Transfer of cell-mediated immunity with cell-free leukocyte extracts: I. Comparison of in vitro and in vivo assays of cell-mediated immune function

Abstract

Delayed hypersensitivity was induced in mice by exposure to the contact sensitizing agent, 2,4-dinitrofluorobenzene. Reactivity was assessed in vivo by measurement of ear thickness and in vitro by lymphocyte transformation and MIF production using the soluble analog, sodium 2,4-dinitrobenzenesulfonate (DNBSO 3Na). The peak of the response occurred 5 days after sensitization in all three assays but thereafter the time response curves diverged. When lymph node cells were used in vitro, the macrophage migration inhibition (MMI) test and lymphocyte transformation became negative at 12 days whereas in vivo delayed hypersensitivity remained detectable for 3 months. On the other hand, significant proliferation of splenic lymphocytes was observed for at least 100 days after sensitization. Furthermore, spleen was a poorer source of cells releasing macrophage inhibition factor (MIF) than lymph nodes at all times of assay. MIF production and lymphocyte transformation were both T-cell dependent as judged by their sensitivity to treatment to anti-Thy-1.2 serum and complement. Purified T cells obtained from nylon wool columns gave a positive response in the MMI test. Their proliferative capacity, however, was markedly reduced despite addition of peritoneal macrophages which suggested that some of the proliferating cells had adherent properties. Confirmation of a difference in the cells responsible for the effect observed in the two in vitro assays was obtained by pretreatment with a panel of sera directed against Ia and Ly determinants. The phenotype of the MIF producing cells was Thy-1 +, Ia −, Ly-1 +, Ly- 2 3 − , Ly-4ndash;7 +, the first four markers of which are the same as those known to be present on the cells responsible for transfer of delayed hypersensitivity in vivo. By contrast cells proliferating in response to DNBSO 3Na in vitro were more heterogeneous since all antisera abrogated transformation to some extent. The conclusion was therefore reached that the two in vitro systems measure different cellular responses and the MMI test is a better correlate of in vivo delayed hypersensitivity than lymphocyte transformation. This information is vital when using the assay for analysis of immune phenomena such as the effect of soluble mediators of delayed hypersensitivity.