T Cell Immune Profiles of Blood and Tumor in Dogs Diagnosed With Malignant Melanoma

Abstract

Investigation of canine T cell immunophenotypes in canine melanomas as prognostic biomarkers for disease progression or predictive biomarkers for targeted immunotherapeutics remains in preliminary stages. We aimed to examine T cell phenotypes and function in peripheral blood mononuclear cells (PBMC) and baseline tumor samples by flow cytometry, and to compare patient (n = 11–20) T cell phenotypes with healthy controls dogs (n = 10–20). CD3, CD4, CD8, CD25, FoxP3, Ki67, granzyme B, and interferon-γ (IFN-γ) were used to classify T cell subsets in resting and mitogen stimulated PBMCs. In a separate patient cohort (n = 11), T cells were classified using CD3, CD4, CD8, FoxP3, and granzyme B in paired PBMC and single cell suspensions of tumor samples. Analysis of flow cytometric data of individual T cell phenotypes in PBMC revealed specific T cell phenotypes including FoxP3+ and CD25+FoxP3- populations that distinguished patients from healthy controls. Frequencies of IFN-γ+ cells after ConA stimulation identified two different patient phenotypic responses, including a normal/exaggerated IFN-γ response and a lower response suggesting dysfunction. Principle component analysis of selected T cell immunophenotypes also distinguished patients and controls for T cell phenotype and revealed a clustering of patients based on metastasis detected at diagnosis. Findings supported the overall hypothesis that canine melanoma patients display a T cell immunophenotype profile that is unique from healthy pet dogs and will guide future studies designed with larger patient cohorts necessary to further characterize prognostic T cell immunophenotypes.

Acupuncture regulates the Th17/Treg balance and improves cognitive deficits in a rat model of vascular dementia

“…Treg cells also exhibit a degree of plasticity and can differentiate into other effector T cell subtypes in particular autoimmune contexts [ 46 , 47 ]. Treg cells include FoxP3 + (CD25 − and CD25 + cells) or CD25 + FoxP3 + cells, with FoxP3 serving as a central transcription factor associated with Treg differentiation and functionality [ 48 , 49 ]. IL-10 is a potent anti-inflammatory cytokine, and FoxP3+ T regs can promote IL-10 secretion and aid in recovery in models of experimental autoimmune encephalomyelitis [ 50 ].…”

Safety profile and effects on the peripheral immune response of fecal microbiota transplantation in clinically healthy dogs

“…Staining protocols were used as previously described 54‐56 . When possible, a minimum of 1 million cells were stained for PBMC.…”

“…[51][52][53] 2.8 | Isolation of canine peripheral blood mononuclear cells from whole blood Whole blood was obtained using EDTA-containing vacutainer tubes and processed as previously described. 54 Peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation using Histopaque 1077 (Sigma-Aldrich, Saint Louis, Missouri, USA). The PBMC pellets were treated with RBC lysis buffer (Biolegend, San Diego, California, USA) and cryopreserved in media containing 45% heat inactivated fetal bovine serum, 45% heat inactivated dog serum (Equitechbio, Kerrville, Texas, USA), and 10% dimethyl sulfoxide.…”

Use of multi-color flow cytometry for canine immune cell characterization in cancer

“…Moreover, methods for simultaneous detection of lymphoid and myeloid cells within neoplastic tissues has not been reported. A recent study described a 5-color flow cytometry panel that enables to reveal differences between T cell subsets in the blood and those in melanoma tissues [ 12 ]. However, this panel is designed to characterize specific T cell subsets, rather than broad categories of immune cell subsets.…”

“…Herein we describe a method for multi-color flow cytometry using a minimum of nine antibodies to detect canine immune cells and validate applicability of this panel in various canine neoplastic samples including blood, lymph node, and solid tumor. To date a majority of studies base their insights into tumor microenvironment on immuno-histochemical studies, or small, limited panels targeting specific types of cells [ 12 – 15 ]. The approach described in this study enables the characterization of multiple immune cell subsets rather T cells only in one tube, which will help to build new insights into tumor microenvironment in translational cross-species studies.…”