Differentiation of exhausted CD8+ T cells after termination of chronic antigen stimulation stops short of achieving functional T cell memory

Abstract

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.

Trial registration: ClinicalTrials.gov NCT02476617.

Conflict of interest statement

COMPETING INTERESTS

AbbVie sponsored the clinical trial (NCT02476617) and provided input to 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.

© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.

Figures

Extended Data Fig. 1. Detection and frequency of HCV-specific CD8+ T cells

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.

Extended Data Fig. 2. HCV-epitope sequences and identification of functional escape mutations

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.

Extended Data Fig. 6. Expression patterns of key transcription factors and co-factors

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.

Figure 1:. Study design, patients and virus-specific CD8+ T cell responses.

a, Overview of the clinical trial phases and longitudinal monitoring of HCV viral load. b, Study-subject virus-specific CD8+ T cell responses detected by MHC class I multimers. Targeted HCV-epitopes were sequenced and grouped as “conserved / exhausted (TEX)” (red circles), “mutated / partially escaped (TP-ESC)” (orange circles), or “mutated / fully escaped (TF-ESC)” (green circles) according to subsequent testing of the T cell recognition of the variant compared to wild-type epitopes through intracellular staining of IFNγ (Extended Data Fig, 2). c, Dot plot histograms displaying CD127, PD-1, CD39 and Eomes expression by HCV-specific TEX, TP-ESC and TF-ESC as well as by EBV-, FLU- and CMV-specific T cells, pre-DAA therapy. 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 Mann-Whitney tests (unpaired, nonparametric, two-sided), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. d, Representative flow cytometry plots of virus-specific CD8+ T cells from patient 104, and related HCV epitope-sequences.

Figure 2:. HCV-specific TEX have a characteristic phenotype that is significantly changed after DAA therapy and antigen removal.

a, Representative flow cytometry dot plots of the changes in CD38 expression pre- and post-DAA therapy. b, Deep immune-profiling analysis of TEX HCV-specific CD8+ T cells (paired samples, n=14) before (red plot) and after (blue plot) DAA-treatment. Data are expressed as percentage of cells expressing the listed markers. Changes in expression level, defined by the median fluorescence intensity (MFI) for PD1, CD95 and TIGIT before and after DAA treatment are also indicated in the right panels. Statistical testing by Wilcoxon tests (paired, nonparametric, two-sided), *P < 0.05, **P < 0.01, ***P < 0.001. c, Phenotypic changes in TF-ESC HCV-specific CD8+T cells targeting mutated epitopes (paired samples, n=8) pre- and post-DAA treatment. Phenotypic changes in CMV- (paired samples, n=8), EBV- (paired samples, n=14), and influenza-specific (paired samples, n=13) CD8+T cells pre- and post-DAA treatment are displayed in d, e and f, respectively. g, Correlation plots displaying the frequencies of the different clonotypes identified by TCR sequencing between pre- and post-DAA therapy. Each dot represents a unique TCR clonotype. h, TCR clonotype distribution in n=8 different T cell populations (TEX, n=3; TP-ESC, n=2; and TF-ESC, n=3) between pre- and post-DAA treatment.

Figure 3:. The phenotypic change of TEX towards TMEM after HCV cure remains incomplete.

a, Comparison of the phenotypic immune signature of post-DAA TEX (n=14) to that of TMEM from spontaneous HCV-resolvers (n=10). b, Comparison of the TEX phenotypic immune signature (n=14) to that of TF-ESC (n=8) pre-DAA therapy. c, Comparison of the TEX phenotypical immune-signature (n=14) to TF-ESC (n=8), post-DAA therapy. a-c, Data are expressed as the percentage of cells expressing the listed markers. Dot plot histograms comparing expression levels of the 37 proteins studied across the different HCV-specific CD8+ T cell populations are presented in Extended Data Fig. 3b. Statistical testing by Mann-Whitney tests (unpaired, nonparametric, two-sided). d, Dot plot histograms displaying CD127, TCF-1, PD-1 and Eomes expression levels 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 is presented in Extended Data Fig. 3c. a-d, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. e, t-SNE 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. Expression levels (MFI) of CD38 and PD-1 are displayed using a color scheme. f, Principal component analysis of the expression levels of the 37 proteins, as detected by flow cytometry, by TEX, TP-ESC, TF-ESC and TFLU, pre- and post-DAA therapy, as well as by resolver TMEM. Respective contributions of the 37 different proteins in driving PC1 and PC2 are depicted in the right panel.

Figure 4:. Functional analysis reveals that TESC, but not TEX after viral cure, display functional properties similar to those of TMEM from HCV natural resolvers.

a, Representative flow cytometry plots of the cytokine production and cytotoxicity capability of HCV-specific CD8+ T cells following ex vivo stimulation with or without cognate antigen. The co-expression patterns and percentage of cells producing IFNγ, TNFα and IL-2 cytokines and expressing CD107a are indicated. b, Dot plot histograms displaying IFNγ, TNFα and IL-2 production as well as CD107a expression across TEX (paired samples, n=8) and TESC (paired samples, n=7) pre- and post-DAA therapy, and in resolver TMEM (n=8). Triangles identify two analyzed TP-ESC populations. 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. c, Overlapping pie-charts describing the polyfunctionality of TEX (paired samples, n=8) and TESC (paired samples, n=7) pre- and post-DAA therapy, as well as TMEM (n=8), after ex vivo stimulation with cognate antigens. d, Frequencies of T cells with one, two and three or more functions as defined by the expression or co-expression of CD107a, IFNγ, TNFα and IL-2 after stimulation with cognate antigens. Statistical testing by Mann-Whitney tests (unpaired, nonparametric, two-sided). b,d, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5:. The TEX phenotypic and functional profile shows limited evolution over time post-HCV cure.

a-b, Temporal dynamics of expression levels of CD38, HLA-DR and PD-1 (a), as well as CD127, EOMES, and CD39 (b), by TEX from four chronic HCV patients treated with DAA. c-d, Functional analysis of CD69 and CD107a (c), as well as IFNγ and TNFα production (d) after ex vivo stimulation with cognate antigen. Expression levels are displayed as percentage of expression except for PD-1, which is expressed as MFI. Expression levels in TMEM from spontaneous resolvers are displayed for comparison.

Figure 6:. Transcriptional analysis confirms broad changes in TEX after removal of antigen, but also identifies exhaustion scars in the transcriptional landscape.

HCV-specific MHC-multimer-sorted CD8+ T cells from patients pre- and post-DAA treatment were compared with those from patients who resolved infection spontaneously. a, b, Venn diagrams showing the number of differentially expressed genes (DEGs) and overlap between TEX pre- vs post-DAA therapy (paired samples, n=6), vs TMEM from resolver patients (n=8) (a), or vs TESC (paired samples n=6) (b). c, Principal component analysis of gene expression profiles from pre-DAA TEX, post-DAA TEX, and resolver TMEM cells. d, Gene set enrichment analysis (GSEA) of the transcriptional signature “reactome PD1 signaling (C2)” and “up in effector vs memory CD8+ T cell (C7)” enriched in pre-DAA TEX cells compared post-DAA (left panel) or in pre-DAA TESC cells compared post-DAA (right panel). e, GSEA of transcriptional signature “reactome translation (C2)” and “down in KLRG1 high vs low effector CD8+ T cells (C7)” enriched negatively in resolver TMEM cells compared to post-DAA TEX cells (left panel) or in pre-DAA TESC cells compared to post-DAA (right panel). f, Plot showing the log2 fold change difference of transcription factors and cofactors between post-DAA TEX and resolver T cells with respect to pre-DAA TEX cells. Transcription factors and cofactors that did not recover are highlighted in yellow font and with a red frame if statistical validation was made on 9 additional TEX populations post-DAA therapy and compared to TMEM (Extended Data Fig. 6c). g, Expression (log2 counts) of TOX, TCF7, ETV1 and NKX3–1 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).