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Significance Statement
Immune therapy is a promising approach for improving the treatment of cancer. However, the major obstacles in its successful application, the tumor-induced mechanisms that lead to immune-evasion, have not been satisfactorily resolved. Analysis of the immune status of a given tumor entity and identification of the obstructed immune checkpoints thus are crucial issues for the development of immune-therapeutic anti-cancer strategies. Due to the limited possibilities for analyzing the respective parameters in humans, suitable animal models should be of great value. However, animal models that allow the analysis of how the growth of naturally arising tumors can be controlled by inducing tumor antigen specific immune responses are scarce.
In this study we tested the suitability of our BALB/c mouse based transgenic WAP-T animal model for mammary carcinoma to study tumor antigen specific CD8+ T-cell responses during tumor growth and progression, as well as parameters which obstruct a successful immune response. In WAP-T mice, tumorigenesis is induced in the adult mammary gland by expression of viral oncogenes (the SV40 tumor antigens) in parity-induced epithelial mammary gland progenitor cells. Tumorigenesis and the ensuing invasive carcinomas have been well characterized in previous studies, and WAP-T mice have been validated by cross-species comparison as a suitable animal model for human triple-negative mammary carcinomas. For this study we extended our WAP-T mouse lines by WAP-TNP mice, in which the transgene additionally codes for the NP118-126-epitope contained within the nucleoprotein of lymphocytic choriomeningitis virus (LCMV), resulting in the expression of a chimeric T-Ag/NP protein (T-AgNP). We thus were able to compare immune responses against the “weak” (i.e. low affinity) T-cell epitopes of SV40 T-Ag expressed by WAP-T mice with those against the immune-dominant LCMV NP-epitope in T-AgNP expressed by WAP-TNP mice.
While immunization of WAP-T mice with SV40 did not induce a measurable CTL immune response, immunization of WAP-TNP mice with LCMV induced a strong response which led to transient tumor cell elimination. Most intriguingly, we found that WAP-TNP mice mount an endogenous immune response (i.e. without immunization) against the LCMV NP-epitope, as elimination of CD8+ T-cells by anti-CD8+ antibodies or by irradiation promoted the outgrowth of tumors in WAP-TNP mice. WAP-TNP tumor mice thus contain NP-epitope specific CD8+ T-cells, which, however, are only weakly active due to expression of the programmed death-1 protein (PD1), an important player in the PD1/PD-L1 axis of immune checkpoints obstructing CTL activity. Consequently, treatment of WAP-TNP tumor with anti-PD1 antibodies largely restored their activity. This is demonstrated by the experiment shown in Fig. 1, where we exchanged endogenous lymphocytes of WAP-TNP tumor mice by lymphocytes derived from different donor mice by adoptive transfer (see scheme in Fig. 1A). Fig. 1B, panel a shows the typical T-AgNP expression in nuclei of WAP-TNP mammary carcinoma cells (red dots). No T-AgNP expressing cells can be seen in tumor areas of WAP-TNP mice that had received lymphocytes from BALB/c mice infected with LCMV due to the presence of a highly active NP-epitope specific CTL population (Fig. 1B, panel b). Similarly, tumor areas from WAP-TNP mice, which had received lymphocytes from anti-PD1 treated WAP-TNP mice, were also devoid of T-AgNP expressing cells (Fig. 1B, panel c). Thus the anti-tumor activity of exhausted PD1 expressing CD8+ T-cells could be largely re-activated.
Comparative analysis of WAP-T and WAP-TNP mice thus is suited to analyze the parameters leading to immune evasion of tumors expressing “weak” (WAP-T) or “strong” (WAP-TNP) tumor antigen epitopes, and to test strategies for overcoming blockades in immune response. Furthermore, WAP-TNP mice show that in the case of tumors expressing a “strong” T-cell epitope, immunization strategies might be developed which, together with strategies blocking immune checkpoints might lead to successful tumor elimination.
Figure Legend
Elimination of T-AgNP expressing WAP-TNP tumor cells in mammary carcinomas via adoptive transfer of NP-specific CTLs from LCMV infected mice or of re-activated exhausted CTLs from WAP-TNP tumor mice. (A) Schematic display of immune therapies and (B) immune histologic analysis of T-AgNP expression in WAP-TNP tumor cells in mammary glands of acceptor mice, whose own immune cells were eliminated by sublethal irradiation with 4 Gy. Strong elimination of T-AgNP expressing cells is seen after transfer of immune cells either from LCMV infected BALB/c (b) as donors or from WAP-TNP tumor mice after “therapy” with anti-PD1 antibodies (c) as donors; untreated WAP-TNP tumor mice served as positive controls (a).
Journal Reference
Oncotarget. 2015 Nov 17;6(36):38487-503.
Bruns M1, Wanger J1, Utermöhlen O2, Deppert W1,3.
[expand title=”Show Affiliations”]1.Heinrich-Pette-Institute, Leibniz-Institute for Experimental Virology, Hamburg, Germany.
2. Institute for Medical Microbiology, Immunology and Hygiene, Medical Center and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.
3.Institute for Tumor Biology, University Medical Center Hamburg-Eppendorf (UKE), University of Hamburg, Hamburg, Germany.
[/expand]Abstract
In Simian virus 40 (SV40) transgenic BALB/c WAP-T mice tumor development and progression is driven by SV40 tumor antigens encoded by inducible transgenes. WAP-T mice constitute a well characterized mouse model for breast cancer with strong similarities to the corresponding human disease. BALB/c mice mount only a weak cellular immune response against SV40 T-antigen (T-Ag). For studying tumor antigen specific CD8+ T-cell responses against transgene expressing cells, we created WAP-TNP mice, in which the transgene additionally codes for the NP118-126-epitope contained within the nucleoprotein of lymphocytic choriomeningitis virus (LCMV), the immune-dominant T-cell epitope in BALB/c mice. We then investigated in WAP-TNP mice the immune responses against SV40 tumor antigens and the NP-epitope within the chimeric T-Ag/NP protein (T-AgNP). Analysis of the immune-reactivity against T-Ag in WAP-T and of T-AgNP in WAP-TNP mice revealed that, in contrast to wild type (wt) BALB/c mice, WAP-T and WAP-TNP mice were non-reactive against T-Ag. However, like wtBALB/c mice, WAP-T as well as WAP-TNP mice were highly reactive against the immune-dominant LCMV NP-epitope, thereby allowing the analysis of NP-epitope specific cellular immune responses in WAP-TNP mice. LCMV infection of WAP-TNP mice induced a strong, LCMV NP-epitope specific CD8+ T-cell response, which was able to specifically eliminate T-AgNP expressing mammary epithelial cells both prior to tumor formation (i.e. in cells of lactating mammary glands), as well as in invasive tumors. Elimination of tumor cells, however, was only transient, even after repeated LCMV infections. Further studies showed that already non-infected WAP-TNP tumor mice contained LCMV NP-epitope specific CD8+ T-cells, albeit with strongly reduced, though measurable activity. Functional impairment of these ‘endogenous’ NP-epitope specific T-cells seems to be caused by expression of the programmed death-1 protein (PD1), as anti-PD1 treatment of splenocytes from WAP-TNP tumor mice restored their activity. These characteristics are similar to those found in many tumor patients and render WAP-TNP mice a suitable model for analyzing parameters to overcome the blockade of immune checkpoints in tumor patients.
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