Orai1 and STIM1 move to the immunological synapse and are up-regulated during T cell activation.
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.
Lioudyno
Maria I
MI
Department of Physiology and Biophysics, University of California, Irvine, CA 92697, USA.
Kozak
J Ashot
JA
Penna
Aubin
A
Safrina
Olga
O
Zhang
Shenyuan L
SL
Sen
Debasish
D
Roos
Jack
J
Stauderman
Kenneth A
KA
Cahalan
Michael D
MD
D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000502:physiology*; D002460:Cell Line; D006801:Humans; D017136:Ion Transport; D008213:Lymphocyte Activation; D008565:Membrane Proteins / Q000502:physiology*; D009363:Neoplasm Proteins / Q000502:physiology*; D000071740:ORAI1 Protein; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000071737:Stromal Interaction Molecule 1; D013601:T-Lymphocytes / Q000276:immunology*; D015854:Up-Regulation
D015220:Calcium Channels; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D002118:Calcium