RESUMEN
Memory B cells (mBCs) are characterized by their long-term stability, fast reactivation, and capability to rapidly differentiate into antibody-secreting cells (ASCs). However, the role of T cells in the differentiation of mBCs, in contrast to naive B cells, remains to be delineated. We study the role of T cells in mBC responses, using CD40L stimulation and autologous T-B co-cultures. Our results showed that increased CD40L levels led to a selective increased proliferation of IgM+ mBC, which did not class-switched, resulting in higher frequencies of IgM+ ASCs and a lower frequency of IgG+ ASCs. The IgG+/IgA+ mBCs were unaffected. We further compared the transcription of immune-related genes in IgM+ and IgG+ pre-plasmablasts cultured at high (500 ng/mL) and low (50 ng/mL) CD40L levels. In response to increased CD40L levels, both populations exhibited a core response to genes related to activation (TRAF1, AKT3, CD69, and CD80). However, they differed in genes related to cytokine/chemokine/homing interactions (CCL3/4/17, LTA, NKX2-3, BCL2 and IL21R) and cell-cell interactions (HLADR, CD40, and ICOSL), which were largely confined to IgG+ cells. Our findings revealed that in co-cultures with a high T-ratio, the response was similar to that found in cultures with high CD40L levels. These results suggest that IgG+ mBCs have a greater capacity for proliferation and T cell interaction, and weaker migration capabilities, leading to a preference for an IgG response over IgM in the short term. This adaptable response could fine-tune the memory repertoire with different functions of IgG versus IgM mBCs.
Asunto(s)
Ligando de CD40 , Inmunoglobulina G , Inmunoglobulina M , Células B de Memoria , Linfocitos T , Ligando de CD40/metabolismo , Ligando de CD40/inmunología , Humanos , Inmunoglobulina M/inmunología , Inmunoglobulina M/metabolismo , Inmunoglobulina G/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Células B de Memoria/inmunología , Células B de Memoria/metabolismo , Comunicación Celular/inmunología , Técnicas de Cocultivo , Memoria Inmunológica , Activación de Linfocitos/inmunología , Células Cultivadas , Diferenciación Celular/inmunología , Proliferación CelularRESUMEN
OBJECTIVES: Autoreactive B cells and interferon (IFN) signature are hallmarks of primary sjögren's syndrome (pSS), but how IFN signaling pathways influence autoantibody production and clinical manifestations remain unclear. More detailed studies hold promise for improved diagnostic methodologies and personalized treatment. METHODS: We analyzed peripheral blood T and B cell subsets from 34 pSS patients and 38 healthy donors (HDs) at baseline and upon stimulation regarding their expression levels of type I and II IFN signaling molecules (STAT1/2, IRF1, IRF9). Additionally, we investigated how the levels of these molecules correlated with serological and clinical characteristics and performed ROC analysis. RESULTS: Patients showed elevated IFN pathway molecules, including STAT1, STAT2 and IRF9 among most T and B cell subsets. We found a reduced ratio of phosphorylated STAT1 and STAT2 in patients in comparison to HDs, although B cells from patients were highly responsive by increased phosphorylation upon IFN stimulation. Correlation matrices showed further interrelations between STAT1, IRF1 and IRF9 in pSS. Levels of STAT1 and IRF9 in T and B cells correlated with the IFN type I marker Siglec-1 (CD169) on monocytes. High levels of STAT1 and IRF9 within pSS B cells were significantly associated with hypergammaglobulinemia as well as anti-SSA/anti-SSB autoantibodies. Elevated STAT1 levels were found in patients with extraglandular disease and could serve as a biomarker for this subgroup (p < 0.01). Notably, IRF9 levels in T and B cells correlated with EULAR Sjögren's syndrome disease activity index (ESSDAI). CONCLUSION: Here, we provide evidence that in active pSS patients, enhanced IFN signaling incl. unphosphorylated STAT1 and STAT2 with IRFs entertain chronic T and B cell activation. Furthermore, increased STAT1 levels candidate as biomarker of extraglandular disease, while IRF9 levels can serve as biomarker for disease activity.
Asunto(s)
Biomarcadores , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón , Factor de Transcripción STAT1 , Síndrome de Sjögren , Humanos , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/diagnóstico , Síndrome de Sjögren/metabolismo , Factor de Transcripción STAT1/metabolismo , Femenino , Fosforilación , Persona de Mediana Edad , Masculino , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/metabolismo , Anciano , Adulto , Linfocitos B/inmunología , Linfocitos B/metabolismo , Autoanticuerpos/inmunología , Autoanticuerpos/sangre , Transducción de Señal , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Lectina 1 Similar a Ig de Unión al Ácido Siálico/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Bone marrow plasma cells (BMPC) are the correlate of humoral immunity, consistently releasing antibodies into the bloodstream. It remains unclear if BMPC reflect different activation environments or maturation of their precursors. Here we define human BMPC heterogeneity and track the recruitment of antibody-secreting cells (ASC) from SARS-CoV-2 vaccine immune reactions to the bone marrow (BM). Trajectories based on single-cell transcriptomes and repertoires of peripheral and BM ASC reveal sequential colonisation of BMPC compartments. In activated B cells, IL-21 suppresses CD19 expression, indicating that CD19low-BMPC are derived from follicular, while CD19high-BMPC originate from extrafollicular immune reactions. In primary immune reactions, both CD19low- and CD19high-BMPC compartments are populated. In secondary immune reactions, most BMPC are recruited to CD19high-BMPC compartments, reflecting their origin from extrafollicular reactivations of memory B cells. A pattern also observable in vaccinated-convalescent individuals and upon diphtheria/tetanus/pertussis recall-vaccination. Thus, BMPC diversity reflects the evolution of a given humoral immune response.
Asunto(s)
Antígenos CD19 , Médula Ósea , Interleucinas , Células Plasmáticas , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Células Productoras de Anticuerpos/inmunología , Antígenos CD19/inmunología , Antígenos CD19/metabolismo , Linfocitos B/inmunología , Médula Ósea/inmunología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/citología , COVID-19/inmunología , COVID-19/virología , Vacuna contra Difteria, Tétanos y Tos Ferina/inmunología , Inmunidad Humoral/inmunología , Interleucinas/inmunología , Interleucinas/metabolismo , Células Plasmáticas/inmunología , SARS-CoV-2/inmunología , Análisis de la Célula Individual , VacunaciónRESUMEN
Systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS) share clinical as well as pathogenic similarities. Although previous studies suggest various abnormalities in different immune cell compartments, dedicated cell-type specific transcriptomic signatures are often masked by patient heterogeneity. Here, we performed transcriptional profiling of isolated CD4, CD8, CD16 and CD19 lymphocytes from pSS and SLE patients upon T cell stimulation, in addition to a steady-state condition directly after blood drawing, in total comprising 581 sequencing samples. T cell stimulation, which induced a pronounced inflammatory response in all four cell types, gave rise to substantial re-modulation of lymphocyte subsets in the two autoimmune diseases compared to healthy controls, far exceeding the transcriptomic differences detected at steady-state. In particular, we detected cell-type and disease-specific down-regulation of a range of pro-inflammatory cytokine and chemokine pathways. Such differences between SLE and pSS patients are instrumental for selective immune targeting by future therapies.
Asunto(s)
Enfermedades Autoinmunes , Lupus Eritematoso Sistémico , Síndrome de Sjögren , Humanos , Síndrome de Sjögren/genética , Síndrome de Sjögren/metabolismo , Linfocitos T/metabolismo , Regulación hacia Abajo/genética , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/metabolismoRESUMEN
OBJECTIVE: Altered composition of the B cell compartment in the pathogenesis of systemic lupus erythematosus (SLE) is characterized by expanded plasmablast and IgD-CD27- double-negative B cell populations. Previous studies showed that double-negative B cells represent a heterogeneous subset, and further characterization is needed. METHODS: We analyzed 2 independent cohorts of healthy donors and SLE patients, using a combined approach of flow cytometry (for 16 healthy donors and 28 SLE patients) and mass cytometry (for 18 healthy donors and 24 SLE patients) and targeted RNA-Seq analysis. To compare B cell subset formation during the acute immune response versus that during autoimmune disease, we investigated healthy donors at various time points after receipt of the BNT162b2 messenger RNA COVID-19 vaccine and patients with acute SARS-CoV-2 infection, using flow cytometry. RESULTS: We found that IgD-CD27+ switched and atypical IgD-CD27- memory B cells, the levels of which were increased in SLE patients, represented heterogeneous populations composed of 3 different subsets each. CXCR5+CD19intermediate , CXCR5-CD19high , and CXCR5-CD19low populations were found in the switched memory and double-negative compartments, suggesting the relatedness of IgD-CD27+ and IgD-CD27- B cells. We characterized a hitherto unknown and antigen-experienced CXCR5-CD19low subset that was enhanced in SLE patients, had a plasmablast phenotype with diminished B cell receptor responsiveness, and expressed CD38, CD95, CD71, PRDM1, XBP1, and IRF4. Levels of CXCR5-CD19low subsets were increased and correlated with plasmablast frequencies in SLE patients and in healthy donors who received BNT162b2, suggesting their interrelationship and contribution to plasmacytosis. The detection of CXCR5-CD19low B cells among both CD27+ and CD27- populations calls into question the role of CD27 as a reliable marker of B cell differentiation. CONCLUSION: Our data suggest that CXCR5-CD19low B cells are precursors of plasmablasts. Thus, cotargeting this subset may have therapeutic value in SLE.
Asunto(s)
Subgrupos de Linfocitos B , COVID-19 , Lupus Eritematoso Sistémico , Antígenos CD19/genética , Antígenos CD19/metabolismo , Subgrupos de Linfocitos B/metabolismo , Vacuna BNT162 , Vacunas contra la COVID-19 , Humanos , Inmunoglobulina D , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/metabolismo , Fenotipo , Receptores CXCR5/genética , Receptores CXCR5/metabolismo , SARS-CoV-2RESUMEN
B- and T-lymphocyte attenuator (BTLA/CD272) is an inhibitory checkpoint molecule expressed on T and B cells. Prior studies reported defective function of BTLA by T cells in patients with systemic lupus erythematosus (SLE), whereas nothing is known about its role on B cells in SLE, a disease with various B cell abnormalities. Peripheral blood mononuclear cells (PBMCs) from 23 healthy donors (HD) and 34 SLE patients were stained for BTLA and its expression on B cells was assessed. PBMCs or CD27-IgD+ naive B cells were stimulated together with an activating anti-BTLA antibody or an inhibitor of spleen tyrosine kinase (SYK) and differentiation as well as the expression of activation markers CD71, PD-1 and CD86 were analyzed. Our phenotypic and functional studies revealed reduced BTLA expression on CD27-IgD+ naïve B cells from SLE patients (p=0.0017) related to anti-dsDNA antibody titers (p=0.0394) and SIGLEC-1/CD169 expression on monocytes (p=0.0196), a type I interferon marker related to disease activity. BTLA engagement was found to control CpG/TLR9 activation limiting plasmablast (p=0.0156) and B cell memory induction (p=0.0078) in normal B cells in contrast to other B cell activation pathways (CD40, BCR). These BTLA functions were impaired in SLE B cells. Inhibition of SYK was found to mimic the effects of BTLA activity in vitro. Thus, is it possible that reduced BTLA expression and function of CD27-IgD+ antigen- and T cell-inexperienced SLE B cells could be overcome by SYK inhibition which should be tested in future studies as potential therapeutic principle.
Asunto(s)
Linfocitos B/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Receptores Inmunológicos/metabolismo , Adulto , Anticuerpos Antinucleares/sangre , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Estudios de Casos y Controles , Diferenciación Celular , Células Cultivadas , ADN/inmunología , Femenino , Humanos , Memoria Inmunológica , Lupus Eritematoso Sistémico/diagnóstico , Lupus Eritematoso Sistémico/inmunología , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Monocitos/metabolismo , Fenotipo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Lectina 1 Similar a Ig de Unión al Ácido Siálico/metabolismo , Quinasa Syk/antagonistas & inhibidores , Quinasa Syk/metabolismo , Adulto JovenRESUMEN
Circulating CD11c+ B cells are a key phenomenon in certain types of autoimmunity but have also been described in the context of regular immune responses (i.e., infections, vaccination). Using mass cytometry to profile 46 different markers on individual immune cells, we systematically initially confirmed the presence of increased CD11c+ B cells in the blood of systemic lupus erythematosus (SLE) patients. Notably, significant differences in the expression of CD21, CD27, and CD38 became apparent between CD11c- and CD11c+ B cells. We observed direct correlation of the frequency of CD21-CD27- B cells and CD21-CD38- B cells with CD11c+ B cells, which were most pronounced in SLE compared to primary Sjögren's syndrome patients (pSS) and healthy donors (HD). Thus, CD11c+ B cells resided mainly within memory subsets and were enriched in CD27-IgD-, CD21-CD27-, and CD21-CD38- B cell phenotypes. CD11c+ B cells from all donor groups (SLE, pSS, and HD) showed enhanced CD69, Ki-67, CD45RO, CD45RA, and CD19 expression, whereas the membrane expression of CXCR5 and CD21 were diminished. Notably, SLE CD11c+ B cells showed enhanced expression of the checkpoint molecules CD86, PD1, PDL1, CD137, VISTA, and CTLA-4 compared to HD. The substantial increase of CD11c+ B cells with a CD21- phenotype co-expressing distinct activation and checkpoint markers, points to a quantitative increased alternate (extrafollicular) B cell activation route possibly related to abnormal immune regulation as seen under the striking inflammatory conditions of SLE which shows a characteristic PD-1/PD-L1 upregulation.
Asunto(s)
Autoinmunidad , Linfocitos B/inmunología , Antígeno CD11c/sangre , Citometría de Flujo , Inmunofenotipificación , Lupus Eritematoso Sistémico/inmunología , Activación de Linfocitos , Síndrome de Sjögren/inmunología , ADP-Ribosil Ciclasa 1/sangre , Linfocitos B/metabolismo , Antígeno B7-H1/sangre , Biomarcadores/sangre , Estudios de Casos y Controles , Humanos , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/diagnóstico , Glicoproteínas de Membrana/sangre , Fenotipo , Receptor de Muerte Celular Programada 1/sangre , Receptores de Complemento 3d/sangre , Síndrome de Sjögren/sangre , Síndrome de Sjögren/diagnóstico , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/sangreRESUMEN
OBJECTIVES: SLE is characterized by two pathogenic key signatures, type I IFN and B-cell abnormalities. How these signatures are interrelated is not known. Type I-II IFN trigger activation of Janus kinase (JAK) - signal transducer and activator of transcription (STAT). JAK-STAT inhibition is an attractive therapeutic possibility for SLE. We assess STAT1 and STAT3 expression and phosphorylation at baseline and after IFN type I and II stimulation in B-cell subpopulations of SLE patients compared with other autoimmune diseases and healthy controls (HD) and related it to disease activity. METHODS: Expression of STAT1, pSTAT1, STAT3 and pSTAT3 in B and T cells of 21 HD, 10 rheumatoid arthritis (RA), seven primary Sjögren's (pSS) and 22 SLE patients was analysed by flow cytometry. STAT1 and STAT3 expression and phosphorylation in PBMCs (peripheral blood mononuclear cells) of SLE patients and HD after IFNα and IFNγ incubation were further investigated. RESULTS: SLE patients showed substantially higher STAT1 but not pSTAT1 in B- and T-cell subsets. Increased STAT1 expression in B-cell subsets correlated significantly with SLEDAI and Siglec-1 on monocytes, a type I IFN marker. STAT1 activation in plasmablasts was IFNα dependent while monocytes exhibited dependence on IFNγ. CONCLUSION: Enhanced expression of STAT1 by B-cell candidates as a key node of two immunopathogenic signatures (type I IFN and B-cells) related to important immunopathogenic pathways and lupus activity. We show that STAT1 is activated upon IFNα exposure in SLE plasmablasts. Thus, Jak inhibitors, targeting JAK-STAT pathways, hold a promise to block STAT1 expression and control plasmablast induction in SLE.
Asunto(s)
Linfocitos B/inmunología , Lupus Eritematoso Sistémico/inmunología , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismo , Linfocitos T/inmunología , Adulto , Anciano , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/fisiopatología , Linfocitos B/efectos de los fármacos , Estudios de Casos y Controles , Diferenciación Celular , Femenino , Humanos , Factores Inmunológicos/farmacología , Técnicas In Vitro , Interferón-alfa/farmacología , Interferón gamma/farmacología , Quinasas Janus/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Lupus Eritematoso Sistémico/fisiopatología , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Fosforilación/efectos de los fármacos , Células Plasmáticas/inmunología , Factor de Transcripción STAT1/efectos de los fármacos , Factor de Transcripción STAT3/efectos de los fármacos , Índice de Severidad de la Enfermedad , Transducción de Señal , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/metabolismo , Síndrome de Sjögren/fisiopatología , Linfocitos T/efectos de los fármacos , Adulto JovenRESUMEN
BACKGROUND: Memory B cell (mBC) induction and maintenance is one of the keys to long-term protective humoral immunity. MBCs are fundamental to successful medical interventions such as vaccinations and therapy in autoimmunity. However, their lifestyle and anatomic residence remain enigmatic in humans. Extrapolation from animal studies serves as a conceptual basis but might be misleading due to major anatomical distinctions between species. METHODS AND FINDINGS: Multicolor immunofluorescence stainings on fixed and unfixed frozen tissue sections were established using primary antibodies coupled to haptens and secondary signal amplification. The simultaneous detection of five different fluorescence signals enabled the localization and characterization of human CD27+CD20+Ki67- mBCs for the first time within one section using laser scanning microscopy. As a result, human tonsillar mBCs were initially identified within their complex microenvironment and their relative location to naïve B cells, plasma cells and T cells could be directly studied and compared to the human splenic mBC niche. In all investigated tonsils (n = 15), mBCs appeared to be not only located in a so far subepithelial defined area but were also follicle associated with a previous undescribed gradual decline towards the follicular mantle comparable to human spleen. However, mBC areas around secondary follicles with large germinal centers (GCs) in tonsils showed interruptions and a general widening towards the epithelium while in spleen the mBC-containing marginal zones (MZ) around smaller GCs were relatively broad and symmetrical. Considerably fewer IgM+IgD+/- pre-switch compared to IgA+ or IgG+ post-switch mBCs were detected in tonsils in contrast to spleen. CONCLUSIONS: This study extends existing insights into the anatomic residence of human mBCs showing structural similarities of the superficial follicular area in human spleen and tonsil. Our data support the debate of renaming the human splenic MZ to 'superficial zone' in order to be aware of the differences in rodents and, moreover, to consider this term equally for the human palatine tonsil.
Asunto(s)
Linfocitos B/metabolismo , Centro Germinal/citología , Tonsila Palatina/citología , Bazo/citología , Adolescente , Adulto , Anciano , Linfocitos B/citología , Microambiente Celular , Niño , Humanos , Persona de Mediana Edad , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
The functions of bone marrow plasma cells (BMPC) beyond antibody production are not fully elucidated and distinct subsets of BMPC suggest potential different functions. Phenotypic differences were identified for human BMPC depending on CD19 expression. Since CD19 is a co-stimulatory molecule of the B-cell-receptor (BCR), and IgA+ and IgM+ BMPC express the BCR on their surface, we here studied whether CD19 expression affects cellular responses, such as BCR signaling and the expression of checkpoint molecules. We analyzed 132 BM samples from individuals undergoing routine total hip arthroplasty. We found that both CD19+ and CD19- BMPC expressed BCR signaling molecules. Notably, the BCR-associated kinase spleen tyrosine kinase (SYK) including pSYK was higher expressed in CD19+ BMPC compared to CD19- BMPC. BCR stimulation also resulted in increased kinase phosphorylation downstream of the BCR while expression of CD19 remained stable afterwards. Interestingly, the BCR response was restricted to IgA+ BMPC independently of CD19 expression. With regard to the expression of checkpoint molecules, CD19- BMPC expressed higher levels of co-inhibitory molecule programmed cell death protein-1 (PD-1) than CD19+ BMPC. IgA+ BMPC characteristically upregulated PD-1 upon BCR stimulation in contrast to other PC subsets and inhibition of the kinase SYK abrogated PD-1 upregulation. In contrast, expression of PD-1 ligand, B and T lymphocyte attenuator (BTLA) and CD28 did not change upon BCR activation of IgA+ BMPC. Here, we identify a distinct characteristic of IgA+ BMPC that is independent of the phenotypic heterogeneity of the subsets according to their CD19 expression. The data suggest that IgA+ BMPC underlie different regulatory principles and/or exert distinct regulatory functions.
Asunto(s)
Células de la Médula Ósea/inmunología , Inmunoglobulina A/inmunología , Células Plasmáticas/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Regulación hacia Arriba/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Autoimmune diseases (AID) such as systemic lupus erythematosus (SLE), primary Sjögren's syndrome (pSS), and rheumatoid arthritis (RA) are chronic inflammatory diseases in which abnormalities of B cell function play a central role. Although it is widely accepted that autoimmune B cells are hyperactive in vivo, a full understanding of their functional status in AID has not been delineated. Here, we present a detailed analysis of the functional capabilities of AID B cells and dissect the mechanisms underlying altered B cell function. Upon BCR activation, decreased spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (Btk) phosphorylation was noted in AID memory B cells combined with constitutive co-localization of CD22 and protein tyrosine phosphatase (PTP) non-receptor type 6 (SHP-1) along with hyporesponsiveness to TLR9 signaling, a Syk-dependent response. Similar BCR hyporesponsiveness was also noted specifically in SLE CD27- B cells together with increased PTP activities and increased transcripts for PTPN2, PTPN11, PTPN22, PTPRC, and PTPRO in SLE B cells. Additional studies revealed that repetitive BCR stimulation of normal B cells can induce BCR hyporesponsiveness and that tissue-resident memory B cells from AID patients also exhibited decreased responsiveness immediately ex vivo, suggesting that the hyporesponsive status can be acquired by repeated exposure to autoantigen(s) in vivo. Functional studies to overcome B cell hyporesponsiveness revealed that CD40 co-stimulation increased BCR signaling, induced proliferation, and downregulated PTP expression (PTPN2, PTPN22, and receptor-type PTPs). The data support the conclusion that hyporesponsiveness of AID and especially SLE B cells results from chronic in vivo stimulation through the BCR without T cell help mediated by CD40-CD154 interaction and is manifested by decreased phosphorylation of BCR-related proximal signaling molecules and increased PTPs. The hyporesponsiveness of AID B cells is similar to a form of functional anergy.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Linfocitos B/inmunología , Agammaglobulinemia Tirosina Quinasa/inmunología , Humanos , Proteínas Tirosina Fosfatasas/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Quinasa Syk/inmunologíaRESUMEN
OBJECTIVE: To assess the expression of programmed death 1 (PD-1), PD ligand 1 (PD-L1), and PD-L2 by B cells from patients with systemic lupus erythematosus (SLE) at baseline and after in vitro stimulation and to analyze their functional relationship to B cell proliferation. METHODS: Peripheral blood mononuclear cells obtained from 29 SLE patients and 27 healthy donors were stimulated with interleukin-2 (IL-2)/IL-10, anti-B cell receptor (anti-BCR), CpG, and CD40L alone or in combination. Expression of PD-1, PD-L1, and PD-L2 on defined B cell subsets as well as on CD3+ T cells was analyzed by flow cytometry at baseline and after 48 hours of stimulation. Additionally, after 48 hours of stimulation, CD71 was evaluated as a proliferation marker on CD19+CD20+ B cells. RESULTS: Increased PD-1 expression was characteristic of unstimulated lupus B cells and T cells. Upon stimulation of B cells with IL-2/IL-10, anti-BCR, CpG, and CD40L for 48 hours, the capacity of SLE B cells to up-regulate PD-L1 expression was substantially diminished (P = 0.0006) along with reduced B cell proliferation (P = 0.0039). Reduced PD-L1 expression was inversely correlated with the presence of the interferon signature (r = -0.8571, P < 0.0001) and the clinical SLE Disease Activity Index score (r = -0.5696, P = 0.0087). CONCLUSION: Post-activated, hyporesponsive lupus B cells are characterized by a phenotype of increased PD-1, functionally diminished PD-L1 up-regulation capacity, and reduced proliferation upon stimulation.
Asunto(s)
Linfocitos B/metabolismo , Antígeno B7-H1/fisiología , Leucocitos Mononucleares/metabolismo , Lupus Eritematoso Sistémico/sangre , Receptor de Muerte Celular Programada 1/fisiología , Proliferación Celular , Citometría de Flujo , Humanos , Activación de Linfocitos , Regulación hacia ArribaRESUMEN
Background: Pentraxin3 (PTX3) is overexpressed in kidneys of patients developing lupus nephritis (LN). Active LN is associated with reduced anti-PTX3 antibodies. However, abnormalities of B cell differentiation against PTX3 have not been characterized in systemic lupus erythematosus (SLE). Objective: Characterization of PTX3-specific (PTX3+) B cells in peripheral blood of SLE patients with or without LN and healthy donors (HD). Patients and Methods: SLE patients without LN, biopsy-proven LN and matched HD were analyzed. Active LN was defined as proteinuria>0.5 g/day or CrCl<60 ml/min/1.73 m2 with active urinary sediment. Peripheral B cells were analyzed for direct PTX3 binding by flow cytometry using PTX3 labeled with cyanine 5 (Cy5) and phycoerythrin (PE). Results: Initially, a flow cytometry based assay to identify PTX3+ B cells was developed by demonstrating simultaneous binding of PTX3-Cy5 and PTX3-PE. Specificity of B cells was validated by blocking experiments using unlabeled PTX3. We could identify circulating PTX3+ B-cells in HD and patients. Notably, LN patients showed a significantly diminished number of PTX3+ B cells (SLE vs. LN p = 0.033; HD vs. LN p = 0.008). This decrease was identified in naïve and memory B cell compartments (naïve: SLE vs. LN p = 0.028; HD vs. LN p = 0.0001; memory: SLE vs. LN p = 0.038, HD vs. LN p = 0.011). Conclusions: Decreased PTX3+ B cells in LN within the naïve and memory compartment suggest their negative selection at early stages of B cell development potentially related to a decreased regulatory function. PTX3+ B cells could candidate for autoantigen-defined regulatory B cells as a striking abnormality of LN patients.
Asunto(s)
Linfocitos B/metabolismo , Proteína C-Reactiva/metabolismo , Lupus Eritematoso Sistémico/sangre , Nefritis Lúpica/sangre , Componente Amiloide P Sérico/metabolismo , Adulto , Autoanticuerpos/sangre , Autoantígenos/metabolismo , Biomarcadores/metabolismo , Proteína C-Reactiva/química , Proteína C-Reactiva/inmunología , Carbocianinas/química , Femenino , Citometría de Flujo/métodos , Humanos , Masculino , Persona de Mediana Edad , Ficoeritrina/química , Componente Amiloide P Sérico/química , Componente Amiloide P Sérico/inmunología , Coloración y EtiquetadoRESUMEN
The bone marrow (BM) is, in addition to being the site of B cell development, a tissue that harbors long-lived plasma cells (PC), the cells that protect the body against foreign antigens by continuous production of antibodies. Nothing is known about the long-term stability and functionality of both B cells and PC in the BM at the individual donor level since repeated sampling possibilities outside of oncology are scarce. Here, we had the opportunity to obtain BM samples from a patient undergoing bilateral total hip arthroplasty half a year apart. We observed that the frequencies of the analyzed B cell and PC subsets were similar despite a time of six months in between and sampling on left and right side of the body. Additionally, B cell receptor stimulation led to comparable results. Our data suggest that composition and functionality of B cells are stable in the BM of adults at the individual donor level.
Asunto(s)
Subgrupos de Linfocitos B/citología , Células de la Médula Ósea/citología , Artroplastia de Reemplazo de Cadera , Subgrupos de Linfocitos B/inmunología , Células de la Médula Ósea/inmunología , Femenino , Fémur/citología , Humanos , Persona de Mediana Edad , Osteoartritis de la Cadera/cirugía , Células Plasmáticas/citología , Células Plasmáticas/inmunología , Factores de TiempoRESUMEN
PURPOSE OF REVIEW: This review summarizes recent insights and current understanding of the role of postactivated B cells in SLE and related pathogenic and potential therapeutic implications. RECENT FINDING: B cells are considered key players in SLE and experience from various B-cell-targeted therapies underlines their clinical relevance. In the last years, new insights have been obtained on B-cell abnormalities within the complex pathophysiology of SLE. These insights involve a revised understanding of BCR signaling, that has been reported to be hyperresponsive in the past, but newer studies suggest a postactivation functiotype in terms of reduced BCR and TLR signaling. Despite comprehensive efforts to delineate B-cell abnormalities on assessing large-scale genomic, epigenomic and proteomic data, understanding functional impairments of cellular interactions and subcellular functions remains crucial. A recently identified enhanced protein tyrosine phosphatase (PTP) activity was found in relation to diminished BCR responses in SLE. This finding together with reduced cytokine production upon TLR9 activation appears to mark postactivated lupus B cells. Other studies identified increased PTP activity in line with a gain-of-function mutation of phosphatase PTPN22, one of the strongest SLE risk alleles. Improved understanding of these B cell abnormalities in SLE holds promise to gain further insights in mechanisms of autoimmunity and pave the way for selective therapies targeting key principles of chronic autoimmunity. SUMMARY: SLE B cells (similar as previously described for lupus T cells) are characterized by a postactivation (exhausted) functiotype mandating consideration for innovative therapies.
Asunto(s)
Autoinmunidad , Linfocitos B/inmunología , Inmunoterapia/métodos , Lupus Eritematoso Sistémico/inmunología , Proteómica/métodos , Humanos , Lupus Eritematoso Sistémico/terapiaRESUMEN
The binding of antigen to the B cell receptor (BCR) results in a cascade of signalling events that ultimately drive B cell activation. Uncontrolled B cell activation is regulated by negative feedback loops that involve inhibitory co-receptors such as CD22 and CD32B that exert their functions following phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). The CD22-targeted antibody epratuzumab has previously been shown to inhibit BCR-driven signalling events, but its effects on ITIM phosphorylation of CD22 and CD32B have not been properly evaluated. The present study therefore employed both immunoprecipitation and flow cytometry approaches to elucidate the effects of epratuzumab on direct phosphorylation of key tyrosine (Tyr) residues on both these proteins, using both transformed B cell lines and primary human B cells. Epratuzumab induced the phosphorylation of Tyr(822) on CD22 and enhanced its co-localisation with SHP-1. Additionally, in spite of high basal phosphorylation of other key ITIMs on CD22, in primary human B cells epratuzumab also enhanced phosphorylation of Tyr(807), a residue involved in the recruitment of Grb2. Such initiation events could explain the effects of epratuzumab on downstream signalling in B cells. Finally, we were able to demonstrate that epratuzumab stimulated the phosphorylation of Tyr(292) on the low affinity inhibitory Fc receptor CD32B which would further attenuate BCR-induced signalling. Together, these data demonstrate that engagement of CD22 with epratuzumab leads to the direct phosphorylation of key upstream inhibitory receptors of BCR signalling and may help to explain how this antibody modulates B cell function.