Comparison of the ELISPOT and cytokine flow cytometry assays for the enumeration of antigen-specific T cells

Journal of Immunological Methods 283 (2003) 141 – 153 www.elsevier.com/locate/jim

Comparison of the ELISPOT and cytokine flow cytometry assays for the enumeration of antigen-specific T cells Annika C. Karlsson a,*, Jeffrey N. Martin b,c,d, Sophie R. Younger a, Barry M. Bredt c,d, Lorrie Epling d, Rollie Ronquillo a, Arjun Varma e, Steven G. Deeks c,d, Joseph M. McCune a,c,d,f, Douglas F. Nixon a,d, Elizabeth Sinclair c,d a

Gladstone Institute of Virology and Immunology, University of California-San Francisco, P.O. Box 419100, San Francisco, CA 94141-9100, USA b Department of Epidemiology and Biostatistics, University of California-San Francisco, San Francisco, CA 94110, USA c San Francisco General Hospital General Clinical Research Center, University of California-San Francisco, San Francisco, CA, USA d Department of Medicine, University of California-San Francisco, San Francisco, CA 94110, USA e Department of Medicine, University of California, Los Angeles, CA 90095, USA f Department of Microbiology and Immunology, University of California-San Francisco, San Francisco, CA 94110, USA Received 14 March 2003; received in revised form 11 August 2003; accepted 2 September 2003

Abstract The enumeration of antigen-specific T cell responses has been greatly facilitated in recent years by the development of methods based on the detection of cytokines. In particular, the enzyme-linked immunospot (ELISPOT) and cytokine flow cytometry (CFC) assays have become popular. Since both assays are likely to continue to be in widespread use, it is important to evaluate whether their results are comparable. In the current study, we compared the results obtained in the ELISPOT and CFC assays using peptide pools corresponding to CMV and HIV-1 proteins in chronically HIV-1-infected individuals. Analysis of T cell responses to peptide pools indicated that the CMV pp65 and HIV-1 Gag CFC and ELISPOT-derived results were statistically correlated. However, the results obtained with each assay differed in important ways: the magnitude of the response was consistently higher in the CFC assay while the CFC assay was less likely than the ELISPOT assay to detect low-level responses. Furthermore, there was a lack of numeric agreement between ELISPOT and CFC results. For studies that require the detection of low-level responses, or definition of responses as positive or negative, the ELISPOT assay may be preferable. In contrast, the CFC has a greater dynamic range and allows for phenotypic discrimination of responding cells, making it the assay of choice for most other applications. D 2003 Elsevier B.V. All rights reserved. Keywords: HIV-1; CMV; Cellular immune responses; CD4+ and CD8+ T cells; ELISPOT; Intracellular cytokine flow cytometry

1. Introduction * Corresponding author. Tel.: +1-415-695-3826; fax: +1-415826-8449. E-mail address: [email protected] (A.C. Karlsson). 0022-1759/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2003.09.001

There is a substantial body of data supporting a protective role of HIV-1-specific cellular immunity in HIV-1 disease (reviewed by McMichael and Row-

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land-Jones, 2001; Gandhi and Walker, 2002). Over the past few years, different methods have been evaluated and used for the enumeration of CD4+ and CD8+ antigen-specific T cell responses. Building upon insights gained from traditional assays, e.g. CTL assays (Brunner et al., 1968; Walker et al., 1987; Nixon et al., 1988) and lymphoproliferative assays (Clerici et al., 1989), new methods for the assessment of antigen-specific immune function have been developed. Two of these assays, the enzymelinked immunospot (ELISPOT) assay (Czerkinsky et al., 1988; Lalvani et al., 1997; Larsson et al., 1999; Currier et al., 2002) and the intracellular cytokine flow cytometry (CFC) assay [reviewed in (Maecker et al., 2000)], are non-isotopic, stimulus– response assays amenable to high-throughput analysis. As a consequence, the ELISPOT and CFC assays have gained popularity as rapid, technically straightforward, and highly sensitive methods for the detection and identification of cytokine production by antigenspecific T cells (Goulder et al., 2001). Because the techniques have been rapidly evolving in disparate laboratories, few direct comparisons of ELISPOT and CFC assays have been reported. Those reports that do exist have exclusively used peptides corresponding to optimal epitopes (Asemissen et al., 2001; Sun et al., 2003). In both the ELISPOT and CFC assays, different approaches to antigen stimulation have been evolving. Lysates of whole protein or virus that are processed by the exogenous pathway and presented by MHC class II have been traditionally used to detect CD4+ specific responses. CD8+-specific responses, which recognize antigens processed by the endogenous pathway and presented by MHC class I, have been detected using viral vector constructs that express viral proteins (Nixon et al., 1988; Bennink and Yewdell, 1990). CD8+ responses have also been measured using 8- to 11-mer peptides that mimic optimal epitopes, and it has become very popular to measure the responses to single epitopes or panels of epitopes (Kern et al., 1998; Betts et al., 2000, 2001b; He et al., 2001; Currier et al., 2002). Use of HLA-restricted peptides has overcome the need for growing high titer viral stocks of expression vectors with the attendant safety concerns for laboratory personnel. However, this approach requires a priori knowledge of the HLA haplotype of subjects and is not easily adapted to

large-scale screening. In addition, the use of optimized epitopes will typically underestimate the total response, since only a few well-defined epitopes are used. Recently, the need for HLA typing and for using separate antigens to detect CD4+ and CD8+ responses has been circumvented by the use of overlapping 15mer peptides, which stimulate both CD4+ and CD8+ T cell responses (Kern et al., 2000; Betts et al., 2001a; Maecker et al., 2001). This approach is ideal for rapid and broad analysis of the cellular immune response in large cohorts of individuals, e.g., during the course of clinical studies as well as during vaccine trials. Few comparative studies have evaluated different stimulation approaches (Moretto et al., 2000; Speller and Warren, 2002; Sun et al., 2003) and no direct comparison has been made between the use of recombinant vaccinia virus constructs and overlapping peptide pools. To understand the relative strengths and weaknesses of the ELISPOT and CFC assays and to determine whether data obtained using one assay are comparable to those obtained using the other, we applied both assays to the same clinically derived specimens and measured the responses to overlapping peptide pools covering the HIV-1 Gag, Env, and Vif proteins as well as the CMV pp65 matrix protein. Antigen-specific CD8+ responses to CMV pp65 and HIV-1 Gag, as detected using overlapping peptide pools and recombinant vaccinia virus constructs, were also compared in the CFC assay.

2. Materials and methods 2.1. Subjects Samples were collected from 20 HIV-1-infected subjects participating in a cohort study of the longterm effects of antiretroviral therapy (the ‘‘Study of the Consequences of the Protease Inhibitor Era’’) who all had developed multiple resistances to both protease and reverse transcriptase inhibitors (Hunt et al., 2003). This study was approved by the University of California Committee on Human Research and informed consent was obtained from all subjects. Fresh whole blood or peripheral blood mononuclear cells (PBMC) freshly separated from whole

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blood by Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation were used. 2.2. Antigens Peptide pools consisting of 15 amino acid long peptides with 11 amino acid overlaps corresponding to: HIV-1 Gag (strain SF2, 127 peptides), HIV-1 Gag (strain HXB2, 122 peptides), HIV-1 Env (strain MN, 172 peptides), HIV-1 Vif (strain HXB2, 45 peptides), and CMV pp65 matrix protein (138 peptides) were used. The peptides were synthesized by standard solid phase chemistry, with free N and C termini. Stocks of peptides were dissolved in DMSO and kept at 70 jC at concentrations of 0.6 –2.1 mg/ml. Final concentrations of 2 Ag/ml of each peptide were used in the assays. All peptides were the kind gift from BD Biosciences (San Jose, CA, USA). Recombinant vaccinia constructs expressing the HIV-1 protein Gag (strain IIIB) and the CMV protein pp65 or no antigen (deletion in the tk gene) were obtained from Therion Biologics (Cambridge, MA, USA). The recombinant vaccinia constructs were used at a multiplicity of infection (MOI) of 2:1. Staphylococcal enterotoxin B (SEB) in a final concentration of 10 Ag/ml was used as a positive control. 2.3. Enzyme-linked immunospot (ELISPOT) assay PBMC were resuspended in RPMI 1640 medium supplemented with 15% fetal calf serum. The production of IFN-g by antigen-specific CD4+ and CD8+ T cells was detected using the ELISPOT assay, as described (Larsson et al., 1999; Papasavvas et al., 2000). Ninety-six-well microtiter plates (Millipore, Bedford, MA, USA) were coated overnight at 4 jC with 5 Ag/ml of the anti-IFN-g monoclonal antibody (mAb), 1-D1K (Mabtech, Stockholm, Sweden). The antibody-coated plates were washed four times with PBS and blocked with RPMI containing 5% pooled human serum for 1 h. The PBMC were plated in triplicate at a concentration of 2  105 cells/well along with peptide pools at a final concentration of 2 Ag/ml for each peptide. The plates were incubated overnight

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(37 jC, 5% CO2), washed four times with PBS, and incubated for 2 h with the anti-IFN-g biotinylated mAb, 7-B6-1 (MabTech), at a final concentration of 1 Ag/ml at 37 jC. The plates were washed in PBS with 0.1% Tween 20 and avidin-bound horseradish peroxidase H (Vectastain Elite kit; Vector Laboratories, Burlingame, CA, USA) was added to the wells for 1 h at room temperature. The plates were washed with 0.1% Tween 20 and developed with stable diaminobenzidine tetrahydrochloride (Research Genetics, Huntsville, AL, USA). Spots were counted using a computer-based evaluation system, the AID EliSpot Reader System (Cell Technology, Jessup, MD, USA) (Fig. 1A). In order to avoid variability due to alterations in the settings, all plates analyzed for this study were read at one time and with the same settings. To assess the reproducibility of the automated ELISPOT plate reader, we confirmed that repeated measurements (n = 10) of the same wells in a plate provided consistent responses to the peptide pools [CMV pp65 mean 832 spot-forming cell (SFC)/well, standard deviation (S.D.) 4.3, coefficient of variation (CV) 0.52%; HIV-Gag (SF2) mean 606 SFC/well, S.D. 5.0, CV 0.83%; HIV-Gag (HXB2) mean 449 SFC/ well, S.D. 4.7, CV 1.04%; HIV-Env (MN) mean 42 SFC/well, S.D. 0.8, CV 1.84%; HIV-Vif (HXB2) 62 SFC/well, S.D. 1.2, CV 1.99%]. All responses (spots) counted by the ELISPOT reader were manually edited to ensure that only ‘‘true’’ positive spots with a fuzzy border and a brown color were included. After correction for background signals, as determined in the absence of any antigen, results were expressed either as spot-forming cells (SFC)/106 PBMC or as percent IFN-g-producing PBMC. The background signal was a median of 8 SFC/106 PBMC (interquartile range: 5 – 17 SFC/106 PBMC). For a sample to be considered positive, a response of at least 50 SFC/106 PBMC and/or twofold higher than background signal was required. In an attempt to minimize the differences in how the results were expressed in the ELISPOT versus CFC assays, we also expressed the ELISPOT results as percent IFN-g-producing CD3+ T cells. To calculate the number of CD3+ T cells used in each ELISPOT assay, PBMC were stained with anti-CD3 peridinin chlorophyll protein (PerCP) and run on a FACS Calibur to determine the percentage of CD3+ T cells in each sample.

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Fig. 1. Responses to CMV pp65 and HIV-Gag (SF2) peptide pools as detected by ELISPOT and CFC assays. The response to no stimulation is also included as a negative control. (A) In the ELISPOT assay, IFN-g-producing T cells were identified in each well (of a 96-well microtiter plate) as spots with a fuzzy border and a brown color. Triplicate wells of plated PBMC (at a concentration of 2  105 cells/well along with antigens and controls) were analyzed and the mean is referred to as SFC/well. (B) In the CFC assay, data were first gated on viable CD3+ T cells and then the percentage of CD69+ IFN-g-producing CD3+ T cells was obtained.

2.4. Cytokine flow cytometry (CFC) The CFC assay was performed using whole blood (peptide pools) or freshly isolated PBMC (recombinant vaccinia constructs). Cells were stimulated with peptides (2 Ag/ml) for 2 h or infected with recombinant vaccinia constructs (MOI 2:1) for 4 h at 37 jC. Negative controls included cells incubated without added peptide or cells infected with recombinant vaccinia constructs with a deletion in the tk gene; stimulation with SEB served as a positive control. Purified anti-CD28/CD49d (BD Biosciences) was included for co-stimulation. Brefeldin A (SigmaAldrich, St. Louis, MI, USA) was added at a concentration of 10 Ag/ml and the cells were incubated for an

additional 4 h (peptides) or 5.5 h (recombinant vaccinia constructs). Whole blood samples stimulated with peptides were incubated in a programmable water bath; following the 37 jC incubation, samples were held overnight at 18 jC. PBMC and cells from whole blood were washed, red blood cells were removed from whole blood using FACSLysing Solution (BD Biosciences), and permeabilized with FACS Permeabilizing Solution (BD Biosciences). After washing, the cells were stained for flow cytometry. Whole blood samples stimulated with peptide pools were stained with fluorescein isothiocyanate (FITC)conjugated anti-IFN g, allophycocyanin (APC)-conjugated anti-CD3, R-phycoerythrin (PE)-conjugated anti-CD69 (BD Biosciences) and phycoerythrin – cya-

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nin 5.1 (PC5)-conjugated anti-CD4 (Immunotech, Beckman Coulter, Miami, FL, USA) for 50 min at room temperature. PBMC stimulated with recombinant vaccinia constructs were stained with FITCconjugated anti-IFN-g, PE-conjugated anti-CD4, peridinin chlorophyll protein (PerCP)-conjugated CD3, and APC-conjugated CD8 (BD Biosciences) for 30 min at 4 jC. Following staining, the cells were washed, fixed in 1% paraformaldehyde, and collected on a FACS Calibur instrument using CellQuest software (BD Biosciences). Peptide-stimulated samples were collected using an Autoloader with Worklist Manager software (BD Biosciences). For acquisition of cells from whole blood samples, a large lymphocyte gate excluding granulocytes was used, and only events falling within the gate were collected. To acquire sufficient CD4+ cells, a gate was drawn on CD3+CD4+ events and at least 12,000 CD4+ events were collected. For PBMC samples, a minimum of 1  105 viable lymphocytes were collected. For the comparison of the ELISPOT and CFC data, the results are expressed as percentage of CD69+ IFN-g-producing CD3+ T cells and data were analyzed using CellQuest (BD Biosciences) or FlowJo (TreeStar, San Carlos, CA). For all samples, a positive gate was set on the nonstimulated control such that all CD3+ T cells that were negative for IFN-g and CD69 were excluded; this gate was applied to each antigen stimulation from the same subject (Fig. 1B). For the comparison of CD8+ T cell responses against either peptide pools or recombinant vaccinia constructs, the percentage of IFN-g-producing T cells was obtained by gating on CD3 + CD4 (peptide pools) or CD3+CD4 CD8+ (recombinant vaccinia constructs) cells. The background signals for the whole blood assay represented a median of 0.23% (interquartile range: 0.08– 0.40%) IFN-g-producing CD3+ T cells and, for the PBMC assay using the recombinant vaccinia constructs, a median of 0.03% (interquartile range: 0.02 – 0.04) IFN-g-producing CD8+ T cells. The background signal (response to the negative control) was subtracted in each experiment. To set a cutoff value for the whole blood assay, we analyzed CD3+ T cell responses to HIV-Gag and CMV pp65 peptide pools in samples from 16 individuals who were seronegative for both HIV-1 and CMV. A sample was considered positive (above the cutoff) when the response was 2 S.D. above the mean background

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corrected peptide response. In response to the HIVGag (SF2) peptide pool, the cutoff value was 0.13% and for the CMV pp65 peptide pool the cutoff value was 0.09% IFN-g-producing CD3+ T cells. Since these values are similar, the higher value was used as the cutoff value for all the peptide pools. 2.5. Statistical methods For comparison between responses to different antigens among participants evaluated with the same testing format (i.e., CFC or ELISPOT), a paired t-test or Wilcoxon sign rank test was used depending upon the distribution of the within-subject differences. For comparisons of the responses to either CMV pp65 or HIV-Gag proteins across the different testing formats (i.e., ELISPOT or CFC), we determined a rank correlation coefficient and examined the distribution of within-subject differences (Bland and Altman, 1986). Because we did not observe the same mean and variability of within-subject differences over the entire range of results for a given protein, a natural log transformation of the ELISPOT and CFC-derived results was performed. This yielded approximately the same distribution of within-subject differences over the majority of the range of results (or allowed for partitioning of within-subject differences into two groups). The within-subject differences on the log scale could then be backcalculated to the native scale and interpreted as the ratio of within-subject CFC to ELISPOT results. The median and range of CFC to ELISPOT ratios were calculated. For comparison of the different testing formats when evaluating HIVEnv and HIV-Vif, responses were categorized as negative or positive. The agreement between the assays was described by (a) the percentage of participants with the same results on both assays (concordance) and (b) the Kappa coefficient. The latter was found to range between 1 and 1, with values between 0.7 and 1.0, indicative of good to excellent agreement (Sackett et al., 1991).

3. Results All subjects had chronic HIV infection and had detectable viral loads (median, log10 4.20 copies/ml; interquartile range, log10 3.60 – 4.85). The median

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CD4+ T cell count was 258 cells/mm3 (interquartile range, 201– 401) and median CD8+ T cell count was 1170 cells/mm3 (interquartile range, 935 – 1613). There was a broad range of HLA types (data not shown). A total of 20 samples from 20 subjects were used in this analysis. 3.1. CMV and HIV-1-specific T cell responses detected using overlapping peptide pool stimulation in the ELISPOT and CFC assays An example of results obtained using overlapping peptide pool stimulation in the ELISPOT and CFC assays is shown in Fig. 1A and B, respectively. For comparison of the within-assay responses to different peptide pools, we evaluated the ELISPOT results expressed in the conventional units of SFC/106 PBMC (Fig. 2A) and the CFC results as percent IFN-g-producing CD3+ T cells (Fig. 2B). For both the ELISPOT and CFC assays, the response to the CMV pp65 peptide pool was greater than to each of the individual HIV-1 peptide pools ( P < 0.05 for the ELISPOT and P < 0.06 for the CFC). Among the HIV-1 peptide pools, both the ELISPOT and CFC assays detected a significantly stronger response against the HIV-Gag (SF2) peptide pool than against either the HIV-Env or HIV-Vif peptide pools ( P < 0.01 for the ELISPOT and P < 0.02 for the CFC, Fig. 2A and B). The ELISPOT and CFC assays diverged in the differentiation of the responses to the two different peptide pools representing HIV-1 Gag. Using the ELISPOT assay, the median response to the SF2 strain was significantly higher than that observed against the HXB2 strain (SF2: median 1963 SFC/ 106 PBMC, interquartile range 1401 –3806; HXB2: median 1238 SFC/106 PBMC, interquartile range 569 – 2598; P = 0.02). In contrast, there were no significant differences when the responses to these peptide pools were assayed using the CFC assay (SF2: median 1.02% IFN-g-producing CD3+ T cells, interquartile range 0.54 – 2.20%; HXB2: median 0.95% IFN-g-producing CD3+ T cells, interquartile range 0.18 –1.75%; P = 0.154). Differences between the ELISPOT and CFC assays were also observed when the responses were classified as positive or negative. For all of the HIV-1 peptide pools, the percentage of subjects with positive

Fig. 2. Magnitude of antigen-specific T cells expressed as (A) SFC/106 PBMC in the ELISPOT assay and (B) the percentage of IFN-g-producing CD3+ T cells in the CFC assay. Only subjects with results obtained using both assays are included. A dashed line indicates the cutoff value for each assay.

responses was higher in the ELISPOT assay than in the CFC assay. In the ELISPOT assay, 100%, 100%, 73%, and 36% showed positive responses to the HIV-Gag (SF2), -Gag (HXB2), -Env, and -Vif peptide pools, respectively. In the CFC assay, positive responses were observed in only 85%, 73%, 36%, and 27% of the subjects, respectively. All subjects showed positive responses to CMV pp65 in both assays. 3.2. Numeric agreement between the ELISPOT and CFC assays To assess the numeric agreement between the ELISPOT and CFC assays, responses from both

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ELISPOT and the CFC-derived results (Fig. 3), there was considerable lack of numeric agreement. Other than for the very smallest responses (i.e., less than

Fig. 3. Correlation between frequencies of antigen-specific T cells obtained using the ELISPOT and the CFC assays. Frequencies of IFN-g production in response to antigenic stimulation with CMV pp65, HIV-Gag (SF2), and HIV-Gag (HXB2) peptide pools were measured in the CFC (x-axis) and the ELISPOT ( y-axis) assays. In each case, responses are expressed as the percentage of IFNg-producing CD3+ T cells.

assays were expressed in terms of percentage of IFNg-producing CD3+ T cells. There was no difference in the relative ranking or ordering of the different antigens in terms of their median responses when the ELISPOT results were expressed as IFN-g-producing CD3+ T cells or SFC/106 PBMC (data not shown). We first evaluated the responses obtained to the CMV pp65 and HIV-Gag peptide pools. While there was a statistically significant correlation between the

Fig. 4. Within-subject differences between the results obtained using the ELISPOT or the CFC assay after stimulation with CMV pp65, HIV-Gag (SF2), and HIV-Gag (HXB2). The within-subject mean of the ELISPOT and CFC results (expressed as percent IFN-gproducing CD3+ T cells) is given on the x-axis and the withinsubject difference between the CFC and ELISPOT results is given on the y-axis. The dashed line indicates no (0%) difference between the results obtained with the two assays.

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0.2%), the CFC result was almost always greater than the ELISPOT result. This is shown in Fig. 4, where the within-subject difference (CFC minus ELISPOT results) was plotted against the within-subject mean of the CFC and ELISPOT results. The dashed horizontal line, in each plot of Fig. 4, is placed at 0% difference, which is what would be observed if the CFC and ELISPOT assays had perfect agreement. In general, both the average within-subject CFC minus ELISPOT difference and the variability in the differences increased over the range of within-subject mean values. To determine if the variability in differences was consistent across the range of within-subject mean CFC and ELISPOT results, the ratios of the CFC to ELISPOT result were calculated (data not shown). If the range of CFC to ELISPOT ratios were small, this would indicate that the variability is consistent at different within-subject mean CFC and ELISPOT results, and the median ratio would be a conversion factor between the two assays. However, for both pp65 and Gag peptides, we found that the range of ratios was large, indicating that the variability between the ELISPOT and CFC assays was not consistent at different within-subject mean CFC and ELISPOT results. Because of the high percentage of persons with negative or nondetectable Env- or Vif-specific results, a comparison between CFC and ELISPOT testing of HIV-1 Env and Vif responses was performed by dichotomizing results into negative or positive (Table 1). The observed concordance (agreement) between the assays in response to HIV-Env was 45.5%; the expected concordance by chance alone is 43.8%. The Kappa coefficient was 0.003, indicative of very limited agreement above that expected by chance. In Table 1 Agreement between the ELISPOT and CFC assays in response to the HIV-Env and HIV-Vif peptide pools CFC: negative

CFC: positive

Total

HIV-Env ELISPOT: negative ELISPOT: positive Total

2 5 7

1 3 4

3 8 11

HIV-Vif ELISPOT: negative ELISPOT: positive Total

5 3 8

2 1 3

7 4 11

Fig. 5. The use of lower number of cells/well can be associated with both an increase and a decrease of the antigen-specific responses, depending on the magnitude of the response using the ELISPOT assay. The responses to peptide pools corresponding to (A) CMV pp65 and (B) HIV-Gag (SF2) were evaluated using twofold dilutions of cells ranging from 2  105 to 0.25  105 cells/well in five subjects. The number of spots detected per well using the standard 2  105 cells is indicated in association with each individual plot in the graph. The ELISPOT responses are expressed as SFC per million PBMC.

response to HIV-Vif, the concordance was 54.5%, which is less than what would be seen by chance (56%). The Kappa coefficient was 0.038, again indicative of very limited agreement. 3.3. The magnitude of antigen-specific responses detected in the ELISPOT assay using various cell inputs Since there is a limit to the number of spots that can be accurately counted in a given well, results in

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Fig. 6. Correlation (right panel) and within-subject differences (left panel) between the antigen-specific responses to recombinant vaccinia construct (rVV) and peptide pools using the CFC assay. The results are expressed as percentages of antigen-specific IFN-g-producing CD8+ T cells and were measured in response to the endogeneously processed rVV and the extracellularly processed peptide pools corresponding to (A) CMV pp65, (B) HIV-Gag (SF2), or (C) HIV-Gag (HXB2).

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the ELISPOT may be prone to underestimation. To investigate this, the antigen-specific T cell responses to CMV pp65 and HIV-Gag (SF2) were measured at different cell dilutions. Specifically, twofold dilutions of cells, ranging from 2  105 to 0.25  105 cells/ well, were tested in five subjects. Using 0.5 or 1  105 cells/well instead of the standard 2  105 cells/well, much higher frequencies (up to 50% increases) of antigen-specific responses were detected in subjects with CMV pp65 peptide pool responses of more than 900 SFC/well (or 4500 SFC/106 PBMC) (Fig. 5A). However, in subjects with CMV pp65 responses less than 900 SFC/well (or 4500 SFC/106 PBMC) and those with responses to HIV-Gag (SF2) peptide pool ranging between 364 and 734 SFC/well (or 1820 – 3670 SFC/106 PBMC), the sensitivity of the assay decreased as a function of input cell number (Fig. 5A and B).

isons, the average peptide pool minus recombinant vaccinia virus difference increased over the range of within-subject mean values. In addition, the variability of the differences increased with increasing withinsubject mean values in each of the three comparisons. To determine if the variability in differences was consistent across the range of within-subject mean peptide pool and vaccinia results, the ratio of peptide pool to recombinant vaccinia virus result was calculated (data not shown). The range of ratios for each recombinant vaccinia versus peptide pool comparison was large, indicating that the variability in differences between the two assays was not consistent at different within-subject mean CFC and ELISPOT results. A single conversion factor could therefore not be established between the two antigen stimulation methods.

4. Discussion 3.4. Numeric agreement between peptide pool and recombinant vaccinia virus stimulation To discern whether stimulation with peptide pools or recombinant vaccinia virus constructs might yield different results in the CFC assay, responses to recombinant vaccinia virus construct expressing CMV pp65 were compared to those elicited by the CMV pp65 peptide pool; similarly, responses to recombinant vaccinia virus construct expressing HIV-Gag (IIIB) were compared to those elicited by peptide pools corresponding to SF2 and HXB2 (analogous to IIIB) HIVGag strains. In each of these comparisons, the peptide pool responses were higher than the responses to the corresponding vaccinia constructs for most, but not all, subjects. A significant correlation was observed between the CD8+ responses to the recombinant vaccinia construct and to the peptide pool for CMV pp65 (r = 0.814, P < 0.01) (Fig. 6A, left panel). Likewise, responses to the vaccinia construct HIV-Gag (IIIB) correlated with those to peptide pools corresponding to both the SF2 (r = 0.736, P < 0.01) and the HXB2 (r = 0.855, P < 0.01) HIV-Gag strains (Fig. 6B and C, left panels, respectively). To examine the numeric agreement between responses to the two types of antigen, the within-subject peptide pool minus recombinant vaccinia differences were plotted against the within-subject means of the peptide pool and vaccinia responses (Fig. 6, right panels). For all three compar-

The ELISPOT and CFC assays have become widely used for the detection of cytokine production by antigen-specific T cells (Czerkinsky et al., 1988; Lalvani et al., 1997; Larsson et al., 1999; Maecker et al., 2000; Currier et al., 2002), but few direct comparisons of the assays have been reported. In the current study, the results obtained in the ELISPOT and CFC assays correlated when using the HIV-1 Gag and CMV pp65 peptide pools. Furthermore, a very good correlation was found by comparing the ability of two different types of antigen stimulation (peptide pools and endogenously processed protein responses after infection with recombinant vaccinia virus constructs) to stimulate CD8+ cell responses using the CFC assay. However, there was a substantial numeric disagreement between both the CFC and ELISPOT assays and the two antigen stimulation techniques. Previous studies comparing CD8+ T cell responses in the CFC and ELISPOT assays have used optimal peptide stimulation. (Sun et al., 2003). High interassay reproducibility and a significant correlation between ELISPOT and CFC were demonstrated (Asemissen et al., 2001). In addition, the frequency of responses was reported to be similar in the two assays (Sun et al., 2003). Sun et al. (2003) also described a higher frequency of antigen-specific responses against optimal peptides compared to recombinant vaccinia constructs using the ELISPOT assay; however, and in

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contrast to the current study, the results did not correlate. Furthermore, the numeric agreement between assays or types of antigen was not examined. Thus, no extensive attempt to directly compare antigen-specific responses in the ELISPOT and CFC assays, or to compare specific T cell responses to recombinant vaccinia virus constructs with peptide pools, has been reported. To evaluate the agreement between the results obtained with the CFC and ELISPOT assays, we examined the distribution of within-subject differences. For a given mean level of response obtained with the CFC and ELISPOT assays, there can be considerable variability in the differences between the assays. For example, a significant difference between the two HIV-Gag peptide pools (SF2 and HXB2) was found with the ELISPOT but not the CFC assay. This observation underscores the fact that, even though there can be a correlation between the results generated with the two assays, they can lead to different conclusions. Although values from the CFC assay were typically higher than for the ELISPOT assay, there was no consistent conversion factor between the results of the two assays. For both assays to give similar conclusions in a comparison of different patient groups, either the sample sizes would have to be substantial or the magnitude of the differences between patient groups would have to be greater than the variability in the differences we observed between the two assays. Several factors might contribute to the higher magnitude of responses observed in the CFC assay as compared with the ELISPOT assay. Since it is very hard to accurately count more than 1000 spots/well, there is an inherent detection limit in the ELISPOT assay. We found that titrating down the number of cells added to each well could circumvent this problem. However, we also observed that a lower cell number could be associated with a decreased sensitivity in the ELISPOT assay, emphasizing the need to optimize the cell input used in the assay. Another reason for the lower frequencies detected in the ELISPOT assay may be that contiguous spots overlap and are counted as one rather than more than one. In this study, the ELISPOT and CFC assays were compared on the basis of protocols that are routinely performed in our laboratories. Notably, there are several features to the protocols that could potentially

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cause a discrepancy in the responses between the two assays: (1) stimulation was performed on whole blood in the CFC assay while purified PBMC were used in the ELISPOT assay; (2) co-stimulation using purified anti-CD28/CD49d was used in the CFC assay but not in the ELISPOT assay; (3) antigen stimulation was carried out for 6 h in the CFC assay and for 14– 16 h in the ELISPOT assay; (4) in the CFC assay, only IFN-g produced by CD3+ T cells is quantitated, while the ELISPOT results include all IFN-g-producing cells within the PBMC sample. Preliminary results indicate that there is no significant difference between the responses obtained using whole blood and freshly isolated PBMC in the case of stimulation with CMV antigens (Holden Maecker, personal communication). Co-stimulation with a combination of anti-CD28/CD49d is needed to acquire optimal CD4+ responses against whole protein antigens. However, this effect is less marked for responses to peptide antigens (Holden Maecker, personal communication). In addition, the backgroundcorrected CD3 responses to HIV-Gag (SF2) were not significantly altered when CD28 and CD49d were omitted from the assay (data not shown). In the CFC assay, measurement of IFN-g production is often optimal after 6 h of simulation and not significantly improved with increased incubation times (Sandberg et al., 2001). With respect to the differences in how the results are expressed, we evaluated the ELISPOT results expressed both as IFN-g-producing CD3+ T cells and as IFN-g-producing PBMC. Very similar results were obtained, with maintenance of a good correlation using the CMV pp65 and HIV-Gag peptide pools, suggesting that the IFN-g-producing cells within the PBMC are primarily CD3+ T cells. Since the use of recombinant vaccinia virus constructs remains widespread, we directly compared responses against proteins expressed after recombinant vaccinia virus infection to those obtained with the peptide pools. From previous studies of HIV-1 infected subjects, we have established that the response to recombinant vaccinia constructs expressing viral proteins is primarily CD8+ T cell mediated (Larsson et al., 1999; Ortiz et al., 1999). Accordingly, the comparison was restricted to CD8 + T cell responses. A significant correlation was found between CMV pp65 and HIV-Gag specific responses obtained using peptide pools and recombinant vac-

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cinia constructs. The responses to peptide pool stimulation were typically higher than to recombinant vaccinia virus constructs. Despite the observed correlations, we found an important degree of numeric disagreement and there was no single factor that could be used to convert between the results of these two antigen stimulation methods. For Gag proteins, agreement was best between the two types of antigen when analogous proteins were used; likewise, the correlation between recombinant vaccinia virus and peptide pool responses was stronger for the analogous proteins. The genetic distance between the SF2 and HXB2 strains is only 3.6%, less than the difference usually observed between individuals infected with different subtype B HIV-1 strains. These observations underscore the importance of choosing antigenic sources that are as closely related as possible to the viral strains circulating in a given area of interest (Lee et al., 2002). This study has confirmed that there is a significant correlation between the results obtained by the CFC and ELISPOT assays using CMV pp65 and HIV-Gag peptide pools. However, the response obtained with each assay differed in important ways: the magnitude of the response was consistently higher in the CFC assay while the CFC assay was less likely than the ELISPOT assay to detect low-level responses (thus, the percentage of positive responses in the ELISPOT assay was higher). In addition, for a given magnitude of response, there was considerable variation in the responses obtained with the ELISPOT and CFC assays. These findings indicate that the results generated with the CFC and ELISPOT assays are not necessarily equivalent. The ELISPOT and CFC assays each have advantages and disadvantages. For most applications, it would appear that the CFC assay is the assay of choice, allowing discrimination and phenotypic analysis of individual cytokine-producing cells across a broad dynamic range. Using fresh whole blood and automation for data collection, the effort required to perform CFC has been greatly reduced. However, when measuring low-level responses, the ELISPOT may be a better choice due to its lower detection limit. The ELISPOT assay is also less expensive to perform, less dependent upon sophisticated instrumentation, and better suited to the analysis of frozen samples, when limited numbers of cells are available. This information is important for

the evaluation of results obtained with either the CFC and ELISPOT assay from basic research projects, clinical studies, and vaccine trials.

Acknowledgements This work was supported in part by grants from the NIH (AI46254 and AI052745 to DFN, AI47062 to JMM, and AI052745 to SGD), the California AIDS Research Center (CC99-SF-001), the UCSF/Gladstone Institute of Virology and Immunology Center for AIDS Research (P30 MH59037) and the General Clinical Research Center at San Francisco General Hospital (5-MO1-RR00083-37). Holden Maecker, BD Biosciences (San Jose, CA), kindly donated the peptides and commented on the manuscript. Douglas F. Nixon is an Elizabeth Glaser scientist of the Elizabeth Glaser Pediatric AIDS Foundation. Joseph M. McCune is a recipient of the Burroughs Wellcome Fund Clinical Scientist Award in Translational Research.

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