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*
Center for Surgery Research and
Lerner Institute, Cleveland Clinic Foundation, Cleveland, OH 44195; and
Providence Portland Medical Center, Earle A. Chiles Research Institute, Portland, OR 97213
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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 our laboratory, a potent source of tumor-immune T cells has been identified to be the lymph node (LN) draining a progressively growing tumor (11, 12). Following a short period of in vitro activation with anti-CD3 and IL-2, these LN cells develop into potent effector cells capable of curing syngeneic mice of tumors established at all tested anatomic locations, including the lung, skin, peritoneal cavity, and brain (13, 14, 15). Studying murine tumors without characterized Ags, we recently identified that the down-regulation of L-selectin expression, a peripheral LN-homing receptor, is associated with T cell acquisition of antitumor immunity (16, 17, 18). The use of activated L-selectin (L-sel)low tumor-draining LN T cells has demonstrated at least a 30-fold increase in therapeutic effects over that of unfractionated LN cells. Although concomitant IL-2 administration enhanced therapeutic potency in some situations, it is important to note that transfer of activated draining LN T cells in sufficient numbers could mediate tumor eradication without coadministration of IL-2.
The therapeutic efficacy of adoptive immunotherapy is subject to both up- and down-regulation by a number of factors. An early example is the use of adjunctive exogenous IL-2 to augment the treatment of pulmonary metastases (7, 19, 20). However, in the model system of intracranial tumors, administration of systemic IL-2 reduced the benefit of adoptive immunotherapy (21). To augment T cell-specific responses, we recently studied the effects of in vivo ligation of costimulatory molecules such as 4-1BB and OX-40R on T cells with specific mAb (22, 23, 24). This approach has shown contrasting results, depending on the immunogenicity as well as anatomical location of the tumor. In our study, the systemic administration of either 4-1BB or OX-40R mAb alone during tumor growth resulted in prolongation of survival and cure of mice with intracranial tumors but not of mice with pulmonary metastases. Furthermore, the therapeutic efficacy of in vivo Ab ligation alone was limited to minimal tumor burdens (i.e., 3 days after tumor inoculation). However, during these studies we observed that the coadministration of 4-1BB mAb enhanced the therapeutic effects of adoptively transferred tumor-specific T cells (22).
In the present study, we compared the effects of conjunctional use of OX-40R mAb and IL-2 on the therapeutic outcomes for treatment of well-established 10-day 3-methylcholanthrene (MCA) 205 pulmonary metastases and intracranial tumors. While the effects of IL-2 depended on the location of the tumor, OX-40R mAb augmented adoptive immunotherapy regardless of the anatomic site of tumor growth. Mechanistic studies revealed distinct modes of action by OX-40R mAb and IL-2 on the transferred tumor-immune T cells, leading variously to enhancement or inhibition of therapeutic effects.
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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 guidelines and were used for experiments at the age of 8–12 wk.
Tumors
The MCA 205 and MCA 207 fibrosarcomas are MCA-induced tumors of B6 origin (25). The tumors have been routinely passed in vivo by serial s.c. transplantation in syngeneic mice and were used within the fifth to the tenth transplantation generation. Single cell suspensions were prepared from solid tumor by digestion with a mixture of 0.1% collagenase, 0.01% DNase, and 2.5 U/ml hyaluronidase (Sigma-Aldrich, St. Louis, MO) for 2 h at room temperature. The cells were filtered through a layer of number 100 nylon mesh, washed, and resuspended in HBSS for inoculation. B16/D5 is a poorly immunogenic subclone of the spontaneously arising B16/BL6 melanoma (26). The B16/D5 tumor does not exhibit a detectable level of MHC class I (H-2 Db and Kb) or class II molecules. These tumor cells were maintained in culture in complete medium (CM). 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-Aldrich). GL261 glioma, originally induced by intracranial implantation of MCA pellets in a B6 mouse, was obtained from the Division of Cancer Treatment Repository (Frederick, MD) (27). The GL261 tumor cells were maintained in continuous culture in CM. Cultured B16/D5 or GL261 tumor cells were harvested after a short incubation at 37°C with a solution containing 0.25% trypsin and 0.02% EDTA (Life Technologies). The tumor cells were washed and resuspended in HBSS for challenging tumor-free mice.
Tumor-draining LN cells and in vitro anti-CD3/IL-2 activation
B6 mice were inoculated s.c. with 1.5 x 106 MCA 205 or MCA 207 tumor cells on both flanks. Twelve days later, tumor-draining inguinal LNs were harvested, and single-cell suspensions were prepared mechanically by teasing with needles and pressing tissue fragments with the blunt end of a 10-ml plastic syringe. Tumor-draining LN cells were activated with anti-CD3 mAb (145-2C11) immobilized on 24-well tissue culture plates at 4 x 106 cells/2 ml/well of CM. After 2 days of activation the 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/ml IL-2. Three days later the cells were harvested, washed, and resuspended in HBSS for adoptive immunotherapy.
Depletion of CD4+ or CD8+ T cell subset
Culture-activated tumor-draining LN cells were depleted of CD4 or CD8 T cells by negative separation using L3T4 or Lyt2 Dynabeads, respectively (Dynal Biotech, Oslo, Norway) according to the manufacturer’s instructions.
Purification of L-sellow T cells from tumor-draining LN
Isolation of L-sellow cells from single-cell suspensions of tumor-draining LN was performed by negative selection using rat T cell enrichment columns (Cytovac Technologies, Edmonton, Canada). Tumor-draining LN cells were preincubated for 20 min at 4°C with the L-selectin hybridoma (Mel-14) ascites fluid at 1/3000 dilution. The cells were washed in Ca2+/Mg2+-free HBSS and then applied to the columns to collect effluent L-sellow cells. Isolated L-sellow cells (90–95%) were CD3+ by direct fluorescent analysis. Alternatively, L-sellow cells were isolated using mouse anti-CD62L-loaded MACS (Miltenyi Biotec, Bergisch Gladbach, Germany). In this case, T cells were first purified using mouse T cell enrichment columns (Cytovac Technologies), then incubated for 15 min at 4°C with MACS anti-CD62L-microbeads according to the manufacturer’s instructions. L-sellow T cells were allowed to flow through the column attached to a magnet. The positive fraction (L-selhigh T cells) was eluted from the column using a plunger while off the magnet. Purified L-sellow or L-selhigh T cells were washed, resuspended in CM at 1 x 106 cells/ml, and activated by the anti-CD3/IL-2 method as described.
Adoptive immunotherapy
To establish pulmonary metastases, mice were given i.v. injection of 3 x 105 MCA 205 or MCA 207 tumor cells suspended in 1 ml of HBSS. To establish intracranial tumors, B6 mice were anesthetized with 0.8 mg of pentobarbital i.p. and inoculated with 1 x 105 MCA 205 or GL261 (1 x 103 for D5) tumor cells suspended in 10 µl of HBSS transcranially using a 27-gauge needle and glass tuberculin syringe (Perfectum; Popper & Sons, New Hyde Park, NY). The needle insertion was perpendicular to the skull and in line with the anterior margin of the ear and the medial half of the right eye. The depth of insertion was controlled by placement of electric wire insulation as a collar over the needle with exposure of the terminal 4 mm. Ten days after tumor inoculation, all mice received 500 cGy sublethal whole body irradiation (WBI) followed by i.v. transfer of activated tumor-draining LN through the tail vein. Animals with pulmonary metastases were sacrificed on day 21, and metastatic tumor nodules on the surface of the lung were estimated after counterstaining with india ink. For animals with intracranial tumors, therapeutic effects were evaluated by survival time.
mAb to OX-40R and adjunct treatment protocol
Hybridoma that produced the anti-murine OX-40R-specific mAb (termed OX-86) was obtained from the European Collection of Animal Cell Culture (Salisbury, U.K.) (28). In most experiments, animals with 10-day established tumors treated by adoptive immunotherapy received two i.p. injections of 150 µg of OX-40R mAb in 1 ml of HBSS 2 days apart, commencing on day of T cell transfer. Human rIL-2 was kindly supplied by Chiron (Emeryville, CA). Adjunct IL-2 treatment was given for 4 days following T cell transfer by i.p. injection of 10,000 U of rIL-2 in 1 ml of HBSS twice daily.
Flow cytometric analysis
The following mAb were used for direct or indirect staining: biotin-conjugated CD134 and FITC- or PE-labeled streptavidin (BD PharMingen, San Diego, CA); OX40L:Fc-Ig protein and FITC-conjugated human IgG (Caltag Laboratories, South San Francisco, CA); CyChrome-labeled anti-TCR, FITC- or PE-labeled anti-CD4, FITC- or PE-labeled anti-CD8, and FITC- or PE-labeled anti-Mel-14 (all from BD PharMingen). Controls were performed with labeled anti-rat isotype-matched Abs and analyses were conducted on a FACSCalibur (BD Biosciences, Mountain View, CA).
In vivo trafficking assay
For analysis of in vivo trafficking, activated tumor-draining LN T cells were labeled with CFSE (Molecular Probes, Eugene, OR). Cells were washed twice with HBSS, and 1 x 107 cells/ml were incubated with CFSE (5 µM) in HBSS for 10 min at 37°C. Labeling was stopped by adding ice-cold HBSS, and cells were washed twice with HBSS containing 5% FCS before adoptive transfer into mice bearing 10-day intracranial tumors. At 24 and 48 h after transfer of labeled cells, brains were harvested and fixed with 4% formalin for 24 h and subsequently placed in 30% sucrose for an additional 24 h. After fixation, the tissue was snap-frozen in N-hexane at -70°C and cut into 8-µm sections on a cryostat. The section was dried and examined under a fluorescent microscope (Olympus, Melville, NY) equipped with a filter combination of bandpass 490 for CFSE detection. In the sections, the area of tumor tissue was identified and the number of labeled cells in the tumor was counted using a x40 objective.
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 brain tumor tissues were
analyzed by the Student 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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We recently demonstrated that OX-40R mAb treatment alone mediated
therapeutic effects against small tumor burdens, probably through the
in situ ligation of tumor-infiltrating OX-40R+
cells (23, 24). Therefore, we investigated the potential
use of OX-40R mAb following adoptive immunotherapy to augment in vivo T
cell antitumor reactivity in comparison to the traditional use of IL-2.
As demonstrated in Table I
, conjunctional
administration of either OX-40R mAb or IL-2 resulted in a significant
enhancement of antitumor reactivity against 10-day established MCA 205
pulmonary metastases. Treatment with activated tumor-draining LN cells
alone at a dose of 50 x 106 was able to
reduce >250 metastatic nodules to 2 ± 2. Similar antitumor
effects were observed when 20 x 106 T cells
were transferred to tumor-bearing mice if either OX-40R mAb or IL-2 was
provided.
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).
It is noteworthy that despite the small proportion of CD4 cells in the
transferred T cell population, depletion of this subset had a
significantly greater impact on the therapeutic effects than did
CD8 T cell depletion. Also, the administration of OX-40R mAb did not
seem to change the requirement for both CD4 and CD8 T cells for tumor
regression because in the absence of mAb administration, depletion of
either T cell subset also resulted in inhibition of therapeutic
responses.
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, two
immunogenically distinct tumors were used. The transfer of activated
draining LN cells derived from the MCA 205 sarcoma mediated antitumor
responses against the MCA 205 but not MCA 207 tumors in the absence or
presence of OX-40R mAb treatment, although conjunctional OX-40R mAb
treatment greatly enhanced the therapeutic efficacy. Similar results
were observed when MCA 207 tumor-draining LN cells were used for the
treatment of the MCA 205 and MCA 207 tumors. Therefore, despite the
ability of OX-40R mAb to augment adoptive immunotherapy, the
specificity of the immunologically mediated reactions was not
altered.
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Our previous studies have demonstrated the feasibility of treating
experimental intracranial tumor by the systemic transfer of activated
tumor-draining LN T cells (14). Clearly, the blood-brain
barrier did not inhibit the ability of transferred cells to traffic and
to infiltrate the tumor mass to initiate immune responses that lead to
tumor rejection (30). Although treatment of early (3-day)
established intracranial tumor was effective, attempts to treat 10-day
advanced intracranial tumor with the same activated T cells proved to
be difficult even with the transfer of >100 x
106 cells (data not shown). We recently
identified that in the draining LN, a small population of T cells
(
20%) with down-regulated L-selectin possessed all the antitumor
reactivity (16). In preliminary experiments, the transfer
of purified L-sellow tumor-draining LN T cells
was able to prolong survival and cure mice bearing 10-day established
intracranial tumors. Therefore, we investigated the possible
enhancement of T cell therapeutic efficacy by conjunctional OX-40R mAb
administration, considering that in previous experiments IL-2
administration reduced the antitumor effects of transferred T cells for
intracranial tumors (21). Titration of cell numbers that
were sufficient to mediate the regression of 10-day established
intracranial MCA 205 tumors revealed that the transfer of 10 x
106 L-sellow draining LN
cells significantly prolonged survival of treated mice, but very few
animals were cured. However, by increasing the cell number to 15
x 106, all mice were cured. The effects of
conjunctional IL-2 or OX-40R mAb treatment were investigated in this
experimental model. As depicted in Fig. 1, treatment of advanced intracranial MCA
205 tumors was greatly enhanced if OX-40R mAb was used conjunctionally.
In this case, as few as 5 x 106 activated
L-sellow tumor-draining LN cells were capable of
curing all mice. In contrast, conjunctional IL-2 administration
resulted in the inhibition of adoptive immunotherapy. The dose and
regimen of IL-2 used in these experiments were identical with those for
adoptive immunotherapy of pulmonary metastases (see Table I
).
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). In either condition,
L-selhigh cells did not affect tumor progression
and all mice succumbed to tumors similar to mice without treatment. In
this experiment, it is clear that 5 x 106
L-sellow cells alone prolonged survival and the
therapeutic effects were augmented in mice also treated with
OX-40R mAb.
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). While mice cured of MCA 205 tumors
rejected challenge with the MCA 205 tumor, all mice challenged with
either the GL261 or B16/D5 tumors succumbed to growing tumors. These
results thus demonstrated that effective adoptive immunotherapy is
accompanied by the development of long-lasting, tumor-specific
immunity. Therefore, the effects of OX-40R mAb are likely the results
of interactions with tumor-specific T cells.
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). In fact, as
little as 37.5 µg of OX-40R mAb given once was capable of augmenting
the antitumor effects of transferred T cells. Thus, the dose for OX-40R
mAb required to optimally potentiate T cell adoptive therapy is far
less than the dose required for treatment of tumor as a single
reagent.
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Previous studies suggest that the expression of OX-40R on T cells
be confined mostly to the CD4 subset, although CD8 T cells may express
the OX-40R if stimulated with mitogens such as Con A (31).
To delineate the mechanism of OX-40R mAb-mediated enhancement of T cell
antitumor function, we analyzed OX-40R expression on LN T cells freshly
harvested, after 2-day activation with anti-CD3 mAb as well as
after 5-day activation with both anti-CD3 and IL-2. It is
noteworthy that only 5-day activated T cells were used for adoptive
immunotherapy experiments. As depicted in Fig. 5, when freshly harvested, the greatest
expression of OX-40R was observed on L-sellow CD4
LN T cells. By contrast, fresh LN CD8 T cells were essentially negative
for OX-40R expression. However, after 2 days of activation with
immobilized anti-CD3 mAb, all T cells demonstrated the expression
of OX-40R, although the expression on L-sellow
cells was greater than that on L-selhigh cells.
After 3 more days of culture with IL-2 (4 U/ml), only CD4 T cells of
the L-sellow phenotype remained positive for
OX-40R. L-selhigh CD4 cells and all CD8 cells
lost their OX-40R expression. This kinetics study suggests that OX-40R
expression on T cells is regulated by the stage of activation, although
its expression on L-sellow CD4 cells appears
constant.
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It has been well documented that, in adoptive immunotherapy, systemically transferred tumor-sensitized T cells must infiltrate the tumor mass to initiate tumor regression (29, 32). To study the impact of IL-2 vs OX-40R mAb treatment on the trafficking of transferred L-sellow LN T cells, we labeled T cells cytoplasmically with the green fluorescence dye, CFSE, which freely diffuses through the cell membrane and interacts with glutathione transferase, forming impermeable thioether adducts, which prevents the leakage (33, 34). Preliminary experiments indicated that CFSE-labeling did not interfere with effector T cell functions, including antitumor reactivity, proliferation, and trafficking.
Mice with 10-day established intracranial MCA 205 tumor were treated
with 15 x 106
L-sellow tumor-draining LN T cells in conjunction
with either IL-2 or OX-40R mAb. As depicted in Fig. 6, direct enumeration of fluorescent
cells on sections of brain tumors 24 and 48 h after cell transfer
revealed that the OX-40R mAb treatment did not result in an increased
number of tumor-infiltrating T cells at either time point. This finding
suggests that the mechanism of OX-40R mAb-mediated enhancement cannot
be explained by its ability to promote T cell trafficking to the tumor.
By contrast, concomitant treatment with IL-2 severely impeded tumor
infiltration by the transferred cells. At both 24 and 48 h after T
cell transfer, the number of transferred cells detected in the tumor
were 10–15% of that detected in the control (see Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The rationale for the use of conjunctional IL-2 treatment in many clinical cellular therapies has been based on observations in animal experiments that IL-2 can stimulate in vivo proliferation of cultured T cells (11, 35, 36, 37). Our own and others’ publications (19, 20) have confirmed the in vivo augmenting effects of conjunctional IL-2 in several T cell immunotherapy models. However, augmentation was principally observed in therapies confined to the treatment of established pulmonary or hepatic metastases. In the subsequent course of analyzing requirements associated with the T cell therapy of established intracranial tumors, we noted that the enhancing effects of exogenous IL-2 for treating tumor metastases in the lung were not evident for the same tumor growing in the brain (21).
We have not yet ruled out the possibility that two distinct immune T cell subpopulations eradicate tumors in the lung and brain respectively, with only the latter subpopulation susceptible to inhibition by conjunctional IL-2. We are currently attempting to isolate immune T cells from regressing pulmonary tumors to confirm their equal capacity to traffic into and mediate rejection of intracranial tumors, as well as their capacity to establish long-term systemic immunological memory in secondary hosts. In other tumor models, inhibition of immunotherapy by systemically delivered IL-2 has also previously been described. In a murine graft-vs-leukemia system, the effects of allogeneic sensitized T cells were inhibited by IL-2 (38). In a study involving the transduction of murine tumors with the B7.1 (CD80) costimulatory molecule with or without additional IL-2 transduction, mice injected with tumor expressing both elements were cured, whereas no protective effects were seen if mice inoculated with B7.1-transduced tumors were given systemic IL-2 (39). Therefore, systemic IL-2 administration can, in certain situations, deter rather than facilitate immunotherapy.
Interaction of the TCR with its specific peptide/MHC counterreceptor is required for T cell activation. However, it is believed that this interaction alone is not sufficient to activate T cells under physiologic conditions (40, 41). Additional accessory signals are thought to contribute to and even be essential for proper activation to take place. These are commonly referred to as costimulatory signals, because by themselves they have no effect on T cells. For several years, attention has been given to the interaction between CD28 on the T cell and B7.1 and B7.2 receptors expressed on APCs (42). Although the expression of CD28 on T cells is constitutive, other inducible membrane molecules, mainly of the TNFR family, have been shown to share the ability to function as costimulators for amplification and maintenance of ongoing T cell responses. We have studied two of these activation-induced costimulatory molecules, 4-1BB and OX-40R, for their roles in the amplification of T cell immune responses during tumor growth (22, 23, 24). Although 4-1BB is expressed on both activated CD4 and CD8 T cells, OX-40R seems to be preferentially expressed on activated CD4 T cells. Although in vivo ligation of either costimulatory molecule on T cells with mAb demonstrated antitumor effects, the observed therapeutic efficacy was limited depending not only upon immunogenicity and burden of tumors, but also upon the location of tumor growth. We reasoned that the failure of treating advanced tumors by boosting costimulatory functions with these mAb might not be due to the inadequacy of activation but rather to inadequate numbers of tumor-sensitized T cells in tumor-bearing hosts. If this were the case, treatment of advanced tumors could be augmented if the mAb were administrated following adoptive transfer of tumor-immune T cells. Experimental results in our studies support this hypothesis.
Perhaps the most intriguing finding of the current work is that, unlike
IL-2 adjunct treatment, augmentation of adoptive immunotherapy by
OX-40R mAb was observed for the treatment of both pulmonary metastases
and solitary tumor nodules in the brain. It is interesting to note that
OX-40R expression was confined to CD4 T cells in the freshly isolated
tumor-draining LN cells as well as in the activated cell population
that was used for adoptive immunotherapy. It is possible that the
target of OX-40R mAb is the transferred CD4 T cells, and ligation of
OX-40R leads to an increase in proliferation and cytokine secretion by
CD4 cells which secondarily amplify CD8 T cell antitumor reactivity.
The ability of tumor-immune CD4 T cells to promote the proliferation
and persistence of CD8 CTL has been well documented in animal studies
(43). Alternatively, transferred CD8 T cells may be
induced to up-regulate the expression of OX-40R upon antigenic
stimulation at the tumor site, and the administered mAb may interact
with these CD8 T cells directly and independently amplify their
activation. This possibility is supported by the observation that
tumor-draining LN CD8 T cells transiently express OX-40R after
activation with anti-CD3 (see Fig. 5). More importantly, adoptively
transferred, CD4-depleted T cells could still be therapeutically
enhanced by conjunctional OX-40R mAb treatment (see Table II).
Additionally supporting this possibility is the observation that both
CD4 and CD8 tumor-infiltrating T lymphocytes express OX-40R
(23).
The augmenting effects of OX-40R mAb in adoptive immunotherapy did not change the requirement for pretreatment of tumor-bearing animals with sublethal WBI. With advanced pulmonary metastases and intracranial tumors, it has frequently been demonstrated that WBI facilitate successful therapeutic efficacy of transferred cells (14, 21, 29). Although the physiological effects of WBI remain to be determined, it has been hypothesized that irradiation may eliminate tumor-induced immunosuppressor T cells that interfere with the reactivity of systematically transferred cells (44). Alternatively, pretreatment with irradiation has been reported to alter the permeability of endothelium cells and thus facilitate trafficking of the transferred T cells (45). Because irradiation alone slightly improved the survival of tumor-bearing animals, it is also possible that exposure of the tumor bed may have created an inflammatory or proapoptotic environment which enhanced antitumor efficacy of transferred cells. It is intriguing that our recent observations indicate that irradiation facilitates proliferation of transferred cells at the tumor site (data not shown).
One of the hallmarks of adoptive immunotherapy is that the transferred
T cells must infiltrate into the tumor mass before an antitumor
response is triggered (29, 32). While systematically
transferred T cells rapidly gain access to tumor metastases in the
lung, the numbers of infiltrating cells in the brain tumor increase
with time. This indicates that the i.v. transferred cells continuously
leave the blood stream and undergo a multistep process including
activation of integrins, adhesion to endothelial ligands, diapedesis,
and a series of undefined subsequent events before their localization
in the brain tumor (46, 47). Thus, the immunoblasts within
the tumor may behave differently from circulating lymphocytes. To
investigate the mechanism of IL-2-induced inhibition, we studied the
trafficking pattern of the transferred T cells. Although IL-2 can cross
the blood-brain barrier and induce vascular leakage in the brain
(48, 49), it can inhibit tumor infiltration by transferred
T cells when used in conjunction with adoptive immunotherapy (see Fig. 6). One possibility is that IL-2 stimulation directly impedes the T
cell’s capacity for transendothelial migration in the brain. However,
it is more likely that the activation state induced by in vivo IL-2
renders T cells incapable of properly migrating and homing to the
tumor. If this is true, delaying IL-2 administration until adequate
intratumoral T cell accumulation has occurred might prove
therapeutically beneficial, particularly for the treatment of tumors
where effector T cell accumulation is initially less brisk, such as
s.c. tumors (18, 29).
The mechanism of OX-40R mAb-mediated enhancement of adoptive T cell immunotherapy is not yet fully characterized. Engaging the OX-40R transmits a potent costimulatory signal that is involved in the specific up-regulation of IL-2 production by Ag-specific T cell lines in vitro (50). Sorting OX-40R positive cells from inflammatory lesions in both autoimmune disease and tumor-draining LN enriches for the Ag-specific T cells while leaving the peripheral repertoire undisturbed, rendering the OX-40R an attractive target to enhance Ag-specific responses (24, 50). To elucidate the significance of OX-40R-OX-40L interactions in long-term T cell immune responses, previous studies demonstrated that in OX-40-deficient mice, IL-2 production and T cell proliferation were diminished (51). Furthermore, OX-40-deficient mice could not generate normal numbers of Ag-specific T cells in the later stage of an immune response, and this led to severely impaired development of immunological memory. To support this notion, an agonist Ab when administered during a primary T cell response promoted a greater number of immune T cells to accumulate and to survive over time as memory cells. In other studies, under tolerizing conditions induced by bacterial superantigen administration, a dramatic increase in T cell survival was observed when LPS combined with OX-40R mAb was given to animals (52). Stimulation through OX-40R on T cells may prevent activation-induced cell death. The therapeutic effects of adoptive immunotherapy under our experimental conditions may not reflect or depend on the extent of immunologic memory, although cured mice did develop a long-term resistance to tumor challenges. Therefore, it is likely that the immediate effects following OX-40R mAb administration are a stimulation of greater proliferation and increased functional activity, while suppressing cell death of the transferred tumor-immune T cells. This is supported by our recent findings demonstrating that an early Ag-specific proliferation of the transferred T cells at the site of tumor was associated with tumor regression (data not shown).
With the use of purified, culture-activated
L-sellow LN T cells, it is possible to cure
virtually all mice of weakly immunogenic, advanced brain tumors
(16, 17, 18). Analysis of OX-40R on LN cells revealed that the
expression of OX-40R was associated with down-regulation of L-selectin,
with the majority of OX-40R-positive cells being
L-sellow. It is intriguing that OX-40R was
detected mostly on CD4 LN T cells at the time of their isolation and at
the time of adoptive immunotherapy (see Fig. 5). These findings raise
an interesting possibility that the L-sellow
OX-40R+ CD4 cells, if purified, could represent a
most potent immune effector cell subpopulation. In fact, we have
already demonstrated that CD4 T cells in the
L-sellow fraction of LN cells alone can mediate
antitumor effects (17).
Systemic IL-2 administration has been associated with significant toxicity in adoptive immunotherapy trials (8). In addition, there are sporadic reports that cancer patients treated with immunotherapy that involved IL-2 developed disease progression in the brain despite tumor regression at other sites (53). These observations have been interpreted as meaning that the CNS may be relatively inaccessible to tumor-immune T cells, and patients with CNS metastases are usually excluded from immunotherapy trials. Results in this paper give a new perspective to the current concept of immunotherapy of primary and metastatic brain tumors and suggest the possibility of enhancing therapeutic effects through modification and activation of costimulatory molecules on tumor-immune T cells.
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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 FF50, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail address: shus{at}ccf.org
3 Abbreviations used in this paper: TIL, tumor-infiltrating lymphocyte; LN, lymph node; L-sel, L-selectin; CM, complete medium; WBI, whole body irradiation; MCA, 3-methylcholanthrene.
Received for publication June 8, 2001. Accepted for publication September 28, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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