| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Immunology |
-inducible Carcinoembryonic Antigen (CEA) CD8+ T-Cell Epitope, Which Mediates Tumor Killing in CEA Transgenic Mice
Laboratory of Tumor Immunology and Biology, Center for Cancer Research/National Cancer Institute, NIH, Bethesda, Maryland 20892
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and mediated perforin-dependent tumor cell lysis. However, the CEA peptide-specific T cells killed CEA-expressing murine colorectal tumor cells only after pretreatment of the targets with murine IFN- (muIFN-), and lysis was H-2Db-restricted and involved the Fas-FasL-mediated cytotoxic pathway. When the CEA peptide-specific T cells were used as in vivo effectors in adoptive T-cell transfer studies, muIFN- treatment of the CEA.Tg mice was again required for T-cell-dependent growth suppression of CEA-expressing metastatic tumors. The results indicate that (a) vaccination of mice carrying the human CEA gene with recombinant vaccinia-CEA generates a CEA epitope-specific, CD8-dependent CTL response, (b) CEA, a normal, tissue-specific antigen, can also serve as a target for antitumor immunity after the adoptive transfer of CEA peptide-specific T cells, and (c) muIFN- might be an effective cancer vaccine adjuvant by virtue of its ability to augment the susceptibility of tumor targets to cell-mediated lysis. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Mice expressing human CEA as a transgene have been developed (15 , 16) and used as experimental preclinical models to evaluate both the impact of immune tolerance to CEA as well as the effectiveness of vaccine strategies in overcoming tolerance and mediating tumor rejection (17 , 18) . CEA expression in the CEA.Tg mice used in this study was primarily confined to the major organs of the gastrointestinal tract (15) . In addition, all CEA.Tg mice had measurable serum CEA levels (17) . Peripheral T-cell tolerance to CEA in the CEA.Tg mice was demonstrated by the absence of any immune response to either endogenous CEA or after vaccination with whole CEA protein in adjuvant (17) . However, when CEA was administered to the CEA.Tg mice in recombinant poxvirus-based vaccines, CEA-transfected syngeneic fibroblasts, or oral CEA DNA vaccine, tolerance to this self, tumor-associated antigen was overcome as evidenced by the development of anti-CEA immunoglobulin antibodies, immunoglobulin heavy chain switching, TH1 type CEA-specific CD4+ responses, and CD8-dependent cytotoxicity (17, 18, 19, 20, 21) . The relative strengths of anti-CEA host immune responses correlated with tumor rejection in the vaccinated CEA.Tg mice (17 , 21) .
This study describes a role for IFN- in sensitizing CEA-expressing tumors both in vitro and in vivo for antigen-specific cell-mediated lysis. An H-2Db-restricted CD8+ CTL line specific for an octamer CEA peptide (CEA526–533) was generated from splenocytes of CEA.Tg mice vaccinated with rV-CEA. The low-avidity cytotoxic T cells efficiently lysed peptide-pulsed targets but not CEA-expressing tumor cells. Incubation of CEA-expressing tumor targets in the presence of muIFN- resulted in cytolysis involving the Fas-FasL pathway. Furthermore, treatment of CEA.Tg mice bearing CEA-expressing metastatic tumors with muIFN- allowed for adoptively transferred CEA peptide-specific T cells to suppress tumor growth. The results present a possible role for IFN- in vaccine strategies because of its ability to render tumor targets susceptible to CTL-mediated lysis.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Murine IFNs and Peptides.
Both muIFN-
and muIFN- were purchased from PBL Biomedical Laboratories (New Brunswick, NJ). muIFN- (specific activity = 4.3 x 106 units/mg) was available at a concentration of 107 units/ml in PBS containing 6% glycerol and stored at -70°C. muIFN- (specific activity = 1.02 x 107 units/mg) was available at a concentration of 107 units/ml in ammonium acetate/NaCl containing 6% glycerol and stored at -70°C. Before injection, each IFN was diluted in saline containing 1% normal mouse serum.
The CEA526–533 peptide (EAQNTTYL) was used to stimulate splenocytes and the CEA peptide-specific T cells. The gp10025–33 peptide (EGSRNQDWL) was used as indicated. All peptides were purchased (>95% pure) from Multiple Peptide Systems, Inc. (San Diego, CA), diluted in DMSO to a stock concentration of 10 mg/ml and stored at -80°C. Subsequent dilutions were done using HBSS.
Target Cells.
The CEA-expressing MC-38 cells, designated MC-38-CEA-2 (H-2b), were produced by transducing the human CEA gene using the retroviral expression vector pBNC (22)
. After cloning, >90% of the MC-38-CEA-2 cells expressed CEA as determined by flow cytometry. Both parental MC-38 and MC-38-CEA-2 cell lines were grown in DMEM containing high glucose and 10% heat-inactivated FBS. L929 fibroblasts (H-2k) transfected to express either H-2Kb or H-2Db were kindly provided by Dr. Jon Yewdell (National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD). L929 and EL-4 cells were maintained in RPMI 1640 supplemented with 10% heat-inactivated FBS.
Flow Cytometry.
T cells were recovered from culture by density centrifugation over a Ficoll-Hypaque gradient. They were pelleted by centrifugation (500 x g) and washed in cold Ca2+-Mg2+-free Dulbecco’s PBS and resuspended in Ca2+-Mg2+-free Dulbecco’s PBS at a concentration of 0.5–1.0 x 106 cells/ml. Approximately 106 cells were incubated with 1 µg of FITC-labeled anti-CD4 [clone RM4–5, rat(DA), IgG2a, 
], anti-CD8 [clone 53–6.7, rat (LOU/Ws1/M), IgG2a, ], or appropriate FITC-labeled control antibody (PharMingen, Inc., San Diego, CA) for 1 h at 4°C. MHC class I and CEA expression on L929 and MC-38-CEA-2 tumor cells were analyzed using the following FITC-labeled antibodies: anti-H-2Kb (clone AF6–88.5, BALB/C, IgG2a, ); anti-H-2Db (clone KH95, BALB/C, IgG2a, ); and anti-CEA [COL-1, (Ref. 23
), IgG2a, ]. One µg of the unlabeled 2.2G2 antibody (CD16) to block Fc receptors was added to all samples. After incubation, the cells were washed and analyzed using a Becton-Dickinson FACSCalibur. Data were gathered from 5,000–10,000 cells using a live gate, stored, and later analyzed.
T-Cell Lines.
The CEA peptide-specific CTL line was generated from splenocytes of a CEA.Tg mouse vaccinated twice s.c. with rV-CEA (107 plaque-forming units). Three weeks after the second vaccination, a single suspension of splenocytes was cultured at 20 x 106 cells/T25 flask in 10 ml of growth medium consisting of RPMI 1640 supplemented with 15 mM HEPES (pH 7.4), 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 units/ml glutamine, 100 µg/ml streptomycin, 100 units/ml penicillin (Life Technologies, Inc., Gaithersburg, MD), 10% heat-inactivated FBS (Hyclone Laboratories, Logan UT), 50 µM 2-mercaptoethanol (Sigma, St. Louis, MO), and 10 IU/ml recombinant human interleukin 2 (Proleukin; Chiron Corp., Emeryville, CA). The concentration of the CEA526–533 peptide during the first IVS was 10 µg/ml and during all other subsequent IVSs was reduced to 1 µg/ml. T-cell cultures were stimulated weekly by combining irradiated splenocytes from B6 mice and T cells (25:1 ratio) with the CEA526–533 peptide and interleukin 2. The gp10025–33 peptide-specific T-cell line (24)
, which recognizes a H-2Db-restricted epitope, was a gift from Dr. Kari Irvine (Surgery Branch, National Cancer Institute, Bethesda, MD).
Cytotoxicity Assays.
CTL activity was measured using either 4-h 51Cr- or overnight 111In-release assays, which in a side-by-side comparison yielded similar results. Conditions for the 111In-release assay have been published (17)
. Target cells (2–4 x 106) were also labeled with 250 µCi of Na2[51Cr]O4 (Amersham Corp., Arlington Heights, IL) in serum-free RPMI 1640 for 30 min at 37°C. Targets consisted of cells pulsed with 1 µg/ml for 30 min with either CEA526–533 or appropriate control peptides. Targets cells were also pretreated with either different amounts of muIFN- or muIFN- for 48 h, which were removed before the addition of the targets to the CTL assays. CTL assays were also carried out using muIFN--treated tumor cells or mouse L cells that were preincubated with different blocking antibodies. Fifty µg of either purified anti-H-2Kb [clone AF6–88.5, BALB/C, IgG2b, , PharMingen, Inc.], anti-H-2Db [clone KH95, BALB/C, IgG2b, , PharMingen, Inc.] or appropriate control antibodies were added to media containing the target cells and incubated for 2 h at 4°C. Those antibodies were removed by extensive washing before the CTL assay. T cells were preincubated at 37°C for 2 h with 50 µg of the following antibodies: (a) antimouse FasL (CD95; clone MFL3, Armenian hamster, IgG, group 1, , PharMingen, Inc.); (b) anti-CD4 [clone GK1.5, rat (Lewis) IgG2b, , PharMingen, Inc.]; (c) anti-CD8 [clone 53–6.7, rat (LOU/Ws1/M) IgG2a, , PharMingen, Inc.]; or (d) appropriate isotype control antibodies. Those antibodies were also included throughout the CTL incubation period. All assays consisted of T cells and effectors combined at predetermined E:T ratios or graded peptide concentrations in 96-well U-bottomed Costar plates and carried out at 37°C. The amount of radioactivity released was measured in a gamma counter (Cobra Autogamma; Packard Instruments, Downers Grove, IL). The percentage of specific lysis was calculated as follows: percentage of specific lysis = [(experimental cpm - spontaneous cpm)/(maximal cpm - spontaneous cpm)] x 100.
Maximum release was obtained by adding Triton X-100 (0.25%) to target cells in the absence of effector cells but in the presence or absence of peptides.
Cytokine Production Assays.
Five million irradiated (2000 rad) B6 splenocytes were incubated in 24-well plates in the presence of graded peptide concentrations for 30 min. T cells were added (ratio 10 APC:1 T cell) to a final volume of 2 ml. Cultures were incubated for 48 h at 37°C; the supernatants were harvested and IFN- levels measured using an IFN- ELISA assay (Endogen, Inc., Cambridge, MA).
T-Cell Proliferation Assay.
The CEA peptide-specific CTL line was cultured in flat-bottomed 96-well plates at a cell density of 2 x 104 cells/well together with 5 x 105 irradiated splenocytes from naive, syngeneic B6 mice (serving as APCs) and graded peptide concentrations of medium at 37°C. After 48 h, the cells were pulsed with [3H]thymidine (1 µCi/well; Amersham Corp.) and harvested 24 h later, and the incorporated radioactivity was measured by liquid scintillation spectroscopy (Wallac, Inc., Gaithersburg, MD).
TCR Vß Chain Analysis.
Expression of specific V and Vß chains of the TCR was assessed by reverse transcription-PCR and flow cytometry, respectively. For V chain determination, 
50 million CEA peptide-specific T cells were purified by Ficoll-Hypaque gradient density centrifugation and total RNA isolated with the Triazol reagent (Life Technologies, Inc.). cDNA synthesis was performed according to the manufacturers’ guidelines using oligodeoxythymidylate primers (Perkin-Elmer/Cetus Corp., Emeryville, CA). Approximately 20 ng of total RNA were included in each 20-µl reaction tube. Complementary DNA synthesis was performed at 42°C for 15 min, followed by heat denaturation of the enzyme at 95°C for 15 min. Identification of individual V TCR chains was performed in a subsequent PCR using oligodeoxyribonucleotides derived from target sequences from specific mouse V genes paired with V common primers. Amplification of target sequences by Taq DNA polymerase in 25 cycles of thermal cycling was achieved by 94°C for 20 s, 55°C for 20 s, and 72°C for 10 s. All target sequences were between 200 and 600 bp. PCRs were performed in a Perkin-Elmer/Cetus thermal cycler 9600. All reaction products were analyzed by 4% agarose gel electrophoresis and those positive were identified by visual inspection under an UV light source after ethidium bromide staining.
For Vß determination, the CEA peptide-specific T cells were harvested, and 106 cells were incubated in the presence of 1 µg of each of a screening panel of FITC-conjugated mouse Vß TCR MAbs consisting of Vßs 2, 3, 4, 5.1/5.2, 6, 7, 8.1/8.2, 8.3, 9, 10b, 11, 12, 13, 14, and 17a (PharMingen, Inc.). Samples, which were incubated for 1 h at 4°C, also contained 1 µg of the 2.2G2 antibody (CD16) to block Fc receptors. After incubation, the T cells were washed twice and immediately analyzed by flow cytometry.
In Vivo Adoptive Immunotherapy Studies.
A metastases model was used to determine whether the CEA peptide-specific CTL line was capable of eliciting antitumor effects in vivo. Briefly, female CEA.Tg mice were anesthetized by i.p. injection of 0.25 ml of a 2.5% solution of avertin. The skin was prepared for surgery with iodine, and a left subcostal incision was made to expose the spleen. MC-38-CEA-2 (5 x 104) cells in 100 µl of HBSS were injected beneath the splenic capsule. Five min later, the splenic pedicle was ligated with a 5.0 silk suture, and the incision was closed with staples. Tumor inoculation was done on day 0, and mice received 20 million CEA or gp100 peptide-specific T cells i.p. on days 3 and 7. Control mice received an equal volume (0.2 ml) of HBSS. Groups of mice also received daily i.p. injections of 5 x 105 IU of muIFN- for 5 consecutive days beginning on day 3. Untreated mice developed metastatic CEA-expressing tumors that were evident by abdominal distension, after which they quickly became moribund and were sacrificed 8–10 weeks after tumor inoculation.
Statistics.
Statistical significance of the T-cell functional data (i.e., proliferation/lysis, etc.) was based on Student’s two-tailed t test. Survival analysis was done using Kaplan-Meier (25)
, and the differences were assessed using the log-rank test (26)
. All Ps are two-sided and not adjusted for the multiplicity of evaluation performed on the data. P < 0.05 was considered significant.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
25% peptide-specific lysis of EL-4 targets was observed at a 40:1 E:T (data not shown). The T cells were maintained by weekly IVS in the presence of 1 µg of CEA526–533 peptide/ml, and after 4 additional weeks, the CEA peptide-specific CTL line was >95% CD8+ cells expressing the V9, V4 and Vß5.1, and 5.2 T-cell receptor subunits. The CEA526–533 peptide supported some transient in vitro growth (2 IVSs) of T cells from nonimmunized mice, but those cells did not produce IFN- or mediate cytolysis of either CEA-expressing MC-38-CEA-2 tumor cells or a variety of cell targets (i.e., EL-4, MC-38) pulsed with the CEA526–533 peptide (data not shown).
In Vitro Characteristics of the CEA Peptide-specific CTL Line.
Fig. 1
summarizes the ability of the CEA peptide CTL line to recognize the CEA526–533 peptide. The gp10025–33 peptide was the negative control. The proliferative response, as measured by [3H]thymidine uptake, of the CEA peptide-specific CTL line after incubation with irradiated APCs pulsed with different amounts of CEA526–533 peptide was dose dependent with incorporation of [3H]thymidine occurring with the addition of 10–10,000 ng of CEA526–533/ml (Fig. 1A)
. Increasing the amount of CEA526–533 peptide resulted in IFN- production by the CEA peptide-specific CTL line (half-maximal production at 300 ng/ml; Fig. 1B
). No measurable amount of muIFN- was produced when the CEA peptide-specific T cells were incubated in the presence of the gp10025–33 peptide. To assess CEA peptide-specific cytolytic activity, the T cells were incubated in the presence of EL-4 cells (E:T, 5:1) pulsed with 5 x 103-10-1 ng/ml of either CEA or gp100 peptides. A dose-dependent cytolytic response resulted with half-maximal lysis occurring with the addition of 20–25 ng of the CEA526–533 peptide (0.25 µM) to the EL-4 targets (Fig. 1C)
, a peptide amount generally associated with low-avidity cytotoxic T cells (27)
. No measurable lysis was observed when the EL-4 cells were pulsed with the gp100 peptide.
|
, 
). MC-38-CEA-2 cytolysis was difficult to demonstrate, even when CTL assays used overnight incubations and E:T ratios of 200:1. Poor presentation of the CEA526–533 epitope by the tumor cells was hypothesized as one possible explanation. Treatment of the parental MC-38 and the MC-38-CEA-2 tumor cells with either muIFN- or muIFN- increased MHC class I and Fas expression levels but not MHC class II or CEA expression levels (Table 1)
. MC-38-CEA-2 cells were treated with doses of muIFN- or muIFN- to elicit equivalent increases in MHC class I (1000 IU/ml, muIFN-; 50 IU/ml, muIFN-) or maximal Fas expression levels (1000 IU/ml of either muIFN- or muIFN-). In an overnight 111In-release assay, muIFN- treatment resulted in the appearance of significant (P < 0.05) MC-38-CEA-2 cell lysis by the CEA peptide-specific CTL line (Fig. 2A)
. No such change in target cell lysis was found after muIFN- treatment (Fig. 2A)
, despite an increased expression of MHC class I and Fas (Table 1)
. Susceptibility of the muIFN--treated MC-38-CEA-2 cells was shown with the exogenous addition of 1 µg of CEA526–533 peptide (Fig. 2A)
. Treatment of the CEA-negative MC-38 cells with either muIFN- or muIFN- did not induce any significant lysis by the CEA peptide-specific CTL line (Fig. 2B)
. MC-38 cytolysis was easily achieved with the exogenous addition of 1 µg of CEA526–533 peptide (Fig. 2B)
.
|
|
illustrates the time course for the lysis of muIFN--treated MC-38-CEA-2 cells by the CEA peptide-specific CTL line. Untreated and muIFN--treated MC-38-CEA-2 cells were incubated in the presence of the CEA peptide-specific T cells (E:T, 50:1) for 4, 8, 16, and 24 h. As reported earlier, lysis of untreated MC-38-CEA-2 cells was unremarkable, <10% throughout the entire time period. In contrast, lysis of the muIFN--treated MC-38-CEA-2 tumor cells was observed after 8 h and steadily increased to 50% by 24 h. In a 4-h 51Cr-release assay, 60% lysis was observed for MC-38-CEA-2 cells pulsed with 1 µg of CEA526–533 peptide. By 8 h, lysis of the CEA-peptide pulsed targets was complete (Fig. 3)
.
|
-treated MC-38-CEA-2 Cells by the CEA Peptide-specific CD8+ CTL Line Was H-2Db-restricted and CD8 Dependent.-treated MC-38-CEA-2 tumor cells by >70% (Fig. 4A)
. The addition of an anti-H-2Kb antibody had no such effect. The addition of an antibody reactive with the CD8 molecule abolished lytic activity, which agrees with previous fluorescence-activated cell sorting data showing a CD8+ T-cell population. As expected, the addition of a CD4-reactive antibody did not reduce cytolysis. Mouse L cells transfected with Kb and Db provided additional support that recognition of targets pulsed with the CEA526–533 peptide occurred via the Db allele. H-2Db-expressing mouse L cells pulsed with CEA526–533 were lysed by the CEA peptide-specific CD8+ CTL line (Fig. 4B)
, whereas no appreciable lysis was found when H-2Kb+ L cells were pulsed with the same peptide. Lysis of targets pulsed with the gp10025–33 peptide was also H-2Db restricted (Fig. 4B)
.
|
-treated CEA-expressing Targets.
illustrate the temporal differences observed when the CEA peptide-specific T cells lysed muIFN--treated versus CEA peptide-pulsed MC-38-CEA-2 targets. The rapid killing (
4 h) of 60% of the tumor targets pulsed with the CEA526–533 peptide would be consistent with a perforin-based cytolytic pathway (28)
, whereas the involvement of a Fas-FasL pathway would be consistent with the more delayed cytolysis of the muIFN--treated targets (29
, 30)
. CEA peptide-specific T cells were incubated in the presence of CMA (which specifically inhibits perforin-dependent cytolysis; Ref. 31
) or anti-FasL antibody and then added to the cytotoxicity assays along with either untreated (± CEA526–533 peptide; Fig. 5A
) or muIFN--treated (Fig. 5B)
MC-38-CEA-2 targets. CMA treatment significantly reduced the ability of the CEA peptide-specific T cells to lyse untreated as well as muIFN--treated MC-38-CEA-2 targets that were pulsed with the CEA526–533 peptide. In contrast, CMA treatment had little effect on the ability of the CEA peptide-specific T cells to lyse muIFN--treated MC-38-CEA-2 targets. Lysis of muIFN--treated MC-38-CEA-2 targets was reduced by 50% with preincubation of the CEA peptide-specific T cells in the presence of an anti-FasL antibody.
|
or gp100 peptide-specific (Fig. 6B)
T cells on days 3 and 7 after tumor inoculation had no significant impact on overall survival. Combining the adoptive transfer of the CEA peptide-specific T cells with concomitant exogenous muIFN- administration decreased morbidity and improved overall survival (P = 0.002; Fig. 6A
). Treatment of tumor-bearing mice with gp100 peptide-specific T cells combined with muIFN- had no such effect (Fig. 6B)
. In one experiment, splenocytes were isolated 5 days after the second adoptive transfer of CEA peptide-specific T cells, and after a 7-day stimulation in vitro (1 µg CEA peptide/ml), the T cells lysed 35% (E:T, 40:1) of muIFN--treated MC-38-CEA-2 cells in an overnight killing assay. At sacrifice, tumors as well as small intestines/colons were removed from each group and examined. No gross morphological or histological changes were observed in CEA-expressing normal intestinal tract of CEA.Tg mice that received CEA peptide-specific T cells and muIFN- (data not shown).
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. In particular, the amount of exogenous peptide needed for the CEA526–533 to elicit cytolysis was consistent with their characterization as low-avidity T cells (27)
. Several CEA-specific epitopes capable of inducing CTLs in vitro and recognizing human CEA-expressing tumor targets have been reported (2
, 14
, 33
, 34)
. The CEA526–533 peptide is not among those epitopes, which underscores the inherent differences of antigen processing/presentation between CEA.Tg mice and humans and that caution should be exercised in translating results between the two species.
In vitro cytotoxicity studies provided some insights into the lytic mechanisms used by the CEA peptide-specific T cells to kill peptide-pulsed and muIFN--treated MC-38-CEA-2 tumor cells. Cytotoxic lymphocytes kill target cells by two distinct mechanisms, the first method is a granule exocytosis pathway mediated by perforin and granzymes (28
, 29)
. The CEA peptide-specific T cells kill MC-38-CEA-2 target cells exogenously pulsed with the CEA526–533 peptide with relative ease and, in most cases, killing was accomplished within 4–6 h. CMA identifies perforin-mediated cytotoxicity because, as an inhibitor of H+-ATPase, it disrupts the action of perforin by specifically increasing the pH of the lytic granules (30)
. CMA pretreatment of the CEA peptide-specific T cells significantly reduced the lysis of the CEA526–533 peptide-pulsed target, suggesting that in the presence of high CEA526–533 peptide levels presented by the MHC Db allele, the cytotoxicity elicited by the CEA peptide-specific T cells was perforin mediated.
Repeated attempts failed to demonstrate any significant killing of untreated MC-38-CEA-2 target cells by the CEA peptide-specific T cells. The initial hypothesis was that pretreatment of the MC-38-CEA-2 tumor cells with either muIFN- or muIFN-, both capable of up-regulating MHC expression (35)
, might render those targets susceptible to lysis. Indeed, both IFNs significantly enhanced surface class I MHC expression on the MC-38-CEA-2 cells (Table 1)
. CEA expression levels remained unchanged because the retrovirus used to transfect the MC-38-CEA-2 cells with CEA contained the CEA cDNA but no regulatory elements, thus, not permitting IFN up-regulation (22)
. Still, in multiple experiments, lysis of the MC-38-CEA-2 cells by the CEA peptide-specific T cells was observed after pretreatment with muIFN- but not muIFN-, indicating that up-regulation of MHC expression on the tumor cell surface could not account for the preferential killing after muIFN--treatment.
The second mechanism by which cytotoxic lymphocytes kill target cells is by the Fas-FasL pathway (36)
. In this study, even after pretreatment of the MC-38-CEA-2 targets with muIFN-, a 10–16 h incubation was required before measurable cytolysis appeared. Moreover, the up-regulation of Fas expression (Table 1)
on the muIFN--treated MC-38-CEA-2 tumor cells and the inhibition of cytolysis by the anti-FasL antibody (Fig. 5B)
provided some support that lysis of the muIFN--treated MC-38-CEA-2 targets involved the Fas-FasL pathway, which is consistent with activation by a self-peptide bound to the MHC class I molecule (37)
. However, one cannot exclude a contribution from tumor necrosis factor (38)
. Whether single or multiple cytotoxic mechanism(s) is responsible for the in vivo inhibition of tumor growth in CEA.Tg mice remains to be determined. Nonetheless, the requirement for the exogenous muIFN- administration suggests that the same mechanism(s) is involved in tumor cell lysis in vitro and in vivo. We are left to speculate why muIFN-, not muIFN-, pretreatment resulted in cytolysis of the MC-38-CEA-2 tumor cells despite both IFNs up-regulating MHC and Fas expression levels on those targets. Several possible explanations provide avenues for additional study. First, muIFN- treatment consistently induced higher Fas levels on MC-38-CEA-2 tumor cells than did muIFN- treatment, and the level of Fas expression has been implicated in the induction of apoptosis (39)
. Second, muIFN- selectively modulates other cellular changes, particularly those associated with alterations in peptide processing and presentation, i.e., polypeptide glycosylation (Note: CEA526–533 octamer contains a site for N-linked glycosylation at position 4) and selective induction of cytoplasmic immunoproteasomes that alter peptide cleavage site usage (40)
. Changes in antigen processing and/or presentation by muIFN- would also explain the time lag required for muIFN--mediated cytolysis. Additional studies will determine whether such changes lead to the endogenous expression of the CEA526–533 CTL epitope by muIFN--treated MC-38-CEA-2 targets, which results in their recognition and lysis by the CEA peptide-specific T cells. We and others have generated CEA-specific host immunity in CEA.Tg mice using a variety of immunogens without any noticeable untoward effects that might signal autoimmunity (17, 18, 19, 20, 21)
. Although the reasons for this remain speculative, one might argue that T cells would have difficulty in locating endogenous CEA at the luminal border of the gastrointestinal tract, and the autoreactive T cells that have escaped thymic deletion do not cause adverse reactions against normal tissues (41)
.
The present results concur with previous findings (17)
that the relative strength of CEA-specific host immunity generated in rV-CEA immunized CEA.Tg mice was, for the most part, weak. The CEA peptide-specific T cells recognize a Db-restricted CTL epitope generated by vaccinating CEA.Tg mice with rV-CEA but not well expressed on CEA-positive tumor cells. In fact, the CEA526–533 CTLs probably are not involved in antitumor immunity in protocols that do not include muIFN- treatment. Pretreatment of the CEA-expressing tumor cells with muIFN- rendered those targets susceptible to lysis by the CEA peptide-specific T cells. One might be left to argue that high-avidity autoreactive T cells generated by immunizing CEA.Tg mice with rV-CEA would undergo thymic deletion and/or peripheral clonal anergy (31)
and what would remain would be low-avidity cytotoxic T cells similar to those found in this study. In recent studies in which the recombinant poxvirus-CEA vaccines have been reengineered to coexpress costimulatory molecules and combined with cytokines (42)
, those vaccines have generated a more robust anti-CEA host immunity and can mediate regression of established tumors in CEA.Tg mice. Future plans include characterizing the CEA-specific cytotoxic T-cell responses in those CEA.Tg mice that received the therapeutic vaccines as well as additionally evaluating the use of IFN- as a cancer vaccine adjuvant in studies using poxvirus-based cancer vaccines.
|
FOOTNOTES |
|---|
1 To whom requests for reprints should be addressed, at Laboratory of Tumor Immunology and Biology, CCR/NCI, Building 10, Room 8B09, Bethesda, MD 20892. 
2 The abbreviations used are: CEA, carcinoembryonic antigen; CEA.Tg, CEA transgenic; rV-CEA, recombinant vaccinia-CEA; muIFN-, murine IFN ; muIFN-, murine IFN ; FBS, fetal bovine serum; FasL, Fas ligand; TCR, T-cell receptor; IVS, in vitro stimulation; APC, antigen-presenting cell; MAb, monoclonal antibody; CMA, Concanamycin A.
Received 1/18/02. Accepted 7/ 2/02.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
interferon. Cancer Res., 49: 2651-2655, 1989.This article has been cited by other articles:
|
|
 |
|
  K. W. Hance, C. J. Rogers, D. A. Zaharoff, D. Canter, J. Schlom, and J. W. Greiner The Antitumor and Immunoadjuvant Effects of IFN-{alpha} in Combination with Recombinant Poxvirus Vaccines Clin. Cancer Res., April 1, 2009; 15(7): 2387 - 2396. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. K. Wansley, M. Chakraborty, K. W. Hance, M. B. Bernstein, A. L. Boehm, Z. Guo, D. Quick, A. Franzusoff, J. W. Greiner, J. Schlom, et al. Vaccination with a Recombinant Saccharomyces cerevisiae Expressing a Tumor Antigen Breaks Immune Tolerance and Elicits Therapeutic Antitumor Responses Clin. Cancer Res., July 1, 2008; 14(13): 4316 - 4325. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. T. Garnett, J. Schlom, and J. W. Hodge Combination of Docetaxel and Recombinant Vaccine Enhances T-Cell Responses and Antitumor Activity: Effects of Docetaxel on Immune Enhancement Clin. Cancer Res., June 1, 2008; 14(11): 3536 - 3544. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Huang, R. Fayad, A. Smock, A. M. Ullrich, and L. Qiao Induction of Mucosal and Systemic Immune Responses against Human Carcinoembryonic Antigen by an Oral Vaccine Cancer Res., August 1, 2005; 65(15): 6990 - 6999. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Okano, M. Merad, K. Furumoto, and E. G. Engleman In Vivo Manipulation of Dendritic Cells Overcomes Tolerance to Unmodified Tumor-Associated Self Antigens and Induces Potent Antitumor Immunity J. Immunol., March 1, 2005; 174(5): 2645 - 2652. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Chakraborty, S. I. Abrams, C. N. Coleman, K. Camphausen, J. Schlom, and J. W. Hodge External Beam Radiation of Tumors Alters Phenotype of Tumor Cells to Render Them Susceptible to Vaccine-Mediated T-Cell Killing Cancer Res., June 15, 2004; 64(12): 4328 - 4337. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. E. Zeytin, A. C. Patel, C. J. Rogers, D. Canter, S. D. Hursting, J. Schlom, and J. W. Greiner Combination of a Poxvirus-Based Vaccine with a Cyclooxygenase-2 Inhibitor (Celecoxib) Elicits Antitumor Immunity and Long-Term Survival in CEA.Tg/MIN Mice Cancer Res., May 15, 2004; 64(10): 3668 - 3678. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. W. Hodge, D. J. Poole, W. M. Aarts, A. Gomez Yafal, L. Gritz, and J. Schlom Modified Vaccinia Virus Ankara Recombinants Are as Potent as Vaccinia Recombinants in Diversified Prime and Boost Vaccine Regimens to Elicit Therapeutic Antitumor Responses Cancer Res., November 15, 2003; 63(22): 7942 - 7949. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Chakraborty, S. I. Abrams, K. Camphausen, K. Liu, T. Scott, C. N. Coleman, and J. W. Hodge Irradiation of Tumor Cells Up-Regulates Fas and Enhances CTL Lytic Activity and CTL Adoptive Immunotherapy J. Immunol., June 15, 2003; 170(12): 6338 - 6347. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. W. Greiner, H. Zeytin, M. R. Anver, and J. Schlom Vaccine-based Therapy Directed against Carcinoembryonic Antigen Demonstrates Antitumor Activity on Spontaneous Intestinal Tumors in the Absence of Autoimmunity Cancer Res., December 1, 2002; 62(23): 6944 - 6951. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. M. Aarts, J. Schlom, and J. W. Hodge Vector-based Vaccine/Cytokine Combination Therapy to Enhance Induction of Immune Responses to a Self-Antigen and Antitumor Activity Cancer Res., October 15, 2002; 62(20): 5770 - 5777. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
) or gp10025–33 (
) are shown. Assays were carried out as described in "Materials and Methods." Cytolytic activity (C) was tested in an overnight 111In-release assay using peptide-pulsed EL-4 cells as targets (E:T, 5:1). Data are the mean ± SE of triplicate wells from a single experiment that was repeated 2–3 times with similar results.
; +CEA526–533 peptide, 
) MC-38-CEA-2 cells. B, cytolytic activity of the CEA526–533 peptide-specific CTL line against untreated (-CEA526–533 peptide, 
), or anti-CD8 (
