RESUMO
Autoimmune hepatitis (AIH) is a chronic, inflammatory liver disease of unknown aetiology which requires lifelong immunosuppression. Most therapeutic and outcome studies of AIH have been conducted predominantly in Caucasian (European Ancestry, EA) cohorts, with the exclusion of African American (AA) patients due to inadequate sample size. It is known that AA patients have a severe phenotype of autoimmune diseases and demonstrate a poor response to conventional medical therapy. Understanding cellular and molecular pathways which determine AIH severity and progression in AA patients is likely to lead to the discovery of novel, personalised and better tolerated therapies. The aim of the study is to determine the distinct effector B cell phenotypes which contribute to disease severity and progression of AIH in AA children as compared to their EA cohorts. PBMCs were isolated from blood samples collected from patients visiting Children's Healthcare of Atlanta (CHOA) and were grouped into AA, (n = 12), EA, (n = 11) and controls (n = 12) and were processed for flow cytometry. Markers of B cell development, maturation and activation were assessed namely CD19, CD21, IgD, CD27, CD38, CD11c, CD24, CD138. AA children with AIH demonstrated an expansion of CD19 + ve, Activated Naïve (aN), (CD19+ IgD-/CD27- Double Negative (DN2) ([CD19+/IgD-/CD27++CD38++) cells. Plasmablasts were significantly higher along with Signalling Lymphocytic activation molecule F7 (SLAMF7). Unswitched memory [CD19+] IgD+CD27+ (USM) B cells were significantly contracted in AA patients with AIH. B cell phenotyping reveals a distinct profile in AA AIH patients with a major skewing towards the expansion of effector pathways which have been previously characterised in severe SLE in AA patients. These results suggest that the quantification and therapeutic target of B cell pathway could contribute substantially to the clinical approach to AIH especially in the AA population.
Assuntos
Linfócitos B , Hepatite Autoimune , Imunoglobulina D , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral , Humanos , Hepatite Autoimune/imunologia , Hepatite Autoimune/sangue , Hepatite Autoimune/patologia , Hepatite Autoimune/diagnóstico , Imunoglobulina D/imunologia , Imunoglobulina D/metabolismo , Criança , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Masculino , Feminino , Adolescente , Linfócitos B/imunologia , Linfócitos B/metabolismo , Pré-Escolar , Imunofenotipagem , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Idade de Início , BiomarcadoresRESUMO
Human B cells can be divided into four main subsets based on differential expression of immunoglobulin (Ig)D and CD27. IgD-CD27- double negative (DN) B cells make up a heterogeneous group of B cells that have first been described in relation to aging and systemic lupus erythematosus but have been mostly disregarded in B cell research. Over the last few years, DN B cells have gained a lot of interest because of their involvement in autoimmune and infectious diseases. DN B cells can be divided into different subsets that originate via different developmental processes and have different functional properties. Further research into the origin and function of different DN subsets is needed to better understand the role of these B cells in normal immune responses and how they could be targeted in specific pathologies. In this review, we give an overview of both phenotypic and functional properties of DN B cells and provide insight into the currently proposed origins of DN B cells. Moreover, their involvement in normal aging and different pathologies is discussed.
Assuntos
Subpopulações de Linfócitos B , Lúpus Eritematoso Sistêmico , Humanos , Linfócitos B , Envelhecimento , Memória Imunológica , Imunoglobulina D/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
B cell maturation and immunoglobulin (Ig) repertoire selection are governed by expression of a functional B cell receptor (BCR). Naïve B cells co-express their BCR as IgM and IgD isotype. However, the role of the additionally expressed IgD on naïve B cells is not known. Here we assessed the impact of IgD on naïve B cell maturation and Ig repertoire selection in 8 individuals from 3 different families with heterozygous loss-of-function or loss-of expression mutations in IGHD. Although naïve B cells from these individuals expressed IgM on their surface, the IGHD variant in heterozygous state entailed a chimeric situation by allelic exclusion with almost half of the naïve B cell population lacking surface IgD expression. Flow cytometric analyses revealed a distinct phenotype of IgD-negative naïve B cells with decreased expression of CD19, CD20 and CD21 as well as lower BAFF-R and integrin-ß7 expression. IgD-negative B cells were less responsive in vitro after engaging the IgM-BCR, TLR7/9 or CD40 pathway. Additionally, a selective disadvantage of IgD-negative B cells within the T2 transitional and mature naïve B cell compartment as well as reduced frequencies of IgMlo/- B cells within the mature naïve B cell compartment lacking IgD were evident. RNA-Ig-seq of bulk sorted B cell populations showed an altered selection of distinct VH segments in the IgD-negative mature naïve B cell population. We conclude that IgD expression on human naïve B cells is redundant for generation of naïve B cells in general, but further shapes the naive B cell compartment starting from T2 transitional B cells. Our observations suggest an unexpected role of IgD expression to be critical for selection of distinct Ig VH segments into the pre-immune Ig repertoire and for the survival of IgMlo/- naïve B cells known to be enriched in poly-/autoreactive B cell clones.
Assuntos
Linfócitos B , Imunoglobulina D , Humanos , Imunoglobulina D/metabolismo , Imunoglobulina M , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Isotipos de Imunoglobulinas/metabolismoRESUMO
Activated naive (aNAV) B cells have been shown to be the precursor of the CD11c+T-bet+ IgD-CD27- double-negative (DN)2 or atypical memory (aMEM) B cells in systemic lupus erythematosus (SLE). To determine factors that maintain resting naive (rNAV) B cells, the transcriptomic program in naive (IGHD+IGHM +) B cells in human healthy control subjects (HC) and subjects with SLE was analyzed by single-cell RNA-sequencing analysis. In HC, naive B cells expressed IL-4 pathway genes, whereas in SLE, naive B cells expressed type I IFN-stimulated genes (ISGs). In HC, aNAV B cells exhibited upregulation of the gene signature of germinal center and classical memory (cMEM) B cells. In contrast, in SLE, aNAV B cells expressed signature genes of aMEM. In vitro exposure of SLE B cells to IL-4 promoted B cell development into CD27+CD38+ plasmablasts/plasma and IgD-CD27+ cMEM B cells. The same treatment blocked the development of CD11c+Tbet+ aNAV and DN2 B cells and preserved DN B cells as CD11c-Tbet- DN1 B cells. Lower expression of IL-4R and increased intracellular IFN-ß in naive B cells was correlated with the accumulation of CD21-IgD- B cells and the development of anti-Smith and anti-DNA autoantibodies in patients with SLE (n = 47). Our results show that IL-4R and type I IFN signaling in naive B cells induce the development of distinct lineages of cMEM versus aMEM B cells, respectively. Furthermore, diminished IL-4R signaling shifted activated B cell development from the DN1 to the DN2 trajectory in patients with SLE. Therapies that enhance IL-4R signaling may be beneficial for ISGhi SLE patients.
Assuntos
Subpopulações de Linfócitos B , Lúpus Eritematoso Sistêmico , Autoanticorpos/metabolismo , Humanos , Imunoglobulina D/metabolismo , Interleucina-4/metabolismo , RNA/metabolismoRESUMO
G protein-coupled receptor 183 (GPR183) has been indicated to mediate the migration and localisation of immune cells in T cell-dependent antibody responses. Systemic lupus erythematosus (SLE) is a canonical autoimmune disease involving B cell-mediated tolerance destruction and excessive pathogenic autoantibody production, in which multiple GPCRs play a role. To date, there has been no systematic study regarding the expression of GPR183 in lymphocyte subsets of SLE patients. In this research, firstly, we observed the expression trends of GRP183 in various T and B cell subsets in human tonsil tissues. These lymphocyte subsets include CD4+, CD8+, naïve T, effector T, Tfh, activated Tfh, Th1, Th2, Th17, Treg, CD19+CD27-, CD19+CD27+, naïve B, germinal centre B, memory B, and plasma cells. Further, compared with healthy controls (HCs), GPR183 expression levels in above peripheral blood lymphocyte subsets of patients with SLE were reduced overall. The differential expression of GPR183 expression between inactive and active SLE patients indicates that GPR183 expression may be concerned with the disease activity of SLE. This was further confirmed through the strong negative correlation with SLEDAI score and positive correlation with serum complement protein C3, C4 and C1q levels. Further receiver operating characteristic (ROC) curve analysis revealed that GPR183 expression in circulating CD27-IgD+ B cells may be beneficial in distinguishing between inactive and active SLE patients. In addition, type I interferon stimulation could down-regulate the expression of GPR183 in peripheral blood T and B cell subsets. Aberrant expression of GPR183 may provide some novel insights into disease activity prediction and underlying pathogenesis of SLE.
Assuntos
Subpopulações de Linfócitos B , Lúpus Eritematoso Sistêmico , Receptores Acoplados a Proteínas G , Subpopulações de Linfócitos T , Complemento C1q/metabolismo , Citometria de Fluxo , Humanos , Imunoglobulina D/metabolismo , Interferon Tipo I/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
In past years ex vivo and in vivo experimental approaches involving human naive B cells have proven fundamental for elucidation of mechanisms promoting B cell differentiation in both health and disease. For such studies, it is paramount that isolation strategies yield a population of bona fide naive B cells, i.e., B cells that are phenotypically and functionally naive, clonally non-expanded, and have non-mutated BCR variable regions. In this study different combinations of common as well as recently identified B cell markers were compared to isolate naive B cells from human peripheral blood. High-throughput BCR sequencing was performed to analyze levels of somatic hypermutation and clonal expansion. Additionally, contamination from mature mutated B cells intrinsic to each cell-sorting strategy was evaluated and how this impacts the purity of obtained populations. Our results show that current naive B cell isolation strategies harbor contamination from non-naive B cells, and use of CD27-IgD+ is adequate but can be improved by including markers for CD45RB glycosylation and IgM. The finetuning of naive B cell classification provided herein will harmonize research lines using naive B cells, and will improve B cell profiling during health and disease, e.g. during diagnosis, treatment, and vaccination strategies.
Assuntos
Subpopulações de Linfócitos B , Subpopulações de Linfócitos B/metabolismo , Separação Celular , Glicosilação , Humanos , Imunoglobulina D/metabolismo , Isotipos de Imunoglobulinas/metabolismo , Imunoglobulina M/metabolismo , Memória Imunológica/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
Glycosylation of CD45RB (RB+) has recently been identified to mark antigen-experienced B cells, independent of their CD27 expression. By using a novel combination of markers including CD45RB glycosylation, CD27 and IgM/IgD isotype expression we segregated human peripheral blood B cell subsets and investigated their IGHV repertoire and in vitro functionality. We observed distinct maturation stages for CD27-RB+ cells, defined by differential expression of non-switched Ig isotypes. CD27-RB+ cells, which only express IgM, were more matured in terms of Ig gene mutation levels and function as compared to CD27-RB+ cells that express both IgM and IgD or cells that were CD27-RB-. Moreover, CD27-RB+IgM+ cells already showed remarkable rigidity in IgM isotype commitment, different from CD27-RB+IgMD+ and CD27-RB- cells that still demonstrated great plasticity in B cell fate decision. Thus, glycosylation of CD45RB is indicative for antigen-primed B cells, which are, dependent on the Ig isotype, functionally distinct.
Assuntos
Subpopulações de Linfócitos B , Antígenos Comuns de Leucócito/imunologia , Subpopulações de Linfócitos B/metabolismo , Glicosilação , Humanos , Imunoglobulina D/metabolismo , Isotipos de Imunoglobulinas/metabolismo , Imunoglobulina M/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
B cell abnormalities are common in systemic lupus erythematosus (SLE), and include expansion of double negative (DN) and age-associated-like B cells (ABC-like). We aimed to investigate rituximab (RTX) effects on DN and ABC-like B-cell subsets and, when possible, also secondary effects on T cells. Fifteen SLE patients, fulfilling the ACR 1982 criteria, starting RTX and followed longitudinally up to two years, were analyzed for B- and T- lymphocyte subsets using multicolor flow cytometry. DN were defined as IgD-CD27- and ABC-like as CD11c+CD21- within the DN gate. Additional phenotyping was performed adding CXCR5 in the B-cell panel. Cellular changes were further analyzed in the context of the generation of anti-drug antibodies (ADA) against RTX and clinical information. The SLE patients were mainly females (86.6%), of median age 36.7 (29.8-49.4) years and disease duration of 6.1 (1.6-11.8) years. Within the DN subset, ABC-like (IgD-CD27-CD11c+CD21-) B cell frequency reduced from baseline median level of 20.4% to 11.3% (p=0.03), at early follow-up. The DN B cells were further subdivided based on CXCR5 expression. Significant shifts were observed at the early follow-up in the DN2 sub-cluster (CD11c+CXCR5-), which reduced significantly (-15.4 percentage points, p=0.02) and in the recently described DN3 (CD11c-CXCR5-) which increased (+13 percentage points, p=0.03). SLE patients treated with RTX are at high risk of developing ADA. In our cohort, the presence of ADA at 6 months was associated with lower frequencies of DN cells and to a more pronounced expansion of plasmablasts at early follow-up. The frequency of follicular helper T cells (TFH, CD4+PD-1+CXCR5+) and of peripheral helper T cells (TPH, CD4+PD-1+CXCR5-) did not change after RTX. A sub-cluster of PD-1highCD4+ T cells showed a significant decrease at later follow-up compared to early follow-up (p=0.0039). It is well appreciated that RTX transiently influences B cells. Here, we extend these observations to cell phenotypes which are believed to directly contribute to autoimmunity in SLE. We show early transient effects of RTX on ABC-like memory B cells, later effects on PD-1high CD4+ cells, and possible implications for RTX immunogenicity. Further insight in such effects and their monitoring may be of clinical relevance.
Assuntos
Autoimunidade , Lúpus Eritematoso Sistêmico , Adulto , Antígeno CD11c/metabolismo , Feminino , Humanos , Imunoglobulina D/metabolismo , Masculino , Pessoa de Meia-Idade , Fenótipo , Receptor de Morte Celular Programada 1/metabolismo , Receptores CXCR5/metabolismo , Rituximab/metabolismo , Rituximab/farmacologia , Rituximab/uso terapêutico , Linfócitos T Auxiliares-IndutoresRESUMO
OBJECTIVE: The apoptotic signaling pathway is obviously disordered in systemic lupus erythematosus (SLE). Natural IgM (nIgM) is important in clearing apoptotic cells and preventing them from triggering deleterious autoimmunity. B-1 and innate-like B (ILBs) cells are the main nIgM producers. Human CD27+IgD+ B cells (un-switched memory B cells) are considered ILBs. However, their functional properties in SLE remain undefined. METHODS: Peripheral blood sample of 50 SLE patients and 50 healthy controls were collected, and twelve SLE patients were assessed in a follow-up study. The amount of CD27+IgD+ B cell in each population was analyzed by flow cytometry. The IgM and IL-10 levels of CD27+IgD+ B cell were assessed by ELISPOT and qRT-PCR, respectively. SPSS 17.0 (SPSS, USA) was employed for data analysis. P < 0.05 indicated statistical significance. RESULT: The amounts of CD27+IgD+ B cell were significantly decreased in SLE patients than healthy control (P < 0.01). CD27+IgD+ B cell amounts were positively correlated with WBC (r = 0.337, P = 0.017), platelet count (r = 0.396, P = 0.004), and serum C3 levels (r = 0.415, P = 0.003) and negatively correlated with serum creatinine levels (r = - 0.285, P = 0.045), SLEDAI(r = - 0.724, P = 0.000), and anti-dsDNA(r = - 0.477, P = 0.000). The IgM and IL-10 levels of CD27+IgD+ B in active SLE were decreased than healthy control (P < 0.001). Moreover, CD27+IgD+ B cells are increased in SLE cases after treatment than before treatment (P < 0.001). CONCLUSION: The amounts of CD27+IgD+ B cell were significantly decreased in SLE patients compared with the healthy population, and CD27+IgD+ B cell was verified to be correlated with clinical and immunological features in SLE patients. CD27+IgD+ B cells had impaired function associated with IgM and IL-10 production in active SLE. Moreover, the amounts of CD27+IgD+ B cells were recovered to the normal level in SLE cases with treatment-related disease remission. Key Points ⢠CD27+IgD+ B cell amounts are significantly decreased in SLE patients than healthy control. ⢠CD27+IgD+ B cells are functionally impaired in producing natural antibody-like IgM and IL-10 in SLE patients. ⢠CD27+IgD+ B cell amounts are correlated with clinical and immunological features in SLE.
Assuntos
Subpopulações de Linfócitos B , Lúpus Eritematoso Sistêmico , Seguimentos , Humanos , Imunoglobulina D/metabolismo , Imunoglobulina M , Interleucina-10/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovitis and the destruction of small joints. Emerging evidence shows that immunoglobulin D (IgD) stimulation induces T-cell activation, which may contribute to diseases pathogenesis in RA. In this study, we investigated the downstream signaling pathways by which IgD activated T cells as well as the possible role of IgD in the T-B interaction. Peripheral blood mononuclear cells were isolated from peripheral blood of healthy controls and RA patients. We demonstrated that IgD activated T cells through IgD receptor (IgDR)-lymphocyte-specific protein tyrosine kinase (Lck)-zeta-associated protein 70 (ZAP70)/phosphatidylinositol 3-kinase (PI3K)/nuclear factor kappa-B (NF-κB) signaling pathways; IgD-induced CD4+ T cells promoted the proliferation of CD19+ B cells in RA patients. A novel fusion protein IgD-Fc-Ig (composed of human IgD-Fc domain and IgG1 Fc domain, which specifically blocked the IgD-IgDR binding) inhibited the coexpression of IgDR and phosphorylated Lck (p-Lck) and the expression levels of p-Lck, p-ZAP70, p-PI3K on CD4+ T cells, and decreased NF-κB nuclear translocation in Jurkat cells. Meanwhile, IgD-Fc-Ig downregulated the expression levels of CD40L on CD4+ T cells as well as CD40, CD86 on CD19+ B cells in RA patients and healthy controls. It also decreased the expression levels of CD40L on CD4+ T cells and CD40 on CD19+ B cells from spleens of collagen-induced arthritis (CIA) mice and reduced IL-17A level in mouse serum. Moreover, administration of IgD-Fc-Ig (1.625-13 mg/kg, iv, twice a week for 4 weeks) in CIA mice dose-dependently decreased the protein expression levels of CD40, CD40L, and IgD in spleens. IgD-Fc-Ig restrains T-cell activation through inhibiting IgD-IgDR-Lck-ZAP70-PI3K-NF-κB signaling, thus inhibiting B-cell activation. Our data provide experimental evidences for application of IgD-Fc-Ig as a highly selective T cell-targeting treatment for RA.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Linfócitos B/efeitos dos fármacos , Imunoglobulina D/uso terapêutico , Ativação Linfocitária/efeitos dos fármacos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Receptores Fc/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Animais , Técnicas de Cocultura , Citometria de Fluxo , Humanos , Imunoglobulina D/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos DBA , Microscopia Confocal , Proteínas RecombinantesRESUMO
We report successful clinical experience using anti-BCMA CAR-T combined with lenalidomide in a patient who was refractory to a previous CAR-T treatment. The patient was a 51-year-old man, and was diagnosed with IgD-λ multiple myeloma(MM) in October 2015. 10 courses of chemotherapy including immunomodulators and proteasome inhibitors were used for remission and autologous hematopoietic stem cell transplantation was performed. MM relapsed after 12 months of remission. His disease continued to progress after multiple chemotherapy regimens, mouse anti-BCMA CAR-T and human-derived anti-BCMA CAR-T therapy. After a conditioning chemotherapy regimen of fludarabine and cyclophosphamide, patient took lenalidomide on day -1 and human-derived anti-BCMA CAR-T cells were infused on the next day. He suffered grade 2 cytokine-releasing syndrome(CRS) and grade 3 myelosuppression after infusion, and were resolved after symptomatic treatment. Very good partial response (VGPR) was achieved 14 days after CAR-T treatment, and had been maintained for more than 8 months. We demonstrated for the first time in patients that anti-BCMA CAR-T cell therapy combined with lenalidomide is feasible and effective in the treatment of RRMM. It provides a new strategy for RRMM patients who do not respond to anti-BCMA CAR-T cell therapy alone, and the adverse event is reversible.
Assuntos
Fatores Imunológicos/farmacologia , Lenalidomida/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/terapia , Antígeno de Maturação de Linfócitos B/uso terapêutico , Ciclofosfamida/administração & dosagem , Transplante de Células-Tronco Hematopoéticas , Humanos , Imunoglobulina D/metabolismo , Imunoterapia Adotiva , Masculino , Pessoa de Meia-Idade , Inibidores de Proteassoma , Receptores de Antígenos Quiméricos , Recidiva , Condicionamento Pré-Transplante , Resultado do Tratamento , Vidarabina/administração & dosagem , Vidarabina/análogos & derivadosAssuntos
Cadeias Leves de Imunoglobulina/metabolismo , Nefropatias/diagnóstico , Nefropatias/etiologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Mieloma Múltiplo/complicações , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores , Biópsia , Medula Óssea/patologia , Síndrome de Fanconi , Humanos , Imunoglobulina D/metabolismo , Cadeias lambda de Imunoglobulina/metabolismo , Túbulos Renais Proximais/ultraestrutura , Imageamento por Ressonância Magnética , Masculino , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/etiologia , Avaliação de Sintomas , Resultado do TratamentoRESUMO
Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by joint inflammation, synovial hyperplasia, cartilage degeneration, bone erosion, and pannus. Immunoglobulin D (IgD) plays an important role in autoimmune diseases although the content of it in vivo is low. Increased concentrations of anti-IgD autoantibodies have been detected in many RA patients. IgD-Fc-Ig fusion protein is constructed by connecting human IgD Fc domain and IgG1 Fc domain, which specifically block the IgD/ IgDR pathway and regulate the function of cells expressing IgDR to treat RA. The expression levels of Wnt5A and Frizzled 5 are higher in RA synovial tissue specimens. The complex of Wnt5A-Fzd5-LRP5/6-CTHRC1 promotes the expression of hypoxia inducible factor-1α by activating nuclear factor kappa-B (NF-κB), leading to high expression of VEGF and participating in angiogenesis. VEGF is the strongest angiogenic factor found so far. Here, we aimed to explore whether IgD participates in synovitis by binding to IgDR and regulating the activation of Wnt5A-Fzd5-CTHRC1-NF-κB signaling pathway in fibroblast synovial cells (FLSs), whether IgD-Fc-Ig fusion protein inhibits VEGF production in FLS of CIA and explore mechanism. We found that IgDR is expressed on MH7A and FLS. IgD promotes VEGF expression by activating Wnt5A-Fzd5-CTHRC1-NF-κB signaling pathway in MH7A and FLS. After activation of Fzd5 with Wnt5A, IgD-Fc-Ig reduced VEGF-A level in the culture supernatant of MH7A stimulation by IgD. The expressions of CTHRC1, Fzd5, p-P65 and VEGF in MH7A and FLSs were down-regulated after IgD-Fc-Ig treatment. IgD-Fc-Ig suppressed the combination of CTHRC1 and Fzd5 as well. By using the animal model, we demonstrated that IgD-Fc-Ig suppress ankle CTHRC1 and Fzd5 production resulted in inhibition of index of joint inflammation of CIA rats, which were consistent with vitro results. Conclusively, IgD-Fc-Ig inhibits IgD and Wnt5A-induced angiogenesis and joint inflammation by suppressing the combination of CTHRC1 and Fzd5. Our results show that IgD-Fc-Ig exerts its suppressive effect on IgD and Wnt5A by Wnt5A-Fzd5-CTHRC1-NF-κB signaling pathway.
Assuntos
Artrite Experimental/imunologia , Imunoglobulina D/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Membrana Sinovial/patologia , Sinovite/imunologia , Proteína Wnt-5a/antagonistas & inibidores , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/patologia , Colágeno/administração & dosagem , Colágeno/imunologia , Fibroblastos , Receptores Frizzled/metabolismo , Glicoproteínas/metabolismo , Humanos , Imunoglobulina D/administração & dosagem , Imunoglobulina D/genética , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Fragmentos Fc das Imunoglobulinas/genética , Masculino , NF-kappa B/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/imunologia , Sinoviócitos , Sinovite/tratamento farmacológico , Sinovite/patologia , Proteína Wnt-5a/metabolismoRESUMO
Antigen-specific B cells (ASBCs) can drive autoimmune disease by presenting autoantigen to cognate T cells to drive their activation, proliferation, and effector cell differentiation and/or by differentiating into autoantibody-secreting cells. Autoantibodies are frequently used to predict risk and diagnose several autoimmune diseases. ASBCs can drive type 1 diabetes even when immune tolerance mechanisms block their differentiation into antibody-secreting cells. Furthermore, anti-histidyl tRNA synthetase syndrome patients have expanded IgM+ Jo-1-binding B cells, which clinically diagnostic IgG Jo-1 autoantibodies may not fully reflect. Given the potential disconnect between the pathologic function of ASBCs and autoantibody secretion, direct study of ASBCs is a necessary step towards developing better therapies for autoimmune diseases, which often have no available cure. We therefore developed a high-throughput screening pipeline to 1) phenotypically identify specific B cell subsets, 2) expand them in vitro, 3) drive them to secrete BCRs as antibody, and 4) identify wells enriched for ASBCs through ELISA detection of antibody. We tested the capacity of several B cell subset(s) to differentiate into antibody-secreting cells following this robust stimulation. IgM+ and/or IgD+, CD27- memory, memory, switched memory, and BND B cells secreted B cell receptor (BCR) as antibody following in vitro stimulation, whereas few plasmablasts responded. Bimodal responses were observed across autoimmune donors for IgM+ CD21lo and IgM- CD21lo B cells, consistent with documented heterogeneity within the CD21lo subset. Using this approach, we detected insulin-binding B cell bias towards CD27- memory and CD27+ memory subsets in pre-symptomatic type 1 diabetes donors. We took advantage of routine detection of Jo-1-binding B cells in Jo-1+ anti-histidyl tRNA synthetase syndrome patients to show that Jo-1-binding B cells and total B cells expanded 20-30-fold using this culture system. Overall, these studies highlight technology that is amenable to small numbers of cryopreserved peripheral blood mononuclear cells that enables interrogation of phenotypic and repertoire attributes of ASBCs derived from autoimmune patients.
Assuntos
Doenças Autoimunes/imunologia , Subpopulações de Linfócitos B/fisiologia , Linfócitos B/imunologia , Imunoglobulina D/metabolismo , Imunoglobulina M/metabolismo , Adulto , Idoso , Autoanticorpos/imunologia , Autoantígenos/imunologia , Feminino , Humanos , Imunoglobulina D/genética , Imunoglobulina M/genética , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
BACKGROUND: The Collaboration to Collect Autologous Transplant Outcomes in Lymphoma and Myeloma (CALM) study has provided an opportunity to evaluate the real-world outcomes of patients with myeloma. The aim of this study was to compare the outcome according to the different subtypes of myeloma using CALM data. PATIENTS: This study compared overall survival (OS), progression-free survival (PFS), and complete remission (CR) and the impact of novel versus non-novel drug containing induction regimens prior to autologous hematopoietic cell transplantation (HCT) of 2802 patients with "usual" and "rare" myelomas. RESULTS: Our data suggest that IgM and non-secretory myeloma have superior PFS and OS compared with IgD myeloma and outcomes comparable to those for usual myeloma. Patients who received novel agent induction had higher rates of CR prior to transplant. Non-novel induction regimens were associated with inferior PFS but no difference in OS. Although not the primary focus of this study, we show that poor mobilization status is associated with reduced PFS and OS, but these differences disappear in multivariate analysis suggesting that poor mobilization status is a surrogate for other indicators of poor prognosis. CONCLUSION: We confirm that IgD myeloma is associated with the worst prognosis and inferior outcomes compared with the other isotypes.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Imunoglobulina D/metabolismo , Condicionamento Pré-Transplante/métodos , Transplante Autólogo/métodos , Adulto , Idoso , Feminino , Transplante de Células-Tronco Hematopoéticas/mortalidade , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo , Intervalo Livre de Progressão , Análise de Sobrevida , Condicionamento Pré-Transplante/mortalidade , Transplante Autólogo/mortalidade , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is a kidney-specific autoimmune disease, but its pathogenesis is not fully known. The present study detected the frequencies of circulating memory B cells and plasmablasts and other clinical parameters in FSGS. METHODS: We monitored 16 primary FSGS patients and 23 healthy controls (HC). Flow cytometry was used to analyze circulating memory B cell and plasmablastspercentages. Serum IgG levels were detected using a cytometric bead array (CBA). RESULTS: The proportions of CD27 + IgD- class-switched memory B cells (P = 0.0002), CD27 + IgD-IgG + class-switched memory B cells (P < 0.0001), CD27hiCD38hi plasmablasts (P < 0.0001) and CD138 + plasma cells (P < 0.0001) were markedlyelevated in FSGS patients, and the frequency of CD38 + IgG + plasmablasts (P < 0.0001) and serum IgG levels (P < 0.0001) were lower compared to HC. In the FSGS patients, the frequency of CD27 + IgD-IgG + class-switched memory B cells negatively correlated with CD38 + IgG + plasmablasts (P = 0.0183, R = -0.3375), serum IgG levels (P = 0.0061, R = -0.4263) and estimated glomerular filtration rate (eGFR) (P = 0.0074, R = -0.4114) but positively correlated with 24-h urinary protein levels (P = 0.0077, R = 0.4085). The proportion of CD38 + IgG + plasmablasts positively correlated with serum IgG levels (P = 0.0151, R = 0.3538). CONCLUSIONS: We speculate that alterations in the frequencies of CD27 + IgD-IgG + class-switched memory B cells and plasmablasts may be responsible for the etiopathogenesis of FSGS.
Assuntos
Linfócitos B/imunologia , Glomerulosclerose Segmentar e Focal/imunologia , Imunoglobulina G/sangue , Plasmócitos/imunologia , ADP-Ribosil Ciclase 1/metabolismo , Adulto , Idoso , Feminino , Citometria de Fluxo , Taxa de Filtração Glomerular , Glomerulosclerose Segmentar e Focal/diagnóstico , Humanos , Switching de Imunoglobulina , Imunoglobulina D/metabolismo , Memória Imunológica , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Regulação para CimaRESUMO
Protective humoral memory forms in secondary lymphoid organs where B cells undergo affinity maturation and differentiation into memory or plasma cells. Here, we provide a comprehensive roadmap of human B cell maturation with single-cell transcriptomics matched with bulk and single-cell antibody repertoires to define gene expression, antibody repertoires, and clonal sharing of B cell states at single-cell resolution, including memory B cell heterogeneity that reflects diverse functional and signaling states. We reconstruct gene expression dynamics during B cell activation to reveal a pre-germinal center state primed to undergo class switch recombination and dissect how antibody class-dependent gene expression in germinal center and memory B cells is linked with a distinct transcriptional wiring with potential to influence their fate and function. Our analyses reveal the dynamic cellular states that shape human B cell-mediated immunity and highlight how antibody isotype may play a role during their antibody-based selection.
Assuntos
Centro Germinativo/metabolismo , Switching de Imunoglobulina/imunologia , Células B de Memória/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Comunicação Celular/imunologia , Diferenciação Celular , Criança , Conjuntos de Dados como Assunto , Centro Germinativo/imunologia , Humanos , Imunoglobulina D/genética , Imunoglobulina D/metabolismo , Imunoglobulina M/genética , Imunoglobulina M/metabolismo , Linfonodos/imunologia , Linfonodos/metabolismo , Tonsila Palatina/imunologia , Tonsila Palatina/metabolismo , Tonsila Palatina/cirurgia , Análise de Célula Única , Baço/imunologia , Baço/metabolismo , Tonsilectomia , Recombinação V(D)J/imunologiaRESUMO
B-cell IgD Low (BDL) B cells have been shown to promote immunological tolerance by inducing proliferation of CD4+Foxp3+ T-regulatory cells (Treg) in a glucocorticoid-induced tumor necrosis factor receptor-related protein ligand (GITRL, Tnfsf18)-dependent manner. BDL cells constitute a small subset of splenic B lymphocytes that, in mice, are characterized by the B220+IgMintCD21intCD23+CD93-IgDlow/- cell surface expression profile. In this chapter, we show the flow cytometry gating strategy developed to identify and purify BDL. In addition, we describe an in vitro assay and two in vivo assays to assess BDL regulatory activity by quantitating Treg expansion/proliferation and indicate how they can be used in mouse models of disease. Collectively, these methods are useful to track and quantitate BDL and Treg numbers and assess their regulatory activity in inflammatory disease models.
Assuntos
Linfócitos B Reguladores/imunologia , Citometria de Fluxo/métodos , Imunoglobulina D/isolamento & purificação , Animais , Linfócitos B Reguladores/metabolismo , Linfócitos T CD4-Positivos/imunologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Tolerância Imunológica , Imunoglobulina D/imunologia , Imunoglobulina D/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Receptores do Fator de Necrose Tumoral/metabolismo , Baço/citologia , Linfócitos T Reguladores/imunologiaRESUMO
Memory B cells have known to play an important role in the pathogenesis of rheumatoid arthritis (RA). With the emergence of B cell-targeted therapies, the modulation of memory B cells appears to be a key therapeutic target. Human peripheral memory B cells can be distinguished based on the phenotypic expression of CD27 and IgD, characterizing the three major B cell subpopulations: CD27+IgD+ pre-switch, CD27+IgD- post-switch, and CD27-IgD- double-negative memory B cells. We evaluated different memory cell populations for activation markers (CD95 and Ki-67) and chemokine receptors (CXCR3 and 4) expressing B cells in active RA, as well as under IL6-R blockade by tocilizumab (TCZ) and TNF-α blockade by adalimumab (ADA). Memory B cells were phenotypically analyzed from RA patients at baseline, week 12, and week 24 under TCZ or ADA treatment, respectively. Using flow cytometry, surface expression of CD95, intracellular Ki-67, and surface expressions of CXCR3 and CXCR4 were determined. Compared with healthy donors (n = 40), the phenotypic analysis of RA patients (n = 80) demonstrated that all three types of memory B cells were activated in RA patients. Surface and intracellular staining of B cells showed a significantly higher percentage of CD95+ (p < 0.0001) and Ki-67+ (p < 0.0001) cells, with numerically altered CXCR3+ and CXCR4+ cells in RA. CD95 and Ki-67 expressions were highest in post-switch memory B cells, whereas CD19+CXCR3+ and CD19+CXCR4+ expressing cells were substantially higher in the pre-switch compartment. In all subsets of the memory B cells, in vivo IL-6R, and TNF-α blockade significantly reduced the enhanced expressions of CD95 and Ki-67. Based on our findings, we conclude that the three major peripheral memory B cell populations, pre-, post-switch, and double-negative B cells, are activated in RA, demonstrating enhanced CD95 and Ki-67 expressions, and varied expression of CXCR3 and CXCR4 chemokine receptors when compared with healthy individuals. This activation can be efficaciously modulated under cytokine inhibition in vivo.
Assuntos
Adalimumab/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Artrite Reumatoide/imunologia , Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Imunoterapia/métodos , Adulto , Idoso , Artrite Reumatoide/tratamento farmacológico , Células Cultivadas , Feminino , Humanos , Imunoglobulina D/metabolismo , Memória Imunológica , Imunomodulação , Interleucina-6/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade , Receptores de Interleucina-6/antagonistas & inibidores , Receptores de Retorno de Linfócitos/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
Protein kinase Cß (PKCß) expressed in mammalian cells as two splice variants, PKCßI and PKCßII, functions in the B cell receptor (BCR) signaling pathway and contributes to B cell development. We investigated the relative role of PKCßII in B cells by generating transgenic mice where expression of the transgene is directed to these cells using the Eµ promoter (Eµ-PKCßIItg). Our findings demonstrate that homozygous Eµ-PKCßIItg mice displayed a shift from IgD+IgMdim toward IgDdimIgM+ B cell populations in spleen, peritoneum and peripheral blood. Closer examination of these tissues revealed respective expansion of marginal zone (MZ)-like B cells (IgD+IgM+CD43negCD21+CD24+), increased populations of B-1 cells (B220+IgDdimIgM+CD43+CD24+CD5+), and higher numbers of immature B cells (IgDdimIgMdimCD21neg) at the expense of mature B cells (IgD+IgM+CD21+). Therefore, the overexpression of PKCßII, which is a phenotypic feature of chronic lymphocytic leukaemia cells, can skew B cell development in mice, most likely as a result of a regulatory influence on BCR signaling.