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. 2020 Feb 25;11:164. doi: 10.3389/fimmu.2020.00164

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Copyright © 2020 Garg, Ott, Mostofa, Chen, Chen, Kroeger, Cao, Mailloux, Agrawal, Schaible, Sarnaik, Weber, Berglund, Mulé and Markowitz.

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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Integration of MPATR output for lymphoid markers and delineation of cellular subsets associated with relapse-free survival (RFS) or treatment effects. (A) Unsupervised gating using the cut-offs for each marker generated from the MPATR violin plots (as shown for node 42) to delineate cellular subsets not easily found in bivariate plots. (B) Cox.1 analysis: Density plots of CD19⁺CD25^−/lo B cells (node 86 = CD19⁺CD25^−/loDAF-FM^{−/lo or+/lo}, hazard ratio = 1.777, p = 0.034) from representative short and long RFS samples prior to ipilimumab treatment. (C) Wilc.1 analysis: Representative density plots of CD3⁺CD8⁺ effector T cells (node 155 = CD3⁺CD8⁺CD25^−/loDAF-FM⁺, RFS ≤ 1 year, mean = −8.666, SD = 0.694; RFS > 1 year, mean = −8.066, SD = 0.956, p = 0.035) at the pre-treatment stage from pateints with short and long RFS. (D) Cox.2 analysis: Naïve or memory CD8⁺ T cells (node 53 = CD3⁺CD8⁺CD127^+/loDAF-FM⁺; hazard ratio = 2.787, p = 0.047) displayed from both RFS group post ipilimumab treatment. (E) Wilc.2 analysis: Density plots of CD8⁺ naïve or memory T-cell subset (node 150 = CD3⁺CD8⁺CD127^+or+/loCD25^−/loDAF-FM⁺, RFS ≤ 1 year, mean = −6.979, SD = 0.583; RFS > 1 year, mean = −7.421, SD = 0.577, p = 0.037) from patient samples post treatment. (F) Wilc.3 analysis: A subset of regulatory T cells (node 159 = CD3⁺CD4⁺CD127^−/loCD25⁺DAF-FM⁺, mean = −0.325, p = 0.029) demonstrating a downward trend after treatment without any association with RFS. (G) Cox.4 analysis: Representative density plots of CD11c⁺ natural killer cells (node 42 = CD56^+/loCD11c⁺CD25^−/loDAF-FM⁺, hazard ratio = 1.659, p = 0.018) showing inverse trends from samples of short and long term RFS in reponse to ipilimumab treatment. (H) Wilc. 4 analysis subset of rare CD4 CD8 double negative T cells (node 185 = CD3⁺ CD127^+or+/loCD25^−/loDAF-FM⁺, RFS ≤ 1 year, median = 0.510; RFS > 1 year, median = −0.084, p = 0.041) increased after treatment in a subset of short-term RFS patients and decreased in the majority of long-term RFS patients.