The in vitro induction of delayed-type hypersensitivity to allo-antigens of the mouse

An in vitro procedure has been developed which allows the induction of delayed-type hypersensitivity (DTH) to major allo-antigens of the mouse. Murine spleen cells cultured with irradiated allogeneic cells develop allo-specific DTH reactivity (DTHR). The variables assessed to provide optimal induction of DTHR were (i) the culture system, (ii) the density of responders and the number of stimulator cells and (iii) the kinetics of induction. The reactivity of the allo-specific cells was assayed in 3 different ways in order to select the most sensitive: (i) by local footpad transfer into a mouse syngeneic with the responder cells together with the eliciting (irradiated) antigen-bearing cells; (ii) by local transfer into the footpad of a mouse syngeneic with the stimulator cells, in which the allo-antigens present in the subcutaneous tissues elicit the response, and (iii) by intravenous transfer into syngeneic or allogeneic mice which are challenged 24 h later in the footpad with spleen cells bearing the haplotype of the stimulator. The second assay is clearly the most sensitive (2X that of the first and greater than 20X that of the last). Observations are reported demonstrating the specificity of the swelling reaction. The kinetics of swelling and its T-cell dependence provide strong grounds for believing that the reaction is due to classical delayed-type hypersensitivity. Furthermore the T cells mediating the swelling are of the phenotype Lyt1+ Lyt2(+/-) and Ia- and are radiation resistant, whereas the ability to produce a swelling reaction is sensitive to 1000 rads. whole body irradiation. The system has been applied to determine both the specificity of T cells mediating DTH to major and minor allo-antigens and whether cytotoxic T cells (CTL) and DTH-active T cells are always induced under the same conditions.