Date of Award

5-2021

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Betty Diamond

Abstract

Systemic Lupus Erythematosus (SLE) is an autoimmune disease that causes significant morbidity and increased mortality in affected people. There is strong evidence supporting a role of B cells, plasma cells (PCs) and autoreactive antibodies in disease pathogenesis and organ damage.

The presence of pathogenic autoreactive antibodies in patients with SLE suggests an impairment of the immune tolerance checkpoints that normally precludes their appearance in healthy people. The mechanism for this alteration in tolerance is controversial. Part of the objective of this thesis was to understand the cellular pathway to autoreactive PCs in SLE and the mechanisms that generate them. We approached this question using a flow cytometric assay developed in our laboratory; this assay allows us to detect with high throughput and at single cell resolution B cells that recognize nuclear antigens (ANA+). Using this method, we showed that the frequency of ANA+ B cells diminishes progressively with B cell maturation and differentiation, and that this process does not differ between patients with SLE and healthy controls, even in the plasmablast subset. These data suggest that selection mechanisms for ANA+ B cells may be intact in SLE. However, notwithstanding that the percentage of ANA+ plasmablasts is similar, there is an increment in the total number of IgG plasmablasts in patients with SLE, with a consequent increment in the total number of ANA+ IgG plasmablasts. These findings suggest enhanced IgG PC differentiation in patients with SLE.

We also tried to understand how tolerance is perturbed in patients who are treated with antibodies that cause B cell depletion. After B cell depletion the circulating levels of BAFF increase and studies in mice showed that this cytokine promotes maturation of autoreactive B cells following B cell depletion. These observations allowed us to hypothesize that belimumab, an antibody that targets soluble BAFF, might help to prevent the emergence of autoreactive B cells in patients with SLE. We evaluated the sequential treatment of rituximab and belimumab vs rituximab alone in patients with lupus nephritis (LN) in CALIBRATE, a randomized, multicenter, open-label clinical trial. Forty-three patients with recurrent or refractory LN were included. We used the same flow cytometric assay to evaluate the percentages of total and autoreactive B cell subsets in peripheral blood from these patients. We showed that the combination regimen diminished maturation of transitional to naïve B cells during B cell reconstitution and enhanced the negative selection of autoreactive B cells. The study was not designed to detect clinical differences.

Considering our results that suggest that PC differentiation is an important phenomenon in patients with SLE, we considered the use of metformin, a commonly used drug with metabolic effects and with recently described immunomodulatory effects, as a potential therapeutic agent to block this increased differentiation. Metformin has been used as treatment for SLE in mouse models and in small trials of SLE patients. We found that metformin reduces the in vitro differentiation of memory B cells into PCs induced by the TLR9 agonist CpG. The mechanistic analysis is still ongoing at the time of writing this thesis, but so far, we have showed that metformin reduces the internalization of CpG into B cells. The computational molecular modeling analysis shows that metformin is also able to bind LAIR1, and we are conducting studies to investigate the relevance of this interaction. IV SLE is a heterogeneous disease, and this is one of the reasons that has been proposed to explain the failure of many clinical trials. We explored this heterogeneity focusing on the origin and generation of PCs. There are two main pathways for differentiation of B cells into PCs, the extrafollicular (EF) and germinal center (GC). Both pathways are relevant in SLE and have substantial differences. There is a comprehensive review of the differences and implications of each of those pathways in Chapter 5. We used our cytometric assay to explore the distribution of autoreactive antigen experienced cells in patients with SLE. We showed that in patients with an expansion of circulating plasmablasts, there are two patterns: one characterized by increased memory B cells and other by a higher expansion of plasmablasts without a concomitant increment in memory cells. These patterns were consistent over time, and were not dependent on disease activity or treatment. They also were associated with different profiles of circulating autoantibodies. Furthermore, these profiles were similar to what we observed in mice that were immunized with antigens that characteristically generate a T-dependent (high memory B cells) or T-independent (higher PCs) response; they were also reminiscent of the distribution of antigen experienced cells in two SLE-mouse models: New Zealand black/white (NZB/W), with a more defined GC response and relatively increased memory cells, and MRL/lpr with a prominent EF response with a higher percentage of PCs. Our results suggest that the generation of autoreactive PCs might be associated with either a predominant GC or EF response, and that patients with SLE can be grouped according to the pathway preferentially activated.

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