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Alterations in the Frequency of Dendritic Cell Subsets in the Peripheral Circulation of Patients with Squamous Cell Carcinomas of the Head and Neck¹

University of Pittsburgh Cancer Institute [T. K. H., T. L. W.], Departments of Pathology [T. L. W.], Surgery [J. M-B., W. J. S.], and Otolaryngology [R. L. F., J. T. J., T. L. W.], University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213

	ABSTRACT

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Patients with advanced squamous cell carcinomas of thehead and neck (SCCHN) are frequently immunocompromised.Dendritic cells (DCs) are potent antigen-presenting cells that play a role in antitumor immune responses. Using multicolor flow cytometry, the percentages of lineage-negative (LIN^-) and DR⁺ DC precursors, as well as their LIN^-DR⁺CD11c⁺ (myeloid) and LIN^-DR⁺CD123⁺ (lymphoid) subsets, were determined in the peripheral blood of 36 patients with SCCHN before surgery. Peripheral blood mononuclear cells of 28 age- and sex-matched healthy individuals were used as controls. The proportions of LIN^-DR⁺ cells were found to be comparable in the circulation of patients and controls. However, the relative level of DR expression in LIN^-DR⁺ DC was lower in patients than in controls, suggesting a difference in the maturity of DC. The relative proportion of LIN^-DR⁺CD123⁺ cells in the LIN^-DR⁺ subset of DC did not differ significantly in patients compared with normal individuals. However, the percentage of myeloid-derived LIN^-DR⁺CD11c⁺ DCs was significantly lower (P < 0.002) in SCCHN patients than in controls. Of the 13 patients who were restudied 6 weeks after surgery, 9 showed an increase of the myeloid-derived LIN^-DR⁺CD11c⁺ DC subset postoperatively. This observation suggests that deficiency in the myeloid-derived DC precursors in patients with SCCHN is related to the presence of tumor and is reversible. An overall decrease in the myeloid-derived subset of DC could contribute to the failure of SCCHN patients to develop effective antitumor immune responses.

	INTRODUCTION

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Patients with advanced SCCHN⁴ are known to have dysfunctions of the immune system (1, 2, 3) . Signaling defects have been described in circulating and tumor-infiltrating T cells of these patients (3) , and a higher proportion of spontaneously apoptotic T cells has been detected in the peripheral blood of patients compared with that of healthy individuals (4) . However, only limited information about DCs and their subsets is available for patients with SCCHN. DCs are potent antigen-presenting cells, which migrate, phagocytose, and process antigens and present them to T cells, thereby playing a crucial role in the initiation and maintenance of T-cell immunity (5) . We have reported previously that ex vivo generation of T cells specific for SCCHN-associated tumor antigens, using autologous DCs pulsed with tumor peptides or whole tumor cells, is not impaired in patients with SCCHN (6 , 7) . For these ex vivo studies, DCs were generated from monocytes in the presence of cytokines (IL-4 and granulocyte macrophage colony stimulating factor), and T cells were cultured with DCs in the presence of IL-2 and IL-7. However, cytokine-driven activity of cultured DCs is unlikely to reflect the functional status of DC populations found in vivo. Attempts have been made to enumerate and characterize DCs in the microenvironment of SCCHN tumors, and the number of DCs present in the tumor has been described as a highly significant prognostic, as well as survival, biomarker (8, 9, 10) . Phenotypic analysis of DCs performed with PBMC of patients with SCCHN and other cancers has indicated that maturational defects might be present (11 , 12) , possibly because of the presence of a granulocyte macrophage colony stimulating factor-dependent immunosuppressive subset of CD34⁺ progenitor cells, which have been reported to impair DC development and differentiation (13) .

Currently, two peripheral blood DC subsets have been described, which are distinguishable by their ability to express CD11c (14 , 15) . The CD11c-negative (CD11c^-) DCs, which express high levels of CD123 also known as IL-3 receptor (16) , are designated as lymphoid-derived DCs, whereas the CD11c⁺CD123^- subset represents myeloid-derived DCs. Although both subsets express high levels of HLA-DR and lack the lineage markers CD3, CD14, CD19, CD20, CD16, and CD56, functional differences between CD11c⁺CD123^- and CD11c^-CD123⁺ DCs have been described (17) . The CD11c⁺ DCs have greater T cell-stimulatory activity (14 , 15) . Furthermore, it has been demonstrated that biologically or chemically activated CD11c⁺, but not CD11c^- (CD123⁺), DCs produce proinflammatory cytokines and up-regulate costimulatory molecules (18) . The lack of the ability to produce cytokines in the CD123⁺ DC subset has also been described by others (19) . More recently, it has been found that CD11c^- CD123⁺ DCs can produce IFN-(/ß) (20, 21, 22) . On interaction with T cells, these lymphoid-derived CD123⁺ DCs (DC2) seem to support the generation of a Th2 response, whereas myeloid-derived CD11c⁺ DCs (DC1) predominantly prime a Th1 response (23) . The distinctive functional phenotype of myeloid versus lymphoid DC subsets suggests that they may be used differentially in immune responses to various antigens, including tumor-associated antigens. However, little is known about the frequency or function of these two subsets of DCs in cancer patients.

In the current study, we investigated the proportions of the two DC subsets in the peripheral circulation of 36 patients with SCCHN and 28 healthy individuals, using multicolor flow cytometry. A cocktail of antibodies was used to identify LIN^- cells, which lack CD3, CD14, CD16, CD19, CD20, and CD56 markers and which are DR⁺. Within the LIN^-DR⁺ cells defined as DCs (14 , 24, 25, 26, 27) , it was possible to distinguish and quantitate by flow cytometry DC1 and DC2 subpopulations of circulating DCs. We also show that the frequency of the myeloid DC subset (DC1) was lower in the peripheral circulation of patients than of controls and that this discrepancy was not present after the patients’ tumors were surgically removed.

	PATIENTS AND METHODS

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Patients.
Peripheral blood was obtained from 28 normal healthy donors and 36 patients with histomorphologically confirmed SCCHN. The patients were evaluated before surgery and in 17 of 36 cases also after surgery. Of these 17 patients, 13 were included in this study based on the requirement for a 6-week interval between surgery and the blood draw for DC evaluations. The patients evaluated after surgery were not treated with chemo or radiotherapy at the time of the study. Venous blood (10 ml) was collected into heparinized tubes from patients or controls; specimens were coded on their delivery to the laboratory and processed for flow cytometry to study proportions of DCs or the DC subsets. The study was approved by the Institutional Review Board at the University of Pittsburgh, and a written informed consent was obtained from each individual. PBMCs were isolated by centrifugation over Ficoll-Hypaque gradients (Amersham Pharmacia Biotech, Piscataway, NJ), washed, counted in the presence of a trypan blue dye, and stained for flow cytometry.

Hospital records and pathology slides were reviewed, and tumors were staged according to the 1997 International Union Against Cancer classification. The clinicopathological characteristics of the patients with SCCHN included in this study are presented in Table 1 .

Flow Cytometry Analysis.
Three-color flow cytometry analysis was performed as described previously (7) , using a FACScan (Becton Dickinson) equipped with a single 488-nm argon ion laser. The investigators performing the analysis were blinded regarding the origin of the samples from patients versus normal controls. At least 100,000 events were acquired for each sample. The acquired data were analyzed using the WinMDI program (Version 2.8, Joseph Trotter, Scripps Inst., La Jolla, CA). The assay variability was <10%, as determined by replicate measurements performed using PBMCs of a normal donor tested repeatedly.

The gating strategy used to identify and quantify LIN^-DR+ cells in PBMCs was as follows: Cells in the lymphocyte-monocyte light scatter gate were evaluated for the expression of lineage markers LIN-FITC and DR-PerCP. Within an extended lymphocyte-monocyte light scatter gate, granulocytes, as well as necrotic cells, were excluded. To identify CD11c⁺ and CD123⁺ subsets among LIN^-DR⁺ cells, the gate was set on LIN^- cells, and the percentages of CD11c⁺PE, as well as CD123⁺PE cells, were evaluated using separate tubes.

Statistical Analysis.
Unpaired or paired two-tailed Student’s t tests, as well as an exact Wilcoxon test, were used for statistical analysis of flow cytometry data. Differences were considered significant when: P < 0.05. ANOVA was used to evaluate effects of disease, age, or gender on the proportions of LIN^-DR⁺ precursors and their subsets.

	RESULTS

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Patients’ Characteristics.
The clinicopathological characteristics of patients with SCCHN and the characteristics of the control group are summarized in Table 1 . The group of healthy controls comprised 18 men and 10 women, with a median age of 61 (range 34–82) years, whereas 36 SCCHN patients included 19 men and 17 women with a median age of 63 (range 36–86) years. The 36 histologically verified squamous cell carcinomas originated in the oral cavity (n = 21), oropharynx (n = 5), hypopharynx (n = 2), and larynx (n = 8), including well (n = 9), moderately (n = 19), and poorly (n = 8) differentiated tumors. As shown in Table 1 , 22 of 36 patients were T1/T2, and only 12 had nodal involvement. In 13 of 36 patients included in this study, a second determination of DCs or DC subsets was performed 6 weeks after surgery. At that time, none of the patients had been treated with radio and/or chemotherapy.

Gating Strategy.
Initially, an extended lymphocyte-monocyte light scatter gate was set, and residual granulocytes, as well as cell debris, were excluded (Fig. 1A) . The staining intensity of cells within this compound gate (R1) was evaluated for LIN markers (FL1) and DR expression (FL3). Typically, four distinct populations were identified: LIN⁺DR⁺, LIN⁺DR^-, LIN^-DR^-, and LIN^-DR⁺ (Fig. 1B) . The LIN^-DR⁺ cell population (R3), generally referred to as DC precursors in the peripheral blood, was gated on (Fig. 1, C and D) and used to discriminate the CD11c⁺ or the CD123⁺ DC subsets.

View larger version (67K): [in this window] [in a new window]   Fig. 1. The gating strategy used to identify LIN-DR+ cells. PBMCs obtained from SCCHN patients or healthy donors were stained with a lineage cocktail of FITC-conjugated MoAb (CD3, CD14, CD16, CD19, CD20, and CD56) plus a PE-conjugated anti-DR MoAb. Cells in the lymphocyte-monocyte light scatter gate (A) were evaluated for expression of lineage markers LIN (FL-1) and DR (FL-2). The number of LIN-DR+ DCs was detected in the top left quadrant (B). LIN- cells (gate R2) were evaluated for the expression of CD11c (C) and CD123 (D) to identify LIN-DR+CD11c+ or CD123+ DC subsets. A representative experiment performed with PBMCs of a normal control is shown.

View larger version (17K): [in this window] [in a new window]   Fig. 2. The proportions of LIN-DR+ DC precursors in the peripheral circulation of patients with SCCHN and normal controls are comparable and are not age dependent. The data for individual patients or controls are displayed relative to age. The spline lines depict the actual relationships between the % LIN-DR+ cells and age in patients and controls.

Percentage of CD11c⁺ Versus CD123⁺ DCs in Patients and Controls.
Next, the expression of CD11c or CD123 on LIN^-DR⁺ cells was determined, as shown in Fig. 1, C and D , to distinguish between LIN^-DR⁺CD11c⁺ (myeloid derived) and LIN^-DR⁺CD123⁺ (lymphoid derived) DC subsets in the circulation of patients and controls. The proportions of LIN^-DR⁺CD11c⁺ or CD123⁺ DCs were normalized to the percentage of LIN^-DR⁺ DC precursors determined for every individual studied. As shown in Table 3 , a significant difference in the relative proportions of CD11c⁺ DCs within the LIN^-DR⁺ DC population was found in patients versus controls (P = 0.002). SCCHN patients had a mean of 38.2% of LIN^-DR⁺CD11c⁺, whereas healthy control individuals had 43.2%. This difference was not age dependent (Fig. 3) or gender dependent (Table 3) . On the other hand, the percentage of LIN^-DR⁺CD123⁺ was not found to be significantly different in SCCHN patients compared with normal controls. Additionally, the percentage of DC1 and DC2 subsets was not found to be significantly different among patients with different tumor sites, tumor sizes, nodal status, or tumor differentiation (Table 4) .

View larger version (19K): [in this window] [in a new window]   Fig. 3. Percentages of CD11c+ cells among LIN-DR+ precursors in the circulation of healthy controls and patients with SCCHN relative to their age. No age-dependent effects were seen (spline lines), but the mean proportions of CD11c+ cells among LIN-DR+ precursors were significantly lower in the patients than normal controls (P < 0.002).

View larger version (19K): [in this window] [in a new window]   Fig. 4. Percentages of CD11c+LIN-DR+ cells among LIN-DR+ precursors in patients with SCCHN studied before and after surgery (n = 13). The observed increases in the proportion of these cells after surgery were found to be significant (P = 0.002).

	DISCUSSION

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Human DCs are phenotypically and functionally heterogeneous. The ability to identify and enumerate DCs and their subsets in tumor tissue and in the peripheral circulation of patients with cancer appears to be fundamental for the understanding of the role of these cells in the host antitumor responses. No single specific marker, which could be used to definitely identify all of the DCs and their precursors, is currently available. Therefore, the use of a combination of MoAb, the so-called "lineage cocktail," plus an anti-DR antibody, are currently used to identify LIN^-DR⁺ DCs by flow cytometry in the peripheral circulation of humans. The LIN^-DR⁺ DCs comprise between 0.1 and 2% of the PBMCs in healthy individuals, and the majority of these cells express either CD11c or CD123 molecules (14 , 16 , 27) . We determined that similar proportions of LIN^-DR⁺ DC precursors were present in the circulation of patients with SCCHN and normal controls (an average of 1.6 and 1.4%, respectively). The analysis of the flow cytometry data indicated that LIN^- cells in patients with SCCHN often expressed lower levels of DR than LIN^- cells in PBMCs obtained from healthy donors, although the difference in DR expression between patient versus control cells did not reach statistical significance. This observation is of interest, because the dim DR cells could represent less mature DCs within the LIN^-DR⁺ population. Somewhat similar observations were made by Melichar et al. (28) , who demonstrated that LIN^-CD4⁺ DCs in the peripheral blood of patients with ovarian cancer had significantly lower DR expression compared with those obtained from normal controls.

In an earlier report (11) , significantly lower proportions of DC precursors were found in the peripheral blood of cancer patients with poorer histological grade, including patients with SCCHN, as compared with normal controls. Importantly, low proportions of DC precursors observed in the peripheral circulation appeared to negatively correlate with the tumor stage in the same study (11) . These observations have not been confirmed in our study. The differences between our results and those of Almand et al. could be related to the selection of patients or to experimental protocols used, e.g., Amand et al. did not specify whether untreated or treated patients were recruited into their study or whether controls were age and sex matched with the patients. Importantly, these investigators enriched PBMCs in DCs by adherence followed by a 24-h incubation before flow cytometry (11 , 12) . Our study was performed on freshly isolated PBMCs, which were used for multicolor flow cytometry after staining for mononuclear cells with a cocktail of lineage-specific, FITC-labeled antibodies. Under these experimental conditions, most T, B, natural killer cells, and monocytes could be gated out, leaving behind LIN^- DCs. This type of "negative selection" by flow cytometry with leukocyte-specific monoclonal antibodies allows for gating out cells of all lineages and retaining only LIN^-DR⁺ precursor cells in the gate. Using this strategy, a meaningful analysis of DC precursor cells and their subsets can be reliably performed without any previous ex vivo manipulations, which could alter the proportions of LIN^-DR⁺ precursor cells. Because the proportion of LIN^- precursor cells is very small, any modifications in the experimental protocol are likely to have profound effects on the data generated.

The most striking observation of the current study was a relative decrease in the percentage of CD11c⁺ DC subsets in the peripheral circulation of SCCHN patients as compared with healthy individuals. This decrease was reproducibly seen in patients of both genders and of all ages and was significant at P < 0.002. LIN^-DR⁺CD11c⁺ DCs were described originally as functionally mature DCs with a strong T-cell stimulatory capacity, and it has been hypothesized that they are derived from tissues, where they have been activated by antigen and are en route to the spleen or lymph nodes to stimulate T-cell responses (14) . According to the current knowledge, it is more likely that these cells represent DC precursors that have not yet encountered antigen and are in transit to the lymph nodes, where they might be expected to both meet the antigen and interact with T cells. Decreased numbers of CD11c⁺ DCs in the circulation of patients with SCCHN could be the consequence of cancer, particularly because we observed a recovery in the relative proportions of the CD11c⁺ DC subset after tumor ablation. Changes in the frequency of CD11c⁺ DCs were paralleled by reciprocal alterations in the frequency of CD11c^-CD123^- DCs in the circulation (data not shown). This observation suggests that in patients, as opposed to normal controls, maturation of CD11c⁺ cells from less mature LIN^-DR⁺CD11c^-CD123^- precursors may be impaired. A decreased frequency of CD11c⁺ DCs in patients with SCCHN could either be because of tumor-induced apoptosis in DCs by direct contact (29) or to inhibition of their differentiation in vivo by soluble factors released from the tumor (30) . To date, several immunosuppressive tumor-derived factors with effects on DCs have been described, but the definite characterization of factors regulating DC survival or differentiation has to await additional studies.

Our findings provide evidence for the complexity of the process of immune suppression operative in patients with SCCHN. This process might affect several distinct subsets of immune cells, including T cells (3 , 4) , natural killer cells (31) , and now also a myeloid-derived DC subset. The interference with maturation of myeloid-derived DC subset, which appears to be linked to the presence of tumor, could well contribute to immunosuppression of SCCHN patients and their failure to establish an effective antitumor immune response.

	ACKNOWLEDGMENTS

 
We thank William Gooding for his contribution to the data analysis.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by NIH Grant PO1-DE 12321 (to T. L. W.) and a postdoctoral training fellowship from the Dr. Mildred Scheel Stiftung für Krebsforschung (Grant D/99/08916 to T. K. H. and Grant D/98/14794 to J. M-B.).

² T. K. H. and J. M-B. contributed equally to this work.

³ To whom requests for reprints should be addressed, at University of Pittsburgh Cancer Institute, W 1041 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15213-2582. Phone: (412) 624-0096; Fax: (412) 624-0264; E-mail: whitesidetl{at}msx.upmc.edu

⁴ The abbreviations used are: SCCHN, squamous cell carcinoma of the head and neck; DC, dendritic cell; IL, interleukin; LIN^-, lineage-negative; MoAb, monoclonal antibody; PBMC, peripheral blood mononuclear cell; PE, phycoerythrin.

Received 12/29/00; revised 3/ 4/02; accepted 3/ 8/02.

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