The molecular mechanisms responsible for the sustained basal motility of T cells within lymph nodes (LNs) remain elusive. To study T cell motility in a LN environment, we have developed a new experimental system based on slices of LNs that allows the assessment of T cell trafficking after adoptive transfer or direct addition of T cells to the slice. Using this experimental system, we show that T cell motility is highly sensitive to pertussis toxin and strongly depends on CCR7 and its ligands. Our results also demonstrate that, despite its established role in myeloid cell locomotion, phosphoinositide 3–kinase (PI3K) activity does not contribute to the exploratory behavior of the T lymphocytes within LN slices. Likewise, although PI3K activation is detectable in chemokine-treated T cells, PI3K plays only a minor role in T cell polarization and migration in vitro. Collectively, our results suggest that the common amplification system that, in other cells, facilitates large phosphatidylinositol 3,4,5-trisphosphate increases at the plasma membrane is absent in T cells. We conclude that T cell motility within LNs is not an intrinsic property of T lymphocytes but is driven in a PI3K-independent manner by the lymphoid chemokine-rich environment.

CD4 ؉ T cells use the chemokine receptor CCR7 to home to and migrate within lymphoid tissue, where T-cell activation takes place. Using primary T-cell receptor (TCR)-transgenic (tg) CD4 ؉ T cells, we explored the effect of CCR7 ligands, in particular CCL21, on T-cell activation. We found that the presence of CCL21 during early time points strongly increased in vitro T-cell proliferation after TCR stimulation, correlating with increased expression of early activation markers. CCL21 costimulation resulted in increased Rasand Rac-GTP formation and enhanced phosphorylation of Akt, MEK, and ERK but not p38 or JNK. Kinase-dead PI3K␦ D910A/D910A or PI3K␥-deficient TCR-tg CD4 ؉ T cells showed similar responsiveness to CCL21 costimulation as control CD4 ؉ T cells. Conversely, deficiency in the Rac guanine exchange factor DOCK2 significantly impaired CCL21-mediated costimulation in TCR-tg CD4 ؉ T cells, concomitant with impaired Rac-but not Ras-GTP formation. Using lymph node slices for live monitoring of T-cell behavior and activation, we found that G proteincoupled receptor signaling was required for early CD69 expression but not for Ca 2؉ signaling. Our data suggest that the presence of CCL21 during early TCR signaling lowers the activation threshold through Ras-and Rac-dependent pathways leading to increased ERK phosphorylation. (Blood. 2009;114:580-588) IntroductionNaive T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes (PLNs), where they screen antigen-presenting cells (APCs), in particular dendritic cells (DCs), for the presence of specific peptide Ag presented on MHC (pMHC) complexes. 1 In proinflammatory conditions, DCs expressing cognate pMHC and costimulatory signals, such as B7 molecules, induce efficient T-cell activation through the T-cell receptor (TCR) and CD28. 2 This leads to an early signaling response characterized by activation of multiple signaling pathways, including tyrosine kinase cascades, sustained increase in intracellular Ca 2ϩ , and activation of phosphoinositide-3-kinase (PI3K), small GTPases of the Ras and Rho family, mitogen-activated protein kinase, nuclear factor of activated T-cell, and nuclear factor-B. Activated T cells subsequently increase surface expression of early activation markers, such as CD69 and CD25, produce interleukin-2 (IL-2), expand clonally, and differentiate into effector cells.Direct observations of lymphocytes and DCs presenting cognate pMHC complexes in explanted PLNs or live mice using 2-photon microscopy have uncovered a dynamic range of cellular interactions within lymphoid tissue. In some settings, T cells almost immediately arrest on encountering DCs. 3 Alternatively, T cells were observed to continue to migrate during the first several hours after entry into lymphoid tissue along the stromal network formed by fibroblastic reticular cells (FRCs), where they underwent brief serial contacts with DCs. 4 This first phase of high motility is reminiscent of naive T-cell migration in the absence of pMHC-loaded DCs. 4,5 Of not...