Scientific Report 2005

Immunology

DNA Vaccine–Induced Long-Lived T-Cell Memory Against Tumor Recurrence

Y. Luo, H. Zhou, J. Krueger, C. Kaplan, S.H. Lee, R. Xiang, R.A. Reisfeld

Long-lived T-cell memory is a major objective of vaccination against tumors because it provides continuous protection against tumor dissemination and recurrence. Key requirements for successful protection against tumors include an increased number of tumor antigen–specific CD8⁺ T cells in an immune host and the capability of CD8⁺ T memory cells to proliferate, secrete inflammatory antitumor cytokines, and repeatedly kill recurring tumor cells more effectively than naive CD8⁺ T cells do.

We achieved these objectives with a DNA vaccine that encodes Fos-related antigen 1 (Fra-1), a murine transcription factor. The antigen was fused to polyubiquitin, modified by cotransformation with a gene encoding secretory murine IL-18, and carried to secondary lymphoid organs (i.e., Peyer’s patches) by attenuated Salmonella typhimurium. This DNA vaccine was effective in 2 different breast carcinoma models and in a non–small lung carcinoma model. In syngeneic mice, it induced a strong antitumor immunity that could be maintained as a long-lived specific immune response and resulted in eradication of tumor metastases. In addition, IL-18 enhanced this immune response by activating both T cells and natural killer cells while upregulating expression of MHC class I antigens and promoting the differentiation of CD4⁺ T cells to type 1 helper T cells.

The vaccine also was effective in a therapeutic setting. It markedly suppressed growth and dissemination of established pulmonary metastases of both D2F2 breast and D121 non–small cell lung carcinomas. The primary mediators of this protective immunity were CD8⁺ T cells, which secreted IFN-γ, a proinflammatory cytokine associated with type 1 helper T cells, and were activated in both lymphoid and nonlymphoid tissues, especially in tumor tissues of lung and liver. Furthermore, our finding that a single vaccination can upregulate CD4⁺ T cells releasing IL-2 in Peyer’s patches supports the contention that the mechanism of tumor protection depends on CD4⁺ T cells and is mediated by effector functions of helper T cells.

We also showed that the vaccine induced the establishment and long-term maintenance of immunologic memory CD8⁺ T cells in successfully vaccinated mice. When passively transferred to mice with severe combined immunodeficiency disease, such memory cells maintained sufficient memory in the absence of tumor antigen to markedly suppress dissemination and growth of a lethal challenge of D2F2 breast tumor cells. Importantly, after vaccination, CD8⁺ T cells in the tumor microenvironment also migrated to nonlymphoid tissues such as lungs, where some of the T cells were found as long-lived, dormant memory T cells ready to respond to reencounter with the same tumor antigen. Strikingly, such lymphocytes released more IFN-γ and contained a higher percentage of CD8⁺ cytotoxic
T lymphocytes and memory CD8⁺ T cells than did their splenic counterparts, suggesting that these CD8⁺ T cells can play a key role in the cellular response against tumor metastases. In fact, these results point to the existence of a population of extralymphoid effector memory T cells poised for an immediate antitumor immune response.

On the basis of data in the literature that T-cell proliferation induced by IL-18 depends on IFN-γ, we examined the effect of IL-18 on T-cell turnover. We found that expression of secretory IL-18 in our vaccine induced the rapid turnover of CD8⁺ T memory cells in mice within 24 hours after immunization and that such cells can be maintained in lymphoid tissues as well as locally in lung tumor tissues. Interestingly, CD8⁺ T cells obtained from the lungs of our vaccinated mice proliferated and released IFN-γ after exposure to antigen in vitro. Consequently, we conclude that persistently activated T cells and memory CD8⁺ T cells in the lung can play a key role in the cellular immune response against tumor metastases.

We also demonstrated in our tumor models that a specific memory T-cell response is induced and maintained in the absence of tumor antigen. We showed that CD8⁺ T cells adoptively transferred from successfully vaccinated mice to syngeneic mice with severe combined immunodeficiency and parked there for 7 or 30 days could maintain an effective and long-lived memory in the absence of both tumor antigen and naive T cells. In fact, 6 of 8 of these successfully reimmunized mice lived 3 times longer than control mice did in the absence of any detectable tumor growth up to 56 days after challenge with tumor cells.

Taken together, our data indicate that an oral DNA vaccine encoding ubiquitinated Fra-1 and IL-18 can protect mice against a lethal challenge of murine breast cancer cells and of non–small cell lung carcinoma cells. Moreover, this vaccine can break T-cell tolerance to the Fra-1 self-antigen and generate a long-lived memory T-cell immune response against recurring breast cancer, which can be maintained constantly in both lymphoid and nonlymphoid organs in mice with severe combined immunodeficiency in the absence of tumor antigen.

Publications

King, D.M., Albertini, M.R., Schalch, H., Hank, J.A., Gan, J., Surfus, J., Mahvi, D., Schiller, J.H., Warner, T., Kim, K.M., Eickhoff, J., Kendra, K., Reisfeld, R.A., Gillies, S.D., Sondel, P. Phase I clinical trial of the immunocytokine EMD 273063 in melanoma patients. J. Clin. Oncol. 22:4463, 2004.

Lo, J.F., Zhou, H., Fearns, C., Reisfeld, R.A., Yang, Y., Lee, J.D. Tid1 is required for T cell transition from double-negative 3 to double-positive stages. J. Immunol. 174:6105, 2005.

Loeffler, M., Kruger, J.A., Reisfeld, R.A. Immunostimulatory effects of low-dose cyclophosphamide are controlled by inducible nitric oxide synthase. Cancer Res. 65:5027, 2005.

Luo, Y., Zhou, H., Mizutani, M., Mizutani, N., Liu, C., Xiang, R., Reisfeld, R.A. A DNA vaccine targeting Fos-related antigen 1 enhanced by IL-18 Induces long-lived T-cell memory against tumor recurrence. Cancer Res. 65:3419, 2005.

Mizutani, N., Luo, Y., Mizutani, M., Reisfeld, R.A., Xiang, R. DNA vaccines suppress angiogenesis and protect against growth of breast cancer metastases. Breast Dis. 20:81, 2004.

Niethammer, A.G., Wodrich, H., Loeffler, M., Lode, H.N., Emmerich, K., Abdollahi, A., Krempien, R., Debus, J., Huber, P.E., Reisfeld, R.A. Multidrug resistance-1 (MDR-1): a new target for T cell-based immunotherapy. FASEB J. 19:158, 2005.

Xiang, R., Mizutani, N., Luo, Y., Chiodoni, C., Zhou, H., Mizutani, M., Ba, Y., Becker, J.C., Reisfeld, R.A. A DNA vaccine targeting survivin combines apoptosis with suppression of angiogenesis in lung tumor eradication. Cancer Res. 65:553, 2005.

Zhou, H., Luo, Y., Lo, J.F., Kaplan, C.D., Mizutani, M., Mizutani, N., Lee, J.D., Primus, F.J., Becker, J.C., Xiang, R., Reisfeld, R.A. DNA-based vaccines activate innate and adaptive antitumor immunity by engaging the NKG2D receptor. Proc. Natl. Acad. Sci. U. S. A. 102:10846, 2005.

Zhou, H., Luo, Y., Mizutani, M., Mizutani, N., Reisfeld, R.A., Xiang, R. T cell-mediated suppression of angiogenesis results in tumor protective immunity. Blood, in press.