scholarly journals Intravascular staining for discrimination of vascular and tissue leukocytes

2014 ◽  
Vol 9 (1) ◽  
pp. 209-222 ◽  
Author(s):  
Kristin G Anderson ◽  
Katrin Mayer-Barber ◽  
Heungsup Sung ◽  
Lalit Beura ◽  
Britnie R James ◽  
...  
Keyword(s):  
Intravascular Staining

scholarly journals Cutting Edge: Intravascular Staining Redefines Lung CD8 T Cell Responses

2012 ◽  
Vol 189 (6) ◽  
pp. 2702-2706 ◽  
Author(s):  
Kristin G. Anderson ◽  
Heungsup Sung ◽  
Cara N. Skon ◽  
Leo Lefrancois ◽  
Angela Deisinger ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Cutting Edge ◽  
Cell Responses ◽  
Intravascular Staining

scholarly journals Tissue Trafficking Kinetics of Rhesus Macaque Natural Killer Cells Measured by Serial Intravascular Staining

2022 ◽  
Vol 12 ◽  
Author(s):  
Ryland D. Mortlock ◽  
Chuanfeng Wu ◽  
E. Lake Potter ◽  
Diana M. Abraham ◽  
David S. J. Allan ◽  
...  
Keyword(s):  
Steady State ◽  
Nk Cells ◽  
Tissue Distribution ◽  
Natural Killer ◽  
Rhesus Macaque ◽  
Peripheral Blood ◽  
Cell Biology ◽  
Nk Cell ◽  
Lymphoid Tissues ◽  
Intravascular Staining

The in vivo tissue distribution and trafficking patterns of natural killer (NK) cells remain understudied. Animal models can help bridge the gap, and rhesus macaque (RM) primates faithfully recapitulate key elements of human NK cell biology. Here, we profiled the tissue distribution and localization patterns of three NK cell subsets across various RM tissues. We utilized serial intravascular staining (SIVS) to investigate the tissue trafficking kinetics at steady state and during recovery from CD16 depletion. We found that at steady state, CD16+ NK cells were selectively retained in the vasculature while CD56+ NK cells had a shorter residence time in peripheral blood. We also found that different subsets of NK cells had distinct trafficking kinetics to and from the lymph node as well as other lymphoid and non-lymphoid tissues. Lastly, we found that following administration of CD16-depleting antibody, CD16+ NK cells and their putative precursors retained a high proportion of continuously circulating cells, suggesting that regeneration of the CD16 NK compartment may take place in peripheral blood or the perivascular compartments of tissues.

Measurement of leukocyte trafficking kinetics in macaques by serial intravascular staining

2021 ◽  
Vol 13 (576) ◽  
pp. eabb4582 ◽  
Author(s):  
E. Lake Potter ◽  
Hannah P. Gideon ◽  
Victor Tkachev ◽  
Giulia Fabozzi ◽  
Alexander Chassiakos ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Nonhuman Primates ◽  
Molecular Mechanisms ◽  
Bacterial Pathogen ◽  
Immune Memory ◽  
Multiparameter Flow Cytometry ◽  
Lymphoid Tissues ◽  
Leukocyte Trafficking ◽  
Intravascular Staining

Leukocyte trafficking enables detection of pathogens, immune responses, and immune memory. Dysregulation of leukocyte trafficking is often found in disease, highlighting its important role in homeostasis and the immune response. Whereas some of the molecular mechanisms mediating leukocyte trafficking are understood, little is known about the regulation of trafficking, including trafficking kinetics and its impact on immune homeostasis. We developed a method of serial intravascular staining (SIVS) to measure trafficking kinetics in nonhuman primates using infusions of fluorescently labeled antibodies to label circulating leukocytes. Because antibody infusions labeled only leukocytes in the blood, cells were “barcoded” according to their location at the time of each infusion, providing positional histories that could be used to infer trafficking kinetics. We used SIVS and multiparameter flow cytometry to quantitate cellular trafficking into lymphoid tissues of healthy animals at homeostasis and to identify perivascular cells that could be unique to nonlymphoid organs. To investigate how these parameters could be influenced during disease, SIVS was used to quantify lymphocyte trafficking in macaques infected with the bacterial pathogen Mycobacterium tuberculosis and to enumerate intravascular leukocytes in lung granulomas. We showed that whereas most cells in lung granulomas were localized there for more than 24 hours, granulomas were dynamic with a slow continual cellular influx, the rate of which predicted clearance of M. tuberculosis from the granulomas. SIVS, in combination with intracellular staining and multiparametric flow cytometry, is a powerful method to quantify the kinetics of leukocyte trafficking in nonhuman primates in vivo.

scholarly journals Airway delivery of both a BCG prime and adenoviral boost drives CD4 and CD8 T cells into the lung tissue parenchyma

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Daryan A. Kaveh ◽  
M. Carmen Garcia-Pelayo ◽  
Naomi C. Bull ◽  
Pedro J. Sanchez-Cordon ◽  
John Spiropoulos ◽  
...  
Keyword(s):  
T Cells ◽  
Lung Tissue ◽  
Cd8 T Cells ◽  
Murine Model ◽  
Vaccine Development ◽  
Lung Parenchyma ◽  
Boost Vaccination ◽  
Parenteral Delivery ◽  
Promising Strategy ◽  
Intravascular Staining

Abstract Heterologous BCG prime-boost regimens represent a promising strategy for an urgently required improved tuberculosis vaccine. Identifying the mechanisms which underpin the enhanced protection induced by such strategies is one key aim which would significantly accelerate rational vaccine development. Experimentally, airway vaccination induces greater efficacy than parenteral delivery; in both conventional vaccination and heterologous boosting of parenteral BCG immunisation. However, the effect of delivering both the component prime and boost immunisations via the airway is not well known. Here we investigate delivery of both the BCG prime and adenovirus boost vaccination via the airway in a murine model, and demonstrate this approach may be able to improve the protective outcome over parenteral prime/airway boost. Intravascular staining of T cells in the lung revealed that the airway prime regimen induced more antigen-specific multifunctional CD4 and CD8 T cells to the lung parenchyma prior to challenge and indicated the route of both prime and boost to be critical to the location of induced resident T cells in the lung. Further, in the absence of a defined phenotype of vaccine-induced protection to tuberculosis; the magnitude and phenotype of vaccine-specific T cells in the parenchyma of the lung may provide insights into potential correlates of immunity.

scholarly journals Mucosal immunity and novel tuberculosis vaccine strategies: route of immunisation-determined T-cell homing to restricted lung mucosal compartments

2015 ◽  
Vol 24 (136) ◽  
pp. 356-360 ◽  
Author(s):  
Rocky Lai ◽  
Sam Afkhami ◽  
Siamak Haddadi ◽  
Mangalakumari Jeyanathan ◽  
Zhou Xing
Keyword(s):  
T Cells ◽  
T Cell ◽  
Health Concern ◽  
Pulmonary Vasculature ◽  
Distribution Profile ◽  
Activated T Cells ◽  
T Cell Homing ◽  
Intravascular Staining ◽  
Induced Immunity ◽  
Mucosal Immunisation

Despite the use of bacille Calmette–Guérin (BCG) for almost a century, pulmonary tuberculosis (TB) continues to be a serious global health concern. Therefore, there has been a pressing need for the development of new booster vaccines to enhance existing BCG-induced immunity. Protection following mucosal intranasal immunisation with AdHu5Ag85A is associated with the localisation of antigen-specific T-cells to the lung airway. However, parenteral intramuscular immunisation is unable to provide protection despite the apparent presence of antigen-specific T-cells in the lung interstitium. Recent advances in intravascular staining have allowed us to reassess the previously established T-cell distribution profile and its relationship with the observed differential protection. Respiratory mucosal immunisation empowers T-cells to home to both the lung interstitium and the airway lumen, whereas intramuscular immunisation-activated T-cells are largely trapped within the pulmonary vasculature, unable to populate the lung interstitium and airway. Given the mounting evidence supporting the safety and enhanced efficacy of respiratory mucosal immunisation over the traditional parenteral immunisation route, a greater effort should be made to clinically develop respiratory mucosal-deliverable TB vaccines.

scholarly journals Identification and Evaluation of Novel Protective Antigens for the Development of a Candidate Tuberculosis Subunit Vaccine

2018 ◽  
Vol 86 (7) ◽  
pp. e00014-18 ◽  
Author(s):  
Elena Stylianou ◽  
Rachel Harrington-Kandt ◽  
Julia Beglov ◽  
Naomi Bull ◽  
Nawamin Pinpathomrat ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Protective Efficacy ◽  
Human Infection ◽  
Selection Strategy ◽  
Development Effort ◽  
Subunit Vaccines ◽  
Content Type ◽  
Protective Antigens ◽  
Preclinical Animal Models ◽  
Intravascular Staining

ABSTRACT The development of a vaccine against tuberculosis (TB), a disease caused by Mycobacterium tuberculosis, is urgently needed. The only currently available vaccine, M. bovis BCG, has variable efficacy. One approach in the global vaccine development effort is focused on boosting BCG using subunit vaccines. The identification of novel antigens for inclusion in subunit vaccines is a critical step in the TB vaccine development pathway. We selected four novel mycobacterial antigens recognized during the course of human infection. A replication-deficient chimpanzee adenovirus (ChAdOx1) was constructed to express each antigen individually, and these vectors were evaluated for protective efficacy in murine M. tuberculosis challenge experiments. One antigen, PPE15 (Rv1039c), conferred significant and reproducible protection when administered alone and as a boost to BCG vaccination. We identified immunodominant epitopes to define the protective immune responses using tetramers and intravascular staining. Lung parenchymal CD4+ and CD8+ CXCR3+ KLRG1− T cells, previously associated with protection against M. tuberculosis, were enriched in the vaccinated groups compared to the control groups. Further work to evaluate the protective efficacy of PPE15 in more stringent preclinical animal models, together with the identification of further novel protective antigens using this selection strategy, is now merited.

scholarly journals Preventive CCL2/CCR2 Axis Blockade Suppresses Osteoclast Activity in a Mouse Model of Rheumatoid Arthritis by Reducing Homing of CCR2hi Osteoclast Progenitors to the Affected Bone

2021 ◽  
Vol 12 ◽  
Author(s):  
Darja Flegar ◽  
Maša Filipović ◽  
Alan Šućur ◽  
Antonio Markotić ◽  
Nina Lukač ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Clinical Score ◽  
Differentiation Potential ◽  
Ccr2 Expression ◽  
Molecule Inhibitor ◽  
Intravascular Staining

Detailed characterization of medullary and extramedullary reservoirs of osteoclast progenitors (OCPs) is required to understand the pathophysiology of increased periarticular and systemic bone resorption in arthritis. In this study, we focused on identifying the OCP population specifically induced by arthritis and the role of circulatory OCPs in inflammatory bone loss. In addition, we determined the relevant chemokine axis responsible for their migration, and targeted the attraction signal to reduce bone resorption in murine collagen-induced arthritis (CIA). OCPs were expanded in periarticular as well as circulatory compartment of arthritic mice, particularly the CCR2hi subset. This subset demonstrated enhanced osteoclastogenic activity in arthritis, whereas its migratory potential was susceptible to CCR2 blockade in vitro. Intravascular compartment of the periarticular area contained increased frequency of OCPs with the ability to home to the arthritic bone, as demonstrated in vivo by intravascular staining and adoptive transfer of splenic LysMcre/Ai9 tdTomato-expressing cells. Simultaneously, CCL2 levels were increased locally and systemically in arthritic mice. Mouse cohorts were treated with the small-molecule inhibitor (SMI) of CCR2 alone or in combination with methotrexate (MTX). Preventive CCR2/CCL2 axis blockade in vivo reduced bone resorption and OCP frequency, whereas combining with MTX treatment also decreased disease clinical score, number of active osteoclasts, and OCP differentiation potential. In conclusion, our study characterized the functional properties of two distinct OCP subsets in CIA, based on their CCR2 expression levels, implying that the CCR2hi circulatory-like subset is specifically induced by arthritis. Signaling through the CCL2/CCR2 axis contributes to OCP homing in the inflamed joints and to their increased osteoclastogenic potential. Therefore, addition of CCL2/CCR2 blockade early in the course of arthritis is a promising approach to reduce bone pathology.

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