Mechanisms for B cell tolerance following deep B cell depletion

Systemic Lupus Erythematosus (SLE) is a challenging autoimmune disease in which the body’s immune system makes antibodies that deposit in various tissues causing inflammation and damage. For patients with severe lupus that has not responded to traditional treatments, genetically modified cellular treatments are ushering in a new era of. This revolutionary approach works by entering tissues and eliminating all B cells including those that are responsible for producing the harmful antibodies that cause tissue injury. Several studies have shown that many patients treated with these chimeric (CAR-T) cells achieve complete, often drug-free, remission. However, despite this success, we still do not fully understand how these treatments achieve such deep remissions, why some patients are resistant or relapse sooner than others, and which specific cells are involved when the disease returns.

Our research aims to address these critical questions using mouse models that naturally develop lupus similar to the human disease. These models allow us to study the immune system in much greater detail than is possible in human studies and to track changes in multiple organs over time. We hypothesize that the long-term success of B cell-depleting therapies is limited by two main factors. First some antibody-producing cells (plasma cells) might survive the treatment because they don’t display the target of the cell therapy on their surface or by hiding in protected areas of the body. Second, other immune cells (like T cells) and a general inflammatory state might persist or reactivate, potentially triggering reactivation of surviving B cells or causing new harmful ones to expand.

In Aim 1 we will investigate where the autoreactive B cells come from when they regenerate after treatment and we will identify and characterize any treatment-resistant antibody-producing cells remaining in different organs. We’ll also examine the activation status of the newly developing B cells and whether they have entered a more tolerant state. In Aim 2, we will explore whether lasting remission is maintained reprogramming of other immune cells (such as T cells) and a reduced inflammatory state, rather than simply the absence of B cells and study how different T cell subsets function after treatment.

By understanding the specific immune conditions that lead to remission or relapse, we hope to make B cell removal treatments even more effective. We also hope to understand whether additional treatment, such as follow-on maintenance treatment, needs to be individually tailored. Our ultimate goal is to control the clinical manifestations of SLE with the least immunosuppression possible.

The advent of anti-CD19 based CAR-T and other deep B cell depletion therapies is beginning to revolutionize the treatment of refractory SLE, inducing complete and often drug-free remissions. However, the precise mechanisms of this therapeutic innovation, the factors that influence the variable duration of the response and the cells involved in relapse remain incompletely understood. Here we will use informative mouse models to determine the origins of autoreactive B cell reconstitution and to define a role for reprogramming of T cells in maintaining durable remission or in driving disease relapse.

We will treat well-established polygenic mouse models of SLE (Sle1.Yaa and NZB/W) with an anti-CD19 bispecific T cell engager to achieve deep tissue B cell depletion, allowing us to perform longitudinal analyses of multiple organs. We hypothesize that the durability of remission following deep B cell depletion is limited by two key factors: 1) the survival of therapy-resistant autoreactive plasma cells (PCs) within protective niches, and 2) the persistence or reactivation of a pathogenic autoimmune environment, including autoreactive T cells and innate immune activation, which could both trigger disease relapse from either surviving or newly generated autoreactive B cells.

In Aim 1 we will determine the contribution of B cell subsets, locales, and intrinsic factors to the re-emergence of pathogenic autoantibodies by performing high-dimensional flow cytometry of B cell subsets. Using a flow cytometry reagent that detects a population highly enriched in autoreactivity we will evaluate the developmental stage and anergic status of the autoreactive repertoire that emerges during B cell reconstitution.  Using flow cytometry, ELISpot assays and immunohistochemistry we will identify and characterize therapy-resistant, autoantibody-secreting PCs that survive depletion across major organs. Finally, we will track changes in the autoreactive immunoglobulin repertoire before and after treatment to understand how tolerance is enforced during B cell reconstitution.

In Aim 2 we will test whether remission following B cell depletion is maintained by durable reprogramming of T cells together with a reduced inflammatory burden, rather than by B cell depletion alone. We will assess the phenotype of memory, T follicular helper (Tfh), and T follicular regulatory (Tfr) cells across tissues using high-dimensional flow cytometry and measure the metabolic status of naive, activated and memory T cells by flow cytometry. We will further test whether autoreactive T cells are still functional after deep B cell depletion by performing co-culture experiments with B cells from reconstituted or control mice. Results from these studies will be used to determine which maintenance therapies may be most helpful to prevent relapse.

Together, our experiments will provide insights into the complex immune environment generated by deep B cell depletion in SLE. Understanding the specific immune conditions that govern therapeutic response and relapse will be crucial for optimizing B cell depletion strategies, enabling more personalized treatment and achieving disease quiescence with less immunosuppression.