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C 2004) Journal of Clinical Immunology, Vol. 24, No. 4, July 2004 (°
Tapasin Decreases Immune Responsiveness to a Model Tumor Antigen HETH R. TURNQUIST,1,2 KARL G. KOHLGRAF,1,2 MARY M. McILHANEY,1 R. LEE MOSLEY,2 MICHAEL A. HOLLINGSWORTH,1,2,3 and JOYCE C. SOLHEIM1,2,3,4
tapasin, TAP, calreticulin, and ERp57 (1). The assembly complex releases the MHC class I molecule once peptide is incorporated into the complex, and the MHC class I molecule proceeds through the golgi and reaches the cell surface by vesicular transport (2–5). Tapasin evidently plays an important role in MHC class I antigen presentation, because tapasin knockout mice express an abnormally low number of surface MHC class I molecules. In addition, MHC molecules that reach the cell surface in tapasin-deficient mice are unstable, and the mice have a diminished T lymphocyte response to viral infection (6, 7). A small number of studies have questioned whether tumors downregulate the expression of tapasin. Melanoma lesions representing different stages of tumor progression were analyzed and it was found that tapasin downregulation was strongly associated with advanced melanoma (8). However, an examination of tapasin expression in tonsillar squamous cell carcinoma did not demonstrate any significant association between tapasin level and the clinical course of the disease (9). In a comparison of three pancreatic tumor cell lines, one line had very low expression of tapasin (10). A separate study analyzed two human pancreatic carcinoma cell lines for tapasin expression, and found very weak tapasin expression in one and moderate expression in the other (11). In this latter study, the inducibility of tapasin expression by γ -interferon in the human pancreatic carcinoma cell lines was also assessed. In one line, tapasin expression remained unchanged after γ -interferon treatment and in the other line tapasin expression barely increased (from very weak to low). As a point of comparison, normal tissues from several different sites were also tested, and all (with the exception of
Accepted: December 22, 2003
The T-cell response against cancer is dependent on the cell surface presentation of tumor-associated or tumor-specific peptides by major histocompatibility complex (MHC) class I molecules. We found that tapasin, a chaperone protein that normally assists in the assembly of MHC class I molecules, is undetectable in an unstimulated pancreatic tumor cell line, Panc02, and only very weakly expressed after γ -interferon stimulation. Transfection of tapasin into the Panc02 cells did not quantitatively increase MHC class I surface expression or detectably affect MHC class I association with peptide and β2 -microglubulin (β2 m). However, we found that transfected tapasin downregulated immune reactivity against a model tumor antigen, MUC1. Although tapasin has been previously shown by others to increase immune recognition of particular antigens, our results suggest that tapasin has a negative impact on the presentation of an immunodominant epitope from a specific model tumor antigen. KEY WORDS: Tapasin; MHC; tumor antigen; antigen presentation; pancreatic cancer.
INTRODUCTION
The presentation of antigenic peptides at the surface of malignant cells by MHC molecules can provoke their recognition and lysis by T lymphocytes. The MHC class I heavy chain, β2 -microglobulin (β2 m), and peptide heterotrimer is formed in the endoplasmic reticulum (ER), where several proteins assist in MHC class I assembly: 1 Eppley Institute for Research in Cancer and Allied Diseases, University
of Nebraska Medical Center, Omaha, Nebraska. of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska. 3 Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska. 4 To whom correspondence should be addressed at Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, Nebraska 681986805; e-mail:
[email protected]. 2 Department
Abbreviations used: TAP, transporter associated with antigen processing; ER, endoplasmic reticulum; DNP, dinitrophenol; CHAPS, 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone.
462 C 2004 Plenum Publishing Corporation 0271-9142/04/0700-0462/0 °
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testis and placenta) exhibited high levels of tapasin even in the absence of γ -interferon stimulation. In our experiments, we determined that tapasin was undetectable in an unstimulated murine pancreatic tumor cell line and that its expression was only slightly upregulated by γ -interferon. Transfection of tapasin into this pancreatic tumor cell line did not perceptibly affect MHC class I association with peptide and β2 m or alter MHC class I surface expression, but it did downregulate the immune recognition of a model tumor antigen expressed in these cells, as shown by both in vitro and in vivo assays. Our observations suggest that tapasin can produce qualitative changes in peptide loading in the absence of quantitative changes in MHC class I assembly and surface expression. Furthermore, in addition to its ability to upregulate the presentation of known epitopes (6, 7), our findings indicate that tapasin can downregulate the presentation of certain immunodominant epitopes.
METHODS
Antibodies Rabbit antisera specific for the N-terminus of tapasin and for TAP were generously contributed by Dr. Ted Hansen (Washington University, St. Louis, MO) (2, 12). The anti-TAP2 hybridoma 435.3 was donated by Dr. Peter Van Endert (Institut National de la Sant´e et de la Recherche M´edicale, Paris, France) (13). Anti-calreticulin serum was purchased from StressGen (Victoria, British Columbia, Canada). The anti-MUC1 antibody HMFG-2, which recognizes the tandem repeat of MUC1, was used for flow cytometry to confirm matched expression of MUC1 in transfectants; it was a gift from Dr. Sandra Gendler (Mayo Clinic, Scottsdale, AZ) (14). Anti-MHC class I antibodies used for flow cytometry and immunoprecipitation were the anti-Kb antibody 06101 (BD-Pharmingen, San Diego, CA) and the anti-Db antibody B22/249 (gift from Dr. Ted Hansen) (15). Antibodies used in the CTL blocking experiment were the anti-Kb antibody Y3 (16), the antiDb antibody B22/249 (15), the anti-Ld antibody 30-5-7 [(17); donated by Dr. Ted Hansen], the anti-CD8 antibody 53-6.72, the anti-CD4 antibody GK1.5, and the antidinitrophenol (DNP) antibody UC8-1B9. The B22/249 antibody has been shown to recognize peptide-occupied and not peptide-free Ld and Lq molecules (18), and presumably also recognizes peptide-occupied forms of Db . The Y3, 53-6.72, GK1.5, and UC8-1B9 antibodies were all obtained from the American Type Culture Collection, Rockville, MD.
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Cell Lines Panc02 is a chemically induced C57BL/6-derived pancreatic tumor cell line (19). The human MUC1 transfectant of Panc02 and the corresponding control transfected with the neo-resistant vector alone have been previously described (20). A tapasin cDNA [(21); a kind gift from Dr. Ping Wang, Queen Mary College, London, England] was cloned into pcDNA3.1(-)Zeo (Invitrogen, Carlsbad, CA) and transfected into Panc02.neo and Panc02.MUC1.neo cells. Clonal tapasin transfectants were selected by limiting dilution, and tapasin expression was confirmed in the clones by Western blotting. Results obtained with one of the tapasin-transfected Panc02.neo clones and one of the tapasin-transfected Panc02.MUC1.neo clones are shown in this report, but similar results were also obtained with separate clones. The tapasin knockout mouse fibroblast cell line, used as a tapasin-negative control, was a gift from Drs. Andres Grandea and Luc Van Kaer (Vanderbilt University, Nashville, TN). The RMA cell line (21) is a lymphoma line used as a positive control for Kb and Db expression. The B16 and EL4 cell lines (from American Type Culture Collection, Manassas, VA) were transfected with the MUC1 cDNA.
Western Blots For Western blots, cells were harvested and lysed by addition of a solution containing 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 20 mM iodoacetamide, and 50 mM 1-chloro-3-tosylamido-7amino-2-heptanone (TLCK). Cellular debris was removed by centrifugation, and supernatant protein concentrations were determined with the Bio-Rad Protein Assay Kit (Hercules, CA). Samples containing 35-µg protein were electrophoresed on 10% acrylamide Tris-glycine gels and transferred to Immobilon-P membranes (Millipore, Bedford, MA). After overnight blocking in a solution of 10% dry milk in 0.05% Tween 20 in PBS, the membranes were incubated in a dilution of antibody for 2 h. The membranes were then washed three times with 0.05% Tween 20/PBS, and incubated for 1 h in a dilution of biotin-conjugated goat antimouse or antirabbit IgG (Caltag Laboratories, San Francisco, CA). Following three 0.05% Tween 20/PBS washes, the membranes were incubated in a dilution of streptavidin-conjugated horseradish peroxidase (Zymed, San Francisco, CA) for 1 h, washed with 0.3% Tween 20/PBS three times, and soaked briefly in enhanced chemiluminescence Western blot developing reagents (Amersham Pharmacia, Piscataway, NJ). The membranes were exposed to Kodak BioMax film.
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Immunoprecipitations
Immunizations and Bulk CTL Generation
For each immunoprecipitation, 1.4 × 107 cells (of matched confluence and high viability) were used. Cells were incubated for 45 min at 37◦ C in a culture medium that lacked methionine and cysteine. A mixture of 35 S-methionine and 35 S-cysteine was added (Easy TagTM EXPRE 35 S35 S Protein Labeling Mix, Perkin Elmer, Boston, MA) and incubation was continued for an additional 60 min. The cells were washed three times in phosphate-buffered saline with 20-mM iodoacetamide (Sigma-Aldrich, St. Louis, MO) and lysed in Tris-buffered saline (pH 7.4) with 1% CHAPS (Sigma, St. Louis, MO), 0.1-mM phenylmethylsulfonyl fluoride, 20-mM iodoacetamide, and excess monoclonal antibody. After 1 h of incubation on ice, the lysates were centrifuged to pellet cell nuclei, and the supernatants were incubated with Protein A-Sepharose beads (Amersham Pharmacia, Piscataway, NJ). The beads were washed with 0.1% CHAPS in Tris-buffered saline (pH 7.4) four times and boiled in 0.125-M Tris (pH 6.8)/2% SDS/12% glycerol/0.02% bromphenol blue to elute the proteins. The eluates were electrophoresed on 4 → 20% SDS-PAGE gels (Invitrogen, Carlsbad, CA). The gels were treated with Amplify (Amersham Pharmacia, Piscataway, NJ), dried, and exposed to BioMax MR film (Eastman Kodak Co., Rochester, NY) at –70◦ C for varied lengths of time.
Groups of C57BL/6 mice were immunized subcutaneously in the left flank with 1 × 106 cells of one of the following types (irradiated with 8000 Rad): Panc02 transfected with the G418- and Zeocin-resistant vectors, i.e. pHβ-Apr1-neo and pcDNA3.1(-)Zeo, respectively (P2.N.Z), Panc02 transfected with the pHβ-Apr1-neo vector and with tapasin in pcDNA3.1(-)Zeo (P2.N.TsnZ), Panc02 transfected with human MUC1 in the pHβApr1-neo vector and pcDNA3.1(-)Zeo (P2.MUC1N.Z), or Panc02 transfected with human MUC1 in the pHβApr1-neo vector and tapasin in the pcDNA3.1(-)Zeo vector (P2.MUC1N.TsnZ). After 24 days, a subset of the mice was sacrificed and spleens were harvested. The splenocytes were restimulated with irradiated cells of the same type used for the immunizations (at 1 × 107 splenocytes/1 × 106 stimulators on day 1 and at 1 × 107 splenocytes/1 × 106 stimulators plus 5% rat Con A supernatant on day 8). On day 15 the splenocytes were tested in a CTL assay as described below.
Flow Cytometry For flow cytometry assays, cells were suspended at 5 × 106 /mL in 0.2% BSA/0.1% sodium azide/PBS. Cells (0.1 mL/aliquot) were distributed into a 96-well U-bottom plate, incubated with excess antibody or with BSA/azide/PBS alone at 4◦ C for 30 min, washed twice, and incubated with phycoerythrin-conjugated, Fc-specific F(ab0 )2 portion of goat antimouse IgG (Jackson ImmunoResearch, Bar Harbor, ME) at 4◦ C for 30 min. The cells were washed twice, resuspended in BSA/azide/PBS, and analyzed with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Cell Quest software (BD Biosciences) was used for statistical analysis.
Generation of a CTL Clone Specific for MUC1 The anti-MUC1 CTL clone was generated by immunizing C57BL/6 mice with B16 (H-2b ) melanoma cells transfected with MUC1. Splenocytes from the immunized mice were re-stimulated with B16.MUC1, and a clone was established that was reactive against B16.MUC1 and EL4.MUC1 (also H-2b ) but not against B16 or EL4.
CTL Assays For CTL assays, targets were labeled with 100 µCi Na2 51 CrO4 for 1.5 h and incubated with effector splenocytes for 6 h (for the experiments with the CTL clone) or 16 h (for the experiments with the bulk CTLs). The % specific lysis = 100 × [(experimental 51 Cr release – control 51 Cr release)/maximum 51 Cr release – control 51 Cr release)], where experimental represents targets mixed with effectors; control represents targets in medium alone (spontaneous release), and maximum represents targets lysed with 1% Triton X-100. Comparisons between levels of % specific lysis for different mouse groups were made using the means comparison for unbalanced data sets (at a P < 0.1) in the JMP 4.0 statistical analysis software package. Orthotopic Tumor Cell Injections A separate subset of the immunized mice was challenged orthotopically at day 25 postimmunization with tumor cells by a method described previously (20). In brief, Panc02.neo or Panc02.MUC1.neo tumor cells were harvested and centrifuged. Pelleted cells were resuspended in DMEM without additives and adjusted to 5 × 106 cells/mL. The mice were anesthetized, their abdomens were surgically opened by an upper medial incision, and the gastric lobe of each pancreas was exposed. A dose of 1 × 105 cells was injected in a volume of 20 µL via a 27G needle. After the injection, the pancreas was returned to the abdominal cavity and the incision was closed. The mice
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were observed for 115 days postchallenge, and the experimental endpoint was defined as the time at which the mice exhibited moribund behavior or a distended abdomen. Mice were sacrificed at the endpoint and the growth of tumors was confirmed by postmortem examination. Differences in survival rate were compared by log-rank statistics, using the GraphPad Prism 2.0 statistical analysis software package.
RESULTS
Analysis of Expression of Tapasin in a Pancreatic Cancer Cell Line The level of tapasin expressed in the Panc02 pancreatic tumor cell line was determined by Western blotting. Tapasin was virtually undetectable in Panc02 cells relative to the murine fibroblast cell line DAP-3 (Fig. 1). After treatment of Panc02 cells with γ -interferon, a small amount of tapasin was detected (Fig. 2), indicating that the Panc02 tapasin gene was functional, but that even with cytokine stimulation the expression of endogenous tapasin was very low. Tapasin Transfection into Panc02 Had Minimal Effect on the Quantity of MHC Class I Molecules at the Cell Surface We assessed the effect of tapasin transfection into Panc02 on the surface expression of MHC class I molecules and on the immune response against Panc02. Panc02.neo and a transfectant of Panc02 that expresses a model tumor antigen, human MUC1, transfected or untransfected with tapasin, were used in these experiments. The surface levels of H-2Kb and H-2Db (the MHC class I molecules naturally expressed by Panc02) were compared on the tapasin-transfected versus untransfected cells by flow cytometry (Fig. 3, top and middle panel). No sig-
Fig. 2. Treatment of Panc02 cells with γ -interferon causes a minimal increase in the expression of tapasin. DAP-3 cells and Panc02.neo (P2.N) cells were untreated (−) or treated with 20 ng/mL γ -interferon for 24 h (+, 24 h) or for 48 h (+, 48 h). Mouse tapasin knockout fibroblast cells were included as a negative control. Cells were lysed and protein was quantified as described in the Materials and Methods. Samples containing equal amounts of protein were electrophoresed on 10% acrylamide Trisglycine gels and transferred to blotting membranes that were probed with antiserum against murine (mu) tapasin.
nificant quantitative change in MHC class I expression resulted from tapasin transfection, as untransfected and tapasin-transfected Panc02.neo and Panc02.MUC1.neo expressed a similar low level of surface MHC class I (Fig. 3 top and middle panels and data not shown). These observations are in contrast to data from analysis of cells from mice expressing tapasin versus no tapasin, which showed that the presence of tapasin increased MHC class I surface expression (6, 7). As expected, transfection of tapasin did not alter MUC1 expression (Fig. 3, bottom panel). To determine the intracellular levels of Db and Kb in Panc02 cells, we performed immunoprecipitation experiments. The inability of tapasin to increase H-2Db surface expression significantly was not due to low intracellular expression of the Db heavy chain or β2 m, because Db and associated β2 m in Panc02.MUC1.neo were readily demonstrable (Fig. 4). Notably, transfection of tapasin into Panc02.MUC1.neo did not increase the amount of β2 m-associated, peptide-occupied (B22/249-precipitable) Db (Fig. 4). In contrast, Kb was virtually undetectable in Panc02.MUC1.neo (data not shown), and we are currently investigating the precise nature of this MHC allele specific defect. Thus Panc02 may have deficiencies in at least two separate components of the MHC class I presentation pathway (i.e., tapasin and Kb ), as has been reported previously for a melanoma cell line (23). Expression of Tapasin Altered Immune Reactivity to the Tumor Cells
Fig. 1. Panc02 cells express virtually no tapasin. The cell types indicated on the figure were lysed and aliquots containing equivalent levels of protein were electrophoresed on 10% acrylamide Tris-glycine gels. DAP-3 and Panc02.neo (P2.N) samples were electrophoresed on one gel, and DAP-3 and Panc02.MUC1.neo (P2.MUC1N) samples on a separate gel. After transfer of the cellular proteins to membranes, the blots were probed separately with antiserum against murine (mu) tapasin.
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As described above, the transfection of tapasin into Panc02.neo and Panc02.MUC1.neo did not increase the number of MHC class I molecules at the cell surface. However, since there is evidence that tapasin expression may affect the presented peptide repertoire (24), we tested the Panc02.MUC1 cells with or without tapasin for their
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Fig. 4. Tapasin expression did not increase the amount of β2 m- and peptide-associated Db in Panc02.MUC1N.TsnZ. Db immunoprecipitates made with antibody B22/249 (which recognizes the peptide-occupied, folded conformation) from lysates of the indicated cell types were electrophoresed on 4 → 20% acrylamide Tris-glycine gels, which were then dried and autoradiographed. The RMA cell line is included as a positive control for Db expression. Arrows indicate bands of the correct molecular weights to be the Db heavy chain (HC) and coprecipitated β2 m.
Fig. 3. Transfection of tapasin did not increase MHC class I surface expression on Panc02.MUC1 cells, nor did it alter MUC1 expression. Histograms displaying flow cytometric data are displayed. The light, solid line at the left on each histogram represents the background staining with secondary antibody alone. The relative mean fluorescence values obtained with the anti-Kb antibody 06101 (top panel), anti-Db antibody B22/249 (middle panel), and anti-MUC1 antibody HMFG-2 (bottom panel) for the Panc02.MUC1.Neo.Zeo cells (dark, solid line) and the Panc02.MUC1.Neo cells transfected with tapasin (dashed line) are indicated. Similar results were obtained with separate transfectants.
ability to present a known model antigen. Specifically, we assessed the recognition of Panc02.MUC1N.TsnZ versus Panc02.MUC1N.Z by a MUC1 specific, H-2Db -restricted CTL clone (Figs. 5 and 6). As shown in Fig. 6, recognition of Panc02.MUC1 cells by the MUC1-specific CTL clone was downregulated by tapasin expression. Thus, the expression of tapasin in these cells depressed the presentation of the undefined antigenic MUC1 peptide recognized by this CTL clone. We also performed a separate experiment in which we generated bulk CTL populations by immunization of C57BL/6 mice with Panc02.neo or Panc02.MUC1.neo cells that expressed or did not express tapasin. The CTLs were re-stimulated once in vitro with the same tumor cell line used for immunization, and tested against Panc02.neo or Panc02.MUC1.neo, each either untransfected or transfected with tapasin (Fig. 7). Consistent with our results with the anti-MUC1 CTL clone (Fig. 6), the bulk CTLs raised against Panc02.MUC1.neo lysed Panc02.MUC1.neo cells more readily when they did not express tapasin (compare E and F on Fig. 7). Likewise, when Panc02.MUC1N.TsnZ was used as the immunogen, Panc02.MUC1.neo was recognized more effectively as a target without tapasin (compare G and H). Expression of tapasin in the MUC1-positive immunizing cells decreased the response against MUC1-positive targets (compare E to G and F to H). Therefore the expression of tapasin in the target or immunizing cells interfered with anti-MUC1 bulk CTL lysis.
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Fig. 5. The CTL clone used in this study was MUC1- and Db -specific and CD8-dependent. The CTL clone was tested against Panc02.MUC1.neo and Panc02.neo in the presence of a variety of blocking antibodies, using a 51 Cr-release assay as described in the Materials and Methods. The legend indicates the specificities of antibodies used for blocking in the assay. (DNP indicates that a negative control antibody specific for dinitrophenol was used.)
In contrast, the expression of tapasin in Panc02.neo cells used as immunogens appeared to result in a minor increase in the reactivity of bulk CTLs against Panc02.neo cells expressing no transfected MUC1 (compare A to C). However, there was no additional increase in CTL recognition when tapasin was present in the target cells as well as in the immunizing cells (compare C to D).
Fig. 6. Recognition of Panc02.MUC1 cells by an anti-MUC1 CTL clone was reduced by tapasin transfection. CTL recognition was assessed by a standard 6-h 51 Cr-release assay as described in the Materials and Methods. The line with triangles (N) indicates Panc02.MUC1.neo.zeo targets, the line with diamonds (¨) indicates Panc02.MUC1.neo targets, the line with X’s (×) indicates Panc02.MUC1.neo.Tsn.zeo, and the line with squares (¥) indicates Panc02.neo targets. Similar results were obtained with a separate tapasin transfectant.
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Thus, the expression of tapasin in either the immunizing or target cells downregulated the anti-MUC1 response. The expression of tapasin in immunizing Panc02.N.TsnZ cells seemed to have a slight positive effect on the response against Panc02.N.TsnZ, although the response was still sufficiently weak as to call into question whether presented endogenous tumor antigens were recognized on these MUC1-negative cells. No significant
Fig. 7. Transfection of tapasin affects immune responsiveness against tumor cells. C57BL/6 mice (groups of 4–5) were immunized subcutaneously with cells, the splenocytes harvested from the immunized mice were restimulated with the same cell type, and bulk splenocyte CTL populations were generated and tested against the immunizing and different target cell types in a 51 Cr-release assay at a 20:1 effector:target ratio (as described in the Materials and Methods). The combinations of immunizing-restimulating cell lines/target cell lines, respectively, used were (A) P2.N.Z/P2.N.Z, (B) P2.N.Z/P2.N.TsnZ, (C) P2.N.TsnZ/ P2.N.Z, (D) P2.N.TsnZ/P2.N.TsnZ, (E) P2.MUC1N.Z/P2.MUC1N.Z, (F) P2.MUC1N.Z/P2.MUC1N.TsnZ, (G) P2.MUC1N.TsnZ/P2.MUC1N.Z, (H) P2.MUC1N.TsnZ/P2.MUC1N.TsnZ. The % specific lysis and statistical comparisons were calculated as described in the Materials and Methods.
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reactivity against Panc02.neo or Panc02.neo expressing tapasin was detected after immunization with Panc02.neo (lysis
