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Published ahead of print on October 5, 2006, doi:10.1165/rcmb.2006-0284OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 36, pp. 360-367, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0284OC

Plasmacytoid Dendritic Cells from Human Lung Cancer Draining Lymph Nodes Induce Tc1 Responses

Alexander Faith, Emma Peek, Joanne McDonald, Zoe Urry, David F. Richards, Carol Tan, George Santis and Catherine Hawrylowicz

Department of Asthma, Allergy and Respiratory Science, King's College; and Department of Thoracic Surgery, Guy's and St Thomas' NHS Foundation Trust, London, United Kingdom

Correspondence and requests for reprints should be addressed to Alexander Faith, Dept. Asthma, Allergy and Respiratory Science, 5th Floor Thomas Guy House, Guy's Hospital, St Thomas St, London SE1 9RT, UK. E-mail: alex.faith{at}kcl.ac.uk


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Dendritic cells (DC) resident in draining lymph nodes (LN) of patients with lung cancer are proposed to have a critical role in stimulating anti-tumor immunity. CpG oligodeoxynucleotides are undergoing clinical trials in patients with lung cancer and are likely to target plasmacytoid-DC. The present study, therefore, investigated the capacity of plasmacytoid-DC from human lung cancer draining LN to respond to CpG for activation of T cell responses relevant to anti-tumor immunity. The phenotype of DC was examined by flow cytometry, and cytokine production by cytometric bead array (CBA) and ELISA. Plasmacytoid-DC, purified by cell sorting, were immature but expressed the toll-like receptor, TLR9. Plasmacytoid-DC responded to the CpG oligodeoxynucleotide, CpG 2216, by production of the proinflammatory cytokines, IFN-{alpha} and IL-6. DC were cocultured with normal, allogeneic T cells, and cytokine production determined by CBA and immunophenotyping. CpG 2216 enhanced IFN-{gamma} production and induced intracellular production of IFN-{gamma} by CD8+ and CD4+, granzyme B by CD8+, and IL-2 by CD4+ T cells, respectively. Ligation of CD40 on plasmacytoid-DC combined with exposure to CpG 2216 also strongly enhanced IFN-{gamma} production. There was no significant difference between the responses of plasmacytoid-DC from patients with lung cancer and patients with benign carcinoid tumors with no pathologic LN involvement. These results indicate that plasmacytoid DC from the draining LN of patients with lung cancer effectively induce Tc1 immunity and could, therefore, represent a novel and attractive target for immunotherapeutic intervention.

Key Words: human lung cancer • dendritic cells • lymph node



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The ability of plasmacytoid dendritic cells to induce anti-tumor T cell responses is unclear. We report that plasmacytoid dendritic cells from lymph nodes of patients with lung cancer stimulate Tc1 responses and may thus represent a target for therapeutic intervention.

 
Lung cancer is one of the most common malignancies in the Western world and is associated with very poor survival rate even after tumor resection (1). The development of effective cancer vaccines, which could aid elimination of residual tumor burden, is dependent on the ability of lung resident dendritic cells (DC), the principal antigen-presenting cell population, to stimulate an effective and sustained T cell response (2, 3).

DC are heterogeneous, but two principal subsets in humans have been identified: myeloid-DC, which include interstitial DC resident in peripheral tissues, and plasmacytoid-DC, mainly resident in draining lymph nodes (LN) and peripheral blood (4). Myeloid-DC from the peripheral blood and draining LN of patients with lung cancer were reduced in frequency and reportedly demonstrated impaired function (5, 6), including increased susceptibility to apoptotic death. This has focused attention on plasmacytoid-DC as a potentially attractive target for immunotherapeutic intervention in lung cancer (7).

Plasmacytoid-DC have been identified as the cells responsible for production of type I interferon in response to virus, and consequent induction of Th1 and Tc1 responses by viral-specific CD4+ and CD8+ T cells, respectively (8, 9). Tc1 responses have also been detected after stimulation of plasmacytoid-DC with unmethylated CpG oligodeoxynucleotides (ODN) (10). CpG ODN are synthetic ligands for the Toll-like receptor 9 (TLR9), which is constitutively expressed by human plasmacytoid-DC and B cells (11). Therapeutic administration of CpG ODN to patients with melanoma and non-Hodgkins lymphoma has recently been shown to enhance anti-melanoma–specific CD8+ T cell responses and type I interferon inducible genes, respectively (12, 13). Although clinical trials of CpG in non–small cell lung cancer (NSCLC) are in progress (14), it is not known whether CpG ODN have direct effects on plasmacytoid-DC from patients with lung cancer.

Plasmacytoid-DC migrate from peripheral blood to draining LN as a result of infection or inflammatory responses (8). Migration of DC to lung draining LN is believed essential to the induction and maintenance of effective anti-tumor T cell immunity (7). The products of tumor cells have, however, been shown to inhibit type I interferon production and antigen-presenting capacity by plasmacytoid-DC (7), and induce down-regulation of TLR9 (15). Moreover, tumor infiltrating plasmacytoid-DC from patients with ovarian carcinoma were reported to promote the induction of CD4+ T cells secreting the anti-inflammatory cytokine, IL-10, and exhibiting regulatory function (Treg) (16). Thus, the capacity of plasmacytoid-DC to eliminate lung tumors could be adversely affected by infiltration of tumor cells into cancer draining LN.

We, therefore, investigated the presence of plasmacytoid-DC in lung cancer draining LN and investigated whether purified LN plasmacytoid-DC were stimulatory for CD4+ and CD8+ T cell responses, both of which are generally believed essential for effective anti-tumor immunity. We hypothesized that exposure of plasmacytoid-DC to CpG ODN would enhance Th1 and Tc1 immunity and compared responses of plasmacytoid-DC from patients with lung cancer and patients with benign carcinoid tumors without pathologic LN involvement.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Patients
Lung draining lymph nodes were obtained after resection from eight patients with early stage NSCLC, Clinical details of the patients studied are given in Table 1. Pathologic examination showed that each patient had tumor infiltration of their LN. Three patients with benign carcinoid tumors and with no pathologic lymph node involvement were included as a control group. Lymph nodes obtained for the study were surplus to diagnostic requirements. The study was approved by Guy's Hospital Ethics Committee. All patients gave written informed consent before inclusion in the study.


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  		TABLE 1. CLINICAL DETAILS OF PATIENTS

 
Monoclonal Antibodies
Biotin-labeled mAb to CD3, CD14, CD16, CD19, and CD56; PerCP-labeled mAb to HLA-DR; and a panel of leukocyte surface markers conjugated to PE, FITC, or allophycocyanin (APC) including CD1a, CD3, CD11c, CD14, CD19, CD40, CD80, CD83, CD86, CD123, HLA-DR, CCR6, and CCR7 were obtained from BD Biosciences (Oxford, UK). PE-labeled BDCA-4 mAb was obtained from Miltenyi Biotec (Bisley, UK). A panel of unconjugated mAb to TLR was obtained from AMS Biotechnology (Abingdon, UK).

Identification of DC Subsets within Lung Draining LN
LN were rinsed with saline to remove residual blood. LN were dissected, teased apart, and digested in Hanks' balanced salt solution with endotoxin-free collagenase (2 mg/ml, Liberase C1; Roche, Burgess Hill, UK) for 1 h at 37°C. The digest was then centrifuged at 200 x g and cultured for 4 h in RPMI 1640 containing 10% fetal calf serum, 2 mM L-glutamine and gentamycin (250 µg/ml), designated complete medium, with the addition of IL-3 (10 ng/ml) (R&D Systems, Abingdon, UK). The digested fragments were filtered through a 100-µm cell strainer (Becton Dickinson, Plymouth, UK) and a single cell suspension obtained. Cells were suspended in PBS containing 0.1% BSA and 0.02% sodium azide. Dendritic cells were identified within LN suspensions by immunophenotyping for lineage-negative (lin–) and MHC class II+ leucocytes. LN cells (0.5 x 106) were stained with biotin-conjugated mAb to CD3, CD14, CD16, CD19, and CD56; Per CP-conjugated mAb to HLA-DR; and PE- and FITC-conjugated combinations of mAb to DC subsets, costimulatory molecules, and maturation markers for 30 min. The cells were washed and then stained with streptavidin-APC (BD Biosciences) for 30 min. Approximately 400,000 events were collected for each sample. Cells were then analyzed using Cell Quest software on a FACSCalibur flow cytometer (BD Biosciences). All steps were performed at 4°C to prevent receptor internalization.

Purification of Plasmacytoid-DC
To obtain sufficient numbers of plasmacytoid-DC for functional studies, individual LN from each patient were pooled and cell suspensions cultured for 16 h in complete medium with the addition of IL-3 (10 ng/ml). Plasmacytoid-DC were positively selected on the basis of BDCA-4 expression by cell sorting (FACSAria; BD Biosciences). Initial experiments demonstrated that depletion of CD3+ and CD19+ cells using magnetic beads according to the manufacturer's instructions (Miltenyi Biotec) before positive selection decreased T and B cell contamination (data not shown). The resulting DC population was 92–95% BDCA-4+ containing < 5% CD3+, CD14+, CD19+, and CD56+ cells.

FACS Analysis of Purified Plasmacytoid-DC
Plasmacytoid-DC were analyzed for surface markers by staining with fluorochrome-conjugated antibodies to (1) CD3, CD11c, CD14, CD19, CD40, CD56, CD80, CD83, and HLA-DR; (2) mAb to TLR2, -3, -4, and -9 followed by staining with an F(ab')2 goat anti-mouse IgG-fluorochrome–conjugated secondary reagent (R&D Systems). Isotype controls included the appropriate fluorochrome-conjugated or unconjugated mouse IgG1 or IgG2a (Sigma, Poole, UK) and were concentration matched for each antibody used. The cells were washed, fixed, and then analyzed by flow cytometry. Expression of TLR9 was detected by intracellular staining. Cytofix/Cytoperm Plus (BD Biosciences) was added and the cells permeabilized. For analysis of specific epitope expression by DC, cells were gated for large FSC cells and 5–10,000 events collected.

Stimulation of Plasmacytoid-DC
LN plasmacytoid-DC were seeded at 5 x 104/well (96-well flat bottom) in complete medium (100 µl). DC were stimulated for 48 h with indicated concentrations of CpG 2006 or CpG 2216 (10) or control GC sequences (InvivoGen, Toulouse, France) in the presence of IL-3 (10 ng/ml). Soluble, recombinant CD40L (1 µg/ml) was obtained from Alexis (Nottingham, UK).

Cytokine Production
The cytokine groups IL-6, IL-8, IL-10, IL-12p70, and TNF-{alpha} or IL-2, IL-4, IL-5, IL-10, and IFN-{gamma} were measured by Cytometric Bead Analysis (CBA; BD Biosciences) using the Human Inflammation Kit or Th1/Th2 kit, respectively, according to the manufacturer's instructions. The lower detection limit of the assay for each cytokine was 5 pg/ml. IFN-{alpha} was detected by ELISA using a matched Ab pair (R&D Systems) and the lower limit of detection was 50 pg/ml.

Co-Culture of Plasmacytoid-DC with T Cells
Peripheral blood was obtained from healthy, nonatopic individuals. PBMC were obtained by density gradient centifugation, labeled with mAb to CD3, and T cells were purified by cell sorting. The resulting cell population was > 98% CD3+. Purified plasmacytoid-DC were washed twice and co-cultured at a DC:T ratio of 1:10 with normal, allogeneic T cells (1 x 105, round bottom 96-well plate; 1 x 106, 24-well plates), in the presence or absence of CpG, control ODN, or CD40L, and supernatants obtained after 6 d.

Intracellular Cytokine Production
All reagents for intracellular staining were obtained from BD Biosciences. PMA (10 ng/ml; Sigma) and ionomycin (250 ng/ml; Calbiochem, Beeston, UK) were added to cultures for the final 4 h and monensin for the final 2 h to allow accumulation of cytokines within the cell. The cells were permeabilized and triple-stained with specific fluorochrome-conjugated mAb to IFN-{gamma}, IL-10, IL-2, IL-4, IL-5, and granzyme B. Appropriate fluorochrome-conjugated isotype controls were included. Live T cells were gated based on forward scatter characteristics and nonincorporation of the dye, 7-amino actinomycin D (7-AAD).

Statistics
The Mann Whitney test (Graphpad Prism; Graphpad Software Inc, San Diego, CA) was used to compare cytokine production between groups.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Identification of DC Subsets in Lung Cancer Draining LN
Single-cell suspensions were prepared from lung draining LN from four patients with NSCLC and three patients with carcinoid tumors, and DC identified by four-color staining. DC were gated (Figure 1; R2) based on their expression of HLA-DR and absence of lineage markers for T, B, NK cells, and monocytes. Results are shown for a patient with lung cancer (Figure 1A) and a patient with carcinoid tumor (Figure 1B). Both myeloid (CD11c+) and plasmacytoid (CD123+) DC were identified in hilar and mediastinal LN from the patients studied (data not shown). Fewer plasmacytoid than myeloid DC (range, 9–31% and 32–68%, respectively), were released from LN and detected in R2 gates after enzymatic digestion. Due to the combination of fluorochrome-labeled antibodies available, costimulatory and maturation markers were detected on the whole R2 gated population. There was virtually no expression of the costimulatory molecules, CD40 and CD80, on LN plasmacytoid-DC, but some cells (range 22–48%) expressed low levels of CD86. The vast majority of DC lacked expression of the maturation marker, CCR7. Ten to forty percent of DC expressed the immature DC marker, CCR6. Very few cells (range, 1–5%) expressed the Langerhans cell marker, CD1a. DC from lung cancer draining LN, therefore, expressed an immature phenotype. There was no significant difference in DC subsets or costimulatory/maturation markers between patients with lung cancer and those with carcinoid tumors (Figure 1 and data not shown).


Figure 1
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  		Figure 1. Detection of myeloid- and plasmacytoid-DC subsets in lung cancer draining lymph nodes. Cells (1 x 105) released from draining LN of (A) a patient with lung cancer and (B) a patient with carcinoid tumor after enzymatic digestion were stained for expression of lineage markers, HLA-DR, DC subset, and costimulatory/maturation markers by four-color staining. Cells were gated on R1 and the lineage HLA-DR+ subset (R2) identified. Expression of DC subset and costimulatory/maturation markers was then detected after gating on R2. Numbers represent percentage of positive cells in each quadrant. Representative experiments of seven are shown.

 
Purified LN Plasmacytoid-DC Express HLA-DR and TLR9
Plasmacytoid-DC were purified from lung draining LN on the basis of expression of the marker, BDCA-4 (Figure 2A). This marker was chosen in preference to BDCA-2, as ligation of BDCA-4 does not affect the ability of plasmacytoid-DC to produce type I interferon (17). In a recent study, plasmacytoid-DC were highly enriched from inguinal LN after purification using an antibody to BDCA-4, but significant T cell contamination was observed (18). BDCA-4 is reportedly expressed at low levels on a germinal center T cell population in tonsil (17). An initial T cell depletion step was, therefore, performed to obviate this problem. The mean numbers of plasmacytoid-DC purified from lung cancer and carcinoid LN were 3.8 x 105 (range, 1.7–6.2 x 105) and 5.0 x 105 (range, 3.7–7.2 x 105), respectively. The lower recovery of cells from some patients with lung cancer precluded performing all the procedures described. Therefore, the number of patients investigated is given in each experimental section.


Figure 2
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  		Figure 2. Plasmacytoid-DC purified from lung cancer draining LN express TLR9 and respond to CpG ODN. Plasmacytoid-DC were purified based on expression of BDCA-4. Purified cells were gated on large forward scatter (FSC) cells (A) costimulatory molecule and maturation marker expression were determined. Numbers represent percentage of positive cells. A representative experiment of 11 is shown. (B) Expression of toll-like receptors: TLR2, -3, and -4 were determined by surface staining and TLR9 by intracellular staining. A representative experiment of six (four patients with lung cancer and two patients with carcinoid tumors) is shown. (C) DC (5 x 104) from five patients with lung cancer were stimulated with CD40L alone or together with CpG 2216 or CpG 2006. Supernatants were harvested after 48 h and IFN-{alpha} production assessed. Results are shown as mean level of cytokine production + 1 SEM.

 
Plasmacytoid-DC were shown not to express lineage markers for monocytes, B, NK, or T cells (data not shown). Contamination by CD11c+ myeloid-DC was < 2% (data not shown). BDCA-4+ DC expressed high levels of HLA-DR. On investigation of cosimulation/maturation markers, purified plasmacytoid-DC did not express CD40, CD80, CD83, or CCR7 (Figure 2A), extending the previous results on the whole DC population, and confirming the immature status of LN plasmacytoid-DC. Plasmacytoid–DC expressed the intracellular receptor, TLR9 (Figure 2B), in contrast to a recent report of plasmacytoid-DC isolated from the murine lung (19). Similar levels of expression of TLR9 were observed in LN plasmacytoid-DC and plasmacytoid-DC purified from normal peripheral blood (data not shown). Plasmacytoid-DC did not express TLR2, -3, or -4, which are restricted to human myeloid DC (20).

LN Plasmacytoid-DC Produce Type I Interferon in Response to CpG 2216
Cytokine production by plasmacytoid-DC in response to the well-characterized CpG sequences, 2216 and 2006, representative of the CpG-A and CpG-B classes, respectively (10), was investigated. Appropriate control sequences were included in the analysis. The results represent the responses for five patients with lung cancer and three patients with carcinoid tumors.

We investigated production by plasmacytoid-DC of IFN-{alpha} and IL-12, cytokines shown to enhance T cell responses (8, 21). Dose responses showed optimal production of IFN-{alpha} by 1 µM of CpG and 1 µg/ml of CD40L (data not shown). CpG 2216 (1 µM) induced 16-fold greater amounts of IFN-{alpha} than CpG 2006, while CD40 ligation, a surrogate for T cell interaction (8), had no effect on IFN-{alpha} production (Table 2). Ligation of CD40, concomitant with stimulation with CpG ODN, has been reported to enhance IFN-{alpha} production (22). Costimulation of plasmacytoid-DC with CD40L and CpG 2216 induced a 2.5-fold increase in IFN-{alpha} production compared with CpG 2216 alone (Figure 2C). A dose response was observed with 1 µM CpG 2216 inducing 2.5- to 3-fold greater quantities of IFN-{alpha} than 0.1 µM CpG 2216. CD40 ligation combined with CpG 2006 stimulation induced low levels of IFN-{alpha} (Figure 2C). In contrast, production of IL-12p70 by plasmacytoid-DC was not detected in response to either CpG sequence alone or in combination with CD40L (Table 2 and data not shown). Constitutive production by plasmacytoid-DC of the proinflammatory cytokines IL-6, IL-8, and TNF-{alpha} was significantly enhanced by CD40 ligation (Table 2). Both CpG enhanced production of IL-8, but only CpG 2216 stimulated the production of IL-6. Production of IL-10 could only be detected at a very low level. Control sequences did not themselves induce or enhance cytokine production by plasmacytoid-DC (data not shown). Similar cytokine production results were obtained from patients with lung cancer and from patients with carcinoid tumors (Figure 2C and data not shown).


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  		TABLE 2. CYTOKINE PRODUCTION BY PLASMACYTOID-DC

 
LN Plasmacytoid-DC Induce Th1 and Tc1 Responses on Exposure to CpG 2216
The capacity of plasmacytoid-DC to induce T cell responsiveness was then investigated in the mixed lymphocyte reaction (MLR), a robust assay of DC functional activity (23). As exposure of peripheral blood plasmacytoid-DC to CpG 2216 reportedly failed to enhance priming of naïve CD8+ T cells (10), we investigated the responses of unfractionated T cells in the MLR. Initial experiments demonstrated optimal cytokine production by a DC:T cell ratio of 1:10 (data not shown). The combined responses induced by plasmacytoid-DC from four patients with lung cancer are shown in Figure 3A. Tumor infiltrating cells were identified in LN from each of the patients studied. In the absence of CpG ODN, plasmacytoid-DC induced low levels of IFN-{gamma} production in the MLR. CpG 2216 significantly enhanced IFN-{gamma} production induced by plasmacytoid-DC. In contrast, although there was a trend to increased IFN-{gamma} production induced by DC exposed to CpG 2006, this did not reach significance. Production of IFN-{gamma} induced by plasmacytoid-DC from a control group of three patients with carcinoid tumors are shown in comparison (Figure 3A). There was no significant difference in the responses between patients with lung cancer and those with carcinoid tumors in the presence or absence of CpG. Low but detectable levels of production of IL-2 and of the Th2 cytokine, IL-5, in response to both CpG were measured in the MLR (IL-2 < 200 pg/ml; IL-5 < 350 pg/ml). Production of IL-10 was < 25 pg/ml for all conditions tested in the study populations.


Figure 3
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  		Figure 3. Effect of CpG ODN on induction of Th1 and Tc1 responses by LN plasmacytoid-DC. (A) Plasmacytoid-DC (1 x 104), purified from draining LN of four patients with lung cancer and three patients with carcinoid tumors, were cocultured with normal, allogeneic T cells (1 x 105) in the presence or absence of CpG 2216 or CpG 2006. Supernatants were harvested after 6 d and cytokine production determined. Cumulative results for IFN-{gamma} production in the MLR were expressed as mean level of IFN-{gamma} + 1 SEM. (B) Plasmacytoid-DC (1 x 105) were cocultured with normal, allogeneic T cells (1 x 106) for 6 d. T cells were then harvested and stimulated with PMA/ionomycin. T cells were surface stained for CD8. and intracellular IFN-{gamma}, granzyme B. and IL-2 production were determined. Numbers represent percentage of positive cells. The isotype controls for IFN-{gamma} (APC) and IL-2, granzyme B (PE) are shown. A representative experiment of six is shown.

 
The relative contributions of CD4+ and CD8+ T cells to responses in the MLR induced by exposure of plasmacytoid-DC to CpG 2216 were then assessed by immunophenotyping (Figure 3B). Both CD4+ and CD8+ T cells expressed IFN-{gamma}, but the frequency of CD8+ T cells producing IFN-{gamma} was greater than that of CD4+ T cells. In contrast, CD4+ T cells were the source of IL-2 production. As a measure of cytotoxic capacity induced by plasmacytoid-DC, expression of the cytotoxic pore-forming molecule, granzyme B (10), was determined. CD8+ but not CD4+ T cells expressed granzyme B. Negligible expression of IL-5, IL-13, and IL-10 (< 3% of cells immunophenotyped) and no expression of IL-4 was detected. The results indicated that after exposure to CpG 2216, LN plasmacytoid-DC had the capacity to promote Th1 and Tc1 immunity.

Combined Stimulation of LN Plasmacytoid-DC with CpG2216 and CD40L Enhances IFN-{gamma} Production Compared with CD40 Ligation Alone
As combined stimulation with CpG 2216 and CD40L had enhanced IFN-{alpha} production by plasmacytoid-DC (Figure 2B), we investigated the effect of this combination of stimuli in the MLR. The combined results for six patients with lung cancer patients and three patients with carcinoid tumors (Figure 4A) demonstrated that ligation of CD40 on plasmacytoid-DC significantly enhanced IFN-{gamma} production compared with untreated plasmacytoid-DC. Combined stimulation with CD40L and CpG 2216 significantly enhanced IFN-{gamma} production compared with CD40 ligation alone. In contrast, no additive effect of stimulation with CpG 2006 was obtained. There was no significant difference between the responses induced by DC from patients with lung cancer and those with carcinoid tumors. Intracellular IFN-{gamma} production by T cells in response to DC alone was enhanced 4-fold and 7-fold, respectively, after exposure of DC to CD40L or the combination of CD40L and CpG 2216 (Figure 4B). Although formal neutralization experiments were not performed, the results indicated that type I interferon production in response to CpG 2216 may enhance the capacity of LN plasmacytoid-DC to induce Tc1 responses.


Figure 4
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  		Figure 4. Effect of stimulation with CD40L and CpG ODN on IFN-{gamma} production induced by LN plasmacytoid-DC. (A) Plasmacytoid-DC (1 x 104), purified from draining LN of six patients with lung cancer and three patients with carcinoid tumors, were cocultured with normal, allogeneic T cells (1 x 105) in the presence or absence of CD40L and CpG 2216 or CpG 2006. Supernatants were harvested after 6 d and cytokine production determined. Cumulative results for IFN-{gamma} production are expressed as mean level of IFN-{gamma} + 1 SEM. (B) Plasmacytoid-DC were cocultured with normal, allogeneic T cells as in the legend to Figure 3B and intracellular IFN-{gamma} was determined. Numbers represent percentage of positive cells. A representative experiment of six is shown.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
We have detected the presence of plasmacytoid-DC in draining LN from patients with lung cancer, purified the plasmacytoid-DC population, and investigated their capacity to stimulate T cell responses. Purified plasmacytoid-DC were poorly stimulatory for T cells. Exposure of purified plasmacytoid-DC to CpG 2216 induced type I interferon production and enhanced Th1 and Tc1 responses compared with unstimulated plasmacytoid-DC. Ligation of CD40 on plasmacytoid-DC also enhanced T cell responses, which were further enhanced after concomitant ligation of CD40 and TLR9. As far as we are aware, this is the first report to successfully purify and establish the functional characteristics of plasmacytoid-DC from human lung draining LN.

Plasmacytoid-DC were detected in lower frequency compared with myeloid-DC in cell suspensions from lung draining LN, corresponding to similar results reported for human hepatic LN (24). After enzymatic release and overnight culture of the LN cell suspension, purified plasmacytoid-DC expressed an immature phenotype indicating that this series of manipulations had not induced maturation of the DC. Lung tumors are known to release factors such as IL-10 and vascular endothelial growth factor (VEGF), which can inhibit maturation of DC (7). Tumors from patients with head and neck squamous cell cancer were shown to release products that down-regulated expression of TLR9 on plasmacytoid-DC (15). Levels of TLR9 on LN plasmacytoid-DC, however, were readily detectable and in this respect, therefore, appeared to be unaffected by tumor products. Moreover, comparison of responses induced by DC from patients with lung cancer and patients with carcinoid tumors did not show any significant difference, indicating an apparent lack of susceptibility of plasmacytoid-DC to the effects of tumor cells infiltrating the LN.

Ligation of the plasmacytoid-DC receptor, TLR9, with CpG 2206 induced type I interferon production and stimulated strong Th1 and Tc1 responses. Type I interferon has been shown to potently enhance human CD8+ T cell responses (10), and to be an essential cofactor in survival and clonal expansion of murine, effector CD8+ T cells (25). Combined stimulation with CpG 2216 and ligation of CD40 enhanced IFN-{alpha} production by plasmacytoid-DC and IFN-{gamma} production by T cells, compared with ligation of CD40 alone. Stimulation by CpG has been shown to induce up-regulation of CD40 on peripheral blood plasmacytoid-DC (21), and ligation of CD40 resulted in an enhanced Th1 response (8). In contrast, CpG 2006 did not induce IFN-{alpha} production, corresponding to previous reports of the responses of normal, peripheral blood plasmacytoid-DC (10, 21). A similar type B CpG ODN has, however, been shown to induce type I interferon–inducible genes when administered to patients with non-Hodgkins lymphoma (13). Interaction with other hematopoietic cells or concomitant drug treatment may stimulate enhanced type I interferon production by plasmacytoid-DC exposed to CpG ODN in vivo.

In contrast to the effects of CpG 2216 on type I interferon production, LN plasmacytoid-DC failed to secrete the prototype Th1-inducing cytokine, IL-12, in response to either CpG 2216 or CpG 2006. We have reported that production of IL-12 by respiratory tract myeloid-DC from patients with rhinitis was impaired compared with matched peripheral blood DC (26). A subset of peripheral blood plasmacytoid-DC expressing a more mature phenotype has been reported to be responsible for IL-12 production in response to type B CpG ODN (22). This subset may be absent from lung draining LN or functionally deficient in patients with lung cancer. Alternatively, a recent report has demonstrated lack of IL-12 production by highly purified plasmacytoid-DC (27). Nevertheless, animal models indicate that effective Th1 immunity can be induced in the respiratory tract independent of IL-12 production by DC (28).

No evidence of IL-10–producing CD4+ or CD8+ T cells in response to LN plasmacytoid-DC, in the presence (29) or absence of CpG ODN, was obtained in the present study. It is possible that culture of the LN cell suspension in IL-3, an established survival and maturation factor for plasmacytoid-DC (30), could affect the capacity of plasmacytoid-DC to induce Treg cells. The ability of freshly isolated plasmacytoid-DC from draining LN to induce Treg cells has still to be investigated. Nevertheless, tumor-infiltrating plasmacytoid-DC from patients with ovarian carcinoma, either ex vivo or after culture in IL-3, were demonstrated to induce IL-10 secreting Treg cells (16). Moreover, although the frequency of alloreactive-specific T cells is greater than that of antigen-specific T cells, and the costimulatory requirements less stringent, this is unlikely to explain our results, as the study quoted above also employed the MLR as the assay of plasmacytoid-DC function.

We, therefore, propose four possible explanations of this result. (1) Removal of plasmacytoid-DC from the LN environment may eliminate signals delivered by tumor cells or stromal components (31), which predispose plasmacytoid-DC to induction of Treg cells. (2) Plasmacytoid-DC may be less susceptible to inhibitory signals within the LN compared with the tumor environment (15). (3) The observed production of IL-6 by plasmacytoid-DC may abrogate the induction of Treg cells (32). Recently, ligation of the membrane bound form of the IL-6 receptor on murine, respiratory CD4+CD25+ Treg cells was reported to inhibit their expansion and functional activity (33). (4) Memory T cells, which represent a high proportion of the alloreactive response (34), may be less susceptible than naïve T cells to induction of a regulatory phenotype by plasmacytoid-DC. As tumor-specific memory T cells recirculate to and may undergo local expansion in cancer draining LN (35), plasmacytoid-DC could potentially reactivate anti-tumor T cell immune responses either directly, through their recently reported capacity to process and present tumor antigens (36), or through activation of myeloid-DC (37).

In conclusion, this study is the first to demonstrate that plasmacytoid-DC from draining LN of patients with lung cancer are capable of responding to CpG ODN and CD40 ligation resulting in enhanced Tc1 responses. These results suggest that novel immunotherapeutic intervention such as intranodal priming of plasmacytoid-DC with CpG ODN and CD40L could contribute to reactivation of anti-tumor T cell responses within the target organ of patients with lung cancer.


   Acknowledgments
 
The authors thank Professor T. Treasure, Dr. R. Cameron, and Dr. L. Lang-Lazdunski, Department of Thoracic Surgery, Guy's and St Thomas' NHS Foundation Trust for their help in obtaining lymph nodes for this study.


   Footnotes
 
This work was supported by funding from King's College, London and the British Lung Foundation.

Originally Published in Press as DOI: 10.1165/rcmb.2006-0284OC on October 5, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form August 4, 2006

Accepted in final form September 26, 2006


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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