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IMMUNOBIOLOGY
From the Department of Surgery, University of
Pittsburgh School of Medicine, and the Division of Biological
Therapeutics, University of Pittsburgh Cancer Institute, PA.
Interleukin-10 (IL-10) is a multifunctional cytokine that can exert
suppressive and stimulatory effects on T cells. It was investigated
whether IL-10 could serve as an immunostimulant for specific
CD8+ cytotoxic T cell (CTL) in vivo after vaccination and,
if so, under what conditions. In tumor prevention models,
administration of IL-10 before, or soon after, peptide-pulsed primary
dendritic cell immunization resulted in immune suppression and enhanced tumor progression. Injection of IL-10, however, just after a booster vaccine significantly enhanced antitumor immunity and vaccine efficacy.
Analysis of spleen cells derived from these latter animals 3 weeks
after IL-10 treatment revealed that the number of CD8+
CD44hi CD122+ T cells had increased and that
antigen-specific proliferation in vitro was enhanced. Although
cytotoxicity assays did not support differences between the various
treatment groups, 2 more sensitive assays measuring antigen-specific
interferon- Dendritic cells (DCs) play a key role in the
initiation of CD8+ cytotoxic T cell (CTL)-mediated immune
responses and have been used successfully in cancer
vaccines.1,2 We have previously reported that
CD8+ CTLs can be generated from naive precursors using DC
preloaded with leukemia antigens as stimulators.3-5
Leukemic cell-derived DCs have been used to successfully treat patients
with chronic myelogenous leukemia.6 To more effectively
treat patients with cancer, vaccines must not only amplify antitumor
CTL responses, they must maintain them to preclude disease recurrence.
The main predictor of a strong response versus a weak CTL appears to be the size of a proliferative burst.7,8 However, for the
promotion of long-term effector CTL activity, the participation of
T-cell growth and antiapoptotic factors, such as interleukin 2 (IL-2), IL-10, IL-12, and IL-15 appear important.9-14 In this
study, we have evaluated whether IL-10 plays a dominant role in
supporting the maintenance of effector CD8+ T-cell function
after initial priming in situ.
IL-10, which is produced by a variety of cells including T lymphocytes,
B lymphocytes, and monocytes, has been identified as a key
immunomodulatory cytokine15,16 capable of mediating both
immunosuppressive and immunostimulant effects.17 In vitro studies showed that IL-10 inhibits antigen-specific activation and
proliferation of human CD4+ T cells, at least in part by
down-regulating the cytokine production (ie, IL-2 and tumor necrosis
factor- Mice and cell lines
Cytokines and peptide
Preparation of dendritic cells Dendritic cells were prepared from bone marrow cultures as previously described.31,32 Briefly, bone marrow was flushed from femurs and tibias and subsequently depleted of erythrocytes with ammonium chloride. Bone marrow cells were depleted of T cells and B cells using a mixture of monoclonal antibodies (anti-CD4/rat immunoglobulin [Ig] G2b, GK1.5; anti-CD8/rat IgG2b, 53-6.7; anti-B220/rat IgG2b obtained from ATCC) and rabbit complement (Cedarlane Laboratories, Ontario, Canada). Cells were cultured in complete medium containing 20% fetal bovine serum supplemented with mGM-CSF (1000 U/mL) and mIL-4 (1000 U/mL). At day 7, nonadherent cells were collected and further purified by first blocking with mouse serum (Sigma, St Louis, MO) and then positively selecting with hamster anti-mouse CD11c magnetic beads (Miltenyi Biotec, Auburn, CA). Cells were washed 3 times with phosphate-buffered saline (PBS; Life Technologies) and were used for subsequent experiments.Tumor protection model: vaccination with peptide-pulsed dendritic cells or cytotoxic T cell adoptive transfer In the preliminary tests, we determined the optimal concentration of OVA peptide as 1 µM peptide for use in our study by comparing vaccines consisting of DCs that had been preincubated with varying concentrations of OVA peptide (1 nM-100 µM) for 2 hours. For our studies, 2 × 106 tumor cells (EG.7-OVA) were selected as a standard because this was the maximal dose that could be effectively protected against by prior vaccination with 1 × 106 DCs. In DC vaccination models, mice were randomized and injected intravenously with 1 × 106 peptide-pulsed DC. One week later, syngeneic tumor cells (2 × 106 for EG.7-OVA) suspended in 0.1 mL Hanks balanced salt solution (HBSS; Life Technologies) were injected subcutaneously on one flank. For studies involving IL-10 administration, 0.2 mL rhIL-10 (40 µg/mouse per day) in HBSS was injected intraperitoneally once daily for 4 consecutive days after DC vaccination models, or for 7 days after CTL adoptive transfer. In CTL adoptive transfer studies (EG.7-OVA and MC38), mice were injected intravenously with activated CTLs (1 × 106 reactive against EG.7-OVA and 1 × 105 reactive against MC38). One week later, syngeneic tumor cells (2 × 106 for EG.7-OVA or 3 × 105 for MC38) suspended in 0.1 mL HBSS were injected subcutaneously in one flank. In both vaccination models, serial micro-caliper measurements of perpendicular tumor diameters were obtained in a masked fashion. For the evaluation of in vivo immune response, the phenotype and the total number of each CD8+ T-cell subpopulation, such as CD8+ CD44+ CD122 (IL-2R )+ T cells or CD8+ CD44+
IFN- -expressing T cells, in the spleen were evaluated. Tumor size
was measured every 2 to 4 days, and tumor volumes were calculated using
the formula: longest diameter × shortest diameter. Tumor volumes were depicted as the mean tumor area in graphic representation of tumor progression-regression. All experiments for protection models
were performed using 5 mice per group, and experiments for the analysis
model used 3 mice per group. Each experiment was confirmed at least
once, as indicated.
Proliferation assay in the time course in the vaccination models Cell proliferation was measured by [3H]-thymidine incorporation in 96-well round-bottom plates (Costar, Cambridge, MA) by using 1 × 106 spleen cells/well, pulsed with and without peptide, or added hIL-2 (100 U/mL), hIL-15 (100 U/mL), and PMA (25 ng/mL) plus ionomycin (0.5 µM) (Sigma) as indicated. After 48 hours, 0.037 MBq/well (1 µCi/well) [3H]-thymidine (NENTH Life Science Products, Boston, MA) was added, and cells were harvested 16 hours later.Flow cytometric and tetramer staining Single-cell suspensions of spleen cells were washed in PBS containing 0.1% bovine serum albumin and 0.02% sodium azide and were incubated for 20 minutes on ice with purified anti-mouse CD16/CD32 (Fc III/II receptor) monoclonal antibody (mAb; 2.4G2; PharMingen,
San Diego, CA) to block Fc receptors. For detecting the
activated-memory CD8+ T cells (CD8+
CD44hi CD122+ T cell), phycoerythrin
(PE)-conjugated anti-CD44 (Pgp-1, Ly24) and fluorescein
isothiocyanate (FITC)-conjugated anti-CD122 (IL-2R) (TM-1)
expression in CD8+ T cells were performed by gating
peridinin chlorophyll protein (Per CP)-conjugated
anti-CD8+ (53-6.7) T cells from spleen cells. Total
CD8+ CD44hi CD122+ cells were
designated as activated-memory CD8+ T cells during time
course experiments. In cases of tetramer staining, anti-mouse CD8a mAb
(Caltag Laboratories, Burlingame, CA) was used for blocking nonspecific
binding. These cells were then stained using FITC-conjugated V5.1,
5.2 mAb (MR9-4), and PE-conjugated MHC class I (H-2Kb)
tetramer complexed with OVA257-264 peptide, which was
provided by the NIAID tetramer facility (Rockville, MD). For detecting OVA-specific activated CD8+ T cells (CD8+
CD44hi tetramer+), FITC-conjugated anti-CD44
and PE tetramer were evaluated in CD8+ T cells by gating
Per CP-CD8+ T cells. Other antibodies used in phenotype
analysis included FITC-conjugated anti-CD4 (GK1.5), anti-CD8a (53-6.7),
anti-CD25 (7D4), anti-CD69 (H1.2F3), anti-CD62L (MEL-14), anti-Ly6C
(AL-21), anti-CD45R/B220 (RA3-6B2), and PE-conjugated anti-CD8a,
anti-NK1.1 (PK136); all were purchased from BD Pharmingen (San
Jose, CA). Samples were acquired on a FACScan flow cytometer, and the
data were analyzed using Cellquest software (Becton Dickinson).
Intracellular cytokine staining and flow cytometry Spleen cells from DC-vaccinated mice or naive mice were cultured at 37°C in 5% CO2 for 24 hours in the presence or absence of 1 × 10 7 M peptide (OVA257-264)
in complete medium. Brefeldin A (Golgistop; PharMingen) was added at a
final concentration of 1 µl/mL during the last 5 hours of the culture
period. After culture, the cells were harvested, washed once in FACS
buffer, and surface stained in FACS buffer with CD8a-FITC for CTL or
with CD8a-Per CP and CD44-FITC for spleen cells. After washing the
unbound antibody, cells were subjected to intracellular cytokine stain
using the Cytofix/Cytoperm kit according to the manufacturer's
instructions (PharMingen). For intracellular interferon (IFN-),
IL-2, and IL-4 staining, we used PE-conjugated monoclonal rat
anti-mouse IFN- antibody (XMG 1.2), anti-mouse IL-2 antibody
(S4B6), and anti-mouse IL-4 antibody (BVD4-1D11) and its isotype
control antibody (rat IgG) (PharMingen). Samples were resuspended in
PBS containing 2% formaldehyde and acquired on either a FACScan
(50 000 gated events acquired per sample) and analyzed using Cellquest
software (Becton Dickinson). Total numbers of IFN--producing cells
in the spleen were evaluated in kinetic time course experiments.
Single-cell enzyme-linked immunospot assay for
interferon- antibody (20 µg/mL) (clone R4-6A2; PharMingen) for 16 hours.
Spleen cells (5 × 106 cells/well) from each group were
incubated for 36 hours either with or without peptide stimulation
(1 × 107 M OVA peptide) or were incubated for 24 hours
with EG.7-OVA or EL4 (1.25 × 105 cells/well) at an
effector-target ratio of 40:1. After culture, the plates were washed
and then incubated with biotinylated anti-mouse IFN- antibody (4 µg/mL) (clone XMG 1.2; PharMingen). Spots were developed using
avidin-peroxidase-complex (Vectastain Elite Kit; Vector Laboratories,
Burlingame, CA) and fresh, prepared substrate buffer (0.4 mg/mL
3-amino-9-ethyl-carbazole in 2.5 mL N, N-dimethyl formamide [Sigma]
and 0.015% H2O2 in 0.2 M sodium acetate and 0.2 N acetic acid). The frequency of peptide-specific CD8+
T cells was calculated based on the percentage of CD8+ T
cells in the responding population. Spots were counted microscopically.
Transfer study of purified naive or activated CD8+ T cells into the syngeneic mice For OVA-specific T-cell transfer studies, magnetically purified naive and activated CD8+ OT-1 (OT-1) T cells were used. After removing adherent cells by 2-hour culture, cells pretreated with mouse serum (Sigma) and then incubated with anti-CD8a mAb labeled microbeads (Miltenyi Biotec) by using 2 beads/cell, and they were isolated on a SuperMACS cell sorter (Miltenyi Biotec). Purity of the eluted cells was more than 97%. This procedure did not activate naive or activated cells (T-cell activation; ie, neither cytokine secretion nor proliferation in vitro was shown). For the generation of CTL (effector cell) from OT-1 transgenic mice, 1 × 106 spleen cells per milliliter from OT-1 mice were stimulated with irradiated (30 Gy), 1 × 106 M OVA peptide-pulsed
syngeneic C57BL/6 spleen cells (1 × 106 cells/mL) in
24-well plates (Costar). After 36 hours, OT-1 T cells were isolated
using anti-CD8a mAb-labeled microbeads, investigated for the phenotype
of activation marker, and tested for cytotoxicity for the use of
transfer studies.
In the OT-1 T-cell transfer studies, we adoptively transferred naive or
activated OT-1 T cells into syngenic C57BL/6 mice on day 0, which was
followed by IL-10 injection. Seven days later, we analyzed the
frequency of specific T cells in spleen using tetramer and anti-V Statistical analysis Statistical analysis in our experiments was performed using standard Student t tests.
Analysis of the efficacy of vaccines consisting of peptide-pulsed dendritic cells, combined with IL-10 treatment For investigation of the optimal phase for the efficacy of IL-10, we modulated the time of administration of IL-10 in the tumor protection models after OVA peptide-pulsed DC vaccination (Figure 1A). Immunized mice in group 5 (IL-10 after EG.7-OVA challenge) exhibited transient tumor growth that regressed after 18 to 20 days; however, mice in other groups, such as groups 2 (no IL-10) and 3 (IL-10 before DC vaccination), displayed gradually progressive tumor growth (Figure 1B). This suggests that vaccination by peptide-pulsed DCs could be enhanced with IL-10 administration in vivo under certain conditions. To further analyze the impact of immunization by DCs and IL-10 in group 5, we characterized the responder splenocytes harvested from this group in comparison to other groups (Figure 2). Proliferation assays with and without IL-10, antigen, cytokine (IL-2 or IL-15), or PMA-ionomycin were assessed using splenocytes harvested from mice 14, 21, and 28 days after DC vaccination. Splenocytes from mice administered IL-10 exhibited good proliferative responses to peptide, especially at later time points (more than 21 days later) (group 1 vs group 4; P < 0.05 on day 14, P < 0.01 on days 21 and 28). Moreover, such splenocytes responded to IL-2/IL-15 (P < 0.01) but not to PMA-ionomycin, suggesting that they might represent memory T cells (Figure 3). In these groups, no statistical phenotypic differences were noted in the fractions of CD4+ T cells, CD8+ T cells, B cells, and NK cells (data not shown). We performed comparative phenotypic analyses of lymphocyte subpopulations by gating for CD8+ T cells, and the numbers of CD8+ CD44hi CD122 (IL-2R)+ T
cells (activated-memory T cells) were determined to be elevated in
groups 3 and 4 on day 21 (groups 1, 2 vs group 3, P < .05; groups 1, 2 vs group 4, P < .05;
group 3 vs group 4, P > .05 on day 21; but
P > .01 for all on days 14 and 28), suggesting that activated CD8+ T cells could be increased by systemic IL-10
administration (Figure 4). In group 3, the progressive decline of size after day 21 may support the
therapeutic efficacy of this enhanced cell population. As depicted in
Figure 5, effector CD8+
CD44hi T cells expressing IFN- on day 21 were detected
by intracellular analysis in group 3 (groups 1, 4 vs group 3, P < .05; group 2 vs group 3, P > .05; on
day 28, groups 1, 4 vs group 3, P < .0001; group 2 vs
group 3, P = .005). In preliminary tests in which
activated OT-1 T cells were assayed for both cytotoxicity and IFN-
production against EG.7-OVA target cells, we confirmed that CTL
function correlated well with IFN- production deduced by ELISPOT
assays (data not shown). In addition, specific CD8+ T-cell
reactivation was also documented in Figure
6 using ELISPOT assays and specific
stimuli (ie, OVA peptide and EG.7-OVA) in groups. On day 21, statistical differences were noted: groups 1, 2 vs group 3, P < .05; group 3 vs group 4, P < .01
for EG.7-OVA; group 1 vs group 3, P < .01; groups 2, 4 vs
group 3, P > .05 for OVA peptide; on day 28, groups 1, 2, 4 vs group 3, P < .01 for EG.7-OVA,
P < .0001 for OVA peptide. This suggests antigen-specific CD8+ effector cells producing IFN- can be rapidly
activated and can mediate therapeutic functions relevant to the
clinical benefit associated with animals in group 3.
Enhancement of antigen-specific CD8+ T cell function by interleukin-10 To study the interaction between rapidly activated CD8+ T cell and IL-10, we established that activated T cells express CD25, CD69, CD44hi, and CD62Llow phenotypes and produce IFN- (45.3%) on short-term (36 hours) culturing of OT-1 T cells with peptide (Figure
7).
To study whether the number and function of these activated T cells was
affected by IL-10 in vivo, we transferred naive or activated OT-1 T
cells into syngeneic C57BL/6 mice. Using a double-staining assay with
FITC-conjugated anti-V
This study provides evidence that effector CD8+ T cells capable of rapid activation (ie, memory) can be maintained by the administration of IL-10 in vivo. To discern the impact of IL-10 on vaccine-induced antitumor T-cell numbers and function, we initially had to establish optimal conditions for our vaccine-tumor challenge models. Using this schema, IL-10 was observed to promote optimal vaccine-associated inhibition of tumor growth when applied just after tumor challenge (ie, antigen boosting) (Figure 1). Minimal differences were shown in the numbers of immune subsets, such as B cells, macrophages, DC, CD4+, and CD8+ T cells in the spleens of treated mice on days 14, 21, and 28 in our investigated groups (data not shown).18-22,33,34 Although recent reports suggest that IL-10 can augment NK function in vivo, we observed no increase in NK cell numbers.35 Subsequent analysis focused on the antitumor activity associated with effector CD8+ T cells. In particular, we compared CD8+ T-cell numbers and function
in the spleens of mice in group 3 (group 5 in Figure 1A is equal to
group 3 in Figure 2) with those of the other control groups in
antigen-specific proliferation assays after initial immunization by DC
peptide (Figure 2). Three weeks after DC vaccination, spleen cells from
mice administered IL-10 exhibited antigen-specific proliferation
(Figure 3). We also noted that the activity of antigen-specific T cells
in mice treated with IL-10 could be enhanced by antigen, IL-2, and
IL-15. Because IL-2 and IL-15 have profound effects on CD8+
T-cell proliferation,9,10 it was suggested that
antigen-specific activated-memory type CD8+ T cells might
be induced to proliferate by IL-10.15,16 After 14, 21, and
28 days, spleen cells from immunized mice were cultured with OVA
peptide and assayed for specific lytic activity and their phenotypes.
IL-10 did not promote discernible differences in the phenotypes of
CD4+ or CD8+ T cells (data not shown). However,
splenocytes obtained from mice 21 days after initial vaccination with
DC (+ OVA peptide) contained the activated-memory type
CD8+ T-cell populations (CD8+
CD44hi CD122+ population). This effector T-cell
population was significantly increased in group 3-treated animals
(Figure 4).12-14,36,37 On day 6, the day before tumor
challenge, the mean number of this T-cell population in vaccinated mice
was 2.3 × 106 (control, 9.3 × 105).
However, we could not demonstrate any differences in cytotoxicity between groups after in vitro restimulation with antigen for 5 days
(data not shown). Therefore, by application of 2 sensitive ex vivo
assays to our studies, we characterized low-frequency, antigen-specific
splenic effector CD8+ T cells (ie, cells capable of
producing IFN- The addition of IL-10 does not appear to have any direct cytopathic
effects on activated CTLs in vitro (data not shown). To address the
possibility that preactivated antigen-specific CD8+ T cells
can survive in the absence of antigen on the provision of IL-10 in
vivo, we adoptively transferred OT-1 T cells expressing T-cell
activation phenotype and IFN- It has been noted that IL-10 can synergize with IL-2 or IL-4 to promote
CD8+ T cell proliferation.34,35,43 However, as
shown here in our studies, cultured OVA-specific cultured
CD8+ T cells from mice primed with OVA peptide did not
produce either IL-2 or IL-4 (Figure 7C); therefore, the mechanism of
this effect was different from the previously cited IL-2- or
IL-4-driven systems. These findings also indicated that IL-10 could
selectively drive the proliferation of the activated CD8+ T
cells while maintaining effector function. Neither unprimed nor
activated OT-1 T cells proliferated in response to low concentrations of IL-10 or in the absence of antigen (data not shown). This may suggest overall that OVA-specific cultured CD8+ T cells may
present cross-reacting peptides to themselves, resulting in apoptosis
(activation-induced cell death) in vitro,22,44,47 unless
cytokines such as IL-10 rescue them from death. This may indicate that
the blockade of activation-induced, caspase-dependent T-cell death by
IL-10 also rescues the function of "undead" T cells. However, it
was unclear whether these "maintained" T cells would respond or be
anergic to subsequent antigen restimulation. Therefore, T-cell
proliferation in vivo after antigen stimulation may support the
functional responsiveness to antigen. It has been recently shown that
resistance to activation-induced cell death can be attributed to high
levels of intracellular FLIP (IL-1 Thus, our in vivo observations indicate that specific CD8+ T-cell proliferation can be modulated by IL-10. This does not preclude additional indirect effects mediated by alternative IL-10 responsive cells, such as CD4+ T cells and NK cells. To investigate the potential involvement of these alternative cell types in our IL-10-dependent system, we performed depletion analyses by administration (intravenous) of specific mAbs (such as rat IgG, anti-CD4 Ab, anti-CD8 Ab, and anti-asialo GM1 mAb) 2 days before transferring purified naive or activated OT-1 T cells (Figure 9A). Seven days after transfer, the number of antigen-specific CD8+ CD44hi T cells (OVA-tetramer positive activated T cells) was observed to increase to the extent observed for control mice (ie, rat IgG-treated hosts) and in all other mAb-treated mice (Figure 9A). The injection of mAbs, such as anti-CD8 mAb, anti-CD4 mAb, or asialo-GM1 mAb before activated OT-1 T-cell transfer, did not change the results obtained in the ELISPOT assays. In the ELISPOT assay, however, the injection of anti-CD4 mAb followed by the administration of IL-10 increased the observed number of spots in response to OVA peptide (P < .005) (Figure 9B), suggesting that high-dose IL-10 may promote a suppressive CD4+ T-cell population, in part composed of CD4 regulatory T cells.51,52 Hence, IL-10 may act as an immune suppressor of CD4+ T cells and monocytes-DCs, and conversely it may act as an immunostimulant for an activated population of CD8+ T cells. These opposing effects of IL-10 may serve as a regulator for immune responsiveness.17,25,53 Adoptively transferred OVA-primed OT-1 T cells (CTLs) were augmented in their antitumor efficacy after the injection of mice with IL-10 (Figure 10). In the additional tumor models, IL-10 administration potentiated the antitumor effectiveness of an adoptively transferred CTL clone specific for the MC38 colorectal adenocarcinoma (Figure 11). Thus, the administration of in vitro activated CTLs, followed by the injection of mice with IL-10, may result in the extended durability of effector CD8+ T cells associated with the observed augmentation in antitumor efficacy. Our findings, along with the previous data, demonstrate that despite the suppressive effects of certain CD4+ T cells, IL-10 stimulates activated CD8+ T cells to proliferate, and it supports memory-type CD8+ T-cell effectors capable of rapidly activation in an antigen-specific manner in situ. Enhanced survival and function of CTLs mediated by concurrent IL-10 administration may boost clinical effectiveness or may be useful after adoptive immunotherapy approaches. Furthermore, when applied in a vaccination strategy, we anticipate that tumor vaccination followed by the systemic administration of rIL-10 may result in enhanced antitumor T-cell efficacy.
We thank Drs K. Murali-Krishna, J. Altman (Emory Vaccine Center, Emory University, Atlanta, GA), T. Takayama, and H. Kanaya (University of Pittsburgh, PA) for providing technical advice, and we thank Dr W. Storkus for peer-reviewing the manuscript.
Submitted February 13, 2001; accepted May 29, 2001.
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: Shin-ichiro Fujii, Laboratory of Cellular Physiology and Immunology, The Rockefeller University, 1230 York Ave, New York, NY, 10021-6399; e-mail: fujiis{at}mail.rockefeller.edu.
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Top
Abstract
Introduction
).
80°C until use.
ie, they
expressed CD25, CD69, and CD44 antigen. (C) They also produced IFN-
