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*
Department of Immunobiology and Laboratory for Experimental and Clinical Immunology, Academic Medical Center, CLB, University of Amsterdam, Amsterdam, The Netherlands; and
Department of Cell Biology, University Medical Center Utrecht, Utrecht, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and TCR
and relatively high levels of CD8. CTLA-4 was absent from freshly
isolated effector cells, whereas a limited number of unstimulated
memory cells expressed this molecule. In line with recent findings on
CD8+CD28- T cells,
CD45RA+CD27- T cells were unique in the
abundant expression of NK cell-inhibitory receptors, both of Ig
superfamily and C-type lectin classes. Binding of NK cell-inhibitory
receptors to classical and nonclassical MHC class I molecules may
inhibit the activation of the cytolytic machinery induced by either Ag
receptor-specific or nonspecific signals in
CD8+CD45RA+CD27- T
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In humans, cell surface marker analysis has been thoroughly used to discern functional distinct subsets of CD8+ T cells. Proliferative responses to viral Ags are predominantly confined to the CD45RA-CD45R0+ subset, which suggests that this population contains memory-type cells (6, 7). In line with this assumption, cytokine secretion profile, activation requirements, and the expression of adhesion and homing receptors on CD45RA-CD45R0+ T cells resemble those of Ag-experienced T cells (8, 9). Next to the CD45RA-CD45R0+ subset, a second subpopulation with features of in vivo priming is found in human peripheral blood. This population is characterized by the absence of the costimulatory molecules CD28 (10) and CD27 (7), the presence of both CD45RA and CD57 Ags (11), and abundant expression of CD11a (12). Because CD8+ T cells within this subset express cytolytic mediators such as perforin, granzyme A and B, and Fas ligand (FasL) 6 mRNA and are able to execute cytotoxicity without prior in vitro stimulation, it is suggested that they represent effector cells in vivo (13). Interestingly, although these effector type cells have poor proliferative potential in vitro, it has been documented that these cells increase with age (14). The recent observation (15) that these cells lack the chemokine receptor CCR7 indicates that they do not recirculate through the secondary lymphoid organs but rather migrate to sites of inflammation. Finally, compatible with selection by specific Ag in vivo is the observation that their TCR Vß repertoire is strongly skewed when compared with either naive or memory CD8+ T cells (16). Indeed, recent studies using HLA/viral-peptide tetrameric complexes have demonstrated that specific CD8+ T cells with CD28- and CD45RA+CD27- phenotypes can be found in EBV, hepatitis C virus, and CMV carriers (17, 18).
Studies with in vitro expanded T cell clones and experiments in mutant
mice have provided evidence that CTL can exert their effector functions
by at least two independent pathways: granule exocytosis and the
Fas/FasL pathway (19). The granule exocytosis pathway
involves secretion of granules containing cytotoxic effector molecules
onto the surface of target cells. Perforin plays a critical role in
this pathway, because it can polymerize to form channel-like structures
in target cell membrane, through which granzymes can enter and
subsequently activate the death machinery (20).
Alternatively, CTLs can use the Fas pathway to kill their target cells.
This cytolytic pathway is mainly based on cell-cell interaction between
Fas, expressed on the target cell, and FasL, expressed on the CTL
(21). Engagement of the Fas receptor results in the
aggregation of its intracellular death domains, followed by the
activation of several caspases with the ultimate death of the target
cell (22). We here show that both granule exocytosis and
FasL pathways are readily operational in circulating effector-type
CD8+ T cells. Furthermore, in search for
potential mechanisms that could control the cytotoxic machinery of
these cells in vivo, we found normal expression of CD3 and CD3
chains and absence of CTLA-4 expression. However, in agreement with
recent finding on CD28- T cells (23, 24), a strong increase in the expression of various classes of
killer-inhibitory receptors was observed when cells maturate into
CD8+CD45RA+CD27-
effector T cells (25).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For the analysis of expression of cell surface and intracellular
molecules, the following mouse anti-human mAbs were used: unlabeled
Fas2 and CD3, CD27 FITC, and CD3 FITC and biotinylated granzyme B
(GB12) (CLB, Amsterdam, The Netherlands); CD45RA PECy5 and TCR FITC
(Serotec Kidlington, Oxford, U.K.); CD94, NKG2a, CD158a, CD158b, and
CD45RA, CTLA-4 (CD152), all PE labeled (Coulter-Immunotech, Miami, FL);
CD8 peridinin chlorophyll protein, CD45R0 APC, CD16 PE, NKB1-FITC
(Becton Dickinson, San Jose, CA); unlabeled FasL (Nok-2) (PharMingen,
San Diego, CA); unlabeled granzyme A, perforin FITC, (Hölzel
Diagnostika, Cologne, Germany); and annexin V FITC (Nexins Research,
Hoeven, The Netherlands). As a conjugate for unlabeled or biotinylated
mAbs, goat F(ab')2 anti-mouse IgG2a PE
(Southern Biotechnology Associates, Birmingham, AL), goat
anti-mouse FITC (CLB), and streptavidin red 670 (Life Technologies,
Gaithersburg, MD) were used, respectively. Isotype-matched mAb served
as controls.
Cells
PBMC were isolated from buffy coats of healthy blood donors by
density centrifugation with Ficoll-Isopaque (Pharmacia Biotech,
Uppsala, Sweden). Subsequently, CD8+ T cells
(>97% TCR
ß+CD8+
cells as assessed by flow cytometry; data not shown) were prepared by
incubating PBMC with anti-CD8 microbeads (Miltenyi Biotec, Bergisch
Gladbach, Germany), followed by positive selection with the VarioMACS
(Miltenyi Biotec) according to the manufacturer’s recommendations. For
subset purification (7), CD8+ T
cells were stained with CD45RA and CD27 and sorted on a FACStar (Becton
Dickinson) into
CD45RA+CD27+ (naive
fraction), CD45RA+CD27-
(effector fraction), and
CD45RA-CD27+ (memory
fraction) populations (>95% purity as assessed by flow cytometry;
data not shown). For NK isolation, PBMC were stained with CD3 and CD16
mAb and sorted on a FACStar into
CD3-CD16+ cells (>96%
purity).
The Fas-sensitive subclone Ramos.FsA of the Burkitt lymphoma Ramos (26) has been previously described.
Cell culture
All culture experiments were performed in IMDM (Life Technologies, Gaithersburg, MD) enriched with 10% heat-inactivated FCS (Euro Biochem, Bierges, Belgium), gentamicin (43 µg/ml), and 2-ME (0.0035%) (culture medium).
Confocal laser microscopy
Purified CD45RA+CD27+, CD45RA+CD27-, and CD3-CD16+ populations were isolated as described above. After adhesion to poly-L-lysine-coated object glasses (30 min at room temperature (RT), cell populations were fixated in PBS containing 3% paraformaldehyde (60 min at RT). Next, cells were incubated in PBS containing 50 mM NH4Cl (5 min at RT) and subsequently permeabilized in PBS/0.1% saponin/0.5% BSA (30 min at RT). After washing, intracellular staining was performed by incubating cells with anti-human granzyme A Ab (45 min at RT). Cells were then washed three times and incubated with goat anti-mouse IgG FITC (30 min at RT). Moviol containing 2.5% diazabicyclo octane (Fluka, Buchs, Switzerland) was used to mount on the cells. For immediate analysis, we made use of a Leitz DMIRB fluorescence microscope (Leica, Voorburg, The Netherlands) interfaced with a Leica TCS4D confocal laser microscope (Leica, Heidelberg, Germany). Images were imported in Adobe Photoshop 4.0 (Adobe Systems, San Jose, CA).
Induction and measurement of granzyme release
CD8+ subpopulations were isolated as described above. Cells were cultured in triplicate in 96-well plates (0.3 x 106 cells/well) in either the presence or the absence of 1 ng/ml PMA and 1 µM ionomycin (both from Sigma, St. Louis, MO). Supernatants were harvested after 1 h culture, and the levels of soluble granzyme A and B were measured by ELISA. Stimulated cells were used for intracellular evaluation of these molecules according to the protocol described below.
Granzyme A and B concentrations in culture supernatants were measured by specific solid phase sandwich ELISA as previously described (27) with some modifications. The granzyme A ELISA was performed using the GA29 mAb (coating Ab; CLB) and the biotinylated GA28 mAb (detection Ab; CLB). The limits of detection of the granzyme A and B ELISA systems were 12 and 3 pg/ml, respectively.
Cytotoxicity assay
To evaluate the contribution of the Fas/FasL pathway by effector CD8+ T cells, a redirected cytotoxic assay was performed as previously described (28) with some modifications. Briefly, to specifically block cytotoxicity mediated by perforin and granzymes, isolated CD8+ T cells were pretreated (or not) for 4 h with 400 nM concanamycin A (CMA) (Sigma), washed once, and cultured in the presence of CMA. Next, cells were cocultured in 24-well plates (106 cells/well) with FsA target cells (0.2 x 106 cells/well) (E:T 5:1). The redirected cytotoxic assay conditions were imitated by incubating the cells for 18 h with anti-CD3 (clone CLB-T3/4.1) to stimulate effector CD8+ T cells and bind to the FcR expressed on Ramos.FSA cells. To evaluate the existence of Fas/FasL-dependent cytotoxicity, blocking Abs (anti-FasL (NOK-2) or anti-Fas (Fas2)) were added to the cultures. Apoptosis of Ramos.FSA cells was evaluated using FITC-labeled annexin V as described previously (26). Ramos.FSA cells were discriminated from CD8+ T cells by gating on forward and side scatter parameters (data not shown).
Flow cytometry
Expression of cell surface molecules. Staining for NK cell-inhibitory receptors (NKRs) was performed by incubating freshly isolated PBMC with saturating amounts of directly labeled CD8, CD45RA, CD27, and an anti-MHC class I killer-inhibitory receptor mAb in PBS containing 0.5% BSA (Bayer, Kankakee, IL) (30 min at 4°C). Expression of the different markers was measured on a FACScalibur (Becton Dickinson) and analyzed with the Cell Quest program (Becton Dickinson). CD8+ T cells were gated, and NKRs were analyzed on the different CD8 subsets.
Expression of cytoplasmic molecules/epitopes. Intracellular content of FasL was measured in freshly isolated CD8+ T cells before and after in vitro stimulation. FasL on stimulated cells was measured in the presence of an inhibitor of protein secretion, resulting in the cytoplasmic accumulation of the synthesized FasL. After cell fixation and permeabilization, intracellular staining was performed according to a protocol originally described by Jung et al. (29) with some modifications. Briefly, isolated CD8+ T cells were stimulated (106 cells/ml) for 4 h with PMA (1 ng/ml) and ionomycin (1 µM) in the presence of the protein secretion inhibitor monensin (1 µM) (all from Sigma). Next, the cells were washed twice in cold PBS-0.5% BSA and stained with CD45RA and CD27 (30 min at 4°C, washed twice with cold PBS, and fixed with PBS containing 4% paraformaldehyde (5 min at 4°C). Fixation was followed by permeabilization with PBS containing 0.1% saponin (Calbiochem, La Jolla, CA) and 0.5% BSA. Nonspecific binding was blocked by incubating the cells in the same buffer supplemented with 10% human pooled serum (CLB) (20 min at 4°C). For all subsequent incubation and washing steps, PBS, 0.1% saponin, 0.5% BSA was used. Cells were then washed once and stained with 5 µg/ml anti-FasL (30 min at 4°C). After another washing step, cells were stained with PE-labeled goat anti-mouse IgG2a isotype-specific mAb (20 min at 4°C). Expression of the different markers was measured on a FACScalibur and analyzed with the Cell Quest program. CD8+ T cells were gated, and FasL was analyzed on the different CD8 subsets.
Expression of CTLA-4 and TCR was performed using a permeabilization
and staining protocol identical with that described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously found by flow cytometry that
CD8+CD45RA+CD27-
effector-type T cells abundantly express components of the
exocytotic cytolysis pathway, i.e., granzyme A, B and perforin (Ref.
7 and data not shown). To test whether in circulating CTL
these cytotoxic enzymes are contained within granules and are thereby
ready for receptor-induced exocytosis, the intracellular distribution
of granzyme A in CD8+ T cells (Fig. 1
a) was studied by confocal
scanning laser microscopy. In accordance with the cytotoxic
capacities of the different subsets, nearly all effector
CD8+ T cells contained granzyme A in a granular
fashion (Fig. 1c), whereas in contrast, naive
(CD45RA+CD27+)
CD8+ T cells did not express granzyme A (Fig. 1b). Moreover, the expression of this molecule in
CD8+CD45RA+CD27-
T cells parallels in both qualitative and quantitative terms the
expression observed in freshly isolated NK cells (Fig. 1d).
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). Moderate levels of granzyme A could
also be detected in supernatants of memory
(CD45RA-CD27+) cells, in
accordance with low levels of expression of granzyme A in this subset
(data not shown). In contrast, no significant release of granzyme B was
observed when naive or memory cells were stimulated in our experimental
conditions. Furthermore, the presence of granzymes in the supernatants
corresponded with a decrease in the intracellular levels on stimulated
cells as evaluated by flow cytometry (data not shown). These results
indicate that the stored granzymes (A and B) are rapidly released from
effector cells after stimulation.
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, left). To assess the
expression of FasL after stimulation, purified
CD8+ T cells were cultured for 4 h in the
presence or absence of PMA and ionomycin. Even in the presence of the
metalloproteinase inhibitor KB8301 only a minimal amount of
extracellular FasL could be detected (data not shown). Stimulation did,
however, result in an increase in the intracellular expression of FasL
in the memory compartment (Fig. 3, right).
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55% when cells were incubated
with CMA showing contribution of the granule exocytosis pathway (data
not shown). The remaining cytotoxic response appeared to be largely
dependent on the triggering of Fas on the Ramos.FSA cells, because
addition of either Fas-blocking mAb (Fas2) or FasL mAbs reduced
apoptosis by 74 and 46%, respectively (Fig. 4). Because combined usage of CMA and
Fas-blocking mAb reduced target cell lysis by 90%, we believe that
the granule exocytosis and FasL pathway account for the majority of
cytolytic activity of these circulating effectors.
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The abundance of cytolytic mediators in circulating effector CTLs
suggests that the activity of these cells must be carefully controlled
to prevent unwanted tissue damage. In a number of in vivo conditions,
such as autoimmunity, tumor growth, and HIV infection
(31, 32, 33, 34), low responsiveness of T cells may be induced by
down-regulation of the expression of TCR, which is an essential
signaling component in the CD3/TCR complex. To investigate whether low
expression of TCR would be involved in the control of the activation
of cytotoxic effector cells, we analyzed the expression of TCR on
the different CD8+ T cell subsets. As shown in
Fig. 5, memory and effector CTLs
expressed comparable high levels of TCR whereas the naive subset
expressed TCR less intensively. Moreover, because effector CTLs also
express relatively high levels of CD3 and the coreceptor CD8 (data
not shown), we conclude that it is unlikely that circulating effectors
are kept at a low level of activation by suboptimal signaling via
TCR/CD3.
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10% of memory CTLs expressed this molecule
(Table I). On activation with
anti-CD2 mAbs, both effector CTLs and memory-type cells had
comparable but moderate expression, i.e., 32 and 50% positive cells,
respectively (Table I). The absence on freshly isolated cells and the
low expression on in vitro activated T cells render it unlikely that
CTLA-4 acts as a major negative regulator of effector CTL
activation.
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, Table II). Interestingly, within the
CD45RA- subset, CD27-
cells had a relatively high NKR expression. The present data
corroborate that the differentiation of human
CD8+ T cells toward effector CTL is accompanied
by an increase in the expression of NKRs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF- that are highly produced by
these cells (7) likely aid in coping adequately with
infections, e.g., by limiting virus replication (37). Although circulating effector T cells are well equipped with these cell death-inducing pathways, they do not, without stimulation, release granzymes or perforin, nor do they express FasL on the plasma membrane. Short term stimulation in vitro, however, induces a very rapid release of the constituents of the exocytosis pathway in culture supernatants. On the other hand, although CD8+CD45RA+CD27- T cells contain FasL mRNA (7) and express limited but discernible amounts of FasL intracellularly, we were unable, even in the presence of specific proteinase inhibitors, to reliably demonstrate FasL on the surface of effector cells. However, the contribution of FasL in the induction of apoptosis in a Fas-sensitive Burkitt lymphoma line demonstrates that, in accordance with previous findings in mice (38, 39), in vivo matured human CTLs can make use of the FasL effector pathway
The strong expression of these cytolytic molecules and the functional availability of these compounds upon activation suggest that the activity of these circulating effectors must be carefully controlled to prevent unwanted damage to healthy cells, e.g., after the recognition of cross-reactive peptides. NKRs, which have been orginally described on NK cells, can after binding to MHC class I molecules transduce inhibitory signals for cellular cytotoxicity (40). Concordant with previous findings on CD28- T cells, we found that CD8+CD45RA+CD27- T cells express a variety of NKR, both of the C-type lectin and Ig superfamily classes (23, 24). In healthy individuals the majority of CD8+CD27- T cells express CD45RA but lack CD28 showing that under physiological conditions CD27- and CD28- subsets represent largely overlapping populations of CD8+ T cells (7). However, when patients with acute viral infections are being analyzed a very considerable portion of CD45RA-CD28- T cells does express CD27 (25, 47). We have postulated that by combined analysis of CD27, CD28, and CD45RA expression, distinct stages of differentiated CD8 T cells can be identified (25). We here show that the differentiation toward CD8+CD45RA+CD27- "effector-type" T cell is not only accompanied by an enhancement of intracellular cytolytic mediators but also coincides with an strong increase in the expression of various NKRs. A complicating finding in appreciating the functional consequences of the expression of these molecules is that the mAb that are being used to detect NKR of the Ig family will also bind to splice variants of these molecules that can act as activating receptors, i.e., killer cell activating receptor (41). Differing with Mingari et al. (23), we find highest expression of these regulating receptors on CD45RA+CD27- and therefore CD28- T cells (7) and not on CD45RA-CD27- T cells. Although it has recently been reported that IL15 can induce CD94/NKG-2A expression on mitogenically activated T cells (42), it is less clear which specific signals do induce the expression of Ig superfamily type NKRs on T cells. T cell cloning experiments have indicated that the expression of NKRs is a stable phenotypic trait. In some donors, very high percentages of both C-type lectin and Ig superfamily NKRs were found specifically in the CD27- T cell fraction. This finding could suggest that concomitant with their differentiation toward differentiated effector cells, CD8+CD45RA+CD27 receive specific signals that induce up-regulation of these cell activation-regulating receptors.
Multiple studies have documented that CD8+CD28-, CD8+CD45RA+CD27-, or NKR+ subsets have a restricted and selected usage of the TCR repertoire suggestive of selection by Ag (10, 16). Indeed, more recently, it was shown that virus-specific and tumor Ag-specific CD8+CD45RA+CD27- T cells can be found in hepatitis C virus- and CMV-infected persons and melanoma patients, respectively (18, 43). Several reports have documented that NKRs can inhibit both superantigen- and Ag-induced activation of T cells (36, 44, 45). Interestingly, Lee et al. (43) reported that despite the presence of Ag-specific CD8+CD45RA+CD27- in the circulation of a melanoma patient, no lysis of melanoma cell line in vitro was observed. Because our data suggest that these cells will also express high levels of NKRs, inhibition of TCR-induced killing can be envisaged. For this reason, it is important to more precisely define the signals that induce expression of NKRs on effector T cells, because these receptors will seriously interfere with vaccination strategies for, e.g., cancer.
The question remains on the physiological function of circulating effector-type cells. The functional relevance of these cells appears to be supported by the fact that circulating effector-type cells increase with age (46). Although the presence of differentiated and functionally competent CTL could provide a first line of defense against invading pathogens to which a protective response has already been mounted once, the expression of NKR will preclude rapid activation by these cells by any dose of peptide. Yet, either with decreasing amounts of MHC molecules (which may limit the inhibitory signal) or with increasing specific peptide concentration (which will augment the stimulatory signal), these cells may become activated. In fact, the integration between these positive and negative signals allows the effector CTL to detect the specific loading of MHC molecules with foreign peptides irrespective of the number of MHC molecules expressed on a target cell.
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Acknowledgments |
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Footnotes |
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2 L.R. was supported by Fundação para a Ciência e a Tecnologia, Portugal (Grant PRAXIS XXI/BD/9156/96).
3 Current address: Department of Molecular Immunology, National Institute of Public Health and Environmental Protection, P.O. Box 1, 3720 BA Bilthoven, The Netherlands.
4 Current address: Institut Biochimi Université de Lausanne, Chemin de Boveresses 155, 1066 Epalinges, Switzerland.
5 Address correspondence and reprint requests to Dr. René A. W. van Lier, Department of Immunobiology, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.
6 Abbreviations used in this paper: FasL, Fas ligand; RT, room temperature; CMA, concanamycin A; NKRs, NK cell-inhibitory receptors.
Received for publication January 18, 2000. Accepted for publication June 6, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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),
CD45RA+CD27+ (
),
CD45RA-CD27+ (
),
CD45RA+CD27- (
) were sorted and stimulated
for 1 h. Granzymes A and B were measured in the supernatants by
ELISA.
