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T Cells1
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Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, and
Multidisciplinary Oncology Center, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland;
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO; and
Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T
cells up-regulate 2B4 in vivo, and here we demonstrate that this
correlates with the acquisition of effector cell properties such as
granzyme B and perforin expression, rapid IFN-
production, and
down-regulation of the lymph node homing chemokine receptor CCR7. In
PBLs from healthy donors, cytomegalovirus-specific effector T cells
were 2B4 positive, whereas naive melanoma Ag (Melan-A/melanoma Ag
recognized by T cells-1)-specific T cells were 2B4 negative. In
melanoma patients, Melan-A-specific T cells up-regulated 2B4 in
parallel with in vivo differentiation. This occurred in PBLs after
vaccination with Melan-A peptides and in tumor-infiltrated lymph nodes,
likely through disease-associated activation of Melan-A-specific T
cells. Thus, 2B4 expression correlates with CD8+ T cell
differentiation in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 2B4 Ag was identified on all NK cells, all 
T cells, a subset
of CD8+ T cells, and all
CD14+ monocytes and basophils. 2B4 shares a
signaling pathway with SLAM, in that they both bind to the
SLAM-associated protein, which is mutated in immunodeficiency patients
with X-linked lymphoproliferative syndrome (9, 10, 11, 12). The
function of 2B4 has primarily been analyzed in NK cells, where 2B4
triggering was found to enhance cytotoxicity (5, 13, 14, 15)
and IFN- production (16). There is evidence that 2B4
also promotes T cell cytotoxicity, because it can mediate
non-MHC-restricted killing, and experiments with CD3 and 2B4 mAbs
suggest that 2B4 can enhance signals through the TCR
(6).
In this study, we analyzed CD8+
TCR+ cells ex vivo. We show that 2B4 was
predominantly expressed by differentiated T cells with high levels of
granzyme B and perforin and rapid production of IFN-. Naive resting
tumor Ag-specific CD8+ T cells were 2B4 negative,
whereas effector cells were 2B4 positive. Thus, in contrast to NK cells
and T cells, which express 2B4 constitutively (5, 6), the expression of this molecule is regulated in
CD8+ T cells and correlates with distinct
cellular differentiation stages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peripheral blood was obtained from healthy donors and patients
with advanced stage malignant melanoma. Tumor-infiltrated lymph nodes
(TILNs) were obtained from patients by surgical dissection. All
patients had no irradiation, chemotherapy, or immunotherapy during
several weeks before blood or lymph node withdrawal, except the
melanoma patient (LAU 337) shown in Fig. 6
, who received immunotherapy
consisting of peptides (Melan-A/MART-126–35
EAAGIGILTV and influenza matrix protein58–66
GILGFVFTL, each 100 µg) plus adjuvant SB-AS2 (600 µl) containing
the saponin QS21 and monophosphoryl lipid A in an oil-in-water
emulsion. Immunotherapy was given i.m. at weeks 0, 4, 8, and 12
(17). SB-AS2 was provided by SmithKline Beecham
(Rixensart, Belgium).
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PBLs were separated from heparinized blood by centrifugation over Ficoll-Paque (Pharmacia, Peapack, NJ), washed three times, and cryopreserved in RPMI 1640, 40% FCS, and 10% DMSO. Vials containing 5–10 x 106 cells were stored in liquid nitrogen. Lymph nodes were dissociated to single-cell suspensions in sterile RPMI 1640 supplemented with 10% FCS, washed, and cryopreserved as indicated above for PBLs. For tetramer analysis, the donors were selected on the basis of HLA-A2 Ag expression. PBLs from patient LAU 337 were obtained before (week -1) and 14 days after two vaccines (week 6) and after four vaccines (week 14).
mAbs and tetramers
mAbs were obtained from BD Biosciences (Mountain View, CA),
except anti-CD28FITC and
anti-TCRPE (Immunotech, Marseille,
France), anti-IFN-FITC and
anti-IL-2FITC (BD PharMingen, San Diego, CA),
anti-perforinFITC (Ancell, Bayport, MN),
anti-granzyme BFITC (Hölzel
Diagnostica, Köln, Germany), and goat
anti-ratAPC (Caltag Laboratories,
Burlingame, CA). The mouse Ab 1C7 (anti-2B4) was coupled with
the fluorescent dye PE-cyanin 5 (18). The rat IgG2a
mAb 3D12 (anti-CCR7) was obtained from R. Förster and M. Lipp
(Max Delbrueck Center for Molecular Medicine, Berlin, Germany)
(19). Tetramer complexes were synthesized as described
(20, 21, 22). The peptides used for the tetramers were
GILGFVFTL (influenza matrix protein58–66),
NLVPMVATV (CMV pp65495–503), and ELAGIGILTV
(Melan-A/MART-126–35, with A to L substitution
at position 2).
Flow cytometry
PBLs were thawed and, where indicated,
CD8+ T cells were purified in two rounds of
positive sorting using a Minimacs device (Miltenyi Biotec, Auburn, CA).
The resulting cells were >98%
CD3+CD8+. Cells (5–10
x 105) were stained with tetramers, FITC, PE,
PerCP, PE-Texas Red, PE-cyanin 5, or APC-labeled mAb conjugates
in 50 µl of PBS, 2% BSA and 0.2% azide, during 30 min at 4°C.
Samples with tetramers were stained by first incubating them with
tetramers (50 µg/ml) for 30 min at room temperature and then adding
the fluorescent Abs for 30 min at 4°C. Cells were washed once in the
same buffer and analyzed immediately in a FACSCalibur (four color) or
in a FACSVantage (five color) machine (BD Biosciences). The
"lymphocyte forward/side scatter gate" was set for a cell
population expressing >98% CD45 and <1% CD14 (as determined by
control samples labeled for CD45 and CD14). The staining for IFN-
and IL-2 was done before and after stimulation with 1 µg/ml PMA and
0.25 µg/ml ionomycin, followed by 10 µg/ml brefeldin A
(Sigma-Aldrich, St. Louis, MO) 1 h later. For all intracellular
stainings, cells were first stained with cell-surface mAbs, washed, and
then fixed and permeabilized in 1 ml of Orthopermeafix (Ortho
Diagnostics Systems, Raritan, NJ) for 30 min at room temperature, and
then washed twice and incubated with mAb conjugates for 30 min at
4°C. Data analysis was performed using CellQuest software (BD
Biosciences, San Jose, CA). The values obtained with
isotype-matched fluorescent control Abs were <1% in all cases.
Statistical comparisons were done using the t test for two
samples with equal variance.
FACS sorting and in vitro T cell stimulation
PBLs from healthy donors were thawed and kept in "CTL medium" overnight. CD8+ T cells were purified by positive sorting using a Minimacs device (Miltenyi Biotec). Using 2B4- and CD8-specific mAbs, FACS sorting with the FACSVantage machine was performed to obtain 2B4-positive and -negative CD8+ T cells. The sorted cells were stimulated with either CD3 (10 µg/ml) and CD28 (1 µg/ml) specific mAbs or PHA (1 µg/ml) and were incubated in the presence of rhIL-2 (150 U/ml), rhIL-7 (10 ng/ml), and irradiated allogeneic feeder cells. After 6 days, the percentages of 2B4 expression were determined through staining with anti-CD8 and anti-2B4 mAbs and flow cytometry.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells express 2B4
Many human CD8+ T cells up-regulate
HLA-DR (MHC class II) upon activation, and such cells can be
found in PBLs from healthy donors and patients. In
CD8+ TCR+ PBLs from a
healthy donor, most HLA-DR-positive T cells expressed 2B4, whereas the
2B4-negative cells were largely HLA-DR negative (Fig. 1a). A marker for effector
cells is neural cell adhesion molecule (CD56), which is expressed by
human CD8+ T cells that are cytolytic ex vivo
(23). We found that most CD56+ cells
were 2B4 positive (Fig. 1b). Recently, the chemokine
receptor CCR7 was identified to be expressed by naive and "central
memory" T cells residing in secondary lymphoid organs. In contrast,
CD8+ effector T cells were CCR7 negative
(19). Interestingly, most CCR7-negative cells expressed
2B4, in contrast to the CCR7-positive cells that were primarily 2B4
negative (Fig. 1c). Results representative for a group of 12
individuals confirmed that HLA-DR+,
CD56+, and CCR7- cells
were significantly increased in 2B4+ as compared
with 2B4- cells (Fig. 1, histograms).
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Effector CD8+ T cells express the cytotoxic
molecules granzyme B and perforin, which are released during cytolytic
effector activity leading to target cell lysis (24, 25).
Intracellular staining in fixed CD8+ PBLs
revealed that high levels of granzyme B and perforin expression were
essentially restricted to 2B4-positive cells (Fig. 2a). This correlation was
statistically significant when analyzed in six individuals (Fig. 2b). However, lower levels of positive perforin staining
were also found in the 2B4-negative cells (Fig. 2a).
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expression in 2B4-positive cells
In contrast to naive cells, effector CD8+ T
cells synthesize IFN- in <3 h after stimulation, whereas IL-2
production is less efficient (19, 26). We stimulated
CD8+ T cells with PMA and ionomycin and stained
intracellularly with FITC-labeled cytokine-specific Abs. Whereas
2B4+ cells rapidly became IFN- positive, it
took longer until the 2B4-negative cells expressed IFN-. In
contrast, IL-2 expression was preferentially found in 2B4-negative
cells (Fig. 2
c).
CD8+ T cell differentiation and 2B4 expression
Upon Ag encounter, CD8+ T cells
differentiate from naive to "memory" and effector cells. During
this process, they down-regulate CD45RA (27) and CCR7
(19), and these two markers are useful to separate naive
cells (RA+CCR7+) from
"memory" cells
(RA-CCR7+) and effector
cells (CCR7-). Fig. 3a shows that these three
populations differed with respect to 2B4 expression, whereby naive
cells were mostly negative, effector cells were positive, and
"memory" cells were intermediate. Analysis of 2B4 expression in 11
individuals revealed a statistically significant difference between the
three populations (Fig. 3b). Because effector cells may
re-express the CD45RA isoform (28), we also compared 2B4
expression in CCR7-RA+ vs
CCR7-RA- cells and found
comparable mean fluorescent intensities (54.8 vs 64.1; data not
shown).
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To test for changes in 2B4 expression upon in vitro stimulation,
we sorted CD8+ T cells from PBLs of healthy
donors in 2B4-negative and -positive populations (Fig. 4). Subsequently, the cells were
stimulated in vitro with anti-CD3 and anti-CD28 mAbs or with
PHA. After 6 days, 2B4 was up-regulated in cells from all cultures. The
mean values from four healthy donors showed that 2B4 up-regulation was
significant (Fig. 4d). Thus, in vitro stimulation of
CD8+ PBLs induced surface expression of
2B4.
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To investigate Ag-specific T cells, we prepared fluorescent
HLA-A*0201 tetramers for ex vivo analysis of T cells specific for the
tumor Ag Melan-A/MART-1 and the viral Ags influenza matrix protein and
CMV protein pp65. In healthy donors, Melan-A-specific T cells are found
at relatively high frequencies, even though they are not activated
(i.e., they are CD45RA+,
CCR7+, CD28+, and
HLA-DR-) (17, 29). These cells are
generally considered naive and resting, and our analysis showed that
most were 2B4 negative (Fig. 5a). In the majority of
healthy adults, influenza-specific cells are prototype "memory"
cells, and we found that they expressed 2B4 at low but measurable
levels. This was in contrast to CMV-specific cells, which were mostly
2B4 positive. CMV-specific CD8+ T cells are
preferentially found as effector cells with high cytolytic activity and
strong IFN- expression upon short-term stimulation (30, 31). The results from six healthy donors confirmed that 2B4
expression differed significantly between the T cells with the three
different Ag specificities (Fig. 5b).
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We have recently described that TILNs in melanoma patients often
contain Melan-A-specific T cells in large numbers as a result of tumor
Ag-driven T cell expansion (22, 29). Here, we analyzed
TILNs ex vivo from nine patients by flow cytometry and found measurable
numbers of Melan-A-specific cells in five of these nine patients. These
tumor Ag-specific cells were preferentially 2B4 positive (Fig. 6a). The summarized data from
Melan-A-specific cells in TILNs revealed that 2B4 was expressed at high
frequencies, in contrast to low expression of CCR7 (Fig. 6a). In contrast, Melan-A-specific T cells were largely
CCR7+ and 2B4- in
disease-free lymph nodes (data not shown). Recent evidence suggests
that increased tumor cell infiltration may correlate with increased
percentages of CCR7-negative T cells in TILNs (32). The
fact that tumor infiltration leads to progressive destruction of the
lymph node tissue architecture and progressive T cell activation and
differentiation to effector cells (22) fits well with the
finding that the CCR7 expression by Melan-A-specific T cells was low,
whereas 2B4 was up-regulated.
Expanded tumor Ag-specific CD8+ T cells are 2B4 positive
In PBLs from patient LAU 337, the percentages of Melan-A-specific
cells increased during immunotherapy. They were 0.1% of
CD8+ T cells 1 wk before peptide vaccination,
0.8% after two vaccine injections, and 2.3% after four vaccine
injections. On the surface of these Melan-A-specific cells, 2B4 was
partially expressed before immunotherapy and increased considerably
upon in vivo peptide immunization (Fig. 6b). Comparable
observations were made in three other melanoma patients with in vivo
CTL differentiation upon peptide immunization (data not shown). Thus,
in Melan-A-specific cells, 2B4 was not expressed in healthy donors
(Fig. 5) but progressively up-regulated with in vivo T cell activation
and differentiation in melanoma patients. Activated cells also
up-regulated HLA-DR (Fig. 6b), especially during the period
of strong cellular proliferation and expansion (17), but
this was only transient, despite subsequent differentiation to effector
cells and persistence at high cell numbers during the following 6 mo
(data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effector T cells were described to have a CD45RA+
CD27- phenotype (28). Our findings
are compatible with this observation, because
CD45RA+ CD27- cells are
primarily 2B4+ (18).
CD27- T cells are generally
2B4+ when analyzed in a larger number of
individuals, and the same is true for CD28- T
cells, which are largely overlapping with the
CD27- subset (data not shown).
CD28-/CD27-
CD8+ T cells produce large amounts of IFN- and
are cytolytic. These cells appear to be terminally differentiated
effector cells (33, 34), and their capacity to proliferate
and differentiate (back) to "memory" cells seems low. However,
CD28-/CD27-
CD8+ T cells are only partly overlapping with
CCR7- cells (data not shown), and further
studies are necessary to characterize the subpopulations defined by
these markers.
To study 2B4 expression in Ag-specific T cells, we applied tetramers and focused on three different Ags. 1) In CMV seropositive individuals, CMV-specific T cells are considerably expanded and persist at high numbers in circulation. They express cytokines and cytolytic function and are thus considered to be effector cells (30, 35). In contrast to CMV, 2) influenza virus infection is cleared after a few weeks, and influenza-specific cells persist in a not activated or only weakly activated state thereafter (20, 36). These so-called "memory" cells remain at increased numbers in circulation and are relatively rapidly reactivated upon Ag encounter, thus protecting from severe re-infections. 3) In healthy donors, Melan-A/MART-1-specific cells present all features of naive cells that have not been activated in vivo, despite the fact that they are present at relatively high numbers in the circulation (29). Although the distinction of naive, "memory," and effector cells is not clear cut and many issues remain unresolved (37, 38), the three populations specific for Melan-A, influenza, and CMV epitopes are among the best representatives to cover the spectrum of CD8+ T cell differentiation stages in humans. Using these three Ag-specific cell populations, our data show significant differences in 2B4 expression, suggesting that differentiation to effector cells is associated with 2B4 up-regulation.
In contrast with healthy individuals, melanoma patients may have Melan-A-specific T cells that are activated in vivo through Melan-A expressed by tumor cells (39, 40) and/or through peptide immunization (17). Such cells show a progressive shift in phenotype consisting of CCR7 down-regulation (32) and 2B4 up-regulation, which persisted even many weeks after immunotherapy, suggesting that this phenotype remains relatively stable. In contrast, HLA-DR expression, which was also up-regulated upon cellular activation, decreased afterward in parallel to reduced proliferative and telomerase activity (17). Thus, the data indicate that HLA-DR expression correlated with ongoing proliferative activity, whereas CCR7 down-regulation and 2B4 up-regulation correlated with differentiation to effector T cells, at least in part independently of the cellular activation status.
The primary ligand of 2B4 is CD48, which is ubiquitously expressed in humans (14, 41). NK cells constitutively express 2B4, and it has been demonstrated that signaling through 2B4 enhances NK cell-mediated cytotoxicity when triggered with specific Abs or with the ligand CD48. The function of 2B4 in NK cells is regulated, at least in part, by the signaling molecule SLAM-associated protein, which is up-regulated after 24 h and rapidly degraded during the second day after activation. Thus, despite the fact that NK cells express 2B4 constitutively, it may only be functional shortly after activation.
The finding that the CD2 family member 2B4 is expressed by effector T cells raises the hypothesis that 2B4 may promote effector cell function. Only little is known on 2B4 function in Ag-specific T cells. A major reason for this is that human Ag-specific T cells are difficult to assess ex vivo, due to the low numbers of available cells. We are currently generating target cells that express the appropriate HLA molecule with or without CD48 to investigate the influence of 2B4 triggering on MHC-restricted killing. As mentioned, many effector T cells down-regulate the costimulatory molecules CD28 (33, 34) and CD27 (28) and can thus no longer receive costimulatory signals known to enhance T cell activation (1, 2, 3). Instead, these cells up-regulate 2B4, which raises the possibility that effector function is promoted by 2B4-dependent signals that may allow efficient T cell activity in the absence of costimulation via CD28 and CD27.
In conclusion, the data establish 2B4 as a useful marker for
CD8+ T cell differentiation, which can be
applied to monitor and study human T cells in disease and vaccination.
The data suggest that 2B4-mediated co-activatory signals may be
involved in the control of cellular activation, expansion, and/or
effector function.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Daniel E. Speiser, Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Hôpital Orthopédique, Niveau 5, Aile Est, Av. Pierre-Decker 4, CH-1005 Lausanne, Switzerland. E-mail address: daniel.speiser{at}hospvd.ch
3 Abbreviations used in this paper: SLAM, signaling lymphocytic activation molecule; TILN, tumor-infiltrated lymph node.
Received for publication April 11, 2001. Accepted for publication September 21, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells through surface 2B4. Eur. J. Immunol. 25:1117.[Medline]
production following 2B4 activation in human NK cells. In Vivo 14:625.[Medline]
secretion by circulating CD8 T lymphocytes: implications of a novel approach for T cell monitoring in infectious and malignant diseases. J. Immunol. 166:7634.This article has been cited by other articles:
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