Cortical sinus probing, S1P1-dependent entry and flow-based capture of egressing T cells.

The cellular dynamics of the egress of lymphocytes from lymph nodes are poorly defined. Here we visualized the branched organization of lymph node cortical sinuses and found that after entry, some T cells were retained, whereas others returned to the parenchyma. T cells deficient in sphingosine 1-phosphate receptor type 1 probed the sinus surface but failed to enter the sinuses. In some sinuses, T cells became rounded and moved unidirectionally. T cells traveled from cortical sinuses into macrophage-rich sinus areas. Many T cells flowed from medullary sinuses into the subcapsular space. We propose a multistep model of lymph node egress in which cortical sinus probing is followed by entry dependent on sphingosine 1-phosphate receptor type 1, capture of cells in a sinus region with flow, and transport to medullary sinuses and the efferent lymph

Chemokine Requirements for B Cell Entry to Lymph Nodes and Peyer's Patches

B cell entry to lymph nodes and Peyer's patches depends on chemokine receptor signaling, but the principal chemokine involved has not been defined. Here we show that the homing of CXCR4−/− B cells is suppressed in CCL19 (ELC)- and CCL21 (SLC)-deficient paucity of lymph node T cells mice, but not in wild-type mice. We also find that CXCR4 can contribute to T cell homing. Using intravital microscopy, we find that B cell adhesion to high endothelial venules (HEVs) is disrupted when CCR7 and CXCR4 are predesensitized. In Peyer's patches, B cell entry is dependent on CXCR5 in addition to CCR7/CXCR4. CXCL12 (SDF1) is displayed broadly on HEVs, whereas CXCL13 (BLC) is found selectively on Peyer's patch follicular HEVs. These findings establish the principal chemokine and chemokine receptor requirements for B cell entry to lymph nodes and Peyer's patches

Transient Receptor Potential 1 Regulates Capacitative Ca2+ Entry and Ca2+ Release from Endoplasmic Reticulum in B Lymphocytes〉

Capacitative Ca2+ entry (CCE) activated by release/depletion of Ca2+ from internal stores represents a major Ca2+ influx mechanism in lymphocytes and other nonexcitable cells. Despite the importance of CCE in antigen-mediated lymphocyte activation, molecular components constituting this mechanism remain elusive. Here we demonstrate that genetic disruption of transient receptor potential (TRP)1 significantly attenuates both Ca2+ release-activated Ca2+ currents and inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release from endoplasmic reticulum (ER) in DT40 B cells. As a consequence, B cell antigen receptor–mediated Ca2+ oscillations and NF-AT activation are reduced in TRP1-deficient cells. Thus, our results suggest that CCE channels, whose formation involves TRP1 as an important component, modulate IP3 receptor function, thereby enhancing functional coupling between the ER and plasma membrane in transduction of intracellular Ca2+ signaling in B lymphocytes

Antigen-Engaged B Cells Undergo Chemotaxis toward the T Zone and Form Motile Conjugates with Helper T Cells

Interactions between B and T cells are essential for most antibody responses, but the dynamics of these interactions are poorly understood. By two-photon microscopy of intact lymph nodes, we show that upon exposure to antigen, B cells migrate with directional preference toward the B-zone–T-zone boundary in a CCR7-dependent manner, through a region that exhibits a CCR7-ligand gradient. Initially the B cells show reduced motility, but after 1 d, motility is increased to approximately 9 μm/min. Antigen-engaged B cells pair with antigen-specific helper T cells for 10 to more than 60 min, whereas non-antigen-specific interactions last less than 10 min. B cell–T cell conjugates are highly dynamic and migrate extensively, being led by B cells. B cells occasionally contact more than one T cell, whereas T cells are strictly monogamous in their interactions. These findings provide evidence of lymphocyte chemotaxis in vivo, and they begin to define the spatiotemporal cellular dynamics associated with T cell–dependent antibody responses

Sphingosine-1-phosphate receptor 2 is critical for follicular helper T cell retention in germinal centers

Follicular helper T (Tfh) cells access the B cell follicle to promote antibody responses and are particularly important for germinal center (GC) reactions. However, the molecular mechanisms of how Tfh cells are physically associated with GCs are incompletely understood. We report that the sphingosine-1-phosphate receptor 2 (S1PR2) gene is highly expressed in a subpopulation of Tfh cells that localizes in GCs. S1PR2-deficient Tfh cells exhibited reduced accumulation in GCs due to their impaired retention. T cells deficient in both S1PR2 and CXCR5 were ineffective in supporting GC responses compared with T cells deficient only in CXCR5. These results suggest that S1PR2 and CXCR5 cooperatively regulate localization of Tfh cells in GCs to support GC responses

Two-photon microscopy analysis of leukocyte trafficking and motility

During the last several years, live tissue imaging, in particular using two-photon laser microscopy, has advanced our understanding of leukocyte trafficking mechanisms. Studies using this technique are revealing distinct molecular requirements for leukocyte migration in different tissue environments. Also emerging from the studies are the ingenious infrastructures for leukocyte trafficking, which are produced by stromal cells. This review summarizes the recent imaging studies that provided novel mechanistic insights into in vivo leukocyte migration essential for immunosurveillance

Molecular and Functional Characterization ofNovel TRP Receptor-activated Ca2+ Channels from Mouse Brain

Receptor-activated Ca2+ influx that occurs as a second phase of phosphatidyl inositol-dependent response, has been recently recognized for its physiological significance. Characterization of mammalian homologues of Drosophila TRP proteins is an important clue to understand molecular mechanisms underlying receptor-activated Ca2+ influx in vertebrate cells. Especially, TRP homologues have been proposed to encode store-operated channels (SOC) which is activated through Ca2+ release from the intracellular Ca2+ store and subsequent depletion of the Ca2+ store. However, the hypothesis is still controversial. In order to establish the correlation of cloned TRP homologues and native channels, it is necessary to investigate the functional properties of TRP homologues using the excellent expression system. He has here isolated cDNAs that encode novel TRP homologues, TRP4, TRP5, TRP6, and TRP7 from the mouse brain. All of these homologues were found to be relatively highly expressed in the brain, but their expression was detected also in various organs other than the brain, with various intensities characteristic of each subtype. Among them, first, he characterized the function of the brain-predominant homologue, TRP5. The recombinant expression of the TRP5 cDNA in human embryonic kidney cells potentiated an extracellular Ca2+-dependent increase of [Ca2+]i evoked by ATP, but not by an inhibitor of ER Ca2+-ATPases, thapsigargin. Whole-cell mode of patch-clamp recordings from TRP5-expressing cells demonstrated that ATP application induced a large inward current in the presence of extracellular Ca2+, which reversed at a positive potential. The TRP5 activity was abolished by omission of intracellular Ca2+ and by treatment with a calmodulin antagonist. High concentration of extracellular Ca2+ activated TRP5 without ATP stimulation. On the other hand, omission of extracellular Ca2+ abolished the activity even in the presence of ATP. These results suggest that TRP5 directs the formation of a highly Ca2+-permeable ion channel which can be activated through receptor-operative pathways other than depletion of Ca2+ from Ca2+ stores, and that the activity of this channel is highly dependent on Ca2+ concentrations inside and outside of cells. Second, the TRP7 function was characterized using the same recombinant expression system. The results show that TRP7 directs the formation of a Ca2+-permeable ion channel with significant basal activity. The activity of TRP7 did not depend on extracellular Ca2+ but was enhanced by receptor-stimulation even with the low concentration of ATP which could not induce Ca2+ release from the intracellular Ca2+ store. The obtained functional characters of the two novel TRP homologues together suggest that mammalian TRP homologues are members of a large family of channels responsible for receptor-activated Ca2+ influx including not only SOC but also other diverse subtypes of channels in the family