RESUMO
High failure rates of the current drug development process are driving exemplary changes toward methodologies centered on human diseasein-vitromodeling. Organoids are self-organized tissue sub-units resembling their organ of origin and are widely acknowledged for their unique potential in recapitulating human physio-pathological mechanisms. They are transformative for human health by becoming the platform of choice to probe disease mechanisms and advance new therapies. Furthermore, the compounds' validation as therapeutics represents another point of the drug development pipeline where organoids may provide key understandings and help pharma organizations replace or reduce animal research. In this review, we focus on gastrointestinal organoid models, which are currently the most advanced organoid models in drug development. We focus on experimental validations of their value, and we propose avenues to enhance their use in drug discovery and development, as well as precision medicine and diagnostics.
Assuntos
Desenvolvimento de Medicamentos , Organoides , Medicina de Precisão , Humanos , Organoides/efeitos dos fármacos , Organoides/citologia , Organoides/metabolismo , Animais , Descoberta de Drogas , Modelos Biológicos , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/metabolismoRESUMO
CD4+ T cells are typically considered as 'helper' or 'regulatory' populations that support and orchestrate the responses of other lymphocytes. However, they can also develop potent granzyme (Gzm)-mediated cytotoxic activity and CD4+ cytotoxic T cells (CTLs) have been amply documented both in humans and in mice, particularly in the context of human chronic infection and cancer. Despite the established description of CD4+ CTLs, as well as of the critical cytotoxic activity they exert against MHC class II-expressing targets, their developmental and memory maintenance requirements remain elusive. This is at least in part owing to the lack of a murine experimental system where CD4+ CTLs are stably induced. Here, we show that viral and bacterial vectors encoding the same epitope induce distinct CD4+ CTL responses in challenged mice, all of which are nevertheless transient in nature and lack recall properties. Consistent with prior reports, CD4+ CTL differentiation is accompanied by loss of TCF-1 expression, a transcription factor considered essential for memory T cell survival. Using genetic ablation of Tcf7, which encodes TCF-1, at the time of CD4+ T cell activation, we further show that, contrary to observations in CD8+ T cells, continued expression of TCF-1 is not required for CD4+ T cell memory survival. Whilst Tcf7-deficient CD4+ T cells persisted normally following retroviral infection, the CD4+ CTL subset still declined, precluding conclusive determination of the requirement for TCF-1 for murine CD4+ CTL survival. Using xenotransplantation of human CD4+ T cells into murine recipients, we demonstrate that human CD4+ CTLs develop and persist in the same experimental conditions where murine CD4+ CTLs fail to persist. These observations uncover a species-specific defect in murine CD4+ CTL persistence with implications for their use as a model system.
Assuntos
Linfócitos T CD8-Positivos , Células T de Memória , Animais , Humanos , Camundongos , Linfócitos T CD4-Positivos , Diferenciação Celular , Linfócitos T Citotóxicos/metabolismoRESUMO
Patients with cancer have higher COVID-19 morbidity and mortality. Here we present the prospective CAPTURE study (NCT03226886) integrating longitudinal immune profiling with clinical annotation. Of 357 patients with cancer, 118 were SARS-CoV-2-positive, 94 were symptomatic and 2 patients died of COVID-19. In this cohort, 83% patients had S1-reactive antibodies, 82% had neutralizing antibodies against WT, whereas neutralizing antibody titers (NAbT) against the Alpha, Beta, and Delta variants were substantially reduced. Whereas S1-reactive antibody levels decreased in 13% of patients, NAbT remained stable up to 329 days. Patients also had detectable SARS-CoV-2-specific T cells and CD4+ responses correlating with S1-reactive antibody levels, although patients with hematological malignancies had impaired immune responses that were disease and treatment-specific, but presented compensatory cellular responses, further supported by clinical. Overall, these findings advance the understanding of the nature and duration of immune response to SARS-CoV-2 in patients with cancer.
RESUMO
BACKGROUND: Differences in humoral immunity to coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), between children and adults remain unexplained, and the effect of underlying immune dysfunction or suppression is unknown. Here, we sought to examine the antibody immune competence of children and adolescents with prevalent inflammatory rheumatic diseases, juvenile idiopathic arthritis (JIA), juvenile dermatomyositis (JDM), and juvenile systemic lupus erythematosus (JSLE) against the seasonal human coronavirus (HCoV)-OC43 that frequently infects this age group. METHODS: Sera were collected from JIA (n = 118), JDM (n = 49), and JSLE (n = 30) patients and from healthy control (n = 54) children and adolescents prior to the coronavirus disease 19 (COVID-19) pandemic. We used sensitive flow-cytometry-based assays to determine titers of antibodies that reacted with the spike and nucleoprotein of HCoV-OC43 and cross-reacted with the spike and nucleoprotein of SARS-CoV-2, and we compared them with respective titers in sera from patients with multisystem inflammatory syndrome in children and adolescents (MIS-C). FINDINGS: Despite immune dysfunction and immunosuppressive treatment, JIA, JDM, and JSLE patients maintained comparable or stronger humoral responses than healthier peers, which was dominated by immunoglobulin G (IgG) antibodies to HCoV-OC43 spike, and harbored IgG antibodies that cross-reacted with SARS-CoV-2 spike. In contrast, responses to HCoV-OC43 and SARS-CoV-2 nucleoproteins exhibited delayed age-dependent class-switching and were not elevated in JIA, JDM, and JSLE patients, which argues against increased exposure. CONCLUSIONS: Consequently, autoimmune rheumatic diseases and their treatment were associated with a favorable ratio of spike to nucleoprotein antibodies. FUNDING: This work was supported by a Centre of Excellence Centre for Adolescent Rheumatology Versus Arthritis grant, 21593, UKRI funding reference MR/R013926/1, the Great Ormond Street Children's Charity, Cure JM Foundation, Myositis UK, Lupus UK, and the NIHR Biomedical Research Centres at GOSH and UCLH. This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK, the UK Medical Research Council, and the Wellcome Trust.
Assuntos
Doenças Autoimunes , COVID-19 , Coronavirus Humano OC43 , Doenças Reumáticas , Adolescente , Adulto , Anticorpos Antivirais , Formação de Anticorpos , COVID-19/complicações , Criança , Humanos , Imunoglobulina G , Nucleoproteínas , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Síndrome de Resposta Inflamatória SistêmicaRESUMO
The ubiquitin-proteasome system maintains protein homoeostasis, underpins the cell cycle, and is dysregulated in cancer. However, the role of individual E3 ubiquitin ligases, which mediate the final step in ubiquitin-mediated proteolysis, remains incompletely understood. Identified through screening for cancer-specific endogenous retroviral transcripts, we show that the little-studied E3 ubiquitin ligase HECTD2 exerts dominant control of tumour progression in melanoma. HECTD2 cell autonomously drives the proliferation of human and murine melanoma cells by accelerating the cell cycle. HECTD2 additionally regulates cancer cell production of immune mediators, initiating multiple immune suppressive pathways, which include the cyclooxygenase 2 (COX2) pathway. Accordingly, higher HECTD2 expression is associated with weaker anti-tumour immunity and unfavourable outcome of PD-1 blockade in human melanoma and counteracts immunity against a model tumour antigen in murine melanoma. This central, multifaceted role of HECTD2 in cancer cell-autonomous proliferation and in immune evasion may provide a single target for a multipronged therapy of melanoma.
Assuntos
Evasão da Resposta Imune , Ubiquitina-Proteína Ligases , Animais , Divisão Celular , Proliferação de Células , Humanos , Lipogênese , Melanoma , Camundongos , ProteóliseRESUMO
A correct identification of seropositive individuals for the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is of paramount relevance to assess the degree of protection of a human population to present and future outbreaks of the COVID-19 pandemic. We describe here a sensitive and quantitative flow cytometry method using the cytometer-friendly non-adherent Jurkat T-cell line that stably expresses the full-length native spike "S" protein of SARS-CoV-2 and a truncated form of the human EGFR that serves a normalizing role. S protein and huEGFRt coding sequences are separated by a T2A self-cleaving sequence, allowing to accurately quantify the presence of anti-S immunoglobulins by calculating a score based on the ratio of fluorescence intensities obtained by double-staining with the test sera and anti-EGFR. The method allows to detect immune individuals regardless of the result of other serological tests or even repeated PCR monitoring. As examples of its use, we show that as much as 28% of the personnel working at the CBMSO in Madrid is already immune. Additionally, we show that anti-S antibodies with protective neutralizing activity are long-lasting and can be detected in sera 8 months after infection.
Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , COVID-19/imunologia , Citometria de Fluxo/métodos , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , COVID-19/virologia , Teste Sorológico para COVID-19/estatística & dados numéricos , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/genética , Feminino , Citometria de Fluxo/estatística & dados numéricos , Células Hep G2 , Humanos , Células Jurkat , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Pandemias , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Patients with cancer have higher COVID-19 morbidity and mortality. Here we present the prospective CAPTURE study, integrating longitudinal immune profiling with clinical annotation. Of 357 patients with cancer, 118 were SARS-CoV-2 positive, 94 were symptomatic and 2 died of COVID-19. In this cohort, 83% patients had S1-reactive antibodies and 82% had neutralizing antibodies against wild type SARS-CoV-2, whereas neutralizing antibody titers against the Alpha, Beta and Delta variants were substantially reduced. S1-reactive antibody levels decreased in 13% of patients, whereas neutralizing antibody titers remained stable for up to 329 days. Patients also had detectable SARS-CoV-2-specific T cells and CD4+ responses correlating with S1-reactive antibody levels, although patients with hematological malignancies had impaired immune responses that were disease and treatment specific, but presented compensatory cellular responses, further supported by clinical recovery in all but one patient. Overall, these findings advance the understanding of the nature and duration of the immune response to SARS-CoV-2 in patients with cancer.
Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , COVID-19/imunologia , Neoplasias/complicações , Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/sangue , COVID-19/mortalidade , Feminino , Seguimentos , Humanos , Imunidade Celular , Masculino , Pessoa de Meia-Idade , Neoplasias/sangue , Neoplasias/imunologia , Estudos Prospectivos , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto JovemRESUMO
CD4+ T cells integrate well-defined signals from the T-cell receptor (TCR) (signal 1) and a host of costimulatory molecules (signal 2) to initiate clonal expansion and differentiation into diverse functional T helper (Th) subsets. However, our ability to guide the expansion of context-appropriate Th subsets by deploying these signals in vaccination remains limited. Using cell-based vaccines, we selectively amplified signal 1 by exclusive presentation of an optimized peptide:MHC II (pMHC II) complex in the absence of classic costimulation. Contrary to expectations, amplified signal 1 alone was strongly immunogenic and selectively expanded high-affinity TCR clonotypes, despite delivering intense TCR signals. In contrast to natural infection or standard vaccines, amplified signal 1, presented by a variety of professional and nonprofessional antigen-presenting cells (APCs), induced exclusively polyfunctional Th1 effector and memory cells, which protected against retroviral infection and tumor challenge, and expanded tumor-reactive CD4+ T cells otherwise rendered unresponsive in tumor-bearing hosts. Together, our findings uncover a default Th1 response to ample signal 1 and offer a means to selectively prime such protective responses by vaccination.
Assuntos
Células Apresentadoras de Antígenos , Ativação Linfocitária , Animais , Linfócitos T CD4-Positivos , Diferenciação Celular , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T , Linfócitos TRESUMO
Zoonotic introduction of novel coronaviruses may encounter preexisting immunity in humans. Using diverse assays for antibodies recognizing SARS-CoV-2 proteins, we detected preexisting humoral immunity. SARS-CoV-2 spike glycoprotein (S)-reactive antibodies were detectable using a flow cytometry-based method in SARS-CoV-2-uninfected individuals and were particularly prevalent in children and adolescents. They were predominantly of the immunoglobulin G (IgG) class and targeted the S2 subunit. By contrast, SARS-CoV-2 infection induced higher titers of SARS-CoV-2 S-reactive IgG antibodies targeting both the S1 and S2 subunits, and concomitant IgM and IgA antibodies, lasting throughout the observation period. SARS-CoV-2-uninfected donor sera exhibited specific neutralizing activity against SARS-CoV-2 and SARS-CoV-2 S pseudotypes. Distinguishing preexisting and de novo immunity will be critical for our understanding of susceptibility to and the natural course of SARS-CoV-2 infection.
Assuntos
Anticorpos Antivirais/sangue , COVID-19/imunologia , Imunidade Humoral , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Animais , COVID-19/sangue , Mapeamento de Epitopos , Feminino , Células HEK293 , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/química , Zoonoses Virais/sangue , Zoonoses Virais/imunologia , Adulto JovemRESUMO
Dendritic cells (DCs) are specialized antigen presenting cells that instruct T cell responses through sensing environmental and inflammatory danger signals. Maintaining the homeostasis of the multiple functionally distinct conventional dendritic cells (cDC) subsets that exist in vivo is crucial for regulating immune responses, with changes in numbers sufficient to break immune tolerance. Using Ptpn22-/- mice we demonstrate that the phosphatase PTPN22 is a highly selective, negative regulator of cDC2 homeostasis, preventing excessive population expansion from as early as 3 weeks of age. Mechanistically, PTPN22 mediates cDC2 homeostasis in a cell intrinsic manner by restricting cDC2 proliferation. A single nucleotide polymorphism, PTPN22R620W, is one of the strongest genetic risk factors for multiple autoantibody associated human autoimmune diseases. We demonstrate that cDC2 are also expanded in mice carrying the orthologous PTPN22619W mutation. As a consequence, cDC2 dependent CD4+ T cell proliferation and T follicular helper cell responses are increased. Collectively, our data demonstrate that PTPN22 controls cDC2 homeostasis, which in turn ensures appropriate cDC2-dependent T cell responses under antigenic challenge. Our findings provide a link between perturbations in DC development and susceptibility to a broad spectrum of PTPN22R620W associated human autoimmune diseases.
Assuntos
Autoimunidade/imunologia , Células Dendríticas/imunologia , Ativação Linfocitária/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 22/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Homeostase/imunologia , Tolerância Imunológica/imunologia , Camundongos , Camundongos Transgênicos , Polimorfismo de Nucleotídeo Único , Proteína Tirosina Fosfatase não Receptora Tipo 22/genéticaRESUMO
Effector T-cells rely on integrins to drive adhesion and migration to facilitate their immune function. The heterodimeric transmembrane integrin LFA-1 (αLß2 integrin) regulates adhesion and migration of effector T-cells through linkage of the extracellular matrix with the intracellular actin treadmill machinery. Here, we quantified the velocity and direction of F-actin flow in migrating T-cells alongside single-molecule localisation of transmembrane and intracellular LFA-1. Results showed that actin retrograde flow positively correlated and immobile actin negatively correlated with T-cell velocity. Plasma membrane-localised LFA-1 forms unique nano-clustering patterns in the leading edge, compared to the mid-focal zone, of migrating T-cells. Deleting the cytosolic phosphatase PTPN22, loss-of-function mutations of which have been linked to autoimmune disease, increased T-cell velocity, and leading-edge co-clustering of pY397 FAK, pY416 Src family kinases and LFA-1. These data suggest that differential nanoclustering patterns of LFA-1 in migrating T-cells may instruct intracellular signalling. Our data presents a paradigm where T-cells modulate the nanoscale organisation of adhesion and signalling molecules to fine tune their migration speed, with implications for the regulation of immune and inflammatory responses.This article has an associated First Person interview with the first author of the paper.
Assuntos
Movimento Celular , Antígeno-1 Associado à Função Linfocitária/metabolismo , Linfócitos T/citologia , Citoesqueleto de Actina/metabolismo , Animais , Adesão Celular , Membrana Celular/metabolismo , Células Cultivadas , Feminino , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação de Sentido Incorreto , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 22/metabolismo , Transdução de SinaisRESUMO
Protective immunity relies upon differentiation of T cells into the appropriate subtype required to clear infections and efficient effector T cell localization to antigen-rich tissue. Recent studies have highlighted the role played by subpopulations of tissue-resident memory (TRM) T lymphocytes in the protection from invading pathogens. The intestinal mucosa and associated lymphoid tissue are densely populated by a variety of resident lymphocyte populations, including αß and γδ CD8+ intraepithelial T lymphocytes (IELs) and CD4+ T cells. While the development of intestinal γδ CD8+ IELs has been extensively investigated, the origin and function of intestinal CD4+ T cells have not been clarified. We report that CCR9 signals delivered during naïve T cell priming promote the differentiation of a population of α4ß7+ IFN-γ-producing memory CD4+ T cells, which displays a TRM molecular signature, preferentially localizes to the gastrointestinal (GI) tract and associated lymphoid tissue and cannot be mobilized by remote antigenic challenge. We further show that this population shapes the immune microenvironment of GI tissue, thus affecting effector immunity in infection and cancer.
Assuntos
Quimiocinas CC/fisiologia , Intestinos/imunologia , Linfócitos T/imunologia , Animais , Feminino , Memória Imunológica , Infecções/imunologia , Interferon gama/biossíntese , Tecido Linfoide/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Receptores CCR/fisiologiaRESUMO
A C1858T single nucleotide polymorphism within PTPN22 (which encodes PTPN22R620W) is associated with an enhanced susceptibility to multiple autoimmune diseases including type 1 diabetes and rheumatoid arthritis. Many of the associated autoimmune diseases have an autoantibody component to their pathology. Fc receptors (FcRs) recognise autoantibodies when they bind to autoantigens and form immune complexes. After immune complex binding and receptor crosslinking, FcRs signal via Src and Syk family kinases, leading to antigen uptake, presentation and cytokine secretion. Ptpn22 encodes a protein tyrosine phosphatase that negatively regulates Src and Syk family kinases proximal to immunoreceptor signalling cascades. We therefore hypothesised that PTPN22 regulates immune complex stimulated FcR responses in dendritic cells (DCs). Bone marrow derived DCs (BMDCs) from wild type (WT) or Ptpn22-/- mice were pulsed with ovalbumin:anti-ovalbumin immune complexes (ova ICs). Co-culture with WT OT-II T cells revealed that ova IC pulsed Ptpn22-/- BMDCs have an enhanced capability to induce T cell proliferation. This was associated with an increased capability of Ptpn22-/- BMDCs to present immune complex derived antigens and to form ova IC dependent DC-T cell conjugates. These findings highlight PTPN22 as a regulator of FcR mediated responses and provide a link between the association of PTPN22R620W with autoantibody associated autoimmune diseases.
Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 22/metabolismo , Animais , Complexo Antígeno-Anticorpo/metabolismo , Células da Medula Óssea/citologia , Proliferação de Células/fisiologia , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Predisposição Genética para Doença/genética , Humanos , Camundongos , Camundongos Knockout , Polimorfismo de Nucleotídeo Único/genética , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Quinase Syk/genética , Quinase Syk/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
A missense C1858T single nucleotide polymorphism within PTPN22 is a strong genetic risk factor for the development of multiple autoimmune diseases. PTPN22 encodes a protein tyrosine phosphatase that negatively regulates immuno-receptor proximal Src and Syk family kinases. Notably, PTPN22 negatively regulates kinases downstream of T-cell receptor (TCR) and LFA-1, thereby setting thresholds for T-cell activation. Alterations to the quality of TCR and LFA-1 engagement at the immune synapse and the regulation of downstream signals can have profound effects on the type of effector T-cell response induced. Here we describe how IFNγ+ Th1 responses are potentiated in Ptpn22-/- T-cells and in T-cells from mice expressing Ptpn22R619W (the mouse orthologue of the human genetic variant) as they age, or following repeated immune challenge, and explore the mechanisms contributing to the expansion of Th1 cells. Specifically, we uncover two LFA-1-ICAM dependent mechanisms; one T-cell intrinsic, and one T-cell extrinsic. Firstly, we found that in vitro anti-CD3/LFA-1 induced Th1 responses were enhanced in Ptpn22-/- T-cells compared to WT, whereas anti-CD3/anti-CD28 induced IFNy responses were similar. These data were associated with an enhanced ability of Ptpn22-/- T-cells to engage ICAM-1 at the immune synapse when incubated on planar lipid bilayers, and to form conjugates with dendritic cells. Secondly, we observed a T-cell extrinsic mechanism whereby repeated stimulation of WT OT-II T-cells with LPS and OVA323-339 pulsed Ptpn22-/- bone marrow derived dendritic cells (BMDCs) was sufficient to enhance Th1 cell development compared to WT BMDCs. Furthermore, this response could be reversed by LFA-1 blockade. Our data point to two related but distinct mechanisms by which PTPN22 regulates LFA-1 dependent signals to enhance Th1 development, highlighting how perturbations to PTPN22 function over time to regulate the balance of the immune response.
Assuntos
Artrite Experimental/imunologia , Células Dendríticas/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 22/imunologia , Células Th1/imunologia , Animais , Anticorpos/farmacologia , Artrite Experimental/genética , Artrite Experimental/patologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Antígenos CD28/antagonistas & inibidores , Antígenos CD28/genética , Antígenos CD28/imunologia , Complexo CD3/antagonistas & inibidores , Complexo CD3/genética , Complexo CD3/imunologia , Proliferação de Células/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/patologia , Regulação da Expressão Gênica , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Bicamadas Lipídicas/química , Bicamadas Lipídicas/imunologia , Lipopolissacarídeos/farmacologia , Antígeno-1 Associado à Função Linfocitária/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/farmacologia , Fragmentos de Peptídeos/farmacologia , Polimorfismo de Nucleotídeo Único , Proteína Tirosina Fosfatase não Receptora Tipo 22/deficiência , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Células Th1/efeitos dos fármacos , Células Th1/patologiaRESUMO
A single nucleotide polymorphism within the PTPN22 gene is a strong genetic risk factor predisposing to the development of multiple autoimmune diseases. PTPN22 regulates Syk and Src family kinases downstream of immuno-receptors. Fungal ß-glucan receptor dectin-1 signals via Syk, and dectin-1 stimulation induces arthritis in mouse models. We investigated whether PTPN22 regulates dectin-1 dependent immune responses. Bone marrow derived dendritic cells (BMDCs) generated from C57BL/6 wild type (WT) and Ptpn22-/- mutant mice, were pulsed with OVA323-339 and the dectin-1 agonist curdlan and co-cultured in vitro with OT-II T-cells or adoptively transferred into OT-II mice, and T-cell responses were determined by immunoassay. Dectin-1 activated Ptpn22-/- BMDCs enhanced T-cell secretion of IL-17 in vitro and in vivo in an IL-1ß dependent manner. Immunoblotting revealed that compared to WT, dectin-1 activated Ptpn22-/- BMDCs displayed enhanced Syk and Erk phosphorylation. Dectin-1 activation of BMDCs expressing Ptpn22R619W (the mouse orthologue of human PTPN22R620W ) also resulted in increased IL-1ß secretion and T-cell dependent IL-17 responses, indicating that in the context of dectin-1 Ptpn22R619W operates as a loss-of-function variant. These findings highlight PTPN22 as a novel regulator of dectin-1 signals, providing a link between genetically conferred perturbations of innate receptor signaling and the risk of autoimmune disease.
Assuntos
Doenças Autoimunes/genética , Células Dendríticas/fisiologia , Lectinas Tipo C/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 22/metabolismo , Células Th17/imunologia , Animais , Células Cultivadas , Técnicas de Cocultura , Humanos , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Polimorfismo de Nucleotídeo Único , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Risco , Transdução de SinaisRESUMO
The PTPN22R620W single nucleotide polymorphism increases the risk of developing multiple autoimmune diseases including type 1 diabetes, rheumatoid arthritis and lupus. PTPN22 is highly expressed in antigen presenting cells (APCs) where the expression of the murine disease associated variant orthologue (Ptpn22R619W) is reported to dysregulate pattern recognition receptor signalling in dendritic cells (DCs) and promote T-cell proliferation. Because T-cell activation is dependent on DC antigen uptake, degradation and presentation, we analysed the efficiency of these functions in splenic and GM-CSF bone marrow derived DC from wild type (WT), Ptpn22-/- or Ptpn22R619W mutant mice. Results indicated no differential ability of DCs to uptake antigen via macropinocytosis or receptor-mediated endocytosis. Antigen degradation and presentation was also equal as was WT T-cell conjugate formation and subsequent T-cell proliferation. Despite the likely presence of multiple phosphatase-regulated pathways in the antigen uptake, processing and presentation pathways that we investigated, we observed that Ptpn22 and the R619W autoimmune associated variant were dispensable. These important findings indicate that under non-inflammatory conditions there is no requirement for Ptpn22 in DC dependent antigen uptake and T-cell activation. Our findings reveal that perturbations in antigen uptake and processing, a fundamental pathway determining adaptive immune responses, are unlikely to provide a mechanism for the risk associated with the Ptpn22 autoimmune associated polymorphism.
Assuntos
Apresentação de Antígeno , Antígenos de Bactérias/química , Células Dendríticas/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 22/imunologia , Linfócitos T/imunologia , Animais , Antígenos de Bactérias/imunologia , Técnicas de Cocultura , Células Dendríticas/citologia , Endocitose/imunologia , Corantes Fluorescentes/química , Expressão Gênica , Sinapses Imunológicas , Listeria monocytogenes/química , Listeria monocytogenes/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/química , Ovalbumina/imunologia , Pinocitose/imunologia , Polimorfismo de Nucleotídeo Único , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Transdução de Sinais , Coloração e Rotulagem , Linfócitos T/citologiaRESUMO
Tris(hydroxypyridinone) chelators conjugated to peptides can rapidly complex the positron-emitting isotope gallium-68 (68Ga) under mild conditions, and the resulting radiotracers can delineate peptide receptor expression at sites of diseased tissue in vivo. We have synthesized a dendritic bifunctional chelator containing nine 1,6-dimethyl-3-hydroxypyridin-4-one groups (SCN-HP9) that can coordinate up to three Ga3+ ions. This derivative has been conjugated to a trimeric peptide (RGD3) containing three peptide groups that target the αvß3 integrin receptor. The resulting dendritic compound, HP9-RGD3, can be radiolabeled in 97% radiochemical yield at a 3-fold higher specific activity than its homologues HP3-RGD and HP3-RGD3 that contain only a single metal binding site. PET scanning and biodistribution studies show that [68Ga(HP9-RGD3)] demonstrates higher receptor-mediated tumor uptake in animals bearing U87MG tumors that overexpress αvß3 integrin than [68Ga(HP3-RGD)] and [68Ga(HP3-RGD3)]. However, concomitant nontarget organ retention of [68Ga(HP9-RGD3)] results in low tumor to nontarget organ contrast in PET images. On the other hand, the trimeric peptide homologue containing a single tris(hydroxypyridinone) chelator, [68Ga(HP3-RGD3)], clears nontarget organs and exhibits receptor-mediated uptake in mice bearing tumors and in mice with induced rheumatoid arthritis. PET imaging with [68Ga(HP3-RGD3)] enables clear delineation of αvß3 integrin receptor expression in vivo.
Assuntos
Quelantes/química , Radioisótopos de Gálio/química , Integrina alfaVbeta3/análise , Oligopeptídeos/química , Tomografia por Emissão de Pósitrons/métodos , Piridinas/química , Animais , Artrite Reumatoide/diagnóstico por imagem , Quelantes/farmacocinética , Feminino , Radioisótopos de Gálio/farmacocinética , Articulações/diagnóstico por imagem , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias/diagnóstico por imagem , Oligopeptídeos/farmacocinética , Piridinas/farmacocinética , Distribuição TecidualRESUMO
Integrins are heterodimeric transmembrane proteins that play a fundamental role in the migration of leukocytes to sites of infection or injury. We found that protein tyrosine phosphatase nonreceptor type 22 (PTPN22) inhibits signaling by the integrin lymphocyte function-associated antigen-1 (LFA-1) in effector T cells. PTPN22 colocalized with its substrates at the leading edge of cells migrating on surfaces coated with the LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1). Knockout or knockdown of PTPN22 or expression of the autoimmune disease-associated PTPN22-R620W variant resulted in the enhanced phosphorylation of signaling molecules downstream of integrins. Superresolution imaging revealed that PTPN22-R620 (wild-type PTPN22) was present as large clusters in unstimulated T cells and that these disaggregated upon stimulation of LFA-1, enabling increased association of PTPN22 with its binding partners at the leading edge. The failure of PTPN22-R620W molecules to be retained at the leading edge led to increased LFA-1 clustering and integrin-mediated cell adhesion. Our data define a previously uncharacterized mechanism for fine-tuning integrin signaling in T cells, as well as a paradigm of autoimmunity in humans in which disease susceptibility is underpinned by inherited phosphatase mutations that perturb integrin function.
Assuntos
Autoimunidade/fisiologia , Molécula 1 de Adesão Intercelular/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 22/imunologia , Linfócitos T , Substituição de Aminoácidos , Animais , Adesão Celular/genética , Adesão Celular/imunologia , Humanos , Molécula 1 de Adesão Intercelular/genética , Antígeno-1 Associado à Função Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/imunologia , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
T-cell protein microclusters have until recently been investigable only as microscale entities with their composition and structure being discerned by biochemistry or diffraction-limited light microscopy. With the advent of super resolution microscopy comes the ability to interrogate the structure and function of these clusters at the single molecule level by producing highly accurate pointillist maps of single molecule locations at ~20nm resolution. Analysis tools have also been developed to provide rich descriptors of the pointillist data, allowing us to pose questions about the nanoscale organization which governs the local and cell wide responses required of a migratory T-cell.
Assuntos
Sinapses Imunológicas/química , Conformação Proteica , Proteínas/química , Linfócitos T/química , Movimento Celular/imunologia , Sinapses Imunológicas/ultraestrutura , Integrinas/química , Microscopia de Fluorescência , Proteínas/ultraestrutura , Linfócitos T/imunologia , Linfócitos T/ultraestruturaRESUMO
Interleukin (IL)-7 is critical for the maintenance of the peripheral T-cell compartment of the adaptive immune system. IL-7 receptor α (IL-7Rα) expression is subject to developmental regulation and new T cells induce expression as they leave the thymus, which is essential for their long-term survival. It is not understood how this expression is regulated. Here, we identify a role for the Nuclear Factor κ-B (NF-κB) signaling pathway in controlling expression of IL-7Rα in new T cells. Perturbations to NF-κB signaling, either by deletion of Inhibitor of Kappa-B Kinase-2 (IKK2) or by inhibiting Rel dimer activity, prevented normal IL-7Rα expression in new T cells. Defective IL-7Rα expression resulted in impaired survival and homeostatic cell division responses by T cells that could be attributed to their failure to express IL-7Rα normally. Surprisingly, NF-κB signaling was only required transiently in new T cells to allow their normal expression of IL-7Rα, because IKK2 deletion in mature T cells had no effect on IL-7Rα expression or their normal homeostatic responsiveness. Therefore, we identify a developmental function for NF-κB signaling in the homeostatic maturation of new T cells, by regulating IL-7Rα expression.