Phenotypic and functional analysis of antigen presenting cells in a murine model of cutaneous carcinogenesis

To elucidate the nature of the cells involved in the induction of immunosuppression when antigen is applied through Langerhans cell depleted skin, due to prior exposure to 9, 10- dimethyl benzanthracene (DMBA) or 2,4,6-trinitrochlorobenzene (TNCB) the phenotype of migrating antigen bearing cells was analysed. The initial experiments were aimed at the identification of the antigen bearing cells migrating from murine skin previously treated with DMBA or TNCB to deplete Langerhans cells. This involved application of the fluorescent contact sensitiser fluorescein isothiocyanate (FITC) to DMBA or TN.CB treated skin, enrichment of low density cells from draining lymph nodes by metrizamide gradient centrifugation and the phenotypic characterisation of the FITC bearing cells by flow cytometry. Two distinct distinct populations were identified on the basis of FITC fluorescence intensity. These were classified as FITChi or FITCl0. intensities of FITC positive cells were detected in draining lymph nodes of acetone-FITC treated mice. DMBA or TNCB treatment prior to FITC application substantially reduced the number of FITChi cells intensity positive (FITChi) cells within the draining lymph nodes, whereas the number of FIT0¬¨‚àû was slightly reduced This demonstrates that the cells present in DMBA or TNCB treated skin had an impaired ability to trap antigen and migrate to the draining lymph node. Single colour phenotypic analysis of FITC positive cells that did migrate from DMBA or TNCB treated skin revealed populations that expressed markers consistent with dendritic cells, ˜í‚â•˜í¬•-TCR+ cells, CD4+ cells, CD8a+ cells or B cells. Markers for dendritic cells, CD4+ cells and CD8a+ cells were detected upon both FITChi and FITC'0 populations whereas populations of cells expressing ˜í‚â•˜í¬•-TCR and B cell markers were restricted to the FITC'0 population. Both FITChi and FITCl0 populations expressed accessory and adhesion molecules at levels consistent with phenotypically mature dendritic cells. Further analysis of the FITC bearing populations by multiple colour flow cytometry revealed that FITChi cells consisted primarily of mature dendritic cells, while the FITCl0 population was more heterogenous. FITCl0 cells expressed CD11c, CD11b, MHC 11, CD8˜í¬±, CD80 and CD86. Analysis of FITC positive cells revealed FITChi dendritic cells from DMBA-FITC and TNCB¬¨‚↠FITC treated mice were defective in the ability to activate FITC specific responder cells. To determine the identity of the effector population induced by DMBA an in vitro assay was developed. The effector cell was identified as a nylon wool adherent cell, that expressed CD11b. This population was found to act in an antigen nonspecific manner in vitro.