Fig 1.
CD39 is highly expressed by virus-specific CD8+ T cells in chronic viral infection.
(A) Expression of CD39 by virus-specific CD8+ T cells. Plots are gated on CD8+. (B) Fraction of total or antigen-specific CD8+ T cells expressing CD39. (C, D) Comparison of CD39 expression by total CD8+ T cells with virus-specific CD8+ T cells from patients with HCV (C) and HIV (D) infections. (E) Fraction of total CD8+ T cells expressing CD39 in healthy, HIV or HCV infected donors. Error bars represent SEM. Statistical significance was assessed by Kruskal-Wallis test (B, E), or Wilcoxon test (C, D). *P <0.05, ***P <0.001, ****P <0.0001.
Fig 2.
CD39 expressed by CD8+ T cells in HCV infection is enzymatically active.
(A) Flow cytometry sorting gates of CD39+ and CD39– CD8+ T cells and CD39+ CD25+ CD4+ Tregs used for rpHPLC analysis of CD39 activity. (B) Summary of CD39 expression level relative to Tregs in the same subjects. (C) ATP hydrolysis by CD8+ T cell populations relative to Tregs. Data represent 6 patients with chronic HCV infection. Error bars represent SEM. Statistical significance was assessed by paired Student’s t-test (B, C). *P <0.05, **P <0.01.
Fig 3.
CD39 expression correlates with PD-1 expression and viral load in chronic viral infection.
(A) CD39 and PD-1 expression in chronic HCV (left) or HIV infection (right). Representative plots demonstrate total (gray) and virus-specific (red) CD8+ T cells. (B) Correlation between CD39 and PD-1 expression by HCV- (left) and HIV-specific (right) CD8+ T cells. (C) Correlation between CD39 expression by virus-specific CD8+ T cells and viral load count in HCV (left) or HIV (right) infection. (D) Correlation between PD-1 expression by virus-specific CD8+ T cells and viral load in HCV (left) or HIV (right) infection. Correlation was assessed by Pearson correlation coefficient (B, C, D). MFI; mean fluorescence intensity.
Fig 4.
Transcriptional analysis of CD39+ and CD39– CD8+ T cells in HCV infection.
(A) Consensus hierarchical clustering of expression profiles from CD39+ (black) and CD39– (grey) CD8+ T cells from 8 HCV infected patients. Clustering is based on the top 10% of genes by variance across the dataset. Sample similarity (1-Pearson correlation coefficient) is annotated with color from low (white) to high (green). (B) Gene set enrichment map displaying Gene Ontology gene sets enriched (FDR < 0.1) in CD39+ CD8+ T cells from (A). Nodes (in red) are sized in proportion to gene set size; connecting line thickness represents extent of gene member overlap between gene sets. (C) Gene set enrichment analysis of a signature of 200 genes up-regulated in exhausted CD8+ T cells from the mouse model of chronic viral infection versus acute infection (day 30 post infection) in the ranked list of genes differentially expressed by CD39+ vs. CD39– CD8+ T cells. Leading edge genes are indicated by orange symbols. (D) Volcano plot of all genes (grey) or exhausted leading edge genes (orange) from (C).
Fig 5.
CD39 is highly up-regulated by exhausted CD8+ T cells in a mouse model of chronic infection.
(A, B) Expression of CD39 and PD-1 by CD44– naive mouse CD8+ T cells (A) and in CD8+ T cells at indicated times following LCMV Armstrong (acute) or Clone 13 (chronic) infection (B). Representative plots show total (black) and H-2Db GP276-286 tetramer-specific CD8+ T cells (red). Summary of results in 5 mice per group is shown in bar-graphs on the right. Statistical significance was assessed with Mann-Whitney test. *P < 0.5, **P < 0.01.
Fig 6.
CD39 identifies terminally exhausted CD8+ T cells in mice with chronic LCMV infection.
(A) Expression of CD39 and CD44+ by mouse CD8+ T cells 30–35 days following LCMV Armstrong (left) or Clone 13 (right) infection. (B, C) Representative histograms (left) of CD127 (B) and PD-1 (C) expression by CD39high and CD39int CD8+ T cells from Clone 13 (red and blue, respectively) and CD39int from Armstrong (filled gray) infected mice on d35 p.i. (left). Fraction of CD127+ (B) and MFI of PD-1 in PD-1+ cells (C) is shown on the right. Results are from 5 mice. (D) Fraction of CD39high and CD39int CD44+ CD8+ T cells expressing different combinations of co-inhibitory receptors PD-1, 2B4, and Lag3. (E) Average number of co-inhibitory receptors expressed by CD39int (left) or CD39high (right) CD8+ T cells at d35 p.i. following LCMV Clone 13 infection. (F) Representative plots of T-bet and Eomes expression in CD39int (left) and CD39high (right) cells as in (A). Summary of results is shown on the right. Data are representative of three experiments of 5 mice per group. Statistical significance was assessed with Student’s t-test (B, C, F) with Holm-Sidak multiple comparison correction (D). **P < 0.01, ****P < 0.0001.
Fig 7.
Terminally exhausted CD8+ T cells marked by high levels of CD39 are most impaired in their effector function.
(A) Representative plots showing the production of IFN-γ and TNFα in CD39int or CD39high CD8+ T cells 36 days following LCMV Clone 13 infection. (B) Quantification of cells in (A) that produce both TNFα and IFN-γ relative to IFN-γ only. (C, D) Cytokine production by P14 cells (C) gated from an infection as in (A) and summary of IFN-γ and TNFα producing cells. (E, F) Mean fluorescence intensity (MFI) of IFN-γ in IFN-γ positive endogenous (E) and transferred P14 cells (F). Statistical significance was assessed with paired Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.