Lymphocyte subset variability between human lymphoid tissues

Abstract

Most adaptive immune lymphocytes are in the lymphoid tissues. However, studies of human lymphocytes to date have primarily investigated cells from the blood, and lymphoid tissues remain poorly studied. This thesis explores the hypothesis that there is local variation of B cell subsets between the tissues that are micro-anatomically different and receive antigenic stimulation via different routes.

Matched samples of appendix, mesenteric lymph nodes (mLNs), and spleen from human cadaver were analysed by mass cytometry (cytometry by time of flight [CyTOF]) for deep phenotypic profiling and tissue-specific subset variation. Populations of B and T cells that had significant abundance differences between the three tissues were identified in an unsupervised manner. The subsets that varied significantly in abundance between tissues included: germinal centre (GC) B cells, marginal zone (MZ) B cells, IgM-only B cells, IgA+ B cells, follicular helper T cells and regulatory T cells, as well as CD4+ and CD8+ effector memory cells. These differentially abundant subsets were further characterised by their different marker expression profiles between tissues, and results were confirmed using a second supervised analysis method. Most notably from the unsupervised analysis and subsequently the supervised analysis, MZ B cells were further divided into two populations based on their tissue-specific abundance and distinct phenotype including differences in CCR7 and BAFF-R expression. IgM-only B cells also included two populations with different phenotype and tissue-specific abundances. Single-cell RNA sequencing was used to further investigate the differentiation, functional, and dissemination differences of these subsets between lymphoid tissues. The two MZ B cell populations were transcriptomically distinct and had significant tissue-specific differential abundances. Within each tissue, B cell clones from the CCR7+ MZ B cell subset were significantly associated with IgM-only subset whereas the other CCR7- MZ B cell subset had a greater association with activated naive and double negative B cells. Between the paired tissues, members of the same B cell clone were observed in multiple tissues. Clones of CCR7+ MZ B cells and IgM-only B cells were more disseminated between tissues than other subsets including the CCR7- MZ B cells. On the other hand, transitional and naive B cells only had significant correlation within the same tissue and not between tissues implying local maturation in tissues.

Overall work in this thesis identifies regional variation in B cell subset abundance, tissue based local B cell differentiation and also clonal links between B cell subsets in distant human tissues sites.