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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+
cells (Tc2) secrete IL-4 and IL-5. We assessed the relative therapeutic
effects of adoptively transferred OVA-specific Tc1 and Tc2
CD8+ cells in mice bearing established OVA-transfected B16
melanoma lung metastases. Both Tc1 and Tc2 subpopulations mediated a
reduction in lung tumor growth that subsequently prolonged survival
times in mice with both early (day 7) and more advanced (day 14) levels
of tumor development. CD8+ T cell populations recovered
from spleens of tumor-bearing mice receiving Tc1 or Tc2 cells showed
markedly enhanced tumor Ag-specific cytolytic and cytokine-releasing
activities that correlated with delays in tumor cell growth and
progression. Initially, both tumor-reactive Tc1 and Tc2 effector cells
accumulated at the tumor site with nearly equal frequency. Tc1 cells
persisted, whereas Tc2 cell numbers progressively diminished over time.
Titration of Tc1 and Tc2 effector cells showed that protection was dose
dependent with the former being 5-fold more effective. Tc2 cells
achieved a comparable reduction in lung tumor cell growth at higher
concentrations of cell transfer. Tc1 effectors from IFN--deficient
mice were less therapeutically effective than wild-type mice, but there
was no significant reduction in activity between corresponding Tc2
populations. We speculate that the effectiveness of Tc1 and Tc2 cells
may depend on different mechanisms. These studies suggest a potential
role for Tc1 and Tc2 CD8+ subpopulations in tumor
regression and immunotherapy. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
and TNF-
, whereas type 2 CD8 T cells (Tc2) predominantly secrete
IL-4, IL-5, and IL-10. Such cytokines not only have diverse inhibitory
effects on tumor cells themselves but also affect the nature and
development of the immune response by other cells toward progressively
growing tumors. Both populations of effector CD8 cells are cytolytic
and kill predominantly by the perforin pathway (6, 7, 8). However, Tc1 but
not Tc2 CD8 effector cells do exhibit some Fas-mediated killing (6, 8).
Aside from their cytolytic properties, these distinct
cytokine-secreting CD8 subpopulations can also have direct effects on
tumor cell populations by noncytolytic mechanisms. Several studies have
shown that cytokines, such as IL-4, IFN-, and TNF-, can modulate
surface Ag expression and growth kinetics among many tumor cell
populations, including several types of disseminated malignancies (7, 9, 10, 11). Such effects may include tumor growth inhibition and enhanced
tumor immunogenicity by induction of elevated levels of surface MHC
class I expression. Alternatively, it has been suggested that cytokines
secreted by such polarized CD8+ T cell subpopulations have
additional secondary effects that influence the expression of adhesion
molecules, chemokine receptors, and other immune cell surface molecules
that may aid in proximal lymphocyte activation, cell trafficking, and
differentiation (2, 9, 11, 12, 13, 14). Such "cellular interplay" can
potentially affect both the nature and outcome of antitumor immune
responses and tumor progression. Therefore, it seems probable that such
CD8+ subpopulations, with their distinct cytokine-secreting
profiles, may have differing modes of inducing effective tumor
immunity.
Studies in animal models have defined many of the underlying
principles, and provided many insights for the development, of adoptive
T cell therapy as a viable modality for the treatment of certain human
cancers. However, studies examining the requirements for effective T
cell function following adoptive transfer suggest that the inability of
such T cells to respond at sites of tumor growth may be a major
determinant of therapeutic efficacy. Several factors may modify
effective antitumor responses by T cells following adoptive transfer,
including proper expression of MHC Ags on tumor cell surfaces, the
recruitment and induction of other anti-tumor effector cell
populations, and the production or availability of adequate amounts of
immunoenhancing cytokines, including IL-2, IFN-, and IL-4 (9, 15, 16). It is likely that the multifunctional properties afforded by Tc1
or Tc2 CD8 effector cells can effectively overcome such barriers and
promote effective antitumor immunity.
Using a poorly immunogenic OVA Ag-expressing B16 melanoma lung
metastases model, we assessed the therapeutic effects of adoptively
transferred OVA Ag-specific Tc1 and Tc2 CD8 effector cells in mice
bearing established pulmonary malignancy. Systemic transfer of
tumor-reactive Tc1 and Tc2 effector cells resulted in the local
accumulation of transferred cells at the tumor site that induced tumor
regression and subsequently enhanced survival times in mice with
established pulmonary tumors. Although therapeutic efficacy appeared
greater in mice receiving Tc1 effector cell transfer, systemic
antitumor responses induced by both Tc1 and Tc2 effector cell
subpopulations were highly tumor Ag specific and appeared to be
mediated by different mechanisms of action. Tc1 effector cells
generated from IFN- knockout mice (OT-1.IFN- mice) were less
therapeutically effective than those from wild-type OT-I mice, whereas
there was no significant reduction in activity between corresponding
Tc2 populations. Furthermore, the level of effectiveness by adoptively
transferred Tc1 and Tc2 effector cells appeared to be influenced by the
stage of tumor development in the lung. We discuss the potential
therapeutic roles of tumor-reactive Tc1 and Tc2 effector cell
subpopulations in effective antitumor immunity and tumor regression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 mice, 6 to 10 wk of age, were obtained from the
Animal Breeding Facility at the Trudeau Institute, Saranac Lake, NY.
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 (17). Homozygous IFN--/- knockout
mice, expressing the TCR V2 transgene (OT-I.IFN-), were generated
by backcrossing OT-I mice onto IFN- knockout mice
(H-2b). Animals were maintained and treated according to
animal care committee guidelines of the National Institutes of Health,
Bethesda, MD, and Trudeau Institute.
Tumor cells
The weakly immunogenic OVA-transfected B16 melanoma tumor cell line (B16-OVA) that is syngeneic to the C57BL/6 background was 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 units/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, CA4) mAbs and complement. 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 RPMI 1640 supplemented with DNase I (50 U/ml; Sigma, St. Louis, MO), collagenase I, type 4197 (250 U/ml; Sigma), and 5% FCS. Following 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.
Generation of OVA-specific CD8 effector T cells
To obtain effector cells to OVA peptide, single-cell suspensions
from spleen and lymph nodes of OT-I 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 (4) and
treating with anti-CD4 (RL172.4), anti-heat-stable Ag (J11D),
anti-MHC class II (D3.137, M5114, CA4) mAbs, and complement. 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 (B6)
mice by anti-Thy-1.2 (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 M) for 30 min at 37°C and treated with
mitomycin C (50 g/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),
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.IL4
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), and anti-IFN- mAb
(20 g/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 5 x 105 B16-OVA melanoma cells to establish pulmonary metastases, and 7 or 14 days following tumor challenge, mice were treated i.v. with various doses of either Tc1 or Tc2 OVA-specific effector T cells. At weekly intervals after therapy, mice were sacrificed for enumeration of pulmonary metastatic nodules. Control groups received no treatment. Metastases on freshly isolated lungs appeared as discrete black pigmented foci that were easily distinguishable from normal lung tissue. The number of pulmonary metastases in treatment and control groups was counted in a blind fashion and reported as the mean ± SEM. Metastatic foci too numerous to count were assigned an arbitrary value of >250.
Assay for cytolytic activity
Cytolytic T cell activity was determined by a standard 51Cr release assay as described previously (4). Briefly, syngeneic EG.7-OVA or EL4 target cells were radiolabeled with 200 µCi of 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 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 was calculated as [(experimental - spontaneous)/(maximum - spontaneous)] x 100.
Results are also expressed as lytic units/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 from supernatants of T cell
cultures following restimulation has been described previously (1, 4).
Briefly, CD8-enriched T cells (1 x 106/ml) from
tumor-bearing mice were restimulated with either mitomycin C-treated
EG.7-OVA or EL4 tumor cells (1 x 106/ml) for 24 and
48 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-g 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 cytochrome-conjugated anti-CD8
(PharMingen, San Diego, CA) and fluorescein-conjugated anti-V2
(PharMingen, clone B20.1) 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 Cell Quest software, and the
percentages and absolute numbers of positive cells were determined.
Statistical analysis
For statistical analysis, the two-tailed Student t 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 Materials
and Methods. Effector cells were harvested, and their Ag-specific
cytolytic potentials were evaluated in a standard 4-h chromium
release assay. As shown in Fig. 1
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both Tc1 and Tc2 effector cells demonstrated tumor Ag-specific
cytolytic activity to OVA-expressing tumor cell targets. At E:T ratios
of 50:1, Tc2 effector cells showed a mean specific lysis of 50.0
± 3.5%, whereas Tc1 cell populations were comparatively lower at
36.0 ± 3.0%. Moreover, when cytotoxicity was assessed in lytic
units/106 effector cells, Tc2 effector cell populations
were nearly 10-fold greater than that of Tc1 effector cell populations
(100 LU/106 effector cells vs 10
LU/106 effector cells, p < 0.001). In
contrast, both Tc1 and Tc2 effector cell populations showed negligible
lytic activity to non-OVA-expressing EL4 parental tumor cell targets,
confirming that killing was highly Ag specific.
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, Tc1 effector cell populations produced
substantial amounts of IFN- with no detectable levels of IL-4 or
IL-5 when cocultured with EG.7-OVA tumor cells. In contrast, Tc2
populations released considerable amounts of IL-5 and IL-4 in similarly
restimulated cultures. Although Tc2 cell populations did acquire the
ability to secrete some IFN- upon restimulation with OVA-expressing
tumor cells, these amounts were nearly 50-fold less than that of Tc1
populations (12,224 ± 649 u/ml vs 260 ± 64 U/ml,
respectively). The secretion of these cytokines by both Tc1 and Tc2
effector cell populations was OVA Ag specific, given that control
mitomycin C-treated EL4 parent cell lines did not stimulate detectable
levels of cytokine release from either effector cell population (Table I).
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, both effector cell populations were
CD8+CD4- and expressed up-regulated levels of
CD44 and CD25. Although both Tc1 and Tc2 effector cells showed a
noticeable down-regulation in the levels of CD62L surface Ag
expression, the former showed slightly higher levels than that of the
latter. Aside from the surface Ag marker profiles characteristic of
effector cell phenotypes, both Tc1 and Tc2 cell populations expressed
down-regulated levels of the transgenic V2 TCR, typical of increased
cell activation. In contrast, naive CD8 T cells were characteristically
CD44low, CD62Lhigh, and CD25low
with higher levels of transgenic V2 TCR expression (Fig. 2).
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Since 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 at
different levels of tumor development were assessed in an experimental
OVA Ag-expressing B16 melanoma lung metastases model. Pulmonary
metastasis were induced in normal syngeneic C57BL/6 mice by i.v.
injection of 5 x 105 B16-OVA melanoma 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 the numbers
of lung metastases were evaluated at weekly intervals. Treatment of
tumor-bearing mice with either Tc1 or Tc2 effector cell populations
caused an effective reduction in the number of pulmonary metastases
when compared with animals receiving no treatment. In animals bearing
7-day established tumors, Tc1 or Tc2 effector cell therapy markedly
reduced established lung tumor colonies by nearly 95% and 80%,
respectively (Table II). The mean number
of lung metastases in mice receiving Tc1 effector cell therapy was
significantly lower than that of untreated tumor-bearing control mice
(3.3 ± 0.3 vs 69.0 ± 17.8; p < 0.01).
Moreover, corresponding groups of tumor-bearing mice receiving Tc2
effector cells showed a similarly significant decrease in the number of
established pulmonary tumor foci. This reduction in pulmonary tumor
growth among animals receiving either effector cell therapy was
observed for up to 30 days post-tumor challenge. Lung tumors among
untreated animals grew progressively, with all mice exhibiting >250
tumor foci by day 30 post-tumor challenge (Table II). To determine
whether successful Tc1- or Tc2-mediated antitumor effects can be
extended to more advanced levels of established pulmonary metastases,
similar numbers of effector cells were transferred into animals bearing
14 day-established B16-OVA pulmonary tumors. Both Tc1 and Tc2 effector
cells were relatively ineffective in reducing metastatic disease during
the first week following therapy with the numbers of lung tumor foci
being nearly equal to that of corresponding tumor-bearing control mice
receiving no therapy (Table II). However, a progressive reduction in
the numbers of lung metastases was noted at wks 2 and 3 following
single-dose effector cell transfer with either Tc1 or Tc2 cells.
Twenty-one days following Tc1 or Tc2 effector cell therapy, mice with
advanced 14-day-established tumors showed a nearly 7- and 13-fold
decrease in the numbers of pulmonary metastases, respectively, when
compared with control groups. The mean number of lung metastases after
35 days post-tumor challenge in mice receiving Tc1 or Tc2 effector cell
transfer on day 14 was significantly lower when compared with
corresponding tumor-bearing control mice (38.5 ± 3.5 and
20.0 ± 3.0, respectively, vs >250; p < 0.001).
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, the mean survival time of
mice receiving Tc1 or Tc2 effector cell therapy 7 days after B16-OVA
tumor challenge was significantly (p < 0.001)
prolonged when compared with that of untreated mice. The mean survival
times of mice receiving Tc1 and Tc2 treatment were 45.7 ± 1.6 and
45.2 ± 2.0 days, respectively. In contrast, the mean survival
time of untreated tumor-bearing control mice was 27.4 ± 0.8 days
post-tumor challenge. Although, Tc1 and Tc2 effector cell-mediated
tumor growth inhibition appeared transient in animals with
7-day-established tumors, survival times were substantially increased
by >65% when compared with that of untreated control mice. When
survival rates among animals with more advanced 14-day-established lung
tumors were accessed, Tc1 and Tc2 effector cell-treated mice showed a
nearly 25–35% increase in survival times when compared with that of
untreated tumor-bearing mice (37.5 ± 1.2 and 34.6 ± 0.7
days, respectively, vs 27.4 ± 0.8 days;
p < 0.002) (Table III). Naive CD8 T cells from OT-I
mice had no effect on survival or reduction in tumor cell growth among
mice bearing established pulmonary metastases (data not shown).
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Since the therapeutic efficacy of adoptive immunotherapy is
proportional to the numbers of transferred immune cells, we
quantitatively analyzed the antitumor effects of Tc1 and Tc2 effector
cell populations by transfer of different effector cell concentrations
and monitoring survival times in mice with established pulmonary
tumors. As shown in Fig. 3, mice
receiving doses of 10–50 x 105 of either Tc1 or Tc2
effector cells showed an increase in survival time when compared with
that of untreated control tumor-bearing animals. Moreover, transfer of
25-fold less Tc1 effector cells, at numbers as low as 0.4 x
105, resulted in a similarly effective prolongation in
survival times among mice bearing 7-day established tumors. In
contrast, groups of mice receiving a similar dose of Tc2 effector cells
showed no therapeutic effect and only when given a 5-fold higher Tc2
effector cell number (2 x 105) did animals start to
show modest increases in survival times. These results suggest that, on
a per cell basis, Tc1 effector cells were nearly 5-fold more effective
than Tc2 effector cells since the former demonstrated greater
therapeutic effects at cell numbers as low as 0.4 x
105 when transferred into tumor-bearing mice. All mice
receiving no treatment succumbed to progressively growing tumor within
32 days post-tumor challenge (Fig. 3). Concomitantly, we assessed the
immunological specificity of OVA Ag-specific Tc1 and Tc2 effector cell
populations. As shown in Fig. 3C, transfer of cell numbers
as high as 50 x 105 of either Tc1 or Tc2 effector
cells into mice challenged with the non-OVA-expressing B16 parent line
showed no detectable therapeutic effect in survival times when compared
with control untreated B16 tumor-bearing mice.
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-deficient Tc1 but not
Tc2 effector cell populations in mice with established pulmonary tumor
Since 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. OVA Ag-specific Tc1 and Tc2
effector cell subpopulations, generated from OT-I.IFN- mice, were
transferred into syngeneic C57BL/6 tumor-bearing mice, and survival
times were monitored as previously described. As shown in Table IV, mean survival times among
tumor-bearing mice receiving IFN--deficient Tc1 or Tc2 effector
cell therapy were substantially prolonged when compared with that of
untreated mice. However, the former was significantly
(p < 0.05) less effective than that of
corresponding groups of tumor-bearing mice receiving wild-type Tc1
effector cell therapy (42.8 ± 1.5 days vs 54.0 ± 3.5 days).
This suggests that IFN- may play a substantial role in Tc1 effector
cell-mediated therapeutic responses. In contrast, heightened tumor
recipient survival times among groups of mice treated with
IFN--deficient Tc2 effector cell populations were not significantly
(p < 0.20) different from that of
corresponding groups of tumor-bearing mice receiving wild-type Tc2
effector cell therapy (45.2 ± 2.2 days vs 58.6 ± 8.3 days),
suggesting that Tc2 effector cell-derived IFN- does not play a
significant role in Tc2 effector cell-mediated therapy (Table IV).
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OVA Ag-specific effector CD8 T 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 distribution of such effector cells following adoptive
transfer in mice bearing pulmonary tumors, multicolor flow cytometry
was performed on lung and spleen tissues, respectively. As shown in
Fig. 4A, the number and
frequency of systemic CD8 T cells expressing the TCR V2 transgene
were highest in spleens 7 days after either Tc1 or Tc2 effector cell
treatment in mice with 7-day-established pulmonary tumors. However, the
frequency and absolute cell numbers of detectable transgene-positive
CD8 T cells progressively declined by wk 2 after therapy and thereafter
decreased by nearly 3.5- and 5-fold by wk 3 for both Tc1 and Tc2
populations, respectively. Similarly, the frequencies and cell numbers
of transgene-positive CD8 T cells in lungs of mice receiving Tc1 and
Tc2 effector cells were greatest at 7 days after therapy. However,
transferred Tc1 effector cells remained similarly elevated at the site
of tumor growth for up to 3 wk posttherapy, whereas Tc2 effector cell
populations progressively diminished to nearly equal levels of
endogenous recipient T cell population numbers by wk 2 and 3 after
therapy. Although equal numbers of Tc1 and Tc2 effector cells were
transferred into mice 7 days after tumor challenge, the cell number and
frequency of the former were consistently greater than that of the
latter in both lungs and spleen at all time points tested in these
animals. Endogenous CD8/TCR V2 T cell population levels never
appeared greater than 1.0% in both spleen and lung tissues of
untreated tumor-bearing control mice at all time points tested (Fig. 4). Similar results were obtained in corresponding groups of mice with
more advanced 14-day-established lung tumors (data not shown).
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Systemic cytolytic activity of T cells from spleens of
tumor-bearing mice following adoptive transfer of OVA Ag-specific Tc1
or Tc2 effector T cells were assessed at weekly intervals following
therapy in a standard 4-h chromium release assay. As shown in Table V, CD8-enriched T cells from spleens
containing both donor and recipient cells of 14-day tumor-bearing mice
treated 7 days earlier with either OVA Ag-specific Tc1 or Tc2 effector
cells showed markedly enhanced Ag-specific CTL responses to
OVA-expressing EG.7 tumor cell targets. Systemic OVA Ag-specific CTL
responses among groups of mice receiving Tc1 therapy were significantly
(p < 0.005) greater than that of corresponding
Tc2 effector cell-treated mice (22.4 ± 0.8 vs 15.3 ± 0.6).
Cytolytic responses to control non-OVA-expressing EL4 tumor cell
targets were negligible, confirming that killing was Ag specific and NK
cell independent. Fourteen days after therapy, CD8-enriched T cells
from spleens of mice receiving Tc1 therapy showed progressively lower
OVA Ag-specific CTL responses that were not detectable by day 21
posttherapy. In contrast, OVA Ag-specific CTL responses among
corresponding T cell populations from mice receiving Tc2 effector cell
therapy appeared to diminish at earlier time points with negligible CTL
activity at days 14 and 21 following therapy. Cytolytic responses by
CD8-enriched T cell populations from spleens of tumor-bearing control
mice receiving no treatment were undetectable at all time points tested
(Table V). Similar results were obtained in separate experiments in
mice with more advanced 14-day established pulmonary disease (data not
shown). Since potential antitumor immune responses may be mediated by
cytokines secreted by effector T cells (9), we analyzed the tumor
Ag-specific cytokine-releasing activities of systemic CD8 T cells from
spleens of tumor-bearing mice receiving Tc1 or Tc2 effector cell
therapy. As shown in Table V, CD8-enriched T cells from spleens of
14-day tumor-bearing mice treated 7 days earlier with OVA Ag-specific
Tc1 effector cells showed enhanced levels of IFN- and IL-4 cytokine
production to EG.7-OVA tumor cells when compared with that of
corresponding untreated tumor-bearing control mice. Although IFN--
and IL-4 cytokine-secreting T cell responses among mice receiving Tc1
effector cell therapy persisted for up to 14 days posttherapy, levels
of both cytokines eventually subsided to undetectable levels by day 21
posttherapy. IL-5 cytokine production among these same animals was
undetectable at all time points tested. Cytokine-releasing activities
to control non-OVA-expressing EL4 tumor cells were negligible,
confirming that T cell-mediated cytokine-secreting potentials were Ag
specific. Although corresponding cell populations from spleens of
tumor-bearing mice receiving Tc2 effector cell therapy showed similarly
elevated levels of IFN- production 1 wk after treatment, IFN-
cytokine levels diminished over time and were eventually undetectable
by days 14 and 21 posttherapy. In contrast, these same animals produced
markedly higher levels of IL-4 and IL-5 at both 7 and 14 days after Tc2
effector cell transfer than did untreated or Tc1-treated mice. However,
both IL-4 and IL-5 cytokine producing levels by these CD8 T cells were
substantially lower and negligible by day 21 posttherapy, respectively.
Cytokine-releasing activity by CD8-enriched T cell populations from
spleens of untreated tumor-bearing control mice were negligible at all
time points tested (Table V). Data shown are one representative
experiment of three that were performed with similar results. Similar
results were obtained in separate experiments in mice with more
advanced 14-day-established pulmonary disease (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effective tumor rejection by adoptively transferred cell populations
may be dependent on several potential direct and indirect mechanisms.
First, both effector CD8 T cell populations can directly eradicate
tumor cells through cognate interactions that may involve either
perforin-mediated or Fas-mediated lytic mechanisms (6, 7, 8, 18, 19). As a
result, tumor-associated Ags may be released and reexpressed by host
APC that may enhance host immune responses at sites proximal and distal
to tumor growth. Second, release of Tc1 and Tc2 cytokines, such as
IFN- and IL-4, have been shown to directly inhibit tumor cell growth
(20, 21, 22), enhance Ag presentation through up-regulation of MHC Class I
among both tumors and host APC (22, 23, 24), and influence expression of
specific immunoenhancing effector cell surface molecules that
effectively facilitate antitumor responses and tumor rejection
(22, 23, 24, 25, 26, 27). Alternatively, other T cell-derived cytokines, such as IL-10,
have been shown to potentiate tumor cell growth (28, 29). Third, Tc1
and Tc2 effector cells can induce Ag nonspecific inflammatory responses
that may indirectly aid in cytolytic and/or cytostatic antitumor
effects. Local release of cytokines, such as Tc2-derived IL-4/IL-5 and
Tc1-derived IFN-, have been shown to mediate the selective
recruitment and localization of macrophages, NK cells, and granulocytes
that may facilitate tumor growth inhibition and/or enhance tumor Ag
presentation (22, 30, 31, 32, 33, 34). In either instance, differential utilization
of discrete cytokine profiles by Tc1 and Tc2 effector cell populations
can induce tumor rejection with different mechanisms and potentially
affect both the nature and outcome of effective antitumor responses and
tumor progression. Investigations to elucidate effector cell-mediated
mechanisms and their contribution to host immunity in tumor eradication
are currently under way.
The therapeutic efficacy of Tc1 and Tc2 cell populations administered to tumor-bearing mice with established pulmonary malignancy was evaluated. Effector cell titration studies revealed that in vivo antitumor effects by both Tc1 and Tc2 effector cells are dose dependent, with the former exhibiting greater therapeutic activity at lower effector cell concentrations. Thus, on a per cell basis, Tc1 effector cells were more efficacious. Differences in therapeutic effects among the two CD8 effector cell populations may be attributed to many possible quantitative and/or qualitative differences among each cell populations in vivo. Since the accumulation of effector cells at the tumor site may be important, we determined the distribution of Tc1 and Tc2 effector cells in lungs of tumor-bearing mice at various intervals following therapy and found a local accumulation of both donor Tc1 and Tc2 CD8 cells. Both Tc1 and Tc2 effector cell populations were detectable at the tumor site with nearly equal frequency and cell number within the first week following therapy. However, while Tc1 effector cells maintained similar frequency levels for up to 3 wk following therapy, Tc2 effector cell numbers and frequencies progressively diminished over time, suggesting that Tc1 effector cell populations appear to be more effective in eliciting antitumor immunity with extended effector cell-to-tumor cell interaction times. Such quantitative differences among effector cell populations at the tumor site may be attributed to local tumor-induced inflammatory responses that may modulate various effector cell surface Ag receptors responsible for localization and trafficking properties of such cells in vivo (35, 36). Conversely, inherent quantitative and qualitative differences in the expression of select cell surface receptors may exist between Tc1 and Tc2 cell populations. As in the case with Th1 and Th2 effector cells, differences in various factors, such as chemokine receptor expression, affinity, and surface density, have been suggested to influence effector T cell recirculation and functional capacity (37, 38, 39). Such differences may exist in corresponding CD8 Tc1 and Tc2 cell populations in response to progressively growing tumor in the lung. We have observed differences, however, in the expression levels of the CD62L adhesion/homing Ag among Tc1 and Tc2 cell populations in vitro, in that the former expressed greater levels than those of the latter. Although its significance remains unclear, it has been suggested that the levels of cell surface CD62L expression may have profound effects on lymphocyte-mediated antitumor function in vivo (40, 41).
Another explanation for differences in effector cell frequencies at the tumor site may be that one or the other of the effector cell populations is intrinsically more sensitive in its ability to be anergized (42, 43) or differentially more susceptible to activation-induced cell death (44). As with Th1/Th2 effector cells (44, 45), different CD8 effector cell populations may preferentially express heightened levels of activation-induced cell death-related cell surface receptors, such as Fas, which when engaged by tumor-derived FasL, could induce T cell death and depletion (46, 47, 48). In either instance, such effector cell reactions may result in rapid exhaustion of select Ag-specific effector cell responses at the site of disease and/or progressively growing tumor in vivo (49, 50, 51). The basis for this difference will be further investigated.
Tumors, such as the B16 melanoma, have been shown to express low levels
of class I surface Ag (52, 53) which could potentially result in the
failure of appropriate T cell Ag recognition and activation. Tc1
effector cells may enhance tumor Ag presentation and T cell
expansion/survival time by the secretion of endogenous cytokines, such
as IFN- and IL-2, that may subsequently maintain T cell survival and
tumor-reactive responses for longer periods of time. The adoptive
transfer of CD8 effectors from OT-I mice crossed to IFN--deficient
mice showed that these Tc1 effectors were less effective than the wild
type, as anticipated. Tc2 CD8 effectors from IFN--deficient mice,
however, were not significantly less effective than cells from OT-I
wild-type mice, suggesting that Tc2 effector cells must act by an
effector cell-derived IFN--independent mechanism. Tc2 effector cells
may facilitate tumor rejection through indirect mechanisms that
initiate and recruit nonspecific antitumor immune responses, such as
that of macrophages, NK cells, and eosinophils which may, in part,
delay progressive tumor growth. Investigations on the role of
individual Tc1/Tc2-derived cytokines in recruiting host antitumor
responses are currently under way.
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 (data not shown), minimal residual disease among these animals was still apparent. These observations were in agreement with those obtained using a different tumor Ag model reported by Prevost-Blondel et al. (54), in which adoptively transferred tumor-reactive TIL cells were able to initiate, but not sustain tumor-specific CTL responses sufficient to clear s.c. tumor challenge. Survival studies in our pulmonary model showed a marked increase in the mean survival rates among mice with early vs late stages of tumor development following effector cell treatment, suggesting that effective single dose Tc1 and Tc2 effector cell therapy may, in part, be dependent on the nature and level of tumor cell development in vivo. Differences in effector cell efficacy among animals with early (day 7) or more advanced (day 14) stages of tumor maturation may be attributed to tumor growth kinetics and tumoricidal activity by effector T cell populations in vivo (55), e.g., the inability of transferred effector cells to keep up with more established tumors that rapidly proliferate and lead to enhanced tumor outgrowth in vivo. The persistence or absence of repeated tumor Ag restimulation may lead to tumor-induced tolerance or anergy of adoptively transferred tumor-reactive effector T cells in tumor-bearing recipients (51, 55). 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 cell outgrowth and progression (56, 57). 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-ß and prostaglandins (9); generation of host T cells with suppressor function (58, 59); discordant structural alterations in TCR-mediated signal transduction pathways, particularly in late tumor-bearing hosts (55, 60, 61); induction of CD95-mediated T cell apoptosis by Fas ligand-expressing tumor cells (46, 47, 48); and down-regulation of tumor cell surface costimulatory factors that result in induction of T cell anergy (62).
In summary, we relate 1) that substantial numbers of systemically transferred Tc1 and Tc2 effector cell populations preferentially accumulated at the site of tumor challenge, however, with differing frequencies of retention and/or prolonged localization, 2) single-dose transfer of tumor-reactive Tc1 and Tc2 effector cells induced tumor regression and subsequently enhanced survival times in mice with established pulmonary tumors, 3) systemic antitumor responses induced by both Tc1 and Tc2 effector cell subpopulations were highly tumor Ag specific and appeared to be mediated by different mechanisms of action, 4) the level of effectiveness by adoptively transferred Tc1 and Tc2 effector cells appeared to be influenced by the stage of tumor development in the lung. Future studies will be directed at elucidating the underlying mechanisms and cellular interactions involved in generating and maintaining long term Tc1 and Tc2 effector cell-mediated tumor immunity.
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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 Ave., Saranac Lake, NY 12983. E-mail address:
3 Abbreviations used in this paper: Tc1, CD8+ CTL-producing type 1 cytokines; Tc2, CD8+ CTL-producing type 2 cytokines; B16-OVA, OVA-expressing B16 melanoma; OT-I.IFN- mice, IFN- knockout mice.
Received for publication December 2, 1998. Accepted for publication March 9, 1999.
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M. J. Dobrzanski, J. B. Reome, J. A. Hollenbaugh, J. C. Hylind, and R. W. Dutton Effector Cell-Derived Lymphotoxin {alpha} and Fas Ligand, but not Perforin, Promote Tc1 and Tc2 Effector Cell-Mediated Tumor Therapy in Established Pulmonary Metastases Cancer Res., January 1, 2004; 64(1): 406 - 414. [Abstract] [Full Text] [PDF]
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M. F. Kircher, J. R. Allport, E. E. Graves, V. Love, L. Josephson, A. H. Lichtman, and R. Weissleder In Vivo High Resolution Three-Dimensional Imaging of Antigen-Specific Cytotoxic T-Lymphocyte Trafficking to Tumors Cancer Res., October 15, 2003; 63(20): 6838 - 6846. [Abstract] [Full Text] [PDF]
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A. E. Oran and H. L. Robinson DNA Vaccines, Combining Form of Antigen and Method of Delivery to Raise a Spectrum of IFN-{gamma} and IL-4-Producing CD4+ and CD8+ T Cells J. Immunol., August 15, 2003; 171(4): 1999 - 2005. [Abstract] [Full Text] [PDF]
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T. Schuler and T. Blankenstein Cutting Edge: CD8+ Effector T Cells Reject Tumors by Direct Antigen Recognition but Indirect Action on Host Cells J. Immunol., May 1, 2003; 170(9): 4427 - 4431. [Abstract] [Full Text] [PDF]
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S. M. Jensen, S. L. Meijer, R. A. Kurt, W. J. Urba, H.-M. Hu, and B. A. Fox Regression of a Mammary Adenocarcinoma in STAT6-/- Mice Is Dependent on the Presence of STAT6-Reactive T Cells J. Immunol., February 15, 2003; 170(4): 2014 - 2021. [Abstract] [Full Text] [PDF]
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M. P. Rubinstein, A. N. Kadima, M. L. Salem, C. L. Nguyen, W. E. Gillanders, M. I. Nishimura, and D. J. Cole Transfer of TCR Genes into Mature T Cells Is Accompanied by the Maintenance of Parental T Cell Avidity J. Immunol., February 1, 2003; 170(3): 1209 - 1217. [Abstract] [Full Text] [PDF]
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M. P. Rubinstein, A. N. Kadima, M. L. Salem, C. L. Nguyen, W. E. Gillanders, and D. J. Cole Systemic Administration of IL-15 Augments the Antigen-Specific Primary CD8+ T Cell Response Following Vaccination with Peptide-Pulsed Dendritic Cells J. Immunol., November 1, 2002; 169(9): 4928 - 4935. [Abstract] [Full Text] [PDF]
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S. O. Gollnick, L. Vaughan, and B. W. Henderson Generation of Effective Antitumor Vaccines Using Photodynamic Therapy Cancer Res., March 1, 2002; 62(6): 1604 - 1608. [Abstract] [Full Text] [PDF]
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 J. Reynolds, V. A. Norgan, U. Bhambra, J. Smith, H. T. Cook, and C. D. Pusey Anti-CD8 Monoclonal Antibody Therapy Is Effective in the Prevention and Treatment of Experimental Autoimmune Glomerulonephritis J. Am. Soc. Nephrol., February 1, 2002; 13(2): 359 - 369. [Abstract] [Full Text] [PDF]
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M. B. Faries, I. Bedrosian, S. Xu, G. Koski, J. G. Roros, M. A. Moise, H. Q. Nguyen, F. H. C. Engels, P. A. Cohen, and B. J. Czerniecki Calcium signaling inhibits interleukin-12 production and activates CD83+ dendritic cells that induce Th2 cell development Blood, October 15, 2001; 98(8): 2489 - 2497. [Abstract] [Full Text] [PDF]
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M. J. Dobrzanski, J. B. Reome, and R. W. Dutton Role of Effector Cell-Derived IL-4, IL-5, and Perforin in Early and Late Stages of Type 2 CD8 Effector Cell-Mediated Tumor Rejection J. Immunol., July 1, 2001; 167(1): 424 - 434. [Abstract] [Full Text] [PDF]
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H. Winter, H.-M. Hu, K. McClain, W. J. Urba, and B. A. Fox Immunotherapy of Melanoma: A Dichotomy in the Requirement for IFN-{{gamma}} in Vaccine-Induced Antitumor Immunity Versus Adoptive Immunotherapy J. Immunol., June 15, 2001; 166(12): 7370 - 7380. [Abstract] [Full Text] [PDF]
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B. K. Helmich and R. W. Dutton The Role of Adoptively Transferred CD8 T Cells and Host Cells in the Control of the Growth of the EG7 Thymoma: Factors That Determine the Relative Effectiveness and Homing Properties of Tc1 and Tc2 Effectors J. Immunol., June 1, 2001; 166(11): 6500 - 6508. [Abstract] [Full Text] [PDF]
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B. Seliger, U. Wollscheid, F. Momburg, T. Blankenstein, and C. Huber Characterization of the Major Histocompatibility Complex Class I Deficiencies in B16 Melanoma Cells Cancer Res., February 1, 2001; 61(3): 1095 - 1099. [Abstract] [Full Text]
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R. J. Hogan, E. J. Usherwood, W. Zhong, A. D. Roberts, R. W. Dutton, A. G. Harmsen, and D. L. Woodland Activated Antigen-Specific CD8+ T Cells Persist in the Lungs Following Recovery from Respiratory Virus Infections J. Immunol., February 1, 2001; 166(3): 1813 - 1822. [Abstract] [Full Text] [PDF]
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K. A. Candido, K. Shimizu, J. C. McLaughlin, R. Kunkel, J. A. Fuller, B. G. Redman, E. K. Thomas, B. J. Nickoloff, and J. J. Mulé Local Administration of Dendritic Cells Inhibits Established Breast Tumor Growth: Implications for Apoptosis-inducing Agents Cancer Res., January 1, 2001; 61(1): 228 - 236. [Abstract] [Full Text]
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C. Vizler, N. Bercovici, A. Heurtier, N. Pardigon, K. Goude, K. Bailly, C. Combadiere, and R. S. Liblau Relative Diabetogenic Properties of Islet-Specific Tc1 and Tc2 Cells in Immunocompetent Hosts J. Immunol., December 1, 2000; 165(11): 6314 - 6321. [Abstract] [Full Text] [PDF]
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 A. E. Dixon, J. B. Mandac, P. J. Martin, R. C. Hackman, D. K. Madtes, and J. G. Clark Adherence of adoptively transferred alloreactive Th1 cells in lung: partial dependence on LFA-1 and ICAM-1 Am J Physiol Lung Cell Mol Physiol, September 1, 2000; 279(3): L583 - L591. [Abstract] [Full Text] [PDF]
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M. J. Dobrzanski, J. B. Reome, and R. W. Dutton Type 1 and Type 2 CD8+ Effector T Cell Subpopulations Promote Long-Term Tumor Immunity and Protection to Progressively Growing Tumor J. Immunol., January 15, 2000; 164(2): 916 - 925. [Abstract] [Full Text] [PDF]
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F. Fallarino and T. F. Gajewski Cutting Edge: Differentiation of Antitumor CTL In Vivo Requires Host Expression of Stat1 J. Immunol., October 15, 1999; 163(8): 4109 - 4113. [Abstract] [Full Text] [PDF]
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