reduced in number, but subsequently increased compared to IL7 alone, resulting in a 4-fold increase in the growth rate for the pre-B cell population. Analysis of purified BM sub-populations indicated that pro-B cells and the earliest BP1-pre-B cells were sensitive to the inhibitory effects of TGFβ1. However, the large BP1+pre-B cells, although initially reduced, were increased in number at days 5 and 7 of culture. These results indicate that TGFβ1 is important for normal B cell developmentin vivo, and that B cell progenitors are differentially affected by the cytokine according to their stage of differentiation.

Pathologic Co-Expression and Physical Interaction of c-MYC and BCL6 in B-Cell Lymphomas.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2-2 ◽  
Author(s):  
Masumichi Saito ◽  
Ryan T. Phan ◽  
Herbert C. Morse ◽  
Laura Pasqualucci ◽  
Riccardo Dalla-Favera
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lymphoma ◽  
Somatic Hypermutation ◽  
B Cell Lymphoma ◽  
Transgenic Animals ◽  
Primary Lymphoma ◽  
Physical Interaction

Abstract Deregulated expression of the proto-oncogenes BCL6 and c-MYC caused by chromosomal translocation or somatic hypermutation is common in non-Hodgkin B cell lymphoma derived from germinal center (GC) B cells, including diffuse large cell lymphoma (DLBCL) and Burkitt lymphoma (BL). Normal GC B cells express BCL6, whereas, surprisingly, they do not express c-MYC, suggesting that the expression of this oncogene in BL and DLBCL (20% of cases) is ectopic (Klein, U. et al. Proc Natl Acad Sci U S A100, 2639–2644, 2003). Here we report that c-MYC is absent in proliferating GC B cells because it is transcriptionally suppressed by BCL6, as demonstrated by the presence of specific BCL6 binding sites in the c-MYC promoter region and by chromatin immunoprecipitation experiments showing that BCL6 is bound to these sites in vivo. Thus, c-MYC escapes BCL6-mediated suppression in lymphoma leading to the co-expression of the two transcription factors, an event never observed in immunohistochemical and gene expression profile analysis of normal GC B cells. Surprisingly, co-immunoprecipitation experiments and in vitro binding experiments indicate that, when co-expressed, BCL6 and c-MYC are physically bound in a novel complex detectable in DLBCL and BL cell lines as well as in primary lymphoma cases. The formation of the BCL6/c-MYC complex has several significant functional consequences on the function of both c-MYC and BCL6: 1) a two fold, BCL6-binding dependent increase in c-MYC half-life, an event that has been shown to contribute to its oncogenic activation; 2) a synergistic increase in the ability of both BCL6 and c-MYC to suppress MIZ1-activated transcription of the p21CIP cell cycle arrest gene; 3) MYC-dependent inhibition of BCL6 acetylation by p300, an event that physiologically inactivates BCL6 via c-MYC-mediated recruitment of HDAC. Notably, the pathologic co-expression of c-MYC and BCL6 was shown to have pathologic consequences in vivo, since double transgenic BCL6/c-MYC mice display accelerated lymphoma development and the appearance of a novel GC-derived tumor phenotype not recognizable in single transgenic animals and containing the pathologic c-MYC/BCL6 complex. Thus, the pathologic co-expression and illegitimate physical interaction of BCL6 and c-MYC leads to an increase in the constitutive activity of both oncogenes. These results identify a novel mechanism of oncogenic function for BCL6 and c-MYC and a novel tumor-specific protein complex of potential therapeutic interest.

scholarly journals IgM and IgD B cell receptors differentially respond to endogenous antigens and control B cell fate

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Mark Noviski ◽  
James L Mueller ◽  
Anne Satterthwaite ◽  
Lee Ann Garrett-Sinha ◽  
Frank Brombacher ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Fate ◽  
Plasma Cells ◽  
Autoreactive B Cells ◽  
Binding Domains ◽  
Bcr Signaling ◽  
And Control

Naive B cells co-express two BCR isotypes, IgM and IgD, with identical antigen-binding domains but distinct constant regions. IgM but not IgD is downregulated on autoreactive B cells. Because these isotypes are presumed to be redundant, it is unknown how this could impose tolerance. We introduced the Nur77-eGFP reporter of BCR signaling into mice that express each BCR isotype alone. Despite signaling strongly in vitro, IgD is less sensitive than IgM to endogenous antigen in vivo and developmental fate decisions are skewed accordingly. IgD-only Lyn−/− B cells cannot generate autoantibodies and short-lived plasma cells (SLPCs) in vivo, a fate thought to be driven by intense BCR signaling induced by endogenous antigens. Similarly, IgD-only B cells generate normal germinal center, but impaired IgG1+ SLPC responses to T-dependent immunization. We propose a role for IgD in maintaining the quiescence of autoreactive B cells and restricting their differentiation into autoantibody secreting cells.

scholarly journals Epstein-Barr Virus LMP2A Enhances B-Cell Responses In Vivo and In Vitro

2006 ◽  
Vol 80 (14) ◽  
pp. 6764-6770 ◽  
Author(s):  
Michelle Swanson-Mungerson ◽  
Rebecca Bultema ◽  
Richard Longnecker
Keyword(s):  
B Cells ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Plasma Cells ◽  
Barr Virus ◽  
Epstein Barr ◽  
Hen Egg ◽  
Hen Egg Lysozyme

ABSTRACT Epstein-Barr virus (EBV) establishes latent infections in a significant percentage of the population. Latent membrane protein 2A (LMP2A) is an EBV protein expressed during latency that inhibits B-cell receptor signaling in lymphoblastoid cell lines. In the present study, we have utilized a transgenic mouse system in which LMP2A is expressed in B cells that are specific for hen egg lysozyme (E/HEL-Tg). To determine if LMP2A allows B cells to respond to antigen, E/HEL-Tg mice were immunized with hen egg lysozyme. E/HEL-Tg mice produced antibody in response to antigen, indicating that LMP2A allows B cells to respond to antigen. In addition, E/HEL-Tg mice produced more antibody and an increased percentage of plasma cells after immunization compared to HEL-Tg littermates, suggesting that LMP2A increased the antibody response in vivo. Finally, in vitro studies determined that LMP2A acts directly on the B cell to increase antibody production by augmenting the expansion and survival of the activated B cells, as well as increasing the percentage of plasma cells generated. Taken together, these data suggest that LMP2A enhances, not diminishes, B-cell-specific antibody responses in vivo and in vitro in the E/HEL-Tg system.

scholarly journals Genome-Wide Histone Acetylation Profiling in Chronic Lymphocytic Leukemia Reveals a Distinctive Signature in Stereotyped Subset #8

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1241-1241
Author(s):  
Maria Tsagiopoulou ◽  
Vicente Chapaprieta ◽  
Nuria Russiñol ◽  
Fotis Psomopoulos ◽  
Nikos Papakonstantinou ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Research Funding ◽  
Plasma Cells ◽  
Somatic Hypermutation ◽  
Memory B Cells ◽  
Site Analysis ◽  
Total Variability ◽  
Cell Subpopulations ◽  
Chromatin Acetylation

In CLL, subsets of patients carrying stereotyped B cell receptors (BcR) share similar biological and clinical features independently of IGHV gene somatic hypermutation status. Although the chromatin landscape of CLL as a whole has been recently characterized, it remains largely unexplored in stereotyped cases. Here, we analyzed the active chromatin regulatory landscape of 3 major CLL stereotyped subsets associated with clinical aggressiveness. We performed chromatin-immunoprecipitation followed by sequencing (ChIP-Seq) with an antibody for the H3K27ac histone mark in sorted CLL cells from 19 cases, including clinically aggressive subsets #1 (clan I genes/IGKV(D)1-39, IG-unmutated CLL (U-CLL)(n=3)], #2 [IGHV3-21/IGLV3-21, IG-mutated CLL (M-CLL)(n=3)] and #8 [IGHV4-39/IGKV1(D)-39, U-CLL(n=3)] which we compared to non-stereotyped CLL cases [5 M-CLL|5 U-CLL]. In addition, a series of 15 normal B cell samples from different stages of B-cell differentiation were analyzed [naive B cells from peripheral blood (n=3), tonsillar naive B cells (n=3), germinal centre (GC) B cells (n=3), memory B cells (n=3), tonsillar plasma cells (n=3)]. Initial unsupervised principal component analysis (PCA) disclosed a distinct chromatin acetylation pattern in CLL, regardless of stereotypy status, versus normal B cells. CLL as a whole was found to be closer to naive and memory B cells rather than GC B cells and plasma cells. Detailed analysis of individual principal components (PC) revealed that PC4, which accounts for 5% of the total variability, segregated subset #8 cases and GC B cells from other CLLs and normal B cell subpopulations. Although PC4 accounts for only a small part of the total variability (5%), this suggests that subset #8 cases may share some chromatin features with proliferating GC B cells, in line with the fact that subset #8 BcR are IgG-switched. We also investigated whether stereotyped CLLs have different chromatin acetylation features compared to non-stereotyped CLLs matched by IGHV somatic hypermutation status and identified 878 Differential Regions (DR) in subset #8 vs. U-CLL, 84 DR in subset #1 vs. U-CLL and 66 DR in #2 compared vs. M-CLL. As subset #8 cases seemed to have the most distinct profile, we further characterized the detected regions. The 435 and 443 regions gaining and losing activation, respectively, mostly targeted promoters (29.5%) and regulatory elements located in introns (31%) and distal intergenic regions (21.8%). Hierarchical clustering based on the 878 DRs enabled the clear discrimination of subset #8 cases from U-CLL and normal B cells; however, it is worth noting that for several of these 878 DRs the acetylation patterns were shared between subset #8 and normal B cell subpopulations rather than subset #8 and U-CLL. Of note, 11/435 regions gaining activity on subset #8 were found within the gene encoding for the EBF1 transcription factor (TF); additional regions were associated with genes significant to CLL pathogenesis, e.g. TCF4 and E2F1. Moreover, 3 DRs losing activity in subset #8 were located within the CTLA4 gene and 2 DRs within the IL21R gene, which we have recently reported as hypermethylated and not expressed in subset #8. Next, we performed TF binding site analysis by MEME/AME suit, separately for regions gaining or losing activity, and identified significant enrichment (adj-p<0.001) on TFs such as AP-1, FOX, GATA, IRF. The regions losing activity in subset #8 showed a higher number of enriched TFs versus those gaining activity (165 vs 93 TFs), particularly displaying enrichment for many HOX family members . However, a cluster of TFs with enrichment on TF binding site analysis, such as FOXO1, FOXP1, MEF2D, PRDM1, RUNX1, RXRA, STAT6, were also located within the 878 DRs discriminating subset #8 from either U-CLL or normal B cell subpopulations. Taken together, subset #8 cases have a distinct chromatin acetylation signature which includes both loss and gain of active elements, shared features with proliferating GC B cells, and specific changes in chromatin activity of several genes and TFs relevant to B cell/CLL biology. These findings further underscore the concept that BcR stereotypy defines subsets of patients with consistent biological profile, while they may also be relevant to the particular clinical behavior of subset #8, known to be associated with the highest risk of Richter's transformation amongst all CLL. Disclosures Stamatopoulos: Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

EBV Can Induce Somatic Hypermutation in Naïve B Cells In Vitro but Ig Class Switching Requires T Cell Help.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2370-2370
Author(s):  
Sridhar Chaganti ◽  
Noelia Begue Pastor ◽  
Gouri Baldwin ◽  
Claire Shannon-Lowe ◽  
Regina Feederle ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germ Line ◽  
Somatic Hypermutation ◽  
Memory B Cells ◽  
Class Switching ◽  
B Cell Subsets ◽  
T Cell Help

Abstract Following primary infection, Epstein-Barr virus (EBV) establishes life long persistence in the host IgD− CD27+ memory B cell compartment rather than the IgD+ CD27+ marginal zone (MZ)-like or the IgD+ CD27− naïve B cell compartments. One possible explanation for such exclusive persistence in memory B cells is that EBV preferentially infects memory B cells. Alternatively, the virus may infect all B cell subsets but then drive MZ and naïve B cells to acquire the Ig isotype-switched phenotype and hypermutated Ig genotype of memory cells. Here we ask whether there is any evidence for one or other hypothesis from in vitro experiments. B cells from healthy donor blood samples were FACS sorted on the basis of IgD/CD27 expression into naïve, MZ, and memory B cell subsets with purities of >99%, >97% and >98% respectively. Analysis of the IgVH sequence further confirmed purity of the FACS sorted B cell subsets. Accordingly, 102 of 105 IgVH sequences amplified from purified naïve B cells were germ-line where as the vast majority of sequences amplified from MZ and memory B cells were mutated. All three B cell subsets expressed equal amounts of CD21 (EBV receptor on B cells), bound similar amounts of virus, and transformed with equal efficiency to establish B lymphoblastoid cell lines (LCLs) in vitro. Naïve B cell transformants upregulated CD27 expression but retained the IgM+, IgD+ phenotype as determined by FACS analysis and RT-PCR; MZ-B derived LCLs likewise were IgM+, IgD+, CD27+; and memory-B derived LCLs were consistently CD27+, IgD− and expressed either IgG, IgA or in some cases IgM. Therefore, EBV infection per se did not induce class switching. However, both naïve and MZ-B derived LCLs could still be induced to switch to IgG in the presence of CD40 ligand and IL-4; signals that are normally provided by T cells in vivo. To assess if EBV infection might drive Ig hypermutation, we carried out IgVH sequence analysis on the naïve-B derived LCL clones. Interestingly, 42 of 114 clonal IgVH sequences amplified from naïve-B derived LCLs had 3 or more mutations and the patterns of mutation seen were consistent with that produced by somatic hypermutation (SHM). Furthermore, within some naïve-B cell derived LCL clones, there were both germ-line and mutated sequences all sharing the same VDJ rearrangement (CDR3 sequence), again implying sequence diversification following EBV transformation of a single naïve B cell. Some intraclonal variation of the already hypermutated IgVH sequence was also noted in memory and MZ-B derived LCLs further suggesting ongoing mutational activity. Consistent with this, activation-induced cytidine deaminase (AID) expression was upregulated in transformants as assessed by real time RT-PCR. Our in vitro data is therefore compatible with a model of EBV persistence where the virus infects all mature B cell subsets but then drives infected naïve B cells to acquire a memory genotype by inducing SHM. In addition, EBV infected naïve and MZ-B cells may undergo Ig class switching to acquire the IgD− CD27+ memory phenotype in the presence of T cell help in vivo. EBV’s ability to induce SHM may also contribute to the lymphomagenic potential of the virus in addition to its B cell transforming and growth promoting properties.

L-Stereoisomer RNA Oligonucleotide Anti-SDF-1 (Nox-A12) Disrupts the Interaction of Multiple Myeloma Cells with the Bone Marrow Milieu In Vivo, Leading to Enhanced Sensitivity to Bortezomib

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 887-887
Author(s):  
Aldo M Roccaro ◽  
Antonio Sacco ◽  
Phong Quang ◽  
AbdelKareem Azab ◽  
Patricia Maiso ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Confocal Microscopy ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Research Funding ◽  
Ex Vivo ◽  
In Vivo Confocal Microscopy ◽  
Advisory Committees

Abstract Abstract 887 Background. Stomal-cell-derived factor 1 (SDF-1) is known to be involved in bone marrow (BM) engrafment for malignant tumor cells, including CXCR4 expressing multiple myeloma (MM) cells. We hypothesized that de-adhesion of MM cells from the surrounding BM milieu through SDF-1 inhibition will enhance MM sensitivity to therapeutic agents. We therefore tested NOX-A12, a high affinity l-oligonucleotide (Spiegelmer) binder to SDF-1in MM, looking at its ability to modulate MM cell tumor growth and MM cell homing to the BM in vivo and in vitro. Methods. Bone marrow (BM) co-localization of MM tumor cells with SDF-1 expressing BM niches has been tested in vivo by using immunoimaging and in vivo confocal microscopy. MM.1S/GFP+ cells and AlexaFluor633-conjugated anti-SDF-1 monoclonal antibody were used. Detection of mobilized MM-GFP+ cells ex vivo has been performed by flow cytometry. In vivo homing and in vivo tumor growth of MM cells (MM.1S-GFP+/luc+) were assessed by using in vivo confocal microscopy and in vivo bioluminescence detection, in SCID mice treated with 1) vehicle; 2) NOX-A12; 3) bortezomib; 4) NOX-A12 followed by bortezomib. DNA synthesis and adhesion of MM cells in the context of NOX-A12 (50–200nM) treated primary MM BM stromal cells (BMSCs), in presence or absence of bortezomib (2.5–5nM), were tested by thymidine uptake and adhesion in vitro assay, respectively. Synergism was calculated by using CalcuSyn software (combination index: C.I. according to Chou-Talalay method). Results. We first showed that SDF-1 co-localizes in the same bone marrow niches of growth of MM tumor cells in vivo. NOX-A12 induced a dose-dependent de-adhesion of MM cells from the BM stromal cells in vitro. These findings were corroborated and validated in vivo: NOX-A12 induced MM cell mobilization from the BM to the peripheral blood (PB) as shown ex vivo, by reduced percentage of MM cells in the BM and increased number of MM cells within the PB of mice treated with NOX-A12 vs. control (BM: 57% vs. 45%; PB: 2.7% vs. 15%). We next showed that NOX-A12-dependent de-adhesion of MM cells from BMSCs lead to enhanced MM cell sensitivity to bortezomib, as shown in vitro, where a synergistic effect between NOX-A12 (50–100 nM) and bortezomib (2.5–5 nM) was observed (C.I.: all between 0.57 and 0.76). These findings were validated in vivo: tumor burden detected by BLI was similar between NOX-A12- and control mice whereas bortezomib-treated mice showed significant reduction in tumor progression compared to the control (P<.05); importantly significant reduction of tumor burden in those mice treated with sequential administration of NOX-A12 followed by bortezomib was observed as compared to bortezomib alone treated mice (P <.05). Similarly, NOX-A12 + bortezomib combination induced significant inhibition of MM cell homing in vivo, as shown by in vivo confocal microscopy, as compared to bortezomib used as single agent. Conclusion. Our data demonstrate that the SDF-1 inhibiting Spiegelmer NOX-A12 disrupts the interaction of MM cells with the BM milieu both in vitro and in vivo, thus resulting in enhanced sensitivity to bortezomib. Disclosures: Roccaro: Roche:. Kruschinski:Noxxon Pharma AG: Employment. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Advisory Board, Research Funding.

scholarly journals The aryl hydrocarbon receptor controls cell-fate decisions in B cells

2016 ◽  
Vol 214 (1) ◽  
pp. 197-208 ◽  
Author(s):  
Bharat Vaidyanathan ◽  
Ashutosh Chaudhry ◽  
William T. Yewdell ◽  
Davide Angeletti ◽  
Wei-Feng Yen ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Aryl Hydrocarbon Receptor ◽  
Cell Fate ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Critical Role ◽  
Cell Fate Decisions ◽  
Aryl Hydrocarbon

Generation of cellular heterogeneity is an essential feature of the adaptive immune system. This is best exemplified during humoral immune response when an expanding B cell clone assumes multiple cell fates, including class-switched B cells, antibody-secreting plasma cells, and memory B cells. Although each cell type is essential for immunity, their generation must be exquisitely controlled because a class-switched B cell cannot revert back to the parent isotype, and a terminally differentiated plasma cell cannot contribute to the memory pool. In this study, we show that an environmental sensor, the aryl hydrocarbon receptor (AhR) is highly induced upon B cell activation and serves a critical role in regulating activation-induced cell fate outcomes. We find that AhR negatively regulates class-switch recombination ex vivo by altering activation-induced cytidine deaminase expression. We further demonstrate that AhR suppresses class switching in vivo after influenza virus infection and immunization with model antigens. In addition, by regulating Blimp-1 expression via Bach2, AhR represses differentiation of B cells into plasmablasts ex vivo and antibody-secreting plasma cells in vivo. These experiments suggest that AhR serves as a molecular rheostat in B cells to brake the effector response, possibly to facilitate optimal recall responses. Thus, AhR might represent a novel molecular target for manipulation of B cell responses during vaccination.

scholarly journals CLL B Cells Develop Resistance to Ibrutinib By Reinvigorating the IL-4R - IL-4 Axis Blocked By Bruton's Tyrosine Kinase Inhibitors Including Acalabrutinib and Zanubrutinib

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 477-477
Author(s):  
Shih-Shih Chen ◽  
Constantine S. Tam ◽  
Alan G. Ramsay ◽  
Priyadarshini Ravichandran ◽  
Natalia C. Couto-Francisco ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Cell Subset ◽  
Advisory Committees

Bruton's tyrosine kinases inhibitors (BTKis) represent major advances in CLL therapy. However resistance to this form of therapy is emerging, and such patients often progress more rapidly. Hence there is an important need for therapies that address resistance. Microenvironmental input like IL-4 is critical for CLL disease progression. Compared with normal B cells, CLL cells exhibit significantly higher levels of surface membrane (sm) IL-4 receptor (IL4-R) and contain increased amounts of pSTAT6, a downstream mediator of IL-4R signaling. IL-4 stimulation of CLL B cells suppresses smCXCR4 and increases smIgM, thus promotes CLL cell retention and expansion. In this study, we aimed to examine if smIL-4R expression, IL4R signaling, and IL-4-producing cells are altered in patients sensitive or resistant to BTKis. To do so, T and B cell subset changes were studied overtime in 12 acalabrutinib-treated CLL patients, 6 zanubrutinib-treated CLL patients, 30 ibrutinib-sensitive and 5 ibrutinib-resistant CLL patients, 4 of which exhibited BTK mutations. Consistent with only ibrutinib inhibiting T-cell kinase (ITK), T-cell subset analyses revealed no changes in Th1, Th2, Th17, Th9, and Th22 cells after zanubrutinib or acalabrutinib treatment. In contrast, a Th1-biased T-cell immunity was observed in patients responsive to ibrutinib. In patients progressing on ibrutinib, significantly reduced Th2 T cells were found during the resistant as well as sensitive periods. In an in vitro T-cell function assay using T cells collected before and after the treatment with each BTKi, only ibrutinib treated patients exhibited a reduced ability of T cells to support CLL B cell survival. We next studied changes in CLL B cells, including numbers of IL-4, -10 and -13 producing B cells after BTKi treatment. IL-13 producing CLL B cells were not changed. IL-10 producing CLL B cells were reduced in both ibrutinib sensitive and resistant patients, but not in zanubrutinib or acalabrutinib treated patients. Importantly, IL-4 producing CLL B cells were significantly decreased in patients treated with all 3 BTKi. Significantly reduced smIL-4R levels, impaired IL-4R signaling, decreased smIgM and increased smCXCR4 were also seen in patients treated with each BTKi. To understand the mechanism responsible for inhibition of IL-4 production in CLL cells treated with BTKis, we stimulated CLL cells through IgM, Toll-like receptor and CD40L, finding that only anti-IgM stimulation significantly increased IL-4 production and p-STAT6 induction. We then explored the function of IL-4. IL-4 enhanced CLL B cell survival in vitro and this action was blocked by all 3 BTKis. Moreover, adhesion of CLL B cells to smIL-4R expressing stromal cells was decreased by IL-4 and IL-4R neutralizing antibodies, especially in M-CLL cases. In in vivo studies transferring autologous T cells and CLL PBMCs into alymphoid mice, we found less CLL B cells in mouse spleens post ibrutinib than zanubrutinib or acalabrutinib treatment. This might be due to the suppressed Th2 cells found only in ibrutinib, while IL-4 producing B cells were reduced in all 3 BTKi treated mice. These results support the idea that IL-4 promotes CLL B cell adhesion and growth in tissues. Finally, we investigated the IL-4/IL-4R axis in ibrutinib-resistant patients. Although IL-4 producing T cells remain reduced during the sensitive and resistant phases, CLL B cell production of IL-4 and expression of and signaling through smIL-4R returned when patients developed ibrutinib-resistance. When comparing paired ibrutinib-sensitive and -resistant CLL B cells collected from 3 patients in a xenograft model that requires T cell help, we found ibrutinib-resistant CLL B cells grew in vivo with only minimal (~15%) numbers of autologous T cells compared to B cells collected from ibrutinib-sensitive phase; this suggested a reduced requirement for T-cell help for growth of ibrutinib-resistant CLL cells. In summary, we found IL-4 is a key survival factor in the CLL microenvironment that also improves leukemia cell adhesion to stromal cells expressing smIL-4R. IL-4 production and signaling can be stimulated in CLL B cells through the B-cell receptor, and are consistently blocked by BTKis. Moreover, the recovered ability of ibrutinib-resistant CLL B cells to produce and respond to IL-4 leads to disease progression, suggesting blocking the IL-4/IL-4R axis is a potential treatment for ibrutinib-resistant CLL patients. Disclosures Chen: Pharmacyclics: Research Funding; Beigene: Research Funding; Verastem: Research Funding; ArgenX: Research Funding. Tam:Abbvie, Janssen: Research Funding; Abbvie, Janssen, Beigene, Roche, Novartis: Honoraria. Ramsay:Celgene Corporation: Research Funding; Roche Glycart AG: Research Funding. Kolitz:Boeringer-Ingelheim: Research Funding; Roche: Research Funding; Astellas: Research Funding. Zhou:BeiGene: Employment. Barrientos:Genentech: Consultancy; Gilead: Consultancy; Janssen: Consultancy; Abbvie: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding. Rai:Pharmacyctics: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees.

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