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1.
PLoS Biol ; 18(9): e3000821, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886672

RESUMO

As a novel alternative to established surface display or combinatorial chemistry approaches for the discovery of therapeutic peptides, we present a method for the isolation of small, cysteine-rich domains from bovine antibody ultralong complementarity-determining regions (CDRs). We show for the first time that isolated bovine antibody knob domains can function as autonomous entities by binding antigen outside the confines of the antibody scaffold. This yields antibody fragments so small as to be considered peptides, each stabilised by an intricate, bespoke arrangement of disulphide bonds. For drug discovery, cow immunisations harness the immune system to generate knob domains with affinities in the picomolar to low nanomolar range, orders of magnitude higher than unoptimized peptides from naïve library screening. Using this approach, knob domain peptides that tightly bound Complement component C5 were obtained, at scale, using conventional antibody discovery and peptide purification techniques.


Assuntos
Anticorpos/química , Dissulfetos/isolamento & purificação , Domínios de Imunoglobulina , Fragmentos de Peptídeos/isolamento & purificação , Domínios e Motivos de Interação entre Proteínas , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Afinidade de Anticorpos , Formação de Anticorpos , Especificidade de Anticorpos , Antígenos/genética , Antígenos/imunologia , Linfócitos B/fisiologia , Bovinos , Complemento C5/química , Complemento C5/genética , Complemento C5/imunologia , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Dissulfetos/química , Dissulfetos/imunologia , Mapeamento de Epitopos/métodos , Humanos , Imunização , Domínios de Imunoglobulina/genética , Modelos Moleculares , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Domínios e Motivos de Interação entre Proteínas/genética
2.
Mol Immunol ; 126: 46-55, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32758676

RESUMO

Immunological memory is a critical characteristic of a successful long-term adaptive immune response. During the initial phases of antigen:B lymphocyte interactions, B cells participate in the germinal center reaction, in which T-B cell interactions take place. CD154 on T cells acts as a ligand that binds to the CD40 receptor on B cells and facilitates the differentiation of B cells to memory B cells. However, cell fate determinants controlled by CD40 signal for cellular differentiation are unclear. In this study, we explored miRNA and miRNA-targets as cell fate determinants in CD40 signaled B cells. We selected candidate miRNAs based on their involvement in the regulation of B cell development, activation, and differentiation. We found that CD40 signal reduced transcript levels of miR150-5p, 17-5p, 146a-5p, 26a-5p and increased levels of miR292a-5p. Gene set enrichment analyses of previously submitted microarray data revealed accordant changes in levels of gene targets of these miRNA. Gene ontology analysis of miRNA-targets showed enrichment of genes participating in pathways such as DNA damage response, RNA/protein metabolism, and cell cycle regulation. Subsequently, studies on candidate miRNA-targets showed a CD40 signal driven differential regulation of Ccnd2, Pten, Traf6, c-Myb, and Btla. Further, 'gain of function' studies using mimics of the downregulated miRNAs, confirmed a predicted reduction in miRNA responsive targets; such as reduction of Ccnd2 levels in mimic treated groups of miR146a-5p, 26a-5p, and 17-5p. In conclusion, our study reveals that CD40 signal modulates levels of selected miRNAs as well as their cognate targets, whose enriched participation in diverse processes may help delineate downstream cell fate decisions.


Assuntos
Linfócitos B/fisiologia , Antígenos CD40/metabolismo , Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , MicroRNAs/metabolismo , Transdução de Sinais/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Biologia Computacional , Conjuntos de Dados como Assunto , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , MicroRNAs/agonistas , Análise de Sequência com Séries de Oligonucleotídeos , Cultura Primária de Células , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
4.
Proc Natl Acad Sci U S A ; 117(33): 20100-20108, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32727902

RESUMO

Mutation of HELLS (Helicase, Lymphoid-Specific)/Lsh in human DNA causes a severe immunodeficiency syndrome, but the nature of the defect remains unknown. We assessed here the role of Lsh in hematopoiesis using conditional Lsh knockout mice with expression of Mx1 or Vav Cre-recombinase. Bone marrow transplantation studies revealed that Lsh depletion in hematopoietic stem cells severely reduced B cell numbers and impaired B cell development in a hematopoietic cell-autonomous manner. Lsh-deficient mice without bone marrow transplantation exhibited lower Ig levels in vivo compared to controls despite normal peripheral B cell numbers. Purified B lymphocytes proliferated normally but produced less immunoglobulins in response to in vitro stimulation, indicating a reduced capacity to undergo class switch recombination (CSR). Analysis of germline transcripts, examination of double-stranded breaks using biotin-labeling DNA break assay, and End-seq analysis indicated that the initiation of the recombination process was unscathed. In contrast, digestion-circularization PCR analysis and high-throughput sequencing analyses of CSR junctions and a chromosomal break repair assay indicated an impaired ability of the canonical end-joining pathway in Lsh-deficient B cells. Our data suggest a hematopoietic cell-intrinsic role of Lsh in B cell development and in CSR providing a potential target for immunodeficiency therapy.


Assuntos
Linfócitos B/fisiologia , DNA Helicases/metabolismo , Imunoglobulinas/metabolismo , Animais , Linhagem Celular , DNA Helicases/genética , Inativação Gênica , Humanos , Imunoglobulinas/genética , Camundongos , Camundongos Knockout , Mutação
5.
N Engl J Med ; 383(3): 218-228, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32668112

RESUMO

BACKGROUND: Rheumatoid arthritis, like many inflammatory diseases, is characterized by episodes of quiescence and exacerbation (flares). The molecular events leading to flares are unknown. METHODS: We established a clinical and technical protocol for repeated home collection of blood in patients with rheumatoid arthritis to allow for longitudinal RNA sequencing (RNA-seq). Specimens were obtained from 364 time points during eight flares over a period of 4 years in our index patient, as well as from 235 time points during flares in three additional patients. We identified transcripts that were differentially expressed before flares and compared these with data from synovial single-cell RNA-seq. Flow cytometry and sorted-blood-cell RNA-seq in additional patients were used to validate the findings. RESULTS: Consistent changes were observed in blood transcriptional profiles 1 to 2 weeks before a rheumatoid arthritis flare. B-cell activation was followed by expansion of circulating CD45-CD31-PDPN+ preinflammatory mesenchymal, or PRIME, cells in the blood from patients with rheumatoid arthritis; these cells shared features of inflammatory synovial fibroblasts. Levels of circulating PRIME cells decreased during flares in all 4 patients, and flow cytometry and sorted-cell RNA-seq confirmed the presence of PRIME cells in 19 additional patients with rheumatoid arthritis. CONCLUSIONS: Longitudinal genomic analysis of rheumatoid arthritis flares revealed PRIME cells in the blood during the period before a flare and suggested a model in which these cells become activated by B cells in the weeks before a flare and subsequently migrate out of the blood into the synovium. (Funded by the National Institutes of Health and others.).


Assuntos
Artrite Reumatoide/sangue , Linfócitos B/fisiologia , Expressão Gênica , Células-Tronco Mesenquimais , Análise de Sequência de RNA/métodos , Adulto , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Feminino , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Gravidade do Paciente , Inquéritos e Questionários , Exacerbação dos Sintomas , Líquido Sinovial/citologia
6.
J Infect Dis ; 222(3): 367-371, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32474608

RESUMO

The viral RNA shedding time (VST) for severe acute respiratory syndrome coronavirus 2 has not been well characterized. Clinical data were collected and compared between patients with short and long VSTs (in the lower and upper quartiles, respectively). The probability of recurrent positive reverse-transcription polymerase chain reaction results decreased sharply to 4.8% after 3 consecutive negative results. A series of ≥3 consecutive negative results was suitable as a criterion for the end of viral RNA shedding. The VST for shedding from the respiratory tract was significantly shorter in patients with normal B-cell counts on admission than in those with decreased B-cell counts (median [interquartile range], 11 [9-13] vs 16 [12-20] days, respectively; P = .001).


Assuntos
Linfócitos B/fisiologia , Betacoronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Sistema Respiratório/virologia , Eliminação de Partículas Virais , Betacoronavirus/imunologia , Estudos de Casos e Controles , China , Citocinas/metabolismo , Feminino , Humanos , Modelos Logísticos , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Pandemias , Modelos de Riscos Proporcionais , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Fatores de Risco , Fatores de Tempo
7.
Nat Genet ; 52(7): 655-661, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32514124

RESUMO

Three-dimensional organization of the genome is important for transcriptional regulation1-7. In mammals, CTCF and the cohesin complex create submegabase structures with elevated internal chromatin contact frequencies, called topologically associating domains (TADs)8-12. Although TADs can contribute to transcriptional regulation, ablation of TAD organization by disrupting CTCF or the cohesin complex causes modest gene expression changes13-16. In contrast, CTCF is required for cell cycle regulation17, embryonic development and formation of various adult cell types18. To uncouple the role of CTCF in cell-state transitions and cell proliferation, we studied the effect of CTCF depletion during the conversion of human leukemic B cells into macrophages with minimal cell division. CTCF depletion disrupts TAD organization but not cell transdifferentiation. In contrast, CTCF depletion in induced macrophages impairs the full-blown upregulation of inflammatory genes after exposure to endotoxin. Our results demonstrate that CTCF-dependent genome topology is not strictly required for a functional cell-fate conversion but facilitates a rapid and efficient response to an external stimulus.


Assuntos
Linfócitos B/fisiologia , Fator de Ligação a CCCTC/fisiologia , Macrófagos/fisiologia , Mielopoese/fisiologia , Antígenos de Diferenciação/metabolismo , Fator de Ligação a CCCTC/genética , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Cromatina/fisiologia , Regulação da Expressão Gênica , Humanos , Conformação Molecular , Mielopoese/genética , Conformação Proteica
8.
Mol Cell ; 78(3): 434-444.e5, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32294471

RESUMO

Gene expression is regulated by the rates of synthesis and degradation of mRNAs, but how these processes are coordinated is poorly understood. Here, we show that reduced transcription dynamics of specific genes leads to enhanced m6A deposition, preferential activity of the CCR4-Not complex, shortened poly(A) tails, and reduced stability of the respective mRNAs. These effects are also exerted by internal ribosome entry site (IRES) elements, which we found to be transcriptional pause sites. However, when transcription dynamics, and subsequently poly(A) tails, are globally altered, cells buffer mRNA levels by adjusting the expression of mRNA degradation machinery. Stress-provoked global impediment of transcription elongation leads to a dramatic inhibition of the mRNA degradation machinery and massive mRNA stabilization. Accordingly, globally enhanced transcription, such as following B cell activation or glucose stimulation, has the opposite effects. This study uncovers two molecular pathways that maintain balanced gene expression in mammalian cells by linking transcription to mRNA stability.


Assuntos
Poli A/genética , RNA Mensageiro/metabolismo , Transcrição Genética , Adenosina/análogos & derivados , Animais , Linfócitos B/fisiologia , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Humanos , Sítios Internos de Entrada Ribossomal , Células MCF-7 , Camundongos Endogâmicos C57BL , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Poli A/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , Receptores CCR4/genética , Receptores CCR4/metabolismo
9.
Phytomedicine ; 69: 153194, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32146299

RESUMO

BACKGROUND: The frequency of allergic diseases is constantly rising. Dysregulated production of isotype E immunoglobulins is one of the key factors behind allergic reactions and its modulation is therefore an important target for pharmacological intervention. Natural products of the pseurotin family were reported to be inhibitors of IgE production in B-cells. Mechanistic details underlying these effects are however not well understood. PURPOSE: In the present study, we synthesized new analogs of natural pseurotins and extensively investigated their inhibitory effects on activation, proliferation and differentiation of B-cells, as well as on the production of IgE. STUDY DESIGN: Effects of two natural pseurotins (pseurotins A and D) and a collection of fully synthetic pseurotin analogs were studied on mouse B-cells stimulated by the combination of IL-4 and E. coli lipopolysaccharide. The IgE production was determined along with cell viability and cell proliferation. The phosphorylation of selected members of the STAT transcription factor family was subsequently investigated. Finally, the in vivo effect of pseurotin D on the ovalbumin-induced delayed type hypersensitivity response was tested in mice. RESULTS: We discovered that several fully synthetic pseurotin analogs were able to decrease the production of IgE in stimulated B-cells with potency comparable to that of pseurotins A and D. We found that the two natural pseurotins and the active synthetic analogs inhibited the phosphorylation of STAT3, STAT5 and STAT6 proteins in stimulated B-cells, resulting in the inhibition of B-cell proliferation and differentiation into the plasma cells. In vivo, pseurotin D decreased ovalbumin-induced foot pad edema. CONCLUSION: Our results advance the current mechanistic understanding of the pseurotin-induced inhibition of IgE production in B-cells by linking the effect to STAT signaling, and associated modulation of B-cell proliferation and differentiation. Together with our finding that structurally simpler pseurotin analogs were able to reproduce the effects of natural pseurotins, the presented work has implications for the future research on these secondary metabolites in the context of allergic diseases.


Assuntos
Linfócitos B/efeitos dos fármacos , Imunoglobulina E/metabolismo , Plasmócitos/citologia , Pirrolidinonas/química , Pirrolidinonas/farmacologia , Animais , Linfócitos B/citologia , Linfócitos B/fisiologia , Diferenciação Celular/efeitos dos fármacos , Edema/induzido quimicamente , Edema/tratamento farmacológico , Escherichia coli/química , Imunoglobulina E/sangue , Imunoglobulina M/sangue , Imunoglobulina M/metabolismo , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Ovalbumina/toxicidade , Fosforilação/efeitos dos fármacos , Plasmócitos/fisiologia , Fatores de Transcrição STAT/metabolismo
10.
PLoS One ; 15(3): e0229170, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32210425

RESUMO

Surface protein CD20 serves as the critical target of immunotherapy in various B-cell malignancies for decades, however its biological function and regulation remain largely elusive. Better understanding of CD20 function may help to design improved rational therapies to prevent development of resistance. Using CRISPR/Cas9 technique, we have abrogated CD20 expression in five different malignant B-cell lines. We show that CD20 deletion has no effect upon B-cell receptor signaling or calcium flux. Also B-cell survival and proliferation is unaffected in the absence of CD20. On the contrary, we found a strong defect in actin cytoskeleton polymerization and, consequently, defective cell adhesion and migration in response to homeostatic chemokines SDF1α, CCL19 and CCL21. Mechanistically, we could identify a reduction in chemokine-triggered PYK2 activation, a calcium-activated signaling protein involved in activation of MAP kinases and cytoskeleton regulation. These cellular defects in consequence result in a severely disturbed homing of B cells in vivo.


Assuntos
Actinas/metabolismo , Antígenos CD20/fisiologia , Linfócitos B/fisiologia , Leucemia de Células B/patologia , Linfoma de Células B/patologia , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Antígenos CD20/genética , Antígenos CD20/metabolismo , Linfócitos B/patologia , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Leucemia de Células B/metabolismo , Linfoma de Células B/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Polimerização , Multimerização Proteica/fisiologia , Transdução de Sinais/imunologia
11.
Proc Natl Acad Sci U S A ; 117(10): 5453-5462, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32098847

RESUMO

Developing lymphocytes diversify their antigen receptor (AgR) loci by variable (diversity) joining (V[D]J) recombination. Here, using the micrococcal nuclease (MNase)-based chromatin accessibility (MACC) assay with low-cell count input, we profile both small-scale (kilobase) and large-scale (megabase) changes in chromatin accessibility and nucleosome occupancy in primary cells during lymphoid development, tracking the changes as different AgR loci become primed for recombination. The three distinct chromatin structures identified in this work define unique features of immunoglobulin H (IgH), Igκ, and T cell receptor-α (TCRα) loci during B lymphopoiesis. In particular, we find locus-specific temporal changes in accessibility both across megabase-long AgR loci and locally at the recombination signal sequences (RSSs). These changes seem to be regulated independently and can occur prior to lineage commitment. Large-scale changes in chromatin accessibility occur without significant change in nucleosome density and represent key features of AgR loci not previously described. We further identify local dynamic repositioning of individual RSS-associated nucleosomes at IgH and Igκ loci while they become primed for recombination during B cell commitment. These changes in chromatin at AgR loci are regulated in a locus-, lineage-, and stage-specific manner during B lymphopoiesis, serving either to facilitate or to impose a barrier to V(D)J recombination. We suggest that local and global changes in chromatin openness in concert with nucleosome occupancy and placement of histone modifications facilitate the temporal order of AgR recombination. Our data have implications for the organizing principles that govern assembly of these large loci as well as for mechanisms that might contribute to aberrant V(D)J recombination and the development of lymphoid tumors.


Assuntos
Linfócitos B/fisiologia , Cromatina/metabolismo , Rearranjo Gênico do Linfócito B , Linfopoese/genética , Receptores de Antígenos/genética , Recombinação V(D)J , Animais , Cromatina/química , Loci Gênicos , Testes Genéticos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias kappa de Imunoglobulina/genética , Linfoma/genética , Camundongos , Camundongos Endogâmicos C57BL , Nuclease do Micrococo , Nucleossomos , Receptores de Antígenos de Linfócitos T alfa-beta/genética
12.
J Immunol ; 204(6): 1535-1542, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-32005754

RESUMO

Mature naive B cells expressing BCRs of the IgM and IgD isotypes respond to Ag in secondary lymphoid organs. However, the vast majority of B cells do not undergo productive Ag encounter and have finite life spans dependent on survival signals propagated by the BCR and the BAFFR. In this study, we show that the E3 ubiquitin ligase Fbw7 is required for the maintenance of mature B cell populations in mice. BCR stimulation of B cells induced substantial apoptosis along with proliferative and growth defects upon the loss of Fbw7. Analysis of B cell proteomes revealed aberrant signaling patterns, including lower Bcl2 and diminished NF-κB signaling. Further, excessive accumulation of Fbw7 substrate c-Myc, increased Bim expression, and loss of PI3K signaling mediated apoptosis downstream of BCR signaling. In accordance, strong prosurvival signals delivered through ectopic expression of BCL2 in B cells could largely rescue apoptotic cells in the absence of Fbw7. Overall, this study reveals an unexpected role for Fbw7 in the survival and fitness of mature B cells.


Assuntos
Linfócitos B/fisiologia , Proteína 7 com Repetições F-Box-WD/metabolismo , Animais , Apoptose/imunologia , Proteína 11 Semelhante a Bcl-2/metabolismo , Proliferação de Células/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Proteína 7 com Repetições F-Box-WD/genética , Feminino , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Ubiquitinação/genética , Ubiquitinação/imunologia
13.
Life Sci ; 245: 117390, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-32007574

RESUMO

AIMS: This study aimed to profile circulating T follicular helper cells (cTfh) and their effect on B cells in rheumatic heart disease (RHD). MATERIALS AND METHODS: Participants were divided into healthy control (HC, n = 30) and RHD (n = 30) groups. Percentages of cTfh subpopulations, based on CD4, CXCR5, CXCR3, CCR6, Foxp3, Ki67, and PD-1 cell markers, and of CD19+ B cell subgroups were measured by flow cytometry and compared between the groups. Also, IL-21 concentration in plasma and mitral valve were quantitated by cytometric bead array, immunofluorescence, and western blotting. KEY FINDINGS: The PD-1+ cTfh, B cells (naive B cells, plasmablasts, and plasma B cells) proportion and cTfh17/cTfh ratios in RHD group were significantly increased, compared to HC (p < 0.01 in all cases), while different types of memory B cells were diminished (p < 0.001). In RHD patients, percentages of PD-1+ cTfh and switched memory B cells were negatively correlated (r = -0.565, p = 0.009); meanwhile, percentages of plasmablasts and PD-1+ cTfh cells were positively correlated (r = 0.594, p = 0.005). Additionally, IL-21 levels in plasma and mitral valve of RHD group were higher than those in HC. Also, IL-21 levels correlated with PD-1+ cTfh(r = 0.557, p = 0.010), cTfh17 (r = 0.567, p = 0.009), and plasmablast (r = -0.5957, p = 0.005) cell proportions, and (cTh2 + cTh17)/cTfh1 ratio (r = -0.547, p = 0.013). SIGNIFICANCE: The activation of PD-1+ cTfh and cTfh17 subtype was highly correlated with plasmablast maturation and IL-21 production in rheumatic heart disease. Thus indicating the prominent role of cTfh and humoral reactivity in the immune pathogenesis of RHD.


Assuntos
Imunidade Humoral , Cardiopatia Reumática/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos B/fisiologia , Western Blotting , Estudos de Casos e Controles , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Masculino , Pessoa de Meia-Idade , Cardiopatia Reumática/etiologia , Linfócitos T Auxiliares-Indutores/fisiologia
14.
Nat Commun ; 11(1): 723, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-32024827

RESUMO

How activated B cells build biosynthetic pathways and organelle structures necessary for subsequent robust antibody secretion is still unclear. The dominant model holds that nascent plasma cells adapt to increased antibody synthesis by activating the unfolded protein response (UPR) under the control of the transcription factor Xbp1. Here, by analyzing gene expression in activated B cells with or without plasma cell-inductive signals, we find that follicular B cells up-regulate a wide array of UPR-affiliated genes before initiating antibody secretion; furthermore, initial transcription of these loci requires the mTORC1 kinase adaptor, Raptor, but not Xbp1. Transcriptomic analyses of resting marginal zone B cells, which generate plasma cells with exceptionally rapid kinetics, reinforce these results by revealing the basal expression of UPR-affiliated mRNA networks without detectable Xbp1 activity. We thus conclude that B cells utilize mTORC1 to prepare for subsequent plasma cell function, before the onset of antibody synthesis.


Assuntos
Anticorpos/metabolismo , Linfócitos B/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Diferenciação Celular , Regulação da Expressão Gênica , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Proteína Regulatória Associada a mTOR/genética , Proteína Regulatória Associada a mTOR/metabolismo , Baço/citologia , Resposta a Proteínas não Dobradas/genética , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo
15.
Mol Immunol ; 119: 69-82, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31986311

RESUMO

SRSF1, a member of the SR protein family, is an important splicing factor and regulator of splicing. Multiple splicing isoforms have been reported for this gene. SRSF1-3, a splicing isoform of SRSF1, is necessary for AID-dependent SHM of IgV genes. However, its precise role in SHM remains enigmatic. Transcriptomic analysis of SRSF1-3 reconstituted cells shows upregulation of transcription factor SATB2 and chromatin regulator UBN1. The increased SATB2 and UBN1 are strikingly enriched in the MAR and promoter regions of the IgL gene, respectively. Furthermore, UBN1 enrichment at the promoter region was coupled with a hundred-fold enhanced occupancy of the histone variant H3.3 at the IgL promoter, that is a hallmark of efficient SHM. The enhanced occupancy of SATB2 at the MAR, UBN1 and histone variant H3.3 at the IgL promoter leads to an increase in IgL transcription, revealing a role of SRSF1-3 in SHM. Thus, SRSF1-3 is likely involved in the regulation of SHM, via upregulation of a crucial transcription factor SATB2, as well as, by overexpression of a chromatin modulator of Ig genes, UBN1, which further assists in the recruitment of the histone variant H3.3. Furthermore, the splicing isoform SRSF1-3 regulates alternate splicing pattern of splicing isoforms for various crucial genes. The present study provides the first evidence that a splicing isoform of an SR protein can regulate the post-transcriptional processing of RNA in vivo.


Assuntos
Regulação da Expressão Gênica , Genes de Imunoglobulinas , Histonas/fisiologia , Região Variável de Imunoglobulina/genética , Processamento de RNA/fisiologia , Fatores de Processamento de Serina-Arginina/fisiologia , Fatores de Transcrição/fisiologia , Processamento Alternativo , Animais , Linfócitos B/fisiologia , Linhagem Celular , Galinhas , Ativação Transcricional
16.
Mol Cell ; 77(2): 384-394.e4, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31806351

RESUMO

HMCES (5hmC binding, embryonic stem cell-specific-protein), originally identified as a protein capable of binding 5-hydroxymethylcytosine (5hmC), an epigenetic modification generated by TET proteins, was previously reported to covalently crosslink to DNA at abasic sites via a conserved cysteine. We show here that Hmces-deficient mice display normal hematopoiesis without global alterations in 5hmC. HMCES specifically enables DNA double-strand break repair through the microhomology-mediated alternative-end-joining (Alt-EJ) pathway during class switch recombination (CSR) in B cells, and HMCES deficiency leads to a significant defect in CSR. HMCES mediates Alt-EJ through its SOS-response-associated-peptidase domain (SRAPd), a function that requires DNA binding but is independent of its autopeptidase and DNA-crosslinking activities. We show that HMCES is recruited to switch regions of the immunoglobulin locus and provide a potential structural basis for the interaction of HMCES with long DNA overhangs generated by Alt-EJ during CSR. Our studies provide further evidence for a specialized role for HMCES in DNA repair.


Assuntos
Linfócitos B/fisiologia , Reparo do DNA por Junção de Extremidades/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Switching de Imunoglobulina/genética , Animais , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Camundongos , Camundongos Endogâmicos C57BL , Translocação Genética/genética
17.
J Neuroimmunol ; 338: 577106, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31715458

RESUMO

Recent studies identified that interferon beta (IFN-ß) treatment skews B-cells towards a regulatory phenotype in multiple sclerosis. To assess B cell involvement during IFN-ß therapy, we compared IFN-ß treatment in a B cell-independent model and a B cell-dependent model of experimental autoimmune encephalomyelitis (EAE). We show that in B cell-independent EAE, IFN-ß ameliorates neuroinflammation. Conversely, in B cell-dependent EAE, IFN-ß has no effect on disease. Effective IFN-ß therapy in B cell-independent EAE was associated with reduced inflammatory T cells in the CNS and skewed splenic B cells towards an immature population and away from a germinal center population. These immune cell populations were unchanged in B cell-dependent EAE. Finally, we found that IFN-ß increased marginal zone B cells in both EAE models. These findings indicate that B cell function impacts IFN-ß efficacy during neuroinflammation.


Assuntos
Linfócitos B/fisiologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Interferon beta/uso terapêutico , Animais , Linfócitos B/efeitos dos fármacos , Encefalomielite Autoimune Experimental/imunologia , Feminino , Interferon beta/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/imunologia
18.
J Exp Med ; 217(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31873727

RESUMO

Germinal centers (GCs) are sites at which B cells proliferate and mutate their antibody-encoding genes in the dark zone (DZ), followed by affinity-based selection in the light zone (LZ). B cell antigen receptor (BCR) signals induce Syk activation followed by rapid phosphatase-mediated desensitization; however, how degradation events regulate BCR functions in GCs is unclear. Here, we found that Syk degradation restrains plasma cell (PC) formation in GCs and promotes B cell LZ to DZ transition. Using a mouse model defective in Cbl-mediated Syk degradation, we demonstrate that this machinery attenuates BCR signaling intensity by mitigating the Kras/Erk and PI3K/Foxo1 pathways, and restricting the expression of PC transcription factors in GC B cells. Inhibition of Syk degradation perturbed gene expression, specifically in the LZ, and enhanced the generation of PCs without affecting B cell proliferation. These findings reveal how long-lasting attenuation of signal transduction by degradation events regulates cell fate within specialized microanatomical sites.


Assuntos
Centro Germinativo/metabolismo , Plasmócitos/metabolismo , Quinase Syk/metabolismo , Animais , Linfócitos B/metabolismo , Linfócitos B/fisiologia , Proliferação de Células/fisiologia , Expressão Gênica/fisiologia , Centro Germinativo/fisiologia , Ativação Linfocitária/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Plasmócitos/fisiologia , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/fisiologia
19.
Immunohorizons ; 3(10): 447-462, 2019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31591252

RESUMO

The FOXP1 transcription factor is expressed throughout B cell development until its extinction just prior to terminal differentiation. Foxp1 nulls die of cardiac defects at midgestation, but adult rescue via fetal liver transfer led to a strong pre-B cell block. To circumvent these limitations and to investigate FOXP1 function at later stages of B cell differentiation, we generated and analyzed floxed (F) Foxp1 alleles deleted at pro-B, transitional (T) 1, and mature B cell stages. Mb-1cre-mediated deletion of Foxp1F/F confirmed its requirement for pro-B to pre-B transition. Cd21- and Cd19cre deletion led to significant reduction of germinal center formation and a second block in differentiation at the T2/marginal zone precursor stage. T-dependent and -independent immunization of FOXP1 mutants led to reduction of Ag-specific IgM, whereas responses of class-switched Abs were unimpaired. Yet, unexpectedly, plasmablast and plasma cell numbers were significantly increased by in vitro BCR stimulation of Foxp1F/F splenic follicular B cells but rapidly lost, as they were highly prone to apoptosis. RNA sequencing, gene set enrichment analysis, and chromatin immunoprecipitation sequencing analyses revealed strong enrichment for signatures related to downregulation of immune responses, apoptosis, and germinal center biology, including direct activation of Bcl6 and downregulation of Aicda/AID, the primary effector of somatic hypermutation, and class-switch recombination. These observations support a role for FOXP1 as a direct transcriptional regulator at key steps underlying B cell development in the mouse.


Assuntos
Linfócitos B/fisiologia , Diferenciação Celular , Fatores de Transcrição Forkhead/fisiologia , Proteínas Repressoras/fisiologia , Animais , Fatores de Transcrição Forkhead/genética , Camundongos Knockout , Proteínas Repressoras/genética
20.
J Immunotoxicol ; 16(1): 173-181, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31589084

RESUMO

Endosulfan is a DDT-era organochlorine pesticide. Due to past and current environmental contamination, investigation of endosulfan exposure is of current importance. Acute high dose exposure precipitates neural/endocrine system damage, but the effects on the immune system and of lower doses are not well-characterized. Two relatively low concentrations of endosulfan (i.e. 0.1 and 17 µM ENDO) were investigated in an in vitro study using human peripheral blood mononuclear cells (PBMC) to understand effects of relatively low doses (0.1-25.0 µM [≈0.04-10 ppm/40-10,000 ppb]) of ENDO upon normal human T- and B-lymphocytes and NK cells. The study here found that 17 µM ENDO inhibited phytohemagglutinin-M (PHA)-induced human PBMC proliferation. It was also seen that senescence and apoptosis among non-stimulated cells was increased, specifically within CD8 and NK populations, and that CD4:CD8 ratios also were increased. Treatment of non-stimulated PBMC with ENDO led to overall increases in production of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-2, -4, and -6, and decreased production of anti-inflammatory IL-10, suggesting an immunosenescence secretory phenotype. Interestingly, when the cells were pre-stimulated with mitogen (PHA), ENDO became inhibitory against the mitogen-induced proliferation and cytokine formation - with the exception of that of TNFα and IL-6, suggesting differential effects of ENDO on activated cells. Thus, at the organismal level, ENDO might also display differential effects during states of autoimmune disease or chronic viral infection in the exposed host.


Assuntos
Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Endossulfano/toxicidade , Inseticidas/toxicidade , Linfócitos T Citotóxicos/efeitos dos fármacos , Adulto , Linfócitos B/efeitos dos fármacos , Linfócitos B/fisiologia , Células Cultivadas , Senescência Celular/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Endossulfano/administração & dosagem , Feminino , Voluntários Saudáveis , Humanos , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Inseticidas/administração & dosagem , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/fisiologia , Masculino , Cultura Primária de Células , Linfócitos T Citotóxicos/fisiologia , Adulto Jovem
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