Skin blister immunocytology A new method to quantify cellular kinetics in vivo

A method is described using tuberculin purified protein derivative (PPD) as a model to follow the in vivo cellular immune response. This combines induction of a local response, formation of skin blisters, and staining of the cells appearing in the sterile exudate over time, using standard cytopreparatory and immunoperoxidase techniques. Skin blisters were induced over sites previously injected intradermally with PPD or control saline using suction over a template on the forearm. The cells which appeared in the exudate at 24, 36, 48 and 72 h were collected on small cellulose filters which were divided into several parts. The cells on the filter segments were then stained using a biotin-avidin immunoperoxidase method with a panel of monoclonal antibodies, and with enzyme histochemical techniques. This allowed quantitative estimation over an extended period of total numbers of each cell type responding as a local expression of cellular immunity. The kinetics of an early, non-specific inflammatory response could be distinguished from the later immune response using total cell counts. Maximal cell counts correlated well with PPD induced induration at 48 h, showing an overwhelming predominance of mononuclear cells. Over 72 h, the number of non-specific esterase (NSE) positive cells (macrophage) declined while Leu4 positive cells (T cells) increased. OKT4 positive cells (T helper) outnumbered OKT8 positive cells (T suppressor) as the response developed. This method enables the direct quantitative assessment of cell populations arriving at the site of an immune response using a simple inexpensive technique which is painless, non-invasive and non-scarring.