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R2 Chain Is Regulated by Intracellular Trafficking in Human T Lymphocytes1
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Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy;
Department of Experimental Medicine and Pathology, "La Sapienza" University, Rome, Italy; and
Hôpital Necker-Enfants Malades, Institut National de la Santé et de la Recherche Médicale, Unité 429, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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R2) of the heterodimeric IFN- receptor on human T
lymphocytes have been investigated. We show that its surface expression
is low, whereas high cytoplasmic levels are found in both resting and
PHA-activated T lymphocytes. This low expression does not prevent
activated T cells from responding to IFN-, because it induces
IFN-regulatory factor 1 expression. Low surface IFN-R2 expression
appears to be due to recycling between cytoplasmic stores and the cell
surface, which does not depend on signals mediated by endogenous
IFN-, because IFN-R2 surface expression is low, and its
internalization is equally observed in patients with inherited
IFN-R1 gene deficiency and in healthy donors. Moreover, IFN-R2
internalization in T lymphoblasts from healthy donors was not affected
by the presence of anti-IFN--neutralizing or
anti-IFN-R1-blocking mAb. In conclusion, these data illustrate a
new mechanism whereby human T cells limit the surface expression of
IFN-R2 in a ligand-independent manner. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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plays an important role in regulating the proliferation, cytotoxic
differentiation (1), and apoptosis (2, 3) of
human T lymphocytes, and their response to it is directly influenced by
modulation of the chains of its membrane receptor (IFN-R) (4, 5). This is formed of at least two chains, namely p90, the
binding chain (IFN-R1), and p35, the accessory signal-transducing
chain (IFN-R2) (6).
IFNR1 is expressed on both lymphoid and nonlymphoid cells and is
almost ubiquitous (7). Its low surface expression on human
resting T cells transiently increases after their stimulation and also
increases progressively when they are protractedly activated. This
enhanced expression correlates with their susceptibility to
activation-induced apoptosis (4). Whereas mRNA and
cytoplasmic IFN-R1 protein are present in both human Th clones, Th2
clones express much more surface IFN-R1 than do Th1 clones. This
lower expression on Th1 clones appears to depend on internalization of
IFN-R1 after its binding with endogenously secreted IFN-
(8).
We have previously shown that mRNA for IFN-R2 and the corresponding
cytoplasmic protein are highly expressed in human Th1 and Th2 clones,
whereas their membrane expression of IFN-R2 is low. Despite this,
binding of IFN- on both clones induces the expression of IFN
response factor 1
(IRF-1)3 and
up-modulates the surface expression of class I glycoprotein of the MHC.
Moreover, when IFN-R2 surface expression is low, the signals
transduced by IFN- do not impair T cell viability
(8).
We have also found that stimulation via TCR in the absence of APC or
deprivation of IL-2 up-regulates IFN-R2 surface expression on both
Th1 and Th2 cells, which undergo apoptosis in the presence of IFN-.
The protein may be utilized autocrinally by Th1 cells, whereas Th2
cells require the presence of exogenous IFN-. This IFN--induced
apoptosis is prevented by mAb-blocking IFN-R1 or neutralizing
IFN- (8).
Elucidation of the mechanisms by which the surface expression of
IFN-R2 is kept low would show how T cells respond to signals that
determine their fate. Here, we investigate regulation of the
cytoplasmic and surface expression of the IFN-R2 chain and the role
of endogenous IFN- in this regulation. Both resting and activated
human T lymphocytes display high cytoplasmic IFN-R2 expression,
whereas its surface expression is very low. However, this does not
prevent activated T cells from responding to IFN-. Low IFN-R2
expression is the result of IFN--independent recycling between
cytoplasmic stores and cell surface, because it is equally observed in
activated T cells from: 1) donors carrying inherited IFN-R1 gene
deficiencies; 2) healthy donors; and 3) healthy and deficient donors
cultured in the presence of anti-IFN--neutralizing or
anti-IFN-R1-blocking mAb.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640, FCS, L-glutamine, penicillin,
streptomycin, gentamicin, and trypan blue dye were from Life
Technologies (Grand Island, NY); PHA, PMA, ionomycin, paraformaldehyde,
2-ME, EDTA, HEPES, PMSF, DTT, pepstatin, aprotinin, leupeptin,
benzamidine, glycerol, paraformaldehyde, propidium iodide, Tris, RNase
A, Tween 20, monensin, saponin, and FITC were from Sigma (St. Louis,
MO); DMSO, PBS, BSA, MgCl2, and sodium azide were
from Merck (Milan, Italy); KCl, NaCl, HCl, and ammonium peroxodisulfate
were from Fluka (Milan, Italy); SDS, acrylamide, bisacrylamide, and
N, N, N', N'-tetramethylethylenediamine were from Bio-Rad
(Rockland, ME); PE-conjugated mouse anti-human CD3 mAb, isotype
control FITC-conjugated mouse IgG2a, FITC-conjugated mouse IgG1, and
PE-conjugated mouse IgG1 were from Dako (Milan, Italy); rabbit
polyclonal anti-IRF-1 Ab was from Santa Cruz Biotechnology (Santa
Cruz, CA); HRP-conjugated goat anti-rabbit IgG was from Amersham
International (Little Chalfont, U.K.); PE-conjugated mouse IgG1
anti-IFN- and anti-CTLA-4 were from PharMingen (San Diego,
CA); recombinant human IL-2 was from EuroCetus (Milan, Italy);
recombinant human IFN- was from Genzyme (Milan, Italy).
mAbs to IFN-R and IFN-
Mouse R99 mAb is an IgG1 that specifically interacts with the
extracellular domain of human IFN-R1 and inhibits the binding of
IFN- (9). Mouse C.11 mAb is an IgG2a that specifically
interacts with the extracellular domain of the human IFN-R2
(8). C.11 mAb and R99 mAb were used in flow cytometry
as FITC-conjugated forms (C.11-FITC and R99-FITC, respectively).
Briefly, 10 mg/ml of each mAb were dialyzed against 0.1 M carbonate
buffer, pH 9.2, and conjugated with FITC in DMSO (1 mg/ml) for 4 h
at room temperature, separated by gel filtration on a Sephadex G-25
column, and dialyzed against PBS. Mouse 123 mAb is an IgG1 that
neutralizes the antiviral activity of IFN- (1).
IFN-R1-deficient patients
Two patients with a different IFN-R1 binding chain mutation
responsible for susceptibility to recurrent mycobacterial infections
(10) were used in this study. The mutation carried by one
patient is an insertion of one nucleotide at position 205 (205instT) in
exon 2 of the IFN-R1 gene (11). Nucleotides were
numbered starting with the ATG sequence initiating the coding region
according to the system of Beaudet and Tsui (12). This
null recessive mutation, located in the 5'-region of the gene encoding
the extracellular domain of the IFN-R1 gene, causes a premature stop
codon upstream from the segment encoding the transmembrane domain and
thus precludes cell surface expression of the receptor (E. Jouanguy and
J. L. Casanova, manuscript in preparation). The other patient
carried a 295del12 mutation. Four amino acids in the first domain of
the extracellular region are thus deleted (positions 99–102). One of
them (W85) is a known contact point for IFN- (13). The
patient is homozygous for this mutation. The amino acid substitution
did not prevent surface expression detected with two different
anti-IFN-R1 mAb (E. Jouanguy and J. L. Casanova, manuscript
in preparation). Impairment of receptor function in both patients was
shown by the fact that addition of scalar doses of IFN- (1 to 10,000
U/ml) to an EBV-transformed B cell line from these patients did not
result in Stat1 translocation and HLA class I up-regulation evaluated
by EMSA and flow cytometry (E. Jouanguy and J. L. Casanova,
manuscript in preparation).
Lymphocyte cultures
PBL from heparinized venous blood obtained from healthy or
IFN-R1-deficient donors were isolated by Lymphoprep (Ficoll type
400; Pharmacia, Uppsala, Sweden) gradient centrifugation and stimulated
(1 x 106/ml) with 2.5 µg/ml PHA. After 3
days, 20 U/ml recombinant human IL-2 were added to T lymphoblasts and
replaced every 3 days. The culture medium was RPMI 1640 containing
penicillin, streptomycin, gentamicin, and 2.5 x
10-5 M 2-ME, supplemented with 10% FCS. All the
in vitro cultures were performed at 37°C in a humidified 5%
CO2 atmosphere.
Western blot analysis
Nuclear cell extracts were prepared from PHA lymphoblasts as follows. Twenty million cells were suspended in 3 packed cell volumes of hypotonic buffer (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl) and protease inhibitors (PMSF, DTT, pepstatin, aprotinin, leupeptin, and benzamidine) and allowed to swell for 10 min on ice. Cells were then dounced 10 times with a Eppendorf minidouncer and transferred to centrifuge tubes. Nuclei were collected by centrifuging for 15 min at 2500 rpm at 4°C. The packed nuclear volume was resuspended in a volume of high salt buffer (20 mM HEPES (pH 7.9), 25% glycerol, 1.5 mM MgCl2, 0.4 M NaCl, 0.2 mM EDTA) and protease inhibitors. Nuclei were allowed to extract for 30 min on ice. Extracted nuclei were separated by centrifuging for 30 min at 14,500 rpm. Extracts (25 or 30 µg of protein) were separated on SDS-PAGE at 140 V on 8% miniprotein gels. Gels were electroblotted onto a polyvinylidene fluoride membrane (Bio-Rad) at 100 V for 1 h, which was then blocked with 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 0.05% Tween 20 (TTBS) and 5% nonfat dry milk overnight. The membranes were then incubated with 1:1000 rabbit polyclonal anti-IRF-1 Ab, washed with TTBS, and reacted with 1:1000 diluted HRP-conjugated goat anti-rabbit IgG. Ag-Ab reactions were visualized by enhanced chemiluminescence reagents according to the manufacturer’s instructions (ECL, Amersham International).
Flow cytometry
Membrane expression of IFN-R2 and IFN-R1 were investigated
on resting and PHA-activated T lymphocytes (T lymphoblasts) from
healthy donors and on T lymphoblasts from IFN-R1-deficient patients
with simultaneous incubation of 1 x 106
cells with C.11-FITC or R99-FITC and PE-conjugated anti-CD3 mAb
for 30 min at 4°C followed by two washes with cold PBS supplemented
with 0.2% BSA and 0.05% sodium azide (PBS-azide). For
intracytoplasmic detection of IFN-R2, 1 x
106 cells were stained with PE-conjugated
anti-CD3 mAb and then fixed with 100 ml cold 4% paraformaldehyde
in PBS and incubated overnight at 4°C. Fixed cells were washed twice
with PBS supplemented with 1% FCS, 0.1% sodium azide, and 0.1%
saponin and labeled with C.11-FITC as described above. Membrane and
cytoplasmic expression were evaluated on
CD3+-gated cells (positive to PE-conjugated
anti-CD3 mAb). In another set of experiments, cytoplasmic
expression of IFN-R2 of PHA-activated T lymphocytes was detected
simultaneously with that of IFN-. T lymphoblasts were cultured in
complete medium containing 2 µM monensin with or without 2.5 µg/ml
PHA, 10 ng/ml PMA, and 500 ng/ml ionomycin to stimulate maximal
cytokine production. After 4 h, 1 x
106 cells were recovered, fixed, and
permeabilized as described above. Then cells were labeled with
C.11-FITC and PE-conjugated anti-IFN-. Membrane and
intracellular protein expression were analyzed with a FACScan flow
cytometer (Becton Dickinson, Mountain View, CA).
Endocytosis experiments
In all endocytosis experiments, mAb were used at concentrations
of 10–20 µg/ml. Uptake of fluorescent mAb was then measured by flow
cytometry or confocal microscopy. To measure uptake of anti-CTLA-4
and C.11, T lymphoblasts from healthy donors and IFN-R1-deficient
patients were incubated with PE- or FITC-conjugated mAb from 1 to
4 h at 37°C or 4°C. In a few cases, endocytosis experiments
were performed by coincubating T lymphoblasts with an excess (100
µg/ml) of either anti-IFN--neutralizing (123) or
anti-IFN-R1(R99)-blocking mAb with C.11-FITC mAb. At
different times, 1 x 106 cells were rapidly
cooled to 4°C and washed twice with PBS-azide. Uptake of fluorescent
mAb was then measured by flow cytometry. To analyze membrane and
cytoplasmic expression of IFN-R2 in serial optical sections, cells
were incubated with C11-FITC at 4°C or at 37°C and then
counterstained with propidium iodide as described (2). To
check the overlap between cytoplasmic IFN-R2 and CTLA-4-containing
vesicles, 1 x 106 lymphoblasts were
incubated with PE-conjugated anti-CTLA-4 at 37°C for 3 h.
Then anti-CTLA-4 labeled cells were washed twice with cold
PBS-azide. For cytoplasmic detection of IFN-R2, cells were fixed and
permeabilized as described above and stained with C.11-FITC.
Internalization of fluorescent mAb was then measured by confocal
microscopy. Confocal microscopy was performed on a Zeiss LFM310 model
confocal microscope (488 nm argon laser and 543 nm helium-neon laser).
Green fluorescence was detected after excitation at 488 nm; red
fluorescence was detected after excitation at 543 nm. Images were
recorded as TIF files and processed (Adobe Photoshop, Mountain View,
CA) to subtract background and enhance lower and middle intensity
fluorescence. Potassium depletion was conducted as described
(14). Briefly, T lymphoblasts were washed and resuspended
in hypotonic medium (RPMI-water, 1:1). After 5 min at 37°C, they were
washed in medium without K+ (100 mM NaCl, 50 mM
HEPES, pH 7.4) and incubated at 37°C in the same medium with 1 mg/ml
BSA, with or without 10 mM KCl. After 25 min under these conditions,
C.11-FITC- or PE-conjugated mAb anti-CTLA-4 were added to the cells
at 37°C. After incubation at 37°C for 10 or 20 min, the cells were
rapidly cooled to 4°C and washed twice with PBS-azide. Cell
surface-associated mAb was then removed by two acid pH treatments (2
min at pH 3.0) as described (15). Time-dependent
endocytosis of both IFN-R2 and CTLA-4 was measured by flow
cytometry. More than 80% of the cells were alive at the end of each
experiment, as assessed by trypan blue exclusion.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R2 on resting and
activated T lymphocytes
Flow cytometry shows that IFN-R2 is barely detectable on the
surface of resting CD3+ PBL,
CD3+ PBL stimulated for 24 h with PHA, and
PHA-activated T lymphocytes (T lymphoblasts) cultured with IL-2 for 5,
10, and 15 days (Fig. 1
A, left
panels). By contrast, it is highly expressed in the cytoplasm of
resting, 24-h PHA-stimulated T lymphocytes and T lymphoblasts (Fig. 1A, right panels).
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, IFN- production
and IFN-R2 expression were simultaneously evaluated by flow
cytometry. Fig. 1
B shows that whereas 89% of T lymphoblasts
expressed IFN-R2 and did not secrete IFN-, 7% were
IFN-R2+ and secreted IFN- constitutively
(Fig. 1B, upper panels). When these cells were
cultured for 4 h in the presence of PHA, PMA, and ionomycin, these
percentages decreased to 58% and rose to 
30%, respectively (Fig. 1B, lower panels). By contrast with mouse Th cells, which
lose IFN-R2 expression after their differentiation into
IFN--producing cells (16, 17), these patterns indicate
that activated human T lymphoblasts still secrete IFN- although high
IFN-R2 chain levels are present in their cytoplasm.
Surface expression of IFN-R2 in T cells from patients carrying
inherited IFN-R1 gene deficiencies
When naive T cells from both wild-type mice and mice lacking the
IFN-R1 gene are differentiated into Th2 cells by IL-4, they express
equal surface levels of IFN-R2. Conversely, it is not expressed by
wild-type cells when they are differentiated into Th1 by IL-12 but
continues to be highly expressed in cells from deficient mice
(17). This indicates that IFN-R2 down-modulation may be
induced by the interaction between IFN- and its receptor. To see
whether low surface IFN-R2 expression in resting T cells and
lymphoblasts (see Fig. 1A) was due to down-modulation caused
by its binding to endogenous IFN-, we evaluated its surface
expression in T lymphoblasts from patients with a complete deficiency
in the IFN-R1 gene (E. Jouanguy and J. L. Casanova, manuscript
in preparation). If autocrine utilization of IFN- down-modulates
IFN-R2, activated T lymphocytes from these patients should express
more surface IFN-R2 than those from normal individuals. As shown in
Fig. 2, T lymphoblasts from healthy
donors displayed high IFN-R1 and almost undetectable IFN-R2
expression on their surface (Fig. 2, left panels). IFN-R2
levels were comparably low both in cells from a patient carrying a
mutation in the IFN- binding site impairing signal transduction
without affecting the ability to express surface IFN-R1 (Fig. 2, middle panels) and in those from another patient with a
mutation abolishing the expression of detectable surface IFN-R1
(Fig. 2, right panels). Cytoplasmic IFN-R2 expression was
as high in these patients as in normal T lymphoblasts (not shown).
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Because T lymphoblasts express low surface and high cytoplasmic
IFN-R2, the ability of the IFN-R complex to transduce signals was
evaluated by examining the nuclear induction of IRF-1 protein, the
transcriptional activation of which is induced by IFN-
(18). T lymphoblasts, which produce low levels of IFN-
(see Fig. 1B), constitutively expressed low levels of
nuclear IRF-1 (Fig. 3). IRF-1 expression
was increased by 1000 U/ml exogenous IFN- and inhibited by R99
mAb, which blocks the interaction of IFN- with IFN-R1
(9). A higher induction of IRF-1 was also observed when T
lymphoblasts were restimulated with PHA. Once again, IRF-1 induction
was caused by secreted IFN-, because it was abolished by R99
(Fig. 3).
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R2 between cytoplasmic stores and the cell
surface
IFN-R2 appears to be mainly expressed and stored in the
cytoplasm (Fig. 1). To determine whether it cycles between cytoplasmic
stores and the cell surface, we followed the accumulation of
FITC-conjugated anti-IFN-R2 (C.11-FITC) at 37°C or at 4°C
(Fig. 4A, upper panels). The
accumulation of C.11-FITC mAb was compared with that of
PE-conjugated-anti-CTLA-4 mAb (Fig. 4A, lower panels).
CTLA-4 is a low expressed surface receptor involved in the inhibition
of T cell functions (19) and recycles between the membrane
and the cytoplasm (20). A time-dependent uptake of C.11
and anti-CTLA-4 mAb was evident at 37°C only (not shown).
Similarly to that of anti-CTLA-4 (Fig. 4B, right panel),
the internalization rate of C.11 mAb was linear over a period of 4
h (Fig. 4B, left panel), and the amount of cell-associated
fluorescence doubled about every 1–2 h. A similar uptake of IFN-R2
by normal T lymphoblasts was observed when the
125I-conjugated-C.11 mAb was used, whereas it was
inhibited (95%) by a 1000-fold excess of cold C.11, indicating that it
was Ag specific (data not shown). Moreover, the increased amount of
cell-associated C.11, labeled with FITC or 125I,
was not released after 4 h incubation by treatment of cells at pH
3.0, suggesting that it had been internalized (data not shown).
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,
upper panels). At 37°C, on the other hand, there were
numerous, diffused fluorescence-tagged vesicles or endosomes (Fig. 5, lower panels).
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and cycling of IFN-R2
Internalization of IFN-R2 induced an at least 2-fold increase
of the cell-associated specific fluorescence of the C.11-FITC mAb
uptake after 4 h of incubation at 37°C (Fig. 4B). The
uptake of C.11-FITC mAb occurred equally on T lymphoblasts from normal
donors, from a patient with complete absence of surface IFN-R1 and
from a patient carrying an IFN-R1 gene mutation affecting the
IFN- binding site, but not surface expression (Table I). This demonstrates that surface
expression of the two IFN-R chains is independently regulated.
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R2 recycling is not influenced by
IFN- endogenously released by activating T cells was obtained by
evaluating the uptake of C.11-FITC mAb on normal T lymphoblasts in the
presence of a large excess of 123 or R99 mAb which neutralize
IFN- or hamper its interaction with IFN-R1, respectively
(1, 8). No differences were observed between healthy
treated and untreated T lymphoblasts (Table I).
Lastly, to rule out that the internalization of IFN-R2 on
IFN-R1-deficient T cells could result from its ability to bind
IFN-, the uptake of C.11-FITC mAb in the presence of a large excess
of IFN--neutralizing 123 mAb was also evaluated. No difference in
C.11 mAb uptake was observed in the presence or absence of 123 mAb
(Table I). These data indicated that IFN-R2 internalization on human
T cell is independent of endogenous IFN- and is not influenced by
the abilty to bind either IFN-R1 or IFN-R2.
Similarities between the cytoplasmic expression of IFN-R2 and
CTLA-4
The possible colocalization of IFN-R2 and CTLA-4 was
investigated by incubating T lymphoblasts for 3 h at 37°C with
PE-conjugated anti-CTLA-4 mAb to label CTLA-4-containing endosomes.
Anti-CTLA-4-labeled cells were then fixed, permeabilized, and stained
with C.11-FITC to detect cytoplasmic IFN-R2 expression. Confocal
microscopy showed that CTLA-4 expression was focal and polarized,
whereas vesicles containing IFN-R2 were widely diffused in the
cytoplasm. Nevertheless, there was a significant overlap between
CTLA-4-containing vesicles and cytoplasmic IFN-R2 (Fig. 6).
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R2 internalization by clathrin-mediated endocytosis
Potassium depletion blocks the assembly of coated pits and
prevents endocytosis of receptors that utilize clathrin for
internalization (14). To discover whether IFN-R2, like
CTLA-4 (20, 21, 22), recycles in clathrin-coated pits, the
time-dependent effects of potassium depletion on both CTLA-4 and
IFN-R2 internalization were examined.
T lymphoblasts were incubated for 20 min at 37°C in the presence of
C.11-FITC or PE-conjugated anti-CTLA-4 mAb in medium containing or
lacking potassium. The surface-associated fluorescence of the recovered
cells was stripped by treating them at pH 3.0, and cell-associated
fluorescence, representing the internalized mAb only, was analyzed by
flow cytometry (Fig. 7). In the presence
of potassium, there was a time-dependent increase of internalization of
both IFN-R2 (Fig. 7, left panel) and CTLA-4 (Fig. 7, right panel), whereas in its absence it was inhibited.
Potassium depletion blocked the internalization of CTLA-4 by 60%
and IFN-R2 by 35%.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R2 is prevalently expressed in the
cytoplasm of both resting and activated human T lymphocytes,
irrespective of their ability to produce IFN-. Its limited surface
expression appears to be due to recycling between cytoplasmic stores
and the cell surface.
Uptake of anti-IFN-R2 mAb without accumulation at the cell
surface, in fact, is evidence of this traffic. Cytoplasmic levels of
anti-IFN-R2 accumulate over time and are higher than those at
the surface, suggesting that endocytosis is rapid compared with the
export of IFN-R2 to the surface. This pattern of IFN-R2
distribution and internalization was equally observed in T cells from
patients in which the IFN-R1 gene is mutated and transduction of the
IFN- signal is impaired. Thus, internalization of IFN-R2 in human
T lymphocytes is a constitutive mechanism that does not require
IFN-. This conclusion was further confirmed by the observation that
IFN-R2 internalization in normal T lymphoblasts was not affected by
blocking the interaction between endogenous IFN- and its receptor.
The possibility that IFN-R2 was internalized after binding with
endogenous IFN- was also ruled out, because in T cells from
IFN-R1-deficient patients the uptake of anti-IFN-R2 mAb was
not affected by coculture with anti-IFN--neutralizing
mAb.
Through this mechanism, a few IFN-R2 molecules are continuously
expressed on the surface of activated T lymphocytes and allow a few
heterodimeric receptors to be engaged by IFN-, resulting in
induction of low levels of IRF-1 without triggering apoptosis.
Nevertheless, IRF-1 expression does not provide insight into the
mechanisms of IFN-R2 internalization, but merely underscores the
fact that the IFN-R complex is functional even if the membrane
levels of IFN-R2 are very low. By contrast, higher levels of IRF-1
are specifically induced by IFN- when T lymphocytes are restimulated
with PHA and express high surface levels of IFN-R2 (4, 8), and this increase is inhibited by anti-IFN-R1 mAb.
Thus, the number of functional receptors seems to be a limiting factor
for IFN--induced apoptosis only, because its triggering requires
both high IFN-R2 surface expression (4, 5, 8, 23) and
IRF-1 induction (24, 25, 26). Because IFN-R2 is
constitutively expressed in their cytoplasm, human T lymphocytes never
lose the ability to respond to IFN-. This suggests that their
proliferation or apoptosis response is critically governed by
modulation of IFN-R2 chain trafficking, and this in turn may be
altered by TCR engagement (4, 8), IL-2 (8),
negative growth regulators (5), or nitric oxide
(23).
Although the present data suggest that an ongoing T cell can respond to
IFN- by activating the Jak-Stat pathway even if its IFN-R2
surface expression is low, the possibility that a weak acting,
non-IRF-1-activating signal may be induced by IFN- binding to
IFN-R cannot be completely ruled out. Because IFN-R2 associates
with IFN-R1 at the cell surface, increasing the affinity of the
IFN- for the IFN-R complex (27), the transduction of
an IFN--mediated low affinity signal by IFN-R2 may be the result
of the modulation of the density of its membrane expression, rather
than a consequence of its ability to bind IFN-. A U937 myeloid cell
clone chronically infected with HIV has been shown to display a
selective defect of IFN- in activating the Jak-Stat pathway and to
induce IRF-1 expression associated with absent surface but abundant
cytoplasmic IFN-R2 expression. In these defective U937 cells,
IFN- induces IFN-stimulated gene factor 3 (28). The
possible existence of a second pathway triggered by IFN- in the
absence of IFN-R2 surface expression in T cells and involving
IFN-stimulated gene factor 3 is currently being investigated in our
laboratory.
Our data on IFN-R2 internalization were obtained with divalent mAb,
which can give rise to patching and capping induced by ligand
multivalency (20). Although we did not use monovalent Fab
fragments to avoid this problem, recycling of IFN-R2 was confirmed
by additional data. Partial inhibition of anti-IFN-R2 mAb uptake
by potassium depletion, in fact, indicates that clathrin-coated pits
are involved in this two-way traffic, suggesting that it is regulated
in the same way as CTLA-4, which is mainly located in endosomes
involved in the well-characterized endocytic pathway including
transferrin and its receptor (29). Our confocal microscopy
data suggest that only a fraction of IFN-R2 molecules are in
vesicles colocalizing with CTLA-4 and corresponding to early endosomes
(30), whereas the rest are probably loaded into vesicles
corresponding to late endosomes (31).
CTLA-4 undergoes clathrin-mediated endocytosis and associates
specifically with AP50, the medium subunit of the clathrin-associated
protein complex AP-2 (22). The sequence
Tyr-x-x-z (where x stands for any amino acid and
z stands for a large hydrophobic amino acid) is a frequent
subtype of consensus internalization motif observed in many receptors
rapidly internalized and delivered to endosomes, including the
transferrin receptor, EGFR, and CTLA-4 (32).
Interestingly, analysis of the amino acid sequence of IFN-R2 gene
revealed the presence of the sequence 273-Tyr-Arg-Gly-Leu-276-COOH
within its cytoplasmic domain. This further suggests that the two
molecules might be associated with the same clathrin-associated protein
complex controlling their internalization.
Limited IFN-R2 distribution seems to be a feature of T cells only.
Its surface expression is high on normal and malignant B and myeloid
cells, which undergo apoptosis when exposed to IFN- (P. Bernabei and
F. Novelli, manuscript in preparation). These data suggest that cell
type-specific IFN-R2 internalization is a homeostatic physiological
function that may modulate the growth and apoptosis of hemopoietic
cells. Like CTLA-4, specific signals within the cytoplasmic domain of
IFN-R2 may be required for selective internalization into coated
vescicles with the involvement of cell type-specific adaptors binding
the cytoplasmic domain (22). Alternatively, the same
specific adaptor could bind different IFN-R2 intracellular domain
isoforms resulting from a cell-specific mRNA splicing. Different
cell-specific IL-12Rß1 isoforms, in fact, have been shown to
transduce different pathways in response to IL-12 (33). We
are currently investigating the molecular mechanisms of IFN-R2
internalization.
Ligand-independent restriction of surface expression by an ongoing T
cell to avoid inhibitory signals is not confined to IFN-R2 but
involves many molecules having a major role in T cell inactivation such
as CTLA-4 (20, 21, 22), Fas ligand (34, 35), and
Fas (36), all of which show a typical cellular
compartmentalization into recycling vesicles containing other molecules
involved in inhibition of T cell function, such as Granzyme B
(20).
Mouse Th cell differentiation is associated with contrasting responses
to cytokines: Th1 cells selectively retain a positive response to
IL-12; and Th2 cells respond negatively to IFN- (37).
The antiproliferative effect of IFN- on mouse Th2 cells
(38) is due to their ability to express IFN-R2
(16). Lack of IFN-R2 makes Th1 cells resistant to
IFN- by preventing transduction of its signals (16),
and this resistance results from cellular desensitization induced by
exposure to IFN- (17).
Differences between human and mouse Th subsets are not restricted to
IFN-R2 expression. Both human subsets secrete IL-10, whereas in the
mouse IL-10 is regarded as a Th2 cytokine (39). Moreover,
during the differentiation of mouse Th2 cells, the ability to transduce
signals mediated by IL-12 is rapidly lost. This would appear to be a
mechanism for their stable commitment to the Th2 phenotype
(37). In contrast, established human Th2 clones can still
be induced by IL-12 to produce IFN- (40). Our data
indicate that in humans down-regulation of surface IFN-R2 is mainly
involved in the prevention of IFN--mediated apoptosis by T cells
rather than an event intrinsically linked to the polarization of cells
to the Th1 lineage (41).
In conclusion, these findings illustrate a new mechanism by which human
T cells limit the apoptotic effect of IFN- in a ligand-independent
manner without preventing it from transducing its signal.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Francesco Novelli, Dipartimento di Scienze Cliniche e Biologiche, Università Torino, Ospedale San Luigi Gonzaga, 10043 Orbassano, Italy. E-mail address:
3 Abbreviation used in this paper: IRF-1, IFN response factor 1.
Received for publication May 26, 1999. Accepted for publication October 12, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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inhibits human T lymphocytes and natural killer cell activation. J. Immunol. 147:1445.[Abstract]
receptor on human T cells control whether IFN- promotes proliferation or apoptosis. J. Immunol. 152:496.[Abstract]
plays a critical role in induced cell death of effector T cells: a possible third mechanism of self tolerance. J. Exp. Med. 172:1735. is correlated with the differential expression of the 
and ß chains of its receptor. J. Immunol. 157:1935.[Abstract]
- and ß-chains of the IFN- receptor and triggers IFN- -mediated apoptosis of activated human T lymphocytes. J. Immunol. 161:2114. receptor: a paradigm for the multichain cytokine receptor. Cytokine Growth Factor Rev. 8:189.[Medline]
receptor in human tissues. Eur. J. Immunol. 22:2403.[Medline]
- and ß-chains of the IFN- receptor on human Th1 and Th2 clones. J. Immunol. 159:206.[Abstract]
receptor: mapping of epitopes recognized by neutralizing antibodies using native and recombinant receptor proteins. J. Biol. Chem. 265:6908.-receptor deficiency in an infant with fatal bacille Calmette-Guérin infection. N. Engl. J. Med. 335:1956. receptor. Cell 55:273.[Medline]
and its soluble high-affinity receptor. Nature 376:230.[Medline]
receptor ß chain and the prevention of interferon- signaling in Th1 cells. Science 269:245. receptor ß chain expression in T helper cell subsets. Science 270:1215. is partially mediated through interferon regulatory factor-1. Blood 86:3373.-dependent and IFN--independent induction of apoptosis. J. Immunol. 163:4182. due to phosphorylation of STAT-1 and STAT-3. Blood 90:4749. signals via a high-affinity multisubunit receptor complex that contains two types of polypeptide chain. Proc. Natl. Acad. Sci. USA 92:5401.- but not of IFN--induced JAK/STAT pathway in a subset of U937 clones prevents the antiretroviral effect of IFN- against HIV. J. Immunol. 162:323. in immune regulation. IFN- inhibits the proliferation of Th2 but not Th1 murine helper T lymphocytes. J. Immunol. 140:4245.[Abstract]
(IFN-) production during differentiation of human T helper (Th) cells and transient IFN- production in established Th2 clones. J. Exp. Med. 179:1273. receptor ß-chain expression. J. Immunol. 158:5627.[Abstract]
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) or without (
) potassium in the presence of
C.11-FITC or PE-anti-CTLA-4 mAb at 37°C as described in
Materials and Methods. Time-dependent endocytosis of
both mAb was measured by flow cytometry. Relative fluorescence was
evaluated as described in the legend of Fig. 4