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Division of Viral Immunology, Center for AIDS Research, Kumamoto University, Kumamoto, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Per–GraA+GraB–PerlowGraA+GraB– PerlowGraA+GraB+PerhighGraA+GraB+. The analysis of the expression of these molecules in the subsets classified by the combination of the expression of CCR7 and CD45RA or by that of CD27, CD28, and CD45RA showed that functional CD8+ T cell subsets could be partially identified by these phenotypic classifications. However, the functional subsets could be precisely identified by the classification using five cell surface markers or three cell surface markers and three cytolytic molecules. Per–GraA–GraB– and Per–/lowGraA+GraB– cells were predominantly found in CCR5–CCR7+ and CCR5high/lowCCR7– subsets, respectively, of CD8+ T cells expressing the CD27+CD28+CD45RA– phenotype, whereas PerlowGraA+GraB+ cells were found in the CCR5lowCCR7– subset of those expressing this phenotype and in a part of the CCR5–/lowCCR7– subset of those expressing the CD27–/lowCD28–CD45RA–/+ phenotype. Ex vivo EBV-specific CD8+ T cells, which were Perlow/–GraA+GraB–/+ cells, hardly or very weakly killed the target cells, indicating that these were not effector T cells. These findings suggest that the Per–GraA–GraB–, Per–/lowGraA+GraB–, and PerlowGraA+GraB+ cells were central memory, early effector memory, and late effector memory T cells, respectively. Per–/lowGraA+GraB– cells gained GraB expression after TCR stimulation, indicating that early effector memory T cells could differentiate into late effector and effector T cells. The present study showed the existence of three memory subsets and the pathway for their differentiation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Previous studies showed that CD8+ T cells change the expression of costimulatory molecules (CD27, CD28, and CD45RA) on their surface according to their differentiation and maturation (7, 9). Therefore, these molecules have been used for phenotypic classification of human CD8+ T cells. Naive, memory, and effector CD8+ T cells express CD27+CD28+CD45RA+, CD27+CD28+CD45RA–, and CD27–CD28–CD45RA+/– phenotypes, respectively. A previous study further demonstrated that CD8+ T cells with the CD27–CD28–CD45RA+/– or CD27lowCD28–CD45RA+/– phenotype have the ability to kill target cells (10).
Chemokine receptors play an important role in lymphocyte trafficking. They are also used to define the functional subsets of human CD8+ T cells. Previous studies revealed that T cells could be classified based on their expressions of CD45RA and the chemokine receptor CCR7 that is associated with their ability to home in on secondary lymph nodes (11). They were classified into CCR7+CD45RA+ (naive), CCR7+CD45RA– (central memory), CCR7–CD45RA– (effector memory), and CCR7–CD45RA+ (effector). The CCR5 chemokine receptor is predominantly expressed on memory and effector memory CD8+ T cells, and its expression decreases during the maturation process from memory to effector CD8+ T cells (6, 10).
Perforin (Per)3 and granzymes A/B (GraA/GraB) are key cytolytic effector molecules that are stored in cytolytic granules in effector CD8+ T cells (12). They can be used as markers for effector CD8+ T cells, because they are the actual functional molecules for killing target cells (13, 14, 15). A recent study showed that about one-half of the total human CD8+ T cell population coexpressed both GraA and GraB, but that there was a small population that expressed either one or the other granzyme in CD8+ T cells (16). These findings imply that human CD8+ T cells do not simultaneously express these two effector molecules at the early stage of their peripheral differentiation. Further analysis of the coexpression of GraA/B and Per is expected to clarify the functional subsets of human CD8+ T cells.
In the present study, we analyzed the coexpression of the effector molecules on the phenotypic subsets of human CD8+ T cells by using advanced multicolor flow cytometry. In addition, we characterized a subset of CD8+ T cells expressing GraA, but not GraB, to clarify the differentiation pathway from naive to effector CD8+ T cells and among memory CD8+ T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Blood samples were taken from healthy adult individuals. PBMCs were isolated from blood by using Ficoll-sodium metrizoate (ICN/Cappel).
Cells
C1R cells expressing HLA-A*1101 (C1R-A*1101) or HLA-A*0201 (C1R-A*0201) were previously generated (17) and were maintained in RPMI 1640 medium supplemented with 10% FCS and 0.15 mg/ml hygromycin B.
Monoclonal Abs
FITC-labeled anti-Per, PE-labeled anti-GraA, Alexa647-labeled anti-GraB, PE-Cy7-labeled anti-CCR7, FITC-labeled anti-CD27, Alexa405-labeled anti-CD45RA, allophycocyanin-labeled anti-CD28, FITC-labeled anti-CD27, allophycocyanin-Cy7-labeled anti-CD27, FITC-labeled anti-CD19 mAbs, and purified anti-CD28 mAbs were obtained from BD Biosciences. ECD-labeled anti-CD3, anti-CD45RA, and anti-CD28 mAbs were purchased from Beckman Coulter. FITC-labeled anti-CD3 mAb came from DakoCytomation. Cascade Blue- and Cascade Yellow-labeled anti-CD8 mAbs were made by conjugating Cascade Blue (Molecular Probes) and Cascade Yellow (Molecular Probes), respectively, with anti-CD8 mAb OKT8.
HLA class I tetramers
HLA class I-peptide tetrameric complexes (tetramers) were synthesized, as previously described (18). The human CMV (HCMV) CTL epitope (HCMV-1 pp65 495–503, NLVPMVATV (19)) and the EBV CTL epitope (EBV-3B 399–408, AVFDRKSDAK (20)) were used for refolding of HLA-A*0201 and HLA-A*1101 molecules, respectively. PE- or allophycocyanin-labeled streptavidin (Molecular Probes) at a molar ratio of 4:1 was used for generation of tetramers.
Flow cytometric analysis
Freshly isolated PBMCs were first stained with anti-CCR5 and anti-CCR7 mAbs for 30 min at room temperature, and subsequently with specific mAbs against surface markers at 4°C for 30 min. IgG isotype controls were used for negative controls. The cells were then washed twice with PBS containing 10% newborn calf serum (NCS; Summit Biotechnology). To determine the intracellular expression of Per, GraA, and GraB, we fixed cells with 4% paraformaldehyde PBS at 4°C for 20 min, and then made them permeable with PBS containing 0.1% saponin (Sigma-Aldrich) and 20% NCS (permeabilizing buffer) at 4°C for 10 min. The cells were stained with anti-Per, anti-GraA, and anti-GraB mAbs at 4°C for 20 min. Finally, the cells were washed three times in the permeabilizing buffer at 4°C. The corresponding IgG isotypes were used as negative controls. In some experiments, we used anti-CD19 mAb to identify B cells as a negative control for expression of the effector molecules.
To assess the expression of the effector molecules in virus-specific CD8+ T cells, we mixed PBMC with the tetramers at a concentration of 0.01–0.02 mg/ml. After incubation at 37°C for 30 min, the cells were washed twice with 10% FCS (Sigma-Aldrich) containing RPMI 1640 and then stained with anti-CD8 mAb. Thereafter, they were fixed, made permeable, and then they were stained with anti-Per, anti-GraA, and anti-GraB mAbs.
The stained cells were analyzed by using a FACSAria (BD Biosciences). For determination of the effector molecule expressing cells in each CD8+ T cell subset, all flow cytometric data were analyzed by using FlowJo software (Tree Star).
Fluorescence-activated cell sorting
To purify CD45RA–CCR5high/lowCD8+ cells, we first isolated CD8+ T cells from PBMCs by using anti-CD8-coated (clone: BW135/80) magnetic beads (Miltenyi Biotec). The isolated CD8+ T cells (>98%) were further purified by the staining of anti-CD8 mAb (clone: OKT8) for FACS sorting. The CD8+ T cells were stained with anti-CD8, anti-CCR5, and anti-CD45RA mAbs, and then CD8highCD45RA–CCR5high and CD8highCD45RA–CCR5low cells were sorted by using FACSAria (BD Biosciences).
CD8+ T cell proliferation assay
Sorted CD8+ T cell subsets were labeled with 0.5 µM CFSE (Molecular Probes) for 15 min at 37°C. CFSE-labeled cells were cultured for 5 days in anti-CD3 (5 µg/ml) and anti-CD28 (5 µg/ml) mAb-coated flat-bottom 96-well plates containing RPMI 1640 medium supplemented with 10% FCS and 200 U/ml human rIL-2 for 5 days. The cells were then stained with anti-CD3, anti-CD8, anti-GraA, and anti-GraB mAbs. To exclude the dead cells, we added 7-aminoactinomycin D (BD Biosciences) after the staining for cell surface markers. To assess the proliferation and the expression profile of GraA/B, cells were analyzed by using a FACSAria (BD Biosciences). For identification of each generation of proliferating cells and percentage of divided, the flow cytometric data were analyzed by use of the proliferation platform of the FlowJo software. Percentage of divided is the percentage of divided cells from the original sample. The percentage of the undivided cells was calculated from the percentage of divided.
Assay for cytotoxic activity
The cytotoxic activity of EBV-specific and HCMV-specific CTLs was measured by the standard 51Cr release assay, as follows. Target cells (2 x 105) were incubated for 60 min with 100 µCi of Na251CrO4 in saline and then washed three times with 10% NCS-containing RPMI 1640. Labeled target cells (2 x 103/well) were added to U-bottom 96-well plates along with the corresponding peptide (1 µM). After a 1-h incubation, CD8+ T cells purified by use of anti-CD8 mAb-coated magnetic beads were added, and the mixtures were then incubated for 6 h at 37°C. At the same time, the EBV-specific CTL clone and bulk CTLs were used for a positive control. The supernatants were then collected and analyzed with a gamma counter. The frequency of CMV-specific CD8+ T cells among the total CD8+ T cell population (1.3%) is 3.25 times higher than that of EBV-specific CD8+ T cells (0.4%). To make an equal percentage of effector cells in the total cell population, we mixed purified autologous CD4+ T cells with CD8+ T cells in the ratio of 2.25:1. Then, the relative cytotoxic activity was measured. Each value of relative specific lysis was calculated by subtracting the specific lysis of the sample target cells prepulsed without peptide from that of target cells prepulsed with 1 µM epitope peptide.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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We investigated the coexpression of three cytolytic functional molecules, i.e., Per and GraA/B in human CD8+ T cells. Total CD8+ T cells from healthy individuals were stained by using mAbs specific for Per, GraA, or GraB. Representative results on the expression of these effector molecules are shown in Fig. 1A. GraA-positive (GraA+) CD8+ T cells and GraB-positive (GraB+) CD8+ T cells each had two major populations, one with a high level of expression (Perhigh) and the other with a low level of it (Perlow). Perlow cells expressed GraA and comprised two subsets, GraB– and GraB+. In contrast, Perhigh cells expressed both GraA and GraB. GraA– cells were hardly detected in the GraB+CD8+ population. These results suggested that CD8+ T cells have at least four populations: Per–GraA–GraB–, PerlowGraA+GraB–, PerlowGraA+GraB+, and PerhighGraA+GraB+. Three-dimensional analysis of the expression of these three molecules confirmed that CD8+ T cells had these four populations, and further suggested the existence of a small population of Per–GraA+GraB– (Fig. 1B). These results were confirmed in CD8+ T cells from seven healthy individuals (data not shown).
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Previous studies using CCR7 and CD45RA markers showed that CD8+ T cells could be divided into four populations: naive (CCR7+CD45RA+), central memory (CCR7+CD45RA–), memory effector (CCR7–CD45RA–), and effector (CCR7–CD45RA+) subsets (11). So, we analyzed the expression of the three effector molecules in each CCR7 CD45RA subset to evaluate whether there was a correlation between the functional and phenotypic categories. A representative result from one healthy individual and the summarized results of analysis of CD8+ T cells from five healthy individuals are shown in Fig. 2, A and B, respectively. Human CD8+ T cells were divided into naive, central memory, effector memory, and effector populations, based on the expression of CD45RA and CCR7. The expression of the three effector molecules was compared among these four classical CD8+ T cell subsets. PerlowGraA+GraB+ and PerhighGraA+GraB+ cells were predominantly found in effector and effector memory subsets, whereas naive CD8+ T cells did not express any of the effector molecules. A large proportion of cells with the central memory phenotype was also negative for the effector molecules (81.2 ± 4.7%), although a small subset was Per–/lowGraA+GraB– (17.3 ± 9.4%). The existence of Per–GraA+GraB– cells was clear in the central memory subset, unlike in the composite figure (Fig. 1A). PerlowGraA+GraB+ cells were evenly found in both the effector memory (18.3 ± 3.2%) and the effector subsets (21.7 ± 4.6%), whereas the percentage of PerhighGraA+BraB+ cells was higher in the effector subset than in the effector memory subset (52.1 ± 11.8 vs 31.3 ± 13.8%). The results indicate that both effector and effector memory CD8+ T cell subsets consist of cells with a heterogeneous distribution of the effector molecules Per and GraA and GraB. Thus, these two phenotypically distinct categories do not represent functionally distinct subsets.
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Expression of the effector molecules in CD27 CD28 CD45RA subsets of human CD8+ T cells
We previously showed that the classification of human CD8+ T cells in terms of CD27, CD28, and CD45RA markers is useful to identify naive, memory, and effector cells (6, 10). To re-evaluate this classification, by using seven-color flow cytometric analysis, we further analyzed the expression of the three effector molecules in subsets classified by their expression of CD27, CD28, and CD45RA. The expression of these effector molecules in each CD27 CD28 subset of CD45RA– and CD45RA+CD8+ T cells from a representative individual is shown in Fig. 3, A and B, respectively. In addition, a summary of data from six individuals is represented in Fig. 3C. CD8+ T cells expressing the CD27high/lowCD28+CD45RA– memory phenotypes included Per–GraA–GraB–, Per–GraA+GraB–, PerlowGraA+GraB–, and PerlowGraA+GraB+ cells, indicating that this population contained central memory and effector memory T cells. The CD27lowCD28+CD45RA– subset included many more PerlowGraA+GraB+ cells (22.8 ± 6.4%) than the CD27highCD28+CD45RA– subset (8.1 ± 3.8%), supporting a previous finding that the former subset was a more mature population than the latter one (10). These cells with the CD27highCD28+CD45RA+ naive phenotype did not express any effector molecules. Because these cells express CCR7 (10), this result is consistent with that for CCR7+CD45RA+ cells shown in Fig. 2. CD8+ T cells expressing the CD27lowCD28–CD45RA+/– phenotype expressed the three effector molecules with the same pattern as those expressing CD27–CD28–CD45RA+/–, the effector phenotype, suggesting that both subsets mostly included effector T cells. Because the CD27–CD28–CD45RA+/– subsets contained much more PerhighGraA+GraB+ cells than CD27lowCD28–CD45RA+/– subsets, the former subsets are thought to be more differentiated cells than the latter ones. Interestingly, both CD27highCD28–CD45RA+ and CD27highCD28–CD45RA– subsets included a large number of Per–GraA–GraB– cells, suggesting that these subsets were closely related to the naive or central memory subset. Taken together, central memory T cells can be hardly discriminated from effector T cells by this phenotypic classification.
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60% of the CCR7+CD28+CD45RA– subset were Per–GraA–GraB– cells, whereas the rest were Per–GraA+GraB– or PerlowGraA+GraB– cells. These results suggested that CCR7+ and CCR7– cells in the CD27high/lowCD28+CD45RA– subsets predominantly included central memory and effector memory T cells, respectively.
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Per–/lowGraA+GraB– cells were found predominantly in the CCR7– subset of CD8+ T cells expressing the effector memory phenotype. Previous studies demonstrated that CCR5 is predominantly expressed in memory CD8+ T cells that do not express CCR7 (21). These findings imply that Per–/lowGraA+GraB– cells may express CCR5. To clarify CCR5 expression on Per–/lowGraA+GraB– cells, we stained PBMC from six healthy individuals with mAbs specific for CD3, CD8, CCR5, GraA, and GraB (Fig. 5, A and B). GraA+GraB– cells are thought to be Per–/lowGraA+GraB– cells, because PerhighGraA+GraB– cells were hardly detected. The result showed that Per–/lowGraA+GraB– cells were found predominantly among the CD8+ T cells expressing a high level of CCR5 (CCR5high) or a part of those expressing a low level of it (CCR5low). CCR5high cells were predominantly found among the CD8+ T cells expressing the CD27high/lowCD28+CD45RA– memory phenotype, whereas CCR5low cells were detected among both CD8+ T cells expressing the CD27high/lowCD28+CD45RA– memory phenotype and those expressing the CD27–CD28–CD45RA– effector phenotype (Fig. 5C). These findings together suggest that Per–/lowGraA+GraB– cells exist among CCR5high/lowCD8+ T cells expressing the CD27high/lowCD28+CD45RA– memory phenotype. Because the results shown in Fig. 5, A and B, included effector T cells, we added CD28 and CD45RA markers to the analysis to exclude effector CD8+ T cells. We stained PBMC from two individuals with mAbs specific for CD28, CD45RA, CD8, CCR5, GraA, and GraB. Because effector CD8+ T cells were included in the CD28–CD45RA– subset, the CD28+CD45RA– subset was analyzed. GraA+GraB– cells were predominantly found in CCR5highCD28+ and CCR5lowCD28+ subsets, and GraA–GraB– in the CCR5–CD28+ subset (Fig. 5D). Conversely, we analyzed CCR5 expression on the GraA+GraB– subset of CD45RA–CD8+ T cells. Most of the GraA+GraB– cells were CCR5highCD28+ and CCR5lowCD28+ (Fig. 5E), confirming that Per–/lowGraA+GraB– cells existed in CCR5high/lowCD8+ T cells expressing the CD27high/lowCD28+CD45RA– memory phenotype. The seven-color flow cytometric analysis using mAbs specific for CD3, CD8, CD45RA, CCR5, CCR7, GraA, and GraB confirmed that GraA+GraB–CD45RA–CD8+ T cells expressed CCR7–CCR5high/low phenotype (Fig. 5F). Thus, Per–/lowGraA+GraB–CD8+ T cells predominated in the CCR7–CCR5high/lowCD27high/lowCD28+CD45RA– effector memory subset. These results taken together show that the memory subset included cells at three different stages, i.e., Per–GraA–GraB–, Per–/lowGraA+GraB–, and PerlowGraA+GraB+, which we consider to correspond to central memory, early effector memory, and late effector memory T cells, respectively. Per–GraA–GraB– and Per–/lowGraA+GraB– predominantly existed in CCR5–CCR7+ and CCR5high/lowCCR7– subsets, respectively, of CD27high/lowCD28+CD45RA– T cells, whereas PerlowGraA+GraB+ did so in the CCR5lowCCR7– subset of CD27high/lowCD28+CD45RA– and CD27lowCD28–CD45RA– T cells.
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We suspected that Per–/lowGraA+GraB– cells differentiated into Perlow/highGraA+GraB– cells during their maturation. To elucidate this possibility, we investigated the in vitro maturation of Per–/lowGraA+GraB– cells. CD8+ T cells with the CCR5highCD45RA– and CCR5lowCD45RA– phenotypes were sorted because the former cells are mostly Per–/lowGraA+GraB–, whereas the latter includes more mature cells. These sorted cells were labeled with CFSE before being stimulated with anti-CD3 and anti-CD28 mAbs for 5 days. After a 5-day culture period, the expression of GraA and GraB in the cells was measured (Fig. 6). The sorted CCR5highCD45RA–CD8+ T cells included GraA+GraB– cells at 94.2% purity. The expression of GraB in the CCR5highCD45RA– cells increased during in vitro proliferation, whereas that of GraA decreased. In contrast, the sorted CCR5lowCD45RA–CD8+ T cells included GraA+GraB+, GraA+GraB–, and GraA–GraB– cells at 59.9, 28.2, and 11.5%, respectively. The expression of GraA in the CCR5low/–CD45RA– cells slightly decreased during in vitro proliferation. That of GraB increased after the first division, but did not change after the second division. These results suggest that the GraA+GraB–CD8+ T cells can obtain GraB expression during their proliferation.
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EBV-specific CD27high/lowCD28+CD45RA– CD8+ T cells express Per–/lowGraA+GraB–/+
Previous studies revealed that HLA-A*1101-restricted EBV-3B 399–408-specific CD8+ T cells in healthy individuals express the CD27+CD28+CD45RA– phenotype and that they also predominantly express CCR5, but not CCR7 (6), suggesting that these EBV-specific CD8+ T cells have characteristics more similar to those of differentiated memory T cells than to those to central memory T cells. To clarify the differentiation stage of the EBV-specific CD8+ T cells, we stained PBMC from two healthy individuals (U-5 and U-27) with HLA-A*1101 tetramers specific for EBV-3B 399–408 as well as with anti-CD8, anti-Per, anti-GraA, and anti-GraB mAbs. The results showed that EBV-specific CD8+ T cells expressed Per–/lowGraA+GraB– and PerlowGraA+GraB+ (Fig. 7A). In contrast, it is well known that HCMV-specific T cells in healthy individuals express the CD27low/–CD28–CD45RA–/+ phenotype and have characteristics of effector T cells (6, 10). Analysis of HCMV-pp65 495–503-specific T cells from a healthy individual showed that they expressed PerhighGraA+GraB+ and PerlowGraA+GraB+ (Fig. 7B).
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EBV 399–408-specific CTLs in bulk culture, which were made by stimulating PBMCs from the same individual with the EBV epitope peptide and then culturing them for 2 wk, expressed PerlowGraA+GraB+ (Fig. 8A). They also showed much stronger cytolytic activity than ex vivo CTLs. These results indicate that the EBV-specific CD8+ T cell population, which included Per–/lowGraA+GraB– and PerlowGraA+GraB+ cells, hardly or very weakly killed target cells, but that they could effectively kill them when activated with epitope peptides. HCMV-pp65 495–503 bulk CTLs were also analyzed (Fig. 8B). These results also revealed that the bulk CTLs, which were stimulated with the HCMV peptide to undergo proliferation and activation, had much stronger cytotoxic activity than ex vivo CTLs. These results indicate that activated CTLs had a strong cytotoxic activity even though they expressed a low level of Per.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Per–GraA+GraB– PerlowGraA+GraB–PerlowGraA+GraB+PerhighGraA+GraB+. Several models of the phenotypic classification of human CD8+ T cells have been proposed (7, 9, 10, 11, 13, 21, 23). However, it is unclear whether these models actually reflect the effector function. To evaluate the proposed phenotypic classifications, we analyzed the expression of three effector molecules among the subsets in two well-known models using both CCR7 and CD45RA (11), and CD27, CD28, and CD45RA (7, 9, 10). Naive and central memory cells were mostly identified by the phenotypic classification using CCR7 and CD45RA. However, the effector memory (CCR7–CD45RA–) and effector (CCR7–CD45RA+) subsets contained both PerlowGraA+GraB+ and PerhighGraA+GraB+ cells, indicating that effector memory cells cannot be discriminated from effector cells by this phenotypic classification. In the other classification, the naive and effector subsets could be discriminated from other subsets by the phenotypic classification using CD27, CD28, and CD45RA. In contrast, the memory subset (CD27high/lowCD28+CD45RA–) included Per–GraA–GraB–, Per–GraA+GraB–, PerlowGraA+GraB–, and PerlowGraA+GraB+ cells, indicating that effector memory cells cannot be discriminated from central memory cells or effector cells by this phenotypic classification. These two phenotypic classifications have often been used in various studies on infectious diseases and cancers to characterize Ag-specific human CD8+ T cells (13, 24, 25, 26, 27, 28, 29). However, the present study demonstrated that the functional subsets of human CD8+ T cells cannot be identified by using these classifications.
A recent study showed that Ag-specific effector CD8+ T cells expressing both GraA and GraB are induced by vaccination, and that 1 year later after immunization the number of specific CD8+ T cells expressing GraA is increased (22), indicating that memory CD8+ T cells preferentially express GraA, but not GraB. This was supported by the present study showing that Per–/lowGraA+GraB– cells were predominantly found among the CD8+ T cells expressing the CD27+CD28+CD45RA– memory phenotype. We further showed that CD8+ T cells with this memory phenotype included three subsets: CCR5high/lowCCR7–, CCR5–CCR7+, and CCR5–CCR7–. It is thought that the CCR5–CCR7+ subset represents central memory cells because this subset expressed Per–GraA–GraB– (Figs. 4B and 5E). In contrast, Per–/lowGraA+GraB– cells were predominantly detected in the CCR5high/lowCCR7– subset (Fig. 5, E and F), suggesting that this subset is mostly composed of early effector memory cells. PerlowGraA+GraB– and PerlowGraA+GraB+ cells were found in the CCR5–CCR7– subset, indicating that this subset included both early and late effector memory cells. Per–/lowGraA+GraB– cells gained the expression of GraB after stimulation with anti-CD3 and anti-CD28 Abs. This result supports the idea that the CCR5highCCR7– subset expressing Per–/lowGraA+GraB– is an intermediate one between the CCR5–CCR7+ central memory subset expressing Per–GraA–GraB– and the late effector memory subset expressing PerlowGraA+GraB+. Thus, the results of the present study indicate that the phenotypic classification using five cell surface markers (CD27, CD28, CD45RA, CCR5, and CCR7) or three cell surface makers (CD27, CD28, and CD45RA) and the three cytolytic molecules is much more useful to identify naive, central memory, early effector memory, late effector memory, and effector CD8+ T cells than the other two classifications (Fig. 9).
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Previous studies using ex vivo PBMC from HIV-1-infected donors and healthy donors showed that EBV-specific and HIV-1-specific CD8+ T cells, which had a low frequency of Per+ cells, failed to kill target cells (6, 14, 33), suggesting that Per expression is required for the killing activity of CD8+ T cells. The study also showed that EBV-specific CD8+ T cells, which are mostly Per+ cells, failed to kill target cells (14), implying that the expression level of Per and that of GraB are important for killing activity of CD8+ T cells. Indeed, the present study showed that the expression of Per was critical for this killing activity of CD8+ T cells. Thus, CD8+ T cells obtained cytotoxic function when they highly expressed Per and GraB. However, EBV-specific CTL clones and lines, which can effectively kill target cells, expressed PerlowGraA+GraB+ (Fig. 8), indicating that CD8+ T cells expressing PerlowGraA+GraB+ also have cytotoxic function when they are activated.
A previous study showed that EBV-specific CD8+ T cells expressed the CCR5+CCR7– or CCR5–CCR7– phenotype and CD27+CD28+CD45RA– one, and failed to kill target cells (6), suggesting that they were effector memory T cells having no cytotoxic function. In contrast, HCMV-specific CD8+ T cells expressed the CCR5–CCR7–CD27–/lowCD28–CD45RA–/+ phenotype and effectively killed target cells (10), suggesting that they were effector T cells. In the present study, we divided memory CD8+ T cells into three subtypes, central (Per–GraA–GraB–), early effector memory (Perlow/–GraA+GraB–), and late effector memory (PerlowGraA+GraB+) T cells. EBV-specific CD8+ T cells included both early effector memory and late effector memory T cells, whereas HCMV-specific CD8+ T cells included both late effector memory and effector T cells. Thus, the present study analyzing three effector molecules afforded a more precise characterization of these T cells, and the result obtained suggests that the presence of PerhighGraA+GraB+ T cells is critical for the killing activity of HCMV-specific CD8+ T cells.
In the present study, we characterized a subset of CD8+ T cells expressing different effector molecules. The classification of human CD8+ T cells by using the three cell surface markers and the three cytolytic molecules or the five cell surface markers reflected their effector function. Further detailed analysis of human memory CD8+ T cell subsets may be expected to allow clarification of the functional differences among the memory subsets. In addition, the use of this phenotypic classification of human CD8+ T cells from patients with various diseases such as HIV-1 infection and other chronic infections will provide important information on the pathogenesis of these diseases and lead to better therapy of them.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This research was supported by a Grant-in-Aid (17047033) for Scientific Research from the Ministry of Education, Science, Sports, and Culture, the government of Japan. H.T. is a Japan Society for the Promotion of Science Research Fellow. 
2 Address correspondence and reprint requests to Dr. Masafumi Takiguchi, Division of Viral Immunology, Center for AIDS Research, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan. E-mail address: masafumi{at}kaiju.medic.kumamoto-u.ac.jp
3 Abbreviations used in this paper: Per, perforin; GraA, granzyme A; GraB, granzyme B; HCMV, human CMV; NCS, newborn calf serum.
Received for publication November 29, 2005. Accepted for publication July 10, 2006.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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 A. Bonehill, A. M.T. Van Nuffel, J. Corthals, S. Tuyaerts, C. Heirman, V. Francois, D. Colau, P. van der Bruggen, B. Neyns, and K. Thielemans Single-Step Antigen Loading and Activation of Dendritic Cells by mRNA Electroporation for the Purpose of Therapeutic Vaccination in Melanoma Patients Clin. Cancer Res., May 15, 2009; 15(10): 3366 - 3375. [Abstract] [Full Text] [PDF]
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 T. Kondo and M. Takiguchi Human memory CCR4+CD8+ T cell subset has the ability to produce multiple cytokines Int. Immunol., May 1, 2009; 21(5): 523 - 532. [Abstract] [Full Text] [PDF]
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A. Harari, F. B. Enders, C. Cellerai, P.-A. Bart, and G. Pantaleo Distinct Profiles of Cytotoxic Granules in Memory CD8 T Cells Correlate with Function, Differentiation Stage, and Antigen Exposure J. Virol., April 1, 2009; 83(7): 2862 - 2871. [Abstract] [Full Text] [PDF]
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E. B. Walker, W. Miller, D. Haley, K. Floyd, B. Curti, and W. J. Urba Characterization of the Class I-Restricted gp100 Melanoma Peptide-stimulated Primary Immune Response in Tumor-Free Vaccine-draining Lymph Nodes and Peripheral Blood Clin. Cancer Res., April 1, 2009; 15(7): 2541 - 2551. [Abstract] [Full Text] [PDF]
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 T. Kondo, H. Takata, F. Matsuki, and M. Takiguchi Cutting Edge: Phenotypic Characterization and Differentiation of Human CD8+ T Cells Producing IL-17 J. Immunol., February 15, 2009; 182(4): 1794 - 1798. [Abstract] [Full Text] [PDF]
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P. K. Chattopadhyay, M. R. Betts, D. A. Price, E. Gostick, H. Horton, M. Roederer, and S. C. De Rosa The cytolytic enzymes granyzme A, granzyme B, and perforin: expression patterns, cell distribution, and their relationship to cell maturity and bright CD57 expression J. Leukoc. Biol., January 1, 2009; 85(1): 88 - 97. [Abstract] [Full Text] [PDF]
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 M. E. Dahl, A. Berson, J. Lora, and M. Fuentes A Novel CCR5-Specific Pharmacodynamic Assay in Whole Blood Using Phosphoflow Cytometry Highlights Different Ligand-Dependent Responses but Similar Properties of Antagonists in CD8+ and CD4+ T Lymphocytes J. Pharmacol. Exp. Ther., December 1, 2008; 327(3): 926 - 933. [Abstract] [Full Text] [PDF]
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H. Radziewicz, C. C. Ibegbu, H. Hon, M. K. Osborn, K. Obideen, M. Wehbi, G. J. Freeman, J. L. Lennox, K. A. Workowski, H. L. Hanson, et al. Impaired Hepatitis C Virus (HCV)-Specific Effector CD8+ T Cells Undergo Massive Apoptosis in the Peripheral Blood during Acute HCV Infection and in the Liver during the Chronic Phase of Infection J. Virol., October 15, 2008; 82(20): 9808 - 9822. [Abstract] [Full Text] [PDF]
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M. A. DeBenedette, D. M. Calderhead, H. Ketteringham, A. H. Gamble, J. M. Horvatinovich, I. Y. Tcherepanova, C. A. Nicolette, and D. G. Healey Priming of a Novel Subset of CD28+ Rapidly Expanding High-Avidity Effector Memory CTL by Post Maturation Electroporation-CD40L Dendritic Cells Is IL-12 Dependent J. Immunol., October 15, 2008; 181(8): 5296 - 5305. [Abstract] [Full Text] [PDF]
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R. Okada, T. Kondo, F. Matsuki, H. Takata, and M. Takiguchi Phenotypic classification of human CD4+ T cell subsets and their differentiation Int. Immunol., September 1, 2008; 20(9): 1189 - 1199. [Abstract] [Full Text] [PDF]
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E. B. Walker, D. Haley, U. Petrausch, K. Floyd, W. Miller, N. Sanjuan, G. Alvord, B. A. Fox, and W. J. Urba Phenotype and Functional Characterization of Long-term gp100-Specific Memory CD8+ T Cells in Disease-Free Melanoma Patients Before and After Boosting Immunization Clin. Cancer Res., August 15, 2008; 14(16): 5270 - 5283. [Abstract] [Full Text] [PDF]
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M. Rehr, J. Cahenzli, A. Haas, D. A. Price, E. Gostick, M. Huber, U. Karrer, and A. Oxenius Emergence of Polyfunctional CD8+ T Cells after Prolonged Suppression of Human Immunodeficiency Virus Replication by Antiretroviral Therapy J. Virol., April 1, 2008; 82(7): 3391 - 3404. [Abstract] [Full Text] [PDF]
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J. D. Mintern, C. Guillonneau, F. R. Carbone, P. C. Doherty, and S. J. Turner Cutting Edge: Tissue-Resident Memory CTL Down-Regulate Cytolytic Molecule Expression following Virus Clearance J. Immunol., December 1, 2007; 179(11): 7220 - 7224. [Abstract] [Full Text] [PDF]
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 M. Pedroza-Seres, M. Linares, S. Voorduin, R.-R. Enrique, R. Lascurain, Y. Garfias, and M. C. Jimenez-Martinez Pars planitis is associated with an increased frequency of effector-memory CD57+ T cells Br J Ophthalmol, October 1, 2007; 91(10): 1393 - 1398. [Abstract] [Full Text] [PDF]
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A. Martino, R. Casetti, A. Sacchi, and F. Poccia Central Memory V{gamma}9V{delta}2 T Lymphocytes Primed and Expanded by Bacillus Calmette-Guerin-Infected Dendritic Cells Kill Mycobacterial-Infected Monocytes J. Immunol., September 1, 2007; 179(5): 3057 - 3064. [Abstract] [Full Text] [PDF]
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). It was compared with that of purified CD8+ T cells alone (•). Each E:T ratio was calculated from the frequency of tetramer+ cells in the total CD8+ T cell population. An EBV-specific CTL clone and EBV-specific CTL line (
) were used as positive controls. Each value of relative specific lysis was calculated by subtracting the specific lysis of the sample target cells without peptide from that of target cells with peptide. D, Correlation between Per expression and cytolytic activity in tetramer+ cells. The effector molecule expression and cytotoxic activity of EBV- and HCMV-1-specific CD8+ T cells from five healthy individuals were analyzed in the same manner as in A–C. The Per expression is represented as NMFI, which is the mean fluorescence intensity (MFI) of cells stained with anti-Per mAb divided by the MFI of cells stained with the isotype control mAb. Then, the Per expression and relative specific lysis at an E:T ratio of 2:1 were plotted. The linear correlation coefficient was r = 0.89. E, Lack of correlation between GraA expression and cytolytic activity in tetramer+ cells. The GraA expression and relative specific lysis at an E:T ratio of 2:1 were plotted. The GraA expression is also presented as NMFI based on the isotype control. The linear correlation coefficient was r = 0.02.
