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The Journal of Immunology, 2001, 167: 147-155.
Copyright © 2001 by The American Association of Immunologists

An Immunodominant MHC Class II-Restricted Tumor Antigen Is Conformation Dependent and Binds to the Endoplasmic Reticulum Chaperone, Calreticulin1

Denise Golgher2,*, Firouzeh Korangy{dagger}, Bin Gao*, Kevin Gorski{dagger}, Elizabeth Jaffee{dagger}, Michael Edidin{ddagger}, Drew M. Pardoll{dagger} and Tim Elliott2,3,*

* Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom; {dagger} Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and {ddagger} Department of Biology, Johns Hopkins University, Baltimore, MD 21218


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
There is accumulating evidence that CD4+ T cell responses are important in antitumor immunity. Accordingly, we generated CD4+ T cells against the murine CT26 colon cancer. Three of three independent CT26-specific CD4+ hybridomas were found to recognize the high m.w. precursor of the env gene product gp90. The CD4+ response was completely tumor specific in that the same glycoprotein expressed by other tumors was not recognized by the CT26-specific hybridomas. The recognition of gp90 by the hybridomas was strictly dependent on the conformation of gp90. Different procedures that disrupted the conformation of the glycoprotein, such as disulfide bond reduction and thermal denaturation, completely abrogated recognition of gp90 by all three hybridomas. In CT26 cells, but not in other tumor cells tested, a large proportion of gp90 was retained in the endoplasmic reticulum, mostly bound to the endoplasmic reticulum chaperone, calreticulin. Although calreticulin was not essential for the stimulation of the gp90-specific hybridomas, most of the antigenic form of gp90 was bound to it. The antigenicity of gp90 correlated well with calreticulin binding, reflecting the fact that specificity of binding of calreticulin to its substrate required posttranslational modifications that were also necessary for the generation of this tumor-specific CD4+ epitope.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The majority of tumor Ags recognized by tumor-specific T cells defined to date are MHC class I restricted (1, 2). Although most of the focus of antitumor immunity has been on CD8+ T cells, different studies indicate that an effective immune response depends on the priming of both CD8+ and CD4+ T cells (3, 4, 5).

CD4+ T cells have been shown to have an important role in antitumor immunity, even for MHC class II-negative tumor cells that are unable to directly present Ags to CD4+ T cells (6, 7, 8). In the case of virally induced murine leukemia, adoptively transferred CD4+ T cells combined with cyclophosphamide treatment are capable of mediating complete eradication of tumors in the absence of any contribution by CD8+ T cells (6). In murine melanomas, CD4+ T cells specific for a melanoma-associated Ag can cure mice in which melanoma cells have already metastasized to the lung (7). Vaccinating mice with a tumor-associated viral MHC class II peptide produces long-lasting protective immunity to virally induced tumors (9). In addition to providing help for CTL induction, CD4+ T cells can activate many other effector cells at metastatic sites, including eosinophils and macrophages (8, 10). Despite the appreciation for the role of CD4+ T cells in antitumor immunity, few MHC class II-restricted tumor Ags have been identified (11, 12, 13, 14, 15, 16). As with Ags recognized by CD8+ T cells, the identification and analysis of tumor Ags recognized by CD4+ T cells will help in the dissection of the mechanism by which a protective immune response can be elicited.

The genetic modification of poorly immunogenic tumors with different cytokine genes has been shown to elicit T cell responses in vivo (17, 18, 19, 20). Tumors transduced with GM-CSF can induce a long-lasting and potent immune response (19). In mice vaccinated with the BALB/c-derived CT26 colon carcinoma transduced with the GM-CSF gene, a class I-restricted tumor Ag was identified by a CT26-specific CD8+ cell line (21). This immunodominant epitope is a nonamer derived from the env gene product of the endogenous ecotropic murine leukemia virus (21). The env gene codes for a precursor protein (gp90), which is processed in the Golgi complex into two viral envelope proteins, gp70 and p15E. p15E anchors gp70 to the plasma membrane through noncovalent and/or disulfide bonds (22, 23, 24). gp90/gp70 has eight potential glycosylation sites that can be differentially used depending on the cell in which the glycoprotein is expressed (25, 26). As with other viral envelope glycoproteins, its conformation is very sensitive to differential glycosylation patterns (26, 27, 28).

In this report, we show that the high m.w. precursor gp90, but not the processed gp70, is the Ag recognized by CD4+ T cells from mice vaccinated with CT26-GM-CSF. Surprisingly, although gp90 with an identical sequence is expressed in other tumor lines, it is only recognized by the CD4+ hybridomas when expressed in CT26 cells. The recognition of gp90 is strictly dependent on its conformation. Biochemical procedures that affect conformation of gp90, such as disulfide bond reduction and thermal denaturation, abolish recognition of gp90 by the hybridomas. We found that, in CT26 cells, a large proportion of the gp90 precursor was retained in the endoplasmic reticulum (ER)4 and was mostly bound to the ER chaperone, calreticulin (CRT). CRT is involved in the ER quality control of glycoprotein folding (29, 30). It assists the folding of glycoproteins while the correct disulfide bonds are formed and the correct conformation is achieved. The binding of CRT to its substrate is dependent on the presence of monoglucosylated N-glycans (29, 30), but it also has polypeptide binding capacity (31). Here we demonstrate that the same procedures that affected conformation of gp90 and, as a consequence, affected its antigenicity, also affected its binding to CRT. Although CRT was not necessary for the recognition of gp90 by the hybridomas, it appeared to be a "marker" of its antigenicity in the sense that what was required for the binding of the substrate to this chaperone was also required for the generation of the CT26-specific epitope.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

The CT26 murine colon carcinoma cell line was originated by intrarectal injections of N-nitroso-N-methylurethane into BALB/c mice (32). CT26-GM-CSF was transduced with a retroviral vector coding for the cytokine GM-CSF according to the method of Dranoff et al. (19). B16 F10 melanoma is a spontaneous C57BL/6-derived tumor (33). A20 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA). All these cell lines were cultured in RPMI 1640 supplemented with 10% FBS, penicillin (50 U/ml), streptomycin (50 µg/ml), 1 mM sodium pyruvate, 2 mM L-glutamine, and nonessential amino acids.

Antibodies

Hybridomas for the gp70-specific mAbs rat IgG2a 35/299 and 35/56 (34) were the generous gift of W. Green (Dartmouth Medical School, Dartmouth, NH). Anti-p15E IgG3 mouse hybridoma CRL-1893 372 was purchased from ATCC. The mAbs were purified from culture supernatants using a standard method of affinity purification on protein G-Sepharose (Pharmacia, Piscataway, NJ). Anti-gp70 goat serum (n-80S00024) was purchased from National Cancer Institute/ViroMed Biosafety Laboratories (Camden, NJ). Anti-hemagglutinin (HA) mAb H18 was the generous gift of W. Gerhardt (Winstar, Philadelphia, PA). Other mAbs used in this study, which were purchased from BD PharMingen (San Diego, CA), are as follows: anti-MHC class I 28.14.8, anti-I-Ad 34.5.3, anti-I-Ed 14.4.4, anti-mouse IgG3a isotype-matched control, and anti-rat IgG2a isotype-matched control. For immunoprecipitation of CRT, polyclonal rabbit serum anti-CRT was obtained from Affinity BioReagents (Golden, CO). For immunoprecipitation of calnexin (CX), polyclonal rabbit serum against CX (SPA-860) was obtained from Bioquote (York, U.K.). For Western blots, polyclonal rabbit serum against human tonsillar CRT was a kind gift from B. Sim (University of Oxford, Oxford, U.K.), and polyclonal rabbit serum against CX was made in our laboratory using as immunogen the C-terminal peptide of human CX cross-linked to keyhole limpet hemocyanin (35).

Mice

Female 6- to 8-wk-old BALB/c mice were obtained from National Cancer Institute (Bethesda, MD). Transgenic-HA mice were a generous gift from Dr. H. von Boehmer (Basel Institute for Immunology, Basel, Switzerland) (36) and were bred to a scid/scid genetic background at the animal facility in the Johns Hopkins Oncology Department (Baltimore, MD).

TCR characterization

The primary TCR characterization was done using a panel of anti-murine V{alpha} and V{beta} Abs provided by Dr. D. Woodland (St. Jude Children’s Hospital, Memphis, TN). The TCR chains that could not be identified through Ab staining due to lack of specific Abs were typed by the Woodland laboratory using a panel of V{alpha} and V{beta} oligos for PCR. The TCR was further characterized by the cloning and sequencing of both chains of cDNAs using the Clontech marathon cDNA amplification kit (Clontech, Laboratories, Palo Alto, CA) and TA cloning vectors (Invitrogen, Carlsbad, CA).

Ag-presenting cells

Splenocytes or A20 cells were used as APCs. To prepare splenocytes, BALB/c spleens were made into single-cell suspensions by macerating into 5 ml tissue culture medium (RPMI 1640 supplemented as described above) in a 6-well plate. The cell suspension was filtered through nylon mesh, and low density white blood cells were isolated using Ficoll-Paque (Pharmacia).

CD4+ T cell hybridomas

To create the CT26-specific hybridoma, BALB/c mice were vaccinated with irradiated CT26-GM-CSF (106/hind leg in100 µl HBSS). Seven days after vaccination, the draining lymph nodes were harvested, depleted of CD8+ T cells using MACS columns, and bulk cultures were established by culturing 4 x 106 T cells with 2 x 106 irradiated splenocytes prepulsed for 16 h with tumor lysate (three cycles of freeze and three cycles of thaw). To pulse splenocytes with tumor lysate, 0.5 ml of the lysate (107/ml) was added to splenocytes derived from one whole spleen in 5 ml tissue culture in a 25-cm2 flask and incubated overnight. rIL-2 (20 U/ml) was added 48 h later to the bulk culture. The fusion to BW 5417 (T cell negative) was performed 24 h after addition of IL-2 using polyethylene glycol (Boehringer Mannheim, Indianapolis IN) and cloned in 96-well flat-bottom plates. The selection medium consisted of hypoxanthine-aminopterin-thymidine (Sigma, St. Louis, MO) in IMDM supplemented with 10% FBS and 10% tumor mixture. The tumor mixture, which consisted of 7.5 g dextrose, 75 ml 50x essential amino acids, 140 ml 100x nonessential amino acids, 100 ml sodium pyruvate, 8.5 g sodium bicarbonate, and 34 µl 2-ME, was added 24 h after cell fusion. The resistant clones were adapted to hypoxanthine-thymidine medium and tested for TCR and CD4 expression by FACScan (BD Biosciences, San Jose, CA). The double-positive hybridomas were tested for CT26 tumor recognition in cytokine release assays. The IL-2 content released in the assays was determined by ELISA. The HA hybridoma was derived as described above but using splenocytes from HA-transgenic mice (36) as fusion partners to BW 5417. The transgenic mice were from a scid/scid genetic background.

Cytokine release assay

Cytokine release assays were done in duplicates by incubating 5 x 105 splenocytes (prepulsed for 2–16 h with the Ag to be tested) or 2 x 105 A20 cells (prepulsed for 2–3 h with the Ag to be tested) with 1 x 105 hybridomas in 96-well flat-bottom plates for ~16 h. The supernatant was tested for IL-2 content using ELISA kits (Endogen, Woburn, MA).

Immunoprecipitation

A total of 5 x 107 cells were lysed using 1 ml lysis buffer (0.01 M Tris-0.14 M NaCl containing 1% Triton X-100, 0.2 U/ml aprotinin, 1 mM PMSF, and 5 mM iodoacetamide) for 1 h at 4°C, centrifuged in a microfuge for 15 min at 3,000 rpm and subsequently for 30 min at 13,000 rpm (at 4°C). The supernatant was kept at -70°C when not immediately used. The cell lysate was precleared by incubation for 1 h with isotype-matched Ab and protein G-Sepharose (Pharmacia) at 4°C under constant agitation. The precleared lysate was then incubated for 1 h or more with Ab and protein G-Sepharose at 4°C. The immunoprecipitates were washed three times in 0.01 M Tris-0.14 M NaCl (Tris-saline buffer) and (if the case) treated with the designated enzymes and incubated overnight with APCs. An aliquot of the sample was taken to be analyzed in Western blots. In two assays (Figs. 2Go and 4Go, A), 2 x 107 cells were lysed using 1 ml lysis buffer (0.05 M Tris containing 1% digitonin (Fluka, Buchs, Swizerland), 0.2 U/ml aprotinin, 1 mM PMSF, and 5 mM iodoacetamide) for 1 h at 4°C and centrifuged in a microfuge for 10 min at 13,000 rpm (at 4°C). The cell lysate was then incubated for 1 h with Ab and protein G-Sepharose at 4°C. The immunoprecipitates were washed three times in PBS.



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  		FIGURE 2. A) Anti-gp70 and anti-HA immunoprecipitates specifically stimulate CT26 and HA hybridomas, respectively. IL-2 release by hybridomas 5, P, 27, and HA when incubated with APCs prepulsed with immunoprecipitates from vaccinia HA-infected CT26 tumor lysates (anti-gp70 (35/299), isotype control, and anti-HA). Samples were done in duplicates and did not differ by >5–10%. This experiment was done at least three times. B, Western blot probed with anti-gp90/70 serum. Immunoprecipitates using different mAbs: gp90 plus gp70 (35/299), gp70 (gp90 depletion plus 35/299), and gp90 (p15E), which were probed in a Western blot using anti-gp90/70 serum. C, CT26-specific hybridomas recognize gp90 but not gp70. IL-2 release by hybridomas 5, P, and 27 when incubated with APCs prepulsed with isotype-matched control immunoprecipitate or immunoprecipitates containing gp90 (p15E), gp70 (gp90 depletion plus 35/299), and gp90 plus gp70 (35/299). D, gp90 is degraded faster than gp70. Anti-gp90 (p15E) and anti-gp90/70 (35/299) immunoprecipitates from CT26 lysate incubated for 0, 30, 60, or 240 min at 37°C were probed in a Western blot with anti-gp90/70serum. E, CT26-specific hybridomas recognize gp90 but not gp70. IL-2 release by hybridoma 27 when incubated with APCs prepulsed with CT26 lysate that had been previously incubated for 0, 30, 60, and 240 min at 37°C. IL-2 release by hybridoma 27 when incubated with unpulsed APCs is shown (No lysate). Similar results were obtained for the other two hybridomas, 5 and P.

 


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  		FIGURE 4. A, gp90/70 from CT26 coprecipitates the ER chaperones CX and CRT. gp90/70 (35/299) or isotype-matched control (NS) immunoprecipitates from CT26 and A20 cells were probed in Western blot using anti-CX and anti-CRT serum. Total cell lysate control is shown for both CT26 and A20 cells. (Three different gp90/gp70-specific mAbs were tested for their ability to immunoprecipitate the complexes, p15E, 35/56, and 35/299; only 35/299 could coprecipitate the complexes. This is probably due to steric hindrance of the epitopes recognized by the other Abs p15E and 35/56 in these complexes). B, The CRT-gp90 and CX-gp90 complexes stimulate the CT26-specific hybridomas. IL-2 release when hybridomas 5, P, and 27 are stimulated with autologous APCs prepulsed with immunoprecipitates of rabbit serum control (NS), CRT, CX, or gp90/70 (35/299). Samples were done in duplicates and did not differ by >5–10%. This experiment was done at least three times. C, CX and CRT bind to gp90 but not gp70. Immunoprecipitates of gp90/70 (35/299), CX, CRT, and control rabbit serum (NS) were probed in Western blot using anti-gp90/70 serum.

 
Enzymes

Immunoprecipitates were incubated in Tris-saline buffer (pH 7) for 1 h at 37°C with 2 µl of endoglycosidase H (EndoH) (New England Biolabs, Beverly MA). The immunoprecipitate was added directly to a 15-ml conical tube containing 2 x 106 A20 cells in 1 ml complete RPMI 1640 and incubated for approximately 2 h.

SDS-PAGE

Electrophoresis of protein samples was done under denaturing conditions using 10% bis-acrylamide gels.

Western blots

SDS-PAGE samples were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). The membranes were blocked for 1 h or more in 5% milk in TBST buffer. Anti-gp70 serum (1:2000) or anti-CRT serum (1:200) was added for 1 h in blocking solution at room temperature under constant agitation. The membrane was washed four times (5 min each time) with TBST buffer and incubated for 45 min with secondary Ab conjugated to the enzyme HRP. After washing as described, the membrane was developed using the ECL reagent (Amersham, Arlington Heights, IL).

35S-labeling

Cells were washed and incubated for 40 min in modified RPMI 1640 (cysteine and methionine free) containing 10% dialyzed FCS at 37°C. After centrifugation, the medium was removed, and modified RPMI 1640 containing [L-35S]methionine and [L-35S]cysteine trans-label (Amersham) at a concentration of 100 µCi/107 cells was added. Cells were labeled for 2 h and 30 min and chased in complete RPMI 1640 medium for 1, 2, and 4 h. Cells were lysed in Triton X-100 lysis buffer, and immunoprecipitation was performed as described above.

DTT treatment

DTT treatment of CT26 cells was done according to the method of Braakman et al. (37). Cells were incubated for 50 min with 5 mM DTT in complete RPMI 1640, washed with 20 mM N-ethylmaleimide (Sigma), and lysed for immunoprecipitation as described above.

Vaccinia virus

The HA vaccinia virus has been described elsewhere (38).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of tumor-specific CD4+ T cell hybridomas

Three T cell hybridomas were generated from CD4+ T cells isolated from lymph nodes of BALB/c mice vaccinated with irradiated CT26-GM-CSF. The TCRs of the three hybridomas (5, P, and 27) were identified using PCR cloning and/or Ab staining (VB1.11 VA3.8 for hybridoma 27, VB8.2 VA3.8 for hybridoma 5, and VB6.11 VA3.3 for hybridoma P), indicating that they represented independent clones (data not shown). In a cytokine release assay, IL-2 release was only detected when the hybridomas were incubated with APCs prepulsed with CT26 tumor lysate (Fig. 1GoA). No significant amount of IL-2 release was stimulated by unpulsed APCs or to APCs prepulsed with lysates of other tumors of diverse origination (A20, murine renal cell carcinoma cell line (RENCA) or B16F10). The hybridomas’ response to CT26 lysate was dose dependent; IL-2 release above background levels (hybridoma incubated with APCs) was seen with as little as 1 µl tumor lysate (1 x 104 cell equivalents) (Fig. 1GoB).



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  		FIGURE 1. Hybridomas 5, P, and 27 specifically recognize CT26 tumor lysate. A, IL-2 release when hybridomas 5, P, and 27 are stimulated with autologous APCs prepulsed with 100 µl of CT26, A20, RENCA, and B16F10 lysate (107 cell equivalents/ml). Samples were done in duplicates and did not differ by >5–10%. This experiment was done at least three times. B, Hybridomas 5, P, and 27 recognize CT26 tumor lysate in a dose-dependent manner. IL-2 release when hybridomas 5, P, and 27 are stimulated with autologous APCs prepulsed with 0, 1, 2, 5, 25, 50, and 100 µl of CT26 lysate (107 cell equivalents/ml). Samples were done in duplicates and did not differ by >5–10%. This experiment was done at least three times.

 
CT26-specific CD4+ T cell hybridomas recognize gp90, the high m.w. precursor of the envelope glycoprotein of endogenous murine leukemia virus.

During our attempts to identify the Ag recognized by the CD4+ T cell hybridomas, we assessed their ability to recognize the envelope glycoprotein of an endogenous murine leukemia virus, gp90/70. We had previously shown that mice vaccinated with CT26-GM-CSF mount a strong CD8+ response to an immunodominant epitope derived from this Ag (21), and it seemed reasonable to assume that CD4+ helper epitopes might reside in the same protein. To determine whether the same glycoprotein contained CD4+ epitopes recognized by any of the CT26-specific CD4+ T cell hybridomas, we assayed the ability of anti-gp90/70 immunoprecipitates to activate the hybridomas. As a control for specificity, CT26 cells were infected with vaccinia virus expressing the glycoprotein HA, and both anti-HA and anti-gp90/gp70 immunoprecipitates from the same cell lysate were used to pulse APCs and tested in a cytokine release assay (Fig. 2GoA). All three CT26 hybridomas responded vigorously to gp90/70 pulsed onto autologous APCs. Fig. 2GoA shows that the HA-specific hybridoma exclusively recognized anti-HA immunoprecipitates, and the CT26-specific hybridomas exclusively recognized anti-gp90/70 immunoprecipitates, confirming that the anti-CT26 response was gp90/70-specific. There was no IL-2 release when the APCs were sensitized with five different isotype-matched control Abs, to an Ab specific for an epitope to gp70 expressed only in AKR mice, or when anti-MHC class I immunoprecipitates were used (data not shown). The gp90/70 epitope was recognized in the context of I-Ad because anti-I-Ad Abs, and not anti-I-Ed Abs, blocked the recognition of anti-gp70 by the hybridomas (data not shown). Ag processing of gp90/70 was necessary for the presentation of the I-Ad-restricted epitope, because fixed APCs were not capable of stimulating the CT26-specific CD4+ hybridomas but could still present a HA peptide to the HA-specific hybridoma (data not shown).

To determine whether the CD4+ hybridomas were responding to the high m.w. precursor gp90 or to the mature processed form gp70, we tested immunoprecipitates containing only gp70, only gp90, or both gp70 and gp90 in a cytokine release assay. Fig. 2GoB shows the Western blot analysis using anti-gp90/70 serum of the samples used in the assay. In CT26 cells, mature gp70 is anchored to the surface through noncovalent bonds to p15E and dissociates during the lysis procedure. Thus, "gp90-only" immunoprecipitates, obtained with a mAb specific to p15E, are likely to contain some p15E as well as full-length gp90, although the anti-gp70 serum used on Western blots can only detect gp90. gp90 plus gp70 immunoprecipitates were obtained using a mAb specific for the N-terminal region common to both gp90 and gp70, 35/299, and gp70 was obtained after depletion of cell lysate of gp90 and subsequent immunoprecipitation with 35/299 (Fig. 2GoB). IL-2 release was observed when hybridomas were incubated with APCs prepulsed with immunoprecipitates containing gp90 and gp90 plus gp70 but not with gp70 alone (Fig. 2GoC). The gp90-only immunoprecipitate induced more IL-2 release on the cytokine release assay than the gp90 plus 70 immunoprecipitate, consistent with the greater abundance of gp90 in this immunoprecipitate (Fig. 2GoB). Again, no significant amounts of IL-2 were produced when cells were stimulated with the APC’s given isotype-matched control immunoprecipitate. These results indicated that gp90, the high m.w. precursor of gp70, was the Ag recognized by the CD4+ hybridomas. Our attempts to biochemically purify gp90 from CT26 cells to confirm that gp90 was the Ag recognized by the CT26-specific hybridomas have thus far been unsuccessful due to rapid spontaneous degradation of gp90 during purification. We have taken advantage of the fact that, in a lysate of CT26, proteolytic loss of gp90 occurs more rapidly than loss of gp70. The rate of loss of antigenicity in these lysates correlated well with the loss of gp90, but not gp70 (Fig. 2Go, D and E).

gp90 from other tumors is not recognized by the CT26-specific CD4+ T cells

The finding that gp90 was the Ag recognized by the CD4+ T cell hybridomas specific for CT26 was intriguing because other tumors such as A20, RENCA, B16 (Fig. 1GoA), and the histologically identical BALB/c tumor C26 (data not shown), which were not recognized, nonetheless expressed gp90/70. This differential recognition was unlikely to be due to differences in expression level because B16, which expresses two times more gp70 than CT26, was not recognized even when 100 times more lysate was added to APCs (see Fig. 1GoA). Similarly, although A20 and RENCA can express, respectively, 2- or 10-fold less gp70 than CT26, their lysate was nonstimulatory even when 100 times more lysate was added to APCs. The B16 env gene is known to be polymorphic, which could account for its failure to be recognized, but gp90/70 derived from A20 and RENCA should be identical in sequence to CT26 because both cell lines are BALB/c derived. To confirm this, we sequenced the full-length env cDNA (encoding gp90) from A20 and RENCA. We found three amino acid differences in gp90 from RENCA, but gp90 from A20 was found to be 100% identical with the CT26 env gene (data not shown). In a further set of experiments, we used recombinant vaccinia virus encoding the complete env gene from CT26 (vaccinia gp90) to infect RENCA and B16, and we tested gp90/70 immunoprecipitate for recognition by CD4+ hybridomas. Although vaccinia gp90-infected RENCA increased surface expression levels of gp70 to that seen on CT26 (data not shown), the hybridomas failed to recognize APCs pulsed with immunoprecipitates of gp90/70 from these infected cells (data not shown). We also found that recombinant full-length or truncated gp90 expressed in Escherichia coli was not recognized by the CT26 hybridomas (data not shown). Taken together, these results suggested that the antigenicity of the gp90 glycoprotein depended on the cell in which it was synthesized, perhaps resulting from a cell-specific posttranslational modification.

To try to understand the differences between antigenic gp90 (expressed in CT26) and nonantigenic gp90 (expressed in other tumors), we compared the biosynthesis of gp90 in A20 and CT26 in a pulse-chase experiment. Fig. 3Go shows that processing of gp90 into gp70, which occurs in the Golgi, was much slower in CT26 when compared with A20. This was an indication that more gp90 in CT26 is retained in the ER than in A20, which we have confirmed by immunofluorescence microscopy (data not shown). The retention of membrane glycoproteins in the ER usually occurs when proteins are incompletely folded. For viral glycoproteins, achieving a correct conformation is dependent on formation of appropriate intramolecular disulfide bond formation and glycosylation (39, 40). We hypothesized that slower processing could lead to high steady-state levels of misfolded protein or of folding intermediates in CT26 cells; hence, we might expect to detect more gp90 associated with components of the ER quality control machinery in CT26 than in A20. Among the ER resident chaperones involved in ER quality control are CX and CRT, which have lectin domains that bind to monoglucosylated N-glycans of immature glycoproteins (29). Accordingly, we tested gp90 immunoprecipitates from CT26 and A20 for the presence of coprecipitating CX and CRT. Fig. 4GoA shows that considerably more CX and CRT coprecipitated with gp90/70 from CT26 than from A20, despite the fact that both cell lines expressed roughly the same amount of these chaperones. To determine whether the chaperone-gp90 complexes were antigenic, we tested CRT and CX immunoprecipitates in a cytokine release assay. To determine the composition of the immunoprecipitates, the samples used in the cytokine release assay were probed for gp90/70 in a Western blot. Fig. 4GoB shows that all three hybridomas were stimulated very well by APCs prepulsed with anti-CRT immunoprecipitates. Anti-CX immunoprecipitates could also induce IL-2 release above backgrounds levels. Both CRT and CX coprecipitated gp90 but not gp70 (Fig. 4GoC), consistent with the fact that gp90 is the ER resident precursor of gp70. The amount of gp90 coprecipitated with CX was much less than that coprecipitated with CRT, consistent with the reduced antigenic potency of the former. No gp90 was detected in immunoprecipitates of the two other ER chaperones, Ig heavy chain binding protein or glucose-regulated protein 96, and these did not stimulate any IL-2 release by the hybridomas (data not shown). We have also tested anti-CRT and anti-CX immunoprecipitates from A20 and B16 in a cytokine release assay, and they did not stimulate any IL-2 release above background levels (data not shown). We were able to show that the vast majority (>95%) of gp90 in CT26 was associated with CRT and could be precleared from lysates with anti-gp70 Abs (Fig. 5Go). After preclearing CT26 lysate of gp90 that was associated with CRT or CX, a small amount of gp90 could still be precipitated with anti-p15E mAb (Fig. 5Go). This "free" gp90 was also antigenic, indicating that the association of gp90 to ER chaperones was not essential for its recognition by the hybridomas. The same results were obtained with sequential immunoprecipitation using anti-CRT Abs (data not shown).



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  		FIGURE 3. gp90 takes longer to be processed into gp70 in CT26 cells. SDS-PAGE of gp90/70 immunoprecipitates from A20 or CT26 cells labeled with 35S for 2 h and 30 min and chased for 0, 1, 2, and 4 h.

 


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  		FIGURE 5. gp90 that is not bound to CRT or CX can still stimulate the CT26-specific hybridomas. IL-2 release by hybridomas 5, P, and 27 when incubated with APCs prepulsed with immunoprecipitates from CT26 tumor lysate: Four serial immunoprecipitations with 35/299 (immunoprecipitates gp90 complexed to CRT or to CX) (lanes 1–4) followed by a final immunoprecipitation with p15E (lane 5). NS is the normal rabbit serum control. Samples were done in duplicates and did not differ by >5–10%. Samples used in the cytokine assay were analyzed in a Western blot probed with anti-gp90/70 serum.

 
Altering the conformation of gp90 affects both binding of gp90 to CRT and its recognition by the CT26 hybridomas

Taken together, these results show that the antigenicity of gp90 did not reside in the gp70 moiety of the mature glycoprotein but instead resided in the unprocessed ER resident molecule, gp90, which was mostly bound to CRT. The fact that recombinant gp90 was not recognized by the hybridomas and that gp90 was only stimulatory when expressed in CT26 suggested that the appropriate epitope was dependent on posttranslational processes that may differ between cell types. Given that CRT binds to misfolded or folding intermediates of glycoproteins, we hypothesized that a CT26-specific conformation of a misfolded gp90 was the antigenic form of the glycoprotein recognized by the hybridomas.

To investigate the importance of gp90 conformation on its antigenicity, we evaluated treatments known to interfere with glycoprotein folding for their ability to affect the antigenicity of gp90. First, we treated CT26 cells with DTT for 50 min, which disrupts disulfide bond formation and affects the conformation of most glycoproteins. Fig. 6GoA shows that anti-p15E immunoprecipitated gp90 in both control and DTT-treated cells. SDS-PAGE run in the absence of a reducing agent confirmed that all gp90 in DTT-treated CT26 was reduced in vivo (data not shown). However, anti-CRT immunoprecipitates coprecipitated gp90 in control cells but not in DTT-treated cells, despite the fact that the same amount of calrecticulin could be immunoprecipitated from both (Fig. 6GoA). Therefore, in vivo, DTT treatment led to disruption of the CRT-gp90 interaction. The negative effect of DTT on the binding of glycoproteins to CRT has been reported for other glycoproteins (41) and is probably due to the fact that completely denatured glycoproteins are not good substrates for the enzyme UDP-Glc-glycoprotein-glycosyltransferase (42). This enzyme adds a glucose residue to high mannose glycans, thereby generating the ligand for the lectin site of CRT. Glycosylation of gp90 is not disrupted in DTT-treated cells, as indicated by the equivalent mobility in SDS gels of gp90 from untreated and treated cells (Fig. 6GoA). Fig. 6GoB shows that the gp90-specific hybridomas were not stimulated by anti-p15E or anti-calrecticulin immunoprecipitates from DTT-treated CT26 when these were tested in the cytokine release assay.



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  		FIGURE 6. Disulfide bond reduction through DTT treatment of CT26 cells disrupts the CRT-gp90 complex. A, Immunoprecipitates of normal rabbit serum (NS), anti-CRT, and anti-gp90 (p15E) from CT26 cells not treated (control) or treated with DTT were analyzed in a Western blot probed for anti-gp90/70 (left panel). Immunoprecipitates of normal rabbit serum (NS) and anti-CRT from CT26 cells not treated (control) or treated with DTT were analyzed in a Western blot probed for anti-CRT . B, Disulfide bond reduction of gp90 abrogates its recognition by the CT26-specific hybridomas. IL-2 release when hybridomas 5, P, and 27 are stimulated with autologous APCs prepulsed with immunoprecipitates of rabbit serum control (NS), gp90 (p15E), CRT from CT26 cells, and gp90 (p15E/DTT) and CRT (CRT/DTT) from CT26 cells treated with DTT. Samples were done in duplicates and did not differ by >5–10%. C, Boiling of CRT-gp90 complex causes release of gp90. Anti-CRT immunoprecipitates were analyzed in a Western blot using anti-CRT serum before and after boiling (CRT and CRT Boiled, left panel). Anti-CRT and anti-gp90 (p15E) immunoprecipitates were analyzed in a Western blot using anti-gp90/70 serum before and after boiling (CRT, CRT Boiled; and gp90, gp90 Boiled). D, Thermal denaturation of gp90 abrogates its recognition by the CT26-specific hybridomas. IL-2 release when hybridomas 5, P, and 27 are stimulated with autologous APCs prepulsed with immunoprecipitates of rabbit serum control (NS), gp90 (p15E), CRT controls, and gp90 (gp90 Boiled) and CRT (CRT Boiled) that have been heated at 100°C for 3 min. Samples were done in duplicates and did not differ by >5–10%.

 
A way of changing the conformation of gp90 without disrupting its disulfide bonds or glycosylation is thermal denaturation. We incubated anti-gp90 or anti-CRT immunoprecipitates at 100°C for 3 min before adding them to APCs for use in a cytokine release assay. Fig. 6GoC shows that boiling immunoprecipitates for 3 min did not drastically affect binding of the immunoprecipitating Abs to either CRT or gp90. However, heat treatment caused dissociation of gp90 from anti-CRT coimmunoprecipitates, which could be washed away. The cytokine release assay showed that, although control anti-gp90 and anti-CRT immunoprecipitates are recognized very well by the hybridomas, boiling of immunoprecipitates completely abrogated recognition by the hybridomas (Fig. 6GoD). The same results were obtained regardless of whether immunoprecipitates were washed after boiling and when boiled tumor lysate was pulsed onto APCs (data not shown).

Both of these treatments affected gp90 conformation and CRT binding while preserving the integrity of glycosylation. To determine whether glycosylation of gp90 contributed anything to its antigenicity, we treated anti-gp90 or anti-CRT immunoprecipitates with EndoH before adding them to APCs. This treatment removed all N-glycans (Fig. 7GoA) but is unlikely to affect gp90 conformation. CRT is not glycosylated and is not affected by EndoH (Fig. 7GoA). Fig. 7GoA shows that, consistent with the lectin-like binding properties of CRT, the majority of gp90 was released from anti-CRT immunoprecipitates by this treatment. Some deglycosylated gp90 was still bound to CRT. When these samples were used in a cytokine release assay, they were not washed after EndoH treatment so that the same amount of gp90, N-glycans, and CRT were in the sample as were in the controls. Fig. 7GoB shows that dissociation of N-glycans by EndoH abrogated the recognition of gp90 by hybridomas 5 and 27 and decreased recognition of gp90 by hybridoma P. The same results were obtained with N-glycanase (data not shown). These results indicated that the presence of N-glycans were important for the recognition of gp90 by the hybridomas.



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  		FIGURE 7. gp90 is partially released from CRT through EndoH treatment. A, Anti-CRT immunoprecipitates were treated with EndoH, washed, and probed in a Western blot for anti-gp90/70 (left panel) or anti-CRT (right panel). Immunoprecipitates from normal rabbit serum (NS) and anti-CRT control not treated with EndoH (-EndoH) are shown. B, Removal of N-glycans from gp90 diminishes its recognition by the CT26-specific hybridomas. IL-2 release when hybridomas 5, P, and 27 are stimulated with autologous APCs prepulsed with immunoprecipitate of control rabbit serum (NS) or with anti-CRT immunoprecipitates treated (+EndoH) or not treated (-EndoH) with EndoH. Samples were done in duplicates and did not differ by >5–10%. This experiment was done at least three times.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we show that mice vaccinated with CT26-GM-CSF generated an MHC class II-restricted T cell response to the product of the env gene of an endogenous murine leukemia virus. This glycoprotein, despite being present in other murine tumors, has a tumor-specific epitope that was only recognized by the CD4+ CT26-specific hybridomas. The hybridomas recognized the high m.w. precursor gp90 but not the mature membrane form, gp70. One possible explanation for this differential reactivity is that the epitope for all three hybridomas lies completely or partially in the p15E portion of gp90, which was lost when gp70 is immunoprecipitated from detergent lysates. This has been shown previously to be the case of CD4+ epitopes of insulin, in which most of the epitopes are derived from preproinsulin and proinsulin and are lost in the mature form of insulin (43). We tried to address this issue by testing overlapping synthetic peptides of 20 aa covering the entire length of the gp90 protein. None of the peptides showed significant IL-2 release by any of the hybridomas (data not shown). Our inability to map the epitope with a synthetic peptide would argue in favor of a posttranslational modification being part of the epitope recognized by the TCR. This would corroborate with the fact that our results indicated that the generation of this epitope seems to be associated with posttranslational events specific to the CT26 cell line. The influence of posttranslational modifications such as disulfide bonds, glycosylation, phosphorylation, cysteinylation, and deamidation in the generation of T cell epitopes has been well documented (44, 45, 46, 47, 48, 49, 50, 51). Although this could be the case, other results shown here argue that this cannot be the only cause of antigenicity. The conformation of gp90 was essential for its antigenicity as much as the presence of the N-glycans. Disulfide bond reduction or thermal denaturation completely abolished the recognition of gp90 by the hybridomas. Because none of these procedures affects glycosylation, it indicates that the antigenicity was dependent on a given conformation and on the presence of carbohydrates. One possibility was that the carbohydrate moiety or a deamidated asparagine contributes directly to T cell recognition by being part of the epitope that can only be generated if a given conformation of gp90 was maintained until it achieved the MHC class II-loading compartment. The importance of conformation/oligomerization in the generation of epitopes from conformation-dependent Ags has also been reported (52, 53, 54). It was not possible to synthesize all different glycopeptides corresponding to all glycosylation sites in gp90, but we tested deamidated 20-mers corresponding to the sites. None of the synthetic deamidated peptides elicited a response by the hybridomas (data not shown).

One clue to the tumor-specific antigenicity of gp90 was revealed by pulse-chase analysis of gp90 processing, in which we found an accumulation of gp90 in the ER of CT26. Most of this was associated with CRT. It is not clear why gp90 should accumulate in CT26. Most glycoproteins, after being translocated into the ER, tend to aggregate until the correct disulfide bonds are formed and they are folded correctly (55). For some glycoproteins, the percentage of glycoprotein aggregates and ER retention can depend on the host cell line (56). One possibility is that gp90 expressed in CT26 undergoes different posttranslational modifications that affect its ability to fold and mature rapidly. It is worth noting that the p15E part of the gp90 molecule contains a cryptic glycosylation site, which may be used when the protein is expressed in CT26. gp90 derived from BALB/c mice has a point mutation that slows down its processing into gp70 and p15E (57, 58). Therefore, another possibility is that glycoprotein handling in CT26 differs quantitatively or qualitatively from that in A20, RENCA, or C26. For example, CT26 may have a poor unfolded protein response (59, 60) compared with other cells. Indeed A20 and RENCA express less gp90/70 than CT26, and RENCA tends to down-regulate the amount of gp90/70 expressed with time in culture. This could be in response to extra demands placed on the ER quality control machinery.

Interestingly, in CT26 cells, most of the gp90 recognized by the hybridomas was bound to CRT and a small amount to CX. CRT has been previously implicated in the priming of CD8+ T cells. Small amounts of peptide could elicit a specific CD8+ response in vivo and in vitro only if they were bound to CRT (61, 62). Results shown in Figs. 6Go and 7Go, in which APCs were exposed to anti-CRT immunoprecipitates that had been manipulated to dissociate CRT and gp90, indicate that CRT is unlikely to bind an antigenic peptide recognized by the hybridomas analogous to the situation seen for CD8+ T cells. To confirm this, we biochemically purified CRT from >109 CT26 under conditions that preserved CRT-peptide interaction and showed that this was nonantigenic (data not shown). In our case, the fact that antigenicity of gp90 correlated well with CRT binding was probably due to the fact that binding to CRT was a "marker" of a specific antigenic form of gp90 and only reflected the fact that the specificity of binding of CRT to its substrate is both conformation- and glycosylation-dependent, as was the generation of the CT26-specific epitope. We also found that time-dependent dissociation of gp90 from CRT in a lysate of CT6 correlated well with the loss of antigenicity of anti-CRT immunoprecipitates in these lysates (data not shown). There is the interesting possibility that the role of CRT in the in vivo priming of these T cells was to increase the half-life of gp90, which might otherwise be degraded rapidly. We are currently investigating this possibility.

Our results provide strong evidence that gp90 is a shared tumor Ag whose differential processing is dependent on posttranslational modification, but our inability to map the epitope recognized by the T cell hybridomas leaves some questions regarding the exact specificity of the T cell hybridomas.

There is now a lot known about tumor Ags recognized by CD8+ T cells, but information on tumor Ags recognized by tumor-specific CD4+ T cells is still scarce. This work describes one such response in "vaccinated" mice and indicates that the generation of some MHC class II-restricted epitopes may depend on factors other then the primary sequence of the Ag or processing differences between APCs. These factors could give rise to a tumor-specific response and might indicate that the identification and study of tumor Ags recognized by MHC class II-restricted T cells could be more complex than for their MHC class I-restricted counterparts.


   Acknowledgments
 
We thank Dr. David Woodland for identification of TCRs and Dr. William Green for the generous gifts of hybridomas.


   Footnotes
 
1 This work was supported by National Institutes of Health grants, the Belfer Foundation, the Wellcome Trust, and gifts from Dorothy Needle and the Topercer family. Back

2 Current address: Cancer Sciences Divsion, University of Southampton School of Medicine, Southampton, SO16 6YD, United Kingdom. Back

3 Address correspondence and reprint requests to Dr. Tim Elliott at the current address: Cancer Sciences Divsion, University of Southampton School of Medicine, Level F (824) Centre Block, Southampton General Hospital, Tremona Road, Southampton, SO16 6YD, U.K. E-mail address: tje{at}soton.ac.uk, or Dr. Drew M. Pardoll, E-mail address: dmpardol{at}jhmi.edu Back

4 Abbreviations used in this paper: ER, endoplasmic reticulum; CRT, calreticulin; CX, calnexin; HA, hemagglutinin; EndoH, endoglycosidase H; RENCA, murine renal adenocarcinoma cell line. Back

Received for publication December 26, 2000. Accepted for publication April 24, 2001.


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M. H. Kershaw, C. Hsu, W. Mondesire, L. L. Parker, G. Wang, W. W. Overwijk, R. Lapointe, J. C. Yang, R.-F. Wang, N. P. Restifo, et al.
Immunization against Endogenous Retroviral Tumor-associated Antigens
Cancer Res., November 1, 2001; 61(21): 7920 - 7924.
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