COLLABORATION OF TCR-MEDIATED, CD4-MEDIATED AND CD28-MEDIATED SIGNALING IN ANTIGEN-SPECIFIC MHC CLASS II-RESTRICTED T-CELLS

A previously developed experimental system was applied to obtain quali tative and quantitative data on the contribution of TCR-, CD4- and CD2 8-mediated signalling in the activation of an antigen specific T-cell hybridoma. All the three signal transducing receptors were stimulated by their natural ligands, and intermediate and late responses of an I- E(d) restricted, CD4(+), influenza HA specific murine T-hybridoma (IP- 12-7) were monitored by measuring the concentration of intracellular c alcium [Ca2+](i) and secreted IL-2. This type of analysis of T-cell ac tivation revealed: (i) calcium mobilization induced by peptide loaded APC requires rapid conjugate formation; (ii) a direct correlation betw een the magnitude of the intermediate and the late responses was obser ved as a consequence of differential TCR ligation modulated by peptide dose or by the presence CD4 (iii) considering the APC/peptide and T/A PC ratios, the concentration dependence of the intermediate and late r esponses was similar in both assays but a substantial difference in th e sensitivity of the two methods was observed; (iv) CD4 mediated signa lling has a co-stimulatory effect predominantly at suboptimal in vitro conditions; and (v) sustained increase of [Ca2+](i) as well as the pr oduction of high concentrations of IL-2 is highly dependent on the CD2 8-B7 interaction. These results demonstrate that distinct peptide dose s and the presence or absence of CD4 result in quantitative changes in T-cell responses, while the degree of CD28 mediated signalling has a qualitative affect on the outcome of T-cell activation, revealed by co mplete or partial inhibition of IL-2 secretion as a result of limited CD28-B7 interaction as well as by alteration in the duration and time kinetics of the calcium response. (C) 1996 Elsevier Science B.V.