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Further Analysis of Interleukin-2 Receptor Subunit Expression on the Different
Human Peripheral Blood Mononuclear Cell Subsets
By Denis David, Lynda Bani, Jean-Louis Moreau, Christophe Demaison, Karine Sun, Ombretta Salvucci,
Takayuki Nakarai, Marianne de Montalembert, Salem Chouaı̈b, Marcel Joussemet,
Jerome Ritz, and Jacques Thèze
We have investigated the expression of the three components of the interleukin-2 receptor (IL-2Ra, IL-2Rb, and
IL-2Rg) on the surface of the various peripheral blood mononuclear cell (PBMC) subsets by flow cytometry analysis. The
PBMC were immediately isolated (ficoll) from blood collected on heparin as anticoagulant. The three IL-2R components are absent or only marginally detectable on CD4 T
lymphocytes. No expression of the IL-2R chains is found for
the B lymphocytes. In most donors, the three chains are not
detectable on CD8 T lymphocytes, but for a few of them,
IL-2Rb or IL-2Rg are clearly expressed. CD56 high (IL-2Ra1)
and CD56 low (IL-2Ra2) natural killer (NK) cells express
IL-2Rb, but not IL-2Rg. IL-2Rg is expressed by monocytes of
all donors although with variable intensity. When blood is
collected on other anticoagulants or when cells are isolated
1 day after collection, IL-2Ra, IL-2Rb, and IL-2Rg are largely
expressed on the surface of most PBMC. This observation
provides a possible explanation for divergent data previously reported on IL-2R expression. Finally, we show that
IL-2Rg, which is not detectable on the cell surface of lymphocytes, is nevertheless expressed and stored as an intracellular component. This result is in agreement with the constitutive expression of the IL-2Rg gene and suggests a specific
regulatory mechanism for IL-2Rg membrane translocation.
r 1998 by The American Society of Hematology.
I
blood sample, with significant induction of the IL-2R subunits
on the cell surface. Our experimental conditions also show that
IL-2Rg is present inside all lymphocyte subsets, but remains
undetectable on their surface.
NTERLEUKIN-2 (IL-2) was one of the first cytokines to be
discovered and characterized.1 IL-2 is a main cytokine in
the immune system, as it has pleiotropic effects on lymphocytes
including T, B, and natural killer (NK) cells, as well as on some
hematopoietic cells,2 and beneficial therapeutic roles of IL-2
have been reported.3-5
The effects of IL-2 are mediated through specific cell surface
receptors (IL-2R)6-8 comprising at least three subunits. The first
IL-2R component to be identified, IL-2Ra (CD25, Tac antigen),9,10 is a 55-kD protein that binds IL-2 with low affinity
(1028 mol/L). The mode of action of IL-2Ra and the role of
IL-2 on IL-2Ra gene expression have been reported.11-13 The
second component IL-2Rb (CD122), a 74-kD protein,14 and the
third component IL-2Rg (CD132), a 64-kD protein,15 belong to
the hematopoietin receptor family (for review, see Thèze16).
IL-2Ra resembles IL-15Ra.17 In humans, two functional receptors can transmit IL-2 signals. The high-affinity receptor (10-11
mol/L) is composed of the three IL-2R subunits. The intermediate receptor (10-9 mol/L) is formed by the association of IL-2Rb
and IL-2Rg. IL-2Rb not only belongs to IL-2R, but is also a
component of IL-15R, and IL-2Rg is shared by several cytokine
receptors: IL-4R, IL-7R, IL-9R, IL-15R (for review, see Thèze18).
Signal transduction requires heterodimerization of IL-2Rb with
IL-2Rg and involves p56lck, Shc, Syk, JAK1, and JAK3 tyrosine
kinases and STAT3 and STAT5 transcriptional activators.18-21
Various conflicting data have been reported concerning the
expression of the three IL-2R subunits on human peripheral
blood mononuclear cells (PBMC) from healthy individuals. For
example, some studies reported no detectable expression of
IL-2Ra on CD4 T lymphocytes,22,23 whereas others showed
positive IL-2Ra expression.24,25 Similar discrepancies have
been reported for IL-2Rb expression on monocytes.25-27 Furthermore, cell-surface expression of IL-2Rg on T lymphocytes were
found positive in some reports,25 but insignificant in others.28,29
We describe here a pattern of IL-2R component expression
using blood collected on heparin and freshly isolated PBMC.
We show that cells involved in acquired immunity express very
low levels of the IL-2R subunits, whereas cells of innate
immunity clearly express either IL-2Rb or IL-2Rg subunit. We
show that this profile is highly sensitive to the conditions of the
Blood, Vol 91, No 1 (January 1), 1998: pp 165-172
MATERIALS AND METHODS
Antibodies and reagents. Monoclonal antibodies (MoAbs) anti–IL2Ra (33B3, IgG2a) and anti–IL-2Rb (CF1, IgG1) were purchased from
Immunotech (Marseille, France). The properties of IgG MoAb anti–IL2Rg (3B5) were previously described.28
Mouse MoAbs anti-CD3 (UCHT1, IgG1), anti-CD4 (MT310, IgG1),
anti-CD8 (DK25, IgG1), anti-CD14 (Tük4, IgG2a), anti-CD19 (HD37,
IgG1), anti-CD20 (B-Ly1, IgG1), anti-CD56 (MOC-1, IgG1), antiCD71 (Ber-T9, IgG1), anti-Bcl-2 (124, IgG1) and isotype-matched
control MoAbs were purchased from DAKO A/S (Glostrup, Denmark).
When indicated these antibodies were labeled with R-Phycoerythrin
(PE). Anti-CD16 (3G8, IgG1) and anti-CD28 (IOT28, IgG1) were
obtained from Immunotech. Fluorescein isothiocyanate (FITC)conjugated Fab fragment goat antimouse IgG (H1L) was purchased
From the Unité d’Immunogénétique Cellulaire, Département
d’Immunologie, Institut Pasteur, Paris, France; Laboratoire d’Immunologie,
Institut Gustave Roussy, Villejuif, France; Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, MA; Site Transfusionnel de
l’Hôpital Necker-Enfants Malades, Paris, France; and Centre de
Transfusion Sanguine «Jean Julliard», Clamart, France.
Submitted February 5, 1997; accepted August 19, 1997.
Supported by grants from ANRS (Agence Nationale de Recherche sur
le SIDA, Paris, France) and National Institutes of Health Grant No.
CA41619. D.D. is a fellow of the SIDACTION (Fondation pour la
Recherche Médicale, Paris France). L.B. and C.D. are supported by the
ANRS and by the AFM (Agence Française contre les Myopathies, Paris,
France), respectively.
L.B. and J.-L. M. contributed equally to this work.
Address reprint requests to Jacques Thèze, MD, PhD, Unité
d’Immunogénétique Cellulaire, Département d’Immunologie, Institut
Pasteur, 25 & 28 rue du Dr Roux, 75724 Paris cedex 15, France.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9101-0014$3.00/0
165
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166
from Jackson Immunoresearch (West Grove, PA). Tri-color (FL3)conjugated anti-CD14 (Tük4) and isotype-matched control MoAb were
obtained from Caltag (Burlingame, CA). All these antibodies were used
at saturating concentrations.
Blood collection and PBMC preparation. Venous blood of healthy
donors (Centre de Transfusion Sanguine «Jean Julliard», Clamart,
France) and of hemochromatosis patients (Site Transfusionnel de
l’Hôpital Necker-Enfants Malades, Paris, France) was typically collected on sodique heparin as anticoagulant. At the day of blood sample,
PBMC were isolated by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). PBMC were then immediately stained and analyzed with threecolor cytometry as described below. When indicated, blood was
collected on either CPD (citrate phosphate dextrose) or ACD (acid
citrate dextrose), anticoagulants with divalent metal ion chelator
activity. In one set of experiments, PBMC were isolated by FicollHypaque from heparinized blood stored one day at 18°C in a dark room.
Flow cytometric analysis. Cells were analyzed by three-color flow
cytometry. PBMC were first incubated for 30 minutes on ice with
MoAbs for IL-2Ra, IL-2Rb, or IL-2Rg. Cells were also incubated with
MoAbs directed against markers specific for the different PBMC
subsets or for activation markers as indicated in the figures. After
washing, cells were incubated with FITC-conjugated Fab fragment goat
antimouse IgG (H1L) under the same conditions. For FL2 (PE) and
FL3 (Tri-color) staining, cells were incubated with CD4-PE and
CD14-FL3, CD20-PE and CD14-FL3, CD56-PE and CD14-FL3, or
with CD8-PE MoAbs and washed again. Cells were then fixed in 1%
paraformaldehyde in phosphate-buffered saline (PBS). A total of two
104 PBMC per sample was acquired for CD4, CD56, and CD8 cells and
of five 104 for CD20 cells. Analysis was performed with a FACScan
flow cytometer using CellQuest 3.1 software (Becton Dickinson,
Mountain View, CA).
Intracellular detection of IL-2Rg and Bcl-2 was performed as
previously described.30 Briefly, cells were fixed in 4% paraformaldehyde in PBS, washed, and permeabilized by a solution of 0.05%
(wt/vol) saponin detergent in PBS. Cells were then incubated with
anti–IL-2Rg chain or anti-Bcl2 MoAbs, washed, and stained with
FITC-conjugated goat antimouse Fab fragment. A cell-surface staining
was then performed as described above to discriminate the different
PBMC subsets. For intracellular IL-2Rg staining, the YT cell line and
an Epstein-Barr virus (EBV)-B cell line derived from an X-linked
severe combined immune deficiency (IL-2Rg mRNA2) child (patient
P2 in Hacein-Bey et al31) were used as positive and negative controls,
respectively.
mRNA detection in PBMC subsets. PBMC subsets were highly
purified by negative selection with magnetic beads (purity . 95%) as
previously described.30 Total RNA was then extracted and cDNA was
synthesized using an oligo(dT) primer. Polymerase chain reaction
(PCR) amplification was performed with already published specific
primers.30 PCR products were hybridized with specific g32-adenosine
triphosphate (ATP)-labeled oligonucleotides30 and staining was recorded with the PhosphoImager using the ImagQuant software.
Western blot analysis. Western blots were performed as previously
described.32,33 Briefly, lysates were prepared from PBMC subsets
purified as described above, by addition of sample buffer (125 mmol/L
Tris-HCl pH6.8, 2% sodium dodecyl sulfate (SDS), 10% b-mercaptoethanol, 10% glycerol, 0.01% bromophenol blue). YT and the IL-2Rg2
B-EBV cell lines were used as positive and negative controls, respectively. The protein samples (50 µg) were electrophoresed under
reducing conditions on an 10% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) gel and tranferred onto Immobilon P
nitrocellulose membranes (Millipore Corp, Bedford, MA). Filters were
blocked for 2 hours at room temperature (RT) with blocking buffer (7%
bovine serum albumin (BSA), 0.1% tween 20, 13 PBS) and subsequently incubated for 2 hours at RT in blocking buffer containing the
anti–IL-2Rg rabbit antiserum at a dilution of 1:1,000.34 Membranes
DAVID ET AL
were washed and incubated for 1.5 hours at RT in blocking buffer
containing 1:1,000 peroxidase-labeled goat antirabbit antibody (Biosys,
Compiègne, France) and extensively washed. ECL Western blotting
detection kit (Amersham International, Burchinghamshire, UK) was
used according to the manufacter’s instruction and the filters were
autoradiographed for a few seconds.
RESULTS
Analysis of IL-2Ra, IL-2Rb, and IL-2Rg expression on
monocytes and CD4 T lymphocytes. Expression of the three
IL-2R subunits on CD4 cells derived from nine healthy
individuals was examined; Fig 1 shows the results obtained
with a representative donor. The intensity of CD4 staining
allowed us to distinguish monocytes (CD4 low, CD141) from
CD4 T lymphocytes (CD4 high, CD281) (Fig 1). It was also
verified that monocytes also expressed CD33 and CD11b
markers and that CD4 lymphocytes were CD31 (data not
shown). Under these experimental conditions, monocytes clearly
expressed IL-2Rg (55% for the donor presented in Fig 1). The
intensity of IL-2Rg expression on monocytes varies according
to the donors (from 15% to 75%), resulting in a mean
expression of 38% in the group. IL-2Ra and IL-2Rb were not
detectable or expressed at very low levels depending on the
donor (mean expression in the group, 2% for IL-2Ra and 4%
for IL-2Rb). Concerning CD4 T lymphocytes, IL-2Ra, IL-2Rb,
and IL-2Rg were not detectable or sometimes detectable at very
low levels depending on the donor with mean expressions of
4%, 2%, and 4%, respectively. Under our experimental conditions, CD4 T lymphocytes were found to be in a resting stage as
Fig 1. Expression of IL-2Ra, IL-2Rb, and IL-2Rg by CD4 T lymphocytes and monocytes. PBMC from blood collected on heparin were
treated by MoAbs 33B3 (anti–IL-2Ra), CF1 (anti–IL-2Rb), and 3B5
(anti–IL-2Rg). Further characterization of the two populations was
achieved by treatment with anti-CD14 and anti-CD28 MoAbs. FITClabeled Fab fragment anti-IgG was used to stain the cells, followed by
PE-conjugated anti-CD4 MoAb. Quadrant settings distinguishing
positive immunofluorescence from background fluorescence were
determined by staining with isotype-matched control MoAbs: solid
horizontal line for lymphocytes and dotted horizontal line for monocytes. The vertical dotted line separates CD4 low (monocytes) from
CD4 high (lymphocytes) cells. The percentage of positive cells for the
different markers is indicated.
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INTERLEUKIN-2 RECEPTOR EXPRESSION BY PBMC
measured by size and by the absence of CD71 expression (data
not shown and see below).
B lymphocytes did not express IL-2Ra, IL-2Rb, and IL-2Rg
at their surface. Using three color-flow cytometry with antiCD20 MoAb on whole PBMC, we could clearly detect B
lymphocytes, which were also CD19, CD231 (see Fig 2 for a
representative analysis). For all donors, IL-2Ra, IL-2Rb, and
IL-2Rg were undetectable (mean expressions , 1%). Similar
results were obtained on PBMC enriched in B lymphocytes
after depletion of monocytes and CD3 T lymphocytes (data not
shown). Comparing the results obtained with CD4 T and B
lymphocytes, the mean expression of IL-2R subunits was
around 4% for CD4 T lymphocytes and always less than 1% for
B lymphocytes. This shows that for a few donors low levels of
IL-2R components on CD4 T lymphocytes were observed,
whereas these chains were never detectable on B lymphocytes
among the different donors.
High expression of IL-2Rb by NK cells. NK cells were
analyzed by three color-flow cytometry on PBMC from nine
healthy donors by studying the expression of NK-specific CD56
marker. In roughly one third of the donors, NK cells appeared
heterogeneous in terms of intensity of the CD56 marker (CD56
low NK population could be distinguished from CD56 high NK
population, Fig 3A). As previously reported,22 these two
populations expressed CD8 marker with comparable intensity,
but CD16 expression was higher in the CD56 low NK
population. IL-2Ra was only present in the CD56 high NK
population, whereas IL-2Rb was present in both. Moreover,
IL-2Rg was not detectable in CD56 low and CD56 high NK
cells (Fig 3A). For donors in which CD56 high NK cells could
not be identified, IL-2Ra and IL-2Rg were not detectable, or
present at very low levels (mean expression, 1% and 3%,
Fig 2. Expression of IL-2Ra, IL-2Rb, and IL-2Rg by B lymphocytes.
PBMC from blood collected on heparin were treated by MoAbs 33B3,
CF1, and 3B5. Characterization of the population was achieved by
treatment with anti-CD19 and anti-CD23 MoAbs. FITC-labeled Fab
fragment anti-IgG was used, followed by PE-conjugated anti-CD4 1
FL3-conjugated anti-CD14 MoAbs. CD141 monocytes were excluded
for easier analysis. Quadrant setting distinguishing positive immunofluorescence from background fluorescence was determined by staining with isotype-matched control MoAbs. The percentage of positive
cells for the different markers is indicated.
167
Fig 3. Expression of IL-2Ra, IL-2Rb, and IL-2Rg by NK cells. (A)
PBMC from blood collected on heparin were treated by MoAbs 33B3,
CF1, and 3B5. Further characterization of the population was achieved
by treatment with anti-CD8 and anti-CD16 MoAbs. FITC-labeled Fab
fragment anti-IgG was used, followed by PE-conjugated anti-CD56
MoAb 1 FL3-conjugated anti-CD14 MoAbs. CD141 cells were excluded for easier analysis. Quadrant setting distinguishing positive
immunofluorescence from background fluorescence was determined
by staining with isotype-matched control MoAbs. Vertical dotted line
separates CD56 low from CD56 high cells. The percentage of positive
cells for the different markers is indicated. (B) NK cells were highly
purified from PBMC as previously described.47 The resulting CD56
population was treated by MoAbs 33B3, CF1, and 3B5 followed by
FITC-labeled Fab fragment anti-IgG. The percentage of positive cells
for the different markers is indicated.
respectively), whereas IL-2Rb was always strongly expressed
(mean expression, 76%). IL-2R expression was also examined
in purified NK cells (Fig 3B): this population was also found
IL-2Ra2, IL-2Rb1, and IL-2Rg2.
Fine analysis of the expression of IL-2Ra, IL-2Rb, and
IL-2Rg by CD8 T lymphocytes. By analyzing PBMC of nine
donors with CD8 marker, two populations could be distinguished (Fig 4). CD8 low cells contained NK cells (ie, cells that
were CD561, IL-2Rb1, CD32, CD282). We examined CD8
high cells, which were also CD3 and CD281 (Fig 4A). On the
CD8 T lymphocytes from the majority of the donors, IL-2Ra,
IL-2Rb, and IL-2Rg could not be detected or were present at
very low levels (,5%) (Fig 4A). However, for the remainig
healthy donors, IL-2Rb or IL-2Rg were strongly expressed (Fig
4B). When these are included in the group of healthy donors, the
data are heterogeneous and the mean expression is then more
than 10% (Figs 5 and 6).
Expression of IL-2Rb and IL-2Rg on these different PBMC
subsets was also analyzed with anti–IL-2Rb MoAbs 341 and
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168
DAVID ET AL
Fig 4. Expression of IL-2Ra, IL-2Rb, and IL-2Rg by CD8 T lymphocytes. (A) PBMC from blood collected on heparin were treated by
MoAbs 33B3, CF1, and 3B5. Further characterization of the population
was achieved by treatment with anti-CD3 and anti-CD28 MoAbs.
FITC-labeled Fab fragment anti-IgG was used, followed by PEconjugated anti-CD8 MoAb. Quadrant setting distinguishing positive
immunofluorescence from background fluorescence was determined
by staining with isotype-matched control MoAbs. The percentage of
positive cells for the different markers is indicated. (B) Staining and
analysis was performed as in (A). The percentage of positive cells for
the different markers is shown for a group of nine individuals.
56135 and with anti–IL-2Rg MoAbs 3G11 and TUGh4,25,28
giving similar results (data not shown).
Changes in IL-2R expression when blood is collected on
Ca21 chelators. As shown in Fig 5A, CD4 T lymphocytes
from the blood of one donor collected on CPD clearly expressed
the three IL-2R chains. The appearance of the three IL-2R
subunits on CD4 T lymphocytes was observed when blood from
different patients was collected on CPD or ACD (Fig 5B, upper
panel). For example, with CPD, the mean expressions of
IL-2Ra, IL-2Rb, and IL-2Rg were 30%, 32%, and 37%,
respectively. When PBMC were isolated 1 day after the blood
sample was taken, a similar pattern of expression was found.
These results were significantly different from basal levels
obtained with heparin and PBMC isolated at day 0. These
results have been verified at the mRNA level (Fig 5C). CD4 T
lymphocytes purified from blood collected on CPD expressed
IL-2Ra and IL-2Rb mRNAs contrary to cells purified from
blood collected on heparin. Results obtained with IL-2Rg
mRNA are not shown because this mRNA is constitutively
transcribed.15,30 At the cell surface level, similar results were
obtained for CD8 T lymphocytes (Fig 5B, lower panel). On
these cells, the IL-2R expression due to the anticoagulant and/or
to the time effects was, however, more important compared with
CD4 T lymphocytes. Surprisingly, expression of CD71 activa-
Fig 5. Expression of the IL-2R by cells from blood collected with
different anticoagulants. (A) Blood of one donor was collected on CPD
and PBMC were isolated the same day as the blood sample. Staining
and analysis of CD4 T lymphocytes was performed as indicated in Fig
1 except that FL3-labeled anti-CD14 MoAb was used. (B) Blood was
collected on sodique heparin (n 5 9) or on CPD (n 5 3) or on ACD (n 5
3), and PBMC were isolated the same day as the blood sample (d 5 0)
or 1 day after (d 5 1) in some cases with sodique heparin (n 5 4).
Staining and analysis was performed as in (A) for CD4 T lymphocytes
and as indicated in Fig 4 for CD8 T lymphocytes. Significant differences are indicated as follows, a, P I .0001; b, .0001 F P I .001; c,
.001 F P I .01; and d, .01 F P F .05 (nonpaired t-test). (C) Blood was
collected on sodique heparin (H) or on CPD (C). CD4 T lymphocytes
and monocytes were purified from PBMC isolated at d50. IL-2Ra and
b specific mRNAs were measured as previously described.30 HPRT
detection is also shown as positive control; N, PCR negative control.
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INTERLEUKIN-2 RECEPTOR EXPRESSION BY PBMC
169
Fig 6. Expression of IL-2R subunits on the different PBMC subsets
in healthy donors and hemochromatosis patients. Blood was collected on sodique heparin and PBMC were isolated the same day as
the blood sample, for healthy donors (HD, n 5 9) and hemochromatosis patients (HHP, n 5 5). Staining and analysis was performed as in
Figs 1 to 4. Significant differences are indicated as in Fig 5.
tion marker was not affected under these conditions (Fig 5B),
with the exception of a small increase when PBMC were
purified 1 day after collection. Similarly, unchanged expression
was observed for CD69 and HLA-DR markers (data not
shown).
With monocytes, the use of other anticoagulants and/or
PBMC isolation at day 1 triggered the appearance of IL-2Ra
and IL-2Rb and the over-expression of IL-2Rg (Table 1). As for
CD4 T lymphocytes, the use of CPD also resulted in IL-2Ra
and IL-2Rb mRNA expression (Fig 5C). For B lymphocytes, a
low but significant appearance of the three IL-2R subunits was
observed (mean expression of 10% in the various cases, Table
1). On the other hand, the pattern of IL-2R subunit expression
on NK cells was not influenced by the experimental conditions
(Table 1). As for T lymphocytes, we found no concomitant
increase in CD71 expression except for B lymphocytes when
PBMC were prepared at day 1 (Table 1). Variations of CD69 in
the three subsets presented in Table 1, of CD16 on monocytes,
and of CD23 on B lymphocytes were insignificant (data not
shown).
Heparin did not impede IL-2R subunit detection. To verify
that heparin did not interfere with IL-2R subunit expression or
detection, we analyzed IL-2R chain expression in some pathologic situations with blood collected on sodique heparin and
PBMC isolated at day 0. Hemochromatosis is due to a defect in
iron metabolism and is easily treated by periodic blood-letting.
As shown in Fig 6, we found in this pathology that the
appearance of the three IL-2R subunits on CD4 T lymphocytes
(mean expressions, 40%, 42%, and 22% for IL-2Ra, IL-2Rb,
and IL-2Rg, respectively), B lymphocytes (13%, 31%, and
26%) and CD8 T lymphocytes (36%, 37%, and 45%). On
monocytes, there was appearance of IL-2Ra and IL-2Rb (26%
and 35%) and an increase in IL-2Rg expression (to 73%). In
hemochromatosis, the profile of IL-2R expression on NK cells
was not affected.
Intracellular expression of IL-2Rg in the different lymphocyte
populations. Due to an apparent contradiction between the
constitutive IL-2Rg gene expression and the absence of IL-2Rg
chain cell-surface detection in the lymphocyte subsets when
blood was collected on sodique heparin and PBMC was purified
at day 0, we analyzed the intracellular expression of this chain
on these subsets. Such experiments were performed for three
Table 1. Expression of CD71 and IL-2R Chains on Monocytes, B Lymphocytes, and NK Cells
Percentage of Positive Cells
IL-2 Receptor
Cell Type
Monocytes
B lymphocytes
NK cells
Anticoagulant/Time
Hep Na (d 5 0)
CPD (d 5 0)
ACD (d 5 0)
Hep Na (d 5 1)
Hep Na (d 5 0)
CPD (d 5 0)
ACD (d 5 0)
Hep Na (d 5 1)
Hep Na (d 5 0)
CPD (d 5 0)
ACD (d 5 0)
Hep Na (d 5 1)
CD71
a
b
g
6.6 6 5.1
1.5 6 1.4
6.9 6 1.4
5.3 6 5.5
2.1 6 1.5
1.1 6 1.5
3.9 6 3.4
7.6 6 1.5†
1.8 6 1.2
0.12 6 0.21
0.11 6 0.12
2.7 6 2.4
2.3 6 3.1
24 6 16*
57 6 19†
6.4 6 3.6‡
0.93 6 1.3
4.1 6 1.4§
14 6 4.8†
5.5 6 0.69†
1.5 6 1.3
0.61 6 0.85
0.33 6 0.58
1.6 6 1.1
4.5 6 5.2
32 6 2.8†
74 6 9.5†
16 6 4.8§
0.55 6 1.1
7.5 6 5.9§
24 6 13†
16 6 12*
76 6 11
83 6 6.6
70 6 12
81 6 17
38 6 23
61 6 19
60 6 23
71 6 15‡
0.81 6 1.3
5.2 6 2.0*
10 6 5.4*
11 6 9.1§
2.9 6 1.8
0.71 6 0.11
0.13 6 0.16
4.3 6 2.2
Staining and analysis of these PBMC subsets have been performed as in Figs 1 to 3.
*.0001 , P # .001.
†P # .0001.
‡.01 , P , .05.
§.001 , P # .01.
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170
DAVID ET AL
donors. The absence of IL-2Rg surface expression in the
different lymphocyte populations for these donors was verified
(data not shown). Figure 7A shows a representative donor. In
the four different PBMC populations we could clearly detect the
intracellular expression of IL-2Rg. This result has been confirmed for a purified population of NK cells (Fig 7A, lower
middle panel). Although the IL-2Rg subunit was present as
intracellular protein in these four lymphocyte subsets, the
IL-2Ra component was not detected and IL-2Rb chain was not
detected in B and T lymphocytes (data not shown). Intracellular
expression of IL-2Rg on the different PBMC subsets has been
verified by Western blot (Fig 7B). PBL (which represents all of
the PBMC subsets with nondetectable IL-2Rg expression on
cell surface) and purified CD4 T lymphocyte extracts showed
the characteristics 64-kD band also found in the YT cell line.
Results obtained with the monocyte population, which express
IL-2Rg on their surface, are also shown.
DISCUSSION
Analysis of freshly prepared PBMC from blood collected on
heparin allowed the identification of two group of cells: (1) T
and B lymphocytes involved in acquired immunity that do not
express the three IL-2R components, or express them at very
low levels, and (2) monocytes and NK cells classically considered as the cells of innate immunity that strongly express
Fig 7. Intracellular expression of IL-2Rg in the different lymphocyte populations. (A) After cell permeabilization, intracellular staining
was performed against IL-2Rg and Bcl-2 protein as positive control.
Two-color flow cytometry was performed on PBMC isolated from
blood collected on sodique heparin for the upper and lower left
panels. Results were confirmed for NK cells on purified CD56 cells as
indicated (lower middle panel). Lower right panel shows a positive
(YT) and a negative B-EBV cell line (B) derived from a XSCID child;
background controls are identical with these two cell lines. (B) Blood
was collected on sodique heparin and PBMC were isolated at d 5 0.
PBL, CD4 T lymphocytes and monocytes were purified and Western
blots performed on their lysates as explained in Materials and
Methods. Lane 1, YT cell line (positive control); lane 2, B-EBV cell line
from a XSCID patient (negative control); lane 3, PBMC; lane 4, PBL;
lane 5, monocytes; and lane 6, CD4 T lymphocytes.
IL-2Rg or IL-2Rb, respectively. On CD8 T lymphocytes and
monocytes, some variations are observed in IL-2Rb and IL-2Rg
expressions between healthy donors (Fig 4). The mRNA profile
from purified cells, detected by reverse transcriptase (RT)-PCR,
is in agreement with these results based on flow cytometry.
Indeed, CD4 T and B lymphocytes express only IL-2Rg
mRNA. NK cells express both IL-2Rb and IL-2Rg mRNAs,
whereas monocytes express only IL-2Rg mRNA. For CD8 T
lymphocytes, IL-2Rg mRNA is found and not IL-2Ra mRNA
whereas, as expected, IL-2Rb mRNA could be expressed,
depending on the donor (Karine Sun, unpublished data). The
pattern of expression of IL-2R subunits at the PBMC cellsurface is highly dependent on the preparation conditions of
blood and cells, whereas activation markers are apparently less
affected by these parameters. The use of freshly prepared
PBMC from heparinized blood allows us to clarify the question
of IL-2Rg expression. Indeed, IL-2Rg is not detectable on the
surface of T, B lymphocytes, and NK cells, but is nevertheless
clearly present as an intracellular protein. Our conditions also
allow monitoring of the appearance of IL-2R subunits in a
pathologic situation.
Various conflicting data have been reported about IL-2R
subunit expression on the different PBMC subsets. On CD4 T
lymphocytes, the data are divergent for the expression of the
three IL-2R chains.22-25,28,29,36,37 Contradictory results have also
been reported for IL-2Rb and IL-2Rg expression on CD8 T
lymphocytes.22,24,25,28,29,36,37 For the other PBMC subsets, the
differences are mainly focused on the expression of one IL-2R
component: IL-2Ra for B lymphocytes,25,36 IL-2Rb for monocytes,25-27,36,38 and IL-2Rg for NK cells.25,28,39 In most of these
reports, the method of blood collection and PBMC preparation
are not always well specified and in some reports, results
obtained from only one donor are presented.
Our results on the role of the used anticoagulant and of the
day of PBMC isolation may provide an explanation for some of
the discrepancies in the published data. Ca21 chelators are
typically used as anticoagulants. As CPD and ACD were Ca21
chelators, it seems paradoxical that use of these products results
in an increase of IL-2R subunit expression, whereas Ca21 is
necessary for cell activation. However, Vanham et al40 have also
shown a strong expression of IL-2Ra and IL-2Rb on both CD4
and CD8 T lymphocytes with blood collected on EDTA, another
Ca21 chelator. Moreover, blood conservation temperature41 and
blood age42 have some effect on the detection of different
markers. These factors are important, as some centers deliver
blood after serological testing, ie, 1 day after blood collection.
As IL-2R expression is very sensitive, we can also hypothesize
that commonly used methods for monocyte purification could
enhance the expression of IL-2Rb, which is often detected.26,27
Furthermore as shown here, IL-2R chain expression displays
some variations among donors on some PBMC subsets, CD8 T
lymphocytes mainly, and this factor may have been neglected in
previous studies.
Under our experimental conditions, we observe a broad
change in the IL-2R expression profile in hemochromatosis
patients, compared with healthy donors (Fig 6). Thus blood
drawing on heparin do not impede the detection of the three
IL-2R subunits on various PBMC populations. Moreover, the
increase of IL-2R expression seen in this pathology, particularly
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
INTERLEUKIN-2 RECEPTOR EXPRESSION BY PBMC
on CD8 T lymphocytes, would have been undetectable with
ACD or CPD. The same situation would have probably
occurred during studies of the activation of purified CD4 T
lymphocytes: in a resting stage, the three IL-2R subunits are not
detectable on the cell-surface, and only appear after anti-CD3
stimulation.30 Our observations have two consequences. First,
blood collected during the treatment of hemochromatosis is a
common source of PBMC for many laboratories in cellular
immunology. The expression of the IL-2R on these cells may
have had some influences on previous results, which could not
be taken into account. Second, the use of this protocol allows
detection of IL-2R expression variations between the resting
and activated stages and between normal and pathologic
situations. This procedure has also been useful in the analysis of
IL-2R expression defects in other pathologies, such as hepatitis
(Denis David, unpublished data).
In this study we have shown that whereas IL-2R chain
expression is very sensitive to the conditions of blood collection
and of PBMC preparation, expression of the activation markers
CD69, CD71, and HLA-DR are only weakly affected by these
conditions. Therefore, specific induction of IL-2R chains may
prime cells to become responsive to IL-2 in the absence of
activation marker expression. In this view, one could speculate
that cell IL-2 sensitivity might also depend on the conditions of
blood collection and PBMC purification. Comparison with
previously published data may be difficult because the anticoagulant used and the time of PBMC purification are not always
indicated, and measurement of activation markers may not be
relevant. As IL-2Rb and IL-2Rg belong to the large hematopoietin receptor family,16 other receptor components of this family
may also display very sensitive patterns of expression.
When blood is collected on heparin and PBMC isolated at
day 0, CD4 T lymphocytes, B lymphocytes, NK cells, and CD8
T lymphocytes do not express IL-2Rg on their surface, or
express at very low levels, depending on the donors (Figs 1 to
4). However, we show that even in these conditions, IL-2Rg is
nevertheless largely present as intracellular protein in these
different PBMC subsets (Fig 7A). This result confirms and
extends our previous data showing that purified resting CD4 T
lymphocytes also express IL-2Rg only inside the cell.30 The
intracellular expressions we show here are consistent with the
fact that IL-2Rg gene promoter has the characteristics of a
constitutively active promoter43 and that IL-2Rg mRNA is
constitutively expressed in PBL15 and in monocytes.44 For
human monocytes, IL-2Rg has been shown to migrate as
several Western blot bands, in addition to the classical 64-kD
band,15 which correspond to different levels of N-linked glycosylation.33 We can thus hypothesize that this protein maturation
process might also exist in the other PBMC subsets where we
have detected IL-2Rg as an intracellular protein. In the different
lymphocyte subsets, IL-2Rg may be unable to translocate to the
cell-surface. In this view, a ‘‘helper’’ protein would be necessary for this process. In some conditions, IL-2Rb or another
protein might play this role. Some examples of proteins stored
inside the cell, which require the presence of such a ‘‘helper’’
protein, have been described.45,46
The present study provides a description of the pattern of
IL-2R subunit expression by T and B lymphocytes, NK cells,
and monocyte populations. These results should provide some
171
insight into the immune mechanisms controlling IL-2 responsiveness to the various cellular components of the immune system
and should be helpful in designing new therapeutic strategies
based on the use of IL-2 or cytokines sharing IL-2Rb and
IL-2Rg with IL-2R.
ACKNOWLEDGMENT
We are indebted to Drs R. Robb, K. Sugamura, W.J. Leonard, M.
Noguchi, and Y. Jacques for kindly providing the antibodies used in
these experiments. We are grateful to Dr R. Weil and S. Herblot for their
valuable advice and to Drs G. de Saint Basile and A. Fischer for their
generous gift of the IL-2Rg2 B-EBV cell line. We thank B. Bénard, M.
Minet, and P. Trumbic for their expert technical assistance. We also
thank Dr F. Saul for kindly reviewing this manuscript.
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1998 91: 165-172
Further Analysis of Interleukin-2 Receptor Subunit Expression on the
Different Human Peripheral Blood Mononuclear Cell Subsets
Denis David, Lynda Bani, Jean-Louis Moreau, Christophe Demaison, Karine Sun, Ombretta Salvucci,
Takayuki Nakarai, Marianne de Montalembert, Salem Chouai?b, Marcel Joussemet, Jerome Ritz and
Jacques Thèze
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