Research Paper
The interferon gamma secretion assay: a reliable tool to study
interferon gamma production at the single cell level
I. Desombere
a,*, P. Meuleman
a, H. Rigole
a, A. Willems
a, J. Irsch
b, G. Leroux-Roels
a aCenter for Vaccinology, Department Clinical Biology, Microbiology and Immunology, Ghent University and Hospital, De Pintelaan, 185, 9000 Ghent, Belgium
b
Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
Received 3 September 2003; received in revised form 23 December 2003; accepted 5 January 2004
Abstract
Different single-cell analyses for the detection of antigen-specific T cells based on antigen-triggered induction of cytokine production (elispot, intracellular cytokine staining, cytokine secretion assay, etc.) have been analyzed. In this paper we present the data of a thorough validation of the IFNgSecretion Assay (ISA, Miltenyi Biotec, Bergisch Gladbach, Germany). In this assay the secreted IFNgis bound to the cell surface and is then stained as an artificial surface molecule and analyzed by flow-cytometry. The introduction of five quality criteria markedly improved the reproducibility of this assay and made it very reliable (intra-assay variability < 5%; inter-assay variability < 20%). Recovery experiments further demonstrated that almost 100% of IFNg+labeled cells could be detected by this technology. In order to analyze which cell subsets contribute to IFNg-production,
we compared the results obtained in different individuals after VZAg-stimulation. Three different IFNg-secretion patterns could be discerned. In Pattern 1 there is a predominant and almost equal contribution of T cells and NK cells with a minor contribution of CD3+CD56+and B cells.Pattern 2, which is most abundant, is characterized by a predominance of NK cells (60 – 70%).Pattern 3differs from the previous one in its minor contribution of NK cells. Here T cells predominate the IFNg
secretion. These results clearly demonstrate that the IFNg+subset distribution after VZAg-stimulation is not uniform and differs individually. Furthermore, the ISA-technology proves to be very useful in vaccine research. This was demonstrated by testing the IFNg+secretion pattern after HBsAg-stimulation in PBMC from HBsAg-vaccinated individuals.
D2004 Elsevier B.V. All rights reserved.
Keywords:Flow cytometry; Cytokine-secretion; Reproducibility; Vaccines; IFNg; VZAg
0022-1759/$ - see front matterD2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2004.01.001
Abbreviations:BC, buffy coat; CPA, cellular proliferation assay; CTL, cytotoxic T lymphocyte; CV, coefficient of variation; Elisa, enzyme-linked immunosorbent assay; Elispot, enzyme-enzyme-linked immunospot; FACS, fluorescence activated cell sorter; HBsAg, Hepatitis B surface antigen; HCV-E1, Envelope 1 protein of the Hepatitis C virus; IC, intracellular cytokine staining; IFNg, interferon-g; ISA, IFNgsecretion assay; Mab, monoclonal antibody; MACS, magnetic activated cell sorting; PBMC, peripheral blood mononuclear cells; PI, propidium iodide; QC, quality control; SD, standard deviation; SEB, Staphylococcus aureus enterotoxin B; SI, stimulation index; Th, T helper; TT, tetanus toxoid; VZAg, varicella-zoster virus antigen.
* Corresponding author. Tel.: +32-9-2404174; fax: +32-9-2406311.
E-mail address:[email protected] (I. Desombere).
1. Introduction
Following appropriate antigen-specific stimulation, lymphocytes rapidly express and secrete cytokines. These cytokines can be measured in the culture supernatant using Elisa, but this method, although very useful for certain applications, lacks the capacity to identify the number and the phenotype of the IFNg -secreting cells. Techniques to investigate cytokine production at a cellular level (single-cell analysis) include Enzyme-linked immunospot (Elispot), intra-cellular cytokine staining (IC), in situ hybridization (mRNA) and cytokine secretion assay. The two flow cytometric assays, IC staining and cytokine secretion assay, are able to identify the phenotype of the cytokine-secreting cell. In this paper we thoroughly analyzed and validated the cytokine secretion assay, which not only allows for the identification but also the physical isolation of viable cytokine-secreting cells.
The cytokine secretion assay was developed in the mid-1990s as a method to analyze and isolate single lymphoid cells based on the molecules they secreted
(Manz et al., 1995). Cells are induced to secrete cytokines by stimulation with a lectin or a recall antigen. The cytokine is retained on the secreting cell by a bispecific antibody molecule consisting of a conjugated pair of monoclonal antibodies. One anti-gen-binding site binds to a cell surface molecule, e.g. CD45 present on all leukocytes, while the other binding site recognizes the cytokine studied (e.g. IFNg). The immobilized cytokine can then be revealed by adding a detection reagent which is a phycoerythrin-labeled anti-cytokine antibody. The phenotype of the cytokine-secreting cell can be deter-mined by labeling selected surface markers with appropriate monoclonal antibodies (e.g. anti-CD3, anti-CD4, etc.). To physically isolate the cytokine-secreting cells from the cell suspension an anti-phy-coerythrin antibody conjugated to superparamagnetic particles is added(Assenmacher et al., 1998; Broster-hus et al., 1999; Oelke et al., 2000).
In this paper we describe the validation of the IFNg
Secretion Assay (ISA, Miltenyi Biotec, Bergisch Gladbach, Germany) without physical isolation or enrichment of the cytokine-producing cells. When performed according to the manufacturer’s instruc-tions and adhering to a series of quality measures, the
ISA proves to be a very reliable assay. This method clearly identifies different IFNg-secretion patterns which vary according to the stimulus used and the subject studied. To further demonstrate the applica-bility of the ISA-technology in vaccine-research, we analyzed the IFNg-secretion pattern after HBsAg-stimulation in PBMC from several subjects before and after HBsAg vaccine administration.
2. Materials and methods
2.1. Isolation and freezing of human peripheral blood mononuclear cells (PBMC)
Buffy coats (BC) were obtained from the Blood Transfusion Center, Red Cross, Ghent, Belgium. PBMC were prepared by standard Ficoll-Isopaque (Lymphoprepk, Nycomed Pharma AS, Oslo,
Nor-way) density gradient centrifugation, washed twice in Hanks’ Balanced Salt Solution (HBSS) without Ca2 + and Mg2 + (Invitrogen, Carlsbad, CA, USA) and frozen (liquid N2, 3107PBMC/cryotube) in
freez-ing solution (10% dimethylsulfoxide (DMSO; Sigma, St. Louis, MO) in Fetal Calf Serum (FCS; Invitro-gen)). After thawing, PBMC were washed twice and resuspended at 3106 cells/ml in complete cell culture medium (cRPMI) consisting of RPMI 1640 supplemented with 25 mM HEPES, 50 U/ml penicil-lin, 50 Ag/ml streptomycin, 2 mM L-glutamine (all
from Invitrogen), 510 5 M 2-ME (Sigma), and 10% heat-inactivated human AB+ serum (BioWhit-taker, Cambrex, NJ, USA). BC1, 2, 3, 4 and 7 were used for validation of the Cytokine Secretion Assay. BC5 and 6 were only used for analysis of IFNg -producing cell subsets after antigen-stimulation. Via-bility of the human PBMC was determined by Propi-dium iodide (PI) exclusion in flow cytometry. Only thawed samples with a viability of z80% were used
in the experiments.
2.2. Antigens used for in vitro stimulation
Human PBMC were stimulated in vitro using three different antigen preparations: Tetanus toxoid (TT)(batch 59-9), obtained from Statens Seruminsti-tut (WHO, Copenhagen, Denmark), Varicella-Zoster antigen (VZAg), obtained from Dade Behring
Mar-burg (MarMar-burg, Germany), and recombinant Hepatitis B surface antigen (HBsAg). The endotoxin content of the purified TT-preparation was below 10 EU/ml, a level which has no direct mitogenic activity in in vitro cell cultures. TT was added to lymphocyte cultures at a final concentration of 2 Ag/ml. VZAg was used at a final dilution of 1/200. This antigen preparation is produced from human tissue cell culture infected with Varicella-Zoster Virus and is lyophilized after inactivation. The endotoxin content is negligible. HBsAg (subtype adw) was produced in Saccharomyces cerevisiae (lot DVP23) and kindly provided by GlaxoSmithKline Biologicals (Rixen-sart, Belgium). The antigen is purified for medical use and is free of endotoxin.
2.3. Antibodies used for FACS-analysis
The flow cytometric analysis was performed using a FACScalibur (Becton Dickinson, Mountain View, CA, USA) and the CellQuestk software (Becton
Dickinson). We used the following MoAbs conjugat-ed to fluorescein isothiocyanate (FITC; FL1) or allo-phycocyanin (APC; FL4) for direct fluorescence of surface markers: anti-CD3 (clone SK7), anti-CD4 (clone SK3), anti-CD8 (clone RPA-T8), anti-CD56 (clone NCAM 16,2), anti-CD19 (clone 4G7)(all from PharMingen, San Diego, CA, USA). Anti-IFNg anti-bodies were labeled with phycoerythrin (PE; FL2). Propidiumiodide (PI; FL3) and peridinin chlorophyl protein (PerCP; FL3)-conjugated anti-CD14 (Phar-Mingen) were used for exclusion of dead cells and lymphocyte gate-setting, respectively. FACS solution was prepared by adding 1% Bovine Serum Albumin (BSA, Sigma) and 0.02 M sodium azide (Merck Eurolab, VWR International, IL, USA) to phosphate buffered saline (PBS, Invitrogen).
2.4. In vitro stimulation of PBMC for IFNcsecretion assay (ISA), for cellular proliferation assay (CPA) and for IFNcproduction in supernatant
PBMC were cultured at 3106 cells per ml in complete medium. To at least 1.5 ml of this suspen-sion VZAg was added at a final concentration of 1/ 200 of the stock solution. Another aliquot of at least 1.5 ml was used for stimulation with TT at a concen-tration of 2Ag/ml and a third aliquot of z1.5 ml was
left without antigen (non-stimulated blank). For the ISA, 1 ml of each PBMC-suspension (3106cells) was transferred to a 24-well plate and incubated for 16 h at 37jC in a 5% CO2-atmosphere.
The lymphoproliferative responses of PBMC were determined as described previously(Desombere et al., 1995). In brief, to 100Al aliquots containing 3106 PBMC/ml in a medium containing VZAg, TT or as blank control, 100Al of complete medium containing the same antigen or no antigen (control) were added. These microcultures were set up in triplicate and kept at 37jC in 5% CO2in air for 6 days. Eighteen hours
before harvesting [3H]TdR (0.5 ACi/well, Amersham International, Buckinghamshire, UK) was added. At that time (day 5), supernatant (100 Al/well) was collected from each well and replaced by 100
Al [3H]TdR medium. The supernatant of the three wells for each condition were pooled and used for IFNg-detection in conventional Elisa’s. The cultures were harvested on day 6 by an automated harvesting device and assayed for [3H]TdR incorporation by liquid scintillation counting in an LKB-Wallac 8100 counter (LKB, Bromma, Sweden). Proliferation data are expressed as x¯(mean of triplicate cultures)FSD,
as Dcpm (mean cpm of Ag-stimulated cultures mean cpm of control cultures), or as SI, which was calculated by the following equation: SI = mean ex-perimental cpm with Ag/mean control cpm without Ag. SI were considered positive when z3.
2.5. IFNcsecretion assay (ISA)
IFNg-secreting cells were detected using the IFNg
secretion assay (Miltenyi Biotec) according to the manufacturer’s instructions. Briefly, after overnight stimulation with or without antigen, the PBMC of each 24-well were transferred to 15 ml tubes and washed with 10 ml cold buffer (300 g, 10 min, 4jC).
The cell pellet was suspended in 80Al cold medium and 20 Al IFNg Catch Reagent (a bi-specific MoAb directed against CD45 and IFNg) was added. After 5 min of incubation (labeling) at 4jC, 1 ml of warm (37 jC) medium was added. The cells were placed at 37 jC on a slow rotating platform to allow cytokine
secretion for 45 min. The cells were immediately placed on ice and then washed with cold buffer (300 g, 10 min, 4jC) and resuspended in 80Al cold buffer. The secreted IFNg, bound to the catch reagent, was
stained with 20 Al PE-conjugated IFNg-specific antibody (IFNg Detection Reagent). After an incu-bation period of 10 min at 4 jC, the cells were
washed with cold buffer, spun down (300 g, 10 min, 4 jC) and resuspended in FACS-buffer. The
cells of each tube were divided over six small tubes and stained for expression of surface markers. Each tube was filled with FACS-buffer and a different cell surface marker combination: tube 1, anti-CD3-FITC and anti-CD4-APC; tube 2, anti-CD3-anti-CD3-FITC and anti-CD8-APC; tube 3, anti-CD56-FITC and CD3-APC; tube 4, CD56-FITC and anti-CD4-APC; tube 5, anti-CD56-FITC and anti-CD8-APC; tube 6, anti-CD19-FITC. All tubes also contained Propidium iodide (PI) and anti-CD14-PerCP. Cells were then ready for flow-cytometric analysis. Since frequencies of antigen-specific, cy-tokine-producing cells are low, 105 living lympho-cytes were counted for each cell surface marker combination. The lymphocyte gate (R1) is based on FL3 and forward scatter properties (Fig. 1, panels A). Dead cells and monocytes are excluded accord-ing to PI and anti-CD14-PerCP stainaccord-ing in FL3.
Fig. 1, panels B represent staining of gated (on R1, living lymphocytes) lymphocytes for secreted IFNg
(PE, FL2) versus CD3 (FITC, FL1) in the VZAg-stimulated and control sample. The Upper Right quadrant of the B-panels thus represent the living CD3+ lymphocytes that produce IFNg. The percent-age indicates the frequency of IFNg+CD3+ cells among CD3+ cells. The C-panels of Fig. 1 repre-sent staining of gated (on R2, CD3+ cells) CD3+ cells for secreted IFNg versus CD4 (APC, FL4) in the VZAg-stimulated and the control sample. The Upper Right quadrants of the C-panels thus repre-sent IFNg+CD3+CD4+ living lymphocytes. The per-centage indicates the frequency of IFNg+CD3+CD4+ cells among CD3+CD4+ lymphocytes.
2.6. Spiking experiments
Accuracy of the ISA was analyzed via ‘recovery’ tests. In these experiments, a known number of IFNg+
cells was added to non-IFNg+cells and the number of recovered IFNg+cells was measured. By using a large dilution series the correlation coefficient (R2) between the measured and the expected numbers of IFNg+
cells was calculated. In order to obtain a uniformly
IFNg+ cell population, T cell clones were incubated with IFNgCatch Reagent and incubated for 5 min on ice. Pure recombinant IFNg (200 ng/ml) was added and a further incubation (30V, 4jC) was performed on
a rotation platform. After washing, PE-conjugated IFNg Detection Reagent was added and cells were incubated for 10 min on ice. Subsequently these cells were washed and tested with the FACScalibur for IFNg-positivity. In this way a population of 100% IFNg+CD4+CD3+ T lymphocytes was generated. In-creasing numbers of these T cells (ranging from 0 to 1105 IFNg+ cells) were added to 3105 non-activated non-IFNg-producing T cells. After anti-CD3 FITC labeling of T cells and PI-addition, the recovery of PE-labeled IFNg+cells was measured by flow cytometry. Living cells were gated (R1) accord-ing to PI-exclusion (FL3/FSC). In the first experiment (‘spiking 1’) 1105cells were counted in R1, in the second, independent experiment (‘spiking 2’) 5104 cells were counted in R1. The percentage of recovered IFNg+ T cells is given by the number of measured positive cells divided by the number of living cells in gate R1 and multiplied by 100.
2.7. IFNc-determination with the IFNc-EASIA
The amount of IFNgsecreted in the culture medi-um of CPA-cultures on day 5 was determined with the IFNg-EASIA (catalogue number KAC1231) from Biosource International (Camarillo, CA, USA). The assay was performed following the manufacturer’s guidelines and the results were expressed in pg/ml (range 1.5 – 1175 pg/ml). When the IFNg content exceeded the detection limit of the assay, dilutions of 1/10, 1/20 or 1/40 were made as needed.
2.8. Study design of the HBsAG vaccination trial
To evaluate the usefulness of the ISA for the study of vaccine induced immune responses, we analyzed the HBsAg-induced IFNg production in PBMC from four subjects who participated in a clinical vaccine trial. In this study, 200 healthy subjects were randomized (4 groups, 50/group) to receive three doses (week 0, 6 and 46) of one of four experimental hepatitis B vaccines in the left deltoid muscle according to a double-blind design. All four vaccines, containing 20 Ag HBsAg that was
adju-vanted in a different way, were manufactured by GlaxoSmithKline Biologicals, Rixensart, Belgium. The humoral immune response was evaluated by measuring the anti-HBs levels at different time points. The study protocol was conducted in
accor-dance with the Declaration of Helsinki as amended in Hong Kong (1989) and approved by the Institu-tional Ethical Review Committee of the University of Ghent. Written informed consent was obtained from all volunteers. Several vaccinated individuals
Fig. 1. IFNgsecretion by living VZAg-specific CD3+and CD3+CD4+T cells. The left panels show IFNg-secretion after VZAg-specific
stimulation, while the right panels represent the unstimulated control sample. Dot plots in the A-panels show staining with PerCP-conjugated anti-CD14/PI (FL3) vs. FSC-height. The lymphocyte gate (R1) is based on dead cell-and monocyte-exclusion. The B-panels represent staining of gated (R1) lymphocytes with PE-conjugated anti-IFNg(FL2) and FITC-conjugated anti-CD3 (FL1). The Upper Right (UR) quadrants thus represent the living CD3+cells that produce IFNg. A second gate (R2*R1) comprises all CD3+cells. The C-panels represent staining of gated (R2) CD3+cells with PE-conjugated anti-IFNg (FL2) and APC-conjugated anti-CD4 (FL4). The UR-C quadrants thus represent living IFNg+CD3+CD4+lymphocytes. The % represents the amount of IFNg+cells among the gated cells.
were selected from this study to test the applicability of the ISA-technology in vaccine-research.
3. Results
3.1. Calculation-methods for the ISA
The flow cytometric analysis of the IFNgSecretion Assay has already been described in detail in the Materials and Methods section, and shown inFig. 1. The lymphocyte gate (R1) for this analysis is based on dead cell-and monocyte-exclusion (PI/anti-CD14-PerCP vs. FSC-Height). In this gate, 105lymphocytes were counted and analyzed for IFNg secretion and phenotype. Three different approaches were used to express the IFNg-producing fraction.Table 1gives an overview of these expression/calculation methods. In the firstcalculation-method (Table 1, column 5)the IFNg-producing cells are expressed as a fraction (%) of 105 lymphocytes counted in R1 ( = 100%). In
method 2 (Table 1, column 6) the IFNg-producing cells are expressed as the fraction of the total number of cells within each subset. The 230 IFNgproducing CD3 cells are not expressed as 0,23% as in method 1 but as 230 in 73912 CD3+cells or 0,311%. Each cell subset (CD3+, CD3+CD4+, etc.) is considered as
100%. Inmethod 3the total number of IFNg produc-ing cells is considered as 100% and contribution of each subset is expressed as a fraction (%) thereof
(Table 1, column 7). The 230 IFNg producing CD3 cells are expressed as 18.21% of the total 1263 IFNg
producing cells (100%).
In general, four colours were available for the flowcytometric analyses (FACS-Calibur with two lasers). FL2 was always used for the IFNg-PE colouring and FL3 for the dead-(PI) and monocyte-(anti-CD14-PerCP) exclusion. By doing so there were only two colours left (FL1 and FL4) for surface marker determination. To determine all possible lymphocyte subsets, we had to perform some extra calculations. An example is shown in Table 2. NK cells (CD56+CD3 ) and T cells (CD56 CD3+) can be calculated by subtracting the CD56+CD3+fraction from the total number of CD56+, or CD3+ cells, respectively. The number of NK/CD8 cells can be calculated by subtracting the number of CD56+CD8+ cells from the total number of NK cells. The number of T helper cells (CD3+CD4+), cytotoxic T cells (CD3+CD8+), NK/CD8+(CD56+CD8+), CD3+CD56+ and B cells (CD19+) are given by direct measure-ments. These results can be represented either as a pie-chart wherein each wedge represents a subset of IFNg-producing cells or as a stacked column chart
Table 1
Different calculation-methods for the ISA
+ VZAg, Blank, VZAg-blank, % IFNg-producing cells no. of cells no. of cells no. of cells
Calculation-method 1 (%)
Calculation-method 2 (%)
Calculation-method 3 (%) Total IFNg-producing cells 1386/100,000 123/100,000 1263 1.263 1.263 100 CD3+IFNg-producing cells 254/73,912 24/73,701 230 0.230 0.311 18.211 CD4+IFNg-producing cells 142/53,311 18/52,974 124 0.124 0.233 9.818 CD3+CD4+IFNg-producing cells 115/53,190 9/52,801 106 0.106 0.199 8.393
CD8+IFNg-producing cells 110/18,519 12/18,684 98 0.098 0.529 7.759
CD3+CD8+IFNg-producing cells 37/18,332 10/18,289 27 0.027 0.147 2.138 CD56+IFNg-producing cells 1026/15,638 58/15,785 968 0.968 6.190 76.643 CD56+CD3+IFNg-producing cells 108/4031 13/3818 95 0.095 2.356 7.522
CD56+CD4+IFNg-producing cells 11/577 9/517 2 0.002 0.347 0.158
CD56+CD8+IFNg-producing cells 91/2479 20/2824 71 0.071 2.864 5.622
CD19+IFNg-producing cells 31/13,672 33/13,504 0 0 0 0
Comparison of % IFNg+cells obtained with calculation-methods 1, 2 and 3. Column 2 represents the number of IFNg-producing cells out of the
number of counted lymphocytes in the VZAg-stimulated cultures. Column 3 represents the IFNg+cells in the control cultures. Column 4
represents the net result between both columns. Columns 5, 6 and 7 represent the % of IFNg-producing cells calculated in three different ways. Calculation-method 1 is based on the total number of lymphocytes counted in R1 (100% = 100,000 lymphocytes). Calculation-method 2 is based on the number of specified cells, e.g. number of IFNg+CD3+cells out of the total number of CD3+cells counted ( = 100%). Percentages for
wherein each column represents a major lymphocyte subset (T, B, NK, CD3+CD56+) and within each column, the contribution of minor subsets can be shown(Fig. 2). CD3+CD56+cells are most probably NKT cells, but, since this was not proven, this subset will be named ‘CD3+CD56+ cells’ throughout the text.
3.2. Exclusion criteria for the ISA-results
In order to generate correct results we introduced a series of quality measures that were strictly followed
(Fig. 3). Since the experiments were performed with cryopreserved cells, it was crucial to analyze the viability of the thawed cells. This was done by electronic gating on living cells (PI exclusion) in flow cytometry. A viability of at least 80% was required ( =quality criterion(QC) 1). Thawed cells with a viability of < 80% were excluded from the experi-ments. A second QC relates to the deviation of the values obtained in the different measurements of IFNg-producing cells. As the quantification and sub-set distribution of IFNgproducing cells are performed in a series of analyses using different FACS tubes and overlapping sets of surface marker antibodies, the IFNgproducing cells are counted several times. The variation between these measurements should not exceed 20% (SD < 20%)(QC 2). A third quality crite-rion concerns the total number of IFNg-producing cells and requires that the sum of the major subsets of IFNg producing cells should not deviate more than
20% from the direct count of IFNg producing cells. This criterion can be written as a formula: [CD3+IFNg++ CD56+IF Ng++ CD19+IFNg+] [CD3+CD56+IFNg+]cTotal IFNg+F20% (QC 3).
CD3+CD56+IFNg+ cells are subtracted to avoid double counting of this population. The fourth QC requires that [CD3+CD4+IFNg++ CD3+CD8+ IFNg++ CD3+CD56+IFNg+] has to be equal to the total number of CD3+IFNg+cells F20% (QC 4). The fifth and final QC requires that at least 20 IFNg
producing cells are observed in an experiment to consider it valid. Indeed, at lower cell numbers the experiment is too imprecise and the coefficient of variation (CV) is unacceptably high. This implies that, in our setting, the minimal frequency of IFNg -secret-ing antigen-specific cells that can be detected with this ISA-technology is 0.02% (20 IFNg+cells out of 100,000 lymphocytes counted).Fig. 3(part 3) further shows that there is often a small discrepancy between the total number of IFNg+cells (IFNg+total,n=1263) and the sum of the different subsets (n= 1103). Several experiments were performed (results not shown), but no missing population could be found. We assume that this discrepancy, which seldomly exceeds 15%, is inherent to the technique used.
3.3. Precision of the ISA
To estimate the inter-assay variability (precision) of the ISA we have stimulated three different BC
Table 2
Subset calculation in ISA
Subset Phenotype Flow cytometric data No. of
IFNg+cells
Percentage from total IFNg-producing cells
T CD3+CD56 [# CD3+] – [# CD56+CD3+] 135 12.2 Th CD3+CD4+CD56 [# CD3+CD4+] 106 9.7 CTL CD3+CD8+CD56 [# CD3+CD8+] 27 2.5 NK CD3 CD56+ [# CD56+] – [# CD56+CD3+] 873 79.2 NK8+ CD3 CD56+CD8+ [# CD56+CD8+] 71 6.5 NK8 CD3 CD56+CD8 [# CD56+] – [# CD56+CD8+] – [# CD56+CD3+] 802 72.7 CD3CD56 CD3+CD56+ [# CD56+CD3+] 95 8.6 B CD19+ [# CD19+] 0 0 Total [T]+[NK]+[CD3CD56]+[B] 1103 100
The numbers of IFNg-producing cells belonging to each lymphocyte subset (‘no. of IFNg+cells’) were calculated based on the raw flowcytometric countings obtained inTable 1(column 4). The total number of IFNg-secreting cells is calculated (1103) and considered as 100%. The relative contribution of each subset to this total is calculated and expressed in percent (right column inTable 2) and can be represented as a pie-chart or a stacked column chart(Fig. 2).
with TT and 5 with VZAg. The number of IFNg -producing cells after stimulation was measured by ISA-technology. These experiments were repeated several times (same cells, same standardized proce-dure, etc.) and after exclusion of unvalid experi-ments (based on the aforementioned QC), a number of valid, replicate results with the same BC were obtained. Table 3 shows the results of the precision experiments obtained with BC2 after VZAg-stimu-lation. The experiment was performed 10 times by
two people (ID and EK) and eight experiments complied with the validation criteria. The percent-age of IFNg+ cells was calculated according to calculation-method 3. Standard deviation and % CV demonstrate that the results of these experi-ments are highly reproducible. Table 4 presents the variation coefficients of Elisa-results, proliferation data, and three different calculation-methods for the cytokine secretion assay. BC1-VZAg, BC2-VZAg, BC3-VZAg, BC4-VZAg and BC7-VZAg present the CV of 8, 8, 10, 3 and 6 replicate experiments, respectively. BC1-TT, BC2-TT and BC3-TT present the results of 6, 4 and 4 replicate validated experi-ments, respectively. Table 4 shows that lymphocyte-proliferation data are best presented as Dcpm (in-stead of SI) and cytokine secretion data are best calculated by calculation-method 3. This calcula-tion-method was used in all experiments. An intra-assay variability of less than 5% (results not shown) and an inter-assay variability ranging from 3.2% to 17.5% after VZAg-stimulation were found. A limited inter-assay variability is essential to interpret results generated on different study days.
Table 4 also demonstrates that reproducibility for measurement of IFNg-production in supernatant (Elisa) turns out to be extremely poor.
We also observed that TT-stimulation induced a lower total number of IFNg-producing cells compared to VZAg-stimulation (data not shown). This explains the higher % CV after TT stimulation. Remarkably, the lymphoproliferative response was as high for TT as for VZAg.
3.4. Accuracy of the ISA
The accuracy of a test is a function of its ability to yield results close to the true value of what is being measured. To define the accuracy of the ISA-test we performed two ‘spiking’ experiments in which defined numbers of IFNg+ T cells were added to a large number of IFNg-negative T cells. The fraction of recovered IFNg+T cells was measured by flowcytometry and a correlation coefficient between expected and measured IFNg+T cells was calculated.
Fig. 4 shows that almost 100% of positive cells could be detected by ISA-technology and an almost perfect correlation between measured and expected data was found. The correlation coefficients for both
Fig. 2. Different formats to represent ISA-results. Panel A shows the ISA-results obtained inTable 2-right column (%) in a pie chart. The whole pie represents the total amount of IFNg-producing cells ( = 100%) obtained in that experiment. Each wedge of the pie represents the contribution of a lymphocyte subset to the whole IFNg-production. Panel B shows the ISA-results (absolute cell numbers) fromTable 2in a stacked column chart. In this chart the contribution of the different NK- and T-populations is shown more in detail. Both charts clearly show that, in this experiment, the majority of IFNg-producing cells are NK cells.
spiking experiments exceeded 0.999 demonstrating that the accuracy of the ISA-test is very high. This analysis also demonstrates that as few as 20 IFNg -positive cells can be detected in a reliable manner. The lower limit of detection of the ISA is therefore 0.02%.
3.5. Correlation between results obtained with the ISA-technology, the lymphocyte-proliferation assay and the Elisa-technology (secreted IFNc)
Four different BC were stimulated with VZAg as described (8 replicate experiments for BC1; 8, 10
Table 3
Precision of the ISA-results
This table shows the results of 8 replicate, validated experiments using buffy coat 2 and an overnight stimulation with VZAg. The total number of IFNg+ cells and the number of CD3+and CD56+IFNg+cells are shown. The percentage of positive cells is calculated according to
calculation-method 3 (100% = total number of IFNg+cells). SD and CV of mean show that the results of these experiments are highly
reproducible (%CV < 20 is grey shaded).
and 3 identical experiments for BC2, BC3 and BC4, respectively) and data for ISA, lymphoproliferation and IFNg secretion in the culture supernatant were obtained. ISA data are calculated according to meth-od 3, lymphoproliferation data are expressed as
Dcpm and IFNgsecretion in supernatant is expressed as pg/ml. To examine possible correlations between the different data, the mean value of the results of the individual replicate experiments were calculated for each BC and correlations were examined between two data sets. The R2-value expresses the correlation coefficient between these two data sets. Only R2
values z0.60 are shown in Table 5. All R2 data
shown in this table present positive correlations (the
a-value which expresses the slope of the linear regression line (y=ax+b) through the four given data points is positive (value not shown)). The R2 -values in Table 5 show that an early IFNg -produc-tion (as measured with the ISA-technology) by CD3+/CD4+T helper cells is strongly correlated with the observed lymphocyte proliferation measured after 6 days suggesting that an early activation of T helper cells is linked to the later proliferation of specific lymphocytes. IFNg secretion in the supernatant, on the other hand, shows a better correlation with the number of IFNg-secreting CD8+cells. These may be CTL (CD3+CD8+) or NK cells (CD56+CD8+). Fur-thermore we observe higher R2 values when prolif-eration data were expressed as Dcpm. These results, together with the lower CV values observed with
Dcpm in Table 4, suggest that lymphocyte-prolifer-ation data should preferentially be expressed as
Dcpm.
3.6. Analysis of cell subsets responsible for IFNc -secretion
In order to analyse which cell subsets contribute to IFNg-production, we compared the results obtained from six different buffy coats after VZAg-stimulation.
Fig. 5 shows a representative experiment for each buffy coat. The pie charts represent all IFNg -produc-ing cells ( = 100%) and the different subsets are calculated by method 3. The fraction of T cells is given by the total % of CD3+cells—the % of CD3+/ CD56+cells. The fraction of NK cells is given by the total % of CD56+cells—the % of CD3+/CD56+cells. The % of CD3+CD56+ and CD19+ (B cells) are measured directly. Three different IFNg-secretion patterns can be discerned. To draw a distinction between the different patterns we introduced the T/ NK ratio:Pattern 1(0.5VT/NKV2) is characterized by an almost equal contribution of T cells (38 – 54%) and NK cells (40 – 50%) with a minor contribution of CD3+CD56+ cells (5 – 9%) and B cells (0 – 3%) as seen in BC1, 2 and 4. Within the T cell population, 2/3 have the Th (CD3+CD4+) phenotype and 1/3 are cytotoxic T cells (CD3+CD8+) (data not shown);
Pattern 2(T/NK < 0.5) is characterized by a predom-inance (67 – 70%) of NK cells. T cells, CD3+CD56+
Table 4
Precision of the ISA-results
Comparison of the coefficients of variation (CV expressed as %) between Elisa-results (pg/ml, IFNg-production in supernatant), lymphocyte-proliferation data (Stimulation Index (SI) andDcounts per minute (Dcpm)), and the three different calculation-methods for the Cytokine Secretion Assay. CV values below 25% are highlighted by grey-shading. nd = not done.
cells and B cells are contributing for 24 – 25%, 5 – 7% and 0,4 – 1% in the IFNgsecretion, respectively. This pattern is observed in BC3 and BC5. Within the T cell population 3/4 have the Th phenotype and 1/4 are CTL. Pattern 3 (T/NK>2) differs from the previous one in its minor contribution of NK cells to IFNg
secretion (11%). Here T cells predominate (66%) and CD3+CD56+and B cells account for 17% and 6% of the IFNg subunit, respectively. Within the T cell subset CD4/CD8 contribute in a 1.4/1 ratio. In pattern
3 the contribution of CTL is substantial. This pattern was seen in BC6. The hierarchy in lymphocyte IFNg -production can be summarized as follows: pattern 1: NK = T>CD3+CD56+>B; pattern 2: NK>T>CD3+ CD56+>B; and pattern 3: T>CD3+CD56+zNK>B,
and clearly shows that the IFNg+ subset distribution after VZAg-stimulation is not uniform and differs individually.
3.7. Influence of antigenic stimulus and initial subset distribution on magnitude and phenotype of IFNc
production
To examine whether the nature of the antigen-ic stimulus has an impact on the magnitude and
Table 5
Correlation between ISA-, IFNgsecretion in culture supernatant and proliferation results after VZAg-stimulation
R2 Elisa Proliferation (supernatant) SI Dcpm ISA-total < 0.60 0.83 ISA-cell subsets CD3+ 0.88 < < CD4+ < 0.78 0.99 CD3+CD4+ < 0.74 0.96 CD8+ 0.96 < < CD3+CD8+ 0.85 < < CD56+ < < < CD56+CD8+ 0.86 < < CD56+CD4+ < < < CD56+CD3+ < < < CD19+ < < < Proliferation SI < 0.75 Dcpm < 0.75
The ISA replica-experiments (overnight stimulation) for four different BC were supplemented by lymphocyte proliferation experiments (harvest on day 6) and cytokine content in supernatant (harvest at day 5). The results of the proliferation experiments can be expressed as Stimulation Index (SI) or as Dcpm. The IFNg -content of the collected supernatant was measured using Elisa. For each buffy coat (BC) the mean of the results of the individual replicate experiments was calculated. The contribution of the different cell subsets involved in IFNg-production is presented by ISA-CD3, ISA-CD4, etc. The R2-values express the correlation
coefficients between the two data sets (means for four different BC). OnlyR2valuesz0.60 are shown. Values below 0.60 are shown as ‘ < ‘. All data represent positive correlations, since the a-value (not shown) which expresses the slope of the linear regression line (y=ax+b) through the four given data points, is positive. Fig. 4. Accuracy of the ISA-test (recovery experiment). Panel A
presents the correlation between the % of expected and the % of recovered IFNg-producing T cells (0 – 100%) in spiking experiment 2. Panel B shows a detail (0 – 30%) of panel A. The IFNg+spiking
population consisted of T-cell clones that were labelled with recombinant IFNg.
the phenotypic distribution of the in vitro IFNg
production, PBMC were stimulated with either VZAg or TT. In BC1, both VZAg and TT induced IFNg production in the CD3+ subset predominantly, and in BC3 the IFNg-producing cells were mainly CD56+, irrespective of the antigen used. In BC2, however, VZAg induces IFNg production in CD56+ cells mainly and TT stimulated IFNg in CD56+ and CD3+ cells almost equally (Table 6). We conclude that, in this case, for VZAg and TT, the hierarchy of IFNg -produc-ing cells is poorly dependent on the antigenic stimulus used.
We further examined whether the subset distri-bution of IFNg-producing cells at 16 h was deter-mined by the distribution of lymphocytes at cell harvest (0 h). BC1 (pattern 1; T/NK = 1.35), BC5 (pattern 2; T/NK = 0.34) and BC6 (pattern 3; T/ NK = 6) were examined for subset distributions at 0 h and at 16 hrs, as shown in Table 7. The subset distributions of the IFNg producing cells at 16
h were determined using calculation method 3. At 0 h and at 16 h, the initial (without antigenic stimulation) lymphocyte subset distribution remains
Fig. 5. Cell subset determination in IFNgsecretion. The cell subsets participating in IFNgsecretion after VZAg stimulation were determined for six different buffy coats. For each buffy coat a representative experiment is shown (EK8 for BC1 and BC2, EK10 for BC3, etc.), wherein the whole pie represents all IFNg-producing cells ( = 100%, calculation-method 3). At least three different IFNgsecretion patterns could be observed: (1) in BC1 and BC4 the T cells and the NK cells account forF50% of IFNg-production each, (2) in BC3 and BC5, IFNg-production is dominated by the NK cells, whereas the T cells only participate for F25%, 3) in BC6, on the other hand, the cytokine-production is dominated by T cells. CD3+CD56+cells also largely participate in this IFNgresponse pattern.
Table 6
Influence of antigen on the subset distribution of IFNgproducing cells
% BC1 BC2 BC3
VZAg TT VZAg TT VZAg TT
CD3+ 57 57 42 53 29 46 CD56+ 43 43 58 47 71 54 Conclusion CD3> CD56 CD3> CD56 CD56> CD3 CD3z CD56 CD56> CD3 CD56> CD3 The ratio (% out of 100) of CD3+and CD56+IFNgproducing cells
(calculation-method 3) were determined for three different buffy coats after either VZAg- or TT-stimulation. In BC1, whatever the stimulus used, the majority of IFNg-secreting cells are from the CD3+ phenotype. In BC3, on the other hand, the majority of
secreting cells belongs to the CD56+phenotype. In BC2,
VZAg-stimulation induces a large CD56+ IFNgsecretion and after TT stimulation no major producing subset could be determined.
the same within each BC (BC1, 5 and 6). In BC1 (pattern 1) and 5 (pattern 2) the subset distribution of the IFNg-producing cells (16 h) differs markedly from the initial subset distribution. In BC6 (pattern 3), the subset distribution pattern of IFNg -produc-ing cells closely resembles the initial distribution at 0 and 16 h. To confirm these findings we correlated the subset distribution of six different BC at 0 h with their different IFNg-producing subsets (mean of replicate measurements for the different BC) after overnight VZAg-stimulation (Fig. 6). The R2 -value expresses the correlation coefficient between the two data sets. Only data sets with R2 val-uesz0.80 are shown in Fig. 6. Except for IFNg
-producing NK cells (CD56+), all R2 data shown in this figures present positive correlations (thea-value which expresses the slope of the linear regression line (y=ax+b) through the six given data points, is positive). The R2-values in Fig. 6 demonstrate that the initial percentage of CD3+CD8+ and CD3+ CD56+ double positive cells predicts the ensuing subset distribution of IFNg-producing cells. The higher the initial fraction of these cells, the higher the number of CD3+ cells and the lower the
number of CD56+(NK) cells that participate in IFNg-production.
3.8. Application of the ISA technique in vaccine research
To demonstrate the usefulness of the ISA-technol-ogy in vaccine research, we selected PBMC from several HBsAg-vaccinated individuals. One represen-tative example (study subject number 164) is shown inFig. 7. PBMC were harvested before (week 0) and after (week 6) HBsAg-vaccination. The dramatic increase in IFNg+-producing cells after in vitro stim-ulation with HBsAg of PBMC obtained at week 0 (negative) and at week 6 (strongly positive, 8750 IFNg+ cells) clearly demonstrates the antigen-speci-ficity of the assay. A positive control (VZAg-stimu-lation) was examined at both time points and did not show this vaccine-induced increase. The number of IFNg-producing cells observed before and after HBsAg vaccination were 1587 and 1556 (among 100,000 viable lymphocytes), respectively. The ma-jority of IFNg-producing cells following in vitro stimulation of PBMC at week 6 were CD56+CD8
Table 7
Initial subset distribution of different IFNg-secretion patterns
Subset distribution
All lymphocytes IFNg-secreting cells 0 h (%) 16 h (%) 16 h (%) T/NK
Buffy coat with Pattern 1 (BC1) Th CD3+CD4+CD56 62 67 36 1.35
CTL CD3+CD8+CD56 15 16 18
CD3CD56 CD3+CD56+ 3 3 5
NK CD3 CD56+ 7 6 40
B CD19+ 13 8 1
Buffy coat with Pattern 2 (BC5) Th CD3+CD4+CD56 56 57 18 0.34
CTL CD3+CD8+CD56 13 13 6
CD3CD56 CD3+CD56+ 1 3 5
NK CD3 CD56+ 16 13 70
B CD19+ 14 14 1
Buffy coat with Pattern 3 (BC6) Th CD3+CD4+CD56 39 38 38 6.0
CTL CD3+CD8+CD56 31 30 28
CD3CD56 CD3+CD56+ 13 11 17
NK CD3 CD56+ 4 3 11
B CD19+ 13 18 6
The initial (0 h) and overnight (16 h) subset distribution of lymphocytes belonging to three different IFNg-secretion patterns is shown. Patterns I, II and III represent the results of representative experiments for BC1, 5 and 6, respectively. T/NK ratio is shown for the different IFNg response patterns.
cells (80%). From the 13.6% T cells, 4.2% are Th (CD3+CD4+) and 9.4% CTL (CD3+CD8+). Similar results were obtained in three other vaccinees.
4. Discussion
Different single-cell analyses for the detection of antigen-specific T cells based on antigen-triggered induction of cytokine production (elispot, intracyto-plasmic cytokine staining, cytokine secretion assay, etc.) have been designed. In this paper we present the results of a thorough validation of the IFNgSecretion Assay (ISA, Miltenyi Biotec). In this assay the secreted IFNgis bound to the cell surface, then stained as an artificial surface molecule and analyzed by flow-cytometry. For the enumeration of IFNg+cells in flow cytometry it is important to set a correct gate on living
lymphocytes. This was done by adding PI (exclusion of dead cells) and anti-CD14-PerCP (exclusion of mono-cytes), two dyes detected in FL3. An CD45 anti-body could not be used for the gate-setting since a bi-specific conjugate of monoclonal antibodies directed against CD45 and IFNg (cytokine catch reagent) is used to link the secreted IFNgto the cell surface.
In our hands the ISA proved to be a reliable assay when we strictly adhered to five quality criteria. Since all experiments were performed with cryopreserved cells, a first criterion concerned the viability of the thawed PBMC. Past experience using different tech-niques has taught us that experiments performed with cells with low viability must not be considered valid. Therefore, we defined a limit for viability of at least 80% as measured by PI-dependent exclusion of dead cells. The second quality criterion concerned the reproducibility of the test. Since the total number of
Fig. 7. Usefulness of the ISA-technology in vaccine-research. The number of IFNg-producing cells after appropriate in vitro antigen (VZAg or HBsAg) stimulation was measured in the same vaccine recipient before (week 0) and after (week 6) HBsAg-vaccination. Before vaccination there is no IFNg-production after HBsAg stimulation, which demonstrates the antigen specificity of the system. After vaccination, 9299 lymphocytes (18.9% T, 77.2% NK, 2.3% CD3+CD56+and 1.6% B) out of 100,000 cells counted produce IFNg. The IFNg-production after
VZAg-stimulation was the same at both time-points and served as an internal control.
Fig. 6. Correlation between initial subset distribution (0 h) and cell subset distribution in IFNg-secretion (16 h) after VZAg-stimulation. The initial cell subset distribution (%) of 6six different BC was correlated with its IFNg-producing cell subset pattern (%) after overnight VZAg-stimulation. The % of cell subsets was based on the total amount of living lymphocytes. TheR2-value expresses the correlation coefficient
between the two data sets. Only data sets withR2values
z0.80 are shown. For CD56+IFNg-producing NK cells there is a negative correlation; all otherR2data present positive correlations (thea-value which returns the slope of the linear regression line (y=ax+b) through the six given
IFNg-producing cells is counted several times within each experiment, we required that the SD of these measurements did not exceed 20%. The third quality criterion required that the sum of the different IFNg -producing cell subsets equaled the total number of IFNg+cells F20%. A fourth quality criterion implied
that the sum of the different CD3+IFNg-producing cell subsets (CD3+CD4+(T-helper), CD3+CD8+(CTL), and CD3+CD56+ equaled the total number of IFNg -producing CD3+ cells F20%. The fifth and final
quality criterion required that at least 20 IFNg pro-ducing cells (out of 100,000 lymphocytes) were counted per experimental set-up. At lower numbers of IFNg producing cells the data were too imprecise (high %CV) and considered invalid. This criterion also implied that the ISA-technique allowed for the detection of cytokine-secreting antigen-specific cells down to frequencies of 0.02% (20 IFNg+cells out of 100,000 lymphocytes counted).
To determine the precision of the ISA, we per-formed several validation experiments, using cells from different subjects. PBMC from each individual were tested 10 times in an identical set-up by two executors. When we enumerated the number of IFNg-secreting cells amongst the living lymphocytes (calculation method 1) following stimulation with VZAg in 10 separate experiments, we obtained a large inter-assay variation with a CV of F40%. Since the method and the cells were the same in these replicate experiments, the main cause of vari-ation must be the quality of the antigenic stimulus in each experiment. The quality of the stimulation probably determines the magnitude of all ensuing phenomena. Although the absolute number of IFNg+
cells is variable between replicate experiments, the cell subset distribution of these IFNg+ cells remains constant from one experiment to another (calcula-tion-method 3). In this calcula(calcula-tion-method the total number of IFNg-secreting cells is considered as 100% and the fractional contribution of each subset to this total is expressed in percent. By doing so, an intra-assay variability of less than 5% and an inter-assay variability ranging between 3.2% and 17.5% after VZAg-stimulation were found. These data were confirmed by a small study conducted at the Milte-nyi laboratories where an intra-assay variability of 13,1% and an inter-assay variability of 21,1% after SEB stimulation were observed (personal
communi-cation J. Irsch.).We therefore conclude that the ISA is a convenient and highly reproducible technique if one adheres to the five quality criteria proposed herein.
Recovery experiments (accuracy) with IFNg+T cell clones showed that there is an almost perfect correla-tion between the measured and the expected cell numbers and the lower limit of detection approaches 20 cells per 100,000.
Studies of the analytical qualities of other meth-ods to study the production of cytokines at the single cell level have rarely if ever been made. It would be useful to have an idea of the specificity, the sensi-tivity, and the precision of Elispot or intracytoplas-mic cytokine detection assays. This would allow for a comparison of data generated with these different approaches. Unfortunately, it is readily apparent from the literature that the level of awareness of the many factors that can potentially influence the results obtained is very low. Such factors are likely to contribute considerably to the disparities seen among similar types of study. Clearly, it is not possible to estimate the impact of all biological and analytical variables on the outcome of a test but a minimal evaluation of the analytical qualities of an assay should at least be available.
When we analyzed the IFNgsubset distribution in 6 different BC, at least three different IFNgsecretion patterns could be observed. In pattern 1there was a predominant and almost equal contribution of T and NK cells with a minor contribution of CD3+CD56+ and B cells. Pattern 2, is characterized by a pre-dominance of NK cells. Pattern 3 differs from the previous one in its minor contribution of NK cells. Here most IFNg producing cells are T cells. These results clearly demonstrate that the IFNg+ subset distribution after VZAg-stimulation is not uniform and differs from one person to another. Since these results, especially the IFNg-secretion by NK cells, could be due to some indirect effect on the NK cells upon freezing and thawing, we compared ISA-results of fresh and frozen PBMC samples and obtained almost identical results (data not shown). Further-more, the presence of IFNg-producing NK cells has been confirmed by colleagues using intracytoplasmic IFNg-staining (Emmanuel Hanon, GlaxoSmithKline, personal communication). The phenomenon is thus not method-dependent. The antigen-specificity of the
phenomenon is shown by the vaccination experi-ment, where there is no HBsAg-driven NK cell activation (IFNg production) in subjects ‘before’ vaccination with HBsAg and clear IFNg secretion once the subject has been primed in vivo with the antigen. Preliminary data obtained in selective cell depletion experiments suggest that this early IFNg -production by NK-cells is T dependent.
Analogous techniques for single-cell analysis, but not for isolation of viable cytokine expressing cells, are Elispot and IC staining. Although side-by-side comparisons of the Elispot assay and IC show com-parable results with respect to the detected frequencies of cytokine-expressing cells, the sensitivity of the Elispot seems much higher (1/300,000 for Elispot; 1/5000 for IC)(Helms et al., 2000; Asemissen et al., 2001). The Elispot assay, which enumerates PBMC releasing IFNgfollowing specific antigen stimulation, is becoming the assay of choice for evaluation cell-mediated immune responses induced by vaccines or natural infections (Yang, 2003). Surprisingly, only few publications describe the precision (reproducibil-ity) of this assay. Frequency analyses performed by Schmittel et al. (1997) reported a high sensitivity and reproducibility (small set-up) for the Elispot. In a comparative study, in which T cell frequencies were analyzed in blinded samples in various European laboratories using the Elispot assay, consistent results were obtained in different laboratories (Scheibenbo-gen et al., 2000). Furthermore, Mwau et al. (2002) described the design and validation of an Elispot assay wherein the overall variation of a positive response of approximately 500 spot-forming units (SFU)/106cells was 21%.
Validation studies for IC are equally rare. Nomura et al. (2000) described ( in 3 experiments using two cell donors) an average intra-assay CV for IFNg of 8.4% and an inter-assay CV of 23.6%. One of the problems encountered IC assays is the fact that the technical difficulty to exclude dead cells may lead to false positive results. The ISA overcomes several limitations of previously and presently used methods. First, the possibility of surface-staining allows for the correct determination of the IFNg+producing subset. IC also allows for the phenotyping of the IFNg -producing subset, but the sensitivity of this method is much lower than for ISA. Elispot, which also determines IFNg at the single-cell level, does not
identify the phenotype of the cytokine-producing cell. However, depending on the experiment design, the ELISPOT-assay can also identify the phenotype of the cytokine-producing cells. The importance of pheno-typing is demonstrated by He et al. (2003). These authors observed different patterns of CD27 expres-sion in influenza virus- and cytomegalovirus-specific CD8+ T cells and thus showed that influenza virus-specific memory and effector CD8+ T cells can be differentiated by phenotypic analysis. In the present paper we demonstrated that after overnight stimula-tion with VZAg, the majority of IFNg-producing cells are NK cells. The ignorance of the producing subset in Elispot can thus give rise to misinterpretation of the data. Secondly, the exclusion of dead cells with PI in ISA, prevents false positive results. Finally, the pos-sibility of an immunomagnetic separation in ISA allows for the physical isolation and enrichment of viable cytokine secreting cells for further culture and functional analysis. This is not possible when using IC staining or Elispot.
Validation experiments for the ISA-technology, however, were seldomly reported. Oelke et al. (2000) described a good correlation between the frequencies obtained from IC and those obtained by ISA (r= 0.83). Direct visualization of the correlation between the staining of secreted and intracellular cytokine in the same cells has been shown by Schef-fold et al. (1998) for IFNg and by Ouyang et al. (2000)for IL4. These results show in a direct way that the cytokine secretion assay technology is as sensitive as IC staining.
Since the techniques available today all have strengths and weaknesses we expect that a combina-tion of two or more assays, based on their comple-mentarity, will generate the best results. For example, the functional heterogeneity of antigen-specific T lymphocyte populations can be analyzed by a combi-nation of tetramer staining for epitope-specificity and the IFNgsecretion assay, Elispot or IC for functional analysis(Pittet et al., 2001; He et al., 2001).
In Elispot, IFNg-producing cells are not pheno-typed, still a lot of authors automatically assume that the cytokine-producing cells are T cells (Helms et al., 2000; Scheibenbogen et al., 2000;Smith et al., 2001; Schmittel et al., 2001; Asemissen et al., 2001). In this paper, however, we demonstrate that there are at least three different IFNg-secretion patterns. At
pres-ent, more than 44 different cell/antigen combinations have been tested (results not shown). From these results we know that pattern 2, in which the majority of IFNg-producing cells are NK cells, is most abundant. This IFNg+ subset distribution was ob-served after overnight stimulation with VZAg-, HBsAg, TT-and HCV-E1 (results not all shown). We are now unravelling the mechanism underlying this antigen-driven IFNg-production by NK-cells. Since the duration of an ISA, IC or Elispot assay (16 – 48 h) is too short for in vitro proliferation and/ or differentiation to occur, it was automatically assumed that the number of positive cells detected, reflects both the frequency of memory cells present in the cell isolate and the commitment to produce IFNg these cells acquired in vivo. It was stated that experiments employing long cell culture periods may introduce bias by selecting only cells capable of extensive division and by inadvertently regulating the phenotype of cytokine synthesis. Although short-term assays, as described in this paper, overcome the bias introduced by long-term cell proliferation they introduce another bias, namely that in short-term assays, up to 70% of IFNg+ cells can be of the CD56+ phenotype. With this knowledge at hand, it becomes obligatory to include CD3, CD4 and/or CD8 for further phenotyping and exact determination of precursor frequencies. In view of these new findings, Elispot-technology becomes less valuable.
An important advantage of IFNg single-cell anal-yses is that it is a direct measurement of a Th1 cell-mediated immune response. As such, it is useful for monitoring the effectiveness with which a vaccine induces cell-mediated immunity. To demonstrate the usefulness of the ISA-technology in vaccine research, we selected PBMC from HBsAg-vaccinated individ-uals and tested their IFNg+ subset distribution after HBsAg-stimulation. The ISA-technique proved to be antigen-specific and sensitive. Since it is important in experimental vaccine trials to know the effect of antigen or adjuvant selection and the ensuing cellular reaction and the phenotype of the activated cells, the ISA or IC-technology becomes extremely valuable. Since the ISA provides reliable results as well with frozen as with fresh PBMC, serial samples obtained in the context of a vaccination study can be kept frozen until analyzed at a single moment thus avoiding assay-to-assay variability.
Acknowledgements
The authors are grateful to Dr. B. Riegler and Dr. M Assenmacher from Miltenyi Biotec for their scientific and technical support in performing the IFNg Secretion Assay. We thank E. Krijnen, A. Van de Putte and Y. Gybels for excellent technical support. We thank Dr B. Vandekerckhove from the Blood Transfusion Center of Oost-Vlaanderen (BTC) for supplying buffy coats. We thank Eurocetus (The Netherlands) for the kind gift of recombinant IL2.
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