T cell exhaustion is associated with failure to clear chronic infections and malignant cells. Defining the molecular mechanisms of T cell exhaustion and reinvigoration is essential to improving immunotherapeutic modalities. Here we confirmed pervasive phenotypic, functional and transcriptional differences between memory and exhausted antigen-specific CD8+ T cells in human hepatitis C virus (HCV) infection before and after treatment. After viral cure, phenotypic changes in clonally stable exhausted T cell populations suggested differentiation toward a memory-like profile. However, functionally, the cells showed little improvement, and critical transcriptional regulators remained in the exhaustion state. Notably, T cells from chronic HCV infection that were exposed to antigen for less time because of viral escape mutations were functionally and transcriptionally more similar to memory T cells from spontaneously resolved HCV infection. Thus, the duration of T cell stimulation impacts exhaustion recovery, with antigen removal after long-term exhaustion being insufficient for the development of functional T cell memory.
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Identification of human progenitors of exhausted CD8+ T cells associated with elevated IFN-γ response in early phase of viral infection
Nature Communications Open Access 07 December 2022
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HCV epitope sequences are deposited in the NCBI Sequence Read Archive under accession numbers SRR11811484–SRR11811504. RNA-seq and TCR sequencing data from this study will be made publicly available through the NCBI Gene Expression Omnibus and/or NCBI database of Genotypes and Phenotypes. The remaining data supporting the findings of this study are available from the corresponding authors upon request.
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We are grateful to the patients participating in the clinical trial. Important contributions were made by the The Harvard Stem Cell Institute-Center for Regenerative Medicine (HSCI-CRM) Flow Cytometry Core Facility at Massachusetts General Hospital (MGH), the NIH Tetramer Core Facility and the University of Oklahoma Medical Center HLA Typing Facility (W. Hildebrand). We also thank all members of the laboratory of G.M.L. for insightful comments, critical reading of the manuscript and advice on figure design. This work was supported by NIH grants U19 AI086230 (to G.M.L., W.N.H., R.T.C., N.H., T.M.A. and A.Y.K.), U01 AI131314 (to G.M.L., L.L.L.-X. and A.Y.K.) and R01 DA046277 (to G.M.L. and A.Y.K.) and fellowships from the German Research Foundation DFG (to H.K.D. and L.M.B.). AbbVie sponsored the clinical trial (NCT02476617).
AbbVie sponsored the clinical trial (NCT02476617) and provided input in the trial design and clinical and biological sample collection schedule. W.N.H. is an employee of Merck and Company and holds equity in Tango Therapeutics and Arsenal Biosciences. All other authors declare no competing interests.
Peer review information Nature Immunology thanks Hazem Ghoneim, Barbara Rehermann and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Zoltan Fehervari was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Screening strategy to detect HCV-specific CD8+ T cells by flow cytometry using pools of Class I MHC multimers labeled with PE or APC. b, Positive detection was followed by individual multimer staining to identify and/or distinguish multiple HCV-specific CD8+ T cell responses. c, Representative flow cytometry dot plots of HCV-specific CD8+ T cells before and after magnetic bead enrichment. d, HCV-specific-multimer positive CD8+ T cell frequencies, pre- and post-DAA treatment or after spontaneous resolution of the infection. Statistical testing by Wilcoxon tests (paired, nonparametric, two-sided for TEX and TESC pre- and post-DAA) or Mann-Whitney tests (unpaired, nonparametric, two-sided when compared to Resolvers), *P < 0.05, **P < 0.01, ***P < 0.001. e, HCV-specific CD8+ T cell responses (recognized epitopes and associated HLA class I restrictions) detected in a cohort of patients with spontaneously resolved HCV infection.
a, Sequences of select HCV epitopes across patients included in this study. Escape mutations are written in red and T cell status, whether the cells have full recognition of the virus (TEX) or are partially (TP-ESC) or fully escaped (TF-ESC) is indicated as determined through functional assays of the recognition of the variant epitopes compared to wild-types by intracellular cytokine detection of IFNγ by flow-cytometry (b).
Extended Data Fig. 3 Phenotypical landscape of TEX and TESC, pre- and post-DAA, as compared to TMEM.
a, Flow cytometry gating strategy and representative flow cytometry dot plots. b, Dot plot histograms displaying the expression levels of the 37 proteins analyzed by flow cytometry across TEX and TF-ESC, pre- and post-DAA therapy, and in resolver TMEM. Statistical testing by Mann-Whitney tests when comparing TEX versus TF-ESC or TMEM (unpaired, nonparametric, two-sided), or by Wilcoxon tests (paired, nonparametric, two-sided) when comparing paired samples pre- versus post-DAA. A schematic representation of the comparison rules and statistical tests used are presented in Extended Data Fig. 3c. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. c, Schematic representation of the comparison rules and statistical tests used to compare expression levels across the different T cell populations of interest. d, Principal component analysis of TEX and TF-ESC, pre- and post-DAA therapy, as well as resolver TMEM, based on the expression levels of CD38, HLA-DR, PD-1, CD39, TIGIT, CCR7, CD45RA, Integrin-Beta-7 and CD62L, and as presented also in Fig. 3e by t-SNE analysis. e, Principal component analysis based on the expression levels of the 37 proteins analyzed by flow-cytometry and expressed by TEX, TP-ESC, TF-ESC and TFLU, pre- and post-DAA therapy, as well as by resolver TMEM, with respective contribution and direction (arrows) of each of the 37 different proteins throughout PC1 and PC2 dimensions.
Extended Data Fig. 4 Phenotypical and functional changes in TEX over a long-term period post-DAA cure.
a, Representative flow cytometry dot plots showing the expression and co-expression patterns of HLA-DR and CD38 (upper panels) as well as PD-1 and CD39 (lower panels) by bulk CD8+ T cells (grey dots) or HCV-specific CD8+ T cells (colored dots), pre- and overtime post- DAA therapy or after spontaneous resolution. b, Representative flow cytometry plots showing the expression and co-expression patterns of CD69 and CD107a (upper panels) as well as IFNγ and TNFα cytokines (lower panels) by HCV-specific CD8+ T cells following ex vivo stimulation with or without cognate antigens, pre- and overtime post- DAA therapy or after spontaneous resolution.
Extended Data Fig. 5 Transcriptional landscape of TEX and TESC, pre- and post-DAA, and as compared to TMEM.
a, Heatmap showing all genes that were differentially expressed between pre-DAA TEX and post-DAA TEX. b, Top 20 recovered genes after DAA treatment which are similar to resolver T cells. c, Heatmap showing the 176 significantly DEGs between pre- and post-DAA that are shared by TEX and TESC, as described in Fig. 6b. d, Heatmap showing the top 20 unrecovered genes after DAA treatment which were significantly different between post-DAA TEX and TMEM cells.
a, Expression (log2 counts) of EOMES, LMCD1, SETD7 and CTH in TEX and TESC pre- and post-DAA (paired samples n = 6) as well as in resolver TMEM cells (n = 8). Box plots show the median (vertical bar), 25th and 75th percentiles (lower and upper bounds of the box, respectively) and 1.5 times the interquartile range (or minimum/maximum values if they fall within that range; end of whiskers). Statistical testing by moderated t-test (two-sided, unadjusted). b, Linear regression analysis to model the relationship in gene count expression of TOX and the other transcription factors and co-factors identified in Fig. 6d, by the different populations of HCV-specific CD8+ T cells, pre- and post-DAA therapy or after spontaneous resolution. Error bands represents the 95% confidence level interval. Pearson correlation coefficient R and significance p (two-sided) values are reported from the linear regression analysis performed with R software. c, Gene count expression of TOX, ETV1, NKX3-1, SETD7, CTH, EOMES and LMCD1, by HCV-specific CD8+ T cells from a validation cohort of additional individual with TEX post-DAA (n = 9), as compared to resolver TMEM cells (n = 8), and following batch effect correction. Statistical testing by Mann-Whitney tests (two-sided), *P < 0.05, **P < 0.01, ***P < 0.001.
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Tonnerre, P., Wolski, D., Subudhi, S. et al. Differentiation of exhausted CD8+ T cells after termination of chronic antigen stimulation stops short of achieving functional T cell memory. Nat Immunol 22, 1030–1041 (2021). https://doi.org/10.1038/s41590-021-00982-6
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