Therapeutic limitations in tumor-specific CD8+ memory T cell engraftment
© Bathe et al; licensee BioMed Central Ltd. 2003
Received: 07 April 2003
Accepted: 28 July 2003
Published: 28 July 2003
Adoptive immunotherapy with cytotoxic T lymphocytes (CTL) represents an alternative approach to treating solid tumors. Ideally, this would confer long-term protection against tumor. We previously demonstrated that in vitro-generated tumor-specific CTL from the ovalbumin (OVA)-specific OT-I T cell receptor transgenic mouse persisted long after adoptive transfer as memory T cells. When recipient mice were challenged with the OVA-expressing E.G7 thymoma, tumor growth was delayed and sometimes prevented. The reasons for therapeutic failures were not clear.
OT-I CTL were adoptively transferred to C57BL/6 mice 21 – 28 days prior to tumor challenge. At this time, the donor cells had the phenotypical and functional characteristics of memory CD8+ T cells. Recipients which developed tumor despite adoptive immunotherapy were analyzed to evaluate the reason(s) for therapeutic failure.
Dose-response studies demonstrated that the degree of tumor protection was directly proportional to the number of OT-I CTL adoptively transferred. At a low dose of OT-I CTL, therapeutic failure was attributed to insufficient numbers of OT-I T cells that persisted in vivo, rather than mechanisms that actively suppressed or anergized the OT-I T cells. In recipients of high numbers of OT-I CTL, the E.G7 tumor that developed was shown to be resistant to fresh OT-I CTL when examined ex vivo. Furthermore, these same tumor cells no longer secreted a detectable level of OVA. In this case, resistance to immunotherapy was secondary to selection of clones of E.G7 that expressed a lower level of tumor antigen.
Memory engraftment with tumor-specific CTL provides long-term protection against tumor. However, there are several limitations to this immunotherapeutic strategy, especially when targeting a single antigen. This study illustrates the importance of administering large numbers of effectors to engraft sufficiently efficacious immunologic memory. It also demonstrates the importance of targeting several antigens when developing vaccine strategies for cancer.
Cytotoxic T lymphocytes (CTL) represent attractive immunotherapeutic effectors for cancer, as they are specific and potent killers of all cells that bear the target antigen in the context of a class I MHC molecule [1–4]. However, like other immunological strategies, they infrequently effect cure of established tumors. Rather, tumor-specific CTL are most likely to succeed in the presence of minimal disease, where the primed immune effectors keep the disease at bay or eliminate residual malignant foci. To utilize CTL in this way, they must survive for long periods after adoptive transfer as memory cells, ready to mount a response at the earliest sign of recurrence.
Recently, we described a mouse model in which tumor-specific CTL generated in vitro persisted long after adoptive transfer into syngeneic mice, with the phenotypic and functional characteristics of memory cells . In this model, we utilized CD8+ T cells originating from OT-I T cell receptor (TCR) transgenic mice that recognize ovalbumin (OVA) in the context of class I MHC molecules. While adoptive transfer of OT-I CD8+ T cells conferred long-term protection against the OVA-expressing E.G7 thymoma, complete protection was not observed. The reasons for the limited therapeutic efficacy of adoptive immunotherapy targeting a single model tumor antigen were explored.
OT-I TCR transgenic mice  were maintained by breeding heterozygous OT-I TCR transgenic mice to wild-type C57BL/6J mice. The progeny were screened by PCR for the expression of the TCR transgene. All recipient mice were C57BL/6J mice aged 6 – 9 weeks, purchased from Jackson laboratories (Bar Harbor, ME). Mice were treated in accordance with the guidelines established by the University of Miami Animal Care and Use Committee.
EL-4, a thymoma that was derived from the C57BL/6 mouse (H-2b), was obtained from the American Type Culture Collection (ATCC; Rockville, MD). E.G7 cells are EL-4 cells transfected with OVA cDNA , and these were a gift from Dr. M. Bevan (University of Washington, Seattle, WA). These cell lines were maintained in complete medium (CM), consisting of RPMI 1640 containing 5% FCS, glutamine (30 μg/mL), penicillin (100 U/mL), streptomycin (100 μg/mL), and β-mercaptoethanol (5 × 10-5 M).
Antibodies and other reagents
OVA257–264 peptide (SIINFEKL)  was synthesized by Research Genetics (Huntsville, AL). Directly conjugated monoclonal antibodies included Cychrom-anti-CD8α, PE-conjugated anti-Vα2-TCR, FITC-Vβ5.1,5.2-TCR (Pharmingen, San Diego, CA), FITC-anti-CD8α (53.6.7). Antibodies for ELISA for measurement of ovalbumin consisted of anti-chicken albumin and horseradish peroxidase-labeled anti-chicken albumin (Rockland, Gilbertsville, PA).
Adoptive immunotherapy model
OT-I CTL were generated by stimulation with 1 nM OVA peptide (OVA257–264), IL-2 (50 U/mL) and IL-4 (175 U/mL), as previously described . On the fifth day of culture, OT-I CTL were injected via tail vein in a volume of 0.5 mL HBSS, into normal C57BL/6J mice. Tumor cells (E.G7 or EL-4, 1 × 106 in 0.2 mL HBSS) were injected subcutaneously in the lower abdomen 21 – 28 days after adoptive transfer of OT-I CTL. The tumor cells were freshly thawed within 6 days of inoculation.
Phenotypical and Functional Analyses
Cell surface phenotypes were determined by flow cytometry, on a FACScan flow cytometer (Becton-Dickinson, San Jose, CA). CTL activity was measured with a 5-h 51Cr-release assay, using E.G7 and EL-4 cells as targets. T cell proliferation was assessed by thymidine uptake assay. The methods for each of these analyses has been previously described .
Adoptively transferred OT-I CTL protect against inoculation with ovalbumin-expressing tumor
On the day of adoptive transfer, OT-I splenocyte cultures typically consisted of greater than 90% CD8+ cells that co-expressed Vα2-TCR and Vβ5.1,5.2-TCR. As previously shown, on the day of injection, the activated cells typified effector T lymphocytes. These effector cells were potently and specifically cytotoxic against ovalbumin-expressing targets in vitro .
Analysis of therapeutic failures
Potential reasons for failure of protection from tumor following adoptive transfer include insufficient effector-to-target ratio, anergy, or inability of effector T cells to recognize tumor as it progresses. To analyze treatment failures, mice were sacrificed at 21 – 28 days for baseline analysis of the function of persistent T cells, or within two days of developing a tumor measuring 7.0 mm in maximal diameter.
Several clones shown to secrete detectable amounts of ovalbumin were expanded. One of these clones was tested in an in vitro cytotoxicity assay using freshly generated OT-I CTL, and it served as an excellent target (Figure 4). Moreover, recipients of 10 × 106 OT-I CTL were challenged with this E.G7 clone. When these freshly cloned tumor cells were utilized as the target, the therapeutic outcome was markedly better compared to mice treated with the old E.G7 cells. Only one mouse in 6 developed tumor and this tumor did not appear until 21 days after tumor inoculation (data not shown). Moreover, OT-I CD8+ T cells primed under various conditions were tested over several experiments; cure rates were generally better when the freshly cloned E.G7 was utilized to inoculate recipients .
Altogether, the data suggest that one mechanism of therapeutic failure, typically seen in animals treated with higher numbers of OVA-specific CTL, is selection of tumor cells that produced insufficient amounts of antigen to be recognized by effectors. This mechanism of therapeutic failure did not appear to be responsible for the earlier tumor growth seen in animals treated with low numbers of CTL. Rather, it is more likely that insufficient effector-to-target ratios were responsible for therapeutic failures in these animals.
Since a large tumor burden is a potential obstacle to successful immunotherapy, it is likely that the role of immunotherapy will be most pronounced in the presence of minimal disease. Clinically, this strategy might then be best applied as adjuvant therapy following resection or after cytoreductive chemotherapy. Therefore, cellular immunotherapy must be designed to respond to recurrence or progression of minimal disease over a long period of time. This would require engraftment of immunologic memory. While our model does not perfectly reflect the clinical situation of minimal residual disease, it does enable the assessment of how effectively tumor-specific T cell memory engraftment prevents tumor emergence. T cells administered well before tumor challenge persisted long after adoptive transfer and they responded readily to re-stimulation by antigen. Moreover, their in vivo activity was demonstrable by their ability to provide long-term protection against tumor expressing the target antigen, in a dose-dependent manner. Unfortunately, even in what appeared to be an idealized experimental situation, responses were incomplete and nondurable. This was secondary to a very powerful selective influence, culminating in the emergence of antigen-loss variants.
According to the immune surveillance theory, cancer arises when the immune system is unable to recognize individual cancer cells, enabling them to escape detection. While this postulate does not completely explain the pathogenesis of cancer, escape from the immune system is an important factor. Indeed, tumor cells escape by a number of mechanisms. Peripheral tolerance to tumor antigens may exist [11, 12]. Immune precursors may be ignorant of tumor because of malpresentation , MHC downregulation [13, 14] or insufficient antigen expression [15, 16]. As tumor evolves, the immune system sequentially and consecutively eliminates cells expressing certain antigens in order of degree of immunodominance [16–19], diminishing tumor immunogenicity. MHC downregulation appears to be particularly detrimental to the anti-tumor immune response, as this results in permanent escape from immune detection .
Even when tumor cells are recognized by effectors, a number of processes may interfere with their clearance. For example, insufficient costimulation or other mechanisms may lead to anergy  or blunted responsiveness of tumor-specific effectors [21, 22]. Induction of apoptosis of potentially hostile lymphocytes by tumor has also been described [23–25]. Thus, in addition to escape from detection by the immune system, tumor may directly influence immune effector function.
Some of the mechanisms that enable escape from the immune system in its natural state may also be responsible for treatment failures following immunotherapy. In the idealized experimental system described in the present work, therapeutic failures predictably occurred when effector-to-target ratios were insufficient in vivo. This problem was easily overcome by administration of greater numbers of activated CTL. However, in the clinical situation, where CTL are generated in non-transgenic individuals, generation of such high numbers of CTL may be problematic. When vaccination strategies are employed (using modified tumor cells or dendritic cells, for example), generating sufficient numbers of CTL may be particularly difficult, even following booster vaccinations. It will therefore be imperative to get a better understanding of the factors that improve survival of tumor-specific CTL and enhance differentiation to memory cells.
A second important limitation of adoptive immunotherapy that became apparent in the presence of higher effector-to-target ratios was selection for antigen loss variants. It may be argued that the model described in the present paper is not truly reflective of the situation seen with a normal cancer cell. That is, the E.G7 cell has been transfected with a foreign gene that has inherent instability and this may be responsible for the antigen loss. On the other hand, data from others have shown that, in general, cancer cells are typically genetically unstable. As seen in the adenoma-colorectal cancer sequence, by the time of clinical manifestation, many tumors have accumulated thousands of mutations [26–28]. As a result, antigen expression would be expected to be heterogeneous in any given tumor. Therefore, in the model presented (where 85% of input cells expressed the target antigen), the heterogeneity of tumor cells is probably considerably less than what might be expected in the real situation, emphasizing the magnitude of the problem of emergence of antigen loss variants.
Targeting a single antigen is not likely to succeed unless that antigen is necessary for the function and survival of the cancer cell. This situation is rare, although one example is BCR-ABL, a constitutively activated tyrosine kinase that causes chronic myeloid leukemia (CML). Targeting such a protein, albeit pharmacologically, has met with therapeutic success . In the absence of an appropriate pharmacological agent, targeting such an antigen immunologically would be expected to be successful. Unfortunately, few such targets have so far been identified and so other strategies are required to overcome the problem of emergence of antigen loss variants. Undoubtedly, targeting multiple antigens will ameliorate part of the problem [30–32], although the probability of success with this approach has not yet been quantified. Another potential solution is the concomitant utilization of approaches with more bystander effect. For example, MHC non-restricted effectors such as NK or LAK cells may be helpful [33, 34], particularly in tumors where loss of MHC expression has occurred. Alternatively, biological response modifiers or cytotoxic agents could be administered concomitantly. The problem of emergence of antigen loss variants is therefore not insurmountable.
We have demonstrated two obstacles to success with tumor-specific CD8+ memory T cell engraftment. Firstly, sufficient persistence of functional tumor-specific CTL over long periods will prove a potential problem in non-transgenic individuals and when vaccination strategies are utilized. Secondly, targeting a single antigen by cellular immunotherapy is not likely to be successful because of selective emergence of antigen loss variants. While we are certainly not the first to describe antigen loss variants [15, 35, 36, 16, 37], this study is important in that it demonstrates unequivocally the magnitude of the problem, even in an idealized experimental system, in the presence of minimal disease burden. This is especially topical given the number of vaccines in development and in clinical trials that are reliant on a single antigen expressed by a given tumor. Clearly, targeting multiple antigens is essential to success. Moreover, treatment with multiple therapeutic modalities may prove to be best.
We thank Aixin Yu and Paul Scibelli for technical assistance. This research was supported by the Department of Defense (DAMD17-98-1-8208) the National Institute of Health (R01-AI40114).
- Liu B, Podack ER, Allison JP, Malek TR: Generation of primary tumor-specific CTL in vitro to immunogenic and poorly immunogenic mouse tumors. J Immunol. 1996, 156: 1117-1125.PubMedGoogle Scholar
- Tsai V, Southwood S, Sidney J, Sakaguchi K, Kawakami Y, Appella E, Sette A, Celis E: Identification of subdominant CTL epitopes of the GP100 melanoma-associated tumor antigen by primary in vitro immunization with peptide-pulsed dendritic cells. J Immunol. 1997, 158: 1796-1802.PubMedGoogle Scholar
- Alexander-Miller MA, Leggatt GR, Berzofsky JA: Selective expansion of high- or low-avidity cytotoxic cytotoxic T lymphocytes and efficacy for adoptive immunotherapy. Proc Natl Acad Sci. 1996, 93: 4102-4107. 10.1073/pnas.93.9.4102.View ArticlePubMedPubMed CentralGoogle Scholar
- Dalyot-Herman N, Bathe OF, Malek TR: Reversal of CD8+ T cell ignorance and induction of anti-tumor immunity by peptide-pulsed antigen presenting cells. J Immunol. 2000, 165: 6731-6737.View ArticlePubMedGoogle Scholar
- Bathe OF, Dalyot-Herman N, Malek TR: IL-2 during in vitro priming promotes subsequent engraftment and successful adoptive tumor immunotherapy bypersistent memory phenotypic CD8+ T cells. J Immunol. 2001, 167: 4511-4517.View ArticlePubMedGoogle Scholar
- Carbone FR, Sterry SJ, Butler J, Rodda S, Moore MW: T cell receptor a-chain pairing determines the specificity of residue 262 within the Kb-restricted, ovalbumin257-264 determinant. Int Immunol. 1992, 4: 861-867.View ArticlePubMedGoogle Scholar
- Moore MW, Carbone FR, Bevan MJ: Introduction of soluble protein into the Class I pathway of antigen processing and presentation. Cell. 1988, 54: 777-785.View ArticlePubMedGoogle Scholar
- Pihlgren M, Dubois PM, Tomkowiak M, Sjogren T, Marvel J: Resting memory CD8+ T cells are hyperreactive to antigenic challenge in vitro. J Exp Med. 1996, 184: 2141-2151.View ArticlePubMedPubMed CentralGoogle Scholar
- Curtsinger JM, Lins DC, Mescher MF: CD8+ memory T cells(CD44high, Ly6C+) are more sensitive than naive cells (CD44low, Ly6C-) to TCR/CD8 signaling in response to antigen. J Immunol. 1998, 160: 3236-3243.PubMedGoogle Scholar
- Veiga-Fernandes H, Walter U, Bourgeois C, McLean A, Rocha B: Response of naive and memory CD8+ T cells to antigen stimulation in vivo. Nature Immunol. 2000, 1: 47-53. 10.1038/76907.View ArticleGoogle Scholar
- Staveley-O'Carroll K, Sotomayor E, Montgomery J, Borrello I, Hwang L, Fein S, Pardoll D, Levitsky H: Induction of antigen-specific T cell anergy: an early event in the course of tumor progression. Proc Natl Acad Sci. 1998, 95: 1178-1183. 10.1073/pnas.95.3.1178.View ArticlePubMedPubMed CentralGoogle Scholar
- Ye X, McCarrick J, Jewett L, Knowles BB: Timely immunization subverts the development of peripheral nonresponsiveness and suppresses tumor development in simian virus 40 tumor antigen-transgenic mice. Proc Natl Acad Sci USA. 1994, 91: 3916-3920.View ArticlePubMedPubMed CentralGoogle Scholar
- Seliger B, Hohne A, Jung D, Kallfelz M, Knuth A, Jaeger E, Bernhard H, Momburg F, Tampe R, Huber C: Expression and function of the peptide transporters in escape variants of human renal cell carcinomas. Exp Hematol. 1997, 25: 608-614.PubMedGoogle Scholar
- Smith ME, Marsh SG, Bodmer JG, Gelsthorpe K, Bodmer WF: Loss of HLA-A,B,C allele products and lymphocyte function-associated antigen 3 in colorectal neoplasia. Proc Natl Acad Sci U S A. 1989, 86: 5557-5561.View ArticlePubMedPubMed CentralGoogle Scholar
- Schreiber K, Wu TH, Kast WM, Schreiber H: Tracking the common ancestry of antigenically distinct cancer variants. Clin Cancer Res. 2001, 7: 871s-875s..PubMedGoogle Scholar
- Jager E, Ringhoffer M, Karbach J, Arand M, Oesch F, Knuth A: Inverse relationship of melanocyte differentiation antigen expression in melanoma tissues and CD8+ cytotoxic-T-cell responses: evidence for immunoselection of antigen-loss variants in vivo. Int J Cancer. 1996, 66: 470-476. 10.1002/(SICI)1097-0215(19960516)66:4<470::AID-IJC10>3.0.CO;2-C.View ArticlePubMedGoogle Scholar
- Dudley ME, Roopenian DC: 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. 1996, 184: 441-447.View ArticlePubMedGoogle Scholar
- Seung S, Urban JL, Schreiber H: A tumor escape variant that has lost one major histocompatibility complex class I restriction element induces specific CD8+ T cells to an antigen that no longer serves as a target. J Exp Med. 1993, 178: 933-940.View ArticlePubMedGoogle Scholar
- Urban JL, Kripke ML, Schreiber H: Stepwise immunologic selection of antigenic variants during tumor growth. J Immunol. 1986, 137: 3036-3041.PubMedGoogle Scholar
- Chamberlain RS, Carroll MW, Bronte V, Hwu P, Warren S, Yang JC, Nishimura M, Moss B, Rosenberg SA, Restifo NP: Costimulation enhances the active immunotherapy effect of recombinant anticancer vaccines. Cancer Res. 1996, 56: 2832-2836.PubMedPubMed CentralGoogle Scholar
- Lee PP, Yee C, Savage PA, Fong L, Brockstedt D, Weber JS, Johnson D, Swetter S, Thompson J, Greenberg PD, Roederer M, Davis MM: Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients. Nat Med. 1999, 5: 677-685. 10.1038/9525.View ArticlePubMedGoogle Scholar
- Kerkmann-Tucek A, Banat GA, Cochlovius B, Zoller M: Antigen loss variants of a murine renal cell carcinoma: implications for tumor vaccination. Int J Cancer. 1998, 77: 114-122. 10.1002/(SICI)1097-0215(19980703)77:1<114::AID-IJC18>3.3.CO;2-#.View ArticlePubMedGoogle Scholar
- Ungefroren H, Voss M, Bernstrff WV, Schmid A, Kremer B, Kaltoff H: Immunological escape mechanisms in pancreatic carcinoma. Ann N Y Acad Sci. 1999, 880: 243-251.View ArticlePubMedGoogle Scholar
- Zaks TZ, Chappell DB, Rosenberg SA, Restifo NP: Fas-mediated suicide of tumor-reactive T cells following activation by specific tumor: selective rescue by caspase inhibition. J Immunol. 1999, 162: 3273-3279.PubMedPubMed CentralGoogle Scholar
- Hahne M, Rimoldi D, Schroter M, Romero P, Schreier M, French LE, Schneider P, Bornand T, Fontana A, Lienard D, Cerotti J, Tschopp J: Melanoma cell expression of Fas (Apo-1/CD95) ligand: implications for tumor immune escape. Science. 1996, 274: 1363-1366. 10.1126/science.274.5291.1363.View ArticlePubMedGoogle Scholar
- Boland CR, Ricciardiello L: How many mutations does it take to make a tumor?. Proc Natl Acad Sci U S A. 1999, 96: 14675-14677. 10.1073/pnas.96.26.14675.View ArticlePubMedPubMed CentralGoogle Scholar
- Stoler DL, Chen N, Basik M, Kahlenberg MS, Rodriguez-Bigas MA, Petrelli NJ, Anderson GR: The onset and extent of genomic instability in sporadic colorectal tumor progression. Proc Natl Acad Sci U S A. 1999, 96: 15121-15126. 10.1073/pnas.96.26.15121.View ArticlePubMedPubMed CentralGoogle Scholar
- Cho KR, Vogelstein B: Genetic alterations in the adenoma--carcinoma sequence. Cancer. 1992, 70: 1727-1731.View ArticlePubMedGoogle Scholar
- Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM, Lydon NB, Kantarjian H, Capdeville R, Ohno-Jones S, Sawyers CL: Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med. 2001, 344: 1031-1037. 10.1056/NEJM200104053441401.View ArticlePubMedGoogle Scholar
- Smith SG: The polyepitope approach to DNA vaccination. Curr Opin Mol Ther. 1999, 1: 10-15.PubMedGoogle Scholar
- Heiser A, Maurice MA, Yancey DR, Coleman DM, Dahm P, Vieweg J: Human dendritic cells transfected with renal tumor TNA stimulate polyclonal T-cell responses against antigens expressed by primary and metastatic tumors. Cancer Res. 2001, 61: 3388-3393.PubMedGoogle Scholar
- Boczkowski D, Nair SK, Nam J, Lyerly HK, Gilboa E: Induction of tumor immunity and cytotoxic T lymphocyte responses using dendritic cells transfected with messenger RNA amplified from tumor cells. Cancer Res. 2000, 60: 1028-1034.PubMedGoogle Scholar
- Lafreniere R, Rosenberg SA: Adoptive immunotherapy of murine hepatic metastases with lymphokine activated killer (LAK) cells and recombinant interleukin 2 (RIL 2) can mediate the regression of both immunogenic and nonimmunogenic sarcomas and an adenocarcinoma. J Immunol. 1985, 135: 4273-4280.PubMedGoogle Scholar
- Yasumura S, Lin W, Hirabayashi H, Vujanovic NL, Herberman RB, Whiteside TL: Immunotherapy of liver metastases of human gastric carcinoma with interleukin 2-activated natural killer cells. Cancer Res. 1994, 54: 3808-3816.PubMedGoogle Scholar
- Prevost-Blondel A, Roth E, Rosenthal FM, Pircher H: Crucial role of TNF-alpha in CD8 T cell-mediated elimination of 3LL-A9 Lewis lung carcinoma cells in vivo. J Immunol. 2000, 164: 3645-3651.View ArticlePubMedGoogle Scholar
- Hoffmann TK, Nakano K, Elder EM, Dworacki G, Finkelstein SD, Appella E, Whiteside TL, DeLeo AB: Generation of T cells specific for the wild-type sequence p53(264-272) peptide in cancer patients: implications for immunoselection of epitope loss variants. J Immunol. 2000, 165: 5938-5944.View ArticlePubMedGoogle Scholar
- Riker A, Cormier J, Panelli M, Kammula U, Wang E, Abati A, Fetsch P, Lee KH, Steinberg S, Rosenberg S, Marincola F: Immune selection after antigen-specific immunotherapy of melanoma. Surgery. 1999, 126: 112-120. 10.1067/msy.1999.98739.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/3/21/prepub
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