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*
Department of Experimental Medicine, Institute of Human Anatomy, University of Genova, Genova, Italy;
National Institute for Cancer Research;
Advanced Biotechnology Center, Department of Immunology, S. Martino Hospital, Genova, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ß+CD3+8+ CTL clones,
activation induced by anti-CD3, anti-CD28, or anti-CD2 mAb
was inhibited by anti-CD152 mAb in a redirected killing assay. In
eight clones inhibition was >40%, in 10 it ranged between 20–40%,
and in four it was <20%. This suggests the existence of a clonal
heterogeneity as well as for the ability of CTLA-4 to inhibit
CD3/TCR-, CD28-, or CD2-mediated CTL activation. To support further
this contention, we used an experimental model based upon Ag-specific
CTL. Eight Ag-specific T cell clones that lyse autologous EBV-infected
B lymphocytes, but are unable to lyse allogeneic EBV-infected B
cell lines, were used in a cytolytic assay in which anti-CD152 mAb
or soluble recombinant receptor (i.e., CTLA-4 Ig) were included. In
this system, at variance from the redirected killing assay,
cross-linking of surface molecules by mAb does not occur. Thus,
addition of anti-CD152 mAb or of CTLA-4 Ig and anti-CD80/CD86
mAb to the assay should result in a blockade of receptor/ligand
interactions. As a consequence, inhibition of a negative signal, such
as that delivered via CD152, should enhance lysis. A >40% increment
of target cell lysis was achieved in three of eight clones studied.
Since it is not equally shared by all CTL clones, this feature also
appears to be clonally distributed. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To exert their effector functions (e.g., cytokine production or target cell killing), T cells must recognize Ags making use of their specific receptors (TCR). However, this event is not sufficient to activate virgin T cells at the time of priming by Ag, and coactivatory signals, such as those mediated by CD28 molecules, are mandatory for T cell activation (5). In the absence of coactivation, paradoxically, Ag recognition leads to anergy (4). This negative functional outcome could result from a prevalence of inhibitory signals.
At variance from the wide range of activatory molecules, only a few inhibitory molecules are known to be expressed by T lymphocytes, namely the killer cell inhibitory receptors for HLA class I and CD94-NKG2A (both types of receptors are typically found on CD3- NK cells) (6), and CD152 (3, 7), which was originally identified as a molecule present on CTL (8, 9). Killer cell inhibitory receptors and CD94-NKG2A are detected on only a small proportion of normal T cells, whereas CD152 is found in all activated T cells (10, 11).
The largest complement of CD152 molecules resides in the post-Golgi/endosomal compartment of activated T lymphocytes (12). When T cells interact with APC presenting a specific Ag or with a cell that has to be killed, CD152 is translocated to the plasma membrane at the site of cell-to-cell contact, where it interacts with CD80/CD86 molecules expressed by APC and, possibly, by putative CTL targets (10, 12, 13, 14). It should be noted that the same ligands, CD80/CD86, are also recognized by the coactivatory molecule CD28 (15, 16, 17). Thus, a first inhibitory mechanism could be provided by competition between CD28 and CD152 for the same ligands. Furthermore, it has been suggested that interaction between CD152 and CD80/CD86 triggers a phosphatase that dephosphorylates molecules of the TCR/CD3 activation cascade (7, 18). The balance between the strength of TCR/CD28 signaling and the down-regulatory activity of CD152 could determine whether T cells are activated or rendered anergic.
Most data on the inhibitory function exerted by CD152 have been gathered in studies of proliferation (4, 11, 19, 20, 21) or cytokine production (22, 23) by naive T lymphocytes during T cell priming. Here, we have investigated the role of CD152 in effector T cells. In particular, we have assessed the ability of CD152 to inhibit the cytolytic functions exerted by CTL clones obtained following PHA stimulation or Ag selection. We provide evidence that CD152 inhibits specific CTL functions. This property is not equally distributed among all CTL clones, thus suggesting that, with respect to this, a clonal heterogeneity exists.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mAb used for immunophenotypic analyses were anti-CD2
(Leu 5b), anti-CD3 (Leu 4), anti-CD4 (Leu 3a), anti-CD8
(Leu 2a), anti-TCR-/ß (anti-WT31; Becton Dickinson, San
Jose, CA), anti-CD45 (ALB12), anti-CD45RA (ALB11),
anti-CD45RO (UCHL.1), anti-CD11a (25.3), anti-CD11b
(Bear1), anti-CD18 (7E4; Immunotech, Luminy-Marseille, France),
anti-CD80 (BB1/B7-1), anti-CD86 (B70/B7-2), anti-CTLA-4
(anti-CD152; PharMingen, Hamburg, Germany), and anti-HLA class
I mAb W6/32 (American Type Culture Collection, Manassas, VA). mAb
CD28.2 (specific for CD28) and mAb CD2.1 and CD2.9 (specific for two
different CD2 epitopes and able, when used in combination, to activate
T cells) were provided by Dr. Daniel Olive (Laboratoire de Immunologie
Functionel et Moleculaire, Marseille, France).
Generation of nonspecific CD8+ T cell clones
PBMC, separated from heparinized venous blood on Ficoll density gradients, were cultured with PHA (5 µg/ml) in 24-well plates (Costar, Cambridge, MA); human rIL-2 (PeproTech, London, U.K.) at a 50 U/ml final concentration was added on days 2 and 4 of culture. After 7 days, cells were collected, run on a Ficoll gradient, depleted of CD4+ T lymphocytes by immunomagnetic negative selection with Dynabeads (Unipath, Milan, Italy), and plated at a limiting dilution of 10/1 cells/well. Cells were restimulated every 7–10 days with 1 x 105 irradiated allogeneic mononuclear cells/well in medium containing 5 µg/ml PHA and 50 U/ml IL-2. T cell clones were analyzed by immunofluorescence and flow cytometry as described below.
EBV-transformed B cell lines
PBMC (2 x 106/ml) were incubated for 2 h at 37°C with 2 ml of EBV-containing supernatant from the cell line B95-8 (American Type Culture Collection). Cells were washed twice with PBS and incubated in complete culture medium at 5 x 105 cells/ml in the presence of 1 µg/ml cyclosporin in 24-well plates. Cultures were expanded in flasks for 20–30 days.
Generation of EBV-specific CD8+ T cell clones
Polyclonal cell lines were generated by stimulating 2 x 106 PBMC with 5 x 105 autologous irradiated (6000 rad) B-EBV cells3 in 24-well plates (24). Responder cells were restimulated in weekly cycles by plating 1 x 106 cells and 3 x 105 irradiated B-EBV cells/well in complete medium containing IL-2 at a 50 U/ml final concentration on days 2 and 4 after restimulation. After the third stimulation, the cell lines were enriched for CD8+ T lymphocytes by negative selection with immunomagnetic beads (Unipath). Clones were produced by plating these cells at a limiting dilution of 10/1 cells/well, in the presence of 3 x 104 irradiated autologous B-EBV cells.
Immunophenotyping
The surface phenotype of T cell clones was determined by indirect immunofluorescence and flow cytometric analysis (FACScalibur, Becton Dickinson, San Jose, CA). The secondary reagent was phycoerythrin- or FITC-labeled goat anti-mouse Ig Ab (Southern Biotechnology Associates, Birmingham, AL). T cells (5 x 104) were incubated with specific mAb for 20 min at 4°C. Cells were washed twice with PBS, and the secondary labeled reagent was added. After incubation, cells were washed and fixed with 1% paraformaldehyde. Control cells were stained with the secondary reagent alone.
B-EBV cells stained with the fusion protein CTLA-4 Ig (a gift from Dr. Antonio Lanzavecchia, Basel Institute for Immunology, Basel, Switzerland) were further incubated with a FITC-labeled polyclonal goat anti-human Fc IgG (Southern Biotechnology Associates) and analyzed by flow cytometry.
Cytotoxicity assays
The abilities of various mAb to trigger or to inhibit the
cytolytic activity of T cell clones were measured in a conventional 4-h
51Cr release assay. Target cells (the murine mastocytoma
cell line P815 or the autologous B-EBV cells) were labeled for 1 h
with 51Cr (100 µCi/106 cells), washed twice
with PBS, resuspended in RPMI with 10% FCS, and plated at 5 x
103 cells/well in 96-well U-bottom plates. In the
redirected killing assay, effector cells were plated in triplicate at
various E:T cell ratios in the presence of one of the following mAb:
anti-CD3, anti-CD28, anti-CD2 (CD2.1 and CD2.9),
anti-CD4, or anti-CD152. To evaluate a possible inhibitory
effect of CD152, effector cells were pretreated for 20 min at 4°C
with the anti-CD152 mAb. After preincubation, target cells (P815)
were added to each well together with the stimulatory mAb
(anti-CD3, anti-CD28, anti-CD2, anti-CD3 and
anti-CD28, and anti-CD3 and anti-CD2). An anti-DR mAb
(Becton Dickinson) of the same isotype as that of anti-CD152 mAb
(i.e., 
2a) was used, in combination with anti-CD3 mAb, as a
control to rule out that the inhibitory effect was due to competition
of the added mAb with the activatory Ab on the P815 cell line.
mAb anti-CD152 or the fusion protein CTLA-4 Ig was included in the cytolytic test performed with autologous 51Cr-labeled B-EBV to block CD80/CD152 or CD86/CD152 interactions. In this assay, a cross-linking of CD152 was obtained using anti-CD152 mAb in combination with goat antiserum to murine Ig (Southern Biotechnology Associates).
After a 4-h culture period, 100 µl of supernatant was collected from each well and analyzed in a gamma counter for 51Cr release. The percent specific lysis was calculated as: [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To ascertain the regulatory role of CD152 in the activity of CTL,
22 cytolytic T cell clones were derived from PBL of normal donors.
Fourteen CTL clones obtained after PHA stimulation were thus
characterized by a random range of specificities. The remaining eight
CTL clones were Ag specific and were selected on the basis of their
ability to lyse autologous, but not allogeneic, B-EBV. All these clones
displayed a homogeneous surface phenotype and expressed CD3, CD8,
TCRß, and CD28, but CD152 was expressed at extremely low levels.
After cross-linking with appropriate mAb, CD152 inhibits CTL activation induced via CD3 in a redirected killing assay
We used the murine P815 cell line as the target cell in a
redirected cytotoxicity assay for two reasons. 1) the presence of Fc
receptors for 1, 2a, and 2b murine Ig isotypes on these cells
provides a useful model for a killing assay that makes use of mouse mAb
against human lymphocyte receptors. In addition, in this assay,
receptors are cross-linked and thus mimic their function. 2) Since P815
cells are of murine origin, ligands expressed by these cells do not
react with human receptors and therefore do not interfere in the
cytolytic assay (see Fig. 1
, left
panel).
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). The presence of mAb specific for
CD152 in combination with the activatory anti-CD3 mAb yielded
inhibition of target cell lysis in the majority of the clones.
According to the level of inhibition, three groups of CTL clones could
be distinguished. In eight clones inhibition was >40%, in 10 it
ranged between 20–40%, and in four it was negligible (<20%; Table I). Six representative clones from the three different groups are shown
in Fig. 2. In clones 25A and Ge3, anti-CD152 mAb yielded a
reduction of the cytolytic activity, obtained by addition of
anti-CD3 mAb, from 88 to 39% and from 62 to 29%, respectively. In
clones Co3 and 10A the reduction of the lysis was from 90 to 58% and
from 82 to 50%, respectively. At variance, in clones 28A and G1B a
significant reduction of lysis was not observed. To rule out that an
inhibitory effect was due to competition of the added mAb with the
activatory mAb on the P815 cell line, we used an anti-DR mAb of the
same isotype as that of anti-CD152 mAb (i.e., 2a) in combination
with anti-CD3 mAb. Under these experimental conditions, inhibition
of the CD3/TCR activation was not observed (Table I).
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In addition, we have monitored the behavior of CTL clones at different
times after Ag-specific or PHA restimulation. As shown in Table II, the inhibitory property of
anti-CD152 mAb is consistently maintained in time, and therefore,
all CTL clones belong stably to the respective arbitrary group.
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Inhibition of CD2- or CD28-induced activation by cross-linked CD152
Since CD152 inhibits CTL activation via CD3/TCR, we next
investigated whether this molecule is able to inhibit other activatory
pathways, such as those mediated by CD2 and CD28. Addition of
anti-CD28 mAb or of mAb CD2.1 and CD2.9 (specific for two different
epitopes of CD2 and able, when used in combination, to activate T
cells) in the redirected killing assay, significantly increased lysis
of P815 target cells. Anti-CD152 mAb, included in the cytolytic assay
in association with these activatory mAb, yielded variable levels of
target cell lysis inhibition. The level of inhibition was comparable to
that provided by anti-CD152 in association with anti-CD3 mAb
(see Fig. 2
). In addition, CD152 mAb
inhibit lysis when anti-CD2 or anti-CD28 mAb are used together
with anti-CD3 mAb. These data indicate that CD152 also inhibits
activatory pathways other than those mediated by CD3/TCR and, again,
that this property is clonally distributed.
Definition of a role for CD152 in the CTL-specific target cell lysis
Eight Ag-specific T cell clones that lyse autologous Ag-presenting
B-EBV lymphocytes, but not unrelated allogeneic B-EBV cells (not
shown), were used in a cytolytic assay. mAb specific for CD152, capable
of blocking the receptor/ligand interaction (rather than cross-linking
the receptor), were added at the onset of the test (see Fig. 1, right panel). In all these clones, lysis of specific targets
was inhibited by addition of anti-CD3 or anti-HLA class I mAb
(not shown). When anti-CD152 mAb was included in the assay, an
increment of >40% of the lysis was achieved in three of eight clones.
The cytolytic activities of these three clones are shown in Fig. 3. A control of the cross-linking
capability of anti-CD152 mAb is provided by addition of goat
anti-murine Ig. Under these experimental conditions, inhibition of
specific lysis was observed. It should be noted that while
cross-linking of CD152 leads to inhibition, blocking of receptor/ligand
interaction yields an enhancement of specific lysis. This feature is
not equally shared by all CTL clones, but it is significantly exhibited
by a proportion (three of eight) of them.
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B-EBV cell lines consistently express on their surface CD80 and
CD86, which are ligands for both CD28 and CD152. Thus, we studied the
role played by these molecules for the specific CTL-mediated lysis.
Soluble CD152 (i.e., CTLA-4 Ig) was included in the cytolytic assay.
This molecule competes with CD152 expressed by CTL clones for the
binding to CD80/CD86 present on B-EBV cell targets. Staining of B-EBV
cell lines by CTLA-4 Ig is significantly reduced by the simultaneous
addition of anti-CD80 and anti-CD86 mAb (see Fig. 4A).
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B). CTL clones used in this assay were the same as those
that showed an increase in specific lysis when anti-CD152 mAb were
added to the cytolytic test.
Identical results were obtained when anti-CD80 (IgM isotype) and
anti-CD86 (IgG2b isotype) mAb were added to the cytolytic assay in
combination (Fig. 4C). Addition of CTLA-4 Ig, anti-CD80,
or anti-CD86 to CTL clones that had shown a minimal increment of
specific lysis induced by anti-CD152 mAb produced a very weak
effect (not shown).
In conclusion, blocking CD152 ligands expressed by target cells leads to an increase in target cell lysis. This suggests that under physiologic conditions also, CD80 and CD86 could protect target cells from the lysis mediated by CTL clones in which an inhibitory function of CD152 is effective.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The majority of the data on the inhibitory function exerted by CD152 have been gathered by studies of proliferation or cytokine production of naive T lymphocytes during T cell priming. In our study we have investigated the role of CD152 during effector T cell functions. Namely, we assessed the ability of CD152 to inhibit the cytolysis exerted by CTL clones derived following PHA stimulation or by Ag selection. It should be noted that these CTL clones were used several weeks after Ag or mitogen stimulation.
In a redirected killing assay, CD152 cross-linking resulted in inhibition of the activation mediated by CD3, CD2, and CD28, also when these pathways were triggered in combination. It is of note that although some reports suggest a possible activatory role of CTLA-4 (25, 26, 27), in the redirected killing assay mAb specific for CTLA-4 failed to induce activation. A possible explanation for this apparently contradictory phenomenon is that the addition of anti-CD152 mAb or soluble CTLA-4 ligand, which are able to interfere with receptor/ligand interaction in the experimental models, leads to an activation resulting from a loss of inhibition. The ability of this inhibitory molecule to negatively regulate activatory pathways distinct from CD3 suggests that CD152 could inhibit the CTL activation that follows interaction between effector and target cells. In fact, this is a complex event that requires the involvement of several and distinct activatory molecules. To kill their target, T cells must recognize Ags specifically, making use of their CD3/TCR receptors. However, this is not sufficient to activate the effector cells, and the involvement of CD2, LFA-1, CD40 ligand, and other activatory molecules is required. Thus, the ability of CD152 to inhibit distinct activatory pathways suggests an involvement of this molecule in vivo during Ag recognition by effector CTL.
To lend support to this hypothesis, experiments with specific CTL were performed since this model mimics a physiologic condition. It should be noted that cross-linking of CD152 obtained by the addition of a goat antiserum to murine Ig to anti-CD152 mAb leads to inhibition of the specific lysis. On the contrary, addition to the cytolytic test of a mAb specific for the inhibitory receptor blocks the receptor-ligand interaction and enhances specific target cell lysis. These data were also supported by experiments performed with soluble CTLA4 Ig and with anti-CD80 and anti-CD86 mAb in which an increment of specific lysis was also observed. Thus, in our experimental model also, CD152 delivers inhibitory signals able to down-regulate the cytolytic activity of CTL clones. It should be noted that for three specific clones in which CD152-mediated inhibition prevails, the fusion protein CTLA-4 Ig increased lysis. This strongly suggests that, at least for these three clones, the fusion protein competes only with CD152 expressed by effector T cells and not with CD28. At variance, data in the literature indicate that during Ag priming of virgin T cells, CTLA-4 Ig induces anergy by subtraction of ligands to CD28 (17, 28). Thus, it is possible to foresee a different role played by CD28 during Ag priming of virgin T cells and during lysis of Ag-presenting target cells by effector CTL.
The ability of CD152 to inhibit cytolytic functions could render tumor-specific T lymphocytes unable to recognize tumor cells, thus providing a mechanism of escape that could allow tumor growth in cancer patients. The expression of ligands (i.e., CD80/CD86) on target cells is crucial for the protection from lysis mediated by CTL clones. Since the expression of these Ags was recently demonstrated on tumor cells, such as malignant B lymphocytes (29), the possibility of blocking interactions between CD80/CD86 and CD152 could provide a mechanism to overcome anergy of tumor Ag-specific CTL.
Inhibition of the cytolytic function of effector CTL by CD152 is not equally shared by all clones. In some clones, inhibition is prevalent; in others, activation predominates. This suggests the existence of a clonal heterogeneity. The mechanisms that regulate inhibition are presently unknown. The level of CD152 surface expression is equally low in all tested clones. The molecule is largely confined to the endosomal cell compartment. It is suggested that at the time of effector-target conjugate formation, endosomes fuse with the cell membrane in the area of cell-to-cell contact, and the molecule is therefore translocated into the plasma membrane (7, 13). A quantitative regulation of this mechanism could responsible for the different levels of inhibition.
The following questions arise from our observations. Is only a proportion of T lymphocytes rendered anergic by this mechanism or, on the contrary, do all T cells, sooner or later, become anergic following chronic antigenic challenge? Although we cannot provide an answer to these questions, this type of regulation could play a crucial role in autoimmunity (21, 30) and in the prevention of uncontrolled proliferation of T lymphocytes, as it occurs in CD152 knockout mice (31).
The lack, in our experimental system, of CTL clones showing complete inhibition, is probably due to the fact that CTL characterized by such an efficient CD152 activity are unable to proliferate and, thus, to undergo clonal expansion. Cell cycle and apoptosis analyses could answer this question. Presently, there is evidence that CD152 is capable of blocking the cell cycle in G1 (32, 33) or, alternatively, in a different model, that it leads to apoptosis of effector cells (34, 35).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ermanno Ciccone, Istituto di Anatomia Umana, Facoltá di Medicina e Chirurgia, Via De Toni 14, 16132 Genova, Italy.
3 Abbreviation used in this paper: B-EBV, EBV-infected B cells.
Received for publication July 2, 1998. Accepted for publication September 24, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, HLA class I;
, TCR;
, CD152;
, CD80/CD86.
