Multi-color flow cytometry for evaluating age-related changes in memory lymphocyte subsets in dogs

Withers, Sita S.; Moore, Peter F.; Chang, Hong; Choi, Jin Wook; McSorley, Stephen J.; Kent, Michael S.; Monjazeb, Arta M.; Canter, Robert J.; Murphy, William J.; Sparger, Ellen E.; Rebhun, Robert B.

doi:10.1016/j.dci.2018.05.022

Cited by 27 publications

(34 citation statements)

References 81 publications

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“…Despite contradictory results, some findings were consistently associated with canine elderly, namely the reduction of blood CD4 + T cells, expansion of the CD8 + cell subset with a subsequent reduction of CD4:CD8 ratio, and a decrease of naïve lymphocytes [97][98][99][100][101][102][103].…”

Section: Immunosenescensementioning

confidence: 99%

Development of Dog Immune System: From in Uterus to Elderly

Pereira

Valério-Bolas

Marques

et al. 2019

Veterinary Sciences

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Immune system recognize and fight back foreign microorganisms and inner modifications that lead to deficient cell and tissue functions. During a dog’s life, the immune system needs to adapt to different physiological conditions, assuring surveillance and protection in a careful and controlled way. Pregnancy alters normal homeostasis, requiring a balance between immunity and tolerance. The embryos and fetus should be protected from infections, while the female dog must tolerate the growing of semi-allografts in her uterus. After birth, newborn puppies are at great risk of developing infectious diseases, because their immune system is in development and immune memory is absent. Passive transfer of immunity through colostrum is fundamental for puppy survival in the first weeks of life, but hampers the development of an active immune response to vaccination. At the end of life, dogs experience a decline in the structure and functional competence of the immune system, compromising the immune responses to novel antigenic challenges, such as infections and vaccines. Therefore, the current article reviews the general processes related to the development of the dog´s immune system, providing an overview of immune activity throughout the dog’s life and its implications in canine health, and highlighting priority research goals.

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Section: Immunosenescensementioning

confidence: 99%

Development of Dog Immune System: From in Uterus to Elderly

Pereira

Valério-Bolas

Marques

et al. 2019

Veterinary Sciences

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“…Whole blood was obtained from healthy donor dogs for isolation of PBMCs to examine binding of PD-1 and PD-L1 antibodies to primary canine lymphocytes and monocytes after stimulation with various activation agents. Blood was processed for PBMCs by density centrifugation as previously described [32]. Briefly, whole blood was diluted in Hank's Buffered Salt Solution (HBSS; Corning, Corning, NY, USA), layered over Histopaque 1077 (Sigma-Aldrich, Saint Louis, MO, USA), and centrifuged at 2000 rpm for 30 minutes at room temperature.…”

Section: Canine Pbmc Isolationmentioning

confidence: 99%

“…For experiments testing ConA stimulation of PD-1 expression on T cell subsets within PBMCs, single cell suspensions were stained with cell surface markers including PacBlue-labeled anti-canine CD4 (clone YKIX302.9., Bio-Rad Laboratories, Hercules, CA), PerCP-EF 710-labeled anti-canine CD8 (YCATE55, Thermo Fisher Scientific) and APC-labeled JC053 (anti-canine PD-1). Cells were then fixed and permeabilized for intracellular staining using Fixation/Permeabilization concentrate and diluent (eBioscience, Carlsbad, CA, USA), and Permeabilization buffer (eBioscience) with FITC-labeled anti-human CD3 (clone CD3-12, Bio-Rad Laboratories) using conditions previously described [32]. For experiments testing PD-L1 expression on PBMC subsets after stimulation with either PBS, PGN, or LPS, single cell suspensions were first stained for viability and then treated with FcR blocking antibody (Thermo Fisher Scientific) 4C for 20 min.…”

Section: Flow Cytometrymentioning

confidence: 99%

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Development of canine PD-1/PD-L1 specific monoclonal antibodies and amplification of canine T cell function

et al. 2020

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Interruption of the programmed death 1 (PD-1) / programmed death ligand 1 (PD-L1) pathway is an established and effective therapeutic strategy in human oncology and holds promise for veterinary oncology. We report the generation and characterization of monoclonal antibodies specific for canine PD-1 and PD-L1. Antibodies were initially assessed for their capacity to block the binding of recombinant canine PD-1 to recombinant canine PD-L1 and then ranked based on efficiency of binding as judged by flow cytometry. Selected antibodies were capable of detecting PD-1 and PD-L1 on canine tissues by flow cytometry and Western blot. Anti-PD-L1 worked for immunocytochemistry and anti-PD-1 worked for immunohistochemistry on formalin-fixed paraffin embedded canine tissues, suggesting the usage of this antibody with archived tissues. Additionally, anti-PD-L1 (JC071) revealed significantly increased PD-L1 expression on canine monocytes after stimulation with peptidoglycan or lipopolysaccharide. Together, these antibodies display specificity for the natural canine ligand using a variety of potential diagnostic applications. Importantly, multiple PD-L1-specific antibodies amplified IFN-γ production in a canine peripheral blood mononuclear cells (PBMC) concanavlin A (Con A) stimulation assay, demonstrating functional activity.

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“…We performed a thorough literature search for previously published panels and vendor websites for the most up-to-date marker availability and cross-reactivity for dogs (1,(9)(10)(11). Quite a few of those did not work in our hands (Table S5).…”

Section: Introductionmentioning

confidence: 99%

OMIP‐065: Dog Immunophenotyping and T‐Cell Activity Evaluation with a 14‐Color Panel

Pantelyushin

Ranninger

Bettschart‐Wolfensberger

et al. 2020

Cytometry Pt A

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Companion dogs are increasingly recognized as large-animal models of diseases such as cancer, infectious-, inflammatory-, or autoimmune diseases. At the same time, compared to human clinics, veterinarians have only a fraction of the treatment options available. To study the immunological aspects of canine diseases and ultimately develop or adapt human treatments for the dog, the methodology also needs to be in place. Such tools include robust and reliable flow cytometric panels. The purpose of the panel described here is to assess the immune cell composition and their functionality in the peripheral blood mononuclear cells (PBMCs) of dogs. Moreover, its "plug and play" composition allows for an in-depth analysis of T-cell responses in ex vivo assays (Table 1). Initially, this panel has been designed for the analysis of cryopreserved PBMCs to allow batched analysis and to reduce interexperimental variation. Withers and colleagues published a comparable and-to our knowledge-currently the most extensive canine panel to date (1). While their study focused on the aging and activation status of T cells in dogs, our panel is designed to look at a broader range of cells with a higher number of markers. This allows a more in-depth analysis of functional extracellular and intracellular markers. In addition, all antibodies in our proposed panel are directly labeled. In combination with suitable lymphocyte isolation protocols, this panel could potentially also be adapted to analyze tissue biopsies from various different organs.

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Multi-color flow cytometry for evaluating age-related changes in memory lymphocyte subsets in dogs

Cited by 27 publications

References 81 publications

Development of Dog Immune System: From in Uterus to Elderly

Development of Dog Immune System: From in Uterus to Elderly

Development of canine PD-1/PD-L1 specific monoclonal antibodies and amplification of canine T cell function

OMIP‐065: Dog Immunophenotyping and T‐Cell Activity Evaluation with a 14‐Color Panel

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