The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Matsuo, M.
Right arrow Articles by Nakayama, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Matsuo, M.
Right arrow Articles by Nakayama, E.
The Journal of Immunology, 1999, 162: 6420-6425.
Copyright © 1999 by The American Association of Immunologists

Expression of Multiple Unique Rejection Antigens on Murine Leukemia BALB/c RL1 and the Role of Dominant Akt Antigen for Tumor Escape1

Mitsutoshi Matsuo*,{dagger}, Hisashi Wada*, Shinichiro Honda*, Isao Tawara*, Akiko Uenaka*, Takashi Kanematsu{dagger} and Eiichi Nakayama2,*

* Department of Parasitology and Immunology, Okayama University Medical School, Okayama, Japan; and {dagger} Department of Surgery II, Nagasaki University School of Medicine, Nagasaki, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Using the pRL1a Ag-loss RL1 tumor variant cell line RM2-1, we demonstrated the presence of tumor Ags other than pRL1a that were recognized by CTLs on RL1 cells. Semiallogeneic CB6F1 or syngeneic BALB/c CTLs generated against RM2-1 lysed RM2-1 and RL1 cells to a similar extent, but no killing was observed with any other tumor or normal cells examined. Clonal analysis and sensitization with reversed phase-HPLC fractions revealed that there were Dd- and Ld-binding peptides recognized by RM2-1 CTLs. Lysis by bulk CTLs stimulated against RL1 and limiting dilution analysis suggested that the pRL1a peptide was dominantly recognized to the RM2-1 peptides by CTLs on RL1 cells. The rejection response against the parental RL1 tumor was much less than that against RM2-1 cells in either CB6F1 or BALB/c mice, suggesting that the presence of altered Akt molecules from which the dominant pRL1a peptide was derived inhibited the rejection response against RL1. Depletion of CD4 T cells caused the regression of RL1 at the doses in which the tumor grew in untreated mice. The generation of pRL1a CTLs was inhibited in RL1-bearing mice. Thus, immunoregulatory CD4 T cells were most likely activated by the altered Akt molecules and inhibited the efficient generation of CTLs against the dominant pRL1a Ag in RL1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Individually distinct (unique) tumor Ags were first identified by tumor transplantation studies of methylcholanthrene-induced fibrosarcomas in syngeneic mice (1, 2, 3, 4). Extremely high polymorphism is characteristic of the Ag: even tumors induced in the same mouse at different sites showed different Ag specificities (5). The Ags belonging to this class were later found in a variety of tumors from different species, including humans. Recent analyses of the molecules of these unique Ags revealed that they resulted mostly from mutational events, and that the antigenic peptides derived from altered molecules were presented on MHC and recognized by T cells (6). Interestingly, most of these genetic changes also appeared to be the cause of the transformation or malignant phenotype (7, 8, 9, 10).

BALB/c radiation leukemia RL1 is an immunogenic tumor. An inocula of 1 x 106 RL1 cells into (BALB/c x C57BL/6)F1 (CB6F1)3 mice initially grew to tumors and then regressed in 2–3 wk (11, 12). CTLs were generated in spleen cells from CB6F1 mice upon in vitro stimulation with mitomycin C (MMC)-treated RL1 cells (12). Expression of the Ag recognized by those CTLs on RL1 was unique. We demonstrated previously by peptide extraction and direct sequencing that the Ag recognized by the CTL was the octamer peptide pRL1a derived from the normally untranslated 5' region of protooncogene c-akt, which became translated by insertion of the murine leukemia virus long terminal repeat (13, 14). Ab blocking, competitive inhibition assays, and limiting dilution analysis revealed that the CTLs generated in CB6F1 spleen cells against RL1 were reactive mostly against the pRL1a peptide (13).

In this study, we investigated tumor Ags other than the pRL1a peptide recognized by CTLs on RL1 cells. For this purpose, we established a pRL1a Ag-loss RL1-variant tumor cell line, RM2-1, by immunoselection. We demonstrated that these cells expressed two tumor Ags recognized by CTLs on Dd and Ld molecules which were also present on the parental RL1 cells, but not on any other tumor or normal cells investigated. These Ag peptides were subdominantly recognized by the CTLs on RL1 cells to pRL1a peptide. We also found evidence that the altered Akt molecule from which the dominant pRL1a peptide was derived stimulated immunoregulatory CD4 T cells and allowed RL1 tumor escape.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

BALB/c and CB6F1 mice were purchased from Japan SLC (Shizuoka, Japan).

Tumors and cell lines

RL1, RL6, and RL8 are radiation-induced leukemias in BALB/c mice (12). RVA, RVC and RVD are leukemias induced by an injection of radiation leukemia virus into neonatal BALB/c mice (15). Meth A and CMS8 are methylcholanthrene sarcomas induced in BALB/c mice (16). MOPC-70A is a mineral oil-induced myeloma in a BALB/c mouse. P815 is a methylcholanthrene-induced mastocytoma in a DBA/2 mouse (17), and P1.HTR is its subline (18). 5-9 is transfected P1.HTR. EL4 is a chemically induced leukemia in a C57BL mouse.

Antibodies

Anti-L3T4 (CD4) mAb, a rat Ab of the IgG2b Ig class that is produced by hybridoma GK1.5 (19), was provided by Dr. F. Fitch (University of Chicago, Chicago, IL). Anti-Lyt-2.2 (CD8) mAb, a mouse Ab of the IgG2a class that is produced by hybridoma 19/178 (12), was provided by Dr. U. Hämmerling (Memorial Sloan-Kettering Cancer Center, New York, NY). Anti-TCR-ß mAb, a hamster Ab of the IgG class that is produced by hybridoma H57-597 (20), was provided by Dr. R. Kubo (National Jewish Center, Denver, CO). Anti-H-2Kd and anti-H-2Dd mAbs are mouse Abs of the IgG2a class that are produced by hybridomas KD40 and DD98, respectively, and were established by the hybridization of P3U1 myeloma and spleen cells from BALB.B mice that had been immunized with BALB/c lymphoid cells. Anti-H-2Ld mAb, a mouse Ab of the IgG2a class that is produced by hybridoma 30-5-7 (21), was provided by Dr. N. Shinohara (Kitasato University School of Medicine, Sagamihara, Japan).

Flow cytometry analysis

Cells (1 x 106) were incubated with the mAbs for 30 min on ice. Next, cells were washed, incubated with an appropriate FITC-conjugated goat anti-mouse IgG, and analyzed in a FACScan (Becton Dickinson, Mountain View, CA).

Acid extraction of whole cells and reversed phase (RP)-HPLC analysis

RL1 ascites cells (5 x 109) were homogenized in 0.1% trifluoroacetic acid (TFA) with a Dounce homogenizer and sonicated (Sonifier W-185; Branson Sonic Power, Danbury, CT) for 3 min. The homogenates were then stirred at a pH of 2.0 in 0.1% TFA for 30 min. The supernatants obtained by centrifugation at 10,000 rpm for 30 min were filtered with a molecular cutoff membrane (m.w. 5000, Millipore, Bedford, MA). These procedures were done at 4°C. The filtrates were lyophylized, resolved in 0.1% TFA, and analyzed by RP-HPLC on a semipreparative C18 column (ODP, 10 x 250 mm, 10-µm particles; Asahipak, Kawasaki, Japan) with a Waters 625L apparatus (Waters/Millipore, Milford, MA). Solvent A was 20% acetonitrile containing 0.1% TFA, and solvent B was 60% acetonitrile containing 0.1% TFA. The gradient for chromatography was 0–100% B over 40 min. The fractions (2.0 ml) collected at 1-min intervals were assayed for sensitization activity on P1.HTR target cells for CTLs.

Western blot analysis

Cells (3.5 x 106) were lysed in sample buffer consisting of 1% SDS, 0.2 M Tris-HCl (pH 6.8), and 50% glycerol with 10 µM leupeptin and 1 µM pepstatin A and were boiled. The supernatants obtained by centrifugation at 10,000 rpm for 30 min were separated by SDS-PAGE, blotted onto a nitrocellulose membrane, and incubated with rabbit antiserum against a synthetic peptide composed of the N-terminal 15-aa residues (SIIPGLPLSLGATDT) of the altered Akt protein coupled to keyhole limpet hemocyanin. Bound Ab was detected by goat anti-rabbit IgG Fc conjugated with alkaline phosphatase (Promega, Madison, WI) using a Bio-Rad substrate kit (Hercules, CA).

Northern blot analysis

Poly(A)+ RNA (2 µg) was electrophoresed in 1.4% agarose-formaldehyde gel and subsequently blotted onto a nylon membrane (Hybond-N+; Amersham International, Tokyo, Japan). The membrane was baked at 80°C for 2 h; prehybridized at 42°C for 4 h in 0.75 M NaCl, 0.075 M sodium citrate, 2x Denhardt’s solution, 40 µg/ml salmon sperm DNA, 0.5% SDS, and 50% formamide; and hybridized for 20 h with the 32P-labeled c-akt 5' probe at 42°C. A purified DNA fragment from SalI (New England Biolabs, Beverly, MA) and PstI (New England Biolabs) digests of cAkt17b (22) (542 bp) was labeled with 32P and used as a probe. Glyceraldehyde-3-phosphate dehydrogenase was used as the control for RNA loading. The membrane was then washed in 2x SSCB (0.3 M NaCl, 0.03 M sodium citrate, and 0.05% SDS) at 42°C for 10 min, 2x SSCB at 65°C for 10 min, 1x SSCB at 65°C for 10 min, and 0.5x SSCB at 65°C for 10 min, and exposed to Kodak XAR-5 film.

Generation of Con A blasts

Spleen cells (2 x 107) were cultured with Con A at a concentration of 5 µg/ml for 3 days.

Establishment and maintenance of CTL clones

Spleen cells (4 x 107) were cultured with 4 x 106 MMC-treated stimulator cells in tissue culture flasks (model 25100, Corning Glass, Corning, NY) at 37°C in a 5% CO2 atmosphere for 5 days. MMC treatment was done by incubating cells with MMC at a concentration of 50 µg/ml at 37°C for 30 min. The culture medium was RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME. Cells (1 x 105) were maintained in 24-well culture plates (76-033-05; Flow Laboratories, McLean, VA) as a bulk CTL cell line by weekly stimulation with 1 x 105 MMC-treated stimulator cells and 5 x 106 MMC-treated splenic feeder cells in the presence of 2% culture supernatant from Chinese hamster ovary cells transfected with human IL-2 gene (CHO-IL-2) as a source of IL-2. For cloning, the cells were diluted to 3–0.3 cells/well and cultured with MMC-treated stimulator cells and 5 x 105 MMC-treated splenic feeder cells in the presence of IL-2 in 96-well culture plates (model 25860, Corning Glass). Clones grown after 10–14 days were maintained by weekly stimulation as described above.

Cell-mediated cytotoxicity assay

Tumor cells and Con A blasts were labeled by incubating 2 x 106 cells with 2 MBq of Na251CrO4 (New England Nuclear, Boston, MA) for 1.5 h at 37°C in a 5% CO2 atmosphere. Next, the cells and blasts were washed and used as target cells. In direct assays, 5 x 103 labeled target cells (100 µl) were incubated with the effector cell suspension (100 µl). In Ab-blocking assays, serially diluted mAb (50 µl) was added to the culture of effector cells (50 µl) and labeled target cells (100 µl). In sensitization assays, 10 µl of each HPLC fraction in 100 µl of medium was added to aliquots (5 x 103) of 51Cr-labeled target cells (100 µl) and incubated for 60 min at room temperature before adding effector cells (100 µl). After incubation for 4 h at 37°C in a 5% CO2 atmosphere, the supernatants (100 µl) were removed and their radioactivity was measured. The percentage of specific lysis was calculated as follows: ([a-b]/[c-b]) x 100, where a is the radioactivity of the supernatant from target cells mixed with effector cells, b is that of the supernatant from target cells incubated alone, and c is that of the supernatant after lysis of target cells with 1% Nonidet P-40.

Immunoselection

RL1 cells were cultured with pRL1a-specific bulk CTLs (1 x 105) in 24-well culture plates with feeder cells for 3 wk, and the remaining tumor cells were allowed to expand. The procedure was repeated several times, and the cells were then cloned by limiting dilution.

Tumor assay

Tumor cells grown in ascites were washed and injected intradermally (i.d.) into the backs of mice with a 27-gauge needle. Tumor growth was measured every 3–4 days at right angles using a caliper.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Establishment of pRL1a Ag-loss RL1 variant cell line RM2-1

RL1 cells were cultured with pRL1a-specific bulk CTLs derived from CB6F1 spleen cells. Repeated exposure of RL1 cells to the CTLs resulted in the variant RL1 tumor cell line RM2-1 being resistant to lysis (Fig. 1Go). Northern blot analysis showed no overexpression of the akt mRNA in RM2-1 cells, unlike in parental RL1 cells. Western blot analysis showed that RM2-1 cells lost the altered Akt molecules expressed in RL1 cells. Flow cytometry analysis showed no diminished expression of H-2Kd, Dd, or Ld Ags on RM2-1 cells.



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 1. Establishment of the pRL1a Ag-loss RL1 variant cell line RM2-1. A, Cytotoxicity of pRL1a-specific CTLs against RL1, RM2-1, and P1.HTR as determined by 4-h 51Cr release assay. B, Northern blot analysis. The expression of akt mRNA in RM2-1 cells was one-eighth that of RL1 cells by densitometry after normalization by glyceraldehyde-3-phosphate dehydrogenase. C, Western blot analysis. A 59-kDa altered Akt molecule was detected in RL1, but not in the RM2-1 lysate. D, flow cytometry analysis. The expression of H-2Kd, Dd, and Ld Ags on RM2-1 and RL1 cells is shown.

 
Immunogenicity of pRL1a-loss RL1 variant RM2-1 cells

pRL1a-loss RL1 variant RM2-1 cells (2.5 x 106) were inoculated into the backs of CB6F1 mice, and tumor growth was examined. As shown in Fig. 2Go, the tumor grew initially and then regressed, all in 2 wk. No tumor recurrence was observed thereafter. The results suggested the presence of tumor rejection Ag(s) other than pRL1a on RM2-1 cells.



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 2. Tumor growth. CB6F1 mice were inoculated i.d. in the back with 2.5 x 106 RM2-1 and 2 x 105 RL1 cells. Values represent means ± SD. Each experimental group consisted of five mice.

 
Generation and clonal analysis of CTLs against pRL1a-loss RL1 variant RM2-1 in CB6F1 spleen cells

Spleen cells from CB6F1 mice that had rejected pRL1a-loss RL1 variant RM2-1 tumor cells were cultured with MMC-treated RM2-1 cells for 5 days, and the generation of cytotoxicity was investigated. As shown in Fig. 3GoA, cytotoxicity against RM2-1 was generated. The cytotoxicity was abrogated by treating effector cells with anti-Lyt-2.2 (CD8) mAb but not with anti-L3T4 (CD4) mAb plus complement. The cytotoxicity was blocked by anti-Lyt-2.2 (CD8) mAb, but not by anti-L3T4 (CD4) mAb in the absence of complement added exogenously to the culture (data not shown).



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 3. Cytotoxicity of CB6F1 bulk (A), clone 7 (Dd-restricted) (B), clone 2 (Ld-restricted) (C) CTLs generated against RM2-1 and that of CB6F1 bulk CTLs generated against RL1 (D). Bulk CTLs were generated in spleen cells from mice that had rejected RM2-1 or RL1 upon stimulation with MMC-treated RM2-1 or RL1 cells, respectively, for 5 days. 5-9 is P1.HTR transfected with RLakt (14). Cytotoxicity was determined by a 4-h 51Cr release assay.

 
Clonal analysis of CB6F1 bulk CTLs generated against RM2-1 was performed by limiting dilution. The cytotoxicity of 2 of 12 clones was blocked by anti-Dd mAb but not by anti-Kd mAb or anti-Ld mAb (Fig. 4Go). Alternatively, the cytotoxicity of 10 of 12 CTL clones was blocked by anti-Ld mAb but not by anti-Kd mAb or anti-Dd mAb. Bulk and cloned CTLs showed cytotoxicity against RM2-1 and also RL1 to a similar extent, but not against RLakt (14)-transfected P1.HTR 5-9 cells, pRL1a-pulsed P1.HTR, five other BALB/c leukemias, two BALB/c fibrosarcomas, a BALB/c myeloma, a DBA/2 mastocytoma, a C57BL leukemia, or BALB/c and C57BL/6 splenic Con A blasts (Fig. 3Go and Table IGo).



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 4. Ab blocking of cytotoxicity by RM2-1 CTL clones. Dilution of mAbs was 1/500. The E:T ratios were 12:1 with bulk CTLs and 3:1 with all five CTL clones. Cytotoxicity was determined by a 4-h 51Cr release assay.

 

View this table:
[in this window]
[in a new window]
  		Table I. Specificity of CB6F1 anti-RM2-1 CTLs

 
The results suggested that there were two antigenic epitopes recognized by the RM2-1 CTLs that were presented on Dd and Ld molecules and that their expression was unique to RM2-1 and parental RL1 cells.

The CTLs generated in spleen cells from CB6F1 mice that had rejected RL1 upon in vitro stimulation with MMC-treated RL1 cells showed less killing to RM2-1 cells than RL1 or pRL1a-pulsed P1.HTR (Fig. 3GoD). This observation confirms that the pRL1a peptide Ag was dominantly recognized by the CTLs on RL1 cells over the peptides detected by the RM2-1 CTLs.

Purification of Ag peptides recognized by RM2-1 CTLs by RP-HPLC

An acid extract from RL1 cells was separated by RP-HPLC, and the sensitization activity of each fraction on P1.HTR target cells for RM2-1 CTLs was investigated. As shown in Fig. 5Go, the sensitization activity for RM2-1 bulk CTLs was eluted in fractions of 19 and 22 min. Dd-restricted CTL clones showed cytotoxicity against the P1.HTR target cells sensitized with the fraction eluted in 22 min. Ld-restricted CTL clones showed cytotoxicity against the P1.HTR target cells sensitized with the fraction eluted in 19 min. The sensitization activity for pRL1a-specific CTLs was eluted in the 24- and 27-min fractions, consistent with previous findings (13).



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 5. RP-HPLC purification of RM2-1 rejection Ag peptides. The acid extract from RL1 cells was separated by RP-HPLC, and the sensitization activity of each fraction was analyzed for CB6F1 and BALB/c bulk RM2-1 CTLs (A), CB6F1 clone 7 (Dd-restricted) (B) and clone 2 (Ld-restricted) (C) RM2-1 CTLs, and pRL1a-specific CB6F1 CTL clone Y-15 (D). Each concentrated fraction (10 µl) was added to the 51Cr-labeled P1.HTR target cells. Cytotoxicity was determined by a 4-h 51Cr release assay. The E:T ratios were 50 in A, 8 in B and C, and 5 in D.

 
Failure of parental RL1 cells to induce effective tumor rejection response in CB6F1 and BALB/c mice

Inocula of ~1 x 107 RM2-1 cells regressed after initial growth, not only in semiallogeneic CB6F1 mice, but also in syngeneic BALB/c mice (Table IIGo). Alternatively, only inocula of <=1 x 106 parental RL1 cells regressed after initial growth in CB6F1 mice. In BALB/c mice, regression of RL1 was observed in some mice inoculated with <2 x 105 cells.


View this table:
[in this window]
[in a new window]
  		Table II. Tumorigenicity of pRL1a-loss variant RM2-1 and parental RL1 cells in semiallogeneic CB6F1 and syngeneic BALB/c mice

 
To investigate the failure of parental RL1 cells to induce an effective tumor rejection response, the effect of an in vivo depletion of CD4 T cells was investigated. As shown in Fig. 6Go, CD4 depletion resulted in regression of the RL1 tumor in CB6F1 and BALB/c mice at doses at which the tumor continued to grow in control CD4 nondepleted mice.



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 6. Effect of in vivo depletion of CD4 T cells on RL1 tumor growth. A total of 5 x 106 and 5 x 105 RL1 cells were inoculated i.d. into the backs of CB6F1 (A) and BALB/c (B) mice, respectively, and 2.5 x 106 RM2-1 cells were similarly inoculated into BALB/c mice (C). Next, the mice were treated with anti-L3T4 (CD4) mAb (right) or with minimum essential medium (control) (left) on day 6. Each line indicates tumor growth in individual mice. D and E, A total of 5 x 105 RL1 and 5 x 106 RM2-1 cells, respectively, were inoculated into BALB/c nu/nu mice, and the mice were either left untreated or treated with anti-L3T4 (CD4) mAb on days 6 and 14. Values represent means ± SD. Each experimental group consisted of five mice.

 
As shown in Fig. 7Go, CTLs were generated against pRL1a and much less efficiently against RM2-1 in spleen cells from CB6F1 and BALB/c mice that had rejected high and low doses of RL1 with and without CD4 depletion, respectively. However, the generation of CTLs against pRL1a was suppressed in spleen cells from CB6F1 and BALB/c mice in which the RL1 tumor was growing progressively (Fig. 7GoC).



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 7. Cytotoxicity of bulk CTLs generated in spleen cells from BALB/c mice that had rejected low dose (2 x 105) RL1 cells (A) or had rejected 5 x 105 RL1 cells by CD4-depletion (B), or from BALB/c mice in which the RL1 tumor was growing progressively (C). Cytotoxicity was determined by a 4-h 51Cr release assay.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using the pRL1a-loss RL1 tumor variant cell line RM2-1, we demonstrated the presence of tumor Ags other than pRL1a that were recognized by CTLs on RL1 cells. RM2-1 tumors regressed after initial growth in both semiallogeneic CB6F1 and syngeneic BALB/c mice when <=1 x 107 cells were inoculated. CTLs were generated in spleen cells from mice that had rejected the tumor by in vitro stimulation with RM2-1 cells. These CTLs lysed RM2-1 and RL1 cells to a similar extent, but no killing was observed with any other tumor or normal cells examined. Clonal analyses revealed that 2 of 12 CTL clones were Dd-restricted and 10 of 12 clones were Ld-restricted. RP-HPLC analysis of the acid extract from RL1 cells showed that the Dd-binding peptide was eluted in 22 min and the Ld-binding peptide in 19 min. These findings suggested that there were two Ag peptides uniquely expressed on RM2-1 and RL1 cells which were recognized by the specific CTLs. This expression was not due to the immunoselection procedure.

The CTLs generated in spleen cells from CB6F1 mice that had rejected the RL1 tumor by in vitro stimulation with RL1 lysed RM2-1 cells less efficiently than RL1 and pRL1a-pulsed P1.HTR. Moreover, the CTL clones obtained from bulk CTLs stimulated against RL1 were mostly reactive against pRL1a. These findings suggested that the pRL1a Ag peptide was dominantly recognized on RL1 cells by the CTLs to the peptides detected by RM2-1 CTLs.

The presence of multiple tumor Ags on individual tumor cells has been demonstrated in murine (23, 24, 25) and human (26, 27, 28) tumors. Using specific CTL clones and tumor variants that displayed selective Ag losses, several independently expressed tumor Ags were defined on a UV-induced tumor (23, 29). Similar findings were obtained with methylcholanthrene-induced fibrosarcomas (25). Human malignant melanoma cells were shown to express multiple Ags, and CTL lines reactive against individual Ags were established (26). The studies with murine tumors showed immunodominance among the Ags on the tumors; in addition, immunoselection of the negative variant of the dominant Ag in vivo or by the specific CTLs revealed the second most dominant Ags recognized by the specific CTLs. It should be noted that loss of the dominant Ag resulted in a tumor rejection response elicited by the second most dominant Ag (29) or in tumor escape from immunosurveillance (24, 30).

This study demonstrated that loss of the dominant Akt Ag converted the RL1 tumor, which grows progressively, into a regressor phenotype. Regardless of the dominant antigenicity of pRL1a (see above), no efficient rejection response was observed with the parental RL1 tumor cells when inoculated into either semiallogeneic CB6F1 or syngeneic BALB/c mice. pRL1a Ag-loss variant RM2-1 cells were efficiently rejected by both groups of mice. These findings suggested that the presence of altered Akt molecules from which the pRL1a peptide was derived inhibited the rejection response against parental RL1. We showed that depletion of CD4 T cells caused a regression of RL1 tumors at doses at which tumors grew in CD4 nondepleted mice. Immunoregulatory CD4 T cells were likely to be activated by the altered Akt molecules and inhibited the efficient generation of CTLs against the pRL1a Ag. A strong CTL response was observed in spleen cells from CB6F1 or BALB/c mice that had rejected RL1 by CD4 depletion. CTLs were reactive predominantly against pRL1a and were much less reactive against the antigenic peptides on RM2-1 similarly as those generated in spleen cells from CB6F1 or BALB/c mice that had rejected low doses of RL1 without CD4 depletion. Cytotoxicity against RL1 cells was also observed in spleen cells from RL1 tumor-bearing CB6F1 and BALB/c mice. However, in this case, no significant CTL response against the pRL1a Ag was observed. These findings suggest that CTL generation against the pRL1a Ag was specifically inhibited in tumor-bearing mice. Akt Ag-specific inhibition of CTL generation was also supported by findings from in vivo studies. RM2-1 and allogeneic EL4 (C57BL) tumor cells inoculated into the opposite flank from that inoculated with RL1 in BALB/c mice were normally rejected (data not shown). The Akt-expressing RM2-1 transfectant will greatly facilitate investigations into the mechanisms of the induction, specificity, and functional role of immunoregulatory CD4 T cells. However, we have thus far failed to obtain RM2-1 transfectants with an expression of Akt at the level of RL1. No progressive tumor growth was observed with RM2-1 transfectants expressing a low level of Akt. These findings suggest that the extremely high expression of Akt in RL1 was the cause of the induction of immunoregulatory CD4 T cells.

A preventive role has been suggested for immunoregulatory CD4 T cells in autoimmune diseases in mice (31, 32) and humans (33, 34). These cells have also been shown to be involved in tumor growth (35, 36, 37). North (35) demonstrated that a depletion of CD4 T cells caused a regression of Meth A fibrosarcoma in syngeneic BALB/c mice. This observation was similar to the findings obtained with other murine tumors, such as P815 mastocytoma and L5178Y lymphoma in syngeneic DBA/2 mice (36, 38). However, the Ags involved in inducing those CD4 T cells are still unknown. Our present findings are consistent with the studies mentioned above, and also suggest that the dominant rejection Ag could be the one that induces the immunoregulatory CD4 T cells which caused the tumor escape.


   Acknowledgments
 
We thank M. Isobe for excellent technical assistance and J. Mizuuchi for preparation of the manuscript.


   Footnotes
 
1 This work was supported in part by a grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture of Japan. Back

2 Address correspondence and reprint requests to Dr. Eiichi Nakayama, Department of Parasitology and Immunology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. E-mail address: Back

3 Abbreviations used in this paper: CB6F1, (BALB/c x C57BL/6)F1; MMC, mitomycin C; RP, reversed phase; TFA, trifluoroacetic acid. Back

Received for publication July 30, 1998. Accepted for publication March 16, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gross, L.. 1943. Intradermal immunization of C3H mice against a sarcoma that originated in an animal of the same line. Cancer Res. 3:326.[Free Full Text]
  2. Foley, E. L.. 1953. Antigenic properties of methylcholanthrene-induced tumors in mice of the strain of origin. Cancer Res. 13:835.
  3. Prehn, R. T., J. M. Main. 1957. Immunity to methylcholanthrene-induced sarcomas. J. Natl. Cancer Inst. 18:769.
  4. Klein, G., H. O. Sjögren, E. Klein, K. E. Hellström. 1960. Demonstration of resistance against methylcholanthrene-induced sarcomas in the primary autochthonous host. Cancer Res. 20:1561.
  5. Globerson, A., M. Feldman. 1964. Antigenic specificity of benzo(a)pyrene-induced sarcomas. J. Natl. Cancer Inst. 32:1229.
  6. Van den Eynde, B., P. van der Bruggen. 1997. T cell-defined tumor antigens. Curr. Opin. Immunol. 9:684.[Medline]
  7. Wölfel, T., M. Hauer, J. Schnider, M. Serrano, C. Wölfel, E. Klehmann-Hieb, E. De Plaen, T. Hankeln, K.-H. Meyer zum Büschenfelde, D. Beach. 1995. A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science 269:1281.[Abstract/Free Full Text]
  8. Robbins, P. F., M. El-Gamil, Y. F. Li, Y. Kawakami, D. Loftus, E. Appella, S. A. Rosenberg. 1996. A mutated ß-catenin gene encodes a melanoma-specific antigen recognized by tumor-infiltrating lymphocytes. J. Exp. Med. 183:1185.[Abstract/Free Full Text]
  9. Rubinfeld, B., P. Robbins, M. El-Gamil, I. Albert, E. Porfiri, P. Polakis. 1997. Stabilization of ß-catenin by genetic defects in melanoma cell lines. Science 275:1790.[Abstract/Free Full Text]
  10. Mandruzzato, S., F. Brasseur, G. Andry, T. Boon, P. van der Bruggen. 1997. A CASP-8 mutation recognized by cytolytic T lymphocytes on a human head and neck carcinoma. J. Exp. Med. 186:785.[Abstract/Free Full Text]
  11. Sato, H., E. A. Boyse, T. Aoki, C. Iritani, L. J. Old. 1973. Leukemia-associated transplantation antigens related to murine leukemia virus. The X.1 system: immune response controlled by a locus linked to H-2. J. Exp. Med. 138:593.[Abstract]
  12. Nakayama, E., H. Shiku, T. Takahashi, H. F. Oettgen, L. J. Old. 1979. Definition of a unique cell surface antigen of mouse leukemia RL1 by cell-mediated cytotoxicity. Proc. Natl. Acad. Sci. USA 76:3486.[Abstract/Free Full Text]
  13. Uenaka, A., T. Ono, T. Akisawa, H. Wada, T. Yasuda, E. Nakayama. 1994. Identification of a unique antigen peptide pRL1 on BALB/c RL1 leukemia recognized by cytotoxic T lymphocytes and its relation to the akt oncogene. J. Exp. Med. 180:1599.[Abstract/Free Full Text]
  14. Wada, H., M. Matsuo, A. Uenaka, N. Shimbara, K. Shimizu, E. Nakayama. 1995. Rejection antigen peptides on BALB/c RL1 leukemia recognized by cytotoxic T lymphocytes: derivation from the normally untranslated 5' region of the c-akt proto-oncogene activated by long terminal repeat. Cancer Res. 55:4780.[Abstract/Free Full Text]
  15. Stockert, E., A. B. DeLeo, P. V. O’Donnell, Y. Obata, L. J. Old. 1979. G(AKSL2): a new cell surface antigen of the mouse related to the dualtropic mink cell focus-inducing class of murine leukemia virus detected by naturally occurring antibody. J. Exp. Med. 149:200.[Abstract/Free Full Text]
  16. DeLeo, A. B., H. Shiku, T. Takahashi, M. John, L. J. Old. 1977. Cell surface antigens of chemically induced sarcomas of the mouse: murine leukemia virus-related antigens and alloantigens on cultured fibroblasts and sarcoma cells: description of a unique antigen on BALB/c Meth A sarcoma. J. Exp. Med. 146:720.[Abstract/Free Full Text]
  17. Dunn, T. B., M. Potter. 1957. A transplantable mast-cell neoplasm in the mouse. J. Natl. Cancer Inst. 18:587.
  18. Wölfel, T., A. Van Pel, E. De Plaen, C. Lurquin, J. L. Maryanski, T. Boon. 1987. Immunogenic (tum-) variants obtained by mutagenesis of mouse mastocytoma P815: detection of stable transfectants expressing tum- antigen with a cytolytic T cell stimulation assay. Immunogenetics 26:178.[Medline]
  19. Dialynas, D. P., Z. S. Quan, K. A. Wall, A. Pierres, J. Quintans, M. R. Loken, M. Pierres, F. W. Fitch. 1983. Characterization of the murine T cell surface molecule, designated L3T4, identified by monoclonal antibody GK1.5: similarity of L3T4 to the human Leu-3/T4 molecule. J. Immunol. 131:2445.[Abstract]
  20. Kubo, R. T., W. Born, J. W. Kappler, P. Marrack, M. Pigeon. 1989. Characterization of a monoclonal antibody which detects all murine {alpha}ß T cell receptors. J. Immunol. 142:2736.[Abstract]
  21. Ozato, K., T. H. Hansen, D. H. Sachs. 1980. Monoclonal antibodies to mouse MHC antigens: antibodies to the H-2Ld antigen, the products of a third polymorphic locus of the mouse MHC. J. Immunol. 125:2473.[Abstract]
  22. Bellacosa, A., T. F. Franke, M. E. Gonzalez-Portal, K. Datta, T. Taguchi, J. Gardner, J. Q. Cheng, J. R. Testa, P. N. Tsichlis. 1993. Structure, expression, and chromosomal mapping of c-akt: relationship to v-akt and its implications. Oncogene 8:745.[Medline]
  23. Wortzel, R. D., C. Phillips, H. Schreiber. 1983. Multiple tumour-specific antigens expressed on a single tumour cell. Nature 304:165.[Medline]
  24. Uyttenhove, C., J. L. Maryanski, T. Boon. 1983. Escape of mouse mastocytoma P815 after nearly complete rejection is due to antigen-loss variants rather than immunosuppression. J. Exp. Med. 157:1040.[Abstract/Free Full Text]
  25. Dudley, M. E., D. C. Roopenian. 1996. Loss of a unique tumor antigen by cytotoxic T lymphocyte immunoselection from a 3-methylcholanthrene-induced mouse sarcoma reveals secondary unique and shared antigens. J. Exp. Med. 184:441.[Abstract/Free Full Text]
  26. Knuth, A., T. Wölfel, E. Klehmann, T. Boon, K.-H. Meyer zum Büschenfelde. 1989. Cytolytic T-cell clones against an autologous human melanoma: specificity study and definition of three antigens by immunoselection. Proc. Natl. Acad. Sci. USA 86:2804.[Abstract/Free Full Text]
  27. Van den Eynde, B., P. Hainaut, M. Hérin, A. Knuth, C. Lemoine, P. Weynants, P. van der Bruggen, R. Fauchet, T. Boon. 1989. Presence on a human melanoma of multiple antigens recognized by autologous CTL. Int. J. Cancer. 44:634.[Medline]
  28. Ioannides, C. G., R. S. Freedman, C. D. Platsoucas, S. Rashed, Y. P. Kim. 1991. Cytotoxic T cell clones isolated from ovarian tumor-infiltrating lymphocytes recognize multiple antigenic epitopes on autologous tumor cells. J. Immunol. 146:1700.[Abstract]
  29. Urban, J. L., R. C. Burton, J. M. Holland, M. L. Kripke, H. Schreiber. 1982. Mechanisms of syngeneic tumor rejection: susceptibility of host-selected progressor variants to various immunological effector cells. J. Exp. Med. 155:557.[Abstract/Free Full Text]
  30. Wortzel, R. D., J. L. Urban, H. Schreiber. 1984. Malignant growth in the normal host after variant selection in vitro with cytolytic T-cell lines. Proc. Natl. Acad. Sci. USA 81:2186.[Abstract/Free Full Text]
  31. Kumar, V., E. E. Sercartz. 1993. The involvement of T cell receptor peptide-specific regulatory CD4+ T cells in recovery from antigen-induced autoimmune disease. J. Exp. Med. 178:909.[Abstract/Free Full Text]
  32. Akhtar, I., J. P. Gold, L. Y. Pan, J. L. Ferrara, X. D. Yang, J. I. Kim, K. N. Tan. 1995. CD4+ ß islet cell-reactive T cell clones that suppress autoimmune diabetes in nonobese diabetic mice. J. Exp. Med. 182:87.[Abstract/Free Full Text]
  33. Saruhan-Direskeneli, G., F. Weber, E. Meinl, M. Pette, G. Giegerich, A. Hinkkanen, J. T. Epplen, R. Hohlfeld, H. Wekerle. 1993. Human T cell autoimmunity against myelin basic protein: CD4+ cells recognizing epitopes of the T cell receptor ß-chain from a myelin basic protein-specific T cell clone. Eur. J. Immunol. 23:530.[Medline]
  34. Chou, Y. K., W. J. Morrison, A. D. Weinberg, R. Dedrick, R. Whitham, D. N. Bourdette, G. Hashim, H. Offner, A. A. Vandenbark. 1994. Immunity to TCR peptides in multiple sclerosis: T cell recognition of Vß 5.2 and Vß 6.1 CDR2 peptides. J. Immunol. 152:2520.[Abstract]
  35. North, R. J.. 1986. Radiation-induced, immunologically mediated regression of an established tumor as an example of successful therapeutic immunomanipulation: preferential elimination of suppressor T cells allows sustained production of effector T cells. J. Exp. Med. 164:1652.[Abstract/Free Full Text]
  36. Awwad, M., R. J. North. 1988. Immunologically mediated regression of a murine lymphoma after treatment with anti-L3T4 antibody: a consequence of removing L3T4+ suppressor T cells from a host generating predominantly Lyt-2+ T cell-mediated immunity. J. Exp. Med. 168:2193.[Abstract/Free Full Text]
  37. Mullen, C. A., J. L. Urban, C. Van Waes, D. A. Rowley, H. Schreiber. 1985. Multiple cancers: tumor burden permits the outgrowth of other cancers. J. Exp. Med. 162:1665.[Abstract/Free Full Text]
  38. Rakhmilevich, A. L., R. J. North, E. S. Dye. 1993. Presence of CD4+ T suppressor cells in mice rendered unresponsive to tumor antigens by intravenous injection of irradiated tumor cells. Int. J. Cancer. 55:338.[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Matsuo, M.
Right arrow Articles by Nakayama, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Matsuo, M.
Right arrow Articles by Nakayama, E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS