CONTROL OF CANCER IMMUNOEDITING BY AUTOLOGOUS CELLULAR VACCINE AND EFFICACY ASSESSMENT OF IFN-alpha-ENGINEERED MESENCHYMAL STROMAL CELLS (MSCs) IN A MOUSE PLASMACYTOMA MODEL

In the mouse Balb/c-derived myeloma model represented by the Sp6 hybridoma, de novo expression of the B7-1 costimulatory molecule obtained by transfection of the relative cDNA (Sp6/B7 transfectants) inhibits tumor growth in vivo and activates a memory immune response mediated by cytotoxic T lymphocytes (CTLs), protective against wild type (WT) Sp6. In the Balb/c genetic background, the MHC-I H-2 Ld molecule is the restriction element presenting the immunodominant epitopes of the two most common mouse tumor associated antigens: P1A and gp70. WT Sp6 and Sp6/B7 show a downregulated cell surface expression of H-2 Ld, still maintaining normal expression levels of H-2 Kd and Dd. Sp6 cells are lysed in vitro by gp70-Ld-restricted CTLs (not by P1A-Ld-restricted), indicating that they are able to present gp70 epitopes in spite of the low Ld expression. Increase of H-2 Ld expression in WT Sp6 and Sp6/B7 by transfection of the relative cDNA (Sp6/Ld and Sp6/B7/Ld transfectants) drives the immune response towards H-2 Ld-restricted antigens, whilst the H-2 Ld-downregulated state of WT Sp6 and Sp6/B7 determines a shift of the immune response towards subdominant antigens presented by H-2 Kd. Nevertheless, the immune response elicited by in vivo immunization with the autologous B7-1 positive cellular vaccine, either Sp6/B7 or Sp6/B7/Ld, is never gp70-specific. Analysis of gp70 expression in peripheral lymphoid organs showed the presence of gp70 specific transcripts in both spleen and lymph nodes of animals immunized with Sp6/B7 and Sp6/B7/Ld, absent in naïve animals. Electron mycroscope analysis of WT Sp6 and transfectants showed the presence of virus-like particles in both cytoplasma and exocytosis vesicles. We may hypothesize that, during immunization phase, a CTL-dependent lysis of Sp6/B7 and/or Sp6/B7/Ld cells migrated to the draining lymph nodes results in the release of a high number of virions, rapidly infecting the whole resident B cell population. The subsequent presentation of the immunodominant viral antigen epitopes by a large number of infected B cells can generate an activation-induced cell death of high affinity TCR-T cells, leading to clonal deletion of gp70-specific CD8+ T cells. In conclusion, Sp6 tumor appears to carry out two immunoescape mechanisms: 1) induction of peripheral tolerance against the gp70 immunodominant antigen; 2) H-2 Ld downregulation, to escape the tumor-specific immune response against the Ld-restricted epitopes of previously subdominant antigens, that the established gp70-peripheral tolerance has eventually made dominant. Our data show that, when tumors adopt these immunoescape mechanisms, autologous whole tumor cell vaccines still remain a potent tool for generating anti-tumor immunity, because they allow to rescue and amplify the protective immune responses against subdominant tumor antigens. Then, we have analyzed the therapeutic potential of mesenchymal stromal cells (MSCs) as cellular vehicle for intra-tumoral delivery of active molecules. Bone marrow derived MSCs (BM-MSCs) may survive and proliferate in the presence of cycling neoplastic cells. Exogenously administered MSCs are actively incorporated in the tumor as stromal fibroblasts, thus competing with the local mesenchymal cell precursors. For this reason, MSCs have been suggested as a suitable carrier for gene therapy strategies, as they can be engineered with genes encoding for biologically active molecules, which can inhibit tumor cell proliferation and enhance the anti-tumor immune response. We used BM-MSCs engineered with the murine interferon-alpha (IFN-a) gene (BM-MSCs/IFN-a) to assess in the mouse myeloma model represented by Sp6 the efficacy of this approach towards neoplastic plasma cells. We found that IFN-a can be efficiently produced and delivered inside the tumor microenvironment. Subcutaneous multiple administration of BM-MSCs/IFN-a significantly hampered the tumor growth in vivo and prolonged the overall survival of mice. The anti-tumor effect was associated with enhanced apoptosis of tumor cells, reduction in microvessel density, and ischemic necrosis. By contrast, intravenous administration of BM-MSCs/IFN-a did not significantly modify the survival of mice, mainly as a consequence of an excessive entrapment of injected cells in the pulmonary vessels. In conclusion, BM-MSCs/IFN-a are effective in inhibiting neoplastic plasma cell growth; however, systemic administration of engineered MSCs still needs to be improved to make this approach potentially suitable for the treatment of multiple myeloma.