The genes for perforin, granzymes A–C and IFN-{gamma} are differentially expressed in single CD8+ T cells during primary activation

Anne Kelso1,2,3, Elaine O. Costelloe1,4, Barbara J. Johnson1,2,5, Penny Groves1, Kathy Buttigieg1 and David R. Fitzpatrick1,6

1 The Queensland Institute of Medical Research, Brisbane, Queensland 4029, Australia 2 Cooperative Research Centre for Vaccine Technology, The Queensland Institute of Medical Research, Brisbane, Queensland 4029, Australia 3 Joint Transplantation Biology Program, The Queensland Institute of Medical Research and The University of Queensland, Brisbane, Queensland 4029, Australia 4 Present address: Institute for Molecular Bioscience, The University of Queensland, St Lucia,Queensland 4072, Australia 5 Present address: Department of Medicine, The University of Queensland, Mater Adult Hospital, South Brisbane, Queensland 4101, Australia 6 Present address: Immunex Corp., 51 University Street, Seattle, WA 98101, USA

Correspondence to: A. Kelso, Cooperative Research Centre for Vaccine Technology, The Queensland Institute of Medical Research, Post Office Royal Brisbane Hospital, Queensland 4029, Australia. E-mail: anneK{at}qimr.edu.au
Transmitting editor: D. Tarlinton


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Here we show that the genes for perforin, the three major T cell granzymes (A–C) and IFN-{gamma} are differentially expressed during primary activation of naive CD8+ T cells, kinetically and at the single-cell level. When CD44lowCD62LhighCD8+ lymph node T cells were activated with IL-2 and immobilized antibodies to CD3, CD8 and CD11a, expression of perforin, granzyme B and IFN-{gamma} mRNAs was induced by day 2, and increased in parallel with perforin-dependent cytolytic activity. Granzyme C and A transcripts were not detected until 1 and 3 days later respectively. Single-cell PCR showed that expression frequencies rose in parallel with total levels of each mRNA, but that individual cells expressed diverse combinations of perforin, granzyme A–C and IFN-{gamma} mRNAs. These expression patterns indicated that the delayed expression of granzymes A and C was not due to late activation of distinct cell subpopulations. Statistical analysis of the data suggested that each gene was differentially regulated at the single-cell level. Individual naive CD8+ T cells gave rise over 7 days to clones that expressed all five products at the clonal level, but also expressed diverse combinations at the single-cell level. We conclude that, during primary activation, CD8+ T cells progressively acquired the ability to express most or all of these genes, and that the variable expression patterns observed among single cells within clones and populations reflected transient rather than heritable differences in expression profile.

Keywords: cytotoxic T lymphocyte, IFN-{gamma}, single-cell analysis


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Exocytosis of perforin-containing granules is believed to be the major cytotoxic pathway by which activated CD8+ T cells destroy virus-infected and tumor cells in vitro and in vivo (13). TCR-dependent interaction with target cells triggers the directional release of cytotoxic T lymphocyte (CTL) granule contents which are then endocytosed by the target cell and induce its death by apoptosis (4). The pore-forming protein perforin plays an essential role in the lytic process, both by insertion into the target cell membrane, and, more importantly, by enabling delivery of other mediators from the endosome to the cytosol and nucleus (5,6).

Among these mediators are granule-associated proteases or granzymes, of which eight have been identified in the mouse (4). The best characterized is granzyme B, which acts through caspase-dependent and -independent pathways to induce rapid DNA fragmentation and is the major protease responsible for target cell apoptosis (4,7). Granzyme A induces single-strand DNA breaks, enhances DNA accessibility to exogenous endonucleases and enables slow cytotoxicity in the absence of the granzyme B/C gene cluster (810). This granzyme also displays other activities, such as activation of IL-1ß (11). Little is known about the activities and functions of the other granzymes, either in the immune response or in other physiological processes, although antisense oligonucleotide inhibition of granzyme C has been reported to block CTL activity of a T cell clone (12). Data from mice in which one or more of the perforin, granzyme A and linked granzyme B/C genes has been inactivated suggest that their functions in immunity to pathogens and tumors and in apoptosis are sometimes distinct from their roles in target cell lysis in vitro (1315). For example, granzyme A-deficient mice display normal CTL activity, and eradicate several intracellular pathogens and syngeneic tumors normally, but are unable to recover from ectromelia virus infection (16,17). A role for perforin has been demonstrated in T cell homeostasis (1820), raising the possibility that other CTL granule components may also serve roles apart from target cell killing.

Previous studies by several groups have shown that, while perforin and the granzymes can be expressed simultaneously by T cell clones and populations activated in vitro, their kinetics and relative levels of expression can vary between clones, and between activation systems in vitro and in vivo (2124). These studies have suggested that the genes encoding perforin and the various granzymes may be differentially regulated in activated CD8+ T cells, perhaps reflecting previously unrecognized specialization of CTL function (24). Such specialization might in turn reflect the different roles of these granule components in various immune and other processes. No studies have been reported, however, investigating whether such specialization could be identified among single CTL.

To address this issue, we have exploited a system of high-efficiency activation of naive CD8+ T cells to become cytolytic IFN-{gamma}-secreting effector cells, then used sensitive PCR-based methods to analyze expression of perforin, the three most abundant granzymes (A–C) and IFN-{gamma} in single T cells, clones and populations over the first week of activation. This is the first reported comparison of the expression of perforin and multiple granzymes in single cells, and shows that these genes are differentially expressed, in both their kinetics of induction and their co-expression patterns, during CTL development in the primary response.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Lymphocyte preparation
Female specific pathogen-free C57BL/6 mice (6–8 weeks old) were purchased from the Animal Resources Centre (Perth, Western Australia, Australia) and held at The Queensland Institute of Medical Research. Single-cell suspensions were isolated from pooled brachial, axillary, lumbar and inguinal lymph nodes (LN) by passage through stainless steel mesh and centrifugation over Ficoll-Paque (Pharmacia, Uppsala, Sweden). Cells were then incubated on ice in balanced salt solution with 5% heat-inactivated FCS and the following rat anti-mouse mAb at predetermined optimal concentrations: phycoerythrin-conjugated anti-CD8{alpha} antibody (53-6.7; Becton Dickinson, Sunnyvale, CA), biotinylated anti-CD44 antibody (IM7.8; PharMingen, San Diego, CA) followed by TriColor-conjugated streptavidin (Caltag, San Francisco, CA), and anti-CD62L antibody (Mel-14; hybridoma SN) followed by FITC-conjugated goat anti-rat Ig (Vector, Burlingame, CA). Propidium iodide was added at 1 µg/ml immediately before cell sorting to allow exclusion of dead or damaged cells. Cell suspensions were separated using a FACS Vantage (Becton Dickinson) flow cytometer with Lysys II or CellQuest (Becton Dickinson) software after gating on viable lymphocytes based on forward/side scatter and exclusion of propidium iodide-stained cells. CD8+CD44lowCD62Lhigh cells were collected by sorting the positive CD8 (26 ± 3% of all cells) and CD62L (94 ± 2% of CD8+ cells) peaks and the lowest 15–30% of the CD44 distribution.

Lymphocyte activation and cloning
Tissue culture wells were first incubated with Protein G-purified mAb to CD3{epsilon} (145-2C11; 10 µg/ml), CD8{alpha} (53.6.7; 10 µg/ml) and CD11a (I21.7/7; 5 µg/ml) in PBS for at least 4 h at 37°C, then washed 3 times to remove unbound antibody. For bulk cultures, purified CD8+CD44lowCD62Lhigh cells were cultured at concentrations from 2 x 104 to 3 x 105 cells/ml (depending on the duration of culture) in mAb-coated wells of 24-well tissue culture plates (Falcon; Becton Dickinson) containing 2 ml supplemented DMEM (25) with 10% heat-inactivated FCS (DMEM-FCS) and 600 IU/ml human recombinant IL-2 (rIL-2; Cetus, Emeryville, CA). For clonal cultures, the automated cell deposition unit attached to the FACS Vantage was used to deposit single CD8+CD44lowCD62Lhigh cells directly into mAb-coated wells of 96-well round-bottom microtiter plates (Falcon; Becton Dickinson) containing 200 µl DMEM-FCS and 600 IU/ml rIL-2. All cultures were incubated at 37°C in a humidified atmosphere of 5% CO2 in air.

51Cr-release assay
Target cells of the H-2d mastocytoma P815 were incubated at 37°C in 5% CO2 for 90 min with 100 µCi of Na51CrO4 (Amersham, NSW, Australia) then washed 3 times. Labeled cells were mixed with soluble anti-CD3 mAb (145–2C11) to give 3 µg/ml final concentration and then added at 5 x 103 target cells/well to round-bottom 96-well plates containing serially diluted effector cells (from 0.01:1 to 100:1 E:T ratio) in triplicate in a final volume of 200 µl DMEM with 5% FCS. The assay was incubated for 4 h at 37°C, 5% CO2, after which 50 µl aliquots of supernatant fluid were transferred to 96-well Lumaplates (Packard, Meriden, CT). The plates were dried overnight and {gamma}-emission was measured using a Topcount microplate scintillation counter (Packard). Percent specific lysis was calculated as: 100 x [(mean sample release – mean spontaneous release)/(mean maximal release – mean spontaneous release)]; maximal release was determined by target cell lysis with Triton X-100. Lytic units were determined from dose–response curves and defined as the reciprocal of the number of effector cells required to achieve 25% specific lysis.

Quantitative competitive PCR (QC-PCR)
RNA was isolated from FACS-purified samples of 1000 cells by mixing with Total RNA Isolating Reagent (ABgene, Epsom, UK) followed by chloroform extraction, isopropanol precipitation and washing with ethanol according to the manufacturer’s instructions, then reverse transcribed using AMV reverse transcriptase (Promega, Madison, WI) (26). mRNA was measured by QC-PCR, based on a previously published protocol in which sample cDNAs were co-amplified with serially diluted competitor cDNA containing small deletions (26). Primers and probes were designed using the OLIGO program (Bresatec, Thebarton, South Australia, Australia). Competitor plasmids were constructed as follows. For perforin, a 656 bp fragment was amplified using the primer combination MPERFEX1 and MPERFEX2 (Table 1) and ligated into pGEM5Zf+ (Promega, Madison, WI). The restriction enzymes AvrII (New England Biolabs, Beverly, MA) and BsrG1 (New England Biolabs) were then used to delete a 123-bp fragment, and the plasmid was re-ligated to form the competitor plasmid. For granzyme A, the primers MGRANAEX1 and MGRANAEX2 amplified a 582-bp fragment that was ligated into pCR2.1 (Invitrogen, Carlsbad, CA). A 174-bp deletion was made using HpaI (New England Biolabs) to create a restriction site. For granzyme B, the primer combination MGRANBEX1 and MGRANBEX2 amplified a 468-bp band that was ligated into pGEM5Zf+ and cut with Bsu36I (New England Biolabs) and BsmI (New England Biolabs) to delete a 150-bp fragment. MGRANCEX1 and MGRANCEX2 amplified a 393-bp granzyme C fragment which was ligated into pGEM5Zf+ and cut with HpaI (New England Biolabs) to create a restriction site and delete a 94-bp fragment. The CD3{epsilon} and IFN-{gamma} primers and competitor plasmids have been described elsewhere (26,27).


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Table 1. PCR and hybridization primer sequences
 
QC-PCR assays were performed by amplifying mixtures of a fixed amount of test cDNA with 5-fold serial dilutions of competitor cDNA ranging over 7 orders of magnitude, using internal primer pairs shown in Table 1, and PCR reaction components and cycling parameters as described (26). Following agarose electrophoresis to verify product size and estimate equivalence points, amplified DNA was quantified by ELISA, using hybridization to internal FITC-labeled probes specific for either the exogenous competitor products or the endogenous DNA products (26). The equivalence point of the products was then expressed as the number of mRNA molecules detected in the sample as a ratio to the number of CD3{epsilon} mRNA molecules detected in the same sample (CD3{epsilon} units). Optimization experiments established that sensitivity of detection of endogenous and competitor cDNA was similar. Sensitivity of the QC-PCR assay for perforin, granzymes A–C, and IFN-{gamma} was 4–20 input competitor molecules by agarose gel electrophoresis and 1–4 molecules by ELISA.

Single-cell PCR
Single cells harvested from bulk cultures were gated for lymphocyte size and low 90° scatter and placed into 96-well plates containing NP-40 lysis buffer (28) using the automated cell deposition unit attached to the FACS Vantage. Single cells from T cell clones were transferred into 96-well plates containing NP-40 lysis buffer by manual micromanipulation using a drawn-out capillary pipette. Plates were snap-frozen on dry ice and stored at –70°C until cDNA synthesis. Reverse transcription and two rounds of nested PCR amplification of 30 and 40 cycles respectively, were performed using external and internal primer pairs as described (Table 1) (27). CD3{epsilon}, perforin, granzymes A–C and IFN-{gamma} were amplified for each reverse-transcribed DNA sample, as was a series of cloned plasmid cDNAs as PCR controls. Because perforin mRNA was detected at low levels per cell (<=10 molecules per positive cell by QC-PCR assay of single cells), cDNA synthesis of single-cell RNA was performed within 24 h of NP-40 lysis and snap-freezing. Multiple two-round PCR assays on single cells showed that reproducibility was high and false negatives rare when 20% of the total cDNA sample was amplified for each of CD3{epsilon} and perforin. Granzyme A–C and IFN-{gamma} mRNAs in positive single cells were more abundant, and reproducible results were obtained when 10% of the sample was used to amplify each of these products. PCR products were then agarose electrophoresed and visualized by ethidium bromide DNA staining. Each PCR run included a set of 10-fold serially diluted cloned cDNAs for CD3{epsilon}, perforin, granzymes A–C and IFN-{gamma}, and a minimum of six negative water control samples. The sensitivity of each reaction was typically <=10–16 g cDNA and information was recorded only where there was no contamination detected. Data obtained for cells at day 5 in Experiment A and at day 7 in Experiment C (see Table 2) were excluded from further analysis because of RNA degradation and DNA contamination respectively.


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Table 2. Gene expression frequencies among single activated CD8+ T cells
 

    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Differential kinetics of perforin, granzyme and IFN-{gamma} expression during primary activation of CD8+ T cells
We have previously shown that culture with immobilized antibodies to CD3, CD8 and CD11a (LFA-1{alpha}) in the presence of IL-2 supports 80–90% of single naive CD8+ T cells to form clones over 7 days (27,29). Most of these clones secrete IFN-{gamma} without IL-4 in response to TCR cross-linking and lyse anti-CD3 antibody-coated target cells in a perforin-dependent manner in 51Cr-release assays (30). To examine the relationship between development of cytolytic activity, and the onset of expression of perforin, granzymes and IFN-{gamma} mRNAs in newly activated CD8+ T cells, bulk cultures were established with CD8+ cells of naive phenotype (CD44lowCD62Lhigh) purified from LN of normal CD57BL/6 mice and analyzed daily from day 2 to 8. As shown in Fig. 1, cytolytic activity was measurable against anti-CD3 mAb-coated 51Cr-labeled target cells from day 2 and was maximal from day 5. Other experiments showed that lysis was low (30- to 100-fold fewer lytic units per 106 cells) in the absence of bridging anti-CD3 antibody or when effector cells were pre-incubated with 100 ng/ml concanamycin A (30), a vacuolar type H+-ATPase inhibitor which blocks perforin-mediated cytotoxicity (data not shown) (31,32). Analysis of perforin, granzyme and IFN-{gamma} expression by QC-PCR revealed different kinetics of expression of each of these mRNA species. Granzyme B and IFN-{gamma} were strongly induced by day 2 and maximal from about day 5, mirroring the rise in cytolytic activity. Although final levels of perforin mRNA were significantly lower, this product was also induced by day 2. The rise in expression of granzymes C and A lagged behind the other mRNA species by 1–3 days and ultimately peaked at day 7. Sensitivity of the competitive RT-PCR assays used for these assays was similar for all three granzyme cDNAs (see Methods).



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Fig. 1. Time course of perforin, granzyme and IFN-{gamma} expression in newly activated CD8+ T cells. CD8+CD44lowCD62Lhigh lymph node cells were cultured with rIL-2 and immobilized mAb to CD3, CD8 and CD11{alpha}. At the indicated times, cells were harvested and assayed for cytolytic activity against anti-CD3 mAb-coated 51Cr-labeled P815 target cells (histograms; mean and SD of triplicate samples). Samples of 1000 cells from the same cultures were assayed for the indicated mRNAs by QC-PCR (lines; geometric mean of duplicate samples). Axis breaks indicate the thresholds of detection of the assays. Similar kinetics and levels of expression were obtained in another experiment.

 
Differential expression of perforin, granzymes and IFN-{gamma} among single CD8+ T cells
The observed lag in the onset of granzyme A and C expression raised the possibility that these mediators were expressed by a distinct subpopulation(s) of cells whose activation or differentiation was delayed compared with cells expressing perforin and granzyme B. We therefore adapted PCR-based techniques previously developed for single-cell analysis of cytokine expression (3335) to detect perforin and granzyme transcripts in single activated CD8+ T cells. CD8+ T cells of naive phenotype were activated in bulk cultures as described above, and sampled at various times for analysis of single cells in two rounds of RT-PCR using nested primers for CD3{epsilon}, perforin, granzymes A–C and IFN-{gamma}. Of 480 cells assayed in total in five independent experiments, 415 (86.5%) yielded a PCR product for CD3{epsilon}. Figure 2 summarizes the patterns of expression obtained for those 415 cells. A high level of reproducibility in expression frequencies was observed between experiments performed at the same time point (Table 2). Frequencies of expression of each gene rose over time with similar kinetics to the rise in average expression levels shown in Fig. 1, suggesting that increasing frequency was a major determinant of these rises. Granzyme B was expressed by most cells from as early as day 3, whereas perforin was maximal by day 4 and granzyme C at day 7. As in Fig. 1, granzyme A expression showed the longest lag. Both granzymes A and C were largely induced within the population of granzyme B-expressing cells, indicating that their delayed expression was not due to late activation of a distinct subset. Frequencies of IFN-{gamma}-expressing cells ranged between ~20 and 40% at all time points. Analysis of the data in Fig. 2 showed that the proportion of cells expressing multiple mRNA species of the five tested here increased progressively with time (Fig. 3). Whereas 92% of cells expressed none, one or two of the five products at day 3, 74% expressed three or more products at day 7.



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Fig. 2. Patterns of perforin, granzyme and IFN-{gamma} expression among single activated CD8+ T cells. CD8+CD44lowCD62Lhigh lymph node cells were cultured as described in Fig. 1. At the indicated times, cells were harvested and assayed individually for perforin (Per), granzymes A–C (GrA–C), IFN-{gamma} and CD3{epsilon} mRNAs by nested RT-PCR. The figure shows data obtained for all CD3{epsilon}+ samples assayed in five experiments (see Table 2). Each row of five boxes represents a cellular phenotype, with detection of a PCR product indicated by shading of the corresponding box. The number of samples with each phenotype is shown at the right and the frequency of expression of each product is shown below the panels.

 


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Fig. 3. Distribution of number of mRNA species detected per cell after various periods of activation. The data in Fig. 2 were used to determine the number of CD3{epsilon}+ cells expressing 0–5 of the PCR products for perforin, granzymes A–C and IFN-{gamma}.

 
Substantial heterogeneity was seen in the patterns of co-expression of the five genes among individual cells at all time points. Despite the progressive rise in individual expression frequencies, only a minority of cells at any time point expressed perforin and all three granzymes, and fewer still co-expressed all these genes with IFN-{gamma}. Nevertheless, the frequency of perforin+ granzyme B+ cells rose from 11% at day 3 to 77% at day 7, and the frequency of perforin+ granzyme B+ IFN-{gamma}+ cells rose from 6 to 31% over the same period. As we have previously observed for cytokine gene expression at the single-cell level (34,35), there were no absolute associations or dissociations between any two products. Analysis of the data by {chi}2 contingency table testing showed that most pairs of products were co-expressed at a frequency close to that expected by chance (Table 3), suggesting that their production was independently regulated. In other instances, co-expression frequencies were significantly higher (or, in one case, lower) than expected by chance at one or more time points. Of these, the most striking were perforin and IFN-{gamma}, which were positively associated at days 3, 5 and 7.


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Table 3. Frequencies of gene co-expression among single activated CD8+ T cells
 
Intraclonal heterogeneity in the expression of perforin, granzymes and IFN-{gamma} by primary T cell clones
The data in Fig. 2 were obtained from single cells selected randomly from bulk cultures. It was therefore unclear whether their heterogeneous expression patterns represented heritable or transient phenotypes. To address this issue, clones were generated from single CD8+ T cells of naive phenotype in cultures of immobilized antibodies and IL-2. Twenty clones of similar size were sampled at days 7 and 10 for analysis of gene expression by nested RT-PCR at the clonal population level. Figure 4A shows that almost all clones expressed all five mRNA species at day 7 and 10. The three clones which did not express perforin or granzyme A on one of the days did so on the other day. Single cells from four clones randomly selected from this panel were also analyzed at day 7 (Fig. 4B). These cells displayed heterogeneous expression patterns, comparable to those presented for cells from bulk cultures in Fig. 2. The observation of variable expression among cells within clones that expressed all five products suggests that this variability reflected transient, rather than heritable, phenotypic differences.



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Fig. 4. Stability and intraclonal heterogeneity in the expression of perforin, granzymes and IFN-{gamma} by primary CD8+ T cell clones. Single CD8+CD44lowCD62Lhigh lymph node cells were cultured with rIL-2 and immobilized mAb to CD3, CD8 and CD11{alpha}. After 7 days, clones were scored microscopically for size. Twenty clones estimated to contain >10,000 cells were randomly selected for analysis. Approximately 1000 cells of each clone was used for nested RT-PCR (A, left panel). Individual cells were harvested from the remaining fraction of four of the clones for single-cell RT-PCR (B). Approximately 10% of the remaining cells of all 20 clones were recultured with rIL-2 and immobilized mAb as above, and then harvested 3 days later (day 10) for re-analysis of 1000 cell samples by nested RT-PCR (A, right panel). Clonal and cellular phenotypes are displayed as described in the legend to Fig. 2. In (B), the number of single cells with each phenotype is shown at the right and the frequency of expression of each product among all cells assayed is shown below the panels.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
This study demonstrates the differential expression of perforin, granzymes A–C and IFN-{gamma} mRNAs in CD8+ T cells at two levels. First, the onset of granzyme C and A expression was consistently delayed compared with cytolytic activity and the expression of perforin, granzyme B and IFN-{gamma} during primary activation. Second, individual T cells differed widely in their patterns of expression of these genes.

The rapid onset of perforin, granzyme B and IFN-{gamma} expression observed in the simple, accessory cell-free activation system used here is consistent with earlier work showing that high-density TCR ligation and either co-stimulation or IL-2 are sufficient to induce CTL activation from naive CD8+ precursors (3638). The differential kinetics of granzyme A and C expression are more surprising. Detection of granzyme C transcripts was previously reported to lag behind lytic activity and granzyme B expression in mitogen- and anti-CD3 antibody-activated splenocyte populations (24). However, since these studies were performed with unfractionated leukocyte populations, it was unclear whether the kinetic differences reflected the activation of different cell lineages or subsets. By contrast, our results were obtained using highly-purified CD8+ T cells of naive phenotype (CD44lowCD62Lhigh) (39) cultured in conditions that enable almost every cell to undergo primary activation and clonal expansion. Tracking of cell divisions in this system has shown that essentially every cell enters its first division within 1–2 days then divides every 6–8 h for several days (P. Groves and A. Kelso, unpublished observations). The differential kinetics we observed are therefore unlikely to be due to heterogeneity in lineage or activation status within the starting population. The expression profiles of single cells analyzed at various time points confirmed that the delayed expression of granzymes A and C was not due to the later activation of a distinct subset of cells. Instead, it appears that individual cells (or their progeny) progressively acquired the ability to express additional granzymes as they underwent clonal expansion. Experiments are in progress to determine whether this is due to an intrinsic differentiation program or additional signals that induce these ‘late’ genes. The presence of distinct regulatory elements in the promoters of some granzyme genes (40,41) supports the latter model.

Single-cell analysis of co-expression patterns of perforin and granzymes has not previously been reported. Several groups have demonstrated the presence of perforin or particular granzyme transcripts or proteins in single cells (42,43), in some cases showing correlations with CTL activity, but these studies have not determined whether different cells expressed distinct combinations of these genes. The single-cell PCR analyses presented here revealed unexpected diversity in perforin/granzyme/IFN-{gamma} profiles of individual cells. No combination of the five genes was invariably co-expressed and, although some pairs were co-expressed more frequently than expected by chance (notably perforin and IFN-{gamma}), statistical analysis suggested that most of these genes were independently regulated. This conclusion does not preclude co-regulation by common signaling pathways or genetic elements but, like the kinetic data discussed above, suggests the involvement of additional gene-specific signals or elements. The importance of genomic position is also unclear. Whereas the genes for perforin and granzyme A have been localized to mouse chromosomes 10 and 13 respectively, granzymes B and C are tightly linked in a cluster with four other granzyme genes on chromosome 14 (44,45). It is not yet known whether this linkage plays a role either in the sequential activation of granzyme B and C expression or in the co-expression of these genes by most cells in 7-day bulk and clonal cultures.

Since at least some perforin and granzyme protein is normally stored in cytoplasmic granules, it is important to note that these stores may be present in the absence of the corresponding mRNA. For example, cells which lacked detectable perforin mRNA at the time of lysis may nevertheless have been functional CTL. We were unable to compare mRNA and protein levels of these molecules in single cells because of the lack of suitable antibodies, although the presence of perforin could be readily demonstrated at the population level by Western blotting (30). The presence of the protein may also not correlate directly with cytolytic activity since perforin and granzymes require proteolytic cleavage by dipeptidyl peptidase I for activity (4648). New tools will therefore be needed to determine whether the diversity we observed in perforin/granzyme mRNA profiles is reflected at the level of protein expression and function among individual cells.

The diversity of single-cell profiles raised the possibility that some of these phenotypes might represent stable subsets, akin to those described for cytokine-producing T cells (49). However, the finding that all of 20 primary clones tested at day 7 expressed all five genes, whereas individual cells from within the clones expressed variable combinations, suggested instead that the diversity we observed was due to transient rather than heritable variations in gene expression. Taken with the rising expression frequencies observed among single cells from bulk cultures, these data are consistent with a model in which T cells progressively acquire the ability to express all of these genes as they clonally expand (e.g. as a result of chromatin remodeling and promoter demethylation), but display transient and largely stochastic differences in mRNA levels at a given time-point. A similar model can describe the development of T cell cytokine profiles (50), even in the presence of type 1 and type 2 polarizing signals that clearly influence the relative levels of expression of each cytokine gene.

While distinct subsets of perforin/granzyme-expressing cells were not detected under the intense T cell activation conditions used in the present study, there is growing evidence that stable differences in expression levels do occur between various T cell responses in vitro and in vivo (2123). Moreover, we have recently found that panels of single CD8+ T cells activated in vivo during influenza virus infection expressed perforin and granzyme C at significantly lower frequencies relative to granzymes A and B than those observed among the polyclonally activated cells studied here (B. J. Johnson, unpublished observations). As presaged by Bleackley et al. (24), the regulation and immunological significance of these different expression patterns between cells, kinetic stages and types of response now become important issues for understanding CTL function in vivo.


    Acknowledgements
 
We thank Grace Chojnowski and Paula Hall for assistance with flow cytometry, and Dr Joe Trapani for valuable advice. This work was supported by the Queensland Cancer Fund, the Cooperative Research Centre for Vaccine Technology and the National Health and Medical Research Council, Australia.


    Abbreviations
 
CTL—cytotoxic T lymphocyte

LN—lymph node

QC-PCR—quantitative competitive PCR


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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