Chemokine-mediated distribution of dendritic cell subsets in renal cell carcinoma

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs), and play a central role in the processing and presentation of antigens to T cells during an immune response [1]. DC progenitors in the bone marrow give rise to circulating precursors that home to the tissue where they reside as immature cells with high phagocytic capacity. Upon tissue damage or exposure to antigens, DCs capture antigens and subsequently migrate to the lymphoid organs, where they select the rare antigen-specific T cells and initiate a cellular immune response [1, 2]. It has been shown that during migration and within secondary or tertiary lymphoid organs, DCs undergo functional maturation from antigen collection and processing to very potent APCs [1, 3, 4]. Immature DCs capture antigens, but weakly stimulate T lymphocytes. In the presence of particular signals, such as lipopolysaccharide (LPS) or various cytokines, immature DCs mature into potent T stimulatory cells, a process that is associated with up-regulation of co-stimulatory molecules (CD80, CD86, CD40, CD83, and DC-LAMP), as well as changes in chemokine receptors expressed on their surface [1‐6]. Immature CD1a+ DCs are CC-chemokine-receptor (CCR) 6-positive and respond to MIP-3α [7]. In contrast, mature DCs are attracted by the chemokine, MIP-3ß, or secondary lymphoid chemokines (SLCs) following de novo expression of CCR7 [6, 8, 9]. A critical characteristic of fully mature DCs is the production of pro-inflammatory cytokines, particularly IL-12, which plays a critical role in the induction of efficient T-helper cell 1 immunity [10], as observed for an efficient anti-tumour T cell response [1, 6].

The involvement of DCs in tumour immunity has clinical importance. The infiltration of DCs into some primary tumour types has been found to be associated with significantly improved patient survival and a reduced incidence of recurrent disease [11, 12]. It is known that tumours avoid surveillance by the immune system through various mechanisms, including the inhibition of the recruitment of DCs at the tumour site, as well as impairment of function of DCs by local production of immunosuppressive cytokines [13]. However, the precise knowledge of the tumour environment, which varies between different tumour types, might be important for the design of optimal immunotherapeutic strategies against cancer [14‐16].

Promising results have been previously reported using DC-based vaccination against immunogenic tumours, such as melanoma or renal cell carcinoma (RCC) [17, 18]. A subset of patients with metastatic RCC develops significant immune and clinical responses after immunotherapy with DC vaccination [1]. In this context mature DCs are thought to play a key role, since they are known to represent the most effective antigen presenting cells for induction of a potent T cell response. In order to give an answer to the question why some patients respond to DC-vaccine based therapies and others not, a detailed knowledge about the cellular T cell immune response in RCC with special regard to antigen-presenting DCs is required. However, detailed studies concerning the type and distribution of DC subsets in RCC are still lacking.

More recently, novel markers have emerged allowing the identification of a broader spectrum of DC subpopulations with respect to their function on formalin-fixed and paraffin-embedded tissue. Thus, we characterized the phenotype, distribution, and maturation of the different DC subsets in RCCs. In addition, we analyzed the local expression of chemokines that are known to play a key role for the recruitment of DC subsets.

A RCC is considered to be one of the most immunoresponsive cancers in humans. During the last few decades, promising new immunologic-based treatment strategies for this tumour entity have been developed [18]. However, the tumour response is observed only in a subset of patients. Thus, detailed knowledge of the mechanisms underlying the immune response at the interface between immune attack and immune suppression might help to improve the promising immunotherapeutic approaches in RCCs. More recently, emphasis has shifted to the use of DC vaccines for active immunization of cancer patients with in vitro-generated DCs, which have been fused with tumour cells or loaded with tumour antigens [19‐21].

Different approaches of RCC vaccines have been explored in the metastatic and adjuvant setting in several studies [22, 23]. Reviewing the current literature, about 20 non-randomised phase 1 or 2 immunotherapeutic trials have been published for DC-vaccines in metastasized RCCs [24‐40]. As a summary, none of these patients developed significant treatment-related toxicity or autoimmunity-related side effects, while approximately 40% of the patients demonstrated clinical tumour regression [18]. Nevertheless, the results of DC vaccination in these non-randomised studies should be viewed with caution because of the relative small number of patients within each trial and the diversity of the vaccination strategies used. Although most groups currently use monocyte-derived DCs for clinical vaccine trials [41], the isolation procedures of the monocytes, the differentiation procedures towards DCs, and the loading and maturation procedures are heterogeneous between the published clinical studies [42]. Thus, the current data suggest that DC vaccination in patients with metastatic RCCs appears to be safe and results in a tumour-specific immune response which can achieve tumour regression in a significant subset of patients [18]. However, for efficient and reliable immunotherapy of tumours, the optimal protocol for DC-based vaccination remains to be clarified further. Therefore, detailed knowledge concerning the immune response in RCCs with special regard to the role of DCs in vivo is required for development of an optimal vaccination strategy. One very important issue in this context represents a detailed knowledge concerning the type and distribution of DC subsets, as well as the mechanisms underlying their migration, maturation, and function in RCCs. Recent data point to a significant role of DC subsets in Th1/Th2 polarization and the induction of the tumour immune response [14, 43]. Thus, the determination of the origin and immune competence of tumour-associated DCs will be of great importance to our understanding of the development of tumour immunity for RCCs [44]. Therefore, we investigated the distribution and maturation of the different DC subtypes in RCCs and corresponding normal kidney tissues. The findings in the current study extend previous investigations for other tumour entities [14, 15, 45, 46]. The results demonstrate a unique compartmentalization of immature and mature DCs within kidneys affected by RCCs.

All tumour samples displayed variable immature DC infiltration. Tumour-infiltrating immature DCs were heterogeneous, and two populations were identified. One population represented the CD1a+/Langerin+ Langerhans-cell type and the other type was CD1a+/Langerin- non-Langerhans cell. Since dermal (interstitial) DCs were found in vitro and in vivo to express CD1a, but not Langerin, we currently conclude that CD1a+/Langerin- tumour-infiltrating DCs represent immature interstitial DCs. The significance of this heterogeneity remains to be established. Earlier studies revealed that interstitial DCs generated in vitro from CD34+ precursors display more potent phagocytic activity than LCs [1, 3].

The observed numbers of immature CD1a+ DCs in the tumour environment was much higher than in normal kidney tissues, suggesting increased homing and infiltration of immature DCs by the tumour. This finding was also been described by Troy et al. [47, 48] nearly a decade ago. This may be best explained by the high levels of intratumoural MIP3-α, a chemokine, which is known to specifically attract immature DCs. Our results show an increased expression of MIP-3α by tumour cells of RCCs by RT-PCR and immunohistochemistry. The immunohistochemistry analysis with anti-MIP3-α is unfortunately not precise enough to allow a correlation between the amount of MIP3-α protein by the tumour cells and the amount of infiltrating immature DCs. However, our finding of an increased expression of MIP-3α mRNA in RCC cell lines, as well as in RCC tissue compared to mRNA derived from normal kidney, underlines a role for an increased expression of MIP-3α in homing or recruitment of immature DCs to RCC tissues. The increased number of immature DCs could also reflect a transient stage due to the high in-and-out migration or the sequestering of immature DCs within the tumour tissue. Another explanation might be tumour-induced maturation arrest of tumour-infiltrating DCs. Studies for different tumour entities have shown that in cancer patients, DCs in the blood, tumour tissues, and draining lymph nodes are often functionally defective and possess poor T cell stimulatory capacity [49]. One possible explanation for this observation might be the expression of tumour-derived factors, such as VEGF, TGF-β, and IL-10, which have been shown to inhibit differentiation or functional maturation of DCs [49‐53]. Therefore, it has been suggested that immaturity of DCs in the tumour tissue may mediate tumour tolerance instead of immune activation caused by induction of T-cell anergy or favouring the development of regulatory T cells [54, 55].

However, in contrast to the observed immaturity of intratumoural DCs, the more important finding might be the presence of numerous mature DCs accumulating specifically within the peri-tumoural areas. This finding suggests that additional factors might be involved in mature DC distribution. Because mature DCs are typically observed in lymphoid organs, where they closely interact with T cells, it is tempting to consider that their presence within the tumour tissue might reflect an ongoing tumour-specific immune response. Thus, the mature DCs could derive from any of the immature DC subset discussed above within the tumour tissues. The peri-tumoural localization of mature DCs observed in our study corresponds to observations in other tumour types, and RCC as well [15, 47]. The preferential localization of mature DCs in lymphocyte-rich peri-tumoural areas of RCCs could be due to the resemblance of these areas to secondary lymphoid organs where mature DCs are normally found.

Interestingly, various studies have shown that the presence of a dense DC infiltration has been associated with prolonged survival and reduced incidence of metastases in patients with various human cancers, such as colorectal, gastric, esophageal, oral, and lung carcinoma [56‐59]. In breast cancer, the number of CD83+ mature DCs, but not the number of CD1a+ or S100+ DCs, has been shown to be of prognostic relevance [60]. In light of their importance in anti-tumour immunity, surprisingly few studies have been aimed at the presence of DCs and its potential clinical correlates in RCCs. For interferon pre-treated RCCs a trend toward a better outcome and better prognosis has been found in patients with a higher number of S100+ DCs within the tumour tissue [61, 62]. Although S100 protein has long been used to indicate DCs, its significance is still under confusion. In our experiments, we found S100 protein to be co-expressed by numerous CD68+ macrophages. Thus, S100 immunohistochemistry was not suitable for evaluation of DC numbers in RCCs (data not shown). In a more recent study, Kobayashi et al. [63] investigated whether or not the local immune environment might be associated with the tumour response following treatment with interferon-α and interleukin-2 in RCCs. Their results demonstrated a higher number of mature CD83+ DCs in tumour tissue of responders following cytokine treatment. In addition, the responders survived longer than non-responders. In contrast, other tumour-associated immune cells, such as CD8+ T-cells or tumour-associated macrophages, were not associated with treatment response or survival outcome. Thus, the presence of high number of CD83+ mature DCs might represent a predictive factor for the clinical outcome in patients with RCCs [63].

These and our observations suggest that the presence of an "immunologic functional unit" in RCCs consisting of antigen-presenting DCs and activated T cells at the tumour border might represent prognostic significance. In this scenario the combination of a low number or absence of DCs with a low density of activated T cells would predict a worse outcome, while a high density of mature DCs and activated T cells predicts a favourable disease outcome. This would also suggest that the presence of mature antigen-presenting DCs at the primary site together with that of activated T cells, represents a functional immune response against RCC progression. Thus, our observations open new insight regarding DC involvement and function concerning the anti-tumour response in RCCs. However, the limited number of RCC tumour samples analyzed in this study and the short follow-up times does not allow us to establish a prognostic significance of the infiltration of tumour by immature or mature DCs. Such determination will require the analysis of a large number of samples in prospective or retrospective studies. Therefore, further analysis of the APC system will provide clues to the understanding of tumour immunity as well as improvement of the efficiency of vaccine strategies in RCCs and other immunogenic tumour entities.