Abstract
Systemic lupus erythematosus (SLE) is an autoimmune disease characterised by the production of autoantibodies, predominantly to nuclear components, and the loss of B cell tolerance. Changes and imbalances in B cell subsets are a key aspect of lupus pathology, however these alterations are not fully understood.Transitional B cells are a population of immature B cells that are the first cells to exit the bone marrow and enter the periphery. Transitional B cells emerge in the peripheral blood as transitional 1 (T1) B cells, which then mature into T2 B cells. Two populations of T2 B cells have been characterised and can be distinguished based on their surface expression of IgM. T2 IgMhi B cells have been linked to a gut-homing, integrin β7+ developmental trajectory that leads to the generation of marginal zone B cells, a subset known to be depleted in lupus nephritis. Several changes in the transitional B cell subset in SLE have also been reported, including the expansion of T1 B cells, depletion of T2 IgMhi B cells as well as dysregulated IL-10 signalling and increased survival in the transitional B cell subset. This study used multiple techniques to understand the diversity of the earliest subsets of B cells in the peripheral blood in lupus nephritis and how this compares to what is seen in healthy individuals, with the aim of improving our understanding the makeup of the peripheral B cell compartment in systemic lupus erythematosus (SLE). Transitional B cells are the cells in which all mature B cell subsets develop from and therefore are of particular interest when it comes to investigating altered peripheral B cell development.
Single-cell RNA-sequencing with CITE-seq antibody staining was used to investigate the gene expression patterns and surface marker expression of CD10+ transitional and CD10- naïve B cells from peripheral blood to gain an insight into how these early, antigen-inexperienced B cells differ in lupus nephritis compared to health with the aim of highlighting key features that may underpin differential B cell development seen in lupus nephritis. This analysis firstly highlighted the heterogeneity of transitional B cells in health and emphasised that whilst CD10+ transitional B cells are typically categorised into T1 and T2 subsets, there are distinct transcriptomic differences between cells belonging to the same subset. In addition, this study identified early, antigen-inexperienced cells with an interferon gene signature, which can be seen in healthy and lupus nephritis bloods.
Following on from the observations made by analysis of single-cell RNA-sequencing data, mass cytometry and flow cytometry were used to explore interferon surface marker expression on blood from healthy donors and lupus nephritis patients. Data generated from these cytometry experiments suggest that transitional B cells express IFITM1 significantly more than all other peripheral B cell subsets in both health and lupus nephritis, and expression of IFITM1 does not differ between health and lupus despite the well-documented interferon signature seen in lupus. IFITM1 expression in transitional B cells correlated with the expression of integrin β7 and IgM, however other interferon markers did not, suggesting a possible link between IFITM1 and the gut-homing B cell developmental trajectory.
Additionally, this study used flow cytometry to compare the proportions of B cell subsets in paired blood samples from lupus nephritis patients at different timepoints during their disease course: when they were flaring, and disease activity was high versus when they were stable and disease activity was lower. This data demonstrated the plasticity of the B cell compartment in peripheral blood. The proportions of subsets were found to be significantly altered in periods of lupus flare compared to health, but in some instances, they returned to levels comparable to health upon disease stability. This data also revealed a reduction in naïve B cells with low IgM expression in lupus nephritis compared to health.
During this study, in a separate project, two subsets of marginal zone B cells were identified by single-cell RNA-sequencing of B cells in tissue from three sites in the gut associated lymphoid tissue. As part of this study, flow cytometry was used to validate the presence of these two marginal zone B cell populations in peripheral blood and investigate the changes in proportions of the two marginal zone subsets in lupus nephritis compared to health. CCR7 and integrin β7 were used to distinguish the marginal zone populations by flow cytometry. Previously it has been established that CD27+IgD+ marginal zone B cells are depleted in lupus nephritis, in this study, it was found that only one of the two novel subsets of marginal zone B cells was depleted in lupus nephritis.
The data presented in this thesis highlight the heterogeneity of transitional B cells in peripheral blood and suggest that early B cells that are naïve to antigen have been influenced by interferon and that this may be key to development of the gut-homing trajectory in both health and lupus nephritis. Additionally, it is evident from the data presented in this study that expression of different interferon inducible protein markers is not all the same and that it may be of use in future studies to explore these proteins individually. Data from paired lupus nephritis samples across different disease activity levels highlighted plasticity in the peripheral blood B cells compartment. Finally, this study has validated the presence of two populations of marginal zone B cells in peripheral blood and has shown that only one of these populations is depleted in lupus nephritis.
Date of Award | 1 Aug 2023 |
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Original language | English |
Awarding Institution |
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Supervisor | Jo Spencer (Supervisor) & David D’Cruz (Supervisor) |