Forkhead box-P3+ regulatory T cells and toll-like receptor 2 co-expression in oral squamous cell carcinoma
Introduction
Oral and oropharyngeal cancer is the sixth most common cancer in the world (Warnakulasuriya 2009). Oral squamous cell carcinoma (OSCC) is the most common form of oral and oropharyngeal cancer, with two-thirds of cases occurring in developing countries (Parkin et al., 2005).
The stroma of OSCC is almost always infiltrated by variable numbers of mononuclear immune cells, mostly lymphocytes, known as tumour infiltrating lymphocytes (TILs) (Rollins, 2006, Uppaluri et al., 2008). This phenomenon was initially thought to be evidence of an anti-tumour response (Yu and Fu, 2006), however functional analysis showed that the infiltrate was richly populated with CD4+ cells, with few NK cells (Snyderman et al., 1991). This population of CD4+ cells was later identified as forkhead box-P3 (FoxP3)+ regulatory T cells (Treg), which in normal circumstances, are important in maintaining immune homeostasis and preventing autoimmune diseases (Schwarz et al., 2008). Studies have shown expansion of this CD4+FoxP3+ Treg population in the tumour microenvironment (TME) of a range of cancers, including in OSCC, possibly induced by the local environment which is rich in interleukin (IL)10 and tumour growth factor (TGF)β (Bergmann et al., 2008, Watanabe et al., 2010, Lim et al., 2014). Malignant oral keratinocytes secrete TGFβ (Gasparoto et al., 2010), strongly indicating that OSCC cells are able to modulate the function of Tregs to their advantage.
For tumour antigens to induce an immunogenic effect, they have to be presented to antigen presenting cells (APCs). The antigen presentation mechanism is commonly chaperoned by a group of molecules belonging to the heat shock protein (HSP) family that interacts with transmembrane proteins such as the toll-like receptor (TLR) family. TLR2 recognises a wide variety of pathogen-associated molecular patterns (PAMPs) from exogenous pathogens, as well as endogenous damage-associated molecular patterns (DAMPs) including HSPs and high-mobility group box 1 protein (Sato et al., 2009). TLR2 expression on immune cells protected mice from developing colorectal cancer (Lowe et al., 2010) and mature dendritic cells (DC) injected with bacterial DNA were able to overcome immune tolerance in colon carcinoma (Heckelsmiller et al., 2002) using the TLR pathway. More keratinocytes in OSCC expressed TLR2 than keratinocytes in control epithelium (Ng et al., 2011). Whether or not this finding denotes the stimulation of an anti-tumour response or survival enhancement in OSCC is yet to be proven.
The expansion of the Treg population in OSCC is increasingly thought to be responsible for suppression of an anti-tumour response, which, in turn, facilitates cancer progression and leads to a poorer prognosis. TLRs, particularly TLR2, play a role in Treg expansion and facilitate their suppressive capacity (Sutmuller et al., 2006). On the other hand, TLR2 can induce loss of Treg function in a murine model with a shift to a pro-inflammatory (Th1) cytokine profile (Sutmuller et al., 2006). More recently it has been shown that TLR2 agonists induce tumour regression by modulating Treg and cytotoxic T cell (Tc) function (Zhang et al., 2011).
The ability of TLRs to modulate Treg and/or the TME has initiated interest in their potential role in cancer immunotherapy. It is not known whether activation of TLR2 receptors on Treg facilitates reversal of suppression of the anti-tumour response.
There are no reports describing the association of Tregs and TLR2 in the OSCC TME. The aim of this study was to investigate the presence and distribution of Tregs and TLR2+ cells in OSCC, as well as to correlate their distribution with the depth of tumour invasion.
Section snippets
Sample selection
The experiment was approved by University of Otago Institutional Ethics Committee (09/111). The database of the Medlab Dental Oral Pathology Diagnostic Service, Faculty of Dentistry, University of Otago was searched for specimens reported as primary OSCC. The original haematoxylin and eosin (H&E) slides were obtained to verify the diagnosis and confirm the suitability of the specimens using the following selection criteria: i) each section must contain a minimum of three regions with more than
IHC staining characteristics
Both OSCC and control groups showed strong intense immunostaining of both single-stained and double-stained positive cells with minimal background in all sections. Single-stained FoxP3+ cells showed intense brown nuclear staining (Fig. 1A) compared with isotype-matched control (Fig. 1B), whereas TLR2+ cells (stained with Fast Red) showed a dusky red ring around the cell membrane with granular cytoplasmic staining (Fig. 1C) compared with isotype-matched control (Fig. 1D). Double-stained FoxP3+
Discussion
TLRs and TLR signalling plays an important role in regulating innate and adaptive immune responses as well as regulating immune responses during carcinogenesis. Their normal biological role is widely recognised as an early protective mechanism against invading pathogens, however their exact function in carcinogenesis remains unclear. TLRs have been implicated in carcinogenesis most particularly in organs constantly exposed to bacteria such as skin, gastric, liver and colorectal cancer (Tye and
Conclusion
In summary, the presence of double-stained FoxP3+TLR2+ cells within the OSCC TME was observed with apparent cell-to-cell contact between TLR2+ and FoxP3+ cells. The presence of FoxP3+TLR2+ cells may represent a DC-dependent pathway capable of inhibiting Treg suppressive activity. This finding may be beneficial to the anti-tumour response; modulation of TLR2-Treg interactions should be explored via assessment of cytokines in the TME. This may hold the key to shifting the balance into triggering
Acknowledgements
The authors would like to thank the New Zealand Dental Association for financial support of this research work. The authors would also like to thank the staff of Oral Pathology Services, University of Otago, Ms Lynda Horne and Mrs Sharla Kennedy for their support and assistance in this research.
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