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* Antigen Presentation Research Group, Faculty of Medicine, Imperial College London, Northwick Park and St. Marks Campus, Harrow, Middlesex, United Kingdom; Departments of
Surgery and
Pathology, Northwest London Hospitals NHS Trust, Harrow, Middlesex, United Kingdom
1Correspondence: Antigen Presentation Research Group, Faculty of Medicine, Imperial College London, Northwick Park, and St. Mark’s Campus, Watford Road, Harrow, Middlesex HA1 3UJ, UK. E-mail: s.knight{at}imperial.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-) were also measured from intracellular accumulation of cytokine after blocking with monensin. All flow cytometry data were collected without compensation and were compensated off-line using the Winlist algorithm (Verity software). This package also provided the subtraction program to calculate percentage positive cells and intensity of staining. PDC (CD11c–, CD123+) expressed more cytokines than did myeloid DC (CD11c+) or CD1a+ putative "migratory" DC (P<0.001). LN PDC from patients with a good prognosis (px; n=11) demonstrated a relative increase in IL-12 and IFN- expression (median IL-10:IL-12 ratio=0.78 and median IL-4:IFN- ratio=0.7), and PDC from LN draining poor px cancer (n=9) showed a relative increase in IL-10 and IL-4 expression (median IL-10:IL-12 ratio=1.31 and median IL-4:IFN- ratio=2.6). The difference in IL-4:IFN- expression between good and poor px cancer groups was significant (P<0.05). Thus, PDC innately producing cytokines were identified in cell suspensions from human LN, and the character of PDC cytokine secretion may differ between two breast cancer prognostic groups. We speculate that a shift towards PDC IL-10 and IL-4 expression could promote tumor tolerance in LN draining poor px breast cancer.
Key Words: flow cytometry • DC subpopulations • intracellular cytokines • tumor immunity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two main DC types in human blood and lymph nodes (LN) are myeloid and plasmacytoid. Myeloid DC (MDC) are found in most peripheral tissues and, following antigen exposure, will mature and migrate to LN and secrete high levels of interleukin (IL)-12 [1
]. The role of plasmacytoid DC (PDC) in directing immune responses is unclear. PDC can be identified by positive surface staining for human leukocyte antigen (HLA)-DR and by bright expression of CD123 (IL-3 receptor 
chain) [5
]. In addition, they lack lineage-specific markers (CD3, CD19, CD56, CD11c, CD13, and CD33) and are found around high endothelial venules (HEV) in the T cell areas of LN [6
]. PDC may be involved in the induction of tolerance by polarizing toward the production of T helper cell type 2 (Th2) cytokines, such as IL-4 and IL-10 [7
]. Conversely, when stimulated by virus, PDC may mature and secrete Th1 promoting cytokines, such as IL-12 and interferon (IFN)- [6
].
PDC have been localized to the peri-tumoral areas of melanomas [8 ] and may be functionally impaired in the tumor tissue and draining LN of patients with head and neck squamous cell carcinoma [9 ]. Cytokines, such as IL-10 and VEGF, found in the vicinity of solid tumors have also been implicated in the inhibition of DC function [10 , 11 ].
This study aimed to identify MDC and PDC subpopulations, and the ongoing cytokines that these cells produce, in tumor-draining LN of patients with breast cancer. Four-color flow cytometry and a sensitive analysis algorithm were used to determine the ongoing intra-cellular cytokine expression of IL-4, IL-10, IL-12, and IFN- in these DC subsets. In our laboratory, this technique has already been used to demonstrate innate cytokine production by DC isolated from gut biopsies/blood. In addition, the exposure of gut/blood DC to LPS or probiotic bacteria resulted in the up-regulation of IL-12 and IL-10, respectively [12
].
Although both MDC and PDC could be easily identified in LN, cytokine expression was primarily found in the PDC subset. In LN draining tumors categorized as poor prognosis (px), we also report a relative increase in IL-10 and IL-4 expression by PDC. Our results suggest that PDC are major producers of DC cytokines in human LN and may influence local tumor immunity in patients with breast cancer.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Peripheral venous blood was also obtained from healthy human volunteers. Informed consent was obtained from all donors, and the project had local ethics committee approval.
Preparation of LN specimens
Fresh LN specimens were obtained directly from theater and transported to the laboratory in sterile growth medium [RPMI 1640 Dutch modification (Sigma Chemical Co., UK) supplemented with 10% (v/v) fetal calf serum (FCS; Gibco Co., Paisley, UK), penicillin (100 units/ml), streptomycin (100 µg/ml) and 2 mM glutamine (ICN Flow, UK)] for immediate processing. Using sterile instruments, the LN was dissected free of perinodal fat and washed in growth medium. The total LN mass was then obtained and recorded. The LN was bisected along the long axis by using a scalpel blade (Swann-Morton, Sheffield, UK). Each half of the LN was pushed through a sterile metal sieve, presoaked in sterile growth medium by using the plunger of a 2 ml syringe. Growth medium (5 ml) was slowly added to rinse through the sieve to create a sterile single cell suspension. The resultant suspension was filtered through a pre-soaked nylon cell strainer (100 um pore size, Falcon, BD Biosciences, San Jose, CA) to remove fat and capsule debris. The total numbers of LN cells in suspension were then counted with a hemocytometer using Trypan blue to exclude nonviable cells.
Monoclonal antibodies (mAb)
The following mAb were used for surface labeling: fluorescein isothiocyanate (FITC)-conjugated CD11c (KB90, Dako, Copenhagen, Denmark) and CD3 (UCHT1, Becton Dickinson, San Jose, CA), phycoerythrin (PE)-conjugated CD3 (UCHT1, Becton Dickinson), CD123 (7G3, Becton Dickinson), and CD1a (NA1/34, Serotec, Oxford, UK), Cy-Chrome-conjugated CD3 (UCHT1, Becton Dickinson), retinal pigment epithelial (RPE)-Cy5-conjugated CD14 (Tuk4, Serotec), Cy-Chrome-conjugated CD16 (1:25 dilution, 3G8, Becton Dickinson), RPE-Cy5-conjugated CD19 (SJ25-C1, Serotec), Cy-Chrome-conjugated CD34 (581, Becton Dickinson), PC5-conjugated CD56 (PC5, N901, Beckman Coulter, Fullerton, CA), allophycocyanin (APC)-conjugated CD3 (UCHT1, Becton Dickinson), and HLA-DR (1:4 dilution, G46-6, Becton Dickinson). Appropriate isotype controls were used (Becton Dickinson).
For cytokine labeling, PE-conjugated IL-4 (#3007, R&D Systems, Minneapolis, MN), IL-10 (JES3-9D7, Serotec), IL-12 (C11.5, Becton Dickinson), and IFN- (D9D10, Serotec) mAb were used. FITC-conjugated B-actin (AC15, Sigma, Dorset, UK) was used as a permeabilization control.
For whole blood cytokine competition experiments, the sources of surface antibodies were FITC-conjugated CD14 (MOP9, Becton Dickinson), ECD-conjugated CD3 (UCHT1, Beckman Coulter), CD19 (J4.119, Beckman Coulter), CD34 (581, Beckman Coulter), and PC5-conjugated HLA-DR (Immu-357, Beckman Coulter). The sources of cytokine antibodies were PE-conjugated IL-4 (3007.11, R&D) and IL-12 (C11.5, Biosource). Appropriate isotype controls were used. The lyophilized cytokines IL-4 and IL-12 (2 µg, PeproTech, Rocky Hill, NJ) were each reconstituted with 20 µl of sterile distilled water, giving a solution equivalent to 100 µg/ml. For blocking experiments, antibodies to IL-4 or IL-12 were preincubated with an equal volume of their respective cytokine or medium to give the following range of final concentrations: 1, 0.5, 0.1, and 0 µg/ml, respectively.
LN cell culture and staining
The method of LN cell culture used to determine innate cytokine production in DC was modified from an existing whole blood protocol developed to measure intracellular cytokines in lymphocytes [14
]. Aliquots (100 µl) of the LN cell suspension were transferred to ventilation-capped 5 ml polystyrene round-bottomed plastic tubes suitable for use in a Becton Dickinson FACSCalibur flow cytometer. To half of the cultures, 100 µl of monensin (sodium salt, Calbiochem, San Diego, CA, final concentration 4.5 µM; in preliminary studies 4.5 mM was found to be the optimum final monensin concentration for use in LN cell suspensions) was added to inhibit the transport of newly synthesized cytokine from the Golgi apparatus. To the other half, 100 µl of growth medium (no-monensin) was added to bring the final volume of all cultures to 200 µl.
The monensin and no-monensin cultures were incubated at 37°C in a humid, 5% CO2 atmosphere for 4 h (in preliminary studies, 4 h was shown to be the optimum incubation period). The cells were then washed with phosphate buffered saline (2 ml) containing sodium azide (0.02%, Sigma), 1 mM EDTA, and 2% v/v FCS (FACS buffer) at 400 g for 5 min, and the cell pellets were resuspended. FCS (20 µl) were added to prevent nonspecific antibody binding 5 min prior to staining with directly conjugated mAb against cell surface markers (15 min in the dark, room temperature). The spectral compensation panel consisted of singly stained CD3 (FITC, PE, CyChrome, APC) for off-line compensation. Surface marker staining was performed prior to fixation because some antibodies have been shown to bind poorly following the fixation and permeabilization steps (15].
The cells were washed with FACS buffer and then fixed by the addition of Cytoperm Reagent A (100 µl, Serotec) for 15 min at room temperature. The cells were washed once more with FACS buffer and were permeabilized by the addition of Cytoperm Reagent B (100 µl, Serotec). PE-conjugated mAb for the cytokines IL-4, IL-10, IL-12, and IFN- were added at 5 µl to monensin and no-monensin culture samples, to stain for 30 min in the dark at room temperature. The cells were washed finally with FACS buffer and resuspended in 1% paraformaldehyde in phosphate buffered saline (400 µl) at 4°C until acquisition within 48 h.
Evaluation of cytokine-specific staining
Blocking experiments were performed to determine the specificity of IL-4 and IL-12 cytokine antibody staining using the whole blood technique [16
].
Aliquots of whole blood (50 µl) were transferred to 5 ml polystyrene round-bottomed plastic tubes suitable for use in the EPICS XL-MCL four-color Flow Cytometer (Beckman Coulter) with a carrousel for automatic acquisition of sample tubes using the Coulter System II Software (version 3.0). Samples were diluted 1:1 with medium alone or medium containing Monensin to give a final concentration of 3 µM and were incubated at 37°C in a humid 5% CO2-in-air atmosphere for 2 h. Antibodies to the surface markers were added directly to each culture and left for 15 min at room temperature. Samples were then washed once with FACs buffer (2 ml). To lyse the RBC, Optilyse C (Immunotech, Marseilles, France; 250 µl) was added, mixed thoroughly, and left for 15 min at room temperature. Samples were then washed once with FACs buffer (2 ml). Leukoperm Reagent A (Serotec; 50 µl) was added to the drained cell pellet, left for 15 min at room temperature, and washed again. For intracellular cytokine detection, cells were permeabilized by the addition of 50 µl Leucoperm Reagent B followed by 10 µl of each of the range of concentrations of antibodies preincubated with cytokine. All samples were then left at room temperature for 30 min, washed with FACs buffer, drained, and resuspended in 500 µl of 4% paraformaldehyde.
Flow cytometry acquisition and data analysis
A Becton Dickinson FACSCalibur flow cytometer (or Beckman Coulter EPICS XL-MC flow cytometer for cytokine competition experiments) set up for 4-color evaluation was used for experimental data acquisition. List mode data from 250,000 events were collected without spectral compensation. Spectral compensation was achieved by data transfer to off-line PCs for analysis using the Color Compensation Toolbox within the WinList Version 5.0 flow-cytometry analysis software (Verity, Topsham, ME). Lymphocytes stained with strongly positive single-color CD3 mAb for each of the four fluorochromes (FITC, PE, CyChrome and APC) were used as the spectral standards.
To enable the calculation of absolute cell numbers of cell populations, we added 20 µl aliquots of a known concentration of fluorescent beads (flow count beads; Beckman Coulter) in proportion (1:5) to the original volume (100 µl) of each cell-suspension sample before data acquisition (17]. Based on the number of events in the cell region of interest compared with number of events in the bead region (Fig. 1A ), the absolute number of cells in the region per microliters of cell suspension was determined.
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The cytokine expression of each DC subset was determined by using the subtraction routine available within Winlist Version 5.0 (Verity Software, Topsham, ME). Single parameter histograms of IL-4, IL-10, IL-12, and IFN- cultured without monensin (no-monensin control samples) were subtracted from the cytokine samples cultured with monensin (test samples; Fig. 2
) to represent the ongoing cytokine expression. Single parameter histograms were subtracted using the same cytokine antibody in the no-monensin control rather than an isotype control antibody. This avoided the potential complications from the labeling of preformed cytokine, and of the differences in the fluorochrome to antibody ratio on isotype controls [18
].
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Based on the percent positive after the subtraction and the total number of cells in the DC subset region of interest, an absolute number of subset DC expressing cytokine was calculated per microliter of original LN cell suspension. By incorporating the original LN mass, total LN cell numbers, and volume of cell suspension, the numbers of DC subset cells expressing cytokines were then calculated per milligram of total LN tissue.
Statistical analysis
Statistical evaluation of grouped data was performed using the nonparametric Kruksal-Wallis one-way analysis of variance on ranks or the 
2 on contingency tables. Results were considered significant if P values were <0.05. Statistical analysis was performed using Sigmastat 3.1 (Systat Software, Point Richmond, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A live cell region was created on a FSC versus SSC histogram (Fig. 1A) and applied to a second HLA-DR versus Lineage histogram (Fig. 1B) . Three cell populations were defined based on expression of HLA-DR and Lineage (Fig. 1B) . A third histogram of HLA-DR versus CD11c was used to identify these populations as DC subsets (Fig. 1C) . In both control and tumor draining LN, PDC were readily identified as a prominent CD123+ population (Fig. 1C) that were also lineage–, HLA-DR+, and CD11c–. MDC were recognized by bright CD11c staining and were lineage– and HLA-DR+ (Fig. 1C) . We have defined the third population as strongly HLA-DR+ and lineage-/+ (Fig. 1C) . In skin draining, axillary and inguinal LN, this HLA-DR+/Lineage–/+ population expressed high levels of CD1a (Fig. 1C) , and hence might represent a heterogeneous population of putative migratory DC (migDC).
With the use of the back-gating feature of WinList 5.0, light-scatter properties suggested that the putative migDC population consisted of large granular cells (Fig. 1A , blue) and that the PDC and MDC populations were smaller, with similar size and granularity (Fig. 1A , green and red, respectively).
PDC were consistent producers of IL-4, IL-10, IL-12, and IFN- in human LN
By acting as an interface between tissue lymph and blood, peripheral LN facilitates interactions between antigen-presenting DC and lymphocytes. LN in vivo thus represent a site of ongoing immune reactions [25
]. We, therefore, sought to examine the function of our defined LN DC subsets by investigating intracellular cytokine expression in their native state without in vitro stimulation. The expression of the Th2 cytokines IL-4 and IL-10, as well as the Th1 cytokines IL-12 and IFN-, were studied [1
]. DC subsets in LN were simultaneously identified and investigated for expression of these cytokines using four-color flow cytometry (Fig. 2)
.
To evaluate the specificity of anti-cytokine antibodies, competition experiments were performed on monocytes and putative whole-blood DC. Two populations were identified as CD14+ CD3– CD19– CD34– HLA-DR+ (monocytes) or CD14– CD3– CD19– CD34– HLA-DR+ (putative DC) and stained for IL-4 and IL-12 intracellular expression in the absence or presence of increasing concentrations of purified IL-4 and IL-12, respectively (0–1 µg/ml). As shown in Figure 3 , IL-4 and IL-12 were expressed by CD14+ populations and each was blocked by 0.01 µg/ml of purified cytokines. Putative DC (CD14–) populations also expressed IL-4 and IL-12 and were also blocked by 0.01 µg/ml of purified cytokine (data not shown). Blocking of the IL-4 antibody with purified IL-12 cytokine, and vice versa, had no effect on staining. At higher cytokine concentrations, such as 1 µg/ml, an increase in positive cells may be observed (Fig. 3) . This effect may represent cellular binding of cytokine-antibody complexes [16 ].
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When compared with both the MDC and migDC subsets, PDC were prominent in consistently expressing ongoing cytokines without stimulation. Collective analysis of all LN studied, using two statistical techniques, established that the PDC subset expressed significantly more IL-4, IL-10, IL-12, and IFN- than MDC or migDC (2 test P<0.001 and Kruksal-Wallis one-way analysis of variance on ranks P<0.05; Fig. 4
).
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(P=0.002) cytokine staining between PDC, MDC and MigDC (Fig. 4)
. In LN draining good px cancer, significant differences in the median values of IL-4 (P<0.001), IL-10 (P=0.01), IL-12 (P<0.001), and IFN- (P=0.02) expression by PDC, MDC, and MigDC were also found.
In LN-draining poor px cancer, although a trend was suggestive of prominent PDC cytokine secretion, the median values of IL-4 and IL-12 expression by PDC, MDC, and MigDC did not differ significantly different. However, significant differences were found in the median values of IL-10 (P=0.049) and IFN- (P=0.035). The lack of a significant difference in IL-4 and IL-12 secretion between DC subsets in LN draining poor px breast cancer may reflect the smaller sample size of this group.
An increase in the IL-10:IL-12 and IL-4:IFN- production ratios by PDC in LN draining advanced breast cancer
We investigated the feasibility of using this new technique for measuring ongoing cytokine production in DC by identifying changes in IL-4, IL-10, IL-12, and IFN- expression by PDC in tumor-draining, axillary LN. As described in Materials and Methods, breast cancer patients were stratified into two prognostic groups.
Human LN, regardless of anatomical location, showed a Log10 normal distribution with respect to size and total cell number (data not shown). Absolute numbers and ratios of IL-10:IL-12 and IL-4:IFN- expressing PDC (per milligram of LN tissue) were calculated and compared among three groups of patients: controls (inguinal and cervical LN), cancer good px, and cancer poor px. By comparing the ratios of cells expressing IL-10:IL-12 and IL-4:IFN-, as well as the absolute cell numbers expressing IL-4, IL-10, IL-12, and IFN-, we examined the balance of cytokine polarization by PDC from individual and grouped patient LN samples.
Despite the small numbers of patients investigated in this preliminary study, the feasibility of this approach was confirmed and some indicators of differences were identified. The median numbers of PDC expressing IL-4, IL-10, and IL-12 were similar in all three patient groups (Fig. 5A
5B
5C
). A trend suggested a decreased number of IFN- expressing PDC from axillary LN draining poor px cancer (Fig. 5D)
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expression ratios with median values of 0.89 and 0.95, respectively (Fig. 6
). In LN draining good px breast cancer, PDC may be observed to demonstrate a trend towards relative increases in IL-12 expression with a median IL-10:IL-12 ratio of 0.78. By contrast, PDC from LN draining poor px breast cancer showed a trend towards a relative increase in IL-10 expression (median ratio of IL-10:IL-12 expression=1.31; Fig. 6A
).
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between the three patient groups (P=0.047; Fig. 6B
). PDC from LN draining good px breast cancer were found to express relatively more IFN- than IL-4 with a median IL-4:IFN- expression ratio of 0.7. Contrary to this observation, PDC from LN draining poor px breast cancer were found to have a relative increase in IL-4 staining with a median IL-4:IFN- expression ratio of 2.6. Of note, in the good px sample group, LN metastases were not detected in any patients. In the poor px sample group 3/9 patients were found to have LN metastases following histological analyses of the operative specimens (data not shown). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and Th2 (IL-4 and IL-10) cytokines in both control and breast cancer draining LN. The regulation of an immune response is as important as its initiation. Local cytokine fields generated by cells within LN should, in most cases, be contained to prevent systemic propagation [26 ]. The escalation of inflammatory responses from the local to the systemic level can be a catastrophic event for patients, as evidenced by the systemic inflammatory response syndrome (SIRS). In the cases where SIRS does not resolve, the circulating levels of cytokines such as IL-8 and IL-18 are significantly elevated compared with those in survivors [27 ].
LN have a highly compartmentalized tissue structure with a complex micro-architecture. The location of PDC in the T cell area of the LN paracortex [6 ] may make them ideal candidates for creating local cytokine fields [26 ] and influencing T cell differentiation. Immature PDC may gain access to LN via HEV by expression of CXCR3 and CD62L [6 ], rather than via afferent lymphatic vessels [30 ]. The problem of how resident LN DC gain exposure to peripheral antigen may be partially overcome by the concept of DC antigen transfer. It has been previously described that antigen transfer and an antigen gradient between DC are required for primary T cell proliferation [31 ]. Recent studies in mice, in the context of DC vaccines, have localized antigen transfer to LN defining an important role for endogenous DC [32 ], such as PDC.
In the steady state, T cells patrol the LN paracortex until they encounter presented antigen [33 ]. It is conceivable that antigen-bearing migDC, such as Langerhans cells, may enter LN via afferent lymphatic vessels and filter through the sinus system to transfer antigen to resident PDC in the paracortex. In healthy individuals, PDC may then produce cytokines in response to the transferred antigen to initiate T cell responses.
Just as pro-inflammatory cytokine fields can propagate from local to systemic environments, a tolerogenic or Th2 type cytokine field could achieve the same systemic penetration [26 ]. In addition, the widespread systemic distribution of tumor antigens may also disrupt existing antigen gradients between lymphoid and blood DC [28 ]. These proposed mechanisms may contribute to the cellular immunodeficiency seen in patients with solid tumors [29 ].
In our hands, the cytokine profiles of PDC from LN draining good px breast cancer were found to be polarized toward a balanced/ Th1 pattern. In contrast, the cytokine expression of PDC from LN draining poor px breast cancer demonstrated a shift towards Th2 polarization. These results indicate that the character of immune responses may differ between two breast cancer prognostic groups and suggest that local DC responses in tumor draining LN might not be uniformly poor [34 ].
The difficulty in interpreting these results lies with determining whether the factors discriminating between good and poor px breast cancer groups are related to time of diagnosis, tumor aggressiveness, or inherent differences in patient immune responses. In addition, the presence of metastatic cancer cells within LN may influence local cytokine polarization and antigen gradients.
If times of diagnosis or tumor qualities are the defining features of px, then the model of immune exhaustion/tolerance may explain why tumors progress locally and metastasize systemically [35 ]. Alternatively, patient-specific local immune responses within the tumor microenvironment might influence individual px. Anti-tumor immune responses might reflect heredity and genetic factors [36 ] or early priming of immune cells to infectious agents, possibly as a neonate or during gestation [37 ].
The information from this small study has demonstrated a suitable methodology for identifying changes in ongoing DC cytokine production in LN-draining human cancers. The initial data require confirmation in a study of larger numbers of patients but indicate that local tumor immune responses might differ among patient prognostic groups, which suggests that PDC cytokine expression might contribute to tolerance of tumor cells in the local environment.
The proposed three-step interaction among tumor cells, migrating DC, and endogenous PDC in local LN might form the crux of tumor immunity and warrants further investigation.
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ACKNOWLEDGEMENTS |
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Received November 3, 2003; revised July 5, 2005; accepted July 6, 2005.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-secreting melanoma specific CD8 lymphocytes and are found in primary melanoma lesions Eur. J. Immunol. 33,1052-1062[CrossRef][Medline]
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