Successful elimination of large established tumors and avoidance of antigen-loss variants by aggressive adoptive T cell immunotherapy

Ken Matsui1, Leigh A. O’Mara1 and Paul M. Allen1

1 Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA

Correspondence to: P. M. Allen; E-mail: allen{at}immunology.wustl.edu
Transmitting editor: J. P. Allison


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Utilization of ex vivo-expanded epitope-specific cytotoxic T lymphocytes has become a clinical standard in the adoptive immunotherapy of tumors. One of the obstacles faced by T cell-based immunotherapy is the development of tumor immune-escape variants. Using our previously reported CMS5 tumor/DUC18 CD8+ TCR transgenic system, we sought to investigate whether large established tumors can be successfully eliminated before the development of escape variants. Using BALB/c mice that were s.c. transplanted with two tumors that had been growing for 8 days (double 8-day tumors), we assessed the in vivo anti-tumor activity of in vitro peptide-stimulated DUC18 T cells. A single infusion of activated DUC18 T cells showed a modest effect against the double 8-day tumors, whereas two and three administrations led to regression of both tumors within 10 days. However, in some mice, the tumors re-grew ~10 days after the regression. We found these tumors to be antigen-loss variants. These relapsed tumor cells progressively grew in DUC18 transgenic mice and did not express tERK-specific message. When four doses of activated DUC18 T cells were infused, the double 8-day tumors were successfully eliminated and the tumors did not grow out in any mice. Our results demonstrate that mono-specific CD8+ T cells can effectively eliminate large established tumors before the development of antigen-loss variants when a high number of T cells is rapidly administered.

Keywords: CMS5, DUC18, mono-specific T cell, tERK


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Identification of tumor-associated antigens, elucidation of molecular interactions between a CD8+ T cell and a target cell, and generation of tumor-specific cytotoxic T cells have greatly facilitated the reality of effective T cell-based immunotherapy (110). Cell-mediated immune responses against tumors have been observed in cancer patients and in experimental animal models (1124). Adoptive T cell immunotherapy has shown a great promise, particularly for those with Epstein–Barr virus-associated malignancies. Immunotherapy of melanoma patients has also met with mild success (2533).

Transferring bulk T lymphocytes of undefined specificity to treat patients was largely unsuccessful and the paucity of anti-tumor responses was likely due to the heterogeneity of cell populations that were utilized (31). More recently, T cells that recognize a defined tumor antigen were in vitro expanded and infused back into melanoma patients (18,30,33). Although more specific, targeting a single epitope can also be problematic due to the propensity of tumors to develop immune-escape variants under immunological pressure (11,3446).

We previously reported a tumor/TCR transgenic system that was based on CMS5, a methylcolanthrene-induced fibrosarcoma from a BALB/c mouse, and C18, a CD8+ cytotoxic T lymphocyte (CTL) clone, that specifically recognizes and kills this tumor (4749). In CMS5, a point mutation in the erk2 kinase gene leads to a single amino acid substitution in the mature protein. The neo-epitope called tERK-I (QYIHSANVL) is specifically recognized by C18 CTL when presented by H-2Kd (48). The DUC18 TCR transgenic mouse expresses the C18 TCR genes and adoptive transfer of naive DUC18 T cells to normal BALB/c mice results in rejection of CMS5 tumors that have been growing in BALB/c mice for up to 5 days (49). With a CD8+ T cell of known antigen specificity, the CMS5/DUC18 model is an ideal system to examine the effectiveness of anti-tumor activity by an epitope-specific T cell.

In this study, we sought to investigate whether a monoclonal population of CD8+ T cells can successfully eliminate a large tumor mass and avoid the development of immune-escape variants. For this purpose, an in vivo system that can reproducibly generate CMS5 escape variants was developed. A CMS5 tumor mass that had been growing for 8 days (8-day tumor) was successfully eliminated by a single infusion of activated DUC18 T cells. Doubling the tumor load by inoculating CMS5 on right and left flanks of recipient mice (double 8-day tumors) required multiple DUC18 T cell infusions. Two and three infusions of activated DUC18 T cells initially led to a complete regression of both tumors; however, the tumors eventually re-grew. These tumors were found to be antigen-loss variants. Importantly, when four infusions of DUC18 T cells were administered, the tumors were completely ablated. Thus, we demonstrate the feasibility of in vitro activated mono-epitope-specific CD8+ T cell utilization to treat large established tumors while avoiding the out-growth of antigen-loss variants.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell lines and reagents
The derivation of the fibrosarcoma cell lines CMS5 and MethA has been previously described (47,50). CMS5 and MethA cells were grown in R10 medium containing RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% FCS (Hyclone, Logan, UT), 2 mM Glutamax (Invitrogen, Carlsbad, CA) and 50 µg/ml gentamycin sulfate (Invitrogen). DUC18 splenocytes were cultured in R10 medium supplemented with 5 x 10–5 M 2-mercaptoethanol. The previously identified epitope tERK-I (QYIHSANVL) was synthesized, purified and characterized as described (48,49). All antibodies used in this study were purchased from BD Biosciences (San Diego, CA).

Mice
All mice were maintained in a specific pathogen-free barrier facility at Washington University School of Medicine. BALB/c AnNCr (referred to as BALB/c) mice were purchased from the National Cancer Institute (Frederick, MD). DUC18 is a TCR transgenic mouse strain on the BALB/c background that expresses an H-2Kd-restricted TCR derived from a C18 CTL clone that specifically recognizes the tERK-I peptide derived from mutated ERK2 kinase protein expressed in CMS5 (48). The generation and characterization of the DUC18 transgenic mouse has been previously described (49). DUC18 TCR expression was determined by FACS analysis (FACSCalibur; Becton Dickinson, San Jose, CA) through CD8+ and Vß8.3 expression.

Preparation of naive and in vitro activated DUC18 T cells
Single-cell suspensions of naive DUC18 T cells were prepared from the spleens of DUC18 transgenic mice on the day of adoptive T cell transfer. Red blood cells were lysed (0.14 M NH4Cl and 0.02 M Tris, pH 7.4) at room temperature and the remaining cells were washed 3 times in HBSS. The cells were resuspended in PBS (1.9 mM NaH2PO4, 8.1 mM Na2HPO4 and 0.9% NaCl) for injection. To stimulate DUC18 T cells in vitro, DUC18 splenocytes were resuspended in R10 2-mercaptoethanol at 5 x 106/ml and the cells were cultured with 0.5 µM tERK-I peptide in 10-cm tissue culture dishes. After 72 h of culture, the cells were split 1:1 with medium alone and incubated for an additional 24 h. On the day of adoptive transfer, activated DUC18 T cells were purified using Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden), washed 3 times in HBSS and resuspended in PBS for injection. For each experiment, the number of DUC18 cells was determined by the percentage of CD8+/Vß8.3 double-positive cells (49). DUC18 T cells constituted 90–95% of total activated splenocytes, and they showed the surface phenotype of effector T cells in that they were CD25high, CD44high and CD62Llow.

In vivo tumor transplantation, adoptive T cell transfer and measurement of tumor growth
Cultured CMS5 cells were harvested by trypsinization, washed 3 times in HBSS and resuspended in PBS at 15 x 106 cells/ml. Three million CMS5 cells were injected into BALB/c mice s.c. on the right and/or left flank. In some experiments, mice were also challenged with the same number of MethA, an irrelevant fibrosarcoma that does not express tERK-I. Tumors were allowed to grow for 4 or 8 days, at which point either naive or tERK-I peptide-stimulated DUC18 T cells were adoptively transferred i.v. Tumors were measured every 2 days using calipers. The measurements are expressed as the product of two orthogonal diameters (49).

Collagenase treatment of ex vivo tumor tissues and transplantation into DUC18 transgenic mice
Tumors were dissected and minced into small pieces, followed by collagenase treatment in HBSS containing 1 mg/ml collagenase (Sigma, St Louis, MO) and 10 µg/ml DNase I for 1.5 h at room temperature. Cells were collected by centrifugation and washed 3 times with HBSS and cultured in R10. Cultured cells were harvested by trypsinization, washed 3 times in HBSS, resuspended in PBS and transplanted into the hind flank of DUC18 transgenic mice as described above. Different tumor cell inoculations were tested, ranging from 0.5 to 2 x 106 cells/mouse.

FACS analysis of tumor cells
Cells from explanted tumor cultures were harvested as described above and incubated with biotin-conjugated anti-H-2Kd antibody (BD Biosciences) on ice for 30 min. The samples were then washed and incubated with streptavidin–phycoerythrin (Caltag, Burlingame, CA) for 30 min on ice. The cells were washed and resuspended in PBS containing 1% paraformaldehyde. FACS analysis was performed using a FACSCalibur. Results were analyzed using CellQuest 3.3 software (Becton Dickinson).

Detection of tERK-specific mRNA
The tERK gene contains a point mutation which creates a SfcI restriction site (CTAAAG to CTACAG, underlined italic letters indicate the site of mutation) (48). We took advantage of this site to determine the presence of tERK mRNA in various tumor cells. Total RNA was prepared from 5 x 106 cells from each tumor cell line or from splenocytes using TRIzol (Invitrogen). For the generation of single-strand cDNA, we performed reverse transcription with 2 µg of total RNA. The samples were mixed with first-strand buffer (50 mM Tris–HCl, pH 8.3, 75 mM KCl and 3 mM MgCl2), 10 mM DTT, 1.25 mM dNTPs and 1.5 µg of random hexamers (Invitrogen, Carlsbad, CA) in a volume of 18 µl. The samples were heated at 65°C for 5 min using a thermocycler (model PTC-100; MJ Research, Watertown, MA). After the temperature was lowered to 37°C, 1 µl Superscript II reverse transcriptase (Invitrogen) and 1 µl RNaseOut ribonuclease inhibitor (Invitrogen) were added, and incubated for 50 min. Enzymes were heat-inactivated at 95°C for 5 min. For the PCR reaction, 1/10th of the single-strand cDNA sample was mixed with PCR buffer (50 mM KCl, 10 mM Tris–HCl, pH 9, 1% Triton® X-100), 2 mM MgCl2, 0.4 µM of forward (TTGGCATCAATGACAT) and reverse (TGTGGCTACGTACTCTGTC) primers, 0.5 mM dNTPs, and Taq DNA polymerase (1 unit) (Promega, Madison, WI). The thermocycling conditions used were 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min. This PCR generates a 320-bp product from the normal ERK2 and tERK cDNAs. The PCR products were resolved in 2% agarose gel, excised and purified using the Geneclean II kit (Quantum Biotechnologies, Carlsbad, CA). The purified products were digested overnight at 25°C with SfcI restriction enzyme (New England Biolabs, Beverly, MA), which will cleave tERK, but not wild-type ERK2 cDNA, and generates 159- and 161-bp fragments. The products were resolved in 2% agarose gel and visualized by ethidium bromide staining.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
DUC18 splenocytes stimulated with tERK-I peptide have increased effector function and retain antigen specificity
To generate sufficient numbers of CD8+ T cells for adoptive immunotherapy in clinical settings, it will require ex vivo expansion of T cells. To reflect this clinical reality, we expanded naive DUC18 T cells in vitro with tERK-I peptide. Cells were stimulated for 24, 48, 72 and 96 h, and these activated DUC18 T cells were assessed for in vivo anti-tumor activity. All conditions augmented the ability of DUC18 T cells to eliminate tumors to a similar degree (data not shown). The 96-h stimulation led to the most enriched preparation of DUC18 T cells and in the highest recovery. Therefore, we utilized these cells for all subsequent studies.

The efficacy and antigen specificity of activated DUC18 T cells were examined by monitoring their effect on CMS5 and MethA, an irrelevant BALB/c-derived fibrosarcoma (50). Three million activated DUC18 T cells completely eliminated 4-day established CMS5 tumors within 6 days of T cell transfer, but had no effect on MethA (Fig. 1A). CMS5 tumor regression was evident within 4 days of T cell transfer. In contrast, naive DUC18 T cells required 6 days to show any effect and 8 days to eliminate these tumors (Fig. 1A). These results, as anticipated, show that activated DUC18 T cells have greater in vivo tumoricidal efficacy while retaining antigen specificity.



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Fig. 1. Ex vivo stimulated DUC18 T cells have increased in vivo anti-tumor efficacy. (A) Groups of BALB/c mice (n = 5) were s.c. transplanted with 3 x 106 CMS5 or MethA cells on the right flank. Four days later, these mice received PBS vehicle control, 3 x 106 naive DUC18 T cells or 3 x 106 activated DUC18 T cells i.v. DUC18 T cells were activated by culturing splenocytes for 96 h in the presence of 0.5 µM tERK-I peptide. Only in this experiment, BALB/c splenocytes were mixed with the activated DUC18 T cells to normalize the total number of cells with that of naive DUC18 splenocytes. (B) Groups of BALB/c mice (n = 5) were s.c. transplanted with 3 x 106 CMS5 cells on the right flank alone (triangles) or on both flanks (circles and squares). Eight days later, the mice received either PBS (circles) or 20 x 106 activated DUC18 T cells (squares and triangles). Tumor growth of each tumor on doubly transplanted mice is shown independently. Tumor growth was measured every 2 days as described in Methods. Means from three independent experiments are shown (total of 15 mice). The bars indicate SEM. Some of the values for the SEM are too small to be visible.

 
Activated DUC18 T cells successfully ablate 8-day CMS5 tumors, but not double 8-day tumors
We wanted to investigate the potency of these activated DUC18 T cells by utilizing them against larger tumors. Our laboratory previously showed that naive DUC18 T cells have no effect on CMS5 tumors that had been growing for >5 days (49). Based on these findings, efficacy of activated DUC18 T cells against 8-day tumors was tested. Although 3 x 106 T cells showed a mild effect, this dose was insufficient to eliminate the tumors (data not shown). Higher doses of 10– 15 x 106 DUC18 T cells were also insufficient to achieve complete tumor elimination (data not shown). It was found that 20 x 106 activated DUC18 T cells were required (Fig. 1B). The tumors in these mice had been completely eliminated, as no tumors grew back in these mice when they were monitored for an additional 2 months. To increase the total tumor load, but keep tumor size and composition the same, CMS5 cells were transplanted into the left and right flanks of BALB/c mice (double 8-day tumors). Eight days later, activated DUC18 T cells were infused. Although both tumors regressed slightly and the growth was temporarily stabilized, they resumed progressive growth (Fig. 1B). Forty million DUC18 T cells also failed to completely eliminate both tumors (data not shown). These results indicate that while tERK-I peptide-stimulated DUC18 T cells possess potent in vivo anti-tumor activity, there is a limitation to their effectiveness.

Regression followed by progressive growth of double 8-day tumors after two DUC18 T cell infusions
Cancer patients generally receive multiple infusions of T cells; therefore, we repeatedly administered 20 x 106 activated DUC18 T cells to examine how tumors on doubly transplanted mice might respond to such treatment. Two doses of activated DUC18 T cells were infused into double 8-day tumor-bearing mice over a period of 2 days and the tumor growth was monitored. Tumor regression in both flanks was observed in 12 of 15 mice within 10 days of the initial infusion (Fig. 2). In the 12 mice, both tumors became completely undetectable. Tumors in the remaining three mice also became very small, ~12 mm2, 10 days post-transfer; however, these tumors began to grow progressively thereafter. Interestingly, within 20 days post-initial T cell transfer, tumors grew back in nine of the 12 mice that had undetectable tumors on day 10 (Fig. 2). In double 8-day tumor-bearing mice, two infusions of T cells resulted in an apparent elimination of the tumors; however, the tumors recurred.



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Fig. 2. Two successive infusions of activated DUC18 T cells lead to regression followed by re-growth in double 8-day tumor-bearing mice. Groups of BALB/c mice (n = 5) were s.c. transplanted with 3 x 106 CMS5 cells on both flanks. Eight days later, the first infusion of PBS (circles) or 20 x 106 activated DUC18 T cells (squares) was administered. Two days later, the second infusion was given. The measured right and left tumor sizes are shown independently. Arrows indicate the days on which PBS or DUC18 T cells were infused. Means from three independent experiments are shown (total of 15 mice). The bars indicate SEM.

 
Relapsed tumor cells grow progressively in DUC18 transgenic mice
We hypothesized that these relapsed tumors are immune-escape variants. If they are indeed escape variants, DUC18 T cells should not recognize these cells. To test this hypothesis, these escape-variant cells were transplanted into DUC18 transgenic mice, which contain an abundant number of CMS5-specific T cells. We have previously shown that DUC18 transgenic mice can successfully reject CMS5 tumor challenge that would otherwise lead to tumor growth in BALB/c mice (49). In vitro cultured relapsed tumor cells were transplanted into DUC18 transgenic mice. Three different relapsed tumors from the mice that received two DUC18 T cell infusions were tested (Fig. 3). The cultured cells had the morphologic and growth characteristics of the original CMS5 cell line (data not shown). Various doses of relapsed tumor cells were transplanted, ranging from 0.5 to 2 x 106 cells, into DUC18 transgenic mice (Fig. 3A, data not shown). These cells grew progressively in DUC18 transgenic mice even when as few as 0.5 x 106 cells were transplanted (Fig. 3A). As expected, parental CMS5 cells failed to grow in DUC18 transgenic mice at any dose tested (Fig. 3A and data not shown). Further, preliminary results showed that when escape-variant cells were transplanted into BALB/c mice and grown for 8 days, activated DUC18 T cells failed to eliminate the tumors (data not shown). These results showed that the relapsed CMS5 tumors could no longer be recognized in vivo by DUC18 T cells.





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Fig. 3. Two infusions of activated DUC18 T cells lead to class I MHC+ antigen-loss variants. (A) Three different relapsed tumors from Fig. 2 were dissected, digested and in vitro cultured as described in Methods. A few days later, 0.5 x 106 relapsed tumor cells or parental CMS5 cells were s.c. transplanted into DUC18 transgenic mice (n = 5). Tumor growth was monitored as before. A representative of three independent experiments is shown. The bars indicate SD. (B) Relapsed tumors and parental CMS5 cells were analyzed by FACS for surface H-2Kd expression. The fluorescence level of an isotype control antibody is shown in the CMS5 panel. (C) Diagrams of the PCR reaction and PCR products. The SfcI recognition sequence (in upper case letters) and its cleavage site (arrow), and the expected fragments generated from the digestion reaction are shown. Bold italic letters indicate the site of mutation. F. primer and R. primer indicate forward and reverse primers respectively. (D and E) RT-PCR analysis of three different 8- and 12-day tumors (D), CMS5 cells, relapsed tumor cells from two (#1–3) and three (#4–6) DUC18 T cell infusion recipients, and BALB/c splenocytes (E) is shown. The PCR products from the different samples were restriction digested by SfcI. The larger size band indicates the presence of wild-type erk2 message and the smaller size band indicates the presence of tERK-specific message.

 
Relapsed tumor cells express high levels of MHC class I
Down-regulation of MHC class I molecules is one of the mechanisms utilized by tumors to escape from immune recognition (35). As tERK-I antigen is presented by H-2Kd molecules (48), class I (H-2Kd) surface expression on the re-grown tumor cells was examined by FACS analysis. Not only did the relapsed tumor cells homogenously express H-2Kd, the expression level was slightly higher than parental CMS5 cells (Fig. 3B). Therefore, the immune escape by the relapsed tumor cells was not due to down-regulation of MHC class I expression.

Eight- and 12-day tumors express tERK-specific mRNA, but the relapsed tumors do not
To ensure that progressively growing CMS5 tumors are still expressing tERK, we examined its expression in 8-day tumors. Although it is not currently possible to distinguish the tERK protein from the normal ERK2 kinase protein, the point mutation in the erk2 kinase gene (48) creates a unique SfcI restriction site (Fig. 3C) and this was utilized to assay the presence of tERK-specific message in the 8-day tumors. RT-PCR was followed by restriction digest mediated by SfcI. The RT-PCR generates a 320-bp product from both tERK and wild-type ERK2 messages. SfcI-mediated digestion produces 159- and 161-bp fragments only from the PCR product generated from tERK-specific message (Fig. 3C). When electrophoresis is performed, these two fragments are seen as a single band. As a result, two products of 320 and 160 bp are observed (Fig. 3D and E). mRNA from three different 8-day tumors was tested by RT-PCR for the presence of tERK-specific message. All three samples expressed the expected smaller size band, indicating that tERK mRNA is expressed in these tumors cells (Fig. 3D). Moreover, we tested for the presence of tERK in 12-day progressively growing CMS5 tumors. Again, they were all positive for tERK (Fig. 3D). These data indicate that tERK expression is maintained in these large progressively growing tumors,.

Although CMS5 was found to be highly resistant to the development of antigen-loss variants in vitro or in vivo (48), the possibility was examined in some of the relapsed tumors from Fig. 2. We also tested the relapsed tumors that developed in mice that had been treated with three DUC18 T cell infusions (Fig. 4A). As before, RT-PCR analysis was carried out and we used cultured CMS5 cells as a control. From CMS5, as expected, both products were observed, indicating the presence of tERK mRNA (Fig. 3E). Importantly, none of the relapsed tumor cells contained the tERK-specific message, as evidenced by the lack of the 160-bp product (Fig. 3E). These data indicate that the relapsed tumor cells do not express detectable levels of mRNA for tERK and suggest that transferred DUC18 T cells may have contributed to the generation of antigen-loss variants.



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Fig. 4. Four infusions of activated DUC18 T cells successfully eliminated double 8-day tumors. (A and B) Groups of BALB/c mice (n = 5) were s.c. transplanted with 3 x 106 CMS5 cells on both flanks. Eight days later, 20 x 106 activated DUC18 T cells were administered. T cell administrations were performed every 2 days The mice received a total of three infusions in (A), whereas four administrations were given to the group of mice in (B). The arrows indicate the days on which the infusions were performed. Left and right tumors were measured, and are shown independently. Means from three independent experiments are shown (total of 15 mice). The bars indicate SEM.

 
Four infusions of activated DUC18 T cells eliminate double 8-day tumors before the development of antigen-loss variants
The above finding that CMS5 cells develop antigen-escape variants during immunotherapy raised the important issue of whether such an escape variant was an inevitable consequence of immunotherapy by mono-specific T cells. We reasoned that if CMS5 tumor masses were rapidly eliminated, it might prevent the appearance of antigen-escape variants. Double 8-day tumors were treated with three or four administrations of activated DUC18 T cells every 2 days (Fig. 4). When three doses were given, tumors in all the mice (n = 15) regressed and became undetectable. Similar to the mice receiving two doses of T cells (Fig. 2), six out of the 15 mice re-grew tumors by the end of the experiment (Fig. 4A). There was, however, a demonstrable improvement in that the number of tumor re-growth incidences decreased and the timing of recurrence was delayed (Fig. 4A) in comparison to the mice that received two DUC18 T cell infusions (Fig. 2). Of interest, the tumors that re-grew in this group were also found to be antigen-loss variants (Fig. 3E). When four doses were administered to the double 8-day tumor-bearing mice, all of the tumors completely regressed and showed no re-growth. To ensure that the CMS5 tumors had been completely eliminated, all of the mice were monitored for an additional 2 months after the T cell treatment (Fig. 4B and data not shown). None of the mice re-grew any tumors. These findings demonstrate that the development of antigen escape-variants can be avoided if a vigorous immunotherapy regimen is followed.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Using the CMS5/DUC18 system, we sought to investigate the efficacy of mono-specific CD8+ T cells in the elimination of established tumors. Whether monoclonal populations of CD8+ T cells can target a single tumor antigen and successfully ablate large established tumors before escape variants develop has not been determined, and the development of escape variants remains as one the major obstacles in immunotherapy. We established a system that requires multiple infusions of DUC18 T cells, a system that closely resembles clinical practice. By using double 8-day tumors, we were able to examine tumor response to multiple infusions of 20 x 106 activated DUC18 T cells. When two or three infusions of activated DUC18 T cells were administered, tumors regressed to an undetectable level, but they re-grew in some mice. These tumors were found to be MHC class I+ antigen-loss variants as they grew in DUC18 transgenic mice and lacked tERK-specific mRNA expression. Most importantly, four administrations of DUC18 T cells led to successful ablation of double 8-day tumors.

In our system, antigen-loss variants developed when two or three DUC18 T cell infusions were given. We did not extensively examine all the mice that had the tumors by the end of our experiments. We investigated six relapsed tumors for the presence of tERK mRNA and all of these were found to be tERK antigen-loss variants. Whether the rest of the tumors are antigen-loss variants or not does not change our general conclusion that large established tumor can be successfully eliminated by mono-specific CD8+ T cells. Our findings show that it is imperative to repetitively administer large numbers of tumor-specific T cells in a short period and this is in agreement with the previously reported study by Speiser et al. (51). Although it is seemingly obvious to achieve greater tumor reduction with more T cell administrations, we demonstrated that an aggressive treatment as such would lead to killing more tumor cells and elimination of large established tumors before tumor immune–escape variants develop.

One of the major limitations in T cell immunotherapy is the development of immune-escape variants. The mechanism of tumor escape can be divided into four major categories: (i) lack or insufficient tumor-rejection antigen recognized by the CTL that are generated, (ii) inhibition of CTL activity by the tumor microenvironment, (iii) failure of CTL to localize to the tumor site and (iv) failure of CTL to be sustained at the tumor site (35). It is likely that all these factors are involved in generating an extremely complex environment in which lymphocytes have to mount anti-tumor responses.

Several studies have been reported on the appearance of tumor-escape variants (38,39,42,43,52,53). In these studies, tumors were transplanted into immunocompetent animals, patients were administered with melanoma-associated antigen peptides to induce host immune responses or escape variants were selected in vitro by utilizing CTL clones. The kinetics of antigen-loss variant development in our system was surprisingly fast. Within 20 days of the initial transfer, the appearance of antigen-loss variants was clearly evident. This could imply that 8-day tumors could have contained a small number of pre-existing tERK-I escape variants. When DUC18 transgenic mice received s.c. inoculations of CMS5 cells grown from 8-day tumors taken from BALB/c mice that did not receive any DUC18 T cells, tumors did not grow in the transgenic mice. Similarly, CMS5 cells that were grown from 12-day tumors were completely rejected in DUC18 transgenic mice (data not shown). These data could be interpreted as such that the selective pressure exerted by immunocompetent hosts was not strong enough to create tERK-I-specific antigen-loss variants in the time frame tested. This would be in agreement with the study reported by Ikeda et al. (48). Alternatively, there may be very low levels of pre-existing antigen-loss variants in our CMS5 cell culture. To this end, we have previously demonstrated that inoculation of CMS5 cells into DUC18 transgenic mice results in complete rejection of tumors and the mice remained tumor-free for 12 months (49). Paradoxically, escape variants do not arise in DUC18 mice despite their slower CMS5 tumor-rejection kinetics (10–12 days) compared to the adoptive transfer system (8 days). We must point out that there is an abundant supply of CMS5-specific T cells in DUC18 mice and the tumor load when giving CMS5 cells into DUC18 mice is not the same as treating 8- or 12-day established tumors. Regardless, at this point, we cannot completely preclude the possibility of pre-existing antigen-loss variants in CMS5 cell culture or in 8- and 12-day established CMS5 tumors. It is conceivable at this time that because the frequency is very low, pre-existing variants are simply eliminated through bystander killing mechanisms. Further, these variants may be at a disadvantage in competing for available nutrients and growth factors during the regular growth phase. However, as DUC18 T cells eliminate tERK-expressing cells, pre-existing antigen-loss variants could have gained growth advantage that led to its out-growth.

How tumors efficiently and rapidly modulate their gene and protein expression is currently not clear. A spontaneous mitotic recombination that results in a loss of heterozygosity has been documented in an in vitro system (54) and this may be the mechanism by which tERK-I antigen-loss variants were generated. Another possibility may be through changes in chromosomal structure that leads to transcriptional repression. However, because the wild-type ERK2 sequence is present in these antigen-loss variants, we cannot explain the specificity of repression at this point. Alternatively, in response to increased environmental stress induced by the adoptively transferred DUC18 T cells, activities of the members of the heat shock protein family may have increased and buffered tERK expression. Buffering of gene expression by heat shock protein has been reported (55). In plants and animals, in response to particular environmental stress, some genes may be revealed or concealed, mediated by heat shock protein and, if some of the changes in gene expression could be selected as a trait, it is possible that such a mechanism may be operative in creating antigen-loss variants because it would be advantageous for tumor cells to adopt a phenotype that would lead to their survival. However, the molecular mechanism of such a selection process is not clear. Also, like the transcriptional repression hypothesis, one cannot explain the specific regulation of mutant ERK2 expression by this mechanism. Perhaps the simplest explanation is that tERK protein is not essential for maintaining the tumorigenic phenotype of CMS5 cells. Therefore, it was advantageous for CMS5 to lose expression of tERK, but because ERK2 kinase is essential for cell growth/signaling/survival, its expression was maintained. Undoubtedly, the elucidation of mechanisms of tumor immune escape can provide a critical insight into the T cell–tumor interaction, which would greatly aid in designing effective therapeutic strategies against cancer.

Currently, data on the optimal infusion regimen and on the number of T cells to be infused are largely undetermined (30,32). Rosenberg et al. utilized an average of 10 x 109 highly active melanoma antigen gp100-specific CD8+ T cells per infusion for each patient and four administrations were performed (33). In this study, a 50% reduction of tumor lesions was observed in responding patients. The number of T cells we administered to mice (4 x 109/kg) was in the same range as that given to patients (0.5 x 109/kg ). DUC18 T cells were transferred every 2 days. In clinical settings, infusion is generally performed over 1–3 weeks (18,33). Unlike TCR transgenic systems, limitations exist in generating a large enough number of T cells for frequent infusions. In one study, however, one infusion per day was performed for almost every day up to five administrations (30). The patients in general did not respond favorably to the treatment, but the number of tumor-specific T cells constituted only 10–30% of 0.5 x 109 transferred cells. Although we cannot accurately compare the total tumor load and tumor status of our system with that of clinical settings, more T cells may be required to be administered as rapidly as possible. One important area of research that lacks any detailed analysis is the migration/homing pattern of adoptively transferred T cells (18,30,33). It would be of great interest to obtain more dynamic patterns of the kinetics of T cell migration. Determining how many of the infused T cells localized to the tumors and how chemokines influence this process are also important issues that remain to be examined.

Using a monoclonal population of CD8+ T cells, we successfully ablated large established tumors and prevented the occurrence of antigen-loss variants. We utilized tumor-specific CD8+ T cells from a TCR transgenic mouse, where the supply of tumor-specific CD8+ T cells is abundant. In clinical settings, the generation of a large number of tumor-specific T cells for frequent infusion may not always be feasible. The tumor-rejection antigen in CMS5 is derived from a mutated self-antigen. For many human tumors, tumor antigens have been identified which are non-mutated self-antigens. Therefore, the T cells may be tolerant, which will have to be circumvented. Also, the CMS5/DUC18 setup is a transplantable system and, in the future, our findings will have to be confirmed in a spontaneous tumor model. It is clear that adoptive T cell immunotherapy has great potential in treating a variety of tumors. However, even in a situation in which tumor-rejection antigen and tumor-specific T cells are known and available, one must be careful in choosing a modality of therapy.


    Acknowledgements
 
We thank Darren Kreamalmeyer, Donna Thompson and Stephen Horvath for their technical assistance. We greatly appreciate critical reading of the manuscript and discussions by Robert D. Schreiber, Silvia Kang, Lyse Norian and Gavin P. Dunn. Finally, we thank Jerri Smith for secretarial support in the preparation of this manuscript. This work is supported by funds from the National Institutes of Health (CA76464).


    Abbreviations
 
CTL—cytotoxic T lymphocyte

ERK2—extracellular signal-regulated kinase 2

tERK-1—tumor-expressing mutated ERK2


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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