28-color, 30 parameter flow cytometry to dissect the complex heterogeneity of tumor infiltrating T cells in human lung adenocarcinoma
E. Lugli1,*, J. Brummelman1, F. Colombo1, F. Clemente1, F. De Paoli1, G. Rotta2, P. Novellis1, G. Veronesi1
1Humanitas Clinical and Research Center; 2BD Biosciences, Milan, Italy
The heterogeneity of tumor-infiltrating lymphocytes and their relative pro- or anti-tumor potential can only be addressed by more powerful single cell approaches. By using antibodies conjugated to new dyes excited by 5 lasers mounted on the FACS Symphony A5 (BD Biosciences), we developed 28-color FACS to profile millions of single T cells from human lung adenocarcinomas. BUV, BV and BB dyes were brighter than many standard dyes regularly used in polychromatic FACS, thereby allowing extreme flexibility in panel development and better sensitivity in detecting dimly expressed proteins. Spreading Error (SE), resulting from errors in the measurement of fluorescence, but not spillover compensation was the main determinant of the success of 28-color panels: indeed, dye combinations with >400% compensation but limited SE could be easily included. We used computational barcoding coupled to single cell PCA and t-SNE to reduce dimensionality and identify correlated or anti-correlated markers and thus putative functional subsets in the blood, the tumor and in the non-tumoral portion of the lung from the same patient (n=16). We thus revealed that PD-1bright exhausted CD8+, enriched at the tumor site, are mainly confined to the CD69+ tissue-resident memory compartment and are T-bet+HLA-DR+, while the same are nearly absent from the early-differentiated CCR7+ compartment. The latter, in turn, appeared to be recruited from the circulation. Pure naïve CD8+, defined by 5 markers, were virtually absent in tumors, hence suggesting that neoantigen priming might not occur in situ. Terminally-differentiated CD57+T-betlowEomeshigh T cells did not include PD-1bright cells, confirming that exhaustion and senescence are divergent states. High-content profiling is currently being performed in dozens of samples and integrated with clinical and metabolic parameters.
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