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Trudeau Institute, Saranac Lake, NY 12983
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, whereas type 2 CD8+ T cells
(Tc2) secrete IL-4, IL-5, and IL-10. Using an OVA-transfected B16 lung
metastases model, we assessed the therapeutic effects of adoptively
transferred OVA-specific Tc1 and Tc2 subpopulations in mice bearing
established pulmonary malignancy. Effector cell-treated mice exhibiting
high (5 x 105) tumor burdens experienced significant
(p < 0.05) delays in mortality compared with those
of untreated control mice, whereas high proportions (70–90%) of mice
receiving therapy with low (1 x 105) tumor burdens
survived indefinitely. Long-term tumor immunity was evident by
resistance to lethal tumor rechallenge, heightened levels of systemic
OVA Ag-specific CTL responses ex vivo, and detection of long-lived TCR
transgene-positive donor cells accompanied by an elevation in the total
numbers of CD8+ CD44high activated and/or
memory T cells at sites of tumor growth. Long-lasting protection by Tc2
and Tc1 effector cells were dependent, in part, on both the level of
tumor burden and effector cell-derived IL-4, IL-5, and IFN-,
respectively. We conclude that Tc1 and Tc2 effector cells provide
immunity by different mechanisms that subsequently potentiate
host-derived antitumor responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunological memory can be defined as an immune response that is
faster and more potent than that of the primary response following
re-exposure to the same Ag (1). In general, induction of
primary antitumor responses by naive CD8+ T cells
requires TCR recognition of relavent tumor-associated Ag in the context
of MHC class I, either expressed endogenously by tumor cells or
exogenously as peptide presented by APCs capable of generating
effective costimulatory signals (2). Such interaction
results in the clonal expansion and differentiation of naive
CD8+ T cells into activated effector cells that
mediate tumor cell killing. It is clear that Ag recognition alone is
not sufficient for optimal T cell activation and that secondary
costimulatory signals are necessary to aviod tolerance or anergy
(3). Most of these activated CD8+
effector T cells eventually die via activation-induced cell death with
some proportion becoming tumor-reactive memory T cells via an undefined
mechanism (1, 4, 5). It has been suggested that generation
of memory cells with tumor-specific activity can be viewed as a process
whereby responding T cells survive rather than die, presumably through
up-regulation or down-regulation of survival (anti-apoptotic) or
death (apoptotic-inducing) signals, respectively (4, 6).
Cytokines such as IL-2, IL-4, IL-15, and IFN- have been shown to
enhance survival and, in some cases, promote clonal expansion of
activated CD8+ T cell subpopulations through the
regulation of such signals (4, 6, 7, 8, 9). Although these
cytokines may be sufficient for T cell activation, survival, and
perhaps memory cell development in vivo, other factors such as the
strength of T effector cell-to-tumor target cell interaction, cell
surface costimulatory molecules, and the quality, duration, and
concentration of recognizable Ag at the site of tumor encounter may be
of equal importance (2, 10, 11).
We and others have shown that cytolytic CD8+
effector T cells fall into two subpopulations based on their
cytokine-secreting profiles following tumor Ag encounter
(12, 13, 14, 15). Type 1 CD8+ T cells
(Tc1)3 secrete IFN-
and TNF-
, whereas type 2 CD8+ T cells (Tc2)
characteristically secrete IL-4, IL-5, IL-10, and IL-13. Aside from
their direct tumorcidal-inducing potentials, Tc1 and Tc2 effector
cell-derived cytokines may further promote and enhance therapeutic
efficacy through either autocrine and/or paracrine mechanisms that
result in effector cell survival and/or memory
CD8+ T cell development. Previously, we have
shown that adoptive transfer of either Tc1 or Tc2
CD8+ effector T cell subpopulations can
effectively induce tumor cell regression and subsequently prolong
survival times in mice bearing established pulmonary malignancy
(12). In the current study, we assessed the therapeutic
effects and generation of long-term tumor-Ag specific T cell memory
responses by tumor-reactive Tc1 and Tc2 effector cell subpopulations in
tumor-bearing mice. Using a previously described OVA-expressing B16
melanoma (B16-OVA) lung metastasis model (12), we show
that adoptively transferred Tc1 and Tc2 effector cell subpopulations
can induce suppression of established B16 melanoma lung metastases and
subsequently establish long-term tumor Ag-specific immunity in a high
proportion of mice with established pulmonary tumor. Collectively,
effector cell-treated mice exhibiting long-term tumor immunity showed
1) resistance to lethal tumor rechallenge with B16-OVA, 2) heightened T
cell-mediated tumor Ag-specific cytolytic responses ex vivo, 3) an
elevation in the total numbers of both CD8+ and
CD4+ T cell populations in the lung, and 4)
detectable levels of long-lived donor CD8+ TCR
V2 transgene-positive cells, accompanied by an elevation in the
total numbers of
CD8+/CD44high activated
and/or memory T cell subpopulations in both spleen and lungs of treated
mice. Moreover, memory responses induced by Tc2 and Tc1 effector cell
therapy were dependent, in part, on both tumor burden and effector
cell-derived IL-4, IL-5, and IFN-, respectively. We discuss the role
of Tc1 and Tc2 CD8+ effector T cell
subpopulations in successful tumor immunity and generation of
long-lasting tumor Ag-specific memory responses in mice with
established disseminated malignancy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 mice, 6–10 wk of age, were obtained from the
Animal Breeding Facility at the Trudeau Institute. The OT-I mouse
strain, on a C57BL/6 background (H-2b), was
originally obtained from Dr. Michael Bevan (University of Washington,
Seattle, WA). These mice express a transgenic TCR V2 specific for
the SIINFEKL peptide of OVA in the context of MHC class I,
H2-Kb (16). Homozygous
IL-4-/- (OT-I.IL-4),
IL-5-/- (OT-I.IL-5), and
IFN--/- (OT-I.IFN-) knockout mice
expressing the TCR V2 transgene were generated by backcrossing OT-I
mice onto designated cytokine gene knockout mice
(H-2b). Animals were maintained and treated
according to animal care committee guidelines of the National
Institutes of Health (Bethesda, MD) and the Trudeau Institute.
Tumor cells
The weakly immunogenic B16-OVA and parental B16 tumor cell lines that are syngeneic to the C57BL/6 background were kindly provided by Drs. Edith Lord and John Frelinger (Rochester, NY). EL4 and the derivative OVA-expressing EG.7-OVA cell lines were obtained from the American Type Culture Collection (Manassas, VA).
Spleen and lung cell preparation
Spleens were collected from mice and single-cell suspensions were prepared, washed twice in HBSS, and resuspended in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 2 mM pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, and 10% heat-inactivated FCS (Life Technologies). CD8-enriched T cells were obtained by treating with anti-CD4 (RL172.4), anti-heat-stable Ag (J11D), and anti-MHC class II (D3.137, M5114, and CA4) mAbs for 30 min at 4°C. Cells were washed and incubated with rabbit (Pel-Freeze, Rogers, AR) and guinea pig (Harlan, Indianapolis, IN) complement for 30 min at 37°C (12). For preparation of single-cell suspensions from lung parenchyma, lungs were flushed in situ with HBSS via cannulation of the heart to remove residual intravascular blood pools. Minced lung tissues were incubated for 1 h at 37°C on a rocker platform in 1.5 ml/lung of RPMI 1640 supplemented with DNase I (50 U/ml; Sigma, St. Louis, MO); collagenase I, type 4197 (250 U/ml; Sigma); and 5% FCS. After incubation, digested lung tissues were mechanically dispersed through stainless-steel mesh screens in RPMI 1640-5% FCS. After three washes in RPMI 1640-5% FCS, lymphoid cells were resuspended in RPMI-10% FCS to attain a cell concentration of 1 x 107 viable cells/ml. Cytospin preparations of cells from lung homogenates were fixed with methanol and stained with eosin and methylene blue (Fisher, Pittsburgh, PA). Cell differential counts were performed on a total of 200–300 cells on coded slides.
Generation of OVA-specific CD8 effector T cells
To obtain effector cells to OVA peptide, single-cell suspensions
from spleen and lymph nodes of designated OVA Ag-specific TCR V2
transgene-positive mice were washed twice in HBSS and resuspended in
RPMI 1640-10% FCS. CD8-enriched T cells were obtained by passing
lymphoid cell suspensions through nylon wool columns and treating with
anti-CD4 (RL172.4), anti-heat-stable Ag (J11D), anti-MHC
class II (D3.137, M5114, and CA4) mAbs, and complement as previously
described (12). Small resting CD8 T cells were harvested
from Percoll Gradients (Sigma) and resuspended to appropriate cell
concentrations in culture media. Naive CD8 cells were typically 90%
pure as demonstrated by immunofluorescent Ab staining. APCs were
enriched from spleens of normal C57BL/B6 mice by anti-Thy1.2
(HO13.14 and F7D5), anti-CD4 (RL172.4), and anti-CD8 (3.155)
mAbs and complement. T cell-depleted APCs were pulsed with OVA peptide
(10 µM) for 30 min at 37°C and treated with mitomycin-C (50
µg/ml; Sigma) for an additional 30 min at 37°C. For Tc1 effector
cell generation, naive CD8 T cells from OT-I trangenic mice (2 x
105 cells/ml) were stimulated with mitomycin
C-treated, OVA peptide-pulsed APCs (6 x 105
cells/ml) in the presence of IL-2 (20 U/ml; X63.IL-2 supernatants
(17)), IL-12 (2 ng/ml; kindly provided by Dr. Stanley
Wolf, Genetics Institute, Cambridge, MA), and anti-IL-4 mAb (200
U/ml; X63.Ag.IL-4 supernatants). Alternatively, for Tc2 effector cell
generation, naive CD8 T cells from OT-I trangenic mice (2 x
105 cells/ml) were stimulated with mitomycin
C-treated, OVA peptide-pulsed APCs (6 x 105
cells/ml) in the presence of IL-2 (20 U/ml), IL-4 (200 U/ml; X63.IL-4
supernatants (17)), and anti-IFN- mAb (20 µg/ml;
XMG1.2). Effector cell cultures were incubated for 4 days with
additional IL-2 (20 U/ml) added to the cultures on day 2 to promote CD8
cell expansion of Tc1 or Tc2 populations.
Adoptive immunotherapy model
Syngeneic B6 mice were injected i.v. with either 5 x 105 or 1 x 105 B16-OVA cells to establish pulmonary metastases. Seven days after tumor challenge, mice were treated i.v. with 2 x 106 Tc1 or Tc2 OVA-specific effector T cells, and survival times were monitored daily (12). Control groups of mice received no treatment. At weekly intervals after therapy, designated mice were sacrificed for enumeration of pulmonary metastatic nodules. Metastases on freshly isolated lungs appeared as discrete black-pigmented foci that were easily distinguishable from normal lung tissue. Alternatively, mice surviving primary tumor challenge were rechallenged with similar cell numbers of either B16-OVA or parental B16 tumor cells, and survival times were monitored as previously described.
Assay for cytolytic activity
Cytolytic T cell activity was determined by a standard 51Cr-release assay as described previously (12). Briefly, syngeneic EG.7-OVA or EL4 target cells were radiolabeled with 200 µCi Na251CrO4 (ICN Radiochemicals, Irvine, CA) for 1 h at 37°C, washed, and resuspended in RPMI 1640-10% FCS. CD8+ T cells were combined with tumor target cells (1 x 104 cells/well) at various E:T cell ratios in 96-well U-bottom plates (Costar, Cambridge, MA) and incubated for 4 h at 37°C with 5% CO2. Culture supernatants were harvested and counted in a Wizard automatic gamma-counter (Wallac, Gaithersburg, MD). Spontaneous release of 51Cr was determined by incubation of targets in the absence of effectors, whereas maximum release of 51Cr was determined by incubation of targets in 1% Triton X-100. Results are expressed as the percent specific release and were calculated as follows: % specific release = [(experimental - spontaneous)/(maximum - spontaneous)] x 100. Results are also expressed as LU/106 effector cells. One lytic unit was defined as the number of effector cells required to cause 30% lysis of 104 target cells.
Assay for T cell cytokine releasing activity
Detection of secreted cytokines in supernatants of T cell
cultures after restimulation has been described previously (12, 13). Briefly, freshly generated effector cells (2 x
105/ml) were harvested, washed, and restimulated
with either mitomycin C-treated EG.7-OVA or EL4 tumor cells (6 x
105/ml) for 24 h in 1-ml volumes. Culture
supernatants were harvested and assessed for cytokine content by
cytokine-specific ELISA. Murine IL-5 and IL-4 were measured with
anti-IL-5 (TRFK5) and anti-IL-4 (TRFK4) mAbs, respectively.
IFN- was detected by anti-IFN- mAbs R46A2 and XMG1.2.
Standard curves were constructed with purified IL-4 (X63.IL-4
supernatants), IL-5 (X63.IL-5 supernatants), and IFN- (X63.IFN-
supernatants). Values for T cells or stimulator cells cultured in media
alone were negligible.
Flow cytometric analysis
Single-cell suspensions of either spleen or processed murine
lung were washed three times in a fluorescent Ab buffer consisting of
1% BSA and 0.02% sodium azide in 0.01 M PBS (pH 7.2). CD8 lymphocytes
expressing the TCR V2 transgene were phenotyped by their expression
of surface markers using direct immunofluorescence staining techniques.
Lymphocytes (106) were mixed with 100 µl of
fluorescent Ab buffer containing 1 µg of both Cy-chrome-conjugated
anti-CD8 (PharMingen, San Diego, CA) and fluorescein-conjugated
anti-V2 (PharMingen; clone B20.1), PE-conjugated CD44
(PharMingen; clone IM7), or CD4 (PharMingen; clone GK1.5) mAbs and
incubated for 20 min on ice. Stained cell preparations were than washed
three times in fluorescent Ab buffer and analyzed by multiparameter
flow cytometry using a Becton Dickinson FACScan (San Jose, CA). Ten
thousand cells were analyzed per sample with dead cells excluded on the
basis of forward light scatter. Surface-marker analysis was performed
using CellQuest Software (Becton Dickinson), and the percentages and
absolute cell numbers of positive cells were determined.
Statistical analysis
For statistical analysis, the two-tailed Student t test or nonparametric Mann-Whitney rank sum test was used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD8+ Tc1 and Tc2 effector T cells were
generated in vitro from OVA-specific TCR transgenic OT-I mice as
described in Material and Methods. As shown in earlier
studies (12), both Tc1 and Tc2 effector cells demonstrated
tumor Ag-specific cytolytic activity to OVA Ag-expressing tumor cell
targets (EG.7-OVA) with the latter being nearly 9-fold greater than
that of the former (100 LU/106 effector cells vs
11 LU/106 effector cells). Furthermore, Tc1
effector cell populations produced substantial amounts of IFN- with
no detectable levels of IL-4 or IL-5 when restimulated with
OVA-expressing EG.7 tumor cells. In contrast, Tc2 populations released
considerable amounts of IL-5 and IL-4 with low, yet detectable, levels
of IFN- upon restimulation with OVA-expressing tumor cells. Flow
cytometric analysis showed that both Tc1 and Tc2 effector cell
populations expressed similar patterns of cell-surface Ag markers that
are characteristic of effector cell phenotype (12). Both
effector cell populations were TCR V2+,
CD8+CD4- and expressed
up-regulated levels of both CD44 and CD25 and down-regulated levels of
CD62L.
Specificity and therapeutic efficacy by adoptively transferred tumor-reactive Tc1 and Tc2 effector cells in mice with established pulmonary tumor.
Because Tc1 and Tc2 effector cell populations showed highly tumor
Ag-specific cytolytic and cytokine-releasing activities in vitro, the
therapeutic efficacy of Tc1 and Tc2 effector cell populations were
assessed in an experimental B16-OVA lung metastases model. Normal
syngeneic C57BL/6 mice (n = 6/group) were injected i.v.
with 5 x 105 B16-OVA tumor cells. Seven
days later, when disseminated micrometastases were established, 2
x 106 OVA Ag-specific Tc1 or Tc2 effector cell
populations were systemically transferred into tumor-bearing mice, and
survival times were monitored daily. As shown in Fig. 1
, pulmonary tumors grew progressively in
untreated mice, whereas tumor growth among corresponding animals
treated with either Tc1 or Tc2 effector cell therapy showed a marked
reduction in the number of lung-associated tumor colonies for up to 30
days after tumor challenge. Concomitantly, survival times of
tumor-bearing mice receiving Tc1 or Tc2 effector cell therapy were
substantially prolonged compared with those of untreated animals (Fig. 2A). Delays in the onset of
mortality among both groups of Tc1 and Tc2 effector cell-treated mice
ranged between 35 and 45 days after tumor challenge with a mean
survival time of 46.2 ± 1.5 and 44.8 ± 2.1 days,
respectively. In contrast, the onset of mortality among untreated
tumor-bearing control mice occurred as early as 25 days with a mean
survival time of 27.1 ± 0.9 days after tumor challenge.
Furthermore, treatment of tumor-bearing mice with similar numbers of
naive pre-effector CD8+ T cells did not
substantially influence survival times compared with those of
corresponding untreated tumor-bearing control mice (Fig. 2A). Next, we assessed the immunological specificity of OVA
Ag-specific Tc1 and Tc2 effector cell populations. As shown in Fig. 2B, transfer of cell numbers as high as 5 x
106 of either Tc1 or Tc2 effector cells into mice
challenged with the non-OVA Ag-expressing B16 parent line showed no
detectable therapeutic effect in survival times compared with those of
control groups of untreated B16 tumor-bearing mice.
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Because large tumor-cell burdens may qualitatively and/or
quantitatively interfere with effective immunotherapy (2),
we investigated the ability of Tc1 and Tc2 effector cells to induce
long-term protective immunity in mice with lower levels of established
pulmonary metastases. Pulmonary tumors were induced by i.v. injection
of 1 x 105 B16-OVA cells into syngeneic B6
mice. Seven days later, when disseminated micrometastases were
established, 2 x 106 systemic OVA
Ag-specific Tc1 or Tc2 effector cells were injected i.v., and survival
times were monitored as previously described. As shown in Fig. 3, treatment with either single-dose Tc1
or Tc2 effector cell therapy prolonged survival times among 75% (6 of
8) and 87% (7 of 8) of tumor-bearing mice, respectively. In contrast,
the mean survival time of untreated tumor-bearing control mice was
45 ± 5.1 days after tumor challenge. Sixty-five days after
initial tumor cell challenge, surviving mice were rechallenged with
1 x 105 B16-OVA tumor cells. All mice,
initially receiving Tc1 or Tc2 effector cell therapy, exhibited
protection and long-term survival for >120 days after tumor cell
rechallenge (Fig. 3). Corresponding groups of untreated control mice
receiving B16-OVA tumor cells died within 40 days after tumor
challenge, suggesting that tumor cell preparations used for rechallenge
experiments were lethal in naive mice (data not shown). Furthermore,
surviving mice challenged with the non-OVA-expressing B16 parent line
showed no evidence of effective tumor rejection (data not shown). This
indicated that protective immunity to lethal tumor-cell challenge was
established in vivo among tumor-bearing mice treated with adoptively
transferred Tc1 and Tc2 effector-cell populations and that antitumor
responses were highly Ag specific. Similar results were obtained in two
other independent experiments.
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Local and systemic T cell populations from lungs and spleens of
mice receiving effector cell therapy nearly 180 days earlier were
enumerated by multicolor flow cytometric analysis. As shown in Table I, the absolute cell numbers and
percentages of systemic CD8+ T cells from mice
(n = 4/group) receiving either Tc1 or Tc2 effector-cell
therapy were similar but significantly enhanced
(p < 0.01) compared with those of
corresponding cell populations in normal mice of similar age. In
spleens of mice receiving either Tc1 or Tc2 effector-cell therapy, the
absolute cell numbers of total CD8+ T cells were
18.6 ± 1.6 x 106 (16.1%) and
23.3 ± 2.7 x 106 (16.3%),
respectively, whereas the absolute cell number of corresponding
CD8+ T cells among normal age-related control
mice was 11.6 ± 3.8 x 106 (11.7%).
In contrast, CD4+ T cell numbers from these same
effector cell-treated animals showed no significant difference when
compared with those of normal control mice (18.6 ± 1.6 and
30.1 ± 2.6 x 106 vs 23.8 ±
3.8 x 106; p < 0.30).
Moreover, the CD4/CD8 T cell ratios in spleens of mice receiving either
Tc1 (1.00) or Tc2 (1.29) effector-cell therapy were comparatively lower
than normal control (2.05) mice in that effector cell-treated mice had
a greater proportion of systemic CD8+ T cells
after therapy (Table I).
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, the absolute
cell number of CD4+ cells among mice receiving
Tc1 effector-cell therapy was 9.9 ± 1.6 x
105 (4.0%), whereas that of
CD8+ cells was 11.8 ± 0.7 x
105 (6.9%). In mice receiving Tc2 effector-cell
therapy, the corresponding absolute cell numbers were comparatively
greater at 26.6 ± 7.9 x 105 (3.7%)
and 43.8 ± 15.3 x 105 (6.4%),
respectively. In contrast, the absolute cell numbers of both
CD4+ and CD8+ T cell
populations among normal control mice were significantly lower
(p < 0.05) at 3.6 ± 0.4 x
105 (3.0%) and 3.5 ± 0.5 x
105 (3.1%), respectively. Consequently, CD4/CD8
ratios among groups of Tc1 and Tc2 effector cell-treated mice were
comparatively lower than those of normal control mice (0.84 and 0.61 vs
1.05) in that mice receiving effector-cell therapy manifested greater
proportions of CD8+ T cells at the site of tumor
challenge after successful tumor immunity.
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A, the absolute cell numbers
and percentages of systemic
CD8+/CD44high cells in
spleens of mice receiving either Tc1 (11.1 ± 0.5 x
106 (65.2%)) or Tc2 (15.7 ± 2.6 x
106 (66.8%)) effector-cell therapy nearly 180
days earlier were significantly higher (p <
0.05) than those of normal age-related control mice (6.0 ±
1.5 x 106 (40.7%)). Similarly, the
absolute cell numbers of corresponding cell subpopulations in lungs of
these same mice were nearly 4- to 6-fold greater than those of normal
control mice (Fig. 4B).
CD8+/CD44high cell numbers
for Tc1 and Tc2 effector cell-treated mice were 7.9 ± 0.4 x
105 (74.4%) and 9.7 ± 2.3 x
105 (78.6%), respectively, whereas corresponding
tissue cell numbers from lungs from normal control mice of similar age
were significantly lower (p < 0.05) at
1.7 ± 0.1 x 105 (43.8%).
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OVA Ag-specific CD8+ T effector cells from
OT-I mice that express the TCR transgene V2 were generated in vitro
and transferred into tumor-bearing mice as previously described. To
assess the local and systemic presence and distribution of such
effector cells after tumor rechallenge, multicolor flow cytometry was
performed on lungs and spleens of effector cell-treated mice. Although
equal numbers of Tc1 and Tc2 effector cells were transferred into mice
nearly 180 days earlier, the percentages and frequencies of TCR V2
transgene-positive cells in both spleens and lungs remained markedly
greater than those of normal age-related control mice. As shown in Fig. 5, A–C, the
percentages of systemic CD8+ T cells expressing
the TCR V2 transgene were nearly 5- to 10-fold greater in spleens of
mice receiving either Tc1 or Tc2 effector-cell treatment compared with
those of normal age-related control mice. Similarly, the frequency and
percentage of CD8+ TCR V2 transgene-positive
cells in lungs of these same animals were comparatively greater than
those of normal control mice (Fig. 5, D–F),
suggesting that both donor Tc1 and Tc2 effector cell populations are
detectable in the spleen and lungs for extended periods of time after
successful long-term tumor immunity to B16-OVA tumor rechallenge.
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To assess tumor-specific T cell functional responses in mice
exhibiting long-term protection, cytolytic activity of systemic
CD8+ T cell populations of effector cell-treated
mice after tumor rechallenge were assessed ex vivo in a standard 5-h
chromium-release assay. As shown in Table III, enriched CD8+
T cells from spleens of surviving mice that received either Tc1 or Tc2
effector-cell therapy over 180 days earlier exhibited OVA
Ag-specific CTL responses to EG.7-OVA tumor-cell targets. Tumor
Ag-specific lytic activity among mice treated with either Tc1 or Tc2
effector-cell therapy ranged from 12 to 45% over different E:T ratios.
In contrast, lytic activity to OVA-non-expressing control EL4 target
cells was negligible, confirming that killing was Ag specific and most
likely not associated to NK cell activity (Table III). Normal
age-matched control mice showed no detectable lytic activity to either
target-cell population (data not shown). These results suggest the
presence of systemic long-lived antitumor CTL memory responses in
tumor-bearing mice receiving adoptively transferred Tc1 or Tc2
effector-cell therapy over 180 days earlier.
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in Tc1- and Tc2-mediated
long-term tumor protection.
Because Tc2 effector-cell populations were found to secrete low,
yet detectable, levels of IFN- upon restimulation with tumor Ag, we
assessed the role of effector cell-derived IFN- in both Tc1 and Tc2
effector cell-mediated antitumor responses. Pulmonary tumors were
induced by i.v. injection of 1 x 105
B16-OVA cells into syngeneic C57BL/6 mice. One week later, 2 x
106 OVA Ag-specific Tc1 or Tc2 effector-cell
subpopulations, generated from OT-I.IFN- knockout mice, were
systemically transferred, and survival times were monitored as
previously described. As shown in Fig. 6A, treatment with either
IFN--deficient or wild-type Tc1 effector cells equally prolonged
survival times among 4 of 6 (67%) tumor-bearing mice. However, after
B16-OVA tumor cell rechallenge on day 60 after therapy, the former was
comparatively less effective than that of the latter. All surviving
mice, initially receiving wild-type Tc1 effector-cell therapy exhibited
tumor protection and long-term survival for >75 days after tumor
rechallenge. In contrast, tumor protection among all surviving mice
receiving IFN--deficient Tc1 effector-cell therapy was completely
eliminated within 45 days after tumor rechallenge. This suggests that
effector cell-derived IFN- may play a substantial role in
establishing effective long-term Tc1 effector cell-mediated tumor
immunity. In contrast, heightened tumor-recipient survival times among
groups of mice treated with IFN--deficient Tc2 effector-cell
populations were not comparatively different to those of corresponding
groups of tumor-bearing mice receiving wild-type Tc2 effector-cell
therapy, suggesting that Tc2 effector cell-derived IFN- does not
play a significant role in the development of Tc2 effector
cell-mediated long-term tumor immunity (Fig. 6B).
Corresponding groups of untreated control mice receiving similar doses
of B16-OVA tumor cells died within 40 days of tumor challenge,
suggesting that tumor-cell preparations used for rechallenge
experiments were lethal in naive mice (data not shown). Results are
representative of two similar experiments.
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To determine whether the type 2 cytokines IL-4 and IL-5 were
required for the induction of effective Tc2 effector cell-mediated
long-term protection, OVA-Ag specific Tc2 effector-cell subpopulations
were generated from either OT-I.IL-4 or OT-I.IL-5 knockout mice and
transferred into syngeneic C57BL/6 mice bearing 7-day established
B16-OVA tumors. As shown in Fig. 7,
survival times among tumor-bearing mice receiving either IL-5- or
IL-4-deficient Tc2 effector cells were significantly prolonged
(p < 0.001) compared with those of untreated
control mice. The mean survival times of mice receiving either IL-5- or
IL-4-deficient Tc2 effector cells were 45.0 ± 4.5 and 53.2
± 2.2 days, respectively. In contrast, the mean survival time of
untreated tumor-bearing control mice was 31.8 ± 0.5 days after
tumor challenge. However, both cytokine gene-deficient effector-cell
populations were less effective in prolonging survival times than those
of corresponding groups of tumor-bearing mice receiving similar doses
of wild-type Tc2 effector-cell therapy. In the latter, effective
protection and long-term survival were exhibited in 6 of 8
tumor-bearing mice (75%) for >100 days after tumor challenge (Fig. 7). In contrast, Tc1 effector-cell populations derived from either IL-4
or IL-5 knockout mice were comparable to those of corresponding
wild-type Tc1 effector-cell populations (data not shown). This suggests
that Tc2 effector cell-derived IL-4 and IL-5 may play a significant and
unique role in Tc2 effector cell-mediated long-term tumor immunity and
protection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Long-term tumor immunity can be defined as a subset of persistent Ag-specific memory T cells that are capable of mediating and coordinating a faster, stronger, and more prolonged response to tumor Ag re-encounter. In the current study, induction of persistent long-term tumor immunity by adoptively transferred tumor-reactive Tc1 and Tc2 effector cells in tumor-bearing mice was supported by 1) successful resistance to lethal tumor rechallenge, 2) heightened levels of systemic OVA-Ag-specific CTL responses ex vivo, 3) elevation in the total numbers of both CD4 and CD8 T cell populations at the site of tumor growth, 4) detection of long-lived transgene-positive donor cells at sites both proximal (lung) and distal (spleen) to tumor growth, and 5) an elevation in the total numbers of activated/memory CD8+ T cell subpopulations coexpressing elevated levels of CD44 surface Ag in both spleen and lungs of treated mice. These results suggest that adoptively transferred tumor-reactive Tc1 or Tc2 effector cells not only persisted as memory and/or activated T cells, but also initiated and potentially modulated host antitumor immune responses in recipients with established pulmonary metastases. Transfer of either polarized Tc1 or Tc2 tumor-reactive CD8 effector-cell populations may effectively regulate established tumor through either direct lytic interaction or by effector cell-derived cytokines that may initiate a cascade of events in the host that result in the recruitment and activation of host immune cells. Subsequently, tumor-associated Ags may be released and re-expressed by host APCs that may aid, in part, with the generation and maintenance of both long-lived donor and host-derived memory T cells. Currently, the mechanisms by which CD8 lymphocytes differentiate into memory cells are unclear. However, it has been suggested that memory T cells can arise directly from progeny of fully differentiated effector T cells or through naive T cells that differentiate along separate developmental stages or pathways (11, 18, 19). Although we are not in a position to preferentially support either view, our data indicate that adoptive transfer of either Tc1 or Tc2 effector-cell subpopulations have the capacity to be long lived and subsequently promote functionally effective T cell memory responses in tumor-bearing mice that are derived, in part, from both host and donor T lymphocytes in vivo. Investigations to further elucidate the role of adoptively transferred Tc1 and Tc2 CD8 effector T cells in the induction and generation of host antitumor immune responses during progressive tumor growth are currently underway.
The mechanisms involved in the development and maintenance of
immunologic memory remain unknown. However, it has been suggested that
successful generation of long-lived tumor-reactive memory T cells is
highly dependent on several factors including the cytokine environment
at the site of tumor challenge (2). Therefore, we assessed
the roles of Tc1 and Tc2 effector cell-derived cytokines in mediating
tumor regression and establishing long-term tumor immunity. Because Tc2
effector-cell populations were found to secrete low, yet detectable,
levels of IFN-, we assessed the role of effector cell-derived
IFN- in both Tc1 and Tc2 effector cell-mediated long-term antitumor
responses. The adoptive transfer of CD8 effector cells from
IFN--deficient OT-I mice showed that these Tc1 effector cells were
therapeutically less effective over time than those of corresponding
cell populations from wild-type mice. Although survival times in mice
treated with the former were substantially prolonged, antitumor effects
were short-lived with all mice experiencing mortality upon tumor
rechallenge. The data suggest that secretion of IFN- by Tc1 effector
cells is a necessary component in the mechanism of tumor eradication
and long-term antitumor protection by these cells. The local secretion
of IFN- by transferred effector cells at the site of tumor growth
may lead to tumor regression by several mechanisms. It has been shown
that IFN- can directly inhibit tumor cell growth (9, 20, 21), enhance recruitment and activation of cells that promote
innate antitumor immune responses (9, 22, 23, 24), and promote
elimination of transformed cells either directly or indirectly through
nonimmune mechanisms such as those involving inhibition of angiogenesis
by mechanisms that are incompletely understood but involve the
IFN--induced angiogenesis-inhibitory chemokines IP-10 and Mig
(25, 26). Moreover, IFN- has been shown to affect tumor
immunogenicity by enhancing tumor Ag presentation that may subsequently
promote immune cell survival and maintain long-term antitumor immunity
(27, 28, 29). Despite the potential role of effector
cell-derived IFN- in Tc1 effector-cell therapy, IFN--deficient
Tc2 CD8 effector-cell populations were not comparatively different than
those of corresponding groups of tumor-bearing mice receiving wild-type
Tc2 effector-cell therapy. These data suggested that Tc2 effector cells
must act by an effector cell-derived IFN--independent mechanism.
Because others have reported that either IL-4 or IL-5, when secreted by
cytokine gene-modified tumor cells, can effectively suppress tumor
growth in vivo (30, 31, 32), we assessed the therapeutic roles
of such type 2-related cytokines in inducing and maintaining effective
Tc2 effector cell-mediated long-term protection in tumor-bearing mice.
Therapy with either IL-4 or IL-5 cytokine-deficient effector-cell
populations was markedly less effective than corresponding wild-type
Tc2 effector-cell populations in prolonging survival times, with all
tumor-bearing mice experiencing mortality within 70 days of primary
tumor challenge. This suggested that Tc2 effector cell-derived IL-4 and
IL-5 may play a substantial role in Tc2 effector cell-mediated tumor
immunity and protection. Local release of IL-4 and IL-5 have been shown
to mediate the selective recruitment, localization, and activation of
nonspecific antitumor responses, such as macrophages, NK cells, and
granulocytes that may facilitate tumor growth inhibition and/or enhance
tumor Ag presentation (24, 33, 34, 35). Alternatively, IL-4
and IL-5 may indirectly modulate local antitumor responses by enhancing
select expression of various chemokines, such as macrophage
inflammatory protein-1 and monocyte chemotactic protein-1, that may
affect both donor and host effector-cell trafficking and activation at
the site of tumor encounter (36). In either instance,
differential utilization of discrete cytokine profiles by Tc1 or Tc2
effector-cell populations appear to induce tumor rejection with
different mechanisms that can potentially affect the nature and outcome
of effective antitumor responses to progressively growing tumors.
Although our observations suggest the existence of a correlation
between effector cell-derived cytokines and heightened in vivo
therapeutic efficacy and survival among mice with established tumor,
other mechanisms such as direct cytolysis by either perforin-mediated
or Fas-mediated pathways must be considered. Investigations to
elucidate cognate effector cell-mediated lytic mechanisms and their
contributions to tumor eradication are in progress.
Survival studies in our pulmonary tumor model showed a marked decrease
in mortality among mice with low vs high levels of established tumor
burden, suggesting that effective single-dose Tc1 and Tc2 effector-cell
therapy is most effective in mice with low-level residual malignancy.
Lower effector-cell efficacy among mice previously receiving a higher
B16-OVA tumor cell burden may be attributed to the inability of
transferred effector cells to keep up with heightened numbers of tumors
that rapidly proliferate and lead to enhanced tumor outgrowth in vivo
(37). Persistence of such high levels of tumor Ag may lead
to tumor-induced tolerance or anergy of adoptively transferred
tumor-reactive effector T cells (37, 38). Alternatively,
tumor-reactive effector T cells may efficiently eradicate tumor cells,
but may select for tumor Ag-negative variants in vivo which would
diminish effector-cell recognition and enhance variant tumor outgrowth
and progression (39, 40). Additionally, other
tumor-related mechanisms contributing to the active immunosuppression
of tumor-reactive effector cells have been described and involve
release of tumor-derived soluble suppressor factors such as TGF-
and
prostaglandins (2), generation of host T cells with
suppressor function (41, 42), discordant structural
alterations in TCR-mediated signal-transduction pathways (particularly
in late tumor-bearing hosts) (37, 43), and induction of
CD95-mediated T cell apoptosis by Fas ligand-expressing tumor cells
(44, 45).
Although metastatic lesions in effector cell-treated mice were greatly
reduced and in fact appeared eliminated in lungs of "cured" mice
receiving lower doses of tumor, minimal residual disease among these
animals was still apparent. Cytospins from single-cell suspensions of
lung homogenates showed low, yet detectable, cell numbers of B16 tumor
present in lungs of both Tc1 and Tc2 effector cell-treated mice at
times >120 days after tumor rechallenge (data not shown). These
observations were in agreement with those described by Vitetta et al.
(46), suggesting that a functional immune system can
potentially induce a state of cancer dormancy whereby tumor cells would
be present but substantially fail to expand for overly extended periods
of time. The underlying mechanisms involved in establishing and
maintaining tumor dormancy appear to involve an interplay between the
immune system and cancer growth. In the case of the latter, tumor
variants may arise that carry mutations responsible for transient
alterations in the surface expression of select growth-factor receptors
and/or tumor-cell signaling pathways that result in a delay in tumor
cell growth and responsiveness (46). Alternatively, others
have shown that CD8+ T cells, but not
CD4+ T cells, are required for the maintenance of
dormancy (47). Moreover, IFN- has been shown to act in
collaboration with CD8+ T cells to mediate, in
part, tumor growth arrest and dormancy in vivo (47).
Although little is known about the changes that occur in both the host
and tumor-cell population that allow tumor escape and regrowth from the
dormant state, our observations suggest that, in the course of
malignancy, tumor-cell presence is not inconsistent with long-term
survival or cure. Further experimentation is warranted to define the
potential roles of various tumor-reactive effector-cell subpopulations
after Tc1 and Tc2 effector-cell therapy in promoting tumor-cell
cytostasis and growth regulation in vivo.
In summary, we show that adoptively transferred Tc1 and Tc2 CD8
effector-cell subpopulations can effectively regulate established B16
melanoma lung metastases and subsequently establish long-term tumor
Ag-specific immunity in large proportions of mice with established
pulmonary tumor. Antitumor responses induced by Tc2 and Tc1 effector
cell therapy were dependent, in part, on both the level of tumor burden
and effector cell-derived IL-4, IL-5, and IFN-, respectively.
Immunoadoptive therapy with such polarized cytokine-secreting
effector-cell subpopulations may offer a new strategy for successful
tumor immunotherapy and provide a practicable means to elicit more
effective T cell-mediated immune responses against various established
tumors in primary and metastatic disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Richard W. Dutton, Trudeau Institute, Algonquin Avenue, Saranac Lake, NY 12983. E-mail address:
3 Abbreviations used in this paper: Tc1, type 1 CD8+ T cells; Tc2, type 2 CD8+ T cells; B16-OVA, OVA-expressing B16 melanoma; OT-I.IFN- mice, OVA TCR+ IFN- knockout mice; OT-I.IL-4 mice, OVA TCR+ IL-4 knockout mice; OT-I.IL-5 mice, OVA TCR+ IL-5 knockout mice.
Received for publication August 31, 1999. Accepted for publication November 4, 1999.
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