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1.
Osteoarthritis Cartilage ; 31(9): 1176-1188, 2023 09.
Article in English | MEDLINE | ID: mdl-37290499

ABSTRACT

OBJECTIVE: Autoantibody (autoAbs) production in osteoarthritis (OA), coupled with evidence of disturbed B-cell homoeostasis, suggest a potential role for B-cells in OA. B-cells can differentiate with T-cell help (T-dep) or using alternative Toll like recptor (TLR) co-stimulation (TLR-dep). We analysed the capacity for differentiation of B-cells in OA versus age-matched healthy controls (HCs) and compared the capacity of OA synovitis-derived stromal cells to provide support for plasma cell (PC) maturation. METHODS: B-cells were isolated from OA and HC. Standardised in vitro models of B-cell differentiation were used comparing T-dep (CD40 (cluster of differentiation-40/BCR (B-cell receptor)-ligation) versus TLR-dep (TLR7/BCR-activation). Differentiation marker expression was analysed by flow-cytometry; antibody secretion (immunnoglobulins IgM/IgA/IgG) by ELISA (enzyme-linked immunosorbent assay), gene expression by qPCR (quantitative polymerase chain reaction). RESULTS: Compared to HC, circulating OA B-cells showed an overall more mature phenotype. The gene expression profile of synovial OA B-cells resembled that of PCs. Circulating B-cells differentiated under both TLR-dep and T-dep, however OA B-cells executed differentiation faster in terms of change in surface marker and secreted more antibody at Day 6, while resulting in similar PC numbers at Day 13, with an altered phenotype at Day 13 in OA. The main difference was reduced early B-cells expansion in OA (notably in TLR-dep) and reduced cell death. Stromal cells support from OA-synovitis allowed better PC survival compared to bone marrow, with an additional population of cells and higher Ig-secretion. CONCLUSION: Our findings suggest that OA B-cells present an altered capacity for proliferation and differentiation while remaining able to produce antibodies, notably in synovium. These findings may partly contribute to autoAbs development as recently observed in OA synovial fluids.


Subject(s)
Osteoarthritis , Synovitis , Humans , Plasma Cells , Osteoarthritis/metabolism , B-Lymphocytes/metabolism , Synovial Membrane , Synovitis/metabolism
2.
Cancers (Basel) ; 15(12)2023 Jun 07.
Article in English | MEDLINE | ID: mdl-37370694

ABSTRACT

Epstein-Barr virus (EBV)-driven B cell neoplasms arise from the reactivation of latently infected B cells. In a subset of patients, EBV was seen to drive a polymorphous lymphoproliferative disorder (LPD) in which B cell differentiation was retained. In this work, spontaneous EBV reactivation following B cell mitogen stimulation was shown to provide a potential model of polymorphic EBV-driven LPD. Here, we developed an in vitro model of plasma cell (PC) differentiation from peripheral blood memory B cells. To assess the frequency and phenotypes of EBV-associated populations derived during differentiation, we analysed eight differentiations during the PC stage with a targeted single-cell gene expression panel. We identified subpopulations of EBV-gene expressing cells with PC and/or B cell expression features in differentiations from all tested donors. EBV-associated cells varied in frequency, ranging from 3-28% of cells. Most EBV-associated cells expressed PC genes such as XBP1 or MZB1, and in all samples these included a quiescent PC fraction that lacked cell a cycle gene expression. With increasing EBV-associated cells, populations with B cell features became prominent, co-expressing a germinal centre (GC) and activating B cell gene patterns. The presence of highly proliferative EBV-associated cells was linked to retained MS4A1/CD20 expression and IGHM and IGHD co-expression, while IGHM class-switched cells were enriched in quiescent PC fractions. Thus, patterns of gene expression in primary EBV reactivation were shown to include features related to GC B cells, which was also observed in EBV-transformed lymphoblastoid cell lines. This suggests a particular association between spontaneously developing EBV-expansions and IgM+ IgD+ non-switched B cells.

3.
J Clin Immunol ; 43(7): 1543-1556, 2023 10.
Article in English | MEDLINE | ID: mdl-37246174

ABSTRACT

BACKGROUND: The human CD19 antigen is expressed throughout B cell ontogeny with the exception of neoplastic plasma cells and a subset of normal plasma cells. CD19 plays a role in propagating signals from the B cell receptor and other receptors such as CXCR4 in mature B cells. Studies of CD19-deficient patients have confirmed its function during the initial stages of B cell activation and the production of memory B cells; however, its role in the later stages of B cell differentiation is unclear. OBJECTIVE: Using B cells from a newly identified CD19-deficient individual, we investigated the role of CD19 in the generation and function of plasma cells using an in vitro differentiation model. METHODS: Flow cytometry and long-read nanopore sequencing using locus-specific long-range amplification products were used to screen a patient with suspected primary immunodeficiency. Purified B cells from the patient and healthy controls were activated with CD40L, IL-21, IL-2, and anti-Ig, then transferred to different cytokine conditions to induce plasma cell differentiation. Subsequently, the cells were stimulated with CXCL12 to induce signalling through CXCR4. Phosphorylation of key downstream proteins including ERK and AKT was assessed by Western blotting. RNA-seq was also performed on in vitro differentiating cells. RESULTS: Long-read nanopore sequencing identified the homozygous pathogenic mutation c.622del (p.Ser208Profs*19) which was corroborated by the lack of CD19 cell surface staining. CD19-deficient B cells that are predominantly naïve generate phenotypically normal plasma cells with expected patterns of differentiation-associated genes and normal levels of CXCR4. Differentiated CD19-deficient cells were capable of responding to CXCL12; however, plasma cells derived from naïve B cells, both CD19-deficient and sufficient, had relatively diminished signaling compared to those generated from total B cells. Additionally, CD19 ligation on normal plasma cells results in AKT phosphorylation. CONCLUSION: CD19 is not required for generation of antibody-secreting cells or the responses of these populations to CXCL12, but may alter the response other ligands that require CD19 potentially affecting localization, proliferation, or survival. The observed hypogammaglobulinemia in CD19-deficient individuals is therefore likely attributable to the lack of memory B cells.


Subject(s)
Antigens, CD19 , Plasma Cells , Humans , Plasma Cells/metabolism , Antigens, CD19/genetics , Antigens, CD19/metabolism , Proto-Oncogene Proteins c-akt/metabolism , B-Lymphocytes , Receptors, Antigen, B-Cell , Adaptor Proteins, Signal Transducing/metabolism , Chemokine CXCL12/genetics , Chemokine CXCL12/metabolism
4.
Methods Mol Biol ; 2675: 271-283, 2023.
Article in English | MEDLINE | ID: mdl-37258770

ABSTRACT

B cells engaging with antigen and secondary signals provided by T cell help, or ligands for Toll-like receptors, undergo a step-wise process of differentiation to eventually produce antibody-secreting plasma cells. During the course of this conversion, the cells transition from a resting, non-growing state to an activated B-cell state engaged in DNA synthesis and mitosis to a terminally differentiated, quiescent cell state with expanded organelles necessary for high levels of secretion. Each of these phases is accompanied by considerable changes in metabolic requirements. To facilitate evaluation of this metabolic reprogramming, methods for the in vitro differentiation of human B cells that incorporates each of the transitional stages are described.


Subject(s)
B-Lymphocytes , Lymphocyte Activation , Humans , T-Lymphocytes , Antibody-Producing Cells , Cell Differentiation , Plasma Cells
5.
Blood Adv ; 7(15): 3874-3890, 2023 08 08.
Article in English | MEDLINE | ID: mdl-36867577

ABSTRACT

Multiple myeloma (MM) shows constitutive activation of canonical and noncanonical nuclear factor κB (NF-κB) signaling via genetic mutations or tumor microenvironment (TME) stimulations. A subset of MM cell lines showed dependency for cell growth and survival on the canonical NF-κB transcription factor RELA alone, suggesting a critical role for a RELA-mediated biological program in MM pathogenesis. Here, we determined the RELA-dependent transcriptional program in MM cell lines and found the expression of the cell surface molecules interleukin-27 receptor-α (IL-27Rα) and the adhesion molecule JAM2 to be responsive to RELA at the messenger RNA and protein levels. IL-27Rα and JAM2 were expressed on primary MM cells at higher levels than on healthy long-lived plasma cells (PCs) in the bone marrow. IL-27 activated STAT1, and to a lesser extent STAT3, in MM cell lines and in PCs generated from memory B cells in an IL-21-dependent in vitro PC differentiation assay. Concomitant activity of IL-21 and IL-27 enhanced differentiation into PCs and increased the cell-surface expression of the known STAT target gene CD38. In accordance, a subset of MM cell lines and primary MM cells cultured with IL-27 upregulated CD38 cell-surface expression, a finding with potential implications for enhancing the efficacy of CD38-directed monoclonal antibody therapies by increasing CD38 expression on tumor cells. The elevated expression of IL-27Rα and JAM2 on MM cells compared with that on healthy PCs may be exploited for the development of targeted therapeutic strategies that modulate the interaction of MM cells with the TME.


Subject(s)
Interleukin-27 , Multiple Myeloma , Humans , Interleukin-27/metabolism , Multiple Myeloma/genetics , NF-kappa B/metabolism , Receptors, Cytokine/metabolism , Tumor Microenvironment , Up-Regulation
6.
Nat Immunol ; 24(3): 474-486, 2023 03.
Article in English | MEDLINE | ID: mdl-36703005

ABSTRACT

The cross-talk between thymocytes and thymic stromal cells is fundamental for T cell development. In humans, intrathymic development of dendritic cells (DCs) is evident but its physiological significance is unknown. Here we showed that DC-biased precursors depended on the expression of the transcription factor IRF8 to express the membrane-bound precursor form of the cytokine TNF (tmTNF) to promote differentiation of thymus seeding hematopoietic progenitors into T-lineage specified precursors through activation of the TNF receptor (TNFR)-2 instead of TNFR1. In vitro recapitulation of TNFR2 signaling by providing low-density tmTNF or a selective TNFR2 agonist enhanced the generation of human T cell precursors. Our study shows that, in addition to mediating thymocyte selection and maturation, DCs function as hematopoietic stromal support for the early stages of human T cell development and provide proof of concept that selective targeting of TNFR2 can enhance the in vitro generation of T cell precursors for clinical application.


Subject(s)
Dendritic Cells , Receptors, Tumor Necrosis Factor, Type II , Humans , Cell Differentiation , Cell Lineage , Interferon Regulatory Factors/metabolism , Receptors, Tumor Necrosis Factor, Type II/metabolism , Thymus Gland/metabolism , Tumor Necrosis Factors/metabolism
7.
J Immunol ; 209(5): 926-937, 2022 09 01.
Article in English | MEDLINE | ID: mdl-36130130

ABSTRACT

Ab-secreting cells survive in niche microenvironments, but cellular responses driven by particular niche signals are incompletely defined. The TNF superfamily member a proliferation-inducing ligand (APRIL) can support the maturation of transitory plasmablasts into long-lived plasma cells. In this study, we explore the biological programs established by APRIL in human plasmablasts. Under conditions allowing the maturation of ex vivo- or in vitro-generated plasmablasts, we find that APRIL drives activation of ERK, p38, and JNK, accompanied by a classical NF-κB response and activation of the AKT/FOXO1 pathway. Time-course gene expression data resolve coordinated transcriptional responses propagated via immediate early genes and NF-κB targets and converging onto modules of genes enriched for MYC targets and metabolism/cell growth-related pathways. This response is shared between APRIL and an alternate TNF superfamily member CD40L but is not a feature of alternative niche signals delivered by IFN-α or SDF1. However, APRIL and CD40L responses also diverge. CD40L drives expression of genes related to the activated B cell state whereas APRIL does not. Thus, APRIL establishes a broad foundation for plasma cell longevity with features of cellular refueling while being uncoupled from support of the B cell state.


Subject(s)
CD40 Ligand , NF-kappa B , Humans , NF-kappa B/metabolism , Plasma Cells/metabolism , Proto-Oncogene Proteins c-akt , Tumor Necrosis Factor Ligand Superfamily Member 13
9.
J Immunol ; 208(2): 514-525, 2022 01 15.
Article in English | MEDLINE | ID: mdl-34911772

ABSTRACT

Upon encounter with Ag, B cells undergo a sequential process of differentiation to become Ab-secreting plasma cells. Although the key drivers of differentiation have been identified, research has been limited by the lack of in vitro models recapitulating the full process for murine B cells. In this study, we describe methodology using BCR or TLR ligation to obtain plasma cells that are phenotypically mature, have exited cell cycle and express a gene signature concordant with long-lived plasma cells. Dependent on the initial stimuli, the transcriptomes also show variation including the enhanced expression of matrisome components after BCR stimulation, suggestive of unique functional properties for the resultant plasma cells. Moreover, using the new culture conditions we demonstrate that alternative promoter choice regulating the expression of the master transcription factor Blimp-1/Prdm1 can be observed; when the canonical B cell promoter for Prdm1 is deleted, differentiating B cells exhibit flexibility in the choice of promoter, dictated by the initiating stimulus, with preferential maintenance of expression following exposure to TLR ligation. Thus our system provides a readily tractable model for furthering our understanding of plasma cell biology.


Subject(s)
Cell Differentiation/immunology , Plasma Cells/cytology , Plasma Cells/immunology , Positive Regulatory Domain I-Binding Factor 1/genetics , Receptors, Antigen, B-Cell/immunology , Animals , Gene Expression Regulation/genetics , Gene Expression Regulation/immunology , Mice , Mice, Inbred C57BL , Promoter Regions, Genetic/genetics , Transcriptome/genetics
10.
Elife ; 102021 12 15.
Article in English | MEDLINE | ID: mdl-34908525

ABSTRACT

CRAC channel regulator 2 A (CRACR2A) is a large Rab GTPase that is expressed abundantly in T cells and acts as a signal transmitter between T cell receptor stimulation and activation of the Ca2+-NFAT and JNK-AP1 pathways. CRACR2A has been linked to human diseases in numerous genome-wide association studies, however, to date no patient with damaging variants in CRACR2A has been identified. In this study, we describe a patient harboring biallelic variants in CRACR2A [paternal allele c.834 gaG> gaT (p.E278D) and maternal alelle c.430 Aga > Gga (p.R144G) c.898 Gag> Tag (p.E300*)], the gene encoding CRACR2A. The 33-year-old patient of East-Asian origin exhibited late onset combined immunodeficiency characterised by recurrent chest infections, panhypogammaglobulinemia and CD4+ T cell lymphopenia. In vitro exposure of patient B cells to a T-dependent stimulus resulted in normal generation of antibody-secreting cells, however the patient's T cells showed pronounced reduction in CRACR2A protein levels and reduced proximal TCR signaling, including dampened SOCE and reduced JNK phosphorylation, that contributed to a defect in proliferation and cytokine production. Expression of individual allelic mutants in CRACR2A-deleted T cells showed that the CRACR2AE278D mutant did not affect JNK phosphorylation, but impaired SOCE which resulted in reduced cytokine production. The truncated double mutant CRACR2AR144G/E300* showed a pronounced defect in JNK phosphorylation as well as SOCE and strong impairment in cytokine production. Thus, we have identified variants in CRACR2A that led to late-stage combined immunodeficiency characterized by loss of function in T cells.


Subject(s)
Calcium Release Activated Calcium Channels/genetics , Calcium Release Activated Calcium Channels/metabolism , Cytokines/biosynthesis , Mutation , Primary Immunodeficiency Diseases/genetics , Primary Immunodeficiency Diseases/physiopathology , Receptors, Antigen, T-Cell/metabolism , Adult , Asian People , Calcium Release Activated Calcium Channels/immunology , Cytokines/genetics , Genetic Variation , Humans , Primary Immunodeficiency Diseases/immunology , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology
11.
Aging Cell ; 20(4): e13341, 2021 04.
Article in English | MEDLINE | ID: mdl-33711204

ABSTRACT

BACKGROUND: Age-related immune deficiencies are thought to be responsible for increased susceptibility to infection in older adults, with alterations in lymphocyte populations becoming more prevalent over time. The loss of humoral immunity in ageing was attributed to the diminished numbers of B cells and the reduced ability to generate immunoglobulin. AIMS: To compare the intrinsic B-cell capacity for differentiation into mature plasma cells (PCs), between young and old donors, using in vitro assays, providing either effective T-cell help or activation via TLR engagement. METHODS: B cells were isolated from healthy individuals, in younger (30-38 years) and older (60-64 years) donors. An in vitro model system of B-cell differentiation was used, analysing 5 differentiation markers by flow cytometry, under T-dependent (TD: CD40/BCR stimulation) or T-independent (TI: TLR7/BCR activation) conditions. Antibody secretion was measured by ELISA and gene expression using qPCR. RESULTS: TI and TD differentiation resulted in effective proliferation of B cells followed by their differentiation into PC. B-cell-executed TI differentiation was faster, all differentiation marker and genes being expressed earlier than under TD differentiation (day 6), although generating less viable cells and lower antibody levels (day 13). Age-related differences in B-cell capacity for differentiation were minimal in TD differentiation. In contrast, in TI differentiation age significantly affected proliferation, viability, differentiation, antibody secretion and gene expression, older donors being more efficient. CONCLUSION: Altogether, B-cell differentiation into PC appeared similar between age groups when provided with T-cell help, in contrast to TI differentiation, where multiple age-related changes suggest better capacities in older donors. These new findings may help explain the emergence of autoantibodies in ageing.


Subject(s)
Aging/immunology , Cell Differentiation/immunology , Lymphocyte Activation , Plasma Cells/cytology , Plasma Cells/immunology , Adult , Age Factors , Blood Donors , CD40 Ligand/metabolism , Cells, Cultured , Female , Flow Cytometry , Humans , Imidazoles/pharmacology , Immunity, Humoral , Immunoglobulin Isotypes/biosynthesis , Male , Middle Aged , Plasma Cells/drug effects , Receptors, Antigen, B-Cell/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Toll-Like Receptor 7/agonists
12.
Life Sci Alliance ; 3(10)2020 10.
Article in English | MEDLINE | ID: mdl-32843533

ABSTRACT

The activated B-cell (ABC) to plasmablast transition encompasses the cusp of antibody-secreting cell (ASC) differentiation. We explore this transition with integrated analysis in human cells, focusing on changes that follow removal from CD40-mediated signals. Within hours of input signal loss, cell growth programs shift toward enhanced proliferation, accompanied by ER-stress response, and up-regulation of ASC features. Clustering of genomic occupancy for IRF4, BLIMP1, XBP1, and CTCF with histone marks identifies a dichotomy: XBP1 and IRF4 link to induced but not repressed gene modules in plasmablasts, whereas BLIMP1 links to modules of ABC genes that are repressed, but not to activated genes. Between ABC and plasmablast states, IRF4 shifts away from AP1/IRF composite elements while maintaining occupancy at IRF and ETS/IRF elements. This parallels the loss of BATF expression, which is identified as a potential BLIMP1 target. In plasmablasts, IRF4 acquires an association with CTCF, a feature maintained in plasma cell myeloma lines. Thus, shifting occupancy links IRF4 to both ABC and ASC gene expression, whereas BLIMP1 occupancy links to repression of the activation state.


Subject(s)
B-Lymphocytes/cytology , Gene Regulatory Networks/genetics , Plasma Cells/cytology , Adult , B-Lymphocytes/metabolism , CD40 Antigens/immunology , CD40 Antigens/metabolism , Cell Differentiation/physiology , Cell Proliferation/physiology , Female , Humans , Interferon Regulatory Factors/metabolism , Lymphocyte Activation/physiology , Male , Positive Regulatory Domain I-Binding Factor 1/metabolism , Signal Transduction , Transcriptional Activation/physiology , X-Box Binding Protein 1/metabolism
13.
Immunity ; 53(2): 353-370.e8, 2020 08 18.
Article in English | MEDLINE | ID: mdl-32735845

ABSTRACT

The formation of mammalian dendritic cells (DCs) is controlled by multiple hematopoietic transcription factors, including IRF8. Loss of IRF8 exerts a differential effect on DC subsets, including plasmacytoid DCs (pDCs) and the classical DC lineages cDC1 and cDC2. In humans, cDC2-related subsets have been described including AXL+SIGLEC6+ pre-DC, DC2 and DC3. The origin of this heterogeneity is unknown. Using high-dimensional analysis, in vitro differentiation, and an allelic series of human IRF8 deficiency, we demonstrated that cDC2 (CD1c+DC) heterogeneity originates from two distinct pathways of development. The lymphoid-primed IRF8hi pathway, marked by CD123 and BTLA, carried pDC, cDC1, and DC2 trajectories, while the common myeloid IRF8lo pathway, expressing SIRPA, formed DC3s and monocytes. We traced distinct trajectories through the granulocyte-macrophage progenitor (GMP) compartment showing that AXL+SIGLEC6+ pre-DCs mapped exclusively to the DC2 pathway. In keeping with their lower requirement for IRF8, DC3s expand to replace DC2s in human partial IRF8 deficiency.


Subject(s)
Antigens, CD34/metabolism , Dendritic Cells/cytology , Hematopoiesis/physiology , Interferon Regulatory Factors/metabolism , Animals , Antigens, CD1/metabolism , Cell Line , Cell Lineage/immunology , Dendritic Cells/immunology , Glycoproteins/metabolism , Hematopoietic Stem Cells/cytology , Humans , Interleukin-3 Receptor alpha Subunit/metabolism , Lipopolysaccharide Receptors/metabolism , Mice , Receptors, Immunologic/metabolism
14.
Blood ; 136(9): 1055-1066, 2020 08 27.
Article in English | MEDLINE | ID: mdl-32518946

ABSTRACT

Molecular dissection of inborn errors of immunity can help to elucidate the nonredundant functions of individual genes. We studied 3 children with an immune dysregulation syndrome of susceptibility to infection, lymphadenopathy, hepatosplenomegaly, developmental delay, autoimmunity, and lymphoma of B-cell (n = 2) or T-cell (n = 1) origin. All 3 showed early autologous T-cell reconstitution following allogeneic hematopoietic stem cell transplantation. By whole-exome sequencing, we identified rare homozygous germline missense or nonsense variants in a known epigenetic regulator of gene expression: ten-eleven translocation methylcytosine dioxygenase 2 (TET2). Mutated TET2 protein was absent or enzymatically defective for 5-hydroxymethylating activity, resulting in whole-blood DNA hypermethylation. Circulating T cells showed an abnormal immunophenotype including expanded double-negative, but depleted follicular helper, T-cell compartments and impaired Fas-dependent apoptosis in 2 of 3 patients. Moreover, TET2-deficient B cells showed defective class-switch recombination. The hematopoietic potential of patient-derived induced pluripotent stem cells was skewed toward the myeloid lineage. These are the first reported cases of autosomal-recessive germline TET2 deficiency in humans, causing clinically significant immunodeficiency and an autoimmune lymphoproliferative syndrome with marked predisposition to lymphoma. This disease phenotype demonstrates the broad role of TET2 within the human immune system.


Subject(s)
DNA-Binding Proteins/deficiency , Germ-Line Mutation , Loss of Function Mutation , Lymphoproliferative Disorders/genetics , Proto-Oncogene Proteins/deficiency , Severe Combined Immunodeficiency/genetics , Allografts , Apoptosis , B-Lymphocyte Subsets/pathology , Cellular Reprogramming Techniques , Codon, Nonsense , DNA Methylation , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Dioxygenases , Fatal Outcome , Female , Hematopoietic Stem Cell Transplantation , Humans , Induced Pluripotent Stem Cells/pathology , Infant, Newborn , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/pathology , Lymphoma, T-Cell, Peripheral/genetics , Lymphoma, T-Cell, Peripheral/pathology , Male , Mutation, Missense , Neoplasms, Multiple Primary/genetics , Pedigree , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/physiology , Severe Combined Immunodeficiency/pathology , T-Lymphocyte Subsets/pathology , Exome Sequencing
15.
Blood Adv ; 4(12): 2821-2836, 2020 06 23.
Article in English | MEDLINE | ID: mdl-32574366

ABSTRACT

Waldenström macroglobulinemia (WM) is a rare malignancy in which clonal B cells infiltrate the bone marrow and give rise to a smaller compartment of neoplastic plasma cells that secrete monoclonal immunoglobulin M paraprotein. Recent studies into underlying mutations in WM have enabled a much greater insight into the pathogenesis of this lymphoma. However, there is considerably less characterization of the way in which WM B cells differentiate and how they respond to immune stimuli. In this study, we assess WM B-cell differentiation using an established in vitro model system. Using T-cell-dependent conditions, we obtained CD138+ plasma cells from WM samples with a frequency similar to experiments performed with B cells from normal donors. Unexpectedly, a proportion of the WM B cells failed to upregulate CD38, a surface marker that is normally associated with plasmablast transition and maintained as the cells proceed with differentiation. In normal B cells, concomitant Toll-like receptor 7 (TLR7) activation and B-cell receptor cross-linking drives proliferation, followed by differentiation at similar efficiency to CD40-mediated stimulation. In contrast, we found that, upon stimulation with TLR7 agonist R848, WM B cells failed to execute the appropriate changes in transcriptional regulators, identifying an uncoupling of TLR signaling from the plasma cell differentiation program. Provision of CD40L was sufficient to overcome this defect. Thus, the limited clonotypic WM plasma cell differentiation observed in vivo may result from a strict requirement for integrated activation.


Subject(s)
Lymphoma, B-Cell , Waldenstrom Macroglobulinemia , B-Lymphocytes , Cell Differentiation , Humans , Plasma Cells , Waldenstrom Macroglobulinemia/genetics
16.
Front Immunol ; 11: 569006, 2020.
Article in English | MEDLINE | ID: mdl-33424831

ABSTRACT

The Schnitzler Syndrome (SchS) is an acquired, autoinflammatory condition successfully treated with IL-1 inhibition. The two main defining features of this late-onset condition are neutrophilic urticarial dermatoses (NUD) and the presence of an IgM monoclonal component. While the former aspect has been extensively studied in this disease setting, the enigmatic paraproteinaemia and its potential consequential effects within SchS, has not previously been thoroughly addressed. Previous studies analyzing clonal B cell repertoires have largely focused on autoimmune disorders such as Systemic Lupus Erythematous (SLE) and hematological malignancies such as Chronic Lymphocytic Leukaemia (CLL), where B-cell clonality is central to disease pathology. The present study uses next-generation sequencing to provide detailed insight into aspects of B cell VDJ recombination and properties of the resulting immunoglobulin chains. An overview of IgH regional dynamics in 10 SchS patients, with a particular focus on CDR3 sequences and VDJ gene usage is reported, highlighting the presence of specific B cell expansions. Protein microarray detected a substantial proportion of autoreactive IgM to nuclear target proteins, though a single universal target was not identified. Together, these genetic and functional findings impart new understanding into this rare disorder.


Subject(s)
B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Clonal Evolution/immunology , Disease Susceptibility , Immunoglobulin M/immunology , Schnitzler Syndrome/etiology , Adult , Alleles , Biomarkers , Carrier Proteins/metabolism , Clonal Evolution/genetics , Disease Susceptibility/immunology , Female , Genetic Predisposition to Disease , Genetic Variation , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin M/genetics , Male , Middle Aged , Phenotype , Protein Binding/immunology , Proteome , Proteomics/methods , Schnitzler Syndrome/diagnosis , Schnitzler Syndrome/metabolism , V(D)J Recombination
17.
Arthritis Rheumatol ; 72(5): 769-779, 2020 05.
Article in English | MEDLINE | ID: mdl-31804007

ABSTRACT

OBJECTIVE: Type I interferon (IFN) responses are broadly associated with autoimmune diseases, including systemic lupus erythematosus (SLE). Given the cardinal role of autoantibodies in SLE, this study was undertaken to investigate whether the findings of a B cell-specific IFN assay correlate with SLE activity. METHODS: B cells and peripheral blood mononuclear cells (PBMCs) were stimulated with type I IFN and type II IFN. Gene expression was analyzed, and the expression of pathway-related membrane proteins was determined. A flow cytometry assay for tetherin (CD317), an IFN-induced protein ubiquitously expressed on leukocytes, was validated in vitro and then clinically against SLE diagnosis, plasmablast expansion, and the British Isles Lupus Assessment Group (BILAG) 2004 score in a discovery cohort (n = 156 SLE patients, 30 rheumatoid arthritis [RA] patients, and 25 healthy controls). A second, longitudinal validation cohort of 80 SLE patients was also evaluated for flare prediction. RESULTS: In vitro, a close cell-specific and dose-response relationship between type I IFN-responsive genes and cell surface tetherin was observed in all immune cell subsets. Tetherin expression on multiple cell subsets was selectively responsive to stimulation with type I IFN compared to types II and III IFNs. In patient samples from the discovery cohort, memory B cell tetherin showed the strongest associations with diagnosis (SLE:healthy control effect size 0.11 [P = 0.003]; SLE:RA effect size 0.17 [P < 0.001]), plasmablast numbers in rituximab-treated patients (R = 0.38, P = 0.047), and BILAG 2004. These associations were equivalent to or stronger than those for IFN score or monocyte tetherin. Memory B cell tetherin was found to be predictive of future clinical flares in the validation cohort (hazard ratio 2.29 [95% confidence interval 1.01-4.64]; P = 0.022). CONCLUSION: Our findings indicate that memory B cell surface tetherin, a B cell-specific IFN assay, is associated with SLE diagnosis and disease activity, and predicts flares better than tetherin on other cell subsets or whole blood assays, as determined in an independent validation cohort.


Subject(s)
Arthritis, Rheumatoid/immunology , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , Bone Marrow Stromal Antigen 2/biosynthesis , Interferon Type I/pharmacology , Interferon Type I/physiology , Lupus Erythematosus, Systemic/immunology , Cohort Studies , Flow Cytometry , Humans , Leukocytes, Mononuclear/drug effects , Longitudinal Studies , Predictive Value of Tests , Symptom Flare Up
18.
Arthritis Rheumatol ; 71(12): 2121-2125, 2019 12.
Article in English | MEDLINE | ID: mdl-31268627

ABSTRACT

OBJECTIVE: To assess the prevalence of the MYD88 L265P mutation and variants within NLRP3 and evaluate the status of oligoclonal hematopoiesis in 30 patients with Schnitzler syndrome (SchS). METHODS: Thirty patients with SchS were recruited from 3 clinical centers. Six patients with known acquired cryopyrin-associated periodic syndromes (aCAPS) were included as controls. Allele-specific oligonucleotide-polymerase chain reaction was used for the detection of the MYD88 L265P variant, next-generation sequencing was applied to analyze NLRP3 and 28 genes associated with myelodysplastic syndrome, and gene scanning was performed for the detection of X chromosome inactivation. RESULTS: Activating NLRP3 mutations were not present in 11 SchS patients who had not been sequenced for this gene previously. The MYD88 L265P variant was present in 9 of 30 SchS patients, and somatic mutations associated with clonal hematopoiesis were identified in 1 of 30 patients with SchS and 1 of 6 patients with aCAPS. Evidence of nonrandom X chromosome inactivation was detected in 1 female patient with SchS and 1 female patient with aCAPS. CONCLUSION: A shared molecular mechanism accounting for the pathogenesis of inflammation in SchS remains elusive. Clonal hematopoiesis is not associated with other somatic mutations found in individuals with SchS or aCAPS.


Subject(s)
Hematopoiesis/genetics , Mutation/genetics , Myeloid Differentiation Factor 88/analysis , NLR Family, Pyrin Domain-Containing 3 Protein/analysis , Schnitzler Syndrome/genetics , Cryopyrin-Associated Periodic Syndromes/genetics , High-Throughput Nucleotide Sequencing , Humans , Myeloid Differentiation Factor 88/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Polymerase Chain Reaction , Prevalence
19.
J Immunol ; 202(4): 1287-1300, 2019 02 15.
Article in English | MEDLINE | ID: mdl-30642980

ABSTRACT

Recurrent mutational activation of the MAP kinase pathway in plasma cell myeloma implicates growth factor-like signaling responses in the biology of Ab-secreting cells (ASCs). Physiological ASCs survive in niche microenvironments, but how niche signals are propagated and integrated is poorly understood. In this study, we dissect such a response in human ASCs using an in vitro model. Applying time course expression data and parsimonious gene correlation network analysis (PGCNA), a new approach established by our group, we map expression changes that occur during the maturation of proliferating plasmablast to quiescent plasma cell under survival conditions including the potential niche signal TGF-ß3. This analysis demonstrates a convergent pattern of differentiation, linking unfolded protein response/endoplasmic reticulum stress to secretory optimization, coordinated with cell cycle exit. TGF-ß3 supports ASC survival while having a limited effect on gene expression including upregulation of CXCR4. This is associated with a significant shift in response to SDF1 in ASCs with amplified ERK1/2 activation, growth factor-like immediate early gene regulation and EGR1 protein expression. Similarly, ASCs responding to survival conditions initially induce partially overlapping sets of immediate early genes without sustaining the response. Thus, in human ASCs growth factor-like gene regulation is transiently imposed by niche signals but is not sustained during subsequent survival and maturation.


Subject(s)
Antibody-Producing Cells/immunology , Chemokine CXCL12/immunology , Transforming Growth Factor beta3/immunology , Cell Survival , Cells, Cultured , Chemokine CXCL12/genetics , Healthy Volunteers , Humans , Transforming Growth Factor beta3/genetics
20.
Sci Rep ; 8(1): 14338, 2018 09 25.
Article in English | MEDLINE | ID: mdl-30254311

ABSTRACT

The unfolded protein response (UPR) and activation of XBP1 is necessary for high secretory efficiency and functional differentiation of antibody secreting cells (ASCs). The UPR additionally includes a branch in which membrane-bound transcription factors, exemplified by ATF6, undergo intramembrane-proteolysis by the sequential action of site-1 (MBTPS1/S1P) and site-2 proteases (MBTPS2/S2P) and release of the cytoplasmic domain as an active transcription factor. Such regulation is shared with a family of CREB3-related transcription factors and sterol regulatory element-binding proteins (SREBPs). Of these, we identify that the CREB3 family member CREB3L2 is strongly induced and activated during the transition from B-cell to plasma cell state. Inhibition of site-1 protease leads to a profound reduction in plasmablast number linked to induction of autophagy. Plasmablasts generated in the presence of site-1 protease inhibitor segregated into CD38high and CD38low populations, the latter characterized by a marked reduction in the capacity to secrete IgG. Site-1 protease inhibition is accompanied by a distinctive change in gene expression associated with amino acid, steroid and fatty acid synthesis pathways. These results demonstrate that transcriptional control of metabolic programs necessary for secretory activity can be targeted via site-1 protease inhibition during ASC differentiation.


Subject(s)
Antibody-Producing Cells/cytology , Proprotein Convertases/metabolism , Serine Endopeptidases/metabolism , Basic-Leucine Zipper Transcription Factors/metabolism , Cell Differentiation , Humans , Plasma Cells/cytology
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