Differential expression of human granzymes A, B, and K in natural killer cells and during CD8+ T cell differentiation in peripheral blood

Authors


Abstract

NK cells and cytotoxic T lymphocytes can induce apoptosis in virus-infected and transformed target cells via the granule exocytosis pathway. The key components of the cytolytic granules are perforin and several serine esterases, termed granzymes. While the cellular distribution of human granzymes A (GrA) and B (GrB) has been well characterized much less is known about the expression pattern of human granzyme K (GrK). In this study GrA, GrB, and GrK expression was analyzed in human peripheral blood lymphocytes using flow cytometry. There was a distinct population of GrK expressing CD8+ T cells with a CD27+/CD28+/CCR5high/CCR7/perforin–/low/IFN-γ+ memory-like phenotype, while all CD56bright NK cells were also positive for GrK. In addition, GrK was also expressed in subpopulations of CD56+ T cells, CD4+ T cells, and TCRγδ+ T cells. In contrast, GrB was primarily expressed in CD56dim NK cells and differentiated memory CD8+ T cells with the CD27–/low/CD28–/low/CCR5–/low/CCR7/CD11b+/perforinhigh phenotype. Only few CD8+ T cells expressed both GrB and GrK. GrA was found to be co-expressed in all GrB- and GrK-expressing T cells. Our findings suggest that granzyme expression during the differentiation process of memory CD8+ T cells might be as follows: GrA+/GrB/GrK+ → GrA+/GrB+/GrK+ → GrA+/GrB+/GrK.

Abbreviations:
GrA/B/K:

granzyme A/B/K

MFI:

mean fluorescence intensity

Introduction

In both adaptive and innate immunity killer lymphocytes like cytotoxic T cells and NK cells, respectively, can induce rapid apoptosis of virus-infected or transformed cells 1. Killer lymphocytes utilize two pathways to kill target cells, both of which require cell-cell contact. The first pathway is induced after cross-linking of death receptors such as Fas on the target cell by their respective ligands expressed on the killer cell 2. The second pathway involves exocytosis of cytotoxic proteins such as perforin and granzymes from secretory granules stored in the effector cell cytoplasm 3. Yet, the precise mechanisms of granule-mediated apoptosis have not been elucidated. There is evidence that granzymes can enter target cells by endocytosis 46, whereas perforin permits granzyme access to their substrates in the target cell by destabilizing endosomes 7, 8.

In humans, five granzymes with differing substrate specificity have been identified 3, 9. Human granzyme A (GrA) and granzyme K (GrK) have trypsin-like activity and cleave after basic residues arginine and lysine, granzyme B (GrB) has been shown to cleave after aspartic acid residues, granzyme H cleaves after hydrophobic residues like phenylalanine, and granzyme M cleaves after methionine or leucine, respectively 9, 10. GrA and GrB have been well characterized. Their crystal structure has been reported 1114 and several intracellular target proteins whose cleavage leads to caspase-dependent (GrB) or independent (GrB, GrA) target cell death have been identified 15, 16.

In contrast to GrA and GrB, there are few data on the cellular distribution and function of GrK, which like GrA is also a trypsin-like granzyme. Human GrK was first discovered and purified from lymphokine-activated killer cells in which it was less abundant than granzymes A or B 17. Subsequently, a similar granzyme was isolated from the rat NK tumor cell line RNK-16 18, which could induce caspase-independent target cell apoptosis with disruption of the mitochondrial potential and DNA fragmentation occurring with similar time kinetics compared to GrA-induced apoptosis 18, 19. The crystal structure of the GrK zymogen has recently been reported 20 and high mRNA levels of GrK were detected in peripheral blood lymphocytes (PBL), lung, spleen, and thymus 21. Furthermore, an inter-α-trypsin inhibitor complex was identified as a potent GrK inhibitor in human plasma 10.

In this study, GrK expression in distinct subsets of PBL was compared to that of GrA and GrB at the single-cell level using a recently raised antibody against human GrK (Bade et al., submitted). CD56bright NK cells and subsets of memory CD8+ T cells and CD56+ T cells were identified as the main sources of GrK. Furthermore, we show that memory CD8+ T cells can be separated into distinct subsets according to their GrA, GrB and GrK expression representing different states of CD8+ T cell maturation in peripheral blood.

Results

Expression of GrA, GrB and GrK in PBL

To detect expression of human GrA, GrB and GrK in PBL at the single-cell level, four-color flow cytometry was performed. First PBMC were stained with antibodies against cell surface molecules, and then fixed, permeabilized, and stained with granzyme-specific antibodies. Gating for CD3+ lymphocytes revealed expression of all granzymes in CD8+ and TCRγδ+ T cell subsets (Fig. 1A). The percentage of GrA+ cells in these two T cell populations was higher compared to the percentage of GrB- and GrK-expressing cells (Fig. 1B). CD4+ T cells from all donors showed a distinct but small population of GrA+ and GrK+ cells, while only a few donors had a significant expression of GrB in CD4+ T lymphocytes. Notably, one donor exhibited high levels of GrA+ and GrB+ CD4+ T cells as indicated in Fig. 1B. In CD3 lymphocytes there was a high expression of GrA and GrB but a low expression of GrK in CD56dim and CD16+ NK cells (Fig. 1A). In contrast, the small population of CD56bright NK cells showed a high expression of GrK, but only an intermediate GrA, and low GrB expression (Fig. 1A). In CD19+ B cells expression of GrA, GrB, and GrK was not detectable (Fig. 1A).

Figure 1.

Expression of GrK in comparison with GrA and GrB in human PBL. (A) Incubation of PBL with a fluorescein- and PE-conjugated isotype control antibody mix containing IgG1, IgG2a, and IgG2b results in positive staining below 1% in all donors. Dot-plots showing granzyme expression in CD4+, CD8+, and TCRγδ+ T cells were gated on CD3+ lymphocytes, while dot-plots showing co-expression of granzymes with surface molecules CD19, CD16, and CD56 were gated on CD3 lymphocytes. (B) Boxplots show granzyme expression in human peripheral blood T cell subsets. The median values obtained by analyzing 7 (CD4+, TCRγδ+ T cells) and 12 (CD8+ T cells) different donors are given.

Differential expression of granzymes in NKT cells defines three subsets

As has previously been shown CD56+ NKT cells exhibit high amounts of GrA and GrB 22. To verify the expression patterns of GrK in comparison to GrA and GrB in NKT cells CD3+ cells were gated and the co-expression of granzymes with the NK cell markers CD56 and CD16, respectively was analyzed. Almost all CD56+ as well as CD16+ T cells expressed GrA in all donors tested. GrB and GrK expression in CD56+ T lymphocytes differed interindividually among donors, while CD16+ T cells were uniformly positive for GrB but virtually negative for GrK (Fig. 2A). There was a significant negative correlation between the percentage of GrB+/CD56+ T cells and GrK+/CD56+ T cells (p=0.002) (Fig. 2B), suggesting alternative expression of either granzyme. To substantiate this hypothesis, co-staining of GrB and GrK in CD56+ T cells was performed, which revealed distinct populations of CD56+ T cells, of which one had a GrB+/GrK phenotype, while the other was GrB/GrK+. Only a small subset of CD56+ T cells expressed both GrB and GrK (Fig. 2C). Most donors had more CD56+ T lymphocytes with a GrB+/GrK phenotype, while only a few had more CD56+ T cells with a GrB/GrK+ phenotype. Representative dot-plots from one donor of each type are given in Fig. 2C.

Figure 2.

Expression of GrA, GrB, and GrK in NKT cells from human peripheral blood. (A) Dot-plots were gated on CD3+ lymphocytes and show co-expression of granzymes with NK cell markers CD56 and CD16, respectively. One representative experiment of eight is shown. (B) Percentages of GrK+CD56+ and GrB+CD56+ T cells in donors are negatively correlated using the Spearman rank sum correlation coefficient. (C) Dot-plots are gated on CD3+CD56+ lymphocytes demonstrating expression of GrB and GrK in NKT cells from two different donors.

Co-expression of GrA, GrB, GrK and perforin in CD8+ T cells

Because GrK was shown to be mainly expressed in CD8+ T cells (Fig. 1) we chose this T cell subpopulation to analyze co-expression of the respective granzymes. All GrB and all GrK expressing CD8+ T cells were shown to co-express GrA, while only a small subset of CD8+ T cells expressed both GrB and GrK (Fig. 3A). Because many CD56+ T lymphocytes are also CD8+, this corroborates the above-mentioned findings in CD56+ T cells.

Figure 3.

Co-expression of GrA, GrB, GrK, and perforin in human peripheral blood CD8+ T cells. (A, B) Dot-plots were gated on CD3+CD8+ lymphocytes and one representative experiment of five is shown. (C) Perforin expression in CD3+CD8+ T cells expressing GrB is much higher than in cells expressing GrK, as indicated by MFI values.

The cytotoxic marker protein perforin, which is co-expressed with granzymes, plays an important role in the induction of GrA- and GrB-mediated apoptosis of target cells 1, 2. Interestingly, co-staining of CD8+ T cells with anti-GrK and anti-perforin revealed that the respective proteins were co-expressed in only 60 ± 9.5% of the GrK+ cells, whereas 79.2 ± 7.2% of GrA+ and 93.4 ± 2.9 GrB+ CD8+ T cells stained positive for perforin (each n=5) (Fig. 3B). Furthermore, perforin expression intensity in GrK expressing CD8+ T cells was lower than in GrB-expressing CD8+ T lymphocytes (Fig. 3C).

Cytokine production profiles of granzyme expressing CD8+ T cells

Cytokine production by granzyme-expressing CD8+ T cells was analyzed in PBMC, which were stimulated for 4 h with PMA and Ca – ionophore in the presence of brefeldin A. Cells were then stained for surface markers, fixed, permeabilized, and stained with antibodies against cytokines and granzymes.

As shown in Fig. 4 and Table 1, the majority of CD8+ T cells expressing GrA, GrB and GrK were also positive for IFN-γ expression, while there was no significant co-expression of IL-4 and granzymes. Low levels of IL-2 expression were observed in GrA- and GrK-expressing CD8+ T cells but not in GrB+ cells.

Figure 4.

Co-expression of cytokines and GrA, GrB, and GrK in human peripheral blood CD8+ T cells. PBMC were isolated and incubated in the presence of PMA, Ca – ionophore, and brefeldin A for 4 h as described in Materials and methods. Dot-plots were gated on CD3+CD8+ lymphocytes. One representative experiment of three is shown.

Table 1. Co-expression of surface molecules and cytokines by granzyme-expressing CD8+ T cells in human peripheral blooda)
  nCD8+CD3+GrA+CD8+CD3+GrB+CD8+CD3+GrK+
  1. a) Data indicate the mean value ± standard deviation of n different donors. bd, below detection limit.

CD27(%)1065.7 ± 17.454.2 ± 19.092.3 ± 4.1
CD28(%)1045.8 ± 18.220.3 ± 10.788.0 ± 4.9
CD45RA(%)1050.1 ± 16.460.7 ± 13.130.8 ± 12.9
CD45R0(%)1056.7 ± 16.848.4 ± 12.878.5 ± 13.8
CD62L(%)547.5 ± 19.444.0 ± 21.953.8 ± 11.6
Activation marker     
CD11b(%)1053.3 ± 13.669.8 ± 10.625.4 ± 6.3
CD25(%)52.2 ± 0.4bd3.3 ± 1.6
HLA-DR(%)57.6 ± 5.98.4 ± 5.813.1 ± 4.1
Chemokine receptors     
CCR5(%)570.5 ± 14.259.9 ± 6.888.9 ± 4.8
CCR7(%)5bdbdbd
NK cell marker     
CD16(%)811.8 ± 8.317.1 ± 10.6bd
CD56(%)836.3 ± 8.828.6 ± 11.831.4 ± 10.5
Cytokines     
IL-2(%)33.1 ± 1.0bd6.9 ± 1.2
IL-4(%)3bdbdbd
IFN-γ(%)376.9 ± 14.674.9 ± 19.583.1 ± 6.7

GrK expression in CD8+ T cells is highly restricted to a subset with a CD27+/CD28+/CCR5high/CCR7 phenotype

CD8+ T cells in peripheral blood of healthy individuals can be divided into naive cells, and different differentiation forms of memory cells using expression patterns of surface molecules such as CD27, CD28, CD45RA, CD45R0, CD62L, CCR5, and CCR7 2328. To further characterize granzyme expression in different states of CD8+ T cell differentiation, the co-expression of these surface molecules with GrA, GrB and GrK was analyzed (Fig. 5, Table 1).

Figure 5.

Expression of GrA, GrB, and GrK in human peripheral blood CD8+ T cell subsets. (A) Dot-plots were gated on CD3+CD8+ lymphocytes. All plots are from one representative donor except plots showing co-expression of granzymes with CD25 and HLA-DR. (B) Analysis of CD27, CD28, and CCR5 expression on GrB and GrK expressing CD8+ T cells from five different donors using MFI values.

The chemokine receptor CCR7, which is expressed only on naive and central memory CD8+ T cells 25, was not detectable on granzyme-expressing CD8+ T cells, while CD62L was expressed on intermediate percentages of the respective cells (Fig. 5A, Table 1). Loss of CD27 and CD28 expression on CD8+ T cells has been associated with the differentiation of memory CD8+ T cells to an effector-like type 23, 24, 27, 28. In our study almost all GrK+ CD8+ T cells also expressed CD27 as well as CD28, which was significantly lower on GrA+ and GrB+ CD8+ T cells (Fig. 5A, Table 1). GrB+ CD8+ T cells, which were positive for CD27 and/or CD28, had a lower expression of these surface molecules compared to GrK+ CD8+ T cells (Fig. 5B). Furthermore, most CD27 and CD28 CD8+ T cells stained positive for GrA and GrB but negative for GrK (Fig. 5A, Table 2).

Table 2. Relative expression of granzymes by human PBL subpopulationsa)
PBL subpopulationGrAGrBGrK
  1. a) –, not detected; +/–, <10%; +, 10–50%; ++, 50–75%; +++, >75%, var, 0–100%. The symbols given, represent percentages found in the respective cell populations of most donors. The high percentages of GrA- and GrB-expressing CD4+ T cells in one donor are indicated by (+) in the respective row.

B cells (CD19+CD3)
NK cells (CD16+CD3)HighHighLow
NK cells (CD56dimCD3)HighHighLow
NK cells (CD56brightCD3)IntermediateLow/–High
NK T cells (CD16+CD3+)++++++
NK T cells (CD56+CD3+)+++varvar
T helper cells (CD4+CD3+)+/– (+)+/– (+)+/–
CD8 T cells (CD8+CD3+)++/+++
TCRγδ cells (TCRγδ+CD3+)++++++
CD8 T cell subpopulations (CD8+CD3+)   
CD11b++++++++
CD16+++++++
CD25+++
CD27++++++–/+
CD28++++++–/+
CD45RA+++–/+
CD45R0+++++++
CD56++++varvar
CD62L++++++
CCR5+++++++
CCR7+++++
CCR7+
HLA-DR++++++++
Perforin+++++++
IL-2+++
IFN-γ+++++++

The chemokine receptor CCR5 has not been detected on naive CD8+ T cells but it is expressed on memory T cells and down-regulated during differentiation to an effector-like phenotype of memory CD8+ T cells 24, 27, 28. In our investigation, almost all GrK+ CD8+ T cells expressed high levels of CCR5, while less CCR5 expression was observed on GrA+ and GrB+ CD8+ T cells (Fig. 5A, Table 1). In addition, mean fluorescence intensity (MFI) values revealed that CCR5 expression intensity on GrK+ CD8+ T cells was higher compared to GrB+ CD8+ T cells (Fig. 5B).

Naive T cells have been shown to express high levels of CD45RA, which shifts to the expression of CD45R0 following T cell activation 29, 30. It has been shown that memory CD8+ T cells, however, can regain CD45RA expression 31. In our study on GrK+ CD8+ T cells, CD45R0 is the predominant CD45R isoform, while more GrB+ CD8+ T cells stained positive for CD45RA (Table 1).

Co-expression of granzymes with activation markers CD11b, CD25, and HLA-DR on CD8+ T cells

Co-expression of the early activation marker CD25 was observed only at low percentages on GrA- and GrK- but not on GrB-expressing CD8+ T cells (Fig. 5A, Table 1). However, almost all CD8+ T cells, which express the late activation marker HLA-DR, were positive for GrA and intermediate percentages of these cells stained positive for GrB and GrK (Fig. 5A, Table 2). CD11b was shown to be primarily expressed on effector-like memory CD8+ T cells 23. While there was a significant (p<0.05) positive correlation between the CD11b+ and GrA+, as well as GrB+ subsets, there was no significant correlation with GrK-expressing cells (data not shown). Approximately 70% of GrB-expressing CD8+ T cells also expressed CD11b, which was present in only about 25% of the GrK-expressing subpopulation (Fig. 5A, Table 1). Furthermore, the CD11b+ subpopulation of CD8+ T cells was highly positive for GrA and GrB, but only about 17% of these cells expressed GrK (Table 2).

Discussion

Here we report the expression of human GrA, GrB and GrK in distinct subsets of cytotoxic lymphocytes, which were characterized using a flow-based assay. GrA, GrB, and GrK expression was detected in T cells, mainly in CD8+, CD56+ and TCRγδ+ subsets, and to a lower extend in CD4+ T cells, corresponding with results found by Bade et al. (Bade, personal communication, and Bade et al., submitted) and Grossman et al. 22. Although there was a high degree of variation of granzyme expression, GrA expression was most pronounced in these subsets. Analysis of CD3 lymphocytes revealed only a low expression of GrK in the CD56dimCD16+ NK cell population, while GrA and GrB were detectable in high concentrations, as has been previously described 22. A high GrK expression was found in the small CD56bright NK cell population, despite the fact that this subpopulation has previously been shown to exhibit only a low cytotoxic potential because of low perforin expression 32. In line, we found only low levels of GrB in CD56bright NK cells.

The divergent expression of GrB and GrK in NK cell populations also finds its reflection in CD56+ NKT cells and CD8+ T cells. In these T cell populations we were able to detect three populations based on granzyme expression, two main populations showing the GrA+/GrB/GrK+ or GrA+/GrB+/GrK phenotype, respectively, and one minor population expressing all three granzymes (Figs. 2, 3).

Our results suggest a differential expression of GrA, GrB, and GrK in different CD8+ T cell subpopulations of possibly different maturation states. CD8+ T cell differentiation in peripheral blood has been defined according to the expression of surface molecules such as CD27, CD28, CD45RA, CD45R0, CD62L, CCR5, and CCR7 2328. CD62L and CCR7 can mediate the transmigration of T cells into the lymph nodes, which appears essential for naive T cells to interact with antigen-presenting cells 3335. Sallusto et al. 25 showed that CCR7 expression is restricted to naive cells and a subset of memory, so-called central memory, T cells. In addition, CD62L expression was present on almost all CCR7+ but only on a limited number of CCR7 T cells in this study. Here we show that granzyme expression appears to be restricted to CCR7 T cells, suggesting that naive and central memory CD8+ T cells do not express GrA, GrB, and GrK (Fig. 5, Table 2). This is underlined by the finding that most granzyme-expressing CD8+ T cells co-express IFN-γ, which could not be detected in naive CD8+ T cells 23, 28. In agreement with the findings of Sallusto et al. 25, CD62L expression was found on intermediate levels on granzyme-expressing CD8+ T cells (Fig. 5, Table 1). Interestingly, almost all CD62L and CCR7 CD8+ T cells expressed GrA but only intermediate percentages expressed GrB and GrK, respectively (Fig. 5, Table 2). This suggests widespread expression of GrA and a more selective regulation of GrB and GrK expression in memory CD8+ T cells. In peripheral blood, Tomiyama et al. 24 described four different subsets of CD8+ memory T cells based on their surface expression of CCR7 and CCR5 (CCR7+/CCR5 → CCR7+/CCR5+ → CCR7/CCR5+ → CCR7/CCR5) with the highest differentiation state in the CCR7/CCR5 phenotype. Several studies have reported that CCR5, as well as the surface molecules CD27 and CD28, are down-regulated during memory CD8+ T cell maturation 23, 24, 2628. Our finding that GrK expression is primarily observed in CCR5high, CD27+, CD28+, CD45R0+ and IFN-γ-expressing CD8+ T cells suggests that GrK expression in CD8+ T cells is highly restricted to a small population of memory CD8+ T cells, and might be lost during differentiation to CD28, CD27, and CD11b+ cells. In contrast, CD8+ T cells which were either CD28, CD27, or CD11b+ were found to be highly positive for GrB. Up-regulation of perforin in CD8+ T cells during differentiation of memory cells to effector-like cells with high ex vivo cytolytic activity has been reported 23, 24, 28. Because only a fraction of GrK+ CD8+ T cells showed low intensity signals for perforin, while virtually all GrB+ CD8+ T cells co-express high levels of perforin, it could be speculated that the small GrB+/GrK+ double-positive population might represent a transitional form of CD8+ T cells from GrB/GrK+ to GrB+/GrK cells. The hypothesis of such a transitional subset is underlined by intermediate expression intensities of CD27, CD28, and CCR5 on some GrB+ CD8+ T cells. These findings in peripheral blood CD8+ T cells are summarized in detail in Fig. 6, where the expression of GrA, GrB, and GrK, respectively, is matched to the expression of the respective surface molecules and perforin. Based on these observations, we speculate that granzyme expression during maturation of memory CD8+ T cells might change from GrA+/GrB/GrK+ to GrA+/GrB+/GrK+ and GrA+/GrB+/GrK.

Figure 6.

Differential expression of GrA, GrB, and GrK in peripheral blood CD8+ T cells of healthy humans – schematic approximation. Different CD8+ T cell subsets were defined by expression of the surface molecules CD27, CD28, CD45RA, CD45R0, CCR5, and CCR7. Width of the bars indicate expression intensity of the respective molecules as determined by MFI values.

In conclusion, in this study we report a restricted expression of GrK in CD56bright NK cells and in a distinct subset of CD8+ memory T cells exhibiting a CD27+/CD28+/CCR5high/CCR7/perforin–/low/IFN-γ+ phenotype, while GrB is primarily expressed in CD56dim NK cells and memory CD8+ T cells with a CD27–/low/CD28–/low/CCR5–/low/CCR7/CD11b+/perforinhigh phenotype. Based on granzyme expression, we speculate that the differentiation of memory CD8+ T cells might include different steps from GrA+/GrB/GrK+ to GrA+/GrB+/GrK+ and GrA+/GrB+/GrK.

Materials and methods

Monoclonal antibodies

Antibodies against TCRγδ (11F2, PE), CCR5 (2D7, PE), CCR7 (3D12, PE), CD3 (SK7, PerCP), CD4 (SK3, PerCP), CD8 (SK1, PerCP), perforin (δG9, PE), and GrA (CB9, PE) were purchased from Becton Dickinson (Heidelberg, Germany). Anti-CD8 (DK25, PE), CD16 (DJ130c, PE), CD19 (HD37, PE), CD25 (ACT-1, PE), CD27 (M-T271, PE), CD28 (CD28.1, PE), CD45R0 (UCHL1, PE), CD56 (C5.9, PE), and HLA-DR (AB3, PE) were obtained from Dako (Hamburg, Germany). From Caltag (Hamburg, Germany) we received the following antibodies against CD3 [S4.1, allophycocyanin (APC)], CD11b (VIM12, PE), CD62L (DREG-56, PE), IL-2 (MQ1–17H12, PE), IL-4 (MP4–25D2, PE), GrB (GB12, APC), and IFN-γ (B27, PE). Anti-GrA (GrA-11, FITC), GrB (GB11, FITC, PE), GrK (GM24C3, FITC), fluorescein- and PE-conjugated IgG1, IgG2a, and IgG2b isotype control antibodies as well as anti-CD45RA (F8–11–13, PE) were from ImmunoTools (Friesoythe, Germany).

Isolation of PBMC

PBMC were isolated from venous blood of healthy donors on a Biocoll-gradient with a density of 1.077 g/L (Biochrom, Berlin, Germany) as previously described 36. Isolated PBMC were washed twice and resuspended in PBS supplemented with 2% heat-inactivated FCS at a concentration of 5 × 106/mL.

Intracellular granzyme staining

After incubation with specific antibodies against surface molecules for 20 min at room temperature isolated PBMC were fixed in paraformaldehyde (4% in PBS for 5 min on ice), washed twice with PBS, and permeabilized with saponin (0.1% in PBS + 2% FCS for 10 min on ice). Subsequently, cells were incubated with granzyme-specific antibodies for 20 min at room temperature, washed twice with saponin (0.1% in PBS + 2% FCS), and then analyzed on a FACSCalibur using CellQuest Pro software (Becton Dickinson, Heidelberg, Germany).

Cytokine expression profiles

To analyze co-expression of granzymes and cytokines, PBMC were isolated as described and cultured at 2 × 106 cells/mL in RPMI 1640 medium (supplemented with L-glutamine and 10% FCS; Gibco Invitrogen, Karlsruhe, Germany) for 4 h at 37°C in the presence of PMA (20 ng/mL; Sigma, Taufkirchen, Germany), Ca – ionophore A23187 (2 µg/mL; Sigma), and brefeldin A (10 µg/mL; Sigma). Cells were then washed twice (PBS + 2% FCS), incubated with antibodies against surface molecules, fixed, permeabilized and incubated with granzyme- and cytokine-specific antibodies as described. Finally, cells were washed (0.1% saponin in PBS + 2% FCS) and analyzed by flow cytometry.

Footnotes

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Ancillary