A novel method for isolation and flow cytometry analysis of intraepithelial lymphocytes from colon biopsies

Investigating the immune responses of the intestine in response to different insults is predominantly limited to indirect methods such as circulating markers of intestinal health or gene expression from dissections. We describe here a validated protocol for the isolation and subsequent flow cytometry analysis of intestinal intraepithelial lymphocytes (IEL) from colonic biopsy samples. Colon biopsy samples were collected with endoscopy forceps from Holstein dairy bull calves at d 2, 28, and 42 of life. The biopsies were put into an isolation solution of Hanks' balanced salt solution, and fetal bovine serum followed by digestion solution. The solution was filtered and the flow-through, containing IEL, was stained with fluorescent antibodies for flow cytometry analysis. Density gradient separation of the isolate yielded higher viability and cleaner samples for flow cytometry analysis. Anti-bovine γ chain of the T cell receptor was used to identify populations of gamma delta (γδ) T cells via flow cytometry. In addition, γδ T cell subsets were identified using an anti-bovine antibody against the coreceptor workshop cluster 1. This method allowed for the precise identification of lymphocyte populations and evaluation of the proportion of different subsets of γδ T cells from intestinal IEL over time. The technique described here will allow the research community to characterize intestinal immune function over time and improve our understanding of how different management and nutritional strategies affect intestinal health

Developmental adaptations of γδ T cells and B cells in blood and intestinal mucosa from birth until weaning in Holstein bull calves

ABSTRACT: This study aimed to characterize the development of systemic and colon tissue resident B and γδ T cells in newborn calves from birth until weaning. At birth, calves have limited capacity to initiate immune responses, and the immune system gradually matures over time. Gamma delta (γδ) T cells are an important lymphocyte subset in neonatal calves that confer protection and promote immune tolerance. A total of 36 newborn calves were enrolled in a longitudinal study to characterize how systemic and colon tissue resident B and γδ T cells develop from birth until weaning. Blood and colon biopsy samples were collected on d 2, 28, and 42 to determine the proportions of various B and γδ T cell subsets by flow cytometry. We classified γδ T cells into different functional subsets according to the level of expression intensity of the coreceptors WC1.1 (effector function) and WC1.2 (regulatory function). Furthermore, naive B cells were classified based on the expression IgM receptor, and activation state was determined based on expression of CD21 and CD32, 2 receptors with opposing signals involved in B cell activation in early life. Additional colon biopsy samples were used for 16S sequencing, and microbial diversity data are reported. At birth, γδ T cells were the most abundant lymphocyte population in blood, accounting for 58.5% of the lymphocyte pool, after which the proportions of these cells declined to 38.2% after weaning. The proportion of γδ T cells expressing WC1.1 decreased by 50% from d 2 to d 28, whereas no change was observed in the expression of WC1.2. In the colon, there was a 50% increase of γδ T cells after weaning and the proportion of WC1.2+ γδ T cells doubled from d 28 to 42. The proportion of IgM+ B lymphocytes in blood increased from 23.6% at birth to 30% after weaning, were the proportion of B cells expressing CD21 increased by 25%, while the proportion of B cells expressing CD32 decreased by 30%. While no changes were observed for the overall proportion of IgM+ B lymphocytes in the colon, there was a 6-fold increase in the proportion of CD21+ B cells from pre- (d 28) to postweaning (d 42). Microbial diversity increased from d 2 of life to 28 and declined abruptly after weaning. The reduction in microbial diversity during weaning was negatively correlated with the increase in all γδ T cell subsets and CD21+ B cells. These data suggest that developmental adaptations after birth coordinate expansion of γδ T cells to provide early systemic protection, as well as to steer immune tolerance, while B cells mature over time. Additionally, the increase of colonic γδ T cells on d 42 suggests a protective role of these cells during weaning