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Journal of Virology, October 2001, p. 9983-9985, Vol. 75, No. 20
Department of Medicine, Division of
Infectious Diseases,1 and Department of
Pathology,2 Center for AIDS
Research, Case Western Reserve University, Cleveland, Ohio 44106
Received 7 March 2001/Accepted 28 June 2001
Subnormal T-cell production of interleukin-2 (IL-2) in human
immunodeficiency virus (HIV) disease has been described; however, it is
not clear whether failure to synthesize IL-2 represents a selective or
global defect in T-cell cytokine production. We evaluated the
intracellular production of gamma interferon (IFN- CD4+
T-cell function in human immunodeficiency virus (HIV) disease is
markedly impaired. CD4+ T cells from HIV-infected
individuals fail to proliferate appropriately following stimulation
with antigen or mitogen (2, 6, 7, 9, 10, 12) and display
enhanced susceptibility to apoptosis (5, 8). These defects
are accompanied by reduced production of the immunoregulatory cytokine
interleukin-2 (IL-2) (2, 7). IL-2 production may be
critical in HIV disease, since this cytokine acts as an important
T-cell growth factor. Moreover, the addition of exogenous IL-2 to
patient T cells enhances T-cell proliferation (12) and
protects from apoptosis (1) in vitro. Thus, production of
IL-2 by patient T cells may be especially important for proper immune function.
Recent progress in intracellular cytokine staining has allowed for the
evaluation of cytokine production by defined T-cell populations on a
single-cell basis. This technique has been used to evaluate immune
responsiveness in HIV-infected patients (3, 4, 11).
Commonly, gamma interferon (IFN- Despite the practical advantages of detecting IFN- Contrary to this suggestion, it has been argued that proliferation
failure may be an artifact of prolonged cell culture, during which
cells may be predisposed to an early apoptotic death (11). The detection of intracellular cytokine avoids these complications, since the assay can be performed in 5 to 6 h ex vivo. Therefore, it could be argued that cytokine responses better reflect the true
nature of immune recognition and function in HIV disease.
To determine whether selective defects in cytokine production might be
present in HIV disease, we tested the capacity of patient CD4+ T cells to produce IL-2 and IFN-
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.20.9983-9985.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Differential Expression of Interleukin-2 and Gamma Interferon in
Human Immunodeficiency Virus Disease
ABSTRACT
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) and IL-2 in
CD4+ cells that were stimulated with staphylococcal
enterotoxin B or cytomegalovirus antigen. Strikingly, IFN- and IL-2
are differentially regulated in T cells of HIV-infected patients such
that the numbers of CD69+ cells or IFN--positive cells
that make IL-2 are proportionally decreased in CD4+ T cells
from HIV-infected patients. These findings demonstrate a selective
defect in IL-2 production and suggest that enumeration of
IFN--producing cells in response to T-cell receptor stimulation, while providing some estimate of antigen-reactive cell frequency, may
not reflect or predict "normal" T-cell function in HIV-infected patients.
TEXT
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) production by patient T cells is
used to measure immune responses to recall antigen, HIV antigen, or
mitogens in vitro. The relative ease of detecting IFN- makes it a
useful and sensitive tool for identifying antigen-reactive cells.
production
in T-cell populations, measuring production of this cytokine may
not provide a complete determination of functionality of cells from HIV-infected patients. T cells from HIV-infected patients with advanced disease have poor proliferation responses to HIV antigens
and yet may respond by producing IFN- (11). We have described poor proliferation in CD4+ T cells from
HIV-infected patients even though these cells express CD25 and CD69
activation markers following T-cell receptor stimulation (13). Thus,
some functional responses, such as IFN- production or expression of
CD25 and CD69, may be maintained in HIV disease whereas others, such as
proliferation, are lost.
in
response to staphylococcal enterotoxin B (SEB), a superantigen with
potent cytokine-inducing potential. Whole blood from HIV-infected
patients or healthy controls was stimulated with SEB and subsequently
treated with brefeldin A to prevent the release of cytokine from
intracellular stores. IFN- and IL-2 production was measured in
CD4+ CD69+ cells by
three-color flow cytometry. CD69 is a marker of T-cell activation and
is commonly used to enrich for antigen-reactive cells in intracellular
cytokine assays. In comparison to healthy donor cells, patient
CD69+ cells tended to have reduced IL-2
responses, but relatively normal IFN- responses, on a per cell
basis (Fig. 1 and Table
1). The addition of anti-CD28 antibody at 1 µg/ml to the stimulation had little effect on cytokine
production in response to SEB (data not shown). Thus, with CD69 as a
denominator of activated cells, it was clear that most patient
responses were suboptimal in IL-2 production but normal in IFN-
production.
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FIG. 1.
Differential production of IL-2 and IFN- in
CD69+ cells. Whole blood was incubated with SEB (Sigma) (5 µg/ml) for 2 h before Golgi-stop reagent (BD Pharmingen)
containing brefeldin A was added. Cells were incubated for an
additional 3 h, at which time the cells were treated with Facs
lyse solution (Becton Dickinson) and frozen at 
86°C overnight.
Frozen cells were thawed and treated with Facs permeabilizing
solution (Becton Dickinson) and then stained with anti-CD69
phycoerythrin-conjugated antibody, anti-CD4 peridinin chlorophyll
protein-conjugated antibody, and either anti-IL-2 or anti-IFN-
fluorescein isothiocyanate-conjugated antibody (Becton Dickinson).
Cells stained with isotype control antibodies were used to
establish quadrants. Unstimulated cells in this case were exposed to
anti-CD28 antibody (1 µg/ml) to control for additional
experiments in which cells were stimulated with SEB plus anti-CD28
antibody (data not shown). The numbers shown indicate the percentages
of CD69+ cells that were IL-2 positive following SEB
stimulation.
TABLE 1.
Expression of IL-2 and IFN- following SEB
stimulationa
The differential production of IFN- and IL-2 in HIV disease was even
more dramatically illustrated by using IFN--producing cells as the
denominator for the enumeration of IL-2-producing cells. In
HIV-infected patients, the proportion of IFN--producing cells that
could simultaneously make IL-2 was markedly reduced compared to cells
from healthy donors (Fig. 2 and Table 1).
Again, addition of anti-CD28 antibody made little difference (data not shown). These observations suggest that CD4+ T
cells activated through T-cell receptor stimulation to produce IFN-
have a decreased capacity to make IL-2 in HIV disease.
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Cytokine production following superantigen stimulation may differ from
that induced by antigen stimulation, since superantigen does not
require processing by professional antigen-presenting cells and can
elicit responses from either naïve or memory cell populations
with the appropriate T-cell receptor variable chain. Therefore, to
determine whether differential production of IL-2 and IFN- might
also be observed in response to antigen, whole blood was exposed to
cytomegalovirus (CMV) antigen and assessed for cytokine production.
Following CMV antigen stimulation, patient cells demonstrated a
proportional reduction in IL-2 production both in the
CD69+ T-cell population and in IFN- producing
cells; however, the IFN- responses of CD69+
cells in patients and in controls were similar (Fig.
3 and Table 2). These
results are consistent with what was found with SEB stimulation
and indicate that the differential expression of IFN- and IL-2 is
also seen in memory CD4+ T cells responding to
antigen in HIV-infected patients.
|
|
While diminished IL-2 expression has been noted in HIV disease
(2, 7), this study clearly demonstrates a selective
impairment in IL-2 expression by CD4+ T cells in
HIV disease on an individual-cell basis following T-cell receptor
activation. Importantly, these analyses were performed under conditions
in which cells were incubated for a short time and maintained in whole
blood. Therefore, differences in production of IL-2 and IFN- cannot
be explained by culture-induced artifacts, as might occur in
proliferation assays of long duration.
Important inferences can be drawn from the results of this study, which
shows that CD4+ T cells from HIV-infected
patients maintain a selective defect in IL-2 production while IFN-
expression is preserved. As IL-2 is a critical mediator of immune
competence in health and disease, this selective defect in IL-2
expression after T-cell receptor stimulation may be a key determinant
of immune dysfunction in HIV infection. Selective impairment of IL-2
production in memory cells also might limit appropriate expansion of
antigen-reactive cells following in vivo challenge. This may in part
explain the reduced vaccine responses that have been observed in
HIV-infected patients (14). Furthermore, while T-cell
production of IFN- may provide an indication that T cells with
defined receptor specificity are present, enumeration of these cells
may not provide an appropriate demonstration of functional immune
competence in HIV disease.
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ACKNOWLEDGMENTS |
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We thank Robert Asaad for assistance in acquiring blood samples.
Scott Sieg is supported by National Research Service grant AI07024-21, from the National Institutes of Health. These studies were also supported by the Case Western Reserve University Center for AIDS Research (grant AI36219).
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Infectious Diseases, University Hospitals of Cleveland, AIDS Clinical Trials Unit, Foley Building, 2061 Cornell Rd., Cleveland, OH 44106. Phone: (216) 844-8175. Fax: (216) 844-5523. E-mail: lederman.michael{at}clevelandactu.org.
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Journal of Virology, October 2001, p. 9983-9985, Vol. 75, No. 20
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.20.9983-9985.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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