RESUMEN
The analysis of antigen-specific T-cell responses has become routine in many laboratories. Functional T-cell assays like enzyme-linked-immuno-spot (ELISPOT), which depend on antigen-specific stimulation, increasingly use peptides to represent the antigen of interest. Besides single peptides, mixtures of peptides (peptide pools) are very frequently applied. Such peptide pools may, for example, represent entire proteins (with overlapping peptides covering a protein sequence) or include noncontiguous peptides such as a collection of T-cell-stimulating peptides. The optimum specification of single peptides or peptide pools for T-cell stimulation assays will depend on the purpose of the test, the target T-cell population, the availability of sample, requirements regarding reproducibility, and, last but not least, the available budget, to mention only the most important factors. Because of the way peptides are produced, they will always contain certain amounts of impurities such as peptides with deletions or truncated peptides, and there may be additional by-products of peptide synthesis. Optimized synthesis protocols as well as purification help reduce impurities that might otherwise cause false-positive assay results. However, specific requirements with respect to purity will vary depending on the purpose of an assay. Finally, storage conditions significantly affect the shelf life of peptides, which is relevant especially for longitudinal studies. The present book chapter addresses all of these aspects in detail. It should provide the researcher with all necessary background knowledge for making the right decisions when it comes to choosing, using, and storing peptides for ELISPOT and other T-cell stimulation assays.
Asunto(s)
Péptidos , Linfocitos T , Secuencia de Aminoácidos , Reproducibilidad de los ResultadosRESUMEN
Currently available COVID-19 vaccines include inactivated virus, live attenuated virus, mRNA-based, viral vectored and adjuvanted protein-subunit-based vaccines. All of them contain the spike glycoprotein as the main immunogen and result in reduced disease severity upon SARS-CoV-2 infection. While we and others have shown that mRNA-based vaccination reactivates pre-existing, cross-reactive immunity, the effect of vector vaccines in this regard is unknown. Here, we studied cellular and humoral responses in heterologous adenovirus-vector-based ChAdOx1 nCOV-19 (AZ; Vaxzeria, AstraZeneca) and mRNA-based BNT162b2 (BNT; Comirnaty, BioNTech/Pfizer) vaccination and compared it to a homologous BNT vaccination regimen. AZ primary vaccination did not lead to measurable reactivation of cross-reactive cellular and humoral immunity compared to BNT primary vaccination. Moreover, humoral immunity induced by primary vaccination with AZ displayed differences in linear spike peptide epitope coverage and a lack of anti-S2 IgG antibodies. Contrary to primary AZ vaccination, secondary vaccination with BNT reactivated pre-existing, cross-reactive immunity, comparable to homologous primary and secondary mRNA vaccination. While induced anti-S1 IgG antibody titers were higher after heterologous vaccination, induced CD4+ T cell responses were highest in homologous vaccinated. However, the overall TCR repertoire breadth was comparable between heterologous AZ-BNT-vaccinated and homologous BNT-BNT-vaccinated individuals, matching TCR repertoire breadths after SARS-CoV-2 infection, too. The reasons why AZ and BNT primary vaccination elicits different immune response patterns to essentially the same antigen, and the associated benefits and risks, need further investigation to inform vaccine and vaccination schedule development.
Asunto(s)
Vacuna BNT162 , COVID-19 , ChAdOx1 nCoV-19 , Reacciones Cruzadas , Humanos , Vacuna BNT162/inmunología , ChAdOx1 nCoV-19/inmunología , COVID-19/prevención & control , Receptores de Antígenos de Linfocitos T , SARS-CoV-2 , VacunaciónRESUMEN
The functional relevance of preexisting cross-immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a subject of intense debate. Here, we show that human endemic coronavirus (HCoV)reactive and SARS-CoV-2cross-reactive CD4+ T cells are ubiquitous but decrease with age. We identified a universal immunodominant coronavirus-specific spike peptide (S816-830) and demonstrate that preexisting spike- and S816-830reactive T cells were recruited into immune responses to SARS-CoV-2 infection and their frequency correlated with antiSARS-CoV-2-S1-IgG antibodies. Spikecross-reactive T cells were also activated after primary BNT162b2 COVID-19 messenger RNA vaccination and displayed kinetics similar to those of secondary immune responses. Our results highlight the functional contribution of preexisting spikecross-reactive T cells in SARS-CoV-2 infection and vaccination. Cross-reactive immunity may account for the unexpectedly rapid induction of immunity after primary SARS-CoV-2 immunization and the high rate of asymptomatic or mild COVID-19 disease courses.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , COVID-19/inmunología , SARS-CoV-2/inmunología , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Enfermedades Asintomáticas , Vacuna BNT162 , Complejo CD3/inmunología , Vacunas contra la COVID-19/inmunología , Reacciones Cruzadas , Femenino , Humanos , Inmunidad , Epítopos Inmunodominantes/inmunología , Masculino , Persona de Mediana Edad , Sistemas de Lectura Abierta , Fragmentos de Péptidos/inmunología , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunación , Adulto JovenRESUMEN
Humoral immunity to the Severe Adult Respiratory Syndrome (SARS) Coronavirus (CoV)-2 is not fully understood yet but is a crucial factor of immune protection. The possibility of antibody cross-reactivity between SARS-CoV-2 and other human coronaviruses (HCoVs) would have important implications for immune protection but also for the development of specific diagnostic ELISA tests. Using peptide microarrays, n = 24 patient samples and n = 12 control samples were screened for antibodies against the entire SARS-CoV-2 proteome as well as the Spike (S), Nucleocapsid (N), VME1 (V), R1ab, and Protein 3a (AP3A) of the HCoV strains SARS, MERS, OC43, and 229E. While widespread cross-reactivity was revealed across several immunodominant regions of S and N, IgG binding to several SARS-CoV-2-derived peptides provided statistically significant discrimination between COVID-19 patients and controls. Selected target peptides may serve as capture antigens for future, highly COVID-19-specific diagnostic antibody tests.
Asunto(s)
Anticuerpos Antivirales/sangre , COVID-19/diagnóstico , Análisis por Matrices de Proteínas/métodos , SARS-CoV-2/inmunología , Proteínas Virales/inmunología , Adulto , Anciano , Secuencia de Aminoácidos/genética , Anticuerpos Antivirales/inmunología , Coronavirus Humano 229E/inmunología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Coronavirus Humano OC43/inmunología , Reacciones Cruzadas/inmunología , Pruebas Diagnósticas de Rutina , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Fosfoproteínas/inmunología , Proteoma/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Adulto JovenRESUMEN
BACKGROUND: Epstein-Barr-Virus (EBV) plays an important role as trigger or cofactor for various autoimmune diseases. In a subset of patients with Chronic Fatigue Syndrome (CFS) disease starts with infectious mononucleosis as late primary EBV-infection, whereby altered levels of EBV-specific antibodies can be observed in another subset of patients. METHODS: We performed a comprehensive mapping of the IgG response against EBV comparing 50 healthy controls with 92 CFS patients using a microarray platform. Patients with multiple sclerosis (MS), systemic lupus erythematosus (SLE) and cancer-related fatigue served as controls. 3054 overlapping peptides were synthesised as 15-mers from 14 different EBV proteins. Array data was validated by ELISA for selected peptides. Prevalence of EBV serotypes was determined by qPCR from throat washing samples. RESULTS: EBV type 1 infections were found in patients and controls. EBV seroarray profiles between healthy controls and CFS were less divergent than that observed for MS or SLE. We found significantly enhanced IgG responses to several EBNA-6 peptides containing a repeat sequence in CFS patients compared to controls. EBNA-6 peptide IgG responses correlated well with EBNA-6 protein responses. The EBNA-6 repeat region showed sequence homologies to various human proteins. CONCLUSION: Patients with CFS had a quite similar EBV IgG antibody response pattern as healthy controls. Enhanced IgG reactivity against an EBNA-6 repeat sequence and against EBNA-6 protein is found in CFS patients. Homologous sequences of various human proteins with this EBNA-6 repeat sequence might be potential targets for antigenic mimicry.
Asunto(s)
Infecciones por Virus de Epstein-Barr/sangre , Infecciones por Virus de Epstein-Barr/inmunología , Síndrome de Fatiga Crónica/sangre , Síndrome de Fatiga Crónica/inmunología , Herpesvirus Humano 4/inmunología , Adulto , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Biomarcadores , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/virología , Síndrome de Fatiga Crónica/complicaciones , Síndrome de Fatiga Crónica/epidemiología , Femenino , Herpesvirus Humano 4/clasificación , Herpesvirus Humano 4/genética , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , Prevalencia , Análisis por Matrices de Proteínas , Carga ViralRESUMEN
Peptide microarrays are ideal tools for a variety of applications ranging from epitope mapping to immune monitoring. Here we present a method for high-throughput screening of biological samples using only standard microtiter plate equipment. Parallel incubation of a large number of samples with a small library of peptides is enabled by printing multiple identical mini-arrays on one microarray slide and further combining four slides to yield an incubation frame possessing the dimensions of a 96-well microtiter plate. Applying conventional lab equipment such as ELISA washers, hundreds of samples can be processed in 1 day yielding approx. 200 data points in triplicates per sample.
Asunto(s)
Péptidos/metabolismo , Análisis por Matrices de Proteínas/métodos , Secuencia de Aminoácidos , Métodos Analíticos de la Preparación de la Muestra , Humanos , Datos de Secuencia Molecular , Péptidos/químicaRESUMEN
The regulation of gene repression by corepressors is a controlled process. Surface-enhanced laser desorption ionization MS proteomic analysis and a yeast two-hybrid screen showed independently that the corepressor Alien interacts with the CREB-binding protein (CBP) coactivator. This interaction was further confirmed by coimmunoprecipitation and glutathione S-transferase pull-down experiments, suggesting that Alien interacts in vivo and in vitro with the histone acetyltransferase (HAT) coactivators CBP and its paralog p300. Acetylation detection experiments indicated that Alien is acetylated in vivo. Furthermore, Alien interacts with the central region of CBP/p300 containing the HAT domain and becomes acetylated in vitro. When an inhibitor of CBP/p300 HAT activity was employed, the Alien-mediated silencing was enhanced. Thus, these findings suggest crosstalk between corepressors and coactivators, and indicate fine-tuning of corepressor function by post-translational modification through corepressor acetylation. STRUCTURED DIGITAL ABSTRACT: p300 binds to Alien α by pull down (View interaction) Alien α physically interacts with CBP by two hybrid (View interaction) Alien α physically interacts with MLK2 by two hybrid (View interaction) p300 acetylates Alien α by acetylation assay (View interaction) Alien α physically interacts with NAP1 by two hybrid (View interaction) Alien α physically interacts with TAFI68 by two hybrid (View interaction) Alien α physically interacts with CBP by anti bait coimmunoprecipitation (View Interaction: 1, 2, 3) Alien α binds to CBP by pull down (View interaction).
Asunto(s)
Proteínas Co-Represoras/fisiología , Proteínas Represoras/fisiología , Factores de Transcripción p300-CBP/fisiología , Acetilación , Complejo del Señalosoma COP9 , Proteína de Unión a CREB/fisiología , Células HEK293 , Histona Acetiltransferasas/metabolismo , Humanos , Factores de Transcripción p300-CBP/antagonistas & inhibidoresRESUMEN
ISWI is an evolutionarily conserved ATPase that catalyzes nucleosome remodeling in different macromolecular complexes. Two mammalian ISWI orthologs, SNF2H and SNF2L, are thought to have specialized functions despite their high sequence similarity. To date, the function of SNF2L in human cells has not been a focus of research. Newly established specific monoclonal antibodies and selective RNA interference protocols have now enabled a comprehensive characterization of loss-of-function phenotypes in human cells. In contrast to earlier results, we found SNF2L to be broadly expressed in primary human tissues. Depletion of SNF2L in HeLa cells led to enhanced proliferation and increased migration. These phenomena were explained by transcriptome profiling, which identified SNF2L as a modulator of the Wnt signaling network. The cumulative effects of SNF2L depletion on gene expression portray the cell in a state of activated Wnt signaling characterized by increased proliferation and chemotactic locomotion. Accordingly, high levels of SNF2L expression in normal melanocytes contrast with undetectable expression in malignant melanoma. In summary, our data document an inverse relationship between SNF2L expression and features characteristic of malignant cells.
Asunto(s)
Movimiento Celular/fisiología , Proliferación Celular , Proteínas de Unión al ADN/metabolismo , Nucleosomas/fisiología , Factores de Transcripción/metabolismo , Vía de Señalización Wnt/fisiología , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Secuencia de Bases , Adhesión Celular/fisiología , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Femenino , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Masculino , Melanocitos/metabolismo , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , ARN Interferente Pequeño/genética , Distribución Tisular , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/inmunología , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Active chromatin remodelling is integral to the DNA damage response in eukaryotes, as damage sensors, signalling molecules and repair enzymes gain access to lesions. A variety of nucleosome remodelling complexes is known to promote different stages of DNA repair. The nucleosome sliding factors CHRAC/ACF of Drosophila are involved in chromatin organization during development. Involvement of corresponding hACF1-containing mammalian nucleosome sliding factors in replication, transcription and very recently also non-homologous end-joining of DNA breaks have been suggested. We now found that hACF1-containing factors are more generally involved in the DNA damage response. hACF1 depletion increases apoptosis, sensitivity to radiation and compromises the G2/M arrest that is activated in response to UV- and X-rays. In the absence of hACF1, γH2AX and CHK2ph signals are diminished. hACF1 and its ATPase partner SNF2H rapidly accumulate at sites of laser-induced DNA damage. hACF1 is also required for a tight checkpoint that is induced upon replication fork collapse. ACF1-depleted cells that are challenged with aphidicolin enter mitosis despite persistence of lesions and accumulate breaks in metaphase chromosomes. hACF1-containing remodellers emerge as global facilitators of the cellular response to a variety of different types of DNA damage.
Asunto(s)
Daño del ADN , Reparación del ADN , Puntos de Control de la Fase G2 del Ciclo Celular , Puntos de Control de la Fase M del Ciclo Celular , Factores de Transcripción/fisiología , Afidicolina/toxicidad , Apoptosis , Línea Celular , Proteínas Cromosómicas no Histona , Fragilidad Cromosómica , Humanos , Interferencia de ARN , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genéticaRESUMEN
The tumor suppressor p33ING1 is involved in DNA repair and cell cycle regulation. Furthermore, p33ING1 is a transcriptional silencer that recognizes the histone mark for trimethylated lysine 4 at histone H3. Interestingly, expression of p33ING1 and p33ING2 is able to induce premature senescence in primary human fibroblasts. The corepressor Alien is involved in gene silencing mediated by selected members of nuclear hormone receptors. In addition, Alien acts as a corepressor for E2F1, a member of the E2F cell cycle regulatory family. Furthermore, recent findings suggest that Alien is complexed with transcription factors participating in DNA repair and chromatin. Here, using a proteomic approach by surface-enhanced laser desorption ionization and mass spectrometry (SELDI-MS) combined with immunological techniques, we show that Alien interacts in vivo with the tumor suppressor p33ING1 as well as with the related tumor suppressor candidate p33ING2. The interaction of Alien with p33ING1 and p33ING2 was confirmed in vitro with GST-pull-down, suggesting a direct binding of Alien to these factors. The binding domain was mapped to a central region of Alien. Functionally, the expression of p33ING1 or p33ING2 enhances the Alien-mediated silencing, suggesting that the interaction plays a role in transcriptional regulation. Thus, the findings suggest that the identified interaction between Alien and the tumor suppressors p33ING1 and p33ING2 reveals a novel cellular protein network.
Asunto(s)
Silenciador del Gen , Proteínas de Homeodominio/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas Nucleares/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Proteínas Represoras/metabolismo , Proteínas Supresoras de Tumor/fisiología , Complejo del Señalosoma COP9 , Línea Celular , Cromatina/metabolismo , Reparación del ADN , Genes Supresores de Tumor , Glutatión Transferasa/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Inmunoprecipitación , Proteína Inhibidora del Crecimiento 1 , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Espectrometría de Masas , Proteínas Nucleares/metabolismo , Péptidos/química , Mapeo de Interacción de Proteínas , Receptores Citoplasmáticos y Nucleares/metabolismo , Transcripción Genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The assembly of nucleosomes into chromatin is essential for the compaction of DNA and inactivation of the DNA template to modulate and repress gene expression. The nucleosome assembly protein 1, NAP1, assembles nucleosomes independent of DNA synthesis and was shown to enhance coactivator-mediated gene expression, suggesting a role for NAP1 in transcriptional regulation. Here, we show that Alien, known to harbor characteristics of a corepressor of nuclear hormone receptors such as of the vitamin D receptor (VDR), binds in vivo and in vitro to NAP1 and modulates its activity by enhancing NAP1-mediated nucleosome assembly on DNA. Furthermore, Alien reduces the accessibility of the histones H3 and H4 for NAP1-promoted assembly reaction. This indicates that Alien sustains and reinforces the formation of nucleosomes. Employing deletion mutants of Alien suggests that different regions of Alien are involved in enhancement of NAP1-mediated nucleosome assembly and in inhibiting the accessibility of the histones H3 and H4. In addition, we provide evidence that Alien is associated with chromatin and with micrococcus nuclease-prepared nucleosome fractions and interacts with the histones H3 and H4. Furthermore, chromatin immunoprecipitation and reimmunoprecipitation experiments suggest that NAP1 and Alien localize to the endogenous CYP24 promoter in vivo, a VDR target gene. Based on these findings, we present here a novel pathway linking corepressor function with nucleosome assembly activity.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Nucleosomas/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/fisiología , Complejo del Señalosoma COP9 , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Histonas/metabolismo , Humanos , Modelos Moleculares , Proteínas Nucleares/genética , Proteína 1 de Ensamblaje de Nucleosomas , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Proteínas Represoras/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , Transcripción Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Repression by nuclear hormone receptors (NHRs) plays an important role in development, immune response and cellular function. We review mechanisms of how NHRs act as repressors of gene transcription either by direct contact with basal transcription factors or through recruitment of cofactors and enzymic activities that modulate chromatin accessibility. We describe also the role and biochemical mechanism of the cognate hormone that switches a NHR from a transcriptional silencer into an activator. This includes data from crystal structure, functional receptor domain analyses and the role of co-repressors in chromatin modification and remodelling. Furthermore, the comparison of negative response elements with classical response elements unravels the role of co-repressors in this context. We also describe the inhibition of the nuclear factor kappaB and Jun/Fos pathway by NHRs, as well as the molecular mechanism of anti-hormone therapies. Anti-hormones are commonly used in breast and prostate cancer therapy to inhibit cancer proliferation through repression of the oestrogen or androgen receptor, respectively. Here we provide a comprehensive overview of the various mechanism of NHR repression.
Asunto(s)
Receptores Citoplasmáticos y Nucleares/fisiología , Proteínas Represoras/fisiología , Animales , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Silenciador del Gen , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Co-Represor 1 de Receptor Nuclear , Co-Represor 2 de Receptor Nuclear , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/fisiología , Proteínas Represoras/genéticaRESUMEN
The thyroid hormone receptors (TR) are able to bind DNA and to repress transcription in the absence of thyroid hormone. This repression function is an important feature of TRs as aberrant silencing can lead to severe diseases and developmental abnormalities. TR utilizes different mechanisms to achieve repression of target genes including the recruitment of cofactors called corepressors and interference with the basal transcriptional machinery. Recent studies have revealed an important role of chromatin in TR silencing involving different histone modifications and the responsible enzymes. Furthermore, the transcriptional properties of TR depend on the type of the TR DNA-binding elements. This review will focus on the molecular basis of gene silencing by TR and diseases caused by aberrant functioning.
Asunto(s)
Silenciador del Gen/fisiología , Receptores de Hormona Tiroidea/fisiología , Animales , Cromatina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Receptores de Hormona Tiroidea/química , Proteínas Represoras/fisiología , Transcripción GenéticaRESUMEN
Alien was previously identified as a corepressor for the thyroid hormone receptor (TR) and DAX-1 which belong both to the superfamily of nuclear receptors. Here, we isolated the mixed lineage kinase 2 (MLK2) as an interacting partner for the corepressor Alien using a yeast two hybrid screen. MLK2 is an upstream activator of JNKs and activation of MLK2-mediated signaling cascades play roles in neurodegenerative and apoptotic mechanisms in the central nervous system. MLK2 has been shown to be localized both in the cytoplasm and cell nucleus. We confirmed the Alien-MLK2 interaction using GST pull-down experiments and also show that MLK2 is able to phosphorylate Alien in immune-kinase assays. Functional analyses revealed that Alien, DAX-1 and thyroid hormone receptor mediated transcriptional silencing is strongly enhanced in the presence of active MLK2. Since MAP kinase signaling pathways are important mediators of cellular responses to a wide variety of stimuli, our data suggest that signaling pathways not only regulate transactivation but also enhancement of transcriptional silencing. This novel cross-talk may represent a link between MLK2-mediated signaling and transcriptional repression of target genes during neuronal differentiation processes.