Development of a 51-marker / 44 colors full spectrum flow cytometry assay for longitudinal immune monitoring in patients with srSLE/LN treated with CD19 CAR T cells

12:15 - 12:35

Day 2 – February 6, 2025

Abstract

Adoptive immunotherapy using chimeric antigen receptor (CAR) T-cells, specifically targeting CD19, has demonstrated successful remissions in treatment-refractory B-cell malignancies such as B-cell lymphoma and leukemia. This therapeutic approach is now being explored for B cell-driven autoimmune diseases, including Systemic Lupus Erythematosus (SLE). SLE is a heterogeneous, chronic autoimmune disorder that, in its severe form, leads to significant tissue and organ damage. SLE is caused by the breakdown of immune tolerance to nucleic acids. The pathogenesis of SLE involves profound phenotypic and functional changes in various immune cell types, including dendritic cells (DCs), monocytes, T cells, regulatory T cells (Tregs), B cells, and long-lived plasma cells, and is characterized by the activation of the type I interferon system.
Flow cytometry-based cellular assays have been developed for longitudinal studies with specific focus areas such as CD19 CAR T cells, B cells, or basic pan-immunophenotyping assays like extended TBNK panels. However, these assays are often limited, employing discrete panels of typically up to 13+ markers, thereby necessitating larger patient sample sizes, that due to the nature of the disease (e.g. lymphopenia) or the treatment regimen (e.g. lymphodepletion) may not be accessible in clinical trial settings. A single, comprehensive in-depth panel encompassing and extending existing immune system monitoring capabilities can offer a better understanding and monitoring of disease mechanisms in patients undergoing CD19 CAR T cell treatments. Such an extended assay would facilitate improved assessment of B cell depletion and reconstitution within a holistic view of the patient’s immune system.
To address these limitations, we report the comprehensive validation of a single-tube, full-spectrum flow cytometry assay comprising 51 markers and 44 colors, along with its corresponding data analysis pipeline, tailored for longitudinal studies in CD19 CAR T cell-treated patients. This panel provides broad coverage of various cell subsets (myeloid cells, T cells, B cells, and natural killer (NK) cells) and can be applied transversally to monitor immune system dysregulations in other disease contexts. Additionally, the panel includes an optimized staining protocol for CD19 CAR T cells and intracellular staining for transcription factors such as FOXP3 and Helios, as well as markers and cell types relevant to SLE pathogenic mechanisms (Tregs, T follicular helper cells, double-negative B cells, plasma cells, plasmacytoid dendritic cells, etc.). Furthermore, we demonstrate the successfully application to clinical-grade cryopreserved peripheral blood mononuclear cells (PBMCs) from CD19 CAR T cell-treated patients.

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