PSVIII-9 Longitudinal characterization of lymphocyte subsets and humoral immune markers in growing Labrador retrievers.

Abstract This study aimed to characterize the development of the immune system from puppyhood to adulthood in Labrador retrievers. Monthly blood samples were collected from 21 puppies (11M/10F) from 1 month to 1 year of age. For flow cytometry, EDTA whole blood was stained with a multi-color panel including LIVE/DEAD lime viability stain, anti-canine CD4 antibody conjugated to super bright 600, anti-canine CD8 antibody conjugated to super bright 702, anti-canine CD3 antibody conjugated to FITC, anti-canine CD21 antibody conjugated to r-PE, anti-canine CD5 antibody conjugated to PerCP-eFluor 710, and super bright staining buffer. Samples were then lysed and analyzed on an Attune NxT flow cytometer. Gates were determined according to unstained samples, and data was recorded as the percentage of lymphocytes, with the exception of CD4+ and CD8+ cells which were recorded as the percent of CD3+ cells. Plasma samples were utilized for D2Dx analysis at 12, 24, 36, and 50 weeks of age. Data was analyzed in SAS using a repeated measures mixed model with fixed effects of age, sex, and age*sex with dog as the repeated subject, and visualized in JMP. As expected, there were significant effects of age, sex, and age*sex on weight (P ≤ 0.003), with puppies growing over the source of the study, and males on average weighing more than females. There were significant age and sex effects (P ≤ 0.024) for the percentage of CD3+ T cells, which increased throughout the study and which was higher in females. There was an age effect (P < 0.001) for the percentage of CD21+ B cells which decreased as the subjects aged. There were age and sex effects (P ≤ 0.043) for CD5dim cells, tentatively identified as natural killer cells, with levels decreasing over time, and males on average having lower levels than females. The percentage of CD3+CD4+ T cells had significant effects of age and age*sex (P ≤ 0.044), with percentages increasing over time. The percentage of CD3+CD8+ T cells had significant age and sex effects (P ≤ 0.022) with percentages decreasing as the subjects aged, and males having overall lower percentages compared to females. The ratio between CD3+CD4+ and CD3+CD8+ cells had a significant age effect (P < 0.001), but unexpectedly increased considerably over the course of the study. D2Dx scores, a measure of non-cellular humoral immunity, increased significantly between 12, 24, and 36 weeks (P < 0.001), but remained stable between week 36 and the end of the study. Overall, these findings provide valuable insights for researchers and clinicians regarding immune system maturation in Labrador retrievers.