Purpose: Cryotherapy of localized prostate, renal, and hepatic primary tumors and metastases is considered a minimally invasive treatment demonstrating a low complication rate in comparison with conventional surgery. The main drawback of cryotherapy is that it has no systemic effect on distant metastases. We investigated whether intratumoral injections of dendritic cells following cryotherapy of local tumors (cryoimmunotherapy) provides an improved approach to cancer treatment, combining local tumor destruction and systemic anticancer immunity. Experimental Designs: The 3LL murine Lewis lung carcinoma clone D122 and the ovalbumintransfected B16 melanoma clone MO5 served as models for spontaneous metastasis. The antimetastatic effect of cryoimmunotherapy was assessed in the lung carcinoma model by monitoring mouse survival, lung weight, and induction of tumor-specific CTLs. The mechanism of cryoimmunotherapy was elucidated in the melanoma model using adoptive transfer of T cell receptor transgenic OT-I CTLs into the tumor-bearing mice, and analysis of Th1/Th2 responses by intracellular cytokine staining in CD4 and CD8 cells. Results: Cryoimmunotherapy caused robust and tumor-specific CTL responses, increased Th1 responses, significantly prolonged survival and dramatically reduced lung metastasis. Although intratumor administration of dendritic cells alone increased the proliferation rate of CD8 cells, only cryoimmunotherapy resulted in the generation of effector memory cells. Furthermore, cryoimmunotherapy protected mice that had survived primary MO5 tumors from rechallenge with parental tumors. Conclusions: These results present cryoimmunotherapy as a novel approach for systemic treatment of cancer. We envisage that cryotherapy of tumors combined with subsequent in situ immunotherapy by autologous unmodified immature dendritic cells can be applied in practice.Minimally invasive therapies are an alternative approach to surgical intervention in the treatment of malignant diseases. Cryoablation, i.e., tissue destruction by repeated deep freezing and thawing, is under the larger category of thermal therapy and, during the past decade, it has become an acceptable clinical tool for the management of dermatologic tumors, hepatocellular carcinoma, renal and prostate tumors, and hepatic colorectal metastases (1, 2). Compared with surgical excision, the main advantages are the potential for less invasiveness resulting in reduced mortality and morbidity, and the ability to perform ablative procedures on outpatients, which decreases the treatment cost. In the case of hepatic colorectal metastases, the use of cryosurgery improves the percentages of resectability (2). A comparative study on domestic pigs showed that the cryoablation of renal parenchyma is beneficial over other necrosis-inducing ablations such as microwave thermoablation, radiofrequency energy, and chemoablation by ethanol, hypertonic saline, and acetic acid gels, in terms of reproducibility, consistency in size and shape, and the ability to monitor b...

Specific immunotherapy of prostate cancer may be an alternative or be complementary to other approaches for treatment of recurrent or metastasized disease. This study aims at identifying and characterizing prostate cancerassociated peptides capable of eliciting specific CTL responses in vivo. Evaluation of peptide-induced CTL activity in vitro was done following immunization of HLA-A2 transgenic (HHD) mice. An in vivo tumor rejection was tested by adoptive transfer of HHD immune lymphocytes to nude mice bearing human tumors. To confirm the existence of peptide-specific CTL precursors in human, lymphocytes from healthy and prostate cancer individuals were stimulated in vitro in the presence of these peptides and CTL activities were assayed. Two novel immunogenic peptides derived from overexpressed prostate antigens, prostatic acid phosphatase (PAP) and sixtransmembrane epithelial antigen of prostate (STEAP), were identified; these peptides were designated PAP-3 and STEAP-3. Peptide-specific CTLs lysed HLA-A2.1 + LNCaP cells and inhibited tumor growth on adoptive immunotherapy. Furthermore, peptide-primed human lymphocytes derived from healthy and prostate cancer individuals lysed peptide-pulsed T2 cells and HLA-A2.1 + LNCaP cells. Based on the results presented herein, PAP-3 and STEAP-3 are naturally processed CTL epitopes possessing anti-prostate cancer reactivity in vivo and therefore may constitute vaccine candidates to be investigated in clinical trials. (Cancer Res 2005; 65(14): 6435-42)

A limitation to antitumor immunity is the dysfunction of T cells in the tumor microenvironment, in part due to upregulation of coinhibitory receptors such as PD-1. Here, we describe that poliovirus receptor-related immunoglobulin domain protein (PVRIG) acts as a coinhibitory receptor in mice. Murine PVRIG interacted weakly with poliovirus receptor (PVR) but bound poliovirus receptorlike 2 (PVRL2) strongly, making the latter its principal ligand. As in humans, murine NK and NKT cells constitutively expressed PVRIG. However, when compared with humans, less PVRIG transcript and surface protein was detected in murine CD8 + T cells ex vivo. However, activated CD8 + T cells upregulated PVRIG expression. In the mouse tumor microenvironment, infiltrating CD8 + T cells expressed PVRIG whereas its ligand, PVRL2, was detected predominantly on myeloid cells and tumor cells, mirroring the expression pattern in human tumors. PVRIG-deficient mouse CD8 + T cells mounted a stronger antigen-specific effector response compared with wildtype CD8 + T cells during acute Listeria monocytogenes infection. Furthermore, enhanced CD8 + T-

The success of adoptive cell transfer in the treatment of metastatic cancer in humans is dependent on the selection of highly active tumor-specific cytotoxic T cells. We report here that CTLs capture membrane fragments from their targets while exerting cytotoxic activity and thus gain a detectable functional signature by which they can be identified. Fluorochrome labeling or biotinylation was used to tag tumor cells. CD8 + T cells were coincubated with the tagged targets, sorted, and functionally evaluated. Our results show that membrane capture by CD8 + lymphocytes is T-cell receptor dependent, epitope specific, and preferentially associated with highly cytotoxic clonal subsets. CTLs that captured membranes from unmodified melanoma exhibited enhanced cytotoxic activity against tumor cell lines and autologous melanoma. In a human melanoma in vivo model, adoptive transfer of membrane-capturing, peptide-specific T cells, but not noncapturing or bulk CD8 + T cells, inhibits tumor progression. Membrane capture is therefore a signature of antigenspecific CTLs endowed with high functional avidity and may have direct relevance in the clinical application of adoptive immunotherapy.

The success of T cell-based cancer immunotherapy is limited by tumor's resistance against killing by cytotoxic T lymphocytes (CTLs). Tumor-immune resistance is mediated by cell surface ligands that engage immune-inhibitory receptors on T cells. These ligands represent potent targets for therapeutic inhibition. So far, only few immune-suppressive ligands have been identified. We here describe a rapid high-throughput siRNA-based screening approach that allows a comprehensive identification of ligands on human cancer cells that inhibit CTL-mediated tumor cell killing. We exemplarily demonstrate that CCR9, which is expressed in many cancers, exerts strong immune-regulatory effects on T cell responses in multiple tumors. Unlike PDL1, which inhibits TCR signaling, CCR9 regulates STAT signaling in T cells, resulting in reduced T-helper-1 cytokine secretion and reduced cytotoxic capacity. Moreover, inhibition of CCR9 expression on tumor cells facilitated immunotherapy of human tumors by tumor-specific T cells in vivo. Taken together, this method allows a rapid and comprehensive determination of immune-modulatory genes in human tumors which, as an entity, represent the ‘immune modulatome’ of cancer.