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BACKGROUND: SARS-CoV-2 has triggered a pandemic and contributes to long-lasting morbidity. Several studies have investigated immediate cellular and humoral immune responses during acute infection. However, little is known about long-term effects of COVID-19 on the immune system. METHODS: We performed a longitudinal investigation of cellular and humoral immune parameters in 106 non-vaccinated subjects ten weeks (10 w) and ten months (10 m) after their first SARS-CoV-2 infection. Peripheral blood immune cells were analyzed by multiparametric flow cytometry, serum cytokines were examined by multiplex technology. Antibodies specific for the Spike protein (S), the receptor-binding domain (RBD) and the nucleocapsid protein (NC) were determined. All parameters measured 10 w and 10 m after infection were compared with those of a matched, noninfected control group (n = 98). RESULTS: Whole blood flow cytometric analyses revealed that 10 m after COVID-19, convalescent patients compared to controls had reduced absolute granulocyte, monocyte, and lymphocyte counts, involving T, B, and NK cells, in particular CD3+CD45RA+CD62L+CD31+ recent thymic emigrant T cells and non-class-switched CD19+IgD+CD27+ memory B cells. Cellular changes were associated with a reversal from Th1- to Th2-dominated serum cytokine patterns. Strong declines of NC- and S-specific antibody levels were associated with younger age (by 10.3 years, p < .01) and fewer CD3-CD56+ NK and CD19+CD27+ B memory cells. Changes of T-cell subsets at 10 m such as normalization of effector and Treg numbers, decline of RTE, and increase of central memory T cell numbers were independent of antibody decline pattern. CONCLUSIONS: COVID-19 causes long-term reduction of innate and adaptive immune cells which is associated with a Th2 serum cytokine profile. This may provide an immunological mechanism for long-term sequelae after COVID-19.
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Imunidade Adaptativa , Anticorpos Antivirais , COVID-19 , Citocinas , Imunidade Inata , SARS-CoV-2 , Células Th1 , Células Th2 , Humanos , COVID-19/imunologia , COVID-19/sangue , SARS-CoV-2/imunologia , Masculino , Citocinas/sangue , Feminino , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Pessoa de Meia-Idade , Células Th2/imunologia , Imunidade Adaptativa/imunologia , Adulto , Células Th1/imunologia , Células Th1/metabolismo , Idoso , Glicoproteína da Espícula de Coronavírus/imunologia , Estudos Longitudinais , Proteínas do Nucleocapsídeo de Coronavírus/imunologiaRESUMO
Virus-like nanoparticles (VNP) are regarded as efficient vaccination platforms and have proven to be useful for the non-anaphylactogenic delivery of allergen-specific immunotherapy in preclinical models previously. Herein, we sought to determine the mode of VNP uptake by antigen presenting cells (APC). Accordingly, we screened a collection of substances known to inhibit different uptake pathways by APC. The human leukemia monocytic cell line THP-1 and the murine dendritic cell line DC 2.4 were examined for the uptake of fluorescently labelled VNP in the presence or absence of inhibitors. The inhibitory effect of candidate substances that blocked VNP uptake in APC lines was subsequently evaluated in studies with primary APC present in splenocyte and lung cell homogenates in vitro and upon intratracheal application of VNP in vivo. The uptake of allergen-specific VNP in vitro and in vivo was mainly observed by macrophages and CD103+ dendritic cells and was sensitive to inhibitors that block macropinocytosis, such as hyperosmolarity induced by sucrose or the polyphenol compound Rottlerin at low micromolar concentrations but not by other inhibitors. Also, T-cell proliferation induced by allergen-specific VNP was significantly reduced by both substances. In contrast, substances that stimulate macropinocytosis, such as Heparin and phorbol myristate acetate (PMA), increased VNP-uptake and may, thus, help modulate allergen-specific T-cell responses. We have identified macropinocytosis as the principal uptake mechanism of APC for allergen-specific VNP in vitro and in vivo, paving the way for further improvement of VNP-based therapies, especially those that can be used for tolerance induction in allergy, in the future.
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BACKGROUND: COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has now become endemic and is currently one of the important respiratory virus infections regularly affecting mankind. The assessment of immunity against SARS-CoV-2 and its variants is important for guiding active and passive immunization and SARS-CoV-2-specific treatment strategies. METHODS: We here devised a novel flow cytometry-based diagnostic platform for the assessment of immunity against cell-bound virus antigens. This platform is based on a collection of HEK-293T cell lines which, as exemplified in our study, stably express the receptor-binding domains (RBDs) of the SARS-CoV-2 S-proteins of eight major SARS-CoV-2 variants, ranging from Wuhan-Hu-1 to Omicron. RESULTS: RBD-expressing cell lines stably display comparable levels of RBD on the surface of HEK-293T cells, as shown with anti-FLAG-tag antibodies directed against a N-terminally introduced 3x-FLAG sequence while the functionality of RBD was proven by ACE2 binding. We exemplify the usefulness and specificity of the cell-based test by direct binding of IgG and IgA antibodies of SARS-CoV-2-exposed and/or vaccinated individuals in which the assay shows a wide linear performance range both at very low and very high serum antibody concentrations. In another application, i.e., antibody adsorption studies, the test proved to be a powerful tool for measuring the ratios of individual variant-specific antibodies. CONCLUSION: We have established a toolbox for measuring SARS-CoV-2-specific immunity against cell-bound virus antigens, which may be considered as an important addition to the armamentarium of SARS-CoV-2-specific diagnostic tests, allowing flexible and quick adaptation to new variants of concern.
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Increasing evidence suggests multilineage cytopenias (also known as Evans syndrome) may be caused by inborn errors of immunity (IEI) with immune dysregulation. We studied a patient with autoimmune haemolytic anaemia and immune thrombocytopenia and identified a germline mutation in SASH3 (c.862C>T;p.Arg288Ter), indicating a recently identified IEI. Immunohistochemistry performed after clinically indicated splenectomy revealed severe hypoplasia/absence of germinal centres. The autoimmune phenotype was associated with an increased CD21low T-bet+ CD11c+ subset along with decreased regulatory T cells, impaired T-cell proliferation and T-cell exhaustion. The younger brother carries the same SASH3 mutation and shares immunophenotypic features but is currently clinical asymptomatic, indicating heterogeneity of SASH3 deficiency.
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Anemia Hemolítica Autoimune , Púrpura Trombocitopênica Idiopática , Trombocitopenia , Masculino , Humanos , Anemia Hemolítica Autoimune/genética , Trombocitopenia/genética , MutaçãoRESUMO
INTRODUCTION: Cervical scrofulous lymphadenitis due to Mycobacterium avium complex (MAC) in immunocompetent adults is a rare disease. The presence of MAC infections demands meticulous clinical evaluation of patients along with detailed phenotypic and functional evaluation of their immune system including next-generation sequencing (NGS) analyses of target genes. METHODS: Exact clinical histories of the index patients both suffering from retromandibular/cervical scrofulous lymphadenitis were obtained along with phenotypic and functional immunological evaluations of leukocyte populations followed by targeted NGS-based sequencing of candidate genes. RESULTS: Immunological investigations showed normal serum immunoglobulin and complement levels, but lymphopenia, which was caused by significantly reduced CD3+CD4+CD45RO+ memory T-cell and CD19+ B-cell numbers. Despite normal T-cell proliferation to a number of accessory cell-dependent and -independent stimuli, the PBMC of both patients elaborated clearly reduced levels of a number of cytokines, including IFN-γ, IL-10, IL-12p70, IL-1α, IL-1ß, and TNF-α upon TCR-dependent T-cell stimulation with CD3-coated beads but also superantigens. The IFN-γ production deficiency was confirmed for CD3+CD4+ helper and CD4+CD8+ cytotoxic T cells on the single-cell level by multiparametric flow cytometry irrespective of whether PMA/ionomycin-stimulated whole blood cells or gradient-purified PBMC was analyzed. In the female patient L1, targeted NGS-based sequencing revealed a homozygous c.110T>C mutation in the interferon-γ receptor type 1 (IFNGR1) leading to significantly reduced receptor expression on both CD14+ monocytes and CD3+ T cells. Patient S2 presented with normal IFNGR1 expression on CD14+ monocytes but significantly reduced IFNGR1 expression on CD3+ T cells, despite the absence of detectable homozygous mutations in the IFNGR1 itself or disease-related target genes. Exogenous addition of increasing doses of IFN-γ resulted in proper upregulation of high-affinity FcγRI (CD64) on monocytes from patient S2, whereas monocytes from patient L1 showed only partial induction of CD64 expression after incubation with high doses of IFN-γ. CONCLUSION: A detailed phenotypic and functional immunological examination is urgently required to determine the cause of a clinically relevant immunodeficiency, despite detailed genetic analyses.
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Linfadenite , Infecção por Mycobacterium avium-intracellulare , Adulto , Humanos , Feminino , Complexo Mycobacterium avium/genética , Complexo Mycobacterium avium/metabolismo , Leucócitos Mononucleares , Infecção por Mycobacterium avium-intracellulare/genética , Infecção por Mycobacterium avium-intracellulare/metabolismo , Citocinas/metabolismo , Linfadenite/metabolismoRESUMO
Background: Treg cells have been shown to be an important part of immune-homeostasis and IL-2 which is produced upon T cell receptor (TCR)-dependent activation of T lymphocytes has been demonstrated to critically participate in Treg development. Objective: To evaluate small molecule inhibitors (SMI) for the identification of novel IL-2/Treg enhancing compounds. Materials and methods: We used TCR-dependent and allergen-specific cytokine secretion of human and mouse T cells, next generation messenger ribonucleic acid sequencing (RNA-Seq) and two different models of allergic airway inflammation to examine lead SMI-compounds. Results: We show here that the reported 3-phosphoinositide dependent kinase-1 (PDK1) SMI BX-795 increased IL-2 in culture supernatants of Jurkat E6-1 T cells, human peripheral blood mononuclear cells (hPBMC) and allergen-specific mouse T cells upon TCR-dependent and allergen-specific stimulation while concomitantly inhibiting Th2 cytokine secretion. RNA-Seq revealed that the presence of BX-795 during allergen-specific activation of T cells induces a bona fide Treg cell type highly similar to iTreg but lacking Foxp3 expression. When applied in mugwort pollen and house dust mite extract-based models of airway inflammation, BX-795 significantly inhibited Th2 inflammation including expression of Th2 signature transcription factors and cytokines and influx into the lungs of type 2-associated inflammatory cells such as eosinophils. Conclusions: BX-795 potently uncouples IL-2 production from Th2 inflammation and induces Th-IL-2 cells, which highly resemble induced (i)Tregs. Thus, BX-795 may be a useful new compound for the treatment of allergic diseases.
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Interleucina-2 , Leucócitos Mononucleares , Camundongos , Animais , Humanos , Interleucina-2/metabolismo , Leucócitos Mononucleares/metabolismo , Células Th2 , Alérgenos/metabolismo , Inflamação/metabolismo , Citocinas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
BACKGROUND: Antibody-based tests are available for measuring SARS-CoV-2-specific immune responses but fast T-cell assays remain scarce. Robust T cell-based tests are needed to differentiate specific cellular immune responses after infection from those after vaccination. METHODS: One hundred seventeen individuals (COVID-19 convalescent patients: n = 40; SARS-CoV-2 vaccinees: n = 41; healthy controls: n = 36) were evaluated for SARS-CoV-2-specific cellular immune responses (proliferation, Th1, Th2, Th17, and inflammatory cytokines, activation-induced marker [AIM] expression) by incubating purified peripheral blood mononuclear cells (PBMC) or whole blood (WB) with SARS-CoV-2 peptides (S, N, or M), vaccine antigens (tetanus toxoid, tick borne encephalitis virus) or polyclonal stimuli (Staphylococcal enterotoxin, phytohemagglutinin). RESULTS: N-peptide mix stimulation of WB identified the combination of IL-2 and IL-13 secretion as superior to IFN-γ secretion to discriminate between COVID-19-convalescent patients and healthy controls (p < .0001). Comparable results were obtained with M- or S-peptides, the latter almost comparably recalled IL-2, IFN-γ, and IL-13 responses in WB of vaccinees. Analysis 10 months as opposed to 10 weeks after COVID-19, but not allergic disease status, positively correlated with IL-13 recall responses. WB cytokine responses correlated with cytokine and proliferation responses of PBMC. Antigen-induced neo-expression of the C-type lectin CD69 on CD4+ (p < .0001) and CD8+ (p = .0002) T cells informed best about the SARS-CoV-2 exposure status with additional benefit coming from CD25 upregulation. CONCLUSION: Along with N- and S-peptide-induced IL-2 and CD69 neo-expression, we suggest to include the type 2 cytokine IL-13 as T-cellular recall marker for SARS-CoV-2 specific T-cellular immune responses after infection and vaccination.
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COVID-19 , Leucócitos Mononucleares , Humanos , Citocinas/metabolismo , Imunidade Celular , Interleucina-13 , Interleucina-2 , Leucócitos Mononucleares/metabolismo , SARS-CoV-2 , VacinaçãoRESUMO
BACKGROUND: Prophylactic vaccination against infectious diseases may induce a state of long-term protection in the otherwise healthy host. However, the situation is less predictable in immunocompromised patients and may require adjustment of vaccination schedules and/or basic therapy. METHODS: A patient in full remission of multiple myeloma since the last three years and on long-term maintenance therapy with pomalidomide, a drug inhibiting angiogenesis and myeloma cell growth, was vaccinated twice with Comirnaty followed by two vaccinations with Vaxzevria. Seroconversion and SARS-CoV-2-specific cellular responses were monitored. RESULTS: No signs of seroconversion or T cellular memory were observed after the first "full immunization" with Comirnaty. Consequently, long-term-maintenance therapy with Pomalidomide was stopped and two additional shots of Vaxzevria were administered after which the patient seroconverted with Spike(S)-protein specific antibody levels reaching 49 BAU/mL, mild S-peptide pool-specific T cell proliferation, effector cytokine production (IL-2, IL-13), and T cellular activation with increased numbers of CD3+CD4+CD25+ T cells as compared to vaccinated and non-vaccinated control subjects. However, despite suspension of immunosuppression and administration of in total four consecutive heterologous SARS-CoV-2 vaccine shots, the patient did not develop neutralizing RBD-specific antibodies. CONCLUSIONS: Despite immunomonitoring-based adjustment of vaccination and/or therapy schedules vaccination success, with clear correlates of protection, the development of RBD-specific antibodies could not be achieved in the immunocompromised patient with current SARS-CoV-2 vaccines. Thus, our report emphasizes the need for improved active and passive immunization strategies for SARS-CoV-2 infections.
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BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the ongoing global COVID-19 pandemic. One possibility to control the pandemic is to induce sterilizing immunity through the induction and maintenance of neutralizing antibodies preventing SARS-CoV-2 from entering human cells to replicate in. METHODS: We report the construction and in vitro and in vivo characterization of a SARS-CoV-2 subunit vaccine (PreS-RBD) based on a structurally folded recombinant fusion protein consisting of two SARS-CoV-2 Spike protein receptor-binding domains (RBD) fused to the N- and C-terminus of hepatitis B virus (HBV) surface antigen PreS to enable the two unrelated proteins serving as immunologic carriers for each other. RESULTS: PreS-RBD, but not RBD alone, induced a robust and uniform RBD-specific IgG response in rabbits. Currently available genetic SARS-CoV-2 vaccines induce mainly transient IgG1 responses in vaccinated subjects whereas the PreS-RBD vaccine induced RBD-specific IgG antibodies consisting of an early IgG1 and sustained IgG4 antibody response in a SARS-CoV-2 naive subject. PreS-RBD-specific IgG antibodies were detected in serum and mucosal secretions, reacted with SARS-CoV-2 variants, including the omicron variant of concern and the HBV receptor-binding sites on PreS of currently known HBV genotypes. PreS-RBD-specific antibodies of the immunized subject more potently inhibited the interaction of RBD with its human receptor ACE2 and their virus-neutralizing titers (VNTs) were higher than median VNTs in a random sample of healthy subjects fully immunized with registered SARS-CoV-2 vaccines or in COVID-19 convalescent subjects. CONCLUSION: The PreS-RBD vaccine has the potential to serve as a combination vaccine for inducing sterilizing immunity against SARS-CoV-2 and HBV by stopping viral replication through the inhibition of cellular virus entry.
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Vacinas contra COVID-19 , COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19/imunologia , Humanos , Imunoglobulina G , Pandemias/prevenção & controle , Coelhos , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
BACKGROUND: The determinants of successful humoral immune response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of critical importance for the design of effective vaccines and the evaluation of the degree of protective immunity conferred by exposure to the virus. As novel variants emerge, understanding their likelihood of suppression by population antibody repertoires has become increasingly important. METHODS: In this study, we analyzed the SARS-CoV-2 polyclonal antibody response in a large population of clinically well-characterized patients after mild and severe COVID-19 using a panel of microarrayed structurally folded and unfolded SARS-CoV-2 proteins, as well as sequential peptides, spanning the surface spike protein (S) and the receptor-binding domain (RBD) of the virus. RESULTS: S- and RBD-specific antibody responses were dominated by immunoglobulin G (IgG), mainly IgG1 , and directed against structurally folded S and RBD and three distinct peptide epitopes in S2. The virus neutralization activity of patients´ sera was highly correlated with IgG antibodies specific for conformational but not sequential RBD epitopes and their ability to prevent RBD binding to its human receptor angiotensin-converting enzyme 2 (ACE2). Twenty percent of patients selectively lacked RBD-specific IgG. Only immunization with folded, but not with unfolded RBD, induced antibodies against conformational epitopes with high virus-neutralizing activity. Conformational RBD epitopes required for protection do not seem to be altered in the currently emerging virus variants. CONCLUSION: These results are fundamental for estimating the protective activity of antibody responses after natural infection or vaccination and for the design of vaccines, which can induce high levels of SARS-CoV-2-neutralizing antibodies conferring sterilizing immunity.
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COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , Epitopos , Humanos , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
The aim of this prospective study was to assess lymphocyte proliferative and cytokine response prior to and following tick-borne encephalitis (TBE) immunization among patients after allogeneic hematopoietic stem cell transplantation (HSCT). Seventeen adult patients 11-13 months after HSCT and eight unvaccinated healthy adults received up to three TBE vaccinations. Following in vitro stimulation with TBE-antigen, lymphocyte proliferation and cytokine secretion (IL-2, IL-10, IL-13, TNF-alpha, IFN-gamma, GM-CSF) were analyzed by thymidine incorporation assay and the Luminex system. Ten patients (59%) showed significant baseline TBE-specific lymphocyte proliferation (stimulation index (SI) > 3) prior to vaccination, but none of the unvaccinated controls (p = 0.002). All patients with a TBE-specific antibody response after two vaccinations (at least 2-fold increase of neutralization test titers) exhibited a strong TBE-specific lymphocyte proliferative response at baseline (SI > 10). Patients with sibling donors had a significantly stronger baseline TBE-specific lymphocyte proliferative and IL-13 cytokine response than patients with unrelated donors (p < 0.05). In conclusion, a relevant proportion of patients showed TBE-specific lymphocyte proliferative and cytokine responses prior to vaccination after HSCT, which predicted the humoral response to the vaccine. Patients with vaccinated sibling donors were more likely to elicit a cellular immune response than patients with unrelated donors of unknown vaccination status.
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CD8+ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control. Here, we identified nonsynonymous mutations in MHC-I-restricted CD8+ T cell epitopes after deep sequencing of 747 SARS-CoV-2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding in a cell-free in vitro assay. Reduced MHC-I binding of mutant peptides was associated with decreased proliferation, IFN-γ production and cytotoxic activity of CD8+ T cells isolated from HLA-matched COVID-19 patients. Single cell RNA sequencing of ex vivo expanded, tetramer-sorted CD8+ T cells from COVID-19 patients further revealed qualitative differences in the transcriptional response to mutant peptides. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8+ T cell surveillance through point mutations in MHC-I-restricted viral epitopes.
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Linfócitos T CD8-Positivos/imunologia , COVID-19 , Epitopos de Linfócito T , Antígenos HLA-A/imunologia , Imunidade Celular , Mutação , SARS-CoV-2 , Linfócitos T CD8-Positivos/patologia , COVID-19/genética , COVID-19/imunologia , COVID-19/patologia , Proliferação de Células , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Interferon gama/imunologia , Peptídeos/genética , Peptídeos/imunologia , SARS-CoV-2/genética , SARS-CoV-2/imunologiaRESUMO
Nuclear factor 'κ-light-chain-enhancer' of activated B cells (NF-κB) signaling is a signaling pathway used by most immune cells to promote immunostimulatory functions. Recent studies have indicated that regulatory T cells (Treg) differentially integrate TCR-derived signals, thereby maintaining their suppressive features. However, the role of NF-κB signaling in the activation of human peripheral blood (PB) Treg has not been fully elucidated so far. We show that the activity of the master transcription factor forkhead box protein 3 (FOXP3) attenuates p65 phosphorylation and nuclear translocation of the NF-κB proteins p50, p65, and c-Rel following activation in human Treg. Using pharmacological and genetic inhibition of canonical NF-κB signaling in FOXP3-transgenic T cells and PB Treg from healthy donors as well as Treg from a patient with a primary NFKB1 haploinsufficiency, we validate that Treg activation and suppressive capacity is independent of NF-κB signaling. Additionally, repression of residual NF-κB signaling in Treg further enhances interleukin-10 (IL-10) production. Blockade of NF-κB signaling can be exploited for the generation of in vitro induced Treg (iTreg) with enhanced suppressive capacity and functional stability. In this respect, dual blockade of mammalian target of rapamycin (mTOR) and NF-κB signaling was accompanied by enhanced expression of the transcription factors FOXP1 and FOXP3 and demethylation of the Treg-specific demethylated region compared to iTreg generated under mTOR blockade alone. Thus, we provide first insights into the role of NF-κB signaling in human Treg. These findings could lead to strategies for the selective manipulation of Treg and the generation of improved iTreg for cellular therapy.
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Fatores de Transcrição Forkhead/imunologia , Haploinsuficiência/imunologia , Subunidade p50 de NF-kappa B/imunologia , Linfócitos T Reguladores/imunologia , Serina-Treonina Quinases TOR/imunologia , Fator de Transcrição RelA/imunologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/imunologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Ativação Linfocitária , Subunidade p50 de NF-kappa B/deficiência , Subunidade p50 de NF-kappa B/genética , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Transdução de Sinais , Sirolimo/farmacologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Tiazóis/farmacologia , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/genéticaAssuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Antivirais/sangue , COVID-19/diagnóstico , Convalescença , Receptores de Coronavírus/metabolismo , SARS-CoV-2/imunologia , Adolescente , Adulto , Idoso , Sítios de Ligação , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: SARS-CoV-2 has triggered a pandemic that is now claiming many lives. Several studies have investigated cellular immune responses in COVID-19-infected patients during disease but little is known regarding a possible protracted impact of COVID-19 on the adaptive and innate immune system in COVID-19 convalescent patients. METHODS: We used multiparametric flow cytometry to analyze whole peripheral blood samples and determined SARS-CoV-2-specific antibody levels against the S-protein, its RBD-subunit, and viral nucleocapsid in a cohort of COVID-19 convalescent patients who had mild disease ~10 weeks after infection (n = 109) and healthy control subjects (n = 98). Furthermore, we correlated immunological changes with clinical and demographic parameters. RESULTS: Even ten weeks after disease COVID-19 convalescent patients had fewer neutrophils, while their cytotoxic CD8+ T cells were activated, reflected as higher HLA-DR and CD38 expression. Multiparametric regression analyses showed that in COVID-19-infected patients both CD3+ CD4+ and CD3+ CD8+ effector memory cells were higher, while CD25+ Foxp3+ T regulatory cells were lower. In addition, both transitional B cell and plasmablast levels were significantly elevated in COVID-19-infected patients. Fever (duration, level) correlated with numbers of central memory CD4+ T cells and anti-S and anti-RBD, but not anti-NC antibody levels. Moreover, a "young immunological age" as determined by numbers of CD3+ CD45RA+ CD62L+ CD31+ recent thymic emigrants was associated with a loss of sense of taste and/or smell. CONCLUSION: Acute SARS-CoV-2 infection leaves protracted beneficial (ie, activation of T cells) and potentially harmful (ie, reduction of neutrophils) imprints in the cellular immune system in addition to induction of specific antibody responses.
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Anticorpos Antivirais/sangue , COVID-19/imunologia , Linfócitos/imunologia , Neutrófilos/metabolismo , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adolescente , Adulto , Idoso , Convalescença , Feminino , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Regulatory T lymphocytes (Treg) play an important role in preventing allergic diseases. We characterized Treg expansion kinetics, marker profiles, and recirculation behavior in allergen-challenged mice, which had been pretreated with IL-2/αIL-2 complexes in the presence or absence of allergen. Moreover, the ability of induced Treg to control airway hyperreactivity and effector functions of lung T cells was determined. METHODS: Humanized TCR/HLA-transgenic allergy mice were treated in vivo with recombinant IL-2 complexed to the anti-IL-2 mAb JES6-1 in the presence or absence of mugwort pollen extract (MPE) on days 0-2. Afterward, they were intranasally challenged with MPE (days 13-15) followed by determination of airway hyperreactivity and lung T cell effector functions. Multiparametric flow cytometry on peripheral blood T cells was performed on a daily basis. RESULTS: IL-2/αIL-2 complexes highly efficiently expanded peripheral Treg cells, while concomitant allergen exposure altered the phenotype of expanded Treg cells. Notably, application of allergen together with IL-2/αIL-2 complexes induced expression of Treg marker molecules CTLA4, NRP1, Helios, and GITR on conventional T cells. Apart from CD25, GARP was identified as the most reliable surface-expressed lineage discrimination marker of Treg expanded in the presence of IL-2/αIL-2 complexes and allergen. Finally, IL-2/αIL-2 complex-expanded Treg cells could be recalled upon allergen challenge, which was associated with suppression of lung-specific Th2 responses long after initial treatment. CONCLUSION: The characterization of reliable surface and transcription markers of IL-2/αIL-2 complex-expanded Treg along with their expansion kinetics and function will help to identify protocols for their long-term expansion in vivo.
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Hipersensibilidade , Linfócitos T Reguladores , Alérgenos , Animais , Tolerância Imunológica , Interleucina-2 , CamundongosRESUMO
Malignant cells acquire physiological mechanisms of immunosuppression to escape immune surveillance. Strategies to counteract this suppression could help to improve adoptive immunotherapy regimen. The intracellular second messenger cyclic AMP (cAMP) acts as a potent immunosuppressive signaling molecule in T-cells and is up-regulated by multiple tumor-relevant suppressive factors including prostaglandin E2 (PGE2), adenosine and the functions of regulatory T-cells. Consequently, we aimed to abrogate cAMP signaling in human T-cells by ectopic overexpression of phosphodiesterase 4A (PDE4A). We could show that retroviral transduction of PDE4A into T-cells led to efficient degradation of cAMP in response to induction of adenylate cyclase. Retroviral transduction of PDE4A into CD4+ and CD8+ T-cells restored proliferation, cytokine secretion as well as cytotoxicity under immunosuppression by PGE2 and A2A-R agonists. PDE4A-transgenic T-cells were also partially protected from suppression by regulatory T-cells. Furthermore, PGE2-mediated upregulation of the inhibitory surface markers CD73 and CD94 on CD8+ T-cells was efficiently counteracted by PDE4A. Importantly, no differences in the functionality under non-suppressive conditions between PDE4A- and control-vector transduced T-cells were observed, indicating that PDE4A does not interfere with T-cell activation per se. Similarly, expression of surface markers associated with T-cell exhaustion were not influenced by PDE4A overexpression in long term cultures. Thus, we provide first in vitro evidence that PDE4A can be exploited as immune checkpoint inhibitor against multiple suppressive factors.
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Linfócitos T CD8-Positivos/imunologia , AMP Cíclico/imunologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/imunologia , Regulação Enzimológica da Expressão Gênica/imunologia , Tolerância Imunológica , Linfócitos T Reguladores/imunologia , AMP Cíclico/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Dinoprostona/genética , Dinoprostona/imunologia , Células HEK293 , HumanosRESUMO
BACKGROUND: In high-risk populations, allergen-specific prophylaxis could protect from sensitization and subsequent development of allergic disease. However, such treatment might itself induce sensitization and allergies, thus requiring hypoallergenic vaccine formulations. We here characterized the preventive potential of virus-like nanoparticles (VNP) expressing surface-exposed or shielded allergens. METHODS: Full-length major mugwort pollen allergen Art v 1 was selectively targeted either to the surface or to the inner side of the lipid bilayer envelope of VNP. Upon biochemical and immunological analysis, their preventive potential was determined in a humanized mouse model of mugwort pollen allergy. RESULTS: Virus-like nanoparticles expressing shielded version of Art v 1, in contrast to those expressing surface-exposed Art v 1, were hypoallergenic as they hardly induced degranulation of rat basophil leukemia cells sensitized with Art v 1-specific mouse or human IgE. Both VNP versions induced proliferation and cytokine production of allergen-specific T cells in vitro. Upon intranasal application in mice, VNP expressing surface-exposed but not shielded allergen induced allergen-specific antibodies, including IgE. Notably, preventive treatment with VNP expressing shielded allergen-protected mice from subsequent sensitization with mugwort pollen extract. Protection was associated with a Th1/Treg-dominated cytokine response, increased Foxp3+ Treg numbers in lungs, and reduced lung resistance when compared to mice treated with empty particles. CONCLUSION: Virus-like nanoparticles represent a novel and versatile platform for the in vivo delivery of allergens to selectively target T cells and prevent allergies without inducing allergic reactions or allergic sensitization.
Assuntos
Alérgenos/imunologia , Hipersensibilidade/imunologia , Hipersensibilidade/prevenção & controle , Nanopartículas , Vacinas de Partículas Semelhantes a Vírus/imunologia , Alérgenos/administração & dosagem , Animais , Antígenos de Plantas/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citocinas/biossíntese , Modelos Animais de Doenças , Epitopos de Linfócito T/imunologia , Feminino , Células HEK293 , Humanos , Imunização , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Proteínas de Plantas/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagemRESUMO
BACKGROUND: Immunoglobulin(Ig)E-associated allergies result from misguided immune responses against innocuous antigens. CD4+ T lymphocytes are critical for initiating and perpetuating that process, yet the crucial factors determining whether an individual becomes sensitized towards a given allergen remain largely unknown. OBJECTIVE: To determine the key factors for sensitization and allergy towards a given allergen. METHODS: We here created a novel human T cell receptor(TCR) and human leucocyte antigen (HLA)-DR1 (TCR-DR1) transgenic mouse model of asthma, based on the human-relevant major mugwort (Artemisia vulgaris) pollen allergen Art v 1 to examine the critical factors for sensitization and allergy upon natural allergen exposure via the airways in the absence of systemic priming and adjuvants. RESULTS: Acute allergen exposure led to IgE-independent airway hyperreactivity (AHR) and T helper(Th)2-prone lung inflammation in TCR-DR1, but not DR1, TCR or wildtype (WT) control mice, that was alleviated by prophylactic interleukin(IL)-2-αIL-2 mAb complex-induced expansion of Tregs. Chronic allergen exposure sensitized one third of single DR1 transgenic mice, however, without impacting on lung function. Similar treatment led to AHR and Th2-driven lung pathology in >90% of TCR-DR1 mice. Prophylactic and therapeutic expansion of Tregs with IL-2-αIL-2 mAb complexes blocked the generation and boosting of allergen-specific IgE associated with chronic allergen exposure. CONCLUSIONS: We identify genetic restriction of allergen presentation as primary factor dictating allergic sensitization and disease against the major pollen allergen from the weed mugwort, which frequently causes sensitization and disease in humans. Furthermore, we demonstrate the importance of the balance between allergen-specific T effector and Treg cells for modulating allergic immune responses.