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The aim of our study was to determine whether granzyme B-expressing regulatory B cells (GZMB+ B cells) are enriched in the blood of transplant patients with renal graft tolerance. To achieve this goal, we analysed two single-cell RNA sequencing (scRNAseq) datasets: (1) peripheral blood mononuclear cells (PBMCs), including GZMB+ B cells from renal transplant patients, i.e., patients with stable graft function on conventional immunosuppressive treatment (STA, n = 3), drug-free tolerant patients (TOL, n = 3), and patients with antibody-mediated rejection (ABMR, n = 3), and (2) ex-vivo-induced GZMB+ B cells from these groups. In the patient PBMCs, we first showed that natural GZMB+ B cells were enriched in genes specific to Natural Killer (NK) cells (such as NKG7 and KLRD1) and regulatory B cells (such as GZMB, IL10, and CCL4). We performed a pseudotemporal trajectory analysis of natural GZMB+ B cells and showed that they were highly differentiated B cells with a trajectory that is very different from that of conventional memory B cells and linked to the transcription factor KLF13. By specifically analysing GZMB+ natural B cells in TOLs, we found that these cells had a very specific transcriptomic profile associated with a reduction in the expression of HLA molecules, apoptosis, and the inflammatory response (in general) in the blood and that this signature was conserved after ex vivo induction, with the induction of genes associated with migration processes, such as CCR7, CCL3, or CCL4. An analysis of receptor/ligand interactions between these GZMB+/- natural B cells and all of the immune cells present in PBMCs also demonstrated that GZMB+ B cells were the B cells that carried the most ligands and had the most interactions with other immune cells, particularly in tolerant patients. Finally, we showed that these GZMB+ B cells were able to infiltrate the graft under inflammatory conditions, thus suggesting that they can act in locations where immune events occur.
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Linfócitos B Reguladores , Granzimas , Transplante de Rim , Humanos , Granzimas/metabolismo , Granzimas/genética , Linfócitos B Reguladores/imunologia , Linfócitos B Reguladores/metabolismo , Diferenciação Celular , Feminino , Masculino , Sistema Imunitário/metabolismo , Pessoa de Meia-Idade , Rejeição de Enxerto/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/imunologiaRESUMO
In this study, essential oils and waste hydrosols of leaves of Ocimum tenuiflorum in four different geographical locations were extracted by hydrodistillation method and using gas chromatography / mass spectrometry (GC/MS) for chemical composition analysis. All four essential oil samples contained the main components (E)-ß-caryophyllene (27.8-49.0%), trans-ß-elemene (20.3-37.1%) and eugenol (9.0-44.0%). Three of the four hydrosol samples had eugenol in absolute content (94.5-98.6%), while the remaining hydrosol sample had two main components, elemicin (77.8%) and eugenol (14.2%). Essential oils and hydrosols demonstrated larvicidal activities against four important disease-transmitting mosquito species including Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Culex fuscocephala with 24-h LC50 values in the range 15.42-56.01 µg/mL and 53.88-97.80 µg/mL for the essential oils and the hydrosols, respectively. Essential oils and hydrosols strongly inhibited the acetylcholinesterase (AChE) enzyme of electric eels with IC50 values in the range of 25.35-107.19 µg/mL. Microemulsion (ME) can be considered as a sustainable pesticide formulation over 300 days and has improved larvicidal activity compared to free essential oil. The O. tenuiflorum in Vietnam can be considered a low-cost source of eugenol, botanical pesticides that control disease-transmitting mosquitoes, as well as having therapeutic potential to be further investigated.
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There is an unmet need for robust and clinically validated biomarkers of kidney allograft rejection. Here we present the KTD-Innov study (ClinicalTrials.gov, NCT03582436), an unselected deeply phenotyped cohort of kidney transplant recipients with a holistic approach to validate the clinical utility of precision diagnostic biomarkers. In 2018-2019, we prospectively enrolled consecutive adult patients who received a kidney allograft at seven French centers and followed them for a year. We performed multimodal phenotyping at follow-up visits, by collecting clinical, biological, immunological, and histological parameters, and analyzing a panel of 147 blood, urinary and kidney tissue biomarkers. The primary outcome was allograft rejection, assessed at each visit according to the international Banff 2019 classification. We evaluated the representativeness of participants by comparing them with patients from French, European, and American transplant programs transplanted during the same period. A total of 733 kidney transplant recipients (64.1% male and 35.9% female) were included during the study. The median follow-up after transplantation was 12.3 months (interquartile range, 11.9-13.1 months). The cumulative incidence of rejection was 9.7% at one year post-transplant. We developed a distributed and secured data repository in compliance with the general data protection regulation. We established a multimodal biomarker biobank of 16,736 samples, including 9331 blood, 4425 urinary and 2980 kidney tissue samples, managed and secured in a collaborative network involving 7 clinical centers, 4 analytical platforms and 2 industrial partners. Patients' characteristics, immune profiles and treatments closely resembled those of 41,238 French, European and American kidney transplant recipients. The KTD-Innov study is a unique holistic and multidimensional biomarker validation cohort of kidney transplant recipients representative of the real-world transplant population. Future findings from this cohort are likely to be robust and generalizable.
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Biomarcadores , Rejeição de Enxerto , Transplante de Rim , Humanos , Transplante de Rim/efeitos adversos , Biomarcadores/urina , Biomarcadores/sangue , Feminino , Masculino , Estudos Prospectivos , Pessoa de Meia-Idade , Adulto , França/epidemiologia , Estudos de Coortes , Transplantados/estatística & dados numéricosRESUMO
Introduction: Human Granzyme B (GZMB) regulatory B cells (Bregs) have suppressive properties on CD4+ effector T cells by a mechanism partially dependent on GZMB. Moreover, these cells may be easily induced in vitro making them interesting for cell therapy. Methods: We characterized this population of in vitro induced GZMB+Bregs using single cell transcriptomics. To investigate their regulatory properties, Bregs or total B cells were also co-cultured with T cells and scRNAseq was used to identify receptor ligand interactions and to reveal gene expression changes in the T cells. Results: We find that Bregs exhibit a unique set of 149 genes differentially expressed and which are implicated in proliferation, apoptosis, metabolism, and altered antigen presentation capacity consistent with their differentiated B cells profile. Notably, Bregs induced a strong inhibition of T cell genes associated to proliferation, activation, inflammation and apoptosis compared to total B cells. We identified and validated 5 receptor/ligand interactions between Bregs and T cells. Functional analysis using specific inhibitors was used to test their suppressive properties and we identified Lymphotoxin alpha (LTA) as a new and potent Breg ligand implicated in Breg suppressive properties. Discussion: We report for the first time for a role of LTA in GZMB+Bregs as an enhancer of GZMB expression, and involved in the suppressive properties of GZMB+Bregs in human. The exact mechanism of LTA/GZMB function in this specific subset of Bregs remains to be determined.
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Linfócitos B Reguladores , Linfotoxina-alfa , Humanos , Granzimas , Ligantes , Linfócitos T CD4-Positivos , Proliferação de CélulasRESUMO
Introduction: The human immune system contains cells with either effector/memory or regulatory functions. Besides the well-established CD4+CD25hiCD127lo regulatory T cells (Tregs), we and others have shown that B cells can also have regulatory functions since their frequency and number are increased in kidney graft tolerance and B cell depletion as induction therapy may lead to acute rejection. On the other hand, we have shown that CD28-CD8+ T cells represent a subpopulation with potent effector/memory functions. In the current study, we tested the hypothesis that kidney allograft rejection may be linked to an imbalance of effector/memory and regulatory immune cells. Methods: Based on a large cohort of more than 1000 kidney graft biopsies with concomitant peripheral blood lymphocyte phenotyping, we investigated the association between kidney graft rejection and the percentage and absolute number of circulating B cells, Tregs, as well as the ratio of B cells to CD28-CD8+ T cells and the ratio of CD28-CD8+ T cells to Tregs. Kidney graft biopsies were interpreted according to the Banff classification and divided into 5 biopsies groups: 1) normal/subnormal, 2) interstitial fibrosis and tubular atrophy grade 2/3 (IFTA), 3) antibody-mediated rejection (ABMR), 4) T cell mediated-rejection (TCMR), and 5) borderline rejection. We compared group 1 with the other groups as well as with a combined group 3, 4, and 5 (rejection of all types) using multivariable linear mixed models. Results and discussion: We found that compared to normal/subnormal biopsies, rejection of all types was marginally associated with a decrease in the percentage of circulating B cells (p=0.06) and significantly associated with an increase in the ratio of CD28-CD8+ T cells to Tregs (p=0.01). Moreover, ABMR, TCMR (p=0.007), and rejection of all types (p=0.0003) were significantly associated with a decrease in the ratio of B cells to CD28-CD8+ T cells compared to normal/subnormal biopsies. Taken together, our results show that kidney allograft rejection is associated with an imbalance between immune cells with effector/memory functions and those with regulatory properties.
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Linfócitos B Reguladores , Linfócitos T Reguladores , Humanos , Aloenxertos/metabolismo , Anticorpos/metabolismo , Linfócitos B Reguladores/metabolismo , Biópsia , Antígenos CD28 , Linfócitos T CD8-Positivos , Rim/patologiaRESUMO
Using essential oils to control vectors, intermediate hosts, and disease-causing microorganisms is a promising approach. The genus Croton in the family Euphorbiaceae is a large genus, with many species containing large amounts of essential oils, however, essential oil studies are limited in terms of the number of Croton species investigated. In this work, the aerial parts of C. hirtus growing wild in Vietnam were collected and analyzed by gas chromatography/mass spectrometry (GC/MS). A total of 141 compounds were identified in C. hirtus essential oil, in which sesquiterpenoids dominated, comprising 95.4%, including the main components ß-caryophyllene (32.8%), germacrene D (11.6%), ß-elemene (9.1%), α-humulene (8.5%), and caryophyllene oxide (5.0%). The essential oil of C. hirtus showed very strong biological activities against the larvae of four mosquito species with 24 h LC50 values in the range of 15.38-78.27 µg/mL, against Physella acuta adults with a 48 h LC50 value of 10.09 µg/mL, and against ATCC microorganisms with MIC values in the range of 8-16 µg/mL. In order to provide a comparison with previous works, a literature survey on the chemical composition, mosquito larvicidal, molluscicidal, antiparasitic, and antimicrobial activities of essential oils of Croton species was conducted. Seventy-two references (seventy articles and one book) out of a total of two hundred and forty-four references related to the chemical composition and bioactivity of essential oils of Croton species were used for this paper. The essential oils of some Croton species were characterized by their phenylpropanoid compounds. The experimental results of this research and the survey of the literature showed that Croton essential oils have the potential to be used to control mosquito-borne and mollusk-borne diseases, as well as microbial infections. Research on unstudied Croton species is needed to search for species with high essential oil contents and excellent biological activities.
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Croton , Culicidae , Inseticidas , Óleos Voláteis , Animais , Óleos Voláteis/química , Croton/química , Vietnã , Mosquitos Vetores , Óleo de Cróton , Inseticidas/químicaRESUMO
Multiple sclerosis is an autoimmune disease of the central nervous system. Yet, the autoimmune targets are still undefined. The extracellular e1 sequence of KCNJ10, the inwardly rectifying potassium channel 4.1, has been subject to fierce debate for its role as a candidate autoantigen in multiple sclerosis. Inwardly rectifying potassium channel 4.1 is expressed in the central nervous system but also in peripheral tissues, raising concerns about the central nervous system-specificity of such autoreactivity. Immunization of C57Bl6/J female mice with the e1 peptide (amino acids 83-120 of Kir4.1) induced anti-e1 immunoglobulin G- and T-cell responses and promoted demyelinating encephalomyelitis with B cell central nervous system enrichment in leptomeninges and T cells/macrophages in central nervous system parenchyma from forebrain to spinal cord, mostly in the white matter. Within our cohort of multiple sclerosis patients (n = 252), 6% exhibited high anti-e1 immunoglobulin G levels in serum as compared to 0.7% in the control cohort (n = 127; P = 0.015). Immunolabelling of inwardly rectifying potassium channel 4.1-expressing white matter glia with the anti-e1 serum from immunized mice increased during murine autoimmune neuroinflammation and in multiple sclerosis white matter as compared with controls. Strikingly, the mouse and human anti-e1 sera labelled astrocytoma cells when N-glycosylation was blocked with tunicamycin. Western blot confirmed that neuroinflammation induces Kir4.1 expression, including its shorter aglycosylated form in murine experimental autoencephalomyelitis and multiple sclerosis. In addition, recognition of inwardly rectifying potassium channel 4.1 using mouse anti-e1 serum in Western blot experiments under unreduced conditions or in cells transfected with the N-glycosylation defective N104Q mutant as compared to the wild type further suggests that autoantibodies target an e1 conformational epitope in its aglycosylated form. These data highlight the e1 sequence of inwardly rectifying potassium channel 4.1 as a valid central nervous system autoantigen with a disease/tissue-specific post-translational antigen modification as potential contributor to autoimmunity in some multiple sclerosis patients.
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We previously established a six-gene-based blood score associated with operational tolerance in kidney transplantation which was decreased in patients developing anti-HLA donor-specific antibodies (DSA). Herein, we aimed to confirm that this score is associated with immunological events and risk of rejection. We measured this using quantitative PCR (qPCR) and NanoString methods from an independent multicenter cohort of 588 kidney transplant recipients with paired blood samples and biopsies at one year after transplantation validating its association with pre-existing and de novo DSA. From 441 patients with protocol biopsy, there was a significant decrease of the score of tolerance in 45 patients with biopsy-proven subclinical rejection (SCR), a major threat associated with pejorative allograft outcomes that prompted an SCR score refinement. This refinement used only two genes, AKR1C3 and TCL1A, and four clinical parameters (previous experience of rejection, previous transplantation, sex of recipient and tacrolimus uptake). This refined SCR score was able to identify patients unlikely to develop SCR with a C-statistic of 0.864 and a negative predictive value of 98.3%. The SCR score was validated in an external laboratory, with two methods (qPCR and NanoString), and on 447 patients from an independent and multicenter cohort. Moreover, this score allowed reclassifying patients with discrepancies between the DSA presence and the histological diagnosis of antibody mediated rejection unlike kidney function. Thus, our refined SCR score could improve detection of SCR for closer and noninvasive monitoring, allowing early treatment of SCR lesions notably for patients DSA-positive and during lowering of immunosuppressive treatment.
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Transplante de Rim , Humanos , Transplante de Rim/efeitos adversos , Imunossupressores/uso terapêutico , Anticorpos , Tacrolimo/uso terapêutico , Soro Antilinfocitário , Expressão Gênica , Rejeição de Enxerto/diagnóstico , Rejeição de Enxerto/genética , Rejeição de Enxerto/prevenção & controle , Antígenos HLA/genética , Isoanticorpos , Estudos RetrospectivosRESUMO
Background: Colorectal cancer (CRC) is the fifth most common cancer with rising prevalence in Vietnam. However, there is no data about the mutational landscape and actionable alterations in the Vietnamese patients. During post-operative surveillance, clinical tools are limited to stratify risk of recurrence and detect residual disease. Method: In this prospective multi-center study, 103 CRC patients eligible for curative-intent surgery were recruited. Genomic DNA from tumor tissue and paired white blood cells were sequenced to profile all tumor-derived somatic mutations in 95 cancer-associated genes. Our bioinformatic algorithm identified top mutations unique for individual patient, which were then used to monitor the presence of circulating tumor DNA (ctDNA) in serial plasma samples. Results: The top mutated genes in our cohort were APC, TP53 and KRAS. 41.7% of the patients harbored KRAS and NRAS mutations predictive of resistance to Cetuximab and Panitumumab respectively; 41.7% had mutations targeted by either approved or experimental drugs. Using a personalized subset of top ranked mutations, we detected ctDNA in 90.5% of the pre-operative plasma samples, whereas carcinoembryonic antigen (CEA) was elevated in only 41.3% of them. Interim analysis after 16-month follow-up revealed post-operative detection of ctDNA in two patients that had recurrence, with the lead time of 4-10.5 months ahead of clinical diagnosis. CEA failed to predict recurrence in both cases. Conclusion: Our assay showed promising dual clinical utilities in residual cancer surveillance and actionable mutation profiling for targeted therapies in CRC patients. This could lay foundation to empower precision cancer medicine in Vietnam and other developing countries.
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Background: The World health organization (WHO) recently recommended standardized all-oral shorter regimens for rifampicin resistant Tuberculosis (RR-TB). For highly resistant Tuberculosis patients such as pre-XDR-TB: RR-TB plus additional resistance to fluoroquinolones (FQ), the 6-9-months bedaquiline (bedaquiline)-based regimens or BDQ-based long regimens are recommended. The role of second-line injectable (SLI) drugs in the treatment of drug resistant TB is restricted because of safety concerns. Nevertheless, it is not well-known how all-oral long regimens (BDQ-long) perform compared to SLI-containing long regimens (BDQ/SLI-long) in terms of safety and effectiveness among patients with highly resistant TB. Method: A prospective observational cohort of patients with RR-TB additionally resistant to fluoroquinolones and/or second-line injectable, treated with either BDQ-long or BDQ/SLI-long regimens according to the guidance of the National Tuberculosis Program of Vietnam, enrolled between December 2015 and June 2017. Results: Of 99 patients enrolled, 42 (42%) patients were treated with BDQ-long and 57 (57%) with BDQ/SLI-long. More than 85% of patients were previously exposed to both FQ and SLI. FQ and SLI resistance were confirmed in 28 (67%) and 41 (98%) in the BDQ-long cohort and 48 (84%) and 17 (30%) in the BDQ/SLI-long cohort, respectively. Treatment success was achieved among 29 (69%) and 46 (81%) patients on the BDQ-long and BDQ/SLI-long regimen, respectively (p = 0.2). For both regimens, median time to first smear/culture sputum conversion was 2 months. All patients experienced at least one adverse event (AE) and 85% of them had at least one severe Adverse events. The median time to a first severe adverse event was 2 months. Among patients treated with BDQ-long a higher proportion of patients had three QT-prolonging drugs in the regimen (26.2% versus 7.0%; p = 0.009). The severe prolonged QTcF was observed in 22 (52.4%) and 22 (38.6%) patients on BDQ-long and BDQ/SLI-long, respectively. Overall, 30 (30%) patients had to either temporary or permanently discontinued or more TB drugs due to AEs. Conclusion: Treatment success was similar for both all-oral and SLI-containing BDQ-based long regimens in highly resistant TB patients. Both regimens had a similar high frequency of AEs. For both BDQ-long and BDQ/SLI-long regimens active AEs monitoring is essential.
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BACKGROUND: The mechanisms regulating CD8+ T cell migration to nonlymphoid tissue during inflammation have not been fully elucidated, and the migratory properties of effector memory CD8+ T cells that re-express CD45RA (TEMRA CD8+ T cells) remain unclear, despite their roles in autoimmune diseases and allotransplant rejection. METHODS: We used single-cell proteomic profiling and functional testing of CD8+ T cell subsets to characterize their effector functions and migratory properties in healthy volunteers and kidney transplant recipients with stable or humoral rejection. RESULTS: We showed that humoral rejection of a kidney allograft is associated with an accumulation of cytolytic TEMRA CD8+ T cells in blood and kidney graft biopsies. TEMRA CD8+ T cells from kidney transplant recipients exhibited enhanced migratory properties compared with effector memory (EM) CD8+ T cells, with enhanced adhesion to activated endothelium and transmigration in response to the chemokine CXCL12. CXCL12 directly triggers a purinergic P2×4 receptor-dependent proinflammatory response of TEMRA CD8+ T cells from transplant recipients. The stimulation with IL-15 promotes the CXCL12-induced migration of TEMRA and EM CD8+ T cells and promotes the generation of functional PSGL1, which interacts with the cell adhesion molecule P-selectin and adhesion of these cells to activated endothelium. Although disruption of the interaction between functional PSGL1 and P-selectin prevents the adhesion and transmigration of both TEMRA and EM CD8+ T cells, targeting VLA-4 or LFA-1 (integrins involved in T cell migration) specifically inhibited the migration of TEMRA CD8+ T cells from kidney transplant recipients. CONCLUSIONS: Our findings highlight the active role of TEMRA CD8+ T cells in humoral transplant rejection and suggest that kidney transplant recipients may benefit from therapeutics targeting these cells.
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Linfócitos T CD8-Positivos , Transplante de Rim , Humanos , Transplantados , Selectina-P/metabolismo , Receptores Purinérgicos P2X4/metabolismo , Rejeição de Enxerto , Memória Imunológica , Proteômica , Antígenos Comuns de Leucócito/metabolismo , Subpopulações de Linfócitos T/metabolismoRESUMO
Malignant pleural mesothelioma (MPM) is an aggressive cancer mainly related to asbestos exposure. Despite recent therapeutic advances, notably immunotherapies, the benefit remains limited and restricted to a small percentage of patients. Thus, a better understanding of the disease is needed to identify new therapeutic strategies. Recently, interleukin 7 receptor (IL-7R) has been described as being expressed by MPM cells and associated with poorer patient survival. Thus, the aim of this work was to study the IL-7R/IL-7 pathway in MPM using patient samples. We found that, although more than 40% of MPM cells expressed IL-7R, IL-7 had no effect on their intracellular signaling. Accordingly, the addition of IL-7 to the culture medium did not affect MPM cell growth. Using The Cancer Genome Atlas (TCGA) database, we showed that high IL7 gene expression in MPM tumors was associated with a higher overall patient survival and an induction of genes involved in the immune response. In pleural effusions (PEs), we found that IL-7 concentration was not a good diagnostic biomarker. However, we observed that high IL-7 levels in PEs were associated with shorter survival of MPM patients, but not of lung cancer patients. The prognostic value of IL-7 was also conserved when only patients with epithelioid mesothelioma, the most common histological type of MPM, were analyzed. Taken together, our study suggests that, although the IL-7R/IL-7 signaling pathway is not functional in MPM cells, IL-7 expression in PEs may have prognostic value in MPM patients.
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Amianto , Neoplasias Pulmonares , Mesotelioma Maligno , Mesotelioma , Neoplasias Pleurais , Humanos , Mesotelioma/genética , Mesotelioma/diagnóstico , Neoplasias Pleurais/genética , Interleucina-7 , Prognóstico , Neoplasias Pulmonares/patologia , Receptores de Interleucina-7 , BiomarcadoresRESUMO
BACKGROUND: CD28-CD8+ T cells represent a differentiated CD8+ T cell subset that is found to be increased in various conditions associated with chronic antigenic stimulation such as aging, chronic viral infections, autoimmune diseases, cancers, and allotransplantation. METHODS: Using multivariate models, we analyzed a large cohort of 1032 kidney transplant patients in whom 1495 kidney graft biopsies were performed concomitant with a peripheral blood leukocyte phenotyping by flow cytometry. We investigated the association between the level of CD28-CD8+ T cells in the blood and the diagnosis of graft rejection according to the recent Banff classification of renal allograft pathology. FINDINGS: We found that antibody-mediated rejection (ABMR) was associated with a significant increase in the percentage as well as the absolute number of CD28-CD8+ T cells in the peripheral blood of kidney transplant patients at the time of biopsy. The confounder-adjusted mean difference of log percentage and log absolute value between the ABMR group and the normal/subnormal histology group were 0.29 (p=0.0004) and 0.38 (p=0.0004), respectively. Moreover, we showed that CD28-CD8+ T cells from the patients diagnosed with ABMR responded more rigorously to TCR and FcγRIIIA (CD16) engagement compared to their CD28+ counterparts as evidenced by an increase in the expression of IFNγ, TNFα, and CD107a. INTERPRETATION: Collectively, our data suggest that differentiated CD28-CD8+ T cells, with increased frequency, number, and function, may participate in the pathobiology of ABMR. Further studies are warranted to clarify the immunological role of this T cell subset in kidney graft rejection. FUNDING: Agence nationale de la recherche (France).
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Antígenos CD28 , Transplante de Rim , Aloenxertos , Anticorpos , Antígenos CD28/metabolismo , Linfócitos T CD8-Positivos , Humanos , Transplante de Rim/efeitos adversos , Receptores de Antígenos de Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Extracellular vesicles (EVs) have been extensively studied in the last two decades. It is now well documented that they can actively participate in the activation or regulation of immune system functions through different mechanisms, the most studied of which include protein-protein interactions and miRNA transfers. The functional diversity of EV-secreting cells makes EVs potential targets for immunotherapies through immune cell-derived EV functions. They are also a potential source of biomarkers of graft rejection through donor cells or graft environment-derived EV content modification. This review focuses on preclinical studies that describe the role of EVs from different cell types in immune suppression and graft tolerance and on the search for biomarkers of rejection.
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Vesículas Extracelulares/imunologia , Vesículas Extracelulares/metabolismo , Transplantes/imunologia , Transplantes/metabolismo , Biomarcadores/metabolismo , Comunicação Celular , Rejeição de Enxerto , Humanos , Sistema Imunitário , Tolerância ao Transplante , Transplantes/fisiopatologiaRESUMO
Exosomes are nano-sized vesicles secreted by most cells that contain a variety of biological molecules, such as lipids, proteins and nucleic acids. They have been recognized as important mediators for long-distance cell-to-cell communication and are involved in a variety of biological processes. Exosomes have unique advantages, positioning them as highly effective drug delivery tools and providing a distinct means of delivering various therapeutic agents to target cells. In addition, as a new clinical diagnostic biomarker, exosomes play an important role in many aspects of human health and disease, including endocrinology, inflammation, cancer, and cardiovascular disease. In this review, we summarize the development of exosome-based drug delivery tools and the validation of novel biomarkers, and illustrate the role of exosomes as therapeutic targets in the prevention and treatment of various diseases.
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Biomarcadores/metabolismo , Doenças Cardiovasculares/prevenção & controle , Sistemas de Liberação de Medicamentos , Exossomos/metabolismo , Inflamação/prevenção & controle , Neoplasias/prevenção & controle , Preparações Farmacêuticas/administração & dosagem , Doenças Cardiovasculares/metabolismo , Humanos , Inflamação/metabolismo , Neoplasias/metabolismoRESUMO
Granzyme B (GZMB)-expressing B cells inhibit CD4+ T-lymphocyte proliferation in a contact- and GZMB-dependent manner, through degradation of TCR zeta or induction of T-cell apoptosis. This regulatory B-cell population is present in human healthy individuals and represents about 1% of circulating B cells. Their small proportion requires the development of expansion methods to enable their study and envision clinical applications. We describe here how to expand GZMB-expressing B cells to obtain more than 90% of highly purified GZMB+ B cells, and the protocol of B/T cells coculture for the evaluation of the suppressive function of the GZMB+ B-cell population.
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Linfócitos B Reguladores/imunologia , Técnicas de Cultura de Células/métodos , Granzimas/isolamento & purificação , Apoptose/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B Reguladores/metabolismo , Proliferação de Células , Granzimas/imunologia , Granzimas/metabolismo , Humanos , Ativação Linfocitária , Linfócitos T Citotóxicos/imunologiaRESUMO
Granzyme B-expressing B cells have been shown to be an important regulatory B cell subset in humans. However, it is unclear which subpopulations of B cells express GZMB under normal conditions and which protocols effectively induce ex vivo expansion of GZMB+ B cells. We found that in the peripheral blood of normal individuals, plasmablasts were the major B cell subpopulation that expressed GZMB. However, when using an in vitro plasmablast differentiation protocol, we obtained only 2% GZMB+ B cells. Nevertheless, using an expansion mixture containing IL-21, anti-BCR, CpG oligodeoxynucleotide, CD40L, and IL-2, we were able to obtain more than 90% GZMB+ B cells after 3 d culture. GZMB+ B cells obtained through this protocol suppressed the proliferation of autologous and allogenic CD4+CD25- effector T cells. The suppressive effect of GZMB+ B cells was partially GZMB dependent and totally contact dependent but was not associated with an increase in effector T cell apoptosis or uptake of GZMB by effector T cells. Interestingly, we showed that GZMB produced by B cells promoted GZMB+ B cell proliferation in ERK1/2-dependent manner, facilitating GZMB+ B cell expansion. However, GZMB+ B cells tended to undergo apoptosis after prolonged stimulation, which may be considered a negative feedback mechanism to limit their uncontrolled expansion. Finally, we found that expanded GZMB+ B cells exhibited a regulatory phenotype and were enriched in CD307bhi, CD258hiCD72hi, and CD21loPD-1hi B cell subpopulations. Our study, to our knowledge, provides new insight into biology of GZMB+ B cells and an efficient method to expand GZMB+ B cells for future cell therapy applications.
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Linfócitos B Reguladores/microbiologia , Granzimas/imunologia , Apoptose/imunologia , Ligante de CD40/imunologia , Diferenciação Celular/imunologia , Proliferação de Células/fisiologia , Células Cultivadas , Humanos , Interleucinas/imunologia , Leucócitos Mononucleares/imunologiaRESUMO
BACKGROUND: Interleukin-7 (IL-7) is the most important cytokine for T-cell homeostasis. IL-7 signals through the IL-7 receptor (IL-7R) which is composed of an alpha chain (IL-7Rα), also called CD127 and a common gamma chain. T lymphocytes, especially T helper type 2, play a crucial role in the pathobiology of allergic asthma. OBJECTIVE: To study the effects of an anti-CD127 monoclonal antibody (mAb) in a murine model of allergic airway inflammation induced by house dust mite (HDM). METHODS: Allergic airway inflammation was induced in mice using a protocol comprising 4 weekly percutaneous sensitizations followed by 2 weekly intranasal challenges with total HDM extracts and treated by intraperitoneal injections of an anti-CD127 mAb. Because CD127 is shared by both IL-7R and the receptor for thymic stromal lymphopoietin (TSLP), a group of mice was also treated with an anti-IL-7 mAb to block only the IL-7 signalling pathway. RESULTS: Anti-CD127 mAb-treated mice showed significantly lower airway resistance in response to methacholine and improvement in lung histology compared with isotype mAb-treated animals. Anti-CD127 mAb treatment significantly decreased the mRNA expression of Th2 cytokines (IL-4, IL-5, and IL-13) and chemokines (CCL5/RANTES) in lung tissue, decreased the secretion of Th2 cytokines (IL-4, IL-5, and IL-13) and chemokines (CXCL1 and CCL11/eotaxin) in bronchoalveolar lavage fluid (BALF), decreased serum HDM-specific IgE, and reduced the number of total leucocytes and leucocyte subpopulations such as eosinophils, macrophages, lymphocytes, T lymphocytes, and ILC2 in BALF and lung tissue. Mice treated with anti-IL-7 mAb also showed less allergic airway inflammation as evidenced by significantly lower airway resistance and fewer leucocytes in BALF and lung tissue compared to mice treated with the corresponding isotype control mAb. CONCLUSION AND CLINICAL RELEVANCE: Targeting the IL-7Rα by an anti-CD127 mAb improves allergic airway inflammation in mice and presents as a potential therapeutic approach for allergic asthma.
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Anticorpos Monoclonais/farmacologia , Asma , Subunidade alfa de Receptor de Interleucina-7 , Transdução de Sinais/efeitos dos fármacos , Células Th2/imunologia , Animais , Anticorpos Monoclonais/imunologia , Asma/tratamento farmacológico , Asma/imunologia , Feminino , Inflamação/tratamento farmacológico , Inflamação/imunologia , Subunidade alfa de Receptor de Interleucina-7/antagonistas & inibidores , Subunidade alfa de Receptor de Interleucina-7/imunologia , Camundongos Endogâmicos BALB C , Pyroglyphidae/imunologia , Transdução de Sinais/imunologiaRESUMO
IL-7 is an important cytokine for T cell lymphopoiesis. Blockade of the IL-7 signaling pathway has been shown to induce long-term graft survival or graft tolerance in murine transplant models through inhibiting T cell homeostasis and favoring immunoregulation. In this study, we assessed for the first time the effects of a blocking anti-human cluster of differentiation 127 (CD127) mAb administered in combination with low-dose tacrolimus or thymoglobulin in a life-sustaining kidney allograft model in baboons. Contrary to our expectation, the addition of an anti-CD127 mAb to the treatment protocols did not prolong graft survival compared to low-dose tacrolimus alone or thymoglobulin alone. Anti-CD127 mAb administration led to full CD127 receptor occupancy during the follow-up period. However, all treated animals lost their kidney graft between 1 week and 2 weeks after transplantation. Unlike in rodents, in nonhuman primates, anti-CD127 mAb treatment does not decrease the absolute numbers of lymphocyte and lymphocyte subsets and does not effectively inhibit postdepletional T cell proliferation and homeostasis, suggesting that IL-7 is not a limiting factor for T cell homeostasis in primates.
Assuntos
Anticorpos Monoclonais/farmacologia , Rejeição de Enxerto/tratamento farmacológico , Sobrevivência de Enxerto/efeitos dos fármacos , Subunidade alfa de Receptor de Interleucina-7/imunologia , Transplante de Rim/efeitos adversos , Depleção Linfocítica/métodos , Receptores de Interleucina-7/antagonistas & inibidores , Animais , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto/imunologia , Papio , Complicações Pós-OperatóriasRESUMO
Humans cannot synthesize N-glycolylneuraminic acid (Neu5Gc) but dietary Neu5Gc can be absorbed and deposited on endothelial cells (ECs) and diet-induced anti-Neu5Gc antibodies (Abs) develop early in human life. While the interaction of Neu5Gc and diet-induced anti-Neu5Gc Abs occurs in all normal individuals, endothelium activation by elicited anti-Neu5Gc Abs following a challenge with animal-derived materials, such as following xenotransplantation, had been postulated. Ten primary human EC preparations were cultured with affinity-purified anti-Neu5Gc Abs from human sera obtained before or after exposure to Neu5Gc-glycosylated rabbit IgGs (elicited Abs). RNAs of each EC preparation stimulated in various conditions by purified Abs were exhaustively sequenced. EC transcriptomic patterns induced by elicited anti-Neu5Gc Abs, compared with pre-existing ones, were analyzed. qPCR, cytokines/chemokines release, and apoptosis were tested on some EC preparations. The data showed that anti-Neu5Gc Abs induced 967 differentially expressed (DE) genes. Most DE genes are shared following EC activation by pre-existing or anti-human T-cell globulin (ATG)-elicited anti-Neu5Gc Abs. Compared with pre-existing anti-Neu5Gc Abs, which are normal component of ECs environment, elicited anti-Neu5Gc Abs down-regulated 66 genes, including master genes of EC function. Furthermore, elicited anti-Neu5Gc Abs combined with complement-containing serum down-regulated most transcripts mobilized by serum alone. Both types of anti-Neu5Gc Abs-induced a dose- and complement-dependent release of selected cytokines and chemokines. Altogether, these data show that, compared with pre-existing anti-Neu5Gc Abs, ATG-elicited anti-Neu5Gc Abs specifically modulate genes related to cytokine responses, MAPkinase cascades, chemotaxis, and integrins and do not skew the EC transcriptome toward a pro-inflammatory profile in vitro.