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. 2021 Nov 16;12:765644. doi: 10.3389/fimmu.2021.765644

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Copyright © 2021 Schilling, Glehr, Kapinsky, Ahrens, Riquelme, Cordero, Bitterer, Schlitt, Geissler, Haferkamp, Hutchinson and Kronenberg

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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The MoT test captures information about tumor staging. Fresh whole EDTA blood from patients with stage II (n = 52), stage III (n = 21) or stage IV (n = 27) melanoma were investigated using the MoT test. SPADE analysis of Lin^- cells was performed in Cytobank with a density-dependent down-sampling target of 10% and a 200-cluster spanning tree. (A–E) Five clusters with significant stage-dependent differences were identified (K.W. test; B.H.-adjusted p-values, FDR = 5%). (F–J) All five significant SPADE clusters were re-exported and analyzed as follows: gating on CD3⁺ T cells; gating on CD8⁺ or CD4⁺ T cells; gating on memory T cell subsets (CD45RA/CD197); gating on CD279 cells and gating on CD14^-/CD16^- cells.