RESUMO
FOXP3 deficiency in mice and in patients with immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome results in fatal autoimmunity by altering regulatory T (Treg) cells. CD4+ T cells in patients with IPEX syndrome and Foxp3-deficient mice were analyzed by single-cell cytometry and RNA-sequencing, revealing heterogeneous Treg-like cells, some very similar to normal Treg cells, others more distant. Conventional T cells showed no widespread activation or helper T cell bias, but a monomorphic disease signature affected all CD4+ T cells. This signature proved to be cell extrinsic since it was extinguished in mixed bone marrow chimeric mice and heterozygous mothers of patients with IPEX syndrome. Normal Treg cells exerted dominant suppression, quenching the disease signature and revealing in mutant Treg-like cells a small cluster of genes regulated cell-intrinsically by FOXP3, including key homeostatic regulators. We propose a two-step pathogenesis model: cell-intrinsic downregulation of core FOXP3-dependent genes destabilizes Treg cells, de-repressing systemic mediators that imprint the disease signature on all T cells, furthering Treg cell dysfunction. Accordingly, interleukin-2 treatment improved the Treg-like compartment and survival.
Assuntos
Diabetes Mellitus Tipo 1/congênito , Diarreia/genética , Fatores de Transcrição Forkhead/deficiência , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças do Sistema Imunitário/congênito , Linfócitos T Reguladores/imunologia , Adolescente , Animais , Estudos de Casos e Controles , Criança , Pré-Escolar , Estudos de Coortes , Conjuntos de Dados como Assunto , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Diarreia/sangue , Diarreia/imunologia , Modelos Animais de Doenças , Citometria de Fluxo , Fatores de Transcrição Forkhead/genética , Doenças Genéticas Ligadas ao Cromossomo X/sangue , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Humanos , Doenças do Sistema Imunitário/sangue , Doenças do Sistema Imunitário/genética , Doenças do Sistema Imunitário/imunologia , Lactente , Masculino , Camundongos , Camundongos Transgênicos , Mutação , RNA-Seq , Análise de Célula Única , Linfócitos T Reguladores/metabolismo , Adulto JovemRESUMO
FoxP3 is an essential transcription factor (TF) for immunologic homeostasis, but how it utilizes the common forkhead DNA-binding domain (DBD) to perform its unique function remains poorly understood. We here demonstrated that unlike other known forkhead TFs, FoxP3 formed a head-to-head dimer using a unique linker (Runx1-binding region [RBR]) preceding the forkhead domain. Head-to-head dimerization conferred distinct DNA-binding specificity and created a docking site for the cofactor Runx1. RBR was also important for proper folding of the forkhead domain, as truncation of RBR induced domain-swap dimerization of forkhead, which was previously considered the physiological form of FoxP3. Rather, swap-dimerization impaired FoxP3 function, as demonstrated with the disease-causing mutation R337Q, whereas a swap-suppressive mutation largely rescued R337Q-mediated functional impairment. Altogether, our findings suggest that FoxP3 can fold into two distinct dimerization states: head-to-head dimerization representing functional specialization of an ancient DBD and swap dimerization associated with impaired functions.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core , Linfócitos T Reguladores , Subunidade alfa 2 de Fator de Ligação ao Core/genética , DNA , Dimerização , Fatores de Transcrição Forkhead/metabolismo , HomeostaseRESUMO
Vertebrate cell identity depends on the combined activity of scores of transcription factors (TF). While TFs have often been studied in isolation, a systematic perspective on their integration has been missing. Focusing on FoxP3+ regulatory T cells (Tregs), key guardians of immune tolerance, we combined single-cell chromatin accessibility, machine learning, and high-density genetic variation, to resolve a validated framework of diverse Treg chromatin programs, each shaped by multi-TF inputs. This framework identified previously unrecognized Treg controllers (Smarcc1) and illuminated the mechanism of action of FoxP3, which amplified a pre-existing Treg identity, diversely activating or repressing distinct programs, dependent on different regulatory partners. Treg subpopulations in the colon relied variably on FoxP3, Helios+ Tregs being completely dependent, but RORγ+ Tregs largely independent. These differences were rooted in intrinsic biases decoded by the integrated framework. Moving beyond master regulators, this work unravels how overlapping TF activities coalesce into Treg identity and diversity.
Assuntos
Fatores de Transcrição Forkhead , Linfócitos T Reguladores , Fatores de Transcrição , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Cromatina/metabolismo , Cromatina/genética , Análise de Célula Única/métodosRESUMO
Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) provokes a potentially fatal pneumonia with multiorgan failure, and high systemic inflammation. To gain mechanistic insight and ferret out the root of this immune dysregulation, we modeled, by in vitro coculture, the interactions between infected epithelial cells and immunocytes. A strong response was induced in monocytes and B cells, with a SARS-CoV-2-specific inflammatory gene cluster distinct from that seen in influenza A or Ebola virus-infected cocultures, and which reproduced deviations reported in blood or lung myeloid cells from COVID-19 patients. A substantial fraction of the effect could be reproduced after individual transfection of several SARS-CoV-2 proteins (Spike and some nonstructural proteins), mediated by soluble factors, but not via transcriptional induction. This response was greatly muted in monocytes from healthy children, perhaps a clue to the age dependency of COVID-19. These results suggest that the inflammatory malfunction in COVID-19 is rooted in the earliest perturbations that SARS-CoV-2 induces in epithelia.
Assuntos
COVID-19/imunologia , Células Epiteliais/imunologia , Monócitos/imunologia , SARS-CoV-2/patogenicidade , Adulto , Linfócitos B/imunologia , COVID-19/patologia , Criança , Técnicas de Cocultura , Ebolavirus/patogenicidade , Células Epiteliais/virologia , Perfilação da Expressão Gênica , Humanos , Inflamação , Vírus da Influenza A/patogenicidade , Pulmão/imunologia , Células Mieloides/imunologia , Especificidade da Espécie , Proteínas Virais/imunologiaRESUMO
The hallmark of severe COVID-19 is an uncontrolled inflammatory response, resulting from poorly understood immunological dysfunction. We hypothesized that perturbations in FoxP3+ T regulatory cells (Treg), key enforcers of immune homeostasis, contribute to COVID-19 pathology. Cytometric and transcriptomic profiling revealed a distinct Treg phenotype in severe COVID-19 patients, with an increase in Treg proportions and intracellular levels of the lineage-defining transcription factor FoxP3, correlating with poor outcomes. These Tregs showed a distinct transcriptional signature, with overexpression of several suppressive effectors, but also proinflammatory molecules like interleukin (IL)-32, and a striking similarity to tumor-infiltrating Tregs that suppress antitumor responses. Most marked during acute severe disease, these traits persisted somewhat in convalescent patients. A screen for candidate agents revealed that IL-6 and IL-18 may individually contribute different facets of these COVID-19-linked perturbations. These results suggest that Tregs may play nefarious roles in COVID-19, by suppressing antiviral T cell responses during the severe phase of the disease, and by a direct proinflammatory role.
Assuntos
COVID-19/etiologia , Linfócitos T Reguladores/fisiologia , Adulto , Idoso , Linfócitos T CD4-Positivos/virologia , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Inflamação/metabolismo , Inflamação/virologia , Interleucina-18/genética , Interleucina-18/metabolismo , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Linfócitos do Interstício Tumoral/fisiologia , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/virologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is caused by mutations in forkhead box P3 (FOXP3), which lead to the loss of function of regulatory T cells (Tregs) and the development of autoimmune manifestations early in life. The selective induction of a Treg program in autologous CD4+ T cells by FOXP3 gene transfer is a promising approach for curing IPEX. We have established a novel in vivo assay of Treg functionality, based on adoptive transfer of these cells into scurfy mice (an animal model of IPEX) and a combination of cyclophosphamide (Cy) conditioning and interleukin-2 (IL-2) treatment. This model highlighted the possibility of rescuing scurfy disease after the latter's onset. By using this in vivo model and an optimized lentiviral vector expressing human Foxp3 and, as a reporter, a truncated form of the low-affinity nerve growth factor receptor (ΔLNGFR), we demonstrated that the adoptive transfer of FOXP3-transduced scurfy CD4+ T cells enabled the long-term rescue of scurfy autoimmune disease. The efficiency was similar to that seen with wild-type Tregs. After in vivo expansion, the converted CD4FOXP3 cells recapitulated the transcriptomic core signature for Tregs. These findings demonstrate that FOXP3 expression converts CD4+ T cells into functional Tregs capable of controlling severe autoimmune disease.
Assuntos
Doenças Autoimunes/prevenção & controle , Linfócitos T CD4-Positivos/imunologia , Ciclofosfamida/farmacologia , Fatores de Transcrição Forkhead/genética , Doenças Genéticas Ligadas ao Cromossomo X/prevenção & controle , Interleucina-2/farmacologia , Linfócitos T Reguladores/imunologia , Animais , Antineoplásicos/farmacologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Imunossupressores/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/efeitos dos fármacosRESUMO
Overactivation of the complement alternative pathway drives the pathogenesis of primary atypical hemolytic uremic syndrome (aHUS). Genetically-determined or acquired dysregulation of the complement is frequently identified in patients with aHUS, pregnancy-related hemolytic uremic syndrome (HUS), and severe hypertension-associated HUS. In contrast, it is still unclear whether self-limited complement activation, which frequently occurs in other forms of HUS, provides key mechanistic clues or results from endothelial damage. Development of novel biomarkers is underway to firmly establish complement-driven pathogenesis. C5 blockade therapy has revolutionized the management of aHUS patients, resulting in a halving of the subpopulation under chronic dialysis over the course of a few years. On the other hand, the efficacy of C5 blockade in secondary forms of HUS, as assessed by small and uncontrolled case series, is less compelling and should be investigated through properly designed prospective clinical trials. The increased risk of meningococcal infection, related to C5 inhibition, must be rigorously addressed with suitable prophylaxis. Treatment duration should be determined based on an individualized benefit/risk assessment.
Assuntos
Síndrome Hemolítico-Urêmica Atípica , Humanos , Estudos Prospectivos , Síndrome Hemolítico-Urêmica Atípica/terapia , Ativação do Complemento , Fatores de Risco , BiomarcadoresRESUMO
Hemolytic diseases are frequently linked to multiorgan failure subsequent to vascular damage. Deciphering the mechanisms leading to organ injury upon hemolytic event could bring out therapeutic approaches. Complement system activation occurs in hemolytic disorders, such as sickle cell disease, but the pathological relevance and the acquisition of a complement-activating phenotype during hemolysis remain unclear. Here we found that intravascular hemolysis, induced by injection of phenylhydrazine, resulted in increased alanine aminotransferase plasma levels and NGAL expression. This liver damage was at least in part complement-dependent, since it was attenuated in complement C3-/- mice and by injection of C5-blocking antibody. We evidenced C3 activation fragments' deposits on liver endothelium in mice with intravascular hemolysis or injected with heme as well as on cultured human endothelial cells (EC) exposed to heme. This process was mediated by TLR4 signaling, as revealed by pharmacological blockade and TLR4 deficiency in mice. Mechanistically, TLR4-dependent surface expression of P-selectin triggered an unconventional mechanism of complement activation by noncovalent anchoring of C3 activation fragments, including the typical fluid-phase C3(H2O), measured by surface plasmon resonance and flow cytometry. P-selectin blockade by an antibody prevented complement deposits and attenuated the liver stress response, measured by NGAL expression, in the hemolytic mice. In conclusion, these results revealed the critical impact of the triad TLR4/P-selectin/complement in the liver damage and its relevance for hemolytic diseases. We anticipate that blockade of TLR4, P-selectin, or the complement system could prevent liver injury in hemolytic diseases like sickle cell disease.
Assuntos
Endotélio Vascular/metabolismo , Heme/metabolismo , Hemólise , Selectina-P/metabolismo , Receptor 4 Toll-Like/metabolismo , Alanina Transaminase/sangue , Anemia Falciforme , Animais , Ativação do Complemento , Complemento C3/metabolismo , Modelos Animais de Doenças , Inativação Gênica , Hemólise/efeitos dos fármacos , Humanos , Lipocalina-2/metabolismo , Fígado/lesões , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenil-Hidrazinas/antagonistas & inibidores , Transdução de Sinais , Receptor 4 Toll-Like/efeitos dos fármacos , Receptor 4 Toll-Like/genéticaRESUMO
Rhabdomyolysis is a life-threatening condition caused by skeletal muscle damage with acute kidney injury being the main complication dramatically worsening the prognosis. Specific treatment for rhabdomyolysis-induced acute kidney injury is lacking and the mechanisms of the injury are unclear. To clarify this, we studied intra-kidney complement activation (C3d and C5b-9 deposits) in tubules and vessels of patients and mice with rhabdomyolysis-induced acute kidney injury. The lectin complement pathway was found to be activated in the kidney, likely via an abnormal pattern of Fut2-dependent cell fucosylation, recognized by the pattern recognition molecule collectin-11 and this proceeded in a C4-independent, bypass manner. Concomitantly, myoglobin-derived heme activated the alternative pathway. Complement deposition and acute kidney injury were attenuated by pre-treatment with the heme scavenger hemopexin. This indicates that complement was activated in a unique double-trigger mechanism, via the alternative and lectin pathways. The direct pathological role of complement was demonstrated by the preservation of kidney function in C3 knockout mice after the induction of rhabdomyolysis. The transcriptomic signature for rhabdomyolysis-induced acute kidney injury included a strong inflammatory and apoptotic component, which were C3/complement-dependent, as they were normalized in C3 knockout mice. The intra-kidney macrophage population expressed a complement-sensitive phenotype, overexpressing CD11b and C5aR1. Thus, our results demonstrate a direct pathological role of heme and complement in rhabdomyolysis-induced acute kidney injury. Hence, heme scavenging and complement inhibition represent promising therapeutic strategies.
Assuntos
Injúria Renal Aguda , Rabdomiólise , Injúria Renal Aguda/etiologia , Animais , Ativação do Complemento , Humanos , Rim , Camundongos , Mioglobina , Rabdomiólise/complicaçõesRESUMO
BACKGROUND: Chronic histiocytic intervillositis (chronic intervillositis) is defined by a diffuse infiltration of monocytes into the intervillous space, which often leads to poor obstetrical outcomes, including recurrent intrauterine growth restriction, miscarriage, and fetal death. The pathogenesis of chronic intervillositis is still poorly defined, and there is an unmet medical need for improved management. OBJECTIVE: This study aimed to demonstrate the role of anti-human leukocyte antigen alloantibodies in the pathogenesis of chronic intervillositis through the application of criteria used in solid-organ transplantation for the diagnosis of antibody-mediated rejection. STUDY DESIGN: A multidisciplinary research study based on thorough immunologic and pathologic investigations was carried out for 2 separate couples who experienced recurrent secondary fetal losses following a first normal pregnancy associated with histologic evidence of chronic intervillositis. RESULTS: Very high levels of complement-fixing, fetus-specific antibodies targeting mismatched human leukocyte antigen alleles, harbored by the 2 paternal haplotypes, were identified in both cases. Polymorphic human leukocyte antigens were expressed on the surface of trophoblastic villi of the inflamed placenta but not in healthy placental tissue. The binding of alloantibodies to paternal human leukocyte antigens induced dramatic activation of the complement classical pathway in trophoblastic villi, leading to C4d deposition and formation of the terminal complex C5b-9. All requirements for the diagnosis of antibody-mediated placental rejection were fulfilled according to the criteria used in the Banff classification of allograft pathology. In silico analysis was performed using a human leukocyte antigen epitope viewer to reconstitute the human leukocyte antigen sensitization history. Reactivity against a single mismatched epitope present in the first-born healthy child accounted for a broad sensitization to human leukocyte antigens, including those harbored by the 2 paternal haplotypes. This finding explained the high rates of chronic intervillositis recurrence during subsequent pregnancies. CONCLUSION: This study provides novel mechanistic insights into the pathogenesis of chronic intervillositis and provides new avenues for individualized counseling and therapeutic options.
Assuntos
Vilosidades Coriônicas/patologia , Retardo do Crescimento Fetal/patologia , Isoanticorpos/sangue , Doenças Placentárias/patologia , Diagnóstico Pré-Natal , Adulto , Diagnóstico Diferencial , Feminino , Humanos , GravidezRESUMO
Acute graft-versus-host disease (GVHD) is a rare but frequently lethal complication after solid organ transplantation. GVHD occurs in unduly immunocompromised hosts but requires the escalation of immunosuppression, which does not discriminate between host and donor cells. In contrast, donor-targeted therapy would ideally mitigate graft-versus-host reactivity while sparing recipient immune functions. We report two children with end-stage renal disease and severe primary immune deficiency (Schimke syndrome) who developed severe steroid-resistant acute GVHD along with full and sustained donor T cell chimerism after isolated kidney transplantation. Facing a therapeutic dead end, we used a novel strategy based on the adoptive transfer of anti-HLA donor-specific antibodies (DSAs) through the transfusion of highly selected plasma. After approval by the appropriate regulatory authority, an urgent nationwide search was launched among more than 3800 registered blood donors with known anti-HLA sensitization. Adoptively transferred DSAs bound to and selectively depleted circulating donor T cells. The administration of DSA-rich plasma was well tolerated and notably did not induce antibody-mediated rejection of the renal allografts. Acute GVHD symptoms promptly resolved in one child. This report provides a proof of concept for a highly targeted novel therapeutic approach for solid organ transplantation-associated GVHD.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Rim , Criança , Doença Enxerto-Hospedeiro/etiologia , Humanos , Imunização Passiva , Transplante de Rim/efeitos adversos , Esteroides , Condicionamento Pré-TransplanteAssuntos
Vacinas contra COVID-19 , COVID-19 , Glomerulonefrite Membranosa , Transplante de Rim , Humanos , Vacina BNT162 , COVID-19/epidemiologia , COVID-19/prevenção & controle , Vacinas contra COVID-19/efeitos adversos , Glomerulonefrite Membranosa/etiologia , Transplante de Rim/efeitos adversos , RNA MensageiroRESUMO
Technologies that facilitate the bulk sequencing of small numbers of cells as well as single-cell RNA sequencing (scRNA-seq) have aided greatly in the study of viruses as these analyses can be used to differentiate responses from infected versus bystander cells in complex systems, including in organoid or animal studies. While protocols for these analyses are typically developed with biosafety level 2 (BSL-2) considerations in mind, such analyses are equally useful for the study of viruses that require higher biosafety containment levels. Many of these workstreams, however, are not directly compatible with the more stringent biosafety regulations of BSL-3 and BSL-4 laboratories ensuring virus inactivation and must therefore be modified. Here we show that TCL buffer (Qiagen), which was developed for bulk sequencing of small numbers of cells and also facilitates scRNA-seq, inactivates both Ebola virus (EBOV) and SARS-CoV-2, BSL-4 and BSL-3 viruses, respectively. We show that additional heat treatment, necessary for the more stringent biosafety concerns for BSL-4-derived samples, was additionally sufficient to inactivate EBOV-containing samples. Critically, this heat treatment had minimal effects on extracted RNA quality and downstream sequencing results.
RESUMO
Mutations of the transcription factor FoxP3 in patients with "IPEX" (immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome) disrupt regulatory T cells (Treg), causing an array of multiorgan autoimmunity. To understand the functional impact of mutations across FoxP3 domains, without genetic and environmental confounders, six human FOXP3 missense mutations are engineered into mice. Two classes of mutations emerge from combined immunologic and genomic analyses. A mutation in the DNA-binding domain shows the same lymphoproliferation and multiorgan infiltration as complete FoxP3 knockouts but delayed by months. Tregs expressing this mutant FoxP3 are destabilized by normal Tregs in heterozygous females compared with hemizygous males. Mutations in other domains affect chromatin opening differently, involving different cofactors and provoking more specific autoimmune pathology (dermatitis, colitis, diabetes), unmasked by immunological challenges or incrossing NOD autoimmune-susceptibility alleles. This work establishes that IPEX disease heterogeneity results from the actual mutations, combined with genetic and environmental perturbations, explaining then the intra-familial variation in IPEX.
Assuntos
Fatores de Transcrição Forkhead , Linfócitos T Reguladores , Animais , Feminino , Humanos , Masculino , Camundongos , Alelos , Fatores de Transcrição Forkhead/genética , Camundongos Endogâmicos NOD , Mutação/genéticaRESUMO
Gene expression programs are specified by higher-order chromatin structure and enhancer-promoter loops (EPLs). T regulatory cell (Treg) identity is dominantly specified by the transcription factor (TF) FoxP3, whose mechanism of action is unclear. We applied chromatin conformation capture with immunoprecipitation (HiChIP) in Treg and closely related conventional CD4+ T cells (Tconv). EPLs identified by H3K27Ac HiChIP showed a range of connection intensity, with some superconnected genes. TF-specific HiChIP showed that FoxP3 interacts with EPLs at a large number of genes, including some not differentially expressed in Treg versus Tconv, but enriched at the core Treg signature loci that it up-regulates. FoxP3 association correlated with heightened H3K27Ac looping, as ascertained by analysis of FoxP3-deficient Treg-like cells. There was marked asymmetry in the loci where FoxP3 associated at the enhancer- or the promoter-side of EPLs, with enrichment for different transcriptional cofactors. FoxP3 EPL intensity distinguished gene clusters identified by single-cell ATAC-seq as covarying between individual Tregs, supporting a direct transactivation model for FoxP3 in determining Treg identity.
Assuntos
Fatores de Transcrição Forkhead/imunologia , Regiões Promotoras Genéticas/genética , Linfócitos T Reguladores/imunologia , Animais , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Kidney disease affects 10% of the world population and is associated with increased mortality. Steroid-resistant nephrotic syndrome (SRNS) is a leading cause of end-stage kidney disease in children, often failing standard immunosuppression. Here, we report the results of a prospective study to investigate the immunological impact and safety of a gluten-free and dairy-free (GF/DF) diet in children with SRNS. The study was organized as a four-week summer camp implementing a strict GF/DF diet with prospective collection of blood, urine and stool in addition to whole exome sequencing WES of DNA of participants. Using flow cytometry, proteomic assays and microbiome metagenomics, we show that GF/DF diet had a major anti-inflammatory effect in all participants both at the protein and cellular level with 4-fold increase in T regulatory/T helper 17 cells ratio and the promotion of a favorable regulatory gut microbiota. Overall, GF/DF can have a significant anti-inflammatory effect in children with SRNS and further trials are warranted to investigate this potential dietary intervention in children with SRNS.
Assuntos
Laticínios/efeitos adversos , Dieta Livre de Glúten , Síndrome Nefrótica/congênito , Adolescente , Biomarcadores/sangue , Biomarcadores/urina , Criança , Pré-Escolar , Citocinas/sangue , Dieta Livre de Glúten/efeitos adversos , Estudos de Viabilidade , Feminino , Microbioma Gastrointestinal , Humanos , Lactente , Mediadores da Inflamação/sangue , Intestinos/microbiologia , Masculino , Síndrome Nefrótica/diagnóstico , Síndrome Nefrótica/dietoterapia , Síndrome Nefrótica/imunologia , Síndrome Nefrótica/microbiologia , Projetos Piloto , Estudo de Prova de Conceito , Estudos Prospectivos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
[This corrects the article DOI: 10.3389/fimmu.2021.624821.].
RESUMO
The use of chimeric antigen receptor (CAR)-engineered regulatory T cells (Tregs) has emerged as a promising strategy to promote immune tolerance. However, in conventional T cells (Tconvs), CAR expression is often associated with tonic signaling, which can induce CAR-T cell dysfunction. The extent and effects of CAR tonic signaling vary greatly according to the expression intensity and intrinsic properties of the CAR. Here, we show that the 4-1BB CSD-associated tonic signal yields a more dramatic effect in CAR-Tregs than in CAR-Tconvs with respect to activation and proliferation. Compared to CD28 CAR-Tregs, 4-1BB CAR-Tregs exhibit decreased lineage stability and reduced in vivo suppressive capacities. Transient exposure of 4-1BB CAR-Tregs to a Treg stabilizing cocktail, including an mTOR inhibitor and vitamin C, during ex vivo expansion sharply improves their in vivo function and expansion after adoptive transfer. This study demonstrates that the negative effects of 4-1BB tonic signaling in Tregs can be mitigated by transient mTOR inhibition.
Assuntos
Receptores de Antígenos Quiméricos/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Serina-Treonina Quinases TOR/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/terapia , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/metabolismo , Humanos , Imunossupressores/farmacologia , Imunoterapia Adotiva/métodos , Células Jurkat , Masculino , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Receptores de Antígenos Quiméricos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Transplante Heterólogo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
Hemopexin is the main plasmatic scavenger of cell-free heme, released in the context of intravascular hemolysis or major cell injury. Heme is indispensable for the oxygen transport by hemoglobin but when released outside of the erythrocytes it becomes a danger-associated molecular pattern, contributing to tissue injury. One of the mechanisms of pro-inflammatory action of heme is to activate the innate immune complement cascade. Therefore, we hypothesized that injection of hemopexin will prevent hemolysis-induced complement activation. Human plasma-derived hemopexin is compatible with the heme clearance machinery of the mice. 100 or 500 mg/kg of hemopexin was injected in C57Bl/6 mice before treatment with phenylhydrazine (inducer of erythrocytes lysis) or with PBS as a control. Blood was taken at different timepoints to determine the pharmacokinetic of injected hemopexin in presence and absence of hemolysis. Complement activation was determined in plasma, by the C3 cleavage (western blot) and in the kidneys (immunofluorescence). Kidney injury was evaluated by urea and creatinine in plasma and renal NGAL and HO-1 gene expression were measured. The pharmacokinetic properties of hemopexin (mass spectrometry) in the hemolytic mice were affected by the target-mediated drug disposition phenomenon due to the high affinity of binding of hemopexin to heme. Hemolysis induced complement overactivation and signs of mild renal dysfunction at 6 h, which were prevented by hemopexin, except for the NGAL upregulation. The heme-degrading capacity of the kidney, measured by the HO-1 expression, was not affected by the treatment. These results encourage further studies of hemopexin as a therapeutic agent in models of diseases with heme overload.
Assuntos
Ativação do Complemento/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Hemopexina/farmacologia , Hemopexina/farmacocinética , Animais , Humanos , Rim/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Intravascular hemolysis of any cause can induce acute kidney injury (AKI). Hemolysis-derived product heme activates the innate immune complement system and contributes to renal damage. Therefore, we explored the role of the master complement regulator Factor H (FH) in the kidney's resistance to hemolysis-mediated AKI. Acute systemic hemolysis was induced in mice lacking liver expression of FH (hepatoFH-/-, ~20% residual FH) and in WT controls, by phenylhydrazine injection. The impaired complement regulation in hepatoFH-/- mice resulted in a delayed but aggravated phenotype of hemolysis-related kidney injuries. Plasma urea as well as markers for tubular (NGAL, Kim-1) and vascular aggression peaked at day 1 in WT mice and normalized at day 2, while they increased more in hepatoFH-/- compared to the WT and still persisted at day 4. These were accompanied by exacerbated tubular dilatation and the appearance of tubular casts in the kidneys of hemolytic hepatoFH-/- mice. Complement activation in hemolytic mice occurred in the circulation and C3b/iC3b was deposited in glomeruli in both strains. Both genotypes presented with positive staining of FH in the glomeruli, but hepatoFH-/- mice had reduced staining in the tubular compartment. Despite the clear phenotype of tubular injury, no complement activation was detected in the tubulointerstitium of the phenylhydrazin-injected mice irrespective of the genotype. Nevertheless, phenylhydrazin triggered overexpression of C5aR1 in tubules, predominantly in hepatoFH-/- mice. Moreover, C5b-9 was deposited only in the glomeruli of the hemolytic hepatoFH-/- mice. Therefore, we hypothesize that C5a, generated in the glomeruli, could be filtered into the tubulointerstitium to activate C5aR1 expressed by tubular cells injured by hemolysis-derived products and will aggravate the tissue injury. Plasma-derived FH is critical for the tubular protection, since pre-treatment of the hemolytic hepatoFH-/- mice with purified FH attenuated the tubular injury. Worsening of acute tubular necrosis in the hepatoFH-/- mice was trigger-dependent, as it was also observed in LPS-induced septic AKI model but not in chemotherapy-induced AKI upon cisplatin injection. In conclusion, plasma FH plays a key role in protecting the kidneys, especially the tubules, against hemolysis-mediated injury. Thus, FH-based molecules might be explored as promising therapeutic agents in a context of AKI.