Fig. 2: Chronic TCR stimulation disrupts proteostasis during T cell exhaustion.
From: Proteotoxic stress response drives T cell exhaustion and immune evasion
a, ImageStream analysis of protein aggregates in Teff and Tex cells 8 days after initial activation in vitro. Numbers are identifiers of representative cells within the samples. Scale bar, 7 µm. b, Flow cytometry quantification of protein aggregates (n = 8 for Teff and n = 7 for Tex, two-tailed t-test). MFI, mean fluorescence intensity. c,d, Protein aggregates in CD8+ T cell subpopulations from mouse MC38 tumours (c) and MB49 tumours (d) (n = 4 for spleens, n = 7 for TILs, one-way analysis of variance (ANOVA)). e, Flow cytometry histogram and bar plot of HPG incorporation in Teff and Tex cells in vitro (n = 4, two-tailed t-test). f, Flow cytometry histogram and bar plot of OPP incorporation in CD8+ T cell subpopulations from mouse MC38 tumours (n = 7, one-way ANOVA). g, Schematic of native PAGE and determination of the proteome by MS. Teff and Tex cells were generated as described in Extended Data Fig. 4d. h, Heatmap showing the fold change of 3,889 proteins in HMW and LMW species. i, Lack of association between aggregation tendency of proteins and their functional pathways. Bar colours depict the migration pattern of proteins as indicated in h. j, Fold changes of indicated protein abundances in HMW and LMW species. k, Schematic of cell lysate fractionation based on protein solubility. l, Immunoblot analysis of granzyme B (GZMB), perforin, gp96 and HSP90α in the soluble fractions of cell lysates separated by native PAGE. m, Immunoblot analysis of the indicated proteins in soluble fractions separated by SDS–PAGE. n, Immunoblot analysis of the indicated proteins in insoluble fractions resolved by SDS–PAGE. For l–n, the values on the left of the blots are kDa. For immunoblot source data, see Supplementary Fig. 1. Data in h–j are representative of two independent experiments. Experiments in l–n were repeated at least three times. Data are presented as the mean ± s.d. (b–f). The diagrams in g and k were created using BioRender (https://www.biorender.com).
