In T lymphocytes, Ca2+ release–activated Ca2+ (CRAC) channels composed of Orai1 subunits trigger Ag-induced gene expression and cell proliferation through the NFAT pathway. We evaluated the requirement of CRAC channel function for lymphocyte homing using expression of a dominant-negative Orai1-E106A mutant to suppress Ca2+ signaling. To investigate homing and motility of human lymphocytes in immunocompromised mouse hosts, we transferred human lymphocytes either acutely or after stable engraftment after a second transfer from the same blood donor. Human and mouse lymphocyte homing was assessed, and cells were tracked within lymph nodes (LNs) by two-photon microscopy. Our results demonstrate that human T and B lymphocytes home into and migrate within the LNs of immunocompromised NOD.SCID mice similar to murine lymphocytes. Human T and B cells colocalized in atrophied or reconstituted mouse LNs, where T cells migrated in a random walk at velocities of 9–13 μm/min and B cells at 6 μm/min. Expression of Orai1-E106A inhibited CRAC channel function in human and mouse T cells, and prevented homing from high endothelial venules into murine LNs. Ca2+ signals induced by CCL21 were also inhibited in T cells expressing Orai1-E106A. With CRAC channels inhibited, the high-affinity form of LFA-1 failed to become active, and T cells failed to migrate across endothelial cells in a transwell model. These results establish a requirement for CRAC channel–mediated Ca2+ influx for T cell homing to LNs mediated by high-affinity integrin activation and chemokine-induced transendothelial migration.
This work was supported by National Institutes of Health (NIH) Grants NS-14609 and GM-41514 (to M.D.C.) and GM-48071 (to I.P.), the NIH Immunology Research Training Program Grant T32-AI-060573 (to M.L.G.), and the American Heart Association Scientist Development Grant 0630117N (to Y.Y.). Human blood was prepared using support from the National Center for Research Resources and the National Center for Advancing Translational Sciences (NIH Grant UL1 TR000153).
The online version of this article contains supplemental material.
- Received August 9, 2012.
- Accepted January 29, 2013.
- Copyright © 2013 by The American Association of Immunologists, Inc.