scholarly journals A novel cancer immunotherapy using tumor-infiltrating B cells in the APCmin/+ mouse model

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245608
Author(s):  
Xinying Wang ◽  
Shohei Asami ◽  
Daisuke Kitamura
Keyword(s):  
B Cells ◽  
Cancer Immunotherapy ◽  
Tumor Antigens ◽  
Plasma Cells ◽  
Somatic Hypermutation ◽  
Serum Antibody ◽  
Good Prognosis ◽  
Repeated Injection ◽  
Lymphoid Structures

Accumulating evidence has suggested a correlation of tumor infiltrating B cells (TiBcs) and a good prognosis of cancer diseases. In some cases, TiBcs appear to have experienced antigen stimulation since they have undergone class-switching and somatic hypermutation and formed tertiary lymphoid structures around tumors together with T cells. Assuming TiBcs include those that recognize some tumor antigens, we sought to investigate their possible usefulness for cell-mediated immunotherapies. To expand usually small number of TiBcs in vitro, we modified our B cell culture system: we transduced B cells with ERT2-Bach2 so that they grow unlimitedly provided with tamoxifen, IL-21 and our original feeder cells. Such cells differentiate into plasma cells and produce antibodies upon withdrawal of tamoxifen, and further by addition of a Bach2-inhibitor in vitro. As a preliminary experiment, thus expanded splenic B cells expressing a transgenic antigen receptor/antibody against hen egg lysozyme were intravenously injected into mice pre-implanted with B16 melanoma cells expressing membrane-bound HEL in the skin, which resulted in suppression of the growth of B16 tumors and prolonged survival of the recipient mice. To test the usefulness of TiBcs for the immunotherapy, we next used APCmin/+ mice as a model that spontaneously develop intestinal tumors. We cultured TiBcs separated from the tumors of APCmin/+ mice as above and confirmed that the antibodies they produce recognize the APCmin/+ tumor. Repeated injection of such TiBcs into adult APCmin/+ mice resulted in suppression of intestinal tumor growth and elongation of the survival of the recipient mice. Serum antibody from the TiBc-recipient mice selectively bound to an antigen expressed in the tumor of APCmin/+ mice. These data suggest a possibility of the novel individualized cancer immunotherapy, in which TiBcs from surgically excised tumor tissues are expanded and infused into the donor patients.

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.

The different process of class switching and somatic hypermutation; a novel analysis by CD27− naive B cells

Blood ◽  
2002 ◽  
Vol 99 (2) ◽  
pp. 567-575 ◽  
Author(s):  
Haruo Nagumo ◽  
Kazunaga Agematsu ◽  
Norimoto Kobayashi ◽  
Koji Shinozaki ◽  
Sho Hokibara ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Messenger Rna ◽  
Plasma Cells ◽  
Somatic Hypermutation ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
V Region

Abstract The relationship between class switch recombination (CSR) and somatic hypermutation has been unclear. By using human CD27− naive B cells, we investigated the somatic hypermutation and producibility of immunoglobulins (Igs) that occur after CSR. Although neither adult CD27− nor cord blood B cells, which showed the unmutated Ig V-region genes, produced IgG, IgM, or IgA in response to conventional stimuli, they produced IgG and IgM but not IgA in the presence of Staphylococcus aureus Cowan strain (SAC) + interleukin-2 (IL-2) + IL-10 + anti-CD40 mAb + CD32 transfectants (CD40/CD32T). The naive B cells also produced IgE when combined with IL-4 + CD40/CD32T. In parallel with IgG production, the expression of mature γ1 and γ 2 transcripts was induced from naive B cells by the stimuli. The CD27 expression on human naive B cells was induced remarkably by CD40 signaling or B-cell receptor engagement, but somatic hypermutation could not be induced. The proliferation and differentiation into plasma cells were induced from naive B cells, whereas most of the plasma cells displayed very low levels of mutations in Ig V-region genes. CD27− naive B cells expressed activation-induced cytidine deaminase messenger RNA by the stimuli later than CD27+memory B cells. Our results demonstrate that CSR, but not noticeable somatic hypermutation, can be induced from CD27− naive B cells upon B-cell receptor engagement and CD40 signaling in cooperation with cytokines, suggesting that CSR and somatic hypermutation processes can occur independently, and the antibodies produced in this in vitro system are low-affinity antibodies.

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.

scholarly journals Optimized Protocols for in Vitro T Cell-Dependent and T Cell-Independent Activation for B Cell Differentiation Studies Using Limited Cells

2021 ◽  
Author(s):  
Casper Marsman ◽  
Dorit Verhoeven
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Cell Formation ◽  
B Cell Differentiation ◽  
Differentiation Potential ◽  
Human B Cell

Background/methods: For mechanistic studies, in vitro human B cell differentiation and generation of plasma cells are invaluable techniques. However, the heterogeneity of both T cell-dependent (TD) and T cell-independent (TI) stimuli and the disparity of culture conditions used in existing protocols makes interpretation of results challenging. The aim of the present study was to achieve the most optimal B cell differentiation conditions using isolated CD19+ B cells and PBMC cultures. We addressed multiple seeding densities, different durations of culturing and various combinations of TD stimuli and TI stimuli including B cell receptor (BCR) triggering. B cell expansion, proliferation and differentiation was analyzed after 6 and 9 days by measuring B cell proliferation and expansion, plasmablast and plasma cell formation and immunoglobulin (Ig) secretion. In addition, these conditions were extrapolated using cryopreserved cells and differentiation potential was compared. Results: This study demonstrates improved differentiation efficiency after 9 days of culturing for both B cell and PBMC cultures using CD40L and IL-21 as TD stimuli and 6 days for CpG and IL-2 as TI stimuli. We arrived at optimized protocols requiring 2500 and 25.000 B cells per culture well for TD and TI assays, respectively. The results of the PBMC cultures were highly comparable to the B cell cultures, which allows dismissal of additional B cell isolation steps prior to culturing. In these optimized TD conditions, the addition of anti-BCR showed little effect on phenotypic B cell differentiation, however it interferes with Ig secretion measurements. Addition of IL-4 to the TD stimuli showed significantly lower Ig secretion. The addition of BAFF to optimized TI conditions showed enhanced B cell differentiation and Ig secretion in B cell but not in PBMC cultures. With this approach, efficient B cell differentiation and Ig secretion was accomplished when starting from fresh or cryopreserved samples. Conclusion: Our methodology demonstrates optimized TD and TI stimulation protocols for more indepth analysis of B cell differentiation in primary human B cell and PBMC cultures while requiring low amounts of B cells, making them ideally suited for future clinical and research studies on B cell differentiation of patient samples from different cohorts of B cell-mediated diseases.

scholarly journals Signatures of B Cell Receptor Repertoire Following Pneumocystis Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Han Sun ◽  
Hu-Qin Yang ◽  
Kan Zhai ◽  
Zhao-Hui Tong
Keyword(s):  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Plasma Cells ◽  
Somatic Hypermutation ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Receptor Repertoire ◽  
Elevated Expression ◽  
Pneumocystis Infection

B cells play vital roles in host defense against Pneumocystis infection. However, the features of the B cell receptor (BCR) repertoire in disease progression remain unclear. Here, we integrated single-cell RNA sequencing and single-cell BCR sequencing of immune cells from mouse lungs in an uninfected state and 1–4 weeks post-infection in order to illustrate the dynamic nature of B cell responses during Pneumocystis infection. We identified continuously increased plasma cells and an elevated ratio of (IgA + IgG) to (IgD + IgM) after infection. Moreover, Pneumocystis infection was associated with an increasing naïve B subset characterized by elevated expression of the transcription factor ATF3. The proportion of clonal expanded cells progressively increased, while BCR diversity decreased. Plasma cells exhibited higher levels of somatic hypermutation than naïve B cells. Biased usage of V(D)J genes was observed, and the usage frequency of IGHV9-3 rose. Overall, these results present a detailed atlas of B cell transcriptional changes and BCR repertoire features in the context of Pneumocystis infection, which provides valuable information for finding diagnostic biomarkers and developing potential immunotherapeutic targets.

scholarly journals RNA-Seq Based Immunoglobulin Repertoire Analysis of Normal Plasma Cells Generated in an in Vitro B Cell Differentiation System

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1051-1051
Author(s):  
Renee C Tschumper ◽  
Collin A Osborne ◽  
Pritha Chanana ◽  
Jaime I Davila ◽  
Denise K Walters ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
In Vitro Model ◽  
Plasma Cells ◽  
B Cell Differentiation ◽  
Rna Seq ◽  
Repertoire Analysis ◽  
V Region ◽  
Vitro Model

Antibody secreting plasma cells (PCs) play an important role in effective humoral immune responses. The low frequency of bone marrow PCs in humans makes it challenging to obtain sufficient numbers of PCs for biologic studies. Previous studies have employed in vitro model systems to generate cells that morphologically, phenotypically, and functionally resemble normal polyclonal PCs. Gene expression profiles of in vitro generated PCs (IVPCs) mirror their normal counterparts, however to date extensive immunoglobulin (Ig) repertoire analysis of IVPCs is lacking. Here, we used a modified 3-step protocol to generate IVPCs and used RNA-seq to explore the transcriptome with emphasis on the Ig repertoire of plasmablasts and PCs. Total B cells were isolated from 3 normal donors and cultured with various cytokines and the B cell activators CpG ODN and CD40L. RNA was obtained from freshly isolated B cells (Day 0; D0) as well as from Day 4 (D4) plasmablasts, and Day 10 (D10) IVPCs. Morphologically, D10 cells exhibited typical PC morphology, including an eccentric nucleus and perinuclear hof. RNA-seq was performed on total RNA from all 3 donors and time points using the Standard TRuSeq v2 library prep and with paired end sequencing on the Illumina HiSeq 4000 platform. Principle component analysis of gene expression data showed that D0, D4 and D10 cells could be clearly segregated across all 3 normal donors. Of importance, transcripts previously described as distinguishing B cells from PCs were found to be differentially expressed including overexpression of CXCR5, CD19, EBF, CD83, PAX5, IRF8 in D0 B cells and overexpression of IRF4, Blimp-1, XBP1, BCMA, SLAMF7, Syndecan-1, CD38 and CD27 in IVPCs, thus validating our in vitro model for generating PCs. Furthermore, expression of cell cycle related transcripts such as CKS1, CDK1, and CCDN2 followed the pattern of low expression in resting B cells, increased expression in plasmablasts, and decreased expression in IVPCs confirming the cells are actively cycling in a manner comparable to cells in vivo. D10 IVPCs also overexpressed transcripts known to be upregulated during the unfolded protein response. As expected from Ig secreting cells, D10 IVPCs had an over-representation of Ig transcripts. At D0, resting B cells had high levels of IgD and IgM heavy chain (HC) transcripts. At D10, IgM transcripts modestly increased with Log2 fold change (FC) = 3 and as expected, IgD levels decreased significantly (Log2 FC = -2.2). IgA and IgG isotype transcripts significantly increased at D10 (Log2 FC > 6.0) with the IgG4 subtype having the greatest Log2 FC at 8.4. Next we focused on the Ig repertoire of D0, D4, and D10 cells. By aligning to known germline Ig sequences in IMGT/V-Quest (www.imgt.org) and then assembling the paired ends of D0, D4 and D10 Ig transcripts, we were able to analyze the Ig repertoire. Since the Ig HC variable (V) region is encoded by V, diversity (D) and joining (J) segments, only fragments that could be confidently determined were considered. All but 3 IGHV transcripts (IGHV3-35, IGHV3-47 and IGHV7-8) and 2 IGHD transcripts (IGHD4-4 and IGHD5-5) were found and all IGHJ segments were represented across the differentiation spectrum. In D0 cells, the number of unique VDJ combinations ranged from 643 to 863 across all 3 normal samples and increased to a range of 2524 to 2867 in D10 IVPCs. When looking at the differential expression of each VDJ combination from D0 to D10, a pairwise t-test for relative frequency showed that there was no significant change greater than 1%, suggesting the repertoire diversity was not skewed, thus proving the conditions for stimulation were not targeting any one starting B cell. Our data also allowed us to track clonal expansions during differentiation as defined by the increasing frequency of sequences with identical nucleotide sequence in the V region and CDR3 (including D and J regions). Hence, a single sequence could be tracked from D0 to D10. Of interest, in a small sampling of the total available sequences, only those B cells with a mutated IGHV region, characteristic of a memory B cell, went on to expand in this system whereas B cells with an unmutated IGHV did not. Our analysis of the Ig repertoire of IVPCs suggests this system provides a functional model to study Ig repertoire along the B cell differentiation process and further delineate the conditions that may result in a clonal expansion, a hallmark of many hematologic malignancies including multiple myeloma. Disclosures No relevant conflicts of interest to declare.

scholarly journals Modulation of spontaneous B-cell differentiation in macroglobulinemia by retinoic acid

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2206-2210 ◽  
Author(s):  
Y Levy ◽  
S Labaume ◽  
MC Gendron ◽  
JC Brouet
Keyword(s):  
Retinoic Acid ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
B Cell Differentiation ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Culture Supernatants

Abstract We previously showed that clonal blood B cells from patients with macroglobulinemia spontaneously differentiate in vitro to plasma cells. This process is dependent on an interleukin (IL)-6 autocrine pathway. We investigate here whether all-trans-retinoic acid (RA) interferes with B-cell differentiation either in patients with IgM gammapathy of undetermined significance (MGUS) or Waldenstrom's macroglobulinemia (WM). RA at a concentration of 10(-5) to 10(-8) mol/L inhibited by 50% to 80% the in vitro differentiation of purified B cells from four of five patients with MGUS and from one of five patients with WM as assessed by the IgM content of day 7 culture supernatants. We next determined whether this effect could be related to an inhibition of IL- 6 secretion by cultured B cells and/or a downregulation of the IL-6 receptor (IL-6R), which was constitutively expressed on patients' blood B cells. A 50% to 100% (mean, 80%) inhibition of IL-6 production was found in seven of 10 patients (five with MGUS and two with WM). The IL- 6R was no more detectable on cells from patients with MGUS after 2 days of treatment with RA and slightly downregulated in patients with WM. It was of interest that B cells susceptible to the action of RA belonged mostly to patients with IgM MGUS, which reinforces our previous data showing distinct requirements for IL-6-dependent differentiation of blood B cells from patients with VM or IgM MGUS.

Transformation of BCR-deficient germinal-center B cells by EBV supports a major role of the virus in the pathogenesis of Hodgkin and posttransplantation lymphomas

Blood ◽  
2005 ◽  
Vol 106 (13) ◽  
pp. 4345-4350 ◽  
Author(s):  
Dörte Bechtel ◽  
Julia Kurth ◽  
Claus Unkel ◽  
Ralf Küppers
Keyword(s):  
B Cells ◽  
Cell Lines ◽  
Germinal Center ◽  
Somatic Hypermutation ◽  
Cell Receptor ◽  
Lymphoproliferative Disease ◽  
Barr Virus ◽  
Classic Hodgkin Lymphoma ◽  
Epstein Barr

In classic Hodgkin lymphoma (HL) and posttransplantation lymphoproliferative disease (PTLD), 2 malignancies frequently associated with Epstein-Barr virus (EBV), the tumor cells often appear to derive from B-cell receptor (BCR)–deficient and therefore preapoptotic germinal center (GC) B cells. To test whether EBV can rescue BCR-less GC B cells, we infected human tonsillar CD77+ GC B cells in vitro with EBV. More than 60 monoclonal lymphoblastoid cell lines (LCLs) were established. Among these, 28 cell lines did not express surface immunoglobulin (sIg). Two of the sIg-negative cell lines carry obviously destructive mutations that have been introduced into originally functional VH gene rearrangements during the process of somatic hypermutation. Quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) showed that in most other lines the sIg deficiency was not simply the result of transcriptional down-regulation, but it was rather due to posttranscriptional defects. These findings strongly support the idea that EBV plays a central role in the pathogenesis of classic HL and PTLD by rescuing BCR-deficient, preapoptotic GC B cells from apoptosis, and that EBV infection renders the cells independent from survival signals normally supplied by a BCR. The monoclonal LCLs represent valuable models for early stages of lymphoma development in classic HL and PTLD.

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