K562 cells (Human myelogenous leukemia cell line, ATCC, Manassas, VA) were cultured at 37°C, 5% CO₂ in tissue culture medium (TCM) consisting of RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 1 mM non-essential amino acids, 2 mM glutamine, 100 U/ml Penicillin, 100 μg/ml Streptomycin, 20 mM HEPES and 1 mM sodium pyruvate (Gibco BRL Life Technologies, Grand Island, NY).

The TALL-104 cell line is a T-cell derived line that is highly cytotoxic for NK-sensitive targets and when grown in the presence of IL-2, can be stimulated to secrete IFN-γ and GrB 2627. The cell line was cultured in Iscove's modified Dulbecco's medium (BioWhittaker) supplemented with 20% FBS (Hyclone), 4 mM glutamine, 100 U/ml Penicillin and 100 μg/ml Streptomycin (Gibco BRL Life Technologies) at 8% to 10% CO₂. Recombinant human IL-2 (100 U/ml; Hoffmann-LaRoche, Nutley, NJ) was added every 2–3 days to ensure optimal growth and maintenance of cytotoxic activity.

Peripheral blood mononuclear cells (PBMC) were isolated from venous blood of normal human volunteers by buoyant density centrifugation over Ficoll-Paque (Pharmacia, Piscataway, NJ). Aliquots of effector cells were cryopreserved in the vapor phase of liquid N₂ for future use in functional testing. The PBMC were thawed and resuspended at 2 × 10⁶ cells/ml in 20 ml of RPMI 1640 (BioWhittaker) containing 10% human AB serum (Mediatech, Herndon, VA), 1 mM non-essential amino acids, 2 mM glutamine, 1 mM pyruvate, 20 mM HEPES, 100 U/ml Penicillin and 100 μg/ml Streptomycin (Gibco BRL Life Technologies). Cell suspensions were stimulated with 100 U/ml of IL-2 (Hoffmann-LaRoche) on day 0 and cultured for 5 to 6 days at 37°C, 5% CO₂. LAK cultures consisted of 30.0 ± 2.1 % NK cells (CD3^-,CD16/CD56⁺) and 26.9 ± 4.9 CD8⁺ T-cells as determined by flow cytometric analysis.

TALL-104 cells were treated with inhibitors of cellular secretion prior to use in the GrB and IFN-γ ELISPOT assays. Inhibitors used included 1,2-bis-(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM, Molecular Probes, Eugene, OR), a cell permeant chelator of intracellular Ca²⁺, and γ,4-dihydroxy-2-[6-hydroxy-1-hep-tenyl]-4-cyclopentanecrotonic acid λ-lactone (brefeldin A; Sigma, St. Louis, MO) which blocks protein secretion. TALL-104 cells were resuspended at 2.5 × 10⁵ cells/ml and 2.5 × 10⁴ cells/ml in PBS without divalent cations for BAPTA-AM and brefeldin A treatment, respectively. Cells were pretreated with the indicated concentrations of BAPTA-AM for 45 min or with 5 μg/ml brefeldin A for 1 h at 37°C, washed twice, assessed for viability by trypan blue exclusion, and resuspended at 2.5 × 10⁴ cells/ml in assay media.

Cytotoxicity of TALL-104 and LAK cells was assessed using the standard ⁵¹Cr-release assay. Briefly, one million target cells were labeled at 37°C for 1 h with 100 μCi Na₂⁵¹CrO₄ (New England Nuclear, Boston, MA). Target cells were washed and resuspended in TCM at 5 × 10⁴ cells/ml. Five thousand target cells per well (100 μl) were added to a 96 well plate (Costar, Cambridge, MA) following the appropriate number of effector cells (100 μl/well). The defined effector:target (E:T) ratios were plated in triplicate. Cytotoxicity assays were performed at 37°C for 4 h. Percent specific lysis was calculated using the following equation:

(ER - SR)/(MR - SR) × 100,

where ER = experimental release, SR = spontaneous release and MR = maximum release.

Granzyme B secretion was measured using the GrB ELISPOT assay as previously described 25. Briefly, MultiScreen-IP plates (PVDF membrane, Millipore, Bedford, MA) were coated overnight at 4°C with 100 μl/well of anti-human GrB antibody (7.5 μg/ml in PBS, clone GB-10, PeliCluster, Cell Sciences, Norwood MA). Effector cells (100 μl/well) were added to triplicate wells at specified concentrations followed by 5 × 10⁴ target cells per well (100 μl). After the specified effector-target cell incubation, the plates were washed and 100 μl/well of biotinylated anti-human GrB detecting antibody (0.25 μg/ml in PBS/1% BSA/0.05% Tween 20, clone GB-11, PeliCluster, Cell Sciences) was added. Plates were incubated for 3 h and 50 μl of Streptavidin-Alkaline Phosphatase (1:1500 in PBS/1% BSA, Gibco BRL Life Technologies) was added for 1 h. Spots were visualized with 100 μl/well of BCIP-NBT phosphatase substrate (KPL, Gaithersburg, MD) and subjected to automated evaluation using the ImmunoSpot Imaging Analyzer system (Cellular Technology Ltd, Cleveland, OH).

For assessing IFN-γ secretion, MultiScreen-IP plates (PVDF membranes, Millipore) were coated overnight at room temperature with 50 μl/well of anti-human IFN-γ antibody (20 μg/ml in PBS, Biosource, International, Camarillo CA) as previously described 25. After effector and target cells were incubated at 37°C, the plates were washed and 50 μl/well of biotinylated anti-human IFN-γ antibody (1.3 μg/ml in PBS/1% BSA/0.05% Tween 20, BD PharMingen, San Jose, CA) was added. Plates were incubated with biotinylated antibody, washed and 50 μl of Streptavidin-Alkaline Phosphatase (1:1500 in PBS/1% BSA, Gibco BRL Life Technologies) was added. Spots were visualized and enumerated as described above.

Degranulation of TALL-104 and LAK cells in response to target cell recognition was assessed by monitoring surface antigen expression of CD107a (lysosomal-associated membrane protein-1), a surface antigen transiently present on the cell surface after release of cytolytic granules. Expression of CD107a has been used as a marker to measure degranulation by flow cytometry 2829.

To distinguish target cells from effector cells, K562 cells were first labeled with PKH67 dye (Sigma) according to the manufacturer's instructions and washed extensively prior to use in the assay. Effector cells (2 × 10⁵) were resuspended in 400 μl of phenol red-free TCM in polystyrene tubes (Falcon, Franklin Lakes, NJ). PKH67-labeled K562 target cells (1 × 10⁵ cells in 50 μl) were then added to appropriate tubes, the tubes were spun for 30 sec at 500 rpm and incubated for the specified periods in a 37°C water bath. Reactions were quenched with cold PBS and then 20 μl of CD107a-PE-Cy5 (PharMingen) was added. Mouse IgG₁-biotin (Beckman-Coulter, Miami, FL) and streptavidin-PE-Cy5 (Caltag, Burlingame, CA) were utilized for controls. Tubes were incubated with antibody for 20 min at room temperature, washed with buffer and analyzed using a FACScan instrument (Becton-Dickinson Immunocytometry Systems, San Jose, CA) equipped with a 15 mW argon-ion laser. The percent of effector cells expressing CD107a was determined by gating the PKH67 negative (effector cells) population. Within the PKH67 negative gate, CD107a expression was determined from histograms based on forward and side scatter analysis. Effector cells run in the absence of target cells were evaluated for baseline expression of CD107a. The percentage of effector cells induced by target cells to express CD107a was calculated by subtracting background (spontaneous) expression from experimental samples.

Statistical analysis was performed using Student's t test and Pearson correlation coefficient (R²).

GrB ELISPOT assays were performed using a constant number of K562 targets and decreasing numbers of TALL-104 effector cells per well. As the number of TALL-104 cells decreased, the resulting number of GrB spots decreased linearly. Limited GrB spots were detected in wells containing TALL-104 alone demonstrating that the ELISPOT assay measured primarily stimulated release of GrB (Fig. 1). K562 alone did not secrete GrB (data not shown). The correlation between the number of spots per well and the number of TALL-104 was highly significant with a Pearson correlation coefficient of R² = 0.98. Therefore, the GrB ELISPOT assay is capable of measuring the frequency of effector cells stimulated to secrete GrB.

In tandem with the GrB ELISPOT assay, TALL-104 cells were also utilized as effectors against K562 targets in the standard ⁵¹Cr-release assay to assess lytic activity. Significant specific lysis was observed at effector:target ratios from 10:1 to 1:1 (Table 1). In contrast to the ELISPOT assay, that can measure the frequency of cytotoxic cells at effector:target ratios as low as 0.02:1, specific lysis was not significant in the ⁵¹Cr-release assay at cell ratios below 1:1 (Table 1). However, when optimal ratios were used in each individual assay, similar trends between the number of GrB spots per well in the ELISPOT and specific lysis in the ⁵¹Cr-release assay were observed. These data demonstrate that the release of GrB is indicative of cytolytic activity.

To measure the secretion kinetics of GrB compared to IFN-γ, TALL-104 cells were incubated with K562 target cells for the indicated times in the ELISPOT assays. Secretion of GrB was compared to IFN-γ because IFN-γ is currently the standard utilized to measure the frequency of cytotoxic cells via the ELISPOT method. GrB secretion was observed within 10 min of effector-target cell interaction with optimal secretion around 3–4 h (Fig. 2). This observation is in contrast to maximum IFN-γ production, which was observed at 24 h of effector-target interaction (data not shown). These data are consistent with rapid effector cell degranulation (within minutes) upon contact with target cells 1819.

Effects of secretion inhibitors on the release of GrB and IFN-γ in the ELISPOT assays as well as expression of CD107a on effector cells were evaluated to determine the possible mechanism of GrB release measured in the ELISPOT assay. TALL-104 cells were treated with inhibitors of cellular secretion and assessed for their ability to release GrB and IFN-γ in the ELISPOT assays. BAPTA-AM chelates intracellular Ca²⁺ and was utilized to block degranulation. Brefeldin A, which blocks protein secretion, was utilized to prevent secretion of newly synthesized proteins.

Loading TALL-104 cells with BAPTA-AM resulted in a dose-dependent inhibition of GrB secretion (Fig. 3). Inhibition of GrB was not attributed to alterations in cell viability as TALL-104 cells loaded with as high as 100 μM of BAPTA-AM remained viable as assessed by trypan blue exclusion (data not shown). Brefeldin A treatment did not alter the secretion of GrB when TALL-104 cells were stimulated with K562 targets but did significantly (p < 0.05) inhibit IFN-γ secretion (Fig. 4). Additionally, increased expression of the degranulation marker, CD107a, on TALL-104 cells was observed when TALL-104 cells were incubated with K562 target cells. Compared to TALL-104 cells alone, the addition of K562 targets induced 13.75% of TALL-104 cells to express CD107a (Fig. 5). Taken together, these data are consistent with synthesis of IFN-γ de novo while GrB is released from preformed granules upon effector-target interaction.

To address the potential clinical relevance of the GrB ELISPOT assay, LAK cells derived from PBMC, rather than the TALL-104 cell line, were utilized as effector cells. As shown in Figure 6, the number of GrB spots per well correlated with the number of LAK cells added, results comparable to those obtained with TALL-104 cells. Only wells containing both LAK and K562 target cells contained a substantial number of spots (Fig. 6).

The secretion kinetics of GrB and IFN-γ by LAK cells were similar to that observed for TALL-104 cells. GrB secretion was seen within 10 min of effector-target cell interaction with optimal secretion around 3 h and maximal IFN-γ production was observed at 24 h of effector-target interaction (Fig. 7). Similarly, stimulated secretion of GrB by LAK cells was associated with increased surface expression of the degranulation marker, CD107a. The correlation between the number of GrB spots and the number of cells expressing CD107a per 100,000 effector cells was significant (R² = 0.89, Fig. 8). These data demonstrate that the GrB ELISPOT assay accurately measures the rapid release of GrB by stimulated LAK cells and can enumerate GrB-secreting effector cells.

LAK cells were utilized in the GrB ELISPOT, IFN-γ ELISPOT and ⁵¹Cr-release assays to evaluate the different assays for measuring innate immunity (Table 2). Both ELISPOT assays were significantly more sensitive than the ⁵¹Cr-release assay. At effector:target ratios of 20:1-5:1, significant specific lysis can be measured by the ⁵¹Cr-release assay but the spots per well in the ELISPOT assays were too numerous to count accurately. Significant IFN-γ and GrB secretion was evident at effector:target ratios as low as 0.02:1 (1000 effectors/well) therefore, the optimal ratios for the ELISPOT assays are below the level of sensitivity of the ⁵¹Cr-release assay (Table 2). However, when the optimal number of LAK cells are used in each individual assay, the amount of GrB and IFN-γ secreting cells in the ELISPOT assays and cytotoxicity in the ⁵¹Cr-release assay have shown cross-correlation with R² values greater than 0.86 for all combinations. When comparing the two ELISPOT assays, the GrB ELISPOT is more rapid and may be a more direct measure of cytotoxic activity than the IFN-γ ELISPOT. Therefore, the GrB ELISPOT assay can elucidate the frequency of GrB secreting cells in primary LAK cultures and can be applied to measure innate immunity in clinically relevant samples.