The activation of T cells in vivo is initiated by the presentation of antigen as well as signaling through the T cell receptor complex (TCR/CD3) and CD28. A variety of events including membrane signaling, induction of tyrosine kinases and an increase in intracellular calcium (Ca2+) all participate in T cell activation.

Physiological T cell activation is achieved in vitro by stimulating PBMCs or purified T cells with anti-CD3/anti-CD28 coated microbeads. T cell activation readouts include cell proliferation (72 hours) and cytokine production (24 hours).

Figure: T cell activation assay. PBMCs were activated with anti-CD3/anti-CD28 coated microbeads for 24 and 72 hours. After 24 hours, cell culture supernatants were evaluated for IFN-g, IL-2, IL-10 and TNF-a using multiplex assay. After 72 hrs, proliferation was determined using a luminescence-based ATP assay. Mean values are shown. Error bars represent standard deviations. 

Which subset of T cells is activated in vivo is dependant upon which MHC molecule is present on the antigen presenting cell. CD4+ T cells are activated when antigenic peptides are presented on the surface along with MHC II markers, whereas cytotoxic T cells recognize antigen via the MHC I marker. Ex vivo assays can be performed to evaluate T cell activation.