There is a great deal of information regarding the minimal requirements for activation of primed T cells. T cell clones can be stimulated to release granule contents and produce lymphokines by anti-CD3 antibody or purified antigen-MHC proteins. T cell hybridomas can also be stimulated to produce lymphokines by anti-CD3 antibody or purified antigen-MHC proteins. Activation of these primed T cell lines can occur in the absence of specialized presenting cells and their accessory molecules. This project is to define the minimal requirements for activation of naive T cells and to evaluate the effects of additional stimuli on the magnitude and type of T cell activation elicited. With the use of a potent peptide-MHC complex, we have been successful in the activation of phenotypically naive CD8 positive transgenic T-cells. These T-cells have been stimulated to proliferate, secrete IL-2 and mature to cytolytic effectors. A large proportion of these transgenic T-cells can be activated by immobilized peptide-MHC complexes in the absence of signals from antigen presenting cells. Through the use of less potent peptide-MHC complexes and purified adhesion costimulatory molecules, we plan to evaluate the effects of isolated costimulatory signals. We have generated a recombinant soluble ICAM-1 molecule for use in these studies. Isolated ICAM-1 and a TCR stimulus are capable of increasing the activation of transgenic CD8 positive T-cells. ICAM-1 can increase proliferation and cytokine production of CD8+ T-cells with varying TCR signal intensity, generated through different peptide-MHC complexes. Using anti-CD3 stimulation of CD8+ and CD4+ T cells, we have demonstrated that ICAM-1 has a differential effect on the two T-cell subsets. We have shown that ICAM-1 enhancement of CD8+ T-cell activation can occur in the absence of an immunological synapse. We have also evaluated the effect of a mucin, similar in structure to CD43, on T-cell stimuli. Using SPR, we have probed the binding of the CD8 molecule to MHC alpha 3 mutants in order to better understand the role of this binding in T-cell activation. In addition, we are planning to generate or obtain other soluble adhesion/costimulatory molecules to evaluate their effects in our system. There functional effects may be correlated to binding using SPR analysis. This system will allow us to evaluate specific changes in naive T cell responses caused by the deletion or addition of lymphokines, adhesion molecules or costimulatory molecules. This can be done in the absence of unknown contributions from antigen presenting cells. The molecular requirements for T-cell activation and the varying phenotypes of this activation are very basic to the generation of successful immunity and the modulation of aberrant immune responses.
