Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2989-2993
CORRESPONDENCE
Interleukin-2 Receptor Subunit Expression and Function on Human
Peripheral T Cells Is Not Dependent on the Anticoagulant
 |
LETTER |
To the Editor:
In a recent report, David et al1 addressed the ongoing
controversy on expression levels of interleukin-2 receptor (IL-2R) 
,
, and 
chains on various mononuclear cells from the peripheral blood. Using freshly isolated peripheral blood mononuclear cells (PBMCs) from (sodium) heparinized blood and fluorescein
isothiocyanate (FITC)-labeled commercial monoclonal antibodies, they
showed that all three IL-2R chains usually are hardly detectable on
either CD4 or CD8 T cells from healthy donors and from hemochromatosis patients. These results are in contrast with the much higher levels of
IL-2R subunits on T cells observed by several investigators, including
ourselves.2-9 David et al1 tentatively explain
the discrepancy by invoking effects of anticoagulant and of storage. Indeed, if Ca2+ chelators were used instead of heparin, the
levels of all three IL-2R chains on T cells apparently increased, and
overnight storage of heparinized blood also seemed to upregulate IL-2R
subunit expression.
These observations are very important, because they not only seem to
settle a long-standing controversy on IL-2R expression, but they also
imply that the use of Ca2+ chelators as anticoagulant
instead of heparin could dramatically influence the sensitivity of the
T cells to IL-2. Because IL-2 and other common -chain triggering
cytokines are central to almost any T-cell function, EDTA or citrate
anticoagulants should be avoided if subsequent functional testing is
envisioned. A lot of immunological research is based on buffy coats,
which routinely are anticoagulated with citrate. In our own studies of
T-cell function during human immunodeficiency virus (HIV) infection, we
have systematically used EDTA blood as starting material, because it is
readily available and because we did not find a functional difference
between lymphocytes derived from blood anticoagulated with heparin,
citrate, or EDTA in preliminary experiments. In view of the findings of
David et al,1 we felt obliged to carefully control the
effect of Ca2+ chelators on IL-2R expression and function
and we did not observe any significant influence of the anticoagulant.
In three separate experiments, blood from five healthy control subjects
(all lab personnel) was drawn at 10 AM in three different tubes from Sarstedt containing either sodium heparin (final
concentration, 0.3 mg/mL), potassium-EDTA (final concentration, 1.6 mg/mL), or sodium-citrate (final concentration, 10.6 mmol/L). The largest part of each tube was immediately
processed for mononuclear cell (PBMC) separation, using Histopaque 1077 (Sigma, Bornem, Belgium), whereas the rest was kept at
room temperature. At 2 PM, 50 µL of whole blood and 50 µL of PBMCs (containing 200,000 cells), derived from each of the
three anticoagulant tubes, were incubated for 20 minutes at 4°C with
0.1 µg of the nonconjugated reference monoclonals anti-Tac
(IL-2R-specific; obtained from Dr Thomas Waldman, National
Institutes of Health, Bethesda, MD) and with 2R-B (IL-2R-specific;
from Dr Takashi Uchiyama, Institute for Virus Research, Kyoto
University, Kyoto, Japan). As an isotypic (IgG1) control, we used
purified 56D3 directed against an irrelevant parasitic antigen
(provided by Dr J. Brandt, Institute of Tropical Medicine, Antwerpen, Belgium). After washing with phosphate-buffered saline (PBS), containing 0.5% bovine serum albumin, 1 µL of
FITC-labeled F(ab
)2 goat antimouse IgG (Tago,
Burlingame, CA) was added for another 20 minutes. After washing again,
the remaining binding sites on the FITC-conjugate were blocked with 5 µL of mouse serum. Next, 5 µL of phycoerythrin (PE)-labeled
anti-CD4 and 5 µL of peridinin-chlorophyll A protein (PercP)-labeled
anti-CD3 (both from Becton Dickinson, Erembodegem,
Belgium) were added for the last 20 minutes. The tubes
with whole blood were then subjected to the Becton Dickinson lysing
solution. All preparations were washed once and fixed with 1%
paraformaldehyde. The samples were analyzed on a FACScan (Becton
Dickinson) using the LYSYS I software.
Based on the scatter and the CD3/CD4 expression, the CD4+
and CD4
T lymphocytes were gated separately and the
distribution of the first fluorescence was represented in a histogram
for each subset. An example of this analysis is shown in Fig
1. It is evident that, within both the
CD4+ and CD4 T-cell populations, the
expression profile of IL-2R is rather broad and tends to be bimodal
(a negative and a positive subpopulation), whereas the curve of
IL-2R is unimodal and shows a shift to the right, which is most
evident in the CD4 subset. We chose to express the
results for both chains as percentage of positive cells, after
establishing a narrow threshold at a relative fluorescence intensity of
10, based on the background of the control monoclonal. A summary of the
results is shown in Table 1. No significant difference
was observed in the level of IL-2R and chains on
CD4+ or CD4 T cells, according to the
anticoagulant used and regardless of whether the cells were stained in
the context of whole blood or PBMCs. Comparing the mean fluorescence
intensity of all gated cells (instead of the percentage of positive
cells) showed similar results and confirmed that the low level of
IL-2R expression on CD4+ T cells significantly differed
from background (data not shown).
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| Fig 1.
Representative binding profiles of a control MoAb, the
anti-IL-2Ra MoAb anti-Tac, and the anti-IL-2Rb MoAb 2RB on
CD4+ and CD4 T cells, assessed in
three-color flow cytometry.
|
|
We next wanted to know whether the anticoagulant influences the
sensitivity to IL-2. To this end, we cultured the three preparations of
PBMCs at a final concentration of 106/mL in RPMI,
supplemented with antibiotics (GIBCO, Paisley, UK) and 10% bovine calf
serum (Hyclone, Logan, UT), in the presence or absence of 100 U/mL IL-2
(Boehringer-Mannheim, Brussels, Belgium) at 37°C in a
5% CO2 atmosphere. After an overnight incubation, the
cells were stained with a combination of either FITC-labeled IgG1 or
anti-CD69-FITC and anti-CD4-PE plus anti-CD3-PercP (all from Becton
Dickinson). As shown in Table 2, the specific
expression of CD69 on CD4+ and CD4 T cells
is similarly upregulated by IL-2, irrespective of the original
anticoagulant. The only (nonsignificant) difference was a lower
IL-2-induced CD69 on CD4 T cells from EDTA blood, as
compared with heparinized or citrated blood. The latter observation
certainly does not plead for a higher sensitivity of T cells from EDTA
blood, which is to be expected if the latter anticoagulant was able to
upregulate IL-2R expression, as suggested by David et al.1
To make absolutely sure that the storage of 4 hours we used in our
standard procedure did not influence the results, we performed two
additional experiments in which either whole blood anticoagulated with
heparin, EDTA, or citrate or the isolated PBMCs were stained for
IL-2R or CD69 either immediately or after 4 hours of incubation at
room temperature. Again, no differences in CD25 or CD69 expression were
observed, regardless of whether completely fresh cells or cells after 4 hours were used, thus excluding spurious activation upon short-term
storage of blood or PBMCs.
In summary, our well-controlled flow cytometric measurements, using a
sensitive indirect labeling technique and reference anti- and
anti- monoclonal antibodies, confirm our own and other investigators' previous findings that both IL-2R and chains are expressed to a significant extent on both resting CD4+
and CD4 T cells.2-9 We further confirmed
that the chain is predominantly present on CD4 T cells, whereas the
chain is more prominently expressed on the CD4
subset, largely corresponding to the CD8+ T cells.
Moreover, our present data clearly show that the level of IL-2R chain
expression is not influenced by the use of a Ca2+-chelating
anticoagulant and is similar in the context of fresh whole blood and
freshly isolated PBMCs. We did not investigate the effect of prolonged
storage, because, in our experience, such a treatment results in a
variable degree of viability loss, which might induce artifacts.
However, short-term storage for 4 hours had no influence on either
IL-2R or CD69. Our culture experiments with added IL-2 further
indicated that the IL-2R function, as measured by upregulation of CD69
on T cells, is also not significantly influenced by the anticoagulant.
In our previous studies, we showed that the CD69 upregulation by IL-2
on CD4 T cells could be blocked by a combination of anti-IL-2R and
monoclonals, pointing to an implication of the high-affinity
receptor. In CD8 T cells, nearly complete blocking could be obtained by
anti- monoclonals alone at high IL-2 concentrations, whereas, at
lower concentration, a synergistic effect of anti- and anti-
monoclonals was obvious.2 Thus, the expression of both and chains was shown to be of functional relevance in both T-cell
subsets. In a separate report, we recently demonstrated IL-2R chain
expression on at least 60% of both CD4 and CD8 T cells from healthy
donors and HIV-infected subjects.10
An incomplete screening of the literature found that at least two other
independent groups of investigators who stained PBMCs isolated from
heparinized blood with the same reference anti-Tac monoclonal but a
different anti- (Mik1-) showed expression levels of IL-2R chains
on peripheral T cells very similar to those we observed.5,6
However, other investigators, also starting from heparinized blood but
using different monoclonals and/or different staining
procedures, reported lower or even no measurable expression of the
IL-2R subunits, indeed.11-13 The seemingly low expression
of IL-2R chains observed by some investigators thus might relate to
technical factors, including the affinity of the antibodies used or the
sensitivity of the cytofluorometer rather than to the kind of
anticoagulant.
In conclusion, we feel that the use of sensitive staining procedures is
crucial to correctly assess IL-2R expression on resting T cells.
Moreover, we are convinced that the anticoagulant does not influence
IL-2R expression or function in peripheral blood T cells.
Guido L. Vanham
Godelieve Penne
Chris Vereecken
Johan Vingerhoets
Luc Kestens
Laboratory of
Immunology
Department of Microbiology
Institute of Tropical
Medicine
Antwerpen, Belgium
| |
ACKNOWLEDGMENT |
Supported by Grants No. 3.0307.95 and 3.0226.96 of the "Fonds voor
Wetenschappelijk Onderzoek Vlaanderen" (Fund for Scientific Research
of Flanders).
| |
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Chouaib S,
Joussemet M,
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Further analysis of interleukin-2 receptor subunit expression on the different human peripheral blood mononuclear cell subsets.
Blood
91:165,
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| |
RESPONSE |
We have recently published in BLOOD the first extensive
survey concerning IL-2R, IL-2R, and IL-2R expression
by human PBMC subsets.1 More than 100 donors were
examined through a 4-year period and the results are consistent with
our published data.1 Throughout the work, care was taken to
study healthy donors and resting PBMCs. The reported results were
obtained by measuring cell surface and intracellular expression of
IL-2R, IL-2R, and IL-2R by flow cytometry. The data were
verified for the three chains by measuring mRNA expression. For
intracellular expression of IL-2R, Western blot analysis was also
performed. Table 1 summarizes the data.
View this table:
[in this window]
[in a new window]
|
Table 1.
Expression of IL-2R Chains and Specific mRNA by PBMCs of
Healthy Adult Donors: Summary of our Data Presented in
BLOOD
|
|
Several reasons may explain why Vanham et al were not able to obtain
the same results concerning cell surface expression of IL-2R by CD4
and CD8 T lymphocytes.
(1) Their donors were apparently not controlled. Under some
circumstances, we also found IL-2R expression by CD4 T cells, for
instance during and after winter and spring seasonal infections. In our
study, donors went through a rigorous medical check-up before blood
collection.
(2) Their assay does not identify a clear negative lymphocyte
population; therefore, we cannot exclude either that their assay is not
reliable or that their cells are activated. Under these conditions, it
may not be surprising that they do not find any influence of
anticoagulants. In our hands, all the PBMC subsets analyzed from
healthy donors were negative for IL-2R expression, and we use cells
from hemochromatosis patients as a positive control to show that our
assay was able to detect IL-2R when expressed. These results were
misinterpreted by Vanham et al. Furthermore, we show that IL-2R
could be in vitro induced in IL-2R-negative resting CD4 T cells
after various stimulations.2
(3) The very poor and inconsistent (high standard deviation) induction
of CD69 by CD4 T cells after IL-2 stimulation shown by Vanham et al
suggests that their cells do not express IL-2R, and not the contrary.
Furthermore, we previously demonstrated that IL-2 reactivity does not
always correlate with IL-2R expression.3
In conclusion, it is commonly accepted that CD25 expression
correlates with T-cell activation and that resting T cells do not
express CD25.4-10 Therefore, we believe that Vanham et al should make a more detailed study before claiming high
expression of CD25 by resting CD4 T cells. They should be
aware that, in the IL-2R system, there are not yet defined reference
reagents and techniques and that their methods may have to be improved and controlled if they intend to modify almost consensual data concerning IL-2R expression.
Denis David
Lynda Bani
Jean-Louis Moreau
Jacques Thèze
Unité d'Immunogénétique
Cellulaire
Département d'Immunologie
Institut
Pasteur
Paris, France
| |
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Bani L,
Moreau J-L,
Demaison C,
Sun K,
Salvucci O,
Nakarai T,
de Montalembert M,
Chouaib S,
Joussemet M,
Ritz J,
Thèze J:
Further analysis of interleukin-2 receptor subunit expression on the different human peripheral blood mononuclear cell subsets.
Blood
91:165,
1998
2.
Bani L,
David D,
Moreau J-L,
Cayota A,
Nakarai T,
Ritz J,
Thèze J:
Expression of the IL-2 receptor subunit in resting human CD4 T lymphocytes: mRNA is constitutively transcribed and the protein stored as an intracellular component.
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Ohashi Y,
Takeshita T,
Nagata K,
Mori S,
Sugamura K:
Differential expression of the IL-2 receptor subunits, p55 and p75 on various populations of primary blood mononuclear cells.
J Immunol
143:3548,
1989
8.
Caligiuri MA,
Zmuidzinas A,
Manley TJ,
Levine H,
Smith KA,
Ritz J:
Functional consequences of interleukin 2 receptor expression on resting human lymphocytes.
J Exp Med
171:1509,
1990
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