title: Characteristics of immunosuppressive regulatory T cells in cutaneous squamous cell carcinomas and role in metastasis.
abstract: BACKGROUND
  Non-melanoma skin cancer is the most common cancer worldwide, and cutaneous squamous cell carcinomas (SCCs) account for substantial morbidity and mortality because of their potential for metastasis. SCCs are surrounded by an immune cell infiltrate containing regulatory T cells (Tregs). The aim of this study was to characterise Tregs in SCCs and investigate whether increased Treg numbers in primary skin SCCs are associated with subsequent metastasis.
  
  
  METHODS
  Lymphocytes were extracted from freshly excised skin SCC tumours and corresponding peripheral blood and normal skin. Flow cytometry was used for T-cell analysis and cell sorting. Tritiated thymidine based lymphocyte proliferation assays and interferon γ (IFNγ) ELISPOT assays were used to assess peritumoral lymphocyte function in vitro. Immunohistochemistry was performed on primary cutaneous SCC sections from tumours that subsequently metastasised and from those that did not after 5-year follow-up.
  
  
  RESULTS
  Increased frequencies of CD3+CD4+CD25hiCD127loFOXP3+ Tregs were found in SCCs (21·5% of CD4+ immune infiltrate, n=60 tumours) compared with corresponding peripheral blood (5·4%) and normal skin (7·6%). SCC Tregs expressed significantly higher levels of the co-stimulatory molecules OX40 (37·2% of FOXP3+ cell population, n=10 tumours) and 4-1BB (12·6%, n=9) than peritumoral non-regulatory T cells and Tregs from peripheral blood and normal skin (p=0·0005). The inhibitory receptor CTLA4 and the transcription factor Helios were expressed at high levels in peritumoral Tregs. SCC Tregs significantly suppressed phytohaemagglutinin-stimulated peritumoral CD4+ T-cell proliferation (p=0·005, n=10), peritumoral CD8+ T-cell proliferation (p=0·015, n=9), and IFNγ secretion by CD4+ effector T cells (p=0·026, n=10). Increased in-vitro proliferation of phytohaemagglutinin-stimulated peritumoral CD4+ T cells was shown after the addition of anti-OX40 antibodies (p=0·0078, n=9 tumours) and anti-4-1BB antibodies (p=0·0039, n=9). Immunohistochemistry showed fewer CD8+ T cells in SCCs that metastasised (n=29) than in non-metastatic SCCs (n=26) (28·5% of immune infiltrate vs 44·6%%, p<0·0001) and more FOX3+ Tregs (28·5% of immune infiltrate vs 49·3%, p<0·0001).
  
  
  CONCLUSIONS
  Our study shows that immunosuppressive Tregs are present in the immune infiltrate of cutaneous SCCs, and contribute to ineffective anti-tumour immune responses, thereby permitting SCC development and promoting metastasis.
  
  
  BACKGROUND
  Wellcome Trust, National Institute for Health Research.
authors: Dermatopharmacology, University of Southampton, Southampton, UK. Electronic address: c.y.lai@soton.ac.uk.
authors: Chester
authors: C
authors: Lai
authors: Dermatopharmacology, University of Southampton, Southampton, UK.
authors: Suzannah
authors: S
authors: August
authors: Dermatopharmacology, University of Southampton, Southampton, UK.
authors: Ramnik
authors: R
authors: Behar
authors: Dermatopharmacology, University of Southampton, Southampton, UK.
authors: Marta
authors: M
authors: Polak
authors: Dermatopharmacology, University of Southampton, Southampton, UK.
authors: Mike
authors: M
authors: Ardern-Jones
authors: Histopathology, University Hospital Southampton NHS Foundation Trust, Southampton, UK.
authors: Jeff
authors: J
authors: Theaker
authors: Cancer Sciences, University of Southampton, Southampton, UK.
authors: Aymen
authors: A
authors: Al-Shamkhani
authors: Dermatopharmacology, University of Southampton, Southampton, UK.
authors: Eugene
authors: E
authors: Healy