Peripheral blood mononuclear cells (PBMCs) are commonly isolated from whole blood samples in clinical trials. Isolated PBMCs can be cryopreserved for use in downstream assays such as flow cytometry, ...single-cell RNA sequencing (scRNA-seq) and enzyme-linked immunosorbent spot (ELISpot) assays to aid understanding of disease biology and treatment effects, and biomarker identification. However, due to logistical practicalities, delays from blood collection to PBMC processing may exceed 24 h, which can potentially affect PBMC function and, ultimately, downstream assay results.
Whole blood samples from 20 healthy adults were collected and incubated at 20–25 °C for 2–48 h before PBMC processing. PBMC viability was measured, and flow cytometry immunophenotyping, scRNA-seq and ELISpot were performed following increasing PBMC processing delays. The RosetteSep™ granulocyte depletion kit was used to evaluate the impact of granulocyte contamination following processing delay. Processed scRNA-seq reads were used to identify cell clusters based on marker genes. scRNA-seq data was further used to determine gene expression correlation and pathway activity score in major PBMC cell types (T cells, B cells, natural killer cells, monocytes and dendritic cells) between PBMC preparations subjected to shorter (2–4 h) and longer (8–48 h) processing delays. ELISpot assays evaluated the impact of processing delays on the number of interferon-γ (IFN-γ) secreting cells from ex vivo stimulated PBMCs.
PBMC viability was reduced after a 48-h processing delay. Flow cytometry showed that granulocyte contamination of PBMCs increased after 24 h. Cluster analysis of scRNA-seq data identified 23 immune cell type gene expression clusters that were not significantly changed upon granulocyte depletion. Gene expression correlations across the major PBMC cell types were < 0.8 after 24 h of delay compared with 2 or 4 h of delay. Inflammatory, proliferation and signaling pathway activities increased, whereas IFN-γ and metabolic pathway activities decreased with increasing PBMC processing delays. The number of IFN-γ secreting cells trended towards a reduction as PBMC processing delays increased.
PBMC processing delays should be minimised when designing clinical trials to reduce outcome variability in downstream assays. Ideally clinical trial sites should have on-site PBMC processing capabilities or be located close to such facilities.
•Delayed PBMC processing can affect PBMC function and downstream assay quality.•We used flow cytometry, scRNA-seq and ELISpot to assess processing delay impact.•Delays of <24 h had minimal impact on PBMC quality and downstream assay outcomes.•Granulocyte removal after processing delay did not greatly impact gene expression.•Clinical trial sites should have on-site PBMC processing or be located close to central laboratories.
In patients with drug hypersensitivity reactions, confirmation of causality frequently facilitates decision on a continuation or withdrawal of a given treatment. Unfortunately, identification of the ...culprit drug often proves difficult. In vivo methods possess well-known disadvantages like low sensitivity of skin tests or the risk of relapse during drug provocation tests. Therefore, laboratory assays are of great interest as they may improve causal diagnosis without putting patients at risk. In this article, the mechanistic principles and methodological issues of the enzyme-linked immunospot (ELISpot) assay were recapped the context of drug hypersensitivity reactions. A review of ELISpot application in causal diagnosis of drug hypersensitivity was based on literature search. The main findings are: (i) ELISpot assay has a good performance in the detection of drug-specific response. (ii) ELISpot results seem to be not substantially impacted by the type of drug or phenotype of the reaction. (iii) Testing within 30 days since the episode of drug hypersensitivity reaction shows a better performance than in later recovery phase. (iv) Data from pediatric population are too scarce to draw any conclusions. (v) Differences in laboratory protocols and in criteria used in the assessment of ELISpot plates along with the issue of the technical feasibility and reproducibility may limit the use of this assay in the routine diagnostic of drug hypersensitivity reactions.
•Immune-monitoring for BKV infection in kidney transplant recipient is needed.•This meta-analysis showed that IFN-γ ELISPOT is a potential tool to monitor the immune response against ...BKV.•Sensitivity, specificity, likelihood ratio, and BKV-specific IFN-γ ELISPOT frequencies are provided.•BKV-specific IFN-γ ELISPOT, together with BK viral load, might guide the immunosuppression adjustment in patients with BKV infection.
BK virus (BKV) infection after kidney transplantation can cause BKV nephropathy (BKVAN) resulting in graft dysfunction and allograft loss. The treatment for BKVAN is reduction of the immunosuppressive load which increases the risk of kidney transplant rejection. There is no biomarker to monitor BKV activity besides BK viral load. The value of the Enzyme-Linked Immunosorbent Spot (ELISPOT) assay as a tool to monitor the recipient's anti-BKV immune response after transplantation was investigated systematically. Electronic databases, including MEDLINE, Scopus, and the Cochrane Central Register of Controlled Trials were searched for studies of ELISPOT evaluating the immune response against BKV. BKV status was categorized as "active BKV infection" and as "resolving BKV infection". Random-effects model meta-analysis was performed to determine the diagnostic performance of the ELISPOT assay, after stratifying patients into groups based on positive and negative ELISPOT results. One-hundred twenty-seven articles were identified of which nine were included. Patients with negative ELISPOT had an increased risk of having active BKV replication (odds ratio of 71.9 (95%-CI 31.0–167.1). Pooled sensitivity was 0.95 (95%-CI 0.89–0.98) and specificity was 0.88 (95%-CI 0.78–0.94). The standardized mean difference of the number of IFN-γ producing cells between patients with active BKV infection compared with patients who had resolving BKV infection was -2.09 (95%-CI -2.50, -1.68). The ELISPOT assay is a useful tool for BKV risk assessment and in combination with BKV load may support clinicians in guiding immunosuppressive therapy in patients with BKV replication.
SARS-CoV-2 infection takes a mild or clinically inapparent course in the majority of humans who contract this virus. After such individuals have cleared the virus, only the detection of ...SARS-CoV-2-specific immunological memory can reveal the exposure, and hopefully the establishment of immune protection. With most viral infections, the presence of specific serum antibodies has provided a reliable biomarker for the exposure to the virus of interest. SARS-CoV-2 infection, however, does not reliably induce a durable antibody response, especially in sub-clinically infected individuals. Consequently, it is plausible for a recently infected individual to yield a false negative result within only a few months after exposure. Immunodiagnostic attention has therefore shifted to studies of specific T cell memory to SARS-CoV-2. Most reports published so far agree that a T cell response is engaged during SARS-CoV-2 infection, but they also state that in 20-81% of SARS-CoV-2-unexposed individuals, T cells respond to SARS-CoV-2 antigens (mega peptide pools), allegedly due to T cell cross-reactivity with Common Cold coronaviruses (CCC), or other antigens. Here we show that, by introducing irrelevant mega peptide pools as negative controls to account for chance cross-reactivity, and by establishing the antigen dose-response characteristic of the T cells, one can clearly discern between cognate T cell memory induced by SARS-CoV-2 infection vs. cross-reactive T cell responses in individuals who have not been infected with SARS-CoV-2.
Vaccination against SARS-CoV-2 with coronavirus vaccines that elicit protective immune responses is critical to the prevention of severe disease and mortality associated with SARS-CoV-2 infection. ...Understanding the adaptive immune responses to SARS-CoV-2 infection and/or vaccination will continue to aid in the development of next-generation vaccines. Studies have shown the important role of SARS-CoV-2-specific antibodies for both disease resolution and prevention of COVID-19 serious sequelae following vaccination. However, antibody responses are short-lived, highlighting the importance of studying antigen-specific B-cell responses to better understand durable immunity and immunologic memory. Since the spike protein is the main target of antibody-producing B cells, we developed a SARS-CoV-2 memory B cell ELISPOT assay to measure the frequencies of spike-specific B cells after COVID-19 infection and/or vaccination. Here, we describe in detail the methodology for using this ELISPOT assay to quantify SARS-CoV-2 spike-specific memory B cells produced by infection and/or vaccination in human PBMC samples. Application of this assay may help better understand and predict SARS-CoV-2 recall immune responses and to develop potential B cell correlates of protection at the methodological level.
Memory B cells (B
) provide the second wall of adaptive humoral host defense upon specific antigen rechallenge when the first wall, consisting of preformed antibodies originating from a preceding ...antibody response, fails. This is the case, as recently experienced with SARS-CoV-2 infections and previously with seasonal influenza, when levels of neutralizing antibodies decline or when variant viruses arise that evade such. While in these instances, reinfection can occur, in both scenarios, the rapid engagement of preexisting B
into the recall response can still confer immune protection. B
are known to play a critical role in host defense, yet their assessment has not become part of the standard immune monitoring repertoire. Here we describe a new generation of B cell ELISPOT/FluoroSpot (collectively ImmunoSpot
) approaches suited to dissect, at single-cell resolution, the B
repertoire ex vivo, revealing its immunoglobulin class/subclass utilization, and its affinity distribution for the original, and for variant viruses/antigens. Because such comprehensive B cell ImmunoSpot
tests can be performed with minimal cell material, are scalable, and robust, they promise to be well-suited for routine immune monitoring.
ELISpot and flow cytometry are two methods often utilized side-by-side for detecting secreted and intracellular cytokines, respectively. Each application has its own advantages and challenges. ...ELISpot is more sensitive compared to ELISA and appears to be more consistent in detecting IL-10 production than flow cytometry. ELISpot can be used for detecting the secretion of multiple cytokines but not from the same cells simultaneously, whereas flow cytometry allows for the concurrent detection of multiple intracellular cytokines by the same cells. Flow cytometry is a convenient technique allowing for the detection of many cytokines at the same time in a population of cells. The restimulation cocktails used for cytokine detection in flow cytometry are hard on cells and lead to decreased cell viability. Using a live dead dye allows for the exclusion of dead cells when analyzing data. We illustrated the differences between ELISpot and flow cytometry by stimulating cells with two toll-like receptor (TLR) agonists, LPS or Pam
CSK
. Both activators increase production of various cytokines, including IL-10, IL-6, and TNF-alpha. The TLR2 antagonist, MMG-11, was used to inhibit this increased cytokine production. We observed some inhibition of IL-6 and IL-10 from Pam
CSK
stimulation in the presence of MMG-11 by flow cytometry. TNF-α remains largely unchanged as its basal expression is high, but there is some reduction in the presence of MMG-11 for both methods. However, IL-10 was difficult to detect by ELISpot given the low seeding density. Overall, both ELISpot and flow cytometry are good methods for detecting secreted and intracellular cytokines, respectively, and should be used as complimentary assays.
Interferon-gamma (IFNγ) ELISpot and FluoroSpot are widely used assays to detect functional cell responses in immunotherapy clinical studies. Recognized for their importance in vaccine development ...studies to quantitate immune responses, these assays have more recently risen to the forefront in cell and gene therapy as well as cancer immunotherapy fields where responses against cancer neoantigens are not easily detectable above assay background. Here, we test a new class of fetal bovine serum (FBS), CultraPure FBS, in ex vivo ELISpot and FluoroSpot assays and cultured FluoroSpot assays following in vitro expansion. Several CultraPure FBS lots that have been specially formulated through the process of lyophilization (lyo-FBS) were compared to liquid CultraPure FBS. We stimulated human PBMCs with antigen-specific peptide pools diluted in media supplemented with liquid CultraPure FBS or lyo-FBS and found equivalent cytokine production with negligible to no assay background with both liquid and lyo-FBS formats. Moreover, the lyo-FBS showed lot-to-lot consistency and 90-day refrigerated (4 °C) stability in both ex vivo direct and in vitro cultured assays. In addition, we present here a method using lyo-FBS for the expansion of low-frequency antigen-specific T cells, mimicking the low frequency seen with cancer neoantigens by utilizing a cultured FluoroSpot assay. Our results demonstrate the presence of Granzyme B, interferon-gamma (IFNγ), and tumor necrosis factor (TNF) production by antigen-specific polyfunctional T cells following a 9-day culture using media supplemented with lyo-FBS.
Enzyme-linked immunospot (ELISPOT) is one of the most important methods to measure the number of specific cells by detecting protein secretion at a single-cell level. However, traditional ELISPOT ...based on enzyme-substrate color development can only detect one target. Therefore, scientists developed multiple-target ELISPOT based on enzyme-substrate coloring. Besides, FluoroSPOT that can detect 2-4 fluorescent signals are developed. Nevertheless, the maximum detection targets of multiple-target ELISPOT and FluoroSPOT are around 4, and the signal amplification system can be further optimized. Fluorescence-based oligo-linked immunospot (FOLISPOT), which utilized DNA-barcoded antibodies to provide a highly multiplexed method with signal amplification, was developed to detect multiple targets simultaneously. In this method, multiple targets can be detected in one round and multiple rounds of detection can be conducted, and thus a large number of targets can be detected. Besides, signal amplification is achieved by DNA complementary pairing and modular orthogonal DNA concatemers, and thus cells secreting limited amounts of proteins can be detected. According to the studies, FOLISPOT can detect more spots than ELISPOT and can detect targets that are undetectable by ELISPOT. Furthermore, FOLISPOT can be utilized to detect more than 6 targets, by allowing sequential detection of multiple targets in one round and sequential detection in multiple rounds.
SARS-CoV-2 continues to threaten global public health, making COVID-19 immunity studies of utmost importance. Waning of antibody responses postinfection and/or vaccination and the emergence of immune ...escape variants have been ongoing challenges in mitigating SARS-CoV-2 morbidity and mortality. While a tremendous amount of work has been done to characterize humoral immune responses to SARS-CoV-2 virus and vaccines, cellular immunity, mediated by T cells, is critical for efficient viral control and protection and demonstrates high durability and cross-reactivity to coronavirus variants. Thus, ELISPOT, a standard assay for antigen-specific cellular immune response assessment, allows us to evaluate SARS-CoV-2-specific T-cell response by quantifying the frequency of SARS-CoV-2-specific cytokine-secreting cells in vitro. We have outlined a detailed procedure to study T-cell recall responses to SARS-CoV-2 in human peripheral blood mononuclear cells (PBMCs) following infection and/or vaccination using an optimized IFN-γ ELISPOT assay. Our methodologies can be adapted to assess other cytokines and are a useful tool for studying other viral pathogen and/or peptide-specific T-cell responses.
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