ICAM-1 Contributes to but Is Not Essential for Tumor Antigen Cross-Priming and CD8+ T Cell-Mediated Tumor Rejection In Vivo
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免疫学杂志 2005年第6期
Abstract
ICAM-1 has been described to provide both adhesion and costimulatory functions during T cell activation. In the setting of antitumor immunity, ICAM-1/LFA-1 interactions could be important at the level of T cell priming by APCs in draining lymph nodes as well as for transendothelial migration and tumor cell recognition at the tumor site. To determine the contribution of ICAM-1 to tumor rejection in vivo, we performed adoptive transfer of 2C TCR-transgenic/RAG2–/– T cells into TCR–/– vs ICAM–/–/TCR–/– recipient animals. ICAM-1-deficient mice successfully rejected HTR.C tumors expressing Ld recognized by the 2C TCR, albeit with a kinetic delay. Inasmuch as HTR.C tumor cells themselves express ICAM-1, a second model was pursued using B16-F10 melanoma cells that lack ICAM-1 expression. These cells were transduced to express the SIYRYYGL peptide recognized by the 2C TCR in the context of Kb, which is cross-presented by APCs in H-2b mice in vivo. These tumors also grew more slowly but were eventually rejected by the majority of ICAM-1–/–/TCR–/– recipients. Delayed rejection in ICAM-1–/– mice was associated with diminished T cell priming as assessed by ELISPOT. In contrast, T cell penetration into the tumor was comparable in wild-type and ICAM-1–/– hosts, and adoptively transferred primed effector 2C cells rejected normally in ICAM-1–/– recipients. Our results suggest that ICAM-1 contributes to but is not absolutely required for CD8+ T cell-mediated tumor rejection in vivo and dominantly acts at the level of priming rather than the effector phase of the antitumor immune response.
Introduction
In addition to costimulation through CD28 by B7-1 or B7-2, accessory molecules have been hypothesized to play an important role in T cell adhesion and activation. LFA-1 is a key accessory molecule expressed on T cells that interacts with ICAM-1 on APCs (1). Costimulation via ICAM-1/LFA-1 interactions has been reported to take on a dominant role in the absence of B7/CD28 costimulation (2) by promoting up-regulation of IL-2R expression (3) as well as of the IL-2 gene itself (4, 5). ICAM-1/LFA-1 interactions have also been reported to contribute to differentiation to a Th1-type 1 phenotype accompanied by a decrease in Gata-3 expression and an increase in T-bet (6). ICAM-1 also may be critical for CD8+ T cells (7, 8). Absence of LFA-1 on CD8+ T cells has been reported to result in decreased proliferation and lytic activity (9). Collectively, these data have argued that ICAM-1/LFA-1 interactions could be important in the initial activation and differentiation of tumor Ag-specific CD8+ T cells.
In addition to a role for ICAM-1 in T cell activation by APCs, ICAM-1 also may be involved at the level of inflamed target tissues to support the effector phase of antitumor immune responses. ICAM-1 deficiency on endothelial cells resulted in impaired transendothelial migration of T cells (10, 11), supporting a potential role for penetration into the tumor microenvironment. In addition, ICAM-1 overexpression on tumor cells caused a reduced tumor growth rate in vivo, correlating with increased lysis by tumor-infiltrating lymphocytes (12, 13, 14). Studies using LFA-1-deficient mice indicated an importance for LFA-1/ICAM-1 interactions for rejection of immunogenic tumors but not for clearance of systemic infections (15), suggesting a particular role in antitumor immunity.
Whether ICAM-1/LFA-1 interactions are required for the initial priming of antitumor CD8+ T cells vs the execution of the effector phase of tumor rejection has not been carefully examined in vivo. To pursue this question, we generated TCR/ICAM-1–/– mice as recipients for T cell adoptive transfer and challenged mice with tumors that either expressed or lacked ICAM-1. Our data suggest that, although host expression of ICAM-1 contributes to the development of CD8+ T cell-mediated tumor rejection, it is not absolutely required. In addition, its quantitative contribution can be attributed to the priming phase rather than the effector phase of the antitumor immune response in vivo.
Materials and Methods
Mice
2C/RAG2–/– and TCR–/– mice (H-2b) have been described previously (16, 17). ICAM1–/– mice were described previously (18) and were intercrossed with TCR–/– mice. The animals had been backcrossed at least seven generations. Phenotype was determined for each TCR–/–/ICAM1–/– animal before use by flow cytometric analysis on peripheral blood for absence of T cells and presence of B cells, and by PCR analysis for presence/absence of the ICAM gene (5'- CTG AGC CAG CTG GAG CTC TCG-3' and 5'- GAG CGG CAG AGC AAA AGA AGC-3') and neo gene (5'- GCC CGG TTC TTT TTG TCA AGA CCG A-3' and 5'- ATC CTC GCC GTC GGG CAT GCG CGC C-3'). Animals were maintained in a specific pathogen-free animal facility at the University of Chicago and used in agreement with our Institutional Animal Care and Use Committee according to the National Institutes of Health guidelines for animal use.
Antibodies
Abs against the following molecules coupled to the indicated fluorochromes were purchased from BD Pharmingen: FITC-anti-ICAM-1, biotin-anti-ICAM-1, FITC-anti-B7-1, biotin-anti-B7-1, FITC-anti-B7-2, biotin-anti-Kb, FITC-goat anti-mouse Ig, anti-IFN-, and biotin-anti-IFN-. Streptavidin-conjugated PE was also obtained from BD Pharmingen. The 2C-TCR was stained using the mAb 1B2 (19) which was either FITC or biotin coupled in our laboratory. MHC class I Ld was stained using supernatant of the hybridoma line 30-5-7s and FITC-goat anti-mouse Ig (BD Pharmingen).
Flow cytometry
Flow cytometric analysis was performed as described previously (20) using FACScan (BD Biosciences) flow cytometers and FlowJo software (TreeStar).
T cell purification
Spleens were harvested from 2C/RAG2–/– mice and prepared into single-cell suspensions. CD8+ T cells were purified by the SpinSep-negative selection separation system according to the manufacturer’s instructions (StemCell Technologies). An aliquot of purified cells was routinely stained with 1B2-FITC and PE-anti-CD8 for analysis by flow cytometry.
Tumor cells
Tumor cell lines were cultured in complete DMEM and 10% FCS. The P815.B71 mastocytoma cell line was generated previously and maintained as described in the presence of geneticin (1 mg/ml) (21). HTR.C is a highly transfectable variant of the P815 mastocytoma cell line described previously that grows well as a solid tumor in vivo (22). The B16-F10 spontaneous melanoma cell line was purchased from American Type Culture Collection and transfected with SIYRYYGL as described below. In some experiments, tumor cell lines were cocultured for 48 h with murine IFN- (20 ng/ml; R&D Systems) before analysis.
SIYRYYGL transduction
B16.SIY and B16.C tumor cell lines were obtained by retroviral transfection according to standard protocols of the B16-F10 melanoma cell line with pLEGFP-SIY or empty pLEGFP vectors, provided by Dr. H. Schreiber (University of Chicago, Chicago, IL), as described previously (23). They were maintained in the presence of geneticin (5 mg/ml).
In vivo tumor experiments
Cultured tumor cells (HTR.C or B16.SIY) were washed with PBS and 106 living cells were injected into 100 μl of PBS via a 27-gauge needle on the left flank of the indicated mice. Purified T cells at numbers between 103 and 5 x 106 (as indicated in the figure legends) were transferred i.v. 1 day before by retro-orbital injection. Tumor size was assessed twice per week using calipers, the longest and the shortest diameters were measured, and a mean was calculated. Data of groups of three to five mice were analyzed at each time point, and a mean and SD were determined using Microsoft Excel software. Measurements were continued for 3–4 wk.
ELISPOT
ELISPOT was conducted using the BD murine IL-2 and IFN- kits according to manufacturer’s protocols. Briefly, ELISPOT plates were coated with capture Ab at the prescribed concentration overnight at 4°C. Plates were then washed and blocked with DMEM supplemented with 10% FCS for 2 h at room temperature. Splenocytes (obtained from mice 20 days after T cell transfer and tumor challenge) were plated at 106 cells/well and were stimulated with 40 nM SIY peptide (SIYRYYGL) or PMA and ionomycin as a positive control. Plates were incubated at 37°C overnight, washed, and coated with detection Ab for 2 h at room temperature. Plates were then washed, coated with avidin-peroxidase for 1 h at room temperature, washed again, and developed by addition of 3-amino-9-ethylcarbazole substrate. Developed plates were dried overnight and analyzed with ImmunoSpot software.
Immunohistochemistry
Tumors were removed from mice 6 days after T cell transfer and tumor challenge. The tissue specimens were immediately embedded in OCT compound (Sakura) and frozen in a bath of dry ice and 2-methylbutane (Fisher). Six-micrometer sections were stained with purified 53-6.7 anti-CD8 Ab (BD Pharmingen), followed by secondary anti-rat Ig Ab, streptavidin-alkaline phosphatase conjugate, and enzyme substrate (Vector Laboratories). Sections were counterstained with hematoxylin and mounted. Digital micrographs were captured using a x40 objective on an Axiovert S 100 with AxioCam (Zeiss).
In vitro priming of 2C cells
Purified T cells from 2C/RAG2–/– mice were stimulated in vitro for 4 days with mitomycin C-treated P815.B71 cells as described elsewhere (21). They were then collected, washed, and stimulated an additional 4 days under identical conditions. These primed effector cells showed uniform up-regulation of CD44 and lysed P815 targets in a 4-h chromium release assay (data not shown). A total of 105 primed cells was transferred per mouse for in vivo experiments.
Results
Absence of ICAM-1 in tumor-bearing host delays rejection of an ICAM-1-expressing tumor
To determine whether host ICAM-1 expression was required for CD8+ T cell-mediated tumor rejection, ICAM–/– mice were intercrossed with TCR–/– mice on a C57BL/6 background. These animals served as recipients for adoptive transfer of CD8+ T cells from 2C TCR-transgenic/RAG2–/– mice. 2C T cells recognize the p2Ca peptide presented on Ld and also a synthetic peptide (SIYRYYGL) presented by Kb. Thus, tumors expressing either of these MHC/Ag combinations can be recognized by 2C cells. The DBA/2-derived HTR.C tumor cell line (22) expressed high levels of Ld (Fig. 1A) and of ICAM-1 (Fig. 1B), but lacked expression of the costimulatory molecules B7-1 and B7-2 (Fig. 1, C and D). Using this model in which only the tumor cells would express ICAM-1, significant deficiencies in tumor rejection would indicate an important role for LFA-1/ICAM-1 interactions with host cells, either during initial T cell activation or for trafficking into the tumor microenvironment.
FIGURE 1. Flow cytometric analysis of HTR.C cells. Surface staining for Ld (A), ICAM-1 (B), B7-1 (C), and B7-2 (D) was performed. The respective Ab stainings (solid black) are compared with control Ig staining (black line).
HTR.C tumors grew progressively and at similar rates in both TCR–/– or ICAM–/–/TCR–/– mice without T cell transfer (Fig. 2). Adoptive transfer of naive 2C T cells led to rejection of the tumors in both groups (Fig. 2). However, tumor rejection in ICAM-1-deficient recipients was delayed, suggesting a contributory but nonmandatory role for host ICAM-1 in this process.
FIGURE 2. Absence of host ICAM-1 delays but does not prevent rejection of HTR.C tumors. Each group of four TCR–/– (, ) or TCR–/–/ICAM–/– () mice were challenged s.c. with HTR.C tumor cells (106) in 100 μl of PBS following i.v. adoptive transfer of 5 x 106 naive 2C/RAG2–/– in 100 μl of PBS (, ) or PBS (, ) 24 h before. Mean tumor diameters were determined at the indicated time points. Similar results were seen in two independent experiments.
Lack of ICAM-1 expression by the tumor cells does not eliminate tumor rejection in vivo
To address the impact of ICAM-1 expression on the tumor cells themselves, we screened a panel of tumor cell lines for absence of ICAM-1. We found that the spontaneous melanoma cell line B16-F10 (H-2b) lacked detectable expression of ICAM-1. To make this cell line recognizable by 2C T cells, we transduced B16-F10 with a retroviral construct encoding the Kb-presented peptide SIYRYYGL fused to GFP (designated B16.SIY) (23). This model also has the advantage that tumor Ag would be cross-presented by host APCs in vivo. These transfectants expressed high levels of Ag as assessed by GFP fluorescence (Fig. 3A). In B16-F10 cells, class I MHC expression has been shown to be defective because of down-regulation of Ag-processing machinery, which can be overcome by exposure to IFN- (24). To ensure that even IFN- treatment does not influence ICAM-1 or Ag expression, we analyzed expression of these molecules after IFN- treatment. Although Kb expression was tremendously up-regulated with IFN- exposure, SIY-GFP expression was comparable and ICAM-1 was not detected (Fig. 3). Similarly, the costimulatory ligand B7-1 was never detected (Fig. 3D). Stimulation of primed 2C T cells with IFN--treated B16.SIY cells resulted in low but significant levels of cytokine production, whereas control-transduced B16 cells were completely nonstimulatory (data not shown).
FIGURE 3. Flow cytometric analysis of B16.SIY melanoma cells. Cells were analyzed either without (black histograms) or with (gray histograms) pretreatment with IFN-. SIY-GFP expression (A), Kb (B), ICAM-1 (C), and B7-1 (D) were assessed by flow cytometry. Control Ig staining is shown in the dotted line. Similar results were seen in two experiments.
B16.SIY might be expected to be a difficult tumor to reject in vivo, since it requires exposure to IFN- for high expression of class I MHC and it lacks ICAM-1. However, this was not the case. As shown in Fig. 4, adoptive transfer of just 105 2C T cells was able to control tumor growth in TCR–/–recipients in vivo, although transfer of 103 T cells was modestly less effective. Thus, ICAM-1 expression on the tumor cells is not mandatory for T cell-mediated tumor rejection. However, in ICAM-1-deficient hosts, tumor rejection was quantitatively less effective, with 105 2C cells controlling tumor growth in the majority of mice but 103 2C cells largely failing. The fraction of mice showing tumor rejection after receiving 103, 104, or 105 2C cells from two independent experiments is depicted in Table I and supports a quantitative deficiency of ICAM-1–/– recipients in the rejection of B16.SIY. Thus, ICAM-1 on host cells showed a contribution to antitumor immunity in this model in which the tumor cells lacked ICAM-1 expression.
FIGURE 4. Rejection of B16.SIY tumors in ICAM-1-deficient recipient mice. Each group of four to five TCR–/– (circles) or TCR–/–/ICAM–/– (squares) mice were reconstituted with either 103 or 105 naive 2C/RAG2–/– in 100 μl of PBS (filled symbols) or PBS (open symbols) administered i.v. One day later, B16.SIY tumor cells (106) were injected s.c. in 100 μl of PBS suspension. The mean tumor diameters were assessed at the indicated time points. Similar results were obtained in two independent experiments.
Table I. Proportion of ICAM-1+/+ and ICAM-1–/– mice rejecting B16.SIY tumors in vivoa
Decreased early T cell priming in ICAM-1-deficient recipients
The modest deficiency in tumor rejection by ICAM-1–/– recipients could have been a consequence of decreased priming of 2C T cells and/or by poorly executed effector function at the level of the tumor microenvironment. To examine T cell priming, 2C/RAG2–/– T cells were adoptively transferred into TCR–/– and ICAM-1–/–/TCR–/– mice, and the next day B16.SIY tumor cells were implanted in the flank as before. At day 20, splenocytes were harvested and analyzed for the frequency of SIY-specific cytokine-producing T cells by ELISPOT. As shown in Fig. 5, a lower frequency of T cells producing IFN- and IL-2 was obtained from ICAM-1-deficient recipients. These results argue for a quantitative reduction in the priming of SIY-specific effector cells in the absence of host ICAM-1.
FIGURE 5. ELISPOT analysis from wild-type (WT) and ICAM-1-deficient mice. 2C/RAG2–/– T cells were transferred into TCR–/– () and ICAM-1–/–/TCR–/– () recipients. One day later, B16.SIY tumor cells (106) were injected s.c. in 100 μl of PBS suspension. On day 20, spleens were harvested and ELISPOT was performed for IL-2 (A) and IFN- (B). A control stimulation with culture medium alone is shown. Similar results were seen in two independent experiments.
Intact effector phase in ICAM-1-deficient recipients
It was conceivable that the effector phase of the antitumor immune response also was blunted in the absence of ICAM-1. We first examined whether CD8+ T cells effectively trafficked into the tumor microenvironment, using the same experimental setup as described previously. In fact, a low frequency of CD8+ cells was found by immunohistochemistry in tumors obtained from both TCR–/– and ICAM-1–/–/TCR–/– mice (Fig. 6). These results suggested that, once primed T cells were generated, they were capable of gaining access to the target tissue.
FIGURE 6. Presence of CD8+ T cells in tumors of wild-type and ICAM-1-deficient recipients. 2C/RAG2–/– T cells were transferred into TCR–/– (upper panels) and ICAM-1–/–/TCR–/– (lower panels) recipients. One day later, B16.SIY tumor cells (106) were injected s.c. and on day 6 the tumors were removed and analyzed by immunohistochemistry for the presence of CD8-expressing cells.
To bypass the requirement for in vivo priming and focus directly on the effector phase of tumor rejection, 2C/RAG2–/– T cells were activated in vitro to differentiate into lytic effector cells before adoptive transfer into TCR–/– and ICAM-1–/–/TCR–/– recipients. As shown in Fig. 7, both types of recipient mice controlled the growth of B16.SIY tumors comparably when primed 2C T cells were used. Thus, absence of ICAM-1 did not appear to adversely affect the effector phase of tumor rejection in vivo.
FIGURE 7. Primed 2C effector cells control tumor growth comparably in wild-type and ICAM-1-deficient recipients. 2C/RAG2–/– T cells were primed to effector cells in vitro as described in Materials and Methods. They were then adoptively transferred (105 cells) into TCR–/– (?, ) or TCR–/–/ICAM–/– (, ) mice. One day later, B16.SIY tumor cells (106) were injected s.c. in 100 μl of PBS suspension. The mean tumor diameters were assessed at the indicated time points.
Discussion
ICAM-1/LFA-1 interactions have been postulated to play crucial roles in enhancing antitumor immune responses (14, 15, 25). Several possible functions for ICAM-1 in this setting have been proposed: 1) costimulation during T cell activation (2), 2) facilitation of transendothelial migration of effector cells (10), and 3) enhancement of lymphocyte adhesion during interaction with tumor cells (12). Our data support a contribution of ICAM-1 expression on host non-T cells in antitumor immunity in vivo, whereas tumor rejection could clearly occur without ICAM-1 expression on the tumor cells. Nonetheless, ICAM-1 expression even on host cells was not absolutely required, since tumor rejection could occur if sufficient numbers of T cells were transferred.
Which host cells must express ICAM-1 to contribute to tumor rejection in vivo is not entirely clear, although it is most likely to be an APC. Priming for T cell effector function was modestly reduced in the absence of host ICAM-1. These data are supported by a previous report suggesting a role of ICAM-1 in the development of type 1 immune responses (6). Since that study showed that this deficiency could be overcome by exogenous cytokines, it is likely that the absence of ICAM-1 plays a minor role when dominant Th1/Tc1-promoting factors are present. In contrast, we found no evidence that the effector phase of antitumor immunity was deficient in ICAM-1–/– recipients, as similar numbers of activated T cells were found within the tumor in both types of mice, and transfer of primed effector T cells effectively rejected tumors in both recipients as well. This conclusion is somewhat at odds with previous reports demonstrating that ICAM-1 can mediate transendothelial migration of T cells (10, 11). In addition, ICAM-1-deficient mice have displayed defective migration of inflammatory cells into tissues, such as in the setting of radiation pneumonitis (26). Because the TCR expressed by 2C T cells are of relatively high affinity, it is conceivable that ICAM-1 could play a more critical role at the effector phase when recognition by a lower affinity TCR is involved.
Forced overexpression of ICAM-1 on tumor cells has been shown to result in slower tumor growth rates in vivo (13). This outcome appears to be attributable to increased interaction between effector CTL and the tumor cell targets (12, 14). However, our current results demonstrate that T cell-mediated tumor rejection can occur in the complete absence of ICAM-1 on tumor cells. It is conceivable that other LFA-1 ligands, such as ICAM-2 or ICAM-3, could contribute to this adhesion. Alternatively, other integrins may make a contribution when LFA-1/ICAM-1 interactions are not available. Finally, the 2C T cells in our model could express ICAM-1. Although this would not allow for increased adhesion to tumor cells, it could facilitate homotypic interactions that theoretically could contribute to improved T cell function.
In summary, our data provide evidence that ICAM-1 contributes to, but is not absolutely required for, CD8+ T cell-mediated tumor rejection in vivo. The major contribution of ICAM-1 is at the level of host non-T cells and during the priming phase of the immune response. Examining the role of host ICAM-1 with lower affinity TCR interactions, and also its possible participation in CD8+ T cell homeostasis, may be of interest in future studies.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Janel Washington for assistance with mouse breeding and Marisa Alegre for her careful reading of this manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was funded by National Institutes of Health Grant P01CA97296 and a Burroughs Wellcome Award in Translational Research. C.B. was supported by the Deutsche Akademie der Naturforscher Leopoldina Grant BMBF-LPD 9901/8-35 with funds from the Bundesministerium fuer Bildung und Forschung. A.K.K. was supported by the Medical Scientist National Research Service Award 5 T32 GM07281.
2 Current address: Department of Hematology and Oncology, University of Regensburg, Regensburg, Germany.
3 Address correspondence and reprint requests to Dr. Thomas F. Gajewski, Department of Pathology, University of Chicago, 5841 South Maryland Avenue, MC2115, Chicago, IL 60637. E-mail address: tgajewsk{at}medicine.bsd.uchicago.edu
Received for publication September 15, 2003. Accepted for publication January 6, 2005.
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ICAM-1 has been described to provide both adhesion and costimulatory functions during T cell activation. In the setting of antitumor immunity, ICAM-1/LFA-1 interactions could be important at the level of T cell priming by APCs in draining lymph nodes as well as for transendothelial migration and tumor cell recognition at the tumor site. To determine the contribution of ICAM-1 to tumor rejection in vivo, we performed adoptive transfer of 2C TCR-transgenic/RAG2–/– T cells into TCR–/– vs ICAM–/–/TCR–/– recipient animals. ICAM-1-deficient mice successfully rejected HTR.C tumors expressing Ld recognized by the 2C TCR, albeit with a kinetic delay. Inasmuch as HTR.C tumor cells themselves express ICAM-1, a second model was pursued using B16-F10 melanoma cells that lack ICAM-1 expression. These cells were transduced to express the SIYRYYGL peptide recognized by the 2C TCR in the context of Kb, which is cross-presented by APCs in H-2b mice in vivo. These tumors also grew more slowly but were eventually rejected by the majority of ICAM-1–/–/TCR–/– recipients. Delayed rejection in ICAM-1–/– mice was associated with diminished T cell priming as assessed by ELISPOT. In contrast, T cell penetration into the tumor was comparable in wild-type and ICAM-1–/– hosts, and adoptively transferred primed effector 2C cells rejected normally in ICAM-1–/– recipients. Our results suggest that ICAM-1 contributes to but is not absolutely required for CD8+ T cell-mediated tumor rejection in vivo and dominantly acts at the level of priming rather than the effector phase of the antitumor immune response.
Introduction
In addition to costimulation through CD28 by B7-1 or B7-2, accessory molecules have been hypothesized to play an important role in T cell adhesion and activation. LFA-1 is a key accessory molecule expressed on T cells that interacts with ICAM-1 on APCs (1). Costimulation via ICAM-1/LFA-1 interactions has been reported to take on a dominant role in the absence of B7/CD28 costimulation (2) by promoting up-regulation of IL-2R expression (3) as well as of the IL-2 gene itself (4, 5). ICAM-1/LFA-1 interactions have also been reported to contribute to differentiation to a Th1-type 1 phenotype accompanied by a decrease in Gata-3 expression and an increase in T-bet (6). ICAM-1 also may be critical for CD8+ T cells (7, 8). Absence of LFA-1 on CD8+ T cells has been reported to result in decreased proliferation and lytic activity (9). Collectively, these data have argued that ICAM-1/LFA-1 interactions could be important in the initial activation and differentiation of tumor Ag-specific CD8+ T cells.
In addition to a role for ICAM-1 in T cell activation by APCs, ICAM-1 also may be involved at the level of inflamed target tissues to support the effector phase of antitumor immune responses. ICAM-1 deficiency on endothelial cells resulted in impaired transendothelial migration of T cells (10, 11), supporting a potential role for penetration into the tumor microenvironment. In addition, ICAM-1 overexpression on tumor cells caused a reduced tumor growth rate in vivo, correlating with increased lysis by tumor-infiltrating lymphocytes (12, 13, 14). Studies using LFA-1-deficient mice indicated an importance for LFA-1/ICAM-1 interactions for rejection of immunogenic tumors but not for clearance of systemic infections (15), suggesting a particular role in antitumor immunity.
Whether ICAM-1/LFA-1 interactions are required for the initial priming of antitumor CD8+ T cells vs the execution of the effector phase of tumor rejection has not been carefully examined in vivo. To pursue this question, we generated TCR/ICAM-1–/– mice as recipients for T cell adoptive transfer and challenged mice with tumors that either expressed or lacked ICAM-1. Our data suggest that, although host expression of ICAM-1 contributes to the development of CD8+ T cell-mediated tumor rejection, it is not absolutely required. In addition, its quantitative contribution can be attributed to the priming phase rather than the effector phase of the antitumor immune response in vivo.
Materials and Methods
Mice
2C/RAG2–/– and TCR–/– mice (H-2b) have been described previously (16, 17). ICAM1–/– mice were described previously (18) and were intercrossed with TCR–/– mice. The animals had been backcrossed at least seven generations. Phenotype was determined for each TCR–/–/ICAM1–/– animal before use by flow cytometric analysis on peripheral blood for absence of T cells and presence of B cells, and by PCR analysis for presence/absence of the ICAM gene (5'- CTG AGC CAG CTG GAG CTC TCG-3' and 5'- GAG CGG CAG AGC AAA AGA AGC-3') and neo gene (5'- GCC CGG TTC TTT TTG TCA AGA CCG A-3' and 5'- ATC CTC GCC GTC GGG CAT GCG CGC C-3'). Animals were maintained in a specific pathogen-free animal facility at the University of Chicago and used in agreement with our Institutional Animal Care and Use Committee according to the National Institutes of Health guidelines for animal use.
Antibodies
Abs against the following molecules coupled to the indicated fluorochromes were purchased from BD Pharmingen: FITC-anti-ICAM-1, biotin-anti-ICAM-1, FITC-anti-B7-1, biotin-anti-B7-1, FITC-anti-B7-2, biotin-anti-Kb, FITC-goat anti-mouse Ig, anti-IFN-, and biotin-anti-IFN-. Streptavidin-conjugated PE was also obtained from BD Pharmingen. The 2C-TCR was stained using the mAb 1B2 (19) which was either FITC or biotin coupled in our laboratory. MHC class I Ld was stained using supernatant of the hybridoma line 30-5-7s and FITC-goat anti-mouse Ig (BD Pharmingen).
Flow cytometry
Flow cytometric analysis was performed as described previously (20) using FACScan (BD Biosciences) flow cytometers and FlowJo software (TreeStar).
T cell purification
Spleens were harvested from 2C/RAG2–/– mice and prepared into single-cell suspensions. CD8+ T cells were purified by the SpinSep-negative selection separation system according to the manufacturer’s instructions (StemCell Technologies). An aliquot of purified cells was routinely stained with 1B2-FITC and PE-anti-CD8 for analysis by flow cytometry.
Tumor cells
Tumor cell lines were cultured in complete DMEM and 10% FCS. The P815.B71 mastocytoma cell line was generated previously and maintained as described in the presence of geneticin (1 mg/ml) (21). HTR.C is a highly transfectable variant of the P815 mastocytoma cell line described previously that grows well as a solid tumor in vivo (22). The B16-F10 spontaneous melanoma cell line was purchased from American Type Culture Collection and transfected with SIYRYYGL as described below. In some experiments, tumor cell lines were cocultured for 48 h with murine IFN- (20 ng/ml; R&D Systems) before analysis.
SIYRYYGL transduction
B16.SIY and B16.C tumor cell lines were obtained by retroviral transfection according to standard protocols of the B16-F10 melanoma cell line with pLEGFP-SIY or empty pLEGFP vectors, provided by Dr. H. Schreiber (University of Chicago, Chicago, IL), as described previously (23). They were maintained in the presence of geneticin (5 mg/ml).
In vivo tumor experiments
Cultured tumor cells (HTR.C or B16.SIY) were washed with PBS and 106 living cells were injected into 100 μl of PBS via a 27-gauge needle on the left flank of the indicated mice. Purified T cells at numbers between 103 and 5 x 106 (as indicated in the figure legends) were transferred i.v. 1 day before by retro-orbital injection. Tumor size was assessed twice per week using calipers, the longest and the shortest diameters were measured, and a mean was calculated. Data of groups of three to five mice were analyzed at each time point, and a mean and SD were determined using Microsoft Excel software. Measurements were continued for 3–4 wk.
ELISPOT
ELISPOT was conducted using the BD murine IL-2 and IFN- kits according to manufacturer’s protocols. Briefly, ELISPOT plates were coated with capture Ab at the prescribed concentration overnight at 4°C. Plates were then washed and blocked with DMEM supplemented with 10% FCS for 2 h at room temperature. Splenocytes (obtained from mice 20 days after T cell transfer and tumor challenge) were plated at 106 cells/well and were stimulated with 40 nM SIY peptide (SIYRYYGL) or PMA and ionomycin as a positive control. Plates were incubated at 37°C overnight, washed, and coated with detection Ab for 2 h at room temperature. Plates were then washed, coated with avidin-peroxidase for 1 h at room temperature, washed again, and developed by addition of 3-amino-9-ethylcarbazole substrate. Developed plates were dried overnight and analyzed with ImmunoSpot software.
Immunohistochemistry
Tumors were removed from mice 6 days after T cell transfer and tumor challenge. The tissue specimens were immediately embedded in OCT compound (Sakura) and frozen in a bath of dry ice and 2-methylbutane (Fisher). Six-micrometer sections were stained with purified 53-6.7 anti-CD8 Ab (BD Pharmingen), followed by secondary anti-rat Ig Ab, streptavidin-alkaline phosphatase conjugate, and enzyme substrate (Vector Laboratories). Sections were counterstained with hematoxylin and mounted. Digital micrographs were captured using a x40 objective on an Axiovert S 100 with AxioCam (Zeiss).
In vitro priming of 2C cells
Purified T cells from 2C/RAG2–/– mice were stimulated in vitro for 4 days with mitomycin C-treated P815.B71 cells as described elsewhere (21). They were then collected, washed, and stimulated an additional 4 days under identical conditions. These primed effector cells showed uniform up-regulation of CD44 and lysed P815 targets in a 4-h chromium release assay (data not shown). A total of 105 primed cells was transferred per mouse for in vivo experiments.
Results
Absence of ICAM-1 in tumor-bearing host delays rejection of an ICAM-1-expressing tumor
To determine whether host ICAM-1 expression was required for CD8+ T cell-mediated tumor rejection, ICAM–/– mice were intercrossed with TCR–/– mice on a C57BL/6 background. These animals served as recipients for adoptive transfer of CD8+ T cells from 2C TCR-transgenic/RAG2–/– mice. 2C T cells recognize the p2Ca peptide presented on Ld and also a synthetic peptide (SIYRYYGL) presented by Kb. Thus, tumors expressing either of these MHC/Ag combinations can be recognized by 2C cells. The DBA/2-derived HTR.C tumor cell line (22) expressed high levels of Ld (Fig. 1A) and of ICAM-1 (Fig. 1B), but lacked expression of the costimulatory molecules B7-1 and B7-2 (Fig. 1, C and D). Using this model in which only the tumor cells would express ICAM-1, significant deficiencies in tumor rejection would indicate an important role for LFA-1/ICAM-1 interactions with host cells, either during initial T cell activation or for trafficking into the tumor microenvironment.
FIGURE 1. Flow cytometric analysis of HTR.C cells. Surface staining for Ld (A), ICAM-1 (B), B7-1 (C), and B7-2 (D) was performed. The respective Ab stainings (solid black) are compared with control Ig staining (black line).
HTR.C tumors grew progressively and at similar rates in both TCR–/– or ICAM–/–/TCR–/– mice without T cell transfer (Fig. 2). Adoptive transfer of naive 2C T cells led to rejection of the tumors in both groups (Fig. 2). However, tumor rejection in ICAM-1-deficient recipients was delayed, suggesting a contributory but nonmandatory role for host ICAM-1 in this process.
FIGURE 2. Absence of host ICAM-1 delays but does not prevent rejection of HTR.C tumors. Each group of four TCR–/– (, ) or TCR–/–/ICAM–/– () mice were challenged s.c. with HTR.C tumor cells (106) in 100 μl of PBS following i.v. adoptive transfer of 5 x 106 naive 2C/RAG2–/– in 100 μl of PBS (, ) or PBS (, ) 24 h before. Mean tumor diameters were determined at the indicated time points. Similar results were seen in two independent experiments.
Lack of ICAM-1 expression by the tumor cells does not eliminate tumor rejection in vivo
To address the impact of ICAM-1 expression on the tumor cells themselves, we screened a panel of tumor cell lines for absence of ICAM-1. We found that the spontaneous melanoma cell line B16-F10 (H-2b) lacked detectable expression of ICAM-1. To make this cell line recognizable by 2C T cells, we transduced B16-F10 with a retroviral construct encoding the Kb-presented peptide SIYRYYGL fused to GFP (designated B16.SIY) (23). This model also has the advantage that tumor Ag would be cross-presented by host APCs in vivo. These transfectants expressed high levels of Ag as assessed by GFP fluorescence (Fig. 3A). In B16-F10 cells, class I MHC expression has been shown to be defective because of down-regulation of Ag-processing machinery, which can be overcome by exposure to IFN- (24). To ensure that even IFN- treatment does not influence ICAM-1 or Ag expression, we analyzed expression of these molecules after IFN- treatment. Although Kb expression was tremendously up-regulated with IFN- exposure, SIY-GFP expression was comparable and ICAM-1 was not detected (Fig. 3). Similarly, the costimulatory ligand B7-1 was never detected (Fig. 3D). Stimulation of primed 2C T cells with IFN--treated B16.SIY cells resulted in low but significant levels of cytokine production, whereas control-transduced B16 cells were completely nonstimulatory (data not shown).
FIGURE 3. Flow cytometric analysis of B16.SIY melanoma cells. Cells were analyzed either without (black histograms) or with (gray histograms) pretreatment with IFN-. SIY-GFP expression (A), Kb (B), ICAM-1 (C), and B7-1 (D) were assessed by flow cytometry. Control Ig staining is shown in the dotted line. Similar results were seen in two experiments.
B16.SIY might be expected to be a difficult tumor to reject in vivo, since it requires exposure to IFN- for high expression of class I MHC and it lacks ICAM-1. However, this was not the case. As shown in Fig. 4, adoptive transfer of just 105 2C T cells was able to control tumor growth in TCR–/–recipients in vivo, although transfer of 103 T cells was modestly less effective. Thus, ICAM-1 expression on the tumor cells is not mandatory for T cell-mediated tumor rejection. However, in ICAM-1-deficient hosts, tumor rejection was quantitatively less effective, with 105 2C cells controlling tumor growth in the majority of mice but 103 2C cells largely failing. The fraction of mice showing tumor rejection after receiving 103, 104, or 105 2C cells from two independent experiments is depicted in Table I and supports a quantitative deficiency of ICAM-1–/– recipients in the rejection of B16.SIY. Thus, ICAM-1 on host cells showed a contribution to antitumor immunity in this model in which the tumor cells lacked ICAM-1 expression.
FIGURE 4. Rejection of B16.SIY tumors in ICAM-1-deficient recipient mice. Each group of four to five TCR–/– (circles) or TCR–/–/ICAM–/– (squares) mice were reconstituted with either 103 or 105 naive 2C/RAG2–/– in 100 μl of PBS (filled symbols) or PBS (open symbols) administered i.v. One day later, B16.SIY tumor cells (106) were injected s.c. in 100 μl of PBS suspension. The mean tumor diameters were assessed at the indicated time points. Similar results were obtained in two independent experiments.
Table I. Proportion of ICAM-1+/+ and ICAM-1–/– mice rejecting B16.SIY tumors in vivoa
Decreased early T cell priming in ICAM-1-deficient recipients
The modest deficiency in tumor rejection by ICAM-1–/– recipients could have been a consequence of decreased priming of 2C T cells and/or by poorly executed effector function at the level of the tumor microenvironment. To examine T cell priming, 2C/RAG2–/– T cells were adoptively transferred into TCR–/– and ICAM-1–/–/TCR–/– mice, and the next day B16.SIY tumor cells were implanted in the flank as before. At day 20, splenocytes were harvested and analyzed for the frequency of SIY-specific cytokine-producing T cells by ELISPOT. As shown in Fig. 5, a lower frequency of T cells producing IFN- and IL-2 was obtained from ICAM-1-deficient recipients. These results argue for a quantitative reduction in the priming of SIY-specific effector cells in the absence of host ICAM-1.
FIGURE 5. ELISPOT analysis from wild-type (WT) and ICAM-1-deficient mice. 2C/RAG2–/– T cells were transferred into TCR–/– () and ICAM-1–/–/TCR–/– () recipients. One day later, B16.SIY tumor cells (106) were injected s.c. in 100 μl of PBS suspension. On day 20, spleens were harvested and ELISPOT was performed for IL-2 (A) and IFN- (B). A control stimulation with culture medium alone is shown. Similar results were seen in two independent experiments.
Intact effector phase in ICAM-1-deficient recipients
It was conceivable that the effector phase of the antitumor immune response also was blunted in the absence of ICAM-1. We first examined whether CD8+ T cells effectively trafficked into the tumor microenvironment, using the same experimental setup as described previously. In fact, a low frequency of CD8+ cells was found by immunohistochemistry in tumors obtained from both TCR–/– and ICAM-1–/–/TCR–/– mice (Fig. 6). These results suggested that, once primed T cells were generated, they were capable of gaining access to the target tissue.
FIGURE 6. Presence of CD8+ T cells in tumors of wild-type and ICAM-1-deficient recipients. 2C/RAG2–/– T cells were transferred into TCR–/– (upper panels) and ICAM-1–/–/TCR–/– (lower panels) recipients. One day later, B16.SIY tumor cells (106) were injected s.c. and on day 6 the tumors were removed and analyzed by immunohistochemistry for the presence of CD8-expressing cells.
To bypass the requirement for in vivo priming and focus directly on the effector phase of tumor rejection, 2C/RAG2–/– T cells were activated in vitro to differentiate into lytic effector cells before adoptive transfer into TCR–/– and ICAM-1–/–/TCR–/– recipients. As shown in Fig. 7, both types of recipient mice controlled the growth of B16.SIY tumors comparably when primed 2C T cells were used. Thus, absence of ICAM-1 did not appear to adversely affect the effector phase of tumor rejection in vivo.
FIGURE 7. Primed 2C effector cells control tumor growth comparably in wild-type and ICAM-1-deficient recipients. 2C/RAG2–/– T cells were primed to effector cells in vitro as described in Materials and Methods. They were then adoptively transferred (105 cells) into TCR–/– (?, ) or TCR–/–/ICAM–/– (, ) mice. One day later, B16.SIY tumor cells (106) were injected s.c. in 100 μl of PBS suspension. The mean tumor diameters were assessed at the indicated time points.
Discussion
ICAM-1/LFA-1 interactions have been postulated to play crucial roles in enhancing antitumor immune responses (14, 15, 25). Several possible functions for ICAM-1 in this setting have been proposed: 1) costimulation during T cell activation (2), 2) facilitation of transendothelial migration of effector cells (10), and 3) enhancement of lymphocyte adhesion during interaction with tumor cells (12). Our data support a contribution of ICAM-1 expression on host non-T cells in antitumor immunity in vivo, whereas tumor rejection could clearly occur without ICAM-1 expression on the tumor cells. Nonetheless, ICAM-1 expression even on host cells was not absolutely required, since tumor rejection could occur if sufficient numbers of T cells were transferred.
Which host cells must express ICAM-1 to contribute to tumor rejection in vivo is not entirely clear, although it is most likely to be an APC. Priming for T cell effector function was modestly reduced in the absence of host ICAM-1. These data are supported by a previous report suggesting a role of ICAM-1 in the development of type 1 immune responses (6). Since that study showed that this deficiency could be overcome by exogenous cytokines, it is likely that the absence of ICAM-1 plays a minor role when dominant Th1/Tc1-promoting factors are present. In contrast, we found no evidence that the effector phase of antitumor immunity was deficient in ICAM-1–/– recipients, as similar numbers of activated T cells were found within the tumor in both types of mice, and transfer of primed effector T cells effectively rejected tumors in both recipients as well. This conclusion is somewhat at odds with previous reports demonstrating that ICAM-1 can mediate transendothelial migration of T cells (10, 11). In addition, ICAM-1-deficient mice have displayed defective migration of inflammatory cells into tissues, such as in the setting of radiation pneumonitis (26). Because the TCR expressed by 2C T cells are of relatively high affinity, it is conceivable that ICAM-1 could play a more critical role at the effector phase when recognition by a lower affinity TCR is involved.
Forced overexpression of ICAM-1 on tumor cells has been shown to result in slower tumor growth rates in vivo (13). This outcome appears to be attributable to increased interaction between effector CTL and the tumor cell targets (12, 14). However, our current results demonstrate that T cell-mediated tumor rejection can occur in the complete absence of ICAM-1 on tumor cells. It is conceivable that other LFA-1 ligands, such as ICAM-2 or ICAM-3, could contribute to this adhesion. Alternatively, other integrins may make a contribution when LFA-1/ICAM-1 interactions are not available. Finally, the 2C T cells in our model could express ICAM-1. Although this would not allow for increased adhesion to tumor cells, it could facilitate homotypic interactions that theoretically could contribute to improved T cell function.
In summary, our data provide evidence that ICAM-1 contributes to, but is not absolutely required for, CD8+ T cell-mediated tumor rejection in vivo. The major contribution of ICAM-1 is at the level of host non-T cells and during the priming phase of the immune response. Examining the role of host ICAM-1 with lower affinity TCR interactions, and also its possible participation in CD8+ T cell homeostasis, may be of interest in future studies.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Janel Washington for assistance with mouse breeding and Marisa Alegre for her careful reading of this manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was funded by National Institutes of Health Grant P01CA97296 and a Burroughs Wellcome Award in Translational Research. C.B. was supported by the Deutsche Akademie der Naturforscher Leopoldina Grant BMBF-LPD 9901/8-35 with funds from the Bundesministerium fuer Bildung und Forschung. A.K.K. was supported by the Medical Scientist National Research Service Award 5 T32 GM07281.
2 Current address: Department of Hematology and Oncology, University of Regensburg, Regensburg, Germany.
3 Address correspondence and reprint requests to Dr. Thomas F. Gajewski, Department of Pathology, University of Chicago, 5841 South Maryland Avenue, MC2115, Chicago, IL 60637. E-mail address: tgajewsk{at}medicine.bsd.uchicago.edu
Received for publication September 15, 2003. Accepted for publication January 6, 2005.
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