Profiling Tumor-Related T Cells by Flow Cytometry

Flow cytometry was performed on PBMCs from baseline, after Cycle 1, after Cycle 3, and after surgery using an antibody panel designed to identify tumor-related T cells, Effector cytotoxic T lymphocytes (CTLs), and pro-apoptotic T cells. To perform flow cytometry analysis of the phenotype and functional potential of immune cells, the following antibodies were used as per published protocols^{43 (link),44 (link)}: CD8-PE-Cy7 (clone RPA-T8, catalog 304006, BD Pharmingen), CD11a-APC (clone HI111, catalog 301212, BioLegend), PD-1 fluorescein isothiocyanate (FITC) (clone EH12.2H7, catalog 32990, BioLegend), CX3CR1-APC/Cy7 (clone 2A9-1, catalog 341616, BioLegend), Bim-PE (clone C34C5, catalog 12186S, Cell Signaling Technology), NKG7 monoclonal antibody (AG1490 Rb mAb 8H3/8K3)-FITC conjugated (Fusion antibodies with a contract with Dong lab). T cells were stained for surface markers before intracellular staining. Data was collected on a CytoFLEX LX (Beckman Coulter, Atlanta, GA) and analysis was performed with the R software version 4.1.1.

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Hieken T.J., Nelson G.D., Flotte T.J., Grewal E.P., Chen J., McWilliams R.R., Kottschade L.A., Yang L., Domingo-Musibay E., Dronca R.S., Yan Y., Markovic S.N., Dimou A., Montane H.N., Erskine C.L., Piltin M.A., Price D.L., Khariwala S.S., Hui J., Strand C.A., Harrington S.M., Suman V.J., Dong H, & Block M.S. (2024). Neoadjuvant cobimetinib and atezolizumab with or without vemurafenib for high-risk operable Stage III melanoma: the Phase II NeoACTIVATE trial. Nature Communications, 15, 1430.

Publication 2024

CD8-PE-Cy7 (clone RPA-T8, FITC) (clone EH12.2H7, CytoFLEX LX

Corresponding Organization :

Other organizations : Mayo Clinic in Arizona, Mayo Clinic in Florida, University of Minnesota, WinnMed, Mayo Clinic

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Variable analysis

independent variables

Cycle 1
Cycle 3
Surgery

dependent variables

Phenotype and functional potential of immune cells
Tumor-related T cells
Effector cytotoxic T lymphocytes (CTLs)
Pro-apoptotic T cells

control variables

Baseline

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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