CD28 sensitizes TCR Ca²⁺ signaling during Ag-independent polarization of plasma membrane rafts.

T cells become polarized during initial interactions with an APC to form an Ag-independent synapse (AIS) composed of membrane rafts, TCR, and TCR-proximal signaling molecules. AISs occur temporally before TCR triggering, but their role in downstream TCR signaling is not understood. Using both human and murine model systems, we studied the signals that activate AIS formation and the effect of these signals on TCR-dependent responses. We show that CD28 produces AISs detectable by spinning disc confocal microscopy seconds following initial interactions between the T cell and APC. AIS formation by CD28 coincided with costimulatory signaling, evidenced by a cholesterol-sensitive activation of the MAPK ERK that potentiated Ca²⁺ signaling in response to CD3 cross-linking. CD45 also enriched in AISs but to modulate Src kinase activity, because localization of CD45 at the cell interface reduced the activation of proximal Lck. In summary, we show that signaling by CD28 during first encounters between the T cell and APC both sensitizes TCR Ca²⁺ signaling by an Erk-dependent mechanism and drives formation of an AIS that modulates the early signaling until TCR triggering occurs. Thus, early Ag-independent encounters are an important window for optimizing T cell responses to Ag by CD28.