Lymphocyte immunophenotype in dogs with immune-mediated hematologic disease

Abstract

Immune-mediated hematologic diseases (IMHD) including immune-mediated hemolytic anemia (IMHA) and immune thrombocytopenia (ITP) can cause severe disease in dogs. The underlying immune system abnormalities associated with these conditions is not known. The hypotheses of this study were that dogs with IMHD would have increased frequencies of CD4+ and CD8 + lymphocytes, decreased frequencies and numbers of T regulatory cells, and increased frequencies of interleukin (IL)-17 + lymphocytes. Fifteen dogs with newly diagnosed IMHA or ITP and 15 healthy control dogs were recruited for this prospective study. Flow cytometry was used to enumerate CD4 + lymphocytes, CD8 + lymphocytes, T regulatory (CD4 + CD25 + Foxp3+) cells, and lymphocytes secreting IL-17 in dogs with IMHD at diagnosis, then 2 and 4 days after starting immunosuppressive treatment. Median proportion of CD4+ (Day 0: 3.4%, Day 2: 3.3%) and CD8+ (Day 0: 1%, Day 2: 0.6%) cells was lower in dogs with IMHD compared to control dogs (CD4 + 22.8%, CD8 + : 13.6%; P < 0.0001 for each). Additionally, T regulatory cells were reduced in IMHD dogs at Day 0 (0.2% versus 0.6% of total lymphocytes, P = 0.0025). Dogs with IMHD had a higher proportion of lymphocytes positive for IL-17 at Day 2 (1.3%) compared to control dogs (0.4%, P = 0.0024). Dogs with IMHD have immune system alterations at diagnosis and during early treatment characterized by a deficiency in T regulatory cells and an increase in IL-17 + lymphocyte. These changes might contribute to the pathogenesis of IMHA and ITP.

Fig 1. Characterization of lymphocyte subsets using flow cytometry.

Lymphocytes were identified based on forward and side scatter characteristics (A) and then gated for further analysis. Gating for positive events was based on unstained samples for each dog, where the negative quadrant was set to include >99.5% of cells. The gating strategy was used to identify CD4+ and CD8 + cells (B). CD4 + cells were gated from the lymphocyte population (C) and then further evaluated for CD25+ and Foxp3 + positivity to show T regulatory cells (D). An unstained control is shown in (E) to compare the IL-17 positive lymphocytes in (F). The total number of IL-17 positive lymphocytes was derived from the CD4- and CD4 + populations that were considered positive for IL-17, depicted in the two upper quadrants of this image.

Fig 2. Proportion of lymphocyte subsets in the total lymphocyte population of health dogs and dogs with IMHD.

Proportion of CD4+ (A), CD8+ (B), CD4 + CD25 + Foxp3+ (T regulatory cells; C), and IL-17+ (D) cells is as a percentage of the total lymphocyte population. All p values are in comparison to the control population. All data distribution is non-parametric. (CD, cluster of differentiation; Foxp3, forkhead box protein 3; IL, interleukin.).