Modulation by IL-2 of CD70 and CD27 Expression on CD8?T
Cells: Importance for the Therapeutic Effectiveness of Cell
Jianping Huang, Keith W. Kerstann, Mojgan Ahmadzadeh, Yong F. Li, Mona El-Gamil,
Steven A. Rosenberg, and Paul F. Robbins2
Proper T cell function relies on the integration of signals delivered by Ag, cytokine, and costimulatory receptors. In this study, the
interactions between IL-2, CD27, and its ligand CD70 and their effects on human T cell function were examined. Unstimulated
CD8?T cells expressed relatively low levels of CD70 and high levels of CD27. Incubation in vitro with high doses of IL-2 (3,000
IU/ml) or administration of IL-2 in vivo resulted in substantial up-regulation of CD70 expression and the concomitant loss of cell
surface CD27 expression on CD8?cells. Withdrawal of IL-2 from activated CD8?T cells that had been maintained in IL-2
resulted in a reversal of the expression of these two markers, whereas reciprocal changes were seen following treatment of PBMCs
with IL-2. The proliferation observed in cells stimulated with IL-2 primarily occurred in a subset of the CD70?CD8?T cells that
up-regulated IL-2 receptor expression but did not occur in CD70?CD8?T cells. Blocking CD70 resulted in a significant reduction
of T cell proliferation induced by high-dose IL-2, indicating that the interaction of CD70 with CD27 played a direct role in T cell
activation mediated by IL-2. Finally, studies conducted on tumor-infiltrating lymphocyte (TIL) samples that were administered
to melanoma patients indicated that the size of the pool of CD27?CD8?T cells in bulk TILs was highly associated (p ? 0.004) with
the ability of these TILs to mediate tumor regression following adoptive transfer. The Journal of Immunology, 2006, 176: 7726–7735.
tion to acting to promote the proliferation and effector function of
CD8?and CD4?T cells, IL-2 has been shown to delay the con-
traction phase of T cells responding to viral infection (1) as well as
play a role in the reversal of T cell anergy or tolerance (2, 3). The
administration of IL-2 to patients with metastatic melanoma and
renal cancer resulted in objective responses in 10–15% of treated
patients (4–7). In addition, the adoptive transfer of tumor-infiltrat-
ing lymphocytes (TIL)3cultured in vitro with IL-2 administered
following lymphodepleting chemotherapy leads to objective clin-
ical responses in ?50% of treated patients refractory to treatment
with IL-2 alone (8, 9).
The engagement of costimulatory molecules on T cells with
their ligands, found predominantly on APCs, also plays a critical
role in enhancing T cell activation. The interaction of the costimu-
latory molecule CD27 with its ligand CD70 plays a key role in T
lymphocyte activation, proliferation, survival, and differentiation
(10–13). In both the human and mouse, CD27 is constitutively
expressed on naive and memory T cells as well as on subsets of
he cytokine IL-2 is a member of a family of cytokines that
signal through the common ?-chain receptor in combina-
tion with unique receptors expressed on T cells. In addi-
activated B cells, NK cells, and hemopoietic progenitor cells; how-
ever, expression of CD27 is down-regulated in late effector stage
T cells (14–17). In contrast, little or no expression of CD70 is
found on quiescent T, B, and dendritic cells; however, activation
has been found to transiently up-regulate expression of this marker
on these cell types (13, 18).
Signaling through CD27 can lead to a modest delay in the con-
traction phase of influenza-specific CD8?T cells in peripheral
blood and spleen after viral infection (10), and several studies in-
dicate that the lack of costimulatory signals can lead to the induc-
tion of T cell anergy as well as apoptosis (19–21). Transfection of
tumors with CD70 leads to enhanced anti-tumor immune re-
sponses (22, 23), and stimulation of CD27 through CD70 has been
shown to support clonal expansion of tumor-specific T cells (24,
25). These observations suggest that engagement of CD27 may
play an important role in regulating responses to a variety of an-
The findings presented in this study provide evidence that sig-
naling mediated by the interaction between the CD27 and CD70
molecules expressed on CD8?T cells may play an important role
in IL-2-mediated T cell activation. Examination of the expression
of CD27 and CD70 on samples of tumor-reactive TILs that were
adoptively transferred as part of a human clinical trial also sug-
gested that the expression of these molecules on CD8?T cells in
TILs may play a role in the response to adoptive immunotherapy.
Materials and Methods
Samples of PBMCs were obtained from patients with metastatic melanoma
after the administration of tumor-reactive TIL or IL-2 in clinical protocols
approved by the Institutional Review Board of the National Cancer Insti-
tute. The in vitro cultured TIL cell lines used to treat patients were estab-
lished from tumor fragments by culturing dissociated cells in 3,000 IU/ml
rhIL-2 and then expanded using anti-CD3 Abs in the presence of PBMC
Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda,
Received for publication December 29, 2005. Accepted for publication March
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.
1This research was supported by the Intramural Research Program of the National
Institutes of Health, National Cancer Institute, Center for Cancer Research.
2Address correspondence and reprint requests to Dr. Paul F. Robbins, Surgery
Branch, National Cancer Institute, National Institutes of Health, Building 10, Room
2B42, 10 Center Drive, Bethesda, MD 20892. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: TIL, tumor infiltrating lymphocyte; rhIL-2F, re-
combinant human IL-2 Fluorokine.
The Journal of Immunology
Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00
Cell culture and activation
All experiments were performed in X-VIVO 15 (Cambrex) containing 5%
human serum medium in the presence or absence of IL-2. PBMC samples
from normal blood donors or IL-2-treated patients were cultured in various
concentrations of IL-2 for up to 12 days, and analysis was performed as
indicated in Results. For the CD70 blocking experiment PBMCs were
thawed or obtained directly from pheresis procedures on patients, the in-
dicated concentrations of Ab and IL-2 were added into 106/ml T cells at the
same time, and cell cycle analysis or FACS analysis was conducted at day
4. The separation of CD27?and CD27?cells was performed by initially
culturing TILs in medium without IL-2 for 2 days. Anti-CD27-PE bead
staining and anti-PE bead staining were subsequentially performed, fol-
lowed by passage through an MS separation column from Miltenyi Biotec.
The CD27?cells were obtained from the cells that had passed through
during CD27?cell selection and were then passed through an LD column
(Miltenyi Biotec). The peptide stimulation experiment was done by pulsing
with indicated concentrations of MART-126–35(27L) for 18 h, and cyto-
kine release was measured by ELISA. The total number of CD27?CD8?
T cells in TIL samples were evaluated by washing cells twice with medium
without IL-2 and culturing the TILs in IL-2-free medium for 2 days, fol-
lowed by Ab staining and FACS analysis.
Antibodies and FACS analysis
Characterization of the expression of TCR ?-chain variable region expres-
sion on TIL was conducted using ?-chain variable region Abs obtained
from Beckman Coulter and Pierce. Anti-CD27, anti-CD70, and anti-CD8
were purchased from BD Biosciences. For evaluation of the levels of IL-2
binding to T cells, cells were stained with streptavidin-FITC using reagents
from the recombinant human IL-2 Fluorokine (rhIL-2F) kit (R&D Sys-
tems) (26). Blocking Ab for human CD70 (clone BU69) was purchased
from Ancell. FACS analysis was performed by using CellQuest software
Total RNA was isolated from CD27?purified and CD27?purified cells,
respectively, using RNeasy columns (Qiagen). RT-PCR was performed
using the ThermoScript RT-PCR system (Invitrogen Life Sciences). Four
microliters of RT-PCR products were used for the quantitative PCR using
a commercially available TaqMan kit and probes and primers for CD27
(Hs00386811) and CD28 (Hs01007422) (Applied Biosystems). A full-
length TCR ?-chain was used for the internal control for all the assays. The
C?-specific primers were GAGGGTCTCGGCCACCTT and AGGCGA
CAGTTCAGGTCAAGA, and the probe was 5?-FAM-TGGCAGAAC
CCCCGCAACCAACCAC-TAMRA-3?. The samples were run on a
Chromo 4 detector with a PTC-200 Peltier thermal cycler (MJ Research)
Cell cycle analysis
T cells (106/ml) under different conditions were washed and fixed in 70%
ethanol for at least 1 h at 4°C, and the cells were washed twice with PBS.
One milliliter of propidium iodide staining solution (50 ?g/ml) was added
to the cell pellet and mixed well, 50 ?l of RNase A stock solution (10
?g/ml) was added, and cells were incubated for 3 h or longer at 4oC,
followed by flow cytometric analysis. A lymphocyte gate and doublet dis-
crimination gate was used, and data were analyzed using ModFit LT ver-
sion 3.1 software (Verity Software House).
cDNA-microarray analysis of CD27?and CD27?T cells from
RNA was isolated from CD27?and CD27?populations of patients’ TILs
as described above in the paragraph titled Real-time RT PCR and indirectly
labeled via a single round of linear amplification with the Amino Allyl
MessageAmp antisense RNA kit (Ambion). The cyanine dye-labeled sam-
ples were combined and hybridized overnight to a 22,000 gene-long oligo
array (version 2.0; Operon Biotechnologies) supplied by the Laboratory of
Molecular Technology (National Cancer Institute, Frederick, MD). Data
image files were obtained using a GenePix 4000B scanner (Axon) and
imported into GeneSpring version 7.0 (Silicon Genetics) for data analysis.
IL-2 induces up-regulation of cell surface CD70 expression on
CD8?cells in vitro and in vivo
To examine the influence of IL-2 on the expression of CD27
and CD70, naive PBMCs were initially cultured in relatively
low-dose (300 IU/ml) or high-dose (3000 IU/ml) rhIL-2. Only
a small percentage of CD8?T cells from peripheral blood ex-
pressed CD70 prior to in vitro culture (Fig. 1A). The frequency
of CD8?cells expressing CD70 was progressively up-regulated
over a period of 12 days of in vitro culture in IL-2 (Fig. 1A,
upper row), which was paralleled by an increase in the level of
CD70 expression on the CD8?T cells (data not shown). The
expression of CD70 on CD8?T cells following IL-2 treatment
increased in all 12 patients who were analyzed and was more
notable in cultures conducted in relatively high doses of IL-2
than in those conducted with low-dose IL-2 (Fig. 1A, upper
row). The up-regulation of CD70 expression on CD8?cells in
vitro was inversely related to the loss of CD27 expression (Fig.
1A, bottom row).
These observations led to attempts to determine whether similar
changes in expression of CD27 and CD70 on CD8?T cells oc-
curred in circulating T cells present in melanoma patients who had
been treated with high-dose IL-2. Before the administration of
IL-2, patient peripheral CD8?T cells expressed relatively low
levels of CD70 in vivo; however, between 3 and 4 days following
IL-2 administration, a significant increase in the percentage of
CD8?T cells that expressed CD70 was observed (Fig. 1B). In the
peripheral blood of patients 1 and 2, expression of CD70 increased
from ?15 and 5%, respectively, to nearly 40% of the CD8?T cells
in peripheral blood, whereas smaller increases were observed for
the samples obtained from three additional patients. The maximum
levels of CD70 expression were observed during a period of lym-
phopenia during which the absolute lymphocyte count dropped
nearly 20-fold on average (from 1662 to 85 ?l/ml) compared with
the pretreatment level. Despite the large decrease in the absolute
lymphocyte count, the absolute counts of CD70?CD8?cells
dropped only 2-fold (from 69 to 31 ?l/ml) from pretreatment lev-
els in peripheral blood. As the lymphocyte count started to recover
in peripheral blood, the percentage of CD70-expressing CD8?T
cells declined to levels similar to those seen before treatment. The
peak of CD70 expression correlated with a nadir in the expression
of CD27 on the surface of CD8?T cells during the lymphopenia
that is routinely observed during IL-2 treatment (Fig. 1B, bottom
row). This was followed by a gradual recovery of peripheral CD8?
T cells that expressed CD27 over a period of 3–6 weeks following
the initial IL-2 infusion when CD70 expression levels were de-
clining. Overall, these results indicated that there was an inverse
relationship between the expression of CD70 and CD27 on CD8?
T cells and that IL-2 played a direct role in influencing the ex-
pression of these markers both in vitro and in vivo.
Effects of IL-2 on expression of CD27 and CD70 on activated
CD8?effector T cells
The observation that IL-2 treatment alone was capable of modu-
lating the expression levels of CD70 and CD27 on CD8?T cells
in peripheral blood led to further studies examining the expression
levels of these markers on in vitro cultured effector CD8?from
patients with melanoma. The majority of CD8?T cells in TILs
grown in IL-2 from 10 patients expressed significant levels of
CD70, whereas only ?20% of the T cells expressed CD27 (data
not shown). The withdrawal of IL-2 for a period of 2 days resulted
in substantial up-regulation in CD27 expression on CD8?T cells
present in some TIL cultures. An example is shown in Fig. 2A. The
overall increase in CD27 expression on TIL following IL-2 with-
drawal for 2 days was statistically significant (p ? 0.01, n ? 10).
The association between the expression of CD27 and CD70 on in
vivo persistent T cells following adoptive transfer was then exam-
ined. Expression of CD27 progressively increased during a period
of 2 mo on two dominant T cell clonotypes derived from TILs that
persisted in the peripheral blood of two patients following adoptive
7727 The Journal of Immunology
transfer. Again, there appeared to be a negative correlation between
CD27 and CD70 expression on the persisting T cells (Fig. 2B). These
observations were consistent with the findings reported above indi-
cating that in vitro culture in the absence of IL-2 resulted in up-reg-
because IL-2 levels present in peripheral blood are only elevated in
vivo for a period of 2–3 days following TIL transfer when high-dose
IL-2 was administered. Taken together, these results suggested that
and CD27 was examined on gated populations of CD8?T cells from PBMCs derived from three melanoma patients obtained before any treatment in the
Surgery Branch of the National Cancer Institute, National Institutes of Health. Cells were tested for CD70 and CD27 expression after culturing in medium
with IL-2 (300 IU/ml or 3,000 IU/ml), and FACS analysis was conducted on days 1, 5, 8, and 12 after culture initiation. A time course of the effects of
IL-2 on up-regulating CD70 expression on CD8?cells (upper row) and the correlation between CD27 and CD70 expression on CD8?cells during IL-2
culture (bottom row) are represented. B, The expression of CD70 and CD27 was examined on gated CD8?T cells obtained from the peripheral blood of
five patients either before treatment or following treatment with high-dose IL-2. The first IL-2 dose was administered on day 1. The time course
demonstrating the modulation of CD70 expression is represented in the upper row, and the time course representing the modulation of CD27 expression
is represented in the bottom row.
IL-2 induces up-regulation of cell surface CD70 expression on CD8?cells from PBMCs in vitro and in vivo. A, The expression of CD70
7728 IL-2 MODULATES CD27-CD70 INTERACTIONS
IL-2 plays a direct role in modulating the expression of both CD27
and CD70 on CD8?T cells.
Preferential proliferation of a subset of CD8?T cells that
express CD70 and bind IL-2
The involvement of IL-2 in regulating expression of CD70 and
CD27 was then investigated further by testing whether IL-2 bind-
ing correlated with the expression of these markers following in
vitro culture of PBMCs in low-dose (300 IU/ml) or high-dose
(3,000 IU/ml) IL-2 for 12 days. IL-2 has been shown to bind to
IL-2R? with low affinity, to the IL-2 R?? complex with interme-
diate affinity, and to the IL-2R??? complex with high affinity (27,
28). Before the initiation of in vitro PBMC cultures, binding of
rhIL-2F to CD8?T cells was not detected (data not shown), and
?10% of the CD8?T cells from peripheral blood expressed CD70
at this time (Fig. 1A, upper row). Incubation of PBMCs with high-
dose IL-2 for five days resulted in the generation of a subpopula-
tion of T cells that was capable of binding rhIL-2F in four of the
five samples that were examined (Fig. 3A), and this binding was
correlated with the expression of CD25 (data not shown). High-dose
IL-2 appeared to be more effective than low-dose IL-2 at enhancing
the levels of IL-2 receptor expression on CD70?CD8?cells, which is
consistent with the observation that essentially all of the unstimulated
peripheral CD8?T cells expressed only the intermediate affinity IL-
2?? receptor complex. In addition, the CD70?T cells did not bind to
IL-2, and only a subset of the CD70?T cells bound detectable levels
of IL-2. This heterogeneous response may result from differences in
the susceptibility of naive vs memory cells to be activated by IL-2
alone, and differences between the responses of PBMCs obtained
from different donors may reflect differences in the pool of activated
vs naive cells present in these samples. When cell cycle analysis was
conducted on gated populations of CD8?T cells, the cells that dem-
onstrated detectable binding to rhIL-2F contained a significantly
higher percentage of cells in S phase than cells that failed to bind to
IL-2 (p ? 0.005; n ? 5) (Fig. 3B).
expression of CD27 and CD70 on in
vitro cultured TIL. A, The infused TIL
from 10 melanoma patients were cul-
tured in medium in the presence or ab-
sence of IL-2 for 2 days and costained
with anti-CD27, anti-CD70, and anti-
CD8 Abs. An example of a TIL culture
is shown; the quadrant was based on
isotype controls for gated populations
of CD8?T cells present within the TIL
(left), and a summary analysis of the
patients is shown (right). B, Flow cy-
tometry of CD27 and CD70 on CD8?
cells present in the peripheral blood of
two patients after adoptive TIL trans-
fer. The gated populations, correspond-
ing to dominant persistent clonotypes
that were identified following adoptive
transfer, were analyzed for expression
of CD27 and CD70. For patient num-
ber 9, cells were gated using a tetramer
of the dominant HLA-A2 MART-1
epitope, and for patient number 3 the
population was gated using an anti-
Effects of IL-2 on the
7729 The Journal of Immunology
A time course study was then conducted to examine the effects
of in vitro culture with IL-2 on the expression of CD27 and CD70.
The results demonstrated that the percentage of CD70?CD8?T
cells that bound to rhIL-2F peaked on day 8 at a time when a
significant loss in the expression of CD27 was observed (Fig. 3C).
Twelve days following culture initiation, the levels of IL-2 binding
had begun to decrease, and the expression of CD27 was either
maintained at a low level or had begun to increase (Fig. 3C and
data not shown).
IL-2 mediates T cell proliferation through the interaction of
CD27 and CD70
It appeared that IL-2 preferentially bound to CD70?CD8?T cells
and promoted significant proliferation of CD8?cells. To deter-
mine whether the interaction of CD70 with CD27 played a direct
role in mediating T cell proliferation, TILs were incubated with a
blocking Ab directed against CD70 in the presence of high-dose
IL-2 (3,000IU/ml) for 2 days. Anti-CD70 Ab blocked CD70 ex-
pression (98.1%) and inhibited proliferation of the cultures by
nearly 40%, (Table I); however, prolonged incubation with anti-
CD70 led to significant apoptosis as compared with control Ab
Table I. Blocking of CD70 expression on TIL-inhibited proliferation of
41.8 ? 1.8
36.0 ? 2.8
0.2 ? 0.0
21.3 ? 0.7
98.1 ? 0.0
37.9 ? 1.5
aPatient TIL consisting of ? 98% CD8?T cells were cultured at a concentration
of 1 ? 106cells/ml with 3,000 IU/ml IL-2 for 2 days with either an anti-CD70 or an
isotype-matched control Ab at a concentration of 6.8 ?g/ml.
bPercentage of Inhibition ? value(anti-IgG)? value(anti-CD70)/value(anti-IgG)?
cThe significance of differences was evaluated using a two-tailed Mann-Whitney test.
dThe expression of CD70 was analyzed by FACS analysis of gated CD8?T cells.
eCell cycle analysis was carried out to determine the extent of proliferation of T
cells cultures. Values indicate the percentage of CD8?T cells in S phase.
express CD70 and are bound to IL-2 was analyzed in PBMCs obtained from five patients following in vitro incubation with either low-dose (300 IU/ml)
or high-dose (3,000 IU/ml) IL-2 for 5 days. B, Cell cycle analysis was conducted on samples of PBMCs that were cultured in 3,000 IU/ml IL-2 for 12 days.
Cells that either bound to fluorescence-labeled rIL-2 (IL-2-FITC?) or did not bind detectable levels of IL-2 (IL-2-FITC?) were gated and analyzed for the
percentage of cells in S phase. The mean ? SD of the percentage of gated populations from five PBMCs was calculated, and the significance of this
difference was determined using the two-tailed Mann-Whitney U test. C, A kinetic analysis of the frequency of cells that bound to IL-2FITC in comparison
to the frequency of cells that expressed CD27 and CD70 was conducted for two PBMC samples (from patients number 6 and number 9) that were incubated
with high-dose IL-2 for a total of 12 days.
Binding of IL-2 to a subset of CD70?CD8?T cells and association of binding with proliferation. A, The frequency of CD8?cells that
7730IL-2 MODULATES CD27-CD70 INTERACTIONS
(data not shown). These results suggested that the ability of IL-2 to
mediate T cell proliferation may be mediated in part through the
interaction of CD27 with CD70.
Preferential secretion of IL-2 and proliferation by CD8?T cells
that express CD27
The ability of CD27?and CD27?T cells to secrete IL-2 following
with CD8?T cells demonstrated preferential secretion of IL-2 by
CD27?T cells in response to a viral epitope (11). Cultures of TILs
containing a high frequency of T cells reactive with the melanoma Ag
MART-1 were incubated in the absence of IL-2 for 4 days, and the
CD27?cells and CD27?cells were then isolated. The results of a
quantitative RT-PCR assay indicated that the T cells lacking cell sur-
face expression of CD27 also contained low levels of mRNA encod-
CD27?T cells secreted significantly higher levels of IL-2 than
CD27?T cells following incubation with target cells pulsed with 0.1
?g/ml MART-1 peptide (Fig. 4A, left panel), whereas IFN-? release
was only slightly higher from CD27?CD8?cells than from
CD27?CD8?cells with ?1 ?g/ml peptide (Fig. 4A, right panel).
The analysis of cells present in S phase also indicated that, when
cultured in the absence of exogenous IL-2 for 2 days, the percentage
of cycling CD27?TIL cells was significantly higher than the per-
centage of cycling CD27?T cells, whereas a significant difference
was not observed between these populations when they were cultured
in the presence of IL-2 (Fig. 4B).
Microarray analysis was then conducted to determine whether
there were significant differences in the gene expression profiles of
the isolated CD27?and CD27?T cells following IL-2 with-
drawal. The results of this analysis indicated that more than one-
third of the top 28 genes that were overexpressed in the CD27?T
cells relative to CD27?T cells were involved in either cell cycle
regulation or DNA replication. Genes previously found to be over-
expressed in tumor cells, such as the minichromosome mainte-
nance proteins and proliferating cell nuclear Ags (29, 30), were
also overexpressed in CD27?T cells. In contrast, the majority of
genes that were up-regulated in the CD27?population have been
culture that was maintained in medium without IL-2 for 4 days. The CD27?and the CD27?T cells were cocultured with a T2 cell pulsed with the indicated
concentrations of the MART-126–35(27L) peptide for 18 h, and cytokine release was tested by ELISA. The data were representative of one of two patients
whose TILs contained a high frequency of MART-1-positive T cells (?90%), and the frequency of MART-1-positive cells was equally distributed in
CD27?and CD27?populations. B, Four TILs were cultured in medium with (3,000 IU/ml) or without IL-2 for 2 days, and cell cycle analysis was conducted
by determining the percentage of cells in the CD27?and CD27?populations that were present in S phase, and the significance of this difference was
determined using the two-tailed Mann-Whitney U test.
Preferential secretion of IL-2 and proliferation of CD8?T cells that express CD27. A, CD27?and CD27?T cells were isolated from a TIL
7731The Journal of Immunology
shown to be associated with activation and apoptosis, whereas
genes associated with proliferation were not up-regulated in these
cells (Table II), which was in line with previous studies showing
that loss of expression of CD27 is a hallmark of late stage effector
Expression of CD27 and CD70 on TIL and relevance for
To evaluate the potential relevance of these observations for adop-
tive immunotherapy, the expression levels of CD27 and CD70
were examined on CD8?TIL cells that were administered to a
series of melanoma patients following nonmyeloablative chemo-
therapy. Previous results demonstrated that ?50% of the patients
treated in this trial responded to therapy (8, 9) and that the in vivo
persistence of transferred TIL cells was associated with clinical
response (31). As shown above, IL-2 can modulate CD27 and
CD70 expression; thus, the majority of TIL samples expressed
very low levels of CD27 when they were cultured in high-dose
IL-2. Cultures of TILs were withdrawn from IL-2 for 2 days, and
comparisons were then conducted between CD8?T cell clono-
types in the TILs that were previously shown to either persist or
not persist in vivo in patients following TIL transfer (31). Persis-
tent clonotypes expressed relatively higher levels of CD27?cells
than nonpersistent clonotypes in the same patient (p ? 0.05; n ?
6) (Fig. 5), although considerable overlap existed between persist-
ing and nonpersisting clonotypes among the patients. Bulk samples
of TILs that were administered to patients who either did or did not
respond to therapy were then withdrawn from IL-2 for 2 days, and
the cellular phenotype was assessed. The results of this analysis
indicatedthatthe mean number
CD70?CD8?T cells in these IL-2-deprived TIL cultures was sig-
nificantly higher in patients that showed an objective clinical re-
sponse to cell transfer (p ? 0.004 and p ? 0.01, respectively; n ?
33) (Table III). Neither the total number of infused T cells nor
CD8?T cells differed significantly between responders and non-
responders (Table III), and no significant differences were ob-
served between the expression of the costimulatory molecule
CD28 and its corresponding ligand CD80 in the same TIL samples
(data not shown). These observations suggest that analysis of the
expression of CD27 and CD70 may be useful for the selection of
effective TILs for use in adoptive immunotherapy.
Signaling mediated through the IL-2R, in conjunction with signals
mediated through the T cell Ag receptor, promotes the prolifera-
tion and effector function of T cells (32). Some of the signals, such
Table II. Genes differentially overexpressed in CD27?or CD27?CD8?cellsa
AssociationRatio Gene Symbol
Cell cycle-related genes
Cytokine production genes
Activation and apoptosis genes
aThe listed genes demonstrated 2-fold or greater differential expression between CD27?and CD27?cells and more than
three times background levels in either channel. Analysis was carried out on samples used in Fig. 4A.
7732IL-2 MODULATES CD27-CD70 INTERACTIONS
as up-regulation of the IL-2R ?-chain, result from cooperative sig-
nals mediated both through the T cell Ag receptor as well as
through the IL-2R itself; however, the relative role of signals de-
livered through these pathways has not been fully elucidated. Hu-
man tumor-reactive effector T cells have been shown to proliferate
extensively in vitro in the presence of high-dose IL-2 alone (33).
In addition, between 15 and 20% of melanoma and renal cancer
patients treated with high-dose IL-2 alone respond to therapy (6),
which may reflect the ability of IL-2 to maintain the proliferation
of T cells that were activated by prior exposure to tumor Ags.
Interactions between the costimulatory receptor CD27 and its
ligand, CD70, have also been found to play a key role in T lym-
phocyte activation, proliferation, survival, and differentiation (10–
13). The intracytoplasmic domain of CD27 triggered by CD70
mediates these effects through activation of Jun kinase and NF-?B,
which is required for optimal effector/memory CD8 T cell gener-
This study explores the effects of IL-2 on the expression of
CD70 and its receptor, CD27, on CD8?T cells, as well as the role
of stimulation mediated through the CD27 pathway in regulating
Table III. Total number of CD8?and CD27?CD8?in TILs cultured without IL-2 for 2 daysa
Total Cell Infused
Mean ? SEM
Mean ? SEM
6.3 ? 0.7
22 ? 3.2
1.5 ? 0.3
54.6 ? 5.83.62 ? 0.64.26 ? 0.6
6.1 ? 1.1
11 ? 2.9
4.07 ? 1.1
34.4 ? 5.3
0.54 ? 0.1
2.44 ? 0.5
aSamples from infusion bags were cultured without IL-2 for 2 days, and the expression of the indicated phenotypic markers
were analyzed. The frequency of CD27?CD8?, CD70?CD8?, or CD8?T cells, as well as the total number of cells corre-
sponding to the indicated phenotype that would be present within each of the administered TIL under these conditions, was
calculated based on the analysis of ungated samples. Quadrants were drawn based on staining obtained with IgG control
antibodies. The significance of differences was determined using the two-tailed Wilcoxon rank sum test.
bResponse was defined as a 50% or greater decrease in the sum of perpendicular diameters of all measurable lesions for at
least 1 mo with no increase in any lesions and no new lesions.
lates with in vivo persistence of dominant TIL clonotypes after adoptively
transfer. Six infusion TILs were cultured in medium without IL-2 for 2–4
days, and CD27 expression on dominant CD8?clonotypes was analyzed
by costaining with anti-TCR ?-chain variable region-specific Abs as well
as an anti-CD27 Ab. The frequency of CD27?cells corresponding to each
of the dominant clonotypes that were identified in a previous study is
indicated (31). The significance of differences was determined by two-
tailed Wilcoxon signed rank test.
Frequency of CD27?cells present in infusion TIL corre-
7733The Journal of Immunology
the proliferation and function of effector T cells. Incubation of
PBMCs with IL-2 in vitro resulted in the up-regulation of CD70
expression on CD8?cells and down-regulation of CD27 expres-
sion. This outcome did not appear to result from the selective death
of CD27?T cells, as significant levels of apoptosis were not ob-
served in the CD27?T cells that were present in these cultures
(data not shown). Cells that possess a similar phenotype were ob-
served in vivo in patients receiving high-dose IL-2 therapy at the
time when IL-2 is present at detectable levels in peripheral blood.
Expression of IL-2R? (CD25) was not detected on CD8?T cells
before the up-regulation of CD70; thus, it appears that relatively
high concentrations of IL-2 can up-regulate CD70 expression by
signaling through the intermediate affinity IL-2R?? receptor,
which has previously been shown to activate T cells (35–37).
Previous investigations demonstrated that CD27 expression is
down-regulated on the surface of activated T cells following in-
teraction with CD70. In CD70 transgenic mice that constitutively
express CD70 on B cells, CD27 was down-regulated because of
the continuous interaction of CD70 with CD27?CD8?T cells
(10). In another report, the incubation of CD8?T cell with anti-
CD70 Ab interfered with the down-regulation of CD27 that was
normally observed following Ag activation (11). The results of the
present study suggest that down-regulation of CD27 expression on
CD8?T cells from peripheral blood following incubation with
IL-2 may have resulted from interactions with CD70 expressed on
T cells, which was associated with the acquisition of effector func-
tions on those cells. This hypothesis was supported by the obser-
vation that the withdrawal of IL-2 from activated effector CD8?T
cells was associated with the up-regulation of CD27 and the down-
regulation of CD70 expression. A similar phenotypic change was
observed on CD8?T cells present in the peripheral blood of IL-
2-treated patients soon after the termination of IL-2 administration.
Additional experiments were then conducted to evaluate the na-
ture of the interaction of CD27 with CD70 expressed on CD8?T
cells. The incubation of in vitro cultured effector CD8?T cells that
expressed CD70 with an anti-CD70 blocking Ab significantly in-
hibited the proliferation of these cells, even in the presence of
high-dose IL-2, but did not alter the expression of markers such as
CD25 (data not shown). These findings suggest that this Ab inter-
fered with delivery of a signal through the CD27 molecule rather
than delivering a direct signal by the cross-linking of CD70 mol-
ecules on the surface of T cells. Studies conducted on PBMCs that
were cultured in vitro with IL-2 indicated that the subset of CD70?
cells that bound to rhIL-2F proliferated more extensively than cells
that failed to bind to the rhIL-2F. The population of CD70?CD8?
T cells that bound high levels of IL-2 also appeared to selectively
down-regulate CD27 expression (data not shown), indicating that
the delivery of a strong signal through the IL-2 receptor may play
an important role in the down-regulation of CD27 expression by
CD70. These observations suggest that engagement of CD27 by
CD70 molecules expressed on T cells represents a down-stream
mediator of the activation of T cells by IL-2.
The expression of CD27 was then examined on TILs that were
adoptively transferred to patients, as this might provide an indica-
tion of the size of the effector/memory pool in these polyclonal
populations of cells. Previous studies had demonstrated that the
differentiation of T cells to end stage effector cells was associated
with stable down-regulation of CD27 expression (14–17). The fact
that IL-2, as well as TCR activation, transiently down-regulated
CD27 expression on TILs hampered evaluation of the stage of
differentiation of cells present in these polyclonal populations.
However, the withdrawal of IL-2 from in vitro TIL cultures that
had been grown continuously in the presence of IL-2 resulted in
up-regulation of the expression of CD27 on a variable percentage
of the T cells present in these cultures. The percentage of T cells
that up-regulated CD27 expression varied widely between TILs,
and for some cultures little or no up-regulation was observed. The
minimal levels of CD27 expression that were observed on some
TILs following IL-2 withdrawal may have resulted from the large
percentages of end stage effector T cells present in these cultures.
Additional studies were then conducted to evaluate the proper-
ties of CD27?CD8?T cell present within TILs. When TILs were
cultured for 2 days in vitro in the absence of IL-2, CD27?CD8?
T cells proliferated to a greater extent than CD27?CD8?T cells.
Previous studies have demonstrated that central memory cells pos-
sess an enhanced ability to secrete IL-2 (38), and results presented
in this study demonstrate that CD27?CD8?cells secreted higher
levels of IL-2 than CD27?CD8?cells following Ag activation.
These factors may help to account for the selective ability of
CD27?CD8?T cells to survive in vivo following adoptive transfer
under conditions where common ?-chain cytokines such as IL-2
are limiting (17, 39). In this study, several genes that are selec-
tively expressed in proliferating cells (29, 30) were up-regulated
selectively in CD27?CD8?T cells isolated from TILs. In contrast,
the most highly expressed genes that were selectively up-regulated
in CD27?CD8?T cells included several genes involved with T
cell activation and apoptosis, characteristics that have been asso-
ciated with late stage effector T cells.
The potential relevance of these findings to clinical observations
was then evaluated. Previous studies (31) suggested that the per-
sistence of adoptively transferred T cells was associated with their
proliferative capacity as well as with their ability to mediate tumor
regression. Examination of individual clonotypes present within
bulk TILs that persisted in patients’ peripheral blood following cell
transfer revealed that these cells, when cultured before transfer in
the absence of IL-2 for 2 days, expressed higher levels of CD27
than clonotypes that did not persist. An evaluation of the bulk TILs
obtained from 33 patients treated with TILs following nonmyeloa-
blative chemotherapy, including 16 responders and 17 nonre-
sponders, demonstrated that the mean of total number of
CD27?CD8?T cells present in the TILs that were administered to
responders were significantly higher than those administered to
nonresponders. The expression of CD70 on cells cultured before
transfer in the absence of IL-2 for 2 days also appeared to be
significantly higher in TILs that were administered to responders
than to nonresponders, implying that the pool of activated mem-
ory/effector cells present in TILs that were administered to re-
sponders was larger than in TILs that were administered to non-
responders. The expression of CD70 in vivo on other cells, such as
B cells and dendritic cells that are present in patients that received
adoptive TIL transfer, may also act to costimulate T cells that
express CD27. The expression of CD70 on the transferred T cells
alone, however, may provide a sufficient signal to promote the in
vivo survival and proliferation of transferred CD8?cells that are
also capable of expressing CD27, as suggested by the in vitro
studies presented in this report as well as by additional studies
(40). In addition, in mouse model systems the expression of CD27
appeared to be associated with T cell accumulation at tissue ef-
fector sites and the survival of activated CD8?T cells in vivo,
whereas expression of CD28 was not associated with T cell mi-
gration (41). It is difficult to determine the relative roles of CD27
and CD70 expression in mediating the function of adoptively
transferred T cells, but the expression of both the costimulatory
receptor and ligand on T cells may enhance the in vivo function of
these cells. These observations provide potential explanations for
the association between CD27 expression in TILs and patient re-
sponse to adoptive immunotherapy.
7734IL-2 MODULATES CD27-CD70 INTERACTIONS
In summary, IL-2 is a crucial cytokine that modulates CD8?T
cell responses in immunotherapy. Our results suggested that IL-2
may function, at least in part, by promoting CD8?T cell effector
function through the interaction of CD27 with CD70. In addition,
evaluation of the pool of T cells present in patients’ TILs that
express CD27 and CD70 may facilitate the identification of effec-
tive TILs for use in patient treatment.
We thank Arnold Mixon and Shawn Farid for assistance with fluorescent
The authors have no financial conflict of interest.
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7735The Journal of Immunology