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Center for Surgery Research, The Cleveland Clinic Foundation, Cleveland, OH 44195
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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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In systemic adoptive immunotherapy, it has been generally assumed that the antitumor efficacy of the transferred cells is, to a large extent, determined by their ability to extravasate across the endothelial cell barrier and migrate into the tissue where tumors are located. However, it has been controversial whether the adoptively transferred cells accumulate specifically at the site of tumor and whether such accumulation is a prerequisite for tumor eradication. In a xenograft model of liver metastases in nude mice using human gastric carcinoma cells and adherent IL-2 activated NK (A-NK) cells, Okada et al. (11) found that, despite nearly complete elimination of metastases, only small numbers of transferred cells were localized in metastases. In contrast, in the murine syngeneic B16 melanoma model, a selective accumulation of transferred A-NK cells in pulmonary metastases was demonstrated (12, 13). Activated NK cells are not MHC restricted and do not have the same capacity of specific recognition of tumor cells as that possessed by CTLs (14). In both animal and human studies, however, selective accumulation of i.v. transferred CTLs at the tumor site was not a consistent finding. Using lipophilic cell-labeling reagents, Wallace et al. (15) reported that there was no differential accumulation, selective retention, or proliferation of adoptively transferred murine tumor-infiltrating lymphocytes (TILs) at the tumor site. They hypothesized that a minute number of antitumor effector cells might be sufficient to initiate the regression of an established metastases. Because of the heterogeneity in function and specificity of the effector cell populations used in most studies, it must be presumed that only a small fraction of the transferred cells has the antitumor reactivity and the potential to mediate tumor regression in vivo. It is, therefore, difficult to establish a correlation between cells infiltrating tumor tissue and cells possessing immunologic specificity toward the tumor. In our previous study, several cloned T cell lines were established from the anti-CD3/IL-2 activated tumor-draining LN T cells by repeated stimulation with tumor cells (16). These long-term cultured T cells demonstrated tumor accumulation that correlated with their specificity of antitumor effects when tested against advanced pulmonary metastases. In the clinical setting, however, cloning is not always feasible and practical because culture duration and manipulation often results in the generation of cells with altered physiologic characteristics. To gain insights into the mechanism of tumor eradication by the systemic transfer of short-term cultured cells, we conducted an investigation with a focus on the early trafficking pattern and the distribution of transferred effector T cells.
T cells from tumor-draining LNs display a heterogeneous expression of
the homing molecule, L-selectin (CD62L). Recent experimental results
identified a small population (
20%) of LN T cells with
down-regulated L-selectin (L-selectin-) to be
the sole immune effector cells mediating the regression of established
tumors (17, 18). In the current study, migration and tumor
infiltration of the transferred cells were also analyzed with
particular reference to the expression of L-selectin. Our results
provide unequivocal evidence indicating that tumor infiltration by
transferred cells reflects the physiologic characteristics, but not the
immunologic specificity, of the activated draining LN T cells. However,
the presence of specifically sensitized T cells in the transferred cell
population is required for initiating tumor eradication.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6N (B6) mice, 6–8 wk old, were purchased from the Biologic Testing Branch, Frederick Cancer Research and Development Center, National Cancer Institute (Frederick, MD). They were maintained in a specific pathogen-free environment according to National Institutes of Health guideline and were used for experiments at the age of 8–12 wk.
Tumors
The MCA 205 fibrosarcoma is a 3-methylcholanthrene (MCA)-induced tumor of B6 origin (9). The tumor has been routinely passed in vivo by serial s.c. transplantation in syngeneic mice and was used within the fifth to the eight transplantation generation. Another similarly induced fibrosarcoma, MCA 207, was used for specificity control. Single cell suspensions were prepared from solid tumors by digestion with a mixture of 0.1% collagenase, 0.01% DNase, and 2.5 U/ml hyaluronidase (Sigma, St. Louis, MO) for 3 h at room temperature. The cells were filtered through a layer of no. 100 nylon mesh, washed, and resuspended in HBSS.
Tumor-draining LN cells
B6 mice were inoculated s.c. with 1.5 x 106 MCA 205 tumor cells on both flanks. Twelve days later, tumor-draining inguinal LNs were harvested, and single cell suspensions were prepared mechanically as described previously (3). LN cells were activated with anti-CD3 mAb (145-2C11) immobilized on 24-well tissue culture plates at 4 x 106 cells/2 ml of complete medium (CM) for 2 days. CM consisted of RPMI 1640 supplemented with 10% heat-inactivated FCS, 0.1 mM nonessential amino acids, 1 µM sodium pyruvate, 2 mM fresh L-glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin, 50 µg/ml gentamicin, 0.5 µg/ml fungizone (all from Life Technologies, Grand Island, NY), and 5 x 10-5 M 2-ME (Sigma). After anti-CD3 activation, cells were harvested, washed, and further cultured in gas-permeable culture bags (Baxter Healthcare, Deerfield, IL) at 2 x 105 cells/ml of CM supplemented with 4 U IL-2/ml. Three days later, cells were harvested, washed, and resuspended in HBSS for adoptive immunotherapy. In some experiments, splenic T cells from normal B6 mice were used as control cells. T cells in the spleen cell suspensions were concentrated by passing through nylon wool columns (Robbins Scientific, Sunnyvale, CA) as previously described (18). These T cells were activated and expanded identically to that of tumor-draining LN cells.
Adoptive immunotherapy
B6 mice were injected i.v. with 3 x 105 MCA 205 or MCA 207 tumor cells suspended in 1 ml of HBSS to initiate pulmonary metastases. Ten days after tumor inoculation, all mice received sublethal WBI (500 cGy) delivered from a 137Cs irradiator (J.C. Shepard & Associates, Glendale, CA). Activated cells were then given i.v. to each mouse at numbers indicated. On day 21, mice were killed and metastatic tumor nodules on the surface of lungs enumerated after counterstaining with India ink (19). Lungs with >250 metastatic nodules were assigned >250, as this was the maximum number of metastases that can be reliably counted.
Therapeutic efficacy of transferred cells was also assessed for the treatment of s.c. tumors. In this system, mice were inoculated s.c. with 6 x 106 MCA 205 tumor cells in 100 µl HBSS. Three days later, mice received sublethal WBI (500 cGy) followed by i.v. transfer of cells. The diameters of s.c. tumors were measured twice weekly with a vernier caliper, and size was recorded as an average of perpendicular measurements and presented in mm as the mean of a group.
Trafficking of tetramethylrhodamine isothiocyanate (TRITC)-labeled cells
For fluorochrome labeling, cells were washed and resuspended at 1 x 106/ml in RPMI 1640 containing 0.5 µg TRITC/ml (Sigma). Following incubation at 37°C in a 5% CO2 atmosphere for 30 min, the cells were washed twice in RPMI 1640 and resuspended in HBSS before adoptive transfer. At a different time point after i.v. transfer of TRITC-labeled cells into tumor-bearing mice, samples of organs and tissues, including the lung, liver, kidney, spleen, brain, skin with s.c. tumors, and LN, were harvested and fixed in 4% formalin for 24 h, before being placed in 30% sucrose for an additional 24 h. These tissues were snap frozen in n-Hexane at -70°C, and 8-µm cryosections were prepared from 10–20 different cutting surfaces. TRITC-labeled cells were identified and counted using a fluorescence microscope (Olympus, New Hyde Park, NY) equipped with a filter combination of BP545 for rhodamine detection. In lungs, the area of metastases was estimated, and the number of TRITC-labeled cells in the tumor, as well as in the surrounding normal tissue, were counted using a 40x objective and a reticle containing 100 squares. The number of TRITC-labeled cells in 20–30 metastases from each lung was averaged and presented as the number of cells per 0.024 mm2 because this was the average size of 10-day pulmonary metastases. Afterward, the sections were counterstained with Meyer’s hematoxylin to confirm the presence of metastases by light microscopy.
For estimation of labeled cells in blood, mice were anesthetized with 0.8 mg pentobarbital i.p. before collecting 100 µl blood from the left ventricle of the heart. RBC were lysed with ammonium chloride-potassium buffer at room temperature for 1 min, washed twice in RPMI 1640, and fixed in 1% paraformaldehyde. The percentage of TRITC-labeled cells was estimated by FACS analysis, and the average number of labeled cells/ml blood was calculated.
Isolation and characterization of adoptively transferred cells in tumors and lymphoid organs
Twenty-four hours after i.v. transfer of TRITC-labeled cells, lungs containing metastatic tumors or skin with s.c. tumor nodules were collected, and single-cell suspensions were prepared by digesting minced tissues in 40 ml of HBSS containing 0.1% collagenase, 0.01% DNase, and 2.5 U/ml hyaluronidase (Sigma) for 1 h at room temperature. Preliminary experimental results indicated that treatment of LN cells with a mixture of these three nonproteolytic enzymes did not interfere with the detection of L-selectin by immunofluorescence staining (data not shown). Therefore, in routine analyses, single cell suspensions from LNs or spleens were prepared mechanically by teasing organs with needles followed by pressing tissue fragments with the blunt end of a plastic syringe. The cells were filtered through a layer of no. 100 nylon mesh, washed twice in RPMI 1640 containing 5% FCS, and leukocytes were separated by centrifugation through a ficoll-hypaque gradient (Histopaque-1119; Sigma). Cells were stained by indirect immunofluorescence for the expression of L-selectin. The membrane fluorescence (FITC) and intracellular staining (TRITC) were analyzed by two-color flow cytometry to characterize L-selectin expression on the transferred cells.
Fractionation of activated LN cells based on L-selectin expression
Activated tumor-draining LN cells were separated into L-selectin+ and L-selectin- cells with the use of Rat T-cell immunocolumns that contained glass beads coated with goat anti-rat Ig (Biotex Laboratories, Edmonton, Canada). Cells were incubated for 20 min at 4°C with the L-selectin hybridoma (mel-14) ascites fluid at 1:3000 dilution. The cells were washed and 2–3 x 108 in 4 ml of HBSS was loaded onto each immunocolumn, according to the manufacturer’s instructions. After 30 min of incubation, the first 15-ml pass-through fraction containing highly purified (>95%) L-selectin- cells was collected. After rinsing the column with 30 ml HBSS containing 2% FCS, the glass beads were transferred to a 50-ml tube, along with 30 ml HBSS. The L-selectin+ cells were dislodged from the beads by vigorous pipetting. In some experiments, immunocolumn was incubated with 2 ml of L-selectin hybridoma ascites fluid at 1:3000 dilution for 1 h. The column was then washed with 30 ml HBSS before 2–3 x 108 uncoated cells in 4 ml were allowed to run through the column bed. Using this method of purification, the recovered L-selectin+ cells had little bound mAb on their surface.
Statistical analysis
The significance of differences in numbers of pulmonary
metastases between groups was analyzed by the Wilcoxon rank-sum test.
Differences of numbers of cells infiltrating tumor tissues were
analyzed by the Student’s t test. A two-tailed p
value of 
0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have shown that freshly harvested LN cells,
draining the progressively growing MCA 205 sarcoma, contained 35%
of CD3+ T cells with equal proportions of
CD4+ and CD8+ cells
(20). After in vitro stimulation with anti-CD3 for 2
days, followed by culture in 4 U/ml of IL-2 for 3 days, these activated
cells were virtually all T cells (>95% Thy1.2 and TCR
ß) with
predominantly CD8 (70%) and some CD4 (20%) T cells. The in vivo
antitumor efficacy of anti-CD3/IL-2 activated tumor-draining LN T
cells has been well documented for the treatment of 3-day tumors
established in the lung and skin, as well as in the brain
(5, 6, 7, 8, 9). To facilitate the study of cell trafficking, we
attempted to treat 10-day established MCA 205 pulmonary metastases. At
the time of treatment, metastatic nodules were macroscopic and clearly
visible. In addition to the systemic transfer of activated cells, mice
were also pretreated with WBI (500 cGy) to facilitate homing and
detection of the transferred cells. This dose of WBI might eliminate
tumor-induced immunosuppression, which at times interfered with the
function of transferred immune cells, especially in the case of therapy
against s.c. tumors (7, 10). In three independent
experiments, a dose-response relation between the number of
tumor-draining LN T cells transferred, and the antitumor efficacy was
established (Table I
). Complete tumor
regression was seen in all experiments after the transfer of 50 x
106 activated LN cells. Significant antitumor
effects were also seen in animals receiving as few as 20 x
106 cells. Mice treated with 50 x
106 activated naive spleen T cells failed to
mediate antitumor effects. Adoptive immunotherapy mediated by
tumor-draining LN T cells was also found to be immunologically
specific. As demonstrated in a criss-cross experiment (Table II), cells derived from LNs draining the
MCA 205 sarcoma were therapeutically effective against the MCA 205
tumor, but not against the antigenically distinct MCA 207 tumor.
Similarly, activated MCA 207 tumor-draining LN T cells were only
effective against the MCA 207, but not the MCA 205, tumor. These
results demonstrate that despite the polyclonal T cell activation
induced by the anti-CD3, draining LN cells mediated tumor-specific
effects, and the specificity was determined during the in vivo
sensitization of the tumor-draining LN. Apparently, the in vitro
activation stimulated the committed cells to functionally mature into
effector cells, which does not require antigenically specific
stimulation.
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It was assumed that because of the therapeutic efficacy and specificity of adoptive immunotherapy, only immunologically specific T cells were able to penetrate and localize in the tumor. To seek direct evidence to support or dispute this assumption, cells labeled with TRITC were used to study their in vivo distribution after systemic transfer. Preliminary experiments indicated that the current labeling method did not affect cells’ functions, including in vivo antitumor activity and specificity (data not shown). It was therefore assumed that the trafficking pattern of the functional cells would be minimally affected by TRITC labeling.
After the transfer of labeled cells, tissue was harvested at different
time points, and quantitative analysis was performed. As shown in Fig. 1d, as early as 2 h after
i.v. transfer of a therapeutic number (50 x
106) of MCA 205 tumor-draining LN cells,
accumulation of fluorescent cells in MCA 205 metastatic nodules was
significantly higher than that in surrounding normal lung parenchyma
(p < 0.01). Within the first 24 h, the
number of tumor-draining LN cells visualized within metastases
increased, with concurrent decrease in numbers of labeled cells in the
surrounding normal tissue. Unexpectedly, this accumulation within MCA
205 metastases of activated tumor-draining LN cells was not
immunologically specific because a similar number of activated MCA 207
tumor-draining LN cells, as well as activated splenic T cells from
naive mice, were found within the tumors (Fig. 1, b and
c). Twenty-four hours after transfer, the accumulation of
naive splenic T cells was, however, significantly lower
(p < 0.01) than that found for MCA 205
tumor-draining LN T cells. Fig. 2 shows
examples of cell distribution in metastatic nodules and normal lung
tissues after the transfer of TRITC-labeled cells. Another control used
for trafficking studies was freshly isolated, noncultured splenic T
cells from normal mice. These fresh T cells failed to localize within
pulmonary metastases (Fig. 1a), indicating that the
anti-CD3/IL-2 activation culture procedure conditioned T cells to
infiltrate into metastatic tumors following systemic adoptive transfer.
These results demonstrated a tumor infiltration pattern independent of
immunologic specificity, and suggest that the mere presence of an
excess of effector cells in the tumor tissue does not always result in
eradication of the tumor.
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Therapeutic efficacy and trafficking of tumor-draining LN T cells into s.c. tumors
After i.v. transfer of cells, the lung represents the first
capillary bed at which cells will travel through, thus the model system
may not reflect a systemic migration pattern. To examine the ability of
cells traveling beyond the lung, we used another model system in which
mice bearing s.c. tumors were treated with the systemic adoptive
immunotherapy. In this situation, a large number (6 x
106) of MCA 205 tumor cells were used to induce a
macroscopic tumor rapidly. Three days later, mice were treated with WBI
followed by i.v. transfer of 50 x 106
activated MCA 205 tumor-draining LN cells. Similar to previous
observations (7), treatment with tumor-draining LN cells
completely suppressed the growth of s.c. tumors (Fig. 3a). Mice treated with the
control anti-CD3/IL-2 activated normal spleen cells had
progressively growing tumors, which were not different from control
mice treated with irradiation only.
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b). Of note is that the number of labeled cells
in s.c. tumors is substantially lower than that found in pulmonary
metastatic nodules (Fig. 2, b and d). These
results indicate that a small number of effector cells were capable of
circulating and penetrating into s.c. tumors, initiating an
immunologically specific antitumor reaction. L-selectin expression and migration pattern of transferred T cells to tumor tissues and lymphoid organs
The homing molecule, L-selectin, is highly expressed on naive T
cells in circulation, and its primary function is to facilitate
lymphocyte binding to specialized high endothelial venules (HEV) in LN
and Peyer’s patches (21). In the inguinal LN draining a
progressive tumor, there was an increase from 5–10% to 15–25% of T
cells with down-regulated L-selectin expression. After in vitro
stimulation with anti-CD3 and IL-2, activated cells contained
40% L-selectin- T cells (17).
In previous studies, the L-selectin- cell
population was found to be attributed to the entire antitumor
reactivity of the tumor-draining LNs, while
L-selectin+ cells did not show any detectable
antitumor effects (17, 18). To determine whether early
migration of transferred T cells to tumor tissues was regulated by
subpopulation differences in the expression of L-selectin, we took a
different approach to study cell trafficking. Mononuclear cell
suspensions were prepared from tumor-bearing lungs, solid s.c. tumors,
and lymphoid organs after adoptive immunotherapy. These cells were
stained for L-selectin. Preliminary experiments indicated that the
detection of L-selectin on T cells was not affected by enzymatic
digestion during cell preparation. By two-color flow cytometric
analyses, transferred cells, gated on the basis of their TRITC
staining, could be analyzed for L-selectin expression. Twenty-four
hours after adoptive transfer, the majority (>95%) of transferred
cells from MCA 205 tumor-bearing lung and solid s.c. tumors were
L-selectin- (Fig. 4A). In LNs of the same
animals, 75% transferred T cells were
L-selectin+, whereas both the
L-selectin+ (40–45%) and
L-selectin- (55–60%) cells were found in
cells isolated from spleens. In addition, phenotype analysis of
infiltrating T cells in pulmonary metastases revealed a composition of
9% CD4 and 46% CD8, which is similar to the phenotype of the
transferred T cells (Fig. 4B). Again, the localization of
L-selectin- cells was not immunologically
specific because similar results were obtained after the transfer of
activated normal spleen T lymphocytes (data not shown). These
observations suggest that only T cells with down-regulated L-selectin
expression could infiltrate into tumors, while the
L-selectin+ cells left the recirculating pool and
entered peripheral lymphoid organs, probably as a consequence of
L-selectin interaction with the specific ligand in these tissues.
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During an acute neutrophil inflammation, there is a requisite role
for L-selectin in the initial attachment of neutrophils to endothelium,
but this binding is followed by a rapid endoproteolytic release of
L-selectin molecules from the cell surface (22). To
exclude the possibility of a similar loss of L-selectin expression due
to in vivo modulation, activated tumor-draining LN cells were separated
into L-selectin- and
L-selectin+ populations before adoptive
immunotherapy. The resulting cells were >95% pure, as confirmed by
the flow cytometric analysis. In two independent experiments, the
transfer of as few as 2 x 106
L-selectin- T cells derived from activated MCA
205 tumor-draining LN T cells resulted in nearly complete eradication
of 10-day established pulmonary MCA 205 metastases, whereas the
transfer of 20 x 106
L-selectin+ T cells did not demonstrate
therapeutic effects (Table III). Analysis
of the T cell phenotype of these two cell populations revealed no
significant difference that could contribute to their antitumor
reactivities. Purified L-selectin- cells were
composed of 23 ± 3.6% CD4 and 59.8 ± 2% CD8 cells,
whereas L-selectin+ cells contained 32.8 ±
2.5% CD4 and 56.2 ± 7.2% CD8 cells. In a criss-cross experiment
(Table IV),
L-selectin- T cells mediated antitumor effects
against the MCA 205, but not the MCA 207, tumor, whereas the
L-selectin- T cells from MCA 207 tumor-draining
LNs demonstrated antitumor reactivity against MCA 207, but not MCA 205,
metastases.
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10 times more
L-selectin- cells than
L-selectin+ cells in the tumors (Table IV, and
Fig. 2, e and f). Again, the infiltration of
effector cells did not reflect the immunological specificity of the
transferred cells because the numbers of
L-selectin- cells from both MCA 205 and MCA 207
tumor-draining LNs in either tumor were similar. These findings suggest
that the physiologic conditions, rather than immunological properties,
of the transferred cells determine early trafficking into the tumor
tissue. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the current study, we analyzed the trafficking pattern of systemically transferred, tumor-specific effector T cells. Unlike many previously described antitumor effector cell populations, the anti-CD3/IL-2 activated LN cells are generated from tumor-bearing mice. These cells, thus, are closely analogous to the cells one would isolate from cancer patients for the development of clinically applicable adoptive immunotherapy. In laboratory studies, we found that, despite their potent in vivo antitumor effects, the anti-CD3/IL-2 activated tumor-draining LN cells lack demonstrable cytotoxic activity against their specific tumor target cells in vitro (20). Also different from previous studies, the current adoptive immunotherapy experiments were conducted in the absence of systemic IL-2. In the past, systemic administration of IL-2 has been an integral and necessary part of the treatment when in vitro cultured cells, such as lymphokine-activated killer and TILs, were used for therapy (24, 25). Therefore, our study is unique in that the influence of the presence of IL-2 in vivo on the trafficking and antitumor efficacy is excluded. The absence of IL-2 administration in cellular therapy has drastically reduced the severe toxicity associated with clinical adoptive immunotherapy (26).
Using the fluorescent dye TRITC to label effector cells, we demonstrated that the short-term cultured LN T cells rapidly accumulated in tumor metastases located in the lung in much higher numbers than in the surrounding normal parenchyma. However, this accumulation alone did not appear to reflect immunologic specificity of the infiltrating cells, since there was an equal extent of infiltration by the nonspecific effector cells as well as normal spleen T cells activated identically. The ability of these cells to infiltrate into tumor tissues appeared to be a characteristic acquired during the in vitro activation because a similar localization of noncultured normal splenic T cells was not observed. Intravenous transfer of cells into mice with lung metastases may be regarded as regional delivery of the cells to the tumor. Using a different model system, preferential infiltration by the anti-CD3/IL-2 activated cells was also evident in the s.c. growing tumors, although the number of fluorescent cells was low. This finding indicates that the transferred cells were capable of circulating systemically through the lung capillaries and redistributing to other tissues. The present results differ considerably from our previously reported findings where long-term T cell lines and clones were found to accumulate in the lung metastases in an immunologically specific manner (16). The difference may also be attributed to the differences in culture conditions. The long-term established, tumor-specific clones were exclusively of CD8 phenotype and displayed in vitro cytotoxic effects parallel to the in vivo therapeutic effects, characteristics not shared by the 5-day anti-CD3/IL-2 activated effector cells used for the current study.
More recently, we have identified a small population of T cells with
down-regulated expression of L-selectin in the tumor-draining LNs to be
the specifically sensitized tumor-immune cells (17, 18).
Consistent with these observations, transferred T cells isolated from
both pulmonary metastases and s.c. tumor nodules were largely
L-selectin- cells. In contrast, the majority of
transferred cells recovered from LNs of the recipient mice were
L-selectin+, suggesting that
L-selectin+ cells were capable of recirculating
through peripheral LNs by interaction with HEV (27). In
the spleen, the migration of lymphocytes involves sinusoidal
endothelia, and the molecular mechanisms are distinct and not yet
characterized. In our study, both L-selectin+ and
L-selectin- transferred cells in equal numbers
were found in the spleen of treated animals. To further rule out the
possibility of in vivo down-regulation of L-selectin expression due to
a variety of factors, including interactions with tumor cells, we
fractionated activated cells into L-selectin+ and
L-selectin- cells before adoptive immunotherapy.
This experimental approach with purified cells clearly demonstrated the
correlation between L-selectin down-regulation and the increased
ability of T cells to penetrate to and localize in the tumor mass.
Since the accumulation of L-selectin- cells in
the tumor is independent of the immunologic specificity of the effector
cells, the physiological properties of activated
L-selectin- cells must have played a dominant
role. Compared with L-selectin+ cells, the
activated L-selectin- LN T cells expressed a
high level of cell adhesion molecules, including LFA-1, CD44, and the
4ß7 integrin (data not
shown). While the precise role of these molecules remains to be
determined, preliminary results suggested the involvement of G
protein-coupled chemokine receptors because in vitro treatment of
effector cells with Pertussis toxin abrogated the therapeutic efficacy
of both unfractionated and L-selectin-
tumor-draining LN cells (S. Mukai, unpublished observations). This
suggests that G protein-coupled receptors of the
i class are required for
L-selectin- cell emigration through tumor
tissues (28).
The demonstration of selective, but not immunologically specific, migration of systemically transferred effector cells to the malignant tissue suggests that the early trafficking of activated LN cells is governed by mechanisms independent of specific Ag recognition. Once the effector cells established a close contact with tumor cells or Ag-presenting cells in the tumor bed, specific interactions involving TCRs must have played a pivotal role in initiating tumor regression. In addition to serving as targets in adoptive immunotherapy models, tumors provide a source of Ags to further stimulate the expansion of immune effector cells. In previous experiments, we transferred immune cells into normal and tumor-bearing recipients, and, 9 days later, spleen cells were harvested and tested for their therapeutic efficacy in adoptive immunotherapy (29). Therapeutic effector cells could only be retrieved from tumor-bearing recipients, indicating that the maintenance and expansion of immune cells were dependent on in vivo Ag stimulation.
In a clinical adoptive immunotherapy study (30), localization of 111In-labeled TILs to tumor sites was evident in 26 of 38 patients. There was a clear association between TIL localization to tumors and clinical responses. A total of 10 of 26 patients who showed localization of TILs at melanoma sites had a complete or partial response to treatment, whereas there were no responses in the 12 patients with no localization. It is intriguing to note that not all patients who had a positive imaging showed clinical responses. These observations support our hypothesis that the infiltration into tumors by the systemically transferred cells is a phenomenon independent of immunologic specificity, although tumor response will additionally require the presence of T cells with tumor specificity.
In conclusion, the present study demonstrates that tumor eradication by the systemic adoptive transfer of specifically sensitized T cells requires two distinct but sequential biological mechanisms. Apparently, the anti-CD3/IL-2 culture system has conditioned T cells to acquire a distinct circulation pattern irrespective of their immunologic specificity. Although migratory properties of activated LN cells are poorly understood, the lack of L-selectin expression and the abundance of adhesion molecules on their surface may facilitate their homing to the tumor tissue while avoiding the normal route of lymphocyte recirculation through secondary lymphoid organs, such as the LNs and Peyer’s patches. Once the cells have infiltrated the tumor tissue, interactions with specific tumor Ags result in clonal expansion of tumor-specific effector cells and triggering lytic mechanisms, leading to the eradication of the tumor.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Suyu Shu, Center for Surgery Research/FF-50, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail address:
3 Abbreviations used in this paper: LN, lymph node; L-selectin+, L-selectinhigh; L-selectin-, L-selectinlow; HEV, high endothelial venules; WBI, whole-body irradiation; A-NK cell, activated NK cell; TIL, tumor-infiltrating lymphocyte; TRITC, tetramethylrhodamine isothiocyanate.
Received for publication December 30, 1998. Accepted for publication April 29, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R. Ganss, E. Ryschich, E. Klar, B. Arnold, and G. J. Hammerling Combination of T-Cell Therapy and Trigger of Inflammation Induces Remodeling of the Vasculature and Tumor Eradication Cancer Res., March 1, 2002; 62(5): 1462 - 1470. [Abstract] [Full Text] [PDF]
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H. N. Le, N. C. Lee, K. Tsung, and J. A. Norton Pre-Existing Tumor-Sensitized T Cells Are Essential for Eradication of Established Tumors by IL-12 and Cyclophosphamide Plus IL-12 J. Immunol., December 15, 2001; 167(12): 6765 - 6772. [Abstract] [Full Text] [PDF]
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J. Kjaergaard, L. Peng, P. A. Cohen, J. A. Drazba, A. D. Weinberg, and S. Shu Augmentation Versus Inhibition: Effects of Conjunctional OX-40 Receptor Monoclonal Antibody and IL-2 Treatment on Adoptive Immunotherapy of Advanced Tumor J. Immunol., December 1, 2001; 167(11): 6669 - 6677. [Abstract] [Full Text] [PDF]
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 T. M. Carlos Leukocyte recruitment at sites of tumor: dissonant orchestration J. Leukoc. Biol., August 1, 2001; 70(2): 171 - 184. [Abstract] [Full Text] [PDF]
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 S. M. Ansell, M. Stenson, T. M. Habermann, D. F. Jelinek, and T. E. Witzig CD4+ T-Cell Immune Response to Large B-Cell Non-Hodgkin's Lymphoma Predicts Patient Outcome J. Clin. Oncol., February 1, 2001; 19(3): 720 - 726. [Abstract] [Full Text] [PDF]
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M. Ahmadzadeh, S. F. Hussain, and D. L. Farber Heterogeneity of the Memory CD4 T Cell Response: Persisting Effectors and Resting Memory T Cells J. Immunol., January 15, 2001; 166(2): 926 - 935. [Abstract] [Full Text] [PDF]
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L. Peng, J. Kjaergaard, G. E. Plautz, D. E. Weng, S. Shu, and P. A. Cohen Helper-Independent, L-Selectinlow CD8+ T Cells with Broad Anti-Tumor Efficacy Are Naturally Sensitized During Tumor Progression J. Immunol., November 15, 2000; 165(10): 5738 - 5749. [Abstract] [Full Text] [PDF]
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G. E. Plautz, S. Mukai, P. A. Cohen, and S. Shu Cross-Presentation of Tumor Antigens to Effector T Cells Is Sufficient to Mediate Effective Immunotherapy of Established Intracranial Tumors J. Immunol., October 1, 2000; 165(7): 3656 - 3662. [Abstract] [Full Text] [PDF]
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 G. E. Plautz, D. W. Miller, G. H. Barnett, G. H. J. Stevens, S. Maffett, J. Kim, P. A. Cohen, and S. Shu T Cell Adoptive Immunotherapy of Newly Diagnosed Gliomas Clin. Cancer Res., June 1, 2000; 6(6): 2209 - 2218. [Abstract] [Full Text]
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