Although remission rates for metastatic melanoma are generally very poor, some patients can survive for prolonged periods following metastasis. We used gene expression profiling, mitotic index (MI), and quantification of tumor infiltrating leukocytes (TILs) and CD3؉ cells in metastatic lesions to search for a molecular basis for this observation and to develop improved methods for predicting patient survival. We identified a group of 266 genes associated with postrecurrence survival. Genes positively associated with survival were predominantly immune response related (e.g., ICOS, CD3d, ZAP70, TRAT1, TARP, GZMK, LCK, CD2, CXCL13, CCL19, CCR7, VCAM1) while genes negatively associated with survival were cell proliferation related (e.g., PDE4D, CDK2, GREF1, NUSAP1, SPC24). Furthermore, any of the 4 parameters (prevalidated gene expression signature, TILs, CD3, and in particular MI) improved the ability of Tumor, Node, Metastasis (TNM) staging to predict postrecurrence survival; MI was the most significant contributor (HR ‫؍‬ 2.13, P ‫؍‬ 0.0008). An immune response gene expression signature and presence of TILs and CD3؉ cells signify immune surveillance as a mechanism for prolonged survival in these patients and indicate improved patient subcategorization beyond current TNM staging.gene expression analysis ͉ immune response ͉ TNM staging ͉ tumor infiltrating leukocytes

The use of recombinant tumor antigen proteins is a realistic approach for the development of generic cancer vaccines, but the potential of this type of vaccines to induce specific CD8 ؉ T cell responses, through in vivo cross-priming, has remained unclear. In this cancer vaccine A key step in the development of generic cancer vaccines is the implementation of vaccination strategies allowing for the consistent induction of immune responses to tumor antigens. In this respect, the choice of appropriate antigens, based on both the frequency and the specificity of their expression in cancer tissues, is of paramount importance. The group of cancer/testis antigens (CTA) (1, 2), including the NY-ESO-1 antigen (3), is emerging among the most promising candidates. Because many CTA are not expressed on the surface of cancer cells but rather intracellularly, it is important that vaccination induces specific CD8 ϩ T cells able to directly recognize antigen-expressing tumor cells. Recombinant proteins can be produced in large scale and at relatively low cost, are commonly used in the development of antiviral vaccines, and are therefore attractive candidate antitumor vaccines. The potential of tumor antigen recombinant protein vaccines, however, relies on their ability not only to elicit antibody (Ab) and CD4 ϩ T cell responses but also to efficiently prime naive CD8 ϩ T cells through cross-priming (4), which generally is inefficient during spontaneous immune responses to tumor antigens (5). Professional antigen-presenting cells (APCs) detect pathogens through a variety of receptors such as the Toll-like receptors (TLR), which recognize pathogen-associated molecular patterns including CpG dinucleotides within defined flanking sequences (CpG ODN) (6). Synthetic CpG ODN able to trigger TLR9 are potent vaccine adjuvants, stimulating T helper type 1 (Th1)-type immunity (7). In humans, they can directly activate B lymphocytes and plasmacytoid dendritic cells and also indirectly activate myeloid dendritic cells (mDCs), increasing antigen cross-presentation and stimulating adaptive immune responses (8)(9)(10).In this study, we have assessed the immune response elicited by repeated vaccination with a NY-ESO-1 recombinant protein (rNY-ESO-1) administered with CpG 7909 in a water-oil emulsion with Montanide ISA-51. We show that cancer patients receiving this vaccine developed integrated Ab and CD4 ϩ T cell responses to NY-ESO-1 at an early phase of the vaccination protocol. A fraction of the patients also developed specific CD8 ϩ T cell responses at a later time point. Assessment of the correlation between the development of Ab and T cell responses suggested that the presence of sufficient levels of NY-ESO-1-specific antibodies was determinant for the cross-priming of CD8 ϩ T cells to occur in vivo. In line with this concept, we found that in vitro cross-presentation of NY-ESO-1 protein to vaccineinduced CD8 ϩ T cells by dendritic cells was enhanced by vaccine-induced Ab.

IntroductionThe concept of tumor "immunosurveillance," whereby the host immune system is thought to protect against the development of primary cancers, has been debated for decades and has been recently resurrected. 1 Evidence in support of tumor immunosurveillance includes observations in mice that lymphocytes and molecules essential for immune function, such as interferon-␥ (IFN␥) and perforin, collaborate to protect against the development of certain cancers. Additional corroboration has come from identification of numerous human tumor-associated or tumor-specific antigens recognized by T cells and from isolation of tumor antigenspecific T cells from metastatic lesions. Furthermore, infiltration of certain human cancers by T cells may correlate with dramatically improved survival. 2 The accumulating evidence in favor of tumor immunosurveillance indicates that immunotherapies or "vaccines" may prove effective for the treatment of cancer. Indeed, numerous published reports have shown that vaccination of cancer patients with killed tumor cells, tumor cell lysates or tumor antigen proteins, peptides or DNA administered with cytokines or adjuvants can produce immunologic and clinical responses. However, the immune responses to these vaccines are often weak, and clinical responses are rarely complete and long lasting. [3][4][5] Dendritic cells (DCs) are bone marrow-derived antigenpresenting cells (APCs) that play a critical role in the induction and regulation of immune responses. It has been proposed that the manipulation of DCs as a "natural" vaccine adjuvant may prove to be a particularly effective way to stimulate antitumor immunity. 6,7 This hypothesis has been supported by experiments in mice. However, published reports of DC-based vaccine trials in humans have yet to demonstrate improved potency of DC vaccines over more traditional vaccine preparations. 5,8,9 In this review we discuss the pitfalls of current DC vaccine approaches in the context of recent advances in DC biology and how improved understanding of DC biology can be applied to develop more effective immunotherapies for cancer. DC biology DC differentiation and subtypesDCs are a heterogeneous population of cells produced in the bone marrow in response to growth and differentiation factors fms-like tyrosine kinase-3 ligand (Flt3L) and granulocyte-macrophage colony-stimulating factor (GM-CSF). There are 3 generally accepted stages of differentiation for all DC subtypes: DC precursors, immature DCs, and mature DCs. 10 In human blood, immature DCs and DC precursors are lineage-negative (CD3 Ϫ CD14 Ϫ CD19 Ϫ CD56 Ϫ ) HLA-DR ϩ mononuclear cells 6 and are traditionally divided into 2 populations by staining with antibodies to CD11c and CD123 (interleukin 3 receptor ␣ [IL-3R␣]). CD11c ϩ CD123 lo DCs have a monocytoid appearance and are called "myeloid DCs" (MDCs), whereas CD11c Ϫ CD123 hi DCs have morphologic features similar to plasma cells and are thus called "plasmacytoid DCs" (PDCs). Although commonly used, this nomenclature is somewhat misleading. E...

T cell-mediated immunity to microbes and to cancer can be enhanced by the activation of dendritic cells (DCs) via TLRs. In this study, we evaluated the safety and feasibility of topical imiquimod, a TLR7 agonist, in a series of vaccinations against the cancer/testis Ag NY-ESO-1 in patients with malignant melanoma. Recombinant, full-length NY-ESO-1 protein was administered intradermally into imiquimod preconditioned sites followed by additional topical applications of imiquimod. The regimen was very well tolerated with only mild and transient local reactions and constitutional symptoms. Secondarily, we examined the systemic immune response induced by the imiquimod/NY-ESO-1 combination, and show that it elicited both humoral and cellular responses in a significant fraction of patients. Skin biopsies were assessed for imiquimod’s in situ immunomodulatory effects. Compared with untreated skin, topical imiquimod induced dermal mononuclear cell infiltrates in all patients composed primarily of T cells, monocytes, macrophages, myeloid DCs, NK cells, and, to a lesser extent, plasmacytoid DCs. DC activation was evident. This study demonstrates the feasibility and excellent safety profile of a topically applied TLR7 agonist used as a vaccine adjuvant in cancer patients. Imiquimod’s adjuvant effects require further evaluation and likely need optimization of parameters such as formulation, dose, and timing relative to Ag exposure for maximal immunogenicity.