Matthew F Krummel
University Of California San Francisco
Project start date: 2002-09-15
Project end date: 2012-12-31
Sponsored Links Excellgen http://Excellgen.com
Myosin Motors In T Cell Synapse Formation And Activation
Matthew F Krummel
University Of California San Francisco 3333 California St., Ste 315 San Francisco, Ca 941430962
Grant 5R01AI052116-05 from National Institute Of Allergy And Infectious Diseases IRG: ALY
Abstract: Cell-cell interactions are of critical importance for expanding the range of the immune response in order to control infection. Yet, the mechanisms that control cell-cell contacts and receptor movement in the immune system remain cryptic. Using high-speed video microscopy, we have been able to demonstrate T cell receptor clustering followed by coalescence of these clusters into the central "synapse". Through simultaneous imaging of intracellular calcium levels, it has become apparent that while initial microclusters are associated with the onset of signaling, a program of cellular re-polarization is necessary for the formation of the central synapse structure and for sustained signaling. The overall goal of my research is to define spatial and temporal maps of the initiating events of immune recognition. The hypothesis underlying this project is that cellular myosin motors triggered by initial TCR signals are crucial for polarization of key membrane receptors into signaling complexes. The specific aims of this project are 1. To define the myosin family members that are uniquely responsible for synapse formation in T cells, using biochemistry, in situ localization techniques and dominant negative mutations. 2. To determine the biochemical roles for myosins in T cell signaling by examining the interaction partners and phosphorylation of myosins upon TCR ligation. 3. To define the overlapping role of myosin-motors with signaling mediated by costimulatory signals and with signaling mediated by chemokines. These studies will not only provide fundamental information about the initial steps in lymphocyte activation, but may also lead to clues about ways to manipulate T cell responses in vitro and in vivo.
Keywords: T cell receptor, T lymphocyte, antigen presentation, biological signal transduction, myosin, calcium flux, chemokine, gene mutation, phosphorylation, receptor binding, laboratory mouse, tissue /cell culture, video microscopy
Project start date: 2002-09-15
Project end date: 2008-01-14
5R01AI052116-05 (2006): $313933
5R01AI052116-04 (2005): $356553
5R01AI052116-03 (2004): $297850
5R01AI052116-02 (2003): $297058
5R01AI052116-08 (2010): $342514
Grants awarded to Matthew F Krummel
REGULATION OF T CELL FUNCTIONS BY THE BREAST CANCER MICROENVIRONMENT
Matthew F Krummel, Associate Professor
University Of California San Francisco, 3333 California St., Ste 315, San Francisco, Ca 94143-0962
Grant 5R01CA134622-02 from National Cancer Institute
Abstract: Why don´t tumor-reactive T cells eliminate tumors? Although priming in the draining lymph node is often suboptimal, more frequently, the immune response is stymied at the tumor itself. This may be as a result of a protection of the tumor from immune surveillance (tolerance) or inadequate repriming and feedback to the local draining lymph node (an interrupted feedback loop). Based on a panel of preliminary data, we hypothesize that a subpopulation of APCs in the tumor microenvironment protect the tumor from CTLs by direct interaction with the T cells, by their failure to mature in response to the interaction, and by cooperation with regulatory T cells. We believe the microenvironment builds an arsenal of these cells and collects the immune system at ´inactivating foci´ that prevent stable and effective surveillance of the tumor by primed CTLs. By simultaneously preventing the I cells from seeing the tumor and suboptimally activating them there, these cells may effectively neutralize the response. We propose to study this in a new model system of human breast cancer, the PyMTCherry-OVA mouse that we have produced and characterized expressly to address this poorly accessible problem. The model allows direct visualization of the tumor AND the tumor-sampling APCs via intrinsic fluorescence. This is significant as we are able to study what is happening to the T cell as it contacts these distinct cell types, specifically via 2-photon microscopy. It is also significant because we are also able to phenotype this very important APC population using multicolor flow cytometry, microarray, and in vitro assays and provide key data about the co-development of APC and I cell population that results in tumor acceptance. It should be noted that the studies proposed are amongst the first of their kind that will be achievable in a non-ectopic tumor model that very closely resemble human breast cancer in many important ways. The spontaneous eteliology of our model is also important, as there are many reasons that ectopic tumors are unlikely to faithfully replicate important aspects of immune-evasion, including the microenvironment, by a primary tumor. Thus, studies in this system are much more likely to recapitulate the features of human disease and the ´normal´ establishment of immune evasion. The results of these experiments are going to reveal the dynamics of I cells over time in the developing hyperplasia through to established carcinomas. They will also definitely reveal the interaction of regulatory I cells with cells of the local I cell response. Finally, and not insignificantly, they will shed considerable light upon a previously inaccessible cell population which may protect the tumor from the immune response and thus permit tumor growth and metastasis
Keywords: ATGN; Address; Adoption; Antigens; Biological Models; Bolus; Bolus Infusion; Breast Cancer Model; Breast Neoplasms; Breast Tumors; CD4 Positive T Lymphocytes; CD4 T cells; CD4 lymphocyte; CD4+ T cell; CD4+ T-Lymphocyte; CD4-Positive Lymphocytes; CD8; CD8B; CD8B1; CD8B1 gene; Cancer Induction; Cancer of Breast; Carcinoma; Causality; Cell Count; Cell Function; Cell Locomotion; Cell Migration; Cell Movement; Cell Number; Cell Process; Cell physiology; Cells; Cells, CD4; Cellular Function; Cellular Migration; Cellular Physiology; Cellular Process; Cherries; Cherry - dietary; Collecting Cell; Cytofluorometry, Flow; Data; Defect; Development; Epithelial Neoplasms, Malignant; Epithelial Tumors, Malignant; Etiology; FLR; Failure (biologic function); Feedback; Flow Cytofluorometries; Flow Cytometry; Flow Microfluorimetry; Fluorescence; Genomics; Human; Human, General; Hyperplasia; Hyperplastic; Imagery; Immune; Immune Surveillance; Immune response; Immune system; Immunologic Surveillance; Immunological Surveillance; Invaded; LYT3; Light; Lymph node proper; Malignant Tumor of the Breast; Malignant neoplasm of breast; Mammals, Mice; Mammary Cancer; Mammary Glands, Human; Mammary Neoplasms; Mammary gland; Man (Taxonomy); Man, Modern; Metastasis; Metastasize; Metastatic Neoplasm; Metastatic Tumor; Mice; Microfluorometry, Flow; Microscopy; Model System; Modeling; Models, Biologic; Motility; Motility, Cellular; Murine; Mus; Nature; Neoplasm Metastasis; Organism; Phenotype; Photons; Photoradiation; Population; Primary Neoplasm; Primary Tumor; Programs (PT); Programs [Publication Type]; Regulation; Regulatory T-Lymphocyte; Reticuloendothelial System, Lymph Node; Role; Sampling; Secondary Neoplasm; Secondary Tumor; Sentinel; Staging; Subcellular Process; Surveillances, Immunologic; Surveillances, Immunological; System; System, LOINC Axis 4; T cell response; T-Cell Activation; T-Cell Subsets; T-Cells; T-Lymphocyte; T-Lymphocyte Subsets; T4 Cells; T4 Lymphocytes; Thymus-Dependent Lymphocytes; Time; Tumor Antigens; Tumor Cell; Tumor Cell Migration; Tumor-Associated Antigen; Visualization; adenoma; base; body system, allergic/immunologic; cancer metastasis; carcinogenesis; cell motility; cell type; disease causation; disease etiology; disease/disorder etiology; disorder etiology; epithelial carcinoma; experiment; experimental research; experimental study; failure; flow cytophotometry; helper T cell; host response; human disease; immunogen; immunoresponse; in vitro Assay; in vivo; living system; lymph gland; lymph nodes; macrophage; malignant breast neoplasm; mammary; mammary cancer model; mammary tumor; mammary tumor model; neoplastic cell; organ system, allergic/immunologic; prevent; preventing; programs; research study; response; social role; thymus derived lymphocyte; tumor; tumor growth; tumor-specific antigen
Relevance: 7. T cells are the primary sentinels of the immune system and they constantly move around to seek out invading organisms and initiate responses against these. Their poor responsiveness to tumors is likely dependent upon specific features of the microenvironment including the cell type that are responsible for presenting antigens to T cells. Here, we will determine the specific defects in immune surveillance in a developing breast-cancer model, tracking the activation of T cells during their responses in the microenvironment
Project start date: 2009-07-17
Project end date: 2011-06-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
PFA/PA: PA-07-070
5R01CA134622-02 (2010): $320588
1R01CA134622-01A1 (2009): $363029
Imaging-Based Analysis Of Tolerance-Induction Mechanisms
Matthew F Krummel
University Of California San Francisco 3333 California St., Ste 315 San Francisco, Ca 941430962
Grant 5R21AI062899-02 from National Institute Of Allergy And Infectious Diseases IRG: ZRG1
Abstract: The nature of the signals that are perceived as T cell activating versus tolerogenic is as yet unclear. A recently revisited readout for T cell activation is receptor clustering at the T celI-APC interface followed by coalescence of these clusters into the central "synapse". We and others have demonstrated that while initial micro-clusters are associated with the onset of signaling, a program of cellular re-polarization is necessary for the formation of the central synapse structure and for sustained signaling. All of the known signaling players in T cell activation that have been examined have been shown to coalesce at the synapse making this site a critical hub for signaling and making participation in this structure a biosensor at some level for participation in signaling. In this proposal, we screen a library of T cell expressed gene-products for their participation in the immunological synapse under varying activation conditions. To do this, we will i.) Construct fluorescent-protein fusion libraries in T cells using gene-trapping and/or cDNA fusions. This technology is already partially developed in our laboratory, ii.) The library will be phenotypically screened for localization and differential localization of fusions to the synapse under varying activation conditions. For this, we will develop and utilize novel medium-high throughput imaging-based single-cell assays including instrumentation and analysis algorithms. In addition to identifying these gene products, our study will open up a technology for larger scale analyses of activating and tolerant signaling. Thus, consistent with this R21 PA, our studies will investigate the unique and innovative use of existing methodology to explore a new area and will develop novel techniques that could have a major impact on the field of biomedical research.
Keywords: T lymphocyte, antigen presenting cell, biological signal transduction, chimeric protein, gene expression profiling, image processing, immune tolerance /unresponsiveness, molecular /cellular imaging, protein localization, protein structure function, computer program /software, genetic library, high throughput technology, leukocyte activation /transformation, bioimaging /biomedical imaging, green fluorescent protein, laboratory mouse, tissue /cell culture
Project start date: 2005-03-01
Project end date: 2008-02-28
5R21AI062899-02 (2006): $143706
1R21AI062899-01 (2005): $172291
COLLABORATIVE INNATE-ADAPTIVE IMMUNE REGULATION OF TUMOR PROGRESSION
Matthew F Krummel
University Of California San Francisco, 3333 California St., Ste 315, San Francisco, Ca 94143-0962
Grant 5U01CA141451-02 from National Cancer Institute
Abstract: The immune system reacts to the evolving tumor, so why does it not eradicate tumors? T cell clones that recognize tumor-specific antigens are expanded in cancer patients, yet tumors are rarely spontaneously eradicated by the immune system. Similarly, immune therapies that boost T cells, though showing some efficacy, are inefficient. It appears that the immune response frequently is stymied in the tumor microenvironment. There, T cells are exposed to inhibitory and stimulatory signals, either in the form of soluble or cell-surface derived stimuli. Much is still unclear about how tumor-reactive immune cells function and behave in the microenvironment of naturally evolving tumors. We have literally been blind to the types of dynamic interactions that occur between T cells and antigen presenting, innate immune cells in tumors. However, the nature of the collaboration of the innate and adaptive arms of the immune system can now be fully addressed in situ, within the tumor microenvironment, using mouse models accessible to imaging. Based on preliminary data, we hypothesize that a population of innate immune cells regulate the cells of the adaptive immune system in the microenvironment, protecting the tumor from immune attack. Capitalizing on our ability to concomitantly image innate and adaptive immune cells in situ in mouse models of cancer, we will undertake an assessment of the types of immune cell interactions that occur in the tumor. We will address how interactions between adaptive T cells and innate antigen presenting cells are influenced by microenvironments and by tumor types and how it evolves with tumor progression. We will further seek to identify pathways involved in the collaboration between the innate and adaptive immune responses. Finally, we will use our models to visualize and define what immune and cytotoxic therapy does to the immune response in real-time. In this latter point, direct imaging will guide the development and optimization of therapies. Throughout, we will also coordinate with clinical researchers to undertake concurrent analyses of human biopsy samples to translate our findings into diagnosis and therapy
Keywords: APC; ATGN; Address; Antigen-Presenting Cells; Antigens; Arm; Biopsy Sample; Biopsy Specimen; Cancer Patient; Cancers; Cell Communication; Cell Communication and Signaling; Cell Function; Cell Interaction; Cell Process; Cell Signaling; Cell physiology; Cell surface; Cell-to-Cell Interaction; Cells; Cellular Function; Cellular Physiology; Cellular Process; Clinical; Collaborations; Cytotoxic Chemotherapy; Cytotoxic Therapy; Data; Development; Diagnosis; Human; Human, General; ITX; Image; Immune; Immune response; Immune system; Immunologic Accessory Cells; Immunologically Directed Therapy; Immunotherapy; In Situ; Intracellular Communication and Signaling; Investigators; Malignant Neoplasms; Malignant Tumor; Man (Taxonomy); Man, Modern; Modeling; Monocytes / Macrophages / APC; Nature; Pathway interactions; Population; Regulation; Research Personnel; Researchers; Signal Transduction; Signal Transduction Systems; Signaling; Stimulus; Subcellular Process; T-Cells; T-Lymphocyte; Thymus-Dependent Lymphocytes; Time; Translating; Translatings; Tumor Antigens; Tumor-Associated Antigen; Upper arm; accessory cell; base; biological signal transduction; blind; body system, allergic/immunologic; cancer progression; host response; imaging; immune therapy; immunogen; immunoresponse; language translation; malignancy; mouse model; neoplasm progression; neoplasm/cancer; neoplastic progression; organ system, allergic/immunologic; pathway; thymus derived lymphocyte; tumor; tumor progression; tumor-specific antigen
Project start date: 2009-09-01
Project end date: 2014-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: RFA-CA-08-018
5U01CA141451-02 (2010): $424356
1U01CA141451-01 (2009): $400975
New Models For Molecular-Level Imaging Of Cell Signaling In Vivo.
Matthew F Krummel, Associate Professor
Pathologyuniversity Of California San Francisco
Grant 5R21RR024895-02 from National Center For Research Resources IRG: ZRG1
Abstract: Intravital imaging has become a vital tool to study cells in their native context and yet there are almost no methods or tools suitable for real-time spatially-resolved analysis of signaling in living tissues. Light-based fluorescence has the best resolution for subcellular resolution of signaling and can be used in real-time. Numerous groups have developed protocols for applying multiphoton fluorescence imaging to target tissues and organs in the mouse. These protocols are currently limited to observation of cell morphology, positioning and motility parameters while progress requires development of probe systems that respond to signaling onset. In vitro, the use of GFP-tagged biomarkers, genetically expressed and used to track molecular behaviors within cells has proven to be extremely useful in tracking cellular activation. However, the situation in vivo has been considered difficult, as a result of considerations of probe intensity, difficulty with interpretation in the absence of fiducials, and regional autofluorescence. We show that, with new improved GaAs detection in 2-photon microscopy, excitation and detection of weak fluorescent protein expression is not the primary hindrance to molecular imaging so long as the molecule of interest is expressed at or near a now-defined level. Instead, the limitations are autofluorescence reduction and strategies that provide fiducial markers to characterize biosensing fusion-proteins. We propose that a collection of multiplexed biosensor arrays will permit the imaging of nuclear import of transcription factors, activation of PI3kinase enzymes and T cell receptors. The genes encoding these will be integrated as P2A-based fusion into the tissue-independent ROSA-26 locus with lox-stop- lox control so that they can be conditionally expressed under the many existing tissue-specific- promoter Cre driver-strains. Based on the design, which mimics our previous success with the T cell receptor first in vitro and now in vivo, these will be functional even in an organ with uneven levels of autofluorescence. Our goal is to develop these tools and subsequently apply them to a T cell interacting with a newly improved model for spontaneous tumor outgrowth. We will also make the mice expressing these biosensors available to the community through non-restrictive sharing and/or the placement of mouse strains into a repository.7. Project Narrative While our science is achieving great success at understanding the behavior of our cells in isolation, it is more difficult to study the way cells work in their native context. This proposal will create new mouse strains that will permit us to look into tissues using microscopy and observe cells being activated to divide and/or remodel the tissue of which they are a part. It is important for us to understand which cells are being activated and deactivated in the context of many diseases of humans, and our results are likely to provide great insight into the nature of many of these
Project start date: 2008-04-01
Project end date: 2010-03-31
Myosin Motors In T Cell Synapse Formation And Activation
Matthew F Krummel
University Of California San Francisco 3333 California St., Ste 315 San Francisco, Ca 941430962
Grant 3R01AI052116-03S1 from National Institute Of Allergy And Infectious Diseases IRG: ALY
Abstract: Cell-cell interactions are of critical importance for expanding the range of the immune response in order to control infection. Yet, the mechanisms that control cell-cell contacts and receptor movement in the immune system remain cryptic. Using high-speed video microscopy, we have been able to demonstrate T cell receptor clustering followed by coalescence of these clusters into the central "synapse". Through simultaneous imaging of intracellular calcium levels, it has become apparent that while initial microclusters are associated with the onset of signaling, a program of cellular re-polarization is necessary for the formation of the central synapse structure and for sustained signaling. The overall goal of my research is to define spatial and temporal maps of the initiating events of immune recognition. The hypothesis underlying this project is that cellular myosin motors triggered by initial TCR signals are crucial for polarization of key membrane receptors into signaling complexes. The specific aims of this project are 1. To define the myosin family members that are uniquely responsible for synapse formation in T cells, using biochemistry, in situ localization techniques and dominant negative mutations. 2. To determine the biochemical roles for myosins in T cell signaling by examining the interaction partners and phosphorylation of myosins upon TCR ligation. 3. To define the overlapping role of myosin-motors with signaling mediated by costimulatory signals and with signaling mediated by chemokines. These studies will not only provide fundamental information about the initial steps in lymphocyte activation, but may also lead to clues about ways to manipulate T cell responses in vitro and in vivo.
Keywords: T cell receptor, T lymphocyte, antigen presentation, biological signal transduction, myosin, calcium flux, chemokine, gene mutation, phosphorylation, receptor binding, laboratory mouse, tissue /cell culture, video microscopy
Project start date: 2002-09-15
Project end date: 2007-02-28
3R01AI052116-03S1 (2004): $33095
1R01AI052116-01 (2002): $147975
GENES ASSOCIATED WITH POSTTHYMIC T CELL DIFFERENTIATION
Matthew F Krummel
Stanford University Stanford, Ca 94305
Grant 5F32AI010120-03 from National Institute Of Allergy And Infectious Diseases IRG: ZRG2
Keywords: T lymphocyte, cell differentiation, immunogenetics, developmental immunology, laboratory mouse
5F32AI010120-03 (1999): $40036
Sponsored Links Excellgen http://Excellgen.com
5F32AI010120-02 (1998): $31492
1F32AI010120-01 (1997): $29600