ZnT8186–194-reactive CD8+ T cell clones from patient D222D.

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ZnT8186–194-reactive CD8+ T cell clones from patient D222D. ZnT8186–194-reactive CD8+ T cell clones from patient D222D. (A) Frozen-thawed PBMCs were cultured with ZnT8186–194 or no peptide and stained with phycoerythrin (PE)/allophycocyanin (APC)–labeled ZnT8186–194 MMrs. (B) ZnT8186–194 and control MMr stains for one clone obtained from single-sorted ZnT8186–194/ZnT8186–194 double-MMr+ cells. (C) Percentage of intracellular TNF-α+ D222D clone 1 cells stimulated for 6 hours with K562-A2 cells pulsed with ZnT8186–194 or Flu MP58–66 peptide. (D) Percent lysis of Far Red–labeled LCL targets pulsed with ZnT8186–194 (top) or Flu MP58–66 peptide (bottom) and cultured for 24 hours with CFSE-labeled D222D clone 3 at increasing E/T ratios. (E) Percent lysis of LCL targets cultured with D222D clone 1, 2, or 3 (mean ± SEM; each clone is depicted in fig. S1, D to F). (F) Lysis of cognate peptide–pulsed LCLs cultured for 4 hours with D222D clone 2 or a MelanA26–35-reactive clone (E/T, 1:1) in the presence of concanamycin A (CMA), brefeldin A (BFA), CMA and BFA, anti-FasL (aFasL), or control IgG1 (immunoglobulin G1). *P = 0.015, **P = 0.009, and ***P < 0.001 by Student’s t test. Results are mean ± SEM of triplicate measurements from one of three experiments. (G) Percentage of surface CD107a+ D222D clone 1 cells stimulated as in (C). For (A), (C), and (G), the gate is on viable CD8+ cells. Slobodan Culina et al. Sci. Immunol. 2018;3:eaao4013 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works