If you have trained in the field of immunology, it may not be uncommon to emerge with a self-characterization as either a “B-cell immunologist” or a “T-cell immunologist”. Historically, this distinction arose from years of specialization and the degrees of separation between T-cell and B-cell biology research. Approaches to therapeutic design have often mirrored that, with either a T-cell or B-cell-specific approach.
However, as a broader understanding of immune networks and coordinated immune responses has emerged, this line is increasingly blurred. Advanced technologies like flow cytometry enable deep interrogation of both T and B cell populations. As a result, for those working in drug development and clinical research, it is more important than ever to have a comprehensive understanding of both T- and B-cell biology and a knowledge of how to leverage advanced technology to distinguish and resolve these populations.
In this two-part blog series, we will consider how flow cytometry is supporting an advanced understanding of B- and T-cell phenotype and function. Specifically, in part one of this series we will highlight KCAS Bio’s newest validated spectral flow cytometry B cell panel, designed to offer an in-depth evaluation of this key immune cell subset.
B cells, at the forefront of Immunology
B cells have a diverse and multifunctional role in immune response. While traditionally associated with antibody production, multifunctional B cells are also important cytokine producers and serve as professional antigen-presenting cells.
In humans, B-cell genesis begins in the bone marrow, where hematopoietic stem cells (HSC) differentiate first into immature naïve B cells and then into transitional B cells. Exiting the bone marrow, transitional B cells move into the periphery and develop into mature naïve B cells. Following recognition of a cognate antigen, the mature naïve B cells are activated, proliferate, and further differentiate into memory B cells, short-lived plasmablasts, or long-lived plasma cells. These cells can rapidly activate, proliferate, and produce antibodies and cytokines. In healthy individuals, the activation is induced by foreign antigens from infection or protective vaccination. The subsequent production of antibodies protects against pathogens and are key for vaccine-mediated immune protection.
B cells in Autoimmunity
While B cells are essential for maintaining immunity, they can also be dysregulated and contribute to pathology and disease. In autoimmune diseases, autoreactive B cells are a critical driver in the complex immune environment found in rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), type 1 Diabetes, and others. A dysregulated B cell response can be characterized by a break in tolerance or molecular mimicry response that results in an attack of “self.” Indeed, in a variety of autoimmune diseases, the pathological impact of tissue-targeting autoantibodies is well documented.
Effective treatments for B cell-driven diseases have included B cell targeted immunotherapies. One such approach has been monoclonal antibody therapies, which are safe and well-tolerated. The production of monoclonal antibody therapies in large-scale manufacturing results in a low risk of product variability. However, the relatively short therapeutic half-life requires multiple administrations, and limited overall B-cell depletion has driven the need to explore therapeutic alternatives (Li, 2024).
Another approach to target B cells is the use of cellular therapies. With recent successes in B cell malignancies, there is a growing interest in understanding how B-cell targeted CAR-T cell therapy can be successfully applied to autoimmune disease. Early studies have shown promise in a variety of B cell-driven diseases such as SLE, Sjogren’s, and myasthenia gravis among others. While CAR-T therapies also have unique challenges, there is a critical need to understand the impact of CAR-T therapy on the B cell subsets in this emerging space.
Comprehensive B cell phenotyping by flow cytometry
Given the dual role that B cells play in health and disease advanced, scalable research tools like high-dimensional flow cytometry are essential. A well-developed flow cytometry phenotyping panel enables precise characterization of B cell subsets and functional state in diverse disease contexts. Phenotyping panels by flow can help support the needs of researchers in the pharmaceutical and biotech industries working on innovative therapies in autoimmunity, inflammation, and oncology.
KCAS BIO Validated B cell Panel
As we discussed in our blog, “All Mixed Up? Implementing Best Practices for Unmixing in Spectral Flow Cytometry”, designing high-complexity panels requires thoughtful fluorophore selection to minimize spectral overlap and maximize signal resolution. Leveraging in-house expertise, KCAS Bio embarked on the design and validation of a spectral flow cytometry panel to facilitate the detection and characterization of diverse B-cell subsets in human PBMC samples.
With a 23-color design, KCAS Bio’s validated B cell panel includes antibodies to detect and discriminate live leukocytes and B cell subsets, including naïve transitional cells, unswitched, switched, and double negative switched memory B cells, plasmablasts, and plasma cells. Recognizing that spectral flow cytometry has an inherent ability to enable deeper discrimination of population subsets, KCAS Bio also added markers to evaluate key sub-populations such as:
- Resting and activated switched memory cells
- Resting and/or activation status of bulk populations: through the inclusion of antibodies to detect expression of Ki-67, CD95, and/or CD86.
Given the key role that B cells play in autoimmunity, we also recognized that the ability to detect B cells with an autoimmune-associated phenotype is critical. Accordingly, antibodies against T-bet, CD11c, CD21, and CXCR5 are included in the 23-color design. In total, more than 60 reportables can be determined from the validated panel.
Sample Selection, Method Precision and Stability:
Precision is a cornerstone of any reliable flow cytometry assay. KCAS Bio’s panels are rigorously validated to ensure reproducibility across experiments and samples. While healthy donor samples often suffice for method validation, cryopreserved PBMCs from patients with systemic lupus erythematosus (SLE) were utilized in this validation to ensure appropriate detection of all key subsets. The repeatability, inter-assay, inter-analyst, and inter-instrument precision were all evaluated in the fit-for-purpose validation. Recognizing the importance of time-sensitive and complex sample workflows, KCAS Bio additionally evaluated the post-fixation sample stability of the method. All of this was conducted to ensure the delivery of robust and reliable data generation with the comprehensive B cell panel method.
Final Thoughts
Recognizing the importance and prevalence of B cell involvement in disease there is a pressing need for advanced and scalable research tools to properly interrogate B cell phenotype, modulation, and function in both healthy and diseased individuals. The evolution of technology like spectral flow cytometry opens new paths for understanding the complexities of the immune response. Thoughtfully designed methods can transform our understanding of B cells in health and in disease.
By leveraging KCAS Bio’s validated Comprehensive B cell panel, researchers can confidently evaluate B cell subsets in human PBMC samples. Whether you are exploring disease processes or assessing a proprietary therapeutic’s impact, the right tools are key to your success.
Want to learn more? Let’s talk! Reach out and schedule a discussion today to learn how we can support your comprehensive B cell phenotyping needs.
References
Li, Y.-R. e. (2024). Frontiers in CAR-T cell therapy for autoimmune diseases. Trends in Pharmacological Sciences, 839-524.