Mechanisms of immune evasion by tumors developing within the eyeDate Added: 6/20/2007 2:42:00 PM
Last Updated: 6/20/2007 2:59:00 PM
Description of projects available to graduate students:
Uveal and cutaneous melanomas express tumor antigens that are immunogenic (1), tumor growth induces tumor-specific CD8+ cytolytic T lymphocyte (CTL) responses (2,3), and vaccination of melanoma patients with melanoma-specific antigens further increases melanoma-specific CD8+ CTL in the circulation and within tumors (4-6). However, tumor regression rarely occurs in vaccinated cancer patients (7) suggesting that tumor-specific CD8+ CTL are somehow inhibited within the tumor microenvironment. Understanding the mechanisms of CTL inhibition within the tumor microenvironment is critical for the success of tumor vaccines and is the focus of this laboratory.
Primary uveal melanomas which are heavily infiltrated with CD68+ myeloid cells are associated with increased mortality (8) and CD68+ myeloid cells also accumulate in uveal melanoma liver metastases (9). The role of myeloid cells (monocytes, macrophages, and granulocytes) in uveal melanoma is not well understood. However, in other malignancies immunosuppressive myeloid cells, termed myeloid derived suppressor cells (MDSC) accumulate systemically and within tumors and inhibit CD8+ CTL responses. We hypothesize that myeloid cells associated with uveal melanomas are MDSC and contribute to tumor growth by suppressing tumor-specific CD8+ CTL responses.
We have developed a transplantable tumor model in mice which is ideal for understanding mechanisms of CD8+ CTL inhibition within the ocular-tumor microenvironment because tumors grow progressively in the eye but are rejected by CD8+ CTL in the skin though both routes prime for robust tumor-specific CD8+ CTL responses systemically (10). Moreover, the failure to eliminate tumors by CD8+ CTL responses is correlated with the accumulation of CD11b+ MDSC within the eye that inhibit CD8+ CTL responses in vitro (10). Nitric oxide production by ocular tumor associated CD11b+ myeloid cells contributes to the suppression of CTL responses in vitro whereas NO production by skin tumor CD11b+ cells contributes to the tumoricidal activity of CD8+ CTL in the skin (11,12). We are currently testing the following hypotheses:
1. That NO produced by CD11b+ associated with skin tumors is tumoricidal whereas NO produced by CD11b+ cells associated with ocular tumors is tumor promoting.
2. That tumor growth induces the expression cytokines and chemokines through a MyD88 dependent pathway which promotes the accumulation of CD11b+ cells within the ocular tumor microenvironment.
3. That ocular tumor-associated CD11b+ cells must process and present antigen to CD8+ CTL to activate CD11b+ cells to produce NO which induces apoptosis of tumor specific CD8+ CTL within the tumor microenvironment
4. That all-trans retinoic acid (ATRA) therapy which converts immunosuppressive CD11b+ cells to tumoricidal-neutrophils will promote primary ocular tumor elimination and augment tumor-specific CD8+ CTL responses.
1. van Dinten, L. C., N. Pul, A. F. van Nieuwpoort, C. J. Out, M. J. Jager, and P. J. van den Elsen. 2005. Uveal and cutaneous melanoma: shared expression characteristics of melanoma-associated antigens. Invest Ophthalmol. Vis. Sci. 46:24.
2. Zippelius, A., P. Batard, V. Rubio-Godoy, G. Bioley, D. Lienard, F. Lejeune, D. Rimoldi, P. Guillaume, N. Meidenbauer, A. Mackensen, N. Rufer, N. Lubenow, D. Speiser, J. C. Cerottini, P. Romero, and M. J. Pittet. 2004. Effector function of human tumor-specific CD8 T cells in melanoma lesions: a state of local functional tolerance. Cancer Res. 64:2865.
3. Kan-Mitchell, J., P. E. Liggett, W. Harel, L. Steinman, T. Nitta, J. R. Oksenberg, M. R. Posner, and M. S. Mitchell. 1991. Lymphocytes cytotoxic to uveal and skin melanoma cells from peripheral blood of ocular melanoma patients. Cancer Immunol. Immunother. 33:333.
4. Rosenberg, S. A., J. C. Yang, D. J. Schwartzentruber, P. Hwu, F. M. Marincola, S. L. Topalian, N. P. Restifo, M. E. Dudley, S. L. Schwarz, P. J. Spiess, J. R. Wunderlich, M. R. Parkhurst, Y. Kawakami, C. A. Seipp, J. H. Einhorn, and D. E. White. 1998. Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nat. Med. 4:321.
5. Rosenberg, S. A., R. M. Sherry, K. E. Morton, W. J. Scharfman, J. C. Yang, S. L. Topalian, R. E. Royal, U. Kammula, N. P. Restifo, M. S. Hughes, D. Schwartzentruber, D. M. Berman, S. L. Schwarz, L. T. Ngo, S. A. Mavroukakis, D. E. White, and S. M. Steinberg. 2005. Tumor progression can occur despite the induction of very high levels of self/tumor antigen-specific CD8+ T cells in patients with melanoma. J. Immunol. 175:6169.
6. Valmori, D., V. Dutoit, M. Ayyoub, D. Rimoldi, P. Guillaume, D. Lienard, F. Lejeune, J. C. Cerottini, P. Romero, and D. E. Speiser. 2003. Simultaneous CD8+ T cell responses to multiple tumor antigen epitopes in a multipeptide melanoma vaccine. Cancer Immun. 3:15.
7. Rosenberg, S. A., J. C. Yang, and N. P. Restifo. 2004. Cancer immunotherapy: moving beyond current vaccines. Nat. Med. 10:909.
8. Makitie, T., P. Summanen, A. Tarkkanen, and T. Kivela. 2001. Tumor-infiltrating macrophages (CD68(+) cells) and prognosis in malignant uveal melanoma. Invest Ophthalmol. Vis. Sci. 42:1414.
9. Toivonen, P., T. Makitie, E. Kujala, and T. Kivela. 2004. Microcirculation and tumor-infiltrating macrophages in choroidal and ciliary body melanoma and corresponding metastases. Invest Ophthalmol. Vis. Sci. 45:1.
10. McKenna, K. C., and J. A. Kapp. 2006. Accumulation of immunosuppressive CD11b+ myeloid cells correlates with the failure to prevent tumor growth in the anterior chamber of the eye. J. Immunol. 177:1599.
11. Hollenbaugh, J. A., J. Reome, M. Dobrzanski, and R. W. Dutton. 2004. The rate of the CD8-dependent initial reduction in tumor volume is not limited by contact-dependent perforin, Fas ligand, or TNF-mediated cytolysis. J. Immunol. 173:1738.
12. Hollenbaugh, J. A., and R. W. Dutton. 2006. IFN-gamma regulates donor CD8 T cell expansion, migration, and leads to apoptosis of cells of a solid tumor. J. Immunol. 177:3004.
Techniques graduate student will learn:
Hypothesis 1: Tumors cells are injected in the skin or eye of anesthetized mice and ocular tumor injections are performed under a dissecting surgical microscope which requires animal care and handling training. Tumor growth in the skin is measured by a caliper and tumor growth in the eye is measured by flow cytometric analysis of collagenase digested eyes stained with flourescently labeled antibodies. The requirement of NO for tumor elimination or tumor growth will be evaluated by utilizing NOS2-/- mice which are genetically deficient in the inducible Nitric oxide synthase enzyme. In some experiments, tumor-specific CD8+ CTL will be generated by in vitro culture and then adoptively transferred into tumor bearing wild type or NOS2-/- mice by intravenous injection. CD11b+ cells are isolated from ocular tumors by magnetic cell separation and flow cytometric cell sorting. Immunosuppressive CD11b+ cells from ocular tumors will be compared and contrasted to inflammatory CD11b+ cells from endotoxin-induced uveitic eyes for differences in cytokine expression measured by the luminex assay, nitric oxide production measured by the Griess reagent assay, reactive oxygen species generation measured by a flow cytometric assay, myeloperoxidase activity, and cell surface molecule expression by flow cytometric analysis.
Hypothesis 2: Tumor cells are injected into the eye of anesthetized mice which are genetically deficient in particular cytokines, cytokine receptors, chemokines, chemokine receptors, or MyD88 which requires animal care and handling training. Tumor growth in the eye and infiltration of CD11b+ myeloid cells is evaluated by flow cytometric analysis of collagenase digested eyes stained with fluorescently labeled antibodies.
Hypothesis 3: Ocular tumor associated CD11b+ cells are isolated from collagenase digested eyes of wild type and beta-2 microglobulin deficient mice by magnetic cell separation and flow cytometric cell sorting. Isolated CD11b+ cells are cocultured with CD8+ CTL and lytic activity is measured by 51Cr release assay, proliferation is measured by CFSE dilution and flow cytometric analysis, and Nitric oxide production is measured by the Griess assay.
Hypothesis 4: ATRA pellets are implanted subcutaneously by a small animal surgery which requires animal care and handling training. Tumor growth is measured in the skin by a caliper and in the eye by flow cytometric analysis of collagenase digested eyes.
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