To increase (tumor) vaccine efficacy, there is an urgent need for phenotypic and functional characterization of human dendritic cell (DC) subsets residing in lymphoid tissues. In this study we identified and functionally tested 4 human conventional DC (cDC) subsets within skin-draining sentinel lymph nodes (SLNs) from early-stage melanoma patients. These SLNs were all tumor negative and were removed on average 44 days after excision of the primary melanoma. As such, they were considered representative of steady-state conditions. On comparison with skin-migrated cDC, 2 CD1a ؉ subsets were identified as most likely skin-derived CD11c int Langerhans cells (LC) with intracellular langerin and E-cadherin expression or as CD11c hi dermal DCs with variable expression of langerin. Two other CD1a ؊ LNresiding cDC subsets were characterized as CD14 ؊ BDCA3 hi CD103 ؊ and CD14 ؉ BDCA3 lo CD103 ؉ , respectively. Whereas the CD1a ؉ skin-derived subsets displayed greater levels of phenotypic maturation, they were associated with lower levels of inflammatory cytokine release and were inferior in terms of allogeneic T-cell priming and IFN␥ induction. Thus, despite their higher maturation state, skin-derived cDCs (and LCs in particular) proved inferior T-cell activators compared with the CD1a ؊ cDC subsets residing in melanoma-draining LNs. These observations should be considered in the design of DC-targeting immunotherapies. (Blood. 2011;118(9):2502-2510) IntroductionDendritic cells (DCs) are the most powerful APCs and play critical roles in keeping the balance between immune tolerance and activation. DCs are therefore also important for starting an efficient antitumor immune response and are seen as promising targeting candidates for tumor immunotherapy strategies. 1,2 Current DCbased immunotherapies generally use ex vivo-generated autologous monocyte-derived or CD34 ϩ hematopoietic precursorderived DCs. 2,3 Despite occasionally observed clinical benefits from DC-based vaccination, there is a large gap between the actual and expected efficacy of such trials on the basis of in vivo animal experiments. 4 Many questions remain as to which DC type to use, how to stimulate them, or where best to administer the DCs to achieve vaccination with mature, migratory, and Th1-inducing DCs that provoke an efficient antitumor immune response. [5][6][7] An ever-increasing insight in specialized functions of murine DC subsets is sadly mirrored by a lack of knowledge on how human DCs relate to mouse DCs and whether subsets that have been identified in mice have a (phenotypically different, but functionally equivalent) counterpart in humans. 4,[8][9][10][11] In particular the interrelationship between nonplasmacytoid, conventional DC (cDC) subsets has been obscure, in large part because of their plasticity and dynamic changes in their differentiation and maturation state, which is accompanied by shifts in associated phenotypic markers. 12 In mice, extensive DC-subset analyses have been performed through the use of transgenic models, the ability ...

Purpose: A decrease in the frequency and activation state of dendritic cells in the sentinel lymph node (SLN) has been observed in early stages of melanoma development. This may hinder the generation of effective antitumorT-cell responses and increase the likelihood of metastatic spread. Immunopotentiation of the melanoma SLN may therefore be a valuable adjuvant treatment option. One way to achieve this is through the use of bacterially derived unmethylated cytosinephosphate-guanine (CpG) DNA sequences that bind Toll-like receptor 9 and activate plasmacytoid dendritic cells (PDC). CpG-activated PDC, in turn, release IFNa and may thus boost T-cell and natural killer cell responses as well as activate conventional myeloid dendritic cells (MDC).

Purpose: Impaired immune effector functions in the melanoma sentinel lymph node (SLN) may allow for early metastatic events. Local administration of PF-3512676 (formerly known as CpG 7909) has shown immunostimulatory effects of both dendritic cell and T-cell subsets in the melanoma SLN. Here, we set out to ascertain whether these PF-3512676-induced immunostimulatory effects translate into higher frequencies of melanoma-specific CD8 + Tcells. Experimental Design: Twenty-four stage I to III melanoma patients were randomized to preoperative local administration of either PF-3512676 or saline. CD8 + T cells from SLN and peripheral blood were tested for reactivity by IFN-g ELISPOT assay against several HLA-A1/A2/ A3-restricted epitopes derived from various melanoma-associated antigens (MAA) in 21 of 24 enrolled patients. Frequencies of natural killer (NK) cells and frequencies and maturation state of dendritic cell subsets in the SLN were determined by flow cytometry. Results: Melanoma-specific CD8 + T-cell response rates against >1MAA epitope in the SLN were 0 of 11 for the saline group versus 5 of 10 for the PF-3512676-administered group (P = 0.012).Of these 5 responding patients, 4 also had a measurable response to >1 MAA epitope in the blood. Increased frequencies in the SLN of both MAA-specific CD8 + T cells and NK cells correlated to CpG-induced plasmacytoid dendritic cell maturation. Conclusions: These data show an increase in melanoma-specific CD8 + T-cell frequencies as well as an increased effector NK cell rate after a single dose of PF-3512676 and thus support the utility of local PF-3512676 administration as adjuvant treatment in early-stage melanoma to try and halt metastatic spread.

In this study we investigated whether the presence of specific populations of tumor infiltrating lymphocytes (TILs) in diagnostic primary melanoma biopsies are related to outcome in clinically stage II melanoma patients. Moreover, we investigated whether the presence of TILs correlates with expression of MHC class I antigen and MHC class II antigen on tumor cells and/or tumor infiltrating antigen presenting cells. Diagnostic primary melanoma samples of 15 patients with an unfavorable outcome were compared with 20 patients with favorable outcome. Patients were matched for age, gender and Breslow thickness. Biopsies were examined for the presence of granzyme B 1 , CD8 1 , CD4 1 and CD56 1 TILs and for expression of MHC class I antigen and MHC class II antigen on tumor and/or tumor infiltrating cells. A favorable clinical outcome was strongly associated with the presence of GrB 1 and CD4 1 TILs, with expression of MHC class I antigen on tumor cells and with expression of MHC class II antigen on intratumoral antigen presenting cells. These data strongly support the notion that in melanoma patients the cellular immune response is a major factor in preventing melanoma cell dissemination. ' 2008 Wiley-Liss, Inc.Key words: TILs; granzyme B; MHC class I; MHC class II; prognosis; CD4; CD8 Even though melanomas account for only 4% of all skin cancers, they cause the greatest number of skin cancer-related deaths worldwide. Over the last few decades an increase in incidence and mortality has been observed in Caucasian populations across the world. 1,2 Clinical outcome in melanoma patients depends on several variables of which tumor thickness is an important factor (according to Breslow). 3 The 5 year survival rate for patients with a Breslow thickness <1.5 mm is more than 90%, whereas survival in patients with a Breslow thickness of >3.5 mm is only 50%. 4 Other important prognostic factors are, amongst others, gender and age. [5][6][7] Fatal outcome in melanoma patients often results from occurrence of distant metastases, which mostly coincide or are preceded by lymph node metastases. In line with this concept, previous studies demonstrated that patients with a melanoma sentinel lymph node (SLN) metastasis have a worse prognosis than patients without a SLN metastasis. 8,9 However, despite known prognostic parameters, outcome often remains unpredictable and further research to identify additional relevant prognostic markers is warranted.It has previously been shown that melanomas can elicit an immune response 10,11 and that melanoma cells can effectively be eradicated in vivo by cytotoxic activity of MHC class I antigen restricted CD8 1 Granzyme B (GrB 1 ) T-cells. 12 Thus, a possible explanation for differences in clinical outcome might be that a proper immune response, although incapable of preventing the primary tumor from growing, is able to prevent the occurrence of lymph node and/or distant metastases. A large number of studies have shown that the cellular immune response plays an important role in the control of melan...