T cell activation is a key event in adaptive immunity. However, factors affecting T cell activation have not been systematically analyzed. Here, we analyzed stimulated CD4 T cells with anti-CD3/CD28 under several conditions to explore the factors affecting T cell activation. We defined stimulated T overlapped with resting T on UMAP as inert T. Inert T expressed activated T specific genes and cytokines, indicating it is a special functional state. Stimulated T derived from peripheral CD4 T has higher fraction of effector T (TEFF) while stimulated T derived from CD4 TNhas higher fraction of proliferation T and interferon highly expressed T (IFNhiT). CD4 T was more likely to differentiate into TEFFand less likely to differentiate into heat shock protein specific T (HSPhiT) and IFNhiT in the presence of CD8 T. Interestingly,CXCR4lowT responded to stimulation more efficiently thanCXCR4hiT. These information facilitates we design stimulation to obtain ideal activated T.
