Download Effects of the Administration of High-Dose Interleukin

Cancer Therapy: Clinical
Effects of the Administration of High-Dose Interleukin-2 on
Immunoregulatory Cell Subsets in Patients with
Advanced Melanoma and Renal Cell Cancer
HansJ.J. van derVliet,1,2 Henry B. Koon,1Simon C.Yue,1Burak Uzunparmak,1Virginia Seery,1Marc A. Gavin,3
AlexanderY. Rudensky,3 Michael B. Atkins,1 Steven P. Balk,1 and Mark A. Exley1
Abstract
Purpose: High-dose recombinant human interleukin-2 (IL-2) therapy is of clinical benefit in a
subset of patients with advanced melanoma and renal cell cancer. Although IL-2 is well known
as aT-cell growth factor, its potential in vivo effects on human immunoregulatory cell subsets are
largely unexplored.
Experimental Design: Here, we studied the effects of high-dose IL-2 therapy on circulating
dendritic cell subsets (DC), CD1d-reactive invariant natural killerT cells (iNKT), and CD4+CD25+
regulatory-typeTcells.
Results: The frequency of both circulating myeloid DC1and plasmacytoid DC decreased during
high-dose IL-2 treatment. Of these, only a significant fraction of myeloid DC expressed CD1d.
Although the proportion of Th1-type CD4 iNKT increased, similarly to DC subsets, the total
frequency of iNKT decreased during high-dose IL-2 treatment. In contrast, the frequency
of CD4+CD25+ T cells, including CD4+Foxp3+ T cells, which have been reported to suppress
antitumor immune responses, increased during high-dose IL-2 therapy. However, there was little,
if any, change of expression of GITR, CD30, or CTLA-4 on CD4+CD25+ T cells in response to
IL-2. Functionally, patient CD25+ T cells at their peak level (immediately after the first cycle of
high-dose IL-2) were less suppressive than healthy donor CD25+ Tcells and mostly failed toTh2
polarize iNKT.
Conclusions: Our data show that there are reciprocal quantitative and qualitative alterations
of immunoregulatory cell subsets with opposing functions during treatment with high-dose IL-2,
some of which may compromise the establishment of effective antitumor immune responses.
Interleukin (IL)-2 was first identified in 1976 and entered
clinical trials in cancer patients after it was shown to exert
potent antitumor activity in several murine tumor models
(1, 2). In highly selected patients with metastatic melanoma or
renal cell cancer (RCC), treatment with high-dose bolus IL-2
has now been shown to produce durable responses (3, 4).
Although IL-2 is well known as a T-cell growth factor and has
Authors’ Affiliations: 1Cancer Biology Program, Division of Hematology and
Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School,
Boston, Massachusetts; 2Department of Internal Medicine, Vrije Universiteit
Medisch Centrum, Amsterdam, the Netherlands; and 3Department of Immunology,
University of Washington, Seattle,Washington
Received 7/7/06; revised 11/10/06; accepted 11/28/06.
Grant support: Netherlands Organization for Scientific Research NWO-TALENT
grant and grant 920-03-142, Dutch Cancer Society (Koningin Wilhelmina Fonds)
academic grant, NIH grants R01 DK066917 and R01 AI42955, Dana-Farber/
Harvard Cancer Center Skin Cancer Specialized Program of Research Excellence
grant P50 CA93683, Harvard Medical School Center for Human Cell Therapy pilot
grant, and Hershey Family Prostate Cancer Research Fund.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Mark A. Exley, Harvard Institutes of Medicine, 330
Brookline Avenue, Boston, MA 02115. Phone: 617-667-0982; E-mail: mexley@
bidmc.harvard.edu.
F 2007 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-06-1662
Clin Cancer Res 2007;13(7) April 1, 2007
been shown to result in the early depletion and subsequent
expansion of lymphoid subsets after in vivo administration (5),
no data are available on the effects of high-dose IL-2 therapy on
immunoregulatory natural killer (NK) T cells and CD4+CD25+
regulatory T cells.
CD1d-restricted invariant NK T cells (iNKT) display a semiinvariant T-cell receptor (Va24/Ja18 in human and Va14/Ja18
in mouse) and are characterized by their capacity to rapidly
produce large amounts of cytokines on triggering. iNKT cells
have been shown to promote antitumor immune responses by
rapidly producing copious amounts of IFN-g, resulting in the
activation of NK cells, dendritic cells (DC), and conventional
CD4+ and CD8+ T cells as well as in the inhibition of tumor
angiogenesis (reviewed in refs. 6, 7). Importantly, the circulating pool of iNKT cells shows quantitative and qualitative
defects in cancer patients (8, 9), which seem to be clinically
relevant, as increased numbers of intratumoral or circulating
iNKT cells are associated with improved prognosis (10, 11).4
In contrast to iNKT cells, CD4+CD25+ regulatory T cells, a
population of CD4+ T cells constitutively expressing the IL-2
receptor a-chain, have been shown to suppress immune responses, including antitumor immune responses, via both contact-dependent (CTLA-4 and CD30) and contact-independent
4
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J.W. Molling, personal communication.
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Effects of IL-2 on Immune Subsets
(IL-10 and transforming growth factor-h) pathways (reviewed in
refs. 6, 12, 13). As elevated numbers of CD4+CD25+ regulatory T
cells have been observed in melanoma and various other types
of cancer (14, 15) and are associated with reduced survival (16),
a recent study evaluated whether depletion of CD4+CD25+
regulatory T cells using recombinant IL-2 diphtheria toxin
(DAB389IL-2) would enhance tumor-specific T-cell responses in
patients with RCC, as indicated by earlier reports in mice (17),
and found that this was indeed the case (18).
As discussed above, both iNKT cells and CD4+CD25+
regulatory T cells seem to play important roles in the regulation
of antitumor immune responses. As CD4+CD25+ regulatory T
cells constitutively express the IL-2 receptor a-chain (CD25)
and IL-2 is an important growth factor for both iNKT cells and
CD4+CD25+ regulatory T cells, both these regulatory T-cell
subsets could be important cellular targets during high-dose
IL-2. Here, we evaluated the in vivo effects of high-dose IL-2 on
circulating iNKT cells, CD4+CD25+ regulatory T cells, and the
DC with which they interact.
Functional evaluation of CD4+CD25+ regulatory T cells. For evaluation of the suppressive properties of CD4+CD25+ regulatory T cells,
CD4+CD25+ and CD4+CD25 T cells were purified using high-speed
fluorescence-activated cell sorting (MoFlo Cytomation, Boulder, CO) or
by CD25 mAb magnetic microbeads at relatively low density to favor
removal of CD25hi cells and cocultured with purified iNKT cell lines
(purity >95%) and a-GalCer – pulsed monocyte-derived DCs for
48 h essentially as described elsewhere (21).
CD25+ cells were obtained by magnetic-activated cell sorting. Cells
were rested overnight after sorting to release beads before use. Assays
were carried out in 96-well plates. CD25 cells (column ‘‘flow
through’’) were used in all wells plus variable levels of autologous
CD25+ cells. For the highest level of CD25+ cells (‘‘high’’), a 1:1 ratio to
CD25 cells was used. At lowest level (‘‘low’’), the relative ratio was
1:16 of CD25+ cells to CD25 cells, respectively. Results of the middle
dilution of CD25+ cells were intermediate and therefore not shown for
clarity.
Supernatants were harvested for detection of IFN-g and IL-4 by
standard capture ELISA with matched antibody pairs in relation to
cytokine standards (Endogen, Cambridge, MA).
Statistical analysis. Statistical analyses were done using paired
Student’s t tests, Wilcoxon matched pairs tests, or ANOVA as
appropriate. P < 0.05 was considered significant.
Materials and Methods
Patients. Patients (mean age, 53 years; range, 30-68 years; 15 males;
10 females) in this study had been diagnosed with either advanced
melanoma (n = 15) or RCC (n = 10). All were treated with high-dose
IL-2 as described (3). Peripheral blood was obtained on four occasions:
before treatment, after the first week of high-dose IL-2, before the
second week of high-dose IL-2, and after the second week of high-dose
IL-2. Informed consent was obtained from all patients, and the study
was approved by the Committee for Clinical Investigation of Beth Israel
Deaconess Medical Center (Boston, MA). Clinical responses were
assessed using previously reported criteria (19).
Flow cytometry. Peripheral blood mononuclear cells were obtained
by Ficoll-Paque (Amersham Pharmacia, Uppsala, Sweden) density
gradient centrifugation of heparinized peripheral blood. Peripheral
blood mononuclear cells were stained using combinations of the
following reagents after an initial incubation with 10% human pooled
serum to reduce nonspecific binding. FITC-labeled CD3, FITC-labeled
CD25, phycoerythrin (PE)-labeled CD56, PE-labeled CD1d, PE-labeled
CD8a, PE-labeled HLA-DR, PE-labeled CD86, PE-labeled CD80,
PE-Cy5-labeled CD19, PE-Cy5-labeled CD25, PE-Cy7-labeled CD4,
PE-Cy7-labeled CD14, FITC-labeled anti-invariant Va24Ja18 T-cell
receptor monoclonal antibody (mAb) 6B11 (9), and the appropriate
isotype controls were obtained from BD PharMingen (San Jose, CA).
PE-labeled Va24 and FITC-labeled Vh11 were obtained from Immuno
tech (Marseille, France). FITC-labeled BDCA-1, BDCA-2, and BDCA-3
were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany).
FITC-labeled anti-CTLA-4, FITC-labeled CD30, and PE-labeled anti-GITR
were obtained from R&D Systems (Minneapolis, MN). The anti-human
Foxp3 polyclonal antibody was obtained by repeated immunization
of rabbits (20). Flow cytometry was done on a Cytomics FC 500
(Beckman Coulter, Fullerton, CA).
Generation of iNKT cell lines. iNKT cells were enriched from
peripheral blood mononuclear cells of healthy adult volunteers by
positive selection using biotinylated anti-invariant T-cell receptor mAb
(6B11) in combination with anti-biotin microbeads (Miltenyi Biotec)
and subsequently expanded using a-galactosylceramide (a-GalCer)loaded (KRN7000, kindly provided by the Pharmaceutical Research
Laboratory, Kirin Brewery, Ltd., Gunma, Japan) and lipopolysaccharidematured monocyte-derived DCs and 100 units/mL recombinant human
IL-2 (National Biological Response Modifier Program, National Cancer
Institute, Frederick, MD). In some cases, iNKT cell lines were further
purified using high-speed sorting (Modular Flow FACS, Cytomation,
Fort Collins, CO).
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Results
Effect of high-dose IL-2 therapy on T cells, NK cells, and B
cells. First, we evaluated the effect of high-dose IL-2 therapy on
the main peripheral blood lymphocyte subsets (Fig. 1). The
frequency of both CD4+ and CD8+ T cells decreased during the
first week of treatment with high-dose IL-2 (P = 0.0001 and
0.009, paired Student’s t test), but levels recovered before the
second week of treatment (P = 0.003 and 0.02). The second
week of high-dose IL-2 induced no significant changes in the
frequency of either CD4+ or CD8+ T cells. High-dose IL-2
induced a substantial increase in the frequency of NK cells
during the first week of IL-2 (P = 0.0005), and their frequency
remained at higher than pretreatment levels thereafter. In
contrast to NK cells, B cells substantially decreased in frequency
during both the first week (P = 0.006) and the second week
(P = 0.02) of high-dose IL-2 treatment.
Effect of high-dose IL-2 therapy on circulating DC subsets. We
analyzed both the frequency and CD1d expression of circulating myeloid DC (mDC) 1, mDC2, and plasmacytoid DC
(pDC), which can each be readily identified using either
BDCA-1 (mDC1), BDCA-2 (pDC), or BDCA-3 (mDC2)
expression, respectively, within CD14 CD19 peripheral blood
mononuclear cells (22). No statistically significant differences
in the frequency of circulating mDC1, mDC2, or pDC (P =
0.06, 0.10, and 0.21, respectively) nor in their expression of
CD1d (P = 0.67, 0.57, and 0.34) were found between patients
with RCC and melanoma. During high-dose IL-2 therapy,
mDC1 and pDC behaved in the same fashion: their frequency
decreased during the first week of high-dose IL-2 (P = 0.001 for
mDC1 and P = 0.024 for pDC), after which there was a
significant recovery (P = 0.009 for mDC1 and P = 0.0089 for
pDC) followed by a trend toward a further decrease during the
second week of high-dose IL-2 (P = 0.097 for both mDC1 and
pDC; Fig. 2). No significant changes were observed in the
frequency of circulating mDC2. CD1d was primarily expressed
by circulating mDC1, although both mDC2 and pDC were
found to express some CD1d. However, the expression of CD1d
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Fig. 1. Frequency of CD4+ Tcells, CD8+ T
cells, NK cells, and B cells during high-dose
IL-2 therapy. Columns, mean frequency of
CD4+ Tcells, CD8+ Tcells, NK cells, and B
cells as a percentage of total lymphocytes
during high-dose IL-2 therapy; bars, SD.
Frequencies were assessed before (pre) and
after (post) the first (1) and second (2)
week of high-dose IL-2.
on mDC1, mDC2, or pDC did not change significantly during
treatment with high-dose IL-2 (Fig. 2).
Effect of high-dose IL-2 therapy on circulating iNKT cells. As
expected from previous reports (8, 9), both the frequency
[median, 0.007% of lymphocytes; interquartile range (IQR),
0.002-0.37%] and absolute number (median, 69/mL peripheral
blood; IQR, 19-245) of iNKT cells were low before treatment.
The frequency of circulating iNKT cells was comparable in
patients with RCC and melanoma (P = 0.16). During both the
first and the second week of treatment with high-dose IL-2, the
Fig. 2. Frequency and CD1d expression of circulating DC subsets during high-dose IL-2 therapy. Columns, mean frequency (top row) and CD1d expression (bottom row)
of circulating mDC1, pDC, and mDC2 during high-dose IL-2 therapy; bars, SD. Analyses were done before (pre) and after (post) the first (1) and second (2) week of
high-dose IL-2.
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Effects of IL-2 on Immune Subsets
iNKT cell frequency decreased further (P = 0.01 and 0.007,
Wilcoxon matched pairs test; Fig. 3A) with a recovery between
the two cycles (P = 0.02). A similar trend was observed when
analyzing changes in the absolute number of circulating iNKT
cells. Importantly, the absolute number of iNKT cells increased
to higher than pretreatment levels 1 week after the first cycle
of high-dose IL-2 (P = 0.02; Fig. 3B). As different subsets of
iNKT cells have been shown to preferentially produce either
Th1-type cytokines (CD4 CD8 double-negative iNKT cells)
or Th2-type cytokines (CD4+ iNKT cells; refs. 23, 24), we
evaluated the contribution of these subsets to the total
circulating iNKT cell pool during high-dose IL-2. Of interest,
we found that there was a significant increase in the proportion
of double-negative iNKT cells after the first week of high-dose
IL-2 (P = 0.02; Fig. 3C). This increase was not sustained,
however, as the proportion of double-negative iNKT cells
decreased in the week thereafter (P = 0.03) and did not increase
significantly during the second week of high-dose IL-2. To
evaluate whether the increase in the proportion of doublenegative iNKT cells corresponded with an increased production of IFN-g by iNKT cells, we additionally purified iNKT
cells from peripheral blood of several individuals using biotinylated 6B11 mAb and magnetic bead sorting with anti-biotin
IgG beads (Miltenyi Biotec) for a subsequent overnight culture
in the presence of CD1d-transfected HeLa cells and a-GalCer.
Although we found low-level IFN-g secretion in most cases,
only in one case could we detect a clear increase in the
percentage of IFN-g – secreting iNKT cells during high-dose IL-2
therapy (data not shown).
Effects of high-dose IL-2 therapy on CD4+CD25+ T cells. Patients
with RCC and melanoma were found to have a similar
frequency of CD4+CD25+ T cells (P = 0.39) before treatment.
High-dose IL-2 resulted in a substantial increase in the
frequency of CD4+CD25+ T cells during both the first and
the second week of therapy (P = 0.0023 and 0.002). However,
the increased frequency was not sustained as it decreased to
pretreatment levels when IL-2 was discontinued (P = 0.0009;
Fig. 4A). The absolute numbers of circulating CD4+CD25+ T
cells similarly increased during the first week of high-dose
IL-2 (P = 0.008) and remained at higher levels throughout
further treatment (Fig. 4B). As CD25 can be expressed by any
T cell on activation, the combination of CD4 and CD25 does
not unequivocally identify CD4+CD25+ regulatory T cells. As
especially CD4+CD25high T cells have been reported to be most
suppressive (25), we evaluated whether the intensity of CD25
expression changed during high-dose IL-2 therapy. The
intensity of CD25 expression closely followed the increase in
the frequency of CD4+CD25+ T cells, suggesting an increase in
regulatory CD4+CD25+ T cells (Fig. 4C). In five patients, we
subsequently analyzed the expression of the transcription factor
Foxp3, which to date is the most specific marker of CD4+CD25+
regulatory T cells (6). In each case, pretreatment levels of
Fig. 3. Response and phenotype of circulating iNKTcells during high-dose IL-2
therapy. Columns, circulating iNKTcell frequency (A , median F IQR), absolute
number of circulating iNKTcells (B, median F IQR), and proportion of doublenegative iNKTcells of the total circulating iNKTcell pool (C, mean and SD) during
high-dose IL-2 therapy. iNKTcells were quantitated as double positive for Va24
Vh11T-cell receptor mAb. Similar results were obtained with anti-invariant
Va24Ja18 T-cell receptor mAb 6B11. Analyses were done before (pre) and after
(post) the first (1) and second (2) week of high-dose IL-2.
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Cancer Therapy: Clinical
CD4+Foxp3+ T cells were very low (data not shown; Fig. 5). In
three patients in whom we observed an increase in CD4+CD25+
T cells, we also observed a substantial increase in CD4+Foxp3+ T
cells to comparable levels during high-dose IL-2 therapy
(representative dot plots are shown in Fig. 5). In contrast, in
two patients in whom the CD4+CD25+ T-cell frequency did not
substantially change during high-dose IL-2, the frequency of
CD4+Foxp3+ T cells similarly remained in the same range (data
not shown).
High-dose IL-2 – induced changes in the cell surface expression of several molecules that have been implicated in the
function of CD4+CD25+ T cells (i.e., GITR, CD30, and CTLA-4)
are depicted in Fig. 6. GITR, CD30, and CTLA-4 were all
expressed by a minority of CD4+CD25+ T cells. Compared with
pretreatment values, the expression of GITR was significantly
increased after the second cycle of high-dose IL-2 (P = 0.009;
Fig. 6A). The expression of CD30 increased during the first cycle
of high-dose IL-2 (P = 0.0007; Fig. 6B), decreased before the
second cycle (P = 0.003), and tended to increase again during
the second cycle (P = 0.10). Surface expression of CTLA-4
tended to increase during both the first and the second cycle of
high-dose IL-2 (P = 0.07 and 0.05; Fig. 6C).
Further experiments addressed the ability of CD25+ T cells
from cancer patients to suppress CD25 T-cell responses.
Purified CD25+ cells were coincubated with autologous CD25
cells in the presence of CD3 mAb, and cytokine responses were
measured. The presence of higher levels of healthy donor
CD25+ cells (1.1:1 relative to CD25 cells) reduced the levels of
both IFN-g and IL-4 produced (Fig. 7A). In contrast, cancer
patient CD25+ cells from the peak of the high-dose IL-2 CD25+
cell response (after week 1) did not significantly influence
CD25 T-cell IFN-g and IL-4 responses (Fig. 7B).
We next evaluated whether the failure of CD25+ T cells from
cancer patients tested to suppress the CD3 response of CD25
cells represented a general suppressive defect. The Th1suppressive properties of the expanded pool of CD4+CD25+ T
cells on iNKT were assayed (Fig. 7C). CD4+CD25+ and
CD4+CD25 T cells were isolated from peripheral blood
mononuclear cells by high-speed sorting and subsequently
added to cocultures of a-GalCer – loaded mature monocytederived DCs and healthy donor – derived iNKT cell lines (iNKT
cell to CD4+CD25+ and iNKT cell to CD4+CD25 T-cell ratio =
2:1) over a period of 2 days. A clear Th2 deviation of the IFN-g/
IL-4 ratio was observed in one of seven donors. In the other
donors, no evidence of suppression or Th2 polarization of
iNKT cells could be observed.
Relation between clinical response and iNKT cells and
CD4+CD25+ T cells during high-dose IL-2 therapy. Finally, we
evaluated whether the pretreatment frequency of iNKT cells
or CD4+CD25+ T cells or whether changes in the frequency
of CD4+CD25+ T cells could be related to clinical outcome.
Clinical responses were assessed at 6 and 12 weeks after
initiation of high-dose IL-2 therapy. The pretreatment
Fig. 4. Response of circulating CD4+CD25+ Tcells during high-dose IL-2 therapy.
Columns, circulating CD4+CD25+ T-cell frequency (A, median F IQR), absolute
number of circulating CD4+CD25+ Tcells (B, median F IQR) during high-dose
IL-2 therapy, and mean fluorescence intensity (MFI) of CD25 expression on
CD4+CD25+ Tcells during high-dose IL-2 therapy (C, median F IQR). Analyses
were done before (pre) and after (post) the first (1) and second (2) week of
high-dose IL-2.
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Effects of IL-2 on Immune Subsets
Fig. 5. Response of circulating CD4+CD25+ regulatoryTcells during high-dose IL-2 therapy. Representative flow cytometric dot plots of one donor showing the association
between changes in the frequency of circulating CD4+CD25+ Tcells and CD4+Foxp3+ Tcells during high-dose IL-2 therapy. Right of each dot plot, frequency of doublepositive cells. Analyses were done before (pre) and after (post) the first (1) and second (2) week of high-dose IL-2.
CD4+CD25+ T-cell frequency was comparable in patients with a
partial response [7.4 F 6.1% (mean F SD)], mixed response
(3.6 F 1.9%), stable disease (4.2 F 2.6%), or progressive
disease (12.8 F 12.0%) at 6 weeks (P = 0.26, one-way
ANOVA). Similarly, the pretreatment iNKT cell frequency was
not predictive of the clinical outcome (P = 0.58 and 0.55 for
response assessment at 6 and 12 weeks, respectively). As highdose IL-2 resulted in an increase in the frequency of
CD4+CD25+ T cells in most, but not all, patients, we also
evaluated whether the fold change in CD4+CD25+ T-cell
frequency (pretreatment versus 1 week after high-dose IL-2)
could be used to predict disease outcome. We found that the
fold change in the frequency of CD4+CD25+ T cells was similar
in patients with partial response (3.7 F 4.0 – fold), mixed
response (3.1 F 0.6 – fold), stable disease (2.8 F 1.6 – fold), or
progressive disease (2.7 F 2.1 – fold) at 6 weeks (P = 0.91).
Similarly, neither the pretreatment CD4+CD25+ T-cell frequency nor the fold change in CD4+CD25+ T cells during the
first week of high-dose IL-2 was statistically significantly
different in patients with a partial response, mixed response,
stable disease, or progressive disease at 12 weeks (P = 0.49 and
0.40, respectively; data not shown).
Discussion
Treatment with high-dose bolus IL-2 can be used in selected
patients with metastatic melanoma or RCC (3, 4). IL-2, best
known as a T-cell growth factor, plays a dominant role in the
biology of iNKT cells and CD4+CD25+ regulatory T cells, both
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of which have been shown to play important roles in the
regulation of antitumor immune responses. Here, we evaluated
the in vivo effects of high-dose IL-2 therapy on iNKT cells,
CD4+CD25+ regulatory T cells, and the DC with which they
interact.
As professional antigen-presenting cells, DCs play critical
roles in the initiation of immune responses that, depending on
the microenvironment and DC subtype (e.g., myeloid versus
lymphoid), can support either Th1- or Th2-type immune
responses (26). We analyzed the frequency and CD1d
expression of three types of circulating DC that can be readily
identified by expression of BDCA-1 (mDC1), BDCA-2 (pDC),
and BDCA-3 (mDC2; ref. 22). We found that CD1d was
predominantly expressed by mDC1, confirming previous data
(27), and that this mDC1-specific expression did not fluctuate
during high-dose IL-2 therapy. The frequency of mDC1 and
pDC, but not mDC2, temporarily decreased during treatment
with high-dose IL-2. Because of the very low frequency of
circulating mDC1 and pDC during high-dose IL-2, we were
unable to assess whether the decreases in DC frequencies were
accompanied by functional changes.
As expected from previous reports (8, 9), we found low
pretreatment circulating iNKT cell numbers in the evaluated
patients. Interestingly, although both the frequency and the
absolute number of iNKT cells decreased further during
treatment with high-dose IL-2, the absolute number of iNKT
cells thereafter recovered to higher than pretreatment levels,
reflecting either redistribution of iNKT cells from tissue to
peripheral blood or an actual expansion of the total iNKT cell
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Fig. 6. Surface expression of GITR, CD30, and CTLA-4 on circulating CD4+CD25+
Tcells during high-dose IL-2 therapy. Columns, expression of GITR, CD30, and
CTLA-4 (median F IQR) on circulating CD4+CD25+ Tcells during high-dose IL-2
therapy. Analyses were done before (pre) and after (post) the first (1) and second
(2) week of high-dose IL-2.
Clin Cancer Res 2007;13(7) April 1, 2007
pool. As the size of the circulating iNKT cell pool predicts
disease outcome in patients with squamous cell head and neck
cancer (11) and immunologic responses to the iNKT cell ligand
a-GalCer (28), this suggests potential value of evaluating the
antitumor effects of a-GalCer after pretreatment of patients
with IL-2. As CD4+ iNKT cells and CD4 CD8 double-negative
iNKT cells represent distinct functional lineages of iNKT cells
(23, 24), we assessed whether the relative proportion of each
subset would change during high-dose IL-2. Although not
sustained, the contribution of proinflammatory doublenegative iNKT cells to the total iNKT cell pool did increase
significantly after the first week of high-dose IL-2, potentially
resulting in a more Th1-biased iNKT cell response when highdose IL-2 therapy would be followed by a-GalCer therapy.
In several patients, we found that purified iNKT cells from
these patients could indeed produce IFN-g when activated by
a-GalCer in the context of CD1d, but a clear increase in iNKT
cell IFN-g secretion was only detected anecdotally.
Strikingly, high-dose IL-2 therapy resulted in an increase in
both the frequency and the absolute number of CD4+CD25+
T cells. These increases of CD4+CD25+ T cells were accompanied by an increase in the level of CD25 expression, indicating the predominant expansion of CD4+CD25high T cells
previously shown to exert the majority of the suppressive
effects attributed to the population of CD4+CD25+ T cells (25).
As CD4+CD25+ regulatory T cells are critically dependent on
the X chromosome – encoded FOXP3 gene (6, 12), we similarly
evaluated the levels of CD4+Foxp3+ T cells during high-dose
IL-2 therapy. Importantly, the increase in the frequency of
CD4+CD25+ T cells was accompanied by a marked increase
in CD4+Foxp3+ T cells to levels comparable with total
CD4+CD25+ T cells, strongly suggesting that high-dose IL-2
indeed results in an increase in CD4+CD25+ regulatory T cells.
We found limited surface expression on CD4+CD25+ T cells
of GITR, CD30, and CTLA-4, molecules implicated in the
function of CD4+CD25+ regulatory T cells. Among these, the
increase in surface expression during high-dose IL-2 was most
prominent for CD30, although the expression of GITR and
CTLA-4 also increased. As IL-2 is not only a vital cytokine for
the thymic generation and peripheral maintenance and
function of CD4+CD25+ regulatory T cells but also seems to
reduce the CD4+CD25+ regulatory T-cell inhibitory effects at
higher doses (29), we additionally tested whether the expanded
pool of CD4+CD25+ T cells could still suppress a-GalCer –
induced iNKT cell activation as previously shown for healthy
donor – derived CD4+CD25+ T cells (21). As we found no
evidence for suppressive effects of the expanded pool of
CD4+CD25+ T cells, one can conclude that, although highdose IL-2 results in the expansion of CD4+CD25+ T cells, it may
also impair their suppressive effects, at least temporarily,
perhaps due to activated conventional CD25+ T cells overwhelming the truly functionally suppressive CD4+CD25+ T
cells. Indeed, in patients with HIV or cancer who are treated
with lower doses of IL-2, an increase in CD4+CD25+ T cells was
also noted, and although these cells were Foxp3+, they were
also poor functional suppressors (30 – 32). In line with this, we
found no relation between either the pretreatment CD4+CD25+
T-cell frequency or the high-dose IL-2 – induced increase in
CD4+CD25+ T-cell frequency and clinical responses. As previous studies showed that patients with higher CD4+CD25+
T-cell frequencies had a worse prognosis compared with
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Effects of IL-2 on Immune Subsets
Fig. 7. Effects of CD4+CD25+ Tcells and
CD4+CD25 Tcells on the IFN-g/IL-4
responses of iNKTcells and CD25 Tcells.
A and B, CD25 Tcells were incubated at
increasing levels with CD25+ Tcells in the
presence or absence of CD3 mAb.
Columns, mean; bars, SD. IL-4 (A) and
IFN-g (B) release were determined as
above.‘‘CD25 lo’’ indicates a ratio of 1:16,
and ‘‘CD25 hi’’ represents a ratio of 1:1. Data
from three individual healthy and three
high-dose IL-2 ^ treated patient donors after
week 1of high-dose IL-2 (peak of CD25+
cells). C, IFN-g/IL-4 ratios were
determined in supernatants collected after a
48-h coculture of CD1d-transfected HeLa
and iNKTcells in the presence of 100 ng/mL
a-GalCer, 20 units/mL recombinant human
IL-2, and either CD4+CD25+ Tcells
(white columns) or CD4+CD25 Tcells
(black columns). CD4+CD25+ and
CD4+CD25 Tcells were isolated from
peripheral blood after 1wk of high-dose IL-2
therapy and added at a ratio of 1:2 iNKT
cells. Data of six individual high-dose
IL-2 ^ treated patient donors.
patients with lower CD4+CD25+ T-cell frequencies, one mecha
nism through which high-dose IL-2 might induce effective
antitumor immunity may be ironically through the (temporary) inhibition of the suppressive effects of CD4+CD25+
T cells. It is likely that the suppressive effects of CD4+CD25+
T cells are indeed only temporarily inhibited, as two recent
articles showed that suppressive effects of CD4+CD25+ T cells
were intact from a few days to 4 weeks after high-dose IL-2
therapy (33, 34). Interestingly, these authors confirmed the
increase in the frequency of circulating CD4+CD25+ T cells in
patients with metastatic melanoma and RCC during high-dose
IL-2 therapy, but Cesana et al. (33) also noted a significant
decrease in CD4+CD25+ regulatory T cells 4 weeks after the
second cycle of high-dose IL-2 in patients achieving an objec
tive clinical response, perhaps reflecting migration of nonsuppressing CD4+CD25+ regulatory T cells to sites of antigenic
stimulation.
In conclusion, our data show reciprocal effects of highdose IL-2 therapy on circulating immunoregulatory iNKT and
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CD4+CD25+ T cells. Although the frequency and absolute
number of iNKT cells decrease during high-dose IL-2 therapy,
this is accompanied by an increase in the proportion of
potentially proinflammatory double-negative iNKT cells and is
followed by an increase in the total number of circulating iNKT
cells. In contrast, both the frequency and the absolute number
of CD4+CD25+ regulatory T cells increase during high-dose IL-2
therapy, and although the frequency returns to baseline levels,
the absolute number remains elevated thereafter. Significantly,
the expanded population of CD4+CD25+ T cells did not
consistently exert suppressive effects, suggesting surprisingly
that high-dose IL-2 therapy may also reverse CD4+CD25+ T-cell
suppressive effects.
Acknowledgments
We thank the patients for providing serial samples, Kirin Brewery for a-GalCer,
and colleagues for helpful discussions.
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Clin Cancer Res 2007;13(7) April 1, 2007
Cancer Therapy: Clinical
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