Dendritic cell generation and CD4+CD25HIGHFOXP3+ regulatory T cells in human head and neck carcinoma during Radio-chemotherapy
© I. Holzapfel Publishers 2011
Received: 7 September 2010
Accepted: 28 September 2010
Published: 24 February 2011
Regulatory T cells (Treg) and dendritic cells (DC) play an important role in tumor immunity and immune escape. However, their interplay and the effects of anti-cancer therapy on the human immune system are largely unknown.
For DC generation, CD14+ monocytes were enriched by immunomagnetic selection from peripheral blood of advanced head and neck squamous cell carcinoma (HNSCC) patients and differentiated into immature DC using GM-SCF and IL-4. DC maturation was induced by addition of TNFα. The frequency of CD4+CD25highF0XP3+ Treg in HNSCC patients was analyzed before and after radio-chemotherapy (RCT) by four-color flow cytometry.
In HNSCC patients, the frequency of Treg (0.33 ± 0.06%) was significantly (p = 0.001) increased compared to healthy controls (0.11 ± 0.02%), whereas RCT had variable effects on the Treg frequency inducing its increase in some patients and decrease in others. After six days in culture, monocytes of all patients had differentiated into immature DC. However, DC maturation indicated by CD83 up-regulation (70.7 ± 5.5%) was successful only in a subgroup of patients and correlated well with lower frequencies of peripheral blood Treg in those patients.
The frequency of regulatory T cells is elevated in HNSCC patients and may be modulated by RCT. Monocyte-derived DC in HNSCC patients show a maturation deficiency ex vivo. Those preliminary data may have an impact on multimodality clinical trials integrating cellular immune modulation in patients with advanced HNSCC.
Keywordshead and neck cancer dendritic cell vaccination regulatory T cell chemotherapy
Radical surgery and various radio-chemotherapy (RCT) regimens represent the current therapy standards for patients with head and neck squamous cell cancer (HNSCC). However, local recurrence and distant metastasis are still the major limiting factors for improving survival rates . Aside from anti-EGFR antibody therapy and targeted therapy with tyrosine kinase inhibitors, newer therapeutic approaches have been focused on dendritic cell (DC)-based immunotherapy . DC are unique antigen-presenting cells characterized by their potent T-cell stimulatory activity and therefore, they are preferentially utilized in cancer vaccines . Immature DC capture antigens in peripheral tissues and migrate to secondary lymphoid organs after danger signal-induced maturation. There, DC present the captured antigens to specific T cells. For cancer vaccination, DC are loaded with specific tumor antigen or mRNA and administered to cancer patients in hope of stimulating an anti-tumor T-cell response . A number of human vaccination studies utilizing DC have been recently published for various tumor entities, however, DC-based vaccinations for therapy of human HNSCC are rare .
The production of DC-based vaccines requires techniques for a large-scale ex vivo generation of clinical-grade DC, and several methods for DC generation have been established . DC can be directly isolated from the peripheral blood, which may be hampered by the low number and functional abnormalities of DC in cancer patients . Currently, Dc culture for therapy utilizes monocytes separated from leukapheresis products . For this purpose, in patients with cancer, monocytes have to be collected before RCT as their number drops rapidly after RCT. Whether HNSCC patients at this disease stage have functionally normal monocytes which can be used to generate DC vaccines is currently unknown.
To date, Dc-based vaccines for cancer have not yet fulfilled their promise, as clinical benefits are only rarely reported in therapeutic vaccination trials, such as in prostate cancer . This lack of clinical benefits might be partly due to the strong immunosuppressive influence of regulatory T cells (treg) in cancer patients with advanced malignancies.
CD4+CD25highFOXP3+ Treg play an important role in various human diseases including the development of allergy , host-versus-graft-reaction in transplantation , and autoimmune diseases . All are associated with a decrease in Treg numbers. In contrast, in cancer patients, Treg are frequently elevated and can inhibit t-cells, DC, natural killer t-cells and even B-cells, thereby probably contributing to tumor immune escape . The potential influence of RCT on Treg has been addressed in several recent studies. In breast cancer, FOXP3+ cells infiltrating the tumor were decreased in frequency after neoadjuvant chemotherapy, and a reduced Treg infiltration correlated with improved responses . AIso in breast cancer, trastuzumab therapy in combination with chemotherapy resulted in a decrease of Treg in peripheral blood . In end-stage cancer patients with various tumor types, a low dose of cyclosphosphamide caused a selective depletion of Treg in peripheral blood . For HNSCC, a study by strauss et al. showed that oncologic therapy induced a significant increase in the frequency and suppressor function of Treg in the peripheral circulation. Surprisingly, HNSCC patients had a higher frequency of Treg after successful therapy compared to patients with active untreated disease .
The present study focuses on Dc generation and the alteration of CD4+CD25highFOXP3+ Treg during RCT in HNSCC patients. Understanding the frequency and function of Treg and DC is necessary to effectively orchestrate the various regimens of surgery, RCT and anti-cancer immune therapy.
Materials and methods
Dendritic cell culture
Number and gender
9 (9 male, 0 female)
13 (11 male, 2 female)
Mean age (range)
63.1 yrs (47-72)
58.8 yrs (47-73)
Tumor site: Larynx
Tumor stage: T1
Generation of dendritic cells
DC were generated following a protocol adapted from Zhou et al. [17, 18]. CD14+ monocytes were immunoselected from peripheral blood mononuclear cells (PBMC) using LS separation columns on a VarioMACS (Miltenyi Biotec, Bergisch Gladbach, Germany). Enriched CD14+ monocytes were cultured in 24-well plates (Greiner, Nurtingen, Germany) at 1 × 106 cells/mL and 2 mL/well of serum-free CellGroDC medium (Cellgenix, Freiburg, Germany) supplemented with 1,000 U/mL GM-CSF (Leukine, Berlex, Richmond, CA) and 1,000 U/mL IL-4 (cellgenix). After 3 and 6 days, half of the medium was replaced by fresh medium containing cytokines. To induce DC maturation, 1,000 U/mL TNFα (cellgenix), GM-CSF and IL-4 were added on day 6, and cultures were continued for 3 days. Monocyte purity was determined by flow cytometry, using CD45-FITC (BD Biosciences, Heidelberg, Germany) and CD14-PE (Beckman-Coulter, Krefeld, Germany) monoclonal antibodies. To monitor DC differentiation and maturation, the frequencies of CD14+CD83neg monocytes, CD14negCD83neg immature DC and CD14negCD83+ mature DC were determined by flow cytometry using CD14-PE and CD83-FITC (Beckman-Coulter) monoclonal antibodies.
Flow cytometric determination of Treg frequencies
Data are presented as geometric means ± standard error of means, and statistical significance was evaluated using the Student's t-test.
Frequency of regulatory T cells
Dendritic cell generation
Tumor induced immunosuppression is a well known phenomenon in cancer patients, for which changes in the Treg compartment may play a central role . In the present study, it was documented, that the frequency of CD4+CD25highFOXP3+ Treg in peripheral blood of HNSCC patients was significantly increased compared to healthy controls, suggesting an active mechanism of tumor-induced immune escape. This finding is in accordance with a previous study in HNSCC  and similar changes have also been reported for other types of cancer .
RCT has been reported to preferentially deplete CD4+ T cells in HNSCC patients . Analyzing the effects of RCT on the Treg population, we observed that in 7/13 patients Treg frequencies within CD4+ T cells increased, whereas 6/13 patients showed a decrease in Treg frequencies after RCT. Presumably, not all chemotherapeutic agents have the same effect on Treg frequency, and T cell subsets may be affected differently by RCT . Studies with a higher number of patients receiving the same drug regimen could provide a more definite result. Of note, a lack of a distinct phenotypic marker for human Treg makes comparison of data obtained by different investigators difficult. Most commonly, Treg are defined as CD4+CD25highFOXP3+, but also the presence of CD39, and the absence of CD127 and CD49d have been used to define Treg .
The results presented here suggest that there might be an association between the Treg frequency and abnormalities in DC maturation. In HNSCC patients with a high frequency of Treg in peripheral blood, monocytes failed to develop to mature DC ex vivo as evident by the lack of CD83 up-regulation. HNSCC patients with a low frequency of Treg show a normal ability of DC maturation as compared to normal donors who had 65 ± 6.8% CD83+ cells after TNFα-induced DC maturation . Whether such effects are long lasting or whether there is normalization when Treg are depleted, is currently unknown.
The defective DC maturation of monocytes obtained from cancer patients possibly reflects tumor-induced suppression, and TNFα used as the only maturation signal may not be sufficient to overcome this suppression. Several factors that negatively influence DC maturation in vitro are described in the literature including IL-10 and TGFβ production by regulatory T cells  or vascular endothelial growth factor produced by human cancer cells . Both mechanisms, a direct effect mediated by tumor cells or a tumor-induced effect mediated by Treg, could cause the decreased DC maturation in HNSCC patients. In another study, high thrombocyte counts in blood samples were found to interfere with DC development , but thrombocyte counts were in a normal range in all participants of this study (data not shown). Also, neither the patients' age nor their level of alcohol and nicotine consumption gave an explanation for the deficiency in DC maturation of certain patients (data not shown). Nevertheless, it should be stressed, that monocytes of all patients differentiated normally to immature DC upon culture with GM-CSF and IL-4, thus, only maturation of DC appears to be affected.
Aiming for an increase in survival rates, future therapy of HNSCC patients will likely focus on the combination of surgery, RCT and immunotherapy. In order to determine an optimal and feasible timing of the various therapeutic modalities, the understanding of depletion and recovery of immuno-relevant cell populations is essential. Therefore, longitudinal studies of cell frequencies, including Treg but also myeloid suppressor cells and TH-17 cells are warranted, all of which may be differently affected by RCT. Having in mind that Treg are highly immunosuppressive, the application of DC-based anticancer vaccination would be most effective when frequencies of Treg are low and those of CD4+ and CD8+ lymphocytes are comparatively high. Whether this point of time can be reached before, during or after RCT needs to be determined. It will also be important to identify regimens of RCT that are most effective for elimination of immunosuppressive Treg and functional normalization of DC, as well as subgroups of patients who will especially benefit from a supportive anticancer vaccination therapy. As the combination of RCT and immunotherapy is hypothesized to have an additive effect in cancer patients, this strategy may provide for the opportunity to employ RCT in a more concerted manner [27, 28].
Forkhead box P3
Granulocyte macrophage colony-stimulating factor
Head and neck squamous cell cancer
Peripheral blood mononuclear cells
Transforming growth factor beta
Tumor necrosis factor alpha
Regulatory T cells.
The authors wish to thank Corinna Peters and Heike Löffler for excellent technical assistance.
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