60 Assessing infiltrating lymphoid activation state in human samples via a new 30-color spectral flow cytometry panel

<h3>Background</h3> Flow cytometry is a common method to assess immune cell infiltration into tumors. Spectral flow cytometers are now being used to develop larger antibody panels to increase the number of immune markers analyzed. With a focus on immunotherapy and adoptive cell therapy, we have developed the 30-color Spectral CompLymphocyte™ Panel using the Cytek Aurora™ Spectral Flow Cytometer (table 1). The panel backbone includes viability and 10 lineage markers to measure live 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. <h3>Methods</h3> We carefully considered each antibody-fluorophore combination in a novel 30-color spectral flow cytometry panel and titrated each antibody to determine the optimal concentration. Human PBMCs and Veri-cells (BioLegend) were used to develop the panel. PBMCs were stimulated with anti-CD3/-CD28 or with IL-15. Changes in expression of activation markers were compared between stimulated and unstimulated PBMCs. Veri-cells were used as stabilized control samples to assess reproducibility across experiments. Additional experiments were performed to assess sample stability and intra-experimental precision, which were found to be within the acceptable range (less than 25% variance). Assay stability was measured by comparing freshly stained cells with cells that had been stored at 4°C for up to 72h. t-SNE analysis was conducted to measure changes in surface activation and exhaustion markers. <h3>Results</h3> Staining of Veri-cells confirmed that the activated T cells showed the expected upregulation of activation markers in stabilized samples. Stimulated and unstimulated PBMC samples were stained and run in two independent experiments to measure the reproducibility of the assays. Stability of 14 different activation markers was assessed on T, B, and NK cells into highly stable (4 markers), moderately stable (7 markers), and unstable (3 markers). PBMC samples with two stimulation conditions were utilized to determine the ability to broadly assess activation changes. Anti-CD3/-CD28 stimulated T cell expansion, resulting in increased expression of activation and exhaustion markers across the board as well as a shift from naïve to effector and memory phenotypes. Low dose IL-15 stimulated NK cell expansion (increasing CD69 expression) and T cell maintenance. <h3>Conclusions</h3> With the use of spectral flow cytometry, we developed a lymphoid panel that measures multiple activity markers in multiple cell types, demonstrating the utility of this panel in future studies for clinical PBMC or tumor samples as well as pre-clinical cell and gene therapy samples.