Orai1 and STIM1 move to the immunological synapse and are up-regulated during T cell activation

Lioudyno MI, Kozak JA, Penna A, Safrina O, Zhang SL, Sen D, Roos J, Stauderman KA, Cahalan MD
Source: Proc Natl Acad Sci USA
Publication Date: (2008)
Issue: 105(6): 2011-6
Research Area:
Immunotherapy / Hematology
Cells used in publication:
Species: human
Tissue Origin: blood
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
T cell, human stim.
Species: human
Tissue Origin: blood
Nucleofector™ I/II/2b
Jurkat or primary T cells wer co-transfected with plasmid clone pcDNA/hSTIM1 and eGFP-tagged WT Orai1 with a 1:2 ratio (5 µg STIM1 : 10 µg Orai1 per sample) or GFP-tagged E106A mutant Orai1. For RNAi a mixture of 4 siRNAs against STIM1 and Orai1 were transfected into human resting T cells.
For efficient development of an immune response, T lymphocytes require long-lasting calcium influx through calcium release-activated calcium (CRAC) channels and the formation of a stable immunological synapse (IS) with the antigen-presenting cell (APC). Recent RNAi screens have identified Stim and Orai in Drosophila cells, and their corresponding mammalian homologs STIM1 and Orai1 in T cells, as essential for CRAC channel activation. Here, we show that STIM1 and Orai1 are recruited to the immunological synapse between primary human T cells and autologous dendritic cells. Both STIM1 and Orai1 accumulated in the area of contact between either resting or super-antigen (SEB)-pretreated T cells and SEB-pulsed dendritic cells, where they were colocalized with T cell receptor (TCR) and costimulatory molecules. In addition, imaging of intracellular calcium signaling in T cells loaded with EGTA revealed significantly higher Ca2+ concentration near the interface, indicating Ca2+ influx localized at the T cell/dendritic cell contact area. Expression of a dominant-negative Orai1 mutant blocked T cell Ca2+ signaling but did not interfere with the initial accumulation of STIM1, Orai1, and CD3 in the contact zone. In activated T cell blasts, mRNA expression for endogenous STIM1 and all three human homologs of Orai was up-regulated, accompanied by a marked increase in Ca2+ influx through CRAC channels. These results imply a positive feedback loop in which an initial TCR signal favors up-regulation of STIM1 and Orai proteins that would augment Ca2+ signaling during subsequent antigen encounter.