stimulation and incubation of whole blood samples for antigen-specific analysis of host immunity to fungal and viral pathogens

<meta charset="utf8"></head><body>Inmunoensayos de sangre total optimizados para analizar la inmunidad fúngica y viral

Quantification and characterization of antigen-specific immunity, especially specific T-cell responses, is pivotal for immunobiology and vaccination research, as well as some diagnostic tests. Traditionally, antigen-specific immunoassays commonly relied on isolated peripheral blood mononuclear cells (PBMCs). However, isolation of these cells is time-consuming and resource-intensive and often requires relatively large blood volumes. Additionally, to prevent granulocyte activation and subsequent T-cell disturbance during pre-analytic storage1 rapid processing of the samples is paramount, which is often not feasible in clinical practice. These limitations hamper the practicability of antigen-specific immunoassays in high-throughput research scenarios and clinical routines. Therefore, the development of easy-to-use and potentially automatable whole blood-based approaches in recent years has opened new areas of immunoassay applications. However, current commercially available systems usually lack optimal co-stimulatory environments for T-cells and are susceptible to pre-analytic delays. For instance, a widely used whole blood-based IFN-&#947; release assay has a 19% positive to negative reversion rate after 6 h of pre-analytic blood storage2. Optimized protocols with dual anti-CD28 and anti-CD49d co-stimulation have been developed to overcome these limitations3,4,5,6.
The protocol presented here allows for accurate and reproducible quantification and characterization of antigen-specific T-cells, assessment of antigen-induced cytokine responses, and other (flow cytometric or transcriptional) functional immune markers from minimal blood volume, i.e., 500 &#181;L of blood per stimulation tube. Further advantages of this protocol include low hands-on time, high resilience to pre-analytic confounders, and preservation of functional inter-cellular interactions in a relatively physiological ex vivo environment. The comparability of whole blood-based flow cytometric antigen-specific&#160;T-cell characterization with data generated from traditional PBMC-based assays has been previously shown in the context of mold-specific T-cell quantification6. Furthermore, direct stimulation of the subjects&#39; blood abrogates the need for supplementation with autologous, allogenic, or even xenogeneic serum that is commonly required for optimal PBMC stimulation. Omission of cell isolation also reduces shear and temperature stress, thereby improving cell viability. Most importantly, whole blood-based assays preserve granulocyte populations that are lost during gradient centrifugation for isolation of PBMCs7. Thereby, this assay setup preserves and captures functional interaction loops between granulocytes and mononuclear cells4.
Of note, this protocol requires only minimal modifications to accommodate different readout modalities and even allows for dual analysis of cytokine release and transcriptional responses from the same stimulation tube. Specifically, while cytokines are analyzed from the culture supernatant after stimulation, the cell pellet can be used for RNA isolation with subsequent transcriptomic analysis. The general workflow for the various readout modalities is summarized in Figure 1.
In recent years, an increasing number of whole blood-based assays has been developed for pathogen-reactive immune monitoring in research and clinical settings, e.g., for Mycobacterium tuberculosis8,9, Bordetella pertussis3, Orientia tsutsugamushi10, and SARS-CoV-25,11,12. For instance, a previously established system has been used for multiple antigens, including M. tuberculosis, Influenza A virus, and SARS-CoV-2, but does not use co-stimulatory factors optimized for T-helper (Th) cell stimulation13,14,15. Even though the blood volume required for these assays is already significantly lower than that used for traditional PBMC-based assays or commercially available whole blood stimulation kits, an even smaller sample volume might be warranted for applications in pediatrics, neonatology, patients in the intensive care unit (ICU), and preclinical research in small animal models. For instance, even terminal blood sampling from mice (e.g., by cardiac puncture) commonly yields a maximum of 0.7-1 mL of blood. Thus, the possibility to further downscale previously established whole blood-based immunoassay protocols4,6 for precise quantification and characterization of antigen-reactive T-cell responses from 250 &#181;L of blood volume per stimulation tube has been evaluated as part of this protocol.

<meta charset="utf8"></head><body>Autor destacado: Avance de la monitorización inmunitaria en pacientes de cuidados intensivos mediante ensayos de sangre total

Ensayo de sangre total con coestimulación dual para el análisis específico de antígenos de la inmunidad del huésped a patógenos fúngicos y virales

Our research focuses on the immune monitoring of patients in the intensive care unit. Some patients develop severe viral pneumonia during mechanical ventilation. We isolate the cause of the viruses, but also characterize the local and systemic immune response using the protocol presented in this video to identify immunological risk factors.Traditional antigen-specific T-cell assays that are based on isolated mononuclear cells are highly susceptible to pre-analytical confounders, time-and resource-intensive, and require large amounts of blood. This poses severe limitations to the implementation of antigen-specific T-cell assays in the clinical research or clinical routine use as prognostic or diagnostic biomarkers. We present a versatile immunoassay protocol, which accommodates various downstream readouts from as little as 250 micros blood per stimulus.This enables applications, even when that volume is limited or several antigens need to be screened. Furthermore, our assay is easy to perform and amenable to several pathogens. In addition to its versatility and the low blood volume required, our protocol used as stimulation environment that was thoroughly and specifically optimized for antigen-induced T-helper cell responses.This was achieved through dual core stimulation, which also makes the protocol more robust towards confounders, such as immunosuppressive pharmacotherapy or pre-analytical delays. We are using this protocol for immune monitoring of patients with ventilation-associated viral pneumonia. Our collaborators in Wuerzburg and Houston are working on a clinical translation of cytomegalovirus-specific immunoassays.Furthermore, they're using this protocol for studies on fungal immunopathogenesis and preclinical immunotherapy studies in mouse models for opportunistic infections.

Estimulación e incubación de muestras de sangre entera para el análisis específico de antígenos de la inmunidad del huésped a patógenos fúngicos y virales

stimulation-incubation-whole-blood-samples-for-antigen-specific

Research

JoVE Journal

Immunology and Infection

Stimulation and Incubation of Whole Blood Samples for Antigen-Specific Analysis of Host Immunity to Fungal and Viral Pathogens

173 vistas. Para empezar, preparar tubos de extracción de sangre de 2,7 mililitros sin anticoagulante, con antígenos y anticuerpos coestimuladores en condiciones estériles. Agregue anti-CD28 y anti-CD49d en cada tubo, incluido el control negativo. Si están congelados, descongele los tubos de estimulación listos para usar a temperatura ambiente aproximadamente de 30 a 60 minutos antes de la estimulación con sangre completa.Recolectar sangre venosa del donante...

Video: Estimulación e incubación de muestras de sangre entera para el análisis específico de antígenos de la inmunidad del huésped a patógenos fúngicos y virales

Whole Blood Assay with Dual Co-Stimulation for Antigen-Specific Analysis of Host Immunity to Fungal and Viral Pathogens

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of advanced antigen-specific immunoassays. Traditionally, such immunoassays, especially antigen-specific T-cell analysis by flow cytometry or enzyme-linked immunosorbent spot assays, often rely on the isolation of peripheral blood mononuclear cells. This process is time-consuming, subject to many pre-analytic confounders, and requires large blood volumes. Whole blood-based assays provide a facile alternative with increased pre-analytic robustness and lower blood volume requirements. Furthermore, whole blood-based assays allow for the preservation of inter-cellular interactions that are not captured by assays using isolated cell subsets. Recently, a refined whole blood immunoassay with dual anti-CD28 and anti-CD49d co-stimulation for comprehensive analysis of both antigen-specific T-cell functions and complex intercellular interactions in response to various fungal and viral antigens has been proposed. This protocol provides guidance for the preparation of stimulation tubes, blood stimulation, and downstream sample processing for flow cytometry, cytokine secretion assays, and transcriptional analyses. This includes a validated and functionally equivalent, previously unpublished, low-volume protocol (250 &#181;L) to make flow cytometric and cytokine-based T-cell monitoring more accessible for studies in pediatric patients or preclinical studies in small animals (e.g., mice). Altogether, these protocols provide a versatile toolbox for complex antigen-specific immune analysis in both clinical and translational research settings.

Whole blood-based immunoassays provide a facile and resource-efficient tool to analyze antigen-specific immunity for diagnostic and research purposes. This article provides an optimized whole blood-based protocol with dual co-stimulation for comprehensive analysis of host immunity to fungal and viral pathogens, including a low-volume version for pediatric patients and small animals.

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of ...

Investigación

Inmunología e Infección

To begin, prepare 2.7 milliliter blood collection tubes without anticoagulant, with antigens and co-stimulatory antibodies under sterile conditions. Add anti-CD28 and anti-CD49d into each tube, including the negative control. If frozen, thaw the ready-to-use stimulation tubes at room temperature about 30 to 60 minutes before whole blood stimulation.Collect venous blood from the donor or patient using blood collection tubes with lithium heparin anticoagulant. Pipette the required amount of heparinized whole blood into the stimulation tubes under a sterile workbench. Carefully invert the tubes about 5 to 10 times.Then, place the tubes in an incubator at 37 degrees Celsius. After four hours of incubation, prepare one milligram per milliliter brefeldin A solution in RPMI medium. In a sterile workbench, uncap the tubes used for flow cytometric analysis.Then transfer 1%of the sample volume of the brefeldin A solution into the stimulation tube. Incubate the capped tubes at 37 degrees Celsius for another 16 to 18 hours.

Preparation of Whole Blood Samples for Flow Cytometry and Cytokine Analysis to Determine Antigen-Specific Host Immunity to Fungal and Viral Pathogens

To begin sample preparation for flow cytometry, take out stimulation tubes containing whole blood samples with Brefeldin A from the incubator. Add 500 microliters of 0.5 molar EDTA solution to each stimulation tube containing Brefeldin A.Transfer the samples into new 15 milliliter centrifuge tubes. Then pipette one milliliter of erythrocyte lysis buffer into each stimulation tube to rinse it.Transfer the buffer and cell suspension into the same centrifuge tube. Centrifuge the tubes at 600 g for seven minutes, and carefully pipette out the supernatant. Then add erythrocyte lysis buffer to the pellet and resuspend it.Incubate samples at room temperature until the samples appear clear or for a maximum of six minutes. Centrifuge the tubes at 600 g for seven minutes. After discarding the supernatant, add one milliliter of HBSS to the pellet.Transfer the cell suspension into two-milliliter reaction tubes. Then centrifuge the tubes at 400 g for five minutes. Discard the supernatant before performing flow cytometric staining.Transfer the samples from the stimulation tubes that do not contain Brefeldin A into 1.5 milliliter tubes. Centrifuge the tubes at 2, 000 g for 20 minutes. Now carefully pipette the supernatant into a fresh 1.5-milliliter tube.If not analyzing the supernatant immediately, cryopreserve the sample at 80 degrees Celsius. When using frozen samples, centrifuge thawed supernatants again at a speed greater than 7, 000 g for five minutes before analysis. Perform pre-dilution of the samples as required by the cytokine assay protocol.Resuspend the cell pellet in one milliliter of RNA protection buffer. Cryopreserve it at 80 degrees Celsius for subsequent RNA isolation. Alternatively, resuspend the cell pellet in 700 microliters of lysis buffer for immediate RNA isolation.

Preparación de muestras de sangre total para citometría de flujo y análisis de citocinas para determinar la inmunidad del huésped específico del antígeno a patógenos fúngicos y virales