Distinct regulation of macromolecular signaling complex formation in Th1 and Th2 effector cells: The role of lipid rafts and the immunological synapse

To investigate whether differences in raft recruitment account for differences in signaling and T cell receptor (TCR) macromolecular complex organization in Th1 and Th2 cells, we questioned whether the signaling proteins found associated with lipid rafts prior to and during activation differ in these two subsets of T cells. We show here that TCR complex members are recruited efficiently to rafts and aggregate with rafts at the site of MHC/peptide contact in Th1 but not Th2 cells. Functionally, these observations are supported by the analysis of calcium mobilization in response to TCR signaling in the presence of a raft disrupting agent, which demonstrates that calcium mobilization is dependent on raft integrity in Th1 but not Th2 cells. Interestingly, while the pattern of signaling in response to a high affinity peptide is similar in Th1 and Th2 cells, there is a marked defect in the response of Th2 cells to low affinity peptides, particularly in downstream events such as calcium mobilization and cell division.; To investigate the role of CD4 as a regulatory molecule which may control these processes, we examined both recruitment of the TCR to lipid rafts and immunological synapse formation in T cells from CD4 deficient mice. We show that the differential raft recruitment of the TCR in Th1 vs. Th2 cells is in fact regulated by CD4, suggesting that CD4-dependent signaling may aid in recruitment and/or aggregation of rafts upon TCR signaling. We then show that the requirement for CD4 in governing immunological synapse formation is dependent on the strength of peptide used to stimulate the TCR: CD4 deficient T cells stimulated with agonist peptides show a partial defect in immunological synapse formation, whereas CD4 deficient cells stimulated with a lower affinity antigenic peptide show more profound defects. These differences correlate with downstream defects in AP-1 and NFκB activation, particularly in response to low affinity peptide stimulation. Finally, we demonstrate that these defects can be rescued by retroviral transduction of wild type, but not tailless or palmitoylation mutant CD4, indicating that both CD4 signaling function and recruitment to rafts are required for efficient immunological synapse formation.