|
|
 Previous Article | Table of Contents | Next Article 
Blood, 15 September 2002, Vol. 100, No. 6, pp. 2159-2167
IMMUNOBIOLOGY
Long-term effects of intermittent interleukin 2 therapy
in patients with HIV infection: characterization of a novel subset of
CD4+/CD25+ T cells
Irini Sereti,
Hector Martinez-Wilson,
Julia A. Metcalf,
Michael W. Baseler,
Claire W. Hallahan,
Barbara Hahn,
Richard L. Hengel,
Richard T. Davey,
Joseph A. Kovacs, and
H. Clifford Lane
From the National Institute of Allergy and Infectious
Diseases, National Institutes of Health (NIH), Bethesda, MD; Science
Applications International Corp, Frederick, NC; Georgetown University,
Washington DC; and the Critical Care Medicine Department, Clinical
Center, NIH, Bethesda, MD.
 |
Abstract |
The long-term immunologic effects of intermittent interleukin 2 (IL-2) therapy were evaluated in a cross-sectional study by comparing 3 groups: HIV-seronegative volunteers, HIV-infected (HIV+)
patients receiving highly active antiretroviral therapy (HAART), and
HIV+ patients receiving HAART and intermittent IL-2.
Whole-blood immunophenotyping was performed to study expression of the
IL-2 receptor chains on T lymphocytes and natural killer cells and to
further characterize CD4+/CD25+ T cells.
Increased CD25 expression, especially in CD4+ T cells but
also in CD8+ T cells, without increases in expression of
the  and  chains of the IL-2 receptor was detected in the IL-2
group. Up to 79% of naive CD4+ T cells (median, 61%) from
patients in the IL-2 group expressed CD25, and the number of naive
CD4+/CD25+ T cells correlated positively with
both the total and naive CD4+ T-cell counts. A discrete
population of CD45 double intermediate RA+/RO+
CD4+ cells was also preferentially expanded in the IL-2
group, and the number of these cells strongly correlated with the total
CD4+ count. Despite increases in CD25 expression, T
lymphocytes from patients treated with IL-2 did not have increased
expression of early (CD69) or late (CD95) activation markers or
evidence of recent proliferation (Ki67). Both
CD4+/CD25+ and
CD4+/CD25 cells from IL-2-treated
HIV+ patients proliferated in response to mitogens,
specific antigens, and T-cell-receptor-mediated stimuli. Thus,
intermittent administration of IL-2 in HIV+ patients leads
to preferential expansion of a unique subset of CD4+ T
cells that may represent a critical population in T-cell homeostasis.
(Blood. 2002;100:2159-2167)
© 2002 by The American Society of Hematology.
| |
Introduction |
Interleukin 2 (IL-2) or T-cell growth factor is
currently approved for the treatment of metastatic renal cell carcinoma
and melanoma. IL-2 has also been tested experimentally in HIV infection in phase I and II studies since the early years of the AIDS epidemic. In randomized controlled clinical trials, intermittent administration of IL-2 in HIV-infected (HIV+) patients has been shown to
lead to substantial and sustained expansion of the CD4+
T-cell pool.1,2 This expansion is enriched for naive
CD4+ T cells and is not associated with increases in HIV-1
viral load.3,4 Subcutaneous administration of IL-2 was
found to be equally effective and better tolerated than intravenous
administration, thus facilitating outpatient
management.5-7 In combination with highly active
antiretroviral therapy (HAART), intermittent administration of IL-2 can
lead to increases in CD4+ cell count even in cases in which
the baseline count is as low as 50 cells/µL.8-12 Phase
III studies with clinical end points (SILCCAT: Subcutaneous IL-2 in HIV
Infected Subjects with Low CD4 Counts under Active Antiretroviral
Therapy and ESPRIT: Evaluation of Subcutaneous Proleukin in a
Randomized International Trial) are under way to assess the clinical
benefit of the increases in CD4+ T cells induced by
IL-2.
IL-2 binds to a cellular receptor that is composed of 3 chains:
the  chain (CD25), chain (CD122), and c or common
cytokine chain (CD132). IL-15 shares both the and c
chains of the IL-2 receptor, whereas the c chain is also
shared by IL-4, IL-7, and IL-9 and is constitutively transcribed in
lymphocytes.13-15 Two forms of a functional receptor
exist: the or intermediate-affinity receptor (Kd
109) and the or high-affinity receptor (Kd
1011).16 The chain (CD25) is necessary
for the formation of the high-affinity receptor but has limited binding
ability on its own (low-affinity receptor [Kd 108]);
also, it has a short intracytoplasmic tail that lacks signaling function.17-19 Although expression of CD25 is classically
induced by antigenic stimulation, IL-2 alone is sufficient to induce
expression of CD25 and progression of T cells through the cell
cycle.20
Despite some disagreement on the exact percentage of human
peripheral blood T cells expressing CD25 likely reflecting differences in the monoclonal antibodies used, staining techniques, or populations under studya consistent finding has been that CD4+ T
cells express higher levels of CD25 and lower levels of CD122 than
CD8+ T cells.19,21-23 Natural killer (NK)
cells typically express very low levels of CD25 (< 5%), whereas most
NK cells are CD122+.24
On the basis of data from prospective controlled studies, it has been
reported that HIV+ patients treated with IL-2 have an
expansion of CD4+ T cells bearing the chain (CD25) of
the IL-2 receptor.1,25 It was previously assumed that
these CD25+ cells coexpress increased levels of the and
c chains of the IL-2 receptor and are thus more
sensitive to IL-2 signaling. Their presumed increased IL-2-binding
potential was thought to contribute to a preferential use of
endogenously produced IL-2, thus sustaining a higher basal
CD4+ T-cell proliferation and enhancing responses to
subsequent administration of IL-2. In the previous studies, however,
the levels of expression of the and chains of the IL-2 receptor
were not tested; therefore, it remained unknown whether the expression
of CD25 was an indication of cells expressing the high-affinity IL-2
receptor and thus being more responsive to IL-2 signaling.
Additionally, the phenotypic characteristics of CD25-bearing
CD4+ T cells in patients treated with IL-2 were not
previously studied in detail with respect to their differentiation or
activation status. Similarly, their functional and proliferative
potentials have never been compared with those of
CD4+/CD25 cells.
To better characterize the effect of IL-2 on expression of the IL-2
receptor by T cells and NK cells, we examined - and -chain expression in HIV-seronegative (HIV) volunteers,
HIV+ patients with high CD4+ counts given
HAART, and HIV+ patients given both HAART and intermittent
IL-2 therapy. The long-term effects of IL-2 treatment on CD25
expression in HIV+ patients were also studied. We tested
the hypothesis that higher levels of expression of the chain would
be associated with higher baseline proliferation rates in T lymphocytes
in the patients who received IL-2. In addition, because of the recent
description of a subset of regulatory
CD4+/CD25+ cells in animal models and
humans,26,27 we sought to identify similarities or
differences between these cells and the
CD4+/CD25+ cells induced by intermittent
administration of IL-2.
| |
Patients and methods |
Study participants
A cross-sectional study compared HIV volunteers
and HIV+ patients who had been receiving HAART for at least
6 months either alone (HAART group) or in combination with intermittent
IL-2 therapy (IL-2+HAART group; Proleukin; Chiron, Emeryville, CA).
Consecutively seen persons in each group who agreed to participate at
the time of routine follow-up visits and on prespecified days of the
week were included in the study. Patients receiving IL-2 were enrolled in institutional review board-approved trials at the National Institutes of Health evaluating the role of IL-2 as an experimental agent in the treatment of HIV+ patients. Informed consent
was provided according to 45 Code of Federal Regulations (CFR)
governing human subjects research. The maintenance regimen
used in these trials is 1.5 to 7.5 million units (U) given
subcutaneously twice a day for 5 days at intervals determined by the
patients' CD4+ T-cell counts. On initiation of IL-2
therapy, cycles are typically administered every 8 weeks until the
CD4+ T-cell count increases to more than 1000/µL or twice
the baseline value. Subsequently, the cycling interval is
individualized to maintain these values and can range from several
months to years. Patients receiving HAART were chosen with the goal of
achieving CD4 cell counts comparable to those in the IL-2
cohort. Viral burden was tested by using USbDNA, version 3 (Bayer Diagnostics, Tarrytown, NY; sensitivity, < 50 copies/mL).
Immunophenotyping
Blood was collected in a heparin-coated Vacutainer and
processed within 4 hours after it was drawn (Becton Dickinson, Franklin Lakes, NJ). The whole-blood lysis technique for surface staining was
used (BD Immunocytometry, San Jose, CA). The following monoclonal antibodies were used: CD25 phycoerythrin (PE) or fluorescein
isothiocyanate, conjugated (FITC; clone 2A3); CD122 PE (clone TU27);
CD4 peridinin chlorophyll protein (PerCP), FITC, or allophycocyanin
(APC; clone SK3); CD8 FITC or PerCP (clone SK1); CD3 FITC, PerCP, or
APC (clone SK7); CD16 FITC (clone NKP15); CD56 FITC (clone NCAM16.2);
CD45RO APC (clone UCHL-1); CD62L PE (clone SK11); CD45RA FITC or PE
(clone L48); CD27 FITC or PE (clone L128); CD95 APC (clone DX2); CD69 APC (clone L78), and IgG1 FITC, PE, or APC (clone X40; all from BD
Immunocytometry). Samples were analyzed with a 4-color multiparameter flow cytometer (FACS Calibur; BD Immunocytometry).
CD3+/CD4+ gating and
CD3+/CD8+ gating were used for CD4+
and CD8+ T cells, respectively, and
CD3/CD16+-56+ gating was used to
identify NK cells (Figure 1).
CD25+ and CD122+ populations were identified
with the use of isotype controls. The statistical comparison of CD132
expression was restricted to the mean fluorescence intensity (MFI)
measurement, which is more accurate when distinct populations are not
easily differentiated by flow cytometry.
View larger version (42K):
[in this window]
[in a new window]
| Figure 1.
Intermittent IL-2 administration leads to
persistent increased CD25 expression on T lymphocytes, with transient
increases in the expression of CD122 and CD132 during cycles of IL-2.
(A) CD25 expression on T cells and NK cells in HIV
controls, HIV+ patients treated with HAART, and
HIV+ patients treated with HAART and IL-2 (contour plots).
Gating was done in CD3+/CD4+ and
CD3+/CD8+ in the lymphocyte gate for
CD4+ and CD8+ T cells, respectively, and in
CD3/CD16+-56+ for NK cells. In
IL-2-treated patients, very distinct
CD4+/CD25+ populations were observed. Of note,
a slightly decreased fluorescence intensity for CD4 was observed in the
CD25+ cells. (B) Increase in CD25 expression on
CD4+ T cells accompanied by transient but not persistent
increases in CD122 and CD132 expression. Histograms for
CD4+ T cells of an IL-2-treated patient before initiation
of IL-2 therapy (solid gray), at the end of an IL-2 cycle (broken
black), and 12 months later (solid black).
|
|
To study expression of CD25 on naive CD4+ cells, gating was
done in the CD4+/CD45RO population.
CD45RO gating was strictly defined so that all
CD45RO cells were also CD45RA bright, CD27+,
and CD62L+ as tested in parallel tubes. Cryopreserved cells
were stained to confirm induction of changes by IL-2 by studying
specimens collected before initiation of IL-2 therapy, and in those
cells, the naive population was defined as
CD45RO/CD27+. Cryopreserved cells were also
stained to confirm the increased percentage of the dull
RA+/RO+ CD4 subset in patients given IL-2.
Approximately 1 to 1.5 × 105 total events and a minimum
of 5000 events in the CD4+ gate were collected per sample.
Intracellular staining for the nuclear antigen Ki67 was performed as
previously described28 with Ki67 PE antibody (clone B56;
BD Biosciences Pharmingen, San Diego, CA) in cryopreserved peripheral
blood mononuclear cells (PBMCs) obtained at the same times the samples
used for the immunophenotypic analyses were collected.
Approximately 1.5 to 2 × 105 total events and a minimum
of 10 000 events in the CD4+ or CD8+ gate were
collected per sample. FlowJo software (Tree Star, San Carlos, CA) was
used for all flow cytometric data analyses.
Proliferation assays of CD4+/CD25+ and
CD4+/CD25 cells
Thirteen patients given IL-2+HAART were recruited for functional
studies of CD25+ and CD25CD4+ T
cells. Separated cell subsets were used for all or some of the
experimental conditions, depending on the yield of the cell separations
(cells from one patient were used exclusively for the suppression
experiment). The median CD4 count in this subgroup was 1022 cells/µL,
the CD4:CD8 ratio was 1, and the median time since the last IL-2 cycle
was 22 months (range, 3-48). Two of the 13 patients had a plasma viral
load higher than 50 copies/mL. Fresh PBMCs were isolated by
Ficoll-Hypaque lymphocyte separation. A fraction of PBMCs was
irradiated for use as antigen-presenting cells and a second fraction
was used as a control for the proliferation assays. CD4+
cells were isolated by the multisort CD4+
positive-selection procedure or the CD4+ T-cell
negative-isolation procedure (Miltenyi Biotec, Auburn, CA) according to
the manufacturer's recommended protocol. The resulting population had
a median of 97% CD4+ cells (range, 91%-99%) and was
further separated into CD25+ and CD25
fractions by CD25 microbeads (Miltenyi Biotec). The median purity of
the separated populations was 93% for the
CD4+/CD25 fraction (range, 85%-98%) and
90% for the CD4+/CD25+ fraction (range,
81%-98%). Separated fractions had similar percentages of naive cells
(32% in the CD25+ fraction and 36% in the
CD25 fraction), but compared with the CD25
fraction, the CD25+ fraction was highly enriched in dull
intermediate RA+/RO+ cells and had a higher
ratio of long-term or central memory cells (62L+,
CD27+) to effector memory (62L,
CD27) cells.
Cells (PBMCs, CD4+/CD25+, and
CD4+/CD25) were cultured in triplicate in
96-well, round-bottomed plates (Nalge Nunc International, Rochester,
NY) at 1.5 × 105 cells/per well in complete medium in a
final volume of 200 µL. Cells were stimulated with irradiated (30 Gy
[3000 rad]) antigen-presenting cells (1 × 105
cells in each well) and the following proliferation stimuli: phytohemagglutinin (PHA; Sigma-Aldrich, St Louis, MO) at a final concentration 3 µg/mL, anti-CD3 (OKT3; Ortho Biotech, Raritan, NJ)
with and without anti-CD28 (BD Biosciences Pharmingen) at 1 µg/mL,
cytomegalovirus (CMV; whole lysate, Bio-Whittaker, Walkersville, MD) at
2.5 µL/mL, tetanus toxoid (TT; Aventis Pasteur, Swiftwater, PA) at 3 µg/mL, pokeweed mitogen (PWM; Gibco BRL Life Technologies, Rockville,
MD) at 1:200, or IL-2 at 100 IU/mL (Aldesleukin; Chiron).
In 6 experiments designed to test specifically for suppression,
5 × 104 CD4+/CD25 cells were
stimulated with equal numbers of irradiated antigen-presenting cells
and anti-CD3, anti-CD3 with anti-CD28, or PHA as described above,
either alone or in the presence of incremental numbers of
CD4+/CD25+ cells, according to the method
described by Thornton and Shevach.29 Plates were incubated
at 37°C with 5% carbon dioxide, and wells were pulsed with 1 µCi
(.037 MBq) tritium-thymidine for 6 hours on the third day
(PHA, anti-CD3 with or without anti-CD28, and IL-2) or fifth day (PWM,
CMV, TT, and IL-2) of incubation. Harvesting was performed in an
automatic plate harvester (Wallac, Gaithersburg, MD). Radioactivity was
measured as tritium-thymidine incorporation in a counter (Wallac).
Wells containing cells with medium alone were used as negative
controls. Net counts per minute (cpm) were calculated by subtracting
the cpm of the wells with the medium (background controls) from the cpm
of the wells with the specific stimuli. In all experiments,
CD25+ and CD25CD4 cells had background cpm of
below 1500.
Statistical methods
All 3-group comparisons were done by using the Kruskal-Wallis
test. When significant differences (P = .05) were
detected, 2-group comparisons were done with the Wilcoxon 2-sample
test. The Student t test was used to compare normally
distributed variables: the percentage of
RA+/RO+ dull intermediate CD4 cells, the
percentage of CD25 expression on the RA+/RO+
dull intermediate CD4 subset, and the percentage of Ki67+ T
cells in the HAART and IL-2+HAART groups. The paired Student t test was used to compare the mean CD25 expression on naive and RA+/RO+ dull CD4 cells and the geometric means
of the proliferative responses of CD25+ and
CD25CD4 cells. The associations between variables were
determined by using the Spearman rank correlation method. Adjustment of
P values for multiple testing was done with the
Bonferroni method.
| |
Results |
Patient characteristics
The characteristics of participants at study entry are shown in
Table 1. Patients in the IL-2 group had
received a minimum of 3 cycles of IL-2 in the past and were tested
several months after the last IL-2 cycle (range, 3-54 months), with 3 of them studied immediately before initiation of an IL-2 cycle. The 3 groups did not differ significantly with respect to their total CD4+ count, percentage of naive and memory CD4+
cells, age, or viral load. Significant differences were observed for
the following factors: CD4:CD8 ratio (HIV versus HAART or
IL-2+HAART group and IL-2+HAART versus HAART group,
P = .01), percentage of CD4+ T cells
(HIV versus HAART group and IL-2+HAART versus HAART
group, P = .005) and percentage and total CD8+
T cells (HAART and IL-2+HAART versus HIV group,
P < .001).
Expression of the chains of the IL-2 receptor on
CD4+ and CD8+ T cells and NK cells
The results of the immunophenotypic analysis of expression of the
3 chains of the IL-2 receptor on T cells and NK cells are shown in
Table 2. We found, as others did
previously,21 that the percentage of CD4+ T
cells expressing CD25 was higher than the percentage of
CD8+ T cells or NK cells expressing CD25 in all 3 groups
(Figure 1A and Table 2). The percentage of CD4+ T
lymphocytes expressing CD25 was higher in the IL-2 group than in the
other 2 groups. The same was observed for MFI of the CD25 population.
No differences were observed in CD25 expression or MFI between the
HAART group and the HIV volunteers. When -chain
(CD122) expression was evaluated, the percentage of CD4+ T
cells expressing CD122 was found to be lower in the IL-2+HAART group
than in the HIV group. Finally, we observed significantly
lower expression of CD132 (MFI) on CD4+ T cells from the
IL-2+HAART group than on CD4+ T cells from the HAART group.
Transient increases in expression of and chains were detected
during IL-2 cycles (Figure 1B).
CD25 expression and MFI were also elevated in CD8+ T cells
from the IL-2 group compared with the other 2 groups. The MFI for CD122
on CD8+ T cells was higher in both the HAART and IL-2+HAART
groups than in HIV controls. No other significant
differences were observed among the groups in expression of CD122 or
CD132 on CD8+ T cells. In the staining experiments in which
both anti-CD25 and anti-CD122 antibodies were used simultaneously, the
percentages of double-positive (CD25+/CD122+)
CD4+ and CD8+ T cells were very low (< 5%) in
all 3 groups (data not shown). No differences among the 3 groups were
detected in the expression of or chains of the IL-2 receptor on
NK cells. Similar to previous observations, we found that the median
percentage of NK cells expressing CD122 (> 90%) was higher than the
median percentage of CD8+ T cells (13%-16%) or
CD4+ T cells (< 2%) expressing CD122.
Enhanced expression of CD25 on CD4+ T cells in patients
treated with IL-2 is persistent and occurs predominantly in the
naive subset of CD4+ T cells
In HIV volunteers, 39% of CD4+ T cells
(range, 9%-61%) were naive and 10% of these cells expressed CD25. In
HIV+ patients treated with HAART, 24% of CD4+
T cells (range, 10%-57%) were naive and 13% of these expressed CD25
(P > .5 versus HIV controls). In
patients given IL-2, 30% of total CD4+ T cells (range,
3%-54%) were naive, and in contrast to findings in the other groups,
61% of naive CD4+ cells were CD25+
(P < .001 versus the HIV and HAART groups).
The absolute counts of naive CD4+/CD25+ cells
reflected the same differences (Figure
2A). No significant differences were
observed in the percentages of memory CD4+ T cells
expressing CD25 (56% in HIV volunteers, 52% in
HAART-treated patients, and 64% in IL-2+HAART-treated patients;
P > .11). As demonstrated by the shaded histograms in Figure 2B, the differences in CD25 expression on naive CD4+
cells of patients receiving IL-2 and participants in the other 2 groups
were striking.
View larger version (33K):
[in this window]
[in a new window]
| Figure 2.
A large fraction of the naive
(CD45RO) CD4+ T cells in IL-2-treated
patients expressed the chain of the IL-2 receptor.
(A) Median percentages and absolute counts of naive CD4+ T
cells are shown. The hatched areas represent the fraction of cells that
were CD25+. (B) A CD25+ population of naive
CD4+ cells was identified in all groups and was largest in
the IL-2+HAART group. Levels of expression of CD25 on memory (solid
black lines) and naive (gray shaded histograms) CD4+ T
cells are shown for all 3 groups. The differences in CD25 expression in
the memory subsets were less consistent than the differences in the
naive subset and were not significant. (C) Correlation between the
number of naive CD4+/CD25+ cells and the total
number of naive CD4+ T cells. The absolute number of naive
CD4+/CD25+ cells was associated with the total
number of naive CD4+ T cells in HIV
volunteers (open triangles), HAART-treated patients (gray circles), and
IL-2+HAART-treated patients (black squares). Spearman rank correlation
coefficients and P values are shown.
|
|
To determine the contribution of the expansion of the naive
CD4+/CD25+ subset to the increases in the
overall size of the CD4+ pool in the HAART and IL-2+HAART
groups, we looked for correlations between the number of these cells
and the numbers of total naive and total CD4+ T cells. In
the HAART group, a strong correlation was found between the number of
naive CD4+/CD25+ cells and the total
CD4+ T-cell count (r = 0.75; P = .03) or the
total number of naive CD4+ T cells (r = 0.83;
P = .001; Figure 2C). Similarly, in the IL-2+HAART group,
a significant correlation was observed between the number of naive
CD4+/CD25+ T cells and the total
CD4+ T-cell counts (r = 0.61; P = .03) or
the absolute number of naive CD4+ T cells (r = 0.77;
P = .001). In HIV volunteers, a weaker
correlation was found between the naive CD4+/CD25+ cell counts and the total naive
CD4+ counts (r = 0.55; P = .08). In this
group, no correlation was found between the number of naive
CD4+/CD25+ cells and the total CD4+
T-cell count.
Cryopreserved cells obtained at various time points before and after
initiation of IL-2 therapy were tested to provide further documentation
of the emergence of naive CD4+/CD25+ cells
after administration of IL-2 and their persistence after an IL-2 cycle.
After initiation of IL-2, a distinct CD25+ population
emerged in the CD45RO/CD27+ (naive)
CD4+ pool (Figure 3). In the
patient whose results are shown in Figure 3, this population has
persisted for 5 years since the last IL-2 cycle (administered at month
28).
View larger version (30K):
[in this window]
[in a new window]
| Figure 3.
A
distinct population of naive CD4+/CD25+ cells
emerged after initiation of IL-2 treatment and remained present for
long periods after IL-2 cycles.
Frozen PBMCs were stained before and at different time points (months)
after initiation of IL-2. Each arrow represents a 5-day IL-2 cycle
(months 2, 4, 6, 8, 10, and 28). Naive CD4+ cells were
defined as CD45RO/CD27+, and the CD25
histograms were gated on these naive CD4+ cells.
|
|
On staining for CD45RA/CD45RO, a CD4+ population that
stained positive but had intermediate fluorescence intensity for both CD45RA and CD45RO isoforms was observed in all groups (Figure 4). This population was distinct from
RA+/RO and RA/RO+
cells and has been previously referred to as a dull RA/RO
double-positive population.30 This CD4+ subset
was particularly prominent in IL-2-treated patients, in whom it was
also observed to express high levels of CD25 (Figure 4A,B). It thus
appeared to represent a phenotypically unique population that had
preferentially expanded in patients given IL-2. In the HAART group,
12.3% of CD4+ cells (95% confidence interval [CI],
10.7%-13.9%) had this phenotype (dull
RA+/RO+). In a previous HIV
cohort (I.S. unpublished data, May 2001), we found that this population represented 10.7% of the CD4 pool. In contrast, in the
IL-2+HAART group, 20.2% (95% CI, 15.8%-24.6%) had this phenotype (P = .01 versus the HAART group).
View larger version (33K):
[in this window]
[in a new window]
| Figure 4.
CD4+ T cells that were dull double-positive for
CD45RA+/CD45RO+ were increased in IL-2-treated
patients and expressed high levels of CD25.
For the results shown in panel A, gating was done on
CD3+/CD4+ cells. A representative example from
each group is shown. In panel B, the histograms of CD25 expression on
the different CD4+ T cell subsets are shown for an
IL-2-treated patient (R1, CD45RA+/RO cells;
R2, dull double-positive CD45RA+/CD45RO+ cells,
and R3, CD45RO+/RA). In panel C, the number
of dull RA+/RO+ CD4+ cells is shown
to be associated with the total CD4+ T-cell count in both
the HAART group (circles) and the IL-2+HAART group (squares). Spearman
rank correlation coefficients and P values are
shown.
|
|
The mean absolute numbers of dull RA+/RO+ cells
were 81/µL (95% CI, 48-114/µL) in the HAART group and 174 (95%
CI, 125-223/µL) in the IL-2+HAART group (P = .01). In
both groups, the total number (but not the percentage) of these cells
correlated strongly with the total CD4+ count (r = 0.99;
P = .002 in the HAART group and r = 0.74;
P = .002 in the IL-2 group; Figure 4C), suggesting that
expansion of these cells plays a role in the CD4+ increases
observed with HAART or IL-2+HAART. When expression of CD25 in the dull
RA+/RO+ CD4 subsets in these 2 groups was
compared, a significant difference was observed (mean values, 75% in
the IL-2+HAART group versus 35% in the HAART group;
P < .001). Finally, in patients treated with IL-2+HAART,
expression of CD25 was higher in the dull
RA+/RO+ CD4 cells than in either the memory or
naive CD4 subsets (both paired differences were significant;
P = .002). This was in contrast to findings in the HAART
group, in which progressive increases in CD25 expression from the naive
to the dull RA+/RO+ to the memory
CD4+ T-cell subsets were observed.
CD25 up-regulation in patients treated with IL-2 is
not accompanied by increased expression of other activation
markers
Staining with early (CD69) and late (CD95) activation markers was
performed to investigate the possibility that increased CD25 expression
reflected an overall state of immune activation similar to that
occurring after antigenic stimulation. The percentage of either
CD4+ or CD8+ T cells expressing CD69 was
similar in all 3 groups (data not shown). CD95 expression on
CD4+ T cells was similar in the 2 HIV+ groups
but was higher than that in the HIV volunteers (56% in
the HIV group versus 83% in the HAART group and versus
80% in the IL-2+ HAART group; P = .02 for both
comparisons). A similar observation was made in the CD8+
T-cell pool (54% versus 87% and versus 83%;
P = .01).
To investigate the possibility that continuous increased levels of
CD4+ T-cell proliferation were sustaining the higher
CD4+ counts in patients treated with IL-2, intracellular
staining for the nuclear antigen Ki67 was performed in a subset of
HIV+ patients who had plasma HIV RNA levels below 50 copies/mL and for whom cryopreserved cells from the study-entry time
point were available (8 of 10 patients in the HAART group and 9 of 9 patients in the IL-2+HAART group). Ki67 is expressed during the late
G1, S, G2, and M phases of the cell cycle and
is considered a marker of recent proliferation. An increased percentage
of Ki67-positive staining cells has been reported in HIV+
patients before initiation of HAART.28 Surprisingly, we
found that patients given IL-2 had a lower fraction of T cells
(CD4+ or CD8+) that stained positive for Ki67
(Figure 5), a result suggesting that
after IL-2 therapy, continuous higher rates of proliferation do not
account for the sustained increases in CD4+ T-cell
counts. Although the differences were not statistically significant (P = .14 for the CD4+ and
P = .48 for the CD8+ T cells), the data
suggest that decreased turnover and increased survival may be
responsible for the increases in CD4+ T cells observed in
patients who have received IL-2.
View larger version (21K):
[in this window]
[in a new window]
| Figure 5.
Persistent increased T-cell proliferation is not the
mechanism that maintains high CD4+ counts in IL-2
recipients.
In patients with an HIV viral load below 50 copies/mL, the percentage
of Ki67+ CD4+ and CD8+ T cells was
not higher in the IL-2+HAART group than in HAART group.
|
|
Both CD4+/CD25+ and
CD4+/CD25 fractions in patients treated with
IL-2 can proliferate in response to mitogenic or T-cell-receptor
(TCR)-mediated signals
A population of anergic CD4+/CD25+ cells
capable of suppressing proliferation of other cells has been reported
to be important in regulation of autoimmunity in both animal models and
humans.27,31 To examine the possibility that the
CD4+/CD25+ population that expands in patients
given IL-2 represents an expansion of
CD4+/CD25+ immunoregulatory cells, we conducted
experiments to study the proliferative capacity of these cells. As
shown in Figure 6A, CD4+/CD25+ cells from patients given IL-2 were
not anergic and showed various degrees of proliferative responses to
all tested stimuli. CD4+/CD25+ cells had weaker
responses than CD4+/CD25 cells to the
mitogens PHA and PWM (P = .03) and to CMV antigen (P = .01). A trend toward higher responses to IL-2 was
also observed (P = .06). Additionally, in all experiments,
the background proliferation of CD4+/CD25
cells was higher than that of CD4+/CD25+ cells
(P < .01). In experiments designed to detect suppression (Figure 6B), no evidence of suppression was found in the response to
TCR stimulation with anti-CD3 or anti-CD3 with anti-CD28.
Interestingly, a blunting of the response to PHA that did not lead to
complete suppression was evident. This observation is under further
investigation.
View larger version (15K):
[in this window]
[in a new window]
| Figure 6.
CD4+/CD25+ cells from
IL-2-treated patients are not anergic.
(A) Separated CD4+/CD25+ (black bars) and
CD4+/CD25 (white bars) subsets from cells of
IL-2-treated patients were stimulated with anti-CD3 with or without
anti-CD28 (n = 12), PHA (n = 12), TT (n = 7), CMV (n = 10), PWM
(n = 11), and IL-2 (n = 8). Geometric means (net cpm), SE bars, and
P values for paired comparisons (paired Student t
test) are shown. (B) CD4+/CD25+ cells from
IL-2-treated patients do not suppress TCR-mediated responses of
CD4+/CD25 cells. In 6 experiments,
incremental numbers of CD4+/CD25+ cells (black
solid squares) were added to a fixed number (5 × 104) of
CD4+/CD25 cells to test for suppression.
Addition of equal numbers of CD4+/CD25 cells
(open circles) was used as a control. Results are expressed as the
percentage of the proliferative response of the
CD4+/CD25 cells cultured alone, and × signs
represent the proliferative response of 5 × 104
CD4+/CD25+ cells cultured alone. Mean values
with SE bars are shown.
|
|
| |
Discussion |
This study clearly demonstrated the emergence of a unique
population of CD4+/CD25+ T cells in
HIV+ patients who had received intermittent IL-2 therapy.
These cells are not anergic, do not proliferate continuously or express
increased levels of the trimeric IL-2 receptor, and may represent a
long-lived population of cells that are predominantly responsible for
the increases in CD4+ T cells observed with IL-2 therapy.
It is likely that these cells represent the product of IL-2-induced
T-cell expansion in the absence of antigenic stimulation.
It was previously reported that naive human T cells can proliferate in
vitro and retain their naive phenotype if stimulated in the absence of
antigen with a combination of IL-2, IL-6, and tumor necrosis factor (TNF-).32,33 In one of these reports,33 Unutmaz et al also observed the presence of a small fraction
of CD25+ naive CD4+ cells in human peripheral
blood and hypothesized that these cells represented the product of
cytokine-driven peripheral expansion of the naive CD4+
pool. A similar population of naive CD4+/CD25+
cells has also been described in human cord blood.34
During administration of IL-2, a transient 6- to 10-fold increase in the proliferation rates of both naive and memory CD4+ and
CD8+ T cells has been observed.35,36
Additionally, during the cycle, serum levels of proinflammatory
cytokines (such as IL-6 and TNF-) increase.37 It thus
appears likely that the naive CD4+/CD25+ cells
that expand preferentially with IL-2 administration are the product of
cytokine-driven proliferation and an in vivo reflection of the
previously described in vitro data. This hypothesis seems even more
plausible given the strong correlation between the absolute numbers of
these cells and the total CD4+ count and the total naive
CD4+ counts in HIV+ patients. The weaker
correlation in the HIV volunteers could reflect the fact
that far fewer of the cells in the CD4+ pool in these
individuals are the products of recent cell division.38 Naive T cells with similar phenotypes have been found in studies in
animals during cytokine-driven homeostatic proliferation in lymphopenic
hosts.39
In our study, we also found a population of dull double-positive
RA+/RO+ CD4+ T cells that
constituted approximately 10% of the total CD4+ pool in
HIV volunteers and patients in the HAART group and was
preferentially expanded in patients given IL-2. This population of
cells expressed very high levels of CD25 in IL-2 recipients and
decreased proportionally with decreases in CD4+ counts. The
source of these cells is unknown. We believe it is most likely that
these cells represent naive cells assuming an intermediate phenotype as
a result of cytokine-driven peripheral expansion similar to what has
been observed in lymphopenic animals.40 It is also
possible that they represent memory cells that are reverting to a naive
phenotype as a result of cytokine-driven proliferation in the absence
of their cognate antigen. In this regard, these cells may be analogous
to the population of dull double-positive
CD45RA+/RO+ CD4+ T cells described
by Hamann et al.30 Those authors concluded that cells of
this phenotype were able to produce cytokines such as IL-4 and
interferon at levels much higher than those produced by naive or
double RA+/RO+ bright cells and that they
contained substantial numbers of TT-specific precursors.
Alternatively, the expanded CD45RA+/RO+
CD4+ cells in IL-2 recipients could represent naive cells
that have recently encountered antigen. According to Picker et al, that transition, when induced by antigen, is a short-lived event and is
associated with bright double-positive
CD45RA+/RO+ CD4+
cells,41 which are found predominantly in lymphoid tissue. The characteristics of the cells observed in the current study, which
were not bright double-positive, were stable, and lacked evidence of
recent proliferation, constitute evidence against this hypothesis.
The current study highlights the fact that increased expression of the
chain may not always reflect a heightened immune-activation state.
As compared with the expression of CD25 on naive and dull CD45RA+/RO+ CD4+ T cells in IL-2
recipients, CD25 expression on memory CD4+ T cells was not
as pronounced and appeared to be less stable over time. Patients
studied several months after an IL-2 cycle had only similar or slightly
increased levels of CD25 on memory CD4+ cells compared with
persons not given IL-2. In agreement with this observation, other
markers of activation, such as CD69 and CD95, were not expressed at
higher levels in patients treated with IL-2. Additionally, despite
persistent long-term increases in CD25 expression, increased expression
of the and chains on CD4+ cells of IL-2-treated
patients occurred only transiently during the period of IL-2
administration. The short half-lives of the and chains on the
cellular surface and the limitations of the flow cytometric technique
in identifying low-level expression preclude definitive statements
about the degree of expression of the trimeric receptor on the
CD4+/CD25+ cells in the patients who received
IL-2. Binding studies will be necessary to clarify this issue.
Intracellular staining for the nuclear activation antigen Ki67 was used
as an indirect measurement of recent proliferation and activation in a
subset of study participants who had an HIV burden of less than 50 copies/mL. No evidence of increased Ki67 expression was detected in the
IL-2 group, suggesting that persistent, increased T-cell proliferation
in response to endogenously produced IL-2 cannot account for the
sustained high CD4+ counts observed in that group. Given
this finding, decreased turnover and prolonged survival of these cells
is a more plausible explanation for this observation. Other data seem
to support this hypothesis.42 Ongoing studies that allow
longitudinal evaluation of cell survival in vivo with tracking of T
lymphocytes labeled with bromodeoxyuridine or deuterium-glucose should
shed additional light on these fundamental areas.43
A CD4+/CD25+ cell population has been
identified in studies in animals and humans as a subset of cells with
regulatory function that are anergic and
immunosuppressive.27,44 These cells have been studied
extensively in animal models and are considered to be of thymic origin.
Once they encounter their cognate antigen, they become anergic (do not
proliferate in response to TCR-mediated signals) and acquire an
immunosuppressive function that is not antigen specific.45
Their ability to suppress the cytotoxic T lymphocytes of
CD8+ T cells has also been reported.46 These
immunoregulatory CD4+/CD25+ cells have been
observed in humans and identified as terminally differentiated memory
cells that do not proliferate in response to anti-CD3, anti-CD3 with
anti-CD28, or mitogens such as PHA and concanavalin A; are prone to
apoptosis; and express CD25 with high fluorescence
intensity.47,48 Despite some similarities (such as the
high MFI for CD25 and the expression of homing molecules such as 62L),
there are important phenotypic differences between these cells and the
CD4+/CD25+ cells in our IL-2-treated group. In
animal models, the immunoregulatory cells have been described as CD45RB
low, indicating a memory phenotype. Similarly, studies in humans found
that these cells were present exclusively in the CD45RO+
fraction of CD4+ T cells.49 In the current
study, the expanded CD4+/CD25+ cells in
patients given IL-2 included a high proportion of naive or dull
intermediate RA+/RO+ cells. In addition, no
evidence of anergy or suppression of TCR responses was detected in the
proliferative responses of the CD25+ and CD25
subsets of CD4+ cells from IL-2 recipients. Together, these
data suggest that the CD25+ cells expanded in the presence
of IL-2 are not the described CD4+/CD25+
suppressor cells, although we cannot exclude the possibility that some
of these clones expand during administration of IL-2.
In summary, in HIV+ patients receiving intermittent
IL-2 therapy, a sustained increase in CD4+ T-cell counts
was observed with a preferential expansion of CD4+ T cells
with a naive phenotype. A high proportion of these cells expressed CD25
but not the other chains of the IL-2 receptor. Preliminary data suggest
that prolonged survival of this population may represent the main
mechanism of the sustained increases in CD4+ count observed
after administration of IL-2. Additional studies focusing on the
origin, functional characteristics, and properties of these cell
subsets will further clarify their role in T-cell homeostasis. The
results of ongoing phase III trials of intermittent IL-2 therapy in HIV
infection that are assessing clinical end points will be critical in
clarifying the clinical implications of our observations.
| |
Acknowledgments |
We thank all the participating patients and the
staff of the National Institute of Allergy and Infectious
Diseases/Critical Care Medicine Department Clinic for their commitment
and enthusiasm, Dr Anthony Fauci for his continued encouragement and
support, and Mary Rust for assistance in the preparation of the manuscript.
| |
Footnotes |
Submitted September 6, 2001; accepted May 8, 2002.
Funded in whole or in part with federal funds under National Cancer
Institute contract NO1-CO-56000. The content of this publication does
not necessarily reflect the views or policies of the Department of
Health and Human Services, nor does mention of trade names, commercial
products, or organization imply endorsement by the US Government.
The US Government has been granted a use patent for intermittent
interleukin-2 therapy including Drs. H. Clifford Lane and Joseph A. Kovacs as inventors.
Partly presented in abstract form at the American Association of
Immunologists meeting, Experimental Biology 2001, Orlando, Florida,
March 31-April 4, 2001. Abstract B-816.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: H. Clifford Lane, Clinical and Molecular
Retrovirology Section, Laboratory of Immunoregulation, National
Institute of Allergy and Infectious Diseases, National Institutes of
Health, Building 10, Room 11S-231, 10 Center Drive, MSC 1876, Bethesda,
MD 20892; e-mail: clane{at}niaid.nih.gov.
| |
References |
1.
Kovacs JA, Vogel S, Albert JM, et al.
Controlled trial of interleukin-2 infusions in patients infected with the human immunodeficiency virus.
N Engl J Med.
1996;335:1350-1356[Abstract/Free Full Text].
2.
Kovacs JA, Baseler M, Dewar RJ, et al.
Increases in CD4 T lymphocytes with intermittent courses of interleukin- 2 in patients with human immunodeficiency virus infection. A preliminary study.
N Engl J Med.
1995;332:567-575[Abstract/Free Full Text].
3.
Emery S, Capra WB, Cooper DA, et al.
Pooled analysis of 3 randomized, controlled trials of interleukin-2 therapy in adult human immunodeficiency virus type 1 disease.
J Infect Dis.
2000;182:428-434[CrossRef][Medline]
[Order article via Infotrieve].
4.
Davey RT Jr, Chaitt DG, Albert JM, et al.
A randomized trial of high- versus low-dose subcutaneous interleukin-2 outpatient therapy for early human immunodeficiency virus type 1 infection.
J Infect Dis.
1999;179:849-858[CrossRef][Medline]
[Order article via Infotrieve].
5.
Levy Y, Capitant C, Houhou S, et al.
Comparison of subcutaneous and intr |
Top
Abstract
Introduction