Melanie P. Matheu

Imaging of Effector Memory T Cells during a Delayed-Type Hypersensitivity Reaction and Suppression by Kv1.3 Channel Block

Effector memory T (Tem) cells are essential mediators of autoimmune disease and delayed-type hypersensitivity (DTH), a convenient model for two-photon imaging of Tem cell participation in an inflammatory response. Shortly (3 hr) after entry into antigen-primed ear tissue, Tem cells stably attached to antigen-bearing antigen-presenting cells (APCs). After 24 hr, enlarged Tem cells were highly motile along collagen fibers and continued to migrate rapidly for 18 hr. Tem cells rely on voltage-gated Kv1.3 potassium channels to regulate calcium signaling. ShK-186, a specific Kv1.3 blocker, inhibited DTH and suppressed Tem cell enlargement and motility in inflamed tissue but had no effect on homing to or motility in lymph nodes of naive and central memory T (Tcm) cells. ShK-186 effectively treated disease in a rat model of multiple sclerosis. These results demonstrate a requirement for Kv1.3 channels in Tem cells during an inflammatory immune response in peripheral tissues. Targeting Kv1.3 allows for effector memory responses to be suppressed while central memory responses remain intact.

Imaging regulatory T cell dynamics and CTLA4-mediated suppression of T cell priming

Foxp3(+) regulatory T cells (Tregs) maintain immune homoeostasis through mechanisms that remain incompletely defined. Here by two-photon (2P) imaging, we examine the cellular dynamics of endogenous Tregs. Tregs are identified as two non-overlapping populations in the T-zone and follicular regions of the lymph node (LN). In the T-zone, Tregs migrate more rapidly than conventional T cells (Tconv), extend longer processes and interact with resident dendritic cells (DC) and Tconv. Tregs intercept immigrant DCs and interact with antigen-induced DC:Tconv clusters, while continuing to form contacts with activated Tconv. During antigen-specific responses, blocking CTLA4-B7 interactions reduces Treg-Tconv interaction times, increases the volume of DC:Tconv clusters and enhances subsequent Tconv proliferation in vivo. Our results demonstrate a role for altered cellular choreography of Tregs through CTLA4-based interactions to limit T-cell priming.Foxp3+ regulatory T cells (Tregs) maintain immune homeostasis through mechanisms that remain incompletely defined. Here, by two-photon imaging, we examine the cellular dynamics of endogenous Tregs. Tregs are identified as two non-overlapping populations in the T-zone and follicular regions of the lymph node. In the T-zone, Tregs migrate more rapidly than conventional T cells (Tconv), extend longer processes, and interact with resident dendritic cells (DC) and Tconv. Tregs intercept immigrant DCs and interact with antigen-induced DC:Tconv clusters, while continuing to form contacts with activated Tconv. During antigen-specific responses, blocking CTLA4-B7 interactions reduces Treg-Tconv interaction times, increases the volume of DC:Tconv clusters, and enhances subsequent Tconv proliferation in vivo. Our results demonstrate a role for altered cellular choreography of Tregs through CTLA4-based interactions to limit T cell priming.

Class IA Phosphoinositide 3-Kinase Modulates Basal Lymphocyte Motility in the Lymph Node

Recruitment of PI3K to the cell membrane is an indispensable step in normal lymphocyte proliferation and activation. In this study we identify PI3K as an important signaling molecule for maintaining basal T and B lymphocyte motility and homing in the intact lymph node. Pharmacological inhibition of PI3K catalytic isoforms exerted broad effects on basal lymphocyte motility, including changes in homing kinetics, localization of B cells within the lymph node, and reduced cell velocities. Lymphocytes deficient in either or both of the class IA PI3K regulatory subunits p85α and p85β also exhibited reduced velocities, with the magnitude of reduction depending upon both cell type and isoform specificity. B cells deficient in p85α exhibited gross morphological abnormalities that were not evident in cells treated with a PI3K inhibitor. Our results show, for the first time, that class IA PI3Ks play an important role in regulating basal lymphocyte motility and that p85α regulatory subunit expression is required to maintain B cell morphology in a manner independent of PI3K catalytic function. Moreover, we demonstrate distinct roles for catalytic domain function and class IA PI3K regulatory domain activity in lymphocyte motility, homing, and homeostatic localization of mature resting B cells.

Generation of bone marrow derived murine dendritic cells for use in 2-photon imaging.

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and <85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Three Phases of CD8 T Cell Response in the Lung Following H1N1 Influenza Infection and Sphingosine 1 Phosphate Agonist Therapy

Influenza-induced lung edema and inflammation are exacerbated by a positive feedback loop of cytokine and chemokine production termed a ‘cytokine storm’, a hallmark of increased influenza-related morbidity and mortality. Upon infection, an immune response is rapidly initiated in the lungs and draining lymph node, leading to expansion of virus-specific effector cells. Using two-photon microscopy, we imaged the dynamics of dendritic cells (DC) and virus-specific eGFP+CD8+ T cells in the lungs and draining mediastinal lymph nodes during the first two weeks following influenza infection. Three distinct phases of T cell and CD11c+ DC behavior were revealed: 1) Priming, facilitated by the arrival of lung DCs in the lymph node and characterized by antigen recognition and expansion of antigen-specific CD8+ T cells; asymmetric T cell division in contact with DCs was frequently observed. 2) Clearance, during which DCs re-populate the lung and T cells leave the draining lymph node and re-enter the lung tissue where enlarged, motile T cells come into contact with DCs and form long-lived interactions. 3) Maintenance, characterized by T-cell scanning of the lung tissue and dissociation from local antigen presenting cells; the T cells spend less time in association with DCs and migrate rapidly on collagen. A single dose of a sphingosine-1-phosphate receptor agonist, AAL-R, sufficient to suppress influenza-induced cytokine-storm, altered T cell and DC behavior during influenza clearance, delaying T cell division, cellular infiltration in the lung, and suppressing T-DC interactions in the lung. Our results provide a detailed description of T cell and DC choreography and dynamics in the lymph node and the lung during influenza infection. In addition, we suggest that phase lags in T cell and DC dynamics induced by targeting S1P receptors in vivo may attenuate the intensity of the immune response and can be manipulated for therapeutic benefit.

Two-photon imaging of remyelination of spinal cord axons by engrafted neural precursor cells in a viral model of multiple sclerosis

Significance Stem cell transplantation has emerged as a promising cell-based therapy for the treatment of demyelinating diseases such as multiple sclerosis (MS). This study provides the first real-time imaging of transplanted stem cell-mediated remyelination in a mouse model of MS. Whereas current treatments solely delay disease progression, transplanted stem cells actively reverse clinical disease in animal models. Using two-photon microscopy and viral-induced demyelination, we describe a technique to visualize cellular migration and remyelination in the mouse spinal cord. Transplanted neural precursor cells physically wrap damaged axons with newly formed myelin, preserving axonal health. Neural precursor cells (NPCs) offer a promising approach for treating demyelinating diseases. However, the cellular dynamics that underlie transplanted NPC-mediated remyelination have not been described. Using two-photon imaging of a newly developed ventral spinal cord preparation and a viral model of demyelination, we describe the motility and intercellular interactions of transplanted mouse NPCs expressing green fluorescent protein (GFP) with damaged axons expressing yellow fluorescent protein (YFP). Our findings reveal focal axonal degeneration that occurs in the ventral side of the spinal cord within 1 wk following intracranial instillation with the neurotropic JHM strain of mouse hepatitis virus (JHMV). Axonal damage precedes extensive demyelination and is characterized by swelling along the length of the axon, loss of YFP signal, and transected appearance. NPCs engrafted into spinal cords of JHMV-infected mice exhibited diminished migration velocities and increased proliferation compared with transplanted cells in noninfected mice. NPCs preferentially accumulated within areas of axonal damage, initiated direct contact with axons, and subsequently expressed the myelin proteolipid protein gene, initiating remyelination. These findings indicate that NPCs transplanted into an inflammatory demyelinating microenvironment participate directly in therapeutic outcome through the wrapping of myelin around damaged neurons.

Dissection and 2-photon imaging of peripheral lymph nodes in mice

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response 1. Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 2. Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37°C.

Toll-like receptor 4-activated B cells out-compete Toll-like receptor 9-activated B cells to establish peripheral immunological tolerance

B-cell–induced peripheral T-cell tolerance is characterized by suppression of T-cell proliferation and T-cell–dependent antibody production. However, the cellular interactions that underlie tolerance induction have not been identified. Using two-photon microscopy of lymph nodes we show that tolerogenic LPS-activated membrane-bound ovalbumin (mOVA) B cells (LPS B cells) establish long-lived, highly motile conjugate pairs with responding antigen-specific OTII T cells but not with antigen-irrelevant T cells. Treatment with anti–CTLA-4 disrupts persistent B-cell–T-cell (B–T) contacts and suppresses antigen-specific tolerance. Nontolerogenic CpG-activated mOVA B cells (CpG B cells) also form prolonged, motile conjugates with responding OTII T cells when transferred separately. However, when both tolerogenic and nontolerogenic B-cell populations are present, LPS B cells suppress long-lived CpG B–OTII T-cell interactions and exhibit tolerogenic dominance. Contact of LPS B cells with previously established B–T pairs resulted in partner-swapping events in which LPS B cells preferentially migrate toward and disrupt nontolerogenic CpG mOVA B-cell–OTII T-cell pairs. Our results demonstrate that establishment of peripheral T-cell tolerance involves physical engagement of B cells with the responding T-cell population, acting in a directed and competitive manner to alter the functional outcome of B–T interactions.

Isolation of CD4+ T cells from mouse lymph nodes using Miltenyi MACS purification.

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are > or = 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Lymphocyte cell motility: the twisting, turning tale of phosphoinositide 3-kinase.

The PI3K (phosphoinositide 3-kinase) family of lipid kinases regulate cell motility in diverse organisms and cell types. In mammals, the main PI3K enzyme activated by chemokine receptor signalling is the class IB isoform, p110gamma. Studies of p110gamma-knockout mice have shown an essential function for this isoform in chemotaxis of neutrophils and macrophages both in vitro and in vivo. However, the roles of p110gamma and other PI3K enzymes and regulatory subunits in lymphocyte motility have been more difficult to discern. Recent studies of adoptively transferred, fluorescently labelled lymphocytes have revealed complex and unexpected functions for PI3K in lymphocyte migration in vivo. In this review we highlight cell-type-specific roles for PI3K catalytic and regulatory subunits in the homing and basal motility of lymphocytes in the intact lymph node.