B cell targeted therapies (BCTT) are used as effective treatment options to eliminate auto-reactive B cells in autoimmune diseases. Adverse effects of BCTT can include hypogammaglobulinemia (HG) with concomitant infection risk. A subset of children developing persistent HG (pHG) post-BCTT may have an undiagnosed inborn error of immunity (IEI) that can potentially be unveiled through deep immunophenotyping of peripheral blood mononuclear cells (PBMCs) from patients. Immune cell subsets that are altered/impaired, but not directly secondary to BCTT, may indicate underlying IEIs.
Patients receiving BCTT at Sidra Medicine were recruited pre-/post-BCTT treatment. The 24-color deep immunophenotyping panel was designed and optimized on the Cytek Aurora flow cytometer. The panel is now being implemented on patient samples along with age-matched healthy donors for phenotype characterization. The gating strategy was refined for effective data analysis using FlowJo Software. Patients developing pHG will be selected for IEI investigation through whole genome sequencing and a primary immunodeficiency (PID) gene panel.
The high-dimensional 24-color panel was successfully optimized on healthy donor samples, including antibody titration. Samples analyzed simultaneously on two flow cytometers in Sidra’s Research and Pathology departments (College of American Pathologists [CAP] accredited) were relatively comparable, demonstrating reproducibility and cross-instrument consistency. From 74 BCTT-treated study patients, 8 (10.8%) developed pHG. In the deep immunophenotyping conducted on a lupus patient with pHG, the B cell subsets displayed a significant reduction in the plasmablast population, which are not directly targeted by the anti-CD20 BCTT treatment. This may suggest an underlying immunodeficiency that requires further investigation through functional and genetic studies for confirmation.
Successful troubleshooting and cross-platform validation of the 24-color panel sets the stage for its implementation in our patient cohorts, where it may uncover immunophenotypic alterations indicative of IEIs. Further examples for the utilization of this extended panel on the suspected IEI cases are still ongoing and will be presented in the upcoming stages.
