Abstract 5485: A new 30-color spectral flow cytometry panel for T cell characterization

Abstract Spectral flow cytometers have facilitated the development of antibody panels with 30 colors or more to provide deep insights into the immune response. With a focus on T cell therapies, Labcorp has developed the 30-color immunophenotyping panel using the Cytek Aurora™ Spectral Flow Cytometer. The panel backbone includes 1 viability dye and 10 lineage markers to profile T cell, NK, and B cell subsets. 19 additional functional markers enable the characterization of activation, exhaustion, cytotoxicity, and proliferation in each subset, plus memory T cell subset delineation. Antibody-fluorophore combinations in a novel 30-color spectral flow cytometry panel were carefully considered and the resulting panel was developed utilizing primary PBMCs cultured with IL-2 and anti-CD3/CD28 to activate T cell subsets. Modulation of the expression levels for each functional marker was validated via comparisons made to unstimulated PBMCs. The most notable changes observed were in the expression of CCR7, CD25, ICOS, and Granzyme B which were expressed at levels between 60-95% of CD8+ T cells. Checkpoints including PD-1, LAG-3, TIM-3, and TIGIT were also upregulated, albeit to a lesser extent (<50% of CD8+ T cells). For memory phenotypes, analysis of CD45RA, CD45RO, CCR7, CD95, CD62L, and CD103 were combined to delineate Tn, Teff, Tte, Tcm, Tem, and Trm subsets. To provide an additional unbiased and wholistic analysis, t-SNE was conducted to deliver a comprehensive interpretation T cell functionality in the context of various exhausted phenotypic profiles. Finally, Nanostring® analysis using the nCounter® CAR-T Characterization panel was performed to further validate the modulation of gene sets underlying the TCR-driven PBMC activation. As expected, the transcriptomic analysis mirrored the flow cytometry immunophenotyping data with regard to activation and immunosuppressive gene modulation downstream of the TCR signaling. Taken together, a lymphoid panel was developed that measured multiple phenotypic and activity markers in T cells. Importantly, our data demonstrate how spectral flow cytometry can be applied to assess T cell therapies in preclinical tumor-bearing mouse models or clinical trials. Citation Format: David Draper, Brogan Yarzabek, Stacey Roys, Anita J. Zaitouna, Philip Lapinski, Amber Rowse, Scott Wise. A new 30-color spectral flow cytometry panel for T cell characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5485.