Introduction
Tumor-specific T-cell activation begins in the primary tumor when dendritic cells (DCs) encounter antigens in the form of apoptotic or necrotic tumor cells. The DCs engulf dying tumor cells and process their antigens into peptides that are presented in the context of MHC class I and class II molecules [
1,
2]. The function of a DC is highly influenced by its level of maturation. Immature DCs are capable of antigen uptake and processing but cannot, unless given the proper cytokine signals, present antigen to T cells [
1,
3,
4]. After receiving the correct cytokine signals, the mature, peptide-loaded DCs migrate from the tumor to the first draining lymph node, referred to as the sentinel lymph node (SLN). In the SLN, naïve T cells are activated by the peptide-loaded mature DCs. These T cells then undergo clonal expansion, gain effector function, and circulate back to the tumor, where their function is to lyse tumor cells. Evidence to support this process comes almost entirely from
in vitro experiments [
1‐
4].
SLN biopsy allows identification of the first lymph node into which a primary tumor drains. In breast cancer, identification of tumor cells in the SLNs is a predictor of the tumor's metastatic potential [
5,
6]. In the present study, we examined SLNs for evidence of immune activation by examining the maturation state of DCs within the SLNs. We defined mature DCs by their expression of the marker CD83 [
7,
8], while immature DCs were identified by their expression of the marker CD1a. We were interested in determining whether the maturation status of DCs in SLNs was associated with the tumor status of the SLN, so we compared DCs in tumor-free SLNs and in tumor-containing SLNs.
Materials and methods
Study population
SLN tissues from women aged 26–87 years who had a SLN biopsy performed at The University of Texas MD Anderson Cancer Center between 1998 and 2001 were included in the study. Each of the patients had received a diagnosis of breast cancer and had undergone SLN biopsy as part of her surgical treatment. Paraffin-embedded SLN tissues from 50 patients, 25 with tumor-free SLNs and 25 with tumor-containing SLNs, were examined. The tumor status of the SLN was determined by H & E and immunohistochemical staining. All samples were banked in the Breast Tumor Bank at The University of Texas MD Anderson Cancer Center.
The study population included six women who received chemotherapy prior to their SLN biopsy: four whose SLNs contained tumors and two whose SLNs were tumor-free. Twelve lymph nodes draining from the unaffected breast of women with breast cancer were similarly processed. All materials were collected under a protocol approved by the MD Anderson Cancer Center Institutional Review Board.
Antibodies
The following antibodies were used for immunohistochemical staining: anti-CD3 (clone PS1; BioGenex, San Ramon, CA, USA), anti-HLA class II (clone CR3/43; DAKO, Carpinteria, CA, USA), anti-CD83 (clone HB15A; Immunotech, Marseille, France), anti-CD1a (clone O10; Immunotech), anti-IL-10 (clone 23738.111; R & D Systems, Minneapolis, MN, USA), and anti-IL-12 (clone 24910.1; R & D Systems). Optimal concentrations were determined empirically, and all antibodies were tested for staining of cells in cytospin preparations of peripheral blood mononuclear cell (PBMC)-derived immature DCs (PBMCs stimulated with granulocyte–macrophage colony-stimulated factor and IL-4 for 6 days) or mature DCs (PBMCs stimulated with granulocyte–macrophage colony-stimulated factor and IL-4 for 6 days followed by tumor necrosis factor alpha for 24 hours).
Immunohistochemistry
Tissue sections, 3 μm thick, were cut from tissue blocks of formalin-fixed, paraffin-embedded SLNs. Immunocytochemical staining of deparaffinized, fixed slides was performed after antigen retrieval by microwave heating in citrate buffer. Slides were incubated with biotinylated goat anti-mouse IgG followed by avidin–biotin–peroxidase complex (Vectastain ABC Kit; Vector Laboratories, Berlingame, CA, USA). The peroxidase was developed by 3-amino-9-ethylcarbazole-9 (AEC substrate kit; Vector Laboratories) and counterstained with Gill's hematoxylin (Vector Laboratories). Isotype-matched antibodies were used as negative controls. The positive control antibody for each group of slides was anti-CD3.
Because of the localized distribution of DCs within the SLNs, each slide was scored by counting positively stained cells in five high-powered fields (hpfs) under × 400 magnification. Results were reported for each SLN as the mean ± standard error of the mean for 5 hpfs. This method has been used by other workers when analyzing the expression of mature DCs in breast tissue [
2,
9]. All slides were counted without knowledge of the SLN status, and results were confirmed through a second reading by a breast cancer pathologist (AS).
Statistical analysis
All data sets were tested for normal distribution. Because most data groups were not normally distributed, all data groups were analyzed by the Wilcoxon rank sum test. P < 0.05 was considered significant.
Discussion
Our experiments were designed to determine whether the SLN was the site of T-cell activation in breast cancer patients. Identification of immunologically mature DCs within the SLN would support this hypothesis. In our experiments, mature DCs were defined by their expression of CD83, a marker that has been shown on DCs capable of antigen presentation and activation of naïve T cells [
7,
8]. Our results show a definite trend toward higher numbers of CD83-positive DCs in tumor-free SLNs than in tumor-containing SLNs. In addition, tumor-free SLNs contained significantly higher numbers of IL-10-expressing cells. Both of these observations support the hypothesis that a tumor-free SLN is immunologically competent and is potentially a site of tumor-specific T-cell activation.
What remains unclear from our data is the type of immune response that occurs in the tumor-free SLNs. Identification of both IL-10-expressing and IL-12-expressing cells suggests two possibilities. The existence of IL-10 in tumors has been associated with immune suppression of a Th1 response and increased tumorigenicity [
11]. But it has been shown convincingly through
in vitro studies that IL-10 specifically affects immature DCs by inhibiting upregulation of MHC class II and costimulatory molecules and expression of CD83-positive cells. Mature CD83-expressing DCs are resistant to the effects of IL-10 [
12]. Less information is available on the expression of IL-12 in tumor-draining lymph nodes. Animal, as well as
in vitro, studies support the role of IL-12 as a proinflammatory cytokine that drives DCs toward activation of Th1 [
13,
14]. Clearly, the existence of both of these two cytokines in the SLN and not in uninvolved lymph nodes supports the hypothesis that an active immune response is occurring in the SLNs. Future functional studies examining both DCs and T cells are needed to determine whether the SLN is a site of immune suppression, as suggested by the expression of IL-10, or of immune activation, as suggested by expression of IL-12.
Our results differ from those reported by Huang and colleagues [
15], who compared S-100-positive DCs in SLNs and non-SLNs. Their conclusion, based on the morphology and density of the paracortical area and the number of paracortical S-100-positive DCs, was that the SLN was immunomodulated compared with the non-SLN. The SLNs examined in their study had reduced paracortical areas, reduced densities of paracortical DCs, and reduced frequencies of S-100-positive DCs with a predominance of immature, poorly dendritic DCs. Because they did not identify the tumor-free SLNs versus the tumor containing SLNs in their study, it is hard to compare our results with theirs. When we group our data and compare all SLNs with lymph nodes draining from the noncancer-containing breast of women with cancer, however, the numbers of DCs in the SLNs and these lymph nodes are not different, suggesting that the SLN is not immunomodulated compared with uninvolved nodes.
Few laboratories have examined the immune status of SLNs of human tumors [
15‐
17]. Most work involving SLNs has been limited to studies of frozen or paraffin-embedded tissue. In one of the few studies in which fresh SLN tissue was available, an attempt was made to determine whether tumor-specific T cells could be categorized as Th1-type or Th2-type cells, based on cytokine profiles [
16]. No classification was possible because the SLN T cells secreted both interferon gamma and IL-10 when stimulated with autologous tumor cells. However, these T cells were clearly tumor specific because they responded to autologous but not allogeneic tumor cells [
16]. A more recent study examining the cytokines produced by Staphylococcal enterotoxin-A-stimulated T cells from SLNs that drain primary melanoma showed both Th1 and Th2 cytokines could be measured by an enzyme-liked immunospot (ELISPOT) assay in tumor-free SLNs, whereas tumor-containing SLNs showed no increase in cytokine secretion [
17]. The implication from this study was that immune responses were downregulated by micrometastases in the SLN. Both reports support our data and suggest that the SLN is the site of antigen-specific activation and that the tumor status of the node may predict the immune response to the tumor.
The pathologic features of the tumor may affect the immune response in the SLN. It is accepted that the tumor microenvironment affects the maturation state of the DCs in the tumor. Our data showed that the number of CD1a-expressing cells in the SLN was associated with tumor grade. When high-grade tumors (grade III) were analyzed, higher numbers of CD1a cells were found in the tumor-containing SLNs. This suggested that the undifferentiated state of the tumor inhibits DC maturation, causing an accumulation of immature DCs unable to activate naïve T cells and resulting in metastasis of the tumor to the draining node. A report by Vitale and colleagues [
18] suggested that the lack of an immune response in the high-grade tumor is explained by the downregulation of MHC class I and TAP-1 and TAP-2 proteins in these tumors.
A recent report by Iwamoto and colleagues [
9] examining CD83-expressing DCs in 130 human breast tumors demonstrated that the presence of tumor-infiltrating CD83-expressing DCs correlated inversely with lymph node metastasis. Their data strongly suggested that CD83 DCs are involved in the initiation of the primary antitumor response. Our data, which showed a trend toward increased numbers of CD83-positive DCs in tumor-free SLNs compared with tumor-containing SLNs, further support this conclusion, and similarly suggest that the maturation state of the DCs predicts the metastatic potential of the tumor. Further experiments to compare the DCs in primary tumors and their SLNs are now underway in our laboratory to confirm the relationship between the maturation state of the DCs, the tumor grade, and the tumor metastatic potential.
Conclusions
In summary, we found a trend toward higher numbers of CD83-positive DCs in tumor-free SLNs than in tumor-containing SLNs. In addition, tumor-free SLNs contain significantly higher numbers of IL-10-expressing cells. Both of these observations support the hypothesis that the tumor-free SLN is immunologically competent and is potentially a site of tumor-specific T-cell activation. To establish the prognostic potential of the DCs in the SLN, we are now undertaking functional studies to compare the immune status of DCs isolated from the primary tumors of patients with tumor-free SLNs and those with tumor-containing SLNs.
Acknowledgements
The authors thank Sandra Kinney for excellent technical assistance. In addition, they are grateful for the technical assistance provided by the staff of the Breast Tumor Bank at The University of Texas MD Anderson Cancer Center. The Breast Tumor Bank is supported through a grant from The Nellie B Connally Breast Cancer Research Fund. Statistical analysis was performed with the help of Marcella Johnson, of the Department of Biostatistics, MD Anderson Cancer Center. This research was supported by Department of the Army Grant DAMD17-00-1-0680 (to NJP) and by funds from the University Cancer Foundation of the University of Texas MD Anderson Cancer Center (to NJP).