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*
Department of Cell Biology and Kaplan Cancer Center, New York University School of Medicine, New York, NY 10016; and
The Institute of Molecular Genetics and Genetic Engineering, Belgrade, Yugoslavia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain, or
p59fyn) in spleen T cells have been described in
murine tumor models that were postulated to be responsible for an
altered pattern of cellular protein tyrosine phosphorylation thought to
reflect defective T cell function (4, 5, 6). Such defects in
the T cell signal transduction machinery are considered to be the
biochemical basis of suppression of immune response that is postulated
to be causally associated with tumor escape from immune destruction
(1, 2, 3). Although interest in the area of tumor-induced immune suppression has increased recently, the mechanism(s) by which tumor growth induces the biochemical changes in T cells that are postulated to underlie T cell dysfunction are largely unknown. Some possibilities include production by tumor (or host cells that infiltrate the tumor microenvironment) of soluble factors that inhibit cell cycle progression, tumor-induced defects in proximal TCR-mediated signal transduction molecules, tumor-induced defects in intracellular signaling molecules, and activation of tumor-infiltrating macrophages that may express immunosuppressive factors such as IL-10, PGE, hydrogen peroxide, or nitric oxide.
The physiological consequences of the tumor-induced T cell defects are
variable. In some murine tumor models TCR-mediated proliferation is
reduced in T cells obtained from spleens (7); in other
models cytokine expression from spleen T cells or induction of CTL
activity in MLR established in vitro is diminished (5, 8).
In several studies the reported diminished function of spleen T cells
in mice bearing late-stage tumors is postulated to correlate with the
development of a particular deficit in one of several molecules
associated with proximal TCR-mediated cell signaling (5, 6, 7, 9). However, two reports persuasively argue that these
biochemical changes in T cells of tumor-bearing mice represent normal
fluctuation not attributable to the effects of tumor growth (8, 10). In addition, several reports argue that, owing to its
unusual sensitivity to proteolysis, the apparent reduction of the
TCR levels in spleen T cells from tumor-bearing mice may be an
experimental artifact resulting from contamination of T cell
preparations with neutrophils and/or macrophages (8, 10, 11). These observations, considered together with an analysis of
T cell function in cancer patients in whom the loss of proteins
involved in proximal TCR-mediated signal transduction was not observed
(12), prompted us to carefully evaluate the function of T
cells in spleens of tumor-bearing mice. Our data demonstrate that
tumor-bearing mice do not have systemic T cell dysfunction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C3H/HeN, C57BL/6, male, and FvBN female mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were housed four per cage in a barrier facility and maintained on a 12-h light, 12-h dark cycle (0700–1900 h) with ad libitum access to food and water. A sentinel program revealed that the mice were murine hepatitis virus negative. Experiments involving animals were conducted with the approval of the New York University Medical School committee on animal research.
Tumors
6-1 tumor was created by expression of plasmids encoding
activated Ha-Ras plus p53 genes in primary murine C3H/HeN embryonic
fibroblasts. The properties of this tumor have been described
previously (13). Lp53 was created by transfection of L929
cells with a plasmid expressing activated murine p53 (13).
MEFSV40TAg was created by transfection of primary C3H/HeN MEF with a
plasmid expressing SV40 large T Ag (14). MCA-38 cells and
MC57G cells were gifts from S. Vukmanovic (New York University Medical
School, New York, NY). EL-4 cells were gifts from A. Menoret
(University of Connecticut, Farmington, CT). UV6138, 6139b, K1735, and
K5222 were gifts from H. Schreiber (University of Chicago, Chicago,
IL). L929 (C3H/HeN) was purchased from American Type Culture Collection
(Manassas, VA). A-20 (BALB/c) was a gift from Y. Liu (Ohio State
University, Columbus, OH). Most adherent tumor cell lines were removed
from tissue culture plastic by incubation in HBSS containing 2 mM EDTA
and were washed three times in HBSS. A-20 cells were grown in
suspension culture, and 6139b were passaged in vivo in syngeneic nude
mice by trocar injection of small tumor fragments (
2
mm3). The viability of the cell lines was
determined by trypan blue dye exclusion, and 2 x
105 cells were injected i.p. in a volume of 0.2
ml of HBSS. Regressor tumor 6139b was injected as small tumor fragments
using a trocar. Control mice received injections of HBSS only.
Transgenic FvBN mice expressing activated ß-catenin under the mouse
mammary tumor virus
(MMTV)3 promoter (an
amino-terminal deletion of 89 residues termed "
BCM") were
created and provided by Alexandra Imbert and Pam Cowin (New York
University Medical School). These mice develop multifocal breast
carcinoma at 4–5 mo of age.
Tissue culture
RPMI 1640 medium (BioWhittaker, Walkersville, MD) was used for isolation and culture of T cells and was supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, 0.002 mM L-glutamine, and 10% FBS (Intergen, Purchase, NY). DMEM was used for culture of tumor cell lines. All tissue culture supplements were supplied by Life Technologies (Grand Island, NY).
Isolation of T cells
T cells were purified using immunomagnetic separation using type
MS+ or VS+ columns
according to the manufacturer’s instructions (MACS, Miltenyi Biotec,
Bergish-Gladbach, Germany). In each experiment aliquots of isolated
cells were analyzed for cell surface expression of various markers by
flow cytometry and were routinely >95% CD3
+.
T cells purified thusly from splenocytes of control mice do not express
activation Ags (CD25 and CD69), do not transcribe IL-2 mRNA, and do not
incorporate tritiated thymidine in proliferation assay unless
stimulated in vitro (15).
Cytofluorometry
For single-color analysis, splenocytes (106) from control or tumor-bearing mice were once washed with FACS buffer (HBSS without phenol red (BioWhittaker), 1% BSA (Sigma, St. Louis, MO), and 0.1% sodium azide (Sigma)) and analyzed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) as previously described (16).
Proliferation assay
Single-cell splenocyte suspensions were prepared and analyzed by measurement of incorporation of tritiated thymidine after stimulation with plate-bound purified anti-TCRß mAb (H57-597, 0.01 mg/ml for 60 min at 37°C) as previously described (17).
Generation of primary CTL in vitro
Responder spleen cells (5 x 106)
from C3H/HeN or transgenic BCM FvBN mice were cultured with the same
number of irradiated stimulator splenocytes from C57BL/6 mice in 2 ml
of complete RPMI 1640 medium. Responder cells from C57BL/6 mice were
similarly stimulated with irradiated C3H/HeN cells. After 5 days cells
were harvested, the viability was assessed by trypan blue exclusion,
the percentage of CD8+ T cells was determined by
flow cytometry of an aliquot (or in some cases, as indicated in the
figure legend, CD8+ T cells purified by magnetic
immunobeading), and CTL activity was determined.
Chromium release assay
CTL activity of splenocytes was determined in standard
51Cr release assays. In brief,
106 target cells (EL-4 or P815 for redirected
assays) were incubated with 0.2 mCi of
Na[51Cr]04 in RPMI 1640
medium for 60 min at 37°C. Cells were washed twice with complete
medium and transferred to round-bottom 96-well plates at 5 x
103 cells/well. Effector cells were added at
varying numbers as indicated in the figure legends in a final volume of
0.2 ml. After a 4-h incubation at 37°C, 0.1 ml of supernatants were
harvested, and released radiolabel was determined by scintillation
counting. Redirected cytolysis assays using anti-CD3 Ab 145-2C11
(at 0.001 mg/ml final concentration) were used to determine the CTL
function of splenocytes from animals bearing MCA-38 tumors (because of
lack of a suitable target cell) exactly as previously described
(18). Purified hamster IgG was used as control Ab for
redirected assay, and lysis of labeled targets in the presence of
effector cells plus control IgG was equivalent to spontaneous release.
Maximal release from target cells was determined by treatment of cells
with 1% Triton X-100, spontaneous release was determined from cultures
of labeled target cells incubated with medium only, and the formula
used for determination of specific lysis was: [(experimental
release - spontaneous release)/(maximal release -
spontaneous release)] x 100.
RNA isolation, RT, and PCR amplification
Total cellular RNA was isolated from T cells without in vitro
stimulation, used to prepare cDNA, and used to program PCR
amplification as described previously (16). Actin was
amplified for 30 cycles; IL-2, IL-10, TNF-
, and IFN-
were
amplified for 33 cycles; and IL-4 was amplified for 38 cycles. The
sizes of the PCR fragments generated were 450 (actin), 247 (IL-2), 383
(IL-4), 354 (TNF-), 186 (IL-10), and 237 (IFN-). The sequences of
the PCR primers used are: actin: sense, 5'-GTG GGG CGC CCC AGG CAC CA;
and antisense, 5'-CTC CTT ATT GTC ACG CAC GAT TTC; IL-2: sense, 5'-GAG
TCA AAT CCA GAA CAT GCC; and antisense, 5'-TCC ACT TCA AGC TCT ACA G;
IL-4: sense, 5'-GAA TGT ACC AGG AGC CAT ATC; and antisense, 5'-CTC AGT
ACT ACG AGT AAT CCA; IL-10: sense, 5'-CAT TTC CGA TAA GGC TTG G; and
antisense, 5'-CGG GAA GAC AAT AAC TG; TNF-: sense, 5'-TTC TGT CTA
CTG AAC TTC GGG GTG ATC GGT CC; and antisense, 5'- GTA TGA GAT AGC AAA
TCG GCT GAC GGT GTG GG; and IFN-: sense, 5'-AAC GCT ACA CAC TGC ATC
TTG G; and antisense, 5'-GAC TTC AAA GAG TCT GAG G. There were no
cytokine PCR products in control reactions in which polymerase was
omitted, as detected by Southern blot analyses.
End labeling, agarose gel electrophoresis, and Southern blotting
Oligonucleotide probes for all cytokine PCR products are
internal to the PCR primers used for amplification (IL-2, 5'-CTC CCC
AGG ATG CTC ACC TTC; IL-4, 5'-AGG GCT TCC GTG CTT CGC A; IL-10, 5'-GGA
CTG CCT TCA GCC AGG TGA AGA CTT; TNF-, 5'-AG CGC GCC AAC GCC CTC CTG
GCC AAC GGC; and IFN-, 5'-GGA GGA ACT GGC AAA AGG A; actin was
visualized by ethidium bromide staining only). Probes were labeled with
[-32P]ATP (7000 mCi/mM; NEN/DuPont, Boston,
MA) and used to analyze PCR-amplified DNA fragments by agarose gel
electrophoresis followed by Southern blotting as previously described
(16).
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Results |
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As a prelude to characterization of systemic T cell function in
tumor-bearing mice we analyzed the effect of progressive tumor growth
on spleen size and cellularity. Naive mice were seeded with tumor
cells, and spleens were isolated and characterized by flow cytometry at
increasing times of tumor growth. An innocula of 2 x
106 6-1 cells, a dose that caused progressive
tumor growth and death after about 8 wk, resulted in tumors of 3 g
after about 30–35 days. The total number of splenocytes increased
progressively in tumor-bearing mice, such that by wk 2 of growth
spleens had about 3-fold more cells than controls (Fig. 1
). The increase was not due to
metastasis of tumor, because spleens of mice bearing 6-1 tumor
expressing neomycin resistance did not contain cells able to grow in
the presence of G-418 in vitro (data not shown).
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BCM) were analyzed for splenomegaly. In all 10
tumor models tested, encompassing four strains of mice and five tumor
types (6-1, MCA-38, K1735, K5222, Lp53, UV6138, MEFSV40TAg, EL4, MC57G,
and BCM), splenomegaly was an inevitable consequence of tumor growth
(data not shown).
The cellular composition of tumor-bearing mouse spleens as a function
of tumor burden was analyzed by flow cytometry. For 6-1 sarcoma, at
early times of tumor growth (1–2 wk) the percentages of
Mac1+ and Gr-1+ were
notably increased, whereas the abundance of other major cell types
(CD4+ or CD8+ T cells and B
cells) was not dramatically affected (data not shown). There was a
progressive accumulation of Mac1+ and
Gr-1+ cells, such that in mice with large tumor
burdens (>4 wk of growth) macrophages and neutrophils together
accumulated to 50–60% of the total splenocytes. At early times of
tumor growth there was only a modest effect on the percent abundance of
CD4+ and CD8+ T cells, but
by 3–4 wk CD3+ T cells were reduced from
25–30% to about 10% (Fig. 2). There
was a similar reduction in abundance of B cells from 50 to 10% by
wk 4 of tumor growth. The mean fluorescence intensity of staining of T
cell subsets did not diminish, but for B cells, as the abundance
decreased, so did the mean fluorescence intensity (from 750 to
340).
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and data
not shown), in all models it can be generalized that with increasing
time of tumor growth spleens accumulate macrophages and neutrophils
simultaneously, with a decrease in T cells and in most cases also in B
cells. Spleen cells of tumor-bearing mice are deficient in proliferative response to TCR ligation in vitro
To determine whether the altered cellular composition of spleens
reflected T cell function, the proliferative responsiveness of
splenocytes from tumor-bearing mice was tested compared with that of
cells from control mice (Fig. 3).
Proliferation assay of 105 splenocytes from mice
bearing 6-1 tumor showed a progressive decrease in the incorporation of
tritiated thymidine as a function of time of tumor growth. At early
times after tumor seeding the proliferative deficit was modest (25%
that in control spleen cells at 2 wk), but in all mice analyzed (>40
individual 6-1 tumor-bearing mice) proliferation was reduced by about
40% by about 3 wk of tumor growth. At later times the deficit was even
greater, approaching 90% at 5 wk. The proliferative deficit was not a
function of the method of T cell activation in vitro (plate-bound
anti-TCR Ab), because the deficit was also noted when soluble
anti-TCR Ab (or anti-CD3) plus anti-CD28 Ab, plate-bound
anti-TCR plus anti-CD28 Abs, or Con A were used to stimulate
cells (data not shown). A similar proliferative deficit was noted in
other tumor models.
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The apparent proliferative deficit in tumor-bearing mice was
anticipated to reflect some phenotypic abnormality in T cell function,
possibly related to escape of tumor from antitumor immune response. To
address this point we asked whether tumor-bearing mice can prime a T
cell immune response in vivo. Tumor-bearing or control mice were
injected with an antigenic protein, KLH, and the development of
anti-KLH T cell immune response was measured by proliferation assay
of splenocytes in vitro. We found that mice bearing tumors for 2 or 3
wk produced anti-KLH T cell immune response equivalent to that of
non-tumor-bearing control mice (Fig. 4).
This finding suggests that the dramatic change in spleen cellular
composition and deficit in spleen T cell proliferation do not inhibit
development of CD4+ T cells reactive with a
soluble antigenic protein after injection.
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We performed experiments designed to test the physiologically
relevant function of T cell immune response in tumor-bearing mice by
asking whether tumor-bearing mice could reject allogeneic tumor
challenge. Mice were seeded with 6-1 tumor cells and after 3 wk of
tumor growth were injected with tumors originating in the H-2B (Fig. 5a) or H-2D (data not shown)
background. Mice syngeneic with the secondary tumors injected at the
same time as mice bearing 6-1 tumor developed tumors with kinetics
typical of the individual tumor. One hundred percent of both
non-tumor-bearing control and tumor-bearing H-2K mice rejected
allogeneic tumor challenges. This finding shows that tumor-bearing mice
are competent to reject allogeneic tumor challenge.
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Because allogeneic tumor is a very strong antigenic stimulus,
rejection of allogeneic tumor may not be sufficiently sensitive to
evaluate immune dysfunction in tumor-bearing mice in which immune
dysfunction may be subtle. Therefore, to evaluate the immune function
of tumor-bearing mice using a milder antigenic stimulus we also tested
whether mice bearing 3-wk-old 6-1 tumor could reject challenge of
syngeneic and non-cross-reactive tumors, which are rejected by mice
with functional immune systems. The goal of this experiment was to test
whether mice bearing tumors can mount a successful antitumor immune
response to antigenically unrelated tumors. The choice of the specific
second tumors, so-called regressor tumors (19), was made
because they are known to require T cells for their rejection in intact
(non-tumor-bearing) mice (20). If mice bearing tumors can
reject secondary tumors, then it can be inferred that the T cells in
the tumor-bearing mouse are not defective in the ability to recognize
and reject antigenic tumors. The growth of regressor tumors was
authenticated by seeding tumors in athymic syngeneic mice
simultaneously with injection into tumor-bearing mice. As was found for
the rejection of allogeneic tumors, mice bearing 6-1 tumors could
reject challenge of three different syngeneic regressor tumors (Fig. 5b) and L929 and 1402RE tumors (data not shown). These
results show that the immune function in tumor-bearing mice is
sufficient to permit rejection of syngeneic, but not cross-reactive,
tumors.
Splenocytes of tumor-bearing mice can develop CD8+ CTL after in vitro priming
The ability to develop spleen-derived CD8+
CTL response after in vitro priming has been found by others to be
defective in tumor-bearing mice (4, 8). Because rejection
of allogeneic and syngeneic tumor in vivo is dependent upon
CD8+ T cells, this assay of systemic immune
competency was tested in various tumor models. After 3 wk of 6-1 tumor
growth (a point at which spleen cellularity is dramatically altered;
Fig. 2) spleens were isolated from mice and primed in vitro with
irradiated allogeneic H-2B splenocytes. After 5 days of coculture
viable cells were isolated and analyzed by flow cytometry. Viable cells
present after in vitro priming were 15% CD8+
using splenocytes from control mice and were 8% using splenocytes from
tumor-bearing mice (day 21). Cells were used as effector cells in a
standard cytolysis assay and were equal to or more active than control
T cells in CTL activity (Fig. 6a). Target lysis was
inhibited by inclusion of anti-CD8 Ab, but not by irrelevant Ig
(data not shown).
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BCM, Fig. 6, b and c; K1735 and EL4 tumors, data not shown).
Splenocytes from mice bearing MCA-38 tumors were stimulated in vitro
with C3H/HeN spleen cells; the others were primed with C57BL/6 spleen
cells. Cytolytic activity induced in spleen cells obtained from MCA-38
tumor-bearing mice was analyzed by redirected CTL assay (using P815
tumor as target), and assay of the other tumor models used direct lysis
of labeled EL4 targets (H-2B as were cells used for in vitro priming).
In all tumor models tested efficient CTL activity was elicited from
spleen cells of tumor-bearing mice. Alloreactivity induced in
splenocytes from tumor-bearing mice was potent; when
CD8+ T cells were purified after priming for use
in CTL assay, significant killing was seen at E:T cell ratios as low as
2.5:1. These experiments demonstrate that although the cellular
composition of spleens in tumor-bearing mice is changed compared with
that in control mice (Fig. 2), the presence of abundant macrophages and
neutrophils and the reduced levels of CD8+ T
cells (even during priming in vitro) does not diminish functional
priming of T cell-mediated cytolytic immune response. Purified spleen CD3+ T cells from tumor-bearing mice are not defective in proliferative response in vitro
The results of the functional analyses of spleen T cell immune
responses (shown above) motivated us to re-examine the proliferative
deficit previously noted in Fig. 3. We reasoned that the presence of
macrophages and neutrophils that accumulate in spleens may have
artifactually influenced T cell proliferation assays, perhaps due to
elaboration of immunosuppressive chemokines and/or cytokines produced
during in vitro culture. Alternatively, because of the progressive
decrease in abundance of T cells as a function of tumor growth, the
absolute number of T cells in spleen preparations of tumor-bearing mice
may have been reduced below a minimum number required to reveal
anti-TCR Ab-induced T cell proliferation in vitro. Therefore, we
modified the proliferation assay to include analysis of the percentage
of CD3+ T cells in splenocyte preparations,
which permitted plating of known numbers of T cells from spleens of
both control and tumor-bearing mice. When proliferation was assessed in
this manner, spleen T cells from tumor-bearing mice were not diminished
in incorporation of tritiated thymidine compared with control T cells
(Fig. 7, A and
B).
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+ and were not contaminated with
Mac1+ cells (Fig. 7A) or
Gr-1+ cells (15). In addition,
incorporation of thymidine in vitro was dependent upon mitogen
activation, demonstrating that isolation does not activate the T cells
(15). Purified T cells obtained from tumor-bearing mice
reproducibly proliferated better than T cells purified from control
mice (Fig. 7B). In another confirmatory experiment
CD3+ T cells purified from spleens of
tumor-bearing mice by FACS also proliferated better than the equivalent
cell population purified from control mice (data not shown).
To generalize this finding we performed proliferation assays using
spleen cells from mice bearing 3-wk tumors of the following types:
K1735, EL4, UV6138, L929/p53 (21), K5222, B16, SV40 large
T Ag-transformed primary murine embryonic fibroblasts
(21), MCA-38, and MC57G. In addition, we tested a
transgenic murine line in which mutant ß-catenin is expressed under
the MMTV promoter and mice develop spontaneous multifocal breast
carcinoma at 4–5 mo of age, BCM. For all tumor models proliferation
of spleen T cells from mice bearing tumors was equivalent to or greater
than that of splenocytes from syngeneic control mice (Table I). Collectively, our analyses do not
indicate a proliferative deficit in spleen T cells of mice bearing
tumors up to 15–20% of body weight.
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The accumulation of cytokine RNA in spleen T cells of
tumor-bearing mice RNA was isolated from purified
Thy1+ cells and analyzed for the expression of
mRNAs encoding various cytokines. Cells were purified by magnetic
immunobeading from tumor-bearing or control spleens as described above,
and an aliquot taken for flow cytometric confirmation of purity (cells
were >95% CD3+; data not shown). The
remainder of the cells were snap-frozen in liquid nitrogen before RNA
isolation. We found that T cells obtained from spleens of tumor-bearing
mice expressed a variety of cytokine RNAs, including IL-2, IL-4, IL-10,
and IFN- (Fig. 8). Cytokine RNAs were
strongly expressed without stimulation in vitro, but not in T cells of
control mice, indicating that the T cells were transcribing these genes
in situ. Importantly, cells from control mice did not express these
RNAs, indicating that the isolation procedure does not induce
transcription of these cytokine RNAs, which is indicative of
activation.
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The cytokine RT-PCR analyses showed that several cytokine
mRNAs were transcribed in T cells of tumor-bearing, but not
control, mice without activation in vitro (Fig. 8). Because spleen T
cells are apparently activated in situ, we considered the possibility
that cytokine production upon further activation may be dampened,
perhaps due to overstimulation in response to tumor growth. To make
this determination T cells from MCA-38 tumor-bearing and control mice
were purified before collection of culture supernatants either with or
without activation in vitro by incubation on anti-TCR-coated
plasticware and analysis of tissue culture supernatants for IL-2
content by CTLL-2 bioassay (22) (Fig. 9A). We found that highly
purified CD4+ T cells of MCA-38 tumor-bearing
mice secreted IL-2 without in vitro activation. The amount of IL-2
secreted ranged from 2 pg to 500 fg/ml/2 x
106 cells/24 h. CD4+ spleen
T cells from the other tumor models did not secrete IL-2 unless
activated.
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BCM
tumors (and 6-1, K1735, and EL-4; data not shown) and stimulated them
with plate-bound anti-TCR plus anti-CD28 Abs before IL-2
analysis by bioassay (Fig. 9B). CD4+
spleen T cells in three of five tumor models tested produced IL-2 upon
activation in vitro at levels close to those of control cells. For
MCA-38 and 6-1 tumor models the production of IL-2 from
CD4+ T cells was slightly less than that for
controls.
IL-2 secretion from T cells from mice bearing BCM tumors required
stimulation in vitro. However, although T cells from the BCM model
(and EL-4; data not shown) did not secrete IL-2 without in vitro
activation, upon stimulation these T cells secreted greater levels of
IL-2 than did syngeneic control cells. Collectively, these analyses
demonstrate that IL-2 production from spleen T cells of tumor-bearing
mice is not diminished compared with that in control animals.
Furthermore, in several models systemic T cells of tumor-bearing mice
secrete IL-2 without deliberate activation in vitro, implying a
heightened activation status in vivo, an idea in concert with our other
findings and which implies functional integrity in situ.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
which, due to its fundamental position in the cascade of cytosolic
signaling events, was postulated to be the basis for defective immune
response, permitting antigenic tumor to grow. Subsequently, several
independent reports described similar results (6, 9).
These findings renewed enthusiasm into the search for a mechanistic
understanding of tumor-induced escape from immune response.
However, it has been proposed that the apparent loss of TCR-proximal
signal-transducing proteins may not accurately reflect expression in T
cells of tumor-bearing mice or be truly tumor-induced functional immune
suppression (8, 10, 26). The possibility of artifactual
proteolysis of TCR-associated signaling proteins generated during T
cell isolation and analysis was considered because of the observation
that when splenic T cells are rigorously purified before detergent
solubilization and immune precipitation analysis there is no loss of
these proteins (8, 10). In support of this contention is
the report that the inclusion of high levels of protease inhibitors
during cell lysis resulted in enhanced recovery of TCR
(11). This issue is particularly acute if the
contaminating cell types are macrophages or neutrophils, both of which
are abundant in the spleen and primary tumors of tumor-bearing mice and
which elaborate powerful protease activities or reactive oxygen
intermediates difficult to inhibit (11). Our data
substantiate the idea of inadvertent cell contamination as the basis
for the conflicting data concerning putative systemic T cell
dysfunction in tumor-bearing mice and extend the conclusions to include
analysis of several T cell functions both in vivo and in vitro to prove
this important point.
Functional impairment of murine systemic T cells resulting from tumor growth has not been observed in our hands. We analyzed several different tumor types, including colon carcinoma, fibrosarcoma, thymoma, melanoma, and spontaneous breast cancer, and found, contrary to our expectation, that purified spleen T cells proliferate better than control T cells. In addition, instead of a functional deficit, T cells are activated in situ as evidenced by the expression of cytokine genes, which is observed at the time of cell isolation without activation in vitro. Furthermore, spleen T cells from tumor-bearing mice can be activated in vitro to produce IL-2 and are therefore not inhibited in cytokine production by tumor growth. Mice that have a modest systemic T cell proliferative deficit (10–20% lower than controls) were observed, but only when tumors were extremely large, so the relevancy to putative tumor-induced down-regulation of antitumor immune response or an analogous situation in human patients is doubtful.
Previous analyses of putative systemic T cell dysfunction were limited to in vitro biochemical experiments and did not address the in vivo function of T cells of tumor-bearing mice as we have (4, 5, 7, 9, 11, 27). Assessment of T cell function in our experiments was made by in vivo assays, including T cell priming to exogenous protein Ag and rejection of allogeneic and syngeneic tumors. These physiological relevant characteristics, considered together with the in vitro analyses (including CD8+ CTL induction), clearly demonstrate that mice bearing sizable tumor burden have normal systemic T cell function in vivo. We have used at least one tumor model used by others (MCA-38) as well as nine other tumors of different histological origin to demonstrate that there is no systemic T cell dysfunction in tumor-bearing mice.
TIL from cancer patients have been reported to have decreased
expression of TCR, which is suggested to correlate with decreased
proliferation and effector phase functions of TIL
(28, 29, 30, 31). If decreased TCR renders TIL unable to
effectively eliminate human tumors because of decreased CTL function,
then understanding the biochemical basis for this phenomenon becomes
important to develop effective immunotherapeutic strategies for the
management of cancer that are dependent upon activation of antitumor T
cell immune response (2). TCR has been recently shown
to be a substrate for cleavage by caspase 3 resultant from activation
of Fas, implying a mechanism by which TIL could be rendered
dysfunctional by cell-to-cell contact in the tumor microenvironment
(32). In this regard, there have been several reports that
PBL T cells of patients with certain cancers also have decreased TCR
levels, implying that systemic T cells are dysfunctional in cancer
patients (33, 34, 35). If TCR is degraded in TIL by a
Fas-dependent mechanism requiring contact with tumor, as suggested by
the data presented by Rabinowich et al. (31), how TCR
in PBL T cells could be degraded is hard to envisage, because this
would mean that the whole body complement of T cells would have to have
contact with the Fas-activating environment of the tumor. In addition,
loss of TCR presumably reflects diminished T cell function, but
cancer patients do not have loss of T cell functions in vivo (except in
very late stages), which would be reflected in enhanced opportunistic
infection, which is not seen. Therefore, the issue of biochemical
defects in PBL T cells of cancer patients and the relevancy to tumor
escape from immune destruction are at present unresolved.
Despite the concerns that our data illustrate in consideration of the
influence of tumor growth on systemic T cell function, tumor-induced
defective immune response is probably common in the early phase of
immune response and is causally related to tumor escape from immune
response because of tumor-induced defective T cell function in
tumor-specific T cells (2, 3). For example, in recent
reports studying human TIL derived from late-stage renal cell carcinoma
patients, the Finke laboratory has shown that diminished T cell
function in vitro is correlated with defective activation of NF-
B,
which is postulated as the likely basis for diminished antitumor T cell
function (36). Those data illustrate a potentially
important difference between cancer patients and tumor-bearing mice in
terms of tumor-induced effects on systemic T cell function.
In a variety of additional experiments that assessed the function of anti-tumor T cells in murine tumor models in which hosts expressed transgenic TCR specific for tumor Ags, T cells were found to be ineffective in killing tumors, albeit for different reasons depending upon the specific model employed. For example, using adoptive transfer of T cells obtained from a transgenic TCR mouse model, Staveley-O’Carroll et al. (37) showed that systemic Ag-specific T cell anergy is induced in CD4+ T cells in mice bearing early-stage A20 lymphoma modified to express influenza hemagglutinin as tumor Ag, although the biochemical basis underlying the anergic phenotype is still unknown. Ag nonspecific T cell responses were not defective in early-stage tumor-bearing mice. In a different transgenic TCR murine tumor model Prevost-Blondel and colleagues recently showed that B-16 melanoma (modified to express an MHC class I-restricted tumor Ag) grew in mice expressing a transgenic TCR reactive with the tumor Ag. TIL that accumulated in primary tumors were highly lytic for tumor in vitro in 18-h lysis assays, strongly implying that CTL function was inhibited in situ (38). Assessment of perforin-mediated cytolysis was not demonstrated, implying that cytolysis was Fas mediated. Finally, Wick and colleagues showed, using a transgenic TCR mouse model, that antitumor T cells do not accumulate at the site of tumor growth, implying that the tumor microenvironment impedes T cell recruitment, perhaps due to down-regulated inflammatory response at the tumor site (39). Collectively, these data highlight the important influence of the tumor microenvironment in both recruitment of T cells to the site of primary tumor and T cell activation (of both priming and effector phase antitumor T cell functions) and serve to focus our attention on the mechanism(s) by which Ag-specific T cell anergy may be induced in situ.
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Acknowledgments |
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BCM mice; Hans Schreiber, Antoine Menoret, Yang Liu,
and Stanislav Vukmanovic for providing several tumor cell lines; and S.
Vukmanovic for advice on the redirected CTL assay. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Alan B. Frey, Department of Cell Biology, Room MSB 690, New York University School of Medicine, 550 First Avenue, New York, NY 10016. E-mail address:
3 Abbreviations used in this paper: MMTV, mouse mammary tumor virus; KLH, keyhole limpet hemocyanin; MEF, murine embryonic fibroblasts; TIL, tumor-infiltrating lymphocytes.
Received for publication July 30, 1999. Accepted for publication December 13, 1999.
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