Driving Precision Insights with Spectral Flow Cytometry: a Focus on T Cells (Part 2)

Christy Bucks, Ph.D. February 17, 2025

Cell-based immunotherapies like Chimeric Antigen Receptor (CAR)-T cell therapy have been transforming the treatment of hematologic malignancies for more than a decade. Several groundbreaking studies (1) have led to a growing interest in applying these cell-based therapies to other T and B cell-mediated diseases. One emerging application of CARs is in the treatment of autoimmune disease.

Our understanding of autoimmune diseases is rapidly expanding, yet effective therapies remain limited. The number of clinical studies evaluating cellular therapies in autoimmune disease reflects the increased interest by industry. According to one publication, by mid-2024, there were more than 65 ongoing clinical trials evaluating anti-CD19 and anti-BCMA CAR-T therapies in autoimmune disease. Additional trials are ongoing to assess anti-CD7 CAR-T’s, CAR-Treg, and novel Chimeric Autoantigen Receptor (CAAR) T cells. Collectively, these trials target more than 25 different autoimmune diseases (Abigail Cheever, 2024).

Given the emerging interest in applying CAR-T-based therapies for treating autoimmune disease and the recognition that these diseases are driven by both T and B cells, there is a need for a deep understanding of T cell subsets. In part one of this blog series, we explored the importance of phenotyping B cells. Here, we take a deeper look at T cells and highlight the importance of reliable cell phenotyping by high-dimensional spectral flow cytometry.

T cells and Autoimmunity

T cells are critical mediators of adaptive immunity in a regulated immune system. Known for their central role in protection from pathogens, activated T cells have a wide array of activities from cytokine production to cytolytic function. When properly engaged, T cells are responsible for resolving viral infections, providing effective memory against future infection, supporting B cell activation and antibody production, or, in the case of regulatory-T cells maintaining immune tolerance.

Conversely, dysregulated T cells have been shown to contribute to the development and progression of autoimmune diseases. Key subsets such as regulatory T cells (Tregs), T helper 1 (Th1), and T helper 17 (Th17) cells are pivotal in maintaining immune homeostasis or, conversely, driving autoimmune pathology. As just one example of T cell involvement in autoimmunity, activating Th2, Th17 and follicular helper T cells is known to support B cell activation and subsequent autoantibody production in Systemic lupus erythematosus. On a broader scale, mediators of disease can be widely attributed to the complex interactions of autoreactive T cells, autoreactive B cells and autoantibodies which are inappropriately targeting “self” antigens. Self-targeting results in a difficult-to-break cycle of chronic inflammation and cell activation, leading to long-term tissue and organ damage.

Although each autoimmune indication is relatively rare, collectively this category impacts as many as 1 out of every 10 people in the US and more than 350 million people globally. Autoimmune diseases have a significant impact on individual quality of life and can have a social and economic burden for those seeking effective treatment. Given T cells’ complex role in autoimmune disease, understanding T cell dynamics is crucial for developing targeted therapies, including biologics, small molecules, and advanced cell therapies.

Development of therapies targeting T cells requires characterization of potentially rare and unique cell subsets. Technologies like spectral flow cytometry enable this type of complete characterization. High-dimensional analysis yields deeper and more precise insights into T-cell phenotypes and functions that can finally shed light on complex mechanisms of action and fine-tuned immune responses. This capability enhances the understanding of T cell biology and can support the development of effective therapies for T cell-driven autoimmune diseases.

A Turnkey Solution: Accelerating Science with a Validated Flow Cytometry Solution

As a solution-focused organization whose mission is to accelerate your science, the experts at KCAS Bio have focused on expanding our library of off-the-shelf flow cytometry methods. In our first blog post of this series, we highlighted our newest B cell panel offering, which provides a method for deep phenotyping of B cell populations. Given the need for high dimensional T cell analysis, we recently completed validation of 21-color spectral flow cytometry method that is entirely focused on discrimination of T cell subpopulations in human PBMC samples. With a comprehensive phenotyping goal, this validated spectral flow cytometry method is designed to enable evaluation of T cell subpopulations such as:

T helper cells
Cytotoxic T cells
Naïve and memory subsets, including central and effector memory T cells
Activated T cells
Follicular Helper T cells
Regulatory T cells
Th1, Th2, and Th17 cells
Proliferating and PD-1+ exhausted cells

KCAS Bio’s off-the-shelf and custom methods are rigorously validated based on the context of use. The comprehensive T-cell panel is no exception. Repeatability, inter-assay, inter-analyst, inter-instrument precision, and post-fixation sample stability were all evaluated in a fit-for-purpose validation. More than 150 reportables can be determined from the validated, comprehensive T-cell panel. This validated method offers an efficient, scalable approach to evaluating samples with reduced costs and timelines when used as an off-the-shelf offering. When used as a starting backbone, this method offers cost and time value to sponsors by allowing efficient customization, enabling programs to move quickly forward.

Final Thoughts

Understanding both B and T cells through specialized technologies enables the identification of novel biomarkers, better patient stratification, and more precise evaluation of therapeutic efficacy. By leveraging technologies like high-dimensional spectral flow cytometry, scientists in all stages of research, whether in discovery, pre-clinical, or clinical stage programs, can gain deeper insights into the phenotype and function of immune cells.

By leveraging KCAS Bio’s validated Comprehensive T cell panel, researchers can confidently evaluate T cell subsets in human PBMC samples. Whether exploring a mechanism of action 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 T cell phenotyping needs.