Adrenergic regulation of the vasculature impairs leukocyte interstitial migration and suppresses immune responses

•Activation of the sympathetic nervous system halts leukocyte locomotion in tissues•Noradrenaline induces adrenergic receptor signaling to reduce lymph node blood flow•Decreased tissue oxygenation induces calcium signaling to control leukocyte motility•Disruption of leukocyte motility contributes to impaired immune responses The sympathetic nervous system (SNS) controls various physiological functions via the neurotransmitter noradrenaline. Activation of the SNS in response to psychological or physical stress is frequently associated with weakened immunity. Here, we investigated how adrenoceptor signaling influences leukocyte behavior. Intravital two-photon imaging after injection of noradrenaline revealed transient inhibition of CD8+ and CD4+ T cell locomotion in tissues. Expression of β-adrenergic receptor in hematopoietic cells was not required for NA-mediated inhibition of motility. Rather, chemogenetic activation of the SNS or treatment with adrenergic receptor agonists induced vasoconstriction and decreased local blood flow, resulting in abrupt hypoxia that triggered rapid calcium signaling in leukocytes and halted cell motility. Oxygen supplementation reversed these effects. Treatment with adrenergic receptor agonists impaired T cell responses induced in response to viral and parasitic infections, as well as anti-tumor responses. Thus, stimulation of the SNS impairs leukocyte mobility, providing a mechanistic understanding of the link between adrenergic receptors and compromised immunity. The sympathetic nervous system (SNS) controls various physiological functions via the neurotransmitter noradrenaline. Activation of the SNS in response to psychological or physical stress is frequently associated with weakened immunity. Here, we investigated how adrenoceptor signaling influences leukocyte behavior. Intravital two-photon imaging after injection of noradrenaline revealed transient inhibition of CD8+ and CD4+ T cell locomotion in tissues. Expression of β-adrenergic receptor in hematopoietic cells was not required for NA-mediated inhibition of motility. Rather, chemogenetic activation of the SNS or treatment with adrenergic receptor agonists induced vasoconstriction and decreased local blood flow, resulting in abrupt hypoxia that triggered rapid calcium signaling in leukocytes and halted cell motility. Oxygen supplementation reversed these effects. Treatment with adrenergic receptor agonists impaired T cell responses induced in response to viral and parasitic infections, as well as anti-tumor responses. Thus, stimulation of the SNS impairs leukocyte mobility, providing a mechanistic understanding of the link between adrenergic receptors and compromised immunity. The sympathetic nervous system (SNS) controls diverse biological processes such as heart rate and blood flow and is responsible for the “fight or flight” response that is provoked by acute stress. Most tissues, including the lymph nodes (LNs) and spleen (Felten et al., 1984Felten D.L. Livnat S. Felten S.Y. Carlson S.L. Bellinger D.L. Yeh P. Sympathetic innervation of lymph nodes in mice.Brain Res. Bull. 1984; 13: 693-699Crossref PubMed Scopus (137) Google Scholar; Sloan et al., 2007Sloan E.K. Capitanio J.P. Tarara R.P. Mendoza S.P. Mason W.A. Cole S.W. Social stress enhances sympathetic innervation of primate lymph nodes: mechanisms and implications for viral pathogenesis.J. Neurosci. 2007; 27: 8857-8865Crossref PubMed Scopus (118) Google Scholar), are innervated by SNS fibers, and highly diverse cell types respond to SNS neurotransmitters through cell surface G protein-coupled α- or β-adrenergic receptors (ARs) (Elenkov et al., 2000Elenkov I.J. Wilder R.L. Chrousos G.P. Vizi E.S. The sympathetic nerve--an integrative interface between two supersystems: the brain and the immune system.Pharmacol. Rev. 2000; 52: 595-638PubMed Google Scholar). Stress-induced activation of the SNS can influence immune responses (Segerstrom and Miller, 2004Segerstrom S.C. Miller G.E. Psychological stress and the human immune system: a meta-analytic study of 30 years of inquiry.Psychol. Bull. 2004; 130: 601-630Crossref PubMed Scopus (1970) Google Scholar). Acute adrenoceptor stimulation increases numbers of neutrophils and natural killer (NK) cells in the blood, which may prepare the body for potential injury or infection (Benschop et al., 1996Benschop R.J. Rodriguez-Feuerhahn M. Schedlowski M. Catecholamine-induced leukocytosis: early observations, current research, and future directions.Brain Behav. Immun. 1996; 10: 77-91Crossref PubMed Scopus (409) Google Scholar). The SNS also activates the cholinergic anti-inflammatory pathway that limits inflammatory responses by macrophages in response to endotoxin (Abe et al., 2017Abe C. Inoue T. Inglis M.A. Viar K.E. Huang L. Ye H. Rosin D.L. Stornetta R.L. Okusa M.D. Guyenet P.G. C1 neurons mediate a stress-induced anti-inflammatory reflex in mice.Nat. Neurosci. 2017; 20: 700-707Crossref PubMed Scopus (101) Google Scholar; Martelli et al., 2014Martelli D. Yao S.T. McKinley M.J. McAllen R.M. Reflex control of inflammation by sympathetic nerves, not the vagus.J. Physiol. 2014; 592: 1677-1686Crossref PubMed Scopus (146) Google Scholar; Rosas-Ballina et al., 2011Rosas-Ballina M. Olofsson P.S. Ochani M. Valdés-Ferrer S.I. Levine Y.A. Reardon C. Tusche M.W. Pavlov V.A. Andersson U. Chavan S. et al.Acetylcholine-synthesizing T cells relay neural signals in a vagus nerve circuit.Science. 2011; 334: 98-101Crossref PubMed Scopus (863) Google Scholar). Tonic SNS activity mediates macrophage polarization in the small intestine (Gabanyi et al., 2016Gabanyi I. Muller P.A. Feighery L. Oliveira T.Y. Costa-Pinto F.A. Mucida D. Neuro-immune interactions drive tissue programming in intestinal macrophages.Cell. 2016; 164: 378-391Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar) and dampens ILC2 responses in the gut and lungs (Moriyama et al., 2018Moriyama S. Brestoff J.R. Flamar A.L. Moeller J.B. Klose C.S.N. Rankin L.C. Yudanin N.A. Monticelli L.A. Putzel G.G. Rodewald H.R. Artis D. β2-adrenergic receptor-mediated negative regulation of group 2 innate lymphoid cell responses.Science. 2018; 359: 1056-1061Crossref PubMed Scopus (186) Google Scholar). Conversely, sympathectomy of mice increases CD8+ T cell responses during influenza A virus infection (Grebe et al., 2009Grebe K.M. Hickman H.D. Irvine K.R. Takeda K. Bennink J.R. Yewdell J.W. Sympathetic nervous system control of anti-influenza CD8+ T cell responses.Proc. Natl. Acad. Sci. U S A. 2009; 106: 5300-5305Crossref PubMed Scopus (69) Google Scholar). These diverse observations underlie the importance of a mechanistic understanding of the impact of SNS stimulation in leukocyte function. Leukocytes traffic continuously around the body and transit from the blood into tissues and back into the circulation via the lymphatics (Mueller et al., 2013Mueller S.N. Gebhardt T. Carbone F.R. Heath W.R. Memory T cell subsets, migration patterns, and tissue residence.Annu. Rev. Immunol. 2013; 31: 137-161Crossref PubMed Scopus (497) Google Scholar). Many leukocytes are highly motile within the parenchyma of tissues, which facilitates interactions between immune cells to induce activation and for leukocytes to locate and eradicate pathogens and tumors. The motility of leukocytes is therefore critical for immunity. The locomotion of immune cells is affected by the physical microenvironment as well as multiple signals, including adhesion and chemotactic factors, metabolites, and tissue oxygenation (Lämmermann and Sixt, 2009Lämmermann T. Sixt M. Mechanical modes of ‘amoeboid’ cell migration.Curr. Opin. Cell Biol. 2009; 21: 636-644Crossref PubMed Scopus (436) Google Scholar). Leukocytes integrate these signals to make rapid decisions that control cell behavior. Activation of neural signaling and release of neurotransmitters may affect the dynamic motile behavior of leukocytes as they navigate within tissues, as neurotransmitter signals might be a rapid way to modulate leukocyte behavior in tissues, in particular during acute stress that involves increased activation of the SNS. Here, we examined the impact of the neurotransmitter noradrenaline (NA) on immune responses. NA triggered a swift loss of motility in T and B lymphocytes and dendritic cells (DCs) in both lymphoid and non-lymphoid tissues. We found that SNS activation and signaling through ARs reduced tissue blood flow and induced hypoxia that was immediately sensed by leukocytes. This rapid change in tissue oxygenation activated Ca2+ signaling in leukocytes and stopped cell locomotion within minutes. Disruption of leukocyte motility impaired the induction of immune responses in mice following viral skin infection and systemic malaria infection and in a murine melanoma model. These findings provide mechanistic insight into the relationship between activation of ARs and impaired immunity. We hypothesized that SNS signals might modify the movement of T cells in tissues and lead to compromised immunity. To investigate this concept, we performed intravital two-photon imaging of fluorescently labeled CD8+ and CD4+ T cells within the inguinal LNs of mice (Figure 1A) and examined the effect of treatment with NA, the main neurotransmitter released by postganglionic sympathetic neurons. T cells moving rapidly in the LN parenchyma by amoeboid migration responded to NA by an abrupt loss of motility, retraction of pseudopodia, and rounding of the cells (Figure 1B; Video S1). This unexpected abrogation of T cell locomotion following a single exposure to NA was transient, beginning within minutes of NA administration and lasting 45–60 min (Figure 1C). https://www.cell.com/cms/asset/b828c2df-4459-45be-85b1-2bbff6e687f8/mmc2.mp4Loading ... Download .mp4 (7.12 MB) Help with .mp4 files Video S1. Administration of noradrenaline (NA) transiently halts T cell interstitial migration in LNs, related to Figure 1CD4+ (magenta) and CD8+ (green) T cells and blood vessels visualized by Evans blue injection (orange) were imaged by intravital 2-photon microscopy. NA was administered via i.v. catheter after 40 min of imaging. Frames were acquired at 30 s intervals. Display rate: 12 frames per second. LNs are innervated by SNS fibers, and NA levels reach micromolar concentrations close to nerve fibers (Felten et al., 1987Felten D.L. Felten S.Y. Bellinger D.L. Carlson S.L. Ackerman K.D. Madden K.S. Olschowki J.A. Livnat S. Noradrenergic sympathetic neural interactions with the immune system: structure and function.Immunol. Rev. 1987; 100: 225-260Crossref PubMed Scopus (681) Google Scholar). We tested the impact of local catecholamine signaling by superfusion of the LNs in live mice. Localized administration of NA to the LNs also rapidly halted the cells (Figure 1D), suggesting that NA release within tissues can affect T cell migration. Elimination of sympathetic nerve activity by chemical sympathectomy with 6-hydroxydopamine (6-OHDA) did not affect T cell motility in LNs (Figure S1A), indicating that tonic SNS activity is not sufficient to halt cells. NA is used clinically as a vasopressor for the treatment of patients with cardiogenic or vasodilatory shock (including septic shock) and is delivered through a venous catheter. To determine the impact of such treatment on T cell motility, we administered a low dose of NA by infusion during imaging. T cells ceased motility within minutes and remained immotile as NA infusion was maintained (Figure 1E; Video S2), indicating that therapeutic treatment with NA might affect leukocyte functions. https://www.cell.com/cms/asset/a2dbe6b9-b9b9-4c1b-a0bf-b7d1e5623650/mmc3.mp4Loading ... Download .mp4 (6.08 MB) Help with .mp4 files Video S2. Low-dose treatment with NA halts T cell interstitial migration in LNs, related to Figure 1CD4+ (magenta) and CD8+ (green) T cells and blood vessels visualized by Evans blue injection (orange) were imaged by intravital 2-photon microscopy. NA was continuously infused via i.v. catheter using an automated syringe pump after 15 min of imaging. Frames were acquired at 30 s intervals. Display rate: 12 frames per second. We also examined if SNS signals could affect the locomotion of leukocytes within non-lymphoid tissues. T cell locomotion in the skin and the sinusoids of the liver was stopped by AR stimulation (Figure 1F), demonstrating that AR signals regulate the motility of diverse subtypes of leukocytes in non-lymphoid as well as lymphoid tissues. In contrast, no measurable effect on T cell motility was induced by the precursor of NA, dopamine, the parasympathetic nervous system neurotransmitter acetylcholine, or the glucocorticoid receptor agonist dexamethasone (Figure 1G). Thus, adrenergic neurotransmitters rapidly and transiently halt leukocyte motility. NA signals through subtypes of α- and βARs. The non-selective βAR agonist isoprenaline (Iso) also compromised T cell motility in LNs (Figure 2A; Video S3), suggesting that βAR stimulation is sufficient to stop T cell migration. After regaining motility, a second dose of Iso was equally effective at stopping the cells, showing that cells were not desensitized to stimulation (Figure 2A). AR stimulation also halted the motility of B cells and CD11c+ antigen-presenting cells within LNs (Figure 2B). To define the βAR subtypes responsible for loss of T cell motility, we administered the β2AR agonists salmeterol and formoterol. These long-acting agonists caused prolonged cessation of T cell motility persisting for >2 h (Figure 2C; Figure S1B; Video S4). In contrast, the β1AR-selective agonist dobutamine, which increases heart rate, did not significantly alter T cell behavior (Figure 2D). Administration of the α1AR-selective adrenoceptor agonist phenylephrine (PE) also resulted in rapid cessation of motility of T cells (Figure 2E; Video S5). In comparison, the α2AR-selective adrenoceptor agonist clonidine induced a less pronounced reduction in T cell motility (Figure S1C). These responses to α- or βAR agonists were receptor specific, as pre-treatment with the β1/β2AR antagonist propranolol prevented the Iso-induced reduction in cell motility but was unable to block responses following the administration of PE (Figure 2F). In contrast, the α1AR antagonist phentolamine inhibited the effect of PE administration on T cell motility (Figure 2G). We conclude that AR stimulation rapidly and reversibly prevents the migration of leukocytes within the tissue parenchyma. https://www.cell.com/cms/asset/f6e9e27c-6f36-4eb9-8af4-92579a87bf61/mmc4.mp4Loading ... Download .mp4 (2.99 MB) Help with .mp4 files Video S3. The β-adrenoceptor agonist isoprenaline transiently halts T cell interstitial migration in LNs, related to Figure 2CD4+ (magenta) and CD8+ (green) T cells and blood vessels visualized by Evans blue injection (orange) were imaged by intravital 2-photon microscopy. Isoprenaline was administered via i.v. catheter after 15 min of imaging and again at 75 min. Frames were acquired at 30 s intervals. Display rate: 12 frames per second. https://www.cell.com/cms/asset/97079021-8379-44a2-bf1c-89e18aa10af4/mmc5.mp4Loading ... Download .mp4 (10.26 MB) Help with .mp4 files Video S4. The β-adrenoceptor agonist salmeterol halts T cell interstitial migration in LNs, related to Figure 2CD4+ (magenta) and CD8+ (green) T cells and blood vessels visualized by Evans blue injection (orange) were imaged by intravital 2-photon microscopy. Salmeterol was administered via i.v. catheter after 15 min of imaging. Frames were acquired at 30 s intervals. Display rate: 12 frames per second. https://www.cell.com/cms/asset/3983a854-3e3a-46c4-9d23-12454d32f4ca/mmc6.mp4Loading ... Download .mp4 (5.53 MB) Help with .mp4 files Video S5. The α-adrenoceptor agonist phenylephrine (PE) transiently halts T cell interstitial migration in LNs, related to Figure 2CD4+ (magenta) and CD8+ (green) T cells and blood vessels visualized by Evans blue injection (orange) were imaged by intravital 2-photon microscopy. PE was administered via i.v. catheter after 15 min of imaging. Frames were acquired at 30 s intervals. Display rate: 12 frames per second. We examined if leukocyte-intrinsic AR signaling was required to inhibit cell motility or if SNS neurotransmitters alter leukocyte behavior by modulating the surrounding tissue microenvironment. We isolated T cells from β2AR-deficient (Adrb2−/−) or wild-type (WT) mice and co-transferred these into either Adrb2−/− or WT recipient mice. Administration of Iso halted the migration of both WT and Adrb2−/− T cells in LNs of WT recipients but did not impede the movement of T cells in Adrb2−/− LNs (Figure 3A). This demonstrates that T cell-extrinsic AR signals impede cell motility. To confirm a role for SNS signaling to the tissue microenvironment, we investigated if AR expression by non-hematopoietic cells regulates T cell motility. We generated bone marrow (BM) chimeric mice by irradiating Adrb2−/− or WT mice and reconstituting these with Adrb2−/− or WT BM cells. Inhibition of T cell motility in response to Iso required expression of the β2AR by non-hematopoietic cells (Figure 3B). Although not ruling out additional effects of AR signaling to leukocytes, these findings show that leukocyte migration was controlled by signals from non-hematopoietic cells in response to AR stimulation. We investigated a role for the vasculature in modulating leukocyte migration, as the SNS plays key roles in modulating blood flow to tissues by acting on smooth muscle cells. We confirmed that NA treatment decreased heart rate and increased blood pressure in the mice (Figure S2A). To test if SNS signals cause vasomotor effects in lymphoid tissues we used intravital two-photon microscopy to study blood vessels in the T cell zone in LNs. We found that NA administration induced rapid constriction of the microvasculature in LNs (Figures 4A and 4B ; Figure S2B), as well as constriction of the arterial vessels supplying the LN (Figure S2C). We next examined the role of βAR signaling. βAR signals dilate arterial vessels, and the resulting change in blood flow can induce constriction of venules in the microvasculature of some tissues (Eckstein and Hamilton, 1959Eckstein J.W. Hamilton W.K. Effects of isoproterenol on peripheral venous tone and transmural right atrial pressure in man.J. Clin. Invest. 1959; 38: 342-346Crossref PubMed Google Scholar; Kim et al., 1989Kim S. Dörscher-Kim J.E. Lipowsky H.H. Quantitative assessment of microcirculation in the rat dental pulp in response to alpha- and beta-adrenergic agonists.Arch. Oral Biol. 1989; 34: 707-712Crossref PubMed Scopus (12) Google Scholar). Following treatment with Iso, we observed significant constriction of the blood vessels in the LNs (Figure 4C; Figure S2B). Furthermore, imaging of latex microbeads in the microcirculation revealed a reduction in flow rate in LN vessels after treatment (Figure 4D). Thus, AR stimulation resulted in an acute reduction in blood flow in LNs. To confirm that physiological inputs from the circulation are required for the AR-mediated regulation of leukocyte locomotion, LNs were explanted from mice into imaging chambers and perfused with oxygenated media. In the absence of blood flow in explanted LNs, treatment with Iso or NA did not reduce cell motility (Figures 4E and S3A), implicating physiological inputs from the circulation in AR-stimulated leukocyte arrest. In support of this, immunofluorescence staining for tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine biosynthesis, revealed branching perivascular nerves that followed the blood vessels supplying the LNs (Figure S3B). This confirms previous observations of innervation in mouse and primate LNs (Felten et al., 1984Felten D.L. Livnat S. Felten S.Y. Carlson S.L. Bellinger D.L. Yeh P. Sympathetic innervation of lymph nodes in mice.Brain Res. Bull. 1984; 13: 693-699Crossref PubMed Scopus (137) Google Scholar; Sloan et al., 2007Sloan E.K. Capitanio J.P. Tarara R.P. Mendoza S.P. Mason W.A. Cole S.W. Social stress enhances sympathetic innervation of primate lymph nodes: mechanisms and implications for viral pathogenesis.J. Neurosci. 2007; 27: 8857-8865Crossref PubMed Scopus (118) Google Scholar; Villaro et al., 1987Villaro A.C. Sesma M.P. Vazquez J.J. Innervation of mouse lymph nodes: nerve endings on muscular vessels and reticular cells.Am. J. Anat. 1987; 179: 175-185Crossref PubMed Scopus (18) Google Scholar) and suggests that SNS innervation controls LN blood flow. To directly examine the role of sympathetic nerves in this process, we established a chemogenetic mouse model using transgenic designer receptors exclusively activated by designer drugs (DREADD) mice that express a modified form of the human M3 muscarinic receptor (R26-LSL-hM3Dq) (Zhu et al., 2016Zhu H. Aryal D.K. Olsen R.H. Urban D.J. Swearingen A. Forbes S. Roth B.L. Hochgeschwender U. Cre-dependent DREADD (designer receptors exclusively activated by designer drugs) mice.Genesis. 2016; 54: 439-446Crossref PubMed Scopus (89) Google Scholar). The mutant hM3Dq expressed in these mice has two amino acid substitutions that abolish receptor affinity for the native ligand acetylcholine but allow activation by the small pharmacologically inert molecule compound 21 (C21). We crossed R26-LSL-hM3Dq mice with TH-cre mice to generate mice (TH-cre.R26-hM3Dq), where cre-mediated expression of the hM3Dq DREADD in TH+ sympathetic nerves allowed us to activate sympathetic nerves via injection of the ligand C21 into mice during intravital imaging (Figure 4F). To determine whether sympathetic nerves were activated after C21 injection, we examined the levels of the immediate-early transcription factor c-Fos, which serves as a reporter of neuronal activity. Robust induction of c-Fos was detected in TH+ sympathetic neuron cell bodies after C21 injection (Figure 4G). Activation of sympathetic nerves resulted in rapid constriction of the microvasculature in the LN within 5 min in TH-cre.R26-hM3Dq mice but not in control R26-hM3Dq mice (Figure 4H). T cell locomotion was swiftly impaired following activation of the SNS in DREADD mice but not in control mice following C21 application (Figure 4I; Video S6). Consequently, these data demonstrate that activation of the SNS induced vasoconstriction and impaired cell motility in LNs. https://www.cell.com/cms/asset/885c91d6-ab1c-4ad9-9031-8e6516b1b78f/mmc7.mp4Loading ... Download .mp4 (4.16 MB) Help with .mp4 files Video S6. Activation of the SNS halts T cells in LNs, related to Figure 4TH-cre.R26-hM3Dq mice were adoptively transferred with naive CD8+ T cells (magenta). Blood vessels were visualized by Evans blue injection (orange) and imaged by intravital 2-photon microscopy. C21 was administered via i.v. catheter after 15 min of imaging. Frames were acquired at 10 s intervals. Display rate: 12 frames per second. Lymphocytes require oxygen for motility in ex vivo-isolated LNs when blood flow is absent (Huang et al., 2007Huang J.H. Cárdenas-Navia L.I. Caldwell C.C. Plumb T.J. Radu C.G. Rocha P.N. Wilder T. Bromberg J.S. Cronstein B.N. Sitkovsky M. et al.Requirements for T lymphocyte migration in explanted lymph nodes.J. Immunol. 2007; 178: 7747-7755Crossref PubMed Scopus (110) Google Scholar). Yet it is less clear how T cells respond to rapid local changes in oxygen tension in vivo. To examine if the effect of AR stimulation on blood flow affected LN oxygenation, mice were injected with pimonidazole, a probe that binds to cells under hypoxic conditions. Iso administration induced cellular hypoxia in T cells, B cells, and macrophages in the LNs (Figures 5A, 5B, and S3C), and this was sustained for >30 min, consistent with the time frame of leukocyte immobility (Figure 2A). The αAR agonist PE induced a similar hypoxia in LNs (Figure 5C). Although hypoxia induced by Iso had dissipated by 4 h, salmeterol induced hypoxia in LNs that was sustained for >4 h (Figure 5D), consistent with the time frame of leukocyte motility observed with this long-acting β2AR agonist (Figure 2C). Hypoxia can induce an increase in intracellular Ca2+ in endothelial cells and contribute to endothelial cell activation (Aley et al., 2005Aley P.K. Porter K.E. Boyle J.P. Kemp P.J. Peers C. Hypoxic modulation of Ca2+ signaling in human venous endothelial cells. Multiple roles for reactive oxygen species.J. Biol. Chem. 2005; 280: 13349-13354Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar; Michiels et al., 2000Michiels C. Arnould T. Remacle J. Endothelial cell responses to hypoxia: initiation of a cascade of cellular interactions.Biochim. Biophys. Acta. 2000; 1497: 1-10Crossref PubMed Scopus (211) Google Scholar). Conversely, it is not known if leukocytes alter Ca2+ levels during hypoxia. To determine if SNS signals induce Ca2+ signaling in LNs and distinguish which cells were involved, we visualized real-time changes in intracellular Ca2+ in response to SNS signals in the LNs of mice expressing the Ca2+ sensor GCaMP6s (Moseman et al., 2016Moseman E.A. Wu T. de la Torre J.C. Schwartzberg P.L. McGavern D.B. Type I interferon suppresses virus-specific B cell responses by modulating CD8+ T cell differentiation.Sci. Immunol. 2016; 1: eaah3565PubMed Google Scholar). In the absence of exogenous stimuli, some leukocytes demonstrated rapid Ca2+ “flashes” within the T cell zones in LNs of GCaMP6s mice (Video S7). Administration of NA or Iso induced a rapid and widespread wave of Ca2+ signaling in leukocytes, beginning within 1–2 min of treatment (Figures 5E and 5F; Figures S3D and S3E; Video S7). This increase in intracellular Ca2+ coincided with loss of motility by T cells (Figures 5F and S3E). Increased intracellular Ca2+ affects actinomyosin-driven motility downstream of T cell receptor (TCR) signaling (Negulescu et al., 1996Negulescu P.A. Krasieva T.B. Khan A. Kerschbaum H.H. Cahalan M.D. Polarity of T cell shape, motility, and sensitivity to antigen.Immunity. 1996; 4: 421-430Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar), while our findings suggest that Ca2+ signaling is also activated by oxygen sensing to control motility. Notably, Iso had no effect on Ca2+ signaling in T cells in explanted LNs that were perfused with oxygenated media (Figure S3F), whereas the Ca2+ ionophore ionomycin induced a rapid increase in intracellular Ca2+ concentration (Figure S3F) and T cell arrest (Figure 4E). To directly assess the role of hypoxia, we superfused the LNs with oxygenated media to circumvent local hypoxia during intravital imaging. Oxygen supplementation prevented the cessation of T cell locomotion and the wave of Ca2+ signaling in leukocytes following NA administration (Figure 5G; Video S8). Thus, SNS signals reduced oxygen delivery to the tissue. This resulted in Ca2+ signaling in leukocytes that impaired cell locomotion. https://www.cell.com/cms/asset/e81754ee-9c74-4ac4-bf24-e0f679ad1621/mmc8.mp4Loading ... Download .mp4 (15.21 MB) Help with .mp4 files Video S7. SNS neurotransmitters induce a Ca2+ wave in LNs, related to Figure 5GCaMP6s mice were adoptively transferred with naive CD8+ T cells (magenta). Blood vessels were visualized by Evans blue injection (orange) and imaged by intravital 2-photon microscopy. NA was administered via i.v. catheter after 15 min of imaging. Two representative examples of the response are shown. Frames were acquired at 10 s intervals. Display rate: 12 frames per second. https://www.cell.com/cms/asset/f32d70b9-5801-40dd-bfd4-98701143974b/mmc9.mp4Loading ... Download .mp4 (11.47 MB) Help with .mp4 files Video S8. Hypoxia induces a Ca2+ wave in LNs that is prevented by oxygen supplementation, related to Figure 5GCaMP6s mice were adoptively transferred with naive CD8+ T cells (magenta). Blood vessels were visualized by Evans blue injection (orange) and imaged by intravital 2-photon microscopy. The LN was superfused with oxygenated media throughout imaging. NA was administered via i.v. catheter after 15 min of imaging. Frames were acquired at 10 s intervals. Display rate: 12 frames per second. We hypothesized that loss of movement would affect the immunosurveillance functions of leukocytes and be detrimental to the induction of protective immunity. To examine this, we used a well-established model of virus infection with herpes simplex virus type 1 (HSV) (Hor et al., 2015Hor J.L. Whitney P.G. Zaid A. Brooks A.G. Heath W.R. Mueller S.N. Spatiotemporally distinct interactions with dendritic cell subsets facilitates CD4+ and CD8+ T cell activation to localized viral infection.Immunity. 2015; 43: 554-565Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Mice with fluorescently labeled XCR1+ DCs (XCR1venus/+) were transferred with HSV glycoprotein B (gB)-specific gBT-I TCR transgenic CD8+ T cells, TRITC was applied to the skin to label skin DCs, and mice were infected with HSV. As we previously described (Hor et al., 2015Hor J.L. Whitney P.G. Zaid A. Brooks A.G. Heath W.R. Mueller S.N. Spatiotemporally distinct interactions wit