Interrogating Adaptive Immunity Using LCMV.
Authors: Dangi T, Chung YR, Palacio N, Penaloza-MacMaster P
Abstract
In this invited article, we explain technical aspects of the lymphocytic choriomeningitis virus (LCMV) system, providing an update of a prior contribution by Matthias von Herrath and J. Lindsay Whitton. We provide an explanation of the LCMV infection models, highlighting the importance of selecting an appropriate route and viral strain. We also describe how to quantify virus-specific immune responses, followed by an explanation of useful transgenic systems. Specifically, our article will focus on the following protocols. © 2020 Wiley Per...
Source: Current Protocols in Immunology - September 19, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Immunopharmacology and Quantitative Analysis of Tyrosine Kinase Signaling.
Authors: Brian BF, Guerrero CR, Freedman TS
Abstract
In this article we describe the use of pharmacological and genetic tools coupled with immunoblotting (Western blotting) and targeted mass spectrometry to quantify immune signaling and cell activation mediated by tyrosine kinases. Transfer of the ATP γ phosphate to a protein tyrosine residue activates signaling cascades regulating the differentiation, survival, and effector functions of all cells, with unique roles in immune antigen receptor, polarization, and other signaling pathways. Defining the substrates and scaffolding interactions of tyrosine kina...
Source: Current Protocols in Immunology - September 18, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Phosphoproteome Analysis in Immune Cell Signaling.
Authors: Rathore D, Nita-Lazar A
Abstract
Immune cell signaling is largely regulated by protein phosphorylation. Stimulation of toll-like receptors (TLRs) by pathogen-associated ligands drives the cascade of immune response, which can be influenced by differences in phosphoprotein abundance. Therefore, the analysis of phosphorylation signatures at a global level is central to understanding the complex and integrated signaling in macrophages upon pathogen attack. Here, we describe a mass spectrometry-based approach to identify and quantify phosphoproteome changes in response to the stimulation of TLR2, TLR4...
Source: Current Protocols in Immunology - September 18, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
In Vitro Assays to Study PD-1 Biology in Human T Cells.
We describe a phospho flow cytometry method to determine how PD-1 ligation alters the level of CD3ζ phosphorylation on Tyr142 , which can be easily applied to other proximal signaling proteins. Basic Protocol 2 describes a plate-bound assay that is useful to examine the long-term consequences of PD-1 ligation such as cytokine production and T cell proliferation. Complementary to that, Basic Protocol 3 describes an in vitro superantigen-based assay to evaluate T cell responses to therapeutic agents targeting the PD-1/PD-L axis, as well as immune synapse formation in the presence of PD-1 engagement. Finally, in Basic Protoc...
Source: Current Protocols in Immunology - August 13, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Characterization of Anaphylatoxin Receptor Expression and C3a/C5a Functions in Anaphylatoxin Receptor Reporter Mice.
Authors: Laumonnier Y, Karsten CM, Köhl G, Köhl J
Abstract
The anaphylatoxins (AT) C3a and C5a are effector molecules of C3 and C5 exerting multiple biologic functions through binding and activation of their cognate G protein-coupled receptors. C3a interacts with the C3a receptor (C3aR), whereas C5a and its primary degradation product C5a-desArg engage C5aR1 and C5aR2. In the past, analysis of AT expression has been hampered by cross reaction of antibodies designed to recognize the different AT receptors. Furthermore, assessment of effects mediated by cell-specific activation has been difficult. Here, fl...
Source: Current Protocols in Immunology - July 28, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Enrichment of Neutrophils and Monocytes From the Liver Following Either Oral or Intravenous Listeria monocytogenes Infection.
This article includes detailed instructions for use of either foodborne or i.v. inoculation of mice and discusses the rationale for choosing either model. Additionally, a protocol is provided for enrichment of neutrophils and monocytes from the infected liver in a manner that allows for determination of bacterial burden while still providing sufficient cells for use in flow cytometric analysis or in vitro assays. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Foodborne L. monocytogenes infection Support Protocol 1: Preparing L. monocytogenes for foodborne infection Basic Protocol 2: Intravenous L. monocytogenes infection...
Source: Current Protocols in Immunology - July 28, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
A Novel In Vitro Mouse Model to Study Mycobacterium tuberculosis Dissemination Across Brain Vessels: A Combination Granuloma and Blood-Brain Barrier Mouse Model.
We present an in vitro mouse model of the BBB to test Mycobacterium tuberculosis (Mtb) dissemination across brain endothelial cells. One-third of the global population is infected with Mtb, and in 1%-2% of cases bacteria invade the CNS through a largely unknown process. The "Trojan horse" theory supports the role of a cellular carrier that engulfs bacteria and carries them to the brain without being recognized. We present for the first time a protocol for an in vitro BBB-granuloma model that supports the Trojan horse mechanism of Mtb dissemination into the CNS. Handling of bacterial cultures, in vivo and in vitro infection...
Source: Current Protocols in Immunology - July 28, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Animal Models of Mycobacteria Infection.
Authors: Chan ED, Verma D, Ordway DJ
Abstract
This manuscript describes the infection of mice and guinea pigs with mycobacteria via various routes, as well as necropsy methods for the determination of mycobacterial loads within target organs. Additionally, methods for cultivating mycobacteria and preparing stocks are described. The protocols outlined are primarily used for M. tuberculosis, but can also be used for the study of other non-tuberculosis mycobacterial species. A wide variety of animal models have been used to test new vaccines, drugs, and the impact of cigarette exposure. © 2020 Wiley Periodic...
Source: Current Protocols in Immunology - June 6, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation and Characterization of Extracellular Vesicles from Cell Culture Conditioned Medium for Immunological Studies.
This article describes protocols for isolation of EVs from cell culture supernatants by differential ultracentrifugation and density gradient centrifugation. It also provides tools and protocols that enable characterization and validation of isolated particles, as well as analysis of interactions between EVs of interest and different subpopulations of human immune cells. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Isolation of extracellular vesicles by differential ultracentrifugation Basic Protocol 2: Isolation of extracellular vesicles by density gradient centrifugation Support Protocol 1: Imaging of extracellular v...
Source: Current Protocols in Immunology - May 28, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
T Cell Receptor Engineered Lymphocytes for Cancer Therapy.
Authors: Rollins MR, Spartz EJ, Stromnes IM
Abstract
T lymphocytes are capable of specific recognition and elimination of target cells. Physiological antigen recognition is mediated by the T cell receptor (TCR), which is an alpha beta heterodimer comprising the products of randomly rearranged V, D, and J genes. The exquisite specificity and functionality of T cells can be leveraged for cancer therapy: specifically, the adoptive transfer of T cells that express tumor-reactive TCRs can induce regression of solid tumors in patients with advanced cancer. However, the isolation and expression of a tumor antigen...
Source: Current Protocols in Immunology - May 22, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Multispecific, Multivalent Antibody-Based Molecules Engineered on the DART ® and TRIDENTTM Platforms.
This article focuses on the design of DART and TRIDENT molecules that target two or three different antigens. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Design and generation of expression plasmids encoding DART and TRIDENT molecules Basic Protocol 2: Expression of DART and TRIDENT molecules by transient transfection of CHO cells Basic Protocol 3: Purification of DART and TRIDENT molecules from CHO cell supernatants.
PMID: 32294319 [PubMed - in process] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - April 17, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Antibody-Based CAR T Cells Produced by Lentiviral Transduction.
Authors: Prommersberger S, Hudecek M, Nerreter T
Abstract
One promising approach to treat hematologic malignancies is the usage of patient-derived CAR T cells. There are continuous efforts to improve the function of these cells, to optimize their receptor, and to use them for the treatment of additional types of cancer and especially solid tumors. In this protocol, an easy and reliable approach for CAR T cell generation is described. T cells are first isolated from peripheral blood (here: leukoreduction system chambers) and afterwards activated for one day with anti-CD3/CD28 Dynabeads. The gene transfer i...
Source: Current Protocols in Immunology - March 11, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Purification of T Cell Populations.
This article describes a procedure for isolating T cell subpopulations using various methods including indirect panning and immunopanning by microarray. In these methods, cells are selected by their capacity to bind to antibody-coated plates (or slides) on the basis of particular cell-surface markers. Such methods can be superior to the antibody/complement lysis method (Alternate Protocol), as they can select additional cell population for analysis. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Isolation of T cell populations by indirect panning Basic Protocol 2: Immunopanning with microarray Alternate Protocol:...
Source: Current Protocols in Immunology - February 11, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation and Analysis of Tumor-Derived Exosomes.
Authors: Ludwig N, Hong CS, Ludwig S, Azambuja JH, Sharma P, Theodoraki MN, Whiteside TL
Abstract
A method for isolation of exosomes from tumor cell supernatants or cancer patients' plasma is presented. Tumor-derived exosomes (TEX) are defined as a subset of extracellular vesicles (EVs) sized at 30 to 150 nm and originating from multivesicular bodies (MVBs). The method utilizes size exclusion chromatography (SEC) for recovery of exosomes from cell-line supernatants or cancer patients' plasma. The recovered exosomes are morphologically intact, aggregate-free, and functionally competent. Their molecular cont...
Source: Current Protocols in Immunology - November 26, 2019 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Characterization and Purification of Mouse Mucosal-Associated Invariant T (MAIT) Cells.
Authors: Chen Z, Wang H, D'Souza C, Koay HF, Meehan B, Zhao Z, Pediongco T, Shi M, Zhu T, Wang B, Kjer-Nielsen L, Eckle SBG, Rossjohn J, Fairlie DP, Godfrey DI, Strugnell RA, McCluskey J, Corbett AJ
Abstract
This unit describes the utility of various mouse models of infection and immunization for studying mucosal-associated invariant T (MAIT) cell immunity: MAIT cells can be isolated from the lungs (or from other tissues/organs) and then identified and characterized by flow cytometry using MR1 tetramers in combination with a range of antibodies. The response kinetics, cytokine profiles, and functional diff...
Source: Current Protocols in Immunology - November 26, 2019 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
