CUTANEOUS BIOLOGY DOI 10.1111/j.1365-2133.2005.07006.x Transfection of CD40 in a human oral squamous cell carcinoma keratinocyte line upregulates immune potency and costimulatory molecules M. Villarroel Dorrego, S.A. Whawell,* P.M. Speight* and A.W. Barrett  Department of Oral Pathology, Oral Medicine and Oral Surgery, Universidad Santa Maria, and Institute of Dental Research, Universidad Central de Venezuela, Caracas, Venezuela *Department of Oral Pathology, School of Clinical Dentistry, University of Sheffield, Sheffield S10 2TA, U.K.  Department of Histopathology, Queen Victoria Hospital, East Grinstead, West Sussex RH19 3DZ, U.K. Correspondence A.W. Barrett. E-mail: bill.barrett@qvh.nhs.uk Accepted for publication 2 August 2005 Key words: costimulatory molecules, oral mucosal immunity, squamous cell carcinoma Conflicts of interest: None declared. This study was carried out at the Oral and Maxillofacial Unit, Eastman Dental Institute for Oral Healthcare Sciences, University College London, London WC1 8LD, U.K. Summary Background There is upregulation of class II molecules of the major histocompati- bility complex (MHC) by keratinocytes in oral squamous cell carcinoma (OSCC) and inflammatory diseases such as lichen planus. The significance of this expres- sion, or whether it is accompanied by upregulation of membrane-bound costi- mulatory molecules, is unknown. Objectives To compare the expression of CD40, CD80, CD86, MHC class II and intercellular adhesion molecule-1 (ICAM-1), and the ability to induce allogeneic T-lymphocyte proliferation in vitro, of a CD40– OSCC cell line, its CD40+ trans- fected derivative and null transfectants. Methods OSCC cell lines and purified T lymphocytes were cocultured and T cell proliferation recorded. Phenotypes were analysed by flow cytometry. Results After T lymphocyte proliferation, which all OSCC cell lines were able to induce, there was upregulation of MHC class II and ICAM-1. However, the CD40+ transfectants were the most immunologically potent and were the only cells to show increased expression of CD86 (as well as further upregulation of CD40 and a statistically insignificant rise in CD80). The effects of blocking antibodies on T-cell proliferation were only statistically significant with the CD40+ transfectants. Conclusions While not essential, expression of CD40 by OSCC cells is necessary for optimal induction of allogeneic T lymphocytes, possibly because of concurrent upregulation of other membrane-bound costimulatory molecules. Recognition in the 1980s that keratinocytes, the cells that form the bulk of the stratified squamous epithelium which covers the skin and mucous membranes of the upper aerodi- gestive and female genital tracts, can express class II molecules of the major histocompatibility complex (MHC), first raised the possibility that these cells might have an immunological function. Interferon (IFN)-c stimulates MHC class II expression by keratinocytes from oral mucosa and skin in vitro 1–9 and in vivo at sites of oral squamous cell carcinoma (OSCC) and inflammatory diseases such as lichen planus. 5–7,9–15 Demon- stration that human epidermal keratinocytes are able to induce, or inhibit, proliferation of T lymphocytes followed, and keratinocytes treated with IFN-c have been reported to act as accessory cells in mitogen- or superantigen-induced T cell proliferation. 16,17 By this time, the importance of the mem- brane-bound costimulatory proteins CD40, CD80 and CD86 in immune potency had become apparent. These molecules are not antigen specific and do not, in isolation from MHC class II, induce T cell proliferation. They are, none the less, essential for effective interactions between antigen-presenting cells (APC) and responder T lymphocytes, and for effective cytotoxic destruction of target cells. 18 CD40 and its ligand CD40L (CD154) are phosphorylated glycoproteins of 48 kDa and 33 kDa, respectively, that belong to the tumour necrosis factor receptor superfamily. 19,20 CD40 is expressed on the membranes of a diverse range of cell types, reflecting its importance in a number of biological functions. In normal epidermis and the stratified squamous epithelium covering oral mucosa, the expression of CD40 is restricted to keratino- cytes and Langerhans cells of the basal and parabasal layers. 9,12,21–25 In vitro, all OSCC cell lines tested have proved to be CD40+. 9,26 CD40L is transiently expressed by activated T and B lymphocytes, basophils, eosinophils and dend- ritic cells. 27–29 It is also present in the basement membrane Ó 2005 British Association of Dermatologists • British Journal of Dermatology 2006 154, pp231–238 231