A whole blood monokine-based reporter assay provides a sensitive and robust measurement of the antigen-specific T cell response
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The ability to measure T-cell responses to antigens is proving critical in the field of vaccine development and for understanding immunity to pathogens, allergens and self-antigens. Although a variety of technologies exist for this purpose IFNγ-ELISpot assays are widely used because of their sensitivity and simplicity. However, ELISpot assays cannot be performed on whole blood, and require relatively large volumes of blood to yield sufficient numbers of peripheral blood mononuclear cells. To address these deficiencies, we describe an assay that measures antigen-specific T cell responses through changes in monokine gene transcription. The biological amplification of the IFNγ signal generated by this assay provides sensitivity comparable to ELISpot, but with the advantage that responses can be quantified using small volumes of whole blood.
Whole blood or peripheral blood mononuclear cells (PBMCs) from healthy controls and immunosuppressed recipients of solid organ transplants were incubated with peptide pools covering viral and control antigens or mitogen for 20 hours. Total RNA was extracted and reverse transcribed before amplification in a TaqMan qPCR reaction using primers and probes specific for MIG (CXCL9), IP-10 (CXCL10) and HPRT. The induction of MIG and IP-10 in response to stimuli was analysed and the results were compared with those obtained by ELISpot.
Antigen-specific T cell responses can be measured through the induction of MIG or IP-10 gene expression in PBMCs or whole blood with results comparable to those achieved in ELISpot assays. The biological amplification generated by IFNγ-R signaling allows responses to be detected in as little as 25 μL of whole blood and enables the assay to retain sensitivity despite storage of samples for up to 48 hours prior to processing.
A monokine-based reporter assay provides a sensitive measure of antigen-specific T cell activation. Assays can be performed on small volumes of whole blood and remain accurate despite delays in processing. This assay may be a useful tool for studying T cell responses, particularly when samples are limited in quantity or when storage or transportation is required before processing.
KeywordsTacrolimus ELISpot Assay Fold Induction Peptide Pool Human Acute Monocytic Leukaemia Cell
Analysis of T-cell responses to antigen has become central to our understanding of immunity against pathogens  and for vaccine development . Over the years, many methods have been developed to assess antigen-specific T cells, progressing from limiting dilution and 51Cr release assays to techniques that measure cytokine production and changes in T-cell phenotype upon activation [3, 4].
Activation of T-cells by antigen leads to the production of cytokines, which in turn mediate downstream effector responses . Of the cytokines secreted, IFNγ is one of the best studied, having been shown to be a robust marker of T cell activation with levels correlating with the efficacy of the immune response [6, 7]. As a result, the detection of intracellular or secreted IFNγ has become an accepted surrogate for functional immune responses to antigen in many circumstances. Of the techniques available to measure IFNγ, ELISpot analyses are one of the most widely used due to their low cost, reliability and high sensitivity for detecting low numbers of antigen-specific cells . However, these assays require purified PBMCs and relatively large volumes of blood to perform.
MIG and IP-10 are inflammatory chemokines predominantly released by monocytes following stimulation by IFNγ [12, 13], which act as part of a feedback loop binding to the chemokine receptor CXCR3 expressed by activated T lymphocytes . As only minimal transcription of these genes occurs in the basal state , the biological amplification of the IFNγ signal by the second messenger signaling pathways provides an opportunity to improve detection when compared with direct measurement of IFNγ , however previous experiments have primarily focused on intracellular detection of MIG at the protein level in CD14+ cells via flow cytometry [11, 16]. A report demonstrating the detection of MIG at the RNA level in association with T-cell responses to a malaria vaccine supports the idea that this could be used as a bioassay of antigen-specific responses , but the potential utility of a monokine-based detection system has not been evaluated systematically.
Therefore, we wanted to know whether an assay measuring the induction of MIG and IP-10 mRNA could be a sensitive and convenient alternative to ELISpot assays or other techniques based on the use of antibody reagents and/or flow cytometry. To this end, we have developed a protocol using qPCR for MIG and IP-10 to quantify antigen-specific T cell responses that can be performed on whole blood or with PBMCs. We show this assay remains sensitive when tested with small volumes of blood, despite delays of up to 48 hours before sample processing and correlates well with the results of IFNγ-ELISpot assays.
Subject recruitment and collection of samples
Patients attending the Oxford Kidney and Transplant Units and healthy controls recruited through advertisement were invited to donate whole blood following written informed consent. Ethical approval for the study was granted by the Berkshire Research Ethics Committee (REC reference 08/H0607/50). Whole blood was collected directly into anticoagulated sodium heparin tubes (BD, Oxford, UK).
Isolation of PBMCs
Cell separation tubes (Sigma, Gillingham, UK) were used to isolate peripheral blood mononuclear cells (PBMCs) from whole blood by density gradient centrifugation. PBMCs were washed twice with sterile PBS (Fisher Scientific, Loughborough, UK) then counted before being re-suspended in RPMI 1640 media, supplemented with 10% Fetal Calf Serum, 1% L-Glutamine, 1% Penicillin-streptomycin solution, 1% Sodium Pyruvate (all from Sigma), 1% HEPES buffer (GibcoBRL, Scotland, UK), and 0.05 mM 2-Mercaptoethanol (Invitrogen, Paisley, UK).
Preparation of peptides and reagents
Peptide pools (15 amino acids in length with overlaps of 11 amino acids) comprising the immunodominant EBV antigens BZLF1 and EBNA1, and the CMV-specific antigens pp65, UL-40, IE1 and IE2 were purchased from JPT (Berlin, Germany). Control peptide pools derived from human alpha-actin-1 (ACTs) protein (negative control) and CEF (CMV, EBV and Influenza) peptides (positive control) were also obtained from JPT (Berlin, German). All peptides were stored at -20°C as lyophilised powders until used. Peptide pools were dissolved in DMSO (Sigma) and diluted with Dulbecco's PBS (GibcoBRL) according to the manufacturer's instructions. The lymphocyte mitogens, Concanavalin A (ConA) and Phytohemagglutinin (PHA) and the calcineurin inhibitor tacrolimus (FK506) were purchased from Sigma (Gillingham, UK). Recombinant human IFNγ was obtained from Peprotech (London, UK).
96-well PVDF plates (Millipore, Watford, UK) were coated with 100 μl of IFNγ monoclonal antibody at 15 μg/ml (clone 1-D1K), (MAbTech, Stockholm, Sweden) and incubated at 4°C overnight. The next day, plates were washed 5 times with PBS (Fisher Scientific) then incubated for 2 hours at 37°C with 200 μl/well of complete media. Peptide pools were added in triplicate to each plate at a final concentration of 1 μg/ml. PHA and ConA were used at final concentrations of 5 μg/ml, also in triplicate. 2 × 105 PBMCs were added to each well and incubated for 18-20 hours at 37°C in 5% CO2. At the end of the incubation period, plates were washed 5 times with PBS/0.05% Tween 20 (Sigma) then left at room temperature for 2 hours after the addition of 100 μl of 1 μg/ml biotinylated IFNγ antibody (clone 7-B6-1) (MAbTech). After washing 5 times 100 μl (1 μg/ml) Streptavidin-ALP (MAbTech) was added for 1 hour at room temperature, then plates were developed with 100 μl/well of 0.22 μm filtered BCIP/NBT (Pierce, Cramlington, UK). Development continued until spots appeared in the positive control wells. Spots were counted using an automated plate reader, AID ELISpot (AID, Strassberg, Germany), with AID ELISpot software Version 3.5, (CADAMA Medical Ltd, Stourbridge, UK).
PBMCs or whole blood were aliquoted into 96-well round-bottom plates (Greiner, Gloucestershire, UK) before the addition of peptide pools, mitogen or recombinant IFNγ (all in triplicate). Plates were incubated for 18-20 hours at 37°C, in 5% CO2 before RNA extraction using an RNeasy Mini Kit (PBMCs) or QIAamp RNA Blood Mini Kit (whole blood) according to the manufacturer's instructions. Extracted RNA was reverse transcribed using Superscript First-Strand Synthesis System for RT-PCR (Invitrogen, Paisley, UK). cDNA quantification was performed in a 10 μl reaction mix using Taqman Fast Universal Mastermix and the Taqman Gene Expression Assay (Applied Biosystems, Warrington, UK). The Taqman Gene Expression assays included primer and probe combinations for MIG (CXCL9) (Assay ID Hs00171065_m1), IP-10 (CXCL10) (Assay ID Hs00171042_m1) and the housekeeping gene hypoxanthine phosphoribosyltransferase 1 (HPRT) (Assay ID Hs99999909_m1). qPCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems). Experimental triplicates were carried through each step of the protocol. qPCR data are expressed as fold induction (RQ value) of MIG or IP-10 normalized to expression of the housekeeping gene HPRT. RQ is a function of the Ct value, where Ct is the number of PCR cycles required to produce a sustainable amplification in the fluorescent signal above the background threshold. ΔCt is the difference between the Ct value for the housekeeping gene and the Ct value for the gene of interest e.g. MIG. ΔΔCt is the ΔCt value of the stimulated sample subtracted from the ΔCt value of the reference (negative control). RQ is calculated as 2-ΔΔCT.
The efficiency (or E value) of the quantitative PCR reactions was calculated from a standard curve of MIG and IP-10 induction following stimulation with increasing concentrations of recombinant IFNγ. The average E values for whole blood and PBMCs were 0.997 and 0.998 respectively (with no difference between MIG and IP-10). The human acute monocytic leukaemia cell line THP-1 (ATCC, Middlesex, UK) was used for in vitro analyses, and as an external control. To facilitate inter and intra-patient comparison RQ values were routinely normalized to the mean fold induction of MIG and IP-10 in response to the negative control.
Statistical analyses were performed with GraphPad PRISM Version 5 (GraphPad Software Inc, California, USA). Differences between groups were analysed by one-way analysis of variance (ANOVA) or independent t-tests. Normality was assessed using the D'Agostino-Pearson omnibus test. Statistical significance was defined as p < 0.05. Data from monokine assay are presented as the mean and 95% confidence interval calculated from mean, maximum and minimum RQ values from experimental triplicates and ELISpot data as the mean ± SEM from triplicate wells.
Recombinant IFNγ induced MIG and IP-10 in culture
Antigen-specific responses can be measured in whole blood or PBMC
Effects of sample storage on assay reproducibility
Sensitivity with reduced numbers of PBMCs or volume of whole blood
Comparison of monokine and IFNγ-ELISpot assays
Having confirmed a highly significant relationship between the results of ELISpot and monokine assay using PBMCs, we proceeded to assess whether a similar relationship might be evident with whole blood. T-cell responses to stimulation with the CEF peptide pool, CMV or EBV peptides as above, were measured in a further 10 samples using 100 μl/well of whole blood for the monokine assay or 2 × 105 PBMCs/well for the ELISpot assay (Figure 6B). The correlation between the fold increase in MIG in the monokine assay and SPU/million PBMCs in the ELISpot assay was r2 = 0.6729 (p < 0.0037). Except in one case, where low-level induction of MIG and IP-10 was seen when the ELISpot was considered negative (21 fold-induction of MIG vs. 7 SPU/million PBMCs), there was concordance between ELISpot and monokine assay results.
Effects of immunosuppression
The ability to accurately quantify antigen-specific responses has become integral to the study of infectious diseases, vaccine responses, autoimmunity and allergy . While a variety of techniques exist to detect T cells that recognize specific epitopes and to analyse the effects of antigen-induced activation on T cell phenotype, many of these methods require sophisticated equipment and significant expertise to perform. An alternative strategy has been the measurement of cytokines produced by antigen-activated T cells, in particular IFNγ, which has been demonstrated to correlate with functional immunity in a range of clinical settings [22, 23].
ELISpot assays have become the standard method to quantify IFNγ production in many laboratories because they are easy to perform, do not require investment in expensive equipment (e.g. flow cytometers) and are sensitive for the detection of even low numbers of antigen-specific cells . However, ELISpot assays have several important limitations: relatively large volumes of blood are required to obtain sufficient PBMCs for analysis; the process of extracting and purifying PBMCs may cause their activation and alter their phenotype [19, 20]; assays cannot be re-run unless stored samples of cells are available.
Due to the high burden of infectious diseases in the developing world, much of the research into T-cell responses to pathogens is focussed on areas where there are limited resources for the extraction and purification of PBMCs and where sample volumes may be restricted by patient age or co-morbidity. In these settings an assay that requires only small volumes of blood and which retains sensitivity despite delays in processing would provide significant benefits. In this study we have described a method to assess antigen-specific T cell responses ex vivo by measuring the up-regulation of MIG and IP10 gene transcription in mononuclear cells, in response to IFNγ released by activated T cells (Figure 1). We analysed responses to both chemokines as a means of internally validating our results and as expected the correlation between MIG and IP10 induction was high. Interestingly, the induction of IP-10 appears to be a more sensitive marker in the presence of low numbers of antigen-specific T cells, possibly reflecting activation of IP-10 gene transcription by cytokines other than IFNγ (Figure 2) .
In contrast with previous studies that have evaluated antigen-specific responses through changes in T-cell cytokines, we have found the monokine assay to have several advantages. The biological amplification of the IFNγ signal in monocytes means that co-stimulatory antibodies are not required ; responses remain stable despite delays in processing (Figure 4), or where samples sizes are small (Figure 5) and sensitivity equivalent to ELISpot is achievable . Furthermore, by enabling the measurements to be made on whole blood, preparation time is reduced and results may be more representative of the response in vivo[28, 29] (Figure 8).
As the assessment of antigen-specific responses in the monokine assay is indirect, one limitation of the assay is its inability to distinguish between a small number of highly activated T cells and a larger number of less activated T cells producing the same amount of IFNγ. Although this necessarily reduces the strength of the relationship between monokine and ELISpot assay results, overall the correlation between these assays is good and significantly, non-responders are clearly identified (Figure 6). Additional benefits of the monkine assay are that by measuring the effects of IFNγ on target cells, rather than the production of IFNγ alone, the monokine assay provides some confirmation of the downstream functionality of the T-cell response and once RNA has been extracted and reverse transcribed, samples can be stored indefinitely.
The biological amplification of the IFNγ signal generated by the endogenous reporter system described provides a sensitive and accurate way to measure antigen-specific T cell activation. The opportunity to perform these assays on whole blood reduces the time and resource requirements for sample processing and may yield results that are more representative of the in vivo situation. In addition, the retention of sensitivity despite delays in processing or where samples are limited in quantity will make this assay a viable alternative to ELISpot in many settings.
The authors would like to thank Professor Paul Klenerman for his helpful comments. This work was funded by the Oxford Comprehensive Biomedical Research Centre.
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