Direct intratumor injection of a disabled infectious single cycle HSV-2 virus encoding the murine GM-CSF gene (DISC/mGM-CSF) into established murine colon carcinoma CT26 tumors induced a significant delay in tumor growth and complete tumor regression in up to 70% of animals. Pre-existing immunity to HSV did not reduce the therapeutic efficacy of DISC/mGM-CSF, and, when administered in combination with syngeneic dendritic cells, further decreased tumor growth and increased the incidence of complete tumor regression. Direct intratumor injection of DISC/mGM-CSF also inhibited the growth of CT26 tumor cells implanted on the contralateral flank or seeded into the lungs following i.v. injection of tumor cells (experimental lung metastasis). Proliferation of splenocytes in response to Con A was impaired in progressor and tumor-bearer, but not regressor, mice. A potent tumor-specific CTL response was generated from splenocytes of all mice with regressing, but not progressing tumors following in vitro peptide stimulation; this response was specific for the gp70 AH-1 peptide SPSYVYHQF and correlated with IFN-γ, but not IL-4 cytokine production. Depletion of CD8+ T cells from regressor splenocytes before in vitro stimulation with the relevant peptide abolished their cytolytic activity, while depletion of CD4+ T cells only partially inhibited CTL generation. Tumor regression induced by DISC/mGM-CSF virus immunotherapy provides a unique model for evaluating the immune mechanism(s) involved in tumor rejection, upon which tumor immunotherapy regimes may be based.

The OX40 ligand (OX40L), a member of TNF superfamily, is a costimulatory molecule involved in T cell activation. It is expressed on antigen presenting cells including dendritic cells (DC) and activated B cells. This molecule has been reported to provide potent costimulation in APC-T cell interactions upon binding to its cognate receptor, OX40 which is expressed by activated T cells. In this study systemic administration of OX40L fusion protein was used in the treatment of established murine subcutaneous colon and breast carcinomas.Intra-peritoneal injection of mOX40L fusion protein significantly inhibited the growth of subcutaneous 3 day established colon (CT26) and breast (4T1) carcinomas which was dose and route dependent. Effective therapy with OX40L required the the presence of tumour for 3 days prior to OX40L, concomitant therapy, given at the same time (day 0) as tumour cells was less effective. Furthermore, therapy with OX40L fusion protein was effective in significantly reducing CT26 experimental lung metastasis. In addition, inhibition of CT26 and 4T1 tumours in response to therapy with OX40L was significantly enhanced by combination treatment with intra-tumour injection of a DISC-HSV vector encoding mGM-CSF. Tumour rejection in response to OX40L therapy was correlated with splenocyte CTL activity against the gp70 CT26 tumour associated antigen. In vivo depletion studies demonstrated the requirement for both CD4+ and CD8+ T cells for effective OX40L therapy.Collectively these results demonstrate the potential role of the OX40L in cancer immunotherapy.3

The major histocompatibility complex class I-restricted CD8 ؉ cytotoxic T-lymphocyte (CTL) effector arm of the adaptive immune response can specifically recognize and destroy tumor cells expressing peptide antigens. Although adoptive T-cell therapy has been successfully used for the treatment of viral and malignant diseases, little is known of the trafficking and fate of adoptively transferred antigenspecific T cells. In the present study, splenocytes derived from mice that rejected their tumors (CT26 or CT26-clone 25 tumors) in response to direct intratumor injection of disabled infectious single-cycle herpes simplex virus (DISC-HSV) encoding murine GM-CSF were restimulated with peptide in vitro. CTLs specific for the AH-1 and ␤-gal peptides expressed by CT26 and CT26-clone 25 tumor cells, respectively, were generated and used for adoptive cellular therapy and trafficking studies. Intravenous administration of AH-1-specific CTLs 3 days following i.v. injection of CT26 cells resulted in significant tumor growth inhibition, whereas administration of control CTLs generated against a bacterial ␤-gal peptide did not inhibit the growth of tumors. Trafficking of AH-1-specific lymphocytes and their interaction with the CT26 tumor microcirculation was analyzed using realtime in vivo microscopy (IVM). AH-1-specific but not ␤-galspecific CTLs adhered and localized in the CT26 tumor microvasculature, but neither population adhered to the endothelium of the normal microcirculation. This study provides direct visual evidence suggesting that AH-1-specific CTLs that mediate a therapeutic response traffic to and localize within the tumor microenvironment. © 2004 Wiley-Liss, Inc. Key words: cytotoxic T lymphocytes; adoptive transfer; traffickingThe immune system consists of a complex network of cells that mediate innate and antigen-specific responses to invading microbes and tumors. Processing and presentation of antigen by antigen-presenting cells (dendritic cells) via peptide antigens bound to cell surface major histocompatibility complex (MHC) antigens is a prerequisite for the stimulation of T-cell responses (cytotoxic and helper T cells). 1,2 Tumor-specific cytotoxic T lymphocytes (CTLs) play an important role against cancer; this has been demonstrated using a number of experimental approaches, including the adoptive transfer of tumor-specific CTLs in animal models and clinical trials. 3,4 Beneficial antitumor effects have been reported using adoptive cellular therapy with lymphokine-activated killer (LAK) cells, autolymphocyte therapy (ALT) and in vitro expanded tumor-infiltrating lymphocytes. [5][6][7] The efficacy of adoptive cellular immunotherapy with cultured lymphocytes activated toward tumor antigens requires effector cells to migrate into tumor tissue and, once resident at the tumor site, to mediate an antitumor rejection response either by direct killing of tumor cells and/or indirectly through cytokine release and recruitment of other cells. Using the A2/K b transgenic mouse model, Sutmuller et al. 3 have shown t...

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