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*
Department of Experimental Medicine, Human Anatomy Section, University of Genova,
Monoclonal Antibody Unit, National Institute for Cancer Research, Genova, Italy;
Department of Biomorphology, Human Anatomy Section, University of Messina, Messina, Italy; and
§
Immunex, Seattle, WA
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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110 kDa under
both reducing and nonreducing conditions (6, 7). The
inhibitory function of this molecule is mediated by tyrosine
phosphorylation of four immunoreceptor tyrosine-based inhibitory
motif-like sequences in its cytoplasmic tail. Tyrosine phosphorylation
leads to the recruitment of Src homology protein-1, Src homology
protein-2, and Src homology 2 domain-containing inositol phosphatase.
Recruitment and activation of these phosphatases down-regulates the
signaling mediated by activatory receptors (3, 8, 9).
Inhibition has been assessed by down-regulation of NK and T lymphocyte
cytolytic functions. Ligands for LIR-1/ILT2 are nonclassical class I
HLA-G protein (10), some alleles of HLA-A, and -B loci and
the human cytomegalovirus UL18 gene product, a viral homologue of
HLA-class I (6). The first domain of LIR-1/ILT2 interacts
with the relatively nonpolymorphic 
3 domain of HLA-class I and the
analogous region of UL18 (11). mAbs specific for
LIR-1/ILT2 have been produced, namely HP-F1, M402, and M405 (8, 12). In addition, mAb VMP55 and GHI/75, defined at the Fifth
Workshop on Leukocyte Ags as specific for CD85 (13, 14),
have been recently shown to recognize the LIR-1/ILT2 inhibitory
receptor (15), because anti-CD85 mAb react with ILT2
transfectants. In addition, peptide maps of the CD85 Ag are identical
with those of ILT2. This study demonstrates that CD85/LIR-1/ILT2 is present in the cytoplasm and on the surface of all T lymphocytes. The receptor is functional and regulates human T cells by inhibiting CD3/TCR-mediated activation of both CD4+ and CD8+ clones, and Ag recognition by CD8+ cells. In addition, CD85/LIR-1/ILT2 controls responses to recall Ags and, in low responders, its blocking by the HP-F1 mAb sharply increases T cell proliferation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBL isolated from heparinized venous blood on Ficoll density gradients, were cultured with PHA (5 µg/ml) in 24-well plates (Costar, Cambridge, MA); hrIL-2 (Pepro Tech EC, London, U.K.) at 50 U/ml final concentration was added on culture days 2 and 4. After 7 days, cells were harvested, depleted of CD4+ or CD8+ T lymphocytes by immunomagnetic negative selection with Dynabeads (Unipath SpA, Milan, Italy), and plated at a limiting dilution of 10 and 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 or 30 U/ml hrIL-2 (respectively for CD8+ and CD4+ T lymphocytes). Phenotype and monoclonality were assessed using anti-CD4, -CD8, and -Vß mAb (see below).
Generation of EBV-specific CD8+ T cell clones
Polyclonal cell lines were produced by stimulating 2 x 106 PBL with 5 x 105 autologous irradiated (6000 rad) B-EBV cells in 24-well culture plates. Responder cells were restimulated weekly by plating 1 x 106 cells and 3 x 105 irradiated B-EBV cells/well in complete media containing 50 U/ml IL-2, on day 2 and 4 after restimulation. After the third stimulation, cell lines were enriched for CD8+ T lymphocytes by negative selection with immunomagnetic beads (Unipath SpA). Clones were produced by plating T cells at a limiting dilution of 10 and 1 cell/well, in the presence of 3 x 104 autologous irradiated B-EBV cells (16). The immunophenotype of the clones was assessed by flow cytometry.
Antibodies
The mAbs 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-CD85 (clone GHI/75) (13, 14),
anti-CD152, (PharMingen, Hamburg, Germany), and anti-HLA-class
I W6/32 (American Type Culture Collection, Manassas, VA). The complete
panel of anti-TCR Vß mAb from the First Human TCR Monoclonal Ab
Workshop was kindly provided by Dr. Marco Londei (Imperial College,
School Medicine, London, U.K.). The anti-phosphotyrosine mAb, clone
PY-20 (Santa Cruz Biotechnology, Santa Cruz, CA), was used for
detection of phosphorylated proteins by Western blot. The anti-ILT2
HP-F1 mAb was kindly provided by Dr. Miguel Lopez-Botet (Servicio de
Inmunologia, Hospital Universitario de la Princesa, Madrid, Spain)
(8). The anti-LIR-1 mAb M402 and M405 were kindly
provided by Immunex (Seattle, WA) (6, 12).
Immunophenotyping
The surface phenotype of T cell clones was determined by flow cytometric analysis (FACScalibur; Becton Dickinson). The secondary reagent was PE- or FITC-labeled goat anti-mouse (GAM) antiserum (Southern Biotechnology Associates, Birmingham, AL). A total of 5 x 104 T cells 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. For surface two-color immunostaining, cells were first incubated with HP-F1 or M402 mAb followed by GAM-PE, and subsequently stained with FITC-conjugated anti-CD3, -CD4 or -CD8 mAb. For cytoplasmic immunostaining, both single- and two-color, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% saponin, before mAb labeling.
Cytoplasmic immunostaining
For detection of intracellular Ag, T cells were fixed for 10 min at 37°C with 4% paraformaldehyde in PBS. Cells were subsequently washed with PBS and stained with the HP-F1 mouse mAb in PBS containing 0.1% saponin. Secondary labeled reagents were tetramethylrhodamine isothiocyanate-conjugated GAM antiserum and Cy3-conjugated donkey anti-goat antiserum (Jackson ImmunoResearch, Technogenetics, Milano, Italy). After staining, T cells were washed with PBS and let adhere to polylisine-coated slides. Cells were then analyzed using a fluorescence (Leica DM/DR, Leica Microsystem, Heerbrugg, Switzerland) and a confocal microscope (Zeiss LSM 510, Hamburg, Germany). Negative controls were provided by T cells stained with secondary reagents alone, and by unrelated cells (COS and squamous epithelial cell lines). Images were recorded as TIF files and processed (Adobe Photoshop; Adobe System, San Jose, CA).
Surface labeling and immunoprecipitation
Cell surface proteins were labeled by lactoperoxidase-catalyzed iodination as described (17). A total of 20 x 106 cells were labeled with 1 mCi 125I for 15 min at room temperature, followed by one wash with 20 mM NaI in PBS. Alternatively, surface proteins were biotin labeled (18). Cells were lysed with 1 ml RIPA lysis buffer (10 mM NaH2PO4, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, with 150 mM NaCl, 1% Triton X-100, 100 µM sodium pervanadate, 1 mM PMSF, and 5 µg/ml leupeptin), and nuclei were discarded by centrifugation at 400 x g. After three 30-min preclearing cycles with protein G-Sepharose beads (GammaBind; Amersham Pharmacia Biotech, Amersham, U.K.), specific absorption was performed by incubating cell extracts with 10 µg/ml HP-F1 mAb and 20 µl protein G-Sepharose beads. Incubation was conducted by overnight rotation at 4°C. Sepharose was then thoroughly washed with lysis buffer, and bound material was eluted in SDS-PAGE sample buffer.
Immunoblotting
Nuclei-free extracts (200–600 µg/lane) or immunoprecipitated
proteins were fractionated by SDS-electrophoresis on 10%
polyacrylamide gels under reducing conditions, and then transferred
electrophoretically onto nitrocellulose membranes (Hybond C Extra;
Amersham). Membranes were blocked by overnight incubation at 4°C with
4% low fat milk in TBST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl,
0.005% Tween 20). The transferred Ags were then detected by incubating
the membranes with 1 to 5 µg/ml mAb in 2% milk-TBST, for 2 h at
room temperature. The TBST buffer was used for washes and as the
diluent of secondary reagents. Biotin-conjugated GAM 
-light chain
and peroxidase-labeled streptavidin were used according to the
manufacturer’s instructions (Southern Biotechnology Associates,
Birmingham, AL). Peroxidase activity was revealed by enhanced
chemiluminescence (Amersham) and autoradiography (Hyperfilm;
Amersham).
mRNA analysis by RT-PCR
Total RNA was extracted from 5 x 106
to 107 cells using the RNA Clean solution (TIB
Molbiol, Genova, Italy), according to the manufacturer’s instructions.
One microgram of total RNA was transcribed into cDNA by incubating it
at 42°C for 1 h with 20 pmol of oligo(dT) (Roche Molecular
Biochemicals, Basel, Switzerland), 500 µM dNTPs (Roche Molecular
Biochemicals), 30 U of RNase inhibitor (5 Prime 
3 Prime, Boulder,
CO), and 200 U Moloney murine leukemia virus reverse transcriptase
(Life Technologies, Gaithersburg, MD) in a total volume of 20
µl.
To check mRNA expression of CD85/LIR-1/ILT2 gene, the following primers were synthesized: ILT2 forward, 5'-ATGACCCCCATCCTCACGGTC-3' and ILT2 reverse, 5'-CTGCACCGAGAGGGAGACTC-3'. As controls, the following primers were used: G3PDH forward, 5'-ACATCGCTCAGAACACCTATGG-3', G3PDH reverse, 5'-GGGTCTACATGGCAACTGTGAG-3'.
One microliter of cDNA was amplified using 20 pmol of forward primer and 20 pmol of reverse primer, 200 µM of dNTPs, 1.5 mM MgCl2, 1.25 U platinum Taq (Life Technologies). The reaction was amplified in a Mastercycler Personal (Eppendorf GmbH, Hamburg, Germany). The following profiles were used: ILT2, one time at 95°C for 2 min; 30–40 times at 94°C for 45 s, 63°C for 30 s, or 72°C for 1 min); one time at 72°C for 5 min; G3PDH, one time at 95°C for 2 min; 30 times at 94°C for 45 s, 60°C for 30 s, or 72°C for 1 min); one time at 72°C for 5 min. Ten microliters of the product were run in a 1.2% agarose gel and stained with ethidium bromide.
Cytotoxicity assays
The ability of various mAb to trigger or to inhibit the cytolytic activity of T cell clones was measured in a conventional 4-h 51Cr release assay. Briefly, target cells (the murine mastocytoma cell line P815, or the autologous and allogeneic B-EBV cells) were labeled for 1 h with 51Cr (100 µCi/106 cells), washed twice with PBS, resuspended in RPMI 1640 with 10% FCS, and plated at 5 x 103 cells per well in 96-well U-bottom plates. In the redirected killing assay, effector cells were plated in triplicate at 2:1 (E:T) ratios in the presence of one of the following mAb: anti-CD3, anti-CD4, HP-F1, and anti-CD152. To evaluate the inhibitory effect of the ILT2, effector cells were pretreated for 10 min at 4°C with the mAb; subsequently, P815 cells were added to each well together with the stimulatory mAb (anti-CD3).
HP-F1 or anti-CD152 mAb were included in the cytolytic test performed with autologous and allogeneic 51Cr-labeled B-EBV cells. In this assay, a cross-linking of HP-F1 or CD152 was obtained using HP-F1 or anti-CD152 mAb followed by GAM antiserum (Southern Biotechnology Associates).
After 4 h, 100 µl of supernatant were collected from each well and analyzed in a gamma counter for 51Cr release. Percent of specific lysis was calculated as: [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100.
Proliferation assays
Proliferative responses were evaluated by culturing 104 CD4+ clonal T lymphocytes in the presence of 105 irradiated PBL (5000 rad) in 0.2 ml complete medium, in 96-well flat-bottom microtiter plates. At the same time, the stimulatory effect of anti-CD3 mAb and the activity of HP-F1 mAb or of anti-CD152 mAb, and of their cross-linking on the proliferation rate were tested.
Cultures were pulsed with 0.5 µCi [3H]thymidine (Amersham) on day 2 and harvested 18 h later. The dry filters were counted in a gamma counter with scintillation fluid. Results are given as mean values of triplicate cultures.
PBL (4 x 105/well) were cultured in complete medium in 96-well flat-bottom microtiter plates. Cell proliferation was evaluated after stimulation with recall Ags (tetanus toxoid, 5 µg/ml; Candida albicans bodies, 3 x 105/ml; purified protein derivative, 10 µg/ml), and anti-CD3 mAb (1 ng/ml) as the positive control. In the same wells, HP-F1 or anti-CD152 mAb were added in the presence or absence of GAM antiserum as cross-linker. Plates were incubated at 37°C for 4 days and [3H]thymidine was present during the last 18 h of culture.
Ca2+ mobilization assay
To determine cytoplasmic free intracellular Ca2+ concentrations, cells were stained with acetoxymethylester of fura-2 (1 µM final concentration) (Sigma, St. Louis, MO). The fluorescence of the cellular suspension (106 cells) was monitored at 37°C with an LS-5 Spectrofluorometer (Perkin-Elmer, Pomona, CA). The excitation wavelength was 345 nm and fluorescence emission was measured at 496 nm. Stimulation was performed with HP-F1, anti-CD3, and anti-CD4 mAb, followed by a GAM antiserum (Jackson ImmunoResearch, West Grove, PA) as a cross-linker. The concentration of free intracellular Ca2+ was calculated by the method of Grynkiewicz (19).
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Results |
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The HP-F1 mAb has been used with the aim of identifying novel
molecules that inhibit T cell functions. The expression of the Ag
recognized by this mAb has been investigated in T cells in various
experimental conditions. The mAb has been shown to be specific for ILT2
in a variety of leukocytes (monocytes, dendritic cells, B lymphocytes,
and NK cells) (8). Two-color immunofluorescence analyses
were performed in PBL from 10 normal donors. Surface
HP-F1+ T lymphocytes were a fraction of all
CD3+ cells (mean 19 ± 7%). Among
CD8+ T lymphocytes, HP-F1+
cells were 23 ± 13%, and among CD4+
lymphocytes HP-F1+ cells were 12 ± 7%. Results
from PBL of one representative donor are shown in Fig. 1
A.
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ß+CD8+
and the large majority displayed a bimodal expression of surface ILT2.
In three clones, the expression of the Ag recognized by the HP-F1 mAb
was detected in all cells (see below, and Fig. 3, clone BE1), and in
five clones surface expression of ILT2 was undetectable by
immunofluorescence. To prove that cell cultures, albeit bimodal for
ILT2 expression, were monoclonal, CD8+ CTL clones
were screened using a panel of mAb specific for TCR Vß segments. All
cell cultures expressed a single Vß segment and were therefore
monoclonal. An example is shown in Fig. 1
B. Three
CD3+8+TCRß+
clones are positive for three distinct Vß segments, although
displaying a bimodal expression of HP-F1.
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C). In clone AK2, the percentage of
HP-F1+ cells ranged between 26% and 44%, and in
clone CO1 it varied from 1% to 35%. Surface expression of ILT2 was also analyzed in CD3+CD4+ clones. Only 4 of 34 clones were positive for surface staining with the HP-F1 mAb and the percentage of positive cells ranged from 17% to 49%.
ILT2 is localized in the cytoplasm of all T lymphocytes
To assess whether or not T cells express ILT2 in their cytoplasm,
seven CD8+ clones with variable surface
expression of ILT2, and six CD4+ clones that were
consistently negative for surface ILT2 were selected. Cells were fixed
with paraformaldehyde, permeabilized with saponin, and stained with the
HP-F1 mAb followed by secondary reagents (see Materials and
Methods). A cytoplasmic staining was detected in all cells of all
T cell clones. Two CD4+ and two
CD8+ clones are shown (Fig. 2A). To further support the
contention that ILT2 is present in the cytoplasm of all T cells,
resting T lymphocytes purified from peripheral blood were stained as
above, and ILT2 was found in the cytoplasm of all cells (not shown). To
reinforce the finding of a cytoplasmic localization of ILT2, FACS
analyses of fixed and permeabilized cells were performed after staining
with the HP-F1 mAb (Fig. 2B). All T cell clones analyzed,
independently of their surface expression, showed the presence of ILT2
in the cytoplasm of all cells. The Ag was also detected in all resting
T lymphocytes, and a negative control was provided by A431 cells (Fig. 2B).
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Because LIR-1, CD85, and ILT2 are coded by the same gene, in
addition to the HP-F1 mAb, three anti-CD85/LIR-1/ILT2 mAb, namely
the anti-CD85 mAb GHI/75, and two anti-LIR-1 mAb, M402 and
M405, were used for surface staining of T cell clones (15
CD4+ and 20 CD8+). Three
CD4+ T cell clones that were negative for surface
expression when the HP-F1 mAb was used, and four representative
CD8+ clones, two with a bimodal expression of
HP-F1, one negative and one positive on all cells, are shown (Fig. 3). A positive control is provided by the
NK cell line NKL, in which all cells are positive when the entire panel
of anti-CD85/LIR-1/ILT2 mAb is used. The pattern of reactivity of
the HP-F1, anti-CD85 and M405 mAb with T cell clones was identical,
as these Abs stained a proportion of cells only. In contrast, the M402
mAb reacted with all cells of all T cell clones. These data indicate
that CD85/LIR-1/ILT2 is expressed on the surface of all T cell clones,
although the amount of the Ag may differ among clones, as shown by
differences in fluorescence intensity.
Expression of CD85/LIR-1/ILT2 in resting peripheral blood T lymphocytes
To evaluate the expression of CD85/LIR-1/ILT2 in resting T
lymphocytes, a comparison between surface vs cytoplasmatic
immunofluorescence with HP-F1 and M402 mAb was performed using PBL from
eight healthy donors (Table I). In all
donors, surface expression of the Ag recognized by the HP-F1 mAb was
consistently lower than that of the Ag recognized by the M402 mAb.
Different percentages of positive cells detected by M402 mAb surface
staining among the different donors are due to the different mean
fluorescence intensity rather than to a bimodal distribution of M402
expression. In contrast, cytoplasmic staining with both mAb showed a
large proportion of positive cells in all donors. In one representative
donor (i.e., BC 4), a comparison between surface vs cytoplasmatic
immunofluorescence with HP-F1 and M402 mAb was performed in a two-color
fluorescence assay to evaluate the distribution of CD85/LIR-1/ILT2 in T
lymphocytes, in CD4+, and in
CD8+ cells (Fig. 4). Surface staining revealed that all T
cells express on their surface the CD85/LIR-1/ILT2 molecule using the
M402 mAb. Moreover, the CD85/LIR-1/ILT2 Ag was detected in the
cytoplasm of all T lymphocytes by the HP-F1 mAb.
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Immunoprecipitation with HP-F1, M402, M405, and anti-CD85 mAb
from a 125I surface labeled clone AK2 and from
the control NKL cell line, which is consistently positive for the
CD85/LIR-1/ILT2 Ag, revealed, under reducing conditions, the same band
of 116 kDa with all mAb (Fig. 5A). In addition, eight T cell
clones (five CD8+ and three
CD4+) were analyzed by Western blot. A band of
116 kDa consistent with the presence of CD85/LIR-1/ILT2 was detected in
all clones using the M402 mAb (Fig. 5B) and the
anti-CD85 and HP-F1 mAb (not shown). Negative controls in this
experiment were provided by the epithelial cell lines A431, HeLa, and
by COS cells. Occasionally, additional bands of 95 and 75 kDa were
observed, which may be the products of CD85/LIR-1/ILT2 proteolysis. It
is of note that in CD4+ clones, relatively to the
protein content, a much fainter band was observed in comparison with
that yielded by the NKL cell line and by the BE1
CD8+ T cell clone (Fig. 5B). The
intensity of the bands detected by Western blotting is thus consistent
with the fluorescence intensity of surface staining yielded by the
M402 mAb.
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C). To reinforce this concept, we
performed sequential immunoprecipitation of surface biotin-labeled
proteins. All surface CD85/LIR-1/ILT2 molecules were removed from clone
BE1 by HP-F1 mAb and thus were undetectable by M402 mAb (Fig. 5D). In the same experiment, M402 immunoprecipitated a
116-kDa band from BE1 cells that were not precleared with HP-F1 mAb
(Fig. 5D, right).
To determine whether or not the band of 116 kDa is phosphorylated,
cells were treated with Na-pervanadate. Ags immunoprecipitated by the
HP-F1 mAb were identified with an anti-phosphotyrosine mAb in the
Western blot assay. In all clones, the 116-kDa band was phosphorylated
(Fig. 5E), and in some clones it was associated with a
phosphorylated band of 75 kDa, that was particularly evident in clone
AK2. Negative controls in this experiment were represented by A431,
HeLa, and COS cell lines.
Analysis of CD85/LIR-1/ILT2 mRNA expression in T cell clones
Expression of mRNA coding for CD85/LIR-1/ILT2 was analyzed by
RT-PCR using ILT2-specific primers. In 12 T cell clones, 6
CD4+ and 6 CD8+, the mRNA
coding for CD85/LIR-1/ILT2 has been detected (Fig. 6, upper panels). It is of
note that, in the NK cell line NKL and in clone BE1, the amplification
required only 30 cycles; in the remaining T cell clones an
amplification product was detected only after 35–40 cycles. To rule
out the presence of feeder contaminants (monocytes, NK cells, B cells),
the T cell clones were analyzed 2 mo after the last restimulation. In
several epithelial cell lines, CD85/LIR-1/ILT2 mRNA was undetectable
(Fig. 6, lower panels).
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To demonstrate that CD85/LIR-1/ILT2 cross-linking by the HP-F1 mAb inhibits CTL activation induced via CD3, 30 cytolytic T cell clones were obtained from PBL of normal donors and tested in a redirected killing assay using the murine P815 cell target. Nineteen CTL clones were obtained following PHA stimulation, and the remaining 11 were Ag (EBV) specific.
All of the CTL clones lysed poorly P815 cells at an E:T ratio of 2:1.
In contrast, addition of anti-CD3 mAb in the assay yielded a
substantial lysis of the target. The HP-F1 mAb, cross-linked by P815
cells, in combination with the activatory anti-CD3 mAb, inhibited
target cell lysis in the majority of the clones. According to the level
of inhibition, three groups of CTL clones could be distinguished. In 17
clones, inhibition was >40%, in 10, it ranged between 40 and 20%,
and, in 3 clones, it was negligible (<20%) (Table II). To rule out that an inhibitory
effect on P815 cell lysis could be due to competition of the HP-F1 mAb
with the activatory mAb, an anti-CD4 mAb of the same isotype as
that of the HP-F1 mAb (i.e., 
1) was used. In these conditions,
inhibition of CD3/TCR activation did not occur (Table II). In addition,
the inhibitory effects of anti-CD152 (CTLA-4) and of HP-F1 mAb were
compared. The same levels of inhibition of CD3-mediated activation by
both molecules were observed for the majority of the clones. An
additive inhibition was never detected when HP-F1 and anti-CD152
mAb were used in combination (not shown). Furthermore, anti-CD85
and M402 mAb were tested to evaluate their ability to inhibit
CD3/TCR-induced activation in three CD8+ clones
(Fig. 7). Although these cross-linked mAb
exerted an inhibitory function, the HP-F1 mAb was most efficient in
this assay.
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Fourteen Ag-specific CD8+ T cell clones that
lyse autologous Ag presenting EBV-infected B-lymphocytes, but not
allogeneic B-EBV cells, were tested in a cytolytic assay. The HP-F1 mAb
that blocks interaction between the inhibitory receptor and its ligand
(rather than cross-linking the receptor) was added at the onset of the
assay. In all of these clones, lysis of specific targets was inhibited
by addition of anti-HLA-class I mAb, or of anti-CD3 mAb. When
the HP-F1 mAb was included in the assay, a >40% increase of lysis was
achieved in 8 of 14 clones. A control of the cross-linking ability of
HP-F1 is provided by addition of GAM antiserum. In these experimental
conditions, inhibition of specific lysis occurred. Similar results were
obtained using anti-CD85 and M402 mAb. CTL clones did not equally
share this property. However, a proportion of them (8/14) was
significantly inhibited (Fig. 8). This
suggests that a clonal heterogeneity also exists as for the ability of
CD85/LIR-1/ILT2 to inhibit CD3/TCR-mediated activation.
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To ascertain the regulatory role of CD85/LIR-1/ILT2 on CD3-induced
proliferation of CD4+ T cells, 30 clones were
obtained from PBL of normal donors following PHA stimulation. The
clones proliferated in the presence of anti-CD3 mAb. Simultaneous
addition in the assay of HP-F1 mAb cross-linked by GAM antiserum
resulted in >40% inhibition of proliferation in 14 of 30 clones; in
11 clones inhibition ranged between 20% and 40%, and in the remaining
5 it was <20% (Table III). At variance,
addition of non cross-linked HP-F1 mAb increased CD3-induced
proliferation. To rule out that the inhibitory effect was due to
competition of the added mAb with the activatory Ab, we used an
anti-CD4 mAb of the same isotype as that of HP-F1 (i.e., 1).
Inhibition of CD3/TCR-mediated activation was not observed when the
anti-CD4 mAb was cross-linked by GAM antiserum (Table III).
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CD85/LIR-1/ILT2 inhibits recall Ag-induced proliferation of PBL
To assess further the inhibitory function of the CD85/LIR-1/ILT2,
PBL from 15 healthy donors were stimulated using the recall Ags tetanus
toxoid, Candida albicans, and purified protein derivative.
Three groups of responders could be identified. In a first group of
four donors, the HP-F1 mAb did not affect significantly proliferative
responses induced by recall Ags. In a second group of seven donors, the
HP-F1 mAb increased proliferation by 20–40% in four individuals and
by >40% in three donors. Decreased proliferation was observed when
the HP-F1 mAb was cross-linked by GAM antiserum (range of decrease
between 20% and 40% in three donors, and >40% in four donors). In a
third group of four donors, a low response to recall Ags was detected,
but this was briskly increased by addition of the HP-F1 mAb (Fig. 9). As a control, the HP-F1 mAb was
cross-linked with GAM antiserum, and this abolished proliferation
completely. The same effects were observed when an anti-CD152 mAb
was used (Fig. 9).
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CD85/LIR-1/ILT2 down-regulates intracellular Ca2+ mobilization induced by CD3/TCR triggering
To provide additional experimental support to the ability of
CD85/LIR-1/ILT2 to inhibit activation mediated by the CD3/TCR complex,
CD3/TCR-induced Ca2+ mobilization was
investigated in conditions in which CD85/LIR-1/ILT2 was cross-linked by
the HP-F1 mAb and GAM antiserum. Three clones which were >40%
inhibited by the cross-linked HP-F1 mAb were selected and the
concentration of intracellular free Ca2+ after
activation via CD3/TCR in the absence or in the presence of HP-F1 mAb
was evaluated. In Fig. 10, a
representative clone is shown. A reduction of CD3/TCR-induced
Ca2+ mobilization was observed in all clones when
the HP-F1 mAb was simultaneously added. Altogether these data strongly
support the contention that CD85/LIR-1/ILT2 is a molecule that strongly
inhibits T cell functions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Both mRNA and protein contents are lower in CD4+ than in CD8+ clones. In clones in which all cells stain for surface HP-F1, the amount of CD85/LIR-1/ILT2 mRNA and protein is comparable to that detected in the NKL cell line.
The lack of relationship between CD85/LIR-1/ILT2 mRNA or protein expression and the degree of inhibition shown in a variety of experimental systems is worth of consideration. The presence of CD85/LIR-1/ILT2 has been clearly demonstrated in T cell clones that display a negligible inhibitory response and, on the contrary, clones with low amounts of CD85/LIR-1/ILT2 may be strongly inhibited. This observation, and a clonal heterogeneity as for the ability of the CD85/LIR-1/ILT2 to inhibit TCR-mediated activation, suggests that a control mechanism capable of modulating the level of inhibition operates. This is similar to that observed for the CD152 molecule which, again, is not equally distributed and effective among all CTL clones (16, 24, 25, 26). It is of note that the HP-F1 mAb, that in our experiments was the most efficient mAb in functional assays, was able to exert inhibition also in HP-F1surface- clones. There are two possible explanations for this finding: 1) surface immunofluorescence is less sensitive when compared with the functional assays, and thus a lower number of molecules is sufficient to determine inhibition; 2) similarly to that observed for CD152, a redistribution from the cytoplasm to the membrane may occur in the course of the functional assays (27).
The anti-CD85/LIR-1/ILT2 mAb display at least two patterns of reactivity, thus suggesting the existence of different epitopes recognized by the Abs. In particular, the HP-F1 mAb showed in some clones a bimodal distribution of surface immunofluorescence. At variance, in cytoplasmic immunofluorescence assays it stained all T lymphocytes. To explain this finding, it may be suggested that CD85/LIR-1/ILT2 molecules, to reach the lymphocyte surface, have to be linked to accessory molecules that could partially mask a epitope recognized by the HP-F1 mAb. Along this line, M402 mAb may recognize a different and unmasked epitope.
Inhibition mediated by CD85/LIR-1/ILT2 deals with relevant functions of T lymphocytes, namely Ag recognition by Ag-specific clones, T cell proliferation and the response to recall Ags. The ability of CD85/LIR-1/ILT2 to inhibit cytolytic functions could render tumor-specific T lymphocytes unable to recognize tumor cells, thus providing an escape mechanism that may favor cancer development. In contrast, inefficient inhibition mechanisms could sustain the onset of autoimmune disease. Furthermore, blocking of the CD85/LIR-1/ILT2-mediated signaling could boost immune responses to Ags that, in turn, might yield a more efficient rejection of tumor cells. By the same token, it is important to emphasize that, in four subjects, we observed low responses to recall Ags that were drastically increased when the HP-F1 mAb was included in the experimental system. This observation may help establish novel strategies to improve response to vaccines.
The presence, in all T lymphocytes, of an inhibitory receptor that broadly recognizes HLA-class I molecules and is able to modulate T cell function, opens new perspectives for studies of T cell anergy. Class I molecules are ubiquitary and they may be potentially functional in all body compartments. In addition, recognition by CD85/LIR-1/ILT2 of the human CMV protein certainly provides the pathogen with a mechanism to escape recognition by the immune system (9). It is of note that the human CMV infection is common in the normal population, but that it does not lead to the development of illness. At variance, in subjects with a compromised immune system, human CMV causes disease. A question arises from these observations, and from the ability of CD85/LIR-1/ILT2 to control efficiently T cell function. Does a control of the inhibition via CD85/LIR-1/ILT2 exist in vivo? Although we do not have an answer to this question, we may hypothesize the existence of a control of CD85/LIR-1/ILT2 surface expression. Alternatively, an activation receptor for HLA-class I could exist that might counterbalance the T cell inhibitory function exerted by CD85/LIR-1/ILT2.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ermanno Ciccone, Department of Experimental Medicine, Institute of Human Anatomy, University of Genova Via De Toni 14, 16132 Genova, Italy.
3 Abbreviation used in this paper: KIR, killer cell inhibitory receptor; LIR, leukocyte Ig-like receptor; ILT, Ig-like transcript; GAM, goat anti-mouse.
Received for publication April 17, 2000. Accepted for publication July 7, 2000.
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