Michael D. Cahalan - US grants

In the immune response the activation of lymphocytes by specific antigens or mitogens begins with membrane events and culminates in cell division. The intracellular signals from surface receptors to the nucleus remain unclear, but may involve ionic channels, integral membrane proteins that gate the flow of ions across the membrane. Using the gigaohm seal patch recording technique on individual T lymphocytes, in parallel with biochemical and immunological studies, the functional requirement for ionic channels in mitogenesis will be assessed. The patch recording technique allows ionic channels in small cells, such as lymphocytes, to be studied with resolution to the single channel level. Expression of ionic channels in resting and activated human T-cells and a clonal T-cell line will be determined through whole cell and isolated patch recording. Protocols will be used to test for the existence of potassium channels, calcium-activated channels, calcium channels, and sodium channels. We will study the modulation of channel properties by mitogenic and non-mitogenic lectins, and by a mitogenic clonospecific antibody against the T-cell receptor. The dependence of mitogen-stimulated cellular events on functioning potassium channels will be examined in detail. The effect of potassium channel blockers on mitogen-stimulated changes in membrane potential, intracellular calcium concentration, and on capping of cell surface receptors will be explored. The surface distribution and gating properties of potassium channels will be determined in capped lymphocytes. Potassium channel blockers will be tested for effects on phospholipid turnover, various aspects of protein synthesis, interleukin-2 production, and the expression of transferrin receptor and HLA-DR antigen on T-cells. Time windowing experiments will determine periods during which T cell activation is most sensitive to potassium channel block. Other known modulators of ionic channels will be tested for effects on thymidine incorporation. Interleukins, suppressor factors, and cyclosporin A will be tested for effects on ionic channel function. Single cell recording in parallel with biochemical experiments will probe the mechanism of clinically important modulators of the immune response, as well as providing clues regarding the role of ionic channels in the transformation of a resting cell to a proliferating state.

chemical structure function; membrane permeability; neurotoxins; neural transmission; anesthetics; myofibrils; muscle contraction; heart rate; antiarrhythmic agent; secretion; T lymphocyte; cell membrane; hormone biosynthesis; membrane structure; antispasmodic agents; synapses; clone cells;

In the immune response the activation of lymphocytes by specific antigens or mitogens begins with membrane events and culminates in cell division. The intracellular signals from surface receptors to the nucleus remain unclear, but may involve ionic channels, integral membrane proteins that gate the flow of ions across the membrane. Using the gigaohm seal patch recording technique on individual T lymphocytes, in parallel with biochemical and immunological studies, the functional requirement for ionic channels in mitogenesis will be assessed. The patch recording technique allows ionic channels in small cells, such as lymphocytes, to be studied with resolution to the single channel level. Expression of ionic channels in resting and activated human T-cells and a clonal T-cell line will be determined through whole cell and isolated patch recording. Protocols will be used to test for the existence of potassium channels, calcium-activated channels, calcium channels, and sodium channels. We will study the modulation of channel properties by mitogenic and non-mitogenic lectins, and by a mitogenic clonospecific antibody against the T-cell receptor. The dependence of mitogen-stimulated cellular events on functioning potassium channels will be examined in detail. The effect of potassium channel blockers on mitogen-stimulated changes in membrane potential, intracellular calcium concentration, and on capping of cell surface receptors will be explored. The surface distribution and gating properties of potassium channels will be determined in capped lymphocytes. Potassium channel blockers will be tested for effects on phospholipid turnover, various aspects of protein synthesis, interleukin-2 production, and the expression of transferrin receptor and HLA-DR antigen on T-cells. Time windowing experiments will determine periods during which T cell activation is most sensitive to potassium channel block. Other known modulators of ionic channels will be tested for effects on thymidine incorporation. Interleukins, suppressor factors, and cyclosporin A will be tested for effects on ionic channel function. Single cell recording in parallel with biochemical experiments will probe the mechanism of clinically important modulators of the immune response, as well as providing clues regarding the role of ionic channels in the transformation of a resting cell to a proliferating state.

microscopy; biomedical equipment resource; biomedical equipment purchase;

biomedical equipment purchase;

In the immune response the activation of lymphocytes by specific antigens or mitogens begins with membrane events and culminates in cell division. The intracellular signals from surface receptors to the nucleus remain unclear, but may involve ionic channels, integral membrane proteins that gate the flow of ions across the membrane. Using the gigaohm seal patch recording technique on individual T lymphocytes, in parallel with biochemical and immunological studies, the functional requirement for ionic channels in mitogenesis will be assessed. The patch recording technique allows ionic channels in small cells, such as lymphocytes, to be studied with resolution to the single channel level. Expression of ionic channels in resting and activated human T-cells and a clonal T-cell line will be determined through whole cell and isolated patch recording. Protocols will be used to test for the existence of potassium channels, calcium-activated channels, calcium channels, and sodium channels. We will study the modulation of channel properties by mitogenic and non-mitogenic lectins, and by a mitogenic clonospecific antibody against the T-cell receptor. The dependence of mitogen-stimulated cellular events on functioning potassium channels will be examined in detail. The effect of potassium channel blockers on mitogen-stimulated changes in membrane potential, intracellular calcium concentration, and on capping of cell surface receptors will be explored. The surface distribution and gating properties of potassium channels will be determined in capped lymphocytes. Potassium channel blockers will be tested for effects on phospholipid turnover, various aspects of protein synthesis, interleukin-2 production, and the expression of transferrin receptor and HLA-DR antigen on T-cells. Time windowing experiments will determine periods during which T cell activation is most sensitive to potassium channel block. Other known modulators of ionic channels will be tested for effects on thymidine incorporation. Interleukins, suppressor factors, and cyclosporin A will be tested for effects on ionic channel function. Single cell recording in parallel with biochemical experiments will probe the mechanism of clinically important modulators of the immune response, as well as providing clues regarding the role of ionic channels in the transformation of a resting cell to a proliferating state.

DESCRIPTION: In addition to regulating electrical activity in the nervous system, ion channels mediate very different cellular mechanisms within the immune system. The earliest events signaling the binding of antigen to surface receptors in T lymphocytes and mast cells include the activation of ion channels and a rise in cytosolic [Ca2+]i. The "[Ca2+]i signal" is generated by the opening of ion channels, and links membrane receptors to gene expression, lymphokine secretion and cell proliferation essential to the immune response. With emphasis on a single-cell approach, the investigators are proposing video-imaging and patch-clamp experiments to clarify mechanisms which link membrane receptors to effector functions such as cell proliferation, gene expression, cell motility, and secretion of immunomodulatory molecules. The proposed experiments on T lymphocytes are divided into two sections corresponding to the sequence of events following antigen stimulation: (1) intracellular signaling from the engagement of membrane receptors to the [Ca2+]i signal; (2) activation of the interleukin-2 gene and other signaling pathways by the rise in [Ca2+]i. One goal of this project is to apply optical techniques to investigate [Ca2+]i signaling, motility, and gene expression in primary T-cells stimulated by physiological ligands. The investigators will use reporter genes to visualize T-cell activation dynamically in order to relate membrane and second-messenger mechanisms to effector function. They are investigating interactions between cells during antigen presentation, as well as intracellular signaling mechanisms that link membrane receptors with gene expression.

Improvements in optical techniques-laser applications, video and confocal microscopy, cell sorting and analysis and the development of fluorescent probes-are revolutionizing cell biology and are now in the process of being translated into the practical of clinical medicine. The primary objective of the Optical Biology Shared Resource is to support all Basic Science and Clinical Research projects that can benefit from access to instrumentation and expertise in optical methods. Optical techniques are broadly classified in terms of diagnostics and therapeutics, and relevant biological systems range from sub-cellular structures to tissues. In order to utilize the unique core technologies effectively, the Shared Resource has been divided into two fundamental components. The first component embodies the service element of our shared facilities and incorporates the substantial number of existing optical methods/instruments available on campus. An array of instrumentation including imaging methods such as confocal and two-photon microscopy and cytometry and sorting are broadly used by members of the Cancer Center on a wide range of projects. Technical instruction, service and support are provided by three Shared Resource facilities and personnel. The Shared Resource Director and co-Coordinators consult on initial experimental design and matching investigators with the proper facility and collaborator. Since a primary driving force behind optical biology is the development of new technology, the second component of the Shared Resource encourages development and utilization of novel optical methods in the basic science and clinical science programs.

With emphasis on a single-cell approach, we are proposing experiments on antigen-specific T lymphocytes using a combination of optical techniques - confocal microscopy, two-photon microscopy, and optical tweezers - to investigate the dynamics of membrane receptor interactions during antigen presentation. Our laboratory is using biophysical techniques to investigate signaling mechanisms, biochemical and optical assays to monitor cell activation, and genetically modified cells to probe molecular mechanisms. The proposed experiments on T lymphocytes and antigen-presenting cells will focus on the dynamics of signaling from the engagement of membrane receptors to the [Ca2+]i signal. One goal of this project is to apply optical techniques to investigate antigen recognition, [Ca2+]i signaling, motility, and gene expression in T cells stimulated by physiological ligands. We will use reporter genes to visualize T-cell activation dynamically in order to relate membrane and se cond-messenger mechanisms to effector function. We are investigating interactions between cells during antigen presentation, as well as intracellular signaling mechanisms that link membrane receptors with gene expression.

Acquisition of a Fluorescence Activated Cell Sorter (FACS)
Abstract - 9977224

One of the most powerful approaches in biology is reductionism, the process of break-ing down complex systems into their component parts, the better to understand their interactions in the whole. A FACS machine allows for the enrichment of specific sub-populations of cells from large, diverse starting populations based on their expression of specific "markers". These surface markers are detected by binding of fluorescently-labeled specific antibodies that glow when exposed to the UV laser in the FACS ma-chine.

A diverse population of researchers at UCI has an equally diverse range of interests. These interests can be grouped into four broad areas. These represent just some of the many research and teaching projects on campus that will benefit from a FACS machine.

1. Isolation of white blood cell (WBC) subsets. Experiments are proposed that require isolating specific subpopulations of WBC to look at their role in protecting indi-viduals from infection by the parasite T. Cruzi. Infection by this organism is a major cause of heart disease in South America. Other investigators will isolate subpoulations of WBC to look at their role in diseases such as Multiple Sclerosis.

2. Isolation of cells in distinct stages of the cell cycle. Experiments will be per-formed to test the effects of specific growth factors on regulating cell division. Regula-tion of cell growth is important both in development and in cancer, which is an uncon-trolled growth of tumor cells. A FACS machine is particularly well-suited to perform these kinds of experiments.

3. Isolation of minority cell populations. In many cases, both within science and without, the rarest examples are often the most intriguing. Several investigators are in-terested in collecting rare, but important, cells from the body for analysis. Some of these cells help protect us against certain diseases, others may carry a disease. The FACS machine is very good at finding that one-in-a-thousand cell that is of interest.

4. Enrichment of transfected cells. Transfected cells are cells that have been genetically altered in the laboratory so that they express new genes. One day it is hoped that people may be helped by giving them back such altered cells to compensate for a genetic abnormality. In the meantime the FACS machine is used in the lab to en-rich for these cells so that they can be analyzed in greater detail

In summary, a myriad of research and teaching projects at UCI will be facilitated and greatly enhanced by the acquisition of a state-of-the-art FACS machine. Unlike "all the king's horses and all the king's men" we believe we can put the pieces back together again, and in so doing gain a greater understanding of complex biological systems.

Contact with antigen-presenting cells initiates an activation cascade within T lymphocytes, including a rise in cytosolic calcium, lymphokine production, and cell division. Calcium imaging combined with an optical trap enabled the T-cell contact requirements and polarity to be investigated at the single-cell level. Anti-CD3 mAb-coated beads induced calcium signaling with ~ ten-fold higher frequency upon contact with the leading edge of the T cell, compared with the trailing edge. Engagement of at least 340 T cell receptors (~1% of the total on the cell) was required to initiate Ca2+ signaling, and the minimal contact area was ~3 (m2. Our results indicate that ~1000 TCRs are required to generate a long-lasting [Ca2+]i signal which might be required for gene expression. We expect this approach can be combined with ligands for accessory molecules or with downstream assays for gene expression in order to examine species and clonal-specific variations in the T cell resp onse and deepen our understanding of the relationship between early activation events and gene expression.

This project focuses on molecular properties and regulation of ion channels in T lymphocytes, taking advantage of parallel advances in electrophysiology, molecular biology and video imaging techniques. Our goal is to understand the role of ion channels in the immune response. Using patch-clamp techniques, we have characterized a diverse set of functionally significant ion channels that are differentially expressed depending on the developmental and activation state. Through the proposed experiments, we plan to continue our studies of three main channel types. A voltage-gated K+ channel, Kv1.3, is functionally important in resting T cells. Using site-directed mutants, we will map the channel's inner vestibule with tethered blockers and characterize the action of progesterone. Ca2+- activated K+ channels, encoded by IKCa1 in human T cells and SKCa2 in Jurkat T cells, regulate membrane potentials during [Ca2+]i signaling. IKCa1 is up-regulated in activated T cells and is required for sustained proliferation. We will probe the mechanism of Ca2+ sensing by pre-bound calmodulin. Dominant- negative constructs will be developed to suppress channel expression. Calcium signaling and gene expression depend crucially on Ca2+ release-activated Ca2+ (CRAC) channels that open when intracellular Ca2+ stores are depleted. We will investigate the activation mechanism of this channel, with single-channel resolution, investigate block by polyamines, and use a dominant-negative strategy to test for candidate genes. Finally, using highly specific and potent blockers developed during the previous grant period, we will test for functional roles of K+ channels in [Ca2+]i signaling, cytokine release, cell proliferation, and chemotaxis. Through the proposed experiments we hope to define mechanisms that regulate ion channels and corresponding cell functions that underlie the immune response.

DESCRIPTION (provided by applicant):Our laboratory has pioneered the use of patch-clamp and optical techniques to investigate the role of ion channels and (Ca) signaling in T lymphocytes. T cells initiate the immune response to foreign antigens following direct contact with primed antigen presenting cells (APC) such as dendritic cells. T cell receptor (TCR) engagement results in a cytosolic Ca (Ca) signal that leads to Ca dependent gene expression, cytokine secretion, proliferation, and differentiation. Our proposed experimental approach using live-cell imaging will parallel this sequence of events. Two-photon microscopy has recently enable visualization of T and B cells deep within intact lymph node, and has revealed a dynamic pattern of motility and cell-cell interaction, offering a unique opportunity for in vivo imaging of immune system function. Thus, our approach will aim to consolidate results from in vitro and in vivo experiments performed on antigen-specific primary T cells from transgenic mice and T cell lines. A variety of introduced and genetically encoded fluorescent reporters will monitor the TCR directly, (Ca), gene expression, and secretory responses in T cells before and after stimulation. A further goal of this project is to compare the functional responses of quiescent, activated, and chronically activated auto reactive T cells in light of their differing ion channel phenotypes.

This project focuses on molecular properties, regulation, and functional roles of ion channels in T lymphocytes. Using patch-clamp techniques, we have characterized a diverse set of functionally significant Ca2+, K+, and CI" ion channels in human and mouse T cells. These channels are differentially expressed depending on the developmental and activation state and have been shown to contribute to T-cell receptor signaling leading to gene expression, secretion of lymphokines, and cell proliferation. Ion channels in the immune system offer promising targets for development of therapeutic agents for immunomodulation, based upon specific channel blockade. Using single cell patch-clamp and [Ca2+]j imaging techniques, together with molecular and biochemical approaches, this grant renewal application will focus on the two types of Ca2+ channel found in T lymphocytes. Ca2+ release-activated Ca2+ (CRAG) channels are opened upon depletion of intracellular Ca2+ stores and are crucial for sustained [Ca2+]i signaling that leads to gene expression in T cells. We recently discovered Stim and STIM1 as essential and conserved components of CRAG channel function in Drosophila and human cells. In Specific Aim 1, we will use RNAi to screen for additional components of the CRAG channel, define the mechanism of STIM1 in CRAG channel activation, and investigate the role of STIM1 in functional T cell responses. It is now clear that the native MIC current in T cells represents tetramers of TRPM7, a protein with functional channel and kinase domains. MIC channel expression is up-regulated during the activation of T cells to proliferate. In Specific Aim 2 we will identify the molecular basis for MIC channel gating, define mechanisms for ion permeation and block, and determine the functional role of MIC channels in T cell activation. Through the proposed experiments in this renewal application, we seek a clearer understanding how CRAG and MIC channels function as ion channels and how they regulate Ca2+ influx in T cells and corresponding cell functions that underlie the immune response.

[unreadable] DESCRIPTION (provided by applicant): Although many molecular details of the signaling pathways in lymphocytes have been elucidated with exquisite detail, less is known about the equally important aspects of how immune cells migrate, locate one another and transmit signals within their native tissues. There is thus a pressing need for techniques allowing real-time observation of single cells and molecules in lymphoid organs. We have pioneered the use of two- photon microscopy to track motility and cell/cell interactions of dendritic cells, T cells and B cells in isolated lymph nodes and in living mice. This proposal for functional imaging has three inter-related Aims. First, we will examine basic motility properties of temperature dependence, effects of tissue hypoxia and regional variations in the lymph node under basal conditions and at the start of an immune response. The control of lymphocyte trafficking by S1P1 receptors will be probed using specific pharmacological tools and by directly imaging the egress step to determine mechanisms of lymphocyte sequestration. We introduce quantum dots as a novel approach to track and modulate dendritic cells. Second, we will focus on immunoregulatory and effector interactions among cells in the lymph node. We introduce regulatory T (Treg) cells and natural killer (NK) cells as new and relevant cells for immuno-imaging, and extend our approach to include human immune cells reconstituted or transferred into mice. Third, we propose to use [Ca2+]i imaging, K+ channel and PI 3-kinase knockout mice, and specific pharmacological tools to investigate molecular signaling pathways that regulate motility and antigen responses in the lymph node. We anticipate that the advances in technology development and new information gained through this project will provide the means to investigate autoimmune diseases and further the mechanistic understanding of clinically used immunosuppressive agents. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Although many molecular details of the signaling pathways in lymphocytes have been elucidated with exquisite detail, less is known about the equally important aspects of how immune cells migrate, locate one another and transmit signals within their native tissues. There is thus a pressing need for techniques allowing real-time observation of single cells and molecules in lymphoid organs. We have pioneered the use of two- photon microscopy to track motility and cell/cell interactions of dendritic cells, T cells and B cells in isolated lymph nodes and in living mice. This proposal for functional imaging has three inter-related Aims. First, we will examine basic motility properties of temperature dependence, effects of tissue hypoxia and regional variations in the lymph node under basal conditions and at the start of an immune response. The control of lymphocyte trafficking by S1P1 receptors will be probed using specific pharmacological tools and by directly imaging the egress step to determine mechanisms of lymphocyte sequestration. We introduce quantum dots as a novel approach to track and modulate dendritic cells. Second, we will focus on immunoregulatory and effector interactions among cells in the lymph node. We introduce regulatory T (Treg) cells and natural killer (NK) cells as new and relevant cells for immuno-imaging, and extend our approach to include human immune cells reconstituted or transferred into mice. Third, we propose to use [Ca2+]i imaging, K+ channel and PI 3-kinase knockout mice, and specific pharmacological tools to investigate molecular signaling pathways that regulate motility and antigen responses in the lymph node. We anticipate that the advances in technology development and new information gained through this project will provide the means to investigate autoimmune diseases and further the mechanistic understanding of clinically used immunosuppressive agents. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): To mount an immune response, T lymphocytes must home from blood into lymph nodes, recognize specific antigens by direct contact with dendritic cells, proliferate, differentiate, exit the lymph node, and migrate to other lymph nodes as memory cells or to tissues as effectors. These processes involve motility, cell recognition dynamics, and Ca2+ signaling - events that were hidden from view until fairly recently. Two-photon (2P) microscopy now permits real-time visualization of living cells deep within lymphoid organs, revealing an elegant cellular choreography under basal conditions and during an immune response. Ion channels in T lymphocytes regulate the triggering, intensity and duration of Ca2+ signaling leading to downstream changes in gene expression and cell proliferation. In particular, a Ca2+ channel (Orai1) and a K+ channel (Kv1.3) are being developed as immunosuppressive targets for treatment of autoimmune disorders. The overall goals of this project are to investigate how the 'default' antigen search strategy of naive T cells is optimized, how tolerogenic B cells and regulatory T cells (Tregs) interfere with naive T cell activation, and how ion channels in T cells can be targeted for in vivo immunosuppression. Studies will be based on 2P imaging of human and murine immune cells in experimental models of the immune response. In Aim 1, new techniques for long-term cell tracking in intact tissues will be developed, and applied in 3 further subaims: (i) To explore whether T cells are attracted locally and dynamically toward DCs; (ii) Characterization of cellular interactions in a model of B cell-delivered gene therapy by tolerance induction, and (iii) Regulatory T cell suppression of naive T cell activation. Aim 2 focuses on the in vivo action of Kv1.3 and Orai1, with three subaims: (i) Development of two novel preparations for human cell immunoimaging. (ii) Imaging of effector T cells that cause inflammation and tissue damage in autoimmune disorders during treatment with a specific blocker of Kv1.3 channels in both human and murine models. (iii) Exploration of the functional roles of the Orai1 channel in vivo by specific inhibition of Ca2+ channel activity. Collectively, the proposed experiments probe the molecular and cellular mechanisms that modulate immune responses, and which represent potential therapeutic targets for improved treatment of inflammatory and autoimmune diseases. PUBLIC HEALTH RELEVANCE: Our studies using two-photon microscopy reveal the dynamics of immune cells in vivo during an immune response. The first goal in this project is to understand how cellular interactions in the lymph node are modified by B cell-delivered therapy to induce immunological tolerance and by regulatory T cells that prevent autoimmunity. The second goal is to investigate immunosuppression mediated by blockade of specific ion channels in T lymphocytes - a potassium channel Kv1.3 and a calcium channel Orai1 - for treatment of autoimmune disorders.

DESCRIPTION (provided by applicant): In the present application, we seek renewed support for the Immunology Research Training Program at the University of California, Irvine (UCI). Made possible through the initial award, the objective of the Immunology Research Training Program is to train scientists to study the immune system and its role in autoimmunity, infection and cancer. These research areas are addressed by the 30 faculty of the UCI Institute for Immunology (IFI), which, under the leadership of Dr. Paolo Casali, will provide the administrative and academic core for this training program. The IFI faculty will capitalize on the strengths of the existing integrated/umbrella Cellular and Molecular Biosciences (CMB) graduate program to articulate a vibrant training program. The strengths of the UCI Immunology Research Training Program include: 1) The highly structured training program and scientifically strong environment emanating from the IFI; 2) The leadership and experience of the Program Director/PI, Dr. Paolo Casali, founder of the IFI, and former director of the NIH T32-supported Immunology Program at Weill Cornell Medical College, NY (1999-2003); 3) the Preceptors/Mentors are established investigators in wide-ranging areas of immunology; 4) the existing pool of predoctoral trainees, recruited from top-tier institutions, and the success of the Immunology Research Training Program in its first four years of existence in recruiting and attracting individuals from diverse populations; 5) The strong training record of UCI Immunology Research Training Program in its first four years of existence, during which 9 pre-doctoral trainees and 2 postdoctoral trainees were supported and have been highly productive. For this second period of support (2010-2015), funding is sought for 6 pre-docs per year to accommodate more highly qualified predocs from our large recruitment pool. Importantly, the Graduate Division at UCI will also provide 1 additional slot upon funding of this application; thus, there is potential for 7 pre-doctoral students to be supported for immunology research. The existing strengths in immunology at UCI will be embellished through the proposed training program, and will promote the institution's long-standing mission to educate highly qualified graduate students in biomedical sciences. RELEVANCE: Immunology is at its core a discipline that holds the keys to unlock some of humankind's most deadly and debilitating ailments. The Immunology Research Training Program at UCI seeks to train and develop the next generation of outstanding immunologists, scientists who will be at the forefront of discovery and application of knowledge about the immune system to ameliorate human disease.

? DESCRIPTION (provided by applicant): In eukaryotic cells calcium ions are the primary means of controlling protein-based molecular machines. The ability to record these calcium signals using fluorescent indictors has provided an entry into molecular mechanism and a means to follow subcellular signaling dynamics. Recent advances in genetically encoded calcium indicators (GECIs) now provide the ability to detect even small, transient, and rapidly fluctuating calcium signals within individual genetically-targeted cells in vivo. Calcium signaling regulates movement patterns that govern cell-cell contact essential for lymphocyte activation; other facets include close coupling to T cell receptor activation, reliance on Orai1, calcium- dependent transcriptional activation, and suppression of calcium signaling by regulatory T cells. There is a need to simultaneously track and read out calcium concentrations using endogenous transgenic expression of GECIs to (1) uncover mechanisms of regulatory T cell suppression, (2) visualize therapeutic modulation of endogenous T cell activation in disease models (3) identify endogenous polyclonal activation events in the lymph node for the first time. Moreover, the need for these capabilities are shared by neuroscientists seeking to measure, modify, and model brain circuit function for the President's Brain Initiative, the Brain Activity Map, and similar projects. We have created a novel; ratiometric GECI we call Salsa6f by fusing the green fluorescent indicator GCaMP6f with the red fluorescent protein tdTomato and have successfully tested it in vitro. This probe permits quantitative readout of Ca2+ concentrations and Ca2+-independent tracking. This proposal seeks to (1) generate a Cre-dependent GECI transgenic mouse strain with Salsa6f through site directed targeting of the ubiquitously expressed Rosa-26 locus, and (2) cross this line to Cre recombinase driver lines and validate Salsa6f function in immune cells, neurons, and glia. After crossing to drive expression in CD4+ T cells and Vav+ B cells, purified cells will be tested for Salsa6f indicator function, expression, and lack of interference in immune cell homing, localization, motility, proliferation, surface marker expression, and cytokine production, as measured in quantitative assays. Similar crosses will drive expression in CaMKII+ neurons and GFAP+ astrocytes. Brains sections from transgenic mice will be assessed for appropriate expression and neuronal and glial morphology. Neurons and glia will be cultured and the speed and sensitivity of Salsa6f measured upon field stimulation and during spontaneous activity. The new capabilities of Salsa6f transgenic mice will enable new approaches to identify, map, and relate the intricate molecular mechanisms and cell behaviors that collectively define immune and nervous system function.

PROJECT SUMMARY/ABSTRACT Multiple sclerosis (MS) and other autoimmune diseases constitute a major healthcare burden at a cost of >$50 billion per year. Autoimmunity arises from a failure of immunoregulation, in which regulatory T cells (Tregs) play a crucial role in balancing immune responses, though their suppressive mechanisms are incompletely understood and little is known about their cellular dynamics. Our overall goal is to identify cellular and molecular immunoregulatory mechanisms that contribute to disease progression and response to therapy. Building on our expertise in two-photon (2-P) imaging at the cellular level, and Ca2+ signaling at the molecular level, we will use the experimental autoimmune encephalomyelitis (EAE) mouse model of MS to investigate cellular interactions and molecular mechanisms underlying disease progression, as well as therapeutic approaches to promote remission. We focus in particular on Tregs, which maintain homeostasis and limit autoimmunity. Our central hypothesis is that Tregs limit autoimmune-mediated demyelination in the EAE model at two levels. (i) At the cellular level, Tregs compete with conventional T cells for access to antigen-presenting dendritic cells (DCs), and restrict egress of differentiated, pathogenic effector T cells (Teffs) from lymph nodes (LN). In Aim 1, we will apply simultaneous 2-P imaging of Tregs, naïve T cells, Teffs, and DCs in the LN to reveal fundamental cell trafficking and interaction dynamics during EAE induction, progression, and remission. Aim 2 extends those studies to the spinal cord where, by additionally imaging oligodendrocytes and neuronal cells, we will elucidate the cellular dynamics of neuroinflammation and demyelination during disease progression and remission. In both Aims, we further propose to define cellular dynamics during therapies that show great promise for treating MS in humans including drugs that target S1P1 receptors to cause lymphocyte sequestration within the LN, and stem cell therapy to promote remyelination. (ii) At the molecular level, Tregs directly contact target lymphocytes to inhibit Ca2+ signaling and suppress their activation. We have previously shown that Ca2+ signaling in T cells is mediated by plasma membrane Orai1 channels and triggered by STIM1 in the endoplasmic reticulum. In Aim 3, we propose to investigate the roles of these proteins employing a novel `toolkit' of genetically-encoded Ca2+ indicators and probes of channel function to monitor cellular Ca2+ signaling in LN and spinal cord. We hypothesize that Treg contact with naïve or effector T cells results in dissolution of Orai1 puncta and transendocytosis of Orai1 channel protein into Tregs. We will evaluate Orai1 as a therapeutic target in MS by visualizing cellular dynamics following administration of a specific Orai1 blocker during EAE and the translational potential of this approach will be validated with human cells. Although this proposal is targeted specifically to MS, our findings and novel immunoimaging approaches will contribute in a broader context to a better understanding of how immune responses are initiated, how immunological tolerance is achieved, how Tregs prevent autoimmunity and dampen immune responses, and how autoimmunity and infectious diseases can be effectively treated.

Store-operated Ca2+ entry (SOCE) underlies numerous cellular processes throughout the body and initiates signaling cascades in T lymphocytes and microglia that cause changes in motility, secretion of cytolytic granules, cytokine release, and cell proliferation. The channels that underlie SOCE have been identified recently through RNA interference (RNAi) screening as a conserved family of four transmembrane-spanning proteins named Orai that are activated by STIM proteins in the ER membrane. Isoforms of these proteins are expressed throughout the body in a tissue-specific manner. Important cellular functions of Orai1 have been identified in lymphocytes, microglia, mast cells, blood platelets, sweat and salivary glands, dentition, vascular smooth muscle, endothelial cells, and skeletal muscle. In the immune system, STIM1 and Orai1 mediate antigen-induced Ca2+ signaling, motility inhibition at the site of antigen presentation, secretion of cytolytic granules by CD8+ T cells and NK cells, and gene expression responses that lead to cytokine release and cell proliferation. More recent studies show that SOCE is a major route of Ca2+ influx in microglia. Additionally STIM1 and Orai1 play a role in mediating functional Ca2+ responses to purinergic P2Y activation including chemotaxis and phagocytosis. STIM and Orai proteins are being developed as targets for treatment of autoimmune diseases and prevention of transplant rejection. Our overall goal is to understand how Orai channels function at the molecular and cellular level. Orai channels in the plasma membrane are unrelated to other known ion channels and have unusual characteristics that distinguish them, including a very high degree of selectivity for Ca2+, low single-channel conductance, and activation by binding of a small cytosolic domain of the STIM protein. Moreover, the human Orai1 and Orai3 proteins differ in their activation requirements and tissue distribution. In this project, we have three goals. We seek to understand: 1) how Orai1 is activated by STIM1 and interacts with adjacent Orai1 channels in puncta to generate localized Ca2+ signals; 2) how local Ca2+ signals modulate T cell motility, turning behavior and stopping during immune surveillance; and 3) how Orai1 regulates Ca2+ signaling network that underlie the complex motility patterns seen in microglia. To accomplish these Aims, we have developed and continue to develop new tools for monitoring local Ca2+ signals that will be broadly applicable. Our studies will include electrophysiological analysis of gating and ion permeation, optical imaging of Ca2+ flux through Orai1 channels, and two-photon imaging of in situ cellular motility. Overall, our project will provide fundamental insights into the Orai1 proteins that are currently being targeted for treatment of autoimmune disorders, chronic inflammatory conditions, and neurodegerative diseases such as Alzheimer?s.