Age-dependent tolerance to an endogenous tumor-associated antigen
Introduction
Many factors decrease immunosurveillance of tumors by cytotoxic T lymphocytes (CTL) impairing effective antitumor responses [reviews [1], [2], [3], [4]]. Tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells reduce effector functions of tumor-specific CTL. In addition, tumor cells secrete inhibitory cytokines and produce other factors associated with chronic inflammation that reduce antitumor responses. Finally, most tumor-associated antigens (TAAs) are derived from non-mutated self proteins, resulting in deletion of CTL with high avidity for TAAs. Thus, the T cells that may be most effective against tumors are deleted during negative selection in the thymus [5], [6], [7].
Experiments using transgenic mice expressing T-cell receptor (TCR) genes and transfected TAAs have elucidated many of the obstacles that prevent the development and function of tumor-reactive T cells. The influence of antigen-specific tolerance varies depending on the number and source of T cells [8], [9]. Examination of transferred cognate transgenic T cells into tumor-bearing hosts has determined the conditions for optimal effector function and may have general clinical applications [reviewed in Ref. [10]], but the function of a diverse T-cell repertoire differs from that of a T-cell clone [11]. In polyclonal systems, T-cell function varies with the diversity of the repertoire, the avidity for antigen-presenting cells, and the precursor population size [12], [13], [14]. Thus, it is not clear that the rules established from experiments using monoclonal T-cell systems with high affinity TCRs are applicable to polyclonal T-cell repertoires. The role of the antigen in controlling the T-cell response to tumors is also unclear. Many studies use transplantable tumors transfected with model antigens so that either transferred T cells or endogenous responses can be monitored [15], [16]. Although the endogenous T-cell responses to TAAs are typically weak and less amenable to experimentation [17] tumor model systems that employ the endogenous T-cell repertoire and natural TAAs may provide more relevant results to guide the design of tumor immunotherapies.
T-cell responses to TAAs encoded by endogenous retroviruses have been detected in both humans and mice [18], [19]. Gamma-, or Type C, retroviruses are the most common retroviral elements in the human genome [20] and several examples have also been identified in mouse tumors [21]. In one example, the gag gene from Friend leukemia virus encodes an H-2Dd-restricted CTL target on the leukemia cell line, FBL-3 [22]. The ecotropic endogenous Murine leukemia virus (MuLV) also encodes TAAs from both gp70 (SU) and p15E (TM) proteins of the env gene. Both I-Ab and I-Eb-restricted peptides from MuLV have been identified in LB27.4 cells, a hybridoma of A20 lymphoma cells (H-2d) and BW5147 cells (H-2b) [23]. A CTL epitope p15E604-611/H-2Kb was also identified from these cells [24] as well as from B16 tumor cells [25]. In CT26 tumor cells, a unique glycoslylated I-Ed-restricted epitope contributes to the CD4+ antitumor response [26], and CD8+ T cells respond to the dominant antigen gp70423–431/H-2Ld (the AH1 antigen) [18]. Gp70 mRNA and T-cell responses to gp70-derived antigens are also detectable in other murine tumor cells and models including the 4T1 mammary carcinoma [27], A20 lymphoma [28], and B16 and S91 melanoma cells [18], [29]. These findings suggest that the shared gp70 antigen is a bona fide TAA, since these epitopes are derived from a non-mutated self-antigen that is up-regulated during the transformation process. Thus, gp70 is an ideal antigen for the study of the importance of self-tolerance in antitumor immune responses directed against TAAs.
To determine the role of gp70 expression in the T-cell response to the CT26 transplantable tumor, we produced a gp70-deficient mouse. In young mice under 6 months of age, we found that gp70-deficient mice elicited more gp70-specific T cells that exhibited greater binding avidity than those elicited in gp70-sufficient mice. These T-cell responses were associated with prevention of tumor growth in about half of the gp70-deficient mice. These results suggest that, in spite of the suppressive factors and heterogeneity of tumors that influence the quality of antitumor responses, successful antitumor CTL responses can be elicited in the absence of endogenous TAA expression in normal tissues. Furthermore, vaccination of gp70-sufficient mice over 8 months of age did not produce detectable TAA-specific T cells, although responses to a foreign antigen were readily detected. We also detected gp70 mRNA expression in normal tissues of gp70-sufficient mice, particularly in mice older than 8 months. We propose that the age-related increase in expression of gp70 and subsequent T-cell tolerance occur with other TAAs and cognate T cells. These results may explain the variable results observed in the clinic with immunotherapy directed against some TAAs and may suggest criteria for selection of some TAAs over others for more successful immunotherapy.
Section snippets
Generation of BALB.B6 env−/− (gp70−/−, gp70-deficient) congenic mice and littermate controls (gp70+/+, gp70-sufficient)
All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of National Jewish Medical and Research Center. CB6F1/Cr [(BALB/cAnNCr × C57BL/6NCr)F1] male and BALB/cAnNCr female mice were purchased from the NCI-Frederick Animal Production Program, bred together, and the offspring were screened by PCR for heterozygosity surrounding the MuLV integration containing a functional gp70 gene at 28.71 Mb of Chr 5 in BALB/c mice using
Derivation of a gp70-deficient mouse on the BALB/c background
Previous work showed that, despite the detection of T-cell responses specific for the CT26 tumor, T cells cannot prevent tumor growth in normal BALB/c recipient mice [32]. Data from other investigators demonstrated that proteins from MuLV are expressed in BALB/c mice [35]. Therefore, we produced a gp70-deficient mouse strain to determine if the detectable, yet ineffective T-cell responses to the CT26 tumor are limited by normal tolerance mechanisms or by antigen non-specific suppressive
Discussion
Using congenic mice lacking the endogenous ecotropic MuLV that encodes the MHC class I and II immunodominant tumor antigens from the CT26 tumor, we showed that (1) endogenous T cells specific for a natural TAA successfully prevent tumor development and (2) changes in expression of TAAs may lead to changes in immunologic tolerance and the efficacy of corresponding immunotherapies. The absence of the genomic interval encoding gp70 resulted in an improved T-cell response against the CT26 tumor
Acknowledgments
We thank Jonathan M. Horowitz for the discussions on MuLV, Brian Kotzin for advice and microsatellite primer sets, the University of Virginia Mouse Genetics Core facility, especially Shama Satinover, for help with design of the mapping strategy to generate the congenic strain as well as technical aspects of microsatellite analysis, and Sarah Rowland for help with tumor protection experiments. This work was supported by NCI CA109560 and a seed grant from the Colorado Cancer League to J.E.S.,
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These authors contributed equally to this work.