Innate stimulation of B cells ex vivo enhances antibody secretion and identifies tumour-reactive antibodies from cancer patients

Abstract Human B cells and their expressed antibodies are crucial in conferring immune protection. Identifying pathogen-specific antibodies following infection is possible due to enhanced humoral immunity against well-described molecules on the pathogen surface. However, screening for cancer-reactive antibodies remains challenging since target antigens are often not identified a priori and the frequency of circulating B cells recognising cancer cells is likely very low. We investigated whether combined ex vivo culture of human B cells with three innate stimuli, interleukin-17 (IL-17), B cell activation factor (BAFF) and the toll like receptor 9 (TLR-9) agonist DNA motif CpG ODN 2006 (CpG), each known to activate B cells through different signalling pathways, promote cell activation, proliferation and antibody production. Combined IL-17+BAFF+CpG prolonged B cell survival and increased proliferation compared with single stimuli. IL-17+BAFF+CpG triggered higher IgG secretion, likely by activating differentiated, memory and class-switched CD19 +CD20 +CD27 +IgD - B cells. Regardless of anti-FOLR antibody seropositive status, IL-17+BAFF+CpG combined with a monovalent tumour-associated antigen (folate receptor alpha (FOLR)) led to secreted antibodies recognising the antigen and the antigen-expressing IGROV1 cancer cells. In a seropositive individual, FOLR stimulation favoured class-switched memory B cell precursors (CD27 -CD38 -IgD -), class-switched memory B cells and anti-FOLR antibody production, while IL-17+BAFF+CpG combined with FOLR, promoted class-switched memory B cell precursors and antibody-secreting (CD138+IgD-) plasma cells. Furthermore, IL-17+BAFF+CpG stimulation of peripheral blood B cells from patients with melanoma revealed tumour cell-reactive antibodies in culture supernatants. These findings suggest that innate signals stimulate B cell survival and antibody production and may help identify low-frequency antigen-reactive humoral responses.

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Concurrent engagement of CD40 and the antigen receptor protects naive and memory human B cells from APO-1/Fas-mediated apoptosis.

Journal of Experimental Medicine ◽

10.1084/jem.183.4.1377 ◽

1996 ◽

Vol 183 (4) ◽

pp. 1377-1388 ◽

Cited By ~ 87

Author(s):

C Lagresle ◽

P Mondière ◽

C Bella ◽

P H Krammer ◽

T Defrance

Keyword(s):

B Cells ◽

B Cell ◽

Cell Survival ◽

Antigen Receptor ◽

Cell Types ◽

Memory B Cells ◽

Apoptotic Pathway ◽

Cell Responses ◽

Human B Cells ◽

B Cell Survival

Naive and memory B cells were isolated from human tonsils and examined for expression of APO-1/Fas and for their sensitivity to the APO-1-dependent apoptosis. APO-1 was found to be constitutively expressed on memory but not on naive B cells. The susceptibility of both cell types to the APO-1 apoptotic pathway was acquired upon CD40 triggering and was correlated with increased expression of the APO-1 receptor. Both naive and memory B cells were protected from the APO-1-mediated death signal after dual ligation of the Ag receptor adn CD40. Our findings suggest that the APO-1 pathway controls the specificity of B cell responses to T-dependent Ags and that occupancy of the Ag receptor dictates the outcome of APO-1-ligation on B cell survival.

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LUBAC Suppresses IL-21-Induced Apoptosis in CD40-Activated Murine B Cells and Promotes Germinal Center B Cell Survival and the T-Dependent Antibody Response

Frontiers in Immunology ◽

10.3389/fimmu.2021.658048 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jingwei Wang ◽

Tianbao Li ◽

Hong Zan ◽

Carlos E. Rivera ◽

Hui Yan ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Survival ◽

Cell Activation ◽

Caspase 8 ◽

B Cell Activation ◽

Caspase 9 ◽

Apoptosis Pathway ◽

B Cell Survival

B cell activation by Tfh cells, i.e., through CD154 engagement of CD40 and IL-21, and survival within GCs are crucial for the T-dependent Ab response. LUBAC, composed of HOIP, SHARPIN, and HOIL-1, catalyzes linear ubiquitination (Linear M1-Ub) to mediate NF-κB activation and cell survival induced by TNF receptor superfamily members, which include CD40. As shown in this study, B cells expressing the Sharpin null mutation cpdm (Sharpincpdm) could undergo proliferation, CSR, and SHM in response to immunization by a T-dependent Ag, but were defective in survival within GCs, enrichment of a mutation enhancing the BCR affinity, and production of specific Abs. Sharpincpdm B cells stimulated in vitro with CD154 displayed normal proliferation and differentiation, marginally impaired NF-κB activation and survival, but markedly exacerbated death triggered by IL-21. While activating the mitochondria-dependent apoptosis pathway in both Sharpin+/+ and Sharpincpdm B cells, IL-21 induced Sharpincpdm B cells to undergo sustained activation of caspase 9 and caspase 8 of the mitochondria-dependent and independent pathway, respectively, and ultimately caspase 3 in effecting apoptosis. These were associated with loss of the caspase 8 inhibitor cFLIP and reduction in cFLIP Linear M1-Ub, which interferes with cFLIP poly-ubiquitination at Lys48 and degradation. Finally, the viability of Sharpincpdm B cells was rescued by caspase inhibitors but virtually abrogated – together with Linear M1-Ub and cFLIP levels – by a small molecule HOIP inhibitor. Thus, LUBAC controls the cFLIP expression and inhibits the effects of caspase 8 and IL-21-activated caspase 9, thereby suppressing apoptosis of CD40 and IL-21-activated B cells and promoting GC B cell survival.

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A BAFF/APRIL-dependent TLR3-stimulated pathway enhances the capacity of rheumatoid synovial fibroblasts to induce AID expression and Ig class-switching in B cells

Annals of the Rheumatic Diseases ◽

10.1136/ard.2011.150219 ◽

2011 ◽

Vol 70 (10) ◽

pp. 1857-1865 ◽

Cited By ~ 68

Author(s):

Michele Bombardieri ◽

Ngar-Woon Kam ◽

Fabia Brentano ◽

Ken Choi ◽

Andrew Filer ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

B Cells ◽

B Cell ◽

Cell Survival ◽

Cell Activation ◽

Cytidine Deaminase ◽

Synovial Fibroblasts ◽

Dermal Fibroblasts ◽

Class Switching ◽

B Cell Survival

ObjectivesTo dissect the role of toll-like receptor (TLR) signalling and B cell survival/proliferating factors in the crosstalk between rheumatoid arthritis synovial fibroblasts (RASF) and B cells.MethodsRASF, rheumatoid arthritis dermal fibroblasts (RADF) and osteoarthritis synovial fibroblasts (OASF) were analysed for the expression of B cell survival/proliferating factors BAFF and APRIL in resting conditions and upon stimulation with TLR2/TLR3/TLR4 ligands. Unswitched IgD+ B cells were co-cultured with RASF/OASF/RADF in the presence/absence of TLR ligands and with/without BAFF/APRIL blocking antibodies. Activation-induced cytidine deaminase (AID) mRNA expression, Iγ-Cμ and Iα-Cμ circular transcripts (CTs; markers of ongoing class-switching to IgG and IgA) and IgM/A/G production were measured to assess functional activation of B cells.ResultsTLR3 and to a lesser extent TLR4, but not TLR2 stimulation, induced up to ∼1000-fold BAFF mRNA and increased soluble BAFF release. APRIL was less significantly regulated by TLR3. Resting and TLR3-stimulated RASF released higher levels of BAFF/APRIL compared with RADF. TLR3 stimulation of RASF but not RADF in co-culture with B cells strongly enhanced AID expression, Iγ-Cμ and Iα-Cμ CTs and class-switching to IgG/IgA. Blockade of BAFF/APRIL signalling completely inhibited TLR3-induced, RASF-dependent expression of AID, CTs and the secretion of IgG/IgA.ConclusionsRASF produce high levels of BAFF and APRIL constitutively and in response to TLR3 stimulation. These factors are critical in directly modulating AID expression, class-switch recombination and IgG/IgA production in IgD+ B cells. Overall, this work highlights a novel and fundamental role for the TLR3/B cell survival factor axis in sustaining B cell activation in the rheumatoid arthritis synovium.

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Normal B Cell Homeostasis Requires B Cell Activation Factor Production by Radiation-resistant Cells

Journal of Experimental Medicine ◽

10.1084/jem.20030789 ◽

2003 ◽

Vol 198 (6) ◽

pp. 937-945 ◽

Cited By ~ 181

Author(s):

Leonid Gorelik ◽

Kevin Gilbride ◽

Max Dobles ◽

Susan L. Kalled ◽

Daniel Zandman ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Survival ◽

Stromal Cells ◽

Cell Activation ◽

B Cell Activation ◽

B Cell Survival ◽

Activation Factor ◽

Radiation Resistant ◽

B Cell Homeostasis

The cellular source of B cell activation factor (BAFF) required for peripheral B cell survival/maturation is unknown. To determine the nature of BAFF-producing cells we established and analyzed reciprocal bone marrow (BM) chimeras with wild-type (WT) and BAFF-deficient mice. The results revealed that BAFF production by radiation-resistant stromal cells is completely sufficient to provide a necessary signal for B cell survival/maturation, as BAFF−/− BM cells transferred into lethally irradiated WT mice gave rise to normal numbers of follicular (FO) and marginal zone (MZ) B cell subpopulations. On the other hand, transfer of WT BM into BAFF−/− lethally irradiated mice resulted only in minimal reconstitution of mature FO B cells and no restoration of MZ B cells. Thus, in the absence of BAFF+/+ stromal cells, BAFF production by BM-derived cells, presumably by macrophages, dendritic cells, and/or neutrophils, was not at all sufficient to support normal B cell homeostasis. Interestingly, immunization of both types of chimeras stimulated high levels of antigen-specific antibody secretion, indicating that either stromal cell– or hematopoietic cell–derived BAFF is sufficient for B cell antibody responses.

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Loss of Bim Allows Precursor B Cell Survival But Not Precursor B Cell Differentiation in the Absence of Interleukin 7

Journal of Experimental Medicine ◽

10.1084/jem.20041129 ◽

2004 ◽

Vol 200 (9) ◽

pp. 1179-1187 ◽

Cited By ~ 31

Author(s):

Paula M. Oliver ◽

Michael Wang ◽

Yanan Zhu ◽

Janice White ◽

John Kappler ◽

...

Keyword(s):

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Cell Survival ◽

B Cell Differentiation ◽

Normal Numbers ◽

Cell Precursor ◽

Interleukin 7 ◽

B Cell Survival ◽

B Cell Precursors

Interleukin (IL)-7 is a stromal cell–derived cytokine required for the survival, proliferation, and differentiation of B cell precursors. Members of the Bcl-2 family of proteins are known to have profound effects on lymphocyte survival, but not lymphocyte differentiation. To distinguish the relative dependence on IL-7 of B cell precursor survival versus B cell differentiation, the combined effects of lack of IL-7 and lack of the proapoptotic Bcl-2 relative, Bim, were studied. Bim is expressed to varying degrees in all B cell precursors and B cells. Lack of Bim compensated for lack of IL-7 in the survival of pro–, pre–, and immature B cells; however, lack of Bim did not substitute for the requirement for IL-7 in B cell precursor differentiation or B cell precursor proliferation. Precursor B cell survival is more dependent on sufficient levels of IL-7 than precursor B cell differentiation because the number of B cells and their precursors were reduced by half in mice heterozygous for IL-7 expression, but were restored to normal numbers in mice also lacking Bim. Hence, Bim and IL-7 work together to control the survival of B cell precursors and the number of B cells that exist in animals.

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AB0024 IN VITRO CHARACTERIZATION OF CENERIMOD, A POTENT AND SELECTIVE SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 (S1P1) MODULATOR IN PREVENTING MIGRATION OF NON-ACTIVATED AND ACTIVATED PRIMARY HUMAN B CELLS IN THE PRESENCE OR ABSENCE OF GLUCOCORTICOIDS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.2482 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 1046.1-1046

Author(s):

L. Schlicher ◽

P. Kulig ◽

M. Murphy ◽

M. Keller

Keyword(s):

B Cells ◽

B Cell ◽

B Lymphocytes ◽

Cell Activation ◽

Surface Expression ◽

Cell Surface Expression ◽

B Cell Activation ◽

Human B Cells ◽

Circulating Lymphocytes

Background:Cenerimod is a potent, selective, and orally active sphingosine 1-phosphate receptor 1 (S1P1) modulator that is currently being evaluated in a Phase 2b study in patients with systemic lupus erythematosus (SLE) (NCT03742037). S1P1 receptor modulators sequester circulating lymphocytes within lymph nodes, thereby reducing pathogenic autoimmune cells (including B lymphocytes) in the blood stream and in inflamed tissues. Extensive clinical experience has become available for the nonselective S1P receptor modulator fingolimod in relapsing forms of multiple sclerosis, supporting this therapeutic concept for the treatment of autoimmune disorders.Objectives:Although the effect of S1P-receptor modulators in reducing peripheral B cells is well documented1,2, the role of the S1P1 receptor on this cell type is only incompletely understood. In this study, the mode of action of cenerimod on primary human B cells was investigated in a series of in vitro experiments, including S1P1 receptor cell surface expression and chemotaxis towards S1P. Moreover, S1P1 expression following B cell activation in vitro was studied. As glucocorticoids (GC) are frequently used in the treatment of patients with autoimmune disorders including SLE, the potential influence of GC on the mode of action of cenerimod was evaluated.Methods:Primary human B lymphocytes from healthy donors were isolated from whole blood. In one set of experiments, cells were treated with different concentrations of cenerimod to measure S1P1 receptor internalization by flow cytometry. In a second set of experiments, isolated B cells were activated using different stimuli or left untreated. Cells were then analysed for S1P1 and CD69 cell surface expression and tested in a novel real-time S1P-mediated migration assay. In addition, the effect of physiological concentrations of GCs (prednisolone and prednisone) on cenerimod activity in preventing S1P mediated migration was tested.Results:In vitro, cenerimod led to a dose-dependent internalization of the S1P1 receptor on primary human B lymphocytes. Cenerimod also blocked migration of nonactivated and activated B lymphocytes towards S1P in a concentration-dependent manner, which is in line with the retention of lymphocytes in the lymph node and the reduction of circulating lymphocytes observed in the clinical setting. Upon B cell activation, which was monitored by CD69 upregulation, a simultaneous downregulation of S1P1 expression was detected, leading to less efficient S1P-directed cell migration. Importantly, physiological concentrations of GC did not affect the inhibitory activity of cenerimod on B cell migration.Conclusion:These results show that cenerimod, by modulating S1P1, blocks B lymphocyte migration towards its natural chemoattractant S1P and demonstrate compatibility of cenerimod with GC. These results are consistent with results of comparable experiments done previously using primary human T lymphocytes.References:[1]Nakamura M et al., Mult Scler. 2014 Sep; 20(10):1371-80.[2]Strasser DS et al., RMD Open 2020;6:e001261.Disclosure of Interests:None declared

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Ras Mediates Effector Pathways Responsible for Pre-B Cell Survival, Which Is Essential for the Developmental Progression to the Late Pre-B Cell Stage

Journal of Experimental Medicine ◽

10.1084/jem.192.2.171 ◽

2000 ◽

Vol 192 (2) ◽

pp. 171-182 ◽

Cited By ~ 39

Author(s):

Hitoshi Nagaoka ◽

Yoshimasa Takahashi ◽

Reiko Hayashi ◽

Tohru Nakamura ◽

Kumiko Ishii ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Survival ◽

Chain Gene ◽

B Cell Differentiation ◽

Antigen Receptors ◽

Cell Stage ◽

Light Chain Gene ◽

B Cell Survival ◽

Effector Pathways

Ras is essential for the transition from early B cell precursors to the pro-B stage, and is considered to be involved in the signal cascade mediated by pre-B cell antigen receptors. To examine the role of p21ras in the late stage of B cell differentiation, we established transgenic mice (TG) expressing a dominant-inhibitory mutant of Ha-ras (Asn-17 Ha-ras) in B lineage cells at high levels after the early B cell precursor stage. Expression of p21Asn-17 Ha-ras was associated with a prominent reduction in the number of late pre-B cells, but had little effect on proliferation of early pre-B cells. Inhibition of p21ras activity markedly reduced the life span of pre-B cells, due, at least in part, to downregulation of the expression of an antiapoptotic protein, Bcl-xL. Thus, the apparent role for p21ras activity in pre-B cell survival may explain the decreased numbers of late pre-B cells in Asn-17 Ha-ras TG. Consistent with this possibility, overexpression of Bcl-2 in Asn-17 Ha-ras TG reversed the reduction in the number of late pre-B cells undergoing immunoglobulin light chain gene (IgL) rearrangement and progressing to immature B cells. These results suggest that p21ras mediates effector pathways responsible for pre-B cell survival, which is essential for progression to the late pre-B and immature B stages.

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B Cell–Activating Factor Promotes B Cell Survival in Ectopic Lymphoid Tissues in Nasal Polyps

Frontiers in Immunology ◽

10.3389/fimmu.2020.625630 ◽

2021 ◽

Vol 11 ◽

Author(s):

Zhe-Zheng Wang ◽

Jia Song ◽

Hai Wang ◽

Jing-Xian Li ◽

Qiao Xiao ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Survival ◽

Stromal Cells ◽

Caspase 3 ◽

Lymphoid Tissues ◽

B Cell Activating Factor ◽

B Cell Survival ◽

Active Caspase

Ectopic lymphoid tissues (eLTs) characterized by B cell aggregation contribute to the local immunoglobulin production in nasal polyps (NPs). B cell-activating factor (BAFF) is vital for B cell survival, proliferation, and maturation. The purpose of this study is to investigate whether BAFF is involved in the B cell survival and eLT formation in NPs. The mRNA and protein levels of BAFF in NP tissues with and without eLTs were detected by PCR and ELISA assay, respectively. The cellular sources of BAFF and active caspase-3-positive B cells in NPs were studied by immunofluorescence staining. B cells purified from NP tissues were stimulated with BAFF and were analyzed by flow cytometry. Stromal cells purified from NP tissues were stimulated with lymphotoxin (LT) α1β2, and BAFF levels in culture supernatants were analyzed by ELISA. Compared with those in control tissues and NPs without eLTs, the BAFF levels were elevated in NPs with eLTs. Abundant BAFF-positive cells and few active caspase-3-positive apoptotic B cells were found in NPs with eLTs, in contrast to those in NPs without eLTs. There was a negative correlation between the numbers of BAFF-positive cells and frequencies of apoptotic B cells in total B cells in NP tissues. BAFF protected nasal polyp B cells from apoptosis in vitro. Stromal cells were an important cellular source of BAFF in NPs with eLTs. LTα1β2 induced BAFF production from nasal stromal cells in vitro. We propose that BAFF contribute to eLT formation in NPs by promoting B cell survival.

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Attenuation of Apoptosis Underlies B Lymphocyte Stimulator Enhancement of Humoral Immune Response

Journal of Experimental Medicine ◽

10.1084/jem.192.7.953 ◽

2000 ◽

Vol 192 (7) ◽

pp. 953-964 ◽

Cited By ~ 313

Author(s):

Richard K.G. Do ◽

Eunice Hatada ◽

Hayyoung Lee ◽

Michelle R. Tourigny ◽

David Hilbert ◽

...

Keyword(s):

Immune Response ◽

T Cell ◽

B Cells ◽

B Cell ◽

Cell Survival ◽

B Lymphocyte ◽

Humoral Immune ◽

B Cell Survival ◽

B Lymphocyte Stimulator

B lymphocyte stimulator (BLyS) is a newly identified monocyte-specific TNF family cytokine. It has been implicated in the development of autoimmunity, and functions as a potent costimulator with antiimmunoglobulin M in B cell proliferation in vitro. Here we demonstrate that BLyS prominently enhances the humoral responses to both T cell–independent and T cell–dependent antigens, primarily by attenuation of apoptosis as evidenced by the prolonged survival of antigen-activated B cells in vivo and in vitro. BLyS acts on primary splenic B cells autonomously, and directly cooperates with CD40 ligand (CD40L) in B cell activation in vitro by protecting replicating B cells from apoptosis. Moreover, although BLyS alone cannot activate the cell cycle, it is sufficient to prolong the survival of naive resting B cells in vitro. Attenuation of apoptosis by BLyS correlates with changes in the ratios between Bcl-2 family proteins in favor of cell survival, predominantly by reducing the proapoptotic Bak and increasing its prosurvival partners, Bcl-2 and Bcl-xL. In either resting or CD40L-activated B cells, the NF-κB transcription factors RelB and p50 are specifically activated, suggesting that they may mediate BLyS signals for B cell survival. Together, these results provide direct evidence for BLyS enhancement of both T cell–independent and T cell–dependent humoral immune responses, and imply a role for BLyS in the conservation of the B cell repertoire. The ability of BLyS to increase B cell survival indiscriminately, at either a resting or activated state, and to cooperate with CD40L, further suggests that attenuation of apoptosis underlies BLyS enhancement of polyclonal autoimmunity as well as the physiologic humoral immune response.

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