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The Journal of Immunology, 2006, 177: 1981-1987.
Copyright © 2006 by The American Association of Immunologists

Induction of Cytotoxic Granules in Human Memory CD8+ T Cell Subsets Requires Cell Cycle Progression1

Yuru Meng, Helena Harlin, James P. O’Keefe and Thomas F. Gajewski2

Department of Pathology and Department of Medicine, University of Chicago, Chicago, IL 60637


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Memory CD8+ T cell responses are thought to be more effective as a result of both a higher frequency of Ag-specific clones and more rapid execution of effector functions such as granule-mediated lysis. Murine models have indicated that memory CD8+ T cells exhibit constitutive expression of perforin and can lyse targets directly ex vivo. However, the regulated expression of cytotoxic granules in human memory CD8+ T cell subsets has been underexplored. Using intracellular flow cytometry, we observed that only a minor fraction of CD45RA–CD8+ T cells, or of CD8+ T cells reactive to EBV-HLA2 tetramer, expressed intracellular granzyme B (GrB). Induction of GrB-containing cytotoxic granules in both CD45RA+ and CD45RA– cells was achieved by stimulation with anti-CD3/anti-CD28 mAb-coated beads, required at least 3 days, occurred after several rounds of cell division, and required cell cycle progression. The strongest GrB induction was seen in the CCR7+ subpopulations, with poorest proliferation being observed in the CD45RA–CCR7– effector-memory pool. Our results indicate that, as with naive T cells, induction of cytotoxic granules in human Ag-experienced CD8+ T cells requires time and cell division, arguing that the main numerical advantage of a memory T cell pool is a larger frequency of CTL precursors. The fact that granule induction can be achieved through TCR and CD28 ligation has implications for restoring lytic effector function in the context of antitumor immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cytotoxic granules play a major role in T cell-mediated effector function against tumors and viral pathogens (1, 2). Naive (TN)3 CD8+ T cells lack granules, but acquire them upon activation, proliferation, and differentiation into effector cells. The currently accepted linear differentiation model of T cell memory based on murine models suggests that the effector cell pool also gives rise to memory CD8+ T cells (3, 4). In the human system, additional phenotypic analysis has identified at least three subsets of Ag-experienced (CD45RA–) CD8+ T cells that have been termed effector T cells (TE), central memory T cells (TCM), and effector memory T cells (TEM) (5, 6, 7, 8, 9). In addition, it has been proposed that as TN CD8+ T cells undergo differentiation, the costimulatory molecules CD28 and CD27 are progressively down-regulated, concomitant with the acquisition of cytolytic capacity (7, 8).

It recently has been reported that circulating TE and TEM CD8+ T cells can express cytotoxic granules (10, 11). However, a careful analysis of those data reveals that only a subset of those cells actually is positive for granule proteins, suggesting that the majority of circulating Ag-experienced human CD8+ T cells might not be directly cytolytic. In addition, the regulation of granzyme B (GrB) expression is incompletely understood in humans (11, 12, 13).

Recent observations have suggested that tumor-infiltrating lymphocytes (TILs) in patients with advanced melanoma (14, 15), as well as in mouse tumor models (16, 17), lack cytolytic activity, despite expressing surface markers of activation. This functional defect has been associated with low expression of cytotoxic granule proteins, such as perforin and GrB (18), and has suggested that one reason for failed T cell-mediated tumor rejection in vivo is the emergence of activated nonlytic effector/memory T cells. Thus, gaining an increased understanding of the regulation of granule acquisition by Ag-experienced CD8+ T cells may provide insights into how to support reinduction of granules in TILs as well. With these issues in mind, we set out to analyze GrB expression in circulating effector/memory phenotype CD8+ T cell populations in healthy individuals, and to determine the requirements for induction of GrB expression and cytolytic activity in memory compared with TN CD8+ T cells. We found that the majority of memory phenotype CD8+ T cells lacked expression of GrB, that CD3/CD28 stimulation induced up-regulation of GrB and functional cytotoxic granules with comparable kinetics in both naive and Ag-experienced cells, and that acquisition of granules required cell cycle progression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents and mAbs

Anti-CD8 FITC, anti-CD8 PE-Cy7, anti-CCR7 PE-Cy7, anti-GrB FITC, and mouse IgG FITC were purchased from BD Pharmingen. Anti-CD45RA PE-Cy5, anti-CD27 allophycocyanin-Cy7, anti-CD28 allophycocyanin, anti-GrB allophycocyanin, mouse IgG PE-Cy7, mouse IgG allophycocyanin-Cy7, and mouse IgG allophycocyanin were obtained from eBioscience. The PE-conjugated EBV-HLA2 tetramer was from Beckman Coulter.

CD8+ purification and memory CD8+ sorting

Cryopreserved PBMC were purchased from BRT Laboratory and were obtained from healthy individuals. CD8+ T cells were purified by positive selection using MACS CD8 Microbeads (Miltenyi Biotec), according to the manufacturer’s instructions. Purified CD8+ cells were stained with anti-CD8 FITC and anti-CD45RA PE-Cy5 mAbs, and sorted into RA+ and RA– CD8+ cells. In some experiments, purified CD8+ cells were stained with anti-CCR7 PE-Cy7 and anti-CD45RA PE-Cy5 mAbs, and sorted into RA+CCR7+, RA+CCR7–, RA–CCR7+, and RA–CCR7– cells. The purity of each sorted subset was typically >97%.

CFSE labeling and cell culture

Freshly sorted cells were labeled with CFSE, as previously described (19). Cells were cultured in RPMI 1640 with 20 U/ml IL-2, 10% human AB serum, 2 mM glutamine, 1% nonessential amino acids, 1% sodium pyruvate, and 50 µg/ml penicillin/streptomycin. Dynal (Dynal Biotech) beads (M450) coated with anti-CD3 and anti-CD28 mAbs were added at ratio 1:1 with T cells. rIL-7 and IL-15 were used in some experiments at 25 ng/ml (R&D Systems). Mimosine (300 µM; Sigma-Aldrich) was added where indicated to inhibit cell cycle progression (20).

FACS analysis

Freshly thawed cells were stained with anti-CCR7 PE-Cy7, anti-CD45RA PE-Cy5, anti-CD27 allophycocyanin-Cy7, anti-CD28 allophycocyanin, or IgG allophycocyanin-Cy7, IgG allophycocyanin isotype-matched controls, and 4',6'-diamidino-2-phenylindole for differential phenotyping. For phenotypes of GrB-expressing cells, thawed cells were first labeled with anti-CCR7 PE-Cy7 and anti-CD45RA PE-Cy5, then stained with anti-GrB-allophycocyanin or IgG-allophycocyanin after fixation and permeabilization. All stained cells were finally washed and analyzed on a multicolor FACS LSRII with FlowJo software (BD Biosciences).

Immunofluorescence microscopy

As previously described (21), freshly sorted or stimulated subclass CD8+ cells were plated onto poly-L-lysine-coated slides, fixed in paraformaldehyde, and permeabilized in Triton X-100, then stained with anti-GrB-FITC or IgG-FITC isotype-matched controls for 60 min at room temperature. After washing and mounting, cells were analyzed using a Zeiss Axiovert 200 microscope with OpenLab software.

Real-time RT-PCR assay

Freshly sorted or stimulated CD8+ T cell subsets were cryopreserved at –80°C until the time of RNA extraction. Total RNA was extracted using GenElute Mammalian Total RNA Miniprep kit (Sigma-Aldrich). DNase I treatment was performed after RNA extraction to remove any residual genomic DNA. RT-PCR and real-time quantitative PCR were performed using the TaqMan RT-PCR kit (Applied Biosystems). The GrB primers were as follows: 5'-tgc aac caa tcc tgc ttc tg; 5'-ccg atg atc tcc cct gca t, with the probe: (FAM) 5'-tgg cct tcc tcc tgc tgc cca (derived from GenBank accession no. NM_004131). The product size was 67 bp (22). The beta-actin primers were: 5'-gga tgc aga agg aga tca ctg; 5'-cga tcc aca cgg agt act tg, with the probe: (FAM) 5'-tca gcc act tcg tgc cgg tct ttc. Amplification of GrB and beta-actin transcripts was performed in the same plate using ABI-PRISM 7700 Sequence Detection System at the default setting. For normalization, the cycle threshold (CT) values of GrB and beta-actin in each sample were determined and the {Delta}CT was used to calculate the relative expression level of GrB to beta-actin.

ELISA

Supernatants were harvested 16 h after stimulation with anti-CD3 and anti-CD28 mAbs or under control conditions. IFN-{gamma} concentrations were assessed by ELISA by comparing to a standard curve and analysis with the Softmax program (Molecular Devices).

Redirected cytotoxicity assay

Freshly sorted or in vitro stimulated CD8+ T cell subsets were used as effectors. FcR-bearing P815 cells were used as targets. Target cells were labeled with 51Cr, and a standard 4-h 51Cr release assay was performed over a range of E:T ratios in the presence or absence of 2 µg/ml anti-CD3 mAb (Ortho Biotech). Supernatants were harvested and counted in a gamma counter. Specific lysis was calculated according to the following formula: percentage of specific release = ((experimental release – spontaneous release)/(maximum release – spontaneous release)) x 100.

Statistical analysis

Comparisons between groups were analyzed using a Student’s t test. Value of p < 0.05 was accepted for significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
GrB is expressed in a minor fraction of circulating memory-phenotype CD8+ T cells in healthy individuals

To begin to analyze the presence of cytotoxic granules in circulating human CD8+ T cell subsets, expression of GrB was assessed by intracellular flow cytometry. A small fraction of GrB+ CD8+ T cells could be detected in the circulation of healthy donors, ranging from 0.90 to 2.84% (Fig. 1A). When these T cells were gated on the Ag-experienced CD45RA– subset, the frequency of GrB+ cells was higher, but was still <5% (Fig. 1B). To focus on an Ag-specific CD8+ T cell population, HLA-A2 tetramers complexed to a peptide derived from EBV were used. Even among CD8+ T cells specific for EBV, a virus that persists in the host following resolution of acute infection, the fraction of GrB+ cells was only the minority (12% in the example in Fig. 1C; range 8–20% in different donors). Using additional phenotyping with anti-CCR7 and CD45RA Abs, the ranges of percentages of subsets were as follows: TN, 13.9–58.7%; TE, 12.2–26.9%; TEM, 16.9–31.5%; and TCM, 5.68–27.7%. It was confirmed that the GrB+ cells observed belonged to the CCR7– TE and TEM subsets (Fig. 1D). Of note, the CD8low cells were CD3 negative and therefore are not T cells and were excluded from all analyses (data not shown). In addition, while these analyses were done on cryopreserved cells, no difference was seen when fresh vs frozen T cells were compared (data not shown). Together, these results indicate that only a minority of Ag-experienced human CD8+ T cells expresses GrB.


Figure 1
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  		FIGURE 1. GrB+CD8+ T cell frequencies and phenotypes in healthy individuals. A, Contour plot of anti-CD8-FITC vs anti-GrB-allophycocyanin of lymphocytes in three healthy donors. B, Dot plot of anti-CD8-PE-Cy7 vs anti-GrB-allophycocyanin of sorted RA+ and RA– CD8+ cells. C, EBV-HLA-A2.1 tetramer-specific CD8+ T cells were gated, and GrB+-expressing cells were analyzed. D, GrB+CD8+ events were gated from the dot plot of anti-CD8-FITC vs anti-GrB-allophycocyanin of lymphocytes, and expression of CCR7 (anti-CCR7-PE-Cy7) and CD45RA (anti-CD45RA-PE-Cy5) was analyzed. Isotype-matched mouse-IgG1-PE-Cy7 and IgG1-PerCP served as controls. Numbers shown represent percentage of positive cells. Data shown are representative of three independent and reproducible experiments.

 
Induction of GrB expression in Ag-experienced CD8+ cells with anti-CD3/anti-CD28 mAbs

TN CD8+ T cells differentiate into a cytolytic effector phenotype following stimulation through the TCR complex and the costimulatory receptor CD28 (23, 24). It was therefore of interest to determine whether Ag-experienced CD8+ T cells also could acquire cytotoxic granules and cytolytic activity following TCR/CD28 engagement. As a first approximation of naive vs Ag-experienced CD8+ T cells, sorting based on surface expression of CD45RA was used. Sorted CD45RA–CD8+ T cells were stimulated for 6 days with beads coated with anti-CD3 and anti-CD28 mAbs. CD45RA+CD8+ T cells were used for comparison, and the cytokines IL-7 and IL-15 were used as a control stimulus to support in vitro T cell survival during the same time period. As shown in Fig. 2A, anti-CD3/anti-CD28 stimulation induced significant up-regulation of intracellular GrB expression in both CD45RA+ and CD45RA– CD8+ T cells (Fig. 2A). Significant GrB up-regulation was not observed upon culture in IL-7 + IL-15, although these cytokines did prevent cells from apoptotic death (data not shown). As before, the CD8low cells were excluded from the analysis. To confirm that the intracellular GrB was contained within cytotoxic granules, immunofluorescence microscopy was performed. Indeed, GrB was present in intracellular vesicles consistent with granules in both stimulated CD45RA+ and CD45RA– CD8+ T cells (Fig. 2B). In addition, real-time RT-PCR analysis confirmed that the induction of GrB expression was reflected at the mRNA level in both CD8+ T cell subsets (Fig. 2C).


Figure 2
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  		FIGURE 2. Induction of GrB in CD45RA+ and CD45RA– CD8+ cells. A, FACS detection of GrB. Dot plot of anti-CD8-PE-Cy7 vs anti-GrB-allophycocyanin of sorted fresh and stimulated RA+ and RA– CD8+ cells. The numbers shown represent percentage of positive cells. Data shown are representative of three independent and reproducible experiments. B, Immunofluorescence images showing GrB+ granules present in stimulated CD8+ T cells. All images were taken using x63 oil lenses. C, Real-time PCR for GrB expression. CT values of GrB and actin in real-time PCR assay were determined. GrB mRNA level was normalized according to the actin level in each sample, and relative expression values are shown. *, p < 0.01 for comparison of day 5 to day 0. Data are shown as mean values ± SEM of three independent and reproducible experiments.

 
It was desirable to determine whether the granules acquired after in vitro stimulation that contained GrB also contained perforin. Therefore, two-color intracellular flow cytometry was performed. As shown in Fig. 3A, cells analyzed ex vivo showed minimal expression of both GrB and perforin, whereas after a 5-day stimulation all the GrB+ cells were also perforin+. To verify that the acquisition of GrB-containing cytotoxic granules was associated with cytolytic potential, a redirected lysis assay was performed. In this assay, soluble anti-CD3 mAb is used along with 51Cr-labeled FcR+ targets to promote TCR-mediated cytolysis of the targets via FcR binding. In contrast to resting CD45RA+ and CD45RA– CD8+ T cells, cells primed for 5 days with anti-CD3/anti-CD28 mAbs exhibited potent cytotoxic activity (Fig. 3B). Hence, the induced acquisition of cytotoxic granules upon TCR/CD28 stimulation was correlated with the development of cytolytic function in Ag-experienced CD45RA–CD8+ T cells, arguing that GrB expression is reflective of true cytotoxic granules.


Figure 3
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  		FIGURE 3. Perforin staining and redirected cytolytic activity of sorted unstimulated and stimulated RA+ and RA– CD8+ T cells. A, FACS detection of perforin (PFP) vs GrB. Dot plots of anti-PFP-FITC vs anti-GrB-allophycocyanin of sorted fresh and stimulated CD45RA–CD8+ cells. Isotype-matched mouse-IgG1-FITC and IgG1-allophycocyanin served as controls. Data shown are representative of three independent and reproducible experiments. B, Cytolytic activity. The assay was performed using P815 cells as targets in the presence of 2 µg/ml anti-CD3 mAb. In the absence of anti-CD3 mAb, no significant cytotoxicity activity was detected.

 
GrB is detectably up-regulated after 3 days of stimulation and requires cell cycle progression

It has been suggested that memory-phenotype CD8+ T cells should gain cytolytic activity more rapidly than do TN CD8+ T cells following TCR-dependent activation. It therefore was of interest to determine the kinetics of intracellular GrB induction in CD45RA+ vs CD45RA– CD8+ T cells. As before, a small subset of CD45RA– cells again was found to express GrB at baseline. A time course revealed that GrB became significantly up-regulated in the majority of cells starting at day 3 in both subsets (Fig. 4A). Acute stimulation with anti-CD3/anti-CD28 mAbs resulted in production of IFN-{gamma} in the supernatant at 24 h despite lack of GrB expression at that time (Fig. 4B).


Figure 4
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  		FIGURE 4. Kinetics of GrB up-regulation in RA+ and RA– CD8+ T cells. A, FACS monitoring of GrB expression. Dot plot of anti-CD8-PE-Cy7 vs anti-GrB-allophycocyanin of sorted fresh CD45RA+ and CD45RA– CD8+ cells and cells stimulated with anti-CD3 and anti-CD28 mAbs. Numbers shown represent percentage of positive cells. One representative experiment of the flow cytometry analysis is shown. B, IFN-{gamma} production was assessed by ELISA. *, p < 0.05 for comparison of mAb stimulation to control.

 
We and others have found that cell division after activation is critical for CD4+ T cell differentiation and acquisition of effector cytokine production (20, 25, 26, 27). Inasmuch as the timing of GrB induction appeared to correlate with when an increase in T cell number was first observed (data not shown), we investigated whether cell division and up-regulation of GrB expression were directly associated. This was achieved by measuring CFSE dilution in concert with intracellular flow cytometry for GrB expression. As shown in Fig. 5A, almost all CD45RA+CD8+ T cells (97.7%) acquired GrB after cell division had occurred. The majority (88.5%) of CD45RA–CD8+ T cells also acquired GrB after cell division, and cells that did not traverse cell cycle did not gain GrB. To determine whether cell division was required for GrB induction, the cell cycle inhibitor mimosine was used to induce G1 arrest. The concentration of mimosine used was verified to inhibit cell proliferation, but not acute activation as reflected by IFN-{gamma} production (data not shown), although other off-target of effects of mimosine cannot be excluded. Indeed, mimosine successfully blocked CFSE dilution and GrB up-regulation in both CD45RA+ and CD45RA– CD8+ cells (Fig. 5B). These results suggest that Ag-experienced CD8+ cells, like TN CD8+ T cells, need to traverse the cell cycle to acquire GrB expression, and that the kinetics of granule acquisition are also comparable.


Figure 5
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  		FIGURE 5. GrB up-regulation and cell cycle progression. A, GrB (anti-GrB-allophycocyanin) expression vs cell division as measured by CFSE dilution in sorted fresh CD45RA+ and CD45RA– CD8+ cells stimulated with anti-CD3 and anti-CD28 mAbs. Data shown are representative of three independent and reproducible experiments. B, GrB up-regulation is blocked by mimosine in RA+ and RA– CD8+ cells. GrB expression vs cell division in control cells and cells stimulated in the presence of 300 µM mimosine.

 
Induction and expansion of GrB+ T cells among CD8+ effector and memory cell subsets

Phenotyping based only on CD45RA expression does not capture the complexity of individual effector and memory subsets. Ag-experienced CD45RA– T cells can be further characterized by expression of additional phenotypic markers, including expression of CCR7, CD27, and CD28 (9, 19). It was of interest to examine the relationship between cell division and GrB up-regulation in each of these subsets. We analyzed the distribution of the CD27 and CD28 in the four different subsets of CD8+ T cells previously defined by expression of CD45RA and CCR7 (5, 8). The majority of CD45RA–CCR7+ TCM cells (73.8%), like the CD45RA+CCR7+ TN cells (81.5%), were CD27+CD28+. In contrast, the CD45RA–CCR7– TEM cells were distributed among four subsets: CD27–CD28– (57.8%), CD27+CD28+ (18.6%), CD27+CD28– (8.7%), and CD27–28+ (15.2%). There were fewer CD27-expressing cells among the CD45RA+CCR7– TE subset (20.4%); however, most of those still expressed CD28 (90.8%) (Fig. 6A).


Figure 6
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  		FIGURE 6. Differential phenotyping and expansion of memory T cell subsets. A, Expression of the CD27 (anti-CD27 allophycocyanin-Cy7) and CD28 (anti-CD28-allophycocyanin) in the subsets of CD8+ T cells defined on the expression of CCR7 (anti-CCR7-PE-Cy7) and RA (anti-CD45RA-PerCP). B, Expression and induction of GrB (anti-GrB-allophycocyanin) in the subsets of CD8+ T cells defined as above. C, IFN-{gamma} production was assessed by ELISA. Data shown are representative of three independent and reproducible experiments.

 
The heterogeneous surface phenotypes of these memory subsets raised the possibility that they may have differential capacities for clonal expansion and cytolytic potential (28). We therefore sorted purified CD8+ cells into CD45RA+CCR7+, CD45RA+CCR7–, CD45RA–CCR7+, and CD45RA–CCR7– cells, labeled them with CFSE, stimulated with anti-CD3/CD28 mAbs, and stained for intracellular GrB. As shown in Fig. 6B, freshly sorted CD45RA–CCR7+ TCM and CD45RA+CCR7+ TN did not express GrB, but acquired GrB expression after cell division. In addition, the magnitude of GrB was higher after stimulation of the TN. Although a fraction of freshly sorted RA–CCR7– TEM cells (18.4%) expressed GrB, the majority of them did not divide and did not acquire additional GrB. This is in agreement with work published by Wolint et al. (29), and argues for poor proliferative potential of this subset. Finally, a higher proportion of freshly sorted RA+CCR7– TE cells expressed GrB (24.6%), and a greater fraction gained GrB after cell division (Fig. 6B). Although the rate of cell division was not identical in all subsets, the up-regulation of GrB was nonetheless associated with successful CFSE dilution.

To confirm that the sorted T cell subsets had the expected functional characteristics, IFN-{gamma} production in response to anti-CD3/anti-CD28 stimulation was assessed by ELISA. As expected, the CD45RA–CCR7– TEM and CD45RA+CCR7– TE subsets secreted significantly higher amounts of IFN-{gamma} after 6-h stimulation with anti-CD3/CD28 mAbs than the TCM and TN subsets (Fig. 6C).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Circulating TN CD8+ cells typically exist in a basal G0 state, and their interaction with APCs leads them to initiate several rounds of division. This selectively expands the numbers of T lymphocytes responsive to the presented Ags, and also leads to the acquisition of effector and memory functions (3, 4). It is currently accepted, based on data generated in murine systems, that the relationship between effector cell and memory cell differentiation is linear and not parallel, such that a surviving subset of effector cells gives rise to the memory CD8+ T cell pool. The relationship between subsets of human memory T cells may be more complex, and there is some controversy regarding their lineage interrelationship. It is generally accepted that both TCM and TEM are ultimately derived from TN, but it is not clear whether they must pass through an effector phase of differentiation before acquiring their memory phenotypes. The continuing proliferation and differentiation of TCM driven by APC interactions and homeostatic cytokines may be responsible for renewing the memory CD8+ pool (9, 30). Costimulation of CD28 by CD80/CD86 has been reported to contribute to the maintenance of memory T cells, and TCM cells express CD28 (31). It also has been observed that, as human CD8+ T cells progress in their differentiation toward the effector state, they lose expression of CD27 and CD28 (19). Thus, these data are thus most consistent with a TN to TCM to TEM to TE differentiation pathway, although more complex multilinear development models are conceivable (9, 30, 32, 33, 34, 35)

In murine systems, long-lived memory CD8+ T cells that arise following viral infection have been shown to express perforin and to exhibit cytolytic activity ex vivo (19). We were thus surprised to find that most Ag-experienced CD45RA– T cells from normal human donors lacked expression of GrB. Our observations suggest that in the human system, most memory-phenotype CD8+ T cells need to be restimulated with Ag and to traverse the cell cycle to acquire cytotoxic granules, just as with naive cells. Thus, the quantitative advantage of memory may be more related to an augmented precursor frequency rather than immediate cytolytic effector function. It is formally possible that different TCR thresholds in naive vs memory states might render memory cells better able to re-express granules, which was not directly examined in our study. The duration of granule expression by distinct CD8+ T cell subsets also could vary and has not been examined in our study. Finally, the level of expression of intracellular GrB per cell and how it relates to cytolytic potential has not yet been clearly defined.

The apparent discrepancy between mouse and human studies might have been a consequence of staining for different granule proteins in mouse and human systems. However, we confirmed our results using anti-perforin Ab, although the intensity of staining of effector cells was lower than with anti-GrB, making GrB staining much more convincing as a marker. Although perforin and GrB were coexpressed, the possibility of variable expression of other granule-associated proteins has not been examined. Still, we also found that poor cytolytic activity correlated with lack of GrB staining, and that GrB was localized to granule-like structures by immunofluorescence microscopy. Thus, detectable up-regulation of intracellular GrB most likely represents a reasonable marker for cytolytic potential of CD8+ T cells, and should be explored further in clinical trials of vaccines and other immunotherapy interventions.

The mechanism by which cell cycle progression is linked to cytotoxic granule generation is complex and most likely multifactorial. The acquired ability to transcribe the genes for perforin and GrB (and perhaps others) in the effector state is thought to require epigenetic modification of those gene loci to render them capable of being receptive to transcriptional activation (20, 36). Thus, like Th1 and Th2 lineage-specific cytokine production (37, 38), the ability of a CD8+ T cell to acquire granule-mediated cytotoxic potential ties cell cycle progression to epigenetic alterations that control locus accessibility. However, the generation of cytotoxic granules is more complex than the secretion of a specific cytokine, in that multiple cytolytic proteins need to package together into newly formed granules, and the granules themselves are packaged in a lipid membrane and also need to interact with the exocytosis machinery (1, 21). It is conceivable that, after all granules in a CD8+ TE have been used via exocytosis, the mechanism to ensure proper generation of fresh granules is to progress through the cell cycle and redifferentiate into an effector phenotype.

The lack of granule expression by most memory-phenotype CD8+ T cells makes teleological sense from the perspective of lymphocyte homeostasis. Recent data have indicated that cytotoxic granule proteins contribute to T cell fratricide following activation (39, 40). Mice deficient in perforin develop autoimmune-like syndromes (41, 42), and serine protease inhibitors also regulate T cell homeostasis (43, 44). In addition, recent results in the human system have suggested that CD4+CD25+ regulatory T cells are immunosuppressive, at least in part, via granule-mediated lysis of T cells and APCs (45, 46). Thus, excessive presence of granules in T cells might paradoxically have a dominant regulatory effect.

CD8+ TILs isolated from murine and human tumors have been reported in some instances to lack expression of perforin and to exhibit poor cytolytic activity ex vivo (16, 17, 18, 47). Although this has been presumed to be a consequence of negative regulatory influences within the tumor microenvironment, our current results suggest that a component of this phenomenon could be explained as a normal phase in the life of an activated CD8+ T cell. Most Ag-experienced CD8+ T cells circulating in normal donors also lack cytotoxic granules, but can acquire such granules following TCR/CD28 stimulation. It is conceivable that ensuring optimal CD28 costimulation and supporting T cell proliferation within the tumor microenvironment may similarly induce new granule generation and lead to more effective T cell-mediated tumor cell killing in vivo. Our results suggest that this ability would be limited to the subsets of T cells that retain expression of CD28, as the effector-memory cells showed poor proliferation in our studies. These notions will be of interest to investigate in preclinical and clinical experiments in vivo in the future.


   Acknowledgments
 
We thank the University of Chicago Cancer Research Center Flow Cytometry Facility for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by National Institutes of Health Grant R01 CA90575, a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research, and a grant from the Ludwig Trust. Back

2 Address correspondence and reprint requests to Dr. Thomas F. Gajewski, University of Chicago, 5841 South Maryland Avenue, MC2115, Chicago, IL 60637. E-mail address: tgajewsk{at}medicine.bsd.uchicago.edu Back

3 Abbreviations used in this paper: TN, naive T cell; TE, effector T cell; CT, cycle threshold; GrB, granzyme B; TCM, central memory T cell; TEM, effector memory T cell; TIL, tumor-infiltrating lymphocyte. Back

Received for publication August 18, 2005. Accepted for publication April 14, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Russell, J. H., T. J. Ley. 2002. Lymphocyte-mediated cytotoxicity. Annu. Rev. Immunol. 20: 323-370. [Medline]
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