Letter to the Editor
Journal of Investigative Dermatology (2010) 130, 898–901; doi:10.1038/jid.2009.307; published online 15 October 2009
Indoleamine 2,3-Dioxygenase+ Cells Correspond to the BDCA2+ Plasmacytoid Dendritic Cells in Human Melanoma Sentinel Nodes
Gianni Gerlini1, Paola Di Gennaro2, Giulia Mariotti2, Carmelo Urso3, Alberto Chiarugi4, Nicola Pimpinelli2 and Lorenzo Borgognoni1
1Plastic Surgery Unit, Regional Melanoma Referral Center, Tuscan Tumor Institute (ITT), Santa Maria Annunziata Hospital, Florence, Italy
2Department of Dermatological Sciences, University of Florence Medical School, Florence, Italy
3Department of Anatomic Pathology, Dermatopathology Section, Santa Maria Annunziata Hospital, Florence, Italy
4Department of Pharmacology, University of Florence Medical School, Florence, Italy
Correspondence: Gianni Gerlini, E-mail: email@example.com
Abbreviations: DC, dendritic cell; IDO, indoleamine 2,3-dioxygenase; pDC, plasmacytoid DC; SLN, sentinel lymph node; TLR, toll-like receptor
TO THE EDITOR
Dendritic cells (DCs) have crucial roles in driving primary immune responses toward immunity or tolerance. While immature DCs prompt tolerance in peripheral tissues, mature DCs drive immunity in lymph nodes (Banchereau et al., 2000). A particular subset of DC, however, is thought to have the ability to induce tolerance regardless of the maturation state (Gilliet and Liu, 2002).
Munn et al. (2002) have described a subset of CD123+/CCR6+ DC in sentinel lymph nodes (SLN) of cancer patients with regulatory function. These cells express high levels of indoleamine 2,3-dioxygenase (IDO), the tryptophan-catabolizing enzyme, which is emerging as a master regulator of tolerance (Munn et al., 1998; Liu et al., 2006).
Indoleamine 2,3-dioxygenase+ cells are present in SLN of patients with melanoma, breast, colon, lung, and pancreatic cancer (Munn and Mellor, 2007). A high number of IDO+ cells occur in 45% of melanoma SLN, with or without metastasis. Importantly, this accumulation correlates with poor outcome (Lee et al., 2003) strongly suggesting a tolerance role for IDO+ cells. They are located in perisinusoidal regions of nodes, surrounding high endothelial venules; they exhibit monocytoid or plasmacytoid morphology and, in metastatic SLN, form clusters around melanoma nests. Therefore, IDO expression seems to be a crucial, tolerogenic mechanism in cancer immunity (Munn and Mellor, 2007). Consistently, IDO overexpression in tumor cells protects them from immune-mediated rejection, whereas the IDO inhibitor 1-methyl tryptophan can revert to tumor immune tolerance (Muller et al., 2005; Hou et al., 2007).
Plasmacytoid DCs (pDCs) are the main source of the antiviral/antitumor Type I IFN (Siegal et al., 1999; Liu, 2001). Paradoxically, in vitro studies show an active role of pDC in tolerance induction (Gilliet and Liu, 2002; Ito et al., 2007). Whether they have any role in cancer immunology has not been established. Yet, pDC, identified by the expression of the specific marker BDCA2 (Dzionek et al., 2001), are present in SLN, with high frequency in those bearing metastasis (Gerlini et al., 2007). Evidence that these pDC show an inactive immunophenotype and do not produce IFN-α hints that they favor tumor tolerance in humans (Gerlini et al., 2007). In keeping with a role for both pDC and IDO in suppression of tumor immunity, IDO is expressed by a subset of mouse pDC in melanoma tumor-draining lymph nodes. These cells prompt antigen-specific anergy, strengthening the hypothesis that IDO+ pDC are indeed tolerogenic (Munn et al., 2004).
The existence and function of human IDO+ pDC is as yet unknown. This is probably because the antibodies available against BDCA2 are not suitable for paraffin-embedded archive material. To understand whether IDO+ DC correspond to pDC, we took advantage of a recently described method for phenotypic analysis of cells from fresh and frozen human SLN, which does not interfere with pathological diagnosis (Vuylsteke et al., 2002; Gerlini et al., 2007). The study was conducted according to the Declaration of Helsinki Principles, and the Institutional Ethics Committee approved all described studies.
Samples were double stained with mouse anti-human BDCA2 and rabbit anti-human IDO. Variable numbers of BDCA2+ pDC were found in all the 15 SLN analyzed by flow cytometry (0.45±0.11%, mean±SD), and the majority of them coexpressed IDO (72.4±12.4 %), as assessed by immunofluorescence (Figure 1a and b). Notably, a subset of BDCA−/IDO+ large cells were also identified (Figure 1a). This finding suggests that other cells express IDO in SLN. As pDC accumulate in metastatic SLN and typically form clusters, frozen sections of metastatic SLN were investigated. Numerous BDCA2+ IDO+ pDC were observed, with some IDO-negative pDC scattered throughout the tissue (31.4±9.5%) (Figure 1b). Functionally, BDCA2+ pDC did not produce IFN-α (data not shown, Gerlini et al., 2007). Importantly, control LN and blood pDC were IDO negative (Figure 1e and see Supplementary material for details), suggesting that IDO expression may be specific to tumor SLN.
Figure 1 – Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact firstname.lastname@example.org or the author
Sentinel lymph node (SLN) BDCA2+ plasmacytoid dendritic cells (pDCs) express indoleamine 2,3-dioxygenase (IDO). Immunofluorescence analyses of pDC in human melanoma SLN (n=15: 4 positive for metastasis and 11 negative). Labeling was performed with mouse anti-human BDCA2, to identify pDC (revealed with goat anti-mouse Alexa Fluor 594 conjugated; red) and with rabbit anti-human IDO (revealed with goat anti-rabbit FITC conjugated; green). Nuclei were labeled with Hoechst 33342 (blue). (a) Immunofluorescence on cells from a metastatic SLN is shown. BDCA2+ pDC coexpressed IDO (yellow), although at a various degree of intensity. A subset of BDCA2−/IDO+ large cells were also identified (indicated by asterisks). Inset, high magnification of a BDCA2+/IDO+ pDC. (b) Immunofluorescence on frozen section from a metastatic SLN is shown. The majority of BDCA2+ cells coexpressed IDO (yellow), with only a few being IDO− (indicated by arrows). Inset, high magnification of BDCA2+/DO+ (yellow) and BDCA2+/IDO− (red) pDC. (c–d) Isotype controls for anti-BDCA2 Ab (mouse IgG1) and anti-IDO Ab (rabbit IgG), in SLN and normal blood cytospins, respectively. No specific stainings were detected. (e) Immunofluorescence on control LN and normal blood cytospins are shown. Control LN and blood BDCA2+ pDC did not express IDO (scale bars: a, b, 75 μm and 30 μm in the insets; c, d, 75 μm; e, 30 μm).
Full figure and legend (293K)
The role of pDC in cancer immunology is still a matter of debate, as these cells able to produce Type I IFN (Siegal et al., 1999; Liu, 2001), as well as promote tolerance (Munn et al., 2004; Gerlini et al., 2007). By showing that most of pDC express the immunosuppressive IDO in SLN of patients with melanoma, our findings strongly suggest that pDC have a tolerogenic role in human cancer. Recently, human pDC have been shown to upregulate IDO in response to HIV infection (Boasso et al., 2007). It is possible, therefore, that metastatic melanoma exploits this mechanism to repress immune effector functions in SLN (Cochran et al., 2006). Taken together, these findings indicate that the identification of IDO+ cells as the classical BDCA2+ pDC of SLN is not only a matter of immunophenotypic detail but has remarkable immunopathological and therapeutical significance.
Currently, different pharmacological strategies have been adopted to circumvent tumor tolerance in SLN. Toll-like receptor (TLR) agonists, such as the TLR9 activator PF-3512676, promotes DC-dependent proliferation of melanoma-specific CD8+ T cells as well as effector natural killer cell responses (Molenkamp et al., 2007, 2008). Similarly, the TLR7 agonist imiquimod reverts functional defect in pDC of SLN, and boosts tumor-specific immune responses in melanoma patients (Molenkamp et al., 2007; Adams et al., 2008). We claim that abrogating the tolerogenic effects of IDO by means of chemicals such as 1-methyl tryptophan (Hou et al., 2007) plus the concomitant use of TLR7/9 agonists might be an innovative pharmacological strategy to revert tumor tolerance in SLN.
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Conflict Of Interest
The authors state no conflict of interest.
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The authors acknowledge Ente Cassa di Risparmio di Firenze.