The mechanisms of CTL-mediated tumor regression in vivo remain to be fully understood. If CTL do mediate tumor regression in vivo by direct cytotoxicity, this may occur via two major effector mechanisms involving the secretion of perforin/granzymes and/or engagement of Fas by Fas ligand (FasL) expressed by the activated CTL. Although the perforin pathway has been considered the dominant player, it is unclear whether Fas-mediated cytotoxicity is additionally required for optimal tumor rejection. Previously, we produced H-2Ld-restricted CTL reactive against the CMS4 sarcoma, which expresses a naturally occurring rejection Ag recognized by these CTL and harbors a cytokine (IFN-γ plus TNF)-inducible, Fas-responsive phenotype. The adoptive transfer of these CTL to syngeneic BALB/c mice with minimal (day 3 established) or extensive (day 10 established) experimental pulmonary metastases resulted in strong antitumor responses. Here we investigated whether a FasL-dependent CTL effector mechanism was important for optimal tumor regression in this adoptive immunotherapy model. The approach taken was to compare the therapeutic efficacy of wild-type to FasL-deficient (gld) CTL clones by adoptive transfer. In comparison with wild-type CTL, gld-CTL efficiently mediated tumor cytolysis and produced comparable amounts of IFN-γ, after tumor-specific stimulation, as in vitro assessments of Ag recognition. Moreover, gld-CTL mediated comparably potent antitumor effects in a minimal disease setting, but were significantly less effective under conditions of an extensive tumor burden. Overall, under conditions of extensive lung metastases, these data revealed for the first time an important role for a FasL-dependent CTL effector mechanism in optimal tumor regression.

In this study, we developed a mouse model of adoptive immunotherapy reflecting immune recognition of syngeneic tumor cells naturally expressing an endogenous rejection Ag. Specifically, in a pulmonary metastases model, we examined the potency and maintenance of an antitumor CD8+ CTL response in vivo, as well as its effectiveness against an “extensive” tumor burden. The approach taken was to first generate tumor-specific CTL from mice challenged with the CMS4 sarcoma coadministered with anti-CTLA4 mAb, which has been shown to facilitate the induction of Ag-specific T cell responses in vivo. An H-2Ld-restricted nonamer peptide, derived from an endogenous murine leukemia provirus was identified as a CMS4-reactive CTL epitope based upon the following: CTL cross-recognition of another syngeneic tumor cell line (CT26 colon carcinoma) previously characterized to express that gene product; sensitization of Ag-negative lymphoblasts or P815 targets with the peptide; and by cold target inhibition assays. In vivo, the adoptive transfer of CMS4-reactive CTL (≥1 × 106) resulted in nearly the complete regression of 3-day established lung metastases. Furthermore, mice that rejected CMS4 following a single adoptive transfer of CTL displayed antitumor activity to a rechallenge 45 days later, not only in the lung, but also at a s.c. distal site. Lastly, the adoptive transfer of CTL to mice harboring extensive pulmonary metastases (>150 nodules) led to a substantial reduction in tumor burden. Overall, these data suggest that the adoptive transfer of tumor-specific CTL may have therapeutic potential for malignancies that proliferate in or metastasize to the lung.

p53 mutations are frequently found in human cancers and are often associated with the overexpression of wild-type (WT) protein or peptide sequences, supporting the notion that WT p53 epitopes may serve as potential targets for tumor immunotherapy. We have developed a cytotoxic T lymphocyte (CTL)/p53 tumor-associated antigen (TAA) model, based on immune recognition of a WT p53 determinant. WT p53-peptide-specific, major histocompatibility complex (MHC) classI-restricted CTL were produced from immunocompetent C57BL/6 (H-2b) mice after immunization with a previously defined WT p53 peptide (p53(232-240)) Epitope-specific CTL were then employed to identify syngeneic tumor cell populations expressing that antigenic determinant. Two syngeneic tumor cell lines, MC38 colon carcinoma and MC57G fibrosarcoma, were demonstrated to express the endogenous WT p53(232-240) determinant naturally, as defined by CD8 + CTL recognition. Cold-target inhibition assays confirmed that CTL-mediated lysis was due to immune recognition of the p53(232-240) peptide epitope. The p53(232-240)-specific CTL line did not lyse syngeneic normal cells (i.e., mitogen-activated splenocytes) in the absence of exogenous peptide, suggesting that the WT-p53-specific CTL could distinguish between tumor cells expressing self-TAA and normal host cells. We have demonstrated, for the first time, that the adoptive transfer of WT-p53-specific CTL to mice with established pulmonary metastasis resulted in antitumor activity in vivo. The ability to generate MHC-class-I-restricted CD8- CTL lines specific for a non-mutated p53 determinant from normal, immunocompetent mice, which display antitumor activity both in vitro and in vivo (by adoptive transfer), may have implications for the immunotherapy of certain p53-expressing malignancies.

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The incidence of pure red cell aplasia (PRCA) in patients with chronic kidney disease associated with the subcutaneous (sc) administration of epoetin alfa (EPREX®) began to increase in 1998. As part of an intensive investigation into the reasons for this increase, an in vivo model was developed to assess the ability of potential causative factors to stimulate an immune response to recombinant human erythropoietin (rHuEPO). Due to species differences in protein sequence and as well as antigen processing and/or presentation, animal models cannot fully predict immunogenicity in humans. However, changes in relative immunogenicity have been readily detected in animal models. Moreover, transgenic animals have been utilized to negate the effects due to sequence differences between species. The EPO sequence is highly homologous between mouse and man. We found it difficult to generate anti-EPO antibodies in both human EPO transgenic mice and two normal mouse strains (Balb/c and BD-F1). To mitigate the complicating factors associated with the transgenic model (e.g. difficulty breeding, improper regulation of EPO production), normal mouse strains were selected for the development of animal models. Mice were injected sc with rHuEPO weekly for 4–6 weeks. Serum samples were analyzed 1–2 weeks following the last injection to ensure complete clearance of the injected protein. Serum samples were screened by an ELISA and/or by a surface plasmon resonance (BIAcore) method. In animals injected with rHuEPO alone, anti-EPO antibodies were either absent or present at very low levels. The addition of an adjuvant to the immunization protocol was able to boost both the rate of occurrence and titer of the immune response and resulted in the generation of anti-EPO antibodies that, in most cases, recognized both human and mouse EPO. Some of the mice exhibited a dramatic reduction in hematocrit, suggesting that the anti-EPO antibodies neutralized endogenous EPO. The development of an anti-EPO antibody response in humans is an extremely rare event that cannot be reproduced in an animal model without significant manipulation of the system (e.g. use of an adjuvant with doses of EPO considerably higher than therapeutic levels). Nevertheless, the fact that anti-EPO antibodies can develop with an appropriate immunization protocol allows for this model to be used to evaluate factors that may potentiate an immune response to rHuEPO.