CD4 T follicular cells and B cells

© 2023 Wiley-VCH GmbH.Regulatory and effector cell responses to Plasmodium vivax, the most common human malaria parasite outside Africa, remain understudied in naturally infected populations. Here, we describe peripheral CD4 + T- and B-cell populations during and shortly after an uncomplicated P. vivax infection in 38 continuously exposed adult Amazonians. Consistent with previous observations, we found an increased frequency in CD4 + CD45RA - CD25 + FoxP3 + T regulatory cells that express the inhibitory molecule CTLA-4 during the acute infection, with a sustained expansion of CD21 - CD27 - atypical memory cells within the CD19 + B-cell compartment. Both Th1- and Th2-type subsets of CXCR5 + ICOS hi PD-1 + circulating T follicular helper (cTfh) cells, which are thought to contribute to antibody production, were induced during P. vivax infection, with a positive correlation between overall cTfh cell frequency and IgG antibody titers to the P. vivax blood-stage antigen MSP1 19 . We identified significant changes in cell populations that had not been described in human malaria, such as an increased frequency of CTLA-4 + T follicular regulatory cells that antagonize Tfh cells, and a decreased frequency of circulating CD24 hi CD27 + B regulatory cells in response to acute infection. In conclusion, we disclose a complex immunoregulatory network that is critical to understand how naturally acquired immunity develops in P. vivax malaria.publishersversionepub_ahead_of_prin

CD19+ B cells in early rheumatoid arthritis

PhD ThesisBackground Rheumatoid Arthritis (RA) is a genetically complex disease which causes inflammation primarily affecting synovial joints. The therapeutic success of B cell depletion in RA has confirmed the clinical relevance of B cells in disease, but their specific role in pathogenesis remains unclear. Aims 1. Identify differences in the transcriptome of CD19+ B cells between RA samples and disease controls 2. Carry out an expression quantitative trait locus (eQTL) analysis of confirmed RA genetic risk loci 3. Identify RA disease-specific eQTLs 4. Immunophenotype peripheral blood B cells Method 242 patients were recruited, RNA and DNA was extracted for subsequent analyses and parallel flow cytometry data obtained. Results A list of differentially expressed genes, without multiple test correction (MTC), was identified between the transcriptome in RA and disease controls. Web-based analysis tools identified downregulation of B cell receptor (BCR) signalling and pathways involved in transcription and RNA processing in the RA group. A list of differentially expressed genes (with MTC) was identified when samples were divided based on chronological age and inflammatory status, not diagnosis. The eQTL analysis at RA risk loci identified 10 cis eQTLs in B cells and a further 21 potential RA-specific eQTLs which lay outside the known RA risk loci. The RA group had an increased frequency of CD19+CD24hiCD38hi cells, a postulated regulatory subset Conclusions Age and inflammatory status have a greater influence on the CD19+ B cell transcriptome than RA in this cohort. The genetic component to gene expression is highlighted by the eQTL findings and will aid the prioritisation of genes for downstream functional work. The disease-specific eQTLs identified may indicate novel mechanisms of disease. The absence of a robust diagnostic gene signature between the disease groups examined may relate to the heterogeneity of the B cells examined, as highlighted by differences in the frequency of CD19+CD24hiCD38hi cells.Wellcome Trus

Differentially activated B cells develop regulatory phenotype and show varying immunosuppressive features: a comparative study

Regulatory B lymphocytes (Bregs) are B cells with well-pronounced immunosuppressive properties, allowing them to suppress the activity of effector cells. A broad repertoire of immunosuppressive mechanisms makes Bregs an attractive tool for adoptive cell therapy for diseases associated with excessive activation of immune reactions. Such therapy implies Breg extraction from the patient’s peripheral blood, ex vivo activation and expansion, and further infusion into the patient. At the same time, the utility of Bregs for therapeutic approaches is limited by their small numbers and extremely low survival rate, which is typical for all primary B cell cultures. Therefore, extracting CD19+ cells from the patient’s peripheral blood and specifically activating them ex vivo to make B cells acquire a suppressive phenotype seems to be far more productive. It will allow a much larger number of B cells to be obtained initially, which may significantly increase the likelihood of successful immunosuppression after adoptive Breg transfer. This comparative study focuses on finding ways to efficiently manipulate B cells in vitro to differentiate them into Bregs. We used CD40L, CpG, IL4, IL21, PMA, and ionomycin in various combinations to generate immunosuppressive phenotype in B cells and performed functional assays to test their regulatory capacity. This work shows that treatment of primary B cells using CD40L + CpG + IL21 mix was most effective in terms of induction of functionally active regulatory B lymphocytes with high immunosuppressive capacity ex vivo

The differential role of regulatory B cells in cancer and allo-immunity

A new wave of research recognizes a distinct subset of B regulatory cells (Breg) that maintain immune tolerance. Breg cells have been shown to exert immunoregulatory functions through the production of interleukin (IL)-10 and appear to play important roles in autoimmunity and in cancer. Despite the extensive body of evidence reinforcing the notion of B cells as potential regulatory cells, some controversy over the paucity of markers that can unequivocally identify Bregs still exists. To study the role of Breg in immune surveillance, I designed a comprehensive multi‐parameter panel of surface antibodies to define B-cell subsets in peripheral blood (PB) and cord blood (CB). The intracellular detection of IL-10 combined with flow cytometric phenotyping presented in my thesis demonstrate the presence of IL-10– producing Bregs with Treg-independent immunosuppressive functions in both the IgM memory (CD19+IgM+CD27+) and transitional (CD19+CD24hiCD38hi) PB B-cell subsets in healthy donors. The regulatory function PB Bregs against CD4+T cells and CD56+NK cells required both cell-cell contact and IL-10 production. Moreover, I demonstrate that Breg populations are expanded in the PB of AML patients and exert potent suppression of NK function mediated through 2B4-CD48 signaling. I further demonstrated the presence of IL-10- producing B cells with Treg-independent immunosuppressive properties in CB with the ability to suppress allogeneic-CD4+T cells through IL-10, as well as cell-cell contact mediated mechanisms involving CTLA-4 and CD80/CD86. I found an early and robust recovery of IL-10+B cells post-CBT. High Breg frequencies in CB may attenuate T-cell responses and contribute to the lower rates of cGVHD. My findings have important clinical implications and suggest that Bregs may be exploited to treat immune-mediated diseases. Whereas, strategies to deplete Bregs for optimal anti-cancer immunotherapy may benefit antitumor activity in AML and other cancers, adoptive transfer of donor-derived Bregs post transplant may offer a potentially effective immunomodulatory therapy for the treatment of GVHD.Open Acces

Populações linfocitárias no diagnóstico da Síndrome de Sjögren

A Síndrome de Sjögren primária (SSP) é uma doença inflamatória crónica, de origem autoimune, que se carateriza pela infiltração linfocitária e lesão das glândulas exócrinas.O diagnóstico diferencial entre uma síndrome seca de origem autoimune (SSP) ou não-autoimune (complexo Sicca) é o principal desafio no diagnóstico da SSP, tendo extrema importância no prognóstico e na terapêutica.Atualmente, os principais elementos para confirmar o componente autoimune da SSP são a presença de anticorpos anti-SSA e anti-SSB e de infiltrados linfocitários focais na biópsia de glândula salivar minor(labial). São desejáveis novos biomarcadores que traduzam a disfunção imune presente na SSP, pois nem sempre é fácil confirmar o diagnóstico, sobretudo nas fases precoces da doençaA hiperatividade das células B, suportada por células T, é responsável por algumas das caraterísticas clínicas e imunológicas mais reconhecíveis da doença, sendo sugerido na literatura que a avaliação de perfisde linfócitos específicos podeconstituir um elemento auxiliar no diagnóstico da SSP.A partir destes pressupostos, este trabalho teve como objetivo principal explorar das subpopulações linfocitárias (caraterizadas por citometria de fluxo), e a sua utilização como biomarcadores auxiliares no diagnóstico e classificação da SSP. Explorámos também a distribuição das subpopulações linfocitárias com os seguintes objetivos secundários: avaliar a sua relação com o status serológico da infeção pelo vírus Epstein-Barr (EBV); avaliar a sua relação com o perfil serológico (anti-SSA) e eventual associação a caraterísticas fenotípicas da SSP; avaliar a relação da morfologia do plexo nervoso corneano sub-basal, quer com o perfil linfocitário, quer com a atividade da doença na SSP.Para este efeito, foram incluídos 57 doentes com SSP classificados de acordo com os critérios do American-European Consensus Group(AECG ) (Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM et al. 2002), 68 doentes com síndrome Sicca sem critérios de SSP, 20 doentes com Artrite Reumatoide (AR) e 24 indivíduos saudáveis, sem queixas secas.A citometria de fluxo foi a metodologia utilizada na avaliação imunofenotípica das populações celulares, considerando também os ensaios funcionais, utilizados para a avaliação da produção de citocinas. Na abordagem dos níveis séricos de anticorpos, em particular no contexto da infeção por EBV, recorreu-se a ensaios imunoenzimáticos clássicos.Resumidamente, a avaliação imunofenotípica implicou a incubação de sangue periférico com um painel pré-validado de anticorpos monoclonais, otimizado para a caraterização de células T (incluindo várias subpopulações dentro das células T CD4 e T CD8, tais como células reguladoras e células foliculares), e de células B (assegurando a caraterização de diferentes estadios desdeas células de transição a células de memória e plasmablastos, ou células reguladoras, através de estratégias distintas como a classificação Bm1-5, frequentemente aplicada neste contexto). Foi ainda estudada a capacidade de as células T secretarem citocinas como a IL-17 e a IL-21, após estimulação com PMA e ionomicina. O painel de anticorpos monoclonais incluiu os seguintes marcadores: CD3, CD4, CD8, CD19, CD24, CD25, CD27, CD38, CD45, CD45RA, CCR6, CCR7, CXCR3, CXCR5, anti-IgD, anti-IgM. Por fim, para a obtenção de contagens absolutas, utilizou-se uma estratégia de plataforma única, com a utilização de tubos TruCountTM, com esferas calibradas.A aquisição das amostras foi efetuada num citómetro BD FACS CaliburTM(BD Biosciences, San Jose, CA, EUA), equipado com dois lasers, permitindo a avaliação de quatro fluorescências distintas. O software de aquisição foi o MultiSetTM(BD Biosciences), e para efeitos de análise, foram usados ainda os softwares Cell Quest 3.3TM(BD Biosciences) e InfynicytTM(Cytognos, Salamanca, Espanha). A análise estatística utilizou o programa Graph Pad Prism (GraphPad Software, San Diego, CA, EUA).Em linha com a literatura, no nosso estudo, a classificação clássica IgD/CD27 verificou a existência de uma redução dos números absolutos e percentagens de células B de memória (sobretudo sem switch) e aumento das células B naïve em doentes com SSP. A avaliação das células B de acordo com a classificação Bm1-Bm5 confirmou também o aumento das subpopulações Bm2 e Bm2 ́, e a diminuição das eBm5 e Bm5. Na razão entre as células Bm2+Bm2' e as eBm5+Bm5 foi possível aplicar cut-offs na discriminação entre SSP e controlos, ainda que com menor sensibilidade e especificidade em relação aos anteriormente descritos.Ainda assim, doentes com SSP com valores mais baixos células B de memória apresentavam maior frequência de caraterísticas fenotípicas da doença, como FS≥1, anticorpos anti-SSA, anticorpos anti-SSB, ANA e FR, e aumento das imunoglobulinas. Estes resultados, que se associam a menores valores de células B de memória e à disfunção autoimune na SSP estão de acordo com a hipótese de que a diferenciação das células B periféricas que migram para as glândulas salivares favorecem a formação de plasmócitos e a consequente produção de autoanticorpos.Verificámos também que os doentes Sicca apresentaram percentagens de células B naïve e de memória semelhantes às de doentes com SSP, e distintas das dos controlos, enquanto que os valores absolutos destas populações foram superiores aos da SSP, mas inferiores aos controlos. Considerando o desafio diagnóstico dos doentes Sicca, o seu perfil linfocitário intermédio entre SSP e controlos, evidente nos doentes Sicca, sugere a presença de disfunção imunológica, podendo alguns deles corresponder a formas ligeiras ou precoces de SSP.As células T foliculares efectoras (Tfh) têm um importante papel na patogénese da SSP, onde foi descrito um aumento das células Tfh circulantes e uma expansão da sua diferenciação nas GS (Li et al. 2012; Szabo et al. 2013). Ora, a marcada expansão de células Tfh e a simultânea presença de células Tfc nas glândulas salivares na SSP pode relacionar-se com mecanismos de resposta a um agente infecioso sialotrópico, como o EBV. Neste estudo, apesar dos números absolutos de células T CXCR5+estarem diminuídos na SSP, encontrámos um aumento das células T produtoras de IL-21, quer CD4+(Tfh), quer CD8+(Tfc). Além disso, as células Tfc estavam aumentadas nos doentes com SSP com maior atividade da doença, tendo sido possível identificar uma correlação positiva entre os números absolutos de células T IL21+CD8+e o ESSDAI, sugerindo um papel destas células na patogénese da doença. Curiosamente, o aumento das células T produtoras de IL-21 lembra o perfil de uma infeção viral crónica, e de facto, em relação aos marcadores serológicos de EBV, os doentes com SSP apresentaram maior prevalência de anti-EBV EA-D IgG, em comparação com doentes com AR e controlos saudáveis.Reconhecendo este aumento de prevalência de anti-EBV EA-D IgG e uma prevalência importante de anti-EBNA IgG em SSP, subdividimos estesdoentes em 3 grupos de acordo com o padrão serológico para EBV. Doentes com serologia positiva para EBV apresentavam ESSDAI superior ao de doentes sem evidência de infeção (G3). Em doentes com perfil serológico de infeção recente/reativação de EBV, a frequência de doentes com PSS ativa era também tendencionalmente superior à dos doentes com infeção antiga. Do ponto de vista celular também se verificaram alterações, com os doentes com evidência de infeção por EBV, em especial infeção recente/reativação, a apresentarem maior representação das subpopulações T pro-inflamatórias Th1 e Tfh1, o que pode indicar a influência viral na diferenciação preferencial destas células. Adicionalmente, doentes com infeção por EBV recente/reativação apresentavam níveis superiores de células B de transição e de plasmablastos, o que pode indicar a influência do EBV na modulação das respostas imunes na SSP e na expressão clínica, conforme sugerido pela manifestação mais precoce da doença e maior frequência de doença ativa nestes indivíduos...

The diverse roles of human B lymphocytes in renal transplantation

Renal transplantation is the treatment of choice for patients with end stage renal disease. Despite the significant advances made over the last fifty years, one predicament that still perplexes the transplant community is late allograft loss. The two major contributing factors include the limitations with the clinical utility of various markers for early diagnosis and lack of appropriate therapy. This thesis deals with the issue of early diagnosis and tries to establish a link between the clinical, histological and immunological phenotype with a view to identify prognostic markers. Firstly, low-grade proteinuria is clinically analysed for its utility to predict graft outcomes. Secondly, a disjunction between the clinical and histological phenotype and more importantly the limited utility of the clinical phenotype to determine the prognosis for a troubled allograft in light of clinical dysfunction is considered. Thirdly, a novel definition of human B regulatory cells is proposed with a view to address the discrepancy in the current literature with regards to their identification. Fourthly, a link between the histological phenotype of late allograft dysfunction is correlated with the frequency and function of regulatory B cells. Here, the functional diversity of the B cells, specifically within a small sub-group of B regulatory cells in relation to histological abnormalities is considered. Finally, the phenotype and functionality of the Bregs are explored for their use as potential markers for allograft outcomes and the utility of a simple phenotype tested in a prospective sample of patients from a randomized controlled trial

Trypanosoma cruzi Induces Regulatory B Cell Alterations in Patients With Chronic Chagas Disease

The clinical evolution of patients with chronic Chagas disease (CCD) is mainly associated with an excessive inflammation and a defective immunomodulatory profile caused by the interaction between T. cruzi and the host. Regulatory B (Breg) cells exert immune suppression mostly through IL-10 production (B10 cells), but also through IL-10-independent mechanisms. Previously, we demonstrated that CCD patients with cardiomyopathy show changes in the ex vivo Breg cell phenotypic distribution although maintain IL-10 production capacity. Here, we sought to identify potential alterations on Breg cells upon in vitro stimulation. Isolated B cells from CCD patients with or without cardiomyopathy and non-infected (NI) donors were stimulated with T. cruzi lysate or CpG + CD40L, and characterized by flow cytometry based on the expression of CD24, CD27, CD38, and the regulatory molecules IL-10 and PD-L1. IL-10 and IL-17 secretion in the supernatant of B cells was evaluated by ELISA. Data showed that T. cruzi stimulation diminished the expression of CD24 and CD38 on CD27− B cells while reducing the percentage of CD24high inside CD27+ B cells. Furthermore, T. cruzi induced a regulatory B cell phenotype by increasing B10 cells and IL-10 secretion in all the groups. The innate-like B10 cells expansion observed in patients with cardiomyopathy would be associated with CD27− B10 cell subsets, while no predominant phenotype was found in the other groups. Patients with cardiomyopathy also displayed higher IL-17 secretion levels in T. cruzi–activated B cells. CpG + CD40L stimulation revealed that B cells from CCD patients and NI donors had the same ability to differentiate into B10 cells and secrete IL-10 in vitro. Additionally, CCD patients showed an increased frequency of CD24−CD27− B cells and a reduction in the percentage of CD24highCD27+ Breg cells, which appeared to be inversely correlated with the presence of T. cruzi DNA in blood. Finally, CCD patients exhibited a higher frequency of PD-L1+ B cells in T. cruzi–stimulated samples, suggesting that IL-10-independent mechanisms could also be tangled in the control of inflammation. Altogether, our results provide evidence about the potential role of Breg cells in the immune response developed against T. cruzi and its contribution to chronic Chagas cardiomyopathy.Fil: Girard, Magalí Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Ossowski, Micaela Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Muñoz Calderon, Arturo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Fernández, Marisa. Instituto Nacional de Parasitología “Dr. Mario Fatala Chabén”; ArgentinaFil: Hernandez Vasquez, Yolanda Maria. Instituto Nacional de Parasitología Dr. Mario Fatala C; ArgentinaFil: Chadi, Raúl. Gobierno de la Ciudad Autónoma de Buenos Aires. Hospital General de Agudos Doctor Ignacio Pirovano; ArgentinaFil: Gomez, Karina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

B cells on the stage of inflammation in juvenile idiopathic arthritis: leading or supporting actors in disease pathogenesis?

Copyright © 2022 Moura and Fonseca. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Juvenile idiopathic arthritis (JIA) is a term that collectively refers to a group of chronic childhood arthritides, which together constitute the most common rheumatic condition in children. The International League of Associations for Rheumatology (ILAR) criteria define seven categories of JIA: oligoarticular, polyarticular rheumatoid factor (RF) negative (RF-), polyarticular RF positive (RF+), systemic, enthesitis-related arthritis, psoriatic arthritis, and undifferentiated arthritis. The ILAR classification includes persistent and extended oligoarthritis as subcategories of oligoarticular JIA, but not as distinct categories. JIA is characterized by a chronic inflammatory process affecting the synovia that begins before the age of 16 and persists at least 6 weeks. If not treated, JIA can cause significant disability and loss of quality of life. Treatment of JIA is adjusted according to the severity of the disease as combinations of non-steroidal anti-inflammatory drugs (NSAIDs), synthetic and/ or biological disease modifying anti-rheumatic drugs (DMARDs). Although the disease etiology is unknown, disturbances in innate and adaptive immune responses have been implicated in JIA development. B cells may have important roles in JIA pathogenesis through autoantibody production, antigen presentation, cytokine release and/ or T cell activation. The study of B cells has not been extensively explored in JIA, but evidence from the literature suggests that B cells might have indeed a relevant role in JIA pathophysiology. The detection of autoantibodies such as antinuclear antibodies (ANA), RF and anti-citrullinated protein antibodies (ACPA) in JIA patients supports a breakdown in B cell tolerance. Furthermore, alterations in B cell subpopulations have been documented in peripheral blood and synovial fluid from JIA patients. In fact, altered B cell homeostasis, B cell differentiation and B cell hyperactivity have been described in JIA. Of note, B cell depletion therapy with rituximab has been shown to be an effective and well-tolerated treatment in children with JIA, which further supports B cell intervention in disease development.The authors would like to acknowledge Sociedade Portuguesa de Reumatologia (SPR) for funding.info:eu-repo/semantics/publishedVersio